Chimeric Particles Research Articles

Efficacy comparison in cap VLPs of PCV2 and PCV3 as swine vaccine vehicle

As one genotype of porcine circovirus (PCV) identified in 2016, PCV3 has brought huge hidden dangers to the global swine industry together with PCV2. Virus-like particles (VLPs) of capsid protein (Cap) of PCV2 serve as an alternative nano-antigen delivery strategy to efficiently induce antiviral immune response against PCV2 and/or other covalently displayed swine pathogens. However, the current understanding is limited on the capability of PCV3 as a nano-vaccine vehicle. Here we systematically compared the characteristics and the immunogenic efficacy of PCV3 Cap (Cap3) and PCV2 Cap (Cap2) in a VLP form. Cap3 VLPs presented higher internalization efficiency into cells and cytokines production compared to those of Cap2. Meanwhile, cross-reactive immunity between Cap3 VLPs and Cap2 VLPs was detected. Furthermore, to evaluate the function of Cap3 VLPs and Cap2 VLPs as vaccine vehicles carrying foreign proteins, the non-structural protein 6 of porcine reproductive and respiratory syndrome virus (PRRSV) was fused to C-terminus of Cap. Cap3-based chimeric particles induced a higher level of nsp6-specific immune response and PRRSV inhibition. Collectively, these self-assembling, Cap-based VLPs offer a compelling platform for enhancing the effectiveness of subunit vaccinations against newly emerging diseases and hold great promise for the development of Cap3-based chimeric subunit vaccines.

Development of a Plant-Expressed Subunit Vaccine against Brucellosis.

Brucellosis is an important bacterial disease of livestock and the most common zoonotic disease. The current vaccines are effective but unsafe, as they result in animal abortions and are pathogenic to humans. Virus-like particles are being investigated as molecular scaffolds for foreign antigen presentation to the immune system. Here, we sought to develop a new-generation vaccine by presenting selected Brucella melitensis T cell epitopes on the surface of Orbivirus core-like particles (CLPs) and transiently expressing these chimeric particles in Nicotiana benthamiana plants. We successfully demonstrated the assembly of five chimeric CLPs in N. benthamiana plants, with each CLP presenting a different T cell epitope. The safety and protective efficacy of three of the highest-yielding CLPs was investigated in a mouse model of brucellosis. All three plant-expressed chimeric CLPs were safe when inoculated into BALB/c mice at specific antigen doses. However, only one chimeric CLP induced protection against the virulent Brucella strain challenge equivalent to the protection induced by the commercial Rev1 vaccine. Here, we have successfully shown the assembly, safety and protective efficacy of plant-expressed chimeric CLPs presenting B. melitensis T cell epitopes. This is the first step in the development of a safe and efficacious subunit vaccine against brucellosis.

A novel three-plasmid packaging system for chimeric SFV/SIN VRPs derived from Semliki Forest virus and Sindbis virus as a candidate gene delivery vector.

Semliki Forest virus (SFV) viral replicon particles (VRPs) have been frequently used in various animal models and clinical trials. Chimeric replicon particles offer different advantages because of their unique biological properties. We here constructed a novel three-plasmid packaging system for chimeric SFV/SIN VRPs. The capsid and envelope of SIN structural proteins were generated using two-helper plasmids separately, and the SFV replicon contained the SFV replicase gene, packaging signal of SIN, subgenomic promoter followed by the exogenous gene, and 3' UTR of SIN. The chimeric VRPs carried luciferase or eGFP as reporter genes. The fluorescence and electron microscopy results revealed that chimeric VRPs were successfully packaged. The yield of the purified chimeric VRPs was approximately 2.5 times that of the SFV VRPs(1.38 × 107 TU/ml vs. 5.41 × 106 TU/ml) (p < 0.01). Furthermore, chimeric VRPs could be stored stably at 4°C for at least 60 days. Animal experiments revealed that mice immunized with chimeric VRPs(luciferase) had stronger luciferase expression than those immunized with equivalent amount of SFV VRPs(luciferase) (p < 0.01), and successfully expressed luciferase for approximately 12 days. Additionally, the chimeric VRPs expressed the RBD of SARS-CoV-2 efficiently and induced robust RBD-specific antibody responses in mice. In conclusion, the chimeric VRPs constructed here met the requirements of a gene delivery tool for vaccine development and cancer therapy.

The development and application of pseudoviruses: assessment of SARS-CoV-2 pseudoviruses

Although most Coronavirus disease (COVID-19) patients can recover fully, the disease remains a significant cause of morbidity and mortality. In addition to the consequences of acute infection, a proportion of the population experiences long-term adverse effects associated with SARS-CoV-2. Therefore, it is still critical to comprehend the virus’s characteristics and how it interacts with its host to develop effective drugs and vaccines against COVID-19. SARS-CoV-2 pseudovirus, a replication-deficient recombinant glycoprotein chimeric viral particle, enables investigations of highly pathogenic viruses to be conducted without the constraint of high-level biosafety facilities, considerably advancing virology and being extensively employed in the study of SARS-CoV-2. This review summarizes three methods of establishing SARS-CoV-2 pseudovirus and current knowledge in vaccine development, neutralizing antibody research, and antiviral drug screening, as well as recent progress in virus entry mechanism and susceptible cell screening. We also discuss the potential advantages and disadvantages.

Efficient generation and characterization of chimeric dengue viral-like particles

Viral-like particles (VLPs) because of their non-infectious and high immunogenic properties have important applications in diagnostics, drug delivery, and vaccine production. They also serve as an attractive model system to study virus assembly and fusion processes. Unlike other flaviviruses, Dengue virus (DENV) is not very efficient in the production of VLPs on the expression of DENV structural proteins. On the other hand, the stem region and transmembrane region (TM) of G protein of Vesicular Stomatitis virus (VSV) alone are sufficient for budding.Here we generated chimeric VLPs replacing regions of stem and transmembrane domain (STEM) or only transmembrane domain (TM) of E protein of DENV-2 with corresponding regions of VSV G protein. Both chimeric proteins secreted VLPs at higher levels than the wild type (2–4 folds) without any significant change in the expression in the cell. Chimeric VLPs could be recognized by a conformational monoclonal antibody, 4G2. They were also found to interact with dengue-infected patient sera effectively thus implying that their antigenic determinants are preserved. In addition, they were able to bind to its putative receptor, heparin with similar affinity as the parent counterpart thus retaining its functional property. However, cell-cell fusion revealed that there is no significant increase in the fusion ability of chimeras as compared to the parent clone, whereas VSV G protein displayed high cell-cell fusion activity. Overall, this study suggests that chimeric dengue VLPs can be taken forward for their likely potential as vaccine production and serodiagnosis.

Process for production of chimeric antigen receptor-transducing lentivirus particles using infection with replicon particles containing self-replicating RNAs

Viral vectors are indispensable for advanced therapeutics such as chimeric T cell antigen receptor (CAR-T) therapy. CAR-T is a revolutionary treatment for blood cancers in patients who have relapsed or refractory disease after standard therapy. The therapeutic success is driving CAR-T's wider adoption, but the prospects for wider use are currently limited by high price, including the price of a packaged CAR vector, and the limited supply of such a vector on the market. The production of large quantities of packaged lentiviral vector using transfection with multiple plasmids in academic settings is associated with technical difficulties. Here we describe a technology for packaging a lentiviral vector using autonomously replicating RNAs that are delivered to production cultures by infection with replicon particles. Chimeric vectors capable of cytoplasmic replication were assembled using a replicative backbone from the RNA-virus Venezuelan equine encephalitis virus, into which the HIV-1 sequences for genomic integration and the CAR transgene were cloned. These vectors are packaged into high-titered infectious alphavirus replicon particles, allowing subsequent infection of large culture volumes to produce lentivirus particles. Using this method, enough vector was generated to produce therapeutic amounts of CAR+ cells in an academic setting.

Glycyrrhizic Acid Derivatives Bearing Amino Acid Residues in the Carbohydrate Part as Dengue Virus E Protein Inhibitors: Synthesis and Antiviral Activity.

Dengue virus (DENV) is one of the most geographically distributed mosquito-borne flaviviruses, like Japanese encephalitis virus (JEV), and Zika virus (ZIKV). In this study, a library of the known and novel Glycyrrhizic acid (GL) derivatives bearing amino acid residues or their methyl/ethyl esters in the carbohydrate part were synthesized and studied as DENV inhibitors in vitro using the cytopathic effect (CPE), viral infectivity and virus yield assays with DENV1 and DENV-2 in Vero E6 and A549 cells. Among the GL conjugates tested, compound hits GL-D-ValOMe 3, GL-TyrOMe 6, GL-PheOEt 11, and GL-LysOMe 21 were discovered to have better antiviral activity than GL, with IC50 values ranging from <0.1 to 5.98 μM on the in vitro infectivity of DENV1 and DENV2 in Vero E6 and A549 cells. Compound hits 3, 6, 11, and 21 had a concentration-dependent inhibition on the virus yield in Vero E6, in which GL-D-ValOMe 3 and GL-PheOEt 11 were the most active inhibitors of DENV2 yield. Meanwhile, the time-of-addition assay indicated that conjugates GL-D-ValOMe 3 and GL-PheOEt 11 exhibited a substantial decrease in the DENV2 attachment stage. Subsequently, chimeric single-round infectious particles (SRIPs) of DENV2 C-prM-E protein/JEV replicon and DENV2 prM-E/ZIKV replicon were utilized for the DENV envelope I protein-mediated attachment assay. GL conjugates 3 and 11 significantly reduced the attachment of chimeric DENV2 C-prM-E/JEV and DENV2 prM-E/ZIKV SRIPs onto Vero E6 cells in a concentration-dependent manner but did not impede the attachment of wild-type JEV CprME/JEV and ZIKV prM-E/ZIKV SRIPs, indicating the inhibition of Compounds 3 and 11 on DENV2 E-mediated attachment. Molecular docking data revealed that Compounds 3 and 11 have hydrophobic interactions within a hydrophobic pocket among the interfaces of Domains I, II, and the stem region of the DENV2 envelope (E) protein. These results displayed that Compounds 3 and 11 were the lead compounds targeting the DENV E protein. Altogether, our findings provide new insights into the structure–activity relationship of GL derivatives conjugated with amino acid residues and can be the new fundamental basis for the search and development of novel flavivirus inhibitors based on natural compounds.

Off-the-shelf CAR natural killer cells secreting IL-15 target spike in treating COVID-19

Engineered natural killer (NK) cells represent a promising option for immune therapy option due to their immediate availability in allogeneic settings. Severe acute diseases, such as COVID-19, require targeted and immediate intervention. Here we show engineering of NK cells to express (1) soluble interleukin-15 (sIL15) for enhancing their survival and (2) a chimeric antigen receptor (CAR) consisting of an extracellular domain of ACE2, targeting the spike protein of SARS-CoV-2. These CAR NK cells (mACE2-CAR_sIL15 NK cells) bind to VSV-SARS-CoV-2 chimeric viral particles as well as the recombinant SARS-CoV-2 spike protein subunit S1 leading to enhanced NK cell production of TNF-α and IFN-γ and increased in vitro and in vivo cytotoxicity against cells expressing the spike protein. Administration of mACE2-CAR_sIL15 NK cells maintains body weight, reduces viral load, and prolongs survival of transgenic mice expressing human ACE2 upon infection with live SARS-CoV-2. These experiments, and the capacity of mACE2-CAR_sIL15 NK cells to retain their activity following cryopreservation, demonstrate their potential as an allogeneic off-the-shelf therapy for COVID-19 patients who are faced with limited treatment options.

Chimeric virus-like particles presenting tumour-associated MUC1 epitope result in high titers of specific IgG antibodies in the presence of squalene oil-in-water adjuvant: towards safe cancer immunotherapy

BackgroundImmunotherapy is emerging as a powerful treatment approach for several types of cancers. Modulating the immune system to specifically target cancer cells while sparing healthy cells, is a very promising approach for safer therapies and increased survival of cancer patients. Tumour-associated antigens are favorable targets for cancer immunotherapy, as they are exclusively expressed by the cancer cells, minimizing the risk of an autoimmune reaction. The ability to initiate the activation of the immune system can be achieved by virus-like particles (VLPs) which are safe and potent delivery tools. VLP‐based vaccines have evolved dramatically over the last few decades and showed great potential in preventing infectious diseases. Immunogenic potency of engineered VLPs as a platform for the development of effective therapeutic cancer vaccines has been studied extensively. This study involves recombinant VLPs presenting multiple copies of tumour-specific mucin 1 (MUC1) epitope as a potentially powerful tool for future immunotherapy.ResultsIn this report VLPs based on the structural protein of Norovirus (NoV VP1) were genetically modified to present multiple copies of tumour-specific MUC1 epitope on their surface. Chimeric MUC1 particles were produced in the eukaryotic Leishmania tarentolae expression system and used in combination with squalene oil-in-water emulsion MF59 adjuvant to immunize BALB/c mice. Sera from vaccinated mice demonstrated high titers of IgG and IgM antibodies which were specifically recognizing MUC1 antigen.ConclusionsThe obtained results show that immunization with recombinant chimeric NoV VP1- MUC1 VLPs result in high titers of MUC1 specific IgG antibodies and show great therapeutic potential as a platform to present tumour-associated antigens.Graphical

Hepatitis E Virus Capsid as a Carrier of Exogenous Antigens for the Development of Chimeric Virus-Like Particles

Introduction: Virus-like particles (VLPs), self-assembled multiprotein structures, can stimulate robust immune responses due to their structural similarity to native virions that allow the presentation of multiple copies of the target epitopes. Utilizing VLPs as vaccine platforms to present exogenous antigens is a promising and challenging approach in the vaccine development field. This study investigates the potential of the truncated hepatitis E virus (HEV) capsid as a VLP platform to present foreign antigens. Methods: The S and M domains of the HEV capsid protein were selected as the optimal carrier (CaSM). The exogenous antigen Seq8 containing 3 neutralizing epitopes from 3 different foot-and-mouth disease virus (FMDV) strains was linked to the C-terminal of CaSM to construct a chimeric VLP (CaSM-Seq8). The chimeric particles were produced in Escherichia coli, and their morphology, physicochemical properties, antigenicity, and immunogenicity were analyzed. Results: Morphological analysis showed that CaSM-Seq8 self-assembled into VLPs similar to CaSM VLPs (∼26 nm in diameter) but smaller than native HEV virions. Further, the thermal stability and the resistance to enzymatic proteolysis of Seq8 were enhanced when it was attached to the CaSM carrier. The antigenicity analysis revealed a more robust reactivity against anti-FMDV antibodies when Seq8 was presented on CaSM particles. Upon injection into mice, FMDV-specific IgGs induced by CaSM-Seq8 appeared earlier, increased faster, and maintained higher levels for a longer time than those induced by Seq8 alone or the inactivated FMDV vaccine. Conclusion: This study demonstrated the potential of utilizing the truncated HEV capsid as an antigen-presenting platform for the development of chimeric VLP immunogens.

Characterization and purification of pentameric chimeric protein particles using asymmetric flow field-flow fractionation coupled with multiple detectors.

Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius (Rh) measured by online DLS with the Rh values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The Rg/Rh ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03323-6.

Stable Display of Artificially Long Foreign Antigens on Chimeric Bamboo mosaic virus Particles.

Plant viruses can be genetically modified to generate chimeric virus particles (CVPs) carrying heterologous peptides fused on the surface of coat protein (CP) subunits as vaccine candidates. However, some factors may be especially significant in determining the properties of chimeras. In this study, peptides from various sources and of various lengths were inserted into the Bamboo mosaic virus-based (BaMV) vector CP N-terminus to examine the chimeras infecting and accumulating in plants. Interestingly, it was found that the two different strains Foot-and-mouth disease virus (FMDV) VP1 antigens with flexible linker peptides (77 or 82 amino acids) were directly expressed on the BaMV CP, and the chimeric particles self-assembled and continued to express FMDV antigens. The chimeric CP, when directly fused with a large foreign protein (117 amino acids), can self-fold into incomplete virus particles or disks. The physicochemical properties of heterologus peptides N-terminus, complex strand structures of heterologus peptides C-terminus and different flexible linker peptides, can affect the chimera accumulation. Based on these findings, using plant virus-based chimeras to express foreign proteins can increase their length limitations, and engineered plant-made CVP-based vaccines have increasing potential for further development as novel vaccines.

Bioengineering of Virus-like Particles for the Prevention or Treatment of Allergic Diseases.

Recent findings on the mechanism of allergen-specific immunotherapy (AIT) have revisited the role of immunoglobulin G (IgG) as the development of specific blocking IgG antibodies appeared critical for the successful suppression of T-helper 2 (Th2)-biased allergic responses. Consequently, any form of molecular AIT-promoting potent allergen-specific neutralizing antibodies would be preferred to conventional administration of allergen extracts. The potent immunogenicity of virus-like particles (VLPs) could be harnessed for that purpose. The particle size (20–200 nm) optimizes uptake by antigen-presenting cells as well as lymphatic trafficking. Moreover, the display of antigens in repetitive arrays promotes potent B cell activation for the development of sustained antibody responses. The presentation of self-antigens on the particle surface was even capable to break B cell tolerance. In this review, we describe the immunomodulatory properties of the 3 VLP-based strategies designed so far for the treatment of allergic disease: VLP packaged with CpG motifs as well as chimeric particles displaying pro-Th2/Th2 cytokines or allergens (full-length or B cell epitopes).

WEEDS-VEGETABLES AND FRUITS ACT AS POTENTIAL BIOMEDICINES AGAINST COVID-19: ENRICHED AGRICULTURE BIODIVERSITY SOCIO-ECONOMY SCIENCE TECHNOLOGY COMMUNICATION BY CONTROLLING PLANT DISEASES

The past two decades have seen several viral-epidemics, causing great loss of human lives. The recent COVID-19 epidemics emerge as serious-global-threat to human civilization, public health, agriculture, travel, socioeconomic, education and even clinical research also. It has no specific therapeutics, and effective treatment options. Though the middle and upper classes are able to manage, but the low income households are really suffering. So Government of India wants to reorient the COVID-19 crisis by developing policy initiative. So it is focused on the consumption of nutritious as well as traditional medicinal weed vegetables and fruits, for preventive measures against human-diseases which are naturally infected with different pathogens caused diseases and significantly decreasing food production. Though pesticides are the most effective means of control, but they are costly and not environment friendly. So, it is emphasized on the multiple-intercropped weed vegetables fruits plants of the 4 plants species viz., amaranth, cucumber, cowpea, and okra plants, and determines the effects on pathogens infected diseases; Root Knot and Mosaic Virus. After harvesting, of these 4 plant species, amaranth and cucumber received maximum infection, forming the “Eco-Friendly-Highly-Economical-Potential-Biomedicines-Cover-Catch-Weed-Vegetable-Fruits-Crop-Plants”, conserving “Biodiversity-Conservations-Sustainable-Climate-Health-Development-Green-Socio-Economic-Implications-Agriculture”, though all are highly susceptible to pathogens and the farmers would be double benefited; by controlling diseases, and by marketing. In biomedicines, weed-vegetables fruits OR the plant-Mosaic-Virus has been developed as antigenic epitopes derived from the vaccine targets-COVID-19 infectious epidemic disease agents, and the chimeric virus particles, use in vaccine formulations or treatments, as one of the most-powerful cost-effective emergency-healthcare-easily-available safe-edible prepare-able easy-applicable future-personalized-potential-biomedicines act as “Preventive-Measures and Safe-Alternative-to-Live-Replicating-COVID-19-Vaccines”, by increasing our natural-immune system, which is one of the milestone events amidst many important changes in the past decade that have necessitated formulation of a new-outlook and strategy for preventing-“Future-Pandemic-Pathogens-of-21st-Century-Advances-Research-Diagnosis-Treatment-Control-Enriching-Food-Security-Biodiversity-Agriculture-Green-Socio-Economy-Medical-Science-and-Technology-Communication-Applications-Innovation-Issues”, based on the theme “Vision 2040” that would help policy-makers.

Okra maybe potential cost-effective personalized-biomedicines social-vaccine against COVID-19: Improved immunity food-security green-economy science-and-technology-communication applications

The “human-challenge”–pandemic-coronavirus-COVID-19 effects on human-civilization, health, travel, socioeconomic, education, and clinical research. India emphasis on okra, the most economically-important number-one essential-consumption-nutritious-vegetable as well as traditional-medicine, the “Nature’s-Gift to human-disease-free healthy-life,” is naturally-infected with different-diseases, significantly reducing food-production. Although pesticides are the most effective means of control, they are not, cost-effective, and environment-friendly. Hence, it has been an urgency to require; new and more efficient solutions, technologies, products to fulfill its food and nutritional requirement, and methods to develop vaccines or social-vaccine against coronavirus-COVID-19. Hence, it is focused on the okra-plant, intercropped with amaranth-plant, to determine the effects on pathogens-infected-diseases; Root-Knot, Yellow-Vein-Mosaic-Virus, and Okra-Enation-Leaf-Curl-Virus in a well-protected-garden. After harvesting, of the two-plant-species, okra received minimum pathogen-infection, forming the “Good-Diseases-Resistant-Eco-Friendly-Highly-Economical-Biomedicinal-Plants,” conserving “Biodiversity-Conservations-Sustainable-Climate-Health and Development with Important-Economic-Implications in Agriculture,” though both are highly susceptible to diseases, and the farmers would be benefited double; by controlling-diseases, and by buying-selling the okra-amaranth. In biomedicines, highly-trace-tolerance-okra-vegetables or the plant virus; Yellow-Vein-Mosaic-Virus, and Okra-Enation-Leaf-Curl-Virus, which has been developed as antigenic epitopes derived from the vaccine targets COVID-19 infectious epidemic disease agents, and the chimeric virus particles (CVPs), may be used in vaccine formulations or treatments, as one of the most powerful potential-personalized-biomedicine as vaccines or social-vaccine against coronavirus-COVID-19, inducing natural-immunity, enriching food-security green-economy science-and-technology-communication applications-issues. It may be the most; cost-effective, easily-available, safe-edible, prepare-able, and applicable-future-potential-biomedicine, and safe alternative to live replicating COVID-19-“Social Vaccines” which mobilized and united us, hope and opportunity opens new-windows for nations to win the 21st-century pandemic-crisis common-enemy, retaining in the old-form.

Recombinant Retroviral Particles: Technology of Poduction and Application as Positive Controls for PCR Diagnostics of Dangerous Viral Infections

Objective. Construction of positive control samples based on recombinant retroviral particles and their application in RT-PCR diagnostic assays for RNA detection of agents of dangerous and particularly dangerous viral infections.Materials and methods. Molecular biological, genetic engineering, and immunological methods were used: polymerase chain reaction, restriction, ligation, cloning, transformation, transfection, flow cytometry.Results and discussion. Technology of positive control samples producing based on recombinant virions has been developed and tested. It includes construction of retroviral vector with cloned diagnostic sequence of the viral genome; obtaining a packaging cell line producing chimeric retroviral particles; determination of recombinant virions titer by flow cytometry and polymerase chain reaction; application of the obtained preparation as a control sample for PCR diagnostics of infectious agents. Positive controls based on retroviral vectors as carriers of genomic RNA fragments of pathogenic viruses were used in the development of PCR diagnostic kits for dangerous and particularly dangerous viral infections. Their application increased the kits quality and made it possible to exclude the work with concentrated hazardous infectious agents (Lassa virus, tick-borne encephalitis virus, lymphocytic choriomeningitis virus, Puumala virus).

A tumor-specific neoepitope expressed in homologous/self or heterologous/viral antigens induced comparable effector CD8+ T-cell responses by DNA vaccination

Somatic mutations in tumors often generate neoproteins that contain MHC-I-binding neoepitopes. Little is known if and how efficient tumor-specific neoantigens activate CD8+ T cells. Here, we asked whether a de novo generated neoepitope, encoded either within an otherwise conserved and ubiquitously expressed self-antigen or in a chimeric HBV core antigen expression platform, providing heterologous helper functions, induces CD8+ T cells in C57Bl/6J mice by DNA immunization. For it, we chose an established Db/Sp244-252/R251H neoepitope generated in the murine Endophilin-B2/SH3GLB2 (EndoB2-Sp) protein by a single amino acid exchange. We showed that a single injection of EndoB2-Sp expression vectors efficiently primed dimer/pentamer+, IFN-γ+ and cytolytic Db/Sp244-252/R251H-specific effector CD8+ T cells in C57Bl/6J mice. Priming of Db/Sp244-252/R251H-specific CD8+ T cells proceeded independent from CD4+ T-cell help in MHC-II-deficient Aα-/- mice. As compared to the homologous EndoB2-Sp vaccine, the selective expression of the Db/Sp244-252/R251H neoepitope in chimeric particle-forming and assembly-deficient HBV core antigens induced comparable frequencies Db/Sp244-252/R251H-specific CD8+ T cells with the same cytolytic effector phenotype. The homologous EndoB2 carrier, but not the nine-residue neoepitope presented on chimeric HBV core particles, induced EndoB2-specific IgG antibody responses. The HBV core expression platform is thus an attractive option to selectively induce neoepitope-specific effector CD8+ T cells by DNA vaccination. These novel findings have practical implications for the design of heterologous/self and heterologous/viral cancer vaccines that prime and/or activate neoepitope-specific CD8+ T cells.

Development of pepper vein banding virus chimeric virus-like particles for potential diagnostic and therapeutic applications.

Monoclonal antibodies have attracted wide attention in therapeutics owing to their high efficacy, low toxicity, and specific targeting. However, antibodies cannot cross the cell membrane barrier. Therefore, their therapeutic potential is limited to surface-exposed antigens or secreted proteins. In the present investigation, we have developed a chimeric virus-like particle (VLP) of pepper vein banding virus (PVBV) and explored the possibility of using it as a delivery vehicle for antibodies against intracellular antigens as well as for future applications in immunodiagnostics. The chimeric PVBV particles were generated by genetically engineering the B domain of Staphylococcus aureus protein A (SpA) at the N-terminus of the PVBV coat protein (CP). The chimeric VLPs purified by sucrose density gradient centrifugation had ~440-fold higher affinity towards IgG antibody when compared to SpA. Interestingly, the unassembled chimeric CP with the B-domain at the N-terminus (BCP) purified by Ni-NTA chromatography was a monomer, and it had ~45-fold higher affinity towards antibodies compared to SpA. Additionally, the chimeric particles were able to bind and deliver antibodies against both intracellular (α-tubulin) and surface-exposed antigens (CD 20). However, the BCP monomer failed to enter mammalian cells. Thus, for the first time, we have demonstrated that the assembled VLPs are essential for internalization. These results demonstrate the potential of the use of chimeric PVBV VLPs in diagnostics and, more importantly, as nanocarriers for intracellular delivery of antibodies.

A recombinant platform for flavivirus vaccines and diagnostics using chimeras of a new insect-specific virus

Flaviviruses such as dengue, yellow fever, Zika, West Nile, and Japanese encephalitis virus present substantial global health burdens. New vaccines are being sought to address safety and manufacturing issues associated with current live attenuated vaccines. Here, we describe a new insect-specific flavivirus, Binjari virus, which was found to be remarkably tolerant for exchange of its structural protein genes (prME) with those of the aforementioned pathogenic vertebrate-infecting flaviviruses (VIFs). Chimeric BinJ/VIF-prME viruses remained replication defective in vertebrate cells but replicated with high efficiency in mosquito cells. Cryo-electron microscopy and monoclonal antibody binding studies illustrated that the chimeric BinJ/VIF-prME virus particles were structurally and immunologically similar to their parental VIFs. Pilot manufacturing in C6/36 cells suggests that high yields can be reached up to 109.5 cell culture infectious dose/ml or ≈7 mg/liter. BinJ/VIF-prME viruses showed utility in diagnostic (microsphere immunoassays and ELISAs using panels of human and equine sera) and vaccine applications (illustrating protection against Zika virus challenge in murine IFNAR-/- mouse models). BinJ/VIF-prME viruses thus represent a versatile, noninfectious (for vertebrate cells), high-yield technology for generating chimeric flavivirus particles with low biocontainment requirements.

Chimeric viruses of the insect-specific flavivirus Palm Creek with structural proteins of vertebrate-infecting flaviviruses identify barriers to replication of insect-specific flaviviruses in vertebrate cells.

Here we report the generation of novel chimeric flaviviruses, which express the prM and E proteins of either dengue or Zika viruses on the genomic backbone of Palm Creek virus (PCV), an insect-specific flavivirus. The chimeric virus particles were antigenically indistinguishable from their parental prM-E donors, but were unable to infect vertebrate cells. An additional chimera (PCV structural genes in the backbone of West Nile virus - WNV/PCV-prME) was also unable to infect vertebrate cells, but transfection with RNA from this virus resulted in detectable RNA replication and translation but no infectious virion production. These data suggest multiple blocks at the entry, RNA replication and assembly/release stages of insect-specific flavivirus (ISF) infection in vertebrate cells. Serial passaging of these chimeric viruses in mosquito cells identified amino acid substitutions that may lead to increased replication efficiency. These chimeric viruses provide unique tools to further dissect the mechanisms of the host restriction of ISFs.