The frequency of interleukin-1ß-producing monocytes is significantly associated with varicella-zoster responses of nursing home residents.

Previous studies have demonstrated that the status of the T cell compartment and inflammation-related factors are associated with the immunogenicity of the varicella-zoster virus (VZV) vaccine in older adults; however, little is known about the roles of other immune cell subsets known to influence the generation and maintenance of immunological memory. Responses to a live-attenuated VZV vaccine were studied in relation to peripheral blood mononuclear cell (PBMC) composition and function in a sample of 30 nursing home residents (aged 80-99 years). Interferon-gamma enzyme-linked immunospot (ELISPOT) was used to measure VZV responses at baseline and 6 weeks following vaccination, and associations were sought with the frequencies of monocytes and T, B and natural killer (NK) cells and the production and secretion of cytokines following their ex-vivo stimulation with different agents. While only the frequency of interleukin (IL)-6+ CD14+ monocytes was inversely associated with post-vaccination VZV response, amounts of IL-1ß, IL-10, IL-17A and tumour necrosis factor (TNF) secreted by PBMCs and the frequency of IL-1ß+ CD14+ monocytes was positively correlated with pre-vaccination VZV response. Furthermore, both bivariate correlation and causal mediation analyses supported the notion that IL-1ß+ CD14+ monocytes were significant mediators of the associations between IL-1ß and TNF secretion by PBMCs and pre-vaccination VZV responses. Our findings implicate a strong cytokine response mediated by inflammatory IL-1ß+ monocytes in coordinating responses of long-lived VZV-reactive memory T cells, but with an opposing effect of IL-6+ CD14+ monocytes. Whether monocyte status promotes or inhibits the induction and/or maintenance of these memory T cells later in life has yet to be determined.

Validation of the Seegene RV15 multiplex PCR for the detection of influenza A subtypes and influenza B lineages during national influenza surveillance in hospitalized adults.

Background. The Serious Outcomes Surveillance Network of the Canadian Immunization Research Network (CIRN SOS) has been performing active influenza surveillance since 2009 (ClinicalTrials.gov identifier: NCT01517191). Influenza A and B viruses are identified and characterized using real-time reverse-transcriptase polymerase chain reaction (RT-PCR), and multiplex testing has been performed on a subset of patients to identify other respiratory virus aetiologies. Since both methods can identify influenza A and B, a direct comparison was performed.Methods. Validated real-time RT-PCRs from the World Health Organization (WHO) to identify influenza A and B viruses, characterize influenza A viruses into the H1N1 or H3N2 subtypes and describe influenza B viruses belonging to the Yamagata or Victoria lineages. In a subset of patients, the Seeplex RV15 One-Step ACE Detection assay (RV15) kit was also used for the detection of other respiratory viruses.Results. In total, 1111 nasopharyngeal swabs were tested by RV15 and real-time RT-PCRs for influenza A and B identification and characterization. For influenza A, RV15 showed 98.0 % sensitivity, 100 % specificity and 99.7 % accuracy. The performance characteristics of RV15 were similar for influenza A subtypes H1N1 and H3N2. For influenza B, RV15 had 99.2 % sensitivity, 100 % specificity and 99.8 % accuracy, with similar assay performance being shown for both the Yamagata and Victoria lineages.Conclusions. Overall, the detection of circulating subtypes of influenza A and lineages of influenza B by RV15 was similar to detection by real-time RT-PCR. Multiplex testing with RV15 allows for a more comprehensive respiratory virus surveillance in hospitalized adults, without significantly compromising the reliability of influenza A or B virus detection.

The Impact of Prior Season Vaccination on Subsequent Influenza Vaccine Effectiveness to Prevent Influenza-related Hospitalizations Over 4 Influenza Seasons in Canada.

BACKGROUND: Recent studies have demonstrated the possibility of negative associations between prior influenza vaccines and subsequent influenza vaccine effectiveness (VE), depending on season and strain. We investigated this association over 4 consecutive influenza seasons (2011-2012 through 2014-2015) in Canada. METHODS: Using a matched test-negative design, laboratory-confirmed influenza cases and matched test-negative controls admitted to hospitals were enrolled. Patients were stratified into 4 groups according to influenza vaccine history (not vaccinated current and prior season [referent], vaccinated prior season only, vaccinated current season only, and vaccinated both current and prior season). Conditional logistic regression was used to estimate VE; prior vaccine impact was assessed each season for overall effect and effect stratified by age (<65 years, ≥65 years) and type/subtype (A/H1N1, A/H3N2, influenza B). RESULTS: Overall, mainly nonsignificant associations were observed. Trends of nonsignificant decreased VE among patients repeatedly vaccinated in both prior and current season relative to the current season only were observed in the A/H3N2-dominant seasons of 2012-2013 and 2014-2015. Conversely, in 2011-2012, during which B viruses circulated, and in 2013-2014, when A/H1N1 circulated, being vaccinated in both seasons tended to result in a high VE in the current season against the dominant circulating subtype. CONCLUSIONS: Prior vaccine impact on subsequent VE among Canadian inpatients was mainly nonsignificant. Even in circumstances where we observed a trend of negative impact, being repeatedly vaccinated was still more effective than not receiving the current season's vaccine. These findings favor continuation of annual influenza vaccination recommendations, particularly in older adults. CLINICAL TRIALS REGISTRATION: NCT01517191.

Influenza vaccines provide diminished protection but are cost-saving in older adults.

Influenza is associated with substantial morbidity and mortality in adults aged over 65 years. Although vaccination remains the most effective method of preventing influenza and its sequellae, current vaccination strategies provide less protection to older adults than to younger persons. Influenza vaccination in community-dwelling older adults is cost-effective, though there is room for improvement. Newer vaccine strategies considered for use in older adults include alternate routes of administration (intradermal or intranasal), addition of adjuvant, and novel methods of antigen presentation. Measuring cell-mediated immune response to new vaccines in addition to antibody response may correlate better with vaccine efficacy in this population. Whilst pandemic influenza A/H1N1 2009 (pH1N1) has largely spared older adults, the impact of pH1N1 vaccination has yet to be determined.

Granzyme B: a marker of risk for influenza in institutionalized older adults.

Risk for influenza increases with age while cellular immune responses decline. This was a prospective study to determine the relationship between cytokine and granzyme B levels in peripheral blood mononuclear cells stimulated with live influenza virus, and subsequent influenza illness. Granzyme B levels were lower in the group who later developed symptomatic laboratory-confirmed influenza (n=10) compared to the group who did not (n=90) (ANOVA, P=0.024). In contrast, none of the cytokine levels were related to the development of influenza. Thus, granzyme B is a potential marker of influenza risk in older adults.

Standing orders for influenza vaccination increased vaccination rates in inpatient settings compared with community rates.

BACKGROUND: Hospitalization of older adults during the period of influenza vaccination in the fall of each year presents a barrier to immunization against influenza. This study evaluates a program using standing orders for influenza vaccination to increase vaccination rates among hospitalized older adults and to determine the effect of vaccination on readmission rates for influenza-like illness. METHODS: An influenza vaccination program using a standing order policy was implemented to vaccinate all consenting persons 65 years and older prior to hospital discharge. This was a prospective, single center, cohort study in a tertiary care university teaching hospital during an 8-week vaccination period in the fall of 1994 and follow-up during the subsequent influenza season. The vaccination status of each patient was recorded as no vaccination, vaccination received in hospital, or vaccination in the community prior to or after the hospitalization. Hospital vaccination rates were compared with the rate of vaccination of older adults in the community. During the subsequent influenza season, the number of subjects reporting symptoms of influenza-like illness (ILI) or who were readmitted to hospital with influenza-related illness was compared in an analysis of vaccinated versus unvaccinated subjects. RESULTS: Seven hundred and sixty-one patients were interviewed, and 332 of these individuals had been vaccinated in the community prior to their hospital admission. Of the remaining 429 unvaccinated patients who were eligible for vaccination in the study, 171 were vaccinated in our immunization program, eight were vaccinated in the community after discharge, and 244 were not vaccinated. We were able to increase the absolute vaccination rate by 22%, when compared with community rates, with our immunization program. The number of subjects with ILI symptoms or readmission to hospital was too small to compare the vaccinated to the unvaccinated group in the study. CONCLUSIONS: An inpatient influenza immunization program using a standing order policy was able to target a particularly high-risk subset of persons 65 years and over who might otherwise have not received influenza vaccination.

Mortality and institutionalization following hip fracture.

OBJECTIVES: To identify determinants of mortality and institutionalization after hip fracture and to identify those older hip fracture patients at high risk of death or institutionalization after hip fracture. DESIGN: Population-based prospective inception cohort study of hip fracture patients; patients were assessed in the hospital and at 3 months following the hip fracture. SETTING: Edmonton area hip fracture patients admitted to one of two Edmonton, Alberta, Canada, acute care centers between July 10, 1996, and August 31, 1997. PARTICIPANTS: Patients were residents of the Edmonton area and over the age of 64. Those who had previously fractured the same hip within the past 5 years or had some pathological condition underlying the hip fracture were excluded. Of 610 eligible patients, 558 contributed some baseline information and were included in the mortality analysis; the institutionalization analysis was restricted to the 338 patients who lived in the community before fracture, survived the 3-month period postfracture, and had completed a 3-month follow-up interview. MEASUREMENTS: The baseline interview was done in the hospital to assess mental status, prefracture physical function, prefracture health perception, and prefracture social support. The 3-month follow-up interview was done by phone to assess physical function, health perception, and social support 3 months postfracture. Demographic and comorbidity information was collected from medical records. RESULTS: Low mental status in hospital was found to increase the chances of mortality and institutionalization, and male gender was found to increase mortality risk fourfold. Each additional 10 years of age increased the risk of institutionalization approximately 2.5 times. Patients with lower postfracture physical function had at least five times the risk of institutionalization compared to patients with high postfracture physical function. CONCLUSIONS: Cognitive impairment, older age, and gender were associated with increased risk of poor outcome following hip fracture. The socioeconomic variables--social support and health perception--did not contribute significant additional information in explaining mortality or institutionalization risk. While demographic factors cannot be modified, physical function 3 months postfracture may be amenable to intervention and may reduce the risk of institutionalization. Intervening to increase postfracture physical function may be particularly beneficial to older patients, or to those who are cognitively impaired.

Incidence and risk factors for delirium and other adverse outcomes in older adults after coronary artery bypass graft surgery.

OBJECTIVE: To determine the incidence and risk factors for delirium after coronary artery bypass graft (CABG) surgery. DESIGN: Prospective cohort. SETTING: Cardiac surgery units of a tertiary care hospital. PARTICIPANTS: Consecutive patients over age 65 years undergoing elective CABG surgery. Exclusion criteria included preoperative sensory or language barriers. INTERVENTIONS: Each patient was assessed within 24 h before surgery for baseline demographic, medical and functional data. Incident delirium (within four postoperative days) was diagnosed by a study physician. Nine potential risk factors for delirium were subjected to univariate and multivariate analysis. MAIN RESULTS: Of 75 consenting patients, three died during or soon after surgery and one was still comatose at follow-up. Of the remaining 71 participants, 23 (32%) experienced delirium. Those with delirium were more likely than those without delirium to have a history of a stroke (21% versus 4%, respectively, P=0.032) and to have had a longer duration of cardiopulmonary bypass (CPB) (113 mins versus 95 mins, respectively, P=0.025). A tendency to have experienced low cardiac output (83% versus 58%, respectively, P=0.061) postoperatively was also noted. Multivariate analysis confirmed past stroke and duration of cardiopulmonary bypass as risk factors. CONCLUSIONS: Delirium in the elderly after CABG surgery is common. Its occurrence may be predisposed by a history of a stroke and precipitated by a longer duration of CPB.

Validity of the confusion assessment method in detecting postoperative delirium in the elderly.

In this prospective cohort of 71 elderly patients undergoing cardiac surgery, each subject was interviewed before and after surgery to detect incident delirium using the Confusion Assessment Method (CAM), the Mini-Mental State Examination (MMSE), the Clock Test, and a health record review. The first 41 were assessed by a physician and the remaining 30 by two study nurses. Delirium was then diagnosed by a physician using DSM-III-R criteria. Delirium was present in 23 subjects (32.4%). The sensitivity of the CAM differed significantly when administered by physicians compared to nurses (1.00 vs. .13). When standard cutoffs were used, neither the MMSE nor the Clock Test were found to be sensitive markers for delirium (.30 and .09, respectively). Recognition of delirium by charting was superior in nurses compared to physicians (.83 vs. .30). We conclude that the sensitivity of markers for delirium, such as the CAM and health record documentation, is dependent on the training background of the operator.

Assessment of markers of the cell-mediated immune response after influenza virus infection in frail older adults.

The purpose of this study was to determine whether measures of the cell-mediated immune response to influenza virus could be used as markers of influenza virus infection. We studied 23 subjects who developed upper respiratory, lower respiratory, or systemic symptoms during a small outbreak of influenza in a nursing home population. Influenza virus culture from nasopharyngeal swabs yielded influenza virus isolates from 7 of the 23 subjects. Only three of the subjects had a fourfold rise in antibody titer to the influenza virus antigen positivity after the infection. Granzyme B and cytokine levels were measured in peripheral blood mononuclear cells (PBMC) obtained from all subjects and stimulated with live influenza virus. Elevated granzyme B levels in virus-stimulated PBMC in combination with lower respiratory tract or systemic symptoms in study subjects was a significant predictor of culture-confirmed influenza virus infection compared to those from whom influenza virus could not be identified. Cytokine levels did not distinguish between the two groups in a similar type of analysis. Granzyme B in combination with the clinical profile of symptoms may be a useful retrospective marker for influenza virus infection.

Responses to influenza vaccination in different T-cell subsets: a comparison of healthy young and older adults.

T-lymphocyte responses to influenza vaccination were measured in healthy young and older adult volunteers. All participants were vaccinated with the 1995-96 trivalent influenza vaccine. Cytokine and granzyme B levels were measured in peripheral blood mononuclear cells (PBMC) cultures after virus stimulation, prior to and 4 and 12 weeks after vaccination. The major findings in the older adult group were the different types of helper T-cell (Th) responses to each of the vaccine strains of virus and a very poor cytotoxic T lymphocyte (as measured by granzyme B) response to vaccination. IL-10, which is produced in a Th-type 2 response, was higher in PBMC stimulated with A/Texas/36/91 (H1N1) compared with A/Johannesburg/33/94 (H3N2); this difference was more marked in the PBMC from older compared with younger adults. In contrast, IL-2, which is produced in a Th-type 1 response, was measured in the same cultures and was significantly higher in A/Johannesburg/33/94-stimulated PBMC. IFN- gamma levels were highest in the PBMC stimulated with B/Harbin/7/94. The greatest age-related difference was the level of granzyme B in all virus-stimulated PBMC from the young compared with the older adult group. The strain of influenza virus contained in the vaccine, as well as the age of the subject, appear to be very important determinants of the T-cell response to vaccination.

Immune response to influenza vaccination in institutionalized elderly: effect on different T-cell subsets.

Humoral and cellular immunological responses to influenza vaccination were measured in volunteers in a long-term care facility. All participants were vaccinated with the commercially available 1994-95 trivalent influenza vaccine and blood samples were collected before and 6 and 12 weeks after vaccination. Cytokine and granzyme B in peripheral blood mononuclear cell (PBMC) cultures after virus stimulation, and serum antibody titres were measured for each of these time points. In general, the measures of the immunological response to vaccination were low and variably significant. The major finding was the difference with respect to post-vaccination measures for the two strains of influenza A contained in the vaccine. Geometric mean antibody titres were significantly higher for A/Texas/36/91 at all time points in the study when compared to A/Shangdong/09/93. There was a corresponding rise for interleukin-10 (IL-10) to the A/Texas/36/91 strain while no increase in IL-10 was observed in A/Shangdong/09/93-stimulated cultures after vaccination. In contrast, granzyme B rose after vaccination only in cultures stimulated with A/Shangdong/09/93. Interferon-gamma levels were also significantly higher in these PBMC cultures. There was a poor interleukin-2 (IL-2) response to both strains of influenza A. These data suggest that different strains or subtypes of influenza A may preferentially enhance T-helper type 1 versus type 2 responses through vaccination in institutionalized seniors.

Humoral and cellular immune responses following vaccination with purified recombinant hemagglutinin from influenza A (H3N2) virus.

Adults were immunized with either baculovirus-expressed, purified recombinant hemagglutinin (rHA) from influenza A/Beijing/32/92 (H3N2) virus or saline placebo and evaluated for humoral and in vitro cellular immune responses. Compared with responses in placebo recipients, vaccinees had greater postvaccination H3(Beijing/32) HA (H3)-specific lymphoproliferation and interleukin (IL)-2, IL-10, and interferon-gamma (IFN-gamma) production. Mean increases in the production of IL-10 (> or = 20-fold) and IL-2 (10-fold) were relatively greater than that of IFN-gamma (4-fold) or IL-4 (no change). Serum H3 antibodies were induced in 80% of rHA recipients, and the rise in antibody titer was significantly correlated with changes in IL-2, IL-10, and IFN-gamma concentrations. Vaccination with rHA only minimally enhanced anti-influenza virus cytotoxic T lymphocyte activity. These data demonstrate that rHA immunization of adults elicits a significant recall response by memory B and T lymphocytes and suggest that the cytokine response to vaccination has a T helper cell type 0-like profile.

Helper and cytotoxic T lymphocyte responses to influenza vaccination in healthy compared to diabetic elderly.

This study was designed to determine the effect of Type II diabetes mellitus in older adults on two measures of the cell-mediated immune response to influenza vaccination. Twenty-two elderly persons with diabetes mellitus were compared to 20 healthy seniors, all of whom were living independently in the community. Peripheral blood mononuclear cells (PBMC) were challenged in vitro with live influenza virus, pre-vaccination and 4 and 12 weeks post-vaccination. PBMC culture supernatants were assayed for IL-2 activity as a measure of the helper T-cell response to vaccination. The cytotoxic T-lymphocyte response was determined using an assay of granzyme B activity in PBMC lysates. Initial analysis of the data showed increased IL-2 production in post-vaccination PBMC cultures from the diabetic group compared to the healthy group. However, when vaccination histories were used in an analysis of variance, it was found that the difference between the two groups was related to vaccination history. Study subjects vaccinated one year prior to participation in this study compared to those who had not been previously vaccinated, demonstrated a significantly suppressed IL-2 response to vaccination. Type II diabetes mellitus had no effect on the IL-2 response to vaccination. The granzyme B response to vaccination was not significantly affected by previous vaccination and results were similar for the healthy and diabetic elderly groups.

The cell-mediated cytotoxic response to influenza vaccination using an assay for granzyme B activity.

The measurement of cytotoxic T lymphocyte (CTL) activity through 51Cr assays is a very labour intensive method for studying cytotoxicity in human CTL due to the necessary preparation of autologous targets for the assay. An assay for granzyme B, one of a family of serine proteinases implicated in the 'lethal hit' that leads to target cell lysis, is an alternative simple measure of CTL activation. We measured granzyme B activity using its both preferred and unique substrate tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester (BAADT) in peripheral blood mononuclear cells (PBMC) obtained from influenza vaccinated subjects, and stimulated with live virus. We found that granzyme B activity increases in parallel and correlates with cytolytic activity as measured by 51Cr release assays in these virus-stimulated PBMC cultures. The assay was then used to measure the cell-mediated cytotoxic response to influenza vaccination in ten healthy elderly subjects. Peak granzyme B activity (day 6) was measured in lysates of PBMC stimulated with influenza virus, obtained from study participants before and after vaccination. We found a significant increase in granzyme B activity from pre-vaccination levels to 4 weeks post vaccination (pre=2.77 U/mg protein, post=7.23 U/mg protein, p=0.002) and a subsequent decline in the activity measured at 12 weeks post vaccination (4.34 U/mg protein, p=0.0007). Due to its substrate specificity which is unique within the family of serine proteases, this assay is highly specific for granzyme B. The assay also avoids the potential hazard of radioactivity (51Cr) in the clinical laboratory and the need for a gamma counter. The assay of granzyme B activity, therefore, provides a simple, specific and responsive method for measuring changes in cell-mediated cytotoxic activity resulting from influenza vaccination.

Multidrug transporter P-glycoprotein 170 as a differentiation antigen on normal human lymphocytes and thymocytes: modulation with differentiation stage and during aging.

P-glycoprotein 170 (P-gp), the multidrug transport pump, excludes drugs from the interior of cells and is inhibited by agents such as cyclosporin A (CsA), verapamil, and FK-506, which are also substrates for the P-gp pump. This work documents the age- and differentiation-related changes in P-gp on T and B lymphocytes from human blood or spleen, and its absence on most thymus and bone marrow cells. Analysis of rhodamine 123 (Rh123) dye efflux, and its inhibition by cyclosporin A, was used as a quantitative measure of functional P-gp, and reactivity with MRK-16 was used as a measure of P-gp surface expression. The dye efflux and phenotypic expression of P-gp+ PBMC appeared equivalent to that of a moderately drug-resistant cell line, although efflux is prolonged. The sensitivity to inhibition by CsA, cyclosporin G (CsG), and PSC833 of P-gp on PBMC, thymocytes, or T-cell lines varied with apparent cell-type specificity. Normal blood and splenic T- or B-cells included 50-80% of cells with surface P-gp (MRK-16+), which mediated CsA-sensitive dye export. The proportion of P-gp+ T- and B-cells was lowest among children under age 10 years, increased in adulthood, and decreased after age 60. Thymus included 30% of P-gp+ cells mediating CsA-sensitive dye export, including CD3-4-8- progenitors and mature CD3hi CD4+8- or CD4-8+ thymocytes. Mature T-cells in cord or adult blood, spleen, and bone marrow included a large proportion (50-60%) with efficient CsA-sensitive dye export, preferentially among the CD45RA+ subset. Monocytes from all tissue sources, and most bone marrow cells, expressed surface P-gp but retained Rh123, suggesting the absence of a functional dye export mechanism. In vitro mitogen-stimulated PBMC T and B lymphocytes lost P-gp function within 4-24 hr, consistent with the observation that P-gp was reduced on antigen-experienced CD45R0+ T-cells in vivo. Drug export by P-gp may protect lymphocytes from toxic effects of CsA, and may contribute to the immunosuppressive effects of such drugs. The developmentally regulated expression of P-gp function on lymphocytes, and its modulation on activated T- or B-cells, suggest an important role in normal immune development.

Vaccine-related determinants of the interleukin-2 response to influenza vaccination in healthy young and elderly adults.

This study was designed as a parallel-groups comparison of trivalent preparations of split-virus vaccine (SVV) and whole-virus influenza vaccine (WVV) in healthy young and elderly adults. Interleukin-2 (IL-2) was measured in the supernatants of peripheral blood mononuclear cell (PBMC) cultures stimulated with live virus preparations of the two strains of influenza A contained in the vaccine. The duration of increased in vitro IL-2 production after vaccination was significantly different between the two strains of virus. A/Beijing/353/89 resulted in an IL-2 response that was quite short (< 7 weeks) while the response to A/Texas/16/89 was much more prolonged (> 12 weeks). In the first 12 weeks after vaccination, there was no difference in IL-2 levels between SVV and WVV recipients in either the young or elderly groups. The duration of the response to A/Texas/16/89 was slightly longer in WVV recipients as measured at 26 weeks postvaccination. The viral strain contained in the vaccine appears to be an important variable in determining the duration of the IL-2 response to vaccination.

Changes in CD45 isoform expression vary according to the duration of T-cell memory after vaccination.

Healthy young (< 40 years) and elderly (< 60 years) adults were immunized with the 1992-1993 preparation of trivalent influenza vaccine, and changes in CD45 isoform expression on peripheral blood lymphocytes were measured in the pre- and postvaccination periods. Fluorescence-activated cell sorter analysis was used to study T-cell subsets in fresh peripheral blood lymphocytes (day 0) and after 6 days of culture with live influenza virus. We have reported previously that the interleukin-2 response to the stimulating strain of virus, A/Texas/16/89, did not decline until 26 weeks postvaccination. In ex vivo CD4+ subsets, this interleukin-2 response was paralleled by a > 10% increase in the proportion of cells expressing the CD45RO+ phenotype following vaccination (p < 0.0001). In vitro stimulation had no effect on CD4+ subsets prior to vaccination but, after vaccination, was associated with a > 10% increase in CD45RA+RO+ cells (P < 0.0001). In addition, we have identified a change in the population of cells that express a CD45 isoform that is neither CD45RA nor CD45RO (CD45RA-RO-). At 26 weeks postvaccination, the proportion of CD45RA-RO- cells in ex vivo CD4+ peripheral blood mononuclear cells increased by approximately 15% from that measured at the earlier postvaccination time points (P < 0.0001). In vitro stimulation with influenza virus resulted in a further 20% increase in the proportion of CD45RA-RO- cells (P < 0.0001). The CD45RA-RO- phenotype may identify a population of cells undergoing apoptosis (programmed cell death) that limits the duration of helper T-cell (CD4+) memory after vaccination.