Therapeutic Prime/Pull Vaccination of HSV-2 Infected Guinea Pigs with the Ribonucleotide Reductase 2 (RR2) and CXCL11 boosts TRM and TEM CD4+/CD8+ T cells to protect from recurrent genital herpes

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes an asymptomatic latent infection of sensory neurons of dorsal root ganglia (DRG). Chemical and physical stress cause intermittent virus reactivation from latently infected DRG and recurrent virus shedding in the genital mucosal epithelium causing genital herpes in symptomatic patients. While T-cells appear to play a role in controlling virus reactivation from DRG and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T-cells into DRG and the vaginal mucosa (VM) remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4+ and CD8+ T cells and its effect on the frequency and severity of recurrent genital herpes in the recurrent herpes guinea pig model. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-2 RR2 protein (Prime) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 (AAV-8) expressing CXCL9, CXCL10, or CXCL11 chemokines to recruit CD4+ and CD8+ T cells into the infected DRG and VM (Pull). Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident (TRM cells) and effector (TEM cells) memory CD4+ and CD8+ T cells in both DRG and VM tissues. This was associated with less virus in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4+ and CD8+ T cells in reducing virus shedding at the vaginal site of infection and the severity of recurrent genital herpes and propose the novel prime-pull vaccine strategy to protect against recurrent genital herpes. Overall design: In the present study, we sought to improve protein-based vaccination by combining it with a prime and pull strategy. The strategy involves conventional parenteral vaccination using HSV-2 specific RR2 protein to elicit systemic T-cell responses (prime), followed by recruitment of activated T cells via administration of an adenovirus expressing chemokine or T cell attractant (pull), for those T cells to establish long-term protective immunity. The adeno-associated virus type 8 (AAV8) was used to express the chemokines as it efficiently targets the enteric nervous system in guinea pigs. The immunization results showed that the primary CD8+T cell responses were of similar magnitudes in the spleen, whereas the frequency and number of CD8+T cells in the vaginal mucocutaneous tissue were significantly higher in vaccinated mice treated with adenovirus-expressing chemokine as compared with the control immunized mice without the "pull". Furthermore, the action of the chemokine pull was not restricted to the genital mucosa, as CD8+ T cell recruitment was also observed in the DRG. Importantly, the prime and pull strategy conferred near complete protection against the primary challenge of genital HSV-2 infection compared with the prime alone. In this study, we extend this application to therapeutic vaccines and demonstrate that the frequency of recurrent disease and recurrent vaginal shedding is reduced most effectively by the combination of prime (protein vaccine) and pull (Adenovirus expressing chemokine). We have studied the (i) genital infection of the immunized and non-immunized guinea pigs with HSV-2 by viral titration, (ii) monitored the genital herpes infection and disease scoring in guinea pigs, (iii) Bulk RNA sequencing on sorted CD8+ T cells, (iv) Differential gene expression, and (v) function of different immune cell markers by FACS to see the immune cell infiltrations into the VM tissue sections. We performed gene expression profiling analysis using RNA-Seq on 4 guinea pig samples. All samples were female herpes subjects categorised into Immunized (n = 2), and Non-Immunized (n = 2).