AbstractThe long-term survival of the koala is under serious threat from multiple factors, including infectious disease agents such as Chlamydia and koala retrovirus (KoRV). KoRV is present in both exogenous and endogenous forms, depending on the geographical location of the population. In the northern half of Australia, it is present as an endogenous infection in all koalas, making a case for an urgent need to develop a therapeutic vaccine that might prevent KoRV-associated pathologies in these koalas. To this end, we determined the therapeutic effects of vaccinating koalas harbouring endogenous KoRV with a recombinant KoRV Env protein combined with a Tri-adjuvant. We found that vaccination led to a significant increase in circulating anti-KoRV IgG levels, as well as increase in neutralising antibodies. Our study also showed that post-vaccination antibodies were able to recognize epitopes on the Env protein that were unrecognised pre-vaccination, as well as resulting in an increase in the recognition of the previously recognised epitopes. The vaccine also induced antibodies that were cross-reactive against multiple KoRV-subtypes. Finally, we found a complete clearance of KoRV-A in plasma from koalas that had detectable levels of KoRV-A pre-vaccination. Similarly, there was a significant reduction in the expression of KoRV-B viral RNA levels post-vaccination. Collectively, this study showed that koalas harbouring endogenous KoRV can benefit from prophylactic vaccination against KoRV using a recombinant KoRV-A Env protein and that the mechanism of this protection might be through the boosting of natural anti-KoRV antibodies and expanding the breadth of the recognised epitopes.