B.P. Sreenivasa

Novel immunogenic baculovirus expressed virus-like particles of foot-and-mouth disease (FMD) virus protect guinea pigs against challenge

Vaccination is a well accepted strategy for control of foot-and-mouth disease (FMD) in endemic countries. Currently, chemically inactivated virus antigens are used for preparation of FMD vaccine. To develop a non-infectious and safe recombinant vaccine, we expressed structural polypeptide of FMDV (O/IND/R2/75) using baculovirus expression system. We show that inclusion of mutated viral 3C protease in frame with the polypeptide (P1-2A), enhanced the yield of structural proteins. The structural proteins retained antigenicity and assembled into empty virus-like particles (VLPs). Immunization of guinea pigs with purified fractions of the VLPs resulted in humoral and cell mediated immune response by 4 weeks. The VLPs elicited comparable humoral immune response and relatively higher cell mediated immune response, when compared to conventional vaccine in guinea pigs. Further, up to 70% of the VLP immunized guinea pigs were protected against challenge with homologous guinea pig adapted virus. Our results highlight the application of recombinant FMDV VLPs in FMD vaccination.

Comparative sequence analysis of the large polymerase protein (L) gene of peste-des-petits ruminants (PPR) vaccine virus of Indian origin

Summary.The complete nucleotide sequence of the large polymerase (L) protein of the peste-des-petits ruminants (PPR) vaccine virus (PPRV Sungri/96) belonging to the Asian lineage was determined. The gene was 6643 nucleotides in length from the gene-start to the gene-end and encoded a polypeptide of 2183 amino acids. The PPRV Sungri/96 has a nucleotide homology of 94.1% for PPRV Nigeria 75/1 to 64.4% for Canine distemper virus. At amino acid level PPRV Sungri/96 has an amino acid identity of 96.2% with PPRV Nigeria 75/1 and 70.4% to 74.8% with other morbilliviruses. All the established domains in L protein characteristic of paramyxoviruses were also found to be present in PPRV Sungri/96. Phylogenetic analysis of different L proteins of morbilliviruses revealed five well-defined clusters as observed previously. The 3′ trailer sequence of PPRV Sungri/96 is of 37 nucleotides long which is very similar to that of other morbilliviruses. To the best of our knowledge this is the first report describing the polymerase gene sequence of PPRV Indian isolate.

Development of a liquid-phase blocking ELISA based on foot-and-mouth disease virus empty capsid antigen for seromonitoring vaccinated animals

In foot-and-mouth disease (FMD) control programme, liquid-phase blocking ELISA (LPBE) is widely used to assay vaccine-induced seroconversion. Currently, the assay utilizes inactivated FMD virus antigen for the detection of antibodies in serum samples. To develop a non-infectious substitute for the antigen in LPBE, we expressed the structural polypeptide of FMDV (serotype A) using a baculovirus expression system, and show that inclusion of viral 3C with reduced protease activity resulted in a higher yield of structural proteins. Structural proteins expressed in insect cells assembled into empty virus-like particles (VLPs) and showed antigenicity comparable to chemically inactivated FMDV. Screening of serum samples from FMD-vaccinated cattle showed that the test performance of VLP-LPBE had a correlation of 0.89 with conventional inactivated virus antigen LPBE. The VLP-LPBE developed here demonstrates the diagnostic application of recombinant FMDV VLPs in monitoring seroconversion following FMD vaccination.

Sequence analysis of capsid coding region of foot-and-mouth disease virus type A vaccine strain during serial passages in BHK-21 adherent and suspension cells.

Sequence variability within the capsid coding region of the foot-and-mouth disease virus type A vaccine strain during serial in vitro passage was investigated. Specifically, two methods of virus propagation were utilized, a monolayer and suspension culture of BHK-21 cells. At three positions (VP2(131) E-K in both monolayer and suspension passages, VP3(85) H-R in late monolayer passages and VP3(139) K-E in only suspension passages), all mapped to surface exposed loops, amino acid substitutions were apparently fixed without reversion till the end of the passage regime. Interestingly, VP2(131, 121) and VP3(85) which form part of the heparan sulphate binding pocket, showed a tendency to acquire positively charged amino acids in either monolayer or suspension environment probably to better interact with the negatively charged cell surface glycosaminoglycans. At three identified antigenically critical positions (VP2(79), VP3(139) and VP1(154)), amino acids substitutions even in the absence of immune pressure were noticed. Hence both random drift and adaptive mutations attributable to the strong selective pressure exerted by the proposed cell surface alternate receptors could play a role in modifying the capsid sequence of cell culture propagated FMDV vaccine virus, which in turn may alter the desired potency of the vaccine formulations.

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.

Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle

Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O, A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutralizing antibody response in indigenous cattle (Bos indicus). Purified Ad5-FMD viruses were inoculated in cattle as monovalent (5×109 pfu/animal) or trivalent (5×109 pfu/animal per serotype) vaccines. Animals vaccinated with monovalent Ad5-FMD vaccines were boosted 63days later with the same dose. After primary immunization, virus neutralization tests (VNT) showed seroconversion in 83, 67 and 33% of animals vaccinated with Ad5-FMD O, A and Asia 1, respectively. Booster immunization elicited seroconversion in all of the animals (100%) in the monovalent groups. When used in a trivalent form, the Ad5-FMD vaccine induced neutralizing antibodies in only 33, 50 and 16% of animals against serotypes O, A and Asia 1, respectively on primo-vaccination, and titers were significantly lower than when the same vectors were used in monovalent form. Neutralizing antibody titers differed by serotype for both Ad5-FMD monovalent and trivalent vaccines, with Asia 1 serotype inducing the lowest titers. Antibody response to Ad5 vector in immunized cattle was also assessed by VNT. It appeared that the vector immunity did not impact the recall responses to expressed FMDV antigens on booster immunization. In summary, the study suggested that the recombinant Ad5-FMD vaccine has a potential use in monovalent form, while its application in multivalent form is not currently encouraging.

Construction and characterization of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus capsid proteins of Indian vaccine strain, O/IND/R2/75.

Aim: Generation of recombinant human adenovirus type 5 expressing foot-and-mouth disease virus (FMDV) capsid protein genes along with full-length 2B, 3B and 3Cpro and its characterization. Materials and Methods: FMD viral RNA isolation, cDNA synthesis, and polymerase chain reaction were performed to synthesize expression cassettes (P1-2AB3BCwt and P1-2AB3BCm) followed by cloning in pShuttle-CMV vector. Chemically competent BJ5183-AD-1 cells were transformed with the recombinant pShuttle-CMV to produce recombinant adenoviral plasmids. HEK-293 cells were transfected with the recombinant adenoviral plasmids to generate recombinant adenoviruses (hAd5/P1-2AB3BCwt and hAd5/P1-2AB3BCm). Expression of the target proteins was analyzed by sandwich ELISA and indirect immunofluorescence assay. The recombinant adenoviruses were purified and concentrated by CsCl density gradient ultracentrifugation. Growth kinetics and thermostability of the recombinant adenoviruses were compared with that of non-recombinant replication-defective adenovirus (dAd5). Results: The recombinant adenoviruses containing capsid protein genes of the FMDV O/IND/R2/75 were generated and amplified in HEK-293 cells. The titer of the recombinant adenoviruses was approximately 108, 109.5 and 1011 TCID50/ml in supernatant media, cell lysate and CsCl purified preparation, respectively. Expression of the FMDV capsid protein was detectable in sandwich ELISA and confirmed by immunofluorescence assay. Growth kinetics of the recombinant adenoviruses did not reveal a significant difference when compared with that of dAd5. A decrement of up to 10-fold at 4°C and 21-fold at 37°C was recorded in the virus titers during 60 h incubation period and found to be statistically significant (p<0.01). Conclusion: Recombinant adenoviruses expressing capsid proteins of the FMDV O/IND/R2/75 were constructed and produced in high titers. In vitro expression of the target proteins in the adenovirus vector system was detected by sandwich ELISA and immunofluorescence assay.

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.

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.

Detection of replication competent adenovirus upon serial passaging of recombinant adenovirus expressing FMDV capsid proteins.

Replication deficient human adenovirus type 5 (hAd5) is an important gene delivery vehicle and has been used in various fields of biomedical sciences such as gene therapy, cancer therapy and vaccination. Inspite of its various useful features, emergence of replication competent adenovirus (RCA) in recombinant virus stocks is a great concern. In the present study, recombinant adenovirus expressing foot-and-mouth disease virus serotype-O capsid proteins was propagated in HEK-293 cells and purified by CsCl density gradient ultracentrifugation. The virus was serially passaged in HEK-293 cells and at passage level (P) 2-4, 6, 8 and 12, tested for the presence of RCA. A vector dose of 2 × 10(8) and 1 × 10(10) TCID50 of the virus was used in cell line bioassay and PCR, respectively. Our results demonstrated that the PCR is more sensitive and rapid technique for the detection of RCA in recombinant adenovirus stocks as early as at P3, whereas the bioassay detected the RCA at P8.