Microtiter enzyme-linked immunosorbent assay using recombinant derived antigens versus Western blot in the confirmation of presence of antibodies against the human immunodeficiency virus type 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 4E10 binds to a linear, highly conserved epitope within the membrane-proximal external region of the HIV-1 envelope glycoprotein gp41. We have delineated the peptide epitope of the broadly neutralizing 4E10 antibody to gp41 residues 671 to 683, using peptides with different lengths encompassing the previously suggested core epitope (NWFDIT). Peptide binding to the 4E10 antibody was assessed by competition enzyme-linked immunosorbent assay, and the Kd values of selected peptides were determined using surface plasmon resonance. An Ala scan of the epitope indicated that several residues, W672, F673, and T676, are essential (>1,000-fold decrease in binding upon replacement with alanine) for 4E10 recognition. In addition, five other residues, N671, D674, I675, W680, and L679, make significant contributions to 4E10 binding. In general, the Ala scan results agree well with the recently reported crystal structure of 4E10 in complex with a 13-mer peptide and with our circular dichroism analyses. Neutralization competition assays confirmed that the peptide NWFDITNWLWYIKKKK-NH2 could effectively inhibit 4E10 neutralization. Finally, to limit the conformational flexibility of the peptides, helix-promoting 2-aminoisobutyric acid residues and helix-inducing tethers were incorporated. Several peptides have significantly improved affinity (>1,000-fold) over the starting peptide and, when used as immunogens, may be more likely to elicit 4E10-like neutralizing antibodies. Hence, this study represents the first stage toward iterative development of a vaccine based on the 4E10 epitope.

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Use of standardized SCID-hu Thy/Liv mouse model for preclinical efficacy testing of anti-human immunodeficiency virus type 1 compounds.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.3.755 ◽

1996 ◽

Vol 40 (3) ◽

pp. 755-762 ◽

Cited By ~ 52

Author(s):

L Rabin ◽

M Hincenbergs ◽

M B Moreno ◽

S Warren ◽

V Linquist ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Small Animal ◽

Nucleoside Analogs ◽

Antiviral Effect ◽

Enzyme Linked Immunosorbent Assay ◽

Immunodeficiency Virus

We have developed standardized procedures and practices for infection of SCID-hu Thy/Liv mice with human immunodeficiency virus type 1 for the prophylactic administration of antiviral compounds and for evaluation of the antiviral effect in vivo. Endpoint analyses included quantitation of viral load by intracellular p24 enzyme-linked immunosorbent assay, DNA PCR for the presence of proviral genomes, flow cytometry to measure the representation of CD4+ and CD8+ cells, and cocultivation for the isolation of virus. Efficacy tests in this model are demonstrated with the nucleoside analogs zidovudine and dideoxyinosine and with the nonnucleoside reverse transcriptase inhibitor nevirapine. This small-animal model should be particularly useful in the preclinical prioritization of lead compounds within a common chemical class, in the evaluation of alternative in vivo dosing regimens, and in the determination of appropriate combination therapy in vivo.

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