S1 Text - Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

Table S1: Immunization Schedule. Llamas 1 and 3 were immunized via intramuscular injection as indicated in the table. Protein injections were in a freshly prepared 4.5-ml water in oil emulsion prepared by vigorously mixing 2 vol U of antigen with 2.5 vol U of the adjuvant Stimune (CEDI Diagnostics); Table S2: IC50 against 77 viruses. VHH were titrated threefold from 50 µg/ml and incubated with the indicated pseudoviruses on TZM-bl assay as described in the materials and methods. No virus inactivation was observed with a negative control VHH or with a pseudovirus bearing a mouse leukemia virus Env. VHH IC50 titers were calculated using the XLFit4 software (IDBS) or the Labkey Neutralizing Antibody Tool (Piehler et al., 2011). Potent neutralization (IC50<1 µg/ml) is color-coded red, intermediate neutralization (1–10 µg/ml) is color-coded yellow and weak neutralization (10–50 µg/ml) is color-coded green, non neutralization (>50 µg/ml) is colour-coded white, strains that weren't tested for particular VHH are indicated by a black dot; Table S3. IC50 values for B9 VHH and S54W mutant against nine viruses. The S54W mutant was generated by site-directed mutagenesis as described in the materials and methods. VHH were titrated threefold from 50 µg/ml and incubated with the indicated pseudoviruses on TZM-bl assay. VHH IC50 titers were calculated using the XLFit4 software (IDBS). Highly potent neutralization (IC50 <0.1 µg/ml) is color-coded dark red, potent neutralization (0.1–1 µg/ml) is color-coded red, intermediate neutralization (1–10 µg/ml) is color-coded yellow, weak neutralization (10–50 µg/ml) is color-coded green; Table S4: Statistical analysis of sequence variation between immunized and naïve llamas. The mean cluster size was found to be significantly larger for naive than immunized llamas. When considering only non-singleton clusters (i.e. clusters of sequences with 2 or more members) the average cluster size was also considerably larger for the naive llamas. The mean number of reads per unique sequence was also higher in the naive compared to the immunized samples highlighting the greater sequence diversity in the immunized samples. This was despite the total number of reads not varying significantly between either set of samples and further reinforced by the immunized animals generating significantly higher numbers of unique sequences per sample; Figure S1: Post-immune sera anti-HIV activity. (A) Threefold serial dilutions of llama sera were tested against the indicated pseudoviruses, starting at a 1∶5 dilution in the 96-well plate the TZM-bl cell-based assay as described in the materials and methods. (B) Threefold serial dilutions of llama sera were tested against the indicated immunogens, starting at a 1∶20 dilution. Binding was detected with HRP-conujugated anti-llama antibody. Serum samples were heat-inactivated to destroy complement by incubation at 56°C for 1 h before use; Figure S2: VHH binding to HIV Env proteins. VHH binding to (A) clade B gp140 BX08, (B) clade B gp41 IIIB and (C) clade A gp140 92UG037 was assessed by ELISA as described in the Materials and Methods. The positive control for gp140 binding was J3 [26] and that for gp41 binding 2H10 [32]; Figure S3. VHH sequence alignments. Alignment of A14, B9, B21, 3E3, their germ line V genes and human V gene VH3-23*04 and VH1-2*02; Figure S4. Shared sequence identity with J3 relative to divergence from germ line for all naïve and immunized llamas. Shared percentage identities with neutralizing VHH J3 and divergence from its inferred V gene Vt were calculated for all unique sequences from the seven control naïve llamas, and the four immunized llama including the J3-source llama 8. Each panel shows percentage identity for all sequences from the indicated llama plotted against divergence from Vt.
(PDF)