Research progress of therapeutic vaccines for treating chronic hepatitis B

ABSTRACT

Hepatitis B virus (HBV) is a member of Hepadnavirus family, which leads to chronic infection in around 5% of patients with a high risk of developing liver cirrhosis, liver failure, and hepatocellular carcinoma.¹ Despite the availability of prophylactic vaccines against hepatitis B for over 3 decades, there are still more than 2 billion people have been infected and 240 million of them were chronic. Antiviral therapies currently used in the treatment of CHB (chronic hepatitis B) infection include peg-interferon, standard α-interferon and nucleos/tide analogs (NAs), but none of them can provide sustained control of viral replication. As an alternative strategy, therapeutic vaccines for CHB patients have been widely studied and showed some promising efficacies in dozens of preclinical and clinical trials. In this article, we review current research progress in several types of therapeutic vaccines for CHB treatment, including protein-based vaccines, DNA-based vaccines, live vector-based vaccines, peptide-based vaccines and cell-based therapies. These researches may provide some clues for developing new treatments in CHB infection.

KEYWORDS:

• chronic hepatitis B
• Hepatitis B virus
• therapeutic vaccine

Introduction

Hepatitis B virus (HBV) is the prototype member of the family Hepadnaviridae, which can infect only humans, apes, tree shrews (Tupaia belangeri) and recently discovered macaques.² It is reported that nearly 2 billion people worldwide have been infected with the HBV sometime, and an estimated 240 million people currently have chronic hepatitis B (CHB) infection. Meanwhile, HBV causes about one million people to die each year from HBV-related lived diseases, such as liver cirrhosis or hepatocellular carcinoma (HCC).^3,4 HBV infection in adult life is often clinically unapparent and most of the acutely infected adults recover spontaneously from the disease and completely clear or control the virus. Though only 5–10% HBV infected adults become persistently infected and chronic carriers, neonatally transmitted HBV infection is rarely cleared and about 90% of infected children become chronically.⁵

Since the successful launch of prophylactic vaccines against HBV infection in 1982, the incidence of HBV infection has dropped significantly.⁶ However, several million people are newly infected with HBV each year in different parts of the world, especially in developing countries. Prophylactic vaccines are ineffective at treatment in already infected HBV carriers or hepatitis patients.⁷ Dramatic improvements and progresses have been made in the development of antiviral drugs. The first approved therapy for chronic hepatitis B was a-interferon (IFN-a), which has both antiviral and immune modulatory effects against HBV. Combining with other antiviral or immunostimulant properties, IFN-a leads to a sustained suppression of HBV replication in nearly a third of patients. Nevertheless, the duration of interferon therapy is limited and the apparent adverse effect profile restricts its long-term application.^{8, 9}

Nucleotide and nucleoside analogs (NAs) are another group of potent orally administered inhibitors of HBV replication, which include lamivudine, adefovir, entercavir, tenofovir, telbivudine, and clevudine. These are effective in leading to a rapid inhibition of HBV replication, improvement of the necroinflammatory activity of liver diseases and lesser extent of fibrosis. However, nucleotide and nucleoside analogs lead to frequent relapse in the short term treatment and resistant viral variants in the long-term treatment, particularly in cirrhotic and immune-suppressed patients.^10-12 Additionally, the long-term safety profile of NAs therapy is still unknown. Previous study suggested that NAs might inhibit human DNA polymerase gamma involved in mitochondrial DNA replication. A reduction in intracellular mitochondrial DNA levels can lead to varying clinical manifestations of mitochondrial toxicity.¹³ In fact, it is reported that long-term clevudine therapy can induce the depletion of mitochondrial DNA and lead to mitochondrial myopathy associated with myonecrosis.¹⁴ Therefore, there is an urgent need to develop alternative approaches for chronic HBV infection to increase therapeutic efficacy as well as to limit viral resistance.

In the past decades, numerous efforts have focused on exploring therapeutic vaccines as possible alternatives to antiviral drugs and a-interferon in HBV chronically infected patients¹⁵ Though the immune determinants of successful clearance of HBV are still not fully understood, both the adaptive and innate immune responses are known to be involved in viral clearance during HBV infection.¹⁶ In fact, the natural history of HBV infection indicated that recovery from HBV infection is associated with restoration of HBV-specific immune response.¹⁷ The control of HBV by long-term treatment with (NAs) seems to be achieved partially by restoration of host immunity.¹⁸ Besides, there is a clear distinction in the profile of the immune responses between patients naturally resolve viral infection and those develop chronic infection. Patients with self-limited acute HBV display multi-specific antiviral CD4 and CD8 T-cell responses with a T-helper type 1 profile of cytokine production. On the contrary, patients suffering from chronic infection exhibit impaired and distorted immunity to HBV.^19-22 Hence, the rationale of immune therapy for the treatment of patients with chronic hepatitis B was provided. In this review, we outline the recent progresses and challenges of several types of therapeutic vaccines, including protein-based vaccines, DNA-based vaccines, live vector-based vaccines, peptide-based vaccines and cell-based therapies (as shown in Tables 1, 2) in treating chronic HBV infection. These preclinical and clinical researches may provide some clues for developing new cure in the treatment of CHB infection.

Table 1. Protein-based vaccines against chronic hepatitis B.

HBsAg-containing particles alone
Vaccine name	Antigen	Adjuvant	Antiviral treatment	Sponsor	Phase	Patients	Clinical outcomes	References
Recombinant Hepatitis B Vaccine	60μgHBsAg	Alum	—	Shenzhen Biokangtai, China	IND preparation	—	—	^23
Recombivax / GenHevac B	HBsAg OR PreS2/S	Alum	—	Unité d'Hépatologie, Hôpital Necker, Paris, France	Clinical exploration	118	No significant difference between vaccinated and unvaccinated groups on the rate of serum HBV DNA negativation and HBeAg seroconversion	^24
YIC	HBsAg/anti-HBs	Alum	—	Beijing Tiantan Biological, China	3	450	No difference between YIC group and placebo group in decreasing HBV DNA and normalization of liver function (p >0.05)	^25-30
HBsAg combines antiviral drugs
GenHevac	preS2/HBsAg	Alum	IL-7, Entecavir/Tenofovir	Cytheris SA	1/2a (Terminated)	24	No clinical results disclosed	ClinicalTrials.gov, Identifier: NCT01027065
Sci-B-Vac	PreS1/PreS2/S	Alum	Lamivudine	Department of Infectious Diseases and Department of Internal Medicine; University of Pharmacy and Medicine, Ho Chi Minh City, Vietnam / SciGen, Ltd, USA	4 (Terminated)	180	Safe, but no significant difference in HBeAg seroconversion and HBeAg loss rate	^31
Hepatitis B therapeutic vaccine	HBsAg	Alum	Entecavir, IFN-α-2b, IL-2	Tongji Hospital, China	4	100	Ongoing, but not recruiting participants; No clinical results disclosed	ClinicalTrials.gov, Identifier: NCT02360592
Pegasys / Euvax B / Baracrude	HBsAg	Alum	Entecavir, PEG-IFN-α-2a	Seoul National University	4	150	Recruiting participants; No clinical results disclosed	ClinicalTrials.gov, Identifier: NCT02097004
HB-AS02V	HBsAg	AS02B	Lamivudine	GSK	3 (Terminated)	195	No significant difference in HBe/anti-HBe seroconversion	^32
HBsAg/HBcAg based anti-HBV vaccines
DV601	HBsAg+HBcAg	ISCOX	Entecavir	Dynvax Technologies Corporation, United States	1b (Terminated)	30	Safe and well tolerated. Immunologic and virologic responses were observed in all dosage groups	^33, ClinicalTrials.gov, Identifier: NCT01023230
NASVAC	HBsAg+HBcAg	Alum	PEG-IFN	CIGB,Cuba	2b/3	160	Antiviral effect of NASVAC was superior to PEG-IFN 24 weeks after the end of treatment	^34
ABX203	HBsAg+HBcAg	Alum	Nucleos(t)ide	ABIVAX,France	2b/3	276	Safety but unlikely to reach the primary end point	ClinicalTrials.gov, Identifier: NCT02249988
TVAX-008	HBsAg+HBcAg	CpG	—	Theravac, China	Preclinical	—	—	^35
Other protein vaccines
GS-4774	Heat-inactivated yeast cells that expressed HBsAg, HBcAg and HBx	—	Oral antiviral (OAV)	Gilead Sciences, United States	2	178	Safe and well tolerated, but no clinical benefit	^36

Table 2. DNA/Live vector/Peptide-based vaccines/Cell-based therapies against chronic hepatitis B.

Other vaccines
Vaccine classification	Vaccine name	Components	Adjuvant	Antiviral treatment	Sponsor	Phase	Patients	Clinical outcomes	References
DNA vaccines	ANRS HB02 (pCMV-S2.S)	DNA vaccine encoding small (S) and middle (preS2+S) proteins	—	–	Institut National de la Santé Et de la Recherche Médicale, Institut Pasteur, France	1/2	70	No serious side effects, but incapable of controlling the virus recurrence and recovery of anti-HBV immune response,	^37, 38 ClinicalTrials.gov, Identifier: NCT00536627
	INO-1800	DNA plasmids encoding HBsAg and HBcAg	—	Nucleos(t)ide	Inovio Pharmaceuticals	1	126	Recruiting participants; No clinical results disclosed	ClinicalTrials.gov, Identifier: NCT02431312
	Dual-plasmid Hepatitis B Virus DNA Vaccine	DNA plasmid encoding PreS2-S	Plasmid IL-2/IFN-γ	Lamivudine	The 458 Hospital of Chinese PLA	2b	222	Fail to show significant difference in drug resistance and viral breakthrough	http://finance.sina.com.cn/roll/2016–03–30/doc-ifxqsxic3629642.shtml
	GX-100 (HB-100)	Therapeutic adenoviral-based DNA vaccine encoding S1/S2/S envelope gene, core, polymerase (Pol) sequences and X proteins	IL-12	Lamivudine	Genexine, Inc.	1 (Terminated)	12	Approaching 50% of patients showed a sustained viral suppression, obvious T-cell responses, especially CD4+ memory T-cell responses without ALT (alanine aminotransferase) increasing	^39
	GX-110 (HB-110)	2nd-generation therapeutic adenoviral-based DNA vaccine encoding S, L, core, polymerase protein	IL-12	Adefovir	Genexine, Inc.	1/2a	27	No adverse effects were observed by HB-110 co-treated with ADV. However, HB-110 in Korean patients exhibited weaker capability of inducing HBV-specific T-cell responses and HBeAg seroconversion than HB-100 in Caucasian patients.	^40
Live vector-based vaccines	TG1050	Non-replicative adenovirus serotype 5 encoding an unique large fusion protein composed of a truncated HBV core, a modified HBV polymerase and 2 HBV envelope domains	—	Tenofovir or Entecavir	Transgene Tasly, China	1/1b	96	Recruiting participants; No clinical results disclosed	ClinicalTrials.gov, Identifier: NCT02428400
	AIC649	Inactivated parapoxvirus ovis (iPPVO) particle preparation	—	Tenofovir	Aicuris	1	—	No clinical results disclosed	^41
Peptide vaccines	Theradigm-HBV (CY-1899)	HBcAg 18–27, tetanus toxoid-derived peptide 830–843, palmitoylated	—	–	Epimmune	2 (Terminated)	90	No serious adverse effects were observed, but mean CTL responses were low in all participants and peak CTL responses never exceeded 10 lytic units (LU) regardless of vaccine dose	^42
	Therapeutic (synthetic peptide) hepatitis B vaccine ϵPA-44,	PreS2 18–24, HBcAg18–27, TT830–843	—	Entecavir	Third Military Medical University, China	2	378	No significant difference between experimental and control group	^43
Cell-based therapies	DCs vaccines	HBsAg plused DCs	—	Lamivudine	Research Institute for Viral Hepatitis, Chongqing University of Medical Sciences	1	19	11 of 19 patients had a clinical response to DC-treatment. 10 of 19 patients had HBeAg seroconversion and the copies of HBV DNA decreased 10^1.77 ± 2.39 averagely	^44
	T cell vaccination	Expressing specific CAR (chimeric antigen receptor)	—	–	–	Clinical exploration	—	—	^45

Protein-based vaccines

HBsAg-containing particles alone

Since the successful launch of prophylactic vaccine in 1982, conventional HBsAg-based vaccine has significantly decreased the incidence of HBV infection. However, nearly 10% population is unable to generate adequate antibody level to hepatitis B surface antigen.⁴⁶ Recently, a China Biotech company (Shenzhen Biokangtai Co., Ltd) has successfully launched a large-dose prophylactic vaccine containing 60μg recombinant HBsAg, which aims to those who were unresponsive to the traditional low-dose vaccine. Preclinical trial indicated 60 μg HBsAg could generate significantly higher common stimulating factors CD80, CD86, and I-E^k than that of the control group in transgenic mice. Besides, the proliferation rate of specific T-lymphocyte and cytokines production in mice vaccinated with 60 μg HBsAg was significantly increased.²³ Now Biokangtai is planning to initiate IND in China for CHB infection treatment. In another trial, 118 patients were enrolled to evaluate the safety and efficacy of prophylactic HBsAg-based vaccines as an immunotherapy for CHB patients. Patients were given 5 intramuscular injections either preS2/S vaccine (GenHevac B, Pasteur-Merieux) or S vaccine (Recombivax, Merck & Co.) or no treatment as a control. Interestingly, HBV vaccines significantly decreased HBV viral load in vaccinated subjects between 6–12 months. A higher rate of serum HBV DNA negativation was achieved in 2 vaccine groups after the first 3 injections, though there was no difference after 5 injections compared with the control group. HBeAg seroconversion rate between vaccinated and unvaccinated subjects were 13.3% and 3.6%, respectively. The clearance of serum HBsAg was not observed in any of the patients.²⁴

An yeast-derived-immunogenic complex (YIC) consists of yeast-derived recombinant HBsAg and human anti-HBs immunoglobulin, was developed by Wen et al., as an possible approach for CHB treatment.²⁵ Phase I and phase IIstudies indicated that YIC was safe and have great potential in treating CHB.^26-28 In the following phaseIIb clinical trial, 242 CHB patients were injected 6 times with either 30 μg YIC, 60 μg of YIC or alum adjuvant as placebo within 24 weeks.²⁹ Though YIC did not reach the primary and secondary endpoints in phase IIb, one group vaccinated with 60 μg of YIC showed a late and promising effect in HBeAg seroconversion. The HBeAg seroconversion rate of 60 μg YIC vaccinated and placebo groups was 21.8% and 9%, respectively (p = 0.03). Therefore, the efficacy of YIC was further evaluated in the following trial. 450 patients were enrolled in phase III clinical trial, all of them were injected 12 times within 24 weeks, alum adjuvant was used as placebo. However, the HBeAg seroconversion rate decreased from 21.8% (phase IIb) to 14.0% (phase III) in the YIC group, while the HBeAg seroconversion rate increased from 9% (phase IIb) to 21.9% (phase III) in placebo group. Besides, there was no difference between YIC group and placebo group in decreasing HBV DNA and normalization of liver function (p > 0.05). Immune fatigue caused by excessive YIC immunization might be the potential reason for this failure.³⁰

These trials indicated that immunotherapy simply based on prophylactic HBsAg alone might generate the immune response and seroconversion in some individuals. However, the induced immune responses were limited in most of the patients. Therapeutic vaccines targeting only HBsAg might not be sufficient to achieve the desired effects.

HBsAg combines antiviral drugs

Though conventional HBsAg-based vaccines were capable of inducing HBV-specific immune response and reducing the viral load, no significant effects were found in the clearance of HBV DNA and HBeAg seroconversion..^{47, 48} Previous studies have showed that lamivudine (LAM) has the capacity to restore specific immune responses in CHB patients.^{49, 50} A combination of antiviral drugs and therapeutic vaccines also exhibited sustained therapeutic effects in animals.^{51, 52} In a phase IV trial, Pre-S1/Pre-S2/S vaccine (Sci-B-Vac) was combined with LAM to evaluate the efficacy of viral suppression in CHB patients. Sci-B-Vac + LAM vaccinated group achieved greater viral suppression and anti-HBs response than those vaccinated with Sci-B-Vac monotherapy and LAM monotherapy, however, this effect disappeared after 18 months. Though this new combination therapy was safe, there was no significant difference in HBeAg seroconversion and HBeAg loss among these groups.³¹

In a phase III trial, 195 CHB patients received either 12 injections of HB-AS02V (HBsAg/AS02B adjuvant) + LAM daily, or LAM only within 52 weeks. Both combination therapy and control group was safe and well tolerated, however, there was no significant difference between them. The HBeAg seroconversion rate and HBeAg loss of combination therapy and control group was 18.8% vs. 16.1% and 21.3% vs. 18.5, respectively.³² Currently, the efficacy and safety of another 2 candidate therapeutic vaccines, hepatitis B vaccine/ interferon-α2b/ interleukin 2/ entecavir and hepatitis B vaccine / PEG-IFN-α2a/ entecavir, are evaluating in phase IV trials. (clinicaltrials.gov, Identifier: NCT02360592, NCT02097004).

Taken together, these trails suggested that the combination of HBsAg and antiviral drugs lead to seroconversion and a rapid inhibition of HBV replication, however, the induced immune responses disappeared shortly after the end of treatment in most of the patients.

HBsAg/HBcAg based anti-HBV vaccines

The rational of incorporating hepatitis B core antigen (HBcAg) as a component of candidate therapeutic vaccine has been well documented: 1) HBcAg could inhibit virus infection by activating the specific CTL response, inducing specific T-cells and production of anti-HBs antibody. 2) HBcAg activates B cells to work efficiently as primary antigen presentation cells (APCs) and has a synergistic effect on antibody production and cellular responses when co-administered with HBsAg. 3) Previous studies suggested that the unbalance of Th1/Th2 might be responsible for the failure of therapeutic vaccines to induce a persistent and sufficient CTL response. HBcAg combines HBsAg could enhance humoral and cell immunity, restoring the balance of Th1/Th2 response.^53-56

One candidate therapeutic vaccine consisting of HBsAg, HBcAg and saponin-based ISCOMATRIX adjuvant, DV-601, was developed by Dynavax Co., Ltd. The experiments on mice suggested that DV-601 could induce HBV-specific T cells and B cells responses, breaking tolerance in HBV transgenic mice without liver damage.⁵⁷ In a phase Ib clinical trial, 30 patients received entecavir daily and 6 injections of DV-601(0.1, 0.25 or 0.5 ml) on Day 1, 15, 29, 57, 71 and 85 within 3 months. The results indicated DV601 was safe and well tolerated. Besides, immunologic and virologic responses were observed in all dosage groups, fulfilling the trial's primary and secondary endpoints.³³

The efficacy of another candidate therapeutic vaccine consisting of HBsAg, HBcAg and traditional alum adjuvant (CIGB Cuba, NASVAC) was also evaluated on both animal and human. Experiments on mice suggested the NASVAC was highly immunogenic and well tolerated. In particular, HBcAg acts as a Th1 adjuvant and has a synergistic effect on antibody production and cellular responses when co-administered with HBsAg.^{55, 58, 59} In the Phase I clinical trial, 19 healthy male adults were enrolled to evaluated the safety profile and immunogenicity of NASVAC for nasal administration. The participants received either NASVAC (50 μg HBsAg and 50 μg HBcAg) or placebo (0.9% physiologic saline) on day 0, 7, 15, 30, and 60, respectively. The vaccine elicited anti-HBc seroconversion in 100% of subjects as early as day 30 of the immunization schedule and all subjects in the placebo group remained seronegative during the trial. These results indicated NASVAC was safe and well tolerated. Only mild side effects was found, such as sneezing (34.1%), rhinorrhea (12.2%).⁶⁰ In the following phase IIa trial, 18 patients received 10 doses NASVAC containing 100μg HBsAg and 100μg HBcAg. Another 10 untreated CHB patients were enrolled into the study as control group. Nine patients of vaccinated group (50%) achieved sustained HBV DNA negative, and the ALT level of all 18 patients (100%) remained persistently normal. The peripheral blood mononuclear cells (PBMC) and antigen-pulsed dendritic cells (DCs) from HBsAg/HBcAg-vaccinated group generated significantly higher levels of various cytokines than control group (p<0.05) after stimulation with HBsAg/HBcAg in vitro. These results indicated that NASVAC was safe and efficiently overcome the immune tolerance in CHB patients.⁶¹ In the following phase IIb/III clinical trial, 160 CHB patients were enrolled, 151 of them completed the trial. 75 patients received 100 μg HBsAg and 100 μg HBcAg 5 times. The other 76 patients received 180 μg PEG-IFN once a week for 48 weeks as control. The number of patients who became HBV DNA negative after receiving NASVAC and PEG-IFN were 37 vs. 48, and the number of patients who expressed normal value of ALT between NASVAC and PEG-IFN group were 46 vs. 36. Although in both groups a virological relapse was found, it was more dramatic in the PEG-IFN group. Besides, the antiviral effect of NASVAC was superior to PEG-IFN 24 weeks after the end of treatment.³⁴ In 2014, NASVAC was licensed to Abivax and renamed as ABX203. In the following multicenter phase IIb/III clinical trial, the efficacy of ABX203 vaccine as an adjunct therapy to nucleos(t)ide analogs (NUCs) was evaluated in maintaining control of hepatitis B disease after cessation of treatment with NUCs in subjects with HBeAg negative CHB. (ClinicalTrials.gov, Identifier: NCT02249988). Though the clinical trial was not officially terminated, a report posted on ABVAX official website stated that ABX203 was safe but unlikely to reach the primary end point of the study. Further development of ABX203, including the addition of an adjuvant, new administration schedules and therapeutic combinations was under review.

Previous study indicated that the unmethylated CpG ODN (CpG oligodeoxynucleotide) could induce humoral and cellular specific immune responses when co-administrated with vaccine antigens by activating TLR9 signal pathway. Besides, CpG ODN could directly active B cells and plasmacytoid dendritic cells, inducing production of Th1 and proinflammatory cytokines.⁶² In a phaseI/II clinical trial, the immunogenicity, safety and tolerability of CpG ODN as an immunoadjuvant combined with recombinant HBsAg-vaccine (Engerix-B, GlaxoSmithKline) was evaluated. The disclosed data indicated CpG ODN was well-tolerated and significantly increasing vaccine immunogenicity when co-administrated with Engerix-B compared with HBsAg-vaccine used only or HBsAg-alum vaccine.^{63, 64} Recently, the safety and efficacy of another novel therapeutic vaccine consisting of HBsAg, HBcAg and adjuvant CpG ODN has been evaluated on transgenic mice. The result suggested that HBsAg/HBcAg/CpG vaccine formulation could generate vigorous HBV-specific humoral and cellular immune responses, overcome tolerance in HBV transgenic mice. Notably, the induction of CpG adjuvant not only restores intense Th1 responsiveness, but also promotes a Th1-biased response against HBcAg and a Th1/Th2 balance response against HBsAg in both C57BL/6 and HBV transgenic mice, which is critical for a potential HBV therapeutic vaccine to achieve desire efficacy.³⁵

Other protein vaccines

GS-4774 is a recombinant yeast-based biological product engineered to express HBV antigens. The product is a heat-killed yeast (saccharomyces cerevisiae) containing a chimera of HBV X, Score, and Core antigens. In a 3-arm, randomized, open-label, dose-escalation (10, 40, 80YU) phaseI trial, 60 healthy adults were enrolled to assess the safety, tolerability and immunogenicity of GS-4774, and the results suggested GS-4774 was safe and well tolerated.⁶⁵ In the following phase II study, 178 patients who were virally suppressed on an oral antiviral (OAV) for one year were randomized (1:2:2:2) to continue OAV alone or receive OAV plus GS-4774 subcutaneously every 4 weeks until week 20. OAV was continued for the remainder of the study. The trial indicated GS-4774 was safe and well tolerated in CHB patients receiving oral antiviral therapy, but did not result in therapeutic benefit.³⁶

DNA-based vaccines

Due to the advantages of inducing both humoral immune responses and strong cellular immune responses including CD8+ and CD4+ T cell responses, DNA-based vaccines against HBV have been investigating by researchers around the world.⁶⁶ In a phase I clinical trial, 10 patients with chronic active viral hepatitis were injected 1 mg of pCMV-S2.S DNA vaccine, encoding HBV small (S) and middle (preS2+S) envelope proteins, at M0, M2, M4 and M10. Immunizations were well tolerated and adverse effects were mild and considered unrelated to the vaccine. Following 3 injections of vaccine, interferon (IFN)-gamma-producing T-cells specific for the preS2 or the S antigen were detectable in 50 and 100% of the patients, respectively. Serum HBV DNA levels decreased in 5 patients and complete clearance was observed in 1 patient, but the effect did not sustained after the final injection. In conclusion, pCMV-S2.S DNA vaccine is safe and capable of activating and restoring the T-cell responses in some CHB carriers, but the action is transitory and weak.^{67, 68}

In another multicenter phase I/II clinical trial, 70 patients treated effectively with NAs for a median of 3 y were enrolled to investigate the efficacy of pCMV-S2.S DNA vaccine in preventing viral recurrence. Participants were randomized in 2 groups: one group administrated 5 intramuscular injections of pCMV-S2.S DNA vaccine at week 0, 8, 16, 40, 44, and the other group did not receive the vaccine as control. NAs were stopped after an additional 48 weeks of treatment in patients who maintained HBV DNA <12 IU/mL with no clinical progression and monthly HBV DNA for 6 months. This trial showed that pCMV-S2.S DNA vaccine was safe, but the relapse occurred in 97% of each group after a median 28 d. The pCMV-S2.S DNA vaccine did not decrease the rate of recurrence or virological breakthrough in HBV-treated patients, and did not restore the anti-HBV immune response despite effective viral suppression by NAs.^{37, 38}

INO-1800 is another candidate HBV therapeutic DNA vaccine initiated by Inovio Pharmaceuticals. In a phase I, randomized, open label, active-controlled, dose escalation trial, 126 NAs treated patients were enrolled to evaluate the safety, tolerability and immunogenicity of dose combination of INO-1800 (DNA plasmid encoding HBsAg and HBcAg) and INO-9112 (DNA plasmid encoding human interleukin 12) delivered by electroporation (EP). Currently, the study is recruiting participants (ClinicalTrials.gov, Identifier: NCT02431312).

A dual-plasmid vaccine (plasmid encoding PreS2-S and the adjuvant plasmid IL-2/IFN-γ) against the HBV mediated by in vivo electroporation, was evaluated in a total of 39 HBeAg-positive CHB patients. The participants were divided into 3 groups: DNA vaccine monotherapy, LAM monotherapy (LAM+placebo) and LAM+DNA vaccine group. DNA vaccine monotherapy group showed a significant elevation of HBV-specific IFN-gamma-secreting T-cell counts in comparison with baseline. Besides, the rate of patients with HBV DNA suppression was higher in LAM+DNA vaccine group than LAM monotherapy group at each visit time point after the final injection, achieving a significant difference between the 2 groups (P = 0.03) at week 60. In conclusion, the trial suggested that the dual-plasmid vaccine was safe and immunologically effective, and the combination of DNA vaccine and LAM achieved a significant higher positive T-cell response rate (P = 0.03) and a lower virological breakthrough (VBT) rate (P = 0.03) than LAM monotherapy in CHB patients.⁶⁹ However, in the following phase IIb trial, the dual-plasmid vaccine failed to achieve significant efficacy in drug resistance and viral breakthrough.

HB-100 is a therapeutic adenoviral-based DNA vaccine, which encodes S1/S2/S envelope gene, core, polymerase (Pol) sequences, X proteins of HBV and human IL-12 as adjuvant. In a phase I study, 12 CHB patients were enrolled to evaluate the safety of intramuscularly administered HB-100 combined with oral antiviral (Adefovir) over a 48-week period. Nearly 50% of patients achieved a sustained viral suppression and an obvious T-cell responses, especially CD4+ memory T-cell responses.³⁹ HB-110 is a 2nd-generation HBV therapeutic adenoviral-based DNA vaccine, containing an IL-12 gene immunofusion. In a phase I trial, HB-110 was delivered by EP to increase the membrane-penetrability and enhancing immune response. 27 CHB patients randomly received either adefovir dipivoxil (ADV) alone or ADV in combination with HB-110. No adverse effects were observed by HB-110 co-treated with ADV. However, HB-110 in Korean patients exhibited weaker capability of inducing HBV-specific T-cell responses and HBeAg seroconversion than HB-100 in Caucasian patients. The high rate of vertical HBV transmission in Asian patients might explain the higher level of immune tolerance than Caucasian. Therefore, therapeutic HBV DNA vaccines should focus on breaking immune tolerance rather than enhancing immunogenicity.^{40, 70}

Live vector-based vaccines

Live vector-based vaccines carry DNA into a host cell for production of specific antigens, stimulating a wide range of immune responses. Besides, unlike the traditional plasmid DNA vaccines, live vector-based vaccines have the potential to actively invade host cells, replicate and activate the immune system like an adjuvant. Parapoxvirus, adenovirus and herpes virus were often used to produce live vector vaccines against HBV and many other diseases.^{71, 72} TG1050 is an adenovirus-based vaccine, encoding a unique large fusion protein composed of a truncated HBV core, a modified HBV polymerase and 2 HBV envelope domains. Preclinical study indicated that TG1050 could induce robust and long-lasting HBV-specific T cells and exert an antiviral effect in HBV-persistent mice.⁷³ The sponsor of TG1050, Transgene Tasly, is initiating a double-blind, randomized, placebo-controlled, multi-cohort Phase 1/1b trial in patients to assess the safety and tolerability of TG-1050. Currently, the study is recruiting participants (ClinicalTrials.gov, Identifier: NCT02428400).

AIC649 is an inactivated parapox virus (iPPVO) which has the capacity to modulate cytokines release and active T-cell responses. HBV transgenic mice administered with AIC649 twice weekly showed antiviral effect similar to those with tenofovir twice daily. In the chronically woodchuck hepatitis virus (WHV) infected woodchucks system, the HBsAg of AIC649-treated group first increased but then decreased even after the termination of treatment to significantly reduced levels.⁴¹ The sponsor of AIC649, AiCuris, is currently testing AIC649 in a phase I study in CHB patients.

Peptide-based vaccines

Peptide-based vaccines incorporate one or more amino acid sequences as antigens, eliciting protective immunity against the microbe or virus. Theradigm-HBV (alternative names: CY 1899), a peptide-based vaccine against HBV, is consist of HBV core protein CTL epitope (HBcAg 18–27), T-helper cell epitope (tetanus toxoid-derived peptide 830–843) and palmitic acid residues. In a pilot trial, 90 CHB patients were administered up to 4 doses vaccines (ranging from 0.05 mg to 15 mg) 6 weeks apart. Although no serious adverse effects were observed, mean CTL responses were low in all participants and peak CTL responses never exceeded 10 lytic units (LU) regardless of vaccine dose. The CTL activity induced by Theradigm-HBV was of a magnitude lower than that observed during spontaneous HBV clearance. This low-level CTL activity could not lead to viral clearance.⁴²

ϵPA-44, another peptide-based therapeutic vaccine against HBV, is consist of immunodominant B cell epitope of PreS2 18–24 region, the CTL epitope of HBcAg18–27 and the universal T helper epitope of tetanus toxoid (TT) 830–843. In vitro study indicated that ϵPA-44 could induce specific CD8+ T cell expansion and vigorous HBV-specific CTL-mediated cytotoxicity in human PBMCs.⁷⁴ However, in the following phase II clinical trials, ϵPA-44 failed to show significant efficacy in treating CHB patients either by administered alone or in combination with entecavir.⁴³ Although peptide-based vaccines were well defined and easy to produce, only weak immune response were induced in the absent of appropriate immunostimulants or adjuvants.

Cell-based therapies

Dendritic cells (DCs) offer an essential link between innate and adaptive immunity, which can induce such contrasting states as immunity and tolerance. The use of antigen-pulsed DCs as immunotherapy for cancer has been well documented.⁷⁵ In one clinical study, 19 patients were given HBsAg-pulsed DCs vaccine subcutaneously twice, 2 of them were co-administrated with LAM 100 mg daily for one year at the same time. 11 of 19 patients had a clinical response to DC-treatment. 10 of 19 patients had HBeAg seroconversion and the copies of HBV DNA decreased 10^{1.77 ± 2.39} averagely.⁴⁴ In another trial, 5 human healthy volunteers with no apparent concomitant diseases were enrolled. A single administration of HBsAg-pulsed DCs leads to upregulation of anti-HBs in 2 anti-HBs positive volunteers and 2 anti-HBs negative volunteers with no physical, biochemical, and immunological abnormalities documented.⁷⁶

Adoptive T cell therapy is an effective treatment of viral infections and has induced regression of cancer in early-stage clinical trials.^{77, 78} Clinical study has demonstrated that the transfer of HBV-specific memory cells from an immune donor through bone marrow transplantation can induce the seroconversion in patients with CHB.⁷⁹ However, the difficulty of obtaining large scale HBV specific T cells from HBV-infected patients hinders the application of this strategy. As alternative approaches, engineer T cells with pre-defined specificity and chimeric antigen receptor (CAR) technologies were developed.⁴⁵ By grafting autologous T cell with chimeric T-cell antigen receptors directed against HBsAg present on HBV-infected cells, chimeric receptors enable primary human T cells to recognize HBsAg-positive hepatocytes, release IFN-γ, IL-2, and lyse HBV replicating cells. Additionally, when coincubated with HBV-infected primary human hepatocytes, these engineered antigen-specific T cells specifically eliminate HBV-infected and thus cccDNA-positive target cells.⁸⁰ Although cell-based therapies in treating CHB are very promising, developing individualized treatment on a large scale is still a problem, given the high cost and the complex on-site requirement for production and application.

Concluding remarks

Hepatitis B virus (HBV) is a major causative agent for public health problem worldwide. People with chronic hepatitis B infection are at higher risk of liver cirrhosis, liver dysfunction and hepatocellular carcinoma.³ Traditional therapies fail to provide sustained containment of viral replication and have risk of liver damage in some patients⁸ As an alternative strategy, immunotherapeutic approaches have become a research hotspot because of its low cost, safety and promising effects in the treatment of CHB patients.⁷ Though cell-based therapies in treating CHB are very promising, the high cost and the complex on-site requirement for production hamper its large scale application. The fields of epitope selection and vaccine design are still at the exploratory stage. Clinical studies suggested peptide vaccines were not able to induce strong cellular immunity responses.,^{42, 74} Previous studies have shown that DNA vaccines are able to induce strong cellular immune responses, overcoming immunotolerance in animals, however, this efficacy is transitory and weak in patients. Since the launch of prophylactic HBV vaccines, many trials, including increasing vaccine doses, changing immunization scheme, combination of traditional antiviral drugs, addition of PreS1/PreS2 antigens or Th1-biased adjuvants, have been made in an attempt to overcome the tolerance in CHB patients. Though intense humoral immunity responses were observed in most of these trials, robust cell-mediated anti-viral immunity has not been achieved in CHB patients.^{23-26, 31, 32, 35, 55, 57, 61} It seems that the selection of a robust Th1-biased adjuvant and combination of appropriate HBV antigens are critical to achieve the desire efficacy.

In addition to the therapeutic vaccine strategies described above, new means like therapeutic antibody against HBV was also evaluated. Zhang et al. found that a novel mAb E6F6 could profoundly suppress the levels of HBsAg and HBV DNA for several weeks in HBV transgenic mice.⁸¹ Eight CHB patients, who had received long-term NAs treatment, were injected with anti-HBsAg immunoglobulin (HBIG) as an additional treatment. After one year of treatment, 3 patients became anti-HBs positive, implying HBIG might benefit CHB patients.⁸² Recently, Yan et al. found that sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver, is the functional receptor for HBV and HDV.⁸³ These findings provided new insight into understanding the mechanism of antibody against HBV and offered a new direction for developing effective treatment strategies for HBV.

At the present stage, all of the therapeutic hepatitis B vaccines listed here are at experimental stage, each of them may have its own advantages and limitations. Nevertheless, with the increasing understanding of the mechanism of CHB infection, the emerging of more efficient Th1-biased adjuvants, and more data achieved from both preclinical and clinical trials, it is possible to work out new ways in designing and developing effective therapeutic vaccines against CHB infection.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.