RECOMBINANT INFLUENZA-A VIRUSES AS LIVE VACCINES FOR MAN

Saturday

RECOMBINANT INFLUENZA-A VIRUSES AS LIVE VACCINES FOR MAN Report to the Medical Research Council’s Committee on Influenza and other Respiratory Virus Vaccines A. S. BEARE

T. S. HALL

Clinical Research Centre, Harvard Wiltshire

Hospital, Salisbury,

The infection of volunteers with five hybrid influenza-A viruses is described. Four of these were produced in Great Britain by recombining an Ao virus, non-infective for man, with a wild Hong Kong like strain. The fifth was the American recombinant, X-31, derived from similar parents and widely used in the manufacture of killed vaccines. All five viruses had the hæmagglutinin and neuraminidase of A2/Hong Kong/68. Two of the viruses were not attenuated and induced symptoms of clinical influenza. The other three were appreciably attenuated and were infective and antigenic. It seems that recombination is a rapid and effective way of producing live vaccine viruses to specification. It is also the quickest known method of attenuation. Introduction are

believed

logically effective, providing they

are

be

epidemiocorrectly administo

tered and their constituent viruses conform to certain standard criteria.1-3 Immunological failures, which are common, are generally attributable to inherent defects in the viruses. Thus, inhibitor-resistant influenza-A strains, which are commonly used as

vaccines,

are

sometimes unobtainable and

are

poorly

infective in a population which has experienced their particular subtype. Viruses adapted to low tempera-

frequently give a poor yield and this must be improved by laboratory manipulations which carry a danger of reversion to virulence. Lately, several workers have suggested the use of temperaturesensitive (ts) conditional lethal mutants as live vaccines.7,8 More interestingly, there have been proposals for the making of vaccine viruses by recombining strains of known properties 7, 9-a suggestion that opens up considerable possibilities. It is nevertheless important that this approach should be made cautiously and that full account should be taken of the possible hazards. We have done some preliminary trials in volunteers with recombinants developed from known human viruses, and have tried to assess how far human

tures 5,6

7737

December I97I

can be predicted from the characters of the parent viruses.

responses

hereditary

Materials and Methods Viruses recombinant of Ao/PR8/34 and A2/Aichi/2/68 (antigenically identical with A2/Hong Kong/68) was first produced by Dr. E. D. Kilbourne (New York). It was obtained by us from the World Influenza Centre, London, and was passed once in embryonated eggs before being given to volunteers. Four other recombinant strains were prepared by Dr. D. McCahon and Dr. G. C. Schild, of the National Institute for Medical Research, from parent viruses provided by ourselves. These were Ao/PR8/34 of unknown passage history which had been passed once in organ cultures of human embryo trachea and once in leucosisfree eggs in Salisbury, and a newly isolated strain of A2/ Hong Kong/68 known as A2/England/939/69 which had been isolated from an influenza patient in Odstock Hospital, Salisbury, directly in leucosis-free eggs in which it received three passages. The Ao/PR8 strain, when given to ten antibody-free volunteers in an individual dose of 10802 50% egg-infecting doses (E.I.D.5o) did not cause infection. Attempts to enhance its virulence for man by prolonged serial passage at high temperatures and by man-to-man passage gave only limited success. We concluded that in its original state it was totally avirulent. In contrast, A2/England/939/69 was considered to be fully virulent and to be a representative wild virus. The parents of X-319 (Ao/PR8 and AJAichi/2/68) were thought to have been similar to those of the British recombinants. Four cloned recombinants of 939 x PR8 were used in the trials and were designated 6, 7, 64c, and 64d. Methods used in their isolation are being described separately. 10 Three characters, besides the antigenic characteristics, were used in their recognition: a high yield in eggs (a feature of PR8), virulence for mice (PR8), and restricted growth at 39 °C (939). All five recombinant viruses possessed the surface antigens of A2/Hong Kong/68 and it was possible to predetermine this in the course of their

X-31,

Summary

LIVE influenza vaccines

II

a

production.9,lo Clinical Observation of Volunteers Surveillance and grading of clinical symptoms have been described previously.ll Arbitrary numerical scores were also allotted based on incidence and duration of pyrexia, coryza, and subjective discomfort, and on increased use of handkerchiefs.5,122

Laboratory Tests of Infection Nasal washings collected on the third and fourth days after virus inoculation were inoculated into the allantoic cavity of 11-day-embryonated hens’ eggs. These were tested for virus haemagglutinin after 2 days’ incubation at 33 °C.

1272 6, 7, 64C, AND 64D (AO/PR8/34 X A., ENGLAND/939.69) (AO’PR8/34XAs/AICHI’2;68)

TABLE I-RESPONSES OF VOLUNTEERS TO VIRUS CLONES AND TO

Numerators

are

x-31

numbers of responses, denominators numbers of

Sera collected before the trial and 2-3 weeks afterwards assayed for haemagglutination-inhibiting (H.I.) antibodies by standard methods.33

were

Results

Seventy volunteers took part in the trials. The individual virus dose was 105-0-106.0 E.I.D-5,,, and all the recombinants were fully infective at this concentration. It was clear from a summary of the results (table i) that clones 6 and 7 were not attenuated, although it seemed possible that clone 6 at least was less virulent than unmodified A2/Hong Kong/68. 12, 13 However, clones 64c and 64d induced only mild local symptoms and seemed to be adequately attenuated without any evident loss of infectivity and antigenicity. X-31 was also appreciably attenuated but possessed more residual virulence than 64c and 64d. Because of the difference in gross characters of the two parent viruses (PR8 and 939) we tried to relate the virulence of the recombinants to their recognisable affinities with the virulent or non-virulent parent. This was not possible (table 11). The viruses need to be examined in greater detail, and additional recombiTABLE

II-COMPARISON OF THE LABORATORY AND VIRULENCE MARKERS OF AO/PR8/34, AND THE RECOMBINANT STRAINS (SEE MCCAHON AND SCHILD 10)

A2/ENGLAND/939/69,

- r—=high, = = low; += intermediate. The parents of X-31 are thought to have been similar to those of the British recombinants.

need to be prepared for genetic analysis, but there is as yet no easy solution to the problem of laboratory markers for human virulence. nants

Discussion

The standards for influenza viruses which are to be used as live vaccines are: availability in high yield,5

specimens tested.

of hsemagglutinins and neuraminidases with those of the current epidemic strains, adequate attenuation without loss of infectivity and antigenicity, and genetic stability. All these properties are genetically determined,9 and the recombination of viruses with suitable basic characters undoubtedly offers the best way of producing vaccine viruses to specifications. Technical problems are few, and recombinants can be selected without the use of plaques. 14

identity

the possible drawbacks ? The be the continued dependence on greatest volunteers for the screening of viruses. All the characters we have described can be examined in the laboratory, except for human virulence, for which a reliable marker system is not yet in sight. Recombination looks like a significant advance on all other techniques for the rapid and efficient production of live vaccine viruses. It can take account simultaneously of such diverse properties as rapid adaptation to particular culture systems and attenuation for man. Obviously, however, it is in its reliability for the production of attenuated viruses that the technique must ultimately be judged. Our results suggest that, providing the parent strains are suitable, a high proportion of recombinant progeny will be adequately attenuated. Kilbourne 9 maintains that the virulence of influenza viruses is governed by several different genes, and that the mating of a highly virulent strain with an avirulent one will probably lead to the formation of recombinants in which its expression is greatly reduced. Theoretically, it should have been impossible to obtain recombinants more virulent than A2/Englandj 939/69, since progeny strains can only possess what has been given to them by their parents. Nevertheless, this possibility cannot altogether be excluded, and Kilbourne himself has occasionally seen new phenotypes with properties not seen in their parents. Finally, we should like to comment on the suitability of PR8 as a parent of recombinants for man. Its obscure passage history and unrecorded loss of human virulence must cause some misgiving. Mackenzie’1 has suggested that a " master strain " of virus could be prepared by the injection of ts lesions into a virus of known pedigree. This would be completely characterised and would be recombined with each new antigenic subtype to produce a new vaccine strain whenever it was required. The suggestion would appear to have much to commend it.

What, then,

are

snag seems to

1273 We thank Dr. D. McCahon and Dr. G. C. Schild for making the recombinant viruses and for helpful discussions; Dr. D. A. J. Tvirell for advice and encouragement; the volunteers for their cooperation; Nurse J. Bailey for help with the surveillance; and Mrs. Kathleen Keast and Mrs. Kay Callow for technical assistance. The unit receives a grant from the World Health

Organisation. Requests for reprints should be addressed to A. S. B. REFERENCES 1.

2.

Smorodintsev, A. A., Alexandrova, G. A., Chalkina, O. M., Selivanov, A. A. in Applied Virology (1st Annual Symposium, Boca Raton, Florida, 1964) (edited by M. Saunders and E. H. Lennette); p. 142. Sheboygan, Wisconsin, 1965. Slepushkin, A. N., Bobyleva, T. K., Russina, A. E., Vitkina, B. S., Ellengorn, N. S., Zhdanov, V. M. Bull. Wld Hlth Org. 1967, 36, 385.

3. Beare, A. S., Bynoe, M. L., Tyrrell, D. A. J. Br. med. J. 1968, iv, 482. 4. Beare, A. S. Unpublished. 5. Beare, A. S., Maassab, H. F., Tyrrell, D. A. J., Slepushkin, A. N., Hall, T. S. Bull. Wld Hlth Org. (in the press). 6. Maassab, H. F. J. Immun. 1969, 102, 728. 7. Mackenzie, J. S. Br. med. J. 1969, iii, 757. 8. Mills, J., Chanock, R. M. J. infect. Dis. 1971, 123, 145. 9. Kilbourne, E. D. Bull. Wld Hlth Org. 1969, 41, 643. 10. McCahon, D., Schild, G. C. J. gen. Virol. (in the press). 11. Tyrrell, D. A. J. Am. Rev. resp. Dis. 1963, 88 (no. 2), 128. 12. Beare, A. S., Schild, G. C., Hall, T. S., Kundin, W. D. Lancet, 1971, i, 305. 13. Beare, A. S., Bynoe, M. L. Br. med. J. 1969, iv, 198. 14. Webster, R. G. Virology, 1970, 42, 63.

CONTROLLED TRIAL OF AZATHIOPRINE IN CROHN’S DISEASE

(b) Abdominal-wall or perianal fistula. (c) Diarrhoea (passage of three or more loose stools per day in patients who had not had bowel surgery). (d) Sigmoidoscopic evidence of mucosal disease in the form of mucosal haemorrhage or ulceration. Or at least two of the following laboratory investigations: (a) Haemoglobin < 12.5 g. per 100 ml. in men or < 10-5 g. per 100 ml. in

(d)

in 1st hour. Serum-albumin <3’0

P. BECK

D. BAINTON H. CAMPBELL

University Hospital of Wales and Medical Research Council Epidemiological Unit, Cardiff The effect of azathioprine in patients with active Crohn’s disease has been evaluated in a double-blind cross-over trial. Fifteen patients were treated with both azathioprine and placebo: only two improved while on azathioprine, six got worse, and in seven there was no change. The drug seemed to be the cause of deterioration in two of the patients. Although the drug may be of some value in a few patients, it should be used with caution and only when other measures have failed. Sum ary

Introduction

SoME have found azathioprine of value in Crohn’s disease.1-3 It has been used in ill patients, and in one controlled trial azathioprine was found to be of value in preventing a relapse after remission had been obtained with steroid therapy.4 We have assessed the drug in a controlled clinical trial in patients with active disease. Patients and Methods

Patients with clear evidence of active Crohn’s disease considered for the trial.55 Tissue obtained at laparotomy or by rectal biopsy suggested the diagnosis in nine patients, while in seven others we relied on radiological and clinical features. The criteria of activity are were

as

follows: At least

one of the following clinical features: (a) Weight loss of more than 10 lb. (4-5 kg.) in the previous

6 months.

mm.

g. per 100 ml.

Patients Sixteen patients were admitted to the trial (table l). Six patients had fistulas (four of which opened on to the abdominal wall, one was vesicocolic, and one patient had multiple perianal fistult). Two patients were dwarfs and had recurrent abdominal colic, one other patient with severe rectal disease had an ulcerated rectal stricture, while the remaining seven patients had weight loss or diarrhoea as a prominent feature. Four of the sixteen had had previous surgery with resection of diseased bowel. In nine patients the small bowel was chiefly involved, whereas in seven the colon was chiefly affected. Treatment with sulphasalazine (salazopyrin) and supplements of iron and folic acid were continued during the trial in those patients already receiving these drugs; two received corticosteroids throughout the trial. The criteria of activity of the disease in each patient are shown in table 11.

Design of Trial The trial

was

Azathioprine

J. RHODES

women.

(b) White-blood-cell count > 12,000 per c.mm. (c) Erythrocyte-sedimentation rate (Westergren) >20

of

was

double-blind and cross-over design. given for 2 months and placebo tablets a

for 2 months, the sequence of the two treatment periods being made on a random basis. Azathioprine was given in a dose of 4 mg. per kg. body-weight for the first 10 days and 2 mg. per kg. body-weight thereafter. To detect any leucopenia or thrombocytopenia the white-blood-cell count and platelet-count were measured twice weekly during the first 2 weeks of each period and weekly thereafter.

Assessment of Change The following measurements were made at the beginning and end of each trial period : body-weight, haemoglobin, white-blood-cell count, erythrocyte-sedimentation rate, and serum-albumin. Patients were also given a diary card and asked to record daily the number of bowel motions and the occurrence of colic. At the end of each 2-month period the patient was asked to describe his condition during that period as excellent, good, fair, bad, or terrible. These comments were taken into account when assessing whether there had been any improvement or deterioration

during the period. Results

Fourteen patients completed the trial and two were withdrawn because of complications (patients 15 and 16). Patient 15 had a severe generalised arthritis with fever on the tenth day of the second period, while on azathioprine. His family doctor discontinued the drug, and an attempt to restart therapy caused an immediate recurrence of the arthritis. Patient 16 had leucopenia, with a white-blood-cell count of 1000 per c.mm. on the tenth day. The drug was discontinued but the count did not return to normal until 6 weeks later. The design of the trial was modified a little in three patients. Patient 3 insisted on changing the therapy after only 6 weeks of the first period, because the