Mutations in hepatitis B virus surface antigen during therapy with nucleic acid polymer REP 2139-Ca have no influence on treatment outcome or its detection by diagnostic assays

Introduction:

Completed and ongoing clinical trials in hepatitis B virus (HBV) mono-infection and chronic HBV/hepatitis delta (HDV) co-infection have demonstrated that therapy with the lead clinical nucleic acid polymer (NAP) candidate, REP 2139, results in multilog reduction or loss of HBsAg in the blood associated with unmasking of anti-HBs and multilog reductions in HBV DNA and HDV RNA.

Objectives:

We have investigated the large, medium and small HBV envelope proteins to find possible differences between responders and non-responders to therapy with NAPs which may influence their interaction with the drug. In addition, we wanted to exclude that the low or negative values of HBsAg observed in the patients are based on mutations in the “a” determinant which may not be detected by standard diagnostic assays.

Materials & methods:

We analysed three samples of each 12 patients (9 responders and 3 non-responders) under therapy with REP 2139-Ca. Samples were obtained prior to or at the beginning of treatment, during the treatment and as close as possible to the end of the treatment with an HBV viral load sufficient for sequencing analysis. After extraction of HBV DNA, single or semi-nested PCR of PreS1, PreS2 and S region was performed. PCR products were purified and sequenced by Supremerun direct sequencing. Sequences were edited with CLC Sequence Viewer and analysed with Geneious.

Results:

We did not observe any discernible differences between pre-S1, pre-S2 and S sequences in responders and non-responders or mutations that appeared exclusively in non-responders. No mutations emerge within the “a” determinant during the treatment. Three variants we observed within “a” determinant, I126S, P127T and A128V, are present in initial samples and persist during the treatment. P120T, the most relevant mutation outside of “a” determinant observed in this study, emerges at the third time point in one patient.

Conclusion:

There is no indication of structural or functional changes in HBV envelope proteins in non-responders that may explain lack of response. We have found no escape mutations that may affect standard diagnostic assays. Observed HBsAg mutations can be recognized by a spectrum of commercial used HBsAg assays, including the HBsAg assay used for evaluation of patients' samples in REP 102 study.