Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

Abstract

Members of the Camelidae family produce immunoglobulins devoid of light chains. We have characterized variable domains of these heavy chain antibodies, the VHH, from llamas immunized with human immunodeficiency virus type 1 (HIV-1) envelope protein gp120 in order to identify VHH that can inhibit HIV-1 infection. To increase the chances of isolating neutralizing VHH, we employed a functional selection approach, involving panning of phage libraries expressing the VHH repertoire on recombinant gp120, followed by a competitive elution with soluble CD4. By immunizing with gp120 derived from an HIV-1 subtype B /C primary isolate, followed by panning on gp120 from HIV-1 isolates of subtypes A, B, and C, we could select for VHH with cross-subtype neutralizing activity. Three VHH able to neutralize HIV-1 primary isolates of subtypes B and C were characterized. These bound to recombinant gp120 with affinities close to the suggested affinity ceiling for in vivo-maturated antibodies and competed with soluble CD4 for this binding, indicating that their mechanism of neutralization involves interacting with the functional envelope spike prior to binding to CD4. The most potent VHH in terms of low 50% inhibitory concentration (IC 50 ) and IC 90 values and cross-subtype reactivity was A12. These results indicate that camelid VHH can be potent HIV-1 entry inhibitors. Since VHH are stable and can be produced at a relatively low cost, they may be considered for applications such as HIV-1 microbicide development. Antienvelope VHH might also prove useful in defining neutralizing and nonneutralizing epitopes on HIV-1 envelope proteins, with implications for HIV-1 vaccine design.

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

“…One of the most direct mechanisms is blocking the receptor binding sites. Such neutralizing mAbs and Nanobodies were previously identified for influenza virus, HIV, herpes simplex virus, rhinovirus, and others [35][36][37][38][39][40][41]. For example, in the case of HIV, with the aid of an extra long CDR3 loop, the neutralizing Nanobody D7 effectively competed for the CD4 binding site on gp120 protein [42].…”

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…Probably the clearest examples of epitopes that are more favorable for binding by sdAbs than conventional antibodies can be found in the envelope glycoprotein trimer of HIV-1: heterologous cross-strain neutralization is extraordinarily difficult to achieve by conventional antibodies, requiring months of chronic infection and multiple rounds of somatic mutation and selection, yet cross-neutralizing camelid heavy chain-only antibodies directed against the CD4-binding site 72-75 and CD4-induced sites 76-78 can be easily elicited by routine immunizations with recombinant protein antigens. Similar examples can be found for other viral pathogens.…”

Single-domain antibody fragments derived from heavy-chain antibodies: a review

“…The therapeutic effect of sdAb fragments is particularly based on the following principles: Firstly, a remarkable preference of sdAb fragments for binding into clefts and cavities on protein surfaces offers the possibility to develop selective therapeutics for efficient inhibition of enzymes (Paalanen et al 2011), neutralization of proteolytic toxins (Hussack et al 2011b) and activity modulation of cell surface proteins, such as receptors, ion channels and leukocyte ecto-enzymes (Wei et al 2011;Altintas et al 2012) involved in cancer and inflammatory diseases. Moreover, sdAb fragments recognize also cryptic epitopes hidden deeply in the clefts of virus capsids and in surface envelopes of parasites (Stijlemans et al 2004;Henderson et al 2007;Forsman et al 2008). Secondly, intracellular expression of sdAb fragments as "intrabodies" is a potential strategy to target intracellular antigens, e.g.…”