Summary of VHH competition with mAb b12 for binding to HIV-1 envelope proteins.

<p>The amount of VHH required to reduce the b12 signal by 50% of its maximum was estimated from the respective competition curves. +++ <0.44 µg/mL; ++ 0.44–12 µg/mL; + >12 µg/mL; -,no competition was observed even at the highest amount of VHH.</p

Alignment of the VHH against the germline V, D and J segments.

<p>Amino acids sequences of HIV-1 envelope protein CD4bs specific VHH aligned against <i>Lama glama</i> V and J germ line genes and D germ line genes of <i>Lama pacos</i>. The reading frame of the most likely D germline genes are marked. Numbering of amino acid according to Kabat <i>et al</i>.. CDRs of VHH are redefined.</p

Summary of VHH binding to HIV-1 envelope proteins.

<p>The amount of VHH required to give half-maximal A₄₉₀ was estimated from the respective binding curves. +++ half-maximal binding at <0.63 µg/mL; ++, 0.63–10 µg/mL; +, >10 µg/mL; -, no binding was observed even at the highest amount of VHH. N.D., not done.</p

Neutralisation potencies of wild type VHH 2E7 and mutated variants in TZM-b1 neutralisation assay.

<p>Comparison of neutralization potencies of VHH 2E7 wild type and 5 mutants against a panel of 4 viruses. IC₅₀ values are given in µg/mL. Not done is marked as Nd.</p

Binding affinities of D7 wild type and D7 mutants to gp120 (IIIB).

<p>Binding affinities of D7 wild type and D7 mutants to gp120 (IIIB).</p

IC₅₀ values of V_HH D7 against HIV-1 IIIB in TZM-bl cells.

*<p>) Experiments were carried out in duplicate wells.</p

Heavy chain antibody response of llama 8 and 9.

<p>Heavy chain antibody response in llama 8 (A) and llama 9 (B) to gp140_UG37 (□), gp140_CN54 (▪), gp120_IIIB (▪) at indicated days following initial immunisation. Sera from llamas were collected, diluted 1000 fold and tested by ELISA for the presence of specific IgG₃ heavy chain antibodies coated recombinant HIV-1 envelope proteins.</p

Solvent accessible areas of CDR3 D7 residues.

<p>Solvent accessible areas of CDR3 D7 residues.</p

Comparison of CDR2 and CDR3 from D7, b12, b13, F105 and m18 indicates different modes of gp120 interaction.

<p>The Cα atoms of the heavy chain variable domains (b12 pdb code 2NY7; F105 pdb code 3HI1; b13 pdb code 3IDX; m18 pdb code 2AJ3) were superimposed and the CDR H2 and H3 are represented in the same orientation. Amino acids are labelled using the one letter code for clarity. (<b>A</b>) All CDR3 loops expose aromatic residues at their apex. (<b>B</b>) The CDR2 of D7 varies from CDR H2 of b12 indicating a different mode of gp120 interaction. Residues implicated in gp120 interaction are highlighted in orange.</p

Structure based sequence alignment of D7 with V_HH A12 and C8 as well as with the V_H domains from the neutralizing antibodies b12, b13, F105 and m18.

<p>The residue numbering is according to Chothia <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010482#pone.0010482-Chothia1" target="_blank">[38]</a> and the CDRs are indicated by coloured bars.</p