Granzyme B: Correlates with protection and enhanced CTL response to influenza vaccination in older adults

Abstract

This study compared serum antibody titers and granzyme B (GrzB) levels in virus-stimulated peripheral blood mononuclear cells following influenza vaccination. Twelve of 239 older adults who subsequently developed laboratory-diagnosed influenza illness (LDI) had significantly lower GrzB levels compared to subjects without LDI (p = 0.004). Eight subjects with LDI in the previous year showed an enhanced GrzB response to vaccination (p = 0.02). Serum antibody titers following vaccination did not distinguish those older adults who developed LDI from those who did not. These results suggest that GrzB levels could be combined with antibody titers to more effectively predict vaccine efficacy in older adults.

Introduction

Influenza is foremost among all infectious diseases in the age-related increase in risk for serious complications and death. Immunosenescence diminishes the ability of older adults to resist influenza (particularly the A/H3N2 strains) and respond to vaccination. Rising hospitalization and death rates have been observed despite widespread influenza vaccination programs [1], [2]. The need to develop more effective influenza vaccines for older adults is widely recognized, but whether or not the aged immune system has the capacity to respond, and what specific components of the immune response would need to be stimulated for improved vaccine efficacy, are poorly understood. Although the limitations of antibody titers as a sole measure of vaccine efficacy in this population are increasingly recognized [3], alternate correlates of protection are not readily available.

The importance of cytotoxic T lymphocytes (CTL) as a major contributor to defense mechanisms against influenza in humans has long been recognized [4]. In peripheral blood mononuclear cells (PBMC) from adults, influenza-specific memory CTL can be stimulated to rapidly develop effector function that is below the sensitivity of traditional ⁵¹Cr-release assays [5]. For this reason, we have developed an assay of granzyme B (GrzB), a key cytolytic mediator released from the CTL into the virus-infected target through a process facilitated by perforin. GrzB then stimulates a cascade of events within the target cell that lead to apoptotic cell death [6], and clearance of virus from the lungs [7]. GrzB activity in influenza-stimulated peripheral blood mononuclear cells correlates with traditional measures of cytolytic activity [8], and has been shown to be the earliest and main contributor to CTL-mediated killing of influenza virus-infected cells in the mouse [9], [10]. We have shown prospectively that prior to influenza illness, GrzB levels are lower in the influenza-stimulated PBMC from older adults who subsequently develop influenza illness compared to those who do not [11], [12]. Further, antibody titers prior to exposure to influenza did not distinguish between those who developed influenza illness from those who did not [12].

To develop an assay of cell-mediated immunity that would have broad application to the older adult population, the study protocol was designed with limited exclusion criteria and recruited a subset of very high-risk older adults with congestive heart failure (CHF). The method is a relatively simple assay of GrzB activity in lysates of PBMC stimulated ex vivo with live influenza virus, as a potential predictor of protection against influenza in vaccinated older adults. The GrzB assay has been further developed and is now standardized against a commercially available GrzB standard so that it can be validated for use across different studies and laboratories.

Herein, we report data showing that GrzB activity not only correlates with protection against influenza but also demonstrates the potential capacity of older adults to mount an enhanced response to influenza. We also show that in the ex vivo response to influenza virus, increased levels of GrzB are largely produced in the T cell (CD3+ or CD3+CD56+) subset with relatively little increase in the activated natural killer (NK) cell (CD3−CD56+) subset. Further, the phenotype of virus-activated GrzB+ T cells is consistent with a virus-specific effector function. Given that the antibody response to influenza vaccination was again shown to have limited ability to predict protection from influenza in older adults [12], measures of both antibody and CTL/GrzB responses are proposed as a more robust evaluation of protection against influenza in this population.