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

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

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

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

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

The gp41CHRTM antigen used for immunization.

<p><b>A</b>) Schematic representation of gp41 and of the regions present in gp41CHRTM. FP, fusion peptide; HR1, N-terminal heptad repeat; C-C loop, cysteine loop, HR2, C-terminal heptad repeat; MPER, membrane proximal external region; TM, transmembrane region. The residue numbers at the domain/region boundaries are given. <b>B</b>) Gel filtration chromatogram of recombinant gp41CHRTM, which elutes at 13.3 ml from the column, similar to the elution profile of a marker protein of 158 kDa. This indicates that gp41CHRTM is most likely trimeric and may have an elongated structure. The inset shows a Coomassie stained SDS-PAGE gel with the gp41CHRTM protein band at the left and a protein marker at the right with the marker protein sizes in kDa indicated at the right.</p

Molecular modeling of the 2H10-gp41 peptide interaction suggests that 2H10 W100 is oriented towards the membrane.

<p><b>A</b>) Molecular model of the gp41 peptide interaction with 2H10 produced by HADDOCK. Salt bridges and hydrogen bonds present in the top model and found in most of the top10 models are shown as dashed lines. <b>B</b>) Superpositioning of the Cα atoms of the gp41 peptide derived from the HADDOCK 2H10-peptide model onto the structure of a late fusion intermediate of gp41 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003202#ppat.1003202-Buzon1" target="_blank">[29]</a> shows that the CDR3 W100 is oriented towards the membrane and found in the same plane as gp41 residues W678, W680 and Y681. Note that there are no clashes between the 2H10 VHH and gp41.</p

Data collection and refinement statistics.

<p>Numbers in parentheses refer to the highest resolution shell. R_merge = Σ|I _ 〈I〉|/〈ΣI〉, where I = observed intensity. R_cryst = Σ|Fo _ Fc|/Σ|Fo|, where |Fo| = observed structure factor. Amplitude and |Fc| = calculated structure factor amplitude. R_free is R_cryst for 3% of reflections excluded from the refinement.</p

Surface plasmon resonance affinity measurements reveal essential and non-essential 2H10 residues for gp41 interaction.

<p><b>A</b>) 2H10 single domain mutants. The curves are labeled with the mutant codes in colors corresponding with the colors of the curves. The mutants R56A, R96A, E98A and R100A do not bind at all to gp140-92UG037. Fitting the curves with the two-state reaction algorithm yielded K_Ds of 8.0 nM for 2H10 WT and 10.1 nM for 2H10-W100A. The other fits, except for those of the non-binding mutants, yielded K_Ds between 5.5 nM for 2H10-S29F and 18.7 nM for 2H10-R71S <b>B</b>) bi-2H10-W100A mutant and wild type bi-2H10 on immobilized gp140-92UG037. Because the maximum responses units are slightly different for the two bi-2H10 molecules, the curves were normalized to the maximum response, to be able to compare the dissociation rates well.</p

Amino acid and DNA sequence of 2H10.

<p><b>A</b>) Amino acid sequence. Secondary structure assignment is based on the crystal structure. β-strands are depicted as blue arrows. CDR regions are indicated with CDR1 to CDR3 and are underlined. Residues depicted in bold were mutated. Mutants that still showed binding are shown in green and mutants that do not bind to the antigen anymore are shown in red. <b>B</b>) DNA sequence alignment of 2H10 with the germline V-gene from which it originated. The asterisks indicate identical nucleotides. The CDR1 and CDR2 coding regions are underlined. Codons with at least two point mutations are in boldface.</p