L. H. Collier

The Development of a Stable Smallpox Vaccine.

1. Requirements for a heat-stable smallpox vaccine are suggested.2. Sheep lymph dried from the frozen state retains its potency at 22 and 37°C. better than aqueous, glycerinated or lanolinated suspensions of lymph. Dried crude lymph is not, however, sufficiently stable for use in tropical areas; it is also liable to vary in stability from batch to batch. No better results were obtained by prolonging secondary desiccation.3. Phenol in low concentration is deleterious to vaccinia virus stored in the dried or liquid state at 22 and 37° C.4. Dried vaccinia virus stored under nitrogen loses potency more rapidly than when sealed in vacuo, even when precautions are taken to rid the nitrogen of traces of oxygen and moisture.5. A number of suspending media were examined for their preservative influence on purified vaccinia virus. None was effective in the liquid state, but all protected the virus against the lethal influence of freeze-drying. In the dried state, these media varied in their ability to protect the virus during storage at 22 or 37° C. The best results at both temperatures were obtained with virus freeze-dried in 5% peptone. This medium also protects the virus well at 45° C. Different types of peptone were equally effective.6. Virus dried in 5% peptone still gave a full quota of successful primary vaccinations of children after storage for 12 months at 22° C., or for 4 months at 37° C.7. Using partially purified sheep virus dried in 5% peptone, a vaccine can be produced which shows a high degree of resistance to heat, is relatively free from bacterial contamination, and is easy to reconstitute after prolonged storage. These results are highly reproducible.

Trachoma vaccine field trials in The Gambia.

The ability of two live trachoma vaccines to protect against naturally acquired infection was tested in young Gambian children. With a mineral oil adjuvant vaccine prepared from a Gambian strain of trachoma (MRC–187) a barely significant measure of protection was demonstrable 6 months after the first dose, but not at 1 year, despite a reinforcing dose given 6 months after the first. In a later trial an aqueous vaccine prepared from the ‘fast-killing’ variants of strains ‘SA–2’ and ‘ASGH’ failed to induce immunity. Two years after vaccination, the proportion of vaccinated children progressing to cicatricial trachoma was less than in the controls, and the average severity of the disease in terms of clinical score was greater; vaccine-induced hypersensitivity may have contributed to this result.Irrespective of whether they had received trachoma vaccine, children with completely normal eyes at the outset were less likely to acquire trachoma than those with slight conjunctival folliculosis or papillary hyperplasia. In children acquiring trachoma, there was a highly significant positive correlation between severity of the disease and the presence of conjunctival inclusions. The pattern of trachoma differed significantly in the two villages used in both trials; the prev alence, severity and proportion of inclusion-positive subjects were all higher in the village with the greater population density.An efficient follow-up organization, use of a slit-lamp for clinical observations, and a scoring system for recording physical signs are all desirable for trachoma vaccine field trials.We are highly indebted to Dr G. Turner (Lister Institute, Elstree, Herts) for his assistance in making the vaccine used for Trial II; to Dr N. M. Lam (Pfizer Ltd.) and Dr C. H. Smith (Evans Medical Ltd.) for making the Trial III vaccine; to Dr I. A. Sutherland (M.R.C. Statistical Unit) for his advice and help with the statistical aspects; to the Pennsylvania Refinery Co. Inc. for a generous gift of Drakeol 6 VR; and to Mr M. Race for his invaluable technical assistance in The Gambia. We are also grateful to the Director and staff of the M.R.C. Laboratories, The Gambia, for various facilities; and to The Gambian Government for per mission to undertake these trials.

The Antigenicity of Ultra-Violet Irradiated Vaccinia Virus

. . . . 534 Introduction Materials and methods Source of virus Infectivity titrations Irradiation Tests for inactivation of virus Bacterial content of irradiated material Immunization experiments Titre of control vaccine lymph in normal rabbits Experimental results Inactivating dose of irradiation Immunization of rabbits The influence of smaller doses of antigen Relation of irradiation to antigenicity Immunization of monkeys . . 6 The duration of immunity Preservation of the antigen Immunization with small amounts of living virus Confirmatory experiment Discussion Summary References

Immunogenicity of experimental trachoma vaccines in baboons. I. Experimental methods, and preliminary tests with vaccines prepared in chick embryos and in HeLa cells.

Parallel titrations of a strain of trachoma (MRC–221) and one of inclusion conjunctivitis (MRC–4) in the baboon conjunctiva and in chick embryos suggest that ten to twenty 50% egg infective doses are equivalent to one 50% baboon infective dose; but that at least 1000 egg infective doses are needed to induce moderate or severe infections in all of a given number of baboons. For vaccine experiments in baboons, a system of scoring physical signs and presence of inclusion bodies was devised; the significance of differences in vaccinated and control animals in their response to conjunctival challenge was determined by analysis of variance. An aqueous suspension of live MRC–4 grown in the yolk sac was given as two subcutaneous doses and one intravenous dose at weekly intervals, and protected all of six baboons challenged with the homologous strain; three similarly spaced subcutaneous doses were less effective. The immunity induced by this vaccine waned considerably during the ensuing 15 months. Vaccine prepared from a live ‘fast-killing’ variant of MRC–4 grown in HeLa cells was less effective than MRC–4 itself in protecting baboons against infection with the parent strain. Although both yolk sac and HeLa cell vaccines induced the formation of antibody fixing complement with trachoma group antigen, the serum titres in individual animals at the time of challenge were unrelated to the degree of protection; during a 15 month observation period there were pronounced falls in the titres of antibody induced either by vaccination or by challenge with egg-grown TRIC agent. We wish to thank Dr I. Sutherland (M.R.C. Statistical Research Unit) for his helpful advice during the early stages of this work. We are also greatly indebted to Mr P. Avis (Pfizer Ltd.) for his advice and for undertaking the statistical computations; and to Miss Anne Smith and Miss Pay Storey (M.R.C. Trachoma Research Unit) for doing the complement fixation tests.

The serum and conjunctival antibody response to trachoma in Gambian children

Ninety-nine young Gambian children were studied for 61 weeks. About half of them had trachoma at the outset, and 80% of the remainder acquired the disease while under observation. IgG trachoma antibody in the serum and IgG and IgA antibodies in the conjunctival secretions (CS) were titrated by an indirect immunofluorescence method. In serum samples obtained in capillary tubes the mean titre was slightly higher than in samples collected on filter paper. Serum antibody at titres >/= 1/10 was invariably associated with a clinical diagnosis of trachoma; it increased both in frequency and titre as the disease progressed, and was present in about half of those with Tr II. In CS, IgG antibody was present less often and at lower titres than in serum, and IgA antibody was detected even less frequently. There was some evidence of correlation between the titres of IgG and IgA antibodies in CS, but none for a relationship between the titres of the antibodies in serum and those in CS. Antibodies were almost never present in the absence of conjunctival follicles, but their titres were unrelated to the degree of follicular hyperplasia; there was no obvious relationship between the serological findings and corneal lesions. In children diagnosed clinically as trachoma, serum antibody was present in almost all those with conjunctival inclusions, and in a proportion of inclusion-negative subjects; the mean titre was much higher in the inclusion-positive group.These findings do not settle whether CS antibodies are made locally, or are derived partly or wholly from the blood. They suggest that the indirect immunofluorescence test may be a useful diagnostic aid in trachoma, particularly in view of the rarity of false positive reactions; but there is at present little to choose between it and complement-fixation tests in terms of sensitivity.

A comparison of the iodine and fluorescent antibody methods for staining trachoma inclusions in the conjunctiva

In terms of the rate of positive diagnoses the indirect fluorescent antibody (FA) test was rather more effective than iodine for demonstrating trachoma (TRIC) inclusions in conjunctival scrapings, but the degree of advantage was not statistically significant. In duplicate scrapings stained at random by one or the other method, FA staining yielded the higher inclusion count significantly more often than did iodine. Some inclusions that failed to stain with FA were found on subsequent staining with Giemsa. A method is described for improving the post-FA Giemsa staining of conjunctival smears stored at subzero temperatures. Given adequate facilities, the FA stain is preferable to iodine for demonstrating TRIC inclusions in the conjunctiva; but the iodine method, properly used, holds advantages for field use.

Criteria for measuring the efficacy of trachoma vaccines in baboons.

Trachoma vaccines are usually assayed by testing their ability to protect monkeys or baboons against subsequent challenge of the conjunctiva with a pathogenic strain of trachoma/inclusion conjunctivitis (TRIC) agent. In such experiments the course of infection in vaccinated baboons was compared in terms of arbitrary scores assigned to a range of clinical signs, and of counts of TRIC inclusions in conjunctival scrapings. Analysis of many such scores indicated that after a large challenge dose of strain MRC-4s, the scores for signs of inflammation reached their maximum earlier than the follicle score; the inflammation score was closely related to the number of inclusions, whereas the follicle score was not. With this system, the optimum periods for eliciting differences between vaccinated and control measures varied according to the sign used; it was later for follicles than for inflammation or inclusions. For assessing the influence of vaccination, the mean of the inflammation scores read weekly for the first 3 weeks after challenge and the mean inclusion score over the same period were equally satisfactory, and either was rather better than the mean of three follicle scores taken over the period 3-6 weeks.For assessing the influence of vaccines or therapeutic agents on experimental trachoma it is important to determine which signs discriminate best between treated and control animals, and the optimum times for measuring them.

Serotypes of trachoma agent isolated in the Gambia: with an observation on the relation between serotype and morphology.

Of 60 TRIC agents isolated from Gambian children with trachoma, 25 were serotype 1 and the remainder type 2. There was a pronounced difference in the proportions of these types in the two villages studied. In the village with a predominance of type 2 strains, TRIC agents remained confined to 2 adjacent compounds over a 14 month observation period. All 19 type 1 strains examined were characterized by the appearance in yolk sac smears of compact aggregates of elementary bodies; such aggregates were seen in only 2 of 35 type 2 strains, and may reflect a chemical difference in the surface of the elementary bodies or in a substance elaborated during their replication.