PART I.

Reference i. To inquire and report from time to time on matters relating to the preparation, testing and standardisation of vaccine lymph.

The Royal Commission on the use of calf lymph.

The kind of lymph in general use at the present time in this country is the outcome of a recommendation of the Royal Commission on Vaccination, 1889-1896.*

The fourth reference to the Commission reads :-" Whether and, if so, by what means vaccination with animal vaccine should be further facilitated as a part of public vaccination." The reply to this, given in paragraph 437 of their final report, reads:" So long as the State with a view to the public interest, compels the vaccination of children, so long even as it employs public money in promoting and encouraging the practice, we think it is under an obligation to provide that the means of obtaining calf lymph for the purpose of vaccination should be within the reach of all." This recommendation found expression in the Vaccination Act, 1898, and the subsequent order of the same year. Instruction 3 of the third schedule to the Vaccination Order, 1898, reads:-" All public vaccinations are to be performed with glycerinated calf lymph, or with such other lymph as may be issued by the Local Government Board. If the parent or other person having the custody of a child requires that it shall be vaccinated with lymph issued by the Local Government Board, the vaccination must be performed with such lymph."

At the present day the use of animal lymph is practically world-wide and arm-to-arm vaccination, although not expressly prohibited, can no longer be practised at the public expense in this country.

The origin of vaccine lymph and its potency.

Vaccine lymph is obtained from susceptible animals which have been inoculated with "seed " lymph. The origin of seed lymph is not always easy to establish; it may be obtained through various channels most of which probably originate from one source" smallpox."

Seed lymph may be obtained :—

(a) From smallpox direct.-Inoculation of the human subject with smallpox virus gives rise to smallpox which, though often mild in degree, retains the power of disseminating the infection. On the other hand, when smallpox virus,

* Final Report of the Royal Commission appointed to inquire into the subject of Vaccination: London, 1896.

sometimes after a preliminary passage through monkeys, is passed through animals such as bovines and rodents, it assumes the properties of vaccinia. In this form the virus, although deprived of the power of generating smallpox, retains its capacity to protect against that disease.

Monkeys inoculated on the skin with smallpox virus show vesiculation on the sixth to eighth day. The vesicles closely resemble those produced by the inoculation of a similar animal with vaccinia virus. If the contents of these vesicles obtained by variolating the monkey are collected and a calf or rabbit inoculated with them, vesiculation indistinguishable from that of ordinary vaccinia may result. Not uncommonly, however, the lesions fail to proceed to vesiculation on the animal inoculated directly from the monkey it may be necessary to pass the material through three calves or rabbits in succession before what is regarded as the reaction typical of vaccinia appears.

Calves or rabbits inoculated directly with smallpox virus not infrequently fail to give a definite reaction on the first attempt. Cows are stated to react better than calves, and in the tropics the direct variolation of cows is frequently successful, probably because there is an abundance of smallpox material available which can be selected at suitable stages. By this means new strains of vaccine lymph are raised, which after passage through calf or rabbit (or both) are used for human vaccination. Such strains give excellent results.

(b) Cowpox.-Material from the vesicles on the teats and udders of cows naturally infected with cowpox, or the hands of milkers inoculated by contact with the infected cows, gives rise on calves or rabbits to the characteristic vesicular eruption indistinguishable from that produced by vaccine. lymph. As seed lymph, however, such strains generally tend to degenerate rather rapidly when passed from calf to calf.

(c) Horse-pox, sheep-pox, goat-pox, fowl-pox, swine-pox. -The relationship of the pock diseases of domestic animals and fowls to one another and to smallpox has not yet been fully elucidated. In this connection research work is in progress in many countries and it is claimed that horse-pox and sheep-pox have yielded a virus indistinguishable from that of vaccinia but there is not any record that strains from these sources are in use at the present time.

(d) Vaccinia in the human being or retro-vaccination.— This is obviously not an original source of seed lymph, as the subject must necessarily have been vaccinated previously; human lymph can be employed to raise a new strain of seed lymph by the inoculation of animals, and this method is still used abroad, but not in this country. Lymph of

this origin, if inoculated on to the skin of a calf or rabbit or other susceptible animal, gives rise to the characteristic vesicular eruption of vaccinia.

Reference may here be made to Jenner's lymph. Jenner sent several samples of his vaccine lymph abroad, and some establishments (e.g. at Vienna and in the United States of America) believe that these samples were the origin of the seed lymph they still use. At Vienna* it is claimed that Jenner's lymph has been carried on from arm to arm ever since his samples were received over 100 years ago.

Maintenance of quality.

Repeated transference from calf to calf tends to cause deterioration in vaccine lymph, the lymph ceases to produce satisfactory vesiculation-the criterion adopted in lymph production -and the vesicles become irregular and ill-formed. Seed lymph therefore cannot be relied upon to keep its original quality if passaged from calf to calf exclusively. The deterioration has been thought by some workers to be due to admixture in increasing quantities with undesirable micro-organisms present in calf skin, with the result that the vaccine virus cannot assert itself in its full vigour. Though this may be a cause in certain cases it is not the only one, for in deteriorated lymph there is not necessarily any increase in the bacterial content nor any departure from the normal flora.

A suitable calf vaccinated with a seed lymph of good quality by linear incisions should show well-developed vesicles continuous along the lines of inoculation, round in shape, uniform in size, pink at the edges, pearly in the centre and slightly umbilicated. The vesicles should be 5-6 mm. in diameter and raised 2-4 mm. above the surface.

If on vaccinating several animals with a sample of seed lymph there is marked departure from the above description in one or two of them, it is probably due to the idiosyncrasy of the individual animals, but should the departure become frequent, the inference is that the seed lymph has deteriorated. In the latter event it is not unusual to obtain new seed lymph from a recognised laboratory or to propagate a new stock in one of the ways previously mentioned. If seed lymph of fair quality is inoculated cutaneously on the rabbit an improvement is noticed in the quality of the strain when subsequently used for calf vaccination. By periodical passage in this manner, the quality of the seed lymph can be maintained for many years. Methods of vaccinating rabbits for this purpose vary in different establishments; some use full-grown rabbits and collect large and fully developed vesicles on the fourth to sixth day, whereas others

Kraus, R., Levaditi, C: Handbuch der Technik und Methodik der Immunitätsforschung. 1908. Vol. I, p. 597.

use rabbits 6-8 weeks old, vaccinating them on the gently abraded, freshly shaved surface and collect as soon soon as the resulting eruption shows signs of vesiculation-from 48 to 78 hours after vaccination. Some prefer to use the lymph fresh from the rabbit, whereas others store it mixed with 50 per cent. glycerine for years and then obtain excellent results with it. The degree to which the seed lymph obtained from the rabbit is diluted before use for calf vaccination varies greatly. Some use it in equal volume or weight with pure glycerine, others dilute it to 1 in 12 or 1 in 18 with 50 per cent. glycerine in sterile water or saline. But whatever the method employed, almost all vaccine establishments use the rabbit by one or other of these methods for the purpose of improving and maintaining the quality of the seed lymph, with great practical advantage to the process of vaccine lymph production.

A regular sequence of animals may be employed, as in the United States (human being, rabbit, calf) and in Java (human being, rabbit, calf, buffalo).

Cultivation of vaccine virus in vitro.

Many workers have attacked the problem of cultivating the virus in vitro, occasionally with some degree of success. The recently published method of Carrel and Rivers (1927) who have obtained definite evidence of proliferation of vaccine virus when grown in vitro in contact with chick embryo cells, has been tested by two of our number, F.R.B. and J.C.G.L., the latter in conjunction with Drs. Maitland and Schütze of the Lister Institute. Starting with a seeding of low potency they have demonstrated an increase in potency amounting to one thousandfold. Tests are still in progress. Should further experience

of the method substantiate the preliminary findings, we may look forward to the possibility of dispensing with the current methods of producing lymph on the large scale. Moreover, like the vaccine obtained by intracerebral passage through the rabbit (the neurovaccine of Levaditi), this cultivated virus would have the merit of being free throughout from all extraneous organisms.

Potency of vaccine lymph.

By potency in this connection is meant the power which vaccine lymph exerts when cutaneously inoculated on the human subject, as indicated by the extent and character of the reaction at the site of inoculation and the degree and duration of immunity against smallpox produced.

Although the vaccine lymph in general use to-day has been derived, as has been seen, from different sources and by several procedures, there seems to be a consensus of opinion that the different strains have similar immunising powers. So far as is known the action in this respect is similar, even with such widely separated strains as one derived by propagation from arm

to arm for over 100 years and another obtained by direct variolation of animals. In very few instances has it been alleged that there was a wholesale failure of vaccine lymph to protect against smallpox, and these are of doubtful authenticity.

The proof of this immunising action rests primarily on Jenner's experiment, published in 1798, in which he demonstrated that a child after successful vaccination was immune to smallpox when inoculated subsequently with the virus of that disease. In the early days of vaccination this experiment was repeated many thousands of times* with similar success, and in this manner convincing evidence was obtained of the efficacy of vaccination in protecting against smallpox. This experiment with full controls has often been performed on animals with the same result.

Further proof of the protection afforded by vaccination against smallpox consists of mass observations on human beings, which show that people vaccinated antecedently within a reasonable time escape smallpox, whereas others not so protected and with the same exposure do not. This observation has been so general throughout the world, and has extended over such a long period of time, that in spite of hostile criticism it has been accepted as a fact in all civilised countries.

Although the practical ability of vaccination to protect individuals and communities against smallpox has been abundantly established, there are many problems in connection with this specific immunising process awaiting elucidation. Evidence is accumulating to show that the clinical forms of smallpox now occurring vary considerably in their virulence, and it is conceivable that there may be other differences in the strains of smallpox virus in different parts of the world. Great advances have been made in recent years in our knowledge of the group of virus diseases to which smallpox appears to belong, and methods for identifying individual strains are now under investigation both in this country and abroad. Should these researches prove satisfactory a similar enquiry should be made into the properties of the various strains of vaccine virus with a view to defining so far as possible their individual protective values and other characters. The steps taken by the Health Committee of the League of Nations, and also in this country, towards the standardisation of vaccine lymph are referred to elsewhere in this report (vide p. 19 et seq.).

A further matter requiring elucidation is the precise mechanism of the immunity resulting from vaccination. It has been suggested, for instance, that the production of vesicles and the immunising action of vaccine lymph are different properties, and that one may be affected without detriment to the other. The question whether, and if so to what extent, it is practicable

Report of the Royal College of Physicians of London, on Vaccination 1807, p. 5.