The binding sites of monoclonal antibodies to the non-reducing end of Francisella tularensis O-antigen accommodate mainly the terminal saccharide.

We have previously described two types of protective B-cell epitopes in the O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis: repeating internal epitopes targeted by the vast majority of anti-OAg monoclonal antibodies (mAbs), and a non-overlapping epitope at the non-reducing end targeted by the previously unique IgG2a mAb FB11. We have now generated and characterized three mAbs specific for the non-reducing end of F. tularensis OAg, partially encoded by the same variable region germline genes, indicating that they target the same epitope. Like FB11, the new mAbs, Ab63 (IgG3), N213 (IgG3) and N62 (IgG2b), had higher antigen-binding bivalent avidity than internally binding anti-OAg mAbs, and an oligosaccharide containing a single OAg repeat was sufficient for optimal inhibition of their antigen-binding. The X-ray crystal structure of N62 Fab showed that the antigen-binding site is lined mainly by aromatic amino acids that form a small cavity, which can accommodate no more than one and a third sugar residues, indicating that N62 binds mainly to the terminal Qui4NFm residue at the nonreducing end of OAg. In efficacy studies with mice infected intranasally with the highly virulent F. tularensis strain SchuS4, N62, N213 and Ab63 prolonged survival and reduced blood bacterial burden. These results yield insights into how antibodies to non-reducing ends of microbial polysaccharides can contribute to immune protection despite the smaller size of their target epitopes compared with antibodies to internal polysaccharide regions.

Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia.

Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg-binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg-binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb-treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B-cell epitopes of F. tularensis OAg.

The integrin-growth factor receptor duet.

Cell adhesion receptors, referred to as integrins, are recognized as key regulators of cellular processes including growth and differentiation. Integrins communicate with growth factor receptors (GFRs) to control specific cellular responses to stimuli originating in the extracellular environment. In this article, we review the role of integrins as molecular switches that modulate GFR activation and specificity. We also examine the reciprocal modulation of integrin functions by GFRs and the mechanisms through which those actions are fine-tuned.

Antibodies to both terminal and internal B-cell epitopes of Francisella tularensis O-polysaccharide produced by patients with tularemia.

Francisella tularensis, the Gram-negative bacterium that causes tularemia, is considered a potential bioterrorism threat due to its low infectivity dose and the high morbidity and mortality from respiratory disease. We previously characterized two mouse monoclonal antibodies (MAbs) specific for the O-polysaccharide (O antigen [OAg]) of F. tularensis lipopolysaccharide (LPS): Ab63, which targets a terminal epitope at the nonreducing end of OAg, and Ab52, which targets a repeating internal OAg epitope. These two MAbs were protective in a mouse model of respiratory tularemia. To determine whether these epitope types are also targeted by humans, we tested the ability of each of 18 blood serum samples from 11 tularemia patients to inhibit the binding of Ab63 or Ab52 to F. tularensis LPS in a competition enzyme-linked immunosorbent assay (ELISA). Although all serum samples had Ab63- and Ab52-inhibitory activities, the ratios of Ab63 to Ab52 inhibitory potencies varied 75-fold. However, the variation was only 2.3-fold for sequential serum samples from the same patient, indicating different distributions of terminal- versus internal-binding antibodies in different individuals. Western blot analysis using class-specific anti-human Ig secondary antibodies showed that both terminal- and internal-binding OAg antibodies were of the IgG, IgM, and IgA isotypes. These results support the use of a mouse model to discover protective B-cell epitopes for tularemia vaccines or prophylactic/therapeutic antibodies, and they present a general strategy for interrogating the antibody responses of patients and vaccinees to microbial carbohydrate epitopes that have been characterized in experimental animals.

Functional and structural characterization of Francisella tularensis O-antigen antibodies at the low end of antigen reactivity.

The O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis (Ft), which is both a capsular polysaccharide and a component of lipopolysaccharide, is comprised of tetrasaccharide repeats and induces antibodies mainly against repeating internal epitopes. We previously reported on several BALB/c mouse monoclonal antibodies (MAbs) that bind to internal Ft OAg epitopes and are protective in mouse models of respiratory tularemia. We now characterize three new internal Ft OAg IgG2a MAbs, N203, N77, and N24, with 10- to 100-fold lower binding potency than previously characterized internal-OAg IgG2a MAbs, despite sharing one or more variable region germline genes with some of them. In a mouse model of respiratory tularemia with the highly virulent Ft type A strain SchuS4, the three new MAbs reduced blood bacterial burden with potencies that mirror their antigen-binding strength; the best binder of the new MAbs, N203, prolonged survival in a dose-dependent manner, but was at least 10-fold less potent than the best previously characterized IgG2a MAb, Ab52. X-ray crystallographic studies of N203 Fab showed a flexible binding site in the form of a partitioned groove, which cannot provide as many contacts to OAg as does the Ab52 binding site. These results reveal structural features of antibodies at the low end of reactivity with multi-repeat microbial carbohydrates and demonstrate that such antibodies still have substantial protective effects against infection.

B-cell epitopes in GroEL of Francisella tularensis.

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.