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.

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.

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.