TICK VACCINE

Abstract

The present invention relates to combinations of tick-derived antigens for use as a tick vaccine.

Claims

1. A combination comprising at least the following components: (a) (i) a metalloprotease 1 from a tick species particularly selected from I. ricinus, I. persulcatus, I. scapularis, and I. pacificus, (ii) a polypeptide having a sequence identity of at least 80%, 85%, 90%, 95% or 98% to a polypeptide according to (i), (iii) an immunogenic fragment of a polypeptide according to (i) or (ii) comprising at least 6, at least 8, at least 10, at least 12 or at least 15 contiguous amino acids of a polypeptide according to (i) or (ii), (iv) a nucleic acid molecule encoding a polypeptide according to (i), (ii) or (iii), and (b) (i) a thrombin inhibitor from a tick species particularly selected from I. ricinus, I. persulcatus, I. scapularis, and I. pacificus, (ii) a polypeptide having a sequence identity of at least 80%, 85%, 90%, 95% or 98% of a protein according to (i), (iii) an immunogenic fragment of a polypeptide according to (i) or (ii) comprising at least 6, at least 8, at least 10, at least 12 or at least 15 contiguous amino acids of a polypeptide according to (i) or (ii), or (iv) a nucleic acid molecule encoding a polypeptide according to (i), (ii) or (iii).

2. The combination of claim 1 comprising at least one further component selected from (c) (i) an immunogenic polypeptide from a tick species selected from I. ricinus, I. persulcatus, I. scapularis, and I. pacificus, (ii) a polypeptide having a sequence identity of at least 80%, 85%, 90% or 95% of a protein according to (i), (iii) an immunogenic fragment of a polypeptide according to (i) or (ii) comprising at least 6, at least 8, at least 10, at least 12 or at least 15 contiguous amino acids of a polypeptide according to (i) or (ii), or (iv) a nucleic acid molecule encoding a polypeptide according to (i), (ii) or (iii).

3. The combination of claim 1, wherein component (a) is a metalloprotease 1 from I. scapularis, as shown in SEQ ID NO: 1 or an immunogenic homologue or an immunogenic fragment thereof or a nucleic acid molecule coding therefor.

4. The combination of claim 1, wherein component (b) is a thrombin inhibitor from I. scapularis, as shown in SEQ ID NO: 2 or an immunogenic homologue or an immunogenic fragment thereof or a nuclei acid molecule coding therefor.

5. The combination of claim 1 which is a composition comprising several components in a single container or a kit comprising individual components in separate containers.

6. The combination of claim 1 for use in medicine.

7. The combination of claim 1 for use in human medicine.

8. The combination of claim 1 for use as a tick vaccine, particularly a human tick vaccine.

9. The combination of claim 1 for use as a vaccine against ticks of the genus Ixodes.

10. The combination of claim 1 for use as a vaccine, against several different tick species of the genus Ixodes.

11. The combination of claim 1 further comprising an adjuvant.

12. The combination of claim 1 for use in preventing a tick-borne pathogen infection.

13. The combination of claim 1 for use in preventing an infection by a pathogen selected from Babesia spec., Borrelia spec., Francisella spec., Rickettsia spec., Anaplasma spec., Ehrlichia spec., tick-borne encephalitis virus (TBEV), Powassan virus, Colorado tick fever virus, Crimean-Congo hemorrhagic fever virus or Heartland virus.

Description

FIGURE LEGENDS

[0084] FIG. 1: Enhanced antigen-specific systemic humoral immune responses.

[0085] Serum obtained from each single mouse collected 14 days after the last immunization were analyzed for anti-thrombin inhibitor IgG, IgG1 and IgG2c antibodies by ELISA. Results are displayed as floating bars with indicating the mean per each group. (A) IgG and B (IgG1 and IgG2a).

[0086] FIG. 2: Increased cellular immune responses displayed by an enhanced number of antigen-specific cytokine producing cells. The quantity of cytokine producing cells were determined by ELISpot. Single splenocyte suspensions isolated 14 days after the last immunization were pooled for each group and re-stimulated with the TI protein. Results are expressed as spot forming units of 10.sup.6 unstimulated and re-stimulated cells. Results are displayed as box and whiskers plots for (A) IFN, (B) IL-2, (C) IL-4, and (D) IL-17.

[0087] FIG. 3: Generation of antigen-specific multifunctional CD4.sup.+ T cells. The quantity of cytokine producing CD4.sup.+ T cells was determined by a multiparametric flow cytometry approach. Spleens obtained 14 days after the last immunization were isolated and prepared from each single mouse and re-stimulated with the TI protein. Results are expressed as frequencies of CD4.sup.+ T cells producing the indicated cytokines and displayed as box and whiskers plots for (A) IFN, (B) TNF-, (C) IL-4, and (D) IL-17.

[0088] FIG. 4: Enhanced antigen-specific systemic humoral immune responses. Serum obtained from each single mouse collected 7 days after the last immunization were analyzed for anti-MP1 IgG, IgG1 and IgG2c antibodies by ELISA. Results are displayed as floating bars with indicating the mean per each group. (A) IgG and B (IgG1 and IgG2c).

[0089] FIG. 5: Increased cellular immune responses displayed by enhanced number of antigen-specific cytokine producing cells. The quantity of cytokine producing cells was determined by ELISpot. Single splenocyte suspensions isolated 7 days after the last immunization were pooled for each group and re-stimulated with the MP1 protein. Results are expressed as spot forming units of 10.sup.6 unstimulated and re-stimulated cells. Results are displayed as box and whiskers plots for (A) IFN, (B) IL-2, (C) IL-4, and (D) IL-17.

[0090] FIG. 6: Generation of antigen-specific multifunctional CD4+ T cells. The quantity of cytokine producing CD4+ T cells was determined by a multiparametric flow cytometry approach. Spleens derived 7 days after the last immunization were isolated and prepared from each single mouse and re-stimulated with the MP1 protein. Results are expressed as frequencies of CD4.sup. T cells producing the indicated cytokines and displayed as box and whiskers plots for (A) IFN, (B) TNF-, (C) IL-4, and (D) IL-17.

[0091] FIG. 7: Enhanced antigen-specific systemic humoral immune responses. Serum obtained from each single mouse collected 7 days after the last immunization were analyzed for anti-TI and anti-MP1 IgG, IgG1 and IgG2c antibodies by ELISA. Results are displayed as floating bars with indicating the mean per each group. (A), (B) IgG and (C), (D) IgG1 and IgG2c.

[0092] FIG. 8: Changes in IgG1/IgG2c ratio following a combined antigen vaccination approach. Serum obtained from each single mouse collected 7 days after the last immunization were analyzed for anti-TI IgG1 and IgG2c antibodies. Results are displayed as floating bars with indicating the mean per each group. Displayed is the ratio of TI-specific IgG1/IgG2c.

EXAMPLES

Example 1: Immunization with the Thrombin Inhibitor from I. scapularis

[0093] A recombinant thrombin inhibitor (TI) produced in E. coli was tested as an antigen to be implemented in anti-tick vaccine formulations. For this purpose, an immunization study in mice was conducted.

1.1 Immunization Protocol

[0094] Female C57BL/6 mice (n=5/group) 8-12 weeks of age were immunized 3 times (prime-2-boost strategy) at day 0, 14, and 28 by intramuscular (i.m.) route. Each animal received a total dose of 100 l containing 25 g or 50 g of the protein TI alone or co-administered with 50 l (1:1 v/v) of either complete Freund's adjuvant (CFA) (1.sup.st immunization) or incomplete Freund's adjuvant (IFA) (2.sup.nd and 3.sup.rd immunization) or alum hydroxide. Control animals received 100 l PBS. Fourteen days after the last immunization, blood was collected for the analysis of antigen-specific serum antibodies and spleens were removed and processed to address cellular immune responses. Details are shown in Table 1.

TABLE-US-00001 TABLE 1 Protein + Total Group Animals Adjuvants/Animal amount 1 Control (PBS) 5 i.m. C57BL/6 / 100 l 2 TI 25 g 5 i.m. C57BL/6 25 g/ 100 l 3 TI 50 g 5 i.m. C57BL/6 50 g/ 100 l 4 TI 25 g + 5 i.m. C57BL/6 25 g/50 l 100 l CFA and IFA** 5 TI 50 g + 5 i.m. C57BL/6 50 g/50 l 100 l CFA and IFA** 6 TI 25 g + Alum 5 i.m. C57BL/6 25 g/50 l 100 l 7 TI 50 g + Alum 5 i.m. C57BL/6 50 g/50 l 100 l

1.2 Detection of Antigen-Specific Humoral Immune Responses

[0095] An Enzyme-linked Immunosorbent Assay (ELISA) was conducted to investigate the induction of IgG antibodies and the corresponding isotypes (IgG1 and IgG2c) against the TI. For this, high binding protein plates were coated with the protein (2 g/ml in 0.05 M carbonate buffer) and incubated over night at 4 C. On the next day, the plates were washed und blocked for 2 h with 3% BSA (bovine serum albumin) in PBS to prevent unspecific binding. Following, 2-fold serial dilutions (starting with 1:1000) of the serum samples were incubated in 3% BSA/PBS for 2 h at 37 C. After washing with 1% BSA/PBS/0.05% Tween the plates were incubated for 1 h with the biotinylated goat-anti mouse IgG, or IgG1 or IgG2c antibodies. Then, the plates were washed again und incubated with for 30 min with peroxidase-conjugated streptavidin. Finally, the reaction was developed by adding ABTS [2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] in 0.1 M citrate-phosphate buffer containing 0.01 H.sub.2O.sub.2 and measured after 5 min at an OD (optical density) of 405 nm. Endpoint titers are expressed as absolute values of the last dilution giving an OD405 nm being 2 times higher than the negative control (Blank).

[0096] The obtained results revealed the induction of TI-specific humoral immune response following the prime-2-boost immunization strategy. More detailed, elevated anti-TI IgG titers were detected in all groups immunized with the protein co-administered with adjuvant as compared to antigen alone or control group (FIG. 1A). The further analysis of the IgG isotypes, IgG1 and IgG2c showed enhanced titers in all mice which received the antigen combined with one of the adjuvants with a slightly higher titer observed for IgG1 as compared to IgG2c (FIG. 1B). Comparing the two different antigen concentrations of 25 g and 50 g used for the immunization study, no major differences were observed for the induction of anti-TI titers IgG, IgG1 and IgG2c titers indicating that already lower amounts of the antigen are sufficient to induce strong humoral immune responses. Since the distribution of IgG1 and IgG2c provides indication for T-helper (Th) cell polarization, the data suggest rather the stimulation of Th-2 than Th-1 cells.

1.3 Detection of Antigen-Specific Cellular Immune Responses

[0097] To gain insight into the quantity of TI-specific induction of cytokine secreting cells (IFN, IL-2, IL-4, IL-17) an Enzyme-linked Immuno Spot Assay (ELISpot) was performed. To this end, plates with hydrophobic high protein binding Immobilon-P-Membrane were coated with anti-IFN, anti-IL-2, anti-IL-4, and anti-IL-17 antibodies diluted in PBS and incubated over night at 4 C. Unspecific binding sites were blocked for 2 h at room temperature with 200 l/well of complete RPMI medium. Then, 5*10.sup.5 splenocytes/well were added and incubated in the absence (unstimulated) or presence of the TI protein (5 g/ml). As positive control served splenocytes stimulated with the mitogen concanavalin A (5 g/ml). The cells were incubated for 18 h (IFN) or 48 h (IL-2, IL-4, IL-17) at 37 C. Afterwards, the plates were washed with 0.01% Tween/PBS and incubated for further 2 h in the presence of the corresponding biotinylated detection antibodies. After another wash step, the cells were incubated with a peroxidase-conjugated streptavidin for 1 h at room temperature. Following, the plates were washed again and the cytokine secreting cells were detected by adding AEC substrate diluted in 0.1 M acetate buffer supplemented with 0.05% H.sub.2O.sub.2. The reaction was stopped depending on the development of the spots by adding distilled water. The samples were analyzed using the ImmunoSpot Image Analyzer software (CTL-Europe GmbH). Results are expressed as Spot-forming units calculated for 1*10.sup.6 cells.

[0098] In vitro stimulation of splenocytes with the antigen displayed a strong increase in the number of IFN, IL-2, IL-4, and IL-17-secreting cells in the groups which were vaccinated with the TI protein co-administered with adjuvant as compared to unstimulated cells and the groups which received the antigen alone or PBS (FIG. 2A-D). Since IFN, IL-2, IL-4, and IL-17-secreting cells are considered as an indicator for Th-1, Th-2 or Th-17 biased immunity, the obtained results suggest the generation of a mixed/balanced Th-cell responses. Interestingly, the elevated number of IL-2 and IL-4 secreting splenocytes were found to be independent of the used antigen concentration, whereas the number of IFN and IL-17 secreting cells were mainly enhanced in the groups which were immunized with the higher amount of TI protein. Thus, the amount of used protein might impact Th-cell polarization.

[0099] Next, a multiparametric flow cytometry approach was applied to address the occurrence and functionality of antigen-specific CD4 and CD8 T cells. Single splenocyte suspension with 5*10.sup.6 cells/well were incubated with either medium alone or with TI (20 g/ml) at 37 C. for 4 h. Afterwards brefeldin (5 g/ml) and monensin (3 g/ml) were added to the cells to prevent cytokine secretion or receptor internalization, respectively, thereby allowing cytokine accumulation inside the cells. Then, the cells were incubated for further 12 h followed by a surface marker (CD3, CD4, CD8, dead cell marker) and intracellular cytokine staining (IFN, IL-2, IL-4, TNF and IL-17). To this end, splenocytes were incubated with the appropriate antibody cocktail diluted in PBS including the dead cell marker for 20 min at 4 C. After washing with PBS, cells were incubated for 30 min with a fixation/permeabilization buffer followed by the intracellular cytokine staining for 20 min. The cells were acquired using the BD LSRFortessa flow cytometry and analyzed using the FlowJo V10 software. Viable singlet lymphocytes were gated for CD3.sup.+CD4.sup.+ or CD3.sup.+CD8.sup.+ and analyzed for the frequency of IFN, IL-2, IL-4, TNF and IL-17 producing T cells.

[0100] The analysis of multifunctional T cells characterized by the simultaneous production of different cytokines following re-stimulation revealed an enhanced frequency of IFN producing CD4.sup.+ T cells isolated from mice immunized with the TI protein alone as compared to the control group (FIG. 3A). The supplementation of the vaccine formulation with complete/incomplete Freund's adjuvant or alum resulted only in a marginal elevated level of IFN producing CD4.sup.+ T cells. However, adjuvantation could boost to some extend the frequencies of TNF and IL-17 producing CD4.sup.+ T cells, whereas the frequency of IL-4 secreting CD4.sup.+ T cells were not affected (FIG. 3B-D). The analysis of multifunctional CD8.sup.+ T cell did not reveal changes in the frequencies of IFN and TNF producing cells. The obtained data confirm the presence of TI-specific multifunctional CD4.sup.+ T cells following immunization and identify CD4.sup.+ T cells as one crucial cytokine-secreting immune cell population observed in FIG. 2.

[0101] In conclusion, the conducted studies demonstrate the generation of TI-specific IgG antibodies and their corresponding isotypes, thereby showing a clear seroconversion following immunization with the vaccine antigen candidate. The obtained data clearly show that immunization with the TI protein not only generates antigen-specific humoral responses, but also activates antigen-specific cellular immunity, especially multifunctional CD4.sup.+ T cells. The dose of antigen as well as the choice of adjuvant can affect/boost the immune response in diverse directions. The studies convincingly depict that the TI protein represents a suitable protein for an anti-tick vaccine.

Example 2: Immunization with Metalloprotease 1 from I. scapularis

[0102] The metalloprotease (MP1) represents a salivary gland protein found to be secreted by the tick specie Ixodes scapularis. A recombinant MP1 produced in E. coli was tested as an antigen to be implemented in anti-tick vaccine formulations. For this purpose, an immunization study in mice was conducted.

2.1 Immunization Protocol

[0103] Female C57BL/6 mice (n=5/group) 8-12 weeks of age were immunized 3 times (prime-2-boost strategy) at day 0, 7, and 21 by intramuscular (i.m.) route. Each animal received a total dose of 100 l containing 25 g of the protein MP1 alone or co-administered with 50 l (1:1 v/v) of either complete (1.sup.st immunization) or incomplete Freund's adjuvant (**2.sup.nd and **3.sup.rd immunization). Since MP1 was solved in 2 M urea, which was diluted to 0.5 M in the vaccine formulation, control animals received 100 l of 0.5 M Urea in PBS. Another control group were only injected with CFA/IFA. Seven days after the last immunization, blood was collected for the analysis of antigen-specific serum antibodies and spleens were removed and processed to address cellular immune responses. Details are shown in Table 2.

TABLE-US-00002 TABLE 2 Protein + Total Group Animals Adjuvants/Animal amount 1 0.5M Urea in 5 i.m. C57BL/6 / 100 l PBS 2 MP1 25 g 5 i.m. C57BL/6 25 g/ 100 l 3 MP1 25 g + 5 i.m. C57BL/6 25 g/50 l 100 l CFA and IFA** 4 CFA/IFA** alone 5 i.m. C57BL/6 /50 l 100 l

2.2 Detection of Antigen-Specific Humoral Immune Responses

[0104] An Enzyme-linked Immunosorbent Assay (ELISA) was conducted to investigate the induction of IgG antibodies and the corresponding isotypes (IgG1 and IgG2c) against the MP1 antigen. For this, high binding protein plates were coated with the protein (2 g/ml in 0.05 M carbonate buffer) and incubated over night at 4 C. On the next day, the plates were washed und blocked for 2 h with 3% BSA (bovine serum albumin) in PBS to prevent unspecific binding. Following, 2-fold serial dilutions (starting with 1:1000) of the serum samples were incubated in 3% BSA/PBS for 2 h at 37 C. After washing with 1% BSA/PBS/0.05% Tween the plates were incubated for 1 h with the biotinylated goat-anti mouse IgG, or IgG1 or IgG2c antibodies. Then, the plates were washed again und incubated with for 30 min with peroxidase-conjugated streptavidin. Finally, the reaction was developed by adding ABTS [2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] in 0.1 M citrate-phosphate buffer containing 0.01 H.sub.2O.sub.2 and measured after 5 min at an OD (optical density) of 405 nm. Antigen-specific endpoint titers are expressed as absolute values of the last dilution giving an OD405 nm being 2 times higher than the negative control (Blank).

[0105] The obtained results revealed the generation of MP1-specific humoral immune responses following a prime-2-boost immunization strategy. More detailed, elevated anti-MP1 IgG titers were detected in all groups immunized with the protein alone or co-administered with adjuvant as compared to the control groups (FIG. 4A). The further analysis of the IgG isotypes, IgG1 and IgG2c showed mainly enhanced IgG1 titers in mice, which either received the antigen alone or combined with CFA/IFA, whereas only a minor increase of IgG2c was observed in general (FIG. 4B). Although mice that were administered with the protein MP1 alone displayed already antigen-specific IgG and IgG1 titer, the amount of antibody titers could be strongly increased by adding the CFA/IFA to the vaccine formulation. Furthermore, the results demonstrate the activation of Th-2 cells but not Th-1 cells, as pointed by the ratio of IgG1 and IgG2c antibodies respectively.

2.3 Detection of Antigen-Specific Cellular Immune Responses

[0106] To gain insight into the quantity of MP1-specific induction of cytokine secreting cells (IFN, IL-2, IL-4, IL-17) an Enzyme-linked Immuno Spot Assay (ELISpot) was performed. To this end, plates with hydrophobic high protein binding Immobilon-P-Membrane were coated with anti-IFN, anti-IL-2, anti-IL-4, and anti-IL-17 antibodies diluted in PBS and incubated over night at 4 C. Unspecific binding sites were blocked for 2 h at room temperature with 200 l/well of complete RPMI medium. Then, 5*10.sup.5 splenocytes/well were added and incubated in the absence (unstimulated) or presence of the MP1 protein (5 g/ml). As positive control served splenocytes stimulated with the mitogen concanavalin A (5 g/ml). The cells were incubated for 18 h (IFN) or 48 h (IL-2, IL-4, IL-17) at 37 C. Afterwards, the plates were washed with 0.01% Tween/PBS and incubated for further 2 h in the presence of the corresponding biotinylated detection antibodies. After another wash step, the cells were incubated with a peroxidase-conjugated streptavidin for 1 h at room temperature. Following, the plates were washed again and the cytokine secreting cells were detected by adding AEC substrate diluted in 0.1 M acetate buffer supplemented with 0.05% H.sub.2O.sub.2. The reaction was stopped depending on the development of the spots by adding distilled water. The samples were analyzed using the ImmunoSpot Image Analyzer software (CTL-Europe GmbH). Results are expressed as Spot-forming units calculated for 1*10.sup.6 cells.

[0107] In vitro stimulation of splenocytes with MP1 antigen displayed an overall strong increase in the number of IFN, IL-2, IL-4, and IL-17-secreting cells. An elevated number of IFN secreting cells were observed for all MP1 restimulated splenocytes independent of the immunization status, thereby indicating an immune modulatory capacity of the protein itself on IFN producing cells. Nevertheless, mice, which received MP1 combined with CFA/IFA, displayed the highest level of IFN.sup.+ cells (FIG. 5A). The number of IL-2, IL-4 and IL-17 producing cells were mainly increased in the restimulated groups immunized with the antigen alone or together with the adjuvant as compared to the control groups. The addition of CFA to the antigen led to further enhanced number of cytokine secreting cells (FIG. 5B-D). The obtained results suggest the induction of a mixed Th-1/Th-2/Th-17 cell response.

[0108] Next, a multiparametric flow cytometry approach was applied to address the occurrence and functionality of antigen-specific CD4 and CD8 T cells. Single splenocyte suspension with 5*10.sup.6 cells/well were incubated with either medium alone or with MP1 (20 g/ml) at 37 C. for 4 h. Afterwards brefeldin (5 g/ml) and monensin (3 g/ml) were added to the cells to prevent cytokine secretion or receptor internalization, respectively, thereby allowing cytokine accumulation inside the cells. Then, the cells were incubated for further 12 h followed by a surface marker (CD3, CD4, CD8, dead cell marker) and intracellular cytokine staining (IFN, IL-2, IL-4, TNF and IL-17). To this end, splenocytes were incubated with the appropriate antibody cocktail diluted in PBS including the dead cell marker for 20 min at 4 C. After washing with PBS, cells were incubated for 30 min with a fixation/permeabilization buffer followed by the intracellular cytokine staining for 20 min. The cells were acquired using the BD LSRFortessa flow cytometry and analyzed using the FlowJo V10 software. Viable singlet lymphocytes were gated for CD3.sup.+CD4.sup.+ or CD3.sup.+CD8.sup.+ and analyzed for the frequency of IFN, IL-2, IL-4, TNF and IL-17 producing T cells.

[0109] The analysis of multifunctional T cells characterized by the simultaneous production of different cytokines following re-stimulation revealed enhanced frequencies of IFN as well as TNF producing CD4.sup.+ T cells isolated from mice immunized with the MP1 protein alone as compared to the control groups (FIG. 6A-B). The supplementation of the vaccine formulation with CFA/IFA could even further increase the level of IFN and TNF a producing CD4.sup.+ T cells. Furthermore, immunization with MP1 alone or in combination with CFA induced the activation of IL-17 secreting CD4 T cells, whereas IL-4 producing CD4 T cells were not detected (FIG. 6C-D). The analysis of multifunctional CD8.sup.+ T cell did not reveal changes in the frequencies of IFN and TNF producing cells. However, the obtained data confirm the presence of MP1-specific multifunctional CD4.sup.+ T cells following immunization and identify CD4.sup.+ T cells as one crucial cytokine-secreting immune cell population observed in FIG. 5. In contrast to the data obtained above, the CD4 T cell analyses suggest rather the induction of a mixed Th-1/Th-17 immune response than a Th-2 one.

[0110] In conclusion, the conducted studies demonstrate the generation of MP1-specific IgG antibodies, especially of IgG1, thereby pointing a clear seroconversion following immunization with the vaccine antigen candidate. The obtained data clearly show that immunization with the MP1 protein not only generates antigen-specific humoral responses, but also activates antigen-specific cellular immunity, especially multifunctional CD4.sup.+ T cells. However, it needs to be considered that the high number of IFN-producing cells shown in FIG. 5A might be, to some extent, due to a slight contamination with bacterial components in course of the production process. Nevertheless, the analysis of the cytokine secreting CD4 T cells convincingly show the antigen-dependent reactivation of T cells. The performed studies strongly depict that the MP1 protein represents a suitable protein for an anti-tick vaccine.

Example 3: Combined Antigen Approach

[0111] The already conducted immunization revealed a high immunogenic potential of the two proteins, thrombin inhibitor (TI) protein and the metalloprotease (MP1), secreted by the tick species Ixodes scapularis, when administered together with an adjuvant. To address whether the combination of these proteins influences antigen-specific immunity, immunization studies in mice were conducted.

3.1 Immunization Protocol

[0112] Female C57BL/6 mice (n=5/group) 8-12 weeks of age were immunized 3 times (prime-2-boost strategy) at day 0, 7, and 21 by intramuscular (i.m.) route. Each animal received a total dose of 100 l containing 25 g of the protein MP1 alone or co-administered with 50 l (1:1 v/v) of either complete (1.sup.st immunization) or incomplete Freund's adjuvant (**2.sup.nd and **3.sup.rd immunization). Seven days after the last immunization, blood was collected for the analysis of antigen-specific serum antibodies. Details are shown in Table 3.

TABLE-US-00003 TABLE 3 Protein + Total Group Animals Adjuvants/Animal amount 1 TI 25 g + MP1 5 i.m. C57BL/6 25 g + 100 l 25 g + Alum 25 g/50 l 2 Alum alone 5 i.m. C57BL/6 50 l 100 l

3.2 Detection of Antigen-Specific Humoral Immune Responses

[0113] An Enzyme-linked Immunosorbent Assay (ELISA) was conducted to investigate the induction of IgG antibodies and the corresponding isotypes (IgG1 and IgG2c) against the TI and MP1 antigen. For this, high binding protein plates were coated with the protein (2 g/ml in 0.05 M carbonate buffer) and incubated over night at 4 C. On the next day, the plates were washed and blocked for 2 h with 3% BSA (bovine serum albumin) in PBS to prevent unspecific binding. Following, 2-fold serial dilutions (starting with 1:1000) of the serum samples were incubated in 3% BSA/PBS for 2 h at 37 C. After washing with 1% BSA/PBS/0.05% Tween the plates were incubated for 1 h with the biotinylated goat-anti mouse IgG, or IgG1 or IgG2c antibodies. Then, the plates were washed again and incubated with for 30 min with peroxidase-conjugated streptavidin. Finally, the reaction was developed by adding ABTS [2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] in 0.1 M citrate-phosphate buffer containing 0.01 H.sub.2O.sub.2 and measured after 5 min at an OD (optical density) of 405 nm. Antigen-specific endpoint titers are expressed as absolute values of the last dilution giving an OD405 nm being 2 times higher than the negative control (Blank).

[0114] The obtained results revealed the generation of TI-specific as well as MP1-specific humoral immune responses following a prime-2-boost immunization strategy. Elevated anti-TI- and MP1 IgG titers were detected in all groups immunized with the protein co-administered with the adjuvant alum (FIG. 7A-B). The further analysis of the IgG isotypes, IgG1 and IgG2c showed mainly enhanced IgG1 titers whereas only a minor increase of IgG2c was observed in general (FIG. 7B-C). Compared to the immunization studies with the corresponding single antigen, no major differences in IgG titers were observed. However, a shift in the IgG1/IgG2c ratio was detected for the TI protein. Here, mice that were only vaccinated with TI and alum displayed a balanced distribution of IgG1 and IgG2c. In contrast, mice that were vaccinated with both antigens showed a strong reduced IgG2c response and a significant shift to IgG1 (FIG. 8).

[0115] The data suggest that the combination of antigens induces an equal strong humoral immune response as compared to single antigen immunization, but can influence the distribution of antibody subtypes. The changes in antibody balances will most probably also affect the generation of antigen-specific cellular immunity. These changes lead to a synergistic effect resulting in an overall improved vaccine response.

LIST OF REFERENCES

[0116] Ali, A., Tirloni, L., Isezaki, M., Seixas, A., Konnai, S., Ohashi, K., da Silva Vaz Junior, I., Termignoni, C., 2014. Reprolysin metalloproteases from Ixodes persulcatus, Rhipicephalus sanguineus and Rhipicephalus microplus ticks. Exp. Appl. Acarol. 63, 559-578. [0117] Becker, M., Felsberger, A., Frenzel, A., Shattuck, W. M. C., Dyer, M., Kugler, J., Zantow, J., Mather, T. N., Hust, M., 2015. Application of M13 phage display for identifying immunogenic proteins from tick (Ixodes scapularis) saliva. BMC Biotechnol. 15, 43. [0118] Choi, E., Pyzocha, N. J., Maurer, D. M., 2016. Tick-Borne Illnesses. Curr Sports Med Rep 15, 98-104. [0119] Connor, D. O., Zantow, J., Hust, M., Bier, F. F., von Nickisch-Rosenegk, M., 2016. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display. PLoS ONE 11, e0148986. [0120] Contreras, M., de la Fuente, J., 2017. Control of infestations by Ixodes ricinus tick larvae in rabbits vaccinated with aquaporin recombinant antigens. Vaccine 35, 1323-1328. [0121] Contreras, M., de la Fuente, J., 2016. Control of Ixodes ricinus and Dermacentor reticulatus tick infestations in rabbits vaccinated with the Q38 Subolesin/Akirin chimera. Vaccine 34, 3010-3013. https://doi.org/10.1016/j.vaccine.2016.04.092 [0122] Coumou, J., Wagemakers, A., Trentelman, J. J., Nijhof, A. M., Hovius, J. W., 2014. Vaccination against Bm86 Homologues in Rabbits Does Not Impair Ixodes ricinus Feeding or Oviposition. PLoS ONE 10, e0123495. [0123] Dai, J., Narasimhan, S., Zhang, L., Liu, L., Wang, P., Fikrig, E., 2010. Tick histamine release factor is critical for Ixodes scapularis engorgement and transmission of the lyme disease agent. PLoS Pathog. 6, e1001205. [0124] Daix, V., Schroeder, H., Praet, N., Georgin, J.-P., Chiappino, I., Gillet, L., de Fays, K., Decrem, Y., Leboulle, G., Godfroid, E., Bollen, A., Pastoret, P.-P., Gem, L., Sharp, P. M., Vanderplasschen, A., 2007. Ixodes ticks belonging to the Ixodes ricinus complex encode a family of anticomplement proteins. Insect Mol. Biol. 16, 155-166. [0125] de la Fuente, J., Moreno-Cid, J. A., Galindo, R. C., Almazan, C., Kocan, K. M., Merino, O., Perez de la Lastra, J. M., Estrada-Pena, A., Blouin, E. F., 2013. Subolesin/Akirin vaccines for the control of arthropod vectors and vectorborne pathogens. Transbound Emerg Dis 60 Suppl 2, 172-178. [0126] Decrem, Y., Mariller, M., Lahaye, K., Blasioli, V., Beaufays, J., Zouaoui Boudjeltia, K., Vanhaeverbeek, M., Cerutti, M., Brossard, M., Vanhamme, L., Godfroid, E., 2008. The impact of gene knock-down and vaccination against salivary metalloproteases on blood feeding and egg laying by Ixodes ricinus. Int. J. Parasitol. 38, 549-560. [0127] Eisen, L., 2018. Pathogen transmission in relation to duration of attachment by Ixodes scapularis ticks. Ticks and Tick-borne Diseases 9, 535-542. [0128] Francischetti, I. M. B., Mather, T. N., Ribeiro, J. M. C., 2003. Cloning of a salivary gland metalloprotease and characterization of gelatinase and fibrin(ogen)lytic activities in the saliva of the Lyme disease tick vector Ixodes scapularis. Biochem. Biophys. Res. Commun. 305, 869-875. [0129] Frimmel, S., Krienke, A., Riebold, D., Loebermann, M., Littmann, M., Fiedler, K., Klaus, C., Suss, J., Reisinger, E. C., 2014. Tick-borne encephalitis virus habitats in North East Germany: reemergence of TBEV in ticks after 15 years of inactivity. Biomed Res Int 2014, 308371. [0130] Ginsberg, H. S., Albert, M., Acevedo, L., Dyer, M. C., Arsnoe, I. M., Tsao, J. I., [0131] Mather, T. N., LeBrun, R. A., 2017. Environmental Factors Affecting Survival of Immature Ixodes scapularis and Implications for Geographical Distribution of Lyme Disease: The Climate/Behavior Hypothesis. PLoS ONE 12, e0168723. [0132] Gomes, H., Moraes, J., Githaka, N., Martins, R., Isezaki, M., Vaz, I. da S., Logullo, C., Konnai, S., Ohashi, K., 2015. Vaccination with cyclin-dependent kinase tick antigen confers protection against Ixodes infestation. Vet. Parasitol. 211, 266-273. [0133] Hofmann, H., Fingerle, V., Hunfeld, K.-P., Huppertz, H.-I., Krause, A., Rauer, S., Ruf, B., Consensus group, 2017. Cutaneous Lyme borreliosis: Guideline of the German Dermatology Society. Ger Med Sci 15, Doc14. [0134] Hust, M., Meysing, M., Schirrmann, T., Selke, M., Meens, J., Gerlach, G.-F., Dubel, S., 2006. Enrichment of open reading frames presented on bacteriophage M13 using hyperphage. Biotechniques 41, 335-42. [0135] Ismail, N., McBride, J. W., 2017. Tick-Borne Emerging Infections: Ehrlichiosis and Anaplasmosis. Clin. Lab. Med. 37, 317-340. [0136] John, M., Raman, M., Ryan, K., 2017. A tiny tick can cause a big health problem. Indian J Ophthalmol 65, 1228-1232. [0137] Jonsson, N. N., Matschoss, A. L., Pepper, P., Green, P. E., Albrecht, M. S., Hungerford, J., Ansell, J., 2000. Evaluation of tickGARD(PLUS), a novel vaccine against Boophilus microplus, in lactating Holstein-Friesian cows. Vet. Parasitol. 88, 275-285. [0138] Kotsyfakis, M., Anderson, J. M., Andersen, J. F., Calvo, E., Francischetti, I. M. B., Mather, T. N., Valenzuela, J. G., Ribeiro, J. M. C., 2008. Cutting edge: Immunity against a silent salivary antigen of the Lyme vector Ixodes scapularis impairs its ability to feed. J. Immunol. 181, 5209-5212. [0139] Kotsyfakis, M., Karim, S., Andersen, J. F., Mather, T. N., Ribeiro, J. M. C., 2007. Selective cysteine protease inhibition contributes to blood-feeding success of the tick Ixodes scapularis. J. Biol. Chem. 282, 29256-29263. [0140] Kgler, J., Nieswandt, S., Gerlach, G. F., Meens, J., Schirrmann, T., Hust, M., 2008. Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display. Appl. Microbiol. Biotechnol 80, 447-58. [0141] Meyer, T., Schirrmann, T., Frenzel, A., Miethe, S., Stratmann-Selke, J., Gerlach, G. F., Strutzberg-Minder, K., Dubel, S., Hust, M., 2012. Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display. BMC Biotechnol. 12, 29. [0142] Miller, R., Estrada-Pena, A., Almazan, C., Allen, A., Jory, L., Yeater, K., Messenger, M., Ellis, D., Perez de Leon, A. A., 2012. Exploring the use of an anti-tick vaccine as a tool for the integrated eradication of the cattle fever tick, Rhipicephalus (Boophilus) annulatus. Vaccine 30, 5682-5687. [0143] Naseem, S., Meens, J., Jores, J., Heller, M., Dubel, S., Hust, M., Gerlach, G.-F., 2010. Phage display-based identification and potential diagnostic application of novel antigens from Mycoplasma mycoides subsp. mycoides small colony type. Vet. Microbiol 142, 285-292. [0144] Nazario, S., Das, S., de Silva, A. M., Deponte, K., Marcantonio, N., Anderson, J. F., Fish, D., Fikrig, E., Kantor, F. S., 1998. Prevention of Borrelia burgdorferi transmission in guinea pigs by tick immunity. Am. J. Trop. Med. Hyg. 58, 780-785. [0145] Nelson, C. A., Saha, S., Kugeler, K. J., Delorey, M. J., Shankar, M. B., Hinckley, A. F., Mead, P. S., 2015. Incidence of Clinician-Diagnosed Lyme Disease, United States, 2005-2010. Emerging Infect. Dis. 21, 1625-1631. [0146] Ribeiro, J. M. C., Alarcon-Chaidez, F., Francischetti, I. M. B., Mans, B. J., Mather, T. N., Valenzuela, J. G., Wikel, S. K., 2006. An annotated catalog of salivary gland transcripts from Ixodes scapularis ticks. Insect Biochem. Mol. Biol. 36, 111-129. [0147] Schetters, T., Bishop, R., Crampton, M., Kopaek, P., Lew-Tabor, A., Maritz-Olivier, C., Miller, R., Mosqueda, J., Patarroyo, J., Rodriguez-Valle, M., Scoles, G. A., de la Fuente, J., 2016. Cattle tick vaccine researchers join forces in CATVAC. Parasit Vectors 9, 105. [0148] Schuijt, T. J., Narasimhan, S., Daffre, S., DePonte, K., Hovius, J. W. R., Van't Veer, C., van der Poll, T., Bakhtiari, K., Meijers, J. C. M., Boder, E. T., van Dam, A. P., [0149] Fikrig, E., 2011. Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display. PLoS ONE 6, e15926. Shapiro, E. D., 2014. Borrelia burgdorferi (Lyme disease). Pediatr Rev 35, 500-509. [0150] Shattuck, W. M. C., Dyer, M. C., Desrosiers, J., Fast, L. D., Terry, F. E., Martin, W. D., [0151] Moise, L., De Groot, A. S., Mather, T. N., 2014. Partial pathogen protection by tick-bite sensitization and epitope recognition in peptide-immunized HLA DR3 transgenic mice. Hum Vaccin Immunother 10, 3048-3059. [0152] Trager, W., 1939. Accquired immunity to ticks. J Parasitol 25, 57-81. Valenzuela, J. G., Charlab, R., Mather, T. N., Ribeiro, J. M., 2000. Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis. J. Biol. Chem. 275, 18717-18723. https://doi.org/10.1074/jbc.M001486200 [0153] Valenzuela, J. G., Charlab, R., Mather, T. N., Ribeiro, J. M., 2000. Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis. J. Biol. Chem. 275, 18717-18723. https://doi.org/10.1074/jbc.M001486200 [0154] Vonesch, N., D'Ovidio, M. C., Melis, P., Remoli, M. E., Ciufolini, M. G., Tomao, P., 2016. Climate change, vector-borne diseases and working population. Ann. 1st. Super. Sanita 52, 397-405. [0155] Wikel, S. K., 1996. Host immunity to ticks. Annu. Rev. Entomol. 41, 1-22. [0156] Willyard, C., 2014. Resurrecting the yuppie vaccine. Nat. Med. 20, 698-701. [0157] Zantow, J., Dubel, S., Hust, M., 2016. Funktionales Proteom-Display zur Identifikation von Biomarkern. Biospektrum 22, 256-259. [0158] Zantow, J., Moreira, G. M. S. G., Dubel, S., Hust, M., 2018. ORFeome Phage Display. Methods Mol. Biol. 1701, 477-495.