npj Vaccines

Effective pre-erythrocytic Plasmodium falciparum vaccines targeting circumsporozoite protein (CSP) must elicit strong and durable antibody responses. We assessed the ability of different classes of adjuvants to enhance immunity of a promising CSP-epitope-targeted virus-like particle (VLP)-based vaccine. Five formulations significantly increased anti-CSP IgG titers and several also enhanced antibody durability. This study identifies promising adjuvants that enhance both the magnitude and longevity of VLP-induced immunity.

Malaria is a highly lethal infectious disease caused by Plasmodium parasites. These parasites are transmitted to vertebrate hosts when mosquitoes of the Anopheles genus probe for a blood meal. Sporozoites, the infectious stage of Plasmodium, transit to the liver within hours of injection into the dermis. Vaccine efforts are hindered by the complexity of the parasites lifecycle and the speed at which the infection is established in the liver. In an effort to enhance immunity against Plasmodium, we produced a virus-like particle (VLP)-based vaccine displaying an epitope of TRIO, an Anopheles salivary protein which has been shown to enhance mobility and dispersal of sporozoites in the dermis. Previous work demonstrated that passive immunization with TRIO offered protection from liver infection and acted synergistically with a Plasmodium targeted vaccine. Immunization of mice with TRIO VLPs resulted in high-titer and long-lasting antibody responses that did not significantly drop for over 18 months post-immunization. TRIO VLPs were similarly immunogenic when combined with an anti-malaria vaccine targeting the L9 epitope of the Plasmodium falciparum circumsporozoite protein.However, when used in a malaria challenge mouse model, TRIO VLPs only provided modest protection from infection and did not boost the protection provided by L9 VLPs.

Malaria-causing Plasmodium parasites have a complex life cycle and present numerous antigen targets that may contribute to protective immune responses. The currently recommended vaccine--RTS,S--functions by targeting the P. falciparum circumsporozoite protein (CSP), which is the most abundant surface protein of the sporozoite form responsible for initiating infection of the human host. Despite showing only moderate efficacy, RTS,S has established a strong foundation for the development of next-generation subunit vaccines. Our previous work characterizing the sporozoite surface proteome identified additional non-CSP antigens that may be useful as immunogens individually or in combination with CSP. In this study, we examined eight such antigens using the rodent malaria parasite P. yoelii as a model system. We demonstrate that despite conferring weak protection individually, co-immunizing each of several of these antigens alongside CSP, could significantly enhance the sterile protection achieved by CSP immunization alone. Thus, our work provides compelling evidence that a multi-antigen pre-erythrocytic vaccine approach may enhance protection compared to CSP-only vaccines. This lays the groundwork for further studies aimed at testing the identified antigen combinations in human vaccination trials that assess efficacy with controlled human malaria infection. ImportanceThe currently approved malaria vaccine targets a single parasite protein (CSP) and only results in partial protection. We tested several additional vaccine targets in combination with CSP to identify those that could enhance protection from infection upon challenge in the mouse malaria model. In identifying several such enhancing vaccine targets, our work indicates that a multi-protein immunization approach may be a promising avenue to achieving higher levels of protection from infection. Our work identified several candidate leads for follow-up in the models relevant for human malaria, and provides an experimental framework for efficiently carrying out such screens for other combinations of vaccine targets.

Previous studies have demonstrated the efficacy and feasibility of an anti-viral vaccine strategy that takes advantage of pre-existing CD4+ helper T (Th) cells induced by Mycobacterium bovis bacille Calmette-Guerin (BCG) vaccination. This strategy uses immunization with recombinant fusion proteins comprised of a cell surface expressed viral antigen, such as a viral envelope glycoprotein, engineered to contain well-defined BCG Th cell epitopes, thus rapidly recruiting Th cells induced by prior BCG vaccination to provide intrastructural help to virus-specific B cells. In the current study, we show that Th cells induced by BCG were localized predominantly outside of germinal centers and promoted antibody class switching to isotypes characterized by strong Fc receptor interactions and effector functions. Furthermore, BCG vaccination also upregulated Fc{gamma}R expression to potentially maximize antibody-dependent effector activities. Using a mouse model of Ebola virus (EBOV) infection, this vaccine strategy provided sustained antibody levels with strong IgG2c bias and protection against lethal challenge. This general approach can be easily adapted to other viruses, and may be a rapid and effective method of immunization against emerging pandemics in populations that routinely receive BCG vaccination.

Background: The isolation of many HIV broadly neutralizing antibodies (bnAbs) from people living with HIV (PLWH) and rigorous characterization of their ontogeny has promoted the goal of reverse engineering their natural development as a strategy for achieving an effective preventive HIV vaccine. We previously described the developmental process of CH103, a CD4-binding site (CD4bs)-specific monoclonal antibody, and the associated evolution of HIV Envelopes (Envs) within the person (CH505) from whom it was isolated. A series of monomeric gp120 protein subunit immunogens representing the transmitted founder (TF) and Envs that evolved during infection and optimally reacted with lineage members at each step of the CH103 clone maturation path were evaluated in this placebo controlled randomized vaccine trial to test for the first time in humans the concept of whether sequential immunization with gp120 monomeric proteins can recapitulate the development of CD4bs B-cell clonal lineages, including CH103. Methods: HIV Vaccine Trials Network 115 (HVTN 115) was a randomized placebo-controlled vaccine trial at US clinical research sites. We tested the safety and immunogenicity of CH505TF gp120 + GLA-SE (Part A), and then the ability of sequential CH505 gp120 proteins (corresponding to CH505s weeks 53 and 78 Envs) + GLA-SE immunizations to induce CD4bs-specific neutralizing antibodies (Part B). We assessed binding and neutralizing antibody responses, antibody dependent cellular cytotoxicity, antibody dependent cellular phagocytosis, T-cell responses and B-cell phenotyping. Results: We enrolled 42 participants between October 2017 and May 2018 for Part A, and 65 participants from December 2020 to October 2022 for Part B. Immunization with the CH505 gp120 proteins adjuvanted with GLA-SE was well tolerated and induced CD4bs-specific B cells and Env-specific plasma antibodies. The plasma neutralizing antibody response was limited to primarily tier 1 autologous and heterologous HIV-1 strains. Blood-derived B-cell repertoire analyses identified CD4bs antibodies that preferentially bound to open-occluded trimeric Envs that exist in an intermediate state between prefusion-closed to CD4-bound open confirmations, consistent with tier 1 HIV neutralizing activity. Conclusions: Together, these results suggest that the low-affinity CH505TF gp120 monomer elicited CD4bs antibodies in the sera and B-cell repertoires of humans. However, our findings also indicate that gp120 monomers are insufficient to induce detectable bnAb precursors to epitopes on native Env trimers. Nonetheless, our data provide a benchmark for comparison with ongoing clinical trials testing high-affinity CH505 Env trimers for induction of CD4bs bnAb precursors.

The success of the poliovirus (PV) vaccines has enabled the near-eradication of wild PV, however, their continued use post-eradication poses a significant threat to maintaining a polio-free world. Recombinant virus-like particles (VLPs) that lack the viral genome remove this risk. Here, we demonstrate the production of PV VLPs for all three serotypes by controlled fermentation using Pichia pastoris. We determined the cryo-EM structure of a new PV-2 mutant, termed SC5a, in comparison to PV-2 SC6b VLPs described previously and investigated the immunogenicity of PV-2 SC5a VLPs. Finally, a trivalent immunogenicity trial using bioreactor-derived VLPs of all three serotypes in the presence of Alhydrogel adjuvant, showed that these VLPs outperform the current IPV vaccine in the standard vaccine potency assay, offering the potential for dose-sparing. Overall, these results provide further evidence that yeast-produced VLPs have the potential to be a next-generation polio vaccine in a post-eradication world.

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and de-centralized production of recombinant protein vaccine antigens. Here, we use CFPS to produce the putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for use as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four-weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Immunization with CT584 generated robust antibody responses but weak cell mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lungs weights and the presence of high numbers of IFUs in the lungs. While CT584 alone may not be the ideal vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens makes it an attractive technique for antigen production.

An effective vaccine against hepatitis C virus (HCV) must elicit the production of broadly neutralizing antibodies (bnAbs) reproducibly against the E1E2 glycoprotein complex. Little is known about how glycan content affects this process. Ideally, glycans would maximize epitope exposure without compromising antigen stability or exposing new epitopes. However, typical recombinant vaccines contain considerable heterogeneity in glycan content, which can affect the antibody response and neutralization potency. Here we employed glycoengineered Chinese hamster ovary (geCHO) cell lines that impart nearly homogeneous glycosylation as a means to test how specific glycan features influence antigenicity and immunogenicity for the secreted HCV E2 ectodomain (sE2). Specific geCHO antigens exhibited a modest but reproducible increase in affinity for some mAbs relative to CHO- and HEK293-produced sE2. Surprisingly, one geCHO sE2 antigen failed to bind the CD81 receptor, indicating the potential for significant glycan effects on biochemical properties. We immunized mice with the four antigens and found the total antibody response to be the same for all groups. However, sera from one geCHO group exhibited a 7-fold improvement in neutralization against the homologous HCV pseudovirus and had the most mice whose sera exhibited neutralization activity against genotypes 1b, 2a, 2b, and 3. Further analysis identified beneficial and deleterious glycan features, and the glycan that correlated the most with decreased potency was relatively small. However, size was not the sole determinant of glycan-driven effects on the antibody response. In summary, glycan content impacts biochemical properties of antigens to varying degrees and such effects can influence immune response quality and uniformity.

Next-generation vaccines are required to address the evolving nature of SARS-CoV-2 and to protect against emerging pandemic threats from other coronaviruses. These vaccines should aim to elicit broad-protection, provide long-lasting immunity and facilitate equitable access for all populations. In this study, a panel of chimeric, full-length spike antigens were developed that incorporate mutations from previous, circulating and predicted SARS-CoV-2 variants. The lead candidate (CoVEXS5) was obtained from a high-yield production process in stable CHO cells with purity of >95%, long-term stability and elicitation of broadly cross-reactive neutralising antibodies when delivered to mice in a squalene emulsion adjuvant (Sepivac SWE). In both mice and hamsters, CoVEXS5 immunisation reduced clinical disease signs, lung inflammation and organ viral titres after SARS-CoV-2 infection, including challenge with the highly immunoevasive Omicron XBB.1.5 subvariant. In mice previously primed with a licenced protein vaccine (NVX-CoV2373), CoVEXS5 could boost T cell immunity, as well as neutralising antibodies levels against viruses from three sarbecoviruses clades. The breadth of sarbecovirus cross-reactivity elicited by CoVEXS5 exceeded that observed after boosting with the NVX-CoV2373 vaccine. These findings highlight the potential of a chimeric spike antigen, formulated in an open-access adjuvant, as a next-generation vaccine candidate to enhance cross-protection against emerging sarbecoviruses in vaccinated populations globally.

Mycobacterium tuberculosis (Mtb) is an opportunistic bacterial pathogen that causes tuberculosis disease (TB) and exerts an extensive burden on global health. The complex intra- and extracellular nature of this bacterium, coupled with different disease stages have made mechanistic studies evaluating the contributions of innate and adaptive host immunity challenging. In this work we leveraged two delivery platforms as prophylactic vaccines to assess immunity and subsequent efficacy against low dose and ultra-low dose aerosol challenge with Mtb H37Rv in C57BL/6 mice. Our proof-of-concept TB vaccine candidate ID91 was produced as a fusion protein formulated with a synthetic TLR4 agonist (glucopyranosyl lipid adjuvant in a stable emulsion) or as a replicating-RNA (repRNA) formulated in a nanostructured lipid carrier (NLC). Results from this work demonstrate that protein subunit- and RNA-based vaccines preferentially elicit cellular immune responses to different ID91 epitopes. In a single prophylactic immunization screen, both platforms reduced pulmonary bacterial burden compared to controls. Excitingly, in prime-boost strategies, groups that received heterologous RNA-prime, protein-boost or combination (simultaneous in different sites) immunizations demonstrated the greatest reduction in bacterial burden and a unique humoral and cellular immune response profile. These data are the first to report that repRNA platforms are a viable system for TB vaccines and should be pursued with high priority Mtb antigens containing CD4+ and CD8+ T cell epitopes.

Polioviruses have caused crippling disease in humans for centuries, prior to the successful development of vaccines in the mid-1900s, which dramatically reduced disease prevalence. Continued use of these vaccines, however, threatens ultimate disease eradication and achievement of a polio-free world. Virus-like particles (VLPs) that lack a viral genome represent a safer potential vaccine, although they require particle stabilization. Using our previously established genetic techniques to stabilize the structural capsid proteins, we demonstrate production of poliovirus VLPs of all three serotypes, from four different recombinant expression systems. We compare the antigenicity, thermostability and immunogenicity of these stabilized VLPs against the current inactivated polio vaccine, demonstrating equivalent or superior immunogenicity. Structural analyses of these recombinant VLPs provide a rational understanding of the stabilizing mutations and the role of potential excipients. Collectively, we have established these poliovirus stabilized VLPs as viable next-generation vaccine candidates for the future.

Repetitive display of the major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) is the basis for two WHO-recommended vaccines: RTS,S/AS01 and R21/Matrix-M. Recently, however, the CIS43 monoclonal antibody that preferentially targets the junctional region of PfCSP has been shown to be highly protective in humans, highlighting its junctional epitope as a key vaccine target. Here, we develop a vaccine based on the tandem repeats of the junctional epitope displayed on a self-assembling nanoparticle, and compare this CIS43-based junctional vaccine alone or in combination with the benchmark R21 vaccine, using both B cell analysis and monoclonal antibody isolation to define targeting of the immune response. Comparable reduction in liver burden was observed following vaccination with junctional and R21 vaccines at a dose of 1 g. At a dose of 0.25 g, a modest reduction of malaria-liver burden with the junctional vaccine was observed compared to R21. Further, combining junctional and R21 vaccines induced modestly enhanced protection compared to either vaccine alone. While the R21 vaccine elicited antibodies primarily against the major repeats, the junctional vaccine elicited antibodies against both junctional and major repeat regions. In vivo-B cell analysis and isolation of monoclonal antibodies confirmed differences in vaccine-induced antibody specificities. Altogether, these data suggest the nanoparticle-formatted tandem-repeated CIS43-junctional vaccine to be a promising approach to broaden immunity against malaria, either as a standalone intervention or in combination with R21. HIGHLIGHTSO_LIDeveloped a self-assembling nanoparticle-displayed junctional vaccine of PfCSP based on tandem repeats of the epitope preferentially targeted by the highly protective CIS43 antibody C_LIO_LIThe CIS43-based junctional vaccine at low doses significantly reduced liver burden following malaria challenge in mice C_LIO_LIFollowing either low or high doses of the junctional vaccine in naive mice, adoptively transferred B cells expressing the CIS43 inferred germline sequence yielded a high frequency of germinal center and ASC responses C_LIO_LIThe CIS43-based junctional vaccine elicits antibodies against junctional and major repeat regions whereas the R21 vaccine elicits responses primarily against the major repeat region C_LIO_LIAt low dose, the CIS43-based junctional vaccine given together with the R21 vaccine showed modestly improved control of liver burden compared to either vaccine alone C_LI

Having varied approaches to the design and manufacture of vaccines is critical in being able to respond to worldwide needs and to newly emerging pathogens. Virus-like particle (VLP) vaccines form the basis of two of the most successful licensed vaccines (against hepatitis B virus (HBV) and human papillomavirus). They are produced by recombinant expression of viral structural proteins, which self-assemble into immunogenic nanoparticles. VLPs can also be modified to present unrelated antigens, and here we describe a universal bolt-on vaccine platform (termed VelcroVax) where the capturing VLP and the target antigen (hapten) are produced separately. We utilise a modified HBV core (HBcAg) VLP, with surface expression of a high-affinity binding sequence (Affimer) directed against a SUMO tag and use this to capture SUMO-tagged gp1 glycoprotein from the arenavirus, Junin virus (JUNV). Using this model system, we have solved high-resolution structures of VelcroVax VLPs, and shown that the VelcroVax-JUNV gp1 complex induces superior humoral immune responses compared to the non-complexed viral protein. We propose that this system could be modified to present a range of haptens and therefore form the foundation of future rapid-response vaccination strategies.

AbstractCreating effective and thermostable vaccines is of significant relevance for public health. The Atomic Layering Thermostable Antigen and Adjuvant (ALTA(R)) platform combines spray drying to stabilize antigens in a sugar matrix followed by coating with atomic layer deposition (ALD) for temporal control over in vivo release. While these technologies have shown preliminary promise for different vaccine antigens, further characterizations of the immune response to ALTA(R) formulated antigens are still needed. Here, the immune response to ALTA(R) formulated antigens is described and compared to a set of adjuvanted liquid vaccine formulations that included Alhydrogel(R), AddaVax, and Alhydrogel(R)+CpG. The humoral and cell-mediated responses were measured by ELISA and flow cytometry. Increased and lasting antigen-specific antibody titers following administration of ALTA(R) containing ovalbumin (OVA) demonstrated robust and durable humoral response. Furthermore, ALTA(R) injected mice produced both IgG2c and IgG1 indicating a balanced Th1/Th2 response. Importantly, ALTA(R) OVA elicited robust humoral response at lower doses of aluminum than Alhydrogel(R). The most striking difference between ALTA(R) and the liquid vaccine formulations tested was a greater OVA-specific CD8+ T cell response observed at all antigen doses tested. Mechanistically, antigen encapsulation within ALTA(R) particles was critical for antibody production and CD8+ T cell responses as well as antigen capture by antigen-presenting cells (APCs) at the site of injection and draining lymph nodes. To test these concepts in a more physiological application, protein and polysaccharide vaccine antigens derived from a facultative intracellular bacterium Burkholderia pseudomallei, the causative agent of melioidosis, were formulated using the ALTA(R) platform. Compared to liquid vaccine formulations, ALTA(R) immunized mice showed enhanced antigen-specific antibody production and IFN-{gamma} secreting T cell responses using lower adjuvant doses of aluminum and CpG. Overall, ALTA(R) formulated protein and polysaccharide antigens elicited strong humoral and cell-mediated immunity suggesting potential broad applicability of the platform to vaccines against various diseases, including against cancer and infections from intracellular pathogens.

Cryptococcus neoformans is a fungus classified by the World Health Organization as a critically important pathogen, posing a significant threat to immunocompromised individuals. In this study, we present the chemical synthesis and evaluation of two semi-synthetic vaccine candidates targeting the capsular polysaccharide glucuronoxylomannan (GXM) of C. neoformans. These semi-synthetic glycoconjugate vaccines contain the identical synthetic decasaccharide (M2 motif) antigen. This motif is present in serotype A strains, which constitute 95% of clinical cryptococcosis cases. This synthetic oligosaccharide was conjugated to two proteins (CRM197 and Anthrax 63 kDa PA) and tested for immunogenicity in mice. The conjugates elicited a specific antibody response that bound to the M2 motif but also exhibited additional cross-reactivity towards M1 and M4 GXM motifs. Both glycoconjugates produced antibodies that bound to GXM in ELISA assays and to live fungal cells. Mice immunized with the CRM197 glycoconjugate produced opsonic antibodies and displayed trends toward increased median survival relative to mice given a mock PBS injection (18 vs 15 days, p = 0.06). While these findings indicate promise, achieving a successful vaccine demands further optimization of the glycoconjugate. It could serve as a component in a multi-valent GXM motif vaccine, enhancing both strength and breadth of immune responses.

Lipopolysaccharide O-antigen is an immunodominant target of protective antibodies. Variation in O-antigen structures limits antibody-mediated cross-protection between closely-related pathogens including Salmonella Typhimurium (STm) and S. Enteritidis (SEn). Bacterial outer membrane vesicles (OMV) are vaccine platforms presenting surface antigens in their natural conformations. To assess how O-antigen lengths impact antibody responses and control of homologous or heterologous infection, mice were immunized with STm-OMV containing wild-type O-antigen unit repeats (wt-OMV), [≤]1 O-antigen unit (wzy-OMV), or no O-antigen units (wbaP-OMV) respectively and challenged with either STm or SEn. Unexpectedly, anti-STm LPS IgG and protection to STm were comparable after immunization with either wt-OMV or wzy-OMV. Anti-porin responses were elevated after immunization with wzy-OMV and wbaP-OMV. A single immunization with any OMV induced minimal cross-protection against SEn, except in blood. In contrast, boosting with O-antigen-expressing OMV enhanced control of SEn infections by >10-fold. These results suggest that i) Antibody to single or variable-length O-antigen units are comparably protective against Salmonella; ii) Antigens other than immunodominant O-antigens may be targets of cross-reactive antibodies that moderate bacterial burdens; iii) Boosting can enhance the level of cross-protection against related Salmonella serovars and iv) High tissue burdens of Salmonella can be present in the absence of detectable bacteraemia.

With the persistence of the SARS-CoV-2 pandemic and the emergence of novel variants, the development of novel vaccine formulations with enhanced immunogenicity profiles could help reduce disease burden in the future. Intranasally delivered vaccines offer a new modality to prevent SARS-CoV-2 infections through the induction of protective immune responses at the mucosal surface where viral entry occurs. Herein, we evaluated a novel protein subunit vaccine formulation containing a resistin-trimerized prefusion Spike antigen (SmT1v3) and a proteosome-based mucosal adjuvant (BDX301) formulated to enable intranasal immunization. In mice, the formulation induced robust antigen-specific IgG and IgA titers, in the blood and lungs, respectively. In addition, the formulations were highly efficacious in a hamster challenge model, reducing viral load and body weight loss. In both models, the serum antibodies had strong neutralizing activity, preventing the cellular binding of the viral Spike protein based on the ancestral reference strain, the Beta (B.1.351) and Delta (B.1.617.2) variants of concern. As such, this intranasal vaccine formulation warrants further development as a novel SARS-CoV-2 vaccine.

The gastrointestinal pathogen Clostridioides difficile, is a major burden for health systems due to high rates of recurrence. C. difficile pathogenesis is mediated by two virulence factors, toxin A (TcdA) and Toxin B (TcdB). Antibodies specific for TcdA and TcdB are correlated with protection from symptomatic recurrence, however, the role for CD4+ T cells is poorly understood in part due to the lack of tools to study the toxin-specific CD4+ T cell response. Our group recently demonstrated the antibody and CD4+ T cell response to C. difficile toxins is impaired via the glucosyltransferase activity of the toxins; however, tools do not exist to study the protective capacity and the phenotype of toxin-specific CD4+ T cells. Therefore, we developed an MHC-II tetramer to identify TcdB-specific CD4+ T cells via flow cytometry. Herein, we identified an immunodominant epitope (TcdB1961-1975) in the CROPs region of TcdB and optimized an MHC-II tetramer for use in tracking and phenotyping TcdB-specific CD4+ T cell responses following multiple different immunization strategies in mice. Utilizing the tetramer, TcdB-specific T follicular helper (Tfh) cells were detected following TcdB-CROPs mRNA-LNP vaccination validating the advantage of the tetramer. Furthermore, using a modular mRNA vector expressing the TcdB1961 peptide covalently bound to the beta chain of MHC-II (MHC-II{beta}) we were able to generate a robust population of TcdB-specific CD4+ T cells. These data outline the generation of new tools for the C. difficile field and lay the groundwork for future studies of toxin-specific CD4+ T cell responses.

The rapid development of vaccines was a critical part of the global response to the COVID-19 pandemic. SARS-CoV-2 (a Sarbecovirus and member of the Betacoronavirus genus responsible for the pandemic) virus was first detected in Wuhan, China in late 2019. Effective mRNA vaccines based on the viral Spike protein were designed from the earliest isolates and available by December of 2020. SARS-CoV-2 has continued to evolve in the human population, accruing neutralizing antibody resistance mutations that have necessitated updating the vaccine periodically to better match contemporary variants. Neutralizing antibody cross-reactivity is generally very limited among the diverse members of the betacoronavirus genus that are of clinical importance in people. Here, we present an alternative vaccine strategy based on eliciting T-cell responses targeting four highly conserved regions shared across the betacoronavirus proteomes. We hypothesized that cross-reactive responses to these regions could temper disease severity. Focusing immune responses on highly conserved epitopes could be beneficial as SARS-CoV-2 continues to evolve, or if a novel betacoronavirus should enter the human population. Vaccination with these highly conserved regions induced robust T-cell responses in mice and rhesus macaques. Vaccinated hamsters were significantly protected against weight loss and lung inflammation after challenge with the SARS-CoV-2 Omicron variant. After a SARS-CoV-2 Delta challenge in rhesus macaques, 3 out of 4 animals in the control group had infectious virus in their bronchoalveolar lavage samples, while the 4 animals in the vaccinated group did not.

Chlamydia trachomatis (Ct) is the most common bacterial sexually transmitted infection globally, and a vaccine is urgently needed to stop transmission and disease. Chlamydial Protease Activity Factor (CPAF) is an immunoprevalent and immunodominant antigen for CD4 T cells and B cells, which makes it a strong vaccine candidate. Due to the tolerogenic nature of the female genital tract (FGT) and its lack of secondary lymphoid tissue, effective induction of protective cell-mediated immunity will likely require potent and safe mucosal adjuvants. To address this need, we produced CPAF in a cell-free protein synthesis platform and adjuvanted it with the TLR9-agonist CpG1826, STING (stimulator of interferon genes) agonist cyclic-di-AMP (CDA), and/or the squalene oil-in-water nanoemulsion, AddaS03. We determined that intranasal immunization with CPAF plus CDA was well tolerated in female mice, induced CD4 T cells that produced IL-17A or IFN{gamma}, significantly reduced bacterial shedding, and shortened the duration of infection in mice intravaginally challenged with Chlamydia muridarum. These data demonstrate the potential for CDA as a mucosal adjuvant for vaccines against Chlamydia genital tract infection.