Hepatitis B Core Antigen

As a backbone for construction of expression vectors we used plasmid pEAQselectK (GenBank: GQ497231.1), in which the backbone region is shorter than in pBINPLUS (Sainsbury et al., 2009b (link)). To make this vector suitable for subsequent cloning steps, we first removed restriction sites AscI and SmaI from its multiple cloning site. DNA of pEAQselectK was treated with AscI and SmaI, then the ends were made blunt using Klenow fragment and the DNA was circularized by self-ligation. The resulting plasmid was named pEf.
A synthetic sequence of the P24 gene was cloned in plasmid pNRGFP (Mardanova et al., 2007 (link)) instead of the gfp gene using BamHI and BsrGI restriction sites. pNRGFP is a binary vector containing 35S promoter – gfp – NosT terminator expression cassette within the T-DNA region. Thereafter, the 35S – P24 – NosT fragment was amplified by PCR using primers BlnI_35S_F and Nos-T_SnaBI_R, and cloned in the pEf vector at SnaBI and BlnI restriction sites. The resultant plasmid was named pEf-p24.
A similar expression vector, pEf-p19, containing the gene encoding the silencing suppressor P19 from TBSV was also constructed. The P19 coding sequence was amplified from pEAQspecialK-GFP-HT (Sainsbury et al., 2009b (link)) using primers P19-BHI-F and P19-SwI-R, and cloned into pEf-p24 instead of P24 using the BamHI and SwaI restriction sites.
Expression vector pA7248AMV-GFP (Mardanova et al., 2009 ) contains the 5′- UTR of the PVX genome, the gene for viral RNA polymerase (RDRP), the subgenomic promoter of the gene for PVX 25K transport protein (Sgp1), the reporter gfp gene downstream of the AMV leader sequence, and the 3′-UTR of the PVX genome. This sequence is inserted between the 35S promoter and NosT terminator within the T-DNA region of pBIN19 binary vector. Unique restriction sites AscI and SmaI located at 5′ and 3′ ends of gfp can be used for cloning of a target gene to replace gfp (Figure 1). The sequence comprising the whole expression cassette between the 35S promoter and NosT was amplified by PCR using pA7248AMV-GFP DNA as a template and primers XhoI_35S_F and Nos-T_Bst1107I_R. Cloning of this fragment at XhoI and Bst1107I restriction sites in pEf resulted in expression vector pEf-GFP. The same sequence was cloned into pEf-p24 at XhoI and Bst1107I restriction sites to make the final pEff-GFP vector. The nucleotide sequence of pEff-GFP vector has been deposited in the GenBank database under accession no KY439904. Structures of expression vectors are shown in Figure 1.
Recombinant vector pEff-M2eHBc was constructed for expression of the hybrid protein M2eHBc consisting of an extracellular domain of M2 protein (M2e) of influenza virus strain A/Duck/Potsdam/1402-6/1986 fused to the N-terminus of hepatitis B core antigen (HBc). The plant-optimized synthetic M2eHBc gene was excised from vector pA7248amvM2epHBc (Ravin et al., 2012 (link)) and cloned into the pEff vector using the AscI and SmaI restriction sites.

We performed a prospective study from December 2018 to January 2020 at the Department of Infectious Disease at Peking Union Medical College Hospital (PUMCH) clinics. We included 32 PLWHs over 18 years old. They were seronegative for HBsAg, HBsAb, antibody to hepatitis B core antigen (anti-HBc), and antibody to hepatitis C virus (anti-HCV), and without a history of HBV vaccination in previous five years. Patients were eligible to participate if they received cART and had CD4⁺ cell count >350 cells/µL and HIV viral load <200 copies/mL for the past 6 months. Exclusion criteria included being pregnant or breastfeeding; acute elevations of liver enzymes within the past three months (alanine aminotransferase (ALT) or aspartate aminotransferase (AST) two or more times the normal upper); having a history of hypersensitivity to any component of the vaccine or other immunocompromised conditions Furthermore HIV.
The enrolled individuals were given 20 ug recombinant HBV DNA vaccine at weeks 0, 4, and 24. The diagram of the participants is shown in Figure S1. Ten participants did not vaccinate for personal reasons. The main reason was that they needed to take three weeks of leave to vaccinate, which they thought would affect their work. Furthermore, they still had concerns about the safety and effectiveness of vaccines. The patients were followed-up every 12 weeks in our clinics; therefore, HBsAb was tested, and full blood was collected to separate PBMC at weeks 0, 4, and 36 in the original plan. However, some patients could not come for follow-up at week 36 due to the outbreak of the COVID-19 epidemic. Thus, they were followed-up between weeks 36 and 48 (Figure S2). Furthermore, HBsAb was tested annually in every patient, whether vaccinated or not, in order to evaluate HBV infection in our clinics. Therefore, we could observe the dynamics of HBsAb titer in longer follow-up duration for enrolled participants. Enzyme-linked immunosorbent assay (Elisa) was used to measure HBsAb, and the cut-off value of this test was 10 IU/mL. The serological response was defined as HBsAb > 10 IU/mL.

Five-week-old female BALB/c mice were used for immunization. Mice were divided into 2 groups (n = 6 per group), with group 1 receiving 100 μL PBS saline buffer (PBS) with adjuvant aluminum hydroxide gel (alum, Sigma, Burlington, MA, USA) as a mock immunization control, and group 2 receiving 100 μL material containing 25 μg of HBcAg-wDIII VLPs in PBS with alum as adjuvant per dosage. Mice were primed on day 0 with subcutaneous injection and were boosted three times on days 21, 42, and 63 with the same dosage and immune protocol as in the prime immunization. Retro-orbital (r.o.) blood samples were collected on day 0 before the immunization (pre-immune sample) and on days 14 (week 2), 35 (week 5), and 56 (week 8) after the 1st immunization. Final blood samples were collected on day 77 (week 11) after mice were humanely euthanized. The spleens were aseptically removed after euthanization for in vitro splenocyte cultures.