Infectious bursal disease(IBD)causes considerable economic losses in the commercial poultry industry worldwide.The principal way to control IBD virus(IBDV),the causative agent of IBD,is still through vaccination progr...Infectious bursal disease(IBD)causes considerable economic losses in the commercial poultry industry worldwide.The principal way to control IBD virus(IBDV),the causative agent of IBD,is still through vaccination programs.Virus-like particles(VLPs)are recognised as a safe and potent recombinant vaccine platform.This research work explores the characterisation and separation of infectious bursal disease virus-like particles(IBD-VLPs)from crude feedstock.Various characteristics were studied with highperformance size-exclusion chromatography(HP-SEC),sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS-PAGE)and transmission electron microscopy(TEM)analyses.Subsequently,the separation of IBD-VLPs using polyethylene glycol(PEG)/sodium citrate-based aqueous two-phase systems(ATPSs)was conducted and optimised.Moreover,a scale-up study of the best ATPS constituted of 15%PEG 6000,11%sodium citrate and 10%crude feedstock was performed to compare the separation performance of IBD-VLPs with and without centrifugation-assisted.The results indicated that the optimised ATPS with centrifugation-assisted for both 5 g and 50 g systems showed good recovery of IBDVLPs of>97%in the interphase between the PEG-rich top and salt-rich bottom phases.These optimised systems also showed high removal efficiencies of impurities of>95%.The results demonstrated that aqueous two-phase extraction could be a promising technology for efficient VLPs separation.展开更多
Objective Combination immunotherapy strategies targeting OX40,a co-stimulatory molecule that can enhance antitumor immunity by modulating the proliferation,differentiation,and effector function of tumor-infiltrating T...Objective Combination immunotherapy strategies targeting OX40,a co-stimulatory molecule that can enhance antitumor immunity by modulating the proliferation,differentiation,and effector function of tumor-infiltrating T cells,have attracted much attention for their excellent therapeutic effects.In this study,we aimed to evaluate the antitumor efficacy of combined anti-OX40 and hepatitis B core viruslike particles(HBc VLPs)therapy using a mouse colon cancer model.Methods Humanized B-h OX40 mice were injected subcutaneously with MC38 colon tumor cells and treated with HBc VLPs+anti-h OX40 antibody.Tumor growth was monitored.Flow cytometric analysis was performed to evaluate the populations of T cell subsets in the tumors.Results The combination of anti-OX40 with HBc VLPs resulted in a significant delay in tumor growth,suggesting that a potent antitumor immunity was induced by the combination therapy.Further studies revealed that HBc VLPs+anti-OX40 treatment induced a significant increase in effector T cells(Teffs)and a significant decrease in regulatory T cells(Tregs)in the tumor microenvironment(TME),which accounted for the synergistic antitumor effect of anti-OX40 in combination with HBc VLPs.Conclusion Combination therapy of anti-h OX40 and HBc VLPs provides synergistic antitumor activity in colon cancer-bearing mice,which may represent a potential design strategy for cancer immunotherapy.展开更多
AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. ...AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS： The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses： Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS： Both single infection by Bac-P1-3CD and coinfection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins selfassembled into clusters of virus-like particles （VLP） resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION： Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.展开更多
Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). MethodsThe ge...Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). MethodsThe gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichiapastorisunder the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualiza-tion. Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoriswith an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immuno-genicity on surface-displayed HEnAg. Conclusion The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.展开更多
Summary: To prepare carboxyl terminus truncated human papillomavirus type 58 L1(HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1...Summary: To prepare carboxyl terminus truncated human papillomavirus type 58 L1(HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing-HPV58L1 protein-were harvested and analyzed by SDS-PAGE and Western blot. The ProBond~TM purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope. Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virions in vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.展开更多
Filoviruses are hemorrhagic fever viruses endemic to parts of Africa and the Philippines. Infection carries with it a mortality rate of up to 90% and currently there are no effective vaccines or therapeutics available...Filoviruses are hemorrhagic fever viruses endemic to parts of Africa and the Philippines. Infection carries with it a mortality rate of up to 90% and currently there are no effective vaccines or therapeutics available to combat infection. However, the filovirus virus-like particles (VLP), which are currently under development, have been shown to be a promising vaccine candidate. They provide protection from infection in the mouse, guinea pig, and nonhuman primate models of infection, eliciting high anti-glycoprotein antibody titers and T cell responses to viral proteins. In this review, we will highlight the development of the filovirus VLP and describe the current understanding of VLP immunogenicity and correlates of protection.展开更多
The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods....The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid （Opt-Cap） gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% （v/v） methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What＇s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles （VLPs）. These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.展开更多
Objective.To test whether intramuscular,intranasal,intrarectal and intravagina l administration of HPV6b L 1 virus-like particlescould induce immune response in mice and to as sess whether intra-muscular and mucosal v...Objective.To test whether intramuscular,intranasal,intrarectal and intravagina l administration of HPV6b L 1 virus-like particlescould induce immune response in mice and to as sess whether intra-muscular and mucosal vaccination against HPV is feasible.Me thods.HPV6b L1proteins self-assembled into VLPs in Sf-9cell in vitro.Mic e were immunized on day0and21with50ìg HPV6b L1VLPs intramuscularly,int ranasally,intrarectally and intravagi-nally respectively.Sera were collected for testing IgG titer after a further7days and3months respec-tively.Results .After immunizations,all mice developed significant anti-HPV6b L1antibody titers in serum by7days after the second immunization.The titer of the serum I gG antibody against HPV6b L1VLPs in the intramuscularly immunized group was h igher than that in the intranasally,intrarectally and intravaginally immunized groups respectively,indicating that both muscular and mucosal administration of HPV6b L1VLPs can stimulate a systemic HPV-specific antibody response.Sera of the mice in the in-tramuscularly immunized group still maintained a high tit er of the serum IgG antibody against HPV6b L1VLPs 3months after the immunizat ion.Conclusion.The results demonstrated that the HPV6b L1VLPs maintain stro ng antigenicity.Immu-nization with HPV6b L1VLPs via intramuscular and mucos al routes,without adjuvant ,can elicit spe-cific antibody in sera.These fin dings suggest that the VLPs are able to induce protective antibodies.展开更多
Objectives： The aim was to construct 2009 pandemic A/HINI influenza VLPs （virus-like particles） and compare the immunogenicity and protection efficacy with the commercial Panenza vaccine in BALB/c mouse model. Meth...Objectives： The aim was to construct 2009 pandemic A/HINI influenza VLPs （virus-like particles） and compare the immunogenicity and protection efficacy with the commercial Panenza vaccine in BALB/c mouse model. Methods： VLPs derived from influenza A/Hong Kong/01/2009 （H 1N 1 ） virus were constructed by Bac-to-Bac baculovirus expression system. VLPs were purified by sucrose density gradient ultracentrifugation and then characterized by Western blotting analysis and transmission electron microscopy. After single dose vaccination with 3 lag of VLPs and equal amount of Panenza vaccine, the immune responses and efficacy of protection induced by VLPs were compared with those elicited by the Panenza vaccine in 6-8 weeks old female BALB/c mice. Key findings： VLPs could induce higher antibody titer as determined by hemagglutinin inhibition and microneutralization assay. Furthermore, we demonstrated that VLPs induced better antibody response to neuraminidase. In addition, VLP vaccinated mice had stronger cell-mediated immune response. As a result, our VLPs conferred 100% protection while the Panenza vaccine only conferred 67% protection. Conclusion： From the results, we concluded that influenza VLPs are highly immunogenic and they are promising to be developed as an alternative strategy to vaccine production in order to control the spread of influenza viruses.展开更多
Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family,which is widespread and causes high fatality.The nucleocapsid of CCHFV is comprised of N prote...Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family,which is widespread and causes high fatality.The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment.In this research,the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus.Under an electron microscope,Virus-Like Particles (VLPs) with various size and morphology were observed in cytoplasmic vesicles in the infected cells.Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation,which was confirmed by Western blot analysis and immuno-electron microscopy (IEM).展开更多
Objective To eliminate the side effects of aluminum adjuvant and His-tag,we constructed chimeric VLPs displaying the epitope of EV71（SP70） without His-tagged.Then evaluating whether the VLPs could efficiently evoke ...Objective To eliminate the side effects of aluminum adjuvant and His-tag,we constructed chimeric VLPs displaying the epitope of EV71（SP70） without His-tagged.Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.Methods The fusion protein was constructed by inserting SP70 into the MIR of truncated HBc Ag sequence,expressed in E.Coli,and purified through ion exchange chromatography and density gradient centrifugation.Mice were immunized with the VLPs and sera were collected afterwards.The specific antibody titers,Ig G subtypes and neutralizing efficacy were detected by ELISA,neutralization assay,and EV71 lethal challenge.IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.Results HBc-SP70 proteins can self-assemble into empty VLPs.After immunization with HBc-SP70 VLPs,the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge.There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not.The specific Ig G subtypes were mainly IgG1 and IgG2 b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.Conclusion The fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation.In the absence of adjuvant,they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant.Furthermore,the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.展开更多
Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects,offering a promising insecticidal component for insect pest control.H...Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects,offering a promising insecticidal component for insect pest control.However,effective delivery systems are required to help neurotoxic peptides pass through the gut barrier into the hemolymph,where they can act.Here,we investigated the potential of a novel nanocarrier,Drosophila X virus-like particle(DXV-VLP),for delivering a neurotoxin from the scorpion Androctonus australis Hector(AaIT)against the invasive pest fruit fly,Drosophila suzuki.Our results show that the fusion proteins of DXV polyproteins with AalT peptide at their Ctermini could be sufficiently produced in Lepidoptera Hi5 cells in a soluble form using the recombinant baculovirus expression system,and could self-assemble into VLPs with similar particle morphology and size to authentic DXV virions.In addition,the AalT peptides displayed on DXV-VLPs retained their toxicity,as demonstrated in injection bioassays that resulted in severe mortality(72%)in adults after 72 h.When fed to adults,mild mortality was observed in the group treated with DXV-AalT(38%),while no mortality occurred in the group treated with AaIT peptide,thus indicating the significant role of DXV-VLPs in delivering AalT peptides.Overall,this proof-of-concept study demonstrates for the first time that VLPs can be exploited to enhance oral delivery of insect-specific neurotoxic peptides in the context of pest control.Moreover,it provides insights for further improvements and potentially the development of neurotoxin-based bioinsecticides and/or transgenic crops for insect pest control.展开更多
Nanoparticles are significant for veterinary vaccine development because they are safer and more effective than conventional formulations.One promising area of research involves self-assembled protein nanoparticles(SA...Nanoparticles are significant for veterinary vaccine development because they are safer and more effective than conventional formulations.One promising area of research involves self-assembled protein nanoparticles(SAPNs),which have shown potential for enhancing antigen-presenting cell uptake,B-cell activation,and lymph node trafficking.Numerous nanovaccines have been utilized in veterinary medicine,including natural self-assembled protein nanoparticles,rationally designed self-assembled protein nanoparticles,animal virus-derived nanoparticles,bacteriophagederived nanoparticles,and plant-derived nanoparticles,which will be discussed in this review.SAPN vaccines can produce robust cellular and humoral immune responses and have been shown to protect against various animal infectious diseases.This article attempts to summarize these diverse nanovaccine types and their recent research progress in the field of veterinary medicine.Furthermore,this paper highlights their disadvantages and methods for improving their immunogenicity.展开更多
Nature has the ingenious capability to design spiky topological features at the macro-and nanoscales,which exhibits fascinating interface adhesive properties by means of multivalent interactions.Following a biomimetic...Nature has the ingenious capability to design spiky topological features at the macro-and nanoscales,which exhibits fascinating interface adhesive properties by means of multivalent interactions.Following a biomimetic approach,such as nanoscale virus particles are highly infectious toward host cells,a range of organic and inorganic spiky particles(virus-like nanostructures)have been precisely engineered for diverse biomedical applications.Generally,organic virus-like particles(VLPs)derived from viral capsids(often termed as virosomes)have been extensively studied and reviewed,but concomitant concerns regarding immunogenicity and risks of mutagenesis limit clinical potential of organic VLPs.In contrast,inorganic VLPs(viral-mimicking topography)possess fascinating physicochemical characteristics,such as excellent electrical,optical,magnetic,mechanical and catalytic properties,which make them particularly suitable for biomedical applications.Alternatively,there is no comprehensive review related to inorganic VLPs engineered with non-viral shell for biomedical applications.Hence,in this review,we present a brief overview on inorganic VLPs,followed by summarizing the construction and properties of virus-like nanostructures,as well as the mechanisms of nano-bio interface interactions initiated by spiky topography.Furthermore,we focus on the recent advances of VLPs for biomedical applications(including biosensing,antibacterial therapy and cancer treatment).Finally,the future outlook and emerging challenges will be presented.This review aims to provide future scope of the rational design of inorganic non-viral vectors,especially with respect to gene-based therapy platforms.展开更多
The virus-like particle（VLPs） vaccine is an ideal HIV-1 vaccine, which can simultaneously induce a neutralizing antibody reaction and cell-mediated immunity effectively. In this study, two kinds of plasmids have bee...The virus-like particle（VLPs） vaccine is an ideal HIV-1 vaccine, which can simultaneously induce a neutralizing antibody reaction and cell-mediated immunity effectively. In this study, two kinds of plasmids have been used, one can express the HIV-1 main structure proteins, Gagpol and Env, and the other contains an antibiotic gene. The two kinds of plasmids have been cotransfected into 293 cells. A stable cell line that can express Gagpol and Env proteins efficiently and lastingly has been screened. It has been confirmed that Gagpol and Env proteins in the cell culture supernatant can be self-assembled into virus-like particles. The authors have detected the secretion of VLPs in the cell medium, defined the peak of the secretion, and followed and monitored the stability of expression.展开更多
Recombinant technology-based vaccines have emerged as a highly effective way to prevent a wide range of illnesses.The technology improved vaccine manufacturing,rendering it more efficient and economical.These vaccines...Recombinant technology-based vaccines have emerged as a highly effective way to prevent a wide range of illnesses.The technology improved vaccine manufacturing,rendering it more efficient and economical.These vaccines have multiple advantages compared to conventional vaccines.The pandemic has heightened awareness of the advantages of these vaccine technologies;trust and acceptance of these vaccines are steadily growing globally.This work offers an overview of the prospects and advantages associated with recombinant vaccines.Additionally,it discusses some of the challenges likely to arise in the future.Their ability to target diverse pathogen classes underscores their contributions to preventing previously untreatable diseases(especially vector-borne and emerging diseases)and hurdles faced throughout the vaccine development process,especially in enhancing the effectiveness of these vaccines.Moreover,their compatibility with emerging vaccination platforms of the future like virus-like particles and CRISPR/Cas9 for the production of next-generation vaccines may offer many prospects.This review also reviewed the hurdles faced throughout the vaccine development process,especially in enhancing the effectiveness of these vaccines against vector-borne diseases,emerging diseases,and untreatable diseases with high mortality rates like AIDS as well as cancer.展开更多
基金Zhejiang University and TalentIntroduction Program of China for Postdoctoral Researcher for the financial supportfinancially supported by the National Key Research&Development Program of China (2021YFE0113300)the National Natural Science Foundation of China (22078286)。
文摘Infectious bursal disease(IBD)causes considerable economic losses in the commercial poultry industry worldwide.The principal way to control IBD virus(IBDV),the causative agent of IBD,is still through vaccination programs.Virus-like particles(VLPs)are recognised as a safe and potent recombinant vaccine platform.This research work explores the characterisation and separation of infectious bursal disease virus-like particles(IBD-VLPs)from crude feedstock.Various characteristics were studied with highperformance size-exclusion chromatography(HP-SEC),sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS-PAGE)and transmission electron microscopy(TEM)analyses.Subsequently,the separation of IBD-VLPs using polyethylene glycol(PEG)/sodium citrate-based aqueous two-phase systems(ATPSs)was conducted and optimised.Moreover,a scale-up study of the best ATPS constituted of 15%PEG 6000,11%sodium citrate and 10%crude feedstock was performed to compare the separation performance of IBD-VLPs with and without centrifugation-assisted.The results indicated that the optimised ATPS with centrifugation-assisted for both 5 g and 50 g systems showed good recovery of IBDVLPs of>97%in the interphase between the PEG-rich top and salt-rich bottom phases.These optimised systems also showed high removal efficiencies of impurities of>95%.The results demonstrated that aqueous two-phase extraction could be a promising technology for efficient VLPs separation.
基金supported by National Major Science and Technology Projects of China 2017ZX10105015-001-002。
文摘Objective Combination immunotherapy strategies targeting OX40,a co-stimulatory molecule that can enhance antitumor immunity by modulating the proliferation,differentiation,and effector function of tumor-infiltrating T cells,have attracted much attention for their excellent therapeutic effects.In this study,we aimed to evaluate the antitumor efficacy of combined anti-OX40 and hepatitis B core viruslike particles(HBc VLPs)therapy using a mouse colon cancer model.Methods Humanized B-h OX40 mice were injected subcutaneously with MC38 colon tumor cells and treated with HBc VLPs+anti-h OX40 antibody.Tumor growth was monitored.Flow cytometric analysis was performed to evaluate the populations of T cell subsets in the tumors.Results The combination of anti-OX40 with HBc VLPs resulted in a significant delay in tumor growth,suggesting that a potent antitumor immunity was induced by the combination therapy.Further studies revealed that HBc VLPs+anti-OX40 treatment induced a significant increase in effector T cells(Teffs)and a significant decrease in regulatory T cells(Tregs)in the tumor microenvironment(TME),which accounted for the synergistic antitumor effect of anti-OX40 in combination with HBc VLPs.Conclusion Combination therapy of anti-h OX40 and HBc VLPs provides synergistic antitumor activity in colon cancer-bearing mice,which may represent a potential design strategy for cancer immunotherapy.
基金Supported by the National Science Council,No.93-2214-E-007-016 Ministry of Economic Affairs,No.93-EC-17A-17S1-0009
文摘AIM： Enterovirus 71 （EV71） has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS： The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses： Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS： Both single infection by Bac-P1-3CD and coinfection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins selfassembled into clusters of virus-like particles （VLP） resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION： Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.
文摘Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). MethodsThe gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichiapastorisunder the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualiza-tion. Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoriswith an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immuno-genicity on surface-displayed HEnAg. Conclusion The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.
文摘Summary: To prepare carboxyl terminus truncated human papillomavirus type 58 L1(HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing-HPV58L1 protein-were harvested and analyzed by SDS-PAGE and Western blot. The ProBond~TM purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope. Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virions in vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.
基金funded by the Chemical-Biological Medical System-Joint Vaccine Acquisition Program (CBMS-JVAP) as well as the Defense Threat Reduction Agency (DTRA) (CBM.VAXV.03.11.RD.009 and A151 A.41)
文摘Filoviruses are hemorrhagic fever viruses endemic to parts of Africa and the Philippines. Infection carries with it a mortality rate of up to 90% and currently there are no effective vaccines or therapeutics available to combat infection. However, the filovirus virus-like particles (VLP), which are currently under development, have been shown to be a promising vaccine candidate. They provide protection from infection in the mouse, guinea pig, and nonhuman primate models of infection, eliciting high anti-glycoprotein antibody titers and T cell responses to viral proteins. In this review, we will highlight the development of the filovirus VLP and describe the current understanding of VLP immunogenicity and correlates of protection.
基金supported by the National Science and Technology Support Program(2015BAD12B05)the China Agricultural Research System(CARS-43-8)+2 种基金the Integration and Demonstration of Key Technologies for Duck Industrial in Sichuan Province,China(2014NZ0030)the Ministry of Education Program of China(20125103110013)the Sichuan Province Research Programs,China(2013HH0042/2013 TD0015/2014-002)
文摘The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid （Opt-Cap） gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% （v/v） methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What＇s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles （VLPs）. These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.
文摘Objective.To test whether intramuscular,intranasal,intrarectal and intravagina l administration of HPV6b L 1 virus-like particlescould induce immune response in mice and to as sess whether intra-muscular and mucosal vaccination against HPV is feasible.Me thods.HPV6b L1proteins self-assembled into VLPs in Sf-9cell in vitro.Mic e were immunized on day0and21with50ìg HPV6b L1VLPs intramuscularly,int ranasally,intrarectally and intravagi-nally respectively.Sera were collected for testing IgG titer after a further7days and3months respec-tively.Results .After immunizations,all mice developed significant anti-HPV6b L1antibody titers in serum by7days after the second immunization.The titer of the serum I gG antibody against HPV6b L1VLPs in the intramuscularly immunized group was h igher than that in the intranasally,intrarectally and intravaginally immunized groups respectively,indicating that both muscular and mucosal administration of HPV6b L1VLPs can stimulate a systemic HPV-specific antibody response.Sera of the mice in the in-tramuscularly immunized group still maintained a high tit er of the serum IgG antibody against HPV6b L1VLPs 3months after the immunizat ion.Conclusion.The results demonstrated that the HPV6b L1VLPs maintain stro ng antigenicity.Immu-nization with HPV6b L1VLPs via intramuscular and mucos al routes,without adjuvant ,can elicit spe-cific antibody in sera.These fin dings suggest that the VLPs are able to induce protective antibodies.
文摘Objectives： The aim was to construct 2009 pandemic A/HINI influenza VLPs （virus-like particles） and compare the immunogenicity and protection efficacy with the commercial Panenza vaccine in BALB/c mouse model. Methods： VLPs derived from influenza A/Hong Kong/01/2009 （H 1N 1 ） virus were constructed by Bac-to-Bac baculovirus expression system. VLPs were purified by sucrose density gradient ultracentrifugation and then characterized by Western blotting analysis and transmission electron microscopy. After single dose vaccination with 3 lag of VLPs and equal amount of Panenza vaccine, the immune responses and efficacy of protection induced by VLPs were compared with those elicited by the Panenza vaccine in 6-8 weeks old female BALB/c mice. Key findings： VLPs could induce higher antibody titer as determined by hemagglutinin inhibition and microneutralization assay. Furthermore, we demonstrated that VLPs induced better antibody response to neuraminidase. In addition, VLP vaccinated mice had stronger cell-mediated immune response. As a result, our VLPs conferred 100% protection while the Panenza vaccine only conferred 67% protection. Conclusion： From the results, we concluded that influenza VLPs are highly immunogenic and they are promising to be developed as an alternative strategy to vaccine production in order to control the spread of influenza viruses.
基金The Knowledge Innovation Program of the Chinese Academy of Sciences,Grant No.KSCX2-YW-N-065the Knowledge Innovation Program of the Chinese Academy of Sciences,Grant No.KSCX2-EW-G-8+1 种基金National Key Basic Research Program(973Program),Grant No.2010CB530103National Basic Research Priorities Program of China,Grant No.2007FY210700
文摘Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family,which is widespread and causes high fatality.The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment.In this research,the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus.Under an electron microscope,Virus-Like Particles (VLPs) with various size and morphology were observed in cytoplasmic vesicles in the infected cells.Sucrose-gradient purification of the cell lysate indicated that the VLPs were mainly located in the upper fraction after ultracentrifugation,which was confirmed by Western blot analysis and immuno-electron microscopy (IEM).
基金supported by the National Science-technology Support Plan Projects 'The development of EV71 genetic engineering vaccine'[2008BAI69B02]
文摘Objective To eliminate the side effects of aluminum adjuvant and His-tag,we constructed chimeric VLPs displaying the epitope of EV71（SP70） without His-tagged.Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.Methods The fusion protein was constructed by inserting SP70 into the MIR of truncated HBc Ag sequence,expressed in E.Coli,and purified through ion exchange chromatography and density gradient centrifugation.Mice were immunized with the VLPs and sera were collected afterwards.The specific antibody titers,Ig G subtypes and neutralizing efficacy were detected by ELISA,neutralization assay,and EV71 lethal challenge.IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.Results HBc-SP70 proteins can self-assemble into empty VLPs.After immunization with HBc-SP70 VLPs,the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge.There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not.The specific Ig G subtypes were mainly IgG1 and IgG2 b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.Conclusion The fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation.In the absence of adjuvant,they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant.Furthermore,the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.
基金supported by the Special Research Fund of Ghent University and Research Foundation Flanders(FWO).CNTT is recipient of a senior postdoctoral fellowship from FWO(grant number 12V5722N).
文摘Insect-specific neurotoxic peptides derived from the venoms of scorpions and spiders can cause acute paralysis and death when injected into insects,offering a promising insecticidal component for insect pest control.However,effective delivery systems are required to help neurotoxic peptides pass through the gut barrier into the hemolymph,where they can act.Here,we investigated the potential of a novel nanocarrier,Drosophila X virus-like particle(DXV-VLP),for delivering a neurotoxin from the scorpion Androctonus australis Hector(AaIT)against the invasive pest fruit fly,Drosophila suzuki.Our results show that the fusion proteins of DXV polyproteins with AalT peptide at their Ctermini could be sufficiently produced in Lepidoptera Hi5 cells in a soluble form using the recombinant baculovirus expression system,and could self-assemble into VLPs with similar particle morphology and size to authentic DXV virions.In addition,the AalT peptides displayed on DXV-VLPs retained their toxicity,as demonstrated in injection bioassays that resulted in severe mortality(72%)in adults after 72 h.When fed to adults,mild mortality was observed in the group treated with DXV-AalT(38%),while no mortality occurred in the group treated with AaIT peptide,thus indicating the significant role of DXV-VLPs in delivering AalT peptides.Overall,this proof-of-concept study demonstrates for the first time that VLPs can be exploited to enhance oral delivery of insect-specific neurotoxic peptides in the context of pest control.Moreover,it provides insights for further improvements and potentially the development of neurotoxin-based bioinsecticides and/or transgenic crops for insect pest control.
文摘Nanoparticles are significant for veterinary vaccine development because they are safer and more effective than conventional formulations.One promising area of research involves self-assembled protein nanoparticles(SAPNs),which have shown potential for enhancing antigen-presenting cell uptake,B-cell activation,and lymph node trafficking.Numerous nanovaccines have been utilized in veterinary medicine,including natural self-assembled protein nanoparticles,rationally designed self-assembled protein nanoparticles,animal virus-derived nanoparticles,bacteriophagederived nanoparticles,and plant-derived nanoparticles,which will be discussed in this review.SAPN vaccines can produce robust cellular and humoral immune responses and have been shown to protect against various animal infectious diseases.This article attempts to summarize these diverse nanovaccine types and their recent research progress in the field of veterinary medicine.Furthermore,this paper highlights their disadvantages and methods for improving their immunogenicity.
基金This work was financially supported by the National Natural Science Foundation of China(82172085)the“Double First-Class”University project(CPU2022QZ14)the Jiangsu Provincial Natural Science Fund for Distinguished Young Scholars(BK20190028).
文摘Nature has the ingenious capability to design spiky topological features at the macro-and nanoscales,which exhibits fascinating interface adhesive properties by means of multivalent interactions.Following a biomimetic approach,such as nanoscale virus particles are highly infectious toward host cells,a range of organic and inorganic spiky particles(virus-like nanostructures)have been precisely engineered for diverse biomedical applications.Generally,organic virus-like particles(VLPs)derived from viral capsids(often termed as virosomes)have been extensively studied and reviewed,but concomitant concerns regarding immunogenicity and risks of mutagenesis limit clinical potential of organic VLPs.In contrast,inorganic VLPs(viral-mimicking topography)possess fascinating physicochemical characteristics,such as excellent electrical,optical,magnetic,mechanical and catalytic properties,which make them particularly suitable for biomedical applications.Alternatively,there is no comprehensive review related to inorganic VLPs engineered with non-viral shell for biomedical applications.Hence,in this review,we present a brief overview on inorganic VLPs,followed by summarizing the construction and properties of virus-like nanostructures,as well as the mechanisms of nano-bio interface interactions initiated by spiky topography.Furthermore,we focus on the recent advances of VLPs for biomedical applications(including biosensing,antibacterial therapy and cancer treatment).Finally,the future outlook and emerging challenges will be presented.This review aims to provide future scope of the rational design of inorganic non-viral vectors,especially with respect to gene-based therapy platforms.
基金the National Natural Science Foundation of China(No.30371317).
文摘The virus-like particle（VLPs） vaccine is an ideal HIV-1 vaccine, which can simultaneously induce a neutralizing antibody reaction and cell-mediated immunity effectively. In this study, two kinds of plasmids have been used, one can express the HIV-1 main structure proteins, Gagpol and Env, and the other contains an antibiotic gene. The two kinds of plasmids have been cotransfected into 293 cells. A stable cell line that can express Gagpol and Env proteins efficiently and lastingly has been screened. It has been confirmed that Gagpol and Env proteins in the cell culture supernatant can be self-assembled into virus-like particles. The authors have detected the secretion of VLPs in the cell medium, defined the peak of the secretion, and followed and monitored the stability of expression.
文摘Recombinant technology-based vaccines have emerged as a highly effective way to prevent a wide range of illnesses.The technology improved vaccine manufacturing,rendering it more efficient and economical.These vaccines have multiple advantages compared to conventional vaccines.The pandemic has heightened awareness of the advantages of these vaccine technologies;trust and acceptance of these vaccines are steadily growing globally.This work offers an overview of the prospects and advantages associated with recombinant vaccines.Additionally,it discusses some of the challenges likely to arise in the future.Their ability to target diverse pathogen classes underscores their contributions to preventing previously untreatable diseases(especially vector-borne and emerging diseases)and hurdles faced throughout the vaccine development process,especially in enhancing the effectiveness of these vaccines.Moreover,their compatibility with emerging vaccination platforms of the future like virus-like particles and CRISPR/Cas9 for the production of next-generation vaccines may offer many prospects.This review also reviewed the hurdles faced throughout the vaccine development process,especially in enhancing the effectiveness of these vaccines against vector-borne diseases,emerging diseases,and untreatable diseases with high mortality rates like AIDS as well as cancer.
