Trigger factor assisted self-assembly of canine parvovirus VP2 protein into virus-like particles in Escherichia coli with high immunogenicity.
ABSTRACT: Canine parvovirus (CPV) has been considered to be an important pathogen, which can cause acute infectious disease in canids. Although current vaccines are effective in preventing CPV infection, safety problems still remain unsolved. In this study, a subunit vaccine against CPV based on virus-like particles (VLPs) with good safety and immunogenicity is reported. Soluble CPV VP2 protein was produced by co-expression of chaperone trigger factor (Tf16) in Escherichia coli (E.coli), and assembled into CPV VLPs which could be affected by NaCl and pH. At 250 mM NaCl pH 8.0, the VLPs co-expressed with Tf16 had similar size (25 nm) and shape with the authentic virus capsid under the transmission electron microscopy (TEM), which is also in accordance with the dynamic light scattering (DLS) data. Immunization with these particles could induce high-titer hemagglutination inhibition (1:12288) and neutralizing antibodies (1:6144) in guinea pigs. Splenic cells of them could secrete IFN-? and IL-4 after stimulation by CPV. Thus, the VLPs produced by the new approach with high yield and immunogenicity could be a potential candidate for CPV vaccine.
Project description:Quantum dots (QDs) have a promising prospect in live-cell imaging and sensing because of unique fluorescence features. QDs aroused significant interest in the bio-imaging field through integrating the fluorescence properties of QDs and the delivery function of biomaterial. The natural tropism of Canine Parvovirus (CPV) to the transferrin receptor can target specific cells to increase the targeting ability of QDs in cell imaging. CPV virus-like particles (VLPs) from the expression of the CPV-VP2 capsid protein in a prokaryotic expression system were examined to encapsulate the QDs and deliver to cells with an expressed transferrin receptor. CPV-VLPs were used to encapsulate QDs that were modified using 3-mercaptopropionic acid. Gel electrophoresis, fluorescence spectrum, particle size, and transmission electron microscopy verified the conformation of a complex, in which QDs were encapsulated in CPV-VLPs (CPV-VLPs-QDs). When incubated with different cell lines, CPV-VLPs-QDs significantly reduced the cytotoxicity of QDs and selectively labeled the cells with high-level transferrin receptors. Cell-targeted labeling was achieved by utilizing the specific binding between the CPV capsid protein VP2 of VLPs and cellular receptors. CPV-VLPs-QDs, which can mimic the native CPV infection, can recognize and attach to the transferrin receptors on cellular membrane. Therefore, CPV-VLPs can be used as carriers to facilitate the targeted delivery of encapsulated nanomaterials into cells via receptor-mediated pathways. This study confirmed that CPV-VLPs can significantly promote the biocompatibility of nanomaterials and could expand the application of CPV-VLPs in biological medicine.
Project description:BACKGROUND:Canine parvovirus (CPV) is now recognized as a serious threat to the dog breeding industry worldwide. Currently used CPV vaccines all have their specific drawbacks, prompting a search for alternative safe and effective vaccination strategies such as subunit vaccine. VP2 protein is the major antigen targeted for developing CPV subunit vaccine, however, its production in baculovirus expression system remains challenging due to the insufficient yield. Therefore, our study aims to increase the VP2 protein production by using an improved baculovirus expression system and to evaluate the immunogenicity of the purified VP2 protein in mice. RESULTS:The results showed that high-level expression of the full length VP2 protein was achieved using our modified baculovirus expression system. The recombinant virus carrying two copies of VP2 gene showed the highest expression level, with a productivity of 186?mg/L, which is about 1.4-1.6 fold that of the recombinant viruses carrying only one copy. The purified protein reacted with Mouse anti-His tag monoclonal antibody and Rabbit anti-VP2 polyclonal antibody. BALB/c mice were intramuscularly immunized with purified VP2 protein twice at 2?week intervals. After vaccination, VP2 protein could induce the mice produce high level of hemagglutination inhibition antibodies. CONCLUSIONS:Full length CPV VP2 protein was expressed at high level and purified efficiently. Moreover, it stimulated mice to produce high level of antibodies with hemmaglutination inhibition properties. The VP2 protein expressed in this study could be used as a putative economic and efficient subunit vaccine against CPV infection.
Project description:The H1N1 influenza pandemic of 2009 stimulated interest in developing safe and effective subunit influenza vaccines using rapid and cost-effective recombinant technologies that can avoid dependence on hens' eggs supply and live viruses for production. Among alternative approaches to subunit vaccine development, virus-like particles (VLPs) represent an attractive strategy due to their safety and immunogenicity. Previously, we have produced a recombinant monomeric hemagglutinin (HA) protein derived from the A/California/04/09 (H1N1) strain of influenza virus in a plant-based transient expression system and demonstrated immunogenicity and safety of this monomeric HA in animal models and human volunteers. In an effort to produce higher potency influenza vaccine in plants, we have designed and generated enveloped VLPs using the ectodomain of HA from the A/California/04/09 strain and heterologous sequences. The resulting H1 HA VLPs (HAC-VLPs) elicited robust hemagglutination inhibition antibody responses in mice at doses lower than 1 µg in the presence or absence of Alhydrogel adjuvant. These results suggest enhanced immunogenicity of recombinant HA in the form of an enveloped VLP over soluble antigen.
Project description:BACKGROUND Coxsackievirus A16 (CA16) is one of the main causative agents of hand, foot, and mouth disease (HFMD), and the development of a safe and effective vaccine has been a top priority among CA16 researchers. MATERIAL AND METHODS In this study, we developed a Pichia pastoris yeast system for secretory expression of the virus-like particles (VLPs) for CA16 by co-expression of the P1 and 3CD proteins of CA16. SDS-PAGE, Western blot, and transmission electron microscopy (TEM) were performed to identify the formation of VLPs. Immunogenicity and vaccine efficacy of the CA16 VLPs were assessed in BABL/c mouse models. RESULTS Biochemical and biophysical analysis showed that the yeast-expressed CA16 VLPs were composed of VP0, VP1, and VP3 capsid subunit proteins, and present spherical particles with a diameter of 30 nm, similar to the parental infectious CA16 virus. Furthermore, CA16 VLPs elicited potent humoral and cellular immune responses, and VLPs-immunized sera conferred efficient protection to neonatal mice against lethal CA16 challenge. CONCLUSIONS Our results demonstrate that VLPs produced in Pichia pastoris represent a safe and effective vaccine strategy for CA16.
Project description:Retrovirus-derived virus-like particles (VLPs) are particularly interesting vaccine platforms, as they trigger efficient humoral and cellular immune responses and can be used to display heterologous antigens. In this study, we characterized the intrinsic immunogenicity of VLPs and investigated their possible adjuvantization by incorporation of Toll-like receptor (TLR) ligands. We designed a noncoding single-stranded RNA (ncRNA) that could be encapsidated by VLPs and induce TLR7/8 signaling. We found that VLPs efficiently induce in vitro dendritic cell activation, which can be improved by ncRNA encapsidation (ncRNAVLPs). Transcriptome studies of dendritic cells harvested from the spleens of immunized mice identified antigen presentation and immune activation as the main gene expression signatures induced by VLPs, while TLR signaling and Th1 signatures characterize ncRNAVLPs. In vivo and compared with standard VLPs, ncRNAVLPs promoted Th1 responses and improved CD8+ T cell proliferation in a MyD88-dependent manner. In an HIV vaccine mouse model, HIV-pseudotyped ncRNAVLPs elicited stronger antigen-specific cellular and humoral responses than VLPs. Altogether, our findings provide molecular evidence for a strong vaccine potential of retrovirus-derived VLPs that can be further improved by harnessing TLR-mediated immune activation.IMPORTANCE We previously reported that DNA vaccines encoding antigens displayed in/on retroviral VLPs are more efficient than standard DNA vaccines at inducing cellular and humoral immune responses. We aimed to decipher the mechanisms and investigated the VLPs' immunogenicity independently of DNA vaccination. We show that VLPs have the ability to activate antigen-presenting cells directly, thus confirming their intrinsic immunostimulatory properties and their potential to be used as an antigenic platform. Notably, this immunogenicity can be further improved and/or oriented by the incorporation into VLPs of ncRNA, which provides further TLR-mediated activation and Th1-type CD4+ and CD8+ T cell response orientation. Our results highlight the versatility of retrovirus-derived VLP design and the value of using ncRNA as an intrinsic vaccine adjuvant.
Project description:Cancers have killed millions of people in human history and are still posing a serious health problem worldwide. Therefore, there is an urgent need for developing preventive and therapeutic cancer vaccines. Among various cancer vaccine development platforms, virus-like particles (VLPs) offer several advantages. VLPs are multimeric nanostructures with morphology resembling that of native viruses and are mainly composed of surface structural proteins of viruses but are devoid of viral genetic materials rendering them neither infective nor replicative. In addition, they can be engineered to display multiple, highly ordered heterologous epitopes or peptides in order to optimize the antigenicity and immunogenicity of the displayed entities. Like native viruses, specific epitopes displayed on VLPs can be taken up, processed, and presented by antigen-presenting cells to elicit potent specific humoral and cell-mediated immune responses. Several studies also indicated that VLPs could overcome the immunosuppressive state of the tumor microenvironment and break self-tolerance to elicit strong cytotoxic lymphocyte activity, which is crucial for both virus clearance and destruction of cancerous cells. Collectively, these unique characteristics of VLPs make them optimal cancer vaccine candidates. This review discusses current progress in the development of VLP-based cancer vaccines and some potential drawbacks of VLPs in cancer vaccine development. Extracellular vesicles with close resembling to viral particles are also discussed and compared with VLPs as a platform in cancer vaccine developments.
Project description:Canine parvovirus (CPV) type 2 emerged in 1978 in the USA and quickly spread among dog populations all over the world with high morbidity. Although CPV is a DNA virus, its genomic substitution rate is similar to some RNA viruses. Therefore, it is important to trace the evolution of CPV to monitor the appearance of mutations that might affect vaccine effectiveness.Our analysis shows that the VP2 genes of CPV isolated from 1979 to 2016 are divided into six groups: GI, GII, GIII, GIV, GV, and GVI. Amino acid mutation analysis revealed several undiscovered important mutation sites: F267Y, Y324I, and T440A. Of note, the evolutionary rate of the CPV VP2 gene from Asia and Europe decreased. Codon usage analysis showed that the VP2 gene of CPV exhibits high bias with an ENC ranging from 34.93 to 36.7. Furthermore, we demonstrate that natural selection plays a major role compared to mutation pressure driving CPV evolution.There are few studies on the codon usage of CPV. Here, we comprehensively studied the genetic evolution, codon usage pattern, and evolutionary characterization of the VP2 gene of CPV. The novel findings revealing the evolutionary process of CPV will greatly serve future CPV research.
Project description:Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential "differentiating infected from vaccinated animals" (DIVA) vaccine candidates for the surveillance and eradication of PPR.
Project description:Virus-like particles (VLPs) represent a promising vaccine against severe acute respiratory syndrome coronavirus (SARS CoV). In this study, recombinant baculovirus vAcS and vAcME were constructed to express the S protein and the M and E proteins of SARS CoV, respectively. Electron microscope analysis demonstrated the formation of VLPs in vAcME and vAcS coinfected insect cells. Mice immunized four times with VLPs developed high antibody titres against SARS CoV. In addition, VLPs elicited cell-mediated immunity as demonstrated by enhanced interferon-gamma and interleukin-4 production. VLPs also conferred protective immunity against the infection of Spike protein pseudotyped murine leukaemia virus. Our findings demonstrate that SARS CoV VLPs are immunogenic and can elicit strong SARS CoV-specific humoral and cellular immune responses in mice. This is the first study describing the immunogenicity of SARS CoV VLPs, providing valuable data for developing a protective vaccine against SARS CoV infection.
Project description:We introduce a new vaccine platform against Marburg virus (MARV) combining the advantages of the immunogenicity of a highly attenuated vaccine vector (Modified Vaccinia Ankara, MVA) with the authentic conformation of virus-like particles (VLPs). Our vaccine, MVA-MARV-VLP, expresses the minimal components of MARV VLPs: the envelope glycoprotein GP and the matrix protein VP40. Electron microscopy confirmed self-assembly and budding of VLPs from infected cells. Prime/boost vaccination of guinea pigs with MVA-MARV-VLP-elicited MARV-specific binding and neutralizing antibody responses. Vaccination also induced Fc-mediated innate immune effector functions including activation of NK cells and antibody-dependent phagocytosis by neutrophils and monocytes. Inoculation of vaccinated animals with guinea pig-adapted MARV demonstrated 100% protection against death and disease with no viremia. Therefore, our vaccine platform, expressing two antigens resulting in assembly of VLPs in the native conformation in vaccinated hosts, can be used as a potent vaccine against MARV.