PERGAMON Vaccine 17 (1999) 82-94
Immune response to influenza vaccination in a large healthy elderly population
E Bernstein*, D Kaye“, E Abrutyn®, P Gross’, M Dorfman*, D.M Murasko* * ®MCP Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia, USA
> Hackensack Medical Center, Hackensack, USA
Received 30 December 1997; received in revised form 4 March 1998; accepted 4 March 1998
Abstract
Elderly individuals not only demonstrate a greater risk of morbidity and mortality from influenza than the young, but also
have greater difficulty mounting a protective response to influenza vaccine The mechanism of the decreased efficacy of influenza vaccination in the elderly is not well understood The present study was designed to assess the interaction between cell-mediated and humoral immune responses to influenza vaccine in a large population (# = 233) of healthy elderly individuals (mean age = 80.7) living in six continuing care retirement communities (CCRCs) While influenza vaccination resulted in significant increases in the mean anti-influenza antibody titres and mean proliferative responses of peripheral blood mononuclear cells to purified subvirion trivalent influenza vaccine one month after vaccination, only 48.9% and 30.0% of subjects had intact humoral and cell-mediated immune responses, respectively No association was observed between intact cell-mediated and humoral responses; 14.7% of subjects had an intact cell-mediated, but not humoral response, and 32.6% of subjects had an intact humoral, but not cell-mediated response However, IFNy production was significantly correlated with both antibody and cell- mediated responses to influenza vaccination, a finding not previously reported in the elderly These results indicate that there is considerable heterogeneity among immune responses of the elderly to influenza vaccination This heterogeneity needs to be a major consideration in evaluation of new vaccine preparations © 1998 Elsevier Science Ltd All rights reserved
Keywords Elderly; Cell-mediated immunity; Humoral immunity; Influenza vaccination
1 Introduction Many vaccinated elderly demonstrate a decreased
antibody response against influenza immunization [8] Influenza infection is a serious public health problem
in the elderly because it is associated with increased morbidity and mortality from pneumonia and other pulmonary and cardiac complications [1,2] The effi- cacy of influenza vaccine is estimated to be between 70 and 90% in young adults when the vaccine strain clo- sely resembles the epidemic strain antigenically [3] However, several studies [4-6] have shown that the effi- cacy of influenza vaccine is much lower in elderly nur- sing home patients Even in healthy elderly, a randomized, double-blind-placebo-controlled trial of influenza vaccination [7] demonstrated that vaccination resulted in a risk reduction of only 50% among sub- jects aged 60 years or older at low risk for influenza
* Corresponding author Tel: (215) 991-8357; Fax: (215) 848-2271; E-mail: Marasko@AUHS.edu
0264-410X/98/$19.00 © 1998 Elsevier Science Ltd All rights reserved PHI: S0264-410X(98)00117-0
which may account for the decreased efficacy While hemagglutination inhibition (HI) antibody titres >40 are considered protective in young subjects [9, 10], sev- eral studies have shown that at least 25% of the
elderly, including those who are healthy and ambulat- ory, do not develop HI antibody titres >40 in response
Trang 2E Bernstein et qL J Vaccime 17 (1999) 62-94 83
that of 72 vaccinated elderly who were later confirmed to have influenza infection, 60% had titres >40 and 31% had titres >640 four weeks after vaccination [14] This suggests that levels of antibody considered predic- tive of protection in the young are not necessarily pre-
dictive of protection in the elderly
The high proportion of immunized elderly who develop influenza is undoubtedly related, at least in part, to the low rate of protective levels of humoral immunity following immunization The role of cell- mediated immunity in protecting aging humans from influenza infection and its sequelae is not fully under- stood Although T-cells cannot prevent infection of host cells, T-cell responses in both humans and mice are correlated with recovery from symptoms or with decreased viral shedding [15—22; reviewed in 23] Since the most consistent and dramatic effect of age upon the immune response is the decrease in T-cell response [24], the increased susceptibility of the elderly to influenza infection may be related to the lack of development of influenza specific T-cell responses
To further characterize the cell-mediated response of the healthy elderly and to investigate the relationship between cell-mediated and antibody responses to influ-
enza vaccination, we evaluated the humoral and cell-
mediated response to influenza vaccination of 233 independently living, healthy elderly adults Our data
indicate that even in this population of healthy elderly,
immune responsiveness after influenza vaccination is low in both the humoral and the cell-mediated arms of the immune system, with the greatest defect seen in cell-mediated immunity Further, while there was no association between cell-mediated and humoral re- sponses to influenza immunization, IFNy production in response to influenza vaccine was correlated with both antibody and cell-mediated responses to vacci- nation Given the low rates of protection offered by
current vaccines, development and testing of new influ-
enza vaccines will be necessary to adequately immunize this elderly population We present criteria for estab-
lishing intact cell-mediated and antibody responses
which can be used to evaluate newer vaccines
Table |
Demographics of study participants
2 Materials and methods
2.0.1 Subjects
Elderly subjects ranging from 67 to 95 years of age, from six local continuing care retirement communities (CCRC), were included in this study The age and gen- der distributions of the subjects are shown in Table 1 There were no significant differences in age, gender, ethnicity, education, or economic status among the subjects from each of the CCRCs None of the sub- jects were taking medications known to alter immune
responsiveness, ¢.g corticosteroids or other immuno-
suppressive agents, and none had a history of con- ditions associated with immune dysfunction All
subjects signed informed consent forms that were
approved by the institutional review committee Collection of prevaccination blood samples was com- pleted up to four weeks prior to vaccination All the subjects were vaccinated in October and November of 1993 with a commercially available subvirion trivalent 1993-1994 influenza vaccine (FLUSHIELD, Wyeth Lab Inc.), containing 15 ug of hemagglutinin of each of the following strains: A/Texas/36/91 (HINI), A/
Beijing/32/92 (H3N2) and B/Panama/45/90
Postvaccination blood samples were obtained 4-6 weeks after vaccination All postvaccination samples were obtained prior to the first confirmed influenza case in the Delaware Valley Greater than 97% of the subjects had been vaccinated in the previous influenza
season
2.0.2 T-cell subset analysis by flow cytometry
T-cell phenotype of whole blood samples obtained preimmunization was determined using a panel of monoclonal antibodies (MAb) including CD4, CD8
and CD45RA (Biosource, Camarillo, CA) Antibodies
used to identify CD4 and CD8 were labeled with phy- coerythrin so that double labeling with the fluorescein- labeled anti-CD45RA antibody was possible The samples were incubated with MAbs on ice for 20 min followed by addition of FACS lysing solution (Becton
Dickinson, San Jose, CA) to lyse red blood cells The
samples were washed twice with phosphate buffered
Number of subjects Mean age (years + S.E.M.) range
Males Females
Facility Total Males Females
Trang 384 E Bernstein ct aL | Vaccine 17 (1999) 82-94
saline (PBS) (Gibco, Gaithersburg, MD) and sus- pended in 1% paraformaldehyde (Polysciences, Inc., Warrington, PA) Cells were quantified using a FACScan@ flow cytometer The data were analysed by Consort 30 analysis software (Becton Dickinson) 2.0.3 Preparation of peripheral blood mononuclear cells
(PBMCs)
Venous blood was collected in EDTA coated vacu-
tainer tubes The PBMCs were separated on Ficoll Hypaque gradients (Sigma, St Louis, MO) and washed twice in RPMI 1640 (BioWhittiker, Walkersville, MD) Cell concentration was adjusted to 2x 10°mI~! in complete medium (RPMI 1640 supplemented with 10% bovine serum replacement (Controlled Process Serum Replacement, type 1 (CPSR1); Sigma) Due to the variability in lymphoproliferation assays, the same lot of each of the reagents (such as sera, influenza vac- cine, mitogens, etc.) was utilized in all experimental cultures so that variation in reagent composition would not contribute to the overall assay variability 2.0.4 Lymphoproliferation assays
Triplicate cultures of freshly isolated 2 x 10° PBMCs per well were stimulated for three days with 8 ng ml -1 of phytohemagglutinin (PHA); 25 pg ml~! of concana- valin A (ConA); or lpg ml! of pokeweed mitogen (PWM) and for five days with 0.45 pg HA ml”! of the 1993/4 trivalent influenza vaccine (FLU; previously
dialyzed against PBS at 5°C for 24h) in round bottom
96-well microtitre plates (Flow Laboratories, McLean,
VA) In preliminary studies the above concentrations of mitogens/antigen were found to provide optimal stimulation in the majority of subjects (data not shown) All cultures were maintained in RPMI 1640 supplemented with 10% CPSR1 serum and incubated at 37°C in 5% CO) During the last 4h of stimulation,
each culture was pulsed with lOHCI of
[methyl-?H]thymidine (ICN, Irvine, CA) The amount of radioactivity incorporated into the cultures was determined by harvesting the contents of each well onto glass fiber filters using a Ph.D cell harvester (Cambridge Technologies, Watertown, MA) and read- ing the filters using a Packard Liquid Scintillation Counter (Meriden, CT) Proliferation in response to mitogens and FLU was expressed as a net proliferation index (NI) [NI = average cpm with stimulus — average cpm with media alone] Proliferation in response to FLU was also expressed as a stimulation index (SI) [SI = average cpm with FLU + average cpm with media alone]
2.0.5 IFNy analysis
Since we previously demonstrated that peak IFNy production occurs after a five day culture with FLU (data not shown), 100 ul of supernatant were taken
from each of the three FLU-stimulated culture wells prior to the addition of [methyl-*H]thymidine and pooled Supernatants were also pooled from each of the three culture wells with media alone The samples were stored at —70°C until analysis The titre of IFNy
was determined by a modification of the microplate
antiviral assay using human foreskin fibroblast cells and encephalomyocarditis virus [25] One unit of bio- logical IFNy activity is defined as the reciprocal of the
dilution of supernatant that inhibits viral cytopathol-
ogy by 50% The IFN produced in this system was demonstrated to be IFNy by the complete abrogation of inhibitory function by treatment with anti-IFNy monoclonal antibody All assays included NIH inter- national reference standards for IFNy Experimental values were corrected according to the NIH reference standard and expressed as units ml~' Since some of the samples had significant background production of IFNy, values reported for IFNy are units of activity found in supernatants from FLU-stimulated cultures minus units of activity in supernatants from non- stimulated cultures Population means of IFNy pro- duction are presented as geometric mean titres (GMT) The changes in IFNy production from pre- to postvac- cination are presented as net IFNy production which is equal to postvaccination IFNy production minus prevaccination IFNy production
2.0.6 Anti-influenza antibody assay
Hemagglutination inhibition (HI) antibody assays
were performed by standard microtitre
techniques [26,27] Controls for nonspecific HI were included in each assay Paired pre- and postimmuniza- tion sera samples from the same individual were tested simultaneously for each of the test antigens The appropriate influenza A and B test antigens for hemag- glutination inhibition were obtained as egy allantoic fluid from the WHO Collaborating Center for Influenza, CDC, Atlanta, GA
2.0.7 Statistical analysis
All statistics were generated using SPSS 4.0 for the VAX Dependent variables exhibiting considerable skew in the raw form were log; ) transformed for use in parametric statistical tests The Mann-Whitney U- Test was used to compare nonparametric dependent variables between groups Comparisons of variables repeated at pre- and postimmunization were analysed by paired tests (Students t-tests on log), transformed variables or the appropriate paired nonparametric test) Geometric means of HI titres and IFNy titres were obtained by log transformed reciprocal HI titres and IFNy [log»(reciprocal titre)] The Spearman rank correlation was used to investigate the strength of re-
lationships between variables Statistical significance
Trang 4E Bernsteim et ai ¡ Vaccie 17 (1999) 82-04 85 3 Results
3.1 Cell-mediated immune response to influenza HA The proliferative response to influenza vaccine before and after vaccination is shown both as net counts (NI) and stimulation indices (SI) in Fig 1 PBMC proliferation in response to stimulation with the same trivalent vaccine (FLU) administered in vivo increased significantly following vaccination (NI p < 0.0005; SI p < 0.001) This increase does not reflect an overall non-specific increase in immune responsive-
hess since postvaccination mitogen-induced prolifer-
ation was not increased relative to prevaccination mitogen responses (data not shown) The preimmuni-
12000
zation profile of T-cell subsets was not indicative of the proliferative response to FLU either before or after vaccination Neither the percentage of CD4* or CD8~* cells, nor of CD4* CD45RA* (naive) cells was correlated with proliferative responses to FLU as assessed by net count or stimulation indices
IFNy production by these healthy elderly subjects was evaluated in response to in vitro stimulation with vaccine Mean IFNy production in response to FLU increased after vaccination (Fig 2) This increase was not significant probably due to the large percentage
(56%) of the population that did not produce any
detectable IFNy (<4uml™') either before or after vaccination However, there was a positive correlation between FLU-induced net proliferation and IFNy pro-
10000 1 8000 †—- 8000 3 4000 4 2000 7 PBNC proliferation to FLU (NI) PRE mNET FLU| POST p<0.001 PBMC proliferation to FLU (SI) | PRE po —
Trang 586 E Bernstein et al.{ Vaccine 17 (1999) 82-94 E °° = Gj = = 4 5 — = ] iG 3 œ + 24 = = G 13 — za Lod a PRE = POST
Fig 2 FLU-induced [FNy production pre- and postinfluenza vaccination Geometric mcan titre + S.E.M IFNy production after five day culture with FLU in all subjects pre- and postinfluenza vaccination
duction postvaccination (r = 0.4971; p < 0.0005) The net increase in IFNy production from pre- to postvac- cination was also significantly associated with postvac- cination FLU-induced net proliferation (r = 0.3971;
p < 0.0005)
We wanted to characterize the immune responses of individuals to influenza vaccination as either intact or non-intact Criteria for an intact immune response to influenza vaccine needs to reflect both achievement of a biologically relevant level of response after vacci- nation and evidence of a specific response to the cur- rent vaccine Unlike the antibody response to influenza vaccination in which an HI titre >40 is generally con- sidered predictive of protection [9,10], there is no established level of cell-mediated immune response that predicts protection after vaccination Further, while a fourfold rise in antibody titre reflects exposure to a specific antigen between two time assessments, there is no level of increase in lymphocyte proliferation from pre- to postvaccination that has been similarly defined Therefore, it was necessary for us to define a level of cell-mediated response that was considered rel- evant We evaluated three approaches to defining intact cell-mediated immune responses to influenza vaccine
An approach used often in the literature is to desig-
nate arbitrarily a value as the minimum for a positive response, e.g a twofold rise in proliferative responses from pre- to postvaccination Using a twofold rise in pre to post SI to FLU as positive, only 31.7% of the elderly population had an intact cell-mediated response (CMI * ) to influenza vaccination However, in review- ing the proliferative responses of subjects considered positive by the above definition, 25% of subjects achieving twofold increases in FLU-induced SI after vaccination demonstrated postvaccination net prolif- erative responses to FLU lower than the mean pro- liferation of unstimulated cells (background) of the population Since the biological relevance of this level
of response was questionable, more stringent criteria were assessed
A second set of criteria required subjects to demon- strate a twofold increase in FLU-induced SI after vac-
cination and a net proliferative response to FLU greater than or equal to the median background pro- liferation (2000 cpm) With this definition, we found 29.9% of the elderly had intact cell-mediated responses to influenza vaccination Detailed evaluation identified 12 subjects considered CMI”* by this definition who had FLU-induced net proliferative responses postvac- cination within one standard deviation of the mean background proliferation In addition, 34% of the sub- jects who demonstrated post:pre FLU-induced SI] ratios >2 also demonstrated similar increases in their mitogen response from pre to postimmunization Their increase in FLU-induced cell-mediated immune re- sponses, therefore, could have been reflective of a non- specific increase in proliferative response and not rep- resent a specific increase in response to the vacci- nation In order to address these concerns even more conservative criteria were assessed
Trang 6E Bernstein et al./ Vaccine 17 (1999} 82-94 87
after immunization compared with those prevaccina- tion: average mitogen ratio = [(net cpm PHA post/net cpm PHA pre) + (net cpm ConA post/net cpm ConA pre) + (net cpm PWM post/net cpm PWM pre)] = 3
The ratio of the net proliferative response to influenza
vaccine post:preimmunization was considered positive if it was greater than the individuals average mitogen post:prevaccination ratio plus one standard deviation of the mean average mitogen ratios of the population By these criteria an individual would be considered to have an intact cell-mediated response (CMI~ ) to influenza vaccination if their net proliferation to FLU postimmunization was greater than mean background proliferation plus one standard deviation (> 5893) and the rise in the net proliferative response to FLU from pre- to postvaccination was greater than the indivi- dual’s non-specific variation between samplings plus one standard deviation of the mean non-specific vari- ation in proliferative response of the population from pre- to postvaccination Using these criteria, 30% of subjects had intact cell-mediated responses
Twenty-nine subjects classified as CMI™~ by the sec- ond definition were considered CMI using the third definition These subjects were excluded by the second definition because they were unable to demonstrate a twofold increase in FLU-induced SI proliferation after vaccination All of these subjects demonstrated high levels of net proliferative responses to influenza vaccine postvaccination, but in each case the individuals’ post:- prevaccination non-specific mitogen response ratios
were low The arbitrary designation of a positive re- sponse to vaccination as a FLU-induced SI prolifer- ation post:pre ratio >2, masked the specific response of some subjects The third definition of an intact cell- mediated response (CMI ‘) was used in all further comparisons because it controls more directly for indi- vidual variation in non-specific proliferative immune
responses
An intact cell-mediated response was observed in
30% of the 233 elderly subjects The range of pre- and
postvaccination proliferative responses to FLU among
CMI~* or CMI™ subjects is shown in Fig 3 CMI”
subjects had significantly greater pre- to postvaccina- tion increases in IFNy production (p < 0.0007) and achieved significantly higher levels of IFNy after vacci- nation (p< 0.01) then their CMI~ counterparts (Fig 4) As well, those subjects with intact cell- mediated responses were significantly more likely to produce detectable levels of IFNy postvaccination (70.0%) than did CMI™~ subjects (50.6%) (`: p<0.0I) There was no diference at the time of im- munization in the percentage of CD4*, CD8”, or
CD4 ~*~ CD45RA ~ (naive) T-cell subsets between sub-
jects who were CMI~ or CMI”
3.2 Humoral immune response to influenza vaccination As seen in Fig 5, HI antibody titres to A/Texas/36/ 91 (HINI), A/Beijing/32/92 (H3N2) and B/Panama/ 45/90 all increased significantly following vaccination
80000 ~-_ © g 700004 9 a > $ 60000} ° E 9 500004 + L 3 a © @ Pre-vaccination | 400004 ° " l Post-vaccination ° a & a 30000 4 Ỹ 5 i a ~ 200004 a Dp % 100004 H fe 0 †
NOT INTACT INTACT
CMI
Trang 788 E Bernstein et al / Vaccine 17 (1999) 82-94 p<0.01 p<0.0007 Geometric Mean Titer + SEM 14 FLU-induced IFNy Production (U/ml) 0ï CMI HB NOT INTACT GB INTACT
Pre-vaccination Post-vaccination Net IFNy (post-pre)
Fig 4 FLU-induced IFNy production among CMI’ and CMI™~ subjects IFNy production before and after vaccination, as well as the net change in IFNy production from pre- to postvaccination, for CM
CMI* subjects have significantly greater geometric mean titres of
I' and CMI™~ subjects are presented as geometric mean titres + S.E.M IFNy production postvaccination (GMT + S.E.M = 4.8 + 0.4) and signifi- cantly greater net changes in IFNy production from pre- to postvaccination (GMT + S.E.M = 3.2 + 0.5) than do CMI™ subjects (3.4 + 0.3 and 1.2 + 0.2, respectively)
(p < 0.0005) HI antibody titres >40 to A/Texas/36/91 (HINI), A/Beijing/32/92 (H3N2), and B/Panama/45/ 90 were seen prior to immunization in 43.8, 19.3 and 36.9% of subjects, and postvaccination in 60.5, 59.7 and 52.8% of subjects, respectively In accord with prior studies [12], our data show that a higher baseline humoral response to the components in previous vac-
cines, i.e A/Texas and B/Panama, correlated with a
smaller increase in titres following vaccination (A/ Texas/36/91: r = —0.4616; p < 0.0005 and B/Panama/ 45/90: r = —0.1806; p < 0.003) Following vaccination, a fourfold rise in titre to A/Texas and B/Panama occurred in only 14.6 and 12.4% of subjects, respect- ively In contrast, 43.3% of subjects had a fourfold 50 p<0.0005 45 p<0.0005 40 35 7 30 1 25 1 201 Geometric Mean HI Titer + SEM p<0.0005 a PRE = POST
AITEXAS A/BEWING B/PANAMA
Flu Strain
Fig 5 Anti-FLU antibodies pre- and postinfluenza vaccination Influenza antibodies as determined by hemagglutination inhibition shown as arithmetic representation of geometric mean titre + S.E.M “HI titres
t-test at p < 0.0005
Trang 8E Bernstein et al { Vaccine 17 (1999) 82 94
Table 2
Association of mitogen-induced proliferation and immune response to influ- enza vaccination
Influenza response" Mitogen-induced response (cpm x 107 3)°
Level* PHA ConA PWM
CMI~ 59.2428 28.4+ 1.4 22.4+ 1.0 CMI” 4701+2.9 193+ 143 20.1 + 1.2 AB" 33.8+3.2 24.8 + 1.6 20.9 + 1.2 AB“ 574+2.9 26.5+ 1.4 22.5+ 1.0
“AB and CMI are as defined in the results section A * +’ indicates an intact response, while a ‘—* indicates a non-intact response to influenza vac- cination
>Mitogen-induced PBMC proliferation at pre-vaccination sampling (mean
epm x 1077*+S8.E.M.)
“Mitogen-induced proliferation was not predictive of intact or non-intact CMI or AB responses by logistic regression (CMI, p < 0.4440; AB,
89
p < 0.4143)
rise in titre to the new component of the trivalent influenza vaccine, A/Beijing
Since an HI titre >40 has been considered predictive of protection after influenza vaccination [9,10], this level was utilized as the criteria for a biologically rel- evant antibody level after vaccination However, since many elderly have HI titres >40 from previous ex-
posures or immunizations, a rise in titre was essential
for assessing immune response to the current vaccine Based on the accepted principle that a fourfold rise in titre between two time points represents recent ex- posure to the antigen being evaluated, a fourfold rise was used as our criteria for response to the current vaccine Therefore, the criteria for an intact humoral response was established as a fourfold rise in HI titre after vaccination to any one of the three vaccine strains included in the 1993-1994 trivalent vaccine and a postvaccination HI titre >40 to any one of the three strains An intact antibody response was observed in 48.9% of subjects There was no difference in preim- munization percentages of CD4”, CD§”, or CD4*CD45RA* T-cell subsets between subjects
Table 3
who did or did not demonstrate intact antibody responses
3.3 Relationship between cell-mediated and humoral responses
Since mitogen-induced proliferation is an indicator of non-specific immune responsiveness, we investigated whether non-specific immune responses would corre- late with specific T-cell responses or humoral responses to influenza vaccination in the elderly As seen in Table 2, mitogen responsiveness did not predict intact cell-mediated or intact antibody responses (logistic re- gression, cell-mediated: p < 0.4440; antibody: p < 0.4143) Since proliferative responses in vitro were induced by the entire trivalent vaccine and the anti- body titres were assessed to each component of the vaccine separately, direct correlations of proliferative responses and antibody titres were not relevant However, comparisons between CMI* and CMI~ subjects showed that subjects who were CMI did not have different percentages of HI antibody titres
Cell-mediated and humoral response to influenza vaccine
Cell-mediated response”
Antibody response“ CMI” CMI”
AB | 34 (15.2%) 73 (32.6%) 107 (48.9%) AB“ 33 (14.7%) 84 (37.5%) 117 (51.1%) 67 (30%) 157 (70%) 224 (100%)
“Intact antibody response (AB ” ) ¡s delned as a fourfold rise in tire postvaccination and a postvaccination titre >40 to any of the three strains
'Intact CMI response (CMI * ) is defined as a rise in the proliferative re- sponse to FLU from pre to postvaccination significantly greater than the individual’s non-specific variation in proliferative response between sam- plings and a post net proliferation greater than background plus one stan- dard deviation (see Materials and Methods)
Trang 990 E Bernstein et al | Vaccine 17 (1999) 82-94
>40 or percentages of fourfold rises in antibody titre for any of the components CMI" subjects demon- strated significantly higher levels of postvaccination HI titres to A/Texas than did those who were CMI”
(CMI*: 44.341.1; CMI7~: 33.8+ 1.1; p< 0.05) but
not to A/Beijing nor B/Panama Further, individuals with intact cell-mediated responses did not have a higher rate of intact humoral responses (Table 3)
As mentioned earlier, intact cell-mediated responses were strongly correlated with IFNy production after vaccination An association between IFNy production and humoral response was also observed As seen In Fig 6, subjects with an intact antibody response (AB~) had higher mean postvaccination IFNy pro- duction that approached significance (p < 0.06) Further, subjects who were AB~ were more likely to
produce detectable levels of IFNy postvaccination
(64.5%) than AB™ subjects (48.6%); (x7; p < 0.02) In
summary, while there was no concordance between
responsiveness to vaccination as defined by intact cell- mediated immune responses and intact humoral re- sponses, IFNy production was positively associated with both an intact humoral and an intact cell- mediated response
4 Discussion
The elderly are particularly vulnerable to influenza Unfortunately, it is precisely in this population that current influenza vaccines are the least efficacious Although influenza immunization in the elderly is not
completely successful in preventing illness, vaccination appears to reduce the severity of illness Previous reports have indicated a shorter duration of illness, a reduction in pneumonia and hospitalizations, and a decrease in mortality rates in immunized groups of elderly [28-35] A recent study [35] of more than 75000 ambulatory elderly conducted over a three-year period found that influenza vaccination 1s associated with a reduction of 39-54% in mortality from all causes Influenza vaccination also significantly reduced the rates of hospitalization for pneumonia and influ- enza, acute and chronic respiratory conditions, and congestive heart failure, resulting in a cumulative sav- ings of about $5 million [35] In a meta-analysis of observational cohort studies, Gross et al [6] reported an overall pooled estimate of vaccine efficacy of 56% for prevention of respiratory illness, 53% for preven- tion of pneumonia, 50% for prevention of hospitaliz-
ation, and 68% for prevention of death from all
causes
Many studies have sought to characterize and explain the decreased protection offered to the elderly by influenza vaccination While the decreased humoral response to influenza vaccination in the elderly is well documented [reviewed ¡ín [6] and [I3], the cell- mediated response has been less well characterized Investigators have reported age-related changes in CTL activity, proliferative responses and cytokine pro- duction in response to in vitro stimulation with influ- enza vaccine From murine studies, the most relevant measurement of T-cell effector responses against influ- enza after vaccination is CTL activity [22] Seven
p<0.06 FLU-induced IFNy Production (U/ml) Geometric Mean Titer + SEM Pre-vaccination Post-vaccination AB Ml NOTINTACT Hl INTACT
Net IFNy (post-pre)
Fig 6 FLU-induced IFNy production among AB' and AB” subjects IFNy production before and after vaccination, as well as the net change in IFNy production from pre- to postvaccination, for AB ~ and AB™ subjects are represented as geometric mean titres + S.E.M AB * subjects had higher geometric mean titres of IFNy production after
Trang 10E Bernstein et al / Vaccine 17 (1999) 82-94 91
studies assessed CTL activity in the elderly after influ- enza vaccination [36-42]; five demonstrated significant
increases in CTL activity following
vaccination [36, 38, 39, 41,42], one saw no change [40],
and one only reported on postvaccination responses not on changes pre- to postvaccination [37] The study without significant vaccination-induced increases in CTL activity used chronically ill institutionalized elderly >60 years of age [40], while the studies demon- strating a significant rise assessed healthy subjects Therefore, the differing results in CTL response to vac- cination may be due to the health status of the study subjects Only two studies compared CTL activity of elderly and young adults; both found that activity was significantly decreased in the elderly [36, 37]
Few studies examined age-associated changes in
antibody and cell-mediated parameters in
parallel [14, 36-40, 43-46] Further, even fewer directly correlated influenza specific CTL activity [36,38] or PBMC proliferation [44, 46] with antibody responses in vaccinated elderly; none of these were able to show a significant correlation Since the number of subjects in these studies were small (<25), it is unclear whether the reported lack of association between cell-mediated and humoral responses to influenza vaccination reflected a type II error or a real disassociation Due to the high degree of heterogeneity among immune re- sponses in elderly humans, large sample sizes are prob- ably necessary to appreciate subtle associations between humoral and cell-mediated responses to influ- enza vaccination Since most studies presented only mean immune responses, the contribution of the het- erogeneity in responses of the elderly is not easily ap- preciated Thus, the present study addressed these concerns by evaluating in parallel both cell-mediated and humoral immunity in a large population of healthy, ambulatory elderly before and after vacci- nation with a purified subvirion trivalent influenza vac- cine
Our study demonstrates that healthy elderly respond to influenza vaccination with significant increases in mean vaccine-induced proliferative responses (Fig 1) These findings are in agreement with previously pub-
lished reports demonstrating increases in vaccine anti-
gen-induced proliferation after influenza immunization [14, 43, 44,46] Although the heterogeneity of the age- related decline in lymphoproliferation to mitogens is well established [47-50], to our knowledge few reports have addressed the heterogeneity of in vitro lympho- proliferative response to influenza antigen, and none in a large population of healthy elderly In an effort to categorize the proliferative responses of all 233 individ- uals in this population, we developed criteria to reflect proliferative responses after vaccination that are bio- logically relevant, that indicate a response to the cur- rent vaccine, and that consider the inherent variability
between pre- and postvaccination samplings Using these criteria, 30% of our elderly population demon- strated an intact cell-mediated response to the current influenza vaccination
A positive humoral response to influenza vacci- nation has generally been defined as attainment of HI titres >40 to the components of the vaccine However, we wanted to use criteria comparable to those we developed for cell-mediated immunity (i.e a level of re- sponse that reflects both a biological relevant level and a response to the current vaccine) Using such criteria, the humoral response rate to influenza vaccination in the elderly was low as well, with only 48.9% of sub- jects having an intact antibody response Even though
antibody titres increased significantly following vacci-
nation (Fig 5), only 50-60% of subjects had antibody titres predictive of protection to any one of the three
strains after immunization As expected, the new com-
ponent of the vaccine A/Beijing/32/92 was the most immunogenic; 43.3% of the subjects experienced a
fourfold rise in titre after vaccination It is important
to note, however, that 40% of the elderly did not
mount a protective response (i.e antibody titre >40) to this newly encountered antigen after vaccination Although low humoral response rates have previously been reported in institutionalized, chronically ill elderly [13], our results show that low humoral re- sponse rates also occur in a large population of ambu- latory, healthy elderly These data confirm prior findings that the humoral response to influenza vacci- nation in the elderly is inadequate and certainly less than the published response rates for young adults [13] Phenotypic analysis of peripheral blood T-cell sub- sets measured prior to vaccination revealed that per- centages of CD4~ , CD8~, or naive (CD45RA+) T cells were not predictive of either cell-mediated or humoral responses to influenza vaccination These findings are in agreement with previous reports [43, 44,46,51] which also showed no association between peripheral blood T-cell subsets and immune responses to influenza vaccination While Degelau [43] and McElhaney [52] demonstrated an age-associated decline in CD45RA * RO™ (naive) cells both pre- and
postvaccination, neither found any association between
Trang 1192 E Bernstein et al | Vaccine 17 (1999) 82-94
responding in culture with measures of intact cell- mediated response to influenza vaccination These stu- dies are currently under way in our laboratory
Unfortunately, the individual components of the tri- valent influenza vaccine were not available for the in vitro proliferation experiments of this study The pro-
liferative responses to influenza vaccine reported,
therefore, reflects PBMC proliferation in response to all three strains included in the trivalent subvirion influenza vaccine Although cell-mediated responses have been shown to have heterotypic specificity to influenza strain subtypes (e.g cytotoxic T lymphocytes can crossreact with various influenza A strains) [23], the relationship between PBMC proliferation to the trivalent influenza vaccine and the humoral immune response to specific components of the vaccine is diffi- cult to decipher However, the data clearly indicate that when the overall response to influenza vaccination is assessed, an intact cell-mediated immune response to vaccination is unrelated to an intact humoral response in this study (Table 3)
Despite the lack of association between intact
humoral and intact cell-mediated responses, the pre-
sent study clearly shows a correlation between IFNy production and both intact cell-mediated and intact humoral responses to the trivalent vaccine These results suggest an important role for IFNy as a poss- ible marker for vaccine efficacy in both the humoral and cell-mediated arms of the immune response These results agree with studies in young subjects demon- strating a significantly increased influenza vaccine- induced PBMC production of IFNy following vacci- nation which was associated with significant increases in antibody titres [53,54] Powers and colleagues demonstrated that immunization of young subjects with purified recombinant hemagglutinin (rHA) resulted in a fourfold rise in rHA-induced IFNy pro- duction after vaccination [53] In addition, the net increase in rHA-induced IFNy production was signifi- cantly associated with the net increase in anti-rHA antibody production from pre- to postvaccination [53] Moreover, Tomoda and colleagues [54] demonstrated that high levels of IFNy production by PBMC of young adults immunized intranasally with a trivalent cold-adapted recombinant influenza virus vaccine were associated with protection from natural influenza vac- cination during an influenza epidemic Interestingly, one study of chronically ill elderly subjects who received intranasal vaccination with a cold-recombi- nant, live-attenuated, influenza virus vaccine, reported that high levels of prevaccination IFNy production by virus-induced PBMC were associated with decreased vaccine virus infection of subjects [38] IFNy pro- duction was not associated with CTL activity against the vaccine virus in this study, possibly due to interfer- ence with the viral infection by the prevaccination level
of IFNy [38] While a major role for IFNy in protec-
tion from influenza disease after vaccination cannot be
supported by current data, our results suggest that IFNy production is a marker for intact cell-mediated
and antibody responses to influenza vaccination in
healthy elderly
Clearly, the best way to evaluate the ability of intact cell-mediated and/or intact antibody responses as defined by our criteria to predict protection from influ- enza is to challenge with influenza virus after vacci- nation Since influenza infection has many serious consequences for morbidity and mortality in the elderly, challenge experiments are not possible Thus, we are limited to influenza surveillance studies in which rates of intact cell-mediated and antibody re- sponses are compared among subjects who are natu- rally infected or uninfected with influenza after immunization We are currently evaluating our criteria of intact cell-mediated and antibody responses as pre- dictors of protection in an ongoing surveillance pro- Ject Meanwhile, the criteria reported here for intact immune responses to influenza vaccination will serve as a useful tool with which to evaluate consistently across time immune responses to new influenza vaccine formulations
Our data demonstrate that even in a healthy elderly population suboptimal response rates to influenza vac- cination are achieved in both arms of the immune sys- tem In order to protect the elderly from the devastating effects of influenza it may be important to focus efforts on vaccine development that boosts both the cell-mediated and humoral responses Some initial efforts have shown success in boosting the elderly
immune response after vaccination For example, new
virosome influenza vaccines [55] and diphtheria toxoid conjugated influenza vaccines [14] have achieved higher antibody response rates and CTL activity in the elderly than vaccination with subvirion vaccines, possibly because of their associated adjuvants’ ability to stimu- late cell-mediated immunity This study demonstrates further that evaluation of vaccines targeted at maxi- mizing cell-mediated immunity is necessary to meet the public health needs not only of the institutionalized elderly population, but also of the healthy elderly Acknowledgements
We are indebted to the contribution of the study nurse, Cathie Gusz, R.N who was integral to the recruitment and management of the subject popu- lation We are grateful to the residents and staff of the participating CCRCs: Medford Leas, Rydal Park,
Cathedral Village, Foulkeways at Gwynedd,
Trang 12E Bernstein et al / Vaccine 17 (1999) 82-94 93
data-base management assistance; Christine Kinsinger for help in manuscript preparation; and Dr Elizabeth M Gardner, Dr Pasquale G Iannuzzelli, and Dr Denise C LaTemple for critical comments We grate-
fully acknowledge that this work is supported by
National Institute of Health Grants AG03934
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[53 =