NOVEL MICROPARTICLE-BASED MICRONEEDLE VACCINE FOR ZIKA VIRUS

Abstract

NOVEL MICROPARTICLE-BASED MICRONEEDLE VACCINE FOR ZIKA VIRUS (Under the guidance of Dr. Martin D’Souza) Zika is an infectious viral disease caused due to the Zika virus. The primary mode of transmission for this virus is through mosquito bites. The common symptoms of the disease include fever, headache, conjunctivitis, muscle pain, and joint pain. Vertical transmission during pregnancy can result in microcephaly, congenital Zika syndrome, and other congenital abnormalities. Zika can also lead to Guillain-Barr Syndrome – an autoimmune disorder affecting the peripheral nervous system. After the global outbreak of Zika in 2015-2016, the World Health Organization declared a public health emergency of international concern. Research related to Zika has been included in the R and D priority list by the WHO. However, there are no approved treatments or vaccines available for Zika. Current vaccine candidates in research include whole-inactivated vaccines, nucleic acid vaccines, vectored vaccines, and subunit vaccines that are administered via conventional intramuscular or subcutaneous routes. These routes are invasive and painful, thereby reducing patient compliance. To explore a less painful alternative, we investigated the feasibility of the transdermal route as a vaccine delivery strategy for the Zika virus using polymeric microparticle-loaded microneedle patches. Integration of the particulate vaccine strategy and the transdermal route of administration together presented the potential to be efficacious in preventing Zika in a patient-compliant manner. The first part of the project included the formulation of poly(lactic-co-glycolic) acid (PLGA) polymeric vaccine microparticles (MP) encapsulating the inactivated Zika virus, along with adjuvant MP encapsulating Alhydrogel® and MPL-A®. We characterized the vaccine MP for size, surface charge, morphology, encapsulation efficiency, and antigen integrity. Further, we evaluated the immunogenicity and cytotoxicity of vaccine MP in vitro in murine dendritic cells. Vaccine MP with adjuvants induced significantly higher production of nitric oxide, a marker of innate immunity, when compared to the untreated cells. In addition, vaccine MP with or without adjuvants induced increased autophagy in murine dendritic cells when compared to inactivated Zika virus, which is critical in antigen presentation. Vaccine MP along with adjuvant MP also enhanced the expression of antigen-presenting molecules – MHC I and MHC II as well as co-stimulatory molecules – CD80 and CD40 in murine dendritic cells. Next, we evaluated in vivo efficacy of vaccine MP with and without adjuvants in a preclinical murine model by measuring the immune response after intramuscular administration. Vaccine MP with adjuvants induced significant IgG, Ig2a, IgG1, and IgG3 titers as compared to the control group of untreated mice. After the challenge with live Zika virus, vaccinated mice showed higher antibody titers and enhanced expression of helper CD4 and CD8 cell surface markers in splenocytes and lymphocytes. Splenocytes of vaccinated mice also showed enhanced expression of IFN-γ required for cytotoxic cell-mediated immune response. Thus, the immunogenic efficiency of the particulate Zika vaccine was established. In the next part of the study, we embedded Zika vaccine MPs with adjuvant MPs in dissolving microneedles (MNs) administered via the transdermal route as a pain-free vaccine strategy. We characterized the MNs for needle length, pore formation, and dissolvability when applied to murine skin. Further, we evaluated the in vivo efficacy of vaccine MPs-loaded MNs with or without adjuvants by measuring immune response after transdermal immunization. Vaccine MPs-loaded dissolving MNs with adjuvants induced significant IgG, IgG1, IgG2a, and IgG3 titers in immunized mice compared to the untreated control group. After the dosing regimen, animals were challenged with the Zika virus, monitored for seven days, and sacrificed to collect spleen and lymph nodes. Lymphocytes and splenocytes from immunized mice showed significant expression of helper (CD4) and cytotoxic (CD8a) cell surface markers than the control group. Thus, the promising results of this study put forth a ‘proof-of-concept’ for a pain-free transdermal vaccine strategy against Zika.