Monoclonal antibody based solid phase competition ELISA to detect FMDV serotype A specific antibodies.

In Foot-and-mouth disease (FMD) enzootic countries, periodic vaccination is the key tool in controlling the disease incidence. Active seromonitoring of the vaccinated population is critical to assess the impact of vaccination. Virus neutralization test (VNT) and enzyme-linked immunosorbent assays (ELISA) are commonly used for antibody detection. Assays like liquid phase blocking ELISA (LPBE) or solid phase competition ELISA (SPCE) are preferred as they do not require handling of live FMDV and are routinely used for seromonitoring or for vaccine potency testing; however, false positives are high in LPBE. Here we report, a monoclonal antibody (mAb) based SPCE as a potential alternate assay for antibody titration. From a panel of 12 mAbs against FMDV serotype A, two mAbs were chosen for the development of SPCE. Based on a set of 453 sera, it was demonstrated that mAb 2C4G11, mAb 6E8D11and polyclonal antibody (pAb) based SPCE had a relative sensitivity of 86.1, 86.1 and 80.3 %; and specificity of 99.6, 99.1 and 99.1 %, respectively. The correlation, repeatability, and level of agreement of the assays were high demonstrating the potential use of mAb in large scale surveillance studies and regular vaccine potency testing.

In-process quality control in foot-and-mouth disease vaccine production by detection of viral non-structural proteins using chemiluminescence dot blot assay.

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-footed animals. Immunization with inactivated virus vaccine is effective to control the disease. Six-monthly vaccination regimen in endemic regions has proven to be effective. To enable the differentiation of infected animals from those vaccinated, non-structural proteins (NSPs) are excluded during vaccine production. While the antibodies to structural proteins (SPs) could be observed both in vaccinated and infected animals, NSP antibodies are detectable only in natural infection. Quality control assays that detect NSPs in vaccine antigen preparations, are thus vital in the FMD vaccine manufacturing process. In this study, we designed a chemiluminescence dot blot assay to detect the 3A and 3B NSPs of FMDV. It is sensitive enough to detect up to 20 ng of the NSP, and exhibited specificity as it does not react with the viral SPs. This cost-effective assay holds promise in quality control assessment in FMD vaccine manufacturing.

Thermostable negative-marker foot-and-mouth disease virus serotype O induces protective immunity in guinea pigs.

Foot-and-mouth disease (FMD) is a contagious viral disease of high economic importance, caused by FMD virus (FMDV), a positive-sense single-stranded RNA virus, affecting cloven-hoofed animals. Preventive vaccination using inactivated virus is in practice to control the disease in many endemic countries. While the vaccination induces antibodies mainly to structural proteins, the presence of antibodies to the non-structural proteins (NSP) is suggestive of infection, a criterion for differentiation of infected from vaccinated animals (DIVA). Also, there is a growing demand for enhancing the stability of the FMD vaccine virus capsid antigen as the strength of the immune response is proportional to the amount of intact 146S particles in the vaccine. Considering the need for a DIVA compliant stable vaccine, here we report generation and rescue of a thermostable and negative marker virus FMDV serotype O (IND/R2/1975) containing a partial deletion in non-structural protein 3A, generated by reverse genetics approach. Immunization of guinea pigs with the inactivated thermostable-negative marker virus antigen induced 91% protective immune response. Additionally, a companion competitive ELISA (cELISA) targeting the deleted 3A region was developed, which showed 92.3% sensitivity and 97% specificity, at cut-off value of 36% percent inhibition. The novel thermostable-negative marker FMDV serotype O vaccine strain and the companion cELISA could be useful in FMDV serotype O enzootic countries to benefit the FMD control program. KEY POINTS: • Thermostable foot-and-mouth disease virus serotype O with partial deletion in 3A. • Inactivated thermostable marker vaccine induced 91% protection in guinea pigs. • Companion cELISA based on deleted region in 3A could potentially facilitate DIVA.

A single amino acid substitution in the VP2 protein of Indian foot-and-mouth disease virus serotype O vaccine strain confers thermostability and protective immunity in cattle.

Foot-and-mouth disease (FMD) is a significant threat to animal health globally. Prophylactic vaccination using inactivated FMD virus (FMDV) antigen is being practised for the control in endemic countries. A major limitation of the current vaccine is its susceptibility to high environmental temperature causing loss of immunogenicity, thus necessitating the cold chain for maintenance of its efficacy. Hence, the FMD vaccine with thermostable virus particles will be highly useful in sustaining the integrity of whole virus particle (146S) during storage at 4°C. In this study, 12 recombinant mutants of Indian vaccine strain of FMDV serotype O (O/IND/R2/1975) were generated through reverse genetics approach and evaluated for thermostability. One of the mutant viruses, VP2_Y98F was more thermostable than other mutants and the parent FMDV. The oil-adjuvanted vaccine formulated with the inactivated VP2_Y98F mutant FMDV was stable up to 8 months when stored at 4°C and induced protective antibody response till dpv 180 after primary vaccination. It is concluded that the VP2_Y98F mutant FMDV was thermostable and has the potential to replace the parent vaccine strain.

A new blocking ELISA for detection of foot-and-mouth disease non-structural protein (NSP) antibodies in a broad host range.

Large-scale monitoring of foot-and-mouth disease (FMD) in livestock is imperative in an FMD control program. Detection of antibodies against non-structural proteins (NSP) of FMD virus (FMDV) is one of the best tools to estimate the prevalence of past infection; availability of such a well-validated test is therefore essential. Using a FMDV 3B protein-specific monoclonal antibody, we have developed a new NSP antibody blocking ELISA (10H9 bELISA) and validated it on large panels of sera from different susceptible species. The diagnostic sensitivity of the ELISA was 95% with a specificity of 98%, similar to the values found using a commercial kit (PrioCHECK FMD NS test). The 10H9 bELISA can be used in a broad range of FMD susceptible species making it a very useful tool in monitoring the foot-and-mouth disease control programs by detection of virus circulation in the vaccinated populations. KEY POINTS: • A new ELISA for detection of foot and mouth disease (FMD) antibodies. • Diagnostic sensitivity of 95% and specificity of 98%. • Tested with panels of validated sera from broad host range.

Long-term protective immunity to goatpox in goats after a single immunization with a live attenuated goatpox vaccine.

In this study, the duration of immunity following a single-dose vaccination using an attenuated live goatpox vaccine (GTPV/Uttarkashi/1978 strain) was evaluated in goatpox-seronegative goats for 52 months. Long-term immunity was evaluated by clinical protection upon virulent virus challenge and serum neutralization assay applied to serum samples. The rise in the level of GTPV-specific antibodies was found to reach a maximum at 21 days post-vaccination, and these antibodies were maintained for 1 to 2 years after immunization, with a steady decline. Upon virulent virus challenge at 12, 24, 42, and 52 months post-vaccination, protection in all the vaccinated animals was evident (100%), whereas, the control animals developed severe clinical disease. This is the first time that the long-term immunity of a live goatpox vaccine has been investigated up to 52 months after vaccination in goats by virulent virus challenge and demonstration of serum neutralization titres. This vaccine has immense potential for controlling and eradicating goatpox from an enzootic region.

Assessment of fitness of foot-and-mouth disease virus A IND 27/2011 as candidate vaccine strain.

Antigenic profiling of recent field outbreak strains of foot-and-mouth disease virus (FMDV) serotype A in India has revealed considerable antigenic drift from the vaccine strain, A IND 40/2000, necessitating the selection of a new strain. The complete genome sequence of A IND 27/2011 was analysed. Vaccine quality attributes of the new candidate strain including potency as an inactivated vaccine in cattle were evaluated. The capsid coding region of A IND 27/2011 showed variation at eight antigenically critical amino acid positions from that of A IND 40/2000. The strain suited well with traits required by a vaccine in terms of its adaptability to adherent and suspension cell line, its immunogenicity, and potency as an inactivated vaccine formulation in cattle. Complete protection was observed upon homologous virus challenge at 4 weeks post-vaccination. Taken together, these data demonstrate the suitability of A IND 27/2011 as an effective vaccine strain of FMDV serotype A.

Induction of antiviral and cell mediated immune responses significantly reduce viral load in an acute foot-and-mouth disease virus infection in cattle.

Foot-and-mouth disease virus (FMDV) causes a severe infection in ruminant animals. Here we present an in-depth transcriptional analysis of soft-palate tissue from cattle experimentally infected with FMDV. The differentially expressed genes from two Indian cattle (Bos indicus) breeds (Malnad Gidda and Hallikar) and Holstein Friesian (HF) crossbred calves, highlighted the activation of metabolic processes, mitochondrial functions and significant enrichment of innate antiviral immune response pathways in the indigenous calves. The results of RT-qPCR based validation of 12 genes was in alignment with the transcriptome data. The indigenous calves showing lesser virus load, elicited early neutralizing antibodies and IFN-γ immune responses. This study revealed that induction of potent innate antiviral response and cell mediated immunity in indigenous cattle, especially Malnad Gidda, significantly restricted FMDV replication during acute infection. These data highlighting the molecular processes associated with host-pathogen interactions, could aid in the conception of novel strategies to prevent and control FMDV infection in cattle.

Monoclonal antibodies against foot-and-mouth disease virus RNA polymerase for detection of virus infection.

Foot-and-mouth disease (FMD) is a major viral disease in farm animals. In the present study, seven monoclonal antibodies (mAbs) were produced against the FMD virus (FMDV)-encoded RNA-dependent RNA polymerase (3D protein) and characterized. Screening of mAb reactivity against three overlapping fragments of the 3D protein expressed in Escherichia coli revealed that the binding sites of all the mAbs were confined to the N-terminal one-third of the 3D protein. A selected mAb was utilized for detecting FMDV in the infected cell culture and tissues obtained from FMDV-infected animals.

Foot-and-mouth disease virus induces PERK-mediated autophagy to suppress the antiviral interferon response.

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes contagious acute infection in cloven-hoofed animals. FMDV replication-associated viral protein expression induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), in turn inducing autophagy to restore cellular homeostasis. We observed that inhibition of BiP (also known as HSPA5 and GRP78), a master regulator of ER stress and UPR, decreased FMDV infection confirming their involvement. Further, we show that the FMDV infection induces UPR mainly through the PKR-like ER kinase (PERK; also known as EIF2AK3)-mediated pathway. Knockdown of PERK and chemical inhibition of PERK activation resulted in decreased expression of FMDV proteins along with the reduction of autophagy marker protein LC3B-II [the lipidated form of LC3B (also known as MAP1LC3B)]. There are conflicting reports on the role of autophagy in FMDV multiplication. Our study systematically demonstrates that during FMDV infection, PERK-mediated UPR stimulated an increased level of endogenous LC3B-II and turnover of SQSTM1, thus confirming the activation of functional autophagy. Modulation of the UPR and autophagy by pharmacological and genetic approaches resulted in reduced numbers of viral progeny, by enhancing the antiviral interferon response. Taken together, this study underscores the prospect of exploring PERK-mediated autophagy as an antiviral target.

Immunogenicity and protective efficacy of 3A truncated negative marker foot-and-mouth disease virus serotype A vaccine.

Foot-and-mouth disease (FMD) is a highly contagious, economically significant disease of cloven-hoofed animals caused by FMD virus (FMDV) of the Picornaviridae family. Vaccination of susceptible animals with inactivated virus vaccine is the standard practice for disease control. The prophylactic use of the inactivated vaccines has reduced the disease burden in many countries endemic to FMD. In the process of implementation of the mass vaccination program and disease eradication, it is essential to differentiate infected from vaccinated animals (DIVA) where a large proportion of the animal population is vaccinated, and disease-free zones are being established, to help in sero-surveillance of the disease. In such a scenario, the use of a negative marker vaccine is beneficial to rule out false-positive results in a disease-free zone. Here we report the construction and rescue of an infectious cDNA clone for FMDV serotype A Indian vaccine strain lacking 58 amino acid residues (87-144 amino acid position) in the carboxy-terminal region of the viral 3A protein. The recombinant deletion mutant virus showed similarity in the antigenic relationship with the parental strain. Immunization of guinea pigs with the inactivated vaccine formulated using the deletion mutant virus induced potent immune response with 100% protective efficacy upon challenge with homologous virus. Further, we show that sera from the guinea pigs infected with the deletion mutant virus did not show reactivity in an indirect ELISA test targeting the deleted portion of 3A protein. We conclude that the recombinant deletion mutant virus vaccine along with the newly developed companion indirect ELISA targeting portion of FMDV 3A protein could be useful in the implementation of a precise DIVA policy in our country when we reach FMD free status with vaccination.

Foot-and-Mouth Disease Virus: Immunobiology, Advances in Vaccines and Vaccination Strategies Addressing Vaccine Failures-An Indian Perspective.

A mass vaccination campaign in India seeks to control and eventually eradicate foot-and-mouth disease (FMD). Biosanitary measures along with FMD monitoring are being conducted along with vaccination. The implementation of the FMD control program has drastically reduced the incidence of FMD. However, cases are still reported, even in regions where vaccination is carried out regularly. Control of FMD outbreaks is difficult when the virus remains in circulation in the vaccinated population. Various FMD risk factors have been identified that are responsible for FMD in vaccinated areas. The factors are discussed along with strategies to address these challenges. The current chemically inactivated trivalent vaccine formulation containing strains of serotype O, A, and Asia 1 has limitations including thermolability and induction of only short-term immunity. Advantages and disadvantages of several new-generation alternate vaccine formulations are discussed. It is unfeasible to study every incidence of FMD in vaccinated animals/areas in such a big country as India with its huge livestock population. However, at the same time, it is absolutely necessary to identify the precise reason for vaccination failure. Failure to vaccinate is one reason for the occurrence of FMD in vaccinated areas. FMD epidemiology, emerging and re-emerging virus strains, and serological status over the past 10 years are discussed to understand the impact of vaccination and incidences of vaccination failure in India. Other factors that are important in vaccination failure that we discuss include disrupted herd immunity, health status of animals, FMD carrier status, and FMD prevalence in other species. Recommendations to boost the search of alternate vaccine formulation, strengthen the veterinary infrastructure, bolster the real-time monitoring of FMD, as well as a detailed investigation and documentation of every case of vaccination failure are provided with the goal of refining the control program.

Baculovirus expression and purification of peste-des-petits-ruminants virus nucleocapsid protein and its application in diagnostic assay.

Peste-des-petits-ruminants (PPR) is a contagious and highly devastating disease of small ruminants. For control of endemic PPR, adequate supply of affordable and reliable diagnostics is critical for effective surveillance, along with the use of highly efficacious live vaccines that are currently available. The nucleocapsid (N) protein of PPR virus (PPRV) is an important candidate antigen for developing specific diagnostic, as it is a major viral protein being highly immunogenic and conserved among the structural proteins. In the present study, we expressed the N protein of PPRV (Sungri/96 strain), in baculovirus expression system and purified using affinity column chromatography. The recombinant protein reacted well with PPRV anti-N monoclonal antibodies and PPRV-specific polyclonal antiserum, suggesting that the expressed protein was authentic and in native form. The recombinant protein was evaluated as antigen in the diagnostic ELISA as reference positive control in place of whole virus antigen. The utility of recombinant PPRV N protein circumvents the need to use live PPRV antigen in the routinely used diagnostics targeting 'N' protein of PPRV, thus allowing large-scale field application of the test.

Host serum microRNA profiling during the early stage of foot-and-mouth disease virus infection.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals, with many outbreaks in the developing world. MicroRNAs (miRNAs) are non-coding RNAs that regulate antiviral defence by post-transcriptional regulation of gene expression. In this study, the host miRNA response following FMDV infection was investigated in cattle, a natural host for FMDV. A significant alteration in serum miRNA expression was detected at early stages of infection. Compared to prior to infection, on day 2 postinfection (PI), 119 miRNAs were upregulated, of which 39 were significantly upregulated (P < 0.05). Gene target prediction and pathway enrichment analysis suggested that upregulated miRNAs target innate immune signalling pathways, suggesting a homeostasis effect, possibly to limit inappropriate immune responses. Further, for the significantly upregulated miRNAs, nine miRNA recognition elements were identified in the genome sequence of FMDV serotype O, which was used for infection. The antiviral effect of four of these miRNAs was confirmed in a cell culture system. These data demonstrate that changes in miRNA expression occur during early pathogenesis, and the identification of possible miRNA targets genes could help in elucidating molecular events involved in virus-host interaction and thus could be useful in developing therapeutic strategies.

IRES mediated expression of viral 3C protease for enhancing the yield of FMDV empty capsids using baculovirus system.

For expression of FMDV empty capsids, high protease activity associated with 3C co-expressed with P1 polyprotein has been reported to adversely affect the yields of capsids. Limiting the levels of 3Cpro relative to P1-2A polypeptide is thus critical to enhance the yields. In this study, FMDV internal ribosome entry site (IRES) sequence which serves as an alternative to the CAP-dependent translation initiation mechanism, was used for controlled translation of 3C protease. Baculovirus expressing bicistronic cDNA cassette containing two open reading frames-FMDV capsid gene (P1-2A) and 3Cpro intervened by IRES was prepared. Analysis of the expression in insect cells infected with baculovirus showed increased accumulation of processed capsids. Recombinant capsids showed higher immunoreactivity similar to the whole virus antigen, when reacted with polyclonal antibodies against the purified whole virus 146S particles. Thus, inclusion of the IRES upstream of 3Cpro facilitated reduced expression of the protease in baculovirus expression system, without causing significant proteolysis, thereby contributing to improved yields of the processed capsid antigens.

Eri silkworm (Samia ricini), a non-mulberry host system for AcMNPV mediated expression of recombinant proteins.

The baculovirus expression system (BVES) based on Autographa californica nucleopolyhedrovirus (AcMNPV) is widely used for the expression of eukaryotic proteins. Several insect cells/larvae that are permissive to AcMNPV have been routinely used as hosts to express heterologous proteins. Domesticated Eri silkworm (Samia ricini), reared in many parts of India, Japan and China, is a non-mulberry silkworm. The present study shows that the Eri silkworm larvae are susceptible to intra-haemocoelical inoculation of AcMNPV. The virus replicates in the larva, as indicated by an increased viral loads in the haemolymph upon injection of a recombinant AcMNPV carrying green fluorescent protein gene. The virus showed localized replication in different tissues including the fat body, haemocytes, tracheal matrix and in the Malphigian tubules. The larval system was successfully used to express heterologous protein, by infecting with a recombinant AcMNPV carrying the 3ABC coding sequence of foot-and-mouth disease virus (FMDV). The study shows that the Eri silkworm larva can be a potential alternative bioreactor, for scaling up of the recombinant proteins employing the baculovirus system.

Protective immune response to liposome adjuvanted high potency foot-and-mouth disease vaccine in Indian cattle.

BACKGROUND: Foot-and-mouth disease (FMD) vaccines applied for prophylactic use in endemic areas provide short-lived immunity requiring regular boosters. Indian FMD control program recommends twice a year vaccination. Development of high potency vaccines that provide better immune response can singificantly contribute to control programme by reducing the frequency of vaccination. The present study explores new adjuvants to enhance the protective efficacy of inactivated trivalent FMD vaccines. METHODOLOGY AND PRINCIPAL FINDINGS: VacciMax(®) is a novel adjuvant which uses a liposome-based oil emulsion platform. Cattle were immunized using VacciMax-A and VacciMax-B FMD vaccines and evaluated for protective efficacy. Similar groups of animals were also boosted after 6 months to study the effect of booster immunisation on protection against homologous challenge. Serum samples from immunized animals were tested by virus neutralization test (VNT) and liquid phase blocking ELISA (LPBE). After challenge, animals were screened for virus load by real-time PCR and reactivity in non-structural protein (3ABC) antibody detection ELISA to corroborate the protection data. A single dose of VacciMax-A formulation elicited higher percentage protection (63%) in VacciMax-A compared to 25% in VacciMax-B upon challenge at one-year post-vaccination. Upon boosting at 6 months also, VacciMax-A group showed higher levels of protection (100%) compared to VacciMax-B (86%), even though both the groups elicited comparable VNT titre (p=0.4964). The results also demonstrated that intramuscular route was preferrable over subcutaneous route of administration. CONCLUSION: The study demonstrates that immunization with VacciMax-A-IM adjuvanted FMD vaccine with high antigen payload under boosting regimen could effectively be used as potent vaccine to maintain herd immunity.

Rescue of infective virus from a genome-length cDNA clone of the FMDV serotype O (IND-R2/75) vaccine strain and its characterization.

We report here the construction and characterization of an infectious cDNA clone of the Indian vaccine strain of foot and mouth disease virus (FMDV) serotype O, IND-R2/75. Viral genome was amplified by reverse transcription-polymerase chain reaction (RT-PCR) in five fragments and subsequently assembled sequentially in a plasmid vector to generate a complete cDNA clone, flanked by the T7 RNA polymerase promoter and poly (A) tail at 5' and 3' ends, respectively. Transfection of BHK-21 cells with the RNA transcribed from this genome-length cDNA construct allowed the recovery of infectious recombinant FMDV particles as evidenced by cytopathic effect in BHK-21 cells. Characterization of the recombinant virus revealed its similarity to the parental strain. Recombinant virus could be distinguished from the parental virus based on the presence of a unique marker sequence in the former, which was incorporated in the cDNA using a silent mutation. The virus showed no significant amino acid changes in the capsid-coding region when serially passaged up to ten times in BHK-21 cells, while retaining the marker sequence.

Animal poxvirus vaccines: a comprehensive review.

The family Poxviridae includes several viruses of medical and veterinary importance. Global concerted efforts combined with an intensive mass-vaccination campaign with highly efficaceious live vaccine of vaccinia virus have led to eradication of smallpox. However, orthopoxviruses affecting domestic animals continue to cause outbreaks in several endemic countries. Different kinds of vaccines starting from conventional inactivated/attenuated to recombinant protein-based vaccines have been used for control of poxvirus infections. Live virus homologous vaccines are currently in use for diseases including capripox, parapox, camelpox and fowlpox, and these vaccines are highly effective in eliciting (with the exception of parapoxviruses) long-lasting immunity. Attenuated strains of poxviruses have been exploited as vectored vaccines to deliver heterologous immunogens, many of them being licensed for use in animals. Worthy of note are vaccinia virus, fowlpox virus, capripoxvirus, parapoxvirus and canary pox, which have been successfully used for developing new-generation vaccines targeting many important pathogens. Remarkable features of these vaccines are thermostability and their ability to engender both cellular and humoral immune responses to the target pathogens. This article updates the important vaccines available for poxviruses of livestock and identifies some of the research gaps in the present context of poxvirus research.

Expression and characterization of bluetongue virus serotype 21 VP7 antigen: C-terminal truncated protein has significantly reduced antigenicity.

In the present study, group-specific antigen VP7 of bluetongue virus (BTV) serotype 21 isolated from cattle in Tochigi prefecture in Japan in 1994 was characterized by sequencing and expression. Gene was amplified from cDNA synthesized on viral dsRNA using reverse-transcriptase-PCR. Nucleotide sequence of this isolate showed high similarity with other published BTV VP7 sequences. Full-length and C-terminal truncated forms of VP7 were expressed in insect cells by a baculovirus gene expression system under control of the viral polyhedrin promoter. Expression of full-length recombinant VP7 was confirmed by immunoprecipitation with VP7 specific monoclonal antibody (8A3B.6, ATCC). Recombinant proteins expressed with or without 6x His-tag showed good expression levels in TN5 cells and reacted well with the monoclonal antibody in the indirect ELISA. However C-terminal truncated VP7 with His-tag failed to react with this monoclonal antibody, while poor antigenicity was evident when it was reacted with infected bovine serum. Reduced antigenicity of the latter suggested that C-terminal truncation affects 8A3B.6 epitope construction probably via inhibition of VP7 trimer structure formation.