Human Papillomavirus Virus-like Particles Research Articles

Interaction studies between human papillomavirus virus-like particles and laminin 332 by affinity capillary electrophoresis assisted by bio-layer interferometry

Human papillomavirus (HPV) interacts, in vitro, with laminin 332 (LN332), a key component of the extracellular matrix. In this study, we performed bio-layer interferometry (BLI) and affinity capillary electrophoresis (ACE) to investigate the binding properties of this interaction. Virus-like particles (VLPs), composed of the HPV16 L1 major capsid protein, were used as HPV model and LN332 as the VLPs binding partner. Using BLI, we quantitatively determined the kinetics of the interaction, via the measurement of VLP binding and release from LN332 immobilized onto the surface of aminopropylsilane biosensors. We found an averaged kon of 1.74 x 104 M−1s−1 and an averaged koff of 1.50 x 10−4 s−1. Furthermore, an ACE method was developed to study the interaction under physiological conditions, where the interactants are moving freely in solution, without any fluorescence labeling. Specifically, a constant amount of HPV16-VLPs was preincubated with increasing LN332 concentrations and then the samples were injected in the capillary electrophoresis instrument. A shift in the migration time of the HPV16-VLP/LN332 complexes, carrying an increasing number of LN332 molecules bound per VLP, was observed. The mobility of the complexes was found to decrease with increasing LN332 concentrations in the sample. It was used to quantify stability constant. From BLI and ACE approaches, we reported an apparent equilibrium dissociation constant in the nanomolar range (8.89 nM and 17.7 nM, respectively) for the complex between HPV16-VLPs and LN332.

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.

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.

Multi-dose Formulation Development for a Quadrivalent Human Papillomavirus Virus-Like Particle-Based Vaccine: Part II- Real-time and Accelerated Stability Studies

This work describes Part 2 of multi-dose formulation development of a Human Papillomavirus (HPV) Virus-Like Particle (VLP) based vaccine (see Part 1 in companion paper). Storage stability studies with candidate multi-dose formulations containing individual or combinations of seven different antimicrobial preservatives (APs) were performed with quadrivalent HPV VLP (6, 11, 16, 18) antigens adsorbed to aluminum-salt adjuvant (Alhydrogel®). Real-time (up to two years, 2-8°C) and accelerated (months at 25 and 40°C) stability studies identified eight lead candidates as measured by antigen stability (competitive ELISA employing conformational serotype-specific mAbs), antimicrobial effectiveness (modified European Pharmacopeia assay), total protein content (SDS-PAGE), and AP concentration (RP-UHPLC). The AH-adsorbed HPV18 VLP component was most sensitive to AP-induced destabilization. Optimal quadrivalent antigen storage stability while maintaining antimicrobial effectiveness was observed with 2-phenoxyethanol, benzyl alcohol, chlorobutanol, and 2-phenoxyethanol + benzyl alcohol combination. Interestingly, for single-AP containing multi-dose formulations, this rank-ordering of storage stability did not correlate with previously reported biophysical measurements of AP-induced antigen destabilization. Moreover, other APs (e.g., m-cresol, phenol, parabens) described by others for inclusion in multi-dose HPV VLP formulations showed suboptimal stability. These results suggest that each HPV VLP vaccine candidate (e.g., different serotypes, expression systems, processes, adjuvants) will require customized multi-dose formulation development.

Multi-Dose Formulation Development for a Quadrivalent Human Papillomavirus Virus-Like Particle-Based Vaccine: Part I - Screening of Preservative Combinations

The development of multi-dose, subunit vaccine formulations can be challenging since antimicrobial preservatives (APs) often destabilize protein antigens. In this work, we evaluated Human Papillomavirus (HPV) Virus-Like Particles (VLPs) to determine if combining different APs used in approved parenteral products, each at lower concentrations than used alone, would maintain both antimicrobial effectiveness and antigen stability. To identify promising AP combinations, two different screening strategies were utilized: (1) empirical one-factor-at-a-time (OFAT) and (2) statistical design-of-experiments (DOE). Seven different APs were employed to screen for two- and three-AP combinations using high-throughput methods for antimicrobial effectiveness (i.e., microbial growth inhibition assay and a modified European Pharmacopeia method) and antigen stability (i.e., serotype-specific mAb binding to conformational epitopes of HPV6, 11, 16 VLPs by ELISA). The OFAT and DOE approaches were complementary, such that initial OFAT results (and associated lessons learned) were subsequently employed to optimize the combinations using DOE. Additional validation experiments confirmed the final selection of top AP-combinations predicted by DOE modeling. Overall, 20 candidate multi-dose formulations containing two- or three-AP combinations were down-selected. As described in Part 2 (companion paper), long-term storage stability profiles of aluminum-adjuvanted, quadrivalent HPV VLP formulations containing these lead candidate AP combinations are compared to single APs.

Analytical and Preformulation Characterization Studies of Human Papillomavirus Virus-Like Particles to Enable Quadrivalent Multi-Dose Vaccine Formulation Development

Introducing multi-dose formulations of Human Papillomavirus (HPV) vaccines will reduce costs and enable improved global vaccine coverage, especially in low- and middle-income countries. This work describes the development of key analytical methods later utilized for HPV vaccine multi-dose formulation development. First, down-selection of physicochemical methods suitable for multi-dose formulation development of four HPV (6, 11, 16, and 18) Virus-Like Particles (VLPs) adsorbed to an aluminum adjuvant (Alhydrogel®, AH) was performed. The four monovalent AH-adsorbed HPV VLPs were then characterized using these down-selected methods. Second, stability-indicating competitive ELISA assays were developed using HPV serotype-specific neutralizing mAbs, to monitor relative antibody binding profiles of the four AH-adsorbed VLPs during storage. Third, concentration-dependent preservative-induced destabilization of HPV16 VLPs was demonstrated by addition of eight preservatives found in parenterally administered pharmaceuticals and vaccines, as measured by ELISA, dynamic light scattering, and differential scanning calorimetry. Finally, preservative stability and effectiveness in the presence of vaccine components were evaluated using a combination of RP-UHPLC, a microbial growth inhibition assay, and a modified version of the European Pharmacopoeia assay (Ph. Eur. 5.1.3). Results are discussed in terms of analytical challenges encountered to identify and develop high-throughput methods that facilitate multi-dose formulation development of aluminum-adjuvanted protein-based vaccine candidates.

Polysorbate 80 and histidine quantitative analysis by NMR in the presence of virus-like particles.

Polysorbate‐80 (PS80) and histidine are common excipients in vaccine and therapeutic protein formulation. A simple quantitative NMR method to measure both PS80 and histidine in human papillomavirus (HPV) virus‐like particle (VLP) vaccine for aqueous and alum‐containing samples is described. The new NMR method is compared to current colorimetric methods for PS80 and RP HPLC for histidine. The new NMR method is comparable to current assays with an advantage of a simpler sample treatment for PS80. The efficiency is also increased because one method can now provide two assay results instead of two separate methods. Furthermore, the NMR method can detect PS80 stability due to hydrolysis and oxidation when PS80 is stored in a stainless steel container by observing a change of its NMR line shape profile.

Targeting urothelial neoplasia using an investigational virus-like drug conjugate.

514 Background: Human papillomavirus virus-like particles (HPV VLP) preferentially target tumor cells via cell surface modified heparan-sulfate proteoglycans (HSPG). The investigational virus-like drug conjugate, belzupacap sarotalocan (AU-011), is currently in phase 2 clinical trials for treatment of primary choroidal melanoma. We previously demonstrated AU-011’s in vivo tumor acute cytotoxicity upon activation with near-infrared light (nIR), resulting in tumor-free survival for at least 100 days and protection from tumor re-challenge in the MB49 murine flank model of bladder cancer. Here we provide additional data supporting further clinical development in urothelial neoplasia as an additional oncology indication. Methods: We examined the cytotoxicity of AU-011 in a panel of six human bladder cancer cell lines in vitro and binding and distribution in human tumor biopsy samples ex vivo. Tumor distribution of AU-011 was also assessed in vivo after intravesical instillation in the orthotopic MB49 murine model. In consideration of the glycocalyx layer often found coating the interior of the bladder wall and the surface of bladder tumors, pre-treatment with hyauronidase was tested both on human biopsy samples and in the murine orthotopic model. Lastly, tumor distribution of AU-011 was evaluated when formulated with the polyamide Syn3. Results: When tested in vitro on a panel of human bladder tumor cell lines representing grades I-IV carcinomas, we found AU-011’s potency to be in the picomolar range (EC50 range 16.6 pM – 62.75 pM). We assessed the VLPs ability to bind ex vivo to a panel of human urothelial tumor biopsies and observed binding in samples representing both invasive T2 and non-invasive Ta papillary tumors. Tumor biopsy samples were pre-treated with hyaluronidase to break down the glycocalyx layer surrounding the tumor, and an increase in signal was observed in the Ta biopsy sample. Similarly, using the orthotopic MB49 murine tumor model with intravesical delivery of AU-011, we observed an enhancement of AU-011 distribution into the tumor when the bladders were pre-treated with hyaluronidase. Additionally, when formulated with Syn3, AU-011 distribution throughout the tumors was augmented without the need for hyaluronidase pre-treatment. Conclusions: We have demonstrated AU-011 targeted cytotoxicity in vitro using a panel of human bladder cancer cell lines indicating that its binding to bladder cancer cells is tumor grade and genetic mutation agnostic. Additionally, tumor binding and distribution was observed ex vivo using human bladder cancer biopsy samples and in vivo in the murine MB49 orthotopic model. Human and murine tumor distribution was improved with a pre-treatment of hyaluronidase or when formulated with Syn3. Collectively, these data support the further investigation of the use of AU-011 for the indication of uroepithelial cancer.

Cryo EM Analysis Reveals Inherent Flexibility of Authentic Murine Papillomavirus Capsids.

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. However, studies have been hampered due to restricted tropism that makes production and purification of high titer virus problematic. This issue has been overcome by developing alternative HPV production methods such as virus-like particles (VLPs), which are devoid of a native viral genome. Structural studies have been limited in resolution due to the heterogeneity, fragility, and stability of the VLP capsids. The mouse papillomavirus (MmuPV1) presented here has provided the opportunity to study a native papillomavirus in the context of a common laboratory animal. Using cryo EM to solve the structure of MmuPV1, we achieved 3.3 Å resolution with a local symmetry refinement method that defined smaller, symmetry related subparticles. The resulting high-resolution structure allowed us to build the MmuPV1 asymmetric unit for the first time and identify putative L2 density. We also used our program ISECC to quantify capsid flexibility, which revealed that capsomers move as rigid bodies connected by flexible linkers. The MmuPV1 flexibility was comparable to that of a HPV VLP previously characterized. The resulting MmuPV1 structure is a promising step forward in the study of papillomavirus and will provide a framework for continuing biochemical, genetic, and biophysical research for papillomaviruses.

Next generation polyphosphazene immunoadjuvant: Synthesis, self-assembly and in vivo potency with human papillomavirus VLPs-based vaccine

Poly[di(carboxylatomethylphenoxy)phosphazene] (PCMP), a new member of polyphosphazene immunoadjuvant family, is synthesized. In vitro assessment of a new macromolecule revealed hydrolytic degradation profile and immunostimulatory activity comparable to its clinical stage homologue PCPP; however, PCMP was characterized by a beneficial reduced sensitivity to the ionic environment. In vivo evaluation of PCMP potency was conducted with human papillomavirus (HPV) virus-like particles (VLPs) based RG1-VLPs vaccine. In contrast with previously reported self-assembly of polyphosphazene adjuvants with proteins, which typically results in the formation of complexes with multimeric display of antigens, PCMP surface modified VLPs in a composition dependent pattern, which at a high polymer-to VLPs ratio led to stabilization of antigenic particles. Immunization experiments in mice demonstrated that PCMP adjuvanted RG1-VLPs vaccine induced potent humoral immune responses, in particular, on the level of highly desirable protective cross-neutralizing antibodies, and outperformed PCPP and Alhydrogel adjuvanted formulations.

Structural characterization of a neutralizing mAb H16.001, a potent candidate for a common potency assay for various HPV16 VLPs

With more human papillomavirus (HPV) virus-like particle (VLP) vaccines to hit the market in future, a monoclonal antibody (mAb) with preferably comparable reactivity against vaccines from different expression systems and bioprocesses is urgently needed for the potency characterization. Among all mAbs against HPV16 collected, rabbit mAb H16.001 is potently neutralizing with the highest affinity, recognizes an immune-dominant epitope, and can comparably react with HPV16 vaccines from various sources. Cryo-electron microscopic (cryo-EM) structure demonstrated that 360 H16.001 Fabs could bind to HPV16 capsid in preferable binding manner without steric hindrance between neighboring Fabs, potentially supporting its identification for VLP structural integrity and utility in monitoring VLP structural probity. This structural analysis indicated that mAb H16.001 afforded unbiased potency characterization for various HPV16 vaccines and was potential for use in vaccine regulation practice. This study also showed a model process for selecting suitable mAbs for potency assays of other vaccines.

Hyaluronidase-based swim-up for semen selection in patients with human papillomavirus semen infection.

Infection by human papillomavirus (HPV) represents one of the most common sexually transmitted diseases in both men and women worldwide. Recently, the detection of HPV virions in the semen of a large percentage of sexually active men has been associated with detrimental effects on both sperm parameters and on assisted reproductive technologies (ART) treatment outcomes. Conventional semen washing procedure used in ART have proved to be ineffective in removing HPV bound to sperm, requiring the identification of more effective and specific methods. In the present study, we assessed the possible use of hyaluronidase for the detachment of HPV from sperm cell surface. Semen samples from five normozoospermic control subjects (CTRL) were incubated with HPV virus-like particles (HPV-VLP) and treated with hyaluronidase by both a modified swim-up procedure (M-SU) and single-cell approach (SCA). The treatment with hyaluronidase was associated with the complete loss of HPV-VLP signal on sperms by both M-SU and SCA. In addition, semen samples from 12 HPV-positive infertile patients were treated with hyaluronidase 80IU/mL by M-SU, resulting in the complete loss of HPV-DNA signal from sperm surface. Finally, the possible impact of hyaluronidase treatment on sperm parameters was assessed on both sperms from the five CTRL subjects and on further five oligo-astheno-terato-zoospermic (OAT) patients, both HPV negative. The treatment with hyaluronidase was equally associated with a slight reduction of sperm viability and progressive motility in both CTRL and OAT. In conclusion, the treatment with hyaluronidase removed efficiently and safely HPV virions bound to spermatozoa.

Adjuvant-containing control arms in pivotal quadrivalent human papillomavirus vaccine trials: restoration of previously unpublished methodology

PurposeTrustworthy reporting of quadrivalent human papillomavirus (HPV) vaccine trials is the foundation for assessing the vaccine’s risks and benefits. However, several pivotal trial publications incompletely reported important methodological details and...

Designing Chimeric Virus-like Particle-based Vaccines for Human Papillomavirus and HIV: Lessons Learned.

Virus-like particles (VLPs) are a type of subunit vaccine which resembles viruses but do not contain any genetic material so that they are not infectious. VLPs maintain the same antigenic conformation to the original virus, and they could be a better vaccine candidate than live-attenuated and inactivated vaccines. In addition, compared to other subunit vaccines such as soluble protein, VLPs can stimulate both innate and adaptive immune responses effectively and safely against several pathogens by the closer morphology to its native virus. They have already been licensed as vaccines against Hepatitis B virus, human papillomavirus (HPV), and several veterinary diseases. Moreover, it has been investigated to prevent other viral infections including HIV. While HIV VLP-based vaccines have been studied over 35 years, none of them has been successful enough to reach even Phase III clinical trials. In this review, we summarize: (i) general features of VLPs; (ii) epidemiological data and current status of vaccine research and development on HPV and HIV; and (iii) previous studies held on HPV VLPs, HIV VLPs, and chimeric HPV/HIV VLPs including production methods and different animal immunization assays. Furthermore, we review present state of human clinical trials with VLPs and consider the potential to develop a successful preventive HIV vaccine using HPV VLP models. Finally, we discuss the benefits, limitations, and challenges of developing chimeric VLP-based HPV/HIV vaccines with recent findings, critical issues to improve VLP-based vaccines, and hot topics for the next 5 years to join the global effort to fight against these two pathogens.

In silico/In vivo analysis of high-risk papillomavirus L1 and L2 conserved sequences for development of cross-subtype prophylactic vaccine

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.

HPV vaccination: Are we overlooking additional opportunities to control HPV infection and transmission?

Human papillomavirus virus-like particles (HPV VLPs) have distinctive immunogenic properties that generate a durable antibody response, producing high-quality neutralizing antibodies. By vaccination, i.e., intramuscular injection of these HPV VLPs, the viral survival strategy of avoiding exposure to the systemic immune system is completely overruled, and large amounts of vaccine-induced systemic antibodies are generated. These systemic circulating antibodies are easily transuded to the genital mucosa and are detectable in female genital secretions. It is well accepted that these antibodies interact with the virions presented by an infected partner and inhibit infection. However, much less attention has been paid to the role of anti-HPV vaccine-induced antibodies in an HPV-infected individual where infectious virions are encountered by neutralizing antibodies in mucosal secretions. There is a clear need to further investigate and document this role. Indeed, if HPV vaccination of HPV-infected women has an effect on HPV transmission, auto-inoculation, and relapse after treatment, this may influence how we model, assess, and implement HPV vaccination programmes.

Mesoscale model of the assembly and cross-linking of HPV virus-like particles

We present a novel kinetic Monte Carlo model to simulate the real process time-scale of the assembly of Human Papillomavirus (HPV) virus-like particles (VLPs) incorporating the formation of intercapsomeric disulfide bonds. The objective was to develop insights into the underlying mechanisms of HPV VLP assembly and cross-linking during in vitro production of the HPV vaccine. The model integrates actual experimental data and detailed information of VLP geometrical structure in microscopic mechanistic steps. The principal novelty of this model is in the concurrent simulation of VLP assembly and cross-linking including a variable for spatial angular arrangement of capsomeres during their assembly that affects the overall rates of VLP assembly and cross-linking. The cross-linking modeled by using the mechanistic probability rules between involved cysteine residues. The model was utilized to better understand the actual process data and check on the hypothesis related to factors affecting the rates of HPV growth and maturation.

Altered antigenicity and immunogenicity of human papillomavirus virus-like particles in the presence of thimerosal

Thimerosal has been widely used as a preservative in human vaccines for decades. Thimerosal, a thiol capping agent with ethyl mercury being the active degradant, could have impacts on the vaccine potency due to potential thiol modification. The effects on the antigenicity and immunogenicity of human papillomavirus (HPV) virus-like particles (VLPs) in the presence of thimerosal was studied. In general, reduced binding activity was observed between HPV antigens and monoclonal antibodies (mAbs) upon thimerosal treatment, accompanied by reduced protein conformational stability. The immunogenicity of a pentavalent vaccine formulation (HPV6, HPV11, HPV16, HPV18 and hepatitis E virus) with or without thimerosal was studied in mice. The functional antibody titres, as well as the binding titres, were determined, showing a substantial decrease for vaccine formulations containing thimerosal for HPV16/18. Similarly, epitope-specific competition assays using specific and functional mAbs as tracers also showed a significant reduction in immunogenicity for HPV16/18 in the presence of thimerosal. Structural alterations in the capsid protein for HPV18 were observed with cryo-electron microscopy and 3-dimensional reconstruction in the comparative structural analysis. The results should alert scientists in formulation development field on the choice for vaccine preservatives, in particular for thiol-containing antigens.

PF-1 The HPV serology standardization initiative: Development of critical assay reagents

Introduction: Human Papillomavirus (HPV) has been linked to numerous cancers including cervical, oropharyngeal, and additional anogenital cancers, and is believed to be associated with approximately 5% of cancers world-wide. Current licensed vaccines have shown great promise in being able to reduce persistent infection and HPV-linked cancers, and are based on the use of virus-like particles (VLPs) which are comprised of type-specific HPV L1-expressing empty capsids. While commendable strides have been made within the HPV research community to focus on understanding how the HPV vaccines work and eradicating this cancer-causing virus, there is still a lack of assay standards, reference reagents and testing guidelines for HPV serology assays. Our laboratory is leading an international HPV Serology standardization initiative to develop a series of standards, multiplex immunoassays, and procedures in order to harmonize evaluation of antibody responses to currently available vaccines in the context of HPV vaccine trials. To reduce costs associated with serology testing, we investigated performance of alternative transfection reagents typically used in critical reagent production. Discussion: Currently licensed HPV vaccines are based on HPV L1 virus-like particles. HPV VLPs are used extensively in HPV research and as critical reagents in the assessment of HPV-induced immune responses. There are a variety of available methods to create HPV VLPs for use in the research setting; however, transfection methods are expensive and time-consuming. Our team is currently producing HPV VLPs for the 9 HPV types included in the currently administered vaccines to be used as assay reference reagents by the serology scientific community to aid in standardization and harmonization of HPV serology testing in vaccine trials. With the aim of reducing costs and saving time in VLP production while maintaining robust HPV particle assembly, we have investigated different transfection approaches in a mammalian-based HPV L1L2 VLP production system. Aspects of consideration include transfection reagents and efficiency, cell viability, materials, and method optimization in the context of final VLP output via HEK293TT transfection with pHPVL1L2. The VLPs are subsequently confirmed by electron microscopy and dynamic light scattering as well as epitope confirmation assays. Results demonstrate that cost-efficient reagents such as PEI and Transporter5 are as efficient as Lipofectamine in producing particles. Similarly, adaptation of various transfection methods and use of large-scale production techniques lead to faster production time with similar output. Conclusions: The development of HPV L1-based standardized reagents and immunoassays allow research laboratories to better evaluate and compare the vaccine-induced immune response across clinical trials. VLPs, the cornerstone of the current vaccine, are used to develop immunoassays and standards by which researchers can gauge the immune response. By reducing costs and time to produce VLPs, it becomes more feasible to create large production sets of VLPs in less time and with less financial burden for immune monitoring laboratories. Funded by NCI Contract No. HHSN261200800001E.

Applying lessons from human papillomavirus vaccines to the development of vaccines against Chlamydia trachomatis

ABSTRACTIntroduction: Chlamydia trachomatis (Ct), the most common bacterial sexually transmitted infection (STI), leads to pelvic inflammatory disease, infertility, and ectopic pregnancy in women. In this Perspective, we discuss the successful human papillomavirus (HPV) vaccine as a case study to inform Ct vaccine efforts.Areas covered: The immunological basis of HPV vaccine-elicited protection is high-titer, long-lasting antibody responses in the genital tract which provides sterilizing immunity. These antibodies are elicited through parenteral administration of a subunit vaccine based on virus-like particles (VLPs) of HPV. We present three lessons learned from the successful HPV vaccine efforts: (1) antibodies alone can be sufficient to provide protection from STIs in the genital tract, (2) the successful generation of high antibody levels is due to the multivalent structure of HPV VLPs, (3) major challenges exist in designing vaccines that elicit appropriate effector T cells in the genital tract. We then discuss the possibility of antibody-based immunity for Ct.Expert commentary: In this Perspective, we present a case for developing antibody-eliciting vaccines, similar to the HPV vaccine, for Ct. Basic research into the mechanisms of Ct entry into host cells will reveal new vaccine targets, which may be antigens against which antibodies are not normally elicited during natural infection.