Protein nanoparticles displaying LYME disease antigens

Abstract

The present invention relates to self-assembling protein nanoparticles displaying an 'outer surface protein A' (OspA) or a mutant 'complement regulator-acquiring surface protein 2' (CspZ) of Borrelia for use in the vaccination against Lyme disease, particularly in combination.

Description

[0001] PCT Application

[0002] Bavarian Nordic A / S

[0003] BN122PCT

[0004] PROTEIN NANOPARTICLES DISPLAYING LYME DISEASE ANTIGENS

[0005] Technical Field

[0006] The present invention relates to the field of recombinant protein-based vaccines. More specifically, the invention relates to self-assembling protein nanoparticles displaying ‘outer surface protein A’ (OspA) or a mutant ‘complement regulator-acquiring surface protein 2’ (CspZ) of Borrelia. The invention particularly relates to the combination of OspA and the mutant CspZ as antigens for use in the vaccination against Lyme disease.

[0007] Background

[0008] Lyme disease, or Lyme borreliosis, is the most common vector borne disease in the northern hemisphere. The causative agents are gram negative bacteria of the genus Borrelia, which are transmitted by Ixodes ticks. Disease causing genospecies include, but are not limited to, Borrelia burgdorferi senso strictu, the primary cause of Lyme disease in the USA, as well as Borrelia afzelii, Borrelia garinii and Borrelia bavariensis which are found across Europe and Asia. Collectively, Lyme disease causing Borrelia are referred to as Borrelia burgdorferi sensu latu.

[0009] Manifestation of Lyme disease varies from individual and species of Borrelia. In the early stages of disease, they often include flu like symptoms like fever, chills, headaches and fatigue as well as the characteristic bull’s eye rash (erythema migrans). Later symptoms occur when the borrelia disseminate from the bite site and move into the blood stream. They can include arthritis, meningitis, facial palsy and heart irregularities. In the early stage, Lyme disease can often be treated successfully by oral antibiotics, while later stage symptoms require high doses of intravenous antibiotic treatment. As the disease phenotype is varied and not all individuals show early-stage symptoms, diagnosis and treatment are problematic. If untreated early on, the Borrelia infection can persist and cause chronic disease with symptoms occurring months or years after the infectious tick bite. These late-stage symptoms include rheumatoid arthritis (Borrelia burgdorferi sensu stricto), persistent infection of the central nervous system (Borrelia garinii) and of the skin (Borrelia afzelii). Antibiotic courses for treatment can be long and are not always effective, particularly in the late stage of infection, but even when all borrelia are successfully eradicated, patients can be left with debilitating symptoms for years afterwards. These characteristics make a compelling case for the development of an efficacious vaccine against Lyme disease. A vaccine targeting the ‘outer surface protein A’ (OspA) of Borrelia burgdorferi sensu stricto (LYMErix) was approved and marketed in the USA by GSK but has been withdrawn from the market since. OspA is expressed by the bacteria when they reside in the tick midgut, where it plays a role in attachment to the basal epithelium and protection from harmful components in the blood meal (Kurokawa et al., 2020). High levels of anti-OspA antibodies in vaccinees, taken up by a feeding tick during the blood meal, can neutralize Borrelia within the tick midgut and thereby completely block transmission to the host. However, as OspA expression is quickly downregulated upon contact with host blood, it is not expressed at significant levels by Borrelia that have successfully been transmitted to the host. Therefore, if antibody levels in the blood meal are not sufficient for complete inactivation of the Borrelia in the tick midgut, anti-OspA antibodies provide no further protection to the vaccinee. This problem is further exacerbated by the fact that OspA as an antigen is not normally encountered by the host immune system, resulting in the absence of any natural boosting of the anti-OspA immune response, most likely necessitating continuous booster immunizations to maintain the high antibody levels required to provide complete transmission blocking activity in the tick midgut.

[0010] Thus, there is still a need for efficacious vaccines against Lyme disease.

[0011] Summary of Invention

[0012] It is an objective of the present invention to provide means and methods useful for inducing vaccinal immune protection against Lyme disease.

[0013] The objective of the present invention is solved by the provision of a combination of two Lyme disease-associated antigens, namely ‘outer surface protein A’ (OspA) and a mutant ‘complement regulator-acquiring surface protein 2’ (CspZ).

[0014] In particular, the invention is defined by the appended claims and by the following aspects and embodiments.

[0015] In a first aspect, the invention provides a fusion protein comprising a Lyme disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle, wherein the Lyme disease-associated antigen is an ‘outer surface protein A’ (OspA) of Borrelia.

[0016] In a second aspect, the invention provides a fusion protein comprising a Lyme disease- associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle, wherein the Lyme disease-associated antigen is a ‘complement regulator-acquiring surface protein 2’ (CspZ) of Borrelia which is a mutant CspZ not binding to complement regulatory protein (CRP) Factor H (FH) of a Borrelia host.

[0017] In another aspect, the invention provides a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising OspA as described herein.

[0018] In yet another aspect, the invention provides a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising mutant CspZ as described herein.

[0019] In a further aspect, the invention provides a combination of a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein and a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein.

[0020] In yet a further aspect, the invention provides a pharmaceutical composition or vaccine comprising a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising OspA as described herein and / or a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising mutant CspZ as described herein, optionally further comprising a pharmaceutically acceptable excipient.

[0021] In yet a further aspect, the invention provides a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising OspA as described herein and / or a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising mutant CspZ as described herein for use in the prevention or treatment of Lyme disease or a condition related to an infection caused by Borrelia.

[0022] In yet a further aspect, the invention provides a method of prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia, comprising administering to a subject a pharmaceutical composition or vaccine comprising a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein and / or a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein.

[0023] In yet a further aspect, the invention provides a method for inducing an immune response to a Lyme-disease associated antigen comprising the step of administering to a subject a pharmaceutical composition or vaccine comprising a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein and / or a selfassembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein. In yet a further aspect, the invention provides a nucleic acid encoding a fusion protein comprising OspA as described herein.

[0024] In yet a further aspect, the invention provides a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein.

[0025] In yet a further aspect, the invention provides a use of a nucleic acid encoding a fusion protein comprising OspA as described herein and / or a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein for the preparation of a pharmaceutical composition or vaccine.

[0026] In yet a further aspect, the invention provides a use of a nucleic acid encoding a fusion protein comprising OspA as described herein and / or a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein for the preparation of a recombinant expression vector.

[0027] In yet a further aspect, the invention provides a recombinant expression vector comprising a nucleic acid encoding a fusion protein comprising OspA as described herein or a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein.

[0028] In yet a further aspect, the invention provides a use of a recombinant expression vector as described herein for the preparation of a self-assembling protein nanoparticle.

[0029] In yet a further aspect, the invention provides a process for preparing a self-assembling protein nanoparticle as described herein comprising the steps of:

[0030] (a) providing a nucleic acid as described herein;

[0031] (b) preparing a recombinant expression vector comprising the nucleic acid provided in step (a);

[0032] (c) transforming expression cells with the recombinant expression vector obtained in step (b) and propagating selected transformants;

[0033] (d) inducing expression of a fusion protein encoded by the nucleic acid provided in step (a);

[0034] (e) harvesting the self-assembling protein nanoparticle.

[0035] In yet a further aspect, the invention provides a combination comprising:

[0036] (i) a first molecule capable of inducing an immune response in a subject against a Lyme disease-associated antigen, or an antigenic part thereof, wherein the Lyme disease-associated antigen is an ‘outer surface protein A’ (OspA) of Borrelia; and (ii) a second molecule capable of inducing an immune response in a subject against a Lyme disease-associated antigen, or an antigenic part thereof, wherein the Lyme disease-associated antigen is a ‘complement regulatoracquiring surface protein 2’ (CspZ) of Borrelia which is a mutant CspZ not binding to complement regulatory protein (CRP) Factor H (FH) of a Borrelia host.

[0037] Brief Description of Drawings / Figures

[0038] Figure 1 illustrates the construct design of DPS-OspA-ST 1 and DPS-CspZ-YA.

[0039] Surface representations of the assembled DPS dodecamer (left, PDB: 1 JTS), monomeric OspA of Borrelia burgdorferi strain B31 (middle, PDB: 2G8C) as well as monomeric CspZ of Borrelia burgdorferi strain B31 (right, PDB: 6ATG) were generated from PDB files using the PyMOL Molecular Graphics System Version 2.5.5. N-termini of the individual proteins are indicated in the structures by asterisks, C-termini are indicated by hash marks. Below, DPS- OspA-ST1 and DPS-CspZ-YA domain composition is depicted as a bar diagram. The checkered box represents sequence elements obtained from the pRSET expression plasmid, containing a 6xHis poly-histidine tag, the T7 gene 10 leader sequence as well as an enterokinase cleavage site. This is followed in both constructs by a DPS subunit, which acts as a multimerization domain for nanoparticle auto-assembly and facilitates assembly of the fusion proteins into homo-dodecamers. The DPS subunit is followed by a glycine-serine linker (striped box) and the respective antigen (OspA-ST1 or CspZ- YA).

[0040] Figure 2 shows a characterization of DPS-OspA-ST 1 and DPS-CspZ-YA.

[0041] (A) Size exclusion chromatogram of DPS-OspA-ST1 (thick line) and DPS-CspZ-YA run over a Superose 6 Increase 10 / 300 GL column. Approximate elution volumes for different molecular weights were determined by running gel filtration standard on the same column; elution volumes of the 670 kDa, 158 kDA, 44 kDa, and 17 kDa size standards are indicated by dashed lines (from left to right). The elution volumes of DPS-OspA-ST 1 and DPS-CspZ-YA, as well as the symmetrical peak shapes indicate successful and uniform assembly of both fusion proteins into dodecameric nanoparticles. (B) Samples of DPS-OspA-ST1 and DPS-CspZ-YA after purification by size exclusion chromatography were subjected to electrophoresis over a 10% SDS polyacrylamide gel, which was stained with Coomassie brilliant blue to visualize the monomeric DPS-OspA-ST1 and DPS-CspZ-YA fusion proteins as well as any proteinaceous contaminants. Figure 3 shows anti-OspA-ST 1 total IgG and anti-CspZ total IgG in serum from vaccinated Balb / c mice.

[0042] Balb / c mice were immunized intramuscularly (IM) on day 0 (prime), day 21 (first boost) and day 42 (second boost) with TBS (control) or a mixture of DPS-OspA-ST1 and DPS-CspZ-YA (and alum as adjuvant). Peripheral blood was withdrawn on days 19, 41 , and 62 after the prime immunization and serum was analyzed to detect serotype (ST) 1 specific anti-OspA IgG as well as anti-CspZ IgG titers by ELISA. Mouse antibodies were detected using anti-mouse IgG (Fc) antibody conjugated to horseradish peroxidase (HRP). (A) Anti-OspA ST1 total IgG titers and (B) anti-CspZ total IgG titers are shown. Arbitrary titers (AU) were calculated via a 4PL-fit curve with an intercept at OD = 0.3. Data shown as mean ± SEM.

[0043] Figure 4 shows the induction of LA-2 competing antibodies in serum of vaccinated Balb / c mice.

[0044] Balb / c mice were immunized intramuscularly (IM) on day 0 (prime), day 21 (first boost) and day 42 (second boost) with TBS (control) or a mixture of DPS-OspA-ST1 and DPS-CspZ-YA (and alum as adjuvant). Peripheral blood was withdrawn on day 62 and serum was used to detect the level of LA-2 specific antibodies by performing an LA-2 competition ELISA. ELISA plates were coated with 4 pg / ml full-length OspA serotype (ST) 1 protein by overnight incubation at 4°C. Plates were washed and blocked for 1 hour at room temperature. Mouse sera were diluted 1 :30 to 1 :960 with the dilution buffer including 0.3% naive mouse serum and tested in duplicates. 100 pl of prediluted samples and controls were added to the plates and incubated for 1 hour at room temperature. After several washes, 100 pl / well of commercially available HRP-conjugated anti-OspA LA-2 antibody (from Absolute Antibody; 390 ng / ml per well) was added to the wells and incubated for 1 hour at room temperature. After incubation and several washes, 100 pl / well tetramethylbenzidine (TMB) substrate was added and the OD450 values were determined. To calculate the amount of antibody in serum equivalent to the amount of LA-2 antibody [ng / ml], a standard reference curve was established with eight serial two-fold dilutions of HRP-conjugated anti-OspA LA-2 antibody (starting concentration was 390 ng / ml). Data shown as mean ± SEM.

[0045] Figure 5 shows the induction of Factor H-blocking antibodies in serum of vaccinated Balb / c mice.

[0046] Balb / c mice were immunized intramuscularly (IM) on day 0 (prime), day 21 (first boost) and day 42 (second boost) with TBS (control) or a mixture of DPS-OspA-ST1 and DPS-CspZ-YA (and alum as adjuvant). Peripheral blood was withdrawn on day 62 after the prime immunization and serum was analyzed to determine Factor H (FH) binding to CspZ protein by FH binding assay. For the FH binding assay, ELISA plates were coated with rabbit anti-His antibody (0.25 pg / ml; Invitrogen) by overnight incubation, followed by blocking. After blocking, 2 pg / ml CspZ protein was added to the wells for 1 hour incubation. Then, serially diluted sera from TBS injected mice or mice immunized with DPS-OspA-ST1 and DPS-CspZ-YA (in the presence of alum) were added to the wells for 1 hour, followed by several washes, and human FH (2 pg / ml; HycultBiotech) was added to the wells for 1 hour incubation. CspZ-bound human FH amount was detected by adding biotin-labelled mouse anti-human FH monoclonal antibody (1 :3000 dilution; Invitrogen), and then streptavidin-HRP. (A) The percentage of FH binding with decreasing amount of serum added is shown as mean ± SEM. (B) The FH binding rate of negative controls was set as 100% and a 4PL-f it curve was calculated for each sample to determine the titer at 50% inhibition. Data shown as mean ± SEM; (n=5).

[0047] Figure 6 shows the efficacy of a combination of DPS-OspA-ST 1 and DPS-CspZ-YA against infected tick challenge.

[0048] C3H / HeN mice were immunized intramuscularly (IM) on day 0 (prime), day 28 (first boost) and day 56 (second boost) with TBS (control) or DPS-OspA-ST 1 , DPS-CspZ-YA or a combination of the two (and alum as adjuvant). On day 77, mice were challenged with 5 Ixodes scapularis flat nymphs infected with Borrelia burgdorferi (B31 -5A4 strain) each. Ticks were removed three days after challenge (day 80), mice were sacrificed, and serum samples, as well as tissue samples from the skin (tick bite site), knee joints, ears and hearts were collected on day 98.

[0049] (A) Serum was analyzed to measure IgG titers against the C6-peptide of VIsE by anti-C6 peptide ELISA as a marker for Borrelia infection. Mouse antibodies were detected using antimouse IgG (Fc) antibody conjugated to HRP. (B) Tissue samples were analyzed for bacterial burden by qPCR. Data shown as mean ± SEM.

[0050] Figure 7 shows anti-OspA-ST 1 total IgG and anti-CspZ-YA total IgG in serum from vaccinated C3H mice.

[0051] C3H / HeN mice were immunized intramuscularly (IM) on day 0 (prime), day 28 (first boost) and day 56 (second boost) with TBS (control) or DPS-OspA-ST1 (and alum) alone, DPS-CspZ-YA (and alum) alone or a mixture of or DPS-OspA-ST 1 and DPS-CspZ-YA (and alum as adjuvant). Peripheral blood was withdrawn on days 42 after two immunizations and on day 70 after three immunizations, and serum was analyzed to detect serotype (ST) 1 specific anti-OspA IgG as well as anti-CspZ-YA IgG titers by ELISA. Mouse antibodies were detected using anti-mouse IgG (Fc) antibody conjugated to HRP. Anti-OspA-ST1 total IgG titers after two (A) and three

[0052] (B) immunizations, as well as anti-CspZ-YA total IgG titers after two (C) and three (D) immunizations. Titers are shown in arbitrary units (AU). Data shown as mean ± SEM. Figure 8 shows the bactericidal activity in serum of vaccinated C3H mice.

[0053] C3H / HeN mice were immunized intramuscularly (IM) on day 0 (prime), day 28 (first boost) and day 56 (second boost) with DPS-OspA-ST 1 (and alum) alone, DPS-CspZ-YA (and alum) alone or a mixture of or DPS-OspA-ST 1 and DPS-CspZ-YA (and alum as adjuvant). Peripheral blood was withdrawn on day 42 after two immunizations and on day 70 after three immunizations, and serum was analyzed to detect bactericidal activity against Borrelia burgdorferi B31 -5A4 cultures. (A) Bactericidal activity to kill 50% of the bacteria (50% borreliacidal titer, BA5o) provided by serum from animals immunized twice. (B) 50% borreliacidal titer provided by serum from animals immunized three times. Data shown as mean ± SEM.

[0054] Brief Description of Sequences

[0055] SEQ ID NO: 1 is a nucleic acid sequence encoding DPS.

[0056] SEQ ID NO: 2 is the amino acid sequence of DPS.

[0057] SEQ ID NO: 3 is a nucleic acid sequence encoding wild-type OspA of Borrelia burgdorferi.

[0058] SEQ ID NO: 4 is the amino acid sequence of wild-type OspA of Borrelia burgdorferi.

[0059] SEQ ID NO: 5 is a nucleic acid sequence encoding an OspA composed of amino acid stretches of OspA from Borrelia burgdorferi and Borrelia afzelii.

[0060] SEQ ID NO: 6 is the amino acid sequence of an OspA composed of amino acid stretches of OspA from Borrelia burgdorferi and Borrelia afzelii.

[0061] SEQ ID NO: 7 is a nucleic acid sequence encoding wild-type CspZ of Borrelia burgdorferi.

[0062] SEQ ID NO: 8 is the amino acid sequence of wild-type CspZ of Borrelia burgdorferi.

[0063] SEQ ID NO: 9 is a nucleic acid sequence encoding a mutant CspZ (“CspZ-YA”) derived from wild-type CspZ of Borrelia burgdorferi.

[0064] SEQ ID NO: 10 is the amino acid sequence of a mutant CspZ (“CspZ-YA”) derived from wild-type CspZ of Borrelia burgdorferi.

[0065] SEQ ID NO: 11 is a nucleic acid sequence encoding a fusion protein comprising amino acids 18-273 of an OspA composed of amino acid stretches of OspA from Borrelia burgdorferi and Borrelia afzelii (see SEQ ID NO: 5, 6) fused to a DPS subunit.

[0066] SEQ ID NO: 12 is the amino acid sequence of a fusion protein comprising amino acids 18-273 of an OspA composed of amino acid stretches of OspA from Borrelia burgdorferi and Borrelia afzelii (see SEQ ID NO: 5, 6) fused to a DPS subunit.

[0067] SEQ ID NO: 13 is a nucleic acid sequence encoding a fusion protein comprising amino acids 21 -21 1 of mutant CspZ-YA (see SEQ ID NO: 9, 10) fused to a DPS subunit.

[0068] SEQ ID NO: 14 is the amino acid sequence of a fusion protein comprising amino acids 21 -211 of mutant CspZ-YA (see SEQ ID NO: 9, 10) fused to a DPS subunit.

[0069] Detailed Description of Invention

[0070] As detailed above, OspA-based Lyme disease vaccines suffer from the necessity to maintain high serum antibody titers in the absence of an anamnestic response following natural exposure to the antigen. An attempt to overcome this issue was made in a vaccine licensed for veterinary use (Vanguard crLyme) marketed by Zoetis. On top of OspA, this vaccine contains a chimeric protein containing antigenic domains of OspC, another outer surface protein, which is expressed by Borrelia in the host rather than in the vector. However, high inherent sequence diversity of OspC and localization of protective epitopes in variable domains of the protein make it a suboptimal target for a broadly protective vaccine.

[0071] The ‘complement regulator acquiring surface protein 2’ (CRASP-2, herein referred to as CspZ) is also expressed by Borrelia when the bacteria reside in the mammalian host, but unlike OspC it shows low sequence variability. During infection, CspZ recruits the complement regulatory proteins (CRP) Factor H (FH) and FH like protein-1 (FHL-1 ) to the Borrelia bacteria surface, thereby protecting the bacteria from inactivation through the host complement system and aiding dissemination. Immunization with wild-type CspZ does not provide protection from Lyme disease, most likely because the association with FH curtails the launch of a protective anti-CspZ antibody response. This can however be overcome by the mutation of two conserved tyrosines within the FH binding site of CspZ, which abolishes the FH-CspZ interaction. Immunization with this CspZ-YA mutant induces a strong and protective anti-CspZ antibody response in mice (Marcinkiewicz et al., 2018; Chen et al., 2022).

[0072] Here we describe the design of two vaccine antigens consisting of either OspA or CspZ-YA fused to the ‘DNA protection during starvation protein’ (DPS) from E. coli, resulting in dodecameric nanoparticles displaying twelve copies of OspA and CspZ-YA, respectively. We further describe the combination of the two vaccine antigens in a multi-stage Lyme disease vaccine targeting Borrelia at two stages of their lifecycle, and we show synergistic effects of this antigen-combination on bacterial killing. Specifically, we could show that using a vaccine comprising DPS-nanoparticle-presented OspA-ST1 and DPS-nanoparticle-presented mutant CspZ-YA in a formulation with alum induced both high levels of OspA-ST1 -specific as well as CspZ-specific antibodies in mice.

[0073] Furthermore, we report the generation of antibodies that are specific for a protective OspA- ST1 epitope recognized by the well described LA-2 antibody. This indicates the efficient induction of antibodies capable of providing protection against Borrelia infection.

[0074] In addition, we also showed that CspZ-YA comprised in our vaccine leads to the efficient induction of CspZ-specific antibodies that can potently block FH binding of Borrelia CspZ.

[0075] Importantly, using a tick challenge model with Borrelia burgdorferi (strain B31 ) infected ticks we demonstrated that the DPS-OspA-ST1 / DPS-CspZ- YA combination was highly protective against Borrelia infection.

[0076] Finally, we could show a synergistic effect of the combination of DPS-OspA-ST1 and DPS- CspZ-YA, as both vaccine antigens within one formulation induced significantly higher serum borreliacidal activity than could be expected from the added borreliacidal activity of each individual vaccine antigen. This suggests a synergistic effect of the presence of antibodies against both antigens in the same serum, providing a strong rationale for including both antigens in a single formulation to obtain a highly efficacious Lyme disease vaccine. We believe that the observed synergy is dependent on the concurrent presence of antibodies against OspA-ST1 and CspZ-YA, irrespective of how these antibodies were induced. Therefore, we believe that the synergistic effect observed with combined DPS-OspA-ST 1 and DPS-CspZ-YA is not limited to the OspA-ST1 and CspZ-YA antigens when presented on protein nanoparticles, but is a more general principle that can be broadened to monomeric OspA-ST 1 and CspZ-YA proteins and even nucleic acids encoding OspA-ST 1 and CspZ-YA (for example, in the form of mRNA or contained in a viral vector) used for vaccination.

[0077] Definitions

[0078] It must be noted that, as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a nucleic acid sequence” includes one or more nucleic acid sequences.

[0079] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or”, a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0080] Throughout this specification and the appended claims, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated feature but not the exclusion of any other feature. When used in the context of an aspect or embodiment in the description of the present invention the term “comprising” can be amended and thus replaced with the term “containing” or “including” or when used herein with the term “having.” Similarly, any of the afore-mentioned terms (comprising, containing, including, having), whenever used in the context of an aspect or embodiment in the description of the present invention include, by virtue, the terms “consisting of” or “consisting essentially of,” which each denotes specific legal meaning depending on jurisdiction.

[0081] When used herein “consisting of” excludes any feature, element, step, or ingredient not specified in the claim. When used herein, “consisting essentially of” does not exclude features, materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0082] An “aspect” refers to a conception of the invention in its broadest sense; it may map to an independent claim. An “embodiment” is a specific version or implementation or a concrete example of the invention; it may map to a dependent claim.

[0083] The term “recombinant” as used herein refers to nucleic acids or proteins not occurring naturally but being the result of genetic engineering.

[0084] The term “construct” as used herein refers to an artificial nucleic acid, peptide or protein being the result of genetic engineering.

[0085] The term “fusion protein” refers to a recombinant protein comprising at least two separate stretches of amino acids, or proteins, e.g., protein domains, that have been joined artificially so that they are transcribed and translated as a single protein. Mostly, the two separate stretches of amino acids are joined using a linker sequence, but they could also be joined directly to each other. As used herein, the term “fusion protein” generally describes a protein resulting from the fusion of an antigen and a protein nanoparticle subunit. Such a fusion protein may be referred to herein as “monomer”, particularly in the context of a protein nanoparticle. The term “protein nanoparticle” as used herein refers to a polymeric assembly of monomeric polypeptides, e.g., a multimer. Such protein nanoparticles are useful for presenting protein antigens multimerized in a regular array.

[0086] The term “self-assembling protein nanoparticle”, as used herein refers to a polymeric assembly of monomeric polypeptides that are capable of directing their self-assembly into the nanoparticle. For example, the ‘DNA protection during starvation protein’ (DPS) from E. coli forms symmetric homo-dodecameric protein nanoparticles ( / .e., 12mers) by self-assembly.

[0087] The term “adjuvant” as used herein refers to a compound capable of enhancing an immune response to an antigen. For example, a pharmaceutical composition containing a vaccine antigen and additionally an adjuvant would elicit an immune response in a subject that is enhanced as compared to pharmaceutical composition containing the vaccine antigen but not the adjuvant.

[0088] By “antigenic part thereof” as used herein is intended a portion or fragment of a protein that can induce the production of an antibody that will bind to it.

[0089] Abbreviations

[0090] AU arbitrary units

[0091] BA bacterial killing activity, also borreliacidal activity

[0092] BA50 50% borreliacidal titer representing the serum dilution rate that effectively kills 50% of bacterial cells

[0093] C6 peptide derived from the invariable region 6 of the 'variable major protein-like sequence, expressed' (VIsE) protein of Borrelia burgdorferi

[0094] CspZ complement regulator-acquiring surface protein 2

[0095] CspZ-YA mutant CspZ with point mutations Y207A and Y211 A

[0096] DPS DNA protection during starvation protein (also known as DNA binding protein from starved cells)

[0097] DPS-OspA-ST1 DPS presenting 12 copies of OspA serotype 1 (ST 1 )

[0098] DPS-CspZ-YA DPS presenting 12 copies of CspZ-YA

[0099] FH human Factor H

[0100] LA-2 ng eq / ml amount of antibody in ng equivalent to LA-2 antibody

[0101] OD optical density

[0102] OspA outer surface protein qPCR quantitative PCR ST serotype

[0103] TBS Tris-buffered saline

[0104] VIsE variable major protein-like sequence, expressed

[0105] Aspects and embodiments

[0106] A. Subject-matter relating to OspA

[0107] A.1 Aspects and embodiments relating to fusion proteins comprising OspA

[0108] In one aspect, provided is a fusion protein comprising a Lyme disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle, wherein the Lyme disease-associated antigen is an ‘outer surface protein A’ (OspA) of Borrelia.

[0109] Herein, such fusion protein sometimes is briefly referred to as “fusion protein comprising OspA”.

[0110] In one embodiment, the fusion protein comprising OspA is a monomer.

[0111] In one embodiment, the fusion protein comprising OspA has the capability to take part in a self-assembly of fusion proteins into a self-assembling protein nanoparticle composed of monomers of the fusion protein.

[0112] In one embodiment of the fusion protein comprising OspA, the subunit of a self-assembling protein nanoparticle is capable of being joined to an antigen (e.g., OspA) such that the antigen is displayed with its N-terminus facing the nanoparticle core.

[0113] In one embodiment of the fusion protein comprising OspA, the subunit of a self-assembling protein nanoparticle is capable of being joined to the N-terminus of an antigen (e.g., OspA) via the C-terminus of the subunit.

[0114] In one embodiment of the fusion protein, the C-terminus of the subunit of the self-assembling protein nanoparticle is joined to the N-terminus of OspA.

[0115] In one embodiment of the fusion protein comprising OspA, the self-assembling protein nanoparticle is a homo-dodecameric nanoparticle, preferably a ‘DNA protection during starvation protein’ (DPS), more preferably DPS from Escherichia coll, even more preferably DPS from E. coll K12.

[0116] In one embodiment of the fusion protein comprising OspA, the amino acid sequence of the subunit of DPS comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 2 or a part of the amino acid sequence as depicted in SEQ ID NO: 2. In case of “a part of the amino acid”, the subunit has maintained the capability to take part in a self-assembly of fusion proteins into a self-assembling protein nanoparticle.

[0117] In one embodiment of the fusion protein comprising OspA, the amino acid sequence of the subunit of DPS comprises or consists of amino acids 11 -167 of the amino acid sequence as depicted in SEQ ID NO: 2.

[0118] In one embodiment of the fusion protein comprising OspA, the amino acid sequence of the subunit of DPS is encoded by nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 2 or a part of the amino acid sequence as depicted in SEQ ID NO: 2.

[0119] In one embodiment of the fusion protein comprising OspA, the amino acid sequence of the subunit of DPS is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 1 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO:1 .

[0120] In one embodiment of the fusion protein, the OspA is of or is derived from Borrelia burgdorferi sensu lato, preferably selected from the group consisting of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, B. spielmanii, and B. bavariensis, more preferably Borrelia burgdorferi sensu stricto and / or Borrelia afzelii.

[0121] In one embodiment of the fusion protein, the OspA comprises stretches of amino acids of Borrelia burgdorferi sensu stricto and Borrelia afzelii, preferably Borrelia burgdorferi strain B31 and Borrelia afzelii strain K78.

[0122] In one embodiment of the fusion protein, the amino acid sequence of OspA comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 6, or a part of the amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 6.

[0123] In one embodiment of the fusion protein, the amino acid sequence of OspA is encoded by nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 6, or a part of the amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 6.

[0124] In one embodiment of the fusion protein, the amino acid sequence of OspA is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 5 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 5.

[0125] In one embodiment of the fusion protein, the amino acid sequence of OspA comprises or consists of amino acids 18-273 of the amino acid sequence as depicted in SEQ ID NO: 6. In one embodiment of the fusion protein, the OspA is fused to the subunit of a self-assembling protein nanoparticle via a peptide linker, preferably a flexible glycine-serine linker.

[0126] In one embodiment, the amino acid sequence of the fusion protein comprising OspA comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 12 or a part of the amino acid sequence as depicted in SEQ ID NO: 12.

[0127] In one embodiment, the amino acid sequence of the fusion protein comprising OspA is encoded by nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 12 or a part of the amino acid sequence as depicted in SEQ ID NO: 12.

[0128] In one embodiment, the amino acid sequence of the fusion protein comprising OspA is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 11 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO: 11 .

[0129] In one aspect, provided is a nucleic acid encoding a fusion protein comprising OspA as described herein.

[0130] In one embodiment, the nucleic acid encoding a fusion protein comprising OspA as described herein comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 11 or a part thereof.

[0131] In one embodiment, the nucleic acid encoding a fusion protein comprising OspA as described herein encodes an amino acid sequence as depicted in SEQ ID NO: 12 or a part thereof.

[0132] In one aspect, provided is a use of a nucleic acid encoding a fusion protein comprising OspA as described herein for the preparation of a recombinant expression vector, preferably based on an E. co / / expression plasmid.

[0133] In one aspect, provided is a recombinant expression vector comprising a nucleic acid encoding a fusion protein comprising OspA as described herein, preferably based on an E. coli expression plasmid.

[0134] In one aspect, provided is a use of a recombinant expression vector comprising a nucleic acid encoding a fusion protein comprising OspA as described herein for the preparation of a selfassembling protein nanoparticle.

[0135] In one aspect, provided is a use of a nucleic acid encoding a fusion protein comprising OspA as described herein for the preparation of a pharmaceutical composition or vaccine. A.2 Aspects and embodiments relating to self-assembling protein nanoparticles comprising OspA

[0136] In one aspect, provided is a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising OspA as described herein (see A.1 above).

[0137] Herein, such self-assembling protein nanoparticle may be briefly referred to as “selfassembling protein nanoparticle comprising OspA”.

[0138] In one embodiment, the self-assembling protein nanoparticle is a homo-dodecameric nanoparticle and preferably comprises or consists of 12 monomers of the fusion protein comprising OspA as described herein.

[0139] In one embodiment, the self-assembling protein nanoparticle is displaying OspA antigenic determinants on its surface.

[0140] In one aspect, provided is a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising OspA as described herein for the preparation of a pharmaceutical composition or vaccine.

[0141] In one aspect, provided is a process for preparing a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising OspA as described herein comprising the steps of:

[0142] (a) providing a nucleic acid encoding a fusion protein comprising OspA as described herein;

[0143] (b) preparing a recombinant expression vector comprising the nucleic acid provided in step (a);

[0144] (c) transforming expression cells, preferably bacterial expression cells, more preferably competent E. coll cells, with the recombinant expression vector obtained in step (b) and propagating selected transformants;

[0145] (d) inducing expression of a fusion protein encoded by the nucleic acid provided in step (a);

[0146] (e) harvesting and preferably purifying the self-assembling protein nanoparticle.

[0147] A.3 Aspects and embodiments relating to vaccines and medical uses

[0148] In one aspect, provided is a pharmaceutical composition or vaccine comprising a selfassembling protein nanoparticle comprising monomers of the fusion protein comprising OspA as described herein, optionally further comprising a pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition or vaccine further comprises an adjuvant, preferably alum.

[0149] In one aspect, provided is a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising OspA as described herein for use in the prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia.

[0150] In one aspect, provided is a pharmaceutical composition or vaccine as described herein for use in the prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia.

[0151] In one aspect, provided is a method of prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia, comprising administering to a subject a pharmaceutical composition or vaccine as described herein, such a pharmaceutical composition or vaccine comprising a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising OspA as described herein.

[0152] In one aspect, provided is a method for inducing or stimulating an immune response to OspA comprising the step of administering to a subject a pharmaceutical composition or vaccine as described herein, such a pharmaceutical composition or vaccine comprising a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising OspA as described herein. The induction of an immune response can be measured by methods known in the art, for example, by detection of antigen-specific antibodies, which will increase in a subject following administration of the pharmaceutical composition or vaccine in comparison to the level of antibodies in the subject prior to said administration.

[0153] In one embodiment of the methods the subject or vaccine recipient (vaccinee) is a potential Borrelia host, preferably is a vertebrate, preferably a mammal, more preferably a farm or companion animal, or a human, most preferably a human.

[0154] A.4 Further aspects and embodiments

[0155] In one aspect, provided is a pharmaceutical composition comprising a recombinant virusbased vector comprising a heterologous nucleic acid operably linked to a promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising OspA as described herein.

[0156] In one embodiment of the pharmaceutical composition, the recombinant virus-based vector is a recombinant poxvirus, preferably a recombinant vaccinia virus, more preferably a recombinant Modified Vaccinia Virus Ankara (MVA). In one embodiment of the pharmaceutical composition, the recombinant virus-based vector is a recombinant virus replicon particle (VRP), preferably derived from Venezuelan Equine Encephalitis Virus (VEEV).

[0157] In one aspect, provided is pharmaceutical composition comprising a recombinant virus-based vector as described herein for use in the prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia.

[0158] In one aspect, provided is a pharmaceutical composition comprising an RNA or DNA molecule comprising a nucleic acid encoding a fusion protein comprising OspA as described herein.

[0159] In one embodiment of the pharmaceutical composition comprising an RNA or DNA molecule, the RNA molecule is a messenger RNA (mRNA) or a self-amplifying RNA (saRNA).

[0160] In one embodiment of the pharmaceutical composition comprising an RNA or DNA molecule, the DNA molecule is an expression plasmid.

[0161] In one aspect, provided is a pharmaceutical composition comprising an RNA or DNA molecule as described herein for use in the prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia.

[0162] B. Subject-matter relating to mutant CspZ

[0163] B.1 Aspects and embodiments relating to fusion proteins comprising mutant CspZ

[0164] In one aspect, provided is a fusion protein comprising a Lyme disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle, wherein the Lyme disease-associated antigen is a ‘complement regulator-acquiring surface protein 2’ (CspZ) of Borrelia which is a mutant CspZ not binding to complement regulatory protein (CRP) Factor H (FH) of a Borrelia host.

[0165] Herein, such fusion protein sometimes is briefly referred to as “fusion protein comprising mutant CspZ”.

[0166] In one embodiment, the fusion protein comprising mutant CspZ is a monomer.

[0167] In one embodiment, the fusion protein comprising mutant CspZ has the capability to take part in a self-assembly of fusion proteins into a self-assembling protein nanoparticle composed of monomers of the fusion protein. In one embodiment of the fusion protein comprising mutant CspZ, the subunit of a selfassembling protein nanoparticle is capable of being joined to an antigen (e.g., mutant CspZ) such that the antigen is displayed with its N-terminus facing the nanoparticle core.

[0168] In one embodiment of the fusion protein comprising mutant CspZ, the subunit of a selfassembling protein nanoparticle is capable of being joined to the N-terminus of an antigen (e.g., CspZ) via the C-terminus of the subunit.

[0169] In one embodiment of the fusion protein, the C-terminus of the subunit of the self-assembling protein nanoparticle is joined to the N-terminus of mutant CspZ.

[0170] In one embodiment of the fusion protein comprising mutant CspZ, the self-assembling protein nanoparticle is a homo-dodecameric nanoparticle, preferably a ‘DNA protection during starvation protein’ (DPS), more preferably DPS from Escherichia coli, even more preferably from E. coli K12.

[0171] In one embodiment of the fusion protein comprising mutant CspZ, the amino acid sequence of the subunit of DPS is as depicted in SEQ ID NO: 2 or is a part of the amino acid sequence as depicted in SEQ ID NO: 2.

[0172] In one embodiment of the fusion protein comprising mutant CspZ, the amino acid sequence of the subunit of DPS comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 2 or a part of the amino acid sequence as depicted in SEQ ID NO: 2. In case of “a part of the amino acid”, the subunit has maintained the capability to take part in a self-assembly of fusion proteins into a self-assembling protein nanoparticle.

[0173] In one embodiment of the fusion protein comprising mutant CspZ, the amino acid sequence of the subunit of DPS comprises or consists of amino acids 11 -167 of the amino acid sequence as depicted in SEQ ID NO: 2.

[0174] In one embodiment of the fusion protein comprising mutant CspZ, the amino acid sequence of the subunit of DPS is encoded by nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 2 or a part of the amino acid sequence as depicted in SEQ ID NO: 2.

[0175] In one embodiment of the fusion protein comprising mutant CspZ, the amino acid sequence of the subunit of DPS is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 1 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO:1 .

[0176] In one embodiment of the fusion protein, the mutant CspZ is of or is derived from Borrelia burgdorferi sensu lato, preferably selected from the group consisting of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, B. spielmanii, and B. bavariensis, more preferably Borrelia burgdorferi sensu stricto, even more preferably Borrelia burgdorferi strain B31.

[0177] In one embodiment of the fusion protein comprising mutant CspZ, the Borrelia host is a vertebrate, preferably a mammal, more preferably a farm or companion animal, or a human, most preferably a human. Particularly, a vaccine recipient (vaccinee) is a potential Borrelia host.

[0178] In one embodiment of the fusion protein, the mutant CspZ comprises two point mutations at position 207 and 211 related to a wild-type CspZ, preferably two point mutations Y207A and Y211A related to a wild-type CspZ. An amino acid sequence of a wild-tpye CspZ of Borrelia burgdorferi B31 is as depicted in SEQ ID NO: 8.

[0179] Herein, such mutant CspZ is also referred to as “CspZ- YA” or “mutant CspZ- YA”.

[0180] In one embodiment of the fusion protein, the amino acid sequence of mutant CspZ comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 10 or a part of the amino acid sequence as depicted in SEQ ID NO: 10.

[0181] In one embodiment of the fusion protein, the amino acid sequence of mutant CspZ is encoded by nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 10 or a part of the amino acid sequence as depicted in SEQ ID NO: 10.

[0182] In one embodiment of the fusion protein, the amino acid sequence of mutant CspZ is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 9 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO: 9.

[0183] In one embodiment of the fusion protein, the amino acid sequence of mutant CspZ comprises or consists of amino acids 21 -236 of the amino acid sequence is as depicted in SEQ ID NO: 10.

[0184] In one embodiment of the fusion protein, the mutant CspZ is fused to the subunit of a selfassembling protein nanoparticle via a peptide linker, preferably a flexible glycine-serine linker.

[0185] In one embodiment, the amino acid sequence of the fusion protein comprising mutant CspZ comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 14 or a part of the amino acid sequence as depicted in SEQ ID NO: 14.

[0186] In one embodiment, the amino acid sequence of the fusion protein comprising OspA is encoded by nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 14 or a part of the amino acid sequence as depicted in SEQ ID NO: 14. In one embodiment, the amino acid sequence of the fusion protein comprising OspA is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 13 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO: 13.

[0187] In one aspect, provided is a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein.

[0188] In one embodiment, the nucleic acid encoding a fusion protein comprising mutant CspZ as described herein comprises or consists of a nucleic acid sequence as depicted in SEQ ID NO: 13 or a part thereof.

[0189] In one embodiment, the nucleic acid encoding a fusion protein comprising mutant CspZ as described herein encodes an amino acid sequence as depicted in SEQ ID NO: 14 or a part thereof.

[0190] In one aspect, provided is a use of a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein for the preparation of a recombinant expression vector, preferably based on an E. co / / expression plasmid.

[0191] In one aspect, provided is a recombinant expression vector comprising a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein, preferably based on an E. coli expression plasmid.

[0192] In one aspect, provided is a use of a recombinant expression vector comprising a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein for the preparation of a self-assembling protein nanoparticle.

[0193] In one aspect, provided is a use of a nucleic acid encoding a fusion protein comprising CspZ as described herein for the preparation of a pharmaceutical composition or vaccine.

[0194] B.2 Aspects and embodiments relating to self-assembling protein nanoparticles comprising mutant CspZ

[0195] In one aspect, provided is a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising mutant CspZ as described herein (see B.2 above).

[0196] Herein, such self-assembling protein nanoparticle sometimes is briefly referred to as “selfassembling protein nanoparticle comprising mutant CspZ”.

[0197] In one embodiment, the self-assembling protein nanoparticle is a homo-dodecameric nanoparticle and preferably comprises or consists of 12 monomers of the fusion protein comprising mutant CspZ as described herein. In one embodiment, the self-assembling protein nanoparticle is displaying mutant CspZ antigenic determinants on its surface.

[0198] In one aspect, provided is a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising mutant CspZ as described herein for the preparation of a pharmaceutical composition or vaccine.

[0199] In one aspect, provided is a process for preparing a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising mutant CspZ as described herein comprising the steps of:

[0200] (a) providing a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein;

[0201] (b) preparing a recombinant expression vector comprising the nucleic acid provided in step (a);

[0202] (c) transforming expression cells, preferably bacterial expression cells, more preferably competent E. coli cells, with the recombinant expression vector obtained in step (b) and propagating selected transformants;

[0203] (d) inducing expression of a fusion protein encoded by the nucleic acid provided in step (a);

[0204] (e) harvesting and preferably purifying the self-assembling protein nanoparticle.

[0205] B.3 Aspects and embodiments relating to vaccines and medical uses

[0206] In one aspect, provided is a pharmaceutical composition or vaccine comprising a selfassembling protein nanoparticle comprising monomers of the fusion protein comprising mutant CspZ as described herein, optionally further comprising a pharmaceutically acceptable excipient.

[0207] In one embodiment, the pharmaceutical composition or vaccine further comprises an adjuvant, preferably alum.

[0208] In one aspect, provided is a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising mutant CspZ as described herein for use in the prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia.

[0209] In one aspect, provided is a pharmaceutical composition or vaccine as described herein for use in the prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia. In one aspect, provided is a method of prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia comprising administering to a subject a pharmaceutical composition or vaccine as described herein, such a pharmaceutical composition or vaccine comprising a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising mutant CspZ as described herein.

[0210] In one aspect, provided is a method for inducing or stimulating an immune response to CspZ comprising the step of administering to a subject a pharmaceutical composition or vaccine as described herein, such a pharmaceutical composition or vaccine comprising a self-assembling protein nanoparticle comprising monomers of the fusion protein comprising mutant CspZ as described herein. The induction of an immune response can be measured by methods known in the art, for example, by detection of antigen-specific antibodies, which will increase in a subject following administration of the pharmaceutical composition or vaccine in comparison to the level of antibodies in the subject prior to said administration.

[0211] In one embodiment of the methods the subject or vaccine recipient (vaccinee) is a potential Borrelia host, preferably is a vertebrate, preferably a mammal, more preferably a farm or companion animal, or a human, most preferably a human.

[0212] B.4 Further aspects and embodiments

[0213] In one aspect, provided is a pharmaceutical composition comprising a recombinant virusbased vector comprising a heterologous nucleic acid operably linked to a promoter, wherein the heterologous nucleic acid encodes a fusion protein comprising mutant CspZ as described herein.

[0214] In one embodiment of the pharmaceutical composition, the recombinant virus-based vector is a recombinant poxvirus, preferably a recombinant vaccinia virus, more preferably a recombinant Modified Vaccinia Virus Ankara (MVA).

[0215] In one embodiment of the pharmaceutical composition, the recombinant virus-based vector is a recombinant virus replicon particle (VRP), preferably derived from Venezuelan Equine Encephalitis Virus (VEEV).

[0216] In one aspect, provided is pharmaceutical composition comprising a recombinant virus-based vector as described herein for use in the prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia. In one aspect, provided is a pharmaceutical composition comprising an RNA or DNA molecule comprising a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein.

[0217] In one embodiment of the pharmaceutical composition comprising an RNA or DNA molecule, the RNA molecule is a messenger RNA (mRNA) or a self-amplifying RNA (saRNA).

[0218] In one embodiment of the pharmaceutical composition comprising an RNA or DNA molecule, the DNA molecule is an expression plasmid.

[0219] In one aspect, provided is a pharmaceutical composition comprising an RNA or DNA molecule as described herein for use in the prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia.

[0220] C. Subject-matter relating to a combination of OspA and mutant CspZ

[0221] C.1 Aspects and embodiments relating to a combination of self-assembling protein nanoparticles comprising OspA or mutant CspZ

[0222] Herein, “a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein” includes references to aspects and embodiments as described in section A above, “a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein” includes references to aspects and embodiments as described in section B above.

[0223] In one aspect, provided is a combination of:

[0224] (i) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein; and

[0225] (ii) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein.

[0226] In one aspect, provided is a use of a combination of:

[0227] (i) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein; and

[0228] (ii) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein; for the preparation of a pharmaceutical composition or vaccine. In one aspect, provided is a pharmaceutical composition or vaccine comprising a combination of:

[0229] (i) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein; and

[0230] (ii) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein; optionally further comprising a pharmaceutically acceptable excipient, preferably further comprising an adjuvant, more preferably alum.

[0231] In one aspect, provided is a combination of:

[0232] (i) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein; and

[0233] (ii) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein; for use in the prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia.

[0234] In one aspect, provided is a pharmaceutical composition or vaccine comprising a combination of:

[0235] (i) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein; and

[0236] (ii) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein; optionally further comprising a pharmaceutically acceptable excipient, preferably further comprising an adjuvant, more preferably alum; for use in the prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia.

[0237] In one embodiment of the medical uses as described herein, a combination of:

[0238] (i) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein; and

[0239] (ii) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein; is superior to either (i) or (ii) alone. In one aspect, provided is a method of prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia, comprising administering to a subject a pharmaceutical composition or vaccine comprising a combination of:

[0240] (i) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein; and

[0241] (ii) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein; wherein administration of the pharmaceutical composition or vaccine to the subject stimulates an immune response and / or reduces the severity of symptoms that would otherwise be expected or prevents infection with Lyme disease.

[0242] In one aspect, provided is a method for inducing or stimulating an immune response to a Lyme-disease associated antigen comprising the step of administering to a subject a pharmaceutical composition or vaccine comprising a combination of:

[0243] (i) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein; and

[0244] (ii) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein.

[0245] The induction of an immune response can be measured by methods known in the art, for example, by detection of antigen-specific antibodies, which will increase in a subject following administration of the pharmaceutical composition or vaccine in comparison to the level of antibodies in the subject prior to said administration. By stimulating an immune response is intended that administration of the pharmaceutical composition or vaccine to a subject results in detectable levels of or increases the amount of antigen-specific antibodies. These antibodies can be detected by methods known in the art and also described herein, including in the working examples.

[0246] In one embodiment of the methods of prevention or treatment or for inducing or stimulating an immune response as described herein, the administration of a combination of:

[0247] (i) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein; and

[0248] (ii) a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein; is superior to either administration of (i) or (ii) alone.

[0249] In one embodiment of the methods the subject or vaccine recipient (vaccinee) is a potential Borrelia host, preferably is a vertebrate, preferably a mammal, more preferably a farm or companion animal, or a human, most preferably a human. In one aspect, provided is a combination of two pharmaceutical compositions or vaccines comprising:

[0250] (i) a first pharmaceutical composition or vaccine comprising a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein; optionally further comprising a pharmaceutically acceptable excipient, preferably further comprising an adjuvant, more preferably alum; and

[0251] (ii) a second pharmaceutical composition or vaccine comprising a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein; optionally further comprising a pharmaceutically acceptable excipient, preferably further comprising an adjuvant, more preferably alum.

[0252] In one aspect, provided is a kit comprising:

[0253] (i) a first pharmaceutical composition or vaccine comprising a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein in a first vial or container; the pharmaceutical composition or vaccine optionally further comprising a pharmaceutically acceptable excipient, preferably further comprising an adjuvant, more preferably alum; and

[0254] (ii) a second pharmaceutical composition comprising a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein in a second vial or container; the pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient, preferably further comprising an adjuvant, more preferably alum.

[0255] C.2 Further aspects and embodiments

[0256] In one aspect, provided is a combination comprising:

[0257] (i) a first molecule capable of inducing an immune response in a subject against a Lyme disease-associated antigen, or an antigenic part thereof, wherein the Lyme disease-associated antigen is an ‘outer surface protein A’ (OspA) of Borrelia; and

[0258] (ii) a second molecule capable of inducing an immune response in a subject against a Lyme disease-associated antigen, or an antigenic part thereof, wherein the Lyme disease-associated antigen is a ‘complement regulatoracquiring surface protein 2’ (CspZ) of Borrelia which is a mutant CspZ not binding to complement regulatory protein (CRP) Factor H (FH) of a Borrelia host.

[0259] In one embodiment of the combination, the first and / or second molecule is selected from the group consisting of a protein, a peptide and a nucleic acid.

[0260] In one embodiment, the first molecule is a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising OspA as described herein.

[0261] In one embodiment, the first molecule is an OspA protein, or an antigenic part thereof.

[0262] In one embodiment, the first molecule is a peptide comprising an antigenic determinant of an OspA protein.

[0263] In one embodiment of the fusion protein, the OspA protein is of or is derived from Borrelia burgdorferi sensu lato, preferably selected from the group consisting of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, B. spielmanii, and B. bavariensis, more preferably Borrelia burgdorferi sensu stricto and / or Borrelia afzelii.

[0264] In one embodiment of the fusion protein, the OspA protein comprises stretches of amino acids of Borrelia burgdorferi sensu stricto and Borrelia afzelii, preferably Borrelia burgdorferi strain B31 and Borrelia afzelii strain K78.

[0265] In one embodiment, the amino acid sequence of OspA comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 6, or a part of the amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 6.

[0266] In one embodiment, the amino acid sequence of OspA is encoded by nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 6, or a part of the amino acid sequence as depicted in SEQ ID NO: 4 or SEQ ID NO: 6.

[0267] In one embodiment, the amino acid sequence of OspA is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 5 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO: 3 or SEQ ID NO: 5.

[0268] In one embodiment, the first molecule is a nucleic acid encoding an OspA protein, or an antigenic part thereof.

[0269] In one embodiment, the first molecule is a nucleic acid encoding a fusion protein comprising OspA as described herein. In one embodiment, the nucleic acid nucleic acid encoding an OspA protein, or an antigenic part thereof, or encoding a fusion protein comprising OspA as described herein, is an RNA or DNA molecule.

[0270] In one embodiment, the nucleic acid nucleic acid encoding an OspA protein, or an antigenic part thereof, or encoding a fusion protein comprising OspA as described herein, is comprised by a recombinant virus-based vector.

[0271] In one embodiment, the nucleic acid encoding an OspA protein, or an antigenic part thereof, or encoding a fusion protein comprising OspA as described herein, is comprised by a recombinant vaccinia virus, more preferably a recombinant Modified Vaccinia Virus Ankara (MVA).

[0272] In one embodiment, the nucleic acid nucleic acid encoding an OspA protein, or an antigenic part thereof, or encoding a fusion protein comprising OspA as described herein, is comprised by a recombinant virus replicon particle (VRP), preferably derived from Venezuelan Equine Encephalitis Virus (VEEV).

[0273] In one embodiment, the nucleic acid nucleic acid encoding an OspA protein, or an antigenic part thereof, or encoding a fusion protein comprising OspA as described herein, is comprised by or consists of a messenger RNA (mRNA) or a self-amplifying RNA (saRNA).

[0274] In one embodiment, the nucleic acid nucleic acid encoding an OspA protein, or an antigenic part thereof, or encoding a fusion protein comprising OspA as described herein, is comprised by an expression plasmid.

[0275] In one embodiment, the second molecule is a self-assembling protein nanoparticle comprising monomers of a fusion protein comprising mutant CspZ as described herein.

[0276] In one embodiment, the second molecule is a mutant CspZ protein, or an antigenic part thereof.

[0277] In one embodiment, the second molecule is a peptide comprising an antigenic determinant of mutant CspZ protein.

[0278] In one embodiment, the mutant CspZ protein is of or is derived from Borrelia burgdorferi sensu lato, preferably selected from the group consisting of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, B. spielmanii, and B. bavariensis, more preferably Borrelia burgdorferi sensu stricto, even more preferably Borrelia burgdorferi strain B31. In one embodiment of the mutant CspZ protein, the Borrelia host is a vertebrate, preferably a mammal, more preferably a farm or companion animal, or a human, most preferably a human.

[0279] In one embodiment, the mutant CspZ protein comprises two point mutations at position 207 and 211 related to a wild-type CspZ, preferably two point mutations Y207A and Y211 A related to a wild-type CspZ. An amino acid sequence of a wild-tpye CspZ of Borrelia burgdorferi B31 is as depicted in SEQ ID NO: 8.

[0280] In one embodiment, the amino acid sequence of mutant CspZ comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 10 or a part of the amino acid sequence as depicted in SEQ ID NO: 10.

[0281] In one embodiment, the amino acid sequence of mutant CspZ is encoded by nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 10 or a part of the amino acid sequence as depicted in SEQ ID NO: 10.

[0282] In one embodiment, the amino acid sequence of mutant CspZ is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 9 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO: 9.

[0283] In one embodiment, the second molecule is a nucleic acid encoding a mutant CspZ protein, or an antigenic part thereof.

[0284] In one embodiment, the second molecule is a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein.

[0285] In one embodiment, the nucleic acid nucleic acid encoding a mutant CspZ protein, or an antigenic part thereof, or encoding a fusion protein comprising mutant CspZ as described herein, is an RNA or DNA molecule.

[0286] In one embodiment, the nucleic acid nucleic acid encoding a mutant CspZ protein, or an antigenic part thereof, or encoding a fusion protein comprising mutant CspZ as described herein, is comprised by a recombinant virus-based vector.

[0287] In one embodiment, the nucleic acid encoding a mutant CspZ protein, or an antigenic part thereof, or encoding a fusion protein comprising mutant CspZ as described herein, is comprised by a recombinant vaccinia virus, more preferably a recombinant Modified Vaccinia Virus Ankara (MVA).

[0288] In one embodiment, the nucleic acid nucleic acid encoding a mutant CspZ protein, or an antigenic part thereof, or encoding a fusion protein comprising mutant CspZ as described herein, is comprised by a recombinant virus replicon particle (VRP), preferably derived from Venezuelan Equine Encephalitis Virus (VEEV).

[0289] In one embodiment, the nucleic acid nucleic acid encoding a mutant CspZ protein, or an antigenic part thereof, or encoding a fusion protein comprising mutant CspZ as described herein, is comprised by or consists of a messenger RNA (mRNA) or a self-amplifying RNA (saRNA).

[0290] In one embodiment, the nucleic acid nucleic acid encoding a mutant CspZ protein, or an antigenic part thereof, or encoding a fusion protein comprising mutant CspZ as described herein, is comprised by an expression plasmid.

[0291] D. Embodiments relating to SEQ ID NOs

[0292] Regarding SEQ ID NOs: 1 to 14, the disclosure considers certain sequence identities.

[0293] In one embodiment, a nucleic acid sequence has at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the nucleic acid sequences as depicted in any of SEQ ID NOs: 1 , 3, 5, 7,

[0294] 9, 1 1 , and 13.

[0295] In one embodiment, an amino acid sequence has at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequences as depicted in any of SEQ ID NOs: 2, 4, 6, 8,

[0296] 10, 12, and 14.

[0297] EXAMPLES

[0298] The following examples serve to further illustrate the disclosure. They should not be understood as limiting the invention, the scope of which is determined by the appended claims.

[0299] EXAMPLE 1 : Material and methods

[0300] 1 .1 Anti-OspA-STI / CspZ IgG ELISA of Balb / c derived sera

[0301] Enzyme-linked immunosorbent assay (ELISA) plates were coated overnight with 100 pl OspA- ST1 or CspZ protein solution per well at a concentration of 2 pg / ml and 1 pg / ml, respectively. For IgG analysis, different dilutions of serum were incubated on OspA-ST1 - or CspZ-coated plates for 1 hour at room temperature, and detection antibody (goat anti-mouse IgG-(Fc) HRP; BioRad) was used to detect OspA-ST1 or CspZ-specific IgG. Plates were developed with tetramethylbenzidine (TMB; ThermoFisher) for 20 minutes at room temperature and the reaction was stopped with 2N H2SO4. Optical density (OD) values were measured at 450 / 620 nm. IgG titers were assigned an arbitrary titer based on interception of the calculated 4PL-fit curve with an OD of 0.3. Serum samples with an OD value below 0.3 were regarded negative and given the arbitrary value of 1 .

[0302] 1 .2 Factor H binding assay

[0303] For the Factor H (FH) binding assay, ELISA plates were coated with rabbit anti-His antibody (0.25 pg / ml; Invitrogen) by overnight incubation, followed by blocking. After blocking, 2 pg / ml CspZ protein was added to the wells for 1 hour incubation. Then, serially diluted sera were added to the wells for 1 hour, and human FH (2 pg / ml ; HycultBiotech) was added to the wells for 1 hour incubation. CspZ-bound human FH amount was detected by adding biotin-labelled mouse anti-human FH monoclonal antibody (1 :3000 dilution; Invitrogen) and then streptavidin- horseradish peroxidase (HRP). The FH binding rate of negative controls was set as 100% and a 4PL-fit curve was calculated for each sample to determine the titer at 50% inhibition.

[0304] 1 .3 LA-2 competition assay

[0305] ELISA plates were coated with 4 pg / ml OspA-ST1 protein by overnight incubation at 4°C. Plates were washed and blocked for 1 hour at room temperature. Mouse sera were diluted 1 :30- 1 :960 with the dilution buffer including 0.3% naive mouse serum and tested in duplicates. 100 pl of prediluted samples and controls were added to the plates and incubated for 1 hour at room temperature. After several washes, 100 pl / well of commercially available HRP- conjugated anti-OspA LA-2 antibody (Absolute Antibody; 390 ng / ml per well) was added to the wells and incubated for 1 hour at room temperature. After incubation and several washes, 100 pl / well TMB substrate was added and the OD450 values were determined. To calculate the amount of antibody in serum equivalent to the amount of LA-2 antibody [ng eq / ml], a standard reference curve was established with eight serial two-fold dilutions of HRP-conjugated anti- OspA LA-2 antibody (starting concentration was 390 ng / ml).

[0306] 1 .4 Analysis of total IgG titers from C3H / HeN mice derived serum samples by kinetic ELISA

[0307] All ELISAs run with C3H / HeN mice derived sera were performed following the same kinetic ELISA protocol but using different coating antigens to detect immune responses against OspA, CspZ and C6-peptide, respectively. For anti-OspA IgG and anti-CspZ IgG ELISA, ELISA plates were incubated with 1 pg of purified OspA protein or purified CspZ protein per well in 100 pl of carbonate coating buffer (100 mM sodium bicarbonate and 100 mM sodium carbonate, pH 9.6) at 4°C overnight. For C6-peptide ELISA, ELISA plates were incubated with 1 pg of synthetic C6 peptide (GenmedSyn) per well in 100 pl of carbonate coating at 4°C overnight. The next day, after washing the ELISA plates three times with PBS-T buffer (phosphate- buffered saline with 0.05% Tween 20), the ELISA plate wells were blocked with 5% BSA (bovine serum albumin; Millipore Sigma) in PBS at room temperature for 1 hour. After washing the ELISA plate wells three times with PBS-T, 50 pl of mouse serum diluted 1 :100, 1 :300, or 1 :900 was added to the wells and the plates were incubated at room temperature for 1 hour. Then, after washing the ELISA plate wells three times with PBS-T, HRP-conjugated goat antimouse IgG (1 :10,000; Bethyl Lab) was added and the ELISA plates were incubated at room temperature for 1 hour. After washing the ELISA plate wells three times with PBS-T buffer, 50 pl of 1 -Step™ TMB ELISA Substrate Solution (ThermoFisher) was added into the wells, and the colorimetric signal was quantified at 620 nm for 10 cycles of 60 seconds kinetic intervals with 10 second shaking duration in a Sunrise absorbance ELISA plate reader (Tecan, Switzerland). For each serum sample, the maximum slope of optical density / minute of all the dilutions of the serum samples was multiplied by the respective dilution factor, and the greatest value was used as arbitrary unit (AU) to represent the respective IgG antibody titers for the experiment. For C6-peptide ELISA, mice were considered seropositive when samples yielded a titer greater than the mean plus 1 .5-fold standard deviation of the anti-C6 peptide IgG titers obtained from the uninfected mice.

[0308] 1 .5 Analysis of bacterial burden from mice bite sites, ear, knee joints, and heart by qPCR Quantitative PCR (qPCR) was performed to quantify B. burgdorferi bacterial burden at the tick bite site, the ear, the knee joint and the heart. DNAwas purified from tissues using EZ-10 Spin Column Animal Genomic DNA Mini-Prep Kit (Bio Basic) by following the vendor’s manual. The quantity and quality of DNA were assessed by measuring the concentration of DNA and the ratio of the UV absorption at 280 nm to 260 nm using a Nanodrop 1000 UV / Vis spectrophotometer. To determine bacterial burdens using qPCR, B. burgdorferi genomic equivalents were calculated using an Applied Biosystems 7500 Real-Time PCR system in conjunction with PowerUp™ SYBR® Green Master Mix (Thermo Fisher Scientific), based on amplification of the B. burgdorferi recA gene using primers BBRecAfp (5' GTGGATCTATTGTATTAGATGAGGCTCTCG-3') and BBRecArp (5' GCCAAAGTTCTGCAACATTAACACCTAAAG-3'). Cycling parameters were 50°C for 2 minutes, 95°C for 10 minutes, and 45 cycles of 95°C for 15 seconds, and 60°C for 1 minute. The number of recA copies was calculated by establishing a threshold cycle standard curve of a known number of recA gene extracted from B31 -5A4, and burdens were normalized to 100 ng of total DNA.

[0309] 1 .6 Analysis of bacterial killing activity (BA5Q values) from serum samples

[0310] Prior to determining the bactericidal activity of the mouse sera, sera were heat treated at 56°C for 30 minutes to inactivate the complement system in these sera. Then, 50 pl of diluted mouse serum (1 :20, 1 :40, 1 :80, 1 :160, 1 :320, 1 :640, 1 :1 ,280, 1 :2,560, 1 : 5,120, or 1 : 10,240) was mixed with 10 pl of complement preserved guinea pig serum (guinea pig complement, MilliporeSigma) as well as B. burgdorferi strain B31 -5A4 (5 * 105cells / ml) in 40 pl of BSK II complete medium and then incubated at 33°C for 24 hours. Surviving bacteria were quantified by counting motile bacterial cells using dark-field microscopy. The survival percentage is the proportion of serum-treated to PBS buffer-treated B. burgdorferi. The 50% borreliacidal titer (BA5o) representing the serum dilution rate that effectively killed 50% of bacterial cells was calculated using dose-response stimulation fitting in GraphPad Prism 9.3.1

[0311] EXAMPLE 2: Generation and characterization of DPS-OspA-ST1 and DPS-CspZ-YA

[0312] The sequence of the ‘DNA protection during starvation protein’ (DPS) from Escherichia coli K12 was obtained from uniport (P0ABT2), as were the sequences of the ‘outer surface protein A’ (OspA) (P0CL66) and the ‘complement regulator-acquiring surface protein 2’ (CRASP-2, CspZ) (050665) of Borrelia burgdorferi B31. To generate DPS-OspA-ST1 , amino acids 11 - 167 of DPS were fused to amino acids 18-273 of OspA, in which amino acids 164-174 were replaced by the homologous sequence from Borrelia afzelii (strain K78) OspA, via a flexible glycine-serine-linker. Amino acids 164-174 of Borrelia burgdorferi OspA share some sequence homology with the human leucocyte function-associated antigen-1 (LFA-1 ), which let to safety concerns after the introduction of LYMErix to the US market. Although these safety concerns have since been refuted, it was still decided to replace this sequence stretch in DPS-OspA- ST1. To generate DPS-CspZ-YA, amino acids 11 -167 of DPS were fused to amino acids 21 - 236 of CspZ via a flexible glycine-serine-linker. Two point mutations (Y207A and Y211 A) were introduced into the CspZ sequence to abolish binding to Factor H, as described in Marcinkiewicz et al., 2018. Within the fusion proteins, DPS acts as a multimerization domain that facilitates assembly of the fusion proteins into homo-dodecameric nanoparticles, displaying OspA-ST1 and CspZ- YA on their surface, respectively (see Figure 1 ). The design of the fusion proteins as described here results in a display of the antigens with their N- terminus facing the nanoparticle core, reflecting closely the expression of the antigens on the surface of Borrelia burgdorferi where the antigens are anchored in the bacterial outer membrane via an N-terminal lipid anchor.

[0313] The sequences for the fusion proteins were synthesized by GeneArt (Invitrogen), subcloned into the pRSET A E. coli expression plasmid (Invitrogen). The resulting plasmids were transformed into BL21 (DE3)pLysS chemically competent E. co / / cells (Invitrogen) by heat shock. The recovered cells were grown over night on LB agar containing ampicillin to select for positive transformants. A single colony for each construct was picked from the plate to generate a glycerol stock for further characterization.

[0314] Optimal temperature and time of induction were determined for both constructs in small scale pilot studies, testing induction temperatures of 37°C, 24°C and 18°C and expression times after induction with 1 mM isopropyl thiogalactopyranosid (IPTG) of 2 hours, 4 hours and 16- 20 hours. Relative expression levels of DPS-OspA-ST1 and DPS-CspZ-YA were determined by subjecting whole cell lysates of culture samples to electrophoresis over 10% SDS- polyacrylamide gels and staining the gels with Coomassie brilliant blue. Based on the results of these pilot studies, for larger scale expression of DPS-OspA-ST1 or DPS-CspZ-YA, an appropriate volume of ampicillin containing LB media (LB Amp) in baffled shaker flasks was inoculated with an overnight starter culture, grown from the respective glycerol stock in LB Amp, to an OD6oo of 0.1 . The culture was then grown at 37°C under vigorous shaking to an OD6OO of 0.6. The temperature was then reduced to 18°C and expression was induced by the addition of IPTG to a final concentration of 1 mM. Cells were grown for 16-20 hours before being harvested by centrifugation.

[0315] For purification, the pellet was resuspended in an appropriate volume of lysis buffer (20 mM NaPO4, 200 mM NaCI, 40 mM imidazole, pH 7.4) and cells were lysed by sonication. Cell debris was removed by centrifugation and the supernatant was further clarified by filtration through a 0.22 pm membrane. The clarified lysate was then loaded onto a 5 ml HisTrap HP column (Cytiva) using an NGC chromatography set up (BioRad) where the nanoparticles were captured via the N-terminal His-tag of the pRSET expression system. The column was washed by increasing the imidazole concentration in the running buffer to 224 mM (40% elution buffer, 20 mM NaPO4, 200 mM NaCI, 500 mM imidazole, pH 7.4), before the nanoparticle was eluted from the column at an imidazole concentration of 408 mM (80% elution buffer). The nanoparticles were then further purified by pooling the elution fractions, concentrating them to a volume of 500 pl using 100 kDa MWCO Amicon spin concentrators (Invitrogen) and running the concentrated eluates over an S200 increase 10 / 300 gl size exclusion column (Cytiva) in TBS (20 mM Tris, 150 mM NaCI, pH 7.6). DPS-OspA-ST1 and DPS-CspZ-YA eluted from the column in single symmetrical peaks at an elution volume of 13.5 ml and 14.1 ml, respectively (Figure 2A). Comparison of these elution volumes with those of proteins of defined molecular weights in a gel filtration standard (BioRad) run over the same column suggested complete and uniform assembly of the fusion proteins into dodecameric nanoparticles (DPS-OspA-ST1 = 49.7 kDa x 12 = 596.4 kDa and DPS-CspZ-YA = 46.8 kDa x 12 = 561.6 kDa). Fractions corresponding to the nanoparticles were pooled, endotoxin was removed using Pierce High Capacity Endotoxin Removal Spin Columns (0.50 ml), and the protein concentration was determined by BCA (Pierce). Purity of the particles was assessed by boiling the samples in Laemmli buffer and electrophoresis over 10% SDS-polyacrylamide gels, followed by staining the gels with Coomassie brilliant blue, upon which a single band at the height of just below 50 kDa and around 45 kDa was detected for DPS-OspA-ST1 and DPS-CspZ-YA, respectively, suggesting high purity of the purified nanoparticles (Figure 2B) .

[0316] EXAMPLE 3: Characterization of antibody responses against a combination of DPS- OspA-ST1 and DPS-CspZ-YA

[0317] To evaluate the immunogenicity of a combination of DPS-OspA-ST1 and DPS-CspZ-YA, BALB / c mice were immunized three times intramuscularly on days 0, 21 and 42 with a formulation containing 1 pg of DPS-OspA-ST1 , 1 pg of DPS-CspZ-YA and 100 pg alum (Alhydrogel adjuvant 2%; InvivoGen) in a total volume of 50 pl TBS (Tris-buffered saline) buffer. Serum was harvested on days 19, 41 , and 62 and analyzed to detect anti-OspA-ST 1 IgG as well as anti-CspZ IgG titers by ELISA. In day 19-serum samples only minor IgG responses were detected against OspA-ST 1 and no responses were detected against CspZ. In contrast to this, IgG responses against both antigens were readily detected after two immunizations in day 41 -serum samples. These responses could be significantly boosted by a third immunization as was evident from the high titers detected for both antigens in day 62- serum samples (Figure 3A, B).

[0318] To assess functionality of the induced anti-OspA antibody responses, the ability of the serum antibodies to compete with the protective monoclonal antibody LA-2 was assessed in a LA-2 competition assay, a well-established surrogate of protection (Schaible et al., 1990). As expected, we did not detect any LA-2 binding antibodies in mice immunized with TBS. Importantly, a substantial amount of LA-2 competing antibodies were detected in serum after three immunizations with a combination of DPS-OspA-ST1 , DPS-CspZ-YA and alum (Figure 4). Our data indicated the efficient induction of antibodies capable of providing protection against Borrelia infection.

[0319] To test the ability of the induced anti-CspZ responses to interfere with the interaction between FH and CspZ, which is an important characteristic of a protective anti-CspZ antibody response (Marcinkiewicz et al., 2020), a FH binding assay was performed (Figure 5). The results indicated that sera from mice immunized with a combination of DPS-OspA-ST1 and DPS- CspZ-YA potently blocked binding of FH to CspZ, with an average 50% inhibition titer of 943 (Figure 5B).

[0320] In conclusion, these results indicated that immunization with a formulation containing DPS- OspA-ST1 and DPS-CspZ-YA in combination induced a strong and functional antibody response against both borrelial antigens. EXAMPLE 4: Assessment of efficacy of a combination of DPS-OspA-ST1 and DPS- CspZ-YA

[0321] To assess the efficacy of a combination of DPS-OspA-ST1 and DPS-CspZ-YA to protect mice from a challenge with Borrelia burgdorferi (strain B31 ) infected ticks and to compare the combination to each vaccine antigen being administered individually, adult female C3H / HeN mice were vaccinated intramuscularly three times on days 0 (prime), 28 (first boost) and 56 (second boost) with a formulation containing 1 pg of DPS-OspA-ST1 , 1 pg of DPS-CspZ-YA or 1 pg of DPS-OspA-ST1 and 1 pg of DPS-CspZ-YA, each formulated with 100 pg alum (Alhydrogel adjuvant 2%; InvivoGen) in TBS buffer. TBS buffer only served as control.

[0322] On day 77, mice were single-housed and challenged with 5 Ixodes scapularis flat nymphs infected with Borrelia burgdorferi (B31 -5A4 strain) each. Ticks were removed three days after challenge (day 80), and on day 98 mice were sacrificed. Blood was collected by cardiac puncture for preparation of final serum samples and tissue was collected from the tick bite sites, ears, knee joints and hearts.

[0323] As a means to assess efficacy of the vaccine, anti-C6 IgG in the serum samples was measured. The C6 peptide was derived from B. burgdorferi VIsE protein (which is not present in the vaccine), and detection of antibodies directed against it is the standard serodiagnosis assay for Lyme disease infection. Out of the mice immunized with the DPS-OspA-ST 1 , DPS- CspZ-YA or a combination of the two, not a single mouse showed a positive response to the C6 peptide, while all mice in the TBS control group had seroconverted, suggesting a successful challenge (Figure 6A).

[0324] Bacterial burden at the tick bite site, the ear, the knee joint and the heart were further quantified by qPCR. Results mirrored and confirmed observations from the C6 peptide ELISA in that no bacterial burden was detected in any sample taken from any of the four sites from mice vaccinated with DPS-OspA-ST 1 , DPS-CspZ-YA or a combination of the two, with detected DNA levels similar to those detected in uninfected mice. In contrast, bacterial DNAwas readily detected in all four sampled sites in unvaccinated, challenged mice (Figure 6B).

[0325] Absence of anti-C6 peptide IgG titers, as well as detectable levels of bacterial DNA in both uninfected and vaccinated mice suggested high efficacy of the DPS-OspA-ST1 , DPS-CspZ- YA or a combination of the two in protection from challenge with Borrelia infected ticks. EXAMPLE 5: Assessment of synergistic effects of antibodies targeting OspA and CspZ

[0326] To assess how the induced antibody responses differ in mice immunized with only DPS-OspA- ST1 or DPS-CspZ-YA, or with a combination of DPS-OspA-ST1 and DPS-CspZ-YA, serum samples collected from mice immunized as described in Example 4 prior to each immunization (days -1 , 27 and 42) and on day 70. The serum was used for measurement of OspA ST1 - specific and CspZ-specific antibody titers by ELISA and for determination of bacterial killing activity.

[0327] After two immunizations, both DPS-OspA-ST1 containing vaccine formulations (containing DPS-OspA either alone or in combination with DPS-CspZ-YA), but not the formulation containing DPS-CspZ-YA only induced a significant anti-OspA-ST1 antibody response when compared to mice immunized with TBS as a negative control (Figure 7A). While there is a trend for the combination of DPS-OspA-ST 1 and DPS-CspZ-YA to induce lower IgG titers, this difference is no longer apparent upon the third immunization (Figure 7B). Similarly, after two immunizations, both DPS-CspZ-YA containing vaccine formulations, but not the one containing DPS-OspA-ST1 only induced a significant anti-CspZ antibody response when compared to mice immunized with TBS as a negative control (Figure 7C). This immune response appeared to be boosted slightly upon a third immunization (Figure 7D). These antibody responses suggested that the two vaccine antigens, i.e., OspA-ST1 and CspZ-YA displayed on DPS nanoparticles, can be combined within a single formulation without any reduction in antibody responses to each antigen when compared to each vaccine antigen being delivered individually.

[0328] While DPS-OspA-ST1 and DPS-OspA-CspZ-YA induced detectable levels of borreliacidal activity after two as well as after three immunizations (BA5o titers of 431 and 873 for DPS- OspA-ST 1 after two and three immunizations, respectively, and BA5o titers of 597 and 71 1 for DPS-CspZ-YA after two and three immunizations, respectively), the combination of both vaccine antigens within one formulation induced significantly higher serum borreliacidal activity at both time points (BA5o titers of 2435 and of 3792 after two and three immunizations, respectively) (Figure 8A, B). Surprisingly, the BA5o titers induced by the combination of DPS- OspA-ST1 and DPS-OspA-CspZ-YA were by a factor of 2- to 3-fold higher than what would have been expected from adding up BA5o titers induced by the individual vaccine antigens. This suggested a synergistic effect of the presence of antibodies against both antigens in the same serum, providing a strong rationale for including both antigens in a single formulation to obtain a highly efficacious Lyme disease vaccine. Final remark: Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.) are hereby incorporated by reference in their entirety. To the extent, the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0329] References

[0330] 1. Kurokawa, C. et al., Interactions between Borrelia burgdorferi and ticks. Nature Reviews Microbiology 18, 587-600 (2020). doi: 10.1038 / s41579-020-0400-5.

[0331] 2. Marcinkiewicz, A. L. et al., Eliminating Factor H-binding activity of Borrelia burgdorferi CspZ combined with virus-like particle conjugation enhances its efficacy as Lyme disease vaccine. Front. Immunol. 9 (2018) doi: 10.3389 / fimmu.2018.00181.

[0332] 3. Marcinkiewicz, A. L. et al., The Factor H-binding site of CspZ as a protective target against multistrain, tick-transmitted Lyme disease. Infect. Immun 88(5) (2020) doi: 10.1 128 / 1 Al.00956- 19.

[0333] 4. Chen, Y.-L. et al., CspZ FH-binding sites as epitopes promote antibody-mediated Lyme Borrelia Clearance. Infection and Immunity 90 (7) (2022) doi: 10.1128 / iai.00062-22.

[0334] 5. Schaible UE. Et al., Monoclonal antibodies specific for the outer surface protein A (OspA) of Borrelia burgdorferi prevent Lyme borreliosis in severe combined immunodeficiency (scid) mice. Proc Natl Acad Sci U S A. 1990 May;87(10):3768-72. doi: 10.1073 / pnas.87.10.3768.

[0335] Sequences

[0336] SEQ ID NO: 1 Nucleic acid sequence encoding E. coli DPS.

[0337] ATGTCCACTGCCAAGCTGGTTAAATCTAAAGCAACCAATCTGCTGTATACCCGTAATGATG

[0338] TTAGCGATAGCGAAAAGAAAGCAACCGTTGAACTGCTGAATCGTCAGGTGATTCAGTTTA

[0339] TTGATCTGAGCCTGATTACCAAACAGGCCCATTGGAATATGCGTGGTGCAAACTTTATTG

[0340] CCGTTCATGAAATGCTGGATGGTTTTCGTACCGCACTGATTGATCATCTGGATACCATGG

[0341] CAGAACGTGCAGTTCAGTTAGGTGGTGTTGCACTGGGTACAACCCAGGTGATTAATAGC

[0342] AAAACACCGCTGAAAAGCTATCCGCTGGATATTCATAATGTTCAGGATCACCTGAAAGAA

[0343] CTGGCAGATCGTTATGCAATTGTTGCCAATGATGTTCGTAAAGCAATTGGCGAAGCAAAA

[0344] GATGATGATACCGCAGATATTCTGACCGCAGCAAGCCGTGATCTGGATAAATTTCTGTGG

[0345] TTTATCGAGAGCAATATTGAA

[0346] SEQ ID NO: 2 Amino acid sequence of E. coli DPS.

[0347] MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWNMRGANFI

[0348] AVHEMLDGFRTALIDHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIHNVQDHLKE

[0349] LADRYAIVANDVRKAIGEAKDDDTADILTAASRDLDKFLWFIESNIE SEQ ID NO: 3 Nucleic acid sequence encoding wild-type Borrelia burgdorferi

[0350] (B31 ) OspA.

[0351] ATGAAGAAATACCTCCTGGGTATCGGCCTGATCCTGGCTCTGATCGCCTGCAAACAGAA TGTTAGCAGCCTGGATGAGAAAAATAGCGTTAGCGTTGATCTGCCTGGTGAAATGAAAG TTCTGGTTAGCAAAGAGAAAAACAAGGACGGCAAATATGATCTGATTGCCACCGTTGATA AACTGGAACTGAAAGGCACCAGCGATAAAAACAATGGTAGTGGTGTTCTGGAAGGTGTG AAAGCAGATAAAAGCAAAGTGAAACTGACCATTAGTGATGATCTGGGTCAGACCACACT GGAAGTTTTTAAAGAAGATGGTAAAACCCTGGTGAGCAAAAAGGTTACCAGCAAAGATAA AAGTAGCACCGAAGAGAAATTCAACGAAAAAGGTGAAGTGAGCGAGAAAATTATCACCC GTGCAGATGGCACCCGTCTGGAATATACCGGCATTAAAAGTGATGGTAGCGGTAAAGCC AAAGAAGTTCTGAAAGGTTACGTGCTGGAAGGCACCCTGACAGCAGAGAAAACAACCC TGGTTGTTAAAGAAGGTACAGTTACCCTGTCCAAAAACATTAGCAAAAGCGGTGAAGTTT CCGTCGAACTGAATGATACCGATAGCAGCGCAGCAACCAAGAAAACCGCAGCATGGAAT AGCGGTACAAGTACCCTGACAATTACCGTGAATAGCAAGAAAACGAAAGATCTGGTGTT CACCAAAGAAAACACCATTACCGTTCAGCAGTATGATAGCAATGGCACCAAACTGGAAG GTAGTGCAGTTGAAATTACGAAACTGGACGAAATCAAAAATGCCCTGAAA

[0352] SEQ ID NO: 4 Amino acid sequence of wild-type Borrelia burgdorferi (B31 ) OspA.

[0353] MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLEL

[0354] KGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEE

[0355] KFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTL

[0356] SKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDS

[0357] NGTKLEGSAVEITKLDEIKNALK

[0358] SEQ ID NO: 5 Nucleic acid sequence encoding Borrelia burgdorferi (B31 ) OspA with amino acids 164-174 replaced by amino acids 164-174 of Borrelia afzelii (K78).

[0359] ATGAAGAAATACCTCCTGGGTATCGGCCTGATCCTGGCTCTGATCGCCTGCAAACAGAA TGTTAGCAGCCTGGATGAGAAAAATAGCGTTAGCGTTGATCTGCCTGGTGAAATGAAAG TTCTGGTTAGCAAAGAGAAAAACAAGGACGGCAAATATGATCTGATTGCCACCGTTGATA AACTGGAACTGAAAGGCACCAGCGATAAAAACAATGGTTCAGGTGTTCTGGAAGGTGTG AAAGCAGATAAAAGCAAAGTGAAACTGACCATTAGTGATGATCTGGGTCAGACCACACT GGAAGTTTTTAAAGAAGATGGTAAAACCCTGGTGAGCAAAAAGGTTACCAGCAAAGATAA AAGTAGCACCGAAGAGAAATTCAACGAAAAAGGTGAAGTGAGCGAGAAAATTATCACCC GTGCAGATGGCACCCGTCTGGAATATACCGGCATTAAAAGTGATGGTAGCGGTAAAGCC AAAGAGGTGCTGAAAAACTTTACCCTGGAAGGTAAAGTGGCCAATGATAAAACAACCCT GGTTGTGAAAGAAGGTACAGTTACCCTGAGTAAAAACATCAGCAAAAGCGGTGAAGTTT CCGTCGAACTGAATGATACCGATAGCAGCGCAGCAACCAAGAAAACCGCAGCATGGAAT AGCGGTACAAGCACCCTGACAATTACCGTGAATAGTAAAAAGACCAAAGACCTGGTGTT CACCAAAGAAAATACCATTACCGTTCAGCAGTATGATAGCAATGGCACCAAACTGGAAGG TTCTGCAGTTGAAATTACGAAACTGGACGAAATCAAAAACGCCCTGAAA

[0360] SEQ ID NO: 6 Amino acid sequence of Borrelia burgdorferi (B31 ) OspA with amino acids 164-174 replaced by amino acids 164-174 of Borrelia afzelii (K78).

[0361] MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLEL KGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEE KFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTL SKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDS

[0362] NGTKLEGSAVEITKLDEIKNALK

[0363] SEQ ID NO: 7 Nucleic acid sequence encoding wild-type Borrelia burgdorferi

[0364] (B31 ) CspZ.

[0365] ATGAAAAAGAGCTTCCTGTCCATCTACATGCTGATTTCCATTTCTCTCCTGAGCTGTGAC GTTAGCCGTCTGAATCAGCGTAACATTAATGAGCTGAAAATCTTCGTGGAAAAAGCCAAG TACTACAGCATTAAACTGGATGCCATTTATAATGAATGCACGGGTGCCTATAACGACATTAT GACCTATAGCGAAGGCACCTTTAGCGATCAGAGCAAAGTTAATCAGGCCATCAGCATCTT CAAAAAGGACAACAAAATCGTGAACAAATTCAAAGAGCTGGAAAAGATCATCGAAGAGTA CAAACCGATGTTTCTGAGCAAACTGATTGATGATTTTGCCATCGAACTGGATCAGGCCGT TGATAATGATGTTAGCAATGCACGTCATGTTGCCGATAGCTATAAAAAGCTGCGTAAAAGC GTTGTGCTGGCCTATATTGAATCCTTTGATGTGATCAGCAGCAAATTCGTGGATAGCAAAT TTGTTGAAGCCAGCAAAAAGTTTGTGAACAAGGCCAAAGAATTTGTGGAAGAGAATGAT CTGATTGCCCTGGAATGTATTGTGAAAACCATTGGCGATATGGTGAATGATCGTGAAATTA ATAGCCGCAGCCGCTATAACAACTTCTATAAGAAAGAAGCCGATTTTCTGGGAGCAGCAG TTGAACTGGAAGGTGCATATAAAGCAATTAAACAGACCCTGCTG

[0366] SEQ ID NO: 8 Amino acid sequence of wild-type Borrelia burgdorferi (B31 ) CspZ.

[0367] MKKSFLSIYMLISISLLSCDVSRLNQRNINELKIFVEKAKYYSIKLDAIYNECTGAYNDIMTYSE

[0368] GTFSDQSKVNQAISIFKKDNKIVNKFKELEKIIEEYKPMFLSKLIDDFAIELDQAVDNDVSNAR

[0369] HVADSYKKLRKSVVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEFVEENDLIALECIVKTIG

[0370] DMVNDREINSRSRYNNFYKKEADFLGAAVELEGAYKAIKQTLL

[0371] SEQ ID NO: 9 Nucleic acid sequence encoding mutant CspZ-YA derived from wild-type Borrelia burgdorferi (B31 ) CspZ.

[0372] ATGAAAAAGAGCTTCCTGTCCATCTACATGCTGATTTCCATTTCTCTCCTGAGCTGTGAC GTTAGCCGTCTGAATCAGCGTAACATTAATGAGCTGAAAATCTTCGTGGAAAAAGCCAAG TACTACAGCATTAAACTGGATGCCATTTATAATGAATGCACGGGTGCCTATAACGACATTAT GACCTATAGCGAAGGCACCTTTAGCGATCAGAGCAAAGTTAATCAGGCCATCAGCATCTT CAAAAAGGACAACAAAATCGTGAACAAATTCAAAGAGCTGGAAAAGATCATCGAAGAGTA CAAACCGATGTTTCTGAGCAAACTGATCGATGATTTTGCCATTGAACTGGATCAGGCCGT TGATAATGATGTGAGCAATGCACGTCATGTTGCAGATAGCTATAAAAAGCTGCGTAAAAG CGTTGTGCTGGCCTATATTGAATCCTTTGATGTGATCAGCAGCAAATTCGTGGATAGCAA ATTTGTTGAAGCCAGCAAAAAGTTTGTGAACAAGGCCAAAGAATTTGTGGAAGAGAATG ATCTGATTGCCCTGGAATGTATCGTTAAAACCATTGGCGATATGGTGAATGATCGCGAAAT TAATAGCCGTAGCCGTGCAAACAATTTTGCAAAGAAAGAAGCAGATTTTCTGGGAGCAG CAGTGGAACTGGAAGGTGCATATAAAGCCATTAAACAGACCCTGCTG

[0373] SEQ ID NO: 10 Amino acid sequence of mutant CspZ-YA derived from wild-type

[0374] Borrelia burgdorferi (B31 ) CspZ.

[0375] MKKSFLSIYMLISISLLSCDVSRLNQRNINELKIFVEKAKYYSIKLDAIYNECTGAYNDIMTYSE

[0376] GTFSDQSKVNQAISIFKKDNKIVNKFKELEKIIEEYKPMFLSKLIDDFAIELDQAVDNDVSNAR HVADSYKKLRKSVVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEFVEENDLIALECIVKTIG DMVNDREINSRSRANNFAKKEADFLGAAVELEGAYKAIKQTLL SEQ ID NO: 11 Nucleic acid sequence encoding a fusion protein comprising amino acids 18-273 of OspA of Borrelia burgdorferi (B31 ) with amino acids 164-174 replaced by amino acids 164-174 of Borrelia afzelii (K78) fused to a DPS subunit.

[0377] GCAACCAATCTGCTGTATACCCGTAATGATGTTAGCGATAGCGAAAAGAAAGCAACCGTT GAACTGCTGAATCGTCAGGTGATTCAGTTTATTGATCTGAGCCTGATTACCAAACAGGCC CATTGGAATATGCGTGGTGCAAACTTTATTGCCGTTCATGAAATGCTGGATGGTTTTCGTA CCGCACTGATTGATCATCTGGATACCATGGCAGAACGTGCAGTTCAGTTAGGTGGTGTT GCACTGGGTACAACCCAGGTGATTAATAGCAAAACACCGCTGAAAAGCTATCCGCTGGA TATTCATAATGTTCAGGATCACCTGAAAGAACTGGCAGATCGTTATGCAATTGTTGCCAAT GATGTTCGTAAAGCAATTGGCGAAGCAAAAGATGATGATACCGCAGATATTCTGACCGCA GCAAGCCGTGATCTGGATAAATTTCTGTGGTTTATCGAGAGCAATATTGAAGGTAGCGGT GGTAGTGGTAAACAGAATGTTAGCAGCCTGGATGAGAAAAATAGCGTTAGCGTTGATCTG CCTGGTGAAATGAAAGTTCTGGTTAGCAAAGAGAAAAACAAGGACGGCAAATATGATCT GATTGCCACCGTTGATAAACTGGAACTGAAAGGCACCAGCGATAAAAACAATGGTTCAG GTGTTCTGGAAGGTGTGAAAGCAGATAAAAGCAAAGTGAAACTGACCATTAGTGATGAT CTGGGTCAGACCACACTGGAAGTTTTTAAAGAAGATGGTAAAACCCTGGTGAGCAAAAA GGTTACCAGCAAAGATAAAAGTAGCACCGAAGAGAAATTCAACGAAAAAGGTGAAGTGA GCGAGAAAATTATCACCCGTGCAGATGGCACCCGTCTGGAATATACCGGCATTAAAAGT GATGGTAGCGGTAAAGCCAAAGAGGTGCTGAAAAACTTTACCCTGGAAGGTAAAGTGGC CAATGATAAAACAACCCTGGTTGTGAAAGAAGGTACAGTTACCCTGAGTAAAAACATCAG CAAAAGCGGTGAAGTTTCCGTCGAACTGAATGATACCGATAGCAGCGCAGCAACCAAGA AAACCGCAGCATGGAATAGCGGTACAAGCACCCTGACAATTACCGTGAATAGTAAAAAGA CCAAAGACCTGGTGTTCACCAAAGAAAATACCATTACCGTTCAGCAGTATGATAGCAATG GCACCAAACTGGAAGGTTCTGCAGTTGAAATTACGAAACTGGACGAAATCAAAAACGCC CTGAAA

[0378] SEQ ID NO: 12 Amino acid sequence of a fusion protein comprising amino acids

[0379] 18-273 of OspA of Borrelia burgdorferi (B31 ) with amino acids 164- 174 replaced by amino acids 164-174 of Borrelia afzelii (K78) fused to a DPS subunit.

[0380] ATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWNMRGANFIAVHEMLDGFRTALI DHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIG EAKDDDTADILTAASRDLDKFLWFIESNIEGSGGSGKQNVSSLDEKNSVSVDLPGEMKVLVS KEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKED GKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLE GKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKK TKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK

[0381] SEQ ID NO: 13 Nucleic acid sequence encoding a fusion protein comprising amino acids 21 -211 of mutant CspZ-YA fused to a DPS subunit.

[0382] GCAACCAATCTGCTGTATACCCGTAATGATGTTAGCGATAGCGAAAAGAAAGCAACCGTT GAACTGCTGAATCGTCAGGTGATTCAGTTTATTGATCTGAGCCTGATTACCAAACAGGCC CATTGGAATATGCGTGGTGCAAACTTTATTGCCGTTCATGAAATGCTGGATGGTTTTCGTA CCGCACTGATTGATCATCTGGATACCATGGCAGAACGTGCAGTTCAGTTAGGTGGTGTT GCACTGGGTACAACCCAGGTGATTAATAGCAAAACACCGCTGAAAAGCTATCCGCTGGA TATTCATAATGTTCAGGATCACCTGAAAGAACTGGCAGATCGTTATGCAATTGTTGCCAAT GATGTTCGTAAAGCAATTGGCGAAGCAAAAGATGATGATACCGCAGATATTCTGACCGCA GCAAGCCGTGATCTGGATAAATTTCTGTGGTTTATCGAGAGCAATATTGAAGGTAGCGGT GGTAGTGGTGTTAGCCGTCTGAATCAGCGTAACATTAATGAGCTGAAAATCTTCGTGGAA

[0383] AAAGCCAAGTACTACAGCATTAAACTGGATGCCATTTATAATGAATGCACGGGTGCCTATA

[0384] ACGACATTATGACCTATAGCGAAGGCACCTTTAGCGATCAGAGCAAAGTTAATCAGGCCA

[0385] TCAGCATCTTCAAAAAGGACAACAAAATCGTGAACAAATTCAAAGAGCTGGAAAAGATCA

[0386] TCGAAGAGTACAAACCGATGTTTCTGAGCAAACTGATCGATGATTTTGCCATTGAACTGG

[0387] ATCAGGCCGTTGATAATGATGTGAGCAATGCACGTCATGTTGCAGATAGCTATAAAAAGC

[0388] TGCGTAAAAGCGTTGTGCTGGCCTATATTGAATCCTTTGATGTGATCAGCAGCAAATTCG

[0389] TGGATAGCAAATTTGTTGAAGCCAGCAAAAAGTTTGTGAACAAGGCCAAAGAATTTGTG

[0390] GAAGAGAATGATCTGATTGCCCTGGAATGTATCGTTAAAACCATTGGCGATATGGTGAAT

[0391] GATCGCGAAATTAATAGCCGTAGCCGTGCAAACAATTTTGCAAAGAAAGAAGCAGATTTT

[0392] CTGGGAGCAGCAGTGGAACTGGAAGGTGCATATAAAGCCATTAAACAGACCCTGCTG

[0393] SEQ ID NO: 14 Amino acid sequence of a fusion protein comprising amino acids

[0394] 21 -21 1 of mutant CspZ-YA fused to a DPS subunit.

[0395] ATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWNMRGANFIAVHEMLDGFRTALI DHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIG EAKDDDTADILTAASRDLDKFLWFIESNIEGSGGSGVSRLNQRNINELKIFVEKAKYYSIKLDAI YNECTGAYNDIMTYSEGTFSDQSKVNQAISIFKKDNKIVNKFKELEKIIEEYKPMFLSKLIDDF AIELDQAVDNDVSNARHVADSYKKLRKSVVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEF VEENDLIALECIVKTIGDMVNDREINSRSRANNFAKKEADFLGAAVELEGAYKAIKQTLL

Claims

PCT ApplicationBavarian Nordic A / SBN122PCTClaims1 . A fusion protein comprising a Lyme disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle, wherein the Lyme disease-associated antigen is an ‘outer surface protein A (OspA) of Borrelia, and wherein the C-terminus of the subunit of the self-assembling protein nanoparticle is joined to the N-terminus of OspA.

2. A fusion protein comprising a Lyme disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling protein nanoparticle, wherein the Lyme disease-associated antigen is a ‘complement regulator-acquiring surface protein 2’ (CspZ) of Borrelia which is a mutant CspZ not binding to complement regulatory protein (CRP) Factor H (FH) of a Borrelia host.

3. The fusion protein of claim 1 or 2, wherein the self-assembling protein nanoparticle is a homo-dodecameric nanoparticle, preferably a ‘DNA protection during starvation protein’ (DPS), more preferably DPS from E. coll.

4. A self-assembling protein nanoparticle comprising monomers of the fusion protein of claim 1 or 3.

5. A self-assembling protein nanoparticle comprising monomers of the fusion protein of claim 2 or 3.

6. A combination of a self-assembling protein nanoparticle of claim 4 and a selfassembling protein nanoparticle of claim 5.

7. A pharmaceutical composition or vaccine comprising a self-assembling protein nanoparticle of claim 4 and / or a self-assembling protein nanoparticle of claim 5, optionally further comprising a pharmaceutically acceptable excipient.

8. The pharmaceutical composition of claim 7, further comprising an adjuvant, preferably alum.

9. A self-assembling protein nanoparticle of claim 4 and / or a self-assembling protein nanoparticle of claim 5 for use in the prevention or treatment of Lyme disease or a condition related to an infection caused by Borrelia.

10. A nucleic acid encoding a fusion protein of claim 1 or 3.

11. A nucleic acid encoding a fusion protein of claim 2 or 3.

12. Use of a nucleic acid of claim 10 and / or 11 for the preparation of a pharmaceutical composition or vaccine.

13. Use of a nucleic acid of claim 10 or 11 for the preparation of a recombinant expression vector, preferably an E. co / / expression plasmid.

14. A recombinant expression vector, preferably an E. co / / expression plasmid, comprising a nucleic acid of claim 10 or 11 .

15. Use of a recombinant expression vector of claim 14 for the preparation of a selfassembling protein nanoparticle.

16. A process for preparing a self-assembling protein nanoparticle of claim 4 or 5 comprising the steps of:(a) providing a nucleic acid of claim 10 or 11 ;(b) preparing a recombinant expression vector comprising the nucleic acid provided in step (a);(c) transforming expression cells with the recombinant expression vector obtained in step (b) and propagating selected transformants;(d) inducing expression of a fusion protein encoded by the nucleic acid provided in step (a);(e) harvesting the self-assembling protein nanoparticle.

17. A combination comprising:(i) a first molecule capable of inducing an immune response in a subject against a Lyme disease-associated antigen, or an antigenic part thereof, wherein the Lyme disease-associated antigen is an ‘outer surface protein A’ (OspA) of Borrelia; and(ii) a second molecule capable of inducing an immune response in a subject against a Lyme disease-associated antigen, or an antigenic part thereof, wherein the Lyme disease-associated antigen is a ‘complement regulatoracquiring surface protein 2’ (CspZ) of Borrelia which is a mutant CspZ not binding to complement regulatory protein (CRP) Factor H (FH) of a Borrelia host.

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