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

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 (gp120Ds2), 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

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

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

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