B-cell epitopes in GroEL of Francisella tularensis.

Zhaohua Lu,Jacqueline Sharon,Vani Kodela,Timothy Morris,Marly I Roche,Barbara A Seaton,Hillary M Perkins,Chiou-Ying Yang,Guillermo Madico,Seshi R Sompuram,Tong Liu,Sheng Li,Michael J Rynkiewicz,Daphne Wang

doi:10.1371/journal.pone.0099847

Abstract

The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other’s binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange–mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL.

Highlights

Francisella tularensis (Ft), the Gram negative facultative intracellular bacterium that causes tularemia, has been classified by the Centers for Disease Control and Prevention as a category A Tier 1 priority pathogen, a likely bioterrorism agent [1,2,3,4]
Binding Properties of the First Three FtGroEL monoclonal antibodies (mAbs) The first FtGroEL mAb we generated, Ab12 (IgG3,k), was derived from a mouse immunized with live live vaccine strain (LVS) and its target antigen was identified by proteome microarray analysis [32]
We generated hybridomas from mice immunized with various regimens of live LVS and GroEL-enriched killed Ft preparations adjuvanted with CpGcontaining oligodeoxynucleotide, which favors Th1 responses with production of IgG2a in mice and IgG1 in humans [80,81]

Summary

Introduction

Francisella tularensis (Ft), the Gram negative facultative intracellular bacterium that causes tularemia, has been classified by the Centers for Disease Control and Prevention as a category A Tier 1 priority pathogen, a likely bioterrorism agent [1,2,3,4]. As few as 10 bacteria can cause respiratory tularemia, the most severe form of the disease, with up to 30% mortality if untreated [1,2,3,4,5]. T cell immunity is essential for protection against Ft [8,9,10,11,12], B cells are required for anti-Ft memory [13], and polyclonal IgG antibodies to Ft can transfer resistance against the bacteria to naıve hosts, including humans [14,15,16,17,18,19,20,21,22,23]. We and others have described O-antigen mAbs that confer or prolong survival in mice infected subcutaneously, intranasally or intradermally with LVS or the highly virulent Ft type A strain SchuS4 [29,31,32,33]. Passive administration of mAbs specific for Ft outer membrane proteins FopA or LpnA was reported to prolong survival of mice infected intradermally with lethal doses of LVS [33] and passive transfer of FopA-immune sera was shown to protect mice against lethal intradermal LVS challenge [35]

Methods

Results

Conclusion