Toward a virosomal respiratory syncytial virus vaccine with a built-in lipophilic adjuvant:A vaccine candidate for the elderly and pregnant women

Respiratory syncytial virus (RSV) represents the single most important cause of severe acute respiratory infection (SARI) and viral bronchiolitis among infants and young children. RSV also causes serious illness among the elderly and immunocompromised individuals. A vaccine against RSV would significantly reduce the health burden caused by RSV infection. In this study, we describe the development and optimization of a virosomal RSV vaccine, specifically aimed at protection of the elderly and of infants, the latter through vaccination of pregnant women. RSV virosomes are reconstituted viral envelopes that contain the membrane glycoproteins F and G of the virus, but lack the viral nucleocapsid. Thus, the particles are incapable of replication and therefore not able to cause disease. Since the major viral antigens F and G are displayed on the virosomal surface, use of virosomes as a vaccine will lead to the induction of virus-neutralizing (VN) antibodies. In the present study, first the production of RSV virosomes and the incorporation of a lipophilic adjuvant have been optimized to eventually fulfill GMP production requirements. Specifically, we investigated the use of a synthetic variant of the adjuvant monophosphoryl lipid A (MPLA). Subsequently, we monitored the virosomes’ long-term stability and we evaluated their immunogenicity in a murine model system. Additionally, we improved the capacity of virosomes to induce VN antibodies by employing thermostable virus strains with enhanced glycoprotein F stability. Finally, we further optimized the vaccine’s concept by switching from virosomes to synthetic liposomes with conjugated recombinant stabilized preF-protein derived from the native viral F glycoprotein. We conclude that RSV virosomes, with a built-in synthetic MPLA adjuvant, represent a promising vaccine candidate against RSV infection

Glycan repositioning of influenza hemagglutinin stem facilitates the elicitation of protective cross-group antibody responses.

The conserved hemagglutinin (HA) stem has been a focus of universal influenza vaccine efforts. Influenza A group 1 HA stem-nanoparticles have been demonstrated to confer heterosubtypic protection in animals; however, the protection does not extend to group 2 viruses, due in part to differences in glycosylation between group 1 and 2 stems. Here, we show that introducing the group 2 glycan at Asn38 to a group 1 stem-nanoparticle (gN38 variant) based on A/New Caledonia/20/99 (H1N1) broadens antibody responses to cross-react with group 2 HAs. Immunoglobulins elicited by the gN38 variant provide complete protection against group 2 H7N9 virus infection, while the variant loses protection against a group 1 H5N1 virus. The N38 glycan thus is pivotal in directing antibody responses by controlling access to group-determining stem epitopes. Precise targeting of stem-directed antibody responses to the site of vulnerability by glycan repositioning may be a step towards achieving cross-group influenza protection.We thank D. Scorpio, A. Taylor, H. Bao, C. Chiedi, M. Dillon, L. Gilliam, and G. Sarbador (VRC) for help with animal studies; H. Andersen (Bioqual, Inc.) for mouse challenge studies; C. Case (Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.) for help with challenge study coordination; A. Kumar (VRC) for producing RSV proteins; and members of Viral Pathogenesis Laboratory and Universal Influenza Vaccine Program (VRC) for helpful discussion. Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health. Electron microscopy data collection and analyses were funded by federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract number HHSN261200800001E, and by Leidos Biomedical Research, Inc. (Y.T. and T.S.)

Immunogenicity and Protective Capacity of a Virosomal Respiratory Syncytial Virus Vaccine Adjuvanted with Monophosphoryl Lipid A in Mice

Respiratory Syncytial Virus (RSV) is a major cause of viral brochiolitis in infants and young children and is also a significant problem in elderly and immuno-compromised adults. To date there is no efficacious and safe RSV vaccine, partially because of the outcome of a clinical trial in the 1960s with a formalin-inactivated RSV vaccine (FI-RSV). This vaccine caused enhanced respiratory disease upon exposure to the live virus, leading to increased morbidity and the death of two children. Subsequent analyses of this incident showed that FI-RSV induces a Th2-skewed immune response together with poorly neutralizing antibodies. As a new approach, we used reconstituted RSV viral envelopes, i.e. virosomes, with incorporated monophosphoryl lipid A (MPLA) adjuvant to enhance immunogenicity and to skew the immune response towards a Th1 phenotype. Incorporation of MPLA stimulated the overall immunogenicity of the virosomes compared to non-adjuvanted virosomes in mice. Intramuscular administration of the vaccine led to the induction of RSV-specific IgG2a levels similar to those induced by inoculation of the animals with live RSV. These antibodies were able to neutralize RSV in vitro. Furthermore, MPLA-adjuvanted RSV virosomes induced high amounts of IFNγ and low amounts of IL5 in both spleens and lungs of immunized and subsequently challenged animals, compared to levels of these cytokines in animals vaccinated with FI-RSV, indicating a Th1-skewed response. Mice vaccinated with RSV-MPLA virosomes were protected from live RSV challenge, clearing the inoculated virus without showing signs of lung pathology. Taken together, these data demonstrate that RSV-MPLA virosomes represent a safe and efficacious vaccine candidate which warrants further evaluation

Lung pathology in mice after immunization and RSV infection.

<p>Mice were immunized and challenged as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036812#pone-0036812-g002" target="_blank">Figure 2</a> and the lungs were harvested, sliced and stained with H&E and assessed for pathology using light microscopy. Panels represent the lungs of (A) FI-RSV, (B) live virus, (C) RSV virosomes, (D) RSV MPLA virosomes (E) buffer immunized mice. Black arrows point to alveolar infiltrates, grey arrows to peribronchial infiltrates and white arrows to perivascular infiltrates. (F) Eosinophils in BAL expressed as percentage of total BAL cells. Data points represent values from individual mice. Statistical differences were calculated using the ANOVA test with Bonferroni correction for multiple testing. ***p<0.001. The data shown are a representative of two individual experiments.</p

Protection against live virus challenge and infiltration of eosinophils.

<p>Mice were vaccinated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036812#pone-0036812-g002" target="_blank">figure 2</a> and challenged with live virus 14 days after the booster vaccination. Four days after challenge, lungs were removed and the viral titer was determined and expressed as TCID₅₀. RSV TCID₅₀ titers from the lungs of challenged animals. Statistical differences were calculated using the Mann-Whitney-U test. *p<0.05. The data shown are a representative of two individual experiments.</p

RSV specific IgG in mice after vaccination with RSV virosomes and RSV-MPLA virosomes.

<p>Mice were vaccinated twice with RSV virosomes, RSV-MPLA virosomes or controls (HNE, live virus and FI-RSV). Each injection contained 5 µg of protein. (A) RSV-specific IgG titers in serum 14 days after prime and 14 days after booster vaccination. (B) RSV-specific IgG1 and IgG2a subtype levels in serum 14 days after booster vaccination. (C) IgE levels were determined at 14 days after booster vaccination. (D) RSV neutralizing antibody titers in serum obtained 5 days after challenge. Bars represent the GMT (panels A and C), mean concentration of RSV-specific IgG1/2a (panel B) or mean neutralization titer (panel D) of 6 mice per group. Error bars represent the SEM. Statistical differences were calculated using the Mann-Whitney-U test. *p<0.05, **p<0.01, ***p<0.001. Statistical differences in IgE levels were calculated with an ANOVA with Bonferroni correction for multiple testing ***p<0.001. The data shown are a representative of two individual experiments.</p