FEMS Microbiology Immunology 47 (1988) 157-162 157 Published by Elsevier

FIM00025

Vaccine induced HSV 1 antibodies fail to correlate with protection against HSV 2 in guinea pigs

M.J. Welch, P.H. Ridgeway and R.J. Phillpotts

Dioision of Biologics, Centre for Applied Microbiology and Research, Porton Down, Wiltshire SP4 0JG, UK

Received 5 May 1988 Revision received 4 July 1988 Revision accepted 5 July 1988

Key words: Herpesvirus; HSV 1; HSV 2; Herpes vaccine

1. SUMMARY

Guinea pigs immunised with HSV l subunit vaccine were bled, and subsequently challenged intravaginally with HSV 2. The clinical response to virus challenge was quantified, and correlations were sought between clinical score and virus- specific serum antibody titre in functional and binding assays (ELISA, neutralization, comple- ment-mediated cytotoxicity and antibody-depen- dent cellular cytotoxicity). No significant relation- ships were found, and it was concluded that reac- tivity in the serological assays chosen did not correlate with protection against HSV 2 genital infection in vaccinated female guinea pigs.

2. I N T R O D U C T I O N

Passively administered polyclonal antiserum can protect mice against challenge with virulent herpes simplex virus (HSV) [1]. Murine monoclonal an-

tibodies, raised against a variety of HSV glycopro- teins can also protect mice against HSV challenge [2], although they vary in potency. Attempts have been made to correlate the ability to protect with activity and in vitro functional assays, i.e., anti- body and complement -media ted cytotoxicity (CMC), ant ibody-dependent cellular cytotoxicity (ADCC), and neutralization with or without com- plement. However, such associations have not been conclusively demonstrated [3-5]. In the present study we vaccinated guinea pigs with a HSV 1 subunit vaccine [6], and subsequently challenged them intravaginally with HSV 2. In an at tempt to relate the severity of illness to antibody level and function, reproducible estimates of disease activity were made and examined for correlation with the results of antibody assays (both functional and binding) performed on sera taken immediately prior to virus challenge.

3. M A T E R I A L S A N D M E T H O D S

Correspondence to: M.J. Welch, Division of Biologics, Centre for Applied Microbiology and Research, Porton Down, Wilt- shire SP4 0JG, U.K.

3.1. Guinea pig immunization and H S V 2 challenge

The vaccine was prepared from a formalin- treated NP40 extract of HSV 1-infected MRC5 cells [6]. Cell debris and viral nucleocapsids were

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removed by ultracentrifugation, and the remaining viral antigens (predominantly glycoproteins) were acetone precipitated, re-solubilized and freeze- dried.

The protocol for the immunization of guinea pigs with HSV 1 subunit vaccine, and their subse- quent challenge with virulent HSV 2 has already been described [7]. Briefly, outbred Dunkin-Hart- ley guinea pigs were immunized subcutaneously with various doses of HSV 1 subunit vaccine adsorbed to alhydrogel, 5 and 3 weeks before intravaginal challenge with HSV 2. The clinical signs of illness (vaginal oedema relative to the prechallenge state and area of the external geni- talia covered by herpetic lesions) were scored daily for the next 10 days. The cumulative score for each guinea pig (index of oedema + combined lesion score, IO + CLS) was taken as an estimate of disease severity [7]. Serum samples, prepared from blood taken by cardiac puncture before the first vaccination and just prior to virus challenge, were inactivated at 56 ° C for 30 rain and stored at - 2 0 ° C .

3.2. ELISA Details of the ELISA method are given in [7].

Antigen prepared by deoxycholate solubilization of HSV 2-infected or uninfected embryo rabbit skin fibroblasts (ERSF) was used to coat the plates. Titres were estimated by linear interpola- tion through an arbitrary endpoint, and corrected by reference to a standard serum included on every plate. The chromogen used to detect per- oxidase activity of the anti-guinea pig I g G - H R P O conjugate was 5-amino salicyclic acid.

An indirect ELISA was used to determine the specific anti-glycoprotein D-1 (gD-1) activity of sera. PVC plates (Titertek) were coated with monoclonal antibody PIM12 (raised in this laboratory to gD-1; P. Ridgeway, personal com- munication) 100 n g / m l in 50 mM bicarbonate buffer, pH 9.5. Glycoprotein D-1 was captured from an HSV 1 antigen, prepared as described above, and used at an optimal dilution of 1 /500 in PBS + 0.1% Tween 20 (PBS-T) with 1 m g / m l of bovine serum albumin (BSA). Dilutions of guinea pig serum were made in PBS-T + 1% heat inactivated normal mouse serum, and bound

guinea pig IgG was detected with HRPO- conjugated rabbit anti-guinea pig IgG (Dako) at an optimal dilution of 1/2000 in PBS-T+ 1 m g / m l BSA. The chromogen was tetramethyl benzidine [8].

3.3. Assay for neutralizing antibody The ability of serum to neutralize HSV 2 in the

absence of complement was determined by a mi- cro-assay. Doubling dilutions of guinea pig serum were prepared in 50 ~tl volumes of MEM + 5% heat inactivated newborn calf serum in Nunc 96- well tissue culture plates and incubated for 2 h at 37 ° C with an equal volume of medium containing 100 50% tissue culture-infective doses (TCID50) of HSV 2. After the addition of 2 × 10 4 ERSF cells in 100/~1 of medium, the plates were incubated at 37°C for a further 3 days (at which time cell destruction in the 100 TCIDs0 virus controls was complete), fixed in 10% formalin in PBS and stained with 0.1% crystal violet in 20% ethanol. The dilution of serum which reduced virus growth by 50% or more was taken as the endpoint dilu- tion from which the neutralization titre was calcu- lated. Controls for cell growth and appearance, virus potency and serum toxicity were included, and a standard reference serum was titrated in each assay.

3.4. Antibody and complement-mediated cytotoxicity (CMC)

The method used was based on that described by McClung et al. [9]. BHK-21 cells were infected over night at 35 ° C with HSV 2 at a multiplicity of infection (m.o.i.) of 5, loaded with 5~Cr (100 /zCi/ml), washed and resuspended in MEM + 10% heat inactivated newborn calf serum at 3 x l0 s cel ls /ml (target cells). Doubling dilutions of guinea pig serum in 50 /tl volumes were prepared in medium (as above) and 100 t~l of target cell sus- pension (3 x 10 4 cells) added. After incubation at 35°C for 1 h, 50 ~tl of an optimal dilution of uninactivated non-immune guinea pig serum (1/2.5, determined using a hyperimmune HSV 2 antiserum) was added to each well as a source of complement, and incubation continued. On the following day the supernatant from each well was harvested with a Skatron harvesting press, and the

radioactivity present estimated in a gamma coun- ter. Each assay included a standard hyperimmune serum, controls for spontaneous release (no test serum) and serum toxicity (no complement), and 100 ktl of target cells lysed with 100 ttl 1% NP40 (to estimate the total release possible and there- fore the efficiency of target cell infection and loading with 51Cr).

The percent specific lysis of target cells was calculated using the formula:

% specific lysis

test release - spontaneous release

maximal release with - spontaneous release hyperimmune serum

× 100

The titration endpoint was arbitrarily set at 20% of the maximum specific lysis (achieved with the standard hyperimmune HSV 2 antiserum) and the dilution of test serum which achieved this and hence the titre, was calculated from linear regres- sion plots of % specific lysis versus serum dilution.

3.5. Antibody-dependent cellular cytotoxicity (ADCC)

This procedure was essentially as for the CMC assay, except that casein-elicited guinea pig peri- toneal macrophages were added as effector cells at an effector : target ratio of 50 : 1 (1.5 × 106/well) in a volume of 50 #1 instead of complement. Spontaneous release was estimated from control wells with 50 #1 of medium in place of a test serum dilution.

3.6. Statistical methods Correlation coefficients were calculated by the

ranking method of Spearman, using the statistical package SPP (Patrick Royston, Clinical Research Centre, Harrow, U.K.).

4. RESULTS

4.1. Clinical results of virus challenge Seventy three guinea pigs were challenged with

HSV 2; 18 were vaccinated with 300 # g / d o s e of mock vaccine (prepared from uninfected cells), 20 with HSV 1 subunit vaccine 300/xg/dose, 18 with

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Vaccine and protein content per dose Fig. 1. Group median index of oedema + combined lesion score (IO + CLS) in guinea pigs given two doses of vaccine five and three weeks before intravaginal challenge with HSV 2.

gamma-irradiated (2.5 Mr) HSV 1 vaccine 300 #g /dose , and 17 with irradiated vaccine 30 /~g/dose. The clinical results of virus challenge are presented in Fig. 1. Mock-vaccinated animals de- veloped a severe illness with prominent vaginal lesions and oedema, and retention of urine and faeces in many animals. Those given HSV 1 sub- unit vaccine were less severely affected, although vaginal lesions and oedema were still clearly dis- cernible in most animals. (A full description of the clinical and pathological aspects of this model may be found in [10]; the statistical analysis of clinical data is considered in [7]). HSV 2 was reisolated from all 73 animals (data not shown). Only those sera from vaccinated animals were reactive in the assays chosen, therefore the mock- vaccinated group was not included in any further analysis.

4.2. Serology Sera taken prior to vaccination gave negative

results in all the assays, and in the ELISA all sera gave negative results with uninfected ERSF anti- gen. Data from the three HSV 1-vaccinated groups were combined and the serological findings are

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Table 1

Guinea pig serum titres (log]0) determined in functional and binding assays after vaccination

Assay procedure HSV 2 gD-1 NT CMC ADCC ELISA ELISA

Postvaccination Mean 3.35 3.61 1.19 1.73 1.70 titre SD 0.78 0.48 0.46 0.32 0.42

N 52 27 50 49 49

NT = neutralization assay; N = number of sera tested.

TABLE 2

Coefficients of correlation between clinical scores (10 day cumulative 10 + CLS) and guinea pig serum titres

Assay HSV 2 gD-I NT CMC ADCC procedure ELISA ELISA

Correlation coefficient with IO +CLS -0.111 -0.181 -0.149 -0.130 0.162

N 52 27 50 49 49

N = number of animals; NT = neutralization test. None of the correlation coefficients are significant.

summarised in Table 1. All the animals responded to vaccinat ion by producing an t ibody reactive in both b ind ing and funct ional assays.

In order to examine the relat ionship between clinical score (10 day cumulat ive IO + CLS) and serum titres in the various tests, correlat ion coeffi- cients were calculated. These are presented in Table 2. Al though all the correlat ion coefficients are negative, suggesting an inverse relat ionship of titre with clinical score, none of them is suffi- ciently large enough to be statistically significant.

TABLE 3

Correlation matrix comparing the response to vaccination as shown by all serological tests

HSV 2 gD-1 NT CMC ELISA ELISA

gD-1 ELISA 0.409(27) *

NT 0.462(50) ** 0.402(26) * CMC 0.073(49) 0.407(25) * 0.137(49) ADCC 0.248(49) 0.423(25) * 0.295(49) * 0.251(49)

The number of serum samples included in each test is given in parentheses. NT = neutralization test. ( * : P < 0.05, * * ; P < 0.001).

A matrix in which all the serological tests were correlated with each other is presented in Table 3.

All the coefficients were positive. The strongest association was found between the HSV 2 ELISA and serum neutra l iza t ion titre ( P < 0.001); neu- tral ization was also significantly correlated with ADCC. ELISA titre to gD-1 was significantly correlated with all the other tests.

5. D I S C U S S I O N

The role of an t ibody in protect ion against HSV infect ion has been investigated extensively in the mur ine model. Protect ion can be achieved with both polyclonal antiviral sera [1] and monoclona l ant ibodies raised to the major virion glycoproteins (gB, gC and gD, see [2]). No clear relationships have emerged l inking funct ion in vitro (neutraliza- tion, CMC, A D C C ) with abili ty to protect [3,4]. Monoc lona l ant ibodies inactive in all of these assays may nevertheless protect against virus chal- lenge [5]. However, neutral iz ing but not non-neu- tralizing monoc lona l an t ibody may prevent the development of a HSV 1-induced zosteriform rash in B A L B / c mice, even when given up to 60 h post challenge, albeit in non-physiological amounts [11].

Gu inea pigs are protected against intravaginal challenge with HSV 2 by passive immuniza t ion with hype r immune rabbi t ant i -HSV 2 IgG (Barry Thorn ton . personal communica t ion) . The guinea pig model of HSV 2 genital infect ion closely re- sembles h u m a n HSV genital infection and we were interested to examine the relationship of an t ibody level (and funct ion) to disease activity in the guinea pig model. To this end, guinea pigs were vaccinated with a HSV 1 subuni t vaccine, and subsequent ly challenged intravaginal ly with

HSV 2. Reproducible scores of disease activity based on the area of the external genitalia covered by herpetic lesions and vaginal oedema [7] were correlated with the titres of sera (taken just prior to virus challenge) determined in a variety of functional and binding assays. The expectation was that if protection was mediated principally by total antibody, then a highly significant associa- tion would be found between clinical score and serum titre in the ELISA (an antibody binding assay). Moreover, if protection was principally the function of one type of antibody, e.g. that capable of mediating ADCC, then similarly, a strong as- sociation would exist between ADCC titres and clinical score.

Although the assays were rigorously performed, with the inclusion of a standard serum to ensure reproducibility, no such relationship of disease activity to any of the functional or binding assay results was found. In particular, there was no evidence to suggest a close relationship between antibody titre to gD-1 and protection, although raised antibody titres to gD have been associated with a reduced frequency of recrudescence in guinea pigs infected intravaginally with HSV 2 [12]. It would therefore seem unlikely that serum antibody plays a major role in vaccine-induced immunity to HSV 2-challenge in our model. Female guinea pigs given the same subunit vac- cine mount a more rapid and pronounced secre- tory antibody response in the genital tract (both IgA and IgG) than unvaccinated animals [13]. It may be that this, or some other as yet undemon- strated effector arm of the immune system, for example delayed type hypersensitivity or T-cell cytotoxicity, correlates better with protection.

A consistent positive correlation was found be- tween ELISA antibody titres to gD-1, and titre in all the other assays. Monoclonal antibodies to gD are capable of neutralizing virus in the absence of complement, and of mediating CMC and ADCC [3,4]. Western blots probed with sera from our vaccinated guinea pigs have shown that there is a more pronounced response to gD than to the other virion glycoproteins (data not shown). Thus this association may be a reflection of the relative abundance a n d / o r high affinity of antibodies raised to gD in these animals, and does not neces-

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sarily imply a more prominent role for gD (as compared to the other glycoproteins) in CMC or ADCC.

A C K N O W L E D G E M E N T S

The helpful advice of and discussions with Barry Thornton are gratefully acknowledged. This pro- ject was funded in part by Porton International.

R E F E R E N C E S

[1] Oakes, J.E. and Rosemund-Hornbeak, H. (1978) Anti- body-mediated recovery from subcutaneous herpes sim- plex virus type 2 infection. Infect. Immun. 21,489-495.

[2] Dix, R.D., Pereira, L. and Baringer, R.J. (1981) Use of monoclonal antibody directed against herpes simplex virus glycoproteins to protect mice against acute virus-induced neurological disease. Infect. Immun. 34, 192 199.

[3] Balachandran, N., Bacchetti, S. and Rawls, W.E. (1982) Protection against lethal challenge of BALB/c mice by passive transfer of monoclonal antibodies to five glyco- proteins of herpes simplex virus type 2. Infect. Immun. 37, 1132-1137.

[4] Kumel, G., Kaerner, H.C., Levine, M., Schroder, C.H. and Glorioso, J.C. (1985) Passive immune protection by herpes simplex virus-specific monoclonal antibodies and monoclonal antibody-resistant mutants altered in patho- genicity. J. Virol. 56, 930-937.

[5] Rector, J.T., Lausch. R.N. and Oakes, J.E. (1982) Use of monoclonal antibodies for analysis of antibody-dependent immunity to ocular herpes simplex virus type 1 infection. Infect. Immun. 38, 168 174.

[6] Skinner, G.R.B., Woodman, C.B.J., Hartley, C.E., Buchan, A., Fuller, A., Durham, J.. Synnon, M., Clay, J.C., Mell- ing, J., Wiblin, C. and Wilkins, J. (1982) Preparation and immunogenicity of vaccine Ac NFU 1 (S ) MRC towards the prevention of herpes genitalis. Br. J. Vener. Dis. 58, 381-386.

[7] Phillpotts, R.J., Welch, M.J., Ridgeway, P.H., Walkland, A.C. and Melling, J. (1988) A test for the relative potency of herpes simplex virus vaccines based upon the female guinea pig model of HSV 2 genital infection. J. Biol. Stand. 16, 109-118.

[8] McClung, H., Seth, P. and Rawls, W.E. (1976) Quantita- tion of antibodies to herpes simplex types I and 2 by complement-dependent antibody lysis of infected cells. Am. J. Epidemiol. 104, 181 191.

[9] Bos, E.S., van der Doelen, A.A., van Rooij, N. and Schuurs, A.H.W.M. (1981) 3,3'5,5' Tetramethyl benzidine as an Ames test negative chromogen for horseradish per- oxidase in enzyme immunoassay. J. Immunoass. 2, 187-204.

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[10] Thornton, B., Baskerville, A., Bailey, N.E., Melling, J.M. and Hambleton, P. (1984) Herpes simplex genital infec- tion of the female guinea pig as a model for the evaluation of an experimental vaccine. Vaccine 2, 141-148.

[11] Simmons, A. and Nash, A.A. (1985) Role of antibody in primary and recurrent herpes simplex virus infections. J. Virol. 53, 944-948.

[12] Bernstein, D.I., Stanberry, E.R., Harrison, C.J., Shukla,

R., Kappes, J.C, and Myers, M.G. (1987) Antibody re- sponse to herpes simplex virus glycoprotein D: effects of acyclovir and relation to recurrence. J. Infect. Dis. 156, 423-429.

[13] McBride, B.W., Ridgeway, P., Phillpotts, R. and Newell, D.G. (1988) Mucosal antibody responses to vaginal infec- tion with herpes simplex virus in pre-vaccinated guinea pigs. Vaccine, in press.