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.

Anti-OX40 Antibody Combined with HBc VLPs Delays Tumor Growth in a Mouse Colon Cancer Model

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.

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。

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.

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.

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.

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).

Inorganic virus-like nanoparticles for biomedical applications:a minireview

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.

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.

铝佐剂对HBoV1 VP2 VLPs诱导小鼠免疫应答的影响

目的:探讨铝佐剂对HBoV1 VP2 VLPs诱导小鼠免疫应答的影响。方法:BABL/c小鼠随机分为VLPs实验组、明矾佐剂实验组、PBS对照组和明矾佐剂对照组。实验组小鼠分别采用HBoV1 VP2 VLPs和HBoV1 VP2 VLPs加明矾佐剂肌肉注射免疫,对照组小鼠同期注射等量明矾佐剂或PBS缓冲液;实验8周后ELISA检测两实验组特异性Ig G抗体效价和活性,ELIspot检测两实验组特异性细胞免疫反应强度,从细胞免疫和体液免疫强度探讨铝佐剂对HBoV1 VP2 VLPs诱导小鼠免疫应答的影响。结果:明矾佐剂降低HBoV1 VP2 VLPs诱导细胞免疫反应强度(P<0.001),增强HBoV1 VP2 VLPs诱导的血清Ig G效价(P<0.01)和Ig G活性(P<0.05)。结论:明矾佐剂使HBoV1 VP2 VLPs诱导的体液免疫反应增强而细胞免疫反应减弱。HBoV1 VP2 VLPs作为预防性疫苗使用时应添加明矾佐剂,作为治疗性疫苗使用时应不加明矾佐剂。

肠道病毒EV71新型VLPs疫苗的制备及鉴定

目的制备肠道病毒71型(EV71)的新型病毒样颗粒(VLPs)疫苗,为手足口病防治奠定基础。方法应用分子克隆技术构建外壳蛋白VP1融合基因cVP1,免疫细胞化学及Western Blot测定该融合基因在HeLa细胞表达情况;将cVP1克隆于杆状病毒表达载体pFastbac1,应用昆虫细胞TN5制备重组杆状病毒cVP1 rBV,再将此重组病毒与本室保存的gag rBV以不同MOI比例共感染昆虫细胞TN5,得到含有VP1的重组嵌合病毒样颗粒cVP1 VLPs,最后以Westem Blot及电镜进行鉴定。结果免疫细胞化学及Western Blot结果显示构建的融合基因cVP1可在真核细胞内表达,并成功制备成cVP1 rBV,该重组杆状病毒和gag rBV共感染昆虫细胞制备的VLPs结构完整。结论成功制备了EV71的新型VLPs疫苗,为后续研究打下了基础。

促吞噬肽功能化的HBc VLPs纳米载体的制备

促吞噬肽(Tuftsin)是机体脾组织产生的生理活性肽,具有强大的免疫调节和免疫治疗潜力。乙型肝炎病毒核心蛋白病毒样颗粒(hepatitis B virus core protein virus-like particles, HBc VLPs)是由HBc自组装形成的空心纳米颗粒,其不仅能应用于药物的递送,还能应用于外源蛋白质的显示。因此,Tuftsin功能化HBc VLPs载体的研究在免疫治疗、分子递送等方面具有重要意义。本研究选用PET43.1-a质粒作为Tuftsin-HBc VLP的表达载体,以大肠杆菌BL21 (DE3)为工程菌进行诱导表达,通过盐析、分子筛层析和离子交换层析技术纯化生产Tuftsin-HBc VLP。利用Western印迹和ELISA分别对Tuftsin-HBc VLP上HBc和Tuftsin进行定性分析。结果显示,Tuftsin-HBc VLP可与抗HBc抗体和抗Tuftsin抗体发生特异性结合。透射电镜观察结果显示,Tuftsin-HBc VLP呈大小均一的球形结构,粒度分析仪测得Tuftsin-HBc VLP的直径约为30 nm。CCK8法显示,Tuftsin-HBc VLP在0~480μg/mL范围内,细胞增殖未见显著变化。上述结果表明,本研究成功制备了可以在原核系统中高效表达且安全有效的Tuftsin-HBc VLP生物纳米递送载体。

密码子优化的RHDV2双VP60基因重组杆状病毒构建及表达蛋白免疫原性分析

[目的]试验旨在优化兔出血症病毒2型(Rabbit hemorrhagic disease virus 2,RHDV2)病毒样颗粒(virus-like particle, VLP)疫苗的制备策略,探究RHDV2 VLP疫苗对家兔的免疫原性,为低成本、高产量RHDV2新型疫苗研发提供新思路。[方法]根据昆虫细胞的密码子偏好性优化合成RHDV2 VP60全基因,将双VP60基因插入真核载体pFastBacTMDual,转化携带Bacmid质粒的大肠杆菌DH10Bac感受态细胞,构建含双VP60基因的重组杆粒Bacmid-VP60-VP60,转染Sf9昆虫细胞,通过Western blotting、间接免疫荧光试验(IFA)及透射电镜对重组杆状病毒Bacmid-VP60-VP60进行表达验证;将优化策略制备的重组蛋白抗原与氢氧化铝佐剂按照9∶1比例制备VLP灭活疫苗,通过安全性检验、最小免疫剂量、免疫持续期等评估优化策略制备的RHDV2 VLP疫苗的保护效果。[结果]试验成功构建重组杆粒Bacmid-VP60-VP60。Western blotting鉴定结果显示,重组杆状病毒转染Sf9细胞表达出大小约60 ku的RHDV2 VP60蛋白。IFA鉴定结果显示,感染重组杆状病毒的Sf9细胞产生了大量的黄绿色荧光,表明重组杆状病毒在Sf9细胞中大量表达VP60蛋白。透射电镜观察结果显示,VP60蛋白折叠成VLP,大小为40 nm左右,呈现球形结构,表面光滑。优化策略制备的RHDV2 VLP疫苗对家兔具有良好的安全性和免疫原性,最小免疫剂量为0.5 mL/只,免疫持续期可达210 d以上。[结论]试验构建了含有密码子优化的双VP60基因的重组杆状病毒,并在昆虫细胞中成功表达RHDV2 VP60蛋白,该蛋白制备的VLP疫苗对家兔具有良好的免疫原性。

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.

Monoclonal neutralizing antibodies against EV71 screened from mice immunized with yeast-produced virus-like particles

Periodic outbreaks of hand, foot and mouth disease(HFMD) occur in children under 5 years old, and can cause death in some cases. The C4 strain of enterovirus 71(EV71) is the main pathogen that causes HFMD in China. Although no drugs against EV71 are available, some studies have shown that candidate vaccines or viral capsid proteins can produce anti-EV71 immunity. In this study, female BABL/c mice(6–8 weeks old) were immunized with virus-like particles(VLPs) of EV71 produced in yeast to screen for anti-EV71 antibodies. Two hybridomas that could produce neutralizing antibodies against EV71 were obtained. Both neutralizing m Abs(D4 and G12) were confirmed to bind the VP1 capsid protein of EV71, and could protect > 95% cells from 100 TCID50 EV71 infection at 25 μg/m L solution(lowest concentration). Those two neutralizing m Abs identified in the study may be promising candidates in development for m Abs to treat EV71 infection, and utilized as suitable reagents for use in diagnostic tests and biological studies.

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.

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.

Zika Virus Baculovirus-Expressed Virus-Like Particles Induce Neutralizing Antibodies in Mice

The newly emerged mosquito-borne Zika virus(ZIKV) strains pose a global challenge owing to its ability to cause microcephaly and neurological disorders. Several ZIKV vaccine candidates have been proposed, including inactivated and live attenuated virus vaccines, vector-based vaccines, DNA and RNA vaccines. These have been shown to be efficacious in preclinical studies in mice and nonhuman primates, but their use will potentially be a threat to immunocompromised individuals and pregnant women. Virus-like particles(VLPs) are empty particles composed merely of viral proteins, which can serve as a safe and valuable tool for clinical prevention and treatment strategies. In this study, we used a new strategy to produce ZIKV VLPs based on the baculovirus expression system and demonstrated the feasibility of their use as a vaccine candidate. The pre-membrane(prM) and envelope(E) proteins were co-expressed in insect cells and selfassembled into particles similar to ZIKV. We found that the ZIKV VLPs could be quickly and easily prepared in large quantities using this system. The VLPs were shown to have good immunogenicity in immunized mice, as they stimulated high levels of virus neutralizing antibody titers, ZIKV-specific IgG titers and potent memory T cell responses. Thus, the baculovirus-based ZIKV VLP vaccine is a safe, effective and economical vaccine candidate for use against ZIKV.

Design and immunogenicity assessment of HIV-1 virus-like particles as a candidate vaccine

The rapid growth of the global HIV/AIDS epidemic makes it a high priority to develop an effective vaccine.Since a live attenuated or inactivated HIV vaccine is not likely to be approved for clinical application due to safety concerns,HIV virus like particles(VLPs) offer an attractive alternative because they are considered safer since they lack viral genome.We got a stable eukaryotic cell line by G418 resistance selection,engineered to express the HIV-1 structure protein Gag and Env efficiently and stably.We confirmed the presence of Gag and Env proteins in the cell culture supernatant and that they could self-assemble into VLPs.These VLPs were found to be able to elicit specific humoral and cellular immune response after immunization without any adjuvant.

Fabrication of virus-like particles with strip-pattern surface：A two-step self-assembly approach

Spherical nanostructures with striped patterns on the surfaces resembling the essential structures of natural virus particles were constructed through a two-step self-assembly approach of polystyrene-boligo（acrylic acid）（PS-b-oligo-AA） and poly（γ-benzyI L-glutamate）-b-poly（ethylene glycol）（PBLG-bPEG） copolymer mixtures in solution.On the basis of difference in hydrophilicity and self-assembly properties of the two copolymers,the two-step self-assembly process is realized.It was found that PS-boligo-AA copolymers formed spherical aggregates by adding a certain amount of water into polymer solutions in the first step.In the second step,two polymer solutions were mixed and water was further added,inducing the self-assembly of PBLG-b-PEG on the surfaces of PS-b-oligo-AA spheres to form striped patterns.In-depth study was conducted for the indispensable defects of striped patterns which are dislocations and ＋1/2 disclinations.The influencing factors such as the mixing ratio of two copolymers and the added water content in the first step on the morphology and defects of the striped patterns were investigated.This work not only presents an idea to interpret mechanism of the cooperative self-assembly behavior,but also provides an effective approach to construct virus-like particles and other complex structures with controllable morphology.