Human Papillomavirus L1 Research Articles

HPV Vaccination Status in HIV-Negative MSM and Its Association with High-Risk HPV Detection Using HPV Serology and Anorectal Swabs

Background/Objective: The aim of this study was to determine the prevalence of high-risk (HR) human papillomavirus (HPV) types by HPV vaccination status and the feasibility of using HPV L1 serology to identify HIV-negative men who have sex with men (MSM) who may be at risk for anal cancer. Methods: This cross-sectional study recruited HIV-negative MSM from a US metropolitan area. The prevalence of HR, quadrivalent, and nonavalent anorectal HPV DNA and HPV L1 serum antibodies was estimated. McNemar’s chi-square and kappa statistics were used to determine significant differences in HPV detection between anorectal DNA swabs and HPV L1 serology. Results: Eighty-two men had adequate anorectal swabs and serology samples for analysis. Men who self-reported receipt of the HPV vaccine (35.6%) had detectable L1 HPV antibodies (93.1%) and a lower prevalence of active anal HPV infections (20.7%) compared to those who reported none. Conclusions: If confirmed in larger prospective studies, a combination of HPV vaccination status or HPV L1 serology and anorectal swabs for HR HPV types could identify HIV-negative MSM who do not need to undergo follow-up anal testing.

Human papillomavirus vaccines: organisation and experience of preclinical studies

INTRODUCTION. Vaccination is the main measure for the primary prevention of human papillomavirus (HPV)-related diseases. The development of novel vaccine candidates is underway worldwide, including in the Russian Federation. At the same time, the clinical introduction of new HPV vaccines is seriously hampered by the lack of clear and unambiguous recommendations for conducting preclinical studies of these vaccines.AIM. This study aimed to analyse regulatory documents on HPV vaccines, to study the experience of conducting preclinical studies, and to summarise the preclinical approaches that could be recommended for developers and applicants seeking approval for new preventive HPV vaccines, including the vaccines being developed in the Russian Federation.DISCUSSION. The authors have analysed regulatory documents issued by the World Health Organisation (WHO), the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), and the Council of the Eurasian Economic Commission (EEC). Additionally, the authors have reviewed the experimental results of preclinical studies of HPV vaccines. The known licensed and pipeline HPV vaccines are similar in terms of their characteristics and constructive features. However, there may be some differences in the HPV serotype coverage and the methods used to produce the HPV L1 capsid protein. To date, studies have confirmed the role of the HPV L1 capsid protein in the development of specific immunity, rendering challenge tests in animal models unnecessary. Papillomatosis modelling may be required for choosing an alternative immunological target or for studying an alternative (non-parenteral) route for vaccine administration. Preclinical study programmes may be supplemented with individual stages of comprehensive assessment of adjuvants and other additives included in novel HPV vaccine compositions.CONCLUSIONS. The authors have studied the international experience and presented a systemic overview of the methods and approaches used in preclinical studies of HPV vaccines. The authors have formulated recommendations for developers for the planning and organisation of preclinical studies of HPV vaccines (including immunogenicity, toxicity, and local tolerance assessments required for licensing new vaccines).

Genotyping and phylogenetic analysis of human Papillomaviruses from women with genital warts

Background and objectives. The pathophysiology of Human Papillomavirus (HPV) infections is dependent on the genotype of virus, the host immune system and the local environmental conditions. HPV infections are linked to a variety of benign and malignant disorders. Anogenital warts is a Sexually Transmitted Infection (STI) primarily caused by Low-Risk HPV (LR-HPV) genotypes 6 and 11. The aims were to Investigation of the prevalence and genotypic distribution of HPV in women with genital warts in Mosul/Iraq, HPV-DNA sequencing and Phylogenetic analysis including minimum spanning trees of amplified HPV L1 gene comparison the present sequences with similar genotypes in GenBank – National Center Biotechnology Information (NCBI) using mega-7-software. Materials and methods. A total of 150 samples were collected from women with genital wart infections. Polymerase Chain Reaction (PCR) was performed using GP+5/GP+6 and MY09/MY11 consensus primers. Results. About 84.6% prevalence of HPV genotypes. The highest frequency (32% and 28.6%) were observed in the 19-28 and 29-38 age groups. Conclusion. Women in Mosul, Iraq, who have genital warts are more likely to have different genotypes of HPV. HPV 11 type was found to be the most common, followed HPV6 and HPV10 as LR HPV, while HPV 16 and HPV45 were the most common as HR HPV. The analysis of the phylogenetic tree revealed a genetic relationship between our isolates and global isolates ranging from (10-100%) bootstrap ratio this indicating phylogenetic relationships (divergent and convergent evolution) this was meant the diversity in origin of the various genotypes common in Mosul, Iraq.

Engineering of Recombinant Human Papillomavirus 16 L1 Protein for Incorporation with para-Azido-L-Phenylalanine.

Human papillomavirus (HPV) L1 capsid protein were produced in several host systems, but few studies have focused on enhancing the properties of the L1 protein. In this study, we aimed to produce recombinant Human papillomavirus (HPV) L1 capsid protein containing para-azido-L-phenylalanine (pAzF) in Escherichia coli. First, we expressed the maltose-binding protein (MBP)-fused HPV16 L1, and 5 residues in HPV16 L1 protein were selected by the in silico modeling for amber codon substitution. Among the variants of the five locations, we identified a candidate that exhibited significant differences in expression with and without pAzF via genetic code expansion (GCE). The expressed recombinant MBP-HPV16L1 protein was confirmed for incorporation of pAzF and the formation of VLPs was tested in vitro.

Vaccination with a Human Papillomavirus L2 Multimer Provides Broad Protection against 17 Human Papillomavirus Types in the Mouse Cervicovaginal Challenge Model.

Human papillomavirus (HPV) is a prevalent cause of mucosal and cutaneous infections and underlying conditions ranging from benign warts to anogenital and oropharyngeal cancers affecting both males and females, notably cervical cancer. Cervical cancer is the fourth leading cause of cancer deaths among women globally and is the most impactful in low- and middle-income countries (LMICs), where the costs of screening and licensed L1-based HPV vaccines pose significant barriers to comprehensive administration. Additionally, the licensed L1-based HPV vaccines fail to protect against all oncogenic HPV types. This study generated three independent lots of an L2-based target antigen (LBTA), which was engineered from conserved linear L2-protective epitopes (aa11-88) from five human alphapapillomavirus genotypes in E. coli under cGMP conditions and adjuvanted with aluminum phosphate. Vaccination of rabbits with LBTA generated high neutralizing antibody titers against all 17 HPV types tested, surpassing the nine types covered by Gardasil®9. Passive transfer of naïve mice with LBTA antiserum revealed its capacity to confer protection against vaginal challenge with all 17 αHPV types tested. LBTA shows stability at room temperature over >1 month. Standard in vitro and in vivo toxicology studies suggest a promising safety profile. These findings suggest LBTA's promise as a next-generation vaccine with comprehensive coverage aimed at reducing the economic and healthcare burden of cervical and other HPV+ cancers in LMICs, and it has received regulatory approval for a first-in-human clinical study (NCT05672966).

The BICD2 dynein cargo adaptor binds to the HPV16 L2 capsid protein and promotes HPV infection.

During entry, human papillomavirus (HPV) traffics from the endosome to the trans Golgi network (TGN) and Golgi and then the nucleus to cause infection. Although dynein is thought to play a role in HPV infection, how this host motor recruits the virus to support infection and which entry step(s) requires dynein are unclear. Here we show that the dynein cargo adaptor BICD2 binds to the HPV L2 capsid protein during entry, recruiting HPV to dynein for transport of the virus along the endosome-TGN/Golgi axis to promote infection. In the absence of BICD2 function, HPV accumulates in the endosome and TGN and infection is inhibited. Cell-based and in vitro binding studies identified a short segment near the C-terminus of L2 that can directly interact with BICD2. Our results reveal the molecular basis by which the dynein motor captures HPV to promote infection and identify this virus as a novel cargo of the BICD2 dynein adaptor.

Cloning, Expression, and Bioinformatics Modeling of Human Papillomavirus Type 52 L1/L2 Chimeric Protein in Escherichia coli BL21 (DE3)

Human papillomavirus (HPV) L1 major capsid protein generates a highly immunogenic virus like particles (VLPs), which have been used as the main component of its prophylactic vaccine. However, the neutralizing antibodies against L1 VLPs are mostly type specific and may not be effective to prevent infection from different strains of HPV. On the other hand, HPV L2 minor capsid protein has low antigenic variation, thus can induce cross-neutralization. This study aims to obtain HPV 52 L1/L2 chimeric protein, which is designed based on HPV type 52 as one of the most circulated high-risk types in Indonesia, to develop a broad-spectrum HPV vaccine. Substitution of HPV 52 H4 helix L1 region with an HPV 52 L2 epitope was carried out using overlap extension PCR. HPV 52 L1/L2 chimeric gene was constructed into pET-SUMO expression vector and expressed in Escherichia coli BL21 (DE3). Bioinformatics modeling suggested that L2 epitope was located inside of the loop region in monomer form, and on the contrary, it was located outside of the pentamer surface. Furthermore, B cell and T cell epitopes predictions were conducted using Immune Epitope Database (IEDB) analysis. The B cell epitopes prediction revealed eleven potential epitopes, whereas the T cell epitopes prediction showed seven potential epitopes for each MHC class I and MHC class II. This study showed that HPV 52 L1/L2 chimeric protein has the potential to induce cross-neutralizing antibodies and can be developed as a promising candidate for a new HPV vaccine.

Characterization and Structural Analysis of the Human Papilloma Virus L1 Protein in Iran

Characterization and Structural Analysis of the Human Papilloma Virus L1 Protein in Iran

High-risk human papillomaviruses l1 gene isolates identified in Western Kazakhstan

Kazakhstani researchers reported a significant prevalence of highly carcinogenic human papillomavirus types in the country.<br /> The article <b>aimed</b> to present HPV L1 gene sequencing developments in women affected with cervical cancer throughout the western part of Kazakhstan with provided findings on the geographic pathways of obtained isolates.<br /> <b>Methods. </b>The HPV L1 gene was amplified using the consensus primers MY09HPV 5’-CGTCCMARRGGAWACTGATC-3’ and MY11HPV 5’ – GCMCAGGGWCATAAYAATGG-3’.  The purified DNA was used as the target for direct nucleotide sequencing. Phylogenetic analyses were conducted using the MegAlign program from the LASERGENE software package (version 6.0; DNA star, Madison, WI) and with MEGA version 5.0 software. A multiple alignment was created through Clustal W software, and the neighbor-joining method was used to construct the phylogenetic tree.<br /> <b>Results.</b> Of 70 HPV samples transported to the Astana shared laboratory for gene L1 sequencing, only ten appeared fit to obtain isolates (14.3%). The viral load of the samples ranged from 3.3 to 8.2, and the range of DNA concentration was from 8.16 to 69.6 ng/uL. HPV16 unique Kazakhstani isolate from Aktobe, having its own branch, and not yet registered in the world genebank, was revealed. An isolate of potentially carcinogenic HPV53 forming a remote cluster with KF436822/1, KU951264.1 - Southwest China, and 97% identity with EU056643.1 - Ireland, and acted as a single agent for invasive cervical cancer was identified.<br /> In general, the sequencing findings indicate the variety of ways for HPV pervasion into the western region of Kazakhstan: North and South America, Europe, and Asia.<br /> <i><i>The study was recorded in the ISRCTN registry, No. 7154910, 02/01/2018.</i></i>

Virus-like particle vaccine displaying an external, membrane adjacent MUC16 epitope elicits ovarian cancer-reactive antibodies

BackgroundMUC16 is a heavily glycosylated cell surface mucin cleaved in the tumor microenvironment to shed CA125. CA125 is a serum biomarker expressed by > 95% of non-mucinous advanced stage epithelial ovarian cancers. MUC16/CA125 contributes to the evasion of anti-tumor immunity, peritoneal spread and promotes carcinogenesis; consequently, it has been targeted with antibody-based passive and active immunotherapy. However, vaccination against this self-antigen likely requires breaking B cell tolerance and may trigger autoimmune disease. Display of self-antigens on virus-like particles (VLPs), including those produced with human papillomavirus (HPV) L1, can efficiently break B cell tolerance.ResultsA 20 aa juxta-membrane peptide of the murine MUC16 (mMUC16) or human MUC16 (hMUC16) ectodomain was displayed either via genetic insertion into an immunodominant loop of HPV16 L1-VLPs between residues 136/137, or by chemical coupling using malemide to cysteine sulfhydryl groups on their surface. Female mice were vaccinated intramuscularly three times with either DNA expressing L1-MUC16 fusions via electroporation, or with alum-formulated VLP chemically-coupled to MUC16 peptides. Both regimens were well tolerated, and elicited MUC16-specific serum IgG, although titers were higher in mice vaccinated with MUC16-coupled VLP on alum as compared to L1-MUC16 DNA vaccination. Antibody responses to mMUC16-targeted vaccination cross-reacted with hMUC16 peptide, and vice versa; both were reactive with the surface of CA125+ OVCAR3 cells, but not SKOV3 that lack detectable CA125 expression. Interestingly, vaccination of mice with mMUC16 peptide mixed with VLP and alum elicited mMUC16-specific IgG, implying VLPs provide robust T help and that coupling may not be required to break tolerance to this epitope.ConclusionVaccination with VLP displaying the 20 aa juxta-membrane MUC16 ectodomain, which includes the membrane proximal cleavage site, is likely to be well tolerated and induce IgG targeting ovarian cancer cells, even after CA125 is shed.

CER818: A Highly Specific and Sensitive HPV L1 High-Risk Serological Lateral Flow Rapid Test for Early Detection of Cervical Cancer and Its Precursor Lesions.

Objective: The objective of the study is to validate a new human papillomavirus (HPV) L1 high-risk specific serological assay in a case-control study. Methods: Serum samples of 138 patients (cervical intraepithelial neoplasia (CIN) 1, 2, and 3 and cervical cancer), 21 vaccinees, and 246 female controls were tested for the presence of HPV L1 high-risk specific antibodies. Results: HPV L1 high-risk antibodies were detected in 100% of the CIN1 and 2, 86.6% of the CIN3 and 82.4% of the cervical cancer cases, 100% of the vaccinees, and 3.9% of the female controls. Area under the curve (AUC) was calculated with 0.91 for controls versus CIN2+, 0.923 for controls versus CIN1+, and 0.968 for controls versus CIN1/2. Conclusion: The HPV L1 high-risk specific serological lateral flow rapid test shows promising data in the field of early detection of HPV high-risk induced cervical cancer and its precursor lesions. This easy-to-use, robust, and affordable approach could offer a chance to reach women in low- or middle-income countries (LMICs) that could not be reached by HPV molecular testing-based cervical cancer screening programs.

Screening and identification of B cell epitope within the major capsid protein L1 of HPV 52, using monoclonal antibodies

The L1 protein of Human papillomavirus (HPV), the main capsid protein, induces the formation of neutralizing antibodies. In this study, HPV52 L1 protein was induced to be expressed. Monoclonal antibody (mAb) 6A7 against L1 protein were screened by cell fusion techniques. Western Blot and immunofluorescence assay (IFA) demonstrated the specificity of the mAb. The L1 protein was truncated for prokaryotic expression (N1∼N7) and Dot-ELISA showed that 6A7 recognized N3 (aa 200–350). The immunodominant regions were truncated again for expression, with 6A7 recognizing N6 (aa 251–305). The N6 proteins were further truncated and then were constructed an four-segment eukaryotic expression vector. IFA showed that 6A7 could recognize amino acid 262–279. Amino acid 262–279 was selected to be truncated into short peptides P1 and P2. Finally, Peptide-ELISA and Dot-ELISA showed that the epitope regions of mAb 6A7 were amino acid 262–273. The mAbs with defined epitopes can lay the foundation for the analysis of antigenic epitope characteristics and promote the development of epitope peptide vaccines.

Expression, purification, and characterization of self-assembly virus-like particles of capsid protein L1 HPV 52 in Pichia pastoris GS115

BackgroundCervical cancer caused by the human papillomavirus (HPV) is one of the most frequent malignances globally. HPV 52 is a high-risk cancer-causing genotype that has been identified as the most prevalent type in Indonesia. Virus-like particles (VLP)-based vaccinations against HPV infection could benefit from self-assembled VLP of L1 capsid protein. ResultThe recombinant HPV 52 L1 was expressed in Pichia pastoris on a shake-flask scale with 0.5% methanol induction in this study. The copy number was used to compare the expression level and stability. The colony that survived on a solid medium containing 2000 μg/ml of Zeocin was selected and cultured to express HPV 52 L1. DNA was extracted from the chosen colony, and the copy was determined using qPCR. HPV 52 L1 protein was then purified through fast performance liquid chromatography. Transmission electron microscopy (TEM) evaluation confirmed the VLP self-assembly. The genomic DNA remained intact after 100 generations of serial cultivation under no selective pressure medium conditions, and the protein produced was relatively stable. However, the band intensity was slightly lower than in the parental colony. In terms of copy number, a low copy transformant resulted in low expression but produced a highly stable recombinant clone. Eventually, the L1 protein expressed in Pichia pastoris can self-assemble into VLP. Therefore, recombinant HPV possesses a stable clone and the ability to self-assemble into VLP. ConclusionThe recombinant L1 HPV 52 protein is successfully expressed in P. pastoris within a size range of approximately 55 kDa and demonstrated favorable stability. The L1 protein expressed in Pichia pastoris successful self-assembled of HPV VLPs, thereby establishing their potential efficacy as a prophylactic vaccine.

Development of a touchdown droplet digital PCR assay for the detection and quantitation of human papillomavirus 16 and 18 from self-collected anal samples

ABSTRACT Broad-spectrum droplet digital polymerase chain reaction (ddPCR) assays can be used to quantitate multiple human papillomavirus (HPV) types. However, the presence of nucleotide mismatches at the primer binding sites may result in different optimal annealing temperatures and, consequently, under detection of some HPV types. Touchdown PCR uses decreasing annealing temperature over the first PCR cycles allowing for specific and efficient amplification of the target sequence. Here, as a proof-of-concept, we assessed if touchdown ddPCR (TD-ddPCR) using broad-spectrum oligonucleotides and genotype-specific probes targeting HPV L1 can be used to quantitate HPV. We tested our hypothesis using HPV positive cell lines and anal samples which potentially contain PCR inhibitors. Using serial dilutions of HPV16 and HPV18 DNA, the TD-ddPCR assays were performed with linearity across the dilution series (Pearson’s correlation r = 0.9999) and detected one copy of both HPV types, with no cross-reactivity to 27 other low-risk and high-risk HPV types. The PCRs were highly reproducible, with inter- and intra-assay coefficient of variations ranging 2.0–6.3% for HPV16 and 4.9–11.6% for HPV18. Testing self-collected anal samples positive for HPV16 ( n = 29) or HPV18 ( n = 33) and those negative for both HPV types ( n = 22) with the ddPCR assay and Seegene Anyplex II HPV28 (Seegene, TD-ddPCR) showed high agreements between the two assays. In conclusion, HPV broad-spectrum primers containing mismatches at their target binding sites and a touchdown thermal cycling approach resulted in sensitive and specific amplification of HPV genome using ddPCR. The assay has potential clinical diagnostic applications for detection, quantitation, and monitoring of anal HPV infections. IMPORTANCE The quantity of the human papillomavirus (HPV) is associated with disease outcome. We designed an accurate and precise digital PCR assay for quantitating HPV in anal samples, a sample type that is typically problematic due to the presence of PCR inhibitors.

Advances on two serological assays for human papillomavirus provide insights on the reactivity of antibodies against a cross-neutralization epitope of the minor capsid protein L2.

A second generation of prophylactic human papillomavirus (HPV) vaccines based on the minor capsid protein L2 has entered clinical trials as promising alternative to meet the gaps left out by the current vaccines concerning type-restricted protection, high costs and low penetrance in immunization programs of lowand middle-income countries. Most of the serological assays available to assess anti-HPV humoral responses are, however, not well suited for measuring vaccine-induced anti-L2 antibody responses. In this work, we have advanced our automated, purely add-on High-Throughput Pseudovirion-Based Neutralization Assay (HT-PBNA) in an L2-oriented approach for measuring antibody-mediated neutralization of HPV types 6/16/18/31/33/52/58. With the optimized settings, we observed 24- to 120-fold higher sensitivity for detection of neutralizing Ab to the L2 protein of HPV6, HPV16, HPV18, and HPV31, compared to the standard HT-PBNA. Alternatively, we have also developed a highly sensitive, cell-free, colorimetric L2-peptide capture ELISA for which the results were strongly concordant with those of the advanced neutralization assay, named HT-fc-PBNA. These two high-throughput scalable assays represent attractive approaches to determine antibody-based correlates of protection for the HPV L2 vaccines that are to come.

Alum and a TLR7 agonist combined with built-in TLR4 and 5 agonists synergistically enhance immune responses against HPV RG1 epitope

To relieve the limitations of the human papillomavirus (HPV) vaccines based on L1 capsid protein, vaccine formulations based on RG1 epitope of HPV L2 using various built-in adjuvants are under study. Herein, we describe design and construction of a rejoined peptide (RP) harboring HPV16 RG1 epitope fused to TLR4/5 agonists and a tetanus toxoid epitope, which were linked by the (GGGS)3 linker in tandem. In silico analyses indicated the proper physicochemical, immunogenic and safety profile of the RP. Docking analyses on predicted 3D model suggested the effective interaction of TLR4/5 agonists within RP with their corresponding TLRs. Expressing the 1206 bp RP-coding DNA in E. coli produced a 46 kDa protein, and immunization of mice by natively-purified RP in different adjuvant formulations indicated the crucial role of the built-in adjuvants for induction of anti-RG1 responses that could be further enhanced by combination of TLR7 agonist/alum adjuvants. While the TLR4/5 agonists contributed in the elicitation of the Th2-polarized immune responses, combination with TLR7 agonist changed the polarization to the balanced Th1/Th2 immune responses. Indeed, RP + TLR7 agonist/alum adjuvants induced the strongest immune responses that could efficiently neutralize the HPV pseudoviruses, and thus might be a promising formulation for an inexpensive and cross-reactive HPV vaccine.

Immunogenicity and safety of an Escherichia coli-produced human papillomavirus (types 6/11/16/18/31/33/45/52/58) L1 virus-like-particle vaccine: a phase 2 double-blind, randomized, controlled trial

The Escherichia coli-produced human papillomavirus (HPV) 16/18 bivalent vaccine (Cecolin) has received prequalification by the World Health Organization based on its high efficacy and good safety profile. We aimed to evaluate the immunogenicity and safety of the second-generation nonavalent HPV 6/11/16/18/31/33/45/52/58 vaccine (Cecolin 9) through the randomized, blinded phase 2 clinical trial. Eligible healthy women aged 18–45 years were randomly (1:1) allocated to receive three doses of 1.0 mL (270 µg) of Cecolin 9 or placebo with a 0-1-6-month schedule. The primary endpoint was the seroconversion rate and geometric mean titer of neutralizing antibodies (nAbs) one month after the full vaccination course (month 7). The secondary endpoint was the safety profile including solicited adverse reactions occurring within 7 d, adverse events (AEs) occurring within 30 d after each dose, and serious adverse events (SAEs) occurring during the 7-month follow-up period. In total, 627 volunteers were enrolled and randomly assigned to Cecolin 9 (n = 313) or placebo (n = 314) group in Jiangsu Province, China. Almost all participants in the per-protocol set for immunogenicity (PPS-I) seroconverted for nAbs against all the nine HPV types at month 7, while two failed to seroconvert for HPV 11 and one did not seroconvert for HPV 52. The incidence rates of total AEs in the Cecolin 9 and placebo groups were 80.8% and 72.9%, respectively, with the majority of them being mild and recovering shortly. None of the SAEs were considered related to vaccination. In conclusion, the E. coli-produced 9-valent HPV (9vHPV) vaccine candidate was well tolerated and immunogenic, which warrants further efficacy studies in larger populations.

A Cas12a-based fluorescent microfluidic system for rapid on-site human papillomavirus diagnostics.

Persistent infection with human papillomavirus (HPV) is the leading cause of cervical cancer, and early diagnosis is crucial for clinical management. However, the easy and rapid on-site diagnostic for HPV genotyping remains challenging. Here, we develop a Cas12a-based fluorescent microfluidic detection system for diagnosing six HPV subtypes (HPV6, HPV11, HPV16, HPV18, HPV31, andHPV33). A panel of crRNAs and recombinase polymerase amplification (RPA) primers targeting the HPV L1 gene was screened for sensitive and specific detection. Furthermore, a one-pot RPA reaction was developed to amplify the six HPV subtypes without cross-reactivity. For on-site detection, we integrated the RPA-Cas12a detection into a microfluidic device, enabling the detection of processed clinical samples within 35 minutes. The assay was validated using 112 clinical swab samples and obtained consistent results with the qPCR assay, with a concordance rate of 99.1%. Overall, our diagnostic method offers a rapid, sensitive, and easy-to-use on-site assay for detecting HPV genotypes and holds promise for improving cervical cancer screening and prevention. KEY POINTS: • The Cas12a-based fluorescent microfluidic detection system for the diagnosis of six HPV subtypes. • A one-pot RPA reaction for amplifying the six HPV subtypes without cross-reactivity. • The RPA-Cas12a-microfluidic system provides results within 35 minutes for on-site detection.

Bac to Bac System Efficiency for Preparing HPV Type 16 Virus-Like Particle Vaccine.

Today, the human papillomavirus (HPV) L1 protein is the main target in the construction of prophylactic HPV vaccines. The production of virus-like particles (VLPs) that closely resemble the natural structure of the HPV16 virus and induce high levels of virus-neutralizing antibodies in animals and humans is facilitated by the expression of HPV16-L1 protein in eukaryotic cells. The Bac-to-Bac system has been previously used to produce high levels of recombinant proteins. In this study, we utilized this expression system to generate HPV16-L1 VLPs in Spodoptra frugipedra (Sf9) insect cells. The wild-type L1 gene of papillomavirus type 16 was selected from Gene Bank and placed in bacmid structure after codon optimization using pFast Bac vector. The recombinant baculovirus containing HPV-16/L1 gene was then provided using the Bac-to-Bac system. It should be mentioned that the vector was transfected into the Sf9 cell. The cells were then lysed and the expression of L1 protein was revealed by SDS-PAGE and confirmed by Western Blot. The L1 purification was performed through Ni-NTA chromatography. The VLP formation of papillomavirus L1 protein was visualized by transmission electron microscopy. The expressed recombinant L1 was ~60 KD on SDS-PAGE which was identified in western blot by a specific anti-L1 monoclonal antibody. The electron microscopy confirmed the assembly of VLPs. Results of this study showed that the production of this protein at the industrial level can be optimized using a baculovirus/Sf9 system. The characteristics and advantages of this system are promising and it is a suitable candidate for protein synthesis.

Safety and immunogenicity of a pichia pastoris-expressed bivalent human papillomavirus (types 16 and 18) L1 virus-like particle vaccine in healthy Chinese women aged 9-45years: A randomized, double-blind, placebo-controlled phase 1 clinical trial.

We evaluated the safety and immunogenicity of high and low doses of a novel pichia pastoris-expressed bivalent (types 16 and 18) human papillomavirus (HPV) virus-like particle vaccine. In this randomized, double-blind, placebo-controlled phase 1 trial, we enrolled 160 healthy females aged 9-45years in Guangxi, China who were randomized (1:1:2) to receive either low (0.5mL) or high (1.0mL) dosages of bivalent HPV vaccine, or placebo (aluminum adjuvant) in a 0, 2, 6months schedule. Adverse events and other significant conditions that occurred within 30days after each vaccination were recorded throughout the trial. Sera were collected at days 0, 60, 180 and 210 to measure anti-HPV 16/18 neutralizing antibodies. A total of 160 participants received at least one dose of the HPV vaccine and 152 completed the three dose vaccination series. Reporting rates of adverse events in placebo, low dose (0.5mL) and high dose (1.0mL) groups were 47.5%, 55.0% and 55.0%, respectively. No serious adverse events occurred during this trial. 100% of the participants who received three doses of the HPV vaccine produced neutralizing antibodies against HPV 16/18 vaccine. For HPV 16 and HPV 18, the geometric mean titers (GMTs) were similar between the low dose group (GMTHPV 16=10816 [95% CI: 7824-14953]), GMTHPV 18=3966 [95% CI: 2693-5841]) and high dose group (GMT HPV 16=14482 [95% CI: 10848-19333], GMT HPV 18=3428 [95% CI: 2533-4639]). The pichia pastoris-expressed bivalent HPV vaccine was safe and immunogenic in Chinese females aged 9-45years. The low dosage (0.5mL) was selected for further immunogenicity and efficacy study.