Purpose: The respiratory syncytial virus (RSV) is one of the major causes of bronchiolitis and pneumonia in infants and immunocompromised adults. It is estimated that approximately 3.4 million children are annually hospitalized due to RSV-related illnesses and 160,000 people die from RSV infection worldwide. Attempts were made to develop a vaccine for RSV using the inactivated form of the virus. However, these attempts failed as the children who were vaccinated developed vaccine-enhanced respiratory disease. As a result, there is a need for a safe and effective vaccine for RSV. One of the major proteins present on the surface of the virus is the fusion protein F, which can be integrated into a virus-like particle (VLP), yielding a highly immunogenic F-VLP antigen. To enhance the immunogenicity of particulate vaccines certain vital compounds called adjuvants are used in combination with vaccine antigens to enhance their ability to produce a stronger immune response. This study also aims at using a transdermal route of administration to exploit the rich population of Langerhans cells present in the epidermis and dermis.
Methods: In this study, the F-VLP antigen was incorporated into a biodegradable polymer matrix and spray dried to form microparticles. The in vitro immunogenicity was evaluated using Dentritic cells in a mechanistic study to evaluate surface co-stimulatory expression of the antigen presenting cells (APCs). The APCs were stimulated with the a numberb of vaccine-adjuvant combinations. Adjuvants such as Alum, Monophosphoryl lipid A, MF59™, R848, flagellin and poly (I:C) were screened to study their efficiency. In order to study the in vivo immunogenicity, vaccine-adjuvant combination was administered to C57BL/6 mice via the transdermal route using microneedles (AdminPatch®). The mice were challenged with RSV A2 (1 x 106 PFU per mouse) via the intra nasal route under isoflurane anesthesia at 12 weeks after boost immunization and body weight changes were monitored 5 days after challenge . The serum IgG was determined in the serum samples collected 1, 3, 5, 7, and 10 weeks post prime and boost administration by enzyme-linked immunosorbent assay (ELISA). The weight of the mice were monitored for 6 days. The expression of CD4 and CD8 markers in the immune organs such as the spleen, lymph nodes were analysed by using the BD Accuri C6 Software using the flow cytometer. Lung histopathology was performed using hematoxylin and eosin (H&E) staining to assess lung inflammation and necrosis post-challenge.
Results: The VLP based microparticulate vaccines with or without adjuvants significantly increase expression of nitric oxide, which is a hallmark of innate immunity. It also resulted in enhanced cell-surface expression of the major histocompatibility complex MHC I and MHCII as well as their costimulatory molecules CD80/86 and CD40 respectively on dendritic cells. In vivo studies using the non-invasive transdermal route demonstrated elevated humoral and cell-mediated immune responses in a mouse model. The immune sera of mice treated with a prime dose of microparticulate F-VLP with MPL (transdermal) showed significantly higher levels of antibody titers compared to FI-RSV immunized (intramuscular), F-VLP solution and microparticulate F-VLP treated mice. Drastic weight loss was observed in naive (PBS control) group and FI-RSV intramuscularly immunized mice (ca. 19%) compared to F-VLP immune mice dosed with adjuvant MPL by the transdermal route (<3%). Lung samples showed influx of inflammatory cells around pulmonary airways and alveoli. Figure 1 shows gross inflammation in lungs of mice intramuscular FI-RSV as a vaccine as compared to transdermal F-VLP MP + MPL. The other groups showed show overt inflammation, which indicates that the F-VLP antigen proved to be a safe vaccine. The transdermally administered microparticles with adjuvant (TD MP+A) group elicited a higher CD4 T cell count from lymph node and spleen cell populations when compared with the control and other test groups, as seen in Figure 2.
Conclusion: The ability of the RSV F-VLP to induce an innate as well as adaptive immune response provides a great foundation for the potential of microparticulate vaccines due to their robust nature and efficient immunogenic properties. Adjuvants such as MF59 and MPL A help in potentiating the immune response. Elevated CD80/CD86 expression may initiate a cytotoxic T-cell mediated response required for elimination of virus-infected cells thus improving the vaccine efficacy. MHC II expression is associated with activation of the adaptive immune response and will elicit a CD4+ T-helper cell mediated response. Expression of co-stimulatory molecule CD40 is essential in enhancing the activation of CD4+ T-cells which are essential in activation of the cellular and humoral immune systems during infection. Also, transdermal route of administration augments the efficacy of the microparticulate vaccine. In the absence of a licensed RSV vaccine, transdermal microparticulate vaccines have immense potential and offer an alternative approach in the development of a vaccine against RSV.
Ipshita Menon– Ms, Mercer University, Atlanta, Georgia
Sucheta D'Sa– Charles River Laboratories, Inc., Ohio
Sang-Moo Kang– Georgia State University, Georgia
Cherilyn D'Souza– Mercer University, Atlanta, Georgia
Martin D'Souza– Professor, Mercer University, Atlanta, Georgia
Ipshita Menon– Ms, Mercer University, Atlanta, Georgia