Characterization of Attenuated HSV-2 Mutants as Potential Vaccine Candidates Against Genital Herpes

Abstract

A prophylactic and therapeutic vaccine against herpes simplex virus type 2 (HSV-2) infection is necessary to reduce the global disease burden of HSV-2 diseases. Subunit, single-cycle, and DNA vaccines have been studied in pre-clinical and clinical trials but have not been approved mostly due to lack of sufficient efficacy. Using targeted mutagenesis, a live, attenuated HSV-2 could be a likely candidate for a protective vaccine. In this study, we constructed and characterized two novel HSV-2 mutant strains, TKBAC /∆UL24 and TKBAC /∆UL39, that have loss-of-function in genes that are associated with viral pathogenesis. Bacterial artificial chromosome (BAC) technology and recombineering were used for construction of these strains and their revertants, TKBAC /UL24R and TKBAC /UL39R. ∆UL24 and ∆UL39 have internal deletions and TKBAC (the parent virus) has an insertion within the viral thymidine kinase gene consisting of the 7.5 kb BAC sequence. Sequencing of ∆UL24 and ∆UL39 confirmed the expected deletions with the HSV-2 UL24 and UL39 genes while the flanking regions remained intact. BAC DNAs were digested with select restriction endonucleases and fractionated by agarose gel electrophoresis. All constructs showed a similar digestion pattern compared to the parent strain, TKBAC, suggesting the mutant genomes were stable. Mutagenized BACS were used to create infectious virus stocks after transfection into Vero cells. We demonstrated that TKBAC /∆UL24 and TKBAC /∆UL39 were replication-competent in Vero cells. An HSV-2 UL24 mutant was shown to be a potential attenuated vaccine candidate in previous studies. The double mutant containing disruption of the TK gene would provide for an even safer attenuated vaccine candidate that would be less like to reactivate from latency and cause disease. Replication characteristics including a reduction in plaque size for TKBAC /∆UL39 suggested that this virus was crippled in vitro, while TKBAC /∆UL24 demonstrated similar replication characteristics to the parent strain. In vitro plaque reduction assays and viral yield assays against acyclovir suggest that TKBAC /∆UL39 is more sensitive to acyclovir compared to the parent strain, based on a lower IC50. Based on previous studies with individual TK or UL39 mutants, the double mutant should be even more deficient in the establishment of and reactivation from latency. Based on these results, TKBAC /∆UL24 and TKBAC /∆UL39 should be considered for further preclinical evaluation (in animal models) as viable candidates for a protective, safe prophylactic and therapeutic vaccine.