Novel in vitro functional assays for the determination of anthrax toxin components.

The characterisation and evaluation of the UK licensed human anthrax vaccine depends on several in vivo tests that determine its safety and potency. Assays for the determination of functionally active and/or immunoreactive toxin components and S-layer proteins have been developed and applied to the characterisation of anthrax vaccine. These technologies may support production of consistent and effective vaccines, and may ultimately reduce the requirements for in vivo testing.

Report of twelve years experience in open study of Skinner herpes simplex vaccine towards prevention of herpes genitalis.

Three hundred and forty-seven subjects at risk for herpes genitalis were vaccinated with Skinner vaccine, NFUAc.HSV1.(S-MRC5), and were followed for an average duration of 2 years representing a total consortship of 664.4 years. Based on survey information obtained during this consortship, there were estimated to be 3076 recurrences which summated to 3.5 years total duration of disease and comprised at least 6794 lesions; there were an estimated 51997 episodes of intercourse including at least 241 episodes of unprotected intercourse in the presence of herpetic lesions. The rate of contraction of herpes genitalis was 6 of 54 consorts (11.1%) who received one vaccination and 7 of 293 (2.4%) who received two, three of four vaccinations. There was no evidence of physical or psychological side effects from vaccination.

Complement-independent neutralising monoclonal antibody with differential reactivity for strains of human cytomegalovirus.

A mouse monoclonal antibody with complement-independent neutralising activity against cytomegalovirus (CMV) and reactive with the 86 kilodalton (kDa) viral glycoprotein H is described. Neutralisation tests against a range of different strains of CMV showed significant crossreactivity, but clear differences were evident between the two prototype viruses AD169 and Davis, and particularly between AD169 and several low-passage recent clinical isolates; CMV present in urine was neutralised weakly if at all.

Antibody reactivity with Skinner HSV vaccine.

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.

Follow-up report on 50 subjects vaccinated against herpes genitalis with Skinner vaccine.

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.

Covalent linkage of carboxypeptidase G2 to soluble dextrans--I. Properties of conjugates and effects on plasma persistence in mice.

The covalent attachment of the therapeutic enzyme carboxypeptidase G2 to soluble dextrans of varying molecular weight resulted in a 5-15-fold increase in plasma persistence in normal and tumour-bearing mice. The molecular weight of the dextran used markedly affected the number of dextran molecules present in the conjugate, resulting in a molecular weight distribution between 6 and 12 X 10(5) daltons. The isoelectric point of the conjugates varied between 4.1 and 4.8 compared to native enzyme 7.8. Conjugates were resistant to proteolysis by trypsin and chymotrypsin, but showed little difference in their affinity for substrate.

Covalent linkage of carboxypeptidase G2 to soluble dextrans--II. In vivo distribution and fate of conjugates.

The in vivo fate of the therapeutic enzyme, carboxypeptidase G2 (CPG2) in native form and covalently-linked to soluble dextrans was studied in the mouse using radiolabelled compounds. Clearance, from the blood, of all compounds tested was found to be as intact, active material, whilst excreted radiolabel was associated in all cases with low molecular weight substances. The clearance and excretion rates of native CPG2 were found to balance, but this was not so for dextran-CPG2 conjugate or CNBr-activated dextran. Tissue distribution studies demonstrated that there was little or no tissue uptake of native CPG2, whereas dextran-CPG2 conjugate, and CNBr-activated dextran were retained in the liver. Within the liver, the CPG2 component of dextran-CPG2 conjugate was degraded more rapidly than the dextran moiety. Blockade of reticulo-endothelial system (RES) led to increased half-lives of dextran CPG2 conjugate and CNBr-activated dextran, demonstrating the involvement of the RES in the clearance of these compounds. Impairment of RES activity did not affect the clearance rate of native CPG2. These results are discussed in relation to the potential use of dextran-CPG2 conjugates in cancer chemotherapy.

Control of herpes simplex virus infections of the genital tract by vaccination.

The apparent increasing incidence of herpes simplex virus infections of the genital tract has focused attention on the efficacy of vaccination in preventing infection or modifying established disease. Results of an 'open trial' using a DNA-free inactivated virus subunit vaccine have shown that vaccination of subjects at risk of contracting infection from their sexual partner reduced the transmission rate from 34% in unvaccinated controls to 0.5%. In a separate study, vaccination of patients who had experienced their first overt attack of herpes genitalis (the initial clinical episode) had significantly fewer recurrences over the follow-up period of 12 months than the unvaccinated control group. The results, we feel, justify a placebo controlled trial.

Indirect enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies against Coxsackie B viruses.

In tests for IgG antibodies against Coxsackie B viruses in man, the enzyme-linked immunosorbent assay (ELISA) was essentially group-specific and, unlike the type-specific neutralisation test, usually failed to detect rises in antibody titre in paired, acute and convalescent, sera. However, in rabbits immunised against Coxsackie B viruses, ELISA demonstrated both group- and type-specific antibody responses. The lack of type-specificity of ELISA in man is probably because repeated infection with enteroviruses--echoviruses and Coxsackie A as well as Coxsackie B--results in masking of the type-specific antibody response by group-specific antibody.

Efficacy of vaccine Ac NFU1 (S-) MRC 5 given after an initial clinical episode in the prevention of herpes genitalis.

A subunit antigenoid vaccine, Ac NFU1 (S-) MRC 5, was used in patients who had had a clinical episode of herpes genitalis. The rate of recurrence was compared with that in unvaccinated patients to determine the efficacy of vaccination in preventing recurrence and spread of the virus in the community. Seven of 22 (31%) vaccinated patients had eight recurrences after the initial clinical episode; in contrast there were 51 recurrences in 17 of 20 (85%) unvaccinated patients. Although further studies are needed, the results indicate that the vaccine may prevent recurrent episodes of herpes genitalis and thereby reduce the dissemination of this virus in the population.

Pathogenesis and immune response of vaccinated and unvaccinated rhesus monkeys to tick-borne encephalitis virus.

The rhesus monkey was used as a model for diseases caused by viruses of the tick-borne encephalitis virus complex to study the efficacy and safety of a commercial killed vaccine. Animals infected intravenously developed a subclinical infection with no histopathological lesions but with transient clinical chemical changes that included elevated transaminase, dehydrogenase, and creatine kinase activities and that declined as an immune response developed. The immune response was detected as neutralizing antibody in serum and serum antibody to several viral proteins. Antibodies to viral envelope protein and two other infected cell-specific polypeptides were also detected. Intranasal infection resulted in a disease resembling that in humans, except that no pyrexia was observed. Clinical chemical changes similar to those in intravenously infected monkeys developed, but most animals died before an immune response was mounted. Using this model, we have demonstrated that a commercial vaccine protects animals against a wild-type virus isolate and that it elicits an effective immune reaction without any evidence of an immune enhancement phenomenon or adverse side effects as judged by clinical observation, clinical chemistry, and histopathology.

The effect of histidine ammonia-lyase on some murine tumours.

The histidine ammonia-lyase from bacterial strain CAMR 5315 was partially purified to assess its effect on the growth of murine tumours. This strain was selected as the source after an extensive screening programme for histidine ammonia-lyases. The enzyme was partially purified by ammonium sulphate fractionation, chromatography on DEAE-cellulose and Sephadex G-150. The enzyme reduced circulating L-histidine levels in Wistar rats and in mice persisted with a half-life of 6-7 h. Neither LDH virus nor chemical modification with ethylacetimidate increased the half-life as observed with L-asparaginase and L-glutaminase. The enzyme was tested in mice against Ehrlich carcinoma, L5178Y lymphoblastic leukaemia, Mc/S sarcoma, B16 melanoma, P8157 mastocytoma, P1798 lymphosarcoma and the Gardner 6C3HED lymphosarcoma. The only tumours to show sensitivity to the enzyme were the Mc/S sarcoma against which a 65% increase in life span was observed at the highest enzyme dose, 1000 U/kg on alternate days over 14 days and the Ehrlich ascites carcinoma where cures were obtained at 250 U/kg on alternate days over 14 days, but only at inocula levels of 10(5) and 10(3) cells/animal respectively.

Preparation and immunogenicity of vaccine Ac NFU1 (S-) MRC towards the prevention of herpes genitalis.

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.

Early experience with "antigenoid" vaccine Ac NFU1(S-) MRC towards prevention or modification of herpes genitalis.

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.

Enhanced plasma persistence of therapeutic enzymes by coupling to soluble dextran.

Conjugation of carboxypeptidase G and arginase, two enzymes of therapeutic interest, to a soluble dextran significantly enhanced plasma persistence in normal and tumour-bearing mice. A prolonged decrease in arginine concentrations in plasma of tumour-bearing mice was demonstrated by using the dextran-linked arginase. Gel filtration of dextran-enzyme conjugate showed that enzyme activity co-chromatographed as a single peak with carbohydrate, and enzyme was shown to be covalently linked to the dextran.