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

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

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 (IC50) and IC90 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

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

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

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

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

Quantification Assays for Total and Polyglutamine-Expanded Huntingtin Proteins

<div><p>The expansion of a CAG trinucleotide repeat in the huntingtin gene, which produces huntingtin protein with an expanded polyglutamine tract, is the cause of Huntington's disease (HD). Recent studies have reported that RNAi suppression of polyglutamine-expanded huntingtin (mutant HTT) in HD animal models can ameliorate disease phenotypes. A key requirement for such preclinical studies, as well as eventual clinical trials, aimed to reduce mutant HTT exposure is a robust method to measure HTT protein levels in select tissues. We have developed several sensitive and selective assays that measure either total human HTT or polyglutamine-expanded human HTT proteins on the electrochemiluminescence Meso Scale Discovery detection platform with an increased dynamic range over other methods. In addition, we have developed an assay to detect endogenous mouse and rat HTT proteins in pre-clinical models of HD to monitor effects on the wild type protein of both allele selective and non-selective interventions. We demonstrate the application of these assays to measure HTT protein in several HD <i>in vitro</i> cellular and <i>in vivo</i> animal model systems as well as in HD patient biosamples. Furthermore, we used purified recombinant HTT proteins as standards to quantitate the absolute amount of HTT protein in such biosamples.</p></div

Specificity of human mutant and mouse HTT detection.

<p>The adeno-associated AAV-shRNA expression vector AAV-SEWB-sh4 was transduced into heterozygous zQ175 mouse primary neurons and humanized mutant (A) or endogenous mouse (B) HTT proteins were evaluated using the expanded polyglutamine human HTT MSD assay (antibody pair pAb146/MW1) or the mouse/rat HTT MSD assay (antibody pair pAb147/MAB2166), respectively. sh4, <i>HTT</i> targeting shRNA. scr6, scramble control shRNA. (C) Neuronal total tau protein levels measured using a commercially available MSD ELISA-based assay kit were monitored as loading control. Data are averages of n = 3 independent samples with correspondent standard deviations. ***, P<0.001. (D) Immunoblot confirming the AAV-mediated knockdown of humanized mutant (mut) and endogenous mouse HTT in transduced heterozygous zQ175 mouse primary neurons (AAV-SEWB-sh4: <i>HTT</i> targeting shRNA; AAV-SEWB-scr6: scrambled control shRNA). Immunoblot was probed for HTT (MAB2166, 1∶1,000; Millipore) or ATP5B as loading control.</p