Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1992-12-31
2001-07-17
Scheiner, Laurie (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C424S188100, C424S208100, C424S093200
Reexamination Certificate
active
06261799
ABSTRACT:
The present invention relates to a method for constructing means needed for the production of new molecules capable of inducing an immunoprotective response against a virus responsible for acquired immunodeficiency syndrome (AIDS).
In an individual, this disease develops following an infection of T-helper lymphocytes by an HIV (human immunodeficiency virus) retrovirus. To date, these retroviruses have been classified in two major types: type HIV-1, which is rife essentially in Europe and North America, and type HIV-2, which is characteristic of African infections. Within a given type, the HIV retro-viruses exhibit some degree of variability from one another, which is characterized, for example, by a difference in cellular trophism or by viral proteins that differ slightly from one another. For this reason, when it is desired to refer to a particular HIV retrovirus, the term “viral strain” is preferably employed.
Generally speaking, HIV retroviruses have the following structure: the genomic RNA molecule and various associated proteins are encapsulated in a capsid of protein nature (nucleocapsid). The whole is protected by a membrane of cellular origin which has incorporated the envelope protein of viral origin.
This envelope protein, in various forms, is considered at the present time to be a potential therapeutic component of a vaccine against AIDS.
Under natural conditions, the envelope protein (env) is initially synthesized in the form of a precursor, containing at its N-terminal end a signal sequence which initiates the passage of the precursor into the endoplasmic reticulum (secretion route). This signal peptide is then removed by proteolytic cleavage. The product of this cleavage is a protein referred to as gp160, which is itself subsequently cleaved into a gp40 small subunit and a gp120 large subunit. The N-terminal end of the gp120 corresponds to the N-terminal end of the gp160, while the C-terminal end of the gp40 corresponds to the C-terminal end of the gp160.
Each of these subunits is secreted out of the infected cell. However, the gp40 remains anchored in the cell membrane via its transmembrane domain. Its C-terminal portion (intracytoplasmic domain) remains in contact with the cytoplasm, while its N-terminal portion (extracytoplasmic domain) is at the cell surface. The gp120 subunit is released outside the cell, where it interacts with the extracellular domain of the gp40 subunit. The two subunits of the envelope protein thus remain associated in the form of a complex.
The amino acid sequence of the precursors of the env proteins of various viral strains are now known. As an example, FIG. 1 presents the sequence of the precursors of the gp160 proteins of the viral strains HIV-1 Bru, HIV-1 MN, HIV-1 ELI, HIV-1 RF, HIV-1 SF2C and HIV-1 SC. Similarly, FIG. 2 presents the sequence of the precursor of the gp160 of the viral strain HIV-2 Rod.
Hereinafter, and to make the text easier to understand, the sequences of the gp160 proteins other than the gp160 of the strain HIV-1 Bru will be described below by reference to the sequence of the gp160 of the strain HIV-1 Bru (hereinafter referred to as gp160-Bru) as follows:
a) gp160-Bru possesses 831 amino acids, these being numbered from 1 to 831. In addition, gp120-Bru corresponds to the sequence of the first 486 amino acids of gp160-Bru, while gp40-Bru corresponds to the sequence beginning with the amino acid at position 487 and ending with the amino acid at position 831.
b) The sequence of a gp160 of any kind is aligned with the sequence of gp160-Bru so as to display maximum homology. For this purpose, gaps may be introduced either into the sequence of gp160-Bru or into the sequence of the gp160 of any kind. By definition, the position of an amino acid in the gp160 of any kind will be specified in identical fashion by the position of the homologous amino acid in gp160-Bru.
c) Consequently, when a gap is introduced into the sequence of the gp160 of any kind, opposite the amino acid at position x, there is no amino acid at position x in the sequence of the gp160. However, this amino acid is considered to possess a virtual presence.
d) When one or more gap(s) is/are introduced into the sequence of gp160-Bru between the amino acid at position x and the amino acid at position x+1, it is considered that, in the gp160 of any kind, the position covers at least two subpositions x
a
, x
b
, . . . , x
n
, each occupied by an amino acid. By definition, the downstream position x
n
represents the position x.
e) When one or more gap(s) is/are introduced into the sequence of gp160-Bru upstream of the amino acid at position 1, it is considered that, in the gp160 of any kind, the position 1 covers at least two subpositions 1
a
, 1
b
, . . . , 1
n
, each occupied by an amino acid. By definition, the NH
2
-terminal upstream position 1
n
represents the position 1.
f) When one or more gap(s) is/are introduced into the sequence of the gp160 of any kind opposite the NH
2
-terminal amino acid(s) of gp160-Bru, it may be noted that, by definition, the amino acid at the NH
2
-terminal position in the gp160 of any kind holds simultaneously the position 1 and its position of homologous amino acid.
g) When one or more gap(s) is/are introduced into the sequence of the gp160 of any kind opposite the COOH-terminal amino acid(s) of gp160-Bru, it may be noted that, by definition, the amino acid at the COOH-terminal position in the gp160 of any kind holds simultaneously the position 841 and its position of homologous amino acid.
At the present time, with regard to a vaccine, envelope protein is considered to be a potentially advantageous candidate from a therapeutic standpoint. However, its multichain structure in the original state constitutes a handicap in terms of feasibility. For this reason, it has been seen to be preferable to use a single-chain protein which would retain most of the epitopes of the gp120 and those of the gp40. This type of protein has already been proposed in Patent Application WO 87/6260. It is, more especially, a non-cleavable and soluble gp160 variant.
All the gp160 proteins of type HIV-1 viral strains possess, at positions 483-486, a so-called major cleavage site, recognized by one or more proteolytic enzymes. This cleavage site is the same for all the gp160 proteins described in FIG. 1, and corresponds to the sequence [SEQ ID NO.:1] Arg-Glu-Lys-Arg (REKR). The proteolytic enzymes cut immediately downstream of this cleavage site to release a gp120 and a gp40.
When this cleavage site is not there, it has been demonstrated that the proteolytic enzymes recognize, albeit with lower efficiency, a so-called minor cleavage site, and to make [sic] a cut immediately downstream of the latter. Here too, this cleavage site is the same for all the gp160 proteins shown in FIG. 1. It corresponds to the sequence Lys-Arg-Arg (KRR) at positions 477-479, according to some authors or, according to others, the sequence [SEQ ID NO.:2] Lys-Ala-Lys-Arg (KAKR) at positions 475-478.
In addition, it may be noted that the gp160 proteins of type HIV-2 viral strains possess only a major cleavage site at positions 475-478, which corresponds to the sequence [SEQ ID NO.:3] Lys-Glu-Lys-Arg (KEKR).
A non-cleavable gp160 variant is an artificial analog of a native gp160. Its amino acid sequence corresponds to that of a native gp160 in which the major cleavage site and/or the minor cleavage site has/have been eliminated.
Such a gp160 variant may be synthesized by means of recombinant DNA techniques. It suffices to isolate a DNA fragment coding for a native gp160, and then to modify the region coding for the major cleavage site by directed mutagenesis so as to obtain a DNA fragment coding for a gp160 variant insensitive to proteolytic action. This latter DNA fragment is subsequently expressed under suitable conditions to give said gp160 variant. Such a gp160 variant which no longer contains the major cleavage site is referred to hereinafter as a type A non-cleavable gp160 variant.
Preferably, the region coding for the minor
Burns Doane Swecker & Mathis L.L.P.
Parkin Jeffrey S.
Scheiner Laurie
Transgene S.A.
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