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The native HIV-1 envelope spike exists as trimers on the virion surface. Therefore antibodies elicited following immunisation with trimeric envelope vaccines may be directed against epitopes found on functional envelope spikes. Novel monoclonal antibodies were produced by priming mice with plasmid DNA expressing either HIV-1 CN54 or ZM96 gp140 sequences, and boosting with CN54 trimeric rgp140. Spleen cells were fused with NS-0 cells and hybridomas were screened for production of antibodies which bound trimeric rgp140. 18 monoclonal antibodies (MAbs) were isolated from 16 colonies, which were characterised by binding in ELISA and Western blotting, neutralisation of pseudotyped viruses and isotype. Specificity for the third variable (V3) region was detected in 5 of these antibodies, suggesting that the trimeric protein did not alter the main focus of the response compared with monomeric protein. 3 MAbs were identified as neutralising in a pseudotype assay, all of which were mapped to the V3 region. Although cross clade binding of antibodies was detected the neutralisation was C clade specific., (Copyright © 2013 Elsevier B.V. All rights reserved.)