Absence of mutations in the HBsAg “a” determinant during REP 2139 therapy validates serum HBsAg reductions observed in the REP 102 protocol

Nucleic acid polymers (NAPs) clear HBsAg from the blood by blocking its release from infected hepatocytes of patients with chronic Hepatitis B. In the REP 102 protocol (NCT02646189) monotherapy with the NAP REP 2139 achieved 2 – 7 log reductions of serum HBsAg accompanied by 3 – 9 log reductions in serum HBV DNA and the appearance of anti-HBs.

HBsAg is a primary diagnostic tool for HBV infection and its detection depends on the “a” determinant region (124 – 147aa). Mutations within this region may impair detection of HBsAg and allow HBV to escape vaccine induced immunity or passive immunoglobulin therapy. Sequence analysis of the “a” determinant region during REP 2139 therapy in the REP 102 protocol was performed to explore the potential role of mutations in the HBsAg response observed during NAP therapy.

PCR products for direct and deep sequencing were prepared by single or semi nested PCR of HBsAg region of HBV DNA. Deep sequencing targeted the major hydrophilic region (including the “a” determinant) and direct sequencing targeted the whole S region of HBsAg. Direct sequencing was performed by Supremerun® and analyzed with Geneious® software. NGS analysis was performed on Illumina® data from all 12 REP 102 patients. The NGS pipeline involved read filtering (Cutadapt), assembly (BWA), variant calling (GATK) and haplotype reconstruction (QuasiRecomb) to detect variants within the samples.

Of 12 patients treated (1 gtA, 4 gtD, 7 gtC), 9 responders (with HBsAg reduction) and 3 non-responders (no HBsAg reduction) were observed. Deep and direct sequencing revealed no mutations were present in the “a” determinant region during REP 2139 therapy in all 12 patients. In the 9 responder patients, 18 different mutations were observed in responders occurring outside the 'a 'determinant region and include C76G, L98V, Q101R, Q101K, M103I, L109P, L109Q, T115N, G119R, R122K, I126S, G145R, F171S, R78Q, P153T, P120T, V184A and T189I. The G145R mutation was reported with a low frequency of 6% and disappeared as REP 2139 therapy progressed.

No mutations in the “a” determinant region were observed during REP 2139 therapy, confirming that HBsAg reductions observed are not due to the evolution of HBsAg variants undetectable by standard HBsAg assays. These studies further validate the important reductions in HBsAg observed during REP 2139 treatment which has been shown to improve the effect of immunotherapy and allow the achievement of functional control of HBV infection.