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WO2025076210A1 - Chlamydia vaccine compositions - Google Patents

Chlamydia vaccine compositions Download PDF

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Publication number
WO2025076210A1
WO2025076210A1 PCT/US2024/049776 US2024049776W WO2025076210A1 WO 2025076210 A1 WO2025076210 A1 WO 2025076210A1 US 2024049776 W US2024049776 W US 2024049776W WO 2025076210 A1 WO2025076210 A1 WO 2025076210A1
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Prior art keywords
cpaf
polypeptide
adjuvant
amino
nnaa
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PCT/US2024/049776
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French (fr)
Inventor
James Rozelle
Jeff Fairman
Taylor POSTON
Lee Antoinette DARVILLE
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University of North Carolina at Chapel Hill
Vaxcyte Inc
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University of North Carolina at Chapel Hill
Vaxcyte Inc
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Publication of WO2025076210A1 publication Critical patent/WO2025076210A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/118—Chlamydiaceae, e.g. Chlamydia trachomatis or Chlamydia psittaci
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response

Definitions

  • urethritis, epididymitis, and orchitis are the most common acute manifestations, and while long termsequelae are not definitely associated with a CT genitourinary infection in males, proctitis, prostatitis, and prostate cancer have been linked to CT particularly in men that have sex with men (MSM). Most infections are asymptomatic (70%) and thus frequently untreated, despite effective antibiotics. Lymphogranuloma venereum (LGV) infections can result in highly debilitating chronic sequelae with bubo formation, fistulas, fibrosis, and rectal stenosis. Among MSM, LGV infections are on the rise. In low- and middle-income countries, CT-induced trachoma is also the main cause of preventable blindness.
  • LGV Lymphogranuloma venereum
  • the CPAF polypeptide may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or more nnAA.
  • Each nnAA may substitute one or more amino acids relative to a reference CPAF protein, of which may independently be one or more of phenylalanine, lysine, and tyrosine.
  • the site of the substitution may be F130 or Y569 relative to the sequence set forth in SEQ ID NO: 1.
  • Each nnAA may comprise 2-amino-3-(4- azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin- 2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2- yl)propanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid.
  • Each nnAA may be pAMF.Each nnAA may comprise a structure as set forth in formula III: wherein:
  • W 5 is selected from Ci-Cio alkylene, -NH-, -O- and -S-;
  • QI is zero or 1
  • W 6 is selected from the group consisting of azido, 1,2,4,5-tetrazinyl optionally C- substituted with a lower alkyl group, and ethynyl, and R 3 is OH or an amino acid residue of the CPAF polypeptide, and R 4 is H or an amino acid residue of the CPAF polypeptide.
  • the STING agonist may comprise di amidobenzimidazole (diABZl), MSA-2ADU-S100, MK-1454, MK-2118, SB11285, BMS- 986301, DMXAA, E7766, GSK3745417 cyclic di-GMP (guanosine 5 '-monophosphate) (CDG), cyclic di -AMP (adenosine 5 '-monophosphate) (CD A), cyclic GMP-AMP (cGAMP), 5,6- Dimethylxanthenone-4-acetic acid, AS03, MK-1454, TMX-202, or a derivative thereof.
  • di amidobenzimidazole di amidobenzimidazole
  • MSA-2ADU-S100 MSA-2ADU-S100
  • MK-1454 MK-2118
  • SB11285285 - 986301 DMXAA
  • E7766 GSK3745417
  • cyclic di-GMP guanosine 5
  • R2 is independently OH or at least one amino acid of the CPAF polypeptide
  • each of Wl, W2, and W3 is independently a single bond or lower alkylene
  • each XI is independently — NH — , — O — , or — S — ;
  • L22 is independently a bond, alkyl or poly(alkyloxy);
  • X is the adjuvant.
  • Ri is independently H, formyl, or at least one amino acid of the CPAF polypeptide
  • W is C or N
  • a CPAF polypeptide conjugate comprising a catalytically inactive CPAF polypeptide and a pharmaceutical composition comprising the conjugate
  • the catalytically inactive CPAF polypeptide may comprise one or more substitutions of one or more wild-type amino acids of a reference CPAF with a non-natural amino acid (nnAA). Each nnAA may be covalently attached to an adjuvant.
  • the CPAF polypeptide conjugate may comprise a CPAF polypeptide comprising one or more substitutions at one or more of amino acids H97, S499, S491, E550, and C492 relative to the sequence set forth in SEQ ID NO: 1.
  • the subsitutions may comprise one or more of H97A, S491A, S499A, C492T, and E550Q.
  • the CPAF polypeptide may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or more nnAA.
  • Each nnAA may substitute one or more amino acids of a reference CPAF protein, which may each independently comprise phenylalanine, lysine, and tyrosine.
  • the site of substitution may be F130 or Y569 relative to the sequence set forth in SEQ ID NO: 1.
  • Each nnAA may comprise 2-amino-3-(4- azidophenyljpropanoic acid (pAF), 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid.
  • pAF 2-amino-3-(4- azidophenyljpropanoic acid
  • pAMF 2-amino-3-(4-(4- (azidomethyl)phenyl)propanoi
  • the nnAA may be pAMF.
  • the nnAA may comprise a structure as set forth in formula III.
  • the CPAF polypeptide conjugate may comprise the amino acid sequence set forth in any one of SEQ ID NOS: 14-22, or a fragment thereof, or a sequence at least about 80% identical thereto.
  • Each nnAA may be covalently attached to an adjuvant.
  • the conjugated adjuvant may be a water-soluble adjuvant.
  • the adjuvant may enhance the Thl-based immune response.
  • the adjuvant may comprise a STING agonist, TLR agonist, or a dopamine receptor agonist.
  • the STING agonist may comprise di amidobenzimidazole (diABZl), MSA- 2ADU-S100, MK-1454, MK-2118, SB11285, BMS-986301, DMXAA, E7766, GSK3745417 cyclic di-GMP (guanosine 5 '-monophosphate) (CDG), cyclic di-AMP (adenosine 5'- monophosphate) (CD A), cyclic GMP-AMP (cGAMP), 5,6-Dimethylxanthenone-4-acetic acid, AS03, MK-1454, TMX-202, or a derivative thereof.
  • the TLR agonist may comprise 2BXy, DOPA, 2Bxy-DOPA, an imidazoquinoline-based agonist, CpG, a CpG derivative, CpG1826, CpG1018, or poly-C-CpG, or a derivative of the foregoing.
  • Each nnAA may be linked to the to the adjuvant via a first handle on the adjuvant.
  • the first handle may comprise propargyl, DIFO, dibenzylcyclooctyne (DBCO), or a DBCO(PEG)n- NH2 moiety, or a derivative thereof.
  • Each nnAA may form a triazole linkage with the adjuvant.
  • the adjuvant may be attached to the first handle.
  • the first handle may comprise CpG-DBCO, STING-DBCO, 2BxY-DBCO, 2BxY-DOPA-DBCO, or CDA-DBCO, and may comprise a structure as set forth in formula Xlld-XIIh respectively.
  • compositions comprising the CPAF polypeptide conjugate disclosed herein and one or more pharmaceutically acceptable excipients, additional soluble adjuvants, or both.
  • the additional soluble adjuvant be any adjuvant.
  • the additional soluble adjuvant may be an enhancer of Th-1 based immunity.
  • the additional soluble adjuvant may be a different adjuvant than the adjuvant covalently attached to the CPAF polypeptide.
  • the one or more pharmaceutically acceptable excipients may be suitable for mucosal delivery.
  • the pharmaceutical composition may be for use as a vaccine against a chlamydia infection in a mammalian subject, or may be for use in the manufacture of a medicament for use as a vaccine.
  • the pharmaceutical composition may comprise one or more additional polypeptide conjugate, wherein the additional polypeptide conjugate comprises a different polypeptide or a different adjuvant, relative to the CPAF polypeptide and the adjuvant.
  • the pharmaceutical composition may comprise a second vaccine against chlamydia or one or more other microorganisms, or combinations thereof.
  • a CPAF polypeptide-adjuvant conjugate may comprise contacting a catalytically inactive CPAF polypeptide with an adjuvant to form a CPAF polypeptide-adjuvant conjugate.
  • the CPAF polypeptide may comprise one or more substitutions of one or more wild-type amino acids of a reference CPAF protein with a non-natural amino acid (nnAA) comprising a second handle.
  • the adjuvant may comprise a first handle comprising an alkynyl group on a cyclooctane ring structure.
  • the CPAF polypeptide may comprise a substitution at one or more of amino acids H97, S499, S491, E550, and C492 relative to a reference CPAF protein.
  • the nnAA may comprise 2-amino-3-(4- azidophenyljpropanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5- (azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.
  • the method may comprise a synthesis of the CPAF polypeptide and incorporation of nnAA using a cell free extract.
  • the nnAA may comprise or may be activated to comprise a second handle.
  • the second handle may comprise an azido group.
  • the adjuvant may comprise or may be activated to comprise a first handle.
  • the first handle may comprise propargyl, DIFO, dibenzylcyclooctyne (DBCO), or a DBCO(PEG)n-NH2 moiety, or a derivative thereof.
  • the first handle may comprise a DBCO or DIFO ring structure comprising an alkynyl group.
  • the adjuvant may be CpG-DBCO, STING-DBCO, 2BxY-DBCO, 2BxY-DOPA-DBCO or CDA-DBCO, and may have a structure as set forth in formula Xlld- Xllh respectively.
  • the method of making a CPAF polypeptide-adjuvant conjugate may comprise contacting of the adjuvant to the CPAF polypeptide to form the CPAF polypeptide-adjuvant conjugate by an azide-alkyne-based click chemistry.
  • the polypeptide-adjuvant conjugate may form a triazole linkage.
  • the method may not require a catalyst.
  • the resulting polypeptide-adjuvant conjugate may comprise a structure as set forth in formula XV.
  • a vaccine and a method of immunizing a subject using the pharmaceutical composition may further comprise a carrier.
  • the vaccine may be for use against a chlamydia infection.
  • the vaccine and the method of immunizing may be for administering via a mucosal route.
  • the subject may be a mammal, which may be a human.
  • FIG. 1A-1C show that CPAF is an immunodominant CD4 T cell antigen with epitopes across the protein.
  • FIG. IB In vitro expanded T cells were stimulated with CPAF followed by intracellular cytokine staining (ICS).
  • ICS intracellular cytokine staining
  • CPAF-specific CD4+ T cells were dominated by IFNy+TNFu+ dual-positive and TNFa+ single-positive responses in women who were positive or negative in cultured IFNY ELISpot. A positive response was defined as > 3x the mock well and > 15 positive events.
  • FIG. 3A-3C show that CPAF is immunogenic in mice genitally infected with C. muridarum.
  • FIG. 3B shows that CPAF is immunogenic in mice genitally infected with C. muridarum.
  • FIG. 3C Frequency of CD44hi memory IFNy+ TNFa+ CD4 and CD8 T cell responses by ICS in CM972+CM001 immune mice after stimulation with CAP overlapping peptides (OLP).
  • FIG. 4 is a schematic showing the proposed mode of action for dual conjugated vaccine. [0046] FIG.
  • CPAFf CPAF full-length polypeptide synthesized using the cell-free system followed by His-tag purification and TEV protease cleavage.
  • the resulting tagless protein was found to separate into multiple N- and C-terminal fragments (CPAFn and CPAFc respectively).
  • FIG. 6A is a schematic of a SPAAC click chemistry used in the conjugation of a polypeptide comprising a nnAA (Ri) comprising an azide group and a DBCO linked adjuvant (R 2 ).
  • FIG. 7A-7C show that intranasal immunization (i.n.) with CPAF-CpG plus CDA elicits robust T cell and antibody responses.
  • Error bars represent mean SFU ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001 by one-way RM ANOVA.
  • FIG. 7C Anti-CPAF antibody titers after i.n. immunization. Error bars represent mean ⁇ SEM. *p ⁇ 0.05 by one-way RM ANOVA.
  • FIG. 8A-8B show that intranasal immunization (i.n.) with CPAF-CpG+CDA+AS03 provides significant protection against a genital challenge in mice.
  • FIG. 8A Chlamydia burden over the course of infection comparing CPAF vaccines and controls. Error bars represent mean ⁇ SEM. Significance determined by two-way RM ANOVA.
  • FIG. 9A-9B show that Intramuscular immunization (i.m.) with conjugated CPAF-2Bxy- Dopa enhances immunogenicity.
  • FIG. 9A Serum cytokines 1 or 18 hours after i.m. injection.
  • FIG. 9B Endpoint titer and IgG2c/IgGl ratio 13 days post-boost. ***p ⁇ 0.001, ****p ⁇ 0.0001 by one-way RM ANOVA.
  • FIG. 11 provides a schematic of the Study design for toxicity and preliminary immunogenicity. Toxicity studies (red) will assay inflammation and pathology, and immunogenicity studies (blue) will assay antibody and T cell responses.
  • FIG. 12 provides a schematic of the purification strategy using a TEV-protease cleavable tag.
  • T-cell dependent antigens proteins that produce strong, long-lived T-cell dependent immune responses
  • T-cell activating features on these immunogenic proteins recruit helper T-cells to B-cells that recognize the attached weak antigen, and so allow a strong, long-lived immune response to an otherwise weakly immunogenic molecule.
  • trachomatis and/or a prior documented infection respond to CPAF with broad epitope recognition in a CD4 dominant fashion by the production of both IFNy and TNFa, and these T cell responses are maintained for at least a year after infection.
  • CPAF as a vaccine immunogen in mice i.n. led to abbreviated infection and reduced pathology with a standard challenge dose (1.5xl0 3 - IxlO 4 bacteria).
  • CPAF vaccines adjuvanted with interleukin- 12 or mice were administered multiple doses of CpG before and after CPAF immunization.
  • the toxicity of IL-12 administration and the implausibility of delivering CpG on consecutive days make these approaches unsuitable for human use.
  • the inventors discovered an unexpected finding that adjuvant-conjugated CPAF enhanced CPAF immunogenicity without reactogenicity.
  • the inventors have manufactured adjuvant-conjugated CPAF polypeptides using a click chemistry-based approach using CPAF polypeptides comprising non-natural amino acids to which adjuvants are attached. These polypeptide conjugates have been combined further with additional adjuvants to develop highly effective compositions.
  • polypeptides of CPAF comprising one or more non-natural amino acids
  • adjuvants that may be conjugated to the CPAF polypeptides
  • CPAF polypeptide-adjuvant conjugates of (1) and (2) CPAF polypeptide-adjuvant conjugates of (1) and (2)
  • vaccine compositions comprising the foregoing
  • methods of making and using the foregoing are (1) polypeptides of CPAF comprising one or more non-natural amino acids; (2) adjuvants that may be conjugated to the CPAF polypeptides, (3) CPAF polypeptide-adjuvant conjugates of (1) and (2), (4) vaccine compositions comprising the foregoing; and (5) methods of making and using the foregoing.
  • a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included.
  • the present disclosure may include a variety of combinations and/or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.
  • any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration.
  • a substantially planar surface means having an exact planar surface or a similar, but not exact planar surface.
  • polypeptide and “protein,” as used interchangeably herein, refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • amino acid sequence that is “derived from” an amino acid sequence disclosed herein can refer to an amino acid sequence that differs by one or more amino acids compared to the reference amino acid sequence, for example, containing one or more amino acid insertions, deletions, or substitutions as disclosed herein.
  • derivative when used herein with reference to a polypeptide, refers to a polypeptide related to a wild-type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity) and/or function.
  • Derivatives, variants, and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, presence of one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), or combinations thereof compared to a wild-type polypeptide.
  • Each amino acid sequence described herein by virtue of its identity or similarity percentage may be substantially identical to a given amino acid sequence.
  • a “substantially identical” sequence may be at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or
  • sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Sequence identity may be calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s.
  • a Needleman and Wunsch global alignment algorithm can be used to align two sequences over their entire length or part thereof (part thereof may mean at least 50%, 60%, 70%, 80%, 90% of the length of the sequence), maximizing the number of matches and minimizes the number of gaps.
  • Default settings can be used and exemplary program is Needle for pairwise alignment (in an aspect, EMBOSS Needle 6.6.0.0, gap open penalty 10, gap extent penalty: 0.5, end gap penalty: false, end gap open penalty: 10 , end gap extent penalty: 0.5 is used) and MAFFT for multiple sequence alignment ( in an aspect, MAFFT v7Default value is: BLOSUM62 [bl62], Gap Open: 1.53, Gap extension: 0.123, Order: aligned , Tree rebuilding number: 2, Guide tree output: ON [true], Max iterate: 2 , Perform FFTS: none is used).
  • Similarity between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Similar algorithms used for determination of sequence identity may be used for determination of sequence similarity. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called conservative amino acid substitutions. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.
  • Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place.
  • the amino acid change is conservative.
  • suppression codon refers to a nucleotide triplet that is introduced into a polynucleotide at a predetermined location and is recognized by a specific tRNA that can recognize a stop codon (e.g., an amber, ochre or opal stop codon) and allows translation to read through the codon to produce the protein, thereby suppressing the stop codon.
  • a stop codon e.g., an amber, ochre or opal stop codon
  • a “non-natural amino acid” refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine; other terms that are used synonymously with the term “non-natural amino acid” is "non-naturally encoded amino acid,” “unnatural amino acid,” “non-naturally occurring amino acid,” and variously hyphenated and non-hyphenated versions thereof.
  • Non-natural amino acids with bio-orthogonal reactive chemical side chains may be used as a chemical “handle” to conjugate various payloads to discrete sites in a protein.
  • Nonlimiting examples of non-natural amino acids include 2-amino-3-(4- azidophenyljpropanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5- (azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.
  • pAF 2-amino-3-(4- azidophenyljpropanoic acid
  • pAMF 2-amino-3-(4-(azidomethyl)phenyl)propanoic
  • T-cell activating epitope refers to a structural unit of molecular structure which is capable of inducing T-cell immunity.
  • B-cell epitope refers generally to those features of a macromolecular structure which are capable of inducing a B cell response.
  • a B-cell epitope need not comprise a peptide, since processing by antigen-presenting cells and loading onto the peptide-binding cleft of MHC is not required for B-cell activation.
  • carrier protein refers to a polypeptide, for example CPAF or a variant or derivative thereof containing a T-cell activating epitope which may be attached to an adjuvant to enhance the humoral response to the conjugated protein-adjuvant composition in a subject.
  • a “native carrier protein” has only naturally occurring amino acids.
  • a “CPAF polypeptide” has at least one non-natural amino acid replaced for a naturally occurring amino acid in the carrier protein.
  • immunologic polypeptide refers to a polypeptide comprising at least one T-cell activating epitope, wherein the T-cell epitope is derived from a protein capable of inducing immunologic memory in animals.
  • PCR amplification methods are described, for example, in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press Inc. San Diego, Calif., 1990.
  • An amplification reaction typically includes the DNA that is to be amplified, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium.
  • dNTPs deoxynucleotide triphosphates
  • reaction buffer typically magnesium.
  • a desirable number of thermal cycles is between 1 and 25.
  • the PCR primers also contain an RNA polymerase promoter site, such as T7 or SP6, to allow for subsequent in vitro transcription.
  • T7 or SP6 RNA polymerase promoter site
  • Methods for in vitro transcription are found in sources such as Van Gelder et al., Proc. Natl. Acad. Sci. U.S.A. 87: 1663-1667, 1990; Eberwine et al., Proc. Natl. Acad. Sci. U.S.A. 89:3010- 3014, 1992.
  • a chlamydial protease-like activity factor (CPAF) polypeptide comprising at least one non-natural amino acid (nnAA).
  • the CPAF polypeptide may comprise a sequence substantially identical to a reference CPAF polypeptide.
  • the CPAF polypeptide may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the reference CPAF polypeptide.
  • the reference CPAF polypeptide may comprise a wild-type CPAF or a fragment thereof, which may be from a species of chlamydia.
  • the species chlamydia may be Chlamydia muridarum, Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae, Chlamydia abortus, Chlamydia caviae, Chlamydia suis, or Chlamydia felis.
  • the chlamydia species is Chlamydia muridarum or Chlamydia trachomatis.
  • the reference CPAF polypeptide may correspond to WP_010229939.1, WP_080129215.1, WP_015506580.1, WP_011006700.1, WP_149305369.1, or WP_014945227.1.
  • the reference CPAF polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 1 or 2.
  • the reference CPAF polypeptide comprises the sequence set forth in SEQ ID NO: 1.
  • the position of amino acid substitutions disclosed herein may be numbered in reference to SEQ ID NO: 1.
  • Non-limiting examples include His-tag, Glutathione S-transferase (GST), Streptavidin- binding peptide (SBP or Strep-tag), Maltose-binding protein (MBP), Flag-tag, HA-tag, Calmodulin-binding peptide (CBP), and SUMO (Small Ubiquitin-like Modifier) tag.
  • GST Glutathione S-transferase
  • SBP or Strep-tag Streptavidin- binding peptide
  • MBP Maltose-binding protein
  • Flag-tag HA-tag
  • SUMO Mall Ubiquitin-like Modifier
  • the tag may comprise one or more protease cleavage sites.
  • protease cleavage sites include enterokinase (EK) cleavage site, thrombin cleavage site, Tobacco Etch Virus (TEV) protease cleavage site, factor Xa cleavage site, proteinase k cleavage site, precision protease cleavage site, or caspase cleavage site.
  • EK enterokinase
  • TEV Tobacco Etch Virus
  • the tag comprises a TEV protease cleavage site, which may comprise the sequence ENLYFQ (SEQ ID NO: 12) followed by any amino acid except for proline or tryptophan.
  • the protease cleavage site may be located at the N- or C-terminus of the CPAF polypeptide.
  • the protease cleavage site may be present between the purification tag and the polypeptide, to facilitate purification and function.
  • the CPAF polypeptide may comprise a His-tag at its N- or C-terminus.
  • the His- tag comprises six consecutive histidines (6xHis).
  • the CPAF polypeptide comprises a His-tag and a TEV protease cleavage site.
  • the CPAF polypeptide TEV cleavage site may be located after or before the His-tag.
  • the TEV cleavage site is located after the His-tag, which may be a 6xHis tag.
  • the CPAF polypeptide comprises the sequence MHHHHHHGGSENLYFQ (SEQ ID NO: 13), which may be located at the N-terminus of the CPAF polypeptide.
  • the CPAF polypeptide may be a full length CPAF polypeptide or a N- or C- terminally processed polypeptide.
  • the CPAF polypeptide may be a fragment of a CPAF protein disclosed herein, for example a N or C-terminal fragment thereof.
  • the CPAF polypeptide may comprise an amino acid sequence set forth in any one of SEQ ID NOS: 4-9, or a sequence substantially identical thereto.
  • the CPAF polypeptide may be a catalytically inactive form of a reference CPAF polypeptide disclosed herein.
  • the inactivity may be partial or complete.
  • the inactive CPAF polypeptide may comprise a substitution in one or more of residues S491, S499, H97, E550, and C492 relative to SEQ ID NO: 1.
  • the substitutions may comprise one or more of H97A, S491A, S499A, C492T, and E55OQ.
  • the CPAF polypeptide, fragment thereof, or inactive form thereof may comprise a sequence provided in Table 1, or a post-translational processing variant thereof.
  • the CPAF polypeptide of SEQ ID NOs: 3, 4, 5, or 11 may be processed to remove the His-Tag.
  • the CPAF polypeptide may have contained a TEV protease cleavage site that was subsequently processed by a TEV protease. Consequently, the CPAF polypeptide may comprise at its N-terminus a residual amino acid from the cleavage site.
  • the amino acid in the 1 st position of the CPAF polypeptide may be any amino acid except proline or tryptophan (Kapust et al. 2002).
  • the N- terminus of N-terminal fragments of CPAF polypeptides provided in Table 1, may exclude the glycine and/or may be a methionine.
  • the CPAF polypeptide may comprise a mutation disclosed herein in one or more of S491, S499, H97, E550, and C492, and comprise one or more nnAA substitutions at one or more lysine, tyrosine, or phenylalanine as described herein.
  • the nnAA are described herein below.
  • the CPAF polypeptide may comprise a mutation in one or more of S491, S499, H97, E55O, and C492, and comprise nnAA substitutions in at least F130 and Y569, wherein the residues are numbered relative to SEQ ID NO: 1.
  • the CPAF polypeptide may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 14-22, or a fragment thereof, or a sequence substantially identical thereto.
  • the disclosed CPAF polypeptide may comprise a sequence at least about 80%, 85%, 90%, 95%, or 99% identical to a sequence set forth in any one of SEQ ID NOs: 14-22.
  • the CPAF polypeptide may also comprise a tag disclosed herein.
  • the CPAF polypeptide may also be a post-translational variant of a sequence in Table 2, such as a variant produced by cleavage with a TEV protease as described above.
  • the nnAA may be any known in the art. Each nnAA may independently be 2-amino-3- (4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4- (azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, or any combination thereof.
  • pAF 2-amino-3- (4-azidophenyl)propanoic acid
  • pAMF 2-amino-3-(4
  • Examples of CPAF polypeptide nnAA include: a non-natural analog of a tyrosine amino acid; a non-natural analog of a glutamine amino acid; a non-natural analog of a phenylalanine amino acid; a non-natural analog of a serine amino acid; a non-natural analog of a threonine amino acid; a non-natural analog of a lysine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with
  • the nnAA residue may comprise a chemical group or a handle (second chemical handle) suitable for a “click” chemistry reaction with a corresponding group on a separate molecule, which may be an adjuvant.
  • Each nnAA may independently be a 2,3-disubstituted propanoic acid bearing an amino substituent at the 2-position and an azido-containing substituent, a 1,2,4,5-tetrazinyl-containing substituent, or an ethynyl-containing substituent at the 3-position.
  • Each nnAA of the CPAF polypeptide may be conjugated to an adjuvant.
  • each nnAA residue comprises an amino acid having the structure of formula I wherein:
  • Ar comprises a 5-membered or 6-membered aromatic ring optionally containing at least one heteroatom
  • W 5 is selected from Ci-Cio alkylene, -NH-, -O- and -S-;
  • QI is zero or 1; and W 6 is selected from azido, 1,2,4,5-tetrazinyl optionally C-substituted with a lower alkyl group, and ethynyl, such that the nnAA residue in the polypeptide has the structure of formula II
  • R 3 is OH or an amino acid residue of the carrier protein
  • R 4 is H or an amino acid residue of the carrier protein
  • Ar comprises a 5-membered or 6-membered aromatic ring optionally containing at least one heteroatom
  • W 5 is selected from C1-C10 alkylene, -NH-, -O- and -S-; QI is zero or 1 ; and
  • W 6 is selected from azido, 1,2,4,5-tetrazinyl optionally C-substituted with a lower alkyl group, and ethynyl.
  • the nnAA may have the structure of formula IV in which R 3 is OH or an amino acid residue of the carrier protein, and R 4 is H or an amino acid residue of the carrier protein.
  • the Ar may not contain any heteroatoms, in which case the preferred linker is an unsubstituted phenylene group.
  • the Ar may contain a nitrogen heteroatom and at least one additional heteroatom selected from N, O, and S. Exemplary nitrogen heterocycles are described infra.
  • QI is 1, W 5 is lower alkylene, and W 6 is azido.
  • the nnAA residue may comprise an azido-containing nnAA.
  • the nnAA residue may comprise an azido-containing nnAA of formula V: wherein:
  • D is — Ar— W3— or — Wl— Yl— C(O)— Y2— W2— ; each of Wl, W2, and W3 is independently a single bond or lower alkylene; each Xi is independently — NH — , — O — , or — S — ; each Y1 is independently a single bond, — NH — , or — O — ; each Y2 is independently a single bond, — NH — , — O — , or an N-linked or C-linked pyrrolidinylene; and one of Zi, Z2, and Z3 is — N — and the others of Zl, Z2, and Z3 are independently — CH — .
  • the nnAA residue may comprise an azido-containing amino acid of formula VI: wherein:
  • the nnAA residue may comprise an azido-containing amino acid selected from the group consisting of 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2- yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, or 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof.
  • the nnAA residue comprise 2-amino-3-(4-(azidomethyl)phenyl)propa
  • V is a single bond, lower alkylene, or -W1-W2-; one of W1 and W2 is absent or lower alkylene, and the other is -NH-, -O-, or -S-; each one of Zi, Z2, and Z3 is -CH- or -N- and the others of Zi, Z2, and Z3 are each independently -CH-; and Xi is independently -NH-, -O-, or -S-;
  • R is lower alkyl; wherein when and V is -NH-, then one of ZI, Z2, and Z3 is -N- provided the compound is not
  • the nnAA may comprise an alkyne.
  • the alkyne may be a propargyl group.
  • a variety of propargyl-containing amino acids, including syntheses thereof, are found in Beatty et al. Angew. Chem. Int. Ed. 2006, 45, 7364 -7367; Beatty et al. J. Am. Chem. Soc. 2005(127): 14150-14151; Nguyen et al. J Am Chem Soc. 2009(131):8720- 8721.
  • Such propargyl-containing amino acids are suitable for incorporation as nnAAs into proteins using cell-based systems.
  • the nnAA may comprise a propargyl selected from the group consisting of homopropargylglycine, ethynylphenylalanine, and N6-[(2-propynyloxy)carbonyl]- L-lysine.
  • the CPAF polypeptide may be produced by any method known for production of polypeptides. Methods suitable for production of polypeptides include, but are not limited to, solid phase chemical peptide synthesis, cell-based recombinant protein expression (in A. coli or a native host), and cell-free protein expression, and any combination thereof (e.g. expressed protein ligation using a combination of synthetic and recombinant peptide components).
  • the nnAA-bearing CPAF polypeptide may be produced by a method that comprises “codon reassignment.”
  • nnAAs that are close structural analogs of the 20 canonical amino acids (e.g. homoallylglycine, fluorinated leucine, azidohomoalanine) are used.
  • the nnAA is loaded onto its corresponding tRNA using wild-type aminoacyl-tRNA synthetases, and the nnAA completely replaces one of the 20 canonical amino acids specified in a template DNA sequence.
  • this generally requires use of a bacterial expression strain that is auxotrophic for the native amino acid being replaced. This strategy is amino acid rather than residue-specific since all AA residues of a certain type are replaced with the nnAA.
  • the nnAA-bearing CPAF polypeptide is produced by a strategy that comprises “nonsense suppression”.
  • the non-natural amino acid is specified in a template DNA sequence by a rare or “nonsense” codon that does not ordinarily specify an amino acid in nature.
  • nonsense suppression approach has been pioneered by Schultz (Noren et al. Science. 1989(244): 182-188.) and Chamberlin (Bain et al. J Am Chem Soc.
  • production of the CPAF polypeptide does not involve the use of an engineered aminoacyl-tRNA synthetase.
  • an orthogonal tRNA alone is isolated and modified at the anti-codon loop to recognize an orthogonal codon (e.g. the amber codon TGA, or another codon or base sequence not commonly used to specify amino acids in translation).
  • the orthogonal engineered tRNA is then acylated in vitro by a suitable chemical method (e.g., the method of Heckler et al. Biochemistry. 1984 Mar 27;23(7): 1468-73.
  • the CPAF polypeptide may be produced by cell-free extract-based protein synthesis.
  • the cell-free extract may comprise an extract of rabbit reticulocytes, wheat germ, or E. coli.
  • the cell-free extract may be supplemented with amino acids, energy sources, energy regenerating systems, or cation cofactors, and any combination thereof.
  • the extract may comprise exogenously supplemented mutant tRNA or mutant aaRS, and any combination thereof.
  • the extract may comprise lysates from E. coli strains genetically encoding mutant tRNA or mutant aaRS, and any combination thereof.
  • the E. coli strains used for lysates are RF-1 attenuated strains.
  • the disclosure provides for methods of producing polypeptides in a cell-free extract containing 2 or more non-natural amino acids.
  • the methods of producing the nnAA-containing polypeptides may involve altering the concentrations of nnAA-specific tRNA, nnAA-specific synthetase, nnAA itself, or translation temperature, and any combination thereof. Such conditions optionally allow for fewer translational errors, improved rate of incorporation of the nnAA, improved activity of chaperones necessary for protein folding with incorporation of the nnAA, decreased activity of cellular factors that interfere with nnAA incorporation, or any combination of the aforementioned mechanisms.
  • the nnAA-specific tRNA concentration may be increased to a concentration above about 20 pM, leading to an increased fraction of soluble or active polypeptide.
  • the tRNA concentration may be increased while the nnAA concentration is kept below about 2mM and the nnAA synthetase is maintained below about 5pM.
  • the CPAF variant- TEV reaction may then be passaged over a Ni-affinity matrix.
  • the TEV protease is His-tagged so that it and any uncleaved CPAF and CPAF fragments, and the non-specifically bound E. coli contaminants, may all bind to the affinity resin while the untagged CPAF variants flow through.
  • the untagged CPAF variants in the flow through may be concentrated and may be used to covalently link the adjuvant, for example in the production of the DBCO-derivatized adjuvant(s) conjugated via a click chemistry reaction disclosed herein. 4.
  • Adjuvants Adjuvants
  • the adjuvant may be derivatized with a chemical handle to facilitate attachment to CPAF polypeptide.
  • the adjuvant may be derivatized with dibenzylcyclooctyne (DBCO) as provided herein to facilitate attachment to a nnAA on the CPAF polypeptide.
  • DBCO dibenzylcyclooctyne
  • the adjuvant may be a water-soluble adjuvant.
  • the adjuvant may be purified natural, synthetic, or recombinantly produced small molecule, or macromolecule or a fragment thereof.
  • the adjuvant may comprise a mineral, peptide adjuvant, polysaccharides or glycans, polynucleotide, polyamino acid, lipid, small molecules or nanoparticles.
  • the adjuvant may be, for example, a Toll-like receptors (TLR) agonist, Stimulator of Interferon Genes receptor (STING) agonist, C-type lectin receptor (CLR) agonist, RIG-I-like receptor (RLR) agonist, a NOD-like receptor (NLR) agonist, or a dopamine receptor agonist.
  • TLR Toll-like receptors
  • STING Stimulator of Interferon Genes receptor
  • CLR C-type lectin receptor
  • RIG-I-like receptor (RLR) agonist RIG-I-like receptor
  • NLR NOD-like receptor
  • dopamine receptor agonist a dopamine receptor agonist
  • TLR agonists include unmethylated CpG DNA, 2Bxy, triacylated lipoproteins, lipoteichoid acid, peptidoglycans, zymosan, PamsCSK ⁇ diacylated lipopeptides, HSPs, HMGB1, uric acid, imidazoquinoline, fibronectin, ECM proteins, dsRNA, poly 1:C, LPS, P-defensin 2, fibronectin EDA, HMGB1, snapin, tenascin C, flagellin, ssRNA, CpG- A, poly GIO, poly G3, profilin, VSV.
  • Non limiting examples of dopamine agonists include L-DOPA, amantadine, apomorphine, 94 bromocriptine, cabergoline, carmoxirole, optically pure (S)-didesmethyl sibutramine, dopexamine, fenoldopam, ibopamine, lergotrile, lisuride, memantine, mesulergine, pergolide, piribedil, pramipexole, quinagolide, ropinirole, roxindole, and talipexole.
  • the adjuvant comprises a STING agonist.
  • the STING agonist comprises a CDN, which may be a CDA.
  • the reactive sites on the adjuvant that serve as the "first chemical handle” may be one or more alkynyl group.
  • Each alkynyl group may be incorporated in a molecular context that increases reactivity.
  • the alkynyl group may be incorporated into a ring, e.g., a cyclooctynyl ring, such as a diaryl-strained cyclooctyne.
  • Reactive sites on the nnAA of the CPAF polypeptide which may be the second chemical handle of the nnAA, may be an azido group as disclosed herein.
  • the reaction in this case is a [3+2] cycloaddition referred to in the art as "strain-promoted azide'alkyne cycloaddition" (SPAAC).
  • SPAAC strain-promoted azide'alkyne cycloaddition
  • the adjuvant may be derivatized with dibenzylcyclooctyne (DBCO) providing the cyclooctynyl ring comprising an alkynyl group to facilitate attachment to the azido group of a nnAA on the CPAF polypeptide.
  • DBCO dibenzylcyclooctyne
  • the adjuvant may be activated using any chemical method described for production of bioconjugates. Such methods include, but are not limited to, periodate oxidation, unmasking of an intrinsic aldehyde , or hydroxyl activation with 1,1 '-carbonyldiimidazole (CDI) followed by nucleophilic addition.
  • Activation of adjuvant include, but are not limited to, periodate oxidation, unmasking of an intrinsic aldehyde , or hydroxyl activation with 1,1 '-carbonyldiimidazole (CDI) followed by nucleophilic addition.
  • Periodate activation The adjuvant may be activated by periodate oxidation. Periodate oxidation may be used to introduce aldehyde groups into an adjuvant, and is useful for the addition of aldehydes to N-terminal residues of polypeptides to produce an activated adjuvant. Periodate cleaves carbon-carbon bonds that possess a primary or secondary hydroxyl or amine on either end, and so activates r amino acids containing the 2-amino alcohol moiety (N-terminal threonine or serine residues). As the aldehyde moiety has a long half-life, adjuvants activated by this method are optionally chromatographically purified and/or lyophilized after activation. [0124] For periodate oxidation of adjuvants:
  • the reaction mixture is incubated; and [0128] (d) (optional) excess periodate is removed.
  • Deionized water or a suitable buffered solution is optionally used for the oxidation reaction.
  • the solution in step (a) is deionized water.
  • the solution in step (a) comprises an effective amount of a buffer with a pKa around physiological pH.
  • the solution in step (a) comprises an effective amount of a buffer with a pKa around physiological pH.
  • Step (a) may be provided as a solution in an aqueous buffer, e.g., having a pH in the range of 5.1 to 5.9 (e.g., 5.2-5.9, 5.3-5.7, 5.4-5.6, 5.4-5.9).
  • the buffer does not comprise an amine group.
  • amine-free buffers include, but are not limited to acetate, formate, and phosphate.
  • an amine buffer is employed in step (a).
  • Suitable amine buffers generally comprise a combination of a tertiary amine or an N- heterocyclic compound with a weak acid (e.g., pyridine and acetic acid; pyridine and formic acid; N-ethylmorpholine and acetic acid; trimethylamine and carbonic acid; triethanolamine and phosphoric acid; etc.), or they can be a zwitterionic amine buffer such as 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), or 3-[4- (2-hydroxyethyl)piperazin-l-yl]propane-l-sulfonic acid (HEPPS).
  • a weak acid e.g., pyridine and acetic acid; pyridine and formic acid; N-ethylmorpholine and acetic acid; trimethylamine and carbonic acid; triethanolamine and
  • the periodate source in step (b) is optionally selected from any periodate source with appropriate stability in aqueous solution.
  • periodate sources include, but are not limited to, sodium periodate, potassium periodate, tetrabutyl ammonium (meta)periodate, barium periodate, sodium hydrogen periodate, sodium (para)periodate, and tetraethylammonium (meta)periodate.
  • the adjuvant may be activated with carbonyldiimidazole (CDI) or carbonyl di tri azole (CDT).
  • CDI and CDT like CDAP, are capable of activating hydroxyl groups on an adjuvant to form a transient reactive moiety; in this case it is an unstable carbamate ( O for CDI and for CDT), which is then optionally reacted with an amine or thiol on a chemical handle or linker to form a carbamate or carbonothioate linkage.
  • the activation may be performed in a dry organic solvent.
  • the CDI/CDT activation may be performed in anhydrous dimethylsulfoxide (DMSO).
  • CDI/CDT activation may be performed by adding a molar excess of CDI/CDT with respect to the adjuvant.
  • CDI/CDT activation may be performed by adding a molar amount of CDI/CDT approximately equal to the molar amount of the adjuvant.
  • Endogenous amines or other nucleophilic moieties e g. a primary amine
  • nucleophilic moieties e g. a primary amine
  • Such nucleophilic moieties can be conveniently reacted with a variety of common electrophilic conjugation reagents like succinate derivatives (e.g. N-hydroxysuccinimide (NHS) or sulfo-NHS esters). It may be sometimes advantageous to treat with a periodate protocol.
  • succinate derivatives e.g. N-hydroxysuccinimide (NHS) or sulfo-NHS esters
  • Bifunctional linkers The activated adjuvant may be conjugated to the nnAA directly, but in one example the activated group on the adjuvant is derivatized to introduce a functional group with better reactivity with the functional group of the nnAA. For instance, an alkynyl group may be introduced.
  • a bifunctional reagent with an amino group and an alkyne group can react with an aldehyde group that has been introduced into an adjuvant (e.g. via reductive amination) thereby leaving a pendant alkyne which can react with the nnAA.
  • bifunctional reagents including amino and dibenzylcyclooctyne (DBCO) functional groups can be used.
  • the adjuvant may be conjugated to the first chemical handle using any chemical method compatible with the activation methods described above. Such methods include, but are not limited to, Schiff-base formation with synthetic antigen aldehydes followed by reductive amination, hydrazone formation, oxime formation, direct nucleophilic addition, and Schiff-base formation with native antigen aldehydes followed by reductive amination. A description of such methods are also provided in US20180333484, the contents of which are incorporated herein in its entirety.
  • the absolute adjuvant concentration in a conjugation reaction with a chemical handle may vary depending on the adjuvant and can be determined.
  • the chemical handle is added at a lower molar ratio with respect to the aldehyde groups on the activated adjuvant, and excess unreacted aldehydes on the activated adjuvant are consumed by further reaction with an excess of an inexpensive aldehyde-reactive nucleophile (e g. ethanolamine), or by treatment with a reducing agent strong enough to reduce aldehydes to hydroxyl groups (e.g. NaBHT).
  • an inexpensive aldehyde-reactive nucleophile e g. ethanolamine
  • a reducing agent strong enough to reduce aldehydes to hydroxyl groups e.g. NaBHT
  • the chemical handle may be conjugated to the adjuvant by Schiff-base formation with synthetic adjuvant aldehydes followed by reductive amination. This results in an end-product that has secondary amine linkage between the chemical handle and the adjuvant: a direct N-C bond between the amine of the chemical handle and a carbon atom on adjuvant.
  • the chemical handle may comprise an amine.
  • the conjugation method comprises: combining the amine-containing handle with periodate-activated adjuvant in DI water or buffered solution containing DMSO; incubating to form a Schiff base; reducing the Schiff base to a secondary amine using sodium cyanoborohydride (NaBHsCN); and optionally quenching unreacted aldehydes with NaBHi.
  • the chemical handle and adjuvant are combined at or near 1 :1 stoichiometry.
  • the chemical handle and adjuvant are combined with a molar excess of chemical handle.
  • the chemical handle and adjuvant are combined with a molar excess of adjuvant.
  • the chemical handle is conjugated to the adjuvant via hydrazone formation.
  • the chemical handle is conjugated to the adjuvant by oxime formation.
  • the chemical handle comprises an aminooxy (-O-NH2) group.
  • a large molar excess of the amine/thiol-containing chemical handle with respect to activated hydroxyl groups on the adjuvant is added.
  • the chemical handle is added at a concentration closer to 1 : 1 molar ratio with respect to the activated hydroxyl groups on the adjuvant.
  • residual CDI/CDT in the reaction is further inactivated by treatment with sodium tetraborate.
  • Reactions with Non-Activated Adjuvants In some aspects the chemical handle is conjugated to an endogenous amine or other nucleophilic moiety (e.g. a primary amine) either naturally present or the result of a deprotection step from an adjuvant described herein.
  • an electrophilic group e.g. an NHS or sulfo-NHS ester
  • a carboxylic acid group on a chemical handle is reacted with a primary amine group on the adjuvant in the presence of standard peptide coupling reagents and conditions to produce an amide linkage between the chemical handle and the adjuvant amine.
  • Alkyne-containing handles In some aspects the chemical handle comprises a moiety that allows for a “click” chemistry reaction with a corresponding group on the nnAA.
  • One such moiety is an alkyne group, which is capable of reacting with the nnAA residue comprising an azido group. In the simplest aspect, this is a propargyl group, such that an alkyne group on an adjuvant comprises a structure of formula IX:
  • L22 is Cl -CIO alkyl
  • Ui is at least one moiety of an adjuvant.
  • an alkyne group on an adjuvant comprises a structure of formula IXa:
  • L22 is -(CH 2 CH 2 0)I-IO-;
  • Ui is at least one moiety of an adjuvant.
  • Li is independently a bond, -NH-, -O-, -S-, -NH(Li2)-, -O(Li2)-, or -S(Li2)-;
  • L12 is independently L22 or L22NH-
  • L22 is independently C1-10 alkyl or -(CH2CH20)MO-;
  • Ui is independently at least one moiety of an adjuvant.
  • structures of formula X and Xa are formed from an adjuvant comprising a nucleophilic group (e.g. a primary amine) and the NHS or sulfo-NHS ester of the corresponding DIFO or DBCO carboxylic acids of structures X and Xa.
  • structures of formula X are formed from an activated adjuvant, and a DBCO derivative such as DBCO-NH2 or DBCO-PEGn-NH 2 .
  • DBCO-PEGn-NH 2 is DBCO-PEG4-NH2.
  • n is at least 1.
  • an adjuvant comprising a DBCO group comprises at least 90% of the adjuvant covalently attached to DBCO.
  • the adjuvant comprises greater than about 90%, or about 92%, or about 94%, or about 96%, or about 98%, or about 99% molecules covalently attached to DBCO. Any unconjugated DBCO or adjuvant may be inactivated with azide and removed by dialysis. Thus, the adjuvant may comprise 100% of the molecules attached to DBCO.
  • the adjuvant comprises DBCO or a derivative thereof.
  • the adjuvant comprises a 2BxY-DOPA-DBCO, a CpG-DBCO, a STING-DBCO, a 2Bxy-DBCO, or a CDA-DBCO.
  • the adjuvant comprising a DBCO handle has a structure as provided below:
  • CDA-DBCO (formula Xllh)
  • the chemical handle comprises a moiety that allows for a “click” chemistry reaction with a corresponding group on nnAA residue of a polypeptide.
  • One such moiety is an azido group, which is capable of reacting with a nnAA residue comprising an alkyne group on a polypeptide.
  • an azido group on an adjuvant comprises a structure of formula XIII: z- N H
  • L22 is a bond, alkyl, or poly(alkyloxy);
  • Ui is independently at least one moiety of an adjuvant.
  • Alkene-containing handles In some aspects the chemical handle comprises a moiety that allows for a “click” chemistry reaction with a corresponding group on nnAA residue of a polypeptide.
  • One such moiety is an alkene group, which is capable of reacting with a nnAA residue comprising an 1,2,4,5-tetrazine group. In the simplest aspects, this is a vinyl group.
  • an alkene group on an antigen comprises a structure of formula XIV:
  • L 22 is Cl -10 alkyl or -(CH 2 CH 2 0)I-IO-;
  • Ui is independently at least one moiety of an adjuvant.
  • CPAF polypeptide-adjuvant conjugates comprising the CPAF polypeptidethe CPAF polypeptide and the adjuvant.
  • the adjuvant may be covalently linked to one or more of the nnAA of the CPAF polypeptide.
  • the adjuvant may not be linked to a natural amino acid of the polypeptide.
  • the adjuvant is only linked to one or more of the nnAAs of the CPAF polypeptide.
  • the adjuvant is only linked to one or more pAMF of the CPAF polypeptide.
  • the adjuvant may only be linked to one or more pAMFs of the CPAF polypeptide comprising a sequence set forth in any one of SEQ ID NOS: 14-22.
  • at least one adjuvant is linked to a nnAA located outside a T-cell epitope of the CPAF polypeptide.
  • no adjuvant is linked to an amino acid located within a T-cell epitope of an immunogenic portion of the CPAF polypeptide.
  • the amino acids selected for conjugation within the CPAF polypeptide may comprise one or more surface- accessible residues based on the crystal structure of the CPAF polypeptide. Additionally, or alternatively, a comprehensive replacement of natural amino acids for nnAAs is performed on the CPAF polypeptide followed by conjugation to assess the utility of specific sites on the polypeptide for conjugation.
  • the adjuvant is conjugated to the CPAF polypeptide indirectly (e.g. by first combining the CPAF polypeptide or adjuvant with a reactive linker, and then combining the CPAF polypeptide-linker or antigen-linker adduct with an adjuvant or CPAF polypeptide, respectively).
  • the adjuvant is conjugated to the CPAF polypeptide directly (e.g. by combining two components comprising the CPAF polypeptide and adjuvant together in one reaction).
  • the adjuvant is conjugated to the CPAF polypeptide using any chemical method suitable for conjugating the non-natural amino acids and chemical handles herein described.
  • a description of such methods are also provided in US20180333484, the contents of which are incorporated herein in its entirety.
  • Such methods include, but are not limited to, copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), and tetrazinealkene ligation. As “click” reactions, all of these reactions are able to be performed in aqueous solution.
  • the adjuvant is conjugated to the CPAF polypeptide by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC).
  • CuAAC copper(I)-catalyzed alkyne-azide cycloaddition
  • the CPAF polypeptide comprises a propargyl-containing nnAA and the adjuvant comprises an azido group.
  • the CPAF polypeptide comprises an azido-containing nnAA and the adjuvant comprises a propargyl group.
  • Suitable conditions for CuAAC conjugation of biomolecules are found, e.g. Presolski et al. Curr Protoc Chem Biol.
  • the reaction is accelerated by the addition of a Cu-coordinating ligand, such as THPA.
  • a reducing agent to maintain the oxidation state of Cu 2+ .
  • Suitable reducing agents include sodium ascorbate, DTT, or TCEP.
  • the adjuvant is conjugated to the CPAF polypeptideCPAF polypeptide by strain-promoted azide-alkyne cycloaddition (SPAAC).
  • SPAAC strain-promoted azide-alkyne cycloaddition
  • the CPAF polypeptide comprises an azido-containing nnAA (second handle) and the adjuvant comprises a alkyne cyclooctyne group (first handle).
  • the CPAF polypeptide comprises a cyclooctyne-containing nnAA and the adjuvant comprises an azido group.
  • SPAAC requires no additional catalysts or cofactors, this reaction is able to be performed in distilled water, 0.9% saline, PBS, or a physiologically buffered solution.
  • the adjuvant is linked to an azido-containing nnAA in the CPAF polypeptide via a structure of formula XV or XVa:
  • Ri is independently H, formyl, or at least one amino acid of the CPAF polypeptide
  • R.2 is independently OH or at least one amino acid of the CPAF polypeptide
  • D is — Ar— W3— or — Wl— Yl— C(O)— Y2— W2— ;
  • each of Wl, W2, and W3 is independently a single bond or lower alkylene
  • each XI is independently — NH — , — O — , or — S — ;
  • each Yi is independently a single bond, — NH — , or — O — ;
  • each Y2 is independently a single bond, — NH — , — O — , or an N-linked or C-linked pyrrolidinylene;
  • one of Zi, Z2, and Z3 is — N — and the others of Zi, Z2, and Z 3 are independently — CH —
  • L22 is independently a bond, alkyl or poly(alkyloxy);
  • X is the adjuvant.
  • the adjuvant is linked to an azido-containing nnAA in the CPAF polypeptide via a structure of formula XVIa or XVIb:
  • Ri is independently H, formyl, or at least one amino acid of the CPAF polypeptide
  • R2 is independently OH or at least one amino acid of the CPAF polypeptide
  • y is at least 1;
  • n is at least 1;
  • X is the adjuvant.
  • X is the adjuvant.
  • FIG. 6A A schematic of an exemplary SPAAC reaction between an azi do-containing nnAA (Ri) in the CPAF polypeptide and a DBCO linked adjuvant (R2) is provided in FIG. 6A and described in Example 6.
  • Tetrazine-alkyne ligation In some aspects, the adjuvant is conjugated to the CPAF polypeptide by tetrazine-alkyne ligation. In one variation of these aspects, the CPAF polypeptide comprises a 1,2,4,5-tetrazine-containing nnAA and the adjuvant comprises an alkene group. Similar to the SPAAC reaction, the tetrazine-alkyne ligation proceeds without the addition of cofactors this and this reaction is able to be performed in distilled water, 0.9% saline, PBS, or a physiologically buffered solution.
  • the disclosure provides for a method for producing a CPAF polypeptide conjugate comprising: (a) providing a nucleic acid encoding polypeptide, wherein the nucleic acid comprises a suppression codon; (b) creating a reaction mixture by combining the nucleic acid with a cell-free bacterial extract comprising 4-azidomethylphenylalanine (pAMF), a tRNA complementary to the suppression codon, and an aminoacyl-tRNA synthetase; (c) incubating the reaction mixture of (b) under conditions sufficient to selectively incorporate pAMF at a site corresponding to the suppression codon in the carrier protein; and (d) conjugating the pAMF to a adjuvant by a [2+3] cycloaddition.
  • pAMF 4-azidomethylphenylalanine
  • the [2+3] cycloaddition comprises the reaction between an azide and an alkyne group.
  • the method additionally comprises purifying the carrier protein immediately after (c).
  • the suppression codon may be selectively substituted at codons encoding L21, L26, L32, L35, L38 L41, L42, L56, L60, L71, L93, L110, L140, L141, L152, L155, L164, L171, L174, L181, L192, L218, L230, L251, L273, L307, L333, L350, L358, L373, L376, L377, L380, L385, L387, L394, L403, L406, L408, L409, L422, L433, L439, L447, L457, L463, L484, L500, L507, L536, L540, L548, L582, L586, L595, K33, K45, K49, K52, K62, K70
  • the reaction mixture in (b) may further comprise biological components necessary for protein synthesis.
  • the tRNA in (b) is capable of being charged with pAMF.
  • the aminoacyl-tRNA synthetase in (b) preferentially aminoacylates the tRNA with pAMF compared to the 20 natural amino acids.
  • the alkyne group comprises a DBCO moiety conjugated to the adjuvant.
  • a “click” reactions for making the disclosed conjugates, using the disclosed chemistries may be performed in an aqueous solution.
  • conjugation conditions may be conducted at various pH, temperature, mixing speed, and addition speed.
  • Reaction concentration, temperature, adjuvant, and protein input ratios may be optimized depending on the scale of production and the reactants.
  • adjuvant ratio may be added to the in 1-20 fold excess of the polypeptide. In an aspect, the adjuvant may be added in about 2 to about 3 fold excess, or about 3 to about 4 fold excess, 4 to about 5 fold excess, 5 to about 6 fold excess, 6 to about 7 fold excess, 7 to about 8 fold excess, 8 to about 9 fold excess, 9 to about 10 fold excess of the polypeptide.
  • the gas-flow rate, pH, and temperature may be optimized based on the scale of production, to obtain maximum soluble yield. For example, for large scale production, Rushton-style impellers with high shear (or similar systems) may be used to disrupt larger gas bubbles from open-pipe or drilled-hole spargers commonly used in microbial fermentation. These impellers are well characterized, with a variety of correlations for Reynolds Number, Power Number, and kLa for transition to manufacturing and commercial scale.
  • pH control The method of making a CPAF polypeptide conjugate may require a controlled pH.
  • the pH may be controlled by using any suitable buffering agent.
  • buffers or pH adjusting agent include Tris, HEPES, MOPS, sodium borate, sodium phosphate, sodium citrate, ammonium sulfate, succinate, citrate/dextrose, sodium bicarbonate and ammonium chloride or combinations thereof.
  • the pH control may be maintained by manual or automated addition of both acid and base to maintain a set-point pH.
  • Non-limiting examples of suitable acids include acetic, boric, citric, lactic, phosphoric and hydrochloric acids; nonlimiting examples of bases include sodium hydroxide, sodium acetate, sodium lactate and tris- hydroxymethylaminomethane.
  • the scaled-up cell-free reaction may utilize IM citric acid and IM potassium hydroxide, because this acid/base pair provides good pH control without excessive volume additions.
  • the initial set point for cell-free protein production may be about 6.5-8.5 pH.
  • the initial set point for cell-free protein production may be about 7-8 pH.
  • the initial set point for cell-free protein production may be about 7.2 ⁇ 0.1. pH may be varied to further optimize reaction conditions.
  • the method of making a CPAF polypeptide conjugate may require carefully controlled temperature.
  • the temperature of the reaction may be between 15°C to about 37°C.
  • the reaction chemistries may be accomplished at a temperature of about 20°C, or about 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.
  • the CPAF polypeptide and or CPAF polypeptide conjugates may need to be further purified prior to use in disclosed formulations.
  • Methods of purification of polypeptides are well known in the art and may include centrifugation, size fractionation, gel filtration and other chromatographic techniques, affinity purification, and mass spectrometry.
  • the CPAF polypeptide may comprise a TEV protease cleavable N-terminal his6-tag for downstream purification of the conjugate.
  • the cell-free reaction may be loaded onto a pre-equilibrated his Trap (GE Healthcare) column to purify the tagged protein, followed by incubation with TEV protease to cleave the tag.
  • the untagged protein may be purified by reloading the protein mixture onto the affinity column, with the target protein collected in the flow through. If post-affinity chromatography yields a purity of the protein solution of ⁇ 95%, additional orthogonal polishing steps can be employed using ion exchange or mixed-mode resins.
  • the CPAF polypeptide conjugate may comprise: d) a substitution corresponding to one or more of H97A, S491A, S499A, C492T, and E550Q, or a combination thereof, wherein the amino acids are numbered with reference to SEQ ID NO: 1; e) one or more nnAA at Fl 30 or Y569 or both; wherein the nnAA is selected from 2- amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino- 3-(6-(azidomethyl)pyridin-3-yl)propanoic
  • Methods size exclusion, diafiltration, dialysis: Following the conjugation reaction, the CPAF polypeptide conjugates of interest are optionally purified according to methods including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, and size exclusion), molecular size exclusion (dialysis, diafiltration, tangential flow filtration, depth filtration) electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), or SDS-PAGE to obtain substantially pure conjugates.
  • chromatography e.g., ion exchange, affinity, hydrophobic interaction, and size exclusion
  • molecular size exclusion dialysis, diafiltration, tangential flow filtration, depth filtration
  • electrophoretic procedures e.g., preparative isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • SDS-PAGE SDS
  • the conjugated protein of interest are optionally quantitated according to methods including, but not limited to, microfluidic electrophoresis, gel electrophoresis, western blotting, immunoassays (e.g., ELISA), and other assays to assess the activity of the conjugated protein.
  • Exemplary physical parameters One important parameter for polypeptide conjugates is the molecular weight of the conjugate. Since conjugates optionally comprise variable numbers of adjuvant molecules conjugated to each protein molecule as well as variable higher-order crosslinking (protein-adjuvant-protein linkages, for example) the output molecular weight of a conjugate is not necessarily predictable from the input molecular weights of the CPAF polypeptides and adjuvants.
  • the adjuvant-to-protein ratio of the purified conjugate is generally expressed in terms of a weight-weight (w/w) ratio or molar ratio.
  • the adjuvant-to- protein ratio of the purified conjugate may vary depending on the number of nnAA present per polypeptide.
  • the adjuvant to polypeptide ratio in the conjugate may be in the range of about 0.8:1 to about 10: 1 molar ratio.
  • the adjuvant to polypeptide in the conjugate may be in the range of a 0.8:1 to 1 : 1, 1 :1 to 2: 1, 2: 1 to 3: 1, 4: 1 to 5: 1, 6: 1 to 7: 1 8: 1 to 9: 1, or 9: 1 to 10: 1 molar ratio.
  • the adjuvant to polypeptide may be in the range of 1 : 1 to 2: 1. Any free adjuvant may be inactivated by azide and removed by dialysis.
  • the adjuvant to polypeptide molar ratio is 1: 1. In another, it is 2: 1.
  • Presence of contaminants An important parameter for polypeptide conjugates is the level of free adjuvant that is not covalently conjugated to the CPAF polypeptide, but is nevertheless present in the conjugate composition.
  • the free adjuvant is noncovalently associated with (i.e., noncovalently bound to, adsorbed to, or entrapped in or with) the polypeptide conjugate.
  • compositions comprising the CPAF polypeptide, which may comprise a CPAF polypeptide-adjuvant conjugate, and one or more pharmaceutically acceptable excipients.
  • the pharmaceutical composition is a vaccine composition.
  • the vaccine composition enhances a Th 1 -based immune response.
  • the pharmaceutical composition may comprise one or more soluble adjuvants in addition to an adjuvant conjugated to the CPAF polypeptide.
  • the soluble adjuvant may be a different adjuvant than the conjugated adjuvant.
  • the soluble adjuvant may be the same adjuvant as the conjugated adjuvant.
  • the soluble adjuvant may be aluminum salt, for example aluminum potassium phosphate, aluminum hydroxyphosphate sulfate, aluminum hydroxide, or aluminum phosphate, and any combination thereof.
  • the adjuvant may be an oil-in-water emulsion.
  • the adjuvant may be AS03, MF59, or AF03, and any combination thereof.
  • the adjuvant may be an agonist of one or more of the Toll-like receptors (TLR), Stimulator of Interferon Genes receptor (STING), C-type lectin receptors (CLR), RIG-I-like receptors (RLR), and NOD-like receptors (NLR).
  • TLR Toll-like receptors
  • STING Stimulator of Interferon Genes receptor
  • CLR C-type lectin receptors
  • RLR RIG-I-like receptors
  • NLR NOD-like receptors
  • Non-limiting examples of receptor agonists based adjuvants include RC529, cGAMP, c- di-AMP, c-di-GMP, 5,6-Dimethylxanthenone-4-acetic acid, AS03, ADU-S100, MK-1454, 2'3'- cGAMP, TMX-202, CL1151, CDA, BMS-986301, diABZl, 2BXy, DOPA, 2BXy-D0PA, an imidazoquinoline based agonist, CpG, and a CpG derivatives selected from the group consisting of CpG1826, CpG1018 and polyC-CpG.
  • compositions include an aluminum salt adjuvant it is preferred that the concentration of Al 3+ in the composition is ⁇ 1.25 mg per dose e.g. ⁇ 1.25 mg per 0.5 ml, and ideally ⁇ 0.85 mg per dose.
  • Conjugates within a composition may be adsorbed to the aluminum salt adjuvant.
  • conjugates can be adsorbed to an aluminum salt individually and then mixed, or can be added into an aluminum salt to achieve sequential adsorption, thereby forming the mixed conjugate composition.
  • Receptor agonists may be added to a disclosed formulation at about 0.1 pg/dose -100 pg/dose.
  • the disclosed formulation may comprise about 0.1 pg/dose to about 1 pg/dose, 1 pg/dose to about 5 pg/dose. 5 pg/dose to about 10 pg/dose, 10 pg/dose to about 20 pg/dose, 20 pg/dose to about 30 pg/dose of one or more agonists, wherein each dose comprises about 15 pg of the adjuvant conjugated CPAF polypeptide.
  • the pharmaceutical composition does not comprise any unconjugated adjuvant.
  • the pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients.
  • the pharmaceutically acceptable excipient may approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, nonessential amino acids, L-arginine hydrochlorate, saccharose, D-trehalose dehydrate, sorbitol, tris (hydroxymethyl) aminomethane and/or urea.
  • physiological conditions such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, nonessential amino acids, L-arginine hydrochlorate, saccharose, D
  • the pharmaceutically acceptable excipient may comprise one or more additives including, for example, diluents, binders, stabilizers, and preservatives.
  • the pharmaceutical excipient may further comprise a tonicity agent to bring osmolality of the composition into an acceptable range.
  • tonicity agents include sodium chloride, dextrose, and glycerin, and any combination thereof.
  • the pharmaceutically acceptable excipient may comprise a surface-active agent (surfactant).
  • the surface-active agent may be polyoxyethylene sorbitan monolaurate (Tween 20) , polyoxyethylene sorbitan monooleate (Tween 80), Brij 35, Triton X-10, Pluronic F127, or sodium dodecyl sulfate (SDS).
  • the surface-active agent is present at a concentration between 0.0003% and 0.3% (w/w).
  • the pH of the pharmaceutical composition may be between 5.5 and 8, and more specifically between 6.5 and 7.5 (e.g. about 7). Stable pH may be maintained by the use of a buffer e.g.
  • the buffer or pH adjusting agent is selected from the group consisting of sodium borate, sodium phosphate, sodium citrate, ammonium sulfate, or succinate, and any combination thereof.
  • suitable buffers include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride.
  • Pharmaceutical compositions may be isotonic with respect to humans.
  • the pharmaceutical composition may also comprise one or several additional salts, such as NaCl.
  • the pharmaceutical composition may be formulated for any mode of delivery. Suitable routes of administration may, for example, include intravenous, subcutaneous, intranasal route, cranial, transmucosal, trans-nasal, intestinal, and/or parenteral delivery. In an aspect, the pharmaceutical composition is formulated for intramucosal delivery.
  • Suitable excipients for intramucosal delivery may include mucoadhesive polymers for example chitosan, alginate, and polyacrylic acid; gelling agents for example carbomer and sodium alginate; surfactants, for example polysorbates (Tween), sodium lauryl sulfate, and lecithin; penetration enhancers, for example atty acids (oleic acid), bile salts (sodium deoxycholate), and cyclodextrins; buffering agents as disclosed herein; preservatives; antioxidants; solubilizing agents, for example cyclodextrins, propylene glycol; thickening agents, for example hydroxypropyl methylcellulose (HPMC); mucin enhancers, for example thiomers and lectins, bio adhesive polymers, for example polycarbophil and sodium carboxymethylcellulose; polymeric nanoparticles and lipid based excipients.
  • mucoadhesive polymers for example chito
  • the pharmaceutical composition may be sterilized by conventional sterilization techniques, or may be sterile fdtered.
  • the resulting aqueous solutions may be packaged and stored in liquid form or lyophilized, the lyophilized preparation being reconstituted with a sterile aqueous carrier prior to administration.
  • the pharmaceutical composition may be packaged and stored as micropellets via a prilling process.
  • the CPAF polypeptide which may be a CPAF polypeptide-adjuvant conjugate, or pharmaceutical composition thereof may be used as a vaccine to protect a subject susceptible to chlamydial infection, may be used to prevent or reduce a chlamydia infection in a subject, or may be used to induce an immune response in a subject to the CPAF polypeptide.
  • Such uses may be carried out by administering a composition disclosed herein to the subject.
  • the administration may be or may be intended to be via a systemic or mucosal route.
  • Suitable subjects may include any vertebrate susceptible to a chlamydia infection.
  • the subject may be a mammal, which may be a human; a companion animal such as a cat, dog, rodent, horses; a research animals such as a rabbit, sheep, pig, dog, primate, mouse, rat, or other rodents; an agricultural animal such as a cow, cattle, pig, goat, sheep, horse, deer, chicken, or other fowl; a zoo animal; or a primates or ape such as a chimpanzee, monkey, or gorilla.
  • the subject may be of any age.
  • the subject may be a human of >11 years of age.
  • the subject may be a human of ⁇ 11 years of age.
  • an immunologically effective amount of a pharmaceutical composition is administered to the subject.
  • the “effective amount” may refer to a dose required to elicit a robust Thl/Th2 response that reduces the likelihood or severity of infectivity of chlamydia during a subsequent challenge.
  • the methods of the invention can be used for the prevention and/or reduction of clinical infections caused by Chlamydia.
  • the amount of CPAF polypeptide administered to a subject or included in a medicament may be an amount that induces an immunoprotective response without significant adverse effects. The amount may vary depending upon the chlamydia species.Each dose may comprise about 100 ng to 10,000 pg of the CPAF polypeptide.
  • each dose may comprise 100, 150, 200, 250, 300, 400, 500, or 750 ng, or 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ⁇ g, or a range of two of the foregoing amounts.
  • each dose comprises about 1 ⁇ g/kg to about 1 mg/kg of the CPAF polypeptide.
  • the dose comprises about 5 ⁇ g/kg to about 500 ⁇ g/kg.
  • the CPAF polypeptide may be administered in an amount from about 0.01 mg/kg to about 20 mg/kg.
  • the particular amount of the CPAF polypeptide that constitutes an amount effective to generate an immune response depends to some extent upon certain factors such as, for example, the particular composition being administered; the particular adjuvant being administered and the amount thereof; the state of the immune system; the method and order of administration of the composition; the species to which the formulation is being administered; and the desired therapeutic result. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.
  • compositions may be administered to the subject at a dosage of CPAF ranging from about 1 ⁇ g to about 25 mg per dose (e.g., about 1 ⁇ g, 5 ⁇ g, 10 ⁇ g, 20 ⁇ g, 30 ⁇ g, 40 ⁇ g, 50 ⁇ g, 100 ⁇ g, 200 ⁇ g, 300 ⁇ g, 400 ⁇ g, 500 ⁇ g, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, or to about 25 mg per dose or any range therein).
  • the CPAF polypeptide may be administered or be intended to be administered to a subject via a parenteral route.A single dose of the pharmaceutical composition may be administered to a subject intravenously.
  • Booster doses may be necessary to impart full protection.
  • one or more additional doses may be administered at suitable intervals.
  • the pharmaceutical composition may be administered to a subject by an intramucosal route..
  • a single dose of the pharmaceutical composition may be administered to a subject via intramucosal route.
  • Booster doses may be necessary to impart full protection.
  • one or more additional doses may be administered at suitable intervals.
  • Optimal amounts of components for a particular pharmaceutical composition can be ascertained by standard studies involving observation of appropriate immune responses in subjects. For example, the dosage for human vaccination may be determined by extrapolation from animal studies to human data. In another aspect, the dosage is determined empirically.
  • a composition disclosed herein can be formulated as a single dose vial, multi-dose vial, an inhaler device, an intranasal delivery device, or as a pre-fdled syringe.
  • the current disclosure also encompasses a kit comprising a disclosed CPAF polypeptide, which may be a CPAF-polypeptide conjugate, or a pharmaceutical composition thereof and at least one container suitable for a delivery method disclosed herein.
  • the container may be suitable for inhalation or intranasal administration.
  • the kits may further comprise instruction for use.
  • the present invention has multiple aspects, illustrated by the following non-limiting examples.
  • the current disclosure encompasses vaccine compositions comprising a catalytically inactive CPAF polypeptide, wherein the catalytically inactive CPAF polypeptide comprises one or more non-canonical amino acids (ncAA), and wherein each ncAA is covalently attached to an adjuvant.
  • ncAA non-canonical amino acids
  • Example 1 CD4 T cell immunity protects against genital Chlamydia infection
  • CT Chlamydia trachomatis
  • IFNg is needed for optimal antibody-mediated immunity against murine genital chlamydial infection. Women with the highest anti-CT IgG responses may be Th2-skewed, resulting in inadequate IFNg responses. This reinforces the importance of eliciting robust and appropriate functional T cell responses, generating a milieu that enables antibody-mediated opsonophagocytosis. Shortlived sterilizing immunity after infection is observed in animal models. Gene-knockout, depletion, and adoptive transfer studies in mice consistently confirm that CD4 T cells are the primary mediators of immunity and that polyfunctional Thl cells (IFNg+ TNFa+ IL-2+) secreting high cytokine levels correlate with protection.
  • TNFg+ TNFa+ IL-2+ polyfunctional Thl cells
  • CD4 T cells are not terminally differentiated and may shift between subsets to alter their cytokine profile (i.e., Thl7 to Thl shift) or exhibit properties of multiple subsets where a more extensive range of cytokines are produced (i.e., bifunctional Thl/17).
  • Thl or Th 1/17 cells produce IFNg that induces cellular responses that starve intracellular chlamydia of tryptophan and enhance phagocyte effector functions.
  • IL-17A independently promotes epithelial integrity and induces the production of antimicrobial peptides.
  • CT serovar D shedding is detected for only 10 days in mice after vaginal inoculation, suggesting CT delivered intravaginally is insufficient for challenge studies when assessing adaptive immune responses.
  • Transcervical inoculation of mice with human strains results in more productive infections, but bypasses the cervix, a significant mucosal and immunological barrier to the upper genital tract.
  • Transcervical inoculation with human serovars induces minimal oviduct pathology, presenting another limitation to this model.
  • preclinical studies in mice frequently use host-adapted C.
  • CM C. trachomatis
  • C. trachomatis C. trachomatis
  • C. muridarum stock populations contain sub-populations of genetic variants expressing phenotypic differences. Inoculation of mice with different plaque-purified clonal isolates from these stocks results in infections of varying severity.
  • CM006 reflects the infection profile and pathology of its multiclonal parent stock and is a valuable challenge strain for vaccine experiments because it yields consistent levels of cervicovaginal shedding and pathology between individual mice.
  • Intravaginal immunization of mice with a plasmid-deficient live-attenuated CM (strain CM972) elicits protective memory CD4 T cells that reduce bacterial burden by 2-logs and completely protects from oviduct damage when challenged with high doses (5x105) of highly virulent strains.
  • a live-attenuated vaccine is not suitable for humans, but this strain can help guide the rational vaccine design and inform the criteria for vaccine efficacy, defining the parameters that may be achieved with the current vaccine candidate.
  • the T-cell lines were rested for 24 hours and tested for their response to pooled peptides by IFNg ELISPOT. Twelve seronegative healthy donors defined a CT-specific T cell response threshold. The mean CT-specific T cell response for these donors was 15 SFU/10 6 cells, with the 97.5 th percentile not exceeding 220 SFU/10 6 cells. Thus, 300 SFU/10 6 were selected to define a CT-specific T cell response. Sixteen of the TRAC participants profiled responded to CPAF. while only 5 responded to CT MOMP (FIG.
  • CPAF a secreted virulence protein
  • CPAF is a highly conserved serine protease abundantly secreted into the host cell cytoplasm late in infection and released extracellularly with host cell lysis.
  • CPAF-dependent processing of host proteins contributes to the generation of new infectious progeny and their release from the host cell; suppression of the T cell chemokine, CXCL10; and inactivation of neutrophils.
  • CPAF was also found to be the 9th most abundant CT transcript detected in cervical samples by RNAseq.
  • Example 4 is also immunodominant in mice
  • ICS confirmed that IFNg+TNFa+ CPAF-specific T cells were fully CD4-restricted and comprised 6.4% of total CD44 hl memory CD4 T cells (Fig. 3B).
  • Adjuvant selection- vaccine adjuvants amplify CD4 T cell responses to CPAF
  • CpG1018 used in the HEPLISAV-B vaccine
  • bacterial agonists such as c- di-AMP (CD A)
  • CDA c- di-AMP
  • FIG. 4 Preliminary data shows that conjugated CpG-CPAF mixed with CDA enhanced immunogenicity without reactogenicity.
  • third-generation synthetic CDA analogs like diamidobenzimidazole (diABZI) can be directly conjugated to immunogens and are potent and well tolerated on mucosal delivery.
  • Imidazoquinoline-based TLR7 agonists generate potent immunostimulatory activity and Thl -biased adaptive immunity in mice and humans. They are effective topical therapeutics but have been too reactogenic for use as adjuvants, but immunogenicity can be retained by linking TLR7 agonists to immunomodulatory compounds, such as dopamine, to reduce pro- inflammatory side effects. Thus DOPA linked agonists are in consideration.
  • Example 6 Adjuvant conjugation and testing
  • Approaches disclosed herein may use CP AF conjugated adjuvants or mixtures of such compositions and soluble adjuvants to provide effective vaccination and a path to generating strong chlamydia-specific T-cell responses without adverse reactions.
  • This approach facilitates the concurrent delivery of vaccine components to an antigen-presenting cell. It overcomes the disassociation of immunogen and adjuvant in mixed formulations and prolongs antigen presentation to induce adaptive responses through intracellular storage and depot effects. This may provide superior efficacy.
  • conjugating agonists to vaccine antigens such as CPAF polypeptides may enhance immunogenicity, reduce the amount of adjuvant required, and/or prevent systemic toxicity.
  • FIG. 6A shows a generalized schematic of the Click chemistry used in the conjugation of a polypeptide comprising a nnAA (Rl) comprising an azide group and a DBCO linked adjuvant (R2).
  • Site-specific conjugation technology used herein may include TLR or STING agonists without compromising critical T cell and B cell epitopes and preserve the capacity of the product to elicit robust immune responses.
  • Agonists were synthesized with reactive dibenzocyclooctyne (DBCO) terminal modifications for reaction with pAMF-containing CPAF mutants. Purity >95%, was confirmed by 1H-NMR and LC-MS prior to conjugation. Synthesis and preliminary characterization of CPAF-agonist conjugates was conducted. Robust design principles for directly conjugating agonists with recombinant proteins using strain-promoted azide-alkyne click chemistry (SPAAC) were used. Near-complete reaction was achieved when a 2.5-10-fold excess of different TLR agonists was mixed with antigen at 35 °C in PBS for 48 h.
  • SPAAC strain-promoted azide-alkyne click chemistry
  • CPAF-pAMF were reacted with agonist-DBCO using SPAAC, and the resulting CPAF-agonist conjugates dialyzed to remove unreacted material. Size-exclusion HPLC was used to confirm the complete removal of unreacted material, while SDS-PAGE was used to confirm reaction efficiency and conservation of CPAF integrity.
  • ⁇ 1 mg of each CPAF-agonist construct was synthesized (CPAF-CpG, CPAF-2BXy, CPAF-2BXy-Dopa, and CPAF-STING). Following synthesis, gel clot assays was used to confirm that endotoxin is absent ( ⁇ 1 EU/mL). If endotoxin remained, Triton X-l 14 extraction was used.
  • mice were immunized i.n. or i.m. twice with i) conjugated inactivated CPAF to CpG (15 mg/dose), ii) CpG-CPAF conjugate combined with the STING agonist ADU-S100 (5 mg/dose), or CpG-CPAF conjugate formulated with ADU-S100 and squalene oil-in-water emulsion AddaS03 (AS03 mimic; 1 : 1 ratio) (FIG. 7A-7C) 21 days apart were used. Immunized mice exhibited no signs of distress, adverse effects, or weight loss over 14 days, when they were sacrificed to evaluate cellular responses to these regimens.
  • soluble adjuvant CDA was added to the formulation to test the impact of using a multi-adjuvant approach.
  • Groups of female C57BL/6 mice were immunized twice with 15 mg doses of CPAF formulated as above 21 days apart.
  • the mice were intravaginally challenged 21 days post-boost with a high inoculum of CM006 (5x105 IFU).
  • CM006 5x105 IFU.
  • Including CDA in the i.n. vaccine significantly enhanced the frequency of IFNg (FIG. 7A) and IL-17A (FIG. 7B) responses compared to CpG-conjugation alone with i.n. immunization. In contrast, i.m.
  • Example 8 Multi-adjuvant CPAF-TLR7-DOPA linkage enhances immunogenicity without increasing IL-1 ⁇
  • CPAF was conjugated to 2BXy-Dopa-DBCO or 2BXy-G4S-DBCO (where G4S is a soluble linker) as in Example 6 and further illustrated in FIG.6A and evaluated for immunogenicity of the TLR7 agonist (2BXy) platform.
  • Example 9 Refinement of the multi-adjuvant CPAF-CpG+CDA+AS03 vaccine [0280]
  • immunogenicity and challenge experiments comparing (i) CpG1018 versus CpG1826 in an intranasal vaccine regimen will be conducted (FIG.10).
  • Mice will be i.n. immunized with 15 mg of CPAF conjugated with CpG1018 or CpG1826 with and without 5 mg of CDA (ADU-S100) in AS03 plus controls on days 0 (prime) and 30 (boost).
  • mice can then be sacrificed 10 days post-boost for immunogenicity assays or challenged 30 days later. A dose- limiting analysis of the benchmark CpG1018 can then be conducted.
  • CPAF-specific T cell responses from splenocytes of individual mice can be measured by ex vivo IFNy and IL-17A ELISpot following stimulation with pools of overlapping peptides (OLPs) spanning CPAF (lOmM, 15aa long with 11 aa overlap), or no stimulation (negative control).
  • OLPs overlapping peptides
  • the proportion of IFNg and/or IL-17A positive CD4 and CD8 T cell responses can be determined by intracellular cytokine staining (ICS).
  • CPAF-specific antibody IgGl/IgG2b/IgG2c/IgG3/IgA
  • Antibody can also be quantified in vaginal lavages.
  • a panel of CPAF-agonist conjugates have been generated and tested for immunostimulation and toxicity in cell-based assays.
  • CPAF-agonist conjugates that induce cytokine production equivalent or superior to unlinked controls can now be advanced for evaluation.
  • Antigen-adjuvant conjugates which cannot be synthesized on a 1 mg scale or for which CPAF integrity cannot be confirmed will be excluded from further analysis.
  • Conjugates that display cellular toxicity at levels >20% of a lipopolysaccharide positive control or those with poor water solubility will also be excluded from further study.
  • Scalable CPAF vaccine candidates will be identified.
  • Some CPAF-agonist conjugates may have poor solubility in water because charge shielding can occur. If so, hydrophilic spacer groups comprised of polyethylene glycol or glycine oligomers will be introduced to the immune agonists, or the pH of the solution can be adjusted during and after conjugation. If conjugates do not induce detectable cytokine production, they will be reformulated and retested.
  • Toxicity assessments of CPAF-agonist conjugates advanced from these studies will be conducted.
  • Benchmark vaccines can be tested along with four classes of vaccines: (1) CPAF-agonist conjugates, (2) CPAF + soluble agonist (i.e., equivalent doses of mixed antigen and agonist), (3) CPAF-agonist conjugates + a second, soluble agonist, or (4) negative controls (CPAF only and PBS) (FIG. 11).
  • Vaccines can be prepared using the dose-sparing quantity of CPAF-agonist conjugates, including AS03 if indicated by the results of further experiments.
  • Sublingual formulations will consist of 0.10% weight/volume (w/v) Carbopol®, 0.75% w/v HPMC, and 15% w/v Pluronic® Fl 2794.
  • a multiplexed serum cytokine assay can quantify systemic cytokine production at 1 and 24 hours post-primary immunization. JFNy and IL-6 may serve as surrogates of a protective immune response; IL- la and MCP-1 may serve as surrogates of an immunotoxic response.
  • Body weight and temperature will be monitored through day 28 to measure overall health. On day 27, sera and nasal washes will be collected to evaluate IgG and IgA responses.
  • Splenocytes can be isolated on day 28, restimulated with CPAF OLPs, and analyzed via ZFNy ELISpot.
  • the lower respiratory tracts can be harvested for histopathology at sacrifice.
  • Safety issues may manifest early, as elevated sentinel cytokines with either the s.l. or i.n. route, or late, as respiratory tract damage with i.n. delivery. While some i.n. vaccines may cause respiratory tract damage that disqualifies them from further testing, none of the vaccines are anticipated to cause late toxicity when delivered s.l. so those candidates can remain, if sufficiently immunogenic.
  • Preliminary data provided herein showed that CPAF- CpG+CDA+AS03 delivered i.n. was well tolerated but lung pathology was not monitored. If bronchial or lung tissue damage is observed, the s.l. route may be well tolerated. Multiple delivery strategies will be tested and optimized.
  • Each dose will be 15mg or a lesser alternative antigen-sparing concentration. Mice will be sacrificed 10 days post-boost. CPAF-specific T cell responses from splenocytes will be measured. T cell responses will be further defined by flow cytometric analysis of CD4 and CD8 antigen-specific proliferation by dilution of carboxyfluorescein succinimidyl ester (CFSE) and ICS for IFNg, IL-17A, TNFa, IL-2, and CD107a. CPAF-specific antibody responses will be measured by ELISA in serum and vaginal lavages (as shown in FIG. 10).
  • CFSE carboxyfluorescein succinimidyl ester
  • mice Parallel groups of vaccinated mice will be used to enumerate memory CPAF-specific T cells in the genital tract by flow cytometry 30 days post-boost based on the expression of LIVE/DEAD, CD45, CD3, CD4, CD8, CD44, IFNg, IL-17A, and TNFa.
  • Candidates will be advanced if IFNg SFUs are at least 50% or greater of the benchmark vaccine because IFNg is an essential mediator of protection, and/or if they elicit at least a two-log reduction in burden and 50% reduction in gross and microscopic hydrosalpinx compared to naive controls.
  • Candidate vaccines incorporating a single agonist will be prioritized over two agonists if they demonstrate equivalent efficacy for anticipated ease of manufacture and future cost savings.
  • Candidates that meet or exceed selection criteria in C57BL/6 mice along with the benchmark vaccine will be tested as described above in female B ALB/c mice because of their different genetic backgrounds, different MHC alleles, and Th2-skewed immune responses. Similar outcomes for protection as observed in C57BL/6 are expected.
  • the top candidate that passes selection criteria in C57BL/6 and BALB/c mice, along with the benchmark vaccine, will be tested intramuscularly.
  • Infertility testing in C57BL/6 and BALB/c mice The top candidate that pass selection criteria in C57BL/6 and BALB/c mice and the benchmark vaccine will be tested for their ability to protect against infertility.
  • Vaccine process development Select candidates will be transitioned to 1) the optimization and scale-up of production of CPAF, the adjuvant, and conjugation; 2) the delineation of an analytical control strategy for starting materials, intermediates, drug substances, and drug product; 3) evaluation of the stability of the final vaccine, and 4) completion of a non- GLP toxicology study.
  • CPAF at >100 mg/L in CFPS with -85% purity.
  • CPAF-CpG conjugates have been produced at the -lOrng scale to generate the preliminary data provided herein.
  • DoE Design of Experiments
  • DoE allows the mathematical modeling of complex reactions with many interdependent factors influencing the performance, such as temperature, pH, input ratios, and agitation speed in the case of conjugation. Optimization of upstream production, downstream purification and conjugation of CPAF will be done using this approach.
  • the DoE approach will be used to determine impeller speed, temperature, and pH resulting in optimal conditions for the soluble yield of CPAF.
  • Rushton-style impellers with high shear to disrupt larger gas bubbles from open-pipe or drilled- hole spargers commonly used in microbial fermentation will be utilized. These impellers are well characterized, with a variety of correlations for Reynolds Number, Power Number, and kLa for transition to manufacturing and commercial scale. We will modulate the gas-flow rate, pH, and temperature to optimize soluble yield.
  • Gas flow rates will also be optimized. Changing the gas flow rate changes the gas-liquid interface and affects the foaming and solubility of the target protein and cell-free components.
  • Cell-free reactions place proteins, cell-free biomolecules, and components in direct contact with the reactor vessel surfaces and gas-liquid interfaces (bubbles), which can denature the protein of interest or proteins essential to the cell-free reaction, such as essential enzymes.
  • a hydrophilic region aqueous liquid
  • a hydrophobic region gas
  • cell-free reaction setups use a blend of pure oxygen and air, which delivers a more significant percentage of oxygen so that DO control can use less overall gas flow.
  • pH control pH control is maintained by the automated addition of both acid and base to maintain a set-point pH. Most scaled-up cell-free reactions utilize IM citric acid and IM potassium hydroxide, because this acid/base pair provides good pH control without excessive volume additions.
  • the initial set point for cell-free protein production is pH 7.2 ⁇ 0.1. The pH will be varied in 0.2 increments to define the design space using DoE.
  • Temperature To define the design space, the temperature will be from 20 °C to 30 °C in two-degree increments.
  • DoE experiments at the 5-10 mg scale will be evaluated based on critical quality attributes.
  • Promising conditions will be scaled up to 20 mg for immunogenicity and suitability assessment.
  • TFF purification and filter sizing will be performed in five 100 mg scale runs.
  • the Mettler-Toledo EasyMax system will be used for 100-200 mg scale reactions with control over agitation, pH, temperature, and reactant addition.
  • the final candidate condition will be scaled to the 200 mg scale to supply material for TFF optimization, filter sizing, analytical assay development, and formulation development.
  • Figure 13 provides a schematic of the formulation development process that will be used. Exemplary desired formulation are stable in an aqueous isotonic solution stored at 4 °C at approximately physiological pH. CPAF compositions with a panel of (GRAS) excipients will be tested initially in a 96-well plate format. Each condition will be evaluated for thermal melt, turbidity, and size via SEC-MALS to measure the structure and potential aggregation of the candidate antigens.
  • GRAS panel of
  • IVRP in vitro relative potency
  • mice Female C57BL/6 mice were immunized twice intranasally with CPAF candidates or PBS control as provided Table 9 below. Notably, ADU-S100 was present at 5 pg and other agonists were conjugated at 1 agonist molecule per molecule of CPAF. After 30 days following the second dose mice were intravaginally challenged with Chlamydia muridarum (strain CM006) as described herein. Mice were monitored for cervicovaginal shedding via endocervical swabs and IFUs were calculated, as depicted in Figure 13. Notably, the CPAF-CL1151 conjugate performed with a 1.5-log improvement compared with PBS controls.

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Abstract

Provided are chlamydial protease-like activity factor (CPAF) polypeptides conjugated to an adjuvant, such as through click chemistry, and uses thereof. Uses include as vaccines for protection against chlamydia infections.

Description

CHLAMYDIA VACCINE COMPOSITIONS
FIELD OF THE INVENTION
[0001] The present invention relates to chlamydial protease-like activity factor (CPAF) polypeptides conjugated to an adjuvant, such as through click chemistry, and uses thereof. The uses include as vaccines for protection against chlamydia infections.
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant Number AI144181 awarded by the National Institutes of Health. The Government has certain rights in the invention.
BACKGROUND OF THE INVENTION
[0003] Chlamydia trachomatis (CT) is a common sexually transmitted bacterial infection, with over 100 million cases annually, with direct treatment costs in the United States exceeding $500 M. In women, chlamydia infects the endocervix but can spread to the endometrium and oviducts to cause symptomatic (-10%), or subclinical (-25%) pelvic inflammatory disease (PID). Reproductive sequelae include chronic pelvic pain (30%), infertility (10%), and ectopic pregnancy (10%). C. trachomatis also enhances the acquisition of other sexually transmitted infections (STIs) and is an independent risk factor for cervical cancer. Prior CT infection increases the risk for fallopian tube carcinomas and epithelial ovarian cancer. In males, urethritis, epididymitis, and orchitis are the most common acute manifestations, and while long termsequelae are not definitely associated with a CT genitourinary infection in males, proctitis, prostatitis, and prostate cancer have been linked to CT particularly in men that have sex with men (MSM). Most infections are asymptomatic (70%) and thus frequently untreated, despite effective antibiotics. Lymphogranuloma venereum (LGV) infections can result in highly debilitating chronic sequelae with bubo formation, fistulas, fibrosis, and rectal stenosis. Among MSM, LGV infections are on the rise. In low- and middle-income countries, CT-induced trachoma is also the main cause of preventable blindness.
[0004] There is therefore an unmet medical need for effective vaccines against chlamydia infection. SUMMARY OF THE INVENTION
[0005] Provided herein is a chlamydial protease-like activity factor (CPAF) polypeptide comprising one or more non-natural amino acids (nnAA). The CPAF polypeptide may be catalytically inactive. The CPAF polypeptide may comprise one or more substitutions at one or more of the amino acids H97, S499, S491, E550, and C492 relative to the sequence set forth in SEQ ID NO: 1, which may be the sequence of C. muridarum CPAFIN\TM9rll&5‘ 3A. The substitutions may comprise one or more of H97A, S491A, S499A, C492T, and E550Q. The CPAF polypeptide may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or more nnAA. Each nnAA may substitute one or more amino acids relative to a reference CPAF protein, of which may independently be one or more of phenylalanine, lysine, and tyrosine. The site of the substitution may be F130 or Y569 relative to the sequence set forth in SEQ ID NO: 1. Each nnAA may comprise 2-amino-3-(4- azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin- 2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyri din-2- yl)propanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid. Each nnAA may be pAMF.Each nnAA may comprise a structure as set forth in formula III:
Figure imgf000004_0001
wherein:
Ar comprises a 5-membered or 6-membered aromatic ring optionally containing at least one heteroatom;
W5 is selected from Ci-Cio alkylene, -NH-, -O- and -S-;
QI is zero or 1;
W6 is selected from the group consisting of azido, 1,2,4,5-tetrazinyl optionally C- substituted with a lower alkyl group, and ethynyl, and R3 is OH or an amino acid residue of the CPAF polypeptide, and R4 is H or an amino acid residue of the CPAF polypeptide.
[0006] The CPAF polypeptide may comprise the amino acid sequence set forth in any one of SEQ ID NOS: 14-22, or a fragment thereof, or a sequence at least about 80% identical thereto. Each nnAA may be covalently attached to an adjuvant to form a polypeptide conjugate. The adjuvant may enhance the Th 1 -based immune response The adjuvant may comprise a STING agonist, TLR agonist or dopamine receptor agonist. The STING agonistmay comprise di amidobenzimidazole (diABZl), MSA-2ADU-S100, MK-1454, MK-2118, SB11285, BMS- 986301, DMXAA, E7766, GSK3745417 cyclic di-GMP (guanosine 5 '-monophosphate) (CDG), cyclic di -AMP (adenosine 5 '-monophosphate) (CD A), cyclic GMP-AMP (cGAMP), 5,6- Dimethylxanthenone-4-acetic acid, AS03, MK-1454, TMX-202, or a derivative thereof. The TLR agonist may comprise 2BXy, DOPA, 2BXy-DOPA, an imidazoquinoline-based agonist, CpG, or a CpG derivative, which may be CpG1826 CpG1018„ or polyC-CpG, or a derivative of the foregoing. The adjuvant covalently attached to the CPAF polypeptide may comprise a dibenzylcyclooctyne (DBCO) group and may comprise a structure as set forth in formula XV, XVa, XVIa, or XVb.
Figure imgf000005_0001
[0010] (Formula XVa)
[0011] wherein:
[0012] Ri is independently H, formyl, or at least one amino acid of the CPAF polypeptide;
[0013] R2 is independently OH or at least one amino acid of the CPAF polypeptide;
[0014] D is — Ar— W3— or — Wl— Yl— C(O)— Y2— W2— ;
Figure imgf000006_0001
[0016] each of Wl, W2, and W3 is independently a single bond or lower alkylene;
[0017] each XI is independently — NH — , — O — , or — S — ;
[0018] each Y 1 is independently a single bond, — NH — , or — O — ;
[0019] each Y2 is independently a single bond, — NH — , — O — , or an N-linked or C-linked pyrrolidinylene;
[0020] one of Zi, Z2, and Z3 is — N — and the others of Zi, Z2, and Z3 are independently — CH —
[0021] L22 is independently a bond, alkyl or poly(alkyloxy); and
[0022] X is the adjuvant.
[0023] The adjuvant may be linked to an azido-containing nnAA via a structure of formula XVIa or XVIb:
Figure imgf000006_0002
Figure imgf000007_0001
[0027] (Formula XVIb)
[0028] wherein, Ri is independently H, formyl, or at least one amino acid of the CPAF polypeptide;
[0029] R.2 is independently OH or at least one amino acid of the CPAF polypeptide;
[0030] W is C or N;
[0031] y is at least 1;
[0032] n is at least 1; and
[0033] X is the adjuvant.
[0034] Also provided herein is a CPAF polypeptide conjugate comprising a catalytically inactive CPAF polypeptide and a pharmaceutical composition comprising the conjugate The catalytically inactive CPAF polypeptide may comprise one or more substitutions of one or more wild-type amino acids of a reference CPAF with a non-natural amino acid (nnAA). Each nnAA may be covalently attached to an adjuvant. The CPAF polypeptide conjugate may comprise a CPAF polypeptide comprising one or more substitutions at one or more of amino acids H97, S499, S491, E550, and C492 relative to the sequence set forth in SEQ ID NO: 1. The subsitutions may comprise one or more of H97A, S491A, S499A, C492T, and E550Q. The CPAF polypeptide may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or more nnAA. Each nnAA may substitute one or more amino acids of a reference CPAF protein, which may each independently comprise phenylalanine, lysine, and tyrosine. The site of substitution may be F130 or Y569 relative to the sequence set forth in SEQ ID NO: 1. Each nnAA may comprise 2-amino-3-(4- azidophenyljpropanoic acid (pAF), 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid. The nnAA may be pAMF. The nnAA may comprise a structure as set forth in formula III. The CPAF polypeptide conjugate may comprise the amino acid sequence set forth in any one of SEQ ID NOS: 14-22, or a fragment thereof, or a sequence at least about 80% identical thereto. Each nnAA may be covalently attached to an adjuvant. The conjugated adjuvant may be a water-soluble adjuvant. The adjuvant may enhance the Thl-based immune response. The adjuvant may comprise a STING agonist, TLR agonist, or a dopamine receptor agonist. The STING agonist may comprise di amidobenzimidazole (diABZl), MSA- 2ADU-S100, MK-1454, MK-2118, SB11285, BMS-986301, DMXAA, E7766, GSK3745417 cyclic di-GMP (guanosine 5 '-monophosphate) (CDG), cyclic di-AMP (adenosine 5'- monophosphate) (CD A), cyclic GMP-AMP (cGAMP), 5,6-Dimethylxanthenone-4-acetic acid, AS03, MK-1454, TMX-202, or a derivative thereof. The TLR agonist may comprise 2BXy, DOPA, 2Bxy-DOPA, an imidazoquinoline-based agonist, CpG, a CpG derivative, CpG1826, CpG1018, or poly-C-CpG, or a derivative of the foregoing.
[0035] Each nnAA may be linked to the to the adjuvant via a first handle on the adjuvant. The first handle may comprise propargyl, DIFO, dibenzylcyclooctyne (DBCO), or a DBCO(PEG)n- NH2 moiety, or a derivative thereof. Each nnAA may form a triazole linkage with the adjuvant. The adjuvant may be attached to the first handle. The first handle may comprise CpG-DBCO, STING-DBCO, 2BxY-DBCO, 2BxY-DOPA-DBCO, or CDA-DBCO, and may comprise a structure as set forth in formula Xlld-XIIh respectively.
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
[0036] The adjuvant linked to the nnAA may comprise a structure as set forth in formula XV, XVa, XVIa or XVb:
[0037] Provided herein are pharmaceutical composition comprising the CPAF polypeptide conjugate disclosed herein and one or more pharmaceutically acceptable excipients, additional soluble adjuvants, or both. The additional soluble adjuvant be any adjuvant. The additional soluble adjuvant may be an enhancer of Th-1 based immunity. The additional soluble adjuvant may be a different adjuvant than the adjuvant covalently attached to the CPAF polypeptide. The one or more pharmaceutically acceptable excipients may be suitable for mucosal delivery. The pharmaceutical composition may be for use as a vaccine against a chlamydia infection in a mammalian subject, or may be for use in the manufacture of a medicament for use as a vaccine. The pharmaceutical composition may comprise one or more additional polypeptide conjugate, wherein the additional polypeptide conjugate comprises a different polypeptide or a different adjuvant, relative to the CPAF polypeptide and the adjuvant. The pharmaceutical composition may comprise a second vaccine against chlamydia or one or more other microorganisms, or combinations thereof.
[0038] Also, provided herein are methods of making a CPAF polypeptide-adjuvant conjugate. The method may comprise contacting a catalytically inactive CPAF polypeptide with an adjuvant to form a CPAF polypeptide-adjuvant conjugate. The CPAF polypeptide may comprise one or more substitutions of one or more wild-type amino acids of a reference CPAF protein with a non-natural amino acid (nnAA) comprising a second handle. The adjuvant may comprise a first handle comprising an alkynyl group on a cyclooctane ring structure. The CPAF polypeptide may comprise a substitution at one or more of amino acids H97, S499, S491, E550, and C492 relative to a reference CPAF protein. The nnAA may comprise 2-amino-3-(4- azidophenyljpropanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5- (azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid. The method may comprise a synthesis of the CPAF polypeptide and incorporation of nnAA using a cell free extract. The nnAA may comprise or may be activated to comprise a second handle. The second handle may comprise an azido group. The adjuvant may comprise or may be activated to comprise a first handle. The first handle may comprise propargyl, DIFO, dibenzylcyclooctyne (DBCO), or a DBCO(PEG)n-NH2 moiety, or a derivative thereof. The first handle may comprise a DBCO or DIFO ring structure comprising an alkynyl group. The adjuvant may be CpG-DBCO, STING-DBCO, 2BxY-DBCO, 2BxY-DOPA-DBCO or CDA-DBCO, and may have a structure as set forth in formula Xlld- Xllh respectively.
[0039] The method of making a CPAF polypeptide-adjuvant conjugate may comprise contacting of the adjuvant to the CPAF polypeptide to form the CPAF polypeptide-adjuvant conjugate by an azide-alkyne-based click chemistry. The polypeptide-adjuvant conjugate may form a triazole linkage. The method may not require a catalyst. The resulting polypeptide-adjuvant conjugate may comprise a structure as set forth in formula XV.
[0040] Provided herein are a vaccine and a method of immunizing a subject using the pharmaceutical composition. The vaccine may further comprise a carrier. The vaccine may be for use against a chlamydia infection. The vaccine and the method of immunizing may be for administering via a mucosal route. The subject may be a mammal, which may be a human. BRIEF DESCRIPTION OF THE DRAWINGS
[0041] Aspects of the present inventive concept are illustrated by way of example in which like reference numerals indicate similar elements and in which:
[0042] FIG. 1A-1C show that CPAF is an immunodominant CD4 T cell antigen with epitopes across the protein. FIG. 1A. PBMC from 30 seropositive women were cultured for 10 days with IL-2 + peptides spanning each antigen (n=21), then tested by IFNy ELISpot. Dashed line indicates > 20% of CT seropositive women responded to a specific antigen. A positive response was defined as the average of mock-subtracted wells > 300 SFU (spot forming units)/ 106 cells and > 4-fold average of mock wells. FIG. IB. In vitro expanded T cells were stimulated with CPAF followed by intracellular cytokine staining (ICS). CPAF-specific CD4+ T cells were dominated by IFNy+TNFu+ dual-positive and TNFa+ single-positive responses in women who were positive or negative in cultured IFNY ELISpot. A positive response was defined as > 3x the mock well and > 15 positive events. FIG. 1C. CPAF was divided into 20 fragments. PBMC stimulated with peptides spanning CPAF were cultured for 10 days, then tested against each fragment in IFNy ELISpot. PBMC from TRAC participants at 1 month (n=6) and 12 months (n=7) (4 overlapped) after enrollment were used.
[0043] FIG. 2 shows that CPAF-specific T cell response is CD4-dominant and maintained over 12 months. Comparison of CPAF-specific CD4+ and CD8+ T cell IFNy+TNFa+ response at 1- mo and 12-mo in women (n=10) after CT infection. Positive response: > 3-fold mock-stimulated wells and > 15 positive events. Significance within and between groups was measured by Wilcoxon signed-rank tests.
[0044] FIG. 3A-3C show that CPAF is immunogenic in mice genitally infected with C. muridarum. FIG. 3A. C57BL/6 mice (n=4) that cleared infections with CM972 followed by CM001 were sacrificed thirty days post clearance and splenocytes were stimulated with overlapping peptide pools (5 ug/ml) to OmcB, CPAF, MOMP, and thirty putative antigens, and responses were determined by IFNy ELISpot. **** p<0.0001 by one-way ANOVA. FIG. 3B. Frequency of CD44hi memory IFNy+ TNFa+ CD4 and CD8 T cell responses by ICS in CM972+CM001 immune mice after stimulation with CAP overlapping peptides (OLP). FIG. 3C. Frequency of CD44hi memory CPAF-specific IFNy+ TNFa+ CD4 T cells in the genital tract of naive and CM972-immune mice (N=4/group) 7 days after CM001 challenge. Error bars represent mean ± SD. [0045] FIG. 4 is a schematic showing the proposed mode of action for dual conjugated vaccine. [0046] FIG. 5 is an SDS-PAGE analysis of the CPAF full-length (CPAFf) polypeptide synthesized using the cell-free system followed by His-tag purification and TEV protease cleavage. The resulting tagless protein was found to separate into multiple N- and C-terminal fragments (CPAFn and CPAFc respectively). Lanes: 1. Size standards; 2. TEV reaction (load); 3. Imidazole elution; 4. Flow through (JR254-84.1 CPAF 2x pAMF final pool)
[0047] FIG. 6A is a schematic of a SPAAC click chemistry used in the conjugation of a polypeptide comprising a nnAA (Ri) comprising an azide group and a DBCO linked adjuvant (R2).
[0048] FIG. 6B shows the structure of some of the next-generation TLR and STING agonists.
[0049] FIG. 7A-7C show that intranasal immunization (i.n.) with CPAF-CpG plus CDA elicits robust T cell and antibody responses. Splenocytes from immunized mice (n=5) were stimulated with CPAF peptides and T cell responses were determined by IFNy (FIG. 7A) and IL-17A (FIG. 7B) ELISpot. Error bars represent mean SFU ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way RM ANOVA. FIG. 7C. Anti-CPAF antibody titers after i.n. immunization. Error bars represent mean ± SEM. *p<0.05 by one-way RM ANOVA.
[0050] FIG. 8A-8B show that intranasal immunization (i.n.) with CPAF-CpG+CDA+AS03 provides significant protection against a genital challenge in mice. FIG. 8A. Chlamydia burden over the course of infection comparing CPAF vaccines and controls. Error bars represent mean ± SEM. Significance determined by two-way RM ANOVA. FIG. 8B. Oviduct dilatation scores in i.n. immunized mice and naive controls. CPAF-CpG+CDA+AS03 versus naive by Kruskal- Wallis with Dunn’s multiple comparisons test (p=NS).
[0051] FIG. 9A-9B show that Intramuscular immunization (i.m.) with conjugated CPAF-2Bxy- Dopa enhances immunogenicity. FIG. 9A. Serum cytokines 1 or 18 hours after i.m. injection. FIG. 9B. Endpoint titer and IgG2c/IgGl ratio 13 days post-boost. ***p<0.001, ****p<0.0001 by one-way RM ANOVA.
[0052] FIG. 10 provides a schematic of the design of mouse immunogenicity (top) and challenge studies (bottom).
[0053] FIG. 11 provides a schematic of the Study design for toxicity and preliminary immunogenicity. Toxicity studies (red) will assay inflammation and pathology, and immunogenicity studies (blue) will assay antibody and T cell responses. [0054] FIG. 12 provides a schematic of the purification strategy using a TEV-protease cleavable tag.
[0055] FIG. 13 depicts inclusion forming units (IFUs) determined for mice immunized with CPAF and subsequently challenged with Chlamydia muridarum.
DETAILED DESCRIPTION
[0056] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects and aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized, and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.
[0057] In protein-conjugate vaccines the immune response to a “weak” antigens is amplified by attachment to a known “strong” protein antigen. In these semi -synthetic biomolecules, proteins that produce strong, long-lived T-cell dependent immune responses (“T-cell dependent antigens”) are typically attached to a “weak” antigen by nonspecific oxidation/reduction chemistry. The T-cell activating features on these immunogenic proteins recruit helper T-cells to B-cells that recognize the attached weak antigen, and so allow a strong, long-lived immune response to an otherwise weakly immunogenic molecule.
[0058] Current methods and building blocks used for protein-conjugate vaccine production hamper the wider application of conjugate vaccines for disease treatment and prevention. First, relatively few strong protein antigens are chemically resistant, nontoxic, and scalable enough to be used as carriers in conjugate vaccines. Second, the oxidation/reduction chemistry generally used for conjugate vaccine production makes it difficult to preserve epitopes on the carrier and antigen needed for maximum immunogenicity. Third, the relatively low efficiency of these oxidation/reduction reactions complicates quality control and purification, especially at commercial scale.
[0059] Recombinant protein production allows the optimization of antigenicity and nontoxicity of carrier proteins, but the existing carrier proteins are difficult to produce in cells and wholly engineered proteins are difficult to produce in high yields. Gentler conjugation reactions minimize the denatured on/obstructi on of carrier and antigen epitopes, but the lower efficiency of these reactions results in less loading of the antigen on the carrier protein and more complicated purification schemes. Importantly, relatively lower antigen to carrier results in a higher likelihood of immune “interference” by antibody responses to the carrier protein itself.
[0060] Chlamydia infection and associated diseases are common sexually transmitted infections, that can have serious consequences if left untreated. Untreated infections can cause permanent damage to a woman’s reproductive system making it hard to get pregnant later. Chlamdyia infections can also result in fatal ectopic pregnancies. Despite high prevalence, infection does provide natural immunity demonstrated by decreased infection concordance between older sex partners and lower bacterial loads in individuals with a history of prior infection. A recent study showed that young women that spontaneously cleared infection were able to resist reinfection, providing further evidence that protective adaptive immunity can be achieved. This makes Chlamydia a good candidate for vaccine development. Although UV-inactivated whole Chlamydia have been used to investigate the induction of protective responses, cost, safety issues, lack of scalability, reproducibility problems, and poor efficacy in early chlamydial trachoma protection trials preclude their use as commercial vaccines. Of candidate immunogens that have been investigated to date, the major outer membrane protein (MOMP) is the most studied, but no viable commercial product is available. Profiling of human antibody and T cell response has revealed chlamydial protease-like activity factor (CPAF) antigen to be immunodominant and immunoprevalent in chlamydia-exposed women. T cells from women with antibodies to C. trachomatis and/or a prior documented infection, respond to CPAF with broad epitope recognition in a CD4 dominant fashion by the production of both IFNy and TNFa, and these T cell responses are maintained for at least a year after infection. These preliminary data indicate this highly conserved antigen is a strong candidate for a human vaccine. Testing CPAF as a vaccine immunogen in mice i.n. led to abbreviated infection and reduced pathology with a standard challenge dose (1.5xl03 - IxlO4 bacteria). However, these studies used CPAF vaccines adjuvanted with interleukin- 12, or mice were administered multiple doses of CpG before and after CPAF immunization. The toxicity of IL-12 administration and the implausibility of delivering CpG on consecutive days make these approaches unsuitable for human use.
[0061] The inventors discovered an unexpected finding that adjuvant-conjugated CPAF enhanced CPAF immunogenicity without reactogenicity. The inventors have manufactured adjuvant-conjugated CPAF polypeptides using a click chemistry-based approach using CPAF polypeptides comprising non-natural amino acids to which adjuvants are attached. These polypeptide conjugates have been combined further with additional adjuvants to develop highly effective compositions. Accordingly, described herein are (1) polypeptides of CPAF comprising one or more non-natural amino acids; (2) adjuvants that may be conjugated to the CPAF polypeptides, (3) CPAF polypeptide-adjuvant conjugates of (1) and (2), (4) vaccine compositions comprising the foregoing; and (5) methods of making and using the foregoing.
1. Definitions
[0062] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present inventive concept or the appended claims.
[0063] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and/or the like in the description mean that the feature and/or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and/or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and/or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and/or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims. [0064] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.
[0065] The terms "comprising," "including" and "having" are used interchangeably in this disclosure. The terms "comprising," "including" and "having" mean to include, but not necessarily be limited to the things so described.
[0066] Lastly, the terms “or” and “and/or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and/or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.
[0067] The terms “polypeptide” and “protein,” as used interchangeably herein, refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.
[0068] An amino acid sequence that is “derived from” an amino acid sequence disclosed herein can refer to an amino acid sequence that differs by one or more amino acids compared to the reference amino acid sequence, for example, containing one or more amino acid insertions, deletions, or substitutions as disclosed herein. The terms “derivative,” “variant,” and “fragment,” when used herein with reference to a polypeptide, refers to a polypeptide related to a wild-type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary), activity (e.g., enzymatic activity) and/or function. Derivatives, variants, and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, presence of one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), or combinations thereof compared to a wild-type polypeptide.
[0069] Each amino acid sequence described herein by virtue of its identity or similarity percentage may be substantially identical to a given amino acid sequence. A “substantially identical” sequence may be at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or
100%, similar or identical to the given nucleotide or amino acid sequence, respectively. The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Sequence identity may be calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s. In the art, "identity" also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose, such as global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, or another suitable method or algorithm. A Needleman and Wunsch global alignment algorithm can be used to align two sequences over their entire length or part thereof (part thereof may mean at least 50%, 60%, 70%, 80%, 90% of the length of the sequence), maximizing the number of matches and minimizes the number of gaps. Default settings can be used and exemplary program is Needle for pairwise alignment (in an aspect, EMBOSS Needle 6.6.0.0, gap open penalty 10, gap extent penalty: 0.5, end gap penalty: false, end gap open penalty: 10 , end gap extent penalty: 0.5 is used) and MAFFT for multiple sequence alignment ( in an aspect, MAFFT v7Default value is: BLOSUM62 [bl62], Gap Open: 1.53, Gap extension: 0.123, Order: aligned , Tree rebuilding number: 2, Guide tree output: ON [true], Max iterate: 2 , Perform FFTS: none is used).
[0070] "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Similar algorithms used for determination of sequence identity may be used for determination of sequence similarity. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called conservative amino acid substitutions. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains.
[0071] For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Exemplary conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Exemplary conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; He to Leu or Vai; Leu to He or Vai; Lys to Arg; Gin or Glu; Met to Leu or He; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Vai to lie or Leu.
[0072] The term “suppression codon” refers to a nucleotide triplet that is introduced into a polynucleotide at a predetermined location and is recognized by a specific tRNA that can recognize a stop codon (e.g., an amber, ochre or opal stop codon) and allows translation to read through the codon to produce the protein, thereby suppressing the stop codon.
[0073] A “non-natural amino acid” (nnAA) refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine; other terms that are used synonymously with the term “non-natural amino acid” is "non-naturally encoded amino acid,” “unnatural amino acid,” “non-naturally occurring amino acid,” and variously hyphenated and non-hyphenated versions thereof. Non-natural amino acids with bio-orthogonal reactive chemical side chains may be used as a chemical “handle” to conjugate various payloads to discrete sites in a protein. Nonlimiting examples of non-natural amino acids include 2-amino-3-(4- azidophenyljpropanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5- (azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.
[0074] The term “adjuvant” refers to any molecule that may be added to vaccines or medications to enhance their effectiveness. The adjuvant is not the active ingredient that directly fights against the disease or condition but rather work to improve the body's immune response to the vaccine or medication. Adjuvants are used in several medical contexts, with the most common application being in vaccines. Adjuvants used in the current disclosure may function by any or one of more means, for example, enhance the immune response, reduce the amount of active ingredient required for action, prolong immunity, improve vaccine effectiveness, and tailor the immune system. As used herein, the adjuvants may be conjugated to a polypeptide antigen (for example CPAF) or be included in a composition comprising CPAF and CPAF-conjugated adjuvant. Adjuvants may include any known or novel adjuvant, including but not limited to aluminium salts, emulsions, liposomes, saponins, VLPs, TLR agonists, STING agonist, minerals, salts, protein adjuvants, polysaccharides or glycans, polynucleotides, polyamino acids, lipids, and small molecules and nanoparticles.
[0075] The term “T-cell activating epitope” refers to a structural unit of molecular structure which is capable of inducing T-cell immunity.
[0076] The term “B-cell epitope” refers generally to those features of a macromolecular structure which are capable of inducing a B cell response. In contrast to a T-cell epitope, a B-cell epitope need not comprise a peptide, since processing by antigen-presenting cells and loading onto the peptide-binding cleft of MHC is not required for B-cell activation.
[0077] As used herein, “carrier protein” refers to a polypeptide, for example CPAF or a variant or derivative thereof containing a T-cell activating epitope which may be attached to an adjuvant to enhance the humoral response to the conjugated protein-adjuvant composition in a subject. A “native carrier protein” has only naturally occurring amino acids. A “CPAF polypeptide” has at least one non-natural amino acid replaced for a naturally occurring amino acid in the carrier protein.
[0078] As used herein, the term “immunogenic polypeptide” refers to a polypeptide comprising at least one T-cell activating epitope, wherein the T-cell epitope is derived from a protein capable of inducing immunologic memory in animals.
[0079] As used herein, the terms “modified,” “replaced,” “enhanced,” and “substituted” are considered synonymous when used to describe residues of a polypeptide, and in all cases refer to the replacement of a non-natural amino acid for a naturally occurring amino acid within a polypeptide chain.
[0080] The compounds of the various aspects disclosed herein, or their pharmaceutically acceptable salts that contain one or more asymmetric centers and give rise to enantiomers, diastereomers, and other stereoisomeric forms that are defined, in terms of absolute stereochemistry, as (R) or (S) or, as (D) or (L) for amino acids. The present disclosure is meant to include all such isomers, as well as their racemic and optically pure forms.
[0081] The chemical naming protocol and structure diagrams used herein are a modified form of the I.U.P.A.C. nomenclature system, using the ACD/Name Version 9.07 software program and/or ChemDraw Ultra Version 11.0.1 software naming program (CambridgeSoft). Except as described below, all bonds are identified in the chemical structure diagrams herein, except for all bonds on some carbon atoms, which are assumed to be bonded to sufficient hydrogen atoms to complete the valency. a. General Methods
[0082] Unless defined otherwise, all technical and scientific terms used herein have the commonly understood meaning. Practitioners are particularly directed to Green, M. R., and Sambrook, J., eds., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012), and Ausubel, F. M., et al., Current Protocols in Molecular Biology (Supplement 99), John Wiley & Sons, New York (2012), and Plotkin, S.A., Orenstein, W.A., and Offit, P.A., Vaccines, 6 ed, Elsevier, London (2013), which are incorporated herein by reference, for definitions and terms. Standard methods also appear in Bindereif, Schon, & Westhof (2005) Handbook of RNA Biochemistry, Wiley-VCH, Weinheim, Germany which describes detailed methods for RNA manipulation and analysis, and is incorporated herein by reference. Examples of appropriate molecular techniques for generating recombinant nucleic acids, and instructions of many cloning exercises are found in Green, M. R., and Sambrook, J., (Id.); Ausubel, F. M., et al., (Id.); Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology (Volume 152 Academic Press, Inc., San Diego, Calif. 1987); and PCR Protocols: A Guide to Methods and Applications (Academic Press, San Diego, Calif. 1990), which are incorporated by reference herein. Examples of appropriate bio- organic techniques for activating and derivatizing biomolecules with chemical handles, and instructions to design such syntheses are found in Hermanson, G.T, Bioconjugate Chemistry, 2nd ed., Elsevier, London (2008). For examples of techniques and components necessary for parenteral administration of biomolecules described herein, practitioners are directed to Remington, Essentials of Pharmaceutics, Pharmaceutical Press, London (2012). Methods for protein purification, chromatography, electrophoresis, centrifugation, and crystallization are described in Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Methods for cell-free synthesis are described in Spirin & Swartz (2008) Cell-free Protein Synthesis, Wiley-VCH, Weinheim, Germany. Methods for incorporation of non-natural amino acids into proteins using cell-free synthesis are described in Shimizu et at (2006) FEBS Journal, 273, 4133-4140.
[0083] PCR amplification methods are described, for example, in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press Inc. San Diego, Calif., 1990. An amplification reaction typically includes the DNA that is to be amplified, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium. Typically, a desirable number of thermal cycles is between 1 and 25. Methods for primer design and optimization of PCR conditions are found in molecular biology texts such as Ausubel et al., Short Protocols in Molecular Biology, 5th Edition, Wiley, 2002, and Innis et al., PCR Protocols, Academic Press, 1990. Computer programs are useful in the design of primers with the required specificity and optimal amplification properties (e.g., Oligo Version 5.0 (National Biosciences)). In some aspects, the PCR primers additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified DNA fragment into specific restriction enzyme sites in a vector. If restriction sites are to be added to the 5' end of the PCR primers, it is preferable to include a few (e.g., two or three) extra 5' bases to allow more efficient cleavage by the enzyme. In some aspects, the PCR primers also contain an RNA polymerase promoter site, such as T7 or SP6, to allow for subsequent in vitro transcription. Methods for in vitro transcription are found in sources such as Van Gelder et al., Proc. Natl. Acad. Sci. U.S.A. 87: 1663-1667, 1990; Eberwine et al., Proc. Natl. Acad. Sci. U.S.A. 89:3010- 3014, 1992.
[0084] Sequence comparisons, such as for the purpose of assessing identities, sequence variants, or where one or more positions of a test sequence fall relative to one or more specified positions of a reference sequence (e.g. SEQ ID NO: 1), are optionally performed by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/, optionally with default settings), the BLAST algorithm (see e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings), and the Smith-Waterman algorithm (see e.g., the EMBOSS Water aligner available at www.ebi.ac.uk/Tools/psa/emboss_water/, optionally with default settings). In some aspects, optimal alignment is assessed using any suitable parameters of a chosen algorithm, including default parameters.
2. CPAF polypeptides
[0085] Provided herein is a chlamydial protease-like activity factor (CPAF) polypeptide comprising at least one non-natural amino acid (nnAA). The CPAF polypeptide may comprise a sequence substantially identical to a reference CPAF polypeptide. The CPAF polypeptide may be at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the reference CPAF polypeptide. The reference CPAF polypeptide may comprise a wild-type CPAF or a fragment thereof, which may be from a species of chlamydia. The species chlamydia may be Chlamydia muridarum, Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae, Chlamydia abortus, Chlamydia caviae, Chlamydia suis, or Chlamydia felis. In one example, the chlamydia species is Chlamydia muridarum or Chlamydia trachomatis. The reference CPAF polypeptide may correspond to WP_010229939.1, WP_080129215.1, WP_015506580.1, WP_011006700.1, WP_149305369.1, or WP_014945227.1. The reference CPAF polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 1 or 2. In one example, the reference CPAF polypeptide comprises the sequence set forth in SEQ ID NO: 1. The position of amino acid substitutions disclosed herein may be numbered in reference to SEQ ID NO: 1.
[0086] The CPAF polypeptide may comprise one or more tags, each of which may comprise one or more of a purification tag, a protease cleavage site, and a signal peptide. Purification tags, also known as affinity tags or fusion tags, are short amino acid sequences that are genetically fused to a target protein. These tags are used to facilitate the purification and detection of the tagged protein. Non-limiting examples include His-tag, Glutathione S-transferase (GST), Streptavidin- binding peptide (SBP or Strep-tag), Maltose-binding protein (MBP), Flag-tag, HA-tag, Calmodulin-binding peptide (CBP), and SUMO (Small Ubiquitin-like Modifier) tag. Each of such tags may be located at the N- or C-terminus of the CPAF polypeptide.
[0087] The tag may comprise one or more protease cleavage sites. Non-limiting examples of protease cleavage sites include enterokinase (EK) cleavage site, thrombin cleavage site, Tobacco Etch Virus (TEV) protease cleavage site, factor Xa cleavage site, proteinase k cleavage site, precision protease cleavage site, or caspase cleavage site. In one example, the tag comprises a TEV protease cleavage site, which may comprise the sequence ENLYFQ (SEQ ID NO: 12) followed by any amino acid except for proline or tryptophan. The protease cleavage site may be located at the N- or C-terminus of the CPAF polypeptide. The protease cleavage site may be present between the purification tag and the polypeptide, to facilitate purification and function. The CPAF polypeptide may comprise a His-tag at its N- or C-terminus. In one example, the His- tag comprises six consecutive histidines (6xHis). In a further example, the CPAF polypeptide comprises a His-tag and a TEV protease cleavage site. The CPAF polypeptide TEV cleavage site may be located after or before the His-tag. In one example, the TEV cleavage site is located after the His-tag, which may be a 6xHis tag. In a more specific example, the CPAF polypeptide comprises the sequence MHHHHHHGGSENLYFQ (SEQ ID NO: 13), which may be located at the N-terminus of the CPAF polypeptide.
[0088] The CPAF polypeptide may be a full length CPAF polypeptide or a N- or C- terminally processed polypeptide. The CPAF polypeptide may be a fragment of a CPAF protein disclosed herein, for example a N or C-terminal fragment thereof. Thus, the CPAF polypeptide may comprise an amino acid sequence set forth in any one of SEQ ID NOS: 4-9, or a sequence substantially identical thereto.
[0089] The CPAF polypeptide may be a catalytically inactive form of a reference CPAF polypeptide disclosed herein. The inactivity may be partial or complete. The inactive CPAF polypeptide may comprise a substitution in one or more of residues S491, S499, H97, E550, and C492 relative to SEQ ID NO: 1. The substitutions may comprise one or more of H97A, S491A, S499A, C492T, and E55OQ.
[0090] The CPAF polypeptide, fragment thereof, or inactive form thereof may comprise a sequence provided in Table 1, or a post-translational processing variant thereof. The CPAF polypeptide of SEQ ID NOs: 3, 4, 5, or 11 may be processed to remove the His-Tag. The CPAF polypeptide may have contained a TEV protease cleavage site that was subsequently processed by a TEV protease. Consequently, the CPAF polypeptide may comprise at its N-terminus a residual amino acid from the cleavage site. The amino acid in the 1st position of the CPAF polypeptide may be any amino acid except proline or tryptophan (Kapust et al. 2002). The N- terminus of N-terminal fragments of CPAF polypeptides provided in Table 1, may exclude the glycine and/or may be a methionine.
Table 1
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
[0091] Relative to a CPAF sequence provided herein, the CPAF polypeptide comprises one or more nnAA. The CPAF polypeptide may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 nnAAs. Each nnAA may substitute a phenylalanine, lysine, or tyrosine residue. The one or more nnAA may be added to the termini or inserted within the CPAF polypeptide. The one or more nnAA may substitute any amino acid residue in the CPAF polypeptide. Each nnAA may substitute L21, L26, L32, L35, L38 L41, L42, L56, L60, L71, L93, L110, LI 40, L141, L152, L155, L164, L171, L174, L181, L192, L218, L230, L251, L273, L307, L333, L350, L358, L373, L376, L377, L380, L385, L387, L394, L403, L406, L408, L409, L422, L433, L439, L447, L457, L463, L484, L500, L507, L536, L540, L548, L582, L586, L595, K33, K45, K49, K52, K62, K70, KI 16, K161, K184, K193, K202, K207, K226, K232, K240, K241, K274, K346, K354, K383, K389, K440, K451, K468, K479, K501, K532, K578, K581, F37, F66, F81, F89, F96, F102, F103, F122, F124, F130, F175, F238, F245, F252, F260, F264, F285, F286, F300, F3O5, F319, F344, F351, F442, F464, F466, F490, F495, F496, F519, F521, F525, F562, or F601, or any combination thereof relative to SEQ ID NO: 1, or may substitute equivalent residues in any one of SEQ ID NOs: 1-11, wherein the residues are numbered based on the sequence as set forth in SEQ ID NO: 1. The one or more nnAA may substitute amino acid residues F130 and Y569 or an equivalent residue in a CPAF polypeptide, wherein the residues are numbered relative to SEQ ID NO: 1.
[0092] The CPAF polypeptide may comprise a mutation disclosed herein in one or more of S491, S499, H97, E550, and C492, and comprise one or more nnAA substitutions at one or more lysine, tyrosine, or phenylalanine as described herein. The nnAA are described herein below.
The CPAF polypeptide may comprise a mutation in one or more of S491, S499, H97, E55O, and C492, and comprise nnAA substitutions in at least F130 and Y569, wherein the residues are numbered relative to SEQ ID NO: 1. The CPAF polypeptide may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 14-22, or a fragment thereof, or a sequence substantially identical thereto. The disclosed CPAF polypeptide may comprise a sequence at least about 80%, 85%, 90%, 95%, or 99% identical to a sequence set forth in any one of SEQ ID NOs: 14-22. The CPAF polypeptide may also comprise a tag disclosed herein. The CPAF polypeptide may also be a post-translational variant of a sequence in Table 2, such as a variant produced by cleavage with a TEV protease as described above.
Table 2
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
[0093] The nnAA may be any known in the art. Each nnAA may independently be 2-amino-3- (4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4- (azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, or any combination thereof. The nnAA residue may comprise a non-natural amino acid described herein, or another that has been identified as compatible with cell-based or cell-free protein synthesis (see, e.g., Schultz et al. Annu Rev Biochem. 2010;79:413-44 particularly pp.418-420; and Chin et al. Annu Rev Biochem. 2014; 83: 5.1-5.30, which are hereby incorporated by reference). Examples of CPAF polypeptide nnAA include: a non-natural analog of a tyrosine amino acid; a non-natural analog of a glutamine amino acid; a non-natural analog of a phenylalanine amino acid; a non-natural analog of a serine amino acid; a non-natural analog of a threonine amino acid; a non-natural analog of a lysine amino acid; an alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid, or any combination thereof; an amino acid with a photoactivatable cross-linker; a spin-labeled amino acid; a fluorescent amino acid; an amino acid with a novel functional group; an amino acid that covalently or noncovalently interacts with another molecule; a metal binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photocaged and/or photoisomerizable amino acid; a biotin or biotin-analog containing amino acid; a glycosylated or carbohydrate modified amino acid; a keto containing amino acid; amino acids comprising polyethylene glycol or polyether; a heavy atom substituted amino acid; a chemically cleavable or photocleavable amino acid; an amino acid with an elongated side chain; an amino acid containing a toxic group; a sugar substituted amino acid, e.g., a sugar substituted serine or the like; a carbon-linked sugar- containing amino acid; a redox-active amino acid; an a-hydroxy containing acid; an amino thio acid containing amino acid; an a, a disubstituted amino acid; a P-amino acid; or a cyclic amino acid other than proline.
[0094] The nnAA residue may comprise a chemical group or a handle (second chemical handle) suitable for a “click” chemistry reaction with a corresponding group on a separate molecule, which may be an adjuvant. Suitable chemical groups for “click” chemistry include, but are not
Figure imgf000031_0001
limited to azide (N3), alkyne (C=C), alkene (C=C) and 1,2,4,5-tetrazine ( N_N ) groups.
Examples of suitable chemical groups (second chemical handle) are discussed below. A disclosure of nnAA and chemical handles suitable for use in the current disclosure can also be found in in US 2018/0333484, the contents of which are incorporated herein in its entirety. [0095] Each nnAA may independently be a 2,3-disubstituted propanoic acid bearing an amino substituent at the 2-position and an azido-containing substituent, a 1,2,4,5-tetrazinyl-containing substituent, or an ethynyl-containing substituent at the 3-position. Each nnAA of the CPAF polypeptide may be conjugated to an adjuvant. In one example, each nnAA residue comprises an amino acid having the structure of formula I
Figure imgf000031_0002
wherein:
Ar comprises a 5-membered or 6-membered aromatic ring optionally containing at least one heteroatom;
W5 is selected from Ci-Cio alkylene, -NH-, -O- and -S-;
QI is zero or 1; and W6 is selected from azido, 1,2,4,5-tetrazinyl optionally C-substituted with a lower alkyl group, and ethynyl, such that the nnAA residue in the polypeptide has the structure of formula II
Figure imgf000032_0001
[0096] in which R3 is OH or an amino acid residue of the carrier protein, and R4 is H or an amino acid residue of the carrier protein.
[0097] Each nnAA CPAF polypeptide may be a 2, 3 -di substituted propanoic acid bearing an amino substituent at the 2-position and an azido-containing substituent, a 1,2,4,5-tetrazinyl- containing substituent, or an ethynyl-containing substituent at the 3-position. The substituent at the 3-position may be an azido-containing substituent, and, wherein the azido-containing substituent may comprise a terminal azido group bound to the carbon atom at the 3-position through a linking group. For example, the linking group may comprise an arylene moiety that is optionally substituted and optionally heteroatom-containing. For instance, the linking group may comprise a 5- or 6-membered arylene moiety containing 0 to 4 heteroatoms and 0 to 4 nonhydrogen ring substituents.
[0098] In one example, the nnAA has the structure of formula III
Figure imgf000032_0002
wherein:
Ar comprises a 5-membered or 6-membered aromatic ring optionally containing at least one heteroatom;
W5 is selected from C1-C10 alkylene, -NH-, -O- and -S-; QI is zero or 1 ; and
W6 is selected from azido, 1,2,4,5-tetrazinyl optionally C-substituted with a lower alkyl group, and ethynyl.
[0099] The nnAA may have the structure of formula IV
Figure imgf000033_0001
in which R3 is OH or an amino acid residue of the carrier protein, and R4 is H or an amino acid residue of the carrier protein. The Ar may not contain any heteroatoms, in which case the preferred linker is an unsubstituted phenylene group. The Ar may contain a nitrogen heteroatom and at least one additional heteroatom selected from N, O, and S. Exemplary nitrogen heterocycles are described infra. In an exemplary aspect, QI is 1, W5 is lower alkylene, and W6 is azido.
[0100] Azido-containing amino acids: The nnAA residue may comprise an azido-containing nnAA. The nnAA residue may comprise an azido-containing nnAA of formula V:
Figure imgf000033_0002
wherein:
D is — Ar— W3— or — Wl— Yl— C(O)— Y2— W2— ;
Figure imgf000033_0003
each of Wl, W2, and W3 is independently a single bond or lower alkylene; each Xi is independently — NH — , — O — , or — S — ; each Y1 is independently a single bond, — NH — , or — O — ; each Y2 is independently a single bond, — NH — , — O — , or an N-linked or C-linked pyrrolidinylene; and one of Zi, Z2, and Z3 is — N — and the others of Zl, Z2, and Z3 are independently — CH — . [0101] The nnAA residue may comprise an azido-containing amino acid of formula VI:
Figure imgf000034_0001
wherein:
W4 is C1-C10 alkylene.
[0102] The nnAA residue may comprise an azido-containing amino acid selected from the group consisting of 2-amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2- yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6- (azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, or 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, and any combination thereof. In one example, the nnAA residue comprise 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF).
[0103] Preparations of azido-containing amino acids according to formulas V and VI are found, for example, in Stafford et al. US20140066598A1, the contents of which are incorporated by reference, including paragraphs [0331]-[0333], The process involves substitution of hydroxyl groups for chloride on derivatives of the corresponding aryl amino acids using thionyl chloride, followed by nucleophilic displacement of the chloride with azide. Suitable aryl side-chain containing amino acids are also acquired commercially.
[0104] 1,2,4-5-Tetrazinyl-containing amino acids: The nnAA may comprise a 1, 2,4,5- tetrazine. The nnAA may comprises a 1,2,4,5-tetrazine of formula VII:
Figure imgf000035_0003
V is a single bond, lower alkylene, or -W1-W2-; one of W1 and W2 is absent or lower alkylene, and the other is -NH-, -O-, or -S-; each one of Zi, Z2, and Z3 is -CH- or -N- and the others of Zi, Z2, and Z3 are each independently -CH-; and Xi is independently -NH-, -O-, or -S-;
R is lower alkyl; wherein when
Figure imgf000035_0001
and V is -NH-, then one of ZI, Z2, and Z3 is -N- provided the compound is not
Figure imgf000035_0002
[0105] Preparation of 1,2,4,5-tetrazine -containing amino acids according to formula VII is found, for example, in Yang et al. US20160251336A1, the contents of which are incorporated by reference, including paragraphs [0341 ]-[0377] . The process involves Negishi coupling of an amino/carboxyl protected derivative of (R)-2-amino-3-iodopropanoic acid with an aminopyridyl bromide to introduce Ar, followed by reaction with a methylthio- 1,2,4,5-tetrazine derivative to introduce the tetrazine moiety into the amino acid.
[0106] Alkyne-containing amino acids: The nnAA may comprise an alkyne. The alkyne may be a propargyl group. A variety of propargyl-containing amino acids, including syntheses thereof, are found in Beatty et al. Angew. Chem. Int. Ed. 2006, 45, 7364 -7367; Beatty et al. J. Am. Chem. Soc. 2005(127): 14150-14151; Nguyen et al. J Am Chem Soc. 2009(131):8720- 8721. Such propargyl-containing amino acids are suitable for incorporation as nnAAs into proteins using cell-based systems. The nnAA may comprise a propargyl selected from the group consisting of homopropargylglycine, ethynylphenylalanine, and N6-[(2-propynyloxy)carbonyl]- L-lysine.
3. Methods for CPAF polypeptide production
[0107] The CPAF polypeptide may be produced by any method known for production of polypeptides. Methods suitable for production of polypeptides include, but are not limited to, solid phase chemical peptide synthesis, cell-based recombinant protein expression (in A. coli or a native host), and cell-free protein expression, and any combination thereof (e.g. expressed protein ligation using a combination of synthetic and recombinant peptide components).
[0108] The nnAA-bearing CPAF polypeptide may be produced by a method that comprises “codon reassignment.” In one variation of this method, nnAAs that are close structural analogs of the 20 canonical amino acids (e.g. homoallylglycine, fluorinated leucine, azidohomoalanine) are used. The nnAA is loaded onto its corresponding tRNA using wild-type aminoacyl-tRNA synthetases, and the nnAA completely replaces one of the 20 canonical amino acids specified in a template DNA sequence. To prevent interference from the native amino acid, this generally requires use of a bacterial expression strain that is auxotrophic for the native amino acid being replaced. This strategy is amino acid rather than residue-specific since all AA residues of a certain type are replaced with the nnAA.
[0109] In another production method, the nnAA-bearing CPAF polypeptide is produced by a strategy that comprises “nonsense suppression”. In this approach the non-natural amino acid is specified in a template DNA sequence by a rare or “nonsense” codon that does not ordinarily specify an amino acid in nature. One variation of the nonsense suppression approach has been pioneered by Schultz (Noren et al. Science. 1989(244): 182-188.) and Chamberlin (Bain et al. J Am Chem Soc. 1989(111): 8013-8014.) and involves the use of the rare stop codon TAG (the “amber” codon) along with its tRNA and its corresponding aminoacyl-tRNA synthetase (aaRS) to incorporate nnAAs into a polypeptide in a site-specific manner.
[0110] The “nonsense suppression” approach may involve isolating a tRNA/aaRS pair, modifying the tRNA at the anti-codon loop to recognize an orthogonal codon (e.g. the amber codon TGA, or another codon or base sequence not commonly used to specify amino acids in translation), and modifying the aaRS to prefer the nnAA over the aminoacyl-tRNAs native amino acid. In some variations of this method, the tRNA/aminoacyl-tRNA synthetase pair is from the same organism as the translation machinery used for polypeptide synthesis. In other aspects, the tRNA/aminoacyl-tRNA synthetase pair is from a different species as the translation machinery used for polypeptide synthesis. Methods to modify the tRNA anticodon loop and aaRS active site have been described, as are examples of engineered orthogonal tRNA/aaRS pairs.
[0111] In another aspect of the “nonsense suppression” approach, production of the CPAF polypeptide does not involve the use of an engineered aminoacyl-tRNA synthetase. In this aspect, an orthogonal tRNA alone is isolated and modified at the anti-codon loop to recognize an orthogonal codon (e.g. the amber codon TGA, or another codon or base sequence not commonly used to specify amino acids in translation). The orthogonal engineered tRNA is then acylated in vitro by a suitable chemical method (e.g., the method of Heckler et al. Biochemistry. 1984 Mar 27;23(7): 1468-73. which involves the use of T4 RNA ligase and mutant tRNAPhe) and supplemented in a cell-free protein synthesis extract. Because this method uses chemically acylated tRNAs, it is only compatible with protein synthesis methods that are cell-free.
[0112] Cell-free protein synthesis: The CPAF polypeptide may be produced by cell-free extract-based protein synthesis. The cell-free extract may comprise an extract of rabbit reticulocytes, wheat germ, or E. coli. The cell-free extract may be supplemented with amino acids, energy sources, energy regenerating systems, or cation cofactors, and any combination thereof. The extract may comprise exogenously supplemented mutant tRNA or mutant aaRS, and any combination thereof. The extract may comprise lysates from E. coli strains genetically encoding mutant tRNA or mutant aaRS, and any combination thereof. The E. coli strains used for lysates are RF-1 attenuated strains. Compatible cell-free protein synthesis systems have been described for the insertion of formulas I, II, and III into recombinant polypeptides (e.g., US8715958B2, US20160257946A1, and US 20160257945A1, the contents of each of which are incorporated herein in their entirety).
[0113] In a further aspect, the disclosure provides for methods of producing polypeptides in a cell-free extract containing 2 or more non-natural amino acids. [0114] The methods of producing the nnAA-containing polypeptides may involve altering the concentrations of nnAA-specific tRNA, nnAA-specific synthetase, nnAA itself, or translation temperature, and any combination thereof. Such conditions optionally allow for fewer translational errors, improved rate of incorporation of the nnAA, improved activity of chaperones necessary for protein folding with incorporation of the nnAA, decreased activity of cellular factors that interfere with nnAA incorporation, or any combination of the aforementioned mechanisms.
[0115] In an aspect, the nnAA-specific tRNA concentration may be increased to a concentration above about 20 pM, leading to an increased fraction of soluble or active polypeptide. In further variations the tRNA concentration may be increased while the nnAA concentration is kept below about 2mM and the nnAA synthetase is maintained below about 5pM.
[0116] In an aspect, the translation mix incubation temperature may be between 20 degrees and 30 degrees Celsius, about 20 degrees Celsius, or below 20 degrees Celsius. In some variations, these temperature modifications are independently combined with modifications to the nnAA- specific tRNA concentrations, nnAA concentrations, or nnAA synthetase concentrations described in the preceding paragraph.
[0117] The nnAA-containing polypeptides disclosed herein may be expressed in the cell-free system with an N terminal 6xHis affinity tag and a TEV protease cleavable linker. Once synthesized, and after the initial affinity capture the CPAF variants may be incubated with TEV protease which cleaves after the TEV signal sequence ENLYFQ (SEQ ID NO: 1, removing the 6x His tag and creating a new N terminus starting with glycine. The amino acid in the 1st position may also be A, M, C, or H (Kapust et al. 2002) and give a different amino acid at the N terminus. The glycine may be omitted and the variants all begin with methionine.
[0118] The CPAF variant- TEV reaction may then be passaged over a Ni-affinity matrix. The TEV protease is His-tagged so that it and any uncleaved CPAF and CPAF fragments, and the non-specifically bound E. coli contaminants, may all bind to the affinity resin while the untagged CPAF variants flow through. The untagged CPAF variants in the flow through may be concentrated and may be used to covalently link the adjuvant, for example in the production of the DBCO-derivatized adjuvant(s) conjugated via a click chemistry reaction disclosed herein. 4. Adjuvants
[0119] Described herein are one or more immunogenic adjuvants that may be conjugated to the CPAF polypeptide via one or more of the nnAA. The adjuvant may be derivatized with a chemical handle to facilitate attachment to CPAF polypeptide. For example, the adjuvant may be derivatized with dibenzylcyclooctyne (DBCO) as provided herein to facilitate attachment to a nnAA on the CPAF polypeptide.
[0120] The adjuvant may be any known adjuvant that can enhance the immune response, reduce the amount of CPAF polypeptide required for action, prolong immunity, improve vaccine effectiveness, or tailor the immune system, for example by modulating the early cytokine production and Thl/Th2 biasing for vaccination. The adjuvant may function to increase T-cell proliferation, reduce TCR threshold, increase IL-2, IL-8, IL10, IFN-y, TNF-a, granzyme,B, or perforin production, abrogate Treg function, promote T-cell retention, increase CD4+ or CD8+ proliferation, induce Thl response, increase cytotoxic activity of y8T-cell, promote Thl7 differentiation, or any combination thereof. The adjuvant may be a water-soluble adjuvant. The adjuvant may be purified natural, synthetic, or recombinantly produced small molecule, or macromolecule or a fragment thereof. The adjuvant may comprise a mineral, peptide adjuvant, polysaccharides or glycans, polynucleotide, polyamino acid, lipid, small molecules or nanoparticles. The adjuvant may be, for example, a Toll-like receptors (TLR) agonist, Stimulator of Interferon Genes receptor (STING) agonist, C-type lectin receptor (CLR) agonist, RIG-I-like receptor (RLR) agonist, a NOD-like receptor (NLR) agonist, or a dopamine receptor agonist. Non-limiting examples of TLR agonists include unmethylated CpG DNA, 2Bxy, triacylated lipoproteins, lipoteichoid acid, peptidoglycans, zymosan, PamsCSK^ diacylated lipopeptides, HSPs, HMGB1, uric acid, imidazoquinoline, fibronectin, ECM proteins, dsRNA, poly 1:C, LPS, P-defensin 2, fibronectin EDA, HMGB1, snapin, tenascin C, flagellin, ssRNA, CpG- A, poly GIO, poly G3, profilin, VSV. Non-limiting examples of STING agonists include diamidobenzimidazole (diABZl), MSA-2ADU-S100, MK-1454, MK-2118, SB11285, BMS- 986301, DMXAA, E7766, GSK3745417, and a cyclic dinucleotide (CDN). The CDN may comprise a cyclic di-GMP (guanosine 5 '-monophosphate) (CDG), cyclic di-AMP (adenosine 5'- monophosphate) (CDA), or cyclic GMP-AMP (cGAMP). Non limiting examples of dopamine agonists include L-DOPA, amantadine, apomorphine, 94 bromocriptine, cabergoline, carmoxirole, optically pure (S)-didesmethyl sibutramine, dopexamine, fenoldopam, ibopamine, lergotrile, lisuride, memantine, mesulergine, pergolide, piribedil, pramipexole, quinagolide, ropinirole, roxindole, and talipexole. In one example, the adjuvant comprises a STING agonist. In a further example, the STING agonist comprises a CDN, which may be a CDA.
[0121] In an aspect, the disclosed adjuvant may be capable of being activated to incorporate at least one first chemical handle. The first chemical handle may be capable of conjugating to a second chemical handle, which may be in one or more of the nnAA. Methods of activating molecules for attachment to nnAA are provided in US20180333484, the contents of which are incorporated herein in its entirety. The activated adjuvant may be combined with at least one of the nnAA bearing the second handle under conditions in which the first and second chemical handles react to form an adjuvant-CPAF polypeptide conjugate. The reaction may comprise a non-catalytic covalent bioconjugation reaction. The reactive sites on the adjuvant that serve as the "first chemical handle" may be one or more alkynyl group. Each alkynyl group may be incorporated in a molecular context that increases reactivity. For instance, the alkynyl group may be incorporated into a ring, e.g., a cyclooctynyl ring, such as a diaryl-strained cyclooctyne.
Reactive sites on the nnAA of the CPAF polypeptide, which may be the second chemical handle of the nnAA, may be an azido group as disclosed herein. As known in the art, the reaction in this case is a [3+2] cycloaddition referred to in the art as "strain-promoted azide'alkyne cycloaddition" (SPAAC). For example, the adjuvant may be derivatized with dibenzylcyclooctyne (DBCO) providing the cyclooctynyl ring comprising an alkynyl group to facilitate attachment to the azido group of a nnAA on the CPAF polypeptide.
[0122] The adjuvant may be activated using any chemical method described for production of bioconjugates. Such methods include, but are not limited to, periodate oxidation, unmasking of an intrinsic aldehyde , or hydroxyl activation with 1,1 '-carbonyldiimidazole (CDI) followed by nucleophilic addition. a. Activation of adjuvant
[0123] Periodate activation: The adjuvant may be activated by periodate oxidation. Periodate oxidation may be used to introduce aldehyde groups into an adjuvant, and is useful for the addition of aldehydes to N-terminal residues of polypeptides to produce an activated adjuvant. Periodate cleaves carbon-carbon bonds that possess a primary or secondary hydroxyl or amine on either end, and so activates r amino acids containing the 2-amino alcohol moiety (N-terminal threonine or serine residues). As the aldehyde moiety has a long half-life, adjuvants activated by this method are optionally chromatographically purified and/or lyophilized after activation. [0124] For periodate oxidation of adjuvants:
[0125] (a) adjuvants are dissolved in a solution;
[0126] (b) a source of periodate is added to the adjuvant from a concentrated stock solution to form an oxidation mixture;
[0127] (c) the reaction mixture is incubated; and [0128] (d) (optional) excess periodate is removed. [0129] Deionized water or a suitable buffered solution is optionally used for the oxidation reaction. In some aspects, the solution in step (a) is deionized water. In some aspects, the solution in step (a) comprises an effective amount of a buffer with a pKa around physiological pH. In some aspects, the solution in step (a) comprises an effective amount of a buffer with a pKa around physiological pH. Step (a) may be provided as a solution in an aqueous buffer, e.g., having a pH in the range of 5.1 to 5.9 (e.g., 5.2-5.9, 5.3-5.7, 5.4-5.6, 5.4-5.9). In some aspects, the buffer does not comprise an amine group. Examples of amine-free buffers include, but are not limited to acetate, formate, and phosphate. In some aspects, an amine buffer is employed in step (a). Suitable amine buffers generally comprise a combination of a tertiary amine or an N- heterocyclic compound with a weak acid (e.g., pyridine and acetic acid; pyridine and formic acid; N-ethylmorpholine and acetic acid; trimethylamine and carbonic acid; triethanolamine and phosphoric acid; etc.), or they can be a zwitterionic amine buffer such as 4-(2-hy droxy ethyl)- 1- piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), or 3-[4- (2-hydroxyethyl)piperazin-l-yl]propane-l-sulfonic acid (HEPPS).
[0130] The periodate source in step (b) is optionally selected from any periodate source with appropriate stability in aqueous solution. Examples of periodate sources include, but are not limited to, sodium periodate, potassium periodate, tetrabutyl ammonium (meta)periodate, barium periodate, sodium hydrogen periodate, sodium (para)periodate, and tetraethylammonium (meta)periodate.
[0131] Carbonyldiimidazole (CDI)Zcarbonylditriazole (CDT) activation: In some aspects the adjuvant may be activated with carbonyldiimidazole (CDI) or carbonyl di tri azole (CDT). CDI and CDT, like CDAP, are capable of activating hydroxyl groups on an adjuvant to form a
Figure imgf000042_0001
transient reactive moiety; in this case it is an unstable carbamate ( O for CDI and
Figure imgf000042_0002
for CDT), which is then optionally reacted with an amine or thiol on a chemical handle or linker to form a carbamate or carbonothioate linkage. The activation may be performed in a dry organic solvent. The CDI/CDT activation may be performed in anhydrous dimethylsulfoxide (DMSO). CDI/CDT activation may be performed by adding a molar excess of CDI/CDT with respect to the adjuvant. CDI/CDT activation may be performed by adding a molar amount of CDI/CDT approximately equal to the molar amount of the adjuvant.
[0132] No chemical activation: Endogenous amines or other nucleophilic moieties (e g. a primary amine) either naturally present or the result of a deprotection step (e.g. as discussed above) may be used to conjugate a given molecule to a chemical handle on a polypeptide. Such nucleophilic moieties can be conveniently reacted with a variety of common electrophilic conjugation reagents like succinate derivatives (e.g. N-hydroxysuccinimide (NHS) or sulfo-NHS esters). It may be sometimes advantageous to treat with a periodate protocol.
[0133] Bifunctional linkers: The activated adjuvant may be conjugated to the nnAA directly, but in one example the activated group on the adjuvant is derivatized to introduce a functional group with better reactivity with the functional group of the nnAA. For instance, an alkynyl group may be introduced. A bifunctional reagent with an amino group and an alkyne group can react with an aldehyde group that has been introduced into an adjuvant (e.g. via reductive amination) thereby leaving a pendant alkyne which can react with the nnAA. For instance, bifunctional reagents including amino and dibenzylcyclooctyne (DBCO) functional groups can be used. b. Conjugation of first chemical handle to adjuvant
[0134] The adjuvant may be conjugated to the first chemical handle using any chemical method compatible with the activation methods described above. Such methods include, but are not limited to, Schiff-base formation with synthetic antigen aldehydes followed by reductive amination, hydrazone formation, oxime formation, direct nucleophilic addition, and Schiff-base formation with native antigen aldehydes followed by reductive amination. A description of such methods are also provided in US20180333484, the contents of which are incorporated herein in its entirety. The absolute adjuvant concentration in a conjugation reaction with a chemical handle may vary depending on the adjuvant and can be determined.
[0135] Reactions with periodate-activated adjuvants: The first chemical handle may be conjugated to an adjuvant activated with periodate as provided herein. The chemical handle comprising a functional group that forms a stable or semi-stable adduct with aldehydes is combined with the periodate activated adjuvant, followed by optional reduction to convert semistable adducts to stable adducts. In an aspect, the chemical handle is added at a large molar excess with respect to the aldehyde groups on the activated adjuvant, such that all the aldehydes are consumed in the chemical handle/adjuvant conjugation reaction. In other variations, the chemical handle is added at a lower molar ratio with respect to the aldehyde groups on the activated adjuvant, and excess unreacted aldehydes on the activated adjuvant are consumed by further reaction with an excess of an inexpensive aldehyde-reactive nucleophile (e g. ethanolamine), or by treatment with a reducing agent strong enough to reduce aldehydes to hydroxyl groups (e.g. NaBHT).
[0136] The chemical handle may be conjugated to the adjuvant by Schiff-base formation with synthetic adjuvant aldehydes followed by reductive amination. This results in an end-product that has secondary amine linkage between the chemical handle and the adjuvant: a direct N-C bond between the amine of the chemical handle and a carbon atom on adjuvant. The chemical handle may comprise an amine. In this aspect, the conjugation method comprises: combining the amine-containing handle with periodate-activated adjuvant in DI water or buffered solution containing DMSO; incubating to form a Schiff base; reducing the Schiff base to a secondary amine using sodium cyanoborohydride (NaBHsCN); and optionally quenching unreacted aldehydes with NaBHi. In some aspects of this method the chemical handle and adjuvant are combined at or near 1 :1 stoichiometry. In some aspects, the chemical handle and adjuvant are combined with a molar excess of chemical handle. In some aspects of this method, the chemical handle and adjuvant are combined with a molar excess of adjuvant. In some aspects sodium cyanoborohydride is substituted for another reducing agent with similar selectivity for reducing C=N bonds such as sodium triacetoxyborohydride.
[0137] In one aspect, the chemical handle is conjugated to the adjuvant via hydrazone formation. In this aspect the chemical handle comprises a hydrazide (-C(=0)-NH-NH2) group. This aspect results in an end product that has a hydrazone (-C(=O)-NH-N=C-) or N' -alkyl hydrazide (- C(=O)-NH-NH-C-) linkage between the chemical handle and the adjuvant carbon. Tn this aspect, the conjugation method comprises: combining a molar excess of the hydrazide-containing chemical handle with the adjuvant in a solution pH 6.0-8.5 and incubating to form a hydrazone (- C(=O)-NH-N=C-). In some further aspects of this method, sodium cyanoborohydride or sodium triacetoxyborohydride is included in the reaction mixture to reduce the N=C bond, which produces an N’-alkyl hydrazide (-C(=O)-NH-NH-C-).
[0138] In one aspect, the chemical handle is conjugated to the adjuvant by oxime formation. In this aspect the chemical handle comprises an aminooxy (-O-NH2) group. This aspect results in an end product that has an oxime (-O-N=C-) linkage between the chemical handle and an adjuvant carbon. In this aspect, the conjugation method comprises: combining a molar excess of the aminooxy-containing chemical handle with the adjuvant in a solution pH 6.0-8.5 and incubating to form an oxime linkage (-O-N=C-). In some further aspects of this method, sodium cyanoborohydride or sodium triacetoxyborohydride is included in the reaction mixture to reduce the N=C bond and improve stability; this produces an N’-alkyl hydroxylamine linkage (-O-N-C- )•
[0139] Reactions with CDI/CDT- Activated Adjuvants: In some aspects the chemical handle is conjugated to the adjuvant activated with CDI/CDT. In these aspects, an unstable carbamate
Figure imgf000044_0001
produced by CDI/CDT activation of adjuvant hydroxyl groups ( O for CDI and
Figure imgf000044_0002
for CDT) is further reacted with a primary amine to produce a stable carbamate (- NH-C(=O)-O-) linkage or primary thiol to produce a stable carbonothioate (-S-C(=O)-O-) linkage between the chemical handle and an adjuvant carbon. In some aspects, a large molar excess of the amine/thiol-containing chemical handle with respect to activated hydroxyl groups on the adjuvant is added. In other aspects, the chemical handle is added at a concentration closer to 1 : 1 molar ratio with respect to the activated hydroxyl groups on the adjuvant. In yet further aspects, residual CDI/CDT in the reaction is further inactivated by treatment with sodium tetraborate. [0140] Reactions with Non-Activated Adjuvants: In some aspects the chemical handle is conjugated to an endogenous amine or other nucleophilic moiety (e.g. a primary amine) either naturally present or the result of a deprotection step from an adjuvant described herein. In one aspect of this, an electrophilic group (e.g. an NHS or sulfo-NHS ester) on a chemical handle is reacted with a primary amine group on the adjuvant to produce an amide linkage (-C(=O)-NH-) between the chemical handle and the adjuvant amine. In another aspect, a carboxylic acid group on a chemical handle is reacted with a primary amine group on the adjuvant in the presence of standard peptide coupling reagents and conditions to produce an amide linkage between the chemical handle and the adjuvant amine.
[0141] Alkyne-containing handles: In some aspects the chemical handle comprises a moiety that allows for a “click” chemistry reaction with a corresponding group on the nnAA. One such moiety is an alkyne group, which is capable of reacting with the nnAA residue comprising an azido group. In the simplest aspect, this is a propargyl group, such that an alkyne group on an adjuvant comprises a structure of formula IX:
Figure imgf000045_0001
[0143] wherein:
[0144] L22 is Cl -CIO alkyl; and
[0145] Ui is at least one moiety of an adjuvant.
[0146] In other aspects an alkyne group on an adjuvant comprises a structure of formula IXa:
Figure imgf000045_0002
[0147]
[0148] wherein:
[0149] L22 is -(CH2CH20)I-IO-; and
[0150] Ui is at least one moiety of an adjuvant.
[0151] In some aspects the alkyne group further comprises additional features that accelerate or facilitate the reaction of the alkyne with an azido group. An example of one such feature is a cyclooctane ring structure, such that an alkyne group on an adjuvant further comprises a DIFO or DBCO group. In some aspects, an alkyne group on an adjuvant comprises a structure of formula X, formula XI, or Xia:
Figure imgf000046_0001
[0153] wherein:
[0154] Li is independently a bond, -NH-, -O-, -S-, -NH(Li2)-, -O(Li2)-, or -S(Li2)-;
[0155] L2 is independently a bond, -C(=O)-, -S(=O)2-, -C(=O)Li2-, -S(=O)2Li2;
[0156] L12 is independently L22 or L22NH-
[0157] L22 is independently C1-10 alkyl or -(CH2CH20)MO-; and
[0158] Ui is independently at least one moiety of an adjuvant.
[0159] In some aspects, structures of formula X and Xa are formed from an adjuvant comprising a nucleophilic group (e.g. a primary amine) and the NHS or sulfo-NHS ester of the corresponding DIFO or DBCO carboxylic acids of structures X and Xa. In some aspects structures of formula X are formed from an activated adjuvant, and a DBCO derivative such as DBCO-NH2 or DBCO-PEGn-NH2. In some aspects, DBCO-PEGn-NH2 is DBCO-PEG4-NH2.
Figure imgf000046_0002
Figure imgf000047_0001
carboxylic acid
[0164] In some aspects the moiety of Ui is at least one amino acid of a polypeptide adjuvant. [0165] In further aspects, an adjuvant comprising an alkyne comprises a structure of formula Xllb or XIIc
Figure imgf000047_0002
[0170] wherein:
[0171] X is independently an amine; and
[0172] n is at least 1.
[0173] In some aspects, an adjuvant comprising a DBCO group comprises at least 90% of the adjuvant covalently attached to DBCO.. In an aspect, the adjuvant comprises greater than about 90%, or about 92%, or about 94%, or about 96%, or about 98%, or about 99% molecules covalently attached to DBCO. Any unconjugated DBCO or adjuvant may be inactivated with azide and removed by dialysis. Thus, the adjuvant may comprise 100% of the molecules attached to DBCO.
[0174] In one example, the adjuvant comprises DBCO or a derivative thereof. In an aspect, the adjuvant comprises a 2BxY-DOPA-DBCO, a CpG-DBCO, a STING-DBCO, a 2Bxy-DBCO, or a CDA-DBCO. In an aspect, the adjuvant comprising a DBCO handle has a structure as provided below:
Figure imgf000048_0001
[0175] CpG-DBCO: (formula Xlld)
[0176]
Figure imgf000048_0002
(formula Xlle)
[0177]
Figure imgf000049_0001
(formula Xllf)
[0178]
[0179]
Figure imgf000049_0002
2BXy-DOPA-DBCO: (formula Xllg)
Figure imgf000050_0001
[0180]
[0181] CDA-DBCO: (formula Xllh)
[0182] Azido-containing handles: In some aspects the chemical handle comprises a moiety that allows for a “click” chemistry reaction with a corresponding group on nnAA residue of a polypeptide. One such moiety is an azido group, which is capable of reacting with a nnAA residue comprising an alkyne group on a polypeptide. In some aspects, an azido group on an adjuvant comprises a structure of formula XIII: z- N H
J-22 U-i [0183] N3 [0184] (XIII)
[0185] wherein L22 is a bond, alkyl, or poly(alkyloxy); and
[0186] Ui is independently at least one moiety of an adjuvant.
[0187] Alkene-containing handles: In some aspects the chemical handle comprises a moiety that allows for a “click” chemistry reaction with a corresponding group on nnAA residue of a polypeptide. One such moiety is an alkene group, which is capable of reacting with a nnAA residue comprising an 1,2,4,5-tetrazine group. In the simplest aspects, this is a vinyl group. In one such aspect, an alkene group on an antigen comprises a structure of formula XIV:
Figure imgf000050_0002
[0189] wherein:
[0190] Ui is independently at least one moiety of an adjuvant.
[0191] In other aspects, an alkene group on an antigen comprises a structure of formula XlVa:
Figure imgf000051_0001
[0193] wherein:
[0194] L22 is Cl -10 alkyl or -(CH2CH20)I-IO-; and
[0195] Ui is independently at least one moiety of an adjuvant.
5. Polypeptide-adjuvant conjugates
[0196] Described herein are CPAF polypeptide-adjuvant conjugates comprising the CPAF polypeptidethe CPAF polypeptide and the adjuvant. The adjuvant may be covalently linked to one or more of the nnAA of the CPAF polypeptide. In an aspect, the adjuvant may not be linked to a natural amino acid of the polypeptide. In an aspect, the adjuvant is only linked to one or more of the nnAAs of the CPAF polypeptide. In some cases, the adjuvant is only linked to one or more pAMF of the CPAF polypeptide. For example, the adjuvant may only be linked to one or more pAMFs of the CPAF polypeptide comprising a sequence set forth in any one of SEQ ID NOS: 14-22. In an aspect, at least one adjuvant is linked to a nnAA located outside a T-cell epitope of the CPAF polypeptide. In another aspect, no adjuvant is linked to an amino acid located within a T-cell epitope of an immunogenic portion of the CPAF polypeptide. The amino acids selected for conjugation within the CPAF polypeptide may comprise one or more surface- accessible residues based on the crystal structure of the CPAF polypeptide. Additionally, or alternatively, a comprehensive replacement of natural amino acids for nnAAs is performed on the CPAF polypeptide followed by conjugation to assess the utility of specific sites on the polypeptide for conjugation.
[0197] In one aspect, the adjuvant is conjugated to the CPAF polypeptide indirectly (e.g. by first combining the CPAF polypeptide or adjuvant with a reactive linker, and then combining the CPAF polypeptide-linker or antigen-linker adduct with an adjuvant or CPAF polypeptide, respectively). In another aspect, the adjuvant is conjugated to the CPAF polypeptide directly (e.g. by combining two components comprising the CPAF polypeptide and adjuvant together in one reaction).
[0198] The exact location of some or all conjugations and other physical features may be reliably inferred from the design of a synthetic scheme, its expected product, and analytical results consistent with that expectation. a. Polypeptide-adjuvant conjugation reaction
[0199] In some aspects, the adjuvant is conjugated to the CPAF polypeptide using any chemical method suitable for conjugating the non-natural amino acids and chemical handles herein described. A description of such methods are also provided in US20180333484, the contents of which are incorporated herein in its entirety.
[0200] Such methods include, but are not limited to, copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), and tetrazinealkene ligation. As “click” reactions, all of these reactions are able to be performed in aqueous solution.
[0201] CuAAC: In some aspects, the adjuvant is conjugated to the CPAF polypeptide by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC). In one variation of this aspect, the CPAF polypeptide comprises a propargyl-containing nnAA and the adjuvant comprises an azido group. In another variation of this aspect, the CPAF polypeptide comprises an azido-containing nnAA and the adjuvant comprises a propargyl group. Suitable conditions for CuAAC conjugation of biomolecules are found, e.g. Presolski et al. Curr Protoc Chem Biol. 2011; 3(4): 153-162, all of which involve the addition of Cu2+. In some aspects, the reaction is accelerated by the addition of a Cu-coordinating ligand, such as THPA. In some aspects the reaction is accelerated by the addition of a reducing agent to maintain the oxidation state of Cu2+. Suitable reducing agents include sodium ascorbate, DTT, or TCEP.
[0202] SPAAC: In some aspects, the adjuvant is conjugated to the CPAF polypeptideCPAF polypeptide by strain-promoted azide-alkyne cycloaddition (SPAAC). In one variation of these aspects, the CPAF polypeptide comprises an azido-containing nnAA (second handle) and the adjuvant comprises a alkyne cyclooctyne group (first handle). In another variation of these aspects, the CPAF polypeptide comprises a cyclooctyne-containing nnAA and the adjuvant comprises an azido group. As SPAAC requires no additional catalysts or cofactors, this reaction is able to be performed in distilled water, 0.9% saline, PBS, or a physiologically buffered solution.
[0203] In some aspects, the adjuvant is linked to an azido-containing nnAA in the CPAF polypeptide via a structure of formula XV or XVa:
Figure imgf000053_0001
[0207] (Formula XVa)
[0208] wherein:
[0209] Ri is independently H, formyl, or at least one amino acid of the CPAF polypeptide;
[0210] R.2 is independently OH or at least one amino acid of the CPAF polypeptide;
[0211] D is — Ar— W3— or — Wl— Yl— C(O)— Y2— W2— ;
Figure imgf000053_0002
[0213] each of Wl, W2, and W3 is independently a single bond or lower alkylene;
[0214] each XI is independently — NH — , — O — , or — S — ;
[0215] each Yi is independently a single bond, — NH — , or — O — ;
[0216] each Y2 is independently a single bond, — NH — , — O — , or an N-linked or C-linked pyrrolidinylene;
[0217] one of Zi, Z2, and Z3 is — N — and the others of Zi, Z2, and Z3 are independently — CH — [0218] L22 is independently a bond, alkyl or poly(alkyloxy); and
[0219] X is the adjuvant.
[0220] In some aspects, the adjuvant is linked to an azido-containing nnAA in the CPAF polypeptide via a structure of formula XVIa or XVIb:
Figure imgf000054_0001
[0225] wherein, Ri is independently H, formyl, or at least one amino acid of the CPAF polypeptide;
[0226] R2 is independently OH or at least one amino acid of the CPAF polypeptide;
[0227] W is C or N;
[0228] y is at least 1;
[0229] n is at least 1; and
[0230] X is the adjuvant. [0231] A schematic of an exemplary SPAAC reaction between an azi do-containing nnAA (Ri) in the CPAF polypeptide and a DBCO linked adjuvant (R2) is provided in FIG. 6A and described in Example 6.
[0232] Tetrazine-alkyne ligation: In some aspects, the adjuvant is conjugated to the CPAF polypeptide by tetrazine-alkyne ligation. In one variation of these aspects, the CPAF polypeptide comprises a 1,2,4,5-tetrazine-containing nnAA and the adjuvant comprises an alkene group. Similar to the SPAAC reaction, the tetrazine-alkyne ligation proceeds without the addition of cofactors this and this reaction is able to be performed in distilled water, 0.9% saline, PBS, or a physiologically buffered solution.
[0233] In one aspect, the disclosure provides for a method for producing a CPAF polypeptide conjugate comprising: (a) providing a nucleic acid encoding polypeptide, wherein the nucleic acid comprises a suppression codon; (b) creating a reaction mixture by combining the nucleic acid with a cell-free bacterial extract comprising 4-azidomethylphenylalanine (pAMF), a tRNA complementary to the suppression codon, and an aminoacyl-tRNA synthetase; (c) incubating the reaction mixture of (b) under conditions sufficient to selectively incorporate pAMF at a site corresponding to the suppression codon in the carrier protein; and (d) conjugating the pAMF to a adjuvant by a [2+3] cycloaddition. In another aspect, the [2+3] cycloaddition comprises the reaction between an azide and an alkyne group. In another aspect, the method additionally comprises purifying the carrier protein immediately after (c). The suppression codon may be selectively substituted at codons encoding L21, L26, L32, L35, L38 L41, L42, L56, L60, L71, L93, L110, L140, L141, L152, L155, L164, L171, L174, L181, L192, L218, L230, L251, L273, L307, L333, L350, L358, L373, L376, L377, L380, L385, L387, L394, L403, L406, L408, L409, L422, L433, L439, L447, L457, L463, L484, L500, L507, L536, L540, L548, L582, L586, L595, K33, K45, K49, K52, K62, K70, KI 16, K161, KI 84, K193, K202, K207, K226, K232, K240, K241, K274, K346, K354, K383, K389, K440, K451, K468, K479, K501, K532, K578, K581, F37, F66, F81, F89, F96, F102, F103, F122, F124, F130, F175, F238, F245, F252, F260, F264, F285, F286, F300, F305, F319, F344, F351, F442, F464, F466, F490, F495, F496, F519, F521, F525, F562, F601 with respect to the polypeptide of SEQ ID NO: 1. The reaction mixture in (b) may further comprise biological components necessary for protein synthesis. In another aspect, the tRNA in (b) is capable of being charged with pAMF. In another aspect, the aminoacyl-tRNA synthetase in (b) preferentially aminoacylates the tRNA with pAMF compared to the 20 natural amino acids. Tn another aspect, the alkyne group comprises a DBCO moiety conjugated to the adjuvant.
[0234] A “click” reactions for making the disclosed conjugates, using the disclosed chemistries may be performed in an aqueous solution. To optimize stability and immunogenicity, conjugation conditions may be conducted at various pH, temperature, mixing speed, and addition speed. Reaction concentration, temperature, adjuvant, and protein input ratios may be optimized depending on the scale of production and the reactants.
[0235] Adjuvant ratio: In an aspect, adjuvant may be added to the in 1-20 fold excess of the polypeptide. In an aspect, the adjuvant may be added in about 2 to about 3 fold excess, or about 3 to about 4 fold excess, 4 to about 5 fold excess, 5 to about 6 fold excess, 6 to about 7 fold excess, 7 to about 8 fold excess, 8 to about 9 fold excess, 9 to about 10 fold excess of the polypeptide. The gas-flow rate, pH, and temperature may be optimized based on the scale of production, to obtain maximum soluble yield. For example, for large scale production, Rushton-style impellers with high shear (or similar systems) may be used to disrupt larger gas bubbles from open-pipe or drilled-hole spargers commonly used in microbial fermentation. These impellers are well characterized, with a variety of correlations for Reynolds Number, Power Number, and kLa for transition to manufacturing and commercial scale.
[0236] Gas flow rate: Changing the gas flow rate changes the gas-liquid interface and affects the foaming and solubility of the target protein and cell-free components. Cell-free reactions place proteins, cell-free biomolecules, and components in direct contact with the reactor vessel surfaces and gas-liquid interfaces (bubbles), which can denature the protein of interest or proteins essential to the cell-free reaction, such as essential enzymes. At a gas-liquid interface, a hydrophilic region (aqueous liquid) meets with a hydrophobic region (gas) and can cause proteins to denature and aggregate. For this reason, cell-free reaction setups use a blend of pure oxygen and air, which delivers a more significant percentage of oxygen so that control can use less overall gas flow. In an aspect, a method of making the polypeptide conjugates may require carefully controlled gas flow rates and ratio of oxygen to other gases. Methods of controlling gas flow and ratios are well known in the art and can be suitably modified depending on the reactants and scale of production.
[0237] pH control: The method of making a CPAF polypeptide conjugate may require a controlled pH. The pH may be controlled by using any suitable buffering agent. Non-limiting examples of buffers or pH adjusting agent include Tris, HEPES, MOPS, sodium borate, sodium phosphate, sodium citrate, ammonium sulfate, succinate, citrate/dextrose, sodium bicarbonate and ammonium chloride or combinations thereof. The pH control may be maintained by manual or automated addition of both acid and base to maintain a set-point pH. Non-limiting examples of suitable acids include acetic, boric, citric, lactic, phosphoric and hydrochloric acids; nonlimiting examples of bases include sodium hydroxide, sodium acetate, sodium lactate and tris- hydroxymethylaminomethane. In an aspect, the scaled-up cell-free reaction may utilize IM citric acid and IM potassium hydroxide, because this acid/base pair provides good pH control without excessive volume additions. The initial set point for cell-free protein production may be about 6.5-8.5 pH. The initial set point for cell-free protein production may be about 7-8 pH. The initial set point for cell-free protein production may be about 7.2 ± 0.1. pH may be varied to further optimize reaction conditions.
[0238] Temperature; The method of making a CPAF polypeptide conjugate may require carefully controlled temperature. In an aspect, the temperature of the reaction may be between 15°C to about 37°C. In an aspect, the reaction chemistries may be accomplished at a temperature of about 20°C, or about 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.
[0239] Downstream purification optimization In an aspect, the CPAF polypeptide and or CPAF polypeptide conjugates may need to be further purified prior to use in disclosed formulations. Methods of purification of polypeptides are well known in the art and may include centrifugation, size fractionation, gel filtration and other chromatographic techniques, affinity purification, and mass spectrometry. For example, in an aspect, the CPAF polypeptide may comprise a TEV protease cleavable N-terminal his6-tag for downstream purification of the conjugate. The cell-free reaction may be loaded onto a pre-equilibrated his Trap (GE Healthcare) column to purify the tagged protein, followed by incubation with TEV protease to cleave the tag. The untagged protein may be purified by reloading the protein mixture onto the affinity column, with the target protein collected in the flow through. If post-affinity chromatography yields a purity of the protein solution of < 95%, additional orthogonal polishing steps can be employed using ion exchange or mixed-mode resins.
[0240] In an aspect, the CPAF polypeptide conjugate produced after practicing the disclosed method may comprise: a) a substitution at any of the amino acid residues H97, S491, S499, C492, E550Q, or a combination thereof, wherein the amino acids are numbered with reference to SEQ ID NO: 1; b) one or more nnAA; wherein the nnAA is selected from 2-amino-3-(4- azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyri din-2 -yl)propanoic acid, 2-amino-3-(4- (azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3- yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid, or any combination thereof; c) an adjuvant covalently linked to the nnAA.
[0241] In an aspect, the CPAF polypeptide conjugate may comprise: d) a substitution corresponding to one or more of H97A, S491A, S499A, C492T, and E550Q, or a combination thereof, wherein the amino acids are numbered with reference to SEQ ID NO: 1; e) one or more nnAA at Fl 30 or Y569 or both; wherein the nnAA is selected from 2- amino-3-(4-azidophenyl)propanoic acid (pAF), 2-amino-3-(4- (azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino- 3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5-azidopentanoic acid, and 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid, or any combination thereof; f) an adjuvant comprising CpG, DOPA, 2BxY, DiABZl, 2BxY-DOPA, CDA or a combination thereof covalently linked to the CPAF polypeptide via the nnAA. b. Conjugate characterization
[0242] Methods (size exclusion, diafiltration, dialysis): Following the conjugation reaction, the CPAF polypeptide conjugates of interest are optionally purified according to methods including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic interaction, and size exclusion), molecular size exclusion (dialysis, diafiltration, tangential flow filtration, depth filtration) electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), or SDS-PAGE to obtain substantially pure conjugates. [0243] The conjugated protein of interest are optionally quantitated according to methods including, but not limited to, microfluidic electrophoresis, gel electrophoresis, western blotting, immunoassays (e.g., ELISA), and other assays to assess the activity of the conjugated protein. [0244] Exemplary physical parameters One important parameter for polypeptide conjugates is the molecular weight of the conjugate. Since conjugates optionally comprise variable numbers of adjuvant molecules conjugated to each protein molecule as well as variable higher-order crosslinking (protein-adjuvant-protein linkages, for example) the output molecular weight of a conjugate is not necessarily predictable from the input molecular weights of the CPAF polypeptides and adjuvants.
[0245] Another important parameter for the conjugate vaccines of the present disclosure is the ratio of the adjuvant to polypeptide. The adjuvant-to-protein ratio of the purified conjugate is generally expressed in terms of a weight-weight (w/w) ratio or molar ratio. The adjuvant-to- protein ratio of the purified conjugate may vary depending on the number of nnAA present per polypeptide. The adjuvant to polypeptide ratio in the conjugate may be in the range of about 0.8:1 to about 10: 1 molar ratio. In an aspect, the adjuvant to polypeptide in the conjugate may be in the range of a 0.8:1 to 1 : 1, 1 :1 to 2: 1, 2: 1 to 3: 1, 4: 1 to 5: 1, 6: 1 to 7: 1 8: 1 to 9: 1, or 9: 1 to 10: 1 molar ratio. In an aspect, the adjuvant to polypeptide may be in the range of 1 : 1 to 2: 1. Any free adjuvant may be inactivated by azide and removed by dialysis. In one example, the adjuvant to polypeptide molar ratio is 1: 1. In another, it is 2: 1.
[0246] Presence of contaminants: An important parameter for polypeptide conjugates is the level of free adjuvant that is not covalently conjugated to the CPAF polypeptide, but is nevertheless present in the conjugate composition. For example, in certain instances, the free adjuvant is noncovalently associated with (i.e., noncovalently bound to, adsorbed to, or entrapped in or with) the polypeptide conjugate.
6. Pharmaceutical compositions
[0247] Described herein are pharmaceutical compositions comprising the CPAF polypeptide, which may comprise a CPAF polypeptide-adjuvant conjugate, and one or more pharmaceutically acceptable excipients. In one aspect, the pharmaceutical composition is a vaccine composition. In some aspects, the vaccine composition enhances a Th 1 -based immune response.
[0248] The pharmaceutical composition may comprise one or more soluble adjuvants in addition to an adjuvant conjugated to the CPAF polypeptide. The soluble adjuvant may be a different adjuvant than the conjugated adjuvant. The soluble adjuvant may be the same adjuvant as the conjugated adjuvant. The soluble adjuvant may be aluminum salt, for example aluminum potassium phosphate, aluminum hydroxyphosphate sulfate, aluminum hydroxide, or aluminum phosphate, and any combination thereof. The adjuvant may be an oil-in-water emulsion. The adjuvant may be AS03, MF59, or AF03, and any combination thereof. The adjuvant may be an agonist of one or more of the Toll-like receptors (TLR), Stimulator of Interferon Genes receptor (STING), C-type lectin receptors (CLR), RIG-I-like receptors (RLR), and NOD-like receptors (NLR). Non-limiting examples of receptor agonists based adjuvants include RC529, cGAMP, c- di-AMP, c-di-GMP, 5,6-Dimethylxanthenone-4-acetic acid, AS03, ADU-S100, MK-1454, 2'3'- cGAMP, TMX-202, CL1151, CDA, BMS-986301, diABZl, 2BXy, DOPA, 2BXy-D0PA, an imidazoquinoline based agonist, CpG, and a CpG derivatives selected from the group consisting of CpG1826, CpG1018 and polyC-CpG. Where a composition includes an aluminum salt adjuvant it is preferred that the concentration of Al3+ in the composition is <1.25 mg per dose e.g. <1.25 mg per 0.5 ml, and ideally <0.85 mg per dose. Conjugates within a composition may be adsorbed to the aluminum salt adjuvant. For a mixed composition, conjugates can be adsorbed to an aluminum salt individually and then mixed, or can be added into an aluminum salt to achieve sequential adsorption, thereby forming the mixed conjugate composition. Receptor agonists may be added to a disclosed formulation at about 0.1 pg/dose -100 pg/dose. For example, the disclosed formulation may comprise about 0.1 pg/dose to about 1 pg/dose, 1 pg/dose to about 5 pg/dose. 5 pg/dose to about 10 pg/dose, 10 pg/dose to about 20 pg/dose, 20 pg/dose to about 30 pg/dose of one or more agonists, wherein each dose comprises about 15 pg of the adjuvant conjugated CPAF polypeptide. In an aspect, the pharmaceutical composition does not comprise any unconjugated adjuvant.
[0249] The pharmaceutical composition may comprise one or more pharmaceutically acceptable excipients. The pharmaceutically acceptable excipient may approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, nonessential amino acids, L-arginine hydrochlorate, saccharose, D-trehalose dehydrate, sorbitol, tris (hydroxymethyl) aminomethane and/or urea. The pharmaceutically acceptable excipient may comprise one or more additives including, for example, diluents, binders, stabilizers, and preservatives. The pharmaceutical excipient may further comprise a tonicity agent to bring osmolality of the composition into an acceptable range. Non-limiting examples of tonicity agents include sodium chloride, dextrose, and glycerin, and any combination thereof.
[0250] The pharmaceutically acceptable excipient may comprise a surface-active agent (surfactant). The surface-active agent may be polyoxyethylene sorbitan monolaurate (Tween 20) , polyoxyethylene sorbitan monooleate (Tween 80), Brij 35, Triton X-10, Pluronic F127, or sodium dodecyl sulfate (SDS). In some embodiments the surface-active agent is present at a concentration between 0.0003% and 0.3% (w/w).
[0251] The pH of the pharmaceutical composition may be between 5.5 and 8, and more specifically between 6.5 and 7.5 (e.g. about 7). Stable pH may be maintained by the use of a buffer e.g. In particular embodiments, the buffer or pH adjusting agent is selected from the group consisting of sodium borate, sodium phosphate, sodium citrate, ammonium sulfate, or succinate, and any combination thereof. Other examples of suitable buffers include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Pharmaceutical compositions may be isotonic with respect to humans. The pharmaceutical composition may also comprise one or several additional salts, such as NaCl.
[0252] The pharmaceutical composition may be formulated for any mode of delivery. Suitable routes of administration may, for example, include intravenous, subcutaneous, intranasal route, cranial, transmucosal, trans-nasal, intestinal, and/or parenteral delivery. In an aspect, the pharmaceutical composition is formulated for intramucosal delivery. Suitable excipients for intramucosal delivery may include mucoadhesive polymers for example chitosan, alginate, and polyacrylic acid; gelling agents for example carbomer and sodium alginate; surfactants, for example polysorbates (Tween), sodium lauryl sulfate, and lecithin; penetration enhancers, for example atty acids (oleic acid), bile salts (sodium deoxycholate), and cyclodextrins; buffering agents as disclosed herein; preservatives; antioxidants; solubilizing agents, for example cyclodextrins, propylene glycol; thickening agents, for example hydroxypropyl methylcellulose (HPMC); mucin enhancers, for example thiomers and lectins, bio adhesive polymers, for example polycarbophil and sodium carboxymethylcellulose; polymeric nanoparticles and lipid based excipients.
[0253] The pharmaceutical composition may be sterilized by conventional sterilization techniques, or may be sterile fdtered. The resulting aqueous solutions may be packaged and stored in liquid form or lyophilized, the lyophilized preparation being reconstituted with a sterile aqueous carrier prior to administration. The pharmaceutical composition may be packaged and stored as micropellets via a prilling process.
7. Methods of Immunization
[0254] The CPAF polypeptide, which may be a CPAF polypeptide-adjuvant conjugate, or pharmaceutical composition thereof may be used as a vaccine to protect a subject susceptible to chlamydial infection, may be used to prevent or reduce a chlamydia infection in a subject, or may be used to induce an immune response in a subject to the CPAF polypeptide. Such uses may be carried out by administering a composition disclosed herein to the subject. Also provided herein are a composition disclosed herein for such uses and use of a composition disclosed herein in the manufacture of a medicament for such uses. The administration may be or may be intended to be via a systemic or mucosal route. Suitable subjects may include any vertebrate susceptible to a chlamydia infection. The subject may be a mammal, which may be a human; a companion animal such as a cat, dog, rodent, horses; a research animals such as a rabbit, sheep, pig, dog, primate, mouse, rat, or other rodents; an agricultural animal such as a cow, cattle, pig, goat, sheep, horse, deer, chicken, or other fowl; a zoo animal; or a primates or ape such as a chimpanzee, monkey, or gorilla. The subject may be of any age. The subject may be a human of >11 years of age. The subject may be a human of <11 years of age.
[0255] In one example, an immunologically effective amount of a pharmaceutical composition is administered to the subject.
[0256] The “effective amount” may refer to a dose required to elicit a robust Thl/Th2 response that reduces the likelihood or severity of infectivity of chlamydia during a subsequent challenge. The methods of the invention can be used for the prevention and/or reduction of clinical infections caused by Chlamydia. The amount of CPAF polypeptide administered to a subject or included in a medicament may be an amount that induces an immunoprotective response without significant adverse effects. The amount may vary depending upon the chlamydia species.Each dose may comprise about 100 ng to 10,000 pg of the CPAF polypeptide. For example, each dose may comprise 100, 150, 200, 250, 300, 400, 500, or 750 ng, or 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 μg, or a range of two of the foregoing amounts. In one example, each dose comprises about 1 μg/kg to about 1 mg/kg of the CPAF polypeptide. In some aspects, the dose comprises about 5 μg/kg to about 500 μg/kg. [0257] In some aspects, the CPAF polypeptide may be administered in an amount from about 0.01 mg/kg to about 20 mg/kg. The particular amount of the CPAF polypeptide that constitutes an amount effective to generate an immune response, however, depends to some extent upon certain factors such as, for example, the particular composition being administered; the particular adjuvant being administered and the amount thereof; the state of the immune system; the method and order of administration of the composition; the species to which the formulation is being administered; and the desired therapeutic result. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors. For example, disclosed compositions may be administered to the subject at a dosage of CPAF ranging from about 1 µg to about 25 mg per dose (e.g., about 1 µg, 5 µg, 10 µg, 20 µg, 30 µg, 40 µg, 50 µg, 100 µg, 200 µg, 300 µg, 400 µg, 500 µg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, or to about 25 mg per dose or any range therein). In some aspects, the CPAF polypeptide may be administered or be intended to be administered to a subject via a parenteral route.A single dose of the pharmaceutical composition may be administered to a subject intravenously. Booster doses may be necessary to impart full protection. Thus, one or more additional doses may be administered at suitable intervals. The pharmaceutical composition may be administered to a subject by an intramucosal route.. A single dose of the pharmaceutical composition may be administered to a subject via intramucosal route. Booster doses may be necessary to impart full protection. Thus, one or more additional doses may be administered at suitable intervals. [0258] Optimal amounts of components for a particular pharmaceutical composition can be ascertained by standard studies involving observation of appropriate immune responses in subjects. For example, the dosage for human vaccination may be determined by extrapolation from animal studies to human data. In another aspect, the dosage is determined empirically. [0259] In one aspect of the methods of the present invention, a composition of the present invention is administered as a single inoculation. In another aspect, the composition is administered twice, three times or four times or more, adequately spaced apart. For example, the composition may be administered at 1, 2, 3-, 4-, 5-, or 6-month intervals or any combination thereof. The dosing schedule can follow that designated for a chlamydia vaccine known in the art.
[0260] A composition disclosed herein can be formulated as a single dose vial, multi-dose vial, an inhaler device, an intranasal delivery device, or as a pre-fdled syringe. Thus in an aspect, the current disclosure also encompasses a kit comprising a disclosed CPAF polypeptide, which may be a CPAF-polypeptide conjugate, or a pharmaceutical composition thereof and at least one container suitable for a delivery method disclosed herein. The container may be suitable for inhalation or intranasal administration. The kits may further comprise instruction for use.
EXAMPLES
[0261] The present invention has multiple aspects, illustrated by the following non-limiting examples.
[0262] The current disclosure encompasses vaccine compositions comprising a catalytically inactive CPAF polypeptide, wherein the catalytically inactive CPAF polypeptide comprises one or more non-canonical amino acids (ncAA), and wherein each ncAA is covalently attached to an adjuvant. The Examples provided herein elucidate the rationale, design, methods of making and using these novel vaccine compositions.
Example 1 CD4 T cell immunity protects against genital Chlamydia infection
[0263] Studies of natural immunity to Chlamydia trachomatis (CT) in humans indicate the importance of T cells for CT infection resolution and protection from reinfection. Women with confirmed prior infection have lower chlamydial loads when reinfected. Women who spontaneously clear infection without treatment exhibit increased CT-specific IFNg+ CD4 T cells and are less likely to be reinfected. HIV-infected women with low CD4 counts experience higher rates of chlamydial PID, and disclosed human studies associate CT-specific IFNg+ CD4 T cells with resistance to reinfection. In contrast, serum and cervical anti-CT IgG correlate with increased risk of incident infection; among women uninfected at enrollment, hazard ratios increased 3.6-fold and 22.6-fold with each unit of serum and cervical IgG, respectively. IFNg is needed for optimal antibody-mediated immunity against murine genital chlamydial infection. Women with the highest anti-CT IgG responses may be Th2-skewed, resulting in inadequate IFNg responses. This reinforces the importance of eliciting robust and appropriate functional T cell responses, generating a milieu that enables antibody-mediated opsonophagocytosis. Shortlived sterilizing immunity after infection is observed in animal models. Gene-knockout, depletion, and adoptive transfer studies in mice consistently confirm that CD4 T cells are the primary mediators of immunity and that polyfunctional Thl cells (IFNg+ TNFa+ IL-2+) secreting high cytokine levels correlate with protection. Most activated CD4 T cells are not terminally differentiated and may shift between subsets to alter their cytokine profile (i.e., Thl7 to Thl shift) or exhibit properties of multiple subsets where a more extensive range of cytokines are produced (i.e., bifunctional Thl/17). Thl or Th 1/17 cells produce IFNg that induces cellular responses that starve intracellular chlamydia of tryptophan and enhance phagocyte effector functions. IL-17A independently promotes epithelial integrity and induces the production of antimicrobial peptides. Vaccine-induced IL-17A is important for protection against other mucosal bacterial pathogens and higher levels of IL-17 induced by a subunit outer membrane protein have been linked to lower chlamydia burden and less disease upon challenge. Cells can traffic from the circulation or respond directly in the infected tissue as a tissue-resident memory (TRM) population. It was rationalized here that adjuvants and routes of immunization that induce Thl or Thl/17 cells that traffic to the genital tract may promote vaccine efficacy.
Example 2
Mouse models of Chlamydia genital infection
[0264] Chlamydia infects a wide range of species, but productive infection is generally hostspecific, with significant limitations to using C. trachomatis (CT) in mice. CT serovar D shedding is detected for only 10 days in mice after vaginal inoculation, suggesting CT delivered intravaginally is insufficient for challenge studies when assessing adaptive immune responses. Transcervical inoculation of mice with human strains results in more productive infections, but bypasses the cervix, a significant mucosal and immunological barrier to the upper genital tract. Transcervical inoculation with human serovars induces minimal oviduct pathology, presenting another limitation to this model. Thus, preclinical studies in mice frequently use host-adapted C. muridarum (CM) since C. trachomatis (CT) is highly susceptible to murine IFNg-induced GTPases. Female mice inoculated vaginally with CM recapitulate immune mediators of protection and fallopian tube pathology observed with severe CT infection in cisgender women. This pathology, specifically oviduct scarring and fibrosis with post-obstructive dilatation or hydrosalpinx, provides a robust model for vaccine testing. C. muridarum stock populations contain sub-populations of genetic variants expressing phenotypic differences. Inoculation of mice with different plaque-purified clonal isolates from these stocks results in infections of varying severity. A clone provided herein, CM006, reflects the infection profile and pathology of its multiclonal parent stock and is a valuable challenge strain for vaccine experiments because it yields consistent levels of cervicovaginal shedding and pathology between individual mice. Intravaginal immunization of mice with a plasmid-deficient live-attenuated CM (strain CM972) elicits protective memory CD4 T cells that reduce bacterial burden by 2-logs and completely protects from oviduct damage when challenged with high doses (5x105) of highly virulent strains. A live-attenuated vaccine is not suitable for humans, but this strain can help guide the rational vaccine design and inform the criteria for vaccine efficacy, defining the parameters that may be achieved with the current vaccine candidate.
Example 3 Antigen Screening
[0265] An antigen discovery pipeline (U19 AI144181) was set up to evaluate antibody and T cell responses in CT-exposed women enrolled in the T cell Response Against Chlamydia (TRAC) study, simultaneously profiling chlamydial gene expression in infected TRAC2 participants. Screening T cells from 30 CT-exposed TRAC participants for reactivity against pooled peptides representing 33 CT proteins identified CPAF as the top CD4 T cell antigen tested to date. T cell responses for 21 antigens are depicted in (FIG. 1A-1B); 12 additional antigens tested negatively in all individuals. For this assay, short-term T cell lines were generated by culturing PBMCs for 10 days with IL-2 and peptides spanning each protein. The T-cell lines were rested for 24 hours and tested for their response to pooled peptides by IFNg ELISPOT. Twelve seronegative healthy donors defined a CT-specific T cell response threshold. The mean CT-specific T cell response for these donors was 15 SFU/106 cells, with the 97.5th percentile not exceeding 220 SFU/106 cells. Thus, 300 SFU/106 were selected to define a CT-specific T cell response. Sixteen of the TRAC participants profiled responded to CPAF. while only 5 responded to CT MOMP (FIG.
1A). When in vitro expanded T cells from 28 of these participants were examined by intracellular cytokine staining (ICS) after stimulation with CPAF peptides, CD4 T cells from all 28 were positive for both IFNg and TNFa (FIG. 2 A), revealing increased sensitivity of ICS over ELISPOT and a 100% response rate for these individuals. Subsequent epitope mapping (N=9) identified CD4 T cell epitopes spanning the entire protein (FIG. 1C). Flow cytometry confirmed that the IFNg response to CPAF was CD4 T cell dominated and durable because CPAF recognition was detected up to a year after antibiotic treatment (FIG. 2).
[0266] Based on these studies, CPAF, a secreted virulence protein, emerged as the leading candidate immunogen. CPAF is a highly conserved serine protease abundantly secreted into the host cell cytoplasm late in infection and released extracellularly with host cell lysis. CPAF- dependent processing of host proteins contributes to the generation of new infectious progeny and their release from the host cell; suppression of the T cell chemokine, CXCL10; and inactivation of neutrophils. CPAF was also found to be the 9th most abundant CT transcript detected in cervical samples by RNAseq. Whole immunoproteomic antibody profiling of sera obtained from 222 TRAC participants revealed that CPAF (CT858) was the most pharmaceutical protein (recognized by 75% of participants with the highest average antibody expression levels among all antigens). Preliminary data disclosed herein also indicated it is a potent CD4 T cell antigen.
Example 4 CPAF is also immunodominant in mice
[0267] A similar screen was performed against 33 antigens using splenocytes isolated from female C57BL/6 mice with robust natural immunity. These mice previously cleared infection with an attenuated strain, CM972, and a subsequent challenge with virulent CM001. Peptides spanning OmcB and MOMP, abundant outer membrane proteins, elicited moderate frequencies of 100 IFNg SFU/106 splenocytes and 150 IFNg SFU/106 splenocytes, respectively, while CPAF restimulation elicited 2100 IFNg SFU/106 splenocytes (Fig. 3A). Consistent with these data, ICS confirmed that IFNg+TNFa+ CPAF-specific T cells were fully CD4-restricted and comprised 6.4% of total CD44hl memory CD4 T cells (Fig. 3B). Profiling CD4 T cells harvested from the genital tracts of naive or CM972-vaccinated mice 7 days post CM001 challenge, it was observed that naive controls lacked detectable CPAF-specific CD4 T cells while >6% of the total CD4 T cell memory population were CPAF-specific in CM972-immunized mice (Fig. 3C). Example 5
Adjuvant selection- vaccine adjuvants amplify CD4 T cell responses to CPAF
[0268] These natural immunity studies prompted the development of CPAF as a monovalent preclinical vaccine. Previous studies in mice using CPAF vaccines adjuvanted with interleukin- 12, or mice administered with multiple doses of CpG before and after CPAF immunization (intranasal: i.n.) have shown abbreviated infection and reduced pathology with a standard challenge dose (1.5x103 - 1x104 bacteria). The toxicity of IL-12 administration and the implausibility of delivering CpG on consecutive days make these approaches unsuitable for human use. Formulations combining CpG with Montanide as an adjuvant in preclinical studies with CT major outer membrane protein (MOMP) have been explored. These yield similar protective results to those presented in the current proposal. However, this adjuvant combination is reactogenic. In one cancer trial (NCT00299728), participants sustained local and systemic side effects, making it unsuitable for an infectious disease vaccine.
[0269] In addition to CpG1018, used in the HEPLISAV-B vaccine, bacterial agonists such as c- di-AMP (CD A) have shown promise as adjuvants with a good safety record for mice and humans. CpG stimulates via the TLR9 endosomal pathway, while CDA activates the cytosolic STING pathway, synergizing for enhanced IL-12 production by eliciting inflammatory cytokines and type I and II interferons to drive robust Thl and Th 1/17 responses (FIG. 4). Preliminary data shows that conjugated CpG-CPAF mixed with CDA enhanced immunogenicity without reactogenicity. Importantly, third-generation synthetic CDA analogs like diamidobenzimidazole (diABZI) can be directly conjugated to immunogens and are potent and well tolerated on mucosal delivery.
[0270] Imidazoquinoline-based TLR7 agonists generate potent immunostimulatory activity and Thl -biased adaptive immunity in mice and humans. They are effective topical therapeutics but have been too reactogenic for use as adjuvants, but immunogenicity can be retained by linking TLR7 agonists to immunomodulatory compounds, such as dopamine, to reduce pro- inflammatory side effects. Thus DOPA linked agonists are in consideration. Example 6 Adjuvant conjugation and testing
[0271] Approaches disclosed herein may use CP AF conjugated adjuvants or mixtures of such compositions and soluble adjuvants to provide effective vaccination and a path to generating strong chlamydia-specific T-cell responses without adverse reactions. This approach facilitates the concurrent delivery of vaccine components to an antigen-presenting cell. It overcomes the disassociation of immunogen and adjuvant in mixed formulations and prolongs antigen presentation to induce adaptive responses through intracellular storage and depot effects. This may provide superior efficacy. Additionally, conjugating agonists to vaccine antigens such as CPAF polypeptides may enhance immunogenicity, reduce the amount of adjuvant required, and/or prevent systemic toxicity. FIG. 6A shows a generalized schematic of the Click chemistry used in the conjugation of a polypeptide comprising a nnAA (Rl) comprising an azide group and a DBCO linked adjuvant (R2). Site-specific conjugation technology used herein may include TLR or STING agonists without compromising critical T cell and B cell epitopes and preserve the capacity of the product to elicit robust immune responses.
[0272] Despite the potential of antigen-adjuvant conjugate systems, non-targeted conjugation chemistries have not advanced to clinical translation because they: (1) result in batch-to-batch variability, (2) alter the physical properties of the antigen risking aggregation, and (3) may block immunodominant epitopes, all preventing potent T cell responses. To prevent these drawbacks, XpressCF+, a cell-free protein synthesis (CFPS) platform was used herein which enables scalable generation of recombinant proteins modified for subsequent high efficiency, sitespecific conjugation of adjuvants via click chemistry (provide patents). Using this platform, p- azido methyl phenylalanine (pAMF) residues were be introduced at solvent-exposed sites on the antigen away from immunodominant epitopes.
[0273] Expression of a His-tagged CPAF using the Xpress CF+ cell free system could be successfully conducted. The polypeptide had an encoded TEV protease site between the His-tag and the CPAF polypeptide. The resulting polypeptide was purified using affinity chromatography. A TEV protease treatment was used to remove the His-tag and the resulting protein was loaded on an SDS-PAGE. Analysis on an SDS-PAGE gel (see FIG. 5) showed the presence of a few fragments that likely correspond to SEQ ID NOS: 4-9 based on the molecular weights. If these size assignments are correct, it is likely that ATS GLK site is more labile than the KSM RGA site. Further analysis using Mass Spectrometry is being conducted and methods of purification of one or more of these fragments are being tested.
[0274] Using the cell-free protein synthesis approach to candidate chlamydial T-cell antigens, recombinant, enzymatically inactive CPAF containing pAMF residues at Fl 52 or Y560 were successfully manufactured and purified at the 250 ml scale and conjugated with CpG for evaluation in the murine immunogenicity and protection studies described above. Preliminary data indicates that this platform enables a targeted approach toward eliciting protective levels of cell-mediated immunity toward CPAF. Similar design principles were used for making conjugates used herein.
[0275] For further optimization, conjugating different TLR and STING agonists to CPAF- containing pAMF residues with the goal of enhancing mucosal immune responses to CPAF and mitigating toxic side effects are explored herein. 2nd generation immune agonists including synthetic small molecule TLR and STING agonist conjugates for vaccination were developed and used in the examples. Immune agonist dimers and trimers that can modulate early cytokine production and Thl/Th2 biasing for vaccination were also synthesized and characterized. Additionally, heterodimers containing a TLR agonist and an NF-KB pathway immunomodulator, dopamine, facilitating adaptive immunity while reducing reactogenicity were also synthesized. From these studies, synthetic strategies for the conjugation of immune agonists to heterodimeric and polymeric scaffolds were developed. With the goal of enhancing cellular immunity and tolerability in subunit Chlamydia vaccines, four exemplary adjuvants were conjugated and are reported herein: CpG (TLR9 agonist), 2BXy (TLR7/8 agonist), 2BXy-Dopa (TLR7/8 agonist- dopamine multimer), and a non-nucleotide STING agonist derived in-house from diABZI91 (FIG. 6). These agonists were selected because they have high water solubility and enhance Thl- biased immunity. Agonists were synthesized with reactive dibenzocyclooctyne (DBCO) terminal modifications for reaction with pAMF-containing CPAF mutants. Purity >95%, was confirmed by 1H-NMR and LC-MS prior to conjugation. Synthesis and preliminary characterization of CPAF-agonist conjugates was conducted. Robust design principles for directly conjugating agonists with recombinant proteins using strain-promoted azide-alkyne click chemistry (SPAAC) were used. Near-complete reaction was achieved when a 2.5-10-fold excess of different TLR agonists was mixed with antigen at 35 °C in PBS for 48 h. Following this strategy, CPAF-pAMF were reacted with agonist-DBCO using SPAAC, and the resulting CPAF-agonist conjugates dialyzed to remove unreacted material. Size-exclusion HPLC was used to confirm the complete removal of unreacted material, while SDS-PAGE was used to confirm reaction efficiency and conservation of CPAF integrity.
[0276] ~1 mg of each CPAF-agonist construct was synthesized (CPAF-CpG, CPAF-2BXy, CPAF-2BXy-Dopa, and CPAF-STING). Following synthesis, gel clot assays was used to confirm that endotoxin is absent (< 1 EU/mL). If endotoxin remained, Triton X-l 14 extraction was used.
[0277] For initial testing groups of female mice were immunized i.n. or i.m. twice with i) conjugated inactivated CPAF to CpG (15 mg/dose), ii) CpG-CPAF conjugate combined with the STING agonist ADU-S100 (5 mg/dose), or CpG-CPAF conjugate formulated with ADU-S100 and squalene oil-in-water emulsion AddaS03 (AS03 mimic; 1 : 1 ratio) (FIG. 7A-7C) 21 days apart were used. Immunized mice exhibited no signs of distress, adverse effects, or weight loss over 14 days, when they were sacrificed to evaluate cellular responses to these regimens.
Example 7
Multi-adjuvant CPAF-CpG+CDA+AS03 Formulation
[0278] Next, soluble adjuvant CDA was added to the formulation to test the impact of using a multi-adjuvant approach. Groups of female C57BL/6 mice were immunized twice with 15 mg doses of CPAF formulated as above 21 days apart. The mice were intravaginally challenged 21 days post-boost with a high inoculum of CM006 (5x105 IFU). Including CDA in the i.n. vaccine significantly enhanced the frequency of IFNg (FIG. 7A) and IL-17A (FIG. 7B) responses compared to CpG-conjugation alone with i.n. immunization. In contrast, i.m. immunization with CPAF-CpG+CDA+AS03 induced comparable IFNg but low IL-17A responses compared to i.n. immunization. Similar antibody titers were observed between groups vaccinated i.n. with CPAF conjugated to CpG with and without CDA or AS03 (FIG. 7C). This shows that mucosal route of delivery may provide the best protection. The i.n. CPAF-CpG+CDA+/-AS03 vaccines elicited significant protection from challenge, reducing cervical burden by >2.5 logs (p<0.0001), compared to naive controls (FIG. 8A), and by >1.5 logs (p<0.0001), compared to unadjuvanted recombinant CPAF (rCPAF). These vaccinated groups cleared infection by day 10-14, while naive mice were still infected with 1x104 bacteria. Despite comparable IFNg T cell immunogenicity, i.m. immunization induced inferior protection compared to i.n. immunization with CPAF-CpG+CDA+AS03 (FIG.8A; p=0.0018). Fewer oviduct hydrosalpinxes were observed in the i.n. CPAF-CpG+CDA+AS03 vaccine group compared to the naïve group (1 of 8 vs.5 of 10, p=0.15), trending towards significance with this high-dose challenge, while similar numbers of hydrosalpinxes occurred in all other groups. Oviduct dilatation scores supported this trend (FIG.8B). These data demonstrate the potential for a multi-adjuvant approach to enhance efficacy of mucosal immunization and a potential role for IL-17A in vaccine-mediated protection. Example 8 Multi-adjuvant CPAF-TLR7-DOPA linkage enhances immunogenicity without increasing IL-1α [0279] Next CPAF was conjugated to 2BXy-Dopa-DBCO or 2BXy-G4S-DBCO (where G4S is a soluble linker) as in Example 6 and further illustrated in FIG.6A and evaluated for immunogenicity of the TLR7 agonist (2BXy) platform. CPAF-2BXy-Dopa and controls (CPAF + 2BXy-Dopa, CPAF-22BXy, CPAF or PBS) were injected i.m. on days 0 and 14 (7μg dose, n=5/group). Systemic cytokine release (FIG.9A) and antibody responses (FIG.9B) were evaluated. Linked CPAF-2BXy-Dopa vaccination enhanced protective early IFNg responses but not toxic IL-1α response, 18 h after injection compared to controls, and induced a strong IgG2c- biased Th1-correlated response. These data demonstrate the potential for a multi-adjuvant approach to enhance the efficacy of mucosal immunization and a potential role for IL-17A in vaccine-mediated protection. Example 9 Refinement of the multi-adjuvant CPAF-CpG+CDA+AS03 vaccine [0280] To test the immunogenicity of the CPAF-CpG+CDA+AS03 formulation, immunogenicity and challenge experiments comparing (i) CpG1018 versus CpG1826 in an intranasal vaccine regimen will be conducted (FIG.10). Mice (n=5/group) will be i.n. immunized with 15 mg of CPAF conjugated with CpG1018 or CpG1826 with and without 5 mg of CDA (ADU-S100) in AS03 plus controls on days 0 (prime) and 30 (boost). Mice can then be sacrificed 10 days post-boost for immunogenicity assays or challenged 30 days later. A dose- limiting analysis of the benchmark CpG1018 can then be conducted. Female C57BL/6 mice (n=5/group) can be immunized i.n. with different doses of CpG and CD A in the presence or absence of AS03, or PBS (see Table 3)
Table 3
Figure imgf000073_0001
[0281] CPAF-specific T cell responses from splenocytes of individual mice can be measured by ex vivo IFNy and IL-17A ELISpot following stimulation with pools of overlapping peptides (OLPs) spanning CPAF (lOmM, 15aa long with 11 aa overlap), or no stimulation (negative control). The proportion of IFNg and/or IL-17A positive CD4 and CD8 T cell responses can be determined by intracellular cytokine staining (ICS). CPAF-specific antibody (IgGl/IgG2b/IgG2c/IgG3/IgA) can be quantified in sera collected at sacrifice. Antibody can also be quantified in vaginal lavages. Immunized mice treated with Depo-Provera can be challenged (n=10) with a standard dose of 1 x 104 IFU of CM006 (virulent CM) 30 days post-boost. The endpoints for challenge experiments can be bacterial shedding monitored by culture of cervical swabs every 3-4 days for 42 days and oviduct pathology can be determined by gross examination and histologically at sacrifice on day 42. A pathologist blinded to study design can evaluate the oviduct tissues histologically and score them for inflammation, dilatation, erosion and fibrosis. Experiments can be repeated for validation with a separate set of 10 mice/group; yielding 20 mice/group for burden and pathology analysis. The standard CPAF-CpG1018 (15 pg) + CDA (5 pg) dose and lowest effective dose from above can also be analyzed in parallel with other CPAF vaccine candidates for toxicity. The optimal regimen identified after these experiments can be used as the benchmark going forward to establish a strategy of superiority for candidate vaccine testing. Example 10
Further development
[0282] A panel of CPAF-agonist conjugates have been generated and tested for immunostimulation and toxicity in cell-based assays. CPAF-agonist conjugates that induce cytokine production equivalent or superior to unlinked controls can now be advanced for evaluation. Antigen-adjuvant conjugates which cannot be synthesized on a 1 mg scale or for which CPAF integrity cannot be confirmed will be excluded from further analysis. Conjugates that display cellular toxicity at levels >20% of a lipopolysaccharide positive control or those with poor water solubility will also be excluded from further study. Scalable CPAF vaccine candidates will be identified.
[0283] Some CPAF-agonist conjugates may have poor solubility in water because charge shielding can occur. If so, hydrophilic spacer groups comprised of polyethylene glycol or glycine oligomers will be introduced to the immune agonists, or the pH of the solution can be adjusted during and after conjugation. If conjugates do not induce detectable cytokine production, they will be reformulated and retested.
[0284] In-vivo formulation, toxicology, and preliminary immunogenicity analysis of candidate vaccines; CPAF-agonist conjugates inducing the most robust CPAF-specific T cell and antibody responses without toxicity will be identified using the approaches outlined. Thus far, adverse side effects were not observed in mice inoculated i.n. with the current candidate vaccines tested. However, emulsion adjuvants such as AS03 may cause nasal irritation that can be avoided by s.l. delivery in saline or a thermoresponsive gel (TRG). TRG-formulated sublingual (s.l.) vaccines enhance contact time between antigen and the oral mucosa and s.l. delivery may reduce incidence of oviduct pathology in mice challenged with Chlamydia.
[0285] Toxicity assessments of CPAF-agonist conjugates advanced from these studies will be conducted. Female C57BL/6 mice (n=5/group) can be vaccinated s.l. or i n. and boosted after 14 days. Benchmark vaccines can be tested along with four classes of vaccines: (1) CPAF-agonist conjugates, (2) CPAF + soluble agonist (i.e., equivalent doses of mixed antigen and agonist), (3) CPAF-agonist conjugates + a second, soluble agonist, or (4) negative controls (CPAF only and PBS) (FIG. 11). Vaccines can be prepared using the dose-sparing quantity of CPAF-agonist conjugates, including AS03 if indicated by the results of further experiments. Sublingual formulations will consist of 0.10% weight/volume (w/v) Carbopol®, 0.75% w/v HPMC, and 15% w/v Pluronic® Fl 2794. A multiplexed serum cytokine assay can quantify systemic cytokine production at 1 and 24 hours post-primary immunization. JFNy and IL-6 may serve as surrogates of a protective immune response; IL- la and MCP-1 may serve as surrogates of an immunotoxic response. Body weight and temperature will be monitored through day 28 to measure overall health. On day 27, sera and nasal washes will be collected to evaluate IgG and IgA responses. Splenocytes can be isolated on day 28, restimulated with CPAF OLPs, and analyzed via ZFNy ELISpot. The lower respiratory tracts can be harvested for histopathology at sacrifice. Safety issues may manifest early, as elevated sentinel cytokines with either the s.l. or i.n. route, or late, as respiratory tract damage with i.n. delivery. While some i.n. vaccines may cause respiratory tract damage that disqualifies them from further testing, none of the vaccines are anticipated to cause late toxicity when delivered s.l. so those candidates can remain, if sufficiently immunogenic. Preliminary data provided herein showed that CPAF- CpG+CDA+AS03 delivered i.n. was well tolerated but lung pathology was not monitored. If bronchial or lung tissue damage is observed, the s.l. route may be well tolerated. Multiple delivery strategies will be tested and optimized.
[0286] Evaluation of 2nd generation candidates: Next-generation CPAF-TLR and -STING conjugates some of which are provided herein and some are under development will be tested. These will be delivered by the safest mucosal route to elicit equivalent or superior protection, compared to benchmark. Strongest candidate vaccines in two strains of female mice will be determined. The best candidate will be tested for its ability to prevent infertility in female mice and for its immunogenicity in male mice to determine if sex is a biological variable that contributes to chlamydial vaccine responsiveness. Potential benefits of additional boosting to promote the durability of protection will also be evaluated.
[0287] Candidates eliciting superior or equivalent protection from burden and disease in challenged female mice of different genetic backgrounds when compared to benchmark will be determined. To identify the most immunogenic vaccine, female C57BL/6 mice (n=5/group) will be immunized i.n. or s.l. on days 0 (prime) and 30 (boost) with CPAF adjuvanted with conjugated TLR and STING agonists (see Table 4). Table 4
Figure imgf000076_0001
[0288] Each dose will be 15mg or a lesser alternative antigen-sparing concentration. Mice will be sacrificed 10 days post-boost. CPAF-specific T cell responses from splenocytes will be measured. T cell responses will be further defined by flow cytometric analysis of CD4 and CD8 antigen-specific proliferation by dilution of carboxyfluorescein succinimidyl ester (CFSE) and ICS for IFNg, IL-17A, TNFa, IL-2, and CD107a. CPAF-specific antibody responses will be measured by ELISA in serum and vaginal lavages (as shown in FIG. 10). Parallel groups of vaccinated mice will be used to enumerate memory CPAF-specific T cells in the genital tract by flow cytometry 30 days post-boost based on the expression of LIVE/DEAD, CD45, CD3, CD4, CD8, CD44, IFNg, IL-17A, and TNFa. Female C57BL/6 mice will be immunized as provided herein and challenged (n=10) with IxlO4 IFU of CM006 30 days post-boost. Endpoints for challenge experiments will be Chlamydia shedding monitored by the culture of cervical swabs every 3-4 days for 42 days and oviduct pathology determined by gross examination and histologically at sacrifice on day 42. All immunogenicity and challenge experiments will be repeated for validation. Candidates will be advanced if IFNg SFUs are at least 50% or greater of the benchmark vaccine because IFNg is an essential mediator of protection, and/or if they elicit at least a two-log reduction in burden and 50% reduction in gross and microscopic hydrosalpinx compared to naive controls. Candidate vaccines incorporating a single agonist will be prioritized over two agonists if they demonstrate equivalent efficacy for anticipated ease of manufacture and future cost savings. Candidates that meet or exceed selection criteria in C57BL/6 mice along with the benchmark vaccine will be tested as described above in female B ALB/c mice because of their different genetic backgrounds, different MHC alleles, and Th2-skewed immune responses. Similar outcomes for protection as observed in C57BL/6 are expected. The top candidate that passes selection criteria in C57BL/6 and BALB/c mice, along with the benchmark vaccine, will be tested intramuscularly.
[0289] Infertility testing in C57BL/6 and BALB/c mice: The top candidate that pass selection criteria in C57BL/6 and BALB/c mice and the benchmark vaccine will be tested for their ability to protect against infertility. Age-matched female C57BL/6 and BALB/c mice (n=12/group) will be immunized as above and challenged with 1x104 IFU of CM006 30 days post-boost. Sham- immunized, nonchallenged mice will serve as fertility controls. Mice can be treated with doxycycline 4 weeks post-challenge. Three weeks post-doxy, female mice will be housed with a proven breeder male for a maximum of 18 days and then repeated if necessary. Pregnancy will be assessed by measuring the weight gained during the mating period, and the mice considered pregnant will be sacrificed 25 days from the start of the second mating, and embryos present in each uterine horn will be counted. Mice with an embryo(s) in both uterine horns will be considered fertile. All experiments will be repeated and differences in the number of embryos will be determined using a Mann-Whitney U test for comparison of two groups or Kruskal Wallis test for more than two groups with a significance level a = 0.05.
[0290] Immunogenicity of the top candidate will be confirmed in male mice. Male C57BL/6 and BALB/c mice (n=5) will be immunized with the most protective vaccine regimen and benchmark vaccine to address sex as a biological variable. Enumeration and phenotyping of CPAF-specific T cells and antibody will be determined as above. Male mice are expected to respond similarly to female mice after immunization due to the broad distribution of epitopes in CPAF. If the immunogenicity in males matches females, future vaccine studies will investigate the potential for preventing infection in male mice or male guinea pigs and non-human primates before human trials.
[0291] Next, the duration of humoral and cell-mediated responses and protection elicited by top candidate, and the contribution of additional boosting can be evaluated. Female C57BL/6 or BALB/c mice (n=10) immunized with the most protective vaccine regimen and benchmark as above will be sacrificed for immunogenicity studies or challenged at 3 and 6-month time-points following the second dose. Mice will be bled monthly for quantification of anti-CPAF antibodies. The efficacy of two versus three dose regimens in both strains of mice after challenge will be compared. [0292] Vaccine process development; Select candidates will be transitioned to 1) the optimization and scale-up of production of CPAF, the adjuvant, and conjugation; 2) the delineation of an analytical control strategy for starting materials, intermediates, drug substances, and drug product; 3) evaluation of the stability of the final vaccine, and 4) completion of a non- GLP toxicology study. Thus far small-scale methods have produced CPAF at >100 mg/L in CFPS with -85% purity. Furthermore, CPAF-CpG conjugates have been produced at the -lOrng scale to generate the preliminary data provided herein.
[0293] A Design of Experiments (DoE) approach will be taken to optimize process. DoE allows the mathematical modeling of complex reactions with many interdependent factors influencing the performance, such as temperature, pH, input ratios, and agitation speed in the case of conjugation. Optimization of upstream production, downstream purification and conjugation of CPAF will be done using this approach. The DoE approach will be used to determine impeller speed, temperature, and pH resulting in optimal conditions for the soluble yield of CPAF. Rushton-style impellers with high shear to disrupt larger gas bubbles from open-pipe or drilled- hole spargers commonly used in microbial fermentation will be utilized. These impellers are well characterized, with a variety of correlations for Reynolds Number, Power Number, and kLa for transition to manufacturing and commercial scale. We will modulate the gas-flow rate, pH, and temperature to optimize soluble yield.
[0294] Gas flow rates will also be optimized. Changing the gas flow rate changes the gas-liquid interface and affects the foaming and solubility of the target protein and cell-free components. Cell-free reactions place proteins, cell-free biomolecules, and components in direct contact with the reactor vessel surfaces and gas-liquid interfaces (bubbles), which can denature the protein of interest or proteins essential to the cell-free reaction, such as essential enzymes. At a gas-liquid interface, a hydrophilic region (aqueous liquid) meets with a hydrophobic region (gas) and can cause proteins to denature and aggregate. For this reason, cell-free reaction setups use a blend of pure oxygen and air, which delivers a more significant percentage of oxygen so that DO control can use less overall gas flow.
[0295] pH control. pH control is maintained by the automated addition of both acid and base to maintain a set-point pH. Most scaled-up cell-free reactions utilize IM citric acid and IM potassium hydroxide, because this acid/base pair provides good pH control without excessive volume additions. The initial set point for cell-free protein production is pH 7.2 ± 0.1. The pH will be varied in 0.2 increments to define the design space using DoE.
[0296] Temperature. To define the design space, the temperature will be from 20 °C to 30 °C in two-degree increments.
[0297] Downstream purification optimization; CPAF with a TEV protease cleavable N- terminal His6-tag will be expressed. The cell-free reaction will be loaded onto a pre-equilibrated His-TrapTM (GE Healthcare) column to purify the tagged protein, followed by incubation with TEV protease to cleave the tag. The untagged protein will be purified by reloading the protein mixture onto the affinity column, with the target protein collected in the flow through (FIG. 12). This approach has been used to produce GMP-quality antigens and is the most expedient way to obtain relatively pure untagged antigens. The eventual goal is to obtain >95% pure untagged CPAF. If post-affinity chromatography yields a purity of the protein solution of < 95%, additional orthogonal polishing steps can be employed using ion exchange or mixed-mode resins.
[0298] Conjugation optimization; Conjugates used to generate the preliminary data were generated using DBCO-derivatized CpG adjuvant (IDT technologies) covalently attached to pAMF-containing CPAF variants using Cu-free CLICK chemistry. Excess DBCO-CpGwas quenched with Na-azide, and dialysis performed to remove azide and unreacted CpG. To optimize stability and immunogenicity, conjugation conditions will be evaluated through single variable experiments, changing buffer pH, temperature, mixing speed, and addition speed. A DoE will then define the operating space for scale-up based on process performance. Reaction concentration, temperature, adjuvant, and protein input ratios will be optimized in the DoE experiments. DoE experiments at the 5-10 mg scale will be evaluated based on critical quality attributes. Promising conditions will be scaled up to 20 mg for immunogenicity and suitability assessment. TFF purification and filter sizing will be performed in five 100 mg scale runs. The Mettler-Toledo EasyMax system will be used for 100-200 mg scale reactions with control over agitation, pH, temperature, and reactant addition. The final candidate condition will be scaled to the 200 mg scale to supply material for TFF optimization, filter sizing, analytical assay development, and formulation development.
[0299] Formulation development and evaluation of vaccine stability: Figure 13 provides a schematic of the formulation development process that will be used. Exemplary desired formulation are stable in an aqueous isotonic solution stored at 4 °C at approximately physiological pH. CPAF compositions with a panel of (GRAS) excipients will be tested initially in a 96-well plate format. Each condition will be evaluated for thermal melt, turbidity, and size via SEC-MALS to measure the structure and potential aggregation of the candidate antigens. Following an initial screen of GRAS excipients for compatibility with each target antigen, a DoE approach will be used with the variables of pH combined with excipient identity and concentration and assessed on accelerated stability (20 °C - 37 °C for 1-3 months) to identify 2-3 candidate formulations.
[0300] Real-time and accelerated stability will be evaluated in parallel with an in vivo assessment of the immunogenicity of the 2 to 3 candidate formulations previously identified. If a suitable formulation target product profile is not identified as defined by a minimum of 6 months at 4 °C, the resulting information will be used in an iterative process to identify a suitable formulation.
[0301] Toxicology assessment: A preliminary safety study in rabbits is proposed to support the continued development of the CPAF vaccine. This preliminary toxicology study is proposed as a precursor for a rabbit GLP toxicity study. This study will incorporate a recovery group to permit the evaluation of the reversibility of any local toxicity. Animals will be sacrificed at 2 or 14 days following the last vaccine administration. The dose level will be based on the contemplated human dose. The following endpoints will be assessed: mortality, clinical signs, body weight, body temperature, hematology, serum clinical chemistry, gross necropsy, and lung histopathology.
Example 11 Clinical trials
[0302] Select disclosed CPAF vaccines will be tested in human trails. Analytical control strategies of each of the adjuvant, CPAF-polypeptide and the conjugate vaccines is provided in Table 5, Table 6 and Table 7. Table 5
Figure imgf000081_0001
Table 6
Figure imgf000082_0001
Table 7
Figure imgf000082_0002
| | | i | | I | | | | | | | 1 | | | | | | 1 |
Figure imgf000083_0001
I
[0303] Preliminary data in mice as indicates significantly improved efficacy of the developed vaccine when delivered intranasally compared to the intramuscular (i.m.) route. Adverse side effects were not observed in mice inoculated i.n. with this vaccine. TRG-formulated sublingual vaccines enhance contact time between the vaccine and the oral mucosa and may reduce incidence of oviduct pathology in mice challenged with C. muridarum. CPAF-conjugated vaccines developed will be tested via the mucosal route, and candidate(s) that induce the best protection will be tested for efficacy when delivered i.m in clinical trials.
[0304] Human clinical trials for efficacy may involve the enrollment of sexually active adults with a target age of 18-25 years since CT is most prevalent in this age group. Both cis- and transgender males and females would be eligible for enrollment, and efficacy would be assessed by NAAT testing of clinically obtained samples. Based on modeling studies, a CT vaccine that achieves at least a 50% reduction in the rate of infection for 10 years will decrease the prevalence of the infection by 50% after 13 years and essentially eradicate the infection in 24 years. A high-coverage vaccine that reduces the chlamydial peak load by 2 logio could eradicate a chlamydial epidemic in ~ 20 years. Thus, a 50% effective vaccine that results in a 2 logio reduction in burden among those who acquire infection relative to individuals in the unvaccinated arm would be highly beneficial by decreasing the prevalence of infection and reproductive morbidities.
[0305] Product characteristics that will be sought in the initial trials are provided in Table 8. Potency will be evaluated using an in vitro relative potency (IVRP) assay strategy. In vitro potency assays have been extensively correlated with in vivo measurements of neutralizing antibody induction by the antigens. Developing IVRP assays for each antigen of the 4-valent and 9-valent HPV vaccines requires developing and characterizing mAbs specific for each vaccine. This will require the production and screening of monoclonal antibodies specific to CPAF. The investigators will regularly monitor anti-CPAF IgG to individual vaccines. IFNy ELISpot assays will be used to determine an appropriate quality to be used in eventual human clinical trials as primary and/or secondary endpoints as appropriate.
Table 8 Parameter Preferred Characteristics Indication Decrease of Chlamydia-infection complications
Figure imgf000085_0001
Example 12 CPAF-conjugated vaccines provide effective vaccination against chlamydia
[0306] This example demonstrates that CPAF polypeptide conjugates disclosed herein provide effective immunization against Chlamydia infection. Female C57BL/6 mice were immunized twice intranasally with CPAF candidates or PBS control as provided Table 9 below. Notably, ADU-S100 was present at 5 pg and other agonists were conjugated at 1 agonist molecule per molecule of CPAF. After 30 days following the second dose mice were intravaginally challenged with Chlamydia muridarum (strain CM006) as described herein. Mice were monitored for cervicovaginal shedding via endocervical swabs and IFUs were calculated, as depicted in Figure 13. Notably, the CPAF-CL1151 conjugate performed with a 1.5-log improvement compared with PBS controls.
Table 9
Figure imgf000086_0001

Claims

CLAIMS What is claimed is:
1. A chlamydial protease-like activity factor (CPAF) polypeptide comprising one or more non-natural amino acids (nnAA).
2. The CPAF polypeptide of claim 1, wherein the CPAF polypeptide further comprises one or more substitutions at one or more of amino acids H97, S499, S491, E550, and C492 relative to the sequence set forth in SEQ ID NO: 1.
3. The CPAF polypeptide of claim 2, wherein the substitutions comprise one or more of H97A, S491A, S499A, C492T, and E550Q.
4. The CPAF polypeptide of claim 1, wherein the CPAF polypeptide comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or more nnAA.
5. The CPAF polypeptide of claim 1, comprising one or more substitutions of one or more wild-type amino acids relative to a reference CPAF protein with a nnAA.
6. The CPAF polypeptide of claim 5, wherein each nnAA substitutes a phenylalanine, lysine, or tyrosine.
7. The CPAF polypeptide of claim 1, wherein each nnAA comprises 2-amino-3-(4- azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyri din-2 -yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2- yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.
8. The CPAF polypeptide of claim 7, wherein each nnAA is pAMF.
9. The CPAF of claim 5, wherein the nnAA are substituted at one or more of Fl 30 and Y569 relative to the sequence set forth in SEQ ID NO: 1.
10. The CPAF polypeptide of claim 5, wherein the CPAF polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 14-22, or a fragment thereof, or a sequence at least about 80% identical thereto.
11. The CPAF polypeptide of claim 1, wherein each nnAA is covalently attached to an adjuvant.
12. The CPAF polypeptide of claim 11, wherein each nnAA comprises a structure of formula III:
Figure imgf000088_0001
wherein:
Ar comprises a 5-membered or 6-membered aromatic ring optionally containing at least one heteroatom;
W5 is selected from Ci-Cio alkylene, -NH-, -O- and -S-;
QI is zero or 1;
W6 is selected from the group consisting of azido, 1,2,4,5-tetrazinyl optionally C- substituted with a lower alkyl group, and ethynyl, and
R3 is OH or an amino acid residue of the CPAF polypeptide, and R4 is H or an amino acid residue of the CPAF polypeptide.
13. The CPAF polypeptide of claim 11, wherein the adjuvant enhances Thl-based immune response.
14. The CPAF polypeptide of claim 13, wherein the adjuvant comprises a STING agonist, a TLR agonist, or a dopamine receptor agonist.
15. The CPAF polypeptide of claim 14, wherein the adjuvant comprises a STING agonist comprising diamidobenzimidazole (diABZl), MSA-2ADU-S100, MK-1454, MK-2118, SB 11285, BMS-986301, DMXAA, E7766, GSK3745417 cyclic di-GMP (guanosine 5'- monophosphate) (CDG), cyclic di-AMP (adenosine 5 '-monophosphate) (CD A), or cyclic GMP- AMP (cGAMP) , 5,6-Dimethylxanthenone-4-acetic acid, AS03, MK-1454, TMX-202, or a derivative thereof.
16. The CPAF polypeptide of claim 13, wherein the adjuvant comprises a TLR agonist comprising 2BXy, DOPA, 2BXy-D0PA, an imidazoquinoline-based agonist, a CpG, a CpG derivative selected from CpG1826 and CpG1018, or a derivative of the foregoing.
17. The CPAF polypeptide of claim 11, where the adjuvant comprises a structure of formula XV:
Figure imgf000089_0001
wherein:
Ri is independently H, formyl, or at least one amino acid of the CPAF polypeptide;
R2 is independently OH or at least one amino acid of the CPAF polypeptide; D is — Ar— W3— or — Wl— Yl— C(O)— Y2— W2— ;
Figure imgf000089_0002
each of Wl, W2, and W3 is independently a single bond or lower alkylene; each Xi is independently — NH — , — O — , or — S — ; each Y 1 is independently a single bond, — NH — , or — O — ; each Y2 is independently a single bond, — NH — , — O — , or an N-linked or C- linked pyrrolidinylene; one of Zi, Z2, and Z3 is — N — and the others of Zi, Z2, and Z3 are independently —CH—.
18. A CPAF polypeptide conjugate comprising a catalytically inactive CPAF polypeptide, wherein the catalytically inactive CPAF polypeptide comprises one or more substitutions of one or more wild-type amino acids of CPAF with a non-natural amino acid (nnAA), and wherein each nnAA is covalently attached to an adjuvant.
19. The CPAF polypeptide conjugate of claim 18, wherein the CPAF polypeptide comprises a substitution at one or more of amino acids H97, S499, S491, E550, and C492.
20. The CPAF polypeptide conjugate of claim 18, wherein the nnAA comprises 2- amino-3-(4- azidophenyljpropanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4- (azidomethyl)pyri din-2- yljpropanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.
21. The CPAF polypeptide conjugate of claim 20, wherein the nnAA is pAMF.
22. The CPAF polypeptide conjugate of claim 20, wherein each nnAA comprises a structure of formula III:
Figure imgf000090_0001
wherein: Ar comprises a 5-membered or 6-membered aromatic ring optionally containing at least one heteroatom;
W3 is selected from C1-C10 alkylene, -NH-, -O- and -S-;
QI is zero or 1 ; W6 is selected from the group consisting of azido, 1 ,2,4,5-tetrazinyl optionally C- substituted with a lower alkyl group, and ethynyl, and
R3 is OH or an amino acid residue of the CPAF polypeptide, and R4 is H or an amino acid residue of the CPAF polypeptide.
23. The CPAF polypeptide conjugate of claim 18, wherein the CPAF polypeptide comprises the sequence set forth in any one of SEQ ID NOS: 14-22, or a fragment thereof, or a sequence at least about 80% identical thereto.
24. The CPAF polypeptide conjugate of claim 18, wherein the conjugated adjuvant is a water-soluble adjuvant.
25. The CPAF polypeptide conjugate of claim 18, wherein the adjuvant enhances Th 1 -based immunity.
26. The CPAF polypeptide conjugate of claim 25, wherein the adjuvant comprises a STING agonist, a TLR agonist, or a dopamine receptor agonist, or a combination thereof.
27. The CPAF polypeptide conjugate of claim 26, wherein the adjuvant comprises the STING agonist, wherein the STING agonist comprises diamidobenzimidazole (diABZl), MSA- 2ADU-S100, MK-1454, MK-2118, SB11285, BMS-986301, DMXAA, E7766, GSK3745417 cyclic di-GMP (guanosine 5 '-monophosphate) (CDG), cyclic di-AMP (adenosine 5'- monophosphate) (CD A), cyclic GMP-AMP (cGAMP), 5,6-Dimethylxanthenone-4-acetic acid, AS03, MK-1454, TMX-202, or a derivative of the foregoing.
28. The CPAF polypeptide conjugate of claim 26, wherein the adjuvant comprises the TLR agonist, wherein the TLR agonist comprises any one or more of 2BXy, DOPA, 2BXy- DOPA, an imidazoquinoline-based agonist, CpG, a CpG derivative selected from CpG1826, CpG1018, and polyC-CpG, and a derivate thereof.
29. The CPAF polypeptide conjugate of claim 18, wherein each nnAA is linked to the adjuvant via a first handle.
30. The CPAF polypeptide conjugate of claim 18, wherein each nnAA forms a triazole linkage with the adjuvant.
31 . The CPAF polypeptide conjugate of claim 29, wherein the first handle comprises propargyl, DIFO, dibenzylcyclooctyne (DBCO), or a DBC0(PEG)n-NH2 moiety, or a derivative thereof.
32. The CPAF polypeptide conjugate of claim 31, wherein the adjuvant forms a triazole linkage with the nnAA.
33. The CPAF polypeptide conjugate of claim 31, wherein the first handle comprises DBCO having a structure of one of formulas (Xlld-XIIh):
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
(Xllh).
34. The CPAF polypeptide conjugate of claim 33, where the adjuvant linked to the nnAA comprises a structure of formula XV:
Figure imgf000094_0002
wherein:
R1 is independently H, formyl, or at least one amino acid of the CPAF polypeptide;
R2 is independently OH or at least one amino acid of the CPAF polypeptide;
D is — Ar— W3— or — Wl— Yl— C(O)— Y2— W2— ;
Figure imgf000094_0003
each of Wl, W2, and W3 is independently a single bond or lower alkylene; each Xi is independently — NH — , — O — , or — S — ; each Yi is independently a single bond, — NH — , or — O — ; each Y2 is independently a single bond, — NH — , — O — , or an N-linked or C-linked pyrrolidinylene; one of Zi, Z2, and Z3 is — N — and the others of Zi, Z2, and Z3 are independently — CH —
35. A pharmaceutical composition comprising the CPAF polypeptide conjugate of claim 18 and one or more pharmaceutically acceptable excipients.
36. The pharmaceutical composition of claim 35, further comprising one or more soluble.
37. The pharmaceutical composition of claim 36, wherein the one or more soluble adjuvants enhance Th 1 -based immunity.
38. The pharmaceutical composition of claim 37, wherein the one or more soluble adjuvants are different from the adjuvant covalently attached to the CPAF polypeptide.
39. The pharmaceutical composition of claim 35, wherein the one or more pharmaceutically acceptable excipients are suitable for mucosal delivery.
40. The pharmaceutical composition of claim 35, for use as a vaccine against a chlamydia infection in a mammalian subject.
41. The pharmaceutical composition of claim 35, further comprising an additional polypeptide conjugate, wherein the additional polypeptide conjugate comprises a different polypeptide or a different adjuvant, relative to the CPAF polypeptide and the adjuvant.
42. The pharmaceutical composition of claim 41, further comprising a second vaccine against chlamydia or one or more other microorganism, or combination thereof.
43. A method of making a CPAF polypeptide-adjuvant conjugate, comprising contacting a catalytically inactive CPAF polypeptide with an adjuvant to form a CPAF polypeptide-adjuvant conjugate, wherein the CPAF polypeptide comprises one or more substitutions of one or more wild-type amino acids of CPAF with a non-natural amino acid (nnAA) comprising a second handle, and wherein the adjuvant comprises a first handle comprising an alkynyl group on a cyclooctane ring structure.
44. The method of claim 43, wherein the catalytically inactive CPAF polypeptide comprises a substitution at one or more of amino acids H97, S499, S491, E550, and C492.
45. The method of claim 43, wherein the nnAA comprises 2-amino-3-(4- azidophenyl)propanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyridin-2-yl)propanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2- yl)propanoic acid, 2-amino-3-(6-(azidomethyl)pyridin-3-yl)propanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.
46. The method of claim 43, wherein the CPAF polypeptide is synthesized using a cell free extract.
47. The method of claim 43, wherein the nnAA comprises the second handle, wherein the second handle comprises an azido group.
48. The method of claim 43, wherein the first handle comprises a DBCO or DIFO ring structure comprising an alkynyl group.
49. The method of claim 48, wherein the adjuvant comprises one of the following structures of one of formulas (XTId-XIIh):
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
50. The method of claim 43, wherein the contacting of the adjuvant to the CPAF polypeptide forms the CPAF polypeptide-adjuvant conjugate by an azide-alkyne-based click chemistry.
51. The method of claim 50, wherein the contacting is done in the absence of a catalyst.
52. The method of claim 50, wherein the polypeptide-adjuvant conjugate forms a triazole linkage.
53. The method of claim 43, wherein the CPAF polypeptide-adjuvant conjugate comprises a structure of formula XV:
Figure imgf000099_0001
wherein:
Ri is independently H, formyl, or at least one amino acid of the enhanced carrier protein;
R.2 is independently OH or at least one amino acid of the enhanced carrier protein; D is — Ar— W3— or — Wl— Yl— C(O)— Y2— W2— ;
Figure imgf000099_0002
each of Wl, W2, and W3 is independently a single bond or lower alkylene; each Xi is independently — NH — , — O — , or — S — ; each Y i is independently a single bond, — NH — , or — O — ; each Y2 is independently a single bond, — NH — , — O — , or an N-linked or C- linked pyrrolidinylene; one of Zi, Z2, and Z3 is — N — and the others of Zi, Z2, and Z3 are independently —CH—;
X is an adjuvant.
54. A vaccine comprising a pharmaceutical composition of claim 35.
55. The vaccine of claim 54, comprising a carrier.
56. The vaccine of claim 54, for use against a chlamydia infection.
57. A method of immunizing a subject against chlamydia, comprising administering bject the pharmaceutical composition of claim 35.
58. A method of immunizing a subject against chlamydia, comprising administering to the subject a pharmaceutical composition comprising a CPAF -polypeptide adjuvant conjugate.
59. The method of claim 58, wherein the administering is via a mucosal route.
60. The method of claim 58, wherein the subject is a mammal.
61. The method of claim 60, wherein the subject is a human.
62. The method of claim 58, wherein the adjuvant is covalently attached to the CPAF polypeptide.
63. The method of claim 58, wherein the CPAF polypeptide is catalytically inactive.
64. The method of claim 58, wherein the CPAF polypeptide comprises a substitution at one or more of amino acids H97, S499, S491, E550, and C492.
65. The method of claim 58, wherein the CPAF polypeptide comprises one or more substitutions of one or more wild-type amino acids with an nnAA.
66. The method of claim 65, wherein the nnAA comprises 2-amino-3-4- azidophenyljpropanoic acid (pAF), 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid (pAMF), 2-amino-3-(5-(azidomethyl)pyri din-2 -yljpropanoic acid, 2-amino-3-(4-(azidomethyl)pyridin-2- yljpropanoic acid, 2-amino-3-(6-(azidomethyl)pyri din-3 -yljpropanoic acid, 2-amino-5- azidopentanoic acid, or 2-amino-3-(4-(azidomethyl)phenyl)propanoic acid.
67. The method of claim 66, wherein the nnAA is pAMF.
68. The method of claim 58, wherein the CPAF polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOS: 14-22, or a fragment thereof, or a sequence at least about 80% identical thereto.
69. The method of claim 58, wherein the pharmaceutical composition comprises one or more soluble adjuvants.
70. The method of any one of claim 58 or 69, wherein the pharmaceutical composition induces a Thl response.
71. The method of claim 58, wherein the conjugated adjuvant or the one or more soluble adjuvants or both are selected from the group consisting of a STING agonist, a TLR agonist, and a dopamine receptor agonist.
72. The method of claim 71, wherein the STING agonist comprises diamidobenzimidazole (diABZl), MSA-2ADU-S100, MK-1454, MK-2118, SB11285, BMS- 986301, DMXAA, E7766, GSK3745417 cyclic di-GMP (guanosine 5 '-monophosphate) (CDG), cyclic di -AMP (adenosine 5 '-monophosphate) (CD A), or cyclic GMP-AMP (cGAMP) , 5,6- Dimethylxanthenone-4-acetic acid, AS03, MK-1454, TMX-202, or a derivative thereof.
73. The method of claim 72, wherein the soluble adjuvant comprises a TLR agonist comprising 2BXy, DOPA, 2BXy-DOPA, imidazoquinoline based agonists, CpG, or a CpG derivative selected from the group consisting of CpG1826 and CpG1018.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090022755A1 (en) * 2005-03-31 2009-01-22 Glaxosmithkline Biologicals Sa Vaccines against chlamydial infection
US20100172927A1 (en) * 2006-10-04 2010-07-08 Mark Alderson Vaccines Against Chlamydial Infection
US20110123491A1 (en) * 2007-10-01 2011-05-26 Board Of Regents, The University Of Texas System Methods and compositions for immunization against chlamydial infection and disease

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090022755A1 (en) * 2005-03-31 2009-01-22 Glaxosmithkline Biologicals Sa Vaccines against chlamydial infection
US20100172927A1 (en) * 2006-10-04 2010-07-08 Mark Alderson Vaccines Against Chlamydial Infection
US20110123491A1 (en) * 2007-10-01 2011-05-26 Board Of Regents, The University Of Texas System Methods and compositions for immunization against chlamydial infection and disease

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