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The Journal of Immunology, 2008, 180, 6222-6228
Copyright © 2008 by The American Association of Immunologists, Inc.
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Bactericidal Action of a Complement-Independent Antibody against Relapsing Fever Borrelia Resides in Its Variable Region1,2

Timothy J. LaRocca, Laura I. Katona, David G. Thanassi and Jorge L. Benach3

Center for Infectious Diseases, Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
A single chain variable fragment (scFv) of CB515, a complement-independent bactericidal monoclonal IgM against a relapsing fever Borrelia, was constructed to investigate the region wherein the unique bactericidal function resides. Monomeric CB515 scFv (26 kDa) was capable of binding its Ag on whole organisms and by immunoblot. This binding was shown to be species and serotype-specific to the 19 kDa variable small protein, recognized by its parent monoclonal IgM. A dose-dependent bactericidal effect of the CB515 scFv was detected by direct enumeration of spirochetes. Spirochetes incubated with the CB515 scFv before inoculation into mice grew into escape mutants, whereas spirochetes incubated with an irrelevant scFv developed as the original infecting serotype. This bactericidal effect, as seen at the ultrastructural level, was due to disruption of the outer membrane and to severe membrane blebbing eventually progressing to lysis. These results indicate that the variable region of CB515 is responsible for this bactericidal activity and that the constant region of the Ab is dispensable.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The Borrelia of both the relapsing fever and Lyme disease groups are extracellular pathogens. As such, they are controlled by Abs, which are largely responsible for the clearance of these organisms. Relapsing fever is an arthropod-borne, spirochetal infection (1, 2). This disease is of world-wide distribution and is caused by many different species of Borrelia that include tick-borne species and one louse-borne species (3, 4). Relapsing fever has clinical similarities to Lyme disease, caused by Borrelia burgdorferi (5). Both diseases share manifestations affecting the skin, joints, heart, and nervous system (1, 3). Relapsing fever, however, differs from Lyme disease in several respects. Tick-borne relapsing fever Borrelia is transmitted by soft-bodied ticks of the Family Argasidae, whereas hard-bodied ticks of the Family Ixodidae are the vectors of Lyme disease Borrelia (6, 7, 8). Relapsing fever follows a more rapid disease evolution and increased severity due to the high density spirochetemia that is a hallmark of this disease (2, 3, 9, 10). Relapsing fever spirochetemia reaches an initial peak, the largest of the infection, which is rapidly cleared by specific Abs and is followed by a series of smaller peaks as the course of infection progresses. These relapsing peaks of spirochetemia, which cause recurrent bouts of fever (for which the disease is named), are due to antigenic variation of the Borrelia (11, 12). This antigenic variation results in the presence of different bacterial serotypes, that are dependent upon the expression of a repertoire of variable major proteins (Vmps)4 (1, 12). The antigenic variation is thought to involve the insertion of a previously silent (archival) vmp gene into an active expression site via recombination (13, 14). This relapsing cycle of fever and spirochetemia can continue throughout the infection but is self-limited (15).

It has been known for some time that lytic complement is dispensable for efficient host defense against Borrelia infections (16, 17). In fact, clearance of relapsing fever spirochetemia can occur in vivo in mice deficient in different complement components (16, 18, 19). This is most likely the result of the binding of regulators of the alternative (Factor H and Factor H-like protein) (20, 21, 22, 23, 24, 25) and classical (C4BP) (26) complement pathways onto the surface of both Lyme disease and relapsing fever Borrelia.

Abs mediate the clearance of the spirochetemia of relapsing fever and are the main purveyors of protection against these spirochetes (10, 18, 19, 27, 28, 29). Serotype-specific IgM Abs are critical for the in vivo clearance of relapsing fever spirochetemia and can do so without the aid of complement (18, 19, 28, 29, 30). Furthermore, CB515, an IgM mAb derived from a murine immune response to relapsing fever and specific for a 19 kDa variable small protein (Vsp), was observed to be bactericidal in the absence of complement in vitro by causing damage to the Borrelia outer membrane (OM) (19). CB515 was also protective against in vivo challenge in B cell- and complement-deficient mice (19). That CB515 is bactericidal in the absence of complement is unusual since, traditionally, Abs require the assistance of a phagocytic cell or complement to exert a bactericidal effect. Complement-independent killing by Abs alone (in vitro) has been characterized before against B. burgdorferi and Borrelia hermsii (10). CB2, H6831 (both directed against B. burgdorferi OspB), and H4825 (against B. hermsii) are monoclonal IgG1, IgG2a, and IgG2a, respectively, that have a bactericidal effect in the absence of complement in vitro (31, 32, 33). Fab fragments of these Abs also exhibited the same bactericidal activity, indicating that the effect was not due to agglutination or complement fixation (31, 32, 33). How these complement-independent bactericidal Abs exert their effects is not known. CB2 and H6831 are highly specific, the epitope for both is within the C terminus of OspB with an absolute requirement for lysine in position 253 (32, 34). Both Abs produce damage to the OM (32, 35). OspB has been observed to undergo structural changes upon CB2 or H6831 binding as determined by limited proteolysis and examination of the crystallized immune complex, but further details regarding these changes are unknown (36, 37).

To determine the origin of the bactericidal function within the complement-independent bactericidal IgM CB515, we constructed a single chain variable fragment (scFv) of the Ab. These molecules are small, monomeric Ab fragments composed of variable heavy (VH) and variable light (VL) chains with no constant region (Fig. 1) (38, 39). ScFvs have been widely studied for their potential use in therapy for cancer and infection (39). In this study, we are interested in using an scFv of CB515 to determine the location of the bactericidal function within this complement-independent bactericidal IgM. As the scFv retained bactericidal activity, we conclude that this unique function resides within the variable region and that the constant region is not required.


Figure 1
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  		FIGURE 1. The CB515 scFv is a monomer. Shown is the amino acid sequence of the CB515 scFv. A modified (M21G) PelB leader sequence (light blue) and 6x HisTag (light green) are at the N and C terminus, respectively. Shown in black is the linker, which connects the VH (green) and VL (blue) regions. The 15 aa length is specific for production of an scFv monomer. CDRs are underlined and in italics.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and bacteria

A patient-derived, virulent strain of relapsing fever Borrelia was used in this study (40). To obtain spirochetes for in vitro assays, 6–8-wk-old C3H/HeN mice (Charles River Laboratories) were inoculated with stocks of the strain frozen at –80°C and sacrificed during the first peak of spirochetemia. Blood was obtained by cardiac puncture and spirochetes were harvested from plasma by centrifugation and washed with fresh Barbour-Stoenner-Kelly-H (BSK-H) (serum-free) medium (Sigma-Aldrich). The escape mutant of this strain was generated as described in the results section. B. hermsii, B. crocidurae, and B. burgdorferi were grown in BSK-H medium at 33°C to stationary phase. Animal procedures were done by protocols approved by the institutional review board.

Cloning of the scFv of CB515

RNA from the VH and VL chains of mAb CB515 from hybridoma cells (19) was reverse transcribed to cDNA using consensus primers from the Ig-Prime kit (Novagen) and AMV reverse transcriptase (Roche). VH and VL chain cDNAs were amplified (Ig-Prime consensus primers), cloned into Perfectly Blunt vectors (Novagen), and transformed into clone-competent NovaBlue singles Escherichia coli (Novagen). After screening, plasmid DNA was isolated (Promega), concentrated, and sequenced. VH and VL chains were amplified from clones and joined by a linker encoding for (Gly4Ser)3 by overlap extension PCR using the forward primer G G C G G C G G C G G C T C C G G A G G T G G T G G A T C C G A C A T T G T G A T G A C C (for partial linker sequence and VL) and the reverse primer C C G G A G C C G C C G C C G C C A G A A C C A C C A C C A C C T G C A G A G A C A G T G A C C A G (for partial linker sequence and VH). The linker-joined CB515 scFv gene was amplified using the forward primer C C A G C C T G C A G G G G C C C A G G T T C A G C and the reverse primer T G G T G G T G C T C G A G C C G T T T T A T T T C C A and was cloned into a pET26b expression plasmid (Novagen) and inserted into DH5{alpha} E. coli (Novagen) for sequencing. After sequencing, the pET26b plasmid was inserted into Rosetta 2 (DE3) E. coli (Novagen) for expression. From the pET26b plasmid, the CB515 scFv gene encodes for a 6x HisTag at its C terminus and a PelB signal sequence at its N terminus, intended to direct the scFv polypeptide to the E. coli periplasm for folding and disulfide bond formation during expression. Sequences for the H and L chain variable regions, and completed CB515 scFv were deposited with GenBank under the accession numbers EU073761, EU073762, and EU073760.

CB515 scFv expression and purification

Rosetta 2 E. coli were grown at 37°C until an OD600 of ~0.6 was reached. Cultures were then induced with 100 µM IPTG for 3 h and harvested. Harvested E. coli were resuspended in periplasmic extraction buffer (20 mM Tris-HCl, 20% sucrose, 5 mM EDTA, 75 µg/ml lysozyme, pH 8.0) and incubated on ice for 40 min, after which 20 mM MgCl2 was added. Soluble fractions were concentrated in Centriprep YM-10 centrifugal filter units (Millipore), pooled, and diluted 5-fold in binding buffer (20 mM NaPO4, 0.5 M NaCl, and 25 mM imidazole (pH 7.4)). The sample was applied to a 1 ml HisTrap HP Ni+ column (GE Healthcare Amersham Biosciences) using an Akta FPLC (GE Heatlthcare Amersham Biosciences). E. coli contaminants were eluted from the column using 101 mM imidazole and the CB515 scFv was eluted in a pure form using 500 mM imidazole.

Size-exclusion chromatography and homology-based Fv modeling

Affinity-purified CB515 scFv was applied to a Superdex 200 16/60 size-exclusion column (GE Healthcare Amersham Biosciences) using an Akta FPLC. The scFv was eluted using 96 ml PBS, and this volume was compared with a standard curve of proteins of known m.w. to determine the quaternary structure and m.w. of the scFv. The Web Ab Modeling (WAM) service (http://antibody.bath.ac.uk/) provided by the University of Bath, U.K., was used to generate a 3-D molecular model of the CB515 Fv. Sequences were aligned according to the WAM specifications and compared with a library of known, crystallized V regions to generate the structure.

Immunoblots

Then, 12.5% SDS-PAGE gels were run with 37.5 µg of whole Borrelia lysate per well, transferred to nitrocellulose, and exposed to CB515 scFv or monoclonal CB515 at a concentration of 1 µg/ml for 1 h. The CB515 scFv was detected with a 2° anti-HisTag mouse IgG (Novagen) followed by a 3° anti-mouse IgG infrared conjugate (Rockland Immunochemicals). A 2° anti-IgM infrared conjugate (Rockland; 1/1000) was used to detect CB515. Reactivity of the immunoblot was observed by scanning in an Odyssey infrared scanner (LI-COR) in the 800 channel.

In vitro bactericidal assays and transmission electron microscopy (TEM) analysis

Relapsing fever Borrelia were harvested from the plasma of C3H/HeN mice as described. Spirochetes from a pooled sample in BSK-H medium were aliquoted into separate tubes to a final volume of 100 µl. The spirochetes were exposed to either the CB515 scFv or an irrelevant scFv (synovial scFv 2–2; a gift from Brigitte Huber and Srimoyee Ghosh, Tufts University, Boston, MA) (41) at the concentrations indicated in the results section. ScFv elute buffer and BSK-H medium alone also served as negative controls. After 15 and 75 min or 2 h of incubation at 33°C, spirochetes were enumerated directly by dark field microscopy. Following the 75 min time points, spirochetes were prepared for negative-stain TEM analysis as previously described (19). Relapsing fever Borrelia analyzed by ultrathin section TEM were exposed to 200 µg/ml of an irrelevant scFv or the CB515 scFv for 75 min at 33°C and prepared into thin sections as previously described (31). Images were captured using a BioTwinG2 TEM (FEI) and digital camera (AMT) at an accelerating voltage of 80 kV.

Fluorescent microscopic imaging

Spirochetes were treated with several concentrations of CB515 scFv for 1 h at 37°C followed by anti-HisTag (Novagen) 2° mouse IgG. Controls included an irrelevant scFv and the parent mAb CB515. The irrelevant scFv, which has a protein A tag, was detected with mouse anti-protein A IgG (Sigma-Aldrich). For both, a 3° FITC-labeled anti-mouse IgG (Sigma- Aldrich) was used. mAb CB515 was detected with a FITC-labeled anti-mouse IgM (Sigma-Aldrich). Fluorescence was observed using an Eclipse E600 fluorescence microscope (Nikon).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The CB515 scFv is a monomer

CB515 scFv was analyzed by size exclusion chromatography to determine the quaternary structure of the fragment. The 15 amino acid linker that joins the VH and VL chains is designed for production of a monomeric scFv (Fig. 1) (39, 42, 43). However, scFvs intended to be monomers are known to form noncovalent multimers for a number of reasons. Production of high concentrations of scFvs in the bacterial periplasm and existence of scFvs in solution at high concentrations (>1 mg/ml) are two factors that promote scFv multimerization (43, 44, 45). Since these are conditions that would occur during hyperexpression of the scFv in E. coli, we analyzed affinity-purified CB515 scFv (Fig. 2a) by size-exclusion chromatography to determine whether the scFv is monomeric, as intended, or multimeric. Elution properties corresponded to an scFv monomer as compared with protein standards (data not shown). Affinity-purified CB515 scFv was also analyzed by MALDI-TOF to obtain an accurate molecular mass for the construct and to confirm the results determined by size exclusion chromatography. Mass spectrometry indicated that the CB515 scFv had a molecular mass of 26 kDa, the expected size of the monomeric fragment (data not shown).


Figure 2
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  		FIGURE 2. Purity and structure of the CB515 scFv. A, Coomassie blue-stained SDS-PAGE gel showing a representative affinity purification of the CB515 scFv. Nonspecific proteins are shown eluted with 101 mM imidazole and the scFv is shown purified to homogeneity (arrow) using 500 mM imidazole. B, The 3-D molecular model of the CB515 Fv shows the VH and VL chains in green and blue, respectively, and CDRs are highlighted illustrating the Ag binding site.

 
A 3-D molecular model of the CB515 Fv was created through homology-based modeling by the WAM service provided by the University of Bath, U.K. (Fig. 2b). The model provides the predicted scFv structure, particularly demonstrating the correct orientation of complementarity-determining regions (CDRs) forming the accessible Ag binding site.

CB515 scFv binds its Ag in immunoblot and is species and serotype-specific

The CB515 scFv was first tested for Ag binding in immunoblot. Nitrocellulose strips containing lysate from the relapsing fever Borrelia were exposed to CB515 scFv or monoclonal CB515 both at a concentration of 1 µg/ml. Binding was detected with infrared-conjugated Abs and the CB515 scFv exhibited binding to the same Ag as its parent Ab (data not shown). The CB515 scFv was tested for binding to an escape mutant of the infecting organisms, as well as B. hermsii, B. crocidurae, and B. burgdorferi to determine its specificity (Fig. 3, a and b). To generate an escape mutant of this strain, spirochetes harvested from C3H/HeN plasma were adjusted to a concentration of 2x104/ml and used to inoculate B cell-deficient B6.129S2-IgH6 mice (The Jackson Laboratory). Monoclonal CB515 (50 µg) was administered i.p. daily, from days 0–5, to eliminate the predominant relapsing fever serotype and allow for the growth of an escape mutant. In immunoblot the CB515 scFv only recognized the 19 kDa Vsp present in the original infecting relapsing fever serotype and did not recognize the escape mutant or the other species, indicating that it is species and serotype-specific (Fig. 3, a and b).


Figure 3
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  		FIGURE 3. The CB515 scFv is species and serotype-specific. A, Coomassie blue-stained SDS-PAGE gel of Borrelia lysates: original infecting serotype (1 ), an escape mutant of this same species (2 ), B. hermsii (3 ), B. crocidurae (4 ), and B. burgdorferi (5 ). B, Corresponding immunoblot using CB515 scFv as a probe. The CB515 scFv only binds the 19 kDa Vsp band in lane 1, confirming that the scFv is species-specific and serotype-specific.

 
CB515 scFv binds its native Ag on relapsing fever Borrelia

Spirochetes were exposed to several concentrations of CB515 scFv, irrelevant scFv, and monoclonal CB515 followed by FITC-labeled conjugates to detect binding to the whole organisms. Fluorescent spirochetes were observed in samples exposed to the CB515 scFv (Fig. 4, a and b), or its parent mAb, CB515 (Fig. 4d), but not in those exposed to an irrelevant scFv (Fig. 4c) or conjugate alone (data not shown).


Figure 4
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  		FIGURE 4. A and B, The CB515 scFv binds to whole cells of relapsing fever Borrelia. C, An irrelevant scFv was added to spirochetes as a negative binding control. D, Monoclonal CB515 was added to slides with fixed spirochetes as a positive control. This demonstrates that the CB515 scFv is capable of binding its native Ag on whole relapsing fever Borrelia. Size bars equal 20 µm.

 
CB515 scFv retains the bactericidal ability of its parent mAb

We next wanted to determine whether or not the CB515 scFv could retain the bactericidal function of its parent Ab. To test this, relapsing fever Borrelia harvested from mouse plasma were exposed to 100, 200, or 300 µg/ml CB515 scFv, or an irrelevant scFv in vitro. Spirochetes, at an initial concentration of 8.3 x 107 spirochetes/ml, were incubated at 33°C and enumerated under dark field microscopy after 15 (Fig. 5a) and 75 min (data not shown) of incubation to determine bactericidal effect. A significant bactericidal effect was observed when spirochetes were exposed to either 200 or 300 µg/ml CB515 scFv as compared with an irrelevant scFv at both time points. At the highest concentration, a 50% reduction in spirochete numbers was observed after 15 min (Fig. 5a). After 75 min of incubation, 200 µg/ml CB515 scFv caused a 50% reduction in spirochete numbers (data not shown). Additionally, spirochetes were also exposed to buffer or BSK-H medium without scFv in triplicate and no effect was seen (data not shown). The dose-dependence of this bactericial effect was investigated by incubating spirochetes, at an initial concentration of 7.2 x 106 spirochetes/ml, with different concentrations of the CB515 scFv, ranging from 100–300 µg/ml, with increasing 10 µg/ml intervals, for 2 h (Fig. 5b). As with previous experiments, the CB515 scFv exhibited a significant bactericidal effect as compared with 300 µg/ml of an irrelevant scFv. These experiments demonstrated a dose-dependent bactericidal effect of the fragment. It is, however, important to note that in these experiments, darkfield microscopy does not distinguish between live or dead microorganisms, so it is possible that many spirochetes counted were dead or in the process of lysis. Since this was a concern, we investigated the bactericidal efficiency of the CB515 scFv. Relapsing fever Borrelia were exposed to 300 µg/ml of either an irrelevant scFv or the CB515 scFv for 2 h at 33°C to determine their capacity for in vivo infection. Naive mice were inoculated with spirochetes from this experiment (105 spirochetes per mouse) and spirochete recovery, which would be indicative of the killing efficiency of the CB515 scFv, was evaluated. The spirochetes caused murine infection in both cases, and were therefore harvested at peak spirochetemia and probed with the CB515 scFv in an immunoblot to determine serotype identity. Spirochetes exposed to an irrelevant scFv grew as the original infecting serotype whereas those exposed to the CB515 scFv are composed of escape mutants (Fig. 5c). This experiment demonstrated that incubation with the CB515 scFv before infecting mice efficiently eliminated those organisms recognized by this fragment.


Figure 5
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  		FIGURE 5. CB515 scFv retains the bactericidal activity of its parent Ab and eliminates a serotype population. In vitro bactericidal assay using 100, 200, or 300 µg/ml CB515 scFv (gray bars) compared with an irrelevant scFv (black bars). A, Results after incubation with CB515 scFv or an irrelevant scFv for 15 min at 33°C. A significant bactericidal effect can be observed using 200 and 300 µg/ml compared with negative controls. Error bars represent SEM of three experiments. One way ANOVA was performed to analyze statistical significance between samples (*, p < 0.01, **, p < 0.001). B, In vitro bactericidal assay using 100–300 µg/ml CB515 scFv (circles) or 300 µg/ml of irrelevant scFv (square) for 2 h at 33°C. The CB515 scFv is bactericidal at all concentrations tested and exhibits a dose-dependent effect. One way ANOVA was performed to analyze statistical significance between samples (p < 0.001). C, Immunoblot showing the killing efficiency of the CB515 scFv. Spirochetes were exposed to 300 µg/ml of an irrelevant scFv (2 3 ) or the CB515 scFv (4 5 ) for 2 h and then used to inoculate naive mice to evaluate the killing efficiency of the CB515 scFv. The blot contains whole cell lysates from harvested spirochetes and was probed with the CB515 scFv to identify serotypes. Those spirochetes exposed to an irrelevant scFv (2 3 ) still exist as the original infecting serotype, whereas those exposed to the CB515 scFv (4 5 ) are composed of escape mutants, demonstrating that the CB515 scFv eliminates those spirochetes to which it is specific. Lane 1 is an immunoblot control.

 
CB515 scFv causes membrane damage to relapsing fever Borrelia

To assess ultrastructural damage that may have occurred to spirochetes and to confirm the bactericidal effect observed with the CB515 scFv, relapsing fever Borrelia exposed to 100, 200, and 300 µg/ml CB515 scFv, or irrelevant scFv at the same concentrations, for 75 min were examined by negative-stained TEM. Borrelia exposed to the irrelevant scFv, at the same concentrations, or to medium alone, appeared intact and unaffected, showing characteristic morphology and membrane structure (Fig. 6a, panels 1 and 2). Borrelia exposed to the CB515 scFv, however, exhibited visible damage. When exposed to 100 µg/ml CB515 scFv, periplasmic flagella were exposed to the extracellular environment, indicating damage to the spirochete OM (Fig. 6a, panels 3 and 4). Damage was more extensive when spirochetes were exposed to 200 µg/ml CB515 scFv and included membrane disruption and blebbing (Fig. 6a, panels 5-8). The most dramatic effect was observed using 300 µg/ml CB515 scFv, where there was even more exposure of periplasmic flagella and severe membrane blebbing (Fig. 6a, panels 9–12). In some cases, complete spirochete degradation appeared to be taking place (Fig. 6a, panels 8–11). These results confirm the dose-dependent bactericidal effect observed in the previous experiments and demonstrate that this effect is achieved through disruption of the Borrelia OM in a process that is identical to that caused by the parent Ab (19). The damaged spirochetes presumably represent a large population of spirochetes counted in the in vitro bactericidal assays, as those experiments only took into account spirochetes that had disappeared and not those that may have been in the process of dying or already dead but not removed. In this respect, the TEM analysis complements the in vitro bactericidal assays.


Figure 6
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  		FIGURE 6. CB515 scFv causes ultrastructural damage to relapsing fever Borrelia. A, Negative-stain TEM analysis. Spirochetes were exposed to (1–2) 300 µg/ml irrelevant scFv, (3–4) 100 µg/ml CB515 scFv, (5–8) 200 µg/ml CB515 scFv, or (9–12) 300 µg/ml CB515 scFv and examined by negative-stain TEM to observe damage. Size bars equal 500 nm. B, Ultrathin section TEM analysis. Spirochetes were exposed to 200 µg/ml (1–2) irrelevant scFv or (3–4) CB515 scFv and examined by ultrathin section TEM. Size bars, 100 nm. PF, Released periplasmic flagella; MB, membrane blebs; DIS, distention of the outer membrane.

 
The damage imparted by the CB515 scFv was also analyzed by ultrathin section TEM (Fig. 6b). Relapsing fever Borrelia that were exposed to an irrelevant scFv (Fig. 6b, panels 1 and 2) appeared normal with an obvious intact OM. Those spirochetes exposed to 200 µg/ml of the CB515 scFv exhibited a disrupted OM, as is evidenced by the release of periplasmic flagella and distention of the OM (Fig. 6b, panels 3 and 4).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have shown that an unmodified scFv retains the bactericidal effect of its parent IgM. This is important for several reasons. Unmodified scFvs are not, by themselves, bactericidal, rather they exert their effects through the introduction of specific modifications that can include antimicrobial peptides and other toxic molecules. Such molecules are referred to as immunotoxins (39, 46, 47, 48, 49). The CB515 scFv does not contain this type of modification, but rather is a direct clone of the variable region of its parent Ab, and so, the lytic activity is a property of this scFv alone. A bactericidal effect of this nature has never been observed to occur solely within the Ab variable region, the domain which is traditionally considered to be important for Ag binding. That the constant region, traditionally thought of as the Ab effector domain, is not required for this effect to occur is a significant finding.

Abs typically do not kill microorganisms without the assistance of complement or phagocytic cells that ingest opsonized pathogens. Bactericidal Abs of both IgG and IgM classes have been characterized for in vitro and in vivo effects against Borrelia (18, 19, 31, 32); yet, the mechanism by which these Abs kill the spirochetes is not known. The bactericidal effect could be a function of the uniqueness of these Abs that target equally unique and vital Ags on the surface of the Borrelia. Our results show that the complement-independent bactericidal function of CB515 is contained within the variable region of the Ab. The monomeric scFv (26kDa) derived from this IgM mAb exerted a dose-dependent bactericidal effect against relapsing fever Borrelia. The CB515 scFv binds the 19 kDa Vsp from this bacterium and is not cross-reactive, indicating species and serotype-specificity. Further, we have shown that the scFv binds its native Ag on whole relapsing fever Borrelia in an immunofluorescence assay. As a result of its specificity, the CB515 scFv was capable of eliminating the entire serotype population to which it is specific. The bactericidal effect is due to OM damage, evidenced by the release of periplasmic flagella and the formation and release of membrane blebs. These results demonstrate that the CB515 scFv is capable of attacking the spirochetes by itself; thus, confirming that the bactericidal effect of CB515 resides within the variable region. The TEM analysis confirmed the results obtained from the in vitro killing assays. This killing activity is also significant in demonstrating the uniqueness of the Ab-Ag relationship that is not shared with other types of microbicidal Abs. Nontraditional mechanisms used by directly antimicrobial Abs are known (50). They range from Ab-catalyzed water oxidation reactions (51, 52, 53) to interference with iron uptake (54, 55). Fungicidal Abs as well as their derivative scFvs can destroy yeast (56, 57) and interfere with other biological functions of Cryptococcus neoformans and Candida albicans (57, 58, 59).

Bactericidal assays using 100 µg/ml (0.11 µM) of CB515 resulted in a 77% reduction in spirochete numbers (19). The scFv, in an identical assay, required 200 µg/ml (7.4 µM) to achieve a 50% reduction, which would correspond to a sizable decrease in activity compared with the parent IgM. This is not surprising as the difference in bactericidal effect can be attributed to a higher number of binding sites on pentameric CB515 in contrast to the monomeric CB515 scFv. In addition, recent studies have shown that the constant regions of Abs directed to infectious organisms induce structural changes in the variable regions that affect binding affinity and fine specificity (60, 61, 62, 63). Although the bactericidal effect was retained in the variable regions, the killing efficiency was reduced compared with the parent Ab. Thus, it is possible that the absence of the constant regions could be responsible for the decrease in bactericidal activity.

We believe that the bactericidal activity of the CB515 scFv and related Abs to Borrelia may be due to a synergistic effect in which the variable region of the Ab and the Ag play integral parts in creating the effect. As the CB515 scFv is so specific, it would appear that there is something about the combination of the variable region with this unique Borrelia Ag that is extraordinarily lethal. In fact, high specificity is shared by all the known bactericidal Abs to either Lyme disease or relapsing fever Borrelia (10, 19, 31, 32, 34). Bactericidal Abs to B. burgdorferi are specific to one amino acid of OspB (32, 34), a lipoprotein that is down-regulated as spirochetes are transmitted from tick to mammal (64). It is of note that this lipoprotein, regardless of its physiological role to the spirochete, is not exposed to immune surveillance as Abs directed against it would be lethal to the spirochetes. Perhaps the profound effects of these bactericidal Abs were a factor in driving the evolution of antigenic variation in relapsing fever, and the differential expression of Ags in the Lyme disease Borrelia, owing to their specificity and lethality. As CB515 and functionally related Abs are so efficient at eliminating spirochetes, one would assume that antigenic variation would be a necessity for relapsing fever Borrelia to persist and prolong infection.


   Acknowledgments
 
We appreciate the assistance of Brigitte Huber and Srimoyee Ghosh (Tufts University, Boston, MA), who provided the irrelevant scFv. We also appreciate the help of David Holthausen (State University of New York, Stony Brook, NY) who provided technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant R37-AI-27044 from the National Institute of Health. Back

2 We used the facilities of the Northeast Biodefense Center U54AI075158 for protein purification. Back

3 Address correspondence and reprint requests to Dr. Jorge L. Benach, Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, 5120 Centers for Molecular Medicine, Stony Brook University, Stony Brook, New York 11794-5120. E-mail address: jbenach{at}notes.cc.sunysb.edu Back

4 Abbreviations used in this paper: Vmp, variable major proteins; scFv, single chain variable fragment; Vsp, variable small protein; BSK-H, Barbour-Stoenner-Kelly-H; WAM, Web Ab Modeling; TEM, transmission electron microscopy; OM, outer membrane; VH, variable heavy; VL, variable low; CDR, complementarity-determining regions. Back

Received for publication December 20, 2007. Accepted for publication February 20, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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