Design and development of a novel vaccine for protection against Lyme borreliosis

Abstract

There is currently no Lyme borreliosis vaccine available for humans, although it has been shown that the disease can be prevented by immunization with an OspA-based vaccine (LYMErix). Outer surface protein A (OspA) is one of the dominant antigens expressed by the spirochetes when present in a tick. The Borrelia species causing Lyme borreliosis in Europe express different OspA serotypes on their surface, B. burgdorferi (serotype 1), B. afzelii (serotype 2), B. garinii (serotypes, 3, 5 and 6) and B. bavariensis (serotype 4), while only B. burgdorferi is present in the US. In order to target all these pathogenic Borrelia species, we have designed a multivalent OspA-based vaccine. The vaccine includes three proteins, each containing the C-terminal half of two OspA serotypes linked to form a heterodimer. In order to stabilize the C-terminal fragment and thus preserve important structural epitopes at physiological temperature, disulfide bonds were introduced. The immunogenicity was increased by introduction of a lipidation signal which ensures the addition of an N-terminal lipid moiety. Three immunizations with 3.0 µg adjuvanted vaccine protected mice from a challenge with spirochetes expressing either OspA serotype 1, 2 or 5. Mice were protected against both challenge with infected ticks and in vitro grown spirochetes. Immunological analyses (ELISA, surface binding and growth inhibition) indicated that the vaccine can provide protection against the majority of Borrelia species pathogenic for humans. This article presents the approach which allows for the generation of a hexavalent vaccine that can potentially protect against a broad range of globally distributed Borrelia species causing Lyme borreliosis.

Figure 1. Localization of the stabilizing disulfide bonds and schematic representation of the heterodimers.

Left panel: Part of the OspA serotype 1 crystal structure showing the monomer protein (Protein Data Bank accession 1OSP [34]). Locations of the five modifications: “A” (aa 141∶ 241), “B” (aa 182∶ 269), “C” (aa 244∶ 259), “D” (aa 165∶ 265) and “E” (aa 182∶ 272) are indicated as dotted lines between the β-carbon atoms of the corresponding residues. Right panel: Schematic illustration of two stabilized monomers joined with a linker sequence to form one heterodimer. The α-helix at the C-terminal end of the first monomer is joined with the β-sheet at the N-terminal domain of the second monomer using a linker sequence derived from B. burgdorferi OspA serotype 1 (L1; aa 43–53 with modification [D53S] and L2; aa 65–74). The heterodimer is expressed with a posttranslationally attached N-terminal lipid moiety, indicated in line drawing. The positions of the disulfide bonds for stabilization “B” in both OspA monomer subunits are indicated in magenta. Abbreviations: N-terminal (N-term.), C-terminal (C-term.).

Figure 2. Protein expression and lipid-phase separation for OspA heterodimers.

Each heterodimer was expressed in two different versions having the respective monomers stabilized with the “A” or the “B” modifications; Lip-D1A2A-His, Lip-D1B2B-His, Lip-D4A3A-His, Lip-D4B3B-His, Lip-D5A6A-His and Lip-D5B6B-His. The expression levels of all six heterodimers were similar when the crude lysates were analyzed. However, heterodimers using stabilization “B” were recovered in higher amounts in the lipid-phase after phase separation, indicating more efficient lipidation and higher solubility. Protein bands representing the heterodimers are indicated with arrows. Abbreviations: Crude lysate (C), Lipid-phase (L).

Figure 3. Immunogenicity of OspA heterodimers.

(A) The effect of adjuvant on the immune response was evaluated. The three heterodimers (Lip-D1B2B-His, Lip-D4B3B-His and Lip-D5B6B-His) were tested individually for immunogenicity (5.0 µg) when formulated with or without adjuvant (0.15% aluminum hydroxide). IgG titers were determined by ELISA using the respective heterodimer as coating antigen. The half-max geometric mean titers (GMT) with 95% confidence intervals (CI) are shown. (B) The serotype specific immune response of the LB-vaccine was studied by immunizing mice with 3.0 µg LB-vaccine (Lip-D1B2B, Lip-D4B3B and Lip-D5B6B in a 1∶1∶1 ratio), or 1.0 µg of the heterodimers or the homologous lipidated full-length OspA, with adjuvant. IgG titers were determined by ELISA using the monomer of the corresponding serotype (ST1–6) as coating antigen. The half-max geometric mean titers (GMT) with 95% CI are shown.