Detection of specific antibodies in gingival crevicular transudate by enzyme-linked immunosorbent assay for diagnosis of human immunodeficiency virus type 1 infection.

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.

Download Full-text

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.

Download Full-text

Fruit-Specific Expression of the Human Immunodeficiency Virus Type 1 Tat Gene in Tomato Plants and Its Immunogenic Potential in Mice

Clinical and Vaccine Immunology ◽

10.1128/cvi.00028-07 ◽

2007 ◽

Vol 14 (6) ◽

pp. 685-692 ◽

Cited By ~ 27

Author(s):

Yuri Jorge Peña Ramírez ◽

Ennio Tasciotti ◽

Abel Gutierrez-Ortega ◽

Alberto J. Donayre Torres ◽

María Teresa Olivera Flores ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Tomato Fruit ◽

Enzyme Linked Immunosorbent Assay ◽

Potential Candidate ◽

Tat Protein ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) Tat protein is considered a potential candidate vaccine antigen. In an effort to design a strategy for noninvasive vaccination against HIV-1, we developed transgenic tomatoes expressing the Tat protein. Two independent plants testing positive in transgene detection analysis were selected and grown to maturity. Monoclonal antibodies against Tat recognized a protein of the expected size. Interestingly, expression of Tat seemed to be toxic to the plant, as in all cases the fruit exhibited underdeveloped reproductive structures and no seeds. Nine groups of 10 pathogen-free BALB/c male mice were primed either orally, intraperitoneally, or intramuscularly with 10 mg of tomato fruit extract derived from transgenic or wild-type plants and with 10 μg of Tat86 recombinant protein. Mice were immunized at days 0, 14, and 28, and given boosters after 15 weeks; sera were drawn 7 days after each booster, and the antibody titer was determined by enzyme-linked immunosorbent assay. All three immunization approaches induced the development of a strong anti-Tat immunological response, which increased over time. Isotype subclass determination showed the presence of mucosal (immunoglobulin A) immunity soon after the beginning of the oral immunization protocol, and the data were confirmed by the presence of anti-Tat antibodies in fecal pellets and in vaginal washes. We also demonstrated that sera from immunized mice inhibited with high efficiency recombinant Tat-dependent transactivation of the HIV-1 long terminal repeat promoter. This neutralization activity might be relevant for the suppression of extracellular Tat activities, which play an important role in HIV disease development.

Download Full-text