Enhancement of protective humoral immune responses against Herpes simplex virus-2 in DNA-immunized guinea-pigs using protein boosting.

Genital Herpes is a common sexually transmitted disease that is caused mostly by Herpes simplex virus type 2 (HSV-2). Its prevalence has increased in developing countries in spite of the availability of valuable antiviral drug therapy. Considering the importance of HSV-2 infections, effective vaccines remain the most likely hope for controlling the spread of HSV diseases. In the present study, the complete HSV-2 glycoprotein D gene was isolated and cloned into different plasmid vectors to construct a DNA vaccine and prepare recombinant subunit vaccines using a baculovirus expression system. The vaccines were tested alone or in combination to evaluate their ability to induce protective immunity in guinea-pigs against genital HSV infections. Immunization elicited humoral responses as measured by neutralization tests and enzyme-linked immunosorbent assay, and immunized animals had less severe genital skin disease as well as reduced replication of the challenging virus in the genital tract during experimental infection. Our results further demonstrate that DNA priming-protein boosting induced a neutralizing antibody titer higher than that obtained with DNA-DNA vaccination. The massive increase of antibody titer following DNA priming-protein boosting might be attributed to a recall of B cell memory.

Production of recombinant gG-1 protein of herpes simplex virus type 1 in a prokaryotic system in order to develop a type-specific enzyme-linked immunosorbent assay kit.

The herpes simplex viruses are important causes of disease worldwide. Herpes simplex virus type 1 (HSV-1) is the primary cause of oral-facial and pharyngeal infections and may cause herpetic whitlow, eye infections as well as severe and sometimes dangerous infections of the eyes and brain. HSV-1 also accounts for 10-15% of all genital herpetic infections. Therefore, laboratory diagnosis of this virus and development of diagnostic serological techniques for HSV-1 is of particular importance. In the present study, pTrc His2A-gG1 plasmid, containing the full-length glycoprotein G (gG) protein, was produced in a prokaryotic system for the first time. Upon confirmation of a 37-kDa gG-1 protein production in a prokaryotic system based on western blotting and monoclonal antibodies, the protein was produced at a large scale and purified by ion-exchange chromatography using DEAE-sepharose. An HSV-1 type-specific diagnostic kit was designed and developed and the specificity and sensitivity of this kit were demonstrated to be 89.5% and 100%, respectively, as compared with a commercially available kit. A significant correlation was shown between the developed kit and the commercial kit.

A serological survey of Herpes Simplex Virus type 1 and 2 immunity in pregnant women at labor stage in Tehran, Iran.

This study was carried out to determine the prevalence of neutralizing antibodies to Herpes Simplex Virus type 1 (HSV-I) and type 2 (HSV-2) in pregnant women at labor stage. Blood samples from umbilical cord of four hundred women aging 16 to 40 years at labor stage were collected. After sera separation quantity of anti HSV-1 and HSV-2 antibodies were measured by serum neutralization test. Antibody quantification was assayed by two fold dilution of sera (from 1/2 to 1/256) with 500 Tissue Culture Infective Dose fifty percent (TCID50) of the HSV-1 and HSV-2, separately. Three hundred sixty three (90.75%) of women had neutralizing antibody against HSV-1. Thirty three (8.25%) tested women were seropositive for HSV-2 antibodies. Our results indicate that there is positive correlation between increase of age and seroprevalence of anti-HSV-2 infection in pregnant women. Furthermore, the pattern of HSV-2 infection is similar with other Sexual Transmitted Disease (STD). These results also show that seroprevalence of anti-HSV-1 antibody in our tested population was remarkable. However, the seroprevalence of anti-HSV-1 antibody in different age groups statistically were not significant. These results also showed that most women before fertility age have infections with HSV-1.

The effect of DNA priming-protein boosting on enhancing humoral immunity and protecting mice against lethal HSV infections.

Herpes simplex virus produces primary and latent infections with periodic recurrency. The prime-boost immunization strategies were studied using a DNA vaccine carrying the full-length glycoprotein D-1 gene and a baculovirus-derived recombinant glycoprotein D, both expressing herpes simplex virus glycoprotein D-1 protein. Immunization with recombinant DNAs encoding antigenic proteins could induce cellular and humoral responses by providing antigen expression in vivo. Higher immune response, however, occurred when the recombinant proteins followed DNA inoculation. While all groups of the immunized mice and positive control group could resist virus challenge, a higher virus neutralizing antibody level was detected in the animals receiving recombinant protein following DNA vaccination.

Development of a multiplex nested consensus PCR for detection and identification of major human herpesviruses in CNS infections.

BACKGROUND: Rapid, sensitive and economical detection and identification of human herpesviruses as causative agents of central nervous system (CNS) infections are of clinical importance. The traditional methods for the detection of herpesviruses in CNS infections all suffer from limitations. PCR has a potential to overcome each of them. OBJECTIVES: The aims of this study were reducing the number of primers in multiplex PCR and increasing the sensitivity of the assay by nested PCR. STUDY DESIGN: A multiplex nested consensus PCR (MNC-PCR) was developed for the simultaneous detection of major human herpesviruses. A pair of conserved primers was designed for detection of HSV-1, HSV-2, CMV and EBV and another pair of conserved primers for nested PCR. For VZV, a different pair of primers was designed and another pair of primers for nested PCR. A reduction in the number of designed primer pairs (from five pairs to two in both stages of PCR) is an advantage in this assay. One hundred forty-seven cerebral spinal fluid (CSF) samples from patients that showed clinical manifestation of CNS infections were tested. Results of MNC-PCR in CSF samples were compared with those of single PCR assay for each individual DNA virus. Sensitivity of the assay was determined with a plasmid containing VZV DNA binding protein gene and another plasmid for HSV-1 DNA polymerase gene. False negative results (due to the presence of inhibitor of DNA amplification in CSF samples) were avoided by the inclusion of beta2-microglobulin primers in the MNC-PCR assay as an internal control. RESULTS: Positive results were obtained in 20 CSF samples (8 HSV-1, 2 HSV-2, 4 CMV, 3 VZV, 3 HSV-1/CMV, CMV/VZV and HSV-1/EBV coinfections). The comparison between single PCR and MNC-PCR showed a marked increase in sensitivity of MNC-PCR test, since six negative samples in single PCR proved positive in MNC-PCR (P<0.005). Sensitivity was determined 1-5 plasmid copies for VZV and 50-100 plasmid copies for HSV-1. CONCLUSIONS: The MNC-PCR assay presented in this study can provide a rapid, sensitive and economical method for detection of viral infections and is applicable to small volumes of CSF samples.

An Accurate Confirmation of Human Immunodeficiency Virus Type 1 (HIV-1) and 2 (HIV-2) Infections with a Dot blot assay Using Recombinant p24, gp41, gp120 and gp36 Antigens.

An immunoblot assay using four recombinant proteins corresponding to human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) gene products was developed to confirm the presence of antibodies to HIV-1 and 2 in sera reactive in screening ELISAs. Serum samples for testing were obtained from healthy seronegative blood donors and from different categories of HIV-infected individuals (asymptomatic HIV-infected, and AIDS). A positive reaction was defined as reactivity against gag (p24) and at least one other env (either gp41 or gp120) HIV gene products; negative result was defined as no reaction with any antigen; and indeterminate result was defined as reactivity with gag (p24) or with env (gp41 or gp120) alone. None of the 180 serum samples from healthy seronegative blood donors gave a positive result, and only 4 of these samples (2.2%) gave indeterminate results. The recombinant HIV Dot blotting assay identified seropositive individuals with a high degree of accuracy; none of the 125 HIV-seropositive subjects had a negative test result. Reactivity with these antigens, demonstrated 100% sensitivity and specificity in distinguishing seronegative from seropositive sera. All seronegative and seropositive samples were tested both with the commercially available ELISA and by Western blot. The recombinant in-house HIV Dot blot assay accurately identified more seropositive and seronegative samples and had fewer indeterminate results than did commercial Western blot (as interpreted by CDC criteria).