HB vaccines and adjuvants
The commercially available S-HBs vaccine, Bimmugen, was purchased from KM Biologics (Kumamoto, Japan). This vaccine uses the yeast-derived S-HBsAg of the HBV GTC strain and contains aluminum hydroxide as an adjuvant. The yeast-derived L-HBsAg (BCL-AG-001) was obtained from Beacle, Inc. (Kyoto, Japan)16,18. The K-type CpG oligodeoxynucleotide adjuvant wrapped by schizophyllan (K3-SPG) was prepared as previously described26,27. A squalene-based oil-in-water nanoemulsion adjuvant (Addavax) was purchased from Invivogen (San Diego, CA). This adjuvant is known to have a formulation similar to that of MF59.
In this study, nine rhesus macaques (3–5 years of age, male or female, and approximately 5 kg body weight) were used as a nonhuman primate model for immunization with HB vaccines. All monkeys were supplied from colonies at the Center for the Evolutionary Origins of Human Behavior, Kyoto University. The monkeys were reared in outdoor group cages with wooded toys provided as environmental enrichment. They were fed apples, potatoes, and commercial monkey diets. They were able to access water ad libitum. They had health records from birth with yearly health checkups. We confirmed that they were free of B virus, simian immunodeficiency virus, simian T-cell leukemia virus, and simian retrovirus. The animal experiments were conducted using protocols and experimental procedures that were approved by the Animal Welfare and Animal Care Committee of Kyoto University (approval number: 2017-158, 2018-122, 2019-144, 2020-105, 2021-159) and were carried out in accordance with the Guidelines for Care and Use of Nonhuman Primates (Version 3) by the Animal Welfare and Animal Care Committee of Kyoto University. This guideline was prepared based on the provisions of the Guidelines for Proper Conduct of Animal Experiments (June 1, 2006; Science Council of Japan) as well as Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions [Notice No. 71 of the Ministry of Education, Culture, Sports, Science and Technology dated June 1, 2006], in accordance with the recommendations of the Weatherall report: The use of nonhuman primates in research (http://www.acmedsci.ac.uk/more/news/the-use-of-nonhuman-primates-in-research/).
HB vaccines were introduced subcutaneously to immunize rhesus macaques under ketamine anesthesia with medetomidine, and then its antagonist atipamezole was administered at the end of the procedure. Blood samples were collected under anesthesia as described above. To assess the potential immediate side effects caused by the vaccines, veterinary observation, including any clinical and hematological abnormalities as well as the presence of skin abnormalities such as flare, rash, or edema, was carefully performed.
HepG2 cells were obtained from the European Collection of Authenticated Cell Cultures (catalog number; EC85011430, ECACC, Salisbury, UK) and cultured in MEM supplemented with 10% fetal calf serum22. Sodium taurocholate cotransporting polypeptide (NTCP)-transduced HepG2 cells; G2/NT18-C and HepG2-NTCPsec+, were used as described previously24,25.
Enzyme-linked immunosorbent assay
The titer of anti-S-HBs antibodies was measured using an Enzygnost Anti-HBs II (Siemens Healthcare Diagnostics, Tokyo, Japan). To measure the titers of anti-preS1 and anti-L-HBsAg, laboratory-made enzyme-linked immunosorbent assays were used33. Briefly, 1 μg/mL of preS1-peptide (N-terminal 12–119 aa of preS1 region, BCL-AGS1-02, Beacle) or 1 μg/mL of L-HBsAg (Beacle) in phosphate-buffered saline (PBS) containing 0.02% NaN3 (PBSN) was adsorbed onto MaxiSorp 96-well plates (Nunc Immunoplate F96 Cert MaxiSorp, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) during overnight incubation at 4 °C. Wells were washed three times with wash buffer (PBS containing 0.05% Tween 20), blocked with 0.2% BSA-supplemented PBSN, and incubated for more than 24 h. The plates were washed three times prior to use. Serum diluted with 0.2% BSA-supplemented PBST containing 0.02% NaN3 (PBSTN) was added to each well, and the plates were incubated overnight at 4 °C. The supernatant was then discarded, and each well was washed three times. HRP-conjugated anti-human IgG (P0214, Dako Cytomation, Glostrup, Denmark) was added at the concentration of 0.2 µg/mL and incubated for 2 h at 37 °C prior to the addition of substrate (TMB Substrate Kit, Thermo Scientific, IL). Thirty minutes later, color development was stopped by the addition of 2 N H2SO4 at 100 µL/well, and then the optical density was measured at 450 nm in a microplate reader (SUNRISE, Tecan, Grödig, Austria).
Production and infection of cell culture-generated HBV
The plasmid for HBVcc was generated with the HBV GTC strain (accession number: AB246345)22. This plasmid encodes a replication-competent HBV molecular clone with a 1.38-fold genome length (Supplementary Fig. 2a). The HBV clone was transfected into HepG2 cells by using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA), and the culture medium was harvested 1 week after transfection. The collected medium was passed through a 0.45-μm filter to remove cell debris and purified by an iodixanol density gradient. The peak fraction of infectivity in the gradient was used as an inoculum. The HBV DNA titer of the inoculum was measured by real-time PCR targeting the HBs region after treatment with DNase (RQ1 RNase-Free DNase, Promega, Madison, WI).
The prepared viruses were mixed with rhesus macaque serum, incubated at 37 °C for 1 h, and inoculated onto HepG2-NTCP-sec+ cells in the presence of 4% PEG8000 and 2% dimethyl sulfoxide for 16 h. Twelve days after infection, infected cells were treated with rabbit polyclonal anti-HBc IgG fraction (catalog number; HBP-023-9, Austral Biologicals, San Ramon, CA) at the concentration of 1.0 µg/mL followed by staining with Alexa Fluor 555-conjugated anti-rabbit IgG (catalog number; A32732, Thermo Fisher Scientific) at the concentration of 1.0 µg/mL after fixation and permeabilization. Nuclei were also stained with 4’,6-diamidino-2-phenylindole. The infected cells were visualized and captured with a BZ-X710 fluorescence microscope (Keyence, Osaka, Japan). To estimate the infection efficiencies, the area size of staining was quantified with the built-in software BZ-3HA ver. 184.108.40.206 (Keyence).
Recombinant HBV reporter virus system
Regarding the recombinant HBV reporter virus (HBV/NL) infection system, HBV-NL plasmids encoding the 1.2-fold HBV genome replacing the HBe/HBc region with NL and HBV-dEdelS plasmids encoding the 1.2-fold HBV genome lacking the encapsidation signal and the start codons of ORF of all HBsAg species were prepared (Supplementary Fig. 2b)22,34. We used the GTC clone (accession number: AB246345) for the generation of these plasmids. One week after the transfection of these plasmids into HepG2 cells, the HBV/NL in the culture medium was harvested. The collected medium was passed through a 0.45-μm filter to remove cell debris and purified by an iodixanol density gradient. The peak fraction of infectivity in the gradient was used as an inoculum, and the same inoculum was used to assess neutralization by serum or purified antibodies induced by all vaccines. The viral titer was measured by real-time PCR with a primer and probe set targeting the NL region after treatment with DNase (RQ1 RNase-Free DNase). One week after infection of HBV/NL into G2/NT18-C cells, the infection efficiencies were estimated by measuring the luciferase activities in infected cells using a Nano-Glo Luciferase Assay System (Promega) after lysis of cells with Passive Lysis Buffer (Promega).
The HBV/NL strain with VEMs was also generated. In this system, all HBsAg species were provided from the HBV-NL plasmid. Thus, the amino acid mutations I126S, G145R, and G145A were introduced into the HBV-NL plasmid by site-directed PCR, and the fragment was swapped by digestion with the restriction enzymes BstEII and BstBI (Supplementary Fig. 2b). The chimeric HBV-NL plasmid encoding the L-HBs region of GTA, GTB, and GTD was also generated by replacing the L-HBs region with those of the GTA (accession number: LC488828), GTB (accession number: AB246341), and GTD strains by digestion with the restriction enzymes BstEII and BstBI (Supplementary Fig. 2b). The sequence of the L-HBs region of the GTD strain is identical to that of HBV derived from HepG2.2.15 cells35. The chimeric HBV-NL plasmids HBV/NL-pS1-GTB, -pS2-GTB, -RBD-GTB, and -exRBD-GTB were also generated by site-directed PCR and fragment swapping. The plasmids for HBV/NL-RBD-GTB with GTC-type amino acids were generated by site-directed PCR. The alignment of the amino acid sequences of the L-HBs region is provided in Supplementary Fig. 5.
Purification of antibodies and antibody adsorption assay
The induced antibodies in serum samples of rhesus macaques were purified by Protein G HP SpinTrap (Cytiva, Tokyo, Japan) following the manufacturer’s instructions. To estimate the amount of purified antibodies, the protein concentrations of the purified antibodies were quantified by a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). For the antibody adsorption assay, S-HBsAg (recombinant HBsAg adr produced in CHO cells, ProSpec, Rehovot, Israel) and L-HBsAg (Beacle) were used.
Human serum samples
Serum samples were obtained with written informed consent without compensation from three individuals (37–42 years old, 1 male and 2 females) who were vaccinated with the commercially available S-HBs vaccine in Japan, Bimmugen. The vaccine-induced antibodies were purified from serum samples and evaluated for the neutralization of HBV reporter viruses. This experiment was approved by the Ethics Committees of the institute (approval number 780 from the National Institute of Infectious Diseases).
Statistical analysis was performed by a ratio paired t-test using GraphPad PRISM 8 software (GraphPad Software, La Jolla, CA). The IC50 values were calculated by generating the dose–response curves with the log (inhibitor) vs. the normalized response model. Results with P values of <0.05 were considered statistically significant.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.