Fixed herpes simplex type 1-infected cells as antigen for in vitro lymphocyte proliferation.

Fixed herpes simplex virus type 1 (HSV-1)-infected Vero cells were used as antigen in the in vitro lymphocyte reactivity (LR) test and compared with extracellular HSV, HSV-infected cell extract and purified virions. The highest LR was measured after an incubation period of lymphocytes with the fixed HSV-infected Vero cells of 5-7 days. The LR appeared to be dependent on the lymphocyte to fixed HSV-infected cell ratio and was found to be optimal at a ratio of 10-20. The fixed HSV-infected cells could be stored at 6 degrees C without detectable loss of LR. Addition of high-titered anti-HSV pooled serum to the lymphocyte cultures with the fixed HSV-infected cells as antigen inhibited the LR. The highest reactivity was found using HSV-negative pooled serum. Lymphocytes from seropositive donors were stimulated by the fixed HSV-infected cells and the purified virions. LR to extracellular HSV and an extract of HSV-infected cells were negative for 5 and 2 out of 13 seropositive donors, respectively. Lymphocytes from seronegative donors were not stimulated by any of the HSV-antigen preparations. Fixed HSV-infected cells, which have the advantage that they are easy to prepare and can be stored at 6 degrees C for several months, are a good alternative to purified HSV-1 virions in the LR test.

[The global eradication of polio by the year 2000]. / Polio de wereld uit, rond het jaar 2000.

The World Health Organization wants to attain global eradication of poliomyelitis in the year 2000. In all countries commissions are installed to document polio-free certification. Four activities are considered vital by the WHO to achieve this goal: (a) strict surveillance of all suspected cases of poliomyelitis, (b) close clinical and virological examination of all children younger than 15 years with acute flaccid paralysis (AFP), (c) laboratory surveillance of poliovirus isolates and (d) surveillance of sewage. The Netherlands has been selected as example country for the certification process. However, the AFP surveillance is not yet optimal, therefore all physicians are asked to join in the effort.

Age at first pregnancy and the viral etiology of cervical neoplasia.

In a retrospective study of cervical neoplasia, the relative risk estimate (with 95% confidence limits) of a first pregnancy before 22 years of age was 2.6 (1.41;5.12), with regard to the herpes simplex virus (HSV) infection 6 (2.05;23.81) and with regard to the cytomegalovirus (CMV) infection 2 (1.07;3.85). There was no relation between gravidity and cervical neoplasia. After eliminating the confounding effect of the HSV and the CMV infection, the relative risk for cervical neoplasia of a first pregnancy before 22 years of age was estimated to be 2.17 (1.24; 3.80). There was no evidence of a tumor-promoting role of low age at first pregnancy in the possible neoplastic outcome of the HSV and CMV infections. The association between low age at first pregnancy and cervical neoplasia presumably results from the association between a low age at first pregnancy and the occurrence of a causal infectious agent other than HSV and CMV.

The negative association between a history of recurrent herpes labialis and cervical neoplasia.

We considered the possibility that herpetic recurrences and herpes virus associated neoplasia are mutually exclusive disorders because they are expressions of different herpes virus-host relationships. We assumed that the human body copes with orofacial and genital herpes infections in the same manner. In our retrospective study, the relative risk of a history of fever blisters for cervical neoplasia was estimated to be 0.49, with 0.34 and 0.69 as the limits of the 95% confidence interval. It is suggested that recurrent herpes labialis is presumably a determinant of an effective immune response in general.

Detection of viral antigens in cerebrospinal fluid of rabbits with experimental herpes simplex virus type 1 encephalitis.

We investigated several methods for the rapid diagnosis of herpes simplex virus induced encephalitis in a rabbit model. The corneas of twenty-two rabbits were infected with herpes simplex virus type 1 (HSV-1) and diagnosis of Herpes encephalitis was made by virus isolation, immunofluorescent and peroxidase staining of brain biopsies, demonstration of anti-HSV IgM in cerebrospinal fluid (CSF) and by an indirect enzyme-linked immunosorbent assay (ELISA), designed for detection of viral antigens. With the last method we were able to demonstrate viral antigens in cerebrospinal fluid six days post infection, before clinical signs of encephalitis appeared. In three rabbits this was before anti-HSV IgM appeared in the CSF. Virus was isolated from brain samples of 67 per cent of the animals which died from Herpes encephalitis. Nine rabbits received cortisone before infection, resulting in markedly lower antibody titers and a higher lethality, 77 per cent, as compared to 46 per cent in nontreated rabbits. For rapid diagnosis of Herpes encephalitis in rabbits, demonstration of herpes simplex virus antigens in CSF by means of an indirect ELISA is superior to the other methods investigated.

Differences in antigenic properties of Fc-binding activity during infection with herpes simplex virus type 1.

The antigenic properties of the Fc receptor induced by herpes simplex virus type 1 (HSV-1) were studied with anti-HSV F(ab')2 and pFc' from infected rabbits. It appeared that the HSV-induced Fc-binding receptor had different antigenic characteristics at different times after infection. The Fc receptor present early in the infection (0.5 hours), during the adsorption period, most probably is the result of a fusion event between the virus envelope and the infected cell. We found that this Fc receptor reacted with anti-HSV F(ab')2 and thus showed HSV-antigenic properties in such a way that binding of anti-HSV F(ab')2 prevented the binding of pFc' fragments. Later on in the infection (5 hours), the Fc-binding activity present on the surface of the infected cell is the result of newly synthesized and in the plasma membrane integrated polypeptides. The Fc-binding activity present on the cell surface of 5 hours infected cells could not be inhibited by anti-HSV F(ab')2 and did not interfere with the binding of pFc' to the Fc receptor.

Detection of viral antigens in cerebrospinal fluid of patients with herpes simplex virus encephalitis.

Thirty-two cerebrospinal fluid (CSF) samples from eighteen patients with confirmed herpes simplex encephalitis (HSE) were assayed by an indirect enzyme-linked immunosorbent assay (ELISA) for the presence of viral antigens. The results are expressed as an antigen ratio distinguishing between herpes simplex virus (HSV) antigens containing samples and negative samples. Judged by this criterion a positive result was obtained in 33% of the patients. Overall, 25% of the CSF samples from HSE patients were positive. In one out of 33 control patients with other neurological disorders a positive antigen ratio was found. Two or more CSF samples were available from eleven patients. In six of these, the second or later samples showed a decreased antigen ratio when compared to the first CSF sample. An increase of the anti-HSV antibody titer was seen in the CSF of five of these six patients. Five out of six patients with a decreasing antigen ratio had an unfavorable outcome of their encephalitis, while a favorable outcome was seen in four of the five patients with an increasing or steady antigen ratio. A decrease of the antigen ratio in the course of HSE can be explained by the presence of immune complexes in CSF and may indicate a poor prognosis.

T cell responses to synthetic peptides of herpes simplex virus type 1 glycoprotein D in naturally infected individuals.

To locate T cell determinants of glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1), proliferation assays of lymphocytes obtained from 10 healthy HSV-seropositive individuals were performed using 34 overlapping gD peptides as antigens. Despite large differences between individual responses to the peptides both in number of stimulating peptides and gD regions, three regions (1-54, 110-214, and 290-314) induced a response in 50% or more of the HSV-seropositives. T cells were less frequently stimulated by peptides of region 210-294. No correlation was found between serological data and proliferative responses to the peptides. The diversity in T cell response to the peptides suggests a lack of immunodominance, implying that a single peptide/region of gD, or a combination of peptides, will not be sufficient to serve as a basis for a future HSV-1 vaccine.

Isolation by high-performance liquid chromatography and partial characterization of a 57,000-dalton herpes simplex virus type 1 polypeptide.

A Nonidet P-40 extract of HSV-1-purified virions was fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). The first peak fraction eluted at 25% organic solvent. Polyacrylamide gel electrophoresis showed that it contained a 57,000-dalton polypeptide. The polypeptide was characterized by determination of the amino acid composition and the N-terminal amino acid sequence. Adsorption of the detergent extract before RP-HPLC showed that the polypeptide reacted with monoclonal antibodies LP1 directed against herpes simplex virus polypeptide VP-16.

Isolation of detergent-extracted Sendai virus proteins by gel-filtration, ion-exchange and reversed-phase high-performance liquid chromatography and the effect on immunological activity.

Virus envelope proteins were isolated from Triton X-100 extracts of purified Sendai virions by gel-filtration, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC). The fusion protein F, the matrix protein M and the tetrameric and dimeric form of the HN protein were isolated by gel-filtration HPLC with a solvent containing 0.1% sodium dodecyl sulphate. HN and F were also isolated by ion-exchange HPLC with 0.1% Triton X-100 in the eluent. Reversed-phase HPLC was performed on a C1 column with acetonitrile as the organic solvent. Especially the F1 and F2 component of the fusion protein F were obtained in pure form. The immunological activity of the proteins after HPLC was determined with an enzyme-linked immunosorbent assay (ELISA). After gel-filtration and ion-exchange HPLC, proteins still reacted with antiserum to the intact virus while proteins purified by reversed-phase HPLC did not react.

Rapid detection of infectious cytomegalovirus in blood with the aid of monoclonal antibodies.

The recently developed early antigen immunofluorescence (IF) method for the detection of infectious cytomegalovirus (CMV) in clinical specimens has hardly been applied on blood samples. We compared the CMV early antigen detection technique with the conventional cell culture method in 415 different buffy coat samples from 85 different immunocompromised patients. Duplicate coverslips were stained with two different monoclonal antibodies 4-6 days after inoculation. The conventional cultures were examined for typical cytopathic effects (CPE) during 10 weeks. Forty samples from 19 patients were positive by the IF technique, most of them with both monoclonal antibodies. Only 22 of these samples were positive in the conventional cell culture assay, on average after 15.8 days. CMV viraemia was detected exclusively by the IF method in 18 samples, 7 of which were from five patients without any further evidence of an active CMV infection. CMV viraemia was detected exclusively by the CPE method in eight samples, on average after no less than 36.6 days. CMV viraemia was not found in blood samples from 10 patients with laboratory proven active CMV infections and 53 patients without any evidence of an active CMV infection. In our hands the early antigen method for the detection of infectious CMV in blood is nearly as specific (at least 98.1%) and clearly much faster and more sensitive than the conventional cell culture method. The early CMV antigen detection method is therefore a very useful tool for the rapid detection of infectious CMV in blood.

Virus neutralizing activity induced by synthetic peptides of glycoprotein D of herpes simplex virus type 1, selected by their reactivity with hyperimmune sera from mice.

Mice were immunized with synthetic peptides covering the first 56 amino acids of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) and a fusion protein, produced in Escherichia coli, containing the first 55 amino acid residues of gD. It was found that mice immunized with peptides composed of amino acid residues 1 to 13, 18 to 30. 22 to 38 and 38 to 56 of gD were not significantly protected against a lethal challenge with HSV-1. Immunization with peptide 9-21 and the gD fusion protein resulted in significant protection. Antisera, from mice immunized with HSV-1, were investigated for reactivity with a series of 57 overlapping gD peptides covering the entire amino acid sequence, except for the membrane-spanning region. All antisera reacted with peptides 9-21, 10-24, 151-165, 216-232, 282-301 and with peptide 340-354 located in the anchoring region of gD, and 15 other peptides were recognized by at least one antiserum. Twelve peptides (10-24, 151-165, 216-232, 244-267, 260-274, 270-284, 260-284, 282-301, 300-314, 340-354, 348-362 and 355-369) reacted most frequently with the hyperimmune sera from mice and were selected for further study. These were conjugated to bovine serum albumin and used to immunize rabbits. Only antisera against peptide 10-24, which covers the same epitope as peptide 9-21, neutralized HSV-1 in vitro.