• KOTLA SIVAREDDY

Title for the Ph.D Thesis

“Study on Immune responses of Cytokine Adjuvented DNA vaccine (genes coding for structural proteins) for FMD delivered by cationic PLG micro particles”.

Objectives:

1. To clone gene coding for structural protein of FMDV in eukaryotic expression vector (pC DNA) under CMV promoter.
2. To clone bovine interleukin 18 gene in suitable cloning vector and express the same in Bacterial/yeast expression system for characterization of expressed recombinant protein.
3. To construct a vaccine construct consisting of FMDV P1 (structural), 3C (Viral protease), and Bovine interleukin 18 genes in eukaryotic expression system under CMV promoter (pC DNA).
4. To study the expression of the vaccine constructs in vitro in BHK-21cells.
5. PLG micro particles’ preparation and characterization.
6. To study the immune responses of the DNA vaccine in experimental animals (guinea pigs / cattle).

Foot-and-mouth disease (FMD) caused by foot-and mouth disease virus (FMDV) is an infectious disease affecting cloven-hoofed animals, and poses a serious threat for animal health and exacts an economic toll on the livestock industry. FMD viral genome is a positive-sense single stranded RNA of approximately 8.5kb. The viral RNA genome is translated as a single polypeptide precursor that is subsequently processed by virus-encoded proteases 2A and 3C to produce the structural and non-structural proteins required for virus assembly and replication. One of the initial polypeptide cleavages, mediated by the 2A protein, is a co-translational cleavage at its own C terminus to release it from the 2B protein. The viral 3C proteinase subsequently processes the structural protein precursor, P1-2A, into the capsid proteins, VP0, VP3, and VP1, and the non structural peptide, 2A. These proteins then self assemble to form empty icosahedral capsid particles that contain 60 copies of each protein. Immunological studies have identified linear and conformational sites that are present on both empty capsids and virions, and antiserum raised against either form has the same serological specificity. Thus, the structural protein precursor, P1-2A, and the 3C protease of FMDV are desirable immune antigens for new vaccine development. In countries where disease eradication has not been achieved, vaccination plays a crucial role in its control. Although inactivated virus vaccines effectively prevent FMD, they have several limitations like short duration of immunity, incomplete viral inactivation and virus escape from vaccine producing facilities. As a result, alternative approaches are being investigated, including the construction of modified live virus, subunit vaccines, synthetic peptides, naked DNA plasmids. DNA vaccination which offers several promising features i.e., DNA is convenient to manufacture and store, its production is safe, sequences from circulating strains can be easily incorporated in the vaccines, and it also allows the discrimination of the infected from the vaccinated animals. Several reports have shown the efficiency of DNA vaccination to induce protective immunity in the mouse model. However, the primary difficulty with DNA vaccination is its poor immunogenicity in target species. Cytokines are being used as molecular adjuvants by co administering with DNA vaccines to improve the efficacy of the vaccine. Cytokines play an important role both in the development of a functional immune system as well as in the responses of the organism to infection. Interleukin18 (IL18) is a potent interferon γ (IFNγ) inducing factor (IGIF), enhances Th1 immune responses. Recent studies have shown that IL18 also promote Th-2 type responses and increases dendritic cell (DC) number in lymph nodes in mice. In addition, IL18 has been used as an adjuvant to DNA vaccines for classical swine fever virus, pseudo rabies virus, porcine reproductive and respiratory syndrome virus.IL18 was co expressed along with FMDV VP1 in Pichia as fusion protein has enhanced humoral responses and marginally the CMI response in mice. Recombinant fowl pox co-expressing FMDV P1 2A3C and IL18 enhanced the immune responses and gave higher protection in swine Many other studies have shown the positive effect of plasmid encoding the IL-18 as a molecular adjuvant on DNA vaccinations. Efficacy of DNA vaccine could be improved by the inclusion of adjuvants and good vaccine delivery systems. Importantly, cationic microparticle with adsorbed DNA induced enhanced immune responses in comparison to naked DNA and this enhancement was apparent in all species evaluated, including nonhuman primates. Cationic PLG microparticles appear to be effective predominantly as a consequence of the efficient delivery of the adsorbed DNA into DC. Following administration, the micro particles are also very effective at recruiting DC to the injection site, and the micro particles also protect adsorbed DNA against degradation in vivo. A second useful property of micro particles is that they can present multiple copies of antigens on their surface, which has been shown to be optimal for B cell activation. The main advantage of this type of association is the efficient immobilisation of plasmid DNA on the microparticle surface without compromising its integrity. Moreover, after administration, the release of cationic DNA complexes from the surface appeared to facilitate the transfection of cells .At this point, it is not completely understood, whether the adsorption on cationic micro particles can protect plasmid DNA from cleavage through endonucleases after administration in vivo. Nevertheless, release of cationic DNA complexes is expected to provide better protection as compared to release of free DNA. Beside their inherent safety and ease of administration, they improve the DNA capture by antigen presenting cells (APC) and stimulate APC maturation. DNA delivery via PLG has been successfully used to vaccinate against several infections in mice, guinea pigs and even in macaques models.

1. AMPLIFICATION and cloning of gene sequence coding for P12A &3C

FMDV serotype ‘Asia1’ gene coding for the polyprotein, P1-2A (2.3 kb) was amplified from viral genome, of the serotype ‘Asia1’ polyprotein gene, using VP4L (Bac) and 2AR (E.coR1) primers. Cloned in to pC DNA at E.co R I, BamHI sites. Ligated and transformed in to DH alpha 5 cells. Transformants are screened by colony PCR by using insert specific primers. Orientation was checked by PCR. Insert release was confirmed by RE digestion by using E.co R I, Bam HI. 3C coding sequences were amplified from clone available in lab. 0.6 Kb was amplified .The purified amplicon was digested and ligated in to p C DNA and transferred in to competent DH5 cells upon screening by PCR and by re digestion positive clones were conformed.

2. Cytokine amplification (IL18) and cloning in prokaryotic / yeast & pC DNA expression vector and characterization of expressed protein.

Interleukin 18 (IL18) modulates immune functions by inducing interferon–γ(IFN-γ) production and promoting Th1 immune responses. In the present study I amplified and cloned the sequence (582 bp) encoding full length bovine IL18 from peripheral blood mononuclear cells (PBMC) stimulated with Phytohaemoglutinin (PHA). Nucleotide and the deduced amino acid sequence of the cloned IL18 showed an identity of 86-98% with IL 18 sequences of the other ruminants compared. The insert was sub cloned in to eukaryotic expression vector (PcDNA) .The specificity of the expressed IL 18 was confirmed by western blotting. The insert was sub cloned in to pET 32a vector and expressed in E.Coli as fusion protein of 42kDa. The specificity of the expressed IL 18 was confirmed by western blotting. The biological activity of the purified protein was analysed for its ability to induce IFN-γ production in PBMC as measured by Enzyme linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qPCR). IL18 anti FMD viral activity was conformed in vitro in BHK-21 cells by using plaque assay; viral replication was quantified by Real time PCR, ELISA and titration assays.

3. Study of the expression of the constructs in vitro in BHK-21 Cells

Expression of cloned P12A3C and IL18 genes were studied in mammalian expression system for confirming the frame and intactness. The P12A3C, IL18 genes cloned under Eukaryotic promoter was transfected in BHK 21 cells with lipid based lipofectamine. Subsequently, the proteins were confirmed by Western blotting by using using anti FMDV serotype ‘Asia’, serum from experimentally infected cattle. IL18 transfected cell lysate showed 18 KDa by using human IL18 Mab.

4. PLG microparticles preparation and characterization

The PLG/CTAB micro particles were prepared using a solvent evaporation technique essentially as described previously and briefly, the micro particles were prepared by emulsifying 10ml of a 6% (w/v) polymer solution in methylene chloride with 1ml of TE buffer at high speed using an soniprep. The primary emulsion was then added to 50ml of distilled water containing CTAB (0.5%, w/v). This resulted in the formation of a water/oil/water emulsion which was stirred at 6000rpm for 12h at room temperature, allowing the methylene chloride to evaporate. The resultingmicro particles were washed in distilled water by centrifugation at 10,000 × g and freeze dried. The plasmid construct was adsorbed onto the microparticles by incubating 100 mg of cationic microparticles with 100 mgs (1 mg/ml solution) of plasmid DNA at 4⁰_C for 6 h. The coated microparticles were then separated washed with TE and freeze-dried. Amount of plasmid adsorbed on PLG particles was quantified by eluting the DNA by 0.2 N NaOH (incubation for 10 h at 4 ⁰C and measuring the Optical Density (OD) at 260 nm. Blank PLG micro particles controls were run simultaneously to deduct background value. The size distribution of the micro particles was determined using a particle size analyzer and electron microscopy.

5.A. Evaluation of the Immunological response of various DNA vaccine constructs in guinea pigs.

Foot and Mouth Disease (FMD) can be controlled by regular vaccination and restricting the movement of animals infected in the endemic countries.. DNA vaccine construct was made with P1-2A3C coding sequences of serotype Asia1 in p C DNA. To evaluate the optimal dose of the construct in guinea pigs, the plasmid was coated on cationic Poly Lacto-co-Glycolide (PLG) micro particles was injected in to guinea pigs at 2,5,10,15,20,30 ug doses intramuscularly. Sera samples collected from the vaccinated animals at 21st dpv were evaluated for immune response by Enzyme linked immunosorbent assay (ELISA), Serum neutralization test (SNT) and MTT assay. Maximum ELISA / SNT titers and MTT stimulation indices were observed at 10 µg dose which also gave 83% protection when the guinea pigs were challenged with homologues virus. 10ug was found to be the optimal dose to guinea pigs.

P12A3CpCDNA and bovine IL-18 pcDNA plasmids were constructed under CMV promoter and the coated with Cationic PLG microparticle, immune response of the co administered constructs was evaluated in guinea pigs. Both the plasmids constructed under CMV promoter and 10µgs each of the plasmids were inoculated intra muscularly in guinea pigs with a booster dose at 21^st day post vaccination (dpv). Both humoral and cellular immune response were analysed by IgG1, IgG2 enzyme linked immunosorbent assay (ELISA), Serum neutralization test (SNT) and MTT assay. Th1, Th2 cytokine profile was analysed by real time PCR and the phenotyping of T cell sub population in the peripheral blood was performed by flowcytometry. The results have sown significantly higher humoral and cell mediated immune responses in P12A3CIL18+PLG group than P12A3C IL18, and inactivated virus vaccine inoculated groups. Similarly, higher CD4, CD8 population and Th1, Th2 cytokine levels were seen in former group. P12A3CIL18+PLG vaccine protected all the six animals when challenged with homologous virus compared to five in inactivated virus vaccine group respectively. These results have shown that the plasmid encoding for P12A3C pcDNA when co inoculated with IL18 and PLG induce higher and protective immune responses, suggesting rBoIL-18 and Micro particles has a potential to enhance the efficacy of vaccine against FMD.

5. B Evaluation of the Immunological response of various DNA vaccine constructs in Cattle.

Healthy male cattle calves of local breed ( Hallikar Breed) of 6 months to one year age group were purchased from local village shandy( cattle market). These animals were housed in healthy animal shed facilities available at IVRI Animal experimental station at Yelahanka , Bangalore. After initial quarantine the animals were bled and the sera were screened for FMDV antibodies for serotype Asia 1 by SNT.

The FMD antibody free animals were divided in to 6 groups of six animals each namely Group I to Group IV. All the group were vaccinated with each construct with 200 ug injected by intramuscularly except conventional vaccine group injected with 2 ml of FMDV Inactivated vaccine. One group kepted for control group (vaccinated with PBS) .After 21 st days of first vaccination with same amount booster dose was injected

P12A3CpCDNA and bovine IL-18 pcDNA plasmids were constructed under CMV promoter and the coated with Cationic PLG microparticle, immune response of the co administered constructs was evaluated in guinea pigs. Both the plasmids constructed under CMV promoter and 200µgs each of the plasmids were inoculated intra muscularly in calves with a booster dose at 21^st day post vaccination (dpv). Both humoral and cellular immune response were analysed by IgG1, IgG2 enzyme linked immunosorbent assay (ELISA), Serum neutralization test (SNT) and MTT assay. Th1, Th2 cytokine profile was analysed by real time PCR (γIFN, IL4, IL2, αIFN, IL12, IL25,TLR-4,TLR3,TLR-2,IL8,IL10) and the phenotyping of T cell sub population (CD4 and CD8) and intracellular cytokine molecules (γIFN, IL4, IL2) in the peripheral blood was performed by flowcytometry. The results have sown significantly higher humoral and cell mediated immune responses in P12A3CIL18+PLG group than P12A3C IL18, and inactivated virus vaccine inoculated groups. Similarly, higher CD4, CD8 population and Th1, Th2 cytokine levels were seen in former group. P12A3CIL18+PLG vaccine protected four out of six animals when challenged with homologous virus compared to 3 in inactivated virus vaccine group respectively. Non structural proteins,ELISA conformed in challenged animals.These results have shown that the plasmid encoding for P12A3C pcDNA when co inoculated with IL18 and PLG induce higher and protective immune responses, suggesting rBoIL-18 and Micro particles has a potential to enhance the efficacy of vaccine against FMD

Journal papers and conference/seminar papers from Doctoral research work

1. Expression of Bovine (Bos indicus) interleukin-18 inEscherichia coli and its biological activity.Kotla Siva Reddy, Dowlathabad. Muralidhar Rao, Hosur Joyappa Dechamma,Veluvarthy V.S. Suryanarayana and Golla Ramalinga Reddy.Published in Microbiology and Immunology 2010; 54: 564–567.

2. Enhancement of DNA vaccine (P12A3C-pcDNA) efficacy against Foot- andMouth Disease by co-administration of Interleukin-18 expressing (IL18pcDNA) plasmid in Guinea Pigs. Siva Reddy .K. Muralidhar Rao.D., Badrinaryana.M. Suryanaryana.VVS. and Reddy G.R. Accepted in FEMS Immunology and Medical Microbiology. Dec -2010 1–9.

3. Dose optimization of Cationic PLG micro particle coated DNA vaccine against Foot and Mouth Disease in Guinea pigs. Siva Reddy, K.,Rashmi., B.R., Muralidhar Rao, D., Dechamma H.J., Banumathi .N., Suryanarayana V.V.S and Reddy .G.R. accepted in J.of Life science.(Article in press)

4. Cytokine profile studied by Real time PCR in FMDV antigen stimulated Bovine PBMC cells. Siva Reddy .K., Muralidhar Rao,D.,PrabhuDas,K., Suryanaryana.VVS., Reddy ,G.R. Accepted in Journal of Biotechnology , Bio engineering and Bio –Informatics. (Article in press).

5. Bos indicus Interleukin 18 complete coding sequence published in NCBI Gen bank .SivaReddy,K., Muralidhar Rao,D., Dechamma,H., Banumathi,N.,Suryanaryana,V. and Reddy,G. Acc.No. FJ985771

6. Enhancement of DNA vaccine (P12A3C-pcDNA) efficacy against Foot- andMouth Disease by co-administration of Interleukin-18 expressing (IL18pcDNA) plasmid in Guinea Pigs. Siva Reddy .K. Muralidhar Rao.D., Badrinaryana.M. Suryanaryana.VVS. and Reddy G.R. Presented in Society for applied biotech biotechnology (SAB) annual conference at Dharmapuri Dec 17,18

7.Bovine Interleukin -18 inhibits Foot-and-Mouth Disease virus Replication in BHK- 21 cells. K. Siva Reddy, D.Murali Dhar Rao, Kakoli Ahmed, H.J Dechamma N.Bhanumathi ,VVS Suryanarayana ,G.R Reddy presented at VIROCON 2010 XIX National Conference” RECENT TRENDS IN VIRAL DISEASE PROBLEMS AND MANAGEMENT” SVU Tirupathi, Mar 18-20 ,2010 .

8. Cationic Micro Particle (PLG) coated DNA vaccination Induces a long term immune response and Protective Immunity against Foot –and-Mouth disease virus. K. Siva Reddy, Rashmi Dechamma N.Bhanumathi ,VVS Suryanarayana ,G.R Reddy Presented at VIROCON 2010 XIX National Conference” RECENT TRENDS IN VIRAL DISEASE PROBLEMS AND MANAGEMENT” SVU Tirupathi, Mar 18-20 ,2010.

9. Dose response studies of ID- p VAC (SECRETORY VECTOR CONSTRUCT) coated on cationic PLG micro particles against FMDV in guinea pigs. Siva Reddy K., Reddy G.R. Presented at SBC Annual conference Impact of Basic and Translational Research on Medicine, Agriculture and Industry, IIT Madras 18-20 DEC -2008.

Communicated Articles

1. Cationic Micro Particle (PLG) coated DNA vaccination induces a long term immune response and Protective Immunity against FMD in GuineaPigs. Siva Reddy ,K ., MuraliDhar Rao,D.,Rashmi, B.R., Dechamma H,J., Banumathi.,N., Suryanarayana V.V.S and Reddy G.R Communicated in to Vet Immunology and Immunopathology(Under review).

2. Bovine Interleukin 18 inhibits Foot and mouth disease virus replication in BHK-21 Cells. K. Siva Reddy, D.MuraliDhar Rao, K.PrabhuDas, VVS Suryanarayana,G.R Reddy communicated in to Biotechnology and Applied Biochemistry.

3. Enhanced efficacy of a Foot and mouth disease DNA vaccine (P12A3CpcDNA) by adsorption onto cationic PLG microparticle in guinea pigs .K. Siva Reddy, D.MuraliDhar Rao, K.PrabhuDas, VVS Suryanarayana ,G.R Reddy communicated into International journal of Immunopharmacology.