Presumed Risk Factors and Biomarkers for Severe Respiratory Syncytial Virus Disease and Related Sequelae: Protocol for an Observational Multicenter, Case-Control Study From the Respiratory Syncytial Virus Consortium in Europe (RESCEU).

Respiratory syncytial virus (RSV) is the leading viral pathogen associated with acute lower respiratory tract infection and hospitalization in children < 5 years of age worldwide. While there are known clinical risk factors for severe RSV infection, the majority of those hospitalized are previously healthy infants. There is consequently an unmet need to identify biomarkers that predict host response, disease severity, and sequelae. The primary objective is to identify biomarkers of severe RSV acute respiratory tract infection (ARTI) in infants. Secondary objectives include establishing biomarkers associated with respiratory sequelae following RSV infection and characterizing the viral load, RSV whole-genome sequencing, host immune response, and transcriptomic, proteomic, metabolomic and epigenetic signatures associated with RSV disease severity. Six hundred thirty infants will be recruited across 3 European countries: the Netherlands, Spain, and the United Kingdom. Participants will be recruited into 2 groups: (1) infants with confirmed RSV ARTI (includes upper and lower respiratory tract infections), 500 without and 50 with comorbidities; and (2) 80 healthy controls. At baseline, participants will have nasopharyngeal, blood, buccal, stool, and urine samples collected, plus complete a questionnaire and 14-day symptom diary. At convalescence (7 weeks ± 1 week post-ARTI), specimen collection will be repeated. Laboratory measures will be correlated with symptom severity scores to identify corresponding biomarkers of disease severity. CLINICAL TRIALS REGISTRATION: NCT03756766.

Recombinant measles virus incorporating heterologous viral membrane proteins for use as vaccines.

Recombinant measles virus (rMV) vectors expressing heterologous viral membrane protein antigens are potentially useful as vaccines. Genes encoding the mumps virus haemagglutinin-neuraminidase (MuV-HN), the influenza virus haemagglutinin (Flu-HA) or the respiratory syncytial virus fusion (RSV-F) proteins were inserted into the genome of a live attenuated vaccine strain of measles virus. Additionally, in this case rMV with the MuV-HN or the influenza HA inserts, chimeric constructs were created that harboured the measles virus native haemagglutinin or fusion protein cytoplasmic domains. In all three cases, sucrose-gradient purified preparations of rMV were found to have incorporated the heterologous viral membrane protein on the viral membrane. The possible utility of rMV expressing RSV-F (rMV.RSV-F) as a vaccine was tested in a cotton rat challenge model. Vaccination with rMV.RSV-F efficiently induced neutralizing antibodies against RSV and protected animals from infection with RSV in the lungs.

A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism.

Respiratory syncytial virus (RSV) causes acute lower respiratory tract infections and is the leading cause of infant hospitalizations. Recently, a promising vaccine antigen based on the RSV fusion protein (RSV F) stabilized in the native prefusion conformation has been described. Here we report alternative strategies to arrest RSV F in the prefusion conformation based on the prevention of hinge movements in the first refolding region and the elimination of proteolytic exposure of the fusion peptide. A limited number of unique mutations are identified that stabilize the prefusion conformation of RSV F and dramatically increase expression levels. This highly stable prefusion RSV F elicits neutralizing antibodies in cotton rats and induces complete protection against viral challenge. Moreover, the structural and biochemical analysis of the prefusion variants suggests a function for p27, the excised segment that precedes the fusion peptide in the polypeptide chain.

Recombinant low-seroprevalent adenoviral vectors Ad26 and Ad35 expressing the respiratory syncytial virus (RSV) fusion protein induce protective immunity against RSV infection in cotton rats.

RSV is an important cause of lower respiratory tract infections in children, the elderly and in those with underlying medical conditions. Although the high disease burden indicates an urgent need for a vaccine against RSV, no licensed RSV vaccine is currently available. We developed an RSV vaccine candidate based on the low-seroprevalent human adenovirus serotypes 26 and 35 (Ad26 and Ad35) encoding the RSV fusion (F) gene. Single immunization of mice with either one of these vectors induced high titers of RSV neutralizing antibodies and high levels of F specific interferon-gamma-producing T cells. A Th1-type immune response was indicated by a high IgG2a/IgG1 ratio of RSV-specific antibodies, strong induction of RSV-specific interferon-gamma and tumor necrosis factor-alpha cytokine producing CD8 Tcells, and low RSV-specific CD4 T-cell induction. Both humoral and cellular responses were increased upon a boost with RSV-F expressing heterologous adenovirus vector (Ad35 boost after Ad26 prime or vice versa). Both single immunization and prime-boost immunization of cotton rats induced high and long-lasting RSV neutralizing antibody titers and protective immunity against lung and nasal RSV A2 virus load up to at least 30 weeks after immunization. Cotton rats were also completely protected against challenge with a RSV B strain (B15/97) after heterologous prime-boost immunization. Lungs from vaccinated animals showed minimal damage or inflammatory infiltrates post-challenge, in contrast to animals vaccinated with formalin-inactivated virus. Our results suggest that recombinant human adenoviral Ad26 and Ad35 vectors encoding the RSV F gene have the potential to provide broad and durable protection against RSV in humans, and appear safe to be investigated in infants.

Impaired immune response to vaccination against infection with human respiratory syncytial virus at advanced age.

UNLABELLED: Elderly humans are prone to severe infection with human respiratory syncytial virus (HRSV). The aging of today's human population warrants the development of protective vaccination strategies aimed specifically at the elderly. This may require special approaches due to deteriorating immune function. To design and test vaccination strategies tailored to the elderly population, we need to understand the host response to HRSV vaccination and infection at old age. Moreover, the preclinical need for testing of candidate vaccines requires translational models resembling susceptibility to the (unadapted) human pathogen. Here, we explored the effects of aging on immunity and protection induced by a model HRSV vaccine candidate in a translational aging model in cotton rats (Sigmodon hispidus) and examined possibilities to optimize vaccination concepts for the elderly. We immunized young and aged cotton rats with a live-attenuated recombinant HRSV vaccine candidate and analyzed the induced immune response to and protection against challenge with HRSV. In old cotton rats, HRSV infection persisted longer, and vaccination induced less protection against infection. Aged animals developed lower levels of vaccine-induced IgG, virus-neutralizing serum antibodies, and IgA in lungs. Moreover, booster responses to HRSV challenge were impaired in animals vaccinated at an older age. However, increased dose and reduced attenuation of vaccine improved protection even in old animals. This study shows that cotton rats provide a model for studying the effects of aging on the immune response to the human respiratory pathogen HRSV and possibilities to optimize vaccine concepts for the elderly. IMPORTANCE: HRSV infection poses a risk for severe disease in the elderly. The aging of the population warrants increased efforts to prevent disease at old age, whereas HRSV vaccines are only in the developmental phase. The preclinical need for testing of candidate human vaccines requires translational models resembling susceptibility to the natural human virus. Moreover, we need to gain insight into waning immunity at old age, as this is a special concern in vaccine development. In this study, we explored the effect of age on protection and immunity against an experimental HRSV vaccine in aged cotton rats (Sigmodon hispidus), a rodent species that provides a model representing natural susceptibility to human viruses. Older animals generate fewer antibodies upon vaccination and require a higher vaccine dose for protection. Notably, during the early secondary immune response to subsequent HRSV infection, older animals showed less protection and a slower increase of the virus-neutralizing antibody titer.

An improved respiratory syncytial virus neutralization assay based on the detection of green fluorescent protein expression and automated plaque counting.

BACKGROUND: Virus neutralizing antibodies against respiratory syncytial virus (RSV) are considered important correlates of protection for vaccine evaluation. The established plaque reduction assay is time consuming, labor intensive and highly variable. METHODS: Here, a neutralization assay based on a modified RSV strain expressing the green fluorescent protein in combination with automated detection and quantification of plaques is described. RESULTS: The fluorescence plaque reduction assay in microplate format requires only two days to complete and is simple and reproducible. A good correlation between visual and automated counting methods to determine RSV neutralizing serum antibody titers was observed. CONCLUSIONS: The developed virus neutralization assay is suitable for high-throughput testing and can be used for both animal studies and (large scale) vaccine clinical trials.

Systemic signature of the lung response to respiratory syncytial virus infection.

Respiratory Syncytial Virus is a frequent cause of severe bronchiolitis in children. To improve our understanding of systemic host responses to RSV, we compared BALB/c mouse gene expression responses at day 1, 2, and 5 during primary RSV infection in lung, bronchial lymph nodes, and blood. We identified a set of 53 interferon-associated and innate immunity genes that give correlated responses in all three murine tissues. Additionally, we identified blood gene signatures that are indicative of acute infection, secondary immune response, and vaccine-enhanced disease, respectively. Eosinophil-associated ribonucleases were characteristic for the vaccine-enhanced disease blood signature. These results indicate that it may be possible to distinguish protective and unfavorable patient lung responses via blood diagnostics.

Gene expression differences in lungs of mice during secondary immune responses to respiratory syncytial virus infection.

Vaccine-induced immunity has been shown to alter the course of a respiratory syncytial virus (RSV) infection both in murine models and in humans. To elucidate which mechanisms underlie the effect of vaccine-induced immunity on the course of RSV infection, transcription profiles in the lungs of RSV-infected mice were examined by microarray analysis. Three models were used: RSV reinfection as a model for natural immunity, RSV challenge after formalin-inactivated RSV vaccination as a model for vaccine-enhanced disease, and RSV challenge following vaccination with recombinant RSV virus lacking the G gene (DeltaG-RSV) as a model for vaccine-induced immunity. Gene transcription profiles, histopathology, and viral loads were analyzed at 1, 2, and 5 days after RSV challenge. On the first 2 days after challenge, all mice displayed an expression pattern in the lung similar of that found in primary infection, showing a strong innate immune response. On day 5 after RSV reinfection or after challenge following DeltaG-RSV vaccination, the innate immune response was waning. In contrast, in mice with vaccine-enhanced disease, the innate immune response 5 days after RSV challenge was still present even though viral replication was diminished. In addition, only in this group was Th2 gene expression induced. These findings support a hypothesis that vaccine-enhanced disease is mediated by prolonged innate immune responses and Th2 polarization in the absence of viral replication.

A highly attenuated recombinant human respiratory syncytial virus lacking the G protein induces long-lasting protection in cotton rats.

BACKGROUND: Respiratory syncytial virus (RSV) is a primary cause of serious lower respiratory tract illness for which there is still no safe and effective vaccine available. Using reverse genetics, recombinant (r)RSV and an rRSV lacking the G gene (DeltaG) were constructed based on a clinical RSV isolate (strain 98-25147-X). RESULTS: Growth of both recombinant viruses was equivalent to that of wild type virus in Vero cells, but was reduced in human epithelial cells like Hep-2. Replication in cotton rat lungs could not be detected for DeltaG, while rRSV was 100-fold attenuated compared to wild type virus. Upon single dose intranasal administration in cotton rats, both recombinant viruses developed high levels of neutralizing antibodies and conferred comparable long-lasting protection against RSV challenge; protection against replication in the lungs lasted at least 147 days and protection against pulmonary inflammation lasted at least 75 days. CONCLUSION: Collectively, the data indicate that a single dose immunization with the highly attenuated DeltaG as well as the attenuated rRSV conferred long term protection in the cotton rat against subsequent RSV challenge, without inducing vaccine enhanced pathology. Since DeltaG is not likely to revert to a less attenuated phenotype, we plan to evaluate this deletion mutant further and to investigate its potential as a vaccine candidate against RSV infection.

Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming.

The B cell lymphoma-6 (Bcl-6) and Bcl-xL proteins are expressed in germinal center B cells and enable them to endure the proliferative and mutagenic environment of the germinal center. By introducing these genes into peripheral blood memory B cells and culturing these cells with two factors produced by follicular helper T cells, CD40 ligand (CD40L) and interleukin-21 (IL-21), we convert them to highly proliferating, cell surface B cell receptor (BCR)-positive, immunoglobulin-secreting B cells with features of germinal center B cells, including expression of activation-induced cytidine deaminase (AID). We generated cloned lines of B cells specific for respiratory syncytial virus and used these cells as a source of antibodies that effectively neutralized this virus in vivo. This method provides a new tool to study B cell biology and signal transduction through antigen-specific B cell receptors and for the rapid generation of high-affinity human monoclonal antibodies.