WO2023250522A2 - Vaccine adjuvants and methods - Google Patents

Abstract

The disclosure provides adjuvant compositions and methods for generating an immune response by administering the adjuvant compositions with vaccines, such as by the intradermal route. Exemplary adjuvant compositions include a double-mutant heat-labile toxin adjuvant derived from an Escherichia coli enterotoxin and a bacterial-derived outer membrane vesicle adjuvant from an attenuated strain of Burkholderia pseudomallei.

Description

VACCINE ADJUVANTS AND METHODS

Statement of Federal Funding

[0001] This invention was made with government support under 272201800045C and AI166756 awarded by the National Institutes of Health. The government has certain rights in the invention.

Statement of Federal Funding

[0002] This application contains, as a separate part of disclosure, a Sequence Listing in computer-readable form (Filename: 58137_SeqListing.xml; 2,731 bytes - XML file dated June 22, 2023) which is incorporated by reference herein in its entirety.

Field

[0003] The disclosure provides adjuvant compositions and methods for generating an immune response by administering the adjuvant compositions with vaccines, such as by the intradermal route. Exemplary adjuvant compositions include a double-mutant heat-labile toxin adjuvant derived from an Escherichia coli enterotoxin and a bacterial-derived outer membrane vesicle adjuvant from an attenuated strain of Burkholderia pseudomallei.

Background

[0004] Despite the availability of vaccines and other therapeutics, infectious diseases continue to plague people in all regions of the world. The vast majority of infections occur in mucosal tissues, and these infections remain one of the leading causes of mortality in children under the age of five. Notably, the majority of lethal infections in children manifest as pneumonia followed closely by diarrhea. The Global Enteric Multicenter Study (GEMS) found that a majority of moderate-to-severe diarrhea was caused by just four pathogens: Rotavirus, Cryptosporidium, Shigella, and Enterotoxigenic E. coli. Of these, a licensed vaccine exists only for Rotavirus. Respiratory infections are even more prevalent in young children and, while there are a variety of vaccines against respiratory pathogens, there remains a significant lack of effective vaccines for some of the most severe pulmonary pathogens. Respiratory Syncytial Virus (RSV) remains one of the most common causes for hospitalization and death in the developing world and yet there is no vaccine currently licensed for RSV. Tuberculosis is predicted to infect one third of the world’s population and yet the current vaccine, Bacille Calmette-Guerin (BCG), exhibits low to moderate protective efficacy against pulmonary disease that varies geographically. The current vaccine against whooping cough includes an acellular component for Pertussis that prevents disease, but does not limit bacterial mucosal colonization or infection spread. Most obviously, the SARS CoV-2 pandemic highlights the need to discover and understand how vaccine design can better target protective immunity to the pulmonary mucosa. While less prevalent in children, sexually transmitted diseases (STDs) are no less important and can have lasting effects in adults. That vaccines can be effective against STDs is evidenced by the highly successful human papillomavirus (HPV) vaccine, which has significantly decreased HPV infection and reduced the incidence of HPV-caused cervical cancer in women; however, there exists a need for new STD vaccines that can target the female reproductive tract (FRT). For example, there is no vaccine for genital herpes or HIV, demonstrating a need for new vaccines that target these pathogens. Further, while bacterial STDs (e.g. gonorrhea, Chlamydia, and syphilis) are currently treated with antibiotics, the increase in antibiotic resistance and the fact that asymptomatic people can unknowingly spread the disease to partners makes vaccine development crucial. It is highly likely that targeting the immune response directly to the FRT mucosa would increase the effectiveness of such vaccines.

[0005] The mucosal immune response is traditionally initiated when antigen-presenting cells (APC) encounter foreign antigen (Ag) in the mucosal compartment e.g. intestinal lumen or airway), where APCs, particularly dendritic cells (DCs), directly sample Ag through surveillance of the mucosal luminal space. These DCs then have the potential to induce a mucosal homing phenotype on T cells. For example, in the intestine, CD103+ DCs sample Ag in the gut, migrate into the mesenteric lymph nodes (MLN), and impart upregulation of gut-specific homing receptors, a4p7 and CCR9, on T cells. These cells are then primed to migrate back into the intestine where they can elicit antimicrobial function. The mechanisms for mucosal homing are less clear for the lung and the female reproductive tract (FRT). There appears to be a remarkable array of potential lung homing molecules; however, there is not yet a clear pulmonary homing signature. Similarly, while there are clearly mucosal T cells in the FRT, the homing markers that drive those cells into the FRT are less obvious, with CD11c or CXCR3 and CCR5 being potential markers of interest. What is increasingly acknowledged is that, like within the gut, specialized tissue-resident DCs take up infectious luminal antigen, migrate to the mucosal tissue draining lymph nodes [mediastinal lymph nodes (MedLN) for lung; iliac lymph nodes (ILN) for the FRT], and activate CD4 or CD8 T cells to migrate back into the mucosal tissues. This “mucosal to mucosal” cycle is the hallmark of inducing immunity in these tissues and has long served as the standard for how mucosal immunity is achieved.

[0006] There remains a need in the art for products and methods for generating immune responses to antigens of interest.

Summary [0007] Vaccines can be administered mucosally to drive the desired immune response at a particular mucosal site; however, this approach has some caveats that can preclude mucosal vaccination. For example, while some vaccines are delivered mucosally (predominantly orally) and are efficacious in developed countries, they often fail to protect children in developing countries making oral vaccination impossible. A prime example of this is the oral polio vaccine which requires many more immunizations to achieve equivalent protective levels of immunity compared to children in developed countries. Multiple factors appear to be responsible for this; however, inadequate colonization of the intestinal mucosa due to ongoing diarrheal disease appears to play a significant role, as does oral tolerance. It is known that the most effective classical “mucosal adjuvants”, such as cholera toxin (CT - derived from the enteric pathogen Vibrio cholerae), delivered either orally or intranasally in mice, can induce potent cell-mediated and antibody (Ab) responses in the mucosal compartment; however, while this approach is attractive in terms of initiating an immune response at pathogen sites of entry, it carries limitations: mucosally-delivered CT and other bacterial-derived toxins have known side effects, such as inducing facial paralysis, when administered intranasally. Whole toxin adjuvants, while immunologically effective, are dangerous when delivered orally which is unsurprising given their enteric pathogen origin. In some cases, mucosal vaccination is limited by the harsh environment of mucosal tissues (e.g., acidity in the stomach) or the impractically of immunization (/.e., intravaginally) and concerns exist about ensuring that the vaccine correctly targets the inductive mucosal immune tissues. With these caveats in mind, the present disclosure provides an intradermal approach. Importantly, while inducing mucosal immunity in mucosal tissues may be achievable by immunizing individually directly into each of these sites, the compositions and methods herein circumvent this need for site-specific immunization by inducing mucosal immunity in all mucosal tissues. The methods involve specifically targeting mucosal surfaces during the effector phase by using adjuvants to manipulate the inductive phase of systemic vaccination. This can be achieved either by injecting a vaccine plus an adjuvant composition provided herein directly into the skin e.g., Figure 1) or muscle, or by administering the vaccine and the adjuvant composition, at indivimucosal sites (e.g., the mouth, the respiratory tract, the gastrointestinal tract, the nose, and/or the female reproductive tract).

[0008] The disclosure provides adjuvant compositions comprising detoxified bacterial endotoxin adjuvant, such as from Escherichia coli, and an outer membrane vesicle adjuvant from a gram-negative bacteria. The adjuvant compositions can comprise an adjuvant of SEQ ID NO: 1 (dmLT) and an attenuated Burkholderia pseudomallei Bp82 bacterial strain-derived outer membrane vesicle (OMV) adjuvant.

[0009] The adjuvant compositions provided can further comprise a vaccine. The vaccine can comprise a polypeptide, a nucleic acid, a polysaccharide, a polysaccharide-polypeptide conjugate, a live-attenuated or inactivated bacterium, a toxoid, a live-attenuated or inactivated virus, a virus-like particle, or a viral vector. The nucleic acid can be a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). The vaccine can comprise or encode a bacterial, viral, or fungal antigen.

[0010] The disclosure provides kits comprising a DMLT adjuvant and an OMV adjuvant.

[0011] The kits can further comprise a vaccine. The vaccine can comprise a polypeptide, a nucleic acid, a polysaccharide, a polysaccharide-polypeptide conjugate, a live-attenuated or inactivated bacterium, a toxoid, a live-attenuated or inactivated virus, a virus-like particle, or a viral vector. The nucleic acid can be a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). The vaccine can comprise or encode a bacterial, viral, or fungal antigen.

[0012] The disclosure provides methods of generating an immune response in a subject comprising administering to the subject an adjuvant composition of the disclosure. Administration can be by the intradermal route. The methods increase an immune response in the subject compared to the immune response in the absence of the adjuvant composition. The immune response can be a pan-mucosal immune response. The immune response can be in the mucosa of the respiratory, digestive or urogenital tracts. The immune response can be in the mucosa of the eye. The immune response can be one or more of: a B cell response, the generation of CD4+ T cells and the generation of CD8+ T cells.

Brief Description of the Drawings

[0013] Figure 1 shows parenterally immunized mice developing T (Aim 1 ) and B (Aim 2) resident memory cells in all mucosal tissues that will protect against a variety of mucosal infections.

[0014] Figure 2 shows tools to detect model Ag-specific T cells and B cells. The CD4 T cell epitope 2W1S was fused to chicken egg ovalbumin (Ova) to create a single immunogenic fusion protein. Immune responses were tracked using 2W1S-specific CD4 T cells, Ova-specific B cells, and Ova-specific CD 8 T cells as Ova also internally contains the CD8 T cell epitope SIINFEKL (SEQ ID NO: 2) for which MHCI tetramers are made.

[0015] Figure 3 shows dmLT Induces Antigen-Specific T Cell Migration to the Mucosa Following Vaccination. WT mice were immunized ID with 2W1S Ag plus dmLT. 10 days later animals were intravenously injected with fluorescently conjugated anti-CD45 to label circulating CD4 T cells and euthanized 3 minutes later. The CLN, FRT, large intestine lamina propria, and lungs were harvested and tissue resident (non-blood) CD4 T cell responses were assessed by FACS analysis using MHC-II tetramers. The gating strategy to identify tissue resident versus circulatory cells is shown (left). Concatenated flow plots show tissue resident 2W1S-specific T cells in each tissue from 5 mice (representative of 2 independent experiments). Numbers represent the percent in the 2W1S-specific gate.

[0016] Figure 4 shows dmLT induces more isotype switched Ova-specific B cells to migrate to mucosal tissue compared to CpG. WT mice were ID injected with Ova plus CpG or dmLT and were boosted 28 days later. 7 days later mice were IV injected with fluorescently conjugated anti-CD45 to label circulating B cells and euthanized 3 minutes later. Lamina propria of the large intestines, and lungs were stained with decoy and tetramer. A) Representative flow plots of each tissue, B) counts of the number of Ova-specific B cells and C) percentages and of GC B cells and isotype switching. *, p<0.05; **, p<0.01 ; ***, p <0.001 . Statistical analysis was performed using a two-way ANOVA with Sidak’s multiple comparison test.

[0017] Figure 5 shows dmLT induces influenza-specific mucosal CD4 T cell responses after ID Immunization. Mice were intradermally immunized with influenza nucleoprotein (NP) ± dmLT. Ten days after immunization, mice were euthanized and draining LN and lung samples isolated for NP-specific T cell analysis. Concatenated flow plots show tissue resident NP-specific T cells in each tissue from 5 mice (representative of 2 independent experiments). Numbers represent the percent in the NP-specific gate

[0018] Figure 6 shows dmLT induces swig influenza-specific mucosal B cell responses after ID Immunization. Mice were intradermally immunized then boosted two weeks later with influenza nucleoprotein (NP) ± dmLT. Two weeks after the last immunization, mice were euthanized and draining LN and lung samples isolated for NP-specific B cell analysis. Concatenated flow plots show tissue resident NP-specific B cells in each tissue from 3 mice (representative of 2 independent experiments). Isotype switching (right) is shown for each NP-specific population in LN and lung.

[0019] Figure 7 shows dmLT enhances Ag-specific lung IgA responses after ID Immunization. Mice were intradermally immunized then boosted two weeks later with influenza nucleoprotein (NP) ± dmLT. Two weeks after the last immunization, mice were euthanized, and serum and BAL samples isolated for antibody analysis. *, p<0.05 using Student’s t-test. Representative of three independent experiments.

[0020] Figure 8 shows human multifunctional CD4 and CD8 T cell responses to OMVs. Human CD4 and CD8 T cells were activated for 14 days with OMV adjuvant primed APCs. Multi-functional CD8 T cell responses were analyzed by Boolean gating. Frequencies of helper T cells are on bottom. Statistics calculated with ANOVA. n=16 human patients.

[0021] Figure 9 shows OMV adjuvant augments T cell Responses During Vaccination. Mice were immunized with CD4 and CD8 model peptide Ag. Two weeks later CD4 (A) and CD8 (B) T cell responses were assessed by FACS analysis using MHC-II and MHC-I tetramers. Total Ag-specific T cell numbers were calculated and compared using One-Way ANOVA (n=4 per group *, p < 0.05; **, p < 0.01). Representative flow plots are shown.

[0022] Figure 10 shows dmLT-OMVs increase the overall expansion of Ag-specific CD4 T cells following vaccination. WT mice were immunized ID with 2W1 S-Ova fusion protein +/- dmLT, OMVs, or a combination. 10 days later animals were euthanized and the cervical lymph nodes and lungs were harvested and CD4 T cell responses were assessed by FACS analysis using MHC-II tetramers. The total number of Ag-specific CD4 T cells in (A) cervical lymph nodes or (B) lungs were calculated using cell counting beads and compared using one-way ANOVA with Tukey’s multiple comparisons test (n=3 per group *p < 0.05, **p < 0.01 , ***p < 0.001 ).

[0023] Figure 11 shows a single injection leads to an enhanced B cell numbers in gut draining lymph nodes. Ears of WT mice were ID injected once with 2W-OVA alone or plus dmLT, OMVs, or the dmLT-OMV combination. Twelve days later MLNs were harvested and stained with or B cell decoy and tetramer plus phenotypic markers of activation. (A) Total Ova-specific B cells, (B) swig B cells, and (C) GC B cells are shown. Groups were compared using one-way ANOVA with Dunnetts post-hoc testing *, p < 0.05, **, p < 0.01 .

[0024] Figure 12 shows an overview of parabiosis experiments to assess resident memory lymphocytes. (1) WT C57BI/6 CD45.1 ⁺ congenic mice are ID immunized with the dmLT-OMVs plus 2W1 S-OVA Ag (Donor/Parabiont #1). Circulating (red) or tissue resident (blue) T and B cells are allowed to form and become memory cells. (2) separate CD45.2⁺ unimmunized mice (Recipient/Parabiont #2) are then surgically attached to Parabiont #1 and the shared blood supply of both mice enables movement of circulatory, but not tissue resident lymphocytes between the Parabionts. (3) After 3 weeks when the blood exchange reaches homeostasis mice are separated and each Parabiont can be assessed for the presence of CD45.1+ cells in the mucosal tissues (T_RM or B_RM) or in the circulation (nonresident). It is predicted that Parabiont #1 will have both resident and non-resident lymphocytes while Parabiont #2 will have only non-resident lymphocytes. Both Parabionts can also be challenged with pathogens to determine whether resident cell contribute to protection. Predicted data and protection outcomes for each possible result are shown.

[0025] Figure 13 shows the most effective immune response to Chlamydia muridarum primary infection and reinfection. CD4 T cells are always required for resolution of infection whereas antibodies can be helpful and CD8 T cells are less important. [0026] Figure 14 shows adjuvants and routes previously used in the art do not trigger a protective mucosal immune response or a CD8 T cell response that is important to protect against many viral infections.

[0027] Figure 15 shows that dmLT can trigger mucosal immunity and T-vant can induce CD8 T cells and the prediction which has not previously been shown or able to be predicted is that the combination of the two adjuvants can induce mucosal immunity that engages all arms of the immune response (CD4, CD8, antibodies).

[0028] Figure 16 shows parenterally delivered combination adjuvants drive CD4 T cells to the female reproductive tract which are known to be protective against many sexually tranmissted pathogens including Chlamydia..

[0029] Figure 17 shows vaccination strategy (ID immunization in flank) for mucosal protection against Chlamydia muridarum. 1 pg dmLT + 0.1 pg T-vant shown in this Figure is the adjuvant combination referred to as “Combo” in subsequent slides. “T-vant” is OMV adjuvant provided herein. “rMOMP” is a recombinant major outer membrane protein antigen from Chlamydia muridarum.

[0030] Figure 18 shows the number of inclusion bodies (indicated by arrows) decreases in the presence of combination adjuvants. Inclusion bodies represent infectious Chlamydia bacteria inside of cells of the female reproductive tract of infected mice. This shows that the combination adjuvant is protective against infection.

[0031] Figure 19 shows the combination adjuvant protects against Chlamydia muridarum infection in mice when compared to a naive mouse that has never been immunized or a control mouse immunized with the Chlamydia recombinant major outer membrane protein (rMOMP) antigen alone plus a nonadjuvanted vehicle.

[0032] Figure 20 also shows the combination adjuvant protects against Chlamydia muridarum infection in a different format than Figure 19 that allows for statistical comparison where the combination adjuvant induced statistically significantly reduced bacteria numbers compared to the control groups and most times assessed after infection.

[0033] Figure 21 shows the combination adjuvant induces the highest number of rMOMP tetramer positive B cells in mice when compared to naive mice or control mouse immunized with the Chlamydia recombinant major outer membrane protein (rMOMP) antigen alone plus a nonadjuvanted vehicle. These B cells specifically recognize the rMOMP protein from Chlamydia.

[0034] Figure 22 shows the combination adjuvant induces higher antigen-specific serum antibody titers post-immunization and challenge. These antibodies are from the serum of combination adjuvant group plus rMOMP or a control group immunized with the Chlamydia recombinant major outer membrane protein (rMOMP) antigen alone followed by infection. Antibody levels were assessed 7 day after infection. The combination group induced the most Chlamydia specific antibodies.

[0035] Figure 23 shows the vaccination strategy (ID immunization in flank or intravaginal immunization)) for mucosal protection against Chlamydia muridarum. 1 pg dmLT + 0.1 pg T- vant shown in this Figure is the adjuvant combination referred to as “Combo” in subsequent slides. “T-vant” is OMV adjuvant provided herein. “rMOMP” is a recombinant major outer membrane protein antigen from Chlamydia muridarum.

[0036] Figure 24 shows immunization with the combination adjuvant delivered either intradermally or intravaginally protects against Chlamydia muridarum infection. Mice were either intradermally or intravaginally prime-boost immunized with 1 pg dmLT + 0.1 pg T-vant + 5 pg recombinant major outer membrane protein from C. muridarum four weeks apart. One week after the final immunization, mice were administered Depo Provera to allow for intravaginal infection. One week later mice were infected with 3 x 10³ C. muridarum bacteria and monitored for a week. As shown mice immunized by either route were protected against infection for at least a week after infection.

[0037] Some conclusions from Figures 13-24 are: the combination adjuvant can drive T cells to the female reproductive tract, intradermal immunization with the combination adjuvant can induce protective mucosal immunity against Chlamydia muridarum, antigenspecific B cells can be found post-immunization and are producing circulating antibodies, and intradermal immunization is as protective as intravaginal immunization.

[0038] Figure 25 shows intradermal Immunization with the combination adjuvant induced the greatest CD8 T cell response to immunization. Mice were intradermally immunized with 1 pg 2W1S-Ova antigen + 1 pg dmLT + 0.1 pg T-vant (prime, boost three weeks later). Two weeks later Ova-specific CD8 T cells were assessed in the draining lymph nodes. The figure shows that the combination adjuvant induced the greatest number of vaccine-specific CD8 T cells in response to vaccination.

[0039] Figure 26 shows intradermal immunization with the combination adjuvant induced tissue resident CD4 T cells based on parabiosis. A CD45.2+ mouse (BL6) was intradermally immunized with 1 pg 2W1S-Ova antigen + 1 pg dmLT + 0.1 pg T-vant (prime, boost three weeks later). Two weeks later the immunized mouse was conjoined to a CD45.1+ mouse (Pep) to create a parabiotic pair. Mice were sacrificed 21 days post-surgery after circulatory exchange was confirmed and 2W1S-specific CD4 T cells (those cells that recognized the antigen) were assessed in the spleen, lung, large intestine, and injection site-draining lymph nodes. As shown in Figure 2, cells were equally distributed in the spleen where cells freely circulate but were confined only to the immunized mouse in the lungs, large intestine, and draining lymph nodes indicating the vaccine specific CD4 T cells did not migrate into tissues to become tissue resident cells. This shows that the combo adjuvant induces robust tissue resident CD4 T cell immunity. N=4 parabiotic pairs

Detailed Description

[0040] The disclosure provides compositions and methods of using the compositions, e.g., for generating an immune response, treating diseases or infections and/or preventing diseases or infections. The compositions comprise combinations of adjuvants from, for example, bacterial enterotoxins and outer membrane vesicles, and optionally further comprise one or more vaccines. As described herein, the adjuvant compositions provided are capable of generating an immune response {e.g., antibodies, CD4 T cells, and/or CD8 T cell response) in mucosal tissue following, for example, intradermal injection(s). Immune responses in other tissues and other routes of administration are contemplated and are described herein. An exemplary composition provided herein comprises a detoxified Escherichia coli ADP-ribosylating enterotoxin (DmLT) adjuvant and an attenuated Burkholderia pseudomallei Bp82 bacterial strain -derived outer membrane vesicle (OMV) adjuvant.

[0041] “Adjuvants” are agents that increase immune responses to a co-administered vaccine leading to greater protection against infection and disease. Adjuvants can not only increase, but can also change, the immune response against co-delivered vaccines to favor different immunological outcomes e.g.., different types of antibodies or more T cells). Most non-living vaccine antigens {e.g., protein subunit vaccines) are not capable, on their own, of generating an immune response against themselves so adjuvants are necessary to induce robust and protective immunity.

[0042] Detoxified Bacterial Enterotoxin Adjuvant

[0043] One component of the adjuvant compositions provided herein is a detoxified enterotoxin adjuvant derived from Escherichia coli.

[0044] The detoxified enterotoxin adjuvant exemplified herein was originally described in U.S. Patent No. 6,033,673. Referred to as “LT(R192G/L211 A)” in that patent and referred to as “dmLT” herein, the detoxified enterotoxin adjuvant is a genetically distinct mutant of the E. coli heat-labile enterotoxin (LT) which through modification of the arginine at position 192 to glycine and the modification of the leucine at position 211 to arginine, has lost the trypsin sensitive site joining the Al and A2 subunits, rendering the molecule non-toxic but still able to act as an immunological adjuvant. The amino acid sequence of the dmLT adjuvant is set out below.

NGDKLYRADSRPPDEIKRSGGLMPRGHNEYFDRGTQMNINLYDHARGTQTGFVRYDDGY VSTSLSLRSAHLAGQSILSGYSTYYIYVIATAPNMFNVNDVLGVYSPHPYEEVSALGGIPYSQ IYGWYRVNFGVIDERLHRNREYRDRYYRNLNIAPAEDGYRLAGFPPDHQAWREEPWIHHA PQGCGNSSGTITGDTCN EETQNLSTIYARKYQSKVKRQIFSDYQSEVDIYNRIRNEL (SEQ ID NO: 1)

[0045] dmLT can be produced by methods standard in the art. For example, plasmid pECD403, described in Example 6.1 of U.S. Patent No. 6, 033,673, can be utilized to produce substantially pure LT(R192G/L211 A) (dmLT) in E. coli. dmLT can be isolated by agarose affinity chromatography from bacteria expressing an dmLT-encoding plasmid. Alternate methods of purification standard in the art can be used to purify dmLT.

[0046] Other detoxified enterotoxins with equivalent immunological activities can be made and used in compositions and methods disclosed herein by those skilled in the art. For example, detoxified enterotoxins with at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1 that retain the immunological activities of dmLT are contemplated.

[0047] Outer Membrane Vesicles (OMVs)

[0048] Another component of the adjuvant compositions provided herein is outer membrane vesicles (OMVs) from gram-negative bacteria.

[0049] Gram-negative bacteria secrete OMVs, which are sections of outer membrane which separate from the cell and encapsulate of portion of periplasmic space. OMVs contain constituents of the outer membrane, such as lipopolysaccharides, phospholipids and proteins, and may contain virulence factors and other cytosolic proteins.

[0050] The OMVs exemplified herein were obtained from the attenuated Bp82 bacterial strain of Burkholderia pseudomallei and were originally described in U.S. Patent Publication 2021/0162033, which describes compositions and methods of using compositions comprising (a) a non-Burkhorlderia antigen and (b) a plurality of outer membrane vesicles (OMVs) derived from one or more organisms of the species Burkholderia pseudomallei, B. mallei, B. oklahomensis, B. thailandensis, B. humptydooensis, or Burkholderia spp. Clades A, B, or C (collectively, the "B. pseudomallei complex" or "Bpc").

[0051] Culturing of Burkholderia pseudomallei and obtaining OMVs from species of Burkholderia are also described in, for example, Nieves et al., Vaccine, 2011 , 29(46): 8381 - 8389, doi:10.1016/j. vaccine.2011 .08.058; Nieves etal., Clin Vaccine Immunol. 2014, 21 (5):747-54. doi: 10.1128/CVI. 00119-14, and Baker etal., Vaccines (Basel). 2017 December; 5(4): 49, doi: 10.3390/vaccines5040049.

[0052] OMVs obtained from gram-negative bacteria other than Burkholderia pseudomallei, such as OMVs from B. mallei, can also be used in adjuvant compositions described herein.

[0053] According to Wikipedia, “Burkholderia” refers to “a group of virtually ubiquitous Gram-negative, obligately aerobic, rod-shaped bacteria that are motile by means of single or multiple polar flagella, with the exception of Burkholderia mallei which is nonmotile.” Baker 2017, supra, reports that B. mallei evolved from B. pseudomallei through genome reduction and that many virulence determinants including surface polysaccharides, outer membrane proteins, secretion systems, and motility proteins, are highly conserved between the two species. Accordingly, the OMVs of B. mallei should have the same effectiveness as adjuvants as those of B. pseudomallei. B. thailandensis is considered to be very close to B. pseudomallei.

[0054] Price etal. (Journal of Medical Microbiology (2016), 65, 992-997, doi: 0.1099/jmm. 0.000312) proposes an expanded B. pseudomallei complex (“Bpc”) based on the phylogenetic relatedness of B. pseudomallei and its nearest neighbors. According to Price et al., the Bpc comprises B. pseudomallei, B.mallei, B. oklahomensis, B. thailandensis, B. humptydooensis (proposed), and three unassigned Burkholderia spp. Clades A (represented by type strain BDU 5), B (represented by type strain BDU 8) and C (represented by type strain MSMB0265). The same expanded membership of the classical Bpc is also set forth in Lowe et al., PLoS ONE 11 (10): e0164006. Doi.org/10.1371/journal.pone.0164006, which states that the three clades have not been assigned a species but are B. thailandensis-Wke strains most related to B. oklahomensis. B. humptydooensis is further described in Tuanyok et al., 2017, Appl Environ Microbiol 83:e02802-16. Doi.org/10.1128/AEM.02802-16. See also, Gee et al., BMC microbiology. 2008;8:54. Epub 2008/04/04. pmid: 18384685; Glass et al., International J Systematic Evolut Microbiol, 2006; 56(9):2171 — 6. Epub 2006/09/08. pmid:16957116; Ginther et al., PLoS neglected tropical diseases, 2015;9(6):e0003892. Epub 2015/06/30. pmid:26121041.

[0055] The American Type Culture Collection (“ATCC”) website showed, as of April 2018, the availability of over 20 species of Burkholderia and a number of strains or isolates of particular species, other than B. pseudomallei or B. mallei. Strains of B. pseudomallei and of B. mallei are available to persons registering with the Biodefence Emerging Infections Research Resources Repository (“BEI”), run by the ATCC under contract from the National Institute of Allergy and Infectious Diseases (registration requires, among other things, demonstrating that the requestor has facilities suitable for the biosafety level of the organisms requested). A review of the BEI’s website indicates that, as of April 2018, it had available over 40 strains of B. pseudomallei, over 10 strains of B. mallei, and 5 strains of B. thailandensis. Bp strain 1026b is available from BEI under accession number NR-4074. Probst etal., Infection and Immunity, 2010, 78(7):3136-3143, describes how to make a ApurM mutant of Bp strain 1026b, resulting in a mutant form of Bp 1026b they term “Bp82”. Torres, U.S. Patent Application Publication 2017/0333543 also describes producing Burkholderia, and particularly B. mallei, whose pathogenicity is attenuated by deletion or disruption of the tonB and hcp1 genes. Burkholderia whose pathogenicity is attenuated as taught in Torres may be used to produce BOMVs for use in compositions and methods provided herein.

[0056] The BEI website further states that the complete genome of B. pseudomallei strain 1106c is available under accession numbers CP000572.1 and CP000573.1 , for chromosomes 1 and 2, respectively. The website states the genome of B. thailandensis strain E264 has been sequenced and is available under assembly no. ASM 1236V. I. B. humptydooensis is available from the ATCC® under accession number BAA-2767™. B. oklahomensis is available from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture, Braunschweig, Germany.

[0057] Compositions

[0058] Adjuvant compositions provided herein comprise a detoxified bacterial endotoxin adjuvant and an OMV adjuvant. The adjuvant compositions can comprise a DmLT adjuvant and a Burkholderia pseudomallei OMV adjuvant.

[0059] The adjuvants provided herein are present in a composition in an amount between the ranges of 0.1-100 pg/dose for each individual adjuvant (dmLT and OMVs). Exemplary doses include, but are not limited to, 0.1 pg/dose dmLT plus 0.1 pg/dose OMVs, 0.1 pg/dose dmLT plus 1.0 pg/dose OMVs, 0.1 pg/dose dmLT plus 10.0 pg/dose OMVs, 0.1 pg/dose dmLT plus 100 pg/dose OMVs, 1.0 pg/dose dmLT plus 0.1 pg/dose OMVs, 1.0 pg/dose dmLT plus 1.0 pg/dose OMVs, 1.0 pg/dose dmLT plus 10 pg/dose OMVs, 1.0 pg/dose dmLT plus 100 pg/dose OMVs, 10 pg/dose dmLT plus 0.1 pg/dose OMVs, 10 pg/dose dmLT plus 1 .0 pg/dose OMVs, 10 pg/dose dmLT plus 10 pg/dose OMVs, 10 pg/dose dmLT plus 100 pg/dose OMVs, 100 pg/dose dmLT plus 0.1 pg/dose OMVs, 100 pg/dose dmLT plus 1 .0 pg/dose OMVs, 100 pg/dose dmLT plus 10 pg/dose OMVs, 100 pg/dose dmLT plus 100 pg/dose OMVs. Doses may be adjusted depending upon the body mass, body area, weight, blood volume of the subject, or route of delivery. For example, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 11 pg, or 12 pg of adjuvant or each adjuvant in 1 ml is administered intradermally. In this regard, the 1 ml dose of adjuvant or each adjuvant may be injected in equal amounts in multiple locations. For example, about 0.01 pg/kg to about 100 mg/kg body weight of adjuvant will be administered, typically by the intradermal, subcutaneous, intramuscular or intravenous route, or by other routes. In another example, the dosage of adjuvant is about 0.1 pg/kg to about 1 mg/kg, and ranges from about 0.1 pg/kg, 0.2 pg/kg, 0.3 pg/kg, 0.4 pg/kg, 0.5 pg/kg, 0.6 pg/kg, 0.7 pg/kg, 0.8 pg/kg, 0.9 pg/kg, 1 pg/kg, 2 pg/kg, 3 pg/kg, 4 pg/kg, 5 pg/kg, 6 pg/kg, 7 pg/kg, 8 pg/kg, 9 pg/kg, 10 pg/kg to about 200 pg/kg. It will be evident to those skilled in the art that the number and frequency of administration will be dependent upon the response of the host. As described herein, the appropriate dose may also depend upon the subject's condition, that is, stage of the disease, general health status, as well as age, gender, and weight, and other factors familiar to a person skilled in the medical art.

[0060] The adjuvant compositions may be in any form which allows for the composition to be administered to a subject. For example, the adjuvant composition may be in the form of a solid, liquid or gas (aerosol). The pharmaceutical compositions may be administered by any route. Typical routes of administration include, without limitation, oral, sublingual, buccal, topical, parenteral (including intradermal, subcutaneous, percutaneous, intravenous, intramuscular, intrasternal, intracavernous, intrameatal, intratumoral, intracranial, intraspinal or intraurethral injection or infusion), rectal, vaginal, intranasal (e.g., as a spray) and intrapulmonary administration. The term parenteral as used herein includes, but is not limited to, iontophoretic (e.g., U.S. 7,033,598; 7,018,345; 6,970,739), sonophoretic (e.g., U.S. 4,780,212; 4,767,402; 4,948,587; 5,618,275; 5,656,016; 5,722,397; 6,322,532; 6,018,678), thermal (e.g., U.S. 5,885,211 ; 6,685,699), passive transdermal (e.g., U.S. 3,598,122; 3,598,123; 4,286,592; 4,314,557; 4,379,454; 4,568,343; 5,464,387; UK Pat. Spec. No. 2232892; U.S. 6,871 ,477; 6,974,588; 6,676,961), microneedle (e.g., U.S.

6,908,453; 5,457,041 ; 5,591 ,139; 6,033,928) administration and also intradermal/subcutaneous injections, intravenous, intramuscular, intrasternal, intracavernous, intrathecal, intranodal, intrameatal, intraurethral, intratumoral injection or infusion techniques. An adjuvant composition as provided herein can be administered intradermally by a technique selected from iontophoresis, microcavitation, sonophoresis or microneedles.

[0061] The adjuvant compositions may further comprise at least one physiologically (or pharmaceutically) acceptable or suitable excipient. Any physiologically or pharmaceutically suitable excipient or carrier (/.e., a non-toxic material that does not interfere with the activity of the active ingredient) known to those of ordinary skill in the art for use in pharmaceutical compositions may be employed in the compositions provided herein. Exemplary excipients include diluents and carriers that maintain stability and integrity of proteins. Excipients for therapeutic use are well known, and are described, for example, in Remington: The Science and Practice of Pharmacy [Gennaro, 21st Ed. Mack Pub. Co., Easton, PA (2005)].

[0062] “Pharmaceutically acceptable carriers” are well known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro Eds. 1985). For example, sterile saline and phosphate buffered saline at physiological pH may be used. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p hydroxybenzoic acid may be added as preservatives. Id. at 1449. In addition, antioxidants and suspending agents may be used. Id.

[0063] “Pharmaceutically acceptable salt” refers to salts of a compounds derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts). The adjuvant compositions provided herein may be used in either the free base or salt forms.

[0064] The adjuvant composition is formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more adjuvants in aerosol form may hold a plurality of dosage units.

[0065] A liquid adjuvant composition as provided herein, whether in the form of a solution, suspension or other like form, may include one or more of the following carriers or excipients: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as squalene, squalane, mineral oil, a mannide monooleate, cholesterol, and/or synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0066] An adjuvant composition provided herein can comprise a stable aqueous suspension of less than 0.2 pM and further comprises at least one component selected from the group consisting of phospholipids, fatty acids, surfactants, detergents, saponins, fluorodated lipids, and the like. Such a stable aqueous formulation may be a micellar formulation.

[0067] An adjuvant composition provided herein can be formulated in a manner which can be aerosolized, either as a powder or liquid formulation.

[0068] It may also be desirable to include other components in an adjuvant composition, such as including but not limited to water-in-oil emulsions, biodegradable oil vehicles, oil-in- water emulsions, liposomes, micellar components, microparticles, biodegradable microcapsules, and liposomes.

[0069] Adjuvant compositions provided herein can comprise a stable oil-in-water emulsion and a metabolizable oil. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as "being capable of being transformed by metabolism" (Dorland's illustrated Medical Dictionary, W. B. Saunders Company, 25th edition (1974)). The oil may be any plant oil, vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts (such as peanut oil), seeds, and grains are common sources of vegetable oils. Synthetic oils may also be used.

[0070] Additional immunostimulatory substances may be included in the adjuvant compositions provided herein and may include N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), glucan, IL-12, GM CSF, interferon-y and IL-12.

[0071] While any suitable carrier known to those of ordinary skill in the art may be employed in the adjuvant compositions provided herein, the type of carrier will vary depending on the mode of administration and whether a sustained release is desired. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the adjuvant compositions provided herein. Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109. In this regard, it is preferable that the microsphere be larger than approximately 25 microns.

[0072] Adjuvant compositions provided herein may also contain diluents such as buffers, antioxidants such as ascorbic acid, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents. Adjuvants may be formulated as a lyophilizate using appropriate excipient solutions e.g., sucrose) as diluents.

[0073] Adjuvant compositions provided herein may further comprise a vaccine comprising or encoding an antigen. [0074] Kits

[0075] Compositions provided herein may be in a kit. In the kit, the components of the adjuvant composition can already be mixed together for administration, or the components can be separate in the kit and administered separately as directed.

[0076] Antigens and Infectious Organisms

[0077] The adjuvant compositions and methods provided herein are intended for use in a subject, including humans and other animals. Vaccines contemplated for inclusion in adjuvant compositions comprise or encode an antigen. The vaccines included in an adjuvant composition herein can comprise or encode, for example, a bacterial, viral or fungal antigen.

[0078] Antigens contemplated by the disclosure, as examples and not by way of limitation, include antigens from pathogenic strains of bacteria (including, but not limited to, Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromotis, Staphylococcus aureus, Bordetella pertussis , Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Salmonella typhi, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp., Helicobacter pylori); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms) either presented to the immune system in whole organism form or in part isolated from media cultures designed to grow said organisms which are well known in the art, or protective antigens from said organisms obtained by genetic engineering techniques or by chemical synthesis. Other illustrative antigens are addressed in the Examples herein and are antigens from pathogenic strains of Citrobacter rodentium (ATCC 51459) or Chlamydia muridarum.

[0079] Other antigens contemplated include, for example, antigens from pathogenic viruses (e.g., Poxviridae, Herpesviridae, Herpes Simplex virus 1 , Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus) (e.g., rabies virus, herpesviruses, such as herpes simplex virus (HSV) type 2, HSV type 1 , human cytomegalovirus, Epstein-Barr virus, and varicella zoster virus (VZV), human papillomavirus (HPV), Human T-cell lymphotropic virus type 1 , rotavirus, norovirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, polio virus, Japanese encephalitis virus, measles virus, mumps virus, rubella virus, yellow fever virus, varicella virus, dengue virus, rotavirus, paniovirus, human immunodeficiency virus-1 , ebolaviruses, such as Ebola Sudan virus, Marburg virus, hantavirus, norovirus, Zika virus, West Nile virus, hantavirus, Lassa virus, Lymphocytic choriomeningitis virus, Nipah virus, Rift Valley fever virus, Middle East Respiratory Syndrome Coronavirus, SARS coronavirus, SARS coronavirus 2, Crimean- Congo hemorrhagic fever virus, enteroviruses, and noroviruses) either presented to the immune system in whole or in part isolated from media cultures designed to grow such viruses which are well known in the art, or antigens therefrom obtained by genetic engineering techniques or by chemical synthesis.

[0080] Examples of vaccines contemplated include, but are not limited to, influenza vaccine, pertussis vaccine, diphtheria and tetanus toxoid combined with pertussis vaccine, hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine, hepatitis E vaccine, Japanese encephalitis vaccine, herpes vaccine, measles vaccine, rubella vaccine, mumps vaccine, mixed vaccine of measles, mumps and rubella, papillomavirus vaccine, parvovirus vaccine, respiratory syncytial virus vaccine, Lyme disease vaccine, polio vaccine, varicella vaccine, gonorrhea vaccine, schistosomiasis vaccine, rotavirus vaccine, mycoplasma vaccine pneumococcal vaccine, meningococcal vaccine, Campylobacter vaccine, helicobacter vaccine, cholera vaccine, enterotoxigenic E. coli vaccine, enterohemmorgagic E. coli vaccine, shigella vaccine, salmonella vaccine and others. These are produced by known common processes. In general, such vaccines comprise either the entire organism or virus grown and isolated by techniques well known to the skilled artisan, or comprise relevant antigens of these organisms or viruses which are produced by genetic engineering techniques or chemical synthesis. [0081] All commercially available vaccines that are adjuvanted, are adjuvanted with adjuvants other than an adjuvant composition provided herein. It is contemplated that the immune response to currently approved vaccines against viruses can be improved by use of an adjuvant composition provided herein as an adjuvant in place of, or in addition to, the adjuvant or adjuvants currently used in the vaccines. Other viruses, such as HIV-1 , ebola, and Zika virus, have vaccines in development that provide some protection against infection or against severe infection. It is anticipated that adjuvanting vaccines for such viruses with an adjuvant composition provided herein will augment the protection provided by the current vaccine candidates. It is noted that, with viruses such as HIV-1 and ebola, a vaccine that provides even partial protection can itself constitute an important public health advance.

[0082] Vaccines in production or development against viruses that can be potentiated by adjuvanting with an adjuvant composition provided herein include, but are not limited to, vaccines against West Nile Virus (see, e.g., U.S. Pat. No. 9,962,435), enteroviruses (see, e.g., U.S. Pat. No. 9,987,350), noroviruses (see, e.g., U.S. Pat. No. 9,867,876), herpes simplex 2 (see, e.g., U.S. Pat. Nos. 9,919,045; 9,895,436, 9,566,325, and 9,555,100), cytomegalovirus (see, e.g., U.S. Pat. No. 9,901 ,632), dengue fever (see, e.g., U.S. Pat. Nos. 9,861 ,692, 9,783,787, 9,463,235, RE 46,641 , and RE 46,631 ), and chikungunya virus (see, e.g., U.S. Pat. No. 9,844,588).

[0083] An adjuvant composition provided herein can also be used to increase the immune response to viral antigens other than those targeted by current vaccines. In this regard, it is noted that the complete genomes for all or most of these viruses are known, as are the coding sequences of the various proteins of these viruses, which can be used to raise immune responses against the viruses. For example, the complete genome sequences of numerous isolates of Ebola virus have been deposited in GenBank, as exemplified by Ebola virus isolate Ebola virus/H. sapiens-tc/COD/1995/Kikwit-9510622 under accession number KU182909. Similarly, the complete genome of isolates of Marburg virus are available on GenBank, as exemplified by the genome of Marburg virus isolate Marburg virus/RML- IRF/M.auratus-lab/AGO/2005/Angola-368-HA, available under accession number KY047764. The complete genome of Nipah virus is available in GenBank under accession number NC_002728. The complete genome of West Nile virus used by the CDC as a reference reagent is available in GenBank under accession number AY646354 (see, e.g., Grinev etal., Genome Announc 2 (5) (2014). The complete genome of isolates of Zika virus are available in GenBank, as exemplified by the sequence for Zika strain Zika virus/Homo sapiens/VEN/UF-2/2016, available under accession number KX893855 (see, e.g., Blohm et al., Genome Announc 5 (17), e00231 -17 (2017)). The complete genome of yellow fever virus is available in GenBank under accession number NC_002031 (see, e.g., Rice, et al., Science 229 (4715), 726-733 (1985)). The complete genome of isolates of Middle East respiratory syndrome coronavirus are available on GenBank, as exemplified by the genome of isolate Camel/UAE/D1243.12/2014 deposited under accession number KP719932.

[0084] As an example, an adjuvant composition provided herein can be used to increase an immune response against proteins or other antigens from the various human herpesviruses. Such herpesviruses include, for herpes simplex viruses (HSV)-1 and -2 and proteins derived from such HSVs, such as HSV-1 and HSV-2 glycoproteins gB, gD and gH. The complete genomes of various strains of HSV-1 have been sequenced and deposited in GenBank. See, e.g., accession number X14112, version X14112.1 , accession number MH999839 (strain K86), accession number MH999841 (strain Ty148). The complete genomes of various strains and isolated of HSV-2 have also been sequenced and deposited in GenBank. See, e.g., accession number KY922726 (complete genome of isolate HSV2- H12212); and accession number NC_001798, version NC_001798.2 (strain HG52).

[0085] The complete genomes for a number of strains of human herpesvirus 3, or Varicella zoster virus (VZV) are also set forth in GenBank. See, e.g., accession number NC_001348 (Davison and Scott, J. Gen. Virol. (1986) 67:1759-1816), accession number DQ674250, for strain NE29_3, and accession number DQ479963, for strain 32, passage 72 (see, Peters et al., J. Virol. 80 (19), 9850-9860 (2006)). Similarly, the complete genomes of various strains of human cytomegalovirus (CMV) have been sequenced and deposited in GenBank (see, e.g., accession number X17403 (strain AD169, Chee et al., Curr. Top. Microbiol. Immunol. 154, 125-169 (1990) and Bankier et al., DNA Seq. 2 (1), 1-12 (1991 )), and accession number NC_006273 (strain Merlin, Gatherer et al., Proc. Natl. Acad. Sci. U.S.A. 108 (49), 19755-19760 (2011 )).

[0086] Antigens from the hepatitis family of viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV), can also be used in the compositions and methods provided herein. With respect to hepatitis A, the complete genome of the wild-type virus was deposited in GenBank in 1987 under accession number M14707 (see, Cohen et al., J. Virol. 61 (1), 50-59 (1987)), and the genomes of various isolates have also been deposited. See, e.g., accession number KP879216 for isolate 18f (see, Lemon et al., J. Virol. 65 (4), 2056- 2065 (1991), and accession number AB279734, for isolate HAJ95-8 (see, Endo etal., Virus Res. 126 (1-2), 116-127 (2007). With respect to hepatitis B, the entire genome of various strains and isolates are set in GenBank, as exemplified by accession numbers AF363962 (strain G683-2) and AB775201 (isolate HB12-0929). The genome of various isolates and strains of HCV are likewise deposited in GenBank. For example, the sequence of the entire genome of HCV genotype 1 is available under accession number NC-004102, which also sets forth annotations of the mature proteins from the literature. Further, the complete sequence for HCV subtype 6a, strain 6a74, can be found in GenBank under accession number DQ480524, while that of HCV subtype 6A, strain 6a67 can be found under accession number DQ480520.

[0087] Antigens derived from other viruses can also be used in compositions and methods provided herein, such as, without limitation, proteins from members of the families Picomaviridae (e.g., polioviruses, etc.); Caliciviridae; Togaviridae e.g., rubella virus, dengue virus, chikungunya virus, etc.); Flaviviridae; Coronaviridae; Reoviridae; Birnaviridae; Filoviridae; Paramyxoviridae e.g., mumps virus, measles virus, respiratory syncytial virus, etc.); Orthomyxoviridae {e.g., influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae; and Retroviradae {e.g., HIV-1 (the complete genome of HIV-1 is set forth in GenBank under accession number NC_001802, along with the sequences of numerous proviral genomes and genomes of isolates) and HIV-2 (the complete genome of HIV-2 is set forth in GenBank under accession number NC_001722, along with numerous proviral genomes and the genomes of numerous isolates and clones); simian immunodeficiency virus (SIV), feline immunodeficiency virus (Fl V), human papillomavirus (HPV), and the tick- borne encephalitis viruses. See, e.g. Burrell et al., Eds., Fenner and White's Medical Virology, 5. sup. th Ed. (Academic Press, London, 2017); Collier et al., Human Virology, 4. sup. th Ed. (Oxford Univ. Press, Oxford, UK, 2017), and Murray et al., Medical Microbiology, 8.sup.th Ed. (Elsevier, Philadelphia, Pa., 2016), each of which is incorporated herein in its entirety, for a description of these and other viral pathogens. The complete genomes of most or all of these viruses, including the various types of papilloma virus associated with warts or cervical cancer, have been sequenced and deposited in GenBank, as exemplified by the complete genome for HPV 16, deposited under accession number NC 001526.

[0088] As noted above, influenza virus is an example of a virus for which the present compositions and methods are expected to be useful. Specifically, the envelope glycoproteins HA and NA of influenza A are of particular interest for generating an immune response. Numerous HA subtypes of influenza A have been identified and sequenced, as exemplified by the sequence of HA segment 4 deposited in GenBank under accession numbers NC-026433 and FJ966974 (see, Garten etal., Science 325 (5937), 197-201 (2009). Proteins or other antigens derived from any of these isolates can also be used in an adjuvant composition provided herein provided herein.

[0089] Adjuvant compositions provided herein also increase a subject's immune response to parasites and can be used as adjuvants in human and veterinary vaccines currently in use or under development against parasitic diseases including, but not limited to, malaria (see, e.g., U.S. Pat. Nos. 9,943,580; 9,616,115; 9,603,916; and 9,592,282. See also, Coelho et al., Advances in malaria vaccine development: report from the 2017 malaria vaccine symposium, npj Vaccines 2, Article number: 34 (2017), doi: 10.1038/s41541 -017-0035-3) or to increase the immune response to the RTS,S/AS01 vaccine currently in trials with a liposome-based adjuvant (see, e.g., Gosling R, von Seidlein L (2016)). Vaccines against other parasitic diseases that can potentiated by use of an OMV such as Bpc OMV and an enterotoxin such as dmTL include diseases caused by helminths, such as schistosomiasis (reviewed in Tebeje, et al., Schistosomiasis vaccines: where do we stand? Parasites & Vectors, 2016, 9:528, see also, e.g., U.S. Pat. No. 9,248,169) and hookworm (see, e.g., U.S. Pat. No. 8,444,994), as well as diseases caused by other parasites, such as leishmaniasis (see, e.g., Gillespie, et al., Status of vaccine research and development of vaccines for leishmaniasis, 2016, Vaccine, 34(26): 2992-2995 and U.S. Pat. Nos. 9,764,015; 8,986,711 ; and 8,968,749), toxoplasma (see, e.g., Liu et al., Hum Vaccin Immunother. 2012 Sep. 1 ;

8(9): 1305-1308, and U.S. Pat. No. 9,802,974), and diseases caused by trypanosomes (see, e.g., Cazorla etal., Expert Rev of Vaccines, 2009, 8(7):921 -931 , doi.org/10.1586/erv.09.45 and LaGreca and Magez, Human Vaccines, 2011 , 7(11 ):1225-33, doi.org/10.4161 /hv.7.11 .18203).

[0090] Further, it is contemplated that an adjuvant composition provided herein will also increase a subject's immune response to antigens from invasive fungi, and can thus be used as adjuvants in vaccines against fungal infections (reviewed in, e.g., Spellberg, Vaccines for invasive fungal infections, F1000 Med Rep. 2011 ; 3: 13, doi: 10.3410/M3-13; Casson: and Casadevall, Recent Progress in Vaccines against Fungal Diseases, Curr Opin Microbiol. 2012 August; 15(4): 427-433). Vaccines comprising antigens raising an immune response to pathogenic fungi are taught in, for example, U.S. Pat. Nos. 9,914,917; 9,364,539; and 8,449,894, and U.S Patent Pub. 2014/271720. Vaccines in which an OMV such as Bpc OMV and an enterotoxin such as dmTL can be used as an adjuvant include vaccines against fungal pathogens, particularly those within one of the following genera: Aspergilius, Pneumocystis, Histoplasma, Coccidioides, Malassezia, Blastomyces, or Candida.

[0091] An adjuvant composition provided herein can also be used to generate immune responses to antigens expressed by cancer cells that are either not expressed on normal tissues, or that are found on tumors of a tissue type that is not of an essential human tissue e.g., prostate antigens), an adjuvant composition provided herein can be used to enhance humoral and cell-mediated immune responses against antigens expressed by these cancer cells. Such antigens include activated oncogenes, fetal antigens, and activation markers. A number of tumor antigens have been explored for use as cancer vaccines, including the various MAGEs (melanoma associated antigen E), including MAGE 1 , 2, 3, 4, etc. (reviewed in Xiao and Chen, World J Gastroenterol. 2004 Jul. 1 ; 10(13): 1849-1853), MART 1 (melanoma antigen recognized by T cells), mutant K-ras (see, e.g., Weden etal., Int J Cancer, 2011 Mar. 1 , 128(5):1120-1128 (https://doi.org/10.1002/ijc.25449), mutant p53, and carcinoembryonic antigen (CEA). A number of universities and companies are now developing cancer vaccines against tumor-associated antigens, as exemplified by U.S. Pat. Nos. 9,932,384; and 9,908,922, growth factors or variants of growth factors that are overexpressed in some cancer types, such as EGFR (see, e.g, U.S. Pat. No. 9,808,516), or modified heat shock proteins (see, e.g, U.S. Pat. No. 9,238,064), or neoantigens derived from the tumors of indivipatients. Some cancer vaccines are intended to raise an immune response to body tissues found on metastatic disease but not normal body tissues. It is expected that any of these cancer vaccines can be potentiated by being adjuvanted with an adjuvant composition provided herein.

[0092] Further, companies are currently engineering T-cells with chimeric antigen receptors ("CARs") for CAR T-cell therapy, and introducing the engineered T-cells into subjects so the engineered cells can recognize and kill tumor cells bearing antigens recognized by the CAR. It is contemplated that an adjuvant composition provided herein can be used as an adjuvant to increase immune responses against cancer antigens, such as those targeted by CAR-T therapy. As a number of tumor antigens have been and are being explored for in CAR-T therapy, it is expected that the person of skill is familiar with the selection of appropriate tumor antigens for use in compositions and methods provided herein.

[0093] An adjuvant composition provided herein can be used to potentiate vaccines against opioid drugs, such as fentanyl, heroin, and oxycodone. These vaccines are typically designed to raise antibodies intended to intercept the opioid in the user's bloodstream before the opioid reaches the user's brain and induces an enjoyable response. One problem in the development of opioid vaccines to date has been with the limited effectiveness with which the vaccines induce an antibody response. It is expected that some compositions comprising an adjuvant composition provided herein, with their ability to induce a robust antibody response, will increase the effectiveness of such vaccines. The vaccine or vaccines can be administered, for example, to those with a past problem with addiction to one or more opioids, thereby reducing their risk of readdiction, or to those currently addicted to an opioid drug to reduce pleasant effects from their use of the drug.

[0094] Fentanyl and some of the other opioids are relatively small molecules and their immunogenicity may be improved by use of a hapten. Methods of conjugating molecules to various opioids are known. For example, oxycodone has been conjugated to various haptens, such as keyhole limpet hemocyanin subunit dimer. (See, e.g., Barrufaldi et al., Mol Pharm. 2018 Nov. 5; 15(11):4947-4962. doi: 10.1021/acs.molpharmaceut.8b00592. Epub 2018 Oct. 10, and Raleigh et al., PLoS One. 2017 Dec. 1 ; 12(12):e0184876. doi: 10.1371/journal.pone.0184876. eCollection 2017). Hwang et al. have reported on the development of a vaccine containing an admixture of heroin and fentanyl-hapten conjugates. See, Hwang et al., ACS Chem Neurosci. 2018 Jun. 20; 9(6):1269-1275. doi: 10.1021/acschemneuro.8b00079. Epub 2018 Mar. 23. See also, Raleigh et al., J Pharmacol Exp Then 2019 February; 368(2):282-291 . doi: 10.1124/jpet.118.253674. Epub 2018 Nov. 8, Hwang and Janda, Biochemistry. 2017 Oct. 24; 56(42):5625-5627. doi: 10.1021/acs.biochem.7b00948. Epub 2017 Oct. 10, and Olson and Janda, EMBO Rep. 2018 January; 19(1):5-9. doi: 10.15252/embr.2O1745322. Epub 2017 Dec. 13.) It is contemplated that a adjuvant composition provided herein can be added to these and similar vaccine formulations to increase their immunogenic effects. The fentanyl or other opioid to be targeted may be chemically conjugated by conventional chemistry to one of the adjuvant components of a adjuvant composition provided herein, such as that used to create the fentanyl-hapten conjugates used in the studies reported above, to act both as a hapten and as an adjuvant to increase the immunogenic effect of the opioid moiety.

[0095] Any particular vaccine of interest can be readily tested to determine if administering it in combination with an adjuvant composition provided herein increases the immune response compared to the vaccine alone, or compared to a combination of the vaccine and a standard adjuvant, such as alum, by testing the combination of choice in standard assays, such as the assays set forth in the Examples herein and in U.S. Patent Publication 2021/0162033.

[0096] The vaccines included in an adjuvant composition herein can comprise a polypeptide, a nucleic acid, a polysaccharide, a polysaccharide-polypeptide conjugate, a live-attenuated or inactivated bacterium, a toxoid, a live-attenuated or inactivated virus, a virus-like particle, or a viral vector.

[0097] Methods

[0098] Adjuvant compositions provided herein are administered to a subject to generate an immune response in the subject. The immune response generated is increased compared to the subject’s immune response in the absence of the adjuvant composition. A subject herein can be a human or other animal. The animal can be a mammal, such as a horse, cow, sheep, pig, dog or cat.

[0099] Adjuvant compositions provided herein may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. [0100] Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains an adjuvant composition described herein and one or more additional active agents, as well as administration of compositions comprising an adjuvant composition provided herein and each active agent in its own separate pharmaceutical dosage formulation. Similarly, compositions comprising an adjuvant composition provided herein and the other active agent can be administered to the patient together in a single parenteral (e.g., any of the parenteral routes known and provided herein, such as, subcutaneous, intradermal, intranodal, intratumoral or intramuscular) dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations. The combination therapies as provided herein can be administered by the same route or may be administered using different routes. Where separate dosage formulations are used, the compositions comprising an adjuvant composition provided herein and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.

[0101] Other Definitions

[0102] As used herein, the term "antigen" refers to a substance that has the ability to evoke an immune response, either by inducing generation of antibodies, by causing a cell- mediated immune response, or by inducing both an antibody and a cell-mediated immune response.

[0103] As used herein, the term "non- Burkholderia antigen" refers to an antigen which is not naturally present in members of the bacterial genus Burkholderia. The "non- Burkholderia antigen" can be expressed from a nucleic acid sequence introduced into a Burkholderia bacterium by genetic engineering. The non- Burkholderia antigen can be expressed from a nucleic acid sequence introduced into a Burkholderia bacterium, which sequence encodes (a) a protein or peptide not naturally present in members of the bacterial genus Burkholderia, and (b) a Burkholderia protein or peptide which is normally present in the outer membrane vesicle of the Burkholderia bacterium into which the nucleic acid sequence has been introduced, wherein (a) and (b) are expressed as a fusion protein or peptide.

[0104] As used herein, "adjuvant" refers to a substance that is capable of “increasing”, that is enhancing, accelerating, or prolonging, an immune response to an antigen, optionally when co-administered with the antigen.

[0105] "Outer membrane vesicles," which are sometimes referred to herein as "OMVs," are spherical buds of the outer membrane filled with periplasmic content commonly produced by Gram-negative bacteria. [See, for example, Schwechheimer and Kuehn, Nature Reviews Microbiology, 2015, 13:605-619; Kaparakis-Liaskos and Ferrero, Nature Reviews Immunology, 2015, 15:375-387; Kuehn and Kesty, Genes & Dev. 2005. 19: 2645-2655; Kulp and Kuehn, Annual Review of Microbiology, 2010, 64:163-184.]

[0106] As used herein, "derived from," with respect to an antigen or OMVs, refers to obtaining an immunogenic component of an antigen by any of a number of means known in the art, such as by isolation of the antigen or OMVsfrom the native organism, or by recombinant expression or synthesis. Antigens derived from a pathogenic organism may be treated before use to reduce undesired effects. For example, "toxoids" are bacterial toxins which have been treated to suppress or eliminate their ability to act as a toxin, while retaining their ability to induce an immune response against the bacteria from which the toxin originated. The term "derived from" also encompasses structures formed by proteins or peptides that have been recombinantly expressed, such as the virus-like particles that selfassemble from recombinantly expressed capsid proteins of viruses such as human papillomavirus.

[0107] As used herein, "co-administration" refers to co-localized administration of two or more agents, such as an antigen and an adjuvant, two or more adjuvants, or two or more adjuvants and one or more antigens or antigens, etc., to the same subject during a treatment period. The two or more agents or adjuvants may be encompassed in a single formulation and thus be administered simultaneously. Alternatively, the two or more agents may be in separate physical formulations and administered separately to the same spot in the subject, either sequentially or simultaneously. The term "administered simultaneously" or "simultaneous administration" means that the administration of the first agent and that of a second agent overlap in time with each other, while the term "administered sequentially" or "sequential administration" means that the administration of the first agent and that of a second agent does not overlap in time with each other, but takes place sufficiently close in time that the first agent has not been taken up or metabolized before administration of the second agent so that antigen-presenting cells "see" the first agent in conjunction with the second agent.

[0108] "Immune response" refers to any detectable response to a particular substance (such as an antigen or adjuvant) by the immune system of a host vertebrate animal, including, but not limited to, innate immune responses e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses e.g., responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system), and humoral immune responses e.g., responses mediated by B cells, such as generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids). Examples of immune responses include an alteration (e.g., increase) in Toll-like receptor activation, lymphokine (e.g., cytokine e.g., Th1 , Th2 or Th17 type cytokines) or chemokine) expression or secretion, macrophage activation, dendritic cell activation, T cell (e.g., CD4+ or CD8+ T cell) activation, NK cell activation, B cell activation (e.g., antibody generation and/or secretion), binding of an antigen (e.g., immunogenic polypolypeptide)) to an MHC molecule, induction of a cytotoxic T lymphocyte ("CTL") response, induction of a B cell response (e.g., antibody production), and, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells and B cells), and increased processing and presentation of antigen by antigen presenting cells. The term "immune response" also encompasses any detectable response to a particular substance (such as an antigen or adjuvant) by one or more components of the immune system of a vertebrate animal in vitro.

[0109] As used herein, a "humoral immune response" refers to an immune response mediated by antibody molecules, while a "cellular immune response" is one mediated by T- lymphocytes, by other white blood cells, or by both. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells ("CTLs"). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHCI) and expressed on the surfaces of cells. CTLs help induce and promote the intracellular destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigenspecific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHCII molecules on their surface. A "cellular immune response" also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells. A composition or vaccine that elicits a cellular immune response may serve to sensitize a vertebrate subject by the presentation of antigen in association with MHC molecules at the cell surface. The cell-mediated immune response is directed at, or near, cells presenting antigen at their surface. In addition, antigen-specific T-lymphocytes can be generated to allow for the future protection of an immunized host. The ability of a particular antigen to stimulate a cell-mediated immune response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art.

[0110] The role for adjuvants in increasing/shaping an immune response to coadministered antigens is well-known. As used herein, an “increase in” or “increasing” an immune response can indicate numerically more of an immune molecule/cell compared to antigen given alone without any adjuvant. For example, the aluminum salt adjuvant (alum) used in many licensed vaccines increases the antibody response against co-administered antigen; however, alum does not increase the T cell response appreciably. This can also indicate not only a numerical increase but a beneficial increase in the functionality of the immune molecules/cells. For example, alum not only numerically increases antibody numbers directed against co-administered antigen, it also increases the affinity of those antibodies leading to enhanced neutralization of pathogens. Adjuvant compositions provided herein increase one or more of, for example, B cell response, CD4+ T cells, CD8+ T cells, and immune responses in mucosal tissues.

[0111] The terms "effective amount" or "immunologically effective amount" of an adjuvant composition and antigen, as provided herein, refer to a nontoxic but sufficient amount of the composition to provide the desired immunological response, and optionally, a corresponding therapeutic effect, or, an amount sufficient to effect treatment of the subject. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the particular macromolecule of interest, mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0112] The phrase "pharmaceutically acceptable," in connection with administration of a substance to a human refers to a substance that is generally safe for human pharmaceutical use. In connection with administration to a non-human animal of a particular species, it refers to a substance that is generally safe and acceptable to a non-human animal of the species in question.

[0113] As used herein, the terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable vehicle" are interchangeable and refer to a fluid vehicle for containing vaccine antigens that can be injected into a host without adverse effects. Suitable pharmaceutically acceptable carriers known in the art include, but are not limited to, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (/.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.

[0114] Other Terminology and Disclosure

[0115] Unless defined otherwise, 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 disclosure belongs. Any methods and materials similar or equivalent to those provided herein can also be used in the practice or testing of the present disclosure.

[0116] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials for the purpose for which the publications are cited.

[0117] As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antigen” includes a plurality of such antigens, and reference to “a cell” or “the cell” includes reference to one or more cells and equivalents thereof (e.g., plurality of cells) known to those skilled in the art, and so forth. Similarly, reference to “a compound” or “a composition” includes a plurality of such compounds or compositions, and refers to one or more compounds or compositions, respectively, unless the context clearly dictates otherwise.

[0118] It is further noted that the claims may be drafted to exclude any element, e.g., any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0119] When a range of values is provided herein, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0120] When steps of a method are described or claimed, and the steps are described as occurring in a particular order, the description of a first step occurring (or being performed) “prior to” (/.e., before) a second step has the same meaning if rewritten to state that the second step occurs (or is performed) “subsequent” to the first step.

[0121] The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary between 1% and 15% of the stated number or numerical range. [0122] The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) is not intended to exclude that, for example, a composition of matter, composition, method, or process, or the like, provided herein, may “consist of” or “consist essentially of” the described features.

[0123] As will be apparent to those of skill in the art upon reading this disclosure, each of the compositions and methods provided and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other compositions and methods without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. This disclosure is intended to provide support for all such combinations.

[0124] As used herein, “may,” “may comprise,” “may be,” “can,” “can comprise” and “can be” all indicate something envisaged by the inventors that is functional and available as part of the subject matter provided.

Examples

[0125] While the following examples describe specific experiments, variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be placed on the invention.

[0126] The experiments described below show the effect of the combination of two different exemplary adjuvants. The first adjuvant used was LT(R192G/L211 A), or doublemutant heat-labile toxin (dmLT) from E. coli enterotoxin containing two mutations in the A subunit. It has no enterotoxic effects yet remains a potent adjuvant when delivered both mucosally and parenterally [Norton etal., Vaccine. 2015;33(16):1909 1915. doi:10.1016/j. vaccine.2015.02.069; Norton etal., Clin Vaccine Immunol. 2011 ;18(4):546 551 . doi:10.1128/cvi.00538-10]. dmLT adjuvanticity is contemplated herein to act through cAMP. dmLT was admixed with a second adjvant, OMVs derived from the attenuated Bp82 bacterial strain of Burkholderia pseudomallei [Propst et al., Infect Immun. 2010;78(7):3136 3143. doi:10.1128/iai.01313-09]. Notably, the OMVs generated from this bacterial strain have a naturally-attenuated lipid A which eliminates toxicity while preserving the full adjuvanticity of OMVs through Toll-like receptor (TLR) 4-dependent and -independent pathways. This adjuvant combination (herein referred to as dmLT-OMV) was compared to an antigen (Ag) alone or an Ag plus each adjuvant individually.

[0127] The engagement of all arms of the adaptive immune response (CD4, CD8, and humoral) is the holy grail of vaccine-mediated immunity. Eliciting this immunity in pathogen portals of entry like the lung, intestines, and FRT is critical. The experiments below show that parenterally delivered dmLT alone can drive mucosal immunity in all tissues assessed and that OMVs alone can drive all arms of the immune system. Further, the experiments demonstrate that dmLT formulated in a combination with OMVs, increases T and B cell immunity and mucosal migration over single adjuvants. This data together supports the foundation of the concept that, when combined, these adjuvants elicit the most potent, and appropriate, mucosal immune response even when delivered parenterally. What makes this adjuvant combination unique is its ability to both enhance and direct the immune response in ways that other adjuvants, including each of these adjuvants individually, cannot. Both dmLT and OMVs can induce a potent B cell response alone but this induction is synergistically strengthened when the two adjuvants are combined where the B cell response is much greater than would be the case if the response for each adjuvant were simply added tougher. The same is true for CD4+ T cells. dmLT does not induce a particularly robust CD8+ T cell response while OMVs do. Thus, combining the two adjuvants leads to an unexpected immune response that engages all arms of the adaptive immune response in a synergistic fashion. Further, the combination of the two adjuvants not only increases all aspects of adaptive immune responses, but the combination is also expected to drive these immune responses into all mucosal sites tested including the lungs, small and large intestines, and female reproductive tract. This can occur even when the adjuvant combination is delivered non-mucosally (parenterally), making this unique among adjuvants. This synergistic enhancement combined with mucosal immune increases has not been observed with any other adjuvants or adjuvant combinations.

Example 1 Model Antigen

[0128] A model antigen was generated for use in the experiments described in Examples 2 and 3.

[0129] The model antigen comprised a fusion protein of a well-characterized mouse MHC class II peptide epitope (SIINFEKL, SEQ ID NO: 2) that is recognized as foreign in C57BL/6 mice called 2W1 S, fused to chicken egg ovalbumin (OVA) that itself serves as both a CD8 T cell and a B cell Ag (hereafter 2W-OVA). Using magnetic bead enrichment of cells, this fusion protein gives us the unique capability to track both endogenous, Ag-specific CD4 and CD8 T cells (2W1S- or OVA-derived SHNFEKL-specific respectively) and OVA-specific B cells using tetramers for each (for overview see Figure 2). This gave the unique ability to follow how both a cellular and humoral immune responses develop, peak, resolve, and form memory in lymphoid and mucosal tissues after immunization using a single Ag.

Example 2 Experiments with dmLT Adjuvant

[0130] dmLT drives Ag-specific CD4 T cell migration into the intestinal lamina propria, FRT, and lung

[0131] To test whether parenterally delivered dmLT induced mucosal migration of vaccine-specific CD4 T cells, mice were immunized intradermally (ID) with 1 pg dmLT plus 2W1S Ag and Ag-specific T cell migration into the lung, intestine, and FRT was tracked using MHC class II tetramers. Notably for these experiments, rigor was added to the testing of the cells for tissue residence by first intravenously injecting a fluorochrome-labeled anti- CD45 Ab (for B cells) or anti-CD4 Ab (for T cells) three minutes prior to euthanasia. This marked all hematopoietic cells in the blood at the time of tissue harvest allowing distinct discrimination of those cells that were tissue resident at the time of tissue harvest versus those that were moving through the circulation and thus were not tissue resident. As shown in Figure 3, dmLT drives tissue resident CD4 T cells into all mucosal tissues of uninfected mice when administered parenterally. This demonstrates that ID-delivered dmLT can induce residence of the cellular immune response into multiple mucosal tissues when delivered ID.

[0132] dmLT drives Ag-specific B cells into the intestines and lungs

[0133] B cells also have the potential to become tissue resident cells where they may serve to prevent local infection at mucosal tissue sites. Because dmLT excelled at inducing mucosal homing of CD4 T cells, whether it could similarly induce mucosal homing of vaccine-specific B cells was tested. To assess this, mice were immunized ID with 1 pg dmLT plus the model Ag OVA and whether OVA-specific B cells were activated and migrated into the mucosa was tested using B cell Ag tetramers. (Figure 2 demonstrates how these Ag tetramers can detect OVA-specific B cells.) As observed with CD4 T cells, compared to CpG, dmLT preferentially induced non-circulatory (tissue-resident) B cell migration into the lungs and intestines (Figure 4). Of note, the tissue resident B cells induced by dmLT were also predominantly isotype switched (swig), meaning they were activated and had begun to express IgG or IgA. This demonstrates that ID-delivered dmLT induces B cells to migrate into multiple mucosal tissues where they begin producing Ab.

[0134] Reponses to pathogen-derived Ags were detected following immunization

[0135] Next, whether an intradermal vaccine adjuvanted with dmLT plus nucleoprotein (NP) derived from influenza A could be detected in mucosal tissues like observed with our model Ag, 2W-OVA was tested. Initially, NP-specific CD4 T cell responses were assessed following a single ID immunization with NP + 1 pg dmLT. As shown in Figure 5, tissue resident CD4 T cells were detected in the draining LN and lungs of mice that received NP + dmLT that were easily visualized using MHC class II NP tetramers. Next this was extended to NP-specific B cells using a NP B cell tetramer. As shown in Figure 6, swig, tissue resident LN and lung NP-specific B cells were detected following NP + dmLT immunization. This data demonstrates detection of pathogen-specific tissue resident memory T and B cells that can be directly compared to the model Ag responses.

[0136] Mucosal IgA and systemic IgG are produced in response to intradermal immunization with dmLT

[0137] IgA responses at the mucosa are important for clearance of mucosal pathogens. To determine whether dmLT induces this response, mice were immunized ID with 1 pg dmLT plus NP and NP-specific Ab responses were assessed both systemically and in the bronchoalveolar lavage fluid (BAL). As shown in Figure 7, Ag-specific mucosal IgA and systemic IgG were found. This data demonstrates that ID administered dmLT induces both systemic and mucosal Ab.

Example 3 Experiment with OMV Adjuvant

[0138] OMVs stimulate APCs and induce potent Ag-specific CD4 and CD8 T cell activation across species

[0139] OMVs are potent activators of APCs that can upregulate costimulatory molecules on DCs⁶¹. They can activate both CD4 and CD8 T cells in a human cell culture system⁷⁶. As shown in Figure 8, OMVs induced expansion of multifunctional CD4 and CD8 human T cells. The ability of OMVs to expand vaccine-specific CD4 and CD8 T cells in vivo was determined. To do this, mice were immunized with 2W1 S and SIINFEKL and 2W1 S-specific CD4 and OVA-specific CD8 T cells were tracked. As shown in Figure 9, OMVs promoted a vigorous expansion of both T cell types. This data shows that OMVs are powerful immune stimulators that can activate APCs and drive activation and expansion of both arms of T cell immunity.

Example 4 Experiments with dmLT-OMVs

[0140] The dmLT and OMV adjuvants have been shown to be safe in multiple species

[0141] dmLT has been tested in phase 1 and 2 clinical trials and shown to be safe [Lundgren et al., Vaccine. 2014;32(52):7077 7084. doi:10.1016/j. vaccine.2014.10.069; El- Kamary et al., Clin Vaccine Immunol. 2013;20(11 ):1764 1770. doi:10.1128/cvi.00464-13]. OMVs have been tested for safety and reactivity in the LPS-sensitive wax moth larvae Galleria melonella, mice, rhesus macaques, and a human surrogate system and have been shown to be non-toxic in all of these models [Higbee et al., Altern Laboratory Animals. 2009;37(1_suppl):19-27. doi:10.1177/026119290903701 s05; Baker et al., Nato Adv Sci Inst Se. 2017;5(4):49. doi:10.3390/vaccines5040049; Petersen et al., Procedia Vaccinol. 2014;8:3842. doi:10.1016/j.provac.2014.07.007; Nieves et al., Clin Vaccine Immunol. 2014;21 (5):747 754. doi:10.1128/cvi.00119-14; Nieves et al., Vaccine. 2011 ;29(46):8381 8389. doi:10.1016/j.vaccine.2011 .08.058]. Neisseria-derived OMVs are a vital component of the FDA-licensed Bexsero vaccine against Neisseria meningitidis serogroup B and millions of doses of Bexsero have been administered to children worldwide, including children under the age of two, with minimal reactogenicity [Ladhani et al., New Engl J Medicine. 2020;382(4):309-317. doi:10.1056/nejmoa1901229]. The adjuvants are also safe when combined, as no adverse reactions were observed in mice at the proposed combination dose (Figure 10 and Figure 11).

[0142] dmLT-OMVs enhance Ag-specific CD4 T cell responses and drives greater Ag-specific B cell activation in the gut-draining lymph nodes

[0143] To test the capacity of the dmLT-OMV adjuvant to drive increased T cell and B cell immunity, mice were immunized with the combination and the injection site-draining lymph nodes were assayed. A single injection of each adjuvant or a combination of the two, plus 2W-OVA antigen, showed an increase in the CD4 T cell response in the combination (Figure 10). Intriguingly, a single injection of the combination was able to drive more Ag-specific isotype switched, germinal center B cells into the gut-draining MLN (Figure 11 ). This data demonstrates that dmLT-OMVs induce a significant mucosal homing of adaptive immune cells compared to single adjuvants with no observed toxicity.

Example 5 Further experiments with dmLT-OMVs

[0144] Effect of dmLT-OMVs on vaccine-specific CD4 T cell activation and mucosal migration

[0145] The experiment above demonstrates the dmLT-OMV adjuvant induces activation and mucosal migration of vaccine-specific CD4 T cells. This experiment will demonstrate CD4 T cells enter all mucosal tissues and that dmLT is essential for this migration. First, WT mice will receive a single intradermal or mucosal immunization with 1 pg each adjuvant alone or in combination plus 2W-OVA Ag. Nine days later (at the peak of the T cell response), CLN, spleen, MLN, MedLN, ILN, Peyer’s patches, small and large intestine lamina propria, lungs, and FRT will be assessed for the presence of 2W1S-specific CD4 T cells using MHCII-2W1 S tetramers made in our lab coupled with magnetic bead enrichment. In order to discriminate between cells in the vasculature and cells in the tissue parenchyma, we will inject fluorochrome labeled CD4 Ab intravenously (IV) as described for the data above (Figure 3 and Figure 4). Mice will be euthanized three minutes after IV injection and single cell preparations will be stained with a different anti-CD4 Ab clone coupled to a different fluorochrome. Cells labeled with both Ab will be considered to be vascular T cells and those that stain with only the second Ab will be considered to be tissue resident and non-circulatory. Next, the mice will be immunized as above, two times three weeks apart, to mimic vaccine induced boosting responses. Cellular migration will be assessed one week following the final booster.

[0146] Effect of dmLT-OMVs on vaccine-specific CD8 T cell activation and mucosal migration

[0147] Immunization outcomes in mice were assessed using MHCI tetramers made using the CD8 OVA peptide SIINFEKL as the Ag loaded into the MHCI tetramers.

[0148] Mice were intradermally immunized with 1 pg 2W1 S-Ova antigen + 1 pg dmLT + 0.1 pg T-vant (prime, boost three weeks later). Two weeks later Ova-specific CD8 T cells were assessed in the draining lymph nodes. Figure 25 shows that the combination adjuvant induced the greatest number of vaccine-specific CD8 T cells in response to vaccination.

[0149] Effect of dmLT-OMVs on T cell effector responses

[0150] To assess how dmLT-OMVs affects Th effector responses, WT mice will be immunized as described above in this Example and 2W1 S-specific CD4 T cells from each tissue will be restimulated with Phorbol 12-myristate 13-acetate (PMA) + ionomycin in the presence of Brefeldin A for 6 hours. Cells will then be fixed and stained intracellularly for the following Th-specific cytokines and transcription factors: IFN-y and T-bet (Th 1 ), IL-4, IL-5 and GATA-3 (Th2), IL-17 and Roryt (Th17), IL-22 and AHR (Th22), IL-9 (Th9), IL-10 and Foxp3 (T_reg) and analyzed by flow cytometry.

[0151] Effect of dmLT-OMVs on Ag-specific CD8 T cell effector functions

[0152] Mice will be immunized as described above in this Example. Ag-specific CD8 T cells will be enriched and restimulated as above. Readouts will include effector cytokines (IFN-y/TNF-a) and the effector molecule granzyme B known to be important for CD8 antiviral immunity.

[0153] Effect of dmLT-OMVs on establishment of mucosal TRM CD4 and CD8 T cells [0154] To assess establishment of T_RM with our adjuvants, mice will be immunized as described above in this Example and mucosal tissues harvested as described, although in this case memory formation will be assessed early (one week) and late (8 weeks) after the final boost in order to allow cells to equalize in tissues such that we can delineate the establishment of long-term memory. Vaccine-specific CD4 and CD8 T cells will be stained for CD69 and CD103 as initial indicators of T_RM cells and, to verify residency, IV staining will also be performed prior to euthanasia. Notably, T_RM cells rarely leave tissues at steady state once established. To firmly ascertain the persistence of T_RM cells, parabiosis experiments will be performed (see Figure 12 for outline of parabiosis experimental design and expected outcomes), the gold standard approach for delineating T_RM cells. Briefly, CD45.1 ⁺ donor mice (Parabiont #1 ) will be immunized as described above in this Example. Eight weeks after the final boost, immunized mice will be surgically conjoined to a naive, CD45.2+ recipient congenic mouse (Parabiont #2). Three weeks after surgery, when anastomosis is achieved and cells have reached circulatory equilibrium between conjoined mice [Kamran et al., J Vis Exp. 2013;(80). doi:10.3791/50556], the mice will be separated and whether CD45.1 ⁺ vaccine-specific CD4 or CD8 T cells exited the Donor/Parabiont #1 and migrated into the mucosal tissues of the naive CD45.2+ mice (Recipient/Parabiont #2) at homeostasis will be assesses. If not, this would establish that the mucosal T cells in the original immunized CD45.1 ⁺ mice are bona fide T_RM cells.

[0155] A CD45.2+ mouse (BL6) was intradermally immunized with 1 pg 2W1 S-Ova antigen + 1 pg dmLT + 0.1 pg T-vant (prime, boost three weeks later). Two weeks later the immunized mouse was conjoined to a CD45.1 + mouse (Pep) to create a parabiotic pair. Mice were sacrificed 21 days post-surgery after circulatory exchange was confirmed and 2W1 S-specific CD4 T cells (those cells that recognized the antigen) were assessed in the spleen, lung, large intestine, and injection site-draining lymph nodes. As shown in Figure 26, cells were equally distributed in the spleen where cells freely circulate but were confined only to the immunized mouse in the lungs, large intestine, and draining lymph nodes indicating the vaccine specific CD4 T cells did not migrate into tissues to become tissue resident cells. This shows that the combo adjuvant induces robust tissue resident CD4 T cell immunity. N=4 parabiotic pairs

Example 6 Still further experiments with dmLT-OMVs [0156] Effect of dmLT-OMVs on Ab responses to ID immunization

[0157] Mice will be intradermally or mucosally immunized with 1 pg dmLT-OMVs plus 15 pg 2W-OVA and then one week, one month, and one year after the final booster, OVA- specific IgM, IgA, lgG1 , lgG2b, lgG2c, lgG3, and IgE will be measured from serum, feces, saliva, bronchoalveolar lavage fluid, and vaginal washes by ELISA.

[0158] Effiect of dmLT-OMVs on B cell tissue localization persistance [0159] Mice will be immunized twice as described above in this Example and then, 6 and 12 months after the last booster, mice will be immunized again with 2W-OVA alone or in conjunction with dmLT-OMVs to test the long-term recall response.

[0160] Effect of dmLT-OMVs on expansion and mucosal migration of Ag-specific B cells

[0161] Mice will be immunized as described above in this Example (prime boost with 2W- OVA Ag) and one week, one month, and one year after the final booster, tissues outlined in above in this Example (plus bone marrow where plasma cells are expected to reside) will be harvested and OVA-specific B cells will be magnetically bead enriched and quantified. GC cells (CD19⁺ CD38- GL7⁺) and istotype switched cells (CD19⁺ IgM- IgD ) will also be tracked as shown in Figure 4. Intracellular staining will be performed to assess the general isotype in OVA-specific B cells (IgG, IgA, etc.). To evaluate how B cells and T cells interact, B cell-T cell conjugates will be measured by flow cytometry and to show the interaction between cytokine producing CD4 T cells and B cells responding to those cytokines¹¹⁷. Cell conjugates will be stained for Ag specificity. Using 2W-OVA as the model Ag allows the assessment of both members of a conjugate pair for specificity for the vaccine Ag (2W1 S-specific T cells conjugated to OVA-specific B cells) and what cytokines the cells are producing.

[0162] Effect of dmLT-OMVs on Ag-specific memory B cell persistance

[0163] To test whether the Ag-specific B cells are long-lived, mice will be immunized as above and isotype-switched OVA-specific B cells will be measured at 6 and 12 months after the final booster. As in Example 5, immunized mice from each group will be boosted 6 months later to determine if memory B cells proliferate in response to a third booster and are isotype switched to IgA. Tissues as described above in this Example will be stained for OVA- specific B cells paired with assessment of Ab isotype switching by flow cytometry. Additionally, in order to better demonstrate the mucosal homing potential of tissue resident cells, cells from each individual mucosal tissue will be isolated and adoptively transferred into naive mice where they can be tracked (CD45.1 + into CD45.2+ mice) as to whether they re-enter the mucosal tissue from whence they were harvested. This will be done for both T cells and B cells.

[0164] Effect of dmLT-OMVs on the presence of mucosal tissue resident B_RM cells [0165] The experiment in Example 4 shows that dmLT induces B_RM cells (Figure 4). To confirm this, parabiosis experiments will be performed as outlined for TRM; however, in this case, all mucosal tissues in Parabiont #2 (Fig. 12) will be examined for isotype switched CD45.1 ⁺ BRM cells from Parabiont #1. If the cells from the Donor/Parabiont #1 (CD45.1 ⁺) do not leave and deposit into the mucosal tissues of the Recipient/Parabiont #2 (CD45.2⁺), then those cells are considered bona fide B_RM cells. To assess the durability of B_RM in the mucosal tissues following immunization, parabiosis experiments will be performed as described above; however, here surgery will be initiated at six months and one year following the initial immunizations of the CD45.1⁺ mouse.

[0166] Effect of dmLT-OMVs on mucosal infections

[0167] C57BL/6 mice will be immunized ID one or three times as before with 1 pg dmLT, OMV, or dmLT-OMV in combination with the following Ags individually for each infection: 2pg heat-inactivated Influenza A virus H1 N1 A/PR/8/34 (PR8); 10⁸ cfu formalin-inactivated Citrobacter rodentium (ATCC 51459); or 5pg recombinant major outer membrane protein (Genscript) for Chlamydia muridarum. Ag alone will serve as the negative control for each infection. One month following the final immunization, mice will be challenged to assess protective efficacy.

[0168] For lung infection, mice will be challenged with a lethal dose (6 x LD₅o) of H1 N1 PR8 influenza. Mice (n=10) will be monitored for weight loss and survival for up to 14d. A separate cohort of mice (n=5 per group) will be sacrificed at days 5 or 10 after infection to examine Ab, B and T cell responses in serum, lungs, MedLN, and spleen. TRM markers in the mucosa will be assessed for each infection. In a separate cohort of mice (n=5), right lungs will be collected for viral loads which will be measured by PCR for the viral M1 gene as well as by plaque assay if needed. Left lungs will be paraffin embedded and lung injury and inflammation will be assessed by H&E.

[0169] For gut infection, mice will be challenged with 2 x 10⁸ cfu of C. rodentium (ATCC 51459) by intragastric lavage. Mice (n=10) will be monitored daily for weight loss, bacterial fecal shedding and morbidity for up to 14d. At the study endpoint, bacterial burdens will be assessed in the colon, liver and spleen. A separate cohort of mice (n=5) will be sacrificed at days 5 and 10 to evaluate colonic hyperplasia as well as Ab, B and T cell responses in the serum, colon, MLN, and spleen.

[0170] For FRT infections, 1 x 10⁵ C. muridarum (ATCC VR-123) will be deposited in the vaginal vault of female mice. Mice will be monitored for 21 days to assess bacterial burdens and/or clearance in the lower genital tract. Ab, T and B cell responses in the serum, FRT, ILN, and spleen will be assayed at days 7, 14, and 21 using a separate cohort of mice (n=5 per timepoint).

[0171] Mice were either intradermally or intravagin ally prime-boost immunized with 1 pg dmLT + 0.1 pg T-vant + 5 pg recombinant major outer membrane protein from C. muridarum four weeks apart. One week after the final immunization, mice were administered Depo Provera to allow for intravaginal infection. One week later mice were infected with 3 x 10³ C. muridarum bacteria and monitored for a week. As shown in Figure 24, mice immunized by either route were protected against infection for at least a week after infection. [0172] Effect of dmLT-OMVs on vaccine-induced resident T or B cells responsible for protection against infection

[0173] How tissue-resident cellular or humoral immunity adds to protection after immunizing parenterally with adjuvant combinations will be tested by immunizing and performing parabiosis as described above (outlined in Figure 12). Parabionts will be separated and allowed to recover for 2 weeks before viral or bacterial challenge. Mice will then be followed to assess protective efficacy in each Parabiont as previously described. To test whether each type of resident memory cell is important for protection, WT mice (Parabiont #1) will be immunized with dmLT-OMVs plus the appropriate protective Ag (2 .g heat-inactivated Influenza A virus H1 N1 A/PR/8/34 (PR8); 10⁸ cfu formalin-inactivated Citrobacter rodentium (ATCC 51459); or 5|ig recombinant major outer membrane protein (Genscript) for Chlamydia muridarum) and then parabiosis will be performed with mice (Parabiont #2) that lack the following: CD4 T cells ^Cd^¹^ mice); CD8 T cells (Cd8a^tm1Mak mice) and B cells (p.MT mice). All strains are commercially available. Mice will then be separated, and each member of the pair will be infected.

[0174] Note regarding sample size in the experiments

[0175] The primary endpoint for sample size is based on a 2-fold difference of a given inflammatory mediator or cell type between a control group vs. an experimental group. Using this, a power of 90% ( = 0.1), two-sided analysis and a type I error rate of 5% (a = 0.05), an a priori sample size calculation for the Student's t-test estimates that each group would require a minimum of 4-6 mice per group. Experimental designs herein employ an n = 5 mice per group for each experiment with each experiment repeated at least once, thus providing for both rigorous assessment of a phenotype and reproducibility.

Claims

Claims We claim:

1 . An adjuvant composition comprising an adjuvant of SEQ ID NO: 1 (dmLT) and an attenuated Burkholderia pseudomallei Bp82 bacterial strain-derived outer membrane vesicle (OMV) adjuvant.

2. The adjuvant composition of claim 1 further comprising a vaccine.

3. The adjuvant composition of claim 2 wherein the vaccine comprises a polypeptide, a nucleic acid, a polysaccharide, a polysaccharide-polypeptide conjugate, a live-attenuated or inactivated bacterium, a toxoid, a live-attenuated or inactivated virus, a virus-like particle, or a viral vector.

4. The adjuvant composition of claim 3 wherein the nucleic acid is a ribonucleic acid (RNA).

5. The adjuvant composition of claim 3 wherein the nucleic acid is a deoxyribonucleic acid (DNA).

6. The adjuvant composition of any of claims 1 -5 wherein the vaccine comprises or encodes a bacterial, viral, or fungal antigen.

7. The adjuvant composition any of claims 1 -6 comprising 0.1-100 pg/dose of DmLT adjuvant.

8. The adjuvant composition of any of claims 1 -7 comprising 0.1-100 pg/dose of OMV adjuvant.

9. A kit comprising a DMLT adjuvant and an OMV adjuvant.

10. The kit of claim 9 further comprising a vaccine.

11 . The kit of claim 10 wherein the vaccine comprises a polypeptide, a nucleic acid, a polysaccharide, a polysaccharide-polypeptide conjugate, a live-attenuated or inactivated bacterium, a toxoid, a live-attenuated or inactivated virus, a virus-like particle, or a viral vector.

12. The kit of claim 11 wherein the nucleic acid is a ribonucleic acid (RNA).

13. The kit of claim 11 wherein the nucleic acid is a deoxyribonucleic acid (DNA).

14. The kit of any of claims 9-13 wherein the vaccine comprises or encodes a bacterial, viral, or fungal antigen.

15. A method of generating an immune response in a subject comprising administering to the subject the adjuvant composition of any of claims 2-8.

16. The method of claim 15 wherein the composition is administered by the intradermal route.

17. The method of claim 15 or 16 wherein the immune response is in the mucosa of the subject.

18. The method of any of claims 15-17 wherein the immune response is pan- mucosal immunity.

19. The method of any of claims 15-17 wherein the mucosa is in one or more of the respiratory, digestive and urogenital tracts.

20. The method of any of claims 15-17 and 19 wherein the mucosa is in the eye.

21 . The method of any of claims 15-20 wherein the immune response is a B cell response.

22. The method of any of claims 15-20 wherein the immune response is the generation of CD4+ T cells.

23. The method of any of claims 15-20 wherein the immune response is the generation of CD8+ T cells.

24. The adjuvant composition of claim 2, wherein the vaccine is a vaccine against Chlamydia muridarum.

25. The kit of claim 9, wherein the vaccine is a vaccine against Chlamydia muridarum.

26. The method of claim 15, wherein the immune response is an immune response against Chlamydia muridarum.