Efficient production of Tymovirus like particles displaying immunodominant epitopes of Japanese Encephalitis Virus envelope protein.

Japanese Encephalitis (JE) is a mosquito borne arboviral infection caused by Japanese Encephalitis Virus (JEV). It is a major cause of viral encephalitis in Asian countries including India. In the present study, we have used a Tymovirus [i.e. Physalis Mottle Virus (PhMV) coat protein (CP)], which forms virus like particles (VLPs) as a template to display immunodominant epitopes of JEV envelope (E) protein. The immunodominant epitopes of JEV were inserted at the N-terminus of the wild type PhMV CP, and these constructs were cloned and expressed in Escherichia coli. The chimeric proteins were purified from the inclusion bodies and evaluated for VLP formation. The purified protein was identified by Western blotting and VLP formation was studied and confirmed by transmission electron microscopy and dynamic light scattering. Finally, the immunogenicity was studied in mice. Our results indicate that the chimeric protein with JEV epitopes assembled efficiently to form VLPs generating neutralizing antibodies. Hence, we report the purified chimeric VLP would be a potent vaccine candidate, which needs to be evaluated in a mouse challenge model.

Development of recombinant single-chain variable fragment against hepatitis A virus and its use in quantification of hepatitis A antigen.

Phage display technology has been utilized for identification of specific binding molecules to an antigenic target thereby enabling the rapid generation and selection of high affinity, fully human antibodies directed towards disease target appropriate for antibody therapy. In the present study, single chain Fv antibody fragment (scFv) to hepatitis A virus (HAV) was selected from phage displayed antibody library constructed from peripheral blood lymphocytes (PBLs) of a vaccinated donor. The variable heavy (V(H)) and light chains (V(L)) were amplified using cDNA as template, assembled into scFv using splicing by overlap extension PCR (SOE PCR) and cloned into phagemid vector as a fusion for display of scFv on bacteriophage. The phage displaying antibody fragments were subjected to three rounds of panning with HAV antigen on solid phase. High affinity antibodies reactive to hepatitis A virus were identified by phage ELISA and cloned into a bacterial expression vector pET20b. The scFv was purified by immobilized metal affinity chromatography (IMAC) on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The binding activity and specificity of the scFv was established by its non-reactivity towards other human viral antigens as determined by ELISA and immunoblot analysis. The scFv was further used in the development of an in-house IC-ELISA format in combination with a commercially available mouse monoclonal antibody for the quantification of hepatitis A virus antigen in human vaccine preparations. The adjusted r² values obtained by subjecting the values obtained by quantification of the NIBSC standards using the commercial and the in-house ELISA kits by regression analysis were 0.99 and 0.95. 39 vaccine samples were subjected to quantification using both the kits. Regressional statistical analysis through the origin of the samples indicated International Unit (IU) values of 0.0416x and 0.0419x, respectively for the commercial and in-house kit respectively.

MVA recombinants expressing the fusion and hemagglutinin genes of PPRV protects goats against virulent challenge.

Peste des Petits Ruminants (PPR) is a highly contagious animal disease caused by the Peste des Petits Ruminants virus (PPRV) belonging to the genus morbillivirus and family Paramyxoviridae. The disease results in high morbidity and mortality in goats, sheep and in some small wild ruminants. The presence of large number of small ruminants reared in endemic areas makes PPR a notorious disease threatening the livelihood of poor farmers. Conventional vaccination using a live, attenuated vaccine gives adequate protection but cannot be used in case of eradication of the disease due to difficulty in differentiation of infected animals from the vaccinated ones.In the present study, we constructed two recombinant viruses using attenuated Modified Vaccinia virus Ankara virus (MVA) namely MVA-F and MVA-H expressing the full length PPRV fusion (F) and hemagglutinin (H) glycoproteins, respectively. Goats were vaccinated intramuscularly with 105 plaque forming units (PFU) each of the recombinant viruses and a live attenuated vaccine (RAKSHA PPR) and challenged 4 months later with PPRV challenge virus (10(3) goat LD(50)). All goats were completely protected from the clinical disease. This study gave an indication that mass vaccination of small ruminants with either of the above or both recombinant inexpensive virus vaccines could help in possible eradication of PPRV from endemic countries like India and subsequent seromonitoring of the disease for differentiation of infected animals from vaccinated ones.

Recombinant haemagglutinin protein of highly pathogenic avian influenza A (H5N1) virus expressed in Pichia pastoris elicits a neutralizing antibody response in mice.

Recombinant avian influenza vaccines offer several advantages over the conventional vaccines. In this study, the haemagglutinin (HA) gene of highly pathogenic avian influenza H5N1 was cloned and expressed as His tagged protein in methylotropic yeast Pichia pastoris. The expression of recombinant HA (rHA) protein was confirmed by SDS-PAGE and western blot analysis. The rHA protein was purified using Ni-NTA affinity chromatography under denaturing conditions and the functions of the protein was assessed by the haemagglutinin assay after refolding. The immunogenicity of the rHA was evaluated by immunizing four groups of mice with different payloads (2.5, 5.0, 10 and 25µg) of purified rHA and the production of rHA specific antibodies were analysed by haemagglutinin inhibition assay (HI) and enzyme-linked immunosorbent assay (ELISA). An antigen specific immune response was observed against rHA indicating that the rHA antigen could be used as a vaccine candidate against avian influenza. These results suggest that this strategy would pave the way for the development of rapid and cost effective method for the production of an avian influenza vaccine.

Antemortem and postmortem examinations of the cattle calf naturally infected with Mycobacterium avium subsp. paratuberculosis.

A male cattle calf was detected as subclinically and naturally infected with Mycobacterium avium subspecies paratuberculosis (MAP) by a series of antemortem and postmortem tests. The MAP infection was identified by strong antibody and cell-mediated immune (CMI) response by a commercial ELISA kit and an intradermal Johnin test, respectively, in the initial antemortem examination. The antemortem status of the calf was further confirmed by MAP-specific interferon gamma (IFN-γ) response. For detection of IFN-γ response, MAP-specific IFN-γ release assays (IGRAs): (a) immuno capture ELISA (IC-ELISA) and (b) ELISPOT was employed. In addition, the presence of intracellular cytokine IFN-γ was detected by flow cytometry. For all cytokine assays, MAP-specific recombinant antigens HSP65 and 35 kDa were employed to overcome the poor sensitivity and specificity resulting from the use of Johnin, the crude protein purified derivative of MAP. Postmortem examination of the MAP-infected/suspected cattle calf did not reveal any pathognomonic gross lesions in the gastro-intestinal tract. Histopathological examination of multiple organs showed the presence of epithelioid cells/macrophages and edematous lesions in the mesenteric lymph nodes suggestive of MAP; however, no granulomas were observed in the intestinal tract. The necropsy samples of rectum and mesenteric lymph nodes were positive for isolation of MAP by culture in the BACTEC™ MGIT™ 960 system, and acid fast bacilli were demonstrated by fluorescence microscopy confirming the infection. Due to differential and complex expression patterns of MAP antigens reported in literature, a combination of assays such as those based on IGRAs and antibody detection is essential. Therefore, the current experimental evidence confirms the efficacy of the approach adopted. However, further studies will be needed to understand the optimal combination MAP-specific antigens for use in IGRAs or antibody assays that can be used for detecting MAP infection in every stage of the disease.

Use of ESAT-6-CFP-10 fusion protein in the bovine interferon-gamma ELISPOT assay for diagnosis of Mycobacterium bovis infection in cattle.

Bovine tuberculosis (BTB) is a chronic bacterial disease, and a major animal health problem with zoonotic implications. Screening of mycobacterial infections in bovines is traditionally done using the single intradermal tuberculin test. Though the test is widely used, it has its own disadvantages and they include its inability to distinguish between pathogenic and non-pathogenic mycobacterial infections owing to its low specificity. Furthermore, the associated operative difficulties of this test have driven the quest for discovery of new antigens and diagnostic assays leading to the development of the interferon (IFN)- test. Presently, combinatorial testing using the skin test and the interferon gamma assays are being used in the diagnosis of BTB in various control and surveillance programs. In this study, we report the cloning, expression and purification of ESAT-6-CFP-10 fusion protein and its further use in the development of the IFN- gamma ELISPOT assay for accurate diagnosis of BTB in cattle. The BTB diagnosis employing the ELISPOT assay was evaluated using peripheral blood mononuclear cells from culture positive and culture negative cattle. The ELISPOT assay showed higher specificity and sensitivity in detecting BTB when a recombinant ESAT-6-CFP-10 fusion protein was used. The present study indicated that the usefulness of the fusion protein can replace the ESAT-6, CFP-10 or combination of both proteins for detecting BTB in IFN-gamma ELISPOT assay.

Multi-antigen print immunoassay for seroepidemiological surveillance of bovine tuberculosis on Indian cattle farms.

Bovine tuberculosis caused by Mycobacterium bovis is a zoonotic disease that is responsible for significant economic losses in many countries. The standard diagnostic method, the tuberculin test (TST) that is used in control programmes has serious shortcomings and, given the complex nature and the economic impact of the disease, a number of other diagnostic methods have been examined. The authors have attempted to characterise antibody response using the multi-antigen print immunoassay (MAPIA). A total of 511 serum samples were collected from farms in India on which bovine tuberculosis was prevalent and on farms with low incidence. These were tested using the MAPIA against a panel of five defined M. bovis recombinant antigens and two purified protein derivatives (bovine PPD and avian PPD) to study the seroprevalence of the disease on Indian cattle farms. Results indicated that the fusion protein of antigen CFP-10:MPB83 showed a positive response in 142 out of 298 serum samples from tuberculosis-prevalent farms, thereby indicating the serological dominance of the proteins post infection. The antigen selected could be used further in the development of a simple, rapid and accurate serological diagnostic test, paired with TST, for use in bovine tuberculosis control programmes.

Generation and Characterization of an scFv Directed against Site II of Rabies Glycoprotein.

Recombinant antibody phage display technology is a vital tool that facilitates identification of specific binding molecules to a target enabling the rapid generation and selection of high affinity, fully human, or mouse antibody product candidates essentially directed towards disease target appropriate for antibody therapy. In this study, a recombinant single-chain Fv antibody fragment (scFv) A11 was isolated from immune spleen cells obtained from mice immunized with inactivated rabies virus (Pasteur strain) using standard methodology and was characterized for its specificity towards the rabies virus glycoprotein. Epitope mapping using peptide libraries and truncated glycoprotein polypeptides suggested that A11 bound to the antigenic site II of rabies glycoprotein against which a majority of rabies virus neutralizing antibodies are directed. The use of the above technology could, therefore, allow development of scFvs with different specificities against the rabies glycoprotein as an alternative to the more cumbersome protocols used for the development of monoclonal antibodies.

Assessment of critical parameters in blood processing for the bovine interferon-gamma ELISPOT assay to detect Mycobacterium bovis infected cattle in India.

In vitro production of bovine interferon gamma (BoIFN-γ) cytokine from bovine peripheral blood mononuclear cells (PBMCs) can be detected using the most sensitive enzyme-linked immunosorbent spot (ELISPOT) assay. ELISPOT assays are dependent on the quantity and quality of PBMC preparations and hence contribute significantly to the performance of this assay. In order to standardise the methods for isolation of PBMCs, we compared two methods for the processing of bovine blood which included aliquots of blood that were stored in a horizontal position without dilution or agitation and aliquots of blood that were immediately diluted 1:1 with complete Rosewell Park Memorial Institute (RPMI) 1640 medium and stored in a horizontal position with gentle agitation. PBMCs were isolated at 2, 4, 6, 8 and 24 h and at 4°C and at 22°C ± 2°C. They were stimulated using tuberculosis-specific antigens, after which the ELISPOT assay was performed. Quantities of spot-forming cells (SFC) created by the release of BoIFN-γ in ELISPOT assays were significantly greater in the samples stored at 22°C ± 2°C than those held +4°C and the intensity of the signals dropped following processing after 6 h. A further drop in SFC was observed in those samples that had been stored undiluted and without agitation. These findings demonstrated that optimisation of PBMC isolation procedures can lead to increased sensitivity in the detection of BoIFN-γ using the ELISPOT assay, thus contributing to an enhanced diagnosis of bovine tuberculosis.

Recombinant mid gut antigen (Bm95) as a vaccine against Indian Rhiphicephalus haemaphysaloides in Bos indicus cattle.

In the present study, we report for the first time the efficacy of recombinant Bm95 mid gut antigen isolated from an Argentinean strain of Rhipicephalus microplus strain A in controlling the tick infestations in India. The synthetic gene for Bm95 optimized for expression in yeast was obtained and used to generate yeast transformants expressing Bm95 which was purified to apparent homogeneity. Liquid chromatography-mass spectrometry analysis of the purified protein confirmed its identity as Bm95. Vaccine was prepared by blending various concentrations of purified Bm95 with aluminium hydroxide as an adjuvant. Immunogenicity studies of the vaccine in rabbits and cattle indicated that the vaccine was highly immunogenic. The efficacy studies of the vaccine was done in cattle. Naïve Bos indicus cattle were vaccinated with the recombinant vaccine and were challenged with the larval, nymphal and adult forms of Rhiphicephalus haemaphysaloides. The vaccine protected the animals from larval, nymph and adult tick challenges with an efficacy of 98.7%, 84.6% and 78.9% respectively. The results obtained from the above studies clearly demonstrated the advantage and possibilities of the use of Bm95 in controlling R. haemaphysaloides infestations in the field.

Recombinant diabody-based immunocapture enzyme-linked immunosorbent assay for quantification of rabies virus glycoprotein.

The potency of rabies vaccines, determined using the NIH mouse protection test, can be directly correlated to the amount of rabies virus glycoprotein (RV GP) present in the vaccine. In an effort to develop a simple and sensitive enzyme-linked immunosorbent assay (ELISA) using recombinant diabody for quantification of RV GP, the variable heavy (V(H)) and light chain (V(L)) domains of an RV GP-specific human monoclonal antibody (MAb) secreted by a human x mouse heterohybridoma (human MAb R16E5) was amplified, linked using splicing by overlap extension PCR (SOE PCR), and expressed as a recombinant diabody (D06) in the pET28a bacterial expression system. The diabody D06 was purified by immobilized metal affinity chromatography on a nickel-nitrilotriacetic acid (NTA) agarose column and characterized. The purified diabody was used in combination with a well-characterized RV GP-specific mouse MAb, M5B4, to develop an immunocapture ELISA (IC-ELISA) for the quantification of RV GP in human rabies vaccine preparations. The maximum detection limit of the IC-ELISA using the M5B4-D06 combination was up to 31.25 ng/ml of RV GP. The specificity of the diabody was established by its nonreactivity toward other human viral antigens as determined by ELISA and toward RV GP as determined by immunoblot transfer assay and competitive ELISA with the parent human MAb R16E5 and MAb M5B4. The adjusted r(2) value obtained by the regression through the origin model was 0.902, and the equation for predicted potency values for M5B4-D06-based IC-ELISA and MAb M5B4 IC-ELISA were 0.5651x and 0.8044x, respectively, where x is the estimate of RV GP from the IC-ELISA in micrograms. Analysis of variance (ANOVA) results showed the estimates of the two methods differed significantly (P < 0.001), while the predicted potencies by the two tests did not differ significantly (P > 0.05). The IC-ELISA can be readily adapted to measure the RV GP content in purified antigen, and a vaccine can be formulated based on the estimated GP.

Development of a recombinant epsilon toxoid vaccine against enterotoxemia and its use as a combination vaccine with live attenuated sheep pox virus against enterotoxemia and sheep pox.

Sheep pox and enterotoxemia are important diseases of sheep, and these diseases cause severe economic losses to sheep farmers. The present study was undertaken to evaluate the potential of formaldehyde-inactivated recombinant epsilon toxin as a vaccine candidate. The potency of the recombinant epsilon toxoid with aluminum hydroxide as an adjuvant in sheep was determined. Vaccinated sheep were protected against enterotoxemia, with potency values of >5 IU being protective. Further, the use of this construct in a combination vaccine against sheep pox resulted in the sheep being protected against both sheep pox and enterotoxemia.

Display of neutralizing epitopes of Canine parvovirus and a T-cell epitope of the fusion protein of Canine distemper virus on chimeric tymovirus-like particles and its use as a vaccine candidate both against Canine parvo and Canine distemper.

Expression of Physalis mottle tymovirus coat protein in Escherichia coli was earlier shown to self-assemble into empty capsids that were nearly identical to the capsids formed in vivo. Amino acid substitutions were made at the N-terminus of wild-type Physalis mottle virus coat protein with neutralizing epitopes of Canine parvovirus containing the antigenic sites 1-2, 4 and 6-7 and T-cell epitope of the fusion protein of Canine distemper virus in various combinations to yield PhMV1, PhMV2, PhMV3, PhMV4 and PhMV5. These constructs were cloned and expressed in E. coli. The chimeric proteins self-assembled into chimeric tymovirus-like particles (TVLPs) as determined by electron microscopy. The TVLPs were purified by ultracentrifugation and injected into guinea pigs and dogs to determine their immunogenicity. Initial immunogenicity studies in guinea pigs indicated that PhMV3 gave a higher response in comparison to the other TVLPs for both CPV and CDV and hence all further experiments in dogs were done with PhMV3. HI was done against different isolates obtained from various parts of the country. Protective titres indicated the broad spectrum of the vaccine. In conclusion the study indicated that the above chimeric VLP based vaccine could be used in dogs to generate a protective immune response against diseases caused by both Canine parvo and Canine distemper virus.

Immune responses against recombinant tick antigen, Bm95, for the control of Rhipicephalus (Boophilus) microplus ticks in cattle.

Immune responses against Bm95 recombinant cattle tick antigen and its protective efficacy for control of Rhipicephalus (Boophilus) microplus ticks were determined in experimental crossbred cow calves. Anti-Bm95 antibody titers, as assessed by indirect ELISA, in immunized calves ranged from 196.1+/-13.7 on day 0 to 7979.9+/-312.5 on day 110 post-primary immunization. The rise in antibody titer was statistically significant (p<0.01) throughout the study period. Besides this, constantly higher lymphoproliferative response (LPR), as assessed by lymphocyte stimulation test, was observed from 10 days post-immunization, but a positive LPR of antigen stimulated cells in immunized animals was recorded only on day 50 and day 70 post-immunization. Following challenge of immunized calves with larvae of R. microplus, significant increase (p<0.01) in rejection percentage, mean number of damaged ticks, mean percentage of dead ticks, and decrease in engorgement weight were recorded in immunized animals. Also, there were significant differences (p<0.01) in preoviposition period, oviposition period, egg mass weight and percent hatchability between the immunized and control calves. The percent reduction in number of adult females in vaccinated calves, reduction in mean weight of egg masses, percent reduction in mean weight and reduction in fertility of engorged females collected from vaccinated calves were determined and the efficacy of Bm95 recombinant cattle tick antigen was 81.27%.

Vaccinia virus entry/fusion complex subunit A28 is a target of neutralizing and protective antibodies.

The vaccinia virus entry/fusion complex (EFC) is comprised of at least eight transmembrane proteins that are conserved in all poxviruses. However, neither the physical structure of the EFC nor the immunogenicity of the individual components has been determined. We prepared soluble forms of two EFC components, A28 and H2, by replacing the transmembrane domain with a signal peptide and adding a polyhistidine tail. The proteins were expressed by baculoviruses, secreted from insect cells, purified by affinity chromatography and used to raise antibodies in rabbits. The antibodies recognized the viral proteins but only the antibody to recombinant A28 bound intact virions and neutralized infectivity. Analyses with a set of overlapping peptides revealed a neutralizing epitope between residues 73 and 92 of A28. Passive immunization of mice with IgG purified from the anti-A28 serum provided partial protection against a vaccinia virus intranasal challenge, whereas IgG from the anti-H2 serum did not.

Correlation of immunogenicities and in vitro expression levels of recombinant modified vaccinia virus Ankara HIV vaccines.

The purpose of the present study was to correlate the in vitro level of HIV Env expression by recombinant modified vaccinia virus Ankara (rMVA) with immunogenicity in mice. A 5-fold difference in Env synthesis was achieved at the translational level by the presence or absence of an out-of-frame initiation codon upstream of the env gene. This perturbation had no effect on the size or processing of Env. In contrast to the variation in Env synthesis, the rMVAs produced similar amounts of HIV Gag, which were expressed from identical cassettes. Mice immunized with the higher Env expressing rMVAs had about 15-fold higher titers of Env antibodies and several fold higher frequencies of Env-specific CD8+ and CD4+ T cells than mice immunized with the low expresser. The greater immune response achieved by high expression was maintained over a 100-fold dose range. Importantly, enhanced Env immune responses did not come at the expense of lower Gag T cell responses. These data suggest that for high immunogenicity, rMVAs should be engineered to produce the most recombinant protein that can be achieved without compromising the growth and stability of the rMVA.

Chimeric tymovirus-like particles displaying foot-and-mouth disease virus non-structural protein epitopes and its use for detection of FMDV-NSP antibodies.

Expression of Physalis mottle tymovirus (PhMV) coat protein (CP) in Escherichia coli (E. coli) was earlier shown to self-assemble into empty capsids that are nearly identical to the capsids formed in vivo. Aminoacid substitutions were made at the N-terminus of wild-type PhMV CP with single or tandem repeats of infection related B-cell epitopes of foot-and-mouth disease virus (FMDV) non-structural proteins (NSPs) 3B1, 3B2, 3AB, 3D and 3ABD of lengths 48, 66, 49, 51 and 55, respectively to produce chimeras pR-Ph-3B1, pR-Ph-3B2, pR-Ph- 3AB, pR-Ph-3D and pR-Ph-3ABD. Expression of these constructs in E. coli resulted in chimeric proteins which self-assembled into chimeric tymovirus-like particles (TVLPs), Ph-3B1, Ph-3B2, Ph-3AB, Ph-3D and Ph-3ABD as determined by ultracentrifugation and electron microscopy. Ph-3B1, Ph-3B2, Ph-3AB and Ph-3ABD reacted with polyclonal anti-3AB antibodies in ELISA and electroblot immunoassay, while wild-type PhMV TVLP and Ph-3D antigens did not react. An indirect ELISA (I-ELISA) was developed using Ph-3AB to detect FMDV-NSP antibodies in sera of animals that showed clinical signs of FMD. Field serum samples from cattle, buffalos, sheep, goats and pigs were examined by using these chimeric TVLPs for the differentiation of FMDV infected animals from vaccinated animals (DIVA). The assay was demonstrated to be highly specific (100%) and reproducible with sensitivity levels (94%) comparable to the Ceditest kit (P>0.05).

Molecular characterization of foot-and-mouth disease virus type C of Indian origin.

Comparison of nucleotide sequences of the partial 1D region of foot-and-mouth disease type C viruses of Indian origin with those of European, South American, and Southeast Asian viruses revealed that the Indian viruses form a distinct genotype. The vaccine strain C IND/51/79 belongs to this genotype and may be a prototype strain of this genotype.