CMV IgG is Associated with Physical Function but not Muscle Density in People with HIV

Abstract

Background:

Cytomegalovirus (CMV) seropositivity is associated with poor outcomes, including physical function impairment, in people without HIV. We examined associations between CMV IgG titer and physical function in virologically suppressed people with HIV (PWH).

Methods:

REPRIEVE is a double-blind randomized trial evaluating pitavastatin for primary prevention of atherosclerotic cardiovascular disease in PWH. This analysis focused on participants enrolled in a sub-study with additional biomarker testing, imaging [coronary CT angiography], and physical function measures at entry. CMV IgG was measured using quantitative enzyme immunoassay, physical function by Short Physical Performance Battery (SPPB), and muscle density and area by CT. Associations between CMV IgG (risk factor) and outcomes were evaluated using the partial Spearman correlation and linear and log-binomial regression.

Results:

Among 717 participants, 82% male, the median CMV IgG was 2716 (Q1, Q3: 807, 6672) IU/mL, all above the limit of quantification. Among 631 participants with imaging, there was no association between CMV IgG and CT-based muscle density or area, controlling for age (r=−0.03 and r=−0.01, respectively; p≥0.38). Among 161 participants with physical function data, higher CMV IgG was associated with poorer overall modified SPPB score (p=0.02), adjusted for age, nadir CD4 and high-sensitivity C-reactive protein (hsCRP).

Conclusions:

Higher CMV IgG titer was associated with poorer physical function, not explained by prior immune comprise, inflammation, or muscle density or area. Further mechanistic studies are needed to understand this association and whether CMV-specific therapy can impact physical function in PWH.

Introduction

In the current age of combination antiretroviral therapy (ART), the life expectancy of people with human immunodeficiency virus (PWH) is now nearing that of the general population(1, 2). Despite the increasing lifespan, PWH are more likely to experience declines in health span, or the years of life free of comorbidity and disability, including a faster decline in physical function. Similarly, PWH may experience an earlier onset of frailty, or vulnerability to stressors. These impairments place older PWH at increased risk for falls, hospitalizations, morbidity and mortality (3–5).

Physical function impairment and frailty among PWH have been associated with decreased muscle mass and muscle quality (6–9), and greater inflammation and immune activation (10–13). One proposed reason for increased inflammation and activation in PWH is asymptomatic cytomegalovirus (CMV) infection (14–16). PWH are nearly universally co-infected with CMV (17, 18). Mucosal CMV DNA shedding is frequent in otherwise healthy PWH even they have preserved immune system (19). After initial infection, CMV establishes latency, and may reactivate in the setting of immunosuppression (such as a low CD4) or secondary to stress and inflammation. Active and asymptomatic CMV infection can lead to immune activation (20–24), and can induce sustained systemic inflammatory responses (25). Asymptomatic CMV infection has been linked to frailty (26, 27), atherosclerosis (28, 29), cognitive decline (30), and all-cause mortality (31) in older adults without HIV. Among PWH, asymptomatic CMV infection has been linked to poorer neurocognitive performance (32, 33) and increased carotid intima thickness (34). Some studies in PWH have found CMV associated with physical function and frailty but were limited in sample size (n= 22–30) (26, 35). Based on these results from smaller studies, our goal was to confirm these findings in a larger relatively healthy, racially, and geographically diverse population of PWH on ART.

Our aim was to investigate the relationship of CMV IgG with physical function and frailty in PWH on ART, leveraging the robust baseline data of the well-characterized and phenotyped REPRIEVE study population (36). We sought to understand whether associations between CMV IgG titer and physical function or frailty were explained by the nadir CD4 or inflammatory biomarkers, accounting for age as a known confounder of physical function impairment, frailty, and CMV immune responses. We also sought to evaluate the associations with muscle quality (density) and quantity (area) and examine whether they could be mediating the association between CMV IgG and physical function.

Methods

Study Participants

PWH, aged 40 to 75 years, receiving stable ART, and with low-to-moderate traditional cardiovascular disease (CVD) risk were recruited into REPRIEVE. Exclusion criteria included known atherosclerotic cardiovascular disease (ASCVD), diabetes if low density lipoprotein (LDL) cholesterol ≥70 mg/dL, glomerular filtration rate (eGFR) less than 60 mL/min/1.73 m², decompensated cirrhosis, active cancer, and ongoing statin use. Participants were randomized to oral pitavastatin calcium 4 mg daily versus matching placebo (36). Of the 3787 REPRIEVE participants enrolled at U.S. sites, 805 were also enrolled in a mechanistic substudy which entailed coronary computed tomography angiogram (CCTA) and blood sampling at baseline and after two years. Substudy exclusion criteria included contrast allergy, arrhythmia precluding CCTA, contraindication to β-blockers for heart rate control during CCTA, and body mass index (BMI) of 40 kg/m² or greater(37). The study was approved by the Mass General Brigham Human Research Committee, and each clinical research site obtained institutional review board/ethics committee approval and any other applicable regulatory entity approvals. Participants were provided with study information, including discussion of risks and benefits and signed the approved declaration of informed consent.

Clinical Data and Physical Function Assessments

Demographic and clinical data were obtained at enrollment. CCTA and assessments of inflammation and immune activation have been previously described (38). All participants completed a baseline self-report of physical function using the Duke Activity Status Index (DASI) (39); we defined impairment as less than maximum score of 58.2 (40). A subset of participants from the mechanistic substudy were enrolled in the PREPARE substudy and underwent physical function and frailty assessments, as previously described (41). Trained personnel at each study site measured physical function by the Short Physical Performance Battery (SPPB) and a modified Short Physical Performance Battery (mSPPB). Physical function evaluations were conducted utilizing the short performance physical battery (SPPB) (42), and the modified SPPB (mSPPB) (43). The SPPB includes 1) balance test with the ability to hold 3 different positions with increased difficulty for 10 seconds (side by side, semitandem, tandem); 2) a 4- m walk at usual pace, measured twice and 3) 5 repeated chair stands. The SPPB score is calculated from 0 (worst) to best (12) and classified as impairment according to standard cutoff (score ≤10). The mSPPB score expands the testing by increasing balance to 30 seconds and adding a 1-leg test, and increasing the chair rises to 10, providing a greater ability to detect impairments in higher-functioning individuals. mSPPB scores range from 0 to 3 (maximal performance), and are calculated as a sum of the 3 components, each divided by the maximal possible performance to derive a ratio between 0 to 1: 1) chair rise rate (stands per second), based on 10 chair stands divided by maximal performance of 1 stand per second; 2) proportion of total standing balance time, calculated as the total time each of the semitandem, tandem, and 1-leg stand positions were held (maximum 30 seconds each), divided by 90 seconds; and 3) gait speed (meters per second), based on an average of the two 4-m walk results; divided by maximal performance (2 m/s). Using the Fried Frailty Phenotype (FFP), participants were classified as frail if they met ≥3 criteria of impaired gait speed, grip strength, exhaustion, low physical activity, and weight loss; pre-frail if they met 1–2 criteria, and non-frail if they did not meet any criteria (48).

CT-Based Muscle Assessment

The mechanistic substudy included a non-contrast CT scan to assess muscle area and attenuation, limited to the heart and lower chest. The lower thoracic paraspinal muscles were identified as the most reliably included muscle group on the CT images (49). A single axial CT slice was identified at the vertebral body interspace closest to the level of the tip of the sternal xyphoid process. The contour of the paraspinal muscles was manually traced using 3D Slicer (50). Lean paraspinal muscle was defined by voxels with an attenuation of 31 to 100 Hounsfield units (HU); intramuscular fat was defined by voxels with an attenuation of −190 to 30 Hounsfield units. Muscle density was calculated as the mean of the attenuation values within all the paraspinal muscle voxels. Cross-sectional muscle area was calculated by dividing the volume of the voxels by the slice thickness of the image. Height-adjusted muscle area was calculated as the muscle area in square centimeters (cm)² divided by the participant height in meters.

Biomarker Measurements

CMV IgG titer (IU/ml) was measured at baseline on the mechanistic substudy participants, using the CMV IgG enzyme immunoassay (EIA) by Genway Biotech (GWB-892399) in the Burdo Laboratory (51). The coefficient of variation for CMV IgG titer was <20% (any quantity >20% was repeated). The ELISA procedure was carried out according to the manufacturer’s instructions and all samples were run in duplicate. The lower limit of quantification of the assay was 1.2 IU/ml. Serum high-sensitivity C-reactive protein (hs-CRP) was performed at Quest Diagnostics.

Statistical Analysis

Partial Spearman correlation was used to examine associations between CMV IgG and physical function, frailty, and muscle density and area, controlling for age associated with CMV IgG as well as with physical function and muscle measures. Regression analyses evaluated associations between CMV IgG (as risk factor) and physical function (as outcome) further adjusting for CD4 nadir (prior immune compromise) and hsCRP (inflammation). Of the biomarkers previously reported associated with CMV IgG in this study population (44), hsCRP was chosen due to observed evidence of association with physical function. Results are presented on forest plots of estimates (relative risk for binary outcomes, interpreted as prevalence in this cross-sectional analysis; difference in continuous outcomes) and two-sided 95% CIs. Parameter estimates are shown per 1 log₁₀ shift in CMV IgG. Inference was guided by two-sided tests at 5% significance level and consistency of trends across models. The analysis was conducted using SAS software (Version 9.4 for Linux. Copyright © 2016 SAS Institute Inc., Cary, NC, USA).

Results

Study population

A total of 717 participants (89% of REPRIEVE substudy) had baseline CMV IgG titer data available at study entry. Of those, 161 participants had data on SPPB and mSPPB, and 159 participants on frailty. Baseline CT muscle measurements were available for 631 participants (Figure 1).

Figure 1: Study Population ¹.

Includes participants with both SPPB and CT data available as well as those with SPPB or CT only, respectively, SPPB was conducted in the PREPARE substudy. CT in the Mechanistic substudy of REPRIEVE.² See Erlandson et al JAIDS 2023 for reasons CT muscle data not available.³ Of the 717 participants with CMV IgG results, 61 participants did not have SPPB or CT data available.

The median age was 51 years, 82% were male and 37% identified as black (Table 1), and the participants generally reflected the larger REPRIEVE US participants (data not shown). There were no substantive differences in the distributions of baseline characteristics in the subsets of those with SPPB and of those with CT muscle data.

Table:

Baseline Characteristics

		CMV analysis population (N=1717)	Subset with CT muscle data (N=631)	Subset with SPPB data (N=161)
Age (years)	Median (Q1, Q3)	51 (46, 55)	51 (46, 55)	51 (46, 55)
	Min, Max	40, 71	40, 71	40, 71
Natal sex	Male	586 (82%)	520 (82%)	138 (86%)
	Female	131 (18%)	111 (18%)	23 (14%)
Race	White	369 (51%)	327 (52%)	86 (53%)
Black or African American		267 (37%)	228 (36%)	61 (38%)
	Asian	10 (1%)	9 (1%)	1 (1%)
	Other	71 (10%)	67 (11%)	13 (8%)
Ethnicity	Hispanic or Latino	178 (25%)	165 (26%)	32 (20%)
	Not Hispanic or Latino	528 (74%)	456 (72%)	128 (80%)
	Unknown	11 (2%)	10 (2%)	1 (1%)
BMI (kg/m2)	Median (Q1, Q3)	27.1 (24.4, 30.4)	27.1 (24.4, 30.3)	27.1 (24.5, 30.8)
Smoking status	Current	173 (24%)	149 (24%)	46 (29%)
Substance use1	Current	16 (2%)	14 (2%)	3 (2%)
History of AIDS-defining event		144 (20%)	120 (19%)	41 (25%)
Nadir CD4 count (cells/mm3)	<200	358 (52%)	312 (51%)	78 (50%)
	200+	333 (48%)	295 (49%)	77 (50%)
CD4 count (cells/mm3)	Median (Q1, Q3)	607 (436, 784)	606 (426, 783)	609 (424, 789)
HIV-1 RNA (copies/mL)	<50	658 (93%)	575 (93%)	147 (92%)
HsCRP (mg/L)	Median (Q1, Q3)	1.8 (0.9, 3.8)	1.8 (0.9, 3.7)	1.5 (0.9, 3.5)
Hypertension2		245 (34%)	209 (33%)	58 (36%)
History of diabetes		2 (0%)	2 (0%)	0 (0%)
History of depression treatment3		325 (45%)	279 (44%)	81 (50%)
Overall DASI score	Impairment	235 (33%)	209 (33%)	56 (35%)
Composite SPPB Score	Impairment	-	-	49 (30%)
Frailty	Non-frail	-	-	90 (57%)
	Pre-frail	-	-	62 (39%)
	Frail	-	-	7 (4%)
Composite mSPPB4	Median (Q1, Q3)	-	-	1.89 (1.74, 2.04)
Chair rise component of mSPPB4	Median (Q1, Q3)	-	-	0.41 (0.35, 0.49)
Balance component of mSPPB4	Median (Q1, Q3)	-	-	1.00 (1.00, 1.00)
Gait speed component of mSPPB4	Median (Q1, Q3)	-	-	0.50 (0.42, 0.59)
Grip strength (kg)
Among Males (N=137)	Median (Q1, Q3)	-	-	37.3 (31.3, 44.0)
Among Females (N=23)	Median (Q1, Q3)	-	-	25.3 (21.3, 31.3)

Frequency (%) for categorical measures, median with lower and upper quartiles (Q1, Q3) for continuous measures. For age, minimum and maximum are also shown. All statistics are calculated out of participants with data collected. Missing data include: Smoking status (n=2), Substance use (n=3), Nadir CD4 count (n=26), HIV-1 RNA (n=11), HsCRP (n=1), History of depression treatment (n=2), Overall DASI score (n=1); and within the subset with SPPB data: Frailty (n=2), Grip strength (n=1).

¹Substance use includes use of cocaine, methamphetamine, and intravenous drugs.

²Hypertension is defined as any of the following: hypertension diagnosis, use of antihypertensive treatment for elevated blood pressure, systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg.

³History of depression treatment is defined as ever being treated for depression with medications per self-report.

⁴Composite mSPPB is on the scale from 0 (worst) to 3 (best performance); the individual components of chair rise, balance and gait speed are from 0 (worst) to 1 (best performance).

CMV IgG distribution

The median CMV IgG was 2,716 (Q1, Q3: 807, 6672) IU/mL; all results were at or above the lower limit of quantification of the assay. There was a modest trend for higher CMV IgG titers with advancing age (p=0.003; Figure 2a), with no apparent difference by known factors associated with physical function, muscle density and area including natal sex, race and BMI (p=0.59, 0.21 and 0.71, controlling for age; Figure 2b–d).

Figure 2: CMV IgG Distribution.

Violin plots showing Kernel estimate for probability density function, and mean (circle), median (white dash), Q1-Q3 (box), 5th and 95th percentiles (whiskers), minimum and maximum (black dashes). For visual purposes, CMV IgG is plotted on the log10 scale. P-values are from tests for Spearman correlation (for age), for partial Spearman correlation controlling for age (for BMI) and from Wilcoxon test stratified by age (for natal sex and race).

Associations between CMV IgG and Physical Function and Frailty

Overall, 33% had physical function impairments by DASI, and 30% by SPPB, among the subset of 161 participants with SPPB data (Table 1). For the physical function outcomes on the continuous scale, higher CMV IgG was associated with worse mSPPB performance (i.e., lower score; Spearman correlation coefficient r=−0.15, p=0.05) that was mostly driven by the chair rise (r=−0.22; p=0.005) and gait speed components (r=−0.18, p=0.026) components. No associations were apparent with the balance component of mSPPB (r=0.07; p=0.39) or with grip strength (r=−0.19, p=0.28). Similar trends were observed for categorical outcomes (Figure S1). Higher CMV IgG titer was associated with physical function impairment on the SPPB (score ≤10; p=0.08) and with pre-frailty and frailty (p=0.10), with no clear trend for DASI (p=0.37).

The observed associations between CMV IgG titer and mSPPB, gait speed, and chair rise and SPPB, controlling for age, were robust to further adjustments for CD4 nadir and hsCRP on the SPPB (p=0.08), composite mSPPB (p=0.02), chair rise (p=0.01), and gait speed (p=0.002), Figure 3.

Figure 3: Association between CMV IgG and Physical Function and Frailty.

Estimates on panel (a) are from log-binomial regression models with physical function as outcome (binary) and CMV IgG as a covariate, estimates of panel (b) are from linear regression models with physical function as outcome (continuous) and CMV IgG as a covariate. Three models are shown throughout: CMV as the only covariate (Unadjusted), adjusted for age (Adjusted 1), and adjusted for age, nadir CD4 and hs-CRP (Adjusted 2), respectively. Estimates are shown for CMV IgG only, per 1 log10 shift in CMV IgG. Impairment is defined as DASI score <58.2 (maximum), and as SPPB score ≤10, respectively. Composite mSPPB is on the scale from 0 (worst) to 3 (best performance); the individual components of chair rise, balance and gait speed are from 0 (worst) to 1 (best performance).

Association between CMV IgG and Muscle Quality

Among the 631 participants with both CMV IgG titer and muscle imaging available, no associations were observed between CMV IgG and paraspinal muscle density (r=−0.03; p=0.38) or muscle area adjusted for height (r=−0.01; p=0.73; Figure S2) (52). The results were similar in the subset of 136 participants with both muscle and physical function measurements (data not shown). As such, there was no ground for mediation of CMV IgG effect through muscle density or area. Indeed, the observed associations between CMV IgG and physical function described above remained unchanged after further adjusting for muscle density and area (data not shown).

Discussion

In this cohort of PWH, we leveraged the mechanistic substudy embedded in the global REPRIEVE trial to evaluate the association between CMV IgG titer and physical function or frailty in a larger study population than has been previously evaluated among PWH (26, 35, 53). Higher CMV IgG titer was associated with frailty and worse physical function, driven mostly by chair rise and gait speed components of the mSPPB. These findings were not explained by nadir CD4 or inflammation. Further, there was no evidence of association between CMV IgG and muscle density or area, and therefore no ground for their mediating the association between CMV IgG and physical function in this cross-sectional analysis.

During primary infection and reactivation, CMV generates a robust inflammatory response. Increased frequency of CMV reactivation thus may contribute to higher cumulative inflammatory burden (54) that may explain the association between prior CMV infection and a variety of comorbidities driven in part by inflammation, including atherosclerosis, vascular dementia (55–57); and frailty (58). CMV IgG titer may represent a marker of cumulative frequency of viral reactivation or antiviral inflammatory response, or both, and CMV titers tend to be higher in older adults (59). CMV seropositivity has been associated with a greater incidence of frailty and mortality among women without HIV aged 70–79 years (27). Similarly, CMV IgG titers in the highest quartile have been associated with higher risk of frailty and a higher risk of mortality in the general population of older adults (26, 27). Thomasini et al, reported both frequency of CMV reactivation as well as CMV viral load levels (as measured by CMV quantitative real-time polymerase chain reaction) to be higher in older women compared with younger women (58). In a study of PWH on ART and people without HIV, CMV-specific T-cell responses strongly influenced chronic immune activation and the magnitude of CD4-IL2 response predicted subsequent frailty in non-frail men (26). In another study, the percentage of CMV specific T-cells was predictive of the onset and maintenance of frailty in men with and without HIV (53).

Among PWH, the prevalence of CMV seropositivity is higher than in the general population, and both CMV IgG and the percentage of CMV-specific T-cells are higher among PWH compared to people without HIV (60, 61) (35). Similar to the general population, CMV IgG titers have been associated with 5-fold greater odds of physical function impairment in PWH in a small-case control study (n=78) (35). Interestingly, the relationship between high CMV IgG titer and impaired physical function was CMV-specific, and not a generalized response to other herpes viruses (herpes simplex virus or varicella zoster virus) that would similarly be expected to reactivate.

Whether CMV contributes directly or indirectly to physical function impairment or frailty remains unclear. We hypothesized that the association between CMV IgG titer and physical function impairment or frailty was explained either by heightened inflammation or prior immune compromise. Chronic stimulation of immune cells by CMV is thought to be a driving force for inflammation, since it evokes a release of pro-inflammatory cytokines such as tumor necrotic factor-alpha (TNF-alpha) that in turn induces asymptomatic CMV reactivation and further upregulation of the inflammatory response (56). In our prior study of PWH, CMV IgG titers were associated with heightened markers of inflammation, and the association of CMV IgG titer with physical function was attenuated and no longer significant when adjusting for interleukin 6 (IL-6) or CD4/CD8 ratio(35). In this study population, the associations between CMV IgG titer and physical function were not attenuated when adjusting for hsCRP and CD4 nadir, suggesting an independent pathway.

We also found no evidence that the association between CMV IgG titer and physical function was mediated by effects of CMV on muscle density or area. To the best of our knowledge, only one study has reported on an association between CMV seropositivity or CMV IgG titer with measures of muscle, as a potential mechanism for the observed associations with frailty and physical function: among older adults (>70 years) without HIV, CMV seropositivity was associated with smaller neck muscle cross-sectional area, after adjusting for IL-6 (62). It is possible that our results may have differed had we imaged different muscle groups. For example, Anderson et al, have shown differences in muscle density across muscle groups. In a cross-sectional analysis, the psoas major muscle had the highest overall muscle density (indicating lowest fat accumulation), whereas the latissimus dorsi had lowest density (indicating highest fat accumulation)(63).

The major strengths of this study include a large, virally suppressed cohort of PWH with a variety of physical function assessments, measures of inflammation and CT-measured muscle density and area, representing racially and ethnically diverse populations. Limitations include the cross-sectional nature and limited number of women enrolled in our study, highlighting the need for inclusion of women in future clinical trials. CMV IgG titer was used as a surrogate for asymptomatic infection but may not be the ideal surrogate marker. While the relatively young age in our cohort may limit generalizability to older populations, this study provided the opportunity to identify potential mechanisms and therapeutic targets in a younger population more amenable to interventions to slow physical function declines. Lastly, the associations identified in this cross-sectional analysis may not reflect causation or directionality, and substantive variability in CMV IgG was observed across all levels of physical function.

In summary, in a large national cohort of PWH, we observed an association between higher CMV IgG titer and impaired physical function and frailty, unexplained by muscle density or mass, nadir CD4 or inflammation. Further studies are needed to investigate the longitudinal relationship between CMV IgG titer with objective assessments of physical function and frailty. Other assays besides CMV IgG titer are needed to better understand the contribution of CMV to physical function and frailty. These assays include quantitative measurement of CMV replication with CMV Quantitative PCR, and CMV Cell Mediated Immune Assays e.g. CMV EliSPOT, CMV lymphocyte proliferation assay which measure CMV cell-mediated immunity reconstitution which helps in controlling CMV viremia. If these relationships persist, interventions designed to prevent or treat asymptomatic CMV infection could improve physical function outcomes in PWH as well as in the general population.