Vaccination against Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) causes infection throughout life, with infants, adults who are severely immunocompromised, and the elderly at special risk of developing lower respiratory tract disease, hospitalisation, and death. The burden of severe disease in the elderly is comparable to seasonal influenza, and there remains no effective anti-viral drugs or vaccine for any target population. The development of a vaccine to confer immunity against severe disease is a major global health priority. A multitude of safe and immunogenic vaccine candidates have failed to induce the protective immunity needed for licensure, and in recent years this has included the largest clinical trials of RSV vaccines in history. The obstacles to vaccine development in elderly populations include an incomplete understanding of the immune responses needed for protection, the effect of aging on induction and maintenance of immunity (natural and vaccine induced immunity), and the high rate of co-morbid disease in older adults. Recent advances in structural biology, new biological platforms for antigen delivery, and insights from experimental challenge models mark the latest developments in over 50 years of research. This continues to be an active and evolving field of scientific discovery with renewed hope for a vaccine in the future.

Priorities for developing respiratory syncytial virus vaccines in different target populations.

The development of an effective vaccine against respiratory syncytial virus (RSV) has been hampered by major difficulties that occurred in the 1960s when a formalin-inactivated vaccine led to increased severity of RSV disease after acquisition of the virus in the RSV season after vaccination. Recent renewed efforts to develop a vaccine have resulted in about 38 candidate vaccines and monoclonal antibodies now in clinical development. The target populations for effective vaccination are varied and include neonates, young children, pregnant women, and older adults. The reasons for susceptibility to infection in each of these groups may be different and, therefore, could require different vaccine types for induction of protective immune responses, adding a further challenge for vaccine development. Here, we review the current knowledge of RSV vaccine development for these target populations and propose a view and rationale for prioritizing RSV vaccine development.

Respiratory syncytial virus: diagnosis, prevention and management.

Respiratory syncytial virus (RSV) is responsible for a large burden of disease globally and can present as a variety of clinical syndromes in children of all ages. Bronchiolitis in infants under 1 year of age is the most common clinical presentation hospitalizing 24.2 per 1000 infants each year in the United Kingdom. RSV has been shown to account for 22% of all episodes of acute lower respiratory tract infection in children globally. RSV hospitalization, that is, RSV severe disease, has also been associated with subsequent chronic respiratory morbidity. Routine viral testing in all children is not currently recommended by the United Kingdom National Institute for Health and Care Excellence (NICE) or the American Academy of Pediatrics (AAP) guidance and management is largely supportive. There is some evidence for the use of ribavirin in severely immunocompromised children. Emphasis is placed on prevention of RSV infection through infection control measures both in hospital and in the community, and the use of the RSV-specific monoclonal antibody, palivizumab, for certain high-risk groups of infants. New RSV antivirals and vaccines are currently in development. Ongoing work is needed to improve the prevention of RSV infection, not only because of the acute morbidity and mortality, but also to reduce the associated chronic respiratory morbidity after severe infection.

Author Correction: Airway response to respiratory syncytial virus has incidental antibacterial effects.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Airway response to respiratory syncytial virus has incidental antibacterial effects.

RSV infection is typically associated with secondary bacterial infection. We hypothesise that the local airway immune response to RSV has incidental antibacterial effects. Using coordinated proteomics and metagenomics analysis we simultaneously analysed the microbiota and proteomes of the upper airway and determined direct antibacterial activity in airway secretions of RSV-infected children. Here, we report that the airway abundance of Streptococcus was higher in samples collected at the time of RSV infection compared with samples collected one month later. RSV infection is associated with neutrophil influx into the airway and degranulation and is marked by overexpression of proteins with known antibacterial activity including BPI, EPX, MPO and AZU1. Airway secretions of children infected with RSV, have significantly greater antibacterial activity compared to RSV-negative controls. This RSV-associated, neutrophil-mediated antibacterial response in the airway appears to act as a regulatory mechanism that modulates bacterial growth in the airways of RSV-infected children.

Agreement between ELISA and plaque reduction neutralisation assay in Detection of respiratory syncytial virus specific antibodies in a birth Cohort from Kilifi, coastal Kenya.

Background: Severe disease associated with respiratory syncytial virus (RSV) infection occurs predominantly among infants under 6 months of age. Vaccines for prevention are in clinical development. Assessment of the vaccine effectiveness in large epidemiological studies requires serological assays which are rapid, economical and standardised between laboratories. The objective of this study was to assess the agreement between two enzyme linked immunosorbent assays (ELISA) and the plaque reduction neutralisation test (PRNT) in quantifying RSV specific antibodies. Methods: Archived sera from 99 participants of the Kilifi Birth Cohort (KBC) study (conducted 2002-2007) were screened for RSV antibodies using 3 methods: ELISA using crude RSV lysate as antigen, a commercial RSV immunoglobulin G (IgG) ELISA kit from IBL International GmbH, and PRNT. Pearson correlation, Bland-Altman plots and regression methods were used in analysis. Results: There was high positive correlation between the IBL RSV IgG ELISA and PRNT antibodies (Pearson r=0.75), and moderate positive correlation between the crude RSV lysate IgG ELISA and PRNT antibodies (r= 0.61). Crude RSV lysate IgG ELISA showed a wider 95% limit of agreement (-1.866, 6.157) with PRNT compared to the IBL RSV IgG ELISA (1.392, 7.595). Mean PRNT titres were estimated within a width of 4.8 log 2PRNT and 5.6 log 2PRNT at 95% prediction interval by IBL RSV IgG and crude RSV lysate IgG ELISA, respectively. Conclusion: Although, the IBL RSV IgG ELISA is observed to provide a reasonable correlate for PRNT assay in detecting RSV specific antibodies, it does not provide an accurate prediction for neutralizing antibody levels. An RSV neutralising antibody level is likely to fall within 2.4 fold higher and 2.4 fold lower than the true value if IBL RSV IgG ELISA is used to replace PRNT assay. The utility of an ELISA assay in vaccine studies should be assessed independent of the PRNT method.

Novel genetically-modified chimpanzee adenovirus and MVA-vectored respiratory syncytial virus vaccine safely boosts humoral and cellular immunity in healthy older adults.

OBJECTIVES: Respiratory syncytial virus (RSV) causes respiratory infection across the world, with infants and the elderly at particular risk of developing severe disease and death. The replication-defective chimpanzee adenovirus (PanAd3-RSV) and modified vaccinia virus Ankara (MVA-RSV) vaccines were shown to be safe and immunogenic in young healthy adults. Here we report an extension to this first-in-man vaccine trial to include healthy older adults aged 60-75 years. METHODS: We evaluated the safety and immunogenicity of a single dose of MVA-RSV given by intra-muscular (IM) injection (n = 6), two doses of IM PanAd3-RSV given 4-weeks apart (n = 6), IM PanAd3-RSV prime and IM MVA-RSV boost 8-weeks later (n = 6), intra-nasal (IN) spray of PanAd3-RSV prime and IM MVA-RSV boost 8-weeks later (n = 6), or no vaccine (n = 6). Safety measures included all adverse events within one week of vaccination and blood monitoring. Immunogenicity measures included serum antibody responses (RSV- and PanAd3-neutralising antibody titres measured by plaque-reduction neutralisation and SEAP assays, respectively), peripheral B-cell immune responses (frequencies of F-specific IgG and IgA antibody secreting cells and memory B-cells by ex vivo and cultured dual-colour ELISpot assays respectively), and peripheral RSV-specific T-cell immune responses (frequencies of IFNγ-producing T-cells by ex vivo ELISpot and CD4+/CD8+/Tfh-like cell frequencies by ICS/FACS assay). RESULTS: The vaccines were safe and well tolerated. Compared with each individual baseline immunity the mean fold-changes in serum RSV-neutralising antibody, appearance and magnitude of F-specific IgG and IgA ASCs and expansion of CD4+/CD8+ IFNγ-producing T-cells in peripheral circulation were comparable to the results seen from younger healthy adults who received the same vaccine combination and dose. There were little/no IgA memory B-cell responses in younger and older adults. Expansion of IFNγ-producing T-cells was most marked in older adults following IM prime, with balanced CD4+ and CD8+ T cell responses. The RSV-specific immune responses to vaccination did not appear to be attenuated in the presence of PanAd3 (vector) neutralising antibody. CONCLUSIONS: PanAd3-RSV and MVA-RSV was safe and immunogenic in older adults and the parallel induction of RSV-specific humoral and cellular immunity merits further assessment in providing protection from severe disease.

Antibodies in lymphocyte supernatants can distinguish between neutralising antibodies induced by RSV vaccination and pre-existing antibodies induced by natural infection.

INTRODUCTION: Respiratory syncytial virus (RSV) is the single most important cause of severe respiratory illness in infants. There is no effective vaccine and the only effective treatment available is the monoclonal antibody palivizumab which reduces the risk of severe RSV disease in prematurely born infants. However, palivizumab is too costly to allow for wide implementation and thus treatment is restricted to supportive care. Despite extensive efforts to develop a vaccine, progress has been hindered by the difficulty in measuring and assessing immunological correlates of RSV vaccine efficacy in the presence of high levels of pre-existing RSV antibodies. METHODS: Here we describe a new method for measuring the functional activity of antibodies induced by vaccination distinct from pre-existing antibodies. Antibodies in lymphocyte supernatants (ALS) from the cultured peripheral blood mononuclear cells (PBMCs) of young adults who had recently been vaccinated with a novel RSV candidate vaccine were directly assayed for virus neutralising activity. An ELISA method was used to measure antibodies in nasal and serum samples and then compared with the adapted ALS based method. RESULTS: There was a wide background distribution of RSV-specific antibodies in serum and nasal samples that obscured vaccine-specific responses measured two weeks after vaccination. No RSV-specific antibodies were observed at baseline in ALS samples, but a clear vaccine-specific antibody response was observed in ALS seven days after the administration of each dose of vaccine. These vaccine-specific antibodies in ALS displayed functional activity in vitro, and quantification of this functional activity was unperturbed by pre-existing antibodies from natural exposure. The results demonstrate a promising new approach for assessing functional immune responses attributed to RSV vaccines.

Untargeted analysis of the airway proteomes of children with respiratory infections using mass spectrometry based proteomics.

The upper airway - which consists mainly of the naso- and oro-pharynx - is the first point of contact between the respiratory system and microbial organisms that are ubiquitous in the environment. It has evolved highly specialised functions to address these constant threats whilst facilitating seamless respiratory exchange with the lower respiratory tract. Dysregulation of its critical homeostatic and defence functions can lead to ingress of pathogens into the lower respiratory tract, potentially leading to serious illness. Systems-wide proteomic tools may facilitate a better understanding of mechanisms in the upper airways in health and disease. In this study, we aimed to develop a mass spectrometry based proteomics method for characterizing the upper airways proteome. Naso- and oropharyngeal swab samples used in all our experiments had been eluted in the Universal Transport Media (UTM) containing significantly high levels of bovine serum albumin. Our proteomic experiments tested the optimal approach to characterize airway proteome on swab samples eluted in UTM based on the number of proteins identified without BSA depletion (Total proteome: Protocol A) and with its depletion using a commercial kit; Allprep, Qiagen (cellular proteome: Protocol B, Ci, and Cii). Observations and lessons drawn from protocol A, fed into the design and implementation of protocol B, and from B to protocol Ci and finally Cii. Label free proteome quantification was used in Protocol A (n = 6) and B (n = 4) while commercial TMT 10plex reagents were used for protocols Ci and ii (n = 83). Protocols Ci and ii were carried out under similar conditions except for the elution gradient: 3 h and 6 h respectively. Swab samples tested in this study were from infants and children with and without upper respiratory tract infections from Kilifi County Hospital on the Kenyan Coast. Protocol A had the least number of proteins identified (215) while B produced the highest number of protein identifications (2396). When Protocol B was modified through sample multiplexing with TMT to enable higher throughput (Protocol Ci), the number of protein identified reduced to 1432. Modification of protocol Ci by increasing the peptide elution time generated Protocol Cii that substantially increased the number of proteins identified to 1875. The coefficient of variation among the TMT runs in Protocol Cii was <20%. There was substantial overlap in the identity of proteins using the four protocols. Our method was were able to identify marker proteins characteristically expressed in the upper airway. We found high expression levels of signature nasopharyngeal and oral proteins, including BPIFA1/2 and AMY1A, as well as a high abundance of proteins related to innate and adaptive immune function in the upper airway. We have developed a sensitive systems-level proteomic assay for the systematic quantification of naso-oro-pharyngeal proteins. The assay will advance mechanistic studies of respiratory pathology, by providing an untargeted and hypothesis-free approach of examining the airway proteome.

Development of respiratory syncytial virus (RSV) vaccines for infants.

2017 will mark the 60th anniversary since the first isolation of RSV in children. In spite of concerted efforts over all these years, the goal of developing an effective vaccine against paediatric RSV disease has remained elusive. One of the main hurdles standing in the way of an effective vaccine is the fact that the age incidence of severe disease peaks within the first 3 months of life, providing limited opportunity for intervention. In addition to this complexity, the spectre of failed historical vaccines, which increased the risk of illness and death upon subsequent natural infection, has substantially increased the safety criteria against which modern vaccines will be assessed. This review traces the history of RSV vaccine development for young infants and analyses the potential reasons for the failure of historic vaccines. It also discusses recent breakthroughs in vaccine antigen design and the progressive evolution of platforms for the delivery of these antigens to seronegative infants.

Defining the vaccination window for respiratory syncytial virus (RSV) using age-seroprevalence data for children in Kilifi, Kenya.

BACKGROUND: Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract disease in early life and a target for vaccine prevention. Data on the age-prevalence of RSV specific antibodies will inform on optimizing vaccine delivery. METHODS: Archived plasma samples were randomly selected within age strata from 960 children less than 145 months of age admitted to Kilifi County Hospital pediatric wards between 2007 and 2010. Samples were tested for antibodies to RSV using crude virus IgG ELISA. Seroprevalence (and 95% confidence intervals) was estimated as the proportion of children with specific antibodies above a defined cut-off level. Nested catalytic models were used to explore different assumptions on antibody dynamics and estimate the rates of decay of RSV specific maternal antibody and acquisition of infection with age, and the average age of infection. RESULTS: RSV specific antibody prevalence was 100% at age 0-<1month, declining rapidly over the first 6 months of life, followed by an increase in the second half of the first year of life and beyond. Seroprevalence was lowest throughout the age range 5-11 months; all children were seropositive beyond 3 years of age. The best fit model to the data yielded estimates for the rate of infection of 0.78/person/year (95% CI 0.65-0.97) and 1.69/person/year (95% CI 1.27-2.04) for ages 0-<1 year and 1-<12 years, respectively. The rate of loss of maternal antibodies was estimated as 2.54/year (95% CI 2.30-2.90), i.e. mean duration 4.7 months. The mean age at primary infection was estimated at 15 months (95% CI 13-18). CONCLUSIONS: The rate of decay of maternal antibody prevalence and subsequent age-acquisition of infection are rapid, and the average age at primary infection early. The vaccination window is narrow, and suggests optimal targeting of vaccine to infants 5 months and above to achieve high seroconversion.

Best practice in the prevention and management of paediatric respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) infection is ubiquitous with almost all infants having been infected by 2 years of age and lifelong repeated infections common. It is the second largest cause of mortality, after malaria, in infants outside the neonatal period and causes up to 200,000 deaths per year worldwide. RSV results in clinical syndromes that include upper respiratory tract infections, otitis media, bronchiolitis (up to 80% of cases) and lower respiratory tract disease including pneumonia and exacerbations of asthma or viral-induced wheeze. For the purposes of this review we will focus on RSV bronchiolitis in infants in whom the greatest disease burden lies. For infants requiring hospital admission, the identification of the causative respiratory virus is used to direct cohorting or isolation and infection control procedures to minimize nosocomial transmission. Nosocomial RSV infections are associated with poorer clinical outcomes, including increased mortality, the need for mechanical ventilation and longer length of hospital stay. Numerous clinical guidelines for the management of infants with bronchiolitis have been published, although none are specific for RSV bronchiolitis. Ribavirin is the only licensed drug for the specific treatment of RSV infection but due to drug toxicity and minimal clinical benefit it has not been recommended for routine clinical use. There is currently no licensed vaccine to prevent RSV infection but passive immunoprophylaxis using a monoclonal antibody, palivizumab, reduces the risk of hospitalization due to RSV infection by 39-78% in various high-risk infants predisposed to developing severe RSV disease. The current management of RSV bronchiolitis is purely supportive, with feeding support and oxygen supplementation until the infant immune system mounts a response capable of controlling the disease. The development of a successful treatment or prophylactic agent has the potential to revolutionize the care and outcome for severe RSV infections in the world's most vulnerable infants.

RSV vaccine use--the missing data.

Respiratory syncytial virus (RSV) infection is the most important cause of hospitalization in infants and is one of the leading global causes of infant mortality and as such its prevention through vaccination is a public health priority. While essential for the successful implementation of vaccine programs, there remains a paucity of data on the epidemiology of the virus in different settings and age groups and limited knowledge about virus transmission and the health-care costs of the disease. Such data are now needed to populate health economic models and to inform optimal approaches to disease control through vaccination.

Chimpanzee adenovirus- and MVA-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults.

Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication-defective viral vectors encoding the RSV fusion (F), nucleocapsid (N), and matrix (M2-1) proteins for the induction of humoral and cellular responses. We performed an open-label, dose escalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intramuscular (IM) and intranasal (IN) administration of the adenovirus-vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralizing antibody titers rose in response to IM prime with PanAd3-RSV and after IM boost for individuals primed by the IN route. Circulating anti-F immunoglobulin G (IgG) and IgA antibody-secreting cells (ASCs) were observed after the IM prime and IM boost. RSV-specific T cell responses were increased after the IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV. Interferon-γ (IFN-γ) secretion after boost was from both CD4(+) and CD8(+) T cells, without detectable T helper cell 2 (TH2) cytokines that have been previously associated with immune pathogenesis following exposure to RSV after the formalin-inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease.

The association between age and the development of respiratory syncytial virus neutralising antibody responses following natural infection in infants.

To determine the age at which infants mount significant neutralising antibody responses to both natural RSV infection and live vaccines that mimic natural infection, RSV-specific neutralising antibodies in the acute and convalescent phase sera of infants with RSV infection were assayed. Age-specific incidence estimates for hospitalisation with severe RSV disease were determined and compared to age-specific neutralising antibody response patterns. Disease incidence peaked at between 2 and 3.9 months of life. Following natural infection, relative to the mean acute phase antibody titre, the mean convalescent titre was lower in the 0-1.9 month age class, no different in the 2-3.9 month age class and greater in all age classes greater than 4 months. These data suggest effective vaccination with live vaccines that mimic natural infection may not be achieved before the age of 4 months. Maternal vaccination may be an alternative to direct infant vaccination in order to protect very young babies.

Group- and genotype-specific neutralizing antibody responses against respiratory syncytial virus in infants and young children with severe pneumonia.

The effect of genetic variation on the neutralizing antibody response to respiratory syncytial virus (RSV) is poorly understood. In this study, acute- and convalescent-phase sera were evaluated against different RSV strains. The proportion of individuals with homologous seroconversion was greater than that among individuals with heterologous seroconversion among those infected with RSV group A (50% vs 12.5%; P = .0005) or RSV group B (40% vs 8%; P = .008). Seroconversion to BA genotype or non-BA genotype test viruses was similar among individuals infected with non-BA virus (35% vs 50%; P = .4) or BA virus (50% vs 65%; P = .4). The RSV neutralizing response is group specific. The BA-associated genetic change did not confer an ability to escape neutralizing responses to previous non-BA viruses.

Genetic relatedness of infecting and reinfecting respiratory syncytial virus strains identified in a birth cohort from rural Kenya.

BACKGROUND: Respiratory syncytial virus (RSV) reinfects individuals repeatedly. The extent to which this is a consequence of RSV antigenic diversity is unclear. METHODS: Six-hundred thirty-five children from rural Kenya were closely monitored for RSV infection from birth through 3 consecutive RSV epidemics. RSV infections were identified by immunofluorescence testing of nasal washing samples collected during acute respiratory illnesses, typed into group A and B, and sequenced in the attachment (G) protein. A positive sample separated from a previous positive by ≥14 days was defined as a reinfection a priori. RESULTS: Phylogenetic analysis was undertaken for 325 (80%) of 409 identified infections, including 53 (64%) of 83 reinfections. Heterologous group reinfections were observed in 28 episodes, and homologous group reinfections were observed in 25 episodes; 10 involved homologous genotypes, 5 showed no amino acid changes, and 3 were separated by 21-24 days and were potentially persistent infections. The temporal distribution of genotypes among reinfections did not differ from that of single infections. CONCLUSIONS: The vast majority of infection and reinfection pairs differed by group, genotype, or G amino acid sequence (ie, comprised distinct viruses). The extent to which this is a consequence of immune memory of infection history or prevalent diversity remains unclear.