Search Results (27)

Background: Prophylactic human papillomavirus (HPV) vaccines are crucial for preventing HPV-related cancers. This study aimed to preclinically evaluate a novel recombinant 9-valent HPV vaccine produced in Escherichia coli (E. coli), which targets HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, and is based on virus-like particles (VLPs) of the HPV major capsid protein L1. Methods: The molecular weight and purity of HPV L1 protein bands were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie Brilliant Blue staining. The morphology and size distribution of VLPs were characterized using cryo-electron microscopy and DLS. The immunogenicity and durability of the recombinant 9-valent HPV vaccine were evaluated in BALB/c mice and Wistar rats. Mice received single or triple immunizations (2-week intervals) of two vaccine batches or Gardasil^®9 (MSD, USA) control at 1/20 human dose. Antibody responses were monitored via ELISA and pseudovirus neutralization assays over 24 weeks. Rats were administered single or triple immunizations (2-week intervals) of high- (1/10), medium- (1/20), or low-dose (1/40) vaccine or Gardasil^®9 control (1/20), with neutralizing antibodies tracked for 16 weeks. Results: Cryo-electron microscopy and DLS revealed that VLPs of each type appeared as uniformly distributed, spherical or ellipsoidal hollow intact particles with a diameter of approximately 45–65 nm. This vaccine demonstrated robust immunogenicity and long-lasting efficacy in BALB/c mice and Wistar rats, with effects comparable to those of the commercially available vaccine Gardasil^®9. Conclusions: The 9-valent HPV vaccine induces robust and persistent immune responses in mice and rats, strongly supporting further clinical trials. It is expected to be an alternative to marketed vaccines and ease the global supply shortage of 9-valent HPV vaccines. Full article

Background/Objectives: Cervical cancer, predominantly driven by persistent infection with high-risk human papillomaviruses (HPVs), is one of the most common malignancies and an important cause of cancer-related mortality among women worldwide. Although existing licensed prophylactic HPV vaccines confer excellent protection, their global use remains suboptimal due to concentrated manufacturing capacity and high production costs. This study aimed to establish a cost-effective multivalent HPV virus-like particle (VLP) vaccine platform. Specifically, we used an enhanced baculovirus expression vector system to produce a 12-valent HPV VLP vaccine to improve antigen yield, thereby reducing manufacturing costs and ultimately improving affordability and availability in low- and middle-income countries. Methods: Optimized expression cassettes and an insect cell culture process were designed to enhance productivity across 12 HPV L1 genotypes. A scalable purification scheme integrating ion-exchange and adsorption chromatography was developed to produce high-purity VLPs with consistent structural integrity. Immunogenicity was assessed in a murine model. Elicited HPV type-specific IgG antibody responses were compared with those induced by the licensed 9-valent HPV vaccine. Results: The assembled 12-valent VLPs were comprehensively characterized using biophysical and immunochemical analyses, confirming structural stability and correct antigenicity. In vivo immunogenicity studies in mice showed strong and serotype-specific IgG responses, comparable or superior to those induced by the licensed 9-valent commercial vaccine. Conclusions: The enhanced baculovirus expression vector system is a versatile and economically sustainable platform for next-generation HPV vaccine production. This technology offers a promising approach to lowering vaccine manufacturing costs and improving global access, particularly in low- and middle-income regions heavily burdened by HPV-associated diseases. Full article

Background: Cervical carcinoma remains a widespread cancer worldwide, primarily caused by persistent infection with high-risk human papillomavirus (HPV). HPV types 16 and 18 account for approximately 70% of cervical cancer cases. Although prophylactic HPV vaccines are commercially available, their high cost and reliance on expensive expression platforms limit their accessibility in developing countries. Objectives: This study aimed to develop a cost-effective, plant-based HPV vaccine candidate by expressing capsomeric HPV-16 and HPV-18 L1 antigens in Brassica oleracea (broccoli). Methods: Modified L1 from HPV types 16 and 18 were designed to retain capsomeric assembly and fused with heat-labile enterotoxin B subunit (LTB). Immunoinformatics analyses were used to assess antigenicity, epitope distribution, and structural characteristics. Codon-optimized genes were cloned using Gateway^® technology and expressed in broccoli via Agrobacterium-mediated transformation. Transgenic plants were validated by PCR and qRT-PCR. Protein accumulation was quantified, and immunogenicity was evaluated in mice. Results: PCR and qRT-PCR confirmed the stable integration of two copies of the LTB-L1 transgenes in broccoli plants. Western blotting detected L1 protein at ~56.5 kDa, indicating the cleavage of the LTB-L1 fusion protein. The correct folding of L1 capsomeres was verified by antigen-capture ELISA. The recombinant proteins accumulated to approximately 0.33% and 0.35% of total soluble protein for HPV-16 and HPV-18, respectively. The immunization of mice with transgenic L1 induced significant humoral immune responses, comparable to those elicited by purified VLPs. Conclusions: The results demonstrate broccoli as a promising platform for the expression of immunogenic HPV L1 capsomeres and highlight its potential for the development of affordable, plant-based HPV vaccines. Full article

Background/Objectives: Virus-like particle (VLP) vaccines based on human papillomavirus (HPV) L1 proteins have high efficacy for preventing cervical cancer and other HPV-associated diseases. The production yields of commercial HPV VLPs remain suboptimal. We aimed to improve HPV VLP production efficiency using a hyper-expression vector system for the expression of L1 proteins of four major HPV serotypes—HPV 6, 11, 16, and 18. Methods: HPV L1 proteins were expressed in Trichoplusia ni (Hi5) insect cells via a hyper-expression baculovirus vector system. Following cell lysis using a microfluidizer, VLPs were purified through a two-step chromatographic process. Particle morphology was characterized using transmission electron microscopy and dynamic light scattering. Immunogenicity was evaluated using a murine model; mice received three intramuscular injections of the purified quadrivalent VLPs. The resulting IgG and neutralizing antibody responses were compared with those elicited by the commercial quadrivalent vaccine, Gardasil. Results: The L1 proteins from HPV 6, 11, 16, and 18 were successfully expressed at high levels in Hi5 cells, forming uniformly sized VLPs with hydrodynamic diameters of 50–60 nm. The average production yield of the quadrivalent VLPs exceeded 40 mg/L, an improvement over conventional yields. The candidate VLPs elicited strong HPV-specific IgG and neutralizing antibody responses in mice, comparable to those induced by Gardasil. Conclusions: The hyper-expression baculovirus vector system enables high-yield production of HPV L1 VLPs with desirable structural and immunogenic properties. This approach holds promise for the cost-effective and scalable manufacturing of next-generation HPV VLP vaccines, facilitating broader global access to HPV immunization. Full article

Antigenicity and immunogenicity define a potent immunogen in vaccinology. Nowadays, there are simplified platforms to produce nanocarriers for small-peptide antigen delivery, derived from various infectious agents for the treatment of a variety of diseases, based on virus-like particles (VLPs). They have good cell-penetrating properties and protective action for target molecules from degradation. Human papillomavirus (HPV) causes anogenital warts and six types of cancer in infected women, men, or children, posing a challenge to global public health. The HPV capsid is composed of viral type-specific L1 and evolutionarily conserved L2 proteins. Produced in heterologous systems, the L1 protein can self-assemble into VLPs, nanoparticles sized around 50–60 nm, used as prophylactic vaccines. Devoid of the viral genome, they are safe for users, offering no risk of infection because VLPs do not replicate. The immune response induced by HPV VLPs is promoted by conformational viral epitopes, generating effective T- and B-cell responses. Produced in different cell systems, HPV16 L1 VLPs can be obtained on a large scale for use in mass immunization programs, which are well established nowadays. The expression of heterologous proteins was evaluated at various transfection times by transfecting cells with vectors encoding codon-optimized HPV16L1 and HPV16L2 genes. Immunological response induced by chimeric HPV16 L1/L2 VLP was evaluated through preclinical assays by antibody production, suggesting the potential of broad-spectrum protection against HPV as a prophylactic nanovaccine. These platforms can also offer promising therapeutic strategies, covering the various possibilities for complementary studies to develop potential preventive and therapeutic vaccines with broad-spectrum protection, using in silico new epitope selection and innovative nanotechnologies to obtain more effective immunobiologicals in combating HPV-associated cancers, influenza, hepatitis B and C, tuberculosis, human immunodeficiency virus (HIV), and many other illnesses. Full article

Background: Cervical cancer is associated with persistent infection of high-risk human papillomaviruses (HPVs). Prophylactic HPV vaccines have been recommended and have significant efficacy in preventing cervical cancer. Multivalent HPV vaccines have a better preventative effect on HPV-related diseases. However, there is currently only one nine-valent HPV vaccine on the market: Gardasil^® 9. The development of new HPV vaccines is still urgent in order to achieve the goal of eliminating cervical cancer as proposed by the WHO. Methods: In this study, we developed a nine-valent recombinant HPV virus-like particle (VLP) vaccine (HPV-9 vaccine) containing HPV type 6, 11, 16, 18, 31, 33, 45, 52, and 58 antigens, with an adjuvant of aluminum phosphate (AlPO₄). The type-specific L1 proteins were recombinantly expressed using Pichia pastoris, followed by self-assembly into VLPs. Immunogenicity studies of the HPV-9 vaccine were performed using rodents (mice and rats) and non-human primates (macaques) as animal models. Results: Immunogenicity studies showed that the HPV-9 vaccine is able to elicit a robust and long-lasting neutralizing antibody response in rodents (mice and rats) and non-human primates (cynomolgus macaque) models. The HPV-9 vaccine shows immunogenicity comparable to that of Walrinvax^® and Gardasil^® 9. Conclusions: In summary, this study provides a comprehensive investigation of the immunogenicity of the HPV-9 vaccine, including its immune persistence. These findings, derived from using models of diverse animal species, contribute valuable insights into the potential efficacy of the vaccine candidate in clinical settings. Full article

During the multi-dose formulation development of recombinant vaccine candidates, protein antigens can be destabilized by antimicrobial preservatives (APs). The degradation mechanisms are often poorly understood since available analytical tools are limited due to low protein concentrations and the presence of adjuvants. In this work, we evaluate different analytical approaches to monitor the structural integrity of HPV16 VLPs adsorbed to Alhydrogel™ (AH) in the presence and absence of APs (i.e., destabilizing m-cresol, MC, or non-destabilizing chlorobutanol, CB) under accelerated conditions (pH 7.4, 50 °C). First, in vitro potency losses displayed only modest correlations with the results from two commonly used methods of protein analysis (SDS-PAGE, DSC). Next, results from two alternative analytical approaches provided a better understanding of physicochemical events occurring under these same conditions: (1) competitive ELISA immunoassays with a panel of mAbs against conformational and linear epitopes on HPV16 VLPs and (2) LC-MS peptide mapping to evaluate the accessibility/redox state of the 12 cysteine residues within each L1 protein comprising the HPV16 VLP (i.e., with 360 L1 proteins per VLP, there are 4320 Cys residues per VLP). These methods expand the limited analytical toolset currently available to characterize AH-adsorbed antigens and provide additional insights into the molecular mechanism(s) of AP-induced destabilization of vaccine antigens. Full article

Bacillus Calmette–Guerin (BCG), the only current vaccine against tuberculosis (TB) that is licensed in clinics, successfully protects infants and young children against several TB types, such as TB meningitis and miliary TB, but it is ineffective in protecting adolescents and adults against pulmonary TB. Thus, it is a matter of the utmost urgency to develop an improved and efficient TB vaccine. In this milieu, virus-like particles (VLPs) exhibit excellent characteristics in the field of vaccine development due to their numerous characteristics, including but not limited to their good safety without the risk of infection, their ability to mimic the size and structure of original viruses, and their ability to display foreign antigens on their surface to enhance the immune response. In this study, the HPV16 L1 capsid protein (HPV16L1) acted as a structural vaccine scaffold, and the extracellular domain of Ag85B was selected as the M. tb immunogen and inserted into the FG loop of the HPV16 L1 protein to construct chimeric HPV16L1/Ag85B VLPs. The chimeric HPV16L1/Ag85B VLPs were produced via the Pichia pastoris expression system and purified via discontinuous Optiprep density gradient centrifugation. The humoral and T cell-mediated immune response induced by the chimeric HPV16L1/Ag85B VLP was studied in female C57BL/c mice. We demonstrated that the insertion of the extracellular domain of Ag85B into the FG loop of HPV16L1 did not affect the in vitro stability and self-assembly of the chimeric HPV16L1/Ag85B VLPs. Importantly, it did not interfere with the immunogenicity of Ag85B. We observed that the chimeric HPV16L1/Ag85B VLPs induced higher Ag85B-specific antibody responses and elicited significant Ag85B-specific T cell immune responses in female C57BL/c mice compared with recombinant Ag85B. Our findings provide new insights into the development of novel chimeric HPV16L1/TB VLP-based vaccine platforms for controlling TB infection, which are urgently required in low-income and developing countries. Full article

Human papillomaviruses (HPVs) are a large family of viruses with a capsid composed of the L1 and L2 proteins, which bind to receptors of the basal epithelial cells and promote virus entry. The majority of sexually active people become exposed to HPV and the virus is the most common cause of cervical cancer. Vaccines are available based on the L1 protein, which self-assembles and forms virus-like particles (VLPs) when expressed in yeast and insect cells. Although very effective, these vaccines are HPV type-restricted and their costs limit broad vaccination campaigns. Recently, vaccine candidates based on the conserved L2 epitope from serotypes 16, 18, 31, 33, 35, 6, 51, and 59 were shown to elicit broadly neutralizing anti-HPV antibodies. In this study, we tested whether E. coli outer membrane vesicles (OMVs) could be successfully decorated with L2 polytopes and whether the engineered OMVs could induce neutralizing antibodies. OMVs represent an attractive vaccine platform owing to their intrinsic adjuvanticity and their low production costs. We show that strings of L2 epitopes could be efficiently expressed on the surface of the OMVs and a polypeptide composed of the L2 epitopes from serotypes 18, 33, 35, and 59 provided a broad cross-protective activity against a large panel of HPV serotypes as determined using pseudovirus neutralization assay. Considering the simplicity of the OMV production process, our work provides a highly effective and inexpensive solution to produce universal anti-HPV vaccines. Full article

To achieve maximum efficacy, vaccines, such as subunit, recombinant, and conjugate vaccines, necessitate the incorporation of immunostimulators/adjuvants. Adjuvants play a vital role in bolstering and extending the strength of the immune response while also influencing its type. As antigen and adjuvant formulations become more intricate, it becomes imperative to establish a well-characterized and robust formulation to ensure consistent and reproducible outcomes in preclinical and clinical studies. In the present study, an HPV bivalent vaccine was developed using a BC02 adjuvant in conjunction with HPV 16 and 18 L1 VLP antigens produced from an E. coli expression system. The study involved evaluating the adjuvant formulation and in vivo immunogenicity in mice. Remarkably, a medium-dose of BCG-CpG-DNA combined with a low-dose of aluminum hydroxide substantially enhanced the immunogenicity of HPV16 and 18 VLPs, resulting in improved cellular and humoral immune responses. Full article

Human papillomavirus (HPV) vaccines based on HPV L1 virus-like particles (VLPs) are already licensed but not accessible worldwide. About 38.0 million people were living with HIV in 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against both viruses are an urgent need. In this study, the HIV-1 P18I10 CTL peptide from the V3 loop of HIV-1 gp120 glycoprotein was inserted into the HPV16 L1 protein to construct chimeric HPV:HIV (L1:P18I10) VLPs. Instead of the traditional baculovirus expression vector/insect cell (BEVS/IC) system, we established an alternative mammalian 293F cell-based expression system using cost-effective polyethylenimine-mediated transfection for L1:P18I10 protein production. Compared with conventional ultracentrifugation, we optimized a novel chromatographic purification method which could significantly increase L1:P18I10 VLP recovery (~56%). Chimeric L1:P18I10 VLPs purified from both methods were capable of self-assembling to integral particles and shared similar biophysical and morphological properties. After BALB/c mice immunization with 293F cell-derived and chromatography-purified L1:P18I10 VLPs, almost the same titer of anti-L1 IgG (p = 0.6409) was observed as Gardasil anti-HPV vaccine-immunized mice. Significant titers of anti-P18I10 binding antibodies (p < 0.01%) and P18I10-specific IFN-γ secreting splenocytes (p = 0.0002) were detected in L1:P18I10 VLP-immunized mice in comparison with licensed Gardasil-9 HPV vaccine. Furthermore, we demonstrated that insertion of HIV-1 P18I10 peptide into HPV16 L1 capsid protein did not affect the induction in anti-L1 antibodies. All in all, we expected that the mammalian cell expression system and chromatographic purification methods could be time-saving, cost-effective, scalable platforms to engineer bivalent VLP-based vaccines against HPV and HIV-1 Full article

In this study, the HIV-1 P18I10 CTL peptide derived from the V3 loop of HIV-1 gp120 and the T20 anti-fusion peptide of HIV-1 gp41 were inserted into the HPV16 L1 capsid protein to construct chimeric HPV:HIV (L1:P18I10 and L1:T20) VLPs by using the mammalian cell expression system. The HPV:HIV VLPs were purified by chromatography. We demonstrated that the insertion of P18I10 or T20 peptides into the DE loop of HPV16 L1 capsid proteins did not affect in vitro stability, self-assembly and morphology of chimeric HPV:HIV VLPs. Importantly, it did not interfere either with the HIV-1 antibody reactivity targeting sequential and conformational P18I10 and T20 peptides presented on chimeric HPV:HIV VLPs or with the induction of HPV16 L1-specific antibodies in vivo. We observed that chimeric L1:P18I10/L1:T20 VLPs vaccines could induce HPV16- but weak HIV-1-specific antibody responses and elicited HPV16- and HIV-1-specific T-cell responses in BALB/c mice. Moreover, could be a potential booster to increase HIV-specific cellular responses in the heterologous immunization after priming with rBCG.HIVA vaccine. This research work would contribute a step towards the development of the novel chimeric HPV:HIV VLP-based vaccine platform for controlling HPV16 and HIV-1 infection, which is urgently needed in developing and industrialized countries. Full article

The spontaneous interaction between human papillomavirus type 16 (HPV16) L1 virus-like particles (VLPs) and non-functionalized gold nanoparticles (nfGNPs) interferes with the nfGNPs’ salt-induced aggregation, inhibiting the red–blue color shift in the presence of NaCl. Electron microscopy and competition studies showed that color-shift inhibition is a consequence of direct nfGNP–VLP interaction and, thus, may produce a negative impact on the virus entry cell process. Here, an in vitro infection system based on the HPV16 pseudovirus (PsV) was used to stimulate the natural infection process in vitro. PsVs carry a pseudogenome with a reporter gene, resulting in a fluorescent signal when PsVs infect a cell, allowing quantification of the viral infection process. Aggregation assays showed that nfGNP-treated PsVs also inhibit color shift in the presence of NaCl. High-resolution microscopy confirmed nfGNP–PsV complex formation. In addition, PsVs can interact with silver nanoparticles, suggesting a generalized interaction of metallic nanoparticles with HPV16 capsids. The treatment of PsVs with nfGNPs produced viral infection inhibition at a higher level than heparin, the canonical inhibitor of HPV infection. Thus, nfGNPs can efficiently interfere with the HPV16 cell entry process and may represent a potential active component in prophylactic formulations to reduce the risk of HPV infection. Full article

We report a unique phenomenon, the opposite color response of a giant polyoxometalate, (NH₄)₄₂[Mo₁₃₂O₃₇₂(CHCOO)₃₀] (H₂O)₇₂ ([Mo₁₃₂]), to the existing states of human papillomavirus (HPV) major capsid protein, L1-pentamer (L1-p), and virus-like particles (VLPs). The color responses originate from the different assembly forms between [Mo₁₃₂] and the capsid protein. The latter were inspected and separated by using CsCl gradient centrifugation, and validated in detail by sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE), dynamic light scattering (DLS), and transmission electron microscopy (TEM) imaging. Furthermore, the intrinsic mechanisms were investigated in-depth by using XPS-based semi-quantitative analysis and well-designed peptides, revealing the critical points of L1 that determine the charge–transfer ratio between Mo(V) to Mo(VI), and consequently, the levels of [Mo₁₃₂] hypochromic in different assemblies. Such a unique phenomenon is significant as it supplies a colorimetry approach to distinguish the existing states of the HPV capsid protein and would be significant in the quality assay of the HPV vaccine and existing states of other viruses in the future. Full article

Human papillomavirus (HPV) 16 capsids have been chosen as a DNA delivery vehicle in many studies. Our preliminary studies suggest that HPV58 capsids could be better vehicles than HPV16 capsids to deliver encapsidated DNA in vitro and in vivo. In the current study, we compared HPV16, HPV58, and the cottontail rabbit papillomavirus (CRPV) capsids either as L1/L2 VLPs or pseudoviruses (PSVs) to deliver externally attached GFP-expressing DNA. Both rabbit and human cells were used to test whether there was a species-specific effect. DNA delivery efficiency was determined by quantifying either GFP-expressing cell populations or mean fluorescent intensities (MFI) by flow cytometry. Interestingly, CRPV and 58-VLPs and PSVs were significantly more efficient at delivering attached DNA when compared to 16-VLPs and PSVs. A capsid/DNA ratio of 2:1 showed the highest efficiency for delivering external DNA. The PSVs with papillomavirus DNA genomes also showed higher efficiency than those with irrelevant plasmid DNA. HPV16L1/58L2 hybrid VLPs displayed increased efficiency compared to HPV58L1/16L2 VLPs, suggesting that L2 may play a critical role in the delivery of attached DNA. Additionally, we demonstrated that VLPs increased in vivo infectivity of CRPV DNA in rabbits. We conclude that choosing CRPV or 58 capsids to deliver external DNA could improve DNA uptake in in vitro and in vivo models. Full article