sCD4-17b bifunctional protein: extremely broad and potent neutralization of HIV-1 Env pseudotyped viruses from genetically diverse primary isolates

Abstract

Background: We previously described a potent recombinant HIV-1 neutralizing protein, sCD4-17b, composed of soluble CD4 attached via a flexible polypeptide linker to an SCFv of the 17b human monoclonal antibody directed against the highly conserved CD4-induced bridging sheet of gp120 involved in coreceptor binding. The sCD4 moiety of the bifunctional protein binds to gp120 on free virions, thereby enabling the 17b SCFv moiety to bind and block the gp120/coreceptor interaction required for entry. The previous studies using the MAGI-CCR5 assay system indicated that sCD4-17b (in concentrated cell culture medium, or partially purified) potently neutralized several genetically diverse HIIV-1 primary isolates; however, at the concentrations tested it was ineffective against several other strains despite the conservation of binding sites for both CD4 and 17b. To address this puzzle, we designed variants of sCD4-17b with different linker lengths, and tested the neutralizing activities of the immunoaffinity purified proteins over a broader concentration range against a large number of genetically diverse HIV-1 primary isolates, using the TZM-bl Env pseudotype assay system. We also examined the sCD4-17b sensitivities of isogenic viruses generated from different producer cell types.

Results: We observed that immunoaffinity purified sCD4-17b effectively neutralized HIV-1 pseudotypes, including those from HIV-1 isolates previously found to be relatively insensitive in the MAGI-CCR5 assay. The potencies were equivalent for the original construct and a variant with a longer linker, as observed with both pseudotype particles and infectious virions; by contrast, a construct with a linker too short to enable simultaneous binding of the sCD4 and 17b SCFv moieties was much less effective. sCD4-17b displayed potent neutralizing activity against 100% of nearly 4 dozen HIV-1 primary isolates from diverse genetic subtypes (clades A, B, C, D, F, and circulating recombinant forms AE and AG). The neutralization breadth and potency were superior to what have been reported for the broadly neutralizing monoclonal antibodies IgG b12, 2G12, 2F5, and 4E10. The activity of sCD4-17b was found to be similar against isogenic virus particles from infectious molecular clones derived either directly from the transfected producer cell line or after a single passage through PBMCs; this contrasted with the monoclonal antibodies, which were less potent against the PMBC-passaged viruses.

Conclusions: The results highlight the extremely potent and broad neutralizing activity of sCD4-17b against genetically diverse HIV-1 primary isolates. The bifunctional protein has potential applications for antiviral approaches to combat HIV infection.

Figure 1

Design and purification of sCD4-17b constructs and related proteins. A) Schematic representation of three sCD4-17b constructs with different L1 linkers, with the total number of L1 amino acids indicated in the construct name (in each case consisting of the appropriate number of repeats of the G₄S motif). Also shown are the constructs representing the individual components sCD4 and 17b SCFv. All constructs include the Ig kappa light chain leader sequence at the amino terminus, and the C9 epitope tag at the carboxy terminus. B) Immunoaffinity purification of sCD4-40-17b, as analyzed by Coomassie Blue staining of reducing SDS-PAGE gels (10 μl per lane for each sample). In this example, C9 peptide elution was performed in conjunction with low pH. The fractions analyzed were the initial concentrated media supernatant (load), flow-through (FT), the 5 wash fractions (W1-W5) and the two elution fractions (E1, E2). Numbers on the left indicate molecular weight markers (kDa). C) Western blot analysis of purified preparations of the indicated sCD4-17b proteins as well as the 17b SCFv and sCD4 proteins (10 μl per lane for each sample). The 1D4 MAb directed against the C-terminal tag on each protein was used for detection.

Figure 2

HIV-1 neutralization of various isolates by different sCD4-17b constructs and related proteins. Assays were performed using the TZM-bl system or where indicated, with the MAGI-CCR5 system. Dose-response analyses were performed with the indicated proteins and HIV-1 Env pseudotypes or infectious virus, as indicated by the symbols above the graphs and the names within the graphs. Each point represents the mean of duplicate samples; error bars indicate SD. A) Comparison of the potencies of sCD4-35-17b and sCD4-40-17b against the 91US054 pseudotype and infectious virus. In the TZM-bl system, the IC₅₀ values against the pseudotype were 11 nM for both sCD4-35-17b and sCD4-40-17b; the value against the infectious virus was 22 nM for sCD4-40-17b. In the MAGI-CCR5 system, the IC₅₀ value against the infectious virus was 30 nM. B) Comparison of the effects against the indicated Env pseudotypes of sCD4-40-17b, sCD4-5-17b (shorter linker) and proteins representing individual moieties (sCD4 alone, or in combination with 17b SCFv). The IC₅₀ values for sCD4-40-17b were 12.3 nM against CAAN5342.A2, 9.8 nM against RW020, and 0.8 nM against YU2; the value for sCD4-5-17b against YU2 was 16.5 nM.

Figure 3

Breadth of sCD4-40-17b activity against pseudotypes from genetically diverse HIV-1 primary isolates. Nearly all entries are primary isolates for which the Env sequences were originally obtained by direct cloning from infected tissue; the exceptions are the laboratory-adapted Ba-L, and 89.6 strains. The IC₅₀ values (μg/ml) represent the mean of multiple independent assays (+/- SEM) (3-5 replicate assays in most cases, 2 in a few instances). The values are color coded according to the IC₅₀ ranges as indicated at the top of the figure, with red<orange<yellow<white. Note that none of the IC₅₀ values are in the white range (least potent). The superscript letters indicate the references describing the individual isolates, as follows: ^a[41], clade A; ^b[39], clade B, tier 2 reference panel; ^c[40], clade C reference panel; ^d [V. Polonis and S. Tovanabutra, personal communication], clades A, D, AE and AG. Further details on various features of these isolates can be found in these cited references. ** Indicates isolates previously found to be insensitive to sCD4-17b in the MAGI-CCR5 assay using infectious virus [29].

Figure 4

Comparison of neutralization breadth of sCD4-40-17b with broadly neutralizing monoclonal antibodies. Each pie graph shows the fraction of isolates within each IC₅₀ range for each agent tested against isolates from the indicated clades. The color-coding for IC₅₀ ranges (indicated at the top of the Figure) is the same as in Figure 3, with red<orange<yellow<white. Note that whereas for sCD4-40-17b none of the clades had isolates with IC₅₀ values in the white range (least potent), each of the MAbs had some or all isolates with IC₅₀ values in the white range for at least some clades; thus when the total isolates where evaluated for "% neutralized", the value was 100 for sCD4-40-17b but considerably less for the MAbs. The pie graphs are based on the indicated number of isolates (#) in each clade, which are the ones designated in Figure 3 with superscript letters. The sCD4-40-17b results are based on the data in Fig. 3; the MAb results are based on findings by others as follows: clade A, 6 isolates [41], 3 isolates [V. Polonis and S. Tovanabutra, personal communication]; clade B, all 12 isolates (reference panel clade B) [39]; clade C, all 12 isolates (reference panel clade C) [40]; all 3 clade D isolates [V. Polonis and S. Tovanabutra, personal communication]; all 3 clade AE and AG isolates [V. Polonis and S. Tovanabutra, personal communication].

Figure 5

HIV-1 sensitivity to sCD4-40-17b: independence from cellular source of virions. IMCs from strains BL01 and 89.6 were used to generate virus particles directly from transfected 293T cells; a second set of viruses was generated by single passage of the 293T-derived particles in PBMCs [30]. Both sets of particles (directly provided to us by M. Louder and J. Mascola) were analyzed for sensitivity to the agents shown. The results are plotted as the IC₅₀ ratios of the PBMC-passaged to the 293T-derived virus particles. In each graph, the dotted line indicates a ratio of 1 (i.e., equal potency against each type of virus particle).