Introduction
Age-related reduced function of the immune system, often referred to as “immunosenescence”, is suggested to be influenced by cytomegalovirus (CMV) infection [
1]. Main features of CMV seropositivity include low percentages of naïve T-cells and reduced diversity in the T cell repertoire, which may impair the ability to respond to heterologous infection or vaccination [
2] and result in lower B-cell functions by lack of T-cell help [
3,
4]. CMV seropositivity has also been identified as a factor of the Immune Risk Profile (IRP) for mortality in the Swedish longevity studies [
5].
CMV prevalence increases with age in the general population from 30% in children to above 90% at the age of 80 and older [
6,
7]. Primary CMV infection and reactivation from latency can cause significant problems when the immune system is compromised or immature, but is usually asymptomatic in healthy individuals [
8]. However, CMV frequently reactivates during life [
9,
10] and can lead to detectable CMV DNA levels, mainly in the elderly [
8,
11]. Control of CMV requires continuous immune surveillance and leads to large numbers of CMV-specific T cells, up to 10–30% of CD8+ T cells in the periphery. The lifelong need to control CMV is by many thought to take its toll and to hamper immune responses to heterologous infections or vaccination [
3]. Indeed, in several mouse models the immune responses to heterologous infections was shown to be negatively affected by CMV [
12‐
14]. However, other studies suggested a positive effect of CMV on the response to heterologous infections [
12,
15].
In humans, the potential effect of CMV-infection on a heterologous immune response is mainly studied in the context of influenza vaccination. Seasonal influenza vaccination is an effective means to prevent influenza infection [
16‐
18]. However, effectiveness of influenza vaccination decreases with age, leaving older adults exposed to an increased risk of influenza infection [
1]. In older adults, influenza infection more often leads to disease-related hospitalization, complications and mortality [
17,
19‐
21]. Influenza vaccines are primarily focused on eliciting a strain-specific antibody response. Antibodies are important as they give rise to so-called sterilizing immunity; the immune status where the host immune response effectively blocks virus infection. The most widely used method to measure strain-specific influenza titers is the hemagglutination-inhibition (HI) assay, which reflects the ability of specific antibodies to bind influenza virus and inhibit viral agglutination of red blood cells [
22,
23]. European medicine agency guidelines describe the analysis and presentation of influenza antibody data for development of influenza vaccines [
24], stating as a minimum requirement that geometric mean titers (GMTs) (with 95% confidence intervals) and pre-/post-vaccination ratios (GMR), and response rates should be reported.
Clarification of the effect of CMV on influenza vaccine responses is of high importance. The current suboptimal immune response to influenza vaccination in elderly will become an increasingly large problem. By 2050, the population of older persons (defined by the United Nations as those aged 60 years and above) is expected to double in size compared to 2015. With an increasing life expectancy, the group of elderly at high-risk for influenza complications will increase quickly and contribute to the rising challenges of public health. As latent CMV infection is highly frequent in the population, it is critical to elucidate whether CMV infection influences influenza vaccination responses, to be able to optimize vaccine strategies in the population.
Several studies investigated the effect of CMV infection on immune responses induced by influenza vaccination. The first study by Trzonkowski et al. reported a negative association between CMV-infection and the response to influenza vaccination [
25]. Some studies confirmed this result [
26], but others did not find an effect of CMV infection on the influenza vaccine response [
27]. In contrast, Furman et al. reported a positive effect of CMV infection on the immune response to influenza vaccination in adults [
28]. To date, no consensus of the effect of latent CMV infection on the antibody response to influenza vaccination has been reached [
4,
29,
30].
Here, we systematically reviewed studies on the effect of CMV infection on the antibody response to influenza vaccination in healthy individuals. The process of systematic reviewing the available evidence in literature was reported in line with the PRISMA criteria (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) [
31]. We extracted three standardized outcome variables of 17 studies, in line with European Medicine Agency (EMA) [
24] and Food and Drug Administration (FDA) guidelines [
32]. Summarizing all extracted data on GMR to influenza vaccination revealed no clear difference between CMV-seropositive and CMV-seronegative individuals. In a meta-analysis, a small (but non-significant) trend was observed that CMV-seropositive participants responded less often to influenza vaccination than CMV-seronegative individuals. We show that this effect is likely explained by publication bias. In addition, we summarized reports on a possible correlation between CMV antibody titers and influenza antibody titers, which showed weak negative correlations between the two. Together, these analyses provide no unequivocal evidence that latent CMV infection affects the influenza antibody response to vaccination.
Discussion
This is the first systematic review investigating the association between latent CMV infection and the immune response to influenza vaccination. Almost two decades ago, CMV was associated with “immunosenescence” [
58]. Since then, multiple studies on CMV-induced immunosenescence have been performed. The idea that CMV decreases the ability of the immune system to respond to other pathogens or vaccination [
59,
60] is mainly based on studies investigating the influenza vaccine response [
61]. Indeed, various studies reported a negative association between latent CMV infection and influenza vaccine responses, while other studies lacked to find an effect of CMV or even reported a positive effect of CMV. Thus, consensus on the effect of CMV is lacking. Nevertheless, an effect of CMV on the influenza vaccine response in the elderly is generally assumed [
29,
61,
62]. By systematically reviewing and integrating the available studies, we here show that there is no unequivocal evidence for an impact of CMV on the influenza vaccine response.
We systematically selected studies on CMV and influenza vaccine responses and extracted three standardized influenza antibody outcome variables. The geometric mean titer ratio (GMR) pre/post-influenza vaccination with (Fig.
4a) and without (Fig.
4b) 95% CI revealed no difference between CMV-seropositive and CMV-seronegative individuals (outcome a). Of note, also when only the post-vaccination geometric mean titer (post-GMT) was summarized, no overall trend for an effect of CMV serostatus was observed (Supplementary figure 5). We primarily assessed the GMR and not the post-GMT since the participants in the studies were not all influenza seronegative before vaccination. Pre-existing immunity is usually present in the case of seasonal influenza vaccination. Thus, post-vaccination titers as outcome will overestimate the vaccine antibody response and are, therefore, less meaningful. Linear regression analysis, as performed in some studies [
42,
63], is the best method to correct for pre-vaccination titers [
46], but this could not be analyzed on the basis of the extracted data of the studies included for this review. Thus, with the GMR, the best outcome available, no effect of CMV seropositivity on the influenza vaccine response is observed.
The meta-analysis of response rate to influenza vaccination (outcome b) (Fig.
5) revealed a small (albeit non-significant) trend that CMV-seropositive participants respond less often to influenza vaccination than CMV-seronegative individuals. Funnel plot analysis suggested that publication bias most likely underlies this trend in the literature (Fig.
6).
Unfortunately, it was not possible to extract a standardized outcome for an association between CMV antibody level and the influenza antibody response to vaccination (outcome c), since the methods of the studies varied and no raw data were available. Overall, the reported correlation results (Table
4) of the studies indicated a small negative association between CMV antibody titers levels and influenza antibody levels after vaccination, suggesting that individuals who experienced multiple CMV reactivations during life may have impaired influenza vaccine responses. The tabulated correlations, however, should be interpreted with caution. CMV antibody levels increase with age and are thought to reflect experienced CMV reactivation or reinfection [
7]. Therefore, high anti-CMV antibody levels may be related to enhanced CMV-induced immunosenescence and impaired influenza vaccine responses [
42]. However, we noticed that in most studies CMV-seronegative individuals were included in the correlation of CMV antibody titers and influenza antibody titers, which may affect the correlation coefficient or the significance of the correlation. Of importance, in only one study CMV antibody levels were correlated with the fold increase in influenza antibody titers [
47]; in all other studies, it was correlated with the post-vaccination titer, thereby overestimating the vaccine response. Together, this questions the importance of the reported weak correlations between CMV antibody levels and influenza antibody titers to vaccination.
To illustrate the controversy in the literature, we also summarized the reported conclusions of various studies on the influenza antibody response (Fig.
3). Two previous reviews directly combined the various results in literature on the effect of latent CMV-infection on the antibody response to influenza vaccination [
4,
29]. Frasca et al. (2015) and Merani et al. (2017) refer to some of the studies included in our systematic review and describe the effect of CMV on influenza antibody vaccine responses as controversial or ambiguous. Despite this, both reviews come to the conclusion that CMV does affect the immune response to influenza vaccination [
4,
29]. In addition, Merani et al. discuss possible methods to reduce the impact of immunosenescence on influenza vaccine responses by anti-CMV strategies [
29]. The controversy in the literature and the difficulty to compare different influenza antibody outcomes in different studies highlight that a systematic approach is necessary.
The strength of our review lies in its systematic approach. This allowed us to synthesize all the available evidence (until 27th June 2017) on this particular question and to eliminate the effect of potential publication bias. Instead of merely summarizing the conclusions in literature, we extracted the published data in three standardized outcome variables of influenza antibody response, separated per age group. Furthermore, whenever possible, we assessed the data of each study per influenza strain. By this, we included multiple records per study and not only the record on which the authors’ conclusion was based. To the best of our knowledge, only two new articles came out that investigated the effect of latent CMV infection on the influenza antibody response since the systematic search of this review (27th June 2017). Merani et al. (sept 2017) concluded that there is no difference in influenza GMR between CMV-seronegative and CMV-seropositive individuals, while CMV-seropositive individuals do show an impaired cellular granzyme B response to influenza virus challenge [
64]. We published in Van den Berg et al. (January 2018) that there is no negative effect of CMV infection on the antibody response to a novel influenza vaccine strain in adults [
65]. Both studies will not change the conclusion of this systematic review that there is no unequivocal evidence for an effect of CMV infection on the antibody response to influenza vaccination.
In this review, the direct association between CMV infection and the influenza antibody vaccine response is investigated. Several mechanisms of a potential negative effect have been postulated, based on the known effects of CMV infection on the immune system and the subsequent potential impact on the influenza vaccine response [
47,
58]. CMV infection leads to increased pro-inflammatory cytokine levels, which in turn are associated with decreased influenza vaccine responses [
29]. Likewise, CMV infection leads to increased differentiation of T cells, which has been associated with poor influenza vaccination responses [
25,
53]. It has also been reported that CMV infection is associated with decreased switched B cell percentages before influenza vaccination, and subsequently lower influenza vaccine responses [
32]. In contrast, De Bourcy et al. (2017) suggested a potential mechanism for the positive effect of CMV on the antibody influenza vaccine response reported in Furman et al.: based on B-cell repertoire analyses, CMV infection is associated with more activated B cells after influenza vaccination.
This systematic review also has some limitations. Ideally, a systematic review is based on randomized controlled trials (RCT), but due to obvious ethical and practical reasons, no RCTs have been conducted to study the relation between CMV infection and influenza vaccine responses. Consequently, only observational studies were included. We assumed that the reported sizes of the study populations were correct for the duration of the studies, even if no statement was made on the number of participants that were lost to follow-up. Furthermore, this systematic review was limited by the number of studies that was found to be eligible for inclusion, which led to a meta-analysis of only 5 studies leading to 13 records. Another limitation is the incomplete correcting for confounders. We only adjusted for pre-vaccination antibody levels by investigating the GMR and partially adjusted for age by separating the results for young and old adults. However, age is associated with different influenza antibody responses, not only because of immunosenescence, but also due to immunologic imprinting and different influenza exposure during lifetime. Thus, merely assigning individuals to a young and old age group might not be sufficient to adjust for age as a confounder. Research on the effect of CMV on the influenza antibody vaccine response is further complicated by different study populations, and different influenza strains, as summarized previously [
29]. There is no biological basis for a differential effect of CMV on different influenza strains, but influenza vaccine responses vary a lot per season and subtype [
66]. Unfortunately, data from Reed et al. could not be incorporated in this review. They reported a negative association between CMV seropositivity and influenza antibody vaccine response in a study including different seasons and influenza strains. Their data could, however, not be extracted for analysis for one of the outcomes of this systematic review, since they were only reported as a result of a multi-factor model.
Universality in reported influenza antibody data in the CMV-immunosenescence field is necessary to reveal the potential effect of CMV on the antibody influenza vaccine response. We recommend further studies investigating the effect of CMV-infection on the influenza antibody vaccine response to follow the EMA guidelines [
24] and as an absolute minimum, to always report the influenza pre-GMT and post-GMT (with 95% CI) and the number of participants per group. It is important to take influenza strain and season into account by measuring and reporting the titers separately per influenza strain. A response rate is also of interest, but should not be the only outcome reported. The response rate can be defined in several ways [
24] and the correlate of protection of 40 is based on adults, making the use in elderly questionable [
24,
67]. In addition, a regression analysis to correct for pre-existing immunity is necessary. Especially when the effect of CMV infection on the influenza antibody response is small, correcting for confounders, like age (as continuous variable), pre-existing immunity, vaccination history, medicine use or comorbidities is highly recommended.
In conclusion, we show that based on the GMR, which in our perception is the best outcome available, there is no evidence for an effect of CMV seropositivity on the influenza antibody vaccine response, and that publication bias probably explains the trend in the literature that CMV-seropositive individuals seem to respond less often to influenza vaccines than CMV-seronegative individuals. We suspect that in the past, several studies did not reach publication because they did not fit the prevailing idea that CMV induces immunosenescence. Our systematic review emphasizes that the effect of CMV infection on a clinically relevant immune function in humans, such as influenza vaccine responses, is not as black-and-white as previously suggested. Further large studies investigating the relation between CMV antibody levels and influenza vaccine responses with enough power to detect a potential small effect of CMV infection are needed, in which also confounding factors in addition to age are taken into account. Only if there is unequivocal evidence for CMV-associated impaired influenza vaccine responses, can we begin to address whether a CMV-impaired vaccine response in the elderly is merely a sign of immunosenescence, or whether CMV is causing immunosenescence.
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