Mutations in the F protein of the live-attenuated respiratory syncytial virus vaccine candidate ΔNS2/Δ1313/I1314L increase the stability of infectivity and content of prefusion F protein.

Respiratory syncytial virus (RSV) is the leading viral cause of bronchiolitis and pneumonia in infants and toddlers, but there currently is no licensed pediatric vaccine. A leading vaccine candidate that has been evaluated for intranasal immunization in a recently completed phase 1/2 clinical trial is an attenuated version of RSV strain A2 called RSV/ΔNS2/Δ1313/I1314L (hereafter called ΔNS2). ΔNS2 is attenuated by deletion of the interferon antagonist NS2 gene and introduction into the L polymerase protein gene of a codon deletion (Δ1313) that confers temperature-sensitivity and is stabilized by a missense mutation (I1314L). Previously, introduction of four amino acid changes derived from a second RSV strain "line 19" (I79M, K191R, T357K, N371Y) into the F protein of strain A2 increased the stability of infectivity and the proportion of F protein in the highly immunogenic pre-fusion (pre-F) conformation. In the present study, these four "line 19" assignments were introduced into the ΔNS2 candidate, creating ΔNS2-L19F-4M. During in vitro growth in Vero cells, ΔNS2-L19F-4M had growth kinetics and peak titer similar to the ΔNS2 parent. ΔNS2-L19F-4M exhibited an enhanced proportion of pre-F protein, with a ratio of pre-F/total F that was 4.5- to 5.0-fold higher than that of the ΔNS2 parent. The stability of infectivity during incubation at 4°C, 25°C, 32°C and 37°C was greater for ΔNS2-L19F-4M; for example, after 28 days at 32°C, its titer was 100-fold greater than ΔNS2. ΔNS2-L19F-4M exhibited similar levels of replication in human airway epithelial (HAE) cells as ΔNS2. The four "line 19" F mutations were genetically stable during 10 rounds of serial passage in Vero cells. In African green monkeys, ΔNS2-L19F-4M and ΔNS2 had similar growth kinetics, peak titer, and immunogenicity. These results suggest that ΔNS2-L19F-4M is an improved live attenuated vaccine candidate whose enhanced stability may simplify its manufacture, storage and distribution, which merits further evaluation in a clinical trial in humans.

A potential bivalent mRNA vaccine candidate protects against both RSV and SARS-CoV-2 infections.

As the world continues to confront severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV) is also causing severe respiratory illness in millions of infants, elderly individuals, and immunocompromised people globally. Exacerbating the situation is the fact that co-infection with multiple viruses is occurring, something which has greatly increased the clinical severity of the infections. Thus, our team developed a bivalent vaccine that delivered mRNAs encoding SARS-CoV-2 Omicron spike (S) and RSV fusion (F) proteins simultaneously, SF-LNP, which induced S and F protein-specific binding antibodies and cellular immune responses in BALB/c mice. Moreover, SF-LNP immunization effectively protected BALB/c mice from RSV infection and hamsters from SARS-CoV-2 Omicron infection. Notably, our study pointed out the antigenic competition problem of bivalent vaccines and provided a solution. Overall, our results demonstrated the potential of preventing two infectious diseases with a single vaccine and provided a paradigm for the subsequent design of multivalent vaccines.

Assessing the protection elicited by virus-like particles expressing the RSV pre-fusion F and tandem repeated G proteins against RSV rA2 line19F infection in mice.

Excessive pulmonary inflammation is the hallmark of respiratory syncytial virus (RSV) infection hindering efficacious RSV vaccine development. Yet, the vast majority of the experimental RSV vaccine studies use laboratory-adapted RSV strains that do not reflect the highly pathogenic and inflammatory nature of the virus found in clinical settings. Here, we re-evaluated the protective efficacy of the virus-like particle (VLP) vaccine co-expressing the pre-fusion (pre-F) protein and G protein with tandem repeats (Gt) reported in our previous study against the recombinant RSV rA2-line19F strain, which inflicts severe mucus production and inflammation in mice. VLP vaccine immunization elicited virus-specific serum antibody responses that mediated RSV rA2-line19F virus neutralization. VLP vaccine immunization promoted Th1 immune response development in the spleens and CD8 + T cell influx into the lungs of mice, which are essential for efficient viral clearance and dampened inflammatory response. When compared to the VLPs expressing only the pre-F antigen, those co-expressing both pre-F and Gt antigens conferred better protection in mice against rA2-line19F challenge infection. Overall, our data suggest that the pre-clinical VLP vaccine co-expressing RSV pre-F and Gt antigens can effectively protect mice against RSV strains that resemble pathogenic clinical isolates.

Genomic characterisation of respiratory syncytial virus: a novel system for whole genome sequencing and full-length G and F gene sequences.

To advance our understanding of respiratory syncytial virus (RSV) impact through genomic surveillance, we describe two PCR-based sequencing systems, (i) RSVAB-WGS for generic whole-genome sequencing and (ii) RSVAB-GF, which targets major viral antigens, G and F, and is used as a complement for challenging cases with low viral load. These methods monitor RSV genetic diversity to inform molecular epidemiology, vaccine effectiveness and treatment strategies, contributing also to the standardisation of surveillance in a new era of vaccines.

mRNA vaccines against respiratory viruses.

PURPOSE OF REVIEW: The successes of the coronavirus disease 2019 (COVID-19) mRNA vaccines have accelerated the development of mRNA vaccines against other respiratory pathogens. The aim of this review is to highlight COVID-19 mRNA vaccine advances and provide an update on the progress of mRNA vaccine development against other respiratory pathogens. RECENT FINDINGS: The COVID-19 mRNA vaccines demonstrated effectiveness in preventing severe COVID-19 and death. H7N9 and H10N8 avian influenza mRNA vaccines have demonstrated safety and immunogenicity in phase 1 clinical trials. Numerous seasonal influenza mRNA vaccines are in phase 1-3 clinical trials. Respiratory syncytial virus (RSV) mRNA vaccines have progressed to phase 2-3 clinical trials in adults and a phase 1 clinical trial in children. A combined human metapneumovirus and parainfluenza-3 mRNA vaccines was found to be well tolerated and immunogenic in a phase 1 trial among adults and trials are being conducted among children. Clinical trials of mRNA vaccines combining antigens from multiple respiratory viruses are underway. SUMMARY: The development of mRNA vaccines against respiratory viruses has progressed rapidly in recent years. Promising vaccine candidates are moving through the clinical development pathway to test their efficacy in preventing disease against respiratory viral pathogens.

Development and qualification of cell-based relative potency assay for a human respiratory syncytial virus (RSV) mRNA vaccine.

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract infections worldwide. A safe and effective RSV vaccine has been an elusive goal but recent advances in vaccine technology have improved the likelihood that a vaccine for the prevention of RSV could be licensed in near future. We have developed an RSV vaccine V171 consisting of four lipids and messenger ribonucleic acid (mRNA) encoding an engineered form of the RSV F protein stabilized in its prefusion conformation. The lipids form lipid nanoparticles (LNP) with mRNA encapsulated during process, which protects the mRNA from degradation and enables the mRNA to be delivered into mammalian cells. Once inside the cells, the mRNA then can be translated into RSV F protein and elicit both humoral and cellular immune responses. Preclinical results and Phase I clinical trial results indicate that this mRNA vaccine targeting RSV F protein is a promising RSV vaccine approach and should be further evaluated in clinical trials. We have developed a cell-based relative potency assay to support the Phase II development of this vaccine. Test articles and a reference standard are tested with serial dilutions in a 96-well plate pre-seeded with Hep G2 cells. Cells were incubated for 16-18 h after transfection and then permeabilized and stained with a human monoclonal antibody specific to RSV F protein, followed by a fluorophore-conjugated secondary antibody. The plate is then analyzed for percentage of transfected cells and relative potency of the test article is calculated by comparing its EC50 to that of a reference standard. This assay takes advantage of the fact that due to the inherent variability in biological test systems an absolute measure of potency is more variable than a measure of activity relative to a standard. Targeting testing relative potency range 25-250 %, our assay showed an R2 close to 1 for linearity, relative bias of 1.05-5.41 %, and intermediate precision of 11.0 %. The assay has been used for testing of process development samples, formulation development samples, as well as drug product intermediate (DPI) and drug product (DP) in support of Phase II development of our RSV mRNA vaccine.

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.

Split-Dose Administration Enhances Immune Responses Elicited by a mRNA/Lipid Nanoparticle Vaccine Expressing Respiratory Syncytial Virus F Protein.

mRNA vaccines have recently received significant attention due to their role in combating the SARS-CoV-2 pandemic. As a platform, mRNA vaccines have been shown to elicit strong humoral and cellular immune responses with acceptable safety profiles for prophylactic use. Despite their potential, industrial challenges have limited realization of the vaccine platform on a global scale. Critical among these challenges are supply chain considerations, including mRNA production, cost of goods, and vaccine frozen-chain distribution. Here, we assess the delivery of lipid nanoparticle-encapsulated mRNA (mRNA/LNP) vaccines using a split-dose immunization regimen as an approach to develop mRNA dose-sparing vaccine regimens with potential to mitigate mRNA supply chain challenges. Our data demonstrate that immunization by a mRNA/LNP vaccine encoding respiratory syncytial virus pre-F (RSV pre-F) over a 9 day period elicits comparable or superior magnitude of antibodies when compared to traditional bolus immunization of the vaccine. The split-dose immunization regimens evaluated in our studies were designed to mimic reported drug or antigen release profiles from microneedle patches, highlighting the potential benefit of pairing mRNA vaccines with patch-based delivery technologies to enable sustained release and solid-state stabilization. Overall, our findings provide a proof of concept to support further investigations into the development of sustained delivery approaches for mRNA/LNP vaccines.

[Prevention of respiratory syncytial virus infection in infants. What has been done and where are we today?] / Prevención de la infección por virus respiratorio sincicial en lactantes. ¿Qué se ha hecho y en qué estamos hoy?

Respiratory syncytial virus (RSV) infection is a frequent cause of morbidity and mortality in children. Recently, great advances have been made in the development of new monoclonal antibodies and vaccines thanks to the recognition of the structural conformation of virus proteins. The objective of this study was to review the advances related to the prevention of RSV infection in the first 6 months of life. Advances in structural biology have shown that the RSV fusion protein (F-Protein) in its prefusion state (Pre-F) is an excellent antigen for developing monoclonal antibodies and vaccines to prevent respiratory syncytial virus (RSV) infections. A new single-dose monoclonal antibody, Nirsevimab, has greater neutralizing power than currently available Palivizumab, and prolonged protection for 5 to 6 months. Nirsevimab has demonstrated an efficacy of 76.8% (95% CI, 49.4 to 89.4) in preventing lower respiratory infection 150 days after vaccination, decreasing the risk of ICU admission by 90.1% (95% CI: 16.4-98.8). Clesrovimab is another single-dose monoclonal antibody that has also shown promising results in phase 1b-2a trials. More recently, a bivalent vaccine against RSV A and B (Bivalent Prefusion F) has also been developed by replicating the F-protein stabilized in its Pre-F state as an antigen, using genetic engineering. This antigen, when administered to pregnant women between 24-36 weeks of gestation, induces high levels of antibodies in the mother with high transplacental transfer to the fetus. This vaccine has demonstrated an efficacy of 81.8% (95% CI: 40.6-96.3) at 90 days and 69.4% (95% CI: 44.3-84.1) at 180 days to prevent severe RSV disease (primary endpoint) without safety events detected so far. Nirsevimab and the Pre-F vaccine for pregnant women confer effective protection through passive immunity against RSV that lasts for the first 5 to 6 months of life and have already been approved for use in Europe by the EMA and in Canada and the United States by the FDA.

Virus-like particles containing a prefusion-stabilized F protein induce a balanced immune response and confer protection against respiratory syncytial virus infection in mice.

Respiratory syncytial virus (RSV) is a serious respiratory pathogen in infants and young children worldwide. Currently, no licensed RSV vaccines are available. In this study, we explored stable prefusion conformation virus-like particles (Pre-F VLPs) as RSV vaccine candidates. RSV fusion (F) protein mutants were constructed to form stabilized Pre-F or postfusion (Post-F) configurations. VLPs containing Pre-F or Post-F protein were generated using a recombinant baculovirus (rBV)-insect cell expression system. The assembly and immunological properties of Pre-F or Post-F VLPs were investigated. Pre-F and Post-F VLPs contained antigenic sites Ø and I of pre- and postfusion conformations, respectively. Compared with Post-F VLPs, immunization with Pre-F VLPs elicited upregulation of IFN-γ, IL-2 and IL-10 and downregulation of IL-4 and IL-5 cytokine production in mice. A high percentage of CD25+ Foxp3+ cells or a low percentage of IL-17A-producing cells among CD4+ T cells was observed in the lungs of mice vaccinated with Pre-F VLPs. Importantly, immunization with Pre-F VLPs induced a high level of RSV neutralizing antibody and a balanced immune response, which protected mice against RSV infection without evidence of immunopathology. Our results suggested that Pre-F VLPs generated from rBV-insect cells represent promising RSV vaccine candidates.

An intranasal recombinant NDV-BRSV Fopt vaccine is safe and reduces lesion severity in a colostrum-deprived calf model of RSV infection.

Human respiratory syncytial virus (HRSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, yet no safe, effective vaccine is commercially available. Closely related bovine RSV (BRSV) causes respiratory disease in young calves, with many similar features to those seen in HRSV. We previously showed that a Newcastle disease virus (NDV)-vectored vaccine expressing the F glycoprotein of HRSV reduced viral loads in lungs of mice and cotton rats and protected from HRSV. However, clinical signs and pathogenesis of disease in laboratory animals following HRSV infection differs from that observed in human infants. Thus, we examined whether a similar vaccine would protect neonatal calves from BRSV infection. Codon-optimized rNDV vaccine (rNDV-BRSV Fopt) was constructed and administered to colostrum-deprived calves. The rNDV-BRSV Fopt vaccine was well-tolerated and there was no evidence of vaccine-enhanced disease in the upper airways or lungs of these calves compared to the non-vaccinated calves. We found two intranasal doses reduces severity of gross and microscopic lesions and decreases viral load in the lungs. Furthermore, serum neutralizing antibodies were generated in vaccinated calves. Finally, reduced lung CXC chemokine levels were observed in vaccinated calves after BRSV challenge. In summary, we have shown that rNDV-BRSV Fopt vaccine is safe in colostrum-deprived calves, and is effective in reducing lung lesions, and decreasing viral load in upper respiratory tract and lungs after challenge.

A live single-cycle RSV vaccine expressing prefusion F protein.

Live-attenuated Respiratory syncytial virus (RSV) vaccines given intranasally have potential to provide comprehensive protection, including lung-resident immunity. It has however proven challenging to impart both sufficient safety and efficacy in a vaccine. To achieve the latter, we used a trans-complementing approach to generate live single-cycle RSV vaccines expressing the prefusion form (preF) of the viral fusion protein (F), either membrane-anchored or secreted. Both viruses were tested for their ability to induce a protective immune response in mice after intranasal prime-boost vaccination. The secreted preF vaccine failed to induce a protective response. The anchored preF vaccine induced anti-preF antibodies and antiviral T cells, and protected mice from lung pathology and viral shedding after challenge. Neither vaccine induced anti-G antibodies, for reasons unknown. In spite of the latter and single-cycle replication, the membrane-anchored preF vaccine was protective and demonstrates potential for development of an efficacious live vaccine with a stable safety phenotype.

Development of mRNA vaccines against respiratory syncytial virus (RSV).

Respiratory syncytial virus (RSV) is a single-stranded negative-sense RNA virus that is the primary etiologic pathogen of bronchitis and pneumonia in infants and the elderly. Currently, no preventative vaccine has been approved for RSV infection. However, advances in the characterization, and structural resolution, of the RSV surface fusion glycoprotein have revolutionized RSV vaccine development by providing a new target for preventive interventions. In general, six different approaches have been adopted in the development of preventative RSV therapeutics, namely, particle-based vaccines, vector-based vaccines, live-attenuated or chimeric vaccines, subunit vaccines, mRNA vaccines, and monoclonal antibodies. Among these preventive interventions, MVA-BN-RSV, RSVpreF3, RSVpreF, Ad26. RSV.preF, nirsevimab, clesrovimab and mRNA-1345 is being tested in phase 3 clinical trials, and displays the most promising in infant or elderly populations. Accompanied by the huge success of mRNA vaccines in COVID-19, mRNA vaccines have been rapidly developed, with many having entered clinical studies, in which they have demonstrated encouraging results and acceptable safety profiles. In fact, Moderna has received FDA approval, granting fast-track designation for an investigational single-dose mRNA-1345 vaccine against RSV in adults over 60 years of age. Hence, mRNA vaccines may represent a new, more successful, chapter in the continued battle to develop effective preventative measures against RSV. This review discusses the structure, life cycle, and brief history of RSV, while also presenting the current advancements in RSV preventatives, with a focus on the latest progress in RSV mRNA vaccine development. Finally, future prospects for this field are presented.

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.

Mucosal immunization with an adenoviral vector vaccine confers superior protection against RSV compared to natural immunity.

Respiratory syncytial virus (RSV) infections are the leading cause of severe respiratory illness in early infancy. Although the majority of children and adults mount immune responses against RSV, recurrent infections are frequent throughout life. Humoral and cellular responses contribute to an effective immunity but also their localization at respiratory mucosae is increasingly recognized as an important factor. In the present study, we evaluate a mucosal vaccine based on an adenoviral vector encoding for the RSV fusion protein (Ad-F), and we investigate two genetic adjuvant candidates that encode for Interleukin (IL)-1ß and IFN-ß promoter stimulator I (IPS-1), respectively. While vaccination with Ad-F alone was immunogenic, the inclusion of Ad-IL-1ß increased F-specific mucosal immunoglobulin A (IgA) and tissue-resident memory T cells (TRM). Consequently, immunization with Ad-F led to some control of virus replication upon RSV infection, but Ad-F+Ad-IL-1ß was the most effective vaccine strategy in limiting viral load and weight loss. Subsequently, we compared the Ad-F+Ad-IL-1ß-induced immunity with that provoked by a primary RSV infection. Systemic F-specific antibody responses were higher in immunized than in previously infected mice. However, the primary infection provoked glycoprotein G-specific antibodies as well eventually leading to similar neutralization titers in both groups. In contrast, mucosal antibody levels were low after infection, whereas mucosal immunization raised robust F-specific responses including IgA. Similarly, vaccination generated F-specific TRM more efficiently compared to a primary RSV infection. Although the primary infection resulted in matrix protein 2 (M2)-specific T cells as well, they did not reach levels of F-specific immunity in the vaccinated group. Moreover, the infection-induced T cell response was less biased towards TRM compared to vaccine-induced immunity. Finally, our vaccine candidate provided superior protection against RSV infection compared to a primary infection as indicated by reduced weight loss, virus replication, and tissue damage. In conclusion, our mucosal vaccine candidate Ad-F+Ad-IL-1ß elicits stronger mucosal immune responses and a more effective protection against RSV infection than natural immunity generated by a previous infection. Harnessing mucosal immune responses by next-generation vaccines is therefore a promising option to establish effective RSV immunity and thereby tackle a major cause of infant hospitalization.

Evaluation of Recombinant Live-Attenuated Respiratory Syncytial Virus (RSV) Vaccines RSV/ΔNS2/Δ1313/I1314L and RSV/276 in RSV-Seronegative Children.

BACKGROUND: This United States-based study compared 2 candidate vaccines: RSV/ΔNS2/Δ1313/I1314L, attenuated by NS2 gene-deletion and temperature-sensitivity mutation in the polymerase gene; and RSV/276, attenuated by M2-2 deletion. METHODS: RSV-seronegative children aged 6-24 months received RSV/ΔNS2/Δ1313/I1314L (106 plaque-forming units [PFU]), RSV/276 (105 PFU), or placebo intranasally. Participants were monitored for vaccine shedding, reactogenicity, and RSV serum antibodies, and followed over the subsequent RSV season. RESULTS: Enrollment occurred September 2017 to October 2019. During 28 days postinoculation, upper respiratory illness and/or fever occurred in 64% of RSV/ΔNS2/Δ1313/I1314L, 84% of RSV/276, and 58% of placebo recipients. Symptoms were generally mild. Cough was more common in RSV/276 recipients than RSV/ΔNS2/Δ1313/I1314L (48% vs 12%; P = .012) or placebo recipients (17%; P = .084). There were no lower respiratory illness or serious adverse events. Eighty-eight and 96% of RSV/ΔNS2/Δ1313/I1314L and RSV/276 recipients were infected with vaccine (shed vaccine and/or had ≥4-fold rises in RSV antibodies). Serum RSV-neutralizing titers and anti-RSV F IgG titers increased ≥4-fold in 60% and 92% of RSV/ΔNS2/Δ1313/I1314L and RSV/276 vaccinees, respectively. Exposure to community RSV during the subsequent winter was associated with strong anamnestic RSV-antibody responses. CONCLUSIONS: Both vaccines had excellent infectivity and were well tolerated. RSV/276 induced an excess of mild cough. Both vaccines were immunogenic and primed for strong anamnestic responses. CLINICAL TRIALS REGISTRATION: NCT03227029 and NCT03422237.

Protective antigenic sites identified in respiratory syncytial virus fusion protein reveals importance of p27 domain.

Respiratory syncytial virus (RSV) vaccines primarily focused on surface fusion (F) protein are under development. Therefore, to identify RSV-F protective epitopes, we evaluated 14 antigenic sites recognized following primary human RSV infection. BALB/c mice were vaccinated with F peptides, F proteins, or RSV-A2, followed by rA2-Line19F challenge. F peptides generated binding antibodies with minimal in vitro neutralization titers. However, several F peptides (including Site II) reduced lung viral loads and lung pathology scores in animals, suggesting partial protection from RSV disease. Interestingly, animals vaccinated with peptides (aa 101-121 and 110-136) spanning the F-p27 sequence, which is only present in unprocessed F0 protein, showed control of viral loads with significantly reduced pathology compared with mock-vaccinated controls. Furthermore, we observed F-p27 expression on the surface of RSV-infected cells as well as lungs from RSV-infected mice. The anti-p27 antibodies demonstrated antibody-dependent cellular cytotoxicity (ADCC) of RSV-infected A549 cells. These findings suggest that p27-mediated immune response may play a role in control of RSV disease in vivo, and F-p27 should be considered for inclusion in an effective RSV vaccine.

Dynamical Differences in Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a leading viral cause of pediatric respiratory infections and early infant mortality. Despite extensive development efforts currently underway, there remain no vaccines available for the prevention of RSV. RSV is an enveloped, negative-strand RNA virus that utilizes two different proteins (G and F) to mediate attachment and entry into host cells. These G and F proteins are the primary determinants of viral strain-specific differences and elicit protective neutralizing antibodies during natural infection in humans. Earlier studies have demonstrated that these proteins play an additional role in regulating the stability of RSV particles in response to temperature and pH. However, it remains unclear how much variability exists in the stability of RSV strains and what contribution changes in temperature and pH make to the clearance of virus during an active infection. In this study, we evaluated the impacts of changes in temperature and pH on the inactivation of four different chimeric recombinant RSV strains that differ exclusively in G and F protein expression. Using these data, we developed predictive mathematical models to examine the specific contributions and variations in susceptibility that exist between viral strains. Our data provide strain-specific clearance rates and temperature-pH landscapes that shed light on the optimal contributions of temperature and pH to viral clearance. These provide new insight into how much variation exists in the clearance of a major respiratory pathogen and may offer new guidance on optimization of viral strains for development of live-attenuated vaccine preparations.

Improved epitope resolution of the prefusion trimer-specific antibody AM14 bound to the RSV F glycoprotein.

Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens.

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-ß,  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.