Rapid and Sparse Labeling of Neurons Based on the Mutant Virus-Like Particle of Semliki Forest Virus / 神经科学通报·英文版

Sparse labeling of neurons contributes to uncovering their morphology, and rapid expression of a fluorescent protein reduces the experiment range. To achieve the goal of rapid and sparse labeling of neurons in vivo, we established a rapid method for depicting the fine structure of neurons at 24 h post-infection based on a mutant virus-like particle of Semliki Forest virus. Approximately 0.014 fluorescent focus-forming units of the mutant virus-like particle transferred enhanced green fluorescent protein into neurons in vivo, and its affinity for neurons in vivo was stronger than for neurons in vitro and BHK21 (baby hamster kidney) cells. Collectively, the mutant virus-like particle provides a robust and convenient way to reveal the fine structure of neurons and is expected to be a helper virus for combining with other tools to determine their connectivity. Our work adds a new tool to the approaches for rapid and sparse labeling of neurons in vivo.

Virus-like Particle Vaccine Containing Toxoplasma gondii Rhoptry Protein 13 Induces Protection against T. gondii ME49 Infection in Mice

Toxoplasma gondii can infect humans worldwide, causing serious diseases in pregnant women and immunocompromised individuals. T. gondii rhoptry protein 13 (ROP13) is known as one of the key proteins involved in host cell invasion. In this study, we generated virus-like particles (VLPs) vaccine expressing T. gondii rhoptry ROP13 and investigated VLPs vaccine efficacy in mice. Mice immunized with ROP13 VLPs vaccine elicited significantly higher levels of T. gondii-specific IgG, IgG1, IgG2a, and IgA antibody responses following boost immunization and challenge infection, whereas antibody inductions were insignificant upon prime immunization. Differing immunization routes resulted in differing antibody induction, as intranasal immunization (IN) induced greater antibody responses than intramuscular immunization (IM) after boost and challenge infection. IN immunization induced significantly higher levels of IgG and IgA antibody responses from feces, antibody-secreting cells (ASCs), CD4⁺ T, CD8⁺ T cells and germinal center B cell responses in the spleen compared to IM immunization. Compared to IM immunization, IN immunization resulted in significantly reduced cyst counts in the brain as well as lesser body weight loss, which contributed to better protection. All of the mice immunized through either route survived, whereas all naïve control mice perished. These results indicate that the ROP13 VLPs vaccine could be a potential vaccine candidate against T. gondii infection.

Advance in human papillomavirus major capsid protein L1-based vaccines / 中华微生物学和免疫学杂志

L1 is the major capsid protein of human papillomavirus (HPV) encoded by late gene 1. Based on the fact that L1 can self-assemble into virus like particle (VLP) with good immunogenicity, it has aroused wide concern in studying the pathogenesis of and vaccines against HPV. Nevertheless, there are a few limitations of present L1-based HPV vaccines. For instance, low expression of the protein and the complexity of purification result in the relatively low yield of vaccines. Type-specific antibody induced by L1 also results in the unsatisfactory cross-protection rate. Furthermore, there is no reported therapeutic effect against HPV-related diseases because of its undefined role in virus eliminating. This review focused on the structure, immunogenicity and role in immune response of L1 and the development of and latest progress in HPV vaccines.

Advance in human papillomavirus major capsid protein L1-based vaccines / 中华微生物学和免疫学杂志

L1 is the major capsid protein of human papillomavirus (HPV) encoded by late gene 1. Based on the fact that L1 can self-assemble into virus like particle (VLP) with good immunogenicity, it has aroused wide concern in studying the pathogenesis of and vaccines against HPV. Nevertheless, there are a few limitations of present L1-based HPV vaccines. For instance, low expression of the protein and the com-plexity of purification result in the relatively low yield of vaccines. Type-specific antibody induced by L1 also results in the unsatisfactory cross-protection rate. Furthermore, there is no reported therapeutic effect against HPV-related diseases because of its undefined role in virus eliminating. This review focused on the struc-ture, immunogenicity and role in immune response of L1 and the development of and latest progress in HPV vaccines.

Virus-Like Particles Expressing Toxoplasma gondii Rhoptry Protein 18 Induces Better Protection Than Rhoptry Protein 4 against T. gondii Infection

Toxoplasma gondii is a ubiquitous protozoan parasite responsible for causing toxoplasmosis. Preventive measures for toxoplasmosis are currently lacking and as such, development of novel vaccines are of urgent need. In this study, we generated 2 virus-like particles (VLPs) vaccines expressing T. gondii rhoptry protein 4 (ROP4) or rhoptry protein 18 (ROP18) using influenza matrix protein (M1) as a core protein. Mice were intranasally immunized with VLPs vaccines and after the last immunization, mice were challenged with ME49 cysts. Protective efficacy was assessed and compared by determining serum antibody responses, body weight changes and the reduction of cyst counts in the brain. ROP18 VLPs-immunized mice induced greater levels of IgG and IgA antibody responses than those immunized with ROP4 VLPs. ROP18 VLPs immunization significantly reduced body weight loss and the number of brain cysts in mice compared to ROP4 VLPs post-challenge. These results indicate that T. gondii ROP18 VLPs elicited better protective efficacy than ROP4 VLPs, providing important insight into vaccine design strategy.

Progress in developing Norovirus vaccines based on virus-like particles / 中华微生物学和免疫学杂志

Human norovirus (NoV) is the major cause of human acute non-bacterial gastroenteritis worldwide. The development of NoV vaccines is limited by the inability to culture cells in vitro and the lack of small animal models. Thus, traditional methods used to prepare live attenuated vaccines are not suitable for preparing NoV vaccines. Subunit vaccines against NoV infection, especially virus-like particle (VLP)-based vaccines, have outstanding advantages in the research and development of NoV vaccines. In this review, we focused on reviewing recent advances in the fields of NoV VLP-based vaccines and the development of NoV VLP-based vaccines using different expression systems as well as identifying the research direction for NoV VLP-based vaccines.

Evaluation of neutralizing antibodies in serum immunized with virus-like particle chimerized HCV series of neutralizing epitopes / 中国免疫学杂志

Objective:New Zealand rabbits were immunized with VLPs-MEpS,VLPs-E2S,and the levels of neutralizing antibodies in serum were determined.Methods: New Zealand rabbits were immunized with 10 μg VLPs-MEpS and VLPs-E2S,serum was collected at diffferent time with a two-weeks interval.The neutralizing antibodies were determined by ELISA.HCV(type 1b) had been prepared and mixed with serum from immunized rabbit before infected Huh7.5 cell.The protection of neutralizing antibodies in serum was assessed.Results: Neutralizing antibodies had been induced in rabbit after immunized with VLPs-MEpS and VLPs-E2S.VLPs-MEpS group had higher titer of antibodies than that of VLPs-E2S group(P<0.05),both group had higher titer of antibodies than that of control groups significantly(P<0.01).VLPs-MEpS group had higher neutralization than that of VLPs-E2S group(P<0.05),the highest neutralization rate was 61.49%.Both groups were higher than control group notably(P<0.01).Conclusion: Protective neutralizing antibodies have been induced in New Zealand rabbit after immunized with VLPs-MEpS and VLPs-E2S.It′s the basement for development of neutralizing antibodies vaccine.

Development of a double antibody sandwich ELISA for detection of Chikungunya virus antigen / 中华实验和临床病毒学杂志

Objective@#To establish a method for detection of chikungunya virus(CHIKV) antigen.@*Methods@#CHIKV virus like particle(VLP), that contains all structural proteins, was prepared by baculovirus expression system. Mice and rabbits were immunized with the VLP to develop antibodies against CHIKV. A double antibody sandwich ELISA was established for detection of CHIKV antigens. The concentrations of the antibodies used and the reaction conditions were optimized. The detection limit and repeatability of the ELISA was evaluated.@*Results@#The sensitivity and specificity was estimated by 10 mimicking CHIKV sera, 90 health person sera, 40 other virus infected sera. It was show that the specificity of DAS-ELISA was 100%, the detection limit was 10 TCID50, the coefficients of variation (CV) within plate was <5%, the CV of different plates was <10%.@*Conclusions@#The double antibody sandwich ELISA established in this study can be used to detect the CHIKV antigen in acute phase serum of patient and provide a method for detection of CHIKV.

Preparation and immunogenicity of influenza virus-like particles using nitrocellulose membrane filtration

PURPOSE: Nitrocellulose membrane–based filtration system (NCFS) is widely used for protein concentration. In this study, we applied NCFS for production of virus-like particle (VLP) as a vaccine candidate and evaluated yield property and immunogenicity. MATERIALS AND METHODS: Influenza VLPs were generated by baculovirus-insect cell protein expression system. NCFS and sucrose gradient ultracentrifugation were used for purification of VLP. Immunogenicity of VLP was evaluated by animal experiment. RESULTS: Influenza VLPs expressing hemagglutinin (HA) and neuraminidase proteins derived from highly pathogenic influenza virus (H5N8) were effectively produced and purified by NCFS. HA activity of VLP which correlated with antigenicity was well conserved during multiple purification steps. This NCFS based purified VLPs induced influenza virus–specific antibody responses. CONCLUSION: Our results indicate that the influenza VLP vaccine could be prepared by NCFS without loss of immunogenicity and elicit antigen-specific immune responses.

Influenza M1 Virus-Like Particles Consisting of Toxoplasma gondii Rhoptry Protein 4

Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.

Norovirus P particle and its application / 中华微生物学和免疫学杂志

The challenges posed by norovirus infections to global health are increasing accompa-nied by the rapid rate of the genetic and antigenic evolution of circulating noroviruses. Due to lack of in vitro culture cells and small animal models, norovirus vaccine cannot be prepared by using traditional techniques. With the in-depth understanding and study of norovirus, the subunit vaccines against norovirus infection based on P particles have been developed and presented the characteristics of easily expressed, low cost, high immunogenicity, stable structure and so on. In addition, norovirus P particle has been used as a subvi-ral nanoparticle for vaccine development against other viruses and for antibody production against chronic dis-ease ( Alzheimer′s disease) , which benefits from the accommodation of foreign antigens in the three loops of P particle. In this review, we describe the progresses in the field of P particle related vaccines for providing suggestions about the research and development of multivalent vaccines in China.

Recent vaccine technology in industrial animals

Various new technologies have been applied for developing vaccines against various animal diseases. Virus-like particle (VLP) vaccine technology was used for manufacturing the porcine circovirus type 2 and RNA particle vaccines based on an alphavirus vector for porcine epidemic diarrhea (PED). Although VLP is classified as a killed-virus vaccine, because its structure is similar to the original virus, it can induce long-term and cell-mediated immunity. The RNA particle vaccine used a Venezuela equine encephalitis (VEE) virus gene as a vector. The VEE virus partial gene can be substituted with the PED virus spike gene. Recombinant vaccines can be produced by substitution of the target gene in the VEE vector. Both of these new vaccine technologies made it possible to control the infectious disease efficiently in a relatively short time.

Human papilloma virus in oral cancer

Cervical cancer is the second most prevalent cancer among women, and it arises from cells that originate in the cervix uteri. Among several causes of cervical malignancies, infection with some types of human papilloma virus (HPV) is well known to be the greatest cervical cancer risk factor. Over 150 subtypes of HPV have been identified; more than 40 types of HPVs are typically transmitted through sexual contact and infect the anogenital region and oral cavity. The recently introduced vaccine for HPV infection is effective against certain subtypes of HPV that are associated with cervical cancer, genital warts, and some less common cancers, including oropharyngeal cancer. Two HPV vaccines, quadrivalent and bivalent types that use virus-like particles (VLPs), are currently used in the medical commercial market. While the value of HPV vaccination for oral cancer prevention is still controversial, some evidence supports the possibility that HPV vaccination may be effective in reducing the incidence of oral cancer. This paper reviews HPV-related pathogenesis in cancer, covering HPV structure and classification, trends in worldwide applications of HPV vaccines, effectiveness and complications of HPV vaccination, and the relationship of HPV with oral cancer prevalence.

Interleukin-12 gene adjuvant increases the immunogenicity of virus-like particles of human papillomavirus type 16 regional variant strain

Objectives: To analyze the immunogenicity of virus-like particles (VLP) of human papillomavirus type 16 (HPV16) isolated in East China and the adjuvant potential of interleukin-12 (IL-12). Methods: The variant HPV16 L1VLP expressed in sf9 insect cells were purified with cesium chloride gradient centrifugation. BALB/c mice were vaccinated with VLP (L1N), VLP with Freund's adjuvant (L1A) or VLP with IL-12 recombinant plasmid (L1P). HPV16 VLP specific IgG and IFN-γ level in the serum were detected by ELISA, and the percentage of CD4+ and CD8+ in spleen cells was detected with flow cytometry. Results: The titers of serum IgG antibodies in vaccinated groups were higher than in negative control and the serum antibodies mainly recognized conformation-dependent HPV16 VLP epitopes. Splenic CD4+ and CD8+ T cell subsets increased after vaccination in every experimental group, and CD8+ increased obviously in L1P group. The ratio of CD4+/CD8+ decreased in L1P group and increased in the other two groups, compared to control group. Vaccination induced specific secretion of IFN-γ in the serum of vaccinated group (p < 0.05), especially in the L1P group. Conclusions: VLP of HPV16 variant strain isolated in East China could induce humoral immunity and cellular immunity in mice, and IL-12 recombinant plasmid can enhance cellular immunity. .

Application of novel nanoparticles carriers in drug or gene delivery / 中国药学杂志

OBJECTIVE: To summary the application of new types of nanoparticles carriers emerged in recent years in drug or gene delivery. METHODS: By sorting, analyzing and summarizing domestic and foreign literatures, the characteristics, in vivo and in vitro properties and the applications in pharmacy of novel nanoparticles carriers such as nano cochleates, virus-like particles, hydrogel nanoparticles, gold nanoshells, carbon nanomaterials, quantum dots and dendrimers were reviewed and elaborated. RESULTS AND CONCLUSION: Nanoparticles possess special physical and chemical properties which could improve the stability and bioavailability of drugs and have a targeting and sustained release effect.

Status and progress of research on mucosal vaccines / 中国药学杂志

OBJECTIVE: Mucosal surfaces are a major portal of entry for many human pathogens that are the cause of infectious diseases. Vaccines that immunize by mucosal routes may induce protective immunity against mucosal pathogens at their sites of entry thus to be more effective and economical. We try to overview the status and progress of research on mucosal vaccines. METHODS: The databases of CNKI and Pubmed were used to search the related articles about mucosal vaccines with key words "mucosal vaccine, mucosal adjuvant, mucosal particulate delivery vectors" in Chinese and English. Articles closely related to mucosal vaccines were selected. RESULTS: Thirty-six articles were included at last. Live-attenuated or inactivated mucosal vaccines and vaccines based on new concepts such as subunit vaccines, virus-like particles and virosomes have been marketed, and related research work are undergoing. Safe and effective mucosal adjuvants and delivery vectors are being sought to enhance the magnitude and quality of the protective immune response. The composition, size, surface chemistry and ligands of particulate carrier systems may influence the efficacy. Great progress has been made in several particulate delivery systems. CONCLUSION: Although the research and development of mucosal vaccines are facing many difficulties and challenges, the progress of research work will bring new opportunities to mucosal vaccines development.

Induction of antibody and interferon-gamma production in mice immunized with virus-like particles of swine hepatitis E virus

Virus-like particles (VLPs) composed of the truncated capsid protein of swine hepatitis E virus (HEV) were developed and immune responses of mice immunized with the VLPs were evaluated. IgG titers specific for the capsid protein of swine HEV were significantly higher for all groups of mice immunized with the VLPs than those of the negative control mice. Splenocytes from mice immunized with the VLPs also produced significantly greater quantities of interferon (IFN)-gamma than interleukin (IL)-4 and IL-10. These newly developed swine HEV VLPs have the capacity to induce antigen-specific antibody and IFN-gamma production in immunized mice.

Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.

Progress and hurdles in the development of influenza virus-like particle vaccines for veterinary use

Virus-like particles (VLPs), which resemble infectious virus particles in structure and morphology, have been proposed to provide a new generation of vaccine candidates against various viral infections. As effective immunogens, characterized by high immunogenicity and safety, VLPs have been employed in the development of human influenza vaccines. Recently, several influenza VLP vaccines have been developed for veterinary use and successfully evaluated in swine, canine, duck, and chicken models. These VLP vaccine candidates induced protective immune responses and enabled serological differentiation between vaccinated and infected animals in conjunction with a diagnostic test. Here, we review the current progress of influenza VLP development as a next-generation vaccine technology in the veterinary field and discuss the challenges and future direction of this technology.

Current status of human papillomavirus vaccines

Cervical cancer is a malignant neoplasm arising from cells that originate in the cervix uteri. It is the second most prevalent cancer among women. It can have several causes; an infection with some type of human papillomavirus (HPV) is the greatest risk factor for cervical cancer. Over 100 types of HPVs have been identified, and more than 40 types of HPVs are typically transmitted through sexual contact and infect the anogenital region. Among these, a number of HPVs types, containing types 16 and 18, are classified as "high-risk" HPVs that can cause cervical cancer. The HPVs vaccine prevents infection with certain species of HPVs associated with the development of cervical cancer, genital warts, and some less common cancers. Two HPVs vaccines are currently on the global market: quadrivalent HPVs vaccine and bivalent HPV vaccine that use virus-like particles as a vaccine antigen. This review discusses the current status of HPVs vaccines on the global market, clinical trials, and the future of HPVs vaccine development.