Bifunctional CD4-DC-SIGN fusion proteins demonstrate enhanced avidity to gp120 and inhibit HIV-1 infection and dissemination

Abstract

Early stages of mucosal infection are potential targets for HIV-1 prevention. CD4 is the primary receptor in HIV-1 infection whereas DC-SIGN likely plays an important role in HIV-1 dissemination, particularly during sexual transmission. To test the hypothesis that an inhibitor simultaneously targeting both CD4 and DC-SIGN binding sites on gp120 may provide a potent anti-HIV strategy, we designed constructs by fusing the extracellular CD4 and DC-SIGN domains together with varied arrangements of the lengths of CD4, DC-SIGN and the linker. We expressed, purified and characterized a series of soluble CD4-linker-DC-SIGN (CLD) fusion proteins. Several CLDs, composed of a longer linker and an extra neck domain of DC-SIGN, had enhanced affinity for gp120 as evidenced by molecular-interaction analysis. Furthermore, such CLDs exhibited significantly enhanced neutralization activity against both laboratory-adapted and primary HIV-1 isolates. Moreover, CLDs efficiently inhibited HIV-1 infection in trans via a DC-SIGN-expressing cell line and primary human dendritic cells. This was further strengthened by the results from the human cervical explant model, showing that CLDs potently prevented both localized and disseminated infections. This is the first time that soluble DC-SIGN-based bifunctional proteins have demonstrated anti-HIV potency. Our study provides proof of the concept that targeting both CD4 and DC-SIGN binding sites on gp120 represents a novel antiviral strategy. Given that DC-SIGN binding to gp120 increases exposure of the CD4 binding site and that the soluble forms of CD4 and DC-SIGN occur in vivo, further improvement of CLDs may render them potentially useful in prophylaxis or therapeutics.

Fig 1

Schematic diagrams of CLD-expressing plasmids and biochemical characterization of purified fusion proteins. (a) Schematic representation of fusion proteins. Abbrevations: sCD4, the N-terminal 183 aa of CD4; mC, the N-terminal 106 aa of CD4; sC, the N-terminal 87 aa of CD4; N, DC-SIGN neck domain; sDC-SIGN, DC-SIGN carbohydrate-recognition domain (CRD); 15, 20, 25, 35, the number of amino acids; s60c, a Cys-to-Ser mutation at aa 60 on CD4. (b) Schematics of CLD-expressing plasmids. All CD4 and DC-SIGN moieties for CLDs were cloned into pET-28a(+) using restriction enzyme sites NdeI, BamHI and HindIII. Restriction enzyme sites and linker sequences were introduced into CD4 and DC-SIGN sequences by PCR. (c) SDS-PAGE and Western blot analysis of C35NDs60c. Purified C35NDs60c was resolved in 12% SDS-PAGE in reducing or nonreducing condition, followed by detection with Coomassie blue staining or Western blotting (reducing condition only). Lane 1, Western blotting; C35NDs60c was detected by MAb 507 against DC-SIGN, followed by an HRP-conjugated secondery antibody; lane 2 and lane 3, Coomassie blue staining of C35NDs60c in reducing and nonreducing conditions, respectively; lane 4, molecular marker in reducing condition. (d) Analytical ultracentrifugation in sedimentation velocity mode. CD4, peaks at 1.9 S and 3.8 S, corresponding to apparent molecular masses of 21.5 kDa and 57.4 kDa; DC-SIGN, peaks at 2.0 S, corresponding to an apparent molecular mass of 22.4 kDa; C35NDs60c, peaks at 6.3 S and 9.2 S, corresponding to apparent molecular masses of 214.8 kDa and 398.2 kDa. One of three independent experiments is shown.

Fig 2

CLDs inhibit HIV-1 capture and transfer by Raji/DC-SIGN cells and iMDDCs. BaL Env-pseudotyed HIV-1 was preincubated with or without inhibitor for 1 h at 37°C before the addition to Raji/DC-SIGN cells or iMDDCs. Cells pretreated with or without mannan were exposed to viruses as controls. After exposure to virues for 2 h at 37°C, cells were extensively washed and either lysed for capture assay or cocultured with U87-CD4.CCR5 cells for transfer assay. (a) HIV-1 captured by Raji/DC-SIGN cells. Medium alone was defined as 100% and its p24 concentration was 1.79 ng/ml. (b) RLU of HIV-1 trans infection from Raji/DC-SIGN cells to U87-CD4.CCR5 cells. Medium alone was defined as 100%. (c) HIV-1 captured by iMDDCs. Medium alone was defined as 100% and its p24 concentration was 2.87 ng/ml. (d) RLU of HIV-1 trans infection from iMDDCs to U87-CD4.CCR5 cells. Medium alone was defined as 100%. Data shown are mean ± SD of three independent experiments, with each condition performed in triplicate.

Fig 3

CLD inhibits localized mucosal HIV-1 infection and dissemination. Human cervical explants were preincubated with or without inhibitors for 1 h at 37°C before exposure to HIV-1_BaL for 2 h at 37°C. After incubation, explants were extensively washed and cultured in the presence of 100 ng/ml of MIP-3β for 48 h. Emigrating cells were collected, washed, and cocultured with PM1 cells. The explants were cultured in separate wells. Data are representative of three independent experiments, with each condition performed in triplicate. Data shown are p24 antigen (mean± SD) released from both (a) cultured explants and (b) PM1 cocultured migratory cells at day 9. p24 in the absence of inhibitor was defined as 100%, and p24 concentrations were 1.56 and 2.74 ng/ml for the cervical explants and PM1 cocultured migratory cells, respectively. Anti-CD4, RPA-T4. Anti-DC-SIGN, MAb 507 and 526.