Some reports indicate that CMV seropositivity may be associated with better antibody response to vaccination in younger adults (58). This is different in studies involving older adults, where CMV seropositivity has been variably found to be associated with beneficial (59), negative (41, 58–62), or negligible effects (58, 63). The overall impact of CMV infection on influenza vaccine responsiveness remains controversial, as it is depends on many variables. Different studies performed with different seasonal vaccines, tested in different populations, at different times, are difficult to compare directly. As such, there have been no studies that have directly linked CMV seropositivity with increased risk of influenza illness in vaccinated older adults.

In addition to lack of consensus on the impact of aging and CMV seropositivity on antibody responses to influenza vaccination, investigating this issue is further complicated by differing responses to strains of the virus. Vaccine efficacy in the elderly against H3N2 is particularly poor compared to H1N1 or B strains (64, 65). One explanation for an apparent lack of responsiveness in the elderly may reside in the manner in which antibody responses are quantified, which is dependent on the immunological history of the individual. Thus, older adults who already have a high-antibody titer prior to vaccination may be classified as non-responders if they do not further increase an already-protective titer. More importantly, comparisons of the antibody response to influenza vaccination in young and older adults have been confounded by the effects of age and exposure history related to prior vaccination (66). In addition, these differing observations may be explained in the context of original antigenic sin, which supports the notion that vaccination re-stimulates immunological memory of past exposure to a similar strain, and may explain the relative protection of older adults against the pandemic H1N1 (pH1N1) strains (67). The theory of vaccine re-stimulation has not been explored in the context of CMV, but highlights the importance of identifying which subtype of influenza is being studied and a consensus as regards to the definition of vaccine “responder.”

Contradictory observations of influenza strain-specific titers post-vaccination between CMV-seropositive and -negative individuals have also been identified. Specifically, CMV⁺ subjects were found to have higher antibody titer to H1N1 (58, 59, 61), while others have observed the opposite (41). Similarly, in some studies, no association was observed between CMV status and H3N2-directed antibodies (63), while others have reported lower H3N2 antibody responses in such subjects (62). Those identifying an improved response to vaccination have hypothesized that CMV infection is accompanied by a higher level of a low-grade chronic inflammation that in turn provides an ongoing stimulation to the immune system in older (68) and younger adults (58).

It should be noted that studies in this area have used different measures of the antibody response to vaccination as a correlate of protection. Specifically, some have reported peak antibody response, while others measured antibody persistence. Although peak antibody titers after vaccination depend mainly on short-lived plasma B-cells, antibody persistence depends on memory B-cells and long-lived plasma cells. As such, antibody persistence may be a more meaningful measure of clinical protection. Some apparent discrepancies in the literature could derive from such different measures.

Other possible reasons for the discrepancies reported in the literature may be related to confounding factors such as medications, as illustrated in a recent study by Reed et al. These investigators identified a poorer antibody response (quantified based on antibody persistence) to vaccination in CMV-seropositive older adults, but only if they were taking β-adrenergic-blocking drugs (69). β-adrenergic blockers, a class of drug commonly used for blood pressure control, may also influence immune responses (70) and could therefore represent a confounding factor resulting in non-consensus of previous studies of antibody responses in older adults. However, it should also be noted that the use of β-blockers may simply reflect non-specifically generally poorer health or could represent an association with other health conditions relating to immunosenescence. The potential impact of drug treatment on vaccine response is further illustrated in the case of statins and antibody responses to influenza vaccination in older adults (71) as well as vaccine efficacy (72). Statins are known to influence immune responses via multiple different mechanisms (73), indicating a need to investigate the relationship further, especially since this class of drug is used by a growing number of older adults (74, 75).

Sequencing of the immunoglobulin heavy chain has been conducted to study B-cell receptor (BCR) repertories. It was found that V (variable), D (diversity), and J (joining) usage is consistent between age groups, although mutations in V genes are associated with CMV seropositivity. Furthermore, mutations in IgM and IgG sequences are higher in the elderly, and more so in those who are CMV seropositive (76). This suggests that repeated antigen exposure with aging and CMV reactivation induces B-cell proliferation and IgG gene mutations.

In a groundbreaking study, de Bourcy et al. used next-generation sequencing technology to study BCR diversity. They showed that while BCR repertoires become more specialized over the lifespan, they also demonstrate decreased capacity for plasticity or adaptability. Relative to the young, older adults have a smaller naïve repertoire and lower intra-lineage diversity, resulting in a reduced ability to mount a diverse response to novel antigens (77). This suggests that annual updates of the strains of influenza contained in the vaccine are less likely to induce antibody responses to new viral variants in older adults.

The association of leukocyte telomere shortening with some aspects of aging has been well documented. In a seminal study of the correlation between telomere length of B-cells and humoral immune responses to influenza vaccination in adults over the age of 70, it was shown that those with longer telomeres (6.3 kb) had superior antibody responses (based on fold-increase of influenza-specific antibody titers) relative to those with shorter telomeres (5.6 kb) (17). While the mechanism involved is still not well understood, telomere length could be used as a marker for immune function and vaccine responsiveness.

Influenza infection induces HA-specific CD4⁺ T-helper cells (87), resulting in a diverse antibody response (88). Th2 responses stimulate antibody production that is driven by the production of cytokines, including IL-4, IL-5, IL-10, IL-13, IL-31, and IL-33 by mast cells and eosinophils, which are responsible for a Th2 response, thereby leading to the activation of B-cell clones and production of influenza-specific IgG1 and IgE (89, 90). The regulatory T-cell (Treg) response results in the expression of IL-10 and TGF-β, which further hinders a Th1 response (91).

Aging is associated with an increasing acquisition of a Th2 bias, specifically with an increase in CD4⁺CD294⁺ (Th2) cells (92). Furthermore, some studies have reported a decline in the total number of CD8⁺ T-cells and increases in T-helper cells reflected in a lower Th1:Th2 ratio (92). IL-10 and other cytokines produced by Th2 or Treg have been associated with reduced cytotoxic T-lymphocyte (CTL) activity in older adults (93) and against ex vivo influenza virus challenge (94). Although Th2-associated cytokines do not help in the recovery from influenza infection (95), these cytokines continue to be expressed at high levels at the site of influenza infection and may be a factor in inflammation and lung damage associated with infection (78).

Aging creates specific challenges to effective CTL activity against infection: (a) T-cell receptor (TCR) diversity reduction, (b) reduced effector function of cells, (c) cell type frequency changes, and (d) general inflammation.

Cytomegalovirus elicits CD4⁺ and CD8⁺ T-cell responses (113, 114), which play an important role in maintaining latency of CMV infection (115). The immune response necessary to maintain latency has two major consequences: driving T-cells to a late-differentiated state associated with immunosenescence (6, 14, 116) and memory inflation (117). The latter develops from chronic antigen exposure as a result of CMV infection (118, 119), but interestingly Epstein–Barr virus does not have the same impact on inflation (120). CMV-specific memory T-cells account for 0.1–40% of the total memory population in the periphery (113, 114). The high frequency of CMV-specific T-cells develop during the first year after infection and either gradually increases or remains constant long term (121–123). Over a lifetime, a large group of T-cells recognizing CMV epitopes emerges, the majority of which may be dysfunctional (124). This contributes to the concept of a restricted “immunological space,” whereby the T-cell populations consisting of dysfunctional clonally expanded and anergic cells are targeted toward a small number of epitopes (125). In addition, aging results in a decrease of the TCR repertoire, which is associated with a poor response to influenza vaccination (126–129). Furthermore, the repertoire may become oligoclonal due to extended lifespan and homeostatic turnover of naïve T-cells (130).

On infection, viral epitopes bind to the major histocompatibility complex (MHC) molecules of APCs, and through the interaction with TCRs activate naïve or memory T-cells to become effector CTLs (131, 132). The majority of the CD8⁺ T-cell epitopes derived from influenza virus are contained within the NP and matrix 1 (M1) proteins. As a result of the homology of these internal proteins and highly conserved epitopes across the different subtypes (A/H1N1 and A/H3N2), the CD8⁺ T-cell response to influenza is cross-reactive among all of the strains of influenza A. Activation of T-cells leads to their migration to the infection site where they recognize influenza virus-infected cells and eliminate them via lytic activity. While CTL killing of influenza-infected cells can be mediated through Fas- (132), and TRAIL- (133) associated pathways, the dominant mechanism appears to be perforin-mediated killing (134). Recent studies suggest that perforin-mediated killing through granzyme B (GrB) apoptotic pathways is the most critical for viral clearance (135).

Studies comparing dosage of trivalent inactivated influenza virus vaccines in older adults have found that people who received high dose vaccinations had significantly higher antibody titers for all three strains (to varying degrees) than those who received standard dose vaccinations (179, 180). High dose vaccine recipients had a greater frequency of pT_FH cells post-vaccination than those receiving a standard dose. Specifically, the expression of CD278 (also referred to as inducible T-cell costimulator: ICOS) on the pT_FH cells was elevated (181), suggesting an increase in their ability to provide B-cell help. Furthermore, as mentioned previously, the frequency of pT_FH cells was a predictor of seroconversion for all three vaccine strains (181). It has also been shown that the longevity of the antibody response is not influenced by vaccine dose (182). An assessment of the impact of vaccine dose on the cellular immune response has been limited to analysis of IFN-γ response, which found no significant difference (182, 183). However, this important issue requires further study.

While the high dose vaccine has been shown to deliver better clinical protection in older adults (184), further investigation as to the mechanism is required. Furthermore, the impact of vaccine dose in more vulnerable older adults, including those who are CMV seropositive also requires investigation.

The elderly might benefit from the increased application of adjuvanted vaccines. For example, glucopyranosyl lipid adjuvant (GLA) is a TLR4 agonist that has been found to be safe and well tolerated (185). GLA, when combined with Fluzone, a split virus vaccine, showed enhanced antibody response as well as a shift to a Th1 cytokine profile in mice (185). In vivo, GLA produces a shift toward a Th1 cell-mediated response to influenza challenge by reducing IL-10 expression along with an increase in GrB activity (186). It has also been shown in vitro that GLA can induce the maturation of human DCs with an associated release of Th1-inducing cytokine and chemokine constellation (187).

Older adults seropositive for CMV have been found to have lower levels of activated DCs than young CMV⁺ adults, as it is believed that the older group has a greater control of infection due to higher CMV-specific antibodies. Due to a lower preactivated state, the DCs of older adults, regardless of CMV status, were also relatively more responsive to TLR4 antagonist (in this case LPS) and were able to produce TNF and IL-6 at the same level, and same cell frequency as younger adults who were CMV seronegative (188). Thus, the use of a TLR4 agonist in influenza vaccines for the CMV-seropositive elderly may be a potential adjuvant to improve vaccine efficacy in this group.

Conserved proteins, NP and M1, share high levels of homology among influenza strains (189, 190) and contain immunodominant MHC Class I and II epitopes (191). At the present time, influenza vaccines are designed to have specific quantities of HA protein, but the quantity of the internal proteins are believed to be low and are not quantified as part of vaccine formulation quality control (192). Evidence suggests that the inclusion of NP and M1 proteins in influenza vaccines would develop a Th1 memory and provide more effective protection in older adults.

Clinical studies of mRNA-based vaccines demonstrated their ability to elicit functional antibodies and T-cell responses (193, 194). Unlike previously considered DNA-based vaccines, there is no concern regarding genome integration, or the need to design nuclear transport mechanisms for mRNA vaccines. RNA-based vaccines allow for cell entry, viral protein translation, and broad immune responses, while also acting as an adjuvant (TLR7/8) (195). Two different forms of RNA-based vaccines are currently being developed against influenza: non-amplifying mRNA (196) and self-amplifying mRNA molecules (197, 198). A modified vaccinia virus Ankara-based vaccine against influenza has been developed which consists of non-replicating RNA encoding both NP and M1 proteins (199). This vaccine was found to be safe and able to stimulate T-cell responses in older adults similar in magnitude to young adults (200). Amplifying mRNA (SAM1 technology) is based on non-viral delivery of antigen-encoding RNA by modified alphavirus single-stranded RNA, which allows for viral RNA replication and greater viral protein translation (193, 194, 197). In a comparison of replicating and non-replicating vaccines, it was found that self-replicating vaccines elicit significantly stronger cellular and humoral immune responses, which was suggested to be the result of greater antigen presentation (201). These findings suggest that self-replicating influenza RNA vaccines may be used to overcome the effects of immune senescence and CMV seropositivity.

A study mapping the prevalence of influenza-specific antibodies in children found that by the age of 6, all children had seroconverted and thus had immunological memory for at least one influenza virus strain (202). Over time, this memory is boosted and diversified by subsequent infections with drifted influenza virus strains. In turn, this memory response (both humoral and cellular) can act to protect against infection with similar stain variants, referred to as cross-protection. Cross-protection was shown in the pH1N1 virus identified during the 2009–2010 and 2013–2014 influenza outbreaks which resulted in high rates of morbidity and mortality. Analysis of the CD8⁺ T-cells specific for the 2009 pH1N1 virus identified epitopes shared with the 1918 pH1N1 strain, as well as stains circulating prior to 1945 (203). These findings corroborate the observation that the severity of the influenza illness in the over-65 age group infected with pH1N1 was considerably lower than other influenza seasons (204), suggesting cross-protection. Furthermore, it was found that the majority of severe cases of pH1N1 infection occurred in young adults (205). This has led to suggestions that vaccines may only be effective due to their ability to act as a booster to memory CD8⁺ T-cells remaining from previous infections, rather than creating a memory response to new targets (137). Furthermore, evidence suggests that T-cells generated in youth can remain protective over decades (206, 207), while those derived later in life are severely impaired (208, 209). Thus, it could be postulated that the most effective way to ensure protection via vaccination in older adults would be to design a vaccine strategy targeted at youth.

Vaccine effectiveness against influenza declines with time after vaccination over the winter season and is most evident in older adults, with efficacy lasting 140 days for H3N2 and 200 days for influenza B (210). Interestingly, vaccine effectiveness appeared to be maintained for over 200 days for subtype H1N1 (210). Influenza vaccines are only administered once a year but the impact of a double dose or booster vaccines is being studied to determine whether efficacy and level of protection can be improved. At present, a clinical trial of vaccines administered bi-yearly in older adults is underway (http://ClinicalTrials.gov: NCT02655874). The benefit of two-dose influenza vaccines has been shown under somewhat different circumstances in studies of solid organ transplant recipients where a second influenza vaccine dose after 5 weeks resulted in higher rates of seroconversion and seroprotection (defined as titer ≥1:40) (211). Frail older adults who were non-responders to an initial dose of influenza vaccine showed a decline in antibody titers to the A/H3N2 strain following a booster dose of influenza vaccine and derived no clinical benefit from this booster strategy (212). These results are consistent with our earlier studies showing a significant IL-10 response to a booster dose vaccination and a decline in the antibody and GrB response to influenza challenge following vaccination, compared to those older adults who received a single dose of influenza vaccine (213). Studies of H5N1 have shown that a two-dose approach using heterosubtypic H5 antigen results in a greater magnitude of T-cell cytokine response with heterosubtypic protection, but this does not seem to be the case in older adults (214). As such, the impact of a two-dose influenza vaccine is not yet clear and requires further investigation to determine effectiveness.

One possible method to improve influenza vaccine efficacy might be an anti-CMV strategy via vaccination or treatment. While the majority of CMV treatment strategies have been developed for the prevention of congenital CMV infection or for the immunocompromised, there is potential for its application in the wider population.

The challenge in CMV vaccine design lays in the extensive genetic diversity of CMV strains due to recombination (215, 216). Several multi-component subunit (217), recombinant live-attenuated (218), and DNA (219) vaccine candidates aimed at achieving broad cross-neutralizing humoral and cellular immune responses are currently under investigation. One clear caveat for the CMV vaccine strategy to be effective is the need for its administration at a young age, and the ability for the vaccine to impart long-lasting immunity. Drug treatments may be a more practical approach in the short-term including those with anti-CMV activity in vitro currently being tested in clinical trials: a protein kinase inhibitor with specific activity against CMV (220); a CMV terminase inhibitor (221); and a broad spectrum antiviral agent (220).