Prevention of infectious diseases in the elderly is essential to establish healthy aging in the rapidly growing aging population. Yet, immunological aging impairs successful vaccination in the elderly (1–3). Timely vaccination of middle-aged adults may be an alternative option to strengthen the memory immunity before reaching old age. Previously, we showed that a primary meningococcal vaccination, containing antigens toward which no or very low prevaccination immunity exists, was highly immunogenic in middle-aged adults (4). Moreover, we described the induction of T cell responses by the tetanus toxoid (TT) carrier protein that are in favor of efficient T cell help (5). Current research focusses on the identification of immune markers in older individuals to be able to predict the vaccine responders and non-responders (6, 7). At present, the discovery of these predictive immune markers at advanced age is challenging and results are not unambiguous.

Potential biomarkers for vaccine responsiveness may relate to shifts in the immune phenotype from naïve to more memory cells during aging. This phenomenon occurs especially in the T cell compartment and is caused by thymus involution (8–13). Accordingly, the responsiveness to a yellow fever vaccine was found positively associated with the numbers of circulating naïve CD4 T cells that had recently left the thymus (14). Infection with persistent viruses, such as cytomegalovirus (CMV), enhances the numbers of late-differentiated T cells and consequently may accelerate immunological aging (15, 16). High numbers of these late-differentiated T cells were negatively associated with influenza and varicella zoster (VZV) vaccine responses (17, 18). In addition, increased numbers of regulatory T (Treg) cells are observed at old age (19, 20) which may underlie the lower responsiveness to the influenza and VZV vaccinations (18, 21).

Age-associated changes in the B cell compartment have also been reported and include a decrease in naïve B cells and a subsequent increase in late-differentiated and exhausted B cells, as well as B cells with inflammatory characteristics (22–24). Several vaccination studies described a positive correlation between the frequencies of prevaccination Ig switched memory B cells and the responsiveness to influenza and hepatitis B vaccines (17, 23, 25–27), whereas late/exhausted (CD27–IgD−) memory B cells were negatively correlated with the response to the influenza vaccine (23). Moreover, B cell expression levels of activation-induced cytidine deaminase (AID) and TFN-α after in vitro stimulation were found predictive for humoral responses after influenza vaccination (23, 26, 28–30).

In addition, several innate immune functions, gene signatures, or miRNA expressions were associated with influenza vaccine responsiveness (25, 31, 32). Moreover, the age-associated increase in inflammatory mediators, also known as “inflammageing” (33–36), as well as modified expression of biochemical markers, such as dehydroepiandosterone sulfate (DHEAs) (37) and vitamin D (38), might affect the immune function at advancing age (39). Also, a range of vaccination responses, for example, to diphtheria, tetanus, and influenza, are substantially influenced by vaccine-specific prevaccination immunity (27, 31).

The studies mentioned provide some promising predictive biomarkers that require validation in other cohort studies. In the present study, we aimed to explore differences in the prevaccination immune phenotype between low and high vaccine responders toward a primary immune response upon a meningococcal vaccination in middle-aged adults.

In this explorative study, we investigated whether the prevaccination immune phenotype was significantly different between the middle-aged adults being either low or high responder after a primary meningococcal vaccination. Interestingly, the numbers of several CD4 T cell subsets differed between the low and high vaccine responders. More specifically, low vaccine responders possessed higher numbers of naïve Treg, memory Treg, naïve CD4 cells, and the subset of post thymically expanded CD4⁺CD45RA⁺CD25^dim T cells, whereas high responders showed high levels of serum IL-1Ra. These results suggest that the prevaccination CD4 signature may be used to identify middle-aged adults who are potential non/low responders to a primary meningococcal vaccine. Identification of these middle-aged adults may help improve timely vaccination strategies, since vaccination schemes, doses, and adjuvant use might be adapted to improve the vaccine responsiveness in these adults.

Numbers of Treg cells are known to increase with advancing age and suggest elevated immune suppression in older adults, although the exact functionality of these Treg cells in aging individuals is still under investigation (19, 48). Accordingly, high numbers of Treg cells were previously associated with low VZV vaccine responses in nursing home elderly (18). Within our study, high absolute numbers of both naïve and memory Treg cells were associated with low vaccine responsiveness. Naïve Treg cells express CCR7 enabling these cells to migrate to lymphoid organs, whereas memory Treg cells home to the sites of inflammation along with effector T cells (49, 50). Accordingly, our results suggest enhanced suppression of the vaccine response both in the lymphoid organs as well as the site of vaccination in low responders. Elevated numbers of Treg cells might suppress T cell responses toward the tetanus carrier in this conjugated meningococcal vaccine and/or inhibit B cell responses directly (51). Our results confirm previous findings of high numbers of memory Treg cells in low vaccine responders to influenza (21), whereas we are the first showing an association between low vaccine responsiveness and high numbers of naïve Treg cells. Of importance, we observed a higher ratio of memory Treg to effector CD4 T cells in the low responders, suggesting a shift in the Treg/Teffector balance, as previously observed with advancing age (21).

The consistent association found between high numbers ofCD4 naïve and the post thymically expanded CD4⁺CD45RA⁺CD25^dim cells and a higher ratio of these naïve cells to the memory CD4 T cell compartment with low vaccine responsiveness was unexpected, since a naïve T cell repertoire is generally accepted to be beneficial in older adults (9, 11). Previously, the CD4⁺CD45RA⁺CD25^dim subset was found to represent a broad and functional reservoir of naïve CD4 T cells, although some contraction in certain TCR Vβ families was observed in comparison to naïve CD4 T cells that recently left the thymus (52). Since no prior studies were available that linked the numbers of CD4⁺CD45RA⁺CD25^dim cells to vaccine responses, our findings indicate the necessity for further research into repertoire size and functionality of these cells. However, the increase in memory Treg cells might be the dominant factor in predicting vaccine response, overruling the presence of a capable naïve CD4 T cell repertoire.

In line with the different studies investigating biomarkers for influenza and hepatitis B vaccine responses in the elderly (17, 23, 25), we observed trends toward lower numbers of switched memory CD27⁺ B cells in the low responders. In contrast, we did not find increased numbers of CD27-memory B cells in the low responders, as reported by others in vaccine recipients aged over 65 years (23). In addition, low numbers of natural effector CD27⁺IgD⁺ B cells that were previously found to decrease with age (24) were observed in the low vaccine responders. Since we previously described that IgM is essential in the antibody functionality against the meningococcal groups (4), lower numbers of these natural effector cells, mainly producing IgM, likely explain the lower functional antibody titers.

Currently, effects of latent CMV infection on vaccine responses are controversial (53). Despite the clear associations between CMV seropositivity and higher numbers of late differentiated T cells, we did not find an association between CMV seropositivity and meningococcal vaccine responsiveness. As this meningococcal vaccine response is primarily B cell mediated, the effect of CMV might be limited. Hence, the effects of CMV on T cell-mediated vaccine responses, i.e., to influenza and VZV vaccination should be further elucidated. Although frequently suggested by others (54–57), we did not observe any effects of sex and BMI on the vaccine responses. Moreover, the effect of chronological age was inconsistent, although low responders to MenC were significantly older than high responders.

Of note, high levels of IL-1Ra were found in the high responders. IL-1Ra is known as the receptor antagonist of the IL-1 family, executing anti-inflammatory functions (58, 59). These results possibly suggest that IL-1Ra acts as an anti-inflammatory counterpart of the “inflammageing” process. Of note, a trend toward high levels of MCP-1 was found in the low responders. MCP-1 is classified as a pro-inflammatory cytokine, attracting monocytes to the site of inflammation, and serum levels were shown to increase with age (60). Consequently, our findings may suggest a higher pro-inflammatory state in the low responders (33, 34). Nevertheless, serum levels of other inflammatory cytokines, such as IL-6 and TFN-α were still low in all participants. Remarkably, trends toward higher levels of sCD40L were found in the low responders for MenC, as compared with lower levels in the low responders for MenW and MenY, which needs further evaluation.

Of importance, associations with the immune phenotype were primarily found between the extremes in the vaccine response, being either low or high responders. The intermediate group showed high variability of immune markers. In addition, our results may imply meningococcal group-specific associations between the prevaccination immune phenotype and vaccine responsiveness. Noteworthy, the difference in functional antibody titers between the low and high vaccine responders was largest for MenC, possibly explaining the higher numbers of immune parameters found associated with the vaccine response for this meningococcal group. Moreover, the participants that were classified as low or high responder did not completely overlap between the different meningococcal groups. This might be explained by the structural differences in the meningococcal polysaccharides by which different meningococcal epitopes will induce distinct immune responses (61), also shown previously for several pneumococcal conjugated polysaccharides (62). Possible structural differences will affect the B cell processing and subsequently the quality and quantity of the T cell help provided by the carrier, that might cause differences in antibody functionality to the various polysaccharides. Currently, differences between meningococcal group-specific polysaccharides conjugated to TT are not known (63). Furthermore despite similar prevaccination functional antibody titers in the low and high vaccine responders, differences in numbers of meningococcal group-specific memory B cells in the bone marrow could have been present due to historical contacts (64) and affect the vaccine response. Nevertheless, the finding of meningococcal group-specific associations between vaccine responsiveness and immune phenotype is remarkable and requires further research. Of note, we previously found that most participants possessed high prevaccination TT-specific antibody levels (4). Importantly, no direct correlation between the antibody responses to TT or the different polysaccharides and the strength or classification of the T cell response induced by the TT-carrier protein was observed (5). In this study, the similar prevaccination TT-specific antibody levels in the low and high responders, suggests that the prevaccination immunity against the TT carrier protein did not largely affect the immune responses.

An important strength of this study is the ability to compare multiple antigens and multiple immune parameters within the same group of participants. Also, the primary nature of the vaccination allowed us to explore the use of biomarkers, without the strong interference of prevaccination meningococcal immunity, as often seen in other studies. Unfortunately, the presence of prevaccination immunity in some participants did interfere with the long-term functional antibody titers (after 1 year). Consequently, we were not able to investigate the associations between prevaccination immune phenotype and the long-term vaccine responsiveness, since exclusion of participants with detectable prevaccination functional antibody titers dramatically reduced the power of the statistical analysis. In addition, information on the genetic background of the participants could have added to the predictive factors in our analysis, since several studies found associations between genetic signatures and vaccine responsiveness (25, 31, 32).

Future studies, analyzing large cohorts, using different vaccines, and using similar biomarker analyzing techniques are warranted to validate the use of the suggested biomarkers. Hence, systems vaccinology, combining data on genetic background, and environmental factors such as diet, stress, and infections, and even microbiome composition is a promising tool to discover these predictive biomarkers (65). Moreover, future research should compare the suitability of biomarkers in cohorts of different ages, in order to determine the predictive values of these markers over the entire lifespan.

In conclusion, our explorative biomarker analysis suggests several associations between the prevaccination immune phenotype and vaccine responsiveness after primary meningococcal vaccination in middle-aged adults. In general, an altered CD4 T cell signature, involving high absolute numbers of naïve Treg, memory Treg, naïve CD4 T cells, and CD45RA⁺CD25^dim T cells might be used as a predictive immune phenotype for low vaccine responsiveness in middle-aged adults. Accordingly, these findings support the development of vaccination strategies to enhance the memory immunity before reaching old age, in the rapidly aging population.

Written informed consent was obtained from all participants prior to enrollment and all procedures were in accordance with the Declaration of Helsinki. The medical ethical committee: Medical Research Ethics Committees United (MEC-U) approved the study and the study was registered at the Dutch trial register (Protocol no. NTR4636).

MH, GB, MZ, A-MB, and AB conceptualized the study. MH planned and performed the clinical work and executed the laboratory experiments. MH and SF performed the statistical analysis. MH, GB, MZ, MD, SF, A-MB, and AB interpreted the data and wrote the manuscript. All authors critically revised the manuscript.

MH, GB, SF, MZ, and A-MB declare no conflict of interest. AB is a consultant for Grunenthal Gmbh (Germany).