A chimeric vaccine protects farmed saltwater crocodiles from West Nile virus-induced skin lesions

West Nile virus (WNV), including the Australian Kunjin strain (WNVKUN), is a mosquito-borne zoonotic virus causing outbreaks globally in various species. While many infected mammals and birds show subclinical disease, some develop severe meningoencephalitis. Crocodilians exhibit varying responses, from subclinical infection in saltwater crocodiles to severe disease in American alligators, including skin lesions ('pix'). In saltwater crocodiles, these 'pix' lesions cause significant economic losses due to hide rejection in the farming industry. Currently, there are WNV vaccines for horses in the USA, but none are licensed for use in Australia or other regions where crocodile farming is prevalent. One inactivated vaccine used off-label in alligators raised safety concerns. Therefore, a safe and effective vaccine for farmed saltwater crocodiles is urgently needed. This research explored the efficacy of a chimeric vaccine platform based on an insect-specific flavivirus (ISF), Binjari virus (BinJV), as a potential solution. This platform exchanges the structural proteins of BinJV with those of pathogenic flaviviruses, creating chimeric virions antigenically similar to the parent virus but replication-defective in vertebrates.

The literature review extensively covers the impact of WNV on various species, highlighting the varying clinical manifestations and economic consequences. Studies on WNV transmission, including mosquito-borne and fecal-oral routes in crocodilians, are reviewed. Existing WNV vaccines approved for veterinary use, primarily in horses, and their off-label use in alligators and associated safety concerns are discussed. Prior research on the BinJV-based chimeric vaccine platform and its effectiveness in preventing flavivirus infections in mouse models are also summarized.

The study utilized saltwater crocodile hatchlings free of maternal antibodies. Two chimeric vaccine candidates, BinJV/WNVKUNproto (using the 1960 prototype isolate) and BinJV/WNV_KUN2011 (using a 2011 isolate), were tested. Groups of hatchlings were immunized subcutaneously (SC) or intramuscularly (IM) with varying doses (2 µg and 10 µg). Immunogenicity was assessed by measuring neutralizing antibody titers at various time points (4 weeks, 8 weeks, 12 weeks, and 7 months post-prime vaccination). Safety was evaluated by checking for viral replication using RT-PCR and qRT-PCR on plasma, cloacal swabs, and pen water samples. A challenge study employed BinJV/WNV_KUN2011 with and without an adjuvant (Advax™) in both live and UV-inactivated forms. Crocodiles were challenged with WNVKUN 4 weeks post-booster vaccination. Viremia, skin lesions, and viral shedding were monitored through qRT-PCR and visual examination. Mosquito surveillance was conducted using CO2-baited traps and FTA cards to rule out natural WNV transmission during the trials. RNA extraction was performed using various methods depending on sample type (plasma, swabs, water, tissues). Quantitative reverse-transcriptase PCR (qRT-PCR) was used for viral RNA quantification, and statistical analysis was performed using two-way ANOVA with Tukey's post-test.

The immunogenicity study showed that most crocodiles (79-100%) seroconverted by 8-12 weeks post-prime. Neutralizing antibody titers increased significantly by 7 months. No significant difference was found between SC and IM routes or different doses within the tested range. Safety studies revealed no evidence of BinJV/WNV replication in vaccinated crocodiles. The challenge study showed complete protection against viremia and skin lesions in vaccinated crocodiles. Mock-vaccinated animals exhibited viremia, with 22.2% developing WNV-induced lesions. The vaccine prevented viral shedding into the water. Mosquito surveillance showed no natural WNV transmission during the trials. There was no statistically significant difference in efficacy between using live or inactivated vaccine or with or without Advax adjuvant. The study showed that the use of the BinJV/WNVKUN vaccine resulted in sterilizing immunity.

The results demonstrate that the BinJV/WNV_KUN vaccine effectively protects saltwater crocodiles against WNV infection and the development of skin lesions. The delayed increase in antibody titers compared to mammals may reflect the slower immune response in reptiles. The vaccine's efficacy was consistent regardless of the dose or administration route, offering flexibility for farmers. While live vaccines showed slightly higher antibody titers, the difference was not statistically significant from the inactivated vaccine, suggesting flexibility in manufacturing and storage. The lack of detectable residual vaccine in vaccinated crocodiles contrasts with previous safety concerns associated with off-label use of other vaccines, emphasizing the safety profile of the BinJV/WNV_KUN vaccine. The observation of sterilizing immunity is particularly noteworthy.

This study provides the first evidence of a vaccine protecting reptiles against viral infection and represents a significant advancement in veterinary medicine. The BinJV/WNV_KUN vaccine is highly effective, safe, and does not pose environmental risks. The vaccine platform shows significant promise for use in other farmed crocodilians and is currently undergoing regulatory assessment. Further research should focus on evaluating the duration of protection and investigating the cellular immune response.

The relatively small sample size in some groups may limit the statistical power of certain comparisons. Ethical considerations restricted frequent blood sampling, potentially leading to underestimation of viraemia in some animals. The challenge study may not fully represent natural infection conditions, and future work could investigate long-term immunity and the vaccine's effectiveness in different crocodile populations and environments.