Recombinant Coat Protein Research Articles

Immunodiagnostic detection of banana bract mosaic virus (BBrMV) infecting abaca through the recombinant coat protein and its sequence diversity analysis in the Philippines

Abaca is an indigenous fiber crop in the Philippines and is an important source of raw fiber worldwide. Banana bract mosaic virus (BBrMV) causes the abaca bract mosaic disease which results in decreased fiber yield and quality. Out of the 261 abaca samples tested, 175 tested positive via ELISA using raw antiserum against the recombinant BBrMV coat protein (CP). To further validate the antisera produced, a total of 48 samples were screened for BBrMV CP fragment via RT-PCR. Out of the 26 positives for RT-PCR, 22 were cloned and sequenced to validate the RT-PCR amplified fragments. Nucleotide analysis showed a high percentage identity (97–98%) towards BBrMV CP Pacific and Indian isolates while phylogenetic analysis revealed clustering of BBrMV CP Philippine abaca isolates with the Pacific group. The results validate the application of polyclonal antibodies against the recombinant coat protein of BBrMV (rCP) for its serological detection in infected abaca.

Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India

Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507 bp partial coat protein gene (pCPG) segment was amplified from infected kinnow leaf tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23 kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC-RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL−1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507 bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL−1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.

Development of Polyclonal Antibodies-Based Serological Method for the Detection of Calanthe Mild Mosaic Virus and Application in Virus Certification Programme.

Calanthe mild mosaic virus (CalMMV) infecting orchids is an important potyvirus which is known to cause mild leaf mosaic and flower colour-breaking symptoms in Calanthe and other orchid plants. The present study reports the production of polyclonal antibodies against CalMMV using bacterially expressed recombinant coat protein as immunogen, which in turn would be useful in routine indexing and screening of orchid germplasm. The coat protein (CP) gene (~ 807bp) of CalMMV isolated from infected orchid sample was cloned in expression vector, pET-28a ( +) that yielded ~ 31kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was confirmed through SDS-PAGE and Western blotting. The His6BP-CalMMV-CP obtained in soluble state after purification was used to immunize New Zealand white rabbit for the production of polyclonal antibodies (PAb). The PAb produced against the purified fusion protein successfully detected CAlMMV in the orchid samples at a dilution of 1:2000 in direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). This study presents the first report of Histidine tag (His6BP) fusion CalMMV-CP-based antibody production and its successful application in the identification of the virus in orchid plants. Outcome of this study will be helpful in routine certification programmes, screening of orchid germplasm and production of CalMMV-free planting materials of orchids.

Rapid detection of apple latent spherical virus infecting Angelica sinensis by gold immunochromatography reverse transcription loop-mediated isothermal amplification assay

Apple latent spherical virus (ALSV; genus: Cheravirus, family: Secoviridae) is capable of infecting Angelica sinensis under natural conditions and causing symptoms of greenish discoloration and mottling, affecting plant growth and causing a reduction in root quality and yield. Virus detection of ALSV is an urgent need for the continued development of angelica industry. Consequently, it is necessary to develop simple, sensitive, and reliable ALSV detection methods. We describe the preparation of polyclonal antibodies with high titer and high specificity using recombinant ALSV coat protein (CP) as the antigen. Using these antibodies, we established and optimized a rapid detection system using colloidal gold immunochromatography assay (GICA) to detect ALSV. A real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) system with a minimum detection line of 2.72 × 103copys/μl was also developed to detect ALSV. This assay was further enhanced using a combination GICA-RT-LAMP assay. This GICA assay successfully detected ALSV in infected plants without cross reactivity recorded from three other plant viruses. The sensitivity of GICA was the same as that of GICA-RT-PCR, whereas the sensitivity of GICA-RT-LAMP was at least 10-fold higher than that of GICA and GICA-RT-PCR. These methods can detect and identify ALSV under field conditions and accurately measure virus concentration in the laboratory.

The efficacy of a chicken antibody for the development of immunoassay‐based rapid detection in sugarcane mosaic virus disease

Sugarcane Mosaic Virus (SCMV) infection is one of the most serious problems that can result in severe yield loss of sugarcane. Since the symptoms of SCMV infection are similar to other biotic and abiotic stress symptoms, the development of a rapid diagnostic with high precision is required. The use of laboratory animals such as rabbits is required for antibody production in immunoassay‐based detection. However, due to its many advantages, specific chicken egg yolk immunoglobulin (IgY) has received considerable attention as an alternative antibody production in immunodiagnostics for infectious diseases. In this study, IgY antibody against SCMV recombinant coat protein (CP) was successfully obtained from chicken blood serum and tested to compare its efficacy against antibody from rabbit (IgG) using immunocapture reverse transcription‐polymerase chain reaction (IC‐RT‐PCR). The result showed that IgY and IgG could detect 0.1 g SCMV infected leaves using 1000‐times‐diluted antibodies. The IgY antibody was also confirmed to be reproducible and potentially applicable in plant disease diagnostics using an antibody‐based detection.

An improved oral vaccine with molecular adjuvant β-defensin protects grouper against nervous necrosis virus infection

Nervous necrosis virus (NNV) is one of the most important fish viral pathogens infecting more than 120 fish species worldwide. Due to the mass mortality rates often seen among larvae and juveniles, few effective vaccines against NNV were developed up to now. Here, the protective effect of recombinant coat protein (CP) from red-spotted grouper nervous necrosis virus (RGNNV) fused with grouper β-defensin (DEFB) as an oral vaccine was evaluated using Artemia as a biocarrier delivery system in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀). Feeding with Artemia encapsulated with E. coli expressing control vector (control group), CP, or CP-DEFB showed no obvious side effects on the growth of groupers. ELISA and antibody neutralization assay showed that CP-DEFB oral vaccination group induced higher anti-RGNNV CP specific antibodies and exhibited higher neutralization potency than the CP and control group. Meanwhile, the expression levels of several immune and inflammatory factors in the spleen and kidney after feeding with CP-DEFB were also significantly increased compared with the CP group. Consistently, after challenge with RGNNV, groupers fed CP-DEFB and CP exhibited 100% and 88.23% relative percentage survival (RPS), respectively. Moreover, the lower transcription levels of viral genes and milder pathological changes in CP-DEFB group were detected compared with the CP and control group. Thus, we proposed that grouper β-defensin functioned as an efficient molecular adjuvant for an improved oral vaccine against nervous necrosis virus infection.

Generation of specific antibody against recombinant major coat protein of Beet necrotic yellow vein virus and its application for detection of rhizomania disease

Beet necrotic yellow vein virus (BNYVV) is one of the main contributors to economic losses in sugar beet production. The present study generated a polyclonal antibody that detects BNYVV. The conserved genomic region of all BNYVV isolates gene encoding the viral major coat protein (BNYVV-CP) was amplified, cloned, sequenced, and expressed in E. coli strain BL21 (DE3). The expressed protein was purified under native conditions by affinity chromatography. The purified BNYVV CP was used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-CP-IgG detected the BNYVV-CP recombinant protein and BNYVV in infected sugar beet by indirect-ELISA, dot immunobinding assay, and western blot analysis blotting. The serological assays strongly proved the sensitivity and specificity of anti-BNYVV-CP-IgG against BNYVV. These results suggest that the generated anti-BNYVV-CP-IgG polyclonal antibodies can be used as reliable and quick means for the BNYVV virus detection under field conditions.

Production of polyclonal antibodies against leek yellow stripe virus (LYSV) coat protein expressed in Escherichia coli and its application in serological diagnostics.

Leek yellow stripe virus (LYSV) is one of the most important potyviruses, associated with garlic throughout the world, including India. LYSV causes stunting and yellow streaks in garlic and leek leaves and with other coinfecting viruses leading to severe symptom expression and yield reduction. In this study, we have made the first reported attempt to produce specific polyclonal antibodies to LYSV using expressed recombinant coat protein (CP), which would be useful for screening and routine indexing of the garlic germplasm. The CP gene was cloned, sequenced, and further subcloned in pET-28a(+) expression vector, which yielded ∼35 kDa fusion protein. The fusion protein was obtained in insoluble fraction after purification and its identity was confirmed by SDS-PAGE and western blotting. The purified protein was used as immunogen for production of polyclonal antisera in New Zealand white rabbit. Antisera raised, was able to recognize the corresponding recombinant proteins in western blotting, immunosorbent electron microscopy and dot immunobinding assay (DIBA). Developed antisera to LYSV (titer 1:2000) was used for screening of 21 garlic accessions in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and 16 accessions were found positive for LYSV, indicating its widespread presence within the collection tested. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in-vitro expressed CP of LYSV and its successful application in diagnosis of LYSV in garlic accessions in India.

Design, cloning and expression of a synthetic gene encoding a grouper iridovirus major capsid protein

Synthetic biology approaches can be adopted to successfully redesign, clone and express recalcitrant proteins from viruses in a bacterial expression system. The grouper iridovirus major capsid proteins (GI-MCP) is a surface coat protein which has the potential for application as an antigen for the development of vaccines which confer immunity against GI. The native amino acid coding region of the GI-MCP does not lend itself to expression in the Escherichia coli (BL21) (DE3) platform due to the presence of internal motifs which represent the start and stop codons as well as secondary structures. This manuscript reports on the redesigning, cloning and expression of a synthetic GI-MCP in E. coli. The native GI-MCP protein coding region was retrieved from the NCBI GenBank. The sequence was codon optimized, internal start and stop codons were modified and potential secondary structures were resolved. Both the native and modified GI-MCP coding DNA sequences were synthesized and ligated onto an expression plasmid, pET22b (+) followed by transformation, cloning and expression in E. coli (BL21) (DE3). Induction of protein expression was carried out at 12°C, 20°C and 37°C to assess protein solubility. The gene encoding the native GI-MCP failed to express under any of the culture conditions, however, the modified synthetic gene encoding the GI-MCP expressed the recombinant iridovirus coat protein (rGI-MCP) under all the conditions. Gene design and synthesis offer an ideal solution for the recombinant expression of viral proteins in bacterial expression systems for the industrial production of viral antigens.

A new single-chain variable fragment (scFv) antibody provides sensitive and specific detection of citrus tristeza virus

Citrus tristeza virus (CTV) is the most economically important virus disease of citrus worldwide. To develop a specific serological assay for CTV, a Tomlinson phage display antibody library of single chain variable fragments (scFv) was screened with a recombinant CTV coat protein (CTV-CP) heterologously expressed in Escherichia coli. The phage clones were checked by ELISA to identify clones with high specificity for CTV-CP. Eight clones were strongly reactive with CTV-CP. Nucleotide sequencing of these clones revealed that all of them contained the same sequence. Thus, the phage-displayed scFv antibody was termed scFvF10. Evaluation of scFvF10 binding to CTV-CP by plate-trapped antigen ELISA (PTA-ELISA) and immunoblotting, showed that it was specific and allowed sensitive detection of CTV-CP. Homology-based molecular modeling and docking analysis confirmed that the interaction between CTV-CP and scFvF10, with a binding energy of -738 kj mol-1, occurred mainly by 12 intermolecular hydrogen bonds. Moreover, triple-antibody sandwich (TAS)-ELISA using scFvF10 as second antibody showed high sensitivity in the detection of CTV infected samples. The CTV detection limit of scFvF10 by PTA-ELISA and TAS-ELISA were 0.05 and 0.01 μg CP/mL, respectively. Our results with different diagnostic assays demonstrated that scFvF10 has the potential to be used as an efficient tool for CTV-infected plant diagnosis.

Restriction-free cloning for molecular manipulation and augmented expression of banana bunchy top viral coat protein

The online version contains supplementary material available at 10.1007/s13205-021-03017-x.

Development of polyclonal antibodies using bacterially expressed recombinant coat protein for the detection of Onion yellow dwarf virus (OYDV) and identification of virus free onion genotypes

Onion yellow dwarf virus (OYDV) belonging to the genus Potyvirus, family Potyviridae, is one of the widely distributed viruses of Allium species worldwide. It causes dwarfing, yellow striping, crinkling and flaccidity of the leaves of onion and garlic. To see the occurrence and incidence of OYDV on Allium crop, an attempt was made to develop antibody based diagnostic assay which would be useful for routine indexing and screening of the germplasm. The total RNA was isolated from the symptomatic leaves of onion and the gene encoding coat protein (CP) was cloned. The nucleotide sequencing analysis of the cloned RT-PCR product revealed ~ 774bp amplicon (OYDV CP) and it was further cloned in pET-28a ( +) expression vector which yielded ~ 30kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was primarily checked on SDS-PAGE and further confirmed by Western blot. The His6BP-OYDV-CP was obtained in soluble state after purification and was used to immunize New Zealand white rabbit for the production of polyclonal antibody (PAb). The produced PAb against the purified fusion protein successfully detected OYDV from onion and garlic samples at 1:2000 dilutions in indirect-enzyme linked immunosorbent assay (DAC-ELISA). Thus, this study presents first report that Histidine tag (His6BP) fusion OYDV-CP based antibody production and its successful application in identification of virus free onion and garlic genotypes.

Development of immunocapture reverse transcription loop‐mediated isothermal amplification assays to detect both lily symptomless virus and cucumber mosaic virus

AbstractLily symptomless virus (LSV) and cucumber mosaic virus (CMV) are two of the most prevalent viruses that infect lily plants around the world. Infections of the edible Lanzhou lily (Lilium davidii var. unicolor) by these viruses seriously decrease crop yield in northwestern China. Development of reliable, practical and sensitive diagnostic tools is essential for surveillance and management of the viruses. Here we describe the development of immunocapture (IC) reverse transcription (RT) loop‐mediated isothermal amplification (LAMP) assays for detecting both viruses. This approach captures viral antigens using antibodies (anti‐rabbit IgG) against recombinant LSV or CMV coat proteins and then detects them by means of RT‐LAMP, without needing to isolate RNA. Optimal conditions for both LAMP reactions were 60°C for 60 min. The detection limit of both assays was 100 times more sensitive than the IC‐RT‐polymerase chain reaction assay. The IC‐RT‐LAMP assay results are also visible to the naked eye using SYBR Green dye. This is the first study to report the detection of LSV and CMV in lily using an IC‐RT‐LAMP assay. We believe that this convenient and sensitive method has excellent potential for enhanced diagnosis of field‐collected samples, especially where laboratory facilities and technical resources are limited or absent.

Development of a triple antibody sandwich enzyme-linked immunosorbent assay for cassava mosaic disease detection using a monoclonal antibody to Sri Lankan cassava mosaic virus

BackgroundCassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia. Accurate yet affordable diagnostics are one of the fundamental tools supporting successful CMD management, especially in developing countries. This study aimed to develop an antibody-based immunoassay for the detection of Sri Lankan cassava mosaic virus (SLCMV), the only cassava mosaic begomovirus currently causing CMD outbreaks in Southeast Asia (SEA).MethodsMonoclonal antibodies (MAbs) against the recombinant coat protein of SLCMV were generated using hybridoma technology. MAbs were characterized and used to develop a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for SLCMV detection in cassava leaves and stems. Assay specificity, sensitivity and efficiency for SLCMV detection was investigated and compared to those of a commercial ELISA test kit and PCR, the gold standard.ResultsA TAS-ELISA for SLCMV detection was successfully developed using the newly established MAb 29B3 and an in-house polyclonal antibody (PAb) against begomoviruses, PAb PK. The assay was able to detect SLCMV in leaves, green bark from cassava stem tips, and young leaf sprouts from stem cuttings of SLCMV-infected cassava plants without cross-reactivity to those derived from healthy cassava controls. Sensitivity comparison using serial dilutions of SLCMV-infected cassava sap extracts revealed that the assay was 256-fold more sensitive than a commercial TAS-ELISA kit and 64-fold less sensitive than PCR using previously published SLCMV-specific primers. In terms of DNA content, our assay demonstrated a limit of detection of 2.21 to 4.08 × 106 virus copies as determined by quantitative real-time PCR (qPCR). When applied to field samples (n = 490), the TAS-ELISA showed high accuracy (99.6%), specificity (100%), and sensitivity (98.2%) relative to the results obtained by the reference PCR. SLCMV infecting chaya (Cnidoscolus aconitifolius) and coral plant (Jatropha multifida) was also reported for the first time in SEA.ConclusionsOur findings suggest that the TAS-ELISA for SLCMV detection developed in this study can serve as an attractive tool for efficient, inexpensive and high-throughput detection of SLCMV and can be applied to CMD screening of cassava stem cuttings, large-scale surveillance, and screening for resistance.

Potato virus Y recombinant coat protein production in Escherichia coli: Applications in antibody production and virus detection

AbstractThe coat protein coding region of Potato virus Y (PVY) was cloned into pGEM‐T, sub‐cloned into plasmid pET‐28a (+) then transformed into Escherichia coli strain BL21 (DE3) pLysS to express the protein. Induction of the recombinant protein was made with isopropyl‐β‐d‐thiogalactopyranoside (IPTG), which has been tested in a variety of concentrations and for varying periods of time. The highest expression level of the protein was achieved with 1 mM IPTG for 4 hr. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) followed by Western blotting has been used to confirm the identity of the expressed protein. The PVY coat protein (30 kDa) was purified under denaturing conditions by affinity chromatography and identity reconfirmed by Western blotting. The expressed protein was used as a recombinant antigen to raise polyclonal antibodies in mice. Purified anti‐PVYCP immunoglobulin (IgG) reacted positively with PVY‐infected leaf samples and the purified expressed CP in plate‐trapped antigen enzyme‐linked immunosorbent assay (PTA‐ELISA). The currently produced antiserum detects the more prevailing strains in the region: PVY N, NTN and Wilga.

Bacteriophage‐mediated therapy of chondrosarcoma by selective delivery of the tumor necrosis factor alpha (TNFα) gene

Chondrosarcoma is a cartilage-forming bone tumor, well known for intrinsic resistance to chemotherapy and radiotherapy. We have designed a targeted chondrosarcoma gene therapy using a bacteriophage (phage) particle to deliver therapeutic genes. Phage has no tropism for mammalian cells, allowing engineered phage to be targeted to specific cell surface receptors in cancer. We modified the phage capsid to display the RGD4C ligand on the pIII minor coat proteins to specifically bind to αvβ3 or αvβ5 integrin receptors. The endosomal escape peptide, H5WYG, was also displayed on recombinant pVIII major coat proteins to enhance gene delivery. Finally, a human tumor necrosis factor alpha (TNFα) therapeutic transgene expression cassette was incorporated into the phage genome. First, we found that human chondrosarcoma cells (SW1353) have high expression of αvβ3, αvβ5 integrin receptors, and both TNFα receptors. Targeted particle encoding a luciferase reporter gene efficiently and selectively mediated gene delivery to these cells. When SW1353 cells were treated with the targeted particle encoding a TNFα transgene, significant cell killing was evident and was associated with high expression of TNFα and apoptosis-related genes. In vivo, mice with established human chondrosarcoma showed suppression of tumors upon repetitive intravenous administrations of the targeted phage. These data show that our phage-based particle is a promising, selective, and efficient tool for targeted chondrosarcoma therapy.

Recombinant turnip yellow mosaic virus coat protein as a potential nanocarrier.

To display a short peptide (GSRSHHHHHH) at the C-terminal end of turnip yellow mosaic virus coat protein (TYMVc) and to study its assembly into virus-like particles (TYMVcHis6 VLPs). In this study, recombinant TYMVcHis6 expressed in Escherichia coli self-assembled into VLPs of approximately 30-32nm. SDS-PAGE and Western blot analysis of protein fractions from the immobilized metal affinity chromatography (IMAC) showed that TYMVcHis6 VLPs interacted strongly with nickel ligands in IMAC column, suggesting that the fusion peptide is protruding out from the surface of VLPs. These VLPs are highly stable over a wide pH range from 3·0 to 11·0 at different temperatures. At pH 11·0, specifically, the VLPs remained intact up to 75°C. Additionally, the disassembly and reassembly of TYMVcHis6 VLPs were studied in vitro. Dynamic light scattering and transmission electron microscopy analysis revealed that TYMVcHis6 VLPs were dissociated by 7moll-1 urea and 2moll-1 guanidine hydrochloride (GdnHCl) without impairing their reassembly property. A 10-residue peptide was successfully displayed on the surface of TYMVcHis6 VLPs. This chimera demonstrated high stability under extreme thermal conditions with varying pH and was able to dissociate and reassociate into VLPs by chemical denaturants. This is the first C-terminally modified TYMVc produced in E. coli. The C-terminal tail which is exposed on the surface can be exploited as a useful site to display multiple copies of functional ligands. The ability of the chimeric VLPs to self-assemble after undergo chemical denaturation indicates its potential role to serve as a nanocarrier for use in targeted drug delivery.

Modulation of Antigen Display on PapMV Nanoparticles Influences Its Immunogenicity.

Background: The papaya mosaic virus (PapMV) vaccine platform is a rod-shaped nanoparticle made of the recombinant PapMV coat protein (CP) self-assembled around a noncoding single-stranded RNA (ssRNA) template. The PapMV nanoparticle induces innate immunity through stimulation of the Toll-like receptors (TLR) 7 and 8. The display of the vaccine antigen at the surface of the nanoparticle, associated with the co-stimulation signal via TLR7/8, ensures a strong stimulation of the immune response, which is ideal for the development of candidate vaccines. In this study, we assess the impact of where the peptide antigen is fused, whether at the surface or at the extremities of the nanoparticles, on the immune response directed to that antigen. Methods: Two different peptides from influenza A virus were used as model antigens. The conserved M2e peptide, derived from the matrix protein 2 was chosen as the B-cell epitope, and a peptide derived from the nucleocapsid was chosen as the cytotoxic T lymphocytes (CTL) epitope. These peptides were coupled at two different positions on the PapMV CP, the N- (PapMV-N) or the C-terminus (PapMV-C), using the transpeptidase activity of Sortase A (SrtA). The immune responses, both humoral and CD8+ T-cell-mediated, directed to the peptide antigens in the two different fusion contexts were analyzed and compared. The impact of coupling density at the surface of the nanoparticle was also investigated. Conclusions: The results demonstrate that coupling of the peptide antigens at the N-terminus (PapMV-N) of the PapMV CP led to an enhanced immune response to the coupled peptide antigens as compared to coupling to the C-terminus. The difference between the two vaccine platforms is linked to the enhanced capacity of the PapMV-N vaccine platform to stimulate TLR7/8. We also demonstrated that the strength of the immune response increases with the density of coupling at the surface of the nanoparticles.

NOVEL APPROACH FOR DESIGNING ROTAVIRUS VACCINE CANDIDATE BASED ON TWO PLANT VIRUSES

Rotavirus А (genus Rotavirus, family Reoviridae) is still the main cause of viral gastroenteritis in children under 5 years. Existing attenuated vaccines have serious disadvantages, including the risk of potential reversion to pathogenic form, and side effects, the most dangerous of which is intussusception. Moreover, they occurred to be less effective in developing countries, where the most rotavirus-associated deaths are recorded. The development of an effective recombinant rotavirus A vaccine is an actual assignment; herewith the selection of effective and safe adjuvant is the key point for that. Plant viruses are very promising for innovative vaccine designing; they possess high immunostimulating properties, safe for humans and mammals and can serve as a carrier for pathogens’ epitopes. Here we suggest an approach for rotavirus A vaccine development that involves two plant viruses: Alternanthera mosaic virus (AltMV) and Tobacco mosaic virus (TMV) as simultaneously epitope carriers and adjuvants. Spherical particles (SPs) generating by the heating of tobacco mosaic virus were used as an adjuvant and platform for presentation of obtained in our previous study chimeric recombinant protein ER6, which is an AltMV coat protein (CP) fused with the epitope RV14 (RLSFQLMRPPNMTP) of rotavirus A antigen VP6. Epitope RV14 are able to induce protective immune response and is conservative for the majority of rotavirus A strains therefore its usage gives hope to the successful overcoming of one of the main difficulties in rotavirus A vaccine development: wide serological diversity. In present work, effective adsorption of ER6 on the SPs surface leading to the SPs-ER6 complex formation without loss of ER6 antigenic specificity was demonstrated. Two immune antisera with specificity to RV14 epitope within ER6 were obtained. The first serum was obtained via anti-ER6 sera depletion with AltMV CP and recombinant AltMV CP (AltMV rCP), which was expressed in Escherichia coli but did not contain RV14 sequence. The second serum was obtained by a direct immunization with synthetic peptide RLSFQLMRPPNMTP. These sera were utilized for studying RV14 within SPs-ER6 complexes. By means of immunofluorescent microscopy, SPs-ER6 complexes were demonstrated to interact with both depleted serum and anti-RV14 serum. Therefore, rotavirus epitope was confirmed to keep its ability to interact with antibodies within obtained complexes. Considering unique adjuvant properties of spherical particles and characteristics of selected epitope obtained SPs-ER6 complexes can be thought as a promising component for recombinant rotavirus A vaccine. Moreover, it can be hoped that the suggested in present work approach, involving the usage of TMV SPs as a platform and adjuvant for chimeric AltMV CP, containing pathogen’s epitope, will be useful not only for rotavirus A vaccine development but for designing of vaccines against other pathogens of humans or farm animals.

НОВЫЙ ПОДХОД К РАЗРАБОТКЕ КАНДИДАТНОЙ ВАКЦИНЫ ПРОТИВ РОТАВИРУСА НА ОСНОВЕ ДВУХ ВИРУСОВ РАСТЕНИЙ

Rotavirus А (genus Rotavirus, family Reoviridae) is still the main cause of viral gastroenteritis in children under 5 years. Existing attenuated vaccines have serious disadvantages, including the risk of potential reversion to pathogenic form, and side effects, the most dangerous of which is intussusception. Moreover, they occurred to be less effective in developing countries, where the most rotavirus-associated deaths are recorded. The development of an effective recombinant rotavirus A vaccine is an actual assignment; herewith the selection of effective and safe adjuvant is the key point for that. Plant viruses are very promising for innovative vaccine designing; they possess high immunostimulating properties, safe for humans and mammals and can serve as a carrier for pathogens’ epitopes. Here we suggest an approach for rotavirus A vaccine development that involves two plant viruses: Alternanthera mosaic virus (AltMV) and Tobacco mosaic virus (TMV) as simultaneously epitope carriers and adjuvants. Spherical particles (SPs) generating by the heating of tobacco mosaic virus were used as an adjuvant and platform for presentation of obtained in our previous study chimeric recombinant protein ER6, which is an AltMV coat protein (CP) fused with the epitope RV14 (RLSFQLMRPPNMTP) of rotavirus A antigen VP6. Epitope RV14 are able to induce protective immune response and is conservative for the majority of rotavirus A strains therefore its usage gives hope to the successful overcoming of one of the main difficulties in rotavirus A vaccine development: wide serological diversity. In present work, effective adsorption of ER6 on the SPs surface leading to the SPs-ER6 complex formation without loss of ER6 antigenic specificity was demonstrated. Two immune antisera with specificity to RV14 epitope within ER6 were obtained. The first serum was obtained via anti-ER6 sera depletion with AltMV CP and recombinant AltMV CP (AltMV rCP), which was expressed in Escherichia coli but did not contain RV14 sequence. The second serum was obtained by a direct immunization with synthetic peptide RLSFQLMRPPNMTP. These sera were utilized for studying RV14 within SPs-ER6 complexes. By means of immunofluorescent microscopy, SPs-ER6 complexes were demonstrated to interact with both depleted serum and anti-RV14 serum. Therefore, rotavirus epitope was confirmed to keep its ability to interact with antibodies within obtained complexes. Considering unique adjuvant properties of spherical particles and characteristics of selected epitope obtained SPs-ER6 complexes can be thought as a promising component for recombinant rotavirus A vaccine. Moreover, it can be hoped that the suggested in present work approach, involving the usage of TMV SPs as a platform and adjuvant for chimeric AltMV CP, containing pathogen’s epitope, will be useful not only for rotavirus A vaccine development but for designing of vaccines against other pathogens of humans or farm animals.