RG1-VLP and Other L2-Based, Broad-Spectrum HPV Vaccine Candidates

Licensed human papillomavirus (HPV) vaccines contain virus-like particles (VLPs) self-assembled from L1 major-capsid proteins that are remarkably effective prophylactic immunogens. However, the induced type-restricted immune response limits coverage to the included vaccine types, and costly multiplex formulations, restrictive storage and distribution conditions drive the need for next generation HPV vaccines. Vaccine candidates based upon the minor structural protein L2 are particularly promising because conserved N-terminal epitopes induce broadly cross-type neutralizing and protective antibodies. Several strategies to increase the immunological potency of such epitopes are being investigated, including concatemeric multimers, fusion to toll-like receptors ligands or T cell epitopes, as well as immunodominant presentation by different nanoparticle or VLP structures. Several promising L2-based vaccine candidates have reached or will soon enter first-in-man clinical studies. RG1-VLP present the HPV16L2 amino-acid 17–36 conserved neutralization epitope “RG1” repetitively and closely spaced on an immunodominant surface loop of HPV16 L1-VLP and small animal immunizations provide cross-protection against challenge with all medically-significant high-risk and several low-risk HPV types. With a successful current good manufacturing practice (cGMP) campaign and this promising breadth of activity, even encompassing cross-neutralization of several cutaneous HPV types, RG1-VLP are ready for a first-in-human clinical study. This review aims to provide a general overview of these candidates with a special focus on the RG1-VLP vaccine and its road to the clinic.

High resolution cryo EM analysis of HPV16 identifies minor structural protein L2 and describes capsid flexibility

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. HPV is epitheliotropic and its replication is tightly associated with terminal keratinocyte differentiation making production and purification of high titer virus preparations for research problematic, therefore alternative HPV production methods have been developed for virological and structural studies. In this study we use HPV16 quasivirus, composed of HPV16 L1/L2 capsid proteins with a packaged cottontail rabbit papillomavirus genome. We have achieved the first high resolution, 3.1 Å, structure of HPV16 by using a local subvolume refinement approach. The high resolution enabled us to build L1 unambiguously and identify L2 protein strands. The L2 density is incorporated adjacent to conserved L1 residues on the interior of the capsid. Further interpretation with our own software for Icosahedral Subvolume Extraction and Correlated Classification revealed flexibility, on the whole-particle level through diameter analysis and local movement with inter-capsomer analysis. Inter-capsomer expansion or contraction, governed by the connecting arms, showed no bias in the magnitude or direction of capsomer movement. We propose that papillomavirus capsids are dynamic and capsomers move as rigid bodies connected by flexible linkers. The resulting virus structure will provide a framework for continuing biochemical, genetic and biophysical research for papillomaviruses. Furthermore, our approach has allowed insight into the resolution barrier that has previously been a limitation in papillomavirus structural studies.

High resolution cryo EM analysis of HPV16 identifies minor structural protein L2 and describes capsid flexibility

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. HPV is epitheliotropic and its replication is tightly associated with terminal keratinocyte differentiation making production and purification of high titer virus preparations for research problematic, therefore alternative HPV production methods have been developed for virological and structural studies. In this study we use HPV16 quasivirus, composed of HPV16 L1/L2 capsid proteins with a packaged cottontail rabbit papillomavirus genome. We have achieved the first high resolution, 3.1Å, structure of HPV16 by using a local subvolume refinement approach. The high resolution enabled us to build L1 unambiguously and identify L2 protein strands. The L2 density is incorporated adjacent to conserved L1 residues on the interior of the capsid. Further interpretation with our own software for Icosahedral Subvolume Extraction and Correlated Classification (ISECC) revealed flexibility, on the whole-particle level through diameter analysis and local movement with inter-capsomer analysis. Inter-capsomer expansion or contraction, governed by the connecting arms, showed no bias in the magnitude or direction of capsomer movement. We propose that papillomavirus capsids are dynamic and capsomers move as rigid bodies connected by flexible linkers. The resulting virus structure will provide a framework for continuing biochemical, genetic and biophysical research for papillomaviruses. Furthermore, our approach has allowed insight into the resolution barrier that has previously been a limitation in papillomavirus structural studies.

Expression analysis of Bombyx mori parvo-like virus VD2-ORF1 gene encoding a minor structural protein

Bombyx mori parvo-like virus is a small, icosahedral virus containing two single-stranded linear DNA molecules (VD1, VD2). To date, little is known about this virus, how to package. VD2-ORF1 encoded a large viral structural protein with predicted molecular mass of 133 kDa, and this protein was named as P133. It is unusual for 20–22 nm icosahedral viruses to posses so large a structural protein and the function of this protein is still unknown. In this study, the transcription of P133 was examined with quantitative real-time PCR, and the results demonstrated that the mRNA of P133 could be detected from 28 h post inoculation and kept increasing until 72 h post inoculation. P133 C-terminus (P133C) and P133 N-terminus (P133N) were cloned and expressed in E. coli BL21; then the resulting polypeptides were used to produce antibody, respectively. Western blot analysis showed that the protein in virions recognized by anti-P133C and anti-P133N antibody had the same molecular weight, indicating that VD2-ORF1 encoded a viral structural protein without leaky scanning.

Minor structural protein VP2 in rabbit hemorrhagic disease virus downregulates the expression of the viral capsid protein VP60

Rabbit hemorrhagic disease virus (RHDV) has two structural proteins: the major capsid protein VP60 and the minor capsid protein VP2. VP2 is speculated to play an important role in the virus life cycle. To investigate the effect of VP2 on VP60 expression, three types of experiment (baculovirus–insect cell system, mammalian–luciferase assay system and in vitro coupled transcription/translation system) were used to express VP60 alone or co-expressed with VP2. Both forms of VP60 were able to form virus-like particles in insect cells. Western blot analysis and dual-luciferase assays demonstrated that the presence of VP2 results in downregulation of the expression of VP60 in vivo. Real-time RT-PCR of mRNA levels showed that downregulation of VP60 occurs at the transcriptional level. The ability of the viral minor structural protein VP2 to regulate capsid protein levels may contribute to effective virus infection.

Feline Calicivirus VP2 Is Essential for the Production of Infectious Virions

ABSTRACT The third open reading frame (ORF3) located at the 3′ end of the genomic RNA of feline calicivirus (FCV) encodes a small (12.2-kDa) minor structural protein of 106 amino acids designated VP2. Point mutations and deletions were introduced into an infectious FCV cDNA clone in order to evaluate the functional importance of ORF3 and its encoded protein, VP2. Deletion of the entire ORF3 sequence was lethal for the virus, and evidence was found for strong selective pressure to produce the VP2 protein. Extended deletions in the 5′ end and small deletions in the 3′ end of ORF3, as well as the introduction of stop codons into the ORF3 sequence, were tolerated by the viral replication machinery, but infectious virus could not be recovered. Infectious virus particles could be rescued from a full-length FCV cDNA clone encoding a nonfunctional VP2 when VP2 was provided in trans from a eukaryotic expression plasmid. Our data indicate that VP2, a protein apparently unique to the caliciviruses, is essential for productive replication that results in the synthesis and maturation of infectious virions and that the ORF3 nucleotide sequence itself overlaps a cis-acting RNA signal at the genomic 3′ end.