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112 CCR5-Specific Human Recombinant Monoclonal Antibodies Protect from HIV-1 Infection
C. Barassi1, S. Pollini2, C. Pastori1, M. Clerici3, G. Rossolini2, and L. Lopalco*1
1San Raffaele Scientific Inst., Milan, Italy; 2Univ. of Siena, Italy; and 3Univ. of Milan, Italy
Background: Primary HIV infection is associated with CCR5-tropic HIV variants (R5-strains). Cells lacking surface CCR5 are resistant to the infection by R5-strains. Among exposed seronegatives (ESNs), 2 kinds of individuals lacking surface CCR5 have been described so far: (i) homozygotes for a defective CCR5 gene, and (ii) individuals producing anti-CCR5 antibodies that cause antigen down-regulation and a CCR5-minus phenotype.
Methods: B-EBV transformed cell lines derived from ESN (subjects exposed to HIV-1 but seronegatives) with anti-CCR5 antibodies, were used to obtain human recombinant antibody to CCR5. A phage display library to clone and produce human recombinant antibodies Fab fragment reactive to CCR5 was used. To verify the specificity of Fab to CCR5 we utilized a radio binding assay on 3T3/CCR5 transfected cell lines. To establish whether Fab fragment to CCR5 affect biological function of CCR5, chemotaxis and calcium flux were assayed. Because anti-CCR5 antibodies in ESN down-modulate CCR5 expression and redux HIV-1 infectivity, the Fab fragment to CCR5 was tested in the inhibition of HIV-p24 production, of viral entry, and in direct binding to CD4+ cells by flow cytometry.
Results: Fifty bacterial clones producing antibodies to CCR5 were selected from a human combinatorial antibody library constructed in a phage-display vector by a panning procedure against a CCR5 peptide recognised by antibodies. Preliminary data show that 1 human monoclonal antibody (LLK24) recognises CCR5, inhibits HIV viral entry, and reduces chemotaxis activity.
Conclusions: Recombinant human CCR5 reactive antibodies may be used in development of innovative and protective vaccines and/or new therapeutic strategies.
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