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      A novel strategy for efficient production of anti-V3 human scFvs against HIV-1 clade C

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          Abstract

          Background

          Production of human monoclonal antibodies that exhibit broadly neutralizing activity is needed for preventing HIV-1 infection, however only a few such antibodies have been generated till date. Isolation of antibodies by the hybridoma technology is a cumbersome process with fewer yields. Further, the loss of unstable or slowly growing clones which may have unique binding specificities often occurs during cloning and propagation and the strongly positive clones are often lost. This has been avoided by the process described in this paper, wherein, by combining the strategy of EBV transformation and recombinant DNA technology, we constructed human single chain variable fragments (scFvs) against the third variable region (V3) of the clade C HIV-1 envelope.

          Results

          An antigen specific phage library of 7000 clones was constructed from the enriched V3- positive antibody secreting EBV transformed cells. By ligation of the digested scFv DNA into phagemid vector and bio panning against the HIV-1 consensus C and B V3 peptides followed by random selection of 40 clones, we identified 15 clones that showed V3 reactivity in phage ELISA. DNA fingerprinting analysis and sequencing showed that 13 out of the 15 clones were distinct. Expression of the positive clones was tested by SDS-PAGE and Western blot. All the 13 anti-V3 scFvs showed cross-reactivity against both the clade C and B V3 peptides and did not show any reactivity against other unrelated peptides in ELISA. Preliminary neutralization assays indicated varying degrees of neutralization of clade C and B viruses. EBV transformation, followed by antigen selection of lines to identify specific binders, enabled the selection of phage from un-cloned lines for scFv generation, thus avoiding the problems of hybridoma technology. Moreover, as the clones were pretested for antigen binding, a comparatively small library sufficed for the selection of a considerable number of unique antigen binding phage. After selection, the phage clones were propagated in a clonal manner.

          Conclusions

          This strategy can be efficiently used and is cost effective for the generation of diverse recombinant antibodies. This is the first study to generate anti-V3 scFvs against HIV-1 Clade C.

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          Most cited references31

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          Global and regional distribution of HIV-1 genetic subtypes and recombinants in 2004.

          To estimate the global and regional distribution of HIV-1 subtypes and recombinants in 2004. A study was conducted in which molecular epidemiological data on HIV-1 subtype distribution in individual countries were combined with country-specific estimates of the number of people living with HIV. HIV-1 subtype data were collected for 23 874 HIV-1 samples from 70 countries, which together accounted for 89% of all people living with HIV worldwide in 2004. The proportions of HIV-1 infections due to various subtypes detected in each country were combined with the number of HIV infected people in the respective countries to generate regional and global HIV-1 subtype distribution estimates. Subtype C accounted for 50% of all infections worldwide in 2004. Subtypes A, B, D and G accounted for 12%, 10%, 3% and 6%, respectively. The subtypes F, H, J and K together accounted for 0.94% of infections. The circulating recombinant forms CRF01_AE and CRF02_AG each were responsible for 5% of cases, and CRF03_AB for 0.1%. Other recombinants accounted for the remaining 8% of infections. All recombinant forms taken together were responsible for 18% of infections worldwide. Combining data on HIV-1 subtype distribution in individual countries with country-specific estimates of the number of people living with HIV provided a good method to generate estimates of the global and regional HIV-1 genetic diversity in 2004. The results could serve as an important resource for HIV scientists, public health officials and HIV vaccine developers.
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            CD4-dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5.

            The beta-chemokine receptor CCR-5 is an essential co-factor for fusion of HIV-1 strains of the non-syncytium-inducing (NSI) phenotype with CD4+ T-cells. The primary binding site for human immunodeficiency virus (HIV)-1 is the CD4 molecule, and the interaction is mediated by the viral surface glycoprotein gp120 (refs 6, 7). The mechanism of CCR-5 function during HIV-1 entry has not been defined, but we have shown previously that its beta-chemokine ligands prevent HIV-1 from fusing with the cell. We therefore investigated whether CCR-5 acts as a second binding site for HIV-1 simultaneously with or subsequent to the interaction between gp120 and CD4. We used a competition assay based on gp120 inhibition of the binding of the CCR-5 ligand, macrophage inflammatory protein (MIP)-1beta, to its receptor on activated CD4+ T cells or CCR-5-positive CD4- cells. We conclude that CD4 binding, although not absolutely necessary for the gp120-CCR-5 interaction, greatly increases its efficiency. Neutralizing monoclonal antibodies against several sites on gp120, including the V3 loop and CD4-induced epitopes, inhibited the interaction of gp120 with CCR-5, without affecting gp120-CD4 binding. Interference with HIV-1 binding to one or both of its receptors (CD4 and CCR-5) may be an important mechanism of virus neutralization.
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              Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.

              A prerequisite for the use of recombinant antibody technologies starting from hybridomas or immune repertoires is the reliable cloning of functional immunoglobulin genes. For this purpose, a standard phage display system was optimized for robustness, vector stability, tight control of scFv-delta geneIII expression, primer usage for PCR amplification of variable region genes, scFv assembly strategy and subsequent directional cloning using a single rare cutting restriction enzyme. This integrated cloning, screening and selection system allowed us to rapidly obtain antigen binding scFvs derived from spleen-cell repertoires of mice immunized with ampicillin as well as from all hybridoma cell lines tested to date. As representative examples, cloning of monoclonal antibodies against a his tag, leucine zippers, the tumor marker EGP-2 and the insecticide DDT is presented. Several hybridomas whose genes could not be cloned in previous experimental setups, but were successfully obtained with the present system, expressed high amounts of aberrant heavy and light chain mRNAs, which were amplified by PCR and greatly exceeded the amount of binding antibody sequences. These contaminating variable region genes were successfully eliminated by employing the optimized phage display system, thus avoiding time consuming sequencing of non-binding scFv genes. To maximize soluble expression of functional scFvs subsequent to cloning, a compatible vector series to simplify modification, detection, multimerization and rapid purification of recombinant antibody fragments was constructed.
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                Author and article information

                Journal
                BMC Biotechnol
                BMC Biotechnol
                BMC Biotechnology
                BioMed Central
                1472-6750
                2012
                15 November 2012
                : 12
                : 87
                Affiliations
                [1 ]Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
                [2 ]Department of Medicine, All India Institute of Medical Sciences, New Delhi, India
                [3 ]National Brain Research Centre, Manesar, India
                [4 ]Present address: Centre for Biodesign, Translational Health Science and Technology Institute, Gurgaon, India
                Article
                1472-6750-12-87
                10.1186/1472-6750-12-87
                3536577
                23153214
                d3704f2c-5252-4093-8441-da5cbe87bab5
                Copyright ©2012 Kumar et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 2 May 2012
                : 17 October 2012
                Categories
                Methodology Article

                Biotechnology
                hiv-1,scfv,clade c,v3
                Biotechnology
                hiv-1, scfv, clade c, v3

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