Immunoglobulin G is the main protective antibody in mouse vaginal secretions after vaginal immunization with attenuated herpes simplex virus type 2

We investigated the protective role of antibodies in vaginal secretions of mice that were immune to vaginal challenge with herpes simplex virus type 2 (HSV-2). Unfractionated vaginal immunoglobulins from immune and nonimmune mice and affinity-purified immunoglobulin G (IgG) and secretory IgA (S-IgA) from immune secretions were adjusted to their concentrations in vivo. Wild-type HSV-2 was incubated in the immunoglobulin preparations for 15 min in vitro, followed by inoculation into vaginae of nonimmune mice. HSV-2 was neutralized by unfractionated antibody and purified IgG from immune secretions but not by unfractionated nonimmune antibody or by purified immune S-IgA. The protective effect of IgG in vivo was investigated by passively transferring purified serum IgG from immune and nonimmune donors to nonimmune recipients before vaginal challenge infection. Immune IgG significantly reduced the percentage of vaginal epithelium infected, concentrations of shed virus protein in the vaginal lumen, and illness scores, even though the viral antibody titers in serum and vaginal secretions of recipient mice at the time of challenge were only 29 and 8%, respectively, of those in actively immunized mice. Additionally, removal of vaginal secretions from immune mice 10 min before vaginal challenge with HSV-2 significantly increased the concentration of shed virus protein in the vaginal lumen after challenge. Collectively, the data indicate that IgG antibody in vaginal secretions of immune mice provides early protection against vaginal challenge infection, probably by neutralizing virus in the vaginal lumen. In contrast, S-IgA antibody contributed relatively little to immune protection of the vagina.     

Genetic immunization with adeno-associated virus vectors expressing herpes simplex virus type 2 glycoproteins B and D

Intramuscular injection of mice with an adeno-associated virus (AAV) vector expressing herpes simplex virus type 2 glycoprotein B led to the generation of both gB-specific major histocompatibility complex class I-restricted cytotoxic T lymphocytes and anti-gB antibody. AAV-mediated immunization was more potent than plasmid DNA or protein in generating antibody responses.     

Mucosal immunity to herpes simplex virus type 2 infection in the mouse vagina is impaired by in vivo depletion of T lymphocytes

Intravaginal (IVAG) inoculation of wild-type herpes simplex virus type 2 (HSV-2) in mice causes epithelial infection followed by lethal neurological illness, while IVAG inoculation of attenuated HSV-2 causes epithelial infection followed by development of protective immunity against subsequent IVAG challenge with wild-type virus. The role of T cells in this immunity was studied by in vivo depletion of these cells with monoclonal antibodies. Three groups of mice were used for each experiment: nonimmune/challenged mice, immune/challenged mice, and immune depleted mice [immune mice depleted of a T-cell subset(s) shortly before challenge with HSV-2]. Mice were assessed for epithelial infection 24 h after challenge, virus protein in the vaginal lumen 3 days after challenge, and neurological illness 8 to 14 days after challenge. Monoclonal antibodies to CD4, CD8, or Thy-1 markedly reduced T cells in blood, spleen, and vagina, but major histocompatibility complex class II antigens were still partially upregulated in the vaginal epithelium after virus challenge, indicating that virus-specific memory T-cell function was not entirely eliminated from the vagina. Nevertheless, immune mice depleted of CD4+ and CD8+ T cells, Thy-1+ T cells, or CD8+ T cells alone had greater viral infection in the vaginal epithelium than nondepleted immune mice, indicating that T cells contribute to immunity against vaginal HSV-2 infection. All immune depleted mice retained substantial immunity to epithelial infection and were immune to neurological illness, suggesting that other immune mechanisms such as virus-specific antibody may also contribute to immunity.     

Migration of lymphoid cells from vaginal epithelium to iliac lymph nodes in relation to vaginal infection by herpes simplex virus type 2

To determine whether lymphocytes and Langerhans cells in vaginal epithelium are migratory, we stained mouse vaginal epithelium, including its lymphoid cells, by intraluminal administration of H33342, a fluorescent, vital dye. Stromal staining was superficial, and no free dye reached the iliac lymph nodes. The numbers and phenotypes of H33342-stained cells that migrated from the vagina to the iliac lymph nodes during the next 48 h were determined in four groups: normal mice, mice infected intravaginally with wild-type herpes simplex virus type 2 (HSV-2), mice that were immune to vaginal HSV-2 infection, and immune mice that received vaginal challenge with HSV-2. H33342-stained cells migrated from the vaginal epithelium to the iliac lymph nodes in all groups and were mainly Thy-1.2+ cells and B220+ cells. The number of migrating Thy-1.2+ cells was similar to the sum of CD4+ and CD8+ cells in all groups and was not significantly different from the number of CD44+ cells, suggesting that most of the migrating T cells were memory cells. B lymphocytes comprised 31, 32, 43, and 68% of the migrating cells in the four groups, respectively. We found no evidence that Langerhans cells or macrophages were migrating. Thus, most MHC class II+ cells in all groups were accounted for by B cells, and migrating cells did not express B7.1 or F4/80 or exhibit indented nuclei or dendritic processes. We suggest that the migrating T cells and B cells probably belonged to a pool of lymphocytes that recirculates from blood to tissues and back to the lymph nodes via their afferent lymphatics.     

Induction of apoptosis in HEp-2 cells by infection with herpes simplex virus type 2

Although herpes simplex virus type 1 (HSV-1) does not induce apoptosis in infected HEp-2 cells, herpes simplex virus type 2 (HSV-2) did induce apoptosis in a small but significant fraction of the same cells. Apoptosis was not observed in Vero or HeLa cells infected with HSV-2. In addition, HSV-2 infection in the presence of cycloheximide induced extensive apoptosis of HEp-2 or HeLa cells.     

Anterograde, transneuronal transport of herpes simplex virus type 1 strain H129 in the murine visual system

Herpes simplex virus (HSV) undergoes retrograde and anterograde axonal transport as it establishes latency and later intermittently reactivates. Most strains of HSV show preferential retrograde transport within the central nervous system (CNS), however. Previous experiments suggest that an exception to this is HSV type 1 (HSV-1) strain H129, since this virus appears to spread primarily in the CNS via anterograde, transneuronal movement. The objective of the present study was to test how specifically this virus spreads in the visual system, a system with well-described neuronal connections. In the present study, the pattern of viral spread was examined following inoculation into the murine vitreous body. Virus was initially detected in the retina and optic tract. Virus then appeared in all known primary targets of the retina, including those in the thalamus (e.g., lateral geniculate complex), hypothalamus (suprachiasmatic nucleus), and superior colliculus (superficial layers). In previous studies, many strains of HSV were shown to infect these structures, even though they spread predominantly in a retrograde direction. However, the H129 strain was unique in then spreading, via anterograde transport, to the primary visual cortex (layer 4 of area 17) via thalamocortical connections. At later times after infection, specific labeling was also detected in other cortical and subcortical areas known to receive projections from the visual cortex. No labeling was ever detected in the contralateral retina, which is consistent with a lack of retrograde spread of HSV-1 strain H129. These results demonstrate the specific anterograde movement of this virus from the retina to subcortical and cortical regions, with no clear evidence for retrograde spread. HSV-1 strain H129 should be generally useful for tracing sensory pathways and may provide the basis for designing a virus vector capable of delivering genetic material via anterograde pathways within the CNS.     

Prevalence of papillomavirus, Epstein-Barr virus, cytomegalovirus, and herpes simplex virus type 2 in urinary bladder cancer

The need to find an alternative to the use of bowel for urinary reconstruction has renewed research interests involving bladder regeneration. Historically, alloplastic and biodegradable materials have demonstrated bladder regeneration; however, high complication rates and unreliable regenerative results have prevented any of these materials from being used clinically. Small-intestinal submucosa (SIS) is an acellular, nonimmunogenic, biodegradable, xenogeneic, collagen-based material that is derived from the submucosa layer of porcine small intestine. SIS has demonstrated regenerative capacities in multiple organ systems, including the aorta, vena cava, ligaments, tendons, abdominal wall, and skin. SIS has also demonstrated long-term reliable regenerative results in the rat and canine bladder-augmentation models. This article reviews the preclinical studies involving the use of SIS for bladder augmentation.     

Distribution of herpes simplex virus type 1 and 2 genomes in the human spinal ganglia

Herpes simplex virus (HSV) is well known for its propensity to cause recurrent oral or genital mucosal infections in humans. HSV-1 is involved primarily in oral lesions, whereas HSV-2 is more frequently involved in genital lesions. Based on this, it is thought that HSV-1 may produce latent infections in trigeminal ganglia, and HSV-2 in the sacral ganglia. However the distribution pattern of latent HSV-1 and HSV-2 infections in spinal ganglia remains unknown. Using the polymerase chain reaction we detected latent herpes HSV-1 and HSV-2 in human spinal ganglia obtained from autopsy material. A pair of primers which were specific for a part of the HSV-1 and HSV-2 DNA polymerase domain were employed. HSV-1 and HSV-2 DNAs were detected in 11 of 40 (28%) and 15 of 40 (38%) cervical ganglia, respectively, 52 of 103 (50%) and 47 of 103 (46%) thoracic ganglia, 16 of 53 (30%) and 17 of 53 (32%) lumbar ganglia, and 3 of 20 (15%) and 3 of 20 (15%) sacral ganglia. These findings suggest that latent HSV-1 and HSV-2 infections have a widespread distribution from the cervical ganglia to sacral ganglia. Importantly this study demonstrated latent HSV-1 infection of both the lumbar and sacral ganglia for the first time.     

In vivo immune evasion mediated by the herpes simplex virus type 1 immunoglobulin G Fc receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcgammaR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcgammaR activity without affecting other gE functions, we constructed a mutant virus, NS-gE339, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcgammaRs. NS-gE339 expresses gE and gI, is FcgammaR-, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcgammaR does not bind murine IgG; therefore, the absence of an FcgammaR should not affect virulence in mice. NS-gE339 causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcgammaR- mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcgammaR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE339-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcgammaR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.     

Epidemiology of herpes simplex virus type 2 infections in a high-risk adolescent population

The seroprevalence of infection with type 2 herpes simplex virus (HSV-2) was determined in 135 adolescents detained in a juvenile detention facility. A total of 16% of enrollees were seropositive for HSV-2. Age of onset of sexual intercourse, number of lifetime partners, frequency of condom use, and history of sexually transmitted diseases did not predict HSV-2 seropositivity.     

Dimerization and activation of the herpes simplex virus type 1 protease

The quaternary state of the herpes simplex virus type 1 (HSV-1) protease has been analyzed in relation to its catalytic activity. The dependence of specific activity upon enzyme concentration indicated that association of the 27-kDa subunits strongly increased activity. Size-exclusion chromatography identified the association as a monomer-dimer equilibrium. Isolation of monomeric and dimeric species from a size-exclusion column followed by immediate assay identified the dimer as the active form of the enzyme. Activation of the protease by antichaotropic cosolvents correlated with changes in the monomer-dimer equilibrium. Thus, dimerization of the enzyme was enhanced in solvents containing glycerol or the anions citrate or phosphate. These are substances previously identified as activators of HSV-1 protease (Hall, D. L., and Darke, P. L. (1995) J. Biol. Chem. 270, 22697-22700). The relative potencies of these cosolvents as enzyme activators correlated with their efficiency in promoting dimerization. Under all solvent conditions examined, the dependence of specific activity upon enzyme concentration was consistent with a kinetic model in which only the dimer is active. Dissociation constants for the HSV-1 protease dimer determined with this model at 15 degrees C, pH 7.5, were 964 and 225 nM in 20% glycerol with 0.2 and 0.5 M citrate present, respectively. The activation of the HSV-1 protease by antichaotropic cosolvents was hereby shown to be similar in nature to the activation of the other well characterized herpesvirus protease, that from human cytomegalovirus.     

Antibodies and antibody-secreting cells in the female genital tract after vaginal or intranasal immunization with cholera toxin B subunit or conjugates

We studied the antibody response including antibody-secreting cells (ASC) in the female genital tract of mice after mucosal immunizations with the recombinant B subunit of cholera toxin (rCTB) perorally, intraperitoneally, vaginally, and intranasally (i.n.). The strongest genital antibody responses as measured with a novel perfusion-extraction method were induced after vaginal and i.n. immunizations, and these routes also gave rise to specific immunoglobulin A (IgA) and IgG ASC in the genital mucosa. Specific ASC in the iliac lymph nodes, which drain the female genital tract, were seen only after vaginal immunization. Progesterone treatment increased the ASC response in the genital tissue after all mucosal immunizations but most markedly after vaginal immunization. We also tested rCTB as a carrier for human gamma globulin (HGG) and the effect of adding cholera toxin (CT) as an adjuvant for the induction of systemic and genital antibody responses to HGG after vaginal and i.n. immunizations. Vaginal immunizations with HGG conjugated to rCTB resulted in high levels of genital anti-HGG antibodies whether or not CT was added, while after i.n. immunization the strongest antibody response was seen with the conjugate together with CT. In summary, vaginal and i.n. immunization give rise to a specific mucosal immune response including ASC in the genital tissue, and vaginal immunization also elicits ASC in the iliac lymph nodes. We have also shown that rCTB can act as an efficient carrier for a conjugated antigen for induction of a specific antibody response in the genital tract of mice after vaginal or i.n. immunization.     

Subcellular localization of the US3 protein kinase of herpes simplex virus type 2

One supposes that herpes simplex virus US3 gene product possessing serine/threonine protein kinase activity is a cytoplasmic enzyme. To determine its subcellular localization during viral replication we prepared an antiserum to a synthetic oligopeptide corresponding to the N-terminal region of the US3 protein of HSV type 2 strain 186. The US3 protein first appeared in the cytoplasm of infected cell at 4 h postinfection but strong fluorescence was detected in the nuclei at 8 h postinfection. At 12 h postinfection fluorescence was mainly detected in the cytoplasm, again. Further, the US3 protein expressed alone was widely distributed throughout the cell, indicating that the US3 protein by itself can be localized in the nuclei even in the absence of any other viral proteins. These observations suggest that the HSV-2 US3 protein kinase may function not only in the cytoplasm but also in the nuclei.     

Role of type specific herpes simplex virus serology in the diagnosis and management of genital herpes

Chromogenic hexapeptides Dnp-Ala-Ala/Ser-Phe-Phe-Ala-Arg-NH2 containing a Phe-Phe bond, which is sensitive to aspartic proteinases, were used as substrates for assaying the activity of pepsin, chymosin, and aspergillopepsin A. The assay was performed after the separation of hydrolyzates on SP-Sephadex by measuring at 360 nm the absorbance of the dinitrophenylpeptide lacking the cationic group, which was formed upon the cleavage of the substrate. The kinetic parameters of the hydrolysis of the substrates were evaluated. It is shown that replacing the Ala residue with Ser in the P2 position does not substantially change the kinetic parameters. The substrates were hydrolyzed by pepsin several times faster than by aspergillopepsin A or chymosin. The method is sensitive and enables the activity of aspartic proteinases to be determined easily.     

Cytotoxic T lymphocyte activity after intravitreal inoculation of herpes simplex virus type I in mice

After inoculating mice with herpes simplex virus (HSV) by a corneal or intravitreal route, cytotoxic T lymphocyte (CTL) activity in the cervical lymph nodes and spleen was assayed. The spleen and cervical lymph nodes were removed at various points till 2 weeks after inoculation, and CTL activity was assayed in a groups: (A) mice intravitreally inoculated with HSV, and (B) mice with topical application of HSV. The reactivity of delayed type sensitivity was determined by the thickness of the mouse ear pinna on the 6th day in both groups. CTL activity in the spleen was at the same level in both groups. Up to 10 days after inoculation CTL activity in the cervical lymph nodes in group (A) was lower than in group (B). The reactivity of delayed type sensitivity in group (A) was lower than in group (B). These results indicate that an anterior chamber associated immune deviation (ACAID)-like phenomenon occurred after HSV inoculation into the vitreous cavity.     

Loss of DNA loop supercoiling and organization in cells infected by herpes simplex virus type 1

In cells infected by herpesviruses, a sequence of nuclear changes during interphase, as well as chromosomal aberrations during mitosis, are commonly observed. These changes suggest the progressive modification of host-cell chromatin. Previous studies have shown that the early chromatin modifications in cells infected by herpes simplex virus type 1 (HSV1) are not due to extensive breakdown of host-cell DNA or disruption of the nucleosomal structure. We have previously shown that infection by HSV1 induces single-stranded breaks in the host-cell DNA early in the course of infection, and that such breaks lead to modifications in the higher-order structure of host-cell chromatin. Here we report that virus-induced DNA breaks produce permanent long-term effects on the state of supercoiling and organization of the nuclear DNA loops, comparable to the DNA loop disorganization produced by high (and irreparable) doses of ultraviolet radiation.     

Association between human immunodeficiency virus and herpes simplex virus type 2 seropositivity among male factory workers in Zimbabwe

To determine the seroprevalence of herpes simplex virus type 2 (HSV-2), to identify correlates of infection, and to describe the correlation with human immunodeficiency virus (HIV) seropositivity, 224 HIV-negative and 191 HIV-positive male factory workers in Zimbabwe were screened for HSV-2-specific antibodies. HSV-2 seroprevalence was 35.7% among HIV-negative subjects and 82.7% among HIV-positive subjects. The weighted estimate of HSV-2 seroprevalence in this population is 44.6%. The correlation between HIV and HSV-2 remained significant after controlling for multiple sex partners, paying for sex, and history of sexually transmitted disease (adjusted odds ratio, 8.0; 95% confidence interval, 4.8-13.1). If the association between HSV-2 and HIV is causal, then the high seroprevalence of HIV and HSV-2 suggests that suppressive HSV-2 treatment should be considered as a strategy to reduce HIV transmission in this population. HSV-2 seroconversion may be a suitable surrogate end point to evaluate HIV prevention interventions.     

Cytotoxicity and accumulation of ganciclovir triphosphate in bystander cells cocultured with herpes simplex virus type 1 thymidine kinase-expressing human glioblastoma cells

The ability of herpes simplex virus type 1 thymidine kinase (HSV-TK)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-TK-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs through the transfer of phosphorylated GCV, there is little direct proof that bystander cells can accumulate GCV nucleotides. We have studied the ability of U251 human glioblastoma cells expressing HSV-TK (U251tk cells) to induce cytotoxicity in neighboring U251 bystander cells that lack the viral kinase (U251beta gal cells) and evaluated whether this bystander cell killing is mediated by GCV nucleotides. The cytotoxicity studies demonstrated that the ratio of HSV-TK-expressing cells:bystander cells was important in determining the sensitivity of both cell types to GCV. U251tk cells cocultured with an equal number of U251beta gal cells (a 50:50 ratio) exhibited a sensitivity to GCV similar to that observed in the absence of bystander cells, with >99.8% cell kill at 1 microm GCV. However, in cultures with 10% U251tk cells and 90% bystander cells (a 10:90 ratio), 1 microM GCV decreased the survival of U251tk cells by only 54%. Strong bystander cell killing was observed at both ratios. In a 50:50 coculture of U251tk and U251beta gal cells, the survival of bystander cells was decreased by >99.5% with 3 microM GCV, whereas 30 microM GCV was required to effect a similar decrease in bystander cell survival when 90% of the culture consisted of U251beta gal cells. To determine whether this bystander cell killing may be mediated by GCV nucleotides, we developed a technique to separate the two cell populations after coculture. A U251 bystander cell line was developed from the parental cell line by transfection with the cDNA coding for green fluorescent protein (U251gfp cells), which permitted the separation of U251gfp cells from nonfluorescing U251tk cells by flow cytometry with cell sorting. With this technique, bystander cells were isolated in a viable state with >97% purity within 1 h after harvest, permitting analysis of the nucleotide pools for the presence of phosphorylated GCV. The results demonstrated that significant levels of the triphosphate of GCV (GCVTP) accumulated in bystander cells within 4 h of coculture, and this accumulation was dependent upon the percentage of HSV-TK-expressing cells as well as the concentration of GCV and the length of incubation. The proportion of GCVTP in bystander cells was consistently 50-80% of that in HSV-TK-expressing cells in the 50:50 or 10:90 cocultures, suggesting a facile transfer of phosphorylated GCV. However, the actual amount of GCVTP was as much as 8-fold lower in both the U251tk and U251beta gal cells cocultured at a ratio of 10:90 compared to those cocultured at a ratio of 50:50, which is consistent with the lesser effect on cell survival. When U251tk and U251gfp cells were cultured with 1-beta-D-arabinofuranosylthymine (araT), the 5'-triphosphate of araT accumulated in the bystander cells, demonstrating that the transfer of phosphorylated compounds between these cell types is not restricted to GCV nucleotides. However, the proportion of araT-5'-triphosphate in bystander cells compared to that in HSV-TK-expressing cells was lower than that for GCVTP, and the amount was not sufficient to decrease survival in the bystander population.     

Contributions of antibody and T cell subsets to protection elicited by immunization with a replication-defective mutant of herpes simplex virus type 1

Replication-defective mutants of herpes simplex virus 1 (HSV-1) elicit immune responses in mice that reduce acute and latent infection after corneal challenge and are protective against development of disease. To understand the basis for the protective immunity induced by this new form of immunization, we investigated the contribution of various components of the immune response to protection against corneal infection and disease. Passive transfer of sera from mice immunized with the replication-defective mutant virus, d301, its parental HSV-1 strain, or uninfected cell lysate was used to examine the role of antibody. Despite posttransfer neutralizing antibody titers equivalent to those in control mice directly immunized with mutant virus, recipients of immune serum showed no reductions in primary replication in the eye, keratitis, or latent infection of the nervous system. However, immune serum protected mice from encephalitis and death. To examine the contribution of T cell subsets to protection, mice were immunized once with mutant virus and then were depleted in vivo of CD4+ or CD8+ T cells prior to corneal challenge. CD4 depletion resulted in higher titers of challenge virus in the eye at 3 to 4 days after challenge compared to control mice. Latent infection of the nervous system was increased by depletion of CD4+ T cells but not by depletion of CD8+ T cells keratitis developed only in a portion of the CD8+ T cell-depleted mice, suggesting that an immunopathologic potential of CD4+ T cells is held in check when immune CD8+ T cells are also present. Taken together, these data support a role for antibody induced by immunization with a replication-defective virus principally in protecting the central nervous system from disease, roles for CD4+ T cells in reducing primary replication in the eye and protecting against latent infection of the nervous system, and a role for CD8+ T cells in regulating the immunopathologic activity of CD4+ T cells.