Hiromi Imamichi

Broad and potent neutralization of HIV-1 by a gp41-specific human antibody

Characterization of human monoclonal antibodies is providing considerable insight into mechanisms of broad HIV-1 neutralization. Here we report an HIV-1 gp41 membrane-proximal external region (MPER)-specific antibody, named 10E8, which neutralizes ∼98% of tested viruses. An analysis of sera from 78 healthy HIV-1-infected donors demonstrated that 27% contained MPER-specific antibodies and 8% contained 10E8-like specificities. In contrast to other neutralizing MPER antibodies, 10E8 did not bind phospholipids, was not autoreactive, and bound cell-surface envelope. The structure of 10E8 in complex with the complete MPER revealed a site of vulnerability comprising a narrow stretch of highly conserved gp41-hydrophobic residues and a critical arginine or lysine just before the transmembrane region. Analysis of resistant HIV-1 variants confirmed the importance of these residues for neutralization. The highly conserved MPER is a target of potent, non-self-reactive neutralizing antibodies, suggesting that HIV-1 vaccines should aim to induce antibodies to this region of HIV-1 envelope glycoprotein.

The Differential Ability of HLA B*5701+ Long-Term Nonprogressors and Progressors To Restrict Human Immunodeficiency Virus Replication Is Not Caused by Loss of Recognition of Autologous Viral gag Sequences

ABSTRACT Although the HLA B*5701 class I allele is highly overrepresented among human immunodeficiency virus (HIV)-infected long-term nonprogressors (LTNPs), it is also present at the expected frequency (11%) in patients with progressive HIV infection. Whether B57+ progressors lack restriction of viral replication because of escape from recognition of highly immunodominant B57-restricted gag epitopes by CD8+ T cells remains unknown. In this report, we investigate the association between restriction of virus replication and recognition of autologous virus sequences in 27 B*57+ patients (10 LTNPs and 17 progressors). Amplification and direct sequencing of single molecules of viral cDNA or proviral DNA revealed low frequencies of genetic variations in these regions of gag. Furthermore, CD8+ T-cell recognition of autologous viral variants was preserved in most cases. In two patients, responses to autologous viral variants were not demonstrable at one epitope. By using a novel technique to isolate primary CD4+ T cells expressing autologous viral gene products, it was found that 1 to 13% of CD8+ T cells were able to respond to these cells by gamma interferon production. In conclusion, escape-conferring mutations occur infrequently within immunodominant B57-restricted gag epitopes and are not the primary mechanism of virus evasion from immune control in B*5701+ HIV-infected patients. Qualitative features of the virus-specific CD8+ T-cell response not measured by current assays remain the most likely determinants of the differential abilities of HLA B*5701+ LTNPs and progressors to restrict virus replication.

In vivo expansion of CD4+CD45RO–CD25+ T cells expressing foxP3 in IL-2-treated HIV-infected patients

Administration of IL-2 to HIV-infected patients leads to expansion of a unique subset of CD4+CD45RO–CD25+ cells. In this study, the origin, clonality, and function of these cells were investigated. Analysis of TCR excision circles revealed that the CD4+CD45RO–CD25+ cells were the product of peripheral expansion but remained polyclonal as determined by TCR repertoire analysis. Phenotypically, these cells were distinct from naturally occurring Tregs; they exhibited intermediate features, between those of memory and naive cells, and had lower susceptibility to apoptosis than CD45RO–CD25– or memory T cells. Studies of intracellular cytokine production and proliferation revealed that cytokine-expanded naive CD25+ cells had low IL-2 production and required costimulation for proliferation. Despite elevated expression of forkhead transcription factor P3 (foxP3), they exerted only weak suppression compared with CD45RO+CD25+high cells (Tregs). In summary, in vivo IL-2 administration to HIV-infected patients leads to peripheral expansion of a population of long-lived CD4+CD45RO–CD25+ cells that express high levels of foxP3 but exert weak suppressive function. These CD4+CD25+ cytokine-expanded naive cells, distinct from antigen-triggered cells and Tregs, play a role in the maintenance of a state of low turnover and sustained expansion of the CD4+ T cell pool.

Cutting Edge: Ku70 Is a Novel Cytosolic DNA Sensor That Induces Type III Rather Than Type I IFN

Cytosolic foreign DNA is detected by pattern recognition receptors and mainly induces type I IFN production. We found that transfection of different types of DNA into various untreated cells induces type III IFN (IFN-λ1) rather than type I IFN, indicating the presence of uncharacterized DNA sensor(s). A pull-down assay using cytosolic proteins identified that Ku70 and Ku80 are the DNA-binding proteins. The knockdown studies and the reporter assay revealed that Ku70 is a novel DNA sensor inducing the IFN-lambda1 activation. The functional analysis of IFNL1 promoter revealed that positive-regulatory domain I and IFN-stimulated response element sites are predominantly involved in the DNA-mediated IFNL1 activation. A pull-down assay using nuclear proteins demonstrated that the IFN-λ1 induction is associated with the activation of IFN regulatory factor-1 and -7. Thus, to our knowledge, we show for the first time that Ku70 mediates type III IFN induction by DNA.

Human Immunodeficiency Virus Type 1 Quasi Species That Rebound after Discontinuation of Highly Active Antiretroviral Therapy Are Similar to the Viral Quasi Species Present before Initiation of Therapy

In an effort to identify the sources of the viruses that emerge after discontinuation of therapy, analyses of human immunodeficiency virus (HIV) quasi species were done for 3 patients with sustained levels of HIV RNA of <50 copies/mL for 1-3 years. The sequences found in the rebounding plasma virus were closely related to those of the actively replicating form of viruses present before the initiation of combination therapy. All quasi species found in the rebounding plasma virus were also present in proviral DNA, cell-associated RNA in peripheral blood mononuclear cells (PBMC), and virion RNA derived from PBMC coculture during periods when plasma HIV RNA levels were <50 copies/mL. These findings suggest that the rapid resurgence of plasma viremia observed after discontinuation of therapy and the viruses cocultured from PBMC are derived from a relatively stable pool of the replicating form of virus rather than from activation of a previously latent pool.

Relative Replication Fitness of a High-Level 3′-Azido-3′-Deoxythymidine-Resistant Variant of Human Immunodeficiency Virus Type 1 Possessing an Amino Acid Deletion at Codon 67 and a Novel Substitution (Thr→Gly) at Codon 69

ABSTRACT The combination of an amino acid deletion at codon 67 (Δ67) and Thr-to-Gly change at codon 69 (T69G) in the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is associated with high-level resistance to multiple RT inhibitors. To determine the relative contributions of the Δ67 and T69G mutations on viral fitness, we performed a series of studies of HIV replication using recombinant variants. A high-level 3′-azido-3′-deoxythymidine (AZT)-resistant variant containing Δ67 plus T69G/K70R/L74I/K103N/T215F/K219Q in RT replicated as efficiently as wild-type virus (Wt). In contrast, the construct without Δ67 exhibited impaired replication (23% of growth of Wt). A competitive fitness study failed to reveal any differences in replication rates between the Δ67+T69G/K70R/L74I/K103N/T215F/K219Q mutant and Wt. Evaluation of proviral DNA sequences over a 3-year period in a patient harboring the multiresistant HIV revealed that the T69G mutation emerged in the context of a D67N/K70R/T215F/K219Q mutant backbone prior to appearance of the Δ67 deletion. To assess the impact of this stepwise accumulation of mutations on viral replication, a series of recombinant variants was constructed and analyzed for replication competence. The T69G mutation was found to confer 2′,3′-dideoxyinosine resistance at the expense of fitness. Subsequently, the development of the Δ67 deletion led to a virus with improved replication and high-level AZT resistance.

Selection Pressure on HIV-1 Envelope by Broadly Neutralizing Antibodies to the Conserved CD4-Binding Site

ABSTRACT The monoclonal antibody (MAb) VRC01 was isolated from a slowly progressing HIV-1-infected donor and was shown to neutralize diverse HIV-1 strains by binding to the conserved CD4 binding site (CD4bs) of gp120. To better understand the virologic factors associated with such antibody development, we characterized HIV-1 envelope (Env) variants from this donor and five other donors who developed broadly neutralizing antibodies. A total of 473 env sequences were obtained by single-genome amplification, and 100 representative env clones were expressed and tested for entry and neutralization sensitivity. While VRC01 neutralizes about 90% of the genetically diverse heterologous HIV-1 strains tested, only selective archival Env variants from the VRC01 donor were sensitive to VRC01 and all of the Env variants derived from the donor plasma were resistant, indicating strong antibody-based selection pressure. Despite their resistance to this broadly reactive MAb that partially mimics CD4, all Env variants required CD4 for entry. Three other CD4bs MAbs from the same donor were able to neutralize some VRC01 escape variants, suggesting that CD4bs antibodies continued to evolve in response to viral escape. We also observed a relatively high percentage of VRC01-resistant Env clones in the plasma of four of five additional broadly neutralizing donors, suggesting the presence of CD4bs-directed neutralizing antibodies in these donors. In total, these data indicate that the CD4bs-directed neutralizing antibodies exert ongoing selection pressure on the conserved CD4bs epitope of HIV-1 Env.

Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy

Significance The presence of “defective” HIV-1 proviruses in HIV-infected patients has been well documented. The current consensus view of the “defective” proviruses is that these are dead-end products that do not give rise to progeny virus and thus collectively represent a “graveyard” of viruses. We describe the presence of defective HIV-1 proviruses capable of transcribing novel unspliced HIV-RNA species in HIV-infected patients on combination antiretroviral therapy. We propose that the proviruses persistently present in combination antiretroviral therapy-treated patients are not defective in a conventional sense, but rather represent incomplete forms of proviruses encoding translationally competent HIV-RNA transcripts. Strategies directed toward curing HIV-1 infection and eliminating the state of persistent immune activation need to include approaches designed to eliminate cells harboring such proviruses. Despite years of plasma HIV-RNA levels <40 copies per milliliter during combination antiretroviral therapy (cART), the majority of HIV-infected patients exhibit persistent seropositivity to HIV-1 and evidence of immune activation. These patients also show persistence of proviruses of HIV-1 in circulating peripheral blood mononuclear cells. Many of these proviruses have been characterized as defective and thus thought to contribute little to HIV-1 pathogenesis. By combining 5′LTR-to-3′LTR single-genome amplification and direct amplicon sequencing, we have identified the presence of “defective” proviruses capable of transcribing novel unspliced HIV-RNA (usHIV-RNA) species in patients at all stages of HIV-1 infection. Although these novel usHIV-RNA transcripts had exon structures that were different from those of the known spliced HIV-RNA variants, they maintained translationally competent ORFs, involving elements of gag, pol, env, rev, and nef to encode a series of novel HIV-1 chimeric proteins. These novel usHIV-RNAs were detected in five of five patients, including four of four patients with prolonged viral suppression of HIV-RNA levels <40 copies per milliliter for more than 6 y. Our findings suggest that the persistent defective proviruses of HIV-1 are not “silent,” but rather may contribute to HIV-1 pathogenesis by stimulating host-defense pathways that target foreign nucleic acids and proteins.

Decreases in Colonic and Systemic Inflammation in Chronic HIV Infection after IL-7 Administration

Despite antiretroviral therapy (ART), some HIV-infected persons maintain lower than normal CD4+ T-cell counts in peripheral blood and in the gut mucosa. This incomplete immune restoration is associated with higher levels of immune activation manifested by high systemic levels of biomarkers, including sCD14 and D-dimer, that are independent predictors of morbidity and mortality in HIV infection. In this 12-week, single-arm, open-label study, we tested the efficacy of IL-7 adjunctive therapy on T-cell reconstitution in peripheral blood and gut mucosa in 23 ART suppressed HIV-infected patients with incomplete CD4+ T-cell recovery, using one cycle (consisting of three subcutaneous injections) of recombinant human IL-7 (r-hIL-7) at 20 µg/kg. IL-7 administration led to increases of both CD4+ and CD8+ T-cells in peripheral blood, and importantly an expansion of T-cells expressing the gut homing integrin α4β7. Participants who underwent rectosigmoid biopsies at study baseline and after treatment had T-cell increases in the gut mucosa measured by both flow cytometry and immunohistochemistry. IL-7 therapy also resulted in apparent improvement in gut barrier integrity as measured by decreased neutrophil infiltration in the rectosigmoid lamina propria 12 weeks after IL-7 administration. This was also accompanied by decreased TNF and increased FOXP3 expression in the lamina propria. Plasma levels of sCD14 and D-dimer, indicative of systemic inflammation, decreased after r-hIL-7. Increases of colonic mucosal T-cells correlated strongly with the decreased systemic levels of sCD14, the LPS coreceptor - a marker of monocyte activation. Furthermore, the proportion of inflammatory monocytes expressing CCR2 was decreased, as was the basal IL-1β production of peripheral blood monocytes. These data suggest that administration of r-hIL-7 improves the gut mucosal abnormalities of chronic HIV infection and attenuates the systemic inflammatory and coagulation abnormalities that have been linked to it.

IL-15 acts as a potent inducer of CD4+CD25hi cells expressing FOXP3

IL‐15 is a member of the γ chain‐dependent cytokines and known to affect innate and CD8+ adaptive immune responses. Despite a growing interest in the use of IL‐15 as an immunotherapeutic agent, the broad spectrum of immunoregulatory functions exerted by IL‐15 has not been fully elucidated. Here, we demonstrate that IL‐15 increases expression of CD25 and forkhead box transcription factor P3 (FOXP3), a master transcriptional regulator of regulatory T cells, in human peripheral CD4+CD25– T cells in the absence of antigenic stimulation. Comparisons involving IL‐2 and IL‐7 revealed that the induction of CD25hi and FOXP3 expression was most prominent with IL‐15 and IL‐2. More modest effects were seen with IL‐7. Despite levels of FOXP3 expression comparable to that of conventional regulatory T cells, cytokine‐induced CD4+CD25hiFOXP3+ cells exerted only weak suppressor activity. Thus, the current study has demonstrated that IL‐15 acts as a potent inducer of CD4+CD25hiFOXP3+ cells in the periphery, and suggests a potential role for IL‐15 in blunting immune activation. This study has provided further insights into the pleiotropic nature of IL‐15 beyond the regulation of CD8+ T cells.