Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1998-04-28
2003-03-25
Minnifield, Nita (Department: 1645)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023400, C536S023700, C435S069100, C435S069700, C435S173300, C435S320100
Reexamination Certificate
active
06538118
ABSTRACT:
FIELD OF THE INVENTION
The present invention is concerned with genetic engineering to effect expression of lipoproteins from vectors containing nucleic acid molecules encoding the lipoproteins. More particularly, the present invention relates to expression of a recombinant lipoprotein wherein the lipidation thereof is from expression of a first nucleic acid sequence and the protein thereof is from expression of a second nucleic acid sequence, the first and second nucleic acid sequences, which do not naturally occur together, being contiguous. The invention further relates to expression of such lipoproteins wherein the first nucleic acid sequence encodes a Borrelia lipoprotein leader sequence. The invention also relates to recombinant lipidated proteins expressed using the nucleic acid sequence encoding the OspA leader sequence, methods of making and using the same compositions thereof and methods of using the compositions. The invention additionally relates to nucleic acid sequences encoding the recombinant lipoproteins, vectors containing and/or expressing the sequences, methods for expressing the lipoproteins and methods for making the nucleic acid sequences and vectors; compositions employing the lipoproteins, including immunogenic or vaccine compositions, such compositions preferably having improved immunogenicity; and methods of using such compositions to elicit an immunological or protective response.
Throughout this specification, various documents are referred to in order to more fully describe the state of the art to which this invention pertains. These documents are each hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
Lyme borreliosis is the most prevalent tick-borne disease in the United States as well as one of the most important tick-borne infectious diseases worldwide. The spirochete
Borrelia burgdorferi
is the causative agent for Lyme disease. Infection with
B. burgdorferi
produces local and systemic manifestations. Local symptoms that appear early after infection are a skin lesion at the site of the tick bite, termed erythema migrans. Weeks to months after infection, systemic manifestations that include rheumatic, cardiac and neurological symptoms appear. The early local phase of
B. burgdorferi
infection is easily treatable with antibiotics. However, the later systemic phases have proved to be more refractory to antibiotics.
Substantial effort has been directed toward the development of a vaccine for Lyme disease. Two distinct approaches have been used for vaccine development. One approach is to use a vaccine composed of whole inactivated spirochetes, as described by Johnson in U.S. Pat. No. 4,721,617. A whole inactivated vaccine has been shown to protect hamsters from challenge and has been licensed for use in dogs.
Due to the concerns about cross-reactive antigens within a whole cell preparation, human vaccine research has focused on the identification and development of non-cross-reactive protective antigens expressed by
B. burgdorferi
. Several candidate antigens have been identified to date. Much of this effort has focused on the most abundant outer surface protein of
B. burgdorferi
, namely outer surface protein A (OspA), as described in published PCT patent application WO 92/14488, assigned to the assignee hereof. Several versions of this protein have been shown to induce protective immunity in mouse, hamster and dog challenge studies. Clinical trials in humans have shown the formulations of OspA to be safe and immunogenic in humans [Keller et al., JAMA (1994) 271:1764-1768]. Indeed, one formulation containing recombinant lipidated OspA as described in the aforementioned WO 92/14488, is now undergoing Phase III safety/efficacy trials in humans.
While OspA is expressed in the vast majority of clinical isolates of
B. burgdorferi
from North America, a different picture has emerged from examination of the clinical Borrelia isolates in Europe. In Europe, Lyme disease is caused by three genospecies of Borrelia, namely
B. burgdorferi, B. garinii
and
B. afzelli
. In approximately half of the European isolates, OspA is not the most abundant outer surface protein. A second outer surface protein C (OspC) is the major surface antigen found on these spirochetes. In fact, a number of European clinical isolates that do not express OspA have been identified. Immunization of gerbils and mice with purified recombinant OspC produces protective immunity to
B. burgdorferi
strains expressing the homologous OspC protein [V. Preac-Mursic et al., INFECTION (1992) 20:342-349; W. S. Probert et al., INFECTION AND IMMUNITY (1994) 62:1920-1926]. The OspC protein is currently being considered as a possible component of a second generation Lyme vaccine formulation.
Recombinant proteins are promising vaccine or immunogenic composition candidates, because they can be produced at high yield and purity and manipulated to maximize desirable activities and minimize undesirable ones. However, because they can be poorly immunogenic, methods to enhance the immune response to recombinant proteins are important in the development of vaccines or immunogenic compositions.
A very promising immune stimulator is the lipid moiety N-palmitoyl-S-(2RS)-2,3-bis-(palmitoyloxy)propyl-cysteine, abbreviated Pam
3
Cys. This moiety is found at the amino terminus of the bacterial lipoproteins which are synthesized with a signal sequence that specifies lipid attachment and cleavage by signal peptidase II. Synthetic peptides that by themselves are not immunogenic induce a strong antibody response when covalently coupled to Pam
3
Cys [Bessler et al., Research Immunology (1992) 143:548-552].
In addition to an antibody response, one often needs to induce a cellular immune response, particularly cytoxic T lymphocytes (CTLs). Pam
3
Cys-coupled synthetic peptides are extremely potent inducers of CTLs, but no one has yet reported CTL induction by large recombinant lipoproteins.
The nucleic acid sequence and encoded amino acid sequence for OspA are known for several
B. burgdorferi
clinical isolates and is described, for example, in published PCT application WO 90/04411 (Symbicom AB) for B31 strain of
B. burgdorferi
and in Johnson et al., Infect. Immun. 60:1845-1853 for a comparison of the ospA operons of three
B. burgdorferi
isolates of different geographic origins, namely B31, ACA1 and Ip90.
As described in WO 90/04411, an analysis of the DNA sequence for the B31 strain shows that the OspA is encoded by an open reading frame of 819 nucleotides starting at position 151 of the DNA sequence and terminating at position 970 of the DNA sequence (see
FIG. 1
therein). The first sixteen amino acid residues of OspA constitute a hydrophobic signal sequence of OspA. The primary translation product of the full length
B. burgdorferi
gene contains a hydrophobic N-terminal signal sequence which is a substrate for the attachment of a diacyl glycerol to the sulfhydryl side chain of the adjacent cysteine residue. Following this attachment, cleavage by signal peptidase II and the attachment of a third fatty acid to the N-terminus occurs. The complete lipid moiety is termed Pam
3
Cys. It has been shown that lipidation of OspA is necessary for immunogenicity, since OspA lipoprotein with an N-terminal Pam
3
Cys moiety stimulated a strong antibody response, while ospA lacking the attached lipid did not induce any detectable antibodies [Erdile et al., Infect. Immun., (1993), 61:81-90].
Published international patent application WO 91/09870 (Mikrogen Molekularbiologische Entwicklungs-GmbH) describes the DNA sequence of the ospC gene of
B. burgdorferi
strain Pko and the OspC (termed pC in this reference) protein encoded thereby of 22 kDa molecular weight. This sequence reveals that OspC is a lipoprotein that employs a signal sequence similar to that used for OspA. Based on the findings regarding OspA, one might expect that lipidation of recombinant OspC would be useful to enhance its immunogenicity; but, as discussed below, the applicants experienced difficulti
Becker Robert S.
Erdile Lorne F.
Gray Maryann B.
Huebner Robert C.
Pyle Derek J.
Connaught Laboratories Inc.
Halloran Patrick J.
Minnifield Nita
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