Identification of immunodominant T-cell eptitopes in matrix protein of highly pathogenic porcine reproductive and respiratory syndrome virus Ya-Xin Wang, PhD Student Outline 1. Background 2. Research Contents 3. Conclusion 4. Acknowledgement Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences Introduction of HP-PRRSV 1. Background In 2006, Highly pathogenic porcine reproductive and respiratory syndrome virus(hp-prrsv) severely affected the pork industry of China. Infections were characterized by high morbidity (characterized by high fever, 50-100%) and mortality (20-100%). Since the initial outbreak, disease spread rapidly to most provinces in China, resulting in more than one million deaths. So we chose the HP-PRRSV HuN4 strain to do the research. 1985 1987 1990 1991 1992 1995 2000 2005 2006 2010 First observed in The United States The first case reported in Europe First isolated in Lelystad Isolated in Lelystad The United States Officially named PRRSV First outbreak on mainland China Highly pathogenic PRRS The course of viral infections in pigs PRRS Immunology Persistent Infection Delayed Protection! Persistent Infection! 1
Pig immune to PRRSV is not very effective Innate Immunity Cytokines Interferons: IFN-α, IFN- β Ribonuclease Other Cytokines: IL-1/TNF NSP2 antagonizes the type I IFN TC Cell CMI IL-2 IL-12, IFN-g Intracellular CTL Th1 Cell Kill Infected Cells IL-2 Delayed IFN-γ secreting cells PRRSV Adaptive Immunity TH Cell Humoral Immunity Th2 Cell Extracellular Absorb Free Virus B Cell Neutralizing Antibody Delayed Neutralizing Antibody B cell epitopes of ATCC VR-2332 strain 112-123aa 839-849aa 1032-1067aa 691-722aa 236-252aa 30-52aa 180-197aa 36-51aa21-47aa 5'cap 1a 2a 4 6 1b 2b 3 5 7 (A)n 3' 456-469aa 117-139aa 752-807aa 1750-1755aa 913-940aa 90-148aa 27-35aa,37-45aa B cell epitopes of CH-1a strain 119-178aa 146-156aa 164-180aa,182-200aa 69-122aa 37-52aa 51-56aa 117-129aa 52-58aa 54-58aa T cell epitopes 175-183aa Ivan Díaza et al. Lelystad virus strain 7-15aa 113-121aa 64-72aa 117-128aa 5'cap 1a 2a 4 6 1b 7 (A)n 3' 2b 3 5 2. Research Contents Vashisht K, et al. NADC-9, NVS-14 virus strain 149-163aa 187-200aa Experimental design PRRS-negative piglets First vaccinated, then challenged with HP-PRRSV PBMCs ELISPOT Further proven experiment Conservation of identified M epitopes Matrix protein of PRRSV Fifteen amino acid residues in length, overlapped by 11 residues Overlapping Peptides The ELISPOT screening Peptides spanning matrix protein were screened for their ability to elicit a recall interferon-γ (IFN-γ). All of the peptides Three rounds, step by step, reduce the scope Individual peptide epitopes 2
First distribution of Peptide pools Second distribution of Peptide pools I2 II2 III2 Pool VI1 Group 6 M26-M29 Group 1 M1-M5 Pool I1 Group 2 M6-M10 IV2 V2 VI2 VII2 M1 M4 M7 M10 M2 M5 M8 M11 M3 M6 M9 M12 Pool V1 28 peptides Pool II1 VIII2 M13 M14 M15 Group 5 M21-M25 Group 3 M11-M15 Group 4 M16-M20 Pool IV1 Pool III1 Negative Control Positive Control peptide stimulated peptide stimulate Data analysis The first round of screening Selection criteria The number of IFN-γ-secreting cells per one million PBMCs Three selection criteria The maximum the average The average per pig Statistical analysis Difference comparison compared (P < 0.05 ) Using SPSS 13.0 statistic software I 1 II 1 III 1 IV 1 V 1 VI 1 means Maximum 29 34 23 37 16 25 27.33 Average 11.33±1.91 16.07±2.60 12.8±1.82 8.27±2.37 8.47±0.95 15.53±1.87 12.08±0.85 NO.1 6.67±2.33 8.33±2.19 8±1.73 6.33±1.85 6.33±0.88 18.33±2.03 9±1.21 NO.2 15±4.62 15.33±0.33 18.67±0.88 23±7.02 11.33±2.60 17±2.08 16.72±1.54 NO.3 6.67±3.05 4.67±0.67 4±1.53 3±1 4±1.15 3.67±1.67 4.33±0.54 NO.4 19±5.13 24.67±4.41 18±3.61 5±1.73 9.67±1.45 23.33±1.20 16.61±2.06 NO.5 9.33±3.48 27.33±4.06 15.33±3.53 4±0.58 11±0.58 15.33±2.03 13.72±1.99 All the data are measured Result I:Four peptide pools I1, II1, III1,VI1 that generated values greater than or equal to the mean for the three selection criteria. Pool VI1 can elicit significantly higher IFN-γ secreting cells than pool IV1 and pools V1 (p<0.05). M1-M15 were selected to do the second round of ELISPOT assay. The second round of screening The last round of screening Selection criteria the number of IFN-γ-secreting cells per one million PBMCs I 2 II 2 III 2 IV 2 V 2 VI 2 VII 2 VIII 2 means Selection criteria the number of IFN-γ-secreting cells per one million PBMCs M2 M3 e M5 M6 e M8 e M8-v e M8-c e M9 M11 M12 e Mean Maximum 20 43 58 29 35 39 49 16 36.1 Average 9.27±1.6 21.6±3.5 34.67±3.7 17.4±1.7 20.8±2.8 20.8±2.5 29.33±3.3 9.6±1.4 20.43±1.2 NO.1 5±1.1 8.67±0.3 33.33±2.7 12±1.5 15±2.9 13±0.6 29.33±2.3 3.67±1.5 15±2.2 Maximum Average Response 31 170 48 198 261 147 128 25 87 171 126.6 12.1±2.7 77.9±19.6 14.1±5.2 92.7±21.1 88.6±29.8 68.1±14.2 66.9±12.7 11.3±2.5 44.1±8.0 75.7±16.4 55.15±5.6 NO.2 18.33±0.8 41.33±0.9 55.33±1.5 15.67±3.8 29±3.46 29.33±2.7 47±1.2 13.67±0.3 31.21±3.1 NO.3 0.33±0.3 12.67±0.7 17.33±1.2 12.33±1.5 4.67±0.9 11.33±0.9 13.33±0.9 3±1.2 9.38±1.2 NO.4 11±1.1 25.33±6.4 43.33±2.3 25.67±1.7 29±2.5 30.67±5.6 37.33±3.2 13.33±0.3 26.96±2.4 NO.5 11.67±0.9 20±8.1 24±1.7 21.33±1.5 26.33±2.7 19.67±4.3 19.67±1.2 14.33±0.9 19.63±1.4 Result II:Five peptide pools III2, IV2, V2, VI2, VII2 are comparatively stronger IFN-γ s than other peptide pools. Statistical analysis showed that the IFN-γ secreting cells stimulated by peptide pools III2, IV2, V2, VI2, VII2 are significantly higher than peptide pools I2 and VIII2. M2, M3, M5, M6, M8, M9, M11, M12 were selected to do the second round of ELISPOT assay. NO.1 10.5±3.5 24±6 6±3 38±3 31.5±6.5 29.5±9.5 34±6 8.5±0.5 29.5±9.5 44±4 25.55±3.14 NO.2 12.5±2.5 44.5±7.5 5±1 46±3 39±1 46±7 43.5±1.5 5.5±1.5 26±3 36±7 30.4±3.81 NO.3 7±0 164±6 44.5±3.5 194±4 259±2 143±4 109±19 14.5±3.5 86±1 152±19 117.3±17.7 6 NO.4 26±5 131.5±8.5 11.5±2.5 138.5±16.5 93±22 84.5±8.5 114±12 24±1 54.5±1.5 112.5±9.5 79±10.52 NO.5 4.5±1.5 25.5±2.5 3.5±1.5 47±1 20.5±1.5 37.5±0.5 34±1 4±1 24.5±2.5 34±6 23.5±3.39 Result III: Individual peptide M3, M6, M8, M12 can elicit high expression of IFN-g. Statistical analysis showed that the number of IFN-γ secreting cells stimulated by these four individual peptide was significantly higher than other peptides. 3
SFC/Million PBMCs Relative Ratio of IFN-γmRNA/CyPA mrna 02/08/2010 140 120 100 Further proven experiment TaqMan fluorescent quantitative Real-time PCR for detecting IFN-γ. 80 60 40 20 0 M2 M3 M5 M6 M8-H M8-V M8-C M9 M11 M12 The average of the eight candidate peptides M8-H----------High-pathgenic strain HuN4 M8-V----------Vaccine strain HuN4-F112 M8-C----------Representative strain Ch-1a 14 12 10 8 6 4 2 0 M3-1 M3-2 M3-3 M3-4 M3-5 M6-1 M6-2 M6-3 M6-4 M6-5 M8-1 M8-2 M8-3 M8-4 M8-5 M12-1 mock-stimulated cell cells stimulated by peptides Relative levels of IFN-γ mrna after the cells were stimulated by the four epitopes through Real-tine PCR. M12-2 M12-3 M12-4 M12-5 Conservation of identified M epitopes Multiple sequence alignment of the epitopes location between 42 American-type PRRSV strains 3. Conclusion Four T-cell epitopes presented in the matrix protein of HP-PRRSV were identified. 4. Acknowledgement The real-time PCR also showed that the cells stimulated by these four peptides can produce high quality of IFN-g, which proof our conclusion from another aspect. The result of the alignment demonstrated a great degree of identity at each site, MTEP6 was fully conserved ; MTEP3 and MTEP12 were conserved with only two amino acid changes; MTEP8 with 3 amino acid changes. 4
Acknowledgement Shanghai Veterinary Research Institute China Animal Health and Epidemiology Center (Shanghai) Department of Swine Infectious Diseases D r. Guang-Zhi Tong D r. Shi-Shan Yuan D r. Yan-Jun Zhou D r. Guo-Xin Li D r. Hai Yu D r. Shan-rui Zhang Yi-Feng Jiang, Ao-Tian Xu Li-Ping Yan, Meng-Meng Wang Y an-zhao Xu, Huan-Huan Liu Ji-Hong Ma, Jun-Wei Hou Wu Tong 5