Characterization of a breakthrough vaccine escape strain associated with overt hepatitis B virus infection.

Hepatitis B virus (HBV) vaccine is composed of the purified hepatitis B surface antigen (HBsAg) that is produced by recombinant DNA technology. The neutralizing antibodies induced by vaccination target mainly the "a" determinant, aa124-147, of the outer viral envelope (HBsAg). In the present work, we demonstrate a case study for vaccinated patient that is infected with a vaccine escape HBV strain (Eg200). Characterization of the isolate Eg200 showed that it belongs to the genotype D and an uncommon sub-genotype in Egypt; D9. The DNA sequence encoding HBsAg was sequenced. Mutational analysis of the HBsAg showed a double mutation in the "a" determinant of this HBV isolate; T125M and P127T. However, such substitutions were found to be conserved to the detected serotype, ayw3, of Eg200 isolate. This case report indicates that continuous characterization of breakthrough vaccine escape strains of HBV is essential to develop the immunization strategies against HBV infection.

Immune response induced by recombinant pres2/S-protein and a pres2-S-protein fused with a core 18-27 antigen fragment of hepatitis B virus compared to conventional HBV vaccine.

Although comprehensive vaccination is the cornerstone of public health programs to control hepatitis B virus (HBV) infections, 5% of people who receive the existing vaccine do not develop proper immunity against HBV. To overcome this challenge, researchers have tried using various protein fragments encoded by the virus genome to achieve better immunization rates. An important antigenic component of HBsAg called the preS2/S or M protein has also received much attention in this area. The gene sequences of preS2/S and Core18-27 peptide were extracted from the GenBank (NCBI). Final gene synthesis was conducted with pET28. Groups of BALB/c mice were immunized with 10 µg/ml of recombinant proteins and 1 µg/ml CPG7909 adjuvant. Serum levels of IF-γ, TNF-α, IL-2, IL-4, and IL-10 were measured by ELISA assay method on spleen cell cultures on day 45, and IgG1, IgG2a, and total IgG titers obtained from mice serum were quantified on days 14 and 45. Statistical analysis did not show any significant difference between the groups regarding IF-γ level. There were, however, significant differences in terms of IL-2 and IL-4 levels between the groups receiving preS2/S-C18-27 with and without adjuvant and the groups receiving both preS2/S and preS2/S-C18-27 (Plus Recomb-Plus Recomb: the group of mice that received both preS2/S and preS2/S-C18-27 simultaneously). The strongest total antibody production was induced by immunization with both recombinant proteins without CPG adjuvant. The groups that received both preS2/S and preS2/S-C18-27, whether with or without adjuvant, were significantly different from those that received the conventional vaccine considering most abundant interleukins. This difference suggested that higher levels of efficacy can be achieved by the use of multiple virus antigen fragments rather than using a single fragment.

The "humanized" N-glycosylation pathway in CRISPR/Cas9-edited Nicotiana benthamiana significantly enhances the immunogenicity of a S/preS1 Hepatitis B Virus antigen and the virus-neutralizing antibody response in vaccinated mice.

The recent SARS-CoV-2 pandemic has taught the world a costly lesson about the devastating consequences of viral disease outbreaks but also, the remarkable impact of vaccination in limiting life and economic losses. Vaccination against human Hepatitis B Virus (HBV), a major human pathogen affecting 290 million people worldwide, remains a key action towards viral hepatitis elimination by 2030. To meet this goal, the development of improved HBV antigens is critical to overcome non-responsiveness to standard vaccines based on the yeast-produced, small (S) envelope protein. We have recently shown that combining relevant immunogenic determinants of S and large (L) HBV proteins in chimeric antigens markedly enhances the anti-HBV immune response. However, the demand for cost-efficient, high-quality antigens remains challenging. This issue could be addressed by using plants as versatile and rapidly scalable protein production platforms. Moreover, the recent generation of plants lacking ß-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO), by CRISPR/Cas9 genome editing, enables production of proteins with "humanized" N-glycosylation. In this study, we investigated the impact of plant N-glycosylation on the immunogenic properties of a chimeric HBV S/L vaccine candidate produced in wild-type and FX-KO Nicotiana benthamiana. Prevention of ß-1,2-xylose and α-1,3-fucose attachment to the HBV antigen significantly increased the immune response in mice, as compared with the wild-type plant-produced counterpart. Notably, the antibodies triggered by the FX-KO-made antigen neutralized more efficiently both wild-type HBV and a clinically relevant vaccine escape mutant. Our study validates in premiere the glyco-engineered Nicotiana benthamiana as a substantially improved host for plant production of glycoprotein vaccines.

Quantitative assessment of LPS-HBsAg interaction by introducing a novel application of immunoaffinity chromatography.

Lipopolysaccharide (LPS), as a stubborn contamination, should be monitored and kept in an acceptable level during the pharmaceutical production process. Recombinant hepatitis B surface antigen (r-HBsAg) is one of the recombinant biological products, which is probable to suffer from extrinsic endotoxin due to its long and complex production process. This research aims to assess the potential interaction between LPS and r-HBsAg by recruiting immunoaffinity chromatography (IAC) as a novel tool to quantify the interaction. Molecular modeling was performed on the HBsAg molecule to theoretically predict its potential binding and interaction sites. Then dynamic light scattering (DLS) analysis was implemented on HBsAg, LPS, and mixtures of them to reveal the interaction. The virus-like particle (VLP) structure of HBsAg and the ribbon-like structure of LPS were visualized by transmission electron microscopy (TEM). Finally, the interaction was quantified by applying various LPS/HBsAg ratios ranging from 1.67 to 120 EU/dose in the IAC. Consequently, the LPS/HBsAg ratios in the eluate were measured from 1.67 to a maximum of 92.5 EU/dose. The results indicated that 77 to 100% of total LPS interacted with HBsAg by an inverse relationship to the incubated LPS concentration. The findings implied that the introduced procedure is remarkably practical in the quantification of LPS interaction with a target recombinant protein.

Process development for cross-flow diafiltration-based VLP disassembly: A novel high-throughput screening approach.

Virus-like particles (VLPs) are particulate structures, which are applied as vaccines or delivery vehicles. VLPs assemble from subunits, named capsomeres, composed of recombinantly expressed viral structural proteins. During downstream processing, in vivo-assembled VLPs are typically dis- and reassembled to remove encapsulated impurities and to improve particle morphology. Disassembly is achieved in a high-pH solution and by the addition of a denaturant or reducing agent. The optimal disassembly conditions depend on the VLP amino acid sequence and structure, thus requiring material-consuming disassembly experiments. To this end, we developed a low-volume and high-resolution disassembly screening that provides time-resolved insight into the VLP disassembly progress. In this study, two variants of C-terminally truncated hepatitis B core antigen were investigated showing different disassembly behaviors. For both VLPs, the best capsomere yield was achieved at moderately high urea concentration and pH. Nonetheless, their disassembly behaviors differed particularly with respect to disassembly rate and aggregation. Based on the high-throughput screening results, a diafiltration-based disassembly process step was developed. Compared with mixing-based disassembly, it resulted in higher yields of up to 0.84 and allowed for integrated purification. This process step was embedded in a filtration-based process sequence of disassembly, capsomere separation, and reassembly, considerably reducing high-molecular-weight species.

Importance of HBsAg recognition by HLA molecules as revealed by responsiveness to different hepatitis B vaccines.

Hepatitis B (HB) vaccines (Heptavax-II and Bimmugen) designed based on HBV genotypes A and C are mainly used for vaccination against HB in Japan. To determine whether there are differences in the genetic background associated with vaccine responsiveness, genome-wide association studies were performed on 555 Heptavax-II and 1193 Bimmugen recipients. Further HLA imputation and detailed analysis of the association with HLA genes showed that two haplotypes, DRB1*13:02-DQB1*06:04 and DRB1*04:05-DQB1*04:01, were significantly associated in comparison with high-responders (HBsAb > 100 mIU/mL) for the two HB vaccines. In particular, HLA-DRB1*13:02-DQB1*06:04 haplotype is of great interest in the sense that it could only be detected by direct analysis of the high-responders in vaccination with Heptavax-II or Bimmugen. Compared with healthy controls, DRB1*13:02-DQB1*06:04 was significantly less frequent in high-responders when vaccinated with Heptavax-II, indicating that high antibody titers were less likely to be obtained with Heptavax-II. As Bimmugen and Heptavax-II tended to have high and low vaccine responses to DRB1*13:02, 15 residues were found in the Heptavax-II-derived antigenic peptide predicted to have the most unstable HLA-peptide binding. Further functional analysis of selected hepatitis B patients with HLA haplotypes identified in this study is expected to lead to an understanding of the mechanisms underlying liver disease.

Computational Design of a Novel VLP-Based Vaccine for Hepatitis B Virus.

Hepatitis B virus (HBV) is a global virus responsible for a universal disease burden for millions of people. Various vaccination strategies have been developed using viral vector, nucleic acid, protein, peptide, and virus-like particles (VLPs) to stimulate favorable immune responses against HBV. Given the pivotal role of specific immune responses of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in infection control, we designed a VLP-based vaccine by placing the antibody-binding fragments of HBsAg in the major immunodominant region (MIR) epitope of HBcAg to stimulate multilateral immunity. A computational approach was employed to predict and evaluate the conservation, antigenicity, allergenicity, and immunogenicity of the construct. Modeling and molecular dynamics (MD) demonstrated the folding stability of HBcAg as a carrier in inserting Myrcludex and "a" determinant of HBsAg. Regions 1-50 and 118-150 of HBsAg were considered to have the highest stability to be involved in the designed vaccine. Molecular docking revealed appropriate interactions between the B cell epitope of the designed vaccine and the antibodies. Totally, the final construct was promising for inducing humoral and cellular responses against HBV.

Heterologous prime-boost immunization with vesiculovirus-based vectors expressing HBV Core antigen induces CD8+ T cell responses in naïve and persistently infected mice and protects from challenge.

Chronic hepatitis B virus (HBV) infections cause more than 800,000 deaths per year and currently approved treatments do not cure the disease. Because a hallmark of acute infection resolution is the presence of functional CD8+ T cells to the virus, activation of the immune system with therapeutic vaccines represents a potential approach for treating chronic hepatitis B. In this study, we evaluated the immunogenicity and efficacy of two attenuated vesiculovirus-based platforms expressing HBV Core antigen, the highly attenuated vesicular stomatitis virus (VSV) N4CT1 and a unique vaccine platform [virus-like vesicles (VLV)] that is based on a Semliki Forest virus replicon expressing the VSV glycoprotein. We found that heterologous prime-boost immunization with VLV and N4CT1 induced Core-specific CD8+ T cell responses in naïve mice. When immunized mice were later challenged with AAV-HBV, functional Core-specific CD8+ T cells were present in the liver, and mice were protected from establishment of persistent infection. In contrast, when mice with pre-established persistent HBV replication received prime-boost immunization, functional Core-specific CD8+ T cells were found in the spleen but not in the liver. These results highlight the importance of investigating the therapeutic value of different HBV antigens alone and in combination using preclinical animal models, and understanding the correlation between anti-HBV efficacy in these models with human infection.

Exosomes as adjuvants for the recombinant hepatitis B antigen: First report.

Over the past few years, exosomes, a class of extracellular vesicles (EVs), have emerged as key players for inter-cellular communication ultimately modulating the behavior of target cells with countless outcomes. Nevertheless, the potential role of exosomes as vaccine adjuvants remains largely unexplored. Herein, we hypothesized that exosomes derived from immune cells may have an immunostimulatory effect and could constitute a good target towards the development of new fine-tuned vaccine adjuvants. To accomplish this goal, exosomes isolated from lipopolysaccharide endotoxin (LPS)-stimulated human monocytic cell line (THP-1) were characterized and tested for their non-specific immunostimulatory activity when administered subcutaneously to healthy mice; additionally, exosomes' vaccine adjuvant ability was also disclosed after their inclusion in vaccine formulations. The results obtained suggested that the isolated exosomes evoked a pro-inflammatory profile in spleen cells of healthy mice through the induction of cytokines such as tumor necrosis factor alpha (TNF-α), chemokine (C-C motif) ligand 5 (CCL5, also known as RANTES) and interleukin 1 beta (IL-1ß). Moreover, subcutaneous vaccination of mice with exosomes combined with a solution of hepatitis B recombinant antigen (HBsAg) or combined with a suspension containing HBsAg loaded poly-ε-caprolactone (PCL)/chitosan nanoparticles (NPs), induced a humoral immune response quite similar to the one achieved with the experimental control group (HBsAg solution without exosomes). However, exosomes triggered an immunomodulator effect on the cellular immune response, highlighted by the enhancement of IFN-γ secretion. To the best of authors knowledge, this is the first report describing extensively the role of unmodified exosomes as adjuvants and co-adjuvants for hepatitis B vaccination strategies.

Vaccination with a DNA vaccine encoding CD317-targeting HBs antigen elicits enhanced immunity in mice.

BACKGROUND: Conventional hepatitis B virus (HBV) vaccines fail to induce protective antibody titers in 5-10% of immune-competent vaccines. Therefore, safe and effective HBV vaccines are still clinically needed. METHODS: In this study, we developed a plasmid DNA vaccine encoding CD317 single-chain fragment variable (α317scFv) linked with the hepatitis B surface antigen (HBsAg) and detected the humoral and cellular immune responses elicited by this vaccine in BALB/c mice. RESULTS: Vaccination with this fusion DNA vaccine in BALB/c mice induced more robust antiviral T cell and antibody immunity against HBsAg. Compared with mice vaccinated with control vaccine encoding HBsAg, the level of serum-circulating anti-HBsAg antibody (HBsAb) was nearly double in fusion DNA-vaccinated mice. More interesting, splenic lymphocytes isolated from fusion DNA-vaccinated mice showed more potent proliferation and IFN-γ production after being re-stimulated with recombinant HBsAg in vitro. And not only that, the cytotoxicity of fusion DNA vaccine-sensitized splenocytes was ∼3-fold higher than that of controls. CONCLUSION: Taken together, our results reveal that the fusion DNA vaccine can induce more effective immunological protection against HBV, and is a promising candidate for preventing HBV infection.

Hepatitis B virus vaccine breakthrough infection: surveillance of S gene mutants of HBV.

Hepatitis B (HB) is a worldwide public health problem, closely related with liver cirrhosis and hepatocellular carcinoma (HCC). The implementation of universal hepatitis B virus (HBV) vaccination programs has led to significant reduction in incidence of acute and chronic HB, liver cirrhosis and HCC. However, this success is now being threatened by the discovery of HBV vaccine breakthrough infection caused by the S gene mutants of HBV, high maternal viral load and virus-induced immunosuppression. An alteration in the antigenicity and immunogenicity of hepatitis B surface antigen (HBsAg) due to S gene mutations may compromise detection of HBsAg (diagnosis-escape mutants), treatment with hepatitis B-specific immunoglobulin (HBIG), and even cause infections in individuals who are antihepatitis B surface antigen (anti-HBs) antibody-positive after immunization (vaccine-escape mutants). By surveilling for S gene mutants of HBV among vaccinated population, we will have a better understanding of the mechanism of HBV vaccine breakthrough infection; potentially providing new ideas for designing better diagnostic assays and effective vaccines for prevention and treatment of HBV. This review attempts to briefly summarize the status and role of S gene mutations, B-cell epitopes and T-cell epitope mutants, and surveillance of mutant HBV variants in a hospital setting.

Host cell protein testing strategy for hepatitis B antigen in Hexavalent vaccine - Towards a general testing strategy for recombinant vaccines.

BACKGROUND: Recombinant proteins expressed in host cell systems may contain host cell proteins (HCP) as impurities. While there is no clear evidence of clinical adverse events attributable to HCP, HCP levels and profiles must be documented to meet regulatory requirements and to understand the consistency of the biological product and manufacturing process. We present a general strategy for HCP characterization applied to a recombinant protein antigen, Hepatitis B surface antigen (HBsAg) used in a multivalent vaccine. METHODS: Polyclonal antisera raised against HCPs in process fractions from a mock preparation of the HBsAg yeast expression host, Hansenula polymorpha, were used to develop a quantitative sandwich ELISA to measure HCP content in batches of purified recombinant HBsAg. Batches were also subjected to SDS-PAGE and LC-MS/MS to identify detectable proteins. Batch consistency was further assessed by SDS-PAGE/densitometry purity analysis and by the ratio of specific HBsAg content (by ELISA) to total protein. RESULTS: Using the quantitative HCP ELISA, the HCP content showed no discernable trend in multiple HBsAg batches manufactured over a 5-year period. All batches were ≥95% pure by SDS-PAGE/densitometry, with consistent HBsAg/total protein ratios. In addition to the expected HBsAg antigen protein, LC-MS/MS analysis of three HBsAg batches identified several yeast proteins, none of which are known to cause adverse reactions in humans. CONCLUSIONS: Analysis of multiple HBsAg batches showed consistent HCP content and identification profiles, as well as product purity and specific antigen content, demonstrating consistent manufacturing process. Recombinant vaccines, unlike therapeutic products, are administered infrequently with only small amounts of protein injected at a time. With limited potential for adverse reactions to small quantities of HCPs in purified recombinant vaccine antigens, and considering the relevant regulatory guidelines, we conclude that once consistent manufacturing process has been demonstrated, routine HCP testing in recombinant vaccine antigens is no longer required.

Analysis of epitope-based vaccine candidates against the E antigen of the hepatitis B virus based on the B genotype sequence: An in silico and in vitro approach.

Chronic hepatitis B virus infection is a worldwide health problem with no current effective strategy to achieve a cure. The Hepatitis B virus (HBV) E antigen (HBeAg) has a negative effect on the immune system and a therapeutic vaccine is a promising strategy in order to treat chronic virus infection. In this study, we analyzed and identified the MHC-I, MHC-II and B cell epitopes of the HBeAg based on a B genotype sequence of HBV using a bioinformatic approach and in vitro experiments. The computational approach provided us with four epitopes (LLWFHISCL, YLVSFGVWI, MQLFHLCLI, TVLEYLVSF) of the specific MHC-I allele HLA-A0201 that conformed to all criteria. Molecular docking and a peptide binding assay showed that epitope TVLEYLVSF had the lowest binding energy and epitope LLWFHISCL had the highest binding affinity to the HLA-A0201 molecule. An interferonγenzyme-linked immunospot assay and cytotoxicity assay revealed that epitope LLWFHISCL had the highest ability to induce and stimulate T cells. Furthermore, we determined four core peptides of MHC-II epitopes and a region of the B cell epitope. The epitopes and region identified in this research may be helpful in designing epitope-based vaccines and boosting the mechanism research of HBeAg and its effect on the immune system.

Use of ELISpot assay to study HBs-specific B cell responses in vaccinated and HBV infected humans.

Hepatitis B surface antibody (HBsAb) plays a critical role in protecting against infection of hepatitis B virus (HBV) and were extensively studied in literature. At the same time, the status of hepatitis B surface antigen (HBs)-specific B cells in both vaccinated and HBV infected people received limited attention. In the current study, we adopted a highly specific B-cell Enzyme Linked ImmunoSpot (ELISpot) assay to analyze HBs-specific B cells in various clinical settings: healthy individuals with the history of HBV vaccination before and after receiving an extra HBV vaccine boost, people chronically infected with HBV (CHB) in various clinical stages, with or without a particular type anti-viral treatment, or whether receiving a dose of HBV vaccine. In all of these cases, B-cell ELISpot assay was used effectively in enumerating the frequency of HBs-specific B cells. While the focus of the current report was to establish the utility of this assay for HBV research, a number of interesting observations were made in this pilot study based on the profiles and dynamics of HBs-specific B cells in various conditions. Such information is useful to guide the future work in designing novel therapeutic strategies against CHB.

A novel chimeric Hepatitis B virus S/preS1 antigen produced in mammalian and plant cells elicits stronger humoral and cellular immune response than the standard vaccine-constituent, S protein.

Chronic Hepatitis B Virus (HBV) infection leads to severe liver pathogenesis associated with significant morbidity and mortality. As no curable medication is yet available, vaccination remains the most cost-effective approach to limit HBV spreading and control the infection. Although safe and efficient, the standard vaccine based on production of the small (S) envelope protein in yeast fails to elicit an effective immune response in about 10% of vaccinated individuals, which are at risk of infection. One strategy to address this issue is the development of more immunogenic antigens. Here we describe a novel HBV antigen obtained by combining relevant immunogenic determinants of S and large (L) envelope proteins. Our approach was based on the insertion of residues 21-47 of the preS1 domain of the L protein (nomenclature according to genotype D), involved in virus attachment to hepatocytes, within the external antigenic loop of S. The resulting S/preS121-47 chimera was successfully produced in HEK293T and Nicotiana benthamiana plants, as a more economical recombinant protein production platform. Comparative biochemical, functional and electron microscopy analysis indicated assembly of the novel antigen into subviral particles in mammalian and plant cells. Importantly, these particles preserve both S- and preS1-specific epitopes and elicit significantly stronger humoral and cellular immune responses than the S protein, in both expression systems used. Our data promote this antigen as a promising vaccine candidate to overcome poor responsiveness to the conventional, S protein-based, HBV vaccine.

Recombinant vaccinia vector-based vaccine (Tiantan) boosting a novel HBV subunit vaccine induced more robust and lasting immunity in rhesus macaques.

This study explored several prime-boost strategies in rhesus macaques using various novel hepatitis B virus (HBV) vaccines that showed promise as prophylactic and therapeutic approaches in our previous study using in a mouse model. The tested vaccines included an HBV particle subunit (HBSS1) vaccine and the recombinant vaccinia (RVJSS1) or adenoviral (rAdSS1) vector-based vaccines containing S (1-223aa) and PreS1 (21-47aa). The strength and maintenance of humoral activity (IgG and neutralizing antibodies) and cellular immunity (interferon-γ production assessed by IFN-γ enzyme-linked immunosorbent spot (ELISpot) assay) were investigated in a longitudinal study following various vaccination protocols until 79weeks post-vaccination. We found that HBSS1/RVJSS1 heterologous prime-boost elicits similar strong humoral immunity but more robust and lasting cellular immunity (CMI) than HBSS1/HBSS1 homologous vaccination in rhesus macaques. Furthermore, HBSS1/RVJSS1/RVJSS1 induced more robust and lasting CMI in macaques than did HBSS1/HBSS1/rAdSS1 vaccination. Therefore, HBSS1/RVJSS1/RVJSS1 is most promising candidates for protecting humans against HBV infection, especially for therapeutic application.

Hepatitis B vaccine alone may be enough for preventing hepatitis B virus transmission in neonates of HBsAg (+)/HBeAg (-) mothers.

BACKGROUND AND AIM: To prospectively evaluate the efficacy of vaccine alone compared with vaccine plus HBIG for preventing HBV transmission in neonates of HBsAg (+)/HBeAg (-) mothers. METHODS: Combined immunization is currently recommended for neonates of HBsAg (+) mothers in China. As a result, a randomized design is infeasible due to ethical reasons. In practice, Guangxi Zhuang Autonomous Region and Jiangsu Province implement vaccine alone and vaccine plus HBIG strategies for neonates born to HBsAg (+)/HBeAg (-) mothers, respectively. We alternatively enrolled neonates of HBsAg (+)/HBeAg (-) mothers from these two regions. Three doses of a recombinant yeast-derived hepatitis B vaccine were given at 0, 1 and 6months with or without HBIG at birth. RESULTS: At 7months, sera were collected from 132 neonates in Guangxi Zhuang Autonomous Region and 752 neonates in Jiangsu Province. Baseline characteristics of both mothers and neonates were comparable in the two regions. No differences were revealed regarding the occurrence of perinatal HBV transmission with or without HBIG at birth [0.1% (1/752) vs. 0.0% (0/132), p=1.000]. The anti-HBs response rates were 97.7% (129/132) and 98.5% (740/751) for the neonates with vaccine alone and with HBIG (p=0.758), respectively. Vaccine alone induced a significantly higher anti-HBs GMC as compared to vaccine plus HBIG at 7months of age (1555.3mIU/mL vs. 654.9mIU/mL, p<0.0001). At 12months of age, protective levels of anti-HBs remained in 97.4% (596/612) and 98.3% (118/120) of the neonates receiving and not receiving HBIG, respectively (p=0.771). The neonates receiving combined prophylaxis had a markedly lower anti-HBs GMC (210.7mIU/mL vs. 297.0mIU/mL, p=0.011). Horizontal HBV transmission occurred in none of the successfully immunized neonates for both compared groups at 12months of age. CONCLUSIONS: Vaccine alone may be enough for preventing HBV transmission in neonates of HBsAg (+)/HBeAg (-) mothers.

Present and future DNA vaccines for chronic hepatitis B treatment.

INTRODUCTION: With at least 240 million hepatitis B virus (HBV) carriers worldwide, being at a high risk of cirrhosis and hepatocellular carcinoma (HCC), chronic hepatitis B remains a major public health issue. Because current antiviral treatments are only virostatic, there is an urgent need for the development of innovative anti-HBV strategies leading to the functional cure. In this context, DNA-based vaccines appear as a promising approach. Area covered: In this review, the authors summarize the pertinent features of DNA vaccines for chronic hepatitis B therapy. They review several technologies that improve DNA vaccines efficacy evaluated in animal models of hepadnaviral infection. They also discuss the clinical trials of therapeutic DNA vaccination initiated in HBV-carrier patients. Expert opinion: Preclinical studies in HBV transgenic mice, DHBV-carrier ducks and WHV-infected woodchucks, have clearly demonstrated a benefit of DNA vaccine-based combination therapies for chronic hepatitis B treatment. However, the results of clinical trials conducted in HBV patients were rather disappointing and frustrating, as DNA-vaccines have not shown the same efficacy in patients as in animal models. We are convinced that the design of innovative clinical trials based on strategies able to increase DNA vaccine immunogenicity will allow to advance in this challenging field.

The Last Ten Years of Advancements in Plant-Derived Recombinant Vaccines against Hepatitis B.

Disease prevention through vaccination is considered to be the greatest contribution to public health over the past century. Every year more than 100 million children are vaccinated with the standard World Health Organization (WHO)-recommended vaccines including hepatitis B (HepB). HepB is the most serious type of liver infection caused by the hepatitis B virus (HBV), however, it can be prevented by currently available recombinant vaccine, which has an excellent record of safety and effectiveness. To date, recombinant vaccines are produced in many systems of bacteria, yeast, insect, and mammalian and plant cells. Among these platforms, the use of plant cells has received considerable attention in terms of intrinsic safety, scalability, and appropriate modification of target proteins. Research groups worldwide have attempted to develop more efficacious plant-derived vaccines for over 30 diseases, most frequently HepB and influenza. More inspiring, approximately 12 plant-made antigens have already been tested in clinical trials, with successful outcomes. In this study, the latest information from the last 10 years on plant-derived antigens, especially hepatitis B surface antigen, approaches are reviewed and breakthroughs regarding the weak points are also discussed.

Adjuvant effect of B domain of staphyloccocal protein A displayed on the surface of hepatitis B virus capsid.

The hepatitis B virus (HBV) capsid-based recombinant particles, which display both major hydrophilic region of HBV surface antigen (HBV-MHR) and B domain of Staphylococcal protein A (SPAB ), were produced using Escherichia coli as expression host. SPAB was used as an adjuvant to elicit the immune response to HBV-MHR, and its adjuvant effect in the immunized mice was estimated with varying the position and amount of SPAB on the HBV capsid particles. Compared to the emulsified aluminum gel (alum gel) that is a currently commercialized vaccine adjuvant, SPAB caused the significantly higher level of anti-HBV immunoglobulin G (IgG) titer and seroconversion rate, and notably SPAB at the most surface-exposed position on the recombinant particle led to the highest immune response. Moreover, SPAB caused much lower ratio of IgG1 to IgG2a compared to alum gel, indicating that helper T-cell 1-mediated immune response (responsible for cytotoxic T-cell stimulation) is relatively more stimulated by SPAB , unlike alum gel that mainly stimulates helper T-cell 2-mediated immune response (responsible for B-cell stimulation). Although HBV-MHR and HBV capsid particle were used as proof-of-concept in this study, SPAB can be used as a highly effective adjuvant with other disease-specific antigens on the surface of other virus-like particles to produce various recombinant vaccines with high potency.