Hum Vaccin Immunother. 2019 Dec 6:1-18. doi: 10.1080/21645515.2019.1689745. [Epub ahead of print]
Designing a therapeutic hepatitis B vaccine to circumvent immune tolerance.
Whitacre DC1,2, Peters CJ1,2, Sureau C3, Nio K4, Li F5, Su L5, Jones JE1, Isogawa M6, Sallberg M7, Frelin L7, Peterson DL8, Milich DR1,2.
Author information
1
Department of Immunology, VLP Biotech, Inc., JLABS San Diego, San Diego, CA, USA.
2
Department of Immunology, Vaccine Research Institute of San Diego, San Diego, CA, USA.
3
Molecular Virology Laboratory, Institut National de la Transfusion Sanguine (INTS), Paris, France.
4
Graduate School of Medicine, Department of Gastroenterology, Kanazawa University, Kanazawa, Ishikawa, Japan.
5
Lineberger Comprehensive Cancer Center, Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
6
Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
7
Department of Laboratory Medicine, Division of Clinical Microbiology, F68, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockhold, Sweden.
8
Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA.
Abstract
An effective prophylactic hepatitis B virus (HBV) vaccine has long been available but is ineffective for chronic infection. The primary cause of chronic hepatitis B (CHB) and greatest impediment for a therapeutic vaccine is the direct and indirect effects of immune tolerance to HBV antigens. The resulting defective CD4+/CD8+ T cell response, poor cytokine production, insufficient neutralizing antibody (nAb) and poor response to HBsAg vaccination characterize CHB infection. The objective of this study was to develop virus-like-particles (VLPs) that elicit nAb to prevent viral spread and prime CD4+/CD8+ T cells to eradicate intracellular HBV. Eight neutralizing B cell epitopes from the envelope PreS1 region were consolidated onto a species-variant of the HBV core protein, the woodchuck hepatitis core antigen (WHcAg). PreS1-specific B cell epitopes were chosen because of preferential expression on HBV virions. Because WHcAg and HBcAg are not crossreactive at the B cell level and only partially cross-reactive at the CD4+/CD8+ T cell level, CD4+ T cells specific for WHcAg-unique T cell sites can provide cognate T-B cell help for anti-PreS1 Ab production that is not curtailed by immune tolerance. Immunization of immune tolerant HBV transgenic (Tg) mice with PreS1-WHc VLPs elicited levels of high titer anti-PreS1 nAbs equivalent to wildtype mice. Passive transfer of PreS1 nAbs into human-liver chimeric mice prevented acute infection and cleared serum HBV from mice previously infected with HBV in a model of CHB. At the T cell level, PreS1-WHc VLPs and hybrid WHcAg/HBcAg DNA immunogens elicited HBcAg-specific CD4+ Th and CD8+ CTL responses.
KEYWORDS:
Immune tolerance to an infectious agent such as the HBV is not a binary event (tolerance vs no tolerance), but rather represents complex host-viral interactions. Because immune tolerance is clonal, all viral antigens and their constituent epitopes are not equally tolerogenic nor are all cell types equally susceptible to tolerance induction. For example, the secreted HBeAg is more tolerogenic than the cellular HBcAg,69 and one HBeAg-specific epitope is more tolerogenic than another epitope on the same HBeAg.70,71 Similarly, one would expect different degrees of T cell tolerance to epitopes on the PreS1, PreS2, and HBsAg envelope antigens because T cell recognition is distinct between these envelope regions.55 At the cellular level, Th cells are often more susceptible to tolerance induction than B cells or CTL.72 These phenomenon represent forms of “split tolerance”. Immune tolerance preferentially occurs in high avidity clones and is mediated by deletional and non-deletional mechanisms. Therefore, low avidity T cells that have escaped deletion most likely constitute the bulk of the T cell repertoire during chronic HBV infection and low avidity T cells are more prone to negative regulation (i.e., checkpoint inhibitors, regulatory T cells, metabolic dysfunction, and clonal exhaustion). Other hosts and viral factors such as age of infection, immune status, antigen load, secreted or cytosolic antigen and phase of infection affect immune tolerance as well.9,10 For vaccine immunotherapy to be effective it must target either non-tolerant clones or non-deleted clones in which tolerance can be reversed or circumvented. Therefore, we targeted PreS1-specific B cells and HBcAg-specific CTL effector cells and bypassed the more tolerant prone Th cells by substituting heterospecific, WHcAg-specific Th cells for HBV-specific Th cells. Although B cell defects have been defined during chronic HBV infection,6 low-level antibodies to most viral antigens are present during the active phases of infection often masked in immune complexes.66 Further, HBc/HBeAg-specific CTL can be detected in the periphery and the liver during chronic infection,73,74 however, similar to B cell antibody production, CTL function is suboptimal and not sufficient to mediate viral clearance. It is anticipated that both of these nascent effector pathways can be enhanced by providing more efficient Th cell function using the WHcAg platform to present neutralizing, PreS1-specific B cell epitopes and HBcAg-specific CTL epitopes.
Accomplishments of the current study include:
Defined eight PreS1 B cell epitopes (highly conserved amongst HBV genotypes).
Inserted each of the eight PreS1 B cell epitopes onto WHcAg carrier VLPs.
Consolidated all eight PreS1 epitopes onto two PreS1-WHc VLPs (VLP-1.6 and VLP-1.9).
Demonstrated that PreS1-WHc VLPs elicited PreS1-specific Ab that recognizes native HBV virions and multiple L/M/S-HBsAg particles of both major serotypes (ad/ay).
Demonstrated that six PreS1-WHc VLPs elicited nAbs specific for N-terminal, central and C-terminal B cell domains of the PreS1 region, however, the central B cell domain elicited superior neutralizing Ab. A previous study of PreS1-HBc VLPs concluded that anti-PreS1 Abs specific for the N- and C-terminus were not viral neutralizing.61 This suggests that the fine specificity of the B cell response to the PreS1 region is variable and can be influenced by the choice of HBcAg or WHcAg carrier.
Demonstrated that PreS1-WHc VLPs circumvented immune tolerance and elicited equivalent levels of nAb to the PreS1 region in wildtype, HBV-Tg, HBc/HBeAg-Tg, and PreS1-Tg mice lineages.
Demonstrated that PreS1-WHc VLPs primed crossreactive, HBcAg-specific CD4+/CD8+ T cells as well as WHcAg-heterospecific CD4+/CD8+ T cells.
Demonstrated that in human-liver chimeric mice passive transfer of PreS1 nAbs prevented an acute HBV infection.
Demonstrated that in human-liver chimeric mice previously infected with HBV (model of chronic infection), PreS1 nAbs cleared serum HBV and may arrest HBV spread in the liver possibly reducing cccDNA levels.16
Produced hybrid WHcAg/HBcAg particles as DNA constructs, which elicited HBcAg-specific CD8+ CTL.
The relative scarcity of PreS1 antigen relative to the major HBsAg is a limiting factor for anti-PreS1 nAb production during a natural HBV infection. The capacity of the highly immunogenic WHcAg carrier to display multiple PreS1 neutralizing B cell epitopes overcomes this limitation. For example, 240 copies of each of the eight PreS1 B cell epitopes are displayed per PreS1-WHc VLP. A combined PreS1-WHc VLP-1.6/VLP-1.9 vaccine formulated in an adjuvant suitable for human use given in a prime/boost protocol with an optimized WHcAg/HBcAg DNA construct would represent a strong candidate therapeutic HBV vaccine. This vaccine would be capable of circumventing immune tolerance and eliciting multiple PreS1 nAb specificities as well as HBcAg-specific CD8+ CTL to target intracellular HBV DNA including cccDNA. Although a PreS1-WHc VLP prime – hybrid WHcAg/HBcAg DNA boost regimen could be given as a monotherapy, combination with an antiviral agent would enhance efficacy by reducing viral load. Inserting multiple neutralizing B cell PreS1 epitopes will mitigate the possibility of nAb escape mutants, which may be problematic when treating an established HBV infection. Bacterial production of PreS1-WHc VLPs together with a WHcAg/HBcAg DNA immunogen would be cost-efficient and compatible with any antiviral treatment for maximum efficacy. The ultimate goal is to produce both PreS1-WHc VLPs and WHcAg-HBcAg hybrid VLP DNA constructs for the dual purposes of eliciting PreS1-specific nAbs and HBcAg-specific CTL. The current study was limited primarily to evaluating the PreS1-specific humoral response in immune tolerant HBV-Tg mice and in human-liver chimeric mice infected with HBV. Future studies will be necessary to evaluate the ability of the WHcAg-HBcAg hybrid DNA constructs produced herein to circumvent HBcAg-specific CTL immune tolerance.
Although therapeutic HBV vaccines have not been successful to date, a number of experimental vaccines show promise in animal models or in the clinic,74,75 especially PreS-containing candidates.76,77 However, most experimental vaccines rely exclusively on the use of HBV-derived antigens, subject to the direct and indirect effects of immune tolerance, unlike the current PreS1-WHc VLP technology. An alternative approach for bypassing immune tolerant, dysfunctional endogenous T cells is the adoptive transfer of “engineered” HBV-specific T cells, which are demonstrating efficacy in animal models and in one clinical trial.78–80 In a recent study, a single adoptive transfer of transfected HBV-specific T cells demonstrated effective reduction in HBV DNA without severe liver disease in an animal model. However, achieving long-term control of HBV infection required the combined use of the PreS1 peptide viral entry-inhibitor Myrcludex B.16,78 We argue that regardless of the efficacy of any and all antiviral therapies, the induction of nAbs should be included to prevent the spread of HBV to uninfected hepatocytes, which may indeed be a requirement for the complete cure of CHB infection.
In addition to use as a therapeutic HBV vaccine, other possible applications for PreS1-WHc VLPs include: Use as a preventative vaccine in low-to-nonresponders to the conventional HBsAg vaccine; vaccination of pregnant HBV+ carrier mothers in order to provide passive transfer of PreS1-specific neutralizing Abs to block transmission during and after birth; an immunotherapy for chronic HDV infection; and prior to immunosuppressive therapy, vaccination of HBV+ liver transplant recipients in order to prevent infection of the new liver.作者: StephenW 时间: 2019-12-8 20:54