February 2011- Clinical Infectious Diseases
A New Approach to a Lyme Disease Vaccine
- Ian Livey1,
- Maria O'Rourke1,
- Andreas Traweger1,
- Helga Savidis-Dacho1,
- Brian A. Crowe1,
- P. Noel Barrett1,
- Xiaohua Yang3,
- John J. Dunn2, and
- Benjamin J. Luft3
- 1Vaccines Research and Development, Baxter Innovations GmbH, Biomedical Research Center, Orth an der Donau, Austria
- 2Biology Department, Brookhaven National Laboratory, Upton
- 3Department of Medicine, State University of New York at Stony Brook, Stony Brook, New York
- Correspondence: Ian Livey, PhD, Baxter Innovations GmbH, Biomedical Research Center, Uferstrasse 15, A-2304 Orth an der Donau, Austria (firstname.lastname@example.org).
A single recombinant outer surface protein A (OspA) antigen designed to contain protective elements from 2 different OspA serotypes (1 and 2) is able to induce antibody responses that protect mice against infection with either Borrelia burgdorferi sensu stricto (OspA serotype-1) or Borreliaafzelii (OspA serotype-2). Protection against infection with B burgdorferi ss strain ZS7 was demonstrated in a needle-challenge model. Protection against B. afzelii species was shown in a tick-challenge model using feral ticks. In both models, as little as .03 μg of antigen, when administered in a 2-dose immunization schedule with aluminum hydroxide as adjuvant, was sufficient to provide complete protection against the species targeted. This proof of principle study proves that knowledge of protective epitopes can be used for the rational design of effective, genetically modified vaccines requiring fewer OspA antigens and suggests that this approach may facilitate the development of an OspA vaccine for global use.
Lyme disease, the most common tick-borne human disease in the Northern Hemisphere, is caused by bacteria belonging to the Borreliaburgdorferi sensu lato (s.l.) complex. Although there are at least 13 named species in this group, most human disease is caused by 3 species:B. burgdorferi sensu stricto (s.s.), B. afzelii, and B. garinii. All 3 species are found in Europe, but only 2 of these species (B. afzelii and B. garinii) are present in Asia and only B burgdorferi ss is found in the United States. Clinical trials in the United States [1, 2] showed that Lyme disease could be prevented by vaccination with OspA, a major surface antigen encoded by all B. burgdorferi s.l. species. However, OspA is antigenically heterogeneous and at least 7 OspA serotypes are associated with the 3 B burgdorferi sl species found in Europe  with a clear association between OspA serotype and species; B. burgdorferi s.s. serotype-1, B. afzeliiserotype-2, and B. garinii serotypes 3 to 7. Since OspA protective immunity is largely type-specific, a candidate vaccine designed to confer broad protection against Lyme disease globally must contain several antigenic variants of OspA [4–6].
It was the aim of the current study to develop a novel OspA lipoprotein that is stable, safe, and immunogenic, as well as a molecule that is able to generate antibodies with broader protective specificities than the OspA antigens found in nature. Rational design of effective, genetically modified OspA vaccines requires knowledge of protective epitopes such as that defined by the monoclonal antibody LA-2, an antibody that was used to predict protective immune responses in clinical trials [1, 7, 8]. X-ray crystallography and nuclear magnetic resonance analyses have mapped the position of the LA-2 epitope to 3 surface-exposed loops at the carboxy terminal domain of OspA [9–11]. This information was used to design a recombinant OspA molecule (rOspA 1/2) comprising the proximal portion of an OspA serotype-1 molecule with the distal portion of an OspA serotype-2 molecule with the aim of combining the protective properties of both of the parent polypeptides in a single molecule (Figure 1). A putative, arthritogenic T-cell epitope (OspA aa165–173) mimicking human leukocyte function-associated antigen-1 (hLFA-1) has been identified for the B. burgdorferi s.s. serotype-1 OspA . This epitope (YVLEGTLTA) is absent from the new OspA molecule (rOspA 1/2), since the sequence in this region of the serotype-1 OspA antigen was substituted with a corresponding (non–hLFA-1) sequence from a serotype-2 OspA molecule. It was predicted that vaccination with this novel antigen would be able to afford protection against infection with B. burgdorferi s.s. andB. afzelii species of Borrelia, which express OspA serotype-1 and OspA serotype-2 molecules, respectively.
The proximal portion of a serotype-1 OspA sequence (green color) is fused to the distal portion of a serotype-2 OspA sequence (red color). The resultant composite molecule contains the first of the 3 surface-exposed loops recognized by protective mAb LA-2. The second and third loops recognized by LA-2 are replaced by equivalent sequences from the serotype-2 molecule. The hLFA-1 epitope in beta sheet 13 from the serotype-1 sequence was replaced with a 25 amino acid sequence (Beta sheet 13, 14 and partial of 15) derived from the serotype-2 sequence.
Design and Construction of rOspA 1/2
To eliminate the risk of introducing adventitious agents, complementary overlapping synthetic oligonucleotides were used to generate DNA fragments that were ligated together and cloned into vector pET30a and the sequence was verified. This approach also enabled codon usage to be optimized for the Escherichia coli host HMS174 (DE3) used to express theospA gene. The novel gene is based on the proximal portion of a serotype-1 OspA sequence (aa 29 to 218, Strain B31; GenBank accession number X14407) fused to the distal portion of a serotype-2 sequence (aa 219 to 273, Strain PKo; accession number S48322). The 25 amino acid fragment from B. burgdorferi s.s. strain B31 (aa 164 to 188-GYVLEGTLTAEKTTLVVKEGTVTLS) was replaced with sequence from B. afzelii strain PKo (aa 164 to 188 NFTLEGKVANDKVTLEVKEGTVTLS). The N-terminal sequence including the leader sequence and the first 11 amino acids were derived from OspB (Strain B31; GenBank accession numberX74810) in order to optimize lipidated protein expression . Other specific amino acid changes were made to improve the immunogenicity and conformational stability of the rOspA 1/2 molecule [11, 13].
The ability of a single rOspA 1/2 to prevent infection with 2 species ofBorrelia, which express different OspA antigens, was assessed in C3H/HeJ mice immunized subcutaneously (days 0 and 28) with purified recombinant OspA antigen (0.1 or .03 μg doses) formulated with .2% (w/v) aluminum hydroxide as adjuvant. Mice were challenged 2 weeks after the booster immunization, either by intradermal injection (needle challenge; 7 x 104 cells) or by the natural route of infection (tick challenge). For the latter experiments, 8 nymphal ticks were applied per mouse and allowed to feed for up to 5 days. The nymphs were collected in the vicinity of Budweis (Czech Republic), an area endemic for Lyme disease. The majority of these ticks were infected with B. afzelii as determined by testing unfed ticks by polymerase chain reaction (PCR) (data not shown). The infectious status of the mice was determined 4 weeks later as outlined in the Diagnostic Procedures section. In the tick-challenge experiments, the presence of Borrelia was confirmed by culture (urinary bladder) and by detection of Borrelia DNA by real-time PCR (heart). Animal experiments were conducted in accordance with Austrian laws on animal experimentation and international guidelines (AAALAC and OLAW) and were reviewed by the Institutional Animal Care and Use Committee and approved by the Austrian regulatory authorities.
The antibody response (μg IgG/mL) to rOspA 1/2 antigen was determined by ELISA using rOspA 1/2 as the coating antigen and an OspA specific monoclonal antibody (prepared in house) with a defined IgG content as a standard.
For the needle-challenge experiments, the presence of antibodies to a conserved epitope in the surface-exposed lipoprotein VlsE protein (C6 ELISA; coated plates from Immunetics® C6 Lyme ELISA™) or to Borreliaantigens other than the OspA immunogen (Western blotting) was used to diagnose infection. Western blotting used a cell lysate prepared from B. burgdorferi s.s. strain ZS7 as this was the challenge organism. Animals were deemed infected if they were positive in both assays.
For the tick-challenge experiments, the C6 ELISA and Western blotting were also done. However, Western blotting used lysates from B. burgdorferi s.s. ZS7, B. afzelii ACA1, and B. garinii KL11 since the identity of the infecting organism was unknown. Animals were considered to have undergone seroconversion only if both assays were positive. In addition,Borrelia infection was assessed by culture from the urinary bladder and by detection of B. burgdorferi s.l. nucleic acids in genomic DNA extracted from heart tissue using a real-time PCR assay targeting the 5′-region ofospA  and a 16S rRNA gene-based assay. For the latter assay, the forward primer (5′-GGATATAGTTAGAGATAATTATTCCCCGTTTG) and the reverse primer (5′-CATTACATGCTGGTAACAGATAACAAGG) amplified a 139 base pair fragment which was detected with a TaqMan® probe (5′ -6FAM- ACAGGTGCTGCATGGT-MGB; Applied Biosystems). Animals were scored as PCR-positive only if a PCR product was detected with both assays. Overall, to judge an animal as infected, mice needed to be positive either by culture, PCR, or serology.
Characterization of Infecting Borrelia
Where possible, the infecting organism was cultured and the OspA sequence and deduced amino acid sequence was determined for OspA residues 38–262 (B. afzelii VS461, GenBank accession number Z29087). This information was compared with OspA reference sequences so that the OspA type and Borrelia species could be inferred. For species which express a single OspA serotype, the OspA sequence for the type strain for the species was chosen as a reference, for example, B. afzelii VS461 orBorrelia valaisiana VS116 (GenBank accession number Z29087;AF095940). As B. garinii has multiple OspA types, OspA sequences forospA genotypes 3–7  were used, namely, strains PBr, PTrob, WABSou, TlsI and T25 (GenBank accession numbers X80256, X80186, X85441,X85440 and X80254), respectively.
For OspA-based typing using real-time PCR, serotype specific assays were developed based on the analysis of previously published nucleotide sequences encoding all known OspA serotypes. To this end, specific primer and probe sets were designed using Primer Express software, version 3.0 (Applied Biosystems), for example, for the detection of OspA serotype-2 strains (fwd-O2: 5’-CAATGAAAAAGGTGAATTGTCTGC; rev-O2: 5’-CATTTCTGTATATTCAAGTTTGGTTCCA; Probe-O2: 5’-NED-AAA ACCATGACAAGAGAAA - MGB) and OspA-type 7 strains (fwd-O7: 5’- CGCCGTCAGCAGTTAGAGTTC; rev-O7: 5’-ATGGATCCGGAAAAGCTAAAGAA; Probe-O7: 5’-NED-TTCAAGAGTAAGGCTTT-MGB).
All assays were run on an ABI Prism 7900HT sequence detection unit using universal cycling conditions.
Prevention of Borrelia burgdorferi ss (OspA serotype-1) Infection by Immunization with rOspA 1/2
All of the mice immunized with low doses of 2 different lots of the rOspA 1/2 antigen developed IgG antibodies specific for the immunogen as determined by ELISA (Table 1). No antibodies were detected in the control mice which had been treated with vaccine formulation buffer containing aluminum hydroxide. To assess the ability of this immune response to prevent infection with B. burgdorferi s.s., a species that encodes a serotype-1 OspA, the mice were injected intradermally with 7 x 104 cells of B. burgdorferi s.s. strain ZS7. All of the control mice treated with buffer containing adjuvant showed serological evidence of infection as demonstrated by C6 ELISA and by Western blotting. None of the mice immunized with the rOspA 1/2 antigen became infected and the sera from these mice were negative by both assays (Table 1). As little as .03 μg of the rOspA 1/2 antigen, when formulated with aluminum hydroxide as adjuvant and administered in a 2-dose immunization regimen, conferred 100% protection (P < .0001, Fisher exact 2-tailed test) against a needle challenge with the virulent B. burgdorferi s.s. strain ZS7.
rOspA 1/2 is Immunogenic and Protects Mice against Infection with B. burgdorferi s.s. strain ZS7 in a Needle-Challenge Model
Prevention of Borrelia afzelii (OspA serotype-2) Infection by Immunization with rOspA 1/2
To assess the ability of immunization with the rOspA 1/2 antigen to prevent infection with B. afzelii, a species that encodes a serotype-2 OspA, mice were immunized, in 2 separate experiments, with the same antigen lots and study design as used in the needle-challenge experiment described above. However, in this case, the immunized mice were challenged with feral ticks (nymphs) known to be infected mainly with B. afzelii. The ability of these feral ticks to transmit B. burgdorferi s.l. to mice was confirmed by challenging non-immunized control animals.
Most of the control mice (total 11/14, 79%) became infected (Table 2). All infected control animals were positive for Borrelia DNA by 2 independent real-time PCR assays (16S rRNA and ospA genes). In 10 of 11 cases, it was possible to isolate Borrelia by culture of the urinary bladder. The remaining mouse was positive by serology and PCR. For 9 of the 10 culture isolates OspA sequences were retrieved and all were typed as B. afzelii (>99% OspA sequence identity). Furthermore, all infecting organisms were typed as B. afzelii by PCR analysis of the DNA extracted from the heart using a real-time PCR assay specifically targeting serotype-2 ospA genes. These data confirm that B. afzelii was the principal Borrelia species being transmitted from the infected feral ticks to their mouse host.
Immunization with rOspA 1/2 Protects against Infection with Tick-Transmitted B. afzelii
Few of the mice immunized with rOspA 1/2 (total 3/32, 9%) became infected (Table 2). Of these 3 mice, 1 was infected as determined by all 3 diagnostic criteria (serology, PCR, and culture), and sequence analysis revealed that the infecting organism was B. garinii serotype-6 (>99% OspA sequence identity). The remaining 2 animals deemed infected were positive by only 2 of the 3 criteria. One mouse was positive by serology and PCR. However, the infecting organism could not be retrieved in culture. Nevertheless, this organism could be typed as B. garinii serotype-7 by PCR analysis of the DNA extracted from the heart using a PCR specific for the serotype-7 ospA gene. The third mouse was PCR and culture positive, but serologically negative. The isolate cultured from this mouse was B. valaisiana as determined by sequencing (OspA sequence identity with B. valaisiana strain VS116). Importantly, none of the immunized mice (0/32) became infected with B. afzelii (Table 2). As little as .03 μg of the rOspA 1/2 antigen, when formulated with aluminum hydroxide as adjuvant and administered in a 2-dose immunization regimen, conferred full protection (Table 2) against B. afzelii transmitted by feral ticks.
Human vaccines for the prevention of Lyme disease are currently not available. To date, vector-borne proteins and several outer membrane proteins have been explored as vaccine candidates  of which OspA is the most extensively studied and only spirochetal antigen tested in phase 3 clinical trials. Previous studies have shown that an OspA epitope (LA-2) correlates with protective immunity after vaccination [1, 8] and titers of LA-2 equivalent serum antibody accurately predict protection from tick transmission of infection . This study has shown that it is possible to design and develop a novel OspA molecule with an altered LA-2 epitope. Towards this end, a single recombinant OspA antigen was produced which comprised the proximal portion of a serotype-1 strain (lacking a putative arthritogenic epitope) and the distal portion of a serotype-2 strain. This recombinant OspA antigen (rOspA 1/2) is immunogenic and able to prevent infection with B. burgdorferi s.l. strains expressing either a serotype-1 (B. burgdorferi s.s.) or a serotype-2 (B. afzelii) OspA molecule in a mouse model. As anticipated, the protection afforded by this novel antigen did not extend to other Borrelia species as demonstrated by the antigen's inability to provide protection against infection with B. gariniiand B. valaisiana strains. Nevertheless, this proof of concept study indicates that it should be possible to design other novel OspA molecules which will give protection against the other Borrelia species which cause human disease and facilitate the development of a vaccine for global use. Potentially, a vaccine containing 2 additional recombinant OspA antigens could also provide protection against infection with B. garinii, a major cause of neuroborreliosis. A combination vaccine based on this strategy would require fewer OspA antigens than required if using “native” OspA molecules and potentially reduce the amount of antigen required for vaccination. This study proves that knowledge of protective epitopes can be used for the rational design of effective, genetically modified vaccines.
We thank Marie Luise Zips, Doris Trzil, Daniela Nowak, Helmut Schmidt and Elisabeth Hitter for excellent technical assistance.
Financial support. Baxter Innovations GmbH.
Supplement sponsorship. This article was published as part of a supplement entitled “The Need for a New Lyme Disease Vaccine,” sponsored by Baxter Laboratories, the Centers for Disease Control, Fort Collins, CO, and Stanley Plotkin.
Potential Conflicts of Interest. I.L., M.O’R., A.T., H.S-D., B.A.C., and N.B. are salaried employees of Baxter Innovations GmbH. J.J.D. and B.J.L. hold the patent for certain intellectual property that was used in the development of the vaccine (Reference 13).
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