The Experts below are selected from a list of 207 Experts worldwide ranked by ideXlab platform
Anton P J Middelberg - One of the best experts on this subject based on the ideXlab platform.
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structural based designed modular Capsomere comprising ha1 for low cost poultry influenza vaccination
Vaccine, 2016Co-Authors: Jarurin Waneesorn, Nani Wibowo, John Bingham, Anton P J MiddelbergAbstract:Abstract Highly pathogenic avian influenza (HPAI) viruses cause a severe and lethal infection in domestic birds. The increasing number of HPAI outbreaks has demonstrated the lack of capabilities to control the rapid spread of avian influenza. Poultry vaccination has been shown to not only reduce the virus spread in animals but also reduce the virus transmission to humans, preventing potential pandemic development. However, existing vaccine technologies cannot respond to a new virus outbreak rapidly and at a cost and scale that is commercially viable for poultry vaccination. Here, we developed modular Capsomere, subunits of virus-like particle, as a low-cost poultry influenza vaccine. Modified murine polyomavirus (MuPyV) VP1 Capsomere was used to present structural-based influenza Hemagglutinin (HA1) antigen. Six constructs of modular Capsomeres presenting three truncated versions of HA1 and two constructs of modular Capsomeres presenting non-modified HA1 have been generated. These modular Capsomeres were successfully produced in stable forms using Escherichia coli , without the need for protein refolding. Based on ELISA, this adjuvanted modular Capsomere (CaptHA1-3C) induced strong antibody response (almost 10 5 endpoint titre) when administered into chickens, similar to titres obtained in the group administered with insect cell-based HA1 proteins. Chickens that received adjuvanted CaptHA1-3C followed by challenge with HPAI virus were fully protected. The results presented here indicate that this platform for bacterially-produced modular Capsomere could potentially translate into a rapid-response and low-cost vaccine manufacturing technology suitable for poultry vaccination.
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Integrated molecular and bioprocess engineering for bacterially‐produced immunogenic modular virus‐like particle vaccine displaying 18 kDa rotavirus antigen
Biotechnology and Bioengineering, 2016Co-Authors: Alemu Tekewe, Anton P J MiddelbergAbstract:A high global burden of rotavirus disease and the unresolved challenges with the marketed rotavirus vaccines, particularly in the developing world, have ignited efforts to develop virus-like particle (VLP) vaccines for rotavirus. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising alternatives in reducing process complexity and cost. In this study, integrated molecular and bioprocess engineering approaches were used to simplify the production of modular murine polyomavirus Capsomeres and VLPs presenting a rotavirus 18kDa VP8* antigen. A single construct was generated for dual expression of non-tagged murine polyomavirus capsid protein VP1 and modular VP1 inserted with VP8*, for co-expression in Escherichia coli. Co-expressed proteins assembled into pentameric Capsomeres in E. coli. A selective salting-out precipitation and a polishing size exclusion chromatography step allowed the recovery of stable modular Capsomeres from cell lysates at high purity, and modular Capsomeres were successfully translated into modular VLPs when assembled in vitro. Immunogenicity study in mice showed that modular Capsomeres and VLPs induced high levels of VP8*-specific antibodies. Our results demonstrate that a multipronged synthetic biology approach combining molecular and bioprocess engineering enabled simple and low-cost production of highly immunogenic modular Capsomeres and VLPs presenting conformational VP8* antigenic modules. This strategy potentially provides a cost-effective production route for modular Capsomere and VLP vaccines against rotavirus, highly suitable to manufacturing economics for the developing world. Biotechnol. Bioeng. 2017;114: 397-406. (c) 2016 Wiley Periodicals, Inc.
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Design strategies to address the effect of hydrophobic epitope on stability and in vitro assembly of modular virus‐like particle
Protein Science, 2016Co-Authors: Alemu Tekewe, Natalie K. Connors, Anton P J MiddelbergAbstract:Virus-like particles (VLPs) and Capsomere subunits have shown promising potential as safe and effective vaccine candidates. They can serve as platforms for the display of foreign epitopes on their surfaces in a modular architecture. Depending on the physicochemical properties of the antigenic modules, modularization may affect the expression, solubility and stability of Capsomeres, and VLP assembly. In this study, three module designs of a rotavirus hydrophobic peptide (RV10) were synthesized using synthetic biology. Among the three synthetic modules, modularization of the murine polyomavirus VP1 with a single copy of RV10 flanked by long linkers and charged residues resulted in the expression of stable modular Capsomeres. Further employing the approach of module titration of RV10 modules on each Capsomere via Escherichia coli co-expression of unmodified VP1 and modular VP1-RV10 successfully translated purified modular capomeres into modular VLPs when assembled in vitro. Our results demonstrate that tailoring the physicochemical properties of modules to enhance modular Capsomeres stability is achievable through synthetic biology designs. Combined with module titration strategy to avoid steric hindrance to interCapsomere interactions, this allows bioprocessing of bacterially produced in vitro assembled modular VLPs.
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non chromatographic preparation of a bacterially produced single shot modular virus like particle Capsomere vaccine for avian influenza
Vaccine, 2015Co-Authors: Nani Wibowo, Yang Wu, J Meers, Anton P J MiddelbergAbstract:Highly pathogenic avian influenza (HPAI) causes significant economic loss, reduced food security and poses an ongoing pandemic threat. Poultry vaccination significantly decreases these problems and recognizes that the health of humans, animals and ecosystems are connected. Low-cost manufacture of poultry vaccine matched quickly to the ever-changing circulating strain is needed for effective vaccination. Here, we re-engineered the process to manufacture bacterially synthesized modular Capsomere comprising influenza M2e, previously shown to confer complete protection in challenged mice, for application in poultry. Modular Capsomere was prepared using a simplified non-chromatographic salting-out precipitation method and its immunogenicity tested in vivo in poultry. Modular Capsomere crudely purified by precipitation (pCapM2e) contained more contaminants than equivalent product purified by chromatography (cCapM2e). Unadjuvanted pCapM2e containing 80 EU of endotoxin per dose was inferior to highly purified and adjuvanted cCapM2e (2 EU per dose). However, addition of adjuvant to pCapM2e resulting in high immunogenicity after only a single dose of vaccination, yet without any local adverse reaction. This finding suggests a strong synergy between adjuvant, antigen and contaminants, and the possible existence of a "Goldilocks" level of contaminants, where high immunogenicity and low reactogenicity can be obtained in a single-shot vaccination. The simplified process offers potential cost and speed advantages to address the needs in influenza poultry vaccination in low-cost veterinary markets. (C) 2015 Elsevier Ltd. All rights reserved.
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A rapid and simple screening method to identify conditions for enhanced stability of modular vaccine candidates
Biochemical Engineering Journal, 2015Co-Authors: Alemu Tekewe, Nani Wibowo, Natalie K. Connors, Frank Sainsbury, Anton P J MiddelbergAbstract:Virus-like particles (VLPs) and Capsomere subunits have been developed as safe and effective vaccine candidates in the fight against infectious diseases. These bioengineered structures are suitable platforms for repetitive high density presentation of foreign epitope modules. However, due to the module’s diverse physicochemical properties, modularisation of Capsomeres and VLPs requires tailoring of the physicochemical environment specific to each module to maintain protein stability against aggregation. Here, we report a high-throughput technique for screening buffer components to stabilize Capsomeres, based on light scattering analysis. This screening method was applied to modular Capsomeres presenting peptide epitopes from the rotavirus spike protein VP8 subunit domain, engineered as a next-generation rotavirus vaccine candidate. Among various additives tested, non-ionic detergents, such as Triton X-100, Tween-80 and Tween-20, were able to stabilize modular Capsomeres, either alone or in combination with l-arginine, as confirmed with high-resolution size exclusion chromatography. Results demonstrate that tailoring the nature of the environment surrounding self-assembling proteins using small organic molecules can enhance the bioprocessing of modular vaccine Capsomeres. The developed screening method potentially provides a powerful approach for rapid tailoring of processing conditions specific to antigenic modules displayed on next-generation recombinant Capsomere and VLP vaccines, for low-cost vaccine delivery at global scale.
Yap P Chuan - One of the best experts on this subject based on the ideXlab platform.
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the economics of virus like particle and Capsomere vaccines
Biochemical Engineering Journal, 2014Co-Authors: Yap P Chuan, Nani Wibowo, Anton P J MiddelbergAbstract:Effective control of infectious diseases relies on new vaccine technologies that can quicken and broaden vaccine delivery. Novel modular virus-like particle (VLP) and Capsomere technologies have been recently reported. These technologies utilize murine polyomavirus (MuPyV) VLPs and Capsomeres as potent delivery systems to carry and display antigenic modules consisting of heterologous peptides, in the form of modular constructs capable of inducing high levels of specific antibodies against bacterial or viral antigens. These constructs are prepared using high-yield microbial synthesis, potentially enabling low-cost, rapid and scalable manufacture of new vaccines. To evaluate this potential, this study analyzes the economics of Capsomere and VLP production using process simulation. Data here show that the unit production cost (UPC) for Capsomere is up to 69% lower than that for VLP at the comparison scale (500. L fermentor), due to a simpler downstream process and a higher product yield. For VLP production, reactive diafiltration assembly was shown to have a UPC 30% lower than dilution assembly. Sensitivity analysis of uncertain process inputs with Monte Carlo simulations revealed a significant influence of final biomass concentration on UPC, contributing up to 50% of variance observed in the UPC probability distribution. Despite such process variability, optimized Capsomere or VLP vaccine production, using a 500-L or 1500-L fermentor respectively, has more than 80% chance of producing vaccine at a cost less than 1 cent per dose based on a conservative assumption of 50. μg protein per vaccine dose. With a 10-kL fermentor, both the Capsomere and VLP processes have productivity that could allow manufacture of 320 million vaccine doses in 2.3 and 4.7 days, respectively. This study confirms with quantitative data the possible economic, speed and scale benefits of the modular Capsomere and VLP vaccine technologies, which can potentially redefine current vaccine distribution landscape and time-scale benchmarks.
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Energetic changes caused by antigenic module insertion in a virus-like particle revealed by experiment and molecular dynamics simulations.
PLOS ONE, 2014Co-Authors: Lin Zhang, Yap P Chuan, Natalie K. Connors, Ronghong Tang, Anton P J MiddelbergAbstract:The success of recombinant virus-like particles (VLPs) for human papillomavirus and hepatitis B demonstrates the potential of VLPs as safe and efficacious vaccines. With new modular designs emerging, the effects of antigen module insertion on the self-assembly and structural integrity of VLPs should be clarified so as to better enabling improved design. Previous work has revealed insights into the molecular energetics of a VLP subunit, Capsomere, comparing energetics within various solution conditions known to drive or inhibit self-assembly. In the present study, molecular dynamics (MD) simulations coupled with the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method were performed to examine the molecular interactions and energetics in a modular Capsomere of a murine polyomavirus (MPV) VLP designed to protect against influenza. Insertion of an influenza antigenic module is found to lower the binding energy within the Capsomere, and a more active state is observed in Assembly Buffer as compared with that in Stabilization Buffer, which has been experimentally validated through measurements using differential scanning calorimetry. Further in-depth analysis based on free-energy decomposition indicates that destabilized binding can be attributed to electrostatic interaction induced by the chosen antigen module. These results provide molecular insights into the conformational stability of Capsomeres and their abilities to be exploited for antigen presentation, and are expected to be beneficial for the biomolecular engineering of VLP vaccines.
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Microbially synthesized modular virus‐like particles and Capsomeres displaying group A streptococcus hypervariable antigenic determinants
Biotechnology and Bioengineering, 2013Co-Authors: Yap P Chuan, Nani Wibowo, Natalie K. Connors, Yang Wu, Fiona K. Hughes, Michael R. Batzloff, Anton P J MiddelbergAbstract:Effective and low-cost vaccines are essential to control severe group A streptococcus (GAS) infections prevalent in low-income nations and the Australian aboriginal communities. Highly diverse and endemic circulating GAS strains mandate broad-coverage and customized vaccines. This study describes an approach to deliver cross-reactive antigens from endemic GAS strains using modular virus-like particle (VLP) and Capsomere systems. The antigens studied were three heterologous N-terminal peptides (GAS1, GAS2, and GAS3) from the GAS surface M-protein that are specific to endemic strains in Australia Northern Territory Aboriginal communities. In vivo data presented here demonstrated salient characteristics of the modular delivery systems in the context of GAS vaccine design. First, the antigenic peptides, when delivered by unadjuvanted modular VLPs or adjuvanted Capsomeres, induced high titers of peptide-specific IgG antibodies (over 1 × 104). Second, delivery by Capsomere was superior to VLP for one of the peptides investigated (GAS3), demonstrating that the delivery system relative effectiveness was antigen-dependant. Third, significant cross-reactivity of GAS2-induced IgG with GAS1 was observed using either VLP or Capsomere, showing the possibility of broad-coverage vaccine design using these delivery systems and cross-reactive antigens. Fourth, a formulation containing three pre-mixed modular VLPs, each at a low dose of 5 μg (corresponding to
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modular engineering of a microbially produced viral Capsomere vaccine for influenza
Chemical Engineering Science, 2013Co-Authors: Nani Wibowo, Yap P Chuan, Anton P J MiddelbergAbstract:Faster and cheaper vaccine manufacture based on modern technologies is increasingly needed to effectively mitigate the burden of disease caused by highly contagious and mutagenic pathogens, such as influenza viruses. This study describes an approach to synthetically engineer a new influenza vaccine system by antigenic modularization of a carrier viral Capsomere, coupled with microbial processing of the sub-unit vaccine. This approach leads to a system optimized with respect to both biological and process criteria. Murine polyomavirus VP1 protein, which self assembles into a pentameric sub-unit Capsomere of a virus-like particle (VLP), was engineered to inhibit VLP assembly and to allow modular insertion of influenza M2e antigen at multiple sites within the protein. The yield, solubility, and immunogenicity of the resulting modular Capsomeres could be optimized by varying the module insertion site and the number of M2e modules per site. This study demonstrated, for the first time, an innovative strategy of inserting multiple antigenic modules, up to 45 M2e modules, in a single Capsomere. Modularization of M2e antigen was shown to improve its immunogenicity by more than an order of magnitude over that attained by immunization with an equivalent mass of non-modularized M2e peptide. Vaccination of mice using modular Capsomeres induced high antigen-specific antibody levels suggestive of protective efficacy. This modular vaccine design approach, inspired by synthetic biology approaches to new system development, is conducive to technologies rapidly adaptable to pathogenic variations.
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molecular energetics in the Capsomere of virus like particle revealed by molecular dynamics simulations
Journal of Physical Chemistry B, 2013Co-Authors: Lin Zhang, Yap P Chuan, Natalie K. Connors, Ronghong Tang, Anton P J MiddelbergAbstract:Virus-like particles (VLPs) are highly organized nanoparticles that have great potential in vaccinology, gene therapy, drug delivery, and materials science. However, the application of VLPs is hindered by obstacles in their design and production due to low efficiency of self-assembly. In the present study, all-atom (AA) molecular dynamics (MD) simulations coupled with the molecular mechanics-Poisson–Boltzmann surface area (MM-PBSA) method are utilized to examine the molecular interactions in the Capsomere of a murine polyomavirus (MPV) VLP. It is found that both low ionic strength and the intraCapsomere disulfide bonds are favorable for maintaining a stable Capsomere. Simulation results examining the effects of solution conditions on the stabilization of a Capsomere were verified by calorimetry experiments. Simulation results of free energy decomposition indicate that hydrophobic interaction is favorable for the formation of a Capsomere, whereas electrostatic interaction is unfavorable. With increasing io...
Andreas G. Lössl - One of the best experts on this subject based on the ideXlab platform.
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expression of hpv 16 l1 Capsomeres with glutathione s transferase as a fusion protein in tobacco plastids an approach for a Capsomere based hpv vaccine
Human Vaccines & Immunotherapeutics, 2014Co-Authors: Syed Waqas Hassan, Martin Müller, Zabta Khan Shinwari, Mohammad Tahir Waheed, Jihong Liu Clarke, Andreas G. LösslAbstract:Human Papillomavirus (HPV) is the main cause of cervical cancer, which is the second most severe cancer of women worldwide, particularly in developing countries. Although vaccines against HPV infection are commercially available, they are neither affordable nor accessible to women in low income countries e.g. Africa. Thus, alternative cost-effective vaccine production approaches need to be developed. This study uses tobacco plants to express pentameric Capsomeres of HPV that have been reported to generate elevated immune responses against HPV. A modified HPV-16 L1 (L1_2xCysM) protein has been expressed as a fusion protein with glutathione-S-transferase (GST) in tobacco chloroplasts following biolistic transformation. In total 7 transplastomic lines with healthy phenotypes were generated. Site specific integration of the GST-L1_2xCysM and aadA genes was confirmed by PCR. Southern blot analysis verified homogenous transformation of all transplastomic lines. Antigen capture ELISA with the conformation-specif...
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stable expression of pentameric Capsomeres hpv 16 l1 with glutathione s transferase in transplastomic tobacco a step towards affordable Capsomere based hpv vaccine
Current Opinion in Biotechnology, 2013Co-Authors: Syed Waqas Hassan, Mehmood Zaffar, Zabta Khan Shinwari, Andreas G. LösslAbstract:Human Papillomavirus (HPV) is a single major source in causing cervical cancer worldwide which is the most common cause of death in females. Around 85% of the cervical cancer cases occur in developing countries where existing vaccines are not available or unaffordable. Hence, alternative platforms are considered necessary for the development of cost-effective vaccines against HPV for their availability in developing countries. Plants offer unique advantages for vaccine production over fermenter-based systems. Pentameric Capsomeres ofHPVhave been reported to generate elevated immune responses against HPV. In present study a modified HPV-16 L1 (L1 2xCysM) protein has been expressed as a fusion protein with glutathione-S-transferase (GST) into tobacco plastids. Seven transplastomic lines of Nicotiana benthamiana were generated using biolistic transformation technique. Site specific integration of the GST-L1 2xCysM and aadA genes was confirmed by PCR. Southern blot analysis verified homoplasmy of all transplastomic lines.AntigencaptureELISAwith theconformationspecific antibody Ritti01, showed protein expression as well as the retention of immunogenic epitopes of L1 protein. GST-L1 expressing tobacco plants were completely normal in their morphology. Taken together, this data contribute another step forward towards thedevelopmentof cost-effectiveplant-madevaccinesagainstHPV for resource poor countries.
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Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of Capsomeres in tobacco: a step towards cost-effective second-generation vaccines
Transgenic Research, 2011Co-Authors: M. Tahir Waheed, Nadja Thönes, Martin Müller, S. Waqas Hassan, N. Mona Razavi, Elke Lössl, Hans-peter Kaul, Andreas G. LösslAbstract:Certain types of human papillomaviruses (HPV) are causatively associated with cervical carcinoma, the second most common cancer in women worldwide. Due to limitations in the availability of currently used virus-like particle (VLP)-based vaccines against HPV to women of developing countries, where most cases of cervical cancer occur, the development of a cost-effective second-generation vaccine is a necessity. Capsomeres have recently been demonstrated to be highly immunogenic and to have a number of advantages as a potential cost-effective alternative to VLP-based HPV vaccines. We have expressed a mutated HPV-16 L1 (L1_2xCysM) gene that retained the ability to assemble L1 protein to Capsomeres in tobacco chloroplasts. The recombinant protein yielded up to 1.5% of total soluble protein. The assembly of Capsomeres was examined and verified by cesium chloride density gradient centrifugation and sucrose sedimentation analysis. An antigen capture enzyme-linked immunosorbent assay confirmed the formation of Capsomeres by using a conformation-specific monoclonal antibody which recognized the conformational epitopes. Transplastomic tobacco plants exhibited normal growth and morphology, but all such lines showed male sterility in the T_0, T_1 and T_2 generations. Taken together, these results indicate the possibility of producing a low-cost Capsomere-based vaccine by plastids.
Edward E Walsh - One of the best experts on this subject based on the ideXlab platform.
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Antigenic presentation of heterologous epitopes engineered into the outer surface-exposed helix 4 loop region of human papillomavirus L1 Capsomeres
Virology Journal, 2009Co-Authors: Yoshihiko Murata, Robert C. Rose, Paula M Lightfoote, Edward E WalshAbstract:Background Human papillomavirus (HPV) L1 capsid proteins can self-assemble into pentamers (Capsomeres) that are immunogenic and can elicit neutralizing antibodies. Structural modelling of L1 inter-pentameric interactions predicts that helix 4 (h4) of each of the five L1 monomers project laterally and outwards from the pentamer. We sought to utilize HPV L1 Capsomeres as a vaccine platform by engineering heterologous epitopes within L1 derivatives deleted for h4 domain. Results We used baculovirus – infected Trichoplusia ni cells and ultracentrifugation to synthesize and purify three 16L1 derivatives: one bearing a short deletion (amino acids 404–436) encompassing the h4 domain, and two others, each bearing a conserved neutralizing epitope of the human respiratory syncytial virus (RSV) fusion (F) protein (residues 255–278 and 423–436) that was substituted for the deleted L1 h4 domain residues. Each of the three Capsomere derivatives was recognized by anti-L1 antibodies, while two bearing the RSV F-derived moieties were recognized by anti-RSV F antibodies. All three L1 derivatives formed ring-like structures that were similar in morphology and size to those described for native 16L1 Capsomeres. When injected into mice, each of the Capsomere derivatives was immunogenic with respect to L1 protein, and immunization with chimeric L1-RSV F pentamers resulted in RSV non-neutralizing antisera that recognized purified RSV F protein in immunoblots. Conclusion HPV L1 monomers bearing heterologous epitopes within the L1 h4 region can self-assemble into Capsomeres that elicit antibody response against such non-HPV encoded epitopes. Thus, the L1 h4 region can function as a novel antigen display site within the L1 pentamer, which in turn may serve as a potential vaccine template.
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Antigenic presentation of heterologous epitopes engineered into the outer surface-exposed helix 4 loop region of human papillomavirus L1 Capsomeres.
Virology Journal, 2009Co-Authors: Yoshihiko Murata, Robert C. Rose, Paula M Lightfoote, Edward E WalshAbstract:Background Human papillomavirus (HPV) L1 capsid proteins can self-assemble into pentamers (Capsomeres) that are immunogenic and can elicit neutralizing antibodies. Structural modelling of L1 inter-pentameric interactions predicts that helix 4 (h4) of each of the five L1 monomers project laterally and outwards from the pentamer. We sought to utilize HPV L1 Capsomeres as a vaccine platform by engineering heterologous epitopes within L1 derivatives deleted for h4 domain.
Martin Sapp - One of the best experts on this subject based on the ideXlab platform.
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human papillomavirus major capsid protein l1 remains associated with the incoming viral genome throughout the entry process
Journal of Virology, 2017Co-Authors: Stephen Digiuseppe, Malgorzata Bienkowskahaba, Lucile G M Guion, Timothy R Keiffer, Martin SappAbstract:ABSTRACT During infectious entry, acidification within the endosome triggers uncoating of the human papillomavirus (HPV) capsid, whereupon host cyclophilins facilitate the release of most of the major capsid protein, L1, from the minor capsid protein L2 and the viral genome. The L2/DNA complex traffics to the trans -Golgi network (TGN). After the onset of mitosis, HPV-harboring transport vesicles bud from the TGN, followed by association with mitotic chromosomes. During this time, the HPV genome remains in a vesicular compartment until the nucleus has completely reformed. Recent data suggest that while most of L1 protein dissociates and is degraded in the endosome, some L1 protein remains associated with the viral genome. The L1 protein has DNA binding activity, and the L2 protein has multiple domains capable of interacting with L1 Capsomeres. In this study, we report that some L1 protein traffics with L2 and viral genome to the nucleus. The accompanying L1 protein is mostly full length and retains conformation-dependent epitopes, which are recognized by neutralizing antibodies. Since more than one L1 molecule contributes to these epitopes and requires assembly into Capsomeres, we propose that L1 protein is present in the form of pentamers. Furthermore, we provide evidence that the L1 protein interacts directly with viral DNA within the capsid. Based on our findings, we propose that the L1 protein, likely arranged as Capsomeres, stabilizes the viral genome within the subviral complex during intracellular trafficking. IMPORTANCE After internalization, the nonenveloped human papillomavirus virion uncoats in the endosome, whereupon conformational changes result in a dissociation of a subset of the major capsid protein L1 from the minor capsid protein L2, which remains in complex with the viral DNA. Recent data suggest that some L1 protein may accompany the viral genome beyond the endosomal compartment. We demonstrate that conformationally intact L1 protein, likely still arranged as Capsomeres, remains associated with the incoming viral genome throughout mitosis and transiently resides in the nucleus until after the viral DNA is released from the transport vesicle.
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surface exposed amino acid residues of hpv16 l1 protein mediating interaction with cell surface heparan sulfate
Journal of Biological Chemistry, 2007Co-Authors: Maren Knappe, Rolf E. Streeck, Sabrina Bodevin, Hanschristoph Selinka, Dorothe Spillmann, Xiaojiang S Chen, Ulf Lindahl, Martin SappAbstract:Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the Capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between Capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.
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Induction of type-specific neutralizing antibodies by Capsomeres of human papillomavirus type 33.
Virology, 2001Co-Authors: Claudia Fligge, Tzenan Giroglou, Rolf E. Streeck, Martin SappAbstract:Abstract The immunogenicity of Capsomeres of human papillomavirus type 33 was evaluated in a dose–response analysis. Capsomeres were obtained free of capsids by expression of L1 carrying the single point mutation C427S. Neutralizing antibodies were detected using an in vitro pseudoinfection assay. Capsomeres induced type-specific, neutralizing antibodies in mice even in the absence of adjuvant. The neutralization titers of immune sera raised without adjuvant were 10- to 20-fold lower than those of antisera to virus-like particles, but virtually identical using Freund's adjuvant. These data indicate that Capsomeres may substitute for virus-like particles in future vaccines when used with an adjuvant appropriate for human vaccination.