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lovo-cel (bb1111; LentiGlobin for sickle cell disease [SCD]) gene therapy (GT) comprises autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified β-globin gene (β A-T87Q ) to produce anti-sickling hemoglobin (HbA T87Q ). The efficacy and safety of lovo-cel for SCD are being evaluated in the ongoing phase 1/2 HGB-206 study (ClinicalTrials.gov: NCT02140554). The treatment process evolved over time, using learnings from outcomes in the initial patients to optimize lovo-cel's benefit-risk profile. Following modest expression of HbA T87Q in the initial patients (Group A, n = 7), alterations were made to the treatment process for patients subsequently enrolled in Group B (n = 2, patients B1 and B2), including improvements to cell collection and lovo-cel manufacturing. After 6 months, median Group A peripheral blood vector copy number (≥0.08 c/dg) and HbA T87Q levels (≥0.46 g/dL) were inadequate for substantial clinical effect but stable and sustained over 5.5 years; both markedly improved in Group B (patient B1: ≥0.53 c/dg and ≥2.69 g/dL; patient B2: ≥2.14 c/dg and ≥6.40 g/dL, respectively) and generated improved biologic and clinical efficacy in Group B, including higher total hemoglobin and decreased hemolysis. The safety of the lovo-cel for SCD treatment regimen largely reflected the known side effects of HSPC collection, busulfan conditioning regimen, and underlying SCD; acute myeloid leukemia was observed in two patients in Group A and deemed unlikely related to insertional oncogenesis. Changes made during development of the lovo-cel treatment process were associated with improved outcomes and provide lessons for future SCD GT studies., (© 2022 The Authors. American Journal of Hematology published by Wiley Periodicals LLC.)

Background: Sickle cell disease is characterized by the painful recurrence of vaso-occlusive events. Gene therapy with the use of LentiGlobin for sickle cell disease (bb1111; lovotibeglogene autotemcel) consists of autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified β-globin gene, which produces an antisickling hemoglobin, HbA T87Q ., Methods: In this ongoing phase 1-2 study, we optimized the treatment process in the initial 7 patients in Group A and 2 patients in Group B with sickle cell disease. Group C was established for the pivotal evaluation of LentiGlobin for sickle cell disease, and we adopted a more stringent inclusion criterion that required a minimum of four severe vaso-occlusive events in the 24 months before enrollment. In this unprespecified interim analysis, we evaluated the safety and efficacy of LentiGlobin in 35 patients enrolled in Group C. Included in this analysis was the number of severe vaso-occlusive events after LentiGlobin infusion among patients with at least four vaso-occlusive events in the 24 months before enrollment and with at least 6 months of follow-up., Results: As of February 2021, cell collection had been initiated in 43 patients in Group C; 35 received a LentiGlobin infusion, with a median follow-up of 17.3 months (range, 3.7 to 37.6). Engraftment occurred in all 35 patients. The median total hemoglobin level increased from 8.5 g per deciliter at baseline to 11 g or more per deciliter from 6 months through 36 months after infusion. HbA T87Q contributed at least 40% of total hemoglobin and was distributed across a mean (±SD) of 85±8% of red cells. Hemolysis markers were reduced. Among the 25 patients who could be evaluated, all had resolution of severe vaso-occlusive events, as compared with a median of 3.5 events per year (range, 2.0 to 13.5) in the 24 months before enrollment. Three patients had a nonserious adverse event related or possibly related to LentiGlobin that resolved within 1 week after onset. No cases of hematologic cancer were observed during up to 37.6 months of follow-up., Conclusions: One-time treatment with LentiGlobin resulted in sustained production of HbA T87Q in most red cells, leading to reduced hemolysis and complete resolution of severe vaso-occlusive events. (Funded by Bluebird Bio; HGB-206 ClinicalTrials.gov number, NCT02140554.)., (Copyright © 2021 Massachusetts Medical Society.)

Background: Betibeglogene autotemcel (beti-cel) gene therapy for transfusion-dependent β-thalassemia contains autologous CD34+ hematopoietic stem cells and progenitor cells transduced with the BB305 lentiviral vector encoding the β-globin (β A-T87Q ) gene., Methods: In this open-label, phase 3 study, we evaluated the efficacy and safety of beti-cel in adult and pediatric patients with transfusion-dependent β-thalassemia and a non-β 0 /β 0 genotype. Patients underwent myeloablation with busulfan (with doses adjusted on the basis of pharmacokinetic analysis) and received beti-cel intravenously. The primary end point was transfusion independence (i.e., a weighted average hemoglobin level of ≥9 g per deciliter without red-cell transfusions for ≥12 months)., Results: A total of 23 patients were enrolled and received treatment, with a median follow-up of 29.5 months (range, 13.0 to 48.2). Transfusion independence occurred in 20 of 22 patients who could be evaluated (91%), including 6 of 7 patients (86%) who were younger than 12 years of age. The average hemoglobin level during transfusion independence was 11.7 g per deciliter (range, 9.5 to 12.8). Twelve months after beti-cel infusion, the median level of gene therapy-derived adult hemoglobin (HbA) with a T87Q amino acid substitution (HbA T87Q ) was 8.7 g per deciliter (range, 5.2 to 10.6) in patients who had transfusion independence. The safety profile of beti-cel was consistent with that of busulfan-based myeloablation. Four patients had at least one adverse event that was considered by the investigators to be related or possibly related to beti-cel; all events were nonserious except for thrombocytopenia (in 1 patient). No cases of cancer were observed., Conclusions: Treatment with beti-cel resulted in a sustained HbA T87Q level and a total hemoglobin level that was high enough to enable transfusion independence in most patients with a non-β 0 /β 0 genotype, including those younger than 12 years of age. (Funded by Bluebird Bio; HGB-207 ClinicalTrials.gov number, NCT02906202.)., (Copyright © 2021 Massachusetts Medical Society.)

Sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) are the most prevalent monogenic disorders worldwide. Trial HGB-205 ( NCT02151526 ) aimed at evaluating gene therapy by autologous CD34 + cells transduced ex vivo with lentiviral vector BB305 that encodes the anti-sickling β A-T87Q -globin expressed in the erythroid lineage. HGB-205 is a phase 1/2, open-label, single-arm, non-randomized interventional study of 2-year duration at a single center, followed by observation in long-term follow-up studies LTF-303 ( NCT02633943 ) and LTF-307 ( NCT04628585 ) for TDT and SCD, respectively. Inclusion and exclusion criteria were similar to those for allogeneic transplantation but restricted to patients lacking geno-identical, histocompatible donors. Four patients with TDT and three patients with SCD, ages 13-21 years, were treated after busulfan myeloablation 4.6-7.9 years ago, with a median follow-up of 4.5 years. Key primary endpoints included mortality, engraftment, replication-competent lentivirus and clonal dominance. No adverse events related to the drug product were observed. Clinical remission and remediation of biological hallmarks of the disease have been sustained in two of the three patients with SCD, and frequency of transfusions was reduced in the third. The patients with TDT are all transfusion free with improvement of dyserythropoiesis and iron overload., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Betibeglogene autotemcel (beti-cel) gene therapy (GT) for patients with transfusion-dependent β-thalassemia uses autologous CD34+ cells transduced with BB305 lentiviral vector (LVV), which encodes a modified β-globin gene. BB305 LVV also contains select HIV sequences for viral packaging, reverse transcription, and integration. This case report describes a patient successfully treated with beti-cel in a phase 1/2 study (HGB-204; #NCT01745120) and subsequently diagnosed with wild-type (WT) HIV infection. From 3.5 to 21 months postinfusion, the patient stopped chronic red blood cell transfusions; total hemoglobin (Hb) and GT-derived HbAT87Q levels were 6.6 to 9.5 and 2.8 to 3.8 g/dL, respectively. At 21 months postinfusion, the patient resumed transfusions for anemia that coincided with an HIV-1 infection diagnosis. Quantitative polymerase chain reaction assays detected no replication-competent lentivirus. Next-generation sequencing confirmed WT HIV sequences. Six months after starting antiretroviral therapy, total Hb and HbAT87Q levels recovered to 8.6 and 3.6 g/dL, respectively, and 3.5 years postinfusion, 13.4 months had elapsed since the patient's last transfusion. To our knowledge, this is the first report of WT HIV infection in an LVV-based GT recipient and demonstrates persistent long-term hematopoiesis after treatment with beti-cel and the ability to differentiate between WT HIV and BB305-derived sequences., (© 2021 by The American Society of Hematology.)

Novel therapies for sickle cell disease (SCD) based on genetically engineered autologous hematopoietic stem and progenitor cells (HSPCs) are critically dependent on a safe and effective strategy for cell procurement. We sought to assess the safety and efficacy of plerixafor when used in transfused patients with SCD for HSC mobilization. Six adult patients with SCD were recruited to receive a single dose of plerixafor, tested at lower than standard (180 µg/kg) and standard (240 µg/kg) doses, followed by CD34 + cell monitoring in peripheral blood and apheresis collection. The procedures were safe and well-tolerated. Mobilization was successful, with higher peripheral CD34 + cell counts in the standard vs the low-dose group. Among our 6 donors, we improved apheresis cell collection results by using a deep collection interface and starting apheresis within 4 hours after plerixafor administration. In the subjects who received a single standard dose of plerixafor and followed the optimized collection protocol, yields of up to 24.5 × 10 6 CD34 + cells/kg were achieved. Interestingly, the collected CD34 + cells were enriched in immunophenotypically defined long-term HSCs and early progenitors. Thus, we demonstrate that plerixafor can be employed safely in patients with SCD to obtain sufficient HSCs for potential use in gene therapy., (© 2018 by The American Society of Hematology.)

Background: Donor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent β-thalassemia. After previously establishing that lentiviral transfer of a marked β-globin (β A-T87Q ) gene could substitute for long-term red-cell transfusions in a patient with β-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent β-thalassemia., Methods: In two phase 1-2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion-dependent β-thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbA T87Q ). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication-competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbA T87Q , transfusion requirements, and average vector copy number., Results: At a median of 26 months (range, 15 to 42) after infusion of the gene-modified cells, all but 1 of the 13 patients who had a non-β 0 /β 0 genotype had stopped receiving red-cell transfusions; the levels of HbA T87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a β 0 /β 0 genotype or two copies of the IVS1-110 mutation, the median annualized transfusion volume was decreased by 73%, and red-cell transfusions were discontinued in 3 patients. Treatment-related adverse events were typical of those associated with autologous stem-cell transplantation. No clonal dominance related to vector integration was observed., Conclusions: Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe β-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526 .).

Background: Pimodivir (formerly JNJ-63623872) is a novel, non-nucleoside polymerase complex inhibitor with in vitro activity against influenza A virus, including pandemic 2009 H1N1, H7N9, H5N1 strains as well as neuraminidase- and amantadine-resistant strains., Methods: Randomized, double-blind, placebo-controlled, Phase IIa study. Healthy volunteers (n=104) were inoculated with an influenza A/Wisconsin/67/2005 (H3N2) challenge virus. 72 received pimodivir and 32 placebo. Pimodivir was dosed for 5 days once daily from 24 h after viral inoculation at four dose levels: 100 mg, 400 mg, loading dose 900/600 mg and loading dose 1,200/600 mg., Results: Pimodivir significantly reduced viral shedding (area under the concentration versus time curve [AUC] measured by 50% tissue culture infective dose [TCID 50 ] or qRT-PCR) versus placebo as measured by cell culture assay in the pooled analysis (Jonckheere-Terpstra dose-response trend test [P=0.036]). Reductions were observed in viral shedding (AUC, duration and peak measured by grade), influenza-like symptoms (AUC, duration and peak measured by grade) and clinical symptoms (duration and peak measured by grade) for all pimodivir groups versus placebo, significantly so for the 1,200/600 mg group. In the 1,200/600 mg group viral shedding (AUC) by qRT-PCR was 0.45 versus 18.4 log 10 copies/ml*day for pooled placebo (P=0.014). Pimodivir was generally safe and well-tolerated with no serious adverse events or adverse events leading to discontinuation., Conclusions: Pimodivir has potential to not only reduce viral load but to have a clinical impact on patients as a novel treatment for influenza A virus infection. Further trials are therefore warranted to assess pimodivir.

For studies on vaccines and therapies for HIV disease, SIV-HIV chimeric viruses harboring the HIV-1 env gene (SHIVenv) remain the best virus in non-human primate models. However, there are still very few SHIVenv viruses that can cause AIDS in non-CD8-depleted animals. In the present study, a recently created CCR5-using SHIVenv_B3 virus with env gene derived from acute/early HIV-1 infections (AHI) successfully established pathogenic infection in macaques. Through a series of investigations on the evolution, mutational profile, and phenotype of the virus and the resultant humoral immune response in infected rhesus macaques, we found that the E32K mutation in the Env C1 domain was associated with macaque pathogenesis, and that the electrostatic interactions in Env may favor E32K at the gp120 N terminus and "lock" the binding to heptad repeat 1 of gp41 in the trimer and produce a SHIVenv with increased fitness and pathogenesis during macaque infections., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: New simian-human immunodeficiency chimeric viruses with an HIV-1 env (SHIVenv) are critical for studies on HIV pathogenesis, vaccine development, and microbicide testing. Macaques are typically exposed to single CCR5-using SHIVenv which in most instances does not reflect the conditions during acute/early HIV infection (AHI) in humans. Instead of individual and serial testing new SHIV constructs, a pool of SHIVenv_B derived from 16 acute HIV-1 infections were constructed using a novel yeast-based SHIV cloning approach and then used to infect macaques., Results: Even though none of the 16 SHIVenvs contained the recently reported mutations in env genes that could significantly enhance their binding affinity to RhCD4, one SHIVenv (i.e. SHIVenv_B3-PRB926) established infection in macaques exposed to this pool. AHI SHIVenv_B viruses as well as their HIVenv_B counterparts were analyzed for viral protein content, function, and fitness to identify possible difference between SHIVenv_B3-PRB926 and the other 15 SHIVenvs in the pool. All of the constructs produced SHIV or HIV chimeric with wild type levels of capsid (p27 and p24) content, reverse transcriptase (RT) activity, and expressed envelope glycoproteins that could bind to cell receptors CD4/CCR5 and mediate virus entry. HIV-1env_B chimeric viruses were propagated in susceptible cell lines but the 16 SHIVenv_B variants showed only limited replication in macaque peripheral blood mononuclear cells (PBMCs) and 174×CEM.CCR5 cell line. AHI chimeric viruses including HIVenv_B3 showed only minor variations in cell entry efficiency and kinetics as well as replicative fitness in human PBMCs. Reduced number of N-link glycosylation sites and slightly greater CCR5 affinity/avidity was the only distinguishing feature of env_B3 versus other AHI env's in the pool, a feature also observed in the HIV establishing new infections in humans., Conclusion: Despite the inability to propagate in primary cells and cell lines, a pool of 16 SHIVenv viruses could establish infection but only one virus, SHIVenv_B3 was isolated in the macaque and then shown to repeatedly infected macaques. This SHIVenv_B3 virus did not show any distinct phenotypic property from the other 15 SHIVenv viruses but did have the fewest N-linked glycosylation sites.

Simian-human immunodeficiency viruses (SHIVs) that mirror natural transmitted/founder (T/F) viruses in man are needed for evaluation of HIV-1 vaccine candidates in nonhuman primates. Currently available SHIVs contain HIV-1 env genes from chronically-infected individuals and do not reflect the characteristics of biologically relevant HIV-1 strains that mediate human transmission. We chose to develop clade C SHIVs, as clade C is the major infecting subtype of HIV-1 in the world. We constructed 10 clade C SHIVs expressing Env proteins from T/F viruses. Three of these ten clade C SHIVs (SHIV KB9 C3, SHIV KB9 C4 and SHIV KB9 C5) replicated in naïve rhesus monkeys. These three SHIVs are mucosally transmissible and are neutralized by sCD4 and several HIV-1 broadly neutralizing antibodies. However, like natural T/F viruses, they exhibit low Env reactivity and a Tier 2 neutralization sensitivity. Of note, none of the clade C T/F SHIVs elicited detectable autologous neutralizing antibodies in the infected monkeys, even though antibodies that neutralized a heterologous Tier 1 HIV-1 were generated. Challenge with these three new clade C SHIVs will provide biologically relevant tests for vaccine protection in rhesus macaques., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: To investigate in this phase 2a study (ZENITH) the safety, tolerability, and antiviral activity of VX-222, a selective, non-nucleoside inhibitor of hepatitis C virus (HCV) NS5B polymerase, combined with various telaprevir-based regimens for treatment of genotype 1 HCV., Methods: In total, 152 treatment-naive patients received VX-222+telaprevir ('DUAL' regimen; n=47), with ribavirin ('TRIPLE' regimen; n=46), or with peginterferon+ribavirin ('QUAD' regimen; n=59) for 12 weeks. Patients with detectable HCV RNA at weeks 2 and/or 8 received peginterferon+ribavirin for 24 (DUAL and TRIPLE) or 12 (QUAD) additional weeks., Results: VX-222 (100 or 400 mg twice daily) was well tolerated, with an increased rate of gastrointestinal adverse events observed with the higher dose. Across VX-222 400-mg twice-daily regimens, the QUAD was associated with the highest frequency of grade 3/4 adverse events. The DUAL was discontinued because of high viral breakthrough before week 12. Sustained virologic response (SVR) 24 weeks after end of treatment (SVR24), including patients treated with 12 or 24 additional weeks of peginterferon+ribavirin, was 67% for TRIPLE (VX-222 400 mg twice daily) and 79 and 90% for QUAD (VX-222 100 and 400 mg twice daily, respectively)., Conclusion: These results provide valuable information regarding the safety, tolerability, and efficacy of telaprevir combined with a non-nucleoside polymerase inhibitor, as dual therapy or with ribavirin without or with peginterferon. Telaprevir and VX-222, alone or with ribavirin without or with peginterferon, were generally well tolerated, with improved tolerability without peginterferon. SVR24 rates achieved with TRIPLE and QUAD regimens containing telaprevir and VX-222 were comparable to those observed with telaprevir-based therapy.

Effective strategies are needed to block mucosal transmission of human immunodeficiency virus type 1 (HIV-1). Here, we address a crucial question in HIV-1 pathogenesis: whether infected donor mononuclear cells or cell-free virus plays the more important role in initiating mucosal infection by HIV-1. This distinction is critical, as effective strategies for blocking cell-free and cell-associated virus transmission may be different. We describe a novel ex vivo model system that utilizes sealed human colonic mucosa explants and demonstrate in both the ex vivo model and in vivo using the rectal challenge model in rhesus monkeys that HIV-1-infected lymphocytes can transmit infection across the mucosa more efficiently than cell-free virus. These findings may have significant implications for our understanding of the pathogenesis of mucosal transmission of HIV-1 and for the development of strategies to prevent HIV-1 transmission.

2-cys peroxiredoxins (Prx), a group of anti-oxidative enzyme proteins, act directly on virally-infected cells to inhibit HIV-1 replication, and indirectly through destruction of HIV infected cells by stimulation of Natural Killer (NK) cell-mediated immune responses. We assayed for antibody-dependent NK cell mediated viral inhibition (ADCVI) using plasma from SIV-infected rhesus macaques. We found that Prx-1 strongly increased ADCVI in a dose-dependent manner, suggesting augmentation of NK cell killing. We also investigated the effect of Prx-1 on NK cell-independent HIV-1 and HIV-2 inhibition. We found that primary HIV isolates were potently inhibited at nM concentrations, regardless of viral clade, receptor usage or anti-retroviral drug resistance. During NK cell independent inhibition, we found that Prx-1 reversed the HIV-1 induced gene expression of Heat shock protein 90 kDa alpha (cystolic), class A member 2, (HSP90), a protein of the stress pathway. Prx-1 highly activated Cyclin-dependent kinase inhibitor 2B (CDKN2B), a gene of the TGF-β pathway, and Baculoviral IAP repeat-containing 2 (Birc-2), an anti-apoptotic gene of the NF-κB pathway. We identified gene-expression networks highly dependent on the NFκB and ERK1/2 pathways. Our findings demonstrate that Prx-1 inhibits HIV replication through NK cell-dependent and NK cell-independent mechanisms.

Background: Although there have been dramatic strides made recently in the treatment of chronic hepatitis C virus infection, interferon-α based therapy remains challenging for certain populations, including those with unfavorable IL28B genotypes, psychiatric co-morbidity, HIV co-infection, and decompensated liver disease. We have recently shown that ATIII, a serine protease inhibitor (serpin), has broad antiviral properties., Results: We now show that ATIII is capable of inhibiting HCV in the OR6 replicon model at micromolar concentrations. At a mechanistic level using gene-expression arrays, we found that ATIII treatment down-regulated multiple host cell signal transduction factors involved in the pathogenesis of cirrhosis and hepatocellular carcinoma, including Jun, Myc and BMP2. Using a protein interactive network analysis we found that changes in gene-expression caused by ATIII were dependent on three nodes previously implicated in HCV disease progression or HCV replication: NFκB, P38 MAPK, and ERK1/2., Conclusions: Our findings suggest that ATIII stimulates a novel innate antiviral host cell defense different from current treatment options.

The RV144 trial demonstrated that an experimental AIDS vaccine can prevent human immunodeficiency virus type 1 (HIV-1) infection in humans. Because of its limited efficacy, further understanding of the mechanisms of preventive AIDS vaccines remains a priority, and nonhuman primate (NHP) models of lentiviral infection provide an opportunity to define immunogens, vectors, and correlates of immunity. In this study, we show that prime-boost vaccination with a mismatched SIV envelope (Env) gene, derived from simian immunodeficiency virus SIVmac239, prevents infection by SIVsmE660 intrarectally. Analysis of different gene-based prime-boost immunization regimens revealed that recombinant adenovirus type 5 (rAd5) prime followed by replication-defective lymphocytic choriomeningitis virus (rLCMV) boost elicited robust CD4 and CD8 T-cell and humoral immune responses. This vaccine protected against infection after repetitive mucosal challenge with efficacies of 82% per exposure and 62% cumulatively. No effect was seen on viremia in infected vaccinated monkeys compared to controls. Protection correlated with the presence of neutralizing antibodies to the challenge viruses tested in peripheral blood mononuclear cells. These data indicate that a vaccine expressing a mismatched Env gene alone can prevent SIV infection in NHPs and identifies an immune correlate that may guide immunogen selection and immune monitoring for clinical efficacy trials.

Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Several serpins have been reported to modulate HIV pathogenesis, or exhibit potent anti-HIV activity in vitro, but the efficacy of serpins as therapeutic agents for HIV in vivo has not yet been demonstrated. In the present study, we show that heparin-activated antithrombin III (hep-ATIII), a member of the serpin family, significantly inhibits lentiviral replication in a non-human primate model. We further demonstrate greater than one log(10) reduction in plasma viremia in the nonhuman primate system by loading of hep-ATIII into anti-HLA-DR immunoliposomes, which target tissue reservoirs of viral replication. We also demonstrate the utility of hep-ATIIII as a potential salvage agent for HIV strains resistant to standard anti-retroviral treatment. Finally, we applied gene-expression arrays to analyze hep-ATIII-induced host cell interactomes and found that downstream of hep-ATIII, two independent gene networks were modulated by host factors prostaglandin synthetase-2, ERK1/2 and NFκB. Ultimately, understanding how serpins, such as hep-ATIII, regulate host responses during HIV infection may reveal new avenues for therapeutic intervention.

Here we have identified HIV-1 B clade Envelope (Env) amino acid signatures from early in infection that may be favored at transmission, as well as patterns of recurrent mutation in chronic infection that may reflect common pathways of immune evasion. To accomplish this, we compared thousands of sequences derived by single genome amplification from several hundred individuals that were sampled either early in infection or were chronically infected. Samples were divided at the outset into hypothesis-forming and validation sets, and we used phylogenetically corrected statistical strategies to identify signatures, systematically scanning all of Env. Signatures included single amino acids, glycosylation motifs, and multi-site patterns based on functional or structural groupings of amino acids. We identified signatures near the CCR5 co-receptor-binding region, near the CD4 binding site, and in the signal peptide and cytoplasmic domain, which may influence Env expression and processing. Two signatures patterns associated with transmission were particularly interesting. The first was the most statistically robust signature, located in position 12 in the signal peptide. The second was the loss of an N-linked glycosylation site at positions 413-415; the presence of this site has been recently found to be associated with escape from potent and broad neutralizing antibodies, consistent with enabling a common pathway for immune escape during chronic infection. Its recurrent loss in early infection suggests it may impact fitness at the time of transmission or during early viral expansion. The signature patterns we identified implicate Env expression levels in selection at viral transmission or in early expansion, and suggest that immune evasion patterns that recur in many individuals during chronic infection when antibodies are present can be selected against when the infection is being established prior to the adaptive immune response.

With the recent demonstration in the RV144 Thai trial that a vaccine regimen that does not elicit neutralizing antibodies or cytotoxic T lymphocytes may confer protection against human immunodeficiency virus type 1 (HIV-1) infection, attention has turned to nonneutralizing antibodies as a possible mechanism of vaccine protection. In the current study, we evaluated the kinetics of the antibody-dependent cell-mediated cytotoxicity (ADCC) response during acute and chronic SIVmac251 infection of rhesus monkeys. We first adapted a flow cytometry-based ADCC assay, evaluating the use of different target cells as well as different strategies for quantitation of activated natural killer (NK) cells. We found that the use of SIVmac251 Env gp130-coated target cells facilitates analyses of ADCC activity with a higher degree of sensitivity than the use of simian immunodeficiency virus (SIV)-infected target cells; however, the kinetics of the measured responses were the same using these different target cells. By comparing NK cell expression of CD107a with NK cell expression of other cytokines or chemokine molecules, we found that measuring CD107a expression is sufficient for evaluating the anti-SIV function of NK cells. We also showed that ADCC responses can be detected as early as 3 weeks after SIVmac251 infection and that the magnitude of this antibody response is inversely associated with plasma viral RNA levels in animals with moderate to high levels of viral replication. However, we also demonstrated an association between NK cell-mediated ADCC responses and the amount of SIVmac251 gp140 binding antibody that developed after viral infection. This final observation raises the possibility that the antibodies that mediate ADCC are a subset of the antibodies detected in a binding assay and arise within weeks of infection.

Antibody-dependent cell-mediated viral inhibition (ADCVI) is an attractive target for vaccination because it takes advantage of both the anamnestic properties of an adaptive immune response and the rapid early response characteristics of an innate immune response. Effective utilization of ADCVI in vaccine strategies will depend on an understanding of the natural history of ADCVI during acute and chronic human immunodeficiency virus type 1 (HIV-1) infection. We used the simian immunodeficiency virus (SIV)-infected rhesus monkey as a model to study the kinetics of ADCVI in early infection, the durability of ADCVI through the course of infection, and the effectiveness of ADCVI against viruses with envelope mutations that are known to confer escape from antibody neutralization. We demonstrate the development of ADCVI, capable of inhibiting viral replication 100-fold, within 3 weeks of infection, preceding the development of a comparable-titer neutralizing antibody response by weeks to months. The emergence of ADCVI was temporally associated with the emergence of gp140-binding antibodies, and in most animals, ADCVI persisted through the course of infection. Highly evolved viral envelopes from viruses isolated at late time points following infection that were resistant to plasma neutralization remained susceptible to ADCVI, suggesting that the epitope determinants of neutralization escape are not shared by antibodies that mediate ADCVI. These findings suggest that despite the ability of SIV to mutate and adapt to multiple immunologic pressures during the course of infection, SIV envelope may not escape the binding of autologous antibodies that mediate ADCVI.

The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was about a one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01-negative monkeys challenged with SIVsmE660, no CD8(+) T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4(+) T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanisms of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies.

The serine protease inhibitors (serpins) are anti-inflammatory proteins that have various functions. By screening a diverse panel of viruses, we demonstrate that the serpin antithrombin III (ATIII) has a broad-spectrum anti-viral activity for HIV-1, HCV and HSV. To investigate the mechanism of action in more detail we investigated the HIV-1 inhibition. Using gene-expression arrays we found that multiple host cell signal transduction pathways were activated by ATIII in HIV-1 infected cells but not in uninfected controls. Moreover, the signal pathways initiated by ATIII treatment, were more than 200-fold increased by the use of heparin-activated ATIII. The most up-regulated transcript in HIV-1 infected cells was prostaglandin synthetase-2 (PTGS2). Furthermore, we found that over-expression of PTGS2 reduced levels of HIV-1 replication in human PBMC. These findings suggest a central role for serpins in the host innate anti-viral response. Host factors such as PTGS2 elicited by ATIII treatment could be exploited in the development of novel anti-viral interventions.

Mucosal transmission of the human immunodeficiency virus (HIV) results in a bottleneck in viral genetic diversity. Gnanakaran and colleagues used a computational strategy to identify signature amino acids at particular positions in Envelope that were associated either with transmitted sequences sampled very early in infection, or sequences sampled during chronic infection. Among the strongest signatures observed was an enrichment for the stable presence of histidine at position 12 at transmission and in early infection, and a recurrent loss of histidine at position 12 in chronic infection. This amino acid lies within the leader peptide of Envelope, a region of the protein that has been shown to influence envelope glycoprotein expression and virion infectivity. We show a strong association between a positively charged amino acid like histidine at position 12 in transmitted/founder viruses with more efficient trafficking of the nascent envelope polypeptide to the endoplasmic reticulum and higher steady-state glycoprotein expression compared to viruses that have a non-basic position 12 residue, a substitution that was enriched among viruses sampled from chronically infected individuals. When expressed in the context of other viral proteins, transmitted envelopes with a basic amino acid position 12 were incorporated at higher density into the virus and exhibited higher infectious titers than did non-signature envelopes. These results support the potential utility of using a computational approach to examine large viral sequence data sets for functional signatures and indicate the importance of Envelope expression levels for efficient HIV transmission.

While the simian immunodeficiency virus (SIV)-infected rhesus monkey is an important animal model for human immunodeficiency virus type 1 (HIV-1) infection of humans, much remains to be learned about the evolution of the humoral immune response in this model. In HIV-1 infection, autologous neutralizing antibodies emerge 2 to 3 months after infection. However, the ontogeny of the SIV-specific neutralizing antibody response in mucosally infected animals has not been defined. We characterized the kinetics of the autologous neutralizing antibody response to the transmitted/founder SIVmac251 using a pseudovirion-based TZM-bl cell assay and monitored env sequence evolution using single-genome amplification in four rhesus animals that were infected via intrarectal inoculations. We show that the SIVmac251 founder viruses induced neutralizing antibodies at 5 to 8 months after infection. Despite their slow emergence and low titers, these neutralizing antibodies selected for escape mutants that harbored substitutions and deletions in variable region 1 (V1), V2, and V4 of Env. The neutralizing antibody response was initially focused on V4 at 5 to 8 months after infection and then targeted V1/V2 and V4 by 16 months. These findings reveal a striking delay in the development of neutralizing antibodies in SIVmac-infected animals, thus raising questions concerning the suitability of SIVmac251 as a challenge strain to screen AIDS vaccines that elicit neutralizing antibodies as a means to prevent virus acquisition. They also illustrate the capacity of the SIVmac quasispecies to modify antigenic determinants in response to very modest titers of neutralizing antibodies.

Although there is increasing evidence that individuals already infected with human immunodeficiency virus type 1 (HIV-1) can be infected with a heterologous strain of the virus, the extent of protection against superinfection conferred by the first infection and the biologic consequences of superinfection are not well understood. We explored these questions in the simian immunodeficiency virus (SIV)/rhesus monkey model of HIV-1/AIDS. We infected cohorts of rhesus monkeys with either SIVmac251 or SIVsmE660 and then exposed animals to the reciprocal virus through intrarectal inoculations. Employing a quantitative real-time PCR assay, we determined the replication kinetics of the two strains of virus for 20 weeks. We found that primary infection with a replication-competent virus did not protect against acquisition of infection by a heterologous virus but did confer relative control of the superinfecting virus. In animals that became superinfected, there was a reduction in peak replication and rapid control of the second virus. The relative susceptibility to superinfection was not correlated with CD4(+) T-cell count, CD4(+) memory T-cell subsets, cytokine production by virus-specific CD8(+) or CD4(+) cells, or neutralizing antibodies at the time of exposure to the second virus. Although there were transient increases in viral loads of the primary virus and a modest decline in CD4(+) T-cell counts after superinfection, there was no evidence of disease acceleration. These findings indicate that an immunodeficiency virus infection confers partial protection against a second immunodeficiency virus infection, but this protection may be mediated by mechanisms other than classical adaptive immune responses.

Cyclosporine is an immunosuppressive drug that is widely used to prevent organ transplant rejection. Known intracellular ligands for cyclosporine include the cyclophilins, a large family of phylogenetically conserved proteins that potentially regulate protein folding in cells. Immunosuppression by cyclosporine is thought to result from the formation of a drug-cyclophilin complex that binds to and inhibits calcineurin, a serine/threonine phosphatase that is activated by TCR engagement. Amino acids within the cyclophilins that are critical for binding to cyclosporine have been identified. Most of these residues are highly conserved within the 15 mammalian cyclophilins, suggesting that many are potential targets for the drug. We examined the effects of cyclosporine on immune cells and mice lacking Ppia, the gene encoding the prototypical cyclophilin protein cyclophilin A. TCR-induced proliferation and signal transduction by Ppia(-/-) CD4(+) T cells were resistant to cyclosporine, an effect that was attributable to diminished calcineurin inhibition. Immunosuppressive doses of cyclosporine failed to block the responses of Ppia(-/-) mice to allogeneic challenge. Rag2(-/-) mice reconstituted with Ppia(-/-) splenocytes were also cyclosporine resistant, indicating that this property is intrinsic to Ppia(-/-) immune cells. Thus, among multiple potential ligands, CypA is the primary mediator of immunosuppression by cyclosporine.

Cyclophilin A (CypA/Ppia) is a peptidyl-prolyl isomerase (PPIase) that binds the immunosuppressive drug cyclosporine. The resulting complex blocks T cell function by inhibiting the calcium-dependent phosphatase calcineurin. To identify the native function of CypA, long suspected of regulating signal transduction, we generated mice lacking the Ppia gene. These animals develop allergic disease, with elevated IgE and tissue infiltration by mast cells and eosinophils, that is driven by CD4+ T helper type II (Th2) cytokines. Ppia(-/-) Th2 cells were hypersensitive to TCR stimulation, a phenotype consistent with increased activity of Itk, a Tec family tyrosine kinase crucial for Th2 responses. CypA bound Itk via the PPIase active site. Mutation of a conformationally heterogeneous proline in the SH2 domain of Itk disrupted interaction with CypA and specifically increased Th2 cytokine production from wild-type CD4+ T cells. Thus, CypA inhibits CD4+ T cell signal transduction in the absence of cyclosporine via a regulatory proline residue in Itk.

Background: In a monochorionic twin gestation, hydrops in one fetus often results from placental transfusion, accompanied by intrauterine growth restriction (IUGR) of the non-hydropic fetus., Case: A 35-year-old G11P9 presented with hydrops and IUGR of one of a monochorionic-diamniotic gestation. Extensive testing failed to reveal etiology for hydrops. The patient was delivered at 30 weeks gestation after non-reassuring fetal monitoring. The hydropic baby could not be adequately ventilated and expired in the early post-partum period. A diagnosis of superior vena cava syndrome and pulmonary hypoplasia secondary to right-sided congenital diaphragmatic hernia (CDH) was made neonatally., Conclusion: Right-sided CDH can be difficult to diagnose by sonography at times because of the echogenic similarity of liver and lung, and may be suspected from signs of pericardial effusion and ascites., (Copyright 2004 S. Karger AG, Basel)

Effector CD4+ T cells rapidly activate high-level cytokine expression following TCR stimulation. Consistent with accelerated protein production in these cells, global mRNA profiles revealed that, after cytokines, the most impressive cluster of activated genes encode rRNA-maturation factors. Activation of these genes was ERK-MAPK dependent, accompanied by increased rRNA transcription and faster maturation kinetics, and much greater in effector CD4+ T cells than in naive cells. Ribosomal protein subunit (RPS) synthesis was also ERK-MAPK dependent and increased to match rRNA production, but without evident increase in RPS mRNA. Instead, stimulation promoted polysome loading of RPS mRNA via cis-acting, 5'-terminal oligopyrimidines. These results demonstrate how, in response to extracellular signals, effector CD4+ T cells coordinately increase multiple ribosomal components to accommodate burgeoning cytokine production.

Cyclophilins (CyPs) are a family of proteins found in organisms ranging from prokaryotes to humans. These molecules exhibit peptidyl-prolyl isomerase activity in vitro, suggesting that they influence the conformation of proteins in cells. CyPs also bind with varying affinities to the immunosuppressive drug cyclosporin A (CsA), a compound used clinically to prevent allograft rejection. The founding member of the family, cyclophilin A (CyPA), is an abundant, ubiquitously expressed protein of unknown function that binds with nanomolar affinity to CsA. Here, we describe the isolation and characterization of mouse Ppia (mPpia), the gene encoding CyPA. Ppia was isolated using a PCR screen that distinguishes the expressed gene from multiple pseudogenes present in the mouse genome. mPpia consists of 5 exons and 4 introns spanning roughly 4.5 kb and maps to chromosome 11 near the centromere. Sequence analysis of a 369-bp fragment from the proximal promoter region of mPpia revealed the presence of a TATA box and sites recognized by several transcriptional regulators, including Sp1, AP-2, GATA factors, c-Myb, and NF-IL-6. This region is sufficient to drive high-level reporter gene expression in transfected cells. Both copies of Ppia were disrupted in murine embryonic stem (ES) cells via gene targeting. Ppia(-/-) ES cells grow normally and differentiate into hematopoeitic precursor cells in vitro, indicating that CyPA is not essential for mammalian cell viability., (Copyright 2000 Academic Press.)