1343 - Identification of Immunogenic HLA-B*27:05 Restricted Peptides of Salmonella Outer Membrane Protein (OMP) in Patients with Reactive Arthritis and Undifferentiated Spondyloarthropathy

Monday, November 7, 2011: 9:00 AM-6:00 PM
Hall F2 - Poster Hall (McCormick Place West)
Ramnath Misra1, Amit Singh1 and Amita Aggarwal2, 1Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India, 2Additional Professor Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Lucknow, India
Presentation Number: 1343

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 μg/ml) and peptide pools (10 µg/ml) were cultured with 1x105 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-γ 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.

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.

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

Peptide No.

Pool No.

Sequence

Protein Details

No.of Patients responded

100

13, 9

QRAEMLPTL

outer membrane efflux-like protein, OprM (53 kDa) (485 aa) NP-459345

7

47

5, 19

SRSGLNIAL

putative outer membrane protein(205 kDa) (1869 aa) NP-461450

3

43

5, 15

LRFLYAKSL

outer membrane usher protein, FimD , (95 kDa) (870 aa) NP-459541

3

33

13, 2

ARCIAPYAL

outer membrane usher protein, FimD, (95kDa) (870 aa) NP-459541

3

55

13, 6

LRLEGTWVK

putative outer membrane protein(51 kDa) (468 aa) NP-459672

2

108

9, 20

QRPAVRVKL

multidrug efflux system subunit MdtB(1040 aa) NP-461072

2

110

9, 22

KLFLTTAAL

putative outer membrane protein  (11 kDa) (94 aa) NP_459160

2

121

19, 10

YRNSDFFGL

outer membrane protein F (40 kDa) (368 aa) NP_459974

2

125

22, 2

YRVGPGDVL

putative outer membrane polysaccharide export protein (41 kDa) (379aa) NP461063

2

 


Keywords: reactive arthritis and spondylarthropathy

Disclosure: R. Misra, None; A. Singh, None; A. Aggarwal, None.