Association of HCV-related mixed cryoglobulinemia with specific mutational pattern of the HCV E2 protein and CD81 expression on peripheral B lymphocytes

Interaction of the hepatitis C virus (HCV) envelope (E) 2 protein with the cellular receptor CD81 leads to B lymphoproliferation in vitro, a major characteristic of mixed cryoglobulinemia (MC).¹  Within E2, 2 CD81 binding sites have been described in vitro comprising the hypervariable region 1(HVR1) and HVR2.^2,3  In patients with chronic hepatitis C, CD81 expression on peripheral B lymphocytes is increased.⁴  We hypothesize, that specific amino acid (aa) sequences within E2 and CD81 expression on B lymphocytes may act as determinants for the development of MC in vivo.

There were 58 consecutive patients with chronic hepatitis C tested for MC. Cryoprecipitates were detectable in 14 (24.0%) of 58 patients and immunofixation showed MC type II in all patients. MC-positive patients did not differ from those without MC regarding age, alanine aminotransferase level, baseline viremia, and estimated duration of infection.

The E2 regions including HVR1 and HVR2/CD81 binding sites were sequenced and the obtained E2 aa sequences were analyzed according to physicochemical properties.⁵  The hydrophobic pattern of the E2 regions was widely conserved and allowed no differentiation between patients with and without MC. Analyses of the solvent accessibility showed that the majority of mutations were located within the HVR1 and HVR2/CD81 binding sites at residues predicted on the surface of E2 (exposed), underlining the functional relevance of these regions (Figure 1A).³  Furthermore, we evaluated the predictability of MC based on the E2 sequences and used adjusted classification algorithms composed of statistical learning procedures, probabilistic models, and empiric likelihood ratio tests.^6,8  These classification algorithms account for mutational frequencies and dependencies between different aa positions. Classification rates (CRs) and Matthews correlation coefficients (MCCs) were calculated using a nested cross-validation procedure, and the importance of individual aa positions was expressed as differences in MCCs in comparison with random permutations at this position. There were 2 HVR1 positions (389 and 398; CR, 67%, MCC, 0.32) and 3 HVR2/CD81-binding site positions (474, 493, and 497; CR, 72%, MCC, 0.29) correlated with the presence of MC (Figure 1A). The identified positions give reason for site-directed mutagenesis studies to evaluate their influence of the E2 binding affinity to CD81.

Fluorescence-activated cell sorter (FACS) analyses showed that CD81 expression on CD19⁺ B lymphocytes, representing the vast majority of B lymphocytes, was significantly higher in patients with MC (923.7 ± 264.8) compared with those without MC (774.5 ± 147.3; P = .045, one-tailed), patients chronically infected with hepatitis B virus (665.4 ± 132.3; P = .010), and healthy controls (476.0 ± 100.5; P < .001; Figure 1B). These results underline the concept that CD81 exerts a specific role in the host interaction with HCV.⁴  Higher CD81 expression was notably found in MC-positive patients infected with HCV-1a isolates possessing high binding affinity to CD81 in vitro (P = .024, data not shown).³  B lymphoproliferation is thought to be driven by HCV-specific antigen in vitro.⁹  Accordingly, HCV core antigen levels of peripheral blood mononuclear cells (PBMCs) were higher in MC-positive patients (4.86 ± 2.41 pg/mL) compared with those without MC (3.63 ± 1.46 pg/mL; P = .047; Figure 1C).

In conclusion, classification algorithms provide information about specific E2 positions correlated with MC. CD81 expression and HCV core antigen levels in PBMCs are increased in patients with MC.