Immunization with HSV-1 glycoprotein C prevents immune evasion from complement and enhances the efficacy of an HSV-1 glycoprotein D subunit vaccine

Abstract

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) binds complement component C3b and inhibits complement-mediated immunity. HSV-1 glycoprotein D (gD-1) is a potent immunogen and a candidate antigen for a subunit vaccine. We evaluated whether combined immunization with gD-1 and gC-1 provides better protection against challenge than gD-1 alone based on antibodies to gC-1 preventing HSV-1-mediated immune evasion. IgG purified from mice immunized with gC-1 blocked C3b binding to gC-1 and greatly increased neutralization by gD-1 IgG in the presence of complement. Passive transfer of gC-1 IgG protected complement intact mice against HSV-1 challenge but not C3 knockout mice, indicating that gC-1 antibody activity in vivo is complement-dependent. Immunizing mice with gD-1 and gC-1 provided better protection than gD-1 alone in preventing zosteriform disease and infection of dorsal root ganglia. Therefore, gC-1 immunization prevents HSV-1 evasion from complement and enhances the protection provided by gD-1 immunization.

Figure 1

Identifying the optimum dose for gD-1 immunization. A. ELISA responses after the third immunization with gD-1 (P<0.01 and <0.001 comparing mock with 50ng or 100ng, respectively). Results represent the mean ± standard error of five serum samples per group. B. Survival in gD-1 immunized mice challenged with 1 × 10⁶ PFU of HSV-1 NS by flank inoculation (P<0.001 comparing mock with each gD-1 group). C and D. Evaluation of inoculation site disease scores (P< 0.01, comparing mock with 100ng group) and zosteriform site disease scores (P<0.001 comparing mock with 50ng or 100ng immunization group). No significance differences were noted comparing the number of mice with zosteriform disease. Results in B-D represent the mean ± standard error of five mice per group.

Figure 2

Antibody responses to gC-1 immunization. A. ELISA responses after the third immunization with gC-1 (P<0.01 and P<0.001 comparing mock with 1μg or 10μg, respectively). B. A schematic showing C3b-rosetting and anti-gC-1 IgG blocking rosetting. Top cartoon: gC-1 is expressed on the surface of HSV-1 infected cells. C3b-coated erythrocytes bind to gC-1 and form rosettes on the infected cells. Bottom cartoon: Immunization with gC-1 induces antibodies that bind to gC-1 and block the binding of C3b-coated erythrocytes. C. Serum was collected after the third immunization with 0.1μg, 1μg or 10μg gC-1 and was added to infected cells. Rosette inhibition was greater in the 10μg than 1μg group (P<0.001), and in the 1μg than the mock group (P<0.001). Results represent the mean standard error of three serum samples per group.

Figure 3

Survival and disease scores of gC-1 immunized mice challenged with 1 × 10⁶ PFU of HSV-1 NS. A. Survival of mice (five per group) (P<0.01 comparing mock with 10μg group). B and C. Evaluation of inoculation site and zosteriform site disease scores (P>0.7 comparing all groups at the inoculation site, P<0.001 comparing mock with 10μg gC-1). Results in B and C represent the mean ± standard error of five mice per group.

Figure 4

Neutralizing antibody responses with or without complement. A. 70 PFU of HSV-1 WT was incubated with 0.1-100μg of MAb DL11, mouse anti-gC-1 IgG, MAb 1C8, or nonimmune IgG in the absence of complement. The results represent the mean ± standard error of 4 separate determinations. B. Complement mediated enhancement of neutralization. 100-150 PFU of HSV-1 WT or HSV-1gCnull was incubated with PBS, anti-gC-1 IgG 100μg/ml, or anti-gD-1 IgG 100μg/ml with or without 2.5% human complement. The results are the mean of two experiments, each done in duplicate. C. Anti-gC-1 enhances neutralization by anti-gD-1 IgG in the presence of complement. HSV-1 WT was incubated with PBS, anti-gC-1 IgG, anti-gD-1 IgG or both at 200μg/ml or 400μg/ml in presence or absence of 2.5% complement. The results are the mean of two experiments done in duplicate. D. Table listing the reduction in HSV-1 WT titers based on results shown in 4C.

Figure 5

Passive transfer of anti-gC-1 IgG. Images of disease scores at the inoculation site (thick arrows) and zosteriform site (thin arrows) on day 7 post challenge of C57Bl/6 mice (A) or C3 knockout mice (B). Inoculation site (C and D) and zosteriform site (E and F) disease scores in C57Bl/6 and C3 knockout mice. Each data point is the mean of five animals ± standard error. P values are not significant at the inoculation site in C57Bl/6 or C3 knockout mice. Zosteriform disease is significantly greater in C57Bl/6 mice that received nonimmune IgG than mice receiving anti-gC-1 IgG or 1C8 IgG (P<0.001). Zosteriform disease is not significantly different in C3 knockout mice that received nonimmune IgG compared with anti-gC-1 IgG (P=0.21) or 1C8 IgG (P=0.17).

Figure 6

Survival and disease scores of mice immunized with 10ng gD-1 alone or in combination with 10μg gC-1 (left panel) or 50ng gD-1 alone or in combination with 10μg gC-1 (right panel). A and B. Survival of mice (A, P<0.001 comparing mock with both immunization groups; P=0.06 comparing 10ng gD-1 with 10ng gD-1 & 10 μ gC-1; B, P<0.001 comparing mock with both immunization groups; P=0.3 comparing 50ng gD-1 with 50ng gD-1 & 10μg gC-1). C and D. Inoculation site disease (P>0.05 comparing all groups). E and F. Zosteriform site disease (E, P<0.02 comparing gD-1 alone with gD-1 & gC-1; F, P<0.001 comparing gD-1 alone with gD-1 & gC-1). A, C and E have 5 mice per group, while B, D, and F have 5 mice in the mock group and 10 mice in the other two immunization groups. G. Table showing the number of mice in each group that developed inoculation site or zosteriform site disease (P<0.001 comparing 50ng gD-1 with 50ng gD-1 & 10μg gC-1).

Figure 6

Survival and disease scores of mice immunized with 10ng gD-1 alone or in combination with 10μg gC-1 (left panel) or 50ng gD-1 alone or in combination with 10μg gC-1 (right panel). A and B. Survival of mice (A, P<0.001 comparing mock with both immunization groups; P=0.06 comparing 10ng gD-1 with 10ng gD-1 & 10 μ gC-1; B, P<0.001 comparing mock with both immunization groups; P=0.3 comparing 50ng gD-1 with 50ng gD-1 & 10μg gC-1). C and D. Inoculation site disease (P>0.05 comparing all groups). E and F. Zosteriform site disease (E, P<0.02 comparing gD-1 alone with gD-1 & gC-1; F, P<0.001 comparing gD-1 alone with gD-1 & gC-1). A, C and E have 5 mice per group, while B, D, and F have 5 mice in the mock group and 10 mice in the other two immunization groups. G. Table showing the number of mice in each group that developed inoculation site or zosteriform site disease (P<0.001 comparing 50ng gD-1 with 50ng gD-1 & 10μg gC-1).

Figure 7

Protection of DRG by gD-1 & gC-1 immunization. A. Viral DRG titers were measured 5 days post challenge with 1 × 10⁶ PFU of HSV-1 NS (P<0.001 comparing mock with gD-1, or mock with gD-1 & gC-1, P<0.01 comparing gD-1 with gD-1 & gC-1). Each data point represents the mean ± standard error of 5 animals per group. The dotted line represents the limit of detection of the assay at 2 PFU. B. RT qPCR was used to detect HSV-1 genomes in DRG. The naïve group was used to determine background values (P<0.001 comparing mock with gD-1, mock with gD-1 & gC-1 or mock with naïve group, P<0.01 comparing gD-1 with gD-1 & gC-1 or naive group, P>0.05 comparing gD-1& gC-1 with naïve group ). Each data point represents the mean ± standard error of five animals per group.