Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
2002-01-02
2004-11-09
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C435S005000, C435S006120, C435S069700, C530S387300, C530S388350
Reexamination Certificate
active
06815201
ABSTRACT:
BACKGROUND OF THE INVENTION
There is presently a dearth of candidate HIV vaccines that are considered suitable for wide-scale testing in humans, particularly when considering vaccines capable of inducing protective humoral immunity. Whereas live, attenuated viruses may provide protection against more pathogenic strains, safety considerations are likely to preclude the widespread use of such vaccines. A difficulty with purified envelope subunit vaccines is that while the best of these have been able to induce neutralizing responses against the vaccine strain and related laboratory-adapted, T cell-tropic HIV-1 strains, these vaccines have generally not induced neutralizing responses to primary viruses and clinical HIV-1 isolates (Hanson, 1994; Mascola et al., 1994; Matthews, 1994). This finding may be related to the general resistance of primary viruses to neutralization by sCD4 (Ashkenazi et al., 1991; Gomatos et al., 1990), monoclonal antibodies (D'Souza et al., 1995; Moore et al., 1995), and immune sera from many HIV-infected patients (Golding et al., 1994). The reason for the difference in sensitivities of primary viruses and lab isolates is not clear. It has been suggested that epigenetic factors related to the cells used to prepare the virus (Sawyer et al., 1994) and to the incorporation of host cell adhesion proteins into virion membranes (Guo and Hildreth, 1995; Hildreth and Orentas, 1989) may be involved, but it appears that structural differences in the envelope proteins of the different viruses may also be important.
Whereas it is known that some people possess potent neutralizing antibodies against primary strains of HIV, such activities are rare. Moreover, the nature of the epitopes that mediate this activity are generally unknown. A major difference between the immune responses of naturally infected individuals and those vaccinated with envelope subunit proteins is that while the humoral responses of the former are directed mostly against conformational epitopes on the viral envelope proteins that are well exposed on native virions (Moore and Ho, 1993), antibodies produced by vaccination with envelop subunit proteins are directed primarily against linear epitopes that are poorly accessible on both monomeric and cell-associated gp120 molecules (VanCott et al., 1995). The natural immune response against HIV-1 has been characterized by isolation and characterization of monoclonal antibodies (mabs) from infected individuals. These studies have utilized cell-adapted laboratory strains of HIV-1, and the mabs that have been described all have preferential neutralizing activity for lab strains over primary viruses. The major neutralization targets recognized in these studies were the V3 loop and the CD4-binding site (Chamat et al., 1992; D'Souza et al., 1994; Gorny et al., 1993; Thali et al., 1992; Tilley and Pinter, 1993).
Whereas it has been reported that some anti-V3 mabs can neutralize primary viruses (Conley et al., 1994), such neutralization is relatively inefficient, requiring 10-100 ug/ml of antibody (D'Souza et al., 1995), considerably more than that required for neutralization of susceptible lab strains. Consistent with these findings are results showing that depletion of anti-V3 antibodies from a human serum resulted in loss of neutralizing activity against the T cell-tropic MN strain, but not against several primary isolates (VanCott et al., 1995). This may be related to other evidence showing that the V3 loop in primary viruses may be buried, and not readily accessible to neutralizing antibodies (Bou-Habib et al., 1994).
A number of human mabs described in the above studies compete for binding of CD4 and have potent neutralizing activities for lab strains of HIV (Cordell et al., 1991; Ho et al., 1991; Tilley et al., 1991). These mabs are directed against conserved, conformational epitopes that are composed of residues scattered over many conserved regions of gp120 (Thali et al., 1992), including residues essential for binding of CD4 itself (Olshevsky et al., 1990). Primary viruses are much less sensitive to neutralization by these mabs than lab strains (Honnen et al., 1996; Moore et al., 1995), similar to their resistance to sCD4 itself, and there have been reports that in some cases these antibodies actually enhance infection by primary HIV-1 isolates (Lee et al., 1997; Schutten et al., 1995; Stamatatos et al., 1997). Several human mabs against other Env epitopes have been identified that have better neutralizing activities for primary isolates (Trkola et al., 1995). These include IgG b12, an anti-CD4-binding site human mab isolated from a combinatorial phage library (Burton et al., 1994), 2F5, directed against a linear epitope in gp41 (Conley et al., 1994; D'Souza et al., 1995; Muster et al., 1994; Trkola et al., 1995), and 2G12, directed against a poorly defined, glycan-dependent epitope in gp120 (Fouts et al., 1997; Trkola et al., 1996). The ability of all three of these mabs to neutralize primary viruses is a reflection of their overall increased potencies, but they also appear to have preferential activity for lab strains over primary viruses (Honnen et al., 1996).
Several studies document the role of the V1/V2 domain as a major antigenic target for HIV-1. A number of rodent mabs have been isolated from animals immunized with recombinant IIIB gp120 that are directed against linear (Fung et al., 1992) and conformational epitopes in the V2 domain (Ho et al., 1991; McKeating et al., 1993; Moore et al., 1993). HIV-infected humans have been shown to produce antibodies against linear epitopes located in both the V2 (Kayman et al., 1994; McKeating et al., 1993; Moore et al., 1993) and V1 regions (Honnen et al., 1996; Pincus et al., 1994). The linear V1 epitopes and some of the linear V2 epitopes mediate type-specific neutralization of IIIB virus and related lab strains.
Many of the anti-V2 neutralizing antibodies that have been described are directed against type-specific epitopes and appear to possess weak neutralizing activities. Thus, the significance of these antibodies for in vivo protection is unclear. Recently, however, several primate mabs have been described which have more interesting neutralizing properties. Particularly strong evidence for the role of the V1/V2 domain in neutralization of HIV-1 comes from recent studies with chimpanzee mab C108G, an antibody directed against a glycan-dependent epitope in V2 (Honnen et al., 1996; Vijh-Warrier et al., 1996; Warrier et al., 1994; Wu et al., 1995). This antibody possesses extremely potent neutralizing activities for both lab strains and primary isolates bearing the C108G epitope, including NL-HX-ADA, a primary-like, macrophage-tropic isolate.
SUMMARY OF THE INVENTION
The invention features a protein which includes a gp120 V1/V2 domain of an HIV-1 strain (or a variant or portion thereof) and not a gp120 V3 domain of an HIV-1 strain, which protein does not substantially bind CD4. For purposes of this invention, the V1/V2 domain is also intended to include the immediate conserved flanking sequences that form the conserved stem of the V1/V2 region. The gp120 V1/V2 domain of the protein displays an epitope which is recognized by an antibody which neutralizes at least one HIV-1 primary isolate with a ND
90
of less than 100 &mgr;g/ml. Useful V1/V2 domains include those of strain Case-A2B and strain SF162. Also included in the invention are fragments and derivatives of the V1/V2 region, including V1/V2 stem analogs in which a GAG triplet is inserted between the ends of the C1 and C2 regions (
FIG. 13
) and V1/V2 proteins containing deletions of various portions of the V1, V2 or the conserved flanking sequences. Such analogs can be based on any desired V1/V2 loop sequence (e.g., Case-A2B or SF162).
In various embodiments: the V1/V2 domain epitope is recognized by an antibody which neutralizes at least at least one HIV-1 primary isolate from each of at least two different clades with a ND
90
of less than 100 &mgr;g/ml; the two different clades are selected from the group consisting of lade
Fish & Richardson P.C.
Park Hankyel T.
The Public Health Research Institute of the City of New York, In
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