Immunity to Influenza in Ferrets

INFECTION ANI) IMMUNITY. June 1974. 1). 985-99) Copyright ( 1974 American Society for Microbiology Vol. 9. No. 6 Printed in U.S.A. Immunity to Influenza in Ferrets X. Intranasal Immunization of Ferrets with Inactivated Influenza A Virus Vaccines C. McLAREN, C. W. POTTER, AND R. JENNINGS Departments of Medical Microbiology and Medicine, University of Sheffield Medical School, Sheffield S1 2RX, England Received for publication 14 January 1974 The response of ferrets after intranasal inoculation of inactivated A/Hong Kong/68 (H3N2) influenza virus vaccines is reported. Normal ferrets given either saline vaccine in drops or freeze-dried vaccine in an aerosol intranasally did not produce detectable serum or nasal hemagglutination inhibiting antibody and were found to be completely susceptible to challenge infection with A/Hong Kong/68 virus. Intranasal saline vaccine did not produce an additive effect on the response of ferrets simultaneously given the same vaccine intramuscularly with adjuvant. Ferrets primed by previous infection with A/PR/8/34 (HON1) influenza virus, however, responded to intranasal immunization with saline A/Hong Kong/68 virus vaccine and produced serum and nasal antibody. These animals were found to be partially resistant to challenge infection, in contrast to similar animals given saline vaccine intramuscularly which were completely resistant to challenge infection. Primed ferrets did not respond after immunization with the freeze-dried aerosol vaccine, but this may have been due to a failure of the aerosol to be inhaled satisfactorily. Local immunity in the upper respiratory tract may play an important role in the immunity of humans and animals to respiratory virus infections (13). Secretory immunoglobulin A antibody is produced in nasal secretions after natural infections with respiratory viruses, and attempts have been made to correlate secretory antibody titers with immunity to challenge inf'ection (2, 4). In addition, a number of studies have been carried out on the local antibody response after parenteral or intranasal administration of inactivated influenza virus vaccines, but with varving results (1, 3, 5, 7, 15, 16). The ferret may be used as a model system for the study of influenza virus infection and of methods of producing immunity to this virus (11). The present study describes the response of ferrets to intranasal inoculation with inactivated influenza virus A/Hong Kong/68 (H3N2) vaccine, given either as drops of a suspension in saline or as a Freon-based aerosol of freeze-dried virus. The vaccines were administered to normal ferrets and to ferrets which had been primed by previous infection with a heterotypic influenza virus (8). In each case, the immunity of the ferrets after immunization was tested by challenge inf'ection with A/Hong Kong/68 virus. MATERIALS AND METHODS Ferrets. Young adult ferrets were obtained from a number of sources. All animals were immunized against canine distemper a number of weeks before use; some animals were primed by intranasal infection with influenza virus A/PR/8/34 (HONI) 5 weeks before immunization (8). Blood samples were obtained by cardiac puncture at the beginning of each experiment, and the sera were tested for the absence of hemagglutination inhibiting (HI) antibody to influenza A viruses. Influenza virus vaccines. Ferrets were immunized with either one of two saline vaccines. One vaccine contained 3 chick cell agglutinating units (CCA) per.5 ml of zonally purified, inactivated influenza A/Aichi/68 (H3N2) virus and was kindly supplied by M. R. Hilleman (Merck Institute of Therapeutic Research, West Point, Pa.); the other saline vaccine contained 4 IU of A/HK/X31/68 (H3N2) and 2 IU of B/Vic/7 Formalin and 3-propiolactone-inactivated viruses and was kindly supplied by D. Breeze (Evans Medical Ltd., Liverpool). Further groups of ferrets were given a spray vaccine containing 36 IU of A/Aichi/68, 12 IU of B/Netherlands/66, and 12 IU of B/Berkeley/71 per metered dose of four puffs of Freon-propelled, freeze-dried vaccine (7). This vaccine was kindly supplied by T. C. G. Smith (Duphar Laboratories, Southampton). Experimental design. Ferrets were immunized as described below, and serum and serial nasal washings were collected and tested for the presence of serum HI 985

986 McLAREN, POTTER, AND JENNINGS INFECT. IMMUNITY antibody and nasal protein and HI antibody, as described previously (8, 11). After immunization, the animals were challenged by intranasal infection with live influenza virus A/Hong Kong/1/68. The degree of immunity to challenge infection was determined by measuring the amount of virus present in nasal secretions 3 days after infection, the change in nasal wash protein concentration, and the titers of HI antibody produced in serum and nasal washings (11). RESULTS Response of normal ferrets to intranasal immunization with saline vaccine. Four ferrets were inoculated intranasally with 15 CCA of inactivated influenza A/Aichi/2/68 virus on day and again on day 1; the vaccine was administered drop by drop to lightly anesthetized animals. A further group of ferrets received four doses of this vaccine (15 CCA each dose) given on days, 1, 14, and 15. None of these animals developed nasal wash or serum HI antibody after immunization, and both groups of ferrets were as susceptible to challenge infection on day 35 with influenza A/Hong Kong/1/68 virus as was a group of nonimmunized control ferrets (Table 1). Thus, similar titers of virus were recovered from all the ferrets 3 days after the challenge infection, and all the animals produced serum HI antibody after challenge. Virus infection caused a three- to fivefold increase in protein concentration and the production of HI and neutralizing antibody in nasal washings collected after virus infection; peak titers of antibody were found in specimens TABLE 1. collected 7 to 9 days after infection and were similar in all three groups (Table 1). A group of ferrets was also inoculated intranasally with 3 CCA of influenza virus A/ Aichi/2/68 vaccine, given as an aerosol, using a DeVilbis atomizer (mean droplet size, 4,m). None of the animals produced serum or nasal wash HI antibody after immunization, and the animals were completely susceptible to challenge infection with A/HK/68 virus. Response of normal ferrets to simultaneous intranasal and parenteral immunization with saline vaccine. A group of ferrets was inoculated intranasally with 3 CCA of A/ Aichi/68 vaccine, given as large drops, and with the same dose of vaccine given intramuscularly at the same time. Further groups of ferrets were similarly immunized, but the intramuscular vaccine was given with either adjuvant 65 (6) or Freund complete adjuvant, whereas other groups received intramuscular vaccine in adjuvant only. Only those animals given vaccine plus adjuvant intramuscularly produced serum HI antibody, and the titer of antibody was not increased in those animals given intranasal vaccine in addition to intramuscular vaccine plus adjuvant. None of the ferrets given vaccine by both intranasal and intramuscular routes, with or without adjuvant, produced detectable nasal-wash HI antibody. After challenge infection with A/HK/68 virus 5 weeks after immunization, the ferrets previously immunized both intranasally and parenterally with saline vaccine were found to be completely susceptible to Response of ferrets to infection with A2/Hong Kong influenza virus after intranasal inoculation with inactivated A2/Aichi/2/68 vaccine Response to challenge infection Vaccine (dose) Ferret Titer of virus no. isolated Change in Change in (log,eid,(a) serum HI titer' nasal HI titere A2/Aichi/2/68 (15 CCA, intranasal X2) 135 5.25 <5-3,2 <5-3 138 4.75 <5-2,4 <5-3 14 6.25 <5-1,2 <5-3 141 5.5 <5-1,2 <5-1 A2/Aichi/2/68 (15 CCA, intranasal X4) 129 4.5 <5-2,4 <5-2 13 5.25 <5-6 <5-15 131 5.75 <5-1,2 <5-4 132 5.5 <5-1,2 <5-15 Nil 142 5.75 <5-8 <5-3 143 6.25 <5-1,6 <5-5 144 5.25 <5-2,4 <5-2 145 5.5 <5-8 <5-2 a EID5, mean egg infective dose per milliliter. 'Change from pre-infection titer to post-infection titer. c Change from pre-infection titer to peak, post-infection titer.

VOL. 9, 1974 infection. Ferrets given intranasal vaccine together with parenteral vaccine in adjuvant were found to possess partial immunity to challenge infection; this immunity was similar to that found in ferrets given intramuscular vaccine in adjuvant alone (1, 12). Response of ferrets previously infected with influenza virus A/PR8 to intranasal or intramuscular immunization with saline Al H1V68 vaccine. Ferrets were infected with influenza virus A/PR/8/34 5 weeks prior to immunization, and all the animals developed serum HI antibody to the infecting virus. Four of these ferrets were inoculated intranasally, under light anesthesia with bivalent saline vaccine containing 4 IU of A/HK/68 virus; 4 IU were given on day and a further 2 IU on the following day (day 1). A second group of animals received 2 IU of vaccine intranasally on days, 1, 7, and 8. Another group of ferrets was given 4 IU of A/HK/68 vaccine intramuscularly on day. After immunization, all the ferrets produced serum HI antibody to A/ HK/68 virus; similar titers of antibody were found in the two groups of ferrets given intranasal vaccine, whereas the animals given vaccine intramuscularly had higher serum HI antibody IMMUNITY TO INFLUENZA IN FERRETS titers (Table 2). Two out of four ferrets given four doses of vaccine intranasally, and one animal given vaccine intramuscularly, produced nasal HI antibody after immunization; none of the ferrets given two doses of vaccine intranasally produced nasal HI antibody (Table 2). Both groups of ferrets which received intranasal saline vaccine showed increased concentrations of protein in nasal washings collected after immunization; the maximum levels were found 7 days after the first dose of vaccine (Fig. 1). All the ferrets were challenged by intranasal inoculation of 3 x 16 mean egg infective doses of influenza virus A/HK/1/68 14 days after the first dose of vaccine. The three ferrets given intramuscular vaccine were found to be resistant to challenge infection; thus, no virus was isolated from nasal washings collected 3 days after challenge, and no increase in serum HI antibody occurred (Table 2). An increase in protein concentration was found in nasal washings collected after challenge infection, but nasal HI antibody to A/HK/68 was produced by only one animal. Virus was recovered from all four ferrets given two doses of intranasal vaccine, although one animal (ferret no. 42) did not show an increase in serum HI antibody titer (Table 2). The ferrets given four doses of intranasal vaccine showed a measurable degree of resistance to challenge infection; thus, virus was found, in reduced titers, in nasal washings from three out TABLE 2. Response of A/PR/8-primed ferrets to intranasal immunization with saline A/HK/68 vaccine and subsequent challenge infection with A/HK/68 virus Response to [ Response to challenge infection on day 12 immunization A/HK/68 vaccine dose D Ferret and route (IU) ay no. A/HK/68 HI titer Virus Serum Nasal wash HI titer A/HK/68 HI Seruma Nasalb ISOlationc titer" A/HK/68 A/PR/8/34 Intranasal 418 < 1-6 -d 4.5 6-64 _ 4 419 < 1-3 - 5.16 3-24 + 2 1 42 <1-12 - 3.5 12-12 - - 421 < 1-6 - 5.5 6-3,84 2 1 Intranasal 2 437 < 1-15 - 1.83 15-48 4-2 1 438 < 1-24 3 2.5 24-12 - _ 2 7 439 < 1-6 - 2.83 6-24 2 8 44 < 1-12 7.5 Nil 12-24 Intramuscular 4 422 < 1-48 - D+e D D D 424 < 1-2,56 7.5 Nil 2,56-48 7.5 _ 425 < 1-24 - Nil 24-48 - _ 426 < 1-48 - Nil 48-6 a Change from pre-immunization/infection titer to post-immunization/infection titer. b Change from pre-immunization/infection titer to peak, post-immunization/infection titer. c Log,, mean egg infective dose per milliliter. d <5- <5. e Animal died before challenge. 987

988 i i.6 1-44 44.4.2 E + McLAREN, POTTER, AND JENNINGS A 4, INFECT. IMMUNITY 1.6 a 4.8.2 i I LT I I I I I I I I I I I 2 4 C 1 12 14 1i 1i 2 22 TINE (DAYS) T ITT II I I -2 o 2 4 6 I 1o 12 14 TINE (DAVS) 16 1i 2 22 D 1. 4,ki ' i i,9 *4.$.4 I I.5 2 1 T.2 I I I I I 1 I I I I I I T I I I I I I I I I I -2 2 4 a 1 12 14 1 1 2 n TIIE IDAIS) FIG. 1. Nasal response of ferrets to immunization with inactivated A/Hong Kong/68 influenza virus and subsequent challenge infection with A/Hong Kong/68 virus. The protein concentration of nasal washings collected from ferrets before and after immunization and challenge infection is shown. Vaccine was given on the days indicated by small arrows, followed by challenge infection on day 12 with A/HK/68 virus (large arrow). (A) Normal ferrets given aerosol vaccine intranasally (four doses); (B) ferrets previously infected with influenza virus A/PR/8/34 and given one dose of saline vaccine intramuscularly; (C) ferrets previously infected with influenza virus A/PR/8/34 and given saline vaccine intranasally (, two doses;, four doses); (D) ferrets previously infected with influenza virus A/PR/8/34 and given aerosol vaccine intranasally (, two doses;, four doses). TIME IDAS) of four animals, and only two of the ferrets showed a fourfold or greater rise in serum HI antibody (Table 2). In both groups of ferrets given saline vaccine intranasally, nasal antibody was produced by only one animal after challenge infection; in one instance (ferret no. 421), both A/PR8 and A/HK/68 HI antibody were detected in nasal washings collected on day 4. Increased concentrations of protein were also measured in nasal washings collected after challenge from both of these groups of ferrets (Fig. 1). Response of ferrets previously infected with influenza virus A/PR8 to intranasal immunization with aerosol A/HK/68 vaccine. A group of ferrets, previously infected with influenza virus A/PR8, were immunized with an aerosol of freeze-dried A/HK/68 virus; the lightly anesthetized animals were given two puffs of vaccine into each nostril and four puffs into the mouth, by using a close-fitting plastic face mask. Thus, each dose contained a total of 72 IU of A/HK/68 virus. Four ferrets received one dose on day and a further dose on day 1, whereas four other ferrets were given a dose of vaccine on days, 1, 7, and 8. A third group of four ferrets, which had not been previously infected with influenza virus A/PR8, were given a dose of vaccine on days, 1, 7, and 8. After immunization, none of the ferrets given powder vaccine produced serum or nasal wash HI antibody, although a detectable increase in the concentration of protein in nasal washings was observed in both groups of ferrets after intranasal vaccine (Fig. 1). The ferrets were all found to be susceptible to challenge infection with A/HK/68 virus given 14 days after the first dose of vaccine. Thus, virus was recovered from nasal washings of all the ferrets, although the titers of virus recov-

VOL. 9, 1974 IMMUNITY TO INFLUENZA IN FERRETS 989 ered from the immunized ferrets previously infected with A/PR8 were lower than from noninfected ferrets given the same vaccine (Table 3). This difference may be due to heterotypic immunity produced by the A/PR8 infection (14; McLaren, unpublished data). All of the animals produced A/HK/68 serum HI antibody after challenge infection; however, the HI antibody titers were lower for A/PR8-prirmed animals given intranasal aerosol vaccine than for normal ferrets given the same vaccine. In addition, A/PR8-primed ferrets given the vaccine produced lower titers of HI antibody in nasal washings collected after challenge infection than did ferrets which had not been previously infected with A/PR8. Only one of four animals (ferret no. 428) given two doses of aerosol vaccine after A/PR8 infection produced HI antibody to A/HK/68 in nasal washings, whereas three of these ferrets produced nasal HI antibody to A/PR8 (Table 3). The ferrets given aerosol vaccine after previous infection with A/PR8 virus produced high concentrations of protein in nasal washings after challenge; the maximum concentration occurred earlier than for nonprimed ferrets given the same vaccine before challenge (Fig. 1). DISCUSSION This study extends previous observations on the response of ferrets to inactivated influenza virus vaccines. We have shown that normal TABLE 3. ferrets given saline inactivated influenza virus intramuscularly fail to produce either serum or nasal antibody (8, 11). In addition, antibody was not produced after intranasal immunization. Intranasal vaccine also failed to produce any additive effect on the response of ferrets simultaneously given the same vaccine intramuscularly with adjuvant, although studies in other systems have indicated that local administration of an antigen may produce a local immunological response to another antigen given parenterally at the same time (9). Ferrets previously infected with A/PR8 influenza virus, however, responded to both intramuscular (8) and intranasal immunization. After instillation of a total of either 6 or 8 IU of inactivated saline vaccine, given in drops to lightly anesthetized, primed ferrets, all of the animals produced serum HI antibody. Those ferrets given four doses of 2 IU of vaccine intranasally were significantly more resistant to challenge infection than animals given a total of 6 IU in two doses, although their serum HI titers were similar; however, two of the animals given the higher vaccine dose produced nasal antibody after immunization, and this may have increased their resistance to challenge infection. This result suggests that the immunological mechanism involved in the priming of the serum antibody response may also be present in the production of local antibody. Response of ferrets to intranasal immunization with aerosol A/HK/68 vaccine and subsequent challenge infection with A/HK/68 virus Responseto Response to ct A/HK/68 Previously infected Ferret immunization c i vaccine vaccine dose Day with no. A/HK/68 HI titer Virus Serum Nasal wash HI titer (IU) ~A/PR'8/34 A/HK/68 HI Seruma Nasal' isolation titer A/HK/68 A/PR/8/34 72 + 427 _d _ d 4.83 < 1-3 _ 15 72 1 + 428 - - 4.5 < 1-8 5 5 + 429 - - 3.83 <1-12 - 7.5 + 43 - - 4.83 <1-8 - - 72 + 431 - - 4.5 < 1-16 6-72 1 + 432 - - 4.5 < 1-6 15 7.5 72 7 + 433 - - 2.5 < 1-64 2-72 8 + 436 - - 3.83 < 1-12 15-72 _ 25 - - 5.5 < 1-1,28 32-72 1 _ 26 - - 4.16 < 1-2,56 8-72 7 _ 3 - - De D D D 72 8 _ 32 - - 6.5 <1-1,28 15 - a Change from pre-immunization/infection titer to post-immunization/infection titer. b Change from pre-immunization/infection titer to peak, post-immunization/infection titer. mean egg infective dose per milliliter. d, <5- <5 (nasal wash), <1- <1 (serum). e Animal died before challenge. c Log1,

99 McLAREN, POTTER, AND JENNINGS Much discussion has taken place on the relative roles of local and humoral antibody in recovery from respiratory infections and the best methods of producing these antibodies. The results of the present study do not give a definite answer to this problem. The primed ferrets given 4 IU of saline vaccine intramuscularly produced higher titers of serum antibody than those given intranasal vaccine and were found to be completely resistant to challenge infection. This may be due to the greater antigenic stimulus of vaccine given intramuscularly compared with intranasal vaccine which is rapidly lost, rather than to the relative role of serum and local antibody. Studies in humans and guinea pigs have also shown that nasal instillation of vaccine is less effective at inducing serum antibody, but produces better local immunity than parenteral immunization (2, 15, 16). In contrast to animals given intranasal saline vaccine after prior infection with A/PR8, those primed animals given an aerosol of freeze-dried vaccine intranasally did not produce serum HI antibody after immunization, and the.ferrets were susceptible to challenge infection. The titers of virus recovered from nasal washings from primed ferrets given the aerosol vaccine were, however, lower than from normal ferrets given the same dose of vaccine. Studies in man with the same aerosol vaccine have shown that only a low percentage of persons produce serum HI antibody after intranasal immunization, but that nasal antibody is produced in a greater number of subjects (7). The failure of the ferret to respond to the aerosol vaccine may be due to noninhalation of the vaccine; a number of animals have been shown to react to the cooling action of the vaccine spray by constriction of their nasal passages (E. A. Marcus, personal communication). ACKNOWLEDGMENTS We wish to thank Sir Charles Stuart-Harris f'or his advice and criticism and M. D. Denton and L. Shepherd for their excellenit technical assistance. The support of' the Medical Research Council is gratef'ully acknowledged. LITERATURE CITED 1. Buscho, R. F., J. C. Perkins, H. L. S. Knopf, A. Z. Kapikian, and R. M. Chanock. 1972. Further characterization of' the local respiratory tract antibody response induced by intratnasal instillation of' inactivated INFECT. IMMUNITY rhinovirus 13 vaccine. J. Immunol. 18:169-177. 2. Downie, J. C.. and C. H. Stuart-Harris. 197. The production of neutralizing activity in serum and nasal secretion f'ollowing immunization with influenza B virus. J. Hyg. 68:233-244. 3. Fedson, D. S., R. V. Fulk, M. A. Huber, M. A. Reisberg, and J. A. Kasel. 1971. Anti-neuraminidase antibody response in serum and nasal secretions following intranasal or subcutaneous inactivated A2/Hong Kong/68 influenza virus vaccine. J. Immunol. 17:73-737. 4. Freestone, D. S., S. Hamilton-Smith, G. C. Schild, R. Buckland, S. Chinn, and D. A. J. Tvrrell. 1972. Antibody responses and resistance to challenge in volunteers vaccinated with live attenuated, detergent split and oil adjuvant A2/Hong Kong/68 (H3N2) influenza vaccines. J. Hyg. 7:531-543. 5. Gwaltnev, J. M., W. P. Edmondson, R. Rothenberg, and P. W. White. 1971. A comparison of subcutaneous, nasal and combined influenza vaccination. 1. Antigenicity. Amer. J. Epidemiol. 93:472-479. 6. Hilleman, M. R., A. F. Woodhour, A. Friedman, and A. H. Phelps. 1972. Studies for safety of' adjuvant 65. Ann. Allergy 3:477-48:3. 7. Liem, K. S., E. A. Marcus. J. Jacobs, and R. van Strick. 1973. The protective effect of intranasal immunization with inactivated influenza virus vaccine. Postgrad. Med. J. 49:175-179. 8. McLaren. C., and C. W. Potter, 1974. Immunity to influenza in ferrets. 7. Effect of previous infection with heterotypic and heterologous influenza viruses on the response of ferrets to inactivated influenza virus vaccines. J. Hyg. 72:91-1. 9. Nash, D. R., and B. Holle. 1973. Local and systemic cellular immune responses in guinea-pigs given antigen parenterally or directly into the lower respiratory tract. Clin. Exp. Immunol. 13:573-583. 1. Potter, C. W., C. McLaren, and S. L. Shore. 197.3. Immunity to influenza in f'errets. 5. Immunization with inactivated virus in adjuvant 65. J. Hyg. 71:97-16. 11. Potter, C. W., J. S. Oxford, S. L. Shore, C. McLaren, and C. H. Stuart-Harris. 1972. Immunity to influenza in ferrets. 1. Response to live and killed virus. Brit. J. Exp. Pathol. 53:15:3-167. 12. Potter, C. W., S. L. Shore, C. McLaren, and C. H. Stuart-Harris. 1972. Immunity to influenza in ferrets. 2. Influence of adjuvants on immunization. Brit. J. Exp. Pathol. 53:168-179. 13. Rossen, R. D.. J. A. Kasel. and R. B. Couch. 1971. The secretory immune system: its relation to respiratory viral infection, p. 194-238. In J. L. Melnick (ed.). Progress in medical virology. S. Karger, Basel. 14. Schulman, J. L., and E. D. Kilbourne. 1965. Induction of' partial specif'ic heterotypic immunity in mice b- a single infection with influenza A virus.,j. Bacteriol. 89:17-174. 15. Waldman. R. H., C. S. Spencer. and J. E. Johnson. 1972. Respiratory and systemic cellular and humoral immune responses to influenza virus vaccine administered parenterally or by nose drops. Cell. Immunol. 3:294-:3. 16. Waldman, R. H., S. H. Wood, E. J. Torres, and P. A. Small. 197. Influenza antibody response following aerosol administration of inactivated virus. Amer. J. Epidemiol. 91:575-584.