Antigen Engineering Approaches for Lyme Disease Vaccines.

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. 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 repertoire of functional antibodies necessary for efficient heterologous protection. This Review summarizes recent antigen engineering 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.

Live-vaccinia virus encapsulation in pH-sensitive polymer increases safety of a reservoir-targeted Lyme disease vaccine by targeting gastrointestinal release.

The incidence of Lyme disease has continued to rise despite attempts to control its spread. Vaccination of zoonotic reservoirs of human pathogens has been successfully used to decrease the incidence of rabies in raccoons and foxes. We have previously reported on the efficacy of a vaccinia virus vectored vaccine to reduce carriage of Borrelia burgdorferi in reservoir mice and ticks. One potential drawback to vaccinia virus vectored vaccines is the risk of accidental infection of humans. To reduce this risk, we developed a process to encapsulate vaccinia virus with a pH-sensitive polymer that inactivates the virus until it is ingested and dissolved by stomach acids. We demonstrate that the vaccine is inactive both in vitro and in vivo until it is released from the polymer. Once released from the polymer by contact with an acidic pH solution, the virus regains infectivity. Vaccination with coated vaccinia virus confers protection against B. burgdorferi infection and reduction in acquisition of the pathogen by naïve feeding ticks.

Nest box-deployed bait for delivering oral vaccines to white-footed mice.

Although a wide range of interventions are available for use in reducing the public health burden of Lyme disease, additional tools are needed. Vaccinating mouse reservoirs may reduce the prevalence of spirochetal infection due to the powerful vector and reservoir competence-modulating effects of anti-outer surface protein A (OspA) antibody. A delivery system for an oral immunogen would be required for field trials of any candidate vaccine. Accordingly, we tested candidate bait preparations that were designed to be environmentally stable, attractive to mice, and non-nutritive. In addition, we determined whether delivery of such baits within nest boxes could effectively target white-footed mice. A peanut butter-scented bait was preferred by mice over a blueberry-scented one. At a deployment rate of 12.5 nest boxes per hectare, more than half of resident mice ingested a rhodamine-containing bait, as demonstrated by fluorescent staining of their vibrissae. We conclude that a peanut butter-scented hardened bait placed within simple wood nest boxes would effectively deliver vaccine to white-footed mice, thereby providing baseline information critical for designing field trials of a candidate oral vaccine.

Disulfide-mediated oligomer formation in Borrelia burgdorferi outer surface protein C, a critical virulence factor and potential Lyme disease vaccine candidate.

Borrelia burgdorferi OspC is an outer membrane lipoprotein required for the establishment of infection in mammals. Due to its universal distribution among B. burgdorferi sensu lato strains and high antigenicity, it is being explored for the development of a next-generation Lyme disease vaccine. An understanding of the surface presentation of OspC will facilitate efforts to maximize its potential as a vaccine candidate. OspC forms homodimers at the cell surface, and it has been hypothesized that it may also form oligomeric arrays. Here, we employ site-directed mutagenesis to test the hypothesis that interdimeric disulfide bonds at cysteine 130 (C130) mediate oligomerization. B. burgdorferi B31 ospC was replaced with a C130A substitution mutant to yield strain B31::ospC(C130A). Recombinant protein was also generated. Disulfide-bond-dependent oligomer formation was demonstrated and determined to be dependent on C130. Oligomerization was not required for in vivo function, as B31::ospC(C130A) retained infectivity and disseminated normally. The total IgG response and the induced isotype pattern were similar between mice infected with untransformed B31 and those infected with the B31::ospC(C130A) strain. These data indicate that the immune response to OspC is not significantly altered by formation of OspC oligomers, a finding that has significant implications in Lyme disease vaccine design.

An octavalent lyme disease vaccine induces antibodies that recognize all incorporated OspC type-specific sequences.

Lyme disease is the most common vector-borne disease in North America and Europe and, if untreated, has significant arthritic, cardiac, dermatological and neurological sequelae. There is no currently available human Lyme disease vaccine. Outer surface protein C, because of its antigenicity, protective ability, and expression characteristics has emerged as a promising second generation vaccine candidate; however, significant sequence heterogeneity has impeded its development. Analyses of OspC sequences have revealed the existence of stable phylogenetic clusters or types, and that the type-defining sequence variation occurs within defined domains of the protein. Recent data indicating that immunodominant, and potentially protective OspC epitopes are located in these hypervariable regions has allowed development of a tetravalent, epitope-based, chimeric vaccine. In this report, we have extended that previously described tetravalent construct to include four additional OspC types. We demonstrate that the construct is highly immunogenic, and elicits type-specific antibodies that recognize each of the eight incorporated OspC type-specific epitopes. Antibody raised to the octavalent construct readily binds to the surface of strains expressing each component OspC type, indicating that the incorporated epitopes are presented on the surface of intact cells. In addition, the construct elicits antibody isotypes associated with complement-dependent bactericidal activity. These results represent an important step forward in the design of a broadly protective polyvalent OspC-based Lyme disease vaccine.

Structure-based design of a second-generation Lyme disease vaccine based on a C-terminal fragment of Borrelia burgdorferi OspA.

Here, we describe a structure-based approach to reduce the size of an antigen protein for a subunit vaccine. Our method consists of (i) determining the three-dimensional structure of an antigen, (ii) identifying protective epitopes, (iii) generation of an antigen fragment that contains the protective epitope, and (iv) rational design to compensate for destabilization caused by truncation. Using this approach we have successfully developed a second-generation Lyme disease vaccine. Outer surface protein A (OspA) from the Lyme disease spirochete Borrelia burgdorferi elicits protective immunity that blocks transmission of Borrelia from the tick vector to the vaccinated animal, and thus has been a focus of vaccine development. OspA has two globular domains that are connected via a unique single-layer beta-sheet. All anti-OspA monoclonal antibodies that block Borrelia transmission bind to conformational epitopes in the C-terminal domain of OspA, suggesting the possibility of using the C-terminal domain alone as a recombinant protein-based vaccine. The removal of ineffective parts from the OspA antigen may reduce side effects and lead to a safer vaccine. We prepared a C-terminal fragment of OspA by removing approximately 45% of residues from the N terminus. Although the fragment retained the native conformation and affinity to a protective antibody, its vaccine efficacy and conformational stability were significantly reduced with respect to full-length OspA. We successfully stabilized the fragment by replacing amino acid residues involved in buried salt-bridges with residues promoting hydrophobic interactions. The mutations promoted the vaccine efficacy of the redesigned fragment to a level comparable to that of the full-length protein, demonstrating the importance of the antigen stability for OspA's vaccine efficacy. Our strategy should be useful for further refining OspA-based vaccines and developing recombinant vaccines for other diseases.

Approaches toward the directed design of a vaccine against Borrelia burgdorferi.

The overall efficacy of a recombinant vaccine for Lyme disease that is effective worldwide will depend upon the selection of one or more immunoprotective target(s) and the frequency of genetic variation, which can alter the antigenicity of the immunoprotective epitopes of the target proteins. Careful delineation of these protective epitopes on target antigens is essential for the development of vaccine candidates as well as for understanding the limitations of such vaccines. Structural models of these targets will provide critical information about conformation and specific residue surface accessibility for defining protective epitopes. Co-crystal structures with Fab fragments of protective antibodies will further delineate critical antigen surfaces. Population genetics will provide vital information on the heterogeneity of these proteins. Detailed epitope mapping will provide the information needed for the bioengineering of antigens needed to expand the specificity of a candidate vaccine.

The roles of turn formation and cross-strand interactions in fibrillization of peptides derived from the OspA single-layer beta-sheet.

We previously demonstrated that a beta-hairpin peptide, termed BH(9-10), derived from a single-layer beta-sheet of Borrelia OspA protein, formed a native-like beta-turn in trifluoroethanol (TFE) solution, and it assembled into amyloid-like fibrils at higher TFE concentrations. This peptide is highly charged, and fibrillization of such a hydrophilic peptide is quite unusual. In this study, we designed a circularly permutated peptide of BH(9-10), termed BH(10-9). When folded into their respective beta-hairpin structures found in OspA, these peptides would have identical cross-strand interactions but different turns connecting the strands. NMR study revealed that BH(10-9) had little propensity to form a turn structure both in aqueous and TFE solutions. At higher TFE concentration, BH(10-9) precipitated with a concomitant alpha-to-beta conformational conversion, in a similar manner to the BH(9-10) fibrillization. However, the BH(10-9) precipitates were nonfibrillar aggregation. The precipitation kinetics of BH(10-9) was exponential, consistent with a first-order molecular assembly reaction, while the fibrillization of BH(9-10) showed sigmoidal kinetics, indicative of a two-step reaction consisting of nucleation and molecular assembly. The correlation between native-like turn formation and fibrillization of our peptide system strongly suggests that BH(9-10) adopts a native-like beta-hairpin conformation in the fibrils. Remarkably, seeding with the preformed BH(10-9) precipitates changed the two-step BH(9-10) fibrillization to a one-step molecular assembly reaction, and disrupted the BH(9-10) fibril structure, indicating interactions between the BH(10-9) aggregates and the BH(9-10) peptide. Our results suggest that, in these peptides, cross-strand interactions are the driving force for molecular assembly, and turn formation limits modes of peptide assembly.

Formation of the single-layer beta-sheet of Borrelia burgdorferi OspA in the absence of the C-terminal capping globular domain.

Borrelia outer surface protein A (OspA) contains a unique single-layer beta-sheet that connects N and C-terminal globular domains. This single-layer beta-sheet segment (beta-strands 8-10) is highly stable in solution, although it is exposed to the solvent on both faces of the sheet and thus it does not contain a hydrophobic core. Here, we tested whether interactions with the C-terminal domain are essential for the formation of the single-layer beta-sheet. We characterized the solution structure, dynamics and stability of an OspA fragment corresponding to beta-strands 1-12 (termed OspA[27-163]), which lacks a majority of the C-terminal globular domain. Analyses of NMR chemical shifts and backbone nuclear Overhauser effect (NOE) connectivities showed that OspA[27-163] is folded except the 12th and final beta-strand. (1)H-(15)N heteronuclear NOE measurements and amide H-(2)H exchange revealed that the single-layer beta-sheet in this fragment is more flexible than the corresponding region in full-length OspA. Thermal-denaturation experiments using differential scanning calorimetry and NMR spectroscopy revealed that the N-terminal globular domain in the fragment has a conformational stability similar to that of the same region in the full-length protein, and that the single-layer beta-sheet region also has a modest thermal stability. These results demonstrate that the unique single-layer beta-sheet retains its conformation in the absence of its interactions with the C-terminal domain. This fragment is significantly smaller than the full-length OspA, and thus it is expected to facilitate studies of the folding mechanism of this unusual beta-sheet structure.

Relative incompetence of european rabbits for Lyme disease spirochaetes.

To determine whether rabbits may serve as reservoir hosts for Lyme disease spirochaetes in Europe, we compared their competence as hosts for Borrelia afzelii, one of the most prevalent European spirochaetal variants, with that of the Mongolian jird. To infect rabbits or jirds, at least 3 nymphal or adult Ixodes ricinus ticks infected with spirochaetes fed to repletion on each animal. Whereas jirds readily acquired tick-borne Lyme disease spirochaetes and subsequently infected vector ticks, rabbits exposed to tick-borne spirochaetes rarely became infectious to ticks. Only the rabbit that was infectious to ticks developed an antibody response. To the extent that I. ricinus ticks feed on European rabbits, these mammals may be zooprophylactic by diverting vector ticks from more suitable reservoir competent hosts.

A tracked approach for automated NMR assignments in proteins (TATAPRO).

A novel automated approach for the sequence specific NMR assignments of 1HN, 13Calpha, 13Cbeta, 13C'/1Halpha and 15N spins in proteins, using triple resonance experimental data, is presented. The algorithm, TATAPRO (Tracked AuTomated Assignments in Proteins) utilizes the protein primary sequence and peak lists from a set of triple resonance spectra which correlate 1HN and 15N chemical shifts with those of 13Calpha, 13Cbeta and 13C'/1Halpha. The information derived from such correlations is used to create a 'master-_list' consisting of all possible sets of 1HN(i), 15N(i)13Calpha(i),13Cbeta(i) 13C'beta(i)/1Halpha(i), 13Calpha(i-1), 13Cbeta(i-1) and 13C'(i-1)/1Halpha(i-1) chemical shifts. On the basis of an extensive statistical analysis of 13Calpha and 13Cbeta chemical shift data of proteins derived from the BioMagResBank (BMRB), it is shown that the 20 amino acid residues can be grouped into eight distinct categories, each of which is assigned a unique two-digit code. Such a code is used to tag individual sets of chemical shifts in the master_list and also to translate the protein primary sequence into an array called pps_array. The program then uses the master_list to search for neighbouring partners of a given amino acid residue along the polypeptide chain and sequentially assigns a maximum possible stretch of residues on either side. While doing so. each assigned residue is tracked in an array called assig_array, with the two-digit code assigned earlier. The assig_array is then mapped onto the pps_array for sequence specific resonance assignment. The program has been tested using experimental data on a calcium binding protein from Entamoeba histolytica (Eh-CaBP, 15 kDa) having substantial internal sequence homology and using published data on four other proteins in the molecular weight range of 18-42 kDa. In all the cases, nearly complete sequence specific resonance assignments (> 95%) are obtained. Furthermore, the reliability of the program has been tested by deleting sets of chemical shifts randomly from the master_list created for the test proteins.

Two distinct ospA genes among Borrelia valaisiana strains.

Borrelia valaisiana is a recently described bacterial species in the Borrelia burgdorferi sensu lato complex. To further characterize this bacterium, the plasmid-encoded ospA genes from eight B. valaisiana isolates were amplified by PCR, cloned and sequenced. All B. valaisiana isolates studied possessed an ospA gene with a size of 822-825 bp. The identity of the predicted amino acid sequences of the OspA proteins among B. valaisiana isolates was 69.1-100%, and ranged from 68.2 to 79.1% between B. valaisiana and other B. burgdorferi sensu lato species. Based on the OspA protein sequences, the eight B. valaisiana isolates could be distinguished into two subgroups. Subgroup I contained six B. valaisiana isolates of which OspA sequences were almost identical, but clearly differed from other LB spirochetes. Subgroup II consisted of two isolates with identical OspA sequences which were only 70% identical to subgroup I B. valaisiana isolates and similarly distant from the OspA sequences of other B. burgdorferi sensu lato genospecies. Phylogenetic analysis indicates that B. valaisiana isolates belonging to subgroups I and II possibly evolved from two distinct ancestors. Our data showed for the first time a major difference in OspA proteins within a well-defined B. burgdorferi sensu lato species at the evolutionary level, suggesting that it is not always reliable to assign Borrelia isolates to a definite species solely based on data from ospA gene sequence analysis.

Characterization of an anti-Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping.

Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.

Structural identification of a key protective B-cell epitope in Lyme disease antigen OspA.

Outer surface protein A (OspA) is a major lipoprotein of the Borrelia burgdorferi spirochete, the causative agent of Lyme disease. Vaccination with OspA generates an immune response that can prevent bacterial transmission to a mammalian host during the attachment of an infected tick. However, the protective capacity of immune sera cannot be predicted by measuring total anti-OspA antibody. The murine monoclonal antibody LA-2 defines an important protective B-cell epitope of OspA against which protective sera have strong levels of reactivity. We have now mapped the LA-2 epitope of OspA using both NMR chemical-shift perturbation measurements in solution and X-ray crystal structure determination. LA-2 recognizes the three surface-exposed loops of the C-terminal domain of OspA that are on the tip of the elongated molecule most distant from the lipid-modified N terminus. The structure suggests that the natural variation at OspA sequence position 208 in the first loop is a major limiting factor for antibody cross-reactivity between different Lyme disease-causing Borrelia strains. The unusual Fab-dominated lattice of the crystal also permits a rare view of antigen flexibility within an antigen:antibody complex. These results provide a rationale for improvements in OspA-based vaccines and suggest possible designs for more direct tests of antibody protective levels in vaccinated individuals.

Characterization of a recombinant outer surface protein A (OspA) vaccine against Lyme disease.

Lyme disease, the most prevalent tick-borne disease in the United States, results from infection with Borrelia burgdorferi sensu stricto. Early studies of Borrelia burgdorferi sensu stricto identified outer surface protein A (OspA), a lipoprotein on the surface of spirochetes that could be the target of protective antibodies to this agent. Pasteur Mérieux Connaught has developed a Lyme vaccine, ImuLyme, using recombinant OspA protein (rOspA). Methods were developed to routinely assess the identity, quantity, structure, purity, biological activity, heterogeneity, stability, and potency of rOspA. In addition, several methods were performed on a series of lots to support the routine testing methods and further our understanding of the physicochemical characteristics of rOspA. These tests were electrospray mass spectrometry, circular dichroism, two-dimensional gel electrophoresis, amino acid analysis, peptide mapping with peptide sequencing, and the application of proteomic methodology to identify trace contaminant host cell proteins. The results of these methods indicate that the rOspA lots are composed of highly purified and properly processed and folded rOspA with trace amounts of E. coli host cell proteins.