Age-related changes in durability and function of vaccine-elicited influenza-specific CD4⁺ T-cell responses

Abstract

The major antigenic component of licensed influenza vaccines, hemagglutinin (HA), elicits predominantly type-specific antibody responses, thus necessitating frequent antigenic updates to the annual vaccine. However, accumulating evidence suggests that influenza vaccines can also induce significant cross-reactive T-cell responses to highly divergent, heterosubtypic HA antigens not included in the vaccine. Influenza vaccines are less effective among the elderly and studies that characterize cross-reactive T-cell immunity in this vulnerable population are much needed. Here, we systematically compare the ex vivo frequency, cytokine profile and phenotype of vaccine-elicited HA-specific T-cell responses among a cohort of young (18–49 years old) and elderly (≥70 years old) vaccinees, as well as the maturation and activation phenotype of total CD4⁺ and CD8⁺ T-cells. IFN-γ production after in vitro expansion and HA-specific Ab titers were also determined. We find that vaccine-elicited ex vivo frequencies of CD4⁺ T-cells elicited by vaccination reactive to any given homo- or heterosubtypic Ag were comparable across the two age groups. While, no differences were observed between age groups in the phenotype of Ag-specific or total CD4⁺ T-cells, PBMC from young adults were superior at producing IFN-γ after short-term Ag-specific culture. Significantly, while vaccine-elicited T-cell responses were durable among the younger vaccinees, they were short-lived among the elderly. These results have important ramifications for our understanding of vaccine-induced changes in the magnitude and functionality of HA-specific CD4⁺ T-cells, as well as age-related alterations in response kinetics.

Introduction

The influenza pandemic of 1918 saw the introduction of an avian-origin H1N1 that is estimated to have caused over 40 million deaths worldwide [1]. The influenza pandemic of 2009 similarly saw the introduction of an antigenically novel H1N1 into the global population and is estimated to have caused over 18,449 deaths globally [2]. In spite of the dominant emergence of this novel swine-origin H1N1 lineage in 2009, multiple instances of lethal avian influenza infections among humans have also occurred over the past decade. While divergent influenza subtypes including H7N7, H9N2, and H7N3 have circulated sporadically, recurrent outbreaks of the highly pathogenic H5N1 have been documented consistently [3], [4], [5]. Though these outbreaks have primarily occurred in south-east Asia, avian influenza infections among humans and birds have also been reported from Central and Western European nations thus increasing the likelihood of a potential H5N1 pandemic emerging in the future [6].

A number of studies in humans [7], [8], [9], [10] as well as in animal model systems [11], [12] suggest that protective immunity against influenza correlates with the prevalence of influenza-specific antibodies, primarily those directed against the viral hemagglutinin (HA). However, humoral immunity to influenza is predominantly type-specific and affords limited protection against heterosubtypic strains of circulating viruses [13]. Thus, the continual drift- and periodic antigenic shift-events that introduce antigenically novel influenza viruses into the global population have required annual changes in influenza vaccines to achieve reliable vaccine-induced protection [14]. However, instances of mismatches between seasonal influenza Ags found in the vaccine and circulating influenza viruses have generally lead to limited vaccine efficacy in some years [13], [15], [16], [17], [18], [19]. Equally, with subtypes such as H5N1 that show future pandemic potential, several distinct heterotypic strains have been involved in the outbreaks to date, thus increasing uncertainty as to which strain or strains may emerge as a potential avian pandemic strain.

With such antigenic plasticity as a backdrop, cellular immune responses are being increasingly recognized as an alternate correlate of immune protection that could provide protection from influenza viruses of disparate antigenic lineages [20]. A number of studies have recently characterized the existence of subtype-independent, cross-reactive cellular immune responses against diverse influenza strains [21], [22], [23], [24], which may provide protection from influenza infection or reduce severity of influenza disease [24], [25]. Importantly, recent studies clearly suggest that among the elderly, a population that bears the brunt of influenza morbidity and mortality, cell mediated immunity provides a better correlate of protection than do serum antibody responses [20]. As with antibody responses, HA is a major component of protective cellular immunity against influenza [26], [27], including heterosubtypic immunity [28].

Accumulating evidence suggests that cross-reactive T-cell responses can be induced de novo or boosted over pre-existing levels following seasonal vaccination with an unmatched HA antigen [23]. Though our earlier study utilizing a cohort of 18–49-year-olds was unable to assess cross-reactive T-cell responses among the truly elderly [29], we found that ageing reduced the magnitude of T-cells cross-reactive to pandemic H1 even in this relatively young cohort. Also, our use of IFN-γ ELISPOT following expansion in vitro precluded the assessment of HA-specific T-cell frequencies, as well as their memory and activation status directly ex vivo. In the present study, polychromatic flow cytometry was used to compare the ex vivo frequency, maturation and activation phenotypes of HA-specific CD4⁺ T-cells elicited following seasonal influenza vaccine administration among young (18–49 years of age) and elderly (≥70 years of age) adults. As our trial was conducted in 2007, we also address the ability of seasonal vaccination to induce cross-reactive T-cells against antigens that vaccinees had not yet been exposed to, such as the avian H5 that has only seen sporadic outbreaks in Asia, and the novel pandemic swine-origin H1 of 2009 that emerged two seasons following sample collection.