Simultaneous administration of live, attenuated influenza A vaccines representing different serotypes

Simultaneous administration of live, attenuated influenza A vaccines representing different serotypes Peter F. Wright, Meena Bhargava, Philip R. Johnson, Juliette Thompson and David T. Karzon Two live attenuated cold-adapted influenza A vaccines representing current H1N1 and H3N2 serotypes were simultaneously administered intranasally to doubly seronegative childrert No clinical illness resulted Characterization of virus shedding demonstrated shedding of both original vaccine strains and of reassortant virus with the H3N1 and HIN2 phenotyp~ A serum immune response to both serotypes was demonstrated The successful simultaneous administration of two influenza A vaccine strains enhances the potential usefulness of this approach to influenza prophylaxsis~

Keywords:Viruses: influenza: live attenuated vaccine: HI NI serotype: H3N2 serotype

Introduction Live, attenuated, intranasally administered influenza vaccines represent a promising approach to the prevention of influenzal illness ~a. In all pediatric studie~ conducted to date, these vaccines have been administered individually as a single strain. Because of the concurrent circulation of several influenza A strains, vaccine recommendations often are for a muitivalent vaccine. It therefore was of practical as well as theoretical, interest to examine in detail the virologic, clinical and immunologic response to simultaneously administered live attenuated influenza vaccines. The vaccines chosen were derived by reassortment between a cold adapted attenuated master strain, influenza A/Ann Arbor 6/60 and currently circulating H I N ! (A/California 10/78, CR37) and H3N2 (A/Washington 897/80, CR48) strains. All six internal genes were derived from the master strain with the surface glycoproteins reflecting the current strains.

Methods The vaccines were administered to 18 young children (aged 12 to 37 months) who were pre-screened by a haemagglutinin inhibiting antibody assay and found to be seronegative to both influenza A H I N I and H3N2 strains. They were thus undergoing their primary exposure to influenza with the administration of the vaccin~ The children were observed for three days before and 10 days after vaccine administration with daily sampling by the nasal wash technique for isolation of influenza virus. Vaccine administration was by the intranasal route with a total volume of 1 ml containing

Department of Pediatrics, Vanderbilt University Nashville, Tennessee, USA (Received 25- October 1985; revised 10 January 1 985)

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I& "5 TC1D of each vaccine. The clinical design of the study was identical to that previously described in which similar vaccines were administered as single serotypes to children ~. To isolate virus, nasal wash material was inoculated onto rhesus monkey kidney (RMK) cells at 39 and 32°C and also canine kidney (MDCK) tubes at 32°C. Laboratory analysis was carried out to characterize the serotype of the haemagglutinin and neuraminidase of the virus being shed. Towards this end. the original nasal wash was titred in a plaque titration in M D C K cells. Plaques were picked at terminal dilutions for characterization of their haemagglutinin and neuraminidase subtypes. The initial R M K tube passage ofvirus was repassaged in the presence of either H I N I or H3N2 antiserum to detect which subtype(s) were being shed by the child. Haemagglutinin characterization was by standard microtitre haemaglutination inhibition (HA1) assay and neuraminidase inhibition was by the standard assay using fetuin as a substrate a. Serologic responses to the vaccine were determined by HAl on serum drawn at 2, 4, 6 and I I weeks after immunization. Additional evidence of seroconversion was sought by testing the4 week sera for antibody using a type specific ELISA assay with a purified haemagglutinin-neuraminidase preparation modified from the assay of Murphy et a/4.

Results The two vaccines were administered simultaneously to 18 children with four uninoculated placebo controls included in the studies. Clinical illness is summarized in Table 1. As in other trials, some background of mild respiratory illness was seen in both vaccines and controls. There was no suggestion of influenzal like symptoms or temporal clustering to suggest that illness was vaccine related.

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Live attenuated influenza vaccines" P.F. Wright et al. Table 1

Number of days on which children exhibited indicated symptoms 16-

No. Days Fever Cough Rhinorrhoea No. With observed >101"F > 8 X >2 g Otitis Vaccinees 18

180

4

12

1

3

33

3

0

0

0

14-

Controls

4

X

12 -

All 18 vaccinees shed both H3 and H! haemagglutinin containing viruses following vaccination. Total virus shed over the course of the replication of the simultaneously administered vaccines is shown in Figure 1. The overall pattern of virus shedding is similar to that previously observed when vaccines were administered individually:. The bimodal peak appears to reflect maximal shedding of first H3 and then HI virus. In Figure2, isolation ofvirus with an H! haemagglutinin and with an H3 haemagglutinin is summarized. This data was derived from virus expressed after inhibition of virus in the initial R M K harvest with HI or H3 antisera which allowed growth of the minority population in the harvest

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Figure 2 Number of children shedding influenza virus of indicated serotype after simultaneous administration of cold-adapted influenza H1 N1 (X) and H3N2 (0) vaccines

XX X

l X

X

X

X X

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X

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A total of 305 plaques were picked at terminal dilutions of the original nasal wash to determine the serotype that was being shed in highest titre and to look for viruses that had become reassortant in the h u m a n host between the two surface glycoproteins. After a single R M K passage, the plaque purified viruses were characterized as to their haemagglutinin, Table 2. The haemagglutinin content of the plaques at a terminal dilution should reflect the predominant virus in the nasal wash. Throughout the

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Table 2

g

Characterization of plaques picked from original nasal

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No. with indicated H X

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Days after voccinotion Figure 1 Titre of virus shed by seronegative children after simultaneous administration of cold-adapted influenza H1 N1 and H3N2 vaccines, e , Geometric mean titre

306

Vaccine, VoL 3, S e p t e m b e r 1 9 8 5

No. picked

1 2 3 4 5 6 7 8 9

9 46 41 38 41 40 34 40 12

10

4

Total

305

Nonviable or mixed plaque

H3

H1

5 5 3 4 3 7 2 2 1

4 14 15 11 5 4 2 2

5 26 23 23 33 29 30 36

28

57

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LJ've attenuated inf/uenza vaccine~" P.F. Wright et al. Table 3

Characterization of plaques from original nasal wash Reassortant plaques

Days

1 2 3 4 5 6 7 8 9 10

Total

Total No. characterized

H3N1

H1 N2

% Reassortant

9 28 33 26 29 27 21 23 12 4

2 2 5 1 -

3 3 3 7 2 4 13 4 4

22 18 24 15 24 7 19 56 33 100

212

10

43

25%

Table 4 Serologic response to bivalent live, attenuated influenza vaccine in 18 children

4 Weeks

H3N2 H1 N1

6 Weeks

HAl

ELISA

HAl

11 10

13 (72%) 16 (89%)

12 12

study and particularly from day five on, HI viruses clearly predominated with 220 plaques having the HI phenotype and 57 having the H3 phenotyp'e. Twenty-eight plaques were nonviable or mixed and were not characterized further. Two hundred and twelve plaques from three trials involving nine children were analysed in further detail by characterizing their neuraminidase to look for evidence of reassortment between the two input viruses, Table3. Each of the nine children shed reassortant virus with a total of 53 of 212 plaques, 25% being reassortant. Nineteen plaques yielded both input strains and were presumably not pure plaques. Thirty-four of the reassortant plaques underwent a second plaque purification with four additional plaques being characterized, each of which had the original reassortant phenotype. H I N 2 was the more c o m m o n phenotype with a total of 43 plaques being recovered from the nine vaccinees. H3NI plaques were seen ten times from four children. The time of isolation of reassortant strains appeared to correlate best with peak shedding of the respective haemagglutinin. Reassortant plaques were seen throughout the period of virus shedding and in some patients became the dominant virus shed with 4/4 plaques picked being reassortant in three patients on their last day of virus shedding. Serological response By HAl assay a four fold or greater serum antibody response was demonstrated to HI in 10/18 children by four weeks and in 12/18 children by six weeks, Table4. H3 haemagglutinin inhibiting antibody rises were seen in 11/18 children at four weeks and in 12/18 children by six weeks. ELISA assay on pre samples demonstrated prevaccine type specific antibody in one child to H3 and in one child to HI (both children exhibited post vaccination antibody rises and shed vaccine virus). Qualitative ELISA antibody in the four week samples demonstrated that

16/18 children developed a readily measurable response to HI, GMT(Iogz) of 10.61; and that 13/16 children had a response to H3, G M T (log2) of 9.17. Local respiratory antibody levels which might have detected additional immunologic responses were not measured in these subjects. Earlier trials have demonstrated a low titred local immune response to live influenza vaccine s.

Discussion The demonstration that two influenza strains can be given simultaneously without significant interference with viral replication or immunogenicity is an important step in the development of live attenuated respiratory vaccines. The children in the study undergoing primary exposure to influenza with the "simultaneous administration of vaccines all shed viruses of both haemagglutinin types. However, they exhibited slightly differing patterns ofvirus replication for HI and H3. Virus shedding preceded HI virus but may have been curtailed in the shedding after day 5 by the appearance of HI virus. A further suggestion of interaction between the two strains is the observation that the frequency of Hi antibody responses were substantially higher than seen when this virus is given individually and conversely that H3 HAl responses occurred with somewhat lower frequencyL Neither of these observations suggest sufficient interactions to limit the simultaneous intranasal administration of at least two influenza strains in doubly seronegative children. Potter et aL 6 have explored the simultaneous administration of two cold adapted vaccine strains to ferrets and adult volunteers. In the ferreL the H3N2 virus (A/Victoria 75 CR22) interfered with an H I N I virus antibody response. In adults HI N1 virus limited the H3 N2 response was then given simultaneously. Clearly in older populations the combination of differing levels of pre-existing antibody and perhaps some viral interference may make the response to both components of a bivalent vaccine less predictable Reassortment of influenza strains is commonly believed to be a mechanism for emergence of new influenza strains and reassortant influenza strains have been isolated with some internal genes of H3N2 with the HINI haemagglutinin and neuraminidase ~. The phenomenon is regularly observed in mixed cultures in vitro. However, the direct proof of this p h e n o m e n o n occurring in man rests on a single r e p o t . Our study demonstrated the ease with which this event occurs in vivo. The simultaneous administration of two vaccines with entirely homologous internal genes may favour reassortment. However, the finding of 25% of plaques at a terminal dilution exhibiting reassortment phenotype was surprising. One implication, assuming there is no growth advantage in the reassortant state, is that a very high percentage of the susceptible nasopharyngeal epithelial cells were infected with both strains. This in turn suggests, in spite of the somewhat lower shedding of vaccine virus than wild type9, that the vaccine induced infection is quite extensive within the susceptible population of cells. The other implication of the direct demonstration of reassortment by vaccine virus is the possibility that vaccine virus might be given to someone actively harbouring wild type influenza of a new strain with a resultant reassortment that might be beneficial to the further spread of the emerging strain. As all ofthe genes of the A/Ann Arbor cold adapted parent share in the attenuation of the strain and as a full complement seem to inhibit child to child spread completeley ~, it is difficult to

V a c c i n e , Vol. 3, S e p t e m b e r 1 9 8 5

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Live a t t e n u a t e d influenza vaccines" P.F. W r i g h t et al.

see any selective advantage to the acquisition of some or all of the attenuated genes. Conversely, however, the acquisition of virulent genes from an older influenza strain by a new vaccine strain might render the latter infectious. This may limit use of the new vaccine in the face of a threatened epidemic when older strains are circulating that might act as virulence donors with resultant vaccine strain spread. Some of these theoretical risks may be addressable in vitro by looking at patterns of reassortant viruses that emerge from crosses that might be predicted to occur in vivo. Clearly in the context of giving two vaccines with identical internal genes no such risk was incurred. The successful simultaneous administration of two influenza A vaccines in young children without enhanced illness and with good induction of immunity allows the further evaluation of these vaccines in high risk individuals and on a wider scale in face of an established emergence of new, potentially pandemic influenza strains.

References 1 Wright, P.E, Okabe, N., McKee, K.T., Maassab, H.F. and Karzon, D.T. Cold-adapted recombinant influenza A virus vaccines in seronegative young children. J. Infect Di~ 1982, 146(1), 71

308

Vaccine, Vol. 3, S e p t e m b e r 1 9 8 5

2 Clements, M. L, Betts, R.F. and Murphy, B.R. Advantage of live attenuated cold-adapted influenza A virus over inactivated vaccine for A/Washington/80 (H3N2) wild type virus infection. Lancet 1984, March 31 3 Aymard-Henry, M., Coleman, M.T., Dowdle, W.R., Laver, W.C~, Schild, G.C and Webster, R.G. Influenza virus neuraminidase and neuraminidase inhibition test procedure. Bull, WHO 1973, 48 199 4 Murphy, ER., Phelan, M.A., Nelson, D.L et al. Haemagglutininspecific enzyme linked immunosorbent assay for antibodies to influenza A and B viruses. J. C/in. Microbiol. 1981, 13, 554 5 Murphy, N., Nelson, D., Wright, P. eta/. Secretoryand systemic immunological response in children infected with live, attenuated influenza A virus vaccine Infect- Immur~ 1982, 36(3),

1102 6 Potter, C, Jennings, R., Clark, A. and All, M. Interference following dual inoculation with influenza A (H3N2) and (H1 N1 ) viruses in ferrets and volunteers. J. Me rl Virol. 1983, 11,77 7 Young, J.E and Palese, P. Evolution of human influenza A viruses in nature: recombination contributes to genetic variation of H1 N1 strains. Proc Natl Acad. Sci USA 1979 76, 6547 8 Nishikawa, F. and Sugiyama, T. Direct Isolation of H1 N2 recombinant virus from a throat swab of a patient simultaneously infected with H1 N1 and H3 N2 influenza A viruses. J. Cli~ Microbiol. 1983 18(2) 425 9 Wright, P.F., Ross, K.B., Thompson, J. and Karzon, D.T. Influenza A infections in young children. New Engl, J. Med. 1977, 296, 829