Genetically edited CD34+ cells derived from human iPS cells in vivo but not in vitro engraft and differentiate into HIV-resistant cells.

Genetic editing of induced pluripotent stem (iPS) cells represents a promising avenue for an HIV cure. However, certain challenges remain before bringing this approach to the clinic. Among them, in vivo engraftment of cells genetically edited in vitro needs to be achieved. In this study, CD34+ cells derived in vitro from iPS cells genetically modified to carry the CCR5Δ32 mutant alleles did not engraft in humanized immunodeficient mice. However, the CD34+ cells isolated from teratomas generated in vivo from these genetically edited iPS cells engrafted in all experiments. These CD34+ cells also gave rise to peripheral blood mononuclear cells in the mice that, when inoculated with HIV in cell culture, were resistant to HIV R5-tropic isolates. This study indicates that teratomas can provide an environment that can help evaluate the engraftment potential of CD34+ cells derived from the genetically modified iPS cells in vitro. The results further confirm the possibility of using genetically engineered iPS cells to derive engraftable hematopoietic stem cells resistant to HIV as an approach toward an HIV cure.

Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30.

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection.

Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction.

Although invasive cytomegalovirus (CMV) disease is uncommon in the era of antiretroviral therapy (ART), asymptomatic CMV coinfection is nearly ubiquitous in HIV infected individuals. While microbial translocation and gut epithelial barrier dysfunction may promote persistent immune activation in treated HIV infection, potentially contributing to morbidity and mortality, it has been unclear whether CMV replication in individuals with no symptoms of CMV disease might play a role in this process. We hypothesized that persistent CMV replication in the intestinal epithelium of HIV/CMV-coinfected individuals impairs gut epithelial barrier function. Using a combination of state-of-the-art in situ hybridization technology (RNAscope) and immunohistochemistry, we detected CMV DNA and proteins and evidence of intestinal damage in rectosigmoid samples from CMV-positive individuals with both untreated and ART-suppressed HIV infection. Two different model systems, primary human intestinal cells differentiated in vitro to form polarized monolayers and a humanized mouse model of human gut, together demonstrated that intestinal epithelial cells are fully permissive to CMV replication. Independent of HIV, CMV disrupted tight junctions of polarized intestinal cells, significantly reducing transepithelial electrical resistance, a measure of monolayer integrity, and enhancing transepithelial permeability. The effect of CMV infection on the intestinal epithelium is mediated, at least in part, by the CMV-induced proinflammatory cytokine IL-6. Furthermore, letermovir, a novel anti-CMV drug, dampened the effects of CMV on the epithelium. Together, our data strongly suggest that CMV can disrupt epithelial junctions, leading to bacterial translocation and chronic inflammation in the gut and that CMV could serve as a target for therapeutic intervention to prevent or treat gut epithelial barrier dysfunction during HIV infection.

Inhibition of Heat Shock Protein 90 Prevents HIV Rebound.

HIV evades eradication because transcriptionally dormant proviral genomes persist in long-lived reservoirs of resting CD4(+) T cells and myeloid cells, which are the source of viral rebound after cessation of antiretroviral therapy. Dormant HIV genomes readily produce infectious virus upon cellular activation because host transcription factors activated specifically by cell stress and heat shock mediate full-length HIV transcription. The molecular chaperone heat shock protein 90 (Hsp90) is overexpressed during heat shock and activates inducible cellular transcription factors. Here we show that heat shock accelerates HIV transcription through induction of Hsp90 activity, which activates essential HIV-specific cellular transcription factors (NF-κB, NFAT, and STAT5), and that inhibition of Hsp90 greatly reduces gene expression mediated by these factors. More importantly, we show that Hsp90 controls virus transcription in vivo by specific Hsp90 inhibitors in clinical development, tanespimycin (17-(allylamino)-17-demethoxygeldanamycin) and AUY922, which durably prevented viral rebound in HIV-infected humanized NOD scid IL-2Rγ(-/-) bone marrow-liver-thymus mice up to 11 weeks after treatment cessation. Despite the absence of rebound viremia, we were able to recover infectious HIV from PBMC with heat shock. Replication-competent virus was detected in spleen cells from these nonviremic Hsp90 inhibitor-treated mice, indicating the presence of a tissue reservoir of persistent infection. Our novel findings provide in vivo evidence that inhibition of Hsp90 activity prevents HIV gene expression in replication-competent cellular reservoirs that would typically cause rebound in plasma viremia after antiretroviral therapy cessation. Alternating or supplementing Hsp90 inhibitors with current antiretroviral therapy regimens could conceivably suppress rebound viremia from persistent HIV reservoirs.

HIV Maintains an Evolving and Dispersed Population in Multiple Tissues during Suppressive Combined Antiretroviral Therapy in Individuals with Cancer.

UNLABELLED: While combined antiretroviral therapy (cART) can result in undetectable plasma viral loads, it does not eradicate HIV infection. Furthermore, HIV-infected individuals while on cART remain at an increased risk of developing serious comorbidities, such as cancer, neurological disease, and atherosclerosis, suggesting that during cART, tissue-based HIV may contribute to such pathologies. We obtained DNA and RNA env, nef, and pol sequences using single-genome sequencing from postmortem tissues of three HIV(+) cART-treated (cART(+)) individuals with undetectable viral load and metastatic cancer at death and performed time-scaled Bayesian evolutionary analyses. We used a sensitive in situ hybridization technique to visualize HIV gag-pol mRNA transcripts in cerebellum and lymph node tissues from one patient. Tissue-associated virus evolved at similar rates in cART(+) and cART-naive (cART(-)) patients. Phylogenetic trees were characterized by two distinct features: (i) branching patterns consistent with constant viral evolution and dispersal among tissues and (ii) very recently derived clades containing both DNA and RNA sequences from multiple tissues. Rapid expansion of virus near death corresponded to wide-spread metastasis. HIV RNA(+) cells clustered in cerebellum tissue but were dispersed in lymph node tissue, mirroring the evolutionary patterns observed for that patient. Activated, infiltrating macrophages were associated with HIV RNA. Our data provide evidence that tissues serve as a sanctuary for wild-type HIV during cART and suggest the importance of macrophages as an alternative reservoir and mechanism of virus spread. IMPORTANCE: Combined antiretroviral therapy (cART) reduces plasma HIV to undetectable levels; however, removal of cART results in plasma HIV rebound, thus highlighting its inability to entirely rid the body of infection. Additionally, HIV-infected individuals on cART remain at high risk of serious diseases, which suggests a contribution from residual HIV. In this study, we isolated and sequenced HIV from postmortem tissues from three HIV(+) cART(+) individuals who died with metastatic cancer and had no detectable plasma viral load. Using high-resolution evolutionary analyses, we found that tissue-based HIV continues to replicate, evolve, and migrate among tissues during cART. Furthermore, cancer onset and metastasis coincided with increased HIV expansion, suggesting a linked mechanism. HIV-expressing cells were associated with tissue macrophages, a target of HIV infection. Our results suggest the importance of tissues, and macrophages in particular, as a target for novel anti-HIV therapies.

HIV DNA Is Frequently Present within Pathologic Tissues Evaluated at Autopsy from Combined Antiretroviral Therapy-Treated Patients with Undetectable Viral Loads.

UNLABELLED: HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma HIV RNA. While combined antiretroviral therapy (cART) can reduce plasma viral load (pVL) to undetectable levels, the degree that HIV is eliminated from other anatomical sites remains unclear. We investigated the HIV DNA levels in 229 varied autopsy tissues from 20 HIV-positive (HIV(+)) cART-treated study participants with low or undetectable plasma VL and cerebrospinal fluid (CSF) VL prior to death who were enrolled in the National Neurological AIDS Bank (NNAB) longitudinal study and autopsy cohort. Extensive medical histories were obtained for each participant. Autopsy specimens, including at least six brain and nonbrain tissues per participant, were reviewed by study pathologists. HIV DNA, measured in tissues by quantitative and droplet digital PCR, was identified in 48/87 brain tissues and 82/142 nonbrain tissues at levels >200 HIV copies/million cell equivalents. No participant was found to be completely free of tissue HIV. Parallel sequencing studies from some tissues recovered intact HIV DNA and RNA. Abnormal histological findings were identified in all participants, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. All brain tissues demonstrated some degree of pathology. Ninety-five percent of participants had some degree of atherosclerosis, and 75% of participants died with cancer. This study assists in characterizing the anatomical locations of HIV, in particular, macrophage-rich tissues, such as the central nervous system (CNS) and testis. Additional studies are needed to determine if the HIV recovered from tissues promotes the pathogenesis of inflammatory diseases, such as HIV-associated neurocognitive disorders, cancer, and atherosclerosis. IMPORTANCE: It is well-known that combined antiretroviral therapy (cART) can reduce plasma HIV to undetectable levels; however, cART cannot completely clear HIV infection. An ongoing question is, "Where is HIV hiding?" A well-studied HIV reservoir is "resting" T cells, which can be isolated from blood products and succumb to cART once activated. Less-studied reservoirs are anatomical tissue samples, which have unknown cART penetration, contain a comparably diverse spectrum of potentially HIV-infected immune cells, and are important since <2% of body lymphocytes actually reside in blood. We examined 229 varied autopsy specimens from 20 HIV(+) participants who died while on cART and identified that >50% of tissues were HIV infected. Additionally, we identified considerable pathology in participants' tissues, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. This study substantiates that tissue-associated HIV is present despite cART and can inform future studies into HIV persistence.

The meningeal lymphatic system: a route for HIV brain migration?

Two innovative studies recently identified functional lymphatic structures in the meninges that may influence the development of HIV-associated neurological disorders (HAND). Until now, blood vessels were assumed to be the sole transport system by which HIV-infected monocytes entered the brain by bypassing a potentially hostile blood-brain barrier through inflammatory-mediated semi-permeability. A cascade of specific chemokine signals promote monocyte migration from blood vessels to surrounding brain tissues via a well-supported endothelium, where the cells differentiate into tissue macrophages capable of productive HIV infection. Lymphatic vessels on the other hand are more loosely organized than blood vessels. They absorb interstitial fluid from bodily tissues where HIV may persist and exchange a variety of immune cells (CD4(+) T cells, monocytes, macrophages, and dendritic cells) with surrounding tissues through discontinuous endothelial junctions. We propose that the newly discovered meningeal lymphatics are key to HIV migration among viral reservoirs and brain tissue during periods of undetectable plasma viral loads due to suppressive combinational antiretroviral therapy, thus redefining the migration process in terms of a blood-lymphatic transport system.

Oral administration of the nucleoside EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) provides rapid suppression of HIV viremia in humanized mice and favorable pharmacokinetic properties in mice and the rhesus macaque.

Like normal cellular nucleosides, the nucleoside reverse transcriptase (RT) inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) has a 3'-hydroxyl moiety, and yet EFdA is a highly potent inhibitor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication with activity against a broad range of clinically important drug-resistant HIV isolates. We evaluated the anti-HIV activity of EFdA in primary human cells and in HIV-infected humanized mice. EFdA exhibited excellent potency against HIVJR-CSF in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), with a 50% inhibitory concentration of 0.25 nM and a selectivity index of 184,000; similar antiviral potency was found against 12 different HIV clinical isolates from multiple clades (A, B, C, D, and CRF01_AE). EFdA was readily absorbed after oral dosing (5 mg/kg of body weight) in both mice and the rhesus macaque, with micromolar levels of the maximum concentration of drug in serum (Cmax) attained at 30 min and 90 min, respectively. Trough levels were at or above 90% inhibitory concentration (IC90) levels in the macaque at 24 h, suggesting once-daily dosing. EFdA showed reasonable penetration of the blood-brain barrier in the rhesus macaque, with cerebrospinal fluid levels at approximately 25% of plasma levels 8 h after single oral dosing. Rhesus PBMCs isolated 24 h following a single oral dose of 5 mg/kg EFdA were refractory to SIV infection due to sufficiently high intracellular EFdA-triphosphate levels. The intracellular half-life of EFdA-triphosphate in PBMCs was determined to be >72 h following a single exposure to EFdA. Daily oral administration of EFdA at low dosage levels (1 to 10 mg/kg/day) was highly effective in protecting humanized mice from HIV infection, and 10 mg/kg/day oral EFdA completely suppressed HIV RNA to undetectable levels within 2 weeks of treatment.

Alpha interferon and HIV infection cause activation of human T cells in NSG-BLT mice.

The development of small animal models for the study of HIV transmission is important for evaluation of HIV prophylaxis and disease pathogenesis. In humanized bone marrow-liver-thymus (BLT) mice, hematopoiesis is reconstituted by implantation of human fetal liver and thymus tissue (Thy/Liv) plus intravenous injection of autologous liver-derived hematopoietic stem progenitor cells (HSPC). This results in reconstitution of human leukocytes in the mouse peripheral blood, lymphoid organs, and mucosal sites. NOD-scid interleukin-2 receptor-negative (IL-2Rγ(-/-)) (NSG)-BLT mice were inoculated intravaginally with HIV and were monitored for plasma viremia by a branched DNA assay 4 weeks later. T-cell activation was determined by expression of CD38 and HLA-DR on human CD4(+) and CD8(+) T cells in mouse peripheral blood at the time of inoculation and 4 weeks later. Additional BLT mice were treated with human alpha interferon 2b (IFN-α2b) (intron A) and assessed for T-cell activation. Productive HIV infection in BLT mice was associated with T-cell activation (increases in CD38 mean fluorescence intensity and both the frequency and absolute number of CD38(+) HLA-DR(+) T cells) that correlated strongly with plasma viral load and was most pronounced in the CD8(+) T-cell compartment. This T-cell activation phenotype was recapitulated in NSG-BLT mice treated with intron A. HIV susceptibility correlated with the number of HSPC injected, yet a number of mice receiving the Thy/Liv implant alone, with no HSPC injection, were also susceptible to intravaginal HIV. These results are consistent with studies linking T-cell activation to progressive disease in humans and lend support for the use of NSG-BLT mice in studies of HIV pathogenesis.

Impaired surfactant production by alveolar epithelial cells in a SCID-hu lung mouse model of congenital human cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the leading viral cause of birth defects and life-threatening lung-associated diseases in premature infants and immunocompromised children. Although the fetal lung is a major target organ of the virus, HCMV lung pathogenesis has remained unexplored, possibly as a result of extreme host range restriction. To overcome this hurdle, we generated a SCID-hu lung mouse model that closely recapitulates the discrete stages of human lung development in utero. Human fetal lung tissue was implanted into severe combined immunodeficient (CB17-scid) mice and inoculated by direct injection with the VR1814 clinical isolate of HCMV. Virus replication in the fetal lung was assessed by the quantification of infectious virus titers and HCMV genome copies and the detection of HCMV proteins by immunohistochemistry and Western blotting. We show that HCMV efficiently replicated in the lung implants during a 2-week period, forming large viral lesions. The virus productively infected alveolar epithelial and mesenchymal cells, imitating congenital infection of the fetal lung. HCMV replication triggered apoptosis near and within the viral lesions and impaired the production of surfactant proteins in the alveolar epithelium. Our findings highlight that congenital and neonatal HCMV infection can adversely impact lung development, leading to pneumonia and acute lung injury. We have successfully developed a small-animal model that closely recapitulates fetal and neonatal lung development and provides a valuable, biologically relevant tool for an understanding of the lung pathogenesis of HCMV as well as other human respiratory viruses. Additionally, this model would greatly facilitate the development and testing of new antiviral therapies for HCMV along with select human pulmonary pathogens.

HIV disease progression correlates with the generation of dysfunctional naive CD8(low) T cells.

HIV infection can result in depletion of total CD4(+) T cells and naive CD8(+) T cells, and in the generation of dysfunctional effector CD8(+) T cells. In this study, we show that naive CD8(+) T cells in subjects with progressive HIV disease express low levels of CD8α and CD8ß chains. Such naive CD8(low) T cells display broad signaling defects across the T-cell receptor complex, and their appearance correlates with generalized up-regulation of major histocompatibility complex class I (MHC-I) antigens on peripheral blood mononuclear cells (PBMCs). To explore a causal link between increased MHC-I up-regulation and the generation of naive CD8(low) T cells, we used the humanized SCID-hu Thy/Liv mouse model to show that HIV infection of the thymus and interferon α (IFNα) treatment alone result in MHC-I up-regulation and in the generation of dysfunctional CD3(high)CD8(+)CD4(-) single-positive 8 (SP8) thymocytes with low expression of CD8. We suggest that dysfunctional naive CD8(low) T cells are generated as a result of IFNα-mediated up-regulation of MHC-I on stromal cells in the thymus and antigen-presenting cells in the periphery, and that dysfunction in this naive compartment contributes to the immunodeficiency of HIV disease. This study is registered at www.clinicaltrials.gov as NCT00187512.

Impaired infectivity of ritonavir-resistant HIV is rescued by heat shock protein 90AB1.

Certain ritonavir resistance mutations impair HIV infectivity through incomplete Gag processing by the mutant viral protease. Analysis of the mutant virus phenotype indicates that accumulation of capsid-spacer peptide 1 precursor protein in virus particles impairs HIV infectivity and that the protease mutant virus is arrested during the early postentry stage of HIV infection before proviral DNA synthesis. However, activation of the target cell can rescue this defect, implying that specific host factors expressed in activated cells can compensate for the defect in ritonavir-resistant HIV. This ability to rescue impaired HIV replication presented a unique opportunity to identify host factors involved in postentry HIV replication, and we designed a functional genetic screen so that expression of a given host factor extracted from activated T cells would lead directly to its discovery by rescuing mutant virus replication in nonactivated T cells. We identified the cellular heat shock protein 90 kDa α (cytosolic), class B member 1 (HSP90AB1) as a host factor that can rescue impaired replication of ritonavir-resistant HIV. Moreover, we show that pharmacologic inhibition of HSP90AB1 with 17-(allylamino)-17-demethoxygeldanamycin (tanespimycin) has potent in vitro anti-HIV activity and that ritonavir-resistant HIV is hypersensitive to the drug. These results suggest a possible role for HSP90AB1 in postentry HIV replication and may provide an attractive target for therapeutic intervention.

Preexposure prophylaxis with albumin-conjugated C34 peptide HIV-1 fusion inhibitor in SCID-hu Thy/Liv mice.

PC-1505 is a C34 peptide derived from the heptad repeat 2 region of HIV-1 gp41 conjugated to human serum albumin for sustained in vivo activity. One single preexposure dose of PC-1505 reduced viral RNA in HIV-1-infected SCID-hu Thy/Liv mice by 3.3 log10 and protected T cells from virus-mediated depletion. In contrast, a single preexposure dose of Truvada reduced viral RNA by only 0.8 log10 and was substantially less effective in preventing T cell depletion.

IFN-alpha-induced upregulation of CCR5 leads to expanded HIV tropism in vivo.

Chronic immune activation and inflammation (e.g., as manifest by production of type I interferons) are major determinants of disease progression in primate lentivirus infections. To investigate the impact of such activation on intrathymic T-cell production, we studied infection of the human thymus implants of SCID-hu Thy/Liv mice with X4 and R5 HIV. X4 HIV was observed to infect CD3(-)CD4(+)CD8(-)CXCR4(+)CCR5(-) intrathymic T-cell progenitors (ITTP) and to abrogate thymopoiesis. R5 HIV, by contrast, first established a nonpathogenic infection of thymic macrophages and then, after many weeks, began to replicate in ITTP. We demonstrate here that the tropism of R5 HIV is expanded and pathogenicity enhanced by upregulation of CCR5 on these key T-cell progenitors. Such CCR5 induction was mediated by interferon-alpha (IFN-alpha) in both thymic organ cultures and in SCID-hu mice, and antibody neutralization of IFN-alpha in R5 HIV-infected SCID-hu mice inhibited both CCR5 upregulation and infection of the T-cell progenitors. These observations suggest a mechanism by which IFN-alpha production may paradoxically expand the tropism of R5 HIV and, in so doing, accelerate disease progression.

Derivation of non-infectious envelope proteins from virions isolated from plasma negative for HIV antibodies.

Natural membrane-bound HIV-1 envelope proteins (mHIVenv) could be used to produce an effective subunit vaccine against HIV infection, akin to effective vaccination against HBV infection using the hepatitis B surface antigen. The quaternary structure of mHIVenv is postulated to elicit broadly neutralizing antibodies protective against HIV-1 transmission. The founder virus transmitted to infected individuals during acute HIV-1 infection is genetically homogeneous and restricted to CCR5-tropic phenotype. Therefore, isolates of plasma-derived HIV-1 (PHIV) from infected blood donors while negative for antibodies to HIV proteins were selected for expansion in primary lymphocytes as an optimized cell substrate (OCS). Virions in the culture supernatants were purified by removing contaminating microvesicles using immunomagnetic beads coated with anti-CD45. Membrane cholesterol was extracted from purified virions with beta-cyclodextrin to permeabilize them and expel p24, RT and viral RNA, and permit protease-free Benzonase to hydrolyze the residual viral/host DNA/RNA without loss of gp120. The resultant mHIVenv, containing gp120 bound to native gp41 in immunoreactive form, was free from infectivity in vitro in co-cultures with OCS and in vivo after inoculating SCID-hu Thy/Liv mice. These data should help development of mHIVenv as a virally safe immunogen and enable preparation of polyclonal hyper-immune globulins for immunoprophylaxis against HIV-1 infection.

Comparative Pharmacokinetics of a Dual Inhibitor of HIV-1, NBD-14189, in Rats and Dogs with a Proof-of-Concept Evaluation of Antiviral Potency in SCID-hu Mouse Model.

We earlier reported substantial progress in designing gp120 antagonists. Notably, we discovered that NBD-14189 is not only the most active gp120 antagonist but also shows antiviral activity against HIV-1 Reverse Transcriptase (RT). We also confirmed its binding to HIV-1 RT by X-ray crystallography. The dual inhibition is highly significant because, intriguingly, this compound bridges the dNTP and NNRTI-binding sites and inhibits the polymerase activity of isolated RT in the enzymatic assay. This novel finding is expected to lead to new avenues in designing a novel class of HIV-1 dual inhibitors. Therefore, we needed to advance this inhibitor to preclinical assessment. To this end, we report the pharmacokinetics (PK) study of NBD-14189 in rats and dogs. Subsequently, we assessed the toxicity and therapeutic efficacy in vivo in the SCID-hu Thy/Liv mouse model. The PK data indicated a favorable half-life (t1/2) and excellent oral bioavailability (%F = 61%). NBD-14189 did not show any measurable toxicity in the mice, and treatment reduced HIV replication at 300 mg/kg per day in the absence of clear evidence of protection from HIV-mediated human thymocyte depletion. The data indicated the potential of this inhibitor as an anti-HIV-1 agent and needs to be assessed in a non-human primate (NHP) model.

Immunotherapeutic Blockade of CD47 Inhibitory Signaling Enhances Innate and Adaptive Immune Responses to Viral Infection.

Paradoxically, early host responses to infection include the upregulation of the antiphagocytic molecule, CD47. This suggests that CD47 blockade could enhance antigen presentation and subsequent immune responses. Indeed, mice treated with anti-CD47 monoclonal antibody following lymphocytic choriomeningitis virus infections show increased activation of both macrophages and dendritic cells (DCs), enhancement of the kinetics and potency of CD8+ T cell responses, and significantly improved virus control. Treatment efficacy is critically dependent on both APCs and CD8+ T cells. In preliminary results from one of two cohorts of humanized mice infected with HIV-1 for 6 weeks, CD47 blockade reduces plasma p24 levels and restores CD4+ T cell counts. The results indicate that CD47 blockade not only enhances the function of innate immune cells but also links to adaptive immune responses through improved APC function. As such, immunotherapy by CD47 blockade may have broad applicability to treat a wide range of infectious diseases.

Cytotrophoblast induction of arterial apoptosis and lymphangiogenesis in an in vivo model of human placentation.

We studied the vascular effects of invasive human cytotrophoblasts in vivo by transplanting placental villi to the fifth mammary fat pads or beneath the kidney capsules of Scid mice. Over 3 weeks, robust cytotrophoblast invasion was observed in both locations. The architecture of the mammary fat pad allowed for detailed analysis of the cells' interactions with resident murine blood vessels, which revealed specific induction of apoptosis in the endothelial cells and smooth muscle walls of the arterioles. This finding, and confirmation of the results in an in vitro coculture model, suggests that a parallel process is important for enabling cytotrophoblast endovascular invasion during human pregnancy. Cytotrophoblast invasion of the kidney parenchyma was accompanied by a robust lymphangiogenic response, while in vitro, the cells stimulated lymphatic endothelial cell migration via the actions of VEGF family members, FGF, and TNF-alpha. Immunolocalization analyses revealed that human pregnancy is associated with lymphangiogenesis in the decidua since lymphatic vessels were not a prominent feature of the nonpregnant endometrium. Thus, the placenta triggers the development of a decidual lymphatic circulation, which we theorize plays an important role in maintaining fluid balance during pregnancy, with possible implications for maternal-fetal immune cell trafficking.

Cellular HIV Reservoirs and Viral Rebound from the Lymphoid Compartments of 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine (EFdA)-Suppressed Humanized Mice.

Although antiretroviral therapy (ART) greatly suppresses HIV replication, lymphoid tissues remain a sanctuary site where the virus may replicate. Tracking the earliest steps of HIV spread from these cellular reservoirs after drug cessation is pivotal for elucidating how infection can be prevented. In this study, we developed an in vivo model of HIV persistence in which viral replication in the lymphoid compartments of humanized mice was inhibited by the HIV reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) to very low levels, which recapitulated ART-suppression in HIV-infected individuals. Using a combination of RNAscope in situ hybridization (ISH) and immunohistochemistry (IHC), we quantitatively investigated the distribution of HIV in the lymphoid tissues of humanized mice during active infection, EFdA suppression, and after drug cessation. The lymphoid compartments of EFdA-suppressed humanized mice harbored very rare transcription/translation-competent HIV reservoirs that enable viral rebound. Our data provided the visualization and direct measurement of the early steps of HIV reservoir expansion within anatomically intact lymphoid tissues soon after EFdA cessation and suggest a strategy to enhance therapeutic approaches aimed at eliminating the HIV reservoir.

Eradication of HIV from Tissue Reservoirs: Challenges for the Cure.

The persistence of HIV infection, even after lengthy and successful combined antiretroviral therapy (cART), has precluded an effective cure. The anatomical locations and biological mechanisms through which the viral population is maintained remain unknown. Much research has focused nearly exclusively on circulating resting T cells as the predominant source of persistent HIV, a strategy with limited success in developing an effective cure strategy. In this study, we review research supporting the importance of anatomical tissues and other immune cells for HIV maintenance and expansion, including the central nervous system, lymph nodes, and macrophages. We present accumulated research that clearly demonstrates the limitations of using blood-derived cells as a proxy for tissue reservoirs and sanctuaries throughout the body. We cite recent studies that have successfully used deep-sequencing strategies to uncover the complexity of HIV infection and the ability of the virus to evolve despite undetectable plasma viral loads. Finally, we suggest new strategies and highlight the importance of tissue banks for future research.