Production and Characterization of High-Affinity Human Monoclonal Antibodies to Human Immunodeficiency Virus Type 1 Envelope Glycoproteins in a Mouse Model Expressing Human Immunoglobulins

Abstract

Human (Hu) monoclonal antibodies (MAbs) against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) are useful tools in the structural and functional analysis of Env, are under development both as potential prophylaxis and as therapy for established HIV-1 infection, and have crucial roles in guiding the design of preventative vaccines. Despite representing more than 50% of infections globally, no MAbs have been generated in any species against C clade HIV-1 Env. To generate HuMAbs to a novel Chinese C clade Env vaccine candidate (primary isolate strain HIV-1 97CN54 ), we used BAB5 mice that express a human immunoglobulin (Ig) M antibody repertoire in place of endogenous murine immunoglobulins. When immunized with HIV-1 97CN54 Env, these mice developed antigen-specific IgM antibodies. Hybridoma fusions using splenocytes from these mice enabled the isolation of two Env-specific IgM HuMAbs: N3C5 and N03B11. N3C5 bound to HIV-1 Env from clades A and C, whereas N03B11 bound two geographically distant clade C isolates but not Env from other clades. These HuMAbs bind conformational epitopes within the immunodominant region of the gp41 ectodomain. N3C5 weakly neutralized the autologous isolate in the absence of complement and weakly enhanced infection in the presence of complement. N03B11 has no effect on infectivity in either the presence or the absence of complement. These novel HuMAbs are useful reagents for the study of HIV-1 Env relevant to the global pandemic, and mice producing human immunoglobulin present a tool for the production of such antibodies.

The production, characterisation and application of monoclonal antibodies generated by immunisation with HIV-1C clade RGP140 envelope protein

“…Previously, it has been reported that immunisation of transgenic mice with purified HIV-1 envelope protein alone (HIV-1 CN54 rgp140) derived from CHO cells resulted in isolation of very few cell lines and 2 clones (Sheppard et al, 2007). By contrast, this study demonstrates that priming with the DNA expression plasmid used to transfect CHO cells and express the HIV-1 envelope protein employed for the immune boosting yielded a higher frequency of hybridoma cell lines expressing anti-clade C HIV-1 antibodies.…”

Expression-System-Dependent Modulation of HIV-1 Envelope Glycoprotein Antigenicity and Immunogenicity

“…Antibodies (Abs), mAbs and other reagents were obtained from the same sources as previously described60, with the exception of: CD4-IgG251 manufactured by Progenics Inc. and obtained from The International AIDS Vaccine Inititative Neutralizing Antibody Consortium Repository; CD4-binding site (CD4bs)-specific mAbs 15e and 21h obtained from the National Institute of Health (NIH) AIDS Research and Reference Reagent Program; the rabbit antiserum to gp120, ARP421 obtained from the Centralised Facility for AIDS Reagents (CFAR, NIBSC, Potters Bar, UK. Donated by S, S. Ranjbar); D7324, the goat polyclonal Ab (pAb) to the gp120-C-terminal region, from Aalto Bio Reagents Ltd (Dublin, Ireland); human mAbs HJ16 (CD4bs), HR10 (V3) and HG68 (V2) from D. Corti and A. Lanzavecchia54.…”

“…ELISAs were performed as described previously60 except the plates were washed six times following the addition of antisera or mAbs and after the addition of the anti-species IgG HRP conjugates. The absorbance of pre-immune serum was subtracted from that of the post vaccine bleeds before calculation of the endpoint titre by interpolation of the point of intersect between the assay cut-off and a sigmoid dose-response curve fitted to the dilution series in GraphPad Prism v5.061.…”

Monoclonal Antibody-Based Candidate Therapeutics Against HIV Type 1

“…75,76 Recently, we have successfully isolated several cross-reactive high-affinity nonneutralizing mAbs by panning a phage-displayed library constructed from an acutely infected patient (W. Chen et al, unpublished work). These antibodies target gp41, one of which significantly competes with a cross-reactive neutralizing antibody, m44, 70 in binding to gp140s.…”

“…74 Instead of eliminating the virus, these antibodies facilitate its production in the body of infected persons by different means including antagonizing neutralizing antibodies. The reported [75][76][77] and more ''bad'' antibodies that are to be identified could be used as reagents to further characterize the neutralizing antibodies of interest.…”