Search

Mumps virus (MuV) is a highly contagious paramyxovirus that is endemic in most regions of the world and continues to cause outbreaks even in highly immunized populations. Outbreaks of mumps in countries with high measles, mumps, and rubella vaccination coverage have been attributed to waning immunity and antigenic differences between the Jeryl Lynn vaccine strain (genotype A) and circulating wild-type viruses. To obtain a subunit vaccine, we used structure-based design to engineer the mumps fusion (F) glycoprotein stabilized in its prefusion conformation (Pre-F) as well as a chimeric immunogen comprising Pre-F linked to mumps hemagglutinin neuraminidase (HN); in mice, both Pre-F antigen and the chimeric antigen elicited potent cross-reactive plaque reducing neutralizing titers to genotypes A, G, and H mumps. A crystal structure of mumps Pre-F at 2.16 Å resolution validated the stabilization strategy, while a post-fusion form of F was engineered as a comparator. Monoclonal antibodies to mumps Pre-F and HN were isolated from immunized mice; 7 of 14 Pre-F-specific antibodies and 9 of 15 HN-specific antibodies were capable of neutralizing genotype G MuV with a range of potencies. Additionally, 7 of 14 Pre-F-specific antibodies neutralized genotype A mumps. Structural and binding analyses of Pre-F-specific antibodies revealed binding to four discrete neutralizing antigenic sites and binding analyses of HN-specific antibodies revealed binding to five discrete neutralizing antigenic sites. Overall, the PreF and the chimeric Pre-F/HN immunogens are promising candidates to boost MMR-elicited immunity to mumps or as a next-generation vaccine., Competing Interests: Competing interests statement:G.B.E.S.-J. is an shareholder of Mevox, Ltd. R.J.L, B.S.G., and G.B.E.S.-J. are co-inventors on US Provisional 62/946,902.

Background: Humoral immune response against the pre-fusion (pre-F) conformation of respiratory syncytial virus (RSV) F protein has been proposed to play a protective role against infection. An RSV pre-F maternal vaccine has been recently approved in several countries to protect young infants against RSV. We aimed to assess serum IgG titers against the pre-F and post-F conformations of RSV F protein and their association with life-threatening RSV disease (LTD) in previously healthy infants., Methods: A prospective cohort study including hospitalized infants <12 months with a first RSV infection was conducted during 2017-2019. Patients with LTD required intensive care and mechanical respiratory assistance. RSV pre-F exclusive and post-F antibody responses were determined by post-F competition and non-competition immunoassays, respectively, and neutralizing activity was measured by plaque reduction neutralization test., Results: Fifty-eight patients were included; the median age was 3.5 months and 41 % were females. Fifteen patients developed LTD. RSV F-specific antibody titers positively correlated with neutralizing antibody titers in acute and convalescent phases but, importantly, they did not associate with LTD. Acute RSV pre-F exclusive and post-F IgG titers negatively correlated with patient age (P = 0.0007 and P < 0.0001), while a positive correlation was observed between the fold changes in RSV F-specific antibody titers between convalescent and acute phase and patient age (P = 0.0014 and P < 0.0001). Infants ≤2 months exhibited significantly lower fold-changes in RSV F-specific and neutralizing antibody titers between convalescence and acute phase than older infants. Additionally, acute RSV antibody titers showed no correlation with nasal RSV load and, furthermore, nasal viral load was not associated with the development of LTD., Conclusions: This study highlights that protection against life-threatening RSV disease is not necessarily antibody-dependent. Further characterization of the immune response against RSV and its role in protection against severe disease is important for the development of the safest possible preventive strategies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Fausto Martin Ferolla reports financial support was provided by Thrasher Research Fund, United States. Eduardo Luis Lopez reports financial support was provided by Fundacion Argentina de Infectologia Pediatrica. Barney S Graham has patent #WO2014160463A1 Prefusion RSV F proteins and their use licensed to Assignee. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objectives: With remarkable progress in the field of respiratory syncytial virus (RSV) prophylaxis, it is critical to understand population immunity against RSV. We aim to describe the RSV pre-F IgG antibodies across all age groups in Southern China and to evaluate the risk factors associated with lower antibody levels., Methods: We performed a community-based cross-sectional sero-epidemiological study in Anhua County, Hunan Province, Southern China, from July 15, 2021, to November 5, 2021. Serum samples were tested for IgG antibodies against the RSV prefusion F (pre-F) protein using an enzyme-linked immunosorbent assay. We estimated the geometric mean titres (GMTs) and seropositivity rates across all age groups. The generalized linear models were built to identify factors associated with antibody levels., Results: A total of 890 participants aged 4 months to older than 89 years were enrolled. The lowest RSV pre-F IgG GMTs were observed in infants and toddlers aged 4 months to younger than 2 years (3.0; 95% CI, 2.6-3.5). With increasing age, the RSV pre-F IgG GMT increased to 4.3 (95% CI, 4.1-4.4) between the ages of 2 and younger than 5 years and then stabilized at high levels throughout life. All the children had serological evidence of RSV infection by the age of 5 years. Age was associated with RSV pre-F antibody levels in children, with an estimated 1.9-fold (95% CI, 0.8-3.6) increase in titre per year before 5 years of age, although it was not significantly associated with antibody levels in adults aged older than 60 years., Discussion: Our findings could provide a comprehensive understanding of the gaps in RSV immunity at the population level and inform the prioritization of immunization platforms., (Copyright © 2024 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: The key correlate of protection of respiratory syncytial virus (RSV) vaccines and monoclonal antibodies (mAbs) is virus neutralization, measured via sera obtained through venipuncture. Dried blood obtained with a finger prick can simplify acquisition, processing, storage, and transport in trials and thereby reduce costs. In this study, we validate an assay to measure RSV neutralization in dried capillary blood., Methods: Functional antibodies were compared between matched serum and dried blood samples from a phase 1 trial with RSM01, an investigational anti-RSV prefusion F mAb. Hep-2 cells were infected with a serial dilution of sample-virus mixture by using RSV-A2-mKate to determine the half-maximal inhibitory concentration. Stability of dried blood was evaluated over time and during temperature stress., Results: Functional antibodies in dried blood were highly correlated with serum (R2 = 0.98, P < .0001). The precision of the assay for dried blood was similar to serum. The function of mAb remained stable for 9 months at room temperature and frozen dried blood samples., Conclusions: We demonstrated the feasibility of measuring RSV neutralization using dried blood as a patient-centered solution that may replace serology testing in trials against RSV or other viruses, such as influenza and SARS-CoV-2. Clinical Trials Registration.  NCT05118386 (ClinicalTrials.gov)., Competing Interests: Potential conflicts of interest. University Medical Center Utrecht (UMCU) has received major funding (>€100 000 per industrial partner) for investigator-initiated studies from AbbVie, MedImmune, AstraZeneca, Sanofi, Janssen, Pfizer, MSD, MeMed Diagnostics, the Bill and Melinda Gates Foundation, Bill & Melinda Gates Medical Research Institute, and the Dutch Lung Foundation. UMCU has received major funding as part of the public-private partnership Innovative Medicines Initiative–funded RESCEU and PROMISE projects with partners GSK, Novavax, Janssen, AstraZeneca, Pfizer, and Sanofi. UMCU has received major funding by Julius Clinical for participating in clinical studies sponsored by MedImmune and Pfizer. UMCU received minor funding (€1000–€25 000 per industrial partner) for consultation and invited lectures by AbbVie, MedImmune, Ablynx, Bavaria Nordic, MabXience, GSK, Novavax, Pfizer, Moderna, Astrazeneca, MSD, Sanofi, and Janssen. L. J. B. and N. I. M. have regular interaction with pharmaceutical and other industrial partners. They have not received personal fees or other personal benefits. L. J. B. is the founding chair of the ReSViNET Foundation. J. T. W. is an employee at the Bill & Melinda Gates Medical Research Institute. A. D.-C. and T. J. R. are supported by funding from the Intramural Research Program of the NIAID. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Human enteroviruses are the most common human pathogen with over 300 distinct genotypes. Previous work with poliovirus has suggested that it is possible to generate antibody responses in humans and animals that can recognize members of multiple enterovirus species. However, cross protective immunity across multiple enteroviruses is not observed epidemiologically in humans. Here we investigated whether immunization of mice or baboons with inactivated poliovirus or enterovirus virus-like-particles (VLPs) vaccines generates antibody responses that can recognize enterovirus D68 or A71. We found that mice only generated antibodies specific for the antigen they were immunized with, and repeated immunization failed to generate cross-reactive antibody responses as measured by both ELISA and neutralization assay. Immunization of baboons with IPV failed to generate neutralizing antibody responses against enterovirus D68 or A71. These results suggest that a multivalent approach to enterovirus vaccination is necessary to protect against enterovirus disease in vulnerable populations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Importance: Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants, infecting all children by age 5. RSV also causes substantial morbidity and mortality in older adults, and a vaccine for older adults based on a prefusion-stabilized form of the viral F glycoprotein was recently approved by the FDA. Here, we investigate a set of antibodies that belong to the same public clonotype and were isolated from individuals vaccinated with a prefusion-stabilized RSV F protein. Our results reveal that these antibodies are highly potent and recognize a previously uncharacterized antigenic site on the prefusion F protein. Vaccination with prefusion RSV F proteins appears to boost the elicitation of these neutralizing antibodies, which are not commonly elicited by natural infection., Competing Interests: J.S.M. and B.S.G. are inventors on a patent entitled "Prefusion RSV F proteins and their use" (US Patent no. 9738689).

Nanoparticles for multivalent display and delivery of vaccine antigens have emerged as a promising avenue for enhancing B cell responses to protein subunit vaccines. Here, we evaluated B cell responses in rhesus macaques immunized with prefusion-stabilized respiratory syncytial virus (RSV) F glycoprotein trimer compared with nanoparticles displaying 10 or 20 copies of the same antigen. We show that multivalent display skews antibody specificities and drives epitope-focusing of responding B cells. Antibody cloning and repertoire sequencing revealed that focusing was driven by the expansion of clonally distinct B cells through recruitment of diverse precursors. We identified two antibody lineages that developed either ultrapotent neutralization or pneumovirus cross-neutralization from precursor B cells with low initial affinity for the RSV-F immunogen. This suggests that increased avidity by multivalent display facilitates the activation and recruitment of these cells. Diversification of the B cell response by multivalent nanoparticle immunogens has broad implications for vaccine design., Competing Interests: Declaration of interests N.P.K., B.F., L.P., and J.M. are named as inventors on patent applications filed by the University of Washington related to the data presented in this work. N.P.K. is a co-founder and shareholder in Icosavax, a company that has licensed the patent applications, and is a member of Icosavax’s Scientific Advisory Board. The King lab has received unrelated sponsored research agreements from Pfizer and GSK. B.S.G. is an inventor on patents for the stabilization of the RSV-F protein (WO2014160463A1, preF RSV-F proteins and their use)., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

After decades of work, several interventions to prevent severe respiratory syncytial virus (RSV) disease in high-risk infant and older adult populations have finally been approved. There were many setbacks along the road to victory. In this review, I will discuss the impact of RSV on human health and how structure-based vaccine design set the stage for numerous RSV countermeasures to advance through late phase clinical evaluation. While there are still many RSV countermeasures in preclinical and early-stage clinical trials, this review will focus on products yielding long-awaited efficacy results. Finally, I will discuss some challenges and next steps needed to declare a global victory against RSV., (© 2023. Springer Nature Limited.)

New vaccine delivery technologies, such as mRNA, have played a critical role in the rapid and efficient control of SARS-CoV-2, helping to end the COVID-19 pandemic. Enveloped virus-like particles (eVLPs) are often more immunogenic than protein subunit immunogens and could be an effective vaccine platform. Here, we investigated whether the genetic delivery of eVLPs could achieve strong immune responses in mice as previously reported with the immunization of in vitro purified eVLPs. We utilized Newcastle disease virus-like particles (NDVLPs) to display SARS-CoV-2 prefusion-stabilized spikes from the WA-1 or Beta variant (S-2P or S-2Pᵦ, respectively) and evaluated neutralizing murine immune responses achieved by a single-gene-transcript DNA construct for the WA-1 or Beta variant (which we named S-2P-NDVLP-1T and S-2Pᵦ-NDVLP-1T, respectively), by multiple-gene-transcript DNA constructs for the Beta variant (S-2Pᵦ-NDVLP-3T), and by a protein subunit-DNA construct for the WA-1 or Beta variant (S-2P-TM or S-2Pᵦ-TM, respectively). The genetic delivery of S-2P-NDVLP-1T or S-2Pᵦ-NDVLP-1T yielded modest neutralizing responses after a single immunization and high neutralizing responses after a second immunization, comparable to previously reported results in mice immunized with in vitro purified S-2P-NDVLPs. Notably, genetic delivery of S-2Pᵦ-NDVLP-3T yielded significantly higher neutralizing responses in mice after a second immunization than S-2Pᵦ-NDVLP-1T or S-2Pᵦ-TM. Genetic delivery also elicited high spike-specific T-cell responses. Collectively, these results indicate that genetic delivery can provide an effective means to immunize eVLPs and that a multiple-gene transcript eVLP platform may be especially efficacious and inform the design of improved vaccines.

Enterovirus D68 (EV-D68) causes severe respiratory illness in children and can result in a debilitating paralytic disease known as acute flaccid myelitis. No treatment or vaccine for EV-D68 infection is available. Here, we demonstrate that virus-like particle (VLP) vaccines elicit a protective neutralizing antibody against homologous and heterologous EV-D68 subclades. VLP based on a B1 subclade 2014 outbreak strain elicited comparable B1 EV-D68 neutralizing activity as an inactivated viral particle vaccine in mice. Both immunogens elicited weaker cross-neutralization against heterologous viruses. A B3 VLP vaccine elicited more robust neutralization of B3 subclade viruses with improved cross-neutralization. A balanced CD4 + T helper response was achieved using a carbomer-based adjuvant, Adjuplex. Nonhuman primates immunized with this B3 VLP Adjuplex formulation generated robust neutralizing antibodies against homologous and heterologous subclade viruses. Our results suggest that both vaccine strain and adjuvant selection are critical elements for improving the breadth of protective immunity against EV-D68.

Zika virus (ZIKV), an arbovirus transmitted by mosquitoes, was identified as a cause of congenital disease during a major outbreak in the Americas in 2016. Vaccine design strategies relied on limited available isolate sequence information due to the rapid response necessary. The first-generation ZIKV mRNA vaccine, mRNA-1325, was initially generated and, as additional strain sequences became available, a second mRNA vaccine, mRNA-1893, was developed. Herein, we compared the immune responses following mRNA-1325 and mRNA-1893 vaccination and reported that mRNA-1893 generated comparable neutralizing antibody titers to mRNA-1325 at 1/20th of the dose and provided complete protection from ZIKV challenge in non-human primates. In-depth characterization of these vaccines indicated that the observed immunologic differences could be attributed to a single amino acid residue difference that compromised mRNA-1325 virus-like particle formation., (© 2023. The Author(s).)

Respiratory syncytial virus is the second most common cause of infant mortality and a major cause of morbidity and mortality in older adults (aged >60 years). Efforts to develop a respiratory syncytial virus vaccine or immunoprophylaxis remain highly active. 33 respiratory syncytial virus prevention candidates are in clinical development using six different approaches: recombinant vector, subunit, particle-based, live attenuated, chimeric, and nucleic acid vaccines; and monoclonal antibodies. Nine candidates are in phase 3 clinical trials. Understanding the epitopes targeted by highly neutralising antibodies has resulted in a shift from empirical to rational and structure-based vaccine and monoclonal antibody design. An extended half-life monoclonal antibody for all infants is likely to be within 1 year of regulatory approval (from August, 2022) for high-income countries. Live-attenuated vaccines are in development for older infants (aged >6 months). Subunit vaccines are in late-stage trials for pregnant women to protect infants, whereas vector, subunit, and nucleic acid approaches are being developed for older adults. Urgent next steps include ensuring access and affordability of a respiratory syncytial virus vaccine globally. This review gives an overview of respiratory syncytial virus vaccines and monoclonal antibodies in clinical development highlighting different target populations, antigens, and trial results., Competing Interests: Declaration of interests UMCU received minor funding from The Bill & Melinda Gates Medical Research Institute. DMW has received consulting fees from Pfizer, Merck, Affinivax, and Matrivax, unrelated to this manuscript; and is a principal investigator with grant support from Pfizer and Merck to Yale University, unrelated to this manuscript. NIM has regular interaction with pharameutical and other industrial partners: UMC Utrecht has received fees for invited lectures by Abbvie, Merck, and Sanofi. LB has regular interaction with pharmaceutical and other industrial partners. BSG is an inventor of patents for RSV vaccines using the stabilised prefusion F protein. AM has received research grants from National Institutes of Health, Janssen, and Merck institution; fees for participation in advisory boards from Janssen, Sanofi-Pasteur, and Merck; and fees for educational lectures from Sanofi-Pasteur and AstraZeneca. TJR was financially supported by the Intramural Program of the National Institute of Allergy & Infectious Diseases, National Institute of Health. BSG was financially supported by the Intramural Program of the National Institute of Allergy & Infectious Diseases, National Institute of Health. UJB was financially supported by the Intramural Program of the National Institute of Allergy & Infectious Diseases, National Institute of Health. UMCU has received major funding (>€100 000 per industrial partner) for investigator-initiated studies from AbbVie, MedImmune, Janssen, Pfizer, the Bill & Melinda Gates Foundation, and MeMed Diagnostics. UMCU has received major cash or in-kind funding as part of the public private partnership IMI-funded RESCEU project from GSK, Novavax, Janssen, AstraZeneca, Pfizer, and Sanofi. UMCU has received major funding by Julius Clinical for participating in the INFORM study sponsored by MedImmune. UMCU has received minor funding for participation in trials by Regeneron and Janssen from 2015-17 (total annual estimate less than €20 000). UMCU received minor funding for consultation and invited lectures by AbbVie, MedImmune, Ablynx, Bavaria Nordic, MabXience, Novavax, Pfizer, and Janssen (total annual estimate less than €20 000). Dr. Bont is the founding chairman of the ReSViNET Foundation., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

There is currently no licensed vaccine for respiratory syncytial virus (RSV). Here, we assess the effect of RSV fusion protein (F) conformation on B cell responses in a post hoc comparison of samples from the DS-Cav1 [prefusion (pre-F)] and MEDI7510 [postfusion (post-F)] vaccine clinical trials. We compared the magnitude and quality of the serological and B cell responses across time points and vaccines. We measured RSV A and B neutralization, F-binding immunoglobulin G titers, and competition assays at week 0 (before vaccination) and week 4 (after vaccination) to evaluate antibody specificity and potency. To compare B cell specificity and activation, we used pre-F and post-F probes in tandem with a 17-color immunophenotyping flow cytometry panel at week 0 (before vaccination) and week 1 (after vaccination). Our data demonstrate that both DS-Cav1 and MEDI7510 vaccination robustly elicit F-specific antibodies and B cells, but DS-Cav1 elicited antibodies that more potently neutralized both RSV A and B. The superior potency was mediated by antibodies that bind antigenic sites on the apex of pre-F that are not present on post-F. In the memory (CD27 + ) B cell compartment, vaccination with DS-Cav1 or MEDI7510 elicited B cells with different epitope specificities. B cells preferentially binding the pre-F probe were activated in DS-Cav1-vaccinated participants but not in MEDI7510-vaccinated participants. Our findings emphasize the importance of using pre-F as an immunogen in humans because of its deterministic role in eliciting highly potent neutralizing antibodies and memory B cells.

An important consequence of infection with a SARS-CoV-2 variant is protective humoral immunity against other variants. However, the basis for such cross-protection at the molecular level is incompletely understood. Here, we characterized the repertoire and epitope specificity of antibodies elicited by infection with the Beta, Gamma and WA1 ancestral variants and assessed their cross-reactivity to these and the more recent Delta and Omicron variants. We developed a method to obtain immunoglobulin sequences with concurrent rapid production and functional assessment of monoclonal antibodies from hundreds of single B cells sorted by flow cytometry. Infection with any variant elicited similar cross-binding antibody responses exhibiting a conserved hierarchy of epitope immunodominance. Furthermore, convergent V gene usage and similar public B cell clones were elicited regardless of infecting variant. These convergent responses despite antigenic variation may account for the continued efficacy of vaccines based on a single ancestral variant., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Recurrent respiratory syncytial virus (RSV) infection requiring hospitalization is rare and the underlying mechanism is unknown. We aimed to determine the role of CD14-mediated immunity in the pathogenesis of recurrent RSV infection., Methods: We performed genotyping and longitudinal immunophenotyping of the first patient with a genetic CD14 deficiency who developed recurrent RSV infection. We analyzed gene expression profiles and interleukin (IL)-6 production by patient peripheral blood mononuclear cells in response to RSV pre- and post-fusion (F) protein. We generated CD14-deficient human nasal epithelial cells cultured at air-liquid interface (HNEC-ALI) of patient-derived cells and after CRISPR-based gene editing of control cells. We analyzed viral replication upon RSV infection., Results: Sanger sequencing revealed a homozygous single-nucleotide deletion in CD14, resulting in absence of the CD14 protein in the index patient. In vitro, viral replication was similar in wild-type and CD14-/- HNEC-ALI. Loss of immune cell CD14 led to impaired cytokine and chemokine responses to RSV pre- and post-F protein, characterized by absence of IL-6 production., Conclusions: We report an association of recurrent RSV bronchiolitis with a loss of CD14 function in immune cells. Lack of CD14 function led to defective immune responses to RSV pre- and post-F protein without a change in viral replication., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Respiratory syncytial virus is a leading cause of morbidity and mortality in children, due in part to their distinct immune system, characterized by impaired induction of Th 1 immunity. Here we show application of cationic adjuvant formulation CAF08, a liposomal vaccine formulation tailored to induce Th 1 immunity in early life via synergistic engagement of Toll-like Receptor 7/8 and the C-type lectin receptor Mincle. We apply quantitative phosphoproteomics to human dendritic cells and reveal a role for Protein Kinase C-δ for enhanced Th1 cytokine production in neonatal dendritic cells and identify signaling events resulting in antigen cross-presentation. In a murine in vivo model a single immunization at birth with CAF08-adjuvanted RSV pre-fusion antigen protects newborn mice from RSV infection by induction of antigen-specific CD8 + T-cells and Th1 cells. Overall, we describe a pediatric adjuvant formulation and characterize its mechanism of action providing a promising avenue for development of early life vaccines against RSV and other respiratory viral pathogens., (© 2022. The Author(s).)

Respiratory syncytial virus (RSV) is a substantial cause of morbidity and mortality globally. A candidate RSV prefusion (pre-F)-stabilized subunit vaccine, DS-Cav1, has previously been shown to elicit potent and durable neutralizing activity in a phase 1 clinical trial in healthy adults. Here, we used fluorescently labeled probes and flow cytometry to evaluate the antigen specificity and phenotype of RSV F-specific B cells longitudinally after DS-Cav1 immunization. Peripheral blood mononuclear cells (PBMCs) collected at time points before the first immunization through the end of the trial at 44 weeks were assessed by flow cytometry. Our data demonstrate a rapid increase in the frequency of pre-F-specific IgG + and IgA + B cells after the first immunization and a modest increase after a second immunization at week 12. Nearly all F-specific B cells down-regulated CD21 and up-regulated the proliferation marker CD71 after the first immunization, with less pronounced activation after the second immunization. Memory B cells (CD27 + CD21 + ) specific for pre-F remained elevated above baseline at 44 weeks after vaccination. DS-Cav1 vaccination also activated human metapneumovirus (HMPV) cross-reactive B cells capable of binding prefusion-stabilized HMPV F protein and increased HMPV F-binding antibodies and neutralizing activity for HMPV in some participants. In summary, vaccination with RSV pre-F resulted in the expansion and activation of RSV and HMPV F-specific B cells that were maintained above baseline for at least 10 months and could contribute to long-term pneumovirus immunity.

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccine-elicited immunity, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring of vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies., Competing Interests: The authors have declared that no competing interests exist.

Nipah virus (NiV) represents a significant pandemic threat with zoonotic transmission from bats-to-humans with almost annual regional outbreaks characterized by documented human-to-human transmission and high fatality rates. Currently, no vaccine against NiV has been approved. Structure-based design and protein engineering principles were applied to stabilize the fusion (F) protein in its prefusion trimeric conformation (pre-F) to improve expression and increase immunogenicity. We covalently linked the stabilized pre-F through trimerization domains at the C-terminus to three attachment protein (G) monomers, forming a chimeric design. These studies detailed here focus on mRNA delivery of NiV immunogens in mice, assessment of mRNA immunogen-specific design elements and their effects on humoral and cellular immunogenicity. The pre-F/G chimera elicited a strong neutralizing antibody response and a superior NiV-specific Tfh and other effector T cell response compared to G alone across both the mRNA and protein platforms. These findings enabled final candidate selection of pre-F/G Fd for clinical development., Competing Interests: Authors SF, VP, EN, SH and AC were employed by company Moderna Inc. YT is employed by Leidos Biomedical Research, Inc., supported in part with funds from the Frederick National Laboratory for Cancer Research, NIH, under contract HHSN261200800001. RL, GS-J, JM, and BG are inventors on patent applications involving Nipah virus vaccine designs. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Loomis, DiPiazza, Falcone, Ruckwardt, Morabito, Abiona, Chang, Caringal, Presnyak, Narayanan, Tsybovsky, Nair, Hutchinson, Stewart-Jones, Kueltzo, Himansu, Mascola, Carfi and Graham.)

Background: Multiple active vaccination approaches have proven ineffective in reducing the substantial morbidity and mortality caused by respiratory syncytial virus (RSV) in infants and older adults (aged ≥65 years). A vaccine conferring a substantial and sustainable boost in neutralising activity is required to protect against severe RSV disease. To that end, we evaluated the safety and immunogenicity of DS-Cav1, a prefusion F subunit vaccine., Methods: In this randomised, open-label, phase 1 clinical trial, the stabilised prefusion F vaccine DS-Cav1 was evaluated for dose, safety, tolerability, and immunogenicity in healthy adults aged 18-50 years at a single US site. Participants were assigned to receive escalating doses of either 50 μg, 150 μg, or 500 μg DS-Cav1 at weeks 0 and 12, and were randomly allocated in a 1:1 ratio within each dose group to receive the vaccine with or without aluminium hydroxide (AlOH) adjuvant. After 71 participants had been randomised, the protocol was amended to allow some participants to receive a single vaccination at week 0. The primary objectives evaluated the safety and tolerability at every dose within 28 days following each injection. Neutralising activity and RSV F-binding antibodies were evaluated from week 0 to week 44 as secondary and exploratory objectives. Safety was assessed in all participants who received at least one vaccine dose; secondary and exploratory immunogenicity analysis included all participants with available data at a given visit. The trial is registered with ClinicalTrials.gov, NCT03049488, and is complete and no longer recruiting., Findings: Between Feb 21, 2017, and Nov 29, 2018, 244 participants were screened for eligibility and 95 were enrolled to receive DS-Cav1 at the 50 μg (n=30, of which n=15 with AlOH), 150 μg (n=35, of which n=15 with AlOH), or 500 μg (n=30, of which n=15 with AlOH) doses. DS-Cav1 was safe and well tolerated and no serious vaccine-associated adverse events deemed related to the vaccine were identified. DS-Cav1 vaccination elicited robust neutralising activity and binding antibodies by 4 weeks after a single vaccination (p<0·0001 for F-binding and neutralising antibodies). In analyses of exploratory endpoints at week 44, pre-F-binding IgG and neutralising activity were significantly increased compared with baseline in all groups. At week 44, RSV A neutralising activity was 3·1 fold above baseline in the 50 μg group, 3·8 fold in the 150 μg group, and 4·5 fold in the 500 μg group (p<0·0001). RSV B neutralising activity was 2·8 fold above baseline in the 50 μg group, 3·4 fold in the 150 μg group, and 3·7 fold in the 500 μg group (p<0·0001). Pre-F-binding IgG remained significantly 3·2 fold above baseline in the 50 μg group, 3·4 fold in the 150 μg group, and 4·0 fold in the 500 μg group (p<0·0001). Pre-F-binding serum IgA remained 4·1 fold above baseline in the 50 μg group, 4·3 fold in the 150 μg group, and 4·8 fold in the 500 μg group (p<0·0001). Although a higher vaccine dose or second immunisation elicited a transient advantage compared with lower doses or a single immunisation, neither significantly impacted long-term neutralisation. There was no long-term effect of dose, number of vaccinations, or adjuvant on neutralising activity., Interpretation: In this phase 1 study, DS-Cav1 vaccination was safe and well tolerated. DS-Cav1 vaccination elicited a robust boost in RSV F-specific antibodies and neutralising activity that was sustained above baseline for at least 44 weeks. A single low-dose of pre-F immunisation of antigen-experienced individuals might confer protection that extends throughout an entire RSV season., Funding: The National Institutes of Allergy and Infectious Diseases., Competing Interests: Declaration of interests MC and BSG are inventors on patents for the stabilisation of the RSV F protein. The other authors declared no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers [half-maximal inhibitory concentration (IC 50 ) 0.3 to 11.1 nanograms per milliliter; IC 80 1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Vaccine-associated enhanced respiratory disease (VAERD) was previously observed in some preclinical models of severe acute respiratory syndrome (SARS) and MERS coronavirus vaccines. We used the SARS coronavirus 2 (SARS-CoV-2) mouse-adapted, passage 10, lethal challenge virus (MA10) mouse model of acute lung injury to evaluate the immune response and potential for immunopathology in animals vaccinated with research-grade mRNA-1273. Whole-inactivated virus or heat-denatured spike protein subunit vaccines with alum designed to elicit low-potency antibodies and Th2-skewed CD4 + T cells resulted in reduced viral titers and weight loss post challenge but more severe pathological changes in the lung compared to saline-immunized animals. In contrast, a protective dose of mRNA-1273 induced favorable humoral and cellular immune responses that protected from viral replication in the upper and lower respiratory tract upon challenge. A subprotective dose of mRNA-1273 reduced viral replication and limited histopathological manifestations compared to animals given saline. Overall, our findings demonstrate an immunological signature associated with antiviral protection without disease enhancement following vaccination with mRNA-1273., Competing Interests: Declaration of interests O.M.A., K.S.C., and B.S.G. are inventors on pending patent applications related to coronavirus vaccines. S.R.L. and R.S.B. have pending patents on recombinant viruses used in this study. A.T.D., D.K.E., and A.C. are current employees and shareholders of Moderna, Inc. Other authors declare no competing interests., (Published by Elsevier Inc.)

Background: Respiratory syncytial virus (RSV) is a major cause of lower respiratory infection among children and no vaccine is available. The stabilized form of the fusion (F) protein - pre-F - is a leading vaccine candidate to target different populations, including pregnant women. This study aimed to determine the magnitude and nature of RSV-directed maternal antibodies (matAbs) in hospitalized children with RSV infection., Methods: Sixty-five paired blood samples were collected from RSV-infected children aged <6 months and their corresponding mothers. All pairs were screened for levels of pre-F and post-F antibodies using ELISA. The neutralizing antibodies (NAbs) in both groups were measured in vitro against mKate RSV-A2 using H28 cells., Results: It was found that 14% of matAbs (log 2 12.8) were present in infants at hospitalization, with an average log 2 EP titer of 10.2 directed to both F-protein conformations. Additionally, 61.4% of maternal NAbs (log 2 EC 50  = 9.4) were detected in infants (log 2 EC 50  = 8.7), which were mostly pre-F exclusive (81%). Pre-F antibodies in children showed a positive correlation with matAbs titers and negative correlations with age and bronchiolitis score., Conclusions: The maintenance of neutralizing activity in infants relative to maternal titers was greater than the maintenance of antibody binding based on ELISA, suggesting that higher-potency antibodies may have a longer half-life than weakly neutralizing antibodies., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) substantially increases serum-neutralizing activity in healthy adults. We sought to determine whether DS-Cav1 vaccination induces a repertoire mirroring the pre-existing diversity from natural infection or whether antibody lineages targeting specific epitopes predominate. We evaluated RSV F-specific B cell responses before and after vaccination in six participants using complementary B cell sequencing methodologies and identified 555 clonal lineages. DS-Cav1-induced lineages recognized the prefusion conformation of F (pre-F) and were genetically diverse. Expressed antibodies recognized all six antigenic sites on the pre-F trimer. We identified 34 public clonotypes, and structural analysis of two antibodies from a predominant clonotype revealed a common mode of recognition. Thus, vaccination with DS-Cav1 generates a diverse polyclonal response targeting the antigenic sites on pre-F, supporting the development and advanced testing of pre-F-based vaccines against RSV., Competing Interests: Declaration of interests B.S.G., J.S.M., and P.D.K. are inventors on a patent entitled “Prefusion RSV F proteins and their use” (US Patent No. 9738689)., (Published by Elsevier Inc.)

SARS-CoV-2 first emerged in the human population in late 2019 in Wuhan, China, and in a matter of months, spread across the globe resulting in the Coronavirus Disease 19 (COVID-19) pandemic and substantial economic fallout. SARS-CoV-2 is transmitted between humans via respiratory particles, with infection presenting a spectrum of clinical manifestations ranging from asymptomatic to respiratory failure with multiorgan dysfunction and death in severe cases. Prior experiences with human pathogenic coronaviruses and respiratory virus diseases in general have revealed an important role for cellular immunity in limiting disease severity. Here, we review some of the key mechanisms underlying cell-mediated immunity to respiratory viruses and summarize our current understanding of the functional capacity and role of SARS-CoV-2-specific T cells following natural infection and vaccination., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Background: Vaccines to prevent coronavirus disease 2019 (Covid-19) are urgently needed. The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines on viral replication in both upper and lower airways is important to evaluate in nonhuman primates., Methods: Nonhuman primates received 10 or 100 μg of mRNA-1273, a vaccine encoding the prefusion-stabilized spike protein of SARS-CoV-2, or no vaccine. Antibody and T-cell responses were assessed before upper- and lower-airway challenge with SARS-CoV-2. Active viral replication and viral genomes in bronchoalveolar-lavage (BAL) fluid and nasal swab specimens were assessed by polymerase chain reaction, and histopathological analysis and viral quantification were performed on lung-tissue specimens., Results: The mRNA-1273 vaccine candidate induced antibody levels exceeding those in human convalescent-phase serum, with live-virus reciprocal 50% inhibitory dilution (ID 50 ) geometric mean titers of 501 in the 10-μg dose group and 3481 in the 100-μg dose group. Vaccination induced type 1 helper T-cell (Th1)-biased CD4 T-cell responses and low or undetectable Th2 or CD8 T-cell responses. Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of the eight animals in the 100-μg dose group by day 2 after challenge, and limited inflammation or detectable viral genome or antigen was noted in lungs of animals in either vaccine group., Conclusions: Vaccination of nonhuman primates with mRNA-1273 induced robust SARS-CoV-2 neutralizing activity, rapid protection in the upper and lower airways, and no pathologic changes in the lung. (Funded by the National Institutes of Health and others.)., (Copyright © 2020 Massachusetts Medical Society.)

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity 1 . This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant 2 SARS-CoV-2 as well as CD8 + T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.

Zika virus (ZIKV) is the cause of a pandemic associated with microcephaly in newborns and Guillain-Barre syndrome in adults. Currently, there are no available treatments or vaccines for ZIKV, and the development of a safe and effective vaccine is a high priority for many global health organizations. We describe the development of ZIKV vaccine candidates using the self-amplifying messenger RNA (SAM) platform technology delivered by cationic nanoemulsion (CNE) that allows bedside mixing and is particularly useful for rapid responses to pandemic outbreaks. Two immunizations of either of the two lead SAM (CNE) vaccine candidates elicited potent neutralizing antibody responses to ZIKV in mice and nonhuman primates. Both SAM (CNE) vaccines protected these animals from ZIKV challenge, with one candidate providing complete protection against ZIKV infection in nonhuman primates. The data provide a preclinical proof of concept that a SAM (CNE) vaccine candidate can rapidly elicit protective immunity against ZIKV., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The emergence of Zika virus (ZIKV) in the Americas stimulated the development of multiple ZIKV vaccine candidates. We previously developed two related DNA vaccine candidates encoding ZIKV structural proteins that were immunogenic in animal models and humans. We sought to identify neutralizing antibody (NAb) properties induced by each vaccine that correlated with protection in nonhuman primates (NHPs). Despite eliciting equivalent NAb titers in NHPs, these vaccines were not equally protective. The transfer of equivalent titers of vaccine-elicited NAb into AG129 mice also revealed nonequivalent protection, indicating qualitative differences among antibodies (Abs) elicited by these vaccines. Both vaccines elicited Abs with similar binding titers against envelope protein monomers and those incorporated into virus-like particles, as well as a comparable capacity to orchestrate phagocytosis. Functional analysis of vaccine-elicited NAbs from NHPs and humans revealed a capacity to neutralize the structurally mature form of the ZIKV virion that varied in magnitude among vaccine candidates. Conversely, sensitivity to the virion maturation state was not a characteristic of NAbs induced by natural or experimental infection. Passive transfer experiments in mice revealed that neutralization of mature ZIKV virions more accurately predicts protection from ZIKV infection. These findings demonstrate that NAb correlates of protection may differ among vaccine antigens when assayed using standard neutralization platforms and suggest that measurements of Ab quality, including the capacity to neutralize mature virions, will be critical for defining correlates of ZIKV vaccine-induced immunity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

This 20-color flow cytometry panel was designed to resolve the cellular heterogeneity of antigen-presenting cells and was optimized for lymph node tissue. Reagents were carefully selected and optimized for identification of B cells (B220), neutrophils (Ly6G), monocytes and macrophages (Ly6C, CD169, F4/80), and dendritic cells (XCR1, CD172a, CD11c, I-A/I-E, CD24, CD64, pDCA-1, CD103, CD11b). Inclusion of additional functional markers involved in cell migration (CCR7), co-stimulation (CD80), and adhesion (ICAM-1) enabled further phenotypic characterization. Finally, this panel has been tested and is compatible with fluorescently labeled antigens such as Alexa Fluor 488 (Ax488) for the study of antigen-bearing cells in vivo. Published 2019. This article is a U.S. Government work and is in the public domain in the USA., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Respiratory syncytial virus (RSV) has eluded active vaccination efforts for more than five decades and continues to cause substantial morbidity and mortality in infants, the immunocompromised, and older adults. Although newer approaches of passive antibody-mediated protection show promise, vaccines aimed at eliciting fusion protein (F)-targeting antibodies have repeatedly failed to meet pre-established, modest-efficacy goals. Newer candidates, including protein-based vaccines, live-attenuated viruses, and gene-based delivery platforms, incorporate structurally defined and stabilized versions of the prefusion form of the F glycoprotein and are advancing rapidly into critical efficacy studies in susceptible target populations. This review discusses the storied history of RSV vaccine development, immunological lessons learned along the way, and critical findings about protein structure that remodeled our understanding of protective immunity to this important pathogen., (Published by Elsevier Inc.)

Technologies that define the atomic-level structure of neutralization-sensitive epitopes on viral surface proteins are transforming vaccinology and guiding new vaccine development approaches. Previously, iterative rounds of protein engineering were performed to preserve the prefusion conformation of the respiratory syncytial virus (RSV) fusion (F) glycoprotein, resulting in a stabilized subunit vaccine candidate (DS-Cav1), which showed promising results in mice and macaques. Here, phase I human immunogenicity data reveal a more than 10-fold boost in neutralizing activity in serum from antibodies targeting prefusion-specific surfaces of RSV F. These findings represent a clinical proof of concept for structure-based vaccine design, suggest that development of a successful RSV vaccine will be feasible, and portend an era of precision vaccinology., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in children and older adults. An effective vaccine must elicit neutralizing antibodies targeting the RSV fusion (F) protein, which exists in two major conformations, pre-fusion (pre-F) and post-fusion (post-F). Although 50% of the surface is shared, pre-F contains highly neutralization-sensitive antigenic sites not present on post-F. Recent advancement of several subunit F-based vaccine trials has spurred interest in quantifying and understanding the protective potential of antibodies directed to individual antigenic sites. Monoclonal antibody competition ELISAs are being used to measure these endpoints, but the impact of F conformation and competition from antibodies binding to adjacent antigenic sites has not been thoroughly investigated. Since this information is critical for interpreting clinical trial outcomes and defining serological correlates of protection, we optimized assays to evaluate D25-competing antibodies (DCA) to antigenic site Ø on pre-F, and compared readouts of palivizumab-competing antibodies (PCA) to site II on both pre-F and post-F. We show that antibodies to adjacent antigenic sites can contribute to DCA and PCA readouts, and that cross-competition from non-targeted sites is especially confounding when PCA is measured using a post-F substrate. While measuring DCA and PCA levels may be useful to delineate the role of antibodies targeting the apex and side of the F protein, respectively, the assay limitations and caveats should be considered when conducting immune monitoring during vaccine trials and defining correlates of protection.

Tissue-resident memory T (T RM ) cells provide first-line defense against invading pathogens encountered at barrier sites. In the lungs, T RM cells protect against respiratory infections, but wane more quickly than T RM cells in other tissues. This lack of a sustained T RM population in the lung parenchyma explains, at least in part, why infections with some pathogens, such as influenza virus and respiratory syncytial virus (RSV), recur throughout life. Intranasal (IN) vaccination with a murine cytomegalovirus (MCMV) vector expressing the M protein of RSV (MCMV-M) has been shown to elicit robust populations of CD8 + T RM cells that accumulate over time and mediate early viral clearance. To extend this finding, we compared the inflationary CD8 + T cell population elicited by MCMV-M vaccination with a conventional CD8 + T cell population elicited by an MCMV vector expressing the M2 protein of RSV (MCMV-M2). Vaccination with MCMV-M2 induced a population of M2-specific CD8 + T RM cells that waned rapidly, akin to the M2-specific CD8 + T RM cell population elicited by infection with RSV. In contrast to the natural immunodominance profile, however, coadministration of MCMV-M and MCMV-M2 did not suppress the M-specific CD8 + T cell response, suggesting that progressive expansion was driven by continuous antigen presentation, irrespective of the competitive or regulatory effects of M2-specific CD8 + T cells. Moreover, effective viral clearance mediated by M-specific CD8 + T RM cells was not affected by the coinduction of M2-specific CD8 + T cells. These data show that memory inflation is required for the maintenance of CD8 + T RM cells in the lungs after IN vaccination with MCMV.

Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels-Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo.

A better understanding of the seroprevalence and specificity of influenza HA stem-directed broadly neutralizing antibodies (bNAbs) in the human population could significantly inform influenza vaccine design efforts. Here, we utilized probes comprising headless, HA stabilized stem (SS) to determine the prevalence, binding and neutralization breadth of antibodies directed to HA stem-epitope in a cross-sectional analysis of the general population. Five group-1 HA SS probes, representing five subtypes, were chosen for this analyses. Eighty-four percent of samples analyzed had specific reactivity to at least one probe, with approximately 60% of the samples reactive to H1 probes, and up to 45% reactive to each of the non-circulating subtypes. Thirty percent of analyzed sera had cross-reactivity to at least four of five probes and this reactivity could be blocked by competing with F10 bNAb. Binding cross-reactivity in sera samples significantly correlated with frequency of H1 + H5 + cross-reactive B cells. Interestingly, only 33% of the cross-reactive sera neutralized both H1N1 and H5N1 pseudoviruses. Cross-reactive and neutralizing antibodies were more prevalent in individuals >50 years of age. Our data demonstrate the need to use multiple HA-stem probes to assess for broadly reactive antibodies. Further, a universal vaccine could be designed to boost pre-existing B-cells expressing stem-directed bNAbs.

The CD103 + subset of lung-migratory dendritic cells (DCs) plays an important role in the generation of CD8 + T cell responses following respiratory infection. Here, we demonstrate that the dependence on CD103 + DCs for stimulation of RSV-specific T cells is both epitope and age-dependent. CD103 + DCs in neonatal mice develop two phenotypically and functionally distinct populations following respiratory infection. Neonatal CD103 + DCs expressing low levels of CD103 (CD103lo DCs) and other lineage and maturation markers including costimulatory molecules are phenotypically immature and functionally limited. CD103lo DCs sorted from infected neonates were unable to stimulate cells of the K d M2 82-90 specificity, which are potently stimulated by CD103hi DCs sorted from the same animals. These data suggest that the delayed maturation of CD103 + DCs in the neonate limits the K d M2 82-90 -specific response and explain the distinct CD8 + T cell response hierarchy displayed in neonatal mice that differs from the hierarchy seen in adult mice. These findings have implications for the development of early-life vaccines, where the promotion of responses with less age bias may prove advantageous. Alternately, specific approaches may be used to enhance the maturation and function of the CD103lo DC population in neonates to promote more adult-like T cell responses.

Young infants are generally more susceptible to viral infections and experience more severe disease than do adults. CD8 + T cells are important for viral clearance, and although often ineffective in neonates they can be protective when adequately stimulated. Using a murine CB6F1/J hybrid model of respiratory syncytial virus (RSV) infection, we previously demonstrated that the CD8 + T cell immunodominance hierarchy to two RSV-derived epitopes, K d M2 82-90 and D b M 187-195 , was determined by the age at infection. To determine whether age-dependent RSV-specific CD8 + T cell responses could be modified through enhanced innate signaling, we used TLR4 or TLR9 agonist treatment at the time of infection, which remarkably changed the neonatal codominant response to an adult-like K d M2 82-90 CD8 + T cell immunodominant response. This shift was associated with an increase in the number of conventional dendritic cells, CD11b + and CD103 + dendritic cells, in the lung-draining lymph node, as well as increased expression of the costimulatory molecule CD86. The magnitude of the K d M2 82-90 CD8 + T cell response in TLR agonist-treated neonates could be blocked with Abs against CD80 and CD86. These studies demonstrate the age-dependent function of conventional dendritic cells, their role in determining immunodominance hierarchy, and epitope-specific CD8 + T cell requirements for costimulation, all of which influence the immune response magnitude. The unique impact of TLR agonists on neonatal T cell responses is important to consider for RSV vaccines designed for young infants.

Structure-based vaccine design has been used to develop immunogens that display conserved neutralization sites on pathogens such as HIV-1, respiratory syncytial virus (RSV), and influenza. Improving the immunogenicity of these designed immunogens with adjuvants will require formulations that do not alter protein antigenicity. Here, we show that nanoparticle-forming thermoresponsive polymers (TRP) allow for co-delivery of RSV fusion (F) protein trimers with Toll-like receptor 7 and 8 agonists (TLR-7/8a) to enhance protective immunity. Although primary amine conjugation of TLR-7/8a to F trimers severely disrupted the recognition of critical neutralizing epitopes, F trimers site-selectively coupled to TRP nanoparticles retained appropriate antigenicity and elicited high titers of prefusion-specific, T H 1 isotype anti-RSV F antibodies following vaccination. Moreover, coupling F trimers to TRP delivering TLR-7/8a resulted in ∼3-fold higher binding and neutralizing antibody titers than soluble F trimers admixed with TLR-7/8a and conferred protection from intranasal RSV challenge. Overall, these data show that TRP nanoparticles may provide a broadly applicable platform for eliciting neutralizing antibodies to structure-dependent epitopes on RSV, influenza, HIV-1, or other pathogens.

Respiratory syncytial virus causes significant morbidity and mortality in both developed and developing countries, and a vaccine that adequately protects from severe disease remains an important unmet need. RSV disease has an inordinate impact on the very young, and the physical and immunological immaturity of early life complicates vaccine design. Defining and targeting the functional capacities of early life immune responses and controlling responses during primary antigen exposure with selected vaccine delivery approaches will be important for protecting infants by active immunization. Alternatively, vaccination of older children and pregnant mothers may ameliorate disease burden indirectly until infants reach about six months of age, when they can generate more effective anti-RSV immune responses., (Published by Elsevier B.V.)

In this study, we compared two lines of transgenic CD8+ T cells specific for the same KdM282-90 epitope of respiratory syncytial virus in the CB6F1 hybrid mouse model. Here we found that these two transgenic lines had similar in vivo abilities to control viral load after respiratory syncytial virus infection using adoptive transfer. Transfer of the TRBV13-2 line resulted in higher levels of IL-6 and MIP1-α in the lung than TRBV13-1 transfer. Interestingly, when large numbers of cells were co-transferred, the lines formed a hierarchy, with TRBV13-2 being immunodominant over TRBV13-1 in the mediastinal lymph node despite no identifiable difference in proliferation or apoptosis between the lines. This hierarchy was not established when lower cell numbers were transferred. The phenotype and frequency of proliferating cells were also cell transfer dose-dependent with higher percentages of CD127loCD62LloKLRG1lo and proliferating cells present when lower numbers of cells were transferred. These results illustrate the importance of cell number in adoptive transfer experiments and its influence on the phenotype and hierarchy of the subsequent T cell response.

Subunit vaccines provide a safe, focused alternative to conventional vaccines. However, these vaccines often require significant adjuvants and are particularly hard to target toward cytotoxic T lymphocyte (CTL) immunity. Viruslike particles (VLPs) provide biomaterial scaffolds with pathogen-like polyvalent structures making them useful platforms for biomimetic antigen delivery to the immune system. Encapsidation of antigens within VLPs has been shown to enhance antigen availability for CD8 T cell responses. Here, we examine the potential to generate complex responses to multiple subunit antigens localized within the same VLP particle. Two proteins of respiratory syncytial virus (RSV) with well-characterized CD8 T cell responses, the matrix (M) and matrix 2 (M2) proteins, were successfully coencapsidated within the P22 VLP. Upon intranasal administration in mice, the particles stimulated CD8 T cell memory responses against both antigens. In addition, vaccination elicited tissue-resident T cell populations. Upon subsequent RSV challenge, P22-M/M2-treated mice displayed significantly reduced lung viral titers. This demonstrates the utility of the P22 VLP in directing immune responses to multiple encapsidated viral antigens, demonstrating the potential of this technology to facilitate immunity to multiple targets simultaneously., Competing Interests: The authors declare no competing financial interest.

The activation of CD8(+) T-cells requires the uptake of exogenous polypeptide antigens and proteolytic processing of these antigens to octamer or nonamer peptides, which are loaded on MHC-I complexes and presented to the T-cell. By using an azide as a bioorthogonal protecting group rather than as a ligation handle, masked antigens were generated-antigens that are not recognized by their cognate T-cell unless they are deprotected on the cell using a Staudinger reduction., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Our previous work has characterized the functional and clonotypic features of two respiratory syncytial virus (RSV) epitope-specific T cell responses in mice. Following single-cell sequencing, we selected T cell receptor sequences to represent both a public and a private clone specific for the dominant K(d)M2(82-90) epitope for the generation of T cell receptor transgenic (TCR Tg) mice. We evaluated cells from these TCR Tg strains for three major functions of CD8+ T cells: proliferation, cytokine production and cytolytic activity. In vitro comparisons of the functional characteristics of T cells from the newly-generated mice demonstrated many similarities in their responsiveness to cognate antigen stimulation. Cells from both TRBV13-1 (private) and TRBV13-2 (public) TCR Tg mice had similar affinity, and proliferated similarly in vitro in response to cognate antigen stimulation. When transferred to BALB/c mice, cells from both strains demonstrated extensive proliferation in mediastinal lymph nodes following RSV infection, with TRBV13-2 demonstrating better in vivo proliferation. Both strains similarly expressed cytokines and chemokines following stimulation, and had similar Granzyme B and perforin expression, however cells expressing TRBV13-1 demonstrated better cytolytic activity than TRBV13-2 cells. These new, well-characterized mouse strains provide new opportunities to study molecular mechanisms that control the phenotype and function of CD8+ T cell responses.

CD103+ and CD11b+ populations of CD11c+MHCIIhi murine dendritic cells (DCs) have been shown to carry antigens from the lung through the afferent lymphatics to mediastinal lymph nodes (MLN). We compared the responses of these two DC populations in neonatal and adult mice following intranasal infection with respiratory syncytial virus. The response in neonates was dominated by functionally-limited CD103+ DCs, while CD11b+ DCs were diminished in both number and function compared to adults. Infecting mice at intervals through the first three weeks of life revealed an evolution in DC phenotype and function during early life. Using TCR transgenic T cells with two different specificities to measure the ability of CD103+ DC to induce epitope-specific CD8+ T cell responses, we found that neonatal CD103+ DCs stimulate proliferation in a pattern distinct from adult CD103+ DCs. Blocking CD28-mediated costimulatory signals during adult infection demonstrated that signals from this costimulatory pathway influence the hierarchy of the CD8+ T cell response to RSV, suggesting that limited costimulation provided by neonatal CD103+ DCs is one mechanism whereby neonates generate a distinct CD8+ T cell response from that of adults.

Infants in the first 6 months of life and older adults (>65 years of age) are disproportionately burdened with respiratory syncytial virus (RSV)-associated morbidity and mortality. While other factors play a role in the risk these groups assume, shortcomings of the immune response make a substantial contribution to the predisposition to severe disease. Ineffectual antibody production with misdirected cytokine responses and excess inflammation in the airways are common to both groups. However, the mechanisms underlying these immune responses differ between infants and older adults and need to be better understood. Preventative approaches to decreasing the burden of disease are preferable to therapeutic intervention and effective vaccination strategies will need to target the strengths of the immune responses in these populations.

Following respiratory syncytial virus infection of adult CB6F1 hybrid mice, a predictable CD8+ T cell epitope hierarchy is established with a strongly dominant response to a K(d)-restricted peptide (SYIGSINNI) from the M2 protein. The response to K(d)M2(82-90) is ∼5-fold higher than the response to a subdominant epitope from the M protein (NAITNAKII, D(b)M(187-195)). After infection of neonatal mice, a distinctly different epitope hierarchy emerges with codominant responses to K(d)M2(82-90) and D(b)M(187-195). Adoptive transfer of naïve CD8+ T cells from adults into congenic neonates prior to infection indicates that intrinsic CD8+ T cell factors contribute to age-related differences in hierarchy. Epitope-specific precursor frequency differs between adults and neonates and influences, but does not predict the hierarchy following infection. Additionally, dominance of K(d)M2(82-90)-specific cells does not correlate with TdT activity. Epitope-specific Vβ repertoire usage is more restricted and functional avidity is lower in neonatal mice. The neonatal pattern of codominance changes after infection at 10 days of age, and rapidly shifts to the adult pattern of extreme K(d)M2(82-90)-dominance. Thus, the functional properties of T cells are selectively modified by developmental factors in an epitope-specific and age-dependent manner.