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bHerpes simplex virus (HSV) and many other viruses, including HIV, initiate infection of host cells by binding to glycosaminoglycan (GAG) chains of cell surface proteoglycans. Although GAG mimetics, such as sulfated oligo-and polysaccharides, exhibit potent antiviral activities in cultured cells, the prophylactic application of these inhibitors as vaginal microbicides failed to protect women upon their exposure to HIV. A possible explanation for this failure is that sulfated oligo-and polysaccharides exhibit no typical virucidal activity, as their interaction with viral particles is largely electrostatic and reversible and thereby vulnerable to competition with GAG-binding proteins of the genital tract. Here we report that the cholestanol-conjugated sulfated oligosaccharide PG545, but not several other sulfated oligosaccharides lacking this modification, exhibited virucidal activity manifested as disruption of the lipid envelope of HSV-2 particles. The significance of the virus particle-disrupting activity of PG545 was also demonstrated in experimental animals, as this compound, in contrast to unmodified sulfated oligosaccharide, protected mice against genital infection with HSV-2. Thus, PG545 offers a novel prophylaxis option against infections caused by GAG-binding viruses. Since the original finding of WuDunn and Spear (1) that ubiquitous and negatively charged glycosaminoglycan (GAG) chains of cell surface proteoglycans provide the binding sites for attachment of herpes simplex virus (HSV) to cells, these oligosaccharides have been reported to assist infection of cells by a number of different viruses, including respiratory syncytial virus (RSV) (2, 3), HIV-1 (4), and Ebola virus (5). In HSV, glycoprotein C (gC) (6) and/or gB (7) mediates virus binding to cell surface GAGs. Sulfated polysaccharides and other polysulfonated compounds that mimic the structure of GAG chains are well-known inhibitors of the virus-GAG interaction in cultured cells (3,8,9). Due to extensive sulfonation, these compounds efficiently outcompete the binding of cell surface GAGs to the viral attachment proteins, thus preventing infection of cells. Notably, the same GAG mimetics can inhibit infectivity of different 9). In spite of potent antiviral activity in cultured cells, these inhibitors, i.e., cellulose sulfate, carrageenan, and PRO2000 (sulfonated poly-naphthalene), failed to protect women against contraction of HIV when tested as intravaginal virucides in several large clinical trials (10-12). Furthermore, there was an indication that one of these GAG mimetics, i.e., cellulose sulfate, increased the risk of contracting HIV (10). Although it is unclear why these compounds lack protective effects in humans (12, 13), some intrinsic features of the virus-GAG interaction, such as reversibility of the binding, may help to elucidate this issue. Virus binding to ubiquitous cell surface components, such as GAGs or sialic acid, has to be neatly balanced to avoid redundant dead-end interactions resulting in trapping of viral particles. Some...

The envelope glycoproteins of herpes simplex virus 1 (HSV-1) and HSV-2, with the exception of glycoprotein G, elicit cross-reactive B-and T-cell responses. Human vaccine trials, using the cross-reactive glycoproteins B and D, have shown no protection against genital HSV-2 infection or disease. In this study, the mature form of glycoprotein G (mgG-2) of HSV-2 was used for immunization of mice, either alone or in combination with adjuvant CpG, followed by an intravaginal challenge with a lethal dose of a fully virulent HSV-2 strain. Mice immunized with mgG-2 plus CpG showed low disease scores and a significantly higher survival rate (73%) than mice immunized with mgG-2 alone (20%) or controls (0%). Accordingly, limited numbers of infectious HSV-2 particles were detected in the spinal cord of mice immunized with mgG-2 plus CpG. The observed protection was associated with a gamma interferon (IFN-␥) response by splenic CD4 ؉ T cells upon antigen restimulation in vitro and in vaginal washes 1 day postinfection. The majority of sera collected from mice immunized with mgG-2 plus CpG showed macrophagemediated antibody-dependent cellular cytotoxicity and antibody-dependent complement-mediated cytolysis, while no neutralization activity was observed. In conclusion, we have shown that immunization with the type-specific mgG-2 protein in combination with CpG could elicit protective immunity against an otherwise lethal vaginal HSV-2 challenge. The mgG-2 protein may therefore constitute a promising HSV-2 vaccine antigen to be considered for future human trials.

T-cell recognition of the secreted and membrane-bound portions of the herpes simplex virus type 2 (HSV-2) glycoprotein G (sgG-2 and mgG-2, respectively) was compared in symptomatic and asymptomatic HSV-2-infected individuals and in HSV-2-seronegative controls and the responses with HSV-1 glycoproteins C and E (gC-1 and gE-1) were compared. CD4+ T cells from HSV-2-infected individuals specifically recognized both sgG-2 and mgG-2, whereas HSV-1-infected and HSV-seronegative controls did not respond to these glycoproteins. The responses to gC-1 and gE-1, on the other hand, were not type specific, as blood mononuclear cells from both HSV-1-and HSV-2-infected individuals responded in vitro. There was an association between the status of the infection (symptomatic versus asymptomatic) and the CD4 + T-cell responsiveness. Symptomatic HSV-2-seropositive individuals responded with significantly lower Th1 cytokine production to sgG-2 and mgG-2 than did asymptomatic HSV-2-infected carriers, especially within the HSV-1-negative cohort. No differences in T-cell proliferation were observed between asymptomatic and symptomatic individuals. The results have implications for studies of HSV-2-specific CD4 + T-cell reactivity in general and for analysis of immunological differences between asymptomatic and symptomatic individuals in particular. INTRODUCTIONHerpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that infects the human genital tract mucosa and is the most common cause of genital ulcer disease in humans (Ahmed et al., 2003;Kinghorn, 1994). The prevalence of HSV-2 antibodies varies depending on the population studied. In the 1990s, 16-33 % of pregnant Swedish women were found to be HSV-2 seropositive (Forsgren et al., 1994;Persson et al., 1995). Most individuals infected with HSV-2 are asymptomatic (Koutsky et al., 1990). However, after information and counselling, some 'asymptomatic' individuals may become aware of having recurrent genital symptoms corresponding to herpetic infection (unrecognized symptomatic genital herpes), although 20-30 % of HSV-2-seropositive individuals seem to remain without symptoms (Frenkel et al., 1993; Langenberg et al., 1989). Both symptomatically and asymptomatically infected individuals shed virus and can thus transmit the disease (Koelle & Wald, 2000;. Overall, HSV-2 is shed for 1-3 days per month (Krone et al., 2000;Wald et al., 1997Wald et al., , 2002 and in 33-68 % of cases this shedding occurs in the absence of concomitant lesions (Krone et al., 2000;Wald et al., 1995).It is not known why genital HSV-2 infection is asymptomatic in some individuals and symptomatic in others, or why the frequency and severity of recurrences vary among symptomatic patients. Prior HSV-1 infection does not protect against HSV-2 infection, although it has been shown to increase the likelihood of asymptomatic HSV-2 seroconversion by a factor of 2?6 (Langenberg et al., 1999). Other factors known to enhance both the incidence and the severity of HSV-mediated disease, as well as the frequenc...

Herpes simplex virus type 2 (HSV-2) is a common sexually transmitted infection in sub-Saharan Africa. Glycoprotein G (gG) of HSV-2 elicits a type-specific antibody response and is widely used for serodiagnosis. gG is cleaved into a secreted portion (sgG-2) and a highly O-glycosylated mature portion (mgG-2). The performances of these two native immunosorbent purified antigens were compared in an enzyme-linked immunosorbent assay (ELISA) format with a commercially available assay (FOCUS2) using sera from blood donors (n ‫؍‬ 194) and individuals (n ‫؍‬ 198) with genital ulcer disease (GUD) from Tanzania. Discordant results were resolved by Western blotting. The HSV-2 seroprevalence for blood donors was estimated as 42%, and that for the GUD cohort was estimated as 78%. The prevalence increased significantly with age for both cohorts and was higher among human immunodeficiency virus (HIV)-positive individuals than among HIV-negative subjects. In the GUD cohort with a high HSV-2 prevalence, all three assays showed statistically similar performances, with sensitivities between 97% and 99% and specificities in the range of 86% to 91%. In contrast, among blood donors with a lower seroprevalence, the mgG-2-based ELISA presented significantly higher specificity (97%) than the sgG-2 ELISA (89%) and FOCUS2 (74%). Overall, the mgG-2 ELISA gave a high performance, with negative and positive predictive values of 96% for blood donors and a negative predictive value of 95% and a positive predictive value of 97% for the GUD cohort. We conclude that native purified mgG-2 showed the highest accuracy for detection of HSV-2 in patient sera from Tanzania and is therefore suitable for seroprevalence studies as well as in clinical settings.Herpes simplex virus type 2 (HSV-2) infections are common and have spread worldwide, with a reported variation in seroprevalence ranging from less than 1% in children to more than 80% in selected adult populations (13,23,27). After primary infection, HSV-2 establishes latency in sensory ganglia, and after reactivation, HSV-2 can be transmitted by clinical lesions or via asymptomatic shedding (33,35). HSV-2 is sexually transmitted and is the most common cause of genital ulcer disease (GUD) in developing countries (1,8,22). The burden of sexually transmitted diseases (STDs) is high in sub-Saharan Africa, and a major problem is that HSV-2 infection facilitates transmission of human immunodeficiency virus (HIV). It has been estimated that the risk of acquiring HIV is doubled for HSV-2-infected individuals (34), and HSV-2-positive patients present higher HIV viral load than HSV-2-negative HIV-infected patients (26). These data emphasize that identification of HSV-2-infected individuals is important not only for control of HSV-2 transmission but also as a strategy for HIV prevention.The definite diagnosis of HSV-2 infection is achieved by virus isolation (VI) or by the PCR technique using samples from clinical lesions. Both of these methods present high specificity, and PCR is the most sensitive. However, t...

The secreted portion of glycoprotein G (sgG-2) of herpes simplex virus type 2 (HSV-2) was evaluated as a novel antigen in an enzyme-linked immunosorbent assay (ELISA) format for detection of type-specific immunoglobulin G (IgG) antibodies in HSV-2-infected patients. The results were compared with those obtained by a commercially available assay, the HerpeSelect 2 ELISA (the FOCUS2 assay). Five different panels of sera were analyzed: panel A consisted of 109 serum samples from patients with a culture-proven HSV-1 infection that were Western blotting (WB) negative for HSV-2; panel B consisted of 106 serum samples from patients with a culture-proven recurrent HSV-2 infection that were WB positive for HSV-2; panel C consisted of 100 serum samples with no detectable IgG antibodies against HSV-1 and HSV-2; panel D consisted of 70 HSV-2 negative "tricky" serum samples containing antinuclear IgG antibodies or IgM antibodies against other viruses or bacteria; and panel E consisted of consecutive serum samples from 21 patients presenting with a first episode of HSV-2-induced lesions. When sera in panels A to C were analyzed, the sgG-2 ELISA and the FOCUS2 assay both showed sensitivities and specificities of >98%. In total, among the samples in panel D, 13 serum samples (19%) were false positive by the FOCUS2 assay and 1 serum sample (1.4%) was false positive by the sgG-2 ELISA. When the sera in panel E were analyzed, the sgG-2 ELISA detected seroconversion somewhat later than WB or the FOCUS2 assay did. We conclude that sgG-2 induces an HSV-2 type-specific antibody response and can be used for type-discriminating serology.

We investigated the role of antibodies against the mature portion of glycoprotein G (mgG-2) of herpes simplex virus 2 (HSV-2) in protective immunity after vaccination. Mice were immunized intramuscularly with mgG-2 and oligodeoxynucleotides containing two CpG motifs plus alum as adjuvant. All C57BL/6 mice survived and presented no genital or systemic disease. High levels of immunoglobulin G subclass 1 (IgG1) and IgG2 antibodies were detected and re-stimulated splenic CD4+ T cells proliferated and produced IFN-γ. None of the sera from immunized mice exhibited neutralization, while all sera exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (ACMC) activity. Passive transfer of anti-mgG-2 monoclonal antibodies, or immune serum, to naive C57BL/6 mice did not limit disease progression. Immunized B‑cell KO mice presented lower survival rate and higher vaginal viral titers, as compared with vaccinated B-cell KO mice after passive transfer of immune serum and vaccinated C57BL/6 mice. Sera from mice that were vaccinated subcutaneously and intranasally with mgG-2 presented significantly lower titers of IgG antibodies and lower ADCC and ACMC activity. We conclude that anti-mgG-2 antibodies were of importance to limit genital HSV‑2 infection. ADCC and ACMC activity are potentially important mechanisms in protective immunity, and could tentatively be evaluated in future animal vaccine studies and in clinical trials.

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