Antigen Engineering Approaches for Lyme Disease Vaccines

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Antigen Engineering Approaches for Lyme Disease Vaccines Jasmin Federizon, Yi-Pin Lin, and Jonathan F Lovell Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00167 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 16, 2019

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Bioconjugate Chemistry

Antigen Engineering Approaches for Lyme Disease Vaccines Jasmin Federizon†, Yi-Pin Lin‡§, and Jonathan F. Lovell†* † Department

of Biomedical Engineering, University at Buffalo, State University of New York, Buffalo, New York, USA 14260 ‡ Division of Infectious Diseases, Wadsworth Center, New York State Department of Health, Albany, New York, USA 12201 § Department of Biomedical Sciences, State University of New York at Albany, Albany, New York, USA 12201 ABSTRACT: 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.

INTRODUCTION Lyme disease, or Lyme borreliosis, has become the most prevalent vector-borne disease in North America and Europe. It is a zoonotic illness that typically manifests with a distinct erythema migrans rash in its early localized stage, but can lead to severe rheumatologic, neurologic, or cardiac complications in its disseminated stage.1 Etiological agents belong to Borrelia burgdorferi sensu lato bacteria classified under the eubacterial spirochete phylum.2-3 The major pathogenic species in North America is Borrelia burgdoferi sensu stricto whereas the main infectious agents in Europe also include other two genospecies, Borrelia afzelii, and Borrelia garinii. Infrequent clinical cases of Borrelia mayonii4 and Borrelia bissettii5 have been identified in the United States, as well as Borrelia spielmanii6 in Europe. These pathogens are primarily transmitted to humans and animals by hard-bodied ticks of the species Ixodes spp. The principal tick vectors are the deer tick, I. scapularis (northeastern and north central US) and I. pacificus (western US); the sheep tick, I. ricinus (Europe); and the taiga tick, I. persulcatus (Asia). Increasing incidence of Lyme borreliosis in some geographical hotspots has been linked to climate change, which drives expansion of the tick’s ecological niche and an increase in tick density.7-9 Without proper interventions to alleviate the escalating risk of infection, Lyme disease will continue to pose a significant and increasing threat to public health, especially in the endemic regions. Prophylactic vaccination against Lyme disease is an appealing countermeasure due to 1) logistical challenges and potential environmental hazard of using acaricides, tick-

specific pesticides; 2) challenges in early diagnostic testing; and 3) incidence of antibiotic-refractory Lyme arthritis. Identification of potential immunogens for vaccine development necessitates proper understanding of the dynamic antigenic composition of Lyme borreliae surface. To thrive in both the arthropod vector and its subsequent reservoir hosts, the obligate bacteria use an array of adaptation strategies including differential expression of plasmid-encoded outer surface proteins (Osp).10-11 Up-regulating the lipoprotein OspA allows the spirochete to colonize and persist in the tick midgut by attaching to a tick receptor of OspA (TROSPA).12 During a blood meal by Borrelia-infected ticks, spirochetes in the midgut change expression patterns by decreasing ospA gene expression and increasing ospC gene expression to facilitate migration to the tick salivary gland and subsequent transmission of spirochetes to the vertebrate host.10, 13-14 The tick salivary protein, Salp15 (Ixodes scapularis), is induced during feeding and binds to OspC, protecting the spirochete against complement-mediated15 and antibody-mediated killing.16 During the early stages of infection, B. burgdorferi OspC inhibits phagocytic clearance and enables the spirochete to colonize the initial infection site of skin.17 After dissemination in mammalian hosts, Lyme borreliae downregulates OspC but upregulates VlsE that provides antigenic variation of surface exposed lipoproteins to evade adaptive immune response.18,19 Additionally, spirochetes produce multiple outer surface proteins, including complement-regulators-acquiring surface proteins (CRASPs20; CspA21, CspZ22, and OspE-related proteins23), CD59-like proteins24, OspC25, and BBK3226, which bind and recruit complement proteins and regulators to inhibit

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complement activation and confer serum resistance and/or bloodstream survival. Strain-specific variation in immune evasion and host infectivity is associated to polymorphism of the outer surface proteins.27 Current methodologies discussed in this review28 can be useful in further elucidating mechanisms exploited by Lyme borreliae to survive in diverse vertebrate hosts. In addition, adhesion to the vertebrate host and the arthropod vector substrates is a vital Lyme pathogenic mechanism necessary for the dissemination, colonization, and persistence of the disease. To mediate interactions with diverse target tissues, Lyme borreliae encodes multiple adhesive surface proteins, or adhesins, which bind to various host cell surface molecules and different components of the extracellular matrix.29-31 Several identified B. burgdorferi adhesins including BBK32, DbpA, RevA, and OspC are considered potential vaccine candidates. Immunization with BBK32, which exhibits independent fibronectin- and glycosaminoglycan (GAG)binding activities,32-33 conferred partial protection against infection and reduced spirochete transmission during feeding.34 Vaccination with RevA, a fibronectin-binding protein produced during mammalian infection,35 induced borreliacidal antibodies in rabbit but not in mouse.36 Nevertheless, passive immunization of the rabbit antisera in mice prevented B. burgdorferi infection.36 Immunoprotection conferred by decorin-binding protein, DbpA, was observed when mice were challenged by needle inoculation37 but not by tick transmission.38 OspC, which also recognizes mammalianderived plasminogen, has multiple functions essential for transmission and establishment of initial infection.39-40 This immunogen has been widely used in canine vaccine development. Initial efforts in vaccine development for Lyme disease focused on the use of OspA, the predominant surface lipoprotein expressed of spirochetes residing in the tick gut. The protective mechanism of OspA-based vaccines involves blockage of spirochete transmission to the host by eradicating the bacteria within the tick vector (Figure 1).41-43 Elimination of Lyme borreliae within feeding ticks can occur without the presence of a functional complement system in the host44-46 but is less efficient than antibody-dependent complement-mediated killing.47 In vivo studies on spirochete-tick interactions showed that anti-OspA antibodies even without borreliacidal activity could inhibit association of spirochetes on the tick gut.48 A study presented that the transmission blocking mechanism may either proceed by directly interfering with the critical interactions involved in the invasion of the salivary glands or by lowering the spirochete density necessary for the transmission process.43 Irrespective of the protective mechanism, protection from an OspA-based transmission blocking vaccine critically depends on the circulating levels of protective antibody in the host blood. The first licensed Lyme disease vaccine developed by GlaxoSmithKline (LYMErix®) consisted of recombinant lipidated OspA (B. burgdorferi s.s. strain ZS7) with aluminum hydroxide as an adjuvant.49 Its vaccine efficacy against symptomatic disease was 76% after three injections at 0,1, and 12 months.49 Low durability of the antibody response necessitated additional boosters to retain high level of protective antibodies. After its release in December 1998, this vaccine was voluntarily withdrawn from the market in February 2002 due to poor sales partly caused by

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unsubstantiated claims of vaccine-induced arthritis, as well as by the requirement for multiple boosting shots.50-51 Licensure of a non-adjuvanted form of OspA (B. burgdorferi s.s. strain B31) vaccine, ImuLyme® by Pasteur Mérieux Connaught, was no longer pursued due to business-related decisions50 despite good vaccine efficacy (92% after three injections) in a phase III clinical trial.52

Figure 1. Protective mechanism of OspA-based transmission blocking vaccines against Lyme disease. Antibodies uptaken from the blood of the immunized host bind to spirochetes and inhibit transmission from the feeding tick vector to the mammalian host.

Renewed interest in Lyme disease vaccines spurred the development of a novel multivalent OspA vaccine manufactured by Baxter.53-55 A phase I/II clinical trial (ClinicalTrials.gov Identifier: NCT01504347) was implemented on an immunization schedule, which entails three monthly priming injections and a booster injection at either 6 months or 9-12 months after initial vaccination.54-55 Results showed that surface-binding and borreliacidal antibody responses receded to near baseline before booster vaccination and markedly increased after,55 suggesting necessity for booster injections to maintain high levels of circulating OspA antibodies required for protection. Unfortunately, this multivalent vaccine was discontinued after completing phase I/II clinical trial and a phase III efficacy study is not being conducted.54-55 Another multivalent vaccine candidate, VLA15 by Valneva, is in phase I clinical trials (ClinicalTrials.gov Identifier: NCT03010228) with an United States Food and Drug Administration (FDA) fast track designation, aiming for accelerated market approval.56-58 Vaccination in this human trial also involves three initial injections at monthly interval with a booster extension administered 13 month after the initial immunization. At present, the Phase I clinical trial is close to completion whereas phase II (ClinicalTrials.gov Identifier: NCT03769194) is currently in the recruitment stage. The only available Lyme disease vaccines currently on the market are for veterinary use (Table 1). Most canine vaccines, with the exception of Recombitek®, have OspC present in the formulation to exploit borreliacidal activity of anti-OspC antibodies outside the tick vector.59 The inability of anti-OspC serum to facilitate spirochete eradication within the tick reflects a mechanism of action distinct from OspA, which is abundantly expressed in the tick gut but is downregulated by the time the spirochetes transit to the salivary glands.41 OspC, on the other

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hand, is upregulated after the spirochetes leave the tick gut and move to the salivary glands.13 Two possible mechanisms of protection have been postulated; either transmitted spirochetes are eliminated by the host immune response or anti-OspC antibodies ingested during a bloodmeal prevent spirochete transmission to the host.59 Table 1. Commercially available canine vaccines against Lyme disease. Name

Manufacture r

Vaccine type

Ref

RECOMBITEK® LYME

Merial

Non-adjuvated monovalent recombinant OspA

[60]

DURAMUNE® LYME

Boehringer Ingelheim (Elanco)

NOBIVAC® LYME

Merck

LYMEVAX®

ZoetisUS

VANGUARD® crLyme

ZoetisUS

Adjuvanted bivalent whole-cell inactivated bacterin (B. burgdorferi, OspA- and OspCexpressing strains)

[61] [62] [63] [64]

Adjuvanted heptavalent chimeric OspC and recombinant OspA

CONSIDERATIONS FOR VACCINE DESIGN

LYME

[65]

DISEASE

Considering the variable gene expression of Lyme borreliae during its enzootic life cycle, a multifaceted approach may be necessary in developing vaccine for Lyme disease to attain optimum protective efficacy. This kind of approach has been adapted in the design of commercial bacterin and subunit vaccines for dogs in which two reciprocally expressed lipoproteins, OspA and OspC, are combined. This mixed formulation of transmission blocking vaccines allows stimulation of both anti-OspA and anti-OspC bactericidal antibodies for broader protection63, 66 against heterologous population of spirochetes during tick feeding.67 Enhanced protective capacity of multi-antigen vaccines was observed with formulations with adhesins such as DbpA and BBK32. Mice antisera raised against a mixed vaccine consisting of OspA and DbpA produced a synergistic protective effect.68 Vaccination of a three-component vaccine comprising of OspC, DbpA, and BBK32 yielded higher efficacy compared to single or double vaccinogens.69 In this multi-antigen approach, tick salivary protein Salp15 can be used to improve protective capacity of Lyme borreliae antigens such as OspA and OspC.70 Close homologue of Salp-15 in I. ricinus, Salp-15 Iric-1, exhibited preferential protection of B. burgdorferi against antibody-mediated killing despite its binding ability to all three European pathogenic species.71 Immunization with the broadly conserved tick histamine release factor (tHRF) partially impaired spirochete transmission72 but its partial sequence homology (40%) to human TPT1 (Genbank accession no.: CAG33317.1) may pose the risk of autoimmunity.73 Incomplete abrogation of spirochete transmission was also observed for tick salivary lectin pathway inhibitor (TSLPI) protein, which inhibits mannose-binding lectin activity.74-75 It is suggested that

a combination of tick proteins might effectively abolish transmission.75-76 Other arthropod vector proteins for future development of tick antigen-based vaccines are discussed in these review articles.11,76 Interestingly, this vector-directed approach offers a new strategic paradigm to block transmission of additional tick-borne pathogens especially those with similar geographic distribution. Development of Lyme disease vaccines is hampered by significant phylogenetic diversity in Lyme borreliae genospecies and by consequential antigenic heterogeneity, which creates opportunities for multivalent vaccines that confer efficient cross-protection against the disease. Sequence analysis of ospA genes revealed diversity between genospecies but homogeneity within B. burgdorferi (serotype 1), B. afzelii (serotype 2), and B. bavariensis (serotype 4). In contrast to these clusters, B. garinii (serotype 3, 5, 6) exhibits higher variability in the intra-species level. Insufficient protection of antisera raised to a single strain in a heterologous challenge suggested that protective immunity of anti-OspA antibodies is serotypespecific, supported by findings that most immunoprotective epitopes reside in less conserved C-terminal domain. The limited protection afforded by monovalent vaccines constitutes a substantial limitation of previous OspA-based vaccines LYMErix® and ImuLyme®, which confer protection against B. burgdorferi strains only. Unlike OspA-based vaccine, cross protection within B. burgdorferi sensu stricto strains was not observed with OspC immunization59, 77 since heterogeneity occurs at both inter- and intra-species levels.78 Diversity is more pronounced in OspC as 21 phyletic clusters (types A-U), with inter-type amino acid sequence variation as high as 30%, have been defined.79-82 Mapping analyses showed that the immunodominant epitopes are located in the inter-type variable domains,82 supporting the strain-specific immunity of antiOspC antibodies.83 This antigenic variability in OspC, as opposed to early postulations, is not due to immune selection pressure.84-85 About 10% of patients with Lyme arthritis in the United States suffers from antibiotic-refractory Lyme arthritis, a condition in which joint inflammation persists after long-term antibiotic therapy.86 Rheumatoid arthritis-associated MHC class II D-locus alleles, HLA-DRB1*0401 in particular, was linked to treatment-resistant Lyme arthritis,87 implicating involvement of T cell reactivity in the persistence of Lyme arthritis. Furthermore, several studies associated OspA immunity to the etiology of antibiotic-refractory Lyme arthritis.88-90 In a study using T-cell lines from patients with treatment-resistant Lyme arthritis, a highly conserved OspA epitope defined by amino acid (aa) residues 84-133 was first implicated in the pathogenesis of treatment-resistant Lyme arthritis.91 More recently, hypothesis on autoimmunity arising from molecular mimicry between an identified OspA T cell epitope and a self-antigen is extensively received. An immunodominant T-cell epitope, Bb OspA165-173, predicted by a computer algorithm, shares partial sequence homology to the light chain of human lymphocyte function-associated antigen-1 (hLFA-1αL332-340), which is also predicted to bind HLADRB1*0401 molecules.92 The absence of the spirochete in synovial fluids support this hypothesis on autoimmunity. Etiologic relationship between OspA and chronic Lyme arthritis raised some concerns that certain individuals with a given HLA haplotype are susceptible to an autoimmune arthritis

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upon OspA vaccination. To investigate alleged arthritogenicity of OspA immunization, the FDA called for a phase IV safety study, which was not completed due to voluntary withdrawal of the LYMErix® vaccine.50 Recent studies suggest that OspA vaccination does not trigger natural events leading to autoimmune Lyme arthritis.93 From a business and marketing standpoint, safety concerns, proven or not, need to be addressed for widespread acceptance of OspA-based vaccines.

ANTIGEN ENGINEERING STRATEGIES FOR LYME DISEASE VACCINES Addressing issues of antigenic heterogeneity, variability of Borrelia’s surface composition, and potential arthritogenicity of spirochetal antigens calls for new tactical approaches to Lyme disease vaccine development. Several novel design strategies that paved the way for the development of next generation Lyme disease vaccines. Different antigen engineering strategies (Figure 2) including mutation, truncation, fusion, and chimera were employed to tailor immune response and safety profile. Rational design of an effective modified Lyme disease vaccine requires knowledge on antigenic structure and location of protective epitopes. The structure of OspA (31 kDa) comprises 21 consecutive antiparallel β-strands followed by a single α-helix, arranged sequentially in a dumbbell shape in which the N- and C-termini globular domains are connected by a unique ‘single-layer’ β-sheet.94-95 Epitope techniques such as X-ray co-crystallography of OspA/Fab complexes, OspA fragments scanning, and nuclear magnetic resonance (NMR) chemical-shift perturbation method96 allow delineation of the protective epitopes on the C-terminal domain and nonprotective epitopes on the N-terminal domain. Agglutination studies corroborated locality of protective epitopes within the C-terminal domain.97 These findings strongly infer accessibility of OspA C-terminal half on the spirochete’s surface. An important monoclonal antibody (mAb) LA-2 (Figure 3) recognizes a conformational epitope encompassing three surface-exposed loops connecting β-strands 16-17, 18-19, and 20-21 of the C-terminal domain.95 Location of LA-2 epitope determined by NMR chemical-shift perturbation method is consistent with the resolved crystal structure of the OspA/LA-2 Fab complex. Studies showed that mAb LA-2 can protect

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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 antiOspA antibodies.99 Other agglutinating mAbs, 33696 and 105.5100 recognize discontinuous segments in the C-terminal domain (Figure 3). Unlike LA-2 epitope, mostly regular secondary structure elements i.e. α-helix and β-strands 19, 20, 21 form the epitope of mAb 336. Only six out of the 25 residues identified by NMR lie on a loop/turn. The epitope of mAb 105.5, on the other hand, is located on β-strands 11, 12, and 13. Recently, an OspA linear epitope spanning residues 221- 240 (part of β-strand 19) was found to be immunogenic in mice.101 Antibodies raised against OspA221- 240 peptide recognized OspA on the cell surface of Lyme borreliae based on immunofluorescence assay and exhibited complementmediated bactericidal activity.

Figure 2. Schematic diagram of some antigen engineering strategies used in developing next generation vaccines against Lyme disease.

Several structural studies have established the predominantly α-helical structure for OspC (23 kDa).102-104 Its overall fold is quite different to the extended β-sheet structure of OspA. OspC structure consists of five α-helices (four long, one short) and two short β-strands. In the dimeric recombinant form of OspC, two long helices, α-helix 1 (aa 43-74) and 5 (aa 169-196) in

Figure 3. Identified B-cell epitopes in OspA and OspC antigens. (A) Location of conformational epitopes in OspA C-terminal domain. (B)


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Epitope-containing regions (highlighted above in dark gray) in various OspC types. Sequence alignment results by ClustalX are represented by an asterisk “*”, period “.”, or colon “:” to denote full, strong, and weak conservation, respectively. Reprinted in part with permission for (A) from Koide et al.100 Copyright© 2005, Elsevier; and for (B) from Earnhart and Marconi105 Copyright© 2007, Landes Bioscience.

each subunit pack together mainly by hydrophobic interactions to form a typical four-helical bundle.102 Buried area in the dimer interface constitutes about 30% of the total surface area of the dimer, suggesting that the biologically active form of OspC must also be dimeric.103 A study demonstrated that the nondenatured form of OspC vaccinogen is vital to convey protection, indicating conformational dependence of protective epitopes in OspC.59, 106 A study also suggested the presence of a dimer-specific protective epitope in OspC.107 Thus far, no conformational epitope in OspC has yet been defined and studied in detail. A study attempted to locate the conformational epitope formed by mAb B5 but did not identify the pertinent residues.106 Though not consistent with the observed typespecificity of OspC protection, studies have demonstrated that bactericidal antibodies from Lyme disease patients recognize a conserved C-terminal region in OspC.108-109 Furthermore, plasminogen-binding activity of OspC was decreased upon deletion of the conserved C-terminal motif but this motif was found to be inessential for OspC function in spirochete transmission.110 Epitope mapping studies based on immunoblot of generated OspC fragments identified linear epitopes in OspC type A i.e. loop 5 (aa 136-150) and α-helix 5 (aa 168-203) residing mostly within the inter-type variable region of OspC.56 Sequence comparative analyses revealed that both epitopes are highly conserved at the intra-type level but not at the inter-type level. Based from structural analyses to determine spatial location of the epitopes, loop 5 epitope was found to be surface exposed on both the monomeric and dimeric forms of OspC protein.82 Antibodies raised against OspC type A loop 5 are type-specific and exhibits complement-mediated bactericidal activity.111 Linear immunodominant epitopes in other OspC types (B, C, D, H, I, K, N) associated with invasive infection were determined to be mainly localized in α-helix 5 and/or loop 5 of OspC (Figure 3).105, 112

Truncation Earlier studies in fragment vaccines based on OspA involved truncation of the speculated arthritogenic epitope (aa 84-133) to eliminate possible side effects of OspA-based vaccines.113-114 The resulting C-terminal fragment spanning residues 133-273 constitutes approximately 55% of full-length OspA. Vaccination of OspA[133-273] with a fusion partner, gluthathione-S-transferase (GST), failed to protect C3H mice from homologous challenge despite having all key epitope residues in the truncated protein.113 Lack of protection was proposed to arise from significant conformational changes induced by the fusion partner to the C-terminal fragment, which then bore altered B-cell epitopes. Generated antibodies from GST-OspA[133-273] immunization did not recognize the native conformation of OspA.113 Nonetheless, changing GST to a simple polyhistidine (6x) peptide prompted a remarked difference in protective capacity. Passive immunization of antisera raised from polyhistidine-OspA[133-273] partially protected C3H mice from Lyme borreliae infection.114 It is apparent from the different immune responses elicited by the fusion proteins that the conformational epitopes in the OspA fragments are unlikely similar. Intriguingly, the presence of the

flexible linker peptide should have prevented conformational changes to the fragmented antigen regardless of the size of the fusion partner. Folding studies on OspA using NMR and differential scanning spectroscopy depicted two distinct cooperative transitions in denaturation mechanism in which the intermediate state, as determined by small angle X-ray scattering, consists of fully folded N-terminal folding unit and a denatured yet compact C-terminal folding unit.115 This suggests that the more stable N-terminal domain may act as a folding template for the C-terminal domain. As inferred from NMR data, the boundary between the two folding units lies within the highly rigid central β-sheet. Disruption of critical interactions in the boundary as result of fragmentation may lead to destabilization of the C-terminal fragment. Studies on structure and folding mechanism steer possible design for a fragmented antigen. In the absence of a resolved crystal structure, previous studies using OspA[133-273] could not have predicted that residue 133 was located within β-strand 10. To preserve the regular secondary structure on the fragmented end, a lipidated C-terminal fragment consisting of aa 130-273 was designed in a more recent study (Figure 4). However, OspA[130-273] exhibits reduced conformational stability and low vaccine efficacy despite having similar LA-2 equivalent antibodies, suggesting that this antigen fragment still displays its native tertiary structure.100 Failure of the fragment vaccine to elicit sufficient protective immunity is attributed to its instability during immunization period. Rational stabilization of the C-terminal fragment by specific point mutations (R139M;E160Y;K189M) of partially buried residues to hydrophobic residues restored vaccine efficacy.100 Improved conformational stability was facilitated by removing unfavorable desolvation of buried salt bridges and reinforcing hydrophobic interactions without perturbing the antigenic LA2 epitope. Structural studies by NMR suggested that imposed mutations only induced localized conformational changes.100 This structure-based design of an OspA C-terminal fragment exemplifies an effective method in reducing antigen size while preserving important B-cell conformational epitopes required to block transmission of spirochetes.

Figure 4. Structure-based design of an OspA-based fragment



vaccine. Ribbon diagram rendered in light grey represents the deleted arthritogenic segment in OspA (PDB ID 1OSP). Stabilizing mutations (shown in green) imposed on truncated OspA (shown in red) are R139M, E160Y, and K189M. Black arrow indicates point of truncation in earlier studies. Schematic representation was made using PyMOL.

Mutation Strategic substitution of residues in the predicted autoreactive T cell epitope represents a viable approach in tackling the problem regarding probable cross-reaction between hLFA-1α and OspA. The three pathogenic genospecies of Lyme borreliae display sequence variation within this putative T-cell epitope, OspA160-168 for B. garinii and OspA165-173 for B. afzelii and B. burgdorferi. DR4-binding algorithm calculated lower MHCbinding scores for B. afzelii and B. garinii compared to B. burgdorferi.116 This species-specific binding affinity correlates with clinical findings that European strains are fairly nonarthritogenic. A mutant Bb OspA (FTK-OspA) was then constructed by replacing residues at positions 165, 166,and 170 with the corresponding residues in B. afzelii i.e. Y165F:V166T:T170K.117 Calculated binding score for FTKOspA using DR4 algorithm was lower compared to wild-type (WT) OspA and Bg OspA160-168. In silico binding result for FTK-OspA is in good agreement with previous mutagenesis studies.118-119 Despite modifications in the T-cell epitope, comparable antibody responses and similar level of LA-2 equivalent antibodies were observed for mutant and wild-type OspA.117 Homologous challenge indicates indistinguishable protective efficacy between FTK- and WT- OspA. It can be inferred that the important LA-2 epitope remains essentially unaltered after mutation of the epitope residues, which are located in β-strand 13. More importantly, human or mouse DR4-restricted, WT-OspA specific T-cells were not stimulated by mutant Bb OspA, suggesting successful elimination of DR4restricted T cell response.117 This computationally aided design of a mutant subunit vaccine enables removal of a potential cross-reactive T-cell epitope without affecting protective efficacy. A mutation strategy utilized in the design of a Lyme disease vaccine based on another surface-exposed lipoprotein, CspZ

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(also known as CRASP-2, BbCRASP-2, or BBH06 in the literature), allows elicitation of protective antibodies by abrogating its binding to host-derived complement downregulators factor H (FH) and factor-H like protein 1 (FHL-1).22 Initial studies using PepSpot reported four linear FH/FH-1 binding regions in CspZ, i.e. aa 34-52 (region 1), aa 70-88 (region 2), aa 127-145 (region 3), aa 202-226 (region 4).120 Alanine substitutions of hydrophobic and charged residues in regions 2 and 4 resulted to significant reduction in CspZ binding capacity, suggesting that both hydrophobic and electrostatic interactions mediate binding to complement regulators.121 Fourteen critical residues identified by alanine scanning approach were mapped to the crystal structure of CspZ. Structure elucidation revealed that impairment of ligand binding by alanine mutation of Tyr 207 and some hydrophilic residues may be due to destabilization of a local fold resulting from disruption of hydrogen bonds.122 Mutation of Tyr211, which is spatially located near the binding surface, led to complete elimination of ligand binding ability of CspZ.121-122 Overall, mutagenesis and structural characterization studies suggest a structurally sensitive binding site. Considering the functional role of CspZ in inactivation of host complement attack as an immune escape mechanism, it holds great potential as a vaccine candidate. However, immunization with wild-type CspZ could not protect mice from infection or prevent spirochete dissemination.123-124 It was speculated in a study that FH/FHL-1 binding renders potential protective epitopes in CspZ inaccessible and thus eliminating complement binding ability will potentially evoke higher level of bactericidal antibodies.22 Indeed, a modified CspZ-based vaccine comprising of a mutant CspZ (CspZ-Y211A/Y207A) conjugated to virus-like particles (VLP) produced more robust borreliacidal antibodies in comparison to the wild-type counterpart of the vaccine. Also, modified VLP-CspZ prevented spirochete colonization and development of Lyme arthritis.22 Inhibiting ligand binding for better accessibility to neutralizing epitopes enhances the immunogenic potential of CspZ. Potential applicability of this strategy to binding proteins with reduced immunogenicity opens a new avenue in antigen development.

Figure 5. Design strategy for the construction of a bivalent chimeric OspA-based vaccine. OspA serotypes 1 and 2 are rendered in colors green and red, respectively. Reprinted with permission from Livey, et al.53 Copyright© 2011, Oxford University Press.


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Fusions Baxter developed a vaccine that comprised three recombinant OspA antigens, each containing protective epitopes from two different OspA serotypes, i.e., OspA serotypes 1 and 2 (B. burgdorferi sensu stricto and B. afzelii), 5 and 3 (both B. garinii), and 6 and 4 (B. garinii and B. bavariensis). The multivalent vaccine was designed to protect against all major disease-causing Borrelia species in the United States (OspA-1), Europe (OspA-1 to -6), and potentially globally.54-55 The risk of T-cell cross-reactivity was addressed by replacing the putative human cross-reactive OspA-1 epitope with the corresponding OspA-2 sequence.54-55 The problem of type specificity of OspA protective immunity can be circumvented by inclusion of several antigenic variants of OspA into one composite molecule. Effectiveness of this approach, however, critically depends on prior knowledge of the location of protective epitopes of OspA and careful combination of the antigenic portions in a single molecule such that conformational epitopes in OspA are preserved. Rational design of a novel antigen construct (rOspA1/2) (Figure 5) consisting of the proximal portion of B. burgdorferi B31 (serotype 1) and distal portion of B. afzelii PKo (serotype 2) demonstrated that a single composite antigen could afford dual protection without eliciting any adventitious effect.53 In the design of this composite antigen construct, a stretch of 25-aa encompassing the putative hLFA1 epitope from serotype 1 was replaced with the corresponding residues in serotype 2 to avoid risk of inducing antibioticrefractory Lyme arthritis. The important LA-2 epitope is a blend of the two serotypes in which the first of the three surfaceexposed loops of the epitope was derived from serotype 1 and the remaining two loops from serotype 2. A double low-dose (0.03 µg) immunization with rOspA1/2 adjuvanted with

aluminum hydroxide was sufficient to provide protection, proving that this fusion strategy can potentially lower the amount of antigen required to elicit protection against the two targeted serotypes.53 Protective immunity of rOspA1/2 suggests that functionality of induced antibodies is not affected by amalgamation and that the antibodies raised from this chimeric vaccinogen can still recognize the native epitopes in each OspA serotype. Extension of this vaccine construct to other Lyme borreliae species was made to generate a cocktail vaccine consisting of three bivalent antigens i.e. OspA-1 (B. burgdorferi) and OspA-2 (B. afzelii), OspA-3 and 5 (both B. garinii), and OspA-4 (B. bavariensis) and OspA-6 (B. garinii). Safety, tolerability, and immunogenicity of this novel multivalent vaccine was evaluated in a Phase I/II clinical trial.5455 However, development of this vaccine has been discontinued. Another hexavalent vaccine (VLA15) developed by Valneva was constructed by forming three heterodimers (OspA1 and 2, OspA4 and 3, and OspA5 and 6) in which each dimer comprises of two OspA C-terminal monomeric fragments connected by a linker sequence based on the two loop regions in B. burgdorferi B31 (Figure 6).56-57 The Valneva approach of using fused OspA fragments allow the C-terminal epitopes, such as LA-2, in each truncated OspA monomer to conserve their original sequence and hence preserve the native antigenic structure. This differs from Baxter vaccine in which the LA-2 epitope in the composite antigen construct consists of fragments from two Lyme borreliae genospecies.53 To compensate for the stability loss resulting from truncation and to retain integrity of the heterodimer structure, a disulfide bridge was introduced in each monomeric constituents in VLA15 by point mutations at positions equivalent to residues 182 and 269 of serotype 2 (labeled “B” in Figure 6).56 Potential risk of inducing chronic Lyme arthritis was also eliminated by replacing the residues in the cross-reactive T-cell



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Figure 6. Generation of a representative heterodimer construct in VLA15 vaccine. Five pre-selected positions of stabilizing disulfide bridges (A, B, C, D, E) were screened out based primarily on purification efficiency and thermal stability. The resulting heterodimer consists of two “B”-stabilized monomers connected by a linker sequence derived from OspA serotype 1 (L1: aa 43-53 with modifications [D53S], L2: aa 65-74). Adapted from reference [56].

Figure 7. OspC homodimerization via N-terminal disulfide linkage. Monomeric analogue (M-OspC) contains a site-directed mutation on position 19 (C19G). Reprinted in part with permission from Edmondson et al.107 Copyright © 2017, American Society for Microbiology.

epitope of B. burdroferi with those in the non-arthritogenic B. afzelii. Moreover, a lipid moiety in each dimer construct at the N-terminal end was added to increase immunogenicity. Importance of the lipid attachment in OspA immunogenicity have been suggested in a study.125 Further refinements via surface electrostatic stabilization of serotype 3 was performed to improve production yield and immunogenicity. In this improved version of VLA15, approximately one-third of Nterminal part of serotype 3 was exchanged with the corresponding residues in OspA B. valaisiana.58 Simultaneous immunization of the three heterodimers (1:1:1 ratio) did not prompt any antagonistic or synergistic effects and produced functional antibodies that recognize all six OspA serotypes.56 Furthermore, immunogenicity and protection capacity of VLA15 is independent of the order of the monomeric fragments in the heterodimer.58 Broad protection of this multivalent vaccine was confirmed in a challenge study using all targeted OspA serotypes.57 VLA15 protective efficacy can be attained at nanogram-level antigen dose.56-57 This proves that this structure-based approach can be an invaluable strategy to generate a highly efficacious OspA-based with broad protection against Lyme disease using reduced amount of vaccinogen. A fusion technique based on disulfide linkage of truncated OspC via the N-terminal cysteine residue (Cys19) improved antigenicity and conferred immunoprotection against infection.107, 126 Truncation of the intrinsic lipidation site in recombinant OspC dramatically improves purification yield but at the expense of lower immunogenicity. Initial OspC deletion construct omits the N-terminal tripalmitoyl-S-glyceryl-cysteine moiety, which spans the first 19 amino acid residues in OspC.102-104, 127 An OspC deletion variant (Figure 7), which retains Cys19 but not the signal sequence aa 1-18, allowed spontaneous formation of an artificial homodimer by covalent linkage of the N-terminal end. Helical content and thermal stability of the dimeric, OspC(Δ1-18), and monomeric,

OspC(Δ1-18, C19G), constructs were comparable yet the former elicited higher antibody response.126 The homodimer constructs (strains B31 and 297) also protected mice against infection by both needle inoculation and tick transmission of the cognate strain whereas the mutant monomeric construct was only partially protective even at high doses.107 However, dimeric OspC(Δ1-18) from N40 strain (E type) exhibited the same protective efficacy as the corresponding monomer construct and conveyed partial protection against heterologous challenge. Nonetheless, it is suggested that the protective effect of OspC homodimerization is due to the stimulation of antibodies that recognize protective epitopes in the dimer interface.107 On the contrary, a study involving an interdimeric disulfide bridge between centrally located Cys130 residues presented insignificant difference in immunogenicity between OspC(Δ1-19) and OspC(Δ1-19, C130A).128

Chimeritope Strain-dependence of OspC protective immunity due to high polymorphism and antigenic hypervariability demands for a multivalent vaccine that conveys wide-ranging protection. In an effort to generate a polyvalent OspC-based vaccine, Baxter created a cocktail vaccine consisting of 14 different lipidated OspC variants.129 However, such vaccine formulation stimulated undesirable reactogenicity possibly due to the large amount of antigen used to induce adequate immune response. To overcome drawbacks of using traditional OspC-based cocktail vaccines, chimeric epitope-based protein vaccine (chimeritope) provides a strategical way to construct a multivalent vaccine by joining multiple B-cell epitopes from several OspC variants in a single construct.101 This effectively diminishes the amount of antigen necessary for broad protection and eliminates irrelevant epitopes that may possibly cause misdirection of antibody response. As such, design of chimeritope vaccines strongly depends on proper identification of protective epitopes in each OspC type. A tetravalent chimeric vaccine against OspC types A, B, K, D (Figure 8A) was created based on the knowledge of linear immunodominant epitopes in OspC. The vaccine construct consists of type A loop 5 epitope and α-helix 5 epitopes of types B, K, D linked by an unstructured, protease-resistant linker sequence.112 The structure of this chimeric vaccine is predicted to be mainly αhelical with high stability index. Generated anti-ABKD antibodies displayed complement-dependent bactericidal activity against all targeted OspC types. Despite weak immunogenicity observed for type D, antibodies showed apparent surface labeling in immunofluorescence assay and displayed comparable bactericidal activity.112 Study on the immunogenicity of other tetravalent chimeric vaccine variants with different epitope organization was further conducted.130 To incorporate other OspC types, an octavalent chimeritope vaccine (OspC-A8.1) (Figure 8B) was generated by linking two tetravalent constructs, one comprising the epitopecontaining regions for types A, B, K, D and the other for types E, N, I, C.105 A highly conserved C-terminal motif was also appended at the C-terminal end despite its inconsistency with


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the observed strain-specific protection of OspC. Immunization with OspC-A8.1 induced type-specific antibodies that recognize OspC on the spirochete surface. Low antibody titers observed for type D and C-terminal motif could be due to possible occlusion of the epitopes by the chimeric construct

itself.105 This epitope-based approach led to the development of the canine Lyme disease vaccine, VANGUARD® crLYME (ZoetisUS), which is a combination of a heptavalent chimeric OspC protein and recombinant OspA protein.65 Recently identified linear epitope in OspA

Figure 8. Protein sequences of tetravalent (A) and octavalent (B) OspC-based chimeric vaccines. The constituent epitope-containing regions of the multivalent vaccines are represented by different shading whereas linker sequences are designated by numbers. Restriction sites are also indicated in the octavalent construct. Reprinted in part with permission for (A) from Earnhart et al.112 Copyright© 2007, Elsevier; and for (B) from Earnhart and Marconi105 Copyright© 2007, Landes Bioscience.

can in principle be incorporated to an OspC chimeritope to produce a multi-antigen chimeritope that could elicit protection through a possible synergistic mechanism.101

Conjugation and Particle-Based Approaches Immunogenicity of Lyme disease vaccinogens can potentially be augmented by conjugating to particulate nanoscaffolds that enhance preferential uptake and processing by antigen-presenting cells. Different vaccine delivery vesicles including VLP and liposomes have been pursued for Lyme disease vaccine development. The regular surface molecule organization in VLP and the versatility and biocompatibility of liposomes make them attractive vaccine platforms for the delivery of antigens. Surface functionalization of nanoparticle platforms allows presentation of antigens in their native

conformation and exposure of an organized array of antigenic epitopes. In a study using bacterial outer membrane vesicles (OMV), surface display of OspA on meningococcal OMV by fusion to factor H binding protein is necessary to induce higher antibody response.131 An antigen delivery system involving OspA-expressing Escherichia coli was employed for oral administration.132 A reservoir-targeted bait vaccine, which intends to disrupt the enzootic cycle of Lyme disease pathogens, induced durable neutralizing antibody response in white-footed mice.133 VLPs and capsid-like particles (CLPs), which are selfassembling non-infectious mimics of virus particles, are highly immunogenic due to highly structured, repetitive arrays of epitopes.134 In the VLP-CspZ construct, bacteriophage Qβderived VLPs (icosahedral geometry triangulation number T=3) were decorated with CspZ (wild type or mutant) via chemical



conjugation using SMPH (succinimidyl-6-[(βmaleimidopropionamido) hexanoate]). Surface display of CspZ on bacteriophage-derived VLPs evoked significantly higher level of bactericidal antibodies compared to the non-particulate recombinant form.22 Other studies employed genetic fusion technology to associate OspA to hepatitis B virus (HBV)-derived CLPs. The nucleocapsid of HBV consists of 120 (T=4, diameter ~34 nm) or 90 (T=3, diameter ~30 nm)135 core protein dimers, which feature a protruding four-helix bundle formed by the helical hairpin (α-helices 3 and 4) from each monomer unit.136 The surface-exposed loop connecting the two helices contains the immunodominant c/e1 epitope (aa 74-84), which prompts 90% of anti-HBV response.137 Insertion of whole antigen in the c/e1 loop without disrupting particle assembly or antigen conformation is hindered by geometric constraints imposed by the core protein topology and thus necessitates close juxtaposition of the N- and C- termini. The unusual elongated structure of OspA poses a substantial challenge as the termini are remotely positioned relative to the insertion sites. The use of long connecting linkers (10 and 22 aa on the N and C proximal side) facilitated insertion of OspA into CLPs but ensued formation of irregular multimeric nanoparticles.138 Despite this undesirable structural heterogeneity, active and passive immunization of CLP-OspA protected mice from infection at a level comparable to lipidated recombinant OspA. Close proximity of the termini in OspC, on the other hand, permits formation of regular OspC-CLPs without the need for extended linkers.139 Structural limitations toward insertion to the nanocarrier were overcame with the advent of Splitcore, a modified CLP platform with a split in the internal sequence (between Pro79 and Ala80) within the c/e1 loop using artificial start and stop codons.140 The Splitcore approach generates separate coreN and coreC fragments, which serve as sterically unrestrained attachment sites, while upholding CLP formation. This concept is analogous to previous studies involving a cleavable TEV protease site in the c/e1 loop.141 With the versatile Splitcore platform, fusion of OspA to either of the acceptor sites formed regular CLPs, which induced OspAspecific antibodies higher than non-particulate lipidated OspA.140 However, coreC-OspA, in which the point of attachment is at the C-terminal end of OspA, failed to confer protection as it has poor accessibility to the important Cterminal epitopes such as LA-2. The Splitcore platform was also utilized in the construction of a combination vaccine consisting of the tick proteins, Salp15, Salp15 Iric-1, and tHRF.73 Other nanoparticle-based approaches to Lyme disease vaccine development involve non-covalent conjugation of OspC to liposomal surface via metallochelation using polyhistidine tag as anchor. Incorporating nickel-chelating lipid, DOGS-NTA-Ni [1,2-dioleoyl-sn-glycero-3-{[N(5amino-1-carboxypentyl)iminodiacetic acid]succinyl}(nickel salt)], into the nanoliposome platform enable facile surface functionalization that is achieved by simple incubation of the non-lipidated OspC with the metallochelating liposomes. The proteoliposomes, which contains novel non-pyrogenic lipophilized derivative of muramyl dipeptide, induced higher anti-OspC antibody isotypes IgG2a and IgG2b compared to alum.142 Further study showed that levels of OspC-specific antibody response vary with the position of the histidine tag in spite of the structural similarities between the N- and C-terminal

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histidine-tagged OspC proteins.143 This difference in immunogenicity arose from possible occlusion of the Cterminal epitopes in the OspC variant with the histidine-tag positioned at the C-terminus. A metallochelating liposome platform containing cobalt porphyrin-phospholipid offers an alternative non-covalent approach to surface functionalization using polyhistidine tags. Since porphyrin moieties, which harbor the chelating metal, are inaccessible from the aqueous milieu and lie within the hydrophobic bilayer, liposomal binding is stable under high concentrations of imidazole and at 50% (v/v) serum for 24 hr, 37 °C.144 This is significant since NTA-Ni lipids have been shown to be unstable in physiological conditions.144-146 Adjuvanticity of this nanoliposomal platform has been demonstrated in a recent study using malaria transmission-blocking vaccine antigens.147

CONCLUDING REMARKS Since the withdrawal of LYMErix nearly 20 years ago, the need for an effective human Lyme disease vaccine has never been higher. The withdrawal of this controversial but scientifically-sound recombinant OspA-based vaccine, along with a relatively limited Lyme disease market size, complicates vaccine development. While most vaccine strategies involve OspA and OspC, several promising new antigens are gaining attention. Antigen engineering strategies such as truncations, mutations, and fusion have been employed for elicitation of protective antibodies against multiple Borrelia species and strains, and elimination of the putative autoreactive OspA Tcell epitope. Novel subunit vaccines have been validated in human clinical trials led by Baxter and Valneva. Given this precedent and ongoing research efforts, development of an efficacious vaccine against Lyme disease will likely benefit from advanced antigen engineering approaches. Valneva’s VLA15 vaccine, which is the only Lyme disease vaccine presently undergoing ongoing human clinical testing, showcases this paradigm. Translating commercially-available and efficacious veterinary vaccines compatible for human use is also an option to accelerate clinical development of human Lyme disease vaccines while de-risking efficacy concerns. Identification of new pathogenic variants of Lyme borreliae in United States and Europe other than the three well-known pathogenic species is motivation for a broader protection range for Lyme disease vaccines. Given the antigenic heterogeneity of vaccinogens and increasing number of Lyme disease-causing pathogens, there is the apparent need for other multivalent approaches that substantially reduce antigen amount without compromising immunogenicity and protective efficacy. Development of a clinical paradigm for new Lyme disease vaccines entails finding an optimum formulation, administration route, and vaccination regimen that maximizes the duration and magnitude of the antibody response, especially for transmission blocking vaccines. Evaluation of the safety profile and immune autoreactogenicity of vaccine formulations is also vital considering past experience with LYMErix®.

AUTHOR INFORMATION Corresponding Author


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* [email protected]

ACKNOWLEDGMENTS This work was supported by the National Institutes of Health (R21AI122964 and DP5OD017898).

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Antigen Engineering Approaches for Lyme Disease Vaccines

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