Here, we report the ability of MVA-NP+M1 to boost influenza-specific T cell responses in older adults. Recombinant MVA vaccines are establishing a good reputation for safety, although the majority of these data relate to younger individuals aged between 18–45 years. Our results with MVA-NP+M1 add to the experience from cancer trials with MVA-5T4 that recombinant MVA is safe in older adults 
. Indeed, no severe or serious adverse reactions were detected in our volunteers.
We also report that MVA-NP+M1 is highly immunogenic in volunteers over the age of 50 years. In one quantitative review 
of trivalent inactivated influenza vaccines, rates of seroprotection and seroconversion among those over 60 years old were four times lower for H1 and B antigens, and twice as low for H3 antigens. In addition, although not powered to detect declining efficacy with age, an age stratification suggested a far lower efficacy rate for those over 70 years 
. Indeed, other studies have suggested that vaccine efficacy appears to be as low as 30–40% in this age group 
. On an individual level, declines in immunological function are unlikely to occur in a linear fashion (chronological age being only a surrogate indicator of biological age) 
However, on a population level, declines in vaccine responsiveness are likely to be observed as average age increases. Indeed, in the oldest age group (70+ years), we observed a reduction in immunogenicity as detected by ex-vivo
IFN-γ ELISpot compared to the youngest age group (50–59 years), with significantly lower responses at 3 and 8 weeks post-vaccination. However, when the 3 age groups were compared to a younger cohort of volunteers (18–45 years) who received the same dose of the MVA-NP+M1 vaccine, no significant differences were detected.
The functional characteristics of the cellular responses produced by vaccination are potentially as important as magnitude 
. Subsets of CD4+
T cells following vaccination with MVA-NP+M1 are capable of secreting both TNF and IL-2, in addition to IFN-γ. Increases in the number of such polyfunctional T cells have been associated with protective immunity in some models of infection 
. We show here that MVA-NP+M1 vaccination can also induce polyfunctional CD4+
T cell responses in older adults, as determined by flow cytometric assessment of CD107a mobilization and the production of IFN-γ, TNF and IL-2.
MVA-NP+M1 is designed to expand T cells that are already present in the memory pool rather than prime naïve T cells de novo. Direct evidence for this mode of action comes from our comparison herein of M1-specific TCR sequences before and after vaccination. This provides a biological rationale for the use of MVA-NP+M1 in elderly individuals due to the impairment of thymic output with age. The absolute number of NP- and M1-specific T cells required for host defence against influenza is not known. However, the median ex vivo IFN-γ ELISpot response observed in the older volunteers peaked one week after vaccination at 1,603 SFU/million PBMC, which represents an 8·5-fold increase compared to the pre-vaccination response.
No vaccine-induced expression of granzyme B, IL-10 or IL-17 was detected in our cohort of older volunteers. However, we did detect significantly higher non-specific levels of granzyme B expression in group 3 (70+ years) compared to group 1 (50–59 years) at weeks 1 and 3 post-vaccination. It has been shown previously that baseline granzyme B expression in CD8+
T cells is higher in ageing volunteers and that these cells are associated with a decreased ability to respond to stimulation with whole influenza virus 
. Degranulation and extracellular release of granzyme B can also cause inflammation and extracellular granzyme B has been implicated in increasing the risk of serious illness in the elderly, including the risk of influenza induced cardiovascular complications 
A high IFN-γ:IL-10 ratio may be associated with protection from influenza 
. The median frequency of NP- and M1-specific T cells that secreted IL-10 was low (below 0·006%) and did not increase after vaccination, whereas there was a significant increase in the number IFN-γ-secreting T cells following vaccination.
The memory phenotype of vaccine-induced CD8+
T cell populations, at least for a subset of M1-specific cells, was remarkably similar to that observed pre-vaccination. Indeed, a marginal decrease in CD27 expression consistent with progressive differentiation post-vaccination was the only detectable change between the time points studied within individual volunteers (data not shown). Thus, minimal differentiation-associated functional variations would be expected. Interestingly, despite vaccine-mediated expansion of pre-existing memory clonotypes, the observed CD27+
phenotype indicates a lack of terminal differentiation and senescence 
. This is encouraging from the perspective that durable T cell immunity may be feasible using this approach of boosting existing T cell memory with an MVA-vectored vaccine.
MVA-vectored vaccines have the advantage that they can be produced on the large scale required for widespread human vaccination. The low level of polymorphism in NP and M1 across influenza A strains means that a vaccine such as MVA-NP+M1 could provide T cell-mediated protection against all influenza A subtypes.
In summary, we have shown that the novel influenza vaccine candidate MVA-NP+M1 is safe and highly immunogenic in adults over 50 years old. Both CD4+ and CD8+ memory T cell responses are boosted, and have the capacity to secrete multiple cytokines. Indeed, despite the apparent reduction in immune responsiveness observed in the oldest volunteers in this study, there was still a significant induction of IFN-γ-secreting cells and a significant increase in the proportion of CD4+ and CD8+ T cells capable of triple cytokine production after vaccination. These enhanced T cell responses could provide heterosubtypic T cell-based immunity against influenza in the elderly.