Pre-exposure Prophylaxis With OspA-Specific Human Monoclonal Antibodies Protects Mice Against Tick Transmission of Lyme Disease Spirochetes

Abstract

Background. Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease worldwide. Serum concentrations of antibodies against outer surface protein A (OspA) were shown to correlate with protection from infection with Borrelia burgdorferi, the primary cause of Lyme disease in the United States.

Methods. Mice transgenic for human immunoglobulin genes were immunized with OspA from B. burgdorferi to generate human monoclonal antibodies (HuMabs) against OspA. HuMabs were generated and tested in in vitro borreliacidal assays and animal protection assays.

Results. Nearly 100 unique OspA-specific HuMabs were generated, and 4 HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as lead candidates on the basis of borreliacidal activity. HuMabs 319-44, 857-2, and 212-55 were borreliacidal against 1 or 2 Borrelia genospecies, whereas 221-7 was borreliacidal (half maximal inhibitory concentration, < 1 nM) against B. burgdorferi, Borrelia afzelii, and Borrelia garinii, the 3 main genospecies endemic in the United States, Europe, and Asia. All 4 HuMabs completely protected mice from infection at 10 mg/kg in a murine model of tick-mediated transmission of B. burgdorferi.

Conclusions. Our study indicates that OspA-specific HuMabs can prevent the transmission of Borrelia and that administration of these antibodies could be employed as preexposure prophylaxis for Lyme disease.

Lyme disease, OspA, human monoclonal antibody, preexposure prophylaxis

Lyme disease is a bacterial infection that is transmitted through the bite of infected Ixodes ticks. Acute Lyme disease in humans is characterized by fever, fatigue, musculoskeletal pain, and erythema migrans, as well as by the potential for neurologic, cardiac, or joint manifestations. The Centers for Disease Control and Prevention has estimated that >300 000 Americans receive a diagnosis of Lyme disease each year [1]. In Europe, approximately 85 000 Lyme disease cases are estimated to occur annually [2]. Worldwide, 3 main genospecies of Borrelia are associated with Lyme disease in humans. Borrelia burgdorferi is the main cause of Lyme disease in North America, while Borrelia garinii and Borrelia afzelii are the prevalent strains that cause the disease in countries of Europe and Asia [3]. Humans can become infected by nymphs or, less commonly, adult ticks that are infected with Borrelia [4].

Animal studies have demonstrated that transmission of Borrelia from tick vector to the mammalian host can be blocked by antibodies against outer surface protein A (OspA), which is involved in the attachment of spirochetes to the tick midgut. Expression of OspA is downregulated when the tick takes a blood meal and migrates from the midgut to the salivary gland [5–7]. A murine monoclonal antibody, LA-2, was discovered as a protective antibody against Borrelia infection. Passive administration of LA-2 or active immunization with an OspA vaccine protected against tick-transmitted infection in mice, hamsters, and dogs [8–10]. Vaccine-induced antibodies to OspA are taken up by the tick and eliminate the bacteria in the tick midgut. Serum OspA antibody level has been shown to correlate with protection against Borrelia infection in animals [9, 10].

Based on the effectiveness of OspA-specific humoral immunity in animal models, human vaccines containing recombinant OspA from B. burgdorferi were developed for prevention of Lyme disease. Large-scale clinical trials demonstrated the effectiveness of a triple-dose OspA vaccine, which protected up to 92% of human volunteers [11]. However, the vaccine was removed from the market owing to multiple reasons, including reactivity with a potential arthritogenic portion of OspA. Currently, no vaccine is available in the United States to prevent human Lyme disease caused by B. burgdorferi, much less a vaccine that also protects against B. garinii and/or B. afzelii.

Here we describe and characterize a panel of 4 OspA-specific human monoclonal antibodies (HuMabs) that are borreliacidal against a broad range of Borrelia genospecies. Our study demonstrates that the lead HuMabs 319-44 and 221-7 can prevent the transmission of Borrelia from ticks to mice and supports exploring administration of anti-OspA antibodies as preexposure prophylaxis to prevent Lyme disease.

MATERIALS AND METHODS

Expression and Purification of OspA Fusion Proteins From Bacteria

The nucleic acid sequences of OspA from B. burgdorferi B31, B. afzelii BO23, and B. garinii PBi (NP_045688, B8DY02, and Q6LBF1, respectively) were cloned into a pET45-His vector in-frame with a histidine tag. OspA B. burgdorferi truncations were generated by polymerase chain reaction (PCR) amplification of desired fragments of the full-length OspA. Primers were designed to remove the native signal sequence (amino acids 1–18) so that it would be expressed as a cytoplasmic protein with improved solubility.

All cloned OspA constructs were transformed into BL21-DE3 Escherichia coli bacteria (Invitrogen), and expression was induced with 1 mM IPTG. Bacteria were lysed, and proteins were purified with Ni-NTA agarose beads (Invitrogen) and eluted with 250 mM imidazole (Sigma).

Mouse Immunization, Hybridoma Generation, and Antibody Cloning

Transgenic mice containing human immunoglobulin genes and inactivated mouse heavy and κ light chain genes (Bristol-Myers Squib) were immunized with 50 µg of B. burgdorferi OspA weekly with the Sigma adjuvant system (Sigma) for 6–10 weeks. Anti-OspA titer in mouse serum was measured by enzyme-linked immunosorbent assay (ELISA). Hybridomas were generated following a standard PEG fusion protocol. Hybridoma supernatants were screened for reactivity to OspA, and positive cell clones were selected for antibody sequencing. The heavy chain and light chain variable regions were amplified from hybridoma cells and cloned into an immunoglobulin G1 (IgG1) expression vector as previously described [12].

ELISA

Dilutions of purified HuMabs were tested in ELISA for reactivity against OspA. Briefly, 96-well plates were coated with OspA, followed by incubation overnight at 4°C. Hybridoma supernatant or purified antibody was added to the 96-well plates and incubated for 1 hour at room temperature. Antibody binding was detected with anti-human alkaline phosphatase secondary antibody and PNPP substrate.

Borreliacidal Assay by Bac-Titer Glo Detection

Serial dilutions of antibodies were made in 100 µL of BSK-H medium containing 10% of guinea pig complement (Sigma) in a Nunc Edge 96-well plate (Thermo Scientific). A total of 100 µL of Borrelia culture (B. burgdorferi B31 [ATCC35210], B. garinii PBi [ATCCBAA-2496], and B. afzelii BO23 [ATCC51992]) at a concentration of 5 × 10⁶ spirochetes/mL was added to each well to mix with antibodies. The 96-well plate was incubated at 37°C for 3 days. The spirochete viability was quantified by luciferase detection with Bac-Titer Glo reagent (Promega) and read in a Victor3 multilabel counter (Applied Biosystems). Assays were performed in triplicate. The resulting fluorescence was plotted, and the half maximal effective concentration (EC₅₀) and standard deviation were calculated.

Spirochete Surface Staining

We incubated 2 × 10⁷ spirochetes with primary antibody (12.5 µg/mL diluted in 1 mL of phosphate-buffered saline [PBS] plus 10% BSK-H) for 1 hour at room temperature. Spirochetes were washed twice in PBS plus 10% BSK-H and incubated with an anti-human or anti-mouse IgG–phycoerythrin secondary antibody (Jackson ImmunoResearch) at a 1:100 dilution for 1 hour at room temperature. Cells were again washed twice, and fluorescence analysis performed using a FACScan. Assays were performed in triplicate, and mean fluorescence intensities were graphed. An irrelevant HuMab was included as a control.

Mouse Challenge With Infected Ticks

Infected ticks were prepared by placing Ixodes larva on B. burgdorferi N40–infected mice with severe combined immunodeficiency for a blood meal. Larvae were harvested and allowed to molt to the nymphal stage before use for challenge. Groups of 5 C3H mice (Charles River Laboratories) were intraperitoneally injected with one of the 4 lead HuMabs (319-44, 212-55, 221-7, or 857-2), LA2, or an irrelevant HuMab (IgG1) at a dose of 1, 2, 5, or 10 mg/kg. The following day, mice were challenged by the placement of 6 infected tick nymphs behind the ear of each mouse. Three weeks after the tick placement, mice were euthanized, and tissue samples from an ear, the bladder, the heart, and a joint were harvested for Borrelia culture. Tissue samples were monitored twice weekly for 4 weeks by dark-field microscopy for evidence of growth of spirochetes. Samples were also analyzed for Borrelia DNA, using OspA-specific PCR analysis. Serum samples were collected at day 21 to analyze the antibody concentration and for serologic investigation by ELISA against lysate of B. burgdorferi (Supplementary Data). Animals were considered uninfected if results of all 3 tests were negative. Protocols were approved by the Institutional Animal Care and Use Committee of Tufts University.

OspA and OspC Quantitative Reverse-Transcription PCR (qRT-PCR)

Total RNA was isolated from 3 detached ticks per mouse and analyzed using the SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen) with OspA- and OspC-specific primers (Supplementary Data). The transcript levels of OspA and OspC were normalized using the Borrelia housekeeping gene flaB and then calculated relative to levels of RNA isolated from unfed nymphs, using the 2^ΔΔCt method.

RESULTS

Generation and Identification of OspA-Specific HuMabs

To generate a panel of anti-OspA HuMabs, mice transgenic for human immunoglobulin heavy and light chain genes (Bristol-Myers Squibb; HuMab mice) were immunized with B. burgdorferi OspA. Serum responses to OspA were measured by antigen-specific ELISA, and splenic fusions were performed to generate hybridomas. A total of 589 hybridoma clones were identified and confirmed to have reactivity with OspA. To select a unique panel of HuMabs, RT-PCR was performed on all 589 hybridomas to determine the heavy chain gene sequences. The heavy chain gene sequences were aligned on the basis of complementarity-determining region 3 (CDR3) of the VH family. A total of 93 HuMabs were considered to have unique CDR3 sequences and selected for further characterization.

Selection of Lead HuMabs With Strong Borreliacidal Activity

To determine whether the selected HuMabs exhibited borreliacidal activity, all 93 HuMabs were purified from the hybridomas and assessed in an in vitro borreliacidal assay against B. burgdorferi B31 in the presence of guinea pig complement. Twenty HuMabs showed borreliacidal activity at a concentration of 10 nM. HuMabs were further titrated to determine the EC₅₀. Four HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as the lead candidates, based on low EC₅₀ values (<2 nM; Figure 1A and Table 1). In addition, 4 HuMabs were tested against 3 other B. burgdorferi strains, including BN40, 297, and HB19. Strong borreliacidal activity was observed against all strains (Supplementary Supplementary Data).

Table 1.

Half Maximal Effective Concentration (EC₅₀) Values of Borreliacidal Activity of Human Monoclonal Antibodies (HuMabs) Against 3 Borrelia Genospecies

HuMAb	EC50 in nM, by Genospecies
	B. burgdorferi	B. afzelii	B. garinii
221-7	0.59 ± 0.33	<0.40	<0.40
857-2	1.74 ± 0.62	<0.40	0.97 ± 0.78
319-44	0.60 ± 0.16	0.65 ± 0.06	>50.00
212-55	3.20 ± 0.40	>50.00	>50.00

Data are mean or mean ± SD.

Figure 1.

Anti–outer surface protein A (OspA) human monoclonal antibodies (HuMabs) show in vitro borreliacidal activity against 3 Borrelia species. Anti-OspA HuMabs 221-7, 857-2, 319-44, and 212-55, and a murine monoclonal antibody, LA-2, ranging in concentration from 0.4 nM to 50 nM, were incubated with spirochetes to detect borreliacidal activities against 3 Borrelia genospecies. The viability of spirochetes was quantified by luciferase detection and normalized with the result for an irrelevant antibody control. Assays were done in triplicate, and the percentage of live spirochetes was plotted for individual Borrelia genospecies, including Borrelia burgdorferi B31 (A), Borrelia afzelii BO23 (B), and Borrelia garinii PBi (C).

Three major genospecies of Borrelia cause Lyme disease in the United States, Europe, and Asia: B. burgdorferi, B. garinii, and B. afzelii. To select a prophylactic antibody reactive with most Borrelia genospecies, 4 lead HuMabs were further assessed in borreliacidal assay against 2 other prevalent strains, B. garinii PBi and B. afzelii BO23. HuMab 221-7 and 857-2 were found to be borreliacidal against both B. garinii PBi and B. afzelii BO23. The EC₅₀ of 221-7 was much lower than that of 857-2 against both strains, particularly B. garinii PBi. HuMab 319-44 was borreliacidal against B. afzelii BO23 but not B. garinii PBi. In contrast, HuMab 212-55 showed no borreliacidal activity against either B. afzelii BO23 or B. garinii PBi (Figure 1B and 1C and Table 1). These data indicate that, among the panel of borreliacidal HuMabs, 221-7 is the most potent antibody with borreliacidal activity (EC₅₀ < 1 nM) against all 3 genospecies.

Epitope Mapping of 4 Lead Borreliacidal HuMabs

To define the antibody-binding epitope of the 4 lead HuMabs (221-7, 857-2, 319-44, and 212-55), a series of truncated B. burgdorferi OspAs were designed on the basis of the nuclear magnetic resonance imaging–derived secondary structure [13]. Seven OspA truncations were generated as bacterial recombinant proteins, and ELISAs were performed to screen HuMab reactivity. Each of the 4 HuMabs was found to recognize a distinct subset of the OspA truncations. HuMabs 221-7 and 857-2 bound to amino acids 71–141 with overlapping epitopes within this region. HuMab 857-2 but not 221-7 recognized amino acids 106–273, suggesting a smaller binding domain within amino acids 106–141. HuMab 212-55 bound to amino acids 142–273 but not amino acids 178–273, indicating an epitope within amino acids 142–177 of OspA (Figure 2).

Figure 2.

Four lead human monoclonal antibodies (HuMabs) recognize distinct epitopes of outer surface protein A (OspA). A, Expressed OspA truncation proteins are schematically depicted, with the residues defined. B, Enzyme-linked immunosorbent assay results illustrating recognition of each OspA truncation protein by HuMabs 221-7, 857-2, 319-44, and 212-55 and murine monoclonal antibody LA-2. Two alanine mutations, D249A and S250A, were introduced to the full-length OspA (amino acids 18–273) to distinguish the epitope recognized by 319-44 and LA-2. Abbreviations: −, no binding; +, binding.

Amino acids 178–273 of OspA contain the binding epitope the protective murine antibody LA-2. HuMab 319-44 was found to bind to the same epitope (amino acids 178–273), based on a binding competition assay (data not shown). To further distinguish the epitope between 319–44 and LA-2, a series of alanine mutations were introduced into amino acids 178–273 and screened for antibody reactivity. Alterations at amino acids D249A and S250A clearly disrupted the binding with 319–44 but not LA-2, demonstrating that residues comprising the 319–44 epitope are unique from those bound by LA-2 (Figure 2).

HuMabs Bind to the Surface of Live Spirochetes

To verify HuMab recognition of OspA expressed by live spirochetes, 3 genospecies of Borrelia spirochetes (B. burgdorferi B31, B. garinii PBi, and B. afzelii BO23) were incubated with each of HuMabs 221-7, 857-2, 319-44, and 212-55, followed by fluorescence-conjugated secondary antibody and FACScan analysis. All HuMabs showed strong binding activity to B. burgdorferi B31. HuMab 221-7 and 857-2 also recognized B. garinii PBi and B. afzelii BO23. HuMab 319-44 displayed weak binding activity with B. afzelii BO23 but not B. garinii PBi. HuMab 212-55 did not recognize either B. garinii PBi or B. afzelii BO23. Overall, the Borrelia staining results were consistent with HuMab borreliacidal activities against the 3 genospecies (Figure 3).

Figure 3.

Human monoclonal antibodies (HuMabs) bind to the surface of 3 Borrelia genospecies. HuMabs binding to spirochetes of 3 Borrelia genospecies, Borrelia burgdorferi B31, Borrelia afzelii BO23, and Borrelia garinii PBi, were measured by flow cytometry. Assays were done in triplicate, and mean fluorescence intensities were plotted for each of the HuMabs 221-7, 857-2, 319-44, and 212-55; the mouse monoclonal antibody LA-2; and an irrelevant antibody control.

Affinity Determination of Lead HuMabs

To explore the OspA-binding differences of HuMabs, the antibody affinity was measured against individual OspAs by surface plasmon resonance (SPR). HuMab 221-7, 857-2, and 212-55 were found to have high affinities to OspA-burgdorferi (dissociation constant [K_D] <1 nM). 319-44 bound to OspA-burgdorferi with a much weaker affinity of 328 nM. 212-55 showed high affinity to OspA-burgdorferi but very low binding activity to OspA-garinii and OspA-afzelii (K_D >400 nM). As expected, 221-7 and 857-2 bound to all 3 OspAs, OspA-burgdorferi, OspA-garinii, and OspA-afzelii, with K_D <10 nM (Supplementary Data).

HuMabs Protected Mice From Infected-Tick Challenge

To determine whether the lead HuMabs were capable of blocking transmission of Borrelia via ticks, the protective efficacy of OspA-specific HuMabs was tested in C3H mice challenged with B. burgdorferi–infected nymphs. The efficacy of protection was assessed by determining whether HuMabs could prevent transmission to uninfected mice. Groups of 5 mice were administered one of the 4 lead HuMabs (319-44, 212-55, 221-7, or 857-2), LA2, or an irrelevant HuMab at a dose of 10 mg/kg by intraperitoneal injection. One day later, mice were challenged with infected nymphs. Three weeks after the challenge, mice tissues were harvested and examined for the presence of B. burgdorferi by culture and OspA-specific PCR. Serum samples were also collected at the time mice were euthanized, for serologic testing and antibody quantification (Figure 4A). Treatment of mice with 10 mg/kg of HuMabs 319-44, 212-55, 221-7, and 857-2 or murine monoclonal antibody LA-2 completely blocked infection, while nearly 100% of control animals that received an irrelevant HuMab were infected (Figure 4B and Supplementary Data).

Figure 4.

Human monoclonal antibodies (HuMabs) protect tick transmission of Borrelia burgdorferi infection in mice. A, Experimental design for the animal challenge experiments. Five C3H mice each were treated with one of the 4 lead HuMabs (antibodies 221-7, 857-2, 319-44, and 212-55), the mouse monoclonal antibody LA-2, or an irrelevant control HuMab at a dose of 1, 2, 5, or 10 mg/kg. The next day, mice were challenged by serving as a blood meal to infected Ixodes tick nymphs. After 21 days, cultured tissues were monitored until day 50 for evidence of growth of spirochetes. B, Protection of mice that received preexposure prophylaxis with 319-44, 212-55, 221-7, 857-2, LA-2, or control at doses of 1, 2, 5, or 10 mg/kg. Abbreviations: NA, not applicable; NS, not significant.

To determine the minimal HuMab dose required to provide protection from transmission, HuMabs 221-7 and 319-44 that showed the best potency in in vitro assays were further tested at lower doses of 1, 2, and 5 mg/kg. HuMab 221-7 protected 100% mice from infection at a dose of 5 mg/kg, protected 90% at a dose of 2 mg/kg, and protected 60% at a dose of 1 mg/kg. HuMab 319-44 protected 100% of mice from infection at a dose of 2 mg/kg and protected 60% at a dose of 1 mg/kg. Interestingly, synergy between the 2 antibodies was not observed when both antibodies were administered together at suboptimal doses despite binding to distinct epitopes (data not shown). These results demonstrate that HuMabs 319-44, 212-55, 221-7, and 857-2 can each prevent the transmission of B. burgdorferi from ticks to mice (Figure 4B). The protection was not assessed against B. garinii and B. afzelii in mouse experiments.

HuMabs Inhibited Reciprocal Expression Switch of OspA to OspC Associated With Borrelia Transmission

Two outer surface proteins of the spirochetes, OspA and OspC, are differentially expressed during transmission. OspA is downregulated when spirochetes escape the midgut and disseminate to the salivary glands of feeding ticks. In contrast, OspC is temporally upregulated during tick feeding and plays an essential role for the early stage of mammalian transmission [6, 14]. To further elucidate the mechanism of protection by HuMabs, the expression of OspA and OspC in infected ticks was examined 4 days after feeding on HuMab-treated or control-treated mice. RNA was extracted from the detached ticks, and qRT-PCR was performed to examine the transcript level of OspA and OspC. In mice treated with 10 mg/kg of HuMab 221-7, the transcript level of OspC was significantly lower than that of the control-treated mice (P < .05). In addition, the level of OspA transcripts in this group remained the same as that in the control-treated group (P > .05). This reciprocal OspC and OspA transcript difference was not evident in mice treated with 1 mg/kg of HuMab 221-7 (Figure 5). This observation is consistent with the infection outcome in which all mice treated with 10 mg/kg of HuMab 221-7 were protected from infection, whereas only 60% treated with 1 mg/kg were protected. These results suggest that blockade of the OspA to OspC gene switch in spirochetes may contribute to HuMab-mediated protection.

Figure 5.

Temporal changes in Borrelia OspA and OspC gene expression in infected ticks is inhibited by feeding on human monoclonal antibody (HuMab)–treated mice. RNA was extracted from Borrelia-infected ticks 4 days after receiving a blood meal from HuMab-treated mice. OspA and OspC transcript levels were determined by quantitative reverse-transcription polymerase chain reaction analysis and normalized with the FlaB housekeeping gene for Borrelia burgdorferi. The results were calculated from a triplicate experiment and are presented as the mean fold change of OspC or OspA transcript levels relative to those for a group of infected unfed ticks. The Borrelia-infected ticks fed on mice pretreated with a protective, 10 mg/kg dose of 221-7 showed significantly lower levels of OspC expression relative to the nonprotective groups (P < .05).

DISCUSSION

We have produced and characterized HuMAbs that recognize unique epitopes of OspA and demonstrate borreliacidal activity in vitro. Some of these HuMabs exhibit cross-reactivity against multiple Borrelia genospecies that are endemic in the United States, Europe, and Asia. More importantly, when passively administered to mice prior to challenge with B. burgdorferi–infected ticks, these HuMabs were capable of preventing transmission completely. In the absence of a vaccine, the seasonal, passive administration of a protective antibody could be an effective strategy for Lyme disease prophylaxis.

The feasibility of using human monoclonal antibodies for preexposure prophylaxis has been shown to be effective with palivizumab for preventing respiratory syncytial virus infection in premature infants. In this pediatric setting, a passively administered monoclonal antibody is both safe and effective and provides immediate immunity of known titer, specificity, and duration [15, 16]. A similar strategy could be used to prevent infections due to pathogens that cause Lyme disease. Prophylactic antibody could be administered in geographic areas of endemicity prior to the tick season. One challenge of such a strategy for Lyme disease prevention is to ensure that the antibody offers protection against a range of genospecies. The HuMabs described herein demonstrated strong borreliacidal potency against strains from all 3 main genospecies. A second challenge is sustaining a protective plasma antibody concentration for the entire tick season. Recent advances in engineering antibodies through modulating affinity for FcRn have been successful in dramatic extension of half-life. Such strategies could be easily used to maintain effective antibody concentrations for >6 months.

OspA has been previously identified as an immune target for preventing Lyme disease. However, earlier antibodies either lacked bactericidal activity or only displayed bactericidal activity against a single Borrelia genospecies. For example, the well characterized C-terminus–binding antibody, LA-2, is bactericidal and but only provides protection against B. burgdorferi. The epitope of LA-2 has been mapped to 3 surface-exposed C-terminus loops, where the sequence is largely genospecies specific [17]. A candidate recombinant vaccine that contains a chimeric sequence of LA-2 epitopes from B. burgdorferi and B. afzelii was able to prevent infection with both genospecies but failed to extend the protection to B. garinii [18]. Interestingly, our study demonstrated that HuMAbs raised against full-length OspA from B. burgdorferi could elicit antibodies that were cross-reactive against other genospecies. Two HuMabs, 221-7 and 857-2, recognizing overlapping epitopes (amino acids 71–141) were bactericidal against strains of all 3 genospecies. Sequence analysis revealed that this epitope was 96% conserved among B. burgdorferi, 99% conserved within B. afzelii, and 67% conserved for B. garinii. Together, our data suggest that HuMabs 221-7 and 857-2 bind to a novel, protective epitope of OspA that may provide protection against a broad range of genospecies worldwide. HuMab 319-44 binds an epitope (amino acids 178–273) similar to but distinct from the murine antibody LA-2. HuMab 319-44 was found to have potency comparable to that of LA-2 in the prevention of infection in animals and could hold promise as a prophylactic human antibody against B. burgdorferi in the United States. Moreover, the epitopes of HuMabs 221-7, 857-2, and 319-44 do not overlap with the arthritogenic epitope (amino acids 165–173) on OspA that is potentially cross-reactive with T cells.

Antibody-mediated bactericidal activity within the tick midgut is thought to be at least partially responsible for interrupting transmission from ticks to their hosts. It has been proposed that both complement-dependent and complement-independent antibody activities may mediate borreliacidal activity. To investigate the role of complement in protection of our HuMabs, a complement-activity-enhancing mutation, E345R [19], was introduced into the Fc region of HuMab 319-44. However, when tested in the mouse tick challenge model at a suboptimal concentration, this mutation did not mediate significant enhancement in protecting transmission (Supplementary Data), suggesting that factors other than complement dependence may contribute to the protection.

In summary, we have identified 4 novel OspA-specific HuMabs that demonstrate potent, in vitro borreliacidal activity against 3 pathogenic genospecies of Borrelia. When used prophylactically, these HuMabs are capable of preventing natural tick transmission of Borrelia infection in mice. Preclinical development is currently underway to define the pharmacokinetics and dosage required to provide long-term protection. Lyme vaccine trials have established that the protection from infection was correlated to LA-2 equivalent antibody titer at a concentration as low as 0.1 µg/mL [20]. Given that our HuMabs show a similar or better potency than LA-2 in the prevention of infection in animal models, these HuMabs may provide cost-effective preexposure prophylaxis for Lyme disease worldwide.

Notes

Acknowledgments. We thank Lisa Cavacini (MassBiologics) for manuscript review and Peter Cheslock (MassBiologics) for statistical analysis.

Disclaimer. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health (NIH).

Financial support. This work was supported by the National Center for Advancing Translational Sciences of the NIH (award UL1-TR001453) and the Defense Advanced Research Projects Agency (DARPA-BAA-13-03).

Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.