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Keywords:

  • bacteria/bacterial immunity;
  • endotoxin/lipopolysaccharide;
  • epitopes;
  • immunoglobulins;
  • vaccines

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

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.


Abbreviations:
C

core

CFU

colony-forming units

Ft

Francisella tularensis

i.n.

intranasal(ly)

i.p.

intraperitoneal(ly)

LD50 or 100

lethal dose 50 or 100 (dose lethal to 50% or 100% of mice)

LPS

lipopolysaccharide

LVS

live vaccine strain

mAb

monoclonal antibody

OAg

O-antigen (O-polysaccharide)

OAgC

OAg-core

OD

optical density

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Francisella tularensis, the Gram-negative facultative intracellular bacterium that causes tularaemia, is a category A potential bioterrorism agent, specifically the highly virulent type A (tularensis) subspecies.1–4 As few as 10 bacteria can cause respiratory tularaemia, the most severe form of the disease, with up to 30% mortality if untreated.1–5 Even when treated with antibiotics, respiratory tularaemia is still associated with considerable morbidity and up to 2% mortality.2–6 Furthermore, the possibility of engineered antibiotic-resistant strains for bioterrorism remains a threat. An attenuated type B live vaccine strain (LVS) partially protects against type A F. tularensis in humans but is not currently licensed because of safety concerns.6,7 The development of potentially safer, subunit F. tularensis vaccines will require both an understanding of the mechanisms involved in the immune response to this organism and identification of protective F. tularensis antigens and epitopes.

Studies of immune protection against F. tularensis have demonstrated a major role for CD8 and T helper type 1 CD4 F. tularensis-specific T cells,8–10 and for the T helper type 1-associated cytokines interleukin -12, interferon-γ and tumour necrosis factor-α.8,9,11,12 B cells were also shown to be required for generation of memory to F. tularensis13 and polyclonal IgG antibodies to F. tularensis were reported to transfer resistance against F. tularensis to naive hosts, including humans.14–23 Identification of the protective B-cell antigens and epitopes will aid in the design of both vaccines and immunotherapeutics against F. tularensis. Furthermore, because of the linked recognition of antigens by B and T cells, identification of protective B-cell epitopes may also shed light on potentially protective T-cell epitopes. This is supported by a recent study that screened F. tularensis-specific T-cell hybridomas of unknown specificity for reactivity against a protein array of F. tularensis serological targets and identified T-cell epitopes in the pathogenicity protein IglB, and the chaperone proteins GroEL and DnaK.24

A known protective F. tularensis B-cell antigen in mice17,21,25–32 and guinea pigs,30 and presumably in humans,14 is lipopolysaccharide (LPS), the main component of the F. tularensis outer membrane, which is identical in structure between type A and type B F. tularensis strains.25,33–37 The F. tularensis LPS (Ft LPS) is comprised of lipid A, a core oligosaccharide (mainly Hex4HexNAcKdo) and an O-polysaccharide [O-antigen (OAg)] consisting of variable numbers of the tetrasaccharide repeat Qui4NFm-GalNAcAN-GalNAcAN-QuiNAc, with Qui4NFm at the non-reducing end.25,33–37

We have previously reported that anti-Ft LPS mouse monoclonal antibodies (mAbs) of the IgG2a isotype, the mouse analogue of human IgG1,38 can confer survival to BALB/c mice infected intranasally with an otherwise lethal dose of LVS.28 Subsequently, we characterized the mouse IgG2a anti-Ft LPS mAbs Ab3, Ab52, Ab54 and FB11, and showed that Ab3, Ab52 and Ab54 target repeating internal OAg epitopes and cross-compete for LPS binding, whereas FB11 targets a unique terminal OAg epitope and does not compete with the other three mAbs for LPS binding.39 Ab52 was found to have by far the highest bivalent avidity of the internal binders, but still 72-fold lower than that of FB11.39 FB11 and Ab52 are therefore considered prototypic terminal-binding and internal-binding anti-Ft LPS mAbs for further analysis.

In the current study, we probed the binding sites of FB11 and Ab52 using Ft LPS oligosaccharides of defined OAg repeat length and localized their target epitopes to one and two tetrasaccharide repeats, respectively. Furthermore, we tested the efficacy of the two mAbs in a mouse model of respiratory tularaemia, and showed that both mAbs reduce bacterial burden, confer survival to mice infected with LVS, and prolong survival of mice infected with the highly virulent F. tularensis type A strain SchuS4.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Bacterial strains and antibodies

Francisella tularensis holarctica strain LVS was obtained from Dr Jeannine Petersen (Centers for Disease Control and Prevention, Fort Collins, CO) and was manipulated under biosafety level 2 (BSL2) containment conditions. Francisella tularensis strain SchuS4 was obtained from BEI Resources, Manassas, VA in accordance with all federal and institutional select agent regulations. All manipulations of SchuS4 were conducted under BSL3 containment conditions. Francisella tularensis cultures were grown as previously described28 on chocolate agar plates at 37°C (for LVS) or 35°C (for SchuS4) for 2·5 days. Bacteria were scraped and resuspended in PBS.

Protein G-purified mouse IgG2a mAb FB11, specific for F. tularensis OAg,40 was purchased from GeneTex® Inc. (Irvine, CA). For administration to mice, the protein was dialysed against PBS to remove the preservative, and sterilized by filtration through a 0·2-μm membrane. Mouse hybridoma cell line CO17-1A,41 producing an IgG2a antibody specific for the human tumour-associated antigen EpCam42, used as isotype control, was obtained from Dr Dorothee Herlyn of the Wistar Institute (Philadelphia, PA). Generation of the hybridoma cell line producing anti-Ft LPS mouse IgG2a mAb Ab52 and purification of Ab52 and CO17-1A were previously reported.39 The concentrations of sterilized FB11, Ab52 and CO17-1A were determined by optical density at 280 nm (OD280; 1 mg/ml IgG equal to 1·4 OD280 nm) and their purity and antigen specificity were confirmed by SDS–PAGE and Western blot analysis on F. tularensis LVS lysate, as previously described.39 BALB/c mouse serum and protein A-purified IgG (both sterile, without preservative) were purchased from Innovative research (Novi, MI).

Competition ELISA

For purification of oligosaccharides, F. tularensis OAg-core (OAgC) was prepared from LVS LPS (Ft LPS), which was purchased from Sussex Research (Ottawa, ON, Canada), by acid hydrolysis36 followed by size exclusion chromatography as described previously.37 Oligosaccharides of defined compositions and OAg repeat lengths were then purified by an additional step of porous graphitized carbon chromatography (Hypercarb™, 4·6 × 150 mm Thermo-Fisher Scientific, Waltham, MA). Samples were loaded in 99% mobile phase A (13 mm formic acid, pH 3·0, adjusted using ammonia), 1% mobile phase B (90% acetonitrile, 10% mobile phase A). A gradient from 5 to 40% B was delivered over 40 min at a flow rate of 0·5 ml/min. The relative molar concentrations were quantified using hydrophilic interaction chromatography-mass spectrometry from the integrated area under the extracted ion chromatograms as described previously.37 Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF) spectra of the purified oligosaccharides used in the current study are shown in Fig. 1.

image

Figure 1.  Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF) spectra of oligosaccharides purified from Francisella tularensis lipopolysaccharide (LPS).

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Enzyme immunoassay/radioimmunoassay easy wash™ certified high-binding 96-well plates (Corning, Corning, NY) were coated with 100 μl per well of F. tularensis OAgC (purchased from Sussex Research) (10 μg/ml for FB11 or 5 μg/ml for Ab52) by overnight air-drying at 37°, then washed and blocked with PBS supplemented with 2% w/v dry non-fat milk and 0·05% Tween-20 (2% MPBST) at room temperature. Serially diluted purified oligosaccharides were pre-mixed with a fixed final concentration of 0·05 μg/ml FB11 or 3·125 μg/ml Ab52 in 2% MPBST in a Fisherbrand® 96-well round-bottom plate (Fisher Scientific, Pittsburgh, PA) at 1000 rpm for 1 hr at room temperature in an Eppendorf MixMate (Eppendorf AG, Hamburg, Germany). The fixed concentrations of FB11 and Ab52 had been pre-determined to yield direct ELISA OD values in the range of 1·9–2·5. Aliquots of pre-mixed oligosaccharide plus mAb (50 μl) were transferred to the coated (and blocked) plates and incubated with shaking at 300 rpm for 1 hr on a C2 platform shaker (New Brunswick Scientific, Edison, NJ) at room temperature. The binding of FB11 or Ab52 to OAgC was determined using horseradish peroxidase-conjugated anti-mouse IgG2a (SouthernBiotech, Birmingham, AL) as previously described.28 Percent inhibition was calculated using the formula [(A0 − A)/A0] × 100, where A0 is the absorbance in the absence of oligosaccharide and A is the absorbance in the presence of oligosaccharide. The relative molar concentrations required for 50% inhibition were calculated based on the equation y = y intercept + Slope*log(x), by Prism non-linear regression analysis. Normalized values were calculated by division relative to the value for the most potent oligosaccharide for each mAb, which was defined as 1.

Protection assays

All animal procedures were approved by the Boston University Institutional Animal Care and Use Committee. BALB/cJ female mice were obtained from Jackson Laboratories (Bar Harbor, ME), at 5–6 weeks of age for LVS studies or 7–8 weeks of age for SchuS4 studies, and inoculated intranasally (i.n.) with F. tularensis bacteria under ketamine/xylazine anaesthesia as previously described.28 For inoculation of mice, the F. tularensis were serially diluted in PBS to the intended colony-forming units (CFU)/ml based on OD600 of the starting stock. LVS was administered in 40 μl as previously described,28 whereas SchuS4 was administered in 10 μl followed by 10 μl of PBS as described by Klimpel et al.23 The actual CFU inoculated per mouse was determined retrospectively after each experiment by plating serial dilutions of the bacterial preparation used for inoculation on chocolate agar plates. Infected mice were treated with mAb or vehicle at various times after infection by intraperitoneal (i.p.) injection. The vehicle was PBS in most experiments or 5% BALB/c mouse serum in PBS when low mAb concentrations were used. Survival was monitored daily for LVS studies and every 12 hr for SchuS4 studies. Kaplan–Meier survival curves were plotted using Graph-Pad Prism 5.0 (GraphPad Software, San Diego, CA) and the log rank test was used to compare groups. P values of < 0·05 were considered statistically significant.

Bacterial burden assays

For LVS studies blood was collected aseptically from the submandibular vein and 20 μl was immediately diluted with 180 μl water. Lung, liver and spleen were aseptically removed, after euthanasia, and each was placed in a pre-weighed 15-ml screw cap tube (Corning, Corning, NJ) containing 1 ml PBS with protease inhibitors (Complete Mini, protease inhibitor cocktail tablets, Roche Diagnostics, Indianapolis, IN, 1 tablet per 10 ml of PBS as suggested by the manufacturer). The tubes were re-weighed to determine organ weight and kept on ice until processed. The total buffer volume required to achieve complete homogenization of each organ (based on previous observation) was determined and additional buffer was added accordingly (spleen: 2 ml PBS/0·1 g; lung: 1 ml PBS/0·1 g; liver: 2 ml PBS/0·1 g). Each organ was homogenized on ice using a T115 homogenizer (Omni International, Marietta, GA) and the homogenate was passed through a 70-μm nylon cell strainer (BD Biosciences, Bedford, MA). A 20-μl aliquot of the flow-through homogenate was diluted in 180 μl water, followed by 10-fold serial dilutions and plating on chocolate agar (100 μl per plate). After incubation at 37°C for 3 days, single colonies were enumerated and used to determine the number of bacteria (CFU) per wet organ weight or ml of blood.

For SchuS4 studies blood was collected from the submandibular vein into a BD Microtainer® tube with Lithium Heparin additive (BD, Franklin Lakes, NJ). When the spleen was used, it was placed in a 50-ml conical tube containing 5 ml PBS, then transferred to a 6-cm Petri dish containing a fresh 5-ml aliquot of PBS and teased with wide-curved forceps to release spleen cells. The suspension was filtered through a 70-μm nylon cell strainer (BD Biosciences) and 5 ml PBS, which was used to wash the Petri dish and then the cell strainer, was added. Undiluted blood and 10-fold serial dilutions of blood or spleen cell suspension were plated on chocolate agar for CFU determination.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

The epitope targeted by FB11 is contained within one OAg tetrasaccharide repeat whereas the epitope targeted by Ab52 spans two OAg tetrasaccharide repeats

To gain further insights into the epitopes targeted by the terminal OAg-binding mAb FB11 and the internal OAg-binding MAb Ab52, we tested the efficacy of three purified oligosaccharides containing Hex4HexNAcKdo core oligosaccharide only (C), or one, two or three OAg repeats attached to core oligosaccharide [(oag)1C, (oag)2C or (oag)3C] at inhibiting the binding of the two mAbs to F. tularensis OAgC in competition ELISA. As shown in Fig. 2, core alone (C) was ineffective at inhibiting the binding of either mAb, confirming the specificities of both for OAg, but all three OAg-containing oligosaccharides showed inhibition. The relative molar concentrations of the three OAgC oligosaccharides necessary for 50% binding inhibition of each of the antibodies were compared and normalized to the lowest value (most potent competitor). As tabulated in Fig. 2, (oag)1C, (oag)2C and (oag)3C had comparable potency at inhibiting the binding of FB11, indicating that the FB11 binding site spans a single OAg tetrasaccharide repeat. However, (oag)2C and (oag)3C were each 155-fold more potent than (oag)1C at inhibiting the binding of Ab52 (Fig. 2), indicating that the Ab52 binding site spans more than one but not more than two OAg tetrasaccharide repeats since the three-repeat oligosaccharide had no advantage over the two-repeat oligosaccharide.

image

Figure 2.  The epitope targeted by FB11 is contained within one OAg tetrasaccharide repeat whereas the epitope targeted by Ab52 spans two OAg tetrasaccharide repeats. Purified lipopolysaccharide (LPS) oligosaccharides were used to compete the binding of FB11 or Ab52 to Francisella tularensis OAgC coated on ELISA plates. Bars denote standard deviation of the mean from two or three experiments. –, 50% inhibition was not reached.

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FB11 and Ab52 reduce bacterial burden in and confer survival to BALB/c mice infected i.n. with LVS

We have previously shown that 200 μg FB11, administered i.p. 1 hr after i.n. LVS infection, can protect BALB/c mice against an otherwise lethal dose of LVS.28 To ascertain that Ab52, which targets a different epitope on Ft LPS, is also protective and to compare the in vivo efficacy of the two mAbs against i.n. LVS infection, we tested the ability of graded concentrations of FB11 or Ab52, administered i.p. 1 hr after infection, to protect mice against 5 × 104 to 7 × 104 CFU [10–14 50% lethal dose (LD50)28] of LVS. As shown in Fig. 3, both FB11 and Ab52 conferred survival to LVS-infected BALB/c mice in a dose-dependent manner, with FB11 conferring a somewhat higher survival rate than Ab52.

image

Figure 3.  FB11 and Ab52 confer survival to Francisella tularensis LVS-infected BALB/c mice in a dose-dependent manner. Five- to six-week old BALB/cJ female mice (n = 4–6) were inoculated intranasally with 5 × 104 to 7 × 104 colony-forming units (CFU) of LVS and injected intraperitoneally 1 hr later with graded doses of FB11 or Ab52. Survival was monitored for 28 days, and data from three experiments were combined. The log rank test was used to compare monoclonal antibody (mAb) -treated groups with vehicle groups. **P < 0·001; ***P < 0·0001. No difference in survival was observed between the groups treated with the 6 μg mAb dose or vehicle control.

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To confirm that the protective effect correlates with reduced bacterial burden, CFU in the lung, liver, spleen and blood of BALB/c mice infected i.n. with 14 LD50 of LVS and treated i.p. with 50 μg FB11 or Ab52 or mock-treated 1 hr after infection were compared in a time–course experiment. Starting with groups of 19 mice, seven mice were pre-assigned to survival monitoring and 12 mice were pre-assigned to bacterial burden determination, two mice for each of six time-points. As shown in Fig. 4(a),five of seven mice survived in the FB11-treated group, four of seven mice survived in the Ab52-treated group, but none of the seven mice survived in the mock (PBS only) -treated group. Bacterial burden determination (Fig. 4b) showed statistically significant reductions of CFU in mAb-treated mice when compared with the PBS-only-treated mice on day 3 in the lung, liver, spleen and blood, and on day 6 in the lung. The lower bacterial burdens in the lung, liver and spleen of the FB11-treated mice compared with the Ab52-treated mice on day 14, although not significantly, correlate with the 72-fold higher bivalent avidity39 and somewhat higher survival rate (Fig. 3) of FB11 relative to those of Ab52. All mAb-treated mice that survived until day 28 had no detectable CFU in the lung, liver and blood, and very few CFU in the spleen (Fig. 4b), indicating that they had cleared the bacterial infection.

image

Figure 4.  FB11 and Ab52 reduce bacterial burden in BALB/c mice infected intranasally with Francisella tularensis LVS. Three groups of 19 BALB/cJ female mice, 5–6 weeks of age, were used in this experiment; seven mice were pre-assigned for survival (a) and the remaining mice were pre-assigned for determination of bacterial burden (b). Mice were inoculated intranasally with 7 × 104 colony-forming units (CFU) of LVS and 1 hr later, injected intraperitoneally with 50 μg FB11 or Ab52, or with PBS only. Survival was monitored for 28 days (a). For determination of bacterial burden, mice were euthanised on days 0 (1 hr after bacterial inoculation), 1, 3, 6, 14 and 28. For each time-point in B, three mice per group (except for the Ab52 group on day 14, when n = 2, and the PBS groups on days 14 and 28, when there were no surviving mice) were used for CFU determination, and means and standard deviations were calculated. Student’s t-test statistical analysis was performed and significant differences from the PBS control are indicated for each monoclonal antibody by color code: FB11, black, top; and Ab52, grey, bottom. *< 0·05; **P < 0·005; ***< 0·0005.

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FB11 and Ab52 reduce bacterial burden in and prolong survival of BALB/c mice infected intranasally with SchuS4

The in vivo efficacy of FB11 and Ab52 against respiratory tularaemia was further evaluated in mice infected i.n. with the highly virulent F. tularensis type A strain SchuS4, which has been shown to have an LD100 of 1 CFU in mice, with a time-to-death of 5–7 days after infection.43 BALB/c mice were infected i.n. with 20–57 CFU of SchuS4 and treated with FB11 or Ab52 at various doses, or with Vehicle, by i.p. injection 2 hr or 19 hr after infection. Because of the short time-to-death of SchuS4-infected mice, survival was monitored every 12 hr. Mice treated with 10 μg of either FB11 or Ab52 2 hr after infection showed significant prolongation of survival (12–24 hr), compared with Vehicle-treated mice (Fig. 5a), and mice treated with 30 or 100 μg 2 hr after infection showed significant but similar prolongation of survival compared with mice treated with 10 μg (24 hr, Fig. 5b), indicating that Ab52 doses higher than 10 μg provide no added benefit under the conditions tested. When the mAbs were administered at 10 μg 19 hr after infection, neither FB11 nor Ab52 showed any efficacy (Fig. 5c), but when the mAb dose was increased to 50 μg, both mAbs prolonged survival by 24 hr, with the Ab52 results being highly significant and the FB11 results almost reaching statistical significance (Fig. 5d).

image

Figure 5.  FB11 and Ab52, administered 2 hr or 19 hr after intranasal infection of BALB/c mice with Francisella tularensis SchuS4, prolong survival. BALB/cJ female mice, 7–8 weeks of age, were inoculated intranasally with SchuS4 and injected intraperitoneally with the indicated monoclonal antibody (mAb) doses or with Vehicle at the indicated times after infection. The log-rank test was used to compare mAb-treated groups with Vehicle-treated groups. P-values are indicated by colour code: FB11, black; Ab52, grey. (a) Combined results from four experiments for Ab52 [n = 22 (7 + 5+5 + 5)], only the first two of which included FB11 [n = 12 (7 + 5)]; the SchuS4 inocula for the four experiments were 20 colony-forming units (CFU), 25 CFU, 52 CFU and 57 CFU. (b) Ab52 at 30 or 100 μg (n = 5) tested in the same (57 CFU) experiment shown in (a). (c,d) FB11 and Ab52 at 10 μg (n = 5) or 50 μg (n = 5 for FB11, n = 10 for Ab52) tested 19 hr after infection with 35 CFU SchuS4.

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To confirm that prolongation of survival by anti-OAg mAb treatment correlates with reduced bacterial burden in mice infected i.n. with SchuS4, CFU in the spleen and blood of BALB/c mice infected i.n. with 116 CFU of SchuS4 and treated i.p. with 50 μg of Ab52 or CO17-1A (IgG2a isotype control) 2 hr after infection were compared at day 3 after infection. Bacterial burden in both spleen and blood showed statistically significant reductions of 27-fold and 147-fold, respectively (Fig. 6a). Subsequent comparison of Ab52 and FB11 for ability to affect blood bacterial burden, under these experimental conditions, showed 82-fold and 48-fold reductions relative to CO17-1A, respectively (Fig. 6b).

image

Figure 6.  FB11 and Ab52 reduce bacterial burden in BALB/c mice infected intranasally with Francisella tularensis SchuS4. Groups of BALB/c mice (n = 2 in a and 5 in b), 7–8  weeks of age, were infected intranasally with 116 colony-forming units (CFU) (a) or 164 CFU (b) of SchuS4 and injected intraperitoneally with 50 μg Ab52, FB11 or CO17-1A (IgG2a isotype control), 2 hr later. Mice were euthanised on day 3 after bacterial infection. Bacterial burdens in spleen and blood were determined by plating teased spleen suspension or heparinized blood on chocolate agar plates for bacterial enumeration (CFU per spleen or per ml blood). The difference between the Ab52 or FB11 and control (CO17-1A) groups was analysed by Student’s t-test. < 0·05 is indicated by asterisk. Bars denote standard deviation from the means.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

In the current study, we used Ft LPS oligosaccharides with one, two or three OAg tetrasaccharide repeats as molecular rulers in competition ELISA to help map the sites of two anti-F. tularensis OAg mouse mAbs: FB11, specific for a terminal OAg epitope, and Ab52, specific for a repeating internal OAg epitope. The results demonstrate that the FB11 epitope is contained within a single OAg tetrasaccharide repeat whereas the Ab52 epitope spans two OAg tetrasaccharide repeats, indicating that two OAg tetrasaccharide repeats are necessary and sufficient to provide the full binding energy for both a terminal and an internal F. tularensis OAg epitope. An oligosaccharide containing free F. tularensis core was shown not to contribute to the OAgC-binding of either FB11 or Ab52, confirming the specificity of both mAbs for OAg only.

We have previously reported that FB11 binds to but does not immunoprecipitate OAgC and neither competes with nor is competed by Ab52 for Ft LPS-binding, demonstrating its specificity for a terminal epitope of OAgC.39 Furthermore, FB11 binds and strongly agglutinates LVS and SchuS4 bacteria but not an OAg-deficient LVS strain,39 and cannot bind to Ft LPS prepared from R-form F. tularensis, which has no OAg chains.40 These findings and the inability of free-core oligosaccharide to compete with OAgC for FB11 binding, shown in the current study, indicate that the FB11 epitope is located at the non-reducing end of OAgC. The 72-fold higher bivalent avidity39 of FB11 compared with Ab52 suggests that FB11 has a cavity-type or pocket-type site that binds to the non-reducing end of OAg head-on, burying the epitope with all-around complementarity.

Cavity- or pocket-type sites for antibodies binding to non-reducing ends in contrast to groove-type or canyon-type sites for antibodies binding to internal epitopes of carbohydrates, including antibodies to LPS OAg from Shigella flexneri, Vibrio cholerae and Brucella abortus, are supported by immunochemical, homology modelling and X-ray crystallographic studies.44–54 These show a maximum of five sugar residues accommodated by cavity-type sites and six to eight sugar residues accommodated by groove-type sites.44–54

Ab52, specific for a repeating internal OAg epitope, is therefore expected to have a groove-type or canyon-type site that only partly buries the epitope. The current results showing that the oligosaccharide with two OAg repeats is 155-fold more potent than the oligosaccharide with one OAg repeat – but not less potent than the oligosaccharide with three OAg repeats, at inhibiting OAgC binding – demonstrate that the Ab52 epitope spans two OAg repeats (ABCDA’B’C’D’). The large increase in inhibitory potency going from an oligosaccharide with one to an oligosaccharide with two OAg repeats suggests that the second repeat adds substantial binding energy and hence is likely to contribute multiple sugar residues, in contrast to the small incremental increases seen with anti-dextran antibodies when increasing the competing oligosaccharide length by one or two sugar residues, respectively.44,48 Supporting this conclusion, computational modelling of F. tularensis OAg in the X-ray crystallographic structure of Ab52 Fab revealed a six-sugar epitope spanning two OAg repeats, with each repeat contributing three residues: BCDA’B’C’ (Rynkiewicz, M., Lu, Z., Hui, J., Sharon, J. and Seaton, B., manuscript submitted).

Both FB11 and Ab52 conferred survival to BALB/c mice infected i.n. with LVS and prolonged survival of BALB/c mice infected i.n. with SchuS4, the protective effect correlating with reduced bacterial burden in the lungs, liver, spleen and blood of the mAb-treated infected mice. Khlebnikov et al.30 first reported that FB11, administered subcutaneously, protected mice and guinea pigs against subsequent subcutaneous challenge with an otherwise lethal dose of LVS or a virulent type B strain, and we reported that 200 μg FB11 conferred survival to BALB/c mice when administered i.p. 1 hr after intranasal infection with 8 LD50 of LVS.28 Another anti-Ft LPS mAb, Ab3, which like Ab52 binds to a repeating internal epitope of F. tularensis OAgC but with much lower bivalent avidity,39 was also protective.28

The importance of antibodies to the OAg of Ft LPS in protection against both LVS and type A F. tularensis strains, shown by these studies with mAbs, are also reflected in reported studies with OAg-deficient LVS mutants, which are impaired in their ability to protect mice from LVS31 or SchuS421 challenge. Similarly, mouse and rabbit sera induced by immunization with an OAg-deficient LVS mutant are impaired in their ability to passively protect mice against LVS infection,21 as is rabbit anti-LVS serum that has been absorbed with OAg.55 Furthermore, subcutaneous immunization of BALB/c mice with adjuvanted F. tularensis OAg–BSA conjugate, or with adjuvanted F. tularensis OAg–tetanus toxoid conjugate combined with intranasal administration of an OAg-deficient LVS mutant, protected BALB/c mice against subsequent aerosol challenge with the F. tularensis type B strain FSC 108,25 or intranasal challenge with the F. tularensis type A strain SchuS4,32 respectively. The enhanced immunity against SchuS4 provided by the humoral immune response in the latter study32 directly demonstrates the critical role of anti-OAg antibodies in defence against respiratory infection with highly virulent type A F. tularensis strains.

In the current study we demonstrated that Ab52 can protect BALB/c mice from death when administered i.p. 1 hr after i.n. infection with an otherwise lethal dose of LVS and confirmed that protection is associated with reduced bacterial burden in organs and blood. The somewhat higher anti-LVS in vivo efficacy of FB11 compared with Ab52 correlates with its higher bivalent avidity for LPS.39 Surprisingly, at first, this correlation does not hold in mice infected with SchuS4, where Ab52 is somewhat better than FB11 at prolonging survival and reducing bacterial burden. The lower anti-SchuS4 efficacy of FB11 can be explained, in part, by the much higher proportion of OAgC chains with a single OAg repeat versus longer OAgC chains in LVS-derived LPS compared with SchuS4-derived LPS,37 to which the terminal-binder FB11, but not the internal-binder Ab52, can bind. Despite this slight advantage of antibodies to repeating internal epitopes of F. tularensis OAg, a recent report showed that i.n. immunization of mice with an OAg polymerase-deficient mutant of LVS, whose LPS chains contain only one OAg repeat and is therefore likely to produce only terminal-binding anti-OAg antibodies, protects mice against subsequent intranasal immunization with either LVS or SchuS4.55

The demonstration in the current study that two OAg tetrasaccharide repeats are necessary and sufficient to provide the full binding energy of antibodies to both internal and terminal protective B-cell epitopes of Ft LPS suggests that the humoral immunity-inducing component of a subunit tularaemia vaccine could be provided by a protein-conjugated oligosaccharide comprised of only two OAg repeats. Furthermore, the prototypic terminal-binding and internal-binding mAbs FB11 and Ab52, which are now characterized and shown to be protective in the mouse model of respiratory tularaemia, could be used as reporters to interrogate human sera from patients with tularaemia and future recipients of tularaemia vaccines in clinical trials – for the presence of antibodies to terminal and internal F. tularensis OAg epitopes.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

We thank Michael Rynkiewicz and Barbara Seaton for discussion and for permission to refer to their unpublished data on the X-ray crystallography/computational modelling study of Ab52. This work was supported by Contract HHSN272200900054C from the National Institutes of Health to JS.

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  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
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