Eric M. Mishkin

Th1Th2-like immunity and resistance to herpes simplex labialis

To investigate potential immunologic mechanisms of resistance to recurrent herpes simplex labialis, we assayed serum antibody titers and cultured peripheral blood mononuclear cell (PBMC) cytokine production among patients with a history of frequent episodes (H+S+), herpes simplex virus (HSV)-seropositive individuals without a history of herpes labialis (H-S+) and HSV-seronegative persons (H-S-). In addition, H+S+ patients were exposed to experimental ultraviolet radiation (UVR) on the lips and the immunologic assay results compared among those who developed experimental lesions and those who did not. H+S+ patients were found to have higher median serum titers of HSV antibody and trends to lower levels of HSV-specific PBMC IFN-gamma and IL-2 than H-S+ control patients (123 vs 66, P = 0.04; 424 vs 548 pg/ml, P = 0.08; 14 vs 26 pg/ml, P = 0.14, respectively). Correlation of the results with the occurrence of experimental lesions showed the inverse: the subgroup of H+S+ patients with UVR-induced lesions had lower titers of antibody and trends to higher levels of IFN-gamma and IL-2 than H+S+ patients who could not be induced (93 vs 149, P = 0.02; 501 vs 347 pg/ml, P = NS; 26 vs 11 pg/ml, P = NS, respectively). The size and duration of UVR-induced lesions showed positive correlations with IFN-gamma and IL-2 levels (r = 0.60-0.67, P = 0.02-0.04). Although the small number of patients limited the power of this study, the overall pattern of the findings suggests that a Th1-like cytokine response (IFN-gamma and IL-2 production) may be associated with resistance to naturally occurring episodes of herpes labialis. The development and severity of experimental UVR-induced herpes labialis appears to be regulated differently and may involve an immunopathologic mechanism.

Baculovirus-expressed herpes simplex virus type 2 glycoprotein D is immunogenic and protective against lethal HSV challenge

Herpes simplex virus type 2 glycoprotein D (gD2) was cloned and expressed in the baculovirus-Spodoptera frugiperda system. Milligram quantities of glycoprotein were recovered from suspension culture and subjected to purification by ion-exchange and immunoaffinity chromatography. The resultant purified gD existed as a homogeneous 57,500 MW monomeric species demonstrating reactivity with anti-gD monoclonal antibodies including those directed at a non-sequential neutralizing epitope of gD. Immunization of Balb/c mice with doses of 0.1-10.0 micrograms of AlPO4-absorbed gD resulted in elicitation of humoral and cellular responses to both HSV1 and HSV2 as well as to purified gD1 and gD2. Immunized mice receiving an infectious dose of 1 x 10(6) p.f.u. of HSV2 via the footpad route were significantly protected against infection at all doses tested when compared with unimmunized AlPO4 and uninoculated control animals.

Combined systemic and mucosal immunization with microsphere-encapsulated inactivated simian immunodeficiency virus elicits serum, vaginal, and tracheal antibody responses in female rhesus macaques.

We determined the efficacy of immunization with microsphere-encapsulated whole inactivated simian immunodeficiency virus (SIV) by combined systemic and mucosal administration to protect female rhesus macaques against vaginal challenge with homologous rhesus PBMC-grown SIVmac251. Animals in one group were primed and boosted intramuscularly. Two groups were primed intramuscularly and boosted either intratracheally or orally. A final group was primed by vaccinia/rgp140 scarification and subdivided for either intratracheal or oral boosting. Strong ELISA titers of circulating SIV-specific IgG and modest IgA responses were elicited in the animals primed intramuscularly. Intratracheal boosting in the intramuscularly primed macaques resulted in high bronchial alveolar wash (BAW) IgG and less pronounced IgA. SIV-specific vaginal wash (VW) IgG was also present in the intramuscular/intramuscular and intramuscular/intratracheal groups. Vaccinia/rgp140 priming gave low ELISA titers to whole SIV, and failed to elicit mucosal antibody regardless of the booster route. No animal in any group developed serum neutralizing antibody to homologous SIVmac251. On vaginal challenge none of the immunized groups was infected at a lesser frequency than the unimmunized controls. These data suggest that the use of microspheres in a combined parenteral and mucosal regimen is an effective method of eliciting IgG and IgA antibody at mucosal surfaces.

Immunomodulatory effects of HSV2 glycoprotein D in HSV1 infected mice: implications for immunotherapy of recurrent HSV infection

Immunological analyses in this laboratory and others have suggested that a nonrecurrent HSV seropositive immune status is more closely correlated with a type 1 T helper cell (Th1) response characterized by elevated levels of interferon-gamma and IL2 rather than high titers of virus-specific antibodies. Effective intervention with an immunotherapeutic vaccine may require modulation of the regulatory network of T helper cells such that there is selective restimulation and expansion of the Th1 response. We have established a murine model for assessing the immunomodulatory capacity of an HSV glycoprotein subunit vaccine in animals with pre-existing herpes immunity. Animals were infected with varying doses of HSV1 and then administered glycoprotein D (gD) vaccine adjuvanted with aluminum phosphate at 3-week intervals. Observed changes in serological and cellular responses indicated that administration of subunit vaccine adjuvanted with aluminum phosphate could shift a dominant Th1 response, induced by sensitization with live HSV, towards a Th2 profile of activity. These data suggest that use of aluminum based adjuvants will not selectively stimulate Th1-associated responses and alternative adjuvants may be required for effective use of subunit vaccine in an immunotherapeutic indication in humans.

Induction of mucosal antibody responses by microsphere-encapsulated formalin-inactivated simian immunodeficiency virus in a male urethral challenge model

Male rhesus macaques were immunized mucosally with microsphere-encapsulated formalin-inactivated simian immunodeficiency virus (SIV) particles in a test of immunogenicity and protection against mucosal SIV challenge. Tracheal boosting of animals that had been primed intramuscularly resulted in strong serum ELISA titers to SIV, and evidence of local IgA responses in broncho-alveolar washes. The bulk of the antibody response was against non-envelope epitopes. No neutralizing antibody was observed, and intraurethral challenge with cell-free rhesus-grown virus showed no evidence of protection against infection. Microsphere-based immunization efficiently raises local and system responses, but the resulting immunity to SIV is apparently not sufficient to protect against mucosal challenge.