npj Vaccines

The World Health Organization has called for malaria vaccines with > 90% efficacy against Plasmodium falciparum (Pf) infection. The live, non-attenuated PfSPZ-CVac (chloroquine) vaccine provides the strongest protection against controlled human malaria infection (CHMI). Its 100% protective efficacy at 12 weeks against genetically distant Pf stems from a highly amplified immune stimulus. After inoculation, each invading sporozoite (SPZ) replicates up to 50,000 times within a hepatocyte. Over six days of development, these parasites express thousands of distinct proteins, presenting a vast and diverse array of antigens to the host immune system. The late liver stage parasites then rupture into the bloodstream, where they are killed by the co-administered drug, chloroquine. To engineer a Pf parasite that develops to the late liver stage like PfSPZ-CVac (chloroquine), but then arrests before transitioning to the blood stage, two genes required for transition from non-pathogenic liver to pathogenic blood stages were identified and deleted, creating late-liver stage arresting, replication-competent (LARC) PfSPZ. In a first-in-human, randomized, double-blind, placebo-controlled clinical trial, we assessed PfSPZ-LARC2 Vaccine in 75 malaria-exposed Burkinabe 1- to 48-year-olds. No vaccine-strain breakthrough blood-stage infections were detected; safety and tolerability profiles were similar in vaccinees and placebo recipients; and the highest immunogenicity was observed in the youngest recipients. Based on these findings, PfSPZ-LARC2 Vaccine has progressed to Phase 2 testing with the goal of rapidly advancing this genetically attenuated whole parasite vaccine to licensure and deployment.

RTS,S/AS01 (RTS,S) is one of two WHO-recommended malaria vaccines for children in moderate-to-high transmission settings. Yet, no validated immune correlate of protection (CoP) has been established for this vaccine. We selected 16 biomarkers to further assess as candidate CoPs, based largely on their ability to well-predict post-challenge outcomes in a previous integrative correlates analysis of two phase 2a controlled human malaria infection (CHMI) studies, Malaria-068 and Malaria-071. We evaluated the predictive power of these 16 biomarkers in two additional phase 2a CHMI studies, Malaria-092 and Malaria-102, to confirm these candidate CoPs in new malaria vaccine trials. In MAL092, malaria-naive volunteers were challenged three months after a series of two or three doses of RTS,S/AS01, and in MAL102, a subset were boosted nine months later and re-challenged after three weeks. The 16 biomarkers included Plasmodium falciparum circumsporozoite protein (CSP)-specific IgG antibodies targeting the CSP NANP-repeat region (CSP IgG ELISA), NANP repeat-specific antibody binding to human Fc gamma receptors (FcgRs), CSP-specific antibody Fc effector functions, total NANP6-specific serum Ig measured by biolayer interferometry (BLI), and NANP6-specific IgG1 antibodies measured by binding antibody multiplex assay. Immunogenicity analyses confirmed that the vaccine regimens elicited robust immune responses, including multiple biomarkers not previously reported on, in both trials. In MAL092, participants clustered into two distinct immunogenicity profiles that overlapped with malaria protection status. In univariate logistic regression analyses, most biomarkers were significantly associated with protection when measured on the day of challenge in both trials. Notably, the same three biomarkers ranked as top correlates in both trials (FCGR2AH NANP6, FCGR2AR NANP6, and FCGR2B NANP6; all adjusted p-values <0.001), with similar effect sizes across studies. Despite these strong associations, no biomarker demonstrated significantly better individual-level predictive performance than CSP IgG ELISA. In MAL092, CSP IgG ELISA achieved an area under the curve (AUC) [95% confidence interval (CI)] of 0.74 (0.63, 0.84), compared with AUCs of 0.76 - 0.78 for the top five biomarkers, all of which reflected NANP6-specific Fc{gamma}R binding (all adjusted p-values > 0.37 versus CSP IgG ELISA). Predictive models trained on data from two prior CHMI studies and applied to MAL092 and MAL102 identified a model based on FCGR2B NANP6 (validation AUC = 0.77 in MAL092; 0.80 in MAL102) as the best-performing univariate model for both studies. The best-performing multivariate predictive model for MAL092 was based on BLI NANP6 Dissociation AUC and ADCP NANP6 (validation AUC = 0.76), and in MAL102, it was based on CSP IgG ELISA, BLI NANP6 Dissociation AUC, and ADCP NANP6 (validation AUC = 0.82). The next approximately 10 highest-ranked models, however, had comparable performance with overlapping 95% CIs, and none were clearly superior to CSP IgG ELISA (validation AUCs 0.74 and 0.71 in MAL092 and MAL102, respectively). Collectively, these findings suggest that the majority of the previously identified candidate CoPs also hold up well in two additional malaria vaccine CHMI trials and underscore the continued utility of the CSP IgG ELISA readout as a robust marker of protection. Further studies are needed to determine whether these candidate CoPs hold in malaria-endemic field settings. Identifying validated CoPs that predict malaria vaccine efficacy would promote the development and approval of more effective and durable next-generation vaccines. These advances could also inform vaccine strategies for other infectious diseases.

Newborns and infants are particularly vulnerable to influenza, largely due to the immaturity of their immune systems. As such, maternal immunization is a valuable strategy to enhance influenza-neutralizing antibodies in both blood and breast milk, ensuring optimal passive immunity transfer to the infant. In our pregnant and lactating ferret maternal-neonatal model, we evaluated antibody responses to an adenovirus type 5-vectored H1N1 hemagglutinin vaccine delivered enterically, intranasally, or intramuscularly. While all three routes elicited a robust antibody response compared to saline-treated controls, intranasal vaccination significantly increased influenza-specific antibodies in dam serum, upper respiratory tract secretions, and milk, and subsequent transfer to suckling kits. Our results suggest that intranasal immunization was the most effective route for inducing functional influenza-specific antibodies in both dam serum and milk, with efficient passive transfer to suckling kits. Leveraging mucosal immune induction may enhance maternal antibody responses, transfer to offspring, and downstream neonatal protection.

SARS-CoV-2 antigenic evolution continues to challenge strain-matched vaccination. For the 2026-27 season, the European Medicines Agency (EMA) identified XFG as the preferred antigen within the JN.1 family and Comirnaty XFG was authorised in the European Union in July, 2026. We studied 22 participants who received 30 microg of mRNA encoding XFG (Comirnaty XFG, BioNTech/Pfizer, Mainz, Germany) in September, 2026 and had paired serum available before vaccination and median 14 days afterwards. Median Wuhan-Hu-1 S1-specific IgG increased from 1600 BAU/mL (IQR 1118-2926) to 2914 BAU/mL (2117-4468), a 1.8-fold increase. Median RBD-binding ED50 increased from 116 (51.4-212) to 305.6 (170.8-434.6) for LP.8.1 and from 354.3 (129.2-558.2) to 832.9 (429-1175) for NB.1.8.1, corresponding to 2.6-fold and 2.4-fold increases, respectively (all p<0.0001). XFG-adapted vaccination increased neutralising activity against each tested variant. The GMT against XFG increased from 180 before vaccination to 894 afterwards (5.0-fold), and that against RV.1.3 increased from 121 to 599 (4.9-fold). GMTs increased from 117 to 386 for PQ.16.1.1 (3.3-fold), from 101 to 316 for XFG.1.1 (3.1-fold), from 629 to 1772 for LP.8.1 (2.8-fold), from 221 to 611 for NB.1.8.1 (2.8-fold), and from 228 to 642 for PJ.2.1 (2.8-fold). Neutralisation of BA.3.2.2 increased more modestly, from GMT 78 to 126 (1.6-fold; p=0.0009). Pre- versus post-vaccination neutralisation titres differed significantly for all variants (p<0.001). Despite the broad increase, post-vaccination neutralisation remained heterogeneous. Relative to LP.8.1, GMTs were 2.0-fold lower for XFG, 2.8-fold lower for PJ.2.1, 2.9-fold lower for NB.1.8.1, 3.0-fold lower for RV.1.3, 4.6-fold lower for PQ.16.1.1, 5.6-fold lower for XFG.1.1, and 14.1-fold lower for BA.3.2.2. These findings provide early human immunogenicity data after XFG-adapted mRNA vaccination. The largest relative increases were observed for XFG and RV.1.3, whereas LP.8.1 remained the most strongly neutralised variant after Comirnaty XFG vaccination and BA.3.2.2 the least. This pattern suggests broad boosting of cross-reactive humoral immunity without elimination of between-variant differences. The small, extensively pre-immunised cohort, short follow-up, absence of a contemporaneous vaccine-comparator group, and use of a pseudovirus assay limit inference. Our data characterise early antibody binding and neutralisation but do not establish durability or clinical effectiveness.

New therapeutic approaches are needed to treat infections due to multidrug resistant (MDR) Gram-negative bacteria. The use of antibody-recruiting molecules (ARMs) is a promising approach to address the MDR problem given their potential to engage both the adaptive and innate immune systems to treat bacterial infections. Here we present two new strategies to create ARMs. First, we show that a 9-mer antimicrobial peptide (K6) can be fused to a 11-mer mimotope peptide recognized by antibodies targeting the Vibrio cholerae O1 O-antigen to produce an ARM peptide (FZ5) that can render E. coli more sensitive to complement killing in the presence of antibodies that recognize this mimotope. FZ5 can drive killing by complement in the presence of rabbit antibodies raised against O1 serogroup V. cholerae as well as antibodies present in human sera elicited by immunization with the live attenuated cholera vaccine PanChol. We also describe a strategy that combines bacterial surface binding with hapten display through the use of homodimers of the antibiotic streptomycin (Str) and anti-Str antibodies elicited by Str-protein conjugate vaccines. When combined with dimeric Str derivatives and human complement, polyclonal anti-streptomycin antisera kills E. coli by targeting the Str hapten. Dimeric forms of Str also significantly enhanced killing of a Str-resistant P. aeruginosa clinical isolate dependent on low levels of human complement and polyclonal anti-Str antibody. Together these data suggest that anti-bacterial ARM molecules based on mimotope display through simple synthetic fusion peptides, or dimeric haptens have promise as new approaches for the treatment of MDR bacterial infections.

Haemophilus influenzae is a Gram-negative bacterium that causes pneumonia, otitis media, and invasive infections such as meningitis and bacteremia. A vaccine that confers protection against disease caused by H. influenzae serotype b is currently available and is highly effective, but infections caused by non-b serotypes and non-typeable strains are still prevalent and increasing, suggesting a need for a broadly cross-protective vaccine that will protect against infection by all H. influenzae strains. To address the need for broad cross-protection, this study focused on the surface lipoprotein component of the bipartite bacterial transferrin receptor, transferrin binding protein B (TbpB), which is universally present H. influenzae, irrespective of encapsulation status, and is essential for bacterial colonization and pathogenesis. To prevent vaccine escape, we assessed the sequence and structural diversity among TbpB variants derived from local and international H. influenzae isolates and observed that these sequences cluster independently of encapsulation status, suggesting that conferring protection against all H. influenzae strains regardless of capsule type or presence of a capsule using a TbpB-based vaccine is feasible. In addition, our diversity analysis also revealed that the C-terminal lobe (C-lobe) of TbpB contains several large, highly variable loops. After immunizing mice with a trivalent vaccine consisting of a representative set of TbpB variants, we found that the resulting antiserum demonstrated modest cross-reactivity against heterologous TbpB variants. However, immunizing with a loopless C-lobe (LCL) that lacks the large, highly variable loops we had identified elicited broad cross-reactivity against a panel of intact TbpB variants. This suggests that while the intact TbpB may only generate a moderately cross-reactive antibody response, a vaccine consisting of a single LCL may be able to elicit a sufficiently cross-reactive humoral response against diverse TbpB variants, and in turn, confer broad cross-protection targeting both encapsulated and non-typeable H. influenzae.

Background Killed whole-cell oral cholera vaccines require multiple doses and have reduced efficacy in young children, limiting their utility. PanChol is a single-dose, oral live-attenuated cholera vaccine engineered from a current seventh pandemic El Tor O1 Vibrio cholerae strain. PanChol was shown to be safe and immunogenic in a previous phase 1a trial. In participants from this first-in-human trial, we aimed to assess the durability of the initial PanChol dose and the safety and immunogenicity of a second dose. Methods This open-label phase 1a booster study was conducted at Brigham and Women's Hospital (Boston, MA). Eligible participants were healthy adults aged 18-55 years without a history of cholera or gastrointestinal disorders and had received a PanChol dose ranging from 105-1010 colony-forming units (CFU) within the previous 322-1111 days. All participants received a booster dose of 2x107 CFU. The primary outcome was safety, including solicited, unsolicited, and serious adverse events following the second dose of PanChol. Secondary endpoints were the magnitude of pre- and post-vaccination serum vibriocidal titers to both Inaba and Ogawa V. cholerae, including seroconversion (4-fold rise in titers over baseline), and stool shedding of PanChol organisms using quantitative and qualitative stool cultures. Comparisons of the amount of vaccine shedding in stool following primary and secondary immunization were used to assess the intestinal mucosal immunity elicited by the initial PanChol dose. All outcomes were assessed in all participants. This trial is registered with ClinicalTrials.gov, NCT05657782, and is complete. Findings Between Aug 25, 2025, and Feb 20, 2026, 20 healthy adults who previously received a single oral dose of PanChol were enrolled to receive a booster dose of 2x107 CFU. Eleven (55%) of 20 participants were male and the median age was 43{middle dot}6 (IQR 30{middle dot}2-49{middle dot}5). Ten (50%) were White and 17 (85%) were not Hispanic or Latino. Nine (45%) of 20 boost recipients reported at least one solicited adverse event; 13 (93%) of 14 events were mild, and all were transient. The most common solicited adverse event was nausea, reported by 4 (20%) participants; all cases were mild. Most unsolicited adverse events were mild; one was grade 3 and was deemed unrelated to vaccination. Two unsolicited adverse events were deemed related to vaccination (mild gassy sensation and heartburn, both of which occurred on day 1 and resolved the same day). In most participants, vibriocidal antibody titers measured on the day of boosting exceeded baseline titers prior to the initial dose (17 (85%) for Inaba and 14 (70%) for Ogawa). Fifteen (75%) of 20 boost recipients seroconverted vibriocidal antibody titers to both serotypes. After the boost, 17 (85%) participants had no or self-limited vaccine shedding, compared with 10% after the first PanChol dose. Interpretation A second oral dose of PanChol had a favorable safety profile and induced vibriocidal seroconversion to both serotypes in 75% of boost recipients. Intestinal colonization of PanChol following the boost serves as a proxy for measuring mucosal immunity targeting V. cholerae. The decrease in in the abundance of PanChol fecal shedding following the second dose, compared with the first, suggests that a single dose of PanChol is sufficient to confer durable protective immunity against a cholera challenge. These observations underscore the need for further clinical development of PanChol. Funding Wellcome Trust.

Malaria is a leading cause of childhood mortality, yet infant immune responses to vaccination remain poorly understood. Here, we characterize 88 monoclonal antibodies (mAbs) directed against the Plasmodium falciparum circumsporozoite protein cloned from African infants immunized with the RTS,S/AS01E malaria vaccine, identifying potent antiparasitic mAbs exhibiting molecular features resembling those observed in adults and minimal somatic hypermutation. Importantly, germline-reverted variants of four mAbs inducing sterile protection in vivo maintained high binding affinity and Fab-dependent parasite killing. Structural analyses revealed that a few amino acid replacements drive homotypic helical assemblies, fine-tuning parasite inhibition and protective efficacy. Thus, despite maturational constraints of early life, infants can generate potent antimalarial antibodies in a near-germline configuration, informing next-generation vaccine development for this vulnerable group.

There is currently no clinical data for the effectiveness of the only licensed Ebola virus disease vaccine (Ervebo) against Bundibugyo virus disease. A number of studies have shown immune cross-reactivity with Bundibugyo virus after Ervebo vaccination. However, antibody recognition of Bundibugyo is around 2.8-fold lower than recognition of Ebola virus. Here, we aimed to infer how a 2.8-fold drop in immune recognition may affect Ervebo protection against Bundibugyo virus disease based on analysis of data on Ervebo immunogenicity and protection from Ebola virus disease. This work has three main components. Firstly, we analysed the timing of vaccine protection in the Ervebo pivotal ring vaccination clinical trial. Secondly, we performed a systematic review and meta-analysis of antibody responses to Ebola virus after Ervebo vaccination over time. Thirdly, we combined these data with previously reported data on the cross-reactivity to Bundibugyo virus to infer protection of Ervebo against Bundibugyo virus disease. We find that the clinical trial data for Ervebo supports vaccine protection beginning earlier than 10 days after vaccination. Secondly, binding antibody levels against Ebola virus on day 28 post vaccination (peak responses) were on average 12.9-fold (95% confidence interval, CI: 6.4 - 26.0) higher than on day 7, 6.3-fold (95% CI: 3.7 -10.8) higher than on day 10, and 2.5-fold (95% CI: 1.4 - 4.4) higher than on day 14 (neutralising antibodies showed similar results). Finally, we consider the scenarios where antibody levels to Ebola virus at day 7 or 10 are putative protective thresholds against Ebola virus disease, and we assume these thresholds also apply for Bundibugyo virus disease. Then, with a 2.8-fold reduction of responses to Bundibugyo compared to Ebola virus, we predict antibody responses to Bundibugyo virus after Ervebo vaccination are likely above these putative protective thresholds.

The transcriptional and cell repertoire changes of lymph node (LN) T cells following influenza vaccination have been under-investigated across different ethnicities. Draining (dLN) and non-draining (ndLN) axillary LN cells from 17 participants with genotypically varied African or Asian ancestry (ISRCTN13657999) were collected by fine needle aspiration (FNA) before, 5 days and 42 days after adjuvanted influenza vaccination and analysed by single-cell RNA-sequencing (scRNA-seq). The early dLN transcriptional profile and cellular dynamics were distinct from the ndLN; BACH2 expressing T cells were highly induced, exhibiting stem-like and unique quiescence signatures. Proliferating T follicular helper cells (Tfh) were enriched in dLN and Th1-like Tfh predominated with highly induced transcriptional changes. The BACH2+ T cell state and Th1-like Tfh cells, transcriptionally distinct from germinal centre (GC) T cells, were co-localised with LAMP3+ dendritic cells in the T cell zone of publicly available LN datasets, a region that was also distinct from GC Tfh niches. Together, these findings indicate that the induction of a robust, proliferative, and transcriptionally diverse Tfh response, alongside a discrete subset of BACH2+ T cells, represents a marked feature of the early T cell zone immune response to adjuvanted influenza vaccine in the dLN.

The longitudinal effects of initial influenza exposure on the magnitude, persistence, and antigen specificity of antibody responses during infancy remain incompletely understood. We investigated humoral antibody responses following primary influenza infection or seasonal vaccination in a prospective US birth cohort with weekly respiratory surveillance for influenza infection. Influenza-specific serum IgG binding to H1 and H3 haemagglutinin (HA) antigens, HA inhibition (HAI), and live-virus neutralizing antibody responses were assessed longitudinally. To minimize influence of maternally derived antibodies, analyses of primary exposure responses were restricted to infants for whom influenza-specific maternal antibodies had declined below the assay detection limit before exposure. Antibody responses were compared following primary infection and vaccination and examined after subsequent annual vaccination. We found that maternal influenza-specific IgG declined rapidly during early infancy. Among infants without detectable pre-exposure influenza-specific antibodies, primary influenza infection was associated with greater post-exposure antibody responses and less measurable decline over follow-up than primary vaccination. These groups differed in age, calendar season, and exposure characteristics, limiting direct attribution of these differences to route of exposure. In infants with previous influenza infection followed by vaccination, responses after subsequent vaccination initially showed greater binding to the previously infecting subtype among the tested HA antigens. With repeated vaccination, responses became more distributed across the tested antigens. Our findings reveal differences in the magnitude, persistence, and antigen specificity of antibody responses according to the nature and sequence of influenza exposure. By prospectively capturing early-life exposures, this study provides longitudinal evidence linking exposure history to subsequent humoral antibody responses.

Group A rotavirus G1P[8] strains, which are targeted by the Rotarix vaccine, have persisted in several countries following the introduction of the Rotarix vaccine. However, the evolutionary origins of the persisting strains remain unclear. Here, we leveraged over two decades of rotavirus surveillance data from Malawi to understand the genomic epidemiology of G1P[8] strains in a high-disease-burden sub-Saharan African setting. We observed a reversion of G1P[8] strains to the conventional Wa-like genotype constellation identified pre-vaccination, following transient circulation of DS-1-like G1P[8] strains. Most G1P[8] lineages that re-emerged post-vaccination circulated before vaccine introduction. Furthermore, new strains emerged post-vaccination through re-assortment of pre-existing viruses. The predominant post-vaccination lineage (MW-VP7-L2) contained an N147D substitution at an antigenically important site of the VP7 glycoprotein and showed evidence of episodic diversifying selection. Children infected with the MW-VP7-L2 lineage had a higher severity score of gastroenteritis than infections with other co-circulating lineages. These findings highlight the importance of long-term genomic surveillance to understand rotavirus transmission, evolution, and disease severity, and to guide public health interventions.

Herpes zoster (HZ), caused by varicella zoster virus (VZV) reactivation, is common in older adults with declining cell-mediated immune protection. A modestly efficacious live attenuated (ZVL) and a highly efficacious recombinant adjuvanted (RZV) zoster vaccine protect against HZ. To uncover the mechanism(s) associated with the superior efficacy of RZV, we developed a controlled human infection model consisting of intradermal administration of attenuated vOka VZV to older adults previously vaccinated with RZV or ZVL. Infiltration of VZV-specific CD4+ and CD8+ T cell clonotypes at the infection site was quicker and higher in RZV than ZVL recipients [&ge;]5 years post-immunization due to the persistence of RZV-induced gE-reactive CD4+ and CD8+ T cell clonotypes in blood. High CD4+ cytotoxic T lymphocyte frequencies in blood on Day 1 post-inoculation predicted control of VZV replication. Persistence of circulating RZV-induced CD4+ and CD8+ CTL and rapid migration to sites of VZV replication are potential mechanisms underlying durable protection against HZ.

Phenuiviruses which include Rift Valley Fever Virus (RVFV), classified by WHO as one of the top priority pathogens with outbreak potential, are emerging arthropod-borne viruses that pose an increasing threat to public health. In line with the prototype pathogen framework advanced by the NIAID, the sandfly-transmitted Toscana virus (TOSV) has been adopted as a prototype to advance understanding of relevant phenuiviruses and inform the development of broadly applicable medical countermeasures. In this study, we sought to isolate and characterize monoclonal antibodies (mAbs) elicited by natural TOSV infection to identify shared antigenic determinants. By employing the conserved envelope Gc glycoprotein as bait for antigen-specific B cell sorting, we recovered more than 1,450 memory B cells and identified 112 Gc-specific neutralizing mAbs. Over one-third of these mAbs exhibited cross-reactivity with RVFV, a genetically distinct phenuivirus. Selected mAbs provided prophylactic protection in a murine model of TOSV infection. Together, our findings establish Gc as an important target of protective neutralizing antibodies and provide insights that may guide the rational design of widely effective interventions, strengthening readiness against future phenuivirus outbreaks.

Protein replacement therapy is highly effective for treating many protein-deficiency disorders, but its long-term clinical benefit is often limited by anti-drug antibody responses that neutralize therapeutic proteins, compromising safety and efficacy. To address these unwanted immune responses, a lipid nanoparticle-based immunotherapy platform has been developed to induce antigen-specific tolerance. Mice treated with antigen associated with either double-chain phosphatidylserine (PS) or lysophosphatidylserine (LysoPS), a single-acyl-chain phospholipid, showed reduced antibody titers following subcutaneous pre-exposure. However, only LysoPS nanoparticles induced tolerance after oral administration, suggesting structural differences between PS and LysoPS can impact tolerogenic potential, depending on the site of delivery. As an apoptotic "eat-me" signal, PS is recognized by phagocytic receptors such as TIM-4 and is associated with LC3-associated phagocytosis. These nanoparticles have been hypothesized to promote apoptotic mimicry to regulate immunity. However, the contribution of TIM-4 to oral tolerance remains unclear. Here, we investigated the role of TIM-4 in LysoPS-mediated oral tolerance using a knockout mouse model. Our data showed that TIM-4-/- mice induced tolerance to ovalbumin encapsulated in LysoPS comparable to that of wild-type mice. We also found that LysoPS nanoparticles did not follow the same surface engagement and intracellular trafficking pathways as PS nanoparticles. These results reveal that, despite sharing the same headgroup, differences in acyl chain number critically shape nanoparticle-immune cell interactions and provide a mechanistic framework that may explain the unique ability of LysoPS nanoparticles to induce oral tolerance.

Porcine epidemic diarrhea virus (PEDV) was introduced into the United States (USA) in 2013 and has circulated ever since. PEDV causes significant losses in the swine industry, with the greatest impact observed during the first years following its introduction into the country. However, the evolutionary dynamics of the virus remain underexplored. Recently, analyses of the Spike protein subunit S1 suggested that the PEDV population currently circulating in the USA represents a fraction of its historical diversity and comprises only two clades restricted to specific geographic regions. These observations were based on a limited set of S1 sequences. Furthermore, our understanding of evolutionary dynamics at the genomic level is even more limited, as complete PEDV genomes have not been generated since 2017. Here, we characterized the evolutionary dynamics of PEDV populations in the USA using complete genomes and assessed the contribution of different evolutionary mechanisms to shaping population structure. We analyzed 338 complete genomes, of which 25 were obtained in this study, together with 623 Spike S1 sequences, of which 41 were obtained in this study. All sequences obtained in this study were assigned to the non-S-INDEL strain. No S-INDEL sequences were recovered in this study, although these viruses continue to circulate in the country. Our analyses revealed that most sequences sampled after 2023 formed a monophyletic clade divergent from sequences collected in previous years, suggesting an ongoing temporal population structuring. Population structure analyses further supported evidence of population differentiation over time. Recombination analyses indicated that sequences belonging to the contemporary clade shared a single recombination event, suggesting that this clade may have emerged through recombination. Time-scaled S1 phylogenies were consistent with previous findings, supporting the circulation of only two contemporary clades; however, one of these clades was detected for the first time outside its previously reported geographic range. Our findings document temporal genetic differentiation in US PEDV populations, consistent with continued viral evolution, and broaden the known geographic distribution of contemporary S1 clades. These results highlight the value of continued genomic surveillance to monitor changes in PEDV diversity and distribution and provide sequence data to inform vaccine research.

Influenza A virus (IAV) defective interfering particles (DIPs) are regarded as promising broad-spectrum antivirals due to their high efficacy demonstrated in animal trials. Previously, an OP7 chimera DIP was generated that showed markedly enhanced antiviral efficacy compared to conventional IAV DIPs. In addition, a high-yield cell culture-based production process for OP7 chimera DIPs was established in perfusion mode. However, due to the technical complexity of this technology, we aimed to combine the advantages of perfusion in terms of yield with that of a simple and less complex batch process. In the present work, we established a fed-batch production process for OP7 chimera DIPs that allows a more straightforward implementation into existing production facilities. Using a feeding regime that was able to overcome the cell density effect, we achieved productive virus infection in stirred tank reactors until a cell density of 10E+6 cells/mL. Furthermore, we obtained a high virus titer of 3.8 log10(HAU/100 uL), corresponding to a 3.4-fold increase compared to a reference batch production. Moreover, the produced material demonstrated a significantly increased interfering bioactivity shown by an in vitro potency assay. Interestingly, this efficacy was similarly high as compared to OP7 chimera DIP material previously produced in perfusion mode, despite twofold lower virus titers. Finally, we demonstrate robustness of OP7 chimera DIP seed virus trains, which is significant step towards later pharmaceutical production. Further, corresponding GMP MDCK-PB2(sus) cell and OP7 chimera virus banks are now available for later biomanufacturing further use. In conclusion, the established fed-batch process offers a valid n additional option for pharmaceutical grade OP7 chimera DIP GMP manufacturing production under GMP conditions required for subsequent clinical testing and for later large-scale manufacturing.

Bundibugyo ebolavirus (BDBV) is causing an ongoing outbreak in the Democratic Republic of Congo (DRC) with no licensed vaccine available. Recent reports have shown the presence of BDBV-cross-reactive antibody responses in humans vaccinated against the related Ebola virus (EBOV). Here, we used an IFN-{gamma} ELISpot assay to assess T cell responses against the EBOV, BDBV and SUDV glycoproteins in clinical trial participants vaccinated with the licensed VSV-EBOV vaccine. We could show that BDBV-cross-reactive T cell responses are detectable in the majority of individuals and that conserved regions within the glycoprotein are targeted. In the absence of a BDBV-specific vaccine, these data support efficacy trials of the VSV-EBOV vaccine during the current outbreak.

Background and Aims: Genetic diversity within the hepatitis B virus (HBV) surface antigen (HBsAg) drives the emergence of immune escape variants, but studying this diversity in vivo has been limited by the difficulty of establishing infection from patient isolates. We aimed to perform unbiased in vivo screening of variants in the HBs antigenic loop for antibody escape and fitness, using synthetic DNA launch of HBV in human hepatocyte chimeric mice. Approach and Results: Direct intrahepatic injection (IHI) of barcoded, recombinant cccDNA established infection from up to 32,000 independent launch events of a genotype A strain and successfully launched clinical consensus sequences across the major genotypes. Using IHI, we performed deep mutational scanning of 4,608 codon variants across the HBs antigenic loop. Under control antibody treatment, diverse variants expanded, revealing differences in variant fitness in the absence of adaptive immune pressure. Anti-HBs monoclonal antibody treatment suppressed wild-type viremia while permitting breakthrough of resistant variants from the library. Sequencing identified strong enrichment for substitutions at G145 (G145Q, G145H, G145R). Notably, G145Q, rather than the clinically dominant G145R, showed the greatest fitness advantage, indicating that G145R's clinical prevalence does not reflect superior intrinsic fitness. Conclusions: Unbiased in vivo screening reveals that clinically observed HBV escape mutations reflect which substitutions are genetically accessible by single nucleotide change and compatible with the overlapping polymerase reading frame, rather than which confer optimal fitness. This approach establishes a platform for high-resolution mapping of viral variant fitness under antiviral selection pressure.

Background: Global oral cholera vaccine (OCV) supply shortages and the retirement of Shanchol necessitate absolute reliance on alternative formulations. We conducted a systematic review and meta-analysis to evaluate the immunogenicity and safety of Euvichol variants compared with the historical Shanchol benchmark. Methodology/Principal Findings: A searched PubMed, Embase, MEDLINE, Scopus, and Cochrane CENTRAL up to May 2026 for randomized controlled trials (RCTs) comparing any Euvichol variant against Shanchol in healthy populations aged [&ge;]1 year. Primary outcomes were vibriocidal seroconversion rates at 14 days post-dose 2 pooled as Risk Ratios (RRs) via Mantel-Haenszel fixed-effects models. Five RCTs comprising 7,253 participants were included. Euvichol formulations demonstrated non-inferior, equivalent seroconversion rates compared with Shanchol for both Vibrio cholerae O1 Inaba (RR 1.00, 95% CI 0.98-1.02; I2=0%) and O1 Ogawa (RR 1.01, 95% CI 0.99-1.04; I2=26%). Log-transformed Geometric Mean Titers confirmed clinical equivalence for Inaba (MD 0.02, 95% CI -0.01 to 0.05) and Ogawa (MD 0.00, 95% CI -0.03 to 0.03). Overall safety was balanced between arms, showing no significant variation in the risk of any solicited adverse event (RR 0.91, 95% CI 0.75-1.11) or specific symptoms including fever (RR 0.84, 95% CI 0.52-1.35). Serious adverse events were rare and uniformly unrelated to vaccination. Conclusions/Significance: Euvichol variants demonstrate immunogenic non-inferiority and interchangeable safety thresholds compared with Shanchol. These findings robustly support the large-scale integration of modern Euvichol-Plus and streamlined Euvichol-S formulations to stabilize global OCV stockpiles.