Editor's note
Thanks to the development of a prophylactic hepatitis B vaccine and the improvement of vaccination strategies, great progress has been made in the prevention of HBV infection and transmission, and more and more newborns and high-risk groups have benefited from it. In recent years, as an emerging treatment strategy for chronic hepatitis B (CHB), the development and exploration of therapeutic vaccines for CHB at home and abroad have not stopped. Professor Cheng Shuquan from the Third People's Hospital of Guilin was invited to write an article to introduce the current research progress of hepatitis B therapeutic hepatitis B vaccine for the majority of colleagues.
Mechanism of action of the hepatitis B therapeutic vaccine
Therapeutic vaccines are drugs that stimulate the body's immune system to produce specific antibodies or immunoactive cells through specific antigens, thereby changing the immune status of the body and enabling it to resist the lesions caused by diseases. It is hoped that through the use of this special vaccine, more effective treatment of CHB can be achieved, which has always been the tireless and urgent goal of the medical community.
The concept of hepatitis B therapeutic vaccine is to eliminate HBV from CHB patients by breaking immune tolerance or enhancing HBV-specific T cell function, activating and enhancing the body's own immune response, so as to achieve the goal of true cure of CHB. For CHB patients, the experience of prophylactic hepatitis B vaccine is no longer suitable for the development of therapeutic vaccines due to long-term and continuous exposure of HBV antigens to the immune system, and the natural antigens of HBV are mistaken by the immune system for endogenous in this process, so only a weak immunogenicity is retained.
The formation of the concept of a therapeutic vaccine for hepatitis B
In 1986, Academician Wen Yumei of Fudan University School of Medicine in mainland China took the lead in proposing the concept of "hepatitis B therapeutic vaccine" in the world, and published a paper in The Lancet in 1995, formally forming the concept of "hepatitis B therapeutic vaccine", and the therapeutic effect of the vaccine has also been formed and gradually attracted the attention of researchers at home and abroad. In 2009, the phase II study of the world's first therapeutic vaccine developed by Academician Wen was very perfect, but unfortunately the clinical results of the phase III study did not meet expectations, and the vaccine was not popularized and applied in clinical practice. Even so, her pioneering work opened a new door for the development of viral immunology and CHB treatment.
After nearly 30 years since then, there have been many pioneering new methods and new advances in the research of hepatitis B therapeutic vaccines, and they have shown extraordinary results in animal experiments and in vitro cell experiments. Theoretically, the corresponding design should indeed be effective in eliminating HBsAg, and a variety of R&D drugs have been disclosed at home and abroad, because there are few commercial companies that have really reached the pharmaceutical grade, so most varieties do not have standardized names, and are only represented by R&D codes. In this paper, the most promising varieties under research in recent years are introduced as follows.
Hepatitis B therapeutic vaccine under development
Overall, based on the deepening understanding of the immune mechanisms that contribute to the formation of chronic HBV infection, a number of novel strategies have been used to enhance the HBV-specific immune response in patients. On the one hand, pathogen recognition receptors (PRRs) agonists, including stimulators of Toll-like receptors (TLRs), tretinoin-inducible gene 1 (RIG-I) and interferon genes (STING), are applied orally, intranasally or subcutaneously to activate host immune cells and hepatocytes/non-hepatocytes, induce the production of interferon (IFN) and the expression of interferon-stimulated genes (ISG) and pro-inflammatory cytokines, and activate the antiviral status of cells. On the other hand, therapeutic vaccines can be used to induce HBV-specific cytotoxic T lymphocytes (CTLs) to enhance T cell function by blocking immune checkpoint action, or to transfer in vitro activated T cells or NKT cells or gene-edited HBV-specific T cells such as chimeric antigen receptor (CAR) or T cell receptor (TCR)-T cells to enhance cellular immunity. The potential value of these strategies has been partially demonstrated over the past few years, but there are still many hurdles to overcome in the clinical application of these methods.
01
HBsAg/HBcAg combination vaccine
As a therapeutic vaccine mainly composed of HBsAg and HBcAg, it can elicit a strong humoral response specific to HBsAg/HBcAg in wild-type C57BL/6 mice and HBV transgenic animal models, promote Th1/Th2 equilibrium response against HBsAg, and Th1 response against HBcAg. The enhanced HBsAg/HBcAg-specific cellular immune response resulted in a significant reduction in serum HBsAg levels in HBV transgenic mice, but no liver damage. The microparticle vaccine composed of HBsAg, HBcAg and adjuvant ISCOMATRIXTM can induce multispecific and multifunctional T cells, especially specific CD8+ T cells against HBcAg, in HBV transgenic mice, and increase the secretion of IFN-γ, TNF-α and IL-2. After 4 vaccinations, 7 of the 8 animals had anti-HBs titers of more than 10000 mlU/mL. After 2 and 4 vaccinations, the HBV DNA titer in the serum of HBV transgenic mice decreased, but did not reach statistical significance. HBcAg-positive hepatocytes also decreased dramatically, but no significant liver damage occurred.
02
Viral vector-based vaccines
Adenovirus vector-based therapeutic vaccines have been tested in mouse and marmot HBV infection models, and good antiviral effects have been obtained, and highly attenuated VSV strains expressing MHB have been successfully induced by intranasal or intramuscular injection of highly attenuated VSV strains expressing MHB, and HBV replication initiated by transduction of adeno-associated viral vectors (AAV-HBV) can be prevented in mouse models after immunization. The VSV-MHB system also induces a significant multispecific T cell response in a mouse model of HBV replication, resulting in decreased serum and hepatic HBV antigen and DNA levels, as well as a transient increase in ALT activity. These experimental data provide evidence for the potential utility of viral vector-based therapeutic vaccines.
03
TherVacB
TherVacB is a DNA-based therapeutic vaccine developed in Germany that uses recombinant HBsAg and HBcAg proteins to immunize and a modified vaccinia virus Ankara vector (MVA) expressing HBV antigen to activate hepatitis B virus-specific B cell and T cell responses. In the mouse model of HBV, it can induce high-potency anti-HBs, cytotoxic T cells and long-term control of HBV, HBV mice inoculated with TherVac B produced strong specific antibody and CD8+ T cell responses, their HBsAg and intrahepatic HBV DNA decreased after a brief and moderate increase in ALT activity, and the intrahepatic HBV-specific CD8+ T cells of HBeAg-negative mice expressed more IFN-γ, suggesting TherVac The enhancement effect of B on T cell function in HBeAg-negative HBV carriers was higher than that in HBeAg-positive carriers. In the mouse model of HBV continuous replication, the combination of small interfering RNA and TherVac B can interfere with the interaction between PD-1 and PDL-1, enhance the function of HBV-specific CD8+ T cells, and exhibit longer-lasting antiviral effects in both peripheral blood and liver, indicating that small interfering RNA combined with TherVac B targeting PD-L1 can enhance the efficacy of therapeutic vaccines and become one of the promising therapeutic strategies for the ultimate cure of CHB. Its phase I.a clinical trial has been officially launched.
04
LNP-mRNA疫苗
Recently, it was reported that China Pharmaceutical University and Shandong University jointly developed an LNP-mRNA vaccine for CHB treatment, which has the advantages of both strong immunogenicity and sustained viral inhibition by encoding HBsAg and using artificial intelligence algorithms to design messenger ribonucleic acid (mRNA) with optimal folding stability and codon use.
The researchers injected 5 μg or 10 μg of mRNA vaccine intramuscularly three times a week, and showed that mice showed a rapid decrease in serum HBsAg. Serum HBsAg was not even detectable 1 week after the completion of the 3-dose dose. On this basis, the anti-HBs titers in the 5 μg and 10 μg groups reached 3624.0 mIU/mL and 4804.6 mIU/mL, respectively, on day 59 after the 3 injections. Mice were re-injected with the pAAV-HBV2.1 plasmid mimicking viral challenge on day 60, and PBS mice still exhibited high levels of serum HBsAg, while the serum HBsAg levels of mice in all vaccine groups did not reach detectable levels and were accompanied by further elevated anti-HBs titers, demonstrating that mice could continue to protect mice from viral reexposure for a certain period of time after this vaccination. In addition, intrahepatic HBVcccDNA, total DNA, total RNA, and serum HBV DNA were also significantly reduced in vaccinated mice, and the expression of HBcAg in liver tissue was significantly reduced. Comparing the efficacy of mRNA vaccines with entecavir (ETV) side by side, it was found that ETV had no ability to induce anti-HBs or eliminate serum HBsAg, and could only reduce the copy level of serum HBV DNA. The researchers believe that the potential immune mechanism of the mRNA vaccine is the elimination of the virus by non-cytotoxicity, the increase in mouse dendritic cell (DC) subsets and macrophages infiltrating the spleen after naïve immunization, accompanied by effective cell maturation, and that three doses of the mRNA vaccine can induce strong levels of Th1-biased CD4 and CD8 T cells, producing IFN-γ or IL-2 that favors viral elimination. In addition, the frequency of HBsAg-specific memory B cells (MBCs) in the spleen was also significantly increased after vaccination. Since both cytotoxic and non-cytotoxic immune responses against HBV are important in the elimination of HBV, the further development of mRNA vaccines deserves attention.
05
CLB-3000
CLB-3000 is a therapeutic hepatitis B vaccine designed to achieve a functional cure for CHB, consisting of two modified HBsAg modified CLB-405 and CLB-505 purified from Pichia pasteur pasteur with aluminum hydroxide as an adjuvant. At the 2023 Hep-DART conference in Mexico, researchers disclosed that the preclinical efficacy of CLB-3000 in a mouse model of persistent HBV infection has been enrolled in human clinical trials for chronic hepatitis B, and subjects are being recruited.
Preclinical efficacy assays of CLB-3000 in a mouse model of persistent HBV infection showed that serum HBsAg decreased to undetectable levels following vaccination with CLB-3000 therapeutic vaccine, as confirmed by liver immunohistochemistry (IHC). Among the mice treated with CLB-3000 and achieved functional cure, 80% of the mice had both a clearance-related anti-HBs response detected, and the epitope antibody specifically targeted the outer rings 1 and 2 of HBsAg, similar to the antibody specificity observed in functionally cured patients. CLB-3000-specific antibody responses were also detected.
CLB-3000 is believed to have high efficacy in a mouse model of chronic hepatitis B, which is characterized by a rapid decline in HBsAg and clearance from serum and liver, and the conversion of serum to clearance-related anti-HBs, consistent with clinical functional cure of related anti-HBs. Repeated injections were well tolerated by animals, with a maximum dose of 500 μg of total antigen.
06
YFV-HBc PLLAV
Currently, the YFV vaccine induces long-acting humoral responses and robust cellular immunogenicity and is considered one of the most effective dosage forms to date. AstriVax is developing a dual-effect vaccine platform technology for the prevention and treatment of HBV, based on plasmid-initiated live attenuated vaccines (PLLAVs) including flaviviruses such as the Yellow Fever Virus (YFV) 17D vaccine strain that can carry foreign antigens. With the therapeutic potential of the platform, AstriVax has initiated the development of a promising and optimal vaccine against HBV infection, in CHB, where inducing a potent and durable CD8+ T cell response is considered key to eliminating infected hepatocytes, which is currently in preclinical studies. The HBV core (HBc) was used as a model antigen insertion into a polyprotein encoding YFV 17D. The investigators conducted a preclinical evaluation of the ability of this recombinant live attenuated vaccine (LAV) to induce cellular immune responses against HBc in a mouse model that allowed YFV replication. In AAV-HBV-transduced C57BL/6 mice, the researchers preliminarily evaluated the effect of induced HBc-specific T cells on viral parameters by employing adoptive transfer and modulating the liver microenvironment with TLR9 agonists.
The results showed that a single injection of YFV-HBc PLLAV induced YFV and HBc-specific T cell responses in uninfected mice. Similarly, immunization with PLLAV-derived YFV-HBc LAV resulted in the detection of a comparable number of IFN-γ spot-forming cells in response to stimulation of YFV and HBc antigenic peptides. Inoculation with YFV-HBc LAV after 15 days of inoculation with HBc protein stock solution adjuvant increased the number of HBc-specific T cells secreting IFN-γ compared with YFV-HBc LAV alone. Isolated spleen cells were then passively immunized against C57BL/6 mice with established AAV-HBV infection and high HBsAg at baseline (>4 logIU/mL), resulting in a significant decrease in serum HBsAg, a transient increase in ALT, and a decrease in transduced HBc-positive hepatocytes when combined with TLR9 agonists.
It was found that YFV PLLAV carrying the HBc antigen induced a robust T cell response against HBc in a mouse model that allowed YFV replication. In addition, studies have demonstrated that adoptive transfer of T cells generated by heterologous primary proliferation can lead to a sustained decline in HBV despite high baseline HBsAg levels. These encouraging early efficacy data demonstrate the potential of YFV vaccination to induce a robust and potent T-cell response, and it is therefore warranted to be further evaluated in human studies as part of a CHB treatment regimen in the future.
In short, the research and development of therapeutic vaccines has become one of the hot topics in the academic community at home and abroad. Although the design and effectiveness of HBV therapeutic vaccines have progressed over the years, these vaccine candidates can stimulate HBV-specific T cells and achieve varying degrees of HBV inhibition in various experimental settings, and many drugs are refreshing or seem to be at the dawn of improving a certain immune function index, but the overall efficacy is still the biggest bottleneck restricting the wide clinical application, and new breakthroughs in new innovative theories and research methods are expected.
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