Compositions and methods for eliciting a protective immune response against Lyme disease - Patents.com

Abstract

The present invention relates to methods of administering compositions comprising OspA fusion proteins of SEQ ID NO:1 (Lip-S1D1-S2D1), SEQ ID NO:2 (Lip-S4D1-S3hybD1), and SEQ ID NO:3 (Lip-S5D1-S6D1) to induce a protective immune response against Lyme disease in a subject, such as for vaccinating a subject against Lyme disease, and for treating, preventing, and / or reducing the risk of Lyme disease.

Description

[Technical field]

[0001] Electronic Sequence Listing Reference The contents of the electronic sequence listing (I042270147WO00-SEQ-JRV.xml; size: 13,099 bytes; and creation date: April 18, 2023) are incorporated herein by reference in their entirety.

[0002] Field The present invention is directed to methods of eliciting a protective immune response against Lyme disease in a subject, and compositions for use therewith. [Background technology]

[0003] background Lyme disease is the most common vector-borne disease in humans across temperate regions of the Northern Hemisphere, affecting hundreds of thousands of people annually in North America (United States and Canada) and Eurasia. In the United States, the Northeast, Mid-Atlantic, and northern Midwest regions are the most affected, with an estimated 476,000 cases diagnosed and treated annually between 2010 and 2018. In Europe, the incidence rate based on reported cases is approximately 65,000-85,000 cases per year. However, this figure is significantly underestimated due to inconsistent case reporting and the fact that Lyme disease often goes undiagnosed. Lyme disease can occur at any age; however, incidence peaks in children aged 5-15 years and adults older than 50 years.

[0004] Lyme disease is caused by the Borrelia burgdorferi spirochete, which is transmitted to humans through the bite of blood-sucking hard ticks. The most common clinical manifestation of Lyme disease is a slowly expanding erythematous skin rash known as erythema migrans (EM). EM appears at the site of the tick bite for several days to several weeks (average about 1-2 weeks) and is often accompanied by symptoms of fatigue, fever, headache, mild neck stiffness, joint pain, or muscle pain. Early Borrelia infections may be missed because objective signs and symptoms are not evident or are not reported. If untreated or inadequately treated with antibiotics, the infection can spread through the bloodstream to other parts of the body, causing serious manifestations affecting the nervous system, joints, or heart.

[0005] The incidence of Lyme disease is increasing and spreading geographically. More reliable preventative measures, such as a vaccine, are needed to further reduce the risk of contracting this potentially devastating disease. Currently, there is no licensed human Lyme disease vaccine and no other human Lyme disease vaccine candidate in clinical development. Thus, there is a large unmet medical need for a safe and effective Lyme disease vaccine. Summary of the Invention

[0006] SUMMARY OF THE PRESENT APPLICATION The present invention provides methods of administering compositions comprising OspA fusion proteins of SEQ ID NO:1 (Lip-S1D1-S2D1), SEQ ID NO:2 (Lip-S4D1-S3hybD1), and SEQ ID NO:3 (Lip-S5D1-S6D1) to induce a protective immune response against Lyme disease in a subject, e.g., to vaccinate the subject against Lyme disease, and to treat, prevent, and / or reduce the risk of Lyme disease. In particular, at least three doses or at least two doses of the composition are administered to adult and / or pediatric subjects to induce a strong immunogenicity profile.

[0007] Thus, in some aspects, the invention provides a method of eliciting a protective immune response against Lyme disease (Lyme borreliosis) in a subject, the method comprising administering to the subject a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1) in a total protein content of the three fusion proteins per dose in the range of 60 μg to 200 μg, wherein the method comprises administering at least three doses of the composition.

[0008] In some aspects, the present invention provides a method for treating, preventing or reducing the risk of Lyme disease (Lyme borreliosis) in a subject, the method comprising administering to the subject a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1) in a total protein content of the three fusion proteins per dose in the range of 60 μg to 200 μg, wherein the method comprises administering at least three doses of the composition.

[0009] In some aspects, the invention provides a method of vaccinating a subject against Lyme disease (Lyme borreliosis), the method comprising administering to the subject a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1) in a total protein content of the three fusion proteins per dose in the range of 60 μg to 200 μg, wherein the method comprises administering at least three doses of the composition.

[0010] In some embodiments, the second dose is administered within a period of at least 6 weeks and up to 3 months after administration of the first dose, and the third dose is administered within a period of at least 5 months and up to 9 months after administration of the first dose. In some embodiments, the second dose is administered within a period of at least 6 weeks and up to 3 months after administration of the first dose, and the third dose is administered within a period of at least 5 months and up to 7 months after administration of the first dose. In some embodiments, the second dose is administered within a period of at least 50 days and up to 70 days after administration of the first dose, and the third dose is administered within a period of at least 5 months and up to 9 months after administration of the first dose. In some embodiments, the second dose is administered within a period of at least 50 days and up to 70 days after administration of the first dose, and the third dose is administered within a period of at least 170 days and up to 190 days after administration of the first dose. In some embodiments, the first dose is administered on day 1, the second dose is administered about 2 months after the first dose, and the third dose is administered about 5 months to about 9 months after administration of the first dose. In some embodiments, the first dose is administered on day 1, the second dose is administered about 2 months after the first dose, and the third dose is administered about 6 months after administration of the first dose.

[0011] In some embodiments, the fourth dose is administered within a period of at least 15 months and up to 21 months, particularly at least 17 months and up to 19 months, after administration of the first dose. In some embodiments, the fourth dose is administered about 18 months after administration of the first dose. In some embodiments, the fourth dose is administered about 12 months after administration of the third dose.

[0012] In some embodiments, the total protein content of the three fusion proteins is 135 μg or 180 μg per dose, preferably 180 μg per dose, where the first dose is administered on day 1, the second dose is administered within a period of at least 50 days and up to 70 days after the first dose, and the third dose is administered within a period of at least 5 months and up to 9 months, or at least 5 months and up to 7 months, or at least 170 days and up to 190 days after the first dose, and optionally, the fourth dose is administered about 18 months after the administration of the first dose. In some embodiments, the fourth dose is administered about 12 months after the administration of the third dose.

[0013] In some embodiments, the subject is an adult subject, wherein the total protein content of the three fusion proteins is 135 μg or 180 μg per dose, preferably 180 μg per dose, wherein the first dose is administered on day 1, the second dose is administered within a period of at least 50 days and up to 70 days after the first dose, and the third dose is administered within a period of at least 5 months and up to 9 months, or at least 5 months and up to 7 months, or at least 170 days and up to 190 days after the first dose, and wherein optionally, the fourth dose is administered about 18 months after administration of the first dose or about 12 months after administration of the third dose.

[0014] In some embodiments, the subject is a pediatric subject, wherein the total protein content of the three fusion proteins is 135 μg or 180 μg per dose, preferably 180 μg per dose, wherein the first dose is administered on day 1, the second dose is administered within a period of at least 50 days and up to 70 days after the first dose, and the third dose is administered within a period of at least 5 months and up to 9 months, or at least 5 months and up to 7 months, or at least 170 days and up to 190 days after the first dose, and wherein optionally, the fourth dose is administered about 18 months after administration of the first dose or about 12 months after administration of the third dose.

[0015] In some embodiments, the subject is a pediatric subject from birth to 4 years old, wherein the total protein content of the three fusion proteins is 67.5 μg or 90 μg per dose, wherein the first dose is administered on day 1, the second dose is administered within a period of at least 50 days and up to 70 days after the first dose, and the third dose is administered within a period of at least 5 months and up to 9 months, or at least 5 months and up to 7 months, or at least 170 days and up to 190 days after the first dose, and wherein optionally, the fourth dose is administered about 18 months after administration of the first dose or about 12 months after administration of the third dose. In some embodiments, further doses are administered every year after the third or any fourth dose has been administered, particularly after 1 year, 2 years, and 3 years.

[0016] In some aspects, the invention provides a method of eliciting a protective immune response against Lyme disease in a subject, the method comprising administering to the subject a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1) in a total protein content of the three fusion proteins per dose in the range of 60 μg to 200 μg, wherein the method comprises administering at least two doses of the composition.

[0017] In some aspects, the present invention provides methods for treating, preventing or reducing the risk of Lyme disease in a subject, the methods comprising administering to a subject a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1) in a total protein content of the three fusion proteins per dose in the range of 60 μg to 200 μg, wherein the methods comprise administering at least two doses of the composition.

[0018] In some aspects, the invention provides a method of vaccinating a subject against Lyme disease, the method comprising administering to the subject a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1) in a total protein content of the three fusion proteins per dose in the range of 60 μg to 200 μg, wherein the method comprises administering at least two doses of the composition.

[0019] In some embodiments, the second dose is administered within a period of at least 5 months and up to 7 months after administration of the first dose. In some embodiments, the second dose is administered within a period of at least 170 days and up to 190 days after administration of the first dose. In some embodiments, the first dose is administered on day 1 and the second dose is administered about 6 months after administration of the first dose.

[0020] In some embodiments, the third dose is administered within a period of at least 15 months and up to 21 months, particularly at least 17 months and up to 19 months, after administration of the first dose, hi some embodiments, the third dose is administered about 18 months after administration of the first dose. In some embodiments, the total protein content of the three fusion proteins is 135 μg or 180 μg per dose, preferably 180 μg per dose, where the first dose is administered on day 1, the second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and where, optionally, the third dose is administered about 18 months after administration of the first dose.

[0021] In some embodiments, the subject is an adult subject, and the total protein content of the three fusion proteins is 135 μg or 180 μg per dose, preferably 180 μg per dose, where the first dose is administered on day 1, the second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and where, optionally, the third dose is administered about 18 months after administration of the first dose.

[0022] In some embodiments, the subject is a pediatric subject, and the total protein content of the three fusion proteins is 135 μg or 180 μg per dose, preferably 180 μg per dose, where the first dose is administered on day 1, the second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and where, optionally, the third dose is administered about 18 months after administration of the first dose.

[0023] In some embodiments, the subject is a pediatric subject from birth to 4 years old, and the total protein content of the three fusion proteins is 67.5 μg or 90 μg per dose, where the first dose is administered on day 1, the second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and optionally, the third dose is administered about 18 months after administration of the first dose. In some embodiments, further doses are administered every year after the second or any third dose is administered, particularly after 1 year, 2 years, and 3 years.

[0024] In some embodiments, the subject is a human subject. In some embodiments, the subject is 5 years of age or older. In some embodiments, the subject is 5-65 years of age. In some embodiments, the subject is an adult subject 18 years of age or older, e.g., 18-65 years of age and / or 50 years of age or older. In some embodiments, the subject is a pediatric subject between birth and 17 years of age. In some embodiments, the subject is a pediatric subject between 5 and 17 years of age, e.g., 5-11 years of age (children) and / or 12-17 years of age (adolescents). In some embodiments, the pediatric subject is between birth and 4 years of age, e.g., 1-4 years of age and / or 2-4 years of age.

[0025] In some embodiments, the total protein content of the three fusion proteins is 135 μg or 180 μg per dose. In some embodiments, the total protein content of the three fusion proteins is 180 μg per dose. In some embodiments, the total protein content of the three fusion proteins is 67.5 μg or 90 μg per dose. In some embodiments, the three fusion proteins comprise at least 60%, preferably at least 70%, more preferably at least 80% of the total protein in the composition. In some embodiments, the composition comprises the three fusion proteins in a 1:1:1 weight ratio (Lip-S1D1-S2D1: Lip-S4D1-S3hybD1: Lip-S5D1-S6D1).

[0026] In some embodiments, the composition elicits an immune response that comprises an antibody response against OspA serotype 1, serotype 2, serotype 3, serotype 4, serotype 5 and / or serotype 6. In some embodiments, the composition elicits an immune response that comprises antibodies against Borrelia serotypes 1, 2, 3, 4, 5 and 6. In some embodiments, the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, wherein the immune response after administration of at least three doses of the composition to an adult subject is greater than the immune response after administration of at least two doses of the composition to an adult subject.

[0027] In some embodiments, the geometric mean titer (GMT) of antibodies to Borrelia serovars after administration of at least three doses of the composition to an adult subject is at least 2.0-fold higher than the GMT after administration of at least two doses of the composition to an adult subject. In some embodiments, the GMT of antibodies to Borrelia serovars after administration of at least three doses of the composition to an adult subject is about 2.0-fold to about 3.0-fold higher than the GMT after administration of at least two doses of the composition to an adult subject.

[0028] In some embodiments, the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, wherein the immune response following administration of a second dose of the composition to a pediatric subject is at least as great as or greater than the immune response following administration of a third dose of the composition to an adult subject.

[0029] In some embodiments, the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, wherein the immune response after administration of at least three doses of the composition to a pediatric subject is greater than the immune response after administration of at least three doses of the composition to an adult subject. In some embodiments, the GMT of antibodies against Borrelia serotypes after administration of at least three doses of the composition to a pediatric subject is at least 2.0-fold greater than the GMT after administration of at least three doses of the composition to an adult subject.

[0030] In some embodiments, the GMT of antibodies against Borrelia serotypes after administration of at least three doses of the composition to a pediatric subject ages 12-17 is about 2.0-fold to about 3.0-fold higher than the GMT after administration of at least three doses of the composition to an adult subject. In some embodiments, the GMT of antibodies against Borrelia serotypes after administration of at least three doses of the composition to a pediatric subject ages 5-11 is about 3.0-fold to about 5.0-fold higher than the GMT after administration of at least three doses of the composition to an adult subject.

[0031] In some embodiments, the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, wherein the immune response after administration of at least two doses of the composition to a pediatric subject is greater than the immune response after administration of at least two doses of the composition to an adult subject. In some embodiments, the GMT of antibodies against Borrelia serotypes after administration of at least two doses of the composition to a pediatric subject is at least 2.0-fold greater than the GMT after administration of at least two doses of the composition to an adult subject.

[0032] In some embodiments, the GMT of antibodies against Borrelia serotypes after administration of at least two doses of the composition to a pediatric subject ages 12-17 is about 4.0-fold to about 6.0-fold higher than the GMT after administration of at least two doses of the composition to an adult subject. In some embodiments, the GMT of antibodies against Borrelia serotypes after administration of at least two doses of the composition to a pediatric subject ages 5-11 is about 5.0-fold to about 8.0-fold higher than the GMT after administration of at least two doses of the composition to an adult subject. In some embodiments, the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and 6, which immune response lasts for at least about 60 days, at least about 180 days, at least about 365 days, or at least about 540 days.

[0033] In some embodiments, the immune response persists above baseline for at least about 180 days after administration of at least two doses or at least three doses of the composition. In some embodiments, the geometric mean fold rise (GMFR) of antibodies to Borrelia serovars at least 180 days after administration of at least three doses of the composition to a pediatric subject aged 5-11 is at least 2.0-fold higher than the baseline GMFR. In some embodiments, the GMFR of antibodies to Borrelia serovars at least 180 days after administration of at least three doses of the composition to a pediatric subject aged 5-11 is about 2.0-fold to 7.0-fold higher than the baseline GMFR. In some embodiments, the composition is administered to the subject in a volume of 0.25 ml, 0.5 ml, or 1.0 ml. In a preferred embodiment, the composition is administered to the subject in a volume of 0.5 ml.

[0034] In some embodiments, the composition comprises at least one of sodium phosphate, sodium chloride, sucrose, and polysorbate 20. In some embodiments, the sodium phosphate is present at a concentration of 5 mM to 50 mM, the sodium chloride is present at a concentration of 100 mM to 200 mM, the sucrose is present at a concentration of 2.5% to 10%, and the polysorbate 20 is present at a concentration of 0.01% to 0.1%. In some embodiments, the composition comprises an adjuvant. In some embodiments, the adjuvant comprises an aluminum adjuvant. In some embodiments, the composition comprises less than 1.25 ppb of copper. In some embodiments, the copper is in ionic form, particularly in the form of Cu+ or Cu2+. In some embodiments, the composition comprises L-methionine. In some embodiments, the L-methionine is present at a concentration of at least 10 mmol / l. In some embodiments, the concentration (mol / l) of L-methionine is at least equal to the concentration of copper in the composition.

[0035] In some embodiments, the composition further comprises a reactive compound, wherein the reactive compound is selected from a redox active compound, a radical building compound, a stabilizing compound, and any combination thereof, particularly wherein the reactive compound is selected from formaldehyde, ethanol, chloroform, trichloroethylene, acetone, Triton-X-100, deoxycholic acid, diethylpyrocarbonate, sulfite, Na2S2O5, β-proprio-lactone, polysorbates such as Tween 20®, Tween 80®, O2, phenol, pluronic type copolymers, and any combination thereof.

[0036] The present invention further provides a composition as described herein for use in eliciting a protective immune response against Lyme disease in a subject. In another aspect, the present invention provides a composition as described herein for use in treating, preventing, or reducing the risk of Lyme disease in a subject. In another aspect, the present invention provides a composition as described herein for use in vaccinating a subject against Lyme disease. In another aspect, the present invention provides a composition as described herein for use as a medicament. In another aspect, the present invention provides a composition as described herein for use as a vaccine. [Brief description of the drawings]

[0037] [Figure 1] Subject enrollment process for Part A (pivotal study phase) of a Phase 2 clinical trial of a multivalent Borrelia vaccine; investigating a three-dose or two-dose primary immunization schedule implemented in a stepwise fashion with decreasing age for three age cohorts. [Figure 2A] Figure 2A) shows the study design (Part A: main study phase, Part B: booster phase) of a Phase 2 clinical trial of a multivalent Borrelia vaccine, investigating a three-dose or two-dose primary immunization schedule and booster vaccination of the vaccine in a study population aged 5-65 years; and Figure 2B) shows the alternative Part B: booster phase. [Figure 2B] Figure 2A) shows the study design (Part A: main study phase, Part B: booster phase) of a Phase 2 clinical trial of a multivalent Borrelia vaccine, investigating a three-dose or two-dose primary immunization schedule and booster vaccination of the vaccine in a study population aged 5-65 years; and Figure 2B) shows the alternative Part B: booster phase. [Diagram 3] Geometric mean titers (GMTs) of OspA serotype-specific IgG antibodies (ST1-6) determined by ELISA at 208 days / 7 months. [Figure 4]Geometric mean titers (GMTs) of OspA serotype-specific IgG antibodies (ST1-6) determined by ELISA by age cohort at 208 days / 7 months. [Diagram 5] Geometric mean titers (GMTs) of OspA serotype-specific IgG antibodies (ST1-6) determined over time by ELISA, Group 1 (3 doses, M 0-2-6).

[0038] [Figure 6A] Inverse cumulative distribution curves of ELISA% of subjects at 208 days / 7 months vs. OspA-specific IgG antibody titers by age cohort: Figure 6A) serotype 1, Figure 6B) serotype 2, Figure 6C) serotype 3, Figure 6D) serotype 4, Figure 6E) serotype 5, and Figure 6F) serotype 6. [Figure 6B] Inverse cumulative distribution curves of ELISA% of subjects at 208 days / 7 months vs. OspA-specific IgG antibody titers by age cohort: Figure 6A) serotype 1, Figure 6B) serotype 2, Figure 6C) serotype 3, Figure 6D) serotype 4, Figure 6E) serotype 5, and Figure 6F) serotype 6. [Figure 6C] Inverse cumulative distribution curves of ELISA% of subjects at 208 days / 7 months vs. OspA-specific IgG antibody titers by age cohort: Figure 6A) serotype 1, Figure 6B) serotype 2, Figure 6C) serotype 3, Figure 6D) serotype 4, Figure 6E) serotype 5, and Figure 6F) serotype 6. [Figure 6D] Inverse cumulative distribution curves of ELISA% of subjects at 208 days / 7 months vs. OspA-specific IgG antibody titers by age cohort: Figure 6A) serotype 1, Figure 6B) serotype 2, Figure 6C) serotype 3, Figure 6D) serotype 4, Figure 6E) serotype 5, and Figure 6F) serotype 6. [Figure 6E] Inverse cumulative distribution curves of ELISA% of subjects at 208 days / 7 months vs. OspA-specific IgG antibody titers by age cohort: Figure 6A) serotype 1, Figure 6B) serotype 2, Figure 6C) serotype 3, Figure 6D) serotype 4, Figure 6E) serotype 5, and Figure 6F) serotype 6. [Figure 6F]Inverse cumulative distribution curves of ELISA% of subjects at 208 days / 7 months vs. OspA-specific IgG antibody titers by age cohort: Figure 6A) serotype 1, Figure 6B) serotype 2, Figure 6C) serotype 3, Figure 6D) serotype 4, Figure 6E) serotype 5, and Figure 6F) serotype 6.

[0039] [Figure 7] Seroconversion rates (SCR) by age cohort at day 208 / 7 months.

[0040] [Figure 8] Geometric mean titers (GMTs) of OspA serotype-specific IgG antibodies (ST1-6) determined by ELISA over time in the M0-2-6 group. [Figure 9] Geometric mean titers (GMTs) of OspA serotype-specific IgG antibodies (ST1-6) determined by ELISA over time in the M 0-6 groups. [Figure 10] Inverse cumulative distribution curves of ELISA% vs. ST1 OspA-specific IgG antibody titers at 365 days / months 12 by age group: Group 1 (M 0-2-6), Group 2 (M 0-6), Group 3 (placebo), Cohort 1 (18-65 years), Cohort 2 (12-17 years), and Cohort 3 (5-11 years).

[0041] [Figure 11] Inverse cumulative distribution curve of ELISA % of subjects in the M 0-2-6 group versus ST1 OspA-specific IgG antibody titers over time. [Figure 12] Inverse cumulative distribution curve of ELISA % of subjects in M ​​0-6 groups versus ST1 OspA-specific IgG antibody titers over time. [Figure 13] Seroconversion rates (SCR) by age cohort at 365 days / 12 months.

[0042] Detailed Description of the Invention The multivalent Lyme borreliosis vaccine of the present invention contains the immunodominant C-terminal portions of the six most common OspA serotypes present in Europe (ST1-ST6) and the United States (ST1) and is designed to induce anti-OspA antibodies to neutralize Borrelia in the tick gut and block its transmission to vertebrate hosts. OspA-based vaccines require high and sustained antibody titers due to their mechanism of action (outside the immunized host body, against bacteria in the tick vector gut). The vaccine and administration schedule of the present invention allows for the generation, and continued maintenance of high antibody titers, of sufficient immune response activity against bacteria in the tick gut for use in treating, preventing, and / or reducing the risk of Lyme borreliosis.

[0043] The Phase 2 trial described herein is the first clinical study with the vaccine of the present invention, which enrolled a pediatric population (ages 5-17 years). The study compares the immunogenicity and safety of the vaccine of the present invention following administration of a two-dose (0 months and 6 months) or three-dose (0 months, 2 months, and 6 months) primary series vaccination in age groups 5-11 years, 12-17 years, and 18-65 years. The vaccine was found to be more immunogenic in pediatric participants (ages 5-17 years) who received the vaccine in either the two-dose or three-dose schedule than in adults who were tested with both vaccination schedules. A strong immunogenicity profile was observed 1 month after the primary vaccination series in study participants ages 5-17 years. The safety and tolerability profile observed in pediatric participants (ages 5-17 years) was similar to the safety profile previously observed in adult participants.

[0044] The present invention provides a method of eliciting a protective immune response against Lyme disease in a subject comprising administering a composition comprising a fusion protein of SEQ ID NO: 1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO: 3 (Lip-S5D1-S6D1).The present invention further provides a method for treating, preventing, or reducing the risk of Lyme disease in a subject comprising administering a composition comprising a fusion protein of SEQ ID NO: 1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO: 3 (Lip-S5D1-S6D1). The present invention also provides a method of vaccinating a subject, comprising administering a composition comprising a fusion protein of SEQ ID NO: 1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO: 3 (Lip-S5D1-S6D1).

[0045] The method may include administering at least three doses or at least two doses of the composition to the subject. In one embodiment, the method includes administering at least three doses of the composition to the subject. Alternatively, the method includes administering at least two doses of the composition to the subject. The subject may be 5 years of age or older. The subject may be an adult subject (18 years of age or older, including 18-65 years of age and / or 50 years of age or older) or a pediatric subject (birth to 17 years of age, including 5-17 years of age, e.g., 5-11 years of age and 12-17 years of age, and birth to 4 years of age, including 1-4 years of age and 2-4 years of age).

[0046] The composition may contain a total protein content of the three fusion proteins in the range of 60 μg to 200 μg per dose. In an embodiment of the present invention, the composition contains a total protein content of the three fusion proteins of 135 μg per dose. In an embodiment of the present invention, the composition contains a total protein content of the three fusion proteins of 180 μg per dose. In an embodiment of the present invention, the composition contains a total protein content of the three fusion proteins of 67.5 μg or 90 μg per dose.

[0047] The method may comprise administering at least three doses of the composition to an adult and / or pediatric subject. In a preferred embodiment, the composition is administered to an adult subject in at least three doses. In an embodiment of the present invention, the composition is administered to a pediatric subject in at least three doses. Thus, the first dose may be administered on day 1, the second dose may be administered within a period of at least 50 days and up to 70 days after the first dose, and the third dose may be administered within a period of at least 5 months and up to 9 months, or at least 5 months and up to 7 months, or at least 170 days and up to 190 days after the first dose, and optionally, the fourth dose may be administered about 18 months after the administration of the first dose or about 12 months after the administration of the third dose.

[0048] Alternatively, the method may comprise administering at least two doses of the composition to an adult and / or pediatric subject.In an embodiment of the present invention, the composition is administered to an adult subject in at least two doses.In an embodiment of the present invention, the composition is administered to a pediatric subject in at least two doses.Thus, the first dose may be administered on day 1, the second dose may be administered within a period of at least 170 days to up to 190 days after the first dose, and optionally, the third dose may be administered about 18 months after the administration of the first dose. The method may include administering further doses annually, particularly after 1 year, 2 years and 3 years.

[0049] In a preferred embodiment, the method comprises administering to an adult subject a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), wherein the total protein content of the three fusion proteins is 180 μg per dose, and the method comprises administering at least three doses of the composition, wherein a first dose is administered on day 1, a second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and optionally, a third dose is administered about 18 months after administration of the first dose.

[0050] In a preferred embodiment, the method comprises administering to a pediatric subject a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), wherein the total protein content of the three fusion proteins is 180 μg per dose, and the method comprises administering at least three doses of the composition, wherein a first dose is administered on day 1, a second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and optionally, a third dose is administered about 18 months after administration of the first dose.

[0051] Alternatively, a preferred embodiment provides the following method: the method comprising administering to an adult subject a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), wherein the total protein content of the three fusion proteins is 180 μg per dose, and the method comprises administering at least two doses of the composition, wherein a first dose is administered on day 1, a second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and optionally a third dose is administered about 18 months after administration of the first dose.

[0052] Alternatively, a preferred embodiment provides the following method: the method comprising administering to a pediatric subject a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), wherein the total protein content of the three fusion proteins is 180 μg per dose, and the method comprises administering at least two doses of the composition, wherein a first dose is administered on day 1, a second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and optionally a third dose is administered about 18 months after administration of the first dose.

[0053] In the present study, in adults, the geometric mean antibody titers (GMTs) for all six serotypes were statistically significantly higher in the three dose group (M 0-2-6) compared to the two dose group (M 0-6) at day 208 / 7 months. The GMTs of antibodies against Borrelia serotypes after administration of at least three doses of the composition to adult subjects are at least 2.0-fold higher and / or about 2.0-fold to about 3.0-fold higher than the GMTs after administration of at least two doses of the composition to adult subjects. See Tables 2 and 3.

[0054] In one embodiment, the GMT of antibodies against Borrelia serotypes after administration of at least three doses of the composition to an adult subject is about 2.5-fold higher for serotype 1, about 2.0-fold higher for serotype 2, about 2.1-fold higher for serotype 4, about 2.4-fold higher for serotype 5, and / or about 2.7-fold higher for serotype 6 than the GMT after administration of at least two doses of the composition to an adult subject. See Tables 2 and 3.

[0055] Additionally, in adults, the geometric mean fold increase (GMFR) for all six serotypes was statistically significantly higher in the three dose group (M 0-2-6) schedule compared to the two dose group (M 0-6). The GMFR of antibodies to Borrelia serotypes after administration of at least three doses of the composition to adult subjects is at least 1.5-fold higher and / or about 1.5-fold to about 3.0-fold higher than the GMT after administration of at least two doses of the composition to adult subjects. See Table 6.

[0056] Thus, in an embodiment of the invention, the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, wherein the immune response after administration of three doses of the composition to an adult subject is greater than the immune response after administration of two doses of the composition to an adult subject. See Example 1, FIG. 3.

[0057] In the present study, already after the second dose of the M 0-2-6 schedule (i.e., day 85) in pediatric subjects (adolescents aged 12-17 and children aged 5-11), the immune response for all six serotypes was as high as after three doses in adults aged 18-65 (i.e., day 208). See Table 4. Thus, in an embodiment of the present invention, the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, where the immune response after administration of a second dose of the composition to a pediatric subject is at least as high as or higher than the immune response after administration of a third dose of the composition to an adult subject. See Example 1, FIG. 5.

[0058] In an embodiment of the invention, the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, wherein the immune response after administration of at least three doses of the composition to a pediatric subject is higher than the immune response after administration of at least three doses of the composition to an adult subject. The GMT of antibodies against Borrelia serotypes after administration of at least three doses of the composition to a pediatric subject is at least 2.0-fold higher than the GMT after administration of at least three doses of the composition to an adult subject. See Table 2.

[0059] For pediatric subjects aged 12-17 years, the GMT of antibodies against Borrelia serotypes after administration of at least three doses of the composition to pediatric subjects aged 12-17 years is about 2.0-fold to about 3.0-fold higher than the GMT after administration of at least three doses of the composition to adult subjects. Additionally, the GMT of antibodies against Borrelia serotypes after administration of at least three doses of the composition to pediatric subjects aged 12-17 years is about 2.6-fold higher for serotype 1, about 2.2-fold higher for serotype 2, about 2.4-fold higher for serotype 3, about 2.6-fold higher for serotype 4, about 2.4-fold higher for serotype 5, and / or about 2.5-fold higher for serotype 6 than the GMT after administration of at least three doses of the composition to adult subjects.

[0060] For pediatric subjects aged 5-11 years, the GMT of antibodies against Borrelia serotypes after administration of at least three doses of the composition to pediatric subjects aged 5-11 years is about 3.0-fold to about 5.0-fold higher than the GMT after administration of at least three doses of the composition to adult subjects. Additionally, the GMT of antibodies against Borrelia serotypes after administration of at least three doses of the composition to pediatric subjects aged 5-11 years is about 3.5-fold higher for serotype 1, about 3.3-fold higher for serotype 2, about 3.1-fold higher for serotype 3, about 3.6-fold higher for serotype 4, about 3.8-fold higher for serotype 5, and / or about 4.1-fold higher for serotype 6 than the GMT after administration of at least three doses of the composition to adult subjects.

[0061] In an embodiment of the invention, the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, wherein the immune response after administration of at least two doses of the composition to a pediatric subject is higher than the immune response after administration of at least two doses of the composition to an adult subject. See Example 1 and Figure 7. The GMT of antibodies against Borrelia serotypes after administration of at least two doses of the composition to a pediatric subject is at least 2.0-fold higher than the GMT after administration of at least two doses of the composition to an adult subject. See Table 2.

[0062] For pediatric subjects aged 12-17 years, the GMT of antibodies against Borrelia serotypes after administration of at least two doses of the composition to pediatric subjects aged 12-17 years is about 4.0-fold to about 6.0-fold higher than the GMT after administration of at least two doses of the composition to adult subjects. Additionally, the GMT of antibodies against Borrelia serotypes after administration of at least two doses of the composition to pediatric subjects aged 12-17 years is about 5.8-fold higher for serotype 1, about 4.3-fold higher for serotype 2, about 5.4-fold higher for serotype 3, about 5.5-fold higher for serotype 4, about 5.5-fold higher for serotype 5, and / or about 4.9-fold higher for serotype 6 than the GMT after administration of at least three doses of the composition to adult subjects.

[0063] For pediatric subjects aged 5-11 years, the GMT of antibodies against Borrelia serotypes after administration of at least two doses of the composition to pediatric subjects aged 5-11 years is about 5.0-fold to about 8.0-fold higher than the GMT after administration of at least two doses of the composition to adult subjects. Additionally, the GMT of antibodies against Borrelia serotypes after administration of at least two doses of the composition to pediatric subjects aged 5-11 years is about 6.7-fold higher for serotype 1, about 5.7-fold higher for serotype 2, about 7.2-fold higher for serotype 3, about 6.3-fold higher for serotype 4, about 6.9-fold higher for serotype 5, and / or about 6.9-fold higher for serotype 6 than the GMT after administration of at least two doses of the composition to adult subjects.

[0064] In embodiments of the invention, the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and 6, and the immune response persists for at least about 60 days, at least about 180 days, at least about 365 days, or at least about 540 days. For example, the immune response persists above baseline for at least about 180 days after administration of at least two doses or at least three doses of the composition. See Tables 9 and 10.

[0065] For pediatric subjects aged 5-11 years, the GMFR of antibodies to Borrelia serotypes at least 180 days after administration of at least three doses of the composition to a pediatric subject aged 5-11 years is at least 2.0-fold higher and / or about 2.0-fold to 7.0-fold higher than the baseline GMFR. Additionally, the GMFR of antibodies to Borrelia serotypes at least 180 days after administration of at least three doses of the composition to a pediatric subject aged 5-11 years is about 2.8-fold higher for serotype 1, about 6.6-fold higher for serotype 2, about 5.4-fold higher for serotype 3, about 4.4-fold higher for serotype 4, about 5.5-fold higher for serotype 5, and / or about 4.3-fold higher for serotype 6 than the baseline GMFR. See Table 11. In an embodiment of the invention, the composition is administered in a volume of 0.25 ml, 0.5 ml, or 1.0 ml. In a preferred embodiment, the composition is administered in a volume of 0.5 mL.

[0066] Lyme disease Lyme borreliosis (also referred to herein as "LB") or Lyme disease (also referred to herein as "LD") is the most commonly reported tick-borne disease in Europe and North America. The disease is caused by infection with the arthropod-borne gram-negative spirochete Borrelia burgdorferi sensu lato and can affect multiple organs or tissues, resulting in skin, cardiac, musculoskeletal, and neurological disorders.

[0067] In most countries, Lyme borreliosis is not a notifiable disease, and therefore accurate data on annual incidence are not available. In Europe, most human infections are caused by four Borrelia genospecies, representing six OspA serotypes (ST): B. afzelii (ST2), B. garinii (ST3, ST5, and ST6), B. burgdorferi sensu stricto (ss) (ST1), and B. bavariensis (ST4). In the United States, B. burgdorferi ss (ST1) is found in almost 100% of cases. Recently, a new genospecies, named Borrelia mayonii, has been described, which was found in several clinical specimens isolated in the north-central and western United States [Pritt BS, et al. Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study, Lancet Infect Dis 2016; 16: 556-64].

[0068] Surface protein A (OspA) is one of the major proteins expressed by Borrelia burgdorferi sensu lato when present in unfed ticks. During tick feeding, the incoming blood sends a signal that downregulates OspA expression, allowing the spirochete to invade the intestinal epithelium and migrate to the salivary glands and then into the host blood [Schwan TG and Piesman J. Temporal changes in outer surface proteins A and C of the Lyme disease-associated spirochete, Borrelia burgdorferi, during the chain of infection in ticks and mice. J Clin Microbiol 2000,38:382-8. J Clin Microbiol 2000,38:382-8]. OspA-based LB vaccines induce antibodies directed against OspA-expressing spirochetes in the tick gut, blocking transmission of the spirochetes to the salivary glands and subsequently to the vertebrate host [de Silva AM and Telford SR 3rd, Brunet LR, Barthold SW, Fikrig E., J Exp Med. 1996, 183(1):271-5].

[0069] The multivalent Lyme borreliosis vaccine of the present invention contains the immunodominant C-terminal portions of the six most common OspA serotypes present in Europe (ST1-ST6) and the United States (ST1) and is designed to induce anti-OspA antibodies to neutralize Borrelia in the tick gut and prevent its transmission to the host. The multimeric Borrelia vaccine has been shown to be highly protective against four Borrelia genospecies (B. burgdorferi ss, B. afzelii, B. bavariensis and B. garinii) containing five clinically relevant OspA serotypes (ST1, ST2, ST4, ST6) in mouse models challenged with either infected ticks or in vitro cultured spirochetes.

[0070] Multimeric Borrelia Vaccine (hereinafter referred to as "MBV") MBV is a multivalent surface protein A (OspA)-based vaccine designed for the prevention of Lyme borreliosis. The vaccine targets the majority of Borrelia strains expressing clinically relevant OspA serotypes (ST) present in the United States (ST1) and Europe (ST1-ST6). The vaccine contains three proteins, each containing the C-terminal halves of two OspA serotypes, which are linked to form three fusion proteins of ~35 kDa (ST1 and ST2, ST4 and ST3, and ST5 and ST6). MBV is used in formulations containing aluminum hydroxide adjuvant. The compositions of the present invention include a fusion protein of SEQ ID NO: 1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO: 3 (Lip-S5D1-S6D1), and are referred to herein as multimeric Borrelia vaccine (hereinafter "MBV").

[0071] The fusion protein (also called heterodimer) is derived from Borrelia surface protein A (OspA). OspA is expressed only when Borrelia are present in the tick vector's gut. Thus, OspA antibodies generated by vaccination do not fight infection internally, but enter the tick's gut when it ingests blood. There, the antibodies are thought to neutralize the spirochetes and block the transfer of bacteria from the tick's midgut to the salivary glands, the route by which Borrelia invade vertebrate hosts. Thus, OspA-specific antibodies prevent the transmission of Borrelia from the tick vector to the human host.

[0072] The fusion protein is a lipidated mutant OspA fragment fusion protein comprising a hybrid C-terminal OspA fragment, where the hybrid fragment consists of a C-terminal domain of Borrelia OspA composed of a fragment from OspA of a Borrelia strain different from B. garinii PBr strain and a second fragment of OspA from B. garinii PBr strain, and differs from the corresponding wild-type sequence at least by the introduction of at least one disulfide bond. The disulfide bond is disulfide bond type 1, where cysteine ​​residues are inserted at positions 183+ / -3 and 270+ / -3 (for further details, see WO 2015 / 104396 A1, which is incorporated herein by reference in its entirety). S3hyb denotes the fusion of amino acids 125-176 of B. valaisiana with amino acids 177-274 of B. garinii (PBr strain). Lip denotes lipidated, indicating the N-terminal addition of glycerol and fatty acid residues. The "LN1" peptide linker is a fusion of two separate loop regions of the N-terminal half of OspA from B. burgdorferi ss strain B31 (aa 65-74 and aa 42-53, and an amino acid exchange from position 53 to D53S) and has the following sequence: GTSDKNNGSGSKEKNKDGKYS (SEQ ID NO: 7).

[0073] In particular, Lip-S1D1-S2D1 is a disulfide bond type 1 fusion protein of OspA serotype 1 and OspA serotype 2, containing an N-terminal CSS for lipidation, an LN1 linker sequence, and N-terminal lipidation. Amino acids 164-174 of OspA serotype 1 are replaced with the non-hLFA-1-like sequence NFTLEGKVAND (SEQ ID NO:8). The sequence is shown in SEQ ID NO:1 below: Lip-S1D1-S2D1-aa SEQ ID NO: 1 LipCSSFNEKGEVSEKIITRADGTRLEYTGIKSDGSGKAKEVLKNFTLEGKVANDKTTLVVKCGTVTLSKNISKSGEVSVELNDTTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTKENTITVQQYDSNGTKLEGSAVEITKLDEICNALKGTSDKNN GSGSKEKNKDGKYSFNEKGELSAKTMTRENGTKLEYTEMKSDGTGKAKEVLKNFTLEGKVANDKVTLEVKCGTVTLSKEIAKSGEVTVALNDTNTTQATKKTGAWDSKTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGTNLEGTAVEIKTLDELCNALK

[0074] Lip-S4D1-S3hybD1 is a fusion protein of OspA serotype 4 and OspA serotype 3, containing amino acids 125-176 of B. valaisiana strain VS116 and amino acids 177-274 of B. garinii strain PBr, serotype 3, bound by disulfide bond type 1, and containing an N-terminal CSS for lipidation, an LN1 linker sequence, and an N-terminal lipidation. The sequence is shown in SEQ ID NO:2: Lip-S4D1-S3hybD1-aa SEQ ID NO: 2 LipCSSFNAKGELSEKTILRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAADKTTLKVTCGTVVLSKHIPNSGEITVELNDSNSTQATKKTGKWDSNTSTLTISVNSKKTKNIVFTKEDTITVQKYDSAGTNLEGNAVEIKTLDELCNALKGTSDKNNG SGSKEKNKDGKYSFNEKGEVSEKILTRSNGTTLEYSQMTDAENATKAVETLKNGIKLPGNLVGGKTKLTVTCGTVTLSKNISKSGEITVALNDTETTPADKKTGEWKSDTSTLTISKNSQKTKQLVFTKENTITVQNYNRAGNALEGSPAEIKDLAELCAALK

[0075] Lip-S5D1-S6D1 are fusion proteins of OspA serotype 6, both with disulfide bond type 1, an N-terminal CSS for lipidation, an LN1 linker sequence, and N-terminal lipidation. The sequence is shown in SEQ ID NO:3: Lip-S5D1-S6D1-aa SEQ ID NO:3 LipCSSFNEKGEISEKTIVRANGTRLEYTDIKSDKTGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVTLSKNISKSGEITVALDDTDSSGNKKSGTWDSGTSTLTISKNRTKTKQLVFTKEDTITVQNYDSAGTNLEGKAVEITTLKELCNALKGTSDKNNG SGSKEKNKDGKYSFNGKGETSEKTIVRANGTRLEYTDIKSDGSGKAKEVLKDFTLEGTLAADGKTTLKVTCGTVVLSKNILKSGEITAALDDSDTTRATKKTGKWDSKTSTLTISVNSQKTKNLVFTKEDTITVQRYDSAGTNLEGKAVEITTLKELCNALK

[0076] The nucleic acid sequence encoding the above fusion protein is as follows: Lip-S1D1-S2D1-nt SEQ ID NO:4

[0077] Lip-S4D1-S3hybD1-nt SEQ ID NO:5

[0078] Lip-S5D1-S6D1-nt SEQ ID NO:6

[0079] Further information regarding fusion proteins and their production can be obtained from WO 2015 / 104396 A1, which is incorporated by reference in its entirety herein, where Lip-S1D1-S2D1, Lip-S4D1-S3hybD1, and Lip-S5D1-S6D1 correspond to SEQ ID NOs: 29, 27, and 33, respectively.

[0080] As detailed above, the fusion protein is a lipidated protein, where the lipid moiety, together with the glycerol group, is also referred to as "Lip". According to the present invention, Lip is a lipid consisting of one to three lipids, e.g., a C glycerol bonded to the glycerol and amino groups of the N-terminal cysteine ​​of the polypeptide of the present invention. 14-20 Alkyl and / or C 14-20 alkenyl, etc., or preferably, Lip is a moiety represented by formula (I): [ka] wherein one of R1, R2 or R3 is C 14 -C 20 alkyl or alkenyl, and each of the others is independently C 14 -C 20 Alkyl or C 14 -C 20 alkenyl, and X is an amino acid sequence bound to the cysteine ​​residue as shown in formula (I). More preferably, Lip and the N-terminal cysteine ​​of the polypeptide are N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy)propylcysteine ​​(referred to herein as "Pam3Cys"), linked to said amino acid sequence of the invention via the carbonyl C of the cysteine. In the above formula (I), R1, R2 and R3 are palmitoyl moieties, and X is an amino acid sequence bound to the cysteine ​​residue.

[0081] The fusion protein is contained in a composition. The composition may be pharma-ceutically acceptable, allowing administration to humans. It may optionally contain any pharma-ceutically acceptable carrier or excipient, such as a buffer substance, a stabilizer or further active ingredient, in particular an ingredient known in connection with pharmaceutical compositions and / or vaccine production. The composition may contain sodium phosphate, sodium chloride, L-methionine, sucrose and polysorbate 20 (Tween-20) at a pH of 6.7+ / -0.2. Preferably, the pharmaceutical composition also contains aluminium hydroxide, preferably at a concentration of 0.15%. The composition may also contain 5mM-50mM sodium phosphate, 100-200mM sodium chloride, 5mM-25mM L-methionine, 2.5%-10% (w / v) sucrose, 0.01%-0.1% (v / v) Tween 20 and 0.1%-0.2% (w / v) aluminium hydroxide. More preferably, the formulation comprises 10 mM sodium phosphate, 150 mM sodium chloride, 10 mM L-methionine, 5% sucrose, 0.05% Tween 20 and 0.15% (w / v) aluminum hydroxide at a pH of 6.7±0.2. In a preferred embodiment, the excipient is L-methionine.

[0082] In some embodiments, the composition comprising the fusion protein is administered to the subject in a volume of about 0.25 milliliters (ml) to about 1.0 ml or more, such as 0.25 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, or 1.0 ml. In one embodiment, the volume administered to the subject ranges from 0.25 ml to 1.0 ml, or 0.25 ml to 0.5 ml, or 0.5 ml to 1.0 ml. In one embodiment, the volume administered to the subject is about 0.25 ml, about 0.5 ml, or about 1.0 ml. In one embodiment, the volume administered to the subject is 0.25 ml, 0.5 ml, or 1.0 ml. In one embodiment, the volume administered to the subject is 0.25 ml. In a preferred embodiment, the volume administered to the subject is 0.5 ml. In one embodiment, the volume administered to the subject is 1.0 ml. In a preferred embodiment, the volume administered to a subject aged 5 years or older (e.g., 5-17 years, 5-11 years, 12-17 years, 18 years or older, 18-65 years, or 50 years or older) is 0.5 ml or 1.0 ml. In a preferred embodiment, the volume administered to a subject aged 5 years or older (e.g., 5-17 years, 5-11 years, 12-17 years, 18 years or older, 18-65 years, or 50 years or older) is 1.0 ml. In a further preferred embodiment, the volume administered to a subject aged 5 years or older (e.g., 5-17 years, 5-11 years, 12-17 years, 18 years or older, 18-65 years, or 50 years or older) is 0.5 ml. In a further preferred embodiment, the volume administered to a subject aged 0-4 years (e.g., 1-4 years, or 2-4 years) is 0.25 ml.

[0083] According to the present invention, the composition is used as a vaccine, in particular against infections caused by Borrelia species, more preferably pathogenic Borrelia species as disclosed herein, more preferably including B. burgdorferi ss, B. afzelii, B. bavariensis and B. garinii, and / or other pathogens whose antigens are included in the vaccine. Preferably, the Borrelia species is selected from B. burgdorferi ss, B. garinii, B. afzelii, B. andersoni, B. bavariensis, B. bissettii, B. valaisiana, B. lusitaniae, B. spielmanii, B. japonica, B. tanukii, B. turdi or B. sinica infections, preferably B. burgdorferi ss, B. afzelii and B. garinii.

[0084] The composition for use according to the invention can be administered to humans as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. The composition can be administered via a systemic or mucosal route. These administrations can include injection via intramuscular, intraperitoneal, intradermal or subcutaneous routes; or mucosal administration to the oral / digestive, respiratory or urogenital system. The vaccine used in the method of the invention can be administered as a combination of three fusion proteins, although its components (e.g. individual fusion proteins) can be administered separately, simultaneously or sequentially, or as a one-component vaccine (i.e. simply Lip-S1D1-S2D1).

[0085] method It is of utmost importance that, because the special mechanism of action of this vaccine occurs outside the human body, relatively high antibody titers are always required throughout the tick season. Furthermore, because OspA is present on the surface of Borrelia bacteria only while they are in the tick midgut and not present after the antibodies enter the human host, the natural boosting effect on pre-existing OspA-specific antibodies during natural infection was expected to be minimal. Thus, high antibody titers are required and the composition should be administered at least three times, or alternatively two times. Accordingly, the composition may be administered two, three, four, five or more times.

[0086] It would be advantageous to determine a dosing schedule (e.g., number of doses and dose range) that provides sustained protection against all Borrelia OspA serotypes for adult and pediatric subjects. In the present invention, a Phase 2 trial is conducted in two parts: a main study phase (Part A) and a booster phase (Part B). The study compares the safety and immunogenicity of two different primary immunization schedules, applying at least three vaccinations (0-2-6 months) or at least two vaccinations (0-6 months). The study includes 600 healthy subjects aged 5-65 years. Subjects with a history of Lyme borreliosis (having been infected with Borrelia) and subjects who have not been infected with Borrelia are enrolled. Adult subjects may be 18 years or older, e.g., 18-65 years, and / or 50 years or older. Pediatric subjects may be from birth to 17 years of age, including those 5-17 years of age (e.g., 5-11 years (children) and / or 12-17 years (adolescents)), and younger children from birth to 4 years of age (e.g., 0-4 years, 1-4 years, and / or 2-4 years).

[0087] According to this dosing schedule, the vaccine may be administered to a subject at least three times, with the total protein content of the three fusion proteins ranging from 60 μg to 200 μg per dose, or more preferably ranging from 120 μg to 200 μg per dose. In some embodiments, the vaccine may be administered to a subject at least three times, with the total protein content of the three fusion proteins ranging from 135 μg or 180 μg per dose, or more preferably 180 μg per dose.

[0088] Alternatively, according to this dosing schedule, the vaccine may be administered to a subject at least twice, with the total protein content of the three fusion proteins ranging from 60 μg to 200 μg per dose, or more preferably ranging from 120 to 200 μg per dose. In some embodiments, the vaccine may be administered to a subject at least twice, with the total protein content of the three fusion proteins ranging from 135 μg or 180 μg per dose, or more preferably 180 μg per dose.

[0089] According to this dosing schedule, the vaccine may be administered to an adult subject at least three times, with the total protein content of the three fusion proteins ranging from 60 μg to 200 μg per dose, or more preferably ranging from 120 μg to 200 μg per dose. In some embodiments, the vaccine may be administered to an adult subject at least three times, with the total protein content of the three fusion proteins ranging from 135 μg or 180 μg per dose, or more preferably 180 μg per dose.

[0090] Further, according to this administration schedule, the vaccine may be administered to a pediatric subject at least three times with a total protein content of the three fusion proteins in the range of 60 μg to 200 μg per dose, or in the range of 120 μg to 200 μg per dose, or in the range of 60 μg to 100 μg per dose. In some embodiments, the vaccine may be administered to a pediatric subject at least three times with a total protein content of the three fusion proteins of 135 μg or 180 μg per dose, or more preferably 180 μg per dose. In some embodiments, the vaccine may be administered to a pediatric subject at least three times with a total protein content of the three fusion proteins of 67.5 μg or 90 μg per dose.

[0091] Alternatively, according to this dosing schedule, the vaccine may be administered to an adult subject at least twice, with the total protein content of the three fusion proteins ranging from 60 μg to 200 μg per dose, or more preferably ranging from 120 to 200 μg per dose. In some embodiments, the vaccine may be administered to an adult subject at least twice, with the total protein content of the three fusion proteins ranging from 135 μg or 180 μg per dose, or more preferably 180 μg per dose.

[0092] Further alternatively, according to this dosing schedule, the vaccine may be administered to a pediatric subject at least twice with a total protein content of the three fusion proteins in the range of 60 μg to 200 μg per dose, or in the range of 120 μg to 200 μg per dose, or in the range of 60 to 100 μg per dose. In some embodiments, the vaccine may be administered to a pediatric subject at least twice with a total protein content of the three fusion proteins of 135 μg or 180 μg per dose, or more preferably 180 μg per dose. In some embodiments, the vaccine may be administered to a pediatric subject at least twice with a total protein content of the three fusion proteins of 67.5 μg or 90 μg per dose.

[0093] For pediatric subjects, typically from birth to 4 years of age (e.g., 0-4 years, 1-4 years, and / or 2-4 years), half the above doses may be applied, e.g., 60-100, or 67.5 μg or 90 μg (half doses), although adult doses, e.g., 120 μg to 200 μg (adult doses), may also be appropriate. Pediatric subjects aged 5-17 years, including children (5-11 years) and adolescents (12-17 years), may be administered the adult dose.

[0094] The vaccines of the invention, e.g., MBV, may be applied according to different primary immunization schedules. These include a primary immunization schedule with at least three doses of the vaccine, such as doses at 0-2-6 months, and a primary immunization schedule with at least two doses of the vaccine, such as doses at 0-6 months. Further doses are booster doses for re-exposure to the vaccine, typically applied every 12 months after the last dose of the primary immunization schedule, e.g., the first booster dose may be given at 18 months. Booster doses increase immunity to protective levels after antibody titers have declined over time (see PCT Publication No. WO 2021 / 207615). Boosters have been shown to be useful in maintaining the high OspA antibody levels required to confer protection.

[0095] Thus, the methods and compositions for use according to the invention described herein are characterized in that, in the case of a schedule in which at least three doses are applied, the second administration is at least 4-6 weeks and up to 3 months after the first administration, and / or the third administration is at least 5 months and up to 9 months after the first administration. Accordingly, the first administration is administered at time 0 (day 1), the second administration is administered within a period of at least 4 weeks and up to 3 months thereafter, and the third administration is administered within a period of at least 5 months and up to 9 months after the first administration. Furthermore, boosters can be administered annually, for example, about 12 months, 24 months, 36 months, etc. after the third administration (i.e., the 6th month immunization).

[0096] The methods and compositions for use according to the invention described herein are further characterized in that, in the case of a schedule in which at least three doses are applied, the second administration is at least 4-6 weeks and up to 3 months after the first administration, and / or the third administration is at least 5 months and up to 7 months after the first administration. Accordingly, the first administration is administered at time 0 (day 1), the second administration is administered at least 4 weeks and up to 3 months thereafter, and the third administration is administered at least 5 months and up to 7 months after the first administration. Furthermore, boosters can be administered annually, for example, about 12 months, 24 months, 36 months, etc. after the third administration (i.e., the 6th month immunization).

[0097] More preferably, the methods and compositions for use according to the present invention are characterized in that the second administration is administered at a period of at least 40 days and up to 80 days, in particular at least 50 days and up to 70 days, more in particular at least 50 days and up to 60 days, in particular 56 or 57 days after the first administration. Also more preferably, the methods and compositions for use according to the present invention are characterised in that the third administration is at least 5 months and up to 9 months, or at least 5 months and up to 7 months, or at least 120 days and up to 240 days, at least 170 days and up to 190 days, in particular 180 days, after the first administration.

[0098] In some cases, one or more further administrations may be necessary or desirable.Accordingly, the fourth administration of the composition may be at least 15 months and up to 21 months after the first administration, particularly at least 17 months and up to 19 months, particularly about 18 months after.Accordingly, the fourth administration of the composition may be about 12 months after the third administration.

[0099] In some embodiments, the methods and compositions for use according to the invention are characterized in that the second administration is about 2 months (about 56 days) after the first administration, wherein the third administration is about 6 months (about 180 days) after the first administration, and the optional fourth administration (first booster vaccination) is about 18 months after the first administration.

[0100] Alternatively, the methods and compositions for use according to the invention described herein are characterized in that in the case of a schedule in which at least two doses are applied, the second administration is at least 5 months and up to 7 months after the first administration. Accordingly, the first administration is administered at time 0 (day 1) and the second administration is administered at least 5 months and up to 7 months after the first administration. Furthermore, boosters can be administered annually, for example, about 12 months, 24 months, 36 months, etc. after the second administration (i.e., the 6th month immunization). Alternatively, more preferably, the methods and compositions for use according to the invention are characterized in that the second administration is administered at a period of at least 120 days and up to 240 days, at least 170 days and up to 190 days, in particular 180 days, after the first administration.

[0101] In some cases, one or more further administrations may be necessary or desirable. Accordingly, a third administration of the composition may be administered at least 15 months and up to 21 months after the first administration, particularly at least 17 months and up to 19 months, and particularly about 18 months. In some embodiments, the methods and compositions for use according to the invention are characterized in that the second administration is about 6 months (about 180 days) after the first administration, and the optional third administration (first booster vaccination) is about 18 months after the first administration.

[0102] Booster doses are typically administered every 12 months, although booster doses can be administered within months of the 12-month timing, for example between 9 and 15 months after the last dose of primary immunization (e.g., 9, 10, 11, 12, 13, 14, or 15 months after the primary pre-immunization), or after a subsequent booster dose (e.g., 9, 10, 11, 12, 13, 14, or 15 months after the first or subsequent booster dose). Booster doses are generally administered prior to tick season, for example from about February to about June in the Northern Hemisphere, and from about September to about December in the Southern Hemisphere.

[0103] The present invention provides a method of eliciting a protective immune response against Lyme disease (Lyme borreliosis) in a human subject, the method comprising administering at least three times to a human adult or pediatric subject a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), in a total protein content of the three fusion proteins in the range of 120 μg to 200 μg per dose, more preferably 135 μg to 180 μg per dose, and most preferably 180 μg per dose. For pediatric subjects (particularly younger children from birth to 4 years of age, e.g., 1-4 years, or 2-4 years), at least three half doses may be applied in the range of 60-100 μg per dose, more preferably 67.5 μg or 90 μg per dose, and most preferably 90 μg per dose (although a full dose may also be suitable).

[0104] Alternatively, the present invention provides a method of eliciting a protective immune response against Lyme disease (Lyme borreliosis) in a human subject, the method comprising administering at least two doses of a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1) to a human adult or pediatric subject in a total protein content of the three fusion proteins in the range of 120 μg to 200 μg per dose, more preferably 135 μg to 180 μg per dose, and most preferably 180 μg per dose. For pediatric subjects (particularly younger children from birth to 4 years of age, e.g., 1-4 years, or 2-4 years), at least two half doses may be applied in the range of 60-100 μg per dose, more preferably 67.5 μg or 90 μg per dose, and most preferably 90 μg per dose (although a full dose may also be suitable).

[0105] In some embodiments, the method comprises inducing in a human subject a protective immune response against Lyme disease caused by Borrelia species, more preferably a pathogenic Borrelia species disclosed herein, more preferably including B. burgdorferi ss, B. afzelii, B. bavariensis, and B. garinii, and / or other pathogens whose antigens are included in the composition. Preferably, the composition is for use in a method of inducing a protective immune response against the following infections: B. burgdorferi ss, B. garinii, B. afzelii, B. andersoni, B. bavariensis, B. bissettii, B. valaisiana, B. lusitaniae, B. spielmanii, B. japonica, B. tanukii, B. turdi, or B. sinica infection, preferably B. burgdorferi ss, B. afzelii, and / or B. garinii.

[0106] The present invention provides a method of treating, preventing or reducing the risk of Lyme disease (Lyme borreliosis) in a human subject, the method comprising administering a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1) to a human adult or pediatric subject at least three times in a total protein content of the three fusion proteins in the range of 120 μg to 200 μg per dose, more preferably 135 μg to 180 μg per dose, and most preferably 180 μg per dose. For pediatric subjects (particularly younger children from birth to 4 years of age, e.g., 1-4 years, or 2-4 years), at least three half doses may be applied in the range of 60-100 μg per dose, more preferably 67.5 μg or 90 μg per dose, and most preferably 90 μg per dose (although a full dose may also be suitable).

[0107] Alternatively, the present invention provides a method of treating, preventing or reducing the risk of Lyme borreliosis (Lyme disease) in a human subject, the method comprising administering at least two doses of a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1) to a human adult or pediatric subject in a total protein content of the three fusion proteins in the range of 120 μg to 200 μg per dose, more preferably 135 μg to 180 μg per dose, and most preferably 180 μg per dose. For pediatric subjects (particularly younger children from birth to 4 years of age, e.g., 1-4 years, or 2-4 years), at least two half doses may be applied in the range of 60-100 μg per dose, more preferably 67.5 μg or 90 μg per dose, and most preferably 90 μg per dose (although a full dose may also be suitable).

[0108] The present invention provides a method of vaccination against Lyme borreliosis (Lyme disease) in a human subject, the method comprising administering to a human adult or pediatric subject at least three doses of a composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), in a total protein content of the three fusion proteins in the range of 120 μg to 200 μg per dose, more preferably 135 μg to 180 μg per dose, and most preferably 180 μg per dose. For pediatric subjects (particularly younger children from birth to 4 years of age, e.g., 1-4 years, or 2-4 years), at least three half doses may be applied in the range of 60-100 μg per dose, more preferably 67.5 μg or 90 μg per dose, and most preferably 90 μg per dose (although a full dose may also be suitable).

[0109] Alternatively, the present invention provides a method of vaccination against Lyme borreliosis (Lyme disease) in a human subject, the method comprising administering at least two doses of a composition comprising the fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), the fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1) and the fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1) to a human adult or pediatric subject, in a total protein content of the three fusion proteins in the range of 120 μg to 200 μg per dose, more preferably 135 μg to 180 μg per dose, and most preferably 180 μg per dose. For pediatric subjects (particularly younger children from birth to 4 years of age, e.g., 1-4 years, or 2-4 years), at least two half doses may be applied in the range of 60-100 μg per dose, more preferably 67.5 μg or 90 μg per dose, and most preferably 90 μg per dose (although a full dose may also be suitable).

[0110] In one embodiment, the compositions and methods of use thereof described herein according to the invention are characterized in that the total protein content of the three fusion proteins for a human subject, particularly an adult subject, is 135 μg per dose, wherein the second dose is about 2 months (about 56 days) after the first dose, wherein the third dose is about 5 months to about 9 months or about 6 months (about 180 days) after the first dose, and wherein the optional fourth dose (first booster vaccination) is about 18 months after the first dose or about 12 months after the third dose.

[0111] In one embodiment, the compositions and methods of use thereof described herein according to the invention are characterized in that the total protein content of the three fusion proteins for human subjects, particularly pediatric subjects, is 135 μg per dose, or for younger pediatric subjects (particularly pediatric subjects from birth to 4 years of age, e.g. 1-4 years, or 2-4 years), may be as little as 67.5 μg per dose, wherein the second administration is about 2 months (about 56 days) after the first administration, wherein the third administration is about 5 months to about 9 months or about 6 months (about 180 days) after the first administration, and wherein the optional fourth administration (first booster vaccination) is about 18 months after the first administration or about 12 months after the third administration.

[0112] In a preferred embodiment, the compositions and methods of use thereof described herein according to the invention are characterized in that the total protein content of the three fusion proteins for a human subject, particularly an adult subject, is 180 μg per dose, wherein the second dose is about 2 months (about 56 days) after the first dose, wherein the third dose is about 5 months to about 9 months or about 6 months (about 180 days) after the first dose, and wherein the optional fourth dose (first booster vaccination) is about 18 months after the first dose or about 12 months after the third dose.

[0113] In a preferred embodiment, the compositions and methods of use thereof described herein according to the invention are characterized in that the total protein content of the three fusion proteins for human subjects, particularly pediatric subjects, is 180 μg per dose, or for younger pediatric subjects (particularly pediatric subjects from birth to 4 years of age, e.g. 1-4 years, or 2-4 years), may be as little as 90 μg per dose, wherein the second administration is about 2 months (about 56 days) after the first administration, wherein the third administration is about 5 months to about 9 months or about 6 months (about 180 days) after the first administration, and wherein the optional fourth administration (first booster vaccination) is about 18 months after the first administration or about 12 months after the third administration.

[0114] Alternatively, in one embodiment, the composition and method of use thereof described herein according to the invention are characterized in that the total protein content of the three fusion proteins for a human subject, in particular an adult subject, is 135 μg per dose, wherein the second dose is about 6 months (about 180 days) after the first dose, and wherein the optional third dose (first booster vaccination) is about 18 months after the first dose.

[0115] Alternatively, in one embodiment, the compositions and methods of use thereof described herein according to the invention are characterized in that the total protein content of the three fusion proteins for human subjects, particularly pediatric subjects, is 135 μg per dose, or may be as little as 67.5 μg per dose for younger pediatric subjects (particularly pediatric subjects from birth to 4 years of age, e.g. 1-4 years, or 2-4 years), wherein the second administration is about 6 months (about 180 days) after the first administration, and wherein the optional third administration (first booster vaccination) is about 18 months after the first administration.

[0116] Alternatively, in a preferred embodiment, the composition and method of use thereof described herein according to the present invention is characterized in that the total protein content of the three fusion proteins for a human subject, in particular an adult subject, is 180 μg per dose, wherein the second dose is about 6 months (about 180 days) after the first dose, and wherein the optional third dose (first booster vaccination) is about 18 months after the first dose.

[0117] Alternatively, in a preferred embodiment, the compositions and methods of use thereof described herein according to the invention are characterized in that the total protein content of the three fusion proteins for human subjects, particularly pediatric subjects, is 180 μg per dose, or may be as little as 90 μg per dose for younger pediatric subjects (particularly pediatric subjects from birth to 4 years of age, e.g. 1-4 years, or 2-4 years), wherein the second dose is about 6 months (about 180 days) after the first dose, and wherein the optional third dose (first booster vaccination) is about 18 months after the first dose.

[0118] Preferably, methods and compositions for use according to the invention are characterized in that the immune response elicited comprises an anti-OspA serotype 1, anti-OspA serotype 2, anti-OspA serotype 3, anti-OspA serotype 4, anti-OspA serotype 5 and / or anti-OspA serotype 6 antibody response. In some embodiments, the antibodies of the immune response exhibit bactericidal activity (e.g., by promoting complement deposition and / or phagocytosis of antibody-bound spirochetes).

[0119] Preferably, the methods and compositions for use according to the invention are characterized in that the immune response elicited comprises antibodies against spirochetes expressing OspA of serotypes 1, 2, 3, 4, 5, and / or 6. In some embodiments, the antibodies have bactericidal activity against spirochetes expressing OspA of serotypes 1, 2, 3, 4, 5, and / or 6.

[0120] Preferably, the methods and compositions for use according to the present invention elicit an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, wherein the immune response following administration of at least three doses of the composition to an adult subject is greater than the immune response following administration of at least two doses of the composition to an adult subject.

[0121] Preferably, the methods and compositions for use according to the present invention elicit an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, wherein the immune response following administration of a second dose of the composition to a pediatric subject is at least as great as, or greater than, the immune response following administration of a third dose of the composition to an adult subject.

[0122] Preferably, the methods and compositions for use according to the present invention elicit an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, wherein the immune response following administration of at least three doses of the composition to a pediatric subject is greater than the immune response following administration of at least two doses of the composition to an adult subject.

[0123] Preferably, the methods and compositions for use according to the present invention elicit an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, wherein the immune response following administration of at least two doses of the composition to a pediatric subject is greater than the immune response following administration of at least two doses of the composition to an adult subject. Preferably, the compositions and methods of using them described herein according to the present invention are characterized in that the immune response against OspA serotypes 1, 2, 3, 4, 5, and 6 persists for at least about 60 days, at least about 180 days, at least about 365 days, or at least about 540 days.

[0124] In one embodiment, the composition or vaccine of the present invention further comprises at least one additional antigen (generally referred to herein as a "combination vaccine"). In a preferred embodiment, the at least one additional antigen is derived from a Borrelia species that causes Lyme borreliosis. In various aspects, the at least one additional antigen is derived from another pathogen, preferably a tick-borne pathogen. In a further aspect, the pathogen causes Rocky Mountain spotted fever, human granulocytic ehrlichiosis (HGE), glandular fever, human monocytic ehrlichiosis (HME), anaplasmosis, boutonne fever, Rickettsia parkeri rickettsiosis, Southern tick spotted fever (STARI), Helvetica spotted fever, 364D rickettsiosis, African spotted fever, relapsing fever, tularemia, Colorado tick fever, tick-borne encephalitis (TBE; also known as FSME), Crimean-Congo hemorrhagic fever, Q fever, Omsk hemorrhagic fever, Kyasanur Forest disease, Powassan encephalitis, Heartland virus disease, or babesiosis. In a further aspect, the disease is Japanese encephalitis. In a further embodiment, the at least one additional antigen is derived from a vector-borne, preferably tick-borne, pathogen selected from the group comprising: Borrelia hermsii, Borrelia parkeri, Borrelia duttoni, Borrelia miyamotoi, Borrelia turicatae, Rickettsia rickettsii, Rickettsia australis, Rickettsia conori, Rickettsia helvetica, Francisella tularensis, Anaplasma phagocytophilum, Ehrlichia sennetsu, Ehrlichia chaffeensis, Neoehrlichia mikurensis, Coxiella burnetii and Borrelia lonestari, Tick-borne encephalitis virus (TBEV, also known as FSME virus), Colorado Tick Fever virus (CTFV), Crimean-Congo hemorrhagic fever virus (CCHFV), Omsk hemorrhagic fever virus (OHFV), Japanese encephalitis virus (JEV) and Babesia spp.

[0125] Preferably, the composition of the present invention is characterized in that the three fusion proteins comprise at least 60%, preferably at least 70%, more preferably at least 80% of the total protein in the composition. It is clear to those skilled in the art that the composition for use according to the present invention may also include additional proteins different from the fusion proteins of SEQ ID NOs: 1, 2 and 3. However, the proportion of fusion proteins should not be below the above-mentioned limits on a weight basis. The other proteins may have a function, for example, to stabilize the composition, or may be impurities.

[0126] In some embodiments, the composition comprises three fusion proteins, preferably Lip-S1D1-S2D1 (SEQ ID NO:1), Lip-S4D1-S3hybD1 (SEQ ID NO:2), and Lip-S5D1-S6D1 (SEQ ID NO:3), in a weight ratio of 1:1:1, 1:2:1, 1:3:1, 1:1:2, 1:1:3, 1:2:2, 1:2:3, 1:3:2, 1:3:3, 2:1:1, 2:1:2, 2:1:3, 2:2:3, 2:2:1, 2:3:1, 2:3:2, 2:3:3, 3:1:1, 3:1:2, 3:1:3, 3:2:1, 3:2:2, 3:2:3, 3:3:1, or 3:3:2. Preferably, the composition according to the invention is characterized in that the composition comprises the fusion proteins in a weight ratio of 1:1:1 (Lip-S1D1-S2D1:Lip-S4D1-S3hybD1:Lip-S5D1-S6D1). Preferably the composition according to the invention is characterised in that the composition comprises an adjuvant, more preferably an aluminium adjuvant.

[0127] The selection of suitable adjuvants to be mixed with bacterial proteins made by the process of the present invention is within the knowledge of those skilled in the art.Suitable adjuvants include aluminum salts such as aluminum hydroxide or aluminum phosphate, but may also be other metal salts such as calcium, magnesium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or an acylated sugar, cationic or anionic derivatized sugar, or polyphosphazene.In a preferred embodiment, the composition is added with an aluminum adjuvant such as aluminum hydroxide.In a further preferred embodiment, the amount of copper contained in the aluminum adjuvant is less than 1.25 ppb in the vaccine composition.

[0128] In one embodiment, the compositions and methods of use thereof described herein according to the invention contain less than 1.25 ppb of copper or a sufficient amount of L-methionine. Aluminum is an adjuvant often used in vaccination. Aluminum adjuvants typically contain impurities, particularly heavy metals such as copper, nickel, and iron. The presence of these, particularly copper, reduces the bioavailability of the OspA protein in the vaccine. Without being bound by theory, it is assumed that the OspA protein binds to aluminum and the heavy metals, particularly copper, prevent its release, thus reducing the bioavailability of the OspA protein in the vaccine. Thus, the compositions, particularly aluminum adjuvants, contain less than 1.25 ppb of copper. The unit ppb (parts per billion) is often used to quantify impurities in the field of mass spectrometry. For aqueous solutions, 1 ppb means that 1 ng of a substance (impurity) is present in 1 g of solution, and 1 ppb corresponds to 1 μg / l (assuming that 1 liter of solution weighs 1 kg). Typically, copper is in the form of ions, particularly Cu. + or Cu 2+In a preferred embodiment, the composition contains less than 1.00 ppb, less than 0.75 ppb, or less than 0.50 ppb copper based on the weight of the aqueous composition. It is known that L-methionine can bind copper. The amount of L-methionine required obviously depends on the amount of copper in the composition. Those skilled in the art will be able to select a suitable amount of L-methionine. L-methionine can be used to complex excess copper in the composition for use in the present invention. A typical suitable concentration of L-methionine in the composition is at least 10 mmol / l. Depending on the amount of copper in the composition, the concentration may be higher, for example at least 20 mmol / l, at least 30 mmol / l, at least 40 mmol / l, or at least 50 mmol / l, or may be lower, for example up to 10 mmol / l, up to 5 mmol / l, or up to 1 mmol / l. Alternatively, the concentration of L-methionine is determined based on the concentration of copper in the composition. In particular, the concentration of L-methionine (mol / l) is at least equal to the concentration of copper in the composition. Alternatively, the concentration of L-methionine in mol / l is at least 2, 3, 4, 5 or 10 times the concentration of copper in the composition.

[0129] In one embodiment, the composition and its method of use described herein further comprises a reactive compound, wherein the reactive compound is selected from the group consisting of redox active compounds, radical building compounds, stabilizing compounds and any combination thereof.Without being bound by theory, antigen degradation of protein vaccines in aqueous compositions containing heavy metal ions present in aluminum salts such as aluminum hydroxide may be explained by a fundamental degradation pathway assuming free radicals, such as sulfite free radicals. Heavy metal catalyzed oxidation is a degradation pathway that leads to covalent modification of proteins. The altered physicochemical properties of oxidized / modified proteins or antigens may lead to loss of biological activity. Redox active compounds are suitable for preventing this modification. Preferably, the reactive compound is selected from the group consisting of formaldehyde, ethanol, chloroform, trichloroethylene, acetone, Triton-X-100, deoxycholic acid, diethylpyrocarbonate, sulfite, Na2S2O5, β-propriolactone, polysorbates such as Tween 20®, Tween 80®, O2, phenol, Pluron-type copolymers, and any combination thereof.

[0130] Previous studies on vaccines, such as a study of MBV (also referred to as Vaccine A), in which three fusion proteins were administered to adult subjects at 0-1-2 months and 0-2-6 months with total protein contents of 90 μg, 135 μg, and 180 μg per dose, are provided in PCT Publication WO 2021 / 207615, which is incorporated by reference in its entirety.

[0131] ELISA For the assessment of immunogenicity described in the Examples, human sera are analyzed by quantitative ELISA for IgG against each OspA serotype (ST1-ST6) separately. A dilution series of serum is added to microtiter plates coated with full-length OspA ST1, ST2, ST3, ST4, ST5, or ST6. The presence of bound IgG is detected with an anti-human IgG enzyme conjugate, followed by the addition of substrate. The optical density of the colored end product is proportional to the amount of protein-specific IgG present in the serum, which can be quantified based on a four-parameter logistic fit and a reference substance curve by parallel line analysis.

[0132] Further aspects of the invention 1. A composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), in a total protein content of the three fusion proteins in the range of 60 μg to 200 μg per dose, for use in a method of eliciting a protective immune response against Lyme disease in a subject, wherein the subject is administered at least three doses of the composition. 2. A composition for use in a method of treating, preventing or reducing the risk of Lyme disease comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), in a total protein content of the three fusion proteins in the range of 60 μg to 200 μg per dose, wherein a subject is administered at least three doses of the composition.

[0133] 3. A composition for use in a method of vaccinating a human subject against Lyme disease comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), in a total protein content of the three fusion proteins in the range of 60 μg to 200 μg per dose, wherein the subject is administered at least three doses of the composition. 4. The composition of any one of aspects 1-3, wherein the second dose is administered within a period of at least 6 weeks and up to 3 months after administration of the first dose, and the third dose is administered within a period of at least 5 months and up to 9 months or at least 5 months and up to 7 months after administration of the first dose.

[0134] 5. The composition of any one of aspects 1-4, wherein the second dose is administered within a period of at least 50 days and up to 70 days after administration of the first dose, and the third dose is administered within a period of at least 170 days and up to 190 days after administration of the first dose. 6. The composition of any one of aspects 1-5, wherein a first dose is administered on day 1, a second dose is administered about 2 months after the first dose, and a third dose is administered about 6 months after administration of the first dose.

[0135] 7. The composition according to any one of aspects 1 to 6, wherein the fourth dose is administered within a period of at least 15 months and up to 21 months after administration of the first dose, in particular within a period of at least 17 months and up to 19 months, or about 12 months after the third dose. 8. The composition of any one of aspects 1-7, wherein the fourth dose is administered about 18 months after administration of the first dose or about 12 months after administration of the third dose.

[0136] 9. The composition of any one of aspects 1-8, wherein the subject is 5 years of age or older, for example 5-65 years of age. 10. The composition of any one of aspects 1-9, wherein the subject is an adult subject aged 18 years or older, for example 18-65 years and / or 50 years or older. 11. The composition according to any one of aspects 1 to 9, wherein the subject is a pediatric subject from birth to 17 years of age. 12. The composition of embodiment 11, wherein the pediatric subject is aged between 5 and 17 years, such as between 5 and 11 years and / or between 12 and 17 years. 13. The composition of embodiment 11, wherein the pediatric subject is from birth to 4 years of age, e.g., 1-4 years and / or 2-4 years of age.

[0137] 14. The composition of any one of aspects 1 to 13, wherein the total protein content of the three fusion proteins is 135 μg or 180 μg per dose. 15. The composition of any one of aspects 1-13, wherein the total protein content of the three fusion proteins is 67.5 μg or 90 μg per dose. 16. A composition according to any one of aspects 1 to 15, wherein the three fusion proteins comprise at least 60%, preferably at least 70%, and more preferably at least 80% of the total protein in the composition. 17. The composition according to any one of aspects 1 to 16, wherein the composition comprises three fusion proteins in a weight ratio of 1:1:1 (Lip-S1D1-S2D1: Lip-S4D1-S3hybD1: Lip-S5D1-S6D1).

[0138] 18. The composition according to any one of aspects 1-17, wherein the total protein content of the three fusion proteins is 180 μg per dose, wherein the first dose is administered on day 1, the second dose is administered within a period of at least 50 days and up to 70 days after the first dose, and the third dose is administered within a period of at least 170 days and up to 190 days after the first dose, and wherein optionally, the fourth dose is administered about 18 months after administration of the first dose or about 12 months after the third dose.

[0139] 19. The composition of any one of aspects 1-18, wherein the subject is an adult subject, wherein the total protein content of the three fusion proteins is 180 μg per dose, wherein the first dose is administered on day 1, the second dose is administered within a period of at least 50 days and up to 70 days after the first dose, and the third dose is administered within a period of at least 170 days and up to 190 days after the first dose, and wherein optionally, the fourth dose is administered about 18 months after administration of the first dose or about 12 months after the third dose.

[0140] 20. The composition of any one of aspects 1-19, wherein the subject is a pediatric subject, wherein the total protein content of the three fusion proteins is 180 μg per dose, wherein the first dose is administered on day 1, the second dose is administered within a period of at least 50 days and up to 70 days after the first dose, and the third dose is administered within a period of at least 170 days and up to 190 days after the first dose, and wherein optionally, the fourth dose is administered about 18 months after administration of the first dose or about 12 months after the third dose.

[0141] 21. The composition of any one of aspects 1-20, wherein the subject is a pediatric subject from birth to 4 years old, wherein the total protein content of the three fusion proteins is 67.5 μg or 90 μg per dose, wherein the first dose is administered on day 1, the second dose is administered within a period of at least 50 days and up to 70 days after the first dose, and the third dose is administered within a period of at least 170 days and up to 190 days after the first dose, and wherein optionally, the fourth dose is administered about 18 months after administration of the first dose or about 12 months after the third dose.

[0142] 22. The composition according to any one of aspects 1 to 21, wherein a further dose is administered every year after the third dose or any fourth dose has been administered, in particular after 1 year, 2 years and 3 years. 23. A composition comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), in a total protein content of the three fusion proteins in the range of 60 μg to 200 μg per dose, for use in a method of eliciting a protective immune response against Lyme disease in a subject, wherein the subject is administered at least two doses of the composition.

[0143] 24. A composition for use in a method for treating, preventing or reducing the risk of Lyme disease comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), in a total protein content of the three fusion proteins in the range of 60 μg to 200 μg per dose, wherein a subject is administered at least two doses of the composition.

[0144] 25. A composition for use in a method of vaccinating a human subject against Lyme disease comprising a fusion protein of SEQ ID NO:1 (Lip-S1D1-S2D1), a fusion protein of SEQ ID NO:2 (Lip-S4D1-S3hybD1), and a fusion protein of SEQ ID NO:3 (Lip-S5D1-S6D1), in a total protein content of the three fusion proteins in the range of 60 μg to 200 μg per dose, wherein the subject is administered at least two doses of the composition. 26. The composition of any one of aspects 23-25, wherein the second dose is administered within a period of at least 5 months and up to 7 months after administration of the first dose.

[0145] 27. The composition of any one of aspects 23-26, wherein the second dose is administered within a period of at least 170 days and up to 190 days after administration of the first dose. 28. The composition of any one of aspects 23-27, wherein the first dose is administered on day 1 and the second dose is administered about 6 months after administration of the first dose. 29. The composition according to any one of aspects 23 to 28, wherein the third dose is administered within a period of at least 15 months and at most 21 months, in particular within a period of at least 17 months and at most 19 months, after administration of the first dose.

[0146] 30. The composition of any one of aspects 23 to 29, wherein the third dose is administered about 18 months after administration of the first dose. 31. The composition of any one of aspects 23 to 30, wherein the subject is 5 years of age or older, for example 5 to 65 years of age. 32. The composition of any one of aspects 23 to 31, wherein the subject is an adult subject aged 18 years or older, for example aged 18 to 65 years and / or aged 50 years or older. 33. The composition of any one of aspects 23 to 31, wherein the subject is a pediatric subject from birth to 17 years of age.

[0147] 34. The composition of aspect 33, wherein the pediatric subject is 5 to 17 years old, such as 5 to 11 years old and / or 12 to 17 years old. 35. The composition of aspect 33, wherein the pediatric subject is from birth to 4 years of age, e.g., 1-4 years and / or 2-4 years of age. 36. The composition of any one of aspects 23 to 35, wherein the total protein content of the three fusion proteins is 135 μg or 180 μg per dose. 37. The composition of any one of aspects 23 to 35, wherein the total protein content of the three fusion proteins is 67.5 μg or 90 μg per dose.

[0148] 38. A composition according to any one of aspects 23 to 37, wherein the three fusion proteins comprise at least 60%, preferably at least 70%, and more preferably at least 80% of the total protein in the composition. 39. The composition according to any one of aspects 23 to 38, wherein the composition comprises three fusion proteins in a weight ratio of 1:1:1 (Lip-S1D1-S2D1: Lip-S4D1-S3hybD1: Lip-S5D1-S6D1).

[0149] 40. The composition of any one of aspects 23-39, wherein the total protein content of the three fusion proteins is 180 μg per dose, wherein the first dose is administered on day 1, the second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and wherein, optionally, the third dose is administered about 18 months after administration of the first dose.

[0150] 41. The composition of any one of aspects 23-40, wherein the subject is an adult subject, wherein the total protein content of the three fusion proteins is 180 μg per dose, wherein the first dose is administered on day 1, and the second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and wherein, optionally, the third dose is administered about 18 months after administration of the first dose.

[0151] 42. The composition of any one of aspects 23-41, wherein the subject is a pediatric subject, wherein the total protein content of the three fusion proteins is 180 μg per dose, wherein the first dose is administered on day 1, the second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and wherein, optionally, the third dose is administered about 18 months after administration of the first dose.

[0152] 43. The composition of any one of aspects 23-42, wherein the subject is a pediatric subject from birth to 4 years of age, wherein the total protein content of the three fusion proteins is 67.5 μg or 90 μg per dose, wherein the first dose is administered on day 1, the second dose is administered within a period of at least 170 days and up to 190 days after the first dose, and wherein, optionally, the third dose is administered about 18 months after administration of the first dose.

[0153] 44. The composition according to any one of aspects 23 to 43, wherein a further dose is administered every year after the second dose or any third dose has been administered, in particular after 1 year, 2 years and 3 years. 45. A composition described in any one of aspects 1 to 44, wherein the composition elicits an immune response comprising an anti-OspA serotype 1, anti-OspA serotype 2, anti-OspA serotype 3, anti-OspA serotype 4, anti-OspA serotype 5 and / or anti-OspA serotype 6 antibody response. 46. ​​The composition of any one of aspects 1-45, wherein the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and 6.

[0154] 47. The composition of any one of aspects 1-46, wherein the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, and wherein the immune response following administration of at least three doses of the composition to an adult subject is greater than the immune response following administration of at least two doses of the composition to an adult subject. 48. The composition of any one of aspects 1-47, wherein the geometric mean titer (GMT) of antibodies against a Borrelia serotype after administration of at least three doses of the composition to an adult subject is at least 2.0-fold higher than the GMT after administration of at least two doses of the composition to an adult subject.

[0155] 49. The composition of embodiment 48, wherein the GMT of antibodies against a Borrelia serotype after administration of at least three doses of the composition to an adult subject is about 2.0-fold to about 3.0-fold higher than the GMT after administration of at least two doses of the composition to an adult subject. 50. The composition of any one of aspects 1-49, wherein the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, and wherein the immune response following administration of a second dose of the composition to a pediatric subject is at least as great as or greater than the immune response following administration of a third dose of the composition to an adult subject.

[0156] 51. The composition of any one of aspects 1-50, wherein the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, and wherein the immune response following administration of at least three doses of the composition to a pediatric subject is greater than the immune response following administration of at least three doses of the composition to an adult subject.

[0157] 52. The composition of embodiment 51, wherein the geometric mean titer (GMT) of antibodies against a Borrelia serotype after administration of at least three doses of the composition to a pediatric subject is at least 2.0-fold higher than the GMT after administration of at least three doses of the composition to an adult subject. 53. The composition of embodiment 52, wherein the GMT of antibodies against a Borrelia serotype after administration of at least three doses of the composition to a pediatric subject aged 12 to 17 years is about 2.0-fold to about 3.0-fold higher than the GMT after administration of at least three doses of the composition to an adult subject.

[0158] 54. The composition of embodiment 52, wherein the GMT of antibodies against a Borrelia serotype after administration of at least three doses of the composition to a pediatric subject aged 5 to 11 years is about 3.0-fold to about 5.0-fold higher than the GMT after administration of at least three doses of the composition to an adult subject. 55. The composition of any one of aspects 1-54, wherein the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and / or 6, and wherein the immune response following administration of at least two doses of the composition to a pediatric subject is greater than the immune response following administration of at least two doses of the composition to an adult subject.

[0159] 56. The composition of embodiment 55, wherein the geometric mean titer (GMT) of antibodies against a Borrelia serotype after administration of at least two doses of the composition to a pediatric subject is at least 2.0-fold higher than the GMT after administration of at least two doses of the composition to an adult subject. 57. The composition of embodiment 56, wherein the GMT of antibodies against a Borrelia serotype after administration of at least two doses of the composition to a pediatric subject aged 12 to 17 years is about 4.0-fold to about 6.0-fold higher than the GMT after administration of at least two doses of the composition to an adult subject.

[0160] 58. The composition of embodiment 56, wherein the GMT of antibodies against a Borrelia serotype after administration of at least two doses of the composition to a pediatric subject aged 5 to 11 years is about 5.0-fold to about 8.0-fold higher than the GMT after administration of at least two doses of the composition to an adult subject. 59. The composition of any one of aspects 1-58, wherein the composition elicits an immune response comprising antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and 6, and the immune response persists for at least about 60 days, at least about 180 days, at least about 365 days, or at least about 540 days.

[0161] 60. The composition of embodiment 59, wherein the immune response persists above baseline for at least about 180 days after administration of at least two doses or at least three doses of the composition. 61. The composition of embodiment 60, wherein at least 180 days after administration of at least three doses of the composition to a pediatric subject aged 5 to 11 years, the geometric mean fold rise (GMFR) of antibodies against Borrelia serotypes is at least 2.0-fold higher than the baseline GMFR.

[0162] 62. The composition of embodiment 61, wherein the GMFR of antibodies against Borrelia serotypes is about 2.0-fold to 7.0-fold higher than the baseline GMFR at least 180 days after administration of at least three doses of the composition to a pediatric subject aged 5 to 11 years. 63. The composition of any one of aspects 1-62, wherein the composition is administered to the subject in a volume of 0.25 ml, 0.5 ml, or 1.0 ml. 64. The composition of any one of aspects 1 to 63, wherein the composition is administered to the subject in a volume of 0.5 ml.

[0163] 65. The composition of any one of aspects 1-64, wherein the composition comprises at least one of sodium phosphate, sodium chloride, sucrose, and polysorbate 20. 66. The composition of any one of aspects 1 to 65, wherein the sodium phosphate is present at a concentration of 5 mM to 50 mM, the sodium chloride is present at a concentration of 100 mM to 200 mM, the sucrose is present at a concentration of 2.5 to 10%, and the polysorbate 20 is present at a concentration of 0.01 to 0.1%.

[0164] 67. The composition of any one of aspects 1 to 66, wherein the composition comprises an adjuvant. 68. The composition of any one of aspects 1-67, wherein the adjuvant comprises an aluminum adjuvant. 69. The composition of any one of the preceding aspects, wherein the composition contains less than 1.25 ppb copper. 70. The composition of embodiment 69, wherein the copper is in ionic form, particularly in the form of Cu+ or Cu2+.

[0165] 71. The composition of any one of aspects 1-70, wherein the composition comprises L-methionine. 72. The composition according to aspect 71, wherein L-methionine is present at a concentration of at least 10 mmol / l. 73. A composition according to any one of aspects 69 to 72, wherein the concentration of L-methionine (mol / l) is at least equivalent to the concentration of copper in the composition.

[0166] 74. The composition of any one of aspects 1-73, wherein the composition further comprises a reactive compound, wherein the reactive compound is selected from the group consisting of redox-active compounds, radical building compounds, stabilizing compounds, and any combination thereof, particularly wherein the reactive compound is selected from the group consisting of formaldehyde, ethanol, chloroform, trichloroethylene, acetone, Triton-X-100, deoxycholic acid, diethylpyrocarbonate, sulfite, Na2S2O5, β-proprio-lactone, polysorbates such as Tween 20®, Tween 80®, O2, phenol, Pluron-type copolymers, and any combination thereof.

[0167] 75. A composition according to any one of aspects 1 to 74 for use in a method for inducing a protective immune response against Lyme disease in a subject. 76. A composition according to any one of aspects 1 to 75 for use in a method for treating, preventing, or reducing the risk of Lyme disease in a subject. 77. A composition according to any one of aspects 1 to 76 for use in a method for vaccinating a subject against Lyme disease. EXAMPLES

[0168] Example 1. Phase 2 clinical trial of multivalent Borrelia vaccine (MBV): Investigation of a 3-dose or 2-dose primary immunization schedule and booster dose of the vaccine in a study population aged 5-65 years The study is a randomized, observer-blinded, placebo-controlled, multicenter, phase 2 trial in healthy subjects aged 5-65 years. The study is conducted to compare a three-dose and a two-dose primary immunization schedule (0-2-6 months or 0-6 months) in adults (18-65 years), adolescents (12-17 years) and children (5-11 years). In this study, MBV will be administered at a dose of 180 μg total protein (i.e., 60 μg per fusion protein) and aluminum hydroxide adjuvant (0.5 mg) in 0.5 mL of buffer. All injections will be administered intramuscularly (IM). The study will evaluate the immune response to a booster vaccination administered approximately 12 months after completion of the extended primary vaccination schedule and the persistence of immunity for up to 3 years after the booster vaccination.

[0169] Enrollment will be staggered in descending age order for three age cohorts (see Figure 1), and the study will have two study parts, Part A: the main study phase, and Part B: the booster phase (see Figures 2A and 2B). As shown in Table 1, in part A (main study phase), a total of approximately 600 subjects aged 5-65 years will be randomly assigned 1:1:1 to three study arms: arm 1 (approximately 200 subjects) will receive three vaccinations with MBV at months 0-2-6; arm 2 (approximately 200 subjects) will receive two vaccinations with MBV at months 0-6 and a placebo injection at month 2 to maintain blinding; arm 3 (approximately 200 subjects) will receive three placebo injections at months 0-2-6. Within each study arm, subjects will be enrolled 2:1:1 into three age cohorts (18-65 years, 12-17 years, and 5-11 years).

[0170] In Part A, all subjects will receive three IM vaccinations at months 0-2-6 (i.e., days 1-57-180). A safety visit will be conducted (phone call for subjects aged 18-65 years and in-person visit for subjects aged 5-17 years) on Day 8 / Visit 1A (i.e., 7 days after first vaccination). An in-person visit is scheduled 1 month after each vaccination for all age cohorts. Blood samples for immunogenicity assessment will be collected at the screening visit, days 85, 180, 194 (in a subset of adult subjects), days 208, 365 / months 12, and 18 months.

[0171] In Part B (booster phase), all eligible subjects will receive a booster dose of MBV or a placebo injection at 18 months. [Table 1-1] [Table 1-2]

[0172] Immunogenicity analysis The primary immunogenicity objective of this study is to evaluate the immune response to MBV administered in a 3-dose or 2-dose primary immunization schedule (months 0-2-6 or 0-6) at day 208 (month 7) in a healthy study population aged 5-65 years. The primary immunogenicity endpoints are IgG geometric mean titers (GMTs) to each OspA serotype ST1-ST6 at day 208 / month 7 (Part A) as determined by ELISA assay.

[0173] The primary immunogenicity analysis will be an overall and pairwise comparison of OspA serotype-specific IgG GMTs in the per-protocol analysis set between study arms at day 208 (i.e., 28 days after the last vaccination) by ANOVA (factorial treatment group, age group). In addition, the GMTs and GMFRs (i.e., increase in IgG antibody titers compared to the respective titers on day 1 (baseline)) for each OspA serotype ST1-ST6 will be compared overall and pairwise at the specified time points between study arms. Immunogenicity assessment, measuring OspA serotype-specific IgG by IgG binding assay, will be performed on samples collected at Visits 0 and 4-8 during the main study phase (Part A) and at Visits 9-16 during the booster phase (Part B).

[0174] Immunogenicity analysis: 208 days / 7th month Human sera were analyzed separately for IgG to each OspA serotype (ST1-ST6) by quantitative ELISA. Figure 3 shows the GMTs of OspA serotypes (ST1-ST6) at 208 days / 7 months for adults (18-65 years), adolescents (12-17 years), and children (5-11 years) receiving either 3 doses of MBV (M 0-2-6), 2 doses of MBV (M 0-6), or placebo. Figure 4 shows the GMTs of OspA serotypes (ST1-ST6) by age cohort at 208 days / 7 months for either 3 doses of MBV (M 0-2-6) or 2 doses of MBV (M 0-6). See Table 3 below.

[0175] Figure 5 shows the GMTs of OspA serotypes (ST1-ST6) over time for each age cohort in Group 1 (three doses of MBV, M 0-2-6) (Visit 1: month 0 / screening, Visit 4: month 4 / day 85, Visit 5: month 6 / day 180, Visit 5A: month 6 / day 194 (adults 18-65 years only), and Visit 6: month 7 / day 208). In adolescents (12-17 years), the GMTs of all six serotypes after two doses of MBV with M 0-2 are as high as those of adults (18-65 years) after three doses of M 0-2-6. In children (5–11 years), the GMTs for all six serotypes after two doses of MBV with M 0-2 were higher than in adults (18–65 years) after three doses of M 0-2-6.

[0176] Table 2 shows the GMTs of OspA-specific IgG by serotype at 208 days / 7 months. Overall, the GMTs ranged from 332.9 [ST1] to 657.7 [ST2] in the M 0-2-6 schedule group and 195.7 [ST1] to 456.6 [ST2] in the M 0-6 schedule group. In adults (18-65 years), the GMTs ranged from 199.1 [ST1] to 418.5 [ST2] in the M 0-2-6 schedule group and 80.9 [ST1] to 211.5 [ST2] in the M 0-6 schedule group. In adolescents (12-17 years), the GMTs ranged from 513.8 [ST1] to 900.0 [ST2] in the M 0-2-6 schedule group and 463.3 [ST6] to 961.3 [ST3] in the M 0-6 schedule group. In children (5-11 years of age), GMTs ranged from 706.6 [ST1] to 1364.1 [ST2] in the M 0-2-6 schedule group and from 543.3 [ST1] to 1293.1 [ST3] in the M 0-6 schedule group.

[0177] [Table 2-1] [Table 2-2]

[0178] In adults, GMTs for all six serotypes were statistically significantly higher in the M 0-2-6 group compared with the M 0-6 group at 208 days / 7 months; see Table 3. Immune responses in adolescents and children were comparable for both primary vaccination schedules. In general, GMTs for all six serotypes were higher in adolescents and children compared with adults. GMTs in adolescents were 2.2-fold [ST2] to 2.6-fold [ST1 / ST4] (3 doses) and 4.3-fold [ST2] to 5.8-fold [ST1] (2 doses) higher than in adults. GMTs in children were 3.1-fold [ST3] to 4.1-fold [ST6] (3 doses) and 5.7-fold [ST2] to 7.2-fold [ST3] (2 doses) higher than in adults. P values ​​are derived from pairwise comparisons obtained from ANOVA analysis (ELISA:ANOVA (factors: study group, age cohort)) for GMT of OspA ST1-6 specific IgG antibodies by visit. [Table 3]

[0179] Table 4 shows a comparison of GMTs for three- and two-dose vaccination schedules (M 0-2-6 and M 0-6) and for different age groups. In the younger age groups (adolescents aged 12-17 years and children aged 5-11 years), the immune response for all six serotypes was already high after the second dose of the M 0-2-6 schedule (i.e., day 85) as after three doses in adults aged 18-65 years (i.e., day 208). [Table 4]

[0180] Figures 6A-6F show inverse cumulative distribution curves of ELISA% of subjects at 208 days / 7 months vs. OspA-specific IgG antibody titers by age cohort: AB) ST1 and ST2, CD) ST3 and ST4, EF) ST5 and ST6. Table 5 shows the geometric mean fold increase (GMFR) in OspA-specific IgG at 208 days / 7 months compared to day 0. Overall, GMFRs ranged from 15.6 [ST1] to 31.5 [ST2] in the M 0-2-6 schedule group and 9.6 [ST1] to 22.8 [ST2] in the M 0-6 schedule group. In adults (18-65 years), GMFRs ranged from 9.2 [ST1] to 19.7 [ST2] in the M 0-2-6 schedule group and 4.0 [ST1] to 10.6 [ST2] in the M 0-6 schedule group. In adolescents (12-17 years), GMFRs ranged from 25.7 [ST1] to 45.3 [ST2] in the M 0-2-6 schedule group and 19.7 [ST6] to 46.9 [ST3] in the M 0-6 schedule group. In children, GMFR ranged from 32.3 [ST1] to 66.9 [ST2] in the M 0-2-6 schedule group and from 25.4 [ST1] to 64.7 [ST3] in the M 0-6 schedule group.

[0181] [Table 5-1] [Table 5-2]

[0182] In adults, the GMFRs for all six serotypes were statistically significantly higher in the M0-2-6 schedule group compared with the M0-6 schedule group; see Table 6. [Table 6]

[0183] Figure 7 shows the seroconversion rates (SCR) at 208 days / 7 months. Seroconversion rates are defined as seroconversion from seronegative to seropositive or a 4-fold or greater increase in IgG antibody titers compared to baseline if the subject tested OspA seropositive at baseline. Overall, SCRs ranged from 92.8% [ST1] to 98.8% [ST3] in the M 0-2-6 schedule group and 83.4% [ST1] to 94.3% [ST2] in the M 0-6 schedule group. In adults (18-65 years), SCRs ranged from 88.9% [ST1] to 98.9% [ST3] in the M 0-2-6 schedule group and 70.4% [ST1] to 90.1% [ST2] in the M 0-6 schedule group. In adolescents (ages 12-17), SCRs ranged from 97.4% [ST1] to 100% [ST2 / 3 / 4 / 5 / 6] on the M 0-2-6 schedule and from 95.0% [ST6] to 97.5% [ST1 / 2 / 3 / 4 / 5] on the M 0-6 schedule. In children (ages 5-11), SCRs ranged from 94.9% [ST6] to 97.4% [ST1 / ST2 / 3 / 4 / 5] on the M 0-2-6 schedule and from 97.2% [ST1] to 100% [ST2 / 3 / 4 / 5 / 6] on the M 0-6 schedule.

[0184] Immunogenicity analysis: 365 days / 12 months (6 months after priming) Immunogenicity assessments were performed on samples collected at Visit 7, 365 days / 12 months and included GMTs (geometric mean titers), GMFRs (geometric mean fold increase), and SCRs (seroconversion rates) of IgG antibodies against each OspA serotype (ST1-ST6). Higher antibody levels were observed in the three-dose 0-2-6 month vaccination schedule than in the two-dose 0-6 month vaccination schedule. Antibody levels remained above baseline for 6 months after completion of the three-dose (0-2-6 months) or two-dose (0-6 months) vaccination schedule. Compared to baseline, GMFRs were 1.9-fold higher for serotype 1 (ST1) and 3.2-fold higher for serotype 2 (ST2) in all age groups in the 0-2-6 month vaccination schedule. The highest GMFRs were reported in the 5-11 year old age group, with GMFR levels ranging from 2.8-fold (ST1) to 6.6-fold (ST2).

[0185] Human sera were analyzed for IgG to each OspA serotype (ST1-ST6) separately by quantitative ELISA. Table 7 shows the GMTs of serotype-specific OspA-specific IgG at 365 days / 12 months for the M 0-2-6 schedule group. Table 8 shows the GMTs of serotype-specific OspA-specific IgG at 365 days / 12 months for the M 0-6 schedule group. Figures 8 and 9 show the GMTs of serotype-specific OspA-specific IgG over time in the M 0-2-6 and M 0-6 schedule groups, respectively. [Table 7] [Table 8]

[0186] Table 9 shows the estimated percent (%) of subjects exceeding the ELISA threshold of 125 U / mL for ST1 at 208 days / 7 months and 365 days / 12 months in the M 0-2-6 (Group 1) and M 0-6 (Group 2) schedule groups. [Table 9]

[0187] Figure 10 shows the inverse cumulative distribution curve of subject ELISA% vs ST1 OspA-specific IgG antibody titer by age at 365 days / 12 months. Figure 11 shows the inverse cumulative distribution curve of subject ELISA% vs ST1 OspA-specific IgG antibody titer over time for the M 0-2-6 schedule group. Figure 12 shows the inverse cumulative distribution curve of subject ELISA% vs ST1 OspA-specific IgG antibody titer over time for the M 0-6 schedule group.

[0188] Table 10 shows the GMFR over time for OspA-specific IgG by ELISA compared to day 0 in subjects aged 5-65 years. [Table 10]

[0189] Table 11 shows the GMFR over time for OspA-specific IgG by ELISA compared to day 0 in subjects aged 5-11 years. [Table 11]

[0190] Figure 13 shows ELISA OspA-specific IgG seroconversion rates (SCR) by serotype at 365 days / 12 months. Seroconversion rates are defined as seroconversion from seronegative to seropositive, or a 4-fold or greater increase in IgG antibody titers compared to baseline, if the subject tested OspA seropositive at baseline.

[0191] Example 2: A Phase 3, Multicenter, Placebo-Controlled, Randomized, Observer-Blinded Study to Evaluate the Efficacy, Safety, Tolerability, Immunogenicity, and Lot Consistency of a Hexavalent OspA-Based Lyme Disease Vaccine in Healthy Participants Aged 5 Years and Older (VALOR) A hexavalent OspA-based Lyme disease vaccine (multivalent Borrelia vaccine (MBV)) is being studied as active immunization for the prevention of Lyme disease caused by Borrelia species (OspA serotypes 1, 2, 3, 4, 5, and 6) in individuals aged 5 years and older.

[0192] Overall design The objective of this study is to establish the efficacy, safety, tolerability, and immunogenicity of MBV in preventing Lyme disease during Lyme disease seasons after a primary vaccination series and / or during subsequent Lyme disease seasons after a primary series and booster vaccination in North America (NA) and Europe. The study is based on immunogenicity evaluation of three independent lots in a subset of participants and also serves as a lot consistency study for operational efficiency.

[0193] Healthy participants aged 5 years and older will be recruited from areas with high prevalence levels of Lyme disease and randomly receive either MBV or placebo (saline). The study will be conducted in areas of North America and Europe where Lyme disease is endemic to ensure adequate geographical representativeness. Site-based randomization will be used to assign individuals to the different groups. Randomization will be stratified by geographic region (Europe and United States) and age (5–11 years, 12–17 years, 18–44 years, 45–64 years, and 65 years and older). Participants in the United States will be randomized to receive three different lots of MBV or placebo, with a randomization ratio of 1:1:1:3 (MBV in lot 1:MBV in lot 2:MBV in lot 3:placebo) for each region / age group. Participants in Europe will be randomly assigned to receive either MBV in lot 1 or placebo in a 1:1 ratio. The overall study randomization ratio between MBV and placebo will remain 1:1.

[0194] The Lyme disease cases in people who received the study vaccine will be compared to those who did not. This will help determine if the study vaccine is safe and effective. If enrolled, participants will be required to visit the study site at least seven times during the study. There will also be at least one phone call. Each participant is expected to participate in the study for up to approximately two and a half years (30 months).

[0195] Dosage and Schedule Participants will receive a primary vaccination series of three doses at approximately months 0, 2, and 5-9, followed by a booster vaccination approximately 12 months later. Participants will receive one dose of MBV (180 μg) or placebo (1:1 ratio) at each vaccination visit. MBV or placebo will be administered intramuscularly, preferably by injecting 0.5 mL into the deltoid muscle of the non-dominant arm: see Table 12. [Table 12]

[0196] The study is designed to demonstrate whether a three-dose extended schedule primary vaccination series administered over a period of five to nine months, followed by a booster vaccination approximately one year (12 months) after completion of the primary series and immediately prior to the start of the second season, can prevent Lyme disease during the second Lyme disease season. The study is further designed to demonstrate whether the primary series protects against Lyme disease during the first Lyme disease season. Participants will be vaccinated such that completion of the primary series occurs before the peak Lyme disease season in Europe or the United States, between approximately April and early May. The primary series will commence between August and early December, with the majority of participants receiving the first dose of the primary series between mid-September and early November. Following the primary series, a booster vaccination will be administered one year later, between approximately March and early May, immediately prior to the start of the second Lyme disease season.

[0197] the purpose: To demonstrate the effectiveness of MBV in preventing confirmed Lyme disease during a Lyme disease season following completion of a primary vaccination series and booster vaccination. To describe the safety profile of MBV, as measured by the proportion of participants reporting local reactions, systemic events, adverse events (AEs), newly diagnosed chronic conditions (NDCMCs), and serious adverse events (SAEs).

[0198] To demonstrate that the immune responses induced by MBV against the six serotypes were comparable across three independent lots. To demonstrate efficacy of MBV in preventing confirmed Lyme disease during Lyme disease seasons following completion of primary vaccination series. To describe the effectiveness of MBV in reducing undiagnosed Lyme disease seroconversion after each season. To describe the effectiveness of MBV in preventing confirmed Lyme disease by geographic region (NA and Europe separately).

[0199] Success indicators: Reduction in the relative risk of confirmed cases of Lyme disease in the MBV group compared to the placebo group [Time period: the Lyme disease season that begins one month after the booster vaccination (28 days after completion of the booster vaccination to October 31)] Clinically and laboratory-confirmed Lyme disease caused by B. burgdorferi sensu lato, as confirmed by an endpoint adjudication committee

[0200] -Proportion of participants who reported local reactions [time period: within 7 days after each study intervention] -Proportion of participants who reported systemic events [period: within 7 days after each study intervention] -Proportion of participants who reported adverse events (AEs) [Period: up to one month after each study intervention] Proportion of participants who reported a newly diagnosed chronic condition (NDCMC) [time period: up to the end-of-study visit] Proportion of participants who reported serious adverse events (SAEs) [time period: up to the end-of-study visit] Geometric mean ratio (GMR) of anti-OspA antibody titers for each serotype (ST1-ST6) from Lot 1 to Lot 2 [Period: 1 month after completion of the primary series]

[0201] Geometric mean ratio (GMR) of anti-OspA antibody titers for each serotype (ST1-ST6) from Lot 1 to Lot 3 [Period: 1 month after completion of the primary series] Geometric mean ratio (GMR) of anti-OspA antibody titers for each serotype (ST1-ST6) from Lot 2 to Lot 3 [Period: 1 month after completion of the primary series] Reduction in the relative risk of confirmed cases of Lyme disease in the MBV group compared to the placebo group [Time period: the Lyme disease season that begins one month after completion of the primary series (28 days after completion of the primary series through October 31)] Clinically and laboratory-confirmed Lyme disease caused by B. burgdorferi sensu lato, as confirmed by an endpoint adjudication committee

[0202] Reduction in the relative risk of Lyme disease-specific seroconversion in otherwise undiagnosed cases of Lyme disease in the MBV group compared to the placebo group [Time period: the Lyme disease season beginning one month after completion of the primary series (28 days after completion of the primary series through October 31)] Seroconversion of non-vaccine antigens measured in a central laboratory

[0203] Reduction in the relative risk of Lyme disease-specific seroconversion in otherwise undiagnosed cases of Lyme disease in the MBV group compared with the placebo group [time period: the Lyme disease season that begins one month after receiving the booster vaccine (28 days after completion of the booster vaccine to October 31)] Seroconversion of non-vaccine antigens measured in a central laboratory

[0204] Vaccine efficacy in participants enrolled at the North American site [Period: Lyme disease season starting one month after completion of the primary series (28 days after completion of the primary series to October 31)] Clinically and laboratory-confirmed Lyme disease caused by B. burgdorferi sensu lato, as confirmed by an endpoint adjudication committee

[0205] Vaccine efficacy in participants enrolled at European sites [Time period: Lyme disease season starting one month after completion of the primary series (28 days after completion of the primary series to October 31)] Clinically and laboratory-confirmed Lyme disease caused by B. burgdorferi sensu lato, as confirmed by an endpoint adjudication committee

[0206] Vaccine efficacy in participants enrolled at the North American site [Period: Lyme disease season starting one month after booster vaccination (28 days after completion of booster vaccination to October 31)] Clinically and laboratory-confirmed Lyme disease caused by B. burgdorferi sensu lato, as confirmed by an endpoint adjudication committee

[0207] Vaccine efficacy in participants enrolled at the European site [Time period: Lyme disease season starting one month after booster vaccination (28 days after completion of booster vaccination to October 31)] Clinically and laboratory-confirmed Lyme disease caused by B. burgdorferi sensu lato, as confirmed by an endpoint adjudication committee

[0208] Study population Main inclusion criteria: In all countries where enrollment of children is permitted, male or female participants aged 5 years or older at the time of enrollment. In countries or sites where enrollment of children is not permitted, male or female participants aged 18 years or older at the time of informed consent.

[0209] Participants who live in areas where Lyme disease is endemic and who engage in lifestyle habits that put them at higher risk for Lyme disease, including but not limited to: People working in B. burgdorferi-infected / tick-infested areas, especially those in occupations that may involve a higher risk of exposure, such as landscaping, forestry, and wildlife and parks management. · Individuals who engage in recreational activities in such areas, such as hiking, camping, fishing, hunting, jogging, or gardening.

[0210] · Individuals who live on land with treelines and have regular contact with these trees. Individuals who have dogs that regularly go outdoors and frequently return with ticks. ·Individuals participating in activities in areas with tall grass, small wooded areas near forests, open fields, lakeside, and riverside. · Any other risk factors determined at the investigator's discretion.

[0211] Key exclusion criteria: · Diagnosis of Lyme disease within the past three months. History of Lyme carditis, neuroborreliosis, arthritis, or other symptoms of disseminated Lyme disease, regardless of when they were diagnosed. Known tick bite within the past 4 weeks.

[0212] Newly developed or unstable underlying medical conditions that may preclude evaluation for Lyme disease, including but not limited to chronic joint pain / arthritis, second / third degree AV block, chronic pain syndromes, and chronic skin conditions that may reduce the ability to detect cutaneous manifestations of Lyme disease. · Autoimmune conditions with symptoms (e.g., arthritis and neurological symptoms) that may preclude evaluation for Lyme disease. ·Chronic systemic doxycycline or minocycline or other tetracycline-based medications for acne, or other chronic suppressive antibiotics used to treat other conditions.

Claims

[Claim 1] The invention described in the present specification.

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