CdL Molecular and Medical Biotechnology

Molecular Farming thesis

2018/2019

Recombivax-HB
Francesco Gualdi VR433226

Introduction

RECOMBIVAX HB® is the brand name of Hepatitis B Vaccine (Recombinant) It is a non-infectious

subunit viral vaccine consisting of surface antigen (HBsAg or Australia antigen) of hepatitis B virus
produced in Saccharomyces cerevisae cells. A portion of the hepatitis B virus gene, coding for HBsAg,
is cloned into yeast and the vaccine for hepatitis B is produced from cultures of this recombinant
yeast strain.

The structure of HBsAg is known to low resolution only. At a basic level, HBsAg is a glycosylated
integral membrane protein that has been localized to ER, pre-Golgi, and late endosomal membranes.
HBsAg comes in three sizes, small, medium, and large, referred to as S-HBsAg, M-HBsAg, and L-HBsAg.
The L-HBsAg has three domains, in order, pre-S1 (108 or 119 residues), pre-S2 (55 residues), and the
S domain (226 residues). M-HBsAg, which is not required for infection, lacks pre-S1. S-HBsAg is
comprised of only the S domain which is composed of four trans-membrane helices. The S domain
forms disulfide crosslinked dimers, allowing formation of homo- and heterodimers. The Structural
Biology of Hepatitis B Virus: Form and Function

Hepatitis B: Epidemiology

The hepatitis B virus (HBV) is a small DNA virus with unusual features similar to retroviruses, based
on sequence compari- son, HBV is classified into eight genotypes, A to H The viral genome encodes
four overlapping open reading
frames (ORFs: S, C, P, and X). The S ORF encodes the viral surface envelope proteins, the HBsAg, and
can be structurally and functionally divided into the pre-S1, pre-S2, and S regions. The core of the C
gene is divided in precore and core regions and is responsable of the production of a viral
nucleocapsid HBCAg or hepatitis B e Antigen (HBeAg) depending on wheter translation is initiated
from.(1)

The polymerase (pol) is a large protein (about 800 amino acids) encoded by the P ORF and is
functionally divided into three domains: the terminal protein domain, which is involved in encapsida-
tion and initiation of minus-strand synthesis; the reverse transcriptase (RT) domain, which catalyzes
genome syn- thesis; and the ribonuclease H domain, which degrades pregenomic RNA and facilitates
replication. The HBV X ORF encodes a 16.5-kd protein (HBxAg) with multiple functions, including
signal transduction, transcriptional activation, DNA repair, and inhibition of protein degradation and
is well estabilished that is necessary for productive HBV infections in vivo and may contribute to its
oncogenic potential.
The HBV replication pathway consist in an initial phase where mature virions attach to host cell
membranes, likely involving the pre-S domain of the surface protein.

Mechanisms of viral disassembly and intracellular transport of the viral genome into the nucleus are
not well understood and probably involve modification of the nucleocapsid core protein; for example
phosporysation of hepatitys B virus core protein at ser87 facilitates core assembly)

After entry of the viral genome into the nucleus the viral DNA is circularized to the covalently closed
circular (cccDNA) form. This form of HBV DNA serves as the template for transcription of several
species of genomic and sub-genomic RNAs and is the stable component of the replication cycle that
is relatively resistant to antiviral action and immune clearance(2). The transcript of cccDNA is acted
by RNA polymerase II and consist of 2 species with different 5’ends: the pregenomic RNA that serve
as mRna for the core proteins and reverse transcriptase and as template for generation of a new Dna
genome by reverse transciption and the precore RNA that give rise to the precore precursor protein.
(3). Rna Polimerase II transcription could contribute, in addition to reverse transcription, to the virus
variability thanks to high error rate. The next crucial step in hepadnaviral replication is the specific
packaging of pgRNA, plus the reverse transcriptase, into newly forming capsids. For this role are of
crucial importance the cis-elements on the pg-Rna, among all the encapsidation signal 𝜀 and P protein
that binds to ℇ. Once the pg-Rna is encapsidated another key function of of the P-𝜀 is the initiation of
reverse transcription in fact the first DNA nucleotide is convalently linked to P protein, firast is
sinthetyzed the (–) strand and then the (+) one. The nucleocapsid then interacts with the envelope
proteins in the endoplasmic reticlum to assemble into mature virions wich are then secreted into the
exracellular milieu.

DISEASE IN WICH THE MOLECULE IS USED

HBV infection leads to a wide spectrum of liver disease ranging from acute hepatitis (including
fulminant hepatic failure) to chronic hepatitis (CHB), cirrosis, and hepatocellular carcinoma (HCC).
The disease is transimitted through exposure to infected blood and bodily fluids (particularly semen
and vaginal secretions). Most infections worlwide are acquired through parental transmission at birth
throgh horizontal transmission and sexual contact and injecting drug. The epidemiology of hepatitis
B can be described in terms of the prevalence of hepatitis B surface antigen (HBsAg). The Global
Burden of Disease Study 2010 shows an increasing burden of mortality attributable to liver disease ,
HBV was estimated to have resulted in 786000 deaths, the vast majority being attributable to liver
cancer and cirrrhosis. As a result HBV ifection was ranked 15th among all causes of himan mortality.
The likelihood of progression to chronicity, following infection with HBV, is predominantly
determined by age at infection: infantsinfected at birth develop chronic infection in 90% of cases
whereas for children aged between 1 and 5, this falls to 25%-30%, and for immunocompetent adults,
the likelihood of progression to chronicity is ~5%. For individues with CHB acquired in life, the course
of ifection often follows a series of phases determined by the interplay between viral replication and
the host’s immune response; these phases are: immune tolerance – immune clearance – immune
control – immune escape. There is a strong correlation of genoype and geography of infections, and
genotype also influencs the progression of viral infection through phases, wich determines infectivity
and age of transmission.
Because of shared metods of transmission between HBV and others blood-borne viruses in many
cases, there is a coinfection with other viruses such as HCV, HDV and HIV. This is correlated with
modification of the naturla history of liver disease and typically poorer outcomes than are observed
in HBV mono-infections.
Hcc is a major adverse outcome of HBV infection, and an important cause of mortality in a global
context, HBV id estimated to cause around a quarter of liver cancer cases in developed countries and
up to 60% in developing countries. HBV genotypes is also related to HCC incidence, for example in
the Asia-pacific region, genotype C is aassociated with more rapid progression to HCC than genotype
B.
Survival following liver cancer diagnosis remains poor even in welll-resourced health systems with
multiple terapeutic options such as liver transplantation: this emphasizes the need for increased
attention to diagnosis, managment and treatment of hepatitis B.
Ensuring high coverage of infant vaccination will have a profound impact on this disease.
(epidemiology of hbv).

MOST RELEVANT MODE OF ACTION

Many epidemiological studies indicate that patients who develop anti-hepatitis B surface antigen
antibodies (anti-HBs) following infection with hepatitis B are immune to the virus upon reexposure.
Active immunization with hepatitis B vaccine stimulates the immune system to produce anti-HBs
without exposing the patient to the risks of active infection. Infection with hepatitis D can occur only
with concurrent hepatitis B infection, so vaccination with recombinant hepatitis B vaccine provides
protection against hepatitis D as well. (Prescriber drug reference)

PRICE OF THE BP

The Recombivax is sold by Merck & Co. That is an american multinational pharaceutical company, one
of the largest in the world, the price for 1 dose is 51$(5mcg/0.5mL intrauscolar suspension for
Pediatric/Adolescent while $73 for adults (10mcg/mL intrauscolar suspension) [drugss.com]

SURVEY OF CLINICAL TRIALS

The Hepatitis B vaccine formulated from HBsAg produced by a recombinant strain of Saccharomyces
cerevisiae has proved to be highly immunogenic and safe. A 10 µg dose of the vaccine produced an
anti-HBs response of ≥ 10 IU/l in 91% or more of healthy adults who completed the three-dose
regimen. Children developed maximum anti-HBs titres with 5µg doses. Younger adults (20-29 years)
responded more rapidly and with figher anti-HBs titres than did older adults (≥50 years). Children
responded faster and with higher anti-HBs levels than younger adults. Clinical reactions reported
after vaccination were mild and transient ( overview of clinical trials od vaccine hbv).

The efficiency of an inactivated hepatitis B vaccine was assessed in a pacebo-controlled randomized,

double blind trial in 1083 homosexual men known to be at high risk for HepatitisB virus infection. The
vaccine was found to be safe, and the incidence of side effects was low. Within 2 months, 77% of the
vaccinated persons had high levels of antibody against the hepatitis B surface antigen. THis rate
increased to 96% after the booster dose and remained essentially unchanged for the duration of the
trial. For the first 18 months of follow-up, hepatitis B or subclinical infection developed in onl 1,4% to
3,4% of the vaccine recipients as compared with 18% to 27% of placebo recipients (P<0.0001). A
significant reduction of incidence was already seen within 75 days after randomization; this
observation suggests that the vaccine may be efficacious even when given after exposure.

STUDY PROTOCOL

Partecipants were recruited from among more than 2500 HBV negative persons identified during
base-line studies who fulfilled the following criteria: exclusive or predominant homosexuality, no
recent symptoms of hepatitis, a blood specimen drawn within 2 weeks of recruitment negative for
HBsAg, anti HBs, and anti-HBc. An ALT(Alanine Aminotransferase) level below 50 IU and willingness
to provide personal informations and reciving 3 sheduled injections and to make at least 10 follow-
up visits for blood tests during the 2-year period of the study.

The vaccine and placebo were packaged in visually indistinguishable 1-ml bvials that were coded with
numbers up to 1500 that were assegnated in a balance of 12:13 or 13:12 balance of vaccine and
placebo in groups of 25 vials.

First injection was given as soon as the results of the screening blood test were available, the second
1 month and the third 6 months later. The elegibility criterie vor the second and third injections were
the same as those for the first.

Ten cisits to the stydy clinic were scheduled for the 2-year follow-up period: once a month for the
first three months, and once every three months thereafter. Blood specimens taken during these
visits were tested for all HBV markers and, when necessary, for non B-markers as well. In order to
tìkeep the stadd and partecipants blind with respect to whether vaccine or placebo had been fiven
anti-HBs results were not made available.

Wherever a partecipent developed clinical, biochemical, or serologic evidence of hepatitis or HBV

infection, he began a special schedule of follow-up visit every 2 weeks until the diagnosis was
confirmed.

CONDUCT OF THE TRIAL

It was estimated that a minimum of 800 partecipants would be required to prove the efficacy of the
vaccine with a power of 0.9 and a level of signifacnce of 0,01. To analyze the efficacy in sub-groups
of partecipants, to adjust for certain independent variables and allow for imprecision in our
projecirions, it was established a target of 1000 to 1100 trial partecipants.

The randomization procedure allocated 549 to the vaccine group and 534 to the placebo group. This
randomization procedure yielded 2 groups that were highly similare to each other for all the variables
analyed, including variables that were stongly correlated with the risk of HBV.

Of the first 1083 sbjects who were given the fist injection of vaccine or placebo, 1046( 96,5%) recieved
the second. Of these, 994 were eligible for the third injection and 928 did recive it. The corresponding
frequencies for the vaccinated subjects were 549, 529 and 473, and for th placebo group 534 520
455.

The partecipants compliance with sheduled follow-up visits was excellent: of the 6332 scheduled
visits and bleedings for regular follow-up, 91,2% were actually performed. And more than 900
additional visits were made bay partecipants in whom HBV-related events developed.
When ALT levels were above upper limit (≥45 IU) in at least 2 sequential blood specimens frawn
within one to three weeks of each other with one of these values above 90 UI was defined as
Hepatitis. Events in wich ALT values were above 45 IU on one occasion ormore but always under 90
UI were classified as “HBV events with evdence of viral damage” the events with serologic evidence
of a viral infection but no abnormally in ALT were defined as “infection only”.

RESULTS

The study demonstrated that three 40µg doses of the Merck vaccine results in the development of
immunity against HBV. The vaccine induced high levels of antibodies directed agianst surface
antigen, buti t did not induce those directed against the core or “e” antigens. Nine months after the
first vaccination, 96% of the vaccinated subjects possessed anti-HBs and remained essentially
unchengend during the sbsequent nine months.

Such a vigorous antibody resonse was associated with a dramatic reduction of the incidence of HBV
events in the vaccine recipients, a reduction affecting both type B hepatitis and all HBV infections.
For HBV end points that met the criteria for hepatitis, there was a 13-fold difference in attack rates
between the vacicne and placebo groups while for end points with detectable HBsAg or HBV infection
with any biochemical evidence of liver damage there was an eightfold difference. High vaccine
efficacy was observed in all subgroups of partecipants: in both young and old, in both those with and
those without a history of viral hepatitis or venereal disease and both whites and non whites and with
different education. It is noteworthy tat the protective effect was greatest in vaccinees with a large
number of different sexual partners (those with the greatest likehood of exposure to the virus).

This study provided strong evidence that the presence of anti-HBs in synonymous with protection.

The incidence of side effects, both local and general, was lower than that reported for other formalin-
inactivated, alum-absorbed vaccines. The vaccine was also found to be safe. The fact that slightly
more vaccine recipients than placebo recipients had non-A non-B and cytomegalovirus hepatitis is in
accordance with expectations: in placebo recipients, episodes of low or moderate elevations in ALT
that were caused by other agents were probablly masked by frequent HBV events.

Considering that the incubation period for most HBV infections in homosexual men is not likely to
be shorter than six or eight weeks for this was supposed that the first three to four months efter
entry the attack rates in vaccine recipients would non differ notably from those in placebo recipients
because of the so called early non-prevenventable cases; actually the number of hepatitis B or HBsAg-
positiveend points was considerably smaller in vaccinees than in placebo. This reduced early
incidence in vaccine recipients would suggest that the vaccine is partially effective even when given
after exposure. After the trial began, strong evidence was obtained that the antibody response
to three 20-µg doses is as good as the response to doses of 40 µg.

The development of the hepatitis B vaccine and the demonstration that such a vaccine prevents
HBV infection represent a major advance in the control of this extremely serious and widespread
disease
(Demostration of efficacy in a controlled clinical trial)

MOLECULAR FARMING OF THE BP

Several genes from different etiologic agents have been cloned, expressed and purified to be tested
as vaccines. There are a variety of expression systems for antigenic protein components, such as
bacteria, yeast, mammalian cells and insect cells, in which the DNA encoding the antigenic
determinant can be inserted and expressed. However, several factors must be taken into account
before selecting the system for antigen expression. The level of expression obtained using each
specific expression vector and promoter, the selection marker of choice, the presence or absence of
post-translational modification by the recombinant vector, among others, are essential features that
interfere in the efficacy of production of recombinant antigens as vaccines. Bacterial expression
systems are the most used due to the ease of handling and to their capacity for high level expression.
However, for antigens in which post-translational modifications (e.g., glycosylation) are necessary,
the use of mammalian or insect cells should be considered

Most of the vaccines under investigation today are based on highly purified recombinant proteins or
subunits of pathogens. The current vaccines are produced by expressing the hepatitis B surface
antigen (HBsAg) in yeast cells. The HBsAg assembles into virus-like particles (VLPs), which are
extremely immunogenic, making the HBV vaccine a very efficacious vaccine. The yeast expression
system may secrete the antigen into the culture supernatant that can facilitate its purification.
Furthermore, yeast cells offer some of the eukaryotic cellular machinery responsible for the post-
translational modification of proteins, being capable of rendering proteins glycosylated.

Even though vaccines based on recombinant proteins offer several advantages when compared with
traditional vaccines, such as safety and production cost, most of them present weak or poor
immunogenicity when given alone, and thereby require the use of adjuvants to elicit a protective and
long-lasting immune response.

The successful use of recombinant proteins as vaccines, including hepatitis B and, more recently,
HPV, was possible due to the use of aluminium salt as adjuvant. The main difficulties for the
development of new adjuvants involve understanding their molecular complexity and the
mechanisms by which they operate to stimulate or induce the immune response.
(production in saccaromyces)
In the past licensed hepatitis B composed of HBsAg particles which are harvested from the
plasmas of chronic carriers and purified. The purification procedures employ inactivation steps
to kill any adventitious agents. Most of these plasma-derived vaccines are quite effective in
inducing protective anti-HBs antibodies.2 However, the human source of the vaccine antigen
will eventually become a limiting factor in the production of sufficient vaccine for world-wide
use.
(Prince AM. Hepatitis B virus vaccine: a current appraisal of human plasma-derived vaccines. Ann Clin
Res 1982; 14: 225-235.)
Hence a second-generation hepatitis B vaccine was developed. The source of antigen for this vaccine
is HBsAg expressed by genetically engineered bakers's yeast, Saccharomyces cerevisiae.
DETAILED DESCRIPTION OF PRODUCTION OF THE BP
(produzione descrizione dettagliata)

Yeast expression system: A partially sinthetic gene encoding tha 226 amino acid hepatitis
B surface antigen protein was coloned into pGPD-1. This plasmid was introduced into S.
cervisae RH128 or 20B-12 where is kept at high copy number and exhibits a plasmid
stability of 95% when cultured under selective conditions.

The vector constructed for HBsAg expression present origins of DNA

replication for both E. coli and yeast, the promoter (GPD) for the highly expressed
yeast glyceraldehyde-3-phosphate dehydrogenase gene, and the yeast W 1 gene to
supply transcription termination signals as well as a selectable marker for yeast cells
containing the plasmid. The 5' end of the HBsAg gene, subtype adw, was replaced
with a chemically synthesized DNA segment that optimized translation initiation in
yeast. S. cerevisiue strain RH218 containing the expression vector pGPD-l(HBs) and
grown in shaker flasks expressed HBsAg at a level of 1-2% of total cell protein as
estimated by SDS-PAGE of [35S]methionine-labeled whole cell proteins.
PGPD-1

Yeast fermentation: The cells are grown in a fed batch process in which the glucose
concentration is controlled to prevent ethano acculation and the specific growth rate is
maintained so that the cuture is fully aerobic.

HBsAg purification: cells are harvested from fermentors, washed and resuspended in
isotonic buffer containinc Triton x-100, and lysed by agitation with glass beads in a DYno-
Mill apparatus. After centrifugation of the lysate, HBsAg is recovered in the supernatant
fraction and subsequently purified: the clarified lysate is subjected to batchwise binding
to colloidal silicate with selective elution. The HBsAg is then further purified by ion
exchange chromatography, ultrafiltration, isopycnic density gradient centrifugation, and
molecular sieve chromatography.

The HBsAg present in the serum of chronic carriers of hepatitis B is predominantly in the
form of 22-nm spherical particles in which the hydrophobic HBsAg is emnedded in a lipid
envelope, while HBsAg produced in yeast cells assumes the form of a lipid particle and it
is not glycosylated. This fact raises the possibility that the HBsAg protein does not enter
the yeast secretory pathway.

The structure of the HBsAg polypeptide in the purified yeast particle was
examined using six different monoclonal antibodies raised against human plasma
derived HBsAg particles. The monoclonal antibodies were preincubated with
various amounts of either purified yeast or human plasma HBsAg particles. The
uncomplexed monoclonal antibodies present in the solution after preincubation were
then quantitated using a bead assay. For all six
monoclonal antibodies examined, the yeast-derived and human plasma HBsAg particle
react in a quantitavely similar manner.

New approaches regarding the molecular farming of this Biopharmaceutical are te

expression of the recombinant protein in Escherichia coli and Pichia pastoris.

BIBLIOGRAFIA

1.B. Venkatakrishnan and A. Zlotnick: The Structural Biology of Hepatitis B Virus: Form and Function,
Annu Rev Virol. Author manuscript

2. T. Jake Liang, Hepatitis B: the virus and disease

3. Juergen Beck, Michael Nassal Hepatitis B virus replication World J Gastroenterol 2007 January 7;
13(1): 48-64

4.

(Expression and purification of hepatitis B surface antigen

S from Escherichia coli; a new simple method)
(Large-scale production of recombinant hepatitis B surface
antigen from Pichia pastori)