Cloning and molecular characterization of a human recombinant IgG Fab binding to the Tat protein of human immunodeficiency virus type 1 (HIV-1) derived from the repertoire of a seronegative patient.
A study aiming at cloning and characterizing natural antibodies to human immunodeficiency virus type 1 (HIV-1) targets is described. In particular, we report the molecular cloning of a Fab molecule binding the HIV-1/Tat protein from a seronegative patient. The Fab was characterized for its binding specificity and investigated in regards to its molecular structure. Furthermore, to evaluate the role played by the heavy and light chains in the binding to the antigen, hybrid Fabs were constructed combining the heavy and the light chain of the natural anti-Tat clone with a control high-affinity Fab derived from the repertoire of the same patient. The results indicate that the natural immunoglobulin under study: (i) is a polyreactive antibody of IgG1 isotype, and not an IgM as usually described for anti-HIV natural clones, (ii) shows a pattern of mutations compatible with an antigen-driven mechanisms, (iii) its heavy chain derives from a V-gene subfamily (V3-23) highly represented in fetal life, and (iv) its heavy chain variable region exhibits several characteristics, including an extremely long, hydrophilic CDR3, that are unusual and theoretically important in determining the polyreactive capacity of the molecule.
Mancini
Nicasio
N
Università Vita-Salute San Raffaele, San Raffaele Diagnostica e Ricerca, IRCCS Istituto Scientifico San Raffaele, Via Olgettina 60, 20132 Milano, Italy.
Perotti
Mario
M
Carletti
Silvia
S
Canducci
Filippo
F
Sampaolo
Michela
M
Clementi
Massimo
M
Burioni
Roberto
R
D003001:Cloning, Molecular; D015696:Gene Products, tat / Q000378:metabolism*; D015483:HIV Antibodies / Q000235:genetics; D018023:HIV Seronegativity / Q000235:genetics* / Q000276:immunology*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D007113:Immunity, Innate; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000378:metabolism*; D007074:Immunoglobulin G / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D015483:HIV Antibodies; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus