E2 Glycoprotein Research Articles

Surface display of classical swine fever virus E2 glycoprotein on gram-positive enhancer matrix (GEM) particles via the SpyTag/SpyCatcher system

The E2 envelope protein is the main protective antigen of classical swine fever virus (CSFV). Importantly, gram-positive enhancer matrix (GEM) particles can work as an immunostimulant and/or carrier system to improve the immune effect of antigens. In this study, the artificially designed E2-Spy was expressed and glycosylated in Pichia pastoris, and subsequently conjugated with SpyCatcher-PA which was expressed in Escherichia coli. The conjugated E2-Spy-PA was displayed on the surface of GEM particles, generating the E2-Spy-PA-GEM complex. Blocking ELISA analysis and neutralization assays showed that both E2-Spy and E2-Spy-PA-GEM complexes induced high levels of anti-CSFV antibodies in mice. Furthermore, statistical analyses indicated that the E2-Spy-PA-GEM complex exhibited enhanced immunogenicity compared with E2-Spy alone.

Optimized expression of classical swine fever virus E2 protein via combined strategy in Pichia pastoris

Precaution of classical swine fever (CSF) is an important mission for the worldwide swine industry. Glycoprotein E2 is the leading antigen candidate for subunit vaccine of classical swine fever virus (CSFV). In this study, two Spy-tagged E2 genes were synthesized in vitro and subcloned into pMCO-AOX vector for intracellular expression in Pichia pastoris after methanol induction. Western blot analysis and semi-quantitative analysis showed that the yield of recombinant E2 protein was improved 17.87 folds by using co-translocational signal peptide cSIG. After the construction of the tandem multiple copy expression vectors, further increase of E2 production was observed by repetitive transforming expression vectors into P. pastoris genome. Finally, the yeast transformants harboring 8 or 16 copies of cSIG-E2-Spy increased the E2 expression level by 27.01-fold or 30.72-fold, respectively. These results demonstrate that utilizing co-translocational signal peptide together with multi-copy integration strategy can increase the production of recombinant E2 protein efficiently.

Infectious vaccine-derived rubella viruses emerge, persist, and evolve in cutaneous granulomas of children with primary immunodeficiencies.

Rubella viruses (RV) have been found in an association with granulomas in children with primary immune deficiencies (PID). Here, we report the recovery and characterization of infectious immunodeficiency-related vaccine-derived rubella viruses (iVDRV) from diagnostic skin biopsies of four patients. Sequence evolution within PID hosts was studied by comparison of the complete genomic sequences of the iVDRVs with the genome of the vaccine virus RA27/3. The degree of divergence of each iVDRV correlated with the duration of persistence indicating continuous intrahost evolution. The evolution rates for synonymous and nonsynonymous substitutions were estimated to be 5.7 x 10−3 subs/site/year and 8.9 x 10−4 subs/site/year, respectively. Mutational spectra and signatures indicated a major role for APOBEC cytidine deaminases and a secondary role for ADAR adenosine deaminases in generating diversity of iVDRVs. The distributions of mutations across the genes and 3D hotspots for amino acid substitutions in the E1 glycoprotein identified regions that may be under positive selective pressure. Quasispecies diversity was higher in granulomas than in recovered infectious iVDRVs. Growth properties of iVDRVs were assessed in WI-38 fibroblast cultures. None of the iVDRV isolates showed complete reversion to wild type phenotype but the replicative and persistence characteristics of iVDRVs were different from those of the RA27/3 vaccine strain, making predictions of iVDRV transmissibility and teratogenicity difficult. However, detection of iVDRV RNA in nasopharyngeal specimen and poor neutralization of some iVDRV strains by sera from vaccinated persons suggests possible public health risks associated with iVDRV carriers. Detection of IgM antibody to RV in sera of two out of three patients may be a marker of virus persistence, potentially useful for identifying patients with iVDRV before development of lesions. Studies of the evolutionary dynamics of iVDRV during persistence will contribute to development of infection control strategies and antiviral therapies.

Descriptive epidemiology of rubella disease and associated virus strains in Uganda.

Rubella virus causes a mild disease; however, infection during the first trimester of pregnancy may lead to congenital rubella syndrome (CRS) in over 80% of affected pregnancies. Vaccination is recommended and has been shown to effectively reduce CRS incidence. Uganda plans to introduce routine rubella vaccination in 2019. The World Health Organization recommends assessing the disease burden and obtaining the baseline molecular virological data before vaccine introduction. Sera collected during case‐based measles surveillance from January 2005 to July 2018 were tested for rubella immunoglobulin M (IgM) antibodies. Sera from confirmed rubella outbreaks from January 2012 to August 2017 were screened using real‐time reverse‐transcription polymerase chain reaction (RT‐PCR); for positive samples, a region within the E1 glycoprotein coding region was amplified and sequenced. Of the 23 196 suspected measles cases serologically tested in parallel for measles and rubella, 5334 (23%) were rubella IgM‐positive of which 2710 (50.8%) cases were females with 2609 (96.3%) below 15 years of age. Rubella IgM‐positive cases were distributed throughout the country and the highest number was detected in April, August, and November. Eighteen (18%) of the 100 sera screened were real‐time RT‐PCR‐positive of which eight (44.4%) were successfully sequenced and genotypes 1G and 2B were identified. This study reports on the seroprevalence and molecular epidemiology of rubella. Increased knowledge of former and current rubella viruses circulating in Uganda will enhance efforts to monitor the impact of vaccination as Uganda moves toward control and elimination of rubella and CRS.

Bioprocess optimization for purification of chimeric VLP displaying BVDV E2 antigens produced in yeast Hansenula polymorpha

Chimeric virus-like particles (VLP) are known as promising tools in the development of safe and effective subunit vaccines. Recently, a technology platform to produce VLP based on the small surface protein (dS) of the duck hepatitis B virus was established. In this study, chimeric VLP were investigated displaying the 195 N-terminal amino acids derived from the glycoprotein E2 of the bovine viral diarrhea virus (BVDV) on their surface. Isolation of the VLP from methylotrophic yeast Hansenula polymorpha was allowed upon co-expression of wild-type dS and a fusion protein composed of the BVDV-derived antigen N-terminally fused to the dS. It was shown the VLP could be purified by a process adapted from the production of a recombinant hepatitis B VLP vaccine. However, the process essentially depended on costly ultracentrifugation which is critical for low cost production. In novel process variants, this step was avoided after modification of the initial batch capture step, the introduction of a precipitation step and adjusting the ion exchange chromatography. The product yield could be improved by almost factor 8 to 93 ± 12 mg VLP protein per 100 g dry cell weight while keeping similar product purity and antigenicity. This allows scalable and cost efficient VLP production.

Chikungunya Virus as the Agent of Emergent Viral Disease

Chikungunya virus belongs to Alphavirus genus of the Togaviridae family. It is a member of Semliki Forest virus antigenic complex that includes antigenic related Semliki Forest, Chikungunya, O’ Nyong-nyong, Ross River viruses. Chikungunya virus is the causative agent of acute febrile illness with myalgia and arthralgia in humans. Since its discovery in 1952, Chikungunya virus caused sporadic and infrequent outbreaks. Since 2004, global Chikungunya outbreaks have occurred. Now Chikungunya is viewed as a global public health issue in many countries, where Aedes mosquito vectors are widespread. Currently, four genotypes of Chikungunya virus (West African, South African, Asian and Indian Ocean) are distinguished. Appearance of different genotypes is associated with adaptive mutations in peplomers of E1 and E2 glycoproteins. It is shown, that a single mutation in E1 glycoprotein (alanin for valin substitution in 226 position) leads to increasing virus virulence (50-100 times). This mutation is instrumental for epidemic potential increase. For virus variants with this mutation, secondary substitutions enhancing viral virulence are described too. А edes aegypti mosquitoes are common vector for all genotypes of Chikungunya virus, А edes albopictus mosquitoes are vector, mainly, for South African and Asian genotypes. They play the leading role in epidemic potential increase over the last decade. The effectiveness of Chikungunya virus transmission by А edes а egypti mosquitoes is 83.3 %, by А edes albopictus mosquitoes - 96.7 %. The А edes albopictus are more widely disseminated than А edes а egypti (about 40 percent of all land territory). Demonstrated is the possibility of transcontinental spread of А edes albopictus mosquitoes by aviation and naval transport. This review highlights the most recent advances in our knowledge of the ecology, epidemiology and molecular biology of Chikungunya virus. These data play an important role in the development of preventive, treatment and vaccination strategies of Chikungunya fever.

Discrete viral E2 lysine residues and scavenger receptor MARCO are required for clearance of circulating alphaviruses.

The magnitude and duration of vertebrate viremia is a critical determinant of arbovirus transmission, geographic spread, and disease severity. We find that multiple alphaviruses, including chikungunya (CHIKV), Ross River (RRV), and o'nyong 'nyong (ONNV) viruses, are cleared from the circulation of mice by liver Kupffer cells, impeding viral dissemination. Clearance from the circulation was independent of natural antibodies or complement factor C3, and instead relied on scavenger receptor SR-A6 (MARCO). Remarkably, lysine to arginine substitutions at distinct residues within the E2 glycoproteins of CHIKV and ONNV (E2 K200R) as well as RRV (E2 K251R) allowed for escape from clearance and enhanced viremia and dissemination. Mutational analysis revealed that viral clearance from the circulation is strictly dependent on the presence of lysine at these positions. These findings reveal a previously unrecognized innate immune pathway that controls alphavirus viremia and dissemination in vertebrate hosts, ultimately influencing disease severity and likely transmission efficiency.

Localized Outbreaks of Epidemic Polyarthritis among Military Personnel Caused by Different Sublineages of Ross River Virus, Northeastern Australia, 2016-2017.

Two outbreaks of epidemic polyarthritis occurred among Australian Defence Force personnel during and following short military exercises in the Shoalwater Bay Training Area, northeastern Australia, in 2016 and 2017. Ross River virus (RRV) IgM was detected in acute-phase serum samples from most patients (28/28 in 2016 and 25/31 in 2017), and RRV was recovered from 4/38 serum samples assayed (1/21 in 2016 and 3/17 in 2017). Phylogenetic analyses of RRV envelope glycoprotein E2 and nonstructural protein nsP3 nucleotide sequences segregated the RRV isolates obtained in 2016 and 2017 outbreaks into 2 distinct sublineages, suggesting that each outbreak was caused by a different strain of RRV. The spatiotemporal characteristics of the 2016 outbreak suggested that some of the infections involved human-mosquito-human transmission without any intermediate host. These outbreaks highlight the importance of personal protective measures in preventing vectorborne diseases for which no vaccine or specific prophylaxis exists.

A DNA Vaccine Expressing Fusion Protein E2-NT(gp96) Induces Hepatitis C Virus Cross-Neutralizing Antibody in BALB/c Mice

Background: Development of an effective prophylactic vaccine is the optimal long-term goal for the eventual control of HCV infection. An effective HCV vaccine should be able to elicit neutralizing antibodies (NAbs). Glycoprotein E2 of HCV is the major target for NAbs. Methods: In this study, we designed and constructed a DNA vaccine (pcDNA-E2-NT(gp96)) encoding a fusion protein composed of HCV E2 ectodomain (genotype 1a) and N-terminal domain of gp96 as a biological adjuvant. Two possible forms of a fusion protein, namely E2-NT(gp96) and NT(gp96)-E2, were made and subjected to in silico modeling and analysis. After the selection of the best form and confirmation its expression capacity in COS-7 cells, recombinant pcDNA-E2-NT(gp96) plasmid was generated by cloning of target genes into pcDNA3.1(+) plasmid. Constructed DNA vaccine immunogenicity was evaluated in BALB/c mice by measurement specific antibodies by ELISA and their neutralization capacity by neutralization assay. Results: In silico modeling and analysis showed that the E2-NT(gp96) structure was more valid than NT(gp96)-E2. Docking result revealed that the selected fusion protein had a high tendency for interaction with the main receptor (CD81) of HCV. GFP expression in COS-7 cells confirmed the E2-NT(gp96) expression capacity. Restriction enzyme digestion and sequencing results confirmed the integrity of the constructed plasmid. ELISA results showed that the pcDNA-E2-NT(gp96) induced high titers of specific antibodies in immunized mice. The sera of immunized mice cross-neutralized JFH1/HCVcc genotype 2a by 55% relative to pre-immune sera. Conclusions: Total results showed that the generated DNA vaccine induced potent immune responses in immunized mice. Therefore, our findings are sufficiently encouraging to propose the pcDNA-E2-NT(gp96) as a promising vaccine candidate for HCV infection.

Prediction of MAYV peptide antigens for immunodiagnostic tests by immunoinformatics and molecular dynamics simulations

The Mayaro virus is endemic to South America, and the possible involvement of Aedes spp. mosquitoes in its transmission is a risk factor for outbreaks of greater proportions. The virus causes a potentially disabling illness known as Mayaro fever, which is similar to that caused by the chikungunya virus. The cocirculation of both viruses, with their clinical and structural similarities, and the absence of prophylactic and therapeutic measures highlight the need for studies that seek to understand the Mayaro virus. Using approaches in silico, we identified an antigenic and specific epitope (p_MAYV4) in domain A of the E2 glycoprotein of the Mayaro virus. This epitope was theoretically predicted to be stable and exposed on the surface of the protein, where it showed key properties that enable its interaction with neutralizing antibodies. These characteristics make it an interesting target for the development of immunodiagnostic platforms. Molecular dynamics simulation-based structural analysis showed that the PHE95 residue in the E1 fusion loop region is conserved among Alphavirus family members. PHE95 interacts with the hydrophobic residues of the E2 glycoprotein to form a cage-shaped structure that is critical to assemble and stabilize the E1/E2 heterodimer. These results provide important insights useful for the advancement of diagnostic platforms and the study of therapeutic alternatives.

Plasticity of the HEK-293 cells, related to the culture media, as platform to produce a subunit vaccine against classical swine fever virus

Classical swine fever (CSF) is a contagious disease that causes a high mortality to domestic and wild pigs. Its causative agent is an enveloped Pestivirus named Classical Swine Fever Virus (CSFV). Due to the huge economic affectations produced by this disease to porcine industry, several vaccines have been developed using principally the CSFV E2 glycoprotein. Recently, a subunit vaccine based on this structural protein of the CSFV fused to the porcine CD154 molecule as immunomodulator named E2-CD154 was assayed by us. This chimeric protein was produced in the Human Embryonic Kidney (HEK-293) cell line. In this work, the growth and the expression profiles of HEK-293 E2-CD154 cells in four commercially available culture media were studied. The oligosaccharide structures in the N-glycosylation patterns of the E2-CD154 protein produced by this cell line in 10 L fermenters with two different culture media were also analyzed. In addition, the neutralizing antibody response generated in mice vaccinated with these antigens was assayed. Our results suggest that the culture media CDM4HEK293 and SFM4HEK293 which are recommended for HEK-293 growth are the best choice to growth the cell clone expressing the E2-CD154 protein. The glycosylation pattern and the neutralizing antibody response generated by the E2-CD154 protein were independent of the culture medium used which demonstrates the high reproducibility and consistency among protein batches produced by HEK-293 cells even in different culture conditions.

Commercial E2 subunit vaccine provides full protection to pigs against lethal challenge with 4 strains of classical swine fever virus genotype 2

Classical swine fever (CSF) still threatens the swine industry in China, with genotype 2 isolates of CSFV dominating the epizootics. In 2018 the first E2 subunit marker vaccine against CSFV (Tian Wen Jing, TWJ-E2®), containing a baculovirus-expressed E2 glycoprotein of a genotype 1.1 vaccine strain, was officially licensed in China and commercialized. To evaluate the cross-protective efficacy of TWJ-E2 against different virulent genotype 2 Chinese field isolates (2.1b, 2.1c, 2.1 h, and 2.2), 4-week-old pigs were immunized with the TWJ-E2 vaccine according to the manufacturer’s instructions and then challenged with genotype 2 strains. A group vaccinated with the conventional C-strain vaccine was included for comparison. TWJ-E2 vaccinated pigs developed higher levels of E2 and neutralizing antibodies than those receiving the commercial C-strain vaccine. All TWJ-E2 and C-strain vaccinated pigs survived challenge without development of fever, clinical signs or pathological lesions. In contrast, all unvaccinated control pigs displayed severe CSF disease with 40–100% mortalities by 24 days post challenge. None of the TWJ-E2 and C-strain vaccinated pigs developed viremia, viral shedding from tonsils, Erns protein in the sera, or viral RNA loads in different tissues after challenge, all of which were detected in the challenged unvaccinated controls. We conclude that vaccination of young pigs with TWJ-E2 provides complete immune protection against genotypically heterologous CSFVs and prevents viral shedding after challenge, with an efficacy at least comparable to that elicited by the conventional C-strain vaccine.

Rapid decrease in titer and breadth of neutralizing anti-HCV antibodies in HIV/HCV-coinfected patients who achieved SVR

The main targets for neutralizing anti-hepatitis C virus (HCV) antibodies (HCV-nAbs) are the E1 and E2 envelope glycoproteins. We have studied the characteristics of HCV-nAbs through a retrospective study involving 29 HIV/HCV-coinfected patients who achieved sustained virological response (SVR) with peg-IFNα + ribavirin anti-HCV therapy. Plasma samples at baseline and week 24 after SVR were used to perform neutralization assays against five JFH1-based HCV recombinant viruses coding for E1 and E2 from genotypes 1a (H77), 1b (J4), 2a (JFH1), 3a (S52) and 4a (ED43). At baseline, the majority of plasma samples neutralized 1a, 1b, 2a, and 4a, but not 3a, genotypes. Twenty-four weeks following SVR, most neutralizing titers declined substantially. Furthermore, titers against 3a and 2a were not detected in many patients. Plasma samples with high HCV-nAb titers neutralized all genotypes, and the highest titers at the starting point correlated with the highest titers at week 24 after SVR. In conclusion, high titers of broad-spectrum HCV-nAbs were detected in HIV/HCV-coinfected individuals, however, those titers declined soon after SVR.

Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins

Hepatitis C virus (HCV) is an enveloped virus and encodes two envelope glycoproteins, E1 and E2. E1 and E2 are transmembrane type I proteins with a N-terminal ectodomain and C-terminal anchor. During their synthesis, E1 and E2 ectodomains are targeted in the endoplasmic reticulum lumen where they are modified by N-linked glycosylation. After their synthesis, E1 and E2 assemble as a non-covalent heterodimer. The N-linked glycosylation is based on the recognition of specific asparagine residue in the context of the consensus sequence Asn-X-Ser/Thr. E1 contains potentially 4 or 5 N-linked glycosylation sites and E2 up to 11. Recent data indicated that some glycans of glycoproteins E1 and E2 play a major role in protein folding and heterodimer formation. Some N-linked glycans of E2 were involved in interactions with CD81, a putative cellular receptor for HCV. It appeared that N-linked glycans of E1 and E2 played an important role of in the viral entry.

Neutralizing antibodies against Mayaro virus require Fc effector functions for protective activity.

Despite causing outbreaks of fever and arthritis in multiple countries, no countermeasures exist against Mayaro virus (MAYV), an emerging mosquito-transmitted alphavirus. We generated 18 neutralizing mAbs against MAYV, 11 of which had "elite" activity that inhibited infection with EC50 values of <10 ng/ml. Antibodies with the greatest inhibitory capacity in cell culture mapped to epitopes near the fusion peptide of E1 and in domain B of the E2 glycoproteins. Unexpectedly, many of the elite neutralizing mAbs failed to prevent MAYV infection and disease in vivo. Instead, the most protective mAbs bound viral antigen on the cell surface with high avidity and promoted specific Fc effector functions, including phagocytosis by neutrophils and monocytes. In subclass switching studies, murine IgG2a and humanized IgG1 mAb variants controlled infection better than murine IgG1 and humanized IgG1-N297Q variants. An optimally protective antibody response to MAYV and possibly other alphaviruses may require tandem virus neutralization by the Fab moiety and effector functions of the Fc region.

Investigation of chronic and persistent classical swine fever infections under field conditions and their impact on vaccine efficacy

BackgroundRecent studies have hypothesized that circulation of classical swine fever virus (CSFV) variants when the immunity induced by the vaccine is not sterilizing might favour viral persistence. Likewise, in addition to congenital viral persistence, CSFV has also been proven to generate postnatal viral persistence. Under experimental conditions, postnatal persistently infected pigs were unable to elicit a specific immune response to a CSFV live attenuated vaccine via the mechanism known as superinfection exclusion (SIE). Here, we study whether subclinical forms of classical swine fever (CSF) may be present in a conventional farm in an endemic country and evaluate vaccine efficacy under these types of infections in field conditions.ResultsSix litters born from CSF-vaccinated gilts were randomly chosen from a commercial Cuban farm at 33 days of age (weaning). At this time, the piglets were vaccinated with a lapinized live attenuated CSFV C-strain vaccine. Virological and immunological analyses were performed before and after vaccination. The piglets were clinically healthy at weaning; however, 82% were viraemic, and the rectal swabs in most of the remaining 18% were positive. Only five piglets from one litter showed a specific antibody response. The tonsils and rectal swabs of five sows were CSFV positive, and only one of the sows showed an antibody response. After vaccination, 98% of the piglets were unable to clear the virus and to seroconvert, and some of the piglets showed polyarthritis and wasting after 36 days post vaccination. The CSFV E2 glycoprotein sequences recovered from one pig per litter were the same. The amino acid positions 72(R), 20(L) and 195(N) of E2 were identified in silico as positions associated with adaptive advantage.ConclusionsCirculation of chronic and persistent CSF infections was demonstrated in field conditions under a vaccination programme. Persistent infection was predominant. Here, we provide evidence that, in field conditions, subclinical infections are not detected by clinical diagnosis and, despite being infected with CSFV, the animals are vaccinated, rather than diagnosed and eliminated. These animals are refractory to vaccination, likely due to the SIE phenomenon. Improvement of vaccination strategies and diagnosis of subclinical forms of CSF is imperative for CSF eradication.

Evolution-Driven Attenuation of Alphaviruses Highlights Key Glycoprotein Determinants Regulating Viral Infectivity and Dissemination

Understanding the fundamental mechanisms of arbovirus transmission and pathogenesis is essential to develop strategies for treatment and prevention. We previously took an invivo evolution-based approach and identified the chikungunya virus E1 glycoprotein residue 80 to play a critical role in viral transmission and pathogenesis. In this study, we address the genetic conservation and function of position 80 and demonstrate that this residue is a key determinant in alphavirus infectivity and dissemination through modulation of viral fusion and cholesterol dependence. In addition, in studying the evolution of position 80, we identified a network of glycoprotein residues, including epidemic determinants, that regulate virus dissemination and infectivity. These studies underscore the importance of taking evolution-based approaches to not only identify key viral determinants driving arbovirus transmission and pathogenesis but also to uncover fundamental aspects of arbovirus biology.

Dendritic Cell Targeting of Bovine Viral Diarrhea Virus E2 Protein Expressed by Lactobacillus casei Effectively Induces Antigen-Specific Immune Responses via Oral Vaccination.

Bovine viral diarrhea caused by bovine viral diarrhea virus (BVDV) is an important disease in cattle, resulting in significant economic losses to the cattle industry worldwide. In order to develop an effective vaccine against BVDV infection, we constructed a dendritic cell (DC)-targeting oral probiotic vaccine (pPG-E2-DCpep/LC W56) using Lactobacillus casei as antigen delivery carrier to express BVDV glycoprotein E2 fused with DC-targeting peptide, and the immunogenicity of orally administered probiotic vaccine was evaluated in mice model. Our results showed that after immunization with the probiotic vaccine, significantly levels of antigen-specific sera IgG and mucosal sIgA antibodies (p < 0.05) with BVDV-neutralizing activity were induced in vivo. Challenge experiment showed that pPG-E2-DCpep/LC W56 can provide effective immune protection against BVDV, and BVDV could be effectively cleared from the intestine of immunized mice post-challenge. Moreover, the pPG-E2-DCpep/LC W56 could efficiently activate DCs in the intestinal Peyer’s patches, and significantly levels of lymphoproliferative responses, Th1-associated IFN-γ, and Th2-associated IL-4 were observed in mice immunized with pPG-E2-DCpep/LC W56 (p < 0.01). Our results clearly demonstrate that the probiotic vaccine could efficiently induce anti-BVDV mucosal, humoral, and cellular immune responses via oral immunization, indicating a promising strategy for the development of oral vaccine against BVDV.

Development of an enzyme-linked immunosorbent assay for Getah virus infection in horses using recombinant E2 protein as an antigen

Getah virus causes fever, skin eruptions, and limb edema in horses. For a high-throughput and time-saving method for serodiagnosis, we explored immunogenic antigens of Getah virus, and established an enzyme-linked immunosorbent assay (ELISA) using a recombinant protein. Western blot analysis using sera from infected horses showed strong reaction with viral antigens around 46 kDa corresponding to E1 or E2 glycoproteins. Recombinant E2 (rE2) protein reacted more strongly with infected horse sera than did rE1 protein in both Western blotting and ELISA. In ELISA using rE2 protein (rE2-ELISA), for all horses experimentally infected with Getah virus (n = 7), optical density (OD) exceeded the cutoff value at 14 days post-infection. ODs in five of nine vaccinated horses also slightly exceeded the cutoff value after vaccination. Among naturally infected horses (n = 28), 24 were seronegative in the acute sera, which turned seropositive in the convalescent sera. For the four horses seropositive in the acute sera, an endpoint method with serial dilutions of paired sera detected a ≥4-fold increase in titer. In conclusion, we established rE2-ELISA that could detect horse antibodies against Getah virus after experimental and natural infections; this should be useful in the diagnosis and surveillance of Getah virus infection.

Expression of Immunodominant Regions of E2 from an Indian Isolate of Classical Swine Fever Virus

Classical swine fever (CSF) is an economically important contagious fatal disease of domestic pigs and wild boars. This disease is caused by classical swine fever virus (CSFV), a member of the Pestivirus genus within the Flaviviridae family. Upon infection, three proteins of CSFV, namely E2, Erns and NS3 induce detectable antibodies. Since E2 is a major glycoprotein that produces neutralizing antibodies and provides protective immunity, it is widely used as a marker for measuring vaccine efficacy and antibody titer. In the present study, immunodominant regions of E2 glycoprotein from Indian field isolate of CSFV were expressed in E. coli. The 336 amino acid long N-terminal ectodomain (full-E2) and the 207 amino acid (aa 173–380) long C-terminal immunodominant region (partial-E2) were expressed and purified as 54kDa and 24kDa recombinant proteins, respectively. Both full-E2 and partial-E2 recombinant proteins were also characterized using MALDI-TOF-TOF analysis as CSFV-E2 structural proteins. The yield of purified full-E2 and partial-E2 recombinant protein was 26 mg and 84 mg/liter culture, respectively. For raising hyperimmune sera against these proteins, chickens and rabbits were immunized with 200μg purified protein and boosted three times with 100μg purified protein intramuscularly. The sera collected one week after last booster were analyzed for CSFV-specific IgG antibody response using purified CSFV as coating antigen in ELISA. The results revealed that full-E2 induced better antibody response in both rabbits and chicken as compared to partial-E2. This study also indicated that these recombinant E2 proteins can be used as diagnostic antigen in ELISA.