The establishment of chronic HBV infection depends on the age of the host at the time of exposure to the virus, particularly in newborns and infants whose immune function has not yet been trained. Therefore, more than 95% of perinatal and infant infections will be chronic, so that vertical transmission from mother to child has become the main transmission route of chronic HBV infection in mainland China, and chronic HBV infection in mainland China also has a typical immune tolerance period with high viral replication and low liver inflammation as the main histological characteristics. However, as the infected person ages and the immunity becomes sound, host antiviral immunity, especially T cell-mediated cytolytic action, controls infection by clearing infected hepatocytes. This leads to persistent inflammatory damage to the liver, as well as a decrease in viral load, viral mutations or epigenetic modifications under immune selection pressure. Driven by the combined effect of host antiviral and viral adaptive mutation, the HBV DNA level of patients progressively decreased, and HBeAg negative conversion or serological conversion was obtained, but HBsAg negative conversion was rarely seen. The reason for this is related to the loss of anti-HBs-specific T cell function due to long-term continuous stimulation of excessive HBsAg.
With the attenuation and apparent silencing of the cccDNA pool with HBeAg negative conversion as the main serological marker, the expression and secretion of HBsAg from the integration source independent of the HBV replication process has become the biggest obstacle to the functional cure of chronic hepatitis B (CHB). It is generally accepted that a sustained and significant decline in HBsAg expression not only helps to relieve the depletion of functional anti-HBs-specific T cells, but also partially recovers and rebuilds. However, the clinical cure of some patients treated with immunomodulatory drugs in the cure-dominant population [1] further strengthens the view that we may accelerate the depletion of the cccDNA pool and the continuous decline of HBsAg levels through direct antiviral therapy with the blessing of host immunity, so that the function of anti-HBs-specific T cells will not continue to be impaired or even partially recovered, and then the patients with chronic hepatitis B can be cured through immunomodulation.
In 2021, Professor Guo Jutao from the Baruch Blumberg Institute in the United States led us to publish an opinion piece [2], which further clearly stated that compared with the decrease in the level of HBsAg in circulating blood, HBsAg in situ in liver tissue is particularly important for the recovery and reconstruction of host anti-HBs cell immune function. The article from the Protzer Laboratory of the Institute of Virology, Faculty of Medicine and Health, Technical University of Munich, Germany [3], highlights the view that "HBsAg (expressed but) is not secreted, which neither contributes to the induction of antiviral T cell responses, nor promotes the clearance of hepatitis B virus in mice". In their conclusions, the authors made it clear that the removal of HBsAg from the blood did not significantly affect the persistence of HBV infection in AAV-HBV mice, nor did it help to improve HBV-specific T cell immunity in mice. Therefore, the authors conclude that in order to break the immune tolerance to HBV in patients with chronic HBV infection (and more precisely to restore dysfunctional host anti-HBV immunity), it is necessary to limit the expression of hepatic viral antigens. That is, the conclusion of this paper supports Professor Guo Jutao's reasoning.
In their previous study, the team used shRNA or siRNA interference targeting the common sequence of HBV transcripts to significantly reduce the expression of viral proteins, including HBsAg, in the hepatocytes of AAV-HBV mice that mimicked chronic HBV infection. Then the therapeutic vaccine (including HBsAg and HBcAg) (please see the original text for details) was administered, and the results resulted in the negative conversion of serum HBsAg in some mice that were originally immune tolerant to AAV-HBV. Since RNA interference led to a decrease in all viral protein antigens before the therapeutic vaccine induced negative transversion of serum HBsAg, which viral protein(s) induced immune tolerance (persistent infection) in AAV-HBV mice? When it comes to HBsAg, is it the circulating blood or the liver in situ that plays a key role? In this follow-up experiment, the authors wanted to make a clear decision about the HBsAg location problem. In the experiments, the authors used a relatively low infectious dose of AAV-1.3xHBV (genotype D) to better immune tolerance of mice to "infection", and secondly, introduced a T193A base mutation into the ORF of the S gene of 1.3xHBV to cause C/S substitution of amino acid at the 65th position of HBs, because it has been reported that this C65S mutation in the S protein will cause HBsAg secretion disorder [4]. The following is a summary of the experimental results of this paper:
Effect of HBsAg secretion on viral persistence and efficacy of TherVacB in mice expressing low levels of HBV
Four weeks after invading mice with wild and S(C65S)-mutated AAV-1.3xHBV recombinant virus, three consecutive doses of TherVacB (a therapeutic vaccine containing HBsAg and HBcAg) were administered, and the disappearance of HBsAg and positive conversion of anti-HBs in the serum of mice other than wild mice were indeed seen (A, C). Similarly, TherVacB treatment has led to the disappearance of HBeAg and the emergence of anti-HBe (B, D). It is worth noting that in the absence of TherVacB treatment, anti-HBs appeared in HBV(C65S) mice, and even anti-HBe was higher than the detection line (D), but this phenomenon was not seen in HBV(wt) mice, suggesting that the C65S mutation in the S gene accelerated the clearance of infected AAV-HBV in mice. The authors confirmed the therapeutic efficacy of TherVacB by stimulating the release of IFNγ from mouse CD8+ T cells with S and Core epitope peptides, and the increase of ALT caused by concomitant therapy. However, in contradiction with the S (C65S) mutation, which accelerates the clearance of infected AAV-HBV in mice, the C65S mutant mouse group was partially detectable at low levels, both at the total RNA level of HBV (H) and on core proteomic staining (I). Unfortunately, the authors did not provide HBs liver histochemistry results from HBV (C65S) mice, so that we can still detect HBsAg with C65S mutations in liver tissue after treatment? Unknown.
Comparing the above results of wild and S(C65S) mutant mice with HBsAg secretion disorders, especially the results of G and F above, the authors concluded that the elimination of HBsAg in circulating blood alone would not benefit the host's anti-HBs immune response. Next, the authors tried siRNA combined with TherVacB therapy, but the results of the second part are not shown here because I do not accept that the short-term blockade of information flow in the central rule will affect the breakdown of immune tolerance of the host (as in the animal model in this article), or the dysfunctional anti-HBV-specific immune recovery in patients with chronic hepatitis B.
Write at the end
I know that many graduate students and even young scientists who are doing hepatitis B-related research are paying attention to our "Film Police Lab" official account, and here are two questions for you to think about:
1. In patients with chronic hepatitis B, is there a significant decrease in serum HBsAg while the expression of HBsAg in liver tissue remains high? If you select yes, what will happen?
2. 文献[4]（doi.org/10.1128/jvi.67.8.4588-4597.1993）描述的essential for secretion of 20-nm particles的Cys-48、Cys-65和Cys-69 的突变，会在慢乙肝患者普遍存在吗？
For question 1, please pay attention to this official account while thinking, the answer will be revealed soon, at least to some extent.
Finally, it should be emphasized that this mouse model is more like an immune tolerance state in which antiviral immunity has not been fully activated, rather than the depletion of host anti-HBV-specific immunity, especially T cell function, caused by the prolonged interaction between virus and host immunity. So, is this strategy effective for patients with chronic hepatitis B, particularly HBeAg-negative chronic hepatitis B? Professor Ju Tao and I share the same view that we can only be cautiously optimistic.
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Bibliography:
[1] Lu FM, Wang J, Chen XM, et al. Zhonghua Gan Zang Bing Za Zhi. 2017; 25(2):105-110. doi:10.3760/cma.j.issn.1007-3418.2017.02.005
[2] Yang S, Zeng W, Zhang J, Lu F, Chang J, Guo JT. Restoration of a functional antiviral immune response to chronic HBV infection by reducing viral antigen load: if not sufficient, is it necessary?. Emerg Microbes Infect. 2021; 10(1):1545-1554. doi:10.1080/22221751.2021.1952851
[3] Michler T, Zillinger J, Hagen P, et al. The lack of HBsAg secretion does neither facilitate induction of antiviral T cell responses nor Hepatitis B Virus clearance in mice. Antiviral Res. Published online April 26, 2024. doi:10.1016/j.antiviral.2024.105896
[4] Mangold CM, Streeck RE. Mutational analysis of the cysteine residues in the hepatitis B virus small envelope protein. J Virol. 1993; 67(8):4588-4597. doi:10.1128/JVI.67.8.4588-4597.1993
Source: Film Police Labs