WO2025076039A1 - Chlamydial protease-like activity factor and adjuvant compositions and uses thereof - Google Patents

Abstract

This invention relates to compositions comprising chlamydial protease-like activity factor (CPAF) or an antigenic fragment thereof and a stimulator of interferon genes (STING) agonist useful to raise an immune response in a subject and vaccinate the subject against a Chlamydia trachomatis infection. In addition, the composition may further include a TLR agonist and/or an oil-in-water emulsion so as to increase the efficacy of the CPAF immunogen. This invention also describes methods of administration of the composition, including mucosal administration.

Description

CHLAMYDIAL PROTEASE-LIKE ACTIVITY FACTOR AND ADJUVANT

COMPOSITIONS AND USES THEREOF

RELATED APPLICATION INFORMATION

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63/587,535, filed October 3, 2023, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING [0002] A Sequence Listing in XML format, entitled 5470-959WO_ST26.xml, 5,295 bytes in size, generated on September 11, 2024, and filed herewith, is hereby incorporated by reference in its entirety for its disclosures.

STATEMENT OF GOVERNMENT SUPPORT

[0003] This invention was made with government support under Grant No. AI144181 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0004] This invention relates to compositions comprising chlamydial protease-like activity factor (CPAF) or an antigenic fragment thereof and a stimulator of interferon genes (STING) agonist, and methods of use of the same in raising an immune response and/or in vaccinating a subject against a Chlamydia trachomatis infection.

BACKGROUND OF THE INVENTION

[0005] Genital Chlamydia trachomatis (CT) infection and associated diseases remain a significant global health burden, with an estimated 131 million new cases occurring annually. In the United States, >1 million women are infected annually, and direct treatment costs exceed $500 million. Antibiotic treatment efficacy is limited, primarily because >70% of infections are asymptomatic and are thus left untreated. Bacterial ascension from the cervix to the endometrium and oviducts induces inflammation that leads to the clinical syndrome of pelvic inflammatory disease (PID) in 10% of infected women; and even women without symptoms may have endometritis or subclinical PID. Both forms of PID can lead to chronic pelvic pain (30%), irreversible tubal damage resulting in infertility (10-20%), and ectopic pregnancy (1- 10%). Recent evidence suggests that ascending infection increases the risk of ovarian cancer. Infection in males, though less severe, can cause urethritis, orchitis, epididymitis, and prostatitis. Despite screening programs, infection rates continue to rise, with a 22% increase in reported CT cases from 2013 to 2018. CT also enhances the acquisition of other sextually transmitted infections (STIs) and is an independent risk factor for cervical cancer.

[0006] Natural immunity in humans indicates the importance of T cells for CT infection resolution and protection from reinfection. Women with a confirmed prior CT infection had lower chlamydial loads when reinfected. Those who spontaneously cleared a CT infection without treatment exhibited increased levels of CT-specific interferon-y producing (IFNy+) CD4 T cells and were less likely to be reinfected altogether. In contrast, HIV-infected women with low CD4 counts experienced higher rates of chlamydial PID.

[0007] Research into a vaccine for chlamydia infections has spanned over 80 years and over several hundred vaccine trials. Multiple antigens, adjuvants, and delivery mechanisms have been tested without success. In particular, over 50 different adjuvants have been tested with both protein-based and whole-cell chlamydia vaccines, but no combination has yet passed clinical trials. Thus, a vaccine-based prevention strategy for CT infections is desirable.

SUMMARY OF THE INVENTION

[0008] The present invention is based on the finding that a composition of CPAF with one or more adjuvants can be used to improve vaccine and immune response efficacy to protect a subject against a CT infection. Further, it was found that CPAF in combination a STING agonist as an adjuvant was sufficient to elicit an immune response as measured by T-cell immunogenicity. Surprisingly, CPAF alone or CPAF with several other tested adjuvants failed to elicit a similarly strong immune response.

[0009] Thus, one aspect of the invention relates to a composition comprising a CPAF or an antigenic fragment thereof and a STING agonist. In some embodiments, the composition comprises at least one additional adjuvant. In some embodiments, the at least one additional adjuvant is a toll-like receptor (TLR) agonist and/or an oil-in-water emulsion. In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In some embodiments, the composition is suitable for mucosal administration.

[0010] Another aspect of the invention is a method of raising an immune response against CT in a subject, said method comprising administering an effective amount of a composition as described herein, thereby raising an immune response. [0011] Another aspect of the invention is a method of vaccinating a subject against a CT infection, said method comprising administering a therapeutically effective amount of the composition as described herein, thereby vaccinating the subject.

[0012] These and other aspects of the invention are set forth in more detail in the description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Fig. 1, panel A is a graph showing the number of peripheral blood mononuclear cell (PBMC) cultures from 30 CT seropositive women expanded using overlapping peptide pools spanning the respective antigen(s) as shown on the X-axis to provide short term cell lines (STCLs), then restimulated with the same pool and measured for a response to the antigens as measured by an IFNy enzyme-linked immunosorbent spot (ELISpot) assay; the dashed grey line represents the mark at which 20% of the PBMC cultures responded to the supplied antigen; a positive response was defined as the average of mock-subtracted wells >300 spot forming units (SFU) per 10⁶ cells (SFU/10⁶ cells) and > 4-fold average of mock wells. Fig. 1, panel B is a heat map graph showing the number of CPAF stimulated, in vitro expanded T cell cultures that were positive for IFNy and/or TNFa responses as measured by intracellular cytokine staining (ICS) after sorting said cultures for those that had a response to CPAF in an IFNy ELISpot assay (ELISpot-pos) or those that did not (ELISpot-neg); a positive response was defined as >3x the mock well and >15 positive spot-forming units (SFUs) or events. Fig. 1, panel C is an image showing the mapping of CD4 T cell epitopes across the entire protein that were identified as producing an antigen response in PBMC cultures where each fragment’s response was measured by IFNy ELISpot assay.

[0014] Fig. 2 is a dot plot graph showing the flow cytometry measurements of CPAF induced IFNy response in CD4 and CD8 T cells at either 1 month (1-M) or 12 months (12-M) after documented genital tract infection with C. trachomatis,' a positive response was defined as >3x the mock well and >15 positive SFUs in the final gate; significance within groups was measured by the Wilcoxon signed-rank test, and between the 1-M CD4 and CD8 groups by Wilcoxon matched paired sign-rank test.

[0015] Fig. 3, panel A is a graph showing the number of SFU/10⁶ in splenocytes isolated from C57BL/6 mice that had previously cleared a CM972 (a live attenuated Chlamydia muridarum (C. muridarum CM) strain) infection followed by a CM001 (a virulent CM strain) infection (CM972+CM001) and sacrificed 30 days post-clearance; splenocytes were stimulated with 5 pg/mL of the stated antigens and the response was measured by IFNy ELIspot assay; **** indicates p-value < 0.0001 by one-way ANOVA. Fig. 3, panel B is a density scatter plot showing the ICS measured IFNy+ TNFa+ responses in CD4 and CD8 T cells isolated from CM972+CM001 mice that were stimulated with either control media or CPAF overlapping peptides (OLP), sequences of 18 amino acids that overlap one another by 15 amino acids. Fig. 3, panel C is a graph showing the percent of naive or CM972 vaccinated CD4 T cells harvested from mouse genital tracts that produced a IFNy+ TNFa+ response 7 days after CM001 challenge; error bars represent the mean ± one standard deviation.

[0016] Fig. 4, panel A is an SDS-PAGE gel image showing the inactive, full-length CPAF and the corresponding N- and C-terminal protein fragments. Fig. 4, panel B is an SDS-PAGE gel image showing the unclipped, full-length K232Q inactive CPAF. Fig. 4, panel C is graph showing the results from analytical size-exclusion chromatography for both CM CPAF variants.

[0017] Fig. 5 is a graph showing the deconvoluted mass spectrum of intact protein analysis by LC-MS showed the three major forms of S491 A CPAF: the peak at 26,927.9 Daltons is the N- terminal clipped fragment; the peak at 41,419.3 Daltons is the C-terminal fragment; and the peak at 68,329.0 Daltons is the full-length protein. The Y-axis indicates the mass spectrometry signal intensity, the X-axis indicates the mass of the protein or protein fragments in Daltons.

[0018] Fig. 6, panels A-C are a series of graphs showing the expression level of IFNy, IL-6, and TNFa in the lung at 3 hours, 24 hours, and 1 week post immunization, as indicated. Fig.

6, panels D-F are a series of images showing hematoxylin and eosin (H&E) staining of lungs from immunized mice (n=5 mice/group) after 3 hours, 24 hours, and 1 week postimmunization, as indicated. For all panels, Group 1 was immunized with CPAF only; Group 2 was immunized with CPAF + CpG + CD A; Group 3 was immunized with CPAF + CpG + CD A + AS03; Group 4 was immunized with CpG + CD A + AS03; and Group 5 was immunized with CPAF + 2-Bxy-Dopa (a TLR7/8 agonist conjugated to dopamine).

[0019] Fig. 7, panel A is a cartoon schematic showing the immunogenicity and challenge experiments. Fig. 7, panel B is a graph showing the antibody titers in mice immunized with CPAF plus CDA alone, triple adjuvant combination, or antigen alone; p=not significant (NS) for all comparisons by one-way ANOVA (n=5 mice/group). Fig. 7, panel C is a graph showing IFNy ELISpot responses in immunized mice for all adjuvant iterations (n=4-5 mice/group); ****pO .0001 by one-way ANOVA. Fig. 7, panel D is a series of ICS graphs showing the CPAF-specific cytokine responses in CPAF + CDA immunized mice (n=5 mice/group). Fig.

7, panel E is a series of graphs showing the frequency of CPAF-specific mono- and poly- functional memory (CD44^hl CD62L ) CD4 and CD8 T cell responses in CPAF + CDA immunized mice; **p<0.01 by Wilcoxon rank-sum test.

[0020] Fig. 8, panel A is a graph showing the course of C. muridarum challenge infection in immunized mice and controls (n=9-10 mice/group). CPAF + CDA vs. PBS (-1.51 log, ****p<0.0001); CPAF + CDA vs. CPAF (-0.85 log, ****p<0.0001); CPAF + CDA vs. CPAF + CpG (-1.10 log, ****p<0.0001); CPAF + CDA vs. CPAF + AS03 (-0.84 log, ****p<0.0001); CPAF + CDA vs. CPAF + CpG + AS03 (-0.69 log, ****p<0.0001); p=NS for CPAF + CDA vs. all vaccine combinations incorporating CDA. Significance determined by two-way repeated-measures ANOVA. Fig. 8, panel B is a graph showing the frequency of oviduct hydrosalpinx after challenge in mice immunized with CPAF plus CDA alone, triple adjuvant combination, CPAF alone, or PBS. Dotted line set to oviduct hydrosalpinx frequency in PBS controls. Fig. 8, panel C is a graph showing the oviduct dilatation scores of immunized mice and controls. Significance determined by Kruskal -Wallis test. *p<0.05, **p<0.01.

[0021] Fig. 9, panel A is a series of graphs showing the flow cytometric gating strategy for detection of CPAF-specific memory CD4 and CD8 T cell responses.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

[0023] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination. [0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0025] Nucleotide sequences are presented herein by single strand only, in the 5’ to 3’ direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three- letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0026] Except as otherwise indicated, standard methods known to those skilled in the art may be used for production of recombinant and synthetic polypeptides, antibodies or antigenbinding fragments thereof, manipulation of nucleic acid sequences, production of transformed cells, the construction of rAAV constructs, modified capsid proteins, packaging vectors expressing the AAV rep and/or cap sequences, and transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 4th Ed. (Cold Spring Harbor, NY, 2012); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0027] All publications, patent applications, patents, nucleotide sequences, amino acid sequences and other references mentioned herein are incorporated by reference in their entirety.

Definitions

[0028] As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0029] As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0030] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. [0031] Furthermore, the term “about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0032] As used herein, the transitional phrase “consisting essentially of’ is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0033] The term “consists essentially of’ (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5’ and/or 3’ or N-terminal and/or C-terminal ends of the recited sequence or between the two ends (e.g., between domains) such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids added together. The term “materially altered,” as applied to polynucleotides of the invention, refers to an increase or decrease in ability to express the encoded polypeptide of at least about 50% or more as compared to the expression level of a polynucleotide consisting of the recited sequence. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activity of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0034] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelvefold, or even fifteen-fold.

[0035] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0036] A “therapeutically effective” or “treatment effective” amount as used herein is an amount that provides some improvement or benefit to the subject. Alternatively stated, a “therapeutically effective” or “treatment effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject (e.g., in the case of raising an immune response, activating or increasing the number of immune cells known to produce an immune response). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0037] By the term “treat,” “treating,” or “treatment of’ (or grammatically equivalent terms) is meant to reduce or to at least partially improve or ameliorate the severity of the subject’s condition and/or to alleviate, mitigate or decrease in at least one clinical symptom and/or to delay the progression of the condition.

[0038] As used herein, the term “prevent,” “prevents,” or “prevention” (and grammatical equivalents thereof) means to delay or inhibit the onset of a disease. The terms are not meant to require complete abolition of disease, and encompass any type of prophylactic treatment to reduce the incidence of the condition or delay the onset of the condition.

[0039] A “prevention effective” amount as used herein is an amount that is sufficient to prevent and/or delay the onset of a disease, disorder and/or clinical symptoms in a subject and/or to reduce and/or delay the severity of the onset of a disease, disorder and/or clinical symptoms in a subject relative to what would occur in the absence of the methods of the invention. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.

[0040] As used herein, the terms “protein” and “polypeptide” are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.

[0041] The term “fragment,” as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention.

[0042] As used herein, an “isolated” polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.

[0043] As used herein, the term “modified,” as applied to a polypeptide sequence, refers to a sequence that differs from a wildtype sequence due to one or more deletions, additions, substitutions, or any combination thereof.

[0044] As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W ., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0045] As used herein, the term “substantially identical” or “corresponding to” means that two nucleic acid or polypeptide sequences have at least 60%, 70%, 80% or 90% sequence identity. In some embodiments, the two nucleic acid or polypeptide sequences can have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of sequence identity.

[0046] An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.

[0047] The term “vaccinate” and “immunize” are used interchangeably herein to mean administering a compound and/or composition (e.g., a vaccine) in a prevention effective amount to a subject. Thus, a vaccine may prevent, delay, and/or reduce the severity of a disease, disorder and/or clinical symptoms in a subject after administration to said subject relative to what would occur in the absence of the methods of the invention.

[0048] The term “adjuvant” is used herein to mean a compound and/or substance that is added to a composition to increase the efficacy and/or speed of action of an immunogen (e.g., an antigen) when said composition is administered to a subject. In some embodiments, increasing the efficacy of an immunogen will increase the immune response in said subject. In some embodiments, the adjuvant may increase the efficacy of the immunogen by at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000-fold or more. In some embodiments, the adjuvant may reduce the amount of immunogen required to produce an immune response in the subject by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the adjuvant may prolong the time over which the immunogen produces an immune response in the subject by at least about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50-fold or more. Those skilled in the art will appreciate that the adjuvant may work through any mechanism to produce the effects listed above, including, but not limited to, stabilizing the immunogen against degradation, increasing the binding affinity of the immunogen to an immune cell, activating other components of the immune system (e.g., cytokine production, interferon production, etc.), and/or altering inflammatory responses.

[0049] “Pharmaceutically acceptable,” as used herein, means a material that is not biologically or otherwise undesirable, z.e., the material can be administered to an individual along with the compositions of this invention, without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. The material would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art (see, e.g., Remington's Pharmaceutical Science,' 21^st ed. 2005). Exemplary pharmaceutically acceptable carriers for the compositions of this invention include, but are not limited to, phosphate buffered saline (PBS), sterile pyrogen-free water, and other sterile pyrogen-free physiological saline solutions.

[0050] The term “administering” or “administration” of a composition of the present invention to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function (e.g., for raising an immune response in a subject or for vaccinating a subject).

[0051] A “subject” of the invention may include any animal in need thereof. In some embodiments, a subject may be, for example, a mammal, a reptile, a bird, an amphibian, or a fish. A mammalian subject may include, but is not limited to, a laboratory animal (e.g., a rat, mouse, guinea pig, rabbit, primate, etc.), a farm or commercial animal (e.g., cattle, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, gerbil, hamster, etc.). In some embodiments, a mammalian subject may be a primate, or a non-human primate (e.g., a chimpanzee, baboon, macaque (e.g., rhesus macaque, crab-eating macaque, stump-tailed macaque, pig-tailed macaque), monkey (e.g., squirrel monkey, owl monkey, etc.), marmoset, gorilla, etc.). In some embodiments, a mammalian subject may be a human. [0052] A “subject in need” of the methods of the invention can be any subject known or suspected of having increased risk of developing an infection as described herein to which raising an immune response and/or vaccinating against said infection may provide beneficial health effects.

[0053] A “sample”, “biological sample”, and/or “ex vivo sample” of this invention can be any biological material, such as a biological fluid, an extract from a cell, an extracellular matrix isolated from a tissue, a cell (in solution or bound to a solid support), a tissue, a tissue homogenate, and the like as are well known in the art.

Compositions

[0054] One aspect of the invention relates to a composition comprising a CPAF or an antigenic fragment thereof and a STING agonist. In some embodiments, the CPAF is from Chlamydia trachomatis. In some embodiments, the CPAF comprises a sequence at least 90% identical (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the sequence of SEQ ID NO: 1 (wild-type C. trachomatis CPAF). In some embodiments, the CPAF comprises antigenic fragments of the CPAF (e.g., antigenic fragments of SEQ ID NO: 1). In some embodiments, the antigenic fragments of the CPAF are from about 5 to about 50 amino acids in length (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 35, or 50 amino acids in length). In some embodiments, the composition comprising the antigenic fragments of the CPAF comprises one or more antigenic fragments (e.g., about 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, or more antigenic fragments). In some embodiments, the amino acid sequence of the one or more antigenic fragments overlap one another (e.g., the antigenic fragments are OLPs) by from about 1 to about 20 amino acids (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 20 amino acids). In some embodiments, the antigenic fragments of the CPAF comprise the sequence of amino acids of residues between 9- 46, 37-74, 64-102, 148-186, 176-221, 295-333, 435-473, 463-501, 491-536, and/or 526-571 numbered according to the sequence of SEQ ID NO: 1. In some embodiments, the CPAF consists of or consists essentially of the sequence of SEQ ID NO: 1. In some embodiments, the CPAF contains one or more amino acid mutations relative to SEQ ID NO: 1 (e.g., up to 3, 4, 5, 6, 7, 8, 9, or 10 mutations). In some embodiments, the one or more amino acid mutations in the CPAF inhibit the proteolytic activity of the CPAF. In some embodiments, the at least one amino acid mutation in the CPAF is a serine to alanine substitution at amino acid position 491 relative to SEQ ID NO: 1 (e.g., a S491A mutation), which inhibits proteolytic activity. In some embodiments, the at least one amino acid mutation in the CPAF is a lysine to glutamine substitution at amino acid position 232 relative to SEQ ID NO: 1 (e.g., a K232Q mutation) which prevents cleavage of the CPAF during in vitro expression and/or purification. In some embodiments, the CPAF is encoded by a nucleic acid comprising a sequence at least 80% identical (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to the sequence of SEQ ID NO:2 (wild-type C. trachomatis CPAF gene). In some embodiments, the CPAF is present in the composition in a concentration of about 0.5 pg/pL to about 100 pg/pL (e.g., about 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 65, 70, 75, 80, 85, 90, 95, or about 100 pg/pL or any range therein).

SEQ ID NO: 1

MKMNRIWLLLLTFSSAIHSPVRGESLVCKNALQDLSFLEHLLQVKYAPKTWKEQYL GWDLVQSSVSAQQKLRTQENPSTSFCQQVLADFIGGLNDFHAGVTFFAIESAYLPYT VQKS SDGRFYF VDIMTF S SEIRVGDELLEVDGAP VQDVL ATL YGSNHKGT AAEES AA LRTLFSRMASLGHKVPSGRTTLKIRRPFGTTREVRVKWRYVPEGVGDLATIAPSIRAP QLQKSMRSFFPKKDDAFHRSSSLFYSPMVPHFWAELRNHYATSGLKSGYNIGSTDGF LPVIGPVIWESEGLFRAYISSVTDGDGKSHKVGFLRIPTYSWQDMEDFDPSGPPPWEE F AKIIQVF S SNTEALIIDQTNNPGGS VLYL YALLSMLTDRPLELPKHRMILTQDEVVD ALDWLTLLENVDTNVESRLALGDNMEGYTVDLQVAEYLKSFGRQVLNCWSKGDIE LSTPIPLFGFEKIHPHPRVQYSKPICVLINEQDFSCADFFPVVLKDNDRALIVGTRTAG AGGFVFNVQFPNRTGIKTCSLTGSLAVREHGAFIENIGVEPHIDLPFTANDIRYKGYSE YLDKVKKLVCQLINNDGTIILAEDGSF

[0055] In some embodiments, the STING agonist is present in the composition in a concentration of about 0.1 pg/pL to about 50 pg/pL (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or about 50 pg/pL or any range therein). The STING agonist can be any STING agonist known in the art. In some embodiments, the STING agonist is a cyclic dinucleotide, e.g., a cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), cyclic di-guanosine monophosphate (c-di-GMP), cyclic di-adenosine monophosphate (c-di- AMP; CDA), 2’3’-c-di-AM(PS)₂ (Rp,Rp) (e.g., ADU-S100), MK-1454, and/or a cyclic dinucleotide that binds the STING protein and activates the expression of type I IFN. In some embodiments, the STING agonist is a small molecule, e.g., 5,6-dimethylxanthenone-4-acetic acid (DMXAA), amidobenzimidazole (ABZI), and/or diamidobenzimidazole (diABZI). In some embodiments, the composition can comprise more than one STING agonist, e.g., 2, 3, 4, or more STING agonists.

[0056] In some embodiments, the composition as described herein further comprises at least one additional adjuvant (i.e., non-STING agonist adjuvant). In some embodiments, the at least one additional adjuvant comprises a TLR agonist and/or an oil-in-water emulsion. In some embodiments, the composition comprises three adjuvants. In some embodiments, the composition comprises more than three adjuvants, e.g., 4, 5, 6, or more adjuvants. In some embodiments, the TLR agonist is present in the composition in a concentration of about 0.5 pg/pL to about 100 pg/pL (e.g., about 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 65, 70, 75, 80, 85, 90, 95, or about 100 pg/pL or any range therein). In some embodiments, the oil-in-water emulsion is present in the composition in an amount of about 10% to about 75% by weight of the composition (e.g., about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or about 75% by weight of the composition or any range therein).

[0057] The TLR agonist can be any TLR agonist known in the art. In some embodiments, the TLR agonist is an endosomal TLR agonist, e.g., a TLR3, TLR7, TLR8, and/or TLR9 agonist. In some embodiments, the TLR agonist is unmethylated cytosine-phosphate-guanine (CpG) DNA (e.g., CpG 1018, CpG 1826, CpG 2006, and/or CpG 2395), 2BXy, loxoribine, Motolimod, and/or an imidazoquinoline or imidazoquinoline derivative (e.g., resiquimod, imiquimod, and/or gardiquimod). In some embodiments, the TLR agonist (e.g., imidazoquinoline or imidazoquinoline derivative) is conjugated to dopamine.

[0058] The oil-in-water emulsion can be any oil-in-water emulsion known in the art. In some embodiments, the oil-in-water emulsion is a squalene-based oil-in-water emulsion. In some embodiments, the oil-in-water emulsion comprises about 1% to about 20% squalene oil by volume of the emulsion (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or about 20% squalene oil). In some embodiments, the aqueous phase of the oil-in-water emulsion is buffered (e.g., the aqueous phase comprises a tris buffer or PBS). In some embodiments, the squalene-based oil-in-water emulsion further comprises one or more vitamin (e.g., vitamin E) and/or surfactant (e.g., polysorbates [e.g., polysorbate 80], sorbitan trioleate, synthetic phosphatidylcholines, eumulgin Bl, sucrose fatty acid sulfate esters, and/or poloxamers). In some embodiments, the squalene-based oil-in-water emulsion is AS03 (e.g., AddaS03), MF59 (e.g., AddaVax), and/or AF03. In some embodiments, the emulsion is a microemulsion, a submicron emulsion, or a nanoemulsion. In some embodiments, the immunogen (e.g., CPAF or an antigenic fragment thereof) and/or other adjuvants (e.g., non-oil-in-water emulsion adjuvants) are incorporated into the oil-in-water emulsion by simple mixing. In some embodiments, the immunogen and/or other adjuvants (e.g., non-oil-in-water emulsion adjuvants) remain in the aqueous phase of the emulsion. In some embodiments, the immunogen and/or other adjuvants (e.g., non-oil-in-water emulsion adjuvants) are encapsulated in the oil droplets of the emulsion.

Methods of Use

[0059] One aspect of the invention relates to a method of raising an immune response against CT in a subject, said method comprising administering an effective amount of a composition as described herein, thereby raising an immune response. In some embodiments, raising an immune response against CT in a subject comprises producing a T cell response in the subject, e.g., a CD4 T cell response, e.g., an IFNy producing CD4 T cell response; and/or producing an antibody response in the subject, e.g., a protective antibody response. In some embodiments, multiple doses of the composition are administered to the subject to raise an immune response in the subject.

[0060] Another aspect of the invention relates to a method of vaccinating a subject against CT, said method comprising administering an effective amount of a composition as described herein, thereby vaccinating the subject. In some embodiments, multiple doses of the composition are administered to the subject to vaccinate the subject. In some embodiments, a second dose of the composition is administered to the subject (e.g., a booster dose). In some embodiments, the second dose is administered from about 7 to about 29 days (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or about 29 days), or from about 1 month to about 12 months (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or about 12 months) after the first administration. Doses may be repeated until a protective response is produced and/or to maintain a protective response. Doses may be repeated on a regular schedule to maintain protection, e.g., yearly.

[0061] In some embodiments, a composition of the present invention is administered to a subject in an amount of about 1 pL to about 1 mL (e.g., about 1 pL, 2 pL, 3 pL, 4 pL, 5 pL, 10 pL, 15 pL, 20 pL, 25 pL, 30 pL, 35 pL, 40 pL, 45 pL, 50 pL, 100 pL, 150 pL, 200 pL, 250 pL, 300 pL, 350 pL, 400 pL, 450 pL, 500 pL, 600 pL, 700 pL, 800 pL, 900 pL, to about 1 mL). Subjects, Pharmaceutical Formulations, and Modes of Administration

[0062] The methods of the present invention find use in both veterinary and medical applications. Suitable subjects include avians, reptiles, amphibians, fish, and mammals. The term “mammal” as used herein includes, but is not limited to, humans, primates, non-human primates (e.g, monkeys and baboons), cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g, rats, mice, hamsters, and the like), etc. Human subjects include neonates, infants, juveniles, and adults. Optionally, the subject is “in need of’ the methods of the present invention, e.g., because the subject is believed at risk for a Chlamydia trachomatis infection. As a further option, the subject can be a laboratory animal and/or an animal model of disease. Preferably, the subject is a human.

[0063] In some embodiments, the human subject is male or female. In some embodiments, the human subject is female. In some embodiments, the human subject is sexually active. In some embodiments, the sexually active human subject has 1 or more sexual partners (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more sexual partners). In some embodiments, the sexually active human subject has one or more new sexual partners (e.g., one or more new sexual partners within the about last 12 months, e.g., about the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months). In some embodiments, the sexually active human subject may have one or more sexual partners who are known to have, or suspected of having, a chlamydia infection. In some embodiments, the human subject is 25 years old or younger (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year old). In some embodiments, the human subject lives in, or is visiting, an area where there is a known or suspected high level of chlamydia infections among the human population. In some embodiments, the human subject is a male who is sexually active with other human males. In some embodiments, the human subject is a neonate wherein said neonate’s biological mother had, or was suspected of having, a chlamydia infection during pregnancy and/or childbirth.

[0064] In particular embodiments, the present invention provides one or more pharmaceutical compositions comprising CPAF or an antigenic fragment thereof and a STING agonist in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, e.g., mucosal administration, the carrier may be either solid or liquid. In some embodiments, the pharmaceutically acceptable carrier is phosphate buffered saline (PBS) or water. In some embodiments, the pharmaceutical composition of the present invention is suitable for mucosal administration, such as where the composition has high mucoadhesion, optionally by the inclusion of a mucoadhesive polymer such as a hydrogel (e.g., chitosan, alginate, cellulose, poly-lactic-coglycolic acid, etc.) and/or a polyethylene glycol (PEG); and/or has physiological pH (e.g., between about 7.3 to about 7.5).

[0065] The compositions of the present invention may be administered to a subject by any suitable method including, but not limited to, intranasal administration (e.g., sprays or gels); oral administration (e.g., lozenges, sprays, or gels), e.g., sublingual sprays or gels; anal administration (e.g., gels or suppositories); ocular administration (e.g., eye drops or gels); intramuscular administration (e.g., intramuscular injection); subcutaneous administration (e.g., subcutaneous injection); subdermal administration (e.g., subdermal injection); intravenous administration (e.g., intravenous injection); and/or vaginal administration (e.g., gels or suppositories). In some embodiments, the administration is mucosal administration.

[0066] The amount of the disclosed compositions administered to a subject will vary from subject to subject, depending on the nature of the disclosed compositions and/or formulations, the species, gender, age, weight and general condition of the subject, the mode of administration, and the like. Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the disclosed compositions are those large enough to produce the desired effect (e.g., to raise an immune response and/or vaccinate a subject). The dosage should not be so large as to outweigh benefits by causing extensive or severe adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like, although some adverse side effects may be expected. The dosage can be adjusted by the individual clinician in the event of any counterindications. Generally, the disclosed compositions and/or formulations are administered to the subject at a dosage of CPAF or an antigenic fragment thereof ranging from about 1 pg to about 25 mg per dose (e.g., about 1 pg, 5 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, or to about 25 mg per dose or any range therein); STING agonist ranging from about 0.25 pg to about 5 mg per dose (e.g., about 0.25 pg, 0.5 pg, 0.75 pg, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 mg, 2 mg, 3 mg, 4 mg, or to about 5 mg per dose or any range therein); TLR agonist ranging from about 1 pg to about 25 mg per dose (e.g., about 1 pg, 5 pg, 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, or to about 25 mg per dose or any range therein); and/or oil-in-water emulsion ranging from about 10% to about 75% by weight of the composition per dose (e.g., about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or about 75% by weight of the composition or any range therein). Dosages above or below the range cited above may be administered to the individual subject if desired.

[0067] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.

EXAMPLES

Example 1

[0068] Chlamydia infects a wide range of species, but productive infection is generally hostspecific. Preclinical studies in mice frequently use host-adapted CM since CT is highly susceptible to the effects of murine ZFNy-induced GTPases⁴⁶. Shedding of CT serovar D is detected for only 10 days in mice after vaginal inoculation³⁷, suggesting CT delivered intravaginally is insufficient for challenge studies when assessing adaptive immune responses. Transcervical inoculation of mice with human strains results in more productive infections⁴⁷, but bypasses the cervix, a significant mucosal and immunological barrier to the upper genital tract. Transcervical inoculation with human serovars induces minimal oviduct pathology, presenting another limitation to this model.

[0069] Female mice inoculated vaginally with CM recapitulate immune mediators of protection and fallopian tube pathology observed with severe CT infection in cisgender women. This pathology, specifically oviduct scarring and fibrosis with post-obstructive dilatation or hydrosalpinx, provides a robust model for vaccine testing. CM stock populations contain subpopulations of genetic variants expressing phenotypic differences⁴⁸. Inoculation of mice with different plaque-purified clonal isolates from these stocks results in infections of varying severity²⁶. Our clone, strain CM006 (PMID 29661927), recapitulates the infection profile and pathology of its multiclonal parent stock and is a valuable challenge strain for vaccine experiments because it yields consistent levels of cervicovaginal shedding and pathology between individual mice. Intravaginal immunization of mice with the plasmid-deficient live- attenuated CM (strain CM972) elicits a protective memory CD4 T cell response that reduces bacterial burden by 2-logs and completely protects from oviduct damage when challenged with high doses (5xl0⁵) of the most virulent strains. A live-attenuated vaccine is not suitable for humans, but this attenuated strain has helped guide our rational vaccine design and informed our criteria for vaccine efficacy, determining the parameters we have achieved with our CPAF benchmark vaccine. [0070] Identification of CPAF as an immunogen for CT,

[0071] The UNC-CVI CT vaccine antigen discovery pipeline evaluated antibody and T cell responses in CT-exposed women enrolled in the T cell Response Against Chlamydia (TRAC) study^{6, 5} while also profiling chlamydial gene expression in infected TRAC2 participants. Screening T cells from 30 CT-exposed TRAC participants for reactivity against pooled peptides representing 33 CT proteins identified CPAF as the top CD4 T cell antigen tested to date. T cell responses for 21 antigens are depicted in Fig. 1, panel A and Fig. 1, panel B; 12 additional antigens tested negatively in all individuals. For this assay, short-term T cell lines were generated by culturing PBMCs for 10 days with peptides spanning each protein, then rested for 24 hrs and tested for their response to pooled peptides by IFNy ELISpot. Twelve seronegative healthy donors defined a CT-specific T cell response threshold. The mean CT- specific T cell response for these donors was 15 SFU/10⁶ cells, with the 97.5th percentile not exceeding 220 SFU/10⁶ cells. Thus, we selected 300 SFU/10⁶ to define a CT-specific T cell response. Sixteen of the TRAC participants profiled in this way responded to CPAF. In contrast, 5 responded to CT major outer membrane protein (MOMP) (Fig. 1, panel A). When in vitro expanded T cells were examined by ICS after stimulation with CPAF peptides, CD4 T cells from 28 of 28 participants tested were positive for both IFNY ^and TNFa (Fig. 1, panel B), revealing increased sensitivity of ICS over ELISpot and a 100% response rate for these individuals. Subsequent epitope mapping (N=9) identified CD4 T cell epitopes spanning the entire protein (Fig. 1, panel C). Flow cytometry confirmed that the IFNy response to CPAF was CD4 T cell dominated and durable because CPAF recognition was detected up to a year after antibiotic treatment (Fig. 2).

[0072] CPAF is a highly conserved serine protease abundantly secreted into the host cell cytoplasm late in infection and released extracellularly after host cell lysis⁴⁹. Data generated with CPAF mutants have proven that CPAF-dependent processing of host proteins contributes to the generation of new infectious progeny and their release from the host cell⁵⁰; suppression of the T cell chemokine, CXCL10⁵¹; and inactivation of neutrophils⁵². CPAF was the 9th most abundant CT transcript detected in cervical samples by RNAseq⁷. Whole immunoproteome antibody profiling of sera obtained from 232 TRAC participants revealed that CPAF (CT858) was the most immunogenic protein (recognized by 75% of participants with the highest average antibody expression levels among all antigens)⁷. Our preliminary data also indicate it is a potent CD4 T cell antigen. Thus, our candidate immunogen is a mutated CPAF with a serine-to- alanine substitution at position 491 that inhibits proteolytic activity and stabilizes the protein for consistency of manufacture³⁰. [0073] CPAF is immunodominant in mice.

[0074] We performed a screen against 33 antigens, described in detail in Table 1 below, using splenocytes isolated from female C57BL/6 mice with robust natural immunity. These mice had cleared infection with an attenuated strain, CM972, and a subsequent challenge with virulent CM001. The antigens tested included sequences related to inclusion membrane proteins (Inc), polymorphic membrane proteins (Pmps), secreted cytosolic proteins (cytosol), and Type III secretion system (T3SS) proteins. Peptides spanning outer membrane complex B (OmcB) and MOMP, both abundant outer membrane proteins, elicited moderate frequencies of 100 SFU/10⁶ splenocytes and 150 SFU/10⁶ splenocytes, respectively, while CPAF restimulation elicited 2100 SFU/10⁶ splenocytes (Fig. 3, panel A). As a control, 30 of the putative antigens were tested alongside CPAF, OmcB, and MOMP as shown. Consistent with these data, ICS confirmed that IFNy+TNFa+CPAF-specific T cells were fully CD4-restricted and comprised 6.4% of total CD44^hl memory CD4 T cells (Fig. 3, panel B). Profiling CD4 T cells harvested from the genital tracts of naive or CM972-vaccinated mice 7 days post CM001 challenge, we observed that naive controls lacked detectable CPAF-specific CD4 T cells while >6% of the total CD4 T cell memory population were CPAF-specific in CM972-immunized mice (Fig. 3, panel C).

Table 1. Antigenicity results of 33 different CT antigens.

[0075] Identifying adjuvant combinations for use with CPAF,

[0076] Testing CPAF as a vaccine immunogen in mice led to abbreviated infection and reduced pathology with a standard challenge dose (1.5xl0³ - IxlO⁴ bacteria). However, these studies used CPAF vaccines adjuvanted with interleukin- 12 (IL-12)¹⁴, or mice were administered multiple doses of CpG before and after CPAF immunization¹³. The toxicity of IL- 12 administration and the implausibility of delivering CpG on consecutive days make these approaches unsuitable for human use. Formulations combining CpG with Montanide as an adjuvant in preclinical studies with CT MOMP yielded similar protective results to those presented herein⁵³. However, this adjuvant combination is reactogenic. In one cancer trial (NCT00299728), participants sustained local and systemic side effects, making it unsuitable for an infectious disease vaccine.

[0077] In addition to CpG1018, used in the HEPLISAV-B vaccine, bacterial agonists such as c-di-AMP (CD A) show promise as adjuvants with a good safety record for mice and humans^{15, 16}. CpG stimulates via the TLR9 endosomal pathway, while CDA activates the cytosolic STING pathway. They synergize for enhanced IL-12 production by eliciting inflammatory cytokines and type I and II interferons to drive robust Thl/17 responses^{18, 19}. Third-generation synthetic CDA analogs like diABZI can be included in vaccines with immunogens and are potent and well tolerated when mucosally delivered⁵⁴. Imidazoquinoline-based TLR7 agonists generate potent immunostimulatory activity and Th 1 -biased adaptive immunity in mice and humans⁵⁵. They are effective topical therapeutics but have been too reactogenic for use as adjuvants. However, we retain immunogenicity by linking TLR7 agonists to immunomodulatory compounds, such as dopamine, while reducing pro-inflammatory side effects⁵⁶. AS03 is a squalene oil-in-water nanoemulsion that provides dose-sparing effects, significantly enhances high-affinity, antigen-specific antibodies, and increases CD4 T cell responses⁵⁷'⁵⁹. Sublingual (s.l.) and/or intranasal (i.n.) immunization with liquid or gel-based vaccines provides a safer and acceptable mucosal route and circumvents formulation challenges when compared to intramuscular (i.m.) immunization. The benefits of i.n. or s.l. delivery includes the potential for self-administration, induction of protection at multiple mucosal sites (genital, throat, anal, oral, and/or nasal), minimized discomfort, and avoidance of the fear of needles, properties that would aid implementation of broad vaccination for this important sexually transmitted pathogen of adolescents and young adults.

Example 2

[0078] Most vaccines against bacterial pathogens induce antibody as a primary mode of protection. However, chlamydial protection is dependent on CD4 T cell-mediated responses⁸' ⁿ. Until the past decade, the development of vaccines against intracellular bacterial pathogens, which require cell-mediated immunity for protection, was hindered by the absence of safe and effective adjuvants that could induce Thl and Thl7 responses¹². Early studies using CPAF as an immunogen demonstrated protection when CPAF was adjuvanted with IL-12 or multiple doses of CpG before and after CPAF immunization^{13, 14} However, the toxicity of IL- 12 administration and the implausibility of delivering CpG on consecutive days make these approaches unsuitable for human vaccination. Bacterial agonists such as CDA show promise as adjuvants with a good safety record for mice and humans¹⁵'¹⁷. CDA activates the cytosolic STING pathway that elicits inflammatory cytokines and interferons to drive robust Thl/17 responses¹⁸'²¹. CpG and AS03 have shown effectiveness in eliciting CD4 T cell responses in humans²²'²⁴

[0079] Due to the good safety and immunogenicity profiles of CD A, CpG, and AS03, we examined the ability of intranasally delivered CPAF, combined with these adjuvants alone and in combination, to protect female mice against intravaginal challenge with Chlamydia muridarum.

[0080] Materials and Methods

[0081] Mice

[0082] Female C57BL/6J (stock #: 000664) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were given food and water ad libitum in an environmentally controlled, pathogen-free room with a cycle of 12 h of light and 12 h of darkness. Mice were age-matched and used between 8 and 12 weeks of age. All experiments were approved by the Institutional Animal Care and Use Committee at the University of North Carolina and the University of Chicago.

[0083] CPAF antigen and adjuvants

[0084] An inactive variant of Chlamydia (Chlamydia muridarum) Protease Activity Factor (CPAF Cm) antigen was expressed using XpressCF® (Sutro Biopharma). The catalytic serine at position 491 was changed to alanine. The CPAF Cm clone was a gift from Jon Harris (Queensland University of Technology). After cell-free expression, the protein was purified by Ni affinity chromatography (Cytiva). CPAF Cm was admixed with CpG1826 (Invivogen) (10 pg/dose) and/or STING agonist 2’3’-c-di-AM(PS)2 (Rp,RP) (Invivogen) (5 pg/dose) in PBS or mixed at a 1 : 1 ratio with the squalene oil-in-water adjuvant AddaS03 (AS03) (Invivogen). CPAF was conjugated to the TLR7/8 agonist 2-Bxy-Dopa⁵⁶ via DBCO-azide click chemistry and used as a comparator for pathology studies.

[0085] SDS-PAGE Gel Electrophoresis

[0086] Proteins were prepared in 2x-Laemmli sample buffer (Bio-Rad, Philadelphia, PA) added with 5% P-mercaptoethanol and denatured by heating at 95 °C for 5 min. The denatured protein samples (20 pl) were loaded onto 12% precasted polyacrylamide gels (Bio-Rad). For western blot analysis, SDS-PAGE-run samples were then transferred into the nitrocellulose membrane using Trans-Blot Turbo Transfer Packs (Bio-Rad). Then the membrane was blocked with PBS containing 0.5% Tween-20 (PBS-T) and 3% BSA, and incubated with anti-His tag antibodies (#MA1-21315, Invitrogen; diluted 1 : 5,000 in PBS-T) or murine anti-CPAF immune sera (pooled from mice immunized with CPAF protein; diluted 1: 1000 in PBS-T) overnight at 4°C. The membrane was washed with PBS-T and incubated with Horseradish Peroxidase (HRP)-labeled goat anti-mouse IgG (#5220-0460, KPL SeraCare, Gaithersburg, MD) diluted 1 : 10,000 in PBS-T for 1 hour at room temperature. The membrane was again washed with PBS-T, revealed with Immobilon Western Chemiluminescent HRP Substrate (Millipore Sigma, Burlington, MA) and analyzed using Gel Doc imaging system (Bio-Rad). Blots and gels derived from the same or side-by-side experiments were processed together.

[0087] Analytical SEC

[0088] Analytical SEC experiments were performed with a Superdex 200 Increase 10/300GL SEC column (Cytiva). The flow rate was 1 ml/min in PBS running buffer. 200 pl of a 1 mg/ml protein sample was injected, and the chromatograms were monitored at A280.

[0089] Mass spectrometry

[0090] The analysis was done on a Sciex X500B QTOF (AB Sciex, Redwood City, CA) coupled with Agilent’s 1290 UHPLC system (Agilent Technologies, Santa Clara, CA). LC- MS and LC-MS-MS approaches were taken to determine and confirmed the clip site of CPAF. A Waters (Waters, Milford, MA) BioResolve RP mAb Polyphenyl Column (450 A, 2.7 pm 2.1x50 mm) column was used for intact protein analysis. Another Waters Cl 8 column (Acquity Premier, Peptide CSH C18 130A 1.7 pM, 2.1x150 mm) was used for the peptide mapping analysis. The acquired MS data were analyzed with BioPharmaView (AB Sciex, Redwood City, CA) Software.

[0091] Mucosal immunization

[0092] Mice were anesthetized intraperitoneally with 250 pL of Nembutal (50 mg/mL) and then immunized intranasally on day 0 with 15 pg adjuvanted CPAF or CPAF alone in a 12 pl volume (6 pl/nare). Mice were intranasally boosted 30 days later with the same vaccine formulation(s).

[0093] ELISpot assay

[0094] Single-cell suspensions of murine splenocytes were prepared by passing cells through 70 pM cell strainers and ACK lysis buffer prior to resuspension in complete media. For analysis of IFNy production, cells (l-2.5* 10⁵ cells/well) were stimulated with a pool of 18-amino acid peptides overlapping by 15 amino acids (Sigma Aldrich) spanning the Chlamydia muridarum CPAF S491A antigen (final concentration of 2.5 pg/ml)²⁵ on PVDF-membrane plates (Millipore) coated with 5 pg/ml anti-mouse IFNy (AN18). After 18-20 hours of stimulation, IFNy spot-forming cells were detected by staining membranes with anti-mouse IFNy biotin (1 pg/ml; R46A2) followed by streptavidin-alkaline phosphatase (1 pg/ml) and developed with NBT/BCIP substrate solution (Thermo Fisher). Spots were enumerated on an AID ELISpot reader.

[0095] Intracellular cytokine staining (ICS)

[0096] For analysis of intracellular cytokines, cells were stimulated as above in a round-bottom 96 well plate at l>< 10⁶ cells/well in the presence of 2.5 pg/ml CPAF pooled peptides (Synpeptide), media alone (negative control), or Cell Activation Cocktail (Biolegend; positive control) for 6 hours at 37 °C with 5% CO2 in the presence of brefeldin A (Biolegend) and monensin (Biolegend). Cells were stained with a Zombie UV fixable viability kit (Biolegend) in PBS. Cells were washed and incubated with Mouse Fc Block (anti-CD16/CD32; BD Pharmingen) followed by staining for surface markers anti-Ly6G BV421 (1A8; Biolegend; dump channel), anti-CD45 Alexa Fluor 700 (30-F11; BioLegend), anti-CD3 FITC (17A2; BD Biosciences), anti-CD4 BV570 (RM4-5; BioLegend), anti-CD8 BUV395 (53-6.7; BD Biosciences), anti-CD44 BV480 (IM7; BD Horizon), and anti CD62L BV650 (MEL-14; Biolegend) in Brilliant Stain Buffer (BD Horizon). Following surface staining, cells were fixed and permeabilized using Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences). Cells were then stained for intracellular cytokines anti-TNFa BV711 (MP6-XT22; BioLegend), anti-IFNy APC Fire-750 (XMG1.2; BioLegend), anti-IL17A PE-Cy7 (TC11- 18H10.1; BioLegend), and anti-IL-5 PE (TRFK5; Biolegend) using Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences). Samples were washed and resuspended in PBS+1%FBS prior to acquisition on a Cytek Aurora spectral cytometer, and data were analyzed with FlowJo version 10 software.

[0097] Antibody ELISA assay

[0098] Serum from vaccinated C57BL/6 mice were assayed for CPAF-specific IgG, IgGl, IgG2b and IgG2c antibody responses. ELISA plates were coated overnight at 4 °C with 10 pg/ml recombinant MBP-CPAF (from John Harris, Queensland University of Technology) diluted in 0.5M NaHCO3. Plates were washed with PBS-Tween. After blocking with 2% BSA PBS-Tween for 1 hour at 37 °C, samples were serially diluted and incubated for 1 hour at 37 °C. Internal controls were generated using reference serum. Plates were washed with PBS- Tween. A goat anti-mouse horseradish peroxidase-conjugated secondary antibody was added (Southern Biotech) and incubated for 1 hour at 37 °C. Goat anti-mouse IgG was added at a 1 :4,000 dilution and IgGl, IgG2b, and IgG2c were added at a 1 :500 dilution. After washing, the plates were developed using TMB substrate solution (ThermoScientific) for a maximum of 20 minutes. The reaction was stopped using 0.5M H2SO4 and OD was read at 450 nm. The cutoff for detecting a specific antibody response was defined by a value greater than the mean+3 standard deviations of the non-specific antibody values.

[0099] Cytokine ELISA assay

[0100] Mouse lungs were harvested using T-PER Tissue Protein Extraction Reagent (Thermo Fisher, Cat# 78510), supplemented with a protease inhibitor. The supernatant of lung homogenates was collected and used for ELISAs. Cytokine levels of TNF-a, IL-6, and IFN-y were measured using ELISA Max kits from BioLegend, per the manufacturer’s instructions. [0101] Strains, cell lines, and culture conditions

[0102] Plaque-purified C. muridarum Nigg strain CM006²⁶ was propagated in mycoplasma- free L929 cells²⁷ and titrated by inclusion-forming units²⁸ using a fluorescently tagged anti- chlamydial lipopolysaccharide monoclonal antibody (Bio-Rad).

[0103] Chlamydia infection quantification

[0104] Female mice at least 8 weeks old were subcutaneously injected with 2.5 mg medroxyprogesterone (Depo-Provera; Upjohn) 5-7 days prior to infection to induce a state of anestrous. Mice were intravaginally inoculated with U 10⁵ inclusion forming units (IFU) CM006 diluted in 30 pl sucrose-sodium phosphate-glutamic acid buffer 28 days post booster immunization. Mice were monitored for cervicovaginal shedding via endocervical swabs²⁸, and IFUs were calculated, as described previously²⁹. Animal welfare was monitored daily.

[0105] Lung histopathology

[0106] Lungs were perfused with PBS (2 mM EDTA) followed by 10% formalin. They were then inflated through the trachea using 10% formalin and fixed for 48 hours before paraffin processing. A pathologist blinded to the study design analyzed H&E-stained slides for gross pathology for inflammation and hemorrhaging.

[0107] Hydrosalpinx and oviduct histopathology

[0108] Genital tract gross pathology, including hydrosalpinx development, was examined and recorded at sacrifice on day 42 post-challenge. The genital tract was collected in its entirety with careful removal of adipose as needed. Samples were placed flat between two sponges in a histology tissue cassette and placed in 10% neutral buffered formalin for at least 72 hours before routine processing to paraffin blocks. Tissue blocks were sectioned to 5 pm onto charged slides and stained with hematoxylin and eosin. Histological samples were evaluated in a masked fashion by a board-certified veterinary pathologist (RSS). Within a single animal, each side was scored independently. An overall assessment of oviduct dilation was reported for each uterine horn. Samples were scored on a 0-5 scale: 0=no finding; l=minimal finding; 2=mild finding; 3=moderate finding; 4=marked finding; 5=severe finding.

[0109] Statistical Analysis

[0110] Differences between the means of experimental groups after infection were calculated using two-way repeated measures (RM) ANOVA. Significant differences in ELISA and ELISpot data were determined by one-way ANOVA. Significant differences in ICS responses were determined by the Wilcoxon Rank Sum Test. Statistical differences in oviduct histopathology were determined by the Kruskal-Wallis test. Prism software (GraphPad) was utilized for statistical analyses, and values of p < 0.05 were considered significant.

Results

[0111] Expression of inactivated C. muridarum CPAF using cell-free protein synthesis

[0112] The S491A mutation in the tail-specific protease domain renders the CPAF protease inactive and incapable of natural auto-processing and proteolysis^{30, 31}. However, cell-free expression yielded full-length CPAF and a clipped form consisting of N- and C-terminal fragments (Fig. 4, panel A). Mass spectrometry revealed the clip site to be at K232 (Fig. 5, Table 2), three amino acids from the native auto-processing site. The K232Q mutation produced a full-length Cm CPAF protein (Fig. 1, panel B). Both Cm CPAF variants were applied to an analytical sizing column and eluted as a single Gaussian peak (Fig. 1, panel C). The full-length wild-type and K232Q variant had the same retention time demonstrating similar folding and dimerization. The clipped form was used for vaccine studies.

Table 2. Clipped N- and C-terminal peptides of CPAF from cell-free expression

[0113] Intranasal immunization with adjuvanted CPAF does not induce lung pathology.

[0114] Female mice were immunized intranasally with CPAF formulated with different adjuvant combinations and protein/vehicle controls. Lungs were harvested at 3-hour, 24-hour, and 1-week time points post-immunization for analysis of inflammatory cytokines and pathology (Fig. 6, panels A-C). Lung homogenate levels of IFNy, IL-6, and TNFa after immunization were not significantly different than PBS vehicle controls across all time points. Further, lung histology did not show signs of inflammation or differences in multi-focal hemorrhaging between the PBS vehicle group and adjuvanted formulations. No signs of necrosis or scar tissue were observed. (Fig. 6, panels D-F)

[0115] Intranasal immunization with CP AF plus CDA induces a memory CD4 T cell response characterized by the production of IL-17 A or IFNy.

[0116] Female C57BL/6 mice immunized intranasally and boosted with a second dose thirty days later were assessed for systemic antibody and T cell responses ten days post-boost (Fig. 7, panel A). Most mice immunized with CPAF alone, CPAF plus CDA, or CPAF plus CpG/CDA/AS03 had low to undetectable serum antibody titers with no significant differences between the groups (Fig. 7, panel B). Thl-biased IgG2b and IgG2c were detected in a few individual mice, while IgGl was not detected. In contrast, mice immunized with CPAF plus CDA alone or in combination with CpG and/or AS03 had elevated levels of CPAF-specific IFNy-producing T cells (mean = 562-676 SFUs) (Fig. 7, panel C), which were not consistently detected in mice immunized with CPAF alone or adjuvanted with CpG and/or AS03. We did not observe any synergistic effect when CpG and/or AS03 were included with CDA. The memory (CD44^hl CD62L ) CPAF-specific T cell response after CPAF + CDA immunization was almost entirely CD4-biased and contained a mixture of CD4 T cells producing either IL- 17A or IFNY with very few IL-17A/IFNy co-producing cells (Fig. 9; Fig. 7, panels D-E). Nearly 4% of memory CD4 T cells produced TNFa alone or in combination with IL-17A or IFNy, while TNFa was the only cytokine significantly detectable over background from CD8 T cells.

[0117] Intranasal immunization with CPAF plus CDA reduces cervical burden in female mice. [0118] Female mice intranasally immunized with CPAF plus CDA alone or combined with CpG and/or AS03 had significant reductions in cervical chlamydial burden after intravaginal challenge compared to PBS controls (Fig. 8, panel A), with CPAF plus CDA immunization resulting in a 1.5 log reduction in cervical burden. We observed no synergistic protection when combining CDA with CpG and/or AS03. The only regimen that significantly reduced bacterial burden and the frequency of oviduct hydrosalpinx, compared to PBS controls, was CPAF plus CDA (Fig. 8). In contrast, the unadjuvanted controls and formulations that included AS03 without CDA exhibited high frequencies of oviduct hydrosalpinx (Fig. 8, panel B).

Discussion [0119] STING pathway agonists have demonstrated an excellent safety profile in mice and humans^{15, 16} and have elicited strong T cell immunogenicity and protection against bacterial pathogens like Mycobacterium tuberculosis^{32, 33} and Bordetella pertussis^{34, 35}. However, their efficacy has not been tested against Chlamydia infection. Without wishing to be bound by any particular theory, we hypothesized that intranasal immunization with the immunodominant secreted antigen CPAF combined with CDA would induce a memory CD4 T cell response and protect female mice against genital Chlamydia infection.

[0120] We expressed full-length inactive CPAF in a cell-free protein synthesis platform amenable to scale-up³⁶. While a clipped form of CPAF was used in these studies, we were recently able to express a full-length form that provides a consistent product for manufacture and will be the focus of future analyses. Mass spectrometry revealed in vitro clipping at the Lysine232 residue that is three amino acids upstream of the natural auto-processing cleavage site^{30, 31}. Mutation of this residue to glutamine prevented clipping to yield a full-length product. Without wishing to be bound by any particular theory, these data suggest the region, e.g., amino acids 235-276 numbered according to SEQ ID NO: 1, between the N- and C-terminal domains of CPAF (i.e., CPAFn and CPAFc, respectively) is especially prone to proteolysis, perhaps both by auto-processing and exogenous proteolytic activity from bacterial cell lysate depending on various factors such as mutations, protein expression system used, and the like.

[0121] Intranasal delivery of CPAF with CDA, CpG, and/or AS03 did not induce any detectable damaging inflammatory responses in the lungs after intranasal immunization, aligning with the known safety records of these adjuvants in mice and humans. We did not observe any significant increases in the levels of IFNy, IL-6, or TNFa in the lungs compared to PBS controls, nor differences in multi-focal hemorrhaging compared to unadjuvanted controls.

[0122] Prime-boost immunization induced low to undetectable frequencies of CPAF-specific IgG. However, the few detectable responses were characterized by Thl -biased IgG2b and IgG2c consistent with the antibody profiles of mice immunized intranasally with CPAF adjuvanted with CpG or IL-12^{13, 14} Recent data showed that B-cells were completely dispensable for protection elicited with chimp adenovirus (ChAD)-vectored CPAF²⁵, and B- cell deficient mice immunized with recombinant CPAF did not exhibit an altered course of clearance³⁷. However, without wishing to be bound by any particular theory, it is possible that Fc-mediated antigen presentation could be enhanced by CPAF-specific IgG, resulting in boosted CD4 T cell responses³⁸. [0123] We found that the inclusion of CDA in the adjuvant formulation was necessary for T- cell immunogenicity. CPAF-specific T cells were characterized by a CD4-dominant response, consistent with other studies using protein immunization⁴⁰. We did not observe a significant CPAF-specific CD8 T cell response, which contrasts sharply with the dominant CD8 T cell response elicited with ChAd.CPAF²⁵. Without wishing to be bound by any particular theory, this difference can be explained by the fact that exogenous recombinant proteins are easily endocytosed by professional antigen-presenting cells for MHC class II presentation to CD4 T cells, while viral vectors predominately release antigens into the infected cell cytosol for presentation on MHC class I to CD8s⁴¹.

[0124] The CD4 response demonstrated a mixed profile of IFNy ± TNFa and IL-17A ± TNFa producing cells, like those achieved in mice with the subcutaneously delivered MOMP -based immunogen CTH522 adjuvanted with CAF01⁴². The immunophenotypic T cell expression of IFNy i TNFa is routinely measured in Chlamydia studies, while detecting IL-17A responses is less common⁴³. It is unclear if there is a need for de facto or ex-Thl7 cells in vaccine-elicited protection against Chlamydia genital infection, as observed for other bacterial pathogens like Klebsiella spp. and Mycobacterium tuberculosis^{44, 45}. We also observed a low frequency of TNFa single-positive CD8 T cells. These monofunctional cells are shown to be dispensable for protection²⁵.

[0125] The vaccine-elicited reductions in chlamydial burden after genital challenge correlated with our T cell immunogenicity data. Mice vaccinated with regimens including CDA exhibited similar clearance kinetics and achieved a 1.5-log reduction in bacterial load compared to PBS controls. These results further confirmed that CDA as the only adjuvant was sufficient for protection. Despite significant reductions in burden, we were not able to significantly prevent the development of hydrosalpinx with our CPAF + CDA regimen. However, a trend for reduced gross hydrosalpinx and oviduct dilatation compared to PBS controls was observed, and this regimen did not result in the levels of oviduct dilatation elicited with unadjuvanted CPAF or non-protective regimens incorporating AS03 with CDA.

[0126] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

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Claims

WHAT IS CLAIMED IS

1. A composition comprising a chlamydial protease-like activity factor (CPAF) or an antigenic fragment thereof and an adjuvant, wherein the adjuvant is a stimulator of interferon genes (STING) agonist.

2. The composition of claim 1, wherein the CPAF is a Chlamydia trachomatis CPAF.

3. The composition of claim 1 or 2, wherein the CPAF contains one or more amino acid mutations relative to SEQ ID NO: 1.

4. The composition of claim 3, wherein at least one amino acid mutation inhibits proteolytic activity of the CPAF.

5. The composition of claim 3 or 4, wherein the at least one amino acid mutation is a S491A substitution.

6. The composition of any one of claims 1-5, wherein: the CPAF or an antigenic fragment thereof is present in the composition in a concentration of about 0.5 pg/pL to about 100 pg/pL and the STING agonist is present in the composition in a concentration of about 0.1 pg/pL to about 50 pg/pL.

7. The composition of any one of claims 1-6, wherein the STING agonist is a cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), cyclic di-guanosine monophosphate (c-di-GMP), cyclic di -adenosine monophosphate (c-di-AMP; CD A), 2’3 ’-c- di-AM(PS)2 (Rp,Rp), MK-1454, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), amidobenzimidazole (ABZI), and/or diamidobenzimidazole (diABZI).

8. The composition of any one of claims 1-7, wherein the composition further comprises at least one additional adjuvant.

9. The composition of claim 8, wherein the at least one additional adjuvant is a toll-like receptor (TLR) agonist and/or an oil-in-water emulsion.

10. The composition of claim 8 or 9, wherein: the TLR agonist is present in the composition in a concentration of about 0.5 pg/pL to about 100 pg/pL and/or the oil-in-water emulsion is present in the composition in an amount of about 10% to about 75% by weight of the composition.

11. The composition of claim 9 or 10, wherein the TLR agonist is unmethylated cytosine- phosphate-guanine (CpG) DNA (e.g., CpG 1018, CpG 1826, CpG 2006, and/or CpG 2395), 2BXy, loxoribine, Motolimod, and/or an imidazoquinoline or imidazoquinoline derivative (e.g., resiquimod, imiquimod, and/or gardiquimod).

12. The composition of claim 11, wherein the imidazoquinoline or imidazoquinoline derivative is conjugated to dopamine.

13. The composition of claim 9 or 10, wherein the oil-in-water emulsion is a squalene- based oil-in-water emulsion.

14. The composition of claim 13, wherein the squalene-based oil-in-water emulsion further comprises one or more vitamin (e.g., vitamin E) and/or surfactant (e.g., polysorbates, sorbitan trioleate, synthetic phosphatidylcholines, eumulgin Bl, sucrose fatty acid sulfate esters, and/or poloxamers).

15. The composition of claim 14, wherein the squalene-based oil-in-water emulsion is AS03 (e.g., AddaS03), MF59 (e.g., AddaVax), and/or AF03.

16. The composition of any one of claims 1-15, which is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

17. The composition of claim 16, wherein the pharmaceutically acceptable carrier is phosphate buffered saline (PBS) or water.

18. The composition of any one of claims 1-17, wherein the composition is suitable for mucosal administration.

19. A method of raising an immune response against Chlamydia trachomatis in a subject, said method comprising administering an effective amount of the composition of any one of claims 1-18, thereby raising an immune response.

20. The method of claim 19, wherein the administering increases interferon gamma (IFNy) producing CD4 T cell levels in the subject.

21 The method of claim 19 or 20, wherein: the CPAF is present in the composition in an amount of about 1 pg to about 25 mg per dose and the STING agonist is present in the composition in an amount of about 0.25 pg to about 5 mg per dose.

22. The method of any one of claims 19-21, wherein the administering comprises mucosal administration (e.g., intranasal and/or intraoral administration).

23. The method of any one of claims 19-22, wherein the subject is a mammal, optionally wherein the subject is human.

24. The method of any one of claims 19-23, wherein the administering comprises administering from about 1 pL to about 500 pL of the composition.

25. A method of vaccinating a subject against a Chlamydia trachomatis infection, said method comprising administering a effective amount of the composition of any one of claims 1-18, thereby vaccinating the subject.

26. The method of claim 25, wherein the administering increases IFNy producing CD4 T cell levels in the subject.

27. The method of claim 25 or 26, wherein: the CPAF is present in the composition in an amount of about 1 pg to about 25 mg per dose and the STING agonist is present in the composition in an amount of about 0.25 pg to about 5 mg per dose.

28. The method of any one of claims 25-27, wherein the administering comprises mucosal administration (e.g., intranasal and/or intraoral administration).

29. The method of any one of claims 25-28, wherein the subject is a mammal, optionally wherein the subject is human.

30. The method of any one of claims 25-29, wherein the administering comprises administering from about 1 pL to about 500 pL of the composition.

31. The method of any one of claims 25-30, wherein a second dose of the composition is administered to the subject.

32. The method of claim 31, wherein the second dose is administered from about 7 to about 35 days (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or about 35 days) after the first administration.