Natural Respiratory Syncytial Virus Research Articles

Prevalence and Genetic Diversity of Respiratory Syncytial Virus Reinfections in Young Thai Children, 2016-2023.

Although a vaccine for respiratory syncytial virus (RSV) is now available for pregnant women and the elderly, RSV remains a significant cause of respiratory illness in children globally. Reinfections by the same or different RSV subgroups in children residing in the tropics are currently under-studied. Therefore, we examined the patterns of RSV infection and reinfection in Thai children aged ≤ 5 years with respiratory symptoms from 2016 to 2023. Screening of 7710 pediatric respiratory specimens identified 1245 RSV-positive samples (16.1%), mostly from the rainy months (July-November). Interestingly, 74 children experienced two infections, and 6 had three infections. Reinfection by different RSV subgroups occurred in 30 children: 21 were initially infected with RSV-B and later with RSV-A, while 9 had the reverse pattern. Reinfections only by either RSV-A or RSV-B were observed in 22 and 2 children, respectively, with one child infected with RSV-A three times. All RSV-A reinfections belonged to the ON1 genotype, while RSV-B reinfections were BA9. Notably, reinfections across different seasons were observed within homologous pairs. These findings suggest a transitory immunity to natural RSV infection and provide the knowledge that may help optimize pediatric vaccination schedule. Ongoing epidemiological data on RSV are essential in monitoring genotype circulation and vaccine effectiveness.

Longer interval between maternal RSV vaccination and birth increases placental transfer efficiency.

Respiratory Syncytial Virus (RSV) is associated with significant neonatal and infant morbidity and mortality. Maternal bivalent RSVpreF RSV vaccination to protect neonates and infants was approved in September 2023 for administration between 32+0 and 36+6 weeks to protect neonates and infants. This approved timeframe is narrower than the 24-36 week window evaluated in the clinical trial, due to the possible association between preterm birth and vaccine administration. Currently, data are lacking on how maternal vaccine timing within the approved window affects the transfer of antibodies from mother to fetus, critical information that could influence clinical practice. We sought to examine how gestational age at vaccination and time elapsed from maternal RSV vaccination to delivery impacted transfer of maternal antibodies measured in the umbilical cord at delivery and in peripheral blood of 2-month infants. We also examined differences in maternal and cord RSV antibody levels achieved by vaccination versus natural RSV infection. A prospective cohort study was conducted at two academic medical centers between September 20, 2023 and March 21, 2024, enrolling 124 individuals who received the RSV vaccine during pregnancy. Infant capillary blood was collected at 2 months of age from 29 of the infants. Maternal and cord IgG levels achieved by RSV vaccination were compared to those associated with maternal natural RSV infection, using banked blood from 20 maternal:cord dyads collected prior to the availability of the maternal RSV vaccine. Levels of IgG against RSV strain A2 and B fusion (F) and attachment (G) proteins and against pertussis toxin (as a comparator antigen from a vaccine routinely administered earlier in pregnancy) were measured using a Binding Antibody Multiplex Assay. Differences in titers between vaccination and natural infection were examined using Wilcoxon rank sum test. Differences in cord:maternal transfer ratios and 2-month infant antibody levels by timing of maternal vaccination were evaluated by Kruskal-Wallis testing. Maternal RSV vaccination resulted in significantly higher maternal and cord anti-F RSV antibody levels than natural infection (5.72 vs 4.82 log 10 MFI, p < 0.0001 maternal; 5.81 vs 5.03 log 10 MFI, p < 0.0001 cord). Maternal vaccination 2-3 weeks and 3-4 weeks prior to delivery was associated with significantly lower cord:maternal transfer ratios than were observed when vaccination occurred > 5 weeks prior to delivery (p = 0.03 for 2-3 weeks, p = 0.007 for 3-4 weeks), and significantly lower transfer ratios than observed for pertussis vaccination administered prior to 30 weeks' gestation (p = 0.008 for 2-3 weeks, p = 0.03 for 3-4 weeks, similar at > 4 weeks). Vaccine administration earlier in the approved 32-36 week window (at least 5 weeks prior to delivery) results in the highest transplacental transfer of maternal antibodies to the neonate. These results should inform the counseling of pregnant individuals on optimal vaccination timing.

Generation of novel respiratory syncytial virus vaccine candidate antigens that can induce high levels of prefusion-specific antibodies

Respiratory syncytial virus (RSV) infection is an acute respiratory infection caused by RSV. It occurs worldwide, and for over 50 years, several attempts have been made to research and develop vaccines to prevent RSV infection; effective preventive vaccines are eagerly awaited. The RSV fusion (F) protein, which has gained attention as a vaccine antigen, causes a dynamic structural change from the preF to postF state. Therefore, the structural changes in proteins must be regulated to produce a vaccine antigen that can efficiently induce antibodies with high virus-neutralizing activity. We successfully discovered several mutations that stabilized the antigen site Ø in the preF state, trimerized it, and improved the level of protein expression through observation and computational analysis of the RSV-F protein structure and amino acid mutation analysis of RSV strains. The four RSV-F protein mutants that resulted from the combination of these effective mutations stably conserved a wide range of preF- and trimeric preF-specific epitopes with high virus-neutralizing activity. Absorption assay using human serum revealed that mutants constructed bound to antibodies with virus-neutralizing activity that were induced by natural RSV infection, whereas they hardly bound to anti-postF antibodies without virus-neutralizing activity. Furthermore, mouse immunization demonstrated that our constructed mutants induced a high percentage of antibodies that bind to the preF-specific antigen site. These characteristics suggest that the mutants constructed can be superior vaccine antigens from the viewpoint of RSV infection prevention effect and safety.

A Historical Perspective on Respiratory Syncytial Virus Prevention: A Journey Spanning Over Half a Century From the Setback of an Inactive Vaccine Candidate to the Success of Passive Immunization Strategy.

The efforts to prevent respiratory syncytial virus (RSV) infection in infants span over half a century. RSV vaccine development began in the 1960s, and it confronted a significant disappointment after testing a formalin-inactivated RSV (FI RSV) vaccine candidate. This inactivated RSV vaccine was not protective. A large number of the vaccinated RSV-naive children, when subsequently exposed to natural RSV infection from wild-type virus in the community, developed severe lung inflammation termed enhanced respiratory disease. This resulted in a halt in RSV vaccine development. In the 1990s, attention turned to the potential for passive protection against severe RSV disease with immunoglobulin administration. This led to studies on using standard intravenous immunoglobulins in high-risk infants, followed by high-titer RSV immunoglobulin preparation and, subsequently, the development of RSV monoclonal antibodies. Over the past 25 years, palivizumab has been recognized as a safe and effective monoclonal antibody as a prevention strategy for RSV in high-risk children. Its high cost and need for monthly administration, however, has hindered its use to ~2% of the birth cohort, neglecting the vast majority of newborns, including healthy full-term infants who comprise the largest portion of RSV hospitalizations and the greatest part of the burden of RSV disease. Still these efforts, helped pave the way for the present advances in RSV prevention that hold promise for mitigating severe RSV disease for all infants.

Ally, adversary, or arbitrator? The context-dependent role of eosinophils in vaccination for respiratory viruses and subsequent breakthrough infections.

Eosinophils are a critical type of immune cell and central players in type 2 immunity. Existing literature suggests that eosinophils also can play a role in host antiviral responses, typically type 1 immune events, against multiple respiratory viruses, both directly through release of antiviral mediators and indirectly through activation of other effector cell types. One way to prime host immune responses toward effective antiviral responses is through vaccination, where typically a type 1-skewed immunity is desirable in the context of intracellular pathogens like respiratory viruses. In the realm of breakthrough respiratory viral infection in vaccinated hosts, an event in which virus can still establish productive infection despite preexisting immunity, eosinophils are most prominently known for their link to vaccine-associated enhanced respiratory disease upon natural respiratory syncytial virus infection. This was observed in a pediatric cohort during the 1960s following vaccination with formalin-inactivated respiratory syncytial virus. More recent research has unveiled additional roles of the eosinophil in respiratory viral infection and breakthrough infection. The specific contribution of eosinophils to the quality of vaccine responses, vaccine efficacy, and antiviral responses to infection in vaccinated hosts remains largely unexplored, especially regarding their potential roles in protection. On the basis of current findings, we will speculate upon the suggested function of eosinophils and consider the many potential ways by which eosinophils may exert protective and pathological effects in breakthrough infections. We will also discuss how to balance vaccine efficacy with eosinophil-related risks, as well as the use of eosinophils and their products as potential biomarkers of vaccine efficacy or adverse events.

A human antibody potently neutralizes RSV by targeting the conserved hydrophobic region of prefusion F.

Respiratory syncytial virus (RSV) continues to pose serious threats to pediatric populations due to the lack of a vaccine and effective antiviral drugs. RSV fusion (F) glycoprotein mediates viral-host membrane fusion and is a key target for neutralizing antibodies. We generated 23 full-human monoclonal antibodies (hmAbs) against prefusion F protein (pre-F) from a healthy adult with natural RSV infection by single B cell cloning technique. A highly potent RSV-neutralizing hmAb, named as 25-20, is selected, which targets a new site Ø-specific epitope. Site-directed mutagenesis and structural modelling analysis demonstrated that 25-20 mainly targets a highly conserved hydrophobic region located at the a4 helix and a1 helix of pre-F, indicating a site of vulnerability for drug and vaccine design. It is worth noting that 25-20 uses an unreported inferred germline (iGL) that binds very poorly to pre-F, thus high levels of somatic mutations are needed to gain high binding affinity with pre-F. Our observation helps to understand the evolution of RSV antibody during natural infection. Furthermore, by in silico prediction and experimental verification, we optimized 25-20 with KD values as low as picomolar range. Therefore, the optimized 25-20 represents an excellent candidate for passive protection against RSV infection.

Prior respiratory syncytial virus infection reduces vaccine-mediated Th2-skewed immunity, but retains enhanced RSV F-specific CD8 T cell responses elicited by a Th1-skewing vaccine formulation.

Respiratory syncytial virus (RSV) remains the most common cause of lower respiratory tract infections in children worldwide. Development of a vaccine has been hindered due the risk of enhanced respiratory disease (ERD) following natural RSV exposure and the young age (<6 months) at which children would require protection. Risk factors linked to the development of ERD include poorly neutralizing antibody, seronegative status (never been exposed to RSV), and a Th2-type immune response. Stabilization of the more antigenic prefusion F protein (PreF) has reinvigorated hope for a protective RSV vaccine that elicits potent neutralizing antibody. While anecdotal evidence suggests that children and adults previously exposed to RSV (seropositive) are not at risk for developing vaccine associated ERD, differences in host immune responses in seropositive and seronegative individuals that may protect against ERD remain unclear. It is also unclear if vaccine formulations that skew towards Th1- versus Th2-type immune responses increase pathology or provide greater protection in seropositive individuals. Therefore, the goal of this work was to compare the host immune response to a stabilized prefusion RSV antigen formulated alone or with Th1 or Th2 skewing adjuvants in seronegative and seropositive BALB/c mice. We have developed a novel BALB/c mouse model whereby mice are first infected with RSV (seropositive) and then vaccinated during pregnancy to recapitulate maternal immunization strategies. Results of these studies show that prior RSV infection mitigates vaccine-mediated skewing by Th1- and Th2-polarizing adjuvants that was observed in seronegative animals. Moreover, vaccination with PreF plus the Th1-skewing adjuvant, Advax, increased RSV F85-93-specific CD8 T cells in both seronegative and seropositive dams. These data demonstrate the importance of utilizing seropositive animals in preclinical vaccine studies to assess both the safety and efficacy of candidate RSV vaccines.

Codon-optimization of the respiratory syncytial virus (RSV) G protein expressed in a vesicular stomatitis virus (VSV) vector improves immune responses in a cotton rat model

Respiratory syncytial virus is an important cause of pneumonia in children, the elderly, and immunocompromised individuals. The attachment (G) protein of RSV generates neutralizing antibodies in natural RSV infection which correlate with protection against disease. The immune response to RSV is typically short-lived, which may be related to the heavy glycosylation of RSV-G. In order to improve its immunogenicity, we expressed G protein mutants in a vesicular stomatitis virus (VSV) vector system and tested their ability to protect cotton rats from RSV challenge. We found that the most protective construct was codon-optimized RSV-G, followed by wild-type G and membrane-bound G. Constructs which expressed the G protein with reduced glycosylation or the secreted G protein provided either partial or no protection. Our results demonstrate that modifications to the G protein are not advantageous in a VSV vector system, and that an intact, codon-optimized G is a superior vaccine candidate.

Role of Differences in Respiratory Syncytial Virus F and G Glycoproteins on Susceptibility to Inactivation by Antimicrobial Peptides LL-37 and Human Beta-Defensins.

Antimicrobial peptides are proteins that have been found to be an important factor in the natural immune response to a variety of pathogens. Respiratory syncytial virus (RSV) is a respiratory pathogen with the capability to cause serious upper and lower respiratory infections in infants and children and is a major viral cause of infant mortality. There is currently no functional vaccine for the virus, as recent efforts have been hindered by the virus's low immunogenicity, its ability to effectively mutate, and underlying instabilities of potential vaccines. Previous studies have shown that antimicrobial peptides may affect viral replication and spread of RSV. Our study evaluates the susceptibility of chimeric strains of RSV that express different fusion (F) and attachment (G) proteins to susceptibilities to inactivation by LL-37 and human beta-defensins (hBDs) hBD-1, hBD-3, and hBD-4. We show that LL-37 and hBD-3 result in dose-dependent, strain-independent inactivation of RSV, whereas treatment with either hBD-1 or hBD-4 appears more variable between strains. This suggests a potential role of the viral structural proteins in mitigating the inhibitory effects of the peptides. This study provides the first evidence of the sensitivity of RSV to several hBDs and indicates a role of LL-37 and beta-defensins in both limiting establishment of natural RSV infections and in the therapeutic treatment of severe RSV disease.

Immunohistochemical Investigation of TNF-a Expression in Sheep and Goat Lung Paraffin Blocks Infected with Natural Respiratory Syncytial Virus (RSV)

Respiratory Syncytial Virus (RSV) is an important cause of sheep and goat respiratory tract disease. RSV usually replicates in the airway epithelium and proinflammatory cytokines and chemokines are induced. In this study, it was aimed to investigate TNF-a expression in natural RSV-infected sheep and goat lung paraffin blocks by immunohistochemical method. The study material consisted of twenty nine lung archive paraffin blocks (nineteen sheep and ten goats), which were admitted to Etlik Veterinary Control and Research Institute from Ankara and surrounding provinces with the suspicion of pneumonia between 2015 and 2020. Histopathological findings such as degeneration and desquamation in the bronchi and bronchial epithelium, fibromuscular hypertrophy, hyperplasia in the peribronchial lymphoid tissue, cell infiltration in the interalveolar septum, and no statistical difference was detected in the sheep and goat lung paraffin block tissues (p&gt; 0.05). Immunohistochemically, RSV replication in bronchial and bronchial epithelium and cell debris, bronchial glands, interalveolar septum inflammatory cells, alveolar macrophages was statistically similar in sheep and goats (p&gt; 0.05). It was determined that the expression of TNF-a was more intensely stained in the lung tissue of goats than sheep (p

Adult Memory T Cell Responses to the Respiratory Syncytial Virus Fusion Protein During a Single RSV Season (2018-2019).

Respiratory Syncytial Virus (RSV) is ubiquitous and re-infection with both subtypes (RSV/A and RSV/B) is common. The fusion (F) protein of RSV is antigenically conserved, induces neutralizing antibodies, and is a primary target of vaccine development. Insight into the breadth and durability of RSV-specific adaptive immune response, particularly to the F protein, may shed light on susceptibility to re-infection. We prospectively enrolled healthy adult subjects (n = 19) and collected serum and peripheral blood mononuclear cells (PBMCs) during the 2018–2019 RSV season. Previously, we described their RSV-specific antibody responses and identified three distinct antibody kinetic profiles associated with infection status: uninfected (n = 12), acutely infected (n = 4), and recently infected (n = 3). In this study, we measured the longevity of RSV-specific memory T cell responses to the F protein following natural RSV infection. We stimulated PBMCs with overlapping 15-mer peptide libraries spanning the F protein derived from either RSV/A or RSV/B and found that memory T cell responses mimic the antibody responses for all three groups. The uninfected group had stable, robust memory T cell responses and polyfunctionality. The acutely infected group had reduced polyfunctionality of memory T cell response at enrollment compared to the uninfected group, but these returned to comparable levels by end-of-season. The recently infected group, who were unable to maintain high levels of RSV-specific antibody following infection, similarly had decreased memory T cell responses and polyfunctionality during the RSV season. We observed subtype-specific differences in memory T cell responses and polyfunctionality, with RSV/A stimulating stronger memory T cell responses with higher polyfunctionality even though RSV/B was the dominant subtype in circulation. A subset of individuals demonstrated an overall deficiency in the generation of a durable RSV-specific adaptive immune response. Because memory T cell polyfunctionality may be associated with protection against re-infection, this latter group would likely be at greater risk of re-infection. Overall, these results expand our understanding of the longevity of the adaptive immune response to the RSV fusion protein and should be considered in future vaccine development efforts.

Antibody responses of healthy adults to the p27 peptide of respiratory syncytial virus fusion protein

The respiratory syncytial virus (RSV) fusion (F) protein undergoes two furin-cleavage events to become fusion competent, resulting in the release of a twenty-seven amino acid peptide (p27). Recent studies indicate that the p27 region of the F protein was an immunodominant antigen in young children. In this study, we evaluated the kinetics of the serum antibody response to the p27 peptide following natural RSV reinfection in adults. Nineteen healthy adults under sixty-five years of age were enrolled during the 2018–2019 RSV season in Houston, TX. Blood was collected at three study visits and RSV infection status was defined by changes in neutralizing antibody resulting in three groups: uninfected (n = 12), acutely infected (n = 4), and recently infected (n = 3). Serum IgG and IgA antibodies against RSV/A and RSV/B p27 peptides were measured by enzyme-linked immunosorbent assays, and serum p27-like antibodies were detected by a p27 competitive antibody assay. Anti-p27 antibodies were detected in all subjects at each study visit. The measured IgG and IgA anti-p27 antibody levels followed the same pattern as other RSV site-specific and neutralizing antibody responses described for this cohort previously: the uninfected group had stable responses for the duration of the study period, the acutely infected group had a significant increase following RSV infection, and the recently infected group had a decrease in anti-p27 antibody during the study period. These results indicate that antibodies to the p27 region of the F protein are generated following natural RSV reinfection and suggest that some of the F protein is potentially in a partially cleaved state on the surface of virions, expanding on the previous assumption that all of p27 is post-translationally released and not present on mature F. Additionally, antibody responses were significantly lower (1.4–1.5-fold) toward RSV/B than to RSV/A p27 at each study visit, despite being an RSV/B dominant outbreak. Understanding the mechanism for the differences in the magnitude of the RSV/A and RSV/B p27 antibody response may enhance our understanding of the intracellular processing of the F protein.

A safe and effective mucosal RSV vaccine in mice consisting of RSV phosphoprotein and flagellin variant.

Respiratory syncytial virus (RSV) is a major cause of serious acute lower respiratory tract infection in infants and the elderly. The lack of a licensed RSV vaccine calls for the development of vaccines with other targets and vaccination strategies. Here, we construct a recombinant protein, designated P-KFD1, comprising RSV phosphoprotein (P) and the E.-coli-K12-strain-derived flagellin variant KFD1. Intranasal immunization with P-KFD1 inhibits RSV replication in the upper and lower respiratory tract and protects mice against lung disease without vaccine-enhanced disease (VED). The P-specific CD4+ Tcells provoked by P-KFD1 intranasal (i.n.) immunization either reside in or migrate to the respiratory tract and mediate protection against RSV infection. Single-cell RNA sequencing (scRNA-seq) and carboxyfluorescein succinimidyl ester (CFSE)-labeled cell transfer further characterize the Th1 and Th17 responses induced by P-KFD1. Finally, we find that anti-viral protection depends on either interferon-γ (IFN-γ) or interleukin-17A (IL-17A). Collectively, P-KFD1 is a promising safe and effective mucosal vaccine candidate for the prevention of RSV infection.

Dynamic Host Immune and Transcriptomic Responses to Respiratory Syncytial Virus Infection in a Vaccination-Challenge Mouse Model.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children. Inactivated RSV vaccine was developed in the late 1960's, but the vaccine-enhanced disease (VED) occurred to vaccinated infants upon subsequent natural RSV infection. The excessive inflammatory immunopathology in the lungs might be involved in the VED, but the underlying mechanisms remain not fully understood. In this study, we utilized UV-inactivated RSV in the prime/boost approach followed by RSV challenge in BALB/c mice to mimic RSV VED. The dynamic virus load, cytokines, histology and transcriptome profiles in lung tissues of mice were investigated from day 1 to day 6 post-infection. Compared to PBS-treated mice, UV-RSV vaccination leads to a Th2 type inflammatory response characterized by enhanced histopathology, reduced Treg cells and increased IL4+CD4 T cells in the lung. Enhanced production of several Th2 type cytokines (IL-4, IL-5, IL-10) and TGF-β, reduction of IL-6 and IL-17 were observed in UV-RSV vaccinated mice. A total of 5582 differentially expressed (DE) genes between PBS-treated or vaccinated mice and naïve mice were identified by RNA-Seq. Eleven conserved high-influential modules (HMs) were recognized, majorly grouped into regulatory networks related to cell cycle and cell metabolism, signal transduction, immune and inflammatory responses. At an early time post-infection, the vaccinated mice showed obvious decreased expression patterns of DE genes in 11 HMs compared to PBS-treated mice. The extracellular matrix (HM5) and immune responses (HM8) revealed tremendous differences in expression and regulation characteristics of transcripts between PBS-treated and vaccinated mice at both early and late time points. The highly connected genes in HM5 and HM8 networks were further validated by RT-qPCR. These findings reveal the relationship between RSV VED and immune responses, which could benefit the development of novel RSV vaccines.

A prospective surveillance study on the kinetics of the humoral immune response to the respiratory syncytial virus fusion protein in adults in Houston, Texas

Respiratory syncytial virus (RSV)-specific serum antibody has been correlated to protection of infection and reduction of severe disease, but reinfection is still frequent. In this study, we evaluated RSV-specific serum antibody activity following natural RSV re-infection to examine the longevity of the humoral immune response in adults. Nineteen healthy adult volunteers under sixty-five years of age were enrolled during the 2018–2019 RSV season in Houston, TX. Blood was collected at three study visits. The kinetics of RSV-neutralizing, RSV F site-specific competitive, and RSV-binding antibodies in serum samples were measured by microneutralization and enzyme-linked immunosorbent assays. Three distinct profiles of RSV-specific antibody kinetics were identified that were consistent with RSV infection status: uninfected, acutely infected, and recently infected. The uninfected group had stable antibody titers for the duration of the study period (185 days). The acutely infected group had lower antibody responses at the beginning of the study, supporting a correlate of infection, followed by a significant antibody response after infection that was maintained for at least 125 days. Unlike the acutely infected group, the recently infected group had a significant precipitous decrease in RSV antibody in only 60 days. This study is the first, to our knowledge, to describe this abrupt loss of RSV-specific antibody in detail. This rapid decline of antibody may present an obstacle for the development of vaccines with lasting protection against RSV, and perhaps other respiratory pathogens. Neutralizing antibody responses were greater to prototypic than contemporaneous RSV strains, regardless of infection status, indicating that original antigenic sin may impact the humoral immune response to new or emerging RSV strains.

Live-attenuated Vaccines Prevent Respiratory Syncytial Virus–associated Illness in Young Children

Rationale: Active immunization is needed to protect infants and young children against respiratory syncytial virus (RSV). Rationally designed live-attenuated RSV vaccines are in clinical development.Objectives: Develop preliminary estimates of vaccine efficacy, assess durability of antibody responses to vaccination and "booster" responses after natural RSV infection, and determine sample sizes needed for more precise estimates of vaccine efficacy.Methods: We analyzed data from seven phase 1 trials of live-attenuated RSV vaccines in 6- to 24-month-old children (n = 239).Measurements and Main Results: The five vaccine regimens that induced neutralizing antibody responses in ≥80% of vaccinees (defined post hoc as "more promising") protected against RSV-associated medically attended acute respiratory illness (RSV-MAARI) and medically attended acute lower respiratory illness (RSV-MAALRI) and primed for potent anamnestic responses upon natural exposure to wild-type RSV. Among recipients of "more promising" RSV vaccines, efficacy against RSV-MAARI was 67% (95% confidence interval [CI], 24 to 85; P = 0.008) and against RSV-MAALRI was 88% (95% CI, -9 to 99; P = 0.04). A greater than or equal to fourfold increase in RSV serum neutralizing antibody following vaccination was strongly associated with protection against RSV-MAARI (odds ratio, 0.26; 95% CI, 0.09 to 0.75; P = 0.014) and RSV-MAALRI; no child with a greater than or equal to fourfold increase developed RSV-MAALRI. Rates of RSV-MAARI and RSV-MAALRI in placebo recipients were 21% and 7%, respectively. Given these rates, a study of 540 RSV-naive children would have 90% power to demonstrate ≥55% efficacy against RSV-MAARI and ≥80% efficacy against RSV-MAALRI; if rates were 10% and 3%, a study of 1,300 RSV-naive children would be needed.Conclusions: Rapid development of a live-attenuated RSV vaccine could contribute substantially to reducing the global burden of RSV disease.

Conserved T-cell epitopes of respiratory syncytial virus (RSV) delivered by recombinant live attenuated influenza vaccine viruses efficiently induce RSV-specific lung-localized memory T cells and augment influenza-specific resident memory T-cell responses

Respiratory syncytial virus (RSV) can cause recurrent infection in people because it does not stimulate a long-lived immunological memory. There is an urgent need to develop a safe and efficacious vaccine against RSV that would induce immunological memory without causing immunopathology following natural RSV infection. We have previously generated two recombinant live attenuated influenza vaccine (LAIV) viruses that encode immunodominant T-cell epitopes of RSV M2 protein in the neuraminidase or NS1 genes. These chimeric vaccines afforded protection against influenza and RSV infection in mice, without causing pulmonary eosinophilia or inflammatory RSV disease. The current study assessed the formation of influenza-specific and RSV-specific CD4 and CD8 T-cell responses in the lungs of mice, with special attention to the lung tissue-resident memory T cell subsets (TRM). The RSV epitopes did not affect influenza-specific CD4 effector memory T cell (Tem) levels in the lungs. The majority of these cells formed by LAIV or LAIV-RSV viruses had CD69+CD103- phenotype. Both LAIV+NA/RSV and LAIV+NS/RSV recombinant viruses induced significant levels of RSV M282 epitope-specific lung-localized CD8 Tem cells expressing both CD69 and CD103 TRM markers. Surprisingly, the CD69+CD103+ influenza-specific CD8 Tem responses were augmented by the addition of RSV epitopes, possibly as a result of the local microenvironment formed by the RSV-specific memory T cells differentiating to TRM in the lungs of mice immunized with LAIV-RSV chimeric viruses. This study provides evidence that LAIV vector-based vaccination can induce robust lung-localized T-cell immunity to the inserted T-cell epitope of a foreign pathogen, without altering the immunogenicity of the viral vector itself.

Immune profiles provide insights into respiratory syncytial virus disease severity in young children.

Respiratory syncytial virus (RSV) is associated with major morbidity in infants, although most cases result in mild disease. The pathogenesis of the disease is incompletely understood, especially the determining factors of disease severity. A better characterization of these factors may help with development of RSV vaccines and antivirals. Hence, identification of a "safe and protective" immunoprofile induced by natural RSV infection could be used as a as a surrogate of ideal vaccine-elicited responses in future clinical trials. In this study, we integrated blood transcriptional and cell immune profiling, RSV loads, and clinical data to identify factors associated with a mild disease phenotype in a cohort of 190 children <2 years of age. Children with mild disease (outpatients) showed higher RSV loads, greater induction of interferon (IFN) and plasma cell genes, and decreased expression of inflammation and neutrophil genes versus children with severe disease (inpatients). Additionally, only infants with severe disease had increased numbers of HLA-DRlow monocytes, not present in outpatients. Multivariable analyses confirmed that IFN overexpression was associated with decreased odds of hospitalization, whereas increased numbers of HLA-DRlow monocytes were associated with increased risk of hospitalization. These findings suggest that robust innate immune responses are associated with mild RSV infection in infants.

Unbiased analysis of peripheral blood mononuclear cells reveals CD4 T cell response to RSV matrix protein

Respiratory syncytial virus (RSV) is the most important cause of respiratory tract illness especially in young infants that develop severe disease requiring hospitalization, and accounting for 74,000–126,000 admissions in the United States (Rezaee et al., 2017; Resch, 2017). Observations of neonatal and infant T cells suggest that they may express different immune markers compared to T-cells from older children. Flow cytometry analysis of cellular responses using “conventional” anti-viral markers (IL2, IFN-γ, TNF, IL10 and IL4) upon RSV-peptide stimulation detected an overall low RSV response in peripheral blood. Therefore we sought an unbiased approach to identify RSV-specific immune markers using RNA-sequencing upon stimulation of infant PBMCs with overlapping peptides representing RSV antigens. To understand the cellular response using transcriptional signatures, transcription factors and cell-type specific signatures were used to investigate breadth of response across peptides. Unexpected from the ICS data, M peptide induced a response equivalent to the F-peptide and was characterized by activation of GATA2, 3, STAT3 and IRF1. This along with upregulation of several unconventional T cell signatures was only observed upon M-peptide stimulation. Moreover, signatures of natural RSV infections were identified from the data available in the public domain to investigate similarities between transcriptional signatures from PBMCs and upon peptide stimulation. This analysis also suggested activation of T cell response upon M-peptide stimulation. Hence, based on transcriptional response, markers were chosen to validate the role of M-peptide in activation of T cells. Indeed, CD4+CXCL9+ cells were identified upon M-peptide stimulation by flow cytometry. Future work using additional markers identified in this study could reveal additional unconventional T cells responding to RSV infections in infants. In conclusion, T cell responses to RSV in infants may not follow the canonical Th1/Th2 patterns of effector responses but include additional functions that may be unique to the neonatal period and correlate with clinical outcomes.

In vitro model for the assessment of human immune responses to subunit RSV vaccines.

Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract disease in infants and young children worldwide and a high priority for vaccine development. Despite over 50 years of research, however, no vaccine is yet available. One block to vaccine development is an incomplete understanding of the aberrant memory response to the formalin-inactivated RSV vaccine (FI-RSV) given to children in the 1960s. This vaccine caused enhanced respiratory disease (ERD) with later natural RSV infection. Concern that any non-live virus vaccine may also cause ERD has blocked development of subunit vaccines for young children. A number of animal FI-RSV studies suggest various immune mechanisms behind ERD. However, other than limited data from the original FI-RSV trial, there is no information on the human ERD-associated responses. An in vitro model with human blood specimens may shed light on the immune memory responses likely responsible for ERD. Memory T cell responses to an antigen are guided by the innate responses, particularly dendritic cells that present an antigen in conjunction with co-stimulatory molecules and cytokine signaling. Our in vitro model involves human monocyte derived dendritic cells (moDC) and allogenic T cell cultures to assess innate responses that direct T cell responses. Using this model, we evaluated human responses to live RSV, FI-RSV, and subunit RSV G vaccines (G-containing virus-like particles, G-VLP). Similar to findings in animal studies, FI-RSV induced prominent Th2/Th17-biased responses with deficient type-1 responses compared to live virus. Responses to G-VLPs were similar to live virus, i.e. biased towards a Th1 and not a Th2/Th17. Also mutating CX3C motif in G gave a more pronounced moDC responses associated with type-1 T cell responses. This in vitro model identifies human immune responses likely associated with ERD and provides another pre-clinical tool to assess the safety of RSV vaccines.