Vaccination. Michigan women and 11 men, ages 26 to 54 years (mean, 39 years), participated with informed consent.

Transcription

1 INFECTION AND IMMUNrrT, May 1981, p /81/553-5$2./ Vol. 32, No. 2 Influence of Prior Cellular Immunity on the In Vitro Lymphocyte Response to Virus Antigens After Influenza WILLIAM A. BRIGGS Department ofmedicine, Wayne State University School ofmedicine and Harper-Grace Hospitals, Detroit, Michigan 4821 The in vitro peripheral blood lymphocyte response to specific influenza virus antigens was studied before and 2 weeks after trivalent influenza vaccination of 16 healthy persons. Changes in serum hemagglutination inhibition antibody titers were also determined. An inverse correlation was found between the prevaccination antibody titers and the log2 mean-fold increase in antibody titers after vaccination (r = -.86, P <.1). An inverse correlation was also found between the prevaccination stimulation index and the ratio of postvaccination to prevaccination stimulation index for each virus strain (A/Victoria, r = -.48, P <.5; A/USSR, r = -.55, P <.2; and B/Hong Kong, r = -.55, P <.2). Similar negative correlations were not consistently found with the nonspecific mitogens phytohemagglutinin, pokeweed mitogen, and concanavalin A. These results suggest that the state of cellular as well as humoral immunity to virus antigens before vaccination influence the magnitude of response after vaccination and that antigen-specific suppressor cell activity may be stimulated by vaccination. The immunological response of humans given influenza vaccine has generally been evaluated by studying changes in serum antibody titers, especially serum hemagglutination inhibition (HI) antibody titers, after vaccination. Tauraso et al. (14) noted that fourfold or greater rises in antibody titer occurred more frequently in subjects with lower antibody titers before vaccination. Hobson et al. (5) reported a striking inverse relationship between the prevaccination antibody titer and the magnitude of the antibody response to vaccination. Although studies of the induction of cytotoxic lymphocytes by influenza virus (7, 8) and influenza vaccination (1), and of lymphocyte proliferative responses to influenza virus antigens in vitro before and after vaccination (2-4, 6, 11-13), have been reported, none has addressed whether the state of prior cellular immunity might influence the response to vaccination, as has been noted for the humoral response. To gain additional insight into the effect of prior immunity on both cellular and humoral responses, and any relationships between the two, both serum HI antibody titers and in vitro proliferative responses of lymphocytes to virus antigens were studied in 16 healthy human adult volunteers receiving trivalent influenza vaccination. 53 MATERIALS AND METHODS The studies were done as part of a protocol approved by the Wayne State University Human Investigation Committee. Sixteen adult volunteers, 5 women and 11 men, ages 26 to 54 years (mean, 39 years), participated with informed consent. All subjects were in good general health, and none had experienced an acute illness or surgical procedure within 2 weeks of the study period. None was taking aspirin, nonsteroidal anti-inflammatory agents, or other substances known to interfere with lymphocyte transformation studies. Each subject was vaccinated by the intramuscular injection of.5 ml of zonal-centrifuged ether-treated subunit vaccine containing 7 ug each of A/USSR/77/ NIB-6 (HlNl), A/Texas/77/X49 (H3N2), and B/ Hong Kong/5/72 antigens (Parke, Davis & Co., Detroit, Mich.). All subjects received vaccine from the same lot. Blood samples were obtained immediately before and 2 weeks after vaccination. A 2-week interval was selected because peak HI antibody responses occur within that time and because of published data showing ximal or near-maximal lymphocyte proliferative responses to influenza virus antigens over such an interval (4). On each occasion, mononuclear cells were isolated from whole blood by density gradient centrifugation (1), and cells were proportioned for cell culture studies. Cell viability was ascertained by trypan blue exclusion. Serum samples were also obtained and stored at -4 C for subsequent determination of HI antibody titers. Lymphocyte transformation during 3-day (nonspecific mitogens) and 6-day (virus antigens) cell cultures were quantitated by using a microassay for incorporation of radiolabeled thymidine. Mononuclear cells suspended in RPMI 164 medium (GIBCO Laboratories, Grand Island, N.Y.) with 15% pooled human serum were placed in triplicate wells (3 x 1' cells/ Downloaded from on September 4, 218 by guest

2 54 BRIGGS well) of microtiter plates with medium alone and with nonspecific mitogens, specific virus antigens, and allantoic fluid protein. The nonspecific mitogens included phytohemagglutinin (PHA; Difco Laboratories, Detroit, Mich.), pokeweed mitogen (PWM, GIBCO), and concanavalin A (ConA; Pharmacia Fine Chemicals, Inc., Piscataway, N.J.). Each was added to cell culture wells in the following concentrations: PHA, 5 and 1 log/ml; PWM, 15 and 3 ug/ml; and ConA, 5, 15, and 3 itg/ ml. These concentrations of reagents had been shown to result in optimal lymphocyte blast transformation. The specific antigens used in this study were purified influenza virus protein antigens provided by the Pharmaceutical Research Division of Warner-Lambert/Parke, Davis Laboratories, Detroit, Mich., derived from three different virus strains grown in embryonated chicken eggs. Briefly, the virus strains were concentrated by zonal centrifugation, extracted with ether, and purified by molecular filtration. Thimerosal and Formalin were added to the antigen concentrates and then subsequently removed by dialysis against phosphate-buffered saline at ph 7.2. The virus antigen concentrates were reconstituted to contain.67, 1.46, and 1.6 mg of protein per ml for the A/USSR, A/ Victoria, and B/Hong Kong strains, respectively. A virus antigen concentrate of A/Texas was not available. Thus, the A/Victoria concentrate was used in the lymphocyte transformation studies, since A/Victoria/ 75/X-47 is cross-immunogenic with A/Texas/77/X-49. Assays of the concentrates for residual thimerosal and Formalin showed that the latter was absent from each, and the thimerosal concentrations were: A/USSR,.11% (wt/vol); A/Victoria,.24% (wt/vol); and B/Hong Kong,.22% (wt/vol). Purified allantoic fluid protein was harvested from 1-day-old noninfected chicken embryos to test the cellular responsiveness of each subject to chicken allantoic proteins or other potential contaminants of the virus antigen concentrates. The purified virus antigens were added to cell culture wells in final concentrations of 1, 15, and 2 ug of protein per ml, concentrations shown to yield optimal stimulation in dose-response studies conducted before the testing of cells from control subjects. Allantoic fluid protein was added to cell culture wells in a final concentration of 15 jg of protein per ml. The cells were cultured at 37 C for either 72 h (nonspecific mitogens) or 6 days (virus antigens and allantoic fluid protein) in an atmosphere containing 5% C2. During the final 4 h of incubation, [3H]thymidine (New England Nuclear Corp., Boston, Mass.; 6.7 Ci/mmol) was added at.8 PCi/well. At the end of the culture period, the cells were harvested from the wells onto glass filter papers (Whatman Inc., Clifton, N.J.), using an ADAPS cell harvester (ADAPS Inc., Dedham, Mass.). The filter papers were dried, and the cell-containing areas were punched out, placed into scintillation fluid, and counted in a liquid scintillation spectrometer for 1 min. The mean cpm from each triplicate was used to express the rate of [3H]thymidine incorporation for each experimental manipulation with cells from individual subjects. Sera were tested for antibody titers to each virus strain by standard HI techniques (9). Before testing, INFECT. IMMUN. individual sera were randomly assigned a code number, and all samples were run simultaneously. The lymphocyte transformation responses to nonspecific mitogens and specific antigens were expressed as difference in cpm (Acpm; cpm of stimulated cells - cpm of nonstimulated cells) and as stimulation index (SI; cpm of stimulated cells/cpm of nonstimulated cells). The change in lymphocyte responses from before to after vaccination was expressed as the net change in Acpm and SI, i.e., Acpmftr - Acpmbefoe and SIafte, - SIbefore, and as the ratio Acpmft.r/Acpmbefor, and SIafter/SIbefore. The HI antibody titers were expressed as the reciprocal of the dilution of serum resulting in 1% hemagglutination. The change in titers associated with vaccination were expressed as the ratio HIfter/Hlbefore. Correlations were sought between pre- and postvaccination lymphocyte responses and HI titers and between vaccination-associated changes in lymphocyte responses and HI titers. In addition, correlations were sought between the changes in lymphocyte responses and HI titers and the prevaccination lymphocyte response and HI titer in individual subjects. For the HI titers, both individual HIfter/HIbefore ratios for each virus strain and log2 mean-fold increase in titer for all strains were plotted against the prevaccination titer. RESULTS Table 1 summarizes the humoral and in vitro cellular immune responses to vaccination of the 16 subjects. Although significant increases in geometric mean HI antibody titer to each virus strain occurred, no significant increases in mean Acpm or SI were seen after vaccination. A significant decrease in mean Acpm to A/Victoria was noted. Cross-immunogenicity of the A/ Texas and A/Victoria strains was confirmed in this study by comparing the HI antibody titers and responses in individual subjects; the correlation between the two strains within subjects was significant (r =.8, P <.1). There was no significant difference in lymphocyte response when cells were cultured in medium alone or with allantoic fluid protein. When individual subject lymphocyte responses were analyzed, there appeared to be an inverse correlation between the magnitude of response before and that after the vaccination. Subjects with SI less than 1 to any of the three virus antigens often showed an increase after vaccination, with the greatest increases seen in subjects starting with an SI less than 5 (Fig. 1). In contrast, subjects with an SI greater than 2 before vaccination showed either no change or, more commonly, a decrease in SI afterward (Fig. 1). In subjects with an SI between 1 and 2 before vaccination, increases, decreases, and no change occurred with equivalent frequency, When all data points for each subject's magnitude of change in cellular response (ratio of SIft^er/SIbefore) to each virus antigen were plotted Downloaded from on September 4, 218 by guest

3 VOL. 32, 1981 TABLE 1. LYMPHOCYTE RESPONSES TO INFLUENZA VACCINATION 55 Serum antibody titers and in vitro lymphocyte blastogenic responses to influenza virus strains before and after vaccinationa HI antibody titer Cellular response Strain SI Acpm (xlo:,) Before After P Before After P Before After P A/USSR < ± ± 5.5 NSb 24.5 ± ± 2.8 NS A/Texasc < ± ± 3.4 NS 23.4 ± ± 4.1 <.5 B/Hong Kong < ± ± 1.3 NS 1.7 ± ± 2. NS a Numbers represent geometric mean for HI antibody titer and mean ± standard error for the cellular responses. b NS, Not significant. 'The cross-reacting strain (A/Victoria) was used in the lymphocyte response studies. 5- C o E 2- l. >. o ( C a. 21 x S- r- 5- E cn 2- I- o A/Victoria * /JSSR x B/Hong Kong Before After Before After FIG. 1. Individual data points for lymphocyte SI to influenza virus antigens before and after vaccination when prevaccination SI were <1 and >2. The increase in mean SI from 4.6 to 13.4 when prevaccination SI was <1 was statistically significant (P <.1). The decrease from 34.6 to 23.3 when prevaccination SI was >2 did not achieve statistical significance (P =.7). (Fig. 2) as a function of the response before vaccination (Slbefor.), an inverse correlation was found for each antigen (A/Victoria, r = -.48, P <.5; A/USSR, r = -.55, P <.2; B/Hong Kong, r = -.55, P <.2). The inverse correlation between cellular response before vaccination and magnitude of response was also analyzed by plotting SI-fterc.c-. 2- c o - L 2-- o 1.- _ la E.2- &_ 1_._ 2- OX o X X x X o A/Victoria * AVUSSR x B/Hong Kong g x x * x * x 4S X x { -. I I I I I I IIIIII Stimulation index (S.I.) before vaccination FIG. 2. Individual data points demonstrating inverse relationship between magnitude of change in lymphocyte SI after vaccination and the SI existing before vaccination (A/Victoria, r = -.48, P <.5; A/USSR, r = -.55, P <.2; B/Hong Kong, r =.5, P <.2). SIbefore versus SIbefore, ratio of Acpmfe,r/ Acpmbefore versus Acpmbefore, and Acpmfer - Acpmbefore versus Acpmbefore (Table 2). The results showed a significant negative correlation in the majority of instances. Changes in cellular response using SI correlated well with those using Acpm within subjects for each antigen (SIfter/SIbefore versus Acpm.ader/Acpmbefore; A/ Victoria, r =.97, P <.1; A/USSR, r =.57, P <.2; B/Hong Kong, r =.93, P <.1). A negative correlation between changes in SI after vaccination and SI before were also found when lymphocytes were cultured with the nonspecific mitogens PHA, ConA, and PWM, but the correlation was significant only in the case of PWM. No consistent correlation pattern was Downloaded from on September 4, 218 by guest

4 56 BRIGGS TABLE 2. Correlations between lymphocyte responses to virus antigens before vaccination and the changes in those responses after vaccination Acpm,rw., Acpmat. - SI.ft.r/SIb,.fo, VS SI.ftr - SIbfr. VS ACpma.f, VS AcpMbef. VS Strain SIbef.o SIbef, Acpmbefor,e Acpmbefoe r P r P r P r P A/Victoria -.48 <.5.62 < NSa A/USSR -.55 < < <.5 B/Hong Kong -.55 < < < <.8 a NS, Not significant. noted when the data used for analysis from these nonspecific mitogenic stimulation studies were Acpm rather than SI. In addition, although the ratio SI for each virus antigen correlated positively and significantly with the ratio SI for PHA and ConA, there were no consistent correlations among the other expressions of changes in cellular response to the virus antigens and those to the nonspecific mitogens. There was a significant inverse correlation between the log2 mean-fold increase in HI antibody titer after vaccination and the antibody titer before vaccination (r = -.86, P <.1; Fig. 3). No consistent correlations were found between HI antibody titers and lymphocyte responses, or changes therein, whether the lymphocyte responses were expressed as SI or as Acpm. DISCUSSION The observation that fourfold or greater increases in HI antibody titer occur mainly in subjects with low or undetectable antibody titers before vaccination and uncommonly in subjects with high prevaccination titers has been reported by others (5, 14). The results of this study showed that an inverse relationship also exists between the magnitude of change in peripheral blood lymphocyte reactivity to viral antigens after vaccination and the degree of reactivity existing before vaccination. Although low levels of lymphocyte responsiveness (SI less than 1) were usually associated with significant enhancement of responsiveness after vaccination, very high levels (SI greater than 2) were more often associated with profound reductions in responsiveness. This inverse correlation between reactivity of lymphocytes to virus antigen before vaccination and the magnitude of change in that reactivity held whether the lymphocyte reactivity was expressed as SI or as Acpm. The changes in response to virus antigens would not appear to represent simply nonspecific generalized alterations in cellular immune responsiveness, since consistent correlations between changes in cellular reactivity to virus antigens and to nonspecific mitogens were not ob- *1 4._c U -u cw i o E CM "s-% _ o '_ ~~~~~~~ I INFECTr. IMMUN. ' HAI ontibody titer before vaccination FIG. 3. Log2 mean-fold increase in serum HI antibody titer in 16 vaccine recipients as a function of prevaccination titer, demonstrating an inverse relationship (r = -.86, P <.2). taimed. On the other hand, the effect of vaccination on the lymphocyte reactivity to virus antigens may have caused some change in reactivity to nonspecific mitogenic stimulation, since significant correlations were obtained between the Acpm to the virus antigens and to PHA and ConA after vaccination, even though no such correlation was present before vaccination. These observations suggest that, although individual humoral and cellular immune responses to specific influenza virus antigens in influenza vaccine may be dissociated from one another, both are influenced similarly by the degree of immunity existing at the time of vaccine administration. The host factors and immunological regulatory mechanisms responsible for an inverse relationship between the degree of prevaccination immunity, manifested either as antibody titer or as lymphocyte reactivity (SI or Acpm), and the direction or magnitude of change in the immune response cannot be identified from this study. It could be, for instance, that antigen-specific suppressor cell activity is stimulated as a result of exposure to the influenza virus proteins administered in the vaccine. On I Downloaded from on September 4, 218 by guest

5 VOL. 32, 1981 the other hand, a decrease in lymphocyte response after vaccination could reflect an increase in antigen-specific B cells in the cultured cells obtained after vaccination, these cells being capable of binding the virus proteins in such a way as to impair T-cell recognition. These results emphasize the need for additional studies of the effects of vaccine administration on the cellular, as well as the humoral, effector limbs of the immune system in humans, including correlations between changes in lymphocyte proliferative responses and generation of cytotoxic lymphocytes. ACKNOWLEDGMENTS Richard Rozek and Kenn Wielechowski provided technical support. Freda Giblin assisted with statistical analysis and manuscript preparation. Serum HI antibody studies were done under the supervision of Joan L. Shillis, Robert G. Brackett, and Frank B. Brandon, Biological Research and Development Department, Warner-Lambert/Parke, Davis, Detroit, Mich. William B. Beardmore supplied the influenza virus antigen concentrates and allantoic fluid protein. This study was supported in part by funds provided by The Skillman Foundation and Warner-Lambert/Parke, Davis. LITERATURE CITED 1. Boyum, A Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Invest. 21(Suppl. 97): Chow, T. C., K. R. Beutner, and P. L. Ogra Cellmediated immune responses to the hemagglutinin and neuraminidase antigens of influenza A virus after immunization in humans. Infect. Immun. 25: Cole, P. J., and M. E. Molyneux Lymphocyte reactivity to influenza virus in man. Immunology 29: Dolin, R., B. R. Murphy, and E. A. Kaplan Lymphocyte blastogenic responses to influenza virus antigens after influenza infection and vaccination in LYMPHOCYTE RESPONSES TO INFLUENZA VACCINATION 57 humans. Infect. Immun. 19: Hobson, D., F. A. Baker, and R. F. Curry Effect of influenza vaccines in stimulating antibody in volunteers with prior immunity. Lancet ii: Jennings, R., R. J. Fenton, M. G. Entegart, and C. W. Potter A contribution of cellular immunity to protection against influenza in man. Med. Microbiol. Immunol. 166: McMichael, A HLA restriction of human cytotoxic T lymphocytes specific for influenza virus. Poor recognition of virus associated with HLA A2. J. Exp. Med. 148: McMichael, A. J., and B. A. Askones Influenza virus-specific cytotoxic T cells in man; induction and properties of the cytotoxic cell. Eur. J. Immunol. 8: Palmer, D. F., M. T. Coleman, W. R. Dowdle, and G. C. Schild Advanced laboratory techniques for influenza diagnosis, p In Procedure guide, immunology series no. 6. Centers for Disease Control, Atlanta, Ga. 1. Quinnan, G. V., E. A. Ennis, C. U. Tuazon, M. A. Welis, G. M. Butchko, R. Armstrong, C. McLaren, J. F. Manischewitz, and S. Kiley Cytotoxic lymphocytes and antibody-dependent complement-mediated cytotoxicity induced by administration of influenza vaccine. Infect. Immun. 3: Ruben, F., and H. Bachmayer Lymphocyte responses to hemagglutinin and neuraminidase subunits of influenza virus. J. Clin. Microbiol. 7: Ruben, F. L., G. G. Jackson, and S. P. Gotoff Humoral and cellular response in humans after immunization with influenza vaccine. Infect. Immun. 7: Rytel, M. W., R. A. Niebojewski, M. A. Rosenkranz, and G. Sedmek Humoral and cell mediated immune response to bivalent influenza A/NJ/76 and A/Vict/75 vaccine in renal allograft recipients. Dev. Biol. Stand. 39: Tauraso, N. M., R. Glechman, F. A. Pedreira, J. Sabbaj, R. Tahwak R, and M. A. Madoff Effect of dosage and route of inoculation upon antigenicity of inactivated influenza virus vaccine (Hong Kong strain) in man. Bull. W.H.O. 41: Downloaded from on September 4, 218 by guest