Immunogenicity of foot-and-mouth disease virus structural polyprotein P1 expressed in transgenic rice

Transgenic plants have become developed as bioreactors for producing heterologous proteins and may even form edible vaccines. In the present study, a transgenic rice expressing the capsid precursor polypeptide (P1) gene of foot-and-mouth disease virus (FMDV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, northern blot, western blot, and ELISA analyses confirmed that the P1 gene was integrated into the transgenic rice and the protein was expressed specifically in the leaves at levels of 0.6-1.3 μg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, FMDV-specific neutralizing antibodies were detected. The immunized mice could clear virus from their sera after FMDV challenge. In addition, FMDV-specific mucosal immune responses were detected in mice after oral immunization with protein extracts from transgenic rice plants. Partial virus clearance was obtained after FMDV challenge. These results indicate the potential of using a transgenic rice-based expression system as an alternative bioreactor for FMDV subunit vaccines.     

Generation and immunogenicity of transgenic potato expressing the GP5 protein of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that has caused huge economic losses in the global swine industry. The advent of molecular farming has provided a cost-effective strategy for the development of transgenic plants as bioreactors to produce recombinant proteins. In this study, transgenic potato expressing GP5 protein of PRRSV was produced by Agrobacterium-mediated transformation, and confirmed using Southern blot and RT-PCR analyses. Recombinant GP5 protein was detected by ELISA and Western blot analyses. Mice immunized with transgenic potato extracts generated both serum and gut mucosal-specific antibodies, although low levels of neutralizing antibodies were elicited. This study provides a new approach for the production of vaccines against PRRSV.     

Mapping of epitopes of VP2 protein of chicken anemia virus using monoclonal antibodies

To map the epitopes of VP2 protein of chicken anemia virus (CAV), VP2 was expressed as a fusion protein in Escherichia coli BL21 (DE3). The Western blot demonstrated that recombinant VP2 protein could be recognized by sera of chickens infected with CAV. Female BALB/c mice were immunized with purified recombinant VP2 produced in E. coli BL21 (DE3) and seven VP2-specific monoclonal antibodies (MAbs) were developed. The results of Western blot showed that all the seven MAbs recognized the recombinant VP2 protein expressed in the baculovirus and reacted with MDCC-MSB1 cells infected with CAV by indirect immunofluorescence assay. The VP2 protein was dissected into 21 overlapping fragments, expressed as fusion peptides in E. coli and used for epitope mapping by pepscan analysis. ELISA and Western blot assays indicated that most of MAbs reacted with the 12th and 13th fragments (amino acids 111-136) and one of them reacted with the 3rd fragment (amino acids 21-36). The linear immunodominant epitope of VP2 was located mainly in amino acid residues 111-126 and 121-136.     

Rotavirus VP6 expressed by PVX vectors in Nicotiana benthamiana coats PVX rods and also assembles into viruslike particles

The rotavirus major inner capsid protein (VP6) has been expressed in Nicotiana benthamiana plants using vectors based on potato virus X (PVX). VP6 was expressed either as a fusion with the PVX coat protein or from an additional subgenomic promoter inserted to enable both VP6 and PVX coat protein to be expressed independently. Both approaches yielded VP6, which retained the ability to form trimers. VP6 expressed from the subgenomic promoter assembled into paracrystalline sheets and tubes. Expression as a fusion protein yielded PVX rods that presented an external "overcoat" of VP6, but unexpectedly, some rotavirus protein also assembled into icosahedral viruslike particles (VLPs). The assembly of viral protein into VLPs suggests that prior display of VP6 on the flexuous PVX rod facilitates the subsequent assembly of VP6 into stable icosahedral particles.     

The antigenicity of the chicken anaemia virus protein VP3 (Apoptin)

Chicken anaemia virus protein VP3 (Apoptin) was cloned and expressed as a recombinant protein and evaluated for its suitability as a serodiagnostic reagent. VP3 was expressed as a fusion protein either with glutathione S -transferase or with a six-histidine tag. Both recombinant proteins reacted specifically with anti-VP3 monoclonal antibodies and with serum from vaccinated chickens by Western blot and by enzyme-linked immunosorbent assay (ELISA). However, when testing sera from birds of different ages and genetic backgrounds, high non-specific reactions were evident and false positives were observed, especially in older birds. This suggests that VP3 is poorly immunogenic during infection and low antibody concentrations are masked by non-specific reactions. Thus, VP3 is not suitable for use as antigen in ELISAs.     

Rotavirus VP6 Expressed by PVX Vectors in Nicotiana benthamiana Coats PVX Rods and Also Assembles into Viruslike Particles

The rotavirus major inner capsid protein (VP6) has been expressed in Nicotiana benthamiana plants using vectors based on potato virus X (PVX). VP6 was expressed either as a fusion with the PVX coat protein or from an additional subgenomic promoter inserted to enable both VP6 and PVX coat protein to be expressed independently. Both approaches yielded VP6, which retained the ability to form trimers. VP6 expressed from the subgenomic promoter assembled into paracrystalline sheets and tubes. Expression as a fusion protein yielded PVX rods that presented an external “overcoat” of VP6, but unexpectedly, some rotavirus protein also assembled into icosahedral viruslike particles (VLPs). The assembly of viral protein into VLPs suggests that prior display of VP6 on the flexuous PVX rod facilitates the subsequent assembly of VP6 into stable icosahedral particles.     

抗MDV-1 VP22羧基端单克隆抗体的制备与免疫学特性鉴定

目的：制备抗血清Ⅰ型马立克氏病病毒（MDV-1）VP22的单克隆抗体（mAb），并鉴定其免疫学特性。方法：在原核系统中表达VP22羧基端区域（94～243aa），获得融合表达产物GST-VP22C。将该表达产物切胶免疫小鼠，利用杂交瘤技术制备抗MDV-1VP22C的mAb，并通过ELISA、间接免疫荧光（IFA）、Western blot鉴定其特性。结果：获得了2株可稳定分泌抗MDV-1VP22C的mAb的杂交瘤细胞，命名为3F7、4FA。IFA鉴定表明，两株mAb均能与感染MDV-1的成纤维细胞反应，其中，3F7mAb染色呈现MDV空斑，而4FAmAb呈现整个单层的细胞核荧光。ELISA和Westernblot分析表明，3F7能与杆状病毒表达的VP22反应，4FA不具备该特性。对3F7mAb进一步鉴定，确定了该mAb针对的具体位置在94～193aa处，是蛋白转导域的预测位置。结论：成功地制备了抗MDV-1VP22C的mAb，为深入研究VP22蛋白的转导功能提供了有用的试剂。     

Recombinant VP9-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Banna virus (genus Seadornavirus)

Banna virus (BAV, genus Seadornavirus, family Reoviridae) is an arbovirus suspected to be responsible for encephalitis in humans. Two genotypes of this virus are distinguishable: A (Chinese isolate, BAV-Ch) and B (Indonesian isolate, BAV-In6969) which exhibit only 41% amino-acid identity in the sequence of their VP9.The VP7 to VP12 of BAV-Ch and VP9 of BAV-In6969 were expressed in bacteria using pGEX-4T-2 vector. VP9 was chosen to establish an ELISA for BAV, based mainly on two observations: (i). VP9 is a major protein in virus-infected cells and is a capsid protein (ii). among all the proteins expressed, VP9 was obtained in high amount and showed the highest immuno-reactivity to anti-BAV ascitic fluid.The VP9s ELISA was evaluated in three populations: French blood donors and two populations (blood donors and patients with a neurological syndrome) from Malaysia, representing the region where the virus was isolated in the past.The specificity of this ELISA was >98%. In mice injected with live BAV, the assay detected IgG-antibody to BAV infection 21 days post-injection, which was confirmed by Western blot using BAV-infected cells.The VP9 ELISA permits to determine the sero-status of a population without special safety precautions and without any requirements to propagate the BAV. This test should be a useful tool for epidemiological survey of BAV.     

Production of a Recombinant Major Inner Capsid Protein for Serological Detection of Epizootic Hemorrhagic Disease Virus

Constructs of the major core protein, designated VP7, from epizootic hemorrhagic disease virus (EHDV) type 1 were made by amino- or carboxyl-terminal fusion of a six-histidine residue tag to the VP7-1 gene. The resulting fusion proteins were produced in a baculovirus expression system and purified by a rapid, one-step procedure using nickel-nitrilotriacetic acid technology. A high level of VP7-1 protein expression was detected with the N-terminal six-histidine tag fusion construct and was comparable to the level of expression observed with an untagged VP7-1 Bam construct. In contrast, the inclusion of a six-histidine tag at the C terminus adversely affected protein expression. The antigenicity of the N-terminal six-histidine tag EHDV VP7-1 product was identical to that observed with the native virus antigen and untagged EHDV VP7-1 recombinant protein, as determined by reactivity with EHDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA) and Western blot. The high production and purity levels that can be attained for the N-terminal six-histidine tag VP7-1 protein and its reactivity with EHDV-specific sera in a competitive ELISA make it a suitable assay reagent.     

Production of a recombinant major inner capsid protein for serological detection of epizootic hemorrhagic disease virus

Constructs of the major core protein, designated VP7, from epizootic hemorrhagic disease virus (EHDV) type 1 were made by amino- or carboxyl-terminal fusion of a six-histidine residue tag to the VP7-1 gene. The resulting fusion proteins were produced in a baculovirus expression system and purified by a rapid, one-step procedure using nickel-nitrilotriacetic acid technology. A high level of VP7-1 protein expression was detected with the N-terminal six-histidine tag fusion construct and was comparable to the level of expression observed with an untagged VP7-1 Bam construct. In contrast, the inclusion of a six-histidine tag at the C terminus adversely affected protein expression. The antigenicity of the N-terminal six-histidine tag EHDV VP7-1 product was identical to that observed with the native virus antigen and untagged EHDV VP7-1 recombinant protein, as determined by reactivity with EHDV specific antibodies in an enzyme-linked immunosorbent assay (ELISA) and Western blot. The high production and purity levels that can be attained for the N-terminal six-histidine tag VP7-1 protein and its reactivity with EHDV-specific sera in a competitive ELISA make it a suitable assay reagent.     

Incidence and prevalence of human group C rotavirus infections in Argentina

The incidence of human group C rotavirus infections among children and adults in Buenos Aires was evaluated by enzyme linked immunosorbent assays (ELISA) based on recombinant group C VP6 protein (Cowden strain). A total of 976 stool samples taken from patients (ages 6 months to 15 years) with acute diarrhea were tested for the presence of group C rotavirus. Among these, only 10 (1.02%) were group C rotavirus positive by enzyme-linked immunosorbent assay (ELISA) confirmed by absorption with group C VP6 antibodies and by RT-PCR for both VP6 and VP7 genes. The average age (5.86 years) was significantly superior to that in group A-infected patients (1.63 years). Previous exposure to this virus was assessed by detecting specific IgG in sera taken from healthy individuals grouped by age. Of 844 sera tested, 425 (50.3%) were group C IgG positive by ELISA, confirmed by Western blot analysis. The rates of IgG positivity for group A and C rotaviruses during the first years of life indicated that infections with group C are frequent in older children (3-5 years), whereas group A infections are prevalent in infants and young children (6-18 months). This study shows that group C rotavirus infections in Argentine children occur later in life than group A and are relatively common in spite of the low detection rate of this virus.     

Production of rotavirus-like particles in tomato (Lycopersicon esculentum L.) fruit by expression of capsid proteins VP2 and VP6 and immunological studies

A number of different antigens have been successfully expressed in transgenic plants, and some are currently being evaluated as orally delivered vaccines. Here we report the successful expression of rotavirus capsid proteins VP2 and VP6 in fruits of transgenic tomato plants. By western blot analysis, using specific antibodies, we determined that the VP2 and VP6 produced in plants have molecular weights similar to those found in native rotavirus. The plant-synthesized VP6 protein retained the capacity to form trimers. We were able to recover rotavirus virus-like particles from tomato fruit (i.e., tomatoes) by centrifugation on a sucrose cushion and to visualize them by electron microscopy. This result indicated that VP2/VP6 can self-assemble into virus-like particles (VLPs) in plant cells, even though only a small proportion of VP2/VP6 assembled into VLPs. To investigate immunogenicity, adult mice were immunized intraperitoneally (i.p.) three times with a protein extract from a transgenic tomatoes in adjuvant. We found that the transgenic tomato extract induced detectable levels of anti-rotavirus antibodies in serum; however, we did not determine the contribution of either the free rotavirus proteins or the VLPs to the induction of the antibody response. These results suggest the potential of plant-based rotavirus VLPs for the development of a vaccine against rotavirus infection.     

Detection of parvovirus B19 IgG: choice of antigens and serological tests.

Serum samples were analysed for the presence of (a) IgG against VP1+VP2 using recombinant native conformational antigens by ELISA test (b) IgG against VP2 using recombinant native conformational antigens by ELISA test and (c) IgG against VP1 and against VP2 using denatured linear antigens by Western blot. Out of the 446 samples examined, 353 were positive for specific B19 IgG and out of these, 98.6 % proved positive in the ELISA assay using conformational VP1+VP2 antigens, 94.6% proved positive in the ELISA assay using conformational VP2 antigens, 89.5% were positive at the Western blot assay using denatured linear VP1 and VP2 antigens, with all proving positive for linear VP1 and only 29.5% out of the positive samples proving positive for linear VP2. Since all samples positive by Western blot proved positive by ELISA, our data show that recombinant capsids obtained either with VP1+VP2 or with VP2 alone, used in ELISA, are very useful for detecting the immune response against both conformational and native linear epitopes of B19 structural proteins although some sera may have antibodies directed exclusively against VP1+VP2 antigens and few may have antibodies directed exclusively against VP2 antigens alone.     

Recombinant VP6-based enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Eyach virus (genus Coltivirus).

BACKGROUND: Eyach virus (EYAV) is a tick-borne virus belonging to genus Coltivirus, family Reoviridae. It was isolated in Germany and France and has been suspected to be responsible for neurological diseases in humans. To date, there has been no relatively rapid and relatively specific serological assay for EYAV. OBJECTIVES: To develop an ELISA for EYAV, suitable for epidemiological and/or diagnostic purposes. This ELISA should allow to distinguish between infections with EYAV and the related Colorado tick fever virus (CTFV). STUDY DESIGN: VP6, VP7 and VP12 of Eyach virus (the three proteins most divergent between EYAV and CTFV) were expressed in bacteria using the pGEX-4T-2 vector. A partial sequence of VP6 (designated pVP6) was chosen to develop an ELISA for detecting anti-EYAV IgG antibodies in serum. This choice was based on two observations: (i) the homologous VP7 protein of CTFV was successfully used as a target for detecting antibodies to CTFV (the VP7 showed the highest reactivity to an anti-CTFV antibody among all CTFV expressed proteins); (ii) to distinguish infection with EYAV from a CTFV infection: the expressed sequence was chosen within a region which is highly divergent (49% of amino acid identity) from the homologous VP7 sequence of CTFV. RESULTS AND CONCLUSIONS: pVP6 was shown to be the most reactive among the three expressed proteins. The elaborated pVP6 ELISA was evaluated with 340 sera of French blood donors, and found to exhibit a specificity of 100% (no false positives). Furthermore, no cross reaction was detected with antibody to CTFV, thus permitting us to distinguish between infections by either virus. The use of this recombinant protein for serological assays is a good alternative to the use of native EYAV antigen due to the extremely low productivity of the virus in cell culture, and the requirement for suckling mice. This ELISA will be useful to clarify the epidemiological status and the suspected pathogenicity of the virus.     

Immunogenicity of a plant-derived edible rotavirus subunit vaccine transformed over fifty generations

Major efforts have been put forth for the development of effective rotavirus vaccines including transgenic plant vaccines. Previous studies have reported that rotavirus VP7 maintains its neutralizing immunity when it is transformed into the potato genome. The present study was aimed at investigating the hereditary stability of VP7-transformed potatoes over fifty generations. The VP7 gene was stably transcribed and expressed in potato cells as detected by RT-PCR and Western blotting. Humeral and mucosal responses were successfully induced in BALB/c mice fed with the fiftieth generation transformed potato tubers. There were no significant differences in serum IgG and fecal IgA between the mice fed with the first and fiftieth generation potatoes (P>0.05). Profiles of cytokines such as IFN-gamma, IL-2, IL-4, IL-5 and TGF-beta in immunized mice showed a naive T-cells bias to Th1 and Th3 polarization. Moreover, specific CTL responses were also detected in C57BL/6 mice fed with transformed potatoes. This research represents a significant step towards the development of rotavirus vaccines derived from a transgenic plant that can be obtained by long-term and large-scale vegetative reproduction. To our knowledge, this is the first finding regarding vaccines derived from plants that can be propagated for many generations.