Small-scale trial of live-attenuated influenza vaccine (A/Hong Kong/68)

Small-scale trial of live-attenuated influenza vaccine (A/Hong Kong/68) R. WEST,' J. A. STEWART, F. M. M. WHITE,' A N D J . C. MCDONALD Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Renmin University of China on 06/06/13 For personal use only.

D ~ , ~ ( I ~ I I~IfI' E P pI iId/e l r ~ i o I(111d o g ~Hc(IIIII,McCill U ~ i i ~ ~ c ) r Mo~rfrcvrl. si/y, P . Q . , Cotlotlo H3A 2 8 4 AND

A . BOUDREAULT A N D V . PAVILANIS Art~icrt~el F r c r p p i r r I t ~ ~ ~ i /Lert~oI-c1c.s-Rctpides, r~/r. P . Q . . Co~~cicltr H7V 187 Accepted November 19. 1976 and V. WEST, R.. J. A. STEWART.F. M. M. WHITE,J. C. MCDONALD,A . BOUDREAULT. PAVILANIS. 1977. Small-scale trial of live-attenuated influenza vaccine (A/Hong Kong/68). Can. J. Microbiol. 23: 253-257. Seventy-one men who were given live-attenuated A/Hong Kong/68 (H3N2) influenza vaccine during November 1973, and 34 men given placebo were examined for changes in antibody level. Overall. 12 of the 71 men (17%) given the vaccine showed a fourfold rise in haernagglutinationinhibition (HI) antibody titre after 14 days. No such rises were seen in the 34 men given placebo. However, 10 of the men showing afoutfold rise were from 19 who had no detectable HI antibody to this v i ~ u sbefore vaccination, representing a conversion rate of 53%. The other two had a H I titre of 1/10 before vaccination. The absence of antibody response. at 14 days, in those with an H I titre of 1/20 o r greater may indicate that this represents a protective level against infection. However, the vaccine virus was PI-obably overattenuated and may have constituted a weaker challenge than might occur with a wild strain. No influenza virus was isolated from either group in the week after vaccination and no evidence of transmission to the placebo group was seen. Mild symptoms. chills, muscle pain, and stiffness were more frequently seen in the 12 persons showing a fourfold rise in antibody than in the rest of the volunteers. et V. WEST. R., J . A. STEWART,F. M. M. WHITE. J. C. MCDONALD,A. BOUDREAULT P A V I L A N I1977. S . Small-scale trial of live-attenuated influenza vaccine (A/Hong Kong168). Can. J . Microbiol. 23: 253-257. On a cherche des variations du taux d'anticorps chez soixante-onze hommes vaccines en Novembre 1973 avec la souche vivante attenuee d e ['influenza A/Hong Kong/68 (H3N2) et 34 hommes traitis avec un placebo. Au total chez 12 des 71 individus vaccines (17%). le taux d'anticorps HI a augment6 de quatre fois aprks 14 jours. Aucune tlOvation de la sorte n'a ete observee chez les 34 individus soumis au placebo. Par contre 10 des individus chez qui le taux d'antlcorps HI a quadruple etaient du groupe des 19 individus sero-ntgatifs au moment de la vaccination, ce qui represente un taux de conversion d e 53%. Les deux autres etaient positifs B 1/10 avant la vaccination. L'absence de riponse imrnunitaire apres 14jours chez ceuxqui avaient un titre de 1/20 ou plus laisse croire que ce titre correspond a un niveau de protection contre I'infection. I1 est cependant probable que le vaccin viral ait CtC trop attenue et qu'ainsi I'epreuve d'immunisation ait kt6 plus faible que celle obtenue avec une souche sauvage. Au cours de la semaine qui a suivi I'immunisation, on n'a pas isole de virus de I'influenza chez I'un ou I'autre des groupes et il n'y a pas eu evidence de transmission au groupe traite avec le placebo. Des symptbmes legers, frissons, douleurs musculaires et courbatures ont kt6 plus frequents chez les I2 individus dont le taux d'anticorps a augment6 de quatre fois comparativement aux autres volontaires. [Traduit par le journal)

Introduction Immunization with live-attenuated influenza vaccines has developed slowly since their first experimental use in 1937 (1-3). Smorodintsev vatand Zhdanov (4) outlined the criteria cine should meet. These were that the vaccine 'Present address: Bureau of Epidemiology, L.C.D.C., Health a n d Welfare Canada, Ottawa, Canada.

virus should be recovered after 2-4 days from 50% or more of vaccinated subjects with no antibody at the time of vaccination, that fourfold or greater rises in antibody should be demonstrated in 50% Or more of susceptible subjects, that the vaccine contain lo6 EID,, per millilitre (or more) of virus, and when vaccinated subjects were reinoculated 2-3 weeks later the virus would fail to survive. They claimed their vaccine

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Renmin University of China on 06/06/13 For personal use only.

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C A N . J . MICROBI OL. VOL. 23, 1977

met these requirements and that it gave protection in the face of an epidemic (5). McDonald et al. (6) in England showed that a vaccine prepared from the Russian Ishka A2 strain met these requirements. A better serological response was obtained with lo7 EID,, per millilitre than lo6 EID,, and the virus was recovered more frequently from those vaccinated with the stronger dose. There were three subjects with mild reactions among 64 vaccinated and virus transmission to two contacts was demonstrated. Further studies in England by Andrews et 01. (7) confirmed these findings and showed that lower doses of a vaccine prepared from A/Scotland/49/47 (HIN1) gave similar results. Beare and Bynoe (8) have reported on methods of attenuation of human influenza viruses and on questions of pathogenicity and immunogenicity in relation to attenuation and passage. Recently reports have been published concerning the use of the A/ England142172 (H3N2) 'Alice' strain vaccine in the United States (9, 10). A dose of lo7., EID,, was given by intranasal inoculation twice with a 2-week interval. Conversion rates of 7045% were achieved in those with initial HI titres of 1/40. Aleksandrova et 01. (I 1) reported that oral vaccination caused no reactions and was adequately immunogenic. However, Boudreault and Pavilanis were unable to confirm this (12). In this communication we describe the response, both immunological and clinical, of volunteers to whom an A/Hong Kong/68 (H3N2) attenuated vaccine was administered by the nasal route.

Materials and Methods Vaccine The A/Hong Kong/68 strain, attenuated by Beare and Bynoe. (8), was passed on COFAL/Marek negative 11day-old embryonated eggs and tested according to the Canadian Food and Drugs regulations for live vaccine. The vaccine contained lo7 EID,, per millilitre of virus. Normal allantoic fluid from the same batch of eggs was used as a placebo. The vaccine and placebo were dispensed in 1-ml amounts in sterile screw-capped bottles, identical in appearance and identified only by serial number. Selectiorz of Subject The Canadian Forces Base St-Jean, Richelain, Quebec, was selected for the trial which began in November 1973. A member of the investigating team described the purpose and nature of the trial to three consecutive weekly intakes of recruits. The men were then

invited to participate and 140 out of a possible 146 (96%) volunteered to do so. One volunteer was not accepted because of a history of a possible allergy to eggs. Assigr~nrentto Vaccination and Control Gro~rps Blood samples were drawn from all volunteers on the day of admission to the study. The serum was separated and sent to the laboratory where it was divided into three aliquots. One aliquot was tested for haemagglutinationinhibition antibody titre to A/Hong Kong/68 and the remaining aliquots stored at -30 OC for later studies. The volunteers were divided into five groups according t o haemagglutination-inhibition antibody titre to A/Hong Kong/68 : Group 1. 2. 3. 4. 5.

Less than 1/10 1/10 but less than 1/20 1/20 but less than 1/40 1/40 but less than 1/80 More than 1/80

Number 28 40 26 32 13 Total 139

Within each group the volunteers were randomly allocated to receive vaccine or placebo in the ratio of two to one. This allocation was made for each weekly intake. Preuaccine Procedure Immediately before vaccination, which took place 2 weeks after initial screening, the volunteers were given a brief examination, which included the following: (a) examination for specific physical signs; including abnormal conjunctiva and pharynx, nasal disorders, rales, rhonchi, and joint pain on motion; (6) a self-administered questionnaire on specific symptoms, including cough, chills, headache, watery eyes, nausea, chest pain on coughing, nasal discharge, and joint pains; (c) a throat swab for virus isolation. Two men who had symptoms of a n upper respiratory illness and one who had a primary smallpox vaccination lesion were excluded from the study. Vaccinatiorl The volunteers were called in military record number order and as each presented himself our statistician selected the appropriate vial from a predetermined random sequence and gave it to the person giving the vaccine. The statistician recorded the volunteer's name and the serial number of the vial which was used. Neither the code number of the vial nor its content was told to the volunteer, the person giving the vaccine, or to any person making clinical or laboratory observations. Procedure after Vaccination After vaccination the volunteers were requested to remain in camp for 8 days, but were not otherwise isolated. During this period the following observations were made. (1) On days 2, 4, and 7, each volunteer recorded the presence or absence of specific symptoms on a form identical with that used before vaccination.

255


WEST ET AL.

(2) Throat swabs were taken from all volunteers on days 2, 4, and 7. (3) Further blood samples were taken 14 and 56 days after vaccination on all volunteers remaining in the study. Of the 136 men who received vaccine or placebo, 31 were lost from the trial before the 14th day (23%) and a further 15 before the 56th day (11%). The reasons for these losses were as follows. 14 days 56 days Released from the military Reassigned to other training Hospitalized Withdrew from the study for personal reasons

8 17 3

I 14 0

3 Total 31

0 15

One of the three hospitalized cases with onset 3 days after vaccination was diagnosed as bacterial tonsillitis (throat swabs collected on days 0, 2, and 4 were virologically negative), the other two were not s~lfferingfrom upper respiratory symptoms. Labornfor.yProced~rres All serum samples were tested by the microhaemagglutination-inhibition (HI) test against 8 HA units of A/Hong Kong/68, A/England/72, A/Port Chalmers/73, B/Massachusetts/66, and B/Hong Kong/72. The serum sanlples were also tested by complement-fixation (CF) test for antibodies to influenza v i ~ u sA, influenza virus B, parainfluenza virus types 1, 2, and 3, and adenovirus. All samples from the same person were tested in the same test by laboratory staff who had no knowledge of the source of the specinlen or of the result of the initial haemagglutination-inhibition screening test. Virus isolation from a 20% random sanlple of throat swabs from those vaccinated was attempted both in primary rhesus monkey kidney tissue culture and by allantoic and amniotic inoculation of 10-day embryonated hens' eggs. One blind passage was carried out in each system. This sample was representative of each weekly intake. Fifteen Inen who received vaccine and six who received placebo were tested on four occasions for virus excretion (i e., days 0, 2, 4, and 7). In addition, all samples from men who showed a fourfold response to the vaccine, and had not been previously examined, in the

random sample were also tested. Altogether 14 initially seronegative vaccines were tested. Men who reported mild symptoms on the questionnaire and contacts other than those randomly selected were not examined.

Results The distribution of symptoms obtained by self-administered questionnaire showed little difference between the two groups (Table 1). However, when the symptoms were analyzed with respect to serologic results a t 14 days, volunteers with a fourfold response to A/Hong Kong/68 experienced more chills after 2 days and more pain in the muscles or stiffness during the week after vaccination (Table 2). Fourteen days after vaccination, 10 out of 19 recruits who had antibody levels of less than 1/10 before vaccination showed fourfold or greater rises to A/Hong Kong/68 compared with 0 out of 7 persons receiving in the 14-day samples of those who had a n initial titre of 1/20 or greater (Table 3). The serologic results at 56 days against A/Hong Kong/68 indicate further increases in titre both in the vaccine and placebo groups (Table 4). Four vaccinated men also showed fourfold rises against the more recent A/England/72 and A/Port Chalmers/73 strains after 14 days. Table 5 gives the number of persons showing a fourfold or greater rise in H I antibody titre to the three influenza A antigens after 14 and 56 days. Table 6 expresses the geometric mean titres for the vaccine and placebo groups at days 0, 14, and 56. N o fourfold rise in B/Massachusetts/66 o r B/Hong Kong/72 H I antibody was observed in the 14-day samples. One rise in B/Massachusetts/ 66 was observed in the 56-day samples. No fourfold rise in C F antibody against influenza A was found in the 14-day samples. However, there were 17 rises against influenza A in the 56-day samples. Of these, 14 were in persons who had received vaccine (22.27,) and

TABLE1. Percentage of persons reporting selected symptoms by vaccine o r placebo -

Day 0

Total No. Cough chills Headache Muscle pains Joint pains

*V,vaccine; P, placebo.

Day 2

-

-

Day 4

Day 7

v*

P

v

P

v

P

v

P

78

37

77

37

77

36

74

35

51.3 7.8 16.7 9.0 2.6

54.1 10.8 16.2 8.1 0.0

41.6 13.0 15.6 9.1 1.3

45.9 8.1 24.3 2.7 0.0

46.8 3.9 14.3 10.4 3.9

58.3 5.6 13.9 5.6 2.8

50.0 8.1 14.9 8.1 5.4

45.7 11.4 17.1 5.7 2.9

CAN. J.

256

MICROBIOL. VOL. 23. 1977

TABLE2. Percentage of persons reporting selected symptoms by fourfold antibody rises to A/Hong Kong/68 vaccine


Day 0

Total No. Cough Chills Headache Muscle pains Joint pains

*-,

< fourfold;

Day 2

Day 4

Day 7

Rise* -

Rise

+

Rise -

Rise

+

Rise -

Rise

+

Rise -

Rise

93

12

93

11

91

12

93

12

48.4 6.5 9.7 6.5 0.0

50.0 16.7 41.7 16.7 8.3

40.9 7.5 15.1 4.3 0.0

36.4 36.4 27.3 36.4 9.1

47.3 3.3 13.2 5.5 1.1

58.3 9.1 25.0 41.7 16.7

47.3 9.7 16.1 6.5 3.2

58.3 8.3 16.7 25.0 16.7

+

+, 2 fourfold. TABLE3. Changes in A/Hong Kong/68 antibody level 14 days after vaccination Prevaccination titre Group

Rise

< 1/10

1/10

Vaccine

None Twofold Fourfold

8 1 10

15 6 2

7 1 0

10 4 0

7 0 0

47 12 12

Placebo

None Twofold Fourfold

7 0 0

10 1 0

4 1 0

7 1 0

3 0 0

31 3 0

1/20

1/40

>1/80 Total

TABLE4. Changes in A/Hong Kong/68 antibody level 56 days after vaccination Prevaccination titre Group

Rise

Vaccine

None Twofold Fourfold N. A,*

Placebo

None Twofold Fourfold N.A.*

'N.A.

=

< 1/10

1/10

1/20

1/40

2 1/80 Total

not available.

three in those who had received placebo (1 1.1%). The C F tests gave no evidence of activity of influenza B virus or parainfluenza viruses within the base. However, 26 recruits did show a fourfold or greater rise in adenovirus C F antibody levels, 42% of these were in the vaccinated, and 58Y, in the controls. Five of these rises were demonstrated in the 14-day samples and 21 only in the 56-day samples. N o virus was isolated from any of the throat swabs from volunteers examined.

Discussion Our results suggest that infection with attenuated virus in the immunologically sus-

ceptible group gave rise to symptoms though these were generally mild and none caused interference with normal duty which included physical training. However, though illness was trivial the number at risk was small. The absence of antibody response in those with a prevaccination titre of 1/20 or greater is an indication of what may be a protective titre, and indirectly of those requiring vaccination. This correlates with the results obtained by Rosenzweig et al. (9) using the 'Alice' vaccine strain. The fourfold conversion of 532 of those with a titre of 1/10 met one of the basic criteria of Smovodintsev and Zhadnov (4). The serological results at 56 days indicate that

WEST ET AL.

TABLE 5. Number of persons showing fourfold or greater rises in HI antibody titre


Vaccine

Placebo

14 days

56 days

14 days

56 days

AjHong Kong/68 A/England/72 A/Port Chalmers/73

12 4 4

25 33 26

0 0 0

6 11 7

Number tested

71

63

34

27

TABLE 6. HI antibody response of all men for whom three samples were obtained (geometric mean titres) Vaccine Day

0

14

0

14

live vaccines deserves t o be tested in Canada in the face of naturally occurring outbreaks. However, o u r serological findings at 56 days after vaccination are not satisfactorily explained, so such efficacy trials should be conducted with close surveillance of recipients, and their contacts, to confirm that there is no risk of accidental virus transmission.

Acknowledgements The authors acknowledge the willingness of all the military recruits to cooperate in the trial. The study was carried out through the permission of Lt. Col. P. Gamache. Warrant Officer J. A. Bergeron helped with arrangements a t the base. Miss K. F. Oram performed the laboratory tests.

Placebo 56

257

56

some virus activity must have occurred amongst the recruits, probably after 14 days. No virus strains were isolated a n d we cannot say whether the infections were caused by vaccine or natural virus. Possible reasons for the generally poor antibody response are that the A/Hong Kong/68 strain used may have been overattenuated and therefore lacking in infectivity, and only a single dose of vaccine was administered-compared with two in recent trials of the A/England/72 'Alice strain.' Lack of convincing evidence of virus replication and transmission are compatible with both possibilities. Limited evidence that A/Hong Kong/68 vaccine will give some protection against A/ England/72 and A/Port Chalmers/73 was obtained. The difference in geometric mean titres between vaccine and placebo groups was small. Absolute levels of antibody titres are dependent o n the techniaue used for measurement. I n our case, the microhaemagglutination-inhibition test was performed with the Autotiter machine with zero incubation period of the virus-serum mixture. These results may be as much as twofold lower than those obtained with manual tests. However, the results are comparable with the same technique using a 1-h incubation period of the virus-serum mixture. The testing of live-attenated influenza vaccines in Canada should continue. It is apparent from this small-scale trial that the protective effect of

1. SMORODINTSEV. A. A , , A. I. DROBYSHEVSKAYA, S. 1937. Sov. M. OSTROVSKAYA, and 0 . I. SHISHKINA. Vrach. Zh. 41: 403. 2. SMORODINTSEV, A. A,, M. D. TUSHINSKY, A. I. DROBYSHEVSKAYA, A. A. KOROVIN, and A. I. OSETROFF. 1937. Investigation on volunteers infected with the influenza virus. Am. J . Med. Sci. 194: 159-170. 0 .AM. . 1938. Arkh. Biol. Nauk. 52: 126. 3. C H A L K I N 4. SMORODINTSEV, A. A., and V. M. ZHDANOV. 1957. Results and immediate tasks of the study of live influenza vaccine. Probl. Virol. (U.S.S.R.), (English Transl.), 2: 65-71. 5. SMORODINTSEV, A. A . 1960. New live vaccines against virus diseases. Am. J. PublicHealth, 50, Pt. 2: 4c-45. 6. MCDONALD, J. C.. A. J. ZUCKERMAN, A. S. BEARE, and D. A. J. TYRRELL. 1962. Trials of live influenza vaccine in the Royal Air Force. Br. Med. J. i: 10361042. 7. ANDREWS, B. C., A. S. BEARE, J. C. MCDONALD, A. J. ZUCKERMAN, and D. A. J. TYRRELL. 1966. Further trials of live influenza vaccine. Br. Med. J. i: 637-640. 8. BEARE, A. S., and M. L. BYNOE. 1969. Attenuation of human influenzaA viruses. Br. Med. J . iv: 198-201. 9. ROSENZWEIG, D. V., D. J. DWYER, and J. E. FERSTENFELD. 1975. Changes in small ailway function after live attenuated influenza vaccination. Am. Rev. Respir. Dis. 111: 399403. 10. RUBIN, R. J., G. R. N O B LL.COREY, ~, W. J. BROWN. JR.. D. BRANDLING-BENNETT. H. S. KAYE,M. T. COLEMAN. M. B. GREGG, and W. R. DOWDLE. 1976. Five attenuated influenza A/England/42/72 (H3N2) virus vaccine: afield trial. J. Infect. Dis. 113: 613-620. 11. ALEKSANDROVA, G. I., A. A. SMORODINTSEV, N. M. R. A. GEFT,V. G. PANBELJAEVA. B. J. VASILEV, T E L E E V . M. A. SEJFER, and A. A. SELIVANOV. 1970. Testing the safety and effectiveness of oral administration of a live influenza vaccine. Bull. W.H.O. 42: 429-436. 12. BOUDREAULT, A , , and V. PAVILANIS. 1973. Human immunization with live attenuated influenza vaccine. 41st Annual meeting of the CPHA Laboratoly Division, Toronto. November 1973.