Kazunari Kondo

Safety and immunogenicity of a peptide containing the cross-neutralization epitope of HPV16 L2 administered nasally in healthy volunteers

Amino acid (aa) 108-120 of L2 protein of human papillomavirus (HPV) type 16 contains a cross-neutralization epitope against genital HPV. We designed a placebo-controlled trial in healthy adults to evaluate the safety and immunogenicity of a synthetic peptide consisting of the aa 108-120 of HPV16 L2 (L2-108/120) region. A total of 13 volunteers were given nasal inoculations with 0.1 (n=5) or 0.5mg (n=5) doses of the peptides or placebo (n=3) without adjuvant at weeks 0, 4, and 12. Sera were collected before inoculation and at 6, 16 and 36 weeks. The inoculation caused no serious local and systemic complications. The inoculation generated anti-L2 antibodies binding to both HPV16 and 52 L1/L2-capsids in four of the five recipients in the 0.5mg group. Sera of the four recipients showed neutralizing activities against HPV16 and 52. Serological responses to the peptides were not found in the 0.1mg group and the placebo group recipients. This study suggests the L2-108/120 peptide is tolerable in humans and has the potential as a broad-spectrum prophylactic vaccine against genital HPV.

Modification of human papillomavirus-like particle vaccine by insertion of the cross-reactive L2-epitopes.

Infection with human papillomavirus 16 (HPV16), which is one of the 15 types of HPV causally associated with cervical cancer and accounts for 50% of the cases, can be prevented in a type‐specific manner by an HPV16 virus‐like particle (VLP) vaccine comprised of particles of the L1 protein alone. We attempted to modify the VLP vaccine by inserting the HPV16 L2‐peptides including cross‐neutralization epitopes into the L1 polypeptide. The chimeric L1 had, between L1 amino acids (aa) 430 and 433, the L2 sequence of aa 18–38, 56–75, or 96–115 (with the replacements of S at aa 101 and T at aa 112 with L and S, respectively). The three chimeric L1s were each expressed from the recombinant baculovirus in insect Sf9 cells, and the resultant VLPs were characterized. The chimeric VLPs were shown to present the L2‐peptides on their surface. By immunizing rabbits with the VLPs, it was shown that they retained capability to induce the antibody neutralizing HPV16 and acquired capability to elicit antibodies cross‐neutralizing the infectious HPV18, 31, 52, and 58 pseudovirions. Although the cross‐neutralizing titers were lower than the type‐specific neutralizing titer, the results suggest that the chimeric VLPs have potential to serve as a vaccine candidate for a broad spectrum of high‐risk HPVs. J. Med. Virol. 80:841–846, 2008.

Biased amplification of human papillomavirus DNA in specimens containing multiple human papillomavirus types by PCR with consensus primers

Genotyping human papillomavirus (HPV) in clinical specimens is important because each HPV type has different oncogenic potential. Amplification of HPV DNA by PCR with the consensus primers that are derived from the consensus sequences of the L1 gene has been used widely for the genotyping. As recent studies have shown that the cervical specimens often contain HPV of multiple types, it is necessary to confirm whether the PCR with the consensus primers amplifies multiple types of HPV DNA without bias. We amplified HPV DNA in the test samples by PCR with three commonly used consensus primer pairs (L1C1/L1C2+C2M, MY09/11, and GP5+/6+), and the resultant amplicons were identified by hybridization with type‐specific probes on a nylon membrane. L1C1/L1C2+C2M showed a higher sensitivity than the other primers, as defined by the ability to detect HPV DNA, on test samples containing serially diluted one of HPV16, 18, 51, 52, and 58 plasmids. L1C1/L1C2+C2M failed to amplify HPV16 in the mixed test samples containing HPV16, and either 18 or 51. The three consensus primers frequently caused incorrect genotyping in the selected clinical specimens containing HPV16 and one or two of HPV18, 31, 51, 52, and 58. The data indicate that PCR with consensus primers is not suitable for genotyping HPV in specimens containing multiple HPV types, and suggest that the genotyping data obtained by such a method should be carefully interpreted. (Cancer Sci 2011; 102: 1223–1227)

Inhibition of nuclear entry of HPV16 pseudovirus-packaged DNA by an anti-HPV16 L2 neutralizing antibody.

Rabbit anti-HPV16 L2 serum (anti-P56/75) neutralizes multiple oncogenic human papillomaviruses (HPVs). We inoculated HeLa cells with HPV16 pseudovirus (16PV) and with anti-P56/75-bound 16PV (16PV-Ab). Both 16PV and 16PV-Ab attached equally well to the cell surface. However, the cell-attached L1 protein of 16PV became trypsin-resistant after incubation at 37°C, whereas approximately 20% of the cell-attached 16PV-Ab L1 remained trypsin-sensitive. Confocal microscopy of HeLa cells inoculated with 16PV revealed packaged DNA in the nucleus at 22h after inoculation; however, nuclear DNA was not detected in cells inoculated with 16PV-Ab. Electron microscopy of HeLa cells inoculated with 16PV showed particles located in multivesicular bodies, lamellar bodies, and the cytosol after 4h; no cytosolic particles were detected after inoculation with 16PV-Ab. These data suggest that anti-P56/75 inhibits HPV infection partly by blocking viral entry and primarily by blocking the transport of the viral genome to the nucleus.

Monoclonal antibodies recognizing cross-neutralization epitopes in human papillomavirus 16 minor capsid protein L2.

Antisera induced by immunization of rabbits with the synthetic peptide P56/75, which has the amino acid (aa) sequence from aa56 to aa75 of HPV16 L2, neutralize pseudovirions and raft-virions of multiple high-risk HPV types, indicating that cross-neutralization epitopes are present in the aa56-75 region. We generated two mouse monoclonal antibodies (MAb): MAb13B and MAb24B recognizing the regions of aa64-73 and aa58-64, respectively. The neutralization assay using pseudovirions of HPV16, 18, 31, 33, 35, 51, 52 and 58 showed that MAb13B neutralized HPV16, 18, and 51, and MAb24B neutralized all the types tested. The mixture of MAb13B and MAb24B neutralized HPV16, 18, and 51 pseudovirions more efficiently than each of the MAbs alone. The data indicate that there are at least two cross-neutralization epitopes in the aa56-75 region and that an antigen capable of presenting the two cross-neutralization epitopes would be a good vaccine candidate for a broad-spectrum of HPVs.

Development of an HPV vaccine for a broad spectrum of high-risk types.

Cervical cancer, the second most frequent gynecological malignancy in the world, is caused by infection with high-risk human papillomaviruses (HPV16, 18, and other 13 types). Vaccines protecting women from infection with high-risk HPVs can reduce prevalence of cervical cancer without costly screening for cervical precancerous lesions. Two vaccines containing HPV16 and 18 virus-like particles (VLPs), which were produced by self-assembly of the major capsid protein L1 of each HPV type, successfully induced type-specific neutralizing antibodies in the recipients of the large scale clinical trials and have been approved by several countries. Although HPV16 and HPV18 account for approximately 50% and 20% of cervical cancer, respectively, the remaining major issue to be addressed is how to prevent infection with other high-risk HPVs. Our and other studies have indicated that the HPV minor capsid protein L2 has several type-common neutralization epitopes and that immunization of animals with peptides having the L2-epitopes protects them against experimental challenge. Recently, we have shown that a type of chimeric VLP, HPV16 VLPs to which the type-common L2-epitope was added, induced in rabbits both the anti-HPV16 L1 neutralizing antibody at a high level and the anti-L2 cross-neutralizing antibody at lower but sufficient levels to be protective shown in the other animal experiments. Thus, this type of chimeric VLP seems likely to be one of the next-generation vaccine candidates for a broad spectrum of high-risk HPV types.

Genetic variation of human papillomavirus type 16 in individual clinical specimens revealed by deep sequencing.

Viral genetic diversity within infected cells or tissues, called viral quasispecies, has been mostly studied for RNA viruses, but has also been described among DNA viruses, including human papillomavirus type 16 (HPV16) present in cervical precancerous lesions. However, the extent of HPV genetic variation in cervical specimens, and its involvement in HPV-induced carcinogenesis, remains unclear. Here, we employ deep sequencing to comprehensively analyze genetic variation in the HPV16 genome isolated from individual clinical specimens. Through overlapping full-circle PCR, approximately 8-kb DNA fragments covering the whole HPV16 genome were amplified from HPV16-positive cervical exfoliated cells collected from patients with either low-grade squamous intraepithelial lesion (LSIL) or invasive cervical cancer (ICC). Deep sequencing of the amplified HPV16 DNA enabled de novo assembly of the full-length HPV16 genome sequence for each of 7 specimens (5 LSIL and 2 ICC samples). Subsequent alignment of read sequences to the assembled HPV16 sequence revealed that 2 LSILs and 1 ICC contained nucleotide variations within E6, E1 and the non-coding region between E5 and L2 with mutation frequencies of 0.60% to 5.42%. In transient replication assays, a novel E1 mutant found in ICC, E1 Q381E, showed reduced ability to support HPV16 origin-dependent replication. In addition, partially deleted E2 genes were detected in 1 LSIL sample in a mixed state with the intact E2 gene. Thus, the methods used in this study provide a fundamental framework for investigating the influence of HPV somatic genetic variation on cervical carcinogenesis.

Thiol-reactive reagents inhibits intracellular trafficking of human papillomavirus type 16 pseudovirions by binding to cysteine residues of major capsid protein L1

BackgroundA human papillomavirus (HPV) virion is composed of capsid proteins L1 and L2. Several cysteine residues are located on L1 of various HPVs at markedly similar relative positions, suggesting their important functions. Although the authentic virions cannot be studied with cultured cells, surrogate pseudovirions consisting of capsid and reporter plasmid are available for studies dealing with infectivity.ResultsHPV type16-pseudovirions (16PVs) were found to lose their infectivity after incubation with thiol-reactive reagents [biotin polyethyleneoxide iodoacetamide (BPEOIA), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), N-ethylmaleimide (NEM), 4-(N-maleimido)benzyl-trimethylammonium iodide (MBTA), and [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET)]. A labelled streptavidin was detected to bind to the complex of BPEOIA and L1 of the 16PVs incubated with BPEOIA. The analysis of molecular mass of trypsin-fragments derived from the complex of the BPEOIA and L1 indicated that BPEOIA bound to at least C146, C225, and C229. No appreciable change of the 16PVs carrying DTNB or NEM was detected by sedimentation analysis or electron microscopy. The 16PVs carrying DTNB or NEM were able to bind to and enter HeLa cells but degraded before they reached the perinuclear region.ConclusionHPV16 L1 C146, C225, and C229 have free thiol, which are accessible to BPEOIA, DTNB, NEM, MBTA, and MTSET. Binding of DTNB or NEM to the thiols may cause conformational changes that result in the inhibition of the entry and trafficking of the 16PVs.

Neutralizing Antibodies against Human Papillomavirus Types 16, 18, 31, 52, and 58 in Serum Samples from Women in Japan with Low-Grade Cervical Intraepithelial Neoplasia

ABSTRACT We have very limited information on serum neutralizing antibody in women naturally infected with the human papillomaviruses (HPVs) that are causally associated with cervical cancer. In this study, serum samples collected from 217 Japanese women with low-grade cervical intraepithelial neoplasia were examined for their neutralizing activities against HPV16, -18, -31, -52, and -58 pseudovirions. Eighty-four patients (39%), 35 patients (16%), 17 patients (8%), and 1 patient were positive for neutralizing antibodies against one, two, three, and four of these types, respectively. Presence of neutralizing antibody did not always correlate with detection of HPV DNA in cervical swabs collected at the time of blood collection. The neutralizing titers of the majority of sera, ranging between 40 and 640, were found to be conserved in the second sera, collected 24 months later, independently of emergence of HPV DNA in the second cervical swabs. The data strongly suggest that HPV infection induces anti-HPV neutralizing antibody at low levels, which are maintained for a long period of time.

Hypermutation in the E2 gene of human papillomavirus type 16 in cervical intraepithelial neoplasia

Persistent infection with oncogenic human papillomavirus (HPV) causes cervical cancer. However, viral genetic changes during cervical carcinogenesis are not fully understood. Recent studies have revealed the presence of adenine/thymine‐clustered hypermutation in the long control region of the HPV16 genome in cervical intraepithelial neoplasia (CIN) lesions, and suggested that apolipoprotein B mRNA editing enzyme, catalytic polypeptide‐like (APOBEC) proteins, which play a key role in innate immunity against retroviral infection, potentially introduce such hypermutation. This study reports for the first time the detection of adenine/thymine‐clustered hypermutation in the E2 gene of HPV16 isolated from clinical specimens with low‐ and high‐grade CIN lesions (CIN1/3). Differential DNA denaturation PCR, which utilizes lower denaturation temperatures to selectively amplify adenine/thymine‐rich DNA, identified clusters of adenine/thymine mutations in the E2 gene in 4 of 11 CIN1 (36.4%), and 6 of 27 CIN3 (22.2%) samples. Interestingly, the number of mutations per sample was higher in CIN3 than in CIN1. Although the relevance of E2 hypermutation in cervical carcinogenesis remains unclear, the observed hypermutation patterns strongly imply involvement of APOBEC3 proteins in editing the HPV16 genome during natural viral infection. J. Med. Virol. 87:1754–1760, 2015.