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Size and morphology distribution of the HPV 33, 45, 52, and 58 L1 VLPs. a High-performance size-exclusion chromatography profiles of HPV 33, 45, 52, and 58 L1 VLPs. b Dynamic light scattering analysis of HPV 33, 45, 52, and 58 L1 VLPs. c Analytical ultracentrifugation sedimentation profiles of the HPV 33, 45, 52, and 58 L1 VLPs
Source publication
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...
Contexts in source publication
Context 1
... GQ479013, DQ080002.1, FJ615303.1, and FJ615305.1, respectively (Fig. S2). Protein expression of full-length and a series of N-terminally truncated HPV 33, 45, 52, and 58 L1 proteins were determined by SDS-PAGE and western blotting (WB) using cell lysates from E. coli. (Fig. 1a); we compared these with the five HPV types previously reported (Fig. S3a, S3b). The results showed that, among these nine HPV types, only HPV 31 and 45 showed maximal expression as full-length L1 proteins. By com- parison, L1 expression levels of the other HPV types in E. coli were increased by N-terminal truncation (Fig. 1a). Taken together, our data indicates that N-terminal resi- dues of the HPV L1 protein ...
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... of truncated HPV 33, 45, 52, and 58 L1 proteins to 5 L-scale fermentation. The purified L1 proteins of HPV 33, 45, 52, and 58 were confirmed for consistency by SDS-PAGE and WB (Fig. 1b). For the purposes of comparison, we also present the results of the purified L1 proteins of HPV 6, 11, 16, 18, and 31 in the Supplementary information ( Fig. S3c and S3d). Collectively, the results show that purified L1 proteins of all nine HPV types give promi- nent bands at an apparent molecular weight of ~55 kDa, and these bands react with an anti-HPV L1 cross- reactive linear mAb ...
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... reducing condi- tions ( Fig. S4), and could self-assemble into VLPs in vitro after removing the reducing agent. HPSEC, DLS, and AUC were employed to examine the particle size and sedimentation coefficients of the HPV VLPs. We found that VLPs of HPV 33, 45, 52, and 58 had a similar retention time, ranging from 12.3 to 12.5 min (HPSEC profiles; Fig. 3a). In DLS, the hydrodynamic radii (particles with encircled layers of water molecules) of the main components of the HPV 33, 45, 52, and 58 VLPs were about 28.6, 31.1, 32.4, and 29.3 nm, respectively (Fig. 3b). AUC experiments also showed similar particle properties, with sedimentation coefficients of 106.3S, 144.9S, 128.5S, and 109.0S ...
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... of the HPV VLPs. We found that VLPs of HPV 33, 45, 52, and 58 had a similar retention time, ranging from 12.3 to 12.5 min (HPSEC profiles; Fig. 3a). In DLS, the hydrodynamic radii (particles with encircled layers of water molecules) of the main components of the HPV 33, 45, 52, and 58 VLPs were about 28.6, 31.1, 32.4, and 29.3 nm, respectively (Fig. 3b). AUC experiments also showed similar particle properties, with sedimentation coefficients of 106.3S, 144.9S, 128.5S, and 109.0S for HPV 33, 45, 52, and 58 VLPs, respectively (Fig. 3c). Overall, the results indicate that the HPV L1 VLP particle size varies in solution among the different HPV types. The results from particle analyses for ...
Context 5
... with encircled layers of water molecules) of the main components of the HPV 33, 45, 52, and 58 VLPs were about 28.6, 31.1, 32.4, and 29.3 nm, respectively (Fig. 3b). AUC experiments also showed similar particle properties, with sedimentation coefficients of 106.3S, 144.9S, 128.5S, and 109.0S for HPV 33, 45, 52, and 58 VLPs, respectively (Fig. 3c). Overall, the results indicate that the HPV L1 VLP particle size varies in solution among the different HPV types. The results from particle analyses for HPV 6, 11, 16, 18, and 31 L1 VLP (Fig. S5) were consistent with previous results [34][35][36] . Overall, the L1 VLPs of nine HPV types qualified for inclusion in the manufactured HPV ...
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Objective
Patients with systemic lupus erythematosus (SLE) on immunosuppression have increased risk of human papillomavirus virus (HPV) infection, which causes premalignant cervical changes and cervical cancer. In the UK, females are invited from the age of 25 for cervical screening (CS).¹ HPV vaccination has been recommended with a 3-dose schedule...
Background:
In a 2012 pilot, 9111 Mongolian girls aged 11-17 years received three doses of the quadrivalent (4vHPV) vaccine, Gardasil®. This is the first study to measure early vaccine effectiveness and assess knowledge and attitudes of young women in Mongolia in relation to the human papillomavirus (HPV), the vaccine and cervical cancer.
Methods...
Objectives
To identify studies evaluating the epidemiology of recurrent respiratory papillomatosis (RRP), including patient demographics, human papillomavirus (HPV) immunology, clinical course, surgical and medical treatments, and psychosocial factors.
Methods
A systematic literature search through PubMed was performed to identify studies evaluati...
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...
This paper highlights the low levels of vaccine coverage and high levels of reported vaccination hesitancy in Yerevan, Armenia, that present profound challenges to the control of disease through routine vaccination programmes. We draw on investigations of hesitancy towards the introduction of new vaccines, using the Human Papillomavirus (HPV) vacci...
Citations
... The N-and C-terminal domains of the L1 protein of HPV35 specimens from study MSM were strongly affected by amino acid changes. These two domains have been shown to significantly contribute to the viral assembly of the HPV capsid and its immunogenicity (N-terminus) [65][66][67] and also the viral-host interactions (C-terminus) [68]. Therefore, the alterations observed in these two domains in the L1 proteins of HPV35 specimens from the MSM population may have an impact on the viral strains fitness by significantly reducing their ability to self-assemble into a functional viral capsid able to bind to cellular receptors, thereby hampering their uptake into the target cells [63]. ...
Human Papillomavirus (HPV)-35 accounts for up 10% of cervical cancers in Sub-Saharan Africa. We herein assessed the genetic diversity of HPV35 in HIV-negative women from Chad (identified as #CHAD) and HIV-infected men having sex with men (MSM) in the Central African Republic (CAR), identified as #CAR. Ten HPV35 DNA from self-collected genital secretions (n = 5) and anal margin samples (n = 5) obtained from women and MSM, respectively, were sequenced using the ABI PRISM® BigDye Sequencing technology. All but one HPV35 strains belonged to the A2 sublineage, and only #CAR5 belonged to A1. HPV35 from #CAR had higher L1 variability compared to #CHAD (mean number of mutations: 16 versus 6). L1 of #CAR5 showed a significant variability (2.29%), suggesting a possible intra-type divergence from HPV35H. Three (BC, DE, and EF) out of the 5 capsid loops domains remained totally conserved, while FG- and HI- loops of #CAR exhibited amino acid variations. #CAR5 also showed the highest LCR variability with a 16bp insertion at binding sites of the YY1. HPV35 from #CHAD exhibited the highest variability in E2 gene (P<0.05). E6 and E7 oncoproteins remained well conserved. There is a relative maintenance of a well conserved HPV35 A2 sublineage within heterosexual women in Chad and MSM with HIV in the Central African Republic.
... Consequently, based on the same production system, the second generation of HPV vaccine with an additional 7 HPV types, a 9vHPV (types 6/11/16/18/31/33/ 45/52/58) vaccine candidate, has been developed and demonstrated to be safe and immunogenic in preclinical studies. 12 This first-in-human phase 1 clinical trial aimed to preliminarily determine the safety and immunogenicity of the candidate E. coli-produced L1 VLP-based 9vHPV vaccine in healthy adults. ...
Background:
A safe and highly efficacious Escherichia coli (E. coli)-produced HPV 16/18 bivalent vaccine has been prequalified by the World Health Organization. Here, we conducted a single-center, open-label, dose-escalation phase 1 clinical trial to evaluate the safety and immunogenicity of the second-generation nonavalent HPV 6/11/16/18/31/33/45/52/58 vaccine.
Method:
Twenty-four eligible volunteers aged 18-45 years were enrolled in January 2019 in Dongtai, China and received 0.5 mL (135 μg) or 1.0 mL (270 μg) of the candidate vaccine with a 0/1/6-month dose-escalation schedule. Local and systemic adverse events (AEs) occurring within 30 days after each vaccination and serious adverse events (SAEs) occurring within 7 months were recorded. Blood samples from each participant were collected before and 2 days after the first and third vaccinations to determine changes in laboratory parameters. Serum IgG and neutralizing antibody (nAb) levels against each HPV type at month 7 were analyzed (ClinicalTrials.gov: NCT03813940).
Findings:
The incidences of total AEs in the 135 μg and 270 μg groups were 66.7% and 83.3%, respectively. All AEs were mild or moderate, and no SAEs were reported. No clinically significant changes were found in paired blood indices before or after any of the vaccinations. All the participants in the per-protocol set except for two who failed to seroconvert for HPV 11 or 58 in the 135 μg group seroconverted at month 7 for both IgG and nAbs.
Interpretation:
The candidate E. coli-produced 9vHPV vaccine has been preliminarily proven to be well tolerated and immunogenic, which encourages further studies in large cohorts with a wider age range.
Funding:
This study was supported by the National Natural Science Foundation of China, Fujian Provincial Natural Science Foundation, Fujian Province Health and Education Joint Research Program, Xiamen Science and Technology Plan Project, Fundamental Research Funds for the Central Universities, CAMS Innovation Fund for Medical Sciences of China, and Xiamen Innovax Biotechnology Co., Ltd.
... Thus, the researchers always introduced N-and/or C-terminal truncation to enhance L1 protein expression in different expression systems. For instance, In E. coli, the proper N-terminal truncations of HPV33, −45, −52, and −58 L1 could improve the soluble protein levels (Wei et al., 2018). In insect cells, the C-terminal truncations of HPV6, −16, and −58 L1 showed advantages in protein expression and purification (Müller et al., 1997;Xie et al., 2013;Sun et al., 2016), and parallel N-and C-terminal truncation of HPV58 L1 enhanced the protein expression and VLP yields (Wang et al., 2022). ...
... In addition to the excellent antigens of HPV vaccines, L1 VLPs are also the most commonly used carriers for displaying foreign epitopes to develop broad-spectrum cVLP vaccines (Schellenbacher et al., 2009;Huber et al., 2015;Huber et al., 2017;Boxus et al., 2016;Chen et al., 2017;Chen et al., 2018), which has aroused interest in developing cost-effective modification strategies for enhancing L1 VLP production. Wei et al. (2018) reported that N-terminal truncations of L1 proteins could affect their soluble expression levels in E. coli, and the expression-sensitive site on the N-terminus varied with HPV types. Several studies indicated that the C-terminally truncated L1 proteins might have advantages in expression and purification (Müller et al., 1997;Xie et al., 2013;Sun et al., 2016). ...
Human papillomavirus (HPV) major capsid protein L1 virus-like particles (VLPs) produced in the baculovirus system showed excellent safety and immunogenicity, but the relatively high production cost stands as a substantial barrier to extensive commercialization, especially in producing multivalent vaccines. Here, a novel method, C-terminal basic amino acid (aa) substitution, was developed for increasing VLP and chimeric VLP (cVLP) production in this system. A series of mutants of five HPV types, including three L1 VLPs (6L1, 11L1, and 52L1) and two L1-L2 cVLPs (16L1-33L2, 58L1-16L2), were constructed. We found that most mutants exhibited higher protein expression in Sf9 cells, among which the yields of the superior mutants, 6L1CS4, 11L1CS3, 52L1m4∆N13CS1, 16L1-33L2 CS1, and 58L1-16L2 CS3, were up to 40, 35, 20, 35, and 60 mg/L, which respectively increased by 4.2-, 7.3-, 5-, 2.5-, and 3.4-fold, and they also showed robust immunogenicity and great stabilities. Additionally, we found that the increased level of steady-state mRNA may play a crucial role in promoting L1 protein expression. Our results demonstrated that this novel method was cost-effective and can be used to reduce the production costs of L1 VLPs and L1-L2 cVLPs to develop broadly protective and affordable multivalent HPV vaccines.
... AeBlue was expressed in soluble form, whereas HPV16 L1 capsid protein was primarily expressed in insoluble form. Insoluble expression of HPV16 L1 capsid protein has been reported in E. coli [60][61][62] but not in S. cerevisiae. In E. coli, N-terminal truncation 60,61 , use of a fusion partner 61 , and overexpression GroEL/GroES chaperones 62 (which accept broader substrates than cytosolic chaperones in S. cerevisiae 63,64 ), improved soluble expression of HPV L1 capsid proteins. ...
Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L ⁻¹ in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.
... Evidence suggests that the first 129 nucleotides in the 5′-end are composed of a strong RNA inhibitory component, and at least 10 and 30 residues were deleted from the N and C-terminus [17]. Truncation of ten residues in the N-terminal generated small L1 11/16 VLP with ~30 nm diameter [23], while 15 amino acid truncation generated L152 VLP with ~ 55 nm diameter [24]. ...
... The size is smaller than the native L1HPV52 because as many as 117 bp (39 aa) in the upstream of the gene were removed. The purpose of this partial deletion is based on a research conducted by Wei M and colleagues in 2018 [24], where they found that removal of 15 aa in the N-terminal of L1 HPV52 can increase their soluble expression in E. coli and in vitro self-assembly. ...
Background
A major discovery in human etiology recognized that cervical cancer is a consequence of an infection caused by some mucosatropic types of human papillomavirus (HPV). Since L1 protein of HPV is able to induce the formation of neutralizing antibodies, it becomes a protein target to develop HPV vaccines. Therefore, this study aims to obtain and analyze the expression of HPV subunit recombinant protein, namely L1 HPV 52 in E. coli BL21 DE3. The raw material used was L1 HPV 52 protein, while the synthetic gene, which is measured at 1473 bp in pD451-MR plasmid, was codon-optimized (ATUM) and successfully integrated into 5643 base pairs (bps) of pETSUMO. Bioinformatic studies were also conducted to analyze B cell epitope, T cell epitope, and immunogenicity prediction for L1HPV52 protein.
Results
The pETSUMO-L1HPV52 construct was successfully obtained in a correct ligation size when it was cut with EcoR I. Digestion by Eco RI revealed a size of 5953 and 1160 bps for both TA cloning petSUMO vector and gene of interest, respectively. Furthermore, the right direction of construct pETSUMO-L1HPV52 was proven by PCR techniques using specific primer pairs then followed by sequencing, which shows 147 base pairs. Characterization of L1 HPV 52 by SDS-PAGE analysis confirms the presence of a protein band at a size of ~55 kDa with 6.12 mg/L of total protein concentration. Observation under by transmission electron microscope demonstrates the formation of VLP-L1 at a size between 30 and 40 nm in assembly buffer under the condition of pH 5.4. Based on bioinformatics studies, we found that there are three B cell epitopes (GFPDTSFYNPET, DYLQMASEPY, KEKFSADLDQFP) and four T cell epitopes (YLQMASEPY, PYGDSLFFF, DSLFFFLRR, MFVRHFFNR). Moreover, an immunogenicity study shows that among all the T cell epitopes, the one that has the highest affinity value is DSLFFFLRR for Indonesian HLAs.
Conclusion
Regarding the achievement on successful formation of L1 HPV52-VLPs, followed by some possibilities found from bioinformatics studies, this study suggests promising results for future development of L1 HPV type 52 vaccine in Indonesia.
... Moreover, we investigated the effect of copy number on L1 production from the integrated expression cassettes. Finally, we constructed 9 recombinant E. coli strains for L1 proteins of HPV types 6,11,16,18,31,33,45,52,58, and demonstrated desirable purity, VLP morphology and antigenicity of the various L1 proteins. ...
... To further characterize the chromosomally integrated expression of other genotypes of the HPV L1 protein, we constructed another 8 recombinant E. coli strains in addition to that for HPV 11 L1 with the aim of creating an HPV 9-valent vaccine. An HPV 9-valent vaccine has been developed previously with well-characterized VLPs using a plasmid-based E. coli expression system [31] and has been tested in phase 3 clinical trials in China (Register no. 2017L04931) offering benefit over lower valency vaccines against HPV. ...
... Plasmids pCas (Addgene #62225) and pTargetF (Addgene #62226) were gifts from Dr. Jiang Yu (Shanghai Institutes for Biological Sciences). Plasmids for expressing the L1 proteins of various HPV types (6,11,16,18,31,33,45,52, 58) were previously constructed and described elsewhere [31]. ...
Background
The various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli .
Results
Of 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci— lpxM, lpxP, lpxL, eptA, gutQ and kdsD —were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity.
Conclusions
Reengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.
... Moreover, we investigated the effect of copy number on L1 production from the integrated expression cassettes. Finally, we constructed 9 recombinant E. coli strains for L1 proteins of HPV types 6,11,16,18,31,33,45,52,58, and demonstrated desirable purity, VLP morphology and antigenicity of the various L1 proteins. ...
... To further characterize the chromosomally integrated expression of other genotypes of the HPV L1 protein, we constructed another 8 recombinant E. coli strains in addition to that for HPV 11 L1 with the aim of creating an HPV 9-valent vaccine. An HPV 9-valent vaccine has been developed previously with well-characterized VLPs using a plasmid-based E. coli expression system [31] and has been tested in phase 3 clinical trials in China (Register no. 2017L04931) offering bene t over lower valency vaccines against HPV. ...
... Plasmids pCas (Addgene #62225) and pTargetF (Addgene #62226) were gifts from Dr. Jiang Yu (Shanghai Institutes for Biological Sciences). Plasmids for expressing the L1 proteins of various HPV types (6,11,16,18,31,33,45, 52, 58) were previously constructed and described elsewhere [31]. ...
Background
The various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli .ResultsOf 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci— lpxM, lpxP, lpxL, eptA, gutQ and kdsD —were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. Conclusion
Reengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.
... Cervical cancer and genital warts are both caused by persistent HPV infection [135]. Gardasil (Merck), Cervarix (GSK), Gardasil-9 (Merck), and Cecolin (Innovax) are the four HPV preventive vaccines now on the market based on self-assembled VLPs that contain just L1 protein [136,137]. ...
... In phase 3 clinical studies, this technology demonstrated adequate safety and performance [140]. Through chimeric design, VLPs might be employed to produce numerous antigens to defend against various strains of the same virus in the future [135]. ...
Nanotechnology and protein engineering helped revolutionize the invention and upgrading of immunization carriers, as well as medicine packaging and delivery systems. Viruses have been considered functioning NPs in the 21st century. This nano-structure may deliver antigens and medications to multiple locations throughout tissues and organs. Gene-free Viral-like particles (VLPs) offer a safer alternative to inactivating or weakening viral strains for traditional vaccines. This allows for the development of VLPs that can contain polyvalent antigenic structures that can also contain antigenic chemicals to target tissues. Also, they're immunogens. VLPs have also been shown to be excellent adjuvants. VLP-based vaccinations without adjuvants have been found to promote humoral and cellular immunity via the MHC class I and II route in some cases.Also used as therapeutic vaccines, presenting patients' own antigens and assisting them in the fight against chronic and metabolic diseases, as well as various types of cancers. Several vaccines created from VLP have been approved or are being tested in the clinical setting. More investigation is needed to fully assess the effectiveness, bad effects, difficulties, and benefits of VLP-based vaccinations when used for different cancers.Despite technical obstacles such as molecules appearing correctly on the particle surface, current research has provided substantial amounts of knowledge that may assist to alleviate these challenges. One option discovered in our lab is to add a sortase recognition motif (LPXTG) to the surface of distinct VLPs that may be exposed. To protect the VLP integrity, it allows proteins to be attached to the VLP surface. Use of VLP external surfaces to transport medications to a specific cell or tissue in order to treat an illness. The VLP's surface can be manipulated in order to optimize the VLP's efficacy. In order to deliver this complex to a specified target, it must be fused with molecules on the surface. Despite the fact that VLP-based vaccinations have been successful in helping to prevent disease, more work is needed to reach the optimum condition.
... HPVs are classified into high-risk (H) and low risk (LR) according to their oncogenic potential 2 . The persistent infection of approximately 15 high-risk HPV types causes almost all cervical cancer cases (CC) and immediate precursor lesions 3 . Of these genotypes, HPV16 and HPV18 account for approximately 70% of global CC cases 4 . ...
... Using this host, a VLP-based vaccine was recently licensed in China 14 . L1 proteins in this vaccine (including HPV16-L1 protein) were produced in truncated form and purified from E. coli soluble fraction 3,15 . However, soluble full-length HPV16-L1 protein production in E. coli has not yet been achieved since L1 protein is mainly detected in the cells induced by the cells' insoluble fractions IPTG 16 . ...
... The auto-induction culture is a strategy that allows accumulating soluble recombinant protein in E. coli cytoplasm [17][18][19] , as well as the employ of low temperature and low inductor concentration [19][20][21] . Therefore, it would be attractive to produce high amounts of soluble full-length HPV16-L1 protein, considering that L1 protein purification from soluble fraction may be a cost-effective alternative since refolding of purified protein is avoided 3 . Thus, the present study aimed to assess soluble full-length L1 protein production from a wild type HPV16-L1 gene in E. coli by manipulating culture conditions. ...
Persistent infection with human papillomavirus type 16 (HPV16) causes the development of cervical cancer. Escherichia coli is a cost-effective host successfully used to develop a second-generation vaccine against HPV, based on the purification of soluble truncated L1 protein variants. Previous attempts to produce soluble full-length HPV16-L1 protein by E. coli have failed. This study was aimed at cloning a Cuban HPV16-L1 gene in E. coli and assessing its expression as a soluble full-length L1 protein by manipulating culture conditions. The L1 gene was amplified from a Cuban patient’s cervical sample and cloned into pET28a and pBAD/Myc-HisA vectors. Production and solubility of L1 protein were evaluated in E. coli TOP10 harboring pBADHPV16-L1 plasmid and E. coli BL21-(DE3), Rosetta-(DE3)/pLysS, and SHuffle® T7 Express lysY strains harboring pETHPV16-L1 plasmid, grown under arabinose (0.2%)- or isopropyl β-D-1-thiogalactopyranoside (IPTG, 100 µM)-induction or Super Broth-based auto-induction for 24 and 48 h. The recombinant plasmids pETHPV16-L1 and pBADHPV16-L1 were constructed. The HPV16-L1 protein was produced insoluble to high levels in conventionally IPTG-induced E. coli-pETHPV16-L1 cells. However, under auto-induction, soluble full-length HPV16-L1 protein was successfully produced at similar levels by E. coli BL21 (DE3), Rosetta (DE3) pLysS and SHuffle® T7 Express lysY cells, reaching up to 7.2 ± 0.5% and 14.3 ± 1.6% of the total proteins in the soluble fraction after growing for 24 and 48 h, respectively. It is concluded that the auto-induction procedure at 18 °C with 30 µM IPTG and 100 rev/min promotes soluble full-length HPV16-L1 protein production by E. coli.
... Persistent HPV infection is the major cause of the cervical cancer and genital warts [135]. There are currently four HPV prophylactic vaccines on the market based on self-assembled VLPs that contain only L1 protein including Gardasil (Merck), Cervarix (GSK), Gardasil-9 (Merck), and Cecolin (Innovax) [136,137]. ...
... This system has shown acceptable safety and performance in phase 3 clinical trials [140]. It has been suggested that in future developments VLPs can be used to express multiple antigens to protect against different strains of the same virus through chimeric design [135]. ...
Virus-like particles (VLPs) are virus-derived structures made up of one or more different molecules with the ability to self-assemble, mimicking the form and size of a virus particle but lacking the genetic material so they are not capable of infecting the host cell. Expression and self-assembly of the viral structural proteins can take place in various living or cell-free expression systems after which the viral structures can be assembled and reconstructed. VLPs are gaining in popularity in the field of preventive medicine and to date, a wide range of VLP-based candidate vaccines have been developed for immunization against various infectious agents, the latest of which is the vaccine against SARS-CoV-2, the efficacy of which is being evaluated. VLPs are highly immunogenic and are able to elicit both the antibody- and cell-mediated immune responses by pathways different from those elicited by conventional inactivated viral vaccines. However, there are still many challenges to this surface display system that need to be addressed in the future. VLPs that are classified as subunit vaccines are subdivided into enveloped and non- enveloped subtypes both of which are discussed in this review article. VLPs have also recently received attention for their successful applications in targeted drug delivery and for use in gene therapy. The development of more effective and targeted forms of VLP by modification of the surface of the particles in such a way that they can be introduced into specific cells or tissues or increase their half-life in the host is likely to expand their use in the future. Recent advances in the production and fabrication of VLPs including the exploration of different types of expression systems for their development, as well as their applications as vaccines in the prevention of infectious diseases and cancers resulting from their interaction with, and mechanism of activation of, the humoral and cellular immune systems are discussed in this review.
