Multimeric LYME disease antigen particles

Abstract

The present invention relates to self-assembling multimeric protein particles displaying Borrelia antigens, namely an encapsulin protein particle displaying an 'outer surface protein A' (OspA) antigen and a DPS (DNA binding protein from starved cells) protein particle displaying a mutant 'complement regulator-acquiring surface protein 2' (CspZ) antigen for use in the vaccination against Lyme disease, particularly in combination.

Description

[0001] PCT Application Bavarian Nordic A / S BN124PCT

[0002] MULTIMERIC LYME DISEASE ANTIGEN PARTICLES

[0003] Technical Field

[0004] The present invention relates to the field of recombinant protein-based vaccines. More specifically, the invention relates to a vaccine against Lyme disease comprising selfassembling multimeric protein particles, namely encapsulin and DPS (DNA binding protein from starved cells), displaying ‘outer surface protein A’ (OspA) and a mutant ‘complement regulator-acquiring surface protein 2’ (CspZ) of Borrelia, respectively. The invention further relates to pharmaceutical compositions comprising the particles and their use in vaccination.

[0005] Background

[0006] 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 sensu stricto, 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 lato.

[0007] 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.

[0008] 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 the 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.

[0009] Thus, there is still a need for efficacious vaccines against Lyme disease.

[0010] Summary of Invention

[0011] It is an objective of the present invention to provide means and methods useful for inducing vaccinal immune protection against Lyme disease.

[0012] The objective of the present invention is solved by the provision of two different selfassembling multimeric protein particles, each displaying another Lyme disease-associated antigen, and their combination in vaccination.

[0013] In particular, the invention is defined by the appended claims and by the following aspects and embodiments.

[0014] 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 multimeric protein particle, wherein the Lyme disease-associated antigen is an ‘outer surface protein A’ (OspA) of Borrelia. Most preferably, the self-assembling multimeric protein particle is an encapsulin.

[0015] 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 multimeric protein particle, wherein the Lyme disease-associated antigen is a ‘complement regulator-acquiring surface protein 2’ (CspZ) of Borrelia which is a mutant CspZ that does not bind to complement regulatory protein (CRP) Factor H (FH) of a Borrelia host. Most preferably, the self-assembling multimeric protein particle is a ‘DNA protection during starvation protein’ (DPS).

[0016] In another aspect, the invention provides a self-assembling multimeric protein particle comprising monomers of the fusion protein comprising OspA as described herein.

[0017] In yet another aspect, the invention provides a self-assembling multimeric protein particle comprising monomers of the fusion protein comprising mutant CspZ as described herein.

[0018] In a further aspect, the invention provides a combination of a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein and a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein.

[0019] In yet a further aspect, the invention provides a pharmaceutical composition or vaccine comprising a self-assembling multimeric protein particle comprising monomers of the fusion protein comprising OspA as described herein and / or a self-assembling multimeric protein particle comprising monomers of the fusion protein comprising mutant CspZ as described herein, optionally further comprising a pharmaceutically acceptable excipient.

[0020] In yet a further aspect, the invention provides a self-assembling multimeric protein particle comprising monomers of the fusion protein comprising OspA as described herein and / or a self-assembling multimeric protein particle 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.

[0021] 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 multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein and / or a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein. 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 multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein and / or a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein.

[0022] In yet a further aspect, the invention provides a nucleic acid encoding a fusion protein comprising OspA as described herein.

[0023] In yet a further aspect, the invention provides a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein.

[0024] 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.

[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 recombinant expression vector.

[0026] 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 multimeric protein particle.

[0027] In yet a further aspect, the invention provides a process for preparing a self-assembling multimeric protein particle as described herein comprising the steps of:

[0028] (a) providing a nucleic acid as described herein;

[0029] (b) preparing a recombinant expression vector comprising the nucleic acid provided in step (a);

[0030] (c) transforming expression cells with the recombinant expression vector obtained in step (b) and propagating selected transformants;

[0031] (d) inducing expression of a fusion protein encoded by the nucleic acid provided in step (a);

[0032] (e) harvesting the self-assembling multimeric protein particle. Brief Description of Drawings / Figures

[0033] Figure 1 illustrates the construct design of Encapsulin-OspA-ST1 , DPS-OspA-ST1 and DPS-CspZ- YA monomers and the resulting protein particles.

[0034] Surface representations of the assembled icosahedral encapsulin homo 60-mer (PDB: 7mu1 ), monomeric OspA of Borrelia burgdorferi strain B31 (PDB: 2G8C) as well as of the assembled DPS dodecamer (PDB: 1 JTS) and monomeric CspZ of Borrelia burgdorferi strain B31 (PDB: 6ATG) were generated from Protein Data Bank (PDB) files using the PyMOL Molecular Graphics System Version 2.5.5. Fusion sites on the assembled homo-oligomers and the respective antigens are indicated by in the structures by asterisks. Below the surface structures, Encapsulin-OspA-ST1 , DPS-OspA-ST1 and DPS-CspZ- YA domain composition, respectively, is depicted as a bar diagram. The checkered box at the DPS N-terminus 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. In all constructs the subunit of the self-assembling homo-oligomer, which acts as the multimerization domain for protein particle auto-assembly and facilitates assembly of the fusion proteins into antigen displaying protein particles is followed by a glycine-serine linker (striped box) and the respective antigen (OspA-ST1 or CspZ- YA). Structures are depicted to scale. Width of the boxes in the bar diagram is proportional to the molecular weight of the respective protein domains.

[0035] Figure 2 shows a characterization of Encapsulin-OspA-ST1 , DPS-OspA-ST1 and DPS- CspZ- YA protein particles.

[0036] (A) Size exclusion chromatogram of Encapsulin-OspA-ST1 , DPS-OspA-ST1 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 and 44 kDa size standards are indicated by dashed lines (from left to right). (B) Samples of Encapsulin-OspA-ST1 , 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 Encapsulin-OspA-ST1 (left lane), DPS-OspA (middle lane) and DPS-CspZ-YA (right lane) fusion proteins as well as any proteinaceous contaminants. Figure 3 shows anti-OspA-ST 1 total IgG in serum of vaccinated mice.

[0037] BALB / c mice were immunized intramuscularly on day 0 (prime), day 21 (first boost) and day 42 (second boost) with Tris-buffered saline (TBS) (“BB2”, negative control) or a formulation containing 1 pg DPS-OspA-ST1 , 1 pg Encapsulin-OspA-ST1 or 1 pg monomeric OspA-ST1 protein. As an adjuvant either 100 pg alum (Alhydrogel adjuvant 2%; InvivoGen) or 50 pg ODN 1018 (CpG 1018®; InvivoGen) was used in the formulation. Peripheral blood was drawn on days 20, 40, and 62 after the prime immunization and serum was analyzed to detect serotype (ST) 1 specific anti-OspA IgG titers by ELISA. Mouse antibodies were detected using antimouse IgG (Fc) antibody conjugated to horseradish peroxidase (HRP). Arbitrary titers (AU) were calculated via a 4PL-fit curve with an intercept at OD = 0.3. Data shown as mean ± SEM.

[0038] Figure 4 shows the induction of LA-2 competing antibodies in serum of vaccinated mice.

[0039] BALB / c mice were immunized as described for Figure 3, i.e., intramuscularly on day 0 (prime), day 21 (first boost) and day 42 (second boost) with TBS (“BB2”, negative control) or a formulation containing 1 pg DPS-OspA-ST1 , 1 pg Encapsulin-OspA-ST1 or 1 pg monomeric OspA-ST1 protein. As an adjuvant either 100 pg alum or 50 pg ODN 1018 was used. Serum obtained on day 40 and day 62 was used to detect the level of LA-2 specific antibodies by performing an LA-2 competition ELISA. 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 1040 ng / ml). Data shown as mean ± SEM.

[0040] Figure 5 shows the bactericidal activity in serum of vaccinated mice.

[0041] BALB / c mice were immunized as described for Figure 3, i.e., intramuscularly on day 0 (prime), day 21 (first boost) and day 42 (second boost) with TBS (“buffer”’, negative control) or a formulation containing 1 pg DPS-OspA-ST1 , 1 pg Encapsulin-OspA-ST1 or 1 pg monomeric OspA-ST1 protein. As an adjuvant either 100 pg alum or 50 pg ODN 1018 was used in the formulation. Serum obtained on day 40 and day 62 was analyzed to detect bactericidal activity against Borrelia burgdorferi B31 -5A4 cultures. Bactericidal activity to kill 50% of the bacteria (50% borreliacidal titer, BA5o) provided by serum from animals immunized twice (“day 40”) or three times (“day 62”). Data shown as mean ± SEM. N.K. = No killing.

[0042] Figure 6 shows the efficacy of immunization against infected tick challenge in mice.

[0043] C3H / HeN mice were vaccinated intramuscularly on days 0 (prime) and 28 (boost) with a formulation containing 5 pg of DPS-OspA-ST1 or 5 pg of Encapsulin-OspA-ST1 , each formulated with 50 pg ODN 1018 in TBS. TBS buffer only served as infection control. On day 49, mice were challenged with 5 Ixodes scapularis flat nymphs infected with Borrelia burgdorferi (B31 -5A4 strain) each. Ticks were removed three days after the challenge (day 52), 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 70. (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 anti-mouse IgG (Fc) antibody conjugated to HRP. A.U. = Arbitrary units. (B) Tissue samples were analyzed for bacterial burden by qPCR. Data shown as mean ± SEM.

[0044] Figure 7 shows the bactericidal activity in serum of vaccinated mice.

[0045] C3H / HeN mice were vaccinated intramuscularly on days 0 (prime) and 28 (boost) with a formulation containing 5 pg of DPS-OspA-ST1 or 5 pg of Encapsulin-OspA-ST1 , each formulated with 50 pg ODN 1018 in TBS buffer. Peripheral blood was withdrawn on day 27 and day 42 after prime immunization, and serum was analyzed to detect bactericidal activity against Borrelia burgdorferi B31 -5A4 cultures. Bactericidal activity to kill 50% of the bacteria (50% borreliacidal titer, BA5o) provided by serum from animals immunized once (A) or twice(B). Data shown as mean ± SEM. NK = No killing.

[0046] Figure 8 shows the anti-OspA-ST 1 antibody response induced by different doses of DPS-OspA-ST1 or Encapsulin-OspA-ST1 protein particles.

[0047] BALB / c mice were immunized intramuscularly on days 0, 21 and 42 with a formulation containing increasing amounts (0.1 pg, 1 pg, 10 pg) of DPS-OspA-ST1 or Encapsulin-OspA- ST1 , adjuvanted with 50 pg ODN 1018. As a negative control, mice received TBS by intramuscular injection on the same days. Serum was collected on days 20 and 41 and analyzed by ELISA to detect anti-OspA-ST1 IgG titers. Mouse antibodies were detected using anti-mouse IgG (Fc) antibody conjugated to HRP. Arbitrary titers (AU) were calculated via a 4PL-fit curve with an intercept at OD = 0.3. Data shown as mean ± SEM.

[0048] Figure 9 shows the bactericidal activity in serum of mice vaccinated with different doses of DPS-OspA-ST1 and Encapsulin-OspA-ST1 protein particles.

[0049] BALB / c mice were intramuscularly immunized on days 0, 21 and 42 with increasing amounts (0.1 pg, 1 pg, 10 pg) of DPS-OspA-ST1 or Encapsulin-OspA-ST1 , adjuvanted with 50 pg ODN 1018. As a negative control, mice received TBS by intramuscular injection on the same days. Serum was collected on days 20 and 41 and analyzed to detect bactericidal activity against Borrelia burgdorferi B31 -5A4 cultures. (A) Bactericidal activity to kill 50% of the bacteria (50% borreliacidal titer, BA50) provided by serum from animals immunized once with Encapsulin- OspA-ST1 + ODN 1018. (B) 50% borreliacidal titer provided by serum from animals immunized twice with Encapsulin-OspA-ST1 + ODN 1018 or DPS-OspA-ST1 + ODN 1018. Data shown as mean ± SEM.

[0050] Figure 10 shows anti-OspA-ST 1 total IgG titers in serum of mice vaccinated with different doses of Encapsulin-OsA-ST1 protein particles.

[0051] BALB / c mice were immunized intramuscularly on days 0, 21 and 42 with a formulation containing 10 pig, 1 pg, 0.2 pg, 0.04 pg, 0.008 pg, or 0.00016 pg Encapsulin-OspA-ST1 , adjuvanted with 10 pg ODN 1018. As a negative control, some mice were intramuscularly injected with TBS on the same days. Peripheral blood was withdrawn on days 20, 41 , and 62 after the prime immunization and serum was analyzed to detect serotype (ST) 1 specific anti- OspA IgG titers by ELISA. Mouse antibodies were detected using anti-mouse IgG (Fc) antibody conjugated to HRP. Arbitrary titers (AU) were calculated via a 4PL-fit curve with an intercept at OD = 0.3. Data shown as mean ± SEM.

[0052] Figure 11 shows the induction of LA-2 competing antibodies in serum of mice vaccinated with different doses of Encapsulin-OsA-ST 1 protein particles.

[0053] BALB / c mice were immunized as described for Figure 10, i.e., intramuscularly on days 0, 21 and 42 with a formulation containing 10 pg, 1 pg, 0.2 pg, 0.04 pg, 0.008 pg, or 0.00016 pg Encapsulin-OspA-ST1 , adjuvanted with 10 pg ODN 1018. TBS served as a negative control. Serum obtained on day 62 was used to detect the level of LA-2 specific antibodies by performing an LA-2 competition ELISA. The starting concentration of HRP-conjugated anti- OspA LA-2 antibody for establishing a standard reference curve was 520 ng / ml. Data shown as mean ± SEM.

[0054] Figure 12 shows anti-OspA-ST1 total IgG and anti-CspZ total IgG in serum of mice vaccinated with a combination of Encapsulin-OspA-ST1 and DPS-CspZ-YA protein particles.

[0055] BALB / c mice were immunized intramuscularly on days 0, 21 and 42 with a formulation containing 1 pg of Encapsulin-OspA-ST1 , 1 pg of DPS-CspZ-YA or a mixture of Encapsulin- OspA-ST1 and DPS-CspZ-YA. 10 pg ODN 1018 was used as an adjuvant. Serum was collected on days 20, 41 , and 62 and analyzed by ELISA to detect anti-OspA-ST1 IgG and anti-CspZ IgG titers. Mouse antibodies were detected using anti-mouse IgG (Fc) antibody conjugated to 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. Figure 13 shows the induction of LA-2 competing antibodies in serum of mice vaccinated with a combination of Encapsulin-OspA-ST1 and DPS-CspZ-YA protein particles.

[0056] BALB / c mice were immunized intramuscularly on days 0, 21 and 42 with a formulation containing 1 pg of Encapsulin-OspA-ST1 , 1 pg of DPS-CspZ-YA or a mixture of Encapsulin- OspA-ST1 and DPS-CspZ-YA. 10 pg ODN 1018 was used as an adjuvant. Peripheral blood was withdrawn on day 62 and serum was analyzed by an LA-2 competition ELISA to assess the level of LA-2 specific antibodies. The starting concentration of HRP-conjugated anti-OspA LA-2 antibody for establishing a standard reference curve was 260 ng / ml. Data shown as mean ± SEM (n=5).

[0057] Figure 14 shows the induction of FH-blocking antibodies in serum of vaccinated mice.

[0058] BALB / c mice were immunized intramuscularly on days 0, 21 and 42 with a formulation containing 1 pg of Encapsulin-OspA-ST1 , 1 pg of DPS-CspZ-YA or a mixture of Encapsulin- OspA-ST1 and DPS-CspZ-YA, adjuvanted with 10 pg ODN 1018. 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. 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 (B) The FH binding rate of negative controls was set as 100% and a 4P L-f it curve was calculated for each sample to determine the titer at 50% inhibition. Data shown as mean ± SEM (n=5).

[0059] Figure 15 shows the induction of CspZ-specific T-cell responses in vaccinated mice.

[0060] BALB / c mice were immunized intramuscularly on days 0, 21 and 42 with a formulation containing 1 pg of Encapsulin-OspA-ST1 , 1 pg of DPS-CspZ-YA or a mixture of Encapsulin- OspA-ST1 and DPS-CspZ-YA, adjuvanted with 10 pg ODN 1018. On day 62 after prime immunization, mice were sacrificed, splenocytes were isolated and analyzed by IFNy ELISPOT to detect CspZ-specific T-cell responses after three immunizations. (A) The number of IFNy+spots are shown for the samples restimulated with the peptide pool 1 , in absence or presence of MHC Class II blocking antibody, respectively. (B) The number of IFNy+spots are shown for the samples restimulated with the peptide pool 3, in absence or presence of MHC Class II blocking antibody, respectively. Data shown as mean ± SEM (n=5). Brief Description of Sequences

[0061] SEQ ID NO: 1 is a nucleic acid sequence encoding encapsulin.

[0062] SEQ ID NO: 2 is the amino acid sequence of encapsulin.

[0063] SEQ ID NO: 3 is a nucleic acid sequence encoding DPS.

[0064] SEQ ID NO: 4 is the amino acid sequence of DPS.

[0065] SEQ ID NO: 5 is a nucleic acid sequence encoding wild-type OspA of Borrelia burgdorferi.

[0066] SEQ ID NO: 6 is the amino acid sequence of wild-type OspA of Borrelia burgdorferi.

[0067] SEQ ID NO: 7 is a nucleic acid sequence encoding an OspA composed of amino acid stretches of OspA from Borrelia burgdorferi and Borrelia afzelii.

[0068] SEQ ID NO: 8 is the amino acid sequence of an OspA composed of amino acid stretches of OspA from Borrelia burgdorferi and Borrelia afzelii.

[0069] SEQ ID NO: 9 is a nucleic acid sequence encoding wild-type CspZ of Borrelia burgdorferi.

[0070] SEQ ID NO: 10 is the amino acid sequence of wild-type CspZ of Borrelia burgdorferi.

[0071] SEQ ID NO: 11 is a nucleic acid sequence encoding a mutant CspZ (“CspZ-YA”) derived from wild-type CspZ of Borrelia burgdorferi.

[0072] SEQ ID NO: 12 is the amino acid sequence of a mutant CspZ (“CspZ-YA”) derived from wild-type CspZ of Borrelia burgdorferi.

[0073] SEQ ID NO: 13 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: 7, 8) fused to an encapsulin subunit.

[0074] SEQ ID NO: 14 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:

[0075] 7, 8) fused to an encapsulin subunit.

[0076] SEQ ID NO: 15 is a nucleic acid sequence encoding a fusion protein comprising amino acids 21 -211 of mutant CspZ-YA (see SEQ ID NO: 11 , 12) fused to a DPS subunit.

[0077] SEQ ID NO: 16 is the amino acid sequence of a fusion protein comprising amino acids 21-211 of mutant CspZ-YA (see SEQ ID NO: 11 , 12) fused to a DPS subunit. SEQ ID NO: 17 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: 7, 8) fused to a DPS subunit.

[0078] SEQ ID NO: 18 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: 7, 8) fused to a DPS subunit.

[0079] Detailed Description of Invention

[0080] 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.

[0081] 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, i.e., Y207A / Y211A (CspZ- Y207A / Y211 A, “CspZ- YA”), 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).

[0082] Here we describe the design of a vaccine antigen consisting of OspA-ST 1 fused to the ‘Type 1 encapsulin shell protein’ (encapsulin) from Thermotogo maritima, resulting in 60-meric protein particles displaying 60 copies of OspA (“Encapsulin-OspA-ST 1 ”). We furthermore describe the design of two vaccine antigens consisting of either OspA-ST1 or CspZ-YA fused to the ‘DNA protection during starvation protein’ (DPS) from E. coli, resulting in dodecameric protein particles displaying twelve copies of OspA-ST1 (“DPS-OspA-ST1 ”) or CspZ-YA (“DPS-CspZ- YA”), respectively.

[0083] Particularly, we describe the combination of Encapsulin-OspA-ST1 and DPS-CspZ- YA protein particles in a multi-stage Lyme disease vaccine targeting Borrelia at two stages of their lifecycle. In previous studies using a combination of DPS-OspA-ST1 and DPS-CspZ- YA protein particles, we had already shown synergistic effects of this antigen-combination (i.e., OspA-ST1 and CspZ-YA) on bacterial killing.

[0084] Here we did show that using a vaccine comprising encapsulin- or DPS-presented OspA-ST1 in a formulation with alum or ODN 1018 as an adjuvant induced higher levels of OspA-ST1 - specific antibodies in mice compared to a monomeric protein vaccine formulated with the same adjuvants. Importantly, using a tick challenge model with Borrelia burgdorferi (strain B31 ) infected ticks we demonstrated that two immunizations with either Encapsulin-OspA-ST 1 or DPS-OspA-ST 1 formulated with ODN 1018 was highly protective against Borrelia infection.

[0085] Moreover, we showed that achieving maximal OspA-ST1 -specific antibody responses in mice required lower doses of Encapsulin-OspA-ST1 compared to DPS-OspA-ST1 , potentially allowing for a significant dose sparing.

[0086] In addition, our results demonstrated that using a vaccine comprising encapsulin-presented OspA-ST 1 and DPS-presented mutant CspZ-YA in a formulation with ODN 1018 induced both high levels of OspA-ST1 -specific as well as CspZ-specific antibodies in mice. No interference was observed when both self-forming protein particles and antigens were combined.

[0087] Furthermore, we report the generation of antibodies that were 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.

[0088] Finally, it was demonstrated that the CspZ-YA included in the vaccine induced efficient production of CspZ-specific antibodies, which could effectively inhibit FH binding to Borrelia CspZ. Additionally, immunization with DPS-CspZ- YA also led to CspZ-specific CD4 and CD8 T cell responses.

[0089] Definitions

[0090] 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. 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.”

[0091] 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.

[0092] 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.

[0093] 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.

[0094] The term “recombinant” as used herein refers to nucleic acids or proteins not occurring naturally but being the result of genetic engineering.

[0095] The term “construct” as used herein refers to an artificial nucleic acid, peptide or protein being the result of genetic engineering.

[0096] 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 particle subunit. Such a fusion protein may be referred to herein as “monomer”, particularly in the context of a protein particle.

[0097] The term “self-assembling multimeric protein particle” as used herein, sometimes briefly referred to herein as “protein particle”, refers to a polymeric assembly of monomeric polypeptides referred to as “subunits” that are capable of directing their self-assembly into the protein particle. Sometimes, as in scientific literature (see, e.g., Zang etal., 2020), such protein particles are referred to as “nanoparticles”, “self-assembling nanoparticles” or “selfassembling protein nanoparticles”.

[0098] The term “self-assembling multimeric protein particle” or “protein particle” may refer to a protein particle in which the assembled monomers are no fusion protein, i.e., not fused to an antigen. In this case, the protein particle does not display an antigen. Alternatively, the term may also refer to a protein particle in which the assembled monomers are fusion proteins of a protein particle subunit and an antigen. In this case, the protein particle is assumed to display the antigen on its surface.

[0099] The term “multimeric antigen particle” as used herein refers to a self-assembling multimeric protein particle displaying or presenting antigens or antigenic determinants on its surface.

[0100] 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.

[0101] 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.

[0102] The term “mutant CspZ” as used herein refers to CspZ (complement regulator-acquiring surface protein 2) not binding to complement regulatory protein (CRP) Factor H (FH) of a potential Borrelia host (who most preferably is a human vaccinee). Preferably, the mutant CspZ contains the mutation of two conserved tyrosines within the FH binding site of CspZ, i.e., Y207A / Y211 A, which abolishes the FH-CspZ interaction. Abbreviations

[0103] AU arbitrary units

[0104] BA bacterial killing activity, also borreliacidal activity

[0105] BA50 50% borreliacidal titer representing the serum dilution rate that effectively kills 50% of bacterial cells

[0106] BB2 basic buffer 2

[0107] C6 peptide derived from the invariable region 6 of the 'variable major protein-like sequence, expressed' (VIsE) protein of Borrelia burgdorferi

[0108] CRP complement regulatory protein

[0109] CspZ complement regulator-acquiring surface protein 2 of Borrelia

[0110] CspZ-YA mutant CspZ with point mutations Y207A and Y211 A

[0111] CV column volume

[0112] DPS DNA protection during starvation protein (also known as ‘DNA binding protein from starved cells’)

[0113] DPS-OspA-ST1 DPS presenting 12 copies of OspA serotype 1 (ST 1 )

[0114] DPS-CspZ-YA DPS presenting 12 copies of CspZ-YA

[0115] ELISA enzyme-linked immunosorbent assay

[0116] Encapsulin-OspA-ST 1 encapsulin presenting 60 copies of OspA serotype 1 (ST1 )

[0117] FH Factor H, a complement regulatory protein (CRP)

[0118] HRP horseradish peroxidase

[0119] IMAC immobilized metal ion affinity chromatography

[0120] IPTG isopropyl thiogalactopyranosid

[0121] LA-2 ng eq / ml amount of antibody in ng equivalent to LA-2 antibody

[0122] OD optical density

[0123] ODN 1018 oligodeoxynucleotide 1018 (CpG 1018®).

[0124] OspA outer surface protein A of Borrelia

[0125] PDB Protein Data Bank qPCR quantitative PCR

[0126] SEC size exclusion chromatography

[0127] ST serotype

[0128] TBS Tris-buffered saline

[0129] TMB tetramethylbenzidine

[0130] VIsE variable major protein-like sequence, expressed Aspects and embodiments

[0131] A. Subject-matter relating to OspA

[0132] A.1 Aspects and embodiments relating to fusion proteins comprising OspA

[0133] In one aspect, provided is a fusion protein comprising a Lyme disease-associated antigen, or an antigenic part thereof, joined or fused to a subunit of a self-assembling multimeric protein particle, wherein the Lyme disease-associated antigen is an ‘outer surface protein A’ (OspA) of Borrelia.

[0134] Herein, such fusion protein sometimes is briefly referred to as “fusion protein comprising OspA”.

[0135] In one embodiment, the fusion protein comprising OspA is a monomer.

[0136] 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 multimeric protein particle composed of monomers of the fusion protein.

[0137] In one embodiment of the fusion protein comprising OspA, the subunit of a self-assembling multimeric protein particle is capable of being joined to an antigen (e.g., OspA) such that the antigen is displayed with its N-terminus facing the protein particle core.

[0138] In one embodiment of the fusion protein comprising OspA, the subunit of a self-assembling multimeric protein particle is capable of being joined to the N-terminus of an antigen (e.g., OspA) via the C-terminus of the subunit.

[0139] In one embodiment of the fusion protein, the C-terminus of the subunit of the self-assembling multimeric protein particle is joined to the N-terminus of OspA.

[0140] In one embodiment of the fusion protein comprising OspA, the self-assembling multimeric protein particle is a homo-multimeric protein particle, preferably a 60-mer protein particle.

[0141] In one embodiment of the fusion protein comprising OspA, the self-assembling multimeric protein particle is an encapsulin, preferably Type 1 encapsulin shell protein from Thermotoga maritima.

[0142] In one embodiment of the fusion protein comprising OspA, the amino acid sequence of the subunit of encapsulin 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 multimeric protein particle.

[0143] In one embodiment of the fusion protein comprising OspA, the amino acid sequence of the subunit of encapsulin comprises or consists of amino acids 2-265 of the amino acid sequence as depicted in SEQ ID NO: 2.

[0144] In one embodiment of the fusion protein comprising OspA, the amino acid sequence of the subunit of encapsulin is encoded by a 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.

[0145] In one embodiment of the fusion protein comprising OspA, the amino acid sequence of the subunit of encapsulin 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 .

[0146] 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.

[0147] 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.

[0148] In one embodiment, OspA is OspA serotype 1 (OspA-ST1 ).

[0149] 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: 6 or SEQ ID NO: 8, or a part of the amino acid sequence as depicted in SEQ ID NO: 6 or SEQ ID NO: 8.

[0150] 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: 6 or SEQ ID NO: 8, or a part of the amino acid sequence as depicted in SEQ ID NO: 6 or SEQ ID NO: 8.

[0151] 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: 5 or SEQ ID NO: 7 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO: 5 or SEQ ID NO: 7. 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: 8.

[0152] In one embodiment of the fusion protein, the OspA is fused to the subunit of a self-assembling multimeric protein particle via a peptide linker, preferably a flexible glycine-serine linker.

[0153] 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: 14 or a part of the amino acid sequence as depicted in SEQ ID NO: 14.

[0154] 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.

[0155] 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.

[0156] Nucleic acid encoding the fusion protein

[0157] In one aspect, provided is a nucleic acid encoding a fusion protein comprising OspA as described herein.

[0158] 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: 13 or a part thereof.

[0159] 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: 14 or a part thereof.

[0160] 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.

[0161] 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.

[0162] In one embodiment, the nucleic acid encoding a fusion protein comprising OspA is used for the preparation of a vaccine other than a protein-based vaccine, e.g., a vaccine based on a virus-based vector, such as a recombinant poxvirus or a recombinant virus replicon particle (VRP), or a vaccine based on a nucleic acid, such as a messenger RNA (mRNA) or a selfamplifying RNA (saRNA). Recombinant expression vector encoding the fusion protein

[0163] 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.

[0164] 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 multimeric protein particle.

[0165] A.2 Aspects and embodiments relating to self-assembling multimeric protein particles comprising OspA

[0166] In one aspect, provided is a self-assembling multimeric protein particle comprising monomers of the fusion protein comprising OspA as described herein (see A.1 above).

[0167] Herein, such self-assembling multimeric protein particle may be briefly referred to as “selfassembling multimeric protein particle comprising OspA”.

[0168] In one embodiment, the self-assembling multimeric protein particle comprising OspA is a homo-multimeric protein particle and preferably comprises or consists of 60 monomers of the fusion protein comprising OspA as described herein.

[0169] In one embodiment of the self-assembling multimeric protein particle comprising OspA, the self-assembling multimeric protein particle is an encapsulin, preferably Type 1 encapsulin shell protein from Thermotoga maritime.

[0170] In one embodiment, the self-assembling multimeric protein particle is displaying OspA antigenic determinants on its surface.

[0171] In one aspect, provided is a use of a self-assembling multimeric protein particle comprising monomers of the fusion protein comprising OspA as described herein for the preparation of a pharmaceutical composition or vaccine.

[0172] In one aspect, provided is a process for preparing a self-assembling multimeric protein particle comprising monomers of the fusion protein comprising OspA as described herein comprising the steps of:

[0173] (a) providing a nucleic acid encoding a fusion protein comprising OspA as described herein;

[0174] (b) preparing a recombinant expression vector comprising the nucleic acid provided in step (a); (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;

[0175] (d) inducing expression of a fusion protein encoded by the nucleic acid provided in step (a);

[0176] (e) harvesting and preferably purifying the self-assembling multimeric protein particle.

[0177] A.3 Aspects and embodiments relating to vaccines and medical uses

[0178] In one aspect, provided is a pharmaceutical composition or vaccine comprising a selfassembling multimeric protein particle comprising monomers of the fusion protein comprising OspA as described herein, optionally further comprising a pharmaceutically acceptable excipient.

[0179] In one embodiment, the pharmaceutical composition or vaccine further comprises an adjuvant.

[0180] In one embodiment of the pharmaceutical composition or vaccine, the adjuvant is alum or a CpG oligodeoxynucleotide (ODN), preferably the CpG ODN is ODN 1018 (CpG 1018®).

[0181] In one aspect, provided is a self-assembling multimeric protein particle 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.

[0182] 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.

[0183] 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 multimeric protein particle comprising monomers of the fusion protein comprising OspA as described herein.

[0184] 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 multimeric protein particle 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. By “increase” is intended that a measure of the immune response (for example, antigen-specific antibodies) is increased in comparison to the level in the subject prior to said administration by at least 10%, 20%, or 50%, or by at least 100%. In other embodiments, “increase” means that a measure of the immune response that was not detectable prior to said administration can be detected following said administration.

[0185] 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.

[0186] A.4 Further aspects and embodiments

[0187] 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.

[0188] 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).

[0189] 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).

[0190] 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.

[0191] 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.

[0192] 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).

[0193] In one embodiment of the pharmaceutical composition comprising an RNA or DNA molecule, the DNA molecule is an expression plasmid. 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.

[0194] B. Subject-matter relating to mutant CspZ

[0195] B.1 Aspects and embodiments relating to fusion proteins comprising mutant CspZ

[0196] In one aspect, provided is a fusion protein comprising a Lyme disease-associated antigen, or an antigenic part thereof, joined or fused to a subunit of a self-assembling multimeric protein particle, wherein the Lyme disease-associated antigen is a ‘complement regulator-acquiring surface protein 2’ (CspZ) of Borrelia which is a mutant CspZ that does not bind to complement regulatory protein (CRP) Factor H (FH) of a Borrelia host.

[0197] Herein, such fusion protein sometimes is briefly referred to as “fusion protein comprising mutant CspZ”.

[0198] In one embodiment, the fusion protein comprising mutant CspZ is a monomer.

[0199] 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 multimeric protein particle composed of monomers of the fusion protein.

[0200] In one embodiment of the fusion protein comprising mutant CspZ, the subunit of a selfassembling multimeric protein particle is capable of being joined to an antigen (e.g., mutant CspZ) such that the antigen is displayed with its N-terminus facing the protein particle core.

[0201] In one embodiment of the fusion protein comprising mutant CspZ, the subunit of a selfassembling multimeric protein particle is capable of being joined to the N-terminus of an antigen (e.g., CspZ) via the C-terminus of the subunit.

[0202] In one embodiment of the fusion protein, the C-terminus of the subunit of the self-assembling multimeric protein particle is joined to the N-terminus of mutant CspZ.

[0203] In one embodiment of the fusion protein comprising mutant CspZ, the self-assembling multimeric protein particle is a homo-dodecameric protein particle, preferably a ‘DNA protection during starvation protein’ (DPS), more preferably DPS from Escherichia coll, even more preferably from E. coll K12.

[0204] 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: 4 or is a part of the amino acid sequence as depicted in SEQ ID NO: 4. 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: 4 or a part of the amino acid sequence as depicted in SEQ ID NO: 4. 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 multimeric protein particle.

[0205] 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: 4.

[0206] 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: 4 or a part of the amino acid sequence as depicted in SEQ ID NO: 4.

[0207] 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: 3 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO: 3.

[0208] 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.

[0209] 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.

[0210] 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-type CspZ of Borrelia burgdorferi B31 is as depicted in SEQ ID NO: 10.

[0211] Herein, such mutant CspZ is also referred to as “CspZ- YA” or “mutant CspZ- YA”.

[0212] 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: 12 or a part of the amino acid sequence as depicted in SEQ ID NO: 12. 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: 12 or a part of the amino acid sequence as depicted in SEQ ID NO: 12.

[0213] 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: 11 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO: 11.

[0214] 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: 12.

[0215] In one embodiment of the fusion protein, the mutant CspZ is fused to the subunit of a selfassembling multimeric protein particle via a peptide linker, preferably a flexible glycine-serine linker.

[0216] 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: 16 or a part of the amino acid sequence as depicted in SEQ ID NO: 16.

[0217] In one embodiment, the amino acid sequence of the fusion protein comprising mutant CspZ is encoded by a nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 16 or a part of the amino acid sequence as depicted in SEQ ID NO: 16.

[0218] In one embodiment, the amino acid sequence of the fusion protein comprising mutant CspZ is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 15 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO: 15.

[0219] Nucleic acid encoding the fusion protein

[0220] In one aspect, provided is a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein.

[0221] 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: 15 or a part thereof.

[0222] 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: 16 or a part thereof. 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.

[0223] 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 pharmaceutical composition or vaccine.

[0224] In one embodiment, the nucleic acid encoding a fusion protein comprising mutant CspZ is used for the preparation of a vaccine other than a protein-based vaccine, e.g., a vaccine based on a virus-based vector, such as a recombinant poxvirus or a recombinant virus replicon particle (VRP), or a vaccine based on a nucleic acid, such as a messenger RNA (mRNA) or a self-amplifying RNA (saRNA).

[0225] Recombinant expression vector encoding the fusion protein

[0226] 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.

[0227] 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 multimeric protein particle.

[0228] B.2 Aspects and embodiments relating to self-assembling multimeric protein particles comprising mutant CspZ

[0229] In one aspect, provided is a self-assembling multimeric protein particle comprising monomers of the fusion protein comprising mutant CspZ as described herein (see B.2 above).

[0230] Herein, such self-assembling multimeric protein particle sometimes is briefly referred to as “self-assembling multimeric protein particle comprising mutant CspZ”.

[0231] In one embodiment, the self-assembling multimeric protein particle is a homo-dodecameric protein particle and preferably comprises or consists of 12 monomers of the fusion protein comprising mutant CspZ as described herein.

[0232] In one embodiment, the self-assembling multimeric protein particle is displaying mutant CspZ antigenic determinants on its surface.

[0233] In one aspect, provided is a use of a self-assembling multimeric protein particle comprising monomers of the fusion protein comprising mutant CspZ as described herein for the preparation of a pharmaceutical composition or vaccine. In one aspect, provided is a process for preparing a self-assembling multimeric protein particle comprising monomers of the fusion protein comprising mutant CspZ as described herein comprising the steps of:

[0234] (a) providing a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein;

[0235] (b) preparing a recombinant expression vector comprising the nucleic acid provided in step (a);

[0236] (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;

[0237] (d) inducing expression of a fusion protein encoded by the nucleic acid provided in step (a);

[0238] (e) harvesting and preferably purifying the self-assembling multimeric protein particle.

[0239] B.3 Aspects and embodiments relating to vaccines and medical uses

[0240] In one aspect, provided is a pharmaceutical composition or vaccine comprising a selfassembling multimeric protein particle comprising monomers of the fusion protein comprising mutant CspZ as described herein, optionally further comprising a pharmaceutically acceptable excipient.

[0241] In one embodiment, the pharmaceutical composition or vaccine further comprises an adjuvant.

[0242] In one embodiment of the pharmaceutical composition or vaccine, the adjuvant is alum or a CpG oligodeoxynucleotide (ODN), preferably the CpG ODN is ODN 1018 (CpG 1018®).

[0243] In one aspect, provided is a self-assembling multimeric protein particle 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.

[0244] 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.

[0245] 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 multimeric protein particle comprising monomers of the fusion protein comprising mutant CspZ as described herein. 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 multimeric protein particle 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. By “increase” is intended that a measure of the immune response (for example, antigen-specific antibodies) is increased in comparison to the level in the subject prior to said administration by at least 10%, 20%, or 50%, or by at least 100%. In other embodiments, “increase” means that a measure of the immune response that was not detectable prior to said administration can be detected following said administration.

[0246] 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.

[0247] B.4 Further aspects and embodiments

[0248] 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.

[0249] 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).

[0250] 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).

[0251] 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.

[0252] 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. 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).

[0253] In one embodiment of the pharmaceutical composition comprising an RNA or DNA molecule, the DNA molecule is an expression plasmid.

[0254] 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.

[0255] C. Subject-matter relating to a combination of OspA and mutant CspZ

[0256] C.1 Aspects and embodiments relating to a combination of self-assembling multimeric protein particles comprising OspA or mutant CspZ

[0257] Herein, “a self-assembling multimeric protein particle 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 multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein” includes references to aspects and embodiments as described in section B above.

[0258] In one aspect, provided is a combination of:

[0259] (i) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein; and

[0260] (ii) a self-assembling protein multimeric particle comprising monomers of a fusion protein comprising mutant CspZ as described herein.

[0261] In one aspect, provided is a use of a combination of:

[0262] (i) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein; and

[0263] (ii) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein; for the preparation of a pharmaceutical composition or vaccine.

[0264] In one aspect, provided is a pharmaceutical composition or vaccine comprising a combination of:

[0265] (i) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein; and

[0266] (ii) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein; optionally further comprising a pharmaceutically acceptable excipient, preferably further comprising an adjuvant.

[0267] In one aspect, provided is a combination of:

[0268] (i) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein; and

[0269] (ii) a self-assembling multimeric protein particle 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.

[0270] In one aspect, provided is a pharmaceutical composition or vaccine comprising a combination of:

[0271] (i) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein; and

[0272] (ii) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein; optionally further comprising a pharmaceutically acceptable excipient, preferably further comprising an adjuvant; for use in the prevention or treatment of Lyme disease, or a condition related to an infection caused by Borrelia.

[0273] In one embodiment of the medical uses as described herein, a combination of:

[0274] (i) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein; and

[0275] (ii) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein; is superior to either (i) or (ii) alone.

[0276] 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:

[0277] (i) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein; and

[0278] (ii) a self-assembling multimeric protein particle 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. 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:

[0279] (i) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein; and

[0280] (ii) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein.

[0281] 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. By “increase” is intended that a measure of the immune response (for example, antigen-specific antibodies) is increased in comparison to the level in the subject prior to said administration by at least 10%, 20%, or 50%, or by at least 100%. In other embodiments, “increase” means that a measure of the immune response that was not detectable prior to said administration can be detected following said administration.

[0282] 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:

[0283] (i) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein; and

[0284] (ii) a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein; is superior to either administration of (i) or (ii) alone. By “superior” is intended that the combination provides greater stimulation of a measure of the immune response than either (i) or (ii) alone.

[0285] 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:

[0286] (i) a first pharmaceutical composition or vaccine comprising a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein; optionally further comprising a pharmaceutically acceptable excipient, preferably further comprising an adjuvant; and

[0287] (ii) a second pharmaceutical composition or vaccine comprising a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein; optionally further comprising a pharmaceutically acceptable excipient, preferably further comprising an adjuvant.

[0288] In one aspect, provided is a kit comprising:

[0289] (i) a first pharmaceutical composition or vaccine comprising a self-assembling multimeric protein particle 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; and

[0290] (ii) a second pharmaceutical composition comprising a self-assembling multimeric protein particle 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.

[0291] In one embodiment of the pharmaceutical composition or vaccine referred to herein (see, e.g., C.1 ), the pharmaceutical composition or vaccine comprises alum or a CpG oligodeoxynucleotide (ODN), preferably the CpG ODN is ODN 1018 (CpG 1018®).

[0292] C.2 Further aspects and embodiments

[0293] In one aspect, provided is a combination comprising:

[0294] (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 that does not bind to complement regulatory protein (CRP) Factor H (FH) of a Borrelia host.

[0295] 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.

[0296] In one embodiment, the first molecule is a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising OspA as described herein.

[0297] In one embodiment, the first molecule is an OspA protein, or an antigenic part thereof.

[0298] In one embodiment, the first molecule is a peptide comprising an antigenic determinant of an OspA protein.

[0299] 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.

[0300] 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.

[0301] In one embodiment, the amino acid sequence of OspA comprises or consists of an amino acid sequence as depicted in SEQ ID NO: 6 or SEQ ID NO: 8, or a part of the amino acid sequence as depicted in SEQ ID NO: 6 or SEQ ID NO: 8.

[0302] 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: 6 or SEQ ID NO: 8, or a part of the amino acid sequence as depicted in SEQ ID NO: 6 or SEQ ID NO: 8.

[0303] In one embodiment, the amino acid sequence of OspA is encoded by a nucleic acid sequence as depicted in SEQ ID NO: 5 or SEQ ID NO: 7 or is encoded by a part of the nucleic acid sequence as depicted in SEQ ID NO: 5 or SEQ ID NO: 7.

[0304] In one embodiment, the first molecule is a nucleic acid encoding an OspA protein, or an antigenic part thereof. In one embodiment, the first molecule is a nucleic acid encoding a fusion protein comprising OspA as described herein.

[0305] 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.

[0306] 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.

[0307] 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).

[0308] 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).

[0309] 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).

[0310] 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.

[0311] In one embodiment, the second molecule is a self-assembling multimeric protein particle comprising monomers of a fusion protein comprising mutant CspZ as described herein.

[0312] In one embodiment, the second molecule is a mutant CspZ protein, or an antigenic part thereof.

[0313] In one embodiment, the second molecule is a peptide comprising an antigenic determinant of mutant CspZ protein.

[0314] 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.

[0315] 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.

[0316] 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: 10.

[0317] In one embodiment, the amino acid sequence of mutant CspZ 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.

[0318] In one embodiment, the amino acid sequence of mutant CspZ is encoded by a 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.

[0319] In one embodiment, the amino acid sequence of mutant CspZ 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 .

[0320] In one embodiment, the second molecule is a nucleic acid encoding a mutant CspZ protein, or an antigenic part thereof.

[0321] In one embodiment, the second molecule is a nucleic acid encoding a fusion protein comprising mutant CspZ as described herein.

[0322] 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.

[0323] 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.

[0324] 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). 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).

[0325] 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).

[0326] 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.

[0327] D. Embodiments relating to SEQ ID NOs

[0328] Regarding SEQ ID NOs: 1 to 18, the disclosure considers certain sequence identities.

[0329] 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,

[0330] 9, 1 1 , 13, 15, and 17.

[0331] 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,

[0332] 10, 12, 14, 16, and 18.

[0333] EXAMPLES

[0334] 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.

[0335] EXAMPLE 1 : Material and methods - Immunological analysis techniques

[0336] 1 .1 Anti-OspA-STI / CspZ IgG ELISA of BALB / c derived sera

[0337] 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 .

[0338] 1 .2 Factor H binding assay

[0339] 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 (for example from TBS injected mice or mice immunized with Encapsulin-OspA-ST1 and DPS- CspZ-YA) 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-horseradish peroxidase (HRP). 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.

[0340] 1 .3 LA-2 competition assay

[0341] ELISA plates were coated with 4 pg / ml OspA-ST 1 protein (full-length) by overnight incubation at 4°C. Plates were washed and blocked for 1 hour at room temperature. Mouse sera were diluted from 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 (a recombinant monoclonal antibody to OspA from hybridoma LA-2; Absolute Antibody) 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 260-1040 ng / ml).

[0342] 1 .4 Analysis of C6 IgG titers from C3H / HeN mice derived serum samples by kinetic ELISA

[0343] Kinetic ELISA protocol was performed to detect immune responses against C6-peptide. 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 anti-mouse 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.

[0344] 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. 1 .6 Analysis of bacterial killing activity (BA5Q values) from serum samples

[0345] 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 x 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.

[0346] 1.7. IFNy ELISPOT

[0347] To detect CspZ-specific T-cell responses, IFNy ELISPOT was performed using the BD™ IFNg ELISPOT Set. ELISPOT plates were coated with unlabeled IFNy capture antibody (1 mg / ml; BD Biosciences) by overnight incubation at 4°C. After washing, 200 pl / well blocking solution was added and incubated for 2 hours at room temperature. Meanwhile, CspZ peptide pools were prepared in T-cell medium (5 pg per peptide / ml). After blocking, 100 pl of different peptide pools covering CspZ protein (5 pg per peptide / ml) and controls as well as 0.5 x 106splenocytes / 100 pl were added to each well and incubated overnight at 37°C in a humidified incubator at 5% CO2.

[0348] After several washes, 100 pl / well of detection antibody (0.5 mg / ml; BD Biosciences) was added and incubated for 2 hours at room temperature. After incubation and several washes, 100 pl of enzyme conjugate (streptavidin-HRP) was added to the wells and incubated for 1 hour at room temperature. After final washes, 100 pl of the final substrate dilution was added to each well and spot development was monitored for 16 minutes. When spots developed, the substrate reaction was stopped by washing the wells with distilled water. The plates were air dried at room temperature for 2 hours and then analyzed using a C.T.L S6 Universal Analyzer machine with ImmunoSpot 6.0.0.2 software. EXAMPLE 2: Generation and characterization of 0spA-ST1 and CspZ-YA displaying protein particles

[0349] 2.1 Generation of Encapsulin-OspA-STI

[0350] The sequence of the Type 1 encapsulin shell protein from Thermotoga maritima (Q9WZP2) and the sequence of the ‘outer surface protein A’ (OspA) of Borrelia burgdorferi strain B31 (P0CL66) were obtained from uniport. To generate Encapsulin-OspA-ST1 , encapsulin was fused to amino acids 18-273 of OspA-ST1 , 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. These safety concerns have since been refuted, however, as a measure of precaution, it was decided to replace this sequence stretch in OspA- ST1.

[0351] Within the fusion protein, encapsulin acts as a multimerization domain that facilitates assembly into homo 60-mer protein particles, displaying OspA-ST1 on their surface (see Figure 1 ). The design of the fusion proteins as described here results in a display of OspA with its N-terminus facing the particle’s surface and the C-terminace facing away from it, reflecting closely the presentation of OspA on the surface of Borrelia burgdorferi where it is anchored in the bacterial outer membrane via an N-terminal lipid anchor.

[0352] The sequence for Encapsulin-OspA-ST was synthesized by GeneArt (Invitrogen), subcloned into the pRSET A E. coll expression plasmid (Invitrogen). The resulting plasmid was 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 was picked from the plate to generate a glycerol stock for further characterization.

[0353] Optimal temperature and time of induction were determined 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 Encapsulin-OspA-ST1 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 those results, for larger scale expression of Encapsulin-OspA-ST1 , 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.

[0354] For purification of Encapsulin-OspA-ST 1 by anion exchange chromatography (AEX) the pellet was resuspended in an appropriate volume of AEX buffer (10 mM sodium phosphate [NaPO4], 20 mM sodium chloride [NaCI], pH 6.0) 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 HiTrap Q HP Anion exchange column (Cytiva) using an NGC chromatography set up (BioRad). Protein particles were captured by the resin due to their negative charge. The column was washed by increasing the NaCI concentration in the running buffer to 118 mM (10% elution buffer, 10 mM sodium phosphate, 1 M sodium chloride, pH 6.0), before the protein particles were eluted from the column at an NaCI concentration of 216 mM (20% elution buffer).

[0355] 2.2 Generation of DPS-OspA-ST 1 and DPS-CspZ-YA

[0356] The sequence of the ‘DNA protection during starvation protein’ (DPS) from Escherichia coli K12 (P0ABT2) and the sequence of the ‘complement regulator-acquiring surface protein 2’ (CRASP-2, CspZ) (050665) of Borrelia burgdorferi B31 were obtained from uniport.

[0357] 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.

[0358] 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 Y211A) were introduced into the CspZ sequence to abolish binding to Factor H, as described in Marcinkiewicz et al., 2018.

[0359] Within the fusion proteins, DPS acts as a multimerization domain that facilitates assembly of the fusion proteins into homo-dodecameric protein particles, displaying OspA-ST1 and CspZ- YAon 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 particle’s surface, 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.

[0360] 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.

[0361] Optimal temperature and time of induction were determined as described above (see Example 2.1 ). 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.

[0362] For purification of DPS-OspA-ST1 or DPS-CspZ-YA, the pellet was resuspended in an appropriate volume of immobilized metal ion affinity chromatography (IMAC) 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 protein particles 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 protein particles were eluted from the column at an imidazole concentration of 408 mM (80% elution buffer).

[0363] 2.3 Further purification and characterization

[0364] Encapsulin-OspA-ST 1 , DPS-OspA-ST 1 and DPS-CspZ-YA each were further purified by size exclusion chromatography (SEC). Elution fractions from preceding chromatography runs were pooled, concentrated to a volume of 500 pl using 100 kDa MWCO Amicon spin concentrators (Invitrogen), and the concentrated eluates were run over an Superose 6 Increase 10 / 300 gL size exclusion column (Cytiva) in Tris-buffered saline (TBS; 20 mM Tris, 150 mM NaCI, pH 7.6).

[0365] Encapsulin-OspA-ST 1 eluted from the column in a peak at an elution volume of just below 0.4 CV (Figure 2A). This elution volume is just above the void volume of the column. The separation range of the Superose 6 column is declared between 5 MDa and 5 kDa. The theoretical molecular weight of Encapsulin-OspA-ST1 is approximately 3.5 MDa (60 x 58,47 kDa = 3508.2 kDa). The elution volume of Encapsulin-OspA-ST1 is therefore in good agreement with an assembled 60-mer protein particle. A second peak is detected at an elution volume of approximately 7.5 CV, which contains contaminants co-purified during AEX.

[0366] DPS-OspA-ST1 eluted from the column in a single symmetrical peak at an elution volume of just above 5,5 CV (Figure 2A). This elution volume was compared to those of proteins of defined molecular weights in a gel filtration standard (BioRad) run over the same column. DPS-OspA-ST1 elutes just before the 669 kDa (Thyroglobulin) marker. The molecular weight of DPS-OspA-ST 1 = 49.7 kDa x 12 = 596.4 kDa, therefore an elution volume just after the 669 kDa band would have been expected. The lower elution volume observed is likely due to the extended, beta-sheet rich fold of OspA.

[0367] DPS-CspZ-YA eluted from the column in a symmetrical peak at an elution volume of just above 0.6 column volume (CV) (Figure 2A), which is close after the elution volume of the 669 kDa marker (Thyroglobulin) of gel filtration standard (BioRad) run over the same column. The theoretical molecular weight of the dodecameric DPS-CspZ-YA protein particle is approximately 560 kDa (46.8 kDa x 12 = 561.6 kDa), which is in good agreement with its elution volume from the Superose6 column. Additional, peaks are detected at approximately 3.5 CV and at just above 5 CV, which likely contain aggregates or higher valency assemblies of the protein particle, respectively.

[0368] The elution volumes of Encapsulin-OspA-ST 1 , DPS-OspA-ST 1 and DPS-CspZ-YA, as well as the symmetrical peak shapes indicated successful and uniform assembly of all fusion proteins into multimeric protein particles (Figure 2A).

[0369] SEC fractions corresponding to the assembled protein particles were pooled, endotoxin was removed using Pierce High Capacity Endotoxin Removal Spin Columns (0.50 ml), and the protein concentration was determined by bicinchoninic acid (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 approximately 55 kDa and just below 50 kDa was detected for Encapsulin-OspA-ST1 and DPS-OspA-ST, respectively, while a band of approximately 45 kDa can be detected for DPS-CspZ, suggesting high purity of the purified protein particles and successful removal of any impurities captured in the first purification step by SEC (Figure 2B). EXAMPLE S: Characterization of antibody responses to DPS-0spA-ST1 or Encapsulin-0spA-ST1 protein particles

[0370] To assess the immunogenic potential of the purified Encapsulin-OspA-ST1 and DPS-OspA- ST1 multimeric protein particles, BALB / c mice were immunized three times intramuscularly, i.e., on days 0, 21 and 42, with a formulation containing 1 pg DPS-OspA-ST1 or 1 pg Encapsulin-OspA-ST1 , adjuvanted with either 100 pg alum (Alhydrogel adjuvant 2%; InvivoGen) or 50 pg ODN 1018 (CpG 1018®; InvivoGen), in a total volume of 50 pl TBS buffer. As control, monomeric OspA-ST 1 protein was intramuscularly injected in combination with the same adjuvants on the same days. Serum was harvested on days 20, 40, and 62 and analyzed for anti-OspA-ST1 IgG titers by ELISA.

[0371] As shown in Figure 3, only minor IgG responses against the OspA-ST 1 antigen were detected in serum samples of day 20 for all mice groups tested. Significant increases in OspA-ST 1 IgG responses were readily detected in DPS-OspA-ST1 and Encapsulin-OspA-ST1 immunized animals after two immunizations (i.e., serum samples of day 40), with a peak response observed following the third vaccination in day 62-serum samples. Monomeric OspA-ST 1 was least efficient in antibody induction across all analyzed time points.

[0372] To evaluate the 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 an LA-2 competition assay, a well-established surrogate of protection (Golde et al., 1997). As expected, no LA-2 binding antibodies were detected in mice immunized with TBS (Figure 4). Importantly, a promising amount of LA-2 competing antibodies were detected in serum already after two immunizations (day 40-serum) with DPS-OspA-ST1 or Encapsulin-OspA-ST1 , combined with alum or ODN 1018. LA-2 antibody titers were boosted after the third immunizations (day 62-serum) for the same groups. Like OspA ST1 IgG, monomeric OspA ST1 did not elicit LA-2 antibodies efficiently.

[0373] Next, the borreliacidal activity of the mouse sera from day 40 and day 62 was determined. As shown in Figure 5, both DPS-OspA-ST1 and Encapsulin-OspA-ST1 elicited detectable levels of borreliacidal activity already after two immunizations (day 40-serum), when adjuvanted with alum or ODN 1018, and this response was strengthened after the third immunization (day 62- serum) (BA50 titers of 427 and 1318 for DPS-OspA-ST 1 + alum, BAsotiters of 813 and 1720 for DPS-OspA-ST1 + ODN 1018, BA50 titers of 515 and 767 for Encapsulin-OspA-ST1 + alum, BA50 titers of 744 and 1677 for Encapsulin-OspA-ST1 + ODN 1018, after two and three immunizations, respectively). In summary, our data showed the efficient induction of OspA-ST1 -specific antibodies by both DPS-OspA-ST1 and Encapsulin-OspA-ST1 immunization, including functional antibodies that correlate with protection against Borrelia infection. Multimeric antigen presentation on the selfassembling multimeric protein particles encapsulin and DPS was considerably more immunogenic than monomeric OspA-ST1 protein.

[0374] EXAMPLE 4: Assessment of efficacy of DPS-OspA-ST1 or Encapsulin-OspA-ST1 protein particles

[0375] To assess the efficacy of purified DPS-OspA-ST1 and Encapsulin-OspA-ST1 to protect mice from a challenge with Borrelia burgdorferi (strain B31 ) infected ticks, adult female C3H / HeN mice were vaccinated intramuscularly on days 0 (prime) and 28 (boost) with a formulation containing 5 pg of DPS-OspA-ST1 or 5 pg of Encapsulin-OspA-ST1 , each formulated with 50 pg ODN 1018 in Tris-buffered saline (TBS). TBS buffer only served as an infection control.

[0376] On day 49, 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 52), and on day 70 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.

[0377] As a means of evaluating the vaccine efficacy, anti-C6 IgG was measured in serum samples by ELISA. The C6 peptide is derived from the B. burgdorferi VIsE protein (which is not present in the vaccine), and detection of antibodies directed against it is the standard serodiagnostic assay for Lyme disease infection. As shown in Figure 6A, none of the mice immunized with purified DPS-OspA-ST1 or Encapsulin-OspA-ST1 adjuvanted with ODN 1018 showed a positive response to the C6 peptide, whereas all TBS-injected mice seroconverted. These data indicate that two immunizations with DPS-OspA-ST1 or Encapsulin-OspA-ST1 provide protection against Lyme disease infection.

[0378] Bacterial burden at the tick bite site, the ear, the knee joint and the heart were further quantified by qPCR. Confirming the results of the C6 peptide ELISA, the qPCR results also showed no bacterial burden in any sample taken from any of the four sites sampled from mice vaccinated with DPS-OspA-ST1 or Encapsulin-OspA-ST1 , and the DNA levels detected were similar to those detected in uninfected mice (Figure 6B). In contrast, bacterial DNA was readily detected in all four sampled sites in unvaccinated, challenged mice. Overall, the absence of anti-C6 peptide IgG titers and bacterial DNA in vaccinated mice demonstrates a high efficacy of the DPS-OspA-ST1 and Encapsulin-OspA-ST1 in protecting against challenge with Borrelia infected ticks.

[0379] EXAMPLE 5: Evaluation of antibodies targeting OspA after immunization with DPS- OspA-ST1 or Encapsulin-OspA-ST1 protein particles

[0380] To assess the efficacy of DPS-OspA-ST 1 or Encapsulin-OspA-ST 1 induced antibodies to elicit killing of borrelia a bacteria killing assay was performed. Adult female C3H / HeN mice were vaccinated intramuscularly on days 0 (prime) and 28 (boost) with a formulation containing 5 pg of DPS-OspA-ST1 or 5 pg of Encapsulin-OspA-ST1 , each formulated with 50 pg ODN 1018 in TBS. TBS buffer only served as an infection control. Serum samples were collected on days 27 and 42.

[0381] As shown in Figure 7A, sera from Encapsulin-OspA-ST 1 immunized mice showed detectable borreliacidal activity after one immunization (serum samples of day 27), while at this time point DPS-OspA-ST1 -induced borreliacidal activity was lower compared to the Encapsulin-OspA- ST1 -induced borreliacidal activity (BA5o titers of 153 and 322 for DPS-OspA-ST1 and Encapsulin-OspA-ST1 , respectively). However, we observed a strong increase in the borreliacidal activity in the serum of both DPS-OspA-ST1 and Encapsulin-OspA-ST1 - immunized animals after the second immunization (day 42-serum; BA50 titers of 983 and 850 for DPS-OspA-ST1 and Encapsulin-OspA-ST1 , respectively) (Figure 7B).

[0382] These data indicate that both DPS-OspA-ST1 and Encapsulin-OspA-ST1 can efficiently induce OspA-targ eting antibodies in C3H mice after two immunizations, providing protection against Borrelia infection. Importantly, serum collected from mice after only one immunization with Encapsulin-OspA-ST1 was more efficient in inducing borrelia killing in vitro compared to DPS-OspA-ST 1 .

[0383] EXAMPLE 6: Assessment of the immunogenicity elicited by different doses of DPS- OspA-ST1 or Encapsulin-OspA-ST1 protein particles

[0384] To understand whether the differences between distinct multimeric particles, e.g. in size and valency, could lead to variances in the efficacy to induce immune responses, an in vivo titration experiment with Encapsulin-OspA-ST1 and DPS-OspA-ST1 was performed. BALB / c mice were immunized intramuscularly on days 0, 21 and 42 with a formulation containing 0.1 pg, 1 pg, or 10 pg of DPS-OspA-ST1 or Encapsulin-OspA-ST1 , adjuvanted with 50 pg ODN 1018, in a total volume of 50 pl TBS buffer. As a negative control, mice received TBS by intramuscular injection on the same days. Serum was collected on days 20 and 41 and analyzed by ELISA to detect anti-OspA-ST1 IgG titers.

[0385] In day 20-serum samples IgG responses against OspA-ST1 could only be detected in Encapsulin-OspA-ST1 immunized mice (Figure 8). After the second immunization (day 41 - serum), a strong boost of antibody responses was observed. However, a clear difference between the immunization with DPS-OspA-ST1 and Encapsulin-OspA-ST1 could be observed: While 0.1 pg DPS-OspA-ST1 elicited no measurable antibody responses on day 41 , 0.1 pg Encapsulin-OspA-ST1 induced high OspA ST1 -specific antibody titers that were comparable to the 1 pg or 10 pg dose. On the other hand, immunization with 10 pg Encapsulin- OspA-ST1 or DPS-OspA-ST1 resulted in comparable anti-OspA IgG titers (Figure 8).

[0386] This data indicates that a higher-valency multimeric protein particle, namely encapsulin, is a more potent inducer of immune responses and therefore may require reduced dosing compared to lower-valency multimeric protein particles like DPS to achieve optimal antibody titers.

[0387] To determine the borreliacidal activity of the mouse sera, serum from days 20 and 41 were analyzed. Interestingly, although low anti-OspA-ST 1 IgG titers were detected in day 20-serum samples, immunization with Encapsulin-OspA-ST1 induced promising BA50 levels at all three doses (0.1 , 1 , 10 pg) (Figure 9A). As expected, the efficacy of bacterial killing was increasing with higher doses. After two immunizations (day 41 -serum) BA50 levels were the highest in the serum from mice immunized with 10 pg Encapsulin-OspA-ST1 + ODN 1018, followed by 10 pg DPS-OspA-ST 1 + ODN 1018 (BA50 titers of 1066 and 967 for 10 pg Encapsulin-OspA-ST 1 + ODN 1018 and DPS-OspA-ST1 + ODN 1018, respectively) (Figure 9B). With lower doses the bacterial killing efficacy decreased with no difference being observed between Encapsulin- OspA-ST1 and DPS-OspA-ST1 at 1 pg or 0.1 pg.

[0388] To evaluate the extent of dose reduction possible to still achieve maximal immune responses with Encapsulin-OspA-ST 1 , BALB / c mice were immunized three times intramuscularly, i.e., on days 0, 21 and 42, with a formulation containing 10 pg, 1 pg, 0.2 pg, 0.04 pg, 0.008 pg, or 0.00016 pg Encapsulin-OspA-ST1 , adjuvanted with 10 pg ODN 1018. As a negative control, some mice were intramuscularly injected with TBS on the same days. Serum was harvested on days 20, 41 , and 62 and analyzed to detect anti-OspA-ST 1 IgG titers by ELISA.

[0389] In day 20-serum samples a distinct anti-OspA ST1 antibody response was already observed in mice that were immunized with 10 pg Encapsulin-OspA-ST1 + ODN 1018, whereas antibody titers in sera of other animal groups were much lower (Figure 10). After the second immunization (day 41 -serum) an efficient boost was observed in nearly all animal groups, except the ones that were immunized with 0.008 pg and 0.00016 pg Encapsulin-OspA-ST1 + ODN 1018, respectively. At this time point, OspA ST 1 IgG titers were very comparable in mice immunized with 10 pg, 1 pg or 0.2 pg Encapsulin-OspA-ST1 + ODN 1018. The highest anti- OspA STI IgG titers were detected in day 62-serum samples of the mice immunized with the highest dose (10 pg) of Encapsulin-OspA-ST1. At this time point a slight boost was seen in most of the mice. No antibody response was detected in mice immunized with 0.00016 pg Encapsulin-OspA-ST1 , indicating that this dose was not sufficient to induce an antibody response against OspA-ST 1 in mice.

[0390] The functionality of the anti-OspA antibody responses elicited by different doses of Encapsulin- OspA-ST1 + ODN 1018 immunizations was tested using day 62-serum samples (collected after three immunizations) by an LA-2 competition assay. As expected, no LA-2 binding antibodies were detected in mice immunized with TBS (Figure 11 ). After three immunizations, distinct LA-2 binding antibody titers were detected in mice immunized with 10 pg, 1 pg, 0.2 pg or 0.04 pg Encapsulin-OspA-ST 1 + ODN 1018, which did not show any significant differences. Compared to these groups, LA-2 antibody titers were much lower in mice immunized with 0.008 pg Encapsulin-OspA-ST1 , while 0.00016 pg Encapsulin-OspA-ST1 did not result in induction of LA-2 binding antibodies even after three immunizations.

[0391] In conclusion, these results showed that two immunizations with a low dose of Encapsulin- OspA-ST1 (-down to 0.2 pg) combined with ODN 1018 can induce strong antibody responses against OspA-ST1 and that a third immunization may be useful if lower doses such as 0.04 pg are planned for the immunizations. Furthermore, not only a high dose such 10 pg, but also a lower dose such as 0.04 pg of Encapsulin-OspA-ST1 can induce a functional antibody response against OspA-ST 1 .

[0392] EXAMPLE 7: Characterization of immune responses against OspA and CspZ elicited by the combination of Encapsulin-OspA-ST1 and DPS-CspZ-YA protein particles

[0393] To investigate the immunogenicity of a combination of Encapsulin-OspA-ST 1 and DPS-CspZ- YA, BALB / c mice were immunized intramuscularly on days 0, 21 and 42 with a formulation containing 1 pg of Encapsulin-OspA-ST1 , 1 pg of DPS-CspZ-YA and 10 pg ODN 1018 in a total volume of 50 pl TBS buffer. Immunizations with 1 pg of Encapsulin-OspA-ST1 or 1 pg of DPS-CspZ-YA (both adjuvanted with ODN 1018) served as controls. Serum was collected on days 20, 41 , and 62 and analyzed by ELISA to detect anti-OspA-ST1 IgG and anti-CspZ IgG titers. IgG responses against OspA-ST1 and CspZ were already detected in the day 20-serum samples (Figure 12A, B). After two immunizations (serum samples of day 41 ), a strong increase in anti-OspA-ST 1 antibody titers was observed, which was not as pronounced as for anti-CspZ antibody titers. In contrast, IgG responses against CspZ were strongly boosted after three immunizations, while the third immunization did not result in a significant boost of anti- OspA-ST1 antibodies. At all time points, antibody induction against OspA and CspZ was comparable between mice immunized with Encapsulin-OspA-ST1 , DPS-CspZ-YA or the combination of Encapsulin-OspA-ST1 and DPS-CspZ-YA.

[0394] To assess the functionality of the induced anti-OspA antibody responses, day 62 serum samples were analyzed by the LA-2 competition assay. As expected, we did not detect any LA-2 binding antibodies in mice immunized with TBS (Figure 13). Importantly, after three immunizations with Encapsulin-OspA-ST1 or the combination of Encapsulin-OspA-ST1 and DPS-CspZ-YA, a substantial amount of LA-2 competing antibodies was detected in the serum (Figure 13). At this time point, the levels of LA-2 antibodies induced by Encapsulin-OspA-ST 1 alone or in combination with DPS-CspZ-YA were quite comparable, indicating that the combination of Encapsulin-OspA-ST1 and DPS-CspZ-YA does not interfere with the efficient induction of antibodies capable of providing protection against Borrelia infection.

[0395] To test the ability of the induced anti-CspZ responses to interfere with the interaction between Factor H (FH) and CspZ, which is an important feature of a protective anti-CspZ antibody response (Marcinkiewicz etal., 2020), a FH binding assay was performed. The results showed that sera from mice immunized not only with DPS-CspZ-YA but also with the combination of Encapsulin-OspA-ST1 and DPS-CspZ-YA potently blocked the binding of FH to CspZ (Figure 14A, B).

[0396] To determine whether immunization with purified DPS-CspZ-YA, used alone or in combination with Encapsulin-OspA-ST1 , elicits CspZ-specific T-cell responses, mice were sacrificed on day 62 after three immunizations, splenocytes were isolated and 0.5 x106cells per sample were restimulated overnight with different CspZ peptide pools consisting of overlapping single peptides. The number of peptide-specific T-cells was determined by counting IFNy-producing T-cells using the IFNy-ELISPOT assay.

[0397] A very weak background response was detected in samples from mice injected with TBS (Figure 15). Importantly, splenocytes from mice immunized with DPS-CspZ-YA alone or in combination with Encapsulin-OspA-ST1 responded to restimulation with CspZ peptide pools 1 and 3, and there was no significant difference in response between these two treatment groups. Interestingly, MHC class II blockade inhibited the response of cells restimulated with pool 1 , whereas it had no effect on cells restimulated with pool 3, indicating that three immunizations with DPS-CspZ-YA+ ODN 1018 alone or the combination of it with Encapsulin- OspA-ST1 can induce both CD4+and CD8+T-cell responses against CspZ.

[0398] Collectively, these data demonstrate that immunization with a formulation containing Encapsulin-OspA-ST1 and DPS-CspZ- YA protein particles alone or in combination induced a potent and functional antibody response against both OspA-ST1 and CspZ- YA. Importantly, no interference between the two antigens or the two different protein particles was observed. This suggests that Encapsulin-OspA-ST1 and DPS-CspZ- YA protein particles 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. Furthermore, DPS-CspZ- YA protein particles are a potent inducer not only of CspZ-specific antibody responses, but also of CspZ-specific T-cell responses.

[0399] 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.

[0400] References

[0401] Chen, Y.-L. et al., CspZ FH-binding sites as epitopes promote antibody-mediated Lyme Borrelia Clearance. Infection Immun 90 (7) (2022). doi: 10.1128 / iai.00062-22.

[0402] Golde, W.T. et al., Reactivity with a specific epitope of outer surface protein A predicts protection from infection with the Lyme disease spirochete, Borrelia burgdorferi. Infection and Immunity 65(3):882-9 (1997); doi: 10.1128 / IAI.65.3.882-889.

[0403] Kurokawa, C. et al., Interactions between Borrelia burgdorferi and ticks. Nature Reviews Microbiology 18, 587-600 (2020); doi: 10.1038 / S41579-020-0400-5.

[0404] 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.

[0405] 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: 10.1128 / 1 Al.00956- 19.

[0406] Zhang, B., et al., A platform incorporating trimeric antigens into self-assembling nanoparticles reveals SARS-CoV-2-spike nanoparticles to elicit substantially higher neutralizing responses than spike alone. Sci Rep 10(1): p. 18149 (2020). Sequences

[0407] SEQ ID NO: 1 Nucleic acid sequence encoding encapsulin.

[0408] ATGGAATTCCTGAAACGTTCTTTCGCCCCACTGACCGAAAAACAGTGGCAGGAGATCGA

[0409] TAATCGTGCTCGCGAAATCTTCAAAACCCAGCTGTACGGTCGCAAATTCGTGGATGTTGA

[0410] AGGTCCATACGGCTGGGAATACGCGGCACATCCTCTGGGCGAAGTCGAAGTTCTGTCC

[0411] GACGAGAACGAGGTGGTTAAATGGGGTCTGCGCAAAAGCCTGCCGCTGATCGAGCTGC

[0412] GTGCGACTTTCACTCTGGATCTGTGGGAACTGGACAACCTGGAACGCGGCAAACCGAA

[0413] CGTCGACCTGTCCTCTCTGGAGGAAACTGTGCGCAAGGTTGCTGAGTTCGAAGATGAA

[0414] GTGATTTTCCGCGGCTGCGAAAAGTCTGGCGTAAAAGGCCTCCTGTCTTTCGAGGAAC

[0415] GTAAGATTGAGTGTGGTTCTACCCCAAAGGATCTCCTGGAAGCGATTGTGCGCGCGCTG

[0416] TCCATCTTCAGCAAGGACGGCATCGAAGGCCCGTATACCCTGGTAATTAACACTGACCG

[0417] CTGGATCAATTTCCTGAAAGAGGAAGCAGGTCACTATCCGCTGGAAAAGCGCGTTGAG

[0418] GAATGCCTGCGCGGTGGCAAAATTATCACGACTCCACGTATTGAAGATGCGCTGGTTGT

[0419] GTCCGAGCGCGGCGGTGACTTCAAGCTGATTCTGGGTCAGGATCTGTCCATCGGCTAT

[0420] GAAGACCGTGAGAAAGACGCAGTCCGTCTGTTCATTACTGAGACCTTTACTTTCCAGGT

[0421] GGTTAACCCGGAAGCACTGATCCTCCTGAAATTC

[0422] SEQ ID NO: 2 Amino acid sequence of encapsulin.

[0423] MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSD ENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIF RGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINF LKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKD

[0424] AVRLFITETFTFQVVNPEALILLKF

[0425] SEQ ID NO: 3 Nucleic acid sequence encoding E. coli DPS.

[0426] ATGTCCACTGCCAAGCTGGTTAAATCTAAAGCAACCAATCTGCTGTATACCCGTAATGATG

[0427] TTAGCGATAGCGAAAAGAAAGCAACCGTTGAACTGCTGAATCGTCAGGTGATTCAGTTTA

[0428] TTGATCTGAGCCTGATTACCAAACAGGCCCATTGGAATATGCGTGGTGCAAACTTTATTG

[0429] CCGTTCATGAAATGCTGGATGGTTTTCGTACCGCACTGATTGATCATCTGGATACCATGG

[0430] CAGAACGTGCAGTTCAGTTAGGTGGTGTTGCACTGGGTACAACCCAGGTGATTAATAGC

[0431] AAAACACCGCTGAAAAGCTATCCGCTGGATATTCATAATGTTCAGGATCACCTGAAAGAA

[0432] CTGGCAGATCGTTATGCAATTGTTGCCAATGATGTTCGTAAAGCAATTGGCGAAGCAAAA

[0433] GATGATGATACCGCAGATATTCTGACCGCAGCAAGCCGTGATCTGGATAAATTTCTGTGG

[0434] TTTATCGAGAGCAATATTGAA

[0435] SEQ ID NO: 4 Amino acid sequence of E. coli DPS.

[0436] MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWNMRGANFI

[0437] AVHEMLDGFRTALIDHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIHNVQDHLKE

[0438] LADRYAIVANDVRKAIGEAKDDDTADILTAASRDLDKFLWFIESNIE

[0439] SEQ ID NO: 5 Nucleic acid sequence encoding wild-type Borrelia burgdorferi

[0440] (B31 ) OspA.

[0441] ATGAAGAAATACCTCCTGGGTATCGGCCTGATCCTGGCTCTGATCGCCTGCAAACAGAA TGTTAGCAGCCTGGATGAGAAAAATAGCGTTAGCGTTGATCTGCCTGGTGAAATGAAAG TTCTGGTTAGCAAAGAGAAAAACAAGGACGGCAAATATGATCTGATTGCCACCGTTGATA AACTGGAACTGAAAGGCACCAGCGATAAAAACAATGGTAGTGGTGTTCTGGAAGGTGTG AAAGCAGATAAAAGCAAAGTGAAACTGACCATTAGTGATGATCTGGGTCAGACCACACT GGAAGTTTTTAAAGAAGATGGTAAAACCCTGGTGAGCAAAAAGGTTACCAGCAAAGATAA AAGTAGCACCGAAGAGAAATTCAACGAAAAAGGTGAAGTGAGCGAGAAAATTATCACCC

[0442] GTGCAGATGGCACCCGTCTGGAATATACCGGCATTAAAAGTGATGGTAGCGGTAAAGCC

[0443] AAAGAAGTTCTGAAAGGTTACGTGCTGGAAGGCACCCTGACAGCAGAGAAAACAACCC

[0444] TGGTTGTTAAAGAAGGTACAGTTACCCTGTCCAAAAACATTAGCAAAAGCGGTGAAGTTT

[0445] CCGTCGAACTGAATGATACCGATAGCAGCGCAGCAACCAAGAAAACCGCAGCATGGAAT

[0446] AGCGGTACAAGTACCCTGACAATTACCGTGAATAGCAAGAAAACGAAAGATCTGGTGTT

[0447] CACCAAAGAAAACACCATTACCGTTCAGCAGTATGATAGCAATGGCACCAAACTGGAAG

[0448] GTAGTGCAGTTGAAATTACGAAACTGGACGAAATCAAAAATGCCCTGAAA

[0449] SEQ ID NO: 6 Amino acid sequence of wild-type Borrelia burgdorferi (B31 ) OspA.

[0450] MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLEL

[0451] KGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEE

[0452] KFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKGYVLEGTLTAEKTTLVVKEGTVTL

[0453] SKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDS

[0454] NGTKLEGSAVEITKLDEIKNALK

[0455] SEQ ID NO: 7 Nucleic acid sequence encoding Borrelia burgdorferi (B31 ) OspA with amino acids 164-174 replaced by amino acids 164-174 of Borrelia afzelii (K78).

[0456] ATGAAGAAATACCTCCTGGGTATCGGCCTGATCCTGGCTCTGATCGCCTGCAAACAGAA TGTTAGCAGCCTGGATGAGAAAAATAGCGTTAGCGTTGATCTGCCTGGTGAAATGAAAG TTCTGGTTAGCAAAGAGAAAAACAAGGACGGCAAATATGATCTGATTGCCACCGTTGATA AACTGGAACTGAAAGGCACCAGCGATAAAAACAATGGTTCAGGTGTTCTGGAAGGTGTG AAAGCAGATAAAAGCAAAGTGAAACTGACCATTAGTGATGATCTGGGTCAGACCACACT GGAAGTTTTTAAAGAAGATGGTAAAACCCTGGTGAGCAAAAAGGTTACCAGCAAAGATAA AAGTAGCACCGAAGAGAAATTCAACGAAAAAGGTGAAGTGAGCGAGAAAATTATCACCC GTGCAGATGGCACCCGTCTGGAATATACCGGCATTAAAAGTGATGGTAGCGGTAAAGCC AAAGAGGTGCTGAAAAACTTTACCCTGGAAGGTAAAGTGGCCAATGATAAAACAACCCT GGTTGTGAAAGAAGGTACAGTTACCCTGAGTAAAAACATCAGCAAAAGCGGTGAAGTTT CCGTCGAACTGAATGATACCGATAGCAGCGCAGCAACCAAGAAAACCGCAGCATGGAAT AGCGGTACAAGCACCCTGACAATTACCGTGAATAGTAAAAAGACCAAAGACCTGGTGTT CACCAAAGAAAATACCATTACCGTTCAGCAGTATGATAGCAATGGCACCAAACTGGAAGG TTCTGCAGTTGAAATTACGAAACTGGACGAAATCAAAAACGCCCTGAAA

[0457] SEQ ID NO: 8 Amino acid sequence of Borrelia burgdorferi (B31 ) OspA with amino acids 164-174 replaced by amino acids 164-174 of Borrelia afzelii (K78).

[0458] MKKYLLGIGLILALIACKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVDKLEL KGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSSTEE KFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGTVTL SKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDS NGTKLEGSAVEITKLDEIKNALK

[0459] SEQ ID NO: 9 Nucleic acid sequence encoding wild-type Borrelia burgdorferi

[0460] (B31 ) CspZ.

[0461] ATGAAAAAGAGCTTCCTGTCCATCTACATGCTGATTTCCATTTCTCTCCTGAGCTGTGAC GTTAGCCGTCTGAATCAGCGTAACATTAATGAGCTGAAAATCTTCGTGGAAAAAGCCAAG TACTACAGCATTAAACTGGATGCCATTTATAATGAATGCACGGGTGCCTATAACGACATTAT GACCTATAGCGAAGGCACCTTTAGCGATCAGAGCAAAGTTAATCAGGCCATCAGCATCTT

[0462] CAAAAAGGACAACAAAATCGTGAACAAATTCAAAGAGCTGGAAAAGATCATCGAAGAGTA

[0463] CAAACCGATGTTTCTGAGCAAACTGATTGATGATTTTGCCATCGAACTGGATCAGGCCGT

[0464] TGATAATGATGTTAGCAATGCACGTCATGTTGCCGATAGCTATAAAAAGCTGCGTAAAAGC

[0465] GTTGTGCTGGCCTATATTGAATCCTTTGATGTGATCAGCAGCAAATTCGTGGATAGCAAAT

[0466] TTGTTGAAGCCAGCAAAAAGTTTGTGAACAAGGCCAAAGAATTTGTGGAAGAGAATGAT

[0467] CTGATTGCCCTGGAATGTATTGTGAAAACCATTGGCGATATGGTGAATGATCGTGAAATTA

[0468] ATAGCCGCAGCCGCTATAACAACTTCTATAAGAAAGAAGCCGATTTTCTGGGAGCAGCAG

[0469] TTGAACTGGAAGGTGCATATAAAGCAATTAAACAGACCCTGCTG

[0470] SEQ ID NO: 10 Amino acid sequence of wild-type Borrelia burgdorferi (B31 ) CspZ.

[0471] MKKSFLSIYMLISISLLSCDVSRLNQRNINELKIFVEKAKYYSIKLDAIYNECTGAYNDIMTYSE

[0472] GTFSDQSKVNQAISIFKKDNKIVNKFKELEKIIEEYKPMFLSKLIDDFAIELDQAVDNDVSNAR

[0473] HVADSYKKLRKSVVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEFVEENDLIALECIVKTIG

[0474] DMVNDREINSRSRYNNFYKKEADFLGAAVELEGAYKAIKQTLL

[0475] SEQ ID NO: 11 Nucleic acid sequence encoding mutant CspZ-YA derived from wild-type Borrelia burgdorferi (B31 ) CspZ.

[0476] ATGAAAAAGAGCTTCCTGTCCATCTACATGCTGATTTCCATTTCTCTCCTGAGCTGTGAC GTTAGCCGTCTGAATCAGCGTAACATTAATGAGCTGAAAATCTTCGTGGAAAAAGCCAAG TACTACAGCATTAAACTGGATGCCATTTATAATGAATGCACGGGTGCCTATAACGACATTAT GACCTATAGCGAAGGCACCTTTAGCGATCAGAGCAAAGTTAATCAGGCCATCAGCATCTT CAAAAAGGACAACAAAATCGTGAACAAATTCAAAGAGCTGGAAAAGATCATCGAAGAGTA CAAACCGATGTTTCTGAGCAAACTGATCGATGATTTTGCCATTGAACTGGATCAGGCCGT TGATAATGATGTGAGCAATGCACGTCATGTTGCAGATAGCTATAAAAAGCTGCGTAAAAG CGTTGTGCTGGCCTATATTGAATCCTTTGATGTGATCAGCAGCAAATTCGTGGATAGCAA ATTTGTTGAAGCCAGCAAAAAGTTTGTGAACAAGGCCAAAGAATTTGTGGAAGAGAATG ATCTGATTGCCCTGGAATGTATCGTTAAAACCATTGGCGATATGGTGAATGATCGCGAAAT TAATAGCCGTAGCCGTGCAAACAATTTTGCAAAGAAAGAAGCAGATTTTCTGGGAGCAG CAGTGGAACTGGAAGGTGCATATAAAGCCATTAAACAGACCCTGCTG

[0477] SEQ ID NO: 12 Amino acid sequence of mutant CspZ-YA derived from wild-type

[0478] Borrelia burgdorferi (B31 ) CspZ.

[0479] MKKSFLSIYMLISISLLSCDVSRLNQRNINELKIFVEKAKYYSIKLDAIYNECTGAYNDIMTYSE

[0480] GTFSDQSKVNQAISIFKKDNKIVNKFKELEKIIEEYKPMFLSKLIDDFAIELDQAVDNDVSNAR HVADSYKKLRKSVVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEFVEENDLIALECIVKTIG DMVNDREINSRSRANNFAKKEADFLGAAVELEGAYKAIKQTLL

[0481] SEQ ID NO: 13 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 an encapsulin subunit.

[0482] ATGGAATTTCTGAAACGTAGCTTTGCACCGCTGACCGAAAAACAGTGGCAAGAAATTGAT AATCGTGCCCGTGAAATCTTTAAAACCCAGCTGTATGGTCGCAAATTTGTTGATGTTGAA GGTCCGTATGGTTGGGAATATGCAGCACATCCGCTGGGTGAAGTTGAAGTTCTGAGTGA TGAAAATGAAGTGGTTAAATGGGGTCTGCGTAAAAGCCTGCCGCTGATTGAACTGCGTG CAACCTTTACACTGGATCTGTGGGAATTAGATAATCTGGAACGTGGTAAACCGAATGTTG ATCTGAGCAGCCTGGAAGAAACCGTTCGTAAAGTTGCAGAATTTGAAGATGAAGTGATTT TTCGCGGTTGTGAAAAATCAGGTGTTAAAGGTCTGCTGAGCTTTGAAGAACGTAAAATTG

[0483] AATGTGGTAGCACCCCGAAAGATCTGCTGGAAGCAATTGTTCGTGCACTGAGCATTTTTA

[0484] GCAAAGATGGTATTGAAGGCCCTTATACGCTGGTGATTAATACCGATCGTTGGATCAACT

[0485] TTCTGAAAGAAGAAGCAGGTCATTACCCGCTGGAAAAACGTGTTGAAGAATGTCTGCGT

[0486] GGTGGTAAAATCATTACCACACCGCGTATTGAAGATGCACTGGTTGTTAGCGAACGTGGT

[0487] GGTGATTTTAAACTGATTCTGGGTCAAGATCTGTCCATCGGTTATGAAGATCGTGAAAAA

[0488] GATGCAGTGCGTCTGTTTATTACCGAAACCTTTACCTTTCAGGTTGTTAATCCGGAAGCG

[0489] CTGATTCTGCTGAAATTTGGTAGCGGTGGTTCAGGTAAACAGAATGTTAGCAGCCTGGAT

[0490] GAGAAAAATAGCGTTAGCGTTGATCTGCCTGGTGAAATGAAAGTTCTGGTTAGCAAAGA

[0491] GAAAAACAAGGACGGCAAATATGATCTGATTGCCACCGTTGATAAACTGGAACTGAAAG

[0492] GCACCAGCGATAAAAACAATGGTTCAGGTGTTCTGGAAGGTGTGAAAGCAGATAAAAGC

[0493] AAAGTGAAACTGACCATTAGTGATGATCTGGGTCAGACCACACTGGAAGTTTTTAAAGAA

[0494] GATGGTAAAACCCTGGTGAGCAAAAAGGTTACCAGCAAAGATAAAAGTAGCACCGAAGA

[0495] GAAATTCAACGAAAAAGGTGAAGTGAGCGAGAAAATTATCACCCGTGCAGATGGCACCC

[0496] GTCTGGAATATACCGGCATTAAAAGTGATGGTAGCGGTAAAGCCAAAGAGGTGCTGAAA

[0497] AACTTTACCCTGGAAGGTAAAGTGGCCAATGATAAAACAACCCTGGTTGTGAAAGAAGG

[0498] TACAGTTACCCTGAGTAAAAACATCAGCAAAAGCGGTGAAGTTTCCGTCGAACTGAATGA

[0499] TACCGATAGCAGCGCAGCAACCAAGAAAACCGCAGCATGGAATAGCGGTACAAGCACC

[0500] CTGACAATTACCGTGAATAGTAAAAAGACCAAAGACCTGGTGTTCACCAAAGAAAATACC

[0501] ATTACCGTTCAGCAGTATGATAGCAATGGCACCAAACTGGAAGGTTCTGCAGTTGAAATT

[0502] ACGAAACTGGACGAAATCAAAAACGCCCTGAAATAA

[0503] SEQ ID NO: 14 Amino acid sequence of a fusion protein comprising amino acids

[0504] 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 an encapsulin subunit.

[0505] MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDE NEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGC EKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGH YPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTF QVVNPEALILLKFGSGGSGKQNVSSLDEKNSVSVDLPGEMKVLVSKEKNKDGKYDLIATVD KLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKEDGKTLVSKKVTSKDKSS TEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKEGT VTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQ YDSNGTKLEGSAVEITKLDEIKNALK

[0506] SEQ ID NO: 15 Nucleic acid sequence encoding a fusion protein comprising amino acids 21 -211 of mutant CspZ-YA fused to a DPS subunit.

[0507] GCAACCAATCTGCTGTATACCCGTAATGATGTTAGCGATAGCGAAAAGAAAGCAACCGTT GAACTGCTGAATCGTCAGGTGATTCAGTTTATTGATCTGAGCCTGATTACCAAACAGGCC CATTGGAATATGCGTGGTGCAAACTTTATTGCCGTTCATGAAATGCTGGATGGTTTTCGTA CCGCACTGATTGATCATCTGGATACCATGGCAGAACGTGCAGTTCAGTTAGGTGGTGTT GCACTGGGTACAACCCAGGTGATTAATAGCAAAACACCGCTGAAAAGCTATCCGCTGGA TATTCATAATGTTCAGGATCACCTGAAAGAACTGGCAGATCGTTATGCAATTGTTGCCAAT GATGTTCGTAAAGCAATTGGCGAAGCAAAAGATGATGATACCGCAGATATTCTGACCGCA GCAAGCCGTGATCTGGATAAATTTCTGTGGTTTATCGAGAGCAATATTGAAGGTAGCGGT GGTAGTGGTGTTAGCCGTCTGAATCAGCGTAACATTAATGAGCTGAAAATCTTCGTGGAA AAAGCCAAGTACTACAGCATTAAACTGGATGCCATTTATAATGAATGCACGGGTGCCTATA ACGACATTATGACCTATAGCGAAGGCACCTTTAGCGATCAGAGCAAAGTTAATCAGGCCA TCAGCATCTTCAAAAAGGACAACAAAATCGTGAACAAATTCAAAGAGCTGGAAAAGATCA TCGAAGAGTACAAACCGATGTTTCTGAGCAAACTGATCGATGATTTTGCCATTGAACTGG ATCAGGCCGTTGATAATGATGTGAGCAATGCACGTCATGTTGCAGATAGCTATAAAAAGC

[0508] TGCGTAAAAGCGTTGTGCTGGCCTATATTGAATCCTTTGATGTGATCAGCAGCAAATTCG

[0509] TGGATAGCAAATTTGTTGAAGCCAGCAAAAAGTTTGTGAACAAGGCCAAAGAATTTGTG

[0510] GAAGAGAATGATCTGATTGCCCTGGAATGTATCGTTAAAACCATTGGCGATATGGTGAAT

[0511] GATCGCGAAATTAATAGCCGTAGCCGTGCAAACAATTTTGCAAAGAAAGAAGCAGATTTT

[0512] CTGGGAGCAGCAGTGGAACTGGAAGGTGCATATAAAGCCATTAAACAGACCCTGCTG

[0513] SEQ ID NO: 16 Amino acid sequence of a fusion protein comprising amino acids

[0514] 21 -21 1 of mutant CspZ-YA fused to a DPS subunit.

[0515] ATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWNMRGANFIAVHEMLDGFRTALI DHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIG EAKDDDTADILTAASRDLDKFLWFIESNIEGSGGSGVSRLNQRNINELKIFVEKAKYYSIKLDAI YNECTGAYNDIMTYSEGTFSDQSKVNQAISIFKKDNKIVNKFKELEKIIEEYKPMFLSKLIDDF AIELDQAVDNDVSNARHVADSYKKLRKSVVLAYIESFDVISSKFVDSKFVEASKKFVNKAKEF VEENDLIALECIVKTIGDMVNDREINSRSRANNFAKKEADFLGAAVELEGAYKAIKQTLL

[0516] SEQ ID NO: 17 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.

[0517] GCAACCAATCTGCTGTATACCCGTAATGATGTTAGCGATAGCGAAAAGAAAGCAACCGTT GAACTGCTGAATCGTCAGGTGATTCAGTTTATTGATCTGAGCCTGATTACCAAACAGGCC CATTGGAATATGCGTGGTGCAAACTTTATTGCCGTTCATGAAATGCTGGATGGTTTTCGTA CCGCACTGATTGATCATCTGGATACCATGGCAGAACGTGCAGTTCAGTTAGGTGGTGTT GCACTGGGTACAACCCAGGTGATTAATAGCAAAACACCGCTGAAAAGCTATCCGCTGGA TATTCATAATGTTCAGGATCACCTGAAAGAACTGGCAGATCGTTATGCAATTGTTGCCAAT GATGTTCGTAAAGCAATTGGCGAAGCAAAAGATGATGATACCGCAGATATTCTGACCGCA GCAAGCCGTGATCTGGATAAATTTCTGTGGTTTATCGAGAGCAATATTGAAGGTAGCGGT GGTAGTGGTAAACAGAATGTTAGCAGCCTGGATGAGAAAAATAGCGTTAGCGTTGATCTG CCTGGTGAAATGAAAGTTCTGGTTAGCAAAGAGAAAAACAAGGACGGCAAATATGATCT GATTGCCACCGTTGATAAACTGGAACTGAAAGGCACCAGCGATAAAAACAATGGTTCAG GTGTTCTGGAAGGTGTGAAAGCAGATAAAAGCAAAGTGAAACTGACCATTAGTGATGAT CTGGGTCAGACCACACTGGAAGTTTTTAAAGAAGATGGTAAAACCCTGGTGAGCAAAAA GGTTACCAGCAAAGATAAAAGTAGCACCGAAGAGAAATTCAACGAAAAAGGTGAAGTGA GCGAGAAAATTATCACCCGTGCAGATGGCACCCGTCTGGAATATACCGGCATTAAAAGT GATGGTAGCGGTAAAGCCAAAGAGGTGCTGAAAAACTTTACCCTGGAAGGTAAAGTGGC CAATGATAAAACAACCCTGGTTGTGAAAGAAGGTACAGTTACCCTGAGTAAAAACATCAG CAAAAGCGGTGAAGTTTCCGTCGAACTGAATGATACCGATAGCAGCGCAGCAACCAAGA AAACCGCAGCATGGAATAGCGGTACAAGCACCCTGACAATTACCGTGAATAGTAAAAAGA CCAAAGACCTGGTGTTCACCAAAGAAAATACCATTACCGTTCAGCAGTATGATAGCAATG GCACCAAACTGGAAGGTTCTGCAGTTGAAATTACGAAACTGGACGAAATCAAAAACGCC CTGAAA SEQ ID NO: 18 Amino acid sequence of a fusion protein comprising amino acids

[0518] 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.

[0519] ATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAHWNMRGANFIAVHEMLDGFRTALI DHLDTMAERAVQLGGVALGTTQVINSKTPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIG EAKDDDTADILTAASRDLDKFLWFIESNIEGSGGSGKQNVSSLDEKNSVSVDLPGEMKVLVS KEKNKDGKYDLIATVDKLELKGTSDKNNGSGVLEGVKADKSKVKLTISDDLGQTTLEVFKED GKTLVSKKVTSKDKSSTEEKFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLE GKVANDKTTLVVKEGTVTLSKNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKK TKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEIKNALK

Claims

PCT ApplicationBavarian Nordic A / SBN124PCTClaims1 . A fusion protein comprising a Lyme disease-associated antigen, or an antigenic part thereof, joined to a subunit of a self-assembling multimeric protein particle, wherein the Lyme disease-associated antigen is an ‘outer surface protein A’ (OspA) of Borrelia.

2. The fusion protein of claim 1 , wherein the self-assembling multimeric protein particle is a homomeric 60-mer protein particle, preferably a homomeric 60-mer encapsulin, more preferably Type 1 encapsulin shell protein from Thermotoga maritima.

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

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

5. The fusion protein of claim 3 or 4, wherein the mutant CspZ comprises two point mutations Y207A and Y211 A related to a wild-type CspZ of Borrelia burgdorferi B31 .

6. The fusion protein of anyone of claims 1 to 5, wherein the C-terminus of the subunit of the self-assembling multimeric protein particle is joined to the N-terminus of the Lyme- disease-associated antigen.

7. A self-assembling multimeric protein particle comprising or consisting of monomers of the fusion protein of anyone of claims 1 , 2 and 6.

8. A self-assembling multimeric protein particle comprising or consisting of monomers of the fusion protein of anyone of claims 3 to 6.

9. A combination of a self-assembling multimeric protein particle of claim 7 and a selfassembling multimeric protein particle of claim 8.

10. A pharmaceutical composition or vaccine comprising a self-assembling multimeric protein particle of claim 7 and / or a self-assembling multimeric protein particle of claim 8, optionally further comprising a pharmaceutically acceptable excipient.

11. The pharmaceutical composition or vaccine of claim 10, further comprising an adjuvant, preferably alum or a CpG oligodeoxynucleotide (ODN).

12. A self-assembling multimeric protein particle of claim 7 and / or a self-assembling multimeric protein particle of claim 8 for use in the prevention or treatment of Lyme disease or a condition related to an infection caused by Borrelia.

13. A nucleic acid encoding a fusion protein of anyone of claims 1 , 2 and 6.

14. A nucleic acid encoding a fusion protein of anyone of claims 3 to 6.

15. Use of a nucleic acid of claim 13 and / or 14 for the preparation of a pharmaceutical composition or vaccine.

16. Use of a nucleic acid of claim 13 or 14 for the preparation of a recombinant expression vector, preferably an E. co / / expression plasmid.

17. A recombinant expression vector, preferably an E. co / / expression plasmid, comprising a nucleic acid of claim 13 or 14.

18. Use of a recombinant expression vector of claim 17 for the preparation of a selfassembling multimeric protein particle.

19. A process for preparing a self-assembling multimeric protein particle of claim 7 or 8 comprising the steps of:(a) providing a nucleic acid of claim 13 or 14;(b) preparing a recombinant expression vector, preferably an E. coli expression plasmid, 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 multimeric protein particle.

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