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A major difficulty with diagnostic virus isolation concerns the relative thermolability of certain viruses, e.g. herpes simplex virus type 2, which may, therefore, lose infectivity during transport to the laboratory. This study describes a system of virus isolation and transport, which depends on direct inoculation at the bedside or clinic, to a monolayer or suspension of susceptible cells with subsequent incubation for 10 h at approximately 32 degrees C, whereupon the newly synthesised virus becomes very stable if the cells are subsequently maintained at room temperature. This system was found to increase the sensitivity of isolation of herpes simplex virus, particularly under conditions of asymptomatic virus excretion or if there was significant delay in transportation of clinical samples to the virus laboratory. It is envisaged that this system will allow clinical self-sampling by the patient with application to epidemiological surveys in both the developed and underdeveloped world.

An outbreak of herpes rugbiorum involved nine players including the scrum half and the full back. The infection was characterized by significant constitutional upset with decreased levels of general fitness and match performance for 1-4 months following the outbreak; one player had herpetic lesions on his right eyelid and corneum. Every infected player, 15 non-infected players and five sociosexual contacts received two vaccinations with intracellular subunit vaccine NFU. Ac. HSV-1 (S-MRC5). None of the players or contacts developed cutaneous herpetic recurrence during a follow-up period of 3 years; the player with ocular disease had one recurrence at 30 months following the original episode. These findings encourage consideration of prophylactic or post-exposure vaccination of participants in rugby or other contact sports with this or other appropriate herpes simplex vaccine.

All herpes simplex virus (HSV) infected cell-specific polypeptides (ICSPs) were synthesized in the presence of lithium at a concentration (60 mM) inhibitory to the production of infectious virus. Yields of certain ICSPs were increased and others, in particular glycoprotein C, decreased. HSV DNA synthesis was completely inhibited; synthesis and in vitro activities of HSV DNA polymerase and thymidine kinase were decreased but to a degree insufficient to account for the complete inhibition of HSV DNA synthesis. HSV DNA synthesis was inhibited to an equivalent degree by either incubation with 60 mM-lithium or by potassium starvation; both procedures decreased intracellular potassium by an equivalent amount as adjudged by X-ray microanalysis. We conclude that lithium inhibits HSV DNA synthesis by displacement of potassium from a potassium-dependent biochemical reaction or by other physiological changes brought about by the loss of cellular potassium. The possibility that lithium also directly inhibits a virus replicative event cannot be excluded.

Optimal neutralisation of type 1 herpes simplex virus was obtained by reacting undiluted human serum with virus for 4 h at 37 degrees C, followed by addition of antihuman globulin for 20 min; under these conditions it was possible to detect neutralising antibody activity in 40 of 45 human sera (88%) previously adjudged to be negative by conventional neutralisation tests.

Type-specific antigens for herpes simplex virus type 1 and 2 were prepared by rigorous absorption of cell extracts with heterotypic immune sera. Type-specificity was demonstrated by immunodiffusion and complement-fixation tests against immune sera prepared in rabbits. Specific type 1 complement-fixing reactivity was detected in eleven of fifteen sera from Roman Catholic nuns and in two convalescent sera from patients with recurrent herpes labialis; these sera had been previously shown to contain neutralising and complement-fixing antibody to herpes simplex virus. Three of the non-reacting sera contained low or absent levels of type-common complement-fixing reactivity and other contained no type-specific neutralising antibody. With the exception of three "acute" sera, specific type 2 complement-fixing reactivity was detected in every convalescent or interim serum obtained from patients with a virologically-proven history of type 2 herpes virus infection. It is suggested that complement-fixation testing using these absorbed type-specific antigens preparations may provide a convenient and rapid method for the identification of type-specific antibody in human sera.

The phenomenon of cell transformation by type 2 herpes simplex virus has been investigated. Primary hamster embryo fibroblasts were exposed to type 2 herpes virus under conditions which would restrict or inhibit the lytic events of virus-cell interaction. Cell lines were established by single-cell cloning. There was evidence of altered cell morphology with altered biological activity in terms of longevity and oncogenicity; there was, however, no evidence of virus specific antigen or incorporation of viral nucleic acid into the host cell genome. Virus specific antigen was only detected in the early passages of an uncloned transformed cell line. We are thus unable to confirm previous studies (vide supra) and are obliged to propose a "hit and run" model for in vitro cell transformation by type 2 herpes simplex virus.

A subunit antigenoid vaccine, Ac NFU1 (S-) MRC, was used to prevent primary herpes genitalis in 60 subjects considered to be at risk of this infection. There was no evidence of serious local or general side effects. Neutralising antibody responses were detected in 59% and 90% of subjects receiving the low and high doses of vaccine respectively; immunoprecipitating antibody was detected at a lower frequency, namely in 23% and 43% of subjects receiving the low and high doses respectively. After a mean follow-up period of 18 months none of the vaccinated subjects contracted herpes genitalis after completing the vaccination course.

Repeated intra-vaginal inoculation of mice with inactivated type 2 herpes simplex virus induced cervical carcinoma in approximately 50% of mice. Prior immunization with subunit vaccine Ac NFU1(S-) BHK reduced the frequency of cervical carcinoma to 19%. Inoculation of mice with a control preparation of uninfected cell extract never induced preinvasive or invasive cervical cancer. There was evidence of an antibody response in every vaccinated and/or innoculated animal. Mice developing cervical cancer had a significantly higher antibody titre to type 2 herpes virus than mice not developing cancer. These results are in general accord with sero-epidemiological studies of preinvasive and invasive cervical carcinoma in human subjects and suggests that this experimental model may be appropriate for further investigation of prevention of human cervical cancer by vaccination.

The preparation and early clinical experience with "antigenoid" vaccine Ac NFU1(S-) MRC is described. A neutralising antibody response against both type 1 and type 2 herpes simplex virus was stimulated in 19 of 21 and 25 of 60 patients who received the high and low dosage of vaccine, respectively: immunoprecipitating antibody against type-common virus antigen "band II", a virus antigen of major importance in neutralization of herpes simplex virus, was detected in 12 of 19 and 2 of consorts who received the high and low dosage vaccine, respectively. To date, after a mean follow-up period of one year, none of the 42 consorts have contracted herpes genitalis: following a mean follow-up period of 6 months, only 1 patient vaccinated after the initial clinical episode has reported a recurrence of herpetic disease. These results compare favourably with the rates of consort transmission and first year recurrences in unvaccinated subjects. Seventeen of 24 patients with recurrent herpetic disease reported an "improvement" in terms of modification of the frequency, severity or extent of herpetic lesions. There were no important side-effects from vaccination: most patients experienced a degree of minor local reaction at the vaccination site for 24-72 hours and 3 patients reported a mild transient "flu-like" syndrome which disappeared within 48 hours of vaccination. It is intended to subject these data to the scrutiny of a placebo-controlled trial with an objective system of clinical assessment.

A rabbit and a mouse model of herpes simplex eye disease have been used to evaluate a sub-unit herpes simplex vaccine. Various immunization schedules were investigated. The vaccine was found to stimulate humoral and cellular immune responses and to offer protection against corneal infection with liver herpes simplex virus.

By treatment of herpes simplex virus-infected cells with virus antiserum with or without complement, the yield of infectious extracellular virus was significantly reduced. This was shown to be due to an immune alteration of the cell membrane which inhibited release of virus particles from the infected cells and not due to neutralization; both type-common and type-specific antigens of herpes simplex virus were involved. The phenomenon was also evident with antisera directed against cell determinants. The experimental findings are presented and their significance in the immunological defense mechanisms of the body and in viral immunotherapy is discussed.

Human sera contain antibody (IVR antibody) which will inhibit the release of herpes simplex virus type 1 from virus-infected cells. This antibody activity was removed by adsorption of sera with virus-infected cell extract. There was a positive correlation between IVR and neutralizing antibody activity, particularly when measured by augmented neutralization test; measurement of IVR antibody was equally as sensitive as measurement of neutralizing antibody by augmented neutralization test. IVR antibody levels provided indication of a history of recurrent herpes labialis, the pattern of antibody response following primary herpetic infection, and indication of response to Skinner herpes vaccine in human subjects. It is suggested that consideration should be given to measurement of IVR antibody in both clinical and epidemiological studies of herpes and other virus infections.

A total of 107 women with abnormal cervical smears showing cytological changes consistent with cervical intraepithelial neoplasia (CIN) 1 or CIN 2 were kept under regular cytological, colposcopic, virological and serological surveillance for an average of 18 months (range 9 months-3 years). Regression of the cervical lesion was noted in 31 (29%) and progression to CIN 3 in nine women (8.4%). We found a positive correlation between the presence of type 2 antibody and progression of CIN 1 and 2 to CIN 3 and a negative association with the presence of type 1 antibody and suggest the antibody status of a woman with CIN 1 or CIN 2 may provide a useful basis for follow-up. We found no association between the outcome of the cervical lesion and active infection with herpes simplex or cytomegalovirus or any other infectious agent or sex-related factors.

A mouse model system was used to investigate the preventive efficacy of a subunit herpes simplex virus (HSV) vaccine on the development of HSV induced cervical carcinoma. Ten groups of mice were vaccinated before receiving repeated intravaginal exposure to HSV-type 2 inactivated by ultraviolet irradiation. At 20 months postvaccination, neutralizing antibody activity to herpes simplex viruses was detected in the sera of the mice which had received the highest vaccine dose. Although three experimental mice and one control mouse developed cervical tumours and five mice developed preinvasive malignant changes, 87% of cervices were of normal or koilocytotic appearance on histological examination. There was therefore no evidence from this study that repeated exposure of mouse cervices to inactivated HSV-2 induced a significant incidence of preinvasive or invasive cervical carcinoma.

The replication of type 2 herpes simplex virus in human endocervical tissue in organ culture was investigated. The temporal profile of virus replication was related to the initial virus inoculum; high input inocula induced a rapid increase in virus titre while lower multiplicities induced a more slow-rising increase in virus titre. Our evidence suggested that explants were capable of initiating and supporting virus replication for at least 2 weeks following establishment of the culture. Virus yields were optimal when explants were cultured at 37 degrees and in serum-supplemented medium. Explants also supported the replication of type 1 herpes simplex virus and a "non-human" herpes simplex virus (pseudo-rabies virus). The optimal conditions for replication of type 2 herpes simplex virus in human endocervical explants have been established and will provide a model permitting precise investigation of lytic or other virus-cervical cell interactions and their possible relationship to herpes virus-induced pre-invasive carcinoma of this organ.

Lithium chloride inhibited the replication of type 1 and type 2 Herpes simplex virus at concentrations which permitted host cell replication. Virus polypeptide and antigen synthesis were unaffected while viral DNA synthesis was inhibited. The replication of two other DNA viruses, pseudorabies and vaccinia virus, was inhibited but there was no inhibition of two RNA viruses, namely, EMC and influenze virus.

Neutralizing antibody activity in cervical mucus to type 1 herpes virus was detected in 24 of 28 patients, and to type 2 herpes simplex virus in 18 of 24 patients. The neutralizing antibody activity resisted heat inactivation for 30 minutes at 56 degrees C, was independent of complement and followed first order kinetics. There was evidence of antibody against both virus types in immunoglobulin fractions IgG and IgA, the latter containing approximately threefold greater neutralizing antibody activity per unit of immunoglobulin concentration. Type 1 and type 2 neutralizing antibody activity showed a positive but weak correlation and type 2 neutralizing antibody activity showed a positive but weak correlation and a type-common immunoprecipitin was identified in all concentrated pooled mucus samples. However, type-specific neutralizing antibody against both virus types was identified in pooled mucus samples by heterologous absorption techniques. There was a relatively higher average type 2 neutralizing antibody activity in the mucus than in the serum and there was no correlation between serum and mucus antibody levels for either virus type. These observations support the concept of an independent local antibody system for herpes simplex virus in the uterine cervix.

Systemic vaccination of rabbits with an inactivated type 1 virus subunit vaccine induced humoral and cell-mediated immune responses. Following ocular infection with type 1 herpes virus corneal ulceration and virus excretion were reduced in the vaccinated rabbits.

Neutralizing activity against herpes simplex virus was significantly reduced if initial virus titers were greater than 10(6) PFU/ml; there was no significant neutralization when initial virus titers approached 10(8) PFU/ml. This was a result of utilization of all available antibody by virus particles and 'free' virus antigen and emphasizes the importance of conducting virus neutralization tests under conditions of antibody excess.

Fifty subjects at risk of herpes genitalis received 109 immunizations with Skinner herpes vaccine and were assessed after a follow-up period of 4-48 months, representing a total follow-up period of 694 patient months. There was no evidence of contraction of herpes genitalis in 49 subjects. The risk of virus transmission and rate of contraction of disease was quantified by construction of two functions, namely a unit of exposure risk calculated per year (UYE) and standard contraction rate (SCR); in this study the SCR was 0.02. There was no evidence of significant side-effects from vaccination. Administration of Alhydrogel adjuvant with vaccine induced temporary granuloma formation in most subjects but was only detectable beyond 1 year of follow-up in one subject, in whom a painless swelling of 0.2 cm was detected 3 years after vaccination. There was no evidence of immunological reactivity to host cell or calf serum antigens in any of the subjects vaccinated.

A vaccine against Herpes simplex virus infection was prepared by Nonidet NP 40 and formalin treatment of a type 1, infected-cell extract; virus particles were removed by ultracentrifugation over sucrose. These procedures were not detrimental to the antigenic quality of the vaccine preparation. The vaccine afforded significant protection to experimental type 2 genital herpes virus infection in mice, as adjudged by clinical observations, cytopathological change, and virus yields.

Pre-pubertal immunisation of mice with a formalin-inactivated type 1 and 2 herpes simplex virus vaccine conferred a level of life-long protection against primary type 2 genital infection. Protection levels were better with type 1 vaccine and strikingly influenced by vaccine dosage where a one-hundred-fold reduction from the standard vaccine dosage diminished protection to insignificant levels. Vaccine efficacy was not significantly affected by the method of virus inactivation, the number of immunisations or the age of the mouse at immunisation. Vaccination conferred better protection than previous type 2 genital infection; this may be a consequence of a higher antigenic dose, more acceptable antigenic presentation or to a perversion of the immune response in a latently infected animal to homologous virus challenge.

Patients with cervical intra-epithelial neoplasia had significantly increased neutralising antibody activity to type 2 herpes simplex virus in the cervical mucus. While patients differed from control subjects with respect to their number of sexual partners and socio-economic class, there were significant differences in neutralising antibody activity for case control comparisons within the same number of sexual partners or socio-economic groupings. The results lend support to the putative association between type 2 herpes simplex virus infection and pre-invasive and invasive carcinoma of the uterine cervix.

The incidence of cervical carcinoma, which had been induced by vaginal application of inactivated herpes simplex virus type 2 (HSV-2) with 20% croton oil, was significantly reduced in mice prevaccinated with the Skinner herpes vaccine. There was evidence of an immunological response in both vaccinated and unvaccinated mice.

Vaccine NFU1 (S-L+) MRC was prepared by high multiplicity infection of serum-deprived human embryonic lung (MRC 5) cells with type 1 Herpes simplex virus. The preparative process removed inoculum virus particles and virus DNA while virus particle and DNA synthesis was inhibited by the presence of lithium chloride in the cell culture medium. The vaccine stimulated neutralising antibody in vaccinated mice and provided long-term protection against intra-vaginal challenge with type 2 herpes virus. The safety of the vaccine was confirmed by inoculation into newborn mice and cell lines of human, mammalian, and rodent origin. There was no evidence of cell transformation in vitro or of oncogenicity or teratogenicity in rodent species. It is intended to investigate the efficiency of this vaccine in human subjects.

The serological relatedness of forty-seven strains of type 1 and type 2 herpes simplex virus was investigated by reciprocal and non-reciprocal neutralization kinetics. Early rabbit antisera divided the virus strains into two distinct groups where confident indentification of virus type was possible. Hyperimmune mouse and rabbit antisera did not divide the two virus types into two distinct non-over-lapping groups. The extent of overlap varied with the particular attribute of the virus being studied. The virus types were best discriminated by their neutralizability by type 1 antisera and least well by their neutralizability by type 2 antisera. The results of reciprocal kinetic neutralization test with hyperimmune mouse antisera were analysed by multi-dimensional cluster analysis. Hyperimmune mouse or rabbit antisera could not be discriminated with respect to their immunogenic type by their absolute neutralization rate constants against either type 1 or type 2 virus, but could be distinguished on a group basis by their relative neutralizability against both virus types (antiserum specificity attribute); however, using this latter criterion, the type of immunogen could only be predicted in seven of the forty antisera under test. 'Early' mouse antisera could also be distinguished as groups by their absolute k-values against type 1 herpes virus. Thus, immunogenic identification, on other than a group basis, was unreliable. The specificity of a given serum was inversely related to its titre. There was a positive correlation between the specificity of a given virus strain and of its corresponding antiserum.

Antibody reactivity against herpes simplex virus (HSV) was investigated in 15 subjects who received three subcutaneous immunisations with Skinner HSV vaccine. Humoral antibody responses were detected against type 1 HSV in every subject and against type 2 HSV in all but one subject; immuno-precipitating antibody responses were infrequently detected. There was no antibody reactivity against host-cell (MRC-5), foetal calf serum or rubella virus antigen. None of the vaccinated subjects developed clinical evidence of herpes genitalis.

Patients with abnormal cervical cytology demonstrated a higher prevalence of type-specific complement-fixing antibody to type 2 herpes simplex virus than patients with negative cervical cytology and patients with carcinoma of other body sites. Case-control differences were apparent irrespective of age, socio-economic class and marital status. By contrast, case groups demonstrated a lower prevalence of subjects with type 1 specific antibody. This raises the possibility that pre-adolescent exposure to type 1 herpes simplex virus may offer some measure of protection against pre-malignant and malignant cervical pathology.

Twenty-seven per cent of 70 patients with a history of recurrent herpes genitalis but no concomitant history of recurrent oral or peri-genital disease, had no detectable neutralising antibody against type 1 or type 2 herpes simplex virus; the prevalence and levels of neutralising antibody were similar to 53 patients with no history of herpetic disease and significantly lower than 67 patients with a history of recurrent herpes genitalis in association with oral or peri-genital disease all of whom had neutralising antibody against both virus types. There were similar differences between groups for immunoprecipitating antibody where 80% of patients were herpes genitalis alone had no detectable immunoprecipitating antibody. The results indicate that the failure to detect immunising and immunoprecipitating antibody in an individual's serum is compatible with a long and even severe history of recurrent herpes genitalis and consequently that the development of neutralising antibody does not necessarily indicate an episode of primary herpetic disease.

Bovine mammillitis virus (BMV) cross-reacted in neutralization and radioimmune assay with herpes simplex virus (HSV) and pre-immunization with BMV protected against challenge by type 2 HSV. There was no evidence to suggest a pathogenic role for BMV as adjudged by a literature search or field enquiry and BMV specific antibody was not detected in 21 human sera or in four sera from personnel engaged in research with BMV; in addition there was no replication or antigen synthesis by BMV in explants of human tissue or cell lines of human origin. It is proposed that BMV might provide an alternative vaccine against HSV infections of human subjects.