Granzyme B: Correlates with protection and enhanced CTL response to influenza vaccination 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.
Section snippets
Study design and participants
This was a prospective study of 239 adults age 60 years and older (median, 75 years old; range, 60–95 years old) recruited in the vicinity of the Greater Hartford Area of Connecticut in each of two consecutive influenza seasons (2003–2004 and 2004–2005). A second subset of subjects from the study conducted during the 2008–2009 season was also studied. All subjects were recruited through written informed consent. The Institutional Review Board of the University of Connecticut Health Center
Influenza outcomes
A total of 239 participants were enrolled in the 2 years of the study; 63 subjects were re-enrolled from the first to the second year. In the first year, nine of 90 subjects developed LDI but five subjects who developed LDI before 4-week post-vaccination were excluded; natural infection in these five subjects enhanced the GrzB response masking the effect of vaccination [12]. Thus only the four remaining cases from the first year were included in this analysis. An additional eight of 149 older
Discussion
The association between declining cellular immune function and loss of influenza vaccine efficacy with aging is well documented but the underlying mechanism is poorly understood. Clearly, additional correlates of protection to that provided by antibody titers are needed for advances in vaccine technology and the development of more efficacious vaccines for the older adult population. Current influenza vaccines contain split-virus particles and thus as killed viruses, are poor stimulators of the
Acknowledgements
This work was funded by the National Institutes of Health (NIH), National Institute on Aging, R01 AG20634, and National Institute of Allergy and Infectious Diseases, R01 AI68265 (J.E.M., Principal Investigator). The study was conducted through the Lowell P. Weicker, Jr. General Clinical Research Center funded by the NIH, National Center for Research Resources (Grant Number MO1 RR06192) at the University of Connecticut Health Center (UCHC), and in collaboration with the UConn Center on Aging.
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