Development and Persistence of Local and Systemic Antibody

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Jn. 1986, p /86/ $02.00/0 Copyright C) 1986, Americn Society for Microbiology Vol. 23, No. 1 Development nd Persistence of Locl nd Systemic Antibody Responses in Adults Given Live Attenuted or Inctivted Influenz A Virus Vccine MARY LOU CLEMENTSlt* AND BRIAN R. MURPHY2 Center for Vccine Development, Deprtment of Medicine, University of Mrylnd School of Medicine, Bltimore, Mrylnd 21201,' nd Lbortory of Infectious Diseses, Ntionl Institute of Allergy nd Infectious Diseses, Bethesd, Mrylnd Received 21 June 1985/Accepted 30 September 1985 An enzyme-linked immunosorbent ssy ws used to mesure nsl-wsh nd serum isotype-specific hemgglutinin ntibody responses in 109 seronegtive (hemgglutintion-inhibiting titer <1:8) dults vccinted intrnslly with live ttenuted A/Wshington/897/80 (H3N2) or A/Cliforni/10/78 (H1N1) colddpted (c) virus or with licensed subvirion vccine subcutneously. Live nd inctivted virus elicited serum immunoglobulin A (IgA) responses in 83 nd 96% of vccinees, respectively, nd elicited serum IgG responses in 72 nd 100% of vccinees. Inctivted virus induced higher titers of serum ntibodies thn did live virus nd stimnulted nsl-wsh IgG response more often thn did live virus (94 versus 59%, P < 0.01). In contrst, only 38% of inctivted virus vccinees hd locl IgA responses compred with 83% of live virus vccinees. Serum IgA nd IgG nd nsl IgG ntibody titers remined elevted bove prevccintion levels for t lest 6 months in most of the live nd inctivted vccine responders, but the men level of locl IgA ntibody induced by infection with live virus vccine, in prticulr, decresed substntilly. Considered in the context of previous work, the finding tht live virus vccine induced reltively long-lsting ntibody in both locl nd serum comprtments suggested tht this vccine my be suitble lterntive to inctivted vccine for use in helthy persons. Commercilly vilble, inctivted influenz virus vccine dministered prenterlly stimultes high levels of serum ntibody nd offers protection ginst influenz A disese, but the resistnce induced is incomplete (18, 26). For this reson, there is interest in intrnslly dministered, live ttenuted influenz A vccines tht stimulte locl nd systemic immunity nd my provide longer-lsting, more complete protection. Live influenz A virus vccines consisting of ttenuted, cold-dpted (c) ressortnt viruses tht possess the six internl RNA segments from the c A/Ann Arbor/6/60 (H2N2) donor virus nd both hemgglutinin (HA) nd neurminidse surfce glycoproteins of epidemic wild-type virus re promising (2, 14, 20, 21, 24). Dt from previous study indicted tht intrnsl vccintion of seronegtive children with live ttenuted c ressortnt HlNl or H3N2 influenz A vccine efficiently stimulted both systemic nd locl ntibody responses (29). Locl HA-specific ntibody responses to live nd inctivted influenz A virus vccines hve been incompletely studied in dults. The present study is the first to evlute the mgnitude nd durtion of the serum nd nsl-wsh immunoglobulin A (IgA), IgG, nd IgM HA ntibody responses evoked by vccintion with live or inctivted HlNl or H3N2 influenz A virus in seronegtive dult volunteers. (This study ws presented in prt t the Second Annul Meeting of the Americn Society of Virology, Mdison, Wis., July 23, 1984, nd t the UCLA Symposi on Moleculr nd Cellulr Biology, Keystone, Colo., April 11, 1985.) * Corresponding uthor. t Present ddress: Deprtment of Interntionl Helth, The Johns Hopkins University School of Hygiene nd Public Helth, Bltimore, MD MATERIALS AND METHODS Vccines. The influenz virus vccines used in this study included two live ttenuted, c ressortnt viruses nd one commercil, trivlent, inctivted subvirion vccine. The live ttenuted A/Wshington/897/80 (H3N2) (ntigeniclly simnilr to A/Bngkok/1/79 virus) nd the A/Cliforni/10/78 (HlNl) (ntigeniclly similr to A/BrzilV11/78 virus) c ressortnt viruses ech possessed the six internl RNA segments derived from the ttenuted A/Ann Arbor/6/60 (H2N2) c donor, wheres the two remining genes (i.e., those tht code for the HA nd neurminidse surfce glycoproteins) were derived from their respective wild-type influenz A virus prents. The production nd sfety testing of these c ressortnt viruses nd their evlution in dults hve been described (12, 13, 28). The commercil vccine ws ether extrcted nd contined 15,ug ech of HA of A/Brzil/11/78 (HlNl), A/Bngkok/1/79 (H3N2), nd B/Singpore/222/79 per 0.5-ml dose (Fluogen; Prke, Dvis & Co., Morris Plins, N.J.). Clinicl studies. Helthy volunteers, 18 to 35 yers old, without history of influenz vccintion nd who were seronegtive [i.e., hving serum hemngglutintioninhibiting ntibody titer of 1:8 or less to either A/Wshington/897/80 (H3N2) or A/Cliforni/10/78 (H1N1) virus] were recruited from college students in Mrylnd. Informed consent ws obtined from the study prticipnts. The study protocol ws pproved by the Clinicl Reserch Subpnel of the Ntionl Institute of Allergy nd Infectious Diseses nd the Humn Volunteer Reserch Committee t the University of Mrylnd. Volunteers who were HlNlseronegtive were rndomly ssigned to receive either two 0.5-ml doses of inctivted virus vccine subcutneously 1 month prt or single dose ( % tissue culture infective doses) of A/Cliforni/10/78 c ressortnt virus

2 VOL. 23, 1986 LIVE AND INACTIVATED INFLUENZA A VIRUS VACCINES 67 TABLE 1. Serum HA-specific IgA, IgG, nd IgM ntibody responses of volunteers who received either live cold-dpted or inctivted virus vccine Serum ELISA nti-ha titer (reciprocl men' log2) on specified dy fter vccintion Vccine group (no. in IgA IgG IgM group) Before % with Before % with Before % with vccintion Dys Dys increse vccintion Dys Dys increse vccintion Dys Dys increse Live HlNl (23) Inctivted HlNl (20)b c l0d d Live H3N2 (31) Inctivted H3N2 (35) ' ' d Stndrd devitions of the mens were similr, rnging from 1.2 to 3.4 log2. b Titers for volunteers vccinted on dys 0 nd 28 were lso mesured on dy 42 nd were 10.1 nd 15.0 in IgA nd IgG isotypes, respectively. Increses for this group were cumultive for both vccintions. c Sttisticlly significnt difference between the men ntibody increse of the live nd inctivted vccine groups, P < by the pired t test. d Sttisticlly significnt difference between the percentges of increses in the live nd inctivted vccine groups, P <.001 by X- test. dministered intrnslly. Volunteers who were H3N2 seronegtive were rndomly ssigned to receive one dose of inctivted virus vccine (0.5 ml) subcutneously or % tissue culture infective doses of A/Wshington/897/80 c ressortnt virus intrnslly. Lbortory studies. Serum nd nsl-wsh specimens were collected before vccintion, nd the immunologicl methods hve been described (29, 30). For detection of IgA, IgG, nd IgM HA ntibodies, serum nd nsl-wsh specimens were tested by enzyme-linked immunosorbent ssy (ELISA) with specific rbbit nti-humn immunoglobulin, s previously described (29). Briefly, U-bottomed polystyrene microtiter pltes (Immulon I; Dyntech Lbortories, Inc., Alexndri, V.) were used with ldder of regents from the solid-to-liquid phse consisting of (i) purified HA, (ii) humn serum or nsl wsh, (iii) rbbit nti-humn IgA, IgG, or IgM, (iv) got nti-rbbit IgG serum conjugted with lkline phosphtse, nd (v) substrte. The A/Cliforni/10/78 (HlNl)- or the A/Bngkok/1/79 (H3N2)- purified HA ws used s the homologous ntigen in the ELISA. The ELISA titer ws expressed s the highest dilution in which the opticl density of the ntigencontining well ws t lest twice the opticl density of the respective control well lcking ntigen. The ELISA nslwsh IgA, IgM, nd IgG HA ntibody titers were corrected to n IgA concentrtion of 10 mg/100 ml, s ws done previously (30). A significnt serum or nsl-wsh ELISA response ws defined s fourfold or greter increse in ntibody titers between prevccintion nd postvccintion specimens. Antibody titers were expressed s reciprocl log2 vlues. The bsence of clinicl history consistent with influenz illness nd ny significnt increse in serum or nsl-wsh ntibody titers of these vccinees within 8 to 28 weeks fter vccintion suggested tht nturl influenz infection did not occur in the volunteers during the study period. Anlysis of dt. Student's t test, the chi-squre test with Ytes' correction, nd Fisher's exct test were performed where pproprite. For correltion of serum nd nsl-wsh ntibody titers, the Spermn rnk correltion test ws used. RESULTS Serum ntibody responses. Both the frequency nd the mgnitude of serum IgA, IgG, nd IgM HA ntibody increses of the two groups of volunteers who were vccinted intrnslly with the live virus vccines of the HlNl or H3N2 subtype were similr (Tble 1). Likewise, the frequency nd mgnitude of serum HA ntibody responses in the groups given one or two doses of inctivted virus vccine were comprble. Almost ll vccine recipients mnifested significnt increse in ELISA serum IgA, IgG, or IgM HA ntibody titers fter vccintion. However, inctivted virus vccine ws more effective thn live virus vccine in inducing serum ntibody responses (Tble 1). When dt of the two groups of live virus vccinees were combined nd compred with those of the volunteers who were vccinted with inctivted virus, inctivted virus vccinees hd significnt increses in serum IgA nd IgG HA ntibodies more frequently (P < 0.001) thn did live virus vccinees (96 versus 83% nd 100 versus 72%, respectively). Also, the mgnitude of serum IgA, IgG, nd IgM ntibody responses ws significntly greter (P < 0.001) in inctivted virus vccinees thn in live virus vccinees. Nsl-wsh ntibody responses. The frequency nd mgnitude of increses in nsl-wsh IgA nd IgG HA ntibodies of the two groups given live HlNl or H3N2 virus vccine were comprble (Tble 2). Also, the frequency nd mgnitude of nsl-wsh IgA nd IgG HA ntibody responses of the inctivted virus vccinees were similr whether they received one or two doses. Immuniztion with either live or inctivted HlNl virus vccine elicited locl IgM ntibody responses more frequently (P < 0.001) thn did vccintion with either H3N2 virus vccine. (Presumbly, the mjority of initilly HlNl-seronegtive volunteers hd not been previously infected with n HlNl virus, wheres the initilly H3N2-seronegtive volunteers hd been previously infected with H3N2 viruses.) Interestingly, inctivted virus vccine ws s effective s live virus vccine in stimulting nsl-wsh HA ntibody responses in the volunteers (Tble 2). Overll, significnt increses in IgA, IgG, or IgM ntibodies were detected in postvccintion nsl wshes of ll but one of those who received inctivted virus vccine. Similrly, live HlNl virus vccintion resulted in significnt increse in t lest one of these immunoglobulin clss-specific HA ntibodies in postvccintion nsl secretions of 22 (96%) of 23 HlNlseronegtive volunteers nd in 27 (87%) of 31 H3N2- seronegtive volunteers. However, the predominnt isotypes of the ntibody elicited by live or inctivted virus vccine differed. Comprison of the combined dt of the two groups of live virus vccinees with those of the inctivted virus vccinees indicted tht inctivted virus vccintion stimulted nsl-wsh IgG HA ntibody responses more frequently thn did live virus vccintion (94 versus

3 68 CLEMENTS AND MURPHY J. CLIN. MICROBIOL. TABLE 2. Nsl-wsh HA-specific IgA, IgG, nd IgM ntibody responses of volunteers who received either live cold-dpted or inctivted virus vccine Nsl-wsh ELISA nti-ha titer (reciprocl men" log2) on specified dy fter vccintion Vccine group (no. in IgA IgG IgM group) Before % with Before % with Before % with vccintion Dys Dys increse vccintion Dys Dys increse vccintion Dys Dys increse Live HlNl (23) Inctivted HlNl (20)" ' 30d c 95d Live H3N2 (31) Inctivted H3N2 (35) c 44d ' 94d Stndrd devitions of the mens were similr, rnging from 0.4 to 2.8 log2. I Titers for volunteers vccinted on dys 0 nd 28 were lso mesured on dy 42 nd were 5.0 nd 6.5 in IgA nd IgG isotypes, respectively. Increses for this group were cumultive for both vccintions. c Sttisticlly significnt difference between the men ntibody increse of the live nd inctivted virus vccine groups, P < by the pired t test. d Sttisticlly significnt difference between the percentge of increses in the live nd inctivted virus vccine groups, p < 0.03 by X- test. 59%, respectively, P < 0.001). In contrst, only 21(38%) of 55 inctivted virus vccine recipients developed nsl-wsh IgA HA ntibody increses fter vccintion compred with 45 (83%) of 54 live virus vccinees (P < 0.001). Reltionship of serum nd nsl-wsh ntibodies. Serum nd nsl-wsh HA ntibody titers of 53 inctivted vccine recipients nd 54 live virus vccinees on dy 28 fter vccintion were compred to determine if there ws correltion between the mgnitude of these ntibody popultions. There ws significnt positive reltionship between the levels of serum nd nsl-wsh IgA nd IgG ntibodies induced by ech type of vccine (Fig. 1). Durtion of serum nd nsl-wsh ntibodies. To study the durtion of the immune response induced by live or inctivted virus vccine, seril serum nd nsl-wsh specimens from vccinees who hd mnifested n increse in systemic or locl IgA or IgG HA ntibody fter vccintion were (D J -i -r C. F CK: L) nu 119 -A. INACT. VIRUS VACCINEES-IgG --B. LIVE VIRUS VACCINEES-IgG 15 17~~~~~~~~I~21.3) 113 d l r x r P <.02 p< g* I I I. _ C. INACT. VIRUS VACCINEES-IgA D LIVE VIRUS VACCINEES-IgA , NASAL WASH ELISA HA ANTIBODY TITER (RECIPROCAL LOG2, FIG. 1. Reltionship between serum nd nsl-wsh HA ntibody titers on dy 28 postvccintion. Correltions were clculted by nonprmetric method (Spermn rnk correltion [r]). _..Lg... e... GoJd.. 9 Oft*.-su 7 * o b... r r / * p <.0_P001p< OOI tested. Only the dt of those vccine responders whose specimens were collected serilly for t lest 16 weeks fter vccintion were nlyzed; these dt were obtined from t lest 80% of the live virus vccine responders nd from 83% of the inctivted vccine responders. The pttern of serum nd nsl-wsh IgA nd IgG HA men ntibody responses in the HlNl vccinees is shown (Fig. 2). Live or inctivted virus vccine induced similr levels of serum IgA nd IgG ntibodies nd nsl IgG ntibody. These ntibody levels peked t 2 weeks fter vccintion with live virus nd 4 weeks fter the second vccintion with inctivted virus. In contrst, the live virus vccine stimulted higher levels of nsl IgA ntibody thn did inctivted virus vccine; this ntibody peked t 2 weeks fter vccintion. The serum IgG nd IgA nd nslwsh IgG HA ntibodies remined significntly elevted (with fourfold or greter titer) bove prevccintion levels for t lest 28 weeks in most of the vccinees in both immuniztion groups, wheres the nsl-wsh IgA ntibody titers remined elevted in bout one-hlf of these vccinees (Tble 3). The levels of serum nd nsl-wsh IgG ntibody remined stble between 2 nd 28 weeks fter vccintion in both vccine groups. However, during this intervl there ws sttisticlly significnt decrese in the levels of both serum nd nsl-wsh IgA ntibodies induced by live HlNl virus vccintion (P < ) nd in the level of serum (but not nsl-wsh) IgA ntibody induced by inctivted virus vccintion (P < 0.002). This decrese in locl IgA ntibody titers ws not sttisticlly significnt becuse of the smll number of specimens tested. The pttern of serum nd nsl-wsh ntibody responses to live or inctivted virus vccine in the H3N2 vccinees is illustrted in Fig. 3. The inctivted virus vccine induced higher levels of serum IgG nd IgA ntibodies nd of nsl IgG ntibody thn did the live virus vccine. As before, the level of nsl IgA ntibody chieved by vccintion with live virus ws higher thn tht chieved by inctivted virus vccintion. The ntibody levels generlly peked t 2 weeks fter vccintion. The IgG nd IgA serum nd nslwsh IgG (but not IgA) ntibodies remined significntly elevted bove prevccintion levels in most of the vccinees in both immuniztion groups for 28 weeks (Tble 3). The levels of serum nd nsl-wsh IgG ntibodies remined stble between 2 nd 28 weeks postvccintion. In contrst, significnt decline in levels of both serum nd nsl-wsh IgA ntibodies occurred between 2 nd 28 weeks fter vccintion with live or inctivted H3N2 vccine (P < 0.003).

4 VOL. 23, 1986 LIVE AND INACTIVATED INFLUENZA A VIRUS VACCINES ( (_) w -..6' c. _ SERUM IgA (15) (15) (15) (14) (15) - - (18) (17) (18) (18) 4 i i 6 D0 i is SERUM IgG NASAL WASH IgG F Cf) WEEKS AFTER VACCINATION (O4 () (I (16) (17) (17) (14) (13) (14) ('7) O le FIG. 2. Serum nd nsl-wsh IgA nd IgG HA men ntibody responses in initilly HlNl-seronegtive dults fter immuniztion with live virus (U) nd inctivted virus (l) vccines. Ech squre represents the men vlue nd ech verticl br represents one stndrd devition. The vlues in prentheses re numbers of volunteers. DISCUSSION The reltive role of specific ntibodies in protection ginst influenz hs not been estblished. However, evidence from studies in nimls nd humns suggests tht both secretory nd serum ntibodies my be meditors of immunity to influenz infection, disese, or both (1, 4, 15, 16, 23, 32, 34). Thus, ntibodies in both comprtments should be mesured when the immunogenicity of new vccine cndidte is ssessed. The present study hs chrcterized locl nd systemic HA-specific ntibody responses in seronegtive dults immunized with live ttenuted nd inctivted influenz A vccines. The immune responses to intrnslly dministered, live virus vccintion differed in two spects from those observed with prenterl, inctivted virus vccintion. (i) Inctivted virus vccine induced greter serum IgA, IgG, nd IgM HA ntibody responses thn did live virus vccine. (ii) Although vccintion with both types resulted in significntly incresed levels of nsl-wsh HA TABLE 3. Persistence of serum nd nsl-wsh HA ntibodies induced by live cold-dpted or inctivted virus vccines Vccine dministered S. 6 W 4, z 2 Proportion (%) of vccine responders with persistently elevted ntibody titers t 28 weeks fter vccintion Serum Nsl wsh IgG IgA IgG IgA Live H3N2 16/22 (73) 22/24 (92) 11/18 (61) 13/23 (56) Inctivted H3N2 27/30 (90) 28/31 (90) 20/28 (71) 6/13 (46) Live HlNl 11/14 (79) 12/16 (75) 12/14 (86) 6/16 (38) Inctivted HlNl 18/18 (100) 15/15 (100) 16/17 (94) 2/6 (33) Persistently elevted titers were defined s fourfold or greter increse between prevccintion nd 28-week postvccintion titers. ntibodies, there ws striking difference in the predominnt isotype of locl ntibody induced by ech type of vccine. Recipients of live virus vccine developed IgA ntibody more frequently, wheres recipients of inctivted influenz vccine developed IgG ntibody more frequently. Interestingly, these respective locl ntibodies remined elevted bove prevccintion levels for t lest 6 months (28 weeks) in more thn one-hlf of the vccinees who hd nsl IgG ntibody response nd in t lest one-third of those who hd nsl IgA ntibody response. The finding tht prenterl vccintion with inctivted influenz vccine stimultes systemic ntibodies in ferrets nd in humns more efficiently thn does intrnsl vccintion with live ttenuted virus hs been reported previously (10, 11, 13, 16, 25, 37). In generl, intrnsl vccintion with ttenuted c influenz A vccine virus does not produce s extensive systemic ntibody response s does intrnsl inocultion of the homologous wild-type virus (14, 31). However, some live ttenuted influenz viruses dministered t high doses cn induce levels of serum ntibodies of the sme mgnitude s those induced by the homologous wild-type virus. For exmple, the A/Wshington/897/80 (H3N2) nd A/Cliforni/10/78 (H1N1) c vccine viruses, when dministered intrnslly t doses comprble to those in the present study, stimulted serum ntibody levels s high s those induced by experimentl infection with wildtype virus (12, 28). Previous studies in primed nd unprimed children nd dults hve demonstrted tht the levels of ntibody in serum decline significntly within 6 to 8 months fter prenterl vccintion with subvirion vccine (9, 11, 22) but not fter intrnsl vccintion with live ttenuted virus or fter nturl infection (11, 18, 36). In the current study, we found tht the serum IgA nd IgG HA ntibody levels in

5 70 CLEMENTS AND MURPHY J. CLIN. MICROBIOL J I 12- L-) o l0- CL 3 8 I- -6 W: 4~ SERUM IgA (31) T) (31' (31) (30) (2 (24) (4) (T) (T4) ) SERUM IgG (31) (3 (30) (22) ) (22) (21) (22) m 8- z I 6- _j 4. LLI z < 2- wl WEEKS AFTER VACCINATION FIG. 3. Serum nd nsl-wsh IgA nd IgG HA men ntibody responses in initilly H3N2-seronegtive dults fter immuniztion with live virus (U) nd inctivted virus (O) vccines. Ech squre represents the men vlue nd ech br represents one stndrd devition. The vlues in prentheses re numbers of volunteers. most vccinees remined elevted bove prevccintion levels for t lest 6 months fter vccintion with either inctivted or live virus. The reson for the persistence of elevted serum ntibody levels fter inctivted virus vccintion in our study is not cler, but differences in vrious studies in ntigenicity of the vccines, immunologicl sttus of the vccinees, nd sensitivity of the ssys used to detect ntibodies my hve been responsible. Live influenz vccines dministered intrnslly, like orl poliovirus vccines, regulrly elicit higher levels of locl secretory IgA or neutrlizing ntibody thn inctivted virus vccines dministered prenterlly (15, 16, 18, 25, 32, 37). In the current study, 83% of live virus recipients developed nsl IgA HA ntibody responses compred with only 38% of inctivted vccine recipients. It is interesting tht prenterlly dministered, nonreplicting (i.e., inctivted) viruses lso stimulte locl ntibody responses. More thn 40 yers go, Frncis (17) observed tht subcutneous injection of dults with influenz vccine enhnced the inctivting cpcity of their nsl secretions. Others confirmed this finding nd identified this substnce s neutrlizing ntibody, minly polymeric IgA ntibody contining secretory component (6, 19, 27, 32, 35). More recently, IgG nd IgM HA-specific ntibody responses to live nd inctivted influenz vccines hve been mesured in nsl-wsh specimens (18, 29, 37). In studies with seronegtive dults, Zhrdnik nd co-workers (37) detected nsl IgG HA ntibody increses by rdioimmunoprecipittion ssy in one-third of inctivted virus recipients but in only 6% of live virus vccinees. Johnson nd his collegues (18) lso found nsl IgG (mesured by ELISA to surfce glycoproteins) in one-third of children who received inctivted influenz vccine nd in 7 of 10 children vccinted with live virus vccine. In contrst, in our study nerly ll (94%) inctivted virus vccinees nd 59% of live virus vccinees developed nsl IgG HA ntibody responses (mesured by ELISA) fter vccintion. These discrepncies in locl IgG responses cn be explined in prt by the different ntibody ssys, dose of virus dministered, nd prevccintion immunologicl sttus of the vccinees. Our findings of grdul decline in secretory IgA HAspecific ntibodies induced by live virus vccintion over 6 months gree with previous studies which showed tht locl IgA ntibody resulting from vccintion is reltively shortlived (8, 19). Our study did not ddress the persistence of locl ntibodies beyond 6 months fter vccintion. However, dt from studies in seronegtive children hve indicted tht nsl IgA HA ntibody could be detected for 1 or more yers in 47% of nturlly infected subjects nd in 53% of live virus vccinees, but in only 5% of inctivted vccine recipients (18). Nsl IgG HA ntibody persisted in 77% of nturlly infected children, 53% of live virus vccines, nd 45% of inctivted virus vccinees (18). The origin of nsl-wsh HA ntibodies induced by live nd inctivted virus vccintion is not completely known. There re t lest three possibilities: (i) locl production of J chin contining IgA dimers (or IgM pentmers) which re ctively trnsported externlly by nsl epithelium possessing secretory component to which polymeric IgA nd IgM bind (3, 5); (ii) locl synthesis of non-j chin-contining immunoglobulin by plsm cells in the nsl mucos followed by its pssive trnsport towrd this lumen; nd (iii) pssive trnsudtion of serum-derived ntibodies. In the cse of intrnsl vccintion with live ttenuted virus, there is considerble evidence tht secretory IgA nd to some extent IgM nd IgG re synthesized by nsl epithelil cells nd ctively secreted loclly (3-5, 7, 8, 29). In erlier studies (7, 29) positive correltion between the levels of

6 VOL. 23, 1986 LIVE AND INACTIVATED INFLUENZA A VIRUS VACCINES 71 nsl nd serum IgA HA ntibodies in live virus vccinees ws observed, suggesting tht these ntibodies might be derived from the sme source. Brown nd collegues (6) found tht IgA HA-specific ntibody in nsl-wsh specimens fter infection with wild-type virus ws composed lmost entirely of polymeric (11S) secretory IgA. Furthermore, the serum IgA HA ntibody from these volunteers contined higher proportion of J-chin-contining polymeric IgA with respect to monomeric IgA thn totl serum IgA, suggesting tht serum IgA HA-specific ntibody induced by live virus vccintion ws of mucosl origin. The findings in the present study re consistent with the concept of mucoslly derived serum nd with nsl-wsh IgA ntibodies in live virus vccinees, i.e., the mgnitude of their serum nd psl-wsh IgA HIA ntibodies were relted nd there ws concomitnt decrese in these ntibody popultions over 6 months. The origin of nsl ntibodies, prticulrly IgG HA ntibody, induced by subcutneously dministered inctivted virus vccine, is intriguing but cnnot be unequivoclly identified by our findings. The positive correltion coefficient between postvccintion levels of nsl nd serum IgG HA ntibodies in our inctivted virus vccinees suggests tht these popultions of ntibody re derived from the sme source. The observtion tht levels of both serum nd nsl-wsh IgG HA ntibodies remined stble up to 6 months fter vccintion while nsl-wsh IgA levels declined supports the concept tht nsl IgG HA ntibody is derived from serum ntibody by pssive trnsudtion. This possibility requires further study. Serum IgG influenz ntibody hs been shown to prevent lethl pneumoni in infected nimls nd to correlte with resistnce to influenz illness in neontes (33, 34); however, the role of nsl-wsh IgG ntibody in protection ginst influenz hs not been defined. A previous study demonstrted reltionship between resistnce to influenz infection nd specific IgG in nsl secretions (15). Clerly, the function of inctivted influenz vccine-induced IgG in nsl secretions should be ivestigted further. Results of erlier stud- hve demonstrted tht, in the bsence of serum ntibodies, locl IgA ntibody is mjor determinnt of resistnce to infection nd illness in volunteers chllenged with wild-type virus (13). Liew nd coworkers (23) recently reported dt suggesting tht specific IgA in the respirtory trct induced by infection of mice with live influenz virus my be the most importnt immunologicl meditor of protection ginst wild-type infection. In n erlier volunteer chllenge study, we showed tht immunity conferred by infection with live c virus vccine ws more effective ginst wild-type virus infection thn ws inctivted virus vccine (13). Becuse live virus vccine stimulted nsl IgA more efficiently thn did the inctivted vccine, it is likely tht nsl IgA HA ntibody plys n importnt role in preventing experimentlly induced influenz. The grdul ttrition of nsl nd serum IgA ntibody induced by live virus vccintion, however, my represent wning in immunity nd my result in n incresed susceptibility to influenz infection nd illness with time. In summry, both live nd inctivted influenz vccines induced ntibodies in the serum nd respirtory trct. Although the reltive protective role of systemic nd locl IgA nd IgG ntibodies hs not been clerly defined, the presence of ntibodies in both comprtments my be importnt in the prevention of infection or disese. The finding tht live, ttenuted, cold-dpted ressortnt influenz A vccine induces reltively long-lsting locl nd systemic ntibodies suggests tht this vccine my be suitble lterntive to the current licensed inctivted vccine for use in helthy children nd dults. ACKNOWLEDGMENTS This reserch ws supported by Public Helth Service contrct number N01-AI from the Ntionl Institute of Allergy nd Infectious Diseses. We thnk Mrgret P. Bridwell, Frncis Cve, Mriett Dorn, nd Ptrici Green from the Student Helth Center, University of Mrylnd t College Prk; Jonthn Steinberg, Shron Crver, Jnice Adms, nd nurses from the Center for Vccine Development; nd Frnk Wood from the Lbortory of Infectious Diseses for specil ssistnce in the conduct of this study. LITERATURE CITED 1. Brber, W. H., nd P. A. Sml, Jr Locl nd systemic immunity to influenz infections in ferrets. Infect. Immun. 21: Belse, R. B., nd L. P. Vn Voris Cold recombinnt influenz A/Cliforni (CR-37) in seronegtive children: infectivity nd efficcy ginst investigtionl chllenge. J. Infect. Dis. 149: Bienenstock, J., nd A. D. Befus Mucosl immunology. Immunology 41: Brndtzeg, P Mucosl nd glndulr distribution of immunoglobulin components. Immunohistochemistry with cold-ethnol fixtion technique. Immunology 26: Brndtzeg, P Immune functions of humn nsl mucos nd tonsils in helth nd disese, p In J. Bienenstock (ed.), Immunology of the lung nd upper respirtory trct. McGrw-Hill Book Co., New York. 6. Brown, T. A., B. R. Murphy, J. Ril, J. J. HUmn, nd J. Mestecky Subclss distribution nd moleculr form of immunoglobulin A hemgglutinin ntibodies in ser nd nsl secretions fter experimentl secondry infection with influenz A virus in humns. J. Clin. Microbiol. 22: Burlington, B. D., M. L. Clements, G. Meiklejohn, M. Pheln, nd B. R. Murphy Hemgglutinin-specific ntibody responses in immunoglobulin G, A, nd M s mesured by enzyme-linked immunosorbent ssy fter primry or secondry infection of humns with influenz A virus. Infect. Immun. 41: Butler, W. T., T. A. Widmnn, R. D. Rossen, R. G. Dougls, Jr., nd R. B. Couch Chnges in IgA nd IgG concentrtions in nsl secretions prior to the ppernce of ntibody during virl respirtory infection in mn. J. Immunol. 105: Cte, T. R., R. B. Couch, J. A. Ksel, nd H. R. Six Clinicl trils of monovlent influenz A/New Jersey/76 virus vccines in dults: rectogenicity, ntibody response, nd ntibody persistence. J. Infect. Dis. 136(Suppl.): Clrk, A., C. W. Potter, R. Jennings, J. P. Nicholl, A. F. Lngnick, G. C. Schild, J. M. Wood, nd D. A. J. Tyrrell A comprison of live nd inctivted influenz A (HIlN1) virus vccines. 1. Short-term immunity. J. Hyg. 90: Clrk, A., C. W. Potter, R. Jennings, J. P. Nicholl, A. F. Lngrick, G. C. Schild, J. M. Wood, nd D. A. J. Tyrrell A comprison of live nd inctivted influenz A (I"lN1) virus vccines. 2. Long-term immunity. J. Hyg. 90: Clements, M. L., R. F. Betts, H. F. Mssb, nd B. R. Murphy Dose response of influenz A/Wshingtori/897/80 (H3N2) cold-dpted ressortnt virus in dult volunteers. J. Infect. Dis. 149: Clements, M. L., R. F. Betts, nd B. R. Murphy Advntge of live ttenuted cold-dpted influenz A virus over inctivted vccine for A/Wshington/80 (H3N2) wild-type virus infection. Lncet i: Clements, M. L., S. O'Donnell, M. M. Levine, R. M. Chnock, nd B. R. Murphy Dose response of A/Alsk/6/77 (H3N2) cold-dpted ressortnt vccine virus in dult volunteers: role of locl ntibody in resistnce to infection with

7 72 CLEMENTS AND MURPHY vccine virus. Infect. Immun. 40: Couch, R. B., J. A. Ksel, H. R. Six, nd T. R. Cte The bsis for itnmunity to influenz in mn, p In D. P. Nyk nd D. F. Fox (ed.), Genetic vrition mong influenz viruses. Acdemic Press, Inc., New York. 16. Fenton, R. J., A. Clrk, nd C. W. Potter Immunity to influenz in ferrets. XIV. Comprtive immunity following infection or immuniztion with live or inctivted vccine. Br. J. Exp. Pthol. 62: Frncis, T., Jr A rtionle for studies in the control of epidemic influenz. Science 97: Ijoskins, T. W., R. R. Dvies, A. J. Smith, C. L. Miller, nd A. Allchin Assessment of inctivted influenz-a vccine fter three outbreks of influenz A t Christ's Hospitl. Lncet i: Johnson, P. R., S. Feldmn, J. M. Thompson, nd P. F. Wright Comprison of long-term systemic nd secretory ntibody responses in seronegtive children given live, ttenuted or inctivted influenz A vccine. J. Med. Virol. 17: Ksel, J. A., R. D. Rossen, R. V. Fulk, D. S. Fedson, R. B. Couch, nd P. Brown Humn influenz: spects of the immune iesponse to vccintion. Ann. Intern. Med. 71: LMontgne, J. R., P. F. Wright, M. L. Clements, H. F. Mssb, nd B. R. Murphy Prospects for live, ttenuted influenz' vccines using ressortnts derived from the A/Ann Arbor/6/60 (H2N2) cold-dpted (c) donor virus, p In W. G. Lver (ed.), The origin of pndemic influenz viruses. Elsevier Science Publishing, Inc., New York. 21. Lzr, A., N. Okbe, nd P. F. Wright Humorl nd cellulr immune responses of seronegtive children vccinted with cold-dpted influenz A/HK/123/77 (H1N1) recombinnt virus. Infect. Immun. 27: Lermn, S. J., P. F. Wright, nd K. D. Ptil Antibody detline in children following A/New Jersey/76 influenz virus immuniztion. J. Peditr. 96: Liew, F. Y., S. M. Russell, G. Appleyrd, C. M. Brnd, nd J. Bele Cross-protection in mice infected with influenz A virus by the respirtory route is correlted with locl IgA ntibody rther thn serum ntibody or cytoxic T cell rectivity. Eur. J. Immunol. 14: Mssb, H. F., N. J. Cox, B. R. Murphy, nd A. P. Kendl Biologicl, genetic nd biochemicl chrcteriztion of cold-dpted recombinnt A/Victori/3/75 virus nd its evlution in volunteers. Interntionl symposium on influenz immuniztion (II), Genev. Dev. Biol. Stnd. 39: Mnn, J. J., R. H. Wldmn, Y. Togo, G. G. Heiner, A. T. Dwkins, nd J. A. Ksel Antibody response in respirtory secretions of volunteers given live nd ded influenz virus. J. Immunol. 100: Meyer, H. M., Jr., H. E. Hopps, P. D. Prkmn, nd F. A. J. CLIN. MICROBIOL. Ennis Review of existing vccines for influenz. J. Clin. Pthol. 70: Mostow, S. R., S. C. Schoenbum, W. R. Dowdle, M. T. Colemn, H. S. Kye, nd J. C. Hierholzer Studies on inctivted influenz vccines. II. Effect of'incresing dosge on ntibody response nd dverse rections in mn. Am. J. Epidemiol. 92: Murphy, B. R., M. L. Clements, H. P. Mdore, J. Steinberg, S. O'Donnell, R. Betts, R. Dolin, nd H. F. Mssb Dose response of influenz A/Cliforni/10/78 (HlNl) cold-dpted ressortnt influenz virus in dult volunteers. J. Infect. Dis. 149: Murphy, B. R., D. L. Nelson, P. F. Wright, E. L. Tierney, M. A. Pheln, nd R. M. Chnock Secretory nd systemic immunologicl response in children infected with live ttenuted influenz A virus vccines. Infect. Immun. 36: Murphy, B. R., M. A. Pheln, D. L. Nelson, R. Yrchon, E. L. Tierney, D. W. Alling, nd R. M. Chnock Hemgglutinin-specific enzyme-linked immunosorbent ssy for ntibodies to influenz A nd B viruses. J. Clin. Microbiol. 13: Murphy, B. R., M. B. Rennels, R. G. Dougls, Jr., R. F. Betts, R. B. Couch, T. R. Cte, Jr., R. M. Chnock, A. P. Kendl, H. F. Mssb, S. Suwngool, S. B. Sotmn, L. A. Cisneros, W. C. Anthony, D. R. Nlin, nd M. M. Levine Evlution of influenz A/Hong Kong/123/77 (HlNl) ts-1a2 nd colddpted recombinnt viruses in seronegtive dult volunteers. Infect. Immun. 29: Ogr, P. L., D. T. Krzon, F. Righthnd, nd M. McGillivry Immunoglobulin response in serum nd secretions fter immuniztion with live nd inctivted polio vccine nd nturl infection. N. Engi. J. Med. 279: Puck, J. M., W. P. Glezen, A. L. Frnk, nd H. R. Six Protection of infnts from infection with influenz A virus by trnsplcentlly cquired ntibody. J. Infect. Dis. 142: Rmphl, R., R. C. Coglino, J. W. Shnds, Jr., nd P. A. Smll, Jr Serum ntibody prevents lethl murine influenz pneumonitis but not trcheitis. Infect. Immun. 25: Wenzel, R. P., J. 0. Hendley, M. A. Snde, nd J. M. Gwltney, Jr Revised ( ) bivlent influenz vccine. Serum nd nsl ntibody responses to prenterl vccintion. J. Am. Med. Assoc. 226: Wright, P. F., N. Okbe, K. T. McKee, Jr., H. F. Mssb, nd D. T. Krzon Cold-dpted recombinnt influenz A virus vccines in seronegtive young children. J. Infect. Dis. 146: Zhrdnik, J. M., J. A. Ksel, R. R. Mrtin, H. R. Six, nd T. R. Cte Immune responses in serum nd respirtory secretions following vccintion with live cold-recombinnt (CR35) nd inctivted A/USSR/77 (HlNl) influenz virus vccine. J. Med. Virol. 11: