Increased Frequency of Virus Shedding by Herpes Simplex Virus 2-Infected Guinea Pigs in the Absence of CD4+ T Lymphocytes.

Reactivation of herpes simplex virus 2 (HSV-2) results in infection of epithelial cells at the neuro-epithelial junction and shedding of virus at the epithelial surface. Virus shedding can occur in either the presence or absence of clinical disease and is usually of short duration, although the shedding frequency varies among individuals. The basis for host control of virus shedding is not well understood, although adaptive immune mechanisms are thought to play a central role. To determine the importance of CD4+ T cells in control of HSV-2 shedding, this subset of immune cells was depleted from HSV-2-infected guinea pigs by injection of an anti-CD4 monoclonal antibody (MAb). Guinea pigs were treated with the depleting MAb after establishment of a latent infection, and vaginal swabs were taken daily to monitor shedding by quantitative PCR. The cumulative number of HSV-2 shedding days and the mean number of days virus was shed were significantly increased in CD4-depleted compared to control-treated animals. However, there was no difference in the incidence of recurrent disease between the two treatment groups. Serum antibody levels and the number of HSV-specific antibody-secreting cells in secondary lymphoid tissues were unaffected by depletion of CD4+ T cells; however, the frequency of functional HSV-specific, CD8+ gamma interferon-secreting cells was significantly decreased. Together, these results demonstrate an important role for CD4+ T lymphocytes in control of virus shedding that may be mediated in part by maintenance of HSV-specific CD8+ T cell populations. These results have important implications for development of therapeutic vaccines designed to control HSV-2 shedding.IMPORTANCE Sexual transmission of HSV-2 results from viral shedding following reactivation from latency. The immune cell populations and mechanisms that control HSV-2 shedding are not well understood. This study examined the role of CD4+ T cells in control of virus shedding using a guinea pig model of genital HSV-2 infection that recapitulates the shedding of virus experienced by humans. We found that the frequency of virus-shedding episodes, but not the incidence of clinical disease, was increased by depletion of CD4+ T cells. The HSV-specific antibody response was not diminished, but frequency of functional HSV-reactive CD8+ T cells was significantly diminished by CD4 depletion. These results confirm the role of cell-mediated immunity and highlight the importance of CD4+ T cells in controlling HSV shedding, suggesting that therapeutic vaccines designed to reduce transmission by controlling HSV shedding should include specific enhancement of HSV-specific CD4+ T cell responses.

Development of an anti-guinea pig CD4 monoclonal antibody for depletion of CD4+ T cells in vivo.

The guinea pig serves as a useful animal model for a number of human diseases and has played an important role during development and testing of experimental vaccines and disease therapies. However, the availability of reagents to examine the immunological response in this species is very limited. Monoclonal antibodies (mAb) specific for cell surface proteins or products of immune cells have been useful tools for characterizing and quantifying immune responses in humans and in murine models of human disease, but very few similar reagents are available for characterizing and manipulating the immune response of guinea pigs. A rat IgG2a mAb specific for guinea pig CD4 has previously been described and was shown to inhibit T cell proliferation, but was inefficient at depleting CD4+ T cells in vivo. We hypothesized that the in vivo CD4+ T cell depletion function of this mAb could be improved by expression of the rat IgG2b heavy chain. We show that the purified mAb from an IgG2b class-switch variant, but not the parental IgG2a mAb, significantly depleted CD4+ T cells from secondary lymphoid tissue of guinea pigs. Further, treatment of guinea pigs with the IgG2b mAb at 2.0 mg/kg resulted in depletion of CD4+ T cells from peripheral blood and spleen. The use of this modified antibody to specifically alter the immune response of guinea pigs should prove useful in a number of guinea pig infectious disease models.

Development of disease and immunity at the genital epithelium following intrarectal inoculation of male guinea pigs with herpes simplex virus type 2.

Most analyses of genital immunity to herpes simplex virus type 2 (HSV-2) have been performed in females, consequently immune protection of the male genital epithelium is incompletely understood. We developed a model of male genital HSV-2 infection resulting from intrarectal inoculation of guinea pigs. Vesicular lesions developed transiently on the perineum and foreskin concurrent with acute virus shedding. Virus shedding and recurrent genital lesions were also detected after establishment of a latent infection. Analysis of perineum and foreskin RNA detected transcripts for IFNγ, proinflammatory and regulatory cytokines, and for genes involved in migration and regulation of leukocytes. HSV-specific T cells were detected in lymphoid and genital tissues after resolution of the primary infection whereas virus-specific antibody secreting cells were detected only in lymphoid tissue. Taken together, the ability to quantify pathogenesis and local immunity in this guinea pig model represent an important advance towards understanding immunity to HSV-2 in males.

A combined carrier-adjuvant system of peptide nanofibers and toll-like receptor agonists potentiates robust CD8+ T cell responses.

Improving CD8+ T cell responses activated by subunit vaccination is crucial for improving vaccine efficacy and safety. Here we report a carrier-adjuvant system composed of self-assembling peptide nanofibers presenting an immunodominant antigen from herpes simplex virus (HSV) and toll-like receptor (TLR) agonists that induces robust effector and memory CD8+ T cell responses in mice. The effector function of vaccine-induced CD8+ T cells was influenced by the type of TLR agonist. The use of CpG (TLR9 agonist) resulted in significantly greater specific in vivo cytotoxicity and trended towards more cells producing both IFN-γ and TNF-α compared to gardiquimod (TLR7 agonist). Prime-boost immunization with peptide nanofibers combined with either adjuvant resulted in development of HSV-specific CD8+ memory T cells further demonstrating the capability of the carrier-adjuvant system to induce strong HSV-specific CD8+ T cell responses. Inclusion of peptide epitope-nanofibers in protein-based subunit vaccines should increase the functional spectrum of the vaccine-elicited immune response and protection.

Detection of herpes simplex virus type 2 (HSV-2) -specific cell-mediated immune responses in guinea pigs during latent HSV-2 genital infection.

Genital infections with herpes simplex virus type 2 (HSV-2) are a source of considerable morbidity and are a health concern for newborns exposed to virus during vaginal delivery. Additionally, HSV-2 infection diminishes the integrity of the vaginal epithelium resulting in increased susceptibility of individuals to infection with other sexually transmitted pathogens. Understanding immune protection against HSV-2 primary infection and immune modulation of virus shedding events following reactivation of the virus from latency is important for the development of effective prophylactic and therapeutic vaccines. Although the murine model of HSV-2 infection is useful for understanding immunity following immunization, it is limited by the lack of spontaneous reactivation of HSV-2 from latency. Genital infection of guinea pigs with HSV-2 accurately models the disease of humans including the spontaneous reactivation of HSV-2 from latency and provides a unique opportunity to examine virus-host interactions during latency. Although the guinea pig represents an accurate model of many human infections, relatively few reagents are available to study the immunological response to infection. To analyze the cell-mediated immune response of guinea pigs at extended periods of time after establishment of HSV-2 latency, we have modified flow-cytometry based proliferation assays and IFN-γ ELISPOT assays to detect and quantify HSV-specific cell-mediated responses during latent infection of guinea pigs. Here we demonstrate that a combination of proliferation and ELISPOT assays can be used to quantify and characterize effecter function of virus-specific immune memory responses during HSV-latency.