A composition containing three OspA fusion proteins for medical use.

Abstract

The present invention relates to compositions comprising the OspA fusion protein of SEQ ID NO: 1 (LipS1D1-S2D1), the OspA fusion protein of SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and the OspA fusion protein of SEQ ID NO: 3 (Lip-S5D1-S6D1) for use in a vaccine or in a method for raising an immune response in humans against Lyme disease.

Description

[Technical Field]

[0001] Field of Invention The present invention relates to a composition comprising an OspA fusion protein with SEQ ID NO: 1 (LipS1D1-S2D1), an OspA fusion protein with SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and an OspA fusion protein with SEQ ID NO: 3 (Lip-S5D1-S6D1), for use in vaccines or in methods for inducing an immune response against Lyme disease in humans. [Background technology]

[0002] Background of the Invention Lyme borreliosis (also known as "LB" herein), also known as Lyme disease (also known as "LD" herein), is an emerging zoonotic disease transmitted by ticks, caused by several genetic strains of the spirochete Borrelia burgdorferi sensu lato (sl) in the broad sense. It is the most common vector-borne disease in Europe and North America [Mead PS, Epidemiology of Lyme Disease, Infect Dis Clin N Am 29 (2015) 187-210 (Non-Patent Literature 1)]. In Europe, 65,000 to 85,000 cases are reported annually, but this number is undoubtedly an underestimate due to inconsistent case reporting and the fact that LB often goes undiagnosed [Rizzoli A, et al. Lyme borreliosis in Europe. Euro Surveill 16. (2011) (Non-patent Literature 2); Lindgren E, Jaenson TGT, Organization WHO Europe (2006) Lyme borreliosis in Europe: Influences of climate and climate change, epidemiology, ecology and adaptation measures, http: / / www.euro.who.int / __data / assets / pdf_file / 0006 / 96819 / E89522.pdf, last evaluated March 2016 (Non-patent Literature 3)].The U.S. Centers for Disease Control and Prevention (CDC) estimates approximately 300,000 cases per year in the United States, nearly 10 times higher than the reported cases [Hinckley AF, et al. Lyme disease testing by large commercial laboratories in the United States. Clin Infect Dis. 2014 Sep 1;59(5):676-81. doi: 10.1093 / cid / ciu397. Epub 2014 May 30 (Non-patent Literature 4); Nelson CA, et al. Incidence of Clinician-Diagnosed Lyme Disease, United States, 2005-2010. Emerg Infect Dis. 2015 Sep;21(9):1625-31. doi: 10.3201 / eid2109.150417 (Non-patent Literature 5)]. The incidence of LB shows a bimodal distribution with respect to age. The two main target populations are children aged 5–14 years and adults aged 50–64 years [Rizzoli A, et al. 2011, see above (Non-Patent Literature 2); http: / / www.cdc.gov / lyme / stats / graphs.html, last evaluated March 2016 (Non-Patent Literature 6)].

[0003] In Europe, most human infections are caused by four Borrelia gene species that present six OspA serotypes (STs): 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 nearly 100% of cases. Recently, a new genetic species called Borrelia mayonii has been described, found in a small number of clinical specimens isolated in the north-midwestern 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 (Non-patent Literature 7)].

[0004] The most common clinical manifestation of LD is a gradually expanding erythematous skin rash called migratory erythema (EM), which is a clear sign of early, localized Borrelia infection. EM appears within days to weeks at the site of the tick bite and is often accompanied by symptoms of fatigue, fever, headache, mild torticollis, arthralgia, or myalgia [Mead et al., 2015, see above (Non-Patent Literature 1)]. Approximately 70% to 80% of LD cases develop EM [Stanek G, et al. Lyme borreliosis, Lancet 2012; 379: 461-73 (Non-Patent Literature 8); Wormser GP, et al, The Clinical Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: Clinical Practice Guidelines by the Infectious Diseases Society of America, Clinical Infectious Diseases 2006; 43:1089-134 (Non-Patent Literature 9)].

[0005] If left untreated or improperly treated with antibiotics, the infection can spread to other parts of the body, potentially leading to the development of serious late-stage symptoms affecting the nervous system (facial nerve paralysis, meningitis, myelitis, and encephalitis), the joints (recurrent or persistent synovitis of the large joints), or the heart (e.g., conduction abnormalities and cardiitis).

[0006] The most common late clinical manifestations of LB, occurring in approximately 30% of patients, include musculoskeletal symptoms such as Lyme arthritis. Lyme arthritis involves recurrent episodes or prolonged joint swelling (synovitis) that develop months after a tick bite, usually affecting one or a few joints, most commonly the knee. Neurological manifestations include Lyme neuroborreliosis, most commonly presented within weeks of infection as cranial nerve damage with facial nerve palsy, possibly involving both sides (bilateral Bell's palsy). In adults, the disease typically presents as painful meningoradiculoneuritis and facial nerve palsy. In contrast, children most frequently develop headache due to meningitis and facial nerve palsy. In children, the duration of symptoms is shorter and the outcome is better. Cardiac manifestations in LD appear to be rare, with Lyme cardiomyitis usually presenting within two months of infection as myocarditis with acute intermittent atrioventricular block. In Europe, more severe skin manifestations (e.g., chronic atrophic acrodermatitis (ACA), Borrelia lymphocytoma) may be due to disseminated infections as late complications [Stanek, et al. 2012, see above (Non-Patent Literature 8)].

[0007] Outer surface protein A (OspA) is one of the major proteins expressed by Borrelia burgdorferi sl when present in mites that are not feeding. During mite feeding, incoming blood signals downregulation of OspA expression, allowing the spirochete to penetrate the intestinal epithelium, reach the salivary glands, and then move into the host bloodstream [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 (Non-patent Literature 10)]. OspA-based LB vaccines induce antibodies in the tick gut that act on spirochetes expressing OspA, blocking the transmission of spirochetes to the salivary glands and subsequently to vertebrate hosts [de Silva AM and Telford SR 3rd, Brunet LR, Barthold SW, Fikrig E., J Exp Med. 1996, 183(1):271-5 (Non-Patent Literature 11)].

[0008] Two vaccines containing OspA(ST1) derived from B. burgdorferi have been previously shown to be effective in preventing LB in humans: LYMErix (SmithKline Beecham) [Steere AC, et al., Lyme Disease Vaccine Study Group, Vaccination against Lyme disease with recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant, N Engl J Med. 1998;339(4):209-15 (Non-patent Literature 12)] and ImuLyme (Pasteur Merieux Connaught) [Sigal LH, et al., A vaccine consisting of recombinant Borrelia burgdorferi outer-surface protein A to prevent Lyme disease, N Engl J Med. 1998;339(4):216-22 (Non-patent Literature 13)]. LYMErix was approved and available to customers from 1998 to 2002, and was voluntarily withdrawn from the market in 2002. Following the approval of LYMErix, a possible relationship between the Lyme vaccine and joint reactions was hypothesized due to the partial homology between OspA ST1 in the vaccine and hLFA-1 (human leukocyte function-associated antigen-1), which was claimed to induce antibiotic-resistant Lyme arthritis in a subset of spontaneously infected patients [Gross DM, et al., Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis, Science. 1998 Jul 31;281(5377):703-6 (Non-patent Literature 14)]. However, the hypothesis could not be proven.On the contrary, a retrospective study of joint complaints reported to the Vaccine Adverse Event Reporting System (VAERS) during the first 19 months after the vaccine's approval, after more than 1,400,000 doses had been distributed, did not show an unusually high number of such complaints [Lathrop SL, et al., Adverse event reports following vaccination for Lyme disease: December 1998-July 2000, Vaccine 20 (2002) 1603-1608 (Non-Patent Literature 15)]. In a Phase III trial of the vaccine, the incidence of transient arthralgia increased but not significantly in vaccinated individuals, but the incidence of arthritis did not increase compared to the placebo group [Steere, et al., 1998, see above (Non-Patent Literature 12)].

[0009] More recently, Baxter conducted a Phase I / II trial of a similar multivalent OspA-based vaccine candidate, which demonstrated safety, good tolerability, and high immunogenicity [Wressnigg N, et al.; Safety and immunogenicity of a novel multivalent OspA vaccine against Lyme borreliosis in healthy adults: a double-blind, randomised, dose-escalation phase 1 / 2 trial, Lancet Infect Dis. 2013 Aug;13(8):680-9 (Non-patent Literature 16); Wressnigg N, et al., A Novel multivalent OspA vaccine against Lyme borreliosis is safe and immunogenic in an adult population previously infected with Borrelia burgdorferi sensu lato, Clin Vaccine Immunol. 2014 Nov;21(11):1490-9 (Non-patent Literature 17)]. However, this vaccine is not currently under further development. Currently, there are no LB vaccines on the market, and no other LB vaccine candidates are in clinical development. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Mead PS, Epidemiology of Lyme Disease, Infect Dis Clin N Am 29 (2015) 187-210 [Non-Patent Document 2] Rizzoli A, et al. Lyme borreliosis in Europe. Euro Surveill 16. (2011) [Non-Patent Document 3] Lindgren E, Jaenson TGT, Organization WHO Europe (2006) Lyme borreliosis in Europe: Influences of climate and climate change, epidemiology, ecology and adaptation measures, http: / / www.euro.who.int / __data / assets / pdf_file / 0006 / 96819 / E89522.pdf, last evaluated March 2016. [Non-Patent Document 4] Hinckley AF, et al. Lyme disease testing by large commercial laboratories in the United States. Clin Infect Dis. 2014 Sep 1;59(5):676-81. doi: 10.1093 / cid / ciu397. Epub 2014 May 30 [Non-Patent Document 5] Nelson CA, et al. Incidence of Clinician-Diagnosed Lyme Disease, United States, 2005-2010. Emerg Infect Dis. 2015 Sep;21(9):1625-31. doi: 10.3201 / eid2109.150417 [Non-Patent Document 6] http: / / www.cdc.gov / lyme / stats / graphs.html, last assessment March 2016 [Non-Patent Document 7] 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 [Non-Patent Document 8] Stanek G, et al. Lyme borreliosis, Lancet 2012; 379: 461-73

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[0011] The present invention's multivalent lyme borreliosis vaccine 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 in the tick's intestines to neutralize Borrelia and block its transmission to the host. The multimeric borrelia vaccine has been shown to be highly protective against four Borrelia gene species (B. burgdolferi ss, B. afzeri, B. bavaliensis, and B. galinii), including five clinically relevant OspA serotypes (ST1, ST2, ST4-ST6), in mouse models using either infected ticks or in vitro grown spirochetes for attack.

[0012] OspA-based vaccines have shown efficacy in humans in the past. Valneva's multivalent Borrelia vaccine was able to prevent infection with the most common B. burgdorferi sl and OspA serotypes present in Europe and the United States.

[0013] OspA-based vaccines require high and sustained antibody titers due to their mechanism of action (outside the body). [Invention 1001] A composition for use in vaccines, 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 aforementioned vaccine, - In human adults or human children, at least three times, with a total protein content of the three fusion proteins ranging from 120 to 200 μg per dose; or - In human children, at least three times, with a total protein content of the three aforementioned fusion proteins in the range of 60-100 μg per dose. The composition to be administered. [Invention 1002] 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) for use in a method for inducing an immune response in humans against Lyme disease, The method described above is - In human adults or human children, at least three times, with a total protein content of the three fusion proteins ranging from 120 to 200 μg per dose; or - In human children, at least three times, with a total protein content of the three aforementioned fusion proteins in the range of 60-100 μg per dose. The composition comprising the step of administering the composition. [Invention 1003] A composition for use of the present invention 1001 or 1002, wherein the second dose is administered at least 6 weeks to at most 3 months after the first dose, and / or the third dose is administered at least 5 months to at most 7 months after the first dose. [Invention 1004] A composition for use in any of the inventions 1001 to 1003, wherein the second dose is administered at least 50 to at most 70 days after the first dose, particularly at least 55 to at most 60 days, especially at 56 days; and / or the third dose is administered at least 170 to at most 190 days after the first dose, particularly at least 175 to at most 185 days, especially at 180 days. [Invention 1005] A composition for use in any of the inventions 1001 to 1004, wherein the fourth dose of the composition is administered during a period of at least 15 months to at most 21 months after the first dose, particularly during a period of at least 17 months to at most 19 months, and especially at 18 months. [Invention 1006] A composition for use in any of the inventions 1001 to 1005, wherein the total protein content of the three fusion proteins is in the range of 135 μg to 180 μg per dose for adult or child human, particularly 135 μg or 180 μg per dose, or the total protein content of the three fusion proteins is in the range of 67.5 μg to 90 μg per dose for young child, particularly 67.5 μg or 90 μg per dose. [Invention 1007] A composition for use in any of the inventions 1001 to 1006, wherein the three fusion proteins constitute at least 60%, preferably at least 70%, and more preferably 80% of all proteins in the composition. [Invention 1008] A composition for use in any of the inventions 1001 to 1007, comprising the fusion protein in a weight ratio of 1:1:1 (Lip-S1D1-S2D1 : Lip-S4D1-S3hybD1 : Lip-S5D1-S6D1). [Invention 1009] A composition comprising an adjuvant for use in any of the inventions 1001 to 1008. [Invention 1010] A composition for use in any of the inventions 1001 to 1009, comprising an aluminum adjuvant, preferably an aluminum adjuvant having a low copper content, preferably an aluminum adjuvant having less than 1.25 ppb of copper. [Invention 1011] A composition for use in any of the inventions 1001 to 1010, wherein the total protein content of the three fusion proteins is 135 μg per dose for adult or child humans, the second dose is administered 56 days after the first dose, the third dose is administered 180 days after the first dose, and any fourth dose is administered 18 months after the first dose. [Invention 1012] A composition for use in any of the inventions 1001 to 1010, wherein the total protein content of the three fusion proteins is 67.5 μg per dose for a young child, the second dose is administered 56 days after the first dose, the third dose is administered 180 days after the first dose, and any fourth dose is administered 18 months after the first dose. [Invention 1013] A composition for use in any of the inventions 1001 to 1010, wherein the total protein content of the three fusion proteins is 180 μg per dose for adult or child humans, the second dose is administered 56 days after the first dose, the third dose is administered 180 days after the first dose, and any fourth dose is administered 18 months after the first dose. [Invention 1014] A composition for use in any of the inventions 1001 to 1010, wherein the total protein content of the three fusion proteins is 90 μg per dose for a young child, the second dose is administered 56 days after the first dose, the third dose is administered 180 days after the first dose, and any fourth dose is administered 18 months after the first dose. [Invention 1015] A composition for use in any of the inventions 1001 to 1010, wherein the total protein content of the three fusion proteins is 135 μg per dose for adult or child humans, the second dose is administered 2 months after the first dose, the third dose is administered 6 months after the first dose, and any fourth dose is administered 18 months after the first dose. [Invention 1016] A composition for use in any of the inventions 1001 to 1010, wherein the total protein content of the three fusion proteins is 67.5 μg per dose for young children, the second dose is administered 2 months after the first dose, the third dose is administered 6 months after the first dose, and any fourth dose is administered 18 months after the first dose. [Invention 1017] A composition for use in any of the inventions 1001 to 1010, wherein the total protein content of the three fusion proteins is 180 μg per dose for adult or child humans, the second dose is administered 2 months after the first dose, the third dose is administered 6 months after the first dose, and any fourth dose is administered 18 months after the first dose. [Invention 1018] A composition for use in any of the inventions 1001 to 1010, wherein the total protein content of the three fusion proteins is 90 μg per dose for a young child, the second dose is administered 2 months after the first dose, the third dose is administered 6 months after the first dose, and any fourth dose is administered 18 months after the first dose. [Invention 1019] A composition for use in any of the inventions 1001 to 1018, wherein further administration is annually after the third administration, particularly after one year, two years, and three years. [Invention 1020] A composition for use in any of the inventions 1001 to 1019, wherein the induced immune response comprises an antibody response of 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 having bactericidal activity. [Invention 1021] A composition for use in any of the inventions 1001 to 1020, wherein the immune response induced comprises antibodies against Borrelia serotypes 1, 2, 3, 4, 5, and 6. [Invention 1022] A composition for use in any of the invention 1001 to 1021, wherein the immune response to Borrelia serotypes 1, 2, 3, 4, 5, and 6 is sustained for at least about 60 days, at least about 180 days, at least about 365 days, or at least about 540 days. [Invention 1023] A composition for use in any of the inventions 1001 to 1022, comprising less than 1.25 ppb of copper or a sufficient amount of L-methionine. [Invention 1024] Copper, especially Cu + or Cu 2+ A composition for use in any of the inventions 1001 to 1023, which is in the form of an ion. [Invention 1025] A composition for use in any of the present inventions 1001 to 1024, wherein L-methionine is present at a concentration of at least 10 mmol / l. [Invention 1026] A composition for any of the uses of the present invention 1023 to 1025, wherein the concentration of L-methionine in mol / l units is at least equivalent to the concentration of copper in the composition. [Invention 1027] The material further comprises a reactive compound, the reactive compound being selected from the group consisting of redox active compounds, radical building compounds, stabilizing compounds, and any combination thereof, in particular, the reactive compound being formaldehyde, ethanol, chloroform, trichloroethylene, acetone, triton-X-100, deoxycollate, diethyl pyrocarbonate, sulfite, Na 2 S 2 O 5 β-proprio-lactone, polysorbate, e.g., Tween 20®, Tween 80®, O 2 A composition for use in any of the invention 1001 to 1026, selected from the group consisting of phenol, pluronic copolymers, and any combination thereof. [Invention 1028] A composition for use in any of the inventions 1001 to 1027, wherein the human adult is 18 years of age or older, for example, between 18 and 65 years of age. [Invention 1029] A composition for use in any of the invention 1001 to 1027, wherein the aforementioned human adult is an elderly subject aged 50 years or older. [Invention 1030] A composition for use in any of the invention 1001 to 1027, wherein the human child is 5 to 17 years old, for example, 12 to 17 years old or 5 to 11 years old. [Invention 1031] A composition for use in any of the invention 1001 to 1027, wherein the human child is 0 to 4 years of age, for example, 1 to 4 years or 2 to 4 years of age. [Invention 1032] A composition for use in any of the inventions 1001 to 1031, administered to human adults or children in volumes of 0.25 ml to 1.0 ml, for example, 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. [Invention 1033] A composition for use in any of the inventions 1001 to 1032, administered to human adults or children in a volume of 0.25 ml, 0.5 ml, or 1.0 ml. [Invention 1034] A composition for use in vaccinating humans, The above method provides a composition comprising a fusion protein with SEQ ID NO: 1 (Lip-S1D1-S2D1), a fusion protein with SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and a fusion protein with SEQ ID NO: 3 (Lip-S5D1-S6D1), - In human adults or human children, at least twice, with a total protein content of the three fusion proteins in the range of 120-200 μg per dose; or - In human children, at least twice, with a total protein content of the three fusion proteins in the range of 60-100 μg per dose. The composition comprising the step of administering it. [Invention 1035] A composition for use in inducing an immune response in humans against Lyme disease, The above method provides a composition comprising a fusion protein with SEQ ID NO: 1 (Lip-S1D1-S2D1), a fusion protein with SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and a fusion protein with SEQ ID NO: 3 (Lip-S5D1-S6D1), - In human adults or human children, at least twice, with a total protein content of the three fusion proteins in the range of 120-200 μg per dose; or - In human children, at least twice, with a total protein content of the three fusion proteins in the range of 60-100 μg per dose. The composition comprising the step of administering it. [Invention 1036] A composition for use of the present invention 1034 or the present invention 1035, wherein the second dose is administered at a period of at least 5 months to at most 7 months after the first dose. [Invention 1037] A composition for use in any of the inventions 1034 to 1036, wherein the second dose is administered at least 170 days to at most 190 days after the first dose, at least 175 days to at most 185 days, and particularly 180 days. [Invention 1038] A composition for use in any of the inventions 1034 to 1037, wherein the third dose of the composition is administered during a period of at least 15 months to at most 21 months after the first dose, particularly during a period of at least 17 months to at most 19 months, and especially at 18 months. [Invention 1039] A composition for use in any of the inventions 1034 to 1038, wherein the total protein content of the three fusion proteins is in the range of 135 μg to 180 μg per dose for adult or child human, particularly 135 μg or 180 μg per dose, or the total protein content of the three fusion proteins is in the range of 67.5 μg to 90 μg per dose for young child, particularly 67.5 μg or 90 μg per dose. [Invention 1040] A composition for use in any of the inventions 1034 to 1039, wherein the three fusion proteins constitute at least 60%, preferably at least 70%, and more preferably at least 80% of all proteins in the composition. [Invention 1041] A composition for use in any of the present inventions 1034 to 1040, comprising the fusion protein in a weight ratio of 1:1:1 (Lip-S1D1-S2D1 : Lip-S4D1-S3hybD1 : Lip-S5D1-S6D1). [Invention 1042] A composition comprising an adjuvant for use in any of the inventions 1034 to 1041. [Invention 1043] A composition comprising an aluminum adjuvant for use in any of the inventions 1034 to 1042. [Invention 1044] A composition for any use of the present invention 1034 to 1043, wherein the total protein content of the three fusion proteins is 135 μg per dose for adult or child human, the second dose is 180 days after the first dose, and an optional third dose is 18 months after the first dose, and optionally every 12 months thereafter. [Invention 1045] A composition for any use of the present invention 1034 to 1043, wherein the total protein content of the three fusion proteins is 67.5 μg per dose for young children, the second dose is 180 days after the first dose, and an optional third dose is 18 months after the first dose, and optionally every 12 months thereafter. [Invention 1046] A composition for any use of the present invention 1034 to 1043, wherein the total protein content of the three fusion proteins is 180 μg per dose for adult or child human, the second dose is 180 days after the first dose, and an optional third dose is 18 months after the first dose, and optionally every 12 months thereafter. [Invention 1047] A composition for any use of the present invention 1034 to 1043, wherein the total protein content of the three fusion proteins is 90 μg per dose for a young child, the second dose is 180 days after the first dose, and an optional third dose is 18 months after the first dose, and optionally every 12 months thereafter. [Invention 1048] A composition for any use of the present invention 1034 to 1043, wherein the total protein content of the three fusion proteins is 135 μg per dose for adult or child human, the second dose is 6 months after the first dose, and an optional third dose is 18 months after the first dose, and optionally every 12 months thereafter. [Invention 1049] A composition for any use of the present invention 1034 to 1043, wherein the total protein content of the three fusion proteins is 67.5 μg per dose for young children, the second dose is 6 months after the first dose, and an optional third dose is 18 months after the first dose, and optionally every 12 months thereafter. [Invention 1050] A composition for any use of the present invention 1034 to 1043, wherein the total protein content of the three fusion proteins is 180 μg per dose for adult or child human, the second dose is 6 months after the first dose, and an optional third dose is 18 months after the first dose, and optionally every 12 months thereafter. [Invention 1051] A composition for any use of the present invention 1034 to 1043, wherein the total protein content of the three fusion proteins is 90 μg per dose for a young child, the second dose is 6 months after the first dose, and an optional third dose is 18 months after the first dose, and optionally every 12 months thereafter. [Invention 1052] A composition for use in any of the invention 1034-1051, wherein further administration is annually after the last primary immunization, or at the beginning of each mite season, particularly after one year, two years, and three years. [Invention 1053] A composition for use in any of the inventions 1034 to 1052, wherein the induced immune response comprises an antibody response of 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 having bactericidal activity. [Invention 1054] A composition for use in any of the invention 1034 to 1053, wherein the immune response induced comprises an antibody having bactericidal activity against spirochetes expressing OspA serotypes 1, 2, 3, 4, 5, and 6. [Invention 1055] A composition for use in any of the Invention 1034 to 1054, wherein the immune response to OspA serotypes 1, 2, 3, 4, 5, and 6 is sustained for at least about 60 days, at least about 180 days, at least about 365 days, or at least about 540 days. [Invention 1056] A composition for use in any of the inventions 1034 to 1055, comprising less than 1.25 ppb of copper or a sufficient amount of L-methionine. [Invention 1057] Copper, especially Cu + or Cu 2+ A composition for use in any of the inventions 1034 to 1056, which is in the form of an ion. [Invention 1058] A composition for use in any of the present invention 1034 to 1057, wherein L-methionine is present at a concentration of at least 10 mmol / l. [Invention 1059] A composition for any use of the present invention 1056 to 1058, wherein the concentration of L-methionine in mol / l units is at least equivalent to the concentration of copper in the composition. [Invention 1060] The reaction further comprises a reactive compound, the reactive compound being selected from the group consisting of redox active compounds, radical constituent compounds, stabilizing compounds, and any combination thereof, in particular, the reactive compound being formaldehyde, ethanol, chloroform, trichloroethylene, acetone, triton-X-100, deoxycholate, diethyl pyrocarbonate, sulfite, Na 2 S 2 O 5 β-proplio-lactone, polysorbate, e.g., Tween 20®, Tween 80®, O 2 A composition for use in any of the invention 1034 to 1059, selected from the group consisting of phenol, pluronic copolymers, and any combination thereof. [Invention 1061] A composition for use in any of the inventions 1034 to 1060, wherein the human adult is 18 years of age or older, for example, between 18 and 65 years of age. [Invention 1062] A composition for use in any of the inventions 1034 to 1060, wherein the aforementioned human adult is an elderly subject who is 50 years of age or older. [Invention 1063] A composition for use in any of the inventions 1034 to 1060, wherein the human child is 5 to 17 years of age, for example, 12 to 17 years or 5 to 11 years of age. [Invention 1064] A composition for use in any of the inventions 1034 to 1060, wherein the human child is 0 to 4 years of age, for example, 1 to 4 years or 2 to 4 years of age. [Invention 1065] A composition for use in any of the inventions 1034 to 1064, administered to human adults or children in volumes of 0.25 ml to 1.0 ml, for example, 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. [Invention 1066] A composition for use in any of the inventions 1034 to 1065, administered to human adults or children in a volume of 0.25 ml, 0.5 ml, or 1.0 ml. [Brief explanation of the drawing]

[0014] [Figure 1]A serum bactericidal assay was used as an in vitro functional assay (see Materials and Methods). [Figure 2] Study design (First-in-human Phase 1 trial) including a 13-month booster (booster extension) in subjects selected from the high-dose group. [Figure 3] Figures 3A-3B. Phase 1 data: Immunogenicity data at day 84 for each serotype, per-protocol (PP) population (i.e., subjects who received all three immunizations according to the protocol at the correct dose and timing), Figure 3A) IgG geometric mean titer (GMT), and Figure 3B) seroconversion rate, defined as the percentage of subjects who achieved a 4-fold or greater increase in IgG titer from baseline. [Figure 4] Figures 4A-4D. Phase 1 data: IgG GMT values ​​for each serotype in Figure 4A) d84, Figure 4B) M13, Figure 4C) M14, and Figure 4D) M19, for the booster PP population (i.e., subjects enrolled in the booster extension phase and receiving the correct booster dose at the correct time according to the protocol). [Figure 5] Figures 5A-5F. Phase 1 data: Serotype-specific IgG GMT values ​​over time, booster PP population, Figure 5A) ST1, Figure 5B) ST2, Figure 5C) ST3, Figure 5D) ST4, Figure 5E) ST5, Figure 5F) ST6. [Figure 6A] Phase 1 data: Comparison of ELISA titer and SBA titer. Figure 6A) Samples with ELISA titers lower than 100 U / mL that rarely tested positive in serotype 1 (ST1)-SBA. A total of 116 valid SBA data points were collected. The limit of quantification (LLOQ) for ST1 SBA was set at 20, and a value of 10 was assigned to negative samples. [Figure 6B]Phase 1 data: Comparison of ELISA titer and SBA titer. Figure 6B) ST2 - Samples with ELISA titers lower than 200 U / mL that rarely tested positive in SBA. A total of 48 valid SBA data points were collected. The LLOQ for ST2 SBA was set to 20, and negative samples were assigned a value of 10. [Figure 6C] Phase 1 data: Comparison of ELISA titer and SBA titer. Figure 6C) ST3 - Samples with ELISA titers lower than 100 U / mL that rarely tested positive in SBA. In total, 122 valid SBA data points were collected. The LLOQ for ST3 SBA was set to 160, and negative samples were assigned a value of 80. [Figure 7] The study design for the first Phase 2 trial of the present invention involves investigating three adjuvant-added vaccine doses A (90 μg, 135 μg, and 180 μg) in the "Run-in phase" and two selected dose groups (135 μg and 180 μg) in the "Main Study phase". [Figure 8] Second Phase 2 Trial: A study design for a second Phase 2 trial of the present invention's polyvalent Borrelia vaccine to investigate alternative immunization schedules. [Figure 9A] Correlation between ELISA titer and SBA titer, day 208, pooled multimerized Borrelia vaccine-treated group, PP population excluding baseline SBA seropositive subjects. (Figure 9A) Spearman correlation coefficient: 0.7083, p-value <.0001. [Figure 9B] Correlation between ELISA titer and SBA titer, day 208, pooled multimerized Borrelia vaccine-treated group, PP population excluding baseline SBA seropositive subjects. (Figure 9B) Spearman correlation coefficient: 0.8217, p-value <.0001. [Figure 9C]Correlation between ELISA titer and SBA titer, day 208, pooled multimerized Borrelia vaccine-treated group, PP population excluding baseline SBA seropositive subjects. (Figure 9C) Spearman correlation coefficient: 0.9131, p-value <.0001. [Figure 9D] Correlation between ELISA titer and SBA titer, day 208, pooled multimerized Borrelia vaccine-treated group, PP population excluding baseline SBA seropositive subjects. (Figure 9D) Spearman correlation coefficient: 0.6487, p-value <.0001. [Figure 9E] Correlation between ELISA titer and SBA titer, day 208, pooled multimerized Borrelia vaccine-treated group, PP population excluding baseline SBA seropositive subjects. (Figure 9E) Spearman correlation coefficient: 0.6554, p-value <.0001. [Figure 9F] Correlation between ELISA titer and SBA titer, day 208, pooled multimerized Borrelia vaccine-treated group, PP population excluding baseline SBA seropositive subjects. (Figure 9F) Spearman correlation coefficient: 0.7818, p-value <.0001. [Figure 10] Seroconversion rates of OspA-specific IgG by serotype, D85, PP population. Significant differences between groups are indicated in parentheses. No significant differences were observed between the 135 μg treatment group and the 180 μg treatment group. [Figure 11] Seroconversion rates of OspA-specific IgG by serotype, D208, PP population. Significant differences between groups are indicated in parentheses. No significant differences were observed between the 135 μg treatment group and the 180 μg treatment group. [Figure 12] Inverse cumulative distribution curve for ELISA - ST1. Percentage of subjects relative to OspA-specific IgG at each time point. Top panel: 135 μg treatment group. Bottom panel: 180 μg treatment group. [Figure 13] Inverse cumulative distribution curve for ELISA - ST2. Percentage of subjects relative to OspA-specific IgG at time. Top panel: 135 μg treatment group. Bottom panel: 180 μg treatment group. [Figure 14]Inverse cumulative distribution curve for ELISA - ST3. Percentage of subjects relative to OspA-specific IgG at time. Top panel: 135 μg treatment group. Bottom panel: 180 μg treatment group. [Figure 15] Inverse cumulative distribution curve for ELISA - ST4. Percentage of subjects relative to OspA-specific IgG at time. Top panel: 135 μg treatment group. Bottom panel: 180 μg treatment group. [Figure 16] Inverse cumulative distribution curve for ELISA - ST5. Percentage of subjects relative to OspA-specific IgG at time. Top panel: 135 μg treatment group. Bottom panel: 180 μg treatment group. [Figure 17] Inverse cumulative distribution curve for ELISA - ST6. Percentage of subjects relative to OspA-specific IgG at time. Top panel: 135 μg treatment group. Bottom panel: 180 μg treatment group. [Figure 18] Figures 18A-18B. Time course of OspA-specific IgG antibody (GMT) and PP population by serotype, as determined by ELISA. Figure 18A) 135 μg group; Figure 18B) 180 μg group. [Figure 19] Enrollment for a Phase 2 clinical trial of a multivalent Borrelia vaccine to investigate a three-dose or two-dose primary immunization schedule implemented in a staggered manner across three age cohorts, from oldest to youngest. [Figure 20] A study design for a Phase 2 clinical trial of a polyvalent Borrelia vaccine to investigate a primary immunization schedule of three or two doses of the vaccine, and a booster dose, in a study population aged 5–65 years. [Modes for carrying out the invention]

[0015] Detailed description of the invention Accordingly, in the first aspect, the present invention provides a composition for use as a vaccine 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 vaccine is administered to human adults at least three times in a total protein content of the three fusion proteins in a dose of 60-200 μg, or more preferably 120-200 μg, or to human children at least three times in a total protein content of the three fusion proteins in a dose of 60-200 μg, or 120-200 μg, or 60-100 μg. Alternatively, the present invention provides a composition for use as a vaccine 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 vaccine is administered to human adults at least twice in doses of 60-200 μg, or more preferably 120-200 μg, of the total protein content of the three fusion proteins, or to human children at least twice in doses of 60-200 μg, or 120-200 μg, or 60-100 μg, of the total protein content of the three fusion proteins. The 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) is also referred to herein interchangeably as "Vaccine A" or the multimerized Borrelia vaccine ("MBV").

[0016] The fusion protein described above (sometimes also called a heterodimer) is derived from Borrelia outer surface protein A (OspA). OspA is expressed by Borrelia only when it is present in the intestines of tick-borne animals. Therefore, OspA antibodies produced by vaccination do not fight infection in the body, but they enter the tick's intestines when the tick ingests blood. There, the antibodies may neutralize the spirochete and block the movement of the bacteria from the tick's midgut to the salivary glands, which is the pathway through which Borrelia enters vertebrate hosts. Thus, OspA-specific antibodies prevent the transmission of Borrelia from tick-borne animals to human hosts.

[0017] The above protein is a lipid-added mutant OspA fragment fusion protein containing a hybrid C-terminal OspA fragment, the hybrid fragment consisting of the C-terminal domain of Borrelia OspA, comprising a fragment derived from OspA of a Borrelia strain different from B. galini PBr strain, and a second fragment of OspA from B. galini PBr strain, and differing from the corresponding wild-type sequence by the introduction of at least one disulfide bond. The disulfide bond is a type 1 disulfide bond with cysteine ​​residues inserted at positions 183+ / -3 and 270+ / -3 (see WO 2015 / 104396 A1 for further details). S3hyb represents a fusion of amino acids 125-176 of B. valaisiana and amino acids 177-274 of B. galini PBr strain. Lip signifies lipid addition and indicates N-terminal addition of glycerol and fatty acid residues. The "LN1" peptide linker has the following sequence: It is a fusion of two separate loop regions in the N-terminal half of OspA from B. burgdolferi ssB31 strain, containing TIFF0007887224000001.tif4128 (with an amino acid exchange at position 53 of aa 65-74 and aa 42-53, D53S).

[0018] In particular, Lip-S1D1-S2D1 is a fusion protein of OspA serotype 1 and OspA serotype 2 with a disulfide bond type 1, and includes an N-terminal CSS, LN1 linker sequence, and N-terminal lipid addition for lipid addition. Amino acids 164-174 of OspA serotype 1 are non-hLFA-1-like sequences. It has been replaced by TIFF0007887224000002.tif4128. The sequence is shown below as SEQ ID NO: 1. TIFF0007887224000003.tif32158

[0019] Lip-S4D1-S3hybD1 is a fusion protein of OspA serotype 4 and OspA serotype 3, containing amino acids 125-176 of B. baraisiana VS116 strain and amino acids 177-274 of B. galinii PBr strain, serotype 3, with a type 1 disulfide bond, and includes an N-terminal CSS, LN1 linker sequence, and N-terminal lipid addition. The sequence is shown below as SEQ ID NO: 2. TIFF0007887224000004.tif32158

[0020] Lip-S5D1-S6D1 is a fusion protein of both OspA serotype 6, featuring a disulfide bond type 1, an N-terminal CSS for lipid addition, an LN1 linker sequence, and N-terminal lipid addition. The sequence is shown below as SEQ ID NO: 2. TIFF0007887224000005.tif32158

[0021] The nucleic acid sequence encoding the above fusion protein is as follows: TIFF0007887224000006.tif37158TIFF0007887224000007.tif234158

[0022] Further information about the fusion proteins and their production can be drawn from WO 2015 / 104396 A1, where Lip-S1D1-S2D1, Lip-S4D1-S3hybD1, and Lip-S5D1-S6D1 correspond to SEQ ID NO: 29, 27, and 33, respectively.

[0023] As detailed above, the fusion proteins are lipid-added proteins in which the lipid moiety is also referred to as "Lip" together with the glycerol group. According to the present invention, Lip contains one to three lipids such as C 14-20 alkyl and / or C 14-20 alkenyl and the amino group of the N-terminal cysteine of the polypeptide of the present invention, or preferably, Lip has the following formula (I) TIFF0007887224000008.tif34128 part, 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 the amino acid sequence added to the cysteine residue shown in formula (I). More preferably, the N-terminal cysteine of the Lip + polypeptide is N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy)propyl cysteine (referred to herein as "Pam3Cys"), and is connected to the amino acid sequence of the present invention via the carbonyl C of cysteine. In the above formula (I), R1, R2, and R3 are palmitoyl moieties, and X is the amino acid sequence added to the cysteine residue.

[0024] The fusion protein is incorporated into the composition. The composition is pharmaceutically acceptable and allows for administration to humans. It may optionally contain any pharmaceutically acceptable carrier or excipient, e.g., buffers, stabilizers, or further active ingredients, in particular components known in connection with the manufacture of pharmaceutical compositions and / or vaccines. 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 aluminum hydroxide, preferably at a concentration of 0.15%. The composition may also contain 5 mM to 50 mM sodium phosphate, 100 to 200 mM sodium chloride, 5 mM to 25 mM L-methionine, 2.5% to 10% sucrose, 0.01% to 0.1% Tween 20, and 0.1% to 0.2% (w / v) aluminum hydroxide. More preferably, the formulation contains 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 pH 6.7 ± 0.2. In a preferred embodiment, the excipient is L-methionine.

[0025] In some embodiments, the composition containing the fusion protein is administered to a subject in a volume of about 0.25 ml to about 1.0 ml or more, for example, 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 is in the range of 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 one 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 more preferred embodiment, the volume administered to subjects aged 0-4 years (e.g., 1-4 years or 2-4 years) is 0.25 ml.

[0026] According to the present invention, the composition is used as a vaccine against infectious diseases caused by pathogenic Borrelia species as disclosed herein, more preferably including Borrelia species, B. burgdorferi ss, B. afzeri, B. bavaliensis, and B. galini, and / or other pathogens whose antigens are included in the vaccine. Preferably, the Borrelia species is selected from B. burgdorferi ss, B. galini, B. afzeri, B. andersoni, B. bavaliensis, B. bissettii, B. baraisiana, B. lusitaniae, B. spielmanii, B. japonica, B. tanukii, B. turdi, or B. sinica, preferably B. burgdorferi ss, B. afzeri, and B. galini.

[0027] It has been found that a specific dosing schedule is advantageous in obtaining sustained protection, particularly against all Borrelia OspA serotypes. This relates to the minimum number of doses and dose range. According to this, the vaccine would be administered to human adults in at least three doses, with a total protein content of the three fusion proteins ranging from 60 to 200 μg per dose, or more preferably in the range of 120 to 200 μg per dose, or to human children in at least three doses, with a total protein content of the three fusion proteins ranging from 60 to 200 μg per dose, or in the range of 120 to 200 μg per dose, or in the range of 60 to 100 μg per dose. In some embodiments, the vaccine is administered to human adults in at least two doses, with a total protein content of the three fusion proteins ranging from 60 to 200 μg per dose, or more preferably from 120 to 200 μg per dose, or to human children in at least two doses, with a total protein content of the three fusion proteins ranging from 60 to 200 μg per dose, or from 120 to 200 μg per dose, or from 60 to 100 μg per dose.

[0028] The initial data demonstrate that vaccine A, for example, vaccine A at doses of the fusion protein with a total protein content of 135 μg and 180 μg per dose, was more effective than a dose of 90 μg (see Example 2). Therefore, the total protein content of the three fusion proteins is in the range of 120–200 μg per dose for human adults (adult dose). For children, half of the above dose, i.e., 60–100 μg (half dose), may be applied, but the adult dose, i.e., 120–200 μg (adult dose), may also be appropriate. In situations where a half dose is administered, human children are typically young children from birth to age 4 (0–4 years), 1–4 years, or 2–4 years. Older children (ages 5–17 years), including adolescents (ages 12–17 years), receive the adult dose.

[0029] Given the unique mode of action of this vaccine, which occurs outside the human body, the need for relatively high antibody titers throughout the tick season is paramount. Furthermore, since OspA exists only on the surface of Borrelia, and unless Borrelia resides in the tick's midgut and somehow invades the human host after antibody production, no natural boost to OspA-specific antibodies present during natural infection can be expected. Therefore, high antibody titers are required, and the composition will be administered at least three times, or alternatively, twice. Accordingly, the composition may be administered two, three, four, five times, or even more times.

[0030] The initial primary immunization is referred herein to as the 0-2-6 month immunization series. Further doses are booster doses for re-exposure to the vaccine and are typically applied every 12 months after the third immunization of the primary immunization (i.e., the first booster at 18 months). Alternatively, the initial primary immunization is referred herein to as the 0-6 month immunization series. Further doses are booster doses for re-exposure to the vaccine and are typically applied every 12 months after the second immunization of the primary immunization (i.e., the first booster at 18 months). The booster doses increase immunity and restore it to a protective level after the titer has decreased over time (see Example 1). In the case of compositions according to the present invention, it has been shown that boosters are required to maintain the high OspA antibody levels necessary to confer protection.

[0031] Booster doses are typically administered every 12 months, but they can also be administered within a few months of the 12-month interval after the final dose of primary immunization (e.g., at 9, 10, 11, 12, 13, 14, or 15 months), or after a subsequent booster dose (e.g., 9, 10, 11, 12, 13, 14, or 15 months after the first or subsequent booster dose).

[0032] The compositions according to the present invention may be administered to humans as injectable compositions, for example, preferably as isotonic, sterile aqueous dispersions. The compositions may be administered via systemic or mucosal routes. These administrations may include injection via intramuscular, intraperitoneal, intradermal, or subcutaneous routes; or via mucosal administration to the oral / gastrointestinal tract, respiratory tract, or urogenital tract. The vaccine of the present invention may be administered as a combination of three fusion proteins, but its components (i.e., individual fusion proteins) may also be administered separately, simultaneously or sequentially, or simply as a vaccine of a single component (i.e., simply Lip-S1D1-S2D1).

[0033] In a second aspect, the present invention provides a composition for use in a method for inducing an immune response in humans against Lyme Borreliosis, 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 method comprising administering the composition to a human at least three times, in the total protein content of the three fusion proteins within the range of 120-200 μg per dose for human adults or children, or half a dose, for example, in the case of human children (particularly young children aged 0-4 years, 1-4 years, or 2-4 years), in the total protein content of the three fusion proteins within the range of 60-100 μg per dose, for at least three times. Alternatively, the present invention provides a composition for use in a method for inducing an immune response in humans against Lyme borreliosis, 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 method comprising administering the composition to a human at least twice, in the total protein content of the three fusion proteins within the range of 120-200 μg per dose for human adults or children, or, in the case of human children (particularly young children aged 0-4 years, 1-4 years, or 2-4 years), for example, at least twice, in the total protein content of the three fusion proteins within the range of 60-100 μg per dose.

[0034] The above definitions and comments made in relation to the first aspect of the present invention also apply to the second aspect of the present invention.

[0035] In a second aspect, the composition is used in a method for inducing an immune response in humans against Lyme disease caused by pathogenic Borrelia species as disclosed herein, more preferably B. burgdorferi ss, B. afzeri, B. bavaliensis, and B. galini, and / or other pathogens whose antigens are contained in the composition. Preferably, the composition is for use in a method for inducing an immune response to infection with B. burgdorferi ss, B. galini, B. afzeri, B. andersoni, B. bavaliensis, B. bisetti, B. baraisiana, B. lusitaniae, B. spielmannii, B. japonica, B. tanukii, B. tsurudii, or B. sinica, preferably B. burgdorferi ss, B. afzeri, and / or B. galini.

[0036] Lyme borreliosis, or Lyme disease, is the most commonly reported tick-borne disease in Europe and North America. The disease is caused by infection with Borrelia burgdorferi (B. burgdorferi sl), a Gram-negative-like spirochete transmitted by arthropods, and can affect multiple organs or tissues, resulting in skin, cardiac, musculoskeletal, and neurological disorders. In most countries, Lyme borreliosis is not a notifiable disease; therefore, accurate data on annual incidence rates are not available. In the United States, the causative agent is B. burgdorferi (B. burgdorferi ss), and Lyme borreliosis is localized in the Northeastern, Mid-Atlantic, and North-Central-Northern states. In 2010, a total of approximately 30,000 cases of Lyme borreliosis were reported to the U.S. Centers for Disease Control and Prevention (CDC). A recent 2013 CDC report, taking diagnostic data from other sources, estimates the actual number of new cases per year in the United States to be closer to 300,000 (http: / / www.cdc.gov / media / releases / 2013 / p0819-lyme-disease.html). In Europe, B. afzeri and B. galini, along with B. burgdorferi ss and B. bavaliensis, contributing to a lesser extent depending on geographical location, are the main causative agents of lyme borreliosis. The prevalence of lyme borreliosis varies considerably across different European countries, with an overall increase from west to east. In much of Europe, the number of reported cases of lyme borreliosis has been increasing since the early 1990s (e.g., Czech Republic, Estonia, Lithuania; see the WHO report, Lyme borreliosis in Europe, 2006), and the geographical distribution of cases has also expanded.

[0037] As detailed above, high antibody titers after primary immunization and booster doses are required to maintain high OspA antibody levels. In particular, a broad primary immunization schedule (e.g., 0-2-6 months) has been found suitable in the context of the present invention (see Example 3). Alternatively, a two-dose primary immunization schedule (e.g., 0-6 months) may be used.

[0038] Therefore, the above composition for use according to the present invention is preferably characterized in that the second dose is administered at least 4 to 6 weeks to at most 3 months after the first dose, and / or the third dose is administered at least 5 months to at most 7 months after the first dose. According to this, the first dose is administered at time zero (day 1), the second dose is administered at least 6 weeks to at most 3 months thereafter, and the third dose is administered at least 5 months to at most 7 months after the first dose. Further boosters may be administered annually after the third vaccine dose (i.e., after the 6-month immunization), for example, at 12 months, 24 months, 36 months, etc. Alternatively, the second dose is administered at least 5 months to at most 7 months after the first dose. According to this, the first dose is administered at time zero (day 1), and the second dose is administered at least 5 months to at most 7 months after the first dose. Further boosters may be administered annually after the second vaccine dose (i.e., after the 6-month immunization), for example, at 12 months, 24 months, 36 months, etc. Therefore, compositions for use according to the present invention are preferably characterized in that further doses are administered annually after the third dose (or alternatively, the second dose in a 0-6 month two-dose primary immunization schedule), particularly at 1 year, 2 years, and 3 years, etc. The booster doses may generally be administered before the tick season, i.e., around February to June in the Northern Hemisphere and around September to December in the Southern Hemisphere.

[0039] More preferably, the composition for use according to the present invention is characterized in that the second dose is administered at least 40 days to at most 80 days after the first dose, particularly at least 50 days to at most 70 days, more particularly at least 50 days to at most 60 days, and especially 56 days.

[0040] Furthermore, more preferably, the composition for use according to the present invention is characterized in that the third dose is administered during a period of at least 120 days to at most 240 days after the first dose, during a period of at least 175 days to at most 185 days, and particularly during 180 days. Alternatively, the composition for use according to the present invention is characterized in that the second dose is administered during a period of at least 120 days to at most 240 days after the first dose, during a period of at least 175 days to at most 185 days, and particularly during 180 days.

[0041] In some cases, one or more additional doses may be required or desirable. Accordingly, a fourth dose of the composition (or alternatively, a third dose in a 0-6 month two-dose primary immunization schedule) may occur at least 15 months to at most 21 months after the first dose, particularly at least 17 months to at most 19 months, and especially at 18 months.

[0042] Preferably, the composition for use according to the present invention is characterized in that, for human adults (including those aged 18 to 65 years, aged 18 years or older), the total protein content of the three fusion proteins is in the range of 135 μg to 180 μg per dose, particularly 135 μg or 180 μg per dose. Doses of the three fusion proteins with total protein content of 135 μg and 180 μg are particularly suitable for providing sustained protection against all Borrelia OspA serotypes (see Examples 2 and 3). Thus, it can be concluded that the effect is present across the full range of total protein content of the three heterodimers, from 135 μg to 180 μg per dose. As detailed above, for young children (birth to 4 years), half the dose may be applied (although the full dose may also be suitable). Therefore, for human children (especially young children from birth to 4 years of age, e.g., 1 to 4 years or 2 to 4 years of age), the total protein content of the three fusion proteins is in the range of 67.5 μg to 90 μg per dose, particularly 67.5 μg or 90 μg per dose. For older children (ages 5 to 17 years), including adolescents (ages 12 to 17 years), the full dose of the three heterodimers with a total protein content of 135 μg to 180 μg per dose can be used.

[0043] Preferably, the compositions for use according to the present invention are characterized in that the three fusion proteins constitute at least 60%, preferably at least 70%, and more preferably 80% of all proteins in the composition. It will be apparent to those skilled in the art that the compositions for use according to the present invention may also include further proteins other than the fusion proteins of SEQ ID NO: 1, 2, and 3. However, the ratio of fusion proteins should not be lower than the above limits based on weight. The other proteins may have a function, for example, in stabilizing the composition, or they may be impurities.

[0044] In one embodiment, the pharmaceutical composition comprises three fusion proteins in a weight ratio of 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 Lip-S1D1-S2D1 (SEQ ID NO: 1), Lip-S4D1-S3hybD1 (SEQ ID NO: 2), and Lip-S5D1-S6D1 (SEQ ID NO: 3). Preferably, the composition for use according to the present invention is characterized in that the composition contains fusion proteins in a weight ratio of 1:1:1 (Lip-S1D1-S2D1 : Lip-S4D1-S3hybD1 : Lip-S5D1-S6D1).

[0045] Preferably, the compositions for use according to the present invention are characterized in that the composition comprises an adjuvant, more preferably an aluminum adjuvant.

[0046] The selection of suitable adjuvants to be mixed with bacterial proteins using 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 those of calcium, magnesium, iron, or zinc, or acylated tyrosine, or acylated sugars, cationic or anionic derivatized sugars, or insoluble suspensions of polyphosphazene. In a preferred embodiment, the composition is to which an aluminum adjuvant such as aluminum hydroxide is added as an adjuvant. In a more preferred embodiment, the amount of copper associated with the aluminum adjuvant is less than 1.25 ppb in the vaccine composition.

[0047] Most preferably, the composition for use according to the present invention has a total protein content of the three fusion proteins of 135 μg per dose for human subjects, particularly adults or children, or only 67.5 μg per dose for human children (particularly young children born to 4 years, 1 to 4 years, or 2 to 4 years), characterized in that the second dose is administered 56 days (2 months) after the first dose, the third dose is administered 180 days (6 months) after the first dose, and any fourth dose (first booster dose) is administered 18 months after the first dose.

[0048] Most preferably, the composition for use according to the present invention is characterized in that the total protein content of the three fusion proteins is 180 μg per dose for human subjects, particularly adults or children, or 90 μg per dose for human children (particularly young children born to 4 years, 1 to 4 years, or 2 to 4 years), the second dose is administered 56 days (2 months) after the first dose, the third dose is administered 180 days (6 months) after the first dose, and any fourth dose (first booster dose) is administered 18 months after the first dose.

[0049] Alternatively, the composition for use according to the present invention may have a total protein content of the three fusion proteins of 135 μg per dose for human subjects, particularly adults or children, or only 67.5 μg per dose for human children (particularly young children born to 4 years, 1 to 4 years, or 2 to 4 years), characterized in that the second dose is administered 180 days (6 months) after the first dose, and any third dose (first booster dose) is administered 18 months after the first dose.

[0050] Alternatively, the composition for use according to the present invention may have a total protein content of the three fusion proteins of 180 μg per dose for human subjects, particularly adults or children, or only 90 μg per dose for human children (particularly young children born to 4 years, 1 to 4 years, or 2 to 4 years), characterized in that the second dose is administered 180 days (6 months) after the first dose, and any third dose (first booster dose) is administered 18 months after the first dose.

[0051] Preferably, the compositions for use according to the present invention are characterized in that the induced immune response comprises an antibody response to anti-OspA serotype 1, anti-OspA serotype 2, anti-OspA serotype 3, anti-OspA serotype 5, and / or anti-OspA serotype 6 having bactericidal activity.

[0052] Preferably, the compositions for use according to the present invention are characterized in that the induced immune response comprises antibodies having bactericidal activity against spirochetes expressing OspA serotypes 1, 2, 3, 4, 5, and 6.

[0053] Preferably, the compositions for use according to the present invention are characterized by an immune response against OspA serotypes 1, 2, 3, 4, 5, and 6 that lasts for at least about 60 days, at least about 180 days, at least about 365 days, or at least about 540 days. The sustained immune response may be tested as described in Example 2.

[0054] In one embodiment, the composition or vaccine of the present invention further comprises at least one additional antigen (collectively referred to herein as the “combined vaccine”). In a preferred embodiment, at least one additional antigen is derived from a Borrelia species that causes Lyme borreliosis. In various aspects, at least one additional antigen is derived from another pathogen, preferably a tick-borne pathogen. In further contexts, the pathogen causes Rocky Mountain spotted fever, human granulocytic ehrlichiosis (HGE), glandular fever (Sennetsu Fever), human monocytic ehrlichiosis (HME), anaplasmosis, Boutonuse fever, Rickettsia parkeri rickettsial disease, Southern Tick-Associated Rash Illness (STARI), Helvetica Spotted Fever, 364D rickettsial disease, African spotted fever, relapsing fever, tularemia, Colorado tick-borne fever, tick-borne encephalitis (TBE; also known as FSME), Crimean-Congo hemorrhagic fever, Q fever, Omsk hemorrhagic fever, Kyasanur Forest disease, Poissant encephalitis, Heartland virus disease, or babesiosis. In further contexts, the disease is Japanese encephalitis.In a further embodiment, at least one additional antigen is transmitted by vector animals, preferably ticks, including Borrelia hermsii, Borrelia parkeri, Borrelia duttoni, Borrelia miyamotoi, Borrelia turicatae, Rickettsia rickettsii, Rickettsia australis, Rickettsia conori, Rickettsia helvetica, Francisella tularensis, Anaplasma phagocytophilum, and Ehrlichia sennets. The pathogens are derived from a group of pathogens including *Ehrlichia sennetsu*, *Ehrlichia chaffeensis*, *Neoehrlichia mikurensis*, *Coxiella burnetii*, and *Borrelia lonestari*, tick-borne encephalitis virus (TBEV, also known as FSME virus), Colorado tick-borne fever virus (CTFV), Crimean-Congo hemorrhagic fever virus (CCHFV), Omsk hemorrhagic fever virus (OHFV), Japanese encephalitis virus (JEV), and species of the genus Babesia.

[0055] In one embodiment, the composition for use according to the present invention provides use in older subjects having a similar immunogenicity profile to that in younger subjects, where the human adults are older subjects aged 50 years or older. The present invention surprisingly provides that older adults (age group 50–65 years) showed similar immune responses compared to younger adults (age group 18–49 years), with no statistically significant difference. See experimental section.

[0056] In one embodiment, the composition for use according to the present invention contains 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, especially copper, reduces the bioavailability of the OspA protein in vaccines. Without being bound by theory, it is assumed that the OspA protein binds to aluminum and heavy metals, especially copper, inhibiting their release and thus reducing the bioavailability of the OspA protein in vaccines. Therefore, the composition, and in particular the aluminum adjuvant, contains less than 1.25 ppb of copper. The unit ppb (parts per million) is often used in the field of mass spectrometry to quantify impurities. In the case of an aqueous solution, 1 ppb means that 1 ng of the substance (impurity) is present in 1 g of the solution, which means that 1 ppb is equal to 1 μg / l (assuming that 1 liter of solution has a weight of 1 kg). Typically, copper, especially Cu + or Cu 2+In this form, it is an ionic form. In a preferred embodiment, the composition contains less than 1.00 ppb, less than 0.75 ppb, or less than 0.50 ppb of copper, based on the weight of the aqueous composition. L-methionine is known to be able to bind to copper. The amount of L-methionine required will obviously depend 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 in compositions for use in the present invention to form a complex with excess copper. A typical suitable concentration of L-methionine in a composition is at least 10 mmol / l. Depending on the amount of copper in the composition, the concentration may be even 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 lower, for example, at most 10 mmol / l, at most 5 mmol / l, or at most 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 in mol / l units is at least equivalent to the concentration of copper in the composition. Alternatively, the concentration of L-methionine in mol / l units is at least 2, 3, 4, 5, or even 10 times the concentration of copper in the composition.

[0057] In one embodiment, the composition for use further comprises a reactive compound, the reactive compound selected from the group consisting of redox-active compounds, radical building compounds, stabilizing compounds, and any combination thereof. Without being bound by theory, the antigenic degradation of protein vaccines in aqueous compositions containing heavy metal ions present in aluminum salts such as aluminum hydroxide can be explained by an underlying degradation pathway that assumes free radicals, such as free radicals of sulfites. Oxidation catalyzed by heavy metals is a degradation pathway that results in covalent modification of proteins. Modified physicochemical properties of oxidized / modified proteins or antigens can result in a loss of biological activity. Redox-active compounds are suitable for inhibiting this modification. Preferably, the reactive compound is selected from the group consisting of formaldehyde, ethanol, chloroform, trichloroethylene, acetone, triton-X-100, deoxycholate, diethyl pyrocarbonate, sulfite, Na2S2O5, beta-proprio-lactone, polysorbate, e.g., Tween 20®, Tween 80®, O2, phenol, pluronic copolymers, and any combination thereof.

[0058] Further aspects of the present invention are as follows: 1. A method for vaccinating humans, A composition containing a fusion protein with SEQ ID NO: 1 (Lip-S1D1-S2D1), a fusion protein with SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and a fusion protein with SEQ ID NO: 3 (Lip-S5D1-S6D1) - In human adults, at least three times, with a total protein content of three fusion proteins ranging from 120 to 200 μg per dose; or - In human children, at least three doses, with a total protein content of three fusion proteins ranging from 120 to 200 μg per dose, or within the range of 60 to 100 μg per dose. A method including the step of administering an agent. 2. A method for inducing an immune response in humans against Lyme disease, A composition containing a fusion protein with SEQ ID NO: 1 (Lip-S1D1-S2D1), a fusion protein with SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and a fusion protein with SEQ ID NO: 3 (Lip-S5D1-S6D1) - In human adults, at least three times, with a total protein content of three fusion proteins ranging from 120 to 200 μg per dose; or - In human children, at least three doses, with a total protein content of three fusion proteins ranging from 120 to 200 μg per dose, or within the range of 60 to 100 μg per dose. A method including the step of administering an agent. 3. The method of embodiment 1 or embodiment 2, wherein the second dose is administered at least 6 weeks to at most 3 months after the first dose, and / or the third dose is administered at least 5 months to at most 7 months after the first dose. 4. Any method of aspects 1 to 3, wherein the second dose is administered at least 50 to at most 70 days after the first dose, particularly at least 55 to at most 60 days, especially at 56 days; and / or the third dose is administered at least 170 to at most 190 days after the first dose, particularly at least 175 to at most 185 days, especially at 180 days. 5. Any method according to embodiment 1 to 4, wherein the fourth dose of the composition is administered at a time of at least 15 months to at most 21 months after the first dose, particularly at a time of at least 17 months to at most 19 months, and especially at 18 months. 6. Any method according to embodiment 1 to 5, wherein the total protein content of the three fusion proteins is in the range of 135 μg to 180 μg per dose for adult or child human, particularly 135 μg or 180 μg per dose, or the total protein content of the three fusion proteins is in the range of 67.5 μg to 90 μg per dose for young child, particularly 67.5 μg or 90 μg per dose. 7. Any method according to embodiment 1 to 6, wherein the three fusion proteins constitute at least 60%, preferably at least 70%, and more preferably 80% of all proteins in the composition. 8. A method according to any one of embodiments 1 to 7, wherein the composition contains a fusion protein in a weight ratio of 1:1:1 (Lip-S1D1-S2D1 : Lip-S4D1-S3hybD1 : Lip-S5D1-S6D1). 9. A method according to any of embodiments 1 to 8, wherein the composition comprises an adjuvant. 10. A method according to any one of embodiments 1 to 9, wherein the composition comprises an aluminum adjuvant. 11. Any method according to embodiments 1 to 10, wherein the total protein content of the three fusion proteins is 135 μg per dose for adult or child humans, the second dose is administered 56 days after the first dose, the third dose is administered 180 days after the first dose, and an optional fourth dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 12. Any method according to embodiments 1 to 10, wherein the total protein content of the three fusion proteins is 67.5 μg per dose for young children, the second dose is administered 56 days after the first dose, the third dose is administered 180 days after the first dose, and an optional fourth dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 13. Any method according to embodiments 1 to 10, wherein the total protein content of the three fusion proteins is 180 μg per dose for adult or child humans, the second dose is administered 56 days after the first dose, the third dose is administered 180 days after the first dose, and an optional fourth dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 14. Any method according to embodiments 1 to 10, wherein the total protein content of the three fusion proteins is 90 μg per dose for young children, the second dose is administered 56 days after the first dose, the third dose is administered 180 days after the first dose, and an optional fourth dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 15. Any method according to embodiments 1 to 10, wherein the total protein content of the three fusion proteins is 135 μg per dose for adult or child humans, the second dose is administered 2 months after the first dose, the third dose is administered 6 months after the first dose, and an optional fourth dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 16. Any method according to embodiments 1 to 10, wherein the total protein content of the three fusion proteins is 67.5 μg per dose for young children, the second dose is administered 2 months after the first dose, the third dose is administered 6 months after the first dose, and an optional fourth dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 17. Any method according to embodiments 1 to 10, wherein the total protein content of the three fusion proteins is 180 μg per dose for adult or child humans, the second dose is administered 2 months after the first dose, the third dose is administered 6 months after the first dose, and an optional fourth dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 18. Any method according to embodiments 1 to 10, wherein the total protein content of the three fusion proteins is 90 μg per dose for young children, the second dose is administered 2 months after the first dose, the third dose is administered 6 months after the first dose, and an optional fourth dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 19. Any method of aspects 1 to 18, wherein further administration is annually after the last primary immunization, or at the beginning of each tick season, in particular after one year, two years, and three years. 20. Any method according to embodiment 1 to 19, wherein the induced immune response includes an antibody response to 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 having bactericidal activity. 21. Any method according to embodiment 1 to 20, wherein the induced immune response comprises an antibody having bactericidal activity against spirochetes expressing OspA serotypes 1, 2, 3, 4, 5, and 6. 22. Any method according to embodiment 1 to 21, wherein an immune response to OspA serotypes 1, 2, 3, 4, 5, and 6 is sustained for at least about 60 days, at least about 180 days, at least about 365 days, or at least about 540 days. 23. Any method according to embodiment 1 to 22, wherein the composition contains less than 1.25 ppb of copper or a sufficient amount of L-methionine. 24. Copper, especially Cu + or Cu 2+ A method in which the form of an ion is as described in any of embodiments 1 to 23. 25. Any method according to embodiment 1 to 24, wherein L-methionine is present at a concentration of at least 10 mmol / l. 26. Any method according to embodiment 23 to 25, wherein the concentration of L-methionine in mol / l units is at least equivalent to the concentration of copper in the composition. 27. Any method according to Embodiments 1 to 26, wherein the composition further comprises a reactive compound, the reactive compound being selected from the group consisting of redox-active compounds, radical constituent compounds, stabilizing compounds, and any combination thereof, in particular, the reactive compound being selected from the group consisting of formaldehyde, ethanol, chloroform, trichloroethylene, acetone, triton-X-100, deoxycholate, diethyl pyrocarbonate, sulfite, Na2S2O5, β-proplio-lactone, polysorbate, e.g., Tween 20®, Tween 80®, O2, phenol, pluronic copolymers, and any combination thereof. 28. Any method according to aspects 1 to 27, wherein the subject is a human adult aged 18 or older, for example, between 18 and 65 years of age. 29. Any method according to aspects 1 to 27, wherein the subject is an elderly human adult who is 50 years of age or older. 30. Any method according to aspects 1 to 27, wherein the subject is a human child aged 5 to 17 years, for example, 12 to 17 years or 5 to 11 years. 31. Any method according to aspects 1 to 27, wherein the subject is a human child aged 0 to 4 years, for example, 1 to 4 years or 2 to 4 years. 32. Any method according to Embodiments 1 to 31, wherein the composition is administered to a human adult or child in a volume of 0.25 ml to 1.0 ml, for example, 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. 33. Any method according to embodiment 1 to 32, wherein the composition is administered to a human adult or child in a volume of 0.25 ml, 0.5 ml, or 1.0 ml. 34. A method for vaccinating humans, A composition containing a fusion protein with SEQ ID NO: 1 (Lip-S1D1-S2D1), a fusion protein with SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and a fusion protein with SEQ ID NO: 3 (Lip-S5D1-S6D1) - In human adults, at least twice, with a total protein content of three fusion proteins in the range of 120-200 μg per dose; or - In human children, at least twice, with a total protein content of three fusion proteins in the range of 120-200 μg per dose or 60-100 μg per dose. A method including the step of administering an agent. 35. A method for inducing an immune response in humans against Lyme disease, A composition containing a fusion protein with SEQ ID NO: 1 (Lip-S1D1-S2D1), a fusion protein with SEQ ID NO: 2 (Lip-S4D1-S3hybD1), and a fusion protein with SEQ ID NO: 3 (Lip-S5D1-S6D1) - In human adults, at least twice, with a total protein content of three fusion proteins in the range of 120-200 μg per dose; or - In human children, at least twice, with a total protein content of three fusion proteins in the range of 120-200 μg per dose or 60-100 μg per dose. A method including the step of administering an agent. 36. The method of embodiment 34 or embodiment 35, wherein the second dose is administered at a period of at least 5 months to at most 7 months after the first dose. 37. Any method according to aspects 34 to 36, wherein the second dose is administered at least 170 days to at most 190 days after the first dose, at least 175 days to at most 185 days, and particularly at 180 days. 38. Any method according to embodiments 34 to 37, wherein the third administration of the composition occurs at a period of at least 15 months to at most 21 months after the first administration, particularly at a period of at least 17 months to at most 19 months, and especially at 18 months. 39. Any method according to embodiments 34 to 38, wherein the total protein content of the three fusion proteins is in the range of 135 μg to 180 μg per dose, particularly 135 μg or 180 μg per dose, or the total protein content of the three fusion proteins is in the range of 67.5 μg to 90 μg per dose for young children, particularly 67.5 μg or 90 μg per dose. 40. Any method according to embodiments 34 to 39, wherein the three fusion proteins constitute at least 60%, preferably at least 70%, and more preferably at least 80% of all proteins in the composition. 41. A method according to any one of embodiments 34 to 40, wherein the composition contains a fusion protein in a weight ratio of 1:1:1 (Lip-S1D1-S2D1 : Lip-S4D1-S3hybD1 : Lip-S5D1-S6D1). 42. Any method according to embodiment 34 to 41, wherein the composition comprises an adjuvant. 43. Any method according to embodiment 34 to 42, wherein the composition comprises an aluminum adjuvant. 44. Any method according to embodiments 34 to 43, wherein the total protein content of the three fusion proteins is 135 μg per dose for adult or child humans, the second dose is administered 180 days after the first dose, and an optional third dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 45. Any method according to embodiments 34 to 43, wherein the total protein content of the three fusion proteins is 67.5 μg per dose for young children, the second dose is administered 180 days after the first dose, and an optional third dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 46. ​​Any method according to embodiments 34 to 43, wherein the total protein content of the three fusion proteins is 180 μg per dose for adult or child humans, the second dose is administered 180 days after the first dose, and an optional third dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 47. Any method according to embodiments 34 to 43, wherein the total protein content of the three fusion proteins is 90 μg per dose for young children, the second dose is administered 180 days after the first dose, and an optional third dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 48. Any method according to embodiments 34 to 43, wherein the total protein content of the three fusion proteins is 135 μg per dose for adult or child humans, the second dose is administered 6 months after the first dose, and an optional third dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 49. Any method according to embodiments 34 to 43, wherein the total protein content of the three fusion proteins is 67.5 μg per dose for young children, the second dose is administered 6 months after the first dose, and an optional third dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 50. Any method according to embodiments 34 to 43, wherein the total protein content of the three fusion proteins is 180 μg per dose for adult or child humans, the second dose is administered 6 months after the first dose, and an optional third dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 51. Any method according to embodiments 34 to 43, wherein the total protein content of the three fusion proteins is 90 μg per dose for young children, the second dose is administered 6 months after the first dose, and an optional third dose is administered 18 months after the first dose, and optionally every 12 months thereafter. 52. Any method of aspects 34 to 51, wherein further administration is annually after the last primary immunization, or at the beginning of each tick season, in particular after one year, two years, and three years. 53. Any method according to embodiment 34 to 52, wherein the induced immune response includes an antibody response to 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 having bactericidal activity. 54. Any method according to embodiment 34 to 53, wherein the induced immune response comprises an antibody having bactericidal activity against spirochetes expressing OspA serotypes 1, 2, 3, 4, 5, and 6. 55. A method according to any one of embodiments 34 to 54, wherein an immune response to OspA serotypes 1, 2, 3, 4, 5, and 6 is sustained for at least about 60 days, at least about 180 days, at least about 365 days, or at least about 540 days. 56. Any method according to embodiment 34 to 55, wherein the composition contains less than 1.25 ppb of copper or a sufficient amount of L-methionine. 57. Copper, especially Cu + or Cu 2+ A method in which the form of an ion is as described in any of embodiments 34 to 56. 58. Any method according to embodiment 34 to 57, wherein L-methionine is present at a concentration of at least 10 mmol / l. 59. Any method according to embodiments 56 to 58, wherein the concentration of L-methionine in mol / l units is at least equivalent to the concentration of copper in the composition. 60. The method according to any one of embodiments 34 to 59, wherein the composition further comprises a reactive compound, the reactive compound being selected from the group consisting of redox-active compounds, radical constituent compounds, stabilizing compounds, and any combination thereof, in particular, the reactive compound being selected from the group consisting of formaldehyde, ethanol, chloroform, trichloroethylene, acetone, triton-X-100, deoxycholate, diethyl pyrocarbonate, sulfite, Na2S2O5, β-proplio-lactone, polysorbate, e.g., Tween 20®, Tween 80®, O2, phenol, pluronic copolymers, and any combination thereof. 61. Any method according to aspects 34 to 60, wherein the subject is a human adult aged 18 years or older, for example, between 18 and 65 years. 62. Any method according to aspects 34 to 60, wherein the subject is an elderly human adult who is 50 years of age or older. 63. Any method according to aspects 34 to 60, wherein the subject is a human child aged 5 to 17 years, for example, 12 to 17 years or 5 to 11 years. 64. Any method according to aspects 34 to 60, wherein the subject is a human child aged 0 to 4 years, for example, 1 to 4 years or 2 to 4 years. 65. Any method according to embodiments 34 to 64, wherein the composition is administered to a human adult or child in a volume of 0.25 ml to 1.0 ml, for example, 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. 66. Any method according to embodiment 34 to 65, wherein the composition is administered to a human adult or child in a volume of 0.25 ml, 0.5 ml, or 1.0 ml.

[0059] The inventors intend that the terms “comprising,” “comprise,” and “comprises” as used herein are optionally interchangeable with the terms “consisting of,” “consist of,” and “consists of,” respectively, in all cases. The term “comprises” means “includes.” Therefore, unless otherwise required by context, the word “comprises,” and variations such as “comprise” and “comprising,” are understood to mean the inclusion of the compound or composition (e.g., nucleic acids, polypeptides, antibodies) or process, or group of compounds or processes, but not the exclusion of any other compound, composition, process, or group thereof. The abbreviation “eg” is derived from the Latin “exempli gratia” (for example) and is used herein to indicate a non-limiting example. Therefore, the abbreviation “eg” is synonymous with the term “for example.”

[0060] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure pertains. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0061] The singular terms “a,” “an,” and “the” include multiple referents unless otherwise explicitly stated in the context. Similarly, the word “or” is intended to include “and” unless otherwise explicitly stated in the context. The term “plural” refers to two or more. Furthermore, it should be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for illustrative purposes only. In addition, numerical limits given for the concentration or level of substances such as antigens may be approximate.

[0062] The present invention is further illustrated by the following figures, tables, and examples from which further features, embodiments, and advantages may be obtained. Therefore, the specific modifications discussed should not be construed as limitations on the scope of the invention. It will be apparent to those skilled in the art that various equivalents can be made, various modifications can be made, and various alterations can be made without departing from the scope of the invention, and therefore it will be understood that such equivalent embodiments should be included herein. [Examples]

[0063] material and method The tick attack method is carried out by: a) applying at least one tick nymph (e.g., Ilex ricinus) infected with Borrelia (e.g., B. afzeri IS1 strain) to a mouse immunized with the polyvalent Borrelia vaccine of the present invention or pre-treated with human serum; b) applying at least one infected tick nymph to a second mouse treated with a suitable placebo (negative) control such as buffer or adjuvant alone; and c) generally comparing the infection rates in the two mice 4 to 6 weeks after the attack. Preferably, a group of mice (e.g., 5 to 10 mice / group) is used for each treatment. The infection status can be assessed using VlsE ELISA on serum and / or qPCR on collected tissue, as described in WO2014006226, or by other suitable methods.

[0064] Needle attack method: The needle attack method is used to inject Borrelia into a desired Borrelia strain (e.g., B. burgdorferi N40 strain) at an infectious dose (ID). 50 This is carried out by subcutaneously introducing the multivalent Borrelia vaccine of the present invention at a dose 20 to 50 times greater than that of mice immunized with the present invention, pre-treated with human serum, or pre-treated with an appropriate placebo (negative) control such as buffer or adjuvant alone, and by comparing the infection rate in the attacked mice. The ID measured in numerous bacteria is also used. 50The attack dose is defined as the dose at which 50% of untreated, attacked mice become infected. The attack dose can vary widely and is strain-dependent; therefore, the toxicity of the strain must be identified first. 50 The determination must be evaluated by attack experiments. Four weeks after the needle attack, blood and tissue samples are collected for a reading method to determine the infection status. The reading method may be, for example, VlsE ELISA on serum and / or qPCR on collected tissue for Borrelia identification, as described in WO2014006226, or other methods.

[0065] Spirochete antibody binding assay 1 × 10 5 ~1 × 10 6Mix the spirochetes with an equal volume of 4% paraformaldehyde and incubate in a 96-well plate (Nunclon 96U, Nunc) at room temperature for 20 minutes. Centrifuge the plate at 2,000 g for 5 minutes and discard the supernatant. Wash the cells with Hanks equilibrium salt solution (HBSS-B) containing 150 μL of 2% BSA, centrifuge as described above, and discard the supernatant. Inactivate the serum by incubating at 56°C for 35 minutes. Dilute the thermoactivated serum in HBSS-B and filter by sterile centrifugation at 17,000 g for 3 minutes using a Costar spin-X centrifuge tube filter (0.22 μm, Corning, USA). Suspend the spirochetes in 100 μL of serum and incubate at room temperature for 45 minutes. Centrifuge the plate at 2,000 g for 5 minutes and discard the supernatant. Wash the cells once with 150 μL of HBSS-B and resuspend them in 100 μL of HBSS-B containing a 1:150 dilution of PE conjugate secondary antibody. Add 1 microliter of a suitable secondary antibody (e.g., PE conjugate anti-mouse or anti-human IgG available from Beckman Coulter, USA) to the cells and incubate in the dark at room temperature for 45 minutes. Wash the spirochetes once with 150 μL of HBSS-B and resuspend them in 200 μL of HBSS containing 10 ug of LDS 751 DNA dye and incubate in the dark at room temperature for 10 minutes. Pellet the stained spirochetes by centrifugation at 2,000 g for 5 minutes and then resuspend them in 200 μL of HBSS. Gate the labeled spirochetes to an LDS 751 positive event and measure them using a CytoFlex (Beckman Coulter) flow cytometer.

[0066] The serum bactericidal assay (SBA) measures the ability of vaccine-induced antibodies to kill bacteria in combination with exogenously added complement (Figure 1). Vaccine-induced antibodies recognize antigens exposed on the surface of spirochetes and recruit complement, activated primarily via the classical pathway, resulting in the formation of membrane attack complexes, lysis, and ultimately the death of the target bacteria. Serum samples are serially diluted 2-fold in BSK-II medium and then mixed in BSK-II medium in a white 96-well assay plate with Borrelia obtained from an overnight culture and guinea pig or baby rabbit complement (source and final concentration depend on serotype, 1–25%). The plate is then incubated at +32°C and 5% CO2 for 3–6 days (depending on Borrelia serotype). The amount of live (metabolically active) Borrelia is determined at the end of the assay by measuring ATP-dependent luminescence using a luciferase-based reaction (BacTiter-Glo, Promega). A human serum pool serves as the positive quality control (QC), and commercially available naive human serum serves as the negative control (NC). To calculate the sample titer, each sample dilution is compared to the corresponding dilution of negative human serum (NC) with a relative light unit set to 100%. The SBA titer of the test sample is the reciprocal of the highest serum dilution that induces a survival rate of 50% or less. For serotypes 1, 2, 4, 5, and 6, values ​​below the limit of quantification of the SBA (titer 20) are replaced by a titer of 10. For serotype 3, values ​​below the limit of quantification of the SBA (titer 160) are replaced by a titer of 80.

[0067] OspA serotype-specific human serum IgG enzyme-conjugated immunosorbent assay (ELISA) The geometric mean titer of IgG for OspA serotype-specific antibodies was determined by ELISA. Plates were coated with full-length lipid-added OspA derived from ST1, ST2, ST3, ST4, ST5, or ST6. Serial dilutions of test serum, reference material (RS), and QC samples were prepared from a human serum pool. A blank control was also used. Bound antibodies were detected with a human IgG-specific secondary antibody conjugate. OspA-specific IgG concentrations were calculated using SoftMax Pro 5.2 GxP software. Parallel line analysis curves were created individually for each sample tested against the reference material. The relative potency given in these curves was used to calculate the IgG concentration (U / mL) of the sample compared to the reference material concentration curve. Modifications were made to the ELISA assay from Phase 1 to Phase 2 (see Table A-1). The limit of quantification of the ELISA used in the Phase 1 study (Example 1) was 20 ELISA units (EU) / mL (also referred to herein as U / mL); values ​​less than 20 EU / mL were replaced with values ​​of 10 EU / mL. The slightly modified ELISA used in the first Phase 2 study (Example 2) had a limit of quantification of 40 EU / mL; values ​​less than 40 EU / mL were replaced with values ​​of 20 EU / mL.

[0068] (Table A-1) Differences in IgG measurement (ELISA) between Phase 1 and Phase 2 clinical trials TIFF0007887224000009.tif85143

[0069] Example 1. Phase 1 clinical trial using a polyvalent Borrelia vaccine. Phase 1 clinical trial design and immunogenicity results To investigate the safety and immunogenicity of the multimerized borrelia vaccine ("MBV") of the present invention at various doses, with or without aluminum hydroxide, a first-in-human, observer-blinded, partially randomized, multicenter, dose-escalation Phase I clinical trial was initiated. The trial was conducted in healthy adults aged 18–39 years who were baseline seronegative for Borrelia burgdorferi in the broad sense (i.e., subjects who had not been previously infected with borrelia). The trial design is shown in Figure 2. Briefly, the safety characteristics and immunogenicity of three intramuscular vaccine doses delivered at 28-day intervals were evaluated in 179 healthy subjects using six different treatments (three dose levels, each with or without adjuvant) (i.e., approximately 30 subjects per treatment group). All formulations contained each of the OspA fragment polypeptides (SEQ ID NO: 1, 2, and 3) in a 1:1:1 weight ratio (for example, the low dose (12 μg) contained 4 μg each of the lipid-added polypeptides defined by SEQ ID NO: 1, 2, and 3).

[0070] Phase 1 study dose group (IM vaccination, d0, d28, d56) - 12 μg, with alum (n=29) - 12 μg, no arum (n=29) - 48 μg, with arum (n=31) - 48 μg, without arum (n=29) - 90 μg, with arum (n=31) - 90 μg, without arum (n=30)

[0071] Immunogenicity was assessed in all groups on days 0, 28, 57, 84, 180, 236, and 365, with peak antibody titer expected on day 84 (i.e., 4 weeks after the third vaccination, primary endpoint analysis). Geometric mean titers for each OspA serotype on day 84 are shown in Figure 3A. Figure 3B shows the seroconversion rate (i.e., the proportion of subjects that seroconverted) for each group, where seroconversion is defined as a 4-fold or greater increase in IgG titer from baseline.

[0072] Based on the results from the analysis at day 84 of the booster extension phase, participants were selected to receive booster vaccinations at approximately 13 months, with or without adjuvant doses of 48 μg and 90 μg, and were followed for a further 6 months for safety and antibody persistence. The administered booster dose was the same as the priming dose and formulation. The booster extension portion of this study was conducted at only one of the three study sites, resulting in a substantially lower participation size of N=64 in the booster phase.

[0073] Phase 1 trial booster extended dose group (IM Boost, 13 months) - 48 μg, with aram (n=15) - 48 μg, without arum (n=16) - 90 μg, with aram (n=16) - 90 μg, without arum (n=17)

[0074] Immunogenicity was assessed in the booster extension group at 13 months (pre-boost), 14 months, and 19 months. The purpose of booster extension was to investigate the safety and immunogenicity of the booster dose of vaccine A, which is applied approximately one year after the last immunization in the primary schedule, up to 6 months after the application of the booster dose. Another use of booster extension was to generate high-titer serum for the development of functional assays and potential proof-of-concept (PoC) studies. Furthermore, the booster enabled the collection of safety and immunogenicity data of the booster dose earlier in clinical development.

[0075] The IgG GMT values ​​for subjects included in the primary analysis of the booster extension phases at day 84 and at 13, 14, and 19 months (booster PP population) are shown in Figures 4A-D and Table 1 below, respectively. The geometric mean rate of increase in IgG titer (GMFR), compared to the pre-booster point in time (13 months) and compared to the peak titer after the primary vaccination series (day 84), as well as the seroconversion rates at day 84, 13 months, and 14 months, are provided in Tables 2 and 3, respectively. For group comparisons of immune responses to each of the six serotypes over the entire duration of the study, the GMT values ​​over the duration of the study for subjects included in the booster extension phase (booster PP population, i.e., subjects enrolled in the booster extension phase and receiving the correct booster dose at the correct timing according to the protocol) are provided in Figures 5A-F.

[0076] (Table 1) Day 84, GMT of serotype-specific IgG titers for M13, M14, and M19, booster PP population TIFF0007887224000010.tif23665

[0077] (Table 2) Geometric mean increase in serotype-specific IgG titers (GMFR) at 14 months and 19 months compared to 13 months (before boost) and 84 days (peak antibody titer after primary immunization series), booster PP population TIFF0007887224000011.tif23673

[0078] (Table 3) Seroconversion rates (SCRs) by serotype for M13, M14, and M19 on day 84, and booster PP population. TIFF0007887224000012.tif25562

[0079] Correlation between ELISA titers and SBA titers in Phase 1 serum samples: Serotype-specific serum bactericidal assays (SBA - see Materials and Methods), which are in vitro correlations of protection for clinical samples, yielded values ​​that closely correlated with ELISA values ​​for serotypes 1, 2, and 3 in individual target serums, as shown in Figures 6A, B, and C, respectively.

[0080] In summary, serotype-specific IgG antibodies were induced at all doses and formulations investigated in Phase 1, with higher immunogenicity observed in higher-dose groups and adjuvant-containing formulations. Booster doses applied approximately one year after completion of the primary vaccination series resulted in a substantial booster response for all six OspA serotypes (i.e., IgG titers were substantially higher 4 weeks after booster dose compared to 4 weeks after completion of primary immunization) and persisted at higher levels up to 19 months (Table 3). Because sustained circulating antibody levels are crucial to the efficacy of OspA-based vaccines, further dose increases and alternative schedules were introduced in Phase 2 with the aim of inducing earlier, higher, and more persistent immune responses.

[0081] Example 2. Phase 2 clinical trial using a polyvalent Borrelia vaccine to investigate higher doses of the vaccine. Design of the Phase 2 clinical trial and summary of preliminary described immunogenicity data from the introduction phase. A follower-blinded, randomized, placebo-controlled, multicenter phase 2 trial is currently underway to investigate the safety and immunogenicity of a higher dose range of multimerized borrelia vaccine with aluminum hydroxide adjuvant in healthy adults aged 18–65 years. Participants enrolled in this trial included those who were baseline seronegative for Borrelia burgdorferi (i.e., those who had not previously been infected with borrelia) and those who were baseline seropositive for Borrelia burgdorferi (i.e., those who had previously been infected with borrelia). The study consisted of a safety introduction phase (subjects aged 18–40 years, N=120) to first investigate the safety of three dose levels of the multimerized borrelia vaccine ("MBV") of the present invention at 90 μg, 135 μg, and 180 μg with and without arum in a smaller population before initiating the main study phase (subjects aged 18–65 years, N=450) in two higher dose groups of 135 μg and 180 μg (see Figure 7). Subjects received three doses of IM vaccine at one-month intervals (i.e., 0-1-2 months). In the main study phase, subjects were enrolled in two age groups (18–49 years and 50–65 years) in a ratio of approximately 2:1. The primary objectives of the Phase 2 study were to evaluate the immunogenicity and safety of the multimerized borrelia vaccine at higher dose levels compared to the first-in-human study in healthy adults aged 18–65 years, and to determine the optimal vaccine dose. All formulations contained each of the OspA fragment polypeptides (SEQ ID NO: 1, 2, and 3) in a 1:1:1 weight ratio (for example, the low dose (90 μg) contained 30 μg each of the lipid-added polypeptides defined by SEQ ID NO: 1, 2, and 3, and the high dose (180 μg) contained 60 μg each of the lipid-added polypeptides defined by SEQ ID NO: 1, 2, and 3).

[0082] In the introductory phase, a total of 120 healthy subjects aged 18–40 years were enrolled in the following four treatment groups (IM vaccination, d1, d29, d57): 90 μg of the present invention's multimerized Borrelia vaccine ("MBV") with arum, 135 μg of MBV with arum, 180 μg of MBV with arum, and placebo (PBS), with approximately 30 subjects per treatment group. Following review of safety data up to day 85 by the Data Safety Monitoring Board (DSMB), these two higher-dose groups (i.e., 135 μg and 180 μg) were selected for further investigation in the main study phase. Serum was collected on days 1, 29, 57, 85 (primary immunogenicity analysis), 180, 236, and 365 and evaluated for immunogenicity by ELISA and SBA (at selected time points in a representative subset of subjects).

[0083] A preliminary descriptive analysis of immunogenicity data from the induction phase revealed that the multivalent Borrelia vaccine was immunogenic at all dose levels tested during the induction phase. Preliminary GMT titers (ELISA) in the 90 μg group with arum were comparable to those obtained in the Phase 1 trial at the same dose level. A dose-response was observed for all serotypes, with the lowest IgG titer in the 90 μg dose group and the highest in the 180 μg dose group. As expected, the increase in dose compared to Phase 1 resulted in an increased peak immune response after primary immunization. However, antibody levels decreased relatively in all dose groups until effectively day 180. Given the decrease in antibody titers observed after the primary series in the Phase 2 induction phase, further development of the multivalent Borrelia vaccine should consider a broader 0-2-6 month schedule. Based on experience with other vaccines, this is expected to result in even higher peak titers and, importantly, better persistence, and may also improve antibody quality.

[0084] In the primary phase, a total of 450 healthy subjects aged 18–65 years were enrolled in the following three treatment groups (IM vaccination, D1, d29, d57): 135 μg with alam (n=approx. 180), 180 μg with alam (n=approx. 180), and placebo (PBS) (n=approx. 90). Initial data analysis was performed, including pooled immunogenicity data (GMT of OspA serotype-specific IgG by ELISA) from day 85 of the primary and induction phases. Antibody functionality at day 85 was evaluated using a serum bactericidal assay (SBA). The data confirmed that preliminary data analysis of induction phase data regarding higher antibody titers, i.e., higher antibody levels after completion of the primary immunization series, was achieved at day 85 in the 135 μg dose group (GMT range of 101.1 [ST1] to 282.2 [ST3]) and the 180 μg dose group (GMT range of 115.8 [ST1] to 308.6 [ST3]) compared to the 90 μg dose group (GMT range of 74.3 [ST1] to 267.4 [ST3]) investigated during the induction phase. The data also revealed that older adults (age group 50-65 years), one of the high-risk groups for Lyme disease, also showed a promising immune response with no statistically significant difference between the two age groups. See Table 4 for a summary of immunogenicity data.

[0085] Furthermore, the Borrelia vaccine as described above (OspA fragment polypeptides (SEQ ID NO: 1, 2, and 3) in a 1:1:1 weight ratio (for example, the low dose (90 μg) contains 30 μg each of the lipid-added polypeptides defined by SEQ ID NO: 1, 2, and 3, and the high dose (180 μg) contains SEQ ID NO: The vaccine (containing 60 μg each of the lipid-added polypeptides defined by 1, 2, and 3) was immunogenic across all dose groups tested. Higher doses used in this study induced higher antibody responses across all serotypes compared to Phase 1. Seroconversion rates (SCRs) at the highest dose ranged from 81.5% (ST1) to 95.8% (ST2). In age groups comparable to those investigated in Phase 1 (18–49 years), SCRs ranged from 85.6% to 97% (previous study: 71.4%–96.4%). Immunological responses in older adults, one of the primary target groups for the Lyme vaccine, are particularly promising. The results did not indicate that prior exposure to Lyme (seropositive) had any impact on immunogenicity or safety.

[0086] The Borrelia vaccine was generally safe across all doses and age groups tested. No related serious adverse events (SAEs) were observed with the Borrelia vaccine in any treatment group in this study. Reactiveness decreased with subsequent vaccination.

[0087] Overall, the tolerability profile, including the rate of fever, appeared to be comparable to other lipid-added recombinant vaccines or lipid-containing formulations.

[0088] (Table 4) GMT and PP population at day 85 of the first Phase 2 trial (0-1-2 month schedule) TIFF0007887224000013.tif96160

[0089] Table 12 shows the seroconversion rate (SCR) on day 85. At the 90 μg dose, the SCR ranged from 65.4% (ST1) to 96.2% (ST3); at the 135 μg dose, the SCR ranged from 80.9% (ST1) to 98.4% (ST2); and at the 180 μg group, the SCR ranged from 81.5% (ST1) to 95.8% (ST3).

[0090] (Table 12) SCR (ELISA) and PP population at day 85 of the first Phase 2 trial (0-1-2 month schedule) TIFF0007887224000014.tif70128

[0091] The functionality of the antibodies has been demonstrated for all serotypes using a serum bactericidal assay (see SBA, materials and methods). The GMT at day 85 is summarized in Table 13.

[0092] (Table 13) GMT (0-1-2 month schedule) for functional antibodies measured by SBA on day 85 TIFF0007887224000015.tif67128

[0093] As shown in Figures 9A-9F, ELISA titers were significantly correlated with their respective SBA titers for each OspA serotype and individual target serum samples. Spearman correlation coefficients ranged from 0.5737 (p<0.0001) for serotype 4 to 0.9030 (p<0.0001) for serotype 3.

[0094] Example 3. Phase 2 clinical trial using a polyvalent Borrelia vaccine to investigate higher doses of vaccine and broader immunization schedules. Overall, this study enrolled 246 healthy subjects aged 18–65 years in an observer-blinded, randomized, placebo-controlled, multicenter phase 2 trial investigating the same dose groups tested in Example 2 with an alternative, broader immunotherapy schedule. Participants received three doses of IM immunization at 0, 2, and 6 months (1-57-180 days) with either a 135 μg or 180 μg dose of a multimerized Borrelia vaccine formulated with Alam (approximately 100 participants each) or a placebo (approximately 50 participants) (i.e., OspA fragment polypeptides (SEQ ID NO: 1, 2, and 3) in a 1:1:1 weight ratio (e.g., the low dose (135 μg) contained 45 μg each of the lipid-added polypeptides defined by SEQ ID NO: 1, 2, and 3, and the high dose (180 μg) contained 60 μg each of the lipid-added polypeptides defined by SEQ ID NO: 1, 2, and 3)). Participants were enrolled in two age groups (18-49 years and 50-65 years) in an approximately 2:1 ratio. In addition, subjects who were baseline seronegative for Borrelia burgdorferi in the broad sense (i.e., subjects who had not been previously infected with Borrelia) and subjects who were baseline seropositive for Borrelia burgdorferi in the broad sense (i.e., subjects who had been previously infected with Borrelia) were enrolled in this study. The primary objective of the Phase 2 study is to evaluate the immunogenicity and safety of the multimerized Borrelia vaccine in healthy adults aged 18–65 years at higher dose levels compared to the first-in-human study and with a broader immunization schedule than that used in the above examples. The data, along with the results obtained in Example 2, will be used to determine the optimal dose and schedule of the vaccine for late-stage clinical development. The study design is shown in Figure 8.

[0095] Serum samples were collected on days 1, 29, 57, 85, 180, 208 (primary immunogenicity analysis), 365, and 545, and immunogenicity was assessed by ELISA and SBA (at selected time points). On day 85 (i.e., after the second vaccination), antibody levels (GMT) ranged from 64.1 [ST1] to 166.4 [ST3] in the 135 μg dose group and from 75.2 [ST1] to 217.7 [ST3] in the 180 μg dose group. Table 10 provides a summary of geometric mean titers (GMT) for the second phase 2 trial, and Table 11 and Figure 10 provide a summary of seroconversion rates at day 85 for the second phase 2 trial. See also Figures 12–17.

[0096] Overall, the onset of the immune response appeared somewhat faster with the higher 180 μg dose; specifically, the GMT at day 85 was higher in the 180 μg dose group compared to the 135 μg group. See Figures 18A and 18B for a comparison of GMT between the 135 μg and 180 μg treatment groups, and Table 10 for GMT values ​​at day 85.

[0097] (Table 10) GMT and PP population at day 85 of the second Phase 2 trial (0-2-6 month schedule) TIFF0007887224000016.tif119160

[0098] (Table 11) SCR and PP population at day 85 of the second Phase 2 trial (0-2-6 month schedule) TIFF0007887224000017.tif148160

[0099] Initial data analysis, including immunogenicity and safety data at day 208 (i.e., one month after the third vaccination), revealed that antibody titers increased further using a broader immunization schedule (0-2-6 month schedule). Peak antibody levels (GMT) at day 208 ranged from 276.4[ST1] to 539.0[ST2] in the 135 μg dose group and from 274.7[ST1] to 596.8[ST3] in the 180 μg dose group (see Table 5), representing a 1.7 to 2.7-fold increase in titer compared to the same treatment groups using the 0-1-2 month vaccination schedule (see Table 6). Consistent with ELISA data, GMT for SBA titers also increased 1.4 to 3.1 times using the 0-2-6 month schedule compared to the 0-1-2 month schedule (Table 14). Table 5 provides an overview of the geometric mean titer (GMT) for the second phase 2 trial, and Table 6 compares the GMT using different vaccination schedules.

[0100] (Table 5) GMT (ELISA) at day 208 of the second Phase 2 trial (0-2-6 month schedule), PP population TIFF0007887224000018.tif80160

[0101] As shown in Table 5, the immunological response in older adults, one of the main target groups for the Lyme vaccine, is particularly promising. Furthermore, the results did not indicate that prior exposure to Lyme (serologically positive) had any impact on immunogenicity or safety (data not shown).

[0102] (Table 6) Comparison of GMT (ELISA) using different immunotherapy schedules TIFF0007887224000019.tif81160

[0103] As in the first phase 2 trial, older adults (age group 50-65 years), one of the high-risk groups for Lyme disease, also showed a promising immune response with no statistically significant difference in GMT between the two age groups (Table 5).

[0104] The seroconversion rate (SCR) on day 208 is shown in Table 7 and Figure 11. In the 135 μg dose group, the SCR ranged from 89.6% (ST6) to 98.7% (ST3), while in the 180 μg group, the SCR ranged from 93.8% (ST1) to 98.8% (ST2, ST4).

[0105] (Table 7) SCR (ELISA) and PP population at day 208 of the second Phase 2 trial (0-2-6 month schedule) TIFF0007887224000020.tif69128

[0106] The antibody functionality has been demonstrated for all serotypes using a serum bactericidal assay (see SBA, materials and methods). The GMT at day 208 is summarized in Table 8.

[0107] (Table 8) GMT for functional antibodies measured by SBA on day 208 TIFF0007887224000021.tif67128

[0108] As shown in Figures 9A-9F, ELISA titers were significantly correlated with their respective SBA titers for each OspA serotype and individual target serum samples. Spearman correlation coefficients ranged from 0.6487 (p<0.0001) for serotype 4 to 0.9131 (p<0.0001) for serotype 3.

[0109] (Table 14) Comparison of GMT(SBA) using different immunotherapy schedules TIFF0007887224000022.tif83160

[0110] The multimeric Borrelia vaccine ("MBA") was generally safe across all doses and age groups tested. No related serious adverse events (SAEs) were observed in this or any trial. Reactogenicity decreased after the first vaccination. Overall, the tolerability profile, including the rate of fever, was comparable to other lipid-adjuvanted recombinant vaccines or lipid-containing formulations. Table 9 provides a comparison of safety data during the Phase 2 trial using the vaccine, and a comparison to other lipid-adjuvanted recombinant vaccines or lipid-containing formulations.

[0111] (Table 9) Comparison of solicited local and systemic adverse events to other lipid-adjuvanted recombinant vaccines or lipid-containing formulations TIFF0007887224000023.tif239105TIFF0007887224000024.tif239100 1 Highlights of Trumenba, Prescription Information 9 / 2017, 2 Highlights of Bexsero, Prescription Information 10 / 2017, 3 PI Lymerix (rate of solicited AEs). A "any" is defined as the cumulative frequency of subjects reporting a reaction as "mild", "moderate", or "severe" within 7 days of vaccination. Mild (2.5 - 5.0 cm); Moderate (>5.0 - 10.0 cm); Severe (>10.0 cm).

[0112] In summary, clinical trials have shown that the multimeric Borrelia vaccine was generally safe across all doses and age groups tested. Immunogenicity increased in Phase 2 using higher vaccine doses (135 μg, 180 μg) and an alternative immunization schedule (0 - 2 - 6 months).

[0113] Example 4: A Phase 2 clinical trial using a multivalent Borrelia vaccine to investigate a primary immunization schedule of 3 or 2 doses of the vaccine, and a booster dose, in a test population aged 5 - 65 years This is a randomized, observer-blind, placebo-controlled, multi-center Phase 2 trial in healthy subjects aged 5 to 65 years. The trial is conducted in two trial parts (Part A: main trial phase, Part B: booster phase). The trial starts with age-order registration in the sentinel cohort. Registration of subjects for Part A starts with the adult cohort, which enables the generation and review of appropriate safety data before the pediatric cohort is started.

[0114] Registration for Part A (main examination phase): Registration is carried out in a manner that shifts the registration for the three age cohorts from the oldest to the youngest (see Figure 19).

[0115] Registered cohort 1 (ages 18-65): Registration starts with 30 sentinel adult subjects (Cohort 1) aged 18 to 65 years. The subjects are randomly assigned 1:1:1 to one of three study groups to receive vaccination with multimeric Borrelia vaccine (「MBV」) (Groups 1 and 2) with aluminum hydroxide as an adjuvant or placebo (Group 3). After these 30 sentinel subjects receive the first vaccination and complete the safety follow-up visit 7 days after vaccination (Visit 1A), which includes review of 7-day eDiary data, the safety data are reviewed by an independent Internal Review Committee (IRC) in an unblinded manner. Based on this data, the IRC gives advice on whether registration of the adolescent age cohort (12 - 17 years, Cohort 2) can be started. Registration of the remaining 270 adult subjects in Cohort 1 continues without restriction during the IRC review.

[0116] Registered cohort 2 (ages 12-17): Upon IRC approval, 30 sentinel adolescents aged 12–17 years (Cohort 2) will be enrolled. These participants will be randomized in a 1:1:1 ratio to one of three trial groups to receive either a multimerized Borrelia vaccine with aluminum hydroxide adjuvant (Groups 1 and 2) or a placebo (Group 3). Again, after these participants complete a safety follow-up visit (Visit 1A) 7 days after their first vaccination, including a review of 7-day eDiary data, the IRC will review the safety data. The IRC will then recommend whether enrollment of the youngest age cohort (ages 5–11) can be initiated. Enrollment of the remaining 120 adolescents in this age cohort will only begin after IRC approval.

[0117] Registered cohort 3 (ages 5-11): Upon IRC approval, 30 sentinel subjects (Cohort 3) aged 5–11 years will be enrolled. These subjects will be randomized in a 1:1:1 ratio to one of three study groups to receive either a multimerized Borrelia vaccine with aluminum hydroxide adjuvant (Groups 1 and 2) or a placebo (Group 3). Again, after these 30 subjects complete a safety follow-up visit (Visit 1A) 7 days after the first vaccination, including review of 7-day eDiary data, the IRC will review the safety data. Enrollment of the remaining 120 subjects in this age cohort will begin only after IRC approval. An external, independent Data Safety Monitoring Committee (DSMB) will review the resulting safety data in an open-label manner at regular intervals and may recommend adjusting, pausing, or suspending the study at any time.

[0118] Registration of targets for Part B (Booster Phase): Multimerized Borrelia vaccine booster (group 1 or 2) All eligible subjects (up to 200; see Table 15) in the study group (Group 1 or Group 2) who have received all immunizations according to the primary immunization schedule selected for further development will proceed to Part B and receive an additional MBV vaccine at 18 months.

[0119] Placebo booster (Group 1 or 2) Approximately half of the subjects in the study group (Group 1 or Group 2) who have received all immunizations according to the alternative primary schedule (i.e., the schedule not selected for further development) and are eligible for the booster phase (i.e., up to 100 subjects maintaining a 2:1:1 age stratification; see Table 15) will proceed to Part B. For this purpose, the first 50 eligible adult subjects (age group 18–65 years), the first 25 eligible adolescent subjects (age group 12–17 years), and the first 25 eligible subjects (age group 5–11 years) will be included in the booster phase as they will be at their 8 / 18 month visit. These subjects will receive a placebo injection at 18 months. The remaining subjects from this study group will be discontinued.

[0120] Placebo booster (Group 3) Approximately half of the subjects in the placebo group (Group 3) who have received all injections according to schedule and are eligible for the booster phase (i.e., up to 100 subjects maintaining a 2:1:1 age stratification; see Table 15) will proceed to Part B. For this purpose, the first 50 eligible adult subjects (age group 18–65 years), the first 25 eligible adolescent subjects (age group 12–17 years), and the first 25 eligible subjects (age group 5–11 years) will be included in the booster phase as they will be at their 8 / 18 month visits. These subjects will receive an additional placebo injection at 18 months. The remaining subjects from this group will be discontinued. The sponsor and study statisticians will be open-label at the time of the primary endpoint analysis, i.e., before enrollment of subjects for Part B. Once the number of enrolled eligible subjects reaches the number of subjects for Part B, the study location will be notified via the electronic system.

[0121] Test design The test will be conducted in two parts: Part A: the main test phase, and Part B: the booster phase. Please refer to Figure 20 for the test design.

[0122] In Part A (the main trial phase), approximately 600 participants aged 5–65 years will be randomized in a 1:1:1 ratio to three trial groups. Group 1 (approximately 200 participants) will receive three doses of the multimerized borrelia vaccine with aluminum hydroxide adjuvant at months 0–2–6. Group 2 (approximately 200 participants) will receive two doses of the multimerized borrelia vaccine with aluminum hydroxide adjuvant at months 0–6 and a placebo injection at month 2, in order to maintain blinding. Group 3 (approximately 200 participants) will receive three placebo injections at months 0–2–6. Within each trial group, participants will be enrolled in three age cohorts (18–65 years, 12–17 years, and 5–11 years) in a 2:1:1 ratio.

[0123] In Part A, all subjects will receive three IM injections of either MBV or placebo at 0–2–6 months (i.e., days 1–57–180). A safety visit will be conducted on day 8 / visit 1A (i.e., 7 days after the first vaccination) (by telephone for subjects aged 18–65, and in person for subjects aged 5–17 years). In-person visits are scheduled for all age cohorts one month after each vaccination. Blood samples for immunogenicity assessment will be collected at screening visits on days 85, 180, 194 (a subset of adult subjects), 208, 365 / 12 months, and 18 months.

[0124] Based on safety and immunogenicity data from day 208 / visit 6 of Part A (i.e., one month after the third immunization), the data will be used to guide the decision regarding the Part B booster.

[0125] In Part B (Booster Phase), eligible subjects from the group that received the primary schedule selected in Part A (either Group 1 or 2) will receive a booster dose of the multimerized Borrelia vaccine with aluminum hydroxide as an adjuvant at 18 months.

[0126] For safety comparison, placebo injections are administered to approximately 100 subjects enrolled in the group vaccinated in Part A according to an alternative vaccination schedule (i.e., either Group 1 or 2 vaccinated according to a primary immunization schedule not selected for further vaccine development), and to approximately 100 subjects in Group 3. In all test groups, it is targeted to maintain a 2:1:1 age stratification (18 - 65 years, 12 - 17 years, and 5 - 11 years). All subjects involved in Part B are followed up at the test visits at 19, 23, 26, 30, 36, 42, 48, and 54 months, and for an additional three years (i.e., up to 54 months).

[0127] Table 15 shows an overview of the test groups and treatments. The treatment administered has an injection volume of 0.5 ml.

[0128] (Table 15) Test group and vaccine TIFF0007887224000025.tif59168TIFF0007887224000026.tif212168 * To maintain blinding, subjects assigned to Group 2 receive placebo at 2 months.

Claims

1. A composition for use in a method for vaccinating humans, 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), Formulated for administration to human adults or human children, containing a total protein content of the three aforementioned fusion proteins in the range of 135 to 200 μg per dose, The composition, administered to a human adult or human child at least two or at least three times.

2. 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) for use in a method for inducing an immune response in humans against Borrelia, which causes Lyme disease, Formulated for administration to human adults or human children, containing a total protein content of the three aforementioned fusion proteins in the range of 135 to 200 μg per dose, The composition, administered to a human adult or human child at least two or at least three times.

3. The composition is administered at least three times. The second dose is administered at least 6 weeks to at most 3 months after the first dose, and / or the third dose is administered at least 5 months to at most 7 months after the first dose, or The second dose is administered at least 50 to at most 70 days after the first dose; and / or the third dose is administered at least 170 to at most 190 days after the first dose, or at least 175 to at most 185 days after the first dose. A composition for use according to claim 1 or 2.

4. The composition for use according to claim 3, wherein the second dose is administered at a time of at least 55 days to at most 60 days after the first dose.

5. The composition for use according to claim 3 or 4, wherein the second dose is administered 56 days after the first dose, and / or the third dose is administered 180 days after the first dose.

6. The composition is administered at least three times. If such a dose is present, the composition for use according to any one of claims 1 to 5, wherein the fourth dose of the composition is administered during a period of at least 15 months to at most 21 months after the first dose.

7. The composition for use according to claim 6, wherein the fourth dose is administered at a period of at least 17 months to at most 19 months after the first dose.

8. The composition for use according to claim 6 or 7, wherein the fourth dose is administered 18 months after the first dose.

9. The second dose is administered within a period of at least 5 months to at most 7 months after the first dose, or The second dose is administered at least 170 to at most 190 days after the first dose, or at least 175 to at most 185 days after the first dose. A composition for use according to claim 1 or 2.

10. The composition for use according to claim 9, wherein the second dose is administered 180 days after the first dose.

11. The composition is administered at least twice, If so, the third administration of the composition occurs at least 15 months to at most 21 months after the first administration. A composition for use according to claim 1 or 2.

12. The composition for use according to claim 11, wherein the third dose is administered at a time period of at least 17 months to at most 19 months after the first dose.

13. The composition for use according to claim 11 or 12, wherein the third dose is administered 18 months after the first dose.

14. A composition for use according to any one of claims 1 to 13, wherein the total protein content of the three fusion proteins is in the range of 135 μg to 180 μg per dose for a human adult or human child.

15. The composition for use according to claim 14, wherein the total protein content of the three fusion proteins is 135 μg or 180 μg per dose for a human adult or human child.

16. The composition for use according to any one of claims 1 to 15, wherein the three fusion proteins constitute at least 60%, at least 70%, or at least 80% of all proteins in the composition.

17. A composition for use according to any one of claims 1 to 16, comprising the fusion protein in a weight ratio of 1:1:1 (Lip-S1D1-S2D1 : Lip-S4D1-S3hybD1 : Lip-S5D1-S6D1).

18. A composition for use according to any one of claims 1 to 17, comprising an adjuvant.

19. The composition is administered at least three times, The total protein content of the three fusion proteins is 135 μg per dose for human adults or human children, the second dose is administered 56 days after the first dose, the third dose is administered 180 days after the first dose, and, if applicable, the fourth dose is administered 18 months after the first dose. A composition for use according to claim 1 or 2.

20. The composition is administered at least three times, The total protein content of the three fusion proteins is 135 μg or 180 μg per dose for adult or child humans, the second dose is administered 2 months after the first dose, the third dose is administered 6 months after the first dose, and, if applicable, the fourth dose is administered 18 months after the first dose. A composition for use according to claim 1 or 2.

21. The composition is administered at least twice, The total protein content of the three fusion proteins is 135 μg or 180 μg per dose for human adults or human children, the second dose is 180 days after the first dose, and if there is a third dose, it is 18 months after the first dose, and optionally every 12 months thereafter. A composition for use according to claim 1 or 2.

22. The composition is administered at least twice, The total protein content of the three fusion proteins is 135 μg or 180 μg per dose for human adults or human children, the second dose is 6 months after the first dose, and if there is a third dose, 18 months after the first dose, and optionally every 12 months thereafter. A composition for use according to claim 1 or 2.

23. A composition for use according to any one of claims 1 to 22, wherein further administration is annually after the last primary immunization or at the beginning of each mite season.

24. The composition for use according to claim 23, administered one year, two years, and three years after the last primary immunization treatment.

25. The composition for use according to any one of claims 1 to 24, wherein the immune response induced by the composition comprises an antibody response of 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 having bactericidal activity.

26. The composition for use according to any one of claims 1 to 25, wherein the immune response induced by the composition comprises antibodies against Borrelia OspA serotypes 1, 2, 3, 4, 5, and 6.

27. The composition for use according to claim 26, wherein the immune response to Borrelia OspA serotypes 1, 2, 3, 4, 5, and 6 is sustained for at least about 60 days, at least about 180 days, at least about 365 days, or at least about 540 days.

28. The composition for use according to any one of claims 1 to 27, wherein the human adult is a subject aged 18 years or older, and / or an elderly subject aged 50 years or older.

29. The composition for use according to claim 28, wherein the human adult is between 18 and 65 years of age.

30. The composition for use according to any one of claims 1 to 27, wherein the human child is aged 5 to 17 years.

31. The composition for use according to claim 30, wherein the human child is 12 to 17 years of age or 5 to 11 years of age.

32. A composition for use according to any one of claims 1 to 31, administered to a human adult or human child in a volume of 0.25 milliliters (ml) to 1.0 ml.

33. The composition for use according to claim 32, administered to a human adult or human child in a volume of 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.

34. The composition for use according to claim 32 or 33, administered to a human adult or human child in a volume of 0.25 ml, 0.5 ml, or 1.0 ml.

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