Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-12-30
2004-02-10
Stucker, Jeffrey (Department: 1648)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C435S006120, C435S320100
Reexamination Certificate
active
06689879
ABSTRACT:
TECHNICAL FIELD
The invention relates generally to modified HIV envelope (Env) polypeptides which are useful as immunizing agents or for generating an immune response in a subject, for example a cellular immune response or a protective immune response. More particularly, the invention relates Env polypeptides such as gp120, gp140 or gp160, wherein at least one of the native &bgr;-sheet configurations has been modified. The invention also pertains to methods of using these polypeptides to elicit an immune response against a broad range of HIV subtypes.
BACKGROUND OF THE INVENTION
The human immunodeficiency virus (HIV-1, also referred to as HTLV-III, LAV or HTLV-III/LAV) is the etiological agent of the acquired immune deficiency syndrome (AIDS) and related disorders. (see, e.g., Barre-Sinoussi, et al., (1983)
Science
220:868-871; Gallo et al. (1984)
Science
224:500-503; Levy et al., (1984)
Science
225:840-842; Siegal et al., (1981)
N. Engl. J. Med
. 305:1439-1444). AIDS patients usually have a long asymptomatic period followed by the progressive degeneration of the immune system and the central nervous system. Replication of the virus is highly regulated, and both latent and lytic infection of the CD4 positive helper subset of T-lymphocytes occur in tissue culture (Zagury et al., (1986)
Science
231:850-853). Molecular studies of HIV-1 show that it encodes a number of genes (Ratner et al., (1985)
Nature
313:277-284; Sanchez-Pescador et al., (1985)
Science
227:484-492), including three structural genes—gag, pol and env—that are common to all retroviruses. Nucleotide sequences from viral genomes of other retroviruses, particularly HIV-2 and simian immunodeficiency viruses, SIV (previously referred to as STLV-III), also contain these structural genes. (Guyader et al., (1987)
Nature
326:662-669; Chakrabarti et al., (1987)
Nature.
The envelope protein of HIV-1, HIV-2 and SIV is a glycoprotein of about 160 kd (gp160). During virus infection of the host cell, gp160 is cleaved by host cell proteases to form gp120 and the integral membrane protein, gp41. The gp41 portion is anchored in the membrane bilayer of virion, while the gp120 segment protrudes into the surrounding environment. gp120 and gp41 are more covalently associated and free gp120 can be released from the surface of virions and infected cells.
As depicted in
FIG. 1
, crystallography studies of the gp 120 core polypeptide indicate that this polypeptide is folded into two major domains having certain emanating structures. The inner domain (inner with respect to the N and C terminus) features a two-helix, two-stranded bundle with a small five-stranded &bgr;-sandwich at its termini-proximal end and a projection at the distal end from which the V1/V2 stem emanates. The outer domain is a staked double barrel that lies along side the inner domain so that the outer barrel and inner bundle axes are approximately parallel. Between the distal inner domain and the distal outer domain is a four-stranded bridging sheet which holds a peculiar minidomain in contact with, but distinct from, the inner, the outer domain, and the V1/V2 domain. The bridging sheet is composed of four &bgr;-strand structures (&bgr;-3, &bgr;-2, &bgr;-21, &bgr;-20, shown in FIG.
1
). The bridging region can be seen in
FIG. 1
packing primarily over the inner domain, although some surface residues of the outer domain, such as Phe 382, reach into the bridging sheet to form part of its hydrophobic core.
The basic unit of the &bgr;-sheet conformation of the bridging sheet region is the &bgr;-strand which exists as a less tightly coiled helix, with 2.0 residues per turn. The &bgr;-strand conformation is only stable when incorporated into a &bgr;-sheet, where hydrogen bonds with close to optimal geometry are formed between the peptide groups on adjacent &bgr;-strands; the dipole moments of the strands are also aligned favorably. Side chains from adjacent residues of the same strand protrude from opposite sides of the sheet and do not interact with each other, but have significant interactions with their backbone and with the side chains of neighboring strands. For a general description of &bgr;-sheets, see, e.g., T. E. Creighton,
Proteins: Structures and Molecular Properties
(W. H. Freeman and Company, 1993); and A. L. Lehninger,
Biochemistry
(Worth Publishers, Inc., 1975).
The gp120 polypeptide is instrumental in mediating entry into the host cell. Recent studies have indicated that binding of CD4 to gp120 induces a conformational change in Env that allows for binding to a co-receptor (e.g, a chemokine receptor) and subsequent entry of the virus into the cell. (Wyatt, R., et al. (1998)
Nature
393:705-711; Kwong, P., et al.(1998)
Nature
393:648-659). Referring again to
FIG. 1
, CD4 is bound into a depression formed at the interface of the outer domain, the inner domain and the bridging sheet of gp120.
Immunogenicity of the gp120 polypeptide has also been studied. For example, individuals infected by HIV-1 usually develop antibodies that can neutralize the virus in in vitro assays, and this response is directed primarily against linear neutralizing determinants in the third variable loop of gp120 glycoprotein (Javaherian, K., et al. (1989)
Proc. Natl. Acad. Sci
. 86:6786-6772; Matsushita, M., et al. (1988)
J. Virol
. 62:2107-2144; Putney, S., et al. (1986)
Science
234:1392-1395; Rushe, J. R., et al. (1988)
Proc. Nat. Acad. Sci. USA
85: 3198-3202.). However, these antibodies generally exhibit the ability to neutralize only a limited number of HIV-1 strains (Matthews, T. (1986)
Proc. Nat. Acad. Sci. USA
. 83:9709-9713; Nara, P. L., et al. (1988)
J. Virol
. 62:2622-2628; Palker, T. J., et al. (1988)
Proc. Natl. Acad. Sci. USA
. 85:1932-1936). Later in the course of HIV infection in humans, antibodies capable of neutralizing a wider range of HIV-1 isolates appear (Barre-Sinoussi, F., et al. (1983)
Science
220:868-871; Robert-Guroff, M., et al. (1985)
Nature
(London) 316:72-74; Weis, R., et al. (1985)
Nature
(London) 316:69-72; Weis, R., et al. (1986)
Nature
(London) 324:572-575).
Recent work done by Stamatatos et al (1998)
AIDS Res Hum Retroviruses
14(13):1129-39, shows that a deletion of the variable region 2 from a HIV-1
SF162
virus, which utilizes the CCR-5 co-receptor for virus entry, rendered the virus highly susceptible to serum-mediated neutralization. This V2 deleted virus was also neutralized by sera obtained from patients infected not only with clade B HIV-1 isolates but also with clade A, C, D and F HIV-1 isolates. However, deletion of the variable region 1 had no effect. Deletion of the variable regions 1 and 2 from a LAI isolate HIV-I
IIIB
also increased the susceptibility to neutralization by monoclonal antibodies whose epitopes are located within the V3 loop, the CD4-binding site, and conserved gp120 regions (Wyatt, R., et al. (1995)
J. Virol
. 69:5723-5733). Rabbit immunogenicity studies done with the HIV-1 virus with deletions in the V1/V2 and V3 region from the LAI strain, which uses the CXCR4 co-receptor for virus entry, showed no improvement in the ability of Env to raise neutralizing antibodies (Leu et al. (1998)
AIDS Res. and Human Retroviruses
. 14:151-155).
Further, a subset of the broadly reactive antibodies, found in most infected individuals, interferes with the binding of gp120 and CD4 (Kang, C. -Y., et al. (1991)
Proc. Natl. Acad. Sci. USA
. 88:6171-6175; McDougal, J. S., et al. (1986)
J. Immunol
. 137:2937-2944). Other antibodies are believed to bind to the chemokine receptor binding region after CD4 has bound to Env (Thali et al. (1993)
J. Virol
. 67:3978-3988). The fact that neutralizing antibodies generated during the course of HIV infection do not provide permanent antiviral effect may in part be due to the generation of “neutralization escapes” virus mutants and to the general decline in the host immune system associated with pathogenesis. In contrast, the presence of pre-existing neutralizing antibodies upon initial HIV-1 exposure will likely have a protective effect.
It is widely though
Barnett Susan
Hartog Karin
Martin Eric
Blackburn Robert P.
Chiron Corporation
Pasternak Dahna S.
Stucker Jeffrey
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