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.

Combined truncations at both N- and C-terminus of human papillomavirus type 58 L1 enhanced the yield of virus-like particles produced in a baculovirus system.

Human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs) produced in baculovirus system are highly immunogenic, but the relatively high production cost limits its application in the development of broad-spectrum vaccines. Here we report a novel method for enhancing VLP production in this system. We incorporated respectively 4, 8 or 13 residues truncation mutations in the N-terminus of L1ΔC, a C-terminal 25-residue-deleted L1 of HPV58, to construct three mutants. After expression in Sf9 cells, L1ΔN4C exhibited 2.3-fold higher protein production, 2.0-fold mRNA expression and lower rate of mRNA decay, compared to L1ΔC. More importantly, L1ΔN4C protein was easily purified by two-step chromatography with a VLP yield of up to 60 mg/L (purity > 99 %), 5-fold that of L1ΔC, whereas L1ΔN8C and L1ΔN13C behaved similarly to L1ΔC either in protein or mRNA expression. Moreover, L1ΔN4C VLPs showed similar binding activities with six HPV58 neutralizing monoclonal antibodies and induced comparable level of neutralizing antibody in mice to that of L1ΔC VLPs. Our results demonstrate that certain N- and C-terminal truncations of HPV58 L1 can enhance VLP yield. This method may be used to reduce production costs of other L1VLPs or chimeric VLPs to developing pan-HPV vaccines using baculovirus system.

Designing a therapeutic and prophylactic candidate vaccine against human papillomavirus through vaccinomics approaches.

OBJECTIVE: Human papillomavirus (HPV) is the main cause of cervical cancer, the 4th prominent cause of death in women globally. Previous vaccine development projects have led to several approved prophylactic vaccines available commercially, all of which are made using major capsid-based (L1). Administration of minor capsid protein (L2) gave rise to the second generation investigational prophylactic HPV vaccines, none of which are approved yet due to low immunogenicity provided by the L2 capsid protein. On the other hand, post-translation proteins, E6 and E7, have been utilized to develop experimental therapeutic vaccines. Here, in silico designing of a therapeutic and prophylactic vaccine against HPV16 is performed. METHODS: In this study, several immunoinformatic and computational tools were administered to identify and design a vaccine construct with dual prophylactic and therapeutic applications consisting of several epitope regions on L2, E6, and E7 proteins of HPV16. RESULTS: Immunodominant epitope regions (aa 12-23 and 78-78 of L2 protein, aa 11-27 of E6 protein, and aa 70-89 of E7 protein) were employed, which offered adequate immunogenicity to induce immune responses. Resuscitation-promoting factors (RpfB and RpfE) of Mycobacterium tuberculosis were integrated in two separate constructs as TLR4 agonists to act as vaccine adjuvants. Following physiochemical and structural evaluations carried out by various bioinformatics tools, the designed constructs were modeled and validated, resulting in two 3D structures. Molecular docking and molecular dynamic simulations suggested stable ligand-receptor interactions between the designed construct and TLR4. CONCLUSION: Ultimately, this study led to suggest the designed construct as a potential vaccine candidate with both prophylactic and therapeutic applications against HPV by promoting Th1, Th2, CTL, and B cell immune responses, which should be further confirmed in experimental studies.

Thermostability of a trivalent, capsomere-based vaccine for human papillomavirus infection.

Currently licensed vaccines require a cold-chain to maintain efficacy. This cold-chain requirement reduces the availability of vaccines in resource-poor areas of the world. Commercially available human papillomavirus (HPV) vaccines protect against the most common HPV types related to cervical cancer; however, their impact is limited in many regions due to cold-chain requirements. The goal of this study was to test the thermostability of an adjuvanted, trivalent HPV L1 capsomere-based vaccine (containing HPV types 16, 18, and 31) that was formulated by using lyophilization to embed the antigens within a solid, glassy matrix. Thermal stabilities were determined by storing the vaccine formulations for 3 months at 50 °C, followed by immunization of BALB/c mice and measurement of antibody responses. Antibody responses to capsomere vaccines formulated with alum were unchanged after storage for 3 months at 50 °C. Neutralizing responses to these vaccines were unchanged by high-temperature storage, and were equivalent to those generated after administration of the commercially available liquid HPV vaccine Gardasil®9.

Design of a multi-epitope vaccine against cervical cancer using immunoinformatics approaches.

Cervical cancer, caused by human papillomavirus (HPV), is the fourth most common type of cancer among women worldwide. While HPV prophylactic vaccines are available, they have no therapeutic effects and do not clear up existing infections. This study aims to design a therapeutic vaccine against cervical cancer using reverse vaccinology. In this study, the E6 and E7 oncoproteins from HPV16 were chosen as the target antigens for epitope prediction. Cytotoxic T lymphocytes (CTL) and helper T lymphocytes (HTL) epitopes were predicted, and the best epitopes were selected based on antigenicity, allergenicity, and toxicity. The final vaccine construct was composed of the selected epitopes, along with the appropriate adjuvant and linkers. The multi-epitope vaccine was evaluated in terms of physicochemical properties, antigenicity, and allergenicity. The tertiary structure of the vaccine construct was predicted. Furthermore, several analyses were also carried out, including molecular docking, molecular dynamics (MD) simulation, and in silico cloning of the vaccine construct. The results showed that the final proposed vaccine could be considered an effective therapeutic vaccine for HPV; however, in vitro and in vivo experiments are required to validate the efficacy of this vaccine candidate.

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.

Simultaneous determination of capsid proteins in nine-valent human papilloma virus vaccines by liquid chromatography tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method was developed to determine nine types of capsid proteins simultaneously in nine-valent human papillomavirus vaccines. Signature peptides were optimized in terms of specificity, repeatability, determination accuracy and sensitivity. As a result, three signature peptides per capsid protein were obtained. The linear calibration curves were achieved in the range of 11.6-373.6 nmol/L (R2  > 0.998). Compared to our previous liquid chromatography-tandem mass spectrometry method, the current method was more sensitive (3.18-fold) and it can be used for quality evaluation of nine-valent human papillomavirus vaccines, unlike the previous method, which could only be used for bivalent human papillomavirus vaccines. Then, they were utilized to determine nine types of capsid proteins in nine-valent human papillomavirus vaccines from four different manufactures. Intraday and interday precision values for the determination of capsid proteins in nine-valent human papillomavirus vaccines were less than 6.8 and 9.1%, respectively. Recovery rates of all capsid proteins investigated were in the range of 80-120%. In addition, the current assay was used for determination of free capsid protein in nine-valent human papilloma virus vaccines, and the results were used to evaluate the adsorption rate of the adjuvant.

Combined prophylactic and therapeutic immune responses against human papillomaviruses induced by a thioredoxin-based L2-E7 nanoparticle vaccine.

Global burden of cervical cancer, the most common cause of mortality caused by human papillomavirus (HPV), is expected to increase during the next decade, mainly because current alternatives for HPV vaccination and cervical cancer screening programs are costly to be established in low-and-middle income countries. Recently, we described the development of the broadly protective, thermostable vaccine antigen Trx-8mer-OVX313 based on the insertion of eight different minor capsid protein L2 neutralization epitopes into a thioredoxin scaffold from the hyperthermophilic archaeon Pyrococcus furiosus and conversion of the resulting antigen into a nanoparticle format (median radius ~9 nm) upon fusion with the heptamerizing OVX313 module. Here we evaluated whether the engineered thioredoxin scaffold, in addition to humoral immune responses, can induce CD8+ T-cell responses upon incorporation of MHC-I-restricted epitopes. By systematically examining the contribution of individual antigen modules, we demonstrated that B-cell and T-cell epitopes can be combined into a single antigen construct without compromising either immunogenicity. While CD8+ T-cell epitopes had no influence on B-cell responses, the L2 polytope (8mer) and OVX313-mediated heptamerization of the final antigen significantly increased CD8+ T-cell responses. In a proof-of-concept experiment, we found that vaccinated mice remained tumor-free even after two consecutive tumor challenges, while unvaccinated mice developed tumors. A cost-effective, broadly protective vaccine with both prophylactic and therapeutic properties represents a promising option to overcome the challenges associated with prevention and treatment of HPV-caused diseases.

Optimized dose of synthetic analogues of Monophosphoryl lipid A as an effective alternative for formulating recombinant human papillomavirus vaccine.

Adjuvants are a crucial component of recombinant vaccines such as the human papillomavirus (HPV) vaccine. Monophosphoryl lipid A (MPL) extracted from Salmonella Minnesota lipopolysaccharide is used as an adjuvant for the HPV vaccine. Due to the limitations in accessibility and reproducibility of MPL, investigating synthetic analogues of MPL (synMPL) is urgently needed to overcome these limitations. In this study, female BALB/c mice were vaccinated by HPV vaccine formulated with synMPL and aluminum hydroxide gel in which the concentration of synMPL ranged from 0 to 100 µg/dose. Anti-HPV L1 VLP antibody was measured for each group through Indirect ELISA and compared with Cervarix and Gardasil vaccines as approved anti-HPV vaccines. SynMPL showed a concentration-dependent increase up to 50 µg/dose in the immunogenicity of the vaccine. Therefore, synMPL at concentration of 50 µg/dose was selected as optimum concentration. The GMT profiling of synMPL-formulated vaccine (named Papilloguard) and Cervarix was not statistically different (Mann-Whitney test). The Gardasil vaccine showed 10-fold lower GMT for anti-HPV 18 L1 VLP antibody but anti-HPV 16 L1 VLP antibody was similar to Cervarix and Papilloguard. The current findings suggest that the synMPL in combination with aluminum hydroxide could be used as a potential adjuvant candidate for human vaccine.

Designing a potent L1 protein-based HPV peptide vaccine: A bioinformatics approach.

BACKGROUND: Oncogenic human papilloma viruses (HPV) are the cause of various types of cancer, specifically cervical cancer. L1 protein is the main protein of HPV capsid which targeted in many vaccine-producing attempts. However, they have not enough coverage on the various high risk HPV types. Therefore, having a low cost potent HPV vaccine to protect against all members of the α-papillomaviridea family will be promising. In this study, L1 protein-based peptide vaccine was designed using immunoinformatics methods which provides physicochemical properties such as stability in room temperature, potential of antigenicity, non-allergic properties and no requirement with eukaryotic host system. RESULTS: The designed vaccine has two HPV conserved epitopes with lengths 18 and 27 amino acids in all members of α-papillomaviridea. These peptides promote humoral and cellular immunity and INF-γ responses. In order to ensure strong induction of immune responses, Flagellin, a Toll like receptor 5(TLR-5) agonist, and a short synthetic toll like receptor 4 (TLR-4) agonist were also joined to the epitopes. Structure of the designed- vaccine was validated using Rampage and ERRAT and a high quality 3D structure of the vaccine protein was provided. Docking studies demonstrated an appropriate and stable interaction between the vaccine and TLR-5. CONCLUSIONS: The vaccine is expected to have a high quality structure and suitable properties including high stability, solubility and a high potential to be expressed in E.coli. High potentiality of the vaccine in inducing humoral and cellular immune responses, may be considered as an anti-tumor vaccine.

Efficacy of the AS04-Adjuvanted HPV16/18 Vaccine: Pooled Analysis of the Costa Rica Vaccine and PATRICIA Randomized Controlled Trials.

BACKGROUND: The AS04-adjuvanted HPV16/18 (AS04-HPV16/18) vaccine provides excellent protection against targeted human papillomavirus (HPV) types and a variable degree of cross-protection against others, including types 6/11/31/33/45. High efficacy against any cervical intraepithelial neoplasia grade 3 or greater (CIN3+; >90%) suggests that lower levels of protection may exist for a wide range of oncogenic HPV types, which is difficult to quantify in individual trials. Pooling individual-level data from two randomized controlled trials, we aimed to evaluate AS04-HPV16/18 vaccine efficacy against incident HPV infections and cervical abnormalities . METHODS: Data were available from the Costa Rica Vaccine Trial (NCT00128661) and Papilloma Trial Against Cancer in Young Adults trial (NCT00122681), two large-scale, double-blind randomized controlled trials of the AS04-HPV16/18 vaccine. Primary analyses focused on disease-free women with no detectable cervicovaginal HPV at baseline. RESULTS: A total of 12 550 women were included in our primary analyses (HPV arm = 6271, control arm = 6279). Incidence of 6-month persistent oncogenic and nononcogenic infections, excluding known and accepted protected types 6/11/16/18/31/33/45 (focusing on 34/35/39/40/42/43/44/51/52/53/54/56/58/59/66/68/73/70/74), was statistically significantly lower in the HPV arm than in the control arm (efficacy = 9.9%, 95% confidence interval [CI] = 1.7% to 17.4%). Statistically significant efficacy (P < .05) was observed for individual oncogenic types 16/18/31/33/45/52 and nononcogenic types 6/11/53/74. Efficacy against cervical abnormalities (all types) increased with severity, ranging from 27.7% (95% CI = 21.7% to 33.3%) to 58.7% (95% CI = 34.1% to 74.7%) for cytologic outcomes (low-grade squamous intraepithelial neoplasia lesion or greater, and high-grade squamous intraepithelial neoplasia lesion or greater, respectively) and 66.0% (95% CI = 54.4% to 74.9%) to 87.8% (95% CI = 71.1% to 95.7%) for histologic outcomes (CIN2+ and CIN3+, respectively). Comparing Costa Rica Vaccine Trial and Papilloma Trial Against Cancer in Young Adults results, there was no evidence of heterogeneity, except for type 51 (efficacy = -28.6% and 20.7%, respectively; two-sided P = .03). CONCLUSIONS: The AS04-HPV16/18 vaccine provides some additional cross-protection beyond established protected types, which partially explains the high efficacy against CIN3+.

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.

The New Plant Expression System for the Development of Vaccines against Papillomaviruses.

To enhance the synthesis of antigenic envelope proteins L1 of high-grade papillomavirus types HPV16, HPV18, HPV31, and HPV45, the sequence of the gene encoding the cucumber mosaic virus replicase (RdRP CMV) was inserted into the genetic construct. This made it possible to increase the production of these antigenic proteins to 25-27 µg/mg total soluble protein.

Evaluation of the thermal stability and the protective efficacy of spray-dried HPV vaccine, Gardasil® 9.

High-risk human papillomavirus (HPV) types are responsible for nearly all cases of cervical cancers. Cervarix® and Gardasil® 9 are the current prophylactic vaccines available that protect against the majority of HPVs associated with cancer. Although these vaccines are highly effective, HPV vaccine implementation has been slow, particularly in low-and-middle income countries. Major barriers to the widespread availability of the HPV vaccines is its cost and the requirement for continuous refrigeration (2-8°C). Here, we used spray drying along with stabilizing excipients to formulate a thermostable Gardasil® 9 vaccine. We evaluated the immunogenicity and protective efficacy of the vaccine in mice immediately after spray drying and following storage for three months at 4°C, 25°C, and 40°C. The immunogenicity studies were performed using Gardasil® 9 as a whole antigen, and not individual HPV types, for ELISA. At the dose tested, the spray dried vaccine conferred protection against HPV following storage at temperatures up to 40°C. In addition to the spray-dried vaccine, our studies revealed that the Gardasil® 9 vaccine, as currently marketed, may be stored and transported at elevated temperatures for up to 3 months without losing efficacy, especially against HPV16. This study is critical, as a thermostable vaccine will decrease vaccine cost associated with cold-chain maintenance and could increase vaccine access and coverage, especially in remote regions of the world.

Pleurotus ferulae polysaccharides improve the antitumor efficacy of therapeutic human papillomavirus dendritic cell-based vaccine.

We previously found that Pleurotus ferulae polysaccharides (PFPS) improved the maturation and function of dendritic cells (DCs). In this study, we investigated the effects of PFPS on the antitumor efficacy of therapeutic human papillomavirus (HPV) DC-based vaccine. PFPS stimulated DCs pulsed with HPV E6/E7 peptides were used to treat tumor mice on day 5 & 12 (HPV + PFPS-DCs early) and day 12 & 19 (HPV + PFPS-DCs late) after TC-1 cell injection. Compared to control group, both HPV + PFPS-DCs early and HPV + PFPS-DCs late strategies significantly inhibited tumor growth, which was significantly correlated with the increased activation status of both CD4+ and CD8+ T cells, the decreased frequencies of myeloid-derived suppressor cells, and the induction of HPV-specific CD8+ T cell responses. The survival of tumor mice was also greatly improved by HPV + PFPS-DCs early. Moreover, HPV + PFPS-DCs early completely inhibited the growth of second challenged TC-1 cells in tumor free mice. The results showed that PFPS improved the antitumor efficacy of therapeutic HPV DC-based vaccine, suggesting that PFPS might be a potential adjuvant for DC-based vaccines. This study provides a potential strategy for developing the therapeutic DC-based vaccine against cervical cancer.

A Novel Human Papillomavirus 16 L1 Pentamer-Loaded Hybrid Particles Vaccine System: Influence of Size on Immune Responses.

Cervical cancer remains the second-most prevalent female malignancy around the world, leading to a great majority of cancer-related mortality that occurs mainly in developing countries. Developing an effective and low-cost vaccine against human papillomavirus (HPV) infection, especially in medically underfunded areas, is urgent. Compared with vaccines based on HPV L1 viruslike particles (VLPs) in the market, recombinant HPV L1 pentamer expressed in Escherichia coli represents a promising and potentially cost-effective vaccine for preventing HPV infection. Hybrid particles comprising a polymer core and lipid shell have shown great potential compared to conventional aluminum salts adjuvant and is urgently needed for HPV L1 pentamer vaccines. It is well-reported that particle sizes are crucial in regulating immune responses. Nevertheless, reports on the relationship between the particulate size and the resultant immune response have been in conflict, and there is no answer to how the size of particles regulates specific immune response for HPV L1 pentamer-based candidate vaccines. Here, we fabricated HPV 16 L1 pentamer-loaded poly(d,l-lactide- co-glycolide) (PLGA)/lecithin hybrid particles with uniform sizes (0.3, 1, and 3 µm) and investigated the particle size effects on antigen release, activation of lymphocytes, dendritic cells (DCs) activation and maturation, follicular helper CD4+ T (TFH) cells differentiation, and release of pro-inflammatory cytokines and chemokines. Compared with the other particle sizes, 1 µm particles induced more powerful antibody protection and yielded more persistent antibody responses, as well as more heightened anamnestic responses upon repeat vaccination. The superior immune responses might be attributed to sustainable antigen release and robust antigen uptake and transport and then further promoted a series of cascade reactions, including enhanced DCs maturation, increased lymphocytes activation, and augmented TFH cells differentiation in draining lymph nodes (DLNs). Here, a powerful and economical platform for HPV vaccine and a comprehensive understanding of particle size effect on immune responses for HPV L1 pentamer-based candidate vaccines are provided.

N-terminal truncations on L1 proteins of human papillomaviruses promote their soluble expression in Escherichia coli and self-assembly in vitro.

Human papillomavirus (HPV) is the causative agent in genital warts and nearly all cervical, anogenital, and oropharyngeal cancers. Nine HPV types (6, 11, 16, 18, 31, 33, 45, 52, and 58) are associated with about 90% of cervical cancers and 90% of genital warts. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent HPV-associated diseases. However, there is only one commercially available HPV vaccine, Gardasil 9, produced from Saccharomyces cerevisiae that covers all nine types, raising the need for microbial production of broad-spectrum HPV vaccines. Here, we investigated whether N-terminal truncations of the major HPV capsid proteins L1, improve their soluble expression in Escherichia coli. We found that N-terminal truncations promoted the soluble expression of HPV 33 (truncated by 10 amino acids [aa]), 52 (15 aa), and 58 (10 aa). The resultant HPV L1 proteins were purified in pentamer form and extensively characterized with biochemical, biophysical, and immunochemical methods. The pentamers self-assembled into virus-like particles (VLPs) in vitro, and 3D cryo-EM reconstructions revealed that all formed T = 7 icosahedral particles having 50-60-nm diameters. Moreover, we formulated a nine-valent HPV vaccine candidate with aluminum adjuvant and L1 VLPs from four genotypes used in this study and five from previous work. Immunogenicity assays in mice and non-human primates indicated that this HPV nine-valent vaccine candidate elicits neutralizing antibody titers comparable to those induced by Gardasil 9. Our study provides a method for producing a nine-valent HPV vaccine in E. coli and may inform strategies for the soluble expression of other vaccine candidates.

Antigenic Peptide Prediction From E6 and E7 Oncoproteins of HPV Types 16 and 18 for Therapeutic Vaccine Design Using Immunoinformatics and MD Simulation Analysis.

Human papillomavirus (HPV) induced cervical cancer is the second most common cause of death, after breast cancer, in females. Three prophylactic vaccines by Merck Sharp & Dohme (MSD) and GlaxoSmithKline (GSK) have been confirmed to prevent high-risk HPV strains but these vaccines have been shown to be effective only in girls who have not been exposed to HPV previously. The constitutively expressed HPV oncoproteins E6 and E7 are usually used as target antigens for HPV therapeutic vaccines. These early (E) proteins are involved, for example, in maintaining the malignant phenotype of the cells. In this study, we predicted antigenic peptides of HPV types 16 and 18, encoded by E6 and E7 genes, using an immunoinformatics approach. To further evaluate the immunogenic potential of the predicted peptides, we studied their ability to bind to class I major histocompatibility complex (MHC-I) molecules in a computational docking study that was supported by molecular dynamics (MD) simulations and estimation of the free energies of binding of the peptides at the MHC-I binding cleft. Some of the predicted peptides exhibited comparable binding free energies and/or pattern of binding to experimentally verified MHC-I-binding epitopes that we used as references in MD simulations. Such peptides with good predicted affinity may serve as candidate epitopes for the development of therapeutic HPV peptide vaccines.

A novel HPV prophylactic peptide vaccine, designed by immunoinformatics and structural vaccinology approaches.

Human papillomavirus (HPV)-caused cervical cancer is the fourth common female cancer globally. Despite availability of three effective vaccines in market, development of HPV prophylactic vaccines is still pursued due to affordability issues and type-restricted protection of the marketed vaccines. Investigational second generation prophylactic HPV vaccines are mostly exploiting epitopes from the virus minor capsid protein (L2), which despite many advantages suffer from low immunogenicity, a common problem of epitope vaccines. Adjuvants such as TLR agonists may overcome this drawback. In this study, different immunoinformatics and computational tools were employed to design a novel peptide vaccine for protection against cervical cancer. Two immunodominant epitope domains (amino acids 10-36 and 65-89) from the L2 protein of HPV 16 with potential to promote Th1, Th2, CTL, B-cell, and INF-gamma responses were selected. Flagellin, as a TLR5 agonist, a short synthetic TLR4 agonist, and two universal T-helper agonists (PADRE and TpD) were added to ensure strong induction of immune responses. Different segments were joined by proper linkers, and the physicochemical, structural, and immunological characteristics of the resultant construct were evaluated. Modeling, refinement, and validation were done to achieve a high quality 3D structure of the vaccine protein. Docking and molecular dynamics (MD) studies demonstrated an appropriate and stable interaction between the vaccine and TLR5 during the simulation period. Totally, a potential vaccine candidate with proper immunological and physicochemical properties was designed for HPV prophylaxis. The designed vaccine is expected to be capable of generating humoral and cellular responses, which are vital for protection against HPV.