key: cord-0006636-8k3wl971
authors: Skinner, G. R. B.; Williams, D. R.; Moles, A. W.; Sargent, A.
title: Prepubertal vaccination of mice against experimental infection of the genital tract with type 2 herpes simplex virus
date: 1980
journal: Arch Virol
DOI: 10.1007/bf01320618
sha: 60ee0e6f4ff94dce64daa2bea069b1f163562380
doc_id: 6636
cord_uid: 8k3wl971

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.

Pre-pube~al 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 striMngly 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.

The feasibility of immunisation against herpes simplex infections was first demonstrated in rabbits by LIPo~uTz (9) . These early studies also provided evidence of the contribution of cell-mediated factors on host immunity and even hinted at possible antigenic distinctiveness between herpes simplex virus strains. More recently, successful immunisation against type 1 herpes virus has been demonstrated with live vaccine in mice (17) and with inactivated vaccine in mice and in rabbits (2, 8) . Successful immunisation against type 2 herpes virus has been demonstrated in mice using a formalin-inactivated type 1 or 2 vaccine (4) and with live type 1 or 2 vaccine where, however, only homologous interactions were investigated (29) . Other studies have suggested that mice immunised with type 1 vaccine were less likely to develop a latent ganglionic type 1, or indeed type 2 herpes virus infection (3, 11) . 0304-8608/S0/0064/0329/$ 02.00 G.R.B. SKINNER, D. tR.. WILLIAMS, A. W. MOLES, and A. SARG~:~T:

There is evidence that herpetic cervieitis, a discommoding venereal disease of increasing prevalence, is associated with pre-invasive and invasive carcinoma of the cervix. This evidence is based on studies of in vitro celt transformation with type 2 herpes simplex virus (5, 12, 22) and on sero-epidemiologieal surveys which have indicated a higher prevalence of neutralizing antibody (13, 1) and type-specific complement-fixing antibody (26) to type 2 herpes virus in patients with pre-invasive and invasive cervical carcinoma ; this latter study also indicated a significantly lower prevalence of type 1 specific antibody in the ease group, suggesting that pro-adolescent exposure to type 1 infections may protect against primary type 2 herpes infection in later life.

Therefore there seems to be a good case for vaccination of female subjects particularly against primary type 2 herpes simplex virus infection. This study has investigated the efficacy of pro-adolescent immunisation of mice using formaldehyde-inactivated herpes simplex type 1 and 2 vaccine preparations. The influence of vaccine dosage, method virus inactivation, virus type and immunisation schedule on vaccine efficacy has been examined and compared with levels of protectioa following "natural" type 2 genital herpes simplex virus infections.

Cells BI-IK-21 (clone 13), a stable line of cells derived from a single clone of baby hamster kidney ceils (10), were used for virus propagation and vaccine production. The cells were maintained in supplemented Eagle's medium (28) containing 10 per cent v/v tryptose phosphate broth and 10 per cent v/v calf serum (ETC).

The HFEM derivative of the Rockefeller strain I-IF (30) and strain 3345, a penile isolate, were used as prototype type 1 and type 2 strains for vaccine preparation and virus challenge (7, t6) . Viruses were titrated by the plaque method of RUSSELL (20) .

Virus Antigen BI-IK-21 cells (10) were infected at high multiplicity (10 PFU/ocll) with type 1 herpes virus and incubated at 37 ° C for 24--36 hours. The infected cells were removed from. the glass, washed, resuspended in saline at a concentration of 107 cells/ml, and disrupted by ultrasonic vibration. As a routine, antigen preparations were tested in immunodiffusior~, against homologous immune sera; under these circumstances they gave multiple precipitin lines. Antigen preparations were stored at --70 ° C.

Virus antigen preparations were thawed and resonicated and formaldehyde was added to a .final eo,~eentration of 0.04 per cent. The antigen preparation was then dialysed against phosphate-buffered saline for 72 hours at 4 ° C, with frequent changes of the dialysing medium. There was no residual virus infectivity following this procedure. For control purposes, a formaldehyde treated uninfected BI-IK21 cell extract was prepared ("control vaccine").

Unless otherwise indicated, Swiss mice at 3 to ~ weeks of age (which, in terms of sexual maturity, corresponds to "adolescence" in these animals) were given a "standard" dose containing the equivalent of l06 infected cells by the subcutaneous route. A second standard dose of vaccine was given after an interval of three weeks.

A cotton pledget was allowed to soak in the virus suspension, appropriately diluted in medium (ETC), and gently introduced into the mouse vagina using small surgical forceps. Mice were routinely challenged with 1 × I0 G PFU of type 2 virus unless otherwise indicated. The pledgers were removed after 16 hours.

Mice were investigated virologically and cytologically at one, two, three and seven days following virus challenge. Specimens for cytologic investigation were obtained by rotating a dry cotton pledger within the mouse vagina. Smears were made and immediately fixed in absolute ethanol.

Having made the cytologic spread, the same cotton pledger was then quickly wetted in sterile medium (ETC), re-inserted into the mouse vagina, twisted for three complete rotations, removed and dropped into 1 ml of medinm. In this way, we attempted to standardize the virus yields obtained by the sampling technique. The pledgetcontaining suspension was frozen and then thawed, sonicated and titrated in BHK 21 cells.

Blood for neutralizing antibody studies was obtained by cardiac puncture of mice under deep ether anaesthesia, tlesidual ether in the serum was blown off with nitrogen. Sera were tested at a five-fold dilution by kinetic neutralisation and "k values" calculated as previously described (25) .

Mortality rates were calculated in terms of mice with cytological or virological evidence of virus infection, thereby excluding animals which were never infected, for example, by immediately extruding the infecting intravaginal pledget. The criteria of virus infection in infected mice were those previously described (31) .

The efficacy of vaccination in pre-sexual maturity mice is shown in Table 1 and Figs. l 4. In mice receiving two immunisations of our "standard vaccine", viz. 106 infected cells equivalent per dose, mortality was reduced by over 50 per cent and average virus yields per mouse were reduced tenfold by the 5th day and Fig. 1 ). The proportion of mice with specific herpetic cytopathie effects (CPE) was consistently lower in mice immunised with the standard vaccine ( Fig. 2) and, similar to virus yields, was most striking after 5 days of infection. The proportion of mice with a non-specific inflammatory response and the extent or degree of specific CPE, quantitated in terms of "plusses" (vide s~pra) wa.s also lower in immunized mice, although with these criteria, the differences remained similar at the various times of testing following infection (Figs. 3 and 4 , respectively).

LOG m Fig. 2 ) and with non-specific inflammatory response (Fig. 3) . In terms of mortality, however, there was signifiea,nt protection with only the type 1 vaccine at 10 -2 dosage (p ~0.01; Table 1 ).

Protection levels provided by virus antigen inactivated by methanol or acetone was not significantly different from formaldehyde-inactivated antigen (data not presented).

Mice were protected for at least 2 years following vaccination. The percentage mortalities in mice immunised with standard type 1 and 2 vaccine were significantly less than in control mice (p ~0.01 ; Fig. 5 ). Virus yields from mice immunized with standard type 1 vaccine were significantly less than in control mice at all times (p~0.01); however, with the standard type 2 vaccine, differences were significant at only 3, 6 and 12 months (9 <:0.05), Minor degrees of protection were observed following vaccination with the lower vaccine dosages (Figs. 5 and 6 ). 

There was no evidence that three doses of any vaccine provided better protection than the usual regime of two vaccinations. Indeed, virus yields by the 7th day of infection were terffold greater following three standard immunisations ( Table 2 ). With one standard immunisation, while the level of protection was less than obtained with two standard immunisations, particularly in terms of average virus yields, it remained, on all criteria, significantly greater than in control mice ( Table 2) .

The influence of age or, more specifically sexual maturity on the efficacy of vaccination was investigated by comparing the results with a group of 20 mice vaccinated in adulthood according to our usual immunization regime. There was no evidence of improved protection; mortality was 38 per cent, 20 per cent of mice had evidence of specific viral CPE by the 7th day of infection and average virus yields were 3 PFU per mouse (el . Tables 1---3) .

In/ection Vaccinated and unvaccinated mice which had survived previous challenge with type 2 herpes virus were re-challenged. Vaccination was clearly the most important factor in determining the level of protection. Previous type 2 genital infection did not improve the degree of protection in vaccinated mice and offered little or no protection at all in unvaccinated mice (Table 3) .

Twenty four-week old mice were investigated for neutralizing antibody against herpes simplex viruses following two standard type 1 vaccinations. Every mouse developed neutralizing antibody with a mean k value of 0.35i0.02 against type 1 virus and 0.19:j:0.014 against type 2 virus. 

The results indicate that mice can be protected from primary type 2 genital infection by pre-pubertal vaccination with a formalin-activated type 1 vaccine (Table 1, Figs. 1--4) . This finding is generally consistent with sero-epidemiologieal studies by RAWLS et al. (19) and N~As et al. (13) in which pre-adolescent type 1 infection appeared to impart a measure of protection against primary type 2 genital infections. It was encouraging that protection, which in general terms is considered to be more short-lived with inactivated than live virus vaccines, continued until two years following vaccination (Figs. 5 and 6 ). Investigation of longer vaccination-challenge intervals was precluded by the natural average twoyear life span of our strain of mice. G.R.B. SKJNNER, D. R. WI/uLIA~S, A. W. MOLES, and A. SAImEN~ :

Vaccination with one-hundredfold and the ten-thousand-fold vaccine dosages, 104 and t0 ~ infected cells equivalent respectively, reduced the degree of protection by all criteria (Table t, Figs. 1-4) . This was particularly apparent following long vaccination, challenge intervals where no significant protection was apparent with these lower vaccine dosages (~Sgs. 5 and 6). The dosage is clearly critical and, in the longer term, the vaccination of human subjects emphasises the necessity to investigate dose-protection relationships in larger animals, for example, primate species. Similar considerations obtain with respect to the number of vaccinations where there seemed, at least in our mouse experimental system, no indication to exceed two standard vaccinations ( Table 2) .

There is little doubt that type 1 standard vaccine offered a higher level of protection than type 2 standard vaccine (Tables 1 and 2, Fig. 6 ). While this is unexpected from the "direction" of the one-way cross-neutrMisation reaction between type 1 and 2 herpes simplex virus (14, 15, t6) , in vitro neutralization tests do not measure cytolytie or opsonic humorM antibody or indeed the variety of cell-mediated immune factors which may be of paramount importance in protection from herpetic infections (18, 6) .

It was possibly surprising that vaccination conferred a significantly greater level of protection against challenge with type 2 virus than did a previous episode of homologous type 2 genital infection where local antibody factors might also contribute to protection against intravaginal virus ehMlenge (Table 3 , Figs. 5 and 6). While it is impossible to quantify the precise virus immunogenie dose to the mouse following contraction and recovery from a type 2 genital infection, it is probably less than is contained in two subcutaneous formaldehyde stabilised vaccinations each containing the antigenic content equivalent of 106 infected cells per mouse; indeed, it was quite clear that reduction of vaccine dosages by onehundredfold dramatically reduced protection levels (Tables 1 and 3, Figs. 1--6) . A second explanation merits speeulation, namely, that establishment of latent ganglionic virus involvement following a live type 2 genital infection inhibits further immunological response to challenge with the (particularly) homologous virus on the basis that "familiarity breeds immunologicM contempt". This concept is presently under investigation.

The vaccine preparation investigated in this study is a "crude" virus antigen preparation and is not under consideration for vaccination of human subjects. However, the operative antigen stimulating protective immunity in human subjects is probably the monopreeipitin antigen "Band II" (24, 21) and this antigen will necessarily be represented in even the most refined prospective vaccine for human subjects against herpes simplex infections. On this account, as far as can be judged from experiments in a rodent species, pre-pubertal vaccination would seem a feasible undertaking and the development of an inactivated virus particle and virus DNA free sub-unit vaccine is presently under investigation (27, 23) .

We wish to "~hank Professor J. Newton, Department of Obstetrics and Gynaecology, University of Birmingham, for generous provision of facilities, and Mr. Kim Ward for technical assistance in the preparation of cytologic material. Mr. A. Moles was in receipt of Mt~C Grant G 975/935.

Seroepidemiologie studies of herpes virus type 2 and carcinoma of the cervix IV

Immunization of mice with inactivated herpes simplex virus

Effect of immunization on the development of latent ganglionic infection in mice challenged intravaginally with herpes simplex virus types 1 and 2

Immunogenic properties of formalin herpes antigen prepared from cell cultures

Oncogenie transformation of hamster cells after exposure to herpes simplex virus type 2

Host defense mechanisms against herpes simplex vimxs. II. Protection conferred by sensitized spleen cells, g. inf

Differentiation between type 1 and type 2 strains of herpes simplex virus by an indirect immunofluorescent technique

Protection from oral herpes simplex virus infection by a nucleic acid-free virus vaccine

Untersuchungen fiber die Atiologie der Krankheiten der Herpesgruppe

Polyoma transformation of hamster cell clones--an investigation of genetic factors affecting cell competence

Efficacy of herpes simplex virus type 1 immunization in protecting against acute and latent infection by herpes simplex virus type 2 in mice

Transformation of rat embryo cells by temperature sensitive mutants of herpes simplex virus

Antibodies to Herpesvirus hominis types 1 and 2 in humans. I. Patients with genital infections

Infection with herpes simplex viruses t and 2

A serological study of Herpesvirus hominis strains by mieroneutralisation tests

Herpes simplex viruses: Discrimination of types and correlation between different characteristics

Latent infection of sensory ganglia with herpes simplex virus: efficacy of immunisation

Mechanism of immunologic resistance to herpes simplex virus 1 (HSV-1) infection

Genital herpes in two social groups

A sensitive and precise plaque assay for herpes virus

The role of type-specific and cross-reacting structural antigens in the neutralisation of herpes simplex virus types 1 and 2

Transformation of primary hamster embryo fibroblasts by type 2 simplex virus: evidence for a 'hit-and-run' mechanism

The preparation, efficacy and safety-of a herpes simplex vaccine for human subjects

Precipitating antibodies to herpes simplex virus in human sera: a prevalence of antibody to common antigen (Band II)

Sero-relatedness of t.ype 1 and type 2 herpes simplex virus; type-specificity of antibody response

Prevalence of type-specific antibody against type 1 and type 2 herpes simplex virus in women with abnormal cervical cytology; evidence towards pre-pubertal vaccination of sero-negative female subjects

Preparation and efficacy of an inactivated sub-unit vaccine (NFU BHK) against type 2 herpes simplex virus infection

Interaction of herpes virus and HeLa cells: comparison of cell killing and infective centre formation

Effect of immunization on acute and latent infection of raginG-uterine tissue with herpes simplex virus types t and 2

1%ecombination with herpes simplex virus

Cytological evaluation of experimental type 2 herpes simplex infection in mice

Received October 22, 1979