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 ration and was found to be optimal at a ration of 10–20. The fixed HSV-infected cells could be stored at 6°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°C for several months, are a good alternative to purified HSV-1 virions in the LR test

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

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

Antibodies against synthetic peptides of herpes simplex virus type 1 glycoprotein D and their capability to neutralize viral infectivity in vitro.

Peptides corresponding to residues 1-13, 9-21, 18-30, 82-93, 137-150, 181-197, 232-243, 235-243, 267-281, 271-281 and 302-315 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were chemically synthesized. These peptides were coupled to carrier proteins, and the resulting conjugates were used to immunize rabbits. An enzyme-linked immunosorbent assay was used to determine antipeptide antibody titers in serum collected after immunization. All peptides appeared to be immunogenic in rabbits. Western immunoblot analysis with detergent extracts of HSV-1-infected Vero cells showed that antibodies against each of the peptides were able to react with the parent glycoprotein under denaturing conditions. Antisera against peptides 1-13, 9-21, and 18-30 neutralized HSV-1 infectivity in vitro, peptide 9-21 being the most successful in this respect. Immunization with a mixture of peptides 9-21 and 267-281 yielded antisera which reacted strongly with glycoprotein gD in Western blot analysis and showed a more solid virus-neutralizing activity in vitro