Human Antibodies against Herpes Simplex Virus 2 Glycoprotein G Do Not Neutralize but Mediate Antibody-Dependent Cellular Cytotoxicity.

Herpes simplex virus 2 (HSV-2) is a sexually transmitted infection affecting 491 million individuals globally. Consequently, there is a great need for both prophylactic and therapeutic vaccines. Unfortunately, several vaccine clinical trials, primarily employing the glycoprotein D of HSV-2 (gD-2), have failed. The immune protection conferred by human anti-HSV-2 antibodies in genital infection and disease remains elusive. It is well-known that gD-2 elicits cross-reactive neutralizing antibodies, i.e., anti-gD-2 antibodies recognize gD in HSV-1 (gD-1). In contrast, anti-glycoprotein G in HSV-2 (mgG-2) antibodies are exclusively type-specific for HSV-2. In this study, truncated versions of gD-2 and mgG-2 were recombinantly produced in mammalian cells and used for the purification of anti-gD-2 and anti-mgG-2 antibodies from the serum of five HSV-2-infected subjects, creating a pool of purified antibodies. These antibody pools were utilized as standards together with purified mgG-2 and gD-2 antigens in ELISA to quantitatively estimate and compare the levels of cross-reactive anti-gD-1 and anti-gD-2 antibodies, as well as anti-mgG-2 antibodies in sera from HSV-1+2-, HSV-2-, and HSV-1-infected subjects. The median concentration of anti-mgG-2 antibodies was five times lower in HSV-1+2-infected subjects as compared with cross-reactive anti-gD-1 and anti-gD-2 antibodies, and three times lower in HSV-2 infected subjects as compared with anti-gD-2 antibodies. The pool of purified anti-gD-2 antibodies presented neutralization activity at low concentrations, while the pool of purified anti-mgG-2 antibodies did not. Instead, these anti-mgG-2 antibodies mediated antibody-dependent cellular cytotoxicity (ADCC) by human granulocytes, monocytes, and NK-cells, but displayed no complement-dependent cytotoxicity. These findings indicate that antibodies to mgG-2 in HSV-2-infected subjects are present at low concentrations but mediate the killing of infected cells via ADCC rather than by neutralizing free viral particles. We, and others, speculate that Fc-receptor mediated antibody functions such as ADCC following HSV-2 vaccination may serve as a better marker of protection correlate instead of neutralizing activity. In an mgG-2 therapeutic vaccine, our findings of low levels of anti-mgG-2 antibodies in HSV-2-infected subjects may suggest an opportunity to enhance the immune responses against mgG-2. In a prophylactic HSV-2 mgG-2 vaccine, a possible interference in cross-reactive immune responses in already infected HSV-1 subjects can be circumvented.

A truncated glycoprotein G vaccine formulated with Advax-CpG adjuvant provides protection of mice against genital herpes simplex virus 2 infection.

Herpes simplex virus type 2 (HSV-2) is a common sexually transmitted disease that affects approximately 500 million individuals globally. There is currently no approved vaccine to prevent HSV-2 infection. EXCT4 is a truncated form of the mature glycoprotein G-2 (mgG-2) that unlike full mature form is secreted by expressing cells enabling it to be rapidly scaled up for production. The current study examined whether EXCT4 immunity in mice could be further enhanced through use of adjuvants. EXCT4 formulated with Advax-CpG adjuvant induced a strong Th1-type immune response characterized by interferon gamma (IFN-γ) and protected animals against a lethal genital challenge with HSV-2. This response was associated with reduced viral load in vaginal washes, spinal cord, and dorsal root ganglia. Together the results provide proof of concept that EXCT4 formulated with Advax-CpG adjuvant is a promising HSV-2 vaccine candidate warranting further investigation.

Vaccination with the Secreted Glycoprotein G of Herpes Simplex Virus 2 Induces Protective Immunity after Genital Infection.

Herpes simplex virus 2 (HSV-2) infects the genital mucosa and establishes a life-long infection in sensory ganglia. After primary infection HSV-2 may reactivate causing recurrent genital ulcerations. HSV-2 infection is prevalent, and globally more than 400 million individuals are infected. As clinical trials have failed to show protection against HSV-2 infection, new vaccine candidates are warranted. The secreted glycoprotein G (sgG-2) of HSV-2 was evaluated as a prophylactic vaccine in mice using two different immunization and adjuvant protocols. The protocol with three intramuscular immunizations combining sgG-2 with cytosine-phosphate-guanine dinucleotide (CpG) motifs and alum induced almost complete protection from genital and systemic disease after intra-vaginal challenge with HSV-2. Robust immunoglobulin G (IgG) antibody titers were detected with no neutralization activity. Purified splenic CD4+ T cells proliferated and produced interferon-γ (IFN-γ) when re-stimulated with the antigen in vitro. sgG-2 + adjuvant intra-muscularly immunized mice showed a significant reduction of infectious HSV-2 and increased IFN-γ levels in vaginal washes. The HSV-2 DNA copy numbers were significantly reduced in dorsal root ganglia, spinal cord, and in serum at day six or day 21 post challenge. We show that a sgG-2 based vaccine is highly effective and can be considered as a novel candidate in the development of a prophylactic vaccine against HSV-2 infection.

The Cholestanol-Conjugated Sulfated Oligosaccharide PG545 Disrupts the Lipid Envelope of Herpes Simplex Virus Particles.

Herpes 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.

Novel rat models to study primary genital herpes simplex virus-2 infection.

In this study we describe that six rat models (SD, WIST, LEW, BN, F344 and DA) are susceptible to intravaginal herpes simplex virus-2 (HSV-2) infection after pre-treatment with progesterone. At a virus dose of 5 × 10(6) PFU of HSV-2, all rat models were infected presenting anti-HSV-2 antibodies, infectious virus in vaginal washes, and HSV-2 DNA genome copies in lumbosacral dorsal root ganglia and the spinal cord. Most of the LEW, BN, F344, and DA rats succumbed in systemic progressive symptoms at day 8-14 post infection, but presented no or mild genital inflammation while SD and WIST rats were mostly infected asymptomatically. Infected SD rats did not reactivate HSV-2 spontaneously or after cortisone treatment. In an HSV-2 virus dose reduction study, F344 rats were shown to be most susceptible. We also investigated whether an attenuated HSV-1 strain (KOS321) given intravaginally, could protect from a subsequent HSV-2 infection. All LEW, BN, and F344 rats survived a primary HSV-1 infection and no neuronal infection was established. In BN and F344 rats, anti-HSV-1 antibodies were readily detected while LEW rats were seronegative. In contrast to naïve LEW, BN, and F344 rats where only 3 of 18 animals survived 5 × 10(6) PFU of HSV-2, 23 of 25 previously HSV-1 infected rats survived a challenge with HSV-2. The described models provide a new approach to investigate protective effects of anti-viral microbicides and vaccine candidates, as well as to study asymptomatic primary genital HSV-2 infection.

Anti-glycoprotein g antibodies of herpes simplex virus 2 contribute to complete protection after vaccination in mice and induce antibody-dependent cellular cytotoxicity and complement-mediated cytolysis.

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 B­cell 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 HSV­2 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.

Glycoprotein G of herpes simplex virus 2 as a novel vaccine antigen for immunity to genital and neurological disease.

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-γ) 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 macrophage-mediated 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.

Seroprevalences of herpes simplex virus type 2, five oncogenic human papillomaviruses, and Chlamydia trachomatis in Katowice, Poland.

Herpes simplex virus type 2 (HSV-2), human papillomaviruses (HPVs), and Chlamydia trachomatis are the most common pathogens causing sexually transmitted infections (STIs). There is limited information about the prevalences of these STIs in Poland. Here, we estimated the occurrence of immunoglobulin G (IgG) antibodies against HSV-2, HPV, and C. trachomatis in 199 blood donors and 110 patients of both genders attending an STI clinic in Katowice in southern Poland. The seroprevalences of HSV-2 were 5% for blood donors and 14% in the STI cohort. The seroprevalences of the five potentially oncogenic HPV types 16, 18, 31, 35, and 51 were 15%, 7%, 5%, 5%, and 17%, respectively, in blood donors and 37%, 8%, 12%, 5%, and 21%, respectively, in the STI cohort. The majority of HPV-infected individuals showed antibodies against more than one type, i.e., had been infected with multiple HPV types. Anti-C. trachomatis IgG antibodies were detected in 6% of blood donors and 13% of individuals attending the STI clinic. The relatively high prevalence of HPV-51 may have implications for future vaccine programs, as the newly introduced HPV vaccines are based on the potentially oncogenic HPV types 16 and 18.

Real-time Taqman PCR targeting 14 human papilloma virus types.

BACKGROUND: Subtyping of human papilloma virus (HPV) may enhance the precision of vaginal cytological assessments and will be important for investigating the effect of the recently introduced vaccine against types 16 and 18. OBJECTIVES AND STUDY DESIGN: To evaluate an in-house real-time PCR targeting HPV types 16-18-31-33-35-39-45-51-52-56-58-59-6-11, by analysing 107 liquid-based cytology specimens representing various degrees of dysplasia. RESULTS: In all, 71 samples were HPV positive, with multiple types present in 37 (52%). Comparison with Roche Linear Array on a subset of 24 of these 71 samples showed a good agreement. One or several types were detected in 17/17 (100%) samples with cervical intraepithelial neoplasia grade 2-3 (CIN 2-3), 16/19 (84%) with CIN 1, 32/43 (74%) with Atypical Squamous Cells of Undetermined Significance (ASCUS), and in 6/28 (21%) with benign cytology. Estimates of mean viral load were lower in CIN 1-3 than in ASCUS ( approximately 4000 vs. approximately 25,000 copies/1000 cells), and clearly lower in samples with benign cytology ( approximately 50 copies/1000 cells). CONCLUSION: The HPV rates in groups with different degrees of dysplasia agrees with previous reports and support a strong link between types 16/18 and severe dysplasia. The high rate of multiple type infection might influence the outcome of HPV vaccination. The possible importance of viral load should be further studied.

Multiphasic encephalomyelitis in a patient with recurrent herpes simplex type 2 meningitis.

We here describe a patient with a history of recurrent HSV-2 meningitis who had been free from symptoms for almost 20 y when he developed an acute encephalomyelitis. The clinical course laboratory and radiological findings support an acute multiphasic disseminated encephalomyelitis induced by a recurrent HSV-2 infection.

Mature glycoprotein g presents high performance in diagnosing herpes simplex virus type 2 infection in sera of different tanzanian cohorts.

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.

CD4(+) T-cell responses to herpes simplex virus type 2 (HSV-2) glycoprotein G are type specific and differ in symptomatic and asymptomatic HSV-2-infected individuals.

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.

Secreted portion of glycoprotein g of herpes simplex virus type 2 is a novel antigen for type-discriminating serology.

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 >or=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.