Respiratory Syncytial Virus Fusion Protein Research Articles

Prefusion-specific antibody-derived peptides trivalently presented on DNA-nanoscaffolds as an innovative strategy against RSV entry

Human respiratory syncytial virus (RSV) is the primary cause of acute lower respiratory tract infections in children and the elderly worldwide, for which neither a vaccine nor an effective therapy is approved. The entry of RSV into the host cell is mediated by stepwise structural changes in the surface RSV fusion (RSV-F) glycoprotein. Recent progress in structural and functional studies of RSV-F glycoprotein revealed conformation-dependent neutralizing epitopes which have become attractive targets for vaccine and therapeutic development. As RSV-F is present on viral surface in a trimeric form, a trivalent binding interaction between a candidate fusion inhibitor and the respective epitopes on each of the three monomers is expected to prevent viral infection at higher potency than a monovalent or bivalent inhibitor. Here we demonstrate a novel RSV entry inhibitory approach by implementing a trimeric DNA nanostructure as a template to display up to three linear peptide moieties that simultaneously target an epitope on the surface of the prefusion RSV-F protein. In order to design synthetic binding peptides that can be coupled to the DNA nanostructure, the prefusion RSV-F-specific monoclonal antibody (D25) was selected. Complementarity-determining region 3 (CDR3) derived peptides underwent truncation and alanine-scanning mutagenesis analysis, followed by systematic sequence modifications using non-canonical amino acids. The most effective peptide candidate was used as a binding moiety to functionalize the DNA nanostructure. The designed DNA-peptide construct was able to block RSV infection on cells more efficiently than the monomeric peptides, however a more moderate reduction of viral load was observed in the lungs of infected mice upon intranasal application, likely due to dissociation or absorption of the underlying DNA structure by cells in the lungs. Taken together, our results point towards the inhibitory potential of a novel trimeric DNA-peptide based approach against RSV and open the possibility to apply this platform to target other viral infections.

Nirsevimab for Prevention of RSV in Healthy Late-preterm and Term Infants

(N Engl J Med. 2022;386:837–846. doi: 10.1056/NEJMoa2110275) Infants experience medically attended illness and hospitalization at elevated rates from respiratory syncytial virus (RSV) infection. As a monoclonal antibody with an extended half-life, Nirsevimab (N) is an emerging treatment that binds to the RSV fusion protein and has enhanced inhibitory potency for RSV in cell-culture and animal models over Palivizumab. Preterm (29 to <35 wk gestational age birth) infants receiving a single dose of N before RSV season showed 70.1% efficacy at protection against RSV with a favorable safety profile. The safety and efficacy of N in late-preterm and term infants entering their first RSV season is investigated in this phase 3 trial.

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)-1β and IFN-β promoter stimulator I (IPS-1), respectively. While vaccination with Ad-F alone was immunogenic, the inclusion of Ad-IL-1β 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-1β was the most effective vaccine strategy in limiting viral load and weight loss. Subsequently, we compared the Ad-F+Ad-IL-1β-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-1β 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.

Nanoparticle formulation of the fusion protein virus like particles of respiratory syncytial virus stimulates enhanced in vitro antigen presentation and autophagy

Respiratory Syncytial Virus (RSV) is one of the leading causes of bronchiolitis and pneumonia in childrenunder one year globally. As a result, RSV poses a severe burden on healthcare services. Thus, a vaccine for RSV is a global need. Utilizing polymeric nanoparticles as a delivery system for vaccine antigen holds a lot of promise. In this study, the virus like particles of RSV fusion protein (F-VLP) was encapsulated in poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NP). The F-VLP NP was formulated using a double emulsion solvent evaporation technique. The optimized NPs had a particle size of 525 ± 10.5 nm and an antigen encapsulation efficiency of 73% ± 10.5. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed that the F-VLP was stable post formulation. The F-VLP NP showed a sustained release of the F-VLP antigen for up to a week. In vitro study revealed that the F-VLP NP were non-cytotoxic, and the cellular uptake of the NPs by dendritic cells was observed within 3 h. The F-VLP NP with adjuvant monophosphoryl lipid A (MPL) NP and without MPL NP showed enhanced expression of antigen presentation molecule major histocompatibility complex (MHC)-I and autophagosomes in dendritic cells. In summary, the sustained release of the antigen from the F-VLP NP and the particulate nature of the vaccine resulted in enhanced antigen presentation and induction of autophagy in antigen-presenting cells (APCs).

Transdermal Immunization with Microparticulate RSV-F Virus-like Particles Elicits Robust Immunity.

No approved vaccines against respiratory syncytial virus (RSV) infections exist to date, due to challenges arising during vaccine development. There is an unmet need to explore novel approaches and a universal strategy to prevent RSV infections. Previous studies have proven the immune efficacy of virus-like particles (VLPs) consisting of RSV fusion (F) protein, yielding a highly immunogenic RSV-F VLP subunit vaccine. In this study, RSV-F VLP (with or without MPL®) was added to a polymer mix and spray-dried, forming microparticles. The formulations were transdermally administered in C57BL/6 mice to evaluate vaccine efficacy. The transdermal delivery of RSV-F VLP + MPL® was more effective in clearing lung viral loads and preventing weight loss after RSV challenge. At the cellular level, MPL® augmented the vaccine response in microparticulate form, which was evidenced by higher serum and lung antibody titers, and lower lung viral titers in the vaccinated groups. These preliminary results validate the effectiveness of the RSV-F VLP microparticulate vaccine via the transdermal route due to its potential to trigger robust immune responses.

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.

Immunogenicity and protective efficacy of adenoviral and subunit RSV vaccines based on stabilized prefusion F protein in pre-clinical models

Respiratory Syncytial Virus (RSV) remains a leading cause of severe respiratory disease for which no licensed vaccine is available. We have previously described the derivation of an RSV Fusion protein (F) stabilized in its prefusion conformation (preF) as vaccine immunogen and demonstrated superior immunogenicity in naive mice of preF versus wild type RSV F protein, both as protein and when expressed from an Ad26 vaccine vector. Here we address the question if there are qualitative differences between the two vaccine platforms for induction of protective immunity. In naïve mice, both Ad26.RSV.preF and preF protein induced humoral responses, whereas cellular responses were only elicited by Ad26.RSV.preF. In RSV pre-exposed mice, a single dose of either vaccine induced cellular responses and strong humoral responses. Ad26-induced RSV-specific cellular immune responses were detected systemically and locally in the lungs. Both vaccines showed protective efficacy in the cotton rat model, but Ad26.RSV.preF conferred protection at lower virus neutralizing titers in comparison to RSV preF protein. Factors that may contribute to the protective capacity of Ad26.RSV.preF elicited immunity are the induced IgG2a antibodies that are able to engage Fcγ receptors mediating Antibody Dependent Cellular Cytotoxicity (ADCC), and the induction of systemic and lung resident RSV specific CD8 + T cells. These data demonstrate qualitative improvement of immune responses elicited by an adenoviral vector based vaccine encoding the RSV preF antigen compared to the subunit vaccine in small animal models which may inform RSV vaccine development.

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.

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.

Development and comparison of three cell-based potency assays for anti-respiratory syncytial virus monoclonal antibody

There is an increasing demand for monoclonal antibody (mAb) therapies to confer passive immunity against viral diseases. Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis, lower respiratory tract infections, and hospitalization in infants. Currently, there is no RSV vaccine but a humanized mAb available for high risk infants. MK-1654 is a fully human mAb with YTE mutation in the fragment crystallizable (Fc) region to extend the half-life in circulation. It binds to a highly conserved epitope of RSV Fusion protein with high affinity and neutralizes RSV infection. A functional cell-based assay is a regulatory requirement for clinical development, commercial release, and stability testing of MK-1654. In this study, we have evaluated three RSV neutralization assays to test the potency of MK-1654, including an imaging-based virus reduction neutralization test (VRNT) and two reporter virus-based assays (RSV-GFP and RSV-NLucP). All three methods showed good dose response curves of MK-1654 with similar EC50 values. RSV-NLucP method was chosen for further development because it is simple and can be easily adapted to quality control testing laboratories. After optimization, the RSV-NLucP assay was pre-qualified with good linearity, relative accuracy, intermediate precision, and specificity, therefore suitable for a cell-based potency assay.

Coexpression of respiratory syncytial virus (RSV) fusion (F) protein and attachment glycoprotein (G) in a vesicular stomatitis virus (VSV) vector system provides synergistic effects against RSV infection in a cotton rat model

Respiratory syncytial virus (RSV) is one of the most important causes of respiratory disease in infants, immunocompromised individuals, and the elderly. Natural infection does not result in long-term immunity, and there is no licensed vaccine. Vesicular stomatitis virus (VSV) is a commonly used vaccine vector platform against infectious diseases, and has been used as a vector for a licensed Ebola vaccine. In this study, we expressed the RSV fusion (F) protein, the RSV F protein stabilized in either a pre-fusion or a post-fusion configuration, the attachment glycoprotein (G), or the G and F proteins of RSV in combination in a VSV vector. Cotton rats were immunized with these recombinants intranasally or subcutaneously to test immunogenicity. RSV F stabilized in either a pre-fusion or a post-fusion configuration proved to be poorly immunogenic and protective when compared to unmodified F. RSV G provided partial protection and moderate levels of neutralizing antibody production, both of which improved with intranasal administration compared to subcutaneous inoculation. The most successful vaccine vector was VSV expressing both the G and F proteins after intranasal inoculation. Immunization with this recombinant induced neutralizing antibodies and provided protection from RSV challenge in the upper and lower respiratory tract for at least 80 days. Our results demonstrate that co-expression of F and G proteins in a VSV vector provides synergistic effects in inducing RSV-specific neutralizing antibodies and protection against RSV infection.

Alveolar-like Macrophages Attenuate Respiratory Syncytial Virus Infection.

Respiratory Syncytial Virus (RSV) is the leading cause of acute lower respiratory infections in young children and infection has been linked to the development of persistent lung disease in the form of wheezing and asthma. Despite substantial research efforts, there are no RSV vaccines currently available and an effective monoclonal antibody targeting the RSV fusion protein (palivizumab) is of limited general use given the associated expense. Therefore, the development of novel approaches to prevent RSV infection is highly desirable to improve pediatric health globally. We have developed a method to generate alveolar-like macrophages (ALMs) from pluripotent stem cells. These ALMs have shown potential to promote airway innate immunity and tissue repair and so we hypothesized that ALMs could be used as a strategy to prevent RSV infection. Here, we demonstrate that ALMs are not productively infected by RSV and prevent the infection of epithelial cells. Prevention of epithelial infection was mediated by two different mechanisms: phagocytosis of RSV particles and release of an antiviral soluble factor different from type I interferon. Furthermore, intratracheal administration of ALMs protected mice from subsequent virus-induced weight loss and decreased lung viral titres and inflammation, indicating that ALMs can impair the pathogenesis of RSV infection. Our results support a prophylactic role for ALMs in the setting of RSV infection and warrant further studies on stem cell-derived ALMs as a novel cell-based therapy for pulmonary viral infections.

Combinatorial F-G Immunogens as Nipah and Respiratory Syncytial Virus Vaccine Candidates.

Nipah virus (NiV) and respiratory syncytial virus (RSV) possess two surface glycoproteins involved in cellular attachment and membrane fusion, both of which are potential targets for vaccines. The majority of vaccine development is focused on the attachment (G) protein of NiV, which is the immunodominant target. In contrast, the fusion (F) protein of RSV is the main target in vaccine development. Despite this, neutralising epitopes have been described in NiV F and RSV G, making them alternate targets for vaccine design. Through rational design, we have developed a vaccine strategy applicable to phylogenetically divergent NiV and RSV that comprises both the F and G proteins (FxG). In a mouse immunization model, we found that NiV FxG elicited an improved immune response capable of neutralising pseudotyped NiV and a NiV mutant that is able to escape neutralisation by two known F-specific antibodies. RSV FxG elicited an immune response against both F and G and was able to neutralise RSV; however, this was inferior to the immune response of F alone. Despite this, RSV FxG elicited a response against a known protective epitope within G that is conserved across RSV A and B subgroups, which may provide additional protection in vivo. We conclude that inclusion of F and G antigens within a single design provides a streamlined subunit vaccine strategy against both emerging and established pathogens, with the potential for broader protection against NiV.

Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein.

ABSTRACTThe fusion glycoprotein (F) is essential for respiratory syncytial virus (RSV) entry and has become an attractive target for anti-RSV drug development. Despite the promising prospect of RSV F inhibitors, issues of drug resistance remain challenging. In this study, we established a dual-luciferase protocol for RSV fusion inhibitor discovery. A small-molecule inhibitor, salvianolic acid R (LF-6), was identified to inhibit virus-cell and cell-cell fusion mediated by the RSV F protein. Sequence analysis of the resultant resistant viruses identified a K394R mutation in the viral F protein. The K394R mutant virus also conferred cross-resistance to multiple RSV fusion inhibitors, including several inhibitors undergoing clinical trials. Our study further showed that K394R mutation not only increased the triggering rate of F protein in prefusion conformation but also enhanced the fusion activity of F protein, both of which were positively correlated with resistance to fusion inhibitors. Moreover, the K394R mutation also showed cooperative effects with other escape mutations to increase the fusion activity of F protein. By substitution of K394 into different amino acids, we found that K394R or K394H substitution resulted in hyperfusiogenic F proteins, whereas F variants with other substitutions exhibited less fusion activity. Both K394R and K394H in F protein exhibited cross-resistance to RSV fusion inhibitors. Collectively, these findings reveal a positive correlation between the membrane fusion activity of F protein and the resistance of corresponding inhibitors. All of the results demonstrate that K394R in F protein confers cross-resistance to fusion inhibitors through destabilizing F protein and increasing its membrane fusion activity.IMPORTANCE Respiratory syncytial virus (RSV) causes serious respiratory tract disease in children and the elderly. Therapeutics against RSV infection are urgently needed. This study reports the discovery of a small-molecule inhibitor of RSV fusion glycoprotein by using a dual-luciferase protocol. The escape mutation (K394R) of this compound also confers cross-resistance to multiple RSV fusion inhibitors that have been reported previously, including two candidates currently in clinical development. The combination of K394R with other escape mutations can increase the resistance of F protein to these inhibitors through destabilizing F protein and enhancing the membrane fusion activity of F protein. By amino acid deletion or substitution, we found that a positively charged residue at the 394th site is crucial for the fusion ability of F protein, as well as for the cross-resistance against RSV fusion inhibitors. These results reveal the mechanism of cross-resistance conferred by the K394R mutation and the possible cross-resistance risk of RSV fusion inhibitors.

Disulfide-stapled design of α-helical bundles to target the trimer-of-hairpins motif of human respiratory syncytial virus fusion protein

Human respiratory syncytial virus (RSV) is the major cause of acute lower respiratory tract infections worldwide in infants and young children. The RSV F glycoprotein is a class I fusion protein that mediates viral entry into host cells and is a major target of neutralizing antibodies. Targeting F glycoprotein has been recognized as a promising antiviral therapeutic strategy against RSV infection. Here, we reported the disulfide-stapled design of α-helical bundle to target the trimer-of-hairpins (TOH) motif of RSV F glycoprotein, which is the central regulatory module that triggers viral membrane fusion event. In TOH motif, three N-terminal heptad repeat (NtHR) helices form a trimeric coiled-coil core and other three C-terminal heptad repeat (CtHR) helices add to the core in an antiparallel manner. Interaction analysis between NtHR and CtHR revealed that the C-terminal tail of CtHR packs tightly against NtHR as compared to the N-terminal and middle regions of CtHR. A core binding site in CtHR C-terminus was identified, which represents a 13-mer chp peptide and can effectively interact with NtHR helix in native ordered conformation but would become largely disordered when splitting from the protein context of CtHR helix. Two chp helices were stapled together in a parallel manner with single, double or triple disulfide bridges, thus systematically resulting in seven disulfide-stapled α-helical bundles. Molecular simulations revealed that the double and triple stapling can effectively stabilize the structured conformation of α-helical bundles, whereas the free conformation of single-stapled bundles still remain intrinsically disordered in solvent. The double-stapled bundle chp-ds[508,516] and the triple-stapled bundle chp-ts[508,512,516] were rationally designed to have high potency; they can form a tight three-helix bundle with NtHR helix, thus potently targeting NtHR–CtHR interactions involved in RSV-F TOH motif through a competitive disruption mechanism.

Hyper-Enriched Anti-RSV Immunoglobulins Nasally Administered: A Promising Approach for Respiratory Syncytial Virus Prophylaxis.

Respiratory syncytial virus (RSV) is a public health concern that causes acute lower respiratory tract infection. So far, no vaccine candidate under development has reached the market and the only licensed product to prevent RSV infection in at-risk infants and young children is a monoclonal antibody (Synagis®). Polyclonal human anti-RSV hyper-immune immunoglobulins (Igs) have also been used but were superseded by Synagis® owing to their low titer and large infused volume. Here we report a new drug class of immunoglobulins, derived from human non hyper-immune plasma that was generated by an innovative bioprocess, called Ig cracking, combining expertises in plasma-derived products and affinity chromatography. By using the RSV fusion protein (F protein) as ligand, the Ig cracking process provided a purified and concentrated product, designated hyper-enriched anti-RSV IgG, composed of at least 15-20% target-specific-antibodies from normal plasma. These anti-RSV Ig displayed a strong in vitro neutralization effect on RSV replication. Moreover, we described a novel prophylactic strategy based on local nasal administration of this unique hyper-enriched anti-RSV IgG solution using a mouse model of infection with bioluminescent RSV. Our results demonstrated that very low doses of hyper-enriched anti-RSV IgG can be administered locally to ensure rapid and efficient inhibition of virus infection. Thus, the general hyper-enriched Ig concept appeared a promising approach and might provide solutions to prevent and treat other infectious diseases.ImportanceRespiratory Syncytial Virus (RSV) is the major cause of acute lower respiratory infections in children, and is also recognized as a cause of morbidity in the elderly. There are still no vaccines and no efficient antiviral therapy against this virus. Here, we described an approach of passive immunization with a new class of hyper-enriched anti-RSV immunoglobulins (Ig) manufactured from human normal plasma. This new class of immunoglobulin plasma derived product is generated by an innovative bioprocess, called Ig cracking, which requires a combination of expertise in both plasma derived products and affinity chromatography. The strong efficacy in a small volume of these hyper-enriched anti-RSV IgG to inhibit the viral infection was demonstrated using a mouse model. This new class of immunoglobulin plasma-derived products could be applied to other pathogens to address specific therapeutic needs in the field of infectious diseases or even pandemics, such as COVID-19.

Designing of Potential Polyvalent Vaccine Model for Respiratory Syncytial Virus by System Level Immunoinformatics Approaches.

Background Respiratory syncytial virus (RSV) infection is a public health epidemic, leading to around 3 million hospitalization and about 66,000 deaths each year. It is a life-threatening condition exclusive to children with no effective treatment. Methods In this study, we used system-level and vaccinomics approaches to design a polyvalent vaccine for RSV, which could stimulate the immune components of the host to manage this infection. Our framework involves data accession, antigenicity and subcellular localization analysis, T cell epitope prediction, proteasomal and conservancy evaluation, host-pathogen-protein interactions, pathway studies, and in silico binding affinity analysis. Results We found glycoprotein (G), fusion protein (F), and small hydrophobic protein (SH) of RSV as potential vaccine candidates. Of these proteins (G, F, and SH), we found 9 epitopes for multiple alleles of MHC classes I and II bear significant binding affinity. These potential epitopes were linked to form a polyvalent construct using AAY, GPGPG linkers, and cholera toxin B adjuvant at N-terminal with a 23.9 kDa molecular weight of 224 amino acid residues. The final construct was a stable, immunogenic, and nonallergenic protein containing cleavage sites, TAP transport efficiency, posttranslation shifts, and CTL epitopes. The molecular docking indicated the optimum binding affinity of RSV polyvalent construct with MHC molecules (-12.49 and -10.48 kcal/mol for MHC classes I and II, respectively). This interaction showed that a polyvalent construct could manage and control this disease. Conclusion Our vaccinomics and system-level investigation could be appropriate to trigger the host immune system to prevent RSV infection.

Articles of Significant Interest in This Issue

Human respiratory syncytial virus (RSV) can cause severe disease. Rossey et al. (e02279-20) report the isolation and characterization of a llama-derived single-domain antibody (VHH, also known as a nanobody) that potently neutralizes RSV in vitro. This VHH binds specifically to the prefusion conformation of the RSV fusion protein, to a site that is located relatively close to the viral membrane mainly covering antigenic site I.

In silico virtual screening of lead compounds for major antigenic sites in respiratory syncytial virus fusion protein

Human respiratory syncytial virus (RSV) is a leading ubiquitous respiratory pathogen in newborn infants, young children, and the elderly, with no vaccine available to date. The viral fusion glycoprotein (RSV F) plays an essential role in the infection process, and it is a primary target of neutralizing antibodies, making it an attractive site for vaccine development. With this in view, there is a persistent need to identify selective antiviral drugs against RSV, targeting the major antigenic sites on the F protein. We aimed to conduct a robust in silico high-throughput drug screening of one million compounds to explore potential inhibitors that bind the major antigenic site Ø and site II on RSV F protein, which are the main target of neutralizing antibodies (NAb). We utilized the three-dimensional crystallographic structure of both antigenic site Ø on pre-F and antigenic II on post-F to screen for potential anti-RSV inhibitors. A library of one million small compounds was docked to explore lead binders in the major antigenic sites by using virtual lab bench CLC Drug Discovery. We also performed Quantitative Structure-Activity and Relationship (QSAR) for the lead best binders known for their antiviral activity. Among one million tested ligands, seven ligands (PubChem ID: 3714418, 24787350, 49828911, 24802036, 79824892, 49726463, and 3139884) were identified as the best binders to neutralizing epitopes site Ø and four ligands (PubChem ID: 865999, 17505357, 24802036, and 24285058) to neutralizing epitopes site II, respectively. These binders exhibited significant interactions with neutralizing epitopes on RSV F, with an average of six H bonds, docking energy of − 15.43 Kcal·mol−1, and minimum interaction energy of − 7.45 Kcal·mol−1. Using in silico virtual screening, we identified potential RSV inhibitors that bind two major antigenic sites on the RSV F protein. Using structure-based design and combination-based drug therapy, identified molecules could be modified to generate the next generation anti-RSV drugs.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein induces genetically and antigenically diverse antibody responses

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.