Stephen W. Wietgrefe

Propagation and Dissemination of Infection after Vaginal Transmission of Simian Immunodeficiency Virus

ABSTRACT In the current global AIDS pandemic, more than half of new human immunodeficiency virus type 1 (HIV-1) infections are acquired by women through intravaginal HIV exposure. For this study, we explored pathogenesis issues relevant to the development of effective vaccines to prevent infection by this route, using an animal model in which female rhesus macaques were exposed intravaginally to a high dose of simian immunodeficiency virus (SIV). We examined in detail the events that transpire from hours to a few days after intravaginal SIV exposure through week 4 to provide a framework for understanding the propagation, dissemination, and establishment of infection in lymphatic tissues (LTs) during the acute stage of infection. We show that the mucosal barrier greatly limits the infection of cervicovaginal tissues, and thus the initial founder populations of infected cells are small. While there was evidence of rapid dissemination to distal sites, we also show that continuous seeding from an expanding source of production at the portal of entry is likely critical for the later establishment of a productive infection throughout the systemic LTs. The initially small founder populations and dependence on continuous seeding to establish a productive infection in systemic LTs define a small window of maximum vulnerability for the virus in which there is an opportunity for the host, vaccines, or other interventions to prevent or control infection.

Persistent HIV-1 replication is associated with lower antiretroviral drug concentrations in lymphatic tissues

Significance We show that HIV continues to replicate in the lymphatic tissues of some individuals taking antiretroviral regimens considered fully suppressive, based on undetectable viral loads in peripheral blood, and that one mechanism for persistent replication in lymphatic tissues is the lower concentrations of the antiretroviral drugs in those tissues compared with peripheral blood. These findings are significant because they provide a rationale and framework for testing the efficacy of new agents and combinations of drugs that will fully suppress replication in lymphatic tissues. More suppressive regimens could improve immune reconstitution, as well as provide the effective regimens needed for functional cure and eradication of infection. Antiretroviral therapy can reduce HIV-1 to undetectable levels in peripheral blood, but the effectiveness of treatment in suppressing replication in lymphoid tissue reservoirs has not been determined. Here we show in lymph node samples obtained before and during 6 mo of treatment that the tissue concentrations of five of the most frequently used antiretroviral drugs are much lower than in peripheral blood. These lower concentrations correlated with continued virus replication measured by the slower decay or increases in the follicular dendritic cell network pool of virions and with detection of viral RNA in productively infected cells. The evidence of persistent replication associated with apparently suboptimal drug concentrations argues for development and evaluation of novel therapeutic strategies that will fully suppress viral replication in lymphatic tissues. These strategies could avert the long-term clinical consequences of chronic immune activation driven directly or indirectly by low-level viral replication to thereby improve immune reconstitution.

Premature Induction of an Immunosuppressive Regulatory T Cell Response during Acute Simian Immunodeficiency Virus Infection

Here we report the results of an investigation into the possibility that one mechanism responsible for the establishment of persistent human immunodeficiency virus infection is an early regulatory T (Treg) cell response that blunts virus-specific responses. Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model, we show that, indeed, viral replication and immune activation in lymphatic tissue drive a premature immunosuppressive response, with dramatic increases in the frequencies of CD4+CD25+FOXP3+ Treg cells, transforming growth factor- beta 1+ cells, interleukin-10+ cells, and indoleamine 2,3-dioxygenase+CD3+ cells. When we compared SIV infection with rhesus cytomegalovirus (RhCMV) infection, we found that the frequency of Treg cells paralleled the magnitude of immune activation during both infections but that the magnitude of immune activation and of the Treg cell response were lower and peaked much later during RhCMV infection. Importantly, the frequency of Treg cells inversely correlated with the magnitude of the SIV-specific cytotoxic T lymphocyte response. We conclude that an early Treg cell response during acute SIV infection may contribute to viral persistence by prematurely limiting the antiviral immune response before infection is cleared.

Cumulative mechanisms of lymphoid tissue fibrosis and T cell depletion in HIV-1 and SIV infections.

The hallmark of HIV-1 and SIV infections is CD4(+) T cell depletion. Both direct cell killing and indirect mechanisms related to immune activation have been suggested to cause the depletion of T cells. We have now identified a mechanism by which immune activation-induced fibrosis of lymphoid tissues leads to depletion of naive T cells in HIV-1 infected patients and SIV-infected rhesus macaques. The T regulatory cell response to immune activation increased procollagen production and subsequent deposition as fibrils via the TGF-β1 signaling pathway and chitinase 3-like-1 activity in fibroblasts in lymphoid tissues from patients infected with HIV-1. Collagen deposition restricted T cell access to the survival factor IL-7 on the fibroblastic reticular cell (FRC) network, resulting in apoptosis and depletion of T cells, which, in turn, removed a major source of lymphotoxin-β, a survival factor for FRCs during SIV infection in rhesus macaques. The resulting loss of FRCs and the loss of IL-7 produced by FRCs may thus perpetuate a vicious cycle of depletion of T cells and the FRC network. Because this process is cumulative, early treatment and antifibrotic therapies may offer approaches to moderate T cell depletion and improve immune reconstitution during HIV-1 infection.

Simian Immunodeficiency virus-Induced Lymphatic Tissue Fibrosis Is Mediated by Transforming Growth Factor β1-positive Regulatory T Cells and Begins in Early Infection

In human immunodeficiency virus (HIV) infection, collagen deposition and fibrosis within the T cell zone disrupt the lymphatic tissue architecture, contributing to depletion of CD4(+) T cells and limiting immune reconstitution. We used relevant animal and in vitro models to investigate the kinetics and possible underlying mechanism(s) of this process. In the lymphatic tissue of simian immunodeficiency virus (SIV)-infected rhesus macaques, we observed parallel increases in immune activation, transforming growth factor (TGF) beta 1-positive regulatory T (T(reg)) cells, and collagen type I deposition by 7 days after inoculation, consistent with the hypothesis that early immune activation elicits a countering T(reg) cell response associated with TGF beta 1 expression and collagen deposition. In support of this hypothesis and the possible role of fibrosis in viral pathogenesis, we show (1) spatial colocalization and temporal concordance in levels of TGF beta 1(+) T(reg) cells and collagen deposition; (2) TGF beta 1(+) inducible T(reg) cell stimulation of primary lymphatic tissue fibroblasts to produce collagen type I in vitro; and (3) high levels of immune activation, TGF beta 1(+) T(reg) cells, and collagen deposition in pathogenic SIV infection of macaques, in contrast to apathogenic SIV infection in sooty mangabeys in which levels of immune activation, TGF beta 1(+) T(reg) cells, and collagen deposition were low. We thus conclude that the response of TGF beta 1(+) T(reg) cells to immune activation in early SIV/HIV infection is a double-edged sword: TGF beta 1(+) T(reg) cells normally have a positive effect by limiting immunopathological and autoreactive immune responses, but they also have a negative effect by dampening the antiviral immune response and, as we show here, causing deleterious effects on CD4(+) T cell homeostasis by inducing collagen deposition in lymphatic tissues.

Large number of rebounding/founder HIV variants emerge from multifocal infection in lymphatic tissues after treatment interruption

Significance Antiretroviral therapy (ART) effectively suppresses HIV replication; however, treatment cannot be stopped, because latently infected CD4+ T cells will rekindle infection. As one estimate of the size of the pool of latently infected cells that must be purged for cure, we asked whether recrudescent infection is the result of reactivation from one or a larger number latently infected cells. We briefly stopped ART in fully suppressed patients to see how widespread new infections were in the lymphoid tissues (LTs) and how diverse rebounding/founder viruses were in peripheral blood. Recrudescent infection was detectable in multiple different LTs, and the population was genetically diverse, consistent with reactivation from a larger number of cells. These findings underscore the challenges facing strategies to eradicate HIV infection. Antiretroviral therapy (ART) suppresses HIV replication in most individuals but cannot eradicate latently infected cells established before ART was initiated. Thus, infection rebounds when treatment is interrupted by reactivation of virus production from this reservoir. Currently, one or a few latently infected resting memory CD4 T cells are thought be the principal source of recrudescent infection, but this estimate is based on peripheral blood rather than lymphoid tissues (LTs), the principal sites of virus production and persistence before initiating ART. We, therefore, examined lymph node (LN) and gut-associated lymphoid tissue (GALT) biopsies from fully suppressed subjects, interrupted therapy, monitored plasma viral load (pVL), and repeated biopsies on 12 individuals as soon as pVL became detectable. Isolated HIV RNA-positive (vRNA+) cells were detected by in situ hybridization in LTs obtained before interruption in several patients. After interruption, multiple foci of vRNA+ cells were detected in 6 of 12 individuals as soon as pVL was measureable and in some subjects, in more than one anatomic site. Minimal estimates of the number of rebounding/founder (R/F) variants were determined by single-gene amplification and sequencing of viral RNA or DNA from peripheral blood mononuclear cells and plasma obtained at or just before viral recrudescence. Sequence analysis revealed a large number of R/F viruses representing recrudescent viremia from multiple sources. Together, these findings are consistent with the origins of recrudescent infection by reactivation from many latently infected cells at multiple sites. The inferred large pool of cells and sites to rekindle recrudescent infection highlights the challenges in eradicating HIV.

Simian Immunodeficiency Virus—Induced Intestinal Cell Apoptosis Is the Underlying Mechanism of the Regenerative Enteropathy of Early Infection

The enteropathic manifestations of the human immunodeficiency virus (HIV) and the simian immunodeficiency virus (SIV) in late infection are usually due to infection by other microbes, but in early infection the viruses themselves cause an enteropathy by heretofore undetermined mechanisms. Here we report that SIV induces massive apoptosis of intestinal epithelial cells lining the small and large bowel, thus identifying apoptosis as the driving force behind the regenerative pathology of early infection. We found that apoptosis of gut epithelium paralleled the previously documented apoptosis and massive depletion of CD4 T cells in gut lamina propria, triggered by established mechanisms of gut epithelial cell apoptosis and, at peak, possibly by virus interactions with GPR15/Bob, an intestinal epithelial cell-associated alternative coreceptor for SIV and HIV-1. Apoptosis in early SIV infection is thus the common theme of the pathological processes that quickly afflict the innate as well as adaptive arms of the gut immune system.

Macrophages sustain HIV replication in vivo independently of T cells

Macrophages have long been considered to contribute to HIV infection of the CNS; however, a recent study has contradicted this early work and suggests that myeloid cells are not an in vivo source of virus production. Here, we addressed the role of macrophages in HIV infection by first analyzing monocytes isolated from viremic patients and patients undergoing antiretroviral treatment. We were unable to find viral DNA or viral outgrowth in monocytes isolated from peripheral blood. To determine whether tissue macrophages are productively infected, we used 3 different but complementary humanized mouse models. Two of these models (bone marrow/liver/thymus [BLT] mice and T cell-only mice [ToM]) have been previously described, and the third model was generated by reconstituting immunodeficient mice with human CD34+ hematopoietic stem cells that were devoid of human T cells (myeloid-only mice [MoM]) to specifically evaluate HIV replication in this population. Using MoM, we demonstrated that macrophages can sustain HIV replication in the absence of T cells; HIV-infected macrophages are distributed in various tissues including the brain; replication-competent virus can be rescued ex vivo from infected macrophages; and infected macrophages can establish de novo infection. Together, these results demonstrate that macrophages represent a genuine target for HIV infection in vivo that can sustain and transmit infection.

Targeted Cytotoxic Therapy Kills Persisting HIV Infected Cells During ART

Antiretroviral therapy (ART) can reduce HIV levels in plasma to undetectable levels, but rather little is known about the effects of ART outside of the peripheral blood regarding persistent virus production in tissue reservoirs. Understanding the dynamics of ART-induced reductions in viral RNA (vRNA) levels throughout the body is important for the development of strategies to eradicate infectious HIV from patients. Essential to a successful eradication therapy is a component capable of killing persisting HIV infected cells during ART. Therefore, we determined the in vivo efficacy of a targeted cytotoxic therapy to kill infected cells that persist despite long-term ART. For this purpose, we first characterized the impact of ART on HIV RNA levels in multiple organs of bone marrow-liver-thymus (BLT) humanized mice and found that antiretroviral drug penetration and activity was sufficient to reduce, but not eliminate, HIV production in each tissue tested. For targeted cytotoxic killing of these persistent vRNA+ cells, we treated BLT mice undergoing ART with an HIV-specific immunotoxin. We found that compared to ART alone, this agent profoundly depleted productively infected cells systemically. These results offer proof-of-concept that targeted cytotoxic therapies can be effective components of HIV eradication strategies.

Defining HIV and SIV Reservoirs in Lymphoid Tissues

A primary obstacle to an HIV-1 cure is long-lived viral reservoirs, which must be eliminated or greatly reduced. Cure strategies have largely focused on monitoring changes in T cell reservoirs in peripheral blood (PB), even though the lymphoid tissues (LT) are primary sites for viral persistence. To track and discriminate viral reservoirs within tissue compartments we developed a specific and sensitive next-generation in situ hybridization approach to detect vRNA, including vRNA+ cells and viral particles (“RNAscope”), vDNA+ cells (“DNAscope”) and combined vRNA and vDNA with immunohistochemistry to detect and phenotype active and latently infected cells in the same tissue section. RNAscope is highly sensitive with greater speed of analysis compared to traditional in situ hybridization. The highly sensitive and specific DNAscope detected SIV/HIV vDNA+ cells, including duplexed detection of vDNA and vRNA or immunophenotypic markers in the same section. Analysis of LT samples from macaques prior to and during combination antiretroviral therapy demonstrated that B cell follicles are an important anatomical compartment for both latent and active viral persistence during treatment. These new tools should allow new insights into viral reservoir biology and evaluation of cure strategies.