Littérature scientifique sur le sujet « RSV - respiratory syncytial virus, memory B cells, antibodies, vaccines »

ABSTRACT The capacity of live or inactivated respiratory syncytial virus (RSV) to induce B-cell memory in respiratory-associated lymphoid tissues of mice was examined. Eight weeks after primary inoculation with either live or inactivated RSV, adult BALB/c mice were challenged with 4 × 105 PFU of RSV. Protection from viral shedding and mucosal production of RSV-specific antibodies were examined at various time points after challenge. We found that primary immunization with live, but not inactivated, RSV induced complete and durable protection upon challenge within the upper and lower respiratory tract. Also, primary immunization with live, but not inactivated, RSV enhanced the production of mucosal RSV-specific immunoglobulin A (IgA) upon challenge. Secondary mucosal IgA responses were characterized by (i) the early production of mucosal IgA by B cells that reside in organized nasal-associated lymphoid tissues, cervical lymph nodes, and bronchial lymph nodes, and (ii) the subsequent production of RSV-specific IgA by mucosal effector tissues, such as the tracheal lamina propria and lung. These findings suggest that primary infection of mice with live RSV might induce mucosal IgA-committed memory B cells. A greater understanding of the characteristics of RSA-specific mucosal memory B cells may facilitate the development of an RSV vaccine.

Abstract Background Respiratory syncytial virus (RSV) infection is a major public health burden for infants and the elderly worldwide. Currently, there are no approved vaccines and only one moderately effective marketed antibody (Synagis®) for the prevention of RSV infection in high-risk infants. Sampling the human antibody repertoire has led to the realization that the RSV fusion (F) protein in its prefusion conformation is the preferred target for potent neutralizing antibodies and thus makes the protein an attractive candidate for vaccine developers. Methods We report the isolation of a potent and broad RSV neutralizing monoclonal antibody (mAb), which was discovered through molecular cloning of cultured RSV postfusion F protein-baited single-sorted human memory B cells. The epitope for the mAb was mapped to RSV F protein using various methods; including X-ray crystallography, alanine scan, and RSV escape mutant generation Results The mAb binds to both RSV pre- and postfusion F proteins at site IV and can neutralize RSV A and B laboratory strains with subnanomolar potency, superior to that of Palivizumab. Antigenic site IV is conserved between pre- and postfusion F proteins in both RSV A and B subgroups, and sequence alignment showed that the mAb-binding site was conserved in >1,000 RSV A and B clinical isolates. In vivo cotton rat studies demonstrated protection of both the upper and lower respiratory tract of antibody-infused animals challenged with either RSV A or RSV B. Conclusion Overall, the fully human mAb we have isolated has great potential to be developed for passive immune-prophylaxis in infants. A prevalent view of the RSV scientific community is that RSV neutralizing mAbs in human sera primarily target the prefusion F protein and predominantly bind antigenic site Ø. In contrast, our finding demonstrates that very broad and potent RSV neutralizing mAb can also recognize sites common to pre- and postfusion F proteins. Furthermore, the RSV F antigenic site IV presents a neutralizing epitope which is highly conserved. Therefore, it is worthwhile to consider site IV, in addition to site Ø, in the design of RSV subunit vaccines. Disclosures K. Vora, Merck: Employee, Salary. Z. Chen, Merck: Employee, Salary. H. P. Su, Merck: Employee, Salary. A. Tang, Merck: Employee, Salary. K. Cox, merck: Employee, Salary. C. Callahan, Merck: Employee, Salary. L. Zhang, Merck: Employee, Salary. S. Patel, Merck: Employee, Salary. D. Nahas, merck: Employee, Salary. M. Citron, merck: Employee, Salary. P. Cejas, merck: Employee, Salary. R. Swoyer, Merck: Employee, Salary. B. Luo, merck: Employee, Salary. M. Eddins, Merck: Employee, Salary. J. Reid, Merck: Employee, Salary. A. Fridman, Merck: Employee, Salary. J. Galli, Merck: Employee, Salary. S. Cosmi, Merck: Independent Contractor, Salary. G. Dhanasekeran, Merck: Employee, Salary. Z. Wen, Merck: Employee, Salary. X. He, Merck: Employee, Salary. D. Wang, Merck: Employee, Salary. G. Heidecker, Merck: Employee, Salary. J. Flynn, Merck: Employee, Salary. J. Cook, merck: Employee, Salary. S. Soisson, Merck: Employee, Salary. D. Casimiro, Merck: Employee, Salary. A. Bett, Merck: Employee, Salary. W. Blair, Merck: Employee, Salary. D. Distefano, Merck: Employee, Salary. C. Haines, Merck: Employee, Salary.

AbstractDespite being a leading cause of severe respiratory disease, there remains no licensed respiratory syncytial virus (RSV) vaccine. Neutralizing antibodies reduce the severity of RSV-associated disease, but are not sufficient for preventing reinfection. In contrast, the role of memory CD8 T cells in protecting against a secondary RSV infection is less established. We recently demonstrated that high-magnitude memory CD8 T cells efficiently reduced lung viral titers following RSV infection, but induced fatal immunopathology that was mediated by IFN-γ. To evaluate the ability of RSV-specific neutralizing antibodies to prevent memory CD8 T cell-mediated immunopathology, mice with high-magnitude memory CD8 T cell responses were treated with neutralizing antibodies prior to RSV challenge. Neutralizing antibody treatment significantly reduced morbidity and prevented mortality following RSV challenge compared with IgG-treated controls. Neutralizing antibody treatment restricted early virus replication, which caused a substantial reduction in memory CD8 T cell activation and IFN-γ production, directly resulting in survival. In contrast, therapeutic neutralizing antibody administration did not impact morbidity, mortality, or IFN-γ levels, despite significantly reducing lung viral titers. Therefore, only pre-existing neutralizing antibodies prevent memory CD8 T cell-mediated immunopathology following RSV infection. Overall, our results have important implications for the development of future RSV vaccines.

Effective vaccines inducing lifelong protection against many important infections such as respiratory syncytial virus (RSV), HIV, influenza virus, and Epstein-Barr virus (EBV) are not yet available despite decades of research. As an alternative to a protective vaccine, we developed a genetic engineering strategy in which CRISPR-Cas9 was used to replace endogenously encoded antibodies with antibodies targeting RSV, HIV, influenza virus, or EBV in primary human B cells. The engineered antibodies were expressed efficiently in primary B cells under the control of endogenous regulatory elements, which maintained normal antibody expression and secretion. Using engineered mouse B cells, we demonstrated that a single transfer of B cells engineered to express an antibody against RSV resulted in potent and durable protection against RSV infection in RAG1-deficient mice. This approach offers the opportunity to achieve sterilizing immunity against pathogens for which traditional vaccination has failed to induce or maintain protective antibody responses.

Abstract Respiratory Syncytial Virus (RSV) infects almost all children under the age of one and is the leading cause of hospitalization among infants. It also causes high rates of infection in the elderly and immunocompromised patient populations. Despite several decades of research with dozens of candidate vaccines being vigorously evaluated in pre-clinical and clinical studies, there is no licensed vaccine available to date. A clinical trial conducted in the early 1960s testing a formaldehyde-inactivated RSV (FIRSV) vaccine resulted in enhanced respiratory disease, following subsequent RSV infection, leading to hospitalization of 80% of the participants and 2 deaths. Since then, other forms of RSV vaccine have also been found to induce enhanced disease in preclinical animal studies. Here, we aimed to develop a vaccine that can effectively protect mice from RSV infection and help identify facets of FIRSV-induced enhanced disease. In this study, BALB/c mice were immunized with an adenoviral vector containing the RSV fusion protein (F) fused with CD40 ligand where the CD40 ligand serves two vital functions as a molecular adjuvant and an antigen-targeting molecule. In contrast to the FIRSV vaccine, the vectored vaccine effectively protected animals against RSV without inducing enhanced respiratory disease. This protection involved a robust induction of neutralizing antibodies and memory CD8 T cells, which were not observed in the FIRSV group. Finally, the vectored vaccine was able to elicit long-lasting protection against RSV, which was mediated by increased levels of effector memory CD8 T cell 3 months post-vaccination.

Respiratory syncytial virus (RSV) infections are a major cause of serious respiratory disease in infants. RSV occurs as two major subgroups A and B, which mainly differ regarding the surface glycoprotein G. The G protein is important for virus attachment and G-specific antibodies can protect against infection. We expressed the surface-exposed part of A2 strain-derived G (A2-G) in baculovirus-infected insect cells and synthesized overlapping peptides spanning complete A2-G. The investigation of the natural IgG response of adult subjects during a period of one year showed that IgG antibodies (i) recognize G significantly stronger than the fusion protein F0, (ii) target mainly non-conformational, sequential peptide epitopes from the exposed conserved region but also buried peptides, and (iii) exhibit a scattered but constant recognition profile during the observation period. The IgG subclass reactivity profile (IgG1 > IgG2 > IgG4 = IgG3) was indicative of a mixed Th1/Th2 response. Two strongly RSV-neutralizing sera including the 1st WHO standard contained high IgG anti-G levels. G-specific IgG increased strongly in children after wheezing attacks suggesting RSV as trigger factor. Our study shows that RSV G and G-derived peptides are useful for serological diagnosis of RSV-triggered exacerbations of respiratory diseases and underlines the importance of G for development of RSV-neutralizing vaccines.

ABSTRACTRespiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease, which causes high rates of morbidity and mortality in infants and the elderly. Models of human RSV pulmonary disease are needed to better understand RSV pathogenesis and to assess the efficacy of RSV vaccines. We assessed the RSV-specific human innate, humoral, and cellular immune responses in humanized mice (mice with a human immune system [HIS mice]) with functional human CD4+T and B cells. These mice were generated by introduction of HLA class II genes, various human cytokines, and human B cell activation factor into immunodeficient NOD scid gamma (NSG) mice by the use of an adeno-associated virus vector, followed by engraftment of human hematopoietic stem cells. During the first 3 days of infection, HIS mice lost more weight and cleared RSV faster than NSG mice. Human chemokine (C-C motif) ligand 3 (CCL3) and human interleukin-1β (IL-1β) expression was detected in the RSV-infected HIS mice. The pathological features induced by RSV infection in HIS mice included peribronchiolar inflammation, neutrophil predominance in the bronchioalveolar lavage fluid, and enhanced airway mucus production. Human anti-RSV IgG and RSV-neutralizing antibodies were detected in serum and human anti-RSV mucosal IgA was detected in bronchioalveolar lavage fluid for up to 6 weeks. RSV infection induced an RSV-specific human gamma interferon response in HIS mouse splenocytes. These results indicate that human immune cells can induce features of RSV lung disease, including mucus hyperplasia, in murine lungs and that HIS mice can be used to elicit human anti-RSV humoral and cellular immunity.IMPORTANCEInfections with respiratory syncytial virus (RSV) are common and can cause severe lung disease in infants and the elderly. The lack of a suitable animal model with disease features similar to those in humans has hampered efforts to predict the efficacy of novel anti-RSV therapies and vaccines for use in humans. A murine model consisting of mice with a human immune system (HIS mice) could be useful for assessment of RSV disease and anti-RSV responses specific to humans. This study investigates an HIS mouse model to imitate human RSV disease and immune responses. We found that RSV lung infection in HIS mice results in an RSV-specific pathology that mimics RSV disease in humans and induces human anti-RSV immune responses. This model could be useful for better understanding of human RSV disease and for the development of RSV therapies.

Respiratory syncytial virus (RSV) can cause bronchiolitis and viral pneumonia in young children and the elderly. Lack of vaccines and recurrence of RSV infection indicate the difficulty in eliciting protective memory immune responses. Tissue resident memory T cells (TRM) can confer protection from pathogen re-infection and, in human experimental RSV infection, the presence of lung CD8+ TRM cells correlates with a better outcome. However, the requirements for generating and maintaining lung TRM cells during RSV infection are not fully understood. Here, we use mouse models to assess the impact of innate immune response determinants in the generation and subsequent expansion of the TRM cell pool during RSV infection. We show that CD8+ TRM cells expand independently from systemic CD8+ T cells after RSV re-infection. Re-infected MAVS and MyD88/TRIF deficient mice, lacking key components involved in innate immune recognition of RSV and induction of type I interferons (IFN-α/β), display impaired expansion of CD8+ TRM cells and reduction in antigen specific production of granzyme B and IFN-γ. IFN-α treatment of MAVS deficient mice during primary RSV infection restored TRM cell expansion upon re-challenge but failed to recover TRM cell functionality. Our data reveal how innate immunity, including the axis controlling type I IFN induction, instructs and regulates CD8+ TRM cell responses to RSV infection, suggesting possible mechanisms for therapeutic intervention.

Respiratory syncytial virus (RSV) is one of the primary causative agents of upper and lower respiratory tract infections in young children, in particular infants. Recently, we reported the protective efficacy of a RSV vaccine formulation consisting of a truncated version of the fusion (F) protein formulated with a Toll-like receptor (TLR) agonist and an immunostimulatory peptide in a carrier system (ΔF/TriAdj). To evaluate the duration of immunity induced by this vaccine candidate, we carried out long-term trials. The ΔF was formulated with triple adjuvant (TriAdj) containing either polyinosinic : polycytidylic acid (polyI : C) or cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) and administered intranasally to mice. One year after the second vaccination all mice were challenged with RSV. Both ΔF/TriAdj formulations mediated the induction of high levels of IgG1, IgG2a and virus-neutralizing antibodies, and IgA in the lungs. Based on the numbers of IFN-γ- and IL-5-secreting cells in the spleen, the immune response was slightly T-helper cell type 1 (Th1)-biased. This was confirmed by the presence of F85–93-specific CD8+ effector T cells in the lungs of both ΔF/TriAdj(polyI : C)- and ΔF/TriAdj(CpG)-immunized mice. Both ΔF/TriAdj formulations induced RSV-specific CD8+ T cells. However, ΔF/TriAdj(polyI : C) generated significantly higher IgG affinity maturation and higher numbers of RSV-specific CD8+ effector memory T cells in lungs and CD8+ central memory T cells in spleen and lymph nodes than ΔF/TriAdj(CpG). After RSV challenge, no virus replication and no evidence of vaccine-induced pathology were detected in mice immunized with either of the ΔF/TriAdj formulations, demonstrating that the duration of immunity induced with these vaccines is at least one year.

ABSTRACTAlthough respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, a safe and effective vaccine is not yet available. Live-attenuated vaccines (LAVs) are the most advanced vaccine candidates in RSV-naive infants. However, designing an LAV with appropriate attenuation yet sufficient immunogenicity has proven challenging. In this study, we implemented reverse genetics to address these obstacles with a multifaceted LAV design that combined the codon deoptimization of genes for nonstructural proteins NS1 and NS2 (dNS), deletion of the small hydrophobic protein (ΔSH) gene, and replacement of the wild-type fusion (F) protein gene with a low-fusion RSV subgroup B F consensus sequence of the Buenos Aires clade (BAF). This vaccine candidate, RSV-A2-dNS-ΔSH-BAF (DB1), was attenuated in two models of primary human airway epithelial cells and in the upper and lower airways of cotton rats. DB1 was also highly immunogenic in cotton rats and elicited broadly neutralizing antibodies against a diverse panel of recombinant RSV strains. When vaccinated cotton rats were challenged with wild-type RSV A, DB1 reduced viral titers in the upper and lower airways by 3.8 log10total PFU and 2.7 log10PFU/g of tissue, respectively, compared to those in unvaccinated animals (P< 0.0001). DB1 was thus attenuated, highly immunogenic, and protective against RSV challenge in cotton rats. DB1 is the first RSV LAV to incorporate a low-fusion F protein as a strategy to attenuate viral replication and preserve immunogenicity.IMPORTANCERSV is a leading cause of infant hospitalizations and deaths. The development of an effective vaccine for this high-risk population is therefore a public health priority. Although live-attenuated vaccines have been safely administered to RSV-naive infants, strategies to balance vaccine attenuation with immunogenicity have been elusive. In this study, we introduced a novel strategy to attenuate a recombinant RSV vaccine by incorporating a low-fusion, subgroup B F protein in the genetic background of codon-deoptimized nonstructural protein genes and a deleted small hydrophobic protein gene. The resultant vaccine candidate, DB1, was attenuated, highly immunogenic, and protective against RSV challenge in cotton rats.

Respiratory syncytial virus (RSV) infections are the major contributor to acute lower respiratory syndrome in newborns. Infections generally result in hospitalization and sometimes in death. A vaccine is not available yet, despite decades of research. Vaccine development is hampered in consequence of a failed vaccine trial in the 1960s which entailed fatal outcomes. An alternative to direct vaccination of children is maternal vaccination for passive immunization of babies before birth. RSV infects every person repeatedly throughout life which implies that pregnant woman carry RSV-directed memory B cell (MBC) repertoires. A successful maternal vaccine would therefore elicit high titer RSV-neutralizing IgGs by reactivation of pre-existing MBCs which would protect newborns during the first months of life. RSV has two neutralizing antigens, of which the fusion protein (RSV F) is the most promising vaccine candidate. RSV F mediates the fusion process of viral and cellular membranes, wherefore it exists in a pre-fusion (pre F) or post-fusion (post F) conformation. Even though there is more interest in the pre F conformation as immunogen, the post F conformation may be equally considered as successful vaccine antigen. The pre F conformation is very metastable and readily switches to the highly stable post F conformation, which implies that a post F-based vaccine would be more cost-efficient to produce. More importantly, several neutralizing epitopes are preserved on the post F conformation and a substantial amount of RSV F-directed MBCs induced by natural infection are actually pre/post F cross-reactive. Since most of the pre/post F cross-reactive MBCs were previously shown to have higher affinities for the post F conformation, a post F-based vaccine may be ideal for reactivation and clonal expansion of MBCs which express neutralizing B cell receptors (BCRs). Every antigen, also RSV post F protein, has its own signatures within BCR repertoires because of preferential selection of BCR characteristics for B cell clonal expansions. In order to understand how RSV F protein shapes BCR repertoires, RSV F-directed BCRs were isolated from a healthy blood donor and three vaccinees who received an RSV post F vaccine. BCR repertoire analysis confirmed the assumption that the pre and post F protein have their own signatures within RSV F-directed BCR repertoires. The different characteristics indicated longer affinity maturation of pre F-reactive MBCs. Furthermore, estimation of clonal relatedness between the pre and post F-binding BCR repertoires from the healthy donor provided indications that a substantial number of the isolated BCRs are actually pre/post F cross-reactive, which confirmed a previous study. Analysis of the BCR repertoires isolated from the RSV post F-vaccinees showed that the vaccine induced a biased MBC response with preferential BCR characteristics. There were several implications that the post F-vaccine expanded primarily pre/post F cross-reactive MBCs. In contrast to the high variability of pre/post F cross-reactive BCRs induced by natural infection, the vaccine-induced MBC response indicated a skewed selection of VH4 gene family-encoded BCRs for clonal expansion and affinity maturation. More importantly, estimation of clonal relatedness revealed convergent MBC responses between the three analyzed subjects, while several MBC lineages shared stereotypic characteristics with pre F-binding BCRs or RSV-neutralizing antibodies. Some of the supposedly pre/post F cross-binding or neutralizing BCR sequences were expressed as mAbs and functionally characterized. RSV pre/post F cross-binding and neutralization activities could be demonstrated for all of the expressed mAbs. This project demonstrated the potential of ‘clonal’ grouping as novel reverse approach to screen BCR repertoires for functional antibodies.