CCL19 and CCL28 Assist Herpes Simplex Virus 2 Glycoprotein D To Induce Protective Systemic Immunity against Genital Viral Challenge

Abstract

Potent systemic immunity is important for recalled mucosal immune responses, but in the defense against mucosal viral infections, it usually remains low at mucosal sites. Based on our previous findings that enhanced immune responses can be achieved by immunization with an immunogen in combination with a molecular adjuvant, here we designed chemokine-antigen (Ag) fusion constructs (CCL19- or CCL28-herpes simplex virus 2 glycoprotein D [HSV-2 gD]). After intramuscular (i.m.) immunization with different DNA vaccines in a prime and boost strategy, BALB/c mice were challenged with a lethal dose of HSV-2 through the genital tract. Ag-specific immune responses and chemokine receptor-specific lymphocytes were analyzed to determine the effects of CCL19 and CCL28 in strengthening humoral and cellular immunity. Both CCL19 and CCL28 were efficient in inducing long-lasting HSV-2 gD-specific systemic immunity. Compared to CCL19, less CCL28 was required to elicit HSV-2 gD-specific serum IgA responses, Th1- and Th2-like responses of immunoglobulin (Ig) subclasses and cytokines, and CCR3⁺ T cell enrichment (>8.5-fold) in spleens. These findings together demonstrate that CCL28 tends to assist an immunogen to induce more potently protective immunity than CCL19. This work provides information for the application potential of a promising vaccination strategy against mucosal infections caused by HSV-2 and other sexually transmitted viruses.IMPORTANCE An effective HSV-2 vaccine should induce antigen (Ag)-specific immune responses against viral mucosal infection. This study reveals that chemokine CCL19 or CCL28 enhanced HSV-2 glycoprotein D ectodomain (gD-306aa)-induced immune responses against vaginal virus challenge. In addition to eliciting robust humoral immune responses, the chemokine-Ag fusion construct also induced Th1- and Th2-like immune responses characterized by the secretion of multiple Ig subclasses and cytokines that were able to be recalled after HSV-2 challenge, while CCL28 appeared to be more effective than CCL19 in promoting gD-elicited immune responses as well as the migration of T cells to secondary lymph tissues. Of importance, both CCL19 and CCL28 significantly facilitated gD to induce protective mucosal immune responses in the genital tract. The above-described findings together highlight the potential of CCL19 or CCL28 in combination with gD as a vaccination strategy to control HSV-2 infection.

Keywords: CCL19; CCL28; HSV-2; glycoprotein D; mucosal immunity.

FIG 1

Construct design, immunization procedures, and humoral immune responses of sera and vaginal lavage fluid. (A) Schematics of chemokine-linker-gD and gD-linker-chemokine constructs. (B) Schedule of i.m. immunization and i.vag. challenge. Mice (n = 10) were immunized twice with pcDNA3.1, pgD + pcDNA3.1, pgD + pCCL19, or pCCL28 chemokine-Ag plasmids in saline solution at 0 and 2 weeks. Two weeks postboost, mice (n = 5) in each group were sacrificed for tissue collection, while the rest were used for the challenge experiments. Postchallenge, the weight and clinical symptoms of all mice were monitored every day for 15 days. Murine sera, vaginal lavage, and sacral ganglia were collected at the indicated time points for subsequent tests. (C) Humoral immune responses induced by pgD in combination with pCCL19 or pCCL28 and pCCL28-IZ-gD. Mice were immunized i.m. at 0 and 2 weeks by electroporation. At 2 weeks (wk) postboost, sera and vaginal lavage fluid were collected and an endpoint titration of gD-specific IgG and IgA was measured by ELISA. Data are the means ± SEM, n = 10 mice per group, from at least two independent experiments with each condition being performed in duplicate. (D) Serum gD-specific IgG was compared for each group at the indicated weeks postboost. (E) Serum gD-specific IgA at 7 weeks postboost was compared for each group. Data shown are the means ± SEM (n = 5 mice/group) from three independent experiments with each condition performed in duplicate. Statistically significant differences were determined by comparison to the pgD + pcDNA3.1 group. NS, not statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

gD-specific IgG and IgA responses and Ig subclasses in serum postchallenge. Serum gD-specific IgG (A) and IgA (B) at indicated days postboost and postchallenge. The endpoint titrations of gD-specific IgG and IgA were determined by ELISA separately. (C) The distribution of gD-specific IgG1, IgG2a, IgG2b, IgG3, and IgM isotypes in murine sera at 2 weeks postboost (upper) and 9 dpi (lower). (D) gD-specific IgG2a/IgG1 ratio of the different groups at 2 weeks postboost and 9 dpi. The quantification of each Ig subclass was measured by ELISA and calculated according to the standard curve obtained using corresponding Ig subclass standards. CCL19, 20 mol; CCL28, 10 mol. Data are the means ± SEM, n = 5 or 7 mice per group, from at least two independent experiments with each condition being performed in duplicate. Statistically significant differences determined by comparison to the pgD + pcDNA3.1 group are indicated. NS, not statistically significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

Anti-HSV-2-neutralizing activities of sera and vaginal lavage fluid from immunized mice. Murine sera collected at the indicated times were tested at dilutions starting with 1:10 (at 2 weeks and 7 weeks postboost) and 1:40 (at 5, 9, and 11 dpi). Vaginal lavage fluid collected at 2 weeks postboost were tested at dilutions starting with 1:5. Plaque numbers were determined for each sample and plotted as percent inhibition. NS, not statistically significant; ***, P < 0.001.

FIG 4

Ag-specific Th1/Th2-associated cytokine production from splenocytes and antisera. (A) Cytokine production in splenocyte supernatants from immunized mice (2 weeks postboost) and antisera after HSV-2 challenge (5 dpi and 9 dpi) was quantified by flow cytometry. (B) Fold changes in the pgD + pCCL28 or pCCL28-IZ-gD groups over the pgD + pCCL19 group. Splenocytes (1 × 10⁷ cells) isolated from all groups postboost were stimulated with purified gD (2 μg/ml) and cocultivated for 5 days, and then the supernatants were collected. (C) Hot map of cytokine fold changes (i.e., pgD + CCL28/pgD + CCL19 and pCCL28-IZ-gD/pgD + CCL19). Group 1, 1-1, and 1-2 is named pgD + CCL28/pgD + CCL19, and 2, 2-1, and 2-2 is named pCCL28-IZ-gD/pgD + CCL19. Antisera were collected at 5 and 9 dpi. The production of Th1 (IL-2, IFN-γ, and TNF-α)- and Th2 (IL-4 and IL-5)-associated cytokines was detected using a CBA kit. Data shown are representative of the mean cytokine concentrations (pg/ml) ± SEM with n = 5 mice per group. Statistically significant differences determined by comparing to the pgD + pcDNA3.1 group are indicated. NS, not statistically significant; *, P < 0.05; ***, P < 0.001.

FIG 5

Responsive immunocyte migration to secondary lymph nodes postimmunization and postchallenge. IgA⁺ cells at colorectal sites mediated by the CCL19 or CCL28 adjuvant in immunized mice. (A) IgA⁺ cells in murine colorectal mucosal samples were stained with DBA and hematoxylin. Data shown are representative immunohistochemistry results (magnification, ×200). (B) Mean counts of IgA⁺ cells of 10 high-power fields for each group. Migration of murine splenocytes and MLNLs for each group in response to murine CCL19 or CCL28 protein. Single lymphocytes were prepared and counted for the chemotactic response to CCL19 or CCL28 using a Transwell system, and the fold changes were calculated compared to the cell number in the lower chamber without CCL19 (C) or CCL28 (D). The frequencies of CCR7^+/− (E) and CCR3⁺ (F) CD3⁺ splenocytes at 2 weeks postboost and 9 dpi were analyzed by flow cytometry. By comparing to the pgD + pcDNA3.1 group, the fold change of CCR7⁻ (G) and CCR3⁺ (H) CD3⁺ splenocyte frequencies in the pCCL19 or pCCL28 adjuvant groups were calculated, and the gating strategies are shown in Fig. S1E. Data shown are the means ± SEM for each group (n = 5 mice/group). Statistically significant differences determined by comparing to the pgD + pcDNA3.1 group are indicated. NS, not statistically significant; ***, P < 0.001.

FIG 6

Protective efficacies of vaccine formulations during lethal vaginal HSV-2 challenge. Nine weeks postboost, mice were challenged i.vag. with a lethal dose of HSV-2. Vaccinated and control groups were monitored for up to 15 days for weight changes, illness features, and mortality. The figure shows weight changes (A), disease severity (B), viral shedding in vaginal lavage fluid (C), and latent viral DNA loads (D). Viral shedding was detected by plaque assay at the indicated time points. The dashed line indicates the detection limit. Latent viral DNA in the sacral ganglia was quantified by real-time PCR at 30 dpi or the date of death. The values 6 × ND, 8 × ND, and 9 × ND indicate the number of mice without latent virus. Data shown are the means ± SEM for each group (n = 5 or 7 mice/group) from at least two independent experiments with each condition performed in duplicate. Statistically significant differences determined by comparing to the pgD + pcDNA3.1 group are indicated. NS, not statistically significant; ***, P < 0.001. ND, no virus detected.