The portfolio of next generation COVID-19 vaccines would benefit from candidates that induce durable systemic and mucosal immune responses that would lessen person-to-person transmission. We constructed an intranasal (IN) replication-competent adenovirus type 4 recombinant platform to express SARS-CoV-2 Spike variants (Ad4-S) and assessed immunogenicity and efficacy in the Syrian hamster model. Although both IN Ad4-S and intramuscular (IM) vaccines (Ad26.CoV2.S and mRNA-1273) induced serum binding antibodies, only Ad4-S induced a robust mucosal response in the nasal cavity. IN Ad4-S vaccination induced serum neutralizing titers equivalent to or greater than IM vaccination but more durable up to 6 months. Upon challenge, IN immunization also resulted in less weight loss, greater breadth and durability of restriction of viral replication, and less lung pathology than IM immunization up to 268 days after immunization. These data support the potential of the IN Ad4 vaccine platform to reduce transmission of SARS-CoV-2 and other respiratory viruses with pandemic potential.

Abstract Purpose: The National Breast Cancer Coalition (NBCC), established in 1991, is a collaboration of activists, survivors, grassroots groups and national organizations united to end breast cancer through action and advocacy. Despite progress in certain areas, neither breast cancer incidence nor global mortality have significantly declined. In response, NBCC launched the Artemis Project in 2010, an advocate-led initiative focused on primary prevention of breast cancer and prevention of metastasis, designed to foster new collaborations among researchers and advocates. The project is a response to the fact that despite billions of dollars invested in research that has led to some effective treatments, we still do not know how to prevent or cure breast cancer. Artemis process: The Artemis Project is a collaboration to identify urgent gaps, set milestones, and co-develop research solutions to primary and metastasis prevention. Unlike traditional research development pipelines, Artemis integrates trained patient advocates as equal partners in priority setting, research design and oversight. Advocates identify the participants and choose the issues. Collaborations form at the annual meeting among researchers and advocates who often would not have met otherwise.The annual Artemis meeting brings participants together to explore new ideas, develop innovative projects towards Artemis’s goals, and outline work plans for 12-18 months after the meeting. Online meetings are held throughout the year for the groups to interact and to report progress. Seed funding for some projects has been provided by NBCC. NBCC schedules webinars throughout the year on relevant topics for Artemis participants. Selected Artemis outcomes: The first Artemis project led to an ongoing initiative to develop a breast cancer prevention vaccine, with a plan in place to move to a Phase I trial by mid-2026. The vaccine, consisting of 6 self-antigens overexpressed in breast cancer, is being developed under a contract with the NCI Prevent Program. DNA Land, an effort stemming from another Artemis project, has recruited over 33,000 individuals who have consented and contributed genomic and matched phenotypic data to underpin bioinformatics studies supporting prevention-focused breast cancer research.Additionally, researchers participating in Artemis have formed many collaborations that have grown into externally funded research efforts, including a project to eliminate disseminated tumor cells. Conclusions: Now 15 years in, the Artemis Project has created a durable, advocate-led ecosystem that unites researchers, clinicians, and advocates to pursue transformative prevention solutions rather than incremental advances. This model is adaptable across disease research areas. Citation Format: Fran M. Visco, Jayanta Debnath, Daniel Douek, Stephen J. Elledge, Silvia C. Formenti, Michele S. Garfinkel, Cyrus M. Ghajar, Patricia K. Haugen, Christopher I. Li, Herbert Kim Lyerly, Michelle Tregear, Alana L. Welm, Frank J. Calzone. The Artemis Project: A patient-led global research consortium advancing prevention-focused breast cancer innovation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3695.

The portfolio of next-generation COVID-19 vaccines would benefit from candidates that induce durable systemic and mucosal immune responses that would lessen person-to-person transmission. We constructed an intranasal (IN) replication-competent adenovirus type 4 recombinant platform to express SARS-CoV-2 spike variants (Ad4-S) and assessed immunogenicity and efficacy in the Syrian hamster model. Although both IN Ad4-S and intramuscular (IM) vaccines (Ad26.CoV2.S and mRNA-1273) induced serum binding antibodies, only Ad4-S induced a robust nasal mucosal response. IN Ad4-S vaccination induced serum neutralizing titers equivalent to or greater than IM vaccination but more durable up to 6 months. Upon challenge, IN immunization also resulted in less weight loss, greater breadth and durability of restriction of viral replication, and less lung pathology than IM immunization up to 268 days after immunization. These data support the potential of the IN Ad4 vaccine platform to reduce the transmission of SARS-CoV-2 and other respiratory viruses with pandemic potential.

Identifying receptors for bat coronaviruses is critical for spillover risk assessment, countermeasure development, and pandemic preparedness. While Middle East respiratory syndrome coronavirus (MERS-CoV) uses DPP4 for entry, the receptors of many MERS-related betacoronaviruses remain unknown. The bat merbecovirus HKU5 was previously shown to have an entry restriction in human cells. Using both pseudotyped and full-length virus, we show that HKU5 uses Pipistrellus abramus bat ACE2 but not human ACE2 or DPP4 as a receptor. Cryo-electron microscopy analysis of the virus-receptor complex and structure-guided mutagenesis reveal a spike and ACE2 interaction that is distinct from other ACE2-using coronaviruses. MERS-CoV vaccine sera poorly neutralize HKU5 informing pan-merbecovirus vaccine design. Notably, HKU5 can also engage American mink and stoat ACE2, revealing mustelids as potential intermediate hosts. These findings highlight the versatility of merbecovirus receptor use and underscore the need for continued surveillance of bat and mustelid species.

Human B cell immunity to the influenza hemagglutinin (HA) stem, a universal vaccine target, is often stereotyped and immunogenetically restricted, posing hurdles to study outside of humans. Here, we show that cynomolgus macaques vaccinated with an HA stem immunogen elicit humanlike public B cell lineages targeting two major conserved sites of vulnerability, the central stem and anchor epitopes. Central stem antibodies were predominantly derived from VH1-138, the macaque homolog of human VH1-69, a VH gene preferentially used in human central stem broadly neutralizing antibodies (bnAbs). Similarly, macaques produced anchor bnAbs containing the canonical NWP motif. Both bnAb lineages were functionally and structurally analogous to their human counterparts, with recognition mediated largely by germline-encoded structural motifs. These findings indicate that the macaque immunoglobulin repertoire can support humanlike public bnAb responses to influenza HA, highlighting the value of the macaque model for translational vaccinology. Moreover, this underscores the utility of homologous germline-encoded immunity, suggesting that immune repertoires of macaques and humans have been similarly shaped during evolution.

BACKGROUND:The Pandemic Response Repository through Microbial and Immune Surveillance and Epidemiology (PREMISE) programme was established to translate knowledge gained from global immunoepidemiological surveillance into a better understanding of population-level dynamics of emerging and re-emerging infections, as well as into the discovery and development of biomedical countermeasures against potential pandemic threats. As proof of principle for this approach, we conducted a longitudinal immunoepidemiological study in children in the USA, focusing on enterovirus D68 (EV-D68) infection dynamics but also capturing surveillance of a broad array of other endemic respiratory pathogens. Serendipitously, our sampling spanned the lifting of widespread COVID-19 non-pharmaceutical interventions (NPIs) in 2022-23, following a unique period during which virus exposure markedly diminished. METHODS:This prospective, multicentre, longitudinal, immunoepidemiological surveillance study enrolled children aged 10 years or younger and weighing at least 8 kg at three US university sites. Blood specimens collected from January to June, 2022 (visit 1; pre-enterovirus season), and from January to June, 2023 (visit 3; post-enterovirus season), were tested in a multiplex assay for antibody binding to EV-D68 (prespecified primary objective) and a panel of 15 other respiratory viruses (exploratory objectives), and for neutralising activity against EV-D68, enterovirus A71, and respiratory syncytial virus (RSV; for antibody binding assay validation). Respiratory mid-turbinate swabs collected from children with symptomatic illness who participated in symptom surveys during July-December, 2022 (visit 2; enterovirus season), underwent metagenomic sequencing for pathogen detection. Serological data for EV-D68 were incorporated into epidemiological models based on case data from national surveillance to predict future transmission dynamics. FINDINGS:Of 488 eligible children approached, 174, with a median age of 3·4 years (IQR 1·9-6·4), were enrolled and followed up longitudinally from January, 2022, to June, 2023. Three children withdrew before study completion and 51 were lost to follow-up between visits 1 and 3. 90 paired serological samples and 73 respiratory swabs were tested. Mean antibody binding and neutralisation titres against all viruses tested increased over the study period, most notably in younger children with lower initial titres. The highest exposure rates (seroconversion or antibody boosting) were seen with SARS-CoV-2 (51 [59%] of 87), EV-D68 (36 [41%] of 87), RSV (36 [41%] of 87), and influenza (35 [40%] of 87), whereas the pathogens most frequently detected by respiratory swab sequencing were EV-D68 (clade B3), rhinovirus A, and rhinovirus C (n=7 each). Incorporating EV-D68 serological data into epidemiological models resulted in an 82% reduction in the range of prediction errors and a 33% reduction in median prediction errors for longer-term EV-D68 circulation dynamics compared with national pathogen surveillance data alone. INTERPRETATION:In this study, we captured immunological evidence of endemic virus re-emergence in children following lifting of pandemic NPIs, which revealed high rates of exposure to endemic respiratory pathogens in a large group of seronegative, predominantly younger, children. This study demonstrates the feasibility and utility of immunoepidemiological surveillance to enable more precise and accurate modelling of pathogen circulation dynamics to predict and prepare for future waves of disease. FUNDING:Intramural Research Program of the National Institute of Allergy and Infectious Diseases-Vaccine Research Center, and the National Cancer Institute, National Institutes of Health.

Advances in sequencing technology have made it possible to capture complex immunogenetic loci at a scale that exceeds the capacity for manual annotation. Here we present the Annotator of Loci for ImmunoGlobulins and T cell Receptors (ALIGaToR), an automated pipeline to transfer genetic annotations from a known reference to a novel genomic assembly. We show that ALIGaToR accurately reproduces manually curated annotations and is capable of transferring labels even between distantly related species. Code and documentation for ALIGaToR, including a script reproducing all analyses in this paper, are available at https://github.com/scharch/aligator.

The rapid emergence of divergent SARS-CoV-2 variants led to a 2023-2024 update of the COVID-19 mRNA vaccine to a monovalent version containing the XBB.1.5 SARS-CoV-2 spike antigen. To determine the durability and breadth of the antibody responses after immunization, we analyzed antibodies and memory B cells from 24 individuals before and after a single XBB.1.5 mRNA vaccine dose for up to 6 months. Using a live virus neutralization assay, we found that the XBB.1.5 vaccine improved the magnitude and breadth of antibody neutralizing activity against the ancestral SARS-CoV-2 strain (WA1), BA.5 strain, and XBB.1.5 Omicron variants. Durable WA1 and XBB.1.5 IgG spike protein binding antibodies were induced with an estimated half-life of 703 and 531 days (at day 120), respectively. There was a greater increase of IgG1 and IgG4 binding antibodies against the XBB.1.5 spike protein compared with the WA1 spike protein postvaccination. A high proportion of antibodies were cross-reactive against both WA1 and XBB.1.5 strains, as determined by serum depletion and memory B cell analysis, and this cross-reactivity was durable. Last, we evaluated the neutralizing activity of these antibodies against more contemporary circulating Omicron strains and observed reduced cross-reactivity to KP.2 and KP.3 compared with XBB.1.5 6 months after vaccination. These data show that the XBB.1.5 COVID-19 vaccine promotes more durable binding and neutralizing antibodies than prior ancestral WA1 or bivalent vaccines. However, divergent Omicron variants with mutations in the spike protein were able to evade these neutralizing antibodies, emphasizing the need for periodic consideration of COVID-19 vaccine reformulation.

The membrane-proximal external region (MPER) of the HIV-1 envelope is a target for broadly neutralizing antibodies (bnAbs), and vaccine-elicited MPER-directed antibodies have recently been reported from a human clinical trial. In this study, we sought to identify MPER-directed nAbs in simian immunodeficiency virus (SIV)-infected rhesus macaques. We isolated four lineages of SIV MPER-directed nAbs from two SIV-infected macaques. The nAbs displayed low potency but up to 90% breadth on a 20-strain SIV panel. Crystal structures of representative nAbs in complex with SIV MPER peptides revealed the SIV antibodies to bind a helical epitope at the N-terminal (proximal) region of the MPER, defining a reproducible multi-donor class encompassing all four lineages. HIV-1 comparison showed that this class of SIV MPER-directed antibodies targets a helical region overlapping that targeted by human vaccine-elicited ones. Thus, a prevalent and reproducible class of SIV bnAbs recognizes an epitope similar to that recently observed in an HIV-1-vaccine trial.

Mucosal vaccines can enhance protection against respiratory viruses. This study describes the immune mechanisms by which intranasal boosting with an unadjuvanted protein increases both B cell immunity and protection in a mouse model of SARS-CoV-2.

Alternative splicing (AS) significantly increases the diversity of gene function by enabling a single gene to produce multiple protein isoforms. U1 and U2AF are proteins responsible for the recognition and splicing of an intron's donor and acceptor sites, respectively. Here, we used RNA-seq to explore these fundamental splicing events to separately calculate the splicing efficiency of U1 and U2AF. The expression levels of splice sites were directly calculated by the number of uniquely mapped reads spanning the splice junction. We found that the scaled expression levels of donor and acceptor sites both follow a type III extreme value Weibull distribution with the same shape parameter of 0.14. These observations significantly extend our previous findings by revealing that AS follows a Weibull distribution at the levels of both the fundamental splicing event and the mature transcript isoform. A simple transform of the shape parameter a, 1/(1+a), ranges between 0 and 1, and positively correlates with the dominance of the major splicing product and therefore represents an index of the splicing machinery efficiency. Importantly, the equal splicing efficiency of U1 and U2AF ensure that their combined efficacy reaches a maximum.

Eliciting broadly neutralizing antibodies (bNAbs) remains a key goal in HIV-1 vaccine research. bNAbs targeting the membrane proximal external region (MPER) on the HIV-1 Envelope are highly cross-reactive and are templates for vaccine design. Prior studies defined early events in MPER bNAb development, however, later stages of development need further investigation. Participant 40512 from the RV217 actue-infection longitudinal cohort was followed 4+ years after detection of viral RNA. From this participant, the VRC42 MPER-directed bNAb lineage was isolated and characterized at day 646. Notably, VRC42 was highly similar to bNAbs isolated from several unrelated individuals, suggesting common pathways exist that elicit MPER bNAbs. In this study, 30 new VRC42 lineage bNAbs were isolated from days 1142-1427 via fluorescent-activated cell sorting with MPER probes, followed by rapid amplification, transfection, and production of immunoglobulins (RATP-Ig); and assessed for neutralization activity. Sequence analysis revealed expansion of the VRC42 lineage, somatic hypermutation rates of 10-18%, and multiple amino acid changes in the heavy chain (HC) and light chain genes. Site-directed mutagenesis of the HC, followed by neutralization testing, identified that changes in the CDRH1, CDRH2, and CDRH3, in different sublineages, increased neutralization potency against auto- and heterologous strains. Understanding the fine details of bNAb development will aid in vaccine design for bNAbs. Vaccines and Immunotherapy (VAC)

Mucosal B cell immunity relies on the constant induction of immunoglobulin A (IgA) in the intestine. In spite of abundant homeostatic IgA, here we present further amplification of IgA plasma cells during intestinal inflammation that was linked to massive clonal expansion of dominant B lineages. We characterized the inducible B cell response during colitis and show the properties of intestinal IgA produced by adaptive and innate B cell subsets. Fab-dependent specific recognition of individual commensal taxa by inflammation-induced IgA was associated with signs of affinity maturation and cross-reactivity to cellular autoantigens. However, despite the principal ability of mucosal B cells to specifically induce microbiota-targeting IgA, the vast majority of inflammation-induced intestinal IgA was microbiota non-reactive, generated upon clonal burst of germline-encoded bystanders. Unpredictable variation in the prevalence of microbiota- and auto-reactive IgA in individual colitic mice was indicative of stochastic selection of random B lineages in the inflammatory environment with potentially unforeseeable pathophysiological consequences.

The continued evolution of SARS-CoV-2 variants capable of subverting vaccine and infection-induced immunity suggests the advantage of a broadly protective vaccine against betacoronaviruses (β-CoVs). Recent studies have isolated monoclonal antibodies (mAbs) from SARS-CoV-2 recovered-vaccinated donors capable of neutralizing many variants of SARS-CoV-2 and other β-CoVs. Many of these mAbs target the conserved S2 stem region of the SARS-CoV-2 spike protein, rather than the receptor binding domain contained within S1 primarily targeted by current SARS-CoV-2 vaccines. One of these S2-directed mAbs, CC40.8, has demonstrated protective efficacy in small animal models against SARS-CoV-2 challenge. As the next step in the pre-clinical testing of S2-directed antibodies as a strategy to protect from SARS-CoV-2 infection, we evaluated the in vivo efficacy of CC40.8 in a clinically relevant non-human primate model by conducting passive antibody transfer to rhesus macaques (RM) followed by SARS-CoV-2 challenge. CC40.8 mAb was intravenously infused at 10mg/kg, 1mg/kg, or 0.1 mg/kg into groups (n = 6) of RM, alongside one group that received a control antibody (PGT121). Viral loads in the lower airway were significantly reduced in animals receiving higher doses of CC40.8. We observed a significant reduction in inflammatory cytokines and macrophages within the lower airway of animals infused with 10mg/kg and 1mg/kg doses of CC40.8. Viral genome sequencing demonstrated a lack of escape mutations in the CC40.8 epitope. Collectively, these data demonstrate the protective efficiency of broadly neutralizing S2-targeting antibodies against SARS-CoV-2 infection within the lower airway while providing critical preclinical work necessary for the development of pan-β-CoV vaccines.

We collected samples from febrile patients in Karachi, Pakistan, in 2021-2022. Sequencing, molecular, and serologic screens revealed dengue serotype 2 and Zika virus. The Zika lineage was inferred to be from Brazil in 2016, indicating unobserved circulation. We conclude that Zika virus contributes to perceived dengue outbreak burden in Pakistan.

Bats are reservoir hosts for numerous well-known zoonotic viruses, but their broader virus-hosting capacities remain understudied. Picornavirales are an order of enteric viruses known to cause disease across a wide range of mammalian hosts, including Hepatitis A in humans and foot-and-mouth disease in ungulates. Host-switching and recombination drive the diversification of Picornavirales worldwide. Divergent Caliciviridae and Picornaviridae (families within the Picornavirales ) have been described in bats across mainland Africa, but surveillance for these viruses has been rare in the Southwest Indian Ocean Islands. Bats live in close proximity to and are consumed widely as a food source by humans in Madagascar, providing opportunities for zoonotic transmission. Prior work in Madagascar has described numerous evolutionarily divergent bat viruses, some with zoonotic potential. Using metagenomic Next Generation Sequencing of urine and fecal samples obtained from three species of endemic Malagasy fruit bats ( Eidolon dupreanum , Pteropus rufus , and Rousettus madagascariensis ), we recovered 13 full-length and 37 partial-length genomic sequences within the order Picornavirales (36 Picornaviridae and 14 Caliciviridae sequences), which we identify and describe here. We find evidence that genetic exchange between mainland African bat and Madagascar bat Picornavirales likely shaped the diversification patterns of these novel sequences through recombination events between closely related Picornavirales ; thus far, high host fidelity appears to have limited these viruses from spilling over into other species.

We report the first identification of Zika virus in Pakistan following genomic and serological analyses of blood samples from 20 patients with febrile illness. In November 2021, an outbreak of dengue-like illness occurred in the metropolitan city of Karachi. Viral genome capture and sequencing of seven patients revealed six cases of dengue virus serotype 2 and two Zika virus infections, including one dengue and Zika virus co-infection. The next year, following severe flooding, 13 suspected dengue patients were screened by real time qRT-PCR and serology, and 92% (12/13) had evidence of current or recent Zika virus infection. Phylogenetic analyses revealed the Zika viruses originated from Brazil. The most recent observed ancestor dates to 2016, suggesting a prior importation event and ongoing circulation. Our results suggest that Zika virus may be circulating and contributing to disease burden during seasonal Dengue outbreak.

Highly pathogenic avian influenza H5Nx viruses are an emerging threat for global health, especially clade 2.3.4.4b H5N1 virus which causes panzootic infections. Here we describe the isolation and characterization of broadly cross-neutralizing monoclonal antibodies (mAbs) against diverse H5Nx viruses from individuals who received a monovalent H5N1 vaccine 15 years ago. By screening over 500 mAbs, we identified 5 mAbs that neutralized the majority of H5 clades including 2.3.4.4b and target three distinct conserved epitopes within the HA globular head. Cryo-electron microscopy structures of these mAbs in complex with HA, deep mutational scanning and neutralization escape studies define the sites of vulnerability of H5 HA. These mAbs mediated stronger prophylactic protection against clade 2.3.4.4b H5N1 infection in mice than the best-in-class mAb targeting the HA stem. Our study identified several highly potent broadly neutralizing H5 mAbs from humans that either alone or in combination provide a pragmatic pandemic preparedness option against the threat of panzootic H5N1 influenza.

Noroviruses infect millions each year, and while effective countermeasures are eagerly sought, none have been reported for the GI genogroup, first described more than 50 years ago. Here, to provide insight into GI norovirus neutralization, we isolated a broad GI antibody, 16E10, from a human blood donor and showed it neutralizes noroviruses in human enteroid cultures and abrogates or reduces infection in rhesus macaques. The cryogenic electron microscopy reconstruction of 16E10 with a norovirus protruding-domain dimer at 2.56-Å resolution reveals an exceptionally large binding surface, overlapping an antibody supersite, distal from host receptor-binding or cofactor-binding sites. Cryogenic electron microscopy reconstructions with virus-like particles (VLPs) showed that 16E10 disrupts protruding domains on the VLP surface and disassembles VLPs, altering viral organization required for avidity. While its epitope was generally conserved, 16E10 recognized multiple sequence-divergent residues, binding to which was enabled by corresponding cavities in the 16E10-norovirus interface. Broad recognition of noroviruses can thus incorporate sequence-divergent residues, through a cavity-based mechanism of diversity tolerance.