Virion incorporation of integrin α4β7 facilitates HIV-1 infection and intestinal homing

Abstract

The intestinal mucosa is a key anatomical site for HIV-1 replication and CD4⁺ T cell depletion. Accordingly, in vivo treatment with an antibody to the gut-homing integrin α4β7 was shown to reduce viral transmission, delay disease progression, and induce persistent virus control in macaques challenged with simian immunodeficiency virus (SIV). We show that integrin α4β7 is efficiently incorporated into the envelope of HIV-1 virions. Incorporated α4β7 is functionally active as it binds mucosal addressin cell adhesion molecule-1 (MAdCAM-1), promoting HIV-1 capture by and infection of MAdCAM-expressing cells, which in turn mediate trans-infection of bystander cells. Functional α4β7 is present in circulating virions from HIV-infected patients and SIV-infected macaques, with peak levels during the early stages of infection. In vivo homing experiments documented selective and specific uptake of α4β7⁺ HIV-1 virions by high endothelial venules in the intestinal mucosa. These results extend the paradigm of tissue homing to a retrovirus and are relevant for the pathogenesis, treatment, and prevention of HIV-1 infection.

Fig. 1. HIV-1 virions efficiently incorporate integrin α4β7

(A) Immunomagnetic capture of virion progeny (HIV-1 SF162) produced by RA-activated human PBMC cells using mAbs directed against a panel of cell surface-expressed lymphocyte antigens. Captured virions from identical viral inputs were quantified for p24_Gag antigen concentrations by AlphaLisa. The data represent the mean (±SD) of three biological replicates performed using PBMC from three different healthy blood donors. (B) Cell-surface staining of HIV-infected RA-activated human PBMC using the same panel of mAbs as in panel A. Mean fluorescence intensity (MFI) values obtained with normalized antibody concentrations are shown. The data represent the mean (±SD) of three biological replicates performed using virus produced by PBMC from three different healthy blood donors. (C) Ratio between virion incorporation and cell-surface expression for each lymphocyte marker, with the calculated ratio for ICAM-1 set at 1, as a reference. To obtain an index of incorporation efficiency, we first normalized the levels of both virion incorporation and cell-surface expression to the respective levels detected with ICAM-1, selected as a reference protein; then, for each protein we calculated the ratio between normalized virion incorporation and normalized cell-surface expression. The incorporation of integrin α4β7 (mAb ACT-1) was significantly higher than that of ICAM-1. * p = 0.025; ** p < 0.01 by paired two-tailed t-test. (D) Parallel analysis of α4β7 cell-surface expression (yellow bars, left y-axis) and virion incorporation (green bars, right y-axis) in cultures of RA-treated and untreated human PBMC infected with HIV-1 SF162. The data represent the mean (±SD) of three biological replicates performed using PBMC and respective progeny virus from three different healthy blood donors.

Fig. 2. Quantitative analysis of α4β7 incorporation by HIV-1 virions

Western blot analysis of α4 and HIV-1 structural proteins (gp120_Env and p24_Gag) incorporated into HIV-1 virions produced by RA-treated human PBMC (RA) vs. untreated human PBMC (Unt.). Virus stocks were concentrated by PEG treatment and centrifugation before detergent lysis. As a control for integrin subunit expression on producer cells, we loaded whole cell lysate from RA-treated CD4⁺ T cells. For semi-quantitative assessment of virion incorporation, we used serial dilutions of recombinant α4β7 and recombinant gp120, respectively, at the indicated amounts loaded per lane (in nanograms). The different molecular weight of recombinant α4 (120 kDa) vs. cell-derived α4 (70kDa) reflect the fact that activated T cells express cleaved α4 (α4_cl) on their surface, while recombinant α4 is uncleaved (α4_un). Semi-quantification of incorporated α4β7 and gp120 was performed by densitometric scanning and interpolation of the results on the curves obtained for the recombinant proteins used as reference. The gel was also developed using pooled sera from HIV-infected individuals (bottom panel) to verify the correct amount of input p24G_ag antigen. This Western blot is representative of two experiments performed with similar results.

Fig. 3. Virion-incorporated integrin α4β7 is functionally competent

Virion capture assays were performed on the HIV-1 SF162 α4β7_hi viral stock (produced by RA-treated PBMC) in the presence or absence of functional inhibitors or activators of α4β7, compared to the untreated control condition. Capture with recombinant human MAdCAM-1, which selectively binds to the functionally active integrin, was compared to capture with the anti-α4β7 mAb ACT-1, which binds independently of the integrin activation state. The divalent cation Mn⁺⁺ induces α4β7 to adopt a high-affinity ligand-binding state; the chelating agent EDTA sequesters divalent cations; and the inhibitory peptide mimetic ELN-4757772 (ELN) specifically occludes the MAdCAM-binding site on α4β7, but does not overlap with the ACT-1 epitope. The data represent the mean (±SE) of duplicate samples.

Fig. 4. Incorporation of α4β7 promotes MAdCAM-mediated HIV-1 capture, trans-infection of bystander target cells and infection of MAdCAM-expressing target cells

(A) HIV-1 virions with incorporated α4β7 were selectively captured by plate-immobilized MAdCAM-1 and transferred to susceptible target cells. Serial dilutions of HIV-1 THRO.18 viral stocks either positive (α4β7⁺) or negative (α4β7⁻) for α4β7 were incubated with plastic-coated rhMAdCAM-1 (MAdCAM+, green lines) or an irrelevant integrin ligand (PECAM+, pink lines), washed extensively to remove unbound virus, and overlaid with target cells (TZM-bl), as illustrated in the accompanying cartoon. (B) HIV-1 virions with incorporated α4β7 are selectively captured by MAdCAM-expressing cells, which mediate trans-infection of bystander target cells. Serial dilutions of HIV-1 THRO.18 viral stocks either positive (α4β7⁺) or negative (α4β7⁻) for α4β7 were incubated with MAdCAM-1-expressing cells (MAdCAM+, green lines) or with untransfected control cells (MAdCAM-, pink lines), washed extensively to remove unbound virus, and cocultured with TZM-bl cells. (C) HIV-1 virions with incorporated α4β7 infect MAdCAM-expressing susceptible cells more efficiently than α4β7⁻ virions. Serial dilutions of HIV-1 BaL pseudoviruses either positive (α4β7⁺) or negative (α4β7⁻) for α4β7 were incubated with TZM-bl cells transfected to express MAdCAM-1 (MAdCAM+, green) or with untransfected control TZM-bl (MAdCAM-, pink). In all the experiments, HIV-1 infection was assessed by measuring activation of the luciferase reporter gene.

Fig. 5. Integrin α4β7 is incorporated into HIV-1 virions circulating in vivo in infected patients

MAdCAM-1⁻ and ICAM-1-mediated capture of cell-free HIV-1 virions circulating in serum from infected patients. Sera on the left side were obtained during the early stage of HIV-1 infection (0-6 months), while those on the right side were obtained during the chronic phase, after 6 months of primary infection. Due to the limited amount of patient serum available, capture could only be performed with the recombinant α4β7 ligand, MAdCAM-1, which permitted to document the functionality of the incorporated integrin, as well as with ICAM-1, used as a control integrin ligand. Captured virions were quantified by real-time PCR using specific HIV-1 primers and probes on serum-extracted RNA; the results are expressed as viral genome equivalents per mL. Statistical analysis was performed using an unpaired 2-tailed t-test. In addition to the statistical comparisons shown in the figure, the difference between MAdCAM-1⁻ and ICAM-1-mediated virus capture in the <6-months group was also statistically significant (p = 0.0278 by paired 2-tailed t-test), while no statistical difference was detected in the >6-months group.

Fig. 6. Integrin α4β7 is incorporated into SIV virions circulating in vivo in infected macaques

(A) MAdCAM-1-mediated SIV virion capture in plasma samples obtained from 12 macaques experimentally infected with SIVmac239 followed longitudinally during the acute and chronic phases of infection. Captured virions were quantified by real-time PCR using specific SIV primers and probes on plasma-extracted RNA; the results are expressed as viral genome equivalents per mL. (B) Statistical comparison showing increased MAdCAM-1-mediated SIV virion capture in plasma samples obtained from infected macaques within the first 14 weeks of infection (acute infection) versus those obtained after 14 weeks of infection (chronic infection) (p = 0.0241). Statistical analysis was performed using an unpaired 2-tailed t-test.

Fig. 7. Incorporation of α4β7 promotes HIV-1 gut homing in vivo

(A) Thick-section images of HIV-1 uptake in vivo by Peyer’s patches, inguinal lymph nodes and spleen of mice injected with green fluorescent α4β7⁻ (left column) and α4β7⁺ (right 3 columns) HIV-1 virions. Normalized viral stocks of reverse transcriptase-deficient fluorescent HIV-1 (NL4.3 Gag EGFP) either negative (α4β7⁻) or positive (α4β7⁺) for α4β7 incorporation were inoculated into the tail base of C57BL/6 mice. After 45 minutes, the animals were euthanized, and the indicated organs were extracted and analyzed by confocal microscopy. The three panels in the left column show α4β7⁻ virus; all the panels on the right columns show α4β7⁺ virus. A primatized anti-α4β7 mAb (ACT-1) or an isotype-control macaque mAb were pre-incubated with the viral stocks prior to inoculation. The sections were stained with anti-CD4 (blue) or anti-CD169 and anti-PNAd (red) antibodies, as indicated; PNAd staining specifically labels high endothelial venules (HEV) in Peyer’s patches. α4β7⁺ virions (green) were selectively and specifically captured by HEV in Peyer’s patches. In spleen tissue, both viruses (green) were visible on CD169⁺ (red) marginal zone macrophages, while no virion uptake was detected in inguinal lymph nodes. Scale bars denote 5 µm. (B) Three-dimensional reconstructions of HEV (red) and HIV-1 virions (green spots) of the three images shown in panel A, top row, right columns, generated with the surface and spot functions of the Imaris program. Scale bars denote 5 µm. (C) Quantification of α4β7⁺ and α4β7⁻ HIV-1 virion uptake in Peyer’s patches, as assessed in the images shown in panel A, top row. Statistical analysis of virus uptake in the presence of anti⁻ α4β7 mAb vs. control mAb was performed by paired 2-tailed t-test.