Human Anti-HIV gp41 (596-606) Recombinant Antibody (clone 7B2) (CAT#: PABL-139)

Figure 1 Antigenicity of monomeric gp120 and various VLPs analyzed by ELISA.

Monomeric gp120 and various VLPs, as indicated, were coated on ELISA wells and probed by MAbs directed to various Env epitopes. Data are representative of at least 3 titrations of each MAb against each VLP. mann., mannose.

Tong, T., Crooks, E. T., Osawa, K., & Binley, J. M. (2012). HIV-1 Virus-Like Particles Bearing Pure Env Trimers Expose Neutralizing Epitopes but Occlude Non-Neutralizing Epitopes.Journal of virology, JVI-06938.

Figure 2 Effects of digests on MAb binding to E168K+N189A WT VLPs by ELISA. Data shown are representative of at least 3 titrations of each MAb.

Tong, T., Crooks, E. T., Osawa, K., & Binley, J. M. (2012). HIV-1 Virus-Like Particles Bearing Pure Env Trimers Expose Neutralizing Epitopes but Occlude Non-Neutralizing Epitopes.Journal of virology, JVI-06938.

Figure 3 MAb binding to undigested and digested UNC WT VLPs was investigated by VLP ELISA. In addition, 2F5 binding to bald VLPs before and after digestion was assessed by ELISA. Data are representative of at least 3 titrations of each MAb against each VLP.

Tong, T., Crooks, E. T., Osawa, K., & Binley, J. M. (2012). HIV-1 Virus-Like Particles Bearing Pure Env Trimers Expose Neutralizing Epitopes but Occlude Non-Neutralizing Epitopes.Journal of virology, JVI-06938.

Figure 4 Effects of protease digests on VLP neutralization sensitivity.

The neutralization sensitivities of undigested E168K+N189A WT VLPs and digested E168K+N189A WT trimer VLPs to various MAbs were assayed using CF2.CD4.CCR5 cells as targets.

Tong, T., Crooks, E. T., Osawa, K., & Binley, J. M. (2012). HIV-1 Virus-Like Particles Bearing Pure Env Trimers Expose Neutralizing Epitopes but Occlude Non-Neutralizing Epitopes.Journal of virology, JVI-06938.

Figure 5 Fab and FcR binding

(A) Linear cross-clade epitope mapping of 7B2 IgG1_AAA by peptide microarray. FcR binding (response units), on-rate (ka) and off rate (kd) by Surface Plasmon Resonance (SPR) of 7B2 IgG1_AAA. (B) Fine mapping of the 7B2 epitope within the gp41 immunodominant loop. Top Graph shows the binding response at saturation (~140 seconds after starting injection of 7B2 Fab) of each Ala-substituted gp41596-606 peptide normalized to wild type and the middle graph shows the normalized off-rate of the same peptides. Data are representative of at least two measurements on adjacent spots in the same sensor chip. Residues that are part of the 7B2 epitope are colored in orange. The Lys601Ala mutant peptide is highlighted in green since it gave a higher binding response and a decreased off-rate. Bottom graph is an example of sensogram showing 7B2 Fab binding to WT and select Alanine mutant gp41596-606 peptides that were used to generate the top and middle graphs. (C) Binding between 7B2 and gp41 peptides in standard and reducing conditions. (D) The structure of the 7B2 Fab-gp41 peptide complex shows detailed polar interactions. Hydrogen bonds between functional groups in the peptide and the heavy chain of the Fab are indicated. (E) Comparison of the gp41 ID loop from our structure (far left) against its structure obtained from NMR (middle left) and its conformation as shown in the BG505.SOSIP.664 structure (middle right) superimposed against the 7B2 paratope. A superposition of all three ID conformations (far right) highlights the conformational variability of this region.

Santra, S., Tomaras, G. D., Warrier, R., Nicely, N. I., Liao, H. X., Pollara, J.,... & Shen, X. (2015). Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques. PLoS pathogens, 11(8), e1005042.

Figure 6 Surface plasmon resonance of mAbs to human and rhesus FcR.

(A) 7B2 IgG1_AAA and Fab to human FcR FcgRI, FcgRIIA, FcgRIIIB and (B) rhesus macaque FcgR3A-1 and _FcgR3A-3. (C) A32 IgG1_AAA and (D) CH22 IgG1_AAA to rhesus macaque FcgR3A-1 and _FcgR3A-3.

Santra, S., Tomaras, G. D., Warrier, R., Nicely, N. I., Liao, H. X., Pollara, J.,... & Shen, X. (2015). Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques. PLoS pathogens, 11(8), e1005042.

Figure 7 7B2 mAb captures infectious SHIV BaL and SF162.

SHIV BaL and SHIV SF162 virus capture by 7B2 and controls were measured by either (A) a plate–based capture assay (relative luciferase unit (RLU) on day 7 post infection shown) (B) or a column-based assay (% virus capture based on SIV gag viral RNA measurement for rVirion and RLU infectivity for iVirion percentages, respectively. The error bar is the SEM of three wells replicates. The dashed line is the positivity cutoff. The level of virion capture in the presence (red bars) and absence (blue bars) of soluble CD4 for HIV-1 SF162 (C) and HIV- BAL (D) are shown. Non-neutralizing mAb A32 and a neutralizing mAb 2G12 were used as negative and positive controls, respectively. Error bars show mean ± SEM from 3 separate experiments. (E) 7B2_AAA does not inhibit infection of rectal explants (gray) but does inhibit infectious transfer from migratory cells that emigrate from mucosal tissue (at the highest concentration (50 μg) (blue). This is reflective of CD4+ T cells being the primary targets of infection. Results are the average of two experiments.

Santra, S., Tomaras, G. D., Warrier, R., Nicely, N. I., Liao, H. X., Pollara, J.,... & Shen, X. (2015). Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques. PLoS pathogens, 11(8), e1005042.

Figure 8 Macrophage neutralization assays.

(A) HIV-1 Bal infected macrophages are inhibited in a dose dependent manner by 7B2-SEK and 7B2-AAA mAbs. Palivizumab did not neutralize (>100 IC90). (B) 7B2 mAb neutralization of HIV-1 SF162 (Subtype B), HIV-1 TV-1 (Subtype C), and HIV-1 Vl191. (C) 7B2_AAA displays a dose dependent inhibition of HIV infection of monocyte-derived macrophages. (D) Neutralization of BaL in peritoneal macrophages. gp41 Env specific Ab (7B2 mAb) neutralizes HIV- infection in human peritoneal macrophages. Virus input was normalized to RNA copies/mL. HIV replication was quantified by measuring the amount of luciferase in macrophage lysates.

Santra, S., Tomaras, G. D., Warrier, R., Nicely, N. I., Liao, H. X., Pollara, J.,... & Shen, X. (2015). Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques. PLoS pathogens, 11(8), e1005042.

Figure 9 Ability of HIV-1 Env-specific mAbs to bind HIV-1 infected cells and mediate ADCC.

(A) Mock infected primary human CD4+ T cells and (B) HIV-1 IMCBaL infected primary human CD4+ T cells were incubated with the indicated mAbs, and binding was detected by secondary staining with a FITC-conjugated anti-human IgG antibody. (C) ADCC activities of A32, CH22, and 7B2 mAbs against HIV-1IMCBaL-infected CEM.NKRCCR5 CD4+ targets cells in the presence of NK cells.

Santra, S., Tomaras, G. D., Warrier, R., Nicely, N. I., Liao, H. X., Pollara, J.,... & Shen, X. (2015). Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques. PLoS pathogens, 11(8), e1005042.

Figure 10 7B2 IgG1_AAA, A32 IgG1_AAA and CH22 IgG1_AAA mAb binding to rhesus FcR on NK cells.

(A) Schematic of experiment: Rhesus PBMC were incubated with mAb and detected with fluorescently labeled antigens specific for the mAb being tested. (B) Rhesus PBMC gated on CD16+ NK cells were analyzed for binding of an HIV-1 gp41 immunodominant region reagent. Representative data: gray curve shows binding in the absence of mAb; the black curve shows binding to a control mAb. The blue curve shifted to the right shows binding of the reagent to 7B2 mAb bound to NK cells. (C-E) Assay of PBMC from animals infused in this study. Mean fluorescence intensity of rhesus NK cells for each animal is shown (grouped here by their actual grouping in the passive infusion study). In each case, PBMC were tested using lots of mAbs used for the infusion study. Antibody-reagent pairs are as follows: C. HIV-1 gp41 immunodominant region peptide tetramer with 7B2 mAb (D) HIV-1 gp120 A244 with A32 mAb (E) HIV-1 gp120 V3 loop peptide tetramer with CH22 mAb.

Santra, S., Tomaras, G. D., Warrier, R., Nicely, N. I., Liao, H. X., Pollara, J.,... & Shen, X. (2015). Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques. PLoS pathogens, 11(8), e1005042.

Figure 11 7B2 IgG1_AAA, A32 IgG1_AAA and CH22 IgG1_AAA mAb concentrations in (A) plasma and (B) rectal secretions.

Concentrations of mAb were measured by a binding assay with the infused antibody as a control for calculating concentration equivalents of Ab binding to Env protein (μg/ml). Visible red blood cells in the rectal weck elutions were observed at time points post infusion for some animals.

Santra, S., Tomaras, G. D., Warrier, R., Nicely, N. I., Liao, H. X., Pollara, J.,... & Shen, X. (2015). Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques. PLoS pathogens, 11(8), e1005042.

Figure 12 Viral loads and CD4 T cell counts following high dose SHIV BaL rectal challenge in rhesus macaques passively infused with 7B2 IgG_AAA, A32 IgG_AAA or CH22 IgG_AAA.

(A) Plasma viral RNA levels and (B) CD4 T cell counts in 7B2 IgG_AAA and palivizumab IgG treated rhesus monkeys following challenge with SHIV-BaL. (C) Plasma viral RNA levels and (D) CD4 counts in A32 IgG_AAA mAb and control palivizumab IgG mAb passively infused rhesus monkeys following challenge with SHIV-BaL. (E) Plasma viral RNA levels and (F) CD4 Counts in rhesus monkeys following challenge with SHIV-BaL after passive administration of CH22 or CH65 IgG mAbs.

Santra, S., Tomaras, G. D., Warrier, R., Nicely, N. I., Liao, H. X., Pollara, J.,... & Shen, X. (2015). Human non-neutralizing HIV-1 envelope monoclonal antibodies limit the number of founder viruses during SHIV mucosal infection in rhesus macaques. PLoS pathogens, 11(8), e1005042.