Hepatitis B Strains
HBV is a mostly double-stranded DNA virus in the Hepadnaviridae family. HBV causes hepatitis in human and related virus in this family cause hepatitis in ducks, ground squirrels and woodchucks. The HBV genome has four genes: pol, env, pre-core and X that respectively encode the viral DNA-polymerase, envelope protein, pre-core protein (which is processed to viral capsid) and protein X. The function of protein X is not clear but it may be involved in the activation of host cell genes and the development of cancer.
The genomes of more than a dozen isolates of hepatitis B virus have been cloned and the complete nucleotide sequences determined. Analysis of the coding potential of the genome reveals four open reading frames (ORFs) which are conserved between all of these isolates.
The first ORF encodes the various forms of the surface protein and contains three in-frame methionine codons which are used for initiation of translation. A second promoter is located upstream of the pre-S1 initiation codon. This directs the synthesis of a 2.4 kb mRNA which is co-terminal with the other surface messages and is translated to yield the large (pre-S1) surface proteins.
The core open reading frame also has two in-phase initiation codons. The “precore” region is highly conserved, has the properties of a signal sequence and is responsible for the secretion of HBeAg.
The third ORF, which is the largest and overlaps the other three, encodes the viral polymerase. This protein appears to be another translation product of the 3.5 kb RNA, and is synthesized apparently following internal initiation of the ribosome.
The amino terminal domain is believed to be the protein primer for minus strand synthesis. There is then a spacer region followed by the (RNA and DNA-dependent) DNA polymerase.
The fourth ORF was designated “x” because the function of its small gene product was not known. However, “x” has now been demonstrated to be a transcriptional transactivator.
…The discovery of variation in the epitopes presented on the surface of the virions and subviral particles identified several subtypes of HBV which differ in their geographical distribution. All isolates of the virus share a common epitope, a, which is a domain of the major surface protein which is believed to protrude as a double loop from the surface of the particle. Two other pairs of mutually exclusive antigenic determinants, d or y and w or r, are also present on the major surface protein. These variations have been correlated with single nucleotide changes in the surface ORF which lead to variation in single amino acids in the protein. Four principal subtypes of HBV are recognized: adw, adr, ayw and ayr. Subtype adw predominates in northern Europe, the Americas and Australasia and also is found in Africa and Asia. Subtype ayw is found in the Mediterranean region, eastern Europe, northern and western Africa, the near East and the Indian subcontinent. In the Far East, adr predominates. But the rarer ayr occasionally may be found in Japan and Papua New Guinea.
The major response of recipients of hepatitis B vaccine is to the common a epitope with consequent protection against all subtypes of the virus. First generation vaccines were prepared from 22 nm HBsAg particles purified from plasma donations from chronic carriers. These preparations are safe and immunogenic but have been superseded in some countries by recombinant vaccines produced by the expression of HBsAg in yeast cells. The expression plasmid contains only the 3′ portion of the HBV surface ORF and only the major surface protein, without pre-S epitopes, is produced. Vaccines containing pre-S2 and pre-S1, as well as the major surface proteins expressed by recombinant DNA technology, are undergoing clinical trials.
The following is a list of the major types of HBV genomes found in the human population:
- 1993: Genetic relatedness of hepatitis B viral strains of diverse geographical origin and natural variations in the primary structure of the surface antigen.
- 1995: Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene.
- 1998: Antigenic diversity of hepatitis B virus strains of genotype F in Amerindians and other population groups from Venezuela.
6 Genotypes (A,B,C,D,E,F) [PMID: 8336122]
- Group A – Orig – N. Europe – Sub-Saharan Africa
- Group B – Confined to – Eastern Asia (China)
- Group C – Far East (Japan)
- Group D – Mediterranean – Near, Mid East, South Asia
- Group E – W. Sub-Saharan Africa, south to Angola
- Group F – New World – Brazil, N. + S. America
121 Strains Exist as Quasispecies
There are four serotypes which are based on subtypes of the hepatitis B surface antigen (HBsAg). These are defined by two mutually exclusive determinant pairs d/y and w/r with a common determinant ‘a’. These subtypes are adw, ayw, adr, and ayr.
Four genomic groups of HBV were later referred as genotypes designated with A-D. Sequencing of the S-gene of HBV is the molecular basis for the assessment of the serological variations of HBsAg within the major four subtypes. Two new genotypes of HBV are designated with E and F. The F genotype diverges from other HBV genomes sequenced by 14%. So far, it is the most divergent HBV genome. Worldwide molecular epidemiology of HBV is based on the variability of the S-gene. The E and F strains appear to originate from aboriginal populations of Africa in the New World.
Hepatitis B virus (HBV) has been classified into seven genotypes A–G. However, recently genotype H, which is phylogenetically closely related to genotype F, has been reported (Arauz-Ruiz et al., 2002 ). These genotypes of HBV show a distinctive geographical distribution and a relevance to clinical severity (Mayert et al., 1999 ; Kobayashi et al., 2002 ; Locarnini, 2002)
(Emerging Infectious Diseases Volume 14, Number 11–November 2008)
We conducted a phylogenetic analysis of 19 hepatitis B virus strains from Laos that belonged to 2 subgenotypes of a new genotype I. This emerging new genotype likely developed outside Southeast Asia and is now found in mixed infections and in recombinations with local strains in a geographically confined region.
Hepatitis B Vaccines:
COMVAX® [HAEMOPHILUS b CONJUGATE (MENINGOCOCCAL PROTEIN CONJUGATE) and HEPATITIS B (RECOMBINANT) VACCINE]
HBsAg is produced in recombinant yeast 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 according to methods developed in the Merck Research Laboratories. The antigen is harvested and purified from fermentation cultures of a recombinant strain of the yeast Saccharomyces cerevisiae containing the gene for the adw subtype of HBsAg. The fermentation process involves growth of Saccharomyces cerevisiae on a complex fermentation medium which consists of an extract of yeast, soy peptone, dextrose, amino acids and mineral salts.
The HBsAg protein is released from the yeast cells by mechanical cell disruption and detergent
extraction, and purified by a series of physical and chemical methods, which includes ion and hydrophobic chromatography, and diafiltration. The purified protein is treated in phosphate buffer with formaldehyde and then coprecipitated with alum (potassium aluminum sulfate) to form bulk vaccine adjuvanted with amorphous aluminum hydroxyphosphate sulfate. The vaccine contains no detectable yeast DNA, and 1% or less of the protein is of yeast origin.
The individual PRP-OMPC and HBsAg adjuvanted bulks are combined to produce COMVAX. Each 0.5 mL dose of COMVAX is formulated to contain 7.5 mcg PRP conjugated to approximately 125 mcg OMPC, 5 mcg HBsAg, approximately 225 mcg aluminum as amorphous aluminum hydroxyphosphate sulfate, and 35 mcg sodium borate (decahydrate) as a pH stabilizer, in 0.9% sodium chloride. The vaccine contains not more than 0.0004% (w/v) residual formaldehyde.
The potency of the PRP-OMPC component is measured by quantitating the polysaccharide
concentration by an HPLC method. The potency of the HBsAg component is measured relative to a standard by an in vitro immunoassay.
ENGERIX-B® [Hepatitis B Vaccine (Recombinant)]
ENGERIX-B [Hepatitis B Vaccine (Recombinant)] is a noninfectious recombinant DNA hepatitis B vaccine developed and manufactured by GlaxoSmithKline Biologicals. It contains purified surface antigen of the virus obtained by culturing genetically engineered Saccharomyces cerevisiae cells, which carry the surface antigen gene of the hepatitis B virus. The surface antigen expressed in Saccharomyces cerevisiae cells is purified by several physicochemical steps and formulated as a suspension of the antigen adsorbed on aluminum hydroxide. The procedures used to manufacture ENGERIX-B result in a product that contains no more than 5% yeast protein. No substances of human origin are used in its manufacture.
Pediatric/Adolescent: Each 0.5-mL dose contains 10 mcg of hepatitis B surface antigen adsorbed on 0.25 mg aluminum as aluminum hydroxide. The pediatric formulation contains sodium chloride (9 mg/mL) and phosphate buffers (disodium phosphate dihydrate, 0.98 mg/mL; sodium dihydrogen phosphate dihydrate, 0.71 mg/mL).
Adult: Each 1-mL adult dose contains 20 mcg of hepatitis B surface antigen adsorbed on 0.5 mg aluminum as aluminum hydroxide. The adult formulation contains sodium chloride (9 mg/mL) and phosphate buffers (disodium phosphate dihydrate, 0.98 mg/mL; sodium dihydrogen phosphate dihydrate, 0.71 mg/mL).
PEDIARIX®[Diphtheria and Tetanus Toxoids and Acellular Pertussis Adsorbed, Hepatitis B (Recombinant) and Inactivated Poliovirus Vaccine Combined]
It contains diphtheria and tetanus toxoids, 3 pertussis antigens (inactivated pertussis toxin [PT] and formaldehyde-treated filamentous hemagglutinin [FHA] and pertactin [69 kiloDalton outer membrane protein]), hepatitis B surface antigen, plus poliovirus Type 1 (Mahoney), Type 2 (MEF-1), and Type 3 (Saukett). The diphtheria toxoid, tetanus toxoid, and pertussis antigens are the same as those in
INFANRIX® (Diphtheria and Tetanus Toxoids and Acellular Pertussis Vaccine Adsorbed). The hepatitis B surface antigen is the same as that in ENGERIX-B® [Hepatitis B Vaccine (Recombinant)].
…The hepatitis B surface antigen (HBsAg) is obtained by culturing genetically engineered Saccharomyces cerevisiae cells, which carry the surface antigen gene of the hepatitis B virus, in synthetic medium. The surface antigen expressed in the S. cerevisiae cells is purified by several physiochemical steps, which include precipitation, ion exchange chromatography, and ultrafiltration.
…The diphtheria, tetanus, and pertussis antigens are individually adsorbed onto aluminum hydroxide; hepatitis B component is adsorbed onto aluminum phosphate. All antigens are then diluted and combined to produce the final formulated vaccine. Each 0.5-mL dose is formulated to contain 25 Lf of diphtheria toxoid, 10 Lf of tetanus toxoid, 25 mcg of inactivated PT, 25 mcg of FHA, 8 mcg of pertactin, 10 mcg of HBsAg, 40 D-antigen Units (DU) of Type 1 poliovirus, 8 DU of Type 2 poliovirus, and 32 DU of Type 3 poliovirus.
TWINRIX® [Hepatitis A Inactivated & Hepatitis B (Recombinant) Vaccine]
TWINRIX® [Hepatitis A Inactivated & Hepatitis B (Recombinant) Vaccine] is a sterile bivalent vaccine containing the antigenic components used in producing HAVRIX® (Hepatitis A Vaccine, Inactivated) and ENGERIX-B® [Hepatitis B Vaccine (Recombinant)].
The purified hepatitis B surface antigen (HBsAg) is obtained by culturing genetically engineered Saccharomyces cerevisiae cells, which carry the surface antigen gene of the hepatitis B virus, in synthetic media containing inorganic salts, amino acids, dextrose, and vitamins. Bulk preparations of each antigen are adsorbed separately onto aluminum salts and then pooled during formulation.
A 1.0-mL dose of vaccine contains 720 ELISA Units of inactivated hepatitis A virus and 20 mcg of recombinant HBsAg protein. One dose of vaccine also contains 0.45 mg of aluminum in the form of aluminum phosphate and aluminum hydroxide as adjuvants, amino acids, sodium chloride, phosphate buffer, polysorbate 20, Water for Injection, traces of formalin (not more than 0.1 mg), and residual MRC-5 cellular proteins (not more than 2.5 mcg). Neomycin sulfate, an aminoglycoside antibiotic, is included in the cell growth media; only trace amounts (not more than 20 ng) remain following purification. The manufacturing procedures used to manufacture TWINRIX result in a product that contains no more than 5% yeast protein.
RECOMBIVAX HB® HEPATITIS B VACCINE (RECOMBINANT)
RECOMBIVAX HB* Hepatitis B Vaccine (Recombinant) is a non-infectious subunit viral vaccine
derived from hepatitis B surface antigen (HBsAg) produced in yeast 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 according to methods developed in the Merck Research Laboratories.
The antigen is harvested and purified from fermentation cultures of a recombinant strain of the yeast Saccharomyces cerevisiae containing the gene for the adw subtype of HBsAg. The fermentation process involves growth of Saccharomyces cerevisiae on a complex fermentation medium which consists of an extract of yeast, soy peptone, dextrose, amino acids and mineral salts. The HBsAg protein is released from the yeast cells by cell disruption and purified by a series of physical and chemical methods. The purified protein is treated in phosphate buffer with formaldehyde and then coprecipitated with alum (potassium aluminum sulfate) to form bulk vaccine adjuvanted with amorphous aluminum
hydroxyphosphate sulfate. The vaccine contains no detectable yeast DNA but may contain not more than 1% yeast protein. The vaccine produced by the Merck method has been shown to be comparable to the plasma-derived vaccine in terms of animal potency (mouse, monkey, and chimpanzee) and protective efficacy (chimpanzee and human).
Pediatric/Adolescent Formulation (Without Preservative), 10 mcg/mL: each 0.5 mL dose contains 5 mcg of hepatitis B surface antigen.
Adult Formulation (Without Preservative), 10 mcg/mL: each 1 mL dose contains 10 mcg of
hepatitis B surface antigen.
Dialysis Formulation (Without Preservative), 40 mcg/mL: each 1 mL dose contains 40 mcg of hepatitis B surface antigen.
All formulations contain approximately 0.5 mg of aluminum (provided as amorphous aluminum
hydroxyphosphate sulfate, previously referred to as aluminum hydroxide) per mL of vaccine. In each formulation, hepatitis B surface antigen is adsorbed onto approximately 0.5 mg of aluminum (provided as amorphous aluminum hydroxyphosphate sulfate) per mL of vaccine. The vaccine is of the adw subtype.
RECOMBIVAX HB is indicated for vaccination of persons at risk of infection from hepatitis B virus
including all known subtypes. RECOMBIVAX HB Dialysis Formulation is indicated for vaccination of adult predialysis and dialysis patients against infection caused by all known subtypes of hepatitis B virus.
Nabi-HB® Hepatitis B Immune Globulin (Human)
Hepatitis B Immune Globulin (Human), Nabi-HB, is a sterile solution of immunoglobulin (5 ± 1%
protein) containing antibodies to hepatitis B surface antigen (anti-HBs). It is prepared from plasma
donated by individuals with high titers of anti-HBs. The plasma is processed using a modified
Cohn 6 / Oncley 9 cold-alcohol fractionation process1,2 with two added viral reduction steps
described below. Nabi-HB is formulated in 0.075 M sodium chloride, 0.15 M glycine, and 0.01%
polysorbate 80, at pH 6.2. The product is supplied as a nonturbid sterile liquid in single dose
vials and appears as clear to opalescent. It contains no preservative and is intended for single
use by the intramuscular route only.
The manufacturing steps for Nabi-HB are designed to reduce the risk of transmission of viral disease.
The solvent/detergent treatment step, using tri-n-butyl phosphate and Triton® X-100, is
effective in inactivating known enveloped viruses such as hepatitis B virus (HBV), hepatitis C
virus (HCV), and human immunodeficiency virus (HIV) 3. Virus filtration, using a Planova® 35
nm Virus Filter, is effective in reducing some known enveloped and non-enveloped viruses4. The
inactivation and reduction of known enveloped and non-enveloped model viruses were validated
in laboratory studies as summarized in the following table…
BayHepB Immune Globulin (Human)
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