npj Vaccines

The 2022 Monkeypox virus (MPXV) outbreak renewed interest in vaccines for orthopoxviruses. Initial development efforts focused on well-established antigen targets, especially A35, B6, and M1. However, orthopoxvirus surfaces are complex, displaying many antigens across two infectious forms, mature virions (MV) and extracellular virions (EV) and targets relevant to protection remain to be comprehensively defined. We leveraged advances in orthopoxvirus protein biology and mRNA vaccine technology to compare immunity to all feasible targets. Mice were immunized with mRNAs encoding each antigen, or antigen complex, and neutralizing antibody responses were measured prior to heterologous challenge with vaccinia virus. Among MV antigens, A28 induced potent complement-mediated neutralizing antibodies, and the A17:G10 complex induced neutralizing antibodies and protected from challenge. For EV antigens, A36 induced neutralizing antibodies and protected from challenge. Our results affirm the consensus strategy focusing on key antigens while highlighting additional targets that could enhance updated MPXV mRNA vaccines. SIGNIFICANCEMonkeypox virus, a member of the Orthopoxvirus genus along with variola virus, has been associated with two recent outbreaks of mpox disease leading to a renewed focus on orthopoxvirus vaccine development. We report an agnostic screen of all monkeypox virus surface antigens where we combined recent advances in structural biology and mRNA technology to evaluate these potential new vaccine targets. We confirmed that historically prioritized antigens M1, A35 and B6 were protective but also discovered new antigens of interest including A28, the A17:G10 complex and A36 that can be the targets of protective immune responses. These findings are critical to inform next-generation vaccine designs should novel orthopoxviruses emerge as human pathogens.

Current SARS-CoV-2 vaccines provide limited breadth of protection, underscoring the need for vaccine strategies that optimize immune responses. Virosomesoffer a modular vaccine platform that enables multivalent antigen display and incorporation of adjuvants which can steer immune responses. We evaluated the immune response in BALB/c mice with virosomes displaying SARS-CoV-2 Wuhan or Delta spike antigens and coupled with various distinct adjuvants. Adjuvant selection differentially influenced both humoral and cellular immune outcomes. The TLR7/8 agonist 3M -052 induced a strong Th1-biased response, characterized by elevated IgG2a/IgG1 ratios and robust type 1 cytokine induction with suppression of Th2-associated cytokines. In contrast, the saponin QS-21 enhanced antibody functional quality, illustrated by improved virus neutralization potency and breadth. Furthermore, the combined incorporation of both 3M-052 and QS-21 induced an elevated Th1-biased response without improving neutralization capacity. In conclusion, different adjuvants added onto our virosome-basedvaccine led to distinct antibody responses and splenic T-cell profiles, reflective of differences in immune programming. This information guides the selection of adjuvants for respiratory virus vaccines.

Bundibugyo virus (BDBV), a member of the orthoebolaviruses in the Filoviridae family, causes severe hemorrhagic disease with high case-fatality rates. Currently, there are no medical countermeasures approved for human use hampering the response to the large ongoing outbreak in the Democratic Republic of the Congo and Uganda. While vesicular stomatitis virus (VSV)-based vaccines have demonstrated fast-acting prophylactic single-dose efficacy against multiple filoviruses, it has yet to be defined for VSV-BDBV. Here, we evaluated the rapid protection by a single-dose vaccination of VSV-BDBV in nonhuman primates (NHPs). Vaccination elicited rapid innate and early adaptive immune responses and conferred complete protection from clinical disease against BDBV challenge within 3 days. Vaccinated NHPs exhibited minimal clinical signs, limited systemic inflammation, and no infectious virus was isolated from the blood at any time. Protection correlated with neutralizing antibodies and Fc effector functionality of the humoral immune response. These findings establish VSV-BDBV as a fast-acting vaccine candidate suitable for outbreak response and highlight immune mechanisms underlying rapid protection.

The innate immune system is rapidly activated following antigen exposure and plays a critical role in shaping the ensuing adaptive immune responses. In this study, we performed bulk RNA sequencing and proteomic profiling in a cohort of 25 rhesus macaques following Ad26.COV2.S immunization to characterize early innate correlates of vaccine immunogenicity and protection. Our results show that innate immune activation occurred as early as day 1 post-vaccination and demonstrated an systemic enrichment of antiviral interferon pathways, interleukin signaling, and innate immune cell signatures. These early transcriptomic signatures correlated positively with humoral and cellular immune responses at 6 weeks following vaccination and correlated inversely with viral loads following SARS-CoV-2 challenge. Similar correlates of immunogenicity were observed in a cohort of 25 adult healthy participants vaccinated with Ad26.COV2.S. Taken together, these findings highlight the importance of early activation of the innate immune system for Ad26.COV2.S vaccine immunogenicity and protective efficacy. Graphical SummaryGraphical summary of the model proposed by our findings. Day 1 innate immune signatures are protective of long-term protection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/731069v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@308acaorg.highwire.dtl.DTLVardef@e7e969org.highwire.dtl.DTLVardef@18d1a18org.highwire.dtl.DTLVardef@ac4fb0_HPS_FORMAT_FIGEXP M_FIG C_FIG IMPORTANCEEarly vaccine induction of innate immunity may be critical for vaccine immunogenicity and protective efficacy. In this study, we evaluated innate immune responses following Ad26.COV2.S vaccination in both rhesus macaques and humans. Early induction of innate immune signatures on day 1 following vaccination correlated with subsequent development of adaptive immune responses. These data suggest that the immune programming that occurs immediately after vaccination dictates immunogenicity weeks or months later.

Pulmonary vaccination has been proposed as a strategy to improve protection against tuberculosis, due to the generation of immune cells that more efficiently survey and eliminate infected cells within the lung. However, uncontrolled replication and excessive inflammation associated with mucosal delivery of live-attenuated vaccines highlight the need for alternative strategies that balance efficacy and safety. Here, we describe a prime-and-pull vaccination approach in which systemic immunity is established by subcutaneous BCG vaccination, followed by the induction of local lung immunity through mucosal delivery of lipid nanoparticle-formulated multi-antigen mRNA. Proof-of-concept studies using a model antigen demonstrated the induction of polyfunctional antigen-specific T cells in the lung with minimal inflammatory cell infiltration, compared with mucosal BCG vaccination. Eight Mycobacterium tuberculosis- and BCG-derived proteins were subsequently selected to generate four multi-antigen mRNA constructs. In vivo vaccination and challenge experiments demonstrated that this prime-and-pull strategy is well tolerated and confers protective immunity against tuberculosis in a murine model. Collectively, these data support a modular mRNA-based prime-and-pull vaccination strategy as a translational approach that bridges the improved immunogenicity and efficacy of mucosal BCG vaccination with the safety profile of parenteral BCG administration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/739464v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@d5f991org.highwire.dtl.DTLVardef@1f3c4f9org.highwire.dtl.DTLVardef@108cae6org.highwire.dtl.DTLVardef@40d60f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Development of an effective HCV vaccine requires the induction of both broadly neutralizing antibodies (bnAbs) and a robust cellular response. One issue that has arisen is that HCV subunit vaccines have limited immunogenicity, thus requiring multivalent formats in order to elicit a robust anti-HCV immune response. Toward that end, nanoparticle vaccines possess the ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both arms of the immune system. Here, we used a soluble, secreted form of E1E2 (sE1E2) to assemble native E1E2 into a nanoparticle platform using a post-purification coupling assembly system. Nanoparticles were assembled by purifying sE1E2 containing a C-terminal SpyTag and an mi3-SpyCatcher fusion separately and covalently coupling the components via incubation. Free sE1E2-SpyTag was removed from nanoparticle preparations via gel filtration. The sE1E2-mi3 nanoparticles are fully competent to bind conformation-dependent bnAbs, indicating retention of a native assembly in the nanoparticle format. Electron microscopy analysis showed a clear incorporation of sE1E2 on the surface of the nanoparticle. Immunogenicity of sE1E2-mi3 nanoparticles was examined relative to sE1E2 alone and membrane-bound E1E2 (mbE1E2) following inoculation of groups of CD1 mice. Assessment of the immunogenicity of the sE1E2-mi3 nanoparticles showed that the nanoparticle assembly has a similar immunogenicity profile to that of mbE1E2 after only a prime and one boost, and overall superior to sE1E2. This proof-of-principle study sets the stage for further exploration of nanoparticles and other multivalent platforms for the development of E1E2-based vaccines. ImportanceHepatitis C virus infects approximately 50 million people, and at present no effective HCV vaccine exists. Due to the high sequence variability of HCV and the resulting difficulty in developing a vaccine that elicits a broadly neutralizing response, multiple efforts are underway to enhance the immunogenicity of HCV vaccine candidates. In this study, we incorporated native soluble, secreted E1E2 (sE1E2) into a 60-mer nanoparticle via the SpyTag-SpyCatcher system and covalent isopeptide bond attachment using the purified components. These nanoparticles are antigenically intact and elicit a neutralizing antibody response at an earlier time point in the immunization regimen than the corresponding subunit vaccine. These studies show that a well-characterized sE1E2 platform compatible with multiple genotypes can be coupled to nanoparticles for use as a vaccine candidate.

Tuberculosis (TB) has been a leading cause of death from a single infectious agent for decades. Bacille Calmette-Guerin (BCG) remains both the primary TB vaccine strategy and the oldest vaccine in circulation, with severe limitations in adult populations. Recently, live attenuated vaccine strategies, or generating safe strains of Mycobacterium tuberculosis (Mtb) through genetic engineering, have shown considerable promise. We previously developed an attenuated strain of Mtb lacking the nucleoid-associated protein Lsr2 ({Delta}lsr2) which is also phthiocerol dimycocerosates (PDIM) deficient and induces an immune response that represents an intermediate stage between the parental Mtb strain and BCG. In this study we examined the immune response of {Delta}lsr2 vaccinated mice in comparison to BCG and found a substantially stronger CD4 and CD8 T cell responses from {Delta}lsr2 vaccinated mice. Complementary, we conducted Mtb protection studies in {Delta}lsr2 and BCG vaccinated mice and guinea pigs, where we found that {Delta}lsr2 provided superior protection in both animals. This improved protection is shown with reduced bacterial burden and improved organ pathology in the lungs and spleen. Taken together, our work shows {Delta}lsr2 serves as a promising vaccine candidate for continued preclinical development.

Circular RNA (circRNA) is an emerging vaccine modality that is proposed to improve stability, reduce reactogenicity and extend antigen expression compared with linear mRNA. However, the relative contributions to vaccine performance of its covalently closed structure, its purity, and of nucleotide modifications remain poorly described. Here, we systematically dissected these parameters in vivo across two antigen systems. We identified RNA quality as a major determinant of circRNA reactogenicity, with differences in innate immune activation tracking with the presence of residual RNA species in less refined preparations. In contrast, highly purified circRNA exhibited markedly reduced reactogenicity compared with linear mRNA, independent of nucleotide modification. Despite these differences, circRNA and mRNA vaccines elicited comparable antibody titres and T cell responses, indicating that reduced innate activation does not enhance adaptive immune magnitude. Notably, incorporating N6-methyladenosine (m6A) did not affect reactogenicity or antigen expression but selectively enhanced antibody quality, increasing binding affinity and neutralisation capacity. CircRNA vaccination also altered the anatomical distribution of germinal centre responses, reducing splenic antigen-specific germinal centre B cells while preserving lymph node responses. Together, these findings show that circRNA vaccine performance is governed by RNA preparation quality and epitranscriptomic tuning rather than innate activation alone.

Rapidly deployable, single-dose vaccines that maintain durability under operational constraints remain an unmet need in outbreak preparedness. Live viral vectors such as Modified Vaccinia Ankara (MVA) offer strong safety profiles, yet their suboptimal immunogenicity often requires multidose regimens, reducing flexibility during emergency response. To address these limitations, we developed a modular vaccine platform that leverages immune checkpoint modulation to enhance immune cell priming without compromising the established safety profile of MVA. This platform, exemplified by the recombinant virus MVA-X, was engineered to express a peptide-based PD-1 antagonist (LD10) that provides localized, transient checkpoint blockade during early antigen presentation. The approach requires no external adjuvants, is compatible with lyophilization and stockpiling, and is readily adaptable to diverse antigens and pathogens. A single immunization with MVA-X produced durable protection that matched or exceeded that of a conventional two-dose MVA regimen against the prototypic orthopoxvirus vaccinia virus. Despite modest and contracting antibody titers, single-dose MVA-X vaccination conferred complete survival following both lethal and high-dose viral challenge at early (Day 55), intermediate (Day 90), and long-term (Day 150) time points. MVA-X also restricted viral replication at the primary site of infection, reduced systemic dissemination, and preserved lung architecture during peak disease. Importantly, MVA-X maintained efficacy in the highly susceptible CAST/EiJ mouse model following challenge with highly pathogenic Clade I monkeypox virus (MPXV). Together, these findings demonstrate that vaccine-intrinsic checkpoint modulation provides a modular strategy for enhancing the potency and durability of attenuated viral vectors while preserving their favorable safety profile, supporting broader application to emerging infectious diseases beyond Mpox.

Vaccination is one of the most effective public health interventions. However, vaccine efficacy varies widely among individuals, as immunity arises from complex interplay between genetic, pathogen, and immunological factors. To date, most systems vaccinology studies have remained pathogen-specific, precluding the discovery of potential shared immune architectures underlying durable antibody responses. To address this gap, we leveraged transcriptomic data from 1,032 participants receiving influenza, hepatitis B, or yellow fever vaccines to develop an interpretable machine learning framework for comparative analysis across diverse vaccine platforms. Pathogen-specific models using Blood Transcriptional Module-based feature aggregation accurately predicted high antibody responders and consistently outperformed gene-level models. Distinct predictive immune architectures identified across vaccines were further resolved for dominant hierarchical immune programs using surrogate decision trees. This approach identified the dominant decision boundaries underlying each vaccine model, highlighting leukocyte migration and Th2 differentiation in Hepatitis B, CD4+ T cells, M2 macrophages, and c-MYC signaling in Influenza, and B-cell receptor signaling with B-cell developmental pathways in Yellow Fever. Cross-pathogen concordance analyses further identified four shared transcriptional modules, suggesting partially conserved immune architectures across diverse vaccines. Together, these findings provide new insights into the immune mechanistic underpinnings of durable vaccine responses across vaccines and provide an interpretable framework for comparative systems vaccinology that may guide the rational design of next-generation vaccines. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/737303v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1b5be56org.highwire.dtl.DTLVardef@e323dborg.highwire.dtl.DTLVardef@470eb4org.highwire.dtl.DTLVardef@116759f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Vaccination remains the most successful preventative measure against viral infection, but methods to stably deter rapidly-evolving pathogens have remained elusive. Vaccines capable of incorporating and anticipating viral evolution could address current challenges in seasonal vaccination efforts against SARS-CoV-2 and influenza where economic and disease burdens remain high despite decades of combined study. Rare epitope suppression (RES) is an underutilized concept within vaccine design, where humoral epitope targeting can be molded using complex antigen pools. Based in mRNA vaccine technology, 'wobble vaccines' represent the novel application of RES to human pathogens designed to anticipate and resist viral evolution. To establish this platform, public SARS-CoV-2 sequencing data was compiled from the first two years of the COVID-19 pandemic to identify high-diversity sites across the receptor binding domain (RBD) of the spike protein. Wobble RBD (WobbRBD) libraries reflecting that entropy were synthesized and incorporated into established self-amplifying (SA) vaccine constructs. Animals immunized with these complex antigen pools showed no obvious adverse effects. By three days-post vaccination, WobbRBD stimulated robust primary immune activation with distinctive characteristics compared to traditional single-strain vaccine modalities. By day 14, germinal centers, class switching, and antibody-secreting cells were induced, creating potent SARS-CoV-2 spike-binding IgG antibodies. Despite similar overall activation profiles, WobbRBD generated significantly increased breadth against SARS-CoV-2 variant spikes in comparison to single-strain controls -- even against future-emerging strains. Taken together, wobble vaccines represent a novel method for anticipating and preventing viral escape with promising applications in SARS-CoV-2, influenza, HIV, and beyond.

Current vaccines for respiratory viruses are primarily administered intramuscularly. Messenger RNA-lipid nanoparticle (LNP)-based intramuscular vaccination for respiratory coronaviruses induces strong systemic IgG antibody responses; they do not consistently elicit IgA in the upper and lower respiratory tracts. Using a synthetic consensus spike protein aimed to broadening immunity against SARS-like viruses, SarbConS, coupled to ferritin nanoparticles co-delivered with mastoparan-7 and an FDA-approved CpG adjuvant, we intranasally boost SARS-CoV-2-immune animals. This intranasal boosting strategy elicits durable mucosal IgA responses in the respiratory tract, along with robust systemic IgG responses, and demonstrates durable protection against SARS-CoV-2 and zoonotic SARS-like viruses from bats and pangolins. Intranasal delivery of mastoparan-7 and CpG with MERS-CoV spike protein similarly elicits MERS-CoV-specific mucosal IgA and protects against MERS-CoV challenge in mice. Moreover, we observe durable protection against these genetically divergent zoonotic SARS-like viral challenges compared to intramuscular mRNA-LNP or unadjuvanted intranasal spike boosters. Intranasal SarbConS-ferritin nanoparticle intranasal vaccination similarly elicited durable mucosal IgA and antigen-specific memory B cell responses in the airways. The protective efficacy of M7-CpG adjuvanted SarbConS ferritin nanoparticle intranasal boosters was abolished in IgA knockout mice, suggesting a requirement for IgA in mediating respiratory mucosal vaccine-mediated protection against coronavirus infection. Altogether, our results demonstrate that respiratory mucosal vaccination can elicit durable and cross-protective mucosal IgA responses against genetically diverse zoonotic coronaviruses with implications for improved mucosal vaccines for highly transmissible respiratory viral pathogens.

Background: Vaccination of people with HIV (PWH) on suppressive antiretroviral therapy (ART) represents a novel approach for evaluating candidate broadly neutralizing antibody (bnAb) immunogens for preventive and therapeutic HIV vaccines. Given pre-existing immunity in PWH, the safety of this approach requires careful assessment prior to broader application. Here, we report on the design and safety of the RENEW-SHCS study which evaluates the immunization of PWH with BG505 SOSIP.v4.1-GT1.1, an immunogen engineered to induce precursors of CD4 binding site (CD4bs)- and V2-apex targeting bnAbs. Methods. RENEW-SHCS is a phase I, open-label, non-randomized vaccination trial evaluating a single dose of the recombinant germline-targeting envelope trimer BG505 SOSIP.v4.1-GT1.1 (GT1.1), adjuvanted with 3M052-AF and Aluminum hydroxide (alum), in PWH on suppressive ART enrolled from the Swiss HIV Cohort Study. Participants were previously classified as bnAb or non-neutralizing antibody (nnAb) inducers, with a target enrollment of 15 per group, and were monitored for safety and immunogenicity for 24 weeks while continuing standard ART. Due to an out-of-specification stability measurement of adjuvant 3M052-AF the trial was paused after 23 immunizations and subjected to an unscheduled interim safety and reactogenicity assessment comprising protocol defined outcome measures (adverse events, clinical laboratory measurements and HIV-1 viral load). Results. Twenty-three participants (10 bnAb and 13 nnAb inducers, median age 59 years, 17 male / 6 female) were vaccinated between March and August 2025 before interruption of the trial. All participants completed follow-up with full protocol adherence. The interim-safety analysis confirmed that no vaccine-related serious adverse events occurred. Solicited local (96%) and systemic (83%) reactions were common, predominantly grade 1-2, transient, and self-limited. Transient laboratory changes occurred but mostly remained within the normal range, with no vaccine-related grade 3 abnormalities. We observed predominantly transient local and systemic reactions, which were similar or milder to the reactogenicity profile reported for immunization of adult people without HIV (PWOH) with GT1.1 adjuvanted with AS01b reported in the IAVI C101 trial. No viral rebound under ART occurred. One participant experienced two viral blips (>50 HIV-1 RNA copies/ml), one before and one 16 weeks after vaccination with subsequent re-suppression. All others maintained viral suppression (<50 copies/ml) throughout follow-up. Conclusion. RENEW-SHCS demonstrated a favorable safety and reactogenicity profile of single dose immunization with GT1.1 in PWH, comparable to that observed in PWOH. The findings of this phase I study support the feasibility of vaccinating ART-treated PWH in trials of preventive and therapeutic HIV vaccine strategies.

The development of effective mucosal vaccination strategies against human immunodeficiency virus type 1 (HIV-1) remains a major challenge. This study investigated whether oral administration of hyperimmune anti-HIV-1 gp120 immunoglobulin Y (IgY) could induce mucosal and systemic immune responses in outbred felines through an anti-idiotypic network mechanism. A controlled immunization study involving 42 cats (18 immunized and 24 controls) was conducted to evaluate mucosal anti-gp120 IgA responses. In addition, a proof-of-concept cohort was used to investigate anti-idiotypic antibody (Ab-3) induction, competitive inhibition, and HIV-1 neutralization. Anti-gp120 IgA antibodies were detected in saliva from immunized animals but were absent or present at low levels in controls, indicating activation of mucosal immunity. All immunized cats developed detectable Ab-3 responses against HIV-1 gp120. Competitive inhibition assays demonstrated specific in hibition of gp120-related interactions, supporting the presence of biologically relevant anti-idiotypic antibodies. Furthermore, sera from immunized animals significantly reduced HIV-1 infectivity in a TZM-bl luciferase-based neutralization assay, with viral inhibition exceeding 60% at selected dilutions. Collectively, these findings demonstrate that oral administration of hyperimmune anti-gp120 IgY can induce mucosal IgA responses, systemic anti-idiotypic antibodies, and functional HIV-1 neutralizing activity. This preclinical proof-of-concept study supports further investigation of IgY-based oral immunization as a potential platform for HIV vaccine development. However, the Ab3, competitive inhibition assay using Ab3, and HIV-1 neutralization studies should be regarded as exploratory proof-of-concept investigations designed to establish biological plausibility rather than definitive efficacy.

Influenza virus infects the respiratory mucosa, highlighting the importance of mucosal immunity for early protection and transmission control. Here, we evaluated whether intranasal (IN) vaccination with recombinant trimeric hemagglutinin protein from A/Michigan/45/2015 (triHA) formulated with a combined mucosal adjuvant, nanoemulsion plus IVT, an RNA-based RIG-I agonist (NE/IVT), could protect guinea pigs against heterologous A/Netherlands/602/2009 challenge and reduce viral transmission. To compare mucosal and parenteral immunization, IN triHA/NE/IVT was benchmarked against IN triHA alone, IM triHA/AddaVax (IM triHA/Advx), and standard IM quadrivalent inactivated influenza vaccine (QIV). We also tested whether IN triHA/NE/IVT could boost IM QIV- primed immunity and included animals previously infected with A/Michigan/45/2015 to model pre-existing infection- induced immunity. Transmission was assessed by co-housing naive sentinels with vaccinated, challenged donors. IN triHA/NE/IVT induced systemic humoral responses comparable to IM triHA/Advx while generating superior nasal mucosal IgA responses. Unexpectedly, IM triHA/AddaVax also induced detectable, albeit lower, mucosal IgG and IgA, contrasting with prior mouse data and highlighting species-specific differences. IN triHA/NE/IVT boosting after IM QIV enhanced serum IgG and mucosal IgA compared with QIV prime-boost alone and increased cross-neutralizing activity against antigenically distinct A/Victoria/4897/2022. Both IN triHA/NE/IVT and IN Michigan/15 prior- infection prevented detectable viral shedding after challenge, and naive sentinels co-housed with IN triHA/NE/IVT- vaccinated donors remained seronegative. Together, these findings support NE/IVT as a potential mucosal platform capable of inducing robust systemic and mucosal immunity and boosting IM vaccine-primed responses.

RTS,S/AS01, an adjuvanted subunit malaria vaccine, induces protective but short-lived anti- Plasmodium falciparum circumsporozoite protein (CSP) antibody titers. To better understand the lack of sustained protection post-vaccination, we analyzed CSP-specific B cells over time in malaria-naive individuals following immunization with RTS,S/AS01. Longitudinal analyses of the cellular responses revealed a shift in the specificity of CSP-specific B cells over time. Early post- vaccine responses were dominated by plamsablasts and class-switched memory B cells (MBCs) specific for the NANP-repeat region of CSP, epitopes associated with protective antibodies. However, the frequency of NANP-repeat-specific MBCs declines, while longer-term memory specific for the C-terminus of CSP persists. Despite being class-switched and affinity matured, C- term monoclonal antibodies derived from these MBCs failed to protect mice against a transgenic parasite challenge. Taken together, these findings suggest that RTS,S/AS01 induces a transient, protective NANP repeat-specific B cell response that is subsequently replaced by memory B cells with non-protective reactivities, potentially explaining its limited long-term efficacy and limited response to subsequent challenges or boosters.

The diversity within H1 and H3 subtype influenza A viruses (IAV) in swine prevents effective vaccine control approaches with inactivated whole-virus vaccines. We addressed the challenge of controlling co-circulating hemagglutinin (HA) clades of swine IAV with the development of a multivalent mRNA-lipid nanoparticle (LNP) vaccine expressing 8 HA proteins to maximize genetic coverage. We applied a computational approach to select eight HA genes that represented 95% of the observed IAV detected in the United States between 2022 and 2025. Piglets were vaccinated and boosted intramuscularly with either individual HA mRNA-LNP or an 8-HA multivalent mRNA-LNP. Serum was collected to evaluate systemic antibody levels. Twenty-one days post-boost, pigs were challenged with a field relevant H1 1A.3.3.3-c3 IAV strain. The 8-HA multivalent mRNA-LNP vaccine induced neutralizing antibodies against all eight antigens and vaccinees were protected against lung lesions, with lesion scores similar to non-challenged animals. Homologous monovalent vaccination significantly reduced IAV detection in nasal secretions and in the lungs. Heterologous monovalent vaccination was not cross-protective but did not induce vaccine-associated enhanced respiratory disease. We provide evidence that monovalent and multivalent mRNA-LNP influenza vaccines elicited neutralizing antibody responses in pigs and protected against viral challenge. The versatility and capacity for rapidly updating the mRNA-LNP vaccine platform make it an appealing tool to improve animal health and minimize the circulation and diversity of IAV in swine. ImportanceInfluenza A virus is an important respiratory pathogen in swine, and zoonotic transmission of swine strains to humans remains a public health risk. Control strategies against IAV in swine herds rely heavily on biosecurity measures and vaccination. However, the antigenic diversity of IAV circulating in swine challenges current vaccination programs, and there is a need for broadly protective vaccines or platforms that can rapidly update components to reflect circulating diversity. mRNA-LNP vaccines have emerged as promising vaccine platforms, offering simultaneous delivery of multiple antigens, rapid development, scalable manufacturing, and potent immunogenicity. In this study, we assessed the immunogenicity and protective capacity of monovalent and multivalent mRNA-LNP vaccines encoding eight representative IAV HA antigens. To our knowledge, this is the first study to objectively select multiple representative endemic swine IAV strains by quantifying genetic diversity within the phylogeny and to apply this selection to rationally design and evaluate a multivalent HA mRNA-based influenza vaccine in the swine model.

Mycobacterium tuberculosis (Mtb) infection kills more people worldwide than any other pathogen. While the Bacille Calmette-Guerin (BCG) vaccine for Mtb has been widely used for over a century, it provides insufficient protection to eradicate this disease. One of our labs has recently established that a protein antigen (H1) can be combined with a STING pathway agonist to achieve strong protection against Mtb in mice, with performance that exceeds that of the BCG vaccine. However, its reliance on a synthetic cyclic dinucleotide (CDN) with relatively poor cell uptake requires higher dosing levels, thus increasing costs. To increase the efficiency of this vaccine and provide a delivery strategy that could also be used in humans, the H1 Mtb antigen and CDN adjuvant were conjugated to genome-free MS2 viral capsids that included cationic mutations to increase cell uptake. Specifically, the H1 antigen was conjugated to the external surface of MS2 using a tyrosinase-mediated oxidative coupling reaction, and the native STING agonist cGAMP was coupled to internal cysteine residues through a reductively cleavable disulfide linker. The resulting MS2-H1 and MS2-cGAMP conjugates were then co-delivered for three doses of vaccination in mice before exposure to Mtb. The MS2-based vaccine platform was observed to have comparable efficacy to the original H1/CDN formulation, but its enhanced uptake properties enabled 57-fold less CDN and 3-fold less H1 antigen. Additionally, this vaccine elicited immune responses that have been previously demonstrated to correlate with protection. The ability of the capsid shells to protect the CDN cargo during transport allowed enzymatically produced, and thus readily accessible, cGAMP to be used instead of more costly CDNs that require many synthetic steps. This, combined with the reduced overall amount of CDN and H1 that was required, could lower the production costs of future vaccines substantially. Finally, the ability of the capsid-based carriers to bypass the membrane transporters for CDNs suggests that this enhanced vaccination platform is likely to exhibit improved human efficacy in future studies.

Introduction: Anaemia and micronutrient deficiencies are common in low- and middle-income countries, where vaccine-induced immune responses are often suboptimal. However, whether pre-vaccination nutritional status influences pneumococcal vaccine immunogenicity in young children remains poorly characterised. Methods: We examined associations between pneumococcal vaccine responses in 670 Kenyan children enrolled in three vaccine trials: PRISM (PCV10; n=195; NCT01028326), FPCV (fractional- and full-dose PCV10/PCV13; n=306; NCT03489018), and PATH-wSP (whole-cell pneumococcal vaccine; n=169; NCT02543892) and baseline anaemia (FPCV and PATH-wSP) and micronutrient status (iron, folate, zinc, and vitamins A, B12, D, and E). Analyses were performed separately for each trial. Primary outcomes were post-vaccination serotype- or antigen-specific IgG concentrations, opsonophagocytic activity (OPA) titres, and composite IgG or OPA z scores. Results: Vitamin B12 and haemoglobin concentrations were positively associated with composite and serotype- or antigen-specific antibody responses in analyses controlling for age, sex, malnutrition and inflammation. In the PRISM trial, PCV10-induced IgG (serotypes 1 and 6B) and OPA (serotypes 1, 4, 14, and 23F) responses were positively associated with vitamin B12 concentrations. Moderate anaemia was associated with lower IgG responses to serotypes 9V and 14 following full-dose PCV13 vaccination (FPCV) and lower antigen-specific IgG responses following the 1 mg PATH-wSP vaccine. No consistent associations were observed for ferritin, folate, zinc, or vitamins A, D, and E. Conclusion: Vitamin B12 deficiency and anaemia were associated with reduced pneumococcal vaccine responses in young Kenyan children. Optimising nutritional status before vaccination could be a strategy to improve vaccine responses in populations where anaemia and micronutrient deficiencies are common.

The rapid evolution of avian influenza A/H5N1, including the recent U.S. clade 2.3.4.4b outbreak, highlights its pandemic potential and the urgent need for durable, broadly protective vaccines. Given the capacity of CD8+ T cells to mediate cross-strain immunity, we investigated whether geographically distinct HLA-A33 allotypes, HLA-A*33:01 in East/Southeast Asia and HLA-A*33:03 in South Asia, differentially shape the influenza immunopeptidome and influence antiviral immunity. Antigen-presenting cells overexpressing HLA-A*33:01 or HLA-A*33:03 were transfected with single A/H5N1 antigens or infected with A/X-31 (H3N2) as a control comparison representing current seasonal influenza virus. We identified novel ligands restricted to HLA-A*33:01 (57 from A/H5N1; 55 from A/X-31) and HLA-A*33:03 (29 from A/H5N1; 45 from A/X-31). Although fewer peptides were recovered for HLA-A*33:03, a larger proportion of A/X-31-derived peptides were predicted as high-affinity binders (74%) compared with HLA-A*33:01 (61%), indicating qualitative differences in antigen presentation. To determine immunogenicity, peripheral blood lymphocytes from HLA-A*33:03-positive, A/H5N1-naive donors were stimulated with four conserved peptides: PB2GTF, PB2KTY, NPSVQ and PB1MTK. All elicited robust CD8 T cell activation despite the absence of prior A/H5N1 exposure, demonstrating cross-recognition by memory T cells primed against seasonal influenza. These findings define HLA-A33-restricted influenza epitopes and reveal allotype-specific presentation features that shape CD8+ T cell immunity. Conserved, immunogenic peptides identified here represent promising candidates for rational design of broadly cross-reactive vaccines to protect HLA-A33-expressing populations against severe A/H5N1 disease. Data are available via ProteomeXchange with identifier PXD078870. Author SummaryAvian influenza A/H5N1 continues to pose a significant pandemic threat because of its ability to infect humans and its potential to acquire sustained human-to-human transmissibility. While current influenza vaccines primarily target rapidly evolving viral surface proteins, CD8+ T cells can recognize more conserved internal viral proteins and may provide broader protection against diverse influenza strains. In this study, we investigated how two common HLA-A33 variants, which are prevalent in South, East, and Southeast Asian populations, present influenza-derived peptides to CD8+ T cells. We identified novel influenza peptides presented by HLA-A*33:01 and HLA-A*33:03. Importantly, several conserved A/H5N1-derived peptides were recognized by memory CD8+ T cells from healthy individuals with no prior exposure to A/H5N1, suggesting that previous infection with seasonal influenza viruses can generate cross-reactive immune responses. Our findings expand the current repository of influenza T cell targets and provide new insights into antiviral immunity in HLA-A33-expressing populations. The conserved and immunogenic peptides identified in this study may help guide the development of broadly protective influenza vaccines and contribute to future pandemic preparedness efforts.