Characterization of antibody responses to annual influenza vaccination over four years in a healthy elderly population

Vaccine 19 (2001) 4610– 4617 www.elsevier.com/locate/vaccine

Characterization of antibody responses to annual influenza vaccination over four years in a healthy elderly population Elizabeth M. Gardner a, Erica D. Bernstein a, Sandra Dran b, Gary Munk b, Peter Gross b, Elias Abrutyn c, Donna M. Murasko a a

Department of Microbiology and Immunology, MCP Hahnemann Uni6ersity School of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA b Department of Internal Medicine, Hackensack Uni6ersity Medical Center, Hackensack, NJ 07601, USA c Department of Medicine, MCP Hahnemann Uni6ersity Schools of Medicine and Public Health, 2900 Queen Lane, Philadelphia, PA 19129, USA Received 9 March 2001; received in revised form 21 March 2001; accepted 15 June 2001

Abstract The effects of yearly influenza immunization on the level of antibody responses were assessed in 92 healthy elderly subjects immunized over four contiguous years (1993–1996) with a trivalent influenza vaccine that included A/Texas annually. Anti-A/ Texas antibodies increased significantly and similarly post-vaccination each year, but returned to comparable baseline levels annually. Percentages of subjects with anti-A/Texas titers ]40 post-vaccination were comparable over four years. Importantly, post-vaccination titers ]40 to A/Texas in 1993–1994 predicted anti-A/Texas titers ] 40 in subsequent years. Thirty percent of individuals produced four-fold rises to any vaccine component the first year it was included in the vaccine, however, this percentage decreased to about 10% after subsequent vaccination with the same component. This study clearly supports the concept that annual immunization with the same influenza vaccine component over multiple years does not significantly decrease antibody titers in a healthy elderly population. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Influenza vaccination; Humoral; Elderly

1. Introduction Influenza is a serious public health problem that disproportionately affects the elderly, as evidenced by increased morbidity and mortality from pneumonia and other pulmonary and cardiac complications [1 – 4]. Although annual influenza vaccination is recommended for all individuals 65 years of age or older [3,4], recent estimates indicate that only about 63% of the elderly in the United States receive influenza immunization in any given year [3,4]. One reason for this inadequacy in vaccine coverage is that a significant risk of influenza disease exists, even for those elderly who have been vaccinated. Statistics indicate that influenza immunization prevents influenza-related illness in 70 – 90% of * Corresponding author. Tel.: + 1-215-991-8357; fax: +1-215-8482271. E-mail address: [email protected] (D.M. Murasko).

healthy individuals B 65 years old when the vaccine and infecting virus are antigenically similar [5–7] whereas only a 50% reduction is seen after influenza immunization of healthy subjects \ 60 years old [8]. Protection rates from illness in institutionalized ‘frail’ elderly have been reported as being as low as 30% [9–11]. Although the exact mechanism involved in reduced vaccine efficacy in the elderly is not known, it is believed that decreased antibody responses play a significant role since reduced mean antibody titers to influenza vaccine in the elderly compared to young subjects have been reported [12 –15]. In addition, while hemagglutination inhibition (HI) antibody titers ]40 are considered protective [5,6,16,17], several studies have shown that at least 25% of the elderly, including those who are healthy and ambulatory, do not develop HI antibody titers ] 40 in response to vaccination [7,12 –14,18 –20]. However, it is still important to recog-

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E.M. Gardner et al. / Vaccine 19 (2001) 4610–4617

nize that influenza vaccination is a highly effective measure to prevent severe influenza illness, secondary bacterial pneumonias, and death in both healthy and frail elderly. The value of repeated annual influenza vaccination of both young and elderly has also been questioned. An early study [21] reported that influenza vaccination of English schoolboys afforded protection only in those immunized for the first time, but not in those vaccinated in previous years. However, a more recent study [22] of adults 16 years and older indicated that influenza vaccination was more efficacious in reducing influenza-related mortality when administered over multiple years than after the first administration. Other studies have shown that repeated influenza vaccinations over a ten-year period in young adults with Cystic Fibrosis [23], or over a three-year period in healthy young adults [24], showed no diminution of antibody titers, although higher pre-existing antibody titers to a given strain did limit increases in antibody titers pre- to post-vaccination in the latter study [24]. This is not surprising since high levels of existing circulating antibodies to a specific antigen have been shown repeatedly to inhibit antibody production upon subsequent immunization with the same antigen, presumably by clearing the antigen before a secondary immune response is produced [25]. Despite current CDC recommendations to vaccinate all elderly ]65 years old [3,4], there is still considerable debate about whether or not annual influenza vaccination is beneficial for the elderly. Some reports have shown no significant change in vaccine efficacy or in post-vaccination titers between subjects, with or without a history of recent influenza immunization [11,26– 30], while others have indicated either detrimental effects [31,32] or significant benefits [33,34] on the level of antibody production or vaccine efficacy with repeated vaccination. The interpretation of these results is further complicated by the evaluation of: (1) small sample groups; (2) different subjects over multiple study years; (3) different vaccine preparations (e.g. whole or split) across years; and (4) different strains of influenza in repeated years. Thus, the current study was specifically designed to circumvent these problems by: (1) using the same large group of 92 healthy elderly subjects; (2) evaluating antibody responses to the same H1N1 strain, A/Texas, over four contiguous years (1993–1996); and (3) assessing antibody titers to A/ Texas collectively at the end of four years, rather than separately after each year. Our data clearly indicate that repeated vaccination with A/Texas over multiple years does not impair antibody responses to influenza vaccination. In addition, we have shown the importance of assaying antibody samples simultaneously if a direct comparison among years is desired.

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2. Materials and methods

2.1. Subjects Healthy elderly subjects living independently in six local continuing care retirement communities (CCRC) were evaluated in this study. Initially, a small cohort of 19 subjects was recruited from the CCRCs in 1992– 1993 for a pilot study to determine optimal parameters for assessments of immune function. The mean age of this cohort was 76.19 9.0 (range 65–91); 68% were women. During the next four years (1993–1996), a larger cohort of 92 subjects (71.7% women), with a mean age of 79.195.0 (range 67–91) in the first year, was evaluated. The antibody responses of the same 92 individuals were evaluated yearly during this four-year study. There were no significant differences in age, gender, ethnicity, education, or economic status among subjects from each of the CCRCs. None of the subjects was taking medications known to alter immune responsiveness, such as corticosteroids or other immunosuppressive agents, and none had a history of conditions associated with immune dysfunction. All subjects signed informed consent forms that were approved by the institutional review committee. Pre-vaccination blood samples were collected up to 4 weeks prior to vaccination. All subjects in the larger cohort were vaccinated annually in October and November of 1993–1996 with the recommended commercially available subvirion trivalent influenza vaccine containing 15 mg hemagglutinin each of the recommended influenza strains in each season (FLUSHIELD, Wyeth Laboratories, Inc.). Individuals in the pilot study were vaccinated similarly in October and November of 1992. Table 1 summarizes the components contained in the vaccine preparations in each of the four study years (1993–1996) and in the initial pilot study (1992–1993). Importantly, the H1N1 strain, A/Texas/ 36/91, was included in the vaccine each year, along with different H3N2 and B strains (Table 1). Post-vaccination blood samples were obtained approximately 6 Table 1 Vaccine’s components in each year of study Year

H1N1

H3N2

B

1992–1993 (Pilot) 1993–1994 (Year 1) 1994–1995 (Year 2) 1995–1996 (Year 3) 1996–1997 (Year 4)

A/Texas/36/91 A/Beijing/353/89

B/Panama/45/90

A/Texas/36/91 A/Beijing/32/92

B/Panama/45/90

A/Texas/36/91 A/Shandong/9/93

B/Panama/45/90

A/Texas/36/91 A/Johannesburg/33/94

B/Harbin/7/94

A/Texas/36/91 A/Nanchang/933/95

B/Harbin/7/94

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weeks after immunization, but prior to the first confirmed case of influenza in the Delaware Valley. The 92 subjects evaluated in this study represent a subgroup included in a larger study whose immune response to influenza vaccine in 1993– 1994 [12] and 1994– 1995 [13,14] was characterized previously.

variables. Chi-square analysis was used to compare humoral response characteristics among groups. Statistical significance was set at P5 0.05 for all tests.

2.2. Anti-influenza antibody assay

3.1. Antibody titers to A/Texas after annual immunization with influenza 6accine

Venous blood was collected in serum separation tubes (Becton Dickinson, Mountain View, CA). Sera were obtained after a 20 min centrifugation, aliquoted, and stored at −70 °C until analysis. Hemagglutination inhibition (HI) antibody assays were performed by standard microtiter techniques, as described previously [35,36]. Controls for non-specific HI were included in each assay. The appropriate influenza test antigens for HI assays were obtained as egg allantoic fluid from the WHO Collaborating Center for Influenza, CDC, Atlanta, GA. For all years, paired pre- and post-immunization sera samples from the same individual were tested for HI antibodies against the three components at the end of each respective year. In addition, paired pre- and post-immunization samples from freshly thawed sera obtained from each individual during years 2 through 4 (a total of six samples) were tested again for anti-A/Texas HI antibodies at the end of the fourth year. However, paired pre- and post-immunization sera from both year 1 and the pilot study (1992– 1993) could not be tested for A/Texas antibodies at the end of year 4 because there was not enough frozen sample to repeat the analysis. HI antibody titers were expressed as geometric mean titers (log2-transformed). Since HI antibody titers ] 40 have been used to predict protection after influenza vaccination [5,6,16,17], this level was utilized as the criterion to define a biologically relevant antibody response to vaccination.

2.3. Statistical analysis All statistics were generated using SPSS 9.0 for Windows. Dependent variables exhibiting considerable skew in the raw form were log10-transformed for use in parametric statistical tests. Geometric means of HI titers were obtained by log-transformed reciprocal HI titers (log2 (reciprocal titer)). The Mann– Whitney UTest was used to compare non-parametric dependent variables between groups. Comparisons of variables repeated at pre- and post-immunization in all four years were analyzed by paired tests (Students t-tests on log2-transformed variables or the appropriate paired non-parametric test). Comparisons of post-vaccination A/Texas GMT across the four years were analyzed by the Friedman paired non-parametric test. The Pearson or Spearman (non-parametric) rank correlation was used to investigate the strength of relationships between

3. Results

The first objective of this study was to determine the effect of annual influenza immunization on both current and subsequent antibody responses to A/Texas over four contiguous years (1993–1996). Initially, antibody responses to A/Texas were evaluated separately at the end of each year of the study, rather than collectively upon completion of the study. As shown in Fig. 1A, antibody responses to A/Texas increased significantly (P5 0.001) from pre- to post-vaccination in all four years of the study. Importantly, although pre- and post-immunization titers were similar in years 1 and 2, the variability increased such that pre- and post-immunization titers were significantly higher in years 3 and 4 compared to years 1 and 2 (P5 0.001). Our initial analysis revealed that this variability could not be explained by differences in age, gender, underlying health conditions, or pre-immunization titers. In addition, all post-vaccination samples were collected approximately 6 weeks after vaccination, the peak response in elderly, indicating that this variability was not due to the time interval between collection of pre- and post-vaccination samples. However, it was still unclear whether these differences across the years reflected levels of response or interassay variability when HI antibody titers from each year were assessed separately. Since frozen aliquots of serum samples from years 2, 3, and 4 were available, they were re-analyzed collectively at the end of year 4 (see Section 2). As is clearly illustrated in Fig. 1B, there was little variability in mean antibody titers when samples from years 2, 3, and 4 were assayed simultaneously at the end of year 4. A paired comparison of samples assayed simultaneously showed that antibody titers to A/Texas increased significantly (P5 0.001) pre- to post-vaccination in years 2, 3, and 4, but there were no significant differences among these three years in anti-A/Texas antibody titers at either pre- or post-immunization. Importantly, mean anti-A/Texas antibody titers of pre-immunization sera were comparable among all three years, indicating a return to a constant baseline before re-immunization. Since year 1 of the four-year study represented the second year that A/Texas was a component of the vaccine, evaluation of initial responses was deemed both important and necessary. Although the pilot study from 1992–1993 represents both a different and a smaller cohort of subjects,

E.M. Gardner et al. / Vaccine 19 (2001) 4610–4617

Fig. 1. Pre- and post-immunization antibody titers to A/Texas. Values are arithmetic equivalents of geometric mean titers (GMT) 9 SEM. Panel (A): Pre- and post-immunization samples were measured in 19 healthy elderly at the end of 1992 –1993 and in 92 healthy elderly at the end of each of the four years. *Antibody titers to A/Texas increased significantly (P 5 0.001) pre- to post-immunization in all years. Pre- and post-immunization titers were comparable between 1993 – 1994 and 1994 – 1995, but were significantly different (P 50.001) among 1993 –1994, 1995 –1996, and 1996 –1997 as well as among 1994 – 1995, 1995 –1996, and 1996 –1997. Panel (B): Pre- and post-immunization samples from years 2, 3, and 4 were tested simultaneously at the end of year 4. *Antibody titers to A/Texas increased significantly (P 50.001) pre- to post-immunization in each year. Preand post-immunization A/Texas antibody titers did not differ among years.

the data clearly indicate a similar significant increase (P5 0.001) in antibody titers pre- to postimmunization.

3.2. Characterization of the antibody response to A/Texas after annual influenza 6accination To further characterize antibody responses to A/ Texas, protective responses (post-vaccination antibody titers ] 40) and response rates (four-fold rises in titers pre- to post-vaccination) after annual vaccination with A/Texas were calculated. Table 2 shows that the percentage of subjects with post-vaccination titers ] 40 to A/Texas following each year’s vaccination was not less

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than 50% in any given year. Although the percentage of individuals who produced post-vaccination titers ]40 was only 50% after the second exposure to A/Texas in 1993–1994, 76–84% of the subjects were able to produce post-vaccination titers ]40 in subsequent years. Again, data from our pilot study in 1992–1993 were included to provide baseline responses prior to the initiation of our four-year study; 84% of the subjects demonstrated post-immunization titers ] 40. Table 2 also shows the percentage of subjects able to produce four-fold rises in antibody titers to A/Texas after annual vaccination. Although 32% of the subjects were able to produce a four-fold rise in the titer to A/Texas in 1992–1993, only 6–12% were able to achieve fourfold rises in all subsequent years. To further characterize whether or not repeated immunization diminished the production of an antibody response to A/Texas in subsequent years, relative rises in antibody titers to A/Texas pre- to post-immunization were calculated. Fig. 2 illustrates that relative rises in antibody responses to A/Texas pre- to post-immunization did not differ over time, despite a slight non-significant downward trend. This slight decrease over time was not due to differences in demographics or health status of subjects. Collectively, these data clearly demonstrate that the production of antibodies to A/ Texas is not inhibited by repeated vaccination over multiple years. To examine the effects of repeated immunization on the antibody response to A/Texas, we developed a model in which we divided elderly subjects into two categories: those who did or did not initially achieve antibody titers ] 40 to A/Texas in 1993–1994. We then calculated the percentage of subjects in each category who had antibody titers ]40 in subsequent years. Table 3 shows that \95% of subjects with post-vaccination A/Texas antibody titers ] 40 in 1993–1994 demonstrated antibody titers ] 40 in all subsequent years. In addition, only 3% of subjects never achieved a titer ]40 in any of the four years of the study (data not shown). Importantly, a similar percentage of subjects, with or without titers ] 40, had difficulty attaining four-fold rises in anti-A/Texas antibody titers after Table 2 Characterization of Anti-A/Texas antibody responses after annual immunization with A/Texas Year

1992–1993 1993–1994 1994–1995 1995–1996 1996–1997

Percentage Titers ]40

Four-fold rise

84 50 84 82 76

32 10 10 12 6

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Fig. 2. Relative rises pre- to post-immunization in antibody titers to A/Texas each year. Values are means 9SEM of 92 healthy elderly subjects. Antibody titers of subjects in 1993 –1994 were measured at the end of that year, whereas antibody titers of subjects in years 2, 3, and 4 were measured concurrently at the end of the study (1996 –1997). There were no significant differences in pre- to post-rises among years, despite a slight downward trend.

vaccination. Further analysis indicated that the demographics and health status of individuals falling into either category were similar, and therefore, did not contribute significantly to whether or not individuals did or did not achieve antibody titers ] 40 or to produce a four-fold rise in antibody titers to A/Texas in subsequent years (data not shown). These data clearly demonstrate that the ability to produce four-fold rises in antibody titers to A/Texas in subsequent years was not significantly affected by antibody titers ] 40 to A/Texas in the first year.

3.3. Characterization of antibody responses to H3N2 and B 6accine components each year The next objective was to characterize antibody responses to the H3N2 and B vaccine components included in the vaccine each year. Table 4 shows that mean antibody titers to the H3N2 and B components increased significantly (P 50.0001) pre- to post-immunization in each year. Interestingly, similar to our observations after repeated immunization with A/Texas, approximately 50– 80% of individuals were able to achieve antibody titers ] 40, irrespective of whether or not they were exposed only once or repeatedly to the same components. In contrast, 30% of the subjects were able to achieve a four-fold rise after the first exposure to new H3N2 (i.e. all four years) or B (i.e. 1995– 1996) components. However, only about 10% were able to

achieve four-fold rises after repeated immunization with the same component.

4. Discussion The present study was undertaken to address the controversy that exists concerning the effect of annual influenza vaccination on the antibody response to influenza, particularly when the same influenza strain is included in multiple years. Interpretation of the inconsistency among previous studies that have addressed Table 3 Association of antibody titers ]40 to A/Texas in 1993–1994 with antibody titers to A/Texas in subsequent years Year

1994–1995 1995–1996 1996–1997

Characteristics in 1993–1994 ]40 (N =46; 50.0%)

B40 (N =46; 50%)

Titer ]40

Four-fold rise

Titer ]40

Four-fold rise

97.8 a 95.7 a 97.8 a

6.5 13.0 4.3

69.6 67.4 54.3

13.0 10.9 8.7

a Subjects with antibody titers ]40 to A/Texas in 1993–1994 had significantly different antibody response profiles in subsequent years compared to those with antibody titers B40 to A/Texas in the same year (Chi-square range: P50.00006 to P50.00001).

E.M. Gardner et al. / Vaccine 19 (2001) 4610–4617

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Table 4 Characterization of antibody responses to H3N2 and B vaccine components each year Year

Strain

Antibody titers a

Percentage

Pre

Post b

Titers ]40

Four-fold rise

1993–1994 1994–1995 1995–1996 1996–1997

A/Beijing/32/92 A/Shangdong/09/93 A/Johannesburg/33/94 A/Nanchang/953/95

16.0 9 1.1 13.79 1.1 12.39 1.1 32.491.1

39.9 91.2 27.3 91.1 27.5 91.1 68.1 91.1

57 43 49 76

39 28 29 32

1993–1994 1994–1995 1995–1996 1996–1997

B/Panama/45/90 B/Panama/45/90 B/Harbin/07/94 B/Harbin/07/94

19.8 9 1.1 23.39 1.1 28.2 91.1 30.791.1

28.1 91.1 36.5 91.1 59.3 91.1 47.5 91.1

48 58 82 68

10 12 29 12

a b

Values are geometric means 9 SEM. Significantly increased compared to pre-immunization titers, P= 0.0001 (paired Wilcoxon test).

this question is complicated by: (1) small sample sizes, which are highly vulnerable to sample bias in studies of immune function in aging individuals; (2) inclusion of different subjects across multiple years; (3) utilization of whole and split virus vaccines in subsequent study years [33,34,37], which makes stringent comparison of results across years impossible; and (4) change in strain composition of the vaccine each year, which is the most difficult confounding variable. Since multiple studies have shown strain differences in the age-related decline in antibody production [37– 39], a relevant comparison across years can only be made when the same influenza strain is present each year. Our retrospective study of a large population of healthy elderly immunized yearly between 1993–1996 with trivalent influenza vaccine that contained A/Texas in all four years enabled us to critically evaluate the primary question of whether or not repeated vaccination with the same antigen inhibited the development of antibody responses in the elderly. The current study indicates that annual vaccination with A/Texas did not impair the ability of the elderly to produce either a statistically significant rise in anti-A/Texas antibody titers or titers ] 40 after vaccination in subsequent years. In fact, our data showed that subjects who produced anti-A/Texas HI titers ]40 in the first year of the study were likely to continue to attain this level of antibody upon subsequent vaccination with A/Texas each year. Thus, our data clearly support the concept that annual influenza vaccination certainly is not detrimental and does not potentiate age-related decreases in antibody production that are observed when the young and elderly are compared. An additional critical finding during our analysis was identifying the importance of performing simultaneous analyses of antibody responses of the same individuals immunized with influenza vaccine over several consecutive years. Our data indicate that the variability of antibody response data is minimal if samples are as-

sessed concurrently at the end of the study, rather than if samples are analyzed separately at the end of each year of the study. The inconsistency of data examining the effects of repeated immunization on the immune response to influenza vaccination, therefore, may reflect interassay variability in the tests used to assess antibody titers, rather than true differences in responses among groups. Thus, our data indicates the need for concurrent analysis of antibody titers, particularly in a vulnerable population such as the elderly, to ensure that appropriate conclusions are made regarding the level and magnitude of serologic responses before and after influenza vaccination. The low percentage of the elderly population able to attain four-fold rises to A/Texas in 1993–1994 through 1996–1997 is not surprising since existing circulating antibodies to a particular antigen have been shown to limit the level of antibody production upon subsequent immunization [25]. This phenomenon was apparent in our study of responses to both A/Texas and B/Harbin. The low percentage of the elderly unable to attain four-fold rises to A/Texas could be attributed to the fact that most of this population was exposed to a similar H1N1 strain when they were young, establishing significant memory to A/Texas prior to vaccination [39]. Importantly, however, repeated vaccination with the same antigen over four years did not have a significant effect on the percentage of subjects in any given year achieving four-fold rises to A/Texas. It is interesting to note that the percentage of individuals achieving a four-fold rise to B/Harbin was higher in the first year it was introduced (1995–1996) than after the second immunization (1996–1997). Although we did not measure responses to B/Harbin over multiple years, we would predict that the response observed in 1996– 1997 would have been maintained and not decreased after a third immunization, as was seen after repeated immunization with A/Texas. Similarly, although the pilot 1992–1993 study represents a different cohort of sub-

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jects, the percentage of subjects demonstrating a fourfold rise to A/Texas in this first year that it was in the vaccine is comparable to the percentage producing a four-fold rise to B/Harbin after the first year it was a vaccine component. Importantly, both the population evaluated in this report and the larger group of elderly subjects from which this subset was drawn who were evaluated in the same years [12– 14] produced comparable four-fold rises to new antigens, i.e. all the H3N2 components. These data suggest that the inability of the elderly to produce four-fold rises after repeated immunization with the same vaccine component may be influenced by both pre-existing antibody titers and a decreased ability to produce a significant humoral response to influenza vaccination. The majority of data showing age-related declines in B-cell memory implicates the phenomenon of ‘original antigenic sin’, in which influenza-primed memory Bcells cross-react with antigens from newly encountered, closely related influenza strains and thus skew the development of the immune response to shared epitopes between the new strain and the priming strain [38]. Theoretically, this increasingly dilutes the availability of highly specific antibodies to the newly encountered strain. Decreases in specificity and avidity of neutralizing antibody titers in the elderly to the current influenza vaccine strains have been hypothesized to be due, at least in part, to the concept of original antigenic sin [37,39]. While our data clearly show that repeated vaccination with the trivalent influenza vaccine does not inhibit the production of antibodies specific to A/Texas over four years of challenge, it does not address the effect of ‘original antigenic sin’ since we did not evaluate the impact of repeated immunization on subsequent vaccinations with a related influenza A H1N1 strains. However, Powers and Belshe [39] demonstrated in a study of young, middle-aged, and elderly humans that the decreased humoral response in the elderly to a new component of the influenza vaccine could not be explained by ‘original antigenic sin’. Together, these data and ours argue that the use of annual influenza vaccination in the elderly has no detrimental effects, and thus, should be continued and encouraged. A titer ] 40 is generally considered predictive of protection from influenza infection [5,6,16,17]. Thus, although an antibody titer ] 40 may not reflect a protective response in all elderly, this level may still be useful in identifying a subset of elderly who may be particularly vulnerable to influenza infection. Although the subgroup of elderly who never achieve a titer ] 40 after annual influenza immunization may be small (3% in the current study), the larger group of elderly who sporadically do not achieve this titer in any given year (20–50%) may also provide critical data that would be integral in identifying and developing more aggressive immunization protocols. New DNA vaccines that in-

corporate cytokines into the vaccines may be appropriate for these individuals. However, it should be recognized that an HI titer ] 40 may not provide the same level of protection from influenza infection in the elderly. Gravenstein et al. [40] showed that of 72 vaccinated elderly later confirmed to have influenza infection, 60% had titers ] 40 and 31% had titers ]640 four weeks after vaccination. Thus, it may be necessary to increase the dose of vaccine to induce a level of response that is more indicative of protection in the elderly. In addition, the inability of antibody titers to effectively predict protection in the elderly may suggest that cell-mediated immunity either increases in importance with increasing age and/or is more dissociated from humoral immunity in the elderly. Clearly, the potential role of cell-mediated immunity in protection from influenza infection in the elderly must be evaluated in future studies. In summary, previous vaccination does not have any significant deleterious effects on antibody response to vaccinations with the same influenza vaccine over three subsequent years. Further, our data demonstrate that the humoral responses to A/Texas after vaccination in any given year return to comparable baseline levels prior to immunization the next year. Finally, subjects who produce anti-A/Texas titers ] 40 in any given year are likely to continue to respond at that level to A/ Texas in following vaccination seasons. While there is no question that the currently available influenza vaccine must be improved for use in the elderly, our data clearly support the concept that repeated vaccination with currently available vaccine preparations over multiple contiguous years does not decrease serologic responses to influenza vaccination.

Acknowledgements We are indebted to the residents and staff of the participating CCRCs: Rydal Park, Cathedral Village, Medford Leas, Foulkeways at Gwynedd, Pennswood Village and Stapely Hall of Germantown. We thank Caroline Fidew for her database management assistance and Marion Dorfman for excellent technical assistance. This work was supported by the National Institutes of Health Grant NIH AG03934.

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