The alpha4beta7 integrin has been shown to serve as a coreceptor for HIV. One anti-alpha4 integrin agent (natalizumab) has been approved for the treatment of multiple sclerosis and Crohn's disease. We found that activation of CD4+ T cells with retinoic acid induced the upregulation of alpha4 and beta7 integrins. However, natalizumab failed to block the replication of HIV-1 strains in lymphoid MT-4 cells or CD4+ T cells at concentrations up to 125 μg/ml. Our results suggest that alpha4 integrins are not essential cofactors for HIV replication.
aRetrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain
bInstitució Catalana de Recerca i Estudis Avancats, Barcelona, Spain
cUnitat de Neurología, Hospital Universitari Germans Trias i Pujol, Badalona, Spain.
Received 26 September, 2008
Accepted 29 October, 2008
Integrins (cell adhesion proteins involved in the attachment to the extracellular matrix)  have been involved in the mechanism of entry and infection of different viruses including foot-and-mouth disease virus, adenoviruses  or human cytomegalovirus . We have found that alphaV containing integrins may affect HIV-1 replication in cells expressing this integrin, including monocyte-derived macrophages (MDMs) . Cell adhesion through alphaV integrins is required for efficient HIV replication in MDM; therefore, antibodies, low molecular weight agents and short interfering RNAs targeting alphaV may block virus replication . More recently, the expression and activation of alpha4beta7 integrin in CD4+ T cells has been associated with CD4-independent binding to cells, HIV entry and replication. The HIV-1 envelope has been shown to bind to and signal through alpha4beta7, specifically involving an epitope in the V2 loop of gp120. Signaling produced by gp120–alpha4beta7 interaction led to LFA-1 activation , a process associated with increased virus production and virus transfer through cell-to-cell contacts. Inhibition of virus entry is a relevant target for antiretroviral therapy . Therefore, targeting the alpha4beta7 integrin could provide an approach for the treatment of HIV-1 infection.
Antagonism of the alpha4 integrin has been validated as a therapeutic approach for the treatment of inflammatory diseases such as multiple sclerosis (MS) and inflammatory bowel disease . At least one agent, Tysabri (natalizumab; Biogen Idec Denmark Manufacturing ApS, Hillerod, Denmark), has been approved for the treatment of MS and Crohn's disease, as migration of lymphocytes to the brain and gut-associated lymphoid tissue is mediated by alpha4beta1 and alpha4beta7 integrins, respectively. Tysabri is available only through the in-hospital prescribing program.
Preclinical drug screening is a necessary step that helps to delineate the potency and cytotoxicity of a candidate drug and to determine the necessary conditions for the study of the mechanism of action of active agents. Therefore, we have evaluated the effect of natalizumab in HIV-1 infection in cell culture. Natalizumab was evaluated for its anti-HIV activity using a standard drug-screening assay that is generally used in our laboratory. Anti-HIV activity in peripheral blood mononuclear cells (PBMCs) was determined after acute infection with HIV-1 strains and evaluation of p24 antigen production after 7-day cultures. To determine cytotoxicity, uninfected cells were harvested on the 7th day postinfection, and cell death was quantified by fluorescence activated cell sorter (FACS) analysis as previously described [8,9].
To evaluate the functionality of natalizumab in cell culture, monocytic U937 cells were left to adhere in flat bottom culture plates pretreated with vascular cell adhesion molecule 1 (VCAM-1, CD106; R&D, Abingdon, UK) in the absence or presence of natalizumab. VCAM-1 is an endothelial ligand for alpha4beta1 and alpha4beta7. After 3 h of culture, wells were washed, and attached cells were mechanically detached and counted in a FACSscalibur flow cytometer (BD, Madrid, Spain). Attachment of U937 cells to VCAM-1-coated wells was blocked by natalizumab in a dose-dependent manner (50% inhibitory concentration, IC50 of approximately 0.1 μg/ml) suggesting that alpha4 integrins could effectively be blocked by natalizumab (Fig. 1a).
PBMCs from healthy donors were cultured in the presence of retinoic acid (Sigma, Madrid, Spain) at a concentration of 10 nmol/l for up to 3 days prior to HIV infection. Expression of integrins, CD4, CCR5 and CXCR4 were evaluated by flow cytometry as described elsewhere [3,9]. Retinoic acid increased the expression of alpha4 and beta7 integrins in uninfected PBMCs from several donors (Fig. 1b). Conversely, natalizumab did not affect the replication of HIV-1 NL4-3 or BaL strains in lymphoid MT-4 cells expressing CXCR4 or CCR5, in conditions in which the reverse transcriptase inhibitor zidovudine, or the chemokine receptor antagonists AMD3100 or Tak-779 inhibited HIV replication (data not shown). Similarly, natalizumab at concentrations up to 125 μg/ml did not block the replication of X4, R5 or dual-tropic HIV-1 strains in PBMC from three different donors preincubated or not with retinoic acid, as measured by p24 antigen detection in cell supernatants (Fig. 1c).
Interaction of gp120 with alpha4beta7 was shown to be mediated by a tripeptide leucine, aspartic acid, valine in the V2 loop of gp120 found in several HIV strains, including 89.6 and 92UG024, a peptide motif that mimics the structure presented by the natural ligands of alpha4beta7 .
From our results, we concluded that natalizumab did not have anti-HIV activity in cell culture under the conditions tested. Other monoclonal antibodies (mAbs) targeting alpha4 integrins have been shown to block virus replication . However, in order to evaluate the antiviral activity of anti-alpha4beta7 mAbs, Arthos et al.  first passaged a virus strain mutated at the tripeptide motif in the V2 loop (mutation D183A) that severely reduced viral fitness in the presence of retinoic acid for 56 days. These conditions appear to induce a number of mutations, including the reversion to the aspartic acid at position 183 and sensitivity to mAbs (HP2/1, 2B4 and Act-1) that antagonized gp120–alpha4 interaction. Our data suggest that HIV-1 strains that have not been subjected to such procedure are naturally resistant to the activity of natalizumab. We cannot exclude that the epitopes of mAbs HP2/1, 2B4 and Act-1 are distinct from that of natalizumab but their anti-HIV properties need to be evaluated under conditions that do not require the manipulation of the virus genomic sequence and selective pressure with retinoic acid prior to evaluation of their antiviral activity. Unfortunately, natalizumab, an approved agent for human clinical treatment, appears to be devoid of anti-HIV activity.
Another important consideration is that natalizumab, when administered with or without combination with interferon-β1a for the treatment of MS, may predispose recipients to progressive multifocal leukoencephalopathy (PML) caused by JC virus (JCV). JCV is a polyomavirus that infects approximately 85% of the adult population worldwide but that usually remains latent, causing disease only when the immune system has been severely weakened. Up to 5% of all HIV-infected persons may develop PML . Potential explanations include an alteration of the central nervous system milieu by HIV, facilitating JC viral entry into the brain and activation of the JCV by HIV proteins and by inflammatory products of HIV infection . Therefore, the evaluation of agents targeting alpha4beta7 will require close scrutiny of viral coinfections.
Alpha4beta7 plays an essential role in the migration of T cells to the gut mucosa, a primary site for HIV infection and T-cell destruction . Blockade of cell migration to the gut could prevent the massive T-cell depletion due to the absence of target cells. Therefore, the role of integrins in HIV infection merits further evaluation, especially regarding their role in T-cell migration, as its blockade could comprise a new potential therapeutic strategy, similar to inflammatory diseases treatments.
We thank the NIH AIDS Reference Reagent Program for providing reagents. The work was supported by the Spanish FIS (project PI060624) and MCI (project BFU2006-0096 and SAF2007-63622-C02-02). All coauthors participated in the study and contributed to the development and critical revision of the manuscript. E.P., E.B. and G.M. performed experiments and analyzed data; B.C. reviewed and corrected the manuscript; C.R.-T. and M.B. provided vital reagents and technical support, designed research and analyzed data; J.A.E. designed research, analyzed data, wrote and submitted and coordinated the study.
1. Marsh M, Helenius A. Virus entry: open sesame. Cell 2006; 124:729–740.
2. Wang X, Huang DY, Huong SM, Huang ES. Integrin alphavbeta3 is a coreceptor for human cytomegalovirus. Nat Med 2005; 11:515–521.
3. Bosch B, Clotet-Codina I, Blanco J, Pauls E, Coma G, Cedeno S, et al
. Inhibition of human immunodeficiency virus type 1 infection in macrophages by an alpha-v integrin blocking antibody. Antiviral Res 2006; 69:173–180.
4. Ballana E, Pauls E, Clotet B, Perron-Sierra F, Tucker G, Este J. Cell adhesion through alphaV-containing integrins is required for efficient HIV-1 infection in macrophages. Blood
2008 [Epub ahead of print]. doi:10.1182/blood-2008-06-161869.
5. Arthos J, Cicala C, Martinelli E, Macleod K, Van Ryk D, Wei D, et al
. HIV-1 envelope protein binds to and signals through integrin [alpha]4[beta]7, the gut mucosal homing receptor for peripheral T cells. Nat Immunol 2008; 9:301–309.
6. Este JA, Telenti A. HIV entry inhibitors. Lancet 2007; 370:81–88.
7. Davenport RJ, Munday JR. Alpha4-integrin antagonism: an effective approach for the treatment of inflammatory diseases? Drug Discov Today 2007; 12:569–576.
8. Moncunill G, Armand-Ugon M, Pauls E, Clotet B, Este JA. HIV-1 escape to CCR5 coreceptor antagonism through selection of CXCR4-using variants in vitro. AIDS 2008; 22:23–31.
9. Moncunill G, Armand-Ugon M, Clotet-Codina I, Pauls E, Ballana E, Llano A, et al
. Anti-HIV activity and resistance profile of the CXC chemokine receptor 4 antagonist POL3026. Mol Pharmacol 2008; 73:1264–1273.
10. Lima MA, Marzocchetti A, Autissier P, Tompkins T, Chen Y, Gordon J, et al
. Frequency and phenotype of JC virus-specific CD8+ T lymphocytes in the peripheral blood of patients with progressive multifocal leukoencephalopathy. J Virol 2007; 81:3361–3368.
11. Berger JR. Progressive multifocal leukoencephalopathy in acquired immunodeficiency syndrome: explaining the high incidence and disproportionate, frequency of the illness relative to other immunosuppressive conditions. J Neurovirol 2003; 9:38–41.
12. Brenchley JM, Price DA, Douek DC. HIV disease: fallout from a mucosal catastrophe? Nat Immunol 2006; 7:235–239.