I REVIEW ARTICLE Purified viral neuraminidase vaccine to control influenza D.J.S. ARORA, PH D

The control of influenza by immunoprophylaxis is difficult because of the antigenic mutability of the influenza virus and the unpredictability of its epidemiologic behaviour. The inactivated whole-virus vaccine currently used is not ideal. Vaccination with pure neuraminidase is suggested. The induced antineuraminidase antibody will restrict viral invasion. Mild illness may or may not occur. On subsequent exposure to influenza virus the individual will produce antihemagglutinin and antineuraminidase antibodies and will be resistant to both infection and illness. Since antigenic changes are less frequent in the viral neuraminidase than in the viral hemagglutinin, the vaccine would be usable for longer periods than the presently used inactivated whole-virus vaccine. La lutte contre l'influenza par immunisation prophylactique s'avere difficile en raison de Ia mutabilit6 antigenique du virus responsable et de l'imprevisibllite de son comportement epidemiologique. Le vaccin a virus entier inactive presentement utilis6 n'est pas ideal. On propose Ia vaccination au moyen de neuraminidase pure. Les anticorps antineuraminidase ainsi produits limiteront l'attaque virale. Une maladie benigne peut apparaitre ou non. Lors d'une exposition subsequente au virus de l'influenza, le sujet produira des anticorps antihemagglutinine et antineuraminidase et r6sistera aussi bien a l'infection qu'& la maladie. Comme les changements antigeniques de Ia neuraminidase virale sont moms frequents que ceux de l'hemagglutinine, le vaccin demeurerait utilisable durant de plus longues periodes que le vaccin a virus entier inactive d'utilisation courante.

Influenza, one of the few infectious diseases that man cannot control, continues to cause much disability, death and financial loss throughout the world. During the past century a number of pandemics of influenza have occurred, the most severe being those of 1889-90, 1918-19 (Spanish flu), 1957-58 (Asian flu) and 196869 (Hong Kong flu). The pandemic of 1918-19 was one of the worst plagues in human history and is believed to have killed about 20 million people, most of whom were between 20 and 50 years old. The viruses causing the early epidemics were never isolated (influenza virus Reprint requests to: Dr. D.J.S. Arora, Centre de recherche en virologie, Institut Armand-Frappier, 531, boul. des Prairies, Ville de Laval, PQ H7N 4Z3

was first isolated from a human in 1933) but are assumed to have been the type A strain of influenza virus. The type B strain does not seem to cause worldwide epidemics, results in a milder illness than the type A strain and has been isolated only from humans, whereas the type A virus causes influenza in swine, horses and several species of birds. Recurrent influenza outbreaks have been associated with H3N2 viruses since 1970. Only the period of 1970 to 1971 was free from influenza virus activity. Successive antigenic variants of the Hong Kong influenza virus, typified by the strains A/England/43/72, A/Port Chalmers/i /73, A/Scotland/43/ 74, A/Victoria/3/75 and A/ Texas/i /77, all H3N2 viruses, have produced outbreaks, almost annually in many countries. Activity of a B/Hong Kong-like

virus has also been seen. The 197677 influenza season commenced with the threat of emergence of the A/New Jersey/76 (H..1N1) strain, which fortunately faded away. An A/Texas! 1/77-like strain, which was prevalent early in the 1977-78 influenza season, was gradually replaced by a new antigenic subtype, A/USSR/90/77 (H1N1). This isolate caused widespread illness in China, Asia and Europe, and was found to be similar to the 1950 isolate A/FW/ 1/50. In Canada the incidence of influenza due to all strains was relatively low during the 1977-78 influenza season. Illness due to A/ USSR/90/77 was minimal and was restricted to persons under 24 years of age. There was a low rate of absenteeism, and about 100 deaths were reported to be due to influenza/pneumonia (Dr. J. Peacock: personal communication, 1978). Why does influenza keep recurring in this way despite all our attempts to control it? Why can we control other virus diseases, such as poliomyelitis, smallpox, measles and yellow fever, by vaccination, while all attempts to control influenza by vaccination have failed? The main reason is that these other viruses are stable; their antigens remain the same year after year. The antigens of influenza viruses, on the other hand, undergo extraordinary changes, so that the immunity established by infection or vaccination against today's virus may give little or no protection against viruses appearing in the future. The type A virus is the only one of the three types of influenza virus that infects humans, animals and

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birds, and the relative importance of the nonhuman reservoir is a matter of controversy. There are two known internal antigens, the ribonucleoprotein and the membrane protein, which are responsible for the A, B and C virus types. The subtypes of influenza A viruses, in which there is so much interest at present, possess two surface antigens, the hemagglutinin and the neuraminidase. Continual change in these two antigens is unique to influenza viruses. Antigenic variation of influenza viruses Two types of antigenic variation are observed among influenza viruses: antigenic shift and antigenic drift. The first is characterized by the sudden appearance of new antigenic variants not related to earlier strains.1 It is thought to be caused by recombination between viruses from different hosts, such as birds and humans.14 Support for this hypothesis came after the isolation of a type A influenza virus from apparently healthy birds and the finding in the serum of these birds of antibodies to the neuraminidase of the virus that caused the 1957 pandemic of Asian flu in humans.5 Major changes in the antigenic structure of the neuraminidase of influenza A viruses appear to be less frequent than such changes in the antigenic structure of the hemagglutinin.6 The other type of antigenic variation consists of minor changes in the antigenicity of viruses that are more or less related to each other. Since this antigenic variation proceeds from earlier strains to later strains it is called antigenic drift.7 Antigenic drift due to alterations in the neuraminidase8 and in the hemagglutinin9 of influenza A viruses has been described. With the use of two different test systems (the hemagglutination inhibition test and the inhibition of neuraminidase activity by antisera produced by immunization with different virus preparations) it was found that the minor alterations in the two antigens occurred independently.10 The immunologic and epidemiologic significance of antigenic drift due to alterations in the neuraminidase has not been defined, but mice immunized with isolated purified

1957 N2 neuraminidase have been found to have greater reductions in pulmonary virus titres following challenge with a virus containing the same neuraminidase than after challenge with a virus containing a different neuraminidase (1968 N2).11 Recent data support the view that the phenomenon of original antigenic sin is also operative in the response to the neuraminidase. (The term original antigenic sin was coined to summarize the characteristic antibody response following sequential experience with viruses of different subtypes.12 With exposure to a virus containing a new hemagglutinin, antibody is produced that reacts in the hemagglutination inhibition test not only with the new strain but also with the virus that provided the original antigenic stimulation.) Age-related profiles similar to those seen with titres of antibody to the hemagglutinin have been obtained for neuraminidase antibodies.13 In addition, immunization with H3N2 virus appears to evoke an increase in the titre of antibody to N1 (PR8) neuraminidase in persons born before 1957 but not in those born later.6 Thus, it is evident that the influenza virus continues to mutate even when there is antibody, whether naturally acquired or vaccineinduced, in the community. These mutations represent changes in the two main surface proteins of the virus, the hemagglutinin and the neuraminidase. The control of influenza The problem of controlling an infectious disease turns on identification of the sources of infection and the means of its transmission to man, and the possibility of increasing the resistance of the individual to infection.14 The principle is the same as that of immunization against any other infectious disease: with vaccination an increase in the production of protective antibodies is induced, as after natural infection. Inactivated virus vaccine This type of vaccine, which is widely used in Canada and the United States, is prepared from infected extraembryonic fluid of chick embryos. The virus is inactivated by treatment with formalde-

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hyde and is purified by high-speed centrifugation with a sucrose density gradient. The vaccine is usually administered subcutaneously. It induces the production of antibody against the viral surface proteins (the hemagglutinin and the neuraminidase), which provides protection if the antibody is present in the serum in a reasonable concentration. Unfortunately there are drawbacks to this type of vaccine: * Purified vaccines available commercially occasionally produce a sharp rise of body temperature, which is unpleasant and a deterrent to immunization.15 * Children may react severely to ordinary inactivated virus vaccine, so that care has to be taken to use diluted material.15 * It has been suggested that the Guillain-Barr. syndrome is associated with influenza vaccination, though this syndrome occurs in only about 1 of every 100000 vaccine recipients.15 A ttenuated live virus vaccine This type of vaccine is given intranasally and induces infection of the nose and throat. However, the vaccine must be guaranteed to produce only minimal reactions, and this requires preliminary testing in volunteers. The vaccine induces the production of antibody in both the nose and the serum, but, as with natural infection, the titre diminishes with time. Reinoculation is therefore required, as with inactivated vaccine. The vaccine must be made from a closely related virus strain and a stable attenuated strain. Now in the laboratory genetic recombination can be made between a new virus and an old attenuated strain. The stability of a strain can be tested by studying the gene pattern of the recombinant. A reversion to virulence of such attenuated strains by recombination with the prevalent wild strain cannot be ruled out. Subunit vaccine Subunit vaccines have been produced by the treatment of virus with Tween (sorbitan polyoxyalkalene derivative) and ether, sodium deoxycholate or tri-N-butylphosphate (TNBP).1618 Treatment of whole virus preparations with sodium deoxycholate or TNBP yields

a preparation with hemagglutinating and neuraminidase activity. No untoward reactions were observed in subjects who received 2200 chick cell agglutination (CCA) units, and protection was demonstrated during an epidemic.19 Doses of 6400 CCA units of influenza virus type A or 4800 CCA units of type B have been given intramuscularly with no local or significant systemic reaction.20 Although subunit vaccine has decreased reactivity it is reportedly less immunogenic in children :21 when only one dose of vaccine was given to unprimed children and young adults subunit vaccine proved to be significantly less immunogenic than whole-virus vaccine. One approach to overcoming the decreased immunizing potential of subunit vaccine in unprimed persons is to administer two doses. Recombinant whole-virus vaccine Immunity to influenza is conferred through the induction of the formation of antibody to either or both of the two external glycoproteins of the causative virus. Antibodies to the hemagglutinin neutralize the virus and prevent infection by interfering with attachment of the virus to host cells. Antibodies

to the neuraminidase are nonneutralizing and therefore do not prevent the acquisition of infection, but they do limit the extent of viral replication by restricting the intercellular spread of virus. Kilbourne and colleagues2' proposed a new approach to vaccination against influenza that was based on the possible role of antineuraminidase antibody in restricting viral invasion. This approach, which is depicted in Fig. 1, was explained by Couch and colleagues as follows: "Induction of antineuraminidase monospecific immunity may be achieved by the administration of viral neuraminidase (N) which is 'relevant' or homotypic to the neuraminidase of the anticipated challenge virus. The immunity so induced will be effectively monospecific (anti-N) and, therefore, will not prevent infection with the challenge virus (HN) but should limit infection so that illness will be mild or will not occur (first natural infection). As a result of this initial attenuated infection, the superior immunity characteristic of infection can be expected so that on subsequent exposures to influenza virus the individual would possess bispecific (anti-H, anti-N) immunity and be resistant to both

NATURAL INFECTION 1st ( HN )

2nd ( HN

anti-N N-specific -> monospecific vaccine immunity infection (no disease)

Anti -H anti-N (boost) bi specific immunity No infection No disease

Time

I

FIG. 1-Approach to vaccination against influenza. See text for explanation. (Reproduced, with permission of one author, Dr. E.D. Kilbourne, and of publisher, the University of Chicago Press, from Couch and colleagues.'.)

infection and illness Thus, the advantage of this approach to immunization would be the occurrence of attenuated infection, and the solid and durable immunity that follows". This approach has been tested by a number of workers, and the use of neuraminidase-specific vaccine has proved to be effective. Couch and colleagues23 immunized mice and humans with a vaccine prepared from inactivated X-32 virus, which contains an "irrelevant" hemagglutinin antigen from A/equine virus (HeqiNeqi) that is not cross-reactive with human influenza A viruses, and a neuraminidase derived from A/HK (H3N2) virus. Thus, the vaccine was effectively monospecific for the neuraminidase (N2) even as a whole-virus vaccine. The results indicated that this vaccine could induce protection against illness caused by infection with an influenza virus containing an antigenically identical neuraminidase, and that the infection induced resistance to subsequent challenge with an antigenically similar virus. Kilbourne24 vaccinated volunteers with X-37 (A/England/43/72 a vaccine of conventional bispecific composition, and an experimental homotypic neuraminidase vaccine, X-3 8 (HeqIN2Eng). Comparative studies showed that the hybrid vaccine was of superior potency in engendering a response to the neuraminidase: a significant antibody response to N2 was observed in 25 % of those vaccinated with X-37 and in 69% of those vaccinated with X-38, and the mean antibody response to N2 was twofold greater in those vaccinated with X-38. Ogra and associates. immunized schoolchildren with a vaccine prepared from an inactivated recombinant influenza virus and specific for the neuraminidase of Port Chalmers influenza A virus (H.1N2m) and with a vaccine prepared from the conventional bispecific Port Chalmers (H:ici.N2cit) strain of influenza virus. Immunization with the HeqIN2e. vaccine resulted in no specific hemagglutination-inhibiting antibody response to the H3. antigen, although a specific antibody response to the N2.1 antigen was observed in 90% of the vaccine recipients. During a subsequent na1578

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tural outbreak of influenza 74.3% of the H3chN2.1 vaccine recipients but only 51.4% of the HeqIN2eh vaccine recipients were protected against illness. Kendal, Noble and Dowdle26 proposed that the antibody response to the neuraminidase can be suppressed if subjects receiving the vaccine have been primed with its hemagglutinin component. Their results supported the hypothesis that the immunogenicity of neuraminidase is greatest in populations primed with the neuraminidase but lacking antibodies to the hemagglutinin of the immunizing virus. In studies from 1963 to 1968 H2N2 vaccines did not induce an antibody response to neuraminidase in more than one quarter to one third of vaccine recipients, but in 1968-69 an H:.N2 vaccine containing a novel hemagglutinin induced the production of induced antibodies to neuraminidase in 66% to 96% of vaccine recipients.27'28 These results show that the hypothesis put forward by Kilbourne and colleagues22 is promising and may provide a means of fighting influenza effectively; however, the design of the experiments just cited calls for a few comments. First, the recombinants used by the various authors contained an "irrelevant" hemagglutinin and the neuraminidase of the challenge virus (H:1N2). Although for experimental purposes the vaccine was neuraminidasemonospecific, it was a whole-virus vaccine, and the influenza virion is not only toxic but also known for the instability of its ribonucleic acid. Second, there is a strong possibility that the neuraminidase incorporated into the recombinants may not represent the entire neuraminidase component of the parent virus.2930 Our studies of A/Aichi/ 68 (H:.N2) virus have shown that this virus contains two types of neuraminidase; one demonstrates only neuraminidase activity, whereas the other demonstrates both neuraminidase and hemagglutinating activity. It appears that the recombinant X-32 virus possesses only one of these neuraminidases.30 Conclusions The findings I have discussed emphasize the need for re-evaluat-

ing the role of neuraminidase in the control of influenza. We think that a vaccine containing only pure neuraminidase may prove to be ideal. On the basis of Kilbourne and colleagues' hypothesis, natural infection after immunization with neuraminidase would result in a durable bispecific immunity to subsequent reinfection with antigenically similar influenza A viruses. The vaccine would be changed only when the neuraminidase in the virus altered, and since 1934 only two types of neuraminidase have been detected. (N1 changed to N2 in 1957.) In the future the immunogenic spectra of neuraminidases from the various strains may help in choosing a combination of neuraminidase antigens likely to provide a potent mixture to fight the unpredictable influenza epidemic. The vaccine would be free of contamination by the viral seed strain or the substrate used for its cultivation. It could be prepared in advance and would be available when needed. It would be acceptable to the public because there would likely be no significant secondary reactions. Children and pregnant women could be vaccinated safely. The proposed pure neuraminidase vaccine may cost more but, when one looks at the benefits it offers, it becomes very appealing and worth trying. I am grateful to Dr. G. Lussier for his interest, to Drs. V. Pavilanis, A. Boudreault and J. Lecomte for reading and discussing the manuscript, and to Miss Dominique D'Ascola for typing the paper. This work was supported in part under national health research and development project 6605-1 359-41 of the Department of National Health and Welfare. References 1. PEREIRA HG: Influenza: antigenic spectrum. Prog Med Virol 11: 46, 1969 2. WEBSTER RG, PEREIRA HG: A com-

mon surface antigen in influenza viruses from human and avian sources. J Gen Virol 3: 201, 1968 3. WEBSTER RO: Estimation of the molecular weights of the polypeptide chains from the isolated hemagglutinin and neuraminidase subunits of influenza viruses. Virology 40: 643, 1970 4. Idem: On the origin of pandemic influenza viruses. Curr Top Microbiol Immunol 59: 75, 1972

5. DowNn JC, LAyER WG: Isolation of a type A influenza virus from an Australian pelagic bird. Virology 51:

259, 1973

of the 2nd international Congress for Virology, MELNICK JL (ed), Karger, Basel, 1972, p 121 23. COUCH RB, KASEL JA, GERIN JL,

ships between the neuraminidases of influenza viruses. J Gen Virol 2:

et al: Induction of partial immunity to influenza by a neuraminidasespecific vaccine. J infect Dis 129: 411, 1974 24. KILBOURNE ED: Comparative efficacy of neuraminidase-specific and conventional influenza virus vaccines in induction of antibody to neuraminidase in humans. I infect Dis 134: 384, 1976

385, 1968

25. OGRA PL, CHOW T, BEUTNER KR,

6. SCHULMAN JL: Immunology of in-

fluenza, in influenza Viruses and influenza, KILBOURNE ED (ed), Acad Pr, New York, 1975, p 373

7. BURNET FM: Principles of Animal Virology, Acad Pr, New York, 1955,

p 380 8. PANIKER CKJ: Serological relation-

9. COLEMAN

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PEREIRA HG, et al: The Hong Kong/68 influenza A, variant. Lancet 2: 1384, 1968 10. SCHULMAN JL, KILBOURNE ED: In-

dependent variation in nature of hemagglutinin and neuraminidase antigens of influenza virus: distinctiveness of hemagglutinin antigen of Hong Kong-68 virus. Proc Natl Acad Sci USA 63: 326, 1969 11. SCHULMAN JL: Effects of immunity on transmission of influenza: experimental studies. Prog Med Virol 12: 128, 1970 12. FRANCIS T JR, DAVENPORT FM, HENNESSY AV: Serological recapitulation of human infection with different strains of influenza virus.

et al: Clinical and immunologic evaluation of neuraminidase-specific influenza A virus vaccine in humans. I infect Dis 135: 499, 1977 26. KENDAL AP, NOBLE GR, DOWDLE WR: Neuraminidase content of influenza vaccines and neuraminidase antibody responses after vaccination of immunologically primed and unprimed populations. I infect Dis 136 (suppi): S415, 1977 27. HENNESSY AV, MINusE E, DAvEN-

PORT FM: Antineuraminidase antibody response of man to influenza virus neuraminidase N2: results obtained with an improved hemagglutination inhibition technique and an enzyme inhibition test. I immunol 109: 213, Aug 1972

Trans Assoc Am Physicians 66: 231,

28. MosTow SR, SCHILD GC, DOWDLE

1953 13. KENDAL AP, MINUSE E, MAASSAB HF, et al: Influenza neuraminidase antibody patterns of man. Am J Epidemiol 98: 96, 1973

WR, et al: Application of the single radial diffusion test for assay of antibody to influenza type A viruses. I Clin Microbiol 2: 531, 1975 29. DESSELBERGER U: Preparation-conditioned changes of the antigenicity of influenza virus neuraminidases. Arch Virol 53: 335, 1977

14. STUART-HARRIS C: The influenza problem. Med Lab Technol 32: 161, 1975

15. Influenza virus. Morb Mortal Wkly Rep 26: 193, 1977 16. ARORA DJS, PAvILANIS V,

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DREAULT A, et al: Antigenic properties of influenza virus proteins. Presented at 3rd International Symposium on Aerobiology, University of Sussex, Brighton, England, Sept 1618, 1969 17. BRANDON FB, Cox F, QUINN E, et al: Influenza immunization. Clinical studies with ether-split subunit studies. Bull WHO 41: 629, 1969 18. FENTERS JD, YAMASHIROYA HM, PETZOLD RF, et al: Enhanced immunogenicity in mice of a purified, tween-ether-treated influenza vac-

cine. Appl Microbiol 20: 544, 1970 19. WARBURTON MF: Desoxycholatesplit influenza vaccines. Bull WHO 41: 639, 1969 20. RUBEN FL, JACKSON GG: A new

subunit influenza vaccine: acceptability compared with standard vaccines and effect of dose on antigenicity. J infect Dis 125: 656, 1972 21. WRIGHT PF, DOLIN A, LA MONTAGNE JR: Summary of clinical trials of influenza vaccines - II. J infect

Dis 134: 633, 1976 22. KILBOURNE ED, SCHULMAN JL, COUCH RB, et al: Orthomyxoviruses and paramyxoviruses, in Proceedings

30. ARORA DJS, SCHUBERT JH, VINCENT

L: The presence of two neuraminidases in an influenza virus. Can J Microbiol (in press)

BOOKS I This list is an acknowledgement of books received. It does not preclude review at a later date. ACUTE CARE. Based on the Proceedings of the Sixth International Symposium on Critical Care Medicine. Edited by B.M. Tavares and R. Frey. 234 pp. Illust. Springer-Verlag New York, Inc., New York, 1979. $42.90, paperbound. ISBN 0-387-09210-2 ARACHIDONIC ACID METABOLISM IN INFLAMMATION AND THROMBOSIS. Proceedings of the "First European Workshop on Inflammation" held in Basel in March 1979. Edited by K. Brune and M. Baggiolini. 301 pp. lIlust. Birkhauser Verlag, Basel; Birkhauser Boston Inc., Cambridge, Massachusetts, 1979. $38. ISBN 3-7643-1095-2

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