British Journal of Obstetrics and Gymecology August 1977. VOI 84. pp 622-629
ANTIBODY ACTIVITY TO TYPE 1 AND TYPE 2 HERPES SIMPLEX VIRUS IN HUMAN CERVICAL MUCUS BY
B . M . COUGHLAN, Research Fellow* Department of Experimental Pathology, University of Birmingham AND
G. R. B. S K I N N E R ,Senior Lecturer Department of Virology, Universit-v of Birmingham Summary 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 "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 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.
herpes simplex virus and its possible association with cervical carcinoma (Rawls et al, 1969; Nahmias et al, 1970; Skinner et al, 1971; Adams et al, 1974; Skinner, 1976), there has been little investigation of the local antibody response to herpes simplex virus in the uterine cervix. In a previous study it was shown that immunoglobulin IgA concentration and, to a lesser extent IgG, was significantly higher in the cervical mucus of patients with abnormal cervical cytology than in patients with normal cervical cytology (Coughlan and Skinner, 1977). It was suggested that this difference might be attributable to a local antibody response to prolonged antigenic stimulus such as might be
THEexistence of an independent local antibody system (Tomasi and Bienenstock, 1968) and its importance in the prevention and control of infection of the respiratory tract (Smith et al, 1966; Cate et al, 1966; Waldman et t d , 1970), intestinal tract (Ogra and Karzon, 1969) and female genital tract (Ogra and Ogra, 1973) is now well established. Neutralizing antibody to herpes simplex virus has been identified in the salivary secretion of patients with recurrent herpes labialis (Douglas and Couch, 1970). However, despite recent interest in cervical infection with type 2 ~
* Present address: Assistqnt Master, National
Maternity
Hospital, Dublin 2.
622
HERPES ANTIBODIES I N CERVICAL MUCUS
provided, for example, by infection with type 2 herpes simplex virus. In the present study, we have examined specimens of cervical mucus from healthy female subjects for antibody activity to type 1 and type 2 herpes simplex virus and attempted to relate the antibody titre to the immunoglobulin concentration in the cervical mucus and to serum antibody titres against thesz viruses.
METHODS Patients studied The patients were randomly selected from a routine gynaecological clinic and were predominantly from social classes I11 and IV. The patients' ages ranged from 22 to 44 years with a mean age of 32 years. Fifteen patients were nulliparous. The cervices were macroscopically normal with no evidence of malignant or other change. None of the patients were receiving hormone therapy or had clinical or laboratory evidence of genital tract infection. Cervical mucus The collection and preparation of cervical mucus has been described (Coughlan and Skinner, 1977). To obtain sufficient volumes of mucus to carry out preliminary experiments with replication, a number of mucus samples from different patients were pooled before testing; these are referred to in the text and tables as 'mucus pool one' and 'mucus pool two', etc. Immunoglobulin concentrations were estimated according to the method of Fahey, as previously described (Coughlan and Skinner, 1977). Virus strains Strain HF, the HFEM derivative of the Rockefeller strain H F (Wildy, 1955) and strain 3345, a penile isolate, isolated at the Venereal Disease Clinic of the General Hospital, Birmingham, were used as type 1 and type 2 strains respectively (Geder and Skinner, 1971 ; Plummer et al, 1974). The Dekking strain of pseudorabies was used. Virus infectivity was assayed by the plaque method of Russell (1 962). Virus antigen BHK 21 cells (MacPherson and Stoker, 1962)
623
were infected at high multiplicity (10 plaqueforming units/cell) with type 1 or type 2 herpes virus and incubated at 37 "C for 36 hours. The infected cells were removed from the glass, washed with phosphate buffered saline, resuspended in sterile water at a concentration of lo8 cells/ml and disrupted ultrasonically. As a routine, antigen preparations were tested in immunodiffusion against homologous immune sera. Under these circumstances they gave multiple precipitin lines. Neutrulization of virus infectivity by cervical mucus Cervical mucus samples were diluted 1/10 in saline and heated at 56 "C for 30 minutes to inactivate proteolytic enzymes and commensal microorganisms. The mucus sample was then mixed with an equal volume of either type 1 or type 2 herpes virus containing lo7 plaqueforming units (pfu)/ml and reacted for 16 hours at 25 "C. Residual infective virus was titred as described above. Virus dilutions were made in Eagle's medium containing 10 per cent heatinactivated calf serum which served to stabilize the virus infectivity during the relatively long virus and antiserum reaction period. The tests were controlled by incubating virus suspensions with non-immune cervical mucus and appropriately diluted heat-inactivated calf serum and rabbit serum under similar conditions of test. In addition, the mucus samples were initially tested against pseudorabies virus which, in terms of neutralization of virus infectivity, is serologically unrelated to herpes simplex virus types 1 and 2 and thus provided a control measure of non-specific virus inactivation by heat-stable enzyme activity, pH extremes and other possible unknown elements. However, as there was no inactivation of pseudorabies virus in a random series of 20 mucus samples, this rather time-consuming control was omitted. Antibody activity in cervical mucus samples was expressed as : (log,, virus titre following incubation with non-immune mucus) minus (log,, virus titre following incubation with cervical mucus under test). The neutralizing antibody in sera was investigated by kinetic neutralization tests and expressed as a kinetic neutralization rate constant (k value). Complement was obtained from Burroughs
624
COUGHLAN AND SKINNER
Wellcome Limited and titred with sensitized sheep erythrocytes. Micro-inimunodiffusion Micro-immunodiffusions were carried out in 1 per cent (w/v) Ionagar (Oxford) in isotonic saline containing 0.1 per cent (w/v) sodium azide (Watson, 1969). Six hexagonally-arranged wells surrounded a central well, each well being separated from each neighbouring well by 3 mm of gel. Plates were read following incubation at room temperature in a humidified chamber for 72 hours. Mucus was concentrated fivefold before testing in immunodiffusion.
RESULTS Studies on specimens from individuals The neutralizing antibody activity in the cervical mucus and serum and the immunoglobulin IgG and IgA concentrations in the cervical mucus of 29 healthy female subjects are shown in Table I. Neutralizing antibody activity against type 1 herpes virus was detected in 24 of 28 patients tested and against type 2 herpes simplex virus in 18 of 24 patients tested. Of the 23 patients in whom antibody titres against both virus types were available, three had detectable neutralizing antibody against only type 1 virus, (No. 2, 15, and 20) and one (No. 19) with neutralizing antibody against only type 2 virus. In five patients (No. 5, 9, 12, 18 and 26) neutralizing antibody was detected in the cervical mucus but not in the sera and, conversely, in one patient (No. 22) antibody was detected in the serum but not in the mucus. Correlation between type I and type 2 neutrtrlizing antibody in the cervicul mucus There was a positive but weak correlation between the type 1 and type 2 neutralizing antibody activity in the mucus samples (r = 0-39; P
ANTIBODY ACTIVITY TO TYPE 1 AND TYPE 2 HERPES SIMPLEX VIRUS IN HUMAN CERVICAL MUCUS BY
B . M . COUGHLAN, Research Fellow* Department of Experimental Pathology, University of Birmingham AND
G. R. B. S K I N N E R ,Senior Lecturer Department of Virology, Universit-v of Birmingham Summary 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 "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 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.
herpes simplex virus and its possible association with cervical carcinoma (Rawls et al, 1969; Nahmias et al, 1970; Skinner et al, 1971; Adams et al, 1974; Skinner, 1976), there has been little investigation of the local antibody response to herpes simplex virus in the uterine cervix. In a previous study it was shown that immunoglobulin IgA concentration and, to a lesser extent IgG, was significantly higher in the cervical mucus of patients with abnormal cervical cytology than in patients with normal cervical cytology (Coughlan and Skinner, 1977). It was suggested that this difference might be attributable to a local antibody response to prolonged antigenic stimulus such as might be
THEexistence of an independent local antibody system (Tomasi and Bienenstock, 1968) and its importance in the prevention and control of infection of the respiratory tract (Smith et al, 1966; Cate et al, 1966; Waldman et t d , 1970), intestinal tract (Ogra and Karzon, 1969) and female genital tract (Ogra and Ogra, 1973) is now well established. Neutralizing antibody to herpes simplex virus has been identified in the salivary secretion of patients with recurrent herpes labialis (Douglas and Couch, 1970). However, despite recent interest in cervical infection with type 2 ~
* Present address: Assistqnt Master, National
Maternity
Hospital, Dublin 2.
622
HERPES ANTIBODIES I N CERVICAL MUCUS
provided, for example, by infection with type 2 herpes simplex virus. In the present study, we have examined specimens of cervical mucus from healthy female subjects for antibody activity to type 1 and type 2 herpes simplex virus and attempted to relate the antibody titre to the immunoglobulin concentration in the cervical mucus and to serum antibody titres against thesz viruses.
METHODS Patients studied The patients were randomly selected from a routine gynaecological clinic and were predominantly from social classes I11 and IV. The patients' ages ranged from 22 to 44 years with a mean age of 32 years. Fifteen patients were nulliparous. The cervices were macroscopically normal with no evidence of malignant or other change. None of the patients were receiving hormone therapy or had clinical or laboratory evidence of genital tract infection. Cervical mucus The collection and preparation of cervical mucus has been described (Coughlan and Skinner, 1977). To obtain sufficient volumes of mucus to carry out preliminary experiments with replication, a number of mucus samples from different patients were pooled before testing; these are referred to in the text and tables as 'mucus pool one' and 'mucus pool two', etc. Immunoglobulin concentrations were estimated according to the method of Fahey, as previously described (Coughlan and Skinner, 1977). Virus strains Strain HF, the HFEM derivative of the Rockefeller strain H F (Wildy, 1955) and strain 3345, a penile isolate, isolated at the Venereal Disease Clinic of the General Hospital, Birmingham, were used as type 1 and type 2 strains respectively (Geder and Skinner, 1971 ; Plummer et al, 1974). The Dekking strain of pseudorabies was used. Virus infectivity was assayed by the plaque method of Russell (1 962). Virus antigen BHK 21 cells (MacPherson and Stoker, 1962)
623
were infected at high multiplicity (10 plaqueforming units/cell) with type 1 or type 2 herpes virus and incubated at 37 "C for 36 hours. The infected cells were removed from the glass, washed with phosphate buffered saline, resuspended in sterile water at a concentration of lo8 cells/ml and disrupted ultrasonically. As a routine, antigen preparations were tested in immunodiffusion against homologous immune sera. Under these circumstances they gave multiple precipitin lines. Neutrulization of virus infectivity by cervical mucus Cervical mucus samples were diluted 1/10 in saline and heated at 56 "C for 30 minutes to inactivate proteolytic enzymes and commensal microorganisms. The mucus sample was then mixed with an equal volume of either type 1 or type 2 herpes virus containing lo7 plaqueforming units (pfu)/ml and reacted for 16 hours at 25 "C. Residual infective virus was titred as described above. Virus dilutions were made in Eagle's medium containing 10 per cent heatinactivated calf serum which served to stabilize the virus infectivity during the relatively long virus and antiserum reaction period. The tests were controlled by incubating virus suspensions with non-immune cervical mucus and appropriately diluted heat-inactivated calf serum and rabbit serum under similar conditions of test. In addition, the mucus samples were initially tested against pseudorabies virus which, in terms of neutralization of virus infectivity, is serologically unrelated to herpes simplex virus types 1 and 2 and thus provided a control measure of non-specific virus inactivation by heat-stable enzyme activity, pH extremes and other possible unknown elements. However, as there was no inactivation of pseudorabies virus in a random series of 20 mucus samples, this rather time-consuming control was omitted. Antibody activity in cervical mucus samples was expressed as : (log,, virus titre following incubation with non-immune mucus) minus (log,, virus titre following incubation with cervical mucus under test). The neutralizing antibody in sera was investigated by kinetic neutralization tests and expressed as a kinetic neutralization rate constant (k value). Complement was obtained from Burroughs
624
COUGHLAN AND SKINNER
Wellcome Limited and titred with sensitized sheep erythrocytes. Micro-inimunodiffusion Micro-immunodiffusions were carried out in 1 per cent (w/v) Ionagar (Oxford) in isotonic saline containing 0.1 per cent (w/v) sodium azide (Watson, 1969). Six hexagonally-arranged wells surrounded a central well, each well being separated from each neighbouring well by 3 mm of gel. Plates were read following incubation at room temperature in a humidified chamber for 72 hours. Mucus was concentrated fivefold before testing in immunodiffusion.
RESULTS Studies on specimens from individuals The neutralizing antibody activity in the cervical mucus and serum and the immunoglobulin IgG and IgA concentrations in the cervical mucus of 29 healthy female subjects are shown in Table I. Neutralizing antibody activity against type 1 herpes virus was detected in 24 of 28 patients tested and against type 2 herpes simplex virus in 18 of 24 patients tested. Of the 23 patients in whom antibody titres against both virus types were available, three had detectable neutralizing antibody against only type 1 virus, (No. 2, 15, and 20) and one (No. 19) with neutralizing antibody against only type 2 virus. In five patients (No. 5, 9, 12, 18 and 26) neutralizing antibody was detected in the cervical mucus but not in the sera and, conversely, in one patient (No. 22) antibody was detected in the serum but not in the mucus. Correlation between type I and type 2 neutrtrlizing antibody in the cervicul mucus There was a positive but weak correlation between the type 1 and type 2 neutralizing antibody activity in the mucus samples (r = 0-39; P
