A Potentially Efficacious Approach for Treating HBV Viremia Using

Combinatorial Cocktail Treatment

Yifei Hu

, Junqing Yang

and Haotian Zhou

3,*

University of California-San Diego, 9500 Gilman Dr, La Jolla, CA 92093, U.S.A.

Lafayette College, Easton, PA 18042, U.S.A.

Northwestern Polytechnical University, Xian, 710129, China

Keywords:

HBV Viremia, Combinatorial Cocktail Treatment, Novel Immunotherapy.

Abstract:

Hepatitis B is one of the most prevalent diseases in the world and it covers a large population across the globe.

The main cause–hepatitis B virus (HBV) has been studied for over 50 years. It targets hepatocytes and utilizes

multiple mechanisms to replicate, survive and can cause acute or chronic infection. HBV viremia causes the

impairment of T cell function which is a hallmark of chronic infection, making it an intractable virus to

eliminate. Currently, common treatments for HBV viremia are using immunomodulatory agents such as

nucleoside analogues. However, these drugs only suppress viral protein production and viral replication.

HBsAg (hepatitis B surface antigen) remains on a certain level in serum and thereby the risk of hepatocellular

carcinoma cannot be eradicated. In this study, we mainly review 3 pieces of researches, each of which

introduces novel immunotherapy for chronic infection of HBV. And we hypothesize that the combinatorial

treatment would potentially increase the efficacy for treating HBV viremia by increasing the number of HBV-

specific CD8 T cells and would enhance the HBV-specific T cell functions, and further suppress the chronic

infection.

1 INTRODUCTION

Hepatitis B is a prevalent viral infection that targets

and injures the liver. As a potentially life-threatening

infection, it continues to be a significant global health

problem. Hepatitis B can cause both acute and

chronic diseases. Moreover, persistent infection

frequently results in decompensated cirrhosis and

hepatocellular carcinoma. According to WHO data,

more than 296 million people lived with chronic

hepatitis B infection in 2019, and the diseased

population increased by 1.5 million per year (World

Health Organization, 2021).

Hepatitis B is caused by HBV, a type of double-

stranded DNA virus in the family of Hepadnaviridae.

Some markers used for serologic detection of HBV

infection have been discovered, including HBsAg,

anti-HBs (antibody to HBsAg), IgM anti-HBc

(antibodies to hepatitis B core antigen), and IgG anti-

HBc (immunoglobulin class G anti-HBc). Moreover,

at least one of them is present during HBV infection,

which assists the detection or quantification of HBV

(Jennifer, 2015). Blood or infected bodily fluids are

two common ways for HBV transmission, and HBV

can survive (remains infectious) in the external

environment for at least seven days.

After HBV enters the blood, an irreversible

process of recognition and combination is followed.

Na+-taurocholate co-transporting polypeptide

(NTCP) plays an essential role in assisting HBV

entering the hepatocyte as the functional cellular

receptor. It binds to HBV, along with its protein shells

(Yan, 2012). Then, the initiation of the cccDNA

(covalently closed circular DNA) biogenesis is

conducted by HBV with the participation of cellular

DNA repair enzymes. After the cccDNA

minichromosome is formed, it provides all essential

viral RNAs and promotes viral replication and protein

production. A high level of proteins in the serum

maintains the chronic infection.

Although hepatitis B is vaccine-preventable, the

vaccine is not effective in infected patients. Current

treatments against hepatitis B include liver transplant,

immunomodulatory agents such as conventional or

pegylated type I interferons (interferon-α), and

nucleoside analogues (direct-acting antivirals).

Interferon-α produces several antiviral effects in

infected cells, including the production of antiviral

Hu, Y., Yang, J. and Zhou, H.

A Potentially Efﬁcacious Approach for Treating HBV Viremia Using Combinatorial Cocktail Treatment.

DOI: 10.5220/0012014000003633

In Proceedings of the 4th International Conference on Biotechnology and Biomedicine (ICBB 2022), pages 97-103

ISBN: 978-989-758-637-8

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

proteins and viral RNA suppression, but could only

result in a cure among a small group of patients

(Janssen, 2005; Lau, 2005). Nucleosides analogues

(NAs), on the other hand, serve as direct-acting

antivirals that can block the synthesis of viral DNA

and thereby suppresses viral replication. Nowadays,

NAs is considered as a well-tolerated drug that

impactfully inhibits the activity of HBV polymerase

with safety. However, nucleotide analogues could

only suppress viral replication. After cessation of

antiviral treatment, it has been found that the antiviral

function is significantly constrained by the

rebounding viremia and increasing resistance

mutations of the drug (Locarnini, 2006; Zoulim,

2009). For these reasons, they can hardly eradicate

HBV, so long-term treatment is required (Lannacone,

2021).

The functional exhaustion of virus-specific CD8

T cells is a significant trait of persistent HBV

infection (Klenerman, 2005). According to previous

researches on individual patients, impairment of

HBV-specific T cells under viral load was revealed.

Multiple essential cellular processes act

simultaneously, forming the functional and

quantitative defects of HBV-specific T cell response

(Lopes, 2008; Bengsch, 2014). In the model of

chronic infection to functional LCMV (lymphocytic

choriomeningitis virus)-specific effector, the

persistence of virus results in a hierarchical

impairment and finally a depletion of T cell functions,

with the sequential reduction of IL-2 production,

cytotoxicity TNF-a, and IFN-c production (Wherry,

2007). According to the conceivable explanation of

the dysfunction of HBV-specific T cells, the

alternative pathway–therapeutic vaccination is

regarded as a more efficient approach to induce

immune responses against HBV viremia.

Nevertheless, it failed to meet the expectations and

does not always show efficiency, according to several

studies (von, 2000; Dikici, 2003; Nevens, 2003).

Furthermore, limited effectiveness is revealed under

viral load conditions since the therapeutic vaccination

cannot potently stimulate the dysfunctional T cells to

produce antibodies (Wherry, 2005; Nisii, 2006).

To better understanding the mechanism and

treatment method of HBV Viremia, this paper will

introduce three key primary researches, each of which

puts forward a novel and potentially effective therapy

either for blocking HBV entry or reinforcing the

antiviral effects of HBV-specific T cells. A

combinatorial treatment is discussed, and the effect is

predicted based on reviewed primary researches.

2 PRIMARY RESEARCH

2.1 Enhancing Virus-Specific

Immunity in Vivo by Combining

Therapeutic Vaccination and PD-

L1 Blockade in Chronic

Hepadnaviral Infection (Jia, 2014)

It is suggested that one of the main causes of the T-

cell exhaustion is the interaction between the

inhibitory receptor programmed death-1 (PD-1) and

its ligands (PD-L1) (Barber, 2006). Figure 1 shows

the interaction structure of PD-1 and PD-L1.

Figure 1. Crystal Structure of the PD-1/PD-L1 Complex. Molecular that colored in blue is the PD-1; molecular that colored

in purple is the PD-L1. They form a dimeric complex (Barber, 2006).

ICBB 2022 - International Conference on Biotechnology and Biomedicine

The expression of PD-1 was found in different

human chronic infections, including HBV. In this

study, the T cell function was improved through

inhibiting the PD-1/PD-L1 in vivo in the WHV

model, and the study designed a combinatorial

therapy that included therapeutic vaccination,

traditional antiviral treatment of nucleosides

analogous ETV, and the blockade of PD-1 receptor

on T-cell, which was shown to be able to

continuously suppress WHV replication comparing

with NAs treatment or with therapeutic vaccination.

This study further examined the effect of this

combinatorial therapy in WHV transgenic mice,

which disrupts the immune tolerance to WHV

antigens and decreases viral load. The result

suggested that this is a potentially new approach for

producing efficacious therapeutic vaccination against

chronic infection of HBV.

2.2 A Practical Approach to

Immunotherapy of Hepatocellular

Carcinoma using T Cells

Redirected Against Hepatitis B

Virus (Koh, 2013)

It is shown that the current treatment for HCC cells

has excellent limitations, which induces exogenous

HBV-specific TCR by viral vectors. The process is

dangerous due to the non-exclusiveness of HBV

antigen. Adoptive T-cell therapy could cause severe

damage to the liver because normal hepatocytes may

also express HBV antigens. Moreover, it could

increase the risk of oncogenes activation, not to

mention the costs and complexity of the process.

These limitations could be overcome using mRNA

electroporation, which could rapidly produce a

significant number of HBV-specific T cells.

2.3 Blocking Entry of Hepatitis B and

D Viruses to Hepatocytes as a Novel

Immunotherapy for Treating

Chronic Infections (Maravelia,

2021)

Nucleoside analogues (NAs) are a commonly used

HBV therapy. By suppressing the reverse

transcriptase (of HBV polymerase), it can inhibit the

production and secretion of viral protein and the

biogenesis of cccDNA, which is the primary cause for

HBV chronicity. However, NAs only limit the

replication of the virus and requires long-term

treatment. It cannot eradicate the HBsAg in serum

and thus cannot avoid the risk of hepatocellular

carcinoma (HCC) (Papatheodoridis, 2011; Zoulim,

2012). During the chronic infection, an important

characteristic is the dysfunction of HBV-specific T

cell response (Chen, 2000; Chen, 2005; Chen, 2004;

Milich, 2016). With the overproduction of small

HBsAg, antibodies to small HBsAg are blocked,

which enhance the middle and large HBsAg,

containing PreS1, PreS2, etc (Short, 2009; Rydell,

2017). PreS1 domain is essential for transporting

HBV to hepatocytes (Ni, 2010; Ni, 2014). Therefore,

in this study, a novel therapy against HBV and

potentially HDV was designed, using a PreS1

sequence linked to the HDV antigen, circumventing

the dependence and participation on HBV-specific T

cell response, which is exhausted in chronically

infected hosts, in the purpose of inducing the

production of endogenous antibodies specific to the

PreS1 antigen. In this therapy, HDV serves as a

heterologous T-cell epitope carrier. It supports the

production of PreS1 antibodies and finally aims to

block the entry of HBV.

Nevertheless, the third approach will not be put to

use in the following research since we intend to

enhance the HBV-specific T cell response instead of

bypassing it. Nevertheless, blocking the entry of HBV

provides us with an innovative way to induce stable

loss of HBsAg that may be applied in the future.

Here, based on the researches above, we aim to

examine whether in vivo blockade of the PD-1

pathway and therapeutic vaccination in combination

with NAs treatment and mRNA electroporation anti-

HBV TCR on T cells could improve the outcome of

chronic HBV treatment and finally educe a solution

of chronic infection of HBV in the mice model. HBV

transgenic mice would first receive entecavir (ETV)

to suppress the viral replication. They would be

treated with therapeutic vaccination, followed by

introducing HBV-specific TCR on T cells (using

mRNA electroporation) and in vivo blockade of the

PD-1 receptor. We hypothesize that this

combinatorial treatment would increase HBV-

specific CD8 T cell number and enhance the immune

response of T cells.

3 MATERIAL AND METHODS

3.1 HBV Transgenic Mice

In this study, 1.3x HBV transgenic mice will be used

as the experiment model. Hepatitis B virus could

perform viral replication and produce HBV-infected

protein in the mice. With this advantage, the

measurement of HBV concentration will be easy to

A Potentially Efﬁcacious Approach for Treating HBV Viremia Using Combinatorial Cocktail Treatment

perform by examining the amount of HBV genome,

HBsAg, and HBeAg in the serum. The amount of

functional T cell concentration in the serum is also

able to be examined, providing a pathway to

determine the efficiency of anti PD-1/PD-L1 and

mRNA electroporation therapies.

3.2 Entecavir and Therapeutic Vaccine

Common and traditional treatments will be set for

multiple control or combinatorial groups to compare

the results and highlight the differences.

3.3 In vivo PD-L1 Blockade

Rat anti-mouse PD-L1 antibody (10F:9G2) will be

received by intraperitoneal injection. (5 times every 3

days.)

3.4 Electroporation of TCR

During electroporation, the host cell from peripheral

blood mononuclear cells will be suspended. TCR

mRNA is then added into the solution as the desired

material to be electroporated into the cell. The

mixture is placed in a cuvette. An electrical circuit is

placed closed around the mixture for the

electroporation process. An electrical pulse at a

specific voltage that only lasts a few microseconds is

discharged through the mixture. This process disturbs

the phospholipid bilayer of the cell membrane that

forms temporary pores around it. The increased

electric potential created during electroporation

across the membrane will allow mRNA to be driven

across the membrane through the pores into the

nucleus of the cell. After the electroporation, the cells

will be resuspended and cultured until analysis. Large

scale electroporation is done similarly, but with larger

amount of TCR mRNA (“Electroporation.” Thermo

Fisher Scientific – US; Potter, 2018; Koh, 2013).

3.5 HBV DNA Quantification

Platinum SYBR Green Kit will be used for real-time

PCR, to quantify the HBV DNA.

3.6 Analysis of cccDNA and HBV

Replication

The QIAamp Tissue Kit will be used to extract entire

DNA from transgenic mice. HBV replication will be

analyzed by Southern blot hybridization, and PCR is

once more to be conducted for determining HBV

cccDNA.

3.7 Evaluation of Glutamic Oxaloacetic

Transaminase Levels

According to standard diagnostic procedures in the

Central Laboratory of the University Hospital Essen,

the level of glutamic oxaloacetic transaminase is to be

measured. And it is thought to be promoted if the

values are greater than 50 international units per ml.

4 EXPERIMENTAL SEQUENCE

The experiment contains 9 groups of transgenic HBV

mice, each group has 5 mice in total with different

combinations of therapies. The experiment will last

25 weeks, and blood will be drawn from the tail of the

mice weekly to test the amount of HBV DNA and

TCR of the mice. All 9 groups of experiments will be

administered at the same time, and detailed data and

observations will be recorded after each treatment is

given to the mice and at the end of each week.

Group A is the mock trial that contains 5

transgenic HBV mice with no treatment, given.

Group B mice will be given ETV (oral gavage)

treatment. The appropriate dosage of ETV is given to

each mouse daily. For the previous 12 weeks, 0.2mg

of ETV will be given to each mice per day. For the

remaining weeks, the amount of ETV will be

increased to 1.5mg per week per mouse.

Group C mice will be given a total of 13 doses of

therapeutic vaccination, in which they will receive a

pre-treatment of cardiotoxin by intramuscular

injection a week before the start of the first dose of

vaccination. After that, they will receive one dose of

vaccination via intramuscular injection every two

weeks for 12 doses.

Group D mice will receive anti-PD1 mAB

treatment. 200 ug of anti-mouse PD-L1 antibody

(10F:9G2) will be received 5 times every 3 days.

Group E mice will receive electroporation

treatment. Peripheral blood will be drawn out from

each mouse to undergo electroporation of mRNA

TCR, then reinjected into the mouse. Electroporation

treatment will be administered at the beginning of

each week, and blood will be drawn at the end of each

week for testing.

Starting from Group F, mice will be administered

with mixtures of treatment. For Group F, mice will be

receiving ETV and therapeutic vaccination. ETV will

be given in the same dosage as Group B mice, with

0.2mg per mouse given daily for the previous 12

weeks and 1.5 mg throughout the week per mouse

given for the remaining weeks. Therapeutic

vaccination is also given to each mice similar to those

ICBB 2022 - International Conference on Biotechnology and Biomedicine

100

of Group C, in which 13 doses of intramuscular

injection treatments will be given. The two kinds of

treatments will be administered simultaneously.

Group G contains three treatments. The mice will

be receiving ETV and therapeutic vaccination in a

similar fashion as Group F, as well as anti-PD1

treatment similar to that of the Group D mice, in

which each mouse will be given 200ug of anti-mouse

PD-L1 antibody 5 times every 3 days. The three

treatments will be administered simultaneously.

Group H contains three treatments. In addition to

ETV and therapeutic vaccination treatment as the

mice from Group F, the Group H mice will also be

receiving electroporation of mRNA TCR treatment

similar to that of group E, in which the mice will be

administered one electroporation treatment per week,

simultaneously with the other two treatment.

The last group, group I, of mice will receive the

combination of all 4 kinds of treatments: the ETV,

therapeutic vaccination, anti-PD1, as well as

electroporation of mRNA TCR. The treatments will

be given independently and simultaneously of each

other, each in the same way as previously described

for the whole duration of 25 weeks.

5 PREDICTED RESULTS

For the mock group, normal viral replication and

damage to hepatocytes are expected.

For ETV treated group, we expect the viral

replication will be reduced as the treatment begin, and

the viral replication will rise again after stopping the

treatment.

For therapeutic vaccination, as mentioned in the

introduction, due to the large viral load in the model,

the effectiveness of the vaccination will be relatively

low, which should be shown as the HBV genome is

not decreased very much.

With known information on HBV, the expression

of PD-1/PD-L1 from HBV could exhaust the T-cells,

so for anti-PD-1 treatment, this inhibition of PD-

1/PD-L1 receptor could help reactivate the

dysfunctional T-cells and help to control the HBV

genome concentration in the blood sample. However,

we expect the unregulated viral replication will

decrease the efficiency of this inhibition process.

From the former study, HBV-specific TCR

mRNA electroporation treatment is expected to

increase the amount of HBV-specific T-cells in a

short period and help suppress the viral replication. In

this group, we also expect the number of active T cells

will increase as the treatment start and decrease

significantly after 72 hours20, and this reduction of

active T cells is assumed as the result of the PD-1

expression of HBV.

In the combinatorial treatment of ETV and

therapeutic vaccination, an increase of effectiveness

of therapeutic vaccination is expected which should

be indicated by the further decrease on HBV genome

compared to ETV group, but as the treatment stops a

bounce of the concentration of HBV DNA are

expected.

As we added anti-PD-1 treatment in the former

group, a prolonged suppression is expected as

indicated before. 18

However, when the mRNA electroporation of

HBV-specific TCR is added to the therapy, a further

decrease in HBV genome concentration in serum is

not very likely, because the new HBV-specific T cells

are still affected by PD-1/PD-L1 which leads to the

dysfunction of T cells.

For the final group, we expect to see the number

of HBV-specific T cells increase drastically, and they

will constantly exist and perform antiviral functions

to consistently decrease the HBV genome. Also,

because of the robust immune response caused by the

excess amount of T cells, the side-effect is possible to

be observed.

6 CONCLUSION

From the prediction of the result of the nine groups,

we conclude that control on the PD-1 receptor is

seemed to be the precursor of a prolonged and

consistent suppression on HBV because groups

without the regulation on PD-1 receptor are not able

to provide the model a constant decrease on HBV.

The combinatorial therapies work and possibly cure

only when the immune system works effectively. A

better understanding and strategy on regulating PD-1

receptors could help treat HBV and other related

viruses with similar mechanisms in later studies.

Also, there is a concern that if the number of T-cells

is too large and does not have a system to regulate

their quantity due to the TCR electroporation and

anti-PD-1 treatment, the immune system could not

activate or inactivate the T cells properly. Some data

suggested that PD-1 is an essential cause for the

exhaustion of T cells. Because immunotolerance is

highly preserved and autoimmunity is impeded as

well. In addition, among patients with cancer, the PD-

1 stoppage with monoclonal antibodies usually leads

to immune-related severe adverse events in various

tissues such as the liver, skin, and lung.

A Potentially Efﬁcacious Approach for Treating HBV Viremia Using Combinatorial Cocktail Treatment

101

ACKNOWLEDGEMENT

Yifei Hu, Junqing Yang, and Haotian Zhou

contributed equally to this work and should be

considered co-first authors.

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