Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2002-10-02
2004-11-23
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S006120, C435S325000, C435S339100, C435S320100, C424S184100, C424S208100
Reexamination Certificate
active
06821723
ABSTRACT:
The present invention relates to a polypeptide antigen which derives from the gp41 protein, and also to its use for immunization against HIV-related infections; these studies were cofinanced by the ANRS [French National Association for AIDS Research].
The development of a method of immunization against HIV is, to lay, one of the priorities of scientific research.
The major obstacles represented by the great genetic variability of the virus and the low exposure to the immune system of neutralizing viral epitopes considerably hinder the development of neutralizing immunity.
The HIV envelope glycoprotein, which is required to confer on the virus its infectious nature, represents the target for neutralizing antibodies. These characteristics have made this glycoprotein a subject of intense investigation.
The envelope glycoprotein (env) of the human immunodeficiency virus-1 (HIV-1) is synthesized from the precursor gp160, which gives, under the action of a protease, the gp120 and gp41 subunits.
The attachment of gp120/gp41 to the cellular receptors induces a change in conformation of gp41, from a latent (nonfusogenic) state to an active-fusion (fusogenic) state. Between these two states, there exists a transient “intermediate” state, during which gp41 is like a membrane-bound protein which is in both the viral and cell membranes (Weissenhorn et al. Nature (1997), 387 (6631), 426-30).
The use of gp41 in its fusogenic conformation, for immunization purposes, is described in WO 00/40616. According to that application, the N-helices can be used alone or in combination with the C-helices so as to reproduce, in the latter case, the fusogenic conformation of gp41.
Binding experiments have made it possible to establish, firstly, that the nonfusogenic latent state is characterized by the inaccessibility of large portions of the ectodomain of gp41. gp120 in fact interacts so as to mask the epitopes. It has, moreover, been shown that inhibition of the change in structure of the intermediate state to the fusogenic state with peptides used as competitors may affect viral infection (Weissenhorn W. et al., Molecular Membrane Biology, 1999, 16, 3-9).
The applicant proposes a novel polypeptide antigen which can be used for therapeutic and prophylactic immunization against HIV-related infections. The applicant has, in fact, demonstrated, for the first time, that a polypeptide which mimics the intermediate state of gp41 is capable of inducing antibodies which neutralize primary isolates of HIV.
The present invention therefore relates to a polypeptide comprising a sequence of formula I:
[N−S1]n−C−[S2−N]m
in which:
N represents the sequence of amino acids 30 to 77 of gp41,
C represents the sequence of amino acids 117 to 154 of gp41,
S1 and S2 are, independently of one another, either absent or represent an amino acid sequence such that the sequence of formula I adopts an alpha-helical conformation as determined by the SOPMA program under the following conditions:
number of conformational states=4
similarity limit=8, and
window width=70
n=0 or 1; m=0 or 1 and m+n=1 or 2.
Preferably, the polypeptide as defined above comprises a sequence of formula I in which m+n=1.
Preferably, S1 is absent or represents the amino acid sequence D, DQ, DQQ, DQQL or DNNMT, and S2 is absent or represents the amino acid sequence W, WA, WAS, WASL or WASLW.
The S1 and S2 amino acid sequences are defined using the one letter code in which D represents aspartic acid, Q represents glutamine, etc.
According to a particular embodiment, the polypeptide comprises a sequence of formula I as defined above, in which N represents SEQ ID No. 26 and C represents SEQ ID No. 27.
According to a preferred embodiment, the polypeptide is selected in the group consisting of SEQ ID No. 28 and SEQ ID No. 31.
According to another embodiment, the polypeptide according to the invention comprises an additional sequence of formula (G)a-S—(H)b in which G represents a glycine residue, H represents a histidine residue S is a serine, a is greater than or equal to 4 and b is greater than or equal to 6. Said sequence is linked via an amide bond to the NH
2
-terminal end or COOH-terminal end of the polypeptide according to the invention.
According to another aspect, the present invention relates to a conjugate comprising a polypeptide according to the invention conjugated to a carrier protein or peptide.
According to another aspect, the present invention relates to a DNA sequence encoding a polypeptide according to the invention or a conjugate according to the invention.
The present invention also relates to an expression vector comprising said DNA sequence, and also to a host cell containing said vector.
A subject of the present invention is also a pharmaceutical composition comprising at least one polypeptide as defined above, at least one conjugate as defined above or at least one expression vector as defined above, a pharmaceutically acceptable excipient and, optionally, an adjuvant.
According to a particular embodiment, the present invention relates to a pharmaceutical composition as defined above which can be administered orally.
A subject of the present invention is therefore also the polypeptide as defined above, for its use as a medicinal product, and in particular for its use in immunizing the human body against HIV-related infections.
Another subject of the present invention relates to the method for preparing a polypeptide as described above, comprising the expression of said polypeptide using a host cell as defined above.
The invention is described in greater detail in the description which follows.
The phenomenon of conformational change of gp41 which precedes the cell and viral membrane fusion is illustrated in FIG.
1
. Attachment of gp120 to the receptor and the coreceptor of the virus causes a first modification which leads to unfolding of the fusion peptide and anchoring thereof in the cell membrane. At this time, an intermediate state forms, in which there is no interaction between the N-helices and the C-helices. The second event illustrated in
FIG. 1
is the folding of gp41, which corresponds to the molecule adopting a thermodynamically more stable conformation. The energy released during this folding allows the lipids of the cell and viral membranes to come close to one another and fuse.
The Applicant has demonstrated, surprisingly, that the polypeptide according to the invention induces specific IgG antibodies which neutralize HIV primary isolates. The induction of antibodies which neutralize primary isolates can be determined using the neutralization test as described in the article by C. Moog et al. (AIDS Research and human retroviruses, Vol. 13(1), 13-27, 1997), to which reference may be made for a complete description of the latter. In the context of the present invention, it is estimated that neutralizing antibodies have been induced by the antigen tested according to the technique of C. Moog when the serum diluted at least to ¼, in the presence of HIV, leads to a 10-fold decrease in the viral titer in comparison to HIV alone, the viral titer being evaluated by the amount of p24 produced in the culture supernatant.
The induction of antibodies which neutralize primary isolates may also be determined using the neutralization test of D. Montefiori as described in J. Infect. Dis. 1996, 173:60-67. In this test, the neutralizing titer is expressed by the percentage decrease in p24 antigen produced in the culture supernatants when the virus is incubated in the presence of serum diluted to ¼, by comparison with the virus in the absence of serum. In the context of the present invention, it is considered that neutralizing antibodies have been induced when the decrease in the level of p24 produced reaches at least 80% with a serum diluted to ¼.
In the context of the present invention, it is considered that the antibodies induced by the polypeptide according to the invention are neutralizing antibodies if neutra
Chevalier Michel
El Habib Raphaëlle
Krell Tino
Sodoyer Regis
Aventis Pasteur S.A.
McDonnell & Boehnen Hulbert & Berghoff
Park Hankyel T.
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