Pandemic A/HIN1 2009 Influenza Virus-like Particles Elicited Higher and Broader Immune Responses than the Commercial Panenza Vaccine

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.

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.

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.

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

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.

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.

Strengthening global health resilience:Marburg virus-like particle vaccines and the One Health approach

The Marburg virus(MARV),belonging to the Filoviridae family,poses a significant global health threat,emphasizing the urgency to develop Marburg virus-like particle(VLP)vaccines for outbreak mitigation.The virus's menacing traits accentuate the need for such vaccines,which can be addressed by VLPs that mimic its structure safely,potentially overcoming past limitations.Early Marburg vaccine endeavors and their challenges are examined in the historical perspectives section,followed by an exploration of VLPs as transformative tools,capable of eliciting immune responses without conventional risks.Noteworthy milestones and achievements in Marburg VLP vaccine development,seen through preclinical and clinical trials,indicate potential cross-protection.Ongoing challenges,encompassing durability,strain diversity,and equitable distribution,are addressed,with proposed innovations like novel adjuvant,mRNA technology,and structure-based design poised to enhance Marburg VLP vaccines.This review highlights the transformative potential of Marburg VLPs in countering the virus,showcasing global collaboration,regulatory roles,and health equity for a safer future through the harmonious interplay of science,regulation,and global efforts.

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.

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.

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.

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.

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.

禽流感病毒样颗粒疫苗研究进展

禽流感是由禽流感病毒(Avian influenza virus,AIV)感染引起的一种禽类传染病,给家禽业造成了巨大的经济损失。AIV因抗原漂移和抗原转换可能会产生新的变异毒株和亚型,因此禽流感是在世界范围内需要被重点关注的禽类传染病。病毒样颗粒(virus-like particles,VLPs)能自组装成类似天然病毒结构的蛋白颗粒,易被免疫系统识别,但无遗传物质,安全性较高,是疫苗制备的新方向。禽流感VLPs是基于AIV蛋白模拟禽流感病毒外表面而构成的蛋白颗粒,具有很强的免疫原性,用其制成的疫苗具有无传染性、稳定和不易失活等优势,是一种安全性较高的疫苗类型。笔者就禽流感VLPs疫苗的研发基础、与疫苗有关的结构蛋白及疫苗的通用性和优缺点进行综述,以期对未来禽流感疫苗的研发和防控提供理论参考。

Virus like particle-based vaccines against emerging infectious disease viruses

Emerging infectious diseases are major threats to human health.Most severe viral disease outbreaks occur in developing regions where health conditions are poor.With increased international travel and business,the possibility of eventually transmitting infectious viruses between different countries is increasing.The most effective approach in preventing viral diseases is vaccination.However,vaccines are not currently available for numerous viral diseases.Viruslike particles(VLPs) are engineered vaccine candidates that have been studied for decades.VLPs are constructed by viral protein expression in various expression systems that promote the selfassembly of proteins into structures resembling virus particles.VLPs have antigenicity similar to that of the native virus,but are non-infectious as they lack key viral genetic material.VLP vaccines have attracted considerable research interest because they offer several advantages over traditional vaccines.Studies have shown that VLP vaccines can stimulate both humoral and cellular immune responses,which may offer effective antiviral protection.Here we review recent developments with VLP-based vaccines for several highly virulent emerging or re-emerging infectious diseases.The infectious agents discussed include RNA viruses from different virus families,such as the Arenaviridae,Bunyaviridae,Caliciviridae,Coronaviridae,Filoviridae,Flaviviridae,Orthomyxoviridae,Paramyxoviridae,and Togaviridae families.

Immunogenicity and virus-like particle formation of rotavirus capsid proteins produced in transgenic plants

The human pathogen, group A rotavirus, is the most prevalent cause of acute infantile and pediatric gastroenteritis worldwide, especially in developing countries. There is an urgent demand for safer, more effective and cheaper vaccines against rotavirus infection. Plant-derived antigens may provide an exclusive way to produce economical subunit vaccines. Virus-like particles, constituting viral capsid proteins without viral nucleic acids, are considered a far safer candidate compared with live attenuated viral vaccines. In this study, the rotavirus capsid proteins VP2, VP6 and VP7 were co-expressed in transgenic tobacco plants, and their expression levels, formation of rotavirus-like particles (RV VLPs) and immunogenicity were extensively studied. Quantitative real-time RT-PCR and Western blot analysis revealed that the expression level of vp6 was the highest while vp7 was expressed at the lowest levels. The RV VLPs were purified from transgenic tobacco plants and analyzed by electron microscopy and Western blot. Results indicated that the plant-derived VP2, VP6 and VP7 proteins self-assembled into 2/6 or 2/6/7 RV VLPs with a diameter of 60-80 nm. When orally delivered into mice with cholera toxin as an adjuvant, the total soluble protein extracted from transgenic tobacco plants induced rotavirus-specific antibodies comparable with those of attenuated rotavirus vaccines, while VP 2/6/7 induced higher serum IgG and fecal IgA titers compared with VP 2/6.

流感病毒样颗粒疫苗研究进展

疫苗接种是预防各种类型流行性感冒的理想方式,但流感病毒血清亚型多,变异性强,给流感疫苗的研发带来一定的挑战。病毒样颗粒(VLP)是含有某种病毒一个或多个结构蛋白的空心颗粒,形态结构上类似完整病毒,具有与完整病毒相似的免疫原性,并通过激活抗原提呈细胞,诱导免疫应答。VLP不含有病毒核酸,不能自主复制,因此无致病性,在疫苗研发中具有广泛的应用前景。现已有H1N1、H3N2、H5N1、H7N1、H5N3及H9N2型流感VLP合成的报道,动物实验证明这些VLP可诱导产生保护性免疫应答。数种二价(多价)混合流感VLP也被合成并证实在不同亚型流感病毒间具有交叉保护作用。佐剂的使用能够增强流感VLP疫苗的免疫原性。目前的研究表明流感VLP疫苗具有高免疫效价、能有效诱导交叉免疫应答和安全性高等特点,但现有研究大多是动物实验和临床前实验。流感VLP疫苗的安全性、效价及免疫途径等方面还有待进一步的研究及临床实验的验证。本文就近年来流感病毒样颗粒疫苗的研究进展,从流感VLP的合成和释放、免疫机制、已合成的流感VLP、佐剂的使用等方面进行综述。

From Monovalent to Multivalent Vaccines, the Exploration for Potential Preventive Strategies Against Hand, Foot, and Mouth Disease(HFMD)

Hand,foot,and mouth disease(HFMD)recently emerged as a global public threat.The licensure of inactivated enterovirus A71(EV-A71)vaccine was the first step in using a vaccine to control HFMD.New challenges arise from changes in the pathogen spectrum while vaccines directed against other common serotypes are in the preclinical stage.The mission of a broad-spectrum prevention strategy clearly favors multivalent vaccines.The development of multivalent vaccines was attempted via the simple combination of potent monovalent vaccines or the construction of chimeric vaccines comprised of epitopes derived from different virus serotypes.The present review summarizes recent advances in HFMD vaccine development and discusses the next steps toward a safe and effective HFMD vaccine that is capable of establishing a crossprotective antibody response.

Orthogonal modular biosynthesis of nanoscale conjugate vaccines for vaccination against infection

Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases.Although nanotechnology has been greatly applied in the field of vaccines,it is seldom used for conjugate vaccine research because it is very difficult to connect polysaccharides and nanocarriers.In this work,an orthogonal and modular biosynthesis method was used to produce nanoconjugate vaccines using the SpyTag/SpyCatcher system.When SpyTag/SpyCatcher system is combined with protein glycosylation technology,bacterial O-polysaccharide obtained from Shigela flexneri 2a can be conjugated onto the surfaces of different virus-like particles(VLPs)in a biocompatible and controlled manner.After confirming the excellent lymph node targeting and humoral immune activation abilities,these nanoconjugate vaccines further induced efficient prophylactic effects against infection in a mouse model.These results demonstrated that natural polysaccharide antigens can be easily connected to VLPs to prepare highly efficient nanoconjugate vaccines.To the best of the researchers1 knowledge,this is the first time VLP-based nanoconjugate vaccines are produced efficiently,and this strategy could be applied to develop various pathogenic nanoconjugate vaccines.

Phage display creates innovative applications to combat hepatitis B virus

Hepatitis B virus (HBV) has killed countless lives in human history. The invention of HBV vaccines in the 20th century has reduced significantly the rate of the viral infection. However, currently there is no effective treatment for chronic HBV carriers. Newly emerging vaccine escape mutants and drug resistant strains have complicated the viral eradication program. The entire world is now facing a new threat of HBV and human immunodeficiency virus co-infection. Could phage display provide solutions to these life-threatening problems? This article reviews critically and comprehensively the innovative and potential applications of phage display in the development of vaccines, therapeutic agents, diagnostic reagents, as well as gene and drug delivery systems to combat HBV. The application of phage display in epitope mapping of HBV antigens is also discussed in detail. Although this review mainly focuses on HBV, the innovative applications of phage display could also be extended to other infectious diseases.

流感疫苗脂质体的制备方法及粒径对免疫原性的影响

目的 采用薄膜分散法和冻融冻干法制备四价流感疫苗脂质体冻干粉,通过考察其粒径、包封率和免疫原性,筛选最佳制备方法;使用最佳制备方法制备不同粒径流感疫苗脂质体,比较包封率和免疫原性,筛选最优脂质体粒径。方法 采用Lowry法测定脂质体包封率;腹腔免疫小鼠,通过脾淋巴增殖实验以刺激指数(SI)为指标进行细胞免疫原性研究;采用血凝抑制实验以免后与免前抗体滴度比(HI)为指标,评价体液免疫原性。结果 冻融冻干法是制备流感疫苗脂质体冻干粉的最佳方法,以最佳方法制备出平均粒径为3.7μm、2.0μm、1.0μm、0.45μm的4种不同粒径脂质体冻干粉,在一个免疫周期内,各实验组能显著刺激脾淋巴细胞增殖,产生细胞免疫,且3.7μm组与其他各组比较差异有统计学意义(P <0.05);各免疫组免后与免前抗体滴度比始终≥4,且3.7μm组高于其他组,表明其能产生较强的体液免疫。结论 冻融冻干法制备的四价流感疫苗脂质体能产生较好的免疫原性,其中以粒径3.7μm的流感疫苗脂质体免疫增强效果最佳。