Immunogenicity, protective efficacy, and non-replicative status of the HSV-2 vaccine candidate HSV529 in mice and guinea pigs

Abstract

HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.

Fig 1. Guinea pigs immunized with HSV529 produce antibodies that neutralize HSV-2 infection in vitro.

Guinea pigs were immunized with HSV529 (10⁴ CCID₅₀, 10⁵ CCID₅₀, or 10⁶ CCID₅₀) by the intramuscular (IM; n = 5 each) or subcutaneous (SC; n = 5 each) route or with PBS (n = 3) by the intramuscular route on days 0 (D0) and 21 (D21). Sera were collected from all animals on days 21 and 29 (D29) and measured for HSV-2 neutralizing activity by preincubating dilutions of heat-inactivated sera with 100 CCID₅₀ of live HSV-2 virus for 1 hour prior to infection of Vero cell cultures. Infected cells were detected with anti-HSV glycoprotein D antibodies. The serum dilution that neutralized 50% of the virus (SN50) was determined by plotting the neutralization activity versus the serum dilutions. Error bars represent standard error of the mean.

Fig 2. Mice immunized with HSV529 produce HSV-2-specific IgG1 and IgG2a antibodies, neutralizing antibodies, and HSV-2-specific splenic lymphocytes secreting IFNγ and IL-5.

BALB/c mice (n = 10/group) were immunized with HSV529 (10⁴ CCID₅₀, 10⁵ CCID₅₀, or 10⁶ CCID₅₀) or PBS by the i.m. route on days 0 and 21. Sera were collected on days 21 (D21; n = 10) and 41 (D41; n = 5). (A) HSV-2-specific IgG1 and IgG2a antibody titers in the sera were determined by ELISA using a lysate prepared from HSV-2-infected Vero cells and secondary antibodies specific for mouse IgG1 and IgG2a. (B) HSV-2 neutralizing antibodies in the sera were measured by preincubating dilutions of heat-inactivated sera with 100 CCID₅₀ of live HSV-2 (strain G) virus for 1 hour prior to infection of Vero cell cultures. Infected cells were detected with anti-HSV glycoprotein D antibodies. The serum dilution that neutralized 50% of the virus (SN₅₀) was determined by plotting the neutralization activity versus the serum dilutions. Splenic lymphocytes secreting IFNγ (C) or IL-5 (D) in response to ex vivo stimulation with heat-inactivated HSV-2 (strain G) were counted using an ELISPOT assay. Error bars represent standard error of the mean.

Fig 3. HSV529 immunization protects mice from the effects of lethal HSV-2 vaginal challenge.

BALB/c mice were immunized with HSV529 (10⁶ CCID₅₀) or PBS by the i.m. route on days 0 and 21. On day 48, mice received medroxyprogesterone (2 mg, s.c.) to prevent reproductive cycling. The next day, mice were challenged with an intravaginal inoculation of HSV-2 (strain G; 10⁵ CCID₅₀). (A) Mean body weight change after HSV-2 challenge. (B) Mean vaginal lesion score after HSV-2 challenge. (C) Percent survival after HSV-2 challenge. (D) HSV-2 viral shedding after challenge. *Dead or euthanized animal. Error bars represent the standard error of the mean.

Fig 4. HSV529 immunization protects guinea pigs from the effects of HSV-2 vaginal challenge.

Guinea pigs were immunized with HSV529 (10⁶ CCID₅₀; n = 30) or PBS (n = 25) by the i.m. route on days 0 and 21. On day 48, 15 animals in each group were challenged with an intravaginal inoculation of HSV-2 (G strain; 10⁵ CCID₅₀). The remaining animals received a mock challenge of PBS. (A) Mean body weight change after HSV-2 challenge. (B) Mean vaginal lesion score after HSV-2 challenge. (C) Percent survival after HSV-2 challenge. (D) HSV-2 viral shedding after challenge. (E) Cumulative number of recurrent lesions per animal. *Dead or euthanized animal. Error bars represent standard error of the mean.

Fig 5. HSV529 immunization protects HSV-1-primed guinea pigs from effects of HSV-2 vaginal challenge.

Guinea pigs were inoculated with HSV-1 (KOS strain; 10⁶ CCID₅₀; n = 30) or PBS (n = 18) by the intranasal route on day 0. All animal inoculated with HSV-1 were positive for HSV-1 at week 5. At weeks 7 and 10, animals inoculated with HSV-1 were immunized with HSV529 (10⁶ CCID₅₀; n = 15) or PBS (n = 14) by the i.m. route. At week 14, all animals except 3 PBS controls were challenged with an intravaginal inoculation of HSV-2 (G strain, 2 x 10⁶ CCID₅₀). (A) Mean body weight change after HSV-2 challenge. (B) Mean vaginal lesion score after HSV-2 challenge. (C) Percent survival after HSV-2 challenge. (D) HSV-2 viral shedding after challenge. (E) Cumulative number of recurrent lesions per animal. *Dead or euthanized animal. Error bars represent standard error of the mean.

Fig 6. HSV529 does not propagate in the brains of 4−6-day-old suckling mice.

Four- to 6-day-old sucking mice received an intracranial injection of vaccine buffer (gray squares), HSV529 (gray triangles, 5 x 10⁵ CCID₅₀), or wild-type (wt) HSV-2 186 syn+-1 (black circles, 10 CCID₅₀). Brains were collected on p.i. days 0 (4 hours p.i.), 2, 4, 6, and 14, and from animals that died during the experiment. The titer of each animal is represented by an individual symbol and the mean titer is represented by a horizontal bar. Virus titers were determined on AV529-19 cells.