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WO2025231483A2 - Protéines immunogènes et acides nucléiques les codant - Google Patents

Protéines immunogènes et acides nucléiques les codant

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Publication number
WO2025231483A2
WO2025231483A2 PCT/US2025/027789 US2025027789W WO2025231483A2 WO 2025231483 A2 WO2025231483 A2 WO 2025231483A2 US 2025027789 W US2025027789 W US 2025027789W WO 2025231483 A2 WO2025231483 A2 WO 2025231483A2
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WO
WIPO (PCT)
Prior art keywords
protein
seq
mrna
class
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/027789
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English (en)
Other versions
WO2025231483A3 (fr
Inventor
Christopher COTTRELL
William Schief
Torben SCHIFFNER
Andreia M. SERRA
Jon Steichen
Olivia SWANSON
Daniel W. Kulp
Gevorg GRIGORYAN
Jianfu ZHOU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dartmouth College
Scripps Research Institute
International AIDS Vaccine Initiative Inc
Original Assignee
Dartmouth College
Scripps Research Institute
International AIDS Vaccine Initiative Inc
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Application filed by Dartmouth College, Scripps Research Institute, International AIDS Vaccine Initiative Inc filed Critical Dartmouth College
Publication of WO2025231483A2 publication Critical patent/WO2025231483A2/fr
Publication of WO2025231483A3 publication Critical patent/WO2025231483A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to proteins and nucleic acids, such as, for example, mRNAs. for immunization regimens, modifications thereof, and/or development of nanoparticles, and/or development of membrane-anchored immunogens, and methods of making and using the same.
  • a vaccine for HIV-1 is urgently needed, as there are approximately 1.5 million new infections each year as of 2020 (www.unaids.org/en/resources/fact-sheet).
  • the target of HIV neutralizing antibodies the trimeric envelope (Env) spike, varies substantially in sequence across different HIV-1 isolates, indicating that a vaccine should induce ‘broadly neutralizing antibodies’ (bnAbs), antibodies capable of neutralizing diverse isolates (Burton and Hangartner, 2016, Annu Rev Immunol 34, 635-659).
  • bnAbs bimeric envelope
  • Potent HIV bnAbs develop in a small percentage of infected individuals, typically over an extended course of infection (Burton and Mascola, 2015, Nat Immunol 16, 571-576; Kwong and Mascola, 2018, Immunity 48, 855-871).
  • HIV bnAbs target at least five major epitopic regions on the Env trimer: V2-apex, V3- glycan, CD4 binding site, gp!20/gp41 interface, and membrane proximal external region (MPER).
  • V2-apex V2-apex
  • V3- glycan CD4 binding site
  • gp!20/gp41 interface CD4 binding site
  • MPER membrane proximal external region
  • HCDR3 heavy chain complementarity determining region 3
  • vaccine priming of HCDR3 -dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals.
  • Germline-targeting priming in particular can aid in the development of vaccines to induce several different classes of bnAbs targeting different epitopes in order to achieve optimal neutralization coverage coverage, increasing the need for effective HCDR3- dominant germline-targeting.
  • the present invention relates to non-naturally occurring proteins, which may be involved in forming immunogenic proteins of the present invention.
  • the invention relates to a non-naturally occurring protein, which may comprise any one of the sequences in Tables 1-4.
  • the non-naturally occurring protein can comprise any one of the sequences in Table 2.
  • the non-naturally occurring protein can comprise any one of the sequences in Table 3.
  • the non-naturally occurring protein can comprise any one of the sequences in Table 4.
  • the protein may have at least 90% or 95% homology or identity with the sequence of the non-naturally occurring protein(s) of the invention.
  • the invention also encompasses trimers, which may comprise any one of the non- naturally occurring protein(s) of the invention.
  • the invention also encompasses nucleic acids encoding the non-naturally occurring protein(s) of the present invention, including nucleic acids that encode protein(s) that may have at least 90% or 95% homology or identity with a nucleotide encoding the sequence of the non- naturally occurring protein(s) of the invention.
  • the invention also encompasses eliciting an immune response which may comprise systemically administering to an animal in need thereof an effective amount of any one of the non- naturally occurring protein(s) or any one of the nucleic acids encoding the non-naturally occurring protein(s) of the present invention, including nucleic acids that may have at least 90% or 95% homology or identity with a nucleotide encoding the sequence of the non-naturally occurring protein(s) of the invention.
  • the nucleic acid is formulated in lipid nanoparticles (LNPs).
  • the animal may be a mammal, advantageously a human.
  • the invention also relates to a non-naturally occurring mRNA, which may encode a protein having the sequence of any one of the sequences in Tables 1-4.
  • the non-naturally occurring mRNA can encode a protein having any one of the sequences of Table 2.
  • the non-naturally occurring mRNA can encode a protein having any one of the sequences of Table 3.
  • the non-naturally occurring mRNA can encode a protein having any one of the sequences of Table 4.
  • the mRNA may encode a protein that has at least 90% or 95% homology or identity with the sequence of any one of the sequences in Tables 1-4. In some embodiments, the mRNA may encode a protein that has at least 90% or 95% homology or identity with the sequence of any one of the sequences in Table 2. In some embodiments, the mRNA may encode a protein that has at least 90% or 95% homology or identity with the sequence of any one of the sequences in Table 3. In some embodiments, the mRNA may encode a protein that has at least 90% or 95% homology or identity with the sequence of any one of the sequences in Table 4.
  • the invention also encompasses eliciting an immune response which may comprise systemically administering to an animal in need thereof an effective amount of any one of the non- naturally occurring mRNAs of the present invention, including mRNAs that may have at least 90% or 95% homology or identity with the non-naturally occurring mRNA(s) of the invention.
  • the mRNA is formulated in lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • FIG. 1 Design and characterization of the core-g28v2 60mer boost immunogen.
  • A Shown is the iterative immunogen design workflow diagram to improve upon the starting HxB2 core-e-2cc N276D immunogen.
  • KD values were measured by SPR for mAbs elicited by eOD-GT8 60mer protein in humans and SE09 mice for first-boost immunogen candidates eOD- GT6v2-cRSF, core-g28v2, and 191084-N276D. Thick lines indicate median values, boxes show 25 and 75% percentile values.
  • the low-capture IgG SPR method may include some avidity for trimeric analytes.
  • FIG. 1 Comparison of protein boost immunogens.
  • A Shown is the immunization scheme for evaluating boost immunogen candidates delivered as proteins plus adjuvant in SE09 mice.
  • B The frequency of antigen ++ MBCs among total MBCs was analyzed by flow cytometry. Each group was sorted with matched antigens, except for the PBS placebo group which was sorted with the core-g28v2 probe.
  • C to E Shown is the frequency of VRCOl-class MBCs among antigen ++ MBCs (C), the frequency of VRCOl-class MBCs among total MBCs (D), and the frequency of VRCOl-class MBCs with human VK1-33 light chains among total MBCs (E).
  • the blue dashed bar in (E) indicates the overall frequency of VRCOl-class MBCs with human VK1-33 light chains among total MBCs within each group. Red bars indicate medians and each point represents an individual mouse for (B to E).
  • F and G The median percent aa SHM in the VH gene (F) and in the VK/VL genes (G) is shown for all VRCOl-class MBCs.
  • H and I The median percent aa SHM in the VH gene (H) and in the VK/VL genes (I) is shown for non-VRCOl -class MBCs. Each point represents the median per mouse and the red bars indicate the median of medians for panels (F to I).
  • Statistical comparisons were made by Kruskal -Wallis test followed by Dunn’s test for multiple comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001; ns, not significant.
  • FIG. 3 mAb SPR and serum antibody binding responses after eOD-GT8 60mer priming and core-g28v2 boosting.
  • A Monovalent KD values were measured by SPR of VRCOl- class mAbs from naive SE09 mice, eOD-GT8 60mer primed sE09 mice, and eOD-GT8 60mer primed, core-g28v260mer boosted SE09 mice for eOD-GT8, core-g28v2, and core-g28v2-N276 + . Each point represents the KD of a single antibody. Red bars indicate median affinities and include non-binders. Geomean affinities were calculated among binders only.
  • FIG. 4 Key VRCOl-class heavy chain residues after protein immunization.
  • A Shown is a ribbon diagram of the VRC01 variable fragment (Fv) with key VRCOl-class heavy chain residues colored green for non-paratope residues and pink for paratope residues.
  • B A molecular surface representation of HIV Env shows contact residues of key VRCOl-class heavy chain paratope residues (red).
  • C Shown is the 90 th percentile number of key VRCOl-class heavy chain residues elicited during immunization experiment described in Fig. 2A.
  • Each point is the 90 th percentile number of key VRCOl-class heavy chain residues for each mouse with the red bars indicating the median of the 90 th percentile values for each group.
  • Statistical comparisons were made by Kruskal -Wallis test followed by Dunn’s test for multiple comparisons. **p ⁇ 0.01; ns, not significant.
  • Residue numbers in panels (A), (B), and (D) use the Kabat antibody numbering scheme (T.T. Wu et al., 1970).
  • E to G KD values between core- g28v2 and mAbs isolated after core-g28v2 60mer boosting were correlated with number of key VRCOl-class heavy chain residues (E), percent Vn SHM (F), and percent VK SHM (G).
  • solid lines show correlations
  • dashed lines show 95% confidence internal
  • S represents slope
  • p represent the P-value from a simple linear regression test, ns, not significant.
  • FIG. 5 Immune response elicited using mRNA/LNP immunization.
  • A Shown is the immunization scheme for evaluating immunogens delivered using mRNA/LNPs in SE09 mice.
  • B The frequency of antigen ++ MBCs among total MBCs was analyzed by flow cytometry.
  • C to E Shown is the frequency of VRCOl-class MBCs among antigen ++ MBCs (C), the frequency of VRCOl-class MBCs among total MBCs (D), and the frequency of VRCOl-class MBCs with human VK1-33 light chains among total MBCs (E). Red bars indicate medians and each point represents an individual mouse for panels (B to E).
  • Statistical comparisons were made by Kruskal- Wallis test followed by Dunn’s test for multiple comparisons. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001; ns, not significant.
  • FIG. 6 Key VRCOl-class heavy chain residues, mAh SPR, and serum antibody binding after mRNA/LNP immunization.
  • A Shown are monovalent KD values measured by SPR of VRCOl-class mAbs from naive SE09 mice, eOD-GT8 60mer mRNA/LNP primed SE09 mice, and eOD-GT8 60mer mRNA/LNP primed, core-g28v2 60mer mRNA/LNP boosted SE09 mice for eOD-GT8, core-g28v2, and core-g28v2-N276 + . Red bars indicate median affinities and include non-binders. Geomean affinities were calculated among binders only.
  • FIG. 1 Shown is serum IgG ELISA binding to eOD-GT8, eOD-GT8-KO, core-g28v2, and core-g28v2-KO for SE09 mice primed with eOD-GT8 60mer mRNA/LNPs (magenta) only or primed and boosted with core- g28v2 60mer mRNA/LNPs (green). Each point represents the ED50 value of serum per mouse. Red bars indicate median EDso values.
  • C Shown are the 90 th percentile values for key VRCOl- class heavy chain residues elicited during mRNA/LNP immunization experiment described in Fig. 5 A.
  • FIG. 7 Post core-g28v2 mAbs bind N276(-) native-like HIV Env trimers and neutralize corresponding pseudoviruses.
  • FIG. 8 Characterization of the Vnl-2 JH2 /VKl-33 hTdT (SE09) mouse model.
  • A Illustration of genetic modifications in the Igh and Igk locus of the Vnl-2 JH2 /VKl-33 hTdT (SE09) rearranging mouse model.
  • the mouse VH81X was replaced with the human VH1-2 and the mouse JHS were replaced with the human JH2.
  • the intergenic control region 1 (IGCR1) regulatory element in the Vn-to-D intervening region was deleted.
  • the mouse VK3-2 was replaced with human VKI- 33 plus a CTCF-binding element (CBE) 50 bp downstream of its recombination signal sequence.
  • CBE CTCF-binding element
  • the human TdT gene was knocked into mouse Rosa locus.
  • B Frequency of VRCOl-class naive B cells in humans (Rantalainen et al., 2020; Ofek et al., 2010; Correia et al., 2014) and in SE09 mice after sorting with eOD-GT8. Solid red lines indicate overall frequency (total VRCOl-class naive B cells among all samples divided by total number of naive B cells sorted among all samples).
  • E Monovalent KD values measured by SPR of VRCOl-class naive precursors isolated from humans and SE09 mice for binding to eOD-GT8.
  • GNL Galanthus nivalis lectin
  • FIG. 11 SPR affinity measurements for VRCOl-class mAbs elicited after priming with eOD-GT8 60mer protein in SE09 mice and humans.
  • A KD values were measured by SPR for mAbs elicited by the indicated first boost candidates.
  • Data from Fig. 1C are included here to facilitate easy comparison among all booster immunogen candidates. Thick lines indicate median values, boxes show 25 and 75% quantiles. *Low-capture IgG SPR method many include some avidity for trimeric analytes.
  • B Table listing immunogens with valency and CD4bs epitope mutations.
  • FIG. 12 Representative FACS gating scheme for the isolation of antigen specific MBCs and BCR sequencing workflow.
  • FIG. 13 Comparison of additional protein booster immunogens.
  • A Shown is the immunization scheme for evaluating additional boost immunogen candidates delivered as adjuvanted proteins in SE09 mice.
  • B The frequency of antigen ++ MBCs among total MBCs was analyzed by flow cytometry. Each group was sorted with matched antigens.
  • C to E Shown is the frequency of VRCOl-class MBCs among antigen ++ MBCs (C), the frequency of VRCOl-class MBCs among total MBCs (D), and the frequency of VRCOl-class MBCs with human VK1-33 light chains among total MBCs (E).
  • FIG. 15 SHM analysis of VRCOl-class BCRs induced by mRNA/LNP immunization.
  • A Median percent amino acid SHM in the VH gene among VRCOl-class MBCs induced after mRNA/LNP immunization.
  • B Median percent amino acid SHM in the VK/VL genes among VRCOl-class MBCs induced after mRNA/LNP immunization.
  • FIG. 16 Comparison of protein plus adjuvant immunization versus immunization with mRNA/LNPs. Select data repeated from Fig. 2D, 2E, and 2F; Fig. 4C; Fig. 5C and 5D; Fig. 6A; and Fig. 15 A.
  • A The frequency of VRCOl-class MBCs among total MBCs after priming with eOD-GT8 60mer in a protein (pt) or mRNA/LNP format.
  • B The frequency of VRCOl-class MBCs with human VK1-33 light chains among total MBCs after priming with eOD- GT8 60mer.
  • FIG. LCDR3 logo plots for VRCOl-class VK1-33 BCRS.
  • a and B Logo plots are shown for BCRs analyzed after eOD-GT8 mRNA priming (A) and after core-g28v2 mRNA boosting (B).
  • Figure 18 Comparison of protein plus adjuvant immunization versus immunization with mRNA/LNPs. Select data repeated from Fig. 2D, 2E, and 2F; Fig. 4C; Fig. 5C and 5D; Fig. 6A; and Fig. S8A.
  • A The frequency of VRCOl-class MBCs among total MBCs after priming with eOD-GT8 60mer and boosting with core-g28v2 60mer.
  • B The frequency of VRCOl-class MBCs with human VK1-33 light chains among total MBCs after priming with eOD-GT8 60mer and boosting with core-g28v2 60mer.
  • (C) Median percent amino acid SHM in the VH gene among all VRC01 -class MBCs after priming with eOD-GT8 60mer and boosting with core-g28v2 60mer.
  • (D) 90th percentile key VRCOl-class heavy chain residues elicited after priming with eOD-GT8 60mer and boosting with core-g28v2 60mer.
  • Statistical comparisons were made by Mann-Whitney. *p ⁇ 0.05, **p ⁇ 0.01; ns, not significant.
  • FIG. 19 (A) Key VRCOl-class heavy chain residues among GT8 mRNA primed. (B) GT8 mRNA primed followed by placebo boost (sorted with GT8). (C) Key VRCOl-class heavy chain residues among GT8 mRNA primed followed by GT8 mRNA boost (sorted with GT8). (D) Key VRCOl-class heavy chain residues among GT8 mRNA primed followed by GT8 mRNA boost (sorted with core).
  • FIG. 23 Pseudovirus neutralization activity of polyclonal IgGs isolated from serum after eOD-GT8 60mer mRNA/LNP priming or boosting with core-g28v2 mRNA/LNPs. No neutralization: NN; IC50 > 50 pg/mL.
  • FIG. 24 SPR studies of boost candidate interactions with VRCOl-class mAbs.
  • A SPR affinity data for VRCOl-class mAbs elicited by eOD-GT8 60mer protein immunization in the Vnl-2 mouse model (Verkoczy et al., 2011).
  • B SPR affinity data for VRCOl-class mAbs elicited by eOD-GT8 60mer protein immunization in humans binding to gp!20 core variants with or without the T278M mutation. Red lines indicate medians.
  • FIG. 26 BG18 type I lineages and structural analysis.
  • A Gene segment assignment and representative HCDR3 sequence of 38 BG18 type I lineages. The D3-41 gene is colored blue and two critical contact residues are colored red. D3-41 in alternate reading frame is colored green.
  • FIG. 1 lower panels show magnified view of HCDR3 interacting with gpl20 for each structure; gp!20 is colored gray and the N332 epitope (N332-GT2 residues Gly324, Asp325, Val326, Arg327, Met328, Ala329, His330, Ile415, Leu416 and Pro417 or the equivalent position in N332-GT5) colored red; HCDR3s are colored blue, orange, purple, green for BG18_GLo, RM_N332_03, RM_N332_36, and RM_N332-32, respectively.
  • N332-GT2 residues Gly324, Asp325, Val326, Arg327, Met328, Ala329, His330, Ile415, Leu416 and Pro417 or the equivalent position in N332-GT5 colored red
  • HCDR3s are colored blue, orange, purple, green for BG18_GLo, RM_N332_03, RM_N332
  • FIG. 27 Affinity maturation of BG18 type I and other N332-GT5-elicited antibodies.
  • A SHM of Vn genes in BG18 type I and other Env + sequences from GC B cells.
  • B SHM of VH genes in BG18 type I and other Env + sequences from memory B cells.
  • C SHM of Vi. genes in BG18 type I and other Env + sequences from GC B cells.
  • D SHM of Vi. genes in BG18 type I and other Env + sequences from memory B cells.
  • each dot represents the median SHM for one animal, and the median of the medians +/- interquartile range for all animals is plotted.
  • the dotted line at 2 xl O' 5 M indicates no binding at the highest concentration tested.
  • the dotted line at 1.6x10 -11 M represents the approximate upper affinity limit of the instrument. Lines indicate the median with interquartile range. Each data point is representative of 1 to 4 technical replicates.
  • the dotted line at 2 xlO' 5 M indicates no binding at the highest concentration tested.
  • the dotted line at 1.6xl0’ n M represents the approximate upper affinity limit of the instrument.
  • FIG. 28 Identification of a broader class of BG18-like antibodies.
  • A HCDR3 length distribution of Env + GC B cells and epitope-specific memory B cells isolated at wk 12 after filtering out the BG18 type I sequences.
  • B SPR KDS of Fabs derived from activated B cell supernatants that were positive for binding to BG505 B23 by ELISA. Each data point represents 1 technical replicate.
  • the dotted line at 2 xl0‘ 5 M indicates no binding at the highest concentration tested.
  • the dotted line at 1.6xl0' n M represents the approximate upper affinity limit of the instrument.
  • C HCDR3 sequence and gene segment assignment of two Fabs that bind with high affinity to the BG505_B23 trimer for which cryo-EM structures were determined.
  • D Cryo-EM model of N332-GT2 in complex with BGI8 GL0 (PDB ID: 6DFH).
  • E Cryo-EM model of Fab RM_N332_07 in complex with N332-GT5.
  • F Cryo-EM model of Fab RM_N332_08 in complex with N332-GT5.
  • G Diagram showing definition of latitudinal, longitudinal, and HC-LC twist angles.
  • RM all 98 datasets of rhesus macaque BCR HC sequences from multiple studies (see methods); RM all IgM (not this study), IgM sequences from “RM all“ with the 4 data sets from this study removed; RM 5’ RACE IgMs, RM IgM sequences using 5 ’RACE and constant region primers; RM 5’UTR IgMs, RM IgM sequences using 5’UTR primers and an IgM constant region primer; RM SP IgMs, RM IgM sequences using signal peptide region primers and constant region primers; RM (this study), the 4 animals that were sequenced in this study.
  • C Difference in D gene usage frequency between humans and macaques shows a slightly higher frequency of D3-3 usage in the human repertoire compared to D3-41 usage in macaques.
  • D Comparison of frequencies between humans and macaque naive sequences across multiple sequence definitions show that NHPs have lower frequencies of BG18-like precursors at all levels.
  • E Criteria used to search for BG18 precursors in humans and RMs.
  • FIG. 30 ELISA response.
  • A Schematic of vaccination and time points of plasma collection. ELISA area under the curve showing plasma response at the indicated time points to
  • N332-GT5-binding B cells are gated as N332-GT5-BV650 + / N332-GT5-BV42P dual binders, and termed “N332-GT5 ++ ” or “Env ++ ” hereafter.
  • B Quantification of GC B cell kinetics. The dotted line separates post-prime and post-boost timepoints.
  • C Frequency of N332 epitopespecific (Env ++ K0‘) GC B cells among Env ++ GC B cells.
  • D Frequency of Env GC B cells as a percentage of total CD20 + B cells.
  • the gray area is set from 0.001% to the median frequency of Env ++ GC B cells observed in the pre-immunization samples.
  • E Frequency of N332 epitopespecific GC B cells as a percentage of total CD20 + B cells. The gray area is set from 0.0001% to calculated limit of detection.
  • F Quantification of Env ++ GC B cells per million lymphocytes recorded. They gray area is set from 1 to the median number of epitope-specific GC B cells observed in the pre-immunization samples.
  • G Number of N332 epitope-specific GC B cells per million lymphocytes recorded. The gray area is set from 1 to the median number of epitope-specific GC B cells observed in the pre-immunization samples. Each circle represents a 1 mb aliquot of FNA cells. Data from FNAs from both the left and right side are graphed individually for each animal. Lines indicate the median with interquartile range.
  • FIG. 32 Memory B cells sorting.
  • A Gating strategy for analysis of week 10 IgD' memory B cells.
  • B Antigen-specific (N332-GT5-AF647 + N332-GT5-BV421 + ) memory B cells (CD20 + IgD ) from Fig. 32A were backgated to assess surface immunoglobulin (Ig) expression by IgD versus IgG MFI signals. The gate revealed that most of the antigen-specific memory B cells expressed IgG, indicating successful identification of antigen-specific memory B cells.
  • FIG. 32A Representative flow plot of antigen-specific memory B cells (gated as in Figure 32A) from a preimmunization PBMC sample compared to the post-immunization timepoint (week 12) from the same animal, confirming specificity of N332-GT5 and N332-GT5-KO probe staining.
  • D Gating strategy for analysis of week 12 IgG + memory B cells.
  • E Frequencies of N332-GT5 ++ B cells among IgD' (week 10) or IgG + (week 12) memory B cells.
  • Figure 33 Schematic for sorting, B cell activation, functional screening and sequencing memory B cells.
  • A Overall strategy for sorting memory B cells, functional characterization and sequencing.
  • B ELISA OD405 for screening supernatants of activated memory B cells for binding to N332-GT5.
  • BG18 is a positive control mAb and Den3 is a negative control mAb.
  • FIG. 35 Frequency of Glu at the (D3-41)+2 position in macaque antibodies.
  • A Frequency of glutamate positioned 2 aa past the end of the D3-41 gene when D3-41 is present in the BG18 reading frame and position (+/- 1 aa) in antibodies with HCDR3s >22 aa compared to antibodies with HCDR3s ⁇ 22 aa and D3-41 positioned anywhere in sequences isolated from GC B cells.
  • B as in (A) but sequences were derived from memory B cells.
  • FIG. 36 Angles of approach for N332/V3 glycan antibodies.
  • A Latitudinal,
  • B longitudinal, and
  • C HC-LC rotation angle and
  • D 3D scatter plot for BG18 (6DFG), BG18_iGLo (6DFH), HMP1 (6NF5), HMP42 (6NFC), RM N332 03, RM N332 32, RM N332 36, RM_N332_07, RM_N332_08 in yellow circles and six other N332-dependent bnAbs: PGT122 (4TVP), DH270.6 (6UM6), BF520.1 (6MN7), PGT128 (5ACO), QA013.2 (7N65), PGT135 (4JM2) in cyan circles.
  • FIG. 37 Epitope footprints of BG18-like antibodies.
  • the epitope footprint for nine BG18-like antibodies (defined as atoms within 5 A of the Fab).
  • the yellow oval encompasses the footprint of BGI8 GL0 and is mapped onto the other eight structures for comparison.
  • gp!20 is colored gray.
  • the five structures from this study are RM_N332_36, RM_N332_03, RM_N332_32, RM N332 07 and RM N332 08.
  • the four structures from are BG18 GL0 (PDB ID: 6DFH), BG18 Mat (PDB ID: 6DFG), HMP1 (PDB ID: 6NF5) and HMP42 (PDB ID: 6NFC).
  • FIG. 38 Other BG18-like antibodies.
  • A Cryo-EM structure of N332-GT2 in complex with BG18 GL0 (PDB ID: 6DFH). Inset shows the interactions of the HCDR3 to the conserved residues in gp!20.
  • B Cryo-EM structure of Fab RM_N332_07 in complex with N332- GT5. Inset shows HCDR3 interactions to gpl20.
  • C Cryo-EM structure of Fab RM N332 08 in complex with N332-GT5. Inset shows HCDR3 interactions to gpl20.
  • Figure 40 Site-specific glycan compositions of BG505 MD65 B23.
  • Oligomannose-type glycans are colored green, hybridtype in hashed pink, complex-type in pink, and the proportion of unoccupied PNGS in grey.
  • the table represents the grouping of the bar graphs, with any glycan composition containing at least one fucose or sialic acid (NeuAc) shown.
  • FIG 43 Structure of immunogenic residues in unliganded N332-GT5.
  • the BG18 epitope on unliganded N332-GT5 is shown with solvent exposed VI loop aa side chains (K137 and R139) that are critical for binding to non-BG18-like competitor antibodies indicated.
  • the three mutations in the B23 trimer relative to N332-GT5 are A135T, K137N and R139T.
  • Figure 44 No indication of a lambda3-based BG18 type II response in rhesus macaques. HCDR3 length distribution of antibodies that use VL3 light chains from all epitopespecific GC B cells combined with and without BG18 type I antibodies removed.
  • FIG. 45 ELISA binding to activated B cell supernatants from N332-GT5+ B cells isolated 2 weeks post boost. Each symbol represents the ELISA signal from one well and they are ordered based on the HCDR3 length of the BCR sequence identified in each well. BG18 type II antibodies would be found in wells that contained BCRs with HCDR3s > 20 aa (shaded gray). The plot on top shows ELISA reactivity to N332-GT5 and N332-GT5-KO. The bottom plot shows ELISA reactivity to the B23 trimer. The BG18 type I antibodies are indicated on the plots [0067] Figure 46. Amino acid sequence alignment of trimers used in this study.
  • BG505_MD65_congly_N332-GT5 The immunogen used in this study, referred to as N332-GT5, is labelled BG505_MD65_congly_N332-GT5 in the sequence alignment. Congly indicates glycosylation sites at positions 241 and 289 were introduced.
  • Figure 47 Affinity of boostl candidates for NHP-elicited BG18-class antibodies isolated post-N332-GT5 prime measured by SPR.
  • FIG. 48 A) NHPs (groups of 6) were primed with N332-GT5+SMNP escalating dose and boosted at week 10 with 1 of 4 heterologous trimers. At week 44 animals were boosted with either N332-GT5 (control group 1) or SF162P3_MD64_B20.1 (groups 2-4). PBMC blood draws were taken at the indicated time points (red circles). B) Memory B cells were sorted and BCR sequenced and BG18 type I sequences were quantified. C) Flow cytometry data indicates whether each BG18 sequence came from a B cell that was stained positive for the boost 1 immunogen or the boost 2 immunogen as indicated on the graph.
  • Figure 49 Affinity of NHP-elicited BG18-class antibodies isolated 2 weeks post boost 1 (week 12) for the boost 1 and boost 2 candidates measured by SPR.
  • the DU156 MD39 is a stabilized “wild type“ trimer.
  • FIG. 50 A) 6 NHPs were immunized with N332-GT6 mRNA-LNPs, boosted at week 8 with Bl l mRNA-LNPs and boosted again at week 16 with SF162P3-B20 mRNA-LNPs. B) Affinity of NHP elicited BG18-class Abs isolated 2 weeks post boost 1 (week 10) for prime and boost immunogens, and other HIV trimers measured by SPR.
  • FIG. 10E8-GT immunogens bind diverse 10E8-class precursors.
  • A SPR- measured monovalent KD values for the scaffold without germline-targeting mutations (MPER) and various 10E8-GT scaffolds (10E8-GT9.2 to 10E8-GT12) binding to mature 10E8, GL-reverted 10E8 (10E8-iGL3), the proposed 10E8 UCA and multiple NGS-derived 10E8-class human precursor heavy chains paired with the GL-reverted 10E8 light chain (NGS). Each symbol represents a different antibody.
  • LOD limit of detection.
  • NB no binding.
  • FIG. 10E8-GT immunogens mimic the interaction of 10E8 with the MPER. Structures, from left to right, of 10E8 bnAb bound to MPER peptide (Huang et al, 2012), 10E8 bnAb bound to T117v2 scaffold (Irimia et al., 2017), 10E8-iGLl bound to 10E8-GT4 scaffold, 10E8 bnAb bound to 10E8-GT10.2 scaffold, NGS precursor 10E8-NGS-03 bound to 10E8- GT10.2 scaffold, and 10E8-iGLl bound tolOE8-GTl 1 scaffold, in which the previously published MPER peptide and T117v2 complexes with 10E8 are shown for comparison.
  • FIG. 10E8-GT scaffolds engage 10E8-class HCDR3s in human blood.
  • A Representative flow cytometry staining of 10E8-GT12 double-positive (10E8-GT12 ++ , signifying binding to two probes with different fluorochromes, left) and epitope-specific 10E8-GT12 ++ 10E8- GT12'KO'(right) naive B cells (CD20 + CD27 gD + IgG‘) from HIV-seronegative donors.
  • NGS datasets (n 14) of heavy chains from HIV-seronegative humans served as unsorted controls where indicated (Briney et al., 2019; Steichen et al., 2019).
  • Exact HCDR3 length for the 10E8 bnAb is indicated by a tick mark at 22 aa.
  • FIG. 10E8-class B cells function in vivo.
  • Figure 56 inRNA-LNP delivery of 10E8-GT12 nanoparticles primes diverse 10E8-class B cells.
  • A Percentage of 10E8-GT10.1 ++ 10E8-GT10.1-K0‘ (epitope specific) CD19 + IgD + naive B cells with 10E8-class HCDR3s for humans and IID3-3/JH6 mice.
  • FIG. 57 10E8-GT immunogens induce 10E8-class responses in non-human primates.
  • A Alignment of known rhesus macaque homologues of the human DH3-3 gene. Differences within the critical YxFW binding motif (orange) are highlighted in red.
  • Top structures shown as cartoon diagrams, aligned on the MPER, with antibody heavy chain in white or yellow, light chain in grey and the scaffold in blue. Antibody constant regions are omitted for clarity.
  • Bottom interaction between the HCDR3 YxFW motif (sticks) and the engineered Dn-gene binding pocket on the scaffold.
  • FIG. 58 10E8-class BCRs induced by 10E8-GT nanoparticles bind epitopescaffold 10E8-B1 containing a near-native 10E8 peptide epitope.
  • C SPR-measured monovalent KD values for 10E8-B 1 binding to inferred-germline antibodies (iGL) or antibodies recovered after immunization of hD3-3/Ju6 mice or macaques with 10E8-GT nanoparticles (post-prime). Each symbol represents a different antibody.
  • FIG. 59 Design and properties of immunogens.
  • A Overview of 10E8-class and LN01 -class antibody categories. HCDR3 motifs are shown as regular expressions that were used to query the database. If multiple amino acids were allowed at the same position, they are shown in square brackets; positions in which all amino acids were allowed are indicated as
  • B Schematic of the development of MPER-GT scaffolds.
  • C Schematic overview of nanoparticle formation by genetic fusion of the immunogen (T2983-GT) to each terminus of the 3- dehydroquinase nanoparticle from Thermus thermophilus (NP) via flexible linkers containing exogeneous T-help peptides derived from Aquifex aeolicus lumazine synthase.
  • the epitope scaffold is shown in light blue, the MPER graft in purple, the linker in green, the nanoparticle in red and glycans in dark blue.
  • D SEC-MALS traces of 10E8-GT NPs. Normalized UV280 absorptions are shown as dotted lines and protein molecular weights of main peaks are shown as solid lines.
  • FIG. 60 Glycosylation sites on 10E8-GT nanoparticles vary in occupancy. Sitespecific glycan analysis was measured using the single site glycan profiling (SSGP) and DeGlyPHER methods. Positions of N-linked glycosylation sites are indicated as relative positions within the epitope-scaffold (ES) or the nanoparticle (NP, indicated with a box). The 24mer contains two independent copies of the scaffold, which cannot be distinguished by either method and therefore averaged values are shown.
  • SSGP single site glycan profiling
  • NP nanoparticle
  • FIG. 61 Data collection, refinement and validation.
  • B Summary statistics of data collection, refinement and validation of the cryo- EM reconstruction of 10E8-GT10.2 in complex with 10E8 and W6-10 Fabs.
  • C Fourier Shell Correlation, (D) angular sampling and (E) map colored according to local resolution (units Angstrom) of the cryo-EM reconstruction of 10E8-GT10.2 in complex with 10E8 and W6-10 Fabs.
  • FIG. 53 Ex vivo evaluation of 10E8-GT scaffolds of Fig. 53
  • A Representative gating scheme.
  • B Enrichment of HCDR3 lengths among epitope-specific B cells over unsorted controls as in Fig. 53d.
  • (F) Percentage of TGVL3 family light chains among 10E8-GT12-sorted BCRs that are either 10E8-class (10E8-class H3) or lack the YxFW motif (non-YxFW). n 6 independent donors, ns not significant, two-sided Wilcoxon test.
  • Figure 63 Poly- and Auto-reactivity of 10E8-class precursors.
  • A Polyspecificity reagent binding as measured by ELISA.
  • PGT121, VRC01 and PGT128 served as negative controls, MPER bnAb 4E10 as a positive control.
  • NGS-1 through -22 correspond to human NGS precursors described in the main text.
  • HC 10E8-class heavy chain
  • NGS next-generation sequencing
  • LC 10E8 light chain
  • B Mean fluorescence intensity (MFI) of antibodies as in (A) in a HEp-2 cell autoreactivity assay.
  • C raw images of data shown in (B).
  • FIG. 64 Immunization of MPER-HuGL18 H B cell adoptive transfer recipient mice with 10E8-GT10.2 timers.
  • B-cell progenitors (B220 + ) were divided into immature (CD43 + ) and mature (CD43‘) cells. Early (CD43 + ) B-cell progenitors were subdivided into Hardy populations A (CD24 BP-1 ), B (CD24 + BP-L), and C (CD24 + BP1 + ).
  • Figure 65 Generation and characterization of 1ID3-3/JH6 mice.
  • A Illustration of genetic modifications in hD3-3/Jn6 mice (not drawn to scale) with hD3-3 and hJn6 segments replacing mouse DQ52 and JH1-4 segments. Sequences of hD3-3, JH6, and flanking regions are shown below the diagram.
  • B Characterization of B220 + B cell and CD3 + T cell populations among lymphocyte/live cell/single cells from homozygous D3-3/J6 mouse spleens by flow cytometry compared to a wild-type 129SVE mouse (WT).
  • C Characterization of IgM + IgD hl naive B cells among B cells as in (B).
  • Figure 66 Immunogenicity of 10E8-GT nanoparticles in hD3-3/Ju6 mice.
  • A Representative gating strategy of splenic B cells sorted for sequencing of BCRs from hD3-3/Jn6 mice six weeks after immunization with 10E8-GT12 24mer.
  • (D) Enrichment ratio for HCDR3 amino acid (aa) length distribution for epitope-specifi c ( 10E8-GT 10.1 ++ 10E8-GT 10.1 -KO’ or 10E8-GT 12 ++ 10E8-GT 12-KO ) IgG + B cells from animals immunized with the indicated 10E8-GT immunogens, relative to HCDR3 amino acid length distribution for epitope-specific (10E8-GT9-KO++10E8-GT9-) IgG + B cells from animals immunized with 10E8-GT9-KO 12mer as in b.
  • HCDR3 lengths longer than 22 were only found in the 10E8-GT-immunized groups, precluding calculation of enrichment scores for longer HCDR3s.
  • F Percentage of HCDR3s containing the YxFW motif among epitope-specific IgG + B cells as in (C). *p ⁇ 0.05, Kruskal-Wallis test with Dunn’s multiple comparison correction.
  • P value control vs. GT10 12mer
  • p value control vs. GT12 24mer
  • H Percentage of epitope-specific (10E8-GT9-K0 ++ 10E8-GT9-,10E8- GT10.1 ++ 10E8-GT10.1-K0; or 10E8-GT12 ++ 10E8-GT12-K0’) with 10E8-class or LNOl-class HCDR3s among IgG + BCRs from day 42 after immunization of hD3-3/Jn6 as in (B). Symbols represent individual animals; bars indicate median values.
  • FIG. 67 Gating strategy used to assess immune responses to 10E8-GT immunogens in NHPs.
  • A Gating scheme for 10E8-GT10.2-specific (10E8-GT10.2 ++ ) and 10E8- GT10.2 epitope-specific ( 10E8-GT 10.2 I 0E8-GT 10.2-KO-) naive B cells.
  • B Gating scheme for 10E8-GT10.2-specific and 10E8-GT10.2 epitope-specific GC B cells.
  • C Gating scheme for 10E8-GT10.2-specific and 10E8-GT10.2 epitope-specific PBMC-memory B cells.
  • FIG. 68 Immunogenicity of 10E8-GT NPs in rhesus macaques.
  • A Percentage of GC B cells (CD38’CD71 + ) among all B cells (CD3 CD20 + ) in fine needle aspirate samples of rhesus macaques 3 or 10 weeks post immunization with 10E8-GT10.2 12mer, as described in Fig. 55
  • C Median percent amino acid (aa) mutations in the VH of epitope-specific (10E8-GT10.2 ++ 10E8-GT10.2-K0- or 10E8- GT12 ++ 10E8-GT12-KO ) BCRs sorted from macaques immunized with 10E8-GT10.2 12mer or 10E8-GT12 12mer at the indicated time-points from GC B cells (CD38'CD7 I ) or PBMC-memory B cells (CD20 + IgD ) among BCRs with 10E8-class HCDR3s (10E8-class) compared to BCRs lacking the YxFW motif (Competitor). Symbols represent individual animals, bars indicate median values.
  • E SPR-measured monovalent KD values for 10E8-GT10.2 binding to selected mutated or unmutated antibodies with 10E8-class HCDR3s (10E8-class) or lacking the YxFW motif (Competitor) induced by 10E8-GT10.2 12mer isolated from PBMC-memory (CD20 + IgD‘).
  • dotted lines indicate median frequencies of VRC01 -class precursors 8 weeks after high dose (HD) or low dose (LD) immunization with eOD-GT8 60mer in the IAVI G001 human phase 1 clinical trial (Jardine et al. 2013), with the caveat that frequencies in G001 were measured among IgG + B cells rather than IgD’ B cells.
  • Open symbols indicate macaques lacking a permissive DH3-41 allele. Lines indicate median values.
  • Figure 69 Antigenicity and immunogenicity of epitope-scaffold 10E8-B1 containing a near-native 10E8 peptide epitope.
  • A SPR-measured monovalent KD values for 10E8-B1 binding to different antibodies containing the indicated number of 10E8-class mutations, including the 10E8 UCA, 10E8-class human naive precursors isolated by human B cell sorting (human naive), partially mature 10E8-class antibodies (intermediates), and mature 10E8. Overlapping data are staggered along the x-axis.
  • FIG. 52 (B) Crystal structure of a 10E8-B1 in complex with 10E8 (right), with structure of 10E8 bound to peptide (left) shown for reference. Colors are as in Fig. 52 (C) SPR-measured monovalent KD values for 10E8-B1 binding to antibodies recovered after immunization of IID3-3/JH6 mice or NHPs with 10E8-GT NPs (post-prime) or inferred germline antibodies (iGL). Each symbol represents a different antibody.
  • LOD Limit of detection. **p ⁇ 0.01, ns: not significant, Kruskal-Wallis test with Dunn’s multiple comparison correction.
  • E-F Cells binding epitope-specifically to priming or boosting immunogen (10E8-GT12 + 10E8-GT12-K0‘ and/or 10E8-B1 + 10E8-B1-KO’) were sorted and their BCRs were sequenced. Symbols indicate different animals.
  • E Frequency of epitope-specific BCRs with 10E8-class HCDR3s among memory B cells.
  • FIG. 71 Timeline for an eOD-GT8 60mer priming, core-g28v2 boosting (Boost#l) at week 5, N276(-) membrane-bound trimers (Boost#2) at week 12 and WT boost (Boost#3) at week 18.
  • Humanized Vnl-2/VKl-33 hTdT mice were initially primed with eOD-GT8 60mer delivered by mRNA/LNPs and boosted with core-g28v2 60mer mRNA/LNP.
  • FIG. 72 Frequencies of antigen specific MBCs after boosting with N276(-) membrane-bound trimers.
  • the frequencies of VRCOl-class memory B cells were determined by antigen-specific cell sorting and single cell lOx Genomics VDJ sequencing conducted six weeks after boosting. A subset of mice was boosted again at week 18 with corresponding wildtype membrane-bound trimers delivered by mRNA/LNPs matched to those received at week 12.
  • FIG. 73 Frequencies of VRCOl-class MBCs after boosting with N276(-) membrane-bound trimers. Boosting with stabilized membrane-bound HIV trimers lacking the N276 glycan at week 12 elicited VRCOl-class memory B cells.
  • Figure 74 Frequencies of antigen specific MBCs after boosting with WT membrane-bound trimers. Subsequent boosting with wildtype membrane-bound HIV Env trimers elicited VRCOl-class memory B cells that bind wildtype trimers. [0096] Figure 75. Sequential vaccination induces VRCOl-class B cells that bind WT trimers. Subsequent boosting with wildtype membrane-bound HIV Env trimers elicited VRCOl - class memory B cells that bind wildtype trimers.
  • FIG. 76 Post 001428-WT Boost3 VRCOl-class mAbs neutralize autologous WT virus.
  • VRCOl-class memory B cells that bind wildtype trimers elicited via boosting with wildtype membrane-bound HIV Env trimers have BCRs capable of neutralizing the autologous wildtype HIV pseudovirus and heterologous pseudoviruses lacking the N276 glycan when expressed as recombinant IgGs.
  • the invention relates to improved HIV antigens, including germline-targeting designs, trimer stabilization designs, combinations of those two, trimers designed with modified surfaces helpful for immunization regimens, other types of trimer modifications (see, for example, examples of trimers with combined germline-targeting mutations and stabilization mutations and additional trimer modifications that add functionality and that can be combined with other types of modifications as described herein) and on development of trimer nanoparticles and membranebound trimers.
  • the invention also encompasses combinations of any of the herein described modifications, such as but not limited to, combinations of stabilization and modified surfaces with nanoparticles or membrane-bound trimers.
  • the invention further relates to mRNAs encoding improved HIV antigens, including germline-targeting designs, trimer stabilization designs, combinations of those two, trimers designed with modified surfaces helpful for immunization regimens, other types of trimer modifications (see, for example, examples of trimers with combined germline-targeting mutations and stabilization mutations and additional trimer modifications that add functionality and that can be combined with other types of modifications as described herein) and on development of trimer nanoparticles and membrane-bound trimers.
  • the HIV envelope protein trimer is the target of broadly neutralizing antibodies (bNAbs).
  • the high mannose patch including the N332-linked glycan at the base of the V3 loop of gpl20, is frequently targeted by bnAbs during natural infection and hence is an appealing vaccine epitope.
  • Germline targeting has potential to initiate the elicitation of N332-dependent bnAbs by vaccination, but no immunogen has been reported to bind germline-reverted precursors of N332-dependent bnAbs.
  • VRCOl-class antibodies are defined as those with a Vnl-2 gene in the heavy chain and a five amino acid CDR3 in the light chain.
  • the VH1-2 mouse employed here was originally developed by Ming Tian in the Fred Alt lab at Harvard and was first reported in Tian et al. Cell 2016. It is a stringent model system for inducing VRCOl-class responses, in which Applicants have measured a VRCOl-class precursor frequency of approximately 1 in 1 million naive B cells, which is similar to the frequency measured in humans as reported in Jardine et al. Science 2016 and Havenar-Daughton et al Science Translational Medicine 2018. eOD-GT8 60mer and derivatives are the only immunogens reported to be capable of priming VRCOl-class responses in this model (Tian et al. Cell 2016; Duan et al Immunity 2018).
  • leader sequence (MGILPSPGMPALLSLVSLLSVLLMGCVAETG) (SEQ ID NO: 1) that is cleaved during expression/secretion and is not present in the final expressed protein product.
  • SEQ ID NO: 1 MGILPSPGMPALLSLVSLLSVLLMGCVAETG
  • the embodiments contained herein are not limited to this particular leader sequence as different leader sequences could be used to serve the same purpose.
  • the invention also encompasses a protein having at least 90% homology or identity with the sequence of the protein of any one of the trimers disclosed herein.
  • the invention also encompasses a protein having at least 95% homology or identity with the sequence of the protein of any one of trimers disclosed herein.
  • the invention also encompasses any nucleic acid encoding the protein of any one of the immunogens disclosed herein.
  • the invention also encompasses a nucleic acid having at least 90% or 95% homology or identity with the sequence of said nucleic acid.
  • the invention also encompasses a mRNA encoding an immunogen.
  • the invention also encompasses an mRNA that encodes a protein having at least 90% or 95% homology or identity with the sequence of said protein.
  • the invention also encompasses eliciting an immune response which may comprise systemically administering to an animal in need thereof an effective amount of any one of the non- naturally occurring protein(s) or any one of the nucleic acids encoding the non-naturally occurring protein(s) of the present invention, including nucleic acids that may have at least 90% or 95% homology or identity with a nucleotide encoding the sequence of the non-naturally occurring protein(s) of the invention.
  • the nucleic acid may be a RNA, advantageously a mRNA.
  • the nucleic acid is formulated in lipid nanoparticles (LNPs).
  • the animal may be a mammal, advantageously a human.
  • the invention also encompasses eliciting an immune response which may comprise systemically administering to an animal in need thereof an effective amount of any one of the mRNAs encoding the non-naturally occurring protein(s) of the present invention, including mRNAs that may have at least 90% or 95% homology or identity thereto.
  • the mRNA is formulated in lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • the invention pertains to the identification, design, synthesis and isolation of mutant trimers disclosed herein as well as nucleic acids encoding the same.
  • the present invention also relates to homologues, derivatives and variants of the sequences of the mutant trimers and nucleic acids encoding the same, wherein it is preferred that the homologue, derivative or variant have at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 97%, at least 98% or at least 99% homology or identity with the sequence of the mutant trimers and nucleic acids encoding the same. It is noted that within this specification, homology to sequences of the mutant proteins and nucleic acids encoding the same refers to the homology of the homologue, derivative or variant to the binding site of the mutant proteins and nucleic acids encoding the same.
  • the invention still further relates to nucleic acid sequences expressing the mutant immunogens disclosed herein, or homologues, variants or derivatives thereof.
  • nucleic acid sequences expressing the mutant immunogens disclosed herein, or homologues, variants or derivatives thereof One of skill in the art will know, recognize and understand techniques used to create such. Additionally, one of skill in the art will be able to incorporate such a nucleic acid sequence into an appropriate vector, allowing for production of the amino acid sequence of mutant proteins and nucleic acids encoding the same or a homologue, variant or derivative thereof.
  • the invention still further relates to mRNA sequences expressing the mutant immunogens disclosed herein, or homologues, variants or derivatives thereof.
  • mRNA sequences expressing the mutant immunogens disclosed herein, or homologues, variants or derivatives thereof.
  • One of skill in the art will know, recognize and understand techniques used to create such mRNAs.
  • the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:
  • isolated or “non-naturally occurring” is used herein to indicate that the isolated moiety (e.g. peptide or compound) exists in a physical milieu distinct from that in which it occurs in nature.
  • the isolated peptide may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs.
  • the absolute level of purity is not critical, and those skilled in the art may readily determine appropriate levels of purity according to the use to which the peptide is to be put.
  • isolated when used a step in a process is to be interpreted accordingly.
  • the isolated moiety will form part of a composition (for example a more or less crude extract containing many other molecules and substances), buffer system, matrix or excipient, which may for example contain other components (including proteins, such as albumin).
  • a composition for example a more or less crude extract containing many other molecules and substances
  • buffer system for example a more or less crude extract containing many other molecules and substances
  • matrix or excipient which may for example contain other components (including proteins, such as albumin).
  • the isolated moiety may be purified to essential homogeneity, for example as determined by PAGE or column chromatography (for example HPLC or mass spectrometry).
  • the isolated peptide or nucleic acid of the invention is essentially the sole peptide or nucleic acid in a given composition.
  • the isolated mRNA of the invention is essentially the sole mRNA in a given composition.
  • a tag may be utilized for purification or biotinylation.
  • the tag for purification may be a his tag.
  • the tag for biotinylation may be an avi-tag.
  • Other tags are contemplated for purification, however, purification may be accomplished without a tag.
  • antibody such as, not limited to, a broadly neutralizing antibody
  • affinity columns are contemplated.
  • lectin columns are contemplated.
  • Native-like soluble trimers can be made by several methods that all involve stabilizing associations between envelope protein subunits. See, e.g., Steichen et al., Immunity. 2016 Sep 20;45(3):483-496. doi: 10.1016/j.immuni.2016.08.016. Epub 2016 Sep 8.PMID: 27617678, Kulp et al . , Nat Commun. 2017 Nov 21 ;8(1): 1655. doi : 10.1038/s41467-017-01549-6. PMID : 29162799 and R.W. Sanders et al., “HIV-1 neutralizing antibodies induced by native-like envelope trimers,” Science, doi: 10.1126/science.aac4223, 2015. [00118] The proteins and compounds of the invention need not be isolated in the sense defined above, however.
  • composition is used herein to define a solid or liquid composition in a form, concentration and level of purity suitable for administration to a patient (e.g. a human patient) upon which administration it may elicit the desired physiological changes.
  • patient e.g. a human patient
  • immunological composition cover any composition that elicits an immune response against the targeted pathogen, HIV.
  • an immunogenic or immunological composition covers any composition that induces a protective immune response against the targeted pathogen or which efficaciously protects against the pathogen; for instance, after administration or injection, elicits a protective immune response against the targeted pathogen or provides efficacious protection against the pathogen. Accordingly, an immunogenic or immunological composition induces an immune response, which may, but need not, be a protective immune response.
  • An immunogenic or immunological composition may be used in the treatment of individuals infected with the pathogen, e.g., to stimulate an immune response against the pathogen, such as by stimulating antibodies against the pathogen.
  • an immunogenic or immunological composition may be a pharmaceutical composition.
  • an immunogen may be an antigen or an epitope of an antigen.
  • a diagnostic composition is a composition containing a compound or antibody, e.g., a labeled compound or antibody, that is used for detecting the presence in a sample, such as a biological sample, e.g., blood, semen, vaginal fluid, etc., of an antibody that binds to the compound or an immunogen, antigen or epitope that binds to the antibody; for instance, an anti-HIV antibody or an HIV immunogen, antigen or epitope.
  • a ‘conservative amino acid change’ is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. lysine, arginine and histidine), acidic side chains (e.g. aspartic acid and glutamic acid), non-charged amino acids or polar side chains (e g. glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine), non-polar side chains (e.g.
  • protein protein
  • peptide polypeptide
  • amino acid sequence amino acid sequence
  • 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 or bioactive component.
  • (a) Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule may be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab fragment of an antibody molecule
  • the fragment of an antibody molecule may be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab’ fragments are obtained per antibody molecule;
  • F(ab’)2 the fragment of the antibody that may be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab’)2 is a dimer of two Fab’ fragments held together by two disulfide bonds;
  • scFv including a genetically engineered fragment containing the variable region of a heavy and a light chain as a fused single chain molecule.
  • General methods of making these fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), which is incorporated herein by reference).
  • Fabs, Fv and scFV may also be made recombinantly, i.e. expressed as Fab, Fv or scFV rather than cleaving an intact IgG.
  • a “neutralizing antibody“ may inhibit the entry of HIV-1 virus for example SF162 and/or JR-CSF with a neutralization index >1.5 or >2.0. Broad and potent neutralizing antibodies may neutralize greater than about 50% of HIV-1 viruses (from diverse clades and different strains within a clade) in a neutralization assay. The inhibitory concentration of the monoclonal antibody may be less than about 25 mg/ml to neutralize about 50% of the input virus in the neutralization assay.
  • An “isolated antibody“ or “non-naturally occurring antibody“ is one that has been separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody is purified: (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody ’ s natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies which may comprise the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
  • the modifier “monoclonal“ is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).
  • the “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example.
  • proteins of the invention may differ from the exact sequences illustrated and described herein.
  • the invention contemplates deletions, additions and substitutions to the sequences shown, so long as the sequences function in accordance with the methods of the invention.
  • particularly preferred substitutions will generally be conservative in nature, i.e., those substitutions that take place within a family of amino acids.
  • amino acids are generally divided into four families: (1) acidic— aspartate and glutamate; (2) basic— lysine, arginine, histidine; (3) non-polar— alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar— glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • nucleotide sequences and “nucleic acid sequences” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, including, without limitation, messenger RNA (mRNA), DNA/RNA hybrids, or synthetic nucleic acids.
  • the nucleic acid may be single-stranded, or partially or completely double-stranded (duplex).
  • Duplex nucleic acids may be homoduplex or heteroduplex.
  • transgene“ may used to refer to “recombinant“ nucleotide sequences that may be derived from any of the nucleotide sequences encoding the proteins of the present invention.
  • the term “recombinant“ means a nucleotide sequence that has been manipulated “by man“ and which does not occur in nature, or is linked to another nucleotide sequence or found in a different arrangement in nature. It is understood that manipulated “by man“ means manipulated by some artificial means, including by use of machines, codon optimization, restriction enzymes, etc.
  • nucleotide sequences may be mutated such that the activity of the encoded proteins in vivo is abrogated.
  • nucleotide sequences may be codon optimized, for example the codons may be optimized for human use.
  • nucleotide sequences of the invention are both mutated to abrogate the normal in vivo function of the encoded proteins, and codon optimized for human use.
  • each of the sequences of the invention, such as the mutant trimers may be altered in these ways.
  • the nucleic acid molecules of the invention have a nucleotide sequence that encodes the antigens of the invention and may be designed to employ codons that are used in the genes of the subject in which the antigen is to be produced.
  • codons that are used in the genes of the subject in which the antigen is to be produced.
  • Many viruses including HIV and other lentiviruses, use a large number of rare codons and, by altering these codons to correspond to codons commonly used in the desired subject, enhanced expression of the antigens may be achieved.
  • the codons used are “humanized" codons, i.e., the codons are those that appear frequently in highly expressed human genes (Andre et al., J. Virol.
  • codon usage provides for efficient expression of the transgenic HIV proteins in human cells. Any suitable method of codon optimization may be used. Such methods, and the selection of such methods, are well known to those of skill in the art. In addition, there are several companies that will optimize codons of sequences, such as Geneart (geneart.com). Thus, the nucleotide sequences of the invention may readily be codon optimized.
  • the invention further encompasses nucleotide sequences encoding functionally and/or antigenically equivalent variants and derivatives of the antigens of the invention and functionally equivalent fragments thereof.
  • These functionally equivalent variants, derivatives, and fragments display the ability to retain antigenic activity. For instance, changes in a DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitution of amino acid residues by amino acid analogs are those which will not significantly affect properties of the encoded polypeptide.
  • Conservative amino acid substitutions are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tyrosine/tryptophan.
  • the variants have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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% or at least 99% homology or identity to the antigen, epitope, immunogen, peptide or polypeptide of interest.
  • sequence identity or homology is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • sequence identity may be determined using any of a number of mathematical algorithms.
  • a nonlimiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993;90: 5873-5877.
  • Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS 1988;4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 may be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.
  • WU-BLAST Woodington University BLAST
  • WU-BLAST version 2.0 executable programs for several UNIX platforms may be downloaded from ftp://blast.wustl.edu/blast/executables.
  • NCBLBLAST version 1.4 This program is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBLBLAST version 1.4 (Altschul & Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al., Journal of Molecular Biology 1990; 215: 403-410; Gish & States, 1993;Nature Genetics 3: 266-272; Karlin & Altschul, 1993;Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated by reference herein).
  • the nucleotide sequences of the present invention may be inserted into “vectors.
  • the term “vector” is widely used and understood by those of skill in the art, and as used herein the term “vector” is used consistent with its meaning to those of skill in the art.
  • the term “vector“ is commonly used by those skilled in the art to refer to a vehicle that allows or facilitates the transfer of nucleic acid molecules from one environment to another or that allows or facilitates the manipulation of a nucleic acid molecule.
  • any vector that allows expression of the proteins of the present invention may be used in accordance with the present invention.
  • the proteins of the present invention may be used in vitro (such as using cell-free expression systems) and/or in cultured cells grown in vitro in order to produce the encoded HIV- proteins, which may then be used for various applications such as in the production of proteinaceous vaccines.
  • any vector that allows expression of the proteins in vitro and/or in cultured cells may be used.
  • any vector that allows for the expression of the proteins of the present invention and is safe for use in vivo may be used.
  • the vectors used are safe for use in humans, mammals and/or laboratory animals.
  • the protein coding sequence should be “operably linked” to regulatory or nucleic acid control sequences that direct transcription and translation of the protein.
  • a coding sequence and a nucleic acid control sequence or promoter are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the nucleic acid control sequence.
  • the “nucleic acid control sequence” may be any nucleic acid element, such as, but not limited to promoters, enhancers, IRES, introns, and other elements described herein that direct the expression of a nucleic acid sequence or coding sequence that is operably linked thereto.
  • promoter will be used herein to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II and that when operationally linked to the protein coding sequences of the invention lead to the expression of the encoded protein.
  • the expression of the transgenes of the present invention may be under the control of a constitutive promoter or of an inducible promoter, which initiates transcription only when exposed to some particular external stimulus, such as, without limitation, antibiotics such as tetracycline, hormones such as ecdysone, or heavy metals.
  • the promoter may also be specific to a particular cell-type, tissue or organ.
  • suitable promoters and enhancers are known in the art, and any such suitable promoter or enhancer may be used for expression of the transgenes of the invention.
  • suitable promoters and/or enhancers may be selected from the Eukaryotic Promoter Database (EPDB).
  • EPDB Eukaryotic Promoter Database
  • the vectors used in accordance with the present invention should typically be chosen such that they contain a suitable gene regulatory region, such as a promoter or enhancer, such that the proteins of the invention may be expressed.
  • Any suitable vector may be used depending on the application.
  • plasmids viral vectors, bacterial vectors, protozoal vectors, insect vectors, baculovirus expression vectors, yeast vectors, mammalian cell vectors, and the like, may be used.
  • Suitable vectors may be selected by the skilled artisan taking into consideration the characteristics of the vector and the requirements for expressing the proteins under the identified circumstances.
  • IgGl and Fab expression vectors may be utilized to reconstitute heavy and light chain constant regions if heavy and light chain genes of the proteins of the present invention are cloned.
  • expression vectors that are suitable for expression on that subject, and that are safe for use in vivo, should be chosen.
  • any vectors that are suitable for such uses may be employed, and it is well within the capabilities of the skilled artisan to select a suitable vector.
  • the vectors used for these in vivo applications are attenuated to vector from amplifying in the subject.
  • plasmid vectors preferably they will lack an origin of replication that functions in the subject so as to enhance safety for in vivo use in the subject.
  • viral vectors preferably they are attenuated or replication-defective in the subject, again, so as to enhance safety for in vivo use in the subject.
  • viral vectors are used.
  • Viral expression vectors are well known to those skilled in the art and include, for example, viruses such as adenoviruses, adeno-associated viruses (AAV), alphaviruses, herpesviruses, retroviruses and poxviruses, including avipox viruses, attenuated poxviruses, vaccinia viruses, and particularly, the modified vaccinia Ankara virus (MVA; ATCC Accession No. VR-1566).
  • viruses when used as expression vectors are innately non-pathogenic in the selected subjects such as humans or have been modified to render them non-pathogenic in the selected subjects.
  • replicationdefective adenoviruses and alphaviruses are well known and may be used as gene delivery vectors.
  • the nucleotide sequences and vectors of the invention may be delivered to cells, for example if the aim is to express the HIV-1 antigens in cells in order to produce and isolate the expressed proteins, such as from cells grown in culture.
  • any suitable transfection, transformation, or gene delivery methods may be used. Such methods are well known by those skilled in the art, and one of skill in the art would readily be able to select a suitable method depending on the nature of the nucleotide sequences, vectors, and cell types used.
  • transfection, transformation, microinjection, infection, electroporation, lipofection, or liposome-mediated delivery could be used.
  • Expression of the proteins may be carried out in any suitable type of host cells, such as bacterial cells, yeast, insect cells, and mammalian cells.
  • the proteins of the invention may also be expressed using including in vitro transcription/translation systems. All of such methods are well known by those skilled in the art, and one of skill in the art would readily be able to select a suitable method depending on the nature of the nucleotide sequences, vectors, and cell types used.
  • a synthetic mutant trimer may be chemically synthesized in whole or part using techniques that are well-known in the art (see, e g., Kochendoerfer, G. G., 2001). Additionally, homologs and derivatives of the polypeptide may be also be synthesized.
  • expression vectors containing nucleic acid molecules that encode the polypeptide or homologs or derivatives thereof under appropriate transcriptional/translational control signals, for expression. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., 1989.
  • Env The HIV envelope protein (Env) is the target of broadly neutralizing antibodies (bnAbs) in natural infection.
  • Env is a membrane protein composed of a trimer of gpl20 and gp41 subunits that contains a high degree of sequence diversity and a surface that is shielded by N- linked glycans.
  • the bnAbs that target Env often have unusual features such as a long complementarity-determining region (CDR) H3, high levels of somatic hypermutation (SHM), and insertions and deletions (INDELS).
  • CDR complementarity-determining region
  • SHM somatic hypermutation
  • INDELS insertions and deletions
  • most of the bnAbs recognize complex epitopes that are typically non-linear and have both protein and glycan components.
  • the sequences provided below are exemplary examples, the stabilizing mutations, modifications, (such as, but not limited to, cleavage-independent modifications), and/or a membrane anchoring strategy (such as, but not limited to, linker plus platelet-derived growth factor receptor (PDGFR)) described herein are applicable to any HIV strain or clade, such as but not limited to, those described below.
  • PDGFR linker plus platelet-derived growth factor receptor
  • the present invention relates to non-naturally occurring proteins, which may be involved in forming immunogenic proteins of the present invention.
  • the invention relates to a non-naturally occurring protein which may comprise any one of the following sequences in Tables 1-4.
  • Table 2 Immunogens inducing VRCOl-class bnAbs
  • Table 3 Immunogens inducing BG18 bnAb precursor response.
  • the protein may have at least 90% or 95% homology or identity with the sequence of the non-naturally occurring protein(s) of the invention.
  • Each of the protein sequences of Tables 1-4 optionally has a leader sequence of MGILPSPGMPALLSLVSLLSVLLMGCVAETG (SEQ ID NO: 54).
  • Some of the protein sequences of Tables 1-4 may comprise a linkl4 sequence of SHSGSGGSGSGGHA (SEQ ID NO: 55).
  • the linkl4 sequence is optional in each of the sequences comprising a link! 4 sequence.
  • trimers which may comprise any one of the non- naturally occurring protein(s) of the invention.
  • the proteins of the invention may comprise an additional cysteine and/or be fused to be a multimerization motif.
  • the proteins of the invention may also comprise a tag for purification or biotinylation, such as a his tag or a avi-tag.
  • the invention also encompasses nucleic acids encoding the non-naturally occurring protein(s) of the present invention, including nucleic acids that may have at least 90% or 95% homology or identity with a nucleotide encoding the sequence of the non-naturally occurring protein(s) of the invention.
  • the nucleic acid may be a mRNA.
  • the nucleic acids of the present invention may be delivered as a therapeutic mRNA.
  • the present invention contemplates expressing the herein disclosed immunogens as mRNAs, advantageously as mRNA vaccines.
  • Exemplary aspects of the invention feature efficacious mRNA vaccines. Described herein are mRNA vaccines designed to achieve particular biologic effects. Exemplary vaccines of the invention feature mRNAs encoding a particular antigen of interest (or and mRNA or mRNAs encoding antigens of interest), optionally formulated with additional components designed to facilitate efficacious delivery of mRNAs in vivo. In exemplary aspects, the vaccines of the invention feature and mRNA or mRNAs encoding antigen(s) of interest, complexed with polymeric or lipid components, or in certain aspects, encapsulated in liposomes, or alternatively, in lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • Chemical modification of mRNAs can facilitate certain desirable properties of vaccines on the invention, for example, influencing the type of immune response to the vaccine.
  • appropriate chemical modification of mRNAs can reduce unwanted innate immune responses against mRNA components and/or can facilitate desirable levels of protein expression of the antigen or antigens of interest. Further description of such features of the invention is provided infra.
  • isolated nucleic acids e.g., modified mRNAs encoding a peptide described herein
  • the nucleic acid may comprise a translatable region and at least two different nucleoside modifications, wherein the nucleic acid exhibits reduced degradation in a cell into which the nucleic acid is introduced, relative to a corresponding unmodified nucleic acid.
  • the degradation rate of the nucleic acid is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to the degradation rate of the corresponding unmodified nucleic acid.
  • the nucleic acid may comprise RNA, DNA, TNA, GNA, or a hybrid thereof.
  • the nucleic acid comprises messenger RNA (mRNA).
  • the mRNA does not substantially induce an innate immune response of the cell into which the mRNA is introduced.
  • the mRNA may comprise at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2- thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2 -thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-
  • the mRNA may comprise at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebul
  • the mRNA may comprise at least one nucleoside selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2- aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbam
  • the mRNA may comprise at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza- guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza- 8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy- guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
  • the nucleic acids provided herein comprise a 5' untranslated region (UTR) and/or a 3'UTR, wherein each of the two different nucleoside modifications are independently present in the 5'UTR and/or 3'UTR.
  • nucleic acids are provided herein, wherein at least one of the two different nucleoside modifications are present in the translatable region.
  • nucleic acids provided herein are capable of binding to at least one polypeptide that prevents or reduces an innate immune response of a cell into which the nucleic acid is introduced.
  • isolated nucleic acids e.g., modified mRNAs described herein which may comprise (i) a translatable region encoding a peptide described herein, (ii) at least one nucleoside modification, and (iii) at least one intronic nucleotide sequence capable of being excised from the nucleic acid.
  • isolated nucleic acids e.g., modified mRNAs described herein
  • isolated nucleic acids which may comprise (i) a translatable region encoding a peptide described herein, (ii) at least two different nucleoside modifications, and (iii) a degradation domain.
  • non-enzymatically synthesized nucleic acids e.g., modified mRNAs described herein
  • modified mRNAs described herein may comprise at least one nucleoside modification, and which may comprise a translatable region encoding a peptide described herein.
  • the non-enzymatically synthesized mRNA may comprise at least two different nucleoside modifications.
  • isolated nucleic acids e.g., modified mRNAs described herein
  • the isolated nucleic acids which may comprise a noncoding region and at least one nucleoside modification that reduces an innate immune response of a cell into which the nucleic acid is introduced, wherein the nucleic acid sequesters one or more translational machinery components.
  • the isolated nucleic acids which may comprise a noncoding region and at least one nucleoside modification described herein are provided in an amount effective to reduce protein expression in the cell.
  • the translational machinery component is a ribosomal protein or a transfer RNA (tRNA).
  • the nucleic acid may comprise a small nucleolar RNA (sno-RNA), microRNA (miRNA), small interfering RNA (siRNA) or Piwi -interacting RNA (piRNA).
  • isolated nucleic acids e.g., modified mRNAs described herein which may comprise (i) a first translatable region, (ii) at least one nucleoside modification, and (iii) an internal ribosome entry site (IRES).
  • IRES is obtained from a picomavirus, a pest virus, a polio virus, an encephalomyocarditis virus, a foot-and-mouth disease virus, a hepatitis C virus, a classical swine fever virus, a murine leukemia virus, a simian immune deficiency virus or a cricket paralysis virus.
  • the isolated nucleic acid further may comprise a second translatable region. In certain embodiments, the isolated nucleic acid further may comprise a Kozak sequence. In some embodiments, the first translatable region encodes a peptide described herein. In some embodiments, the second translatable region encodes peptide described herein. In some embodiments, the first and the second translatable regions encode peptides described herein.
  • compositions which may comprise: (i) an effective amount of a synthetic messenger ribonucleic acid (mRNA) encoding peptide described herein; and (ii) a pharmaceutically acceptable carrier, wherein i) the mRNA may comprise pseudouridine, 5'methyl-cytidine, or a combination thereof, or ii) the mRNA does not comprise a substantial amount of a nucleotide or nucleotides selected from the group consisting of uridine, cytidine, and a combination of uridine and cytidine, and wherein the composition is suitable for repeated administration (e.g., intravenous administration) to a mammalian subject in need thereof.
  • mRNA synthetic messenger ribonucleic acid
  • a pharmaceutically acceptable carrier wherein i) the mRNA may comprise pseudouridine, 5'methyl-cytidine, or a combination thereof, or ii) the mRNA does not comprise a substantial amount of a nucleotide or nucle
  • compositions which may comprise and/or consisting essentially of: (i) an effective amount of a synthetic messenger ribonucleic acid (mRNA) encoding peptide described herein; (ii) a cell penetration agent; and (iii) a pharmaceutically acceptable carrier, wherein i) the mRNA may comprise pseudouridine, 5'methyl- cytidine or a combination thereof, or ii) the mRNA does not comprise a substantial amount of a nucleotide or nucleotides selected from the group consisting of uridine, cytidine, and a combination of uridine and cytidine, and wherein the composition is suitable for repeated administration (e.g., intravenous administration) to an animal (e.g., mammalian) subject in need thereof.
  • mRNA synthetic messenger ribonucleic acid
  • nucleic acid in its broadest sense, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
  • exemplary nucleic acids for use in accordance with the present invention include, but are not limited to, one or more of DNA, RNA, hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc., described in detail herein.
  • modified nucleic acids containing a translatable region encoding a peptide described herein, and one, two, or more than two different nucleoside modifications.
  • the modified nucleic acid exhibits reduced degradation in a cell into which the nucleic acid is introduced, relative to a corresponding unmodified nucleic acid.
  • the degradation rate of the nucleic acid is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, compared to the degradation rate of the corresponding unmodified nucleic acid.
  • nucleic acids include ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or a hybrid thereof.
  • the modified nucleic acid includes messenger RNAs (mRNAs).
  • mRNAs messenger RNAs
  • the nucleic acids of the invention do not substantially induce an innate immune response of a cell into which the mRNA is introduced.
  • modified nucleosides include pyridin-4-one ribonucleoside, 5- aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine,
  • modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l- methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine,
  • modified nucleosides include 2-aminopurine, 2,6- diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-
  • the invention provides a modified nucleic acid containing a degradation domain, which is capable of being acted on in a directed manner within a cell.
  • modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl- guanosine, 7-methylinosine, 6-methoxy -guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio- guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
  • nucleic acid Other components of nucleic acid are optional, and are beneficial in some embodiments.
  • a 5' untranslated region (UTR) and/or a 3'UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications.
  • nucleoside modifications may also be present in the translatable region.
  • nucleic acids containing a Kozak sequence are also provided.
  • nucleic acids encoding a peptide described herein, and containing an internal ribosome entry site are provided herein.
  • An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA.
  • An mRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (“multicistronic mRNA”).
  • multicistronic mRNA When nucleic acids are provided with an IRES, further optionally provided is a second translatable region.
  • IRES sequences that can be used according to the invention include without limitation, those from picornaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).
  • picornaviruses e.g., FMDV
  • CFFV pest viruses
  • PV polio viruses
  • ECMV encephalomyocarditis viruses
  • FMDV foot-and-mouth disease viruses
  • HCV hepatitis C viruses
  • CSFV classical swine fever viruses
  • MLV murine leukemia virus
  • SIV simian immune deficiency viruses
  • CrPV cricket paralysis viruses
  • the mRNA of the present invention is formulated as a lipid nanoparticle (LNP) formulation, such as a PEG lipid, which are useful in pharmaceutical compositions, cosmetic compositions, and drug delivery systems, e.g., for use in LNP formulations.
  • LNP lipid nanoparticle
  • the LNPs described in US Patent Publication Nos. 20220047518 and 20200254086 are useful for the delivery of an agent (e.g., therapeutic agent such as a nucleic acid) to a subject.
  • an agent e.g., therapeutic agent such as a nucleic acid
  • lipid nanoparticles are provided.
  • a lipid nanoparticle comprises lipids including an ionizable lipid (such as an ionizable cationic lipid), a structural lipid, a phospholipid, and mRNA.
  • an ionizable lipid such as an ionizable cationic lipid
  • a structural lipid such as an ionizable lipid
  • a phospholipid such as an ionizable cationic lipid
  • mRNA mRNA.
  • a lipid nanoparticle comprises an ionizable lipid, a structural lipid, a phospholipid, and mRNA.
  • the LNP comprises an ionizable lipid, a PEG-modified lipid, a phospholipid and a structural lipid.
  • the LNP has a molar ratio of about 20-60% ionizable lipid: about 5-25% phospholipid: about 25-55% structural lipid; and about 0.5-15% PEG-modified lipid. In some embodiments, the LNP comprises a molar ratio of about 50% ionizable lipid, about 1.5% PEG-modified lipid, about 38.5% structural lipid and about 10% phospholipid. In some embodiments, the LNP comprises a molar ratio of about 55% ionizable lipid, about 2.5% PEG lipid, about 32.5% structural lipid and about 10% phospholipid.
  • the ionizable lipid is an ionizable amino or cationic lipid and the phospholipid is a neutral lipid, and the structural lipid is a cholesterol.
  • the LNP has a molar ratio of 50:38.5: 10: 1.5 of ionizable lipid: cholesterokDSPC: PEG2000-DMG.
  • the invention is a composition for or method of vaccinating a subject comprising administering to the subject a nucleic acid vaccine comprising one or more RNA polynucleotides having an open reading frame encoding a first antigenic polypeptide wherein a dosage of between 10 pg/kg and 400 pg/kg of the nucleic acid vaccine is administered to the subject.
  • the dosage of the RNA polynucleotide is 1-5 pg, 5-10 pg, 10-15 pg, 15-20 pg, 10-25 pg, 20-25 pg, 20-50 pg, 30-50 pg, 40-50 pg, 40-60 pg, 60-80 pg, 60-100 pg, 50-100 pg, 80-120 pg, 40-120 pg, 40-150 pg, 50-150 pg, 50-200 pg, 80-200 pg, 100-200 pg, 120- 250 pg, 150-250 pg, 180-280 pg, 200-300 pg, 50-300 pg, 80-300 pg, 100-300 pg, 40-300 pg, 50- 350 pg, 100-350 pg, 200-350 pg, 300-350 pg, 320-400 gg, 40-380 gg, 40-100 gg, 100-400 gg,
  • the nucleic acid vaccine is administered to the subject by intradermal, intraperitoneal or intramuscular injection.
  • the administration is an intramuscular injection.
  • the nucleic acid vaccine is administered to the subject on day zero.
  • a second dose of the nucleic acid vaccine is administered to the subject on day twenty one.
  • a dosage of 25 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 50 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 75 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject.
  • a dosage of 150 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 400 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, a dosage of 200 micrograms of the RNA polynucleotide is included in the nucleic acid vaccine administered to the subject. In some embodiments, the RNA polynucleotide accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node. In other embodiments the nucleic acid vaccine is chemically modified and in other embodiments the nucleic acid vaccine is not chemically modified.
  • an efficacious vaccine produces an antibody titer of greater than 1 :40, greater that 1 : 100, greater than 1 :400, greater than 1 : 1000, greater than 1 :2000, greater than 1 :3000, greater than 1 :4000, greater than 1 :500, greater than 1 :6000, greater than 1 :7500, greater than 1 :10000.
  • the antibody titer is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination.
  • the titer is produced or reached following a single dose of vaccine administered to the subject.
  • the titer is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.)
  • antigen-specific antibodies are measured in units of pg/ml or are measured in units of IU/L (International Units per liter) or mIU/ml (milli International Units per ml).
  • an efficacious vaccine produces >0.5 pg/ml, >0.1 pg/ml, >0.2 pg/ml, >0.35 pg/ml, >0.5 pg/ml, >1 pg/ml, >2 pg/ml, >5 pg/ml or >10 pg/ml.
  • an efficacious vaccine produces >10 mIU/ml, >20 mIU/ml, >50 mIU/ml, >100 mIU/ml, >200 mIU/ml, >500 mIU/ml or >1000 mIU/ml.
  • the antibody level or concentration is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination.
  • the level or concentration is produced or reached following a single dose of vaccine administered to the subject.
  • the level or concentration is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a booster dose.)
  • antibody level or concentration is determined or measured by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • antibody level or concentration is determined or measured by neutralization assay, e.g., by microneutralization assay.
  • Heterobifunctional crosslinkers such as, for example, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, which link the epsilon amino group on the D-lysine residues of copolymers of D- lysine and D-glutamate to a sulfhydryl side chain from an amino terminal cysteine residue on the peptide to be coupled, may be used as well.
  • Chemical conjugation also includes anything covalently bonded directly via side chain bonds or via a linker or spacer group.
  • the nanoparticle formulations may be a carbohydrate nanoparticle which may comprise a carbohydrate carrier and a modified nucleic acid molecule (e.g., mmRNA).
  • the carbohydrate carrier may include, but is not limited to, an anhydride- modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication No. W02012109121; herein incorporated by reference in its entirety).
  • Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP).
  • Ionizable cationic lipids such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin- MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity.
  • the rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat.
  • ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation.
  • the ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain.
  • the internal ester linkage may replace any carbon in the lipid chain.
  • the average diameter of the nanoparticle employed in the compositions of the invention can be at least one member selected from the group consisting of about 20 nanometers, about 25 nanometers, about 30 nanometers, about 40 nanometers, about 50 nanometers, about 75 nanometers, about 100 nanometers, about 125 nanometers, about 150 nanometers, about 175 nanometers and about 200 nanometers.
  • the average diameter of the particle is at least one member selected from the group consisting of between about 10 to about 200 nanometers, between about 0.5 to about 5 microns and between about 5 to about 10 microns.
  • the average diameter of the microparticle is selected from the group consisting of about 0.1 pm, about 0.2 pm, about 0.4 pm, about 0.5 pm, about 1 pm and about 2 pm.
  • Nanoparticles for use in the compositions of the invention can be made from lipids or other fatty acids (see, for example, U.S. Pat. Nos. 5,709,879; 6,342,226; 6,090,406; Lian, et al., J. of Pharma. Sci. 90:667-680 (2001) and van Slooten, et al., Pharm Res. 17:42-48 (2000)) and nonlipid compositions (see, for example, Kreuter, J. Anat. 189:503-505 (1996), the teachings of all of which are hereby incorporated by reference in their entirety).
  • the compositions can be bilayer or multilamellar liposomes and phospholipid based. Polymerized nanoparticles, as described, for example, in U.S. Pat. No. 7,285,289, the teachings of which are incorporated by reference in their entirety.
  • Metallic oxide nanoparticles for use in the compositions of the invention can be chemically substituted with at least one reactive moiety capable of forming a thioether bond employing conventionally techniques as described herein and in U.S. Pat. No. 6,086,881, the teachings of which are hereby incorporated by reference in their entirety.
  • the antigen described herein can be coupled in a single step onto the metallic oxide particles by the formation of at least one thioether bond or it may be synthesized or assembled stepwise onto the metallic oxide particles after the initial thioether bond formation.
  • the chemical derivatization reagents for the metallic oxide particles can include organosilane reagents that provide thioalkane functionality or other groups that may readily be converted into thiols or thiol-reactive moieties.
  • Organosilane reagents which may be utilized for this purpose may be, but are not limited to, 3- mercaptopropyltrimethoxysilane, 3 -aminopropyltri ethoxy silane, 3 -iodopropyltrimethoxy silane, 2-chloroethyltrichlorosilane, 3-glycidoxypropyltrimethoxysilane, vinyltrichlorosilane and 3- acryloxypropyltrimethoxysilane.
  • Moieties that include one or more disulfide components may also be joined to the metallic oxide particle surface and thereby provide the corresponding reactive moiety able to enter into and form a thioether bond and juncture.
  • Exemplary nanoparticles for use in the compositions of the invention include at least one member selected from the group consisting of poly (D,L-lactide-co-glycolide, also referred to as “poly(lactic-co-glycolic acid) and bi sacy 1 oxy propyl cy steine .
  • Nanoparticles for use in the compositions of the invention can be made of inorganic material.
  • Nanoparticles for use in the compositions of the invention can be made of a polymer material, such as at least one member selected from the group consisting of polystyrene, brominated polystyrene, polyacrylic acid, polyacrylonitrile, polyamide, polyacrylamide, polyacrolein, polybutadiene, poly caprolactone, polycarbonate, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, polymethylmethacrylate, polylactide, polyglycolide, poly(lactide- co-glycolide), polyanhydride, polyorthoest
  • these therapeutics may be a chemical compound, a composition which may comprise a polypeptide of the present invention and/or antibody elicited by such a chemical compound and/or portion thereof or a pharmaceutically acceptable salt or a composition which may comprise a polypeptide of the invention, and may be administered alone or as an active ingredient in combination with pharmaceutically acceptable carriers, diluents, and vehicles, as well as other active ingredients.
  • the compounds or compositions may be administered orally, subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques.
  • mice are treated generally longer than the mice or other experimental animals which treatment has a length proportional to the length of the disease process and drug effectiveness.
  • the doses may be single doses or multiple doses over a period of several days, but single doses are preferred.
  • animal experiments e.g., rats, mice, and the like, to humans, by techniques from this disclosure and documents cited herein and the knowledge in the art, without undue experimentation.
  • the mRNAs of the present invention are administered in combinations of a prime dose followed by one or more boost doses over time.
  • mRNA doses of about 100 pg are advantageous, however, dosages of about 10 pg to about 1000 pg, about 20 pg to about 900 pg, about 30 pg to about 800 pg, about 40 pg to about 700 pg, about 50 pg to about 600 pg, about 60 pg to about 500 pg, about about 70 pg to about 400 pg, about 80 pg to about 300 pg, or about 900 pg to about 200 pg, are contemplated. Varying combinations are presented below as non-limiting examples.
  • the treatment generally has a length proportional to the length of the disease process and drug effectiveness and the patient being treated.
  • a therapeutic of the present invention When administering a therapeutic of the present invention parenterally, it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
  • the pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • the carrier may be a solvent or dispersing medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, may also be used as solvent systems for compound compositions.
  • various additives which enhance the stability, sterility, and isotonicity of the compositions including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added.
  • Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • isotonic agents for example, sugars, sodium chloride, and the like.
  • Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the compounds.
  • Sterile injectable solutions may be prepared by incorporating the compounds utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired.
  • a pharmacological formulation of the present invention may be administered to the patient in an injectable formulation containing any compatible carrier, such as various vehicles, adjuvants, additives, and diluents; or the compounds utilized in the present invention may be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, iontophoretic, polymer matrices, liposomes, and microspheres.
  • any compatible carrier such as various vehicles, adjuvants, additives, and diluents
  • the compounds utilized in the present invention may be administered parenterally to the patient in the form of slow-release subcutaneous implants or targeted delivery systems such as monoclonal antibodies, iontophoretic, polymer matrices, liposomes, and microspheres.
  • a pharmacological formulation of the compound and composition which may comprise a polypeptide utilized in the present invention may be administered orally to the patient.
  • Conventional methods such as administering the compounds in tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like are usable.
  • Known techniques, which deliver the compound orally or intravenously and retain the biological activity, are preferred.
  • a formulation of the present invention may be administered initially, and thereafter maintained by further administration.
  • a formulation of the invention may be administered in one type of composition and thereafter further administered in a different or the same type of composition.
  • a formulation of the invention may be administered by intravenous injection to bring blood levels to a suitable level. The patient's levels are then maintained by an oral dosage form, although other forms of administration, dependent upon the patient's condition, may be used.
  • the vaccine may be administered as a single dose, or the vaccine may incorporate set booster doses.
  • booster doses may comprise variants in order to provide protection against multiple clades of HIV.
  • one or more boost immunogens may be from HIV pseudo viruses (PS Vs) or derivatives or mutations or a portion thereof.
  • the quantity to be administered will vary for the patient being treated and whether the administration is for treatment or prevention and will vary from a few micrograms to a few milligrams for an average 70 kg patient, e.g., 5 micrograms to 5 milligrams such as 500 micrograms, or about 100 ng/kg of body weight to 100 mg/kg of body weight per administration and preferably will be from 10 pg/kg to 10 mg/kg per administration.
  • the antigen is present in an amount on, the order of micrograms to milligrams, or, about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %.
  • any composition to be administered to an animal or human including the components thereof, and for any particular method of administration, it is preferred to determine therefor: toxicity, such as by determining the lethal dose (LD) and LDso in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable immunological response, such as by titrations of sera and analysis thereof for antibodies or antigens, e.g., by ELISA and/or REFIT analysis.
  • toxicity such as by determining the lethal dose (LD) and LDso in a suitable animal model e.g., rodent such as mouse
  • LDso lethal dose
  • concentration of components therein e.g., a suitable immunological response
  • Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein.
  • an adjuvant or additive is commonly used as 0.001 to 50 wt % solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and most preferably about 0.05 to about 5 wt %.
  • Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations may be ascertained without undue experimentation.
  • compositions which may comprise a therapeutic of the invention include liquid preparations for orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric, mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratory mucosa) etc., administration such as suspensions, syrups or elixirs; and, preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as sterile suspensions or emulsions.
  • orifice e.g., oral, nasal, anal, vaginal, peroral, intragastric, mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratory mucosa) etc.
  • administration such as suspensions, syrups or elixirs
  • parenteral subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration
  • compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like.
  • a suitable carrier diluent, or excipient
  • the compositions may also be lyophilized.
  • the compositions may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
  • Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection or orally. Viscous compositions, on the other hand, may be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or nasal mucosa.
  • suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form), or solid dosage form (e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-fdled form).
  • liquid dosage form e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form
  • solid dosage form e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-fdled form.
  • Solutions, suspensions and gels normally contain a major amount of water (preferably purified water) in addition to the active compound. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as NaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, (e.g., methylcellulose), colors and/or flavors may also be present.
  • pH adjusters e.g., a base such as NaOH
  • emulsifiers or dispersing agents e.g., a base such as NaOH
  • buffering agents e.g., preservatives
  • wetting agents e.g., methylcellulose
  • jelling agents e.g., methylcellulose
  • colors and/or flavors may also be present.
  • the compositions may be isotonic, i.e., it may have the same osmotic pressure as blood and lacrimal fluid.
  • compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • sodium chloride is preferred particularly for buffers containing sodium ions.
  • Viscosity of the compositions may be maintained at the selected level using a pharmaceutically acceptable thickening agent.
  • Methylcellulose is preferred because it is readily and economically available and is easy to work with.
  • suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Viscous compositions are normally prepared from solutions by the addition of such thickening agents.
  • a pharmaceutically acceptable preservative may be employed to increase the shelf-life of the compositions.
  • Benzyl alcohol may be suitable, although a variety of preservatives including, for example, parabens, thimerosal, chlorobutanol, or benzalkonium chloride may also be employed.
  • a suitable concentration of the preservative will be from 0.02% to 2% based on the total weight although there may be appreciable variation depending upon the agent selected.
  • compositions should be selected to be chemically inert with respect to the active compound. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems may be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.
  • compositions of this invention are prepared by mixing the ingredients following generally accepted procedures.
  • the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.
  • the pH may be from about 3 to 7.5.
  • Compositions may be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the composition form used for administration (e.g., solid vs. liquid). Dosages for humans or other mammals may be determined without undue experimentation by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.
  • Suitable regimes for initial administration and further doses or for sequential administrations also are variable, may include an initial administration followed by subsequent administrations; but nonetheless, may be ascertained by the skilled artisan, from this disclosure, the documents cited herein, and the knowledge in the art.
  • HIV vaccine first-boost candidate immunogen promoted maturation of VRC01 -class antibodies in a humanized mouse model
  • a protective human immunodeficiency virus (HIV) vaccine will likely need to induce broadly neutralizing antibodies (bnAbs).
  • Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRCOl-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, applicants designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost following eOD-GT8 60mer priming.
  • HIV human immunodeficiency virus
  • bnAbs HIV broadly neutralizing antibodies
  • Env HIV envelope glycoprotein
  • bnAbs can provide sterilizing protection in non-human primate models (A. Pegu et al., 2019) and can protect humans against infection (Y. Huang et al., 2022; L. Corey et al., 2021).
  • bnAb elicitation is widely considered essential for an effective HIV vaccine.
  • bnAb elicitation faces at least two major challenges.
  • bnAb germline precursors typically have no detectable affinity for wildtype Env (X. Xiao et al., Viruses 2009; X. Xiao et al., Biochem Biophys Res Commun 390 2009; D. S. Dimitrov et al., 2010; M. Pancera et al., 2010; T. Zhou et al., 2010; M. Bonsignori et al., 201 1 ; J. F. Scheid et al., 201 1 ; S. Hoot et al., 2013; J. Jardine et al., 2013; A. T. McGuire et al., 2013; J. G.
  • Germline-targeting vaccine design (J. Jardine et al., 2013; A. T. McGuire et al., 2013; J. G. Jardine et al., 2016; J. M. Steichen et al., 2016; A. Escolano et al., 2016; M. Medina- Ramirez et al., 2017; J. M. Steichen et al., 2019; B. Briney et al., 2016; M. Tian et al., 2016; K. R. Parks et al., 2019; X. Chen et al., 2021) offers a potential solution to these challenges.
  • a priming immunogen is designed to induce responses from diverse bnAb precursors for any single bnAb class, in order to prime the desired responses in all or most vaccine recipients.
  • a series of booster immunogens are then designed to be successively closer in structure to the native glycoprotein, such that each boost should engage B cells produced by the prior immunization and select for additional mutation toward bnAb development.
  • the IAVI G001 phase 1 trial provided clinical proof-of-principle for the priming step in the germline-targeting strategy: immunization with the germline-targeting priming immunogen eOD-GT8 60mer as protein with AS01B adjuvant was found to induce bnAb precursors of the VRC01 class in 97% of vaccine recipients (A. C. deCamp et al., 2023; D. J. Leggat et al., 2022; K. W. Cohen et al., 2023 ).
  • VRC01 -class bnAbs bind the Env CD4-binding site (CD4bs) and prevent HIV Env from binding its primary receptor on human CD4+ T cells (J. F. Scheid et al., 2011; X. Wu et al., 2010; X. Wu et al., 2011; T. Zhou et al., 2015; J. Huang et al., 2016; M. M. Sajadi et al., 2018; J. Umotoy et al., 2019).
  • Prior work has shown that passively administered VRC01 IgG can protect humans from infection with diverse VRC01 -sensitive viruses (Y. Huang et al., 2022; L.
  • the eOD-GT8 60mer is a self-assembling nanoparticle designed to bind to diverse VRCOl-class naive human precursors with substantial affinity and avidity (J. G. Jardine et al., 2015) and in pre-clinical studies was shown to prime VRCOl-class B cell responses in multiple knock-in and adoptive transfer mouse models (B. Briney et al., 2016; M. Tian et al., 2016; J. G. Jardine et al., 2015; P. Dosenovic et al., 2015; R. K.
  • the next critical test of the germline-targeting strategy is to determine whether a suitably designed first-boost immunogen can advance maturation toward bnAb development.
  • a suitably designed first-boost immunogen can advance maturation toward bnAb development.
  • pre-clinical studies with mRNA immunogens must be performed.
  • mAbs VRC01 -class and CD4bs-specific non- VRC01 -class monoclonal antibodies
  • the results supported the initiation of the IAVI G002 human clinical trial (NCT0500133) testing eOD-GT8 60mer priming and core-g28v2 60mer boosting delivered by mRNA lipid nanoparticles (LNPs).
  • the main objectives of this study were to evaluate first-boost immunogens following eOD-GT8 60mer priming for their capacity to induce increases in VRC01 -class B cell frequency, SHM, key mutations, and affinity and to compare adjuvanted protein versus mRNA immunization for delivery of self-assembling nanoparticle immunogens for prime and boost.
  • the number of mice in each group was limited by mouse availability and the costs of analysis; however, the number of mice used was judged to be sufficient to detect clear differences between groups. Experiments were conducted once with group sizes ranging from 5 to 15 mice. Mice were randomly assigned to groups. Blinding was not used. Samples were excluded from analysis if fewer than 50,000 CD 19+ B cells were detected during fluorescence activated cell sorting (FACS), indicating poor viability.
  • FACS fluorescence activated cell sorting
  • Boost candidates were developed and tested to follow eOD-GT8 60mer priming using a relatively permissive knockin mouse model (VRCOlgH, (J. G. Jardine et al., 2015)). Boost candidates were BG505-GT3-core 60mer and BG505-GT3-SOSIP (B. Briney et al., 2016) and also HxB2 core-e-2cc N276D 60mer.
  • Kymab mice have extremely low frequencies of VRCOl-class precursors and therefore produce very low frequencies of VRCOl-class responses to eOD-GT8 60mer (D. Sok et al., 2016), so it was hypothesized that the Kymab model was not well-suited for evaluating boosters for VRC01 -class responses. However, it was also hypothesized that the Kymab model might give insights into how the human antibody repertoire might respond to eOD-GT8 60mer in terms of off-target non-VRCOl -class responses and their cross-reactivity to boost candidates.
  • eOD-GT6v2 a series of eOD-GT6 variants termed eOD-GT6v2, eOD-GT6v3, and eOD-GT6v4 (D. J. Leggat et al., 2022).
  • These GT6 variants eliminated 2, 3, and 4 GT mutations, respectively. It was hypothesized that these changes to the VRC01 epitope would reduce reactivity to GT8-induced, non-VRCOl -class, CD4bs-specific Abs.
  • eOD-GT6v2 was noted to have better affinities for GT8-induced VRCOl-class Abs from the Vnl- 2 mouse.
  • Rosetta was used to computationally resurface eOD-GT6 outside the CD4bs and outside the existing glycosylation sites. This resulted in resurfaced variants of the above-mentioned GT6 variants, including resurfaced eOD-GT6v2, termed eOD-GT6v2-cRSF.
  • the aim was to develop a core with the CD4bs as native-like as possible that would still allow binding to GT8-induced VRC01 -class Abs from the VH1-2 mouse; with the non-CD4bs surface differing as much as possible from eOD-GT8 (by resurfacing) and as non-immunogenic as possible (by V3-loop minimization and as much glycan- masking as possible); and with good thermal stability and nanoparticle expression.
  • Readouts included: (i) fundamental biophysical readouts of expression amount, solution multimeric state by SECMALS, and thermal stability; (ii) surface plasmon resonance (SPR) affinities for mature VRC01 -class antibodies; and (iii) SPR affinities for GT8-induced VRC01 -class antibodies from the VH1-2 mouse (antibodies were from Tian et al. 2016 (M.
  • Rosetta KIC D. J. Mandell et al., 2009
  • Rosetta KIC D. J. Mandell et al., 2009
  • One thousand models were generated for each loop length.
  • the final candidates, selected based on loop closure score and total energy, were produced in vitro and evaluated for expression amount, stability and binding to a panel of VRCOl-class bnAbs (VRC01, 12a21, 12al2, CHA31, PGV04, 3BNC60, 3BNC117, VRC07, PG19, PGV20) and the non-nAb B6.
  • the starting v3 loop sequence “RPNNGGSGSGGNMRQ” (SEQ ID NO: 56) was substituted with a 6-residue loop sequence “AGNGTA” (SEQ ID NO: 57), which incorporated an engineered glycosylation site, referred to as position “300*”, with the star signifying that this position is present only on an artificial truncated loop.
  • This modified V3-loop was incorporated into the final core-g5, core-g28, and core-g28v2 designs.
  • glycan masking two glycans (N206 and N246) from a previously reported hyperglycosylated core called 6G (J. Ingale et al., 2014) were included. To identify additional sites for glycosylation, the focus was on surface residues located at least 5 A away from the CD4bs (HxB2 numbering 92-97, 276-282 and 455-476) and at least 5 A away from the asparagine residues within existing glycosylation sites. Using Rosetta, the energy difference resulting from the introduction of the Nx(S/T) sequon were evaluated, where x is any amino acid except proline. Mutations that did not destabilize the protein were deemed suitable for experimental evaluation.
  • core-g5 which includes the final V3-loop minimization design and resurfacing, there are 8 engineered glycosylation sites (at positions 63, 82, 113, 202, 206, 246, 300, and 441 in HxB2 numbering) and 16 native glycosylation sites (at positions 88, 230, 234, 241, 262, 289, 295, 332, 339, 356, 386, 392, 397, 406, 448 and 463 in HxB2 numbering).
  • core-g28v2 which includes the final V3-loop minimization design and resurfacing in the context of the TH6 extended core to increase potential shared T-helper epitopes with HIV Env trimers
  • there are a total of 28 glycosylation sites with 12 engineered sites (at positions 113, 120, 300*, 344, 402, 409, 413, 419, 423, 434, 439, and 442 in HxB2 numbering) and 16 native sites (at positions 88, 230, 234, 241, 262, 289, 295, 332, 339, 356, 386, 392, 397, 406, 448, and 463 in HxB2 numbering).
  • Core-g28 was the same as core-g28v2, except that core- g28 contained an engineered glycosylation site at 399 instead of the native glycosylation site at 397. It was found that core-g28v2 60mer demonstrated superior nanoparticle formation (Fig. 10), with a higher fraction of nanoparticles and greater homogeneity, compared to core-g28 60mer, which was the primary reason that the core-g28v2 was selected over core-g28 for clinical testing. Subsequently, it was also found that core-g28v2 monomer exhibited superior binding to GT8- elicited antibodies from the G001 study.
  • a positionspecific scoring matrix was generated by aligning HIV Env trimer sequences from the LANL database (www.hiv.lanl.gov); only amino acids present in native Env trimers were permitted, and an energy bonus was given to the native amino acid to reduce the likelihood of introducing destabilizing mutations.
  • Most resurfaced designs were tested as fully resurfaced proteins, but for selected positions within the edge or near the CD4bs, individual point mutations were tested. Resurfacing modifications were found to affect thermal stability and nanoparticle formation propensity, with some designs reducing stability by as much as 10° and correspondingly lacking the ability to form nanoparticles, and other designs improving both thermal stability and nanoparticle formation.
  • the core-g5 design included a total of 13 resurfacing mutations, and the core-g28 and core-g28v2 designs each included a total of 8 resurfacing mutations.
  • All lumazine synthase nanoparticles tested in this study utilized the previously reported d41m3 version of lumazine synthase. This version includes two engineered disulfides and three mutations to disable the enzymatic active site (D. Sok et al., 2016).
  • eOD-GT8 60mer was evaluated previously for delivery by an RNA replicon platform, the d41m3 version was found to perform better than the original version (M. Melo et al., 2019).
  • the eOD-GT8 KO used here was eOD-GT8 KO11 (with mutations 280R, 365L, and 371R) (D. Sok et al., 2016).
  • the core-g28v2 KO used here was core-g28v2 KOI lb. This version has the same mutations as KOI 1 but also included D368R.
  • a de novo model for core-g28v2 was generated using AlphaFold2. Man9 glycans were added and relaxed using Rosetta (J. Jumper et al., 2021; J. Adolf-Bryfogle et al., 2021; J. W. Labonte et al., 2017). Figures were made using UCSF Chimera (E. F. Pettersen et al., 2004).
  • His-tagged and His-Avi-tagged monomeric and trimeric antigens were produced by transient transfection of HEK-293F cells (Thermo Fisher) and purified by immobilized metal ion affinity chromatography (IMAC) using HisTrap excel columns (Cytiva) followed be sizeexclusion chromatography (SEC) using either Superdex 75 10/300 GL or Superdex 200 Increase 10/300 GL columns (Cytiva).
  • IMAC immobilized metal ion affinity chromatography
  • SEC sizeexclusion chromatography
  • Nanoparticle 60mer immunogens were produced by transient transfection of HEK- 293F cells (Thermo Fisher). Immunogens were then purified by Galanthus nivalis lectin affinity chromatography (Vectorlabs) followed by SEC using a Superose 6 16/600 PG column (Cytiva). Immunogen preps confirmed to contain ⁇ 5 EU/mg of endotoxin using an Endosafe instrument (Charles River).
  • Engineered N-linked glycosylation sites are frequently under-occupied.
  • site specific glycan profiling was conducted as previously described (S. Baboo et al., 2021). The degree of glycan occupancy and proportion of glycans that were complex and oligomannose/hybrid type were determined.
  • mice were euthanized with compressed CO2 (100%) in a clear chamber to allow for visualization of respiration and subsequent death through respiratory cessation.
  • Blood was collected from the chest cavity prior to the removal of the spleen and lymph nodes (mesenteric, inguinal, and popliteal (RNA injections only, left leg only).
  • Tissues were placed in 3 mb resuspension buffer (lx PBS Ca/Mg++ free, ImM EDTA, 25mM HEPES, pH 7.0, 1% heat- inactivated fetal bovine serum [FBS]) in a 15 m polypropylene tube on ice.
  • Tissues were disassociated using the rough ends of two sandblasted microscope slides in a 5mL petri dish, then returned to the same 15 mb polypropylene tube for centrifugation (460xg for 5 minutes at 4°C). Red blood cell lysis was performed using 1 mb of ACK buffer (Quality Biological, Cat#l 18-156- 721) for 2 minutes on ice in a 15 mb polypropylene tube. Lysis was halted by adding 14 mb resuspension buffer per sample.
  • Streptavidin (SA) conjugated-baits were prepared by combining biotinylated monomeric baits or Env trimer baits with fluorescent SA at room temperature for at least 1 hour in the dark. Wild-type baits were complexed with SA- Alexa Fluor 647 (Invitrogen, cat# S21374) and SA-brilliant violet (BV) 421 (BioLegend, cat# 405225). Knockout (KO) baits, if used, were conjugated with SAphycoerythrin (PE)-cyanine (Cy) 7 (BioLegend, cat# 405206).
  • Monomeric baits were conjugated with SA at a 4: 1 (bait: SA) ratio and used at a final bait concentration of 200 nM for staining.
  • Env trimer baits were conjugated with SA at a 2: 1 (bait: SA) ratio and used at 100 nM.
  • Isolated B cells were transferred over to 15 mL conical tubes, washed once with FACS buffer, and stained with 100 pL antibody cocktail mix consisting of PE anti-CD19 (BD Biosciences, cat# 553786), BV786 anti-IgM (BD Biosciences, cat#743328), Peridinin chlorophyll protein (PerCP)-Cy5.5 anti-IgD (BD Biosciences, cat# 564273), allophycocyanin (APC)-Cy7 anti- F4/80 (BioLegend, cat # 123118), APC-Cy7 anti-CDl lc (BD Biosciences, cat# 561241), APC- Cy7 anti-Ly-6C (BD Biosciences, cat# 557661), APC-H7 anti-CD8a (BD Biosciences, cat# 560182), and APC-H7 anti-CD4 (BD Biosciences, cat# 560181).
  • PE anti-CD19 BD Biosciences, cat# 553786
  • Samples were fdtered through a 35 pm mesh-cap FACS tube (Falcon, cat# 352235) prior to being loaded on the sorter.
  • a maximum of 15,000 cells were sorted using purity mode into a PCR plate well containing 20 pL of 0.2 pm filtered FBS.
  • Event rates were typically maintained at about 1000 events per second and no more than 1500 events per second to ensure high sorting efficiencies.
  • Sorted samples were prepared for BCR sequencing by the 10X Genomics Single Cell Immune Profiling platform. After cell sorting, DPBS was added up to near top of the sample collection well (about 100 pL) and gently mixed to dilute the FBS catch buffer. The plate was sealed and cells were centrifuged for 2 minutes at 2000 rpm, after which the excess buffer was removed except for about 38 pL required for the 10X Genomics GEM reaction. Samples were processed according to manufacturer’s user guide for Chromium Next GEM Single Cell 5’ Reagent Kits v2 (Dual Index) with Feature Barcoding, with two main modifications. The number of PCR cycles in the cDNA amplification step were determined by assuming that only 20% of the total number of cells sorted would be recovered.
  • Raw sequencing data were demultiplexed, processed into assembled VDJ contigs and counts matrix files, and assigned to specific animal IDs based on TotalSeq-C antibody hashtag counts using Cell Ranger (v6.1) and scab as previously described (J. Hurtado et al., 2022).
  • Gene assignment, annotation, and formatting into Adaptive Immune Receptor Repertoire (AIRR) format F. Breden et al. 2017 et al., 2017
  • SADIE Sequencing Analysis and Data library for Immunoinformatics Exploration
  • VRC01 -class identification and mutational analysis was performed using the SADIE renumbering module by numbering each sequence using Kabat numbering and checking for the following key VRCOl-class residues: (i) germline: 47W, 50W, 55G, 71R; (ii) nonparatope: K19R, G31D/A, Y33I/V/T, M34I/L, S76E/D, S82aK/R; and (iii) paratope: N52K/R, N53/R/Q/K/L/M/V/E, S54Y/G/H/F/R, G56A, T57V, Q61R/H/G, K62Q/G, T73V/I, S74Y, Trpio 3 -5.
  • the frequency of VRCOl-class memory B cells was calculated by multiplying the frequency of antigen-specific MBCs among all MBCs processed by FACS and the frequency of VRCOl-class BCRs among all sequenced heavy /light pairs.
  • the frequency of VRCOl-class with human VK1-33 light chains was calculated by multiplying the frequency of antigenspecific MBCs among all MBCs processed by FACS and the frequency of VRC01- class VK1 ' 33 BCRs among all sequenced heavy /light pairs.
  • ELISA plates (Corning 96-Well Half-Area Plates, Catalog # 3690) were directly coated with ELISA antigens at 2 pg/mL on Day 1. Plates were incubated overnight at 4°C. Plates were washed three times with PBST (PBS + 0.2% tween 20) and blocked with PBST containing 5% [00276] skim milk (BD Difco Skim Milk Catalog # 232100) and 1% FBS (Thermo Fisher, Catalog # 16000044) for 1 hour at room temperature on Day 2.
  • PBST PBS + 0.2% tween 20
  • the ligand solution concentrations were 1 to 5 pg/mL for the standard method used to assess monovalent analytes and 0.1 to 0.2 pg/mL for the low-capture IgG method used to assess binding to trimeric analytes. In both methods, the contact time was 3 to 5 minutes.
  • Raw sensograms were analyzed using Kinetics software (Carterra), interspot and blank double referencing, Langmuir model.
  • Analyte concentrations were quantified on NanoDrop 2000c Spectrophotometer using Absorption signal at 280 nm. Analyte samples were buffer-exchanged into the running buffer using dialysis.
  • Polyclonal IgG was isolated from mouse serum samples using Protein G Sepharose 4 Fast Flow resin (Cytiva, 17-0618-05). Resin was washed 3 times in lx PBS and resuspended at 1 : 1 ratio resin to PBS. 50 pL of resin/PBS mixture was added to 200 pL heat-inactivated mouse serum and incubated with agitation overnight at room temperature. Samples were loaded into empty spin columns (Pierce, 89868) and centrifuged at 5000xg for 2 minutes. Resin was washed 3 times with lx PBS. IgGs were eluted into 50 pL IM Tris pH 8 using 50 pL 0. IM glycine pH 2.7. IgGs were concentrated and buffer exchanged into IX TBS using 30 kDa Amicon Ultra 0.5 mL centrifugal filters (Millipore, UFC503096).
  • Loaded biosensors were dipped into kinetics buffer for 1 minute to acquire a baseline and then moved to wells containing VRC01 Fab at 2 pM in kinetics buffer.
  • the VRC01 Fab was allowed to saturate the core-g28v2 load biosensors for 2 minutes.
  • the biosensors were then moved to wells containing competitor IgGs at 500 nM in kinetics buffer for 2 minutes.
  • Control binding experiments were conducted in which no VRC01 Fab was used. Competition was apparent by a lack of signal for IgG binding when VRC01 Fab was used compared to the signal acquired when no VRC01 Fab was used. All BLI experiments were conducted at 25°C.
  • Pseudovirus neutralization assays were performed as previously described (M. Li et al., 2005 et al., 2005) with minor modifications. Briefly, single cycle infectious pseudoviruses were generated by co-transfecting HEK293T cells with Envs of interest with an Env deficient HIV-1 backbone plasmid pSG3DEnv using Fugene 6 (Promega, E2692), or PEI MAX (Polysciences, Inc, 24765-1). Viruses were harvested 72 hours post-transfection and kept frozen at -80°C until use.
  • mAbs were serially diluted in D10 media (Dulbecco's Modified Eagle Medium (Gibco, 10313-021), 10% FBS (Omega Scientific, FB-02), lx PenStrep (Gibco, 15070-063), and lx GlutaMAX (Gibco, 35050- 061)).
  • D10 media Dulbecco's Modified Eagle Medium (Gibco, 10313-021), 10% FBS (Omega Scientific, FB-02), lx PenStrep (Gibco, 15070-063), and lx GlutaMAX (Gibco, 35050- 061)).
  • Viruses where prepared by thawing at 37°C, and concentrated or diluted with D10 according to previously defined titers.
  • DEAE-dextran Spectrum Chemical Mfg.
  • the prepared mAb dilutions and viruses were distributed into 96 well round bottom plates (Corning, 3788) at a 1 : 1 v/v ratio and incubated at 37°C for 1 hour. Following the 1 hour incubation, the supernatant was carefully aspirated from the prepared TZM-bl plates, then 25 pL of appropriate mAb:virus mixtures were added to the target cells and returned to the incubator. 24 hours later, 75 pL of D10 were added to each well and further incubated for an additional 48 hours.
  • TZM-bl cells were lysed in 45 pL/well of IX Cell Lysis Buffer (Promega, cat# E4550) for 15 minutes, after which 30 pL/well of substrate was added (Promega, cat# E4550) and luminescence was read on a BioTek Synergy Hl Plate Reader.
  • Half-maximal inhibitory concentration (IC50) values were interpolated from the One site Fit logIC50 model in Prism 9 (GraphPad). All curve fits were constrained between 0 to 100% neutralization. Both TZMbl cells and HEK293T cells were cultured and maintained in DIO media in a 37°C humidity and CO2 controlled incubator.
  • VH1-2/VK1-33 rearranging mice harbor VRC01 -class precursors with frequencies and affinities approximating those in humans.
  • VRC01 -class antibodies are defined by their use of the human immunoglobulin heavy chain (HC) V gene alleles VH1-2*02 or *04 and any light chain (LC) complementarity determining region (LCDR) 3 with a length of five amino acids (aa) (J. Jardine et al., 2013, A. P. West et al., 2012). Most of the interactions between VRCOl-class bnAbs and HIV Env come from the HC complementarity determining region (HCDR) 2, with the HCDR3 providing a minor supporting role (T. Zhou et al., 2010, T. Zhou et al., 2015, J. Huang et al., 2016), allowing VRCOl-class bnAbs to have diverse HCDR3 lengths and sequences.
  • HCDR human immunoglobulin heavy chain
  • LCDR light chain
  • aa light chain
  • Most of the interactions between VRCOl-class bnAbs and HIV Env come from the HC complementarity
  • Vnl-2 JH2 /VKl-33 hTdT mouse (referred to as SE09 from this point forward) has the human IGHV1-2*O2 and human IGHJ2*01 alleles knocked into the mouse IGH locus and the human IGKVl-33*01 allele knocked into the mouse IGK locus (Fig. 7A) (S. Luo et al., 2023).
  • the SE09 mouse has a knockout of the intergenic control region 1 (IGCR1), resulting in dominant usage of the human IGHV1-2*O2 allele, and a human terminal deoxynucleotidyl transferase (TdT) knock-in that increases the percentage of LCs with 5 aa LCDR3s (Fig. 7A) (S. Luo et al., 2023).
  • the knock-in genes participate in normal V(D)J recombination, resulting in a diverse repertoire of CDR3 lengths and sequences, and the human VH1-2 HCS and VK1-33 LCS are expressed in approximately 45% and 2% of naive B cells, respectively (S. Luo et al., 2023).
  • the frequency of VRCOl-class naive B cells was higher in the SE09 mouse (1 in 13,600) compared to humans (1 in 228,000) (Fig. 7B) (J. G. Jardine et al., 2016, J. H. Lee et al., 2021, C. Havenar-Daughton et al., 2018).
  • KD geomean dissociation constant
  • SE09 mouse provided a model system with a diverse B cell repertoire containing VRCOl-class naive precursors with comparable affinities to human VRCOl-class naive precursors that were present at physiologically relevant frequencies.
  • First-boost candidates were designed to boost maturation of VRCOl-class BCRs primed by eOD-GT8 60mer.
  • An ideal first-boost immunogen to follow eOD-GT8 60mer priming would be the most native-like protein that can bind to eOD-GT8-induced VRCOl-class BCRs and have no measurable affinity for eOD-GT8-induced non-VRCOl -class BCRs specific for the CD4bs.
  • eOD- GT6 and more native variants of eOD-GT6 have been shown to bind to eOD-GT8-primed VRCOl- class BCRs but to have limited binding to eOD-GT8-primed non-VRCOl -class BCRs (D. J. Leggat et al., 2022).
  • eOD-GT6 variants could potentially function as a first boost, immunogens that are more native-like in structure are preferred because they have the potential to shepherd B cells further down the path towards producing bnAbs.
  • Structural platforms that would be more native-like than eOD-GT6 variants would need to include both core-gpl20- and trimer-based immunogens, in which trimer-based immunogens would be the closest in structure to the native glycoprotein.
  • HxB2 gpl20 core lacking the N276 glycan HxB2 core-e-2cc N276D
  • booster immunogens have been suggested or used in previous studies, including but not limited to: a stabilized native HxB2 gpl20 core lacking the N276 glycan (J. G. Jardine et al., 2016, D. J. Leggat et al., 2022), a chimeric gpl20 core immunogen termed cl3 lacking the N276 glycan (M. Medina-Ramirez et al., 2017, M. Tian et al., 2016), and a BG505 gpl20 core lacking the N276 glycan with additional germline-targeting mutations (A. Escolano et al., 2016).
  • eOD-GT6v2-cRSF a resurfaced version of eOD-GT6v2 (D. J. Leggat et al., 2022), core-g28v2, 191084-N276D stabilized Env trimer (from isolate 191084_B7_19, hereafter abbreviated as 191084), C13.G4.2 core (X. Chen et al., 2021), and BG505-core-VRC01-GT3.3 (B. Briney et al., 2016) (Fig. 1C, Fig. 8 and 11).
  • core-g28v2 showed moderate affinity for eOD-GT8 elicited mAbs (Fig. 1C), could assemble into a high avidity 60mer nanoparticle (Fig. 9), and contained a more native CD4bs than eOD-GT6v2-cRSF and the other gpl20 core immunogens (Fig. 8).
  • VRCOl-class B cells were boosted with protein immunogens.
  • SE09 mice were primed with eOD-GT8 60mer protein, received first-boost immunogens at week 6, and responses were evaluated at weeks 6 and 12.
  • the primary readouts were the frequencies, degrees of mutation, and affinities of VRCOl-class responses.
  • Splenocytes and draining lymph node cells from week 6 (post-prime) or week 12 (postboost) were sorted for antigen-specific CD19 + /IgMTgD‘ B cells and subjected to paired single-cell BCR sequencing using lOx Genomics followed by bioinformatic analyses (Fig. 11).
  • MCCs antigen-specific memory B cells
  • mice were primed with eOD-GT860mer protein and then boosted with either core-g28v2 60mer, eOD-GT6v2-cRSF 60mer, 191084-N276D soluble native Env trimer, or phosphate buffered saline (PBS) as a placebo (Fig. 2A).
  • Antigen-specific MBCs were detected in all immunization groups, although frequencies were very low for both the placebo and 191084-N276D groups (Fig. 2B).
  • Substantial fractions of the antigen-specific MBCs were VRCOl-class for both the core-g28v2 and eOD-GT6v2-cRSF boost groups (Fig.
  • VRC01-class K1 ’ 33 responses offered the most direct comparison to human responses. Priming with eOD-GT8 60mer protein led to a 712-fold expansion of VRC01- class VK1 ' 33 MBCs relative to the naive IgD + IgM + VRC01-class VK1 ' 33 precursor pool in SE09 mice (Fig. 2E and Fig. 7C). For comparison, in the G001 clinical trial, the frequency of VRC01-class VK1 ' 33 IgG + B cells was 45-fold or 317-fold higher than the naive VRC01-class XK1 ' 33 precursor frequency at eight weeks after the first or second dose of eOD-GT8 60mers, respectively (D.
  • Boosting with eOD-GT6v2-cRSF 60mer elicited similar frequencies of VRCOl-class MBCs and VRC01-class VK1 ' 33 MBCs compared to core-g28v2 60mer (Fig. 2D and E).
  • VH and VK/VL somatic hypermutation (SHM) among VRCOl-class MBCs did not increase after boosting with eOD-GT6v2-cRSF 60mer relative to the post-prime response (Fig. 2F and G).
  • boosting with core-g28v2 60mer produced VRCOl-class MBCs with higher VH and VK/VL SHM compared to the post-prime response and compared to boosting with eOD-GT6v2- cRSF 60mer (Fig. 2F and G).
  • the core-g28v260mer boost generated similar VRCOl -class MBC frequencies but higher SHM in VRC01 -class MBCs (and similar SHM in non-VRCOl -class MBCs) compared to the eOD-GT6v2-cRSF 60mer boost, and as core-g28v2 had a more nativelike structure than eOD-GT6v2-cRSF (Fig. 8), applicants concluded that core-g28v2 60mer was superior to eOD-GT6v2-cRSF 60mer as a first-boost immunogen candidate.
  • BCR sequences from the eOD-GT8 60mer prime and core-g28v2 60mer boost immunization groups were expressed as mAbs and assessed for affinity to eOD-GT8, core-g28v2, and core-g28v2 with theN276 glycan (Fig. 3A).
  • the geomean affinity of post-prime VRCOl-class mAbs for eOD-GT8 increased by over 1000-fold relative to the affinity of the VRCOl-class naive precursors from SE09 mice (Fig. 3 A).
  • Approximately half of the post-prime VRCOl-class mAbs assayed had measurable affinity for core-g28v2, with geomean affinity among binders of approximately 12 pM (Fig.
  • ELISA enzyme-linked immunosorbent assay
  • Boosting with core-g28v2 60mer elicited serum antibodies with higher 50% effective dilution (ED50) values for eOD-GT8 and core-g28v2 relative to the corresponding epitope-knockout versions, indicating that the core-g28v2 boost induced a strong CD4bs-specific response (Fig. 3B), as intended by the core-g28v2 design.
  • ED50 effective dilution
  • the representative VRCOl-class bnAbs had a median of 13 key residues, and a range of +8 to + 16 (Fig. 4C and Fig. 13 A), illustrating that additional maturation will be required to elicit bnAbs.
  • Paratope key VRCOl-class residues at positions within the HCDR2 (L. Mesin et al., 2020; T. T. Wu et al., 1970; D. J. Mandell et al., 2009) were detected after boosting with core-g28v2 60mer, but not after priming with eOD-GT8 60mer, whether followed by a placebo boost (Fig. 4D) or not (Fig. 13B), illustrating a direct effect of the boost.
  • mRNA/LNP vaccine platforms can provide excellent immunogenicity and safety with rapid timelines for entering clinical trials, as illustrated by the SARS-CoV-2 mRNA vaccines (F. P. Polack et al., 2020; L. R. Baden et al., 2021).
  • SARS-CoV-2 mRNA vaccines F. P. Polack et al., 2020; L. R. Baden et al., 2021).
  • eOD-GT8 60mer priming one or two immunizations
  • core-g28v2 60mer boosting after one or two eOD-GT8 60mer priming immunizations
  • applicants tested a placebo boost and a core-g28v260mer priming group Fig. 5A.
  • eOD-GT8-specific MBCs that were VRC01 -class was not significantly different for one eOD-GT860mer immunization followed by placebo compared to two immunizations with eOD-GT8 60mer (Fig. 5C), indicating that the second eOD-GT8 60mer mRNA immunization did not cause substantial additional priming of VRC01 -class precursors.
  • One or two eOD-GT8 60mer mRNA immunizations induced similar frequencies of VRC01 -class and VRC01-class VK1 ' 33 MBCs (Fig. 5D and E), and the VRC01 -class MBCs in both cases had minimal VH and VK/L SHM (Fig. 14A and B).
  • the core-g28v2 60mer mRNA boost was effective for expanding and maturing VRCOl-class responses after a single eOD-GT8 60mer mRNA prime, and there was no benefit to using a double eOD-GT8 60mer mRNA prime.
  • Boosting with core-g28v2 60mer mRNA after a single eOD-GT8 60mer mRNA prime induced more key VRC01 -class HCDR2 residues compared to priming alone (Fig. 19A) or priming followed by a placebo boost (Fig. 6D and Fig. 19B), as was true for protein immunization.
  • Applicants also found that boosting with core-g28v2 60mer mRNA after a single eOD-GT8 60mer mRNA prime induced more key VRC01 -class HCDR2 residues compared to two immunizations with eOD-GT8 60mer mRNA (Fig. 19C and D).
  • the core-g28v2 60mer mRNA boost induced a similar pattern of responses as protein, with significantly higher ED50 values for eOD-GT8 and core-g28v2 compared to the epitope KO versions (p ⁇ 0.001), indicating that the majority of the response elicited after boosting with mRNA was CD4bs-specific (Fig. 6B), as intended by the core-g28v2 design.
  • Fig. 6B CD4bs-specific
  • core-g28v2-binding BCRs isolated after boosting with core-g28v2 60mer mRNA were expressed as mAbs and assessed for affinity to core-g28v2 and core-g28v2 KO (Fig. 2 IB). More than fifty percent of post core-g28v2 non- VRCOl -class mAbs had >50-fold reduction in affinity for g28v2-KO compared to core-g28v2, indicating that they too were CD4bs-specific competitors (Fig. 21B).
  • determining if post-core- g28v2 VRC01 -class antibodies can bind to such trimers would further support the use of core- g28v2 60mer as a first boost and would identify potential second-boost candidates.
  • N276 glycan was removed from these trimers by introducing N276D, N276Q, or T278M mutations.
  • VRC01 -class mAbs from both protein- and mRNA-immunized animals bound to N276-lacking trimers with similar affinities (Fig. 7A and B).
  • Higher percent binders and higher affinity was noted for the N276D version of 191084 over the N276Q version and for the T278M version of HIV_001428_2 (hereafter referred to as 001428) over N276Q, indicating preferences for D276 and M278 (Fig. 7A and B).
  • D276 and M278 are present in core-g28v2, and D276 is also present in eOD-GT8, likely contributing to the mutation preference.
  • Fifteen of the VRC01- class mAbs elicited after boosting with core-g28v2 60mer mRNA were assessed for neutralization against a panel of N276-lacking and corresponding wildtype pseudoviruses (Fig. 7C). Neutralization was detected against N276D, T278M, and N276Q pseudoviruses, but not against wildtype pseudoviruses (Fig. 7C).
  • VRC01 -class B cells primed by eOD-GT8 60mer and boosted by core-g28v2 60mer acquired additional key VRC01 -class residues and gained affinity for heterologous HIV Env trimers lacking the N276 glycan and also for a core- g28v2 variant containing the N276 glycan.
  • the key VRC01 -class residues within the HCDR2 induced by core-g28v2 60mer boosting are known to be important for neutralization breadth and potency (J. G. Jardine et al., 2016).
  • core-g28v2 60mer was an effective boost after a single priming immunization by eOD-GT8 60mer in this mouse model even though the applicants’ SPR analysis showed that only 56% or 28% of post-GT8 mAbs at week 6 (the timepoint for the core-g28v2 60mer boost) had detectable affinity for core-g28v2, for protein or mRNA priming, respectively, and among those binders, the geomean affinities for core-g28v2 were 12 pM and 8 pM, respectively.
  • VRC01 -class precursor frequency in the SE09 mouse was 17 times higher than in humans; however, the VRC01 -class precursors were still rare (1 in 13,600 naive B cells), highly diverse with different CDR3s and light chains, and had affinities for eOD-GT8 that were comparable to human VRC01 -class precursors.
  • Boosting MBCs in mice to re-enter the germinal center is highly inefficient (L. Mesin et al., 2020) and likely requires a higher precursor frequency present at priming compared to humans to generate sufficient MBCs as targets for boosting.
  • the elevated frequencies of B cells with human Vnl-2 and VK1-33 in the SE09 mouse reduced the genetic diversity of potential competitor B cells.
  • mRNA vaccine technology will likely prove essential for HIV vaccine development, as the favorable immunogenicity combined with increased speed and lower cost of producing clinical material should improve the feasibility and timelines of clinical trials testing multiple immunogens in series. For that reason, applicants compared adjuvanted protein immunization, as in IAVI G001, to mRNA immunization, and applicants found that mRNA performed at least as well, if not slightly better than, protein. This not only provided preclinical support for the IAVI G002 clinical trial evaluating eOD-GT8 60mer mRNA priming followed by core-g28v2 60mer mRNA boosting, but also demonstrated the feasibility of using mRNA to deliver self-assembling nanoparticle immunogens in vivo.
  • Germline-targeting priming and sequential heterologous boosting with protein immunogens in highly permissive mouse models has been shown to induce increased SHM (B. Briney et al., 2016; M. Tian et al., 2016; K. R. Parks et al., 2019, K. O. Saunders et al., 2019) and to produce bnAbs (J. M. Steichen et al., 2016; A. Escolano et al., 2016; X. Chen et al., 2021).
  • mice Humanized Vnl-2/VKl-33 hTdT mice were initially primed with eOD-GT8 60mer delivered by mRNA/LNPs and boosted with core-g28v2 60mer mRNA/LNP ( Figure 71). Subsequentially, these mice were boosted at week 12 with one of four mRNA/LNP immunogens encoding for stabilized membrane-bound HIV Env trimers lacking the N276 glycan (1HD2- N276Q-gpl51, lHD2-T278M-gpl51, 001428-N276Q-gpl51, or 001428-T278M-gpl51).
  • VRCOl-class memory B cells were determined by antigen-specific cell sorting (Figure 72) and single cell lOx Genomics VDJ sequencing conducted six weeks after boosting. A subset of mice was boosted again at week 18 with corresponding wildtype membrane-bound trimers delivered by mRNA/LNPs matched to those received at week 12.
  • Example 2 Vaccine priming by targeting BG18 Vaccine priming of rare HIV broadly neutralizing antibody precursors in non-human primates
  • Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to human immunodeficiency virus (HIV) and other pathogens.
  • bnAbs broadly neutralizing antibodies
  • HBV human immunodeficiency virus
  • antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3 -dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals.
  • HCDR3 heavy chain complementarity determining region 3
  • N332-GT5 an HIV envelope trimer designed to target precursors of the HCDR3 -dominant bnAb BG18, primed bnAb-precursor B cells in 8 of 8 rhesus macaques to substantial frequencies and with diverse lineages, in germinal center and memory B cells.
  • the results demonstrate proof of principle for HCDR3 -dominant bnAb- precursor priming in outbred animals and suggest that N332-GT5 has promise to induce similar responses in humans.
  • This strategy aims to induce bnAbs by first priming rare bnAb-precursor B cells and then guiding B cell affinity maturation with a series of rationally designed boosting immunogens (A. Escolano et al. et al., 2016; B. Briney et al. et al., 2016; M. Tian et al. et al., 2016; J. G. Jardine et al. et al., 2016).
  • the germline-targeting strategy if it is to be employed for induction of other bnAbs to HIV or other pathogens, it must work with HCDR3-dominant antibodies.
  • vaccines will need to induce several different classes of bnAbs targeting different epitopes in order to achieve optimal neutralization coverage, increasing the need for effective HCDR3 -dominant germlinetargeting.
  • the BG18 type I precursors which show greater HCDR3 similarity to BG18 than type II precursors, occurred at a very low frequency in humans, approximately 1 in 50 million human naive B cells. This frequency was too low to be tested in mouse models and more than 150-fold lower than the VRCOl-class precursor frequency in humans (1 in 300,000) for which consistent bnAb-precursor priming was observed in the IAVI G001 trial.
  • consistent priming in humans will likely require activation of BG18 precursors with diverse HCDR3s, heavy chain V genes and light chains, whereas the mouse experiments validated priming of precursors with a single BG18 inferred- germline heavy chain bearing exact HCDR3 junctions from the bnAb itself.
  • V. Vigdorovich et al. et al., 2016 was concatenated into single datasets for each animal and processed along with datasets from Vigdorovich et al. (V. Vigdorovich et al. et al., 2016) using Immcantation (J. A. Vander Heiden et al. et al., 2014; N. T. Gupta et al. et al., 2015).
  • sequences were filtered to include only reads that were observed more than twice. The resulting filtered fastq files served as inputs for the next processing step.
  • RPMI Recovered cells were counted and stained with a B cell staining panel (eBioscience Fixable Viability Dye eFluor 506 (Invitrogen), mouse anti-human CD3 APC-Cy7 (SP34-2, BD Biosciences, mouse anti -human CD 14 APC-Cy7 (M5E2, BioLegend), mouse anti-human CD 16 APC-eFluor780 (eBioCB16, Thermo Fisher Scientific), mouse anti -human CD20 PerCP-Cy5.5 (2H7, BioLegend), mouse anti-human CD27 PE-Cy7 (0323, BioLegend), goat anti-human IgD FITC (polyclonal, Southern Biotech), mouse anti-human IgG BV786 (G18-145, BD Biosciences), mouse anti-human IgM BV605 (G20-127, BD Biosciences)).
  • eBioscience Fixable Viability Dye eFluor 506 Invitrog
  • Reverse transcription (RT) was performed using Clontech SMART er cDNA template switching which involves 5' CDS oligo(dT) (12 pM) being added to RNA and incubated at 72°C for 3 minutes and 4°C for at least 1 minute.
  • the RT mastermix was made using 5x RT Buffer (250 mM Tris-HCl (pH 8.3), 375 mM KC1, 30 mM MgC12), Dithiothreitol, DTT (20 mM), dNTP Mix (10 mM), RNase Out (40 U/pL), SMARTer II A Oligo (12 pM), Superscript II RT (200 U/pL) and was added to the reaction and incubated at 42°C for 90 minutes and 70°C for 10 minutes.
  • First-strand cDNA was purified using AMPure XP beads (Beckman Coulter). Following RT and purification, two PCR rounds were carried out to generate immunoglobulin amplicon libraries that were compatible with Illumina sequencing.
  • Each sample contained 2X KAPA HiFi HS RT PCR Master Mix 2x, Nuclease-free water, 10 pM of P5_Seq BC_XX 5PIIA oligo, 10 pM of P7_ i7_XX RhlgM oligo and were combined with amplified Immunoglobulin from the first round PCR and amplified for 7 cycles using real-time PCR monitoring.
  • the P5_Seq BC XX 5PIIA primers contain a randomized stretch of four to eight random nucleotides followed by a barcode sequence and this step was followed by purification with AMPure XP beads.
  • SMARTer II A Oligo: AAGCAGTGGTATCAACGCAGAGTACATrGrGrG (SEQ ID NO: 59)
  • P5 Seq BC XX 5PIIA CACGACGCTCTTCCGATCT 4-8xN AACCACTA AAGCAGTGGTATCAACGCAGAGT (SEQ ID NO: 62)
  • P7_i7_XX_RhIgM_Discover CAAGCAGAAGACGGCATACGAGAT CGATCGAA GGGGCATTCTCACAGGAGACGAGGGGGAAAAG (SEQ ID NO: 63)
  • P5_Graft P5_seq AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC T (SEQ ID NO: 64)
  • Applicants analyzed NGS datasets of 1.1 billion human BCR heavy chain sequences from 14 human donors that were previously described (J. M. Steichen et al. et al., 2019; B. Briney et al., 2019; I. R. Willis et al. et al., 2022), as well as 95.4 million macaque BCRs from 60 macaques, using the Spark analytics engine on the AWS EMR platform (EMR 6.4.0). Applicants utilized the precursor definitions provided in Fig. 29E and performed the analysis using PySpark scripts. Each node was configured with Spark, lupyterEnterpriseGateway, Hadoop, and JupyterHub via the EMR node configuration interface.
  • a precursor frequency was estimated by taking the number of BCR sequences that met a specific query definition and dividing it by the total number of BCRs for each donor. Applicants then multiplied these numbers by 1,000,000 and plotted them as frequencies per million. Median per species was shown on log scale graphs. All plots were generated using GraphPad Prism.
  • SMNP escalating-dose immunization group 4 RMs (2 females and 2 males) were immunized with 50 pg MD39 and 375 pg SMNP each side. All immunizations were given subcutaneously (s.c.) in the left and right mid-thighs. For priming, a 7- dose 12-day escalating dose strategy was used (K. M. Cirelli et al. et al., 2019) and a bolus boost immunization was given at week 10. Data from the MD39 group have been previously published (J. H. Lee et al. et al., 2022).
  • IG human immunoglobulin loci targeted enrichment protocol
  • PBMCs peripheral blood mononuclear cells
  • DNeasy kit Qiagen
  • DNA 1-2 ug was then sheared using g-tubes (Covaris) and size selected using the YYYY Kbp Marker SI -Improved Recovery cassette definition on the Blue Pippin (Sage Science). Size-selected DNA was End Repaired and A-tailed using the standard KAPA library protocol (Roche), followed by the ligation of sample-specific sequence barcodes and universal primers.
  • PCR amplification was performed for 8-9 cycles using PrimeSTAR GXL polymerase (Takara), and the resulting products were further size-selected and purified using 0.7X AMPure PB beads ( Pacific Biosciences).
  • Targetenrichment hybridization was performed using IGH/K/L-specific oligonucleotide probes (Roche).
  • Target fragments were recovered using streptavidin beads (Life Technologies), followed by a second round of PCR amplification for 16-18 cycles using PrimeSTAR GXL (Takara).
  • HiFi reads for each animal were mapped to the RheMaclO genome reference.
  • To genotype IGHD3-41 phased single nucleotide variants representing two distinct alleles were resolved from HiFi reads spanning the IGHD3-41 gene. At least 10 representative HiFi reads were required to include a given allele in the genotype of an animal.
  • ELISA plates (CorningTM 96-Well Half-Area Plates, Catalog # 3690) were precoated with anti-His antibody (Ipg/ml; Genscript) or PGT128 Fab (Ipg/ml) on Dayl.
  • the V3-peptide (Ipg/ml) was directly coated on plates on Dayl. Plates were incubated overnight at 4°C. Plates were washed with PBST (PBS + 0.2% tween 20) and HIV trimers were captured for 2h at room temperature on Day 2.
  • PBST blocking buffer
  • AUC Area Under Curve
  • LN FNAs Lymph node fine needle aspirates
  • iLNs left and right draining inguinal LNs
  • pen/strep penicillin/streptomycin
  • Ammonium-Chloride-Potassium (ACK) lysing buffer was used if the sample was contaminated with red blood cells.
  • LN FNA samples were frozen down and stored in liquid nitrogen until analysis.
  • Frozen FNA or PBMC samples were thawed and recovered in 10% FBS in RPMI. The recovered cells were counted and stained with the appropriate staining panel.
  • Fluorescent antigen probes were prepared by mixing fluorophore-conjugated streptavidin (SA) with incremental amounts of either biotinylated N332-GT5 or N332-GT5-KO in lx PBS at room temperature (RT) over the course of 45 min.
  • the KO probe, N332-GT5-KO was first added to the cells for 20 minutes, followed by the addition of WT N332-GT5 for 30 minutes, and then with the surface antibodies for 30 minutes at 4°C, similar to previously described protocols (K. M. Cirelli et al.
  • LN FNA samples were sorted for both Env+ (N332-GT5-BV421+N332-GT5-BV650+) and Env+KO' (N332-GT5- BV421+N332-GT5-BV650+/N332-GT5-KO-PE-) GC B cells.
  • the indexed V(D)J, Feature Barcode and Gene Expression libraries of sorted LN FNA or PBMC samples were prepared following the protocol for Single Indexed 10X Genomics V(D)J 5' v.1.1, with Feature Barcoding kit (10X Genomics). Custom primers designed to target RM BCR constant regions were used at concentrations previously described (J. H. Lee et al. et al., 2022).
  • LN FNA data inclusion in GC B cell gating a threshold of 250 total B cells in the sample was used.
  • Env-specific GC B cell gating a threshold of 75 total GC B cells was used.
  • the limit of detection (LOD) was calculated as the median of [3/(number of B cells recorded)] from the LN FNA samples at the pre-immunization timepoint. Samples DHHW left iLN and DHIC left iLN at week 10 were excluded from antigen-specific GC B cell analysis. Week 3 and week 4 samples were gated on all live cells.
  • Cell Ranger v.3.0.2 was used for full-length VDJ read assembly.
  • a custom RM germline VDJ reference was generated using databases published previously (K. M. Cirelli et al. et al., 2019; N. Vazquez Bemat et al. et al., 2021; C. A. Cottrell et al. et al., 2020).
  • the constants. py fde in the Cell Ranger python library was modified to increase the maximum CDR3 length to 110 nucleotides.
  • N332-GT5-KO-binding in GC B cells and memory B cells (MBCs) were determined by the PE-hashtag read counts, which were assessed by flow cytometry for each timepoint.
  • PE-hashtag thresholds were defined per tissue and timepoint based on the flow cytometry analyses. For GC B cells from timepoints of week 3 to 7, a threshold of 100 PE- hashtag read counts was used. A sequence with a read count equal to or less than 100 PE-hashtag read counts was considered as epitope-specific (N332-GT5 + KO ). For week 10 and 13, a threshold of 200 PE-hashtag read counts was used. For MBCs, a threshold of 300 PE-hashtag read count was used. Filtered VDJ contigs from the VDJ pipeline were used in further analysis.
  • the Change-0 pipeline was used to parse the 10X V(D)J sequence output from Cell Ranger into an AIRR community standardized format, to allow for more downstream analysis using the Immcantation Portal.
  • Clonal lineages were calculated for each animal using DefineClones.py with the appropriate clustering threshold value as determined by the disToNearest command from the SHazaM package.
  • Inferred germline V and I sequences from the RM reference were added with CreateGermline.py.
  • the germline D gene sequences and N nucleotide additions were masked from analysis since these cannot be accurately predicted.
  • the total number of mutations (within V- and J-genes) for each heavy chain (HC) was determined by counting the number of nucleotide changes between the observed sequence and the predicted germline sequence with the observedMutations command within SHazaM. For analysis of total HC mutations, all productive HC contigs were analyzed.
  • MS40L-low D. R. Burton et al., 2017
  • irradiated 3T3msCD40L feeder cells J. Jardine et al. et al., 2013
  • MS40L-low cells were maintained in Iscove's Modified Dulbecco's medium with GlutaMAX (IMDM) (Gibco), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Omega Scientific), 100 Units/ml Penicillin, 100 pg/ml Streptomycin (1% Pen-Strep) (Gibco), and 55 pM 2- Mercaptoethanol (2-ME) (Gibco) before sorting.
  • IMDM Iscove's Modified Dulbecco's medium with GlutaMAX
  • FBS heat-inactivated Fetal Bovine Serum
  • Penicillin 100 pg/ml Streptomycin
  • 2-ME 2- Mercaptoethanol
  • MS40L-low cells were maintained in B cell activation media: RPMI-1640 with GlutaMAX supplemented with 10% FBS, 55 pM 2 -ME, 1% Pen-Strep, 10 mM HEPES (Gibco), 1 mM Sodium Pyruvate (Gibco), 1% MEM NEAA (Gibco), while irradiated 3T3msCD40L cells were thawed on day of sorting and maintained in IMDM supplemented with 10% FBS, IX MycoZap Plus-PR (Lonza).
  • B cell activation media RPMI-1640 with GlutaMAX supplemented with 10% FBS, 55 pM 2 -ME, 1% Pen-Strep, 10 mM HEPES (Gibco), 1 mM Sodium Pyruvate (Gibco), 1% MEM NEAA (Gibco), while irradiated 3T3msCD40L cells were thawed
  • HEK293T cells (ATCC) were used to produce viruses and maintained in complete Dulbecco's modified Eagle's medium (DMEM) (Gibco), supplemented with 10% FBS, 2 mM L-glutamine (Gibco), and 1% Pen-Strep.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS FBS
  • 2 mM L-glutamine Gibco
  • Pen-Strep 1% Pen-Strep.
  • TZM-bl cells (NIH AIDS Reagents Program) (RRID:CVCL_B478) were maintained in complete DMEM and used as target cells in pseudovirus neutralization assays. All cell lines were maintained at 37°C in a humidified atmosphere of 5% CO2.
  • N332-GT5 epitope-specific memory B cells Isolation of N332-GT5 epitope-specific memory B cells by flow cytometry for culture
  • Fluorescently labeled antigens used for sorting were generated on the day of the sort by incubating 4 pM biotinylated N332-GT5 WT with streptavidin Alexa Fluor 647 (N332-GT5- AF647) (Invitrogen, cat# S21374) and streptavidin Alexa Fluor 488 (N332-GT5-AF488) (Invitrogen, cat# SI 1223) separately; and 4 pM biotinylated N332-GT5 KO with BV421 streptavidin (N332-GT5 KO-BV421) (BD Biosciences, cat# 563259) at a 2:1 molar ratio at room temperature for 1 hour in the dark.
  • N332- GT5-AF647 and N332-GT5-AF488 WT antigens were added and incubated for an additional 30 min at 4°C in the dark. All antibodies were added at a 1 : 100 dilution in lOOpL and antigens at 100 nM final. Finally, 1 :300 LIVE/DEAD fixable cell dye (Invitrogen, cat# L34957) was added and incubated for 15 min at 4°C in the dark, washed, and resuspended to the desired volume.
  • Double positive N332-GT5 WT, KO negative epitope-specific memory B cells (N332-GT5 ++ /N332-GT5- KO’ IgMlgG + B cells) were single-cell sorted into 96-well plates pre-seeded with appropriate feeder cells using a BD FACSMelody Cell Sorter. Post-sort analyses were done with FlowJo 10.7.2 (FlowJo, LLC). [00366] Single memory B cell culture and Activation
  • MS40L-low feeder cells (MS40L-low cultures) were pre-seeded 24 hours before in 96-well plates at a density of 3 x 103 cells/well in 100 pL B cell activation media and supplemented with 100 pL 2X cytokines on the day of sort: 20 ng/mL IL-4 (Peprotech, cat# 200-04), 20 ng/mL IL-21 (Peprotech, cat# 200-21), 200 ng/mL IL-2 (Peprotech, cat# 200-02), 200 ng/mL BAFF (Peprotech, cat# 310- 13).
  • Irradiated 3T3msCD40L feeder cells were seeded the day of sorting at a density of 4 x 104 cells/well in IMDM complete medium and supplemented with 50 ng/mL each of IL-4, IL-21, IL-2, 100 ng/mL Anti -rhesus IgG (H+L) (Bio-Rad, cat# AAI42) and cultured for 14 days (J. Huang et al. et al., 2013). B cell culture supernatants from days 12, 14, 15, and 18 were harvested and used for IgG and antigen ELISA. After media removal, B cells in 96-well plates were frozen at -80°C without any additional buffer and used for B cell receptor sequence analysis.
  • B cell culture supernatants from days 12, 14, 15, and 18 were used to screen for B cell activation (IgG expression) and antigen binding. Briefly, 96-well half area high binding plates (Corning cat# 3690) were coated overnight at 4°C with AffiniPure F(ab’)2 Fragment Goat Antihuman IgG (H+L) (Jackson Immunoresearch, cat# 109-006-088) diluted 1 :500 in PBS, or 6x- His tag monoclonal antibody diluted 1 :500 in PBS (Invitrogen, cat# MAI-21315).
  • Plates were washed 3x, and detected with alkaline phosphatase (AP)-conjugated anti-Human IgG Fc fragment specific secondary (Jackson Immunoresearch, cat# 109-055-098) diluted 1 : 1000 in 1% BSA/PBS and incubated for 1 hour at RT. Plates were developed with phosphatase substrate (Sigma-Aldrich, cat# S0942), and absorbance was measured at 405 nm.
  • AP alkaline phosphatase
  • Jackson Immunoresearch cat# 109-055-098
  • Antigen-specific activated B cells were selected for B cell receptor sequence analysis. mRNA extraction, cDNA, and nested PCR reactions were done as previously described (F. Zhao et al. et al., 2020). Briefly, frozen cells were thawed, lysed, and mRNA extracted using TurboCapture 96 mRNA kit (QIAGEN, cat# 72251) according to the manufacturer’s protocol. mRNA was reverse transcribed, and cDNA was subjected to nested PCR reactions for Ig heavy and light chain variable regions.
  • PCR products were analyzed with 2% 96 E-gel (Thermofisher, cat# G720802), and wells with PCR products corresponding to Ig heavy and light chain were cleaned with SPRI beads (Beckman Coulter, cat# B23319). Cleaned PCR products were sequenced directly and/or selected for Gibson cloning (NEB, cat# E2621X) prior to Sanger sequencing.
  • a BG18 type I precursor was defined if it had less than four mutations from perfect regular expression match and was found in index position 4,5 or 6 in the HCDR3 sequence. In addition, applicants recorded if a glutamate followed +2 positions from the end of the matching regular expression.
  • the clustering module of SADIE library (github.com/jwillis0720/Sadie. git) was used to cluster both the BG18 Type I and Type I alternate using the following criteria.
  • the sequences were only clustered on the heavy chain and were first grouped by animal and HCDR3 length. Within each group, a distance was computed for all antibodies. The distance was calculated as a Levenshtein distance between the HCDRls + HCDR2s + HCDR3s.
  • the somatic pad option was used in SADIE which a distance of 1 was subtracted for every common somatic amino acid mutation (D. J. Leggat et al. et al., 2022).
  • the final distance matrix was used for agglomerative clustering using average linkage and a distance cutoff of 3. The final clusters were annotated in the dataframe.
  • the clustering module of SADIE was also used in the clustering of off-target sequences defined as those sequences with an N332-GT5 KO antigen count of ⁇ 100 (D. J. Leggat et al. et al., 2022). These sequences had distance matrix constructed such that both heavy and light chain were considered where the distance between every antibody was computed across all six CDR chains. Single-linkage agglomerative clustering was used with a cutoff of 5 to get final cluster assignments. Large clusters that were found in multiple animals and multiple weeks were prioritized for synthesis and testing with SPR.
  • Pseudoviruses were produced in HEK293T cells (RRID:CVCL_0063) co-transfected using FuGENE 6 (Promega, cat# E2691) with pseudovirus Env-expressing plasmid and Env- deficient backbone plasmid (PSG3AEnv). Pseudoviruses were harvested 72 hours posttransfection, sterile filtered (0.45 pm), and concentrated (EMD Millipore, cat# UFC905024). Equal volumes of serially diluted monoclonal antibodies at appropriate concentrations were incubated with HIV pseudovirus in half-area 96-well plates (Greiner, cat# 675083) at 37°C for 1 hour.
  • TZM-bl cells 50 pL of TZM-bl cells at 200,000 cells/mL with or without DEAE-dextran (5 pg/mL final concentration) were added to each well containing the antibody-virus mixture and incubated at 37°C for 72 hours in a humidified atmosphere of 5% CO2. After incubation, culture media was removed, and cells were lysed with 45 pL/well lx Luciferase Culture Lysis buffer (Promega, cat# El 531) for 20 min at RT. Neutralization was measured by adding 30 pL luciferase reagent/well (Promega, cat# El 500) and measuring luminescence.
  • IC50 was calculated using a nonlinear regression curve fit, sigmoidal, 4PL equation constrained from 0-100% in GraphPad Prism 9.3.1. IC50 is reported as the mean IC50 ⁇ SD of two biological replicates.
  • the N332-GT5 trimer immunogen contained two modifications compared to what has been described previously (J. M. Steichen et al. et al., 2019) (Fig. 46).
  • the trimer was stabilized with a set of mutations called MD65, which is defined as the MD39 stabilizing mutations (J. M. Steichen et al. et al., 2016) plus four additional stabilizing mutations (V505T, V513A, V518S, L520D).
  • Second, glycosylation sequons were added to fill the 241 and 289 glycan holes as described previously (D. W. Kulp et al. et al., 2017).
  • Trimers in the pHLsec vector were cotransfected with furin in a 2:1 ratio into 293F cells (RRID:CVCL_D603) cultured in FreeStyle media using either 293Fectin or PEI as a transfection reagent. Proteins were harvested from the supernatant after 7 days incubation at 37°C and untagged trimers were purified by 2G12 antibody affinity chromatography using a HiTrap NHS-activated HP column (Cytiva, Cat#17-0717-01) run on an AKTA Pure 25L HPLC (Cytiva, Cat# 29-0182-24).
  • C-terminal His-tagged trimers were purified using a HIS-TRAP column, starting with a wash buffer (20mM Imidazole, 500 mM NaCl, 20mM Na2HPO4) and mixing in elution buffer (500 mM Imidazole, 500 mM NaCl, 20 mM Na2HPO4) using a linear gradient. Trimers were polished by size exclusion chromatography (SEC) using a Superdex 200 16/600 size exclusion chromatography column (Cytiva, Cat 28-9893- 35) run on an AKTA Pure 25L HPLC. Final proteins were diluted in lx TBS and stored at -80°C.
  • proteins were expressed with a His-tag and avi-tag (GTKHHHHHHGGSGGSGLNDIFEAQKIEWHE) (SEQ ID NO: 68), purified using a HIS- TRAP column followed by SEC, and biotinylated using a BirA biotin-protein ligase reaction kit (Avidity, Cat# BirA500) according to the manufacturer instructions.
  • the N332-GT5 and N332- GT5-KO sorting probes did not contain the 241/289 glycosylation sequons.
  • Fab and antibody purification [00387] Paired HC and LC Fab variable region sequences from select NHP affinity matured mAbs were gene synthesized and inserted into human Fab HC constant region expressing vector pFabCW and human lambda or kappa expressing vectors pCW-CLig-hL2 or pCW-CLig-hk, respectively. Fabs were expressed in 500 mL FreeStyleTM 293F cell cultures or 30 mb ExpiCHOTM cell cultures (Thermo Fisher Scientific, Cat# A29133).
  • HC and 150 pg of LC plasmids were mixed with 225 pg polyethylenimine (PEI; 1:3 DNA:PEI ratio) in 5 mL of Opti-MEMTM reduced serum medium (Thermo Fisher Scientific, Cat# 31985070) for 30 min, then added to 293F cells. Supernatant was collected after 5-6 days. ExpiCHOTM cell cultures were transfected according to manufacturer instructions, using 12.5 pg HC and 31.2 pg LC plasmids. Supernatant was collected 8 days post transfection.
  • PEI polyethylenimine
  • Regeneration was accomplished using phosphoric acid 1.7% or 0.85% with a 180-s contact time and injected four times per cycle.
  • ProteOn Manager software Bio-Rad was used to analyze raw sensograms, including interspot and column double referencing, and to perform either Equilibrium fits or Kinetic fits with Langmuir model, or both, when applicable.
  • N332-GT5 was incubated with mouse polyclonal Fabs, purified as above and concentrated to 2.6 mg/ml. Later data processing (below) revealed that a majority of trimers were unliganded. Cryo grids were prepared using a Vitrobot Mark IV (Thermo Fisher Scientific). The temperature was set to 4°C and humidity was maintained at 100% during the freezing process. The blotting force was set to 1 and wait time was set to 10 s. Blotting time was varied from 5 to 6 s.
  • Quantifoil R 1.2/1.3 Cu, 300-mesh; Quantifoil Micro Tools GmbH
  • UltrAuFoil 1.2/1.3 Al, 300-mesh; Quantifoil Micro Tools GmbH
  • grids were used and treated with Ar/O2 plasma (Solarus plasma cleaner, Gatan) for 8 sec before sample application.
  • 0.5 pL of detergent was mixed with 3.5 pL of samples and 3 pL of the mixture was immediately loaded onto the grid. Following blotting, the grids were plunge-frozen into liquid nitrogen-cooled liquid ethane.
  • N332-GT5 (partially complexed with mouse polyclonal antibodies) data collection occurred at the Pacific Northwest Center for Cryo-EM (PNCC) using a Thermo Fisher Scientific Krios and a Gatan K3 direct electron detector (300 keV, 0.40075 A/pixel super-resolution mode).
  • EPU Thermo Fisher
  • Relion 3.1 J. Zivanov et al., 2020
  • Micrographs were binned during motion correction, with a resulting pixel size of 0.8015 A/pixel and imported in cryoSPARC.
  • CTF correction was performed using cryoSPARC Patch CTF.
  • particle picking was performed using blob picker initially followed by template picker.
  • particles were downscaled to 1.044 A/pix during extraction to reduce box size and increase speed of downstream jobs.
  • Arctica datatsets were processed at the native 1.15 A/pix size, and the Krios dataset was processed at the binned 0.8015 A/pix size.
  • Multiple rounds of 2D classification and 3D ab-initio reconstruction were performed prior to 3D non-uniform refinement with global CTF refinement.
  • For the unligandedN332-GT5 dataset many rounds of 2D classification were performed to remove the subpopulation of particles with mouse polyclonal Fab. Final refinements were performed with C3 symmetry and global resolution estimated by FSC 0.143.
  • Fab Fv homology models were generated using SAbPred ABodyBuilder-ML (B. Abanades et al., 2022). Model building was performing by docking homology models of trimer and Fab Fv in UCSF Chimera (E. F. Pettersen et al. et al., 2004), manually building and refinement in Coot 0.9.8 (A. Casanal et al., 2020) and real space refinement using Rosetta (P. Conway et al., 2014) and Phenix (P. V. Afonine et al. et al., 2018). Final models were validated using MolProbity and EMRinger in the Phenix suite.
  • the center of mass for three N332 epitopes at the trimer 3-fold axis was aligned to coordinates (0, 0, 0) and a third point (the center of mass for the CA of L587 in all three protomers) was aligned to coordinates (0, 0, -50.88).
  • the latitudinal angle was the angle formed by the z-axis and a vector from the N332 epitope center of mass to the HC center of mass (CA atoms for 6 beta strands, residues 21-24, 34-39, 46-52, 67- 71, 77-82, 89-92 for BG18_iGL0) in the x-z plane.
  • the longitudinal angle was the angle formed by the x-axis and the same vector connecting the N332 epitope to the HC in the x-y plane.
  • the HC-LC twist angle was the angle between the x-axis and a vector connecting the HC center of mass to the LC center of mass (CA atoms for 6 beta strands, residues 18-24, 34-37, 45-48, 62-66, 70-76, 84-88 for BG18_iGL0) in the x-y plane.
  • BG505_B23 was found to have low glycan occupancy by mass spectrometry analysis and therefore three new trimers were designed using three different approaches.
  • BG505 B38 was designed by reverting four VI loop amino acids from BG505 B23 back to the WT BG505 amino acid.
  • BG505 SOSIP has been shown to have better glycan occupancy in the VI loop than what the applicants observed with BG505_B23, which indicated that the germline targeting mutations in the VI loop were causing reduced glycan occupancy (L. Cao et al. et al., 2017). Therefore, reverting more GT mutations back to the WT amino acid should potentially improve the glycosylation.
  • BG505 B46 reported here, had a superior binding profile to BG18 type I Fabs.
  • BG505 B48 was designed to have improved N137 glycan occupancy using an optimize sequence described previously (S. S.
  • BG505 B48 does not contain an N133 glycosylation sequon. All three approaches produced trimers with near complete glycan occupancy in the VI loop when expressed in 293F cells (Fig. 39). Amino acid sequences of BG505 B23, BG505 B46, and BG505 B48 are provided in Fig. 46.
  • Method 1 DeGlyPHER (M. Caskey et al., 2017) was used to ascertain site-specific glycan occupancy and processivity on the examined glycoproteins.
  • Proteinase K treatment and deglycosylation HIV Env glycoprotein was exchanged to water using Microcon Ultracel PL-10 centrifugal filter. Glycoprotein was reduced with 5 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HC1) and alkylated with 10 mM 2- Chloroacetamide in 100 mM ammonium acetate for 20 min at room temperature (RT, 24°C). Initial protein-level deglycosylation was performed using 250 U of Endo H for 5 pg trimer, for 1 h at 37°C. Glycorotein was digested with 1 :25 Proteinase K (PK) for 30 min at 37°C.
  • TCEP-HC1 tris(2-carboxyethyl)phosphine hydrochloride
  • PK was denatured by incubating at 90°C for 15 min, then cooled to RT. Peptides were deglycosylated again with 250 U Endo H for 1 h at 37°C, then frozen at -80°C and lyophilized. 100 U PNGase F was lyophilized, resuspended in 20 pl 100 mM ammonium bicarbonate prepared in H2 18 O, and added to the lyophilized peptides. Reactions were then incubated for 1 h at 37°C, subsequently analyzed by LC-MS/MS.
  • LC-MS/MS Samples were analyzed on an Q Exactive HF-X mass spectrometer. Samples were injected directly onto a 25 cm, 100 pm ID column packed with BEH 1.7 pm C18 resin. Samples were separated at a flow rate of 300 nL/min on an EASY-nLC 1200 UHPLC. Buffers A and B were 0.1% formic acid in 5% and 80% acetonitrile, respectively. The following gradient was used: 1-25% B over 160 min, an increase to 40% B over 40 min, an increase to 90% B over another 10 min and 30 min at 90% B for a total run time of 240 min. Column was reequilibrated with solution A prior to the injection of sample.
  • Peptides were eluted from the tip of the column and nanosprayed directly into the mass spectrometer by application of 2.8 kV at the back of the column.
  • the mass spectrometer was operated in a data dependent mode. Full MSI scans were collected in the Orbitrap at 120,000 resolution. The ten most abundant ions per scan were selected for HCD MS/MS at 25 NCE. Dynamic exclusion was enabled with exclusion duration of 10 s and singly charged ions were excluded.
  • Method 2 Glycan structure identification was employed to detect specific glycoforms.
  • Three aliquots of each sample were denatured for Ih in 50 mM Tris/HCl, pH 8.0 containing 6 M of urea and 5 mM dithiothreitol (DTT). Next, Env proteins were reduced and alkylated by adding 20 mM iodoacetamide (IAA) and incubated for Ih in the dark, followed by a Ih incubation with 20 mM DTT to eliminate residual IAA.
  • IAA iodoacetamide
  • the alkylated Env proteins were buffer- exchanged into 50 mM Tris/HCl, pH 8.0 using Vivaspin columns (3 kDa) and two of the aliquots were digested separately overnight using trypsin, chymotrypsin (Mass Spectrometry Grade, Promega) or alpha lytic protease (Sigma Aldrich) at a ratio of 1 :30 (w/w). The next day, the peptides were dried and extracted using Cl 8 Zip-tip (MerckMilipore).
  • the peptides were dried again, re-suspended in 0.1% formic acid and analyzed by nanoLC-ESI MS with an Ultimate 3000 HPLC (Thermo Fisher Scientific) system coupled to an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific) using stepped higher energy collision-induced dissociation (HCD) fragmentation.
  • Peptides were separated using an EasySpray PepMap RSLC C18 column (75 pm x 75 cm). A trapping column (PepMap 100 C18 3pM 75pM x 2cm) was used in line with the LC prior to separation with the analytical column.
  • the LC conditions were as follows: 280 minute linear gradient consisting of 4-32% acetonitrile in 0.1% formic acid over 260 minutes followed by 20 minutes of alternating 76% acetonitrile in 0.1% formic acid and 4% Acn in 0.1% formic acid, used to ensure all the sample had eluted from the column.
  • the flow rate was set to 300 nL/min.
  • the spray voltage was set to 2.5 kV and the temperature of the heated capillary was set to 55 °C.
  • the ion transfer tube temperature was set to 275 °C.
  • the scan range was 375-1500 m/z.
  • the AGC target for MSI was set to standard and injection time set to auto which involves the system setting the two parameters to maximize sensitivity while maintaining cycle time.
  • Glycopeptide fragmentation data were extracted from the raw file using Byos (Version 3.5; Protein Metrics Inc.). The MS data was searched using the Protein Metrics 305 N-glycan library with sulfated glycans added manually. The relative amounts of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted chromatographic areas for different glycotypes with an identical peptide sequence. All charge states for a single glycopeptide were summed. The precursor mass tolerance was set at 4 ppm and 10 ppm for fragments. A 1% false discovery rate (FDR) was applied. The relative amounts of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted ion chromatographic areas for different glycopeptides with an identical peptide sequence. Glycans were categorized according to the composition detected.
  • HexNAc(2)Hex(10+) was defined as M9Glc
  • HexNAc(2)Hex(9-5) was classified as M9 to M3. Any of these structures containing a fucose were categorized as FM (fucosylated mannose).
  • HexNAc(3)Hex(5-6)X was classified as Hybrid with HexNAc(3)Hex(5-6)Fuc(l)X classified as Fhybrid.
  • Complex-type glycans were classified according to the number of HexNAc subunits and the presence or absence of fucosylation.
  • compositional isomers are grouped, so for example a triantennary glycan contains HexNAc 5 but so does a biantennary glycans with a bisect.
  • Core glycans refer to truncated structures smaller than M3.
  • M9glc- M4 were classified as oligomannose-type glycans.
  • Glycans containing at least one sialic acid or were categorized as NeuAc and at least one fucose residue in the “fucose” category.
  • N332-GT5 was designed to target BG18 precursors recombined from human genes
  • BG18 type I human precursors as antibodies with HCDR3s of the same length as BG18 [23 amino acids (aa)], the same heavy chain D gene (D3-3) in the same reading frame and position within the HCDR3, and the same heavy chain joining gene (JH6) (Fig. 25 A).
  • BG18 type I precursors HCDR3 length > 22 aa with a D3-41 gene (homologous to human D3-3) in the same reading frame and in a similar position as D3-3 in BG18 (allowing a shift of ⁇ 1 aa).
  • HCDR3 lengths > 22 aa are five-times more frequent in humans compared to macaques, which can account for the majority of the observed precursor frequency differences between the species (Fig. 25B and Fig. 29).
  • these frequencies are substantially higher than the frequencies of N332- GT5-binding precursors (approximately 1 in 50 million in humans), because they do not account for HCDR3 junction determinants or light chain features that affect binding to N332-GT5.
  • the data indicate RMs are likely a more stringent model for N332-GT priming than humans due to a lower BG18-like B cell precursor frequency.
  • N332-GT5 trimer-specific (N332- GT5+/N332-GT5+ labeled with different fluorophores, “N332-GT5 ++ ” hereafter) and N332 epitope-specific (N332-GT5 ++ /N332-GT5-KO ) GC B cells were sorted from FNA samples, and BCR sequences were recovered by single cell RNA sequencing (Figs. 31 and 32).
  • Class switched IgD' memory B cells were also sorted and sequenced from week 10 PBMC samples using this workflow (Figs. 31 and 32).
  • IgG MBCs from week 12 (two weeks post boost) PBMCs that were specific for the N332 epitope (N332-GT5 ++ /N332-GT5-K0‘) were singlecell sorted into 96-well plates and cultured for three weeks, and then BCR sequences were obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA sequencing for wells that were confirmed N332-GT5-positive by supernatant ELISA (Figs. 32 and 33).
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • N332-GT5 trimer-binding B cells From N332-GT5 trimer-binding B cells, applicants obtained a total of 23,467 heavy chain (HC) and 22,352 heavy chain-light chain paired BCR sequences from GCs, and 1,511 HC with 869 paired BCR sequences from memory B cells. Applicants determined the number and frequency of BG18 type I sequences among GC B cells at each time point and among IgD‘ memory B cells 10 weeks post prime or IgG + memory B cells two weeks post boost (Fig. 25E-J).
  • BG18 type I sequences in trimer-specific GC B cells in 7 of 8 animals at a median frequency among responders of 0.93%> when all time points were combined (Fig. 25F).
  • Applicants also detected BG18 type I responses in PBMCs from 7 of 8 animals, with the one PBMC non-responder being positive in the GC B cell samples, meaning that overall, applicants detected BG18 type I responses in all 8 animals.
  • the median BG18 type I frequency among trimer-specific B cells from responders was 1 .2% in IgD' MBCs at 10 weeks post prime, and 3.4% in IgG + MBCs two weeks post boost (Fig. 251).
  • the BG18 type I frequency was 0.021% two weeks after the boost, which was 3.6-fold higher than the frequency among IgD" MBCs of responders at the time the boost was given at week 10 (0.006%) (Fig. 25 J).
  • VRC01 -class IgG + memory B cells were detected at frequencies of 0.088% and 0.13% for low dose and high dose groups, respectively, within an order of magnitude ofthe BG18-like responses here.
  • N332-GT5 consistently induced BG18- like type I B cell responses in RMs.
  • N332-GT5 molecule was designed to engage antibodies containing a specific HCDR3 motif (derived from BG18) but bearing diverse VH and VL gene segments (J. M. Steichen et al. et al., 2019). To determine if that germline-targeting goal was met, applicants carried out a lineage analysis of the BG18 type I antibodies. The sequences were clustered by animal ID, HCDR3 length, and VH gene usage.
  • the animals were found to have two alleles (*01 and *01_S8240), and all three possible genotypes were represented (*01/*01, *01/*01_S8240, and *01_S8240/*01_S8240).
  • the two alleles each differed from human D3-3 by one amino acid in the BG18 reading frame, and they differed from each other by two amino acids.
  • BG18 type I lineages were derived from both alleles of D3-41, with *01 representing 24 lineages and *01 S8240 representing 14 lineages.
  • Applicants employed cryo-electron microscopy (cryo-EM) to determine whether N332-GT5-induced BG18 type I BCRs engaged the N332-GT5 trimer with a BG18-like binding mode as predicted by the sequence analysis.
  • Applicants determined structures of three BG18 type I Fabs, each containing distinct genetic features, in complex with the N332-GT5 trimer, and applicants also determined the structure of the unliganded trimer for comparison (Figs. 26B-E).
  • the first Fab, RM_N332_03 had a canonical BG18 type I sequence that used the same gene families as BG18 (VH4, VL3, D3-41).
  • the second, RM_N332_36 also used a VL3 light chain (LC) but used D3-41 in a different reading frame and position to produce an HCDR3 closely resembling that ofBG18.
  • the third Fab, RM N332 32 had aBG18 type I HCDR3 sequence but used a kappa chain V gene, V l, the most common kappa chain V-gene family among RM-derived BG18 type I antibodies. Structural analysis confirmed that all three Fabs had a binding mode similar to that of the BG18 inferred germline (BGI8 GL0), with the HCDR3 engaging the base of V3 and the LC straddling the VI loop (Figs. 26B-E).
  • the two Fabs that used VL3 LCS showed especially high similarity in binding orientation to BG18_GL0 in complex with N332-GT2, whereas the RM_N332_32 Fab with a kappa LC showed a somewhat rotated binding orientation relative to BG18 GL0, possibly the result of using a kappa LC (Fig. 36).
  • the epitope footprint (Fig. 37) and HCDR3 conformation (Figs. 26B-E) of all three Fabs showed good similarity to BG18 GL0.
  • BG18 type I antibodies with other D genes and/or reading frames may be relevant to human vaccination as well since applicants were able to substitute several alternate D gene sequences into the BG18 inferred germline while maintaining high affinity binding to N332-GT5.
  • Affinity maturation is a requirement for bnAb development, and induction of on-path affinity maturation is thus a key requirement of germline targeting strategies.
  • the BG18 type I BCRs from GCs acquired SHM that increased from a median of 1% VH aa mutation at week 3 to 7% at week 10 (Fig. 27A).
  • BG18 type I BCRs from PBMCs also showed SHM that increased from 3% VH aa mutation at week 10 to 8% at week 12 (two weeks post boost) (Fig. 27B) and there were similar levels of VL mutations in the LC (Figs. 27C-D).
  • BG18 type I antibodies showed similar levels of SHM as other Env+ antibodies (Figs. 27A-D).
  • BG18 type I Fabs isolated at later time points could bind to trimers that have a more native-like epitope than the priming immunogen
  • BG505 B46, BG505 B48 and BG505 B38 were designed using three different approaches described in methods. All three newly designed trimers showed near complete occupancy of the N137 glycosylation site, and BG505 B46 and BG505 B38 showed near complete occupancy of theN133 glycosylation site (BG505_B48 lacks the N133 site) (Fig. 39).
  • trimers with high VI glycan occupancy showed detectable binding to 65%, 57% and 48% of the BG18 type I Fabs for BG505 B46, BG505 B48, and BG505 B38, respectively.
  • Their median KDS ranged from 1 pM to >20 pM (Fig. 27F).
  • 65% of BG18 type I antibodies isolated at weeks 7 and 10 post prime could bind to at least one trimer containing all glycans in the N332 epitope, a necessary requirement of an N332-dependent bnAb.
  • BG18 type I antibodies showed the ability to neutralize pseudoviruses expressing BG505-based envelope proteins with N332-GT5 mutations introduced only to the VI loop (Fig. 41).
  • the other epitope-specific Fabs showed high affinity for N332-GT5 (median KD of 79 pM) and greatly reduced affinity for N332-GT5-KO (Fig. 27F).
  • the other epitope-specific Fabs showed either no binding or greatly reduced binding to the four trimers with VI loop glycosylation sites restored (Fig. 27F), indicating that they are dependent on the VI glycan hole.
  • BG18 type II B cells Priming of BG18 type II B cells would be significant as this would indicate that a larger and more diverse pool of BG18-related precursors contribute to an N332-GT5-induced response.
  • BG18 type I antibodies Fig. 44
  • BG18 type II antibodies may occur at low frequency in macaques.
  • applicants screened the activated supernatants of N332-GT5-sorted week 12 memory B cells against the BG505 B23 trimer (Fig. 45), a molecule that is highly selective for binding to BG18 type I antibodies over other epitope-specific antibodies (Fig. 27F).
  • This screen identified BG505_B23-binding supernatants, and sequence analysis revealed these wells to contain antibodies with a wide distribution of HCDR3 lengths.
  • HCDR3s > 20 aa there were both BG18 type I and non-BG18 type I sequences (Fig. 45).
  • Applicants produced a subset of Fabs with HCDR3s > 20 aa that were not BG18 type I sequences and found that 7 of 8 showed detectable binding to the BG505 B23 trimer by SPR and 3 of 8 showed high-affinity binding (KD ⁇ 100 nM; Fig. 28B).
  • RM N332 07 showed a binding orientation that was highly similar to BG18, with the LC straddling the V 1 loop and the HCDR3 making interactions to conserved residues at the base of V3, including R327, H330 and the N332 glycan (Fig. 28D-E and Figs. 53A-B).
  • an antibody having no recognizable BG18 sequence features other than a long HCDR3 can bind to the N332-GT5 trimer in a manner that is highly homologous to BG18.
  • the Fab RM N332 08 also showed a similar orientation, with the LC straddling the VI loop and the HCDR3 having partial overlap with the BG18 epitope at the base of V3 but with additional contacts to the VI loop (Fig. 28F and Fig. 38C).
  • Applicants define these antibodies as BG18 type III, having a long HCDR3 (> 20 aa) and a binding angle of approach and epitope-footprint similar to BG18, but lacking the HCDR3 sequence features found in BG18 type I sequences and not using VL3-25, VL3-1 , or VL3-10 LCs that define a BG18 type II sequence.
  • N332-GT5 was designed to induce diverse human BG18-class type I precursors.
  • N332-GT5 proved capable of inducing such precursors in RMs with different immunoglobulin genes and rarer precursor frequencies, which altogether suggest that N332-GT5 has substantial potential to prime diverse BG18 type I precursors consistently in humans.
  • BG18 Boost 1 candidates BG505_MD65_congly_N332B46_m, BG505_MD39_congly_N332B54_m, and 001428_MD39_L14_N332B50_m2 were tested for binding affinity to NHP-elicited BG18-class antibodies isolated after priming with N332-GT5 trimer+SMNP.
  • NHPs were immunized with N332-GT6 mRNA-LNPs and 8 weeks later boosted with BG505-B11 mRNA- LNPs.
  • BG18-class antibodies isolated 2 weeks post boost 1 were produced and their affinities were measured by SPR.
  • Vaccination induces broadly neutralizing antibody precursors to HIV gp41
  • a key barrier to development of vaccines that induce broadly neutralizing antibodies (bnAbs) against HIV and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells.
  • the high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif.
  • HCDR3 long heavy chain complementarity determining region 3
  • Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens; protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques; and mRNA-encoded nanoparticles triggered similar responses in mice.
  • germline-targeting epitope-scaffold nanoparticles can elicit rare bnAb-precursor B cells with pre-defined binding specificities and HCDR3 features.
  • MPER bnAbs face challenges, including the recessed location of the MPER at the base of the Env trimer (Huang et al., 2012; Rantalainen et al., 2020) the need to induce antibodies with long HCDR3s bearing specific sequence motifs, and the lack of affinity of most MPER bnAb precursors for their peptide epitopes (Klein et al., 2013; Soto et al., 2016; Zhang et al., 2019).
  • MPER bnAbs 2F5 and 4E10 immune tolerance mechanisms block the induction of MPER bnAbs 2F5 and 4E10, potentially due to lipid reactivity (Haynes et al., 2005), raising concerns that other more potent MPER bnAbs, such as 10E8, might also face tolerance barriers (Verkoczy et al., 2010; Verkoczy et al., 2011; Chen et al., 2013; Doyle-Cooper et al., 2013).
  • germline-targeting epitope-scaffold nanoparticle priming immunogens to induce 10E8-class HCDR3 -dominant bnAb-precursor responses. These immunogens represent candidates for human vaccination and demonstrate design and evaluation processes that could be applied to other bnAb targets.
  • MPER bnAb precursor frequencies were estimated from publicly available NGS data of -1.1x109 heavy chain sequences from 14 HIV-seronegative donors as previously described (Steichen et al., 2019; Willis et al., 2022).
  • T117v2 scaffold was not well-suited for this goal since both N- and C-termini of the scaffold are near the epitope, hence genetic fusion of the epitope-scaffold to a self-assembling protein would result in poor exposure of the epitope on the nanoparticle.
  • generation 10E8-GT8 applicants switched from T117v2 to T298, a previously described circularly permuted variant of T117 withN- and C-termini opposite the epitope (Correia et al., 2011).
  • N-linked glycosylation sites onto the scaffold in order to focus B-cell responses on the MPER epitope.
  • Applicants initially explored the introduction of single artificial N-linked glycosylation sites into irrelevant surfaces of the epitopescaffold of 10E8-GT8 1. Sites that decreased affinity for 10E8-iGL2 by no more than 1.3-fold and decreased expression yields by no more than 40% were selected for further investigation.
  • Applicants next tested combinations of multiple glycosylation sites on 10E8-GT8.1 and obtained 10E8-GT8.2 with four N-linked glycosylation sites. Applicants further increased the number of N- linked glycosylation sites with each subsequent generation of immunogens.
  • the key residues of 10E8 are the Dn-gene-encoded residues at the tip of the CDRH3, which were present in all 10E8-class precursors used in this study. Applicants hypothesized that additional interactions of the scaffold with these key residues might increase the affinity and breadth for precursors that share these features.
  • Applicants combined this new Dn binding loop into a combinatorial yeast library with other promising residues from the remaining combinatorial NNK-patch screenings and enriched for binding to 10E8-class precursors.
  • Enrichment with 10E8-UCA resulted in 10E8-GT9.1 that bound to the 10E8 UCA and several NGS precursors.
  • Reversion of each MPER mutation revealed that mutation N132I (N677I in Hxb2 numbering) had no impact on binding affinity, hence this mutation was subsequently removed.
  • Applicants therefore further refined the scaffold for increased expression by removing exposed hydrophobic patches, adding additional glycosylation sites, and further improving the D-gene binding pocket by incorporating mutation V42A, discovered from yeast display of an error-prone PCR library of GT10.1 that was simultaneously enriched with 10E8-UCA Fab (directly conjugated to Alexa Fluor 647) and NGS- 57 (stained with PE-conjugated anti-human Fey secondary antibody; Jackson ImmunoResearch).
  • 10E8-GT10.2 Fusion of the resulting protein, termed 10E8-GT10.2, to the glycosylated 3-dehydroquinase from Thermus thermophilus via a linker that incorporated the PADRE56 epitope yielded homogenous particles of the expected molecular weight, termed 10E8-GT10.2 12mer (n.b. unexpectedly, the addition of PADRE to the linker in the GT10.2 12mer increased expression levels substantially).
  • 10E8-GT9 and 10E8-GT10 immunogens bound to several NGS precursors, they bound much more weakly to mature and artificial intermediate 10E8-lineage members compared to previous immunogen generations. Applicants therefore transferred the MPER of 10E8-GT8, the most advanced version that retained strong binding to mature and intermediate 10E8 variants, onto 10E8-GT10.2, moved an N-linked glycosylation site into the scaffold surface patch engaged by 10E8-NGS-03, and added mutation W680N to the MPER graft. The resulting construct, termed 10E8-GT11, bound with high affinity to mature 10E8 (KD 1.4 nM) but interacted only weakly with 10E8-class precursors.
  • yeast display was performed by identifying beneficial mutations using an error-prone PCR library, followed by screening of a combinatorial library that combined identified mutations using NGS precursors.
  • 10E8-GT12 24mers by fusing 10E8-GT12 to each terminus of the 3- dehydroquinase nanoparticle protomers.
  • 10E8-GT12 24mers rather than using linkers with PADRE, applicants included exogeneous T-help peptides derived from Aquifex aeolicus lumazine synthase that were found to be broadly immunogenic in humans (Cohen et al., 2023).
  • Applicants also developed antigens with more native 10E8-epitope grafts, to serve as candidate boost immunogens to follow the prime, and to serve as tools to probe the maturation of 10E8-class antibodies induced by the prime.
  • applicants resurfaced T298 using the dTERMen algorithm (Zhou et al., 2020), and applicants eliminated remaining hydrophobic surface patches manually. Similar to the original T117 (Correia et al., 2010) and T298 (Correia et al., 2011) scaffolds, many designs formed dimers or aggregates in solution.
  • His-tagged proteins were purified from clarified supernatants using immobilized metal affinity chromatography followed by size-exclusion chromatography (SEC), nanoparticles were purified using Galanthus nivalis lectin affinity chromatography (Vector laboratories) followed by SEC, and antibodies were purified by protein A affinity chromatography followed by buffer-exchange into tris-buffered saline. High- throughput expression of antibodies was performed in 96-well plates, using the ExpiCho system (Thermo Scientific) and purified by protein A affinity purification as previously described (Wang et al., 2020).
  • Sorting probes were expressed with a C-terminal AviHis tag (GSGGSGLNDIFEAQKIEWHEGSGGHHHHHH**, where denotes a stop codon) (SEQ ID NO: 78) and purified by metal-affinity chromatography and SEC as described above.
  • Matching KO probes for each immunogen were generated that incorporated five knock-out mutations (672A, 673R, 675R, 680E, 683D; Hxb2 numbering) in the MPER.
  • Purified proteins were biotinylated by BirA enzymatic reaction (Avidity, Inc) according to the manufacturer’s protocol and purified by SEC.
  • Immunogen candidates were characterized by SEC coupled with Multi-angle light scattering (SEC-MALS) on a Dawn 18 instrument (Wyatt Labs) and Optilab dRI detector using ASTRA 7.1.1.3 software as previously described (Willis et al., 2022). Protein stability was determined by dynamic scanning calorimetry (DSC) on a MicroCai VP-Capillary DSC (Malvern Instruments) as described previously (Willis et al., 2022).
  • DSC dynamic scanning calorimetry
  • Regeneration solution was 1.7% phosphoric acid injected three times for 60 seconds per each cycle.
  • Solution concentration of ligands was around 1 pg/ml and contact time was 5 min.
  • Raw sensograms were analyzed using Carterra Kinetics software (Carterra), interspot and blank double referencing, Langmuir model.
  • Carterra Kinetics software Carterra
  • interspot and blank double referencing Langmuir model.
  • Langmuir model for fast off-rates (>0.009 1/s) applicants used automated batch referencing that included overlay y-aline and higher analyte concentrations.
  • slow off-rates ⁇ 0.009 1/s
  • applicants used manual process referencing that included serial y-aline and lower analyte concentrations.
  • a custom R-script was used to remove datasets with maximum response signals smaller than signals from negative controls.
  • KD values were determined on a ProteOn XPR36 (Bio-Rad) using GLC Sensor Chip (Bio-Rad) and ProteOn Manager software or Biacore 4000 with CM5 Series S Sensor Chips as described previously (Willis et al., 2022). The same analyte-ligand pair would produce similar KD values on all systems tested within a factor of two.
  • SSGP was performed as described previously (Allen et al., 2021). Briefly, proteins were denatured, reduced and alkylated, followed by enzymatic digestion using trypsin, chymotrypsin or alpha lytic protease. Peptides were analysed by nanoLC-ESI MS with an Ultimate 3000 HPLC (Thermo Fisher Scientific) system coupled to an Orbitrap Eclipse mass spectrometer (Thermo Fisher Scientific). Peptides were separated using an Easy Spray PepMap RSLC Cl 8 column (75 pm x 75 cm) with an in-line trapping column (PepMap 100 C18 3pM, 75pM x 2cm). Data was analyzed using protein metrics Byos software (version 3.5). The relative amounts of each glycan at each site as well as the unoccupied proportion were determined by comparing the extracted ion chromatographic areas for different glycopeptides with an identical peptide sequence. Glycans were categorized according to the composition detected.
  • DeGlyPher was performed as described previously (Baboo et al., 2021). Briefly, proteins were deglycosylated with Endo H, digested with proteinase K, deglycosylated again with Endo H, followed by lyophilization and resupension in PNGase F-containing H2 18 O. Samples were analyzed on an Q Exactive HF-X mass spectrometer. Protein and peptide identification were performed using the Integrated Proteomics Pipeline (IP2) using the automated GlycoMSQuant (Baboo et al., 2021) implementation.
  • IP2 Integrated Proteomics Pipeline
  • 10E8-GT4/10E8 iGL, 10E8-GT 10.1 /NGS precursor, and 10E8-GT11/10E8 iGLl complexes were adjusted to 8 to 10 mg/ml in 100 mM Hepes, 150 mM NaCl, pH 7.4 buffer.
  • Purified 10E8-GT10.2/NHP W3-01 and 10E8-Bl/mature 10E8 complexes were adjusted to 10 mg/ml in TBS buffer (20 mM Tris and 150 mM sodium chloride), pH 7.6 and 7.4, respectively.
  • the complexes were screened for crystallization on a HTP robotic CrystalMation system (Rigaku) against the 384 conditions of the JCSG 1-4 Core Suite (NeXtal; Rigaku Reagents) in sitting drop format with 0.1 pl of protein and 0.1 pl of reservoir solution. Crystals were harvested, soaked in reservoir solution containing respective cryoprotectant listed below, flash cooled, and stored in liquid nitrogen until data collection. 10E8-GT4/10E8 iGL crystals grew in 10% glycerol, 0.1 M HEPES pH 7.5 5%, PEG 3000 with 26% (v/v) glycerol as cryoprotectant.
  • 10E8-GT10.1/NGS precursor crystals grew in 0.1 M Imidazole pH 8, 40% PEG400 with 40% (v/v) PEG 400 acting as cryoprotectant.
  • 10E8-GT11/10E8 iGLl crystals grew in 0.095 M sodium citrate, 19% 2- propanol, 5% glycerol, 19% PEG4000 with 26% (v/v) glycerol as cryoprotectant.
  • 10E8- GT10.2/NHP W3-01crystals grew in 0.2M ammonium dihydrogen phosphate and 20% PEG3350 with 10% (v/v) ethylene glycol as cryoprotectant.
  • 10E8-Bl/mature 10E8 crystals grew in 0.2M calcium chloride and 20% PEG3350 with 20% (v/v) glycerol as cryoprotectant.
  • Diffraction data were collected at cryogenic temperature (100 K) at the respective synchrotron beamlines indicated in Fig. 61.
  • the diffraction data were processed with HKL200060.
  • the 10E8-GT4/10E8 iGL, 10E8- GT10.1/NGS precursor, and 10E8-GT11/10E8 iGLl complex structure were solved by molecular replacement (MR) with Phaser61 using the 10E8 Fab and T117v2 scaffold structures from PDB 5T6L as search models.
  • MR molecular replacement
  • the scaffold structure from PDB: 5T80 and a VH-VL model generated by Repertoire Builder (sysimm.org/rep_builder/) for the NHP-W3-01 Fab were used.
  • the 10E8-Bl/mature 10E8 complex structure was subsequently determined by MR with Phaser using the 10E8 iGLl Fab and 10E8-GT11 scaffold structures from the 10E8-GT11/10E8 iGLl complex structure as a search model.
  • 10E8-GT10.2 120 pg was incubated with the on-target mature 10E8 Fab (300 pg) and an off-target W6-10 Fab (300 pg) in equal molar ratio (1 : 1 : 1) overnight at room temperature. The complex was then purified over a Superdex 200 Increase column (GE Healthcare) and concentrated to 2.5 mg/ml.
  • Movie frames were collected using EPU image acquisition software (Thermo Fisher Scientific) at a nominal magnification of x 190,000 with a Thermo Fisher Scientific Falcon 4 detector mounted on a Thermo Fisher Scientific Glacios operating at 200 kV. Counting mode was used, with a total exposure dose of 53 e ⁇ / 2. 4,249 micrographs were motion, dose and CTF corrected using cryoSPARC Live imported into cryoSPARC (Punjani et al., 2017) (Fig. 61). Template Picker was used to pick 956,668 particles, which were then extracted and 2D-classified.
  • the particles in selected 2D classes were further filtered by Ab-initio reconstruction using Cl symmetry, resulting in 56,628 particles subjected to Non-Uniform Refinement.
  • the final reconstruction is estimated at ⁇ 4.0 A resolution using Fourier Shell Correlation and 0.143 cutoff (Fig. 61).
  • LRS Leukoreduction
  • Cryopreserved PBMCs were thawed and recovered in RPMI media containing 10% FBS, supplemented with lx penicillin/streptomycin (pen/strep) and lx GlutaMAX (R10).
  • Fluorescently labeled antigen probes were prepared by mixing fluorophore-conjugated streptavidin (SA) with small volumes of biotinylated antigen probes in lx PBS at room temperature (RT), with additions every 15 to 20 minutes for a total of 45 minutes to 1 hour, depending on the human naive B cell screening experiment.
  • the purified cells were counted and then stained with a mix of tetramer probes (the other 10E8-GT10.1 probe and a 10E8-GT10.1-KO probe). Without washing, the antibody master mix was added to the cells for another 30 minutes. Anti-human TotalSeqC hashtag antibodies (BioLegend) were also added at this time, at a concentration of 0.1 pg per million cells. Cells were then washed twice in R10 prior to sorting on a FACSAria II (BD Biosciences).
  • 10E8-GT12 human naive B cell screening total B cells were enriched by negative selection using the EasySep human B cell isolation kit (StemCell). Purified B cells were then counted and incubated with the fluorescently labeled antigen probes. First, 10E8-GT12-KO probe was added for 15 minutes at 4°C, and then incubated with wild-type 10E8-GT12 probes for an additional 15 minutes. Without washing, Fc Block (BD Biosciences) was added for 5 minutes, and then stained with surface antibodies for an additional 30 minutes at 4°C. Cells were washed twice with FACS buffer (PBS + 2% FBS + ImM EDTA) and sorted on a FACSymphony S6 (BD Biosciences).
  • FACS buffer PBS + 2% FBS + ImM EDTA
  • 10E8-GT10.1 (10X Genomics): Alexa Fluor 647 Streptavidin (Invitrogen), BV421 streptavidin (BioLegend), PhycoLink Streptavidin-RPE (ProZyme), mouse anti-human CD 19 PE- Cy7 (HIB19, Thermo Fisher Scientific), mouse anti -human CD3 APC-eFluor 780 (UCHT1, Thermo Fisher Scientific), mouse anti-human CD14 APC-eFluor780 (61D3, Thermo Fisher Scientific), mouse anti-human CD 16 APC-eFluor780 (eBioCB16, Thermo Fisher Scientific), mouse anti-human IgG APC-Cy7 (HP6017, BioLegend), Propidium Iodide (PI, Thermo Fisher Scientific), and TotalSeq-C anti-human Hashtag antibody 5 (LNH-94 and 2M2, BioLegend).
  • Single cell BCR amplification was performed similarly to previously described protocols (lardine et al., 2016; Havenar-Daughton et al.; 2018; Steichen et al., 2019). Briefly, Single B cells were sorted directly into 10-20 pL of lysis buffer. Lysed cells were immediately frozen on dry ice then moved to -80°C for storage. First-strand cDNA synthesis was done using SuperScript II RT (Invitrogen) following instructions stated. Heavy and light chain gene transcripts were amplified using a modified nested PCR protocol (T. Tiller et al., 2008).
  • pooled primers were used at 25nM, with fifty cycle PCR reactions (polymerase activation 98 °C for 30 sec; denaturation, 98 °C for 15 sec; annealing, 62 °C for 20 sec; extension, 72 °C for 35 sec).
  • pooled primers were used at 250nM and 25 cycle PCR reactions were used.
  • Phusion Taq polymerase (ThermoFisher, F53OL) was used at 0.5 units/reaction for all reactions. Primers were used from previously published protocols (T. Tiller et al., 2008). PCR products were run out on 2% agarose E-gels (Life Technologies). Reactions with 300-400 bp products were sequenced in both directions. Sequencher 5.0 was used to align sequences. IMGT/V-QUEST was used for VDJ assignments.
  • Sorted cells were prepared for 10X single cell V(D)J sequencing similar to previously published protocols (Lee et al., 2021).
  • 10E8-GT10.1 human naive B cell screening single indexed V(D)J and Feature Barcode libraries were generated following the user guide for the Chromium Single Cell V(D)J Reagent Kits with Feature Barcoding technology (Legacy version, 10X Genomics). All libraries were pooled and sequencing was performed on aNovaSeq Sequencer (Illumina). The V(D)J contigs were assembled and annotated using Cell Ranger v3.0.2, using an Ig library compiled from IMGT references. The constants.
  • py fde was modified to increase the maximum CDR3 length to 110 nucleotides.
  • a python script was used to associate hashtag read counts with the productive assembled V(D)J sequences, compiling the data into a tabular format (Lee et al., 2021).
  • V(D)J libraries were prepared using the Dual Indexed 10X Genomics V(D)J 5’ v.2 according to the manufacturer’s protocol (10X Genomics).
  • Raw sequencing data was processed using CellRanger v6.1.2.
  • V(D)J contigs were generated and aligned to the pre-built human reference (refdata-cellranger-vdj-GRCh38-alts- ensembl-7.0.0).
  • V(D)J output was further processed following the Immcantation pipeline 71. Briefly, contigs were annotated using IgBlast on the IMGT database and only productive sequences were kept for downstream analysis.
  • HEp-2 cell staining assay was performed using kits purchased from Aesku Diagnostics (Oakland, CA), according to the manufacturer's instructions. These Aesku slides use optimally fixed human epithelial (HEp-2) cells (ATCC) as a substrate and affinity-purified, FITC- conjugated goat anti-human IgG for detection. Briefly, 2.5 pg or 25 pl of 100 pg/ml mAb and controls were added to wells and incubated on HEp-2 slides in a moist chamber at room temperature for 30 minutes. After incubation, the slides were removed from the incubator chamber and rinsed with PBS buffer.
  • HEp-2 human epithelial cells
  • a stream of PBS buffer was run along the midline of the slide, allowing the buffer to run off the lower edge of the slide.
  • 25 pl of FITC-conjugated goat anti-human IgG was immediately applied to each well, and the slide was returned to the incubator chamber.
  • the slides were allowed to incubate at room temperature in a moist chamber for another 30 minutes. Subsequently, the slides were washed in the same manner as described above and then mounted on coverslips using the provided mounting medium.
  • MPER-HuGL18 H mice were generated following published protocols (Lin et al., 2018; Wang et al., 2021).
  • the targeting vector 4E10 was modified by the incorporation of human rearranged MPER HuGL-18 VDJ (heavy chain construct) sequences downstream of the promoter region and by elongation of the 5’ and 3’ homology regions using the Gibson assembly method (NEB).
  • the targeting vector DNA was confirmed by Sanger sequencing (Eton Bioscience Inc.).
  • fertilized mouse oocytes were microinjected with a donor plasmid containing the prerearranged MPER HuGL-18 IGH with the mouse VHI558 promoter, two pairs of single guided RNAs (sgRNAs, 25 ng/mL) targeting the H locus; and AltR-Cas9 protein (50 ng/mL) and injection buffer (Wang et al., 2021).
  • sgRNAs single guided RNAs
  • AltR-Cas9 protein 50 ng/mL
  • injection buffer Wang et al., 2021
  • CD45.2 + B cells from MPER-HUGL18 H donor KI mice were enriched using the Pan B Cell Isolation Kit II (Miltenyi Biotec), counted, diluted to desired cell numbers in PBS and adoptively transferred retro-orbitally into CD45.1+ recipient mice as reported previously (Abbott et al., 2018). All experiments were performed under the approval by the Institutional Animal Care and Use Committee (IACUC) of Harvard University and the Massachusetts General Hospital (MGH) (Animal Study Protocols 2016N000022 and 2016N000286) and conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
  • IACUC Institutional Animal Care and Use Committee
  • MGH Massachusetts General Hospital
  • mice Six weeks after immunization, mice were sacrificed and whole spleens were mechanically dissociated to generate single-cell suspensions.
  • ACK lysis buffer was used to remove red blood cells and splenocytes were then resuspended in FACS buffer (2% FBS/PBS), Fc-blocked (clone 2.4G2, BD Biosciences) and stained for viability with Live/Dead Blue (Thermo Fisher Scientific) for 20 min at 4°C.
  • 10E8-GT10 probes (described above), as well as antibodies against CD4-APCeF780, CD8-APC-eF780, Gr-l-APC-eF780, F4/80-APC-eF780, B220-BUV395, CD95-PE-Cy7, CD38-A700, CD45.1-PerCPCy5.5, CD45.2-PE and IgD-BV421 were used.
  • Cells were acquired by a BD LSRFortessa (BD Biosciences) for flow cytometric analysis and sorted using a BD FACS Aria II instrument (BD Biosciences). Data was analyzed using FlowJo software (Tree Star). B cells were single-cell dry-sorted into 96-well PCR plates, rapidly frozen on dry ice and stored at -80°C until processing.
  • PCR products were analyzed on precast E-Gels 96 2% with SYBR Safe (Thermo Fisher Scientific) and wells with bands of the correct size were submitted to GENEWIZ company for Sanger sequencing.
  • Heavy chain products were sequenced using the heavy chain reverse primer from PCR-2 (5'- GCTCAGGGAARTAGCCCTTGAC-3’ (SEQ ID NO: 79) and the light chain was sequenced using the light chain reverse primer (5'-TGGGAAGATGGATACAGTT-3' (SEQ ID NO: 80) from PCR-2. Reads were quality-checked, trimmed, aligned, and analyzed using the Geneious software (Biomatters Ltd, New Zealand).
  • IMGT/V-QUEST www.imgt.org
  • IMGT/V-QUEST (www.imgt.org) (Brochet et al., 2008; Giudicelli et al., 2011) was used for mouse/Human Ig gene assignments.
  • ES clone with hD3-3/Jn6 was injected into Rag2 deficient blastocysts to yield chimeric mice (Chen et al., 1993), which were subsequently crossed with 129SVE mice to give germline transmission.
  • chimeric mice Choen et al., 1993
  • 129SVE mice For B cell analysis shown in Fig.
  • splenocytes from wild-type mice were stained with the following antibodies: Plot B, FITC anti-B220, APC anti-Thyl.2; Plot C, FITC anti-B220, PE anti-IgM, APC anti-IgD, Plot D, FTIC anti-B220, APC anti-CD93, PE/Cy7 anti-CD23, PerCP/Cy5.5 anti-CD21, Plot E, FITC anti-B220, biotin anti-Igk, APC Streptavidin, PE anti-Igl. Dead cells were gated out by Sytox blue staining.
  • Flow cytometry was performed in a AttuneNxT instrument, and data were analyzed with FlowJolO software.
  • IgH repertoire analysis shown in Fig. 65f-g genomic DNA was isolated from splenocytes of I D3-3/JH6 mice.
  • Repertoire analysis was performed with HTGTS-rep-seq technique (Hu et al., 2016; Lin et al., 2016).
  • Library synthesis was initiated with a primer downstream of hJn6: 5Biosg/CAA CCT GCA ATG CTC AGG AA.
  • Illumina MiSeq adaptors were added to the ends of library DNA, and sequencing was performed on a MiSeq instrument. Sequencing data were analyzed with HTGTS- rep-seq pipeline.
  • mice Homozygous hD3-3/Jn6 mice (>6 weeks old) were randomly distributed into groups. Mice were injected with lOpg (50 pl total volume) of Modema mRNA LNPs (Corbett et al., 2020; Baden et al., 2021) encoding 10E8-GT12 24mer I.M. under anesthesia (5% isoflurane induction) in the left quadriceps muscle. All primes and subsequent boosts were done in the same location. Protein injections (20pg, 50pl total volume) were performed SubQ (tail base), utilizing 5 pg of SMNP (Silva et al., 2021).
  • BD Insulin syringes
  • Mice were euthanized with compressed CO2 (100%) in a clear chamber to allow for visualization of respiration and subsequent death via respiratory cessation.
  • Blood was collected from the chest cavity prior to the removal of the spleen and lymph nodes (mesenteric, inguinal, and popliteal (RNA injections only, left leg only).
  • Tissues were placed in 3mL FACS buffer (lx PBS Ca/Mg++ free, ImM EDTA, 25mM HEPES, pH 7.0, 1% heat-inactivated FBS) in a 15mL polypropylene tube on ice.
  • Tissues were disassociated using the rough ends of two sandblasted microscope slides in a 5mL petri dish, then returned to the same 15mL polypropylene tube for centrifugation (460xg for 5 minutes at 4°C).
  • RBC lysis was performed using ImL of ACK buffer (Quality Biological) for 2 minutes on ice in a 15mL polypropylene tube. Lysis was halted by adding 14mL FACS buffer per sample. Post lysis and centrifugation (460xg for 5 minutes), cells were resuspended in 3mL Bambanker freezing medium (Bulldog Bio) prior to filtration through a cotton-plugged, borosilicate pasteur pipette into a borosilicate glass test tube.
  • Bulldog Bio Bambanker freezing medium
  • ImL filtered-cell solution was subsequently divided into three cryovials/mouse, which were precooled in a styrofoam rack on dry ice. Cells were stored at -80°C for 2-7 days prior to long-term storage in liquid nitrogen. Sera were collected by spinning the blood at 14,000 RPM for 30 minutes. Sera were stored at -20. All work followed IACUC guidelines associated with animal protocol number 20-0001.
  • splenocytes and lymphocytes were thawed in 10 mL 50:50 heat inactivated fetal bovine serum (FBS; Omega Scientific, cat# FB-02): RPMI (Gibco, cat# 61870-036) pre-warmed to 37 °C.
  • FBS heat inactivated fetal bovine serum
  • RPMI fetal bovine serum
  • Unimmunized splenocytes for naive B cell sorting were used fresh after processing. Cells were spun down at 400xg for 5 min.
  • cells were resuspended in 3 mL FAC S buffer (l%v/v heat inactivated FBS, 1 mMEDTA (Invitrogen, cat# 15575-038), 1 mM HEPES (Gibco, cat# 15630-080) in DPBS (Corning, cat # 21- 031-CV)), and enumerated. After counting, cells were subjected to B cell isolation using the Stemcell EasySep Mouse Pan-B Cell Isolation Kit (Stemcell, cat #19844A) according to manufacturer provided instructions.
  • FAC S buffer l%v/v heat inactivated FBS, 1 mMEDTA (Invitrogen, cat# 15575-038), 1 mM HEPES (Gibco, cat# 15630-080) in DPBS (Corning, cat # 21- 031-CV)
  • Streptavidin (SA) conjugated-baits were prepared by combining biotinylated monomeric baits or Env trimer baits with fluorescent SA at RT for at least 1 hr in the dark. Wildtype baits were complexed with SA-AlexaFluor 647 (Invitrogen, cat# S21374) and SA-BV421 (Biolegend, cat# 405225). Knockout (KO) baits were conjugated with Total Seq-C hash-tagged SA-PE (Biolegend, cat# 405261). Baits were conjugated with SA at a 4:1 (bait:SA) ratio and used at a final bait concentration of 200 nM for staining.
  • Isolated B cells were transferred over to 15 mb conical tubes, washed once with FACS buffer, and stained with 100 pL antibody cocktail mix consisting of FITC anti-CD19 (Biolegend, cat# 152404), BV786 anti-IgM (BD, cat#743328), PerCP-Cy5.5 anti-IgD (BD, cat# 564273), APC-Cy7 anti-F4/80 (Biolegend, cat # 123118), APC-Cy7 anti-CDl lc (BD, cat# 561241), APC- Cy7 anti-Ly-6C (BD, cat# 557661), APC-H7 anti-CD8a (BD, cat# 560182), and APC-H7 anti- CD4 (BD, cat# 560181).
  • Sorted samples were prepared for BCR sequencing by the 10X Genomics Single Cell Immune Profiling platform. After cell sorting, DPBS was added up to near top of the sample collection well (-100 pL) and gently mixed to dilute the FBS catch buffer. The plate was sealed and cells were spun down for 2 min at 2000 rpm, after which the excess buffer was removed except for ⁇ 38pL required for the 10X Genomics GEM reaction. Samples were processed according to manufacturer’s user guide for Chromium Next GEM Single Cell 5’ Reagent Kits v2 (Dual Index) with Feature Barcoding, with two previously described main modifications (Hurtado et al., 2022).
  • the number of PCR cycles in the cDNA amplification step were determined by assuming that only 20% of the total number of cells sorted would be recovered. This modification was made based on the observation that on average, the number of unique paired-BCR sequences recovered from the 10X Genomics platform was typically -20% of the total number of cells sorted. In the second modification, the number of PCR cycles for each of the V(D)J amplification steps were increased to 10 cycles if the number of cells sorted (according to the sorter) was fewer than 1000 cells.
  • Raw sequencing data were demultiplexed, processed into assembled VDJ contigs and counts matrix files, and assigned to specific animal IDs based on Total Seq-C antibody hashtag counts using Cell Ranger (v6.1) and scab as previously described (Hurtado et al., 2022).
  • Epitope knockout positive cells were identified based on UMI counts of the hash-tagged PE-streptavidin probe using scab (Hurtado et al., 2022).
  • Adaptive Immune Receptor Repertoire (AIRR) format (B reden et al., 2017) for paired heavy and light chain antibody sequences was performed using Sequencing Analysis and Data library for Immunoinformatics Exploration (SADIE) with a custom hD3-3/Jn6 mouse germline reference database (Leggat et al., 2022). SADIE outputs were summarized using custom python scripts. Animals with low cell viability ( ⁇ 25000 total live B cells) or low sequence recovery ( ⁇ 20 total sequences, where indicated ⁇ 100 total sequences) were excluded. In addition, all non-IgG sequences were discarded. Sequences matching criteria listed in Fig. 59a were counted and frequencies plotted in Graphpad Prism 9.5.1. Multi-panel figures were assembled using Adobe Illustrator.
  • Immunizations were given subcutaneously (s.c.) in the left and right deltoids with a total dose of 50 pg 10E8-GT10.2 12mer nanoparticle and 375 pg SMNP (Silva et al., 2021) per side.
  • s.c. subcutaneously
  • 4 macaques were used in the study, with 2 females and 2 males.
  • Macaques were immunized s.c. in the left and right mid-thighs with a total dose of 50 pg MD39 and 375 pg SMNP (Silva et al., 2021) per side.
  • a 7-dose 12-day escalating dose strategy was used (Cirelli et al., 2019). Data from the MD39 group have been previously published (Lee et al., 2022).
  • LN FNAs Lymph node fine needle aspirates
  • Draining LNs were identified by palpation and performed by a veterinarian.
  • a 22- gauge needle attached to a 3-mL syringe was passed into the LN up to 5 times.
  • Samples were dispensed into RPMI media containing 10% fetal bovine serum (FBS) and lx pen/strep.
  • Ammonium Chloride-Potassium (ACK) lysing buffer was used if the sample was contaminated with red blood cells.
  • LN FNA samples were frozen and stored in liquid nitrogen until analysis.
  • IGHD3.41 targeted long-read Pacific Biosciences single molecule real-time sequencing data generated for each macaque in the study cohort. Sequencing data was generated by adapting the published human immunoglobulin (IG) loci targeted enrichment protocol (Rodriguez et al., 2020; Gibson et al., 2023).
  • a custom oligo probe panel was designed (“HyperExplore”, Roche) using IG heavy chain (IGH), kappa (IGK), and lambda (IGL) genomic region sequences from the RM genome reference build (RheMaclO) and alternative haplotype assemblies from (Cirelli et al., 2019) as sequence targets.
  • High molecular weight genomic DNA was isolated from peripheral blood mononuclear cells (PBMCs) collected from each macaque using the DNeasy kit (Qiagen). DNA (1-2 pg) was then sheared using g-tubes (Covaris) and size selected using a Blue Pippin instrument (Sage Science).
  • Size-selected DNA was End Repaired and A-tailed using the standard KAPA library protocol (Roche), followed by the ligation of sample-specific sequence barcodes and universal primers.
  • PCR amplification was performed for 8-9 cycles using PrimeSTAR GXL polymerase (Takara), and the resulting products were further size-selected and purified using 0.7X AMPure PB beads ( Pacific Biosciences).
  • Target-enrichment hybridization was performed using IGH/K/L- specific oligonucleotide probes (Roche).
  • Target fragments were recovered using streptavidin beads (Life Technologies), followed by a second round of PCR amplification for 16-18 cycles using PrimeSTAR GXL (Takara).
  • HiFi reads for each macaque were mapped to the RheMaclO genome reference.
  • To genotype IGHD3-3 phased single nucleotide variants representing two distinct alleles were resolved from HiFi reads spanning the IGHD3-3 gene. At least 10 representative HiFi reads were required to include a given allele in the genotype of an animal.
  • Frozen macque FNA or PBMC samples were thawed and recovered in R10. The cells were counted and then stained with the appropriate staining panel. Fluorescently labeled antigen tetramer probes were prepared by incrementally mixing fluorophore-conjugated streptavidin (SA) with small volumes of biotinylated antigen probes in lx PBS at RT over the course of 45 minutes.
  • SA fluorophore-conjugated streptavidin
  • the knockout probe, 10E8-GT10.2-KO or 10E8-GT12-KO was first added to the cells for 20 minutes, followed by the addition of either 10E8-GT10.2 or 10E8-GT12 for 30 minutes, and then with the surface antibodies for 30 minutes at 4°C, similar to previously described protocols (Cirelli et al., 2019; Lee et al., 2022).
  • anti-human TotalSeq-C hashtag antibodies BioLegend
  • 10% FBS in RPMI (RIO) supplemented with lx pen/strep and lx GlutaMAX was used as the FACS buffer.
  • Cell Ranger v3.0.2 was used for assembly of full-length VDJ reads.
  • a custom RM germline VDJ reference was generated using previously published databases (Cirelli et al., 2019; Cottrell et al., 2020; Vazquez Bemat et al., 2021). The constants.
  • py file within the Cell Ranger python library was modified to increase the CDR3 maximum length to 110 nucleotides.
  • Cell Ranger v3.1 was used to obtain the gene expression matrix from these sequenced GEX libraries.
  • the libraries were aligned to the Ensemble MmullO reference genome, with the addition of mitochondrial genes from Mmul9.
  • Sequences were de-multiplexed by the hashtag read counts using the MULTIseqDemux command in Seurat v4 (Hao et al., 2021). Sequences were further processed using the same pipeline described above for experiments in DJ mice, except using the default macaque germline reference database and searching for macaque homologues of 10E8-class and LN01 -class Vn-genes.
  • 10E8 bnAb activity further requires a PP motif in the junction between D and J genes within the HCDR3, which could have arisen either during V(D)J recombination or somatic hypermutation (SHM); and germline-encoded HCDR1 and framework region 2 (HFR2) residues and somatically mutated HCDR2 residues within the VH3-15 gene.
  • HMM somatic hypermutation
  • HFR2 germline-encoded HCDR1 and framework region 2
  • the 10E8 light chain contributes to binding of membrane-associated Env by contacting the virion lipid membrane and conformationally stabilizing the HCDR3 (Irimia et al., 2017).
  • the range of germline light chains that have potential to acquire mutations to mediate such contacts is unclear but could be large.
  • human light chains within the VL3 family employed by 10E8 were paired with VH3-15 heavy chains at a frequency of ⁇ 1 :7.5, suggesting the frequency of 10E8-class heavy chains-light chain precursors was approximately 1:510,000.
  • 10E8-class precursors are present in healthy humans at substantial frequency.
  • T117v2 (Irimia et al., 2017), for further optimization due to its favorable thermal stability, solubility and presentation of surfaces adjacent to the MPER graft that could be engineered to increase contacts with the YxFW motif in the 10E8 HCDR3.
  • Applicants then carried out a multi-state design and selection process aimed at developing T117v2-based immunogens with the following features: 10 pM affinity or better for the 10E8 UCA and as many NGS precursors as possible, to enable priming of diverse 10E8-class precursors (Abbott et al., 2018; Steichen et al., 2019; Leggat et al., 2022); affinity gradient for 10E8-class antibodies with highest affinity for mature 10E8, to favor affinity maturation towards mature 10E8 in vivo, (Jardine et al., 2016; Steichen et al., 2019; Leggat et al., 2022); multivalent display of epitope-scaffolds on single-component self-assembling nanoparticles, to facilitate mRNA lipid nanoparticle (mRNA-LNP) delivery, improve trafficking to lymph nodes (Tokatlian et al., 2018) and increase B cell responses (Jardine et al., 2016); and
  • Applicants also added N-linked glycosylation sites to scaffold surfaces outside the MPER graft to reduce off-target responses (Duan et al., 2018). Site-specific glycosylation analysis by mass spectrometry indicated that approximately 50% of glycosylation sites were occupied (Fig. 60). Applicants thus developed self-assembling nanoparticles presenting 10E8-GT scaffolds with broad affinity for 10E8-class precursors.
  • the overall structures of the epitope-scaffold and the MPER helix were similar to the original T117v2 scaffold complexed with 10E8, with backbone root mean square deviation (bb-rmsd) values of 0.73, 0.89, 0.75 and 0.62 A for the 10E8- GT antigens, respectively (Fig. 53).
  • the antibody engaged the epitopescaffold at an angle closely resembling the interaction of mature 10E8 with the MPER peptide (Fig.
  • the 10E8-scaffolds stabilized the MPER in the 10E8-bound conformation and consistently engaged the hydrophobic tip of 10E8-class HCDR3s in a manner closely resembling the interaction of mature 10E8 with gp41.
  • 10E8-GT scaffolds isolate 10E8-class human naive B cells
  • naive BCRs To assess the repertoire of bona fide human naive BCRs able to respond to the 10E8- GT immunogens (Jardine et al., 2016; Havenar-Daughton et al., 2018; Steichen et al., 2019), applicants characterized naive BCRs from the blood of HIV-seronegative human donors that bound the 10E8-GT epitope-scaffolds (Fig. 62). On average, 10E8-GT9.2, 10E8-GT10.1 and 10E8-GT12 bound to 0.05%, 0.8% and 0.7% of naive CD20 + CD27TgD + IgG’ B cells (hereafter naive B cells), respectively (Fig.
  • epitope-scaffold-binding naive B cells 81%, 94% and 97% did not bind matching 10E8 epitope-knockout versions of the respective 10E8-GT constructs, and are hereafter referred to as “epitope-specific BCRs” (Fig. 54c, Fig. 62a).
  • epitope-specific BCRs were enriched for the crucial 10E8 D-gene binding motif YxFW in the HCDR3, with 47%, 81%, and 87% of epitope-specific BCRs containing the motif for 10E8-GT9.2, 10E8-GT10.1 and 10E8-GT12, respectively, compared to 1.4% for unsorted BCRs (Fig. 62d).
  • Applicants also searched 10E8-GT-binding sequences for signatures of other MPER bnAb lineages, including precursors of LN01 -class MPER bnAbs, which are genetically and structurally distinct from 10E8-class bnAbs but share key features such as an Dn3-3-encoded “F W” motif at the tip of a long (20 amino acid) HCDR313.
  • immunogens were optimized for engagement of 10E8-class precursors, applicants detected LN01 -class HCDR3s in all six samples of 10E8-GT12-sorted naive B IgM B cells, with a median frequency of 3.3% (Fig. 62g, h).
  • the total frequency of 10E8-GT12-binding LNOl-class IgH precursors among naive B cells was 1 :41,000, which was only slightly lower than the frequency of 10E8-like IgH precursors (1: 15,000; Fig. 54g).
  • 10E8-class B cells function in vivo
  • 10E8-GT 12mers induce 10E8-class BCRs in rhesus macaques
  • rhesus macaques Two of five known homologues of human Dn3-3in indian rhesus macaques (DH3- 41 *01_S8240 and DH3-41 *01_S4389) encode the YxFW motif, whereas the remaining alleles encode YxIW (Fig. 57a) (Vazquez Bemat et al., 2021), permitting testing of 10E8-GT immunogens in some rhesus macaques.
  • BCR sequencing of sorted 10E8-GT10.2 epitope-specific naive CD20 + IgGTgD + B cells from unimmunized rhesus macaques indicated that sorted BCRs were enriched for long HCDR3s (Fig. 57b).
  • BCRs with 10E8-class HCDR3s (length of 21- 24aa with a YxFW motif at the equivalent position within the HCDR3 as 10E8) were detected in 8 of 9 macaques with a median frequency of 0.0078% among naive B cells (Fig. 57c), 18-fold lower than their frequency of 0.14% in the human naive B cell repertoire, which made rhesus macaques a viable, though challenging, model to assess 10E8-GT immunogens.
  • Applicants used an escalating-dose regimen (Cirelli et al., 2019) to immunize 8 rhesus macaques with a total of 100 pg 10E8-GT10.2 12mer and 750 pg SMNP (Silva et al., 2021) adjuvant delivered through 7 immunizations of increasing doses over 14 days (Fig. 57d).
  • Control macaques (n 4) were immunized using the same protocol with a stabilized soluble HIV-1 Env trimer that lacked the MPER epitope (BG505 MD39 gpl40) (Steichen et al., 2016).
  • Applicants also immunized six rhesus macaques with a total of 100 pg 10E8-GT12 12mers and 375 pg SMNP-QS21 using the same dose-escalation strategy (Fig. 57d). Sequencing of epitope-specific PBMC memory CD20 + IgD' B cells at week 10 revealed that 3 of the 3 macaques which carried at least one permissive DH allele and with sufficient sequencing depth showed strong enrichment of 10E8-class HCDR3s, comparable to the 10E8-GT10.2 12mer immunizations (Fig. 57e).
  • Induced BCRs acquire affinity for a boosting candidate
  • Germline-targeting priming immunogens should consistently induce bnAb-precursor memory and/or GC B cells susceptible to boosting by immunogens more similar to the native viral protein (native-like) than the priming immunogen (Leggat et al., 2022).
  • 10E8-class bnAb precursors induced by 10E8-GT nanoparticles in either IID3-3/JH6 mice or NHPs had no neutralizing activity against HIV pseudoviruses, which was expected because the 10E8 epitope on the priming immunogens was substantially modified from wild-type and lacked steric constraints imposed by the membrane and ectodomain of HIV Env.
  • 10E8-B1 bound to 10% of 10E8-class antibodies induced by 10E8-GT nanoparticles, including 10E8-GT10 12mer or 10E8-GT12 24mer in IID3-3/JH6 mice (Fig. 58c) and to 25% of 10E8-class antibodies primed by 10E8-GT10 12mer in macaques (Fig. 58c). Binding of 10E8-B1 to germline-reverted 10E8-class antibodies from macaques was significantly weaker than to the matching antibodies primed by 10E8-GT10 12mer in macaques (Fig. 58c), indicating that binding by 10E8-B1 was due to SHM acquired by these antibodies.
  • 10E8-GT nanoparticle immunization selected for affinity maturation that conferred affinity for an antigen with a more native-like 10E8 epitope.
  • epitope-scaffolds can be designed to induce responses from rare, HCDR3 -dominant bnAb precursors and select for a degree of favorable maturation in those precursors, extending the functionality of the epitope-scaffold approach (Correia etal., 2010; Ofek et al., 2010; Correia et al., 2014; Krebs et al., 2019; Sesterhenn et al., 2020).
  • the 10E8-GT epitope-scaffolds also induced precursors for a related yet genetically distinct class of bnAb, LN01, demonstrating the capacity for multi -bnAb precursor priming without obvious interference from tolerance mechanisms, consistent with the low poly- or autoreactivity exhibited by 10E8- and LNOl-class lineages (Huang et al., 2012; Pinto et al., 2019. [00551]
  • Germline-targeting vaccine design posits that bnAbs can be elicited by first priming bnAb precursors with the necessary bnAb-associated genetic and structural features and then employing a series of boosters of increasing similarity to the native glycoprotein to select for the necessary SHM to produce bnAbs. Hence, additional work is needed to develop sequential heterologous boosting regimens to induce 10E8-class bnAbs.
  • an epitope-scaffold nanoparticle with a more native-like MPER epitope such as 10E8-B1
  • membrane-bound envelope protein(s) to select for maturation to enable 10E8-like and LNOl-like BCRs to engage the native MPER peptide and its surroundings on the membrane-anchored Env glycoprotein.
  • the data further encourage the development of germlinetargeting epitope-scaffold nanoparticles to induce bnAb precursors and initiate bnAb induction for other epitopes that are sterically occluded or poorly immunogenic in the context of native viral glycoproteins, such as the MPER of Filoviridae (Schoeder et al., 2022), the influenza A haemagglutinin anchor3 or the relatively conserved S2 subunit in betacoronaviruses (Dacon et al., 2023).
  • B cells with 108-class BCRs were ⁇ 30-fold more frequent among IgM /IgD' B cells in animals boosted with 10E8-B1 24mer than in mock-boosted animals (Fig. 69d).
  • 10E8-GT12 24mer priming was essential to induce 10E8-class BCRs since 10E8-class HCDR3s were undetectable in five out of six animals that were boosted with 10E8-B1 24mer but primed with PBS.
  • One group of six macaques was immunized with an escalating-dose regimen (Cirelli, K. M. et al., 2019; Tam, H. H. et al., 2016; Lee, J. H. et al., 2022) in which a total of 100 pg 10E8-GT1224mer protein and 750 pg SMNP adjuvant were delivered through 7 immunizations of increasing doses over 14 days (Fig. 70a), followed by boosting at bolus injection of 100 pg 10E8-B1 24mer protein and 750 pg SMNP at week 10.
  • PBMC- memory B cells were analyzed by flow cytometry, epitope-specific B cells were sorted and their BCRs sequenced (Fig. 70b-f).
  • a non-naturally occurring protein comprising the sequence of:

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Abstract

L'invention concerne des protéines et des acides nucléiques pour des régimes d'immunisation, en particulier, des immunogènes qui peuvent amorcer des lymphocytes B précurseurs de bnAb rares et guider leur maturation vers des bnAb capables de neutraliser diverses souches de VIH, des modifications de celles-ci et/ou le développement de nanoparticules et/ou le développement d'immunogènes ancrés par membrane, et des procédés de fabrication et d'utilisation de ceux-ci. L'invention concerne également des trimères de surface cellulaire qui se lient aux anticorps largement neutralisants et/ou aux acides nucléiques codant ces derniers.
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