Therapeutic mucosal vaccination of herpes simplex virus type 2 infected guinea pigs with an adenovirus-based vaccine expressing the ribonucleotide reductase 2 and glycoprotein D induces local tissue-resident CD4+ and CD8+ TRM cells associated with protection against recurrent genital herpes

Abstract

Introduction: The reactivation of herpes simplex virus 2 (HSV-2) from latency causes viral shedding that develops into recurrent genital lesions. The role of tissue-resident T cells and the nature of viral antigens associated with protection against recurrent genital herpes remain to be fully elucidated.

Methods: In this preclinical study, we investigated the protective therapeutic efficacy, in the guinea pig model of recurrent genital herpes, of five recombinant adenovirus-based therapeutic vaccine candidates (rAd-Ags), each expressing different HSV-2 envelope and tegument proteins: RR1 (UL39), RR2 (UL40), gD (glycoprotein D), VP16 (UL48), or VP22 (UL49). We compared the frequency and function of dorsal root ganglia (DRG)- and vaginal mucosa (VM)-resident CD4+ and CD8+ T cells induced by each vaccine and their effect on the frequency and severity of recurrent genital herpes.

Results: HSV-2 latent-infected guinea pigs immunized with rAd-RR2 and rAd-gD vaccines showed high frequencies of DRG- and VM-tissue-resident IFN-g-producing CD4+ and CD8+ TRM cells associated with significant reductions in viral shedding and genital herpetic lesions.

Discussion: Taken together, these preclinical results provide new insights into the T cell mechanisms of protection against recurrent genital herpes and confirm the tegument RR2 protein and glycoprotein D as viable candidate antigens to be incorporated in future genital herpes therapeutic vaccines.

Keywords: CD4 + T cells; CD8 + T cells; genital herpes; therapeutic; vaccine; vaginal mucosa.

Figure 1

Protection against genital herpes infection in HSV-2 infected guinea pigs following therapeutic immunization with five adenovirus-based vaccine candidates (A) Timeline of HSV-2 infection, virological and immunological analysis. Guinea pigs were infected intravaginally on day 0 with 5 X 10⁵ PFU of HSV-2 (MS strain). Once the acute infection was resolved latently infected animals were divided into 7 groups and then immunized intravaginally on day 15 with 10¹⁰ genomic copies of AD-5 expressing HSV-2 antigens (RR2, RR1, gD, VP22 and Vp16). The replication defective dl529 mutant vaccine was used as a positive control. From day 25 to 56 post-infection, the guinea pigs were monitored daily for the severity of genital herpes lesions, scored on a scale of 0-4 and vaginal swabs were collected to detect virus shedding and to quantify HSV-2 DNA copy numbers. (B) Cumulative scoring of vaginal lesions observed during recurrent infection (C) Cumulative positive days with recurrent genital lesions. (D) HSV-2 DNA copy numbers detected in the VM of vaccinated and mock-vaccinated guinea pig groups. (E) HSV-2 DNA copy numbers detected in the DRG of vaccinated and mock-vaccinated guinea pig groups. (F) Representative images of genital lesions in guinea pigs vaccinated with Ad-RR2, Ad-RR1, Ad-VP22, Ad-gD, Ad-VP16,dl-529 and Mock. * = P<0.05, ** = P≤0.01, *** = P≤0.001, **** = P≤0.0001.

Figure 2

Formation of antibodies in HSV-2 infected guinea pigs following therapeutic immunization with five adenovirus-based vaccine candidates (A) Levels of antigen specific IgG detected in the guinea pigs vaccinated with Ad5 expressing RR2, RR1, gD, VP22 and VP16 antigens by ELISA. The sera were evaluated at 1:1000 dilution. (B) Neutralization assay among the vaccinated and mock-vaccinated groups showing vaccine-induced serum-neutralization. Comparison of the HSV-2 MS strain specific neutralizing antibodies induced in the guinea pigs vaccinated with Ad5 expressing RR2, RR1, gD, VP22 and VP16 antigens. (C); left panel) Representative ELISpot images showing average frequencies of IFN-γ producing cell spots from mononuclear cells from VM tissue (1 x 10⁵ cells per well) of HSV-2 infected guinea pigs treated with different HSV-2 proteins namely RR2, RR1, VP22, gD, VP16. dl529 were used as a positive control. (C); right panel) The bar diagrams show the average/mean numbers (+ SD) of IFN-γ-spot forming cells (SFCs) after stimulation with HSV-2 proteins in VM tissues of different groups of guinea pigs. A strong response is defined for mean SFCs > 25 per 1 x 10⁵ stimulated mononuclear cells. * = P<0.05, ** = P≤0.01, *** = P≤0.001, **** = P≤0.0001, ns, non significant.

Figure 3

Increased frequencies of CD4⁺CD44⁺ T cells and CD8⁺CD44⁺ T cells in the DRG and VM of HSV-2 infected guinea pigs following therapeutic immunization with five adenovirus- based vaccine candidates: (A) Representative FACS data (left panel) and average frequencies (right panel) of CD4⁺CD44⁺ T cells detected in the DRG, VM of vaccinated and mock-vaccinated animals. (B) Representative FACS data (left panel) and average frequencies (right panel) of CD8⁺CD44⁺ T cells detected in the DRG, and VM of vaccinated and mock-vaccinated animals. * = P<0.05, ** = P≤0.01, *** = P≤0.001, **** = P≤0.0001, ns, non significant.

Figure 4

Increased frequencies of CD8⁺CD103⁺ T cells and CD8⁺CRTAM T cells in the DRG and VM of HSV-2 infected guinea pigs following therapeutic immunization with five adenovirus-based vaccine candidates: (A) Representative FACS data (left panel) and average frequencies (right panel) of CD8⁺CD103⁺ T cells detected in the DRG, and VM of vaccinated and mock-vaccinated animals. (B) Representative FACS data (left panel) and average frequencies (right panel) of CD8⁺CRTAM T cells detected in the DRG, and VM of vaccinated and mock-vaccinated animals. * = P<0.05, ** = P≤0.01, *** = P≤0.001, ns, non significant.

Figure 5

Increased frequencies of CD4⁺Ki-67 T cells and CD8⁺Ki-67 T cells in the DRG and VM of HSV-2 infected guinea pigs following therapeutic immunization with five adenovirus-based vaccine candidates: (A) Representative FACS data (left panel) and average frequencies (right panel) of CD4⁺Ki-67 T cells detected in the DRG, and VM of vaccinated and mock-vaccinated animals. (B) Representative FACS data (left panel) and average frequencies (right panel) of CD8⁺Ki-67 T cells detected in the DRG, and VM of vaccinated and mock-vaccinated animals. * = P<0.05, ** = P≤0.01, *** = P≤0.001, ns, non significant.

Figure 6

Increased frequencies of CD4⁺IFN-γ T cells and CD8⁺ IFN-γ T cells in the DRG and VM of HSV-2 infected guinea pigs following therapeutic immunization with five adenovirus-based vaccine candidates: (A) Representative FACS data (left panel) and average frequencies (right panel) of CD4⁺ IFN-γ T cells detected in the DRG, and VM of vaccinated and mock-vaccinated animals. (B) Representative FACS data (left panel) and average frequencies (right panel) of CD8⁺ IFN-γ T cells detected in the DRG, and VM of vaccinated and mock-vaccinated animals.