Single mucosal immunization of recombinant adenovirus-based vaccine expressing F1 protein fragment induces protective mucosal immunity against respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV. The fusion (F) protein of RSV is a potentially important target for protective antiviral immune responses. Here, we studied the immune responses elicited by recombinant replication-deficient adenovirus (rAd)-based vaccines expressing the soluble F1 fragment of F protein (amino acids 155–524) in murine model. The expression of secreted F1 fragment by rAd was significantly increased by codon optimization. Strong mucosal IgA response was induced by single intranasal immunization of codon-optimized vaccine, rAd/F1co, but not by rAd/F1wt. A single intranasal immunization with rAd/F1co provided potent protection against subsequent RSV challenge. Interestingly, neither serum Ig nor T-cell response directed to F protein was detected in the rAd/F1co-immune mice, suggesting that protective immunity by rAd/F1co is mainly mediated through mucosal IgA induction. Indeed, co-delivery of cholera toxin B subunit significantly enhanced mucosal IgA responses by the optimized vaccine, which correlates with protective efficacy. Taken together, our data demonstrate that a single intranasal administration of rAd/F1co is sufficient for the protection and represents a promising prophylactic vaccination regimen against RSV infection.     

A recombinant varicella vaccine harboring a respiratory syncytial virus gene induces humoral immunity

The varicella-zoster virus (VZV) Oka vaccine strain (vOka) is highly efficient and causes few adverse events; therefore, it is used worldwide. We previously constructed recombinant vOka (rvOka) harboring the mumps virus gene. Immunizing guinea pigs with rvOka induced the production of neutralizing antibodies against the mumps virus and VZV.Here, we constructed recombinant vOka viruses containing either the respiratory syncytial virus (RSV) subgroup A fusion glycoprotein (RSV A–F) gene or RSV subgroup B fusion glycoprotein (RSV B–F) gene (rvOka-RSV A–F or rvOka-RSV B–F). Indirect immunofluorescence and Western blot analyses confirmed the expression of each recombinant RSV protein in virus-infected cells. Immunizing guinea pigs with rvOka-RSV A–F or rvOka-RSV B–F led to the induction of antibodies against RSV proteins. These results suggest that the current varicella vaccine genome can be used to generate custom-made vaccine vectors to develop the next generation of live vaccines.     

Long-lasting balanced immunity and protective efficacy against respiratory syncytial virus in mice induced by a recombinant protein G1F/M2

Respiratory syncytial virus (RSV) is the primary cause of serious lower respiratory tract illness in young children. We have engineered a recombinant candidate vaccine G1F/M2, consisting of a cytotoxic T lymphocyte (CTL) epitope of RSV-M2 protein and a domain of RSV-G protein. In this study, the long-term immunogenicity and protective effect were evaluated. In G1F/M2-immunized mice, special antibodies lasted for more than 19 weeks, and the IgG1/IgG2a ratio remained a balanced level till the end of the study, suggesting mixed Th1/Th2 type of responses. Concomitantly, G1F/M2 elicited long-lived RSV-specific CTL activity that was detectable at 12 weeks after the final immunization. Stronger CTL responses were induced with immunization once more at 13 weeks after the last immunization in G1F/M2-primed mice than those in F/M2-primed mice. These results suggest that G1F/M2-induced long-lasting balanced humoral and cellular immunity responses, and immunological memory in mice. Furthermore, following RSV challenge, long-term protective efficacy was observed. RSV replication in lungs of G1F/M2-primed mice elicited also mixed Th1/Th2 responses, a property that is considered advantageous for the safety of an RSV vaccine. Therefore, G1F/M2 is a promising RSV subunit vaccine.     

A recombinant anchorless respiratory syncytial virus (RSV) fusion (F) protein/monophosphoryl lipid A (MPL) vaccine protects against RSV-induced replication and lung pathology

We previously demonstrated that the severe cytokine storm and pathology associated with RSV infection following intramuscular vaccination of cotton rats with FI-RSV Lot 100 could be completely abolished by formulating the vaccine with the mild TLR4 agonist and adjuvant, monophosphoryl lipid A (MPL). Despite this significant improvement, the vaccine failed to blunt viral replication in the lungs. Since MPL is a weak TLR4 agonist, we hypothesized that its adjuvant activity was mediated by modulating the innate immune response of respiratory tract resident macrophages. Therefore, we developed a new vaccine preparation with purified, baculovirus expressed, partially purified, anchorless RSV F protein formulated with synthetic MPL that was administered to cotton rats intranasally, followed by an intradermal boost. This novel formulation and heterologous “prime/boost” route of administration resulted in decreased viral titers compared to that seen in animals vaccinated with F protein alone. Furthermore, animals vaccinated by this route showed no evidence of enhanced lung pathology upon RSV infection. This indicates that MPL acts as an immune modulator that protects the host from vaccine-enhanced pathology, and reduces RSV replication in the lower respiratory tract when administered by a heterologous prime/boost immunization regimen.     

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.     

A respiratory syncytial virus (RSV) vaccine based on parainfluenza virus 5 (PIV5)

Human respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease and hospitalizations in infants and young children. It also causes significant morbidity and mortality in elderly and immune compromised individuals. No licensed vaccine currently exists. Parainfluenza virus 5 (PIV5) is a paramyxovirus that causes no known human illness and has been used as a platform for vector-based vaccine development. To evaluate the efficacy of PIV5 as a RSV vaccine vector, we generated two recombinant PIV5 viruses – one expressing the fusion (F) protein and the other expressing the attachment glycoprotein (G) of RSV strain A2 (RSV A2). The vaccine strains were used separately for single-dose vaccinations in BALB/c mice. The results showed that both vaccines induced RSV antigen-specific antibody responses, with IgG2a/IgG1 ratios similar to those seen in wild-type RSV A2 infection. After challenging the vaccinated mice with RSV A2, histopathology of lung sections showed that the vaccines did not exacerbate lung lesions relative to RSV A2-immunized mice. Importantly, both F and G vaccines induced protective immunity. Therefore, PIV5 presents an attractive platform for vector-based vaccines against RSV infection.     

DNA immunization against respiratory syncytial virus (RSV) in infant rhesus monkeys

A DNA vaccine was tested in infant Rhesus macaques to evaluate its safety, immunogenicity and protective efficacy. Monkeys were vaccinated and challenged with a clinical isolate of human RSV. Vaccinated animals developed humoral and cellular responses following inoculation with plasmid DNA encoding the fusion (F) and nucleoprotein (N), from closely related bovine RSV. Vaccinated monkeys had decreased RSV in their lungs post-infection, and there was a qualitative difference in histopathology observed between vaccinated and unvaccinated animals. The combined result of safety and immunogenicity in a neonatal primate model is encouraging, suggesting the feasibility of DNA vaccines against RSV in infants.     

Enhanced immune protection by a liposome-encapsulated recombinant respiratory syncytial virus (RSV) vaccine using immunogenic lipids from Deinococcus radiodurans

The radiation-resistant bacterium, Deinococcus radiodurans contains a variety of phospho-, glyco- and phosphoglycolipids, the structures of which appear to be largely unique in nature. We show here that such lipids are immunogenic when administered as liposomes intranasally in mice, as evidenced by the induction of serum antibodies which recognize D. radiodurans lipids but not other lipids by thin layer chromatographic immunostaining. By modifying a liposomal vaccine against respiratory syncytial virus (RSV) we find that vaccine efficacy is markedly enhanced by the inclusion of lipids isolated from D. radiodurans. Using dioleoylphosphatidylcholine (DOPC) or D. radiodurans lipids, liposomes were prepared which encapsulated a soluble fragment of the RSV G protein (G(128-188)) fused with a portion of the bacterial thioredoxin (Trx) protein. Mice immunized intranasally with D. radiodurans liposomes showed markedly greater protection against RSV challenge over mice immunized with DOPC liposomes. Enhanced vaccine efficacy was achieved using liposomes prepared from either whole D. radiodurans lipids or from a single isolated phosphoglycolipid previously identified as alpha-galactosylphosphatidylglyceroylalkylamine (lipid 7). Mice immunized and protected against RSV challenge were free of pulmonary eosinophilic infiltration, an undesirable consequence of many RSV vaccines. The results provide further support for liposome-based vaccines for RSV and underline the importance of lipid composition in liposome formulations.     

The immunogenicity, protective efficacy and safety of BBG2Na, a subunit respiratory syncytial virus (RSV) vaccine candidate, against RSV-B

Respiratory syncytial virus (RSV) is divided into subgroups A and B, based primarily on variation within the G glycoprotein. A safe vaccine that protects against both would be the ideal. BBG2Na is a recombinant subunit RSV vaccine candidate derived in part from the G protein of RSV-A. Interestingly, BBG2Na formulated in alum protected against RSV-B challenge at early time points following vaccination in mice. Over 6 months, however, BBG2Na-induced immunogenicity and protective efficacy progressively diminished, such that few animals were considered protected at the end. To study the safety of BBG2Na relative to RSV-B challenge, we established a novel enhanced immunopathology mouse model. We confirmed that RSV-B challenge of formalin-inactivated RSV-A (FI-RSV-A)-immunized BALB/c mice results in enhanced pulmonary pathology. Therefore, this phenomenon is neither subgroup-specific nor dependent on a previously incriminated Th epitope in the RSV-A G protein. In stark contrast, BBG2Na did not induce any signs of enhanced pulmonary pathology. In conclusion, our data indicate that BBG2Na, formulated in alum, induces safe and protective immune responses against RSV-B challenge in mice. However, the duration of protective immunity will probably be insufficient to prevent RSV-B infection for the duration of the RSV epidemic season.     

Vaccination with recombinant modified vaccinia virus Ankara expressing bovine respiratory syncytial virus (bRSV) proteins protects calves against RSV challenge

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and calves. Bovine RSV (bRSV) is a natural pathogen for cattle, and bRSV infection in calves shares many features with the human infection. Thus, bRSV infection in cattle provides the ideal setting to evaluate the safety and efficacy of novel RSV vaccine strategies. Here, we have evaluated the efficacy and safety of modified vaccinia virus Ankara (rMVA)-based vaccine candidates, expressing the bovine RSV-F protein, either or not in combination with the G protein, in colostrums-deprived SPF calves born by caesarean section. Vaccination induced bRSV-specific IgG and CD8 T cell responses. Importantly, no IgE responses were detected. After bRSV challenge, rMVA vaccinated calves experienced less severe symptoms of lower respiratory tract disease compared to the mock-immunized control group. Immunized animals showed reduced pulmonary virus loads, and no eosinophilic infiltration or enhanced respiratory distress. In conclusion, candidate rMVA/bRSV vaccines induced protective and safe immune responses in calves.     

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.     

Primary care physicians’ perspectives on respiratory syncytial virus (RSV) disease in adults and a potential RSV vaccine for adults

Background

Deaths attributable to respiratory syncytial virus (RSV) among adults are estimated to exceed 11,000 annually, and annual adult hospitalizations for influenza and RSV may be comparable. RSV vaccines for older adults are in development. We assessed the following among primary care physicians (PCPs) who treat adults: (1) perception of RSV disease burden; (2) current RSV testing practices; and (3) anticipated barriers to adoption of an RSV vaccine.

Liposome encapsulation of a soluble recombinant fragment of the respiratory syncytial virus (RSV) G protein enhances immune protection and reduces lung eosinophilia associated with virus challenge

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children and infants. Previous animal studies have shown that immunizing intramuscularly or intraperitoneally with the RSV G protein has elicited protective as well as harmful immune responses upon RSV challenge. In an RSV immunization strategy designed to target the respiratory tract directly (the site of RSV replication), we immunized BALB/c mice intranasally with a liposome-encapsulated, prokaryotically expressed thioredoxin fusion protein consisting of amino acids 128-229 of the RSV G protein (Trx-G(128-229)). Upon intranasal challenge with RSV, a 100 to 500-fold reduction in lung RSV replication was observed in mice immunized with liposome-encapsulated Trx-G(128-229) compared to a sham-immunized control group. Analysis of bronchoalveolar lavage fluids revealed an influx of eosinophils (18% of total cells) in mice immunized with Trx-G(128-229) alone. Such eosinophilic infiltration was diminished (to 4.5% of total cells), however, in mice immunized with liposome-encapsulated Trx-G(128-229). Histological analysis of lung tissue revealed an accumulation of cells around the bronchioles and vessels in mice immunized with Trx-G(128-229) alone followed by RSV challenge which was not increased further in mice immunized with liposome-encapsulated Trx-G(128-229). These results show that intranasal immunization of BALB/c mice with Trx-G(128-229), when encapsulated in liposomes, can reduce the level of RSV replication in the lung as well as specifically reduce the degree of eosinophilic infiltration compared to mice immunized with Trx-G(128-229) alone. This demonstrates the potential of liposomes and particular recombinant fragments of the RSV G protein as an effective combination in RSV vaccine studies.     

Immunization of cotton rats with the fusion (F) and large (G) glycoproteins of respiratory syncytial virus (RSV) protects against RSV challenge without potentiating RSV disease

A formalin-inactivated respiratory syncytial virus (RSV) vaccine tested 22 years ago failed to protect infant vaccinees against RSV infection or disease. Instead, lower respiratory tract disease was enhanced during subsequent infection by RSV. Enhancement of pulmonary pathology is also observed when cotton rats are immunized with formalin-inactivated RSV and subsequently infected with this virus. A major question that must be addressed for each new paramyxovirus vaccine is whether the immunogen possesses the capacity to potentiate disease. In the present study, we evaluated a newly developed purified F and G glycoprotein vaccine over a wide dosage range for immunogenicity, efficacy and capacity to potentiate pulmonary pathology in cotton rats. In addition, a formalin-inactivated RSV vaccine, which served as a positive control for enhancement of pulmonary pathology, was evaluated simultaneously. The results of these comparisons indicate that the purified F and G glycoprotein vaccine was highly immunogenic and was efficacious even in animals that developed low levels of serum-neutralizing antibodies. Furthermore, the F and G vaccine did not induce potentiation of pulmonary pathology. In contrast, formalin-inactivated RSV potentiated RSV pulmonary histopathology, but there was a sparing of potentiation at high and low doses. Both the formalin-inactivated RSV and purified F and G preparations induced a high level of serum antibodies capable of binding to purified F and G glycoproteins but both sets of antibodies had significantly reduced neutralizing activity. These results are encouraging because they suggest that purified paramyxovirus glycoproteins might be used safely as a vaccine. However, it would be desirable to use a glycoprotein preparation that stimulates antibodies that have a high level of neutralizing activity similar to those characteristic of adult human convalescent sera.     

Correlates of immunity to respiratory syncytial virus (RSV) associated-hospitalization: establishment of minimum protective threshold levels of serum neutralizing antibodies

OBJECTIVE: To determine if respiratory syncytial virus (RSV) specific, serum antibody titers correlate with protection against RSV associated-hospitalization at all ages. DESIGN: Participants who were enrolled in a trial to determine the frequency of specific virus infections associated with hospitalization [J. Am. Med. Assoc. 283 (2000) 499] were included in our analysis if they were enrolled from July 1991 to June 1993, had a culture for virus isolation, and provided blood samples at hospitalization and 14-60 days later. RSV infection was defined by a positive culture and/or serology. Microneutralization, ELISA to the fusion (F) protein and Western blot were the serological assays that were used to determine correlates of immunity. RESULTS: One hundred and seventy-five individuals, 1 month to 89 years old, out of 538 patients hospitalized with an acute respiratory infection met the criteria for analysis. RSV associated-hospitalization occurred in 11 (40.7%) of 27 infants (<1 year), 8 (38.1%) of 21 young children (1 to <5 years), and 15 (11.8%) of 127 children and adults (> or =5 years). At the time of hospitalization, geometric mean neutralizing antibody titers (log(2)) to RSV/A and RSV/B, and geometric mean binding antibody titer (log(2)) to F protein were significantly higher in patients with non-RSV associated-hospitalization compared to those with RSV associated-hospitalization (RSV/A: 7.9 versus 6.1, P<0.001; RSV/B: 9.4 versus 7.3, P<0.001; ELISA-F, 13.9 versus 12.6, P=0.01). For every 1 log(2) increase in titer of neutralizing antibodies to RSV/A and RSV/B, and binding antibody to F protein there was a significant increase in the likelihood of not having an RSV associated-hospitalization by 22.3, 25, and 24.4% respectively. A minimal protective threshold titer of > or =6.0 (odds ratio 3.5; 95% CI 1.4-9.1) and > or =8.0 log(2) (odds ratio 2.9; 95% CI 1.1-7.7) against RSV associated-hospitalization was established for neutralizing antibodies to RSV/A and RSV/B; a threshold titer could not be established for binding antibody to F protein. CONCLUSION: Participants with naturally acquired serum neutralizing antibody levels at least equal to the minimal protective threshold titer were approximately three times more likely not to have an RSV associated-hospitalization. We speculate that achieving a minimal protective threshold antibody titer through active immunization will significantly reduce RSV associated-hospitalization among all ages.     

Modelling the impact of respiratory syncytial virus (RSV) vaccine and immunoprophylaxis strategies in New Zealand

BackgroundMathematical models of respiratory syncytial virus (RSV) transmission can help describe seasonal epidemics and assess the impact of potential vaccines and immunoprophylaxis with monoclonal antibodies (mAb).MethodsWe developed a deterministic, compartmental model for RSV transmission, which was fitted to population-based RSV hospital surveillance data from Auckland, New Zealand. The model simulated the introduction of either a maternal vaccine or a seasonal mAb among infants aged less than 6 months and estimated the reduction in RSV hospitalizations for a range of effectiveness and coverage values.ResultsThe model accurately reproduced the annual seasonality of RSV epidemics in Auckland. We found that a maternal vaccine with effectiveness of 30–40% in the first 90 days and 15–20% for the next 90 days could reduce RSV hospitalizations by 18–24% in children younger than 3 months, by 11–14% in children aged 3–5 months, and by 2–3% in children aged 6–23 months. A seasonal infant mAb with 40–60% effectiveness for 150 days could reduce RSV hospitalizations by 30–43%, 34–48% and by 14–21% in children aged 0–2 months, 3–5 months and 6–23 months, respectively.ConclusionsOur results suggest that either a maternal RSV vaccine or mAb would effectively reduce RSV hospitalization disease burden in New Zealand. Overall, a seasonal mAb resulted in a larger disease prevention impact than a maternal vaccine.     

Immunization onto shaved skin with a bacterial enterotoxin adjuvant protects mice against respiratory syncytial virus (RSV)

This study evaluated the potential of the skin as a non invasive route for RSV vaccination using two G protein-derived molecules, G2Na and G5 in mice. G2Na contains T and B-cell epitopes whether G5 is a pure B-cell epitope. In contrast to G5, G2Na coadministered with CT three times at 1 month interval onto 1cm of square area shaved skin, elicited a consistent serum anti-G2Na and anti-CT IgG response. The anti-G2Na IgG response was dominated by IgG1 isotype, an indirect marker of a Th2 type of response. Dramatic reduction and decrease of RSV titers in lung tissues and in the nasal tract, respectively, following intranasal virus challenge revealed biological relevance of the transcutaneous immunization in the context of RSV vaccine. These results suggest that the transcutaneous route may offer a promising potential for novel RSV vaccine strategy, simple, painless and economical.     

Respiratory syncytial virus (RSV) fusion protein expressed by recombinant Sendai virus elicits B-cell and T-cell responses in cotton rats and confers protection against RSV subtypes A and B

The respiratory syncytial virus (RSV) is a serious pediatric pathogen for which there is currently no clinically approved vaccine. This report describes the design and testing of a new RSV vaccine construct (rSV-RSV-F), created by the recombination of an RSV F sequence with the murine parainfluenza virus-type 1 (Sendai virus, SV) genome. SV was selected as the vaccine backbone for this study, because it has previously been shown to elicit high-magnitude, durable immune activities in animal studies and has advanced to human safety trials as a xenogenic vaccine for human parainfluenza virus-type 1 (hPIV-1). Cells infected with the recombinant SV expressed RSV F protein, but F was not incorporated into progeny SV virions. When cotton rats were inoculated with the vaccine, high-titer RSV-binding and neutralizing antibodies as well as interferon-γ-producing T-cells were induced. Most striking was the protection against intra-nasal RSV challenge conferred by the vaccine. The rSV-RSV-F construct was also tested as a mixture with a second SV construct expressing the RSV G protein, but no clear advantage was demonstrated by combining the two vaccines. As a final analysis, the efficacy of the rSV-RSV-F vaccine was tested against an array of RSV isolates. Results showed that neutralizing and protective responses were effective against RSV isolates of both A and B subtypes. Together, experimental results encourage promotion of this recombinant SV construct as a vaccine candidate for the prevention of RSV in humans.