Immunological Evaluation of Biodegradable Particle Based Nanoparticles Encapsulating OMP Antigen as Potential Vaccine Candidate

Abstract

Advanced vaccine research approaches need to explore on biodegradable nanoparticles (NPs) based vaccine carrier that can serve as antigen delivery systems as well as immuno-stimulatory action to induce both innate and adaptive immune response. The biodegradable polymeric particles like polylactide-co-glycolide (PLGA) or polylactide (PLA), not only work as a delivery system but also, provide adjuvant activity and marks in the development of long -lasting immunity. In this PhD dissertation work, the immunogenicity of PLA and PLGA NPs encapsulating outer membrane protein (Omp) antigen (Aeromonas hydrophila and Vibrio cholerae) were evaluated. Initially, Various NPs formulations of PLA and PLGA loaded with model protein (Bovine Serum Albumin) or drug (Clindamycin hydrochloride) were prepared by solvent evaporation method varying drug/protein: polymer concentration and optimized for size, encapsulation efficiency, drug loading, morphology etc. A. hydrophila Omp antigen loaded PLA-Omp (223.5± 13.19 nm) and PLGA-Omp (166.4± 21.23 nm) NPs were prepared using double emulsion method by efficiently encapsulating the antigen reaching the encapsulation efficiency 44 ± 4.58 % and 59.33± 5.13 % respectively. Despite low antigen loading in PLA-Omp, it exhibited considerably slower antigen release in vitro than PLGA-Omp NPs. Upon intraperitoneal immunization in fish, Labeo rohita with all antigenic formulations (PLA-Omp NP, PLGA-Omp NP, FIA-Omp, PLA NP, PLGA NP, PBS as control), significantly higher bacterial agglutination titre and haemolytic activity were observed in the case of PLA-Omp and PLGA-Omp immunized groups compared to rest of the groups at both 21 days and 42 days.The antigen specific antibody response was significantly increased and persisted up to 42 days of post immunization by PLA-Omp, PLGA-Omp, FIA-Omp.PLA-Omp NPs showed a better immune response (higher bacterial agglutination titre, haemolytic activity, specific antibody titre, higher percent survival upon A. hydrophila challenge) than PLGA-Omp in L. rohita confirming its better efficacy. Comparable antibody response of PLA-Omp and PLGA-Omp with FIA-Omp treated groups suggested that PLA and PLGA could be a replacement for Freund’s adjuvant (for stimulating antibody response) to overcome many side effects offering long lasting immunity. Similarly, V. cholerae Omp antigen loaded PLA-Omp (196.24± 34.25 nm) and PLGA-Omp (165.34± 3.5 nm) NPs were prepared using double emulsion method by efficiently encapsulating the antigen reaching the encapsulation efficiency 57.85 ± 4.15 % and 69.18± 1.68 respectively. After intraperitoneal immunization in BALB/c mice, the type and strength of immune responses elicited (cellular and humoral) by formulated NPs were evaluated. The antibody titre (IgG1, IgG2a) were significantly higher (P<0.05; Kruskal-Wallis test and post hoc Dunn multiple comparisons) in PLA-Omp NPs, PLGA-Omp NPs treated groups than respective PLA and PLGA NPs treated groups from 7 days to 56 days as confirmed by ELISA tests. Also, PLA-Omp NPs and PLGA-Omp NPs induced significantly higher (P<0.05; Kruskal-Wallis test and post hoc Dunn multiple comparisons) antigen specific IgG titers than Omp antigen treated groups at all-time intervals except 0 day. From the spleen cell analysis (Cell surface phenotype through FACs study), a viii significantly higher cellular activation was observed in the case of PLA-Omp and PLGA-Omp NPs in comparison to Omp antigen alone. Enhanced immune responses elicited by PLA-Omp and PLGA-Omp NPs might be attributed to strong memory T cell response (higher effector memory T cell), efficient induction of dendritic cell (DC) activation (higher MHC I, MHC II, CD 86 expression) and follicular helper T cell differentiation in spleen favoring the generation of antibody responses. Another study aimed at developing a DNA vaccine as well as a delivery system to boost antigenic outer membrane protein (Omp) that would act as potential vaccine candidate was conducted. NPs based delivery systems for plasmid DNA (pDNA) (which encode conserved Omp) administration might be keys to improve the transfection efficiency in vivo even at a lower dose. The conserved antigenic protein sequences [omp(211-382), omp(211-382)opt, omp(703-999) and omp(703-999)opt] of outer membrane protein were identified using bioinformatics tools. The sequences were cloned into a pVAX-GFP expression vector and successfully transformed into E. coli (DH5α). The large scale pDNA production was achieved with shake flask cultures and the pDNA was purified by hydrophobic interaction chromatography (HIC). The formulated PLGA-chitosan NP/plasmid DNA nano complex of ~200 nm (199.25 ± 22.29 nm and 205.25 ±33.59) was transfected in the into CHO cells that confirmed improved transfection efficiency (fluorescence intensity measurement corresponding to GFP expression level from the FACs study) at a lower dose. The DNA entrapment assay demonstrated the possible protection of pDNA inside the pDNA-NP complex. The protection from enzymatic digestion that NP complex confers to pDNA was evaluated and confirmed by gel electrophoresis after treatment with DNase. Further, physio-chemical characterizations of formulated nano complex and extensive transfection studies have proved the functionality of the system in vitro. Overall, the successful formulations of NP based antigen delivery system with all desirable physiochemical characteristics were obtained under highly controlled conditions and reproducibility. The immunological evaluation studies suggest that PLA/PLGA NPs based delivery system could be a novel antigen carrier for fish and mice ensuring its application for commercial value and product development. Further work based on present optimized results can be taken forward for next level vaccine design and approval.