Characterisation and separation of infectious bursal disease virus-like particles using aqueous two-phase systems

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.

Carboxyl Terminus Truncated Human Papillomavirus Type 58 L1 Protein Maintains Its Bioactivity and Ability to Form Virus-like Particles

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.

Efficient Humoral and Cellular Immune Responses Induced by a Chimeric Virus-like Particle Displaying the Epitope of EV71 without Adjuvant

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.

The codon-optimized capsid gene of duck circovirus can be highly expressed in yeast and self-assemble into virus-like particles

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.

Construction of HIV-1 Virus-like Particle Vaccine

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.

Establishment of a Human Malignant T Lymphoma Cell Line Carrying a Retrovirus-like Particles with RT Activity

We have established an IL-2 independent malignant lymphoma line (CM-1) from peripheral T lymphocytes donated by a femalc patient with nervous systcm disease, the binlogical characteristics of CM-1 cells was studied in this paper. Another T lymphocytes,such as peripheral T lymphocytes donated by a maIe patient with multiple sclerosis, could be transformed into a malignant lymphoma line by using filtered supernatant of the CM-1 cultured medium, thus the CM-2 cell line u'as estabIished. The CM-1 and CM-2 cells were transplanted by subcutaneous inoculation into nude mice, and could cause the occurrenceof typical maIignant lymphoma. The observation of eIectron micrographs suggested the existence of virions in the CM-1 and CM-2 cells, and these virions were similar toretrovirus in the ultra-structure characteristics. lt was found that this virus possesses reverse transcriptase activity. ResuIts obtained from serological assay, molecular hybridization and PCR excluded the existence of other human viruses, which were commonly usedin our laboratory. The unknown virus possesses strong transformation activity, and probably is a new retro virus. Meanwhile, the work on the clone and sequence analysis ofthis virus are being carried out.

Construction and Production of FluorescentPapillom avirus-like Particles

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted widespread interest since it was demonstrated to be fluorescent in vivo when expressed in other organisms. In order to investigate papillomavirus life cycle which hampered by the unavailability of conventional cell culture system, we constructed a chimeric bovine papillomavirus (BPV) type 1 virus like particles (VLPs) containing GFP. It was found that fluorescent VLPs could be assembled from L2 protein in which GFP is inserted into the N terminal region of L2 (aa 88). The fluorescent VLPs could also be assembled from a GFP/L2 fusion protein in which part of the L2 sequence had been deleted. In vitro , fluorescent VLPs could bind to CV 1 cells, and this VLP/cell interaction could be analyzed by FACS assay. These results demonstrated that GFP could incorporate into BPV1 VLPs without disruption of the VLP structure. Fluorescent VLPs might be a useful tool for study of papillomavirus virus/cell interaction.

Expression,purification and characterization of enterovirus-71 virus-like particles

瞄准：Enterovirus 71 (EV71 ) 作为为在亚太区域与严重神经病学的疾病联系的手，脚和嘴疾病的最近的爆发负责的病因学的代理人被含有。方法：集会过程被假设发生经由一由病毒的朊酶 3CD 的 P1 先锋的安排解朊的处理。测试这个假设，我们构造了 3 recombinant baculoviruses：表示 P1 的 Bac-P1；表示 3CD 的 Bac-3CD；并且 Bac-P1-3CD 共同表示 P1 和 3CD。结果：由 Bac-P1-3CD 的两单个感染和由 Bac-P1 和 Bac-3CD 的合作感染导致了 P1 的正确劈开产出单个蛋白质 VP0， VP1 和 VP3，当以前的途径产出更高的 VLP 生产时。在房间，进类似于真 EV71 粒子总数的像病毒的粒子(VLP ) 的簇自我装配的结构的蛋白质。在 ultracentrifugation 纯化以后，驱散的 VLP 与在尺寸，外观，作文和表面 epitopes 的真病毒难区分，作为由标记的 SDS 页，西方的污点，传播电子显微镜学和免疫黄金决定了。结论：我们的数据第一次，在 EV71 结构的蛋白质采用一个处理和类似于脊髓灰质炎病毒汇编的汇编小径的昆虫房间建议那。粒子形态学和作文的保藏建议 VLP 可以是一个珍贵疫苗的候选人阻止 EV71 流行病。

PRODUCTION IN PICHIAPASTORISAND CHARACTERIZATION OF GENETIC ENGINEERED CHIMERIC HBV/HEV VIRUS-LIKE PARTICLES

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.

Characterization of a Putative Filovirus Vaccine:Virus-Like Particles

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.

A Virus-type Specific Serological Diagnosis of Flavivirus Infection Using Virus-like Particles

Many flaviviruses are emerging and reemerging pathogens,such as West Nile virus(WNV) ,dengue virus(DENV) ,yellow fever virus(YFV) ,and Japanese encephalitis virus. Serological assay is the dominant method for diagnosis of flavivirus infections in human. Because antibodies generated during flavivirus infections cross-react with other flavivirus members,plaque reduction neutralization test(PRNT) is the only available assay to determine the infecting flavivirus type. Since PRNT requires culturing raw viruses,it must be performed in biosafety level-3 or level-4 containment for many flaviviruses,and takes more than ten days to complete. To overcome these problems,we have developed flavivirus viral-like particles(VLPs) that could be used to replace raw viruses in the neutralization assay. The VLPs were prepared by trans packaging a luciferase-reporting replicon with viral structural proteins. This novel assay involves three simple steps:(i) VLPs from a panel of flaviviruses are incubated with flavivirus-infected sera at 37℃ for 1 h;(ii) the neutralized VLPs are used to infect Vero cells;and(iii) the infected cells are measured for luciferase activities at 22 h post-infection. The virus type whose VLP is most efficiently neutralized by the serum specimen(as quantified by the luciferase activities) is the etiologic agent. As a proof-of-concept,we show that a WNV-infected mouse serum neutralized the WNV VLP more efficiently and selectively than the DENV and YFV VLPs. Our results demonstrate that the VLP neutralization assay maintains the "gold standard" of the classic PRNT;importantly,it shortens the assay time from >10 days to <1 day,and can be performed in biosafety level-2 facility.

Production of CCHF Virus-Like Particle by a Baculovirus-Insect Cell Expression System

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).

HPV 6b L1VIRUS-LIKE PARTICLES ELICIT HUMORAL IMMUNITY IN MICE

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.

Preparation and immunoprotective effects of a virus-like particle candidate vaccine of the dominant epidemic D3 genotype coxsackievirus A6 in China

Coxsackievirus A6 of the D3a genotype(CVA6 D3a)is a primary pathogen causingmainland of China's hand,foot,and mouth disease(HFMD).Viral‐like particle(VLP)vaccines represent a potential candidate vaccine to prevent HFMD.This study collected Anti‐CVA6 D3a VLPs serum from BALB/c female mice immunized using CVA6 D3a VLPs.The neutralizing antibody levels were compared against the representative 14‐JX2018(D3a)and N4‐YN2015(D3b)strains between the antisera of different immune pathways.The immunoprotective effect of anti‐CVA6 D3a VLPs against these strains was monitored using pathological sections and immuno-histochemical results of lung and skeletal muscle tissues in seven‐day‐old Institute of Cancer Research(ICR)mice.Immunological protection against different branches of viruses was evaluated in 7‐day‐old(serum passive immune protection)and 14‐day‐old(VLPs active immune protection)neonatal ICR mice models.Serum‐neutralizing antibody levels were positively correlated with the number of immunizations and higher against 14‐JX2018 than against N4‐YN2015.Furthermore,these levels were significantly higher with abdominal injection than intramuscular injection.The immunized serum of 7‐day‐old ICR mice inoculated three times was 100%protected against CVA6 D3a 14‐JX2018(lethal titer:106.25 TCID 50)and CVA6 D3b N4‐YN2015(lethal titer:105.25TCID 50)fatal attacks,respectively.For ICR mice that have completed two active immunizations for 14 days,both CVA6 D3a 14‐JX2015(challenge titer:108.25 TCID 50)and CVA6 D3b N4‐YN2015(challenge titer:107.25 TCID 50)were used for the challenge,and the mice were able to survive.Overall,the CVA6 D3a VLPs prepared in this study are a potential vaccine candidate for CVA6,as it has the optimal protective effect against both CVA6 D3a and D3b evolutionary branches viruses.

Development of a novel virus-like particle-based vaccine for preventing tick-borne encephalitis virus infection

Tick-borne encephalitis virus(TBEV)is an important tick-borne pathogen that poses as a serious public health concern.The coverage and immunogenicity of the currently available vaccines against TBEV are relatively low;therefore,it is crucial to develop novel and effective vaccines against TBEV.The present study describes a novel strategy for the assembly of virus-like particles(VLPs)by co-expressing the structural(core/prM/E)and non-structural(NS2B/NS3Pro)proteins of TBEV.The efficacy of the VLPs was subsequently evaluated in C57BL/6 mice,and the resultant IgG serum could neutralize both Far-Eastern and European subtypes of TBEV.These findings indicated that the VLP-based vaccine elicited the production of cross-subtype reactive antibodies.The VLPs provided protection to mice lacking the type I interferon receptor(IFNAR^(-/-))against lethal TBEV challenge,with undetectable viral load in brain and intestinal tissues.Furthermore,the group that received the VLP vaccine did not exhibit significant pathological changes and the inflammatory factors were significantly suppressed compared to the control group.Immunization with the VLP vaccine induced the production of multiple-cytokine-producing antiviral CD4+T cells in vivo,including TNF-α^(+),IL-2^(+),and IFN-γ^(+)T cells.Altogether,the findings suggest that noninfectious VLPs can serve as a potentially safe and effective vaccine candidate against diverse subtypes of TBEV.

An RBD virus-like particle vaccine for SARS-CoV-2 induces cross-variant antibody responses in mice and macaques

Dear Editor,Since early 2020,severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has spread globally infecting over 500 million people and causing over 6 million deaths.The massive worldwide immunisation programmes and the arising clinical trial data present a unique opportunity to compare the vaccines and various delivery platforms(i.e.viral vector platform,mRNA platform and virus-like particle platform).

Chimeric Newcastle Disease Virus-like Particles Containing DC-Binding Peptide-Fused Haemagglutinin Protect Chickens from Virulent Newcastle Disease Virus and H9N2 Avian Influenza Virus Challenge

Newcastle disease virus(NDV)and H9N2 subtype Avian influenza virus(AIV)are two notorious avian respiratory pathogens that cause great losses in the poultry industry.Current inactivated commercial vaccines against NDV and AIV have the disadvantages of inadequate mucosal responses,while an attenuated live vaccine bears the risk of mutation.Dendritic cell(DC)targeting strategies are attractive for their potent mucosal and adaptive immune-stimulating ability against respiratory pathogens.In this study,DC-binding peptide(DCpep)-decorated chimeric virus-like particles(cVLPs),containing NDV haemagglutinin–neuraminidase(HN)and AIV haemagglutinin(HA),were developed as a DC-targeting mucosal vaccine candidate.DCpep-decorated cVLPs activated DCs in vitro,and induced potent immune stimulation in chickens,with enhanced secretory immunoglobulin A(sIgA)secretion and splenic T cell differentiation.40μg cVLPs can provide full protection against the challenge with homologous,heterologous NDV strains,and AIV H9N2.In addition,DCpep-decorated cVLPs could induce a better immune response when administered intranasally than intramuscularly,as indicated by robust s IgA secretion and a reduced virus shedding period.Taken together,this chimericVLPs are a promising vaccine candidate to control NDV and AIV H9N2 and a useful platform bearing multivalent antigens.

Induction of virus-neutralizing antibodies and T cell responses by dengue virus type 1 virus-like particles prepared from Pichia pastoris

Background Dengue is currently a significant global health problem but no vaccines are available against the four dengue serotypes virus infections. The development of safe and effective vaccines has been hampered by the requirement of conferring complete protection against all four dengue serotypes and the lack of a convenient animal model. Virus-like particles （VLPs） have emerged as a promising subunit vaccine candidate. One strategy of vaccine development is to produce a tetravalent dengue subunit vaccine by mixing recombinant VLPs, corresponding to all four dengue virus serotypes. Towards this end, this study aimed to establish a Pichia pastoris （P. pastoris） expression system for production of dengue virus type 1 （DENV-1） VLPs and evaluate the humoral and cellular immune response of this particle in mice. Methods A recombinant yeast P. pastoris clone containing prM and E genes of DENV-1 was constructed and DENV-1 VLPs expressed by this clone were analyzed by sucrose density gradient centrifugation, Western blotting, and transmission electron microscope. Groups of mice were immunized by these particles plus adjuvant formulations, then mice were tested by ELISA and neutralization assay for humoral immune response, and by lymphocyte proliferation and cytokine production assays for a cellular immune response. Results Our data demonstrated that recombinant DENV-1 VLPs consisting of prM and E protein were successfully expressed in the yeast P. pastoris. Sera of VLPs immunized mice were shown to contain a high-titer of antibodies and the neutralization assay suggested that those antibodies neutralized virus infection in vitro. Data from the T lymphocyte proliferation assay showed proliferation of T cell, and ELISA found elevated secretion levels of interferon IFN-γ and IL-4. Conclusions P. pastoris-expressed DENV-1 VLPs can induce virus neutralizing antibodies and T cell responses in immunized mice. Using P. pastoris to produce VLPs offers a promising and economic strategy for dengue virus vaccine development.

Virus-like particle vaccinology,from bench to bedside

Virus-like particles(VLPs)have become key tools in biology,medicine and even engineering.After their initial use to resolve viral structures at the atomic level,VLPs were rapidly harnessed to develop antiviral vaccines followed by their use as display platforms to generate any kind of vaccine.Most recently,VLPs have been employed as nanomachines to deliver pharmaceutically active products to specific sites and into specific cells in the body.Here,we focus on the use of VLPs for the development of vaccines with broad fields of indications ranging from classical vaccines against viruses to therapeutic vaccines against chronic inflammation,pain,allergy and cancer.In this review,we take a walk through time,starting with the latest developments in experimental preclinical VLP-based vaccines and ending with marketed vaccines,which earn billions of dollars every year,paving the way for the next wave of prophylactic and therapeutic vaccines already visible on the horizon.