Altered ospa of borrelia burgdorferi
Abstract
A polypeptide comprising an amino acid sequence of an OspA protein from Borrelia burgdorferi, from the remainder 139 to the remainder 273 of an OspA protein from Borrelia burgdorferi, in which the sequence includes all the modifications constituted by the residue 139 which is methionine, the remainder 160 which is tyrosine and the remainder 189 which is methionine, in which the numbering corresponds to the numbering of SEQ ID NO: 7.

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9 claims: 7 independent, 2 dependent
- 1ES 2 298 249 T3 REIVINDICACIONES 1. Un polipéptido que comprende una secuencia de aminoácidos de una proteína OspA de Borrelia burgdorferi, desde el resto 139 hasta el resto 273 de una proteína OspA de Borrelia burgdorferi, en el que la secuencia incluye todas las modificaciones constituidas por el resto 139 que es metionina, el resto 160 que es tirosina y el resto 189 que es metionina, en el que la numeración se corresponde con la numeración de la SEC ID N°:7.
- 2El polipéptido de la reivindicación 1, en el que:(a) el polipéptido ha aumentado su estabilidad conformacional en comparación con el polipéptido OspA no modificado correspondiente;(b) el polipéptido comprende los restos 131 a 273 de la proteína OspA de Borrelia burgdorferi;(c) el polipéptido comprende los restos 17 a 273 de la proteína OspA de Borrelia burgdorferi y (d) el polipéptido se obtiene a partir de una proteína OspA de una cepa sensu stricto de Borrelia burgdorferi.
- 3Un polinucleótido que codifica una secuencia de aminoácidos de una proteína OspA de Borrelia burgdorferi desde el resto 139 hasta el resto 273, en el que la secuencia codifica todas las modificaciones constituidas por:codón 139 que codifica metionina, codón 160 que codifica tirosina y codón 189 que codifica metionina, en el que la numeración se corresponde con la numeración de la SEC ID N°: 7.
- 4El polinucleótido de la reivindicación 3, en el que:(a) el polipéptido codificado comprende los restos 131 a 273 de la proteína OspA de Borrelia burgdorferi;o (b) el polipéptido codificado comprende los restos 17 a 273 de la proteína OspA de Borrelia burgdorferi;o (c) el polipéptido codificado se corresponde con la proteína OspA de una cepa sensu stricto de Borrelia burgdorferi.
- 5Un polinucleótido seleccionado del grupo constituido por las SEC ID N°:103 y 115.
- 6Un procedimiento para generar un polipéptido OspA de Borrelia burgdorferi modificado con una estabilidad conformacional aumentada en comparación con el polipéptido OspA de Borrelia burgdorferi no modificado correspondiente, que comprende:a) seleccionar un polinucleótido que codifica un polipéptido OspA de Borrelia burgdorferi que incluye los restos 139, 160 y 189, en el que la numeración se corresponde con la numeración de la SEC ID N°: 7 b) modificar el polinucleótido de tal modo que el resto 139 sea metionina, el resto 160 sea tirosina y el resto 189 sea metionina;y c) expresar dicho polinucleótido modificado;generando de este modo un polipéptido OspA de Borrelia burgdorferi modificado con una estabilidad conformacional aumentada en comparación con el polipéptido OspA de Borrelia burgdorferi no modificado correspondiente.
- 7Uso de un polipéptido OspA modificado para la fabricación de un medicamento para inmunizar a un mamífero, por ejemplo, un ser humano, contra la enfermedad de Lyme, en el que el polipéptido OspA modificado comprende una secuencia de aminoácidos de una proteína OspA de Borrelia burgdorferi, desde el resto 139 hasta el resto 273 de la proteína OspA de Borrelia burgdorferi, en el que la secuencia incluye todas las modificaciones constituidas por el resto 139 que es metionina, el resto 160 que es tirosina, el resto 189 que es metionina y combinaciones de los mismos, en la que el polipéptido ha aumentado su estabilidad conformacional en comparación con un polipéptido OspA de tipo silvestre correspondiente, en la que la numeración se corresponde con la numeración de la SEC ID N°:7.
- 8Un polipéptido quimérico que comprende:a) una secuencia de aminoácidos de un primer polipéptido OspA desde el resto 1 hasta el resto 164 de una primer cepa de Borrelia burgdorferi;b) una secuencia de aminoácidos de un segundo polipéptido OspA desde el resto 165 hasta el resto 179 de una segunda cepa de Borrelia burgdorferi, en la que dicha segunda cepa es una cepa diferente de dicha primera cepa;c) una secuencia de aminoácidos de un tercer polipéptido OspA desde el resto 180 hasta el resto 216 de una tercera cepa de Borrelia burgdorferi;en la que dicha tercera cepa es una cepa diferente de dicha segunda cepa;d) una secuencia de aminoácidos de un cuarto polipéptido OspA desde el resto 217 hasta el resto 273 de una cuarta cepa de Borrelia burgdorferi, en la que dicha cuarta cepa es una cepa diferente de dicha tercera cepa;ES 2 298 249 T3 en el que la secuencia incluye todas las modificaciones constituidas por el resto 139 que es metionina, el resto 160 que es tirosina, el resto 189 que es metionina y combinaciones de los mismos, en el que la numeración se corresponde con la numeración de la SEC ID N°: 7.
- 9Un polipéptido seleccionado del grupo constituido por las SEC ID N°:104 ó 116.
Independent claims9
379 paragraphs in 29 sections, as filed
ES 2 298 249 T3
DESCRIPTION
Modified OspA from Borrelia burgdorferi.
Related requests
This application claims the benefit of United States Provisional Application No. 60 / 226,484, filed August 18, 2000, the teachings of which are incorporated herein by reference in their entirety.
Government funding
The invention was supported, in whole or in part, by grant 2R01AI37256-05A1 from the National Institute of Allergy and Infectious Diseases. The (United States) Government has certain rights in the invention.
Background of the invention
Lyme disease (Lyme borreliosis) is the most common tick-borne infectious disease in North America and Europe, and has been found in Russia, Japan, China, and Australia. Lyme disease begins at the site of a tick bite, producing a primary infection that spreads throughout the body to secondary sites during the course of infection. The causative bacterial agent of this disease is the spirochete Borrelia burgdorferi, which was isolated and cultured for the first time in 1982 (Burgdorferi, WA et al., Science 216: 13171319 (1982); Steere, AR et al., N. Engl J. Med., 308: 733-740 (1983)).
Three pathogenic Borrelia genospecies have been described, B. burgdorferi sensu stricto (B. burgdorferi or B. bss), B. afzelii and B. garinii (Baranton, G., et al., Int. J. Syst. Bacteriol., 42: 378-383 (1992)). These are members of a species complex, B. burgdorferi sensu lato, made up of at least 10 different genospecies (Piken, RN et al., J. Invest. Dermatol., 110: 211-214 (1998); Prostic, D. et al., Int. J. Syst. Bacteriol., 44: 743-752 (1994); Valsangiacomo, CT et al., Int. J. Syst. Bacteriol., 47: 1-10 (1997)). All three genospecies, B. burgdorferi sensu stricto, B. afzelii, and B. garinii, are thought to be pathogenic and all are found in Europe.
B. burgdorferi has an outer membrane whose main protein constituents are outer surface proteins A and B (OspA and OspB). OspA is a basic lipoprotein of approximately 31 kd, which is encoded in a large linear plasmid together with OspB, a basic lipoprotein of approximately 34 kd (Szczepanski, A., and JL Benach, Microbiol. Rev., 55: 21 (1991) ). The immune response to these outer surface proteins tends to occur late in disease, if at all (Craft, JE et al., J. Clin. Invest. 78: 934-939 (1986); Dattwyler, RJ and BJ Luft, Rheum. Clin. North Am., 15: 727-734 (1989)). Furthermore, patients acutely and chronically infected with B. burgdorferi respond variably to different antigens, including OspA, OspB, OspC, OspD, p39, p41, and p93.
Currently, Lyme disease is treated with a variety of antibiotics, for example, tetracyclines, penicillin, and cephalosporins. However, such treatment is not always effective in clearing the infection. Treatment is often delayed due to an inappropriate diagnosis, with the detrimental effect that the infection progresses to a chronic condition, in which antibiotic treatment is frequently useless. One of the contributing factors to delayed treatment is the lack of effective diagnostic tools.
Attempts have been made to generate vaccines against Lyme borreliosis. However, a recombinant OspA vaccine may require frequent booster immunizations. An additional concern of OspA-based vaccines is the recent identification of a potential autoreactive OspA domain, with a high degree of similarity to a region of human leukocyte function-associated antigen 1 (hLFA-1) (Gross, DM et al. , Science, 281: 703-706 (1998)).
Therefore, it will be advantageous to develop modified OspA proteins that have decreased cross-reactivity with hLFA-1, to reduce the potential side effects of an OspA vaccine. The development of OspA proteins, with decreased cross-reactivity with hLFA-1, that retain or have increased immunoreactivity against more than one member of the Borrelia complex is also desirable. To be useful as vaccines, the conformations of these modified proteins must be stable enough to retain certain structural features of OspA, which are required to elicit a protective immune response. OspA proteins with these characteristics will allow improvements in the diagnosis and / or vaccination against all, or most, of the Borrelia that cause Lyme disease.
Analysis of the immune status of individuals immunized with OspA revealed that the total quantitative response is not predictive of protection, but rather that reactivity with a specific epitope of the OspA lipoprotein directly correlates with protective immunity. The anti-OspA monoclonal antibody, lA-2 (Kramer et al., 1990) defines a lipoprotein epitope that is apparently necessary for protective immunity after OspA vaccination. For example, passive immunization of mice with this antibody leads to protection against spirochete infection (Schaible et al., 1993). Furthermore, the immunization of mice and canines with OspA, which
ES 2 298 249 T3 results in significant serum LA-2 equivalent antibody titers, accurately predicting protection against transmission of tick infection (Golde, 1997). Insufficient levels of LA-2 equivalent antibody result in a lack of protection, as opposed to high serum antibody titers against OspA (Johnson et al., 1995).
Summary of the invention
The present invention relates to modified forms of OspA from Borrelia burgdorferi that have increased conformational stability, yet retain, at least in part, the antigenicity of wild-type OspA. In some embodiments, the modified OspA polypeptide has decreased cross-reactivity with hLFA-1, compared to the corresponding unmodified OspA polypeptide. Modified OspA polypeptides can comprise almost all or only a portion of the native OspA polypeptide. In some embodiments, the modified OspA polypeptide can be part of a combination that includes one or more other proteins, such as, for example, other Borrelia burgdorferi polypeptides including OspA, OspB, OspC, OspD, p93, and p41. In other embodiments, the modified OspA polypeptide can be part of a chimeric protein, such as those described in US Patent No. 6,248,562, the teachings of which are incorporated herein by reference in their entirety.
The modified OspA polypeptides of the present invention comprise an amino acid sequence of the Borrelia burgdorferi OspA protein from about residue 139 to about residue 273, wherein the sequence includes all modifications selected from the group consisting of: residue 139 changed by methionine, residue 160 changed to tyrosine, residue 189 changed to methionine, and combinations thereof. In other embodiments, the modified OspA polypeptides of the present invention comprise an amino acid sequence of a Borrelia burgdorferi OspA protein from about residue 131 to about residue 273, or from about residue 17 to about residue 273. OspA polypeptides of the present invention may comprise longer or shorter fragments of the OspA protein. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7 (OspA of B31).
The polypeptides of the present invention include polypeptides selected from the group consisting of SEQ ID NOs: 104 and 116.
The present invention also relates to polynucleotides encoding the amino acid sequences described herein, such as polynucleotides encoding Borrelia burgdorferi OspA polypeptides from about residue 131 to about residue 273, wherein the sequence encodes all the modifications selected from the group consisting of: codon 139 encoding methionine, codon 160 encoding tyrosine, codon 189 encoding methionine, and combinations thereof. The polynucleotide encoding OspA polypeptides of the present invention can encode longer or shorter fragments of the OspA protein. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7.
The polynucleotides of the present invention include a polynucleotide selected from the group consisting of: SECID N °: 103 and 115.
The present invention also relates to a method for generating a modified Borrelia burgdorferi OspA polypeptide with increased conformational stability, compared to the corresponding unmodified Borrelia burgdorferi OspA polypeptide. The method comprises selecting a polynucleotide that encodes a Borrelia burgdorferi OspA polypeptide, which includes residues 139, 160 and 189, in which the numbering corresponds to the numbering of SEQ ID NO: 7. The polynucleotide is modified such that the following modifications are present: residue 139 is changed to methionine, residue 160 is changed to tyrosine, and residue 189 is changed to methionine. The modified polynucleotide is expressed, thereby generating a modified Borrelia burgdorferi OspA polypeptide with increased conformational stability, compared to the corresponding unmodified Borrelia burgdorferi OspA polypeptide.
The present invention also relates to an expression vector comprising an isolated DNA encoding a modified Borrelia OspA protein. The present invention also includes a host cell comprising a recombinant nucleic acid encoding a modified OspA protein, as described herein.
The present invention also relates to a method of administering the modified Borrelia OspA polypeptides described herein. In one embodiment, the method comprises administering the modified OspA polypeptide in a physiologically acceptable vehicle to an individual. As a result of the administration of the modified OspA protein, the individual develops at least some immune response against the protein. As an example, the individual generates a humoral immune response, in which the individual produces antibodies that recognize at least a portion of said polypeptide. In a preferred embodiment, the individual generates an immunoprotective response, for example, by generating antibodies that recognize the LA-2 epitope.
The present invention can also be used in a method of administering a nucleic acid encoding a modified OspA polypeptide described herein. In one embodiment, the method comprises administering the nucleic acid in a physiologically acceptable vehicle to an individual. As a result of the administration of the nucleic acid, the modified OspA polypeptide is expressed, at least transiently, and the individual develops at least some immune response, preferably an immunoprotective response against the OspA protein.
ES 2 298 249 T3 encoded by nucleic acid. As an example, the individual generates a humoral immune response, in which the individual produces antibodies that recognize at least a portion of the modified OspA polypeptide produced from the nucleic acid. In a preferred embodiment, the individual generates an immunoprotective response, for example, by generating antibodies that recognize the LA-2 epitope.
The invention also includes methods for using the proteins described herein in diagnostic assays. In one embodiment, the method can be used to detect the presence of OspA-specific antibodies in a sample from a host of interest. The method comprises contacting a sample of the host of interest with the modified protein under conditions in which antibodies, if present in the host sample, bind to the modified protein forming antigen-antibody complexes. The antigen-antibody complexes are then detected using standard procedures known in the art.
The present invention can form a diagnostic kit comprising the modified polypeptides described herein. The kit comprises a modified Borrelia burgdorferi OspA protein, as described herein. The kit also includes reagents to detect the antibody-antigen complexes that form between the modified OspA protein and the antibodies that are present in the user supplied host sample.
As a result of the present invention, OspA proteins, or fragments thereof, that have increased conformational stability while retaining at least some antigenicity, or that have reduced cross-reactivity with hLFA-1, are available for use in research, vaccines. and / or diagnostic tests. Furthermore, as a result of the present invention, nucleic acids, which encode OspA polypeptides, which have reduced cross-reactivity with hLFA-1, are available for use in research and vaccines. The modified OspA polypeptides of the present invention are expected to make improved vaccines having fewer side effects.
For a better interpretation of the present invention, together with other and additional objects, reference is made to the following description and the accompanying drawings.
Brief description of the drawings
Figure 1 summarizes peptides and antigenic domains localized by proteolytic and chemical cleavage of OspA.
Figure 2 is a comparison of the antigenic domains represented in Figure 1, for OspA in nine strains of B. burgdorferi.
Figure 3 is a graph depicting a plot of weighted polymorphism versus amino acid position in 14 OspA variants. The peaks noted are: a) amino acids 132-145; b) amino acids 163-177; c) amino acids 208-221. The lower dotted line at the 1.395 polymorphism value demarcates statistically significant excesses of polymorphism at p = 0.05. The upper dotted line at 1,520 is the same, except that the first 29 amino acids from the original analysis of the monomorphic terminal end have been removed .
Figure 4 depicts the amino acid alignment from residue 200 to 220 for OspA from strains B31 and K48, as well as for targeted mutants 613, 625, 640, 613/625, and 613/640. Arrow indicates Trp216. Amino acid changes are underlined.
Figure 5 represents a phylogenetic tree for the Borrelia strains described in Table I. The strains are as follows: 1 = B31; 2 = PKaI; 3 = ZS7; 4 = N40; 5 = 25015; 6 = K48; 7 = DK29; 8 = PHei; 9 = Ip90; 10 = PTrob; 11 = ACAI; 12 = PGau; 13 = Ip3; 14 = PBo; 15 = Pko.
Figures 6A and 6B depict the nucleic acid sequence of OspA-B31 (SEQ ID NO: 6) and the sequence of the encoded protein (SEQ ID NO: 7).
Figures 7A, 7B and 7C depict the nucleic acid sequence of OspA-K48 (SEQ ID NO: 8) and the encoded protein sequence (SEQ ID NO: 9).
Figures 8A, 8B and 8C depict the nucleic acid sequence of OspA-PGau (SEQ ID NO: 10) and the encoded protein sequence (SEQ ID NO: 11).
Figures 9A and 9B depict the nucleic acid sequence of an OspA gene (SEQ ID NO: 127) and its encoded protein sequence (SEQ ID NO: 128).
Figures 10A, 10B and 10C depict the nucleic acid sequence of the OspA-K48 / OspA-PGau chimera (SEQ ID NO: 28) and the sequence of the encoded chimeric protein (SEQ ID NO: 29).
Figures 11A, 11B and 11C depict the nucleic acid sequence of the OspA-B31 / OspA-PGau chimera (SEQ ID NO: 30) and the sequence of the encoded chimeric protein (SEQ ID NO: 31).
ES 2 298 249 T3
Figures 12A and 12B depict the nucleic acid sequence of the OspA-B31 / OspA-K48 chimera (SEQ ID NO: 32) and the sequence of the encoded chimeric protein (SEQ ID NO: 33).
Figures 13A, 13B and 13C depict the nucleic acid sequence of the OspA-B31 / OspA-25015 chimera (SEQ ID NO: 34) and the sequence of the encoded chimeric protein (SEQ ID NO: 35).
Figures 14A, 14B and 14C depict the nucleic acid sequence of the OspA-K48 / OspA-B31 / OspAK48 chimera (SEQ ID No. 36) and the encoded chimeric protein sequence (SEQ ID No. 37).
Figures 15A, 15B and 15C represent the nucleic acid sequence of the chimera OspA-B31 / OspA-K48 / OspAB31 / OspA-K48 (SEQ ID NO: 38) and the sequence of the encoded chimeric protein (SEQ ID NO : 39).
Figures 16A, 16B and 16C depict the nucleic acid sequence of the OspA-B31 / OspB-B31 chimera (SEQ ID NO: 40) and the sequence of the encoded chimeric protein (SEQ ID NO: 41).
Figures 17A, 17B, 17C, 17D, 17E, 17F, 17G, 17H, 17I, 17J, 71K, 17L, 17M, 17N, 17O and 17P represent an alignment of nucleic acid sequences for OspA-B31 (SEQ ID NO. NO: 6), OspA-pKa1 (SEQ ID NO: 42), OspA-N40 (SEQ ID NO: 43), OspA-ZS7 (SEQ ID NO: 44); OspA-25015 (SEQ ID NO: 12), OspA-pTrob (SEQ ID NO: 45), OspA-K48 (SEQ ID NO: 8), OspA-Hei (SEQ ID NO: 46); OspA-DK29 (SEQ ID No: 21), OspA-Ip90 (SEQ ID No: 22), OspA-pBo (SEQ ID No: 23), OspA-Ip3 (SEQ ID No: 24), OspA- Pko (SEQ ID NO: 25), OspA-ACAI (SEQ ID NO: 26) and OspA-PGau (SEQ ID NO: 10). Nucleic acids that are identical to the major nucleic acid sequence (here, OspA-B31) are represented by a period (.); different nucleic acids are shown in lower case letters.
Figures 18A and 18B depict the nucleic acid sequence of the OspA-Tro / OspA-Bo chimera (SEQ ID NO: 47) and the sequence of the encoded chimeric protein (SEQ ID NO: 48).
Figures 19A and 19B depict the nucleic acid sequence of the OspA-PGau / OspA-Bo chimera (SEQ ID NO: 49) and the sequence of the encoded chimeric protein (SEQ ID NO: 50).
Figures 20A and 20B depict the nucleic acid sequence of the OspA-B31 / OspA-PGau / OspAB31 / OspA-K48 chimera (SEQ ID NO: 53) and the encoded chimeric protein sequence (SEQ ID NO: 54 ).
Figures 21A and 21B depict the nucleic acid sequence of the OspA-PGau / OspA-B31 / OspAK48 chimera (SEQ ID NO: 51) and the encoding chimeric protein sequence (SEQ ID NO: 52).
Figure 22 is a bar graph showing the reactivity (measured by ELISA) of sera from mice immunized with the indicated Borrelia protein (OspA or OspC) or with recombinant chimeric protein (OspC2-OspA) (X-axis) against antigens. OspA or OspC indicated (legend) from strain B31 (Borrelia burgdorferi sensu stricto).
Figure 23 is a bar graph showing the reactivity (measured by ELISA) of sera from mice immunized with the indicated Borrelia protein (OspA or OspC) or with recombinant chimeric protein (OspC2-OspA) (X-axis) against antigens. OspA or OspC indicated (legend) from strain B31 (Borrelia burgdorferi sensu stricto). For ELISA results against the OspA antigen of B31, a purified fragment of OspA of B31 (amino acids 18-139) was added in excess to the serum, such that the detected immune response was specific to the C-terminal region of OspA .
Figure 24 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (lipOspA / Bo, lipOspAB / P or OspC-OspAB / P) (X-axis) against the indicated OspA antigens (legend ) of the strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and Pgau (Borrelia afzelii).
Figure 25 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (lipOspAP / Bo, lipOspAB / P or OspC-OspAB / P) (X-axis) against the indicated OspA (legend) of the strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and Pgau (Borrelia afzelii). In all cases, a purified OspA fragment of B31 (amino acids 18-139) was added in excess to the serum, such that the detected immune response is specific for the C-terminal region of OspA.
Figure 26 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB31, OspC2-OspAB31 or lip OspC-B31) (X-axis) against the indicated OspC antigen (legend) of strain B31 (Borrelia burgdorferi sensu stricto).
Figure 27 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB31, OspC2-OspAB31 or Lip OspA K / T) (X-axis) against the indicated OspA antigens (legend ) of strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and Pgau (Borrelia afzelii).
Figure 28 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB / P, OspCB31-OspABPBP or OspCB31-OspAB31) (X-axis) against
ES 2 298 249 T3 the indicated OspA antigens (legend) of the strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and Pgau (Borrelia afzelii).
Figure 29 is a bar graph showing the reactivity of sera from mice immunized with the indicated Borrelia chimeric protein (OspCB31-OspAB / P, OspCB31-OspABPBP or OspCB31-OspAB31) (X-axis) against the indicated OspA (legend) of the strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and Pgau (Borrelia afzelii). In all cases, a purified OspA fragment of B31 (amino acids 18139) was added in excess to the serum, such that the detected immune response is specific for the C-terminal region of OspA.
Figures 30A, 30B and 30C represent the nucleic acid sequence of the chimera OspC-B31 (bp 55-633) / OspA-B31 (bp 52-822) (SEQ ID NO: 55) and the sequence of the chimeric protein encoded (SeC ID No: 56).
Figures 31A, 31B and 31C represent the nucleic acid sequence of the chimera OspC-B31 (bp 55-624) / OspA-B31 (bp 52-822) (SEQ ID NO: 57) and the sequence of the chimeric protein encoded (SEQ ID No. 58).
Figures 32A, 32B and 32C depict the nucleic acid sequence of the OspC-C2 chimera (bp 55-612) / OspAB31 (bp 52-822) (SEQ ID NO: 59) and the encoded chimeric protein sequence ( SEQ ID NO: 60).
Figures 33A, 33B and 33C represent the nucleic acid sequence of the chimera OspC-B31 (bp 55-633) / OspA-B31 (bp 52-651) / OspA-K48 (bp 652-820) (SEQ ID NO. : 61) and the encoded chimeric protein sequence (SEQ ID NO: 62).
Figures 34A, 34B and 34C represent the nucleic acid sequence of the chimera OspC-C2 (bp 55-612) / OspAB31 (bp 52-651) / OspA-K48 (bp 652-820) (SEQ ID NO: 63 ) and the sequence of the encoded chimeric protein (SEQ ID NO: 64).
Figures 35A, 35B and 35C represent the nucleic acid sequence of the chimera OspC-B31 (bp 55-633) / OspA-B31 (bp 52-651) / OspA-Pko (bp 652-820) (SEQ ID NO. : 65) and the encoded chimeric protein sequence (SEQ ID NO: 66).
Figures 36A, 36B and 36C represent the nucleic acid sequence of the OspC-C2 chimera (bp 55-612) / OspAB31 (bp 52-651) / OspA-Pko (bp 652-820) (SEQ ID NO: 67 ) and the sequence of the encoded chimeric protein (SEQ ID NO: 68).
Figures 37A, 37B and 37C represent the nucleic acid sequence of the chimera OspC-B31 (bp 55-633) / OspA-K48 (bp 52-654) / OspA-Tro (bp 655-819) (SEQ ID NO. : 69) and the sequence of the encoded chimeric protein (SEQ ID NO: 70).
Figures 38A, 38B and 38C represent the nucleic acid sequence of the OspC-C2 chimera (bp 55-612) / OspAK48 (bp 52-654) / OspA-Tro (bp 655-819) (SEQ ID NO: 71 ) and the sequence of the encoded chimeric protein (SEQ ID NO: 72).
Figures 39A, 39B and 39C represent the nucleic acid sequence of the chimera OspC-C12 (bp 55-612) / OspA-B31 (bp 88-450) / OspA-Pko (bp 451-537) / OspA-B31 ( bp 538-822) (SEQ ID NO: 73) and the encoded chimeric protein sequence (SEQ ID NO: 74).
Figures 40A, 40B and 40C represent the nucleic acid sequence of the chimera OspC-Pko (bp 55-639) / OspA-B31 (bp 88-450) / OspA-Pko (bp 451-537) / OspA-B31 ( bp 538-651) / OspA-K48 (bp 652-825) (SEQ ID NO: 75) and the encoded chimeric protein sequence (SEQ ID NO: 76).
Figures 41A, 41B and 41C represent the nucleic acid sequence of the OspC-Tro chimera (bp 55-624) / OspA-B31 (bp 88-450) / OspA-Pko (bp 451-537) / OspA-B31 ( bp 538-651) / OspA-Pko (bp 652-822) (SEQ ID NO: 77) and the encoded chimeric protein sequence (SEQ ID NO: 78).
Figures 42A and 42B represent the nucleic acid sequence of the chimera OspC-B31 (bp 55-633) / OspAB31 (bp 394-820) (SEQ ID NO: 79) and the sequence of the encoded chimeric protein (SEQ ID N °: 80).
Figures 43A and 43B represent the nucleic acid sequence of the chimera OspC-B31 (bp SS-631) / OspAB31 (bp 394-651) / OspA-K48 (bp 652-820) (SEQ ID NO: 81) and the sequence of the encoded chimeric protein (SEQ ID NO: 82).
Figures 44A and 44B represent the nucleic acid sequence of the chimera OspC-B31 (bp 55-633) / OspAB31 (bp 394-651) / OspA-Pko (bp 652-820) (SEQ ID NO: 83) and the sequence of the encoded chimeric protein (SEQ ID NO: 84).
Figures 45A and 45B represent the nucleic acid sequence of the chimera OspC-B31 (bp 55-633) / OspAK48 (bp 394-654) / OspA-Tro (bp 655-819) (SEQ ID NO: 85) and the sequence of the encoded chimeric protein (SEQ ID NO: 86).
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Figures 46A, 46B and 46C represent the nucleic acid sequence of the chimera OspC-B31 (bp 55-633) / OspA-B31 (bp 88-450) / OspA-Pko (bp 451-537) / OspA-B31 ( bp 541-651) / OspA-Pko (bp 652-822) (SEQ ID NO: 87) and the encoded chimeric protein sequence (SEQ ID NO: 88).
Figures 47A, 47B and 47C represent the nucleic acid sequence of the OspC-C2 chimera (bp 55-612) / OspAB31 (bp 88-450) / OspA-Pko (bp 451-537) / OspA-B31 (bp 541 -651) / OspA-Pko (bp 652-822) (SEQ ID NO: 89) and the encoded chimeric protein sequence (SEQ ID NO: 90).
Figures 48A and 48B depict the nucleic acid and encoded protein sequence of a modified OspA R139M (SEQ ID NO: 95 and 96).
Figures 49A and 49B depict the nucleic acid and encoded protein sequence of a modified OspA E160Y (SEQ ID NO: 97 and 98).
Figures 50A and 50B depict the nucleic acid and encoded protein sequence of a modified OspA R139M and E160Y (SEQ ID NO: 99 and 100).
Figures 51A and 51B depict the nucleic acid and encoded protein sequence of a modified OspA E160Y (SEQ ID NO: 101 and 102).
Figures 52A and 52B depict the nucleic acid and encoded protein sequence of a modified OspA R139M, E160Y and K189M (SEQ ID NO: 103 and 104).
Figures 53A and 53B depict the nucleic acid and encoded protein sequence of a modified OspA Y165F (SEQ ID NO: 105 and 106).
Figures 54A and 54B depict the nucleic acid and encoded protein sequence of a modified OspA Y165F and V166T (SEQ ID NO: 107 and 108).
Figures 55A and 55B depict the nucleic acid and encoded protein sequence of a modified OspA V166T (SEQ ID NO: 109 and 110).
Figures 56A and 56B depict the nucleic acid and encoded protein sequence of a modified OspA V166T and T170K (SEQ ID NO: 111 and 112).
Figures 57A and 57B depict the nucleic acid and encoded protein sequence of a modified OspA Y165F, V166T and T170K (SEQ ID NO: 113 and 114).
Figures 58A and 58B depict the nucleic acid and encoded protein sequence of a modified OspA R139M, E160Y, K189M, Y165F, V166T and T170K (SEQ ID NO: 115 and 116).
Detailed description of the invention
The present invention relates to modified forms of OspA from Borrelia burgdorferi that have increased conformational stability while retaining antigenicity, as indicated, for example, by the ability to bind to the LA-2 monoclonal antibody. In some embodiments, the modified OspA polypeptides also have decreased cross-reactivity with hLFA-1. Modified OspA polypeptides can comprise all (except for the modifications described herein) or a portion, such as the C-terminal portion, of a wild-type OspA polypeptide. Applicants have discovered that some forms of the OspA protein, such as truncated versions of OspA, do not elicit a potent immunoprotective response when administered to an animal, even though the OspA polypeptide has the immunoprotective sequence of the LA-2 epitope.
The structure of recombinant OspA has been determined at 1.95 A resolution in a binary complex with the Fab fragment of the non-protective mouse mAb184.1, which is reactive with the terminal end of OspA (Li et al., Proc. Natl. Acad. Sci. USA, 94: 3584-3589 (1997)). The OspA polypeptide folds into 21 consecutive antiparallel β-strands followed by a C-terminal α-helix. The structure is conveniently described as two separate folded domains, an N-terminal sandwich domain and a C-terminal barrel domain, connected by a long central β sheet. A set of modified polypeptides, which are described herein, are designed to remove buried charges and / or salt bridges in the C-terminal portion of OspA and replace them with residues that promote hydrophobic interactions.
Accordingly, in one embodiment, the modified OspA polypeptides of the present invention comprise a Borrelia burgdorferi modified OspA protein or polypeptides from about residue 139 to about residue 273, wherein the sequence includes all modifications selected from the group consisting of : residue 139 changed to methionine, residue 160 changed to tyrosine and residue 189 changed to methionine. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7. The modified OspA polypeptide has a methionine in the rest of position 139, a tyrosine in the rest of position 160 and a methionine in the rest of position 189. In other embodiments, the modified OspA polypeptides have both increased conformational stability and reduced cross-reactivity with the hLFA-1 protein.
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For modifications at positions 139, 160, and 189, the modified OspA sequence can be from any Lyme borreliosis strain of Borrelia burgdorferi, such as Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii strains. Strains of Borrelia burgdorferi are well known to those skilled in the art. For example, Borrelia burgdorferi sensu stricto strains include B31, Borrelia afzelii strains include Pgau and Pko, and Borrelia garinii strains include K48.
The modified OspA polypeptide includes all of the modifications described herein. In this embodiment, the modified OspA polypeptide has a methionine at the rest of position 139, a tyrosine at the rest of position 160, a methionine at the rest of position 189, a phenylalanine at the rest of position 165, a threonine at the remainder of position 166 and a lysine at the remainder of position 170.
This invention also relates to polypeptides comprising SEQ ID NO: 104 or 116. The modified OspA polypeptides of the invention may be partially or substantially purified (eg, purified to homogeneity) and / or substantially free of other proteins.
The present invention also relates to polynucleotides encoding the amino acid sequences described herein. As defined herein, the term "polynucleotide" refers to a nucleotide multimer or oligomer that is composed of deoxyribonucleotides or ribonucleotides, or a combination thereof, having from few, for example, 2-20, to many, eg, 20 to several thousand or more, nucleotides. And thus, polynucleotides include nucleic acids of any length and further include oligonucleotides and polynucleotides of both natural and synthetic origin.
The polynucleotides of the present invention include polynucleotides encoding Borrelia burgdorferi OspA polypeptides from about residue 139 to about residue 189, wherein the sequence encodes all modifications selected from the group consisting of: codon 139 encoding methionine, codon 160 encoding methionine encodes tyrosine, codon 189 encoding methionine, and combinations thereof. The numbering of the residues corresponds to the numbering of SEQ ID NO: 7. As described above for polypeptides, in the case of modifications at positions 139, 160 and 189, the polynucleotide encoding the OspA sequence Modified can be from any strain of Lyme borreliosis from Borrelia burgdorferi.
The polynucleotides of the present invention include polynucleotides selected from the group consisting of SECID N °: 103 and 115.
The modified OspA polypeptides of the present invention can be obtained from OspA molecules comprising fragments, derivatives, analogs, variants and mutants of the OspA protein (modified OspA) or they can be fragmented, derivatized or otherwise modified after the modifications are inserted. described in this document. These modified OspA molecules possess the antigenic activity of OspA.
The present invention also relates to a method for generating a modified Borrelia burgdorferi OspA polypeptide with increased conformational stability, compared to the corresponding unmodified Borrelia burgdorferi OspA polypeptide. The method comprises selecting a polynucleotide that encodes a Borrelia burgdorferi OspA polypeptide that includes residues 139, 160 and 189, in which the numbering corresponds to the numbering of SEQ ID NO: 7. The polynucleotide is modified such that residue 139 is methionine, residue 160 is tyrosine, and residue 189 is methionine. The modified polynucleotide is expressed, thereby generating a modified Borrelia burgdorferi OspA polypeptide with increased conformational stability, compared to the corresponding unmodified Borrelia burgdorferi OspA polypeptide. Procedures for modifying a polynucleotide are described below and in the Exemplification and are well known to those of skill in the art. Also described below and in the Exemplification are methods for expressing the modified polypeptides of the invention and well known to those of skill in the art.
OspA β13 strand residues 165-173 have been implicated in the induction of Lyme-related arthritis (Gross DM et al., Science 181: 703-706 (1998)). This region has homology to residues 332-340 of hLFA1, suggesting that this protein is cross-reactive with the T cell epitope (YVLEGTLTA-B31 (SEQ ID NO: 129) and YVIEGTSKQ-hLFA-1 (SEQ ID NO : 130), respectively). Although it is generally thought that B. burgdorferi sensu stricto is more arthrogenic than other Borrelia strains, a recent study of ospA alleles in synovial fluid from Lyme arthritis patients indicates that B. garinii and B. afzelii can also cause arthritis (Eiffert, LF et al., Scand J. Infect. Dec. 30: 265-268 (1998)).
One way to remove the cross-reactive sequence is to replace the β-13 region of OspA-B31 (YVLEGTLTA (SEQ ID NO: 129)) with an analogous region from a strain that does not have the same sequence, such as from a strain by B. Afzelii, eg, Pgau or Pko (US Patent Application entitled "Recombinant Constructs of Borrelia burgdorferi" by Luft et al., filed August 7, 2001, the teachings of which are incorporated herein by reference at its entirety).
In another embodiment, a modified OspA polypeptide is generated that has reduced cross-reactivity with hLFA-1, while retaining the ability to bind LA-2. In that embodiment, for example, residue 130 is methionine, residue 160 is tyrosine, residue 165 is phenylalanine, residue 166 is threonine, residue 170 is lysine, and residue 189 is methionine. Polynucleotides encoding Borrelia burgdorferi OspA polypeptides can be selected, as described herein.
ES 2 298 249 T3
In one embodiment, the modified OspA polypeptide includes minimal sequence, which includes the positions of the modifications. For example, the modified polypeptide may comprise an OspA from about residue 139 to about residue 189, where the numbering corresponds to SEQ ID NO: 7. The modified OspA polypeptides of the present invention also include fragments larger than OspA. For example, modified OspA polypeptides include, but are not limited to, modified OspA polypeptides that comprise an OspA from about residue 160 to about residue 170, an OspA from about residue 150 to 180, an OspA from about residue 131 to 273 or an OspA from about remainder 17 to 273. Described below are procedures for generating and expressing variable-size fragments of OspA, incorporating one or more of the modifications described herein, and well known to those of skill in the art.
As described herein, the OspA sequence that is used to generate the modified OspA polypeptide may be a chimeric OspA polypeptide itself, having two or more segments derived from OspA proteins of different Borrelia genospecies or strains. The size of the modified OspA polypeptide can vary depending on the method used to generate the modified polypeptide and / or the purpose for which it is generated, and such modified chimeric OspA polypeptides can include fragments of OspA. The modified polypeptide can be a part of a larger polypeptide, including additional OspA sequences at the N-terminus, the C-terminus, or at both ends. A fragment of an OspA protein can include polypeptides that are only a part of the full-length OspA protein. Such OspA fragments typically include at least one of the modified residues described herein and possess at least part of the antigenicity of wild-type OspA. OspA fragments can be produced by amino and / or carboxyl terminal deletions, as well as by internal deletions. Fragments can also be produced by enzymatic digestion. Such modified OspA molecules can be tested for their antigenic activity, as described herein or using procedures known in the art.
In some embodiments, the modified OspA polypeptide can be part of a combination with one or more other proteins, such as other Borrelia burgdorferi polypeptides, including, but not limited to, OspA, OspB, OspC, OspD, p93, and p41. In other embodiments the modified OspA polypeptide can be part of a larger molecule, such as a chimeric polypeptide, for example, as described in US Patent No. 6,248,562 and US Patent Application entitled " Recombinant Constructs of Borrelia burgdorferi ”by Luft et al., Filed August 7, 2001. Such larger polypeptides can include amino acid sequences from other proteins including, but not limited to, other Borrelia burgdorferi proteins and / or other proteins useful for generating fusion proteins for immunodiagnostic and / or vaccine procedures. Additional components can be incorporated, for example markers (a radioisotope, an epitope marker (signal) (eg, a hemagglutinin (HA) epitope or a hexahistidine signal), an affinity marker (eg biotin or avidin) , a spin marker, an enzymatic marker, a fluorescent group or a chemiluminescent group), in the modified OspA polypeptides of the invention, to facilitate the isolation and / or purification of the polypeptide. For example, a hexahistidine signal allows easy purification by nickel chromatography. These and other components can also be incorporated into the modified OspA polypeptides of the invention to increase the half-life of the polypeptides. Procedures for incorporating said components into the polypeptides of the invention are well known to those skilled in the art.
In one embodiment, the modified OspA polypeptide of the invention is a chimeric polypeptide. In a particular embodiment, the modified OspA polypeptide comprises the following: a) an amino acid sequence of a first OspA polypeptide from approximately residue 1 to approximately residue 164 of a first strain of Borrelia burgdorferi; b) an amino acid sequence of a second OspA polypeptide from about residue 165 to about residue 179 of a second Borrelia burgdorferi strain, wherein said second strain is a different strain from said first strain; c) an amino acid sequence of a third OspA polypeptide from about residue 180 to about residue 216 of a third strain of Borrelia burgdorferi, wherein said third strain is a different strain from said second strain; d) an amino acid sequence of a fourth OspA polypeptide from about residue 217 to about residue 273 of a fourth strain of Borrelia burgdorferi, wherein said fourth strain is a different strain from said third strain; wherein the sequence includes all modifications selected from the group consisting of residue 139 which is methionine, residue 160 which is tyrosine, residue 189 which is methionine, and combinations thereof, wherein the numbering corresponds to the numbering of SEQ ID NO: 7.
The polypeptides described herein can be isolated from naturally occurring sources, chemically synthesized, or produced recombinantly. The modified OspA polypeptides of the present invention can be obtained from naturally occurring OspA molecules or from nucleic acids encoding said molecules. The OspA polypeptides of the present invention may comprise fragments, derivatives, analogs, variants, and mutants of an OspA protein (modified OspA) and / or may be fragmented, derivatized, or otherwise modified after the modifications described herein are inserted ( also referred to as modified OspA). Said modified OspA molecules possess at least some OspA antigenic activity. According to the invention, the amino acid sequence of the modified OspA polypeptides of the invention may be that of a naturally occurring protein or may comprise additional modifications. Such additional modifications include conservative and / or non-conservative amino acid substitutions, additions of one or more amino acids, and / or deletions of one or more amino acids. Such additional modifications will also retain at least some activity of the encoded protein or polypeptide. For example, the polypeptide or protein mo
Dified ES 2 298 249 T3 will additionally have similar or improved conformational stability, similar or improved immunoprotective activity, or reduced cross-reactivity with hLFA-1, compared to the corresponding modified OspA polypeptide (i.e., OspA polypeptide comprising one or more than the modifications described in this document but not including the additional modification (s)).
For example, the additional modification or modifications preferably retain the three-dimensional configuration of an antibody binding site of the native protein, such as the LA-2 binding site. The presence or absence of biological activity (s) can be determined by various functional assays, as described herein, or using procedures that are known in the art, for example, recognition using an ELISA assay or elicitation of a response. immune (eg, an immunoprotective response) in an animal. Appropriate amino acid modifications, which are included within the scope of the invention, can be made based on various criteria including hydrophobicity, basic or acidic character, charge, polarity, size, presence or absence of a functional group (e.g., -SH or a glycosylation site) and aromatic character, provided that the resulting molecule has at least one of the modifications described herein and retains increased conformational stability and / or reduced cross-reactivity with hLFA-1. The assignment of various amino acids to similar groups based on the above properties will be readily apparent to the skilled person; Additional appropriate amino acid changes can also be found in Bowie (Science, 247: 13061310 (1990)).
OspA "variants" and "mutants" can be generated using in vitro and / or in vivo techniques well known to those of skill in the art, eg, site-specific mutagenesis and oligonucleotide mutagenesis. Manipulations of the OspA polypeptide sequence can also be performed at the protein level. Chemical modifications can be made using known techniques including, but not limited to, specific chemical cleavage using cyanogen bromide, trypsin, and / or papain. OspA can also be structurally modified and / or denatured, for example, using heat. In general, the mutations can be conservative or non-conservative amino acid substitutions, amino acid insertions, or amino acid deletions.
For example, a nucleic acid (eg, DNA) encoding a modified OspA polypeptide can be prepared by site-directed mutagenesis of the nucleic acid (eg, DNA) encoding a wild-type OspA. Site-specific (site-specific) mutagenesis allows the production of OspA variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation (e.g., modification, deletion, or insertion), as well as a sufficient number of nucleotides adjacent to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the desired mutation. Typically, a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 complementary residues on both sides of the mutation of the sequence being modified. In general, site-directed mutagenesis techniques are well known in the art, as exemplified in publications such as Edelman et al., DNA, 2: 183, 1983. For example, a site-specific mutagenesis technique can employ a phage vector that exists in both single and double-stranded forms. Typical vectors useful in site-directed mutagenesis include vectors such as phage M13, for example, as described in Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam, 1981. This and other phage vectors are commercially available and their use is well known to those of skill in the art. A versatile and efficient procedure for the production of oligonucleotide-directed site-specific mutations in DNA fragments, using vectors derived from M13, was published in Zoller, MJ and Smith, M., Nucleic Acids Res., 10: 6487-6500, 1982. In addition, plasmid vectors containing a single-stranded phage origin of replication can be employed to obtain single-stranded DNA (see, eg, Veira et al., Meth Enzymol., 153: 3 (1987)).
Alternatively, nucleotide substitutions can be introduced by synthesis of the appropriate DNA fragment in vitro and its amplification using art-known PCR procedures.
In general, site-specific mutagenesis can be performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence encoding the relevant protein. A primer oligonucleotide carrying the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al., Proc Natl Acad Sci USA, 75: 5765, 1978. This primer can then be hybridized to the single-stranded vector containing the protein sequence and subjected to DNA polymerization enzymes, for example, the Klenow fragment of E. coli polymerase I, to complete the synthesis of the strand carrying the mutation. In this way, a heteroduplex is formed in which one strand encodes the original, non-mutated sequence and the second strand carries the desired mutation. This heteroduplex vector can then be used to transform appropriate host cells, such as JM 101 cells, and clones that include recombinant vectors carrying the mutated sequence arrangement can be selected. Thereafter, the mutated region can be removed and placed in an appropriate expression vector for production of the protein.
The PCR technique can be used to generate variants of the OspA amino acid sequence. When using small amounts of DNA template as starting material in a PCR, primers that differ slightly in sequence from the corresponding region in a DNA template can be used to generate relatively large amounts of a specific DNA fragment that differs from the template sequence only at positions where the primers differ from the template. For the introduction of a mutation into a DNA plasmid, one of the primers can be designed to overlap the position of the mutation and to contain the mutation; the sequence of the other primer is preferably identical to a sequence extension of the opposite strand of the plasmid, but this
ES 2 298 249 T3 sequence can be located in any region of the plasmid DNA. However, it is preferred that the sequence of the second primer is located within 500 nucleotides after that of the first, so that the entire amplified DNA region that binds to the primers can be easily sequenced at the end. PCR amplification, using a primer pair such as the one just described, results in a population of DNA fragments that differ in the mutation end position determined by the primer.
The produced DNA fragments carrying the desired mutation can be used to replace the corresponding region in the plasmid that served as the PCR template, using standard DNA technology. Mutations at different positions can be introduced simultaneously by using a second mutant primer or performing a second PCR with different mutant primers and ligation of the two resulting PCR fragments simultaneously into the vector fragment, in a ligation of three (or more) parts.
A further procedure for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene, 34: 315, 1985. The starting material can be the plasmid (or vector) comprising the OspA DNA to mutate. The codon or codons within the OspA to be mutated are identified. Unique restriction endonuclease sites must exist on either side of the identified mutation site (s). If no such restriction sites exist, they can be generated using the oligonucleotide-mediated mutagenesis procedure described above to introduce them into appropriate locations on the OspA DNA, or they can be generated using PCR and the desired primers, as described in the Exemplification. After restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double stranded oligonucleotide, encoding the DNA sequence between restriction sites, but containing the desired mutation (s), is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is called a cassette. This cassette is designed to have 3 'and 5' ends that are compatible with the ends of the linearized plasmid, so that it can be directly ligated with the plasmid. The plasmid now contains the mutated OspA DNA sequence and can be subcloned and / or expressed to produce the modified OspA protein or polypeptide.
Nucleic acid molecules encoding an OspA (eg, polynucleotides) of the present invention have at least one of the modifications described herein and generally hybridize under high stringency hybridization conditions with an OspA nucleic acid. encoding a polynucleotide, or a fragment thereof, of a Borrelia burgdorferi sensu stricto strain, for example SEQ ID NO: 7. In one embodiment, the OspA-encoding nucleic acid molecules (eg, polynucleotides) of the present invention hybridize under high stringency hybridization conditions to a polynucleotide encoding an OspA, or fragment thereof, from Borrelia afzelii, by Example, SEQ ID NO: 10. In another embodiment, the nucleic acid molecules encoding an OspA (eg, polynucleotides) of the present invention hybridize under high stringency hybridization conditions with a polynucleotide encoding an OspA, or with a fragment thereof, from Borrelia garinii, for example, SEQ ID NO: 8. Therefore, the polynucleotides and polypeptides of the present invention include modified versions of OspA, as described herein.
Appropriate selective stringency conditions are well known to those skilled in the art, or can be found in standard texts such as Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. For example, stringent hybridization conditions include a sodium ion concentration of no more than 1 M and a temperature of at least 25 ° C. In one embodiment, 5X SSPE conditions (750nM NaCl, 50mM Na phosphate, 5mM EDTA, pH 7.4) and a temperature of 25-30 ° C, or equivalent conditions, are suitable for specific hybridization. Equivalent conditions can be determined by varying one or more of the parameters, as is known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used. Nucleic acid molecules capable of hybridization are useful as probes and primers for diagnostic applications.
Accordingly, the invention relates to nucleic acid molecules that have considerable identity to the nucleic acid molecules encoding the modified OspA polypeptides described herein, wherein the nucleic acid encodes one or more of the modifications that are described in this document; Nucleic acid molecules having at least about 90%, more preferably, at least about 95%, and most preferably at least about 98% identity to the nucleic acid molecules described herein are particularly preferred. , wherein the nucleic acid encodes at least one of the modifications described herein. Sequence identity can be determined using commercially or publicly available sequence alignment algorithms, using, for example, the default parameters.
Therefore, included in the present invention are DNA molecules that comprise a sequence that is different from the naturally occurring nucleic acid molecule but that, due to the degeneracy of the genetic code, encodes the same protein or polypeptide. The invention also includes variations of the nucleic acid molecules of the invention, such as the encoding portions, analogs or derivatives of the encoded protein or polypeptide. Said variations may be of natural origin, as in the case of allelic variation, or of non-natural origin, such as those induced by various mutagens and mutagens, provided that the nucleic acid molecule encodes at least one of the modifications described. herein and that the encoded protein has increased conformational stability and / or decreased cross-reactivity with hLFA-1 (compared to the corresponding unmodified protein), that are conferred through the modifications described in this
ES 2 298 249 T3 document. Intended variations include, but are not limited to, the addition, deletion, and / or substitution of one or more nucleotides, which can result in conservative or non-conservative amino acid changes, including additions and deletions. Preferably, said nucleotide or amino acid variations are silent; that is, they do not alter one or more characteristics or the activity of the encoded modified OspA protein or polypeptide. As used herein, the activities of the encoded protein or polypeptide include, but are not limited to, binding function, antigenic function, and conformational stability.
The invention also provides expression vectors that contain a nucleic acid sequence described herein, operably linked with at least one regulatory sequence. Many of these vectors are commercially available, and other suitable vectors can easily be prepared by the skilled person. The term "operably linked" is intended to mean that the nucleic acid molecule is linked to a regulatory sequence in such a way as to allow expression of the nucleic acid sequence. Regulatory sequences are recognized in the art and are selected to produce the encoded polypeptide or protein. Accordingly, the term "regulatory sequence" includes promoters, enhancers, and other expression control elements that are described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). For example, the native regulatory sequences or the native regulatory sequences of the transformed host cell can be employed. It will be understood that the design of the expression vector may depend on factors such as the selection of the host cell to be transformed and / or the type of protein to be expressed. For example, the polypeptides of the present invention can be produced by ligation of the cloned gene, or a portion thereof, into a vector suitable for expression in prokaryotic cells, eukaryotic cells, or both (see, for example, Broach, et al. ., Experimental Manipulation of Gene Expression, ed. M. Inouye (Academic Press, 1983), p. 83; Molecular Cloning: A Laboratory Manual, 2<sup>to</sup> Ed., Ed. Sambrook et al. (Cold Spring Harbor Laboratory Press, 1989), Chapters 16 and 17). Typically, expression constructs contain one or more selectable markers, including, but not limited to, the gene encoding dihydrofolate reductase and genes that confer resistance to neomycin, tetracycline, ampicillin, chloramphenicol, kanamycin, or streptomycin.
Prokaryotic and eukaryotic host cells transfected by the described vectors are also provided in this invention. For example, cells that can be transfected with the vectors of the present invention include, but are not limited to, bacterial cells such as E. coli (eg, K12, BL21, and DH5a strains of E. coli), Streptomyces, Pseudomonas, Serratia marcescens, and Salmonella typhimurium, insect cells (baculovirus) including Drosophila, fungal cells such as yeast cells, plant cells, and mammalian cells, such as thymocytes, Chinese hamster ovary (CHO) cells ) and COS cells.
Therefore, a nucleic acid molecule comprising, for example, SEQ ID NO: 6 with at least one of the specific modifications described herein, or a nucleic acid molecule encoding, for example, la SEQ ID NO: 7 with at least one of the specific modifications described herein, can be used to produce a recombinant form of the protein by microbial or eukaryotic cellular procedures. The ligation of the nucleic acid molecule (eg, a polynucleotide) into a gene construct, such as an expression vector, and transformation or transfection into hosts, whether eukaryotic (yeast, birds, insects, plants, or mammals) or prokaryotes (bacterial cells), are standard procedures used in the production of other well-known proteins. Similar procedures, or modifications thereof, may be employed to prepare recombinant proteins in accordance with the present invention by microbial means or tissue culture technology. Accordingly, the invention relates to the production of encoded proteins or polypeptides by recombinant technology.
The proteins and polypeptides of the present invention can be isolated or purified (eg, to homogeneity) from recombinant cell culture by a variety of procedures. These include, but are not limited to, anion or cation exchange chromatography, ethanol precipitation, affinity chromatography, and high performance liquid chromatography (HPLC). The particular method used will depend on the properties of the polypeptide and the selection of the host cell; proper procedures will be readily apparent to those of skill in the art.
The present invention also relates to pharmaceutical compositions comprising polypeptides and other compounds that are described herein. For example, a polypeptide or protein of the present invention can be formulated with a physiologically acceptable medium to prepare a pharmaceutical composition. The particular physiological medium can include, but is not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, or liquid polyethylene glycol), and dextrose solutions. The optimal concentration of the active ingredient (s) in the selected medium can be determined empirically, according to well-known procedures, and will ultimately depend on the desired pharmaceutical formulation. Methods for the introduction of exogenous polypeptides to the treatment site include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and intranasal administration. Other suitable methods for introduction may also include gene therapy, rechargeable or biodegradable devices, and polymeric slow release devices. The pharmaceutical compositions of this invention can also be administered as part of combination therapy with other agents.
The modified OspA proteins described herein can be produced in such a way that they are highly soluble, overproduced in E. coli, and are not lipidized. In addition, modified OspA proteins can be designed to begin or end with a suitable affinity signal (eg, a His signal) for
ES 2 298 249 T3 facilitate purification. The recombinant proteins described herein have been constructed to maintain high levels of antigenicity and improved conformational stability.
The modified OspA proteins of the present invention are advantageous in that they retain at least some specific reactivity against monoclonal and / or polyclonal antibodies against wild-type Borrelia proteins, are immunogenic, and inhibit the growth or induce lysis of Borrelia in vitro. . The proteins are particularly useful in immunodiagnostic assays. For example, proteins of the present invention can be used as reagents in assays to detect the presence of antibodies against native Borrelia in potentially infected individuals. These proteins can also be used as immunodiagnostic reagents, such as in dot blots, Western blots, enzyme-linked immunosorbent assays, or agglutination assays. The modified OspA proteins of the present invention can be produced by known techniques, such as by recombinant methodology, polymerase chain reaction, or mutagenesis.
Furthermore, the proteins of the present invention are useful as vaccine immunogens against Borrelia infection. One or more of the modified proteins can be combined with a physiologically acceptable carrier and administered to a vertebrate animal by conventional procedures (eg, intravenously or intramuscularly).
The modified forms of the OspA proteins described herein were obtained by genetic engineering, such that at least one immunoprotective domain of the protein was maintained. As described herein, the term "antigen" refers to the ability of a compound to bind to products of an immune response, such as antibodies, T cell receptors, or both. Such responses can be measured using standard antibody detection assays, such as standard ELISA or T-cell activation assays. In a preferred embodiment, the modified forms of OspA described herein elicit an immunoprotective response, for example, by producing antibodies that recognize the LA-2 epitope.
It is understood that the nucleic acids encoding the polypeptides comprising the modified OspA protein may include additional nucleotides or fewer nucleotides to simplify the construction of the gene encoding the chimeric polypeptide, for example, to allow the use of convenient restriction endonuclease sites or to allow ligation of gene fragments such that a contiguous coding region is generated. Based on the guidelines provided herein, one of ordinary skill in the art will be readily able to add or remove nucleotides from the ends of gene fragments encoding OspA protein polypeptides, to generate the modified OspA proteins of the present invention without experimentation. or using just routine experimentation. The modified OspA polypeptides of the present invention may or may not be lipidated.
To test for antigenicity of modified OspA polypeptides, mice can be immunized with OspA polypeptides or proteins containing the polypeptide sequences in aluminum hydroxide. The mice are then bled and assayed for antibody responses to OspA obtained from various Borrelia strains. In further experiments, these immunized mice can be challenged with Borrelia burgdorferi-infected ticks and transmission of infection can be assessed, as described in the Exemplification, using chimeric OspA, OspC and OspC / OspA molecules. The results of such exposure to ticks reveal whether the animal has developed a protective immune response. For example, an immunized animal, which does not seroconvert in response to subsequent tick challenge, has likely generated an immunoprotective response to immunization.
The immunogenic compositions of the present invention can be used to immunize animals, including humans. By immunization is meant to elicit specific immunogenic responses, preferably protective immune responses, as described above. As described herein, an immunogenic response includes responses that result in at least some level of immune response in the treated animal, wherein the animal had previously been treated with a composition comprising at least one modified OspA polypeptide of the present invention.
Immunity, as described herein, is understood to refer to the ability of the treated animal to resist infection, to resist systemic infection, or to overcome an infection, such as a systemic infection, more easily or more rapidly. , compared to unimmunized or untreated individuals. Immunity may also include an enhanced ability of the treated individual to suffer an infection with reduced clinical symptoms or without clinical symptoms of systemic infection. The individual can be treated with the modified OspA proteins of the present invention proactively, for example, once a year or, alternatively, after suffering a tick bite.
In one embodiment, the modified OspA protein of the present invention, together with suitable excipients and / or adjuvants, is administered to an animal such that the animal develops an immune response against the OspA polypeptide of the composition. The pharmaceutical composition can also be administered with other components suitable for in vitro and / or in vivo use. These additional components include buffers, carrier proteins, adjuvants, preservatives, and combinations thereof. In a preferred embodiment, the individual generates an immunoprotective response, eg, by generating antibodies that recognize the LA-2 epitope.
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The present invention also relates to a physiological composition comprising a modified OspA protein. The composition is useful for administration to an animal to generate an immune response or in the diagnostic procedures described herein.
For use as a vaccine, the composition of the present invention may include suitable adjuvants, well known in the art, to enhance the immunogenicity, potency, or half-life of chimeric proteins in the treated animal. Adjuvants and their use are well known in the art (see, for example, PCT Publication WO 96/40290, the teachings of which are incorporated herein by reference in their entirety). The composition can be prepared by known vaccine preparation procedures. For example, the modified OspA polypeptides described herein can be isolated and / or purified using known techniques, such as by size exclusion chromatography, ion exchange chromatography, affinity chromatography, preparative electrophoresis, selective precipitation, or combinations of the above. themselves. The prepared proteins can be mixed with other suitable reagents, as described above, in which the protein is in a suitable concentration. The dosage of the protein will vary and will depend on the age, weight and / or physical condition of the animal to be treated. The optimal dosage can be determined by routine optimization techniques, using suitable animal models.
The composition to be used as a vaccine can be administered by any suitable technique. In one embodiment, administration is by injection, eg, subcutaneously, intramuscularly, intravenously, or by intraperitoneal injection. In another embodiment, the composition is administered to a mucosa, for example, by exposing the nasal mucosa to nasal drops containing the proteins or chimeric proteins of the present invention. In another embodiment, the immunogenic composition is administered by oral administration. In another embodiment of the present invention, chimeric proteins are delivered by DNA immunization using nucleic acids encoding a modified OspA polypeptide.
The present invention also relates to a diagnostic kit comprising the modified OspA polypeptides described herein. The kit also includes reagents for detecting antibody-antigen complexes that form between OspA protein and antibodies that are present in a sample, eg, a user-supplied host sample.
The present invention also relates to methods for detecting an immune response against Lyme disease-causing Borrelia in a host sample. The method comprises contacting a host sample with a modified OspA protein, such that anti-OspA antibodies, if present in said sample, bind to said OspA protein. The amount of antibodies that have bound to said OspA protein are measured, thus detecting an immune response against Borrelia causing Lyme disease.
Exemplification
Example 1
Purification of protein A from the outer surface of Borrelia burgdorferi and analysis of antibody-binding domains
This example details a procedure for the purification of large amounts of native outer surface protein A (OspA) to homogeneity and describes the mapping of antigenic specificities of various anti-OspA MAbs. OspA was purified to homogeneity taking advantage of its resistance to trypsin digestion. Intrinsic labeling with palmitic acid-C<sup>14</sup> confirmed that OspA was lipidated and partial digestion localized lipidation at the amino-terminal cysteine of the molecule.
The reactivity of seven murine anti-OspA monoclonal antibodies against nine different Borrelia isolates was determined by Western blot analysis. The reactivity of the modified OspA polypeptides described herein is analyzed using similar procedures. Intact, lipidated or non-lipidated OspA, and modified OspA can also be tested using similar procedures. The purified OspA was cleaved by enzymatic or chemical cleavage and the monoclonal antibodies were able to define four distinct immunogenic domains (see Figure 1). Domain 3, which included OspA residues 190-220, was reactive with protective antibodies known to agglutinate the organism in vitro, and included various specificities, some of which were not limited to a B. burgdorferi genotype.
A. Purification of native OspA
Detergent solubilization of B. burgdorferi detaches outer surface proteins and produces partially purified preparations containing both OspA and outer surface protein B (OspB) (Barbour, AG et al., Infect. Immun., 52 (5 ): 549-554 (1986); Coleman, JL et al., J Infect. Dis., 155 (4): 756-765 (1987); Cunningham; TM et al., Ann. NY Acad. Sci., 539 : 376-378 (1988), Brandt, ME et al., Infect Immun., 58: 983-991 (1990), Sambri, V. and R. Cevenini, Microbiol., 14: 307-314 (1991)). Although both OspA and OspB are sensitive to proteinase K digestion, unlike OspB, OspA is resistant to trypsin cleavage (Dunn, J. et al., Prot. Exp. Purif., 1: 159-168 (1990 ); Barbour, AG et al., Infect. Immun., 45: 94-100 (1984)). The relative insensitivity to trypsin is surprising in view of the fact that OspA has a high (16% for B31) content of lysine and can be related to the relative configuration of OspA and B in the outer membrane.
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Borrelia intrinsic radiolabeling
Lipoprotein labeling was performed as described in Brandt et al. (Brandt et al., Infect. Immun., 58: 983991 (1990)). Palmitic acid-C was added<sup>14</sup> (ICN, Irving, California) to BSK II medium to a final concentration of 0.5 pCi per milliliter (ml). The organisms were cultured at 34 ° C in this medium until a density of 10<sup>8</sup> cells per ml.
Purification of OspA protein from Borrelia strain B31
Borrelia burgdorferi, labeled with palmitic acid-C<sup>14</sup> or unlabeled, and washed as described (Brandt, ME et al., Infect. Immun., 58: 983-991 (1990)). Whole organisms were treated with trypsin a according to the protocol of Barbour et al., (Infect. Immun., 45: 94-100 (1984)) with some modifications. The pellet was suspended in phosphate buffered saline (PBS, 10 mM, pH 7.2), containing 0.8% tosyl-L-phenylalaninechloromethyl ketone (TPCK) treated trypsin (Sigma, St. Louis, Missouri), the latter at a ratio of 1 pg to 10<sup>8</sup> cells. The reaction was carried out at 25 ° C for 1 hour, after which the cells were centrifuged. The pellet was washed in PBS with 100 pg / ml phenylmethylsulfonyl fluoride (PMSF). Separation of the pellet with Triton X-114 was performed as described in Brandt et al., (Brandt et al., Infect. Immun., 58: 983-991 (1990)). After trypsin treatment, cells were resuspended in ice cold 2% (v / v) Triton X-114 in PBS at 10<sup>9 </sup>cells per ml. The suspension was kept stirred overnight at 4 ° C and the insoluble fraction was removed as a pellet after centrifugation at 10,000 X g for 15 minutes at 4 ° C. The supernatant (soluble fraction) was incubated at 37 ° C for 15 minutes and centrifuged at room temperature at 1000 X g for 15 minutes to separate the aqueous and detergent phases. The aqueous phase was decanted and ice cold PBS was added to decrease the Triton phase, mixed and heated to 37 ° C and centrifuged again at 1000 X g for 15 minutes. Washing was repeated two more times. Finally, the detergent was removed from the preparation using a Bio-beads SM2 spin column (BioRad, Melville, New York) as described in (Holloway, PW Anal. Biochem., 53: 304-308 (1973)).
Ion exchange chromatography was performed as described in Dunn et al. (Dunn et al., Prot. Exp. Purif., 1: 159-168 (1990)) with minor modifications. Crude OspA was dissolved in buffer A (1% Triton X-100, 10 mM phosphate buffer (pH 5.0)) and loaded onto SP Sepharose resin (Pharmacia, Piscataway, NJ), pre-equilibrated with buffer A at 25 ° C. After washing the column with 10 bed volumes of buffer A, bound OspA was eluted with buffer B (1% Triton X-100, 10 mM phosphate buffer (pH 8.0)). OspA fractions were detected by protein assay using the BCA method (Pierce, Rockford, Illinois) or as radioactivity when intrinsically labeled material was fractionated. Triton X-100 was removed using a Biobeads SM2 spin column.
This procedure purifies OspA from an outer surface membrane preparation. In the absence of trypsin treatment, OspA and B were the main components of the soluble fraction obtained after Triton separation of strain B31. In contrast, when Triton extraction was performed after trypsin treatment, the OspB band was not visualized. Further purification of OspA-B31 on a SP Sepharose column resulted in a single band by SDS-PAGE. The yield after removal of the detergent was approximately 2 mg per liter of culture. This OspA purification procedure, as described herein for strain B31, can also be used for other Borrelia isolates. For strains such as strain K48, which lacks OspB, trypsin treatment can be omitted.
OspA-B31 lipidation site
OspA was purified from C-palmitic acid-labeled strain B31<sup>14</sup> , as described above, and was partially digested with endoproteinase Asp-N. After digestion, a new lower molecular weight band was visualized by SDS-PAGE and discovered by direct amino-terminal sequencing starting at Asp<sub>25</sub>. This band had no indications of radioactivity by autoradiography. OspA and B contain a signal sequence (LXYC) similar to the consensus described for E. coli lipoproteins and it has been predicted that the lipidation site of OspA and B will be the amino-terminal cysteine (Brandt, ME et al., Infect. Immun., 58: 983-991 (1990)). The results presented in this document support this prediction.
B. Comparison of OspA Antibody-Binding Regions in Nine Borrelia burgdorferi Strains
The availability of the amino acid sequence for OspA from several different isolates, together with peptide mapping and Western blot analysis, allowed the identification of antigenic domains recognized by monoclonal antibodies (MAbs) and allowed the inference of amino acid residues. key responsible for specific reactivity to antibodies.
Borrelia burgdorferi strains
Nine strains of Borrelia, including seven European strains and two North American strains, were used in this study of antibody-binding domains of various proteins. Information concerning the strains is summarized in Table I, below.
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TABLE I
Representative strains of Borrelia
<td>Strain</td><td>Locality and source</td><td>Strain references</td>
<td>K48</td><td>Czechoslovakia, Ixodes rícinus</td><td>None</td>
<td>PGau</td><td>Germany, ACA human</td><td>Wilske, B. et al., J. Clin. Microbiol. 32: 340-350 (1993)</td>
<td>DK29</td><td>Denmark, human MS</td><td>Wilske, B. et al.</td>
<td>Pko</td><td>Germany, Human EM</td><td>Wilske, B. et al.</td>
<td>PTrob</td><td>Germany, human skin</td><td>Wilske, B. et al.</td>
<td>Ip3</td><td>Khabarovsk, Russia, /. persulcatus</td><td>Asbrink, E. et al., Acta Derm. Venereol., 64: 506-512 (1984)</td>
<td>lp90</td><td>Khabarovsk, Russia, /. persulcatus</td><td>Asbrink, E. et al.</td>
<td> 25015</td><td>Millbrook, NY, /. persulcatus</td><td>Barbour, AG et al., Curr. Microbiol., 8: 123-126 (1983)</td>
<td>B31</td><td>Shelter Island, NY, /. scapularis</td><td>Luft, BJ et al., Infect. Immun, 60: 4309-4321 (1992); ATCC 35210</td>
<td>PKaI</td><td>Germany, Human CSF</td><td>Wilske, B. et al</td>
<td>ZS7</td><td>Freiburg, Germany, /. ricinus</td><td>Wallich, R. et al., Nucí. Acids Res., 17: 8864 (1989)</td>
<td>N40</td><td>Westchester Co., NY</td><td>Fikrig, E. et al., Science, 250: 553-556 (1990)</td>
<td>PHei</td><td>Germany, Human CSF</td><td>Wilske, B. et al.</td>
<td>ACAI</td><td>Sweden, human ACA</td><td>Luft, BJ et al., FEMS Microbiol. Lett. 93: 73-68 (1992)</td>
<td>PBo</td><td>Germany, Human CSF</td><td>Wilske, B. et al.</td>
ACA = patient with chronic atrophic acrodermatitis;
EM = patient with erythema migrans; CSF = cerebrospinal fluid from Lyme disease patient
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R. Johnson from the University of Minnesota supplied the K48, PGau and DK29 strains; B. Wilske and V. PreacMursic, from the Pettenkhofer Institute, Munich, Germany, provided Pko and pTrob; and L. Mayer, Center for Disease Control, Atlanta, Georgia, provided Ip3 and Ip90. J. Anderson of the Connecticut Department of Agriculture provided the North American strains, including strain 25015; and strain B31 (ATCC 35210).
Monoclonal antibodies
Seven monoclonal antibodies (MAbs) were used in this study. Five of the MAbs (12, 13, 15, 83 and 336) were produced from hybridomas cloned and subcloned as previously described (Schubach, WH et al., Infect. Immun., 59 (6): 1911-1915 (1991)). MAb H5332 (Barbour, AG et al., Infect. Immun., 41: 795-804 (1983)) was obtained courtesy of Dr. Alan Barbour, University of Texas, and MAb CIII.78 (Sears, JE et al., J. Immunol., 147 (6): 1995-2000 (1991)) was obtained courtesy of Richard A. Flavell, Yale University. MAbs 12 and 15 were generated against sonicated complete B3; MAb 336 was raised against full PGau; and MAbs 13 and 83 were generated against a truncated form of OspA cloned from strain K48 and expressed in E. coli using the T7 RNA polymerase system (McGrath, BC et al., Vaccines, Cold Spring Harbor Laboratory Press, Plainview, New York, pp. 365-370 (1993)). All MAbs were typed as immunoglobulins G (IgG).
Procedures for protein cleavage, Western blotting, and amino-terminal sequencing
Prediction of the various cleavage sites was achieved by knowledge of the primary amino acid sequence, derived from complete OspA nucleotide sequences, many of which are currently available (see Table II, below). Cleavage sites can also be predicted based on the OspA peptide sequence, which can be determined by conventional techniques after isolation and purification of OspA by the procedure described above. Several OspA isolates were excised to determine the location of monoclonal antibody binding of the proteins.
The cleavage of OspA with hydroxylamine-HCl (HA), N-chlorosuccinimide (NCS) and cyanogen bromide followed the procedures described by Bornstein (Biochem. 9 (12): 2408-2421 (1970)), Shechter et al., ( Biochem., 15 (23): 5071-5075 (1976)) and Gross (enHirs, CHW (ed.): Methods in Enzymology, (NY Acad. Press), 11: 238-255 (1967)), respectively. Protease cleavage, by endoproteinase Asp-N (Boehringer Mannheim, Indianapolis, Indiana), was performed as described in Cleveland D. W. et al. (J. Biol. Chem., 252: 1102-1106 (1977)). Ten micrograms of OspA were used for each reaction. The ratio of enzyme to OspA was approximately 1 to 10 (w / w).
The cleavage-generated proteins and peptides were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, UK, Nature (London) 227: 680-685 (1970)) and electroblotted onto polyvinylidene difluoride membranes ( PVDF) from Immobilon (Ploskal, MG et al., Biotechniques, 4: 272-283 (1986)). They were detected by amide black staining or immunostaining with murine MAbs, followed by alkaline phosphatase-conjugated goat anti-mouse IgG. Specific binding was detected using a 5-bromo-4-chloro-3-indolyl phosphate (BCIP) / nitroblue tetrazolium (NBT) developing system (KPL Inc., Gathersburg, Maryland).
Furthermore, amino-terminal analysis of the amino acid sequence was performed on various cleavage products, as described in Luft et al., (Infect. Immun., 57: 3637-3645 (1989)). The amide black stained bands were extracted from the PVDF blots and sequenced by Edman degradation using a Biosystems model 475A sequencer with a model 120A PTH analyzer and a model 900A control / data analyzer.
Cleavage products of protein A isolates from the outer surface
A purified OspA-B31, labeled with palmitic acid-C<sup>14</sup>, was cleaved with hydroxylamine-HCl (HA) into two peptides, designated HA1 and HA2 (data not shown). The HA1 band migrated at 27 kd and retained its radioactivity, indicating that the peptide included the lipidation site at the N-terminus of the molecule (data not shown). Starting from the predicted cleavage point, HA1 would correspond to residues 1 to 251 of OspAB31. HA2 had a MW of 21.6 kd by SDS-PAGE, demonstrating amino-terminal analysis of the sequence beginning at Gly72, ie, residues 72 to 273 of OspA-B31. In contrast, HA cleaved OspA-K48 into three peptides, designated HA1, HA2, and HA3, with apparent MWs of 22 kd, 16 kd, and 12 kd, respectively. Amino-terminal sequencing showed that HA1 started at Gly72 and HA3 started at Gly142. HA2 was found to have a blocked amino terminus, as observed for the full-length OspA protein. OspA-K48 HA1, 2, and 3 were predicted to be residues 72-274, 1-141, and 142-274, respectively.
N-chlorosuccinimide (NCS) cleaves tryptophan (W), ie at residue 216 of OspA-B31 or at residue 217 of OspA-K48 (data not shown). NCS cleaved OspA-B31 into 2 fragments, NCS1, with a MW of 23 kd, protein residues 1-216, and NCS2, with a MW of 6.2 kd, residues 217 to 273 (data not shown ). Similarly, K48 OspA cleaved into two fragments, NCS1, of residues 1-217, and NCS2, of residues 218 to 274 (data not shown).
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Cyanogen bromide (CNBr) cleavage of OspA occurs at the carboxy terminus of methionine, residue 39. The major fragment, CNBr1, has a MW of 25.7 kd, residues 39-274, by amino-terminal analysis of the amino acid sequence (data not shown). CNBr2 (approximately 4kd) could not be visualized by amide black staining; instead, slightly stained bands of approximately 20 kd MW were observed. These bands reacted with anti-OspA MAbs and were most likely degradation products due to formic acid cleavage.
Determination of antibody-binding domains for anti-OspA monoclonal antibodies
The cleavage products of OspA-B31 and OspA-K48 were analyzed by Western blotting to assess their ability to bind to six different MAbs. Preliminary Western blot analysis of the cleavage products demonstrated that strains K48 and DK29 have similar reactivity patterns, as do IP3, PGau, and Pko. OspA from the PTrob strain was immunologically different from the others, being recognized only by MAb 336. MAb 12 recognized only the two North American strains, B31 and 25015. When the isolates were divided into genogroups, it was surprising that all MAbs, except MAb 12, were cross-reactive with multiple genogroups.
MAb12, specific for OspA-B31, bound both HA1 and HA2 of OspA-B31. However, NCS cleavage of OspA-B31 at the Trp216 residue generated fragments that do not react with MAb12, suggesting that the relevant domain is close to or structurally dependent on the integrity of this residue (data not shown). MAb 13 bound only OspA-K48 and peptides containing the amino terminus of that molecule (eg HA2; NCS1). It did not join CNBr1 remnants 39-274. Therefore, the domain recognized by MAb13 is at the amino terminus of OspA-K48, close to Met38.
MAb15 reacts with OspA from both B31 and K48 strains and with peptides containing the N-terminus of OspA, such as HA1 from OspA-B31 and NCS1, but not with peptides HA2 from OspA-B31 and HA1 from OspA-B31. OspA-K48 (data not shown). Both peptides include residue 72 at the C-terminus of the molecules. MAb15 bound to CNBr1 of OspA-K48, indicating that the domain for this antibody is at residues 39 to 72, specifically, close to Gly72 (data not shown).
MAb83 binds OspA-K48 and peptides containing the C-terminal portion of the molecule, such as HA1. It does not bind to HA2 of OspA-K48, most likely because the C-terminus of HA2 of OspA-K48 ends at 141. Similar to MAb12 and OspA-B31, the binding of MAb 83 and CIII.78 it is removed by cleavage of OspA into the tryptophan moiety. Therefore, the binding of MAbs 12, 83 and CIII.78 to OspA depends on the structural integrity of the Trp residue.<sub>2</sub>i6, which appears to be critical for antigenicity. It is also clear that, although these MAbs bind to a common antigenic domain, the exact epitopes they recognize are different from one another, given the degree of variability of cross-reactivity with these MAbs between strains.
Although there is a similar loss of binding activity of MAb336 with cleavage at Trp2i6, this MAb does not bind to OspA-B31 hA1, suggesting that the domain for this antibody includes the carboxy-terminal end of the molecule, including residues 251 to 273. Low MW peptides, such as hA3 (10 kd) and NCS2 (6 kd) from OspAK48, do not bind to this MAb on Western blots. To confirm this observation, the binding of the 6 MAbs was tested with a recombinant p3A / EC fusion construct containing a trpE leader protein fused to residues 217 to 273 of OspA-B31 (Schubach, WH et al., Infect. Immun ., 59 (6): 1911-1915 (1991)). Only MAb336 reacted with this construct (data not shown). Peptides and antigenic domains localized by OspA fragmentation are summarized in Figure 1.
Example 2
Site-directed mutagenesis within hypervariable domains A (residues 120-140), B (residues 150-180), and C (residues 200216 or 217)
Site-directed mutagenesis was performed to replace recombinant OspA domain residues 204-219 from B31 with analogous residues from a European OspA variant, K48. In the OspA region between residues 204 and 219, there are seven amino acid differences between OspA-B31 and OspA-K48. Three oligonucleotides were generated, each containing nucleotide changes incorporating the K48 amino acids at their analogous positions in the OspAB31 protein. The oligonucleotides used to generate the targeted mutants were:
5'-CTTAATGACTCTGACACTAGTGC-3 '(No. 613, which changes threonine at position 204 to serine and serine at position 206 to threonine (Thr204-Ser and Thr206-Ser)) (SEQ ID No.: 1);
5'-GCTACTAAAAAAACCGGGAAATGGAATTCA-3 '(No. 625, which changes alanine at 214 for glycine and alanine at 215 for lysine (Ala214-Gly and Ala215-Lys)) (SEQ ID No.: 2); Y
5'-GCAGCTTGGGATTCAAAAACATCCACTTTAACA-3 '(No. 640, which changes asparagine at 217 to aspartate and glycine at 219 to lysine (Asn217-Asp and Gly219-Lys)) (SEQ ID No.: 3).
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Site-directed mutagenesis was carried out by performing pairwise mutagenesis of the above oligonucleotides.
Three targeted mutants were generated, each with two changes: OspA 613 (Thr204-Ser and Thr206-Ser), OspA 625 (Ala214-Gly and Ala215-Lys) and 640 (Asn217-Asp and Gly219-Lys). There were also two proteins with four changes: OspA 613/625 (Thr204-Ser, Thr206-Ser, Ala214-Gly and Ala215-Lys) and OspA 613/640 (Thr240-Ser, Thr206-Ser, Asn217-Asp and Gly219-Lys ).
Specificity of antibody binding to epitopes of the non-mutated hypervariable region
Monoclonal antibodies that bind spirochetes, including several that are neutralizing in vitro, recognize epitopes that map in the hypervariable region near Trp216 (Barbour, AG et al., Infect. And Immun., 41: 759 (1983); Schubach, WH et al., Infect. and Immun., 59: 1911 (1991)). Western blot analysis demonstrated that chemical cleavage of OspA from strain B31 at Trp216 suppresses the reactivity of the protein with the binding MAb 105, a monoclonal raised against B31 spirochetes. The reagent, n-chlorosuccinimide (NCS), cleaves OspA at Trp216, forming a 23.2 kd fragment and a 6.2 kd peptide that is not retained on the Immobilon P membrane after transfer. The uncleaved material binds to MAb 105; however, the 23.2 kd fragment is not reactive. Similar Western blots with a TrpE-OspA fusion protein, containing the carboxy-terminal portion of the OspA protein, demonstrated that the small 6.2 kd fragment also does not bind to MAb 105 (Schubach, WH et al., Infect . and Immun., 59: 1911 (1991)).
Monoclonal antibodies H5332 and H3TS (Barbour, AG et al., Infect. And Immun., 41: 759 (1983)) have been shown by immunofluorescence to decorate the surface of fixed spirochetes (Wilske, B. et al., World J Microbiol., 7: 130 (1991)). These monoclonal antibodies also inhibit the growth of the organism in culture. Epitope mapping with fusion proteins has confirmed that the epitopes that bind to these MAbs are conformationally determined and reside in the carboxy half of the protein. MAb H5332 is cross-reactive with all known phylogenetic groups, while MAb H3TS and MAb 105 appear to be specific for the B31 strain against which they were generated. As for MAb 105, the reactivity of H5332 and H3TS against OspA is abrogated by protein fragmentation at Trp216. MAb 336 was raised against all spirochetes of the PGau strain. It is cross-reactive with OspA from group 1 (the group to which B31 belongs) but not with group 2 (of which K48 is a member). Previous studies using fusion and chemical cleavage proteins have indicated that this antibody recognizes an OspA domain in the region between residues 217 and 273. All of these MAbs agglutinate the B31 spirochete.
Western blot analysis of antibody binding to mutated hypervariable regions
MAbs were used for Western blot analysis of the targeted OspA mutants induced in E. coli using the T7 expression system (Dunn, JJ et al., Protein Expression and Purification, 1: 159 (1990)). E. coli cells carrying plasmids pET9c, which had an insert of a targeted OspA mutant, were induced to a medium log phase of growth with IPTG for four hours at 37 ° C. Cell lysates were made by heating to boiling an aliquot of the induced cultures in SDS gel loading dye, and then this material was loaded onto a 12% SDS gel (BioRad mini-Protean II) and subjected to to electrophoresis. The proteins were then transferred to Immobilon-P membranes (Millipore) at 70V, 2 hours at 4 ° C, using the BioRad mini-transfer system. Western analyzes were performed as described in Schubach et al., (Infect. Immun., 59: 1911 (1991)).
Western blot analysis indicated that only mutant 625 (Ala214-Gly and Ala215-Lys) retained binding to the monoclonal binder H3TS. However, the 613/625 mutant, which has additional modifications at the amino-terminus of Trp216 (Ser204-Thr and Thr206-Ser) did not bind to this monoclonal. OspA both 640 and 613/640, which have the Asn217-Asp and Gly219-Lys changes on the carboxy-terminal side of Trp216, also did not bind to MAb H3TS. This indicated that the B31 OspA epitope that binds to H3TS is made up of the amino acid side chains on either side of Trp216.
Mutant 613/625 did not bind to MAbs 105 and H5332, while other mutants retained their ability to bind to these MAbs. This is important in light of data obtained using fusion proteins, which indicate that MAb 105 behaves more like MAb H3TS in terms of its serotype specificity and its binding to OspA (Wilske, B. et al., Med. Microbiol. Immunol., 181: 191 (1992)). Protein 613/625 has, in addition to differences in residues Thr204 and Ser206, immediately amino-terminal changes to Trp216 (Ala214-Gly and Ala215-Lys). Nullification of the reactivity of MAbs 105 and H5332 with this protein indicated that the OspA epitopes that bind to these monoclonal antibodies are constituted by residues on the amino-terminal side of Trp216.
The two proteins that carry the Asn217-Asp and Gly219-Lys replacements on the carboxy-terminal side of Trp216 (OspA 640 and 613/640) retained binding to MAbs 105 and H5332; however, they did not react with MAb 336, a monoclonal that has been mapped, with TrpE-OspA fusion proteins and by chemical cleavage, into a more carboxy-terminal domain. This result may explain why MAb 336 did not recognize OspA type K48 (Group 2).
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It is clear that amino acids Ser204 and Thr206 play an important role in the binding epitopes of the OspA region of B31 that flanks Trp216. The replacement of these two residues modified the OspA epitopes that bind to MAbs 105, H3TS and H5332. The ability of the 640 changes alone to suppress the reactivity of MAb 336 indicated that Thr204 and Ser206 are not involved in direct interaction with MAb 336.
The results indicated that the OspA epitopes that are available for spirochete-binding MAbs are comprised, at least in part, by amino acids in the immediate vicinity of Trp216. Since the analysis of circular dichroism indicated that the OspA structures of B31 and K48 differ very little in this domain, it is unlikely that the changes made by mutation have radically modified the overall structure of the OspA protein (France, LL et al., Biochem. Biophys. Acta., 1120: 59 (1992); and France et al., Biochem, Biophys Acta, filed (1993)). The discovery that mutant recombinant OspAs exhibit the same high solubility and purification properties as the parental B31 protein supports this hypothesis (data not shown).
In summary, the amino acid side chains at positions Ser204 and Thr206 are important for many of the binding epitopes. However, a limited set of conservative changes at these sites were not sufficient to suppress the binding of all binding MAbs. These results suggest that the OspA binding epitopes are distinct, although they may have some overlap. The results also support the hypothesis that the surface-exposed epitope near Trp216, which is thought to be important for immune recognition and neutralization, is a conformationally determined complex domain of OspA.
Example 3
Borrelia strains and proteins
A. Genes encoding Borrelia proteins
The modified OspA polypeptides of the present invention can be part of a combination with other proteins or they can be linked to other proteins to form a chimeric protein. The other polypeptides of the combination or chimera can be obtained from any Borrelia. Representative proteins include OspA, OspB, OspC, OspD, p12, p39, p41 (fla), p66, and p93. Nucleic acid sequences encoding various Borrelia proteins are available (see examples in Table II); alternatively, nucleic acid sequences encoding Borrelia proteins can be isolated and characterized using procedures such as those described below.
TABLE II
Nucleic Acid Sequence References of Various Proteins from Various Borrelia Strains
<td>Strain</td><td>OspA</td>
<td>K48</td><td>X62624 (SID 8)</td>
<td>PGau</td><td>X62387 (SID 10)</td>
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<td>DK29</td><td>X63412 (SID 21)</td>
<td>Pko</td><td>X65599 (SID 25)</td>
<td>PTrob</td><td>X65598 (SID 45)</td>
<td>Ip3</td><td>X70365 (SID 24)</td>
<td>lp90</td><td>Kryuchechnikov, VN et al., J. Microbiol. Epid. Immunobiol. 12: 41-44 (1988) (SID 22)</td>
<td> 25015</td><td>Fikrig, ES et al., J. Immunol. 7: 2256-2260 (1992) (SID 12)</td>
<td>B31</td><td>Bergstrom, S. et al., Mol. Microbiol. 3: 479- 486 (1989) (SID 6)</td>
<td>PKal</td><td>X69606 (SID 42)</td>
<td>ZS7</td><td>Jonsson, M. et al., Infect. Immun. 60: 1845- 1853 (1992) (SID 44)</td>
<td>N40</td><td>Kryuchechnikov, VN et al. (SID 43)</td>
<td>PHei</td><td>X65600 (SID 46)</td>
<td>ACAI</td><td>Kryuchechnikov, VN et al. (SID 26)</td>
<td>PBo</td><td>X65605 (SID 23)</td>
<td colspan="2">Numbers prefixed with "X" are GenBank database accession numbers. SID = SEQ ID No.</td>
B. Isolation of Borrelia genes
Nucleic acid sequences, encoding full-length lipidated proteins, were isolated from known Borrelia strains using the polymerase chain reaction (PCR), as described below. In addition, nucleic acid sequences were generated that encode truncated proteins (proteins in which the lipidation signal has been removed, such as by removing the nucleic acid sequence that encodes the first 18 amino acids, resulting in non-lipidated proteins) . Other proteins were generated that encoded polypeptides from a particular gene (that is, that encoded a segment of the protein that has a different number of amino acids than the protein in nature). Using procedures similar to those described below, primers can be generated from known nucleic acid sequences encoding Borrelia proteins and used to isolate other genes encoding Borrelia proteins. Primers can be designed to amplify an entire gene, as well as to amplify a nucleic acid sequence that encodes truncated protein sequences, as described below for OspC, or nucleic acid sequences that encode a polypeptide derived from a protein of Borrelia. Primers can also be designed to incorporate unique restriction enzyme cleavage sites into the amplified nucleic acid sequences. Then, sequence analysis of the amplified nucleic acid sequences can be performed using standard techniques.
Cloning and sequencing of OspA genes and relevant nucleic acid sequences
Borrelia OspA sequences were isolated as follows: 100 µm reaction mixtures containing 50 mM KCl, 10 mM TRIS-HCl (pH 8.3), MgCl<sub>2</sub> 1.5 mM, 200 pM of each NTP, 2.5 units of DNA
ES 2 298 249 T3 TaqI polymerase (Amplitaq, Perkin-Elmer / Cetus) and 100 pmol each of the 5 'and 3' primers (described below). Amplification was performed in a Perkin-Elmer / Cetus thermal cycler, as described (Schubach, WH et al., Infect. Immun., 59: 1811-1915 (1991)). The amplicon was visualized on an agarose gel by ethidium bromide staining. Twenty nanograms of the chloroform-extracted PCR product were cloned directly into the PC-TA vector (Invitrogen) following the manufacturer's instructions. Recombinant colonies containing the amplified fragment were selected, plasmids were prepared and the nucleic acid sequence of each OspA was determined by the dideoxy chain termination technique using the Sequenase kit (United States Biochemical). Direct sequencing was performed with M13 primers, followed by OspA-specific primers obtained from the sequences, previously obtained with M13 primers.
Because the 5 'and 3' ends of the OspA gene are highly conserved (Fikrig, ES et al., J. Immunol., 7: 2256-2260 (1992); Bergstrom, S. et al., Mol. Microbiol 3: 479-486 (1989); Zumstein, G. et al., Med. Microbiol. Immunol., 181: 57-70 (1992)), the 5 'and 3' primers for cloning can be based on any of known OspA sequences. For example, the following primers based on the OspA nucleic acid sequence from strain B31 were used:
5'-GGAGAATATATTATGAAA-3 '(-12 to +6) (SEQ ID NO: 4); and 5'-CTCCTTATTTTAAAGCG-3 '(+826 to + 809) (SEQ ID NO: 5). (Schubach, WH et al., Infect. Immun, 59: 1811-1915 (1991)).
OspA genes isolated in this way include those from strains B31, K48, PGau, and 25015; nucleic acid sequences are represented in the sequence listing as SEQ ID NO: 6 (OspA-B31), SEQ ID NO: 8 (OspA-K48); SEQ ID NO: 10 (OspA-PGau) and SEQ ID NO: 12 (OspA-25015). An alignment of these and other OspA nucleic acid sequences is shown in Figure 17. The amino acid sequences of the proteins encoded by these nucleic acid sequences are represented as SEQ ID NO: 7 (OspA-B31), SEQ ID NO. 9 (OspA-K48), SEQ ID NO: 11 (OspA-PGau) and SEQ ID NO: 13 (OspA-25015).
The following primers were used to generate specific nucleic acid sequences for the OspA gene:
5'-GTCTGCAAAAACCATGACAAG-3 '
5'-GTCATCAACAGAAGAAAAATTC-3 '
5'-CCGGATCCATATGAAAAAATATTTATTGGG-3 '(Positive Strand Primer No. 369) (Positive Strand Primer No. 357) (Positive Strand Primer No. 607) (SEQ ID No. 14) (SEQ ID No. 15 ) (SEQ ID No. 16)
5'-CCGGGATCCATATGGCTAAGCAAAATGTTAGC-3 '(positive strand primer No. 584) (SEQ ID No. 17)
5'-GCGTTCAAGTACTCCAGA-3 '(negative strand primer No. 200) (SEQ ID No. 18)
5'-GATATCTAGATCTTATTTTAAAGCGTT-3 '(negative strand primer No. 586) (SEQ ID No. 19);
<sup>Y</sup>
5'-GGATCCGGTGACCTTTTAAAGCGTTTTTAAT-3 '(negative strand primer No. 1169) (SEQ ID No. 20)
C. Protein expression from Borrelia genes
The nucleic acid sequences described above can be incorporated into expression plasmids using standard techniques and transfected into compatible host cells to express the proteins encoded by the nucleic acid sequences. As an example, the expression of the p12 gene and the isolation of the p12 protein are explained.
Amplification of the p12 nucleic acid sequence was directed with primers that included an NdeI restriction site in the nucleic acid sequence. The PCR product was phenol / chloroform extracted and ethanol precipitated. The precipitated product was digested and ligated into an expression plasmid as follows: 15 µl (approximately 1 pg) of PCR DNA was combined with 2 µl of 10X restriction buffer for NdeI (Gibco / BRL), 1 µl of NdeI (Gibco / BRL) and 2 µl of distilled water and incubated overnight at 37 ° C. This mixture was subsequently combined with 3 µl of 10X buffer (buffer 3, New England BioLabs), 1 µl of BamHI (NEB) and 6 µl of distilled water and incubated at 37 ° C for two hours. The resulting material was purified by preparative gel electrophoresis using low melting point agarose, the band was visualized under high wavelength ultraviolet light and excised from the gel. The piece of gel was treated with Gelase using the conditions recommended by the manufacturer (Epicenter Technologies). The resulting DNA pellet was resuspended in 25-50 µl of 10 mM TRIS-Cl (pH 8.0) and 1 mM EDTA (TE). An aliquot of this material was ligated with the expression vector pET9c (Dunn, JJ et al., Protein Expression and Purification, 1: 159 (1990)).
ES 2 298 249 T3
To ligate the material with the expression vector pET9c, 20-50 ng of purified and cut p12 nucleic acid sequences were combined, as described above, with 5 µl of 10 One-Phor-All (OPA) buffer (Pharmacia ), 30-60 ng of pET9c cut with Ndel and BamHI, 2.5 µl of 20 mM ATP, 2 µl of T4 DNA ligase (Pharmacia) diluted 1: 5 in 1X OPA buffer and enough distilled water to bring the mixture to a final volume of 50 pl. The mixture was incubated at 12 ° C overnight.
The resulting ligations were transformed into DH5-alpha competent cells and plated on nutrient agar plates, containing 50 pg / ml kanamycin, and incubated overnight at 37 ° C. DH5-alpha is used as a “storage strain” for T7 expression clones because it is deficient in RecA, so recombination and concatenation are not a problem, and because it lacks the T7 RNA polymerase gene, necessary to express the cloned gene. The use of this strain allows the cloning of potentially toxic gene products, while minimizing the probability of deletion and / or rearrangement of the desired genes. Other cell lines having similar properties can also be used.
Kanamycin resistant colonies were individually purified on nutrient agar plates supplemented with kanamycin at 50 pg / ml. One colony of each isolate was inoculated into 3-5 ml of liquid medium containing 50 pg / ml kanamycin and incubated at 37 ° C without shaking. Plasmid DNA was obtained from 1 ml of each isolate using a hot alkaline lysis procedure (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)).
The plasmid DNA was digested with EcoRI and BgIII as follows: 15 µl of plasmid DNA were combined with 2 µl of 10X buffer 3 (NEB), 1 µl of EcoRI (NEB), 1 µl of BgIII (NEB) and 1 µl of distilled water and incubated for two hours at 37 ° C. The entire reaction mixture was electrophoresed on an analytical agarose gel. Plasmids carrying the p12 insert were identified by the presence of a band corresponding to 925 base pairs (full length of p12) or 875 base pairs (non-lipidated p12). One or two plasmid DNAs from the non-lipidated, full-length p12 clones were used in pET9c to transform BL21 DE3 pLysS for kanamycin resistance, as described in Studier et al., (Methods in Enzymology, Goeddel, D. (Ed .), Academic Press, 185: 60-89 (1990)). One or two transformants of the full-length, non-lipidated clones were individually purified on nutrient plates containing 25 pg / ml chloramphenicol (to maintain pLysS) and 50 pg / ml kanamycin at 37 ° C. One colony of each isolate was inoculated in liquid medium supplemented with chloramphenicol and kanamycin and incubated overnight at 37 ° C. The overnight culture was subcultured the next morning in 500 ml of liquid broth with chloramphenicol (25 pg / ml) and kanamycin (50 pg / ml) and grown aerobically at 37 ° C on an orbital shaker with aeration. until the absorbance at 600 nm reached a value of 0.4-0.7. Isopropyl-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM for induction and the culture was incubated for 3-4 hours at 37 ° C, as above. Induced cells were pelleted by centrifugation and resuspended in 25 ml of 20 mM NaPO4 (pH 7.7). A small aliquot was removed for analysis by gel electrophoresis. The expressing clones produced proteins that migrated to the 12 kd position.
A crude cell lysate was prepared from the culture, as described for recombinant OspA in Dunn, JJ et al., (Protein Expression and Purification, 1: 159 (1990)). The crude lysate was first passed through a Q-Sepharose column (Pharmacia) that had been pre-equilibrated in buffer A: NaPO<sub>4</sub> 10 mM (pH 7.7), 10 mM NaCl and 0.5 mM PMSF. The column was washed with NaPO<sub>4</sub> 10 mM, 50 mM NaCl and 0.5 mM PMSF then p12 was eluted in NaPO<sub>4 </sub>10 mM and 0.5 mM PMSF with a 50-400 mM NaCl gradient. P12 eluted approximately midway through the 100-200 mM NaCl gradient. Peak fractions were pooled and dialyzed against NaPO<sub>4 </sub>10 mM (pH 7.7), 10 mM NaCl and 0.5 mM PMSF. The protein was then concentrated and applied to a Sephadex G50 gel filtration column of approximately 50 ml bed volume (Pharmacia), in NaPO<sub>4</sub> 10 mM, 200 mM NaCl and 0.5 mM PMSF. Typically, p12 would elute shortly after the volume exclusion marker. Peak fractions were determined by running small aliquots of all fractions on a gel. The maximum of p12 was pooled and stored in small aliquots at -20 ° C.
Example 4
Generation of chimeric nucleic acid sequences and chimeric proteins
A. General protocol for the generation of chimeric nucleic acid sequences
The megaprimer site-directed mutagenesis procedure, and a modification, was used to generate chimeric nucleic acid sequences (Sarkar and Sommer, Biotechniques, 8 (4): 404-407 (1990); Aiyar, A. and J. Leis, Biotechniques , 14 (3): 366-369 (1993)). A 5 'primer is used for the first genomic template and a 3' fusion oligo is used to amplify the desired region. The fusion primer consists of a 3 'end of the first template (DNA encoding the proximal amino polypeptide of the fusion protein) coupled to a 5' end of the second template (DNA encoding the proximal carboxy polypeptide of the protein of fusion).
PCR amplifications are performed using Taq DNA polymerase, 10X PCR buffer and MgCl<sub>2</sub> (Promega Corp., Madison, WI) and dNTP Ultrapure (Pharmacia, Piscataway, NJ). One pg of genomic template with 1.5 µl of 10 pM 5 'oligo and 5 µl of 10 pM fusion oligo are combined with the following reagents at the final concentrations indicated: 10X Mg-free buffer (1X), MgCl<sub>2</sub> (2mM), dNTP mix (200 pM of each dNTP), Taq DNA polymerase
ES 2 298 249 T3 (2.5 units) and water to bring the mixture to a final volume of 100 µl. A Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) is used to amplify under the following conditions: 35 cycles at 95 ° C for 1 minute, 55 ° C for two minutes, and 72 ° C for three minutes. This procedure results in a "mega primer."
The resulting mega primer is run on a 4% low melting agarose gel in 1X TAE. The mega primer band is cut from the gel and purified using the Promega Magic PCR Preps DNA Purification System. The purified mega primer is then used in a second PCR step. One pg of genomic template 2, approximately 0.5 pg of the mega primer and 5 µl of oligo 3 '10 pM are added to a pool of 10X buffer, MgCl<sub>2</sub>, dNTP and Taq, at the same final concentrations as indicated above, and made up to 100 µl of volume with water. The PCR conditions are the same as indicated above. The fusion product resulting from this amplification is also purified using the Promega Magic PCR Preps DNA Purification System.
The fusion product is then ligated with a TA vector and transformed into E. coli using the Invitrogen (San Diego, CA) TA Cloning Kit. Approximately 50 ng of fusion PCR product is ligated with 50 ng of vector pCRII with 1X ligation buffer and 4 units of T4 ligase and brought to a volume of 10 µl with water. This bound product mixture is incubated at 12 ° C overnight (approximately 14 hours). Two µl of the ligation product mixture are added to 50 µl of competent INC F 'cells and 2 µl of beta-mercaptoethanol. The cells are then incubated for 30 minutes, followed by a heat shock treatment at 42 ° C for 60 seconds and an ice chill for two minutes. Then, 450 µl of heated SOC medium is added to the cells, resulting in a transformed cell culture that is incubated at 37 ° C for one hour with light shaking. 50 µl of the transformed cell culture is plated onto LB + 50 µg / µl ampicillin plates and incubated overnight at 37 ° C. Individual white colonies are picked and added to individual cultures, containing 3 ml of LB with ampicillin (50 pg / pl), overnight.
Individual overnight cultures are prepared using Promega's Magic Miniprep DNA purification system. A small amount of the resulting DNA is cut out using a restriction digest as a check. Next, DNA sequencing is performed to check the sequence of the fusion nucleic acid sequence, using the United States Biochemical (Cleveland, OH) Sequenase Version 2.0 sequencing kit. Three to five pg of plasmid DNA are used per reaction. 2 µl of 2M NAOH / 2 mM EDTA are added to the DNA and the volume is made up to 20 µl with water. The mixture is then incubated at room temperature for five minutes. 7 µl of water, 3 µl of 3m NaAc and 75 µl of ethanol are added. The resulting mixture is vortexed and incubated for ten minutes at -70 ° C and then microcentrifuged. After a ten minute microcentrifugation, the supernatant is removed by aspiration and the pellet is dried on a speed vac type centrifugal evaporator for 30 seconds. Then, 6 µl of water, 2 µl of hybridization buffer and 2 µl of the appropriate 10 µM oligo are added. This mixture is incubated for 10 minutes at 37 ° C and then allowed to stand at room temperature for 10 minutes. Subsequently, 5.5 µl of the labeling combination (described above) is added to each sample of the mixture, which is incubated at room temperature for an additional five minutes. Then, 3.5 µl of labeled DNA is added to each sample, which is then incubated for five minutes at 37 ° C. 4pl of stop solution is added to each well. The DNA is denatured at 95 ° C for two minutes and then placed on ice.
The clones with the desired fusion nucleic acid sequences are then recloned in the reading frame of the pET expression system, in lipidated (full length) and non-lipidated (truncated, i.e. without the first 17 amino acids) form. . The product is amplified using restriction sites contained in the PCR primers. The vector and product are cut with the same enzymes and ligated together with T4 ligase. The resulting plasmid is transformed into E. coli competent, using standard transformation techniques. Colonies are selected, as described above, and positive clones transformed into expressing cells, such as E. coli BL21, for protein expression with IPTG for induction. The expressed protein, in its bacterial culture lysate form and / or in purified form, is then injected into mice for the production of antibodies. Mice are bled and sera are collected for agglutination, in vitro growth inhibition, and complement dependent and independent lysis assays.
A specific example of chimeric OspA is as follows. Other OspA chimeras can be generated using the same procedure with suitable primers.
OspA-K48 / OspA-PGau
A chimera of OspA from strain K48 (OspA-K48) and OspA from strain PGau (OspA-PGau) was generated using the procedure described above. This chimeric nucleic acid sequence included bps 1-654 of OspAK48, followed by bps 655-820 of OspA-PGau. The primers used included: OspA amino-terminal sequence primer No. 607 (SEQ ID No. 16); the fusion primer, 5'-AAAGTAGAAGTTTTTGAATCCCATTTTCCAGTTTTTTT-3 '(negative strand primer No. 668-654) (SEQ ID No. 27); OspA carboxy-terminal sequence primer No. 586 (SEQ ID No. 19); and the sequence primers No. 369 (SEQ ID No. 14) and No. 357 (SEQ ID No. 15). The chimeric nucleic acid sequence is presented as SEQ ID NO: 28; the chimeric protein encoded by this chimeric nucleic acid sequence is reported as SEQ ID NO: 29.
ES 2 298 249 T3
C. Purification of proteins generated by chimeric nucleic acid sequences
The chimeric nucleic acid sequences described above, as well as the chimeric nucleic acid sequences produced by the methods described above, are used to produce chimeric proteins encoded by the nucleic acid sequences. Conventional procedures, such as those described above in Example 3, as regards expression of Borrelia gene proteins, can be used to express the proteins in a compatible host organism. The chimeric proteins can then be isolated and purified using standard techniques.
Nucleic acids encoding modified versions of OspA can be used to generate OspA chimeras. In addition, nucleic acids encoding OspA chimeras can be used to generate the modified OspA polypeptides of the present invention.
If the chimeric protein is soluble, it can be purified on a sepharose column. Insoluble proteins can be solubilized in guanidine and purified on a Ni column.<sup>2+</sup>; alternatively, they can be solubilized in NaPO<sub>4 </sub>10 mM with 0.1-1% TRIXON X 114 and subsequently purified on an S column (Pharmacia). Lipidated proteins were generally purified by the latter procedure. Solubility was determined by separating both the soluble and insoluble fractions of the cell lysate on a 12% PAGE gel and verifying the localization of the protein by Coomassie staining or by Western blotting with monoclonal antibodies directed against a polypeptide. chimeric protein antigen.
Example 5
Generation of chimeric nucleic acids and chimeric proteins OspC / OspA
A. General protocol for the generation of chimeric nucleic acid sequences
A large number of chimeric nucleic acid sequences encoding proteins comprising at least a first and a second Borrelia burgdorferi polypeptide were generated. These chimeric nucleic acid sequences were produced such that the chimeric protein they encoded comprised a Borrelia burgdorferi OspC polypeptide upstream of (or N-terminal to) a Borrelia burgdorferi OspA polypeptide. The chimeric nucleic acid sequences were also produced such that the nucleic acid encoding one polypeptide was in the same reading frame as the nucleic acid sequence encoding the next polypeptide in the chimeric protein.
The general cloning strategy used to construct the chimeric nucleic acid sequences was as follows. The desired OspC fragment was amplified using a 5 'primer, which contained a suitable restriction site for cloning the resulting product into a vector of interest, and a 3' primer, which contained a suitable restriction site for ligation of the fragment of OspC with the OspA fragment. The OspC product was cloned into a suitable vector. For the OspA portion of the chimeric nucleic acid, the desired OspA fragment was amplified using a 5 'primer, which contained a restriction site for ligation of the resulting OspA fragment with the OspC fragment, and a 3' primer, which it contained a suitable restriction site for cloning the resulting OspA product into the vector with the OspC product. The use of a restriction site to allow ligation of the OspC and OspA fragments results in the insertion of 0 to about 3 amino acids between the OspC and OspA fragments.
A specific example of such a construction is the following. It is understood that other suitable restriction sites can be used without any experimentation or with only routine experimentation. The resulting OspC / OspA chimera may therefore have the addition of 0 to about 3 amino acids or more between the OspC and OspA fragments, depending on the restriction site used.
For the OspC portions of the chimeric nucleic acids, the desired fragments of OspC genes from various strains or genospecies were PCR amplified using a 5 'primer, which contained an NdeI site, and a 3' primer, which contained an NcoI site. and a BamHI site. The amplified OspC product was then cloned into the NdeI and BamHI sites of the expression vector pET9c, driven by the T7 promoter. For the OspA portion of the chimeric nucleic acid, desired fragments of OspA genes from a strain of interest, or a genospecies of interest, were PCR amplified using a 5 'primer, containing an NcoI site, and a primer 3 ', which contained a BamHI site. This portion of OspA could then be cloned directly into the NcoI and BamHI sites of the vector pET9c, which contained the desired OspC sequence, thereby producing the desired OspC-OspA construct. By including the NcoI restriction site sequence in the primers, a nine nucleotide linker sequence was produced encoding the amino acids Ser-Met-Ala at the junction between the N-terminal sequence of OspC and the C-terminal sequence. of OspA. The use of the restriction enzyme NcoI (CCATGG) in this cloning strategy was a suitable selection since Borrelia is an AT-rich organism that has only a few NcoI sites in its genome. One of skill in the art will know that different restriction sites can be used and will know how to generate the OspC / A chimeric constructs for use in the subsequent modifications described herein, without any experimentation or with only routine experimentation.
ES 2 298 249 T3
As an example, OspC-OspA chimeric nucleic acids, containing non-lipidated B31 OspC, were generated using the following primers:
(5'OspC-NdeI): 5'-GT CAT ATG GCT TGT AAT AAT TCA GGG AAA GA-3 '(SEQ ID NO: 91); and (3'OspC-NcoI): 5'-T TTC CAT GGA AGG TTT TTT TGG ACT TTC TG-3 '(SEQ ID NO: 92).
For the chimeric OspC-OspA nucleic acids, containing non-lipidated B31 OspA, the following primers were used:
(5'OspA-NcoI): 5'-TT TCC ATG GCC AAG CAA AAT GTT AGC AGC C-3 '(SEQ ID NO: 93); and (3'OspA-BamHI): 5'-TAA GGA TCC TTA TTT TAA AGC GTT TTT-3 '(SEQ ID NO: 94).
Lipidated versions of OspC / OspA chimeras can be constructed by designing primers to amplify the portion of the template that includes the lipidation signal sequence or by generating a nucleic acid construct with a suitable lipidation signal sequence. The leader sequence comprising a lipidation signal can be, for example, from a gene encoding OspA, OspB or OspC polypeptides.
B. Protein expression
As described in the previous examples, it is possible to express and purify Borrelia proteins or polypeptides, for example, OspA polypeptides, OspC polypeptides, chimeric OspC / OspA polypeptides, and polypeptides comprising the OspA modifications described herein. document. This is accomplished by incorporating the desired nucleic acid sequence, encoding the protein of choice, into an expression plasmid, for example, using standard techniques. This expression plasmid can then be transfected into a compatible host cell to express the desired protein.
For example, purified chimeric OspA, OspC or OspC / OspA proteins, which were used to immunize mice and in the ELISA assays described below, were generated and purified by cloning the OspA nucleic acid sequences, OspC or chimeric OspC / OspA in the open reading frame of the expression plasmid pET. The expression plasmid was then transfected into the compatible expression cell line of Escherichia coli strain BL21 (pLysS) or B834 (DE3). BL21 or B834 cells were cultured in 10 ml of LB medium (5 g / l NaCl, 10 g / l tryptone, 5 g / l yeast extract, 25 mg / l chloramphenicol, and 50 mg / l ampicillin) at 37 ° C with agitation. When the optical density at 600λ reached 0.3-0.4 units, recombinant protein expression was induced by the addition of IPTG (isopropyl β-D-thiogalactopyranoside) to a final concentration of 0.5 mM and the cells were they cultivated for an additional three hours. The cultures were harvested by centrifugation at 3800 xg for five minutes. Cells were resuspended in NaPO<sub>4</sub> 20 mM, pH 7.7, and stored at -20 ° C overnight. Once thawed, the crude extracts were incubated with DNase (2 pg / ml) in the presence of MgCl<sub>2</sub> 2.5 mM at room temperature for thirty minutes and then centrifuged at 14,000 rpm (Eppendorf 5417C) for five minutes.
To purify the OspC proteins described below, crude extracts from cells expressing OspC were loaded onto an anion exchange column (Q Sepharose Fast Flow, 2.2 x 10 cm, Pharmacia) that had been pre-equilibrated with Tris- 20 mM Cl at pH 9.3. The column was washed in the same buffer (20 mM Tris-Cl, pH 9.3) that eluted the OspC protein. The wash fractions containing OspC were concentrated using an Amicon 10K and then dialyzed with a solution containing 20 mM NaPO4, pH 8.0, and 250 mM NaCl. The partially purified OspC was then passed through a Ni metal affinity column.<sup>2+</sup> (Chelating Sepharose Fast Flow 2.2 x 10cm) balanced with NaPO<sub>4</sub> 20 mM, pH 8.0, and 250 mM NaCl. The column was washed using a decreasing pH gradient of 20 mM sodium acetic acid and 250 mM NaCl and the bound OspC eluted at a pH of about 5.7. The OspC fractions were then concentrated by ultrafiltration and stored at -70 ° C.
For the purification of OspA proteins, the same procedure was followed except that the dialysis step, after separation with Amicon 10 K, was carried out in NaPO<sub>4</sub> 20 mM, pH 6.0. The partially purified OspA was then applied to a cation exchange column (S Sepharose Fast Flow 2.2 x 10 cm, Pharmacia) equilibrated with 20 nM NaPO4, pH 6.0. The column was washed using an increasing NaCl gradient from 0 to 100 mM. Fractions containing OspA were concentrated by ultrafiltration and stored at -70 ° C.
As noted above, both lipidated and non-lipidated (eg, truncated, lacking the first 17 amino acids) forms of the chimeric OspC, OspA, and OspC / OspA proteins were generated.
The techniques described above were also used to express proteins comprising the modified OspA polypeptides of the present invention.
ES 2 298 249 T3
C. Immunization of mice and serological characterization using ELISA (Enzyme Linked Immunosorbent Assay)
Immunization of mice
C3H-J or ICR mice were immunized with 3 pg of lipidated OspC / OspA chimeric protein or with 6 pg of non-lipidated OspC / OspA chimera in 100 ml of aluminum hydroxide adjuvant (concentration of 1.8 mg / ml) by injection subcutaneously (SC). As a negative control, mice were immunized with only 100 ml of aluminum hydroxide adjuvant. All mice received a total of three injections which were given at two week intervals. One week after the final immunization, blood was drawn from each mouse (including negative control mice) and serum was analyzed for IgG reactivity, using the ELISA procedure described below to determine the presence of antibodies. anti-OspA against three different purified OspA proteins (Borrelia burgdorferi sensu stricto (B31), Borrelia garinii (K48) and Borrelia afzelii (PGau)). Sera were tested at a 1: 1000 dilution.
Mice were immunized with the chimeric proteins described in Table III.
TABLE III
Chimeric proteins used to immunize mice
<td>Name</td><td>Description (amino acids)</td><td>SEC m N °: (nucleic acid)</td><td>SEQ ID NO: (polypeptide)</td><td>Fig. N °:</td>
<td>OspA</td><td>OspA-B31 (18-273)</td><td> 6</td><td> 7</td><td> 22, 23</td>
<td>OspC</td><td>OspC-B31 (19-211)</td><td> ★</td><td> *</td><td> 22, 23</td>
<td>OspC2-OspA</td><td>OspC-C2 (19-204) / OspA-B31 (18-273)</td><td> 59</td><td> 60</td><td> 22, 23</td>
<td><sup>1</sup>lipOspAP / Bo</td><td>OspA-PGau (1-217) / OspA-Bo (218-273)</td><td> 49</td><td> 50</td><td> 24, 25 _</td>
ES 2 298 249 T3
<td><sup>1</sup>lipOspAB / P</td><td>OspA-B31 (1-216) / OspA-Pko (217-273)</td><td> *</td><td> *</td><td> 24, 25</td>
<td>OspC- OspAB / P</td><td>OspC-B31 (19-211) / OspA-B31 (18-216) / OspA-Pko (217-273)</td><td> 65</td><td> 66</td><td> 24, 25, 27, 28, 29</td>
<td>OspCB31- OspAB31</td><td>OspC-B31 (19-211) / OspA-B31 (18-273)</td><td> 55</td><td> 56</td><td> 26, 27, 28, 29</td>
<td>OspC2- OspAB31</td><td>OspC-C2 (19-204) / OspA-B31 (18-273)</td><td> 59</td><td> 60</td><td> 26, 27</td>
<td>Ίίρ OspA K / T</td><td>OspA-K48 (1-217) / OspA-Tro (218-273)</td><td> *</td><td>Y</td><td> 27</td>
<td><sup>1</sup>lip OspC-B31</td><td>OspC-B31 (1-211)</td><td> *</td><td> ★</td><td> 26</td>
<td>OspCB31- OspABPBP</td><td>OspC-B31 (19-211) / OspA-B31 (30-150) / OspA-Pko (151-179) / OspA-B31 (180-216) (190 N deletion) / OspA-Pko (217-273) B31 / B31 / Pko</td><td> 87</td><td> 88</td><td> 28, 29</td>
<sup>1</sup>"Lip refers to the chimeric protein containing its native N-terminal lipidation signal." Serological characterization using ELISA (Enzyme Linked Immunosorbent Assay)
Immobilization of antigen on ELISA plates
A purified recombinant OspC or OspA protein solution from each of the Borrelia burgdorferi strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and PGau (Borrelia afzelii) was added to sodium phosphate buffer, pH 9.0, and It was used to coat a commercial microwell plate (MaxiSorp®, Nunc). The coating procedure was as follows: 100 µl of a solution containing the appropriate OspA or OspC protein (prepared at a concentration of 250 ng / ml in the following coating buffer was added: 100 mM Bis-Tris-propane, pH 9.7) to each well of a microtiter plate that was incubated for one hour at 37 ° C. The antigen solution was removed from the wells, the plate was washed three times with phosphate buffered saline (PBS) at pH 9.0 and 300 pi of blocking buffer solution (3% milk powder, polyoxyethylene sorbitan) was added. 0.1% (referred to in this document as Tween 20<sup>TM</sup>) and 0.02% NaN3 in 100nM Bis-Tris-propane, pH 9.7). After one hour of incubation at 37 ° C, the plates were washed four times with TBS-Tween 20 wash buffer.<sup>TM</sup> (20 mM TrisCl pH 7.5, 136 mM NaCl, Tween 20<sup>TM</sup> 0.1% and 0.02% NaN3) and then allowed to dry. The plates were then wrapped in plastic and stored at 4 ° C until use.
ES 2 298 249 T3
ELISA Assays (Enzyme Linked Immunosorbent Assay)
The standard procedure for ELISA assays was as follows: mouse serum was diluted 1: 1000 in sample dilution buffer (1% milk powder, 136 mM NaCl, Tween 20<sup>TM</sup> 0.1%, 0.02% NaN3 in 20 mM Tris-Cl, pH 7.5) and 100 μ of the diluted serum was added to the wells of the ELISA microtiter plate, which had been coated with the antigen as described above. After incubation for 1 hour at 37 ° C, the samples were removed and the plates were washed four times in TBS-Tween<sup>TM</sup> (20 mM Tris-Cl, pH 7.5; 136 mM NaCl; Tween 20<sup>TM</sup> 0.1% and 0.02% NaN3). For the secondary antibody, alkaline phosphatase-conjugated goat anti-mouse antiserum specific for IgM (Fc) or IgG (Fab) (Jackson Immuno Research Laboratories) was diluted 1: 750 in sample dilution buffer (1% milk powder, NaCl 136 mM, Tween 20<sup>TM</sup> 0.1%, NaN<sub>3</sub> 0.02% in 20 mM Tris-Cl, pH 7.5) and 100 µ of the diluted secondary antibody was added to each well. After incubation for thirty minutes at 37 ° C, the plates were washed three times with TBS-Tween<sup>TM</sup> and 100 joules of phosphatase substrate solution (5 mg of p-nitrophenylphosphate tablets dissolved in 1X diethanolamine substrate buffer were added to each well to produce a 2 mg / ml solution - Kirkegaard Perry Laboratory). The plates were incubated for thirty minutes at 37 ° C and 100 µ of stop solution (5% EDTA) was added to each well. The absorbance at 405 nm was read on a microplate reader (Dynatech). A sample was considered positive if it produced a mean absorbance higher than the mean of the negative controls plus three standard deviations.
Previous work has shown that it is the carboxy-terminal region of OspA that contains the antigenic sites that provide the immunoprotective response. Therefore, in addition to the ELISA assay described above, a modified ELISA (referred to herein as the protective ELISA assay) was performed, in which the N-terminal region (amino acids 18-139) of purified OspA from B31 was used. in excess to block any antibody present in mouse serum that had specificity for this N-terminal region of OspA. These protective ELISA assays were performed as described above, except that 80 jug / ml of a purified OspA fragment of B31 (amino acids 18-139) was added to the diluted mouse serum prior to adding the sera to the wells of ELISA microtiter plate coated with antigen.
ELISA test results
Using the ELISA assays described above, it was demonstrated that mice immunized with a non-lipidated OspC / OspA chimeric protein (OspC2-OspA composed of OspC (amino acids 19-204 of strain C2) / OspA (amino acids 18-273 of strain B31) (SEQ ID NO: 60) produced an immune response for both OspA and OspC that was comparable to the immune response generated against control proteins OspA non-lipidated (OspA amino acids 18-273 of strain B31) and OspC non-lipidated (OspC - amino acids 19-211 of strain B31) (Figure 22). As indicated in Figure 22 and described above, mice were immunized with OspA, OspC, or OspC2-OspA proteins and serum immune responses were measured against B31 OspA antigen (dotted bars) and OspC antigen. of B31 (filled bars).
Using the protective ELISA assay described above, it was also demonstrated that mice immunized with the same chimeric protein OspC / OspA non-lipidated (OspC2-OspA composed of OspC (amino acids 19-204 of strain C2) / OspA (amino acids 18-273 strain B31) (SEQ ID NO: 60) produced an immune response against the C-terminal portion of OspA that was comparable to the immune response generated against the C-terminal portion of a non-lipidated OspA control protein (OspA - amino acids 18-273 of strain B31) (Figure 2. 3). As indicated in Figure 23, the mice were immunized with OspA, OspC or OspC2-OspA proteins and the immune responses of the sera were measured against the OspA antigen of B31. The protective antibody response against the OspA antigen of B31 is indicated by the dotted bars.
Therefore, these results clearly demonstrate that non-lipidated OspC / OspA chimeric proteins are capable of inducing immune responses in mice that are comparable to the immune response generated against non-lipidated OspC and OspA control proteins.
It was previously thought that the lipidation signals that are present in the outer surface proteins of Borrelia burgdorferi were necessary for immunogenicity and that the OspC and OspA proteins lacking this lipidation signal were less or non-immunogenic. To evaluate this idea, mice were immunized with a non-lipidated OspC / OspA chimeric protein (OspC-OspAB / P composed of OspC (amino acids 19-211 of strain B31) / OspA (amino acids 18-216 of strain B31) / OspA (amino acids 217-273 of the Pko strain) (SEQ ID NO: 66), as well as with two lipidated OspA proteins, lipOspAP / Bo (composed of OspA (amino acids 1-217 of the PGau strain) / OspA (amino acids 218-273 of the Bo strain)) and lipOspAB / P (composed of OspA ( amino acids 1-216 of strain B31) / OspA (amino acids 217-273 of strain Pko)), and subjected to ELISA assays. Mice immunized with the non-lipidated OspC / OspA chimeric protein (OspC-OspAB / P) produced an immune response against OspA from each of the Borrelia burgdorferi strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and PGau ( Borrelia afzelii), which was equivalent to or superior to the immune response generated against the two lipidated OspA control proteins (lipOspAP / Bo and lipOspAb / P) (Figure 24).
Similar results to these were obtained using the protective ELISA assay described above. Mice immunized with the chimeric protein OspC / OspA non-lipidated (OspC-OspAB / P) produced an immune response against the C-terminal region of OspA of each of the Borrelia burgdorferi strains B31 (Borrelia burgdorferi sensu stricto), K48 ( Borrelia garinii) and PGau (Borrelia afzelii), which was equivalent to or greater than the immune response generated
ES 2 298 249 T3 against the C-terminal region of OspA of the two lipidated OspA control proteins (lipOspAP / Bo and lipOspAb / P) (Figure 25).
In addition to comparisons between non-lipidated OspC / OspA chimeric proteins and lipidated OspA control proteins, experiments were also performed to compare non-lipidated OspC / OspA chimeric proteins with a lipidated OspC control protein (Figure 26). Mice that were immunized with the non-lipidated OspC / OspA chimeric protein OspC31-OspAB31 (composed of OspC (amino acids 19-211 of strain B31) / OspA (amino acids 18273 of strain B31) (SEQ ID NO: 56) or with the non-lipidated OspC / OspA chimeric protein OspC2-OspAB31 (composed of OspC (amino acids 19-204 of strain C2) / OspA (amino acids 18-273 of strain B31) (SEQ ID NO: 60) produced an immune response against OspC obtained from Borrelia burgdorferi strain B31 that was comparable to the immune response produced by a lipidated OspC control protein (lipOspC-B31 - composed of OspC (amino acids 1-211 of strain B31 )) (Figure 26).
Therefore, these results clearly demonstrate that non-lipidated OspC / OspA chimeric proteins are capable of inducing immune responses against OspA and OspC that are comparable to the immune response generated against OspA and OspC using lipidated OspA or OspC control proteins. The use of non-lipidated forms of these proteins as vaccine immunogens or as diagnostic antigens is highly desirable because the product yield is much higher and the proteins are much easier to purify. For these reasons, the production of these proteins is less expensive.
The chimeric OspC / OspA proteins of the present invention are also capable of generating immune responses against OspA proteins that are obtained from strains that are not represented in the chimeric protein. Mice immunized with the chimeric proteins OspC / OspA OspCB31-OspAB31 (SEQ ID NO: 56) and OspC2-OspAB31 (SEQ ID NO: 60) are not only capable of generating immune responses that recognize OspA obtained from the strain B31 (Borrelia burgdorferi sensu stricto), but also recognize OspA obtained from strain K48 (Borrelia garinii) and from strain PGau (Borrelia afzelii) (Figure 27). For comparative purposes, the mice were also immunized with the lipidated OspA chimeric protein lipOspAK / T (composed of OspA (amino acids 1-217 of the K48 strain) / OspA (amino acids 218-273 of the Tro strain)) (Figure 27).
Sera from mice immunized with other chimeric OspC / OspA proteins also show additional antibody responses against OspA obtained from strain B31 (Borrelia burgdorferi sensu stricto), strain K48 (Borrelia garinii), and strain PGau (Borrelia afzelii ). Therefore, Figure 28 presents the ELISA results of mice immunized with OspCB31-OspAB / P (SEQ ID NO: 66), OspCB31-OspABPBP (SEQ ID NO: 88) or OspCB31-OspAB31 (SEQ ID NO: : 56). In each case, sera from the mice immunized against OspA obtained from each of the strains B31 (Borrelia burgdorferi sensu stricto), K48 (Borrelia garinii) and PGau (Borrelia afzelii) were tested. In all cases, a strong immune response was generated (Figure 28). As with the OspC / OspA chimeric proteins described above, the three OspC / OspA chimeric proteins used to immunize the mice in Figure 27 also elicited a strong immune response against the C-terminal region of OspA, when examined using the protective ELISA assay. described above (Figure 29).
The techniques described above were also used to immunize mice and to serologically characterize the immune response against the proteins comprising the modified OspA polypeptides of the present invention.
Exposure of immunized mice to ticks
C3H-J or JCR mice, which had been immunized as described above, were also challenged to infected nymphs in the laboratory or to field nymphs. The immunized mice were placed in isolation cages and each mouse received 5-10 nymphs. All nymphs were collected and counted after 6 days. Four weeks after challenge, mice were bled and sera were analyzed using commercially available Western blot strips against Borrelia burgdorferi sensu stricto strain B31 (MarDx strips) and / or Borrelia garinii (MRL strips). . Eight weeks after challenge, mice were bled, sera were retested by Western blot, and ear and bladder samples were cultured. As a positive control, mice that had been immunized with aluminum hydroxide adjuvant only, as described above, were subjected to the same challenge.
The results of the tick challenge studies (Table IV) demonstrate that while immunization with lipidated OspC protein was unable to protect mice, as demonstrated by a positive Western blot signal (in 4 out of 5 mice), immunization with two different OspC / OspA chimeric proteins (SEQ ID NO: 56 and SEQ ID NO: 62) did provide protection, as indicated by the absence of signal in the Western blot (in 0 of 8 mice and 0 of 3 mice) (Table IV). Sham positive controls demonstrated that tick challenge was successful in all cases, as demonstrated by 100% positive signal on Western blots (Table IV). The results of the tick challenge experiments are shown in Table IV.
ES 2 298 249 T3
TABLE IV
Effect of vaccination on the transmission of Borrelia by ticks
<td>Vaccine candidate</td><td>Mouse</td><td>Tick nymph</td><td>Seroconversion (Western blots) in vaccinated</td><td>Seroconversion (Western blots) in sham</td>
<td>OspC1- OspAB31</td><td>C3H-J</td><td>Long Island</td><td> 0+/8</td><td> 8+/8</td>
<td>OspC2- OspAB31</td><td>C3H-J</td><td>long Island</td><td> 0+/3</td><td> 4+/4</td>
<td>Lip OspC12</td><td>ICR</td><td>long Island</td><td> 4+/5</td><td> 54-/5</td>
The techniques described above are also used to measure the ability of mice immunized with proteins comprising the modified OspA polypeptides of the present invention to resist or respond to transmission of Borrelia by ticks.
Example 6
Generation of OspA M1, M2, M3, J1, J2 and J3 constructs using site-directed mutagenesis and PCR
All constructs were prepared using the Stratagene's QuikChange site-directed mutagenesis kit. Site-directed mutagenesis was performed using Pfu Turbo II DNA polymerase and a thermocycler. The Pfu Turbo DNA polymerase replicates the two strands of the plasmid with high fidelity and without displacing the mutant oligonucleotide primers. The basic procedure used a supercoiled double stranded DNA vector (pET 9c for all constructs) with an insert of interest and two synthetic oligonucleotide primers containing the desired mutation (s). The oligonucleotide primers, each complementary to opposite strands of the vector, were extended during thermocycling by Pfu Turbo DNA polymerase. Incorporation of the oligonucleotide primers resulted in a mutated plasmid containing staggered nicks. After thermocycling, the linear product was treated with the restriction enzyme Dpn I, which is specific for methylated DNA. DNA isolated from most E. coli strains is methylated and therefore susceptible to Dpn I digestion. Thus, the Dpn I digestion destroyed the original DNA template, leaving only nicked plasmid DNA (which was not methylated) that contained the desired mutation (s). This nicked DNA vector (containing the desired mutation (s)) was then transformed into competent E. coli which were plated on plates containing antibiotic. Colonies containing the plasmid (encoding antibiotic resistance in addition to the modified OspA polypeptide) were cultured and the plasmids purified and sequenced to confirm that they possessed the desired mutation (s).
1) Mutants M1, M2 and M3
The mutations described herein are made using nucleic acids (eg, polynucleotides) that encode OspA polypeptides or fragments of any strain of Borrelia causing Lyme disease, such as Borrelia burgdorferi sensu stricto, Borrelia afzelii, or Borrelia garinii. As an example, the generation of M3 mutations in an OspA chimera, designated "BPBP", is described below. The generation of the OspA BPBP M3 construct was as follows. BPBP is a chimeric OspA polypeptide in which residues 1-164 are from OspA from B31, residues 165-179 are from OspA from Pko or PGau, residues 180-216 are from OspA from B31 and residues 217-273 are from OspA from Pko (where numbering is as shown in SEQ ID NO: 7).
The first stage in generating this build was to obtain the BPBP M1 build. The M1 mutation is constituted by a mutation in codon 139 (aga) of OspA from the amino acid arginine to the amino acid metio
ES 2 298 249 T3 nina (codon atg). A PCR reaction was prepared, as described above, containing a polynucleotide encoding BPBP as a DNA template and the following oligonucleotide primers:
a) 5 'R139M:
5 'gaa aaa ata aca atg gca gac gga acc 3' (SEQ ID NO: 117).
b) 3 'R139M:
5 'ggt tcc gtc tgc cat tgt tat tat ttt ttc 3' (SEQ ID NO: 118)
The PCR reaction also contained reaction buffer, 10 ng of the BPBP DNA template, 125 ng of each oligonucleotide primer, and the dNTP mix. The parameters for the PCR were those described in Table V.
TABLE V
Parameters for PCR
<img file="ES2298249T3_D0001.tif" />
After PCR, the product was transformed into competent E. coli XL1-Blue cells. The colonies formed on the selective agar plates (containing kanamycin) were cultured and the plasmids were purified and sequenced to confirm that they possessed the desired mutation. Then, the BPBP M1 construct was used as a template to generate the plasmid BPBP M2, which in addition to the R139M mutation also contained a mutation that changes the glutamic acid codon (gag) at position 160 for a codon (tat) that encodes tyrosine. The oligonucleotide primers used to generate this plasmid were:
a) 5 'E160Y
5 'gga aaa gct aaa tat gtt tta aaa ggc 3' (SEQ ID NO: 119)
b) 3 'E160Y
5 'gcc ttt taa aac ata ttt agc ttt tcc 3' (SEQ ID NO: 120)
The parameters for the PCR were those described in Table VII. The final mutation, M3, which modifies a lysine residue at position 189 to a methionine residue, was made using BPBP M2 as a DNA template. The oligonucleotide primers used to generate this plasmid were:
a) 5 'K189M
5 'gtt act tta agc atg aat att tca aaa tc 3' (SEQ ID NO: 121)
b) 3 'K189M
5 'ga ttt tga att cat gct taa agt aac 3' (SEQ ID NO: 122)
This final mutation produced the desired construct, BPBP M3.
2) J1, J2 and J3 mutants
Mutants J1, J2, and J3 were generated using the same protocol described for the generation of mutants M1, M2, and M3. Such mutants can be generated using nucleic acids (eg, polynucleotides) encoding OspA polypeptide fragments from any strain of Borrelia causing Lyme disease, such as Borrelia burgdorferi sensu stricto, Borrelia afzelii, or Borrelia garinii.
ES 2 298 249 T3
The oligonucleotide primers that were used to generate the J1 mutation (containing a Y165F mutation (codon tat to ttt) and a V166T mutation (codon gtt to act)) were as follows:
<td>a) 5 'B31 YV-FT 5 'gag gtt tta aaa ggc ttt act ctt gaa gga act c 3'</td><td>(SEQ ID NO: 123)</td>
<td>b) 3 'B31 YV-FT 5 'gag ttc ctt caa gag taa agc ctt tta aaa cct g 3'</td><td>(SEQ ID NO: 124)</td>
The oligonucleotide primers that were used to generate the J2 mutation (containing a T170K mutation (codon act to aag)) were the following:
<td>a) 5 'B31 TK 5 'tct tga agg aaa get aac tgc tg 3'</td><td>(SEQ ID NO: 125)</td>
<td>b) 3 'B31 TK 5 'cag cag tta gct ttc ctt caa ga 3'</td><td>(SEQ ID NO: 126)</td>
To generate the J3 mutant (containing a Y165F mutation (codon tat to ttt), a V166T mutation (codon gtt to act) and a T170K mutation (codon act to aag)), the template containing the J1 mutations was used with the oligonucleotide primers to generate the J2 mutation (5 'B31 TK (SEQ ID NO: 125) and B31 TK (SEQ ID NO: 126)).
The modified OspA polypeptides described herein are expressed, used to immunize mice, and characterized using an ELISA as described in the previous Experiments.
Contents29
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Numbers
- Publication
- 2298249
- Publication, DOCDB
- 2298249
- Publication, EPODOC
- ES2298249T
- Application
- 1964163
- Application, DOCDB
- 01964163
- Application, EPODOC
- ES20010964163T
Titles2
- Spanish
- OSPA MODIFICADA DE BORRELIA BURGDORFERI.
- English
- MODIFIED OSPA OF BORRELIA BURGDORFERI.
Classification
- CPC, 4
- C07K14/20
- A61K39/00
- C07K2319/00
- Y02A50/30
- IPC, 6
- C07K14 20
- A61K39 00
- A61K39 02
- C07K19 00
- C12N1 21
- C12N15 31