Hepatitis C virus and alanine aminotransferase kinetics following B-lymphocyte depletion with rituximab: evidence for a significant role of humoral immunity in the control of viremia in chronic HCV liver disease

The mechanisms involved in the clearance from plasma of approximately 10¹² copies of hepatitis C virus (HCV) produced daily¹  are unclear. The use of the anti-CD20 monoclonal antibody rituximab, which reversibly depletes B cells, in HCV-related mixed cryoglobulinemia²  afforded an opportunity to study the potential role of humoral immunity.

We evaluated chronologic changes in plasma HCV RNA, and alanine aminotransferase (ALT) levels, in a 40-year-old male with chronic genotype 1 HCV-related cryoglobulinemia after 2 courses of rituximab (375 mg/m²  per week for 4 weeks, 70 weeks apart). Antiviral therapy, including pegylated interferon, reduced baseline viral load (VL) from 122 000 IU/mL to less than 7000 IU/mL. Therapy was maintained throughout. VL increased from less than 7000 IU/mL to 252 893 IU/mL within 2 weeks of starting rituximab infusions. It continued to rise progressively to 379 000 IU/mL by week 23 (Figure 1). B-cell markers, including CD20, CD19, CD21, IgM, IgD, IgG, and CD72,³  were absent from peripheral blood up to week 13. Thereafter, B-cell frequency increased to 8% by week 32. Although there is evidence for HCV replication in B cells,⁴  the continued rise in VL after B-cell depletion argues against this as a source of HCV RNA increase.

Following reappearance of B cells, VL decreased starting from week 23 to less than 615 IU/mL by week 63. A second 4-week course of rituximab infusions at week 70 similarly resulted in a prompt, more than 2-log rise in VL to 202 000 IU/mL (Figure 1).

On both occasions, rituximab increased ALT levels transiently. This suggests increased de novo infection⁵  and that B cells may play a protective role, thus accounting for the more rapid disease progression in immunodeficiency disorders.⁶  A second ALT level flare at week 27 coincided with the reappearance of B cells and HCV RNA decline. We speculate that this might indicate antibody-dependent cellular cytotoxicity.

Titers of anti-E1/E2 HCV neutralizing antibodies (nAbs) were measured in plasma using pseudotype viruses expressing heterologous HCV envelope glycoproteins.⁷  Levels were unaffected by B-cell depletion. As others have noted,⁸  plasma IgM fell by more than 50% and continues to be depressed; IgG and IgA remain stable. Stimulated B cells account for most of the IgM circulating in plasma. Furthermore, circulating HCV in chronic infection is predominantly bound to IgM.⁹  In this patient on interferon treatment, the rapid rituximab-induced increase in VL could reflect loss of nonneutralizing IgM antibodies secreted by stimulated naive or memory B cells. Possible mechanisms for HCV clearance might include virus opsonization and complement activation.¹⁰ 

HVR1 sequences were cloned from polymerase chain reaction (PCR) products at various times between weeks 0 and 32. The serine in position 408, present at low frequencies for the first 4 weeks, increased to 55% at week 4 and to 100% at week 6, decreasing slightly thereafter. Reduced HVR1 sequence diversity with depletion of B cells suggests that humoral immunity can exert immune selective pressure on HCV envelope.

These preliminary data are consistent with a significant effect of B cells on the control of plasma hepatitis C viremia.