W. A. Blyth

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.

Interactions of TRIC agents with macrophages and BHK-21 cells observed by electron microscopy.

TRIC agents do not multiply in mouse peritoneal macrophages in culture but have a toxic effect on them, whereas they multiply readily in BHK-21 cells. Sections of macrophages and of BHK-21 cells fixed during the first 90 min after inoculation were examined by electron microscopy. Macrophages ingested all forms of the organism, which were eventually degraded in lysosomes. However, elementary bodies were distinguished from other TRIC particles by the delay in their transfer to lysosomes. BHK-21 cells ingested elementary bodies selectively, and in these cells the organisms were neither found in lysosomes nor degraded. Instead they showed morphological changes that probably represented an early stage of development.

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.

TOXICITY OF THE AGENTS OF TRACHOMA AND INCLUSION CONJUNCTIVITIS.

SUMMARY: The toxicities of strains of trachoma and inclusion conjunctivitis viruses differing in virulence for the chick embryo were compared at different times during their growth in the chick embryo yolk sac. As measured by the ability to kill mice and to induce skin lesions in guinea-pigs, toxicity increased until the time at which embryos began to die. All strains possessed similar particle: toxin ratios. It is considered unlikely that the differing virulence of the strains depends on differences in the amount of toxin per elementary body.

Some consequences of the multiple infection of cell cultures by TRIC organisms

In BHK 21 cells infected with more than one TRIC organism the inclusions coalesce so that 30 hr. after infection only one inclusion per cell remains. Six hours after infection the cells are as susceptible to further infection as uninfected cells.

Cultivation of TRIC agents: a comparison between the use of BHK-21 and irradiated McCoy cells

BHK-21 cells and HeLa cells were as sensitive as irradiated McCoy cells for titrating both fast- and slow-growing strains of TRIC agents. BHK-21 cells were as efficient as irradiated McCoy cells for isolating TRIC agents from trachoma and from the genital tract of patients with non-gonococcal urethritis. More inclusions were formed in BHK-21 than in irradiated McCoy cells.

GROWTH IN THE CHICK EMBRYO OF STRAINS OF TRACHOMA AND INCLUSION BLENNORRHOEA VIRUS OF DIFFERING VIRULENCE.

SUMMARY: The growth in the chick embryo yolk sac of trachoma and inclusion conjunctivitis (TRIC) strains which differ in virulence for the chick embryo was measured in terms of ELD 50 inclusion forming units in HeLa cells and total particles. Observed differences in rates of growth are consistent with the assumption that greater virulence depends on a higher rate of multiplication in the chick embryo. All strains were equally labile when heated at 37° in vitro but only the more virulent kill chick embryos at 37°.

Interactions of TRIC agents with macrophages: effects on lysosomal enzymes of the cell.

TWO CHANGES WERE OBSERVED IN THE ACID PHOSPHATASE OF MACROPHAGES THAT HAD INGESTED INFECTIVE TRIC ORGANISMS: the proportion of extralysosomal enzyme rose, while the total amount in the cells fell. Both effects were directly related to the number of organisms ingested and increased with time. When macrophages were inoculated with more than 50 organisms per cell, changes were obvious within a few hours; with 2-10 organisms per cell changes were detectable only after 18 hr. or more. Enzyme appeared in the culture medium as the amount in the cells decreased. Ingestion of organisms killed by heat or treated with antibody did not induce such changes. In infected BHK-21 cells, no changes in acid phosphatase were detected at any stage of the developmental cycle of the organism.

Neutralization of TRIC organisms by antibody: enhancement by antisera prepared against immunoglobulins

The neutralizing activities of fowl, rabbit and mouse antisera prepared against TRIC agents were enhanced, in some cases over a 100-fold, by the addition of antiserum against the appropriate IgG. Significant non-specific inactivation of the organisms was observed in the absence of antiserum against TRIC agents, in reaction mixtures containing normal serum and antiserum against IgG. Since the extent of this inactivation showed a prozone, control tests with dilutions of normal serum equivalent to the full range of dilutions of the antiserum tested are necessary to show that enhancement of neutralization is specific.