Aeromonas hydrophila OmpW PLGA Nanoparticle Oral Vaccine Shows a Dose-Dependent Protective Immunity in Rohu (Labeo rohita)

Abstract

Aeromonas hydrophila is a Gram-negative bacterium that causes high mortality in different fish species and at different growth stages. Although vaccination has significantly contributed to the decline of disease outbreaks in aquaculture, the use of oral vaccines has lagged behind the injectable vaccines due to lack of proven efficacy, that being from primary immunization or by use of boost protocols. In this study, the outer membrane protein W (OmpW) of A. hydrophila was cloned, purified, and encapsulated in poly d,l-lactide-co-glycolic acid (PLGA) nanoparticles (NPs) for oral vaccination of rohu (Labeo rohita Hamilton). The physical properties of PLGA NPs encapsulating the recombinant OmpW (rOmpW) was characterized as having a diameter of 370–375 nm, encapsulation efficiency of 53% and −19.3 mV zeta potential. In vitro release of rOmpW was estimated at 34% within 48 h of incubation in phosphate-buffered saline. To evaluate the efficacy of the NP-rOmpW oral vaccine, two antigen doses were orally administered in rohu with a high antigen (HiAg) dose that had twice the amount of antigens compared to the low antigen (LoAg) dose. Antibody levels obtained after vaccination showed an antigen dose dependency in which fish from the HiAg group had higher antibody levels than those from the LoAg group. The antibody levels corresponded with post challenge survival proportions (PCSPs) and relative percent survival (RPS) in which the HiAg group had a higher PCSP and RPS than the LoAg group. Likewise, the ability to inhibit A. hydrophila growth on trypticase soy agar (TSA) by sera obtained from the HiAg group was higher than that from the LoAg group. Overall, data presented here shows that OmpW orally administered using PLGA NPs is protective against A. hydrophila infection with the level of protective immunity induced by oral vaccination being antigen dose-dependent. Future studies should seek to optimize the antigen dose and duration of oral immunization in rohu in order to induce the highest protection in vaccinated fish.

Secondary immune response of rainbow trout following repeated immersion vaccination

“…Antigens are presented to central lymphoid organs following uptake whereupon protective immune responses are initiated (Chilmonczyk, ; Tatner, ; Tatner & Manning, ; Zapata, Diez, Cejalvo, Gutierrez‐de Frias, & Cortes, ) and it may be hypothesized that this process may be less effective in immersion vaccinated fish. Thus, the antigen concentration in a vaccine is positively correlated with protection and antibody production of fish (Dubey et al., ; Marana et al., ), which will support the notion that immunization efficacy by immersion is lower than injection. This is supported by investigations showing that administration of vaccine by injection significantly improve survival rate and/or raised antibody levels compared to the immersion vaccinated and unvaccinated control groups (Chettri et al., , ).…”

An Overview of Vaccination Strategies and Antigen Delivery Systems for Streptococcus agalactiae Vaccines in Nile Tilapia (Oreochromis niloticus)

“…Maiti et al [46] used the SIT to evaluate the ability of antibodies induced by an outer membrane protein A (OmpA) vaccine in carp ( Cyprinus carpio ) to inhibit Edwardsiella tarda growth in vitro while Rauta and Nayak [47] used the SIT to determine the ability of antibodies from rohu ( Labeo rohita ) vaccinated using a recombinant OmpA vaccine to inhibit Aeromonas hydrophila growth in vitro. Dubey et al [48], used the SIT to show the ability of antibodies produced by a recombinant OmpA antigen encapsulated in chitosan nanoparticles in rohu to inhibit A. hydrophila growth in vitro in a dose-dependent manner in which fish orally vaccinated with a high antigen dose (HiAg) (8 μg/g) showed a higher inhibition capacity of A. hydrophila than fish vaccinated with a low antigen (LoAg) (4 μg/g) dosage vaccine, which corresponded with post challenge survival proportions in which the HiAg dose group showed a higher RPS (79.99%) than the LoAg dose (RPS = 37.33%), demonstrating that the SIT can be used as an in vitro measure of vaccine efficacy, able to determine the protective ability of antibodies generated by vaccination. In tilapia, Zhang et al [34] used the SIT to evaluate the ability of antibodies induced by the Sip protein administered using the Poly[(methyl methacrylate)-co-(methyl acrylate)-co-(methacrylic acid)]-poly( d , l -lactide-co-glycolide) (PMMMA-PLGA) to inhibit S. agalactiae replication in vitro.…”

“…In our previous studies, we have shown that antigen dose can be optimized to correspond with vaccine protection in vaccinated fish [39,48]. Similarly, Li et al [8] showed an antigen dose-dependent immune protection in fish vaccinated with vaccine doses of 10 5 , 10 6 , 10 7 , 10 8 and 10 9 CFU/fish that corresponded with increasing RPS = 10.15%, 35.48%, 50.74%, 64.52% and 67.74% for fish challenged at 15 days post vaccination, respectively.…”

Edwardsiella tarda OmpA Encapsulated in Chitosan Nanoparticles Shows Superior Protection over Inactivated Whole Cell Vaccine in Orally Vaccinated Fringed-Lipped Peninsula Carp (Labeo fimbriatus)

“…To evaluate the in vivo release of rOmpA, we used the serum inhibition test to evaluate the ability of antibodies induced by rOmpA released in vaccinated fish to inhibit the growth of E. tarda on TSA. The serum inhibition test is an in vitro vaccine efficacy measure used to determine the ability of antibodies generated by vaccination to inhibit bacterial growth in vitro [11,57]. Given that in some cases the bacterial strain used for vaccine production is also used as the challenge strain for measuring the RPS of vaccinated fish, in such cases the serum inhibition test serves as an in vitro measure of vaccine efficacy, used to determine the protective ability of antibodies generated by the vaccine strain challenged using its homologous strain [11,57].…”

“…The serum inhibition test is an in vitro vaccine efficacy measure used to determine the ability of antibodies generated by vaccination to inhibit bacterial growth in vitro [11,57]. Given that in some cases the bacterial strain used for vaccine production is also used as the challenge strain for measuring the RPS of vaccinated fish, in such cases the serum inhibition test serves as an in vitro measure of vaccine efficacy, used to determine the protective ability of antibodies generated by the vaccine strain challenged using its homologous strain [11,57]. As seen from our findings, the NP-rOmpA group had a higher inhibitory capacity than the IWC-ET vaccinated fish, suggesting that the NP-OmpA produced higher levels of protective antibodies than the IWC-ET vaccine.…”