Llama Antibody Fragments Recognizing Various Epitopes of the CD4bs Neutralize a Broad Range of HIV-1 Subtypes A, B and C

Abstract

Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120 Ds2 ), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B′/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.

Role of Human Immunodeficiency Virus Type 1 Envelope Structure in the Induction of Broadly Neutralizing Antibodies

“…Recently, camelid Abs, which consist of a unique heavy chain and have some properties superior to those of classical antibodies, have been described. Llama heavy chain antibody fragment (VHH) that neutralizes and recognizes epitopes on CD4bs has been characterized (131). The VHH antibodies are small enough to interfere during formation of the viral synapsis and thus inhibit the crucial early steps of infection.…”

“…The VHH antibodies are small enough to interfere during formation of the viral synapsis and thus inhibit the crucial early steps of infection. Other animals producing Abs with the same properties, such as sharks and cartilaginous fishes (131,132), could provide new insights into anti-HIV vaccine development.…”

Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies

“…C. difficile TcdA and TcdB (List Biologicals, Campbell, CA) were inactivated prior to immunizations by alkylation of the catalytic domain with UDP-2=,3=-dialdehyde (Sigma-Aldrich, St. Louis, MO) (25). Immunizations and library construction were carried out as previously described (26). The prolonged llama immunizations were approved and performed according to the guidelines of Utrecht University Animal Ethical Committee (approval ID 2007.III.01.013/vervolg2).…”

“…The binding specificities of the selected VHH antibodies for TcdB were screened in the first round by using induced periplasmic extracts from the individual E. coli clones. Periplasmic extracts were prepared according to standard protocols (26) and tested for binding to TcdB (2 g/ml) immobilized on a microtiter plate as previously described (30). The bound VHH antibodies were detected using a rabbit anti-llama IgG antibody (a kind gift from Unilever Research, Vlaardingen, Netherlands) in combination with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Dako, Glostrup, Denmark).…”

Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications

“…[83][84][85][86][87] Owing to their small size and extended CDR3, nanobodies also showed special advantages as therapeutics for infectious disease, including the infection of viruses, bacteria, and parasites, over conventional antibodies that usually obstruct the access of hidden and essential epitopes on pathogens. [88][89][90][91][92][93][94][95][96] The added value of the nanobodies as targeting therapeutics stems from their capacity to distinguish the cognate target from closely related variants. Most of the small organic antagonists or even the conventional antibodies to a larger extent cannot reach such high specificity.…”