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Western blot analysis of B. burgdorferi-specific antibodies. (A) mAbs, IS, and NMS used for reconstitution of scid mice, tested on strains B31 and ZS7 of B. burgdorferi. (B) Sera from individual scid mice reconstituted with the respective mAbs, IS, or NMS, tested on strain ZS7 (strain used for infection of mice) and on strain B31. The following mAbs or sera were used: mAb LA-5 (lane 1), mAb LA-2 (lane 2), mAb LA-10 (lane 3), mAb LA-21 (lane 4), anti-B31 IS (lane 5), anti-ZS7 IS (lane 6) and NMS (lane 7). (C) Amount (Ag/ml) of B. burgdorferi-specific antibody(ies) in reconstituted scid mice during the observation period (one representative experiment). Curves: 1, mAb LA-10; 2, mAb LA-21; 3, mAb LA-2; 4, mAb LA-5; 5, anti-B31; 6, PBS.
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We have recently shown that viable Borrelia burgdorferi organisms induce a chronic infection associated with arthritis and carditis in severe combined immunodeficiency (scid) mice but not in immunocompetent mice. The disease is similar to that found in patients suffering from Lyme disease. We now show that B. burgdorferi-specific immune mouse sera...
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Context 1
... study the influence ofB. burgdorferi-specific antibodies on the development of the disease in scid mice, heteroge- neous antibody and mAb preparations were used for passive- transfer experiments. The specificities of the individual an- tibody preparations as revealed by Western blot analyses on antigen preparations of strains ZS7 and B31 as well as the isotypes of mAbs are documented in Table 1 and Fig. ...
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... first dose of each antibody preparation was passively transferred to scid mice i.p. at the time of the bacterial inoculation (1 x 108 spirochetes s.c. in the tail). Further injections of increasing amounts of antibody preparations were given at half-weekly intervals for 3 weeks to achieve increasing antibody titers in the host. The results show that roughly similar concentrations (1-5 ,g of antibodies per ml) of the injected polyclonal IS or mAbs, except for anti-B31 IS (0.5 ,g of antibodies per ml), could be maintained in the sera of treated scid mice throughout the observation period (Fig. 1C). In additional experiments even higher titers of polyclo- nal antibodies or mAbs (up to 25 ,g/ml) were found in the sera of reconstituted scid mice, which were comparable to those found in the IS used for reconstitution (=60 ug/ml). Western blot analyses of individual sera revealed that the specificity of the antibody recovered from the sera of treated scid mice (Fig. 1B) was identical to those of the injected mAb or polyclonal IS preparation (Fig. ...
Context 3
... first dose of each antibody preparation was passively transferred to scid mice i.p. at the time of the bacterial inoculation (1 x 108 spirochetes s.c. in the tail). Further injections of increasing amounts of antibody preparations were given at half-weekly intervals for 3 weeks to achieve increasing antibody titers in the host. The results show that roughly similar concentrations (1-5 ,g of antibodies per ml) of the injected polyclonal IS or mAbs, except for anti-B31 IS (0.5 ,g of antibodies per ml), could be maintained in the sera of treated scid mice throughout the observation period (Fig. 1C). In additional experiments even higher titers of polyclo- nal antibodies or mAbs (up to 25 ,g/ml) were found in the sera of reconstituted scid mice, which were comparable to those found in the IS used for reconstitution (=60 ug/ml). Western blot analyses of individual sera revealed that the specificity of the antibody recovered from the sera of treated scid mice (Fig. 1B) was identical to those of the injected mAb or polyclonal IS preparation (Fig. ...
Context 4
... first dose of each antibody preparation was passively transferred to scid mice i.p. at the time of the bacterial inoculation (1 x 108 spirochetes s.c. in the tail). Further injections of increasing amounts of antibody preparations were given at half-weekly intervals for 3 weeks to achieve increasing antibody titers in the host. The results show that roughly similar concentrations (1-5 ,g of antibodies per ml) of the injected polyclonal IS or mAbs, except for anti-B31 IS (0.5 ,g of antibodies per ml), could be maintained in the sera of treated scid mice throughout the observation period (Fig. 1C). In additional experiments even higher titers of polyclo- nal antibodies or mAbs (up to 25 ,g/ml) were found in the sera of reconstituted scid mice, which were comparable to those found in the IS used for reconstitution (=60 ug/ml). Western blot analyses of individual sera revealed that the specificity of the antibody recovered from the sera of treated scid mice (Fig. 1B) was identical to those of the injected mAb or polyclonal IS preparation (Fig. ...
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The authors describe the histopathologic evolution of Lyme disease in severe combined immunodeficiency (scid) and normal C.B-17 and C57BL/6 mice inoculated with Borrelia burgdorferi. Starting on day 7 after inoculation, all scid mice infected subcutaneously in the tail with a low-passage European tick isolate of B. burgdorferi had clinical evidence...
Citations
... One underexplored method for preventing bovine anaplasmosis is to block pathogen transmission. This approach was used successfully with the first licensed vaccine to prevent Lyme disease (5,6). However, our lack of understanding of the molecular interface between the tick and the pathogen limits our ability to develop such tools targeting A. marginale. ...
Anaplasma marginale is an obligate, intracellular, tick-borne bacterial pathogen that causes bovine anaplasmosis, an often severe, production-limiting disease of cattle found worldwide. Methods to control this disease are lacking, in large part due to major knowledge gaps in our understanding of the molecular underpinnings of basic host–pathogen interactions. For example, the surface proteins that serve as adhesins and, thus, likely play a role in pathogen entry into tick cells are largely unknown. To address this knowledge gap, we developed a phage display library and screened 66 A . marginale proteins for their ability to adhere to Dermacentor andersoni tick cells. From this screen, 17 candidate adhesins were identified, including OmpA and multiple members of the Msp1 family, including Msp1b, Mlp3, and Mlp4. We then measured the transcript of ompA and all members of the msp1 gene family through time, and determined that msp1b , mlp2 , and mlp4 have increased transcript during tick cell infection, suggesting a possible role in host cell binding or entry. Finally, Msp1a, Msp1b, Mlp3, and OmpA were expressed as recombinant protein. When added to cultured tick cells prior to A. marginale infection, all proteins except the C-terminus of Msp1a reduced A. marginale entry by 2.2- to 4.7-fold. Except OmpA, these adhesins lack orthologs in related pathogens of humans and animals, including Anaplasma phagocytophilum and the Ehrlichia spp., thus limiting their utility in a universal tick transmission-blocking vaccine. However, this work greatly advances efforts toward developing methods to control bovine anaplasmosis and, thus, may help improve global food security.
... Bin 0 constitutes a region of the N-terminus of OspA that is normally buried in the bacterial outer membrane and only accessible when OspA is released from the cell surface (26,43). Bins 1 and 2 encompass OspA's central β-sheet (strands 8-13), while bin 3 is situated within OspA's C-terminus (β-strands [16][17][18][19][20][21] and projects outward from the bacterial surface. MAbs capable of blocking B. burgdorferi tick-to-mouse transmission have been identified in bins 1, 2, and 3 (32,33). ...
Camelid-derived, single-domain antibodies (VHHs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed VHH library directed against the candidate Lyme disease vaccine antigen, outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 from Borrelia burgdorferi sensu stricto strain B31, in combination with the canine vaccine RECOMBITEK Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment (“panning”) against recombinant OspA, yielding 21 unique VHHs within two epitope bins, as determined through competition enzyme linked immunosorbent assays (ELISAs) with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry. Six of the monovalent VHHs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including B. burgdorferi agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The VHHs displayed unique reactivity profiles with the seven OspA serotypes associated with B. burgdorferi genospecies in the United States and Europe consistent with there being unique epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.
... In doing so, we hope to break the disease transmission cycle between wild mice and ticks, and reduce disease prevalence in hard-hit areas like Nantucket and Martha's Vineyard (Buchthal et al., 2019). By combining high-efficiency estrous detection with CRISPR/Cas9 genome engineering, we are poised to generate wild P. leucopus which heritability express anti-Lyme antibodies derived from locally sourced mice (Schaible et al., 1990). Engineered Peromyscus generated via camera-based estrous-tracking may serve as a proof of concept for engineering rodents to prevent zoonoses ranging from Lassa fever to hantavirus pulmonary syndrome. ...
CRISPR/Cas9 technology has revolutionized the production of animal models by reducing experimental timelines, slashing costs and streamlining gene editing, leading to a rapid expansion in the number of unique models for the study of human disease and gene function. However, most non-model animals, many of which are important in cancer and aging research, remain recalcitrant to genome engineering due to our limited understanding of their reproductive biology. Many wild rodents that transmit human diseases remain particularly challenging to engineer due to low pregnancy rates, the lack of external copulatory plugs, and susceptibility to premature termination of pregnancy. Here, we present low-cost activity-based estrous tracking for the efficient generation of timed pregnant and pseudopregnant white-footed mice and extend this tracking method to both lab mice and hamsters. Leveraging this technology, we demonstrate the generation of engineered Peromyscus leucopus , the primary reservoir for Lyme disease-causing bacteria and a putative model organism for studies of aging. These tools have broad implications for biomedical research and ecological engineering.
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... assessment of vaccinated individuals revealed that breakthrough infections were associated with IgG titers below a certain threshold, as defined by a competitive enzymelinked immunosorbent assay (ELISA) with the protective mouse monoclonal antibody (MAb) LA-2 (9,24,35). Having a surrogate measure of immunity was critical in vaccine evaluation, since Johnson and colleagues had reported previously that protection against B. burgdorferi infection in hamsters did not necessarily correlate with total anti-OspA serum IgG titers (27). ...
Lyme disease vaccines based on recombinant Outer surface protein A (OspA) elicit protective antibodies that interfere with tick-to-host transmission of the disease-causing spirochete Borreliella burgdorferi. Another hallmark of OspA antisera and certain OspA monoclonal antibodies (MAbs) is their capacity to induce B. burgdorferi agglutination in vitro, a phenomenon first reported more than 30 years ago but never studied in molecular detail. In this report, we demonstrate that transmission-blocking OspA MAbs, individually and in combination, promote dose-dependent and epitope-specific agglutination of B. burgdorferi. Agglutination occurred within minutes and persisted for hours. Spirochetes in the core of the aggregates exhibited evidence of outer membrane (OM) stress, revealed by propidium iodide uptake. The most potent agglutinator was the mouse MAb LA-2, which targets the OspA C terminus (β-strands 18 to 20). Human MAb 319-44, which also targets the OspA C terminus (β-strand 20), and 857-2, which targets the OspA central β-sheet (strands 8 to 10), were less potent agglutinators, while MAb 221-7, which targets β-strands 10 to 11, had little to no measurable agglutinating activity, even though its affinity for OspA exceeded that of LA-2. Remarkably, monovalent Fab fragments derived from LA-2, and to a lesser degree 319-44, retained the capacity to induce B. burgdorferi aggregation and OM stress, a particularly intriguing observation considering that "LA-2-like" Fabs have been shown to experimentally entrap B. burgdorferi within infected ticks and prevent transmission during feeding to a mammalian host. It is therefore tempting to speculate that B. burgdorferi aggregation triggered by OspA-specific antibodies in vitro may in fact reflect an important biological activity in vivo.
... Transmission of B. burgdorferi from ticks to vertebrate hosts can be blocked in animal models by polyclonal antibodies raised against the bacterial outer surface protein A (OspA) as well as by a mAb (LA-2) directed against a specific OspA epitope (6). Based on the effectiveness of OspA-specific humoral immunity in animal models, human vaccines containing recombinant OspA of B. burgdorferi were developed for the prevention of Lyme disease. ...
Disrupting transmission of Borrelia burgdorferi (B. burgdorferi ) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human monoclonal antibody, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human monoclonal antibody for tick season presents a significant challenge for a pre-exposure prophylaxis strategy. Here, we describe the optimization of 2217 by amino acid substitutions (LS, M428L and N434S) into the Fc domain. The LS mutation led to a twofold-increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA reveals that 2217 binds a novel epitope that is highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective pre-exposure prophylaxis in Lyme-endemic regions with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk. .
... 27,33 The LA-2 antibody has been identified as protective against Borreliella burgdorferi infection in passive transfer and active immunization studies targeting OspA protein. 33,43,44 319-44 mAb has demonstrated the equivalent capability of mediating protection against Borreliella burgdorferi infection in antibody transfer experiments as the LA-2 mAb. 19,20 In the context of the present study, antibodies from sera of pLD1 immunized guinea pigs provided inhibition of 319-44 mAb OspA binding. ...
Lyme disease is the most common vector-borne disease in North America. The etiological agent is the spirochete Borreliella burgdorferi, transmitted to mammalian hosts by the Ixodes tick. In recent years there has been an increase in the number of cases of Lyme disease. Currently, there is no vaccine on the market for human use. We describe the development of a novel synthetically engineered DNA vaccine, pLD1 targeting the outer-surface protein A (OspA) of Borreliella burgdorferi. Immunization of C3 H/HeN mice with pLD1 elicits robust humoral and cellular immune responses that confer complete protection against a live Borreliella burgdorferi bacterial challenge. We also assessed intradermal (ID) delivery of pLD1 in Hartley guinea pigs, demonstrating the induction of robust and durable humoral immunity that lasts at least 1 year. We provide evidence of the potency of pLD1 by showing that antibodies targeting the OspA epitopes which have been associated with protection are prominently raised in the immunized guinea pigs. The described study provides the basis for the advancement of pDL1 as a potential vaccine for Lyme disease control.
... We have applied a similar approach to the surface-exposed C-terminal fragment of OspA, which contains most of the epitopes associated with protection (21)(22)(23). On the surface of the C-terminal fragment, several adjacent regions (patches) were defined, mainly corresponding to the mapped binding sites of monoclonal antibodies (MAbs) described in the literature, as well as to additional binding regions reported for the C-terminal fragment of OspA ST1 (21,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). The surface-exposed amino acids within these patches were exchanged to represent different OspA serotypes, which, in sum, accommodated multiple OspA serotypes on one protein. ...
... As a next step, the surface of the OspA ST2 C-terminal fragment was partitioned into surface areas ("patches") with the potential to harbor possible B-cell epitopes (Fig. 1). This was based mainly on OspA ST1 data, such as the binding sites of monoclonal antibodies at atomic resolution (Fab LA-2 crystal structure of complex; PDB ID 1FJ1), nuclear magnetic resonance (NMR) data (chemical shift perturbation maps), point mutation analyses and low resolution data, and binding analyses to subdomains/ fragments or peptide scanning results (21,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). For the definition of patches, which individually should be able to represent a diversity of serotypes, several assumptions were made, as follows. ...
... MAbs LA-2 and CIII.78 have been shown to provide protection in mice, which points to the importance of these epitopes (21,28), whereas information regarding protection for the other MAbs was not publicly available. The crystal structure of OspA (ST1) in a complex with MAb LA-2 enabled a precise definition of the antibody footprint and was used for the definition of the main area of patch I (24,28,34,35). MAb 336, which was generated with OspA from the B. afzelii strain PGau, is capable of binding to strains of other species, namely, B. burgdorferi and B. bavariensis (23). ...
The development of vaccines for prevention of diseases caused by pathogenic species can encounter major obstacles if high sequence diversity is observed between individual strains. Therefore, the development might be restricted either to conserved antigens, which often are rare, or to multivalent vaccines, which renders the production more costly and cumbersome. In light of this complexity, we applied a structure-based surface shaping approach for the development of a Lyme borreliosis (LB) vaccine suitable for the USA and Europe. The surface of the C-terminal fragment of outer surface protein A (OspA) was divided into distinct regions mainly based on binding sites of monoclonal antibodies. In order to target the six clinically most relevant OspA serotypes (ST) in a single protein, exposed amino acids of the individual regions were exchanged to corresponding amino acids of a chosen OspA serotype. Six chimeric proteins were constructed and based on their immunogenicity four of these chimeras were tested in mouse challenge models. Significant protection could be demonstrated for all four proteins following challenge with infected ticks (OspA ST1, OspA ST2, and OspA ST4), as well as with in vitro grown spirochetes (OspA ST1 and OspA ST5). Two of the chimeric proteins were linked to form a fusion protein, which provided significant protection against in vitro grown spirochetes (OspA ST1) and infected ticks (OspA ST2). This article presents the proof-of-concept study for a multivalent OspA vaccine targeting a wide range of pathogenic LB Borrelia species with a single recombinant antigen for prevention of Lyme borreliosis.
... Assessment of the epitope availability using a whole-liposome immunoprecipitation method confirmed surface exposure and epitope intactness of the LA-2 epitope on the liposome-bound OspA (Fig. 2B). LA-2 is a protective monoclonal antibody against OspA that binds the C-terminus domain of OspA [10,48]. In this experiment, the LA-2 antibody could immunoprecipitate CoPoP liposomes functionalized with OspA, based on the detection of additional PoP added to the liposomes. ...
Outer surface protein A (OspA) is a Borrelia lipoprotein and an established Lyme disease vaccine target. Admixing non-lipidated, recombinant B. burgdorferi OspA with liposomes containing cobalt porphyrin-phospholipid (CoPoP) resulted in rapid, particulate surface display of the conformationally intact antigen. Particleization was serum-stable and led to enhanced antigen uptake in murine macrophages in vitro. Mouse immunization using CoPoP liposomes that also contained a synthetic monophosphoryl lipid A (PHAD) elicited a Th1-biased OspA antibody response with higher IgG production compared to other vaccine adjuvants. Antibodies were reactive with intact B. burgdorferi spirochetes and Borrelia lysates, and induced complement-mediated borreliacidal activity in vitro. One year after initial immunization, mice maintained high levels of circulating borreliacidal antibodies capable of blocking B. burgdorferi transmission from infected ticks to human blood in a feeding chamber.
... In addition to being safer, subunit vaccines can be manufactured in a well-controlled process and can be freeze-dried, which allows better lot-to-lot consistency and nonrefrigerated storage or transport, respectively (107). Importantly, the feasibility of generating an efficacious LD subunit vaccine using surface epitopes of B. burgdorferi is strongly supported by the protective efficacy of the OspA LA-2 epitope (109), whose monoclonal antibodies protected SCID mice from clinical signs of LD (110). ...
Lyme disease (LD), the most prevalent vector-borne illness in the United States and Europe, is caused by Borreliella burgdorferi ( Bb ). No vaccine is available for humans. Dogmatically, Bb can establish a persistent infection in the mammalian host (e.g., mice) due to a surface antigen, VlsE. This antigenically variable protein allows the spirochete to continually evade borreliacidal antibodies. However, our recent study has shown that the Bb spirochete is effectively cleared by anti- Bb antibodies of New Zealand White rabbits despite the surface expression of VlsE. Besides homologous protection, the rabbit antibodies also cross-protect against heterologous Bb and significantly reduced pathology of LD arthritis in persistently infected mice. Thus, this finding that NZW rabbits develop a unique repertoire of very potent antibodies targeting the protective surface epitopes, despite abundant VlsE, prompted us to identify specificities of the rabbit protective antibodies and their respective targets. By applying subtractive reverse vaccinology, which involved random peptide phage display libraries coupled with the next generation sequencing and our computational algorithms, repertoires of non-protective (early) and protective (late) rabbit antibodies were identified and directly compared. Consequently, putative surface epitopes that are unique to the rabbit protective sera have been mapped. Importantly, the relevance of newly identified protection-associated epitopes for their surface exposure has been strongly supported by prior empirical studies. This study is significant because it now allows us to systematically test the putative epitopes for their protective efficacy with an ultimate goal of selecting the most efficacious targets for development of a long-awaited LD vaccine.
... Studies showed that mAb LA-2 can protect severe combined immunodeficiency mice from Lyme borreliae infection. 98 LA-2 equivalent antibody, not the total anti-OspA antibody titer, is correlated to the protective capacity of anti-OspA antibodies. 99 Other agglutinating mAbs, 336 96 and 105.5, 100 recognize discontinuous segments in the C-terminal domain (Figure 3). ...
Increasing rates of Lyme disease necessitate preventive measures such as immunization to mitigate the risk of contracting the disease. At present, there is no human Lyme disease vaccine available on the market, although there was in the past. Since the withdrawal of the first and only licensed Lyme disease vaccine based on lipidated recombinant OspA, vaccine and antigen research has aimed to overcome its risks and shortcomings. Replacement of the putative cross-reactive T-cell epitope in OspA via mutation or chimerism addresses the potential risk of autoimmunity. Multivalent approaches in Lyme disease vaccines have been pursued to address sequence heterogeneity of Lyme borreliae antigens and to induce a reper-toire of functional antibodies necessary for efficient heterologous protection. This review summarizes recent antigen engi-neering strategies that have paved the way for the development of next generation vaccines against Lyme disease, some of which have reached clinical testing. Bioconjugation methods that incorporate antigens to self-assembling nanoparticles for immune response potentiation are also discussed.
