Human Antibodies against Herpes Simplex Virus 2 Glycoprotein G Do Not Neutralize but Mediate Antibody-Dependent Cellular Cytotoxicity

Abstract

Herpes simplex virus 2 (HSV-2) is a sexually transmitted infection affecting 491 million individuals globally. Consequently, there is a great need for both prophylactic and therapeutic vaccines. Unfortunately, several vaccine clinical trials, primarily employing the glycoprotein D of HSV-2 (gD-2), have failed. The immune protection conferred by human anti-HSV-2 antibodies in genital infection and disease remains elusive. It is well-known that gD-2 elicits cross-reactive neutralizing antibodies, i.e., anti-gD-2 antibodies recognize gD in HSV-1 (gD-1). In contrast, anti-glycoprotein G in HSV-2 (mgG-2) antibodies are exclusively type-specific for HSV-2. In this study, truncated versions of gD-2 and mgG-2 were recombinantly produced in mammalian cells and used for the purification of anti-gD-2 and anti-mgG-2 antibodies from the serum of five HSV-2-infected subjects, creating a pool of purified antibodies. These antibody pools were utilized as standards together with purified mgG-2 and gD-2 antigens in ELISA to quantitatively estimate and compare the levels of cross-reactive anti-gD-1 and anti-gD-2 antibodies, as well as anti-mgG-2 antibodies in sera from HSV-1+2-, HSV-2-, and HSV-1-infected subjects. The median concentration of anti-mgG-2 antibodies was five times lower in HSV-1+2-infected subjects as compared with cross-reactive anti-gD-1 and anti-gD-2 antibodies, and three times lower in HSV-2 infected subjects as compared with anti-gD-2 antibodies. The pool of purified anti-gD-2 antibodies presented neutralization activity at low concentrations, while the pool of purified anti-mgG-2 antibodies did not. Instead, these anti-mgG-2 antibodies mediated antibody-dependent cellular cytotoxicity (ADCC) by human granulocytes, monocytes, and NK-cells, but displayed no complement-dependent cytotoxicity. These findings indicate that antibodies to mgG-2 in HSV-2-infected subjects are present at low concentrations but mediate the killing of infected cells via ADCC rather than by neutralizing free viral particles. We, and others, speculate that Fc-receptor mediated antibody functions such as ADCC following HSV-2 vaccination may serve as a better marker of protection correlate instead of neutralizing activity. In an mgG-2 therapeutic vaccine, our findings of low levels of anti-mgG-2 antibodies in HSV-2-infected subjects may suggest an opportunity to enhance the immune responses against mgG-2. In a prophylactic HSV-2 mgG-2 vaccine, a possible interference in cross-reactive immune responses in already infected HSV-1 subjects can be circumvented.

Keywords: ADCC; CDC; concentrations of anti-gD-2 and anti-EXCT4-mgG-2 antibodies; herpes simplex virus 1 and 2 infection; neutralization activity.

Figure 1

(A–C) Quantification of antibodies in clinical sera. Anti-EXCT4-mgG-2 antibodies, cross-reactive anti-gD-1 and gD-2 antibodies in sera of HSV-1+2-infected, HSV-2-infected, HSV-1-infected and HSV-negative subjects were evaluated using purified EXCT4-mgG-2 and gD-2 antigens in ELISA. Pools of purified anti-EXCT4-mgG-2 and anti-gD-2 antibodies with known concentrations were included for generation of standard curves. For statistical analyses the Mann–Whitney nonparametric test was used. The median values are marked with a horizontal line.

Figure 2

Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of purified anti-EXCT4-mgG-2 antibodies (Abs). Pool 1 and pool 2 of anti-EXCT4-mgG-2 antibodies were each purified from five different HSV-2-infected sera. Pool 1 antibodies were evaluated for ADCC by human granulocytes (A), monocytes (B), and NK-cells (C). The dashed horizontal lines mark the cut-off which was defined as mean reactivity of target and effector cells without antibodies plus 2 SD. The dashed vertical lines in (A–C) define the cut-off concentrations of antibodies. An HSV-2-positive serum was diluted 1:400 and used as positive control for the different effector cells including an HSV-negative control serum for granulocytes (D). Total ADCC from peripheral blood mononuclear cells (PBMC), with an HSV-2-positive and with an HSV-negative serum, at 1:400 dilutions, and for comparison pool 1 and pool 2 of purified anti-EXTC4-mgG-2 antibodies, used at concentration of 3 µg/mL, were evaluated (E). Mean values from pool 1 were calculated from duplicate wells in two experiments (A–D) and from three experiments (E), except for pool 2 antibodies which was evaluated in one experiment. An HSV-2-positive serum, and pool 1 of purified anti-EXCT4-mgG-2 antibodies, and Helix pomatia lectin (HPA) purified anti-mgG-2 antibodies, in three concentrations, were evaluated in CDC in two experiments (F). The bars represent mean values +/− SD (A–C) and +SD (D–F).