L1 and L2 gene polymorphisms in HPV-58 and HPV-33: implications for vaccine design and diagnosis.
ABSTRACT: Cervical cancer is associated with infection by certain subtypes of human papillomavirus (HPV). The L1 protein comprising HPV vaccine formulations elicits high-titre neutralizing antibodies and confers protection against specific HPV subtypes. HPV L2 protein is an attractive candidate for cross-protective vaccines. HPV-33 and HPV-58 are very prevalent among Chinese women.To study the gene intratypic variations and polymorphisms of HPV-33 and HPV-58?L1/L2 in Sichuan China, HPV-33 and HPV-58?L1 and L2 genes were sequenced and compared with other genes submitted to GenBank. Phylogenetic trees were constructed by maximum-likelihood and the Kimura 2-parameters methods (MEGA 6). The secondary structure was analyzed by PSIPred software, and HPV-33 and HPV-58 L1 homology models were created by SWISS-MODEL software. The selection pressures acting on the L1/L2 genes were estimated by PAML 4.8.Among 124 HPV-33?L1 sequences 20 single nucleotide mutations were observed included 8/20 non-synonymous and 12/20 synonymous mutations. The 101 HPV-33?L2 sequences included 12 single nucleotide mutations comprising 7/12 non-synonymous and 5/12 synonymous mutations. The 223 HPV-58?L1 sequences included 32 single nucleotide mutations comprising 9/32 non-synonymous and 23/32 synonymous mutations. The 201 HPV-58?L2 sequences comprised 26 single nucleotide mutations including 9/26 non-synonymous and 17/26 synonymous mutations. Selective pressure analysis showed that most of the common non-synonymous mutations showed a positive selection. HPV-33 and HPV-58?L2 were more stable than HPV-33 and HPV-58?L1.HPV-33 and HPV-58?L2 were better candidates as clinical diagnostic targets compared with HPV-33 and HPV-58?L1. Clinical diagnostic probes and second-generation polyvalent vaccines should be designed on the basis of the unique sequence of HPV-33 and 58?L1/L2 variations in Sichuan, to improve the accuracy of clinical detection and the protective efficiency of vaccines.
Project description:HPV account for most of the incidence of cervical cancer. Approximately 90% of anal cancers and a smaller subset (<50%) of other cancers (oropharyngeal, penile, vaginal, vulvar) are also attributed to HPV. The L1 protein comprising HPV vaccine formulations elicits high-titre neutralizing antibodies and confers type restricted protection. The L2 protein is a promising candidate for a broadly protective HPV vaccine. In our previous study, we found the most prevalent high-risk HPV infectious serotypes were HPV-16 and HPV-58 among women of Southwest China. To explore gene polymorphisms and intratypic variations of HPV-16 and HPV-58 L1/L2 genes originating in Southwest China, HPV-16 (L1: n = 31, L2: n = 28) and HPV-58 (L1: n = 21, L2: n = 21) L1/L2 genes were sequenced and compared to others described and submitted to GenBank. Phylogenetic trees were then constructed by Neighbor-Joining and the Kimura 2-parameters methods (MEGA software), followed by an analysis of the diversity of secondary structure. Then selection pressures acting on the L1/L2 genes were estimated by PAML software. Twenty-nine single nucleotide changes were observed in HPV-16 L1 sequences with 16/29 non-synonymous mutations and 13/29 synonymous mutations (six in alpha helix and two in beta turns). Seventeen single nucleotide changes were observed in HPV-16 L2 sequences with 8/17 non-synonymous mutations (one in beta turn) and 9/17 synonymous mutations. Twenty-four single nucleotide changes were observed in HPV-58 L1 sequences with 10/24 non-synonymous mutations and 14/24 synonymous mutations (eight in alpha helix and four in beta turn). Seven single nucleotide changes were observed in HPV-58 L2 sequences with 4/7 non-synonymous mutations and 3/7 synonymous mutations. The result of selective pressure analysis showed that most of these mutations were of positive selection. This study may help understand the intrinsic geographical relatedness and biological differences of HPV-16/HPV-58 and contributes further to research on their infectivity, pathogenicity, and vaccine strategy.
Project description:Cancer of the cervix is associated with infection by certain types of human papillomavirus (HPV). The gene variants differ in immune responses and oncogenic potential. The E6 and E7 proteins encoded by high-risk HPV play a key role in cellular transformation. HPV-33 and HPV-58 types are highly prevalent among Chinese women. To study the gene intratypic variations, polymorphisms and positive selections of HPV-33 and HPV-58 E6/E7 in southwest China, HPV-33 (E6, E7: n = 216) and HPV-58 (E6, E7: n = 405) E6 and E7 genes were sequenced and compared to others submitted to GenBank. Phylogenetic trees were constructed by Maximum-likelihood and the Kimura 2-parameters methods by MEGA 6 (Molecular Evolutionary Genetics Analysis version 6.0). The diversity of secondary structure was analyzed by PSIPred software. The selection pressures acting on the E6/E7 genes were estimated by PAML 4.8 (Phylogenetic Analyses by Maximun Likelihood version4.8) software. The positive sites of HPV-33 and HPV-58 E6/E7 were contrasted by ClustalX 2.1. Among 216 HPV-33 E6 sequences, 8 single nucleotide mutations were observed with 6/8 non-synonymous and 2/8 synonymous mutations. The 216 HPV-33 E7 sequences showed 3 single nucleotide mutations that were non-synonymous. The 405 HPV-58 E6 sequences revealed 8 single nucleotide mutations with 4/8 non-synonymous and 4/8 synonymous mutations. Among 405 HPV-58 E7 sequences, 13 single nucleotide mutations were observed with 10/13 non-synonymous mutations and 3/13 synonymous mutations. The selective pressure analysis showed that all HPV-33 and 4/6 HPV-58 E6/E7 major non-synonymous mutations were sites of positive selection. All variations were observed in sites belonging to major histocompatibility complex and/or B-cell predicted epitopes. K93N and R145 (I/N) were observed in both HPV-33 and HPV-58 E6.
Project description:Human papillomaviruses (HPVs) are the most common sexually transmitted infections worldwide. Ninety percent of infected individuals clear the infection within two years; however, in the remaining 10% of infected individuals, the infection(s) persists and ultimately leads to cancers (anogenital cancers and head and neck cancers) and genital warts. Fortunately, three prophylactic vaccines have been approved to protect against HPV infections. The most recent HPV vaccine, Gardasil-9 (a nonavalent vaccine), protects against seven HPV types associated with ~90% of cervical cancer and against two HPV types associated with ~90% genital warts with little cross-protection against non-vaccine HPV types. The current vaccines are based on virus-like particles (VLPs) derived from the major capsid protein, L1. The L1 protein is not conserved among HPV types. The minor capsid protein, L2, on the other hand, is highly conserved among HPV types and has been an alternative target antigen, for over two decades, to develop a broadly protective HPV vaccine. The L2 protein, unlike the L1, cannot form VLPs and as such, it is less immunogenic. This review summarizes current studies aimed at developing HPV L2 vaccines by multivalently displaying L2 peptides on VLPs derived from bacteriophages and eukaryotic viruses. Recent data show that a monovalent HPV L1 VLP as well as bivalent MS2 VLPs displaying HPV L2 peptides (representing amino acids 17-36 and/or consensus amino acids 69-86) elicit robust broadly protective antibodies against diverse HPV types (6/11/16/18/26/31/33/34/35/39/43/44/45/51/52/53/56/58/59/66/68/73) associated with cancers and genital warts. Thus, VLP-based L2 vaccines look promising and may be favorable, in the near future, over current L1-based HPV vaccines and should be explored further.
Project description:Cervical cancer caused by infection with human papillomaviruses (HPVs) is the fourth most common cancer in women globally, with the burden mainly in developing countries due to limited healthcare resources. Current vaccines based on virus-like particles (VLPs) assembled from recombinant expression of the immunodominant L1 protein are highly effective in the prevention of cervical infection; however, these vaccines are expensive and type-specific. Therefore, there is a need for more broadly protective and affordable vaccines. The HPV-16 L2 peptide sequences 108-120, 65-81, 56-81, and 17-36 are highly conserved across several HPV types and have been shown to elicit cross-neutralizing antibodies. To increase L2 immunogenicity, L1:L2 chimeric VLPs (cVLP) vaccine candidates were developed. The four L2 peptides mentioned above were substituted into the DE loop of HPV-16 L1 at position 131 (SAC) or in the C-terminal region at position 431 (SAE) to generate HPV-16-derived L1:L2 chimeras. All eight chimeras were transiently expressed in Nicotiana benthamiana via Agrobacterium tumefaciens-mediated DNA transfer. SAC chimeras predominantly assembled into higher order structures (T = 1 and T = 7 VLPs), whereas SAE chimeras assembled into capsomeres or formed aggregates. Four SAC and one SAE chimeras were used in vaccination studies in mice, and their ability to generate cross-neutralizing antibodies was analyzed in HPV pseudovirion-based neutralization assays. Of the seven heterologous HPVs tested, cross-neutralization with antisera specific to chimeras was observed for HPV-11 (SAE 65-18), HPV-18 (SAC 108-120, SAC 65-81, SAC 56-81, SAE 65-81), and HPV-58 (SAC 108-120). Interestingly, only anti-SAE 65-81 antiserum showed neutralization of homologous HPV-16, suggesting that the position of the L2 epitope display is critical for maintaining L1-specific neutralizing epitopes.
Project description:Licensed human papillomavirus (HPV) vaccines, based on virus-like particles (VLPs) self-assembled from major capsid protein L1, afford type-restricted protection against HPV types 16/18/6/11 (or 16/18 for the bivalent vaccine), which cause 70% of cervical cancers (CxCas) and 90% of genital warts. However, they do not protect against less prevalent high-risk (HR) types causing 30% of CxCa, or cutaneous HPV. In contrast, vaccination with the minor capsid protein L2 induces low-level immunity to type-common epitopes. Chimeric RG1-VLP presenting HPV16 L2 amino acids 17-36 (RG1 epitope) within the DE-surface loop of HPV16 L1 induced cross-neutralizing antisera. We hypothesized that RG1-VLP vaccination protects against a large spectrum of mucosal and cutaneous HPV infections in vivo. Immunization with RG1-VLP adjuvanted with human-applicable alum-MPL (aluminum hydroxide plus 3-O-desacyl-4'-monophosphoryl lipid A) induced robust L2 antibodies (ELISA titers 2,500-12,500), which (cross-)neutralized mucosal HR HPV16/18/45/37/33/52/58/35/39/51/59/68/73/26/69/34/70, low-risk HPV6/11/32/40, and cutaneous HPV2/27/3/76 (titers 25-1,000) using native virion- or pseudovirion (PsV)-based assays, and a vigorous cytotoxic T lymphocyte response by enzyme-linked immunospot. In vivo, mice were efficiently protected against experimental vaginal challenge with mucosal HR PsV types HPV16/18/45/31/33/52/58/35/39/51/59/68/56/73/26/53/66/34 and low-risk HPV6/43/44. Enduring protection was demonstrated 1 year after vaccination. RG1-VLP is a promising next-generation vaccine with broad efficacy against all relevant mucosal and also cutaneous HPV types.
Project description:Globally, human papillomavirus (HPV)?56 accounts for a small proportion of all high?risk HPV types; however, HPV?56 is detected at a higher rate in Asia, particularly in southwest China. The present study analyzed polymorphisms, intratypic variants, and genetic variability in the long control regions (LCR), E6, E7, and L1 of HPV?56 (n=75). The LCRs, E6, E7 and L1 were sequenced using a polymerase chain reaction and the sequences were submitted to GenBank. Maximum?likelihood trees were constructed using Kimura's two?parameter model, followed by secondary structure analysis and protein damaging prediction. Additionally, in order to assess the effect of variations in the LCR on putative binding sites for cellular proteins, MATCH server was used. Finally, the selection pressures of the E6?E7 and L1 genes were estimated. A total of 18 point substitutions, a 42?bp deletion and a 19?bp deletion of LCR were identified. Some of those mutations are embedded in the putative binding sites for transcription factors. 18 single nucleotide changes occurred in the E6?E7 sequence, 11/18 were non?synonymous substitutions and 7/18 were synonymous mutations. A total 24 single nucleotide changes were identified in the L1 sequence, 6/24 being non?synonymous mutations and 18/24 synonymous mutations. Selective pressure analysis predicted that the majority of mutations of HPV?56 E6, E7 and L1 were of positive selection. The phylogenetic tree demonstrated that the isolates distributed in two lineages. Data on the prevalence and genetic variation of HPV?56 types in southwest China may aid future studies on viral molecular mechanisms and contribute to future investigations of diagnostic probes and therapeutic vaccines.
Project description:Human Papillomavirus (HPV), a non-enveloped, double-stranded DNA virus, is responsible for 5% of human cancers. The HPV capsid consists of major and minor structural proteins, L1 and L2. L1 proteins form an icosahedral shell with building blocks of the pentameric capsomere, and one L2 molecule extends outward from the central hole of the capsid. Thus, L2 is concealed within L1 and only becomes exposed when the capsid interacts with host cells. The low antigenic variation of L2 means that this protein could offer a target for the development of a pan-HPV vaccine. Toward this goal, here we describe an anti-L2 monoclonal antibody, 14H6, which broadly neutralizes at least 11 types of HPV, covering types 6, 11, 16, 18, 31, 33, 35, 45, 52, 58 and 59, in pseudovirion--based cell neutralization assay. The mAb 14H6 recognizes a minimal linear epitope located on amino acids 21 to 30 of the L2 protein. Alanine scanning mutagenesis and sequence alignment identified several conserved residues (Cys22, Lys23, Thr27, Cys28 and Pro29) that are involved in the 14H6 binding with L2. The epitope was grafted to several scaffolding proteins, including HPV16 L1 virus-like particles, HBV 149 core antigen and CRM197. The resultant chimeric constructs were expressed in Escherichia coli and purified with high efficiency. Immunization with these pan-HPV vaccine candidates elicited high titers of the L2-specific antibody in mice and conferred robust (3-log) titers of cross-genotype neutralization, including against HPV11, 16, 18, 45, 52, 58 and 59. These findings will help in the development of an L2-based, pan-HPV vaccine.
Project description:Intratypic variations of HPV-18 are known to differ in the persistence of the infection, frequency of carcinogenesis and the progression of precursor lesions to advanced cervical cancer. This study was designed to analyze sequence variations of HPV-18 isolates in order to discover novel HPV-18 variants and to evaluate the variations among infected women in southwest China. Cervical biopsies from 56 HPV-18-positive women with cervical neoplasia were assayed by PCR amplification and sequencing of all eight genes (E1, E2, E4, E5, E6, E7, L1, L2) of the HPV-18 genome. The most frequently observed variation was a C to G transversion at nucleotide 287 of E6, which was found in 48.2% of samples. Analysis of E7 revealed only one specimen as having sequence variations. In addition, we have identified several novel variations: A551C in E6, G6906A in L1, and C4915T and C5147A in L2. The mutations in E6 and L2 are silent, while the E7 mutation results in a single amino acid change. This study complements and expands on previous descriptions of HPV-18 variants. The sequence variation data presented here provides a foundation for future research on HPV-induced oncogenesis and may prove valuable for developing diagnostic probes and in the design of HPV vaccines for targeted populations.
Project description:The neutralizing antibodies elicited by human papillomavirus (HPV) major capsid protein L1 virus-like particle (VLP)-based vaccines are largely type-specific. An HPV vaccine inducing cross-neutralizing antibodies broadly will be cost-effective and of great value. To this end, we constructed HPV16L1-58L2 chimeric VLP (cVLP) by displaying HPV58 L2 aa.16-37 on the DE surface region of HPV16 L1. We found that vaccination with the HPV16L1-58L2 cVLP formulated with alum plus monophosphoryl lipid A (Alum-MPL) adjuvant elicited robust neutralizing antibodies in both mice and rabbits against all tested HPV types including HPV16/31/33/35/52/58 (genus ?9), HPV18/39/45/59/68 (genus ?7), HPV6/11 (genus ?10), HPV2/27/57 (genus ?4), and HPV5 (genus ?1). Importantly, the cross-neutralizing antibody response was maintained at least 82 weeks in mice or 42 weeks in rabbits, and complete protection against HPV58 was observed at week 85 in mice. Our data demonstrate that HPV16L1-58L2 cVLP is an excellent pan-HPV vaccine candidate.
Project description:Human papillomavirus (HPV) is the most common sexually transmitted infection in the world and the main cause of cervical cancer. Nowadays, the virus-like particles (VLPs) based on L1 proteins have been considered as the best candidate for vaccine development against HPV infections. Two commercial HPV (Gardasil and Cervarix) are available. These HPV VLP vaccines induce genotype-limited protection. The major impediments such as economic barriers especially gaps in financing obstructed the optimal delivery of vaccines in developing countries. Thus, many efforts are underway to develop the next generation of vaccines against other types of high-risk HPV. In this study, we developed DNA constructs (based on L1 and L2 genes) that were potentially immunogenic and highly conserved among the high-risk HPV types. The framework of analysis include (1) B-cell epitope mapping, (2) T-cell epitope mapping (i.e., CD4+ and CD8+ T cells), (3) allergenicity assessment, (4) tap transport and proteasomal cleavage, (5) population coverage, (6) global and template-based docking, and (7) data collection, analysis, and design of the L1 and L2 DNA constructs. Our data indicated the 8-epitope candidates for helper T-cell and CTL in L1 and L2 sequences. For the L1 and L2 constructs, combination of these peptides in a single universal vaccine could involve all world population by the rate of 95.55% and 96.33%, respectively. In vitro studies showed high expression rates of multiepitope L1 (~57.86%) and L2 (~68.42%) DNA constructs in HEK-293T cells. Moreover, in vivo studies indicated that the combination of L1 and L2 DNA constructs without any adjuvant or delivery system induced effective immune responses, and protected mice against C3 tumor cells (the percentage of tumor-free mice: ~66.67%). Thus, the designed L1 and L2 DNA constructs would represent promising applications for HPV vaccine development.