Human respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are frequent drivers of morbidity and mortality in susceptible populations. The primary target of neutralizing antibodies is the fusion (F) glycoprotein on the surface of the RSV and hMPV virion. As a result of the structural conservation between RSV and hMPV F, three antigenic regions are known to induce cross-neutralizing responses: sites III, IV, and V. Leveraging LIBRA-seq, we identify five RSV/hMPV cross-reactive human antibodies. One antibody, RM 5-1, potently neutralizes all tested viruses from the major subgroups of RSV and hMPV and provides protection against RSV and hMPV in a mouse challenge model. Structural analysis reveals that RM 5-1 utilizes an uncommon genetic signature to bind an epitope that spans sites Ø, II, and V. These findings highlight the molecular and structural elements influencing RSV and hMPV cross-reactivity as well as the potential of antibody RM 5-1 for translational development.

IntroductionThe four serotypes of dengue virus (DENV1-4) are the leading cause of arboviral diseases in humans. Currently, developing a safe and effective DENV vaccine remains a challenge. Previously we reported potently neutralizing human monoclonal antibodies (mAbs) preferentially recognize mature DENV particles, on which there is limited access to the fusion loop (FL) epitope and no premembrane (prM) protein. As FL and prM antibodies are weakly- or non-neutralizing and contribute to antibody-dependent enhancement, mature DENV particles represent an ideal DENV vaccine candidate. Several inactivated flavivirus vaccines, generated using formalin, ultraviolet or other inactivation methods, have progressed through preclinical and clinical studies. Little is known about how different inactivation methods affect viral epitopes and the quality of antibodies induced.MethodsWe investigated epitopes on Tween 20-inactivated mature DENV1 particles by testing a panel of well-characterized human mAbs and membrane integrity by sucrose-gradient ultracentrifugation and protein K digestion. We examined the mechanisms of Tween 20 inactivation by measuring RNA copy numbers, virus binding to cells and acid exposure, and antibody responses induced by Tween 20-inactivated mature DENV1 particles in mice.ResultsTween 20 inactivation better preserved the epitopes recognized by potently neutralizing mAbs compared with other methods. Sucrose-gradient ultracentrifugation and protein K digestion revealed no disruption of membrane integrity by Tween 20. Mechanistically, Tween 20 treatment caused reduced virus binding to cells and RNA degradation, which was reverted by pre-treatment with RNAseOUT, suggesting the involvement of extracellular RNase, and prevented the envelope protein conformational changes induced by acid exposure. Moreover, Tween 20-inactivated mature DENV1 particles induced a neutralizing antibody response to all four DENV serotypes characterized by competition with several potently neutralizing mAbs and limited recognition of FL.ConclusionOur results suggest that Tween 20-inactivated mature particles are a promising strategy for DENV vaccine development.

Despite robust antibody responses, immunity induced by acellular pertussis vaccine (DTaP) wanes over time and risk of pertussis seems to be lower in children who receive whole-cell vaccine (DTP) as their first dose. To interrogate the early immunologic response to pertussis vaccine, we enrolled 56 healthy infants who received either DTP or DTaP at 2-, 4-, 6-, and 18-months of age. RNA-sequencing and ribosome profiling of PBMC were performed prior to vaccination (Day 1) and on either Day 2 or Day 8. Pathway enrichment analysis on Days 2 and 8 showed enrichment of TLR-signaling and FcϒR-mediated phagocytosis among DTP recipients. DTP also led to increases in IRAK-4 and IL-1ß. After booster vaccination, a higher frequency of PT-specific B-cells was observed in DTP- vs. DTaP recipients. These data provide insights into the early immunologic responses to pertussis vaccine and may guide next-generation pertussis vaccine development.

H7N9 avian influenza virus is a zoonotic influenza virus of public health concern, with a 39% mortality rate in humans. H7N9-specific prevention or treatments for humans have not been approved. We previously isolated a human monoclonal antibody (mAb) designated H7-235 that broadly reacts to diverse H7 viruses and neutralizes H7N9 viruses in vitro. Here, we report the crystal structure of H7 HA1 bound to the fragment antigen-binding region (Fab) of recombinant H7-235 (rH7-235). The crystal structure revealed that rH7-235 recognizes residues near but outside of the receptor binding site (RBS). Nevertheless, the rH7-235 IgG potently inhibits hemagglutination mediated by H7N9 viruses due to avidity effect and Fc steric hindrance. This mAb prophylactically protects mice against weight loss and death caused by challenge with lethal H7N9 viruses in vivo. rH7-235 mAb neutralizing activity alone is sufficient for protection when used at a high dose in a prophylactic setting. This study provides insights into mechanisms of viral neutralization by protective, broadly reactive anti-H7 antibodies, informing the rational design of therapeutics and vaccines against H7N9 influenza virus.

ABSTRACT Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing a context for future vaccine trials and emphasizing the need for further characterization of protective responses toward conserved epitopes. (201 words)IMPORTANCESeasonal influenza viruses cause hundreds of thousands of deaths each year and up to a billion infections; under the proper circumstances, influenza A viruses with pandemic potential could threaten the lives of millions more. The variable efficacies of traditional influenza virus vaccines and the desire to prevent pandemic influenzas have motivated work toward finding a universal flu vaccine. Many promising universal flu vaccine candidates currently focus on guiding immune responses to highly conserved epitopes on the central stem of the influenza hemagglutinin viral fusion protein. To support the further development of these stem-targeting vaccine candidates, in this study, we use negative stain electron microscopy to assess the prevalence of central stem-targeting antibodies in individuals who were exposed to influenza antigens through traditional vaccination and/or natural infection during the 2018–2019 flu season.

Following SARS-CoV-2 Omicron BA.1, subsequent Omicron sub-lineages have continued to emerge, challenging the development of intervention and prevention strategies, including monoclonal antibodies and vaccines. To better understand the pathogenic effects caused by Omicron BA.5 infection, we developed a mouse-adapted virus with overt disease burden in BALB/c mice. Acute disease was characterized by significant weight loss and lung dysfunction following high-dose challenges. In survivor animals that were followed through 107 days post-infection, subpleural fibrosis with associated tertiary lymphoid structures was noted. Serum from these mice demonstrated potent neutralization against BA.5, with substantially reduced neutralization titers against early epidemic, zoonotic, and more recent contemporary XBB.1.5 variants. Intervention with pre-clinical monoclonal antibodies revealed that robust protection from BA.5-induced lung disease was possible after prophylactic administration. Together, this model enables the investigation of therapeutic approaches for both acute and post-acute sequelae of COVID-19.

The first-ever recent Marburg virus (MARV) outbreak in Tanzania and recent emergences in Rwanda, Ghana and Equatorial Guinea underscore the importance of therapeutic or vaccine development against the virus, for which none are approved. mRNA vaccines were proven successful in a pandemic-response to severe acute respiratory syndrome coronavirus-2, making it an appealing platform to target pathogenic emerging viruses. Here, we develop 1-methyl-pseudouridine-modified mRNA vaccines formulated in lipid nanoparticles (LNP) targeting the glycoproteins (GP) of MARV and the closely-related Ravn virus (RAVV). Vaccination of female guinea pigs elicits robust binding and neutralizing antibodies and confers complete protection against homologous and heterologous virus replication, disease and death. Characterization of antibody responses identifies disparities in the binding and functional profiles between the two viruses and regions in GP that are broadly reactive. The glycan cap is highlighted as an immunoreactive site for orthomarburgviruses, inducing antibody responses that are virus dependent. Profiling the antibody responses against the two viruses provides insight into how antigenic differences may affect the response towards conserved GP regions, which would otherwise be predicted to be cross-reactive, and has implications for the future design of broadly protective vaccines. The results support the use of mRNA-LNPs against pathogens of high consequence.

Although virus-like particle (VLP) vaccines were shown to be effective against several viruses, their advantage over vaccines that include envelope protein only is not completely clear, particularly for mRNA-encoded VLPs. We conducted a side-by-side comparison of the immunogenicity and protective efficacy of mRNA vaccines encoding the Marburg virus (MARV) full-length glycoprotein (GP) delivered alone or as a VLP. Electron microscopy confirmed VLP formation when MARV GP and matrix protein VP40 were coexpressed. We vaccinated guinea pigs with a 2-component mRNA vaccine encoding GP and VP40 (VLP) or GP alone. At the highest dose, both vaccines protected fully, although the VLP vaccine elicited a slightly lower humoral response than did the GP-only mRNA vaccine. However, at low doses, GP-only mRNA conferred 100% protection, whereas the VLP vaccine conferred only partial protection. In mice, VLP mRNA induced a moderate preference for GP-specific CD8+ T cell responses, whereas the GP-only mRNA somewhat favored CD4+ T cell responses. Guinea pig whole-blood RNA-Seq revealed that the VLP vaccine downregulated genes associated with various biological and metabolic processes, including the NF-κB signaling pathway, whereas the GP-only vaccine upregulated IFN signaling. Overall, the VLP mRNA vaccine was less immunogenic and protective, whereas the GP-only mRNA vaccine conferred robust protection with a dose of as little as 1 μg in guinea pigs.

Nipah virus recurrently spills over to humans, causing fatal infections. The viral receptor-binding protein (RBP or G) attaches to host receptors and is a major target of neutralizing antibodies. Here we use deep mutational scanning to measure how all amino-acid mutations to the RBP affect cell entry, receptor binding, and escape from neutralizing antibodies. We identify functionally constrained regions of the RBP, including sites involved in oligomerization, along with mutations that differentially modulate RBP binding to its two ephrin receptors. We map escape mutations for six anti-RBP antibodies, and find that few antigenic mutations are present in natural Nipah strains. Our findings offer insights into the potential for functional and antigenic evolution of the RBP that can inform the development of antibody therapies and vaccines.

The sudden rise of the SARS-CoV-2 virus and the delay in development of effective therapeutics for mitigation made evident a need for ways to screen compounds that can block infection and prevent further pathogenesis and spread. However, identifying effective drugs that are efficacious against viral infection and replication with minimal toxicity for the patient can be difficult. Monoclonal antibodies were shown to be effective, but as the SARS-CoV-2 mutated, these antibodies became ineffective. Small-molecule antivirals were identified using pseudovirus constructs to recapitulate infection in nonhuman cells, such as Vero E6 cells. However, the impact was limited due to poor translation of these compounds in the clinical setting. This is partly due to the lack of similarity of screening platforms to the in vivo physiology of the patient and partly because drugs effective in vitro showed dose-limiting toxicities. In this study, we performed two high-throughput screens in human lung adenocarcinoma cells with authentic SARS-CoV-2 virus to identify both monoclonal antibodies that neutralize the virus and clinically useful kinase inhibitors to block the virus and prioritize minimal host toxicity. Using high-content imaging combined with single-cell and multidimensional analysis, we identified antibodies and kinase inhibitors that reduce viral infection without affecting the host. Our screening technique uncovered novel antibodies and overlooked kinase inhibitors (i.e., PIK3i, mTORi, and multiple RTKi) that could be effective against the SARS-CoV-2 virus. Further characterization of these molecules will streamline the repurposing of compounds for the treatment of future pandemics and uncover novel mechanisms viruses use to hijack and infect host cells.

No licensed vaccines or therapies exist for patients infected with Nipah virus (NiV), although an experimental human monoclonal antibody (mAb) cross-reactive to the NiV and Hendra virus (HeV) G glycoprotein, m102.4, has been tested in a phase 1 trial and has been provided under compassionate use for both HeV and NiV exposures. NiV is a highly pathogenic zoonotic paramyxovirus causing regular outbreaks in humans and animals in South and Southeast Asia. The mortality rate of NiV infection in humans ranges from 40% to more than 90%, making it a substantial public health concern. The NiV G glycoprotein mediates host cell attachment, and the F glycoprotein facilitates membrane fusion and infection. We hypothesized that a mAb against the prefusion conformation of the F glycoprotein may confer better protection than m102.4. To test this, two potent neutralizing mAbs against NiV F protein, hu1F5 and hu12B2, were compared in a hamster model. Hu1F5 provided superior protection to hu12B2 and was selected for comparison with m102.4 for the ability to protect African green monkeys (AGMs) from a stringent NiV challenge. AGMs were exposed intranasally to the Bangladesh strain of NiV and treated 5 days after exposure with either mAb (25 milligrams per kilogram). Whereas only one of six AGMs treated with m102.4 survived until the study end point, all six AGMs treated with hu1F5 were protected. Furthermore, a reduced 10 milligrams per kilogram dose of hu1F5 also provided complete protection against NiV challenge, supporting the upcoming clinical advancement of this mAb for postexposure prophylaxis and therapy.

Development of an efficacious universal influenza vaccines remains a long-sought goal. Current vaccines have shortfalls such as mid/low efficacy and needing yearly strain revisions to account for viral drift/shift. Horses undergo bi-annual vaccines for the H3N8 equine influenza virus, and surveillance of sera from vaccinees demonstrated very broad reactivity and neutralization to many influenza strains. Subsequently, vaccinating mice using the equine A/Kentucky/1/1991 strain or recombinant hemagglutinin (HA) induced similar broadly reactive and neutralizing antibodies to seasonal and high pathogenicity avian influenza strains. Challenge of vaccinated mice protected from lethal virus challenges across H1N1 and H3N2 strains. This protection correlated with neutralizing antibodies to the HA head, esterase, and stem regions. Vaccinated ferrets were also protected after challenge with H1N1 influenza A/07/2009 virus using whole viral or HA. These data suggest that equine H3N8 induces broad protection against multiple influenzas using a unique antigen that diverges from other universal vaccine approaches.

Influenza B viruses (IBVs) comprise a substantial portion of the circulating seasonal human influenza viruses. Here, we describe the isolation of human monoclonal antibodies (mAbs) that recognized the IBV neuraminidase (NA) glycoprotein from an individual following seasonal vaccination. Competition -binding experiments suggested the antibodies recognized two major antigenic sites. One group, which included mAb FluB-393, broadly inhibited IBV NA sialidase activity, protected prophylactically in vivo , and bound to the lateral corner of NA. The second group contained an active site mAb, FluB-400, that broadly inhibited IBV NA sialidase activity and virus replication in vitro in primary human respiratory epithelial cell cultures and protected against IBV in vivo when administered systemically or intranasally. Overall, the findings described here shape our mechanistic understanding of the human immune response to the IBV NA glycoprotein through the demonstration of two mAb delivery routes for protection against IBV and the identification of potential IBV therapeutic candidates.

Immune imprinting is a phenomenon in which prior antigenic experiences influence responses to subsequent infection or vaccination1,2. The effects of immune imprinting on serum antibody responses after boosting with variant-matched SARS-CoV-2 vaccines remain uncertain. Here we characterized the serum antibody responses after mRNA vaccine boosting of mice and human clinical trial participants. In mice, a single dose of a preclinical version of mRNA-1273 vaccine encoding Wuhan-1 spike protein minimally imprinted serum responses elicited by Omicron boosters, enabling generation of type-specific antibodies. However, imprinting was observed in mice receiving an Omicron booster after two priming doses of mRNA-1273, an effect that was mitigated by a second booster dose of Omicron vaccine. In both SARS-CoV-2-infected and uninfected humans who received two Omicron-matched boosters after two or more doses of the prototype mRNA-1273 vaccine, spike-binding and neutralizing serum antibodies cross-reacted with Omicron variants as well as more distantly related sarbecoviruses. Because serum neutralizing responses against Omicron strains and other sarbecoviruses were abrogated after pre-clearing with Wuhan-1 spike protein, antibodies induced by XBB.1.5 boosting in humans focus on conserved epitopes targeted by the antecedent mRNA-1273 primary series. Thus, the antibody response to Omicron-based boosters in humans is imprinted by immunizations with historical mRNA-1273 vaccines, but this outcome may be beneficial as it drives expansion of cross-neutralizing antibodies that inhibit infection of emerging SARS-CoV-2 variants and distantly related sarbecoviruses.

Zika virus (ZIKV) is a mosquito-borne flavivirus that caused a global pandemic in 2016-2017 with continued ongoing transmission at low levels in sev eral countries. In the absence of an approved ZIKV vaccine, neutralizing monoclonal antibodies (mAbs) provide an option for the prevention and treatment of ZIKV infection. Previous studies identified a potent neutralizing human mAb ZIKV-117 that reduced fetal infection and death in mice following ZIKV challenge. In this study, we report exqui site potency of ZIKV-117-LALA-YTE, which has been engineered to reduce Fc receptor binding and to extend half-life, in a titration study in rhesus macaques to protect against ZIKV challenge. We show complete protection at a dose of 0.016 mg/kg ZIKV-117-LALAYTE, which resulted in median serum concentrations of 0.13 mu g/mL. The high potency of this mAb supports its potential clinical development as a novel biotherapeutic interven tion for ZIKV.

ABSTRACT Immune imprinting is a phenomenon in which an individual’s prior antigenic experiences influence responses to subsequent infection or vaccination. Here, using antibody depletion and multiplexed spike-binding assays, we characterized the type-specificity and cross-reactivity of serum antibody responses after mRNA vaccination in mice and human clinical trial participants. In mice, a single priming dose of a preclinical version of mRNA-1273 vaccine encoding Wuhan-1 spike minimally imprinted serum responses elicited by Omicron boosters, enabling a robust generation of type-specific antibodies. However, substantial imprinting was observed in mice receiving an Omicron booster after two priming doses of mRNA-1273, an effect that was mitigated by a second booster dose of Omicron mRNA vaccine. In humans who received two BA.5 or XBB.1.5 Omicron-matched boosters after two or more doses of the prototype mRNA-1273 vaccine, spike-binding and neutralizing serum antibodies cross-reacted with circulating Omicron variants as well as more distantly related sarbecoviruses. Because the serum neutralizing response against Omicron strains and other sarbecoviruses was completely abrogated after pre-clearing with the Wuhan-1 spike protein, antibodies induced by XBB.1.5 boosting in humans focus on conserved epitopes shaped and shared by the antecedent mRNA-1273 primary series. Our depletion analysis also identified cross-reactive neutralizing antibodies that recognize distinct epitopes in the receptor binding domain (RBD) and S2 proteins with differential inhibitory effects on members of the sarbecovirus subgenus. Thus, although the serum antibody response to Omicron-based boosters in humans is dominantly imprinted by prior immunizations with prototype mRNA-1273 vaccines, this outcome can be beneficial as it drives expansion of multiple classes of cross-neutralizing antibodies that inhibit infection of emerging SARS-CoV-2 variants and extend activity to distantly related sarbecoviruses.

The rodent-borne Andes virus (ANDV) causes a severe disease in humans. We developed an ANDV mRNA vaccine based on the M segment of the viral genome, either with regular uridine (U-mRNA) or N1-methylpseudouridine (m1Ψ-mRNA). Female mice immunized by m1Ψ-mRNA developed slightly greater germinal center (GC) responses than U-mRNA-immunized mice. Single cell RNA and BCR sequencing of the GC B cells revealed similar levels of activation, except an additional cluster of cells exhibiting interferon response in animals vaccinated with U-mRNA but not m1Ψ-mRNA. Similar immunoglobulin class-switching and somatic hypermutations were observed in response to the vaccines. Female Syrian hamsters were immunized via a prime–boost regimen with two doses of each vaccine. The titers of glycoprotein-binding antibodies were greater for U-mRNA construct than for m1Ψ-mRNA construct; however, the titers of ANDV-neutralizing antibodies were similar. Vaccinated animals were challenged with a lethal dose of ANDV, along with a naïve control group. All control animals and two animals vaccinated with a lower dose of m1Ψ-mRNA succumbed to infection whereas other vaccinated animals survived without evidence of virus replication. The data demonstrate the development of a protective vaccine against ANDV and the lack of a substantial effect of m1Ψ modification on immunogenicity and protection in rodents.