Plasma proteome perturbation for CMV DNAemia in kidney transplantation

Abstract

Background: Cytomegalovirus (CMV) infection, either de novo or as reactivation after allotransplantation and chronic immunosuppression, is recognized to cause detrimental alloimmune effects, inclusive of higher susceptibility to graft rejection and substantive impact on chronic graft injury and reduced transplant survival. To obtain further insights into the evolution and pathogenesis of CMV infection in an immunocompromised host we evaluated changes in the circulating host proteome serially, before and after transplantation, and during and after CMV DNA replication (DNAemia), as measured by quantitative polymerase chain reaction (QPCR).

Methods: LC-MS-based proteomics was conducted on 168 serially banked plasma samples, from 62 propensity score-matched kidney transplant recipients. Patients were stratified by CMV replication status into 31 with CMV DNAemia and 31 without CMV DNAemia. Patients had blood samples drawn at protocol times of 3- and 12-months post-transplant. Additionally, blood samples were also drawn before and 1 week and 1 month after detection of CMV DNAemia. Plasma proteins were analyzed using an LCMS 8060 triple quadrupole mass spectrometer. Further, public transcriptomic data on time matched PBMCs samples from the same patients was utilized to evaluate integrative pathways. Data analysis was conducted using R and Limma.

Results: Samples were segregated based on their proteomic profiles with respect to their CMV Dnaemia status. A subset of 17 plasma proteins was observed to predict the onset of CMV at 3 months post-transplant enriching platelet degranulation (FDR, 4.83E-06), acute inflammatory response (FDR, 0.0018), blood coagulation (FDR, 0.0018) pathways. An increase in many immune complex proteins were observed at CMV infection. Prior to DNAemia the plasma proteome showed changes in the anti-inflammatory adipokine vaspin (SERPINA12), copper binding protein ceruloplasmin (CP), complement activation (FDR = 0.03), and proteins enriched in the humoral (FDR = 0.01) and innate immune responses (FDR = 0.01).

Conclusion: Plasma proteomic and transcriptional perturbations impacting humoral and innate immune pathways are observed during CMV infection and provide biomarkers for CMV disease prediction and resolution. Further studies to understand the clinical impact of these pathways can help in the formulation of different types and duration of anti-viral therapies for the management of CMV infection in the immunocompromised host.

Fig 1. Study schematics.

Proteomics analysis of plasma samples collected from kidney transplant recipients to identify CMV-associated proteins in kidney transplantation.

Fig 2. CMV DNAemia specific protein signatures exist pre-CMV DNAemia timepoint and post-CMV.

[A] Plasma protein profiles for pre- and post-CMV DNAemia samples CMV DNAemia positive and negative cohort were analyzed using linear discriminant analysis (LDA). LDA was able to separate samples based on CMV status among CMV DNAemia positive and CMV DNAemia negative cohort. Within CMV DNAemia-positive cohort the samples were separated based on post-CMV infection time. Among samples collected from CMV DNAemia-positive individuals, samples collected 1-week and 1-month post-infection were interspersed suggesting the signal of CMV infection persists until 1-month post-infection. [B] Plasma samples were collected at pre-CMV infection time from CMV DNAemia positive and matching CMV DNAemia negative cohort. The analysis resulted in a set of 17 proteins whose levels were either increased (n = 6) or decreased (n = 11). The significance of the changes is demonstrated by a Volcano plot. The most significantly increased protein was Lysine Methyltransferase 2C (KMT2C) with 3.38 fold increase (p = 0.05) in CMV DNAemia positive samples and most significantly decreased protein was Immunoglobulin Lambda Variable 7–43 (IGLV7-43) with 2.17 fold decrease (p = 0.01). [C] We analyzed samples collected at 1-week and 1-month post-CMV infection from CMV DNAemia-positive cohort and compared them against proteins profiles generated from CMV DNAemia-negative cohort that included baseline and 1-year time-point. This analysis resulted in significant changes in 16 proteins, with an increase in their level with CMV infection (n = 11) and a decrease in their level with CMV DNAemia (n = 5). Changes in individual proteins are presented as a volcano plot.

Fig 3. Temporal proteome changes due to CMV DNAemia.

[A] Protein profiles of baseline samples of CMV DNAemia-positive patients were compared with protein profiles of 1-week post-infection samples which resulted in ten significantly changed proteins. [B] CMV DNAemia-induced plasma proteomic profile over time was analyzed. A summary of protein changes due to CMV DNAemia is presented for three timepoints, namely pre-CMV DNAemia, immediately post-CMV DNAemia, and at recovery or 1-yr post-CMV DNAemia.

Fig 4. CMV DNAemia-specific proteins show increase in their level in 1-week post-DNAemia.

Protein profiles immediately after CMV DNAemia were enriched with ceruloplasmin (CP), vaspin (SERPINA12), Lipopolysaccharide-binding protein (LBP), C4b-binding protein alpha chain (C4BPA), Complement Factor H (CFH), immune complex protein (IGHV3-72).

Fig 5. CMV DNAemia specific protein signatures exist pre-CMV DNAemia timepoint and post-CMV.

[A] Using the protein profiles of DNAemia positive and DNAemia negative patients, we identified a classifier that provided a sensitivity of 85% and specificity of 91% in identifying CMV Dnaemia with a ROC AUC of 0.96. This classifier also identified a subset of 37 proteins that contributed with higher significance in distinguishing CMV DNAemia positive from CMV DNAemia negative samples [B] XGBoost-Boruta algorithm was applied to detect the top essential protein features. This process reduced the 241-protein list down to 5 proteins and increased AUC by 1%. This set of proteins included HBG1, KMT2C, IGLV7-43, IGKV3D-15, and CP [C] XGBoost machine learning was used on 70% samples to identify CMV DNAemia specific protein signature of 5 proteins. When tested for its performance on 41 samples (30%) with 21 CMV DNAemia positive and 20 CMV DNAemia negative status. The validation of the panel of proteins to identify CMV DNAmeia positive status irrespective of post-CMV DNAemia time distinguished CMV positives with high accuracy of 88% and 95% specificity.