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 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.

Ribavirin as a curative and prophylactic agent against foot and mouth disease virus infection in C57BL/6 suckling and adult mice model.

Despite the availability of control measures for foot-and-mouth disease (FMD), the application of antiviral agents is imperative due to certain limitations in the prevention and control of FMD. This study pertains to systematic in vivo investigation of ribavirin as a prophylactic/curative agent, both in suckling and adult C57BL/6 mice against foot-and-mouth disease virus (FMDV) infection. In the adult mice, antiviral efficacy was assessed based on standard clinical score, body weight, and viral load. Only 13.33 to 33.33% of adult mice exhibited disease-specific symptoms following treatment and infection and vice versa, respectively, indicating the antiviral efficacy of the ribavirin. Further, the distribution of virus in different vital organs following ribavirin treatment and virus infection, and vice versa using SYBR green-based real-time PCR is reported. In the blood sample, the viral RNA was detected as early as two days post-infection and there was a significant reduction in virus titer (1000 to 10,000-folds) in the treatment groups compared to the infection control group. Animals receiving ribavirin had significantly lower organ virus titers at 2, 4, 6, 9, and 14 days post-challenge (dpc) than placebo-treated. In suckling mice, the treatment groups were 100% protected/cured compared to the control group. Thus, our data demonstrate that ribavirin may provide a feasible therapeutic approach to prevent as well as to treat FMDV infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00746-8.

In vitro antiviral efficacy of pleconaril and ribavirin on foot-and-mouth disease virus replication.

Antiviral therapy is a promising strategy to control acute viral infections. FMDV causes an acute infection and the vaccination provides a protective immunity 7 days post immunization. If the infection is uncontained, then it affects the entire herd. In such circumstances, if antiviral drug is administered the infection can be checked in a herd. Ribavirin is known to cure persistently infected BHK21 cells with FMD virus. However, there have been no systematic studies on antiviral activity of ribavirin against FMDV at different time points with the application of ELISA, PCR or real-time PCR. Pleconaril is known to inhibit enteroviruses and rhinoviruses but has not been explored on FMDV. Hence, the present study evaluates the in vitro antiviral efficacy of pleconaril and ribavirin on FMDV replication. The maximum non-toxic concentrations (MNTC) of pleconaril and ribavirin for BHK21 cells respectively were 7.81 µg/50 µL and 15.62 µg/50 µL. Thus, drug concentrations below MNTC were tested for their antiviral activity against serial tenfold diluted FMDV O, A and Asia 1 serotypes. Pleconaril did not inhibit FMDV serotype O replication at 7.5 µg/50 µL based on CPE inhibition assay and this was further confirmed using sandwich ELISA, PCR/real-time PCR. On the other hand, ribavirin at 15.62 µg/50 µL inhibited the in vitro replication of FMDV O, A and Asia 1 and the inhibition was confirmed by serotype specific sandwich ELISA, PCR and real-time PCR assays. The inhibition was directly proportional to the concentration of ribavirin. Therefore, ribavirin could be explored for its in vivo efficacy as a potential therapeutic in the prevention of early spread of FMDV infection in a herd.

Sequence analysis of haemagglutinin gene of camelpox viruses shows deletion leading to frameshift: Circulation of diverse clusters among camelpox viruses.

Orthopoxviruses (OPVs) have broad host range infecting a variety of species along with gene-specific determinants. Several genes including haemagglutinin (HA) are used for differentiation of OPVs. Among poxviruses, OPVs are sole members encoding HA protein as part of extracellular enveloped virion membrane. Camelpox virus (CMLV) causes an important contagious disease affecting mainly young camels, endemic to Indian subcontinent, Africa and the Middle East. This study describes the sequence features and phylogenetic analysis of HA gene (homologue of VACV A56R) of Indian CMLV isolates. Comparative analysis of CMLV HA gene revealed conserved nature within CMLVs but considerable variability was observed between various species of OPVs. Most Indian CMLV isolates showed 99.5%-100% and 96.3%-100% identity, at nucleotide (nt) and amino acid (aa) levels respectively, among themselves and with CMLV-M96 strain. Importantly, Indian CMLV strains along with CMLV-M96 showed deletion of seven nucleotides resulting in frameshift mutation at C-terminus of HA protein. Phylogenetic analysis displayed distinct clustering among CMLVs which might point to the circulation of diverse CMLV strains in nature. Despite different host specificity of OPVs, comparative sequence analysis of HA protein showed highly conserved N-terminal Ig V-set functional domain with tandem repeats. Understanding of molecular diversity of CMLVs and structural domains of HA protein will help in the elucidation of molecular mechanisms for immune evasion and design of novel antivirals for OPVs.

Evaluation of a recombinant major envelope protein (F1L) based indirect- ELISA for sero-diagnosis of orf in sheep and goats.

Orf or contagious ecthyma, is a highly contagious transboundary disease of sheep and goats. For sero-diagnosis of orf, recombinant antigen based assays are considered as alternatives to conventional approaches such as serum neutralization test (SNT) and counter-immuno-electrophoresis (CIE). A major envelope protein of orf virus (ORFV), F1L, is highly immunogenic and is a candidate for use in these assays. In this study, the F1L gene of the ORFV-59/05 strain encoding a recombinant mature F1L protein (1M-D302 aa) with a C- terminal truncation, was produced as a fusion protein (∼50 kDa) in Escherichia coli. The immunogenic potential of purified rF1L was confirmed by detecting specific anti-F1L antibody responses in sera collected from immunized rabbits and guinea pigs using ELISA and SNT. An indirect-ELISA based on rF1L was developed and optimized. In comparison to SNT by ROC analysis in the detection of ORFV specific antibodies, this new assay exhibited a diagnostic specificity of 94.04% and 92.53% with sheep and goat sera, respectively, while the sensitivity was 89.22% and 94.25%, for sheep and goat sera. No cross reactivity was noted with sera collected from small ruminants infected with other transboundary diseases (goatpox, sheeppox, peste des petits ruminants, foot-and-mouth disease and bluetongue). Furthermore, the rF1L-ELISA applied to screen the vaccinated/challenged goat sera resulted in better detection (30%) than by SNT (28%) in spite of lower levels of antibodies which could be due to predominant cell mediated immune response in vaccinated animals. This study highlighted the potential utility of rF1L protein as a safe and novel diagnostic reagent in comparison to live virus antigen, in the development of sero-diagnostic assay for surveillance of ORFV infection in sheep and goats.

Chemokine CCL20 plasmid improves protective efficacy of the Montanide ISA™ 206 adjuvanted foot-and-mouth disease vaccine in mice model.

This study aimed to investigate the chemokine CCL20, a macrophage inflammatory protein-3 alpha, for adjuvant potential in inactivated foot-and-mouth disease (FMD) vaccine. Groups of mice were injected intramuscularly with either murine CCL20 DNA or CCL20 protein two days ahead of the immunization with Montanide ISA206 adjuvanted inactivated FMD vaccine and humoral and cellular immune responses were measured in post-vaccinal sera. We demonstrated that the mice immunized with CCL20 plasmid plus FMD vaccine showed earlier and significantly (p < 0.05) higher neutralizing antibody responses compared to the mice vaccinated with CCL20 protein plus FMD vaccine. In fact, CCL20 as a protein did not show any adjuvant effect and the immune responses induced in this group were comparable to that of the mice vaccinated with FMD vaccine alone. All the vaccination groups showed serum IgG1 and IgG2 antibody responses; however, the mice vaccinated with CCL20 plasmid plus FMD vaccine showed significantly (p < 0.05) higher IgG1 and IgG2 responses and the responses remained high at all-time points post vaccination, although not always statistically significant. Upon restimulation of the vaccinated splenocytes with the inactivated FMD viral antigen, significantly (p < 0.05) higher IFN-γ and IL-2 levels in culture supernatants were found in animals vaccinated with the CCL20 plasmid plus FMD vaccine, which is indicative of the TH1 type of cellular immunity. On challenge with the homologous FMD virus on 28th day post immunization, CCL20 plasmid plus FMD vaccine showed complete protection (100%) while animals immunized with CCL20 protein plus FMD vaccine or FMD vaccine alone showed 66% protection. In summary, we show that prior injection of CCL20 plasmid improved protective efficacy of the inactivated FMD vaccine and thus offers a valuable strategy to modulate the efficacy and polarization of specific immunity against inactivated vaccines.

Comparative sequence and structural analysis of Indian orf viruses based on major envelope immuno-dominant protein (F1L), an homologue of pox viral p35/H3 protein.

Orf virus (ORFV), a member of the genus Parapoxvirus in the family Poxviridae, is the cause of orf, a highly contagious zoonotic viral disease that affects mainly sheep and goats. In the present study, the sequence and phylogenetic analysis of Indian ORFV isolates (n = 15) from natural outbreaks in sheep and goats belonging to different geographical regions were analysed on the basis of F1L gene along with homology modelling of F1L protein. Multiple sequence alignments revealed highly conserved C-terminus and heterogeneity of N-terminus region of F1L among all orf viruses studied. Further, a comparative sequence alignment indicated conservation of various motifs such as glycosaminoglycan (GAG), Asp/Glu-any residue-Asp (D/ExD) and a Cx3C chemokine like motif among all poxviruses and unique motifs (proline rich region [PRR] and Lys-Gly-Asp [KGD]), in parapoxviruses including ORFV isolates irrespective of geography and host species. Phylogenetically, two major clusters were noticed which included Indian orf isolates along with foreign isolates. Structurally, ORFV F1L resembled the topology as exhibited by its homologue vaccinia virus H3 protein with mixed ß/α folds and ligand binding specificity in docking models. We noted that despite differences in host cell specificity and pathogencity, poxvirus proteins especially ORFV F1L protein and its homologues presumed to share similarities as they are highly conserved irrespective of species and countries of origin. Further, the study also indicated the possibilities of differentiation of ORFV strains based on N-terminal heterogeneity despite highly conserved C-terminal region with conserved motifs.

Comparison of different inactivation methods on the stability of Indian vaccine strains of foot and mouth disease virus.

In this study, the efficiency of binary ethyleneimine (BEI) in combination with formaldehyde (FA) and glutaraldehyde (GTA) in inactivating the Indian FMDV vaccine strains is compared. The acceptable safety of virus inactivation was faster and the inactivation rates were increased many-folds with combination of inactivants than BEI alone. FMDV A was inactivated rapidly than the other two serotypes with BEI + FA combination. Inactivation plots were linear for all the serotypes irrespective of inactivation process. Further, the integrity studies on 146S using serotype specific ELISA indicated no significant change in the antigenic mass of all the serotypes throughout the inactivation process. However, the loss of 146S antigen occurred in the subsequent steps of downstream processing. Further, the studies on intactness of viral RNA using real time PCR indicated the amplification of 1D gene sequences in all the preparations of timed samples irrespective of serotypes/inactivation process. Further, inactivated virus preparation (146S) was more stable at lower temperatures for all the serotypes/inactivation process. Among the combinations of inactivants, BEI + FA out performed compared to BEI + GTA and BEI in terms of inactivation rates, 146S yield and its storage stability, irrespective of the serotypes.

Co-administration of recombinant major envelope proteins (rA27L and rH3L) of buffalopox virus provides enhanced immunogenicity and protective efficacy in animal models.

Buffalopox virus (BPXV) and other vaccinia-like viruses (VLVs) are causing an emerging/re-emerging zoonosis affecting buffaloes, cattle and humans in India and other countries. A27L and H3L are immuno-dominant major envelope proteins of intracellular mature virion (IMV) of orthopoxviruses (OPVs) and are highly conserved with an ability to elicit neutralizing antibodies. In the present study, two recombinant proteins namely; rA27L (21S to E110; ∼30 kDa) and rH3L(1M to I280; ∼50 kDa) of BPXV-Vij/96 produced from Escherichia coli were used in vaccine formulation. A combined recombinant subunit vaccine comprising rA27L and rH3L antigens (10 µg of each) was used for active immunization of adult mice (20µg/dose/mice) with or without adjuvant (FCA/FIA) by intramuscular route. Immune responses revealed a gradual increase in antigen specific serum IgG as well as neutralizing antibody titers measured by using indirect-ELISA and serum neutralization test (SNT) respectively, which were higher as compared to that elicited by individual antigens. Suckling mice passively administered with combined anti-A27L and anti-H3L sera showed a complete (100%) pre-exposure protection upon challenge with virulent BPXV. Conclusively, this study highlights the potential utility of rA27L and rH3L proteins as safer candidate prophylactic antigens in combined recombinant subunit vaccine for buffalopox as well as passive protective efficacy of combined sera in employing better pre-exposure protection against virulent BPXV.

Functional characterization of recombinant major envelope protein (rB2L) of orf virus.

Orf, or contagious ecthyma, a highly contagious transboundary disease of sheep and goats, is caused by a double-stranded DNA virus (ORFV) belonging to the genus Parapoxvirus of the family Poxviridae. The ORFV genome encodes the major envelope proteins B2L and F1L, which have been found to be highly immunogenic and have multiple functional characteristics. In order to investigate the functional properties of the B2L protein, in this study, the B2L gene of ORFV strain 59/05, encoding recombinant mature B2L (aa 1M-D334), was produced as a fusion protein in Escherichia coli. The functional characteristics of purified rB2L fusion protein (~60 kDa) were evaluated in vivo and in vitro, showing that this protein had lipase and immunomodulatory activities. Immunization trials involving laboratory animals (mice, rabbits and guinea pigs) using either constant or graded doses of rB2L fusion protein with or without adjuvants (FCA, alum) as well as co-administration with candidate rErns-Ag protein of classical swine fever virus (CSFV) indicated that the rB2L protein is immunogenic and has immunomodulatory properties. This study shows the potential utility of the rB2L protein as a safe and novel adjuvant in veterinary vaccine formulations.

Immunogenicity and protective efficacy of recombinant major envelope protein (rH3L) of buffalopox virus in animal models.

Buffalopox virus, a zoonotic Indian vaccinia-like virus, is responsible for contagious disease affecting mainly buffaloes, cattle and humans. H3L gene, encoding for an immunodominant major envelope protein of intracellular mature virion of orthopoxviruses, is highly conserved and found to elicit neutralizing antibodies. Therefore in the present study, the immunogenicity and protective efficacy of the recombinant H3L protein of buffalopox virus in laboratory animal models has been evaluated. A partial H3L gene encoding for the C-terminal truncated ectodomain of H3L protein (1M to I280) of BPXV-Vij/96 strain was cloned, over-expressed and purified as histidine-tagged fusion protein (50 kDa) from Escherichia coli using Ni-NTA affinity chromatography. The purified rH3L protein was further used for active immunization of guinea pig (250 µg/dose) and adult mice (10 µg and 50 µg/dose) with or without adjuvants (alum, Freund's Complete Adjuvant and CpG). Subsequently, a gradual increase in antigen specific serum IgG as well as neutralizing antibody titres measured by using indirect-ELISA and serum neutralization test respectively, was noted in both guinea pigs and mouse models. Suckling mice immunized passively with anti-H3L serum showed 80% pre-exposure prophylaxis upon challenge with virulent buffalopox virus strain. An indirect-ELISA based on rH3L protein showed no cross-reactivity with hyperimmune sera against sheeppox virus (SPPV), goatpox virus (GTPV), orf virus (ORFV), foot- and- mouth disease virus (FMDV), peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) during the course of study. The study highlights the potential utility of rH3L protein as a safer prophylactic and diagnostic reagent for buffalopox.

Synergistic effect of high-mobility group box-1 and lipopolysaccharide on cytokine induction in bovine peripheral blood mononuclear cells.

High-mobility group box 1 (HMGB1) is one of the potent endogenous adjuvants released by necrotic and activated innate immune cells. HMGB1 modulates innate and adaptive immune responses in humans and mice by mediating immune cells crosstalk. However, the immuno-modulatory effects of HMGB1 in the bovine immune system are not clearly known. In this study, the effect of bovine HMGB1 alone or in combination with LPS on the expression kinetics of cytokines upon in vitro stimulation of bovine peripheral blood mononuclear cells (PBMCs) was investigated by quantitative PCR assay. The biological activity of bovine HMGB1 expressed in this prokaryotic expression system was confirmed by its ability to induce nitric oxide secretion in RAW 264.7 cells. The present results indicate that HMGB1 induces a more delayed TNF-α response than does LPS in stimulated PBMCs. However, IFN-γ, IFN-ß and IL-12 mRNA transcription peaked at 6 hr post stimulation after both treatments. Further, HMGB1 and LPS heterocomplex up-regulated TNF-α, IFN-γ and IL-12 mRNA expression significantly than did individual TLR4 agonists. The heterocomplex also enhanced the expression of TLR4 on bovine PBMCs. In conclusion, the data indicate that HMGB1 and LPS act synergistically and enhance proinflammatory cytokines, thereby eliciting Th1 responses in bovine PBMCs. These results suggest that HMGB1 can act as an adjuvant in modulating the bovine immune system and thus lays a foundation for using HMGB1 as an adjuvant in various bovine vaccine preparations.

Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells.

Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria. They not only represent a potential platform for development of novel vaccines but also provide a tool for efficient adjuvant and antigen delivery system. In the present study, we investigated the interaction between BGs of Escherichia coli (E. coli) and bovine monocyte-derived dendritic cells (MoDCs). MoDCs are highly potent antigen-presenting cells and have the potential to act as a powerful tool for manipulating the immune system. We generated bovine MoDCs in vitro from blood monocytes using E. coli expressed bovine GM-CSF and IL-4 cytokines. These MoDCs displayed typical morphology and functions similar to DCs. We further investigated the E. coli BGs to induce maturation of bovine MoDCs in comparison to E. coli lipopolysaccharide (LPS). We observed the maturation marker molecules such as MHC-II, CD80 and CD86 were induced early and at higher levels in BG stimulated MoDCs as compared to the LPS stimulated MoDCs. BG mediated stimulation induced significantly higher levels of cytokine expression in bovine MoDCs than LPS. Both pro-inflammatory (IL-12 and TNF-α) and anti-inflammatory (IL-10) cytokines were induced in MoDCs after BGs stimulation. We further analysed the effects of BGs on the bovine MoDCs in an allogenic mixed lymphocyte reaction (MLR). We found the BG-treated bovine MoDCs had significantly (p<0.05) higher capacity to stimulate allogenic T cell proliferation in MLR as compared to the LPS. Taken together, these findings demonstrate the E. coli BGs induce a strong activation and maturation of bovine MoDCs.

Evaluation of thermo-stability of bluetongue virus recombinant VP7 antigen in indirect ELISA.

This study shows the thermo-stability of lyophilized and purified recombinant VP7 bluetongue virus (BTV) protein in the presence of two sugar stabilizers (trehalose and mannitol) at different temperature. Truncated VP7 protein purified by nickel affinity column was lyophilized in the presence of trehalose and mannitol at 60 mM final concentration and then exposed to different temperature like 4, 25, 37 and 45 °C for various periods like 5 months, 7 weeks, 7 days and 48 h, respectively. After thermal treatment, the reactivity of the protein was evaluated in indirect ELISA. At 4 and 25 °C, the protein was stable up to 5 months and 7 weeks, respectively, irrespective of stabilizers used. At 37 °C, it was stable up to 3 days with both the stabilizers, after which it lost its stability and reactivity. At 45 °C, the protein was stable up to 30 and 24 h with trehalose and mannitol stabilizers, respectively. Both stabilizers found suitable for stability of the protein. However, trehalose appeared to have better stabilizing effect, particularly at higher temperatures than the mannitol. Trehalose could be used as stabilizer for freeze-drying the recombinant VP7 protein if an indirect ELISA kit based on the purified rVP7 protein is supplied to different laboratories of the country for detection of BTV antibody in sheep.

Structural analysis and immunogenicity of recombinant major envelope protein (rA27L) of buffalopox virus, a zoonotic Indian vaccinia-like virus.

Buffalopox virus (BPXV), an Indian variant of vaccinia virus (VACV), is a zoonotic agent and affects buffaloes, cattle and humans. A27L is one of the conserved major immuno-dominant envelope proteins of orthopox viruses (OPVs) involved in viral entry/maturation and elicits neutralizing antibodies. In this study, the A27L gene of BPXV-Vij/96 strain encoding recombinant mature A27L (21S to E110) and C-terminal truncated A27L-LZD (21S to N84aa) proteins were cloned and over-expressed in Escherichia coli as fusion proteins. Structurally, A27L of BPXV was similar to that of VACV and found to contain four regions including a potential coiled-coil motif (CCM) in the centre (43 to 84aa). Oligomerization of recombinant A27L fusion protein (∼30 kDa) leads to the formation of dimer/trimers/tetramers under non-reducing conditions. Further, the purified rA27L protein was used for active immunization of rabbit (250 µg/rabbit) and adult mice (10 µg and 50 µg/mice) with or without adjuvants (FCA, alum and CpG). Immune response measured by using indirect-ELISA and SNT revealed a gradual increase in antigen specific serum IgG as well as neutralization antibody titers. Upon challenge with virulent BPXV strain, a protection of 60% was observed in suckling mice passively administered with anti-rA27L sera. No cross-reactivity of rA27L protein with hyperimmune sera against ORFV, GTPV, SPPV, PPRV, FMDV and BTV was noticed in indirect-ELISA. The study indicated that the rA27L protein is a safe and potential prophylactic as well as diagnostic antigen for buffalopox.

Immunological evaluation of mannosylated chitosan nanoparticles based foot and mouth disease virus DNA vaccine, pVAC FMDV VP1-OmpA in guinea pigs.

A DNA vaccine for foot and mouth disease (FMD) based on mannosylated chitosan nanoparticles was evaluated in guinea pigs. The DNA construct was comprised of FMD virus full length-VP1 gene and outer membrane protein A (Omp A) gene of Salmonella typhimurium as a Toll-like receptor (TLR)-ligand in pVAC vector. Groups of guinea pigs immunized either intramuscularly or intra-nasally were evaluated for induction of virus neutralizing antibodies, Th1(IgG2) and Th2 (IgG1) responses, lymphocyte proliferation, reactive nitrogen intermediate production, secretory IgA for naso-mucosal immune response and protection upon homotypic type O virulent FMD virus challenge. The results indicate the synergistic effect of OmpA on the immunogenic potential of FMD DNA vaccine construct delivered using mannosylated chitosan nano-particles by different routes of administration. These observations suggest the substantial improvement in all the immunological parameters with enhanced protection in guinea pigs.