Recombinant Vaccine Production in Yeast - Methylotrophic yeast provide balanced production of the membrane and protein components of a recombinant viral particle. - BioPharm International


Recombinant Vaccine Production in Yeast
Methylotrophic yeast provide balanced production of the membrane and protein components of a recombinant viral particle.

BioPharm International

Human HBV can be grouped into eight genotypes (A–H) which differ by at least 8%.20–22 Genotype F, which is found in Brazil, Colombia, and Polynesia is the most divergent genotype20,23 and is grouped into genotypes F1 and F2.24 The genome of hepadnaviruses codes for four groups of proteins on the minus strand, namely (1) the core protein (HBcAg) which forms the nucleocapsid, and a shorter form (HBeAg), which is secreted by the host cell; (2) the three hepatitis B surface antigen (HBsAg) proteins of different size having different initiation sites of transcription but share the sequence of the small surface protein (SHBs) of 24 kDa at their carboxy-terminus which is the most abundant representative of the HBsAg proteins; minority proteins are the middle HBs of 32 kDa and the large surface protein (LHBs) of 39 kDa (3) the DNA polymerase, which is also a reverse transcriptase with a primer function and an RNAse H domain; and (4) a protein X (HBx) with unknown function for the virus and a plethora of reported properties in vitro.

The hepadnaviridae are round, enveloped viruses of 42–52 nm in diameter25 which appear as 45 nm in negative staining. The viral genome is packed, together with the viral polymerase and a cellular kinase, into a capsid with a diameter of 34 nm.26 In the serum of chronic carriers, the viral surface protein is found as DNA-free spherical or filamentous particles in excess over infectious virions.27

Figure. 1
The hepatitis B virus is taken up by an unknown mechanism or receptor by the hepatocytes. Somewhere in the cytoplasm, the viral envelope is removed and the free nucleocapsid moves to the nuclear pores28 where the HBV DNA-genome leaves the capsid and translocates into the nucleus. In the nucleus, the short plus strand is completed (by the attached viral DNA polymerase), leading to a covalently closed circular double stranded DNA (cccDNA). This cccDNA serves as template for transcription of viral RNAs in the nucleus by the cellular RNA-polymerase II. The different mRNA species are exported from the nucleus into the cytoplasm, where translation occurs. Synthesis of the HBsAg takes place at the endoplasmic reticulum (ER) and the HBsAg is anchored in the ER-membrane. The core protein and the viral polymerase are translated by free ribosomes in the cytoplasm from the largest mRNA (3.5 kb). These two proteins form a complex with their mRNA whereby the core protein encapsidates the viral pre-genome in the cytoplasm. The encapsidated viral RNA is reverse-transcribed into the complete DNA minus strand by the viral polymerase and synthesis of the incomplete plus strand occurs. Then the viral capsid is enveloped at the ER with the HBs-containing membrane, and finally, the infectious virus is secreted.29,30 Subtypes of HBV are determined by different epitopes on particles formed by SHBs and sometimes are distinguished by single amino acid exchanges in the HBsAg sequence. According to the Paris workshop on HBV surface antigen subtypes, eight serotypes exist (adr, ayr, ayw1, ayw2, ayw3, ayw4, adw2, and adw4).

The hepatitis B virus is transmitted parenterally by infected blood or blood products, through mucosal routes, during organ transplantation, or perinatally during birth. The virus mainly infects liver tissue. Hepatitis B virus infections can be transient or chronic. About 90–95% of infected adults recover completely after apparent or inapparent hepatitis and are regarded as cured. Damage to hepatocytes infected by HBV is not caused by the virus itself—HBV per se is not cytopathic—but by the host immune response.

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