Non-structural Protein Antibodies Research Articles

Differentiation of West Nile Virus-Infected Animals from Vaccinated Animals by Competitive ELISA Using Monoclonal Antibodies Against Non-Structural Protein 1

Antibodies against non-structural protein 1 (NS1) are considered to be the most reliable indicator of a present or past infection by West Nile virus (WNV) in animals. In this study, an in-house competitive enzyme-linked immunosorbent assay (NS1-cELISA) utilizing baculovirus-expressed NS1 and monoclonal antibodies against NS1 was established for the detection of antibody responses to NS1 in WNV-infected animals. The assay was validated by the simultaneous detection of early antibody responses to NS1 and the structural envelope protein in animals infected with WNV, or inoculated with inactivated WNV. NS1-cELISA detected WNV antibodies at 6 days post-infection (dpi) in a WNV-infected rabbit (percent inhibition [PI] value of 84.0), and at 10 dpi in a WNV-infected chicken (PI value of 67.0). The NS1-cELISA was able to detect WNV antibodies in sera from all WNV-infected rabbits at 10 dpi (PI value of 79.2±18.0), and from three of four WNV-infected chickens at 14 dpi (PI value of 73.7±22.8). The results of this study demonstrate that the antibody response to NS1 is similar to that against envelope protein in WNV-infected rabbits and chickens, whereas animals inoculated with inactivated WNV develop antibody responses only to the envelope protein but not to NS1. The NS1-cELISA developed here has the potential to be a useful tool for monitoring WNV circulation (i.e., the prevalence of specific antibodies against WNV NS1), by assaying serum samples from regions in which an inactivated vaccine control strategy has been implemented.

Serological and clinical surveillance studies to validate reported foot-and-mouth disease free status in Tsirang district of Bhutan

Serological and clinical studies were conducted between March 2009 and August 2010 to validate the foot-and-mouth disease free status of Tsirang district of Bhutan as determined by the country's passive surveillance system. Randomised (first survey) and targeted (third survey) samplings, with subsequent follow-up samplings (second and fourth), were conducted on FMD-susceptible animals to detect the disease at a design prevalence of 25% and 20% at the individual animal-level and village-level, respectively. Sera from cattle, goats, pigs, and sheep were tested for the presence of non-structural protein (NSP) antibodies using two commercial (PrioCHECK® FMDV NS and CHEKIT®-FMD-3ABC-bo-ov) and one in-house NSP kit (c-ELISA, AAHL, Australia). The overall seropositivity (all species) at the animal-level was 3% (95% CI: 1.7, 4.8) and 3.5% (95% CI: 2.1, 5.4), for the randomised and targeted surveys, respectively. Except for one goat from the first survey, none of the small ruminants and pigs had NSP antibodies. The seropositives from the first and targeted surveys were distributed among 13 and 16 of 20 villages sampled, respectively. All repeat testing from the initial seropositive animals and their herd mates, for both the first and third surveys, were negative in the NSP tests 6–8 months later. Using the hypergeometric exact probability formula for two-stage analyses, the results enabled rejection of the null hypothesis and supported conclusion that the population was free from disease at the minimum expected prevalence of 20% at the 95.53% and 99.46% confidence levels, for the randomised and targeted surveys, respectively. Clinical surveillance also showed absence of disease or clinical signs suggestive of FMD. The few seropositives were likely to be false positives due to factors such as imperfect specificities of the tests and possible NSP-residues in the vaccines. The study has paved the way for initiation of zoning approaches for the progressive control of FMD in Bhutan.

Development of a Synthetic Peptide ELISA Assay for the Detection of Foot-and-Mouth Disease Virus Nonstructural Protein Antibodies

In order to develop an ELISA assay with synthetic peptides for the detection of antibody to the nonstructural proteins of foot-and-mouth disease virus, specific peptides were synthesized by a solid-phase method according to FMDV NSPs B-cell epitopes, and were conjugated to carrier protein BSA. An ELISA system was developed to detect FMDV NSPs antibody with the conjugated proteins as the coating antigen. The optimal coating concentration of the antigen was determined as 2.5 μg mL −1. The comparative study of this assay with UBI NSP ELISA kit and national commercial 3ABC ELISA kit in the detection of 199 serum samples showed that they were very coincident, and the identity rates were 96.48 and 97.48%, respectively. The development of ELISA using the synthetic peptides as coating antigen is specific, reproducible, stable, and easy, and can be used to differentiate FMDV infected pigs from immunized pigs.

Non-structural protein 1 (NS1) antibody-based assays to differentiate West Nile (WN) virus from Japanese encephalitis virus infections in horses: Effects of WN virus NS1 antibodies induced by inactivated WN vaccine

Antibodies to non-structural protein 1 (NS1) of West Nile virus (WNV) have been used to differentiate WNV infection from infection by serologically cross-reactive flaviviruses, including Japanese encephalitis virus (JEV), in horses. However, since the inactivated West Nile (WN) vaccine has been reported to induce NS1 antibodies, there is concern about the reliability of using NS1-based assays for testing vaccinated horses. Therefore, the effect of inactivated WN vaccine-induced antibodies on an epitope-blocking ELISA and complement-dependent cytotoxicity (CDC) assay were investigated. Both assays are based on NS1 antibodies and were established previously to differentiate WNV from JEV infections in horses. Groups of three horses were vaccinated with two or three doses of a commercial inactivated WN vaccine and NS1 antibodies were detected by a conventional ELISA after the second vaccination. Vaccine-induced NS1 antibodies were also detected by blocking ELISA and a CDC assay and affected the ability of these assays to differentiate WNV from JEV infections. However, the effect was less significant in the CDC assay, where use of a low serum concentration ensured effective differentiation. The more efficient detection of infection-induced antibodies over vaccine-induced antibodies by the CDC assay was potentially attributable to the different IgG isotype profiles of these antibodies.

Complement-Dependent Cytotoxicity Assay for Differentiating West Nile Virus from Japanese Encephalitis Virus Infections in Horses

A complement-dependent cytotoxicity (CDC) assay was established to measure antibodies to the West Nile virus (WNV) nonstructural protein 1 (NS1) in horses. Sera collected from a WNV-infected horse mediated lysis of WNV NS1-expressing cells in a dose-dependent manner at higher percentages than sera from a Japanese encephalitis virus (JEV)-infected horse. The percentages of specific lysis for sera diluted 1:10 to 1:80 were <19.8% (assay cutoff) for almost all of the 100 JEV-infected or uninfected horses tested, in contrast to 55 to 76% in WNV-infected horses. Experimental infection revealed that horses became anti-WNV NS1 antibody positive 10 days after WNV infection. This study demonstrated the utility of this assay for differentiating WNV from JEV infections in horses.

Anti-dengue virus nonstructural protein 1 antibodies recognize protein disulfide isomerase on platelets and inhibit platelet aggregation

Hemorrhagic syndrome is a hallmark of severe dengue diseases. We previously suggested a mechanism of molecular mimicry in which antibodies against dengue virus (DV) nonstructural protein 1 (NS1) cross-react with platelets. In the present study, we demonstrate that protein disulfide isomerase (PDI) on the platelet surface is recognized by anti-DV NS1 antibodies. Anti-DV NS1 obtained from hyperimmunized mouse sera inhibited PDI activity and platelet aggregation, and both inhibitory effects were prevented when anti-DV NS1 antibodies were preabsorbed with PDI. Anti-PDI antibodies bound to a peptide consisting of amino acid residues 311–330 (P311–330) of NS1. This peptide was a predicted epitope analyzed by homologous sequence alignments between DV NS1 and PDI. The platelet binding activities of anti-PDI and anti-DV NS1 antibodies were both reduced by P311–330 preabsorption. Similar to the findings using anti-DV NS1, antibodies against P311–330 bound to PDI and platelets, followed by inhibition of PDI activity and platelet aggregation. Furthermore, the cross-reactivity of dengue hemorrhagic fever patient sera with platelets was reduced when patient sera were preabsorbed with PDI or P311–330. Dengue hemorrhagic fever patient sera also inhibited platelet aggregation, while PDI or P311–330 reduced this inhibitory effect. In conclusion, anti-DV NS1 antibodies cross-react with PDI on platelet surface causing inhibition of platelet aggregation, which may provide implications in dengue disease pathogenesis.

Anti-human parvovirus B19 nonstructural protein antibodies in patients with rheumatoid arthritis

Background Previous studies have reported the association between the development of NS1-specific IgG and arthropathy after the infection of human parvovirus B19 (B19). However, the role of anti-B19-NS1 IgG in RA is still unclear. This study investigated the role of anti-B19-NS1 antibody in patients with rheumatoid arthritis (RA). Methods B19-VP IgM and IgG antibodies, nested PCR, B19-NS1 IgM and IgG antibodies, and anti-cyclic citrullinated peptide (CCP) antibodies were assessed by ELISA and Western blot in this study. Results Significantly higher prevalence of B19-NS1 IgM and IgG antibodies in patients with recent B19 infection was observed as well as the higher prevalence of B19-NS1 IgM and IgG antibodies in RA patients with seronegative diagnostic patterns. However, no significant variation of both B19-NS1 IgM and IgG was detected in RA patients with different B19 diagnostic patterns. Additionally, significantly higher presence of anti-CCP IgG was observed in RA patients with B19-NS1 IgM. Conclusions This study suggests the possibility of anti-B19-NS1 IgM as an indicator for RA diagnosis and indicates the suspense of the higher prevalence of anti-B19-NS1 antibody in RA patients with seronegative B19 diagnostic patterns. However, these results provide clues in understanding the association of anti-B19-NS1 antibody in RA patients.

Proteomic Analysis of Endothelial Cell Autoantigens Recognized by Anti-Dengue Virus Nonstructural Protein 1 Antibodies

We previously showed the occurrence of autoimmune responses in dengue virus (DV) infection, which has potential implications for the pathogenesis of dengue hemorrhagic syndrome. In the present study, we have used a proteomic analysis to identify several candidate proteins on HMEC-1 endothelial cells recognized by anti-DV nonstructural protein 1 (NS1) antibodies. The target proteins, including ATP synthase beta chain, protein disulfide isomerase, vimentin, and heat shock protein 60, co-localize with anti-NS1 binding sites on nonfixed HMEC-1 cells using immunohistochemical double staining and confocal microscopy. The cross-reactivity of anti-target protein antibodies with HMEC-1 cells was inhibited by NS1 protein pre-absorption. Furthermore, a cross-reactive epitope on NS1 amino acid residues 311-330 (P311-330) was predicted using homologous sequence alignment. The reactivity of dengue hemorrhagic patient sera with HMEC-1 cells was blocked by synthetic peptide P311-330 pre-absorption. Taken together, our results identify putative targets on endothelial cells recognized by anti-DV NS1 antibodies, where NS1 P311-330 possesses the shared epitope.

Liver injury caused by antibodies against dengue virus nonstructural protein 1 in a murine model

Clinical manifestations of severe dengue diseases include thrombocytopenia, vascular leakage, and liver damage. Evidence shows that hepatic injury is involved in the pathogenesis of dengue infection; however, the mechanisms are not fully resolved. Our previous in vitro studies suggested a mechanism of molecular mimicry in which antibodies directed against dengue virus (DV) nonstructural protein 1 (NS1) cross-reacted with endothelial cells and caused inflammatory activation and apoptosis. In this study, the pathogenic effects of anti-DV NS1 antibodies were further examined in a murine model. We found, in liver sections, that anti-DV NS1 antibodies bound to naive mouse vessel endothelium and the binding activity was inhibited by preabsorption of antibodies with DV NS1. Active immunization with DV NS1 resulted in antibody deposition to liver vessel endothelium, and also apoptotic cell death of liver endothelium. Liver tissue damage was observed in DV NS1-immunized mice by histological examination. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased in mice either actively immunized with DV NS1 protein or passively immunized with antibodies obtained from DV NS1-immunized mice. Furthermore, histological examination revealed mononuclear phagocyte infiltration and cell apoptosis in mice passively immunized with antibodies obtained from mice immunized with DV NS1. Increased AST and ALT levels were observed in mice passively immunized with purified immunoglobulin G (IgG) from dengue patients compared with normal control human IgG-immunized mice. The increased AST and ALT levels were inhibited when dengue patient serum IgG was preabsorbed with DV NS1. In conclusion, active immunization with DV NS1 protein causes immune-mediated liver injury in mice. Passive immunization provides additional evidence that anti-DV NS1 antibodies may play a role in liver damage, which is a pathologic manifestation in dengue virus disease.

Japanese Encephalitis Viral Infection Remains Common in Japan

To the Editor: Japanese encephalitis (JE) virus is a flavivirus that causes about 15,000 deaths in Asia every year. Mosquitoes and swine play important roles in the spread of JE virus in the paradomestic environment, the former as a vector and the latter as an amplifier. In the 1950s, approximately 5000 cases, about 20% fatal, were reported yearly in Japan, but recently fewer than 10 cases are reported each year. Reasons for the marked decrease in JE in Japan include decreases in the number of rice fields which are habitats of vector mosquitoes (mostly Culex tritaeniorhynchus), subsequent decreases in the mosquito population, and increased distance between the habitats of people and swine. However, urban residents are still bitten by mosquitoes during the summer, with the exception of northern Japan. Furthermore, 50–100% of Japanese pigs younger than 8 months have antibodies against the JE virus in summer and autumn, indicating that most Japanese pigs can still be JE virus producers. The extremely low reported incidence of JE in human patients in Japan, despite the high prevalence of antibodies against the JE virus in swine and continued mosquito bites in humans, constitutes a paradoxical situation. To explain this paradox, we proposed a hypothesis that human JE viral infections still occur, but produce only mild symptoms. To test this hypothesis, we measured titers of the nonstructural protein-1 (NS1) antibody,1 which increases only with natural JE viral infection, and titers of the HI antibody, which increases with both vaccination and JE viral natural infection, in blood samples obtained from 50 volunteers and patients, aged 1–88 years, at Eijudo Clinic, located in an urban area of eastern Tokyo. Blood samples were collected for 2–3 years from each subject. Results are shown in Figure 1. The black bars indicate changes in NS1 antibody titers in each serum series, and the gray bars show changes in HI antibody concentration. The adjacent bars are results for a single person. The bars are arranged horizontally by subject age. The NS1 titers indicate that the incidence of JE viral infection has increased with subject age, whereas HI titers, which reflect vaccination, have slowly fallen with subject age. Two of 50 subjects showed rapid and marked increases in the NS1 titer. One was a 57-year-old woman who took a 3-day trip from June 30 through July 2, 2002, to Okinawa, a southern Japanese island where 3 American soldiers contracted JE with severe sequelae several years ago.2 The NS1 titer on June 21 was <1:10 but by July 21 had increased to 1:20 and remained high for 1 year. During this time the subject exhibited no symptoms. Another subject was a 85-year-old woman who had remained at home for several years. The NS1 titer was <1:10 on July 8, 2002, but the titers on July 15 and September 8, 2002, were 1:80. From July 8 through 15 she had a low-grade fever, but no infectious focus was identified.FIGURE 1.: Range of NS1 and HI antibody titers against Japanese encephalitis. The black bars indicate changes in NS1 antibody titers in each serum series, and the grey bars show changes in HI antibody concentrations. The adjacent bars are results for a single person. Sera collected from each person for 2–3 years.We have shown rising NS1 antibody titers with subject age in a series of blood samples obtained at a clinic in eastern Tokyo and described 2 subjects with rapidly increasing NS1 titers. These results and a previous report3,4 suggest that human JE viral infection remains prevalent in Japan, although symptoms can be mild or absent. Therefore, vaccination against JE remains extremely important, although the Japanese Government halted strong recommendation of routine JE vaccination in 2005.5 Teiichi Matsunaga, MD, PhD Eijudo ClinicKatsushika-ku, Tokyo, Japan Mizue Shoda, MSc Eiji Konishi, PhD Department of International Health Kobe Univ. Graduate Sch. of Health Sciences Kobe, Japan

FMD Vaccines: Reflections on Quality Aspects for Applicability in European Disease Control Policy

Most foot-and-mouth disease (FMD) vaccines used around the world are inactivated vaccines for prophylactic or emergency use, generally manufactured by the same basic methodology outlined in the OIE Manual and, for Europe, in the European Pharmacopoeia, and for the EU Member States in compliance with Directive 2001/82/EC of the European Parliament and of the Council of 6 November 2001 on the Community code relating to veterinary medicinal products as amended by Directive 2004/28/EC. Most of the requirements that apply to all immunological veterinary medicinal products apply equally to FMD vaccines. There are, however, some unique features of the disease and vaccines used against it that require a different approach to fulfil the requirements of the relevant legislation, if a vaccinate-to-live policy will be applied with 'authorized' vaccines. Several aspects of vaccine efficacy and safety are elaborated with emphasis on quality assurance/quality control (QA/QC). The purity of the vaccine in respect of the presence of non-structural protein antibodies could be checked indirectly by serology after vaccination. The viability of a vaccine bank approach was greatly aided by the principle of storing inactivated concentrated FMD viral antigen (Ag) over liquid nitrogen for subsequent formulation into vaccine. A worldwide Ag bank network might be an option for the far future and a solution to the problem of covering many different FMDV serotypes and strains. The producers should respect the strict FMD biosecurity rules worked out by the FAO EUFMD and described in Council Directive 2003/85/EC. Making the experience related to vaccine QA/QC available to all countries will reduce the risk of an FMD outbreak within these countries and consequently will reduce the FMD risk around the world.

Use of epidemiologic risk modeling to evaluate control of foot-and-mouth disease in southern Thailand

To assess the impacts of the introduction of foot-and-mouth disease (FMD) and various FMD control programs in southern Thailand. A native population of 562,910 cattle and 33,088 buffalo as well as 89,294 animals legally transported into southern Thailand. A quantitative risk assessment was used to ascertain the probability of FMD introduction, and an intrinsic dynamic model was used to assess impacts. Value for the transmission rate (beta) was estimated. Five scenarios created to assess the impacts of nonstructural protein (NSP) testing, mass vaccination, and culling were examined. Impacts were assessed through an examination of the estimated annual cumulative incidence (ACI) of FMD. The ACIs of various scenarios were compared by use of the Tukey Studentized range technique. beta was estimated at 0.115. Approximately 35,000 cases of FMD would be expected from the baseline situation. A 30% reduction of ACI was detected with the introduction of NSP antibody testing. Prophylactic vaccination resulted in an 85% reduction of ACI. Concurrent use of NSP antibody testing and vaccination reduced the ACI by 96%, and the addition of an eradication policy resulted in a slightly greater decrease in the ACI (98%). The study used epidemiologic models to investigate FMD control interventions. Results suggested that vaccination has more impact than the use of NSP testing. Use of the NSP test reduced ACI during peak seasons, whereas vaccination diminished the underlying incidence. The best mitigation plan was an integrated and strategic use of multiple control techniques.

Utilization of Complement-Dependent Cytotoxicity To Measure Low Levels of Antibodies: Application to Nonstructural Protein 1 in a Model of Japanese Encephalitis Virus

Enzyme-linked immunosorbent assay (ELISA) and related assays are representative of methods currently used for antibody tests. However, they occasionally produce nonspecific reactions, thus making it difficult to reliably measure low levels of specific antibodies. To find a test method that minimizes nonspecific reactions, we introduced the principle of antibody-mediated complement-dependent cytotoxicity (CDC) into an antibody assay. The procedure has three steps: (i) the mixing of test samples with a suspension of cells expressing the antigen of interest on their surfaces, (ii) the addition of rabbit complement, and (iii) the measurement of lactose dehydrogenase (LDH) activities by adding a chromogenic substrate to the reaction mixture. When the specific antibodies exist in the sample, complement activation triggered by antibody binding on the surface of the antigen-expressing cells may lyse the cells, releasing LDH into the medium. Mouse and rabbit sera hyperimmune to nonstructural protein 1 (NS1) of Japanese encephalitis virus (JEV) lysed NS1-expressing cells in a dose-dependent manner. Evaluations using sera from horses naturally infected with JEV showed that the CDC assay had quantitative correlation and qualitative agreement with previously established NS1 antibody-detecting immunostaining and ELISA methods. The assay method also detected NS1 antibodies in sera of mice 2 days after experimental infection with JEV; specific, but not natural, immunoglobulin M antibodies were detected. Since almost all sera examined in this study showed no nonspecific reactions, the CDC assay was shown to be a reliable method for measuring low levels of specific antibodies.

Cedivac-FMD can be used according to a marker vaccine principle

In this study, we investigated whether Cedivac-FMD, an emergency vaccine against foot-and-mouth disease (FMD), is suitable for use conjointly with a screening program intended to confirm freedom from disease in vaccinated herds based on evidence of virus replication in vaccinates. Different sets of sera were tested using the Ceditest ® FMDV-NS ELISA for the detection of antibodies against non-structural proteins (NSPs) of FMD virus. During a vaccine safety study, serum samples were collected from 10 calves, 10 lambs and 10 piglets following administration of a double dose and a repeat dose of high payload trivalent Cedivac-FMD vaccine . All serum samples collected both 2 weeks following the administration of a double dose as well as those collected 2 weeks after the single dose booster (given 2 weeks after the double dose) were negative in the Ceditest ® FMDV-NS ELISA. In a series of vaccine potency experiments, serum samples were collected from 70 vaccinated cattle prior to and following exposure to infectious, homologous FMD virus. When testing cattle sera collected 4 weeks after vaccination with a regular dose of monovalent >6 PD 50 vaccines, 1 of 70 animals tested positive in the NSP antibody ELISA. After infection with FMD virus, antibodies to NSP were detected in 59 of 70 vaccinated cattle and 27 of 28 non-vaccinated control animals within 7 days. Cedivac-FMD vaccines do not induce NSP antibodies in cattle, pigs or sheep following administration of a double dose or a repeat dose. FMD-exposed animals can be detected in a vaccinated group within 7–14 days. Because Cedivac-FMD does not induce NSP antibodies, the principle of ‘marker vaccine’ applies.

Use of a Standardized Bovine Serum Panel To Evaluate a Multiplexed Nonstructural Protein Antibody Assay for Serological Surveillance of Foot-and-Mouth Disease

Liquid array technology has previously been used to show proof of principle of a multiplexed nonstructural protein serological assay to differentiate foot-and-mouth disease virus-infected and vaccinated animals. The current multiplexed assay consists of synthetically produced peptide signatures 3A, 3B, and 3D and the recombinant protein signature 3ABC in combination with four controls. To determine the diagnostic specificity of each signature in the multiplex, the assay was evaluated against a naive population (n = 104) and a vaccinated population (n = 94). Subsequently, the multiplexed assay was assessed by using a panel of bovine sera generated by the World Reference Laboratory for foot-and-mouth disease in Pirbright, United Kingdom. This serum panel has been used to assess the performance of other singleplex enzyme-linked immunosorbent assay (ELISA)-based nonstructural protein antibody assays. The 3ABC signature in the multiplexed assay showed performance comparable to that of a commercially available nonstructural protein 3ABC ELISA (Cedi test), and additional information pertaining to the relative diagnostic sensitivity of each signature in the multiplex was acquired in one experiment. The encouraging results of the evaluation of the multiplexed assay against a panel of diagnostically relevant samples promote further assay development and optimization to generate an assay for routine use in foot-and-mouth disease serological surveillance.

Reduction of foot-and-mouth disease (FMD) virus load in nasal excretions, saliva and exhaled air of vaccinated pigs following direct contact challenge

In future, a policy of “vaccinate-to-live” may be included in the repertoire of foot-and-mouth disease (FMD) control measures and in support of this approach, we have investigated the hypothesis that vaccine-induced reduction in virus replication and excretion from pigs can be correlated to the severity of clinical signs of FMD by measuring excretion of virus in natural secretions and aerosols. The other aims of this study were to verify the existence of sub-clinical infection in vaccinated pigs, to evaluate the correlation between this and seroconversion to foot-and-mouth disease virus (FMDV) non-structural protein antibodies and to re-examine the occurrence of FMDV persistence in the oro-pharynx of pigs. Therefore, pigs were vaccinated (O1 Manisa) and challenged (O1 UKG) in a manner calculated to produce a broad range of clinical outcomes and were monitored for a minimum of another 33 days post-challenge. Eighty-one percent of the early (10 days vaccinated) challenged pigs and 25% of the late (29 days vaccinated) challenged pigs were clinically infected and all other vaccinated pigs were sub-clinically infected. Although vaccination could not provide complete clinical or virological protection, it reduced the severity of the disease, virus excretion and production of non-structural FMDV antibodies in vaccinated and subsequently infected pigs. As hypothesised, vaccine-induced reduction of virus replication and excretion was found to be correlated to the severity of clinical disease. RNA copies, but no live virus was detected from the pharyngeal and soft palate tissues of a minority of vaccinated and infected pigs beyond the acute stage of the infection.

Alpha interferon is a powerful adjuvant for a recombinant protein vaccine against foot-and-mouth disease virus in swine, and an effective stimulus of in vivo immune response

The adjuvant effect of porcine interferon-alpha (PoIFN-alpha) was examined in swine vaccinated with a recombinant FMD protein vaccine named IgG-FMDV, which contains the swine IgG single heavy chain constant region and an immunogenic peptide of serotype O FMDV. The PoIFN-alpha gene was cloned into pcDNA3 vector and the recombinant plasmid was incorporated into cationic liposomes by a dehydration and rehydration procedure to use as an adjuvant, injected together with low-dose IgG-FMDV. This procedure resulted in strong induction of FMDV-specific neutralizing antibody and significant T-cell-mediated immune responses, whereas only a modest humoral and cellular response was observed with low-dose vaccine alone. As an adjuvant for the protein vaccine, PoIFN-alpha induced strong inflammatory cytokines production in vivo and the results denoted that IFN-adjuvant and our vaccines could drive the immune response toward Th1 type responses. The data of ELISA suggests that the recombinant protein vaccine synergizes with the IFN-adjuvant to produce endogenous IFN in vivo. In response to viral challenge, all control animals developed viremia and lesions, whereas all animals received IFN-adjuvant+IgG-FMDV were protected and nonstructural protein antibody in this group could not be detected by 14 days post-challenge (dpc). Our studies indicate that porcine IFN-alpha is a powerful adjuvant for recombinant FMD protein vaccine and could aid in vaccination against FMDV in swine.

Immune responses of pigs to commercialized emulsion FMD vaccines and live virus challenge

The immune response to structural and non-structural proteins (NSPs) was studied on sequential serum samples in swine from O/Taiwan/97 FMDV challenge studies, outbreaks and after vaccination. The results showed that pigs vaccinated with a commercial vaccine prior to or after infection maintained high neutralizing antibody titers with gradual decline from peak titers over the duration of this study. However, neutralizing antibody titers in non-vaccinated pigs only reached moderate levels 2–4 weeks post infection and remained low thereafter. For the 3B and 3ABC NSP antibody ELISA responses, there were gradually decreasing levels of NSP antibody over time. In multiple vaccinations, all pigs showed significant increases in neutralizing antibodies after booster vaccination. For the 3B NSP antibody ELISA after vaccination, the mean S/P ratios for pigs vaccinated with all three FMD vaccines were all below the 0.23 cut-off value set by the manufacture, but some sera from individual vaccinated pigs gave results above this cut-off after primary or secondary vaccination. However, with the 3ABC NSP antibody ELISA, all sera from vaccinated pigs had negative results for NSP antibody for all time points.

Comparison of sensitivity and specificity in three commercial foot-and-mouth disease virus non-structural protein ELISA kits with swine sera in Taiwan

Three commercialized ELISA kits for the detection of antibodies to the non-structural proteins (NSPs) of FMD virus were compared, using sera from uninfected, vaccinated, challenged and naturally infected pigs. The kinetics of the antibody response to NSPs was compared on sequential serum samples in swine from challenge studies and outbreaks. The results showed that ELISA A (UBI) and ELISA B (CEDI) had better sensitivity than that of the 3ABC recombinant protein-based ELISA C (Chekit). The peak for detection of antibodies to NSPs in ELISA C was significantly delayed in sera from natural infection and challenged swine as compared to the ELISA A and B. The sensitivity of the three ELISAs gradually declined during the 6-month post-infection as antibodies to NSP decline. ELISA kits A and B detected NSP antibody in 50% of challenged pigs by the 9–10th-day and 7–8th-day post-challenge, respectively. ELISA B and C had better specificity than ELISA A on sequential serum samples obtained from swine immunized with a type O FMD vaccine commercially available in Taiwan. Antibody to NSPs before vaccination was not detected in swine not exposed to FMD virus, however, antibody to NSPs was found in sera of some pigs after vaccination. All assays had significantly lower specificity when testing sera from repeatedly vaccinated sows and finishers in 1997 that were tested after the 1997 FMD outbreak. However, when testing sera from repeatedly vaccinated sows or finishers in 2003–2004, the specificity for ELISAs A, B and C were significantly better than those in 1997. This effect was less marked for ELISA A. The ELISA B was the best test in terms of the highest sensitivity and specificity and the lowest reactivity with residual NSP in vaccinates.

Development and Evaluation of an Indirect Enzyme-Linked Immunosorbent Assay for Detection of Foot-and-Mouth Disease Virus Nonstructural Protein Antibody using a Chemically Synthesized 2B Peptide as Antigen

Forty peptides were synthesized corresponding to hydrophilic clusters of amino acids within the sequences of foot-and-mouth disease virus (FMDV) nonstructural proteins (NSP). Six peptides were studied in more detail and the most promising, a 2B peptide, was evaluated in enzyme-linked immunosorbent assay (ELISA) using sera from naive, vaccinated, and vaccinated-and-challenged cattle as well as bovine sera from field outbreaks. The performance of the new NSP peptide ELISA was compared to that of 4 commercial NSP ELISA kits. Antibody to 2B was detectable from the end of the first week to the second week after infection in most of the nonvaccinated animals and by the second to third week in vaccinated-and-challenged animals. The sensitivity of the 2B peptide ELISA was comparable to the 3ABC Ceditest (Ceditest FMDV-NS, Cedi Diagnostics B.V.; Chung et al., 2002). With some modification and further validation, this 2B test could be useful as a screening or conformational NSP test in postvaccination surveillance for FMD.