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#26658766   2015/12/15 Save this To Up

Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses.

We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency.

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#25552056   2015/01/01 Save this To Up

[The lysate and recombinant antigens in ELISA-test-systems for diagnostic of herpes simplex].

The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc.").

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#18006033   2008/02/15 Save this To Up

Disruption of the U(L)41 gene in the herpes simplex virus 2 dl5-29 mutant increases its immunogenicity and protective capacity in a murine model of genital herpes.

The herpes simplex virus 2 dl5-29 replication-defective mutant virus has been shown to induce protective immunity in mice and both prophylactic and therapeutic immunity in guinea pigs. In an attempt to improve the efficacy of dl5-29 we disrupted its U(L)41 gene, producing the triple mutant virus dl5-29-41L. dl5-29-41L has a decreased ability to inhibit host cell protein synthesis and a reduced cytopathic effect on cultured cells. When used to immunize mice, dl5-29-41L elicited significantly stronger neutralizing antibody responses and significantly stronger CD4(+) and CD8(+) cellular immune responses than dl5-29. The enhanced immune responses corresponded with increased protective capacity in a murine model of genital herpes. The protective immunity elicited by either virus was very durable, protecting mice for at least 7 months. Furthermore, we show that cell lysate preparations of both viruses were significantly more efficacious than the corresponding extracellular virus preparations.

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#17942525   2007/12/19 Save this To Up

Common infectious agents in multiple sclerosis: a case-control study in children.

Environmental factors, in particular infections, have been linked with the risk of developing multiple sclerosis (MS). The association of Epstein-Barr virus infection with childhood onset of MS has been recently recognized. As other infections characteristically experienced during childhood have not yet been studied in larger cohorts of paediatric MS, we conducted a study on 152 German children with MS (age at onset <16 years) and matched controls in the hope of gaining evidence for their possible aetiological role in MS. Patterns of antibody responses were determined to a range of infections which, in prior studies principally on adult patients, had revealed possible associations with MS. In this study on children the serology of several infections showed associations with MS. In the exceptional case of Chlamydia pneumoniae there was a significantly higher prevalence of IgM antibody but, more typically, as in the case of influenza A, measles, parainfluenza 2, varicella/zoster viruses and particularly to the herpes simplex virus type 2 (HSV-2) lysate antigen, there were significantly higher concentrations of IgG antibody. Additional investigations, however, make it highly unlikely that a relevant number of children have experienced infections with HSV-2. In general this study supports and emphasizes a complex infectious and immunologic background of MS.

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#15122800   2004/05/03 Save this To Up

Acute retinal necrosis six years after herpes simplex encephalitis: an elusive immune deficit suggested by insufficient test sensitivity.

A patient presented with acute retinal necrosis of the left eye. Demonstration of herpes simplex virus (HSV) DNA in the aqueous humour confirmed the diagnosis. Negative results of HSV type-specific antibody tests based on gG antigens suggested a primary HSV infection. However, the patient had a past history of laboratory-confirmed herpes simplex encephalitis 6 years ago. Using antibody tests based on whole viral lysate antigens, he was seropositive from the onset, and immunoblot testing confirmed a lack of anti-gG reactivity. To be able to assess whether this might be related to the apparent inability of his immune system to suppress clinically symptomatic HSV infection, serial samples were tested by an HSV neutralisation test and a whole-blood flow cytometric assay to determine the frequency of HSV-specific CD4 lymphocytes. However, this did not yield evidence of obvious immunodeficiency; the patient reacted similarly to known positive controls by both assays. Although type-specific HSV serological tests based on gG are generally more specific than those based on whole viral lysate antigens, they have a somewhat lower sensitivity, as a certain percentage of HSV-infected individuals do not develop antibodies against gG, and others may suffer a secondary loss of anti-gG reactivity. Thus there is a risk of missing individual infected patients. Unless this potential problem is recognised, serious consequences might possibly result. We therefore urge virologists and clinicians to exercise great care if highly specific antibody assays based on recombinant proteins are employed.

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#10790110   2000/06/21 Save this To Up

Rapid phenotypic characterization method for herpes simplex virus and Varicella-Zoster virus thymidine kinases to screen for acyclovir-resistant viral infection.

A rapid phenotypic screening method for herpes simplex virus (HSV) and varicella-zoster virus (VZV) thymidine kinase (TK) genes was developed for monitoring acyclovir-resistant viruses. This method determines the biochemical phenotype of the TK polypeptide, which is synthesized in vitro from viral DNA using a procedure as follows. The TK gene of each sample virus strain is amplified and isolated under the control of a T7 promoter by PCR. The PCR products are transcribed with T7 RNA polymerase and translated in a rabbit reticulocyte lysate. Using this method, enzymatic characteristics and the size of the TK polypeptides encoding HSV and VZV DNA were defined in less than 2 days without virus isolation. The assay should be a powerful tool in monitoring drug-resistant viruses, especially in cases in which virus isolation is difficult.

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#10534733   2000/01/12 Save this To Up

Whole cell lysate enzyme immunoassays vs. recombinant glycoprotein G2-based immunoassays for HSV-2 seroprevalence studies.

Seroepidemiology studies of herpes simplex virus type 2 (HSV-2) infections have been difficult to carry out because antibodies to HSV type 1 (HSV-1) show an extensive cross-reactivity with HSV-2 antigens. Many kits available currently are not entirely type specific for serodiagnosis of HSV-2 infections and therefore do not allow reliable discrimination of past exposure to these closely related alphaherpes viruses. Attempts to develop type-specific antigens have focused on the envelope glycoproteins, particularly glycoprotein G (gG). A cross-sectional study was carried out to examine the seroprevalence of antibodies to HSV-2 among healthy university students, using different methods: a whole cell lysate enzyme-linked immunosorbent assay (ELISA), two different ELISAs, and a newly developed immunoblot assay, the last three based on recombinant gG2. HSV-2 prevalence was 24 times higher with the whole cell lysate ELISA (31%; 95% confidence interval [CI]: 27-35%) than the ELISAs and the immunoblot assay based on recombinant gG2 (1.3%; 95% CI: 0.1-2.5%), thus showing the inaccuracy of commercial tests based on whole-antigen preparations for epidemiological studies. Laboratories should be cautious and ensure that commercial tests for HSV typing are based on type-specific glycoproteins.

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#9163470   1997/07/10 Save this To Up

Detection and typing of herpes simplex viruses by using recombinant immunoglobulin fragments produced in bacteria.

Thirty-seven bacterial clones producing human recombinant monoclonal antibody Fab fragments (rFabs) reactive to herpes simplex virus (HSV) antigens were selected from a human combinatorial antibody library constructed in a phage-display vector by a panning procedure against an HSV lysate. Thirty-four of the HSV-specific rFabs were able to specifically recognize HSV-infected cells in indirect immunofluorescence (IF) assays; of these, 25 recognized cells infected by either HSV type 1 (HSV-1) or HSV-2, while 9 recognized only HSV-1-infected cells. One HSV type-common rFab (rFab H37) and one HSV-1-specific rFab (rFab H85) were further evaluated as reagents for viral detection and typing by IF staining in 134 HSV-positive (72 HSV-1 and 62 HSV-2) viral cultures from clinical specimens. The results obtained with these two rFabs were fully consistent with those obtained with a commercial preparation of fluorescein-labeled anti-HSV type-specific murine monoclonal antibodies. The detection sensitivity with the type-common rFab in indirect IF assays was higher overall than that provided by the type-specific murine monoclonal antibodies. Preparations of rFabs suitable for IF staining can be easily and inexpensively obtained in a clinical microbiology laboratory from Escherichia coli cultures. Similar HSV-specific rFabs, therefore, could be advantageous for in vitro diagnostic purposes.

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#8627770   1996/06/21 Save this To Up

Locations of herpes simplex virus type 2 glycoprotein B epitopes recognized by human serum immunoglobulin G antibodies.

Herpes simplex virus type 2 (HSV-2) glycoprotein B (gB-2) gene segments were expressed as recombinant proteins in Escherichia coli. gB-2 recombinant proteins were reacted with human serum immunoglobulin G (IgG) antibodies in Western immunoblot assays. Initially, samples were tested for the presence of HSV-1-specific antibodies and HSV-2-specific antibodies by using HSV-infected cell lysates as antigen targets in Western blot assays. Serum samples that contained HSV-2-specific IgG (n = 58), HSV-1-specific IgG (n = 33), or no detectable HSV antibodies (n = 31) were tested for reactivities with the gB-2 recombinant proteins. In 58 of 58 samples that contained HSV-2-specific IgG, antibodies were present that reacted strongly with a gB-2 amino-proximal segment between amino acids (aa) 18 and 75. Three of 33 serum samples that contained HSV-1- and not HSV-2-specific IgG (as defined by the HSV lysate Western blot assay) reacted with this segment. Both HSV-2 antibodies and HSV-1 antibodies reacted strongly with a carboxy-terminal gB-2 segment between aa 819 and 904; a second minor cross-reactive region was mapped to a gB-2 segment between aa 564 and 626. The gB-2 segment from aa 18 to 75 may constitute a useful reagent for the virus type-specific serodiagnosis of HSV-2 infections. Further studies will be required to determine the relative sensitivities and specificities of the assay for gB-2 aa 18 to 75, HSV gG assays, and HSV lysate Western blot assays for detecting virus type-specific antibody responses in acute and chronic HSV-2 infections.

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#7603340   1995/08/10 Save this To Up

Cloning and characterization of human recombinant antibody Fab fragments specific for types 1 and 2 herpes simplex virus.

Twenty-six bacterial clones producing human recombinant Fab fragments specific for Herpes Simplex virus (HSV) antigens were obtained from an IgG1k human antibody combinatorial library displayed on filamentous phage, following panning against an HSV lysate. All the Fabs reacted against the HSV lysate in enzyme-linked immunosorbent assay and were able to recognize both type 1 and type 2 HSV in an indirect immunofluorescence assay (IFA). DNA sequencing of the heavy chain variable regions showed that these Fabs were different from those already described. One of these Fabs (Fab19) was purified and subjected to further characterization. Purified Fab19 was able to specifically recognize several different HSV-1 and HSV-2 strains (including 2 reference strains and 12 clinical isolates) in IFA. It was also able to neutralize the infectivity of both HSV-1 and HSV-2 strains, although the neutralizing activity was somewhat lower against HSV-2. In fact, 100% neutralization of infectivity was observed at a Fab concentration of 2 micrograms/100 TCD50 for the majority of HSV-1 strains, while a concentration of 8 micrograms/100 TCD50 was needed for 100% neutralization of all the HSV-2 strains tested. Owing to the above properties, Fab19 appears to be useful for diagnostic purposes and might also prove useful for in vivo immunoprophylaxis and therapy of HSV infections.

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