Limited effect of duration of CMV infection on adaptive immunity and frailty: insights from a 27-year-long longitudinal study

Abstract

Objectives: Cytomegalovirus infection is thought to affect the immune system and to impact general health during ageing. Higher CMV-specific antibody levels in the elderly are generally assumed to reflect experienced viral reactivation during life. Furthermore, high levels of terminally differentiated and CMV-specific T cells are hallmarks of CMV infection, which are thought to expand over time, a process also referred to as memory inflation.

Methods: We studied CMV-specific antibody levels over ~ 27 years in 268 individuals (aged 60-89 years at study endpoint), and to link duration of CMV infection to T-cell numbers, CMV-specific T-cell functions, frailty and cardiovascular disease at study endpoint.

Results: In our study, 136/268 individuals were long-term CMV seropositive and 19 seroconverted during follow-up (seroconversion rate: 0.56%/year). CMV-specific antibody levels increased slightly over time. However, we did not find an association between duration of CMV infection and CMV-specific antibody levels at study endpoint. No clear association between duration of CMV infection and the size and function of the memory T-cell pool was observed. Elevated CMV-specific antibody levels were associated with the prevalence of cardiovascular disease but not with frailty. Age at CMV seroconversion was positively associated with CMV-specific antibody levels, memory CD4⁺ T-cell numbers and frailty.

Conclusion: Cytomegalovirus-specific memory T cells develop shortly after CMV seroconversion but do not seem to further increase over time. Age-related effects other than duration of CMV infection seem to contribute to CMV-induced changes in the immune system. Although CMV-specific immunity is not evidently linked to frailty, it tends to associate with higher prevalence of cardiovascular disease.

Keywords: CMV‐specific antibodies; T‐cell response; ageing; cardiovascular disease; cytomegalovirus infection; frailty.

Figure 1

Overview of the CMV antibody levels of all participants during time. (a) Study design. Participants (n = 135 men, n = 133 women) donated blood 6 times in ~ 27 years. In 2016–2017, an extra sample was taken for extensive phenotyping of leucocyte subsets. (b) CMV antibody levels were measured every 5 years and presented per age category at study endpoint separately for men and woman. Green lines show the antibody trajectories of CMV^‐ participants, purple lines those of LT CMV⁺ participants, and the bold blue lines those of the participants that seroconverted during follow‐up (ST CMV⁺ participants). Dotted horizontal line shows the cut‐off value for seropositivity.

Figure 2

CMV‐specific antibody levels followed over time. (a) Antibody levels of all CMV⁺ individuals (n = 155). The trend line is based on local polynomial regression through the data of long‐term (LT) CMV⁺ participants (n = 136). (b) Fold increase in CMV‐specific antibody levels over 25 years for each individual. Dashed line shows no increase or decrease. (c) Individual CMV‐specific antibody levels during time of the CMV seroconverters (n = 19) (blue lines) compared to the average trend line of the long‐term CMV⁺ individuals. The first CMV⁺ time point of CMV seroconverters after CMV seroconversion (<5.5 years after CMV conversion) is highlighted by a larger blue dot. (d) Duration of CMV infection: CMV‐specific antibody levels of recently seroconverted individuals (max < 5.5 years after CMV conversion, n = 19) compared with those of long‐term CMV⁺ individuals (> 25 years CMV⁺, n = 136). (e) Age at seroconversion: Antibody levels of individuals that seroconverted at younger age (≤ 38 years of age, n = 26) or older age (≥ 45 years of age, n = 18, mean age 58.5 ± 8.1, shortly after CMV seroconversion (max < 5.5 years)). (f) Antibody levels associated with age at CMV seroconversion. For the selection of LT CMV⁺ individuals, CMV⁺ individuals of round 1 were included that were ≤ 38 years of age and age of seroconversion was set at 38 years. (g) Antibody levels of all CMV⁺ individuals at endpoint. Interval represents geometric mean level ± geometric standard deviation. (h) Variable importance when predicting CMV antibody levels at study endpoint with a random forest algorithm. % increase in MSE: proportion increase in mean squared error in the model when a single variable is randomly shuffled. Slope of Ab level: log‐linear variation in CMV‐specific antibody levels after first CMV⁺ measurement, until time point 6. Boxplots show median with interquartile range. * P < 0.05, **P < 0.01. The dotted line in a and c is the upper boundary of the cut‐off for CMV seropositivity.

Figure 3

CMV‐induced changes in the CD8⁺ T‐cell pool are established early after CMV infection. (a) Absolute numbers of CD8⁺ T‐cell subsets compared between CMV^‐ (n = 113), ST CMV⁺ (n = 19), and LT CMV⁺ individuals (n = 136). (b) Relationship of duration of CMV infection with CD8⁺ T_EMRA cells numbers. (c) CD8⁺ T_EMRA cells numbers at study endpoint in individuals that seroconverted at younger age (≤ 38 years of age, n = 26) compared to those who converted recently at an older age (≥ 45 years of age, n = 18, mean age 58.5 ± 8.1). (d–g) Numbers and percentages of CMV‐specific CD8⁺ T cells (d, e), percentage of T_EMRA cells of total CD8⁺ cells (f) and percentage of expression of KLRG‐1 (G) in HLA‐A2 positive individuals for pp65‐epitope NLVPMVATV compared between ST CMV⁺ (n = 8) and LT CMV⁺ (n = 4) individuals (g). (h, i) Percentages of IFNγ producing CD8⁺ T cells (h) and polyfunctional CD8⁺ T cells producing IFNγ, TNFα, Mip, CD107 and/or IL2 (i) after CMV‐specific peptide stimulation in ST CMV⁺ and LT CMV⁺ individuals (n = 27). Boxplots show median with interquartile range. * P < 0.05, **** P < 0.0001.

Figure 4

CMV‐induced changes in the CD4⁺ T‐cell pool are established early after CMV infection and are most pronounced in older CMV seroconverters. (a) Absolute numbers of CD4⁺ T‐cell subsets compared between CMV^‐ (n = 113), ST CMV⁺ (n = 19), and LT CMV⁺ (n = 136) individuals. (b) Relationship of duration of CMV infection with CD4⁺ T_EMRA cells numbers. Correlation in ST CMV⁺ individuals is indicated by ρ‐ and P‐values. (c) CD4⁺ T_EMRA cells numbers at study endpoint between individuals that seroconverted at younger age (≤ 38 years, n = 26) or older age (≥ 45 years, n = 18, mean age 58.5 ± 8.1). (d, e) Correlation of CD4⁺ T_EMRA cells numbers with their percentages producing granzyme B and perforin (D) and IFNy (e) after CMV peptide stimulation (n = 27). (f, g) Percentages of CD4⁺ T cells producing IFNγ (f) and being polyfunctional producing IFNγ, TNFα, Mip‐1β, CD107 and/or IL2 (G) after CMV‐specific peptide stimulation in CMV^‐, ST CMV⁺ and LT CMV⁺ individuals. Boxplots show median with interquartile range. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 5

CMV infection is not associated with frailty, but is related to prevalence of cardiovascular disease. (a) Comparison of frailty index score between CMV^‐ (n = 110), short‐term (ST) CMV⁺ (n = 18) and long‐term (LT) CMV⁺ (n = 132) participants with available frailty index score data at time point T6. (b, c) The relation of frailty with (b) the duration of CMV infection (n = 18) and (c) age of seroconversion in ST CMV⁺ individuals (seroconverted before frailty index score assessment, n = 16). (d, e) Relationships are shown, respectively, between frailty and CMV‐specific antibody levels at study endpoint (d) and between frailty and fold change in CMV‐specific antibody levels over 26 years (e). (f, g) Difference in increase in frailty index between CMV^‐ participants (n = 110) and those who seroconverted recently after measurement point T5 (n = 4) (f) and between women aged 60–65 years that were CMV^‐ (n = 11) or converted recently after T5 (n = 3) (g). Increase in frailty index: difference between frailty index as measured between T5 and T6. (h, i) Comparison of cardiovascular disease (CVD) prevalence with CMV‐specific antibody levels (h) and fold change in 25 years (i) in these antibody levels in individuals that were CMV⁺ at study endpoint (n = 165) (Prevalence of CVD is indicated in Table 2). Boxplots show median with interquartile range.

Figure 6

Model of establishment of the size of the immune response to control latent CMV infection. Both establishment of the viral latent reservoir after primary infection (1) and the subsequent viral reactivation in time (2) will affect the CMV‐specific immune response. Different factors may influence both phases; those based on our data are presented in black rectangles. The figure was created with BioRender.com.