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Targeting α4β7 integrin reduces mucosal transmission of simian immunodeficiency virus and protects gut-associated lymphoid tissue from infection
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"Our findings are in accord with numerous studies that implicate α4β7 integrin in promoting mucosal transmission of HIV and SIV13, 14, 15, 16. The substantial decrease in the frequency of mucosal infection that we observed in α4β7-mAb-treated animals implicates α4β7 as a key determinant of SIV transmission. Evidence of lower tissue proviral loads and sparing of vital T cell subsets, even after treatment was discontinued, underscores the potential utility of α4β7-directed intervention in SIV and HIV disease. Recent reports from clinical trials of new drugs targeting α4β7 indicate their safety and efficacy in treating gastrointestinal inflammatory disorders17, 18, 19, 20 and raise the possibility that such therapies might prove efficacious in preventing and ameliorating HIV disease."
"Although we found no statistical difference between plasma viral RNA loads in control versus treated groups (Fig. 1a,b), the amount of proviral DNA in colorectal tissues differed significantly (Fig. 1d,e). Gastrointestinal tissue (GIT) biopsies from six treated and infected animals harbored, on average, median 3.5 copies of proviral DNA per ng of DNA, whereas biopsies from ten untreated and infected controls exhibited a median 12.8 copies per ng DNA (P = 0.006) (Fig. 1f). We confirmed that disparity in a survey of tissues collected from four viremic animals from both α4β7-mAb- and IgG control-treated groups post-mortem. Proviral DNA was 5- to 25-fold more abundant in DNA sampled from jejunum, ileum and colon of control as compared to treated infected animals (Fig. 1g), indicating that α4β7-mAb mediated reductions in GALT infection. In contrast, proviral loads in spleen and various other lymph nodes revealed no consistent disparity between the two groups (Fig. 1h). Although proviral DNA was undetectable in most other solid organs, it was detected in ecto- and endocervical tissues from three of three treated, infected animals, but not in ecto- and endocervical tissues of three control infected animals, sampled post-mortem (Fig. 1i). Thus, for animals that became infected despite treatment, α4β7-mAb was associated with a persistence of infected cells at the portal of infection and a substantial decrease in SIV-infected cells within GALT (Fig. 1d). α4β7-mAb-treated animals that became infected maintained higher CD4+ T cell counts in blood (P < 0.001; Fig. 2a), cytobrush specimens (P = 0.0052; Fig. 2b) and gut tissues (P < 0.0001; Fig. 2c) but had no effect on other cell lineages, confirming that α4β7-mAb treatment did not markedly alter the frequencies of major immune cell types in cervicovaginal tissues."
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Nature Medicine (2014) - Siddappa N Byrareddy1,8, Brianne Kallam1,8, James Arthos2, Claudia Cicala2, Fatima Nawaz2, Joseph Hiatt2, Ellen N Kersh3, Janet M McNicholl3, Debra Hanson3, Keith A Reimann4, Markus Brameier5, Lutz Walter5, Kenneth Rogers6, Ann E Mayne1, Paul Dunbar1, Tara Villinger1, Dawn Little1, Tristram G Parslow1, Philip J Santangelo7, Francois Villinger1,6, Anthony S Fauci2 & Aftab A Ansari1
α4β7 integrin-expressing CD4+ T cells preferentially traffic to gut-associated lymphoid tissue (GALT) and have a key role in HIV and simian immunodeficiency virus (SIV) pathogenesis. We show here that the administration of an anti-α4β7 monoclonal antibody just prior to and during acute infection protects rhesus macaques from transmission following repeated low-dose intravaginal challenges with SIVmac251. In treated animals that became infected, the GALT was significantly protected from infection and CD4+ T cell numbers were maintained in both the blood and the GALT. Thus, targeting α4β7 reduces mucosal transmission of SIV in macaques.
In this study, we evaluated the efficacy of α4β7-mAb therapy in preventing transmission. We employed a nonhuman primate model based on repeated low-dose intravaginal challenges, which more faithfully mimics HIV transmission. We found that intravenous administration of α4β7-mAb reduced surface exposure of α4β7 on CD4+ T cells in the cervicovaginal canal and either prevented or delayed infection. When prevention failed, both viral DNA loads in GALT and the rate of peripheral CD4+ T cell depletion were markedly reduced as compared to in the control IgG-treated group. The results of these studies suggest that α4β7 antagonists might be useful for prophylaxis or treatment of HIV infection.
CD4+ T cells residing in GALT are a predominant target of HIV and SIV during the acute phase of infection, and their depletion has been implicated in HIV and SIV disease1, 2, 3. CD4+ and CCR5+ T cells that traffic to GALT typically express α4β7 integrin, which functions as a gut-homing receptor4. α4β7 mediates gut-homing by binding to mucosal vascular addressin cell adhesion molecule (MAdCAM), a ligand expressed on venules that service GALT5. Of note, some strains of HIV and SIV bind to α4β7 (refs. 6,7,8). These findings suggest that the α4β7+ subset of CD4+ T cells may play a key part in transmission and that targeting could disrupt early steps in infection and possibly interfere with disease progression.
To explore that possibility, we used a recombinant rhesus anti-α4β7 monoclonal antibody (mAb) (α4β7-mAb)9. Previously, in a high-dose intravenous or intrarectal challenge study, we showed that α4β7-mAb treatment before and after challenge mediated a significant decrease in gut tissue proviral DNA and a one- to two-log reduction in mean plasma viral loads in both studies10, 11. Notably, whereas 10 of 12 controls died of AIDS within 2 years, all ten treated animals remained healthy with CD4+ T cell counts >500/μl 5 years after infection10, 11.
In this study, we evaluated the efficacy of α4β7-mAb therapy in preventing transmission. We employed a nonhuman primate model based on repeated low-dose intravaginal challenges, which more faithfully mimics HIV transmission. We found that intravenous administration of α4β7-mAb reduced surface exposure of α4β7 on CD4+ T cells in the cervicovaginal canal and either prevented or delayed infection. When prevention failed, both viral DNA loads in GALT and the rate of peripheral CD4+ T cell depletion were markedly reduced as compared to in the control IgG-treated group. The results of these studies suggest that α4β7 antagonists might be useful for prophylaxis or treatment of HIV infection.
The protocol used for the entire study is illustrated in Supplementary Figure 1. We conducted baseline studies designed to optimize the collection and analyses of cervical tissue (cytobrush) and gut tissue samples in 24 uninfected female macaques (phase 1). The results are summarized in Supplementary Figure 2a,b. We then divided the macaques into two groups (n = 8), each of which was administered two sequential intravenous injections, at 3-week intervals, of either rhesus recombinant α4β7-mAb or a recombinant isotype-matched control at 50 mg per kg body weight (mg/kg), as previously described10. We monitored plasma and cervicovaginal lavage (CVL) fluid α4β7-mAb concentrations10 and confirmed that this regimen maintained plasma levels at or above 25 μg/ml and detectable levels in the CVL fluid (Supplementary Fig. 3a,b). We collected peripheral blood, rectal biopsies and cytobrush samples weekly and analyzed them to determine the efficiency and kinetics of the binding of the α4β7-mAb. Cells from all three compartments were bound by the α4β7-mAb (Supplementary Fig. 2c) but did not significantly alter the proportion of T cells expressing the α4 integrin subunit on the same cells (Supplementary Fig. 2d), indicating that treatment did not eliminate α4β7+ cells but rather masked the relevant epitope.
To determine whether masking α4β7 affects susceptibility to infection, the 24 female macaques were divided into two equivalent groups on the basis of the frequencies of CD4+α4β7+ T cells and regulatory T cells in blood, gut tissues and cervicovaginal cells (Supplementary Fig. 4), age and reproductive history and genetic characteristics (Supplementary Tables 1 and 2). We gave each group five injections of either α4β7-mAb or the control mAb at 3-week intervals. Three days after the first injection, we challenged the animals intravaginally with a low dose of SIVmac251. Under the predetermined protocol, we rechallenged the animals every week until 10 of 12 control animals became infected and then compared their plasma viral RNA loads (Fig. 1a,b). By week 5, 10 of 12 control animals became viremic, and we discontinued the challenges. In contrast, only 1 of 12 treated animals became viremic by week 5. An additional five animals developed viremia by week 8, whereas 6 of 12 remained uninfected. All uninfected animals were subsequently found not to be intrinsically resistant to infection (Supplementary Fig. 5a,b).
We used Kaplan-Meier analysis (Fig. 1c) to verify that α4β7-mAb treatment significantly increased the number of challenges required for infection (P = 0.002, log-rank test). The hazard ratio was 4.3, with 95% confidence limits of 1.5-12.2 and P = 0.007 using the proportional-hazards regression model. In all, 71 challenges were needed to infect 6 of 12 mAb treated macaques, whereas only 44 challenges sufficed to infect 10 of 12 controls, implying a 2.7-fold decreased infection risk with each challenge in the α4β7-mAb-treated group (P < 0.05 by Fisher's exact test).
Although we found no statistical difference between plasma viral RNA loads in control versus treated groups (Fig. 1a,b), the amount of proviral DNA in colorectal tissues differed significantly (Fig. 1d,e). Gastrointestinal tissue (GIT) biopsies from six treated and infected animals harbored, on average, median 3.5 copies of proviral DNA per ng of DNA, whereas biopsies from ten untreated and infected controls exhibited a median 12.8 copies per ng DNA (P = 0.006) (Fig. 1f). We confirmed that disparity in a survey of tissues collected from four viremic animals from both α4β7-mAb- and IgG control-treated groups post-mortem. Proviral DNA was 5- to 25-fold more abundant in DNA sampled from jejunum, ileum and colon of control as compared to treated infected animals (Fig. 1g), indicating that α4β7-mAb mediated reductions in GALT infection. In contrast, proviral loads in spleen and various other lymph nodes revealed no consistent disparity between the two groups (Fig. 1h). Although proviral DNA was undetectable in most other solid organs, it was detected in ecto- and endocervical tissues from three of three treated, infected animals, but not in ecto- and endocervical tissues of three control infected animals, sampled post-mortem (Fig. 1i). Thus, for animals that became infected despite treatment, α4β7-mAb was associated with a persistence of infected cells at the portal of infection and a substantial decrease in SIV-infected cells within GALT (Fig. 1d). α4β7-mAb-treated animals that became infected maintained higher CD4+ T cell counts in blood (P < 0.001; Fig. 2a), cytobrush specimens (P = 0.0052; Fig. 2b) and gut tissues (P < 0.0001; Fig. 2c) but had no effect on other cell lineages, confirming that α4β7-mAb treatment did not markedly alter the frequencies of major immune cell types in cervicovaginal tissues.
We next wanted to explore potential mechanisms of the protection we observed in α4β7-mAb-treated animals. Analysis of SIV-specific humoral and cellular responses failed to show any detectable immune responses that could have contributed to the SIV infection resistance of these 6 infection-resistant animals, although samples from the SIV-infected animals analyzed in parallel showed readily detectable SIV-specific humoral and cellular responses. Thus, the observed protection was not mediated by cellular or humoral immune responses. α4β7-mAb treatment may have reduced transmission by inhibiting α4β7 binding to MAdCAM, thereby reducing homing of α4β7+CD4+ T cells to GALT, and/or by interfering with any potential interactions between α4β7 and SIVmac251 envelope protein that could mediate infection. To this end we found that α4β7-mAb inhibits binding of α4β7 to both MAdCAM and a gp120 derived from SIVmac251 (Fig. 2d-g). In addition, we determined that MAdCAM and SIVmac251 gp120 compete for binding to α4β7 (Fig. 2h). Thus, the α4β7-mAb possesses the capacity, in at least two ways, to interfere with intravaginal transmission of SIVmac251.
The present study demonstrates that intravenous administration of α4β7-mAb shortly before and for several weeks after multiple low-dose intravaginal SIV challenges in rhesus macaques substantially decreased the likelihood of viral transmission. Furthermore, in those treated animals that did become infected, GALT viral loads were markedly reduced. Macaques receiving α4β7-mAb prophylactically were, on average, 63% less likely to become infected following any single intravaginal challenge than were controls. The profound destruction of gut CD4+ T cells that typically occurs in acute SIV or HIV infection was prevented in treated but infected animals, which could contribute to the amelioration of the underlying causes of AIDS1. Our earlier studies10, 11 of α4β7-mAb employed high dosages of SIVmac239 that were designed to infect all animals with a single inoculation. Under those conditions, α4β7-mAb markedly impeded disease progression and mortality. The present study was designed to understand the role of α4β7+ T cells in sexual transmission by using repeated low-dose intravaginal challenges. Targeting α4β7+ T cells not only impeded intravaginal transmission but also reduced proviral DNA loads in GALT long after treatment was terminated, despite sustained viremia.
Treatment with α4β7-mAb did not greatly alter the numbers of CD4+ T cells within the cervicovaginal compartment, consistent with the absence of MAdCAM in the female genital tract (FGT) under normal conditions (MAdCAM is induced in the FGT by sexually transmitted diseases known to increase the susceptibility to HIV12). However, α4β7-mAb masked >99.9% of the α4β7 heterodimers on cells in cervicovaginal compartments (Supplementary Fig. 2c). Masking α4β7 in the FGT might prevent transmission by suppressing the spread of a nascent infection into the largest depot of vulnerable target T cells in the body by interfering with the physical interaction of virus with α4β7+CD4+ cells, or by disrupting cell-cell interactions necessary for efficient viral transmission. In any case, these results suggest that SIVmac251 utilizes α4β7+CD4+ cells at some key early point in transmission. This conclusion is further supported by the delayed infection observed in a number of treated animals. Of note, one of these animals did not become viremic until 3 weeks after challenges were discontinued. In addition, we detected proviral DNA in cervical tissues of the SIV-infected α4β7-mAb-treated but not the control IgG-treated macaques in samples collected from these animals at autopsies performed either early or 16-18 weeks after infection (Fig. 1i).
Our findings are in accord with numerous studies that implicate α4β7 integrin in promoting mucosal transmission of HIV and SIV13, 14, 15, 16. The substantial decrease in the frequency of mucosal infection that we observed in α4β7-mAb-treated animals implicates α4β7 as a key determinant of SIV transmission. Evidence of lower tissue proviral loads and sparing of vital T cell subsets, even after treatment was discontinued, underscores the potential utility of α4β7-directed intervention in SIV and HIV disease. Recent reports from clinical trials of new drugs targeting α4β7 indicate their safety and efficacy in treating gastrointestinal inflammatory disorders17, 18, 19, 20 and raise the possibility that such therapies might prove efficacious in preventing and ameliorating HIV disease.
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