Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization

Abstract

Llamas (Lama glama) naturally produce heavy chain-only antibodies (Abs) in addition to conventional Abs. The variable regions (VHH) in these heavy chain-only Abs demonstrate comparable affinity and specificity for antigens to conventional immunoglobulins despite their much smaller size. To date, immunizations in humans and animal models have yielded only Abs with limited ability to neutralize HIV-1. In this study, a VHH phagemid library generated from a llama that was multiply immunized with recombinant trimeric HIV-1 envelope proteins (Envs) was screened directly for HIV-1 neutralization. One VHH, L8CJ3 (J3), neutralized 96 of 100 tested HIV-1 strains, encompassing subtypes A, B, C, D, BC, AE, AG, AC, ACD, CD, and G. J3 also potently neutralized chimeric simian-HIV strains with HIV subtypes B and C Env. The sequence of J3 is highly divergent from previous anti-HIV-1 VHH and its own germline sequence. J3 achieves broad and potent neutralization of HIV-1 via interaction with the CD4-binding site of HIV-1 Env. This study may represent a new benchmark for immunogens to be included in B cell-based vaccines and supports the development of VHH as anti-HIV-1 microbicides.

Figure 1.

Llama 8 immune response evaluation. (A and B) Serial dilutions of llama sera obtained on days 0 and 122 were incubated on ELISA plates preimmobilized with Gp140_UG37 (A) or Gp140_CN54 (B) recombinant ENV. Binding was assessed as described in Materials and methods. All samples were assayed in triplicate. (C and D) Sera taken on days 0 and 122 were preincubated with the 92UG037 pseudovirus (C) or CN54 viral isolate (D), and neutralization activity was assessed as described in Materials and methods. All samples were assayed in duplicate. Error bars represent standard deviation from the mean. (E) ID50 titers (µg/ml) for sera taken on day 122 against both homologous viruses and the indicated heterologous viruses. Experiments were repeated in duplicate and on two independent occasions, except for the heterologous virus neutralization assays which were performed once in duplicate.

Figure 2.

Breadth of J3 HIV-1 neutralization. Percentage of virus strains neutralized by J3 stratified by subtype as described in Materials and methods. To aid comprehension, the columns have been subdivided to show the percentage of strains from each subtype that were neutralized with IC50 values falling into the intervals indicated in the key. The neutralization activity of each VHH was assayed in duplicate or triplicate, and negative controls included mouse leukemia and rabies pseudoviruses. The majority of the panel of viruses were assayed once.

Figure 3.

Comparison of breadth of neutralization achieved by VRCO1/2, b12, and CD4-Ig. Percent neutralization for the matched subset of viruses assayed against by J3, VRC01/2, b12, and CD4-Ig (using data published by Wu et al. [2010]) was stratified by clade.

Figure 4.

J3 binding to trimeric and monomeric Env in ELISA. (A and B) VHH binding to subtype A Gp140_UG37 (A) and CRF BC Gp140_CN54 (B) was assessed by ELISA. Recombinant Envs were immobilized, triplicate serial dilutions of VHH A12, J3, and a negative control VHH were added, and binding was detected as described in the Materials and methods. (C and D) Monomeric subtype B Gp120_IIIB (C) and D368R or wild-type monomeric subtype B Gp120_YU2 (D). All error bars represent standard deviation from the mean. All data are representative of at least two independent experiments

Figure 5.

J3 competition for the CD4-binding site of HIV-1 Env in ELISA. (A) Dose-dependent competition of VHH A12, J3, and a negative control VHH with sCD4 for binding to Gp140_UG37. Recombinant Env was immobilized, and duplicate 10-fold serial dilutions of the indicated VHH were added, followed by 0.1 µg/ml sCD4. sCD4 binding to Env was detected as described in Materials and methods. (B) Dose-dependent competition of VHH A12, J3, and a negative control VHH with mAb b12 for binding to Gp140_UG37. Recombinant Env was immobilized, and duplicate 10-fold serial dilutions of the indicated VHH were added, followed by 1 µg/ml b12. Binding to Env was detected as described in Materials and methods. (C) Dose-dependent competition of VHH J3 and A12 with biotinylated J3 for binding to Gp140_UG37. Recombinant Env was immobilized, and triplicate fivefold serial dilutions of the indicated mAb or VHH were added (including a negative control mAb 2F5), followed by 0.5 µg/ml of biotinylated J3. Biotinylated J3 binding was detected as described in Materials and methods. All error bars represent standard deviation from the mean. All data are representative of at least two independent experiments.

Figure 6.

J3 amino acid and germline sequences. (A) J3 amino acid sequence as determined by purified pCAD51 A12 DNA and J3 germline determined based on sequence data (unpublished data) and DNA analysis of the area on which the V and J genes encoding VHH are located on the L. glama and L. pacos genomes. Residues that deviate from germline outside the CDRs are highlighted in yellow, and those inside CDR1 and CDR2 are highlighted in red. Note that the portion of the VHH sequence arising from the D genes (mapping to CDR3) is too small to enable alignment via ClustalW or BLAST with D genes from L. glama or L. pacos, and no significant association is found, thus the gap in the germline depicted. (B) VHH binding to subtype A Gp140_UG37 was assessed by ELISA. Recombinant Envs were immobilized, triplicate serial dilutions of mutant, wild-type J3, or a negative control VHH were added, and binding was detected as described in Materials and methods. All error bars represent standard deviation from the mean. All data are representative of at least two independent experiments.

Figure 7.

Phylogenetic relationship of heavy chain V genes from llamas and humans. (A) J3 and A12 sequences were aligned with human germline IgVH sequences through the VQUEST tool of IMGT (http://www.IMGT.org; GenBank/EMBL/DDBJ accession no. AJ879486). (B) J3 sequence was aligned with human germline IgVH sequences through the VQUEST tool of IMGT (GenBank accession no. AJ879486). Residues that differ in the VHH sequence as compared with the human germline are underlined.