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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.

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.

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.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.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 (p0·0001 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 (p0·0001). 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 (p0·0001). 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 (p0·0001). 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 (p0·0001). 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.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.The National Institutes of Allergy and Infectious Diseases.

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 1/20thof 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.

Zika virus (ZIKV) deoxyribonucleic acid vaccine VRC5283 encoding viral structural genes has been shown to be immunogenic in humans. Recognizing that antigenically related flaviviruses cocirculate in regions with ZIKV activity, we explored the degree of antibody cross-reactivity elicited by this vaccine candidate using genetically diverse flaviviruses. The antibody response of vaccinated individuals with no evidence of prior flavivirus infection or vaccine experience had a limited capacity to bind heterologous viruses. In contrast, vaccine-elicited antibodies from individuals with prior flavivirus experience had a greater capacity to bind, but not neutralize, distantly related flaviviruses. These findings suggest that prior flavivirus exposure shapes the humoral immune response to vaccination.

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.

Background The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and spread globally, prompting an international effort to accelerate development of a vaccine. The candidate vaccine mRNA-1273 encodes the stabilized prefusion SARS-CoV-2 spike protein. Methods We conducted a phase 1, dose-escalation, open-label trial including 45 healthy adults, 18 to 55 years of age, who received two vaccinations, 28 days apart, with mRNA-1273 in a dose of 25 μg, 100 μg, or 250 μg. There were 15 participants in each dose group. Results After the first vaccination, antibody responses were higher with higher dose (day 29 enzyme-linked immunosorbent assay anti–S-2P antibody geometric mean titer [GMT], 40,227 in the 25-μg group, 109,209 in the 100-μg group, and 213,526 in the 250-μg group). After the second vaccination, the titers increased (day 57 GMT, 299,751, 782,719, and 1,192,154, respectively). After the second vaccination, serum-neutralizing activity was detected by two methods in all participants evaluated, with values generally similar to those in the upper half of the distribution of a panel of control convalescent serum specimens. Solicited adverse events that occurred in more than half the participants included fatigue, chills, headache, myalgia, and pain at the injection site. Systemic adverse events were more common after the second vaccination, particularly with the highest dose, and three participants (21%) in the 250-μg dose group reported one or more severe adverse events. Conclusions The mRNA-1273 vaccine induced anti–SARS-CoV-2 immune responses in all participants, and no trial-limiting safety concerns were identified. These findings support further development of this vaccine. (Funded by the National Institute of Allergy and Infectious Diseases and others; mRNA-1273 ClinicalTrials.gov number, NCT04283461).

Background Respiratory syncytial virus (RSV) is a leading cause of viral pneumonia and bronchiolitis during the first 6 months of life. Placentally transferred antibodies can prevent severe RSV illness, and maternal immunization may reduce illness in young infants. Identification of protective antibody levels facilitates the advancement of vaccine candidates and maternal immunization. Methods We conducted a nested case-control study with 587 Malian mother–infant pairs, followed from birth to age 6 months. RSV cases were infants who developed influenza-like illness (ILI) or pneumonia and were RSV-positive by polymerase chain reaction. Cases were matched to healthy controls and RSV-negative ILI controls. RSV-A and RSV-B neutralizing antibodies were measured in maternal, cord blood, and infant sera at age 3 and 6 months. Results Maternal antibodies were efficiently transferred to infants. Maternal and infant RSV titers were strongly correlated. Infant antibody titers against RSV-A were 3 times higher than those against RSV-B. At birth, infants who remained healthy had significantly higher RSV-A and RSV-B titers compared with infants who subsequently contracted RSV. RSV-A inhibitory concentration (IC)80 titer >239 or RSV-B titer >60 at birth was significantly associated with being a healthy control compared with an RSV case within the first 3 months of life. RSV-A IC80 titers in cord blood were associated with decreased episodes of pneumonia. Conclusions Maternally acquired RSV antibodies were associated with protection of infants against community-detected cases of RSV-ILI and pneumonia. RSV titers in cord blood can predict whether an infant will be infected with RSV or remain uninfected., Maternally acquired respiratory syncytial virus (RSV) neutralizing antibodies were associated with infant protection against RSV up to 3 months. These findings indicate that RSV titers at birth can predict whether an infant will be infected with RSV or remain uninfected in early life.

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., Graphical abstract, As vaccine-enhanced disease to respiratory viruses has been previously observed, a thorough safety evaluation of COVID-19 vaccines in preclinical animal models is essential. Here, DiPiazza and Leist et al. provide evidence for antiviral protection in the absence of lung disease following SARS-CoV-2 challenge in mice immunized with research-grade mRNA-1273.

SARS-CoV-2 mutations may diminish vaccine-induced protective immune responses, and the durability of such responses has not been previously reported. Here, we present a comprehensive assessment of the impact of variants B.1.1.7, B.1.351, P.1, B.1.429, and B.1.526 on binding, neutralizing, and ACE2-blocking antibodies elicited by the vaccine mRNA-1273 over seven months. Cross-reactive neutralizing responses were rare after a single dose of mRNA-1273. At the peak of response to the second dose, all subjects had robust responses to all variants. Binding and functional antibodies against variants persisted in most subjects, albeit at low levels, for 6 months after the primary series of mRNA-1273. Across all assays, B.1.351 had the greatest impact on antibody recognition, and B.1.1.7 the least. These data complement ongoing studies of clinical protection to inform the potential need for additional boost vaccinations., One-Sentence Summary: Most mRNA-1273 vaccinated individuals maintained binding and functional antibodies against SARS-CoV-2 variants for 6 months.

A Zika self-amplifying mRNA vaccine delivered by a flexible bedside mixing formulation induces protective immunity in NHPs., 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.

Summary A severe acute respiratory syndrome coronavirus (SARS-CoV-2) vaccine is needed to control the global coronavirus infectious disease (COVID-19) public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins1. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody responses to wild-type (D614) and D614G mutant2 SARS-CoV-2 and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in Phase 3 efficacy evaluation.

Licensed vaccines or therapeutics are rarely available for pathogens with epidemic or pandemic potential. Developing interventions for specific pathogens and defining generalizable approaches for related pathogens is a global priority and inherent to the UN Sustainable Development Goals. Nipah virus (NiV) poses a significant epidemic threat, and zoonotic transmission from bats-to-humans with high fatality rates occurs almost annually. Human-to-human transmission of NiV has been documented in recent outbreaks leading public health officials and government agencies to declare an urgent need for effective vaccines and therapeutics. Here, we evaluate NiV vaccine antigen design options including the fusion glycoprotein (F) and the major attachment glycoprotein (G). A stabilized prefusion F (pre-F), multimeric G constructs, and chimeric proteins containing both pre-F and G were developed as protein subunit candidate vaccines. The proteins were evaluated for antigenicity and structural integrity using kinetic binding assays, electron microscopy, and other biophysical properties. Immunogenicity of the vaccine antigens was evaluated in mice. The stabilized pre-F trimer and hexameric G immunogens both induced serum neutralizing activity in mice, while the post-F trimer immunogen did not elicit neutralizing activity. The pre-F trimer covalently linked to three G monomers (pre-F/G) induced potent neutralizing antibody activity, elicited responses to the greatest diversity of antigenic sites, and is the lead candidate for clinical development. The specific stabilizing mutations and immunogen designs utilized for NiV were successfully applied to other henipaviruses, supporting the concept of identifying generalizable solutions for prototype pathogens as an approach to pandemic preparedness.

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 KdM282–90 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 KdM282–90-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.

Cytomegalovirus vectors are promising delivery vehicles for vaccine strategies that aim to elicit effector CD8+ T cells. To determine how the route of immunization affects CD8+ T-cell responses in the lungs of mice vaccinated with a murine cytomegalovirus vector expressing the respiratory syncytial virus matrix (M) protein, we infected CB6F1 mice via the intranasal or intraperitoneal route and evaluated the M-specific CD8+ T-cell response at early and late time points. We found that intranasal vaccination generated robust and durable tissue-resident effector and effector memory CD8+ T-cell populations that were undetectable after intraperitoneal vaccination. The generation of these antigen-experienced cells by intranasal vaccination resulted in earlier T-cell responses, interferon gamma secretion, and viral clearance after respiratory syncytial virus challenge. Collectively, these findings validate a novel approach to vaccination that emphasizes the route of delivery as a key determinant of immune priming at the site of vulnerability.

An effective vaccine for RSV is an unrealized public health goal. Recently, a single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) was shown to substantially increase serum neutralizing activity in healthy adults. To understand the induced B-cell repertoire at the single-cell level, we evaluated RSV F-specific B-cell responses before and after vaccination in six participants 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 previously defined antigenic sites on the pre-F trimer. Among the six vaccinees, we identified 34 public clonotypes. Structural analysis of two antibodies from a predominant clonotype revealed a common mode of recognition. Collectively, these findings demonstrate that vaccination with DS-Cav1 generates a diverse polyclonal response targeting all known antigenic sites present on pre-F.

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 immunogenicity1. 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 mutant2 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.

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 (ID50) 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.).

Over six decades after respiratory syncytial virus (RSV) was recognized as a significant human pathogen, an effective vaccine against it has remained elusive. RSV vaccine development has been hindered by early vaccine approaches that caused enhanced illness and the immunological challenges associated with populations that have the highest burden of severe RSV disease: the very young and the elderly. Recent developments in the understanding of the structure of the RSV fusion protein and dissection of neutralizing antibody responses have provided new insights into previous vaccine failures and have identified key aspects of protection, reinvigorating the RSV vaccine field. Several approaches tailored to protect high-risk target populations are competing in the current pipeline, raising the prospects for licensure of candidate vaccines for active immunization against RSV in the foreseeable future.

Zika virus (ZIKV) infection of pregnant women is associated with congenital Zika syndrome (CZS) and no vaccine is available, although several are being tested in clinical trials. We tested the efficacy of ZIKV DNA vaccine VRC5283 in a rhesus macaque model of congenital ZIKV infection. Most animal vaccine experiments have a set pathogen exposure several weeks or months after vaccination. In the real world, people encounter pathogens years or decades after vaccination, or may be repeatedly exposed if the virus is endemic. To more accurately mimic how this vaccine would be used, we immunized macaques prior to conception, and then exposed them repeatedly to ZIKV during early and mid-gestation. In comparison to unimmunized animals, vaccinated animals had a significant reduction in peak magnitude and duration of maternal viremia, early fetal loss, fetal infection, and placental and fetal brain pathology. Vaccine-induced neutralizing antibody titers on the day of first ZIKV exposure were negatively associated with the magnitude of maternal viremia, and the absence of prolonged viremia was associated with better fetal outcomes. These data support further clinical development of ZIKV vaccine strategies to protect against negative fetal outcomes.

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.

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.

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 &Oslash, 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.

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.

A DNA vaccine candidate for Zika The ongoing Zika epidemic in the Americas and the Caribbean urgently needs a protective vaccine. Two DNA vaccines composed of the genes that encode the structural premembrane and envelope proteins of Zika virus have been tested in monkeys. Dowd et al. show that two doses of vaccine given intramuscularly completely protected 17 of 18 animals against Zika virus challenge. A single low dose of vaccine was not protective but did reduce viral loads. Protection correlated with serum antibody neutralizing activity. Phase I clinical trials testing these vaccines are already ongoing. Science , this issue p. 237

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.

Building a better RSV vaccine Respiratory syncytial virus (RSV) causes severe respiratory disease, especially in infants and the elderly. However, attempts to produce effective human vaccines have largely been unsuccessful. Structure-based design has been used to generate an RSV fusion glycoprotein stabilized in its prefusion conformation (DS-Cav1). This immunogen is highly effective in mice and macaques. Crank et al. now report the results of a phase I vaccine clinical trial using the stabilized prefusion DS-Cav1 molecule. Four weeks after immunization, these vaccines elicited substantially more high-quality antibody titers than those typically generated using earlier RSV immunogens. The findings provide a proof of concept for how structural biology can contribute to precision vaccine design. Science , this issue p. 505

The emergence of Zika virus in Brazil and its association with microcephaly and Guillain-Barre syndrome led to accelerated vaccine development efforts. Based on prior flavivirus vaccine development programs, knowledge of flavivirus particle structure, definition of E dimers as the key antigenic target, and deep understanding of neutralizing mechanisms, multiple vaccine strategies have advanced to the stage of clinical evaluation with unprecedented speed. These include nucleic acid (DNA and messenger RNA), whole-inactivated virus, live-attenuated or chimeric virus, and protein or viruslike particle vaccines. Within a year from the declaration by the World Health Organization of Zika virus as a Public Health Emergency of International Concern, multiple vaccine candidates entered clinical trials, now totaling 7 products with an additional 40-plus candidate vaccines in preclinical development. The rapid progress in vaccine development demonstrates the capacity of governments, public health organizations, and the scientific community to respond to pandemic threats when sufficient prior knowledge exists, emergency funding is made available, and interagency cooperation is achieved and serves as a paradigm for preparing for future emerging infectious diseases.

Summary Background The Zika virus epidemic and associated congenital infections have prompted rapid vaccine development. We assessed two new DNA vaccines expressing premembrane and envelope Zika virus structural proteins. Methods We did two phase 1, randomised, open-label trials involving healthy adult volunteers. The VRC 319 trial, done in three centres, assessed plasmid VRC5288 (Zika virus and Japanese encephalitis virus chimera), and the VRC 320, done in one centre, assessed plasmid VRC5283 (wild-type Zika virus). Eligible participants were aged 18–35 years in VRC19 and 18–50 years in VRC 320. Participants were randomly assigned 1:1 by a computer-generated randomisation schedule prepared by the study statistician. All participants received intramuscular injection of 4 mg vaccine. In VRC 319 participants were assigned to receive vaccinations via needle and syringe at 0 and 8 weeks, 0 and 12 weeks, 0, 4, and 8 weeks, or 0, 4, and 20 weeks. In VRC 320 participants were assigned to receive vaccinations at 0, 4, and 8 weeks via single-dose needle and syringe injection in one deltoid or split-dose needle and syringe or needle-free injection with the Stratis device (Pharmajet, Golden, CO, USA) in each deltoid. Both trials followed up volunteers for 24 months for the primary endpoint of safety, assessed as local and systemic reactogenicity in the 7 days after each vaccination and all adverse events in the 28 days after each vaccination. The secondary endpoint in both trials was immunogenicity 4 weeks after last vaccination. These trials are registered with ClinicalTrials.gov, numbers NCT02840487 and NCT02996461. Findings VRC 319 enrolled 80 participants (20 in each group), and VRC 320 enrolled 45 participants (15 in each group). One participant in VRC 319 and two in VRC 320 withdrew after one dose of vaccine, but were included in the safety analyses. Both vaccines were safe and well tolerated. All local and systemic symptoms were mild to moderate. In both studies, pain and tenderness at the injection site was the most frequent local symptoms (37 [46%] of 80 participants in VRC 319 and 36 [80%] of 45 in VRC 320) and malaise and headache were the most frequent systemic symptoms (22 [27%] and 18 [22%], respectively, in VRC 319 and 17 [38%] and 15 [33%], respectively, in VRC 320). For VRC5283, 14 of 14 (100%) participants who received split-dose vaccinations by needle-free injection had detectable positive antibody responses, and the geometric mean titre of 304 was the highest across all groups in both trials. Interpretation VRC5283 was well tolerated and has advanced to phase 2 efficacy testing. Funding Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Zika virus infection during pregnancy can cause congenital abnormities or fetal demise. The persistence of Zika virus in the male reproductive system poses a risk of sexual transmission. Here we demonstrate that live-attenuated Zika virus vaccine candidates containing deletions in the 3′ untranslated region of the Zika virus genome (ZIKV-3′UTR-LAV) prevent viral transmission during pregnancy and testis damage in mice, as well as infection of nonhuman primates. After a single-dose vaccination, pregnant mice challenged with Zika virus at embryonic day 6 and evaluated at embryonic day 13 show markedly diminished levels of viral RNA in maternal, placental, and fetal tissues. Vaccinated male mice challenged with Zika virus were protected against testis infection, injury, and oligospermia. A single immunization of rhesus macaques elicited a rapid and robust antibody response, conferring complete protection upon challenge. Furthermore, the ZIKV-3′UTR-LAV vaccine candidates have a desirable safety profile. These results suggest that further development of ZIKV-3′UTR-LAV is warranted for humans., Zika virus infection can result in congenital disorders and cause disease in adults, and there is currently no approved vaccine. Here Shan et al. show that a single dose of a live-attenuated Zika vaccine prevents infection, testis damage and transmission to the fetus during pregnancy in different animal models.

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, TH1 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.

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, KdM282–90 and DbM187–195, 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 KdM282–90 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 KdM282–90 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.

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.

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.

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., Author Summary Respiratory syncytial virus (RSV) infection is most severe in infants under six months and the most common cause of hospitalization for lower respiratory tract infection in children under five years. Disease is a consequence of virus- and T-cell-mediated pathology. Adaptive immune responses to viral respiratory infections are initiated by dendritic cells (DCs) that traffic to lymph nodes from the infected lungs. We compared the phenotype and function of two lung-migratory DC populations, identified by high expression of MHC Class II and CD103+ (CD103+DCs) or CD11b+ (CD11b+DCs), in mice infected in early life or as adults. We found that DC subsets undergo dramatic quantitative and qualitative changes during the first weeks of life, and CD103+DCs from neonatal mediastinal lymph nodes induced a fundamentally different CD8+ T-cell response profile than CD103+DCs from adults. The adult response pattern required CD28-mediated costimulatory signals, which is a limiting functional property of neonatal CD103+DCs. Thus, the ability of neonatal CD103+DCs to provide sufficient costimulation to neonatal CD8+ T-cells can influence the immunodominance hierarchy and functional properties of neonatal CD8+ T-cell responses compared to those of adults. A better understanding of deficiencies in early life immunity will guide vaccine approaches that induce disease-sparing immune responses in infants.

CD8 T cells are involved in pathogen clearance and infection-induced pathology in respiratory syncytial virus (RSV) infection. Studying bulk responses masks the contribution of individual CD8 T cell subsets to protective immunity and immunopathology. In particular, the roles of subdominant responses that are potentially beneficial to the host are rarely appreciated when the focus is on magnitude instead of quality of response. Here, by evaluating CD8 T cell responses in CB6F1 hybrid mice, in which multiple epitopes are recognized, we found that a numerically subdominant CD8 T cell response against DbM187 epitope of the virus matrix protein expressed high avidity TCR and enhanced signaling pathways associated with CD8 T cell effector functions. Each DbM187 T effector cell lysed more infected targets on a per cell basis than the numerically dominant KdM282 T cells, and controlled virus replication more efficiently with less pulmonary inflammation and illness than the previously well-characterized KdM282 T cell response. Our data suggest that the clinical outcome of viral infections is determined by the integrated functional properties of a variety of responding CD8 T cells, and that the highest magnitude response may not necessarily be the best in terms of benefit to the host. Understanding how to induce highly efficient and functional T cells would inform strategies for designing vaccines intended to provide T cell-mediated immunity., Author Summary CD8 T cells play a key role in RSV clearance, immunopathology and disease. Therefore, CD8 T cells can help or harm the host depending on their timing, magnitude, and function. The CD8 T cell response represents a heterogeneous population of cells with phenotypically and functionally diverse subsets, and needs to at least be studied at the level of epitope specificity to understand how to diminish the risk of immunopathology. Studying the bulk response masks distinct contributions of individual CD8 T subsets to immunity and immunopathology. Focusing on CD8 T cell response with the highest magnitude overlooks role of subdominant responses. Here, we studied response to different epitopes and revealed that a numerically subdominant CD8 T cell response against DbM187 epitope of the virus matrix protein controlled virus replication efficiently with limited pulmonary inflammation and illness compared to the previously well-characterized and numerically dominant KdM282 T cell response. Our data show that selectively boosting of epitope-specific CD8 T cell responses may be more beneficial than indiscriminant boosting of all available epitopes to achieve rapid viral clearance while limiting immunopathology. This work has implications for antigen design of vaccines intended to induce T-cell-mediated immunity.