Enhancement of Capsid Gene Expression: Preparing the Human Papillomavirus Type 16 Major Structural Gene L1 for DNA Vaccination Purposes

Abstract

Expression of the structural proteins L1 and L2 of the human papillomaviruses (HPV) is tightly regulated. As a consequence, attempts to express these prime-candidate genes for prophylactic vaccination against papillomavirus-associated diseases in mammalian cells by means of simple DNA transfections result in insufficient production of the viral antigens. Similarly, in vivo DNA vaccination using HPV L1 or L2 expression constructs produces only weak immune responses. In this study we demonstrate that transient expression of the HPV type 16 L1 and L2 proteins can be highly improved by changing the RNA coding sequence, resulting in the accumulation of significant amounts of virus-like particles in the nuclei of transfected cells. Data presented indicate that, in the case of L1, adaptation for codon usage accounts for the vast majority of the improvement in protein expression, whereas translation-independent posttranscriptional events contribute only to a minor degree. Finally, the adapted L1 genes demonstrate strongly increased immunogenicity in vivo compared to that of unmodified L1 genes.The human papillomaviruses (HPV) comprise a heterogeneous group of epitheliotropic DNA viruses. It is assumed that each of the more than 80 described HPV genotypes represents also a separate serotype (23, 24). The papillomavirus life cycle requires the infection of differentiating epithelia. In this environment, expression of the viral genes is controlled by the cell differentiation program (5, 17). Infections by human papillomaviruses are the major cause of uterine cancer in humans (22,43,44). It is estimated that worldwide half a million new cases of cervical cancer are caused by these viruses every year. The most important HPV type in this respect is HPV type 16 (HPV-16), accounting for approximately 50% of all cases of cervical cancer. Since the recognition of HPV infection as a major health burden, efforts have been undertaken to interrupt the cycle of papillomavirus infections in order to prevent virusinduced disease. Most promising for the prevention of papillomavirus-associated cancer seems to be the development of subviral vaccines that evoke protective immunity by the induction of neutralizing, capsid-directed antibodies. In fact, viruslike particles (VLP) based on the viral capsid protein L1 or L1 plus L2 are currently being developed for prophylactic and therapeutic vaccination against papillomavirus infections (19,26,27). Because they require costly production and purification protocols, it is predictable that it will require a long time for VLP-based vaccines to become affordable in the lessdeveloped countries, which suffer most from papillomaviruscaused cancer. For the same reasons, production and purification of VLP-based vaccines likely have to be restricted to a very limited number of HPV serotypes. As an alternative approach, capsid-specific neutralizing antibodies could be induced by simple DNA vaccination strategies. Since production of DNA vaccines are standardized, it is feasible to produce vaccines against ...

Nuclear Localization but Not PML Protein Is Required for Incorporation of the Papillomavirus Minor Capsid Protein L2 into Virus-Like Particles

“…1A). Similar results were obtained when HPV16 L2 was expressed after transfection of codon-optimized (humanized) L2 (19) (data not shown), suggesting that accumulation of L2 (i) is not due to the vaccinia virus expression system and (ii) is not restricted to HPV33, as already implicated by previous studies (11,19). These data indicate that L2 aggregate formation in the nucleus occurs independently of PML protein.…”

Mutational Inactivation of Two Distinct Negative RNA Elements in the Human Papillomavirus Type 16 L2 Coding Region Induces Production of High Levels of L2 in Human Cells

“…HIV-1 Rev and RRE can override the effect of papillomavirus negative regulatory RNA sequences, as has been shown previously for HPV-1 (33), HPV-16 (31), and bovine papillomavirus type 1 (2). However, others have failed to see an effect of Rev/RRE or CTE (19). Although in the latter case, the complete absence of RNA analysis of the expression levels prevented a more detailed analysis of the data, it is likely that the failure to detect L1 protein may be a result of variations in transfection efficiencies or expression levels of L1 or Rev.…”

“…The presence of these distinct, inhibitory RNA elements explains the lack of L1 and L2 production from the HPV-16 L1 and L2 coding sequences. Others have changed rare codons in the HPV-16 L1 and L2 genes and obtained production of L1 and L2 protein in human cells (19). These authors speculated that the induction of L1 and L2 production was the result of an increase in translation due to an "optimization" of the mRNA coding sequence (19).…”

Surface-exposed Amino Acid Residues of HPV16 L1 Protein Mediating Interaction with Cell Surface Heparan Sulfate

“…Plasmids and Mutagenesis-Expression plasmids carrying codon-modified HPV16 L1 and L2 structural genes have been reported previously (28). A marker plasmid coding for a dimeric green fluorescent protein (2) was co-transfected as a control for efficient transfection and as a reporter plasmid in infectivity assays.…”