Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.


Identification of Immunogenic HLA-B27:05 Restricted Peptides of Salmonella Outer Membrane Protein (OMP) in Patients with Reactive Arthritis and Undifferentiated Spondyloarthropathy.

Misra1,  Ramnath, Singh1,  Amit, Aggarwal2,  Amita

Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India
Additional Professor Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Lucknow, India

Background/Purpose:

We have earlier reported that Salmonella OMPs are major immunogenic targets of synovial fluid lymphocytes of patients with Salmonella induced ReA/uSpA1. Since these group of patients have genetic predisposition to HLA-B*27 and its subtype HLA-B*27:05, we sought to identify HLA-B*27:05 binding Salmonella OMP peptides which are immunogenic in patients with ReA/uSpA.

Methods:

Crude lysates of E coli, Salmonella typhimurium, Shigella flexneri, Y enterocolitica and OMP of S typthimurium were prepared. 125 HLA-B*27:05 binding Salmonella OMP peptides identified using Propred1 software were synthesized and grouped in 23 pools. These panel of antigens (5 and 10 mg/ml) and peptide pools (10 mg/ml) were cultured with 1×105 synovial fluid or peripheral blood mononuclear cells (SF/PBMC) from 23 patients with ReA/uSpA, 10 patients with RA and PBMC of 10 healthy individuals (controls) for 5 days in 96 well culture plate. Lymphocyte proliferation was assayed by tritiated thymidine uptake and IFN-g levels in culture supernatant. A stimulation index (SI) of >2.5 was considered significant. Individual peptides from pools having significant response were retested against cryopreserved SFMC/PBMC to identify immunogenic peptides. HLA-B*27 and its subtypes were done by PCR using allele specific primers. A BLASTp program was used to search for similar peptides from protein bank of arthritogenic bacteria and human.

Results:

SFMC of 19/23 patients with ReA/uSpA showed a significant proliferative response to Salmonella OMP but there was minimal response in PBMC (1/10) of ReA/uSpA, SFMC of RA (1/10) or PBMC from control (1/10). Nine Salmonella OMP peptides QRAEMLPTL, SRSGLNIAL, LRFLYAKSL, RLEGTWVKL, ARCIAPYAL, KLFLTTAAL, YRNSDFFGL, QRPAVRVKL and YRVGPGDVL showed significant response (Table 1). Response to QRAEMLPTL was mainly restricted to HLA-B*27:05 positive (6/7) patients. All immunogenic peptides had sequence similarity with peptides derived from arthritogenic bacterial proteins while 5 immunogenic peptides had similarity with peptides from evolutionary conserved human proteins.

Table 1. Sequence of individual immunogenic peptides, its protein and number of patients SFMC responded

Peptide No.Pool No.SequenceProtein DetailsNo. of Patients responded
10013, 9QRAEMLPTLouter membrane efflux-like protein, OprM (53 kDa) (485 aa) NP-4593457
475, 19SRSGLNIALputative outer membrane protein (205 kDa) (1869 aa) NP-4614503
435, 15LRFLYAKSLouter membrane usher protein, FimD, (95 kDa) (870 aa) NP-4595413
3313, 2ARCIAPYALouter membrane usher protein, FimD, (95 kDa) (870 aa) NP-4595413
5513, 6LRLEGTWVKputative outer membrane protein (51 kDa) (468 aa) NP-4596722
1089, 20QRPAVRVKLmultidrug efflux system subunit MdtB (1040 aa) NP-4610722
1109, 22KLFLTTAALputative outer membrane protein (11 kDa) (94 aa) NP_4591602
12119, 10YRNSDFFGLouter membrane protein F (40 kDa) (368 aa) NP_4599742
12522, 2YRVGPGDVLputative outer membrane polysaccharide export protein (41 kDa) (379 aa) NP4610632

Conclusion:

A set of 9 novel HLA-B*27:05 binding peptides of Salmonella OMP were immunogenic to SFMC of patients with ReA/uSpA. Immune response to these peptide targets could be useful for diagnosis in undifferentiated spondyloarthropathy.

References:

1.Singh, R, Shasany, AK & Aggarwal, A et al. Low molecular weight proteins of outer membrane of Salmonella typhimurium are immunogenic in Salmonella induced reactive arthritis revealed by proteomics. Clin Exp Immunol. 2007;148:486–93.

To cite this abstract, please use the following information:
Misra, Ramnath, Singh, Amit, Aggarwal, Amita; Identification of Immunogenic HLA-B27:05 Restricted Peptides of Salmonella Outer Membrane Protein (OMP) in Patients with Reactive Arthritis and Undifferentiated Spondyloarthropathy. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :1343
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