Mohammad M. Sajadi

Virologic Response Following Combined Ledipasvir and Sofosbuvir Administration in Patients With HCV Genotype 1 and HIV Co-infection

IMPORTANCE There is an unmet need for interferon- and ribavirin-free treatment for chronic hepatitis C virus (HCV) infection in patients co-infected with human immunodeficiency virus (HIV). OBJECTIVE To evaluate the rates of sustained virologic response (SVR) and adverse events in previously untreated patients with HCV genotype 1 and HIV co-infection following a 12-week treatment of the fixed-dose combination of ledipasvir and sofosbuvir. DESIGN, SETTING, AND PARTICIPANTS Open-label, single-center, phase 2b pilot study of previously untreated, noncirrhotic patients with HCV genotype 1 and HIV co-infection conducted at the Clinical Research Center of the National Institutes of Health, Bethesda, Maryland, from June 2013 to September 2014. Patients included those receiving antiretroviral therapy with HIV RNA values of 50 copies/mL or fewer and a CD4 T-lymphocyte count of 100 cells/mL or greater or patients with untreated HIV infection with a CD4 T-lymphocyte count of 500 cells/mL or greater. Serial measurements of safety parameters, virologic and host immune correlates, and adherence were performed. INTERVENTIONS Fifty patients with HCV genotype 1 never before treated for HCV were prescribed a fixed-dose combination of ledipasvir (90 mg) and sofosbuvir (400 mg) once daily for 12 weeks. MAIN OUTCOMES AND MEASURES The primary study outcome was the proportion of patients with sustained viral response (plasma HCV RNA level <12 IU/mL) 12 weeks after end of treatment. RESULTS Forty-nine of 50 participants (98% [95% CI, 89% to 100%]) achieved SVR 12 weeks after end of treatment, whereas 1 patient experienced relapse at week 4 following treatment. In the patient with relapse, deep sequencing revealed a resistance associated mutation in the NS5A region conferring resistance to NS5A inhibitors, such as ledipasvir. The most common adverse events were nasal congestion (16% of patients) and myalgia (14%). There were no discontinuations or serious adverse events attributable to study drug. CONCLUSIONS AND RELEVANCE In this open-label, uncontrolled, pilot study enrolling patients co-infected with HCV genotype 1 and HIV, administration of an oral combination of ledipasvir and sofosbuvir for 12 weeks was associated with high rates of SVR after treatment completion. Larger studies that also include patients with cirrhosis and lower CD4 T-cell counts are required to understand if the results of this study generalize to all patients co-infected with HCV and HIV. TRIAL REGISTRATION clinicaltrials.gov Identifier:NCT01878799.

Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain; and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. Thus, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.

Discordant memory B cell and circulating anti-Env antibody responses in HIV-1 infection.

Long-lived memory B cells (BMem) provide an archive of historic Ab responses. By contrast, circulating Abs typically decline once the immunogen is cleared. Consequently, circulating Abs can underestimate the nature of cognate humoral immunity. On the other hand, the BMem pool should provide a comprehensive picture of Ab specificities that arise over the entire course of infection. To test this hypothesis, we compared circulating Ab and BMem from natural virus suppressors who control HIV-1 without therapy and maintain a relatively intact immune system. We found high frequencies of BMem specific for the conserved neutralizing CD4 induced or CD4 binding site epitopes of gp120, whereas low Ab titers to these determinants were detected in contemporaneous plasma. These data suggest that plasma Ab repertoires can underestimate the breadth of humoral immunity, and analyses of BMem should be included in studies correlating Ab specificity with protective immunity to HIV-1.

Structural Definition of an Antibody-Dependent Cellular Cytotoxicity Response Implicated in Reduced Risk for HIV-1 Infection

ABSTRACT The RV144 vaccine trial implicated epitopes in the C1 region of gp120 (A32-like epitopes) as targets of potentially protective antibody-dependent cellular cytotoxicity (ADCC) responses. A32-like epitopes are highly immunogenic, as infected or vaccinated individuals frequently produce antibodies specific for these determinants. Antibody titers, as measured by enzyme-linked immunosorbent assay (ELISA) against these epitopes, however, do not consistently correlate with protection. Here, we report crystal structures of CD4-stabilized gp120 cores complexed with the Fab fragments of two nonneutralizing, A32-like monoclonal antibodies (MAbs), N5-i5 and 2.2c, that compete for antigen binding and have similar antigen-binding affinities yet exhibit a 75-fold difference in ADCC potency. We find that these MAbs recognize overlapping epitopes formed by mobile layers 1 and 2 of the gp120 inner domain, including the C1 and C2 regions, but bind gp120 at different angles via juxtaposed VH and VL contact surfaces. A comparison of structural and immunological data further showed that antibody orientation on bound antigen and the capacity to form multivalent antigen-antibody complexes on target cells were key determinants of ADCC potency, with the latter process having the greater impact. These studies provide atomic-level definition of A32-like epitopes implicated as targets of protective antibodies in RV144. Moreover, these studies establish that epitope structure and mode of antibody binding can dramatically affect the potency of Fc-mediated effector function against HIV-1. These results provide key insights for understanding, refining, and improving the outcome of HIV vaccine trials, in which relevant immune responses are facilitated by A32-like elicited responses. IMPORTANCE HIV-1 Env is a primary target for antibodies elicited during infection. Although a small number of infected individuals elicit broadly neutralizing antibodies, the bulk of the humoral response consists of antibodies that do not neutralize or do so with limited breadth but may effect protection through Fc receptor-dependent processes, such as antibody-dependent cellular cytotoxicity (ADCC). Understanding these nonneutralizing responses is an important aspect of elucidating the complete spectrum of immune response against HIV-1 infection. With this report, we provide the first atomic-level definition of nonneutralizing CD4-induced epitopes in the N-terminal region of the HIV-1 gp120 (A32-like epitopes). Further, our studies point to the dominant role of precise epitope targeting and mode of antibody attachment in ADCC responses even when largely overlapping epitopes are involved. Such information provides key insights into the mechanisms of Fc-mediated function of antibodies to HIV-1 and will help us understand the outcome of vaccine trials based on humoral immunity.

Epidemiologic characteristics and natural history of HIV-1 natural viral suppressors.

Objective:The objective of this study was to detail the epidemiologic characteristics and natural history of HIV-1 natural viral suppressors (NVSs), a cohort of HIV-1-infected individuals who are able to suppress viral replication to undetectable levels in the absence of therapy. Design and Methods:HIV-1 patients who met the NVS criteria were enrolled into a prospective study. The incidence and prevalence of NVS were calculated by performing a chart review on all patients seen in 1 clinic in a 10-year period. Cumulative probability of progression-free survival was calculated by Kaplan-Meier product limit method. Results:Forty individuals enrolled in the study. The median year of diagnosis was 1994, and individuals demonstrated a median 6.7 years of HIV-1 viral suppression and CD4 count of 795 cells per microliter. NVS had an incidence of 1.1% [95% confidence interval (CI), 0.0 to 2.1] and prevalence of 1.5% (95% CI, 0.8 to 2.1). Only 1 patient (2.5%) has progressed. Within the first 10 years for follow-up having met the definition of NVS, 95.1% (95% CI 86.5% to 100%) of the NVS continued to control their viral loads to undetectable levels. Conclusions:The NVS cohort has demonstrated remarkable stability and a low rate of progression over many years. Detailed evaluations of viral-host immune regulatory factors associated with persistent HIV-1 natural viral suppression, and loss of such suppression, has the potential to provide important new insight in HIV pathogenesis and future immune regulatory targeted preventive and therapeutic research.

Natural viral suppressors of HIV-1 have a unique capacity to maintain γδ T cells

Objective:To evaluate Vγ2Vδ2 T cells in a group of HIV-infected patients who suppress HIV replication without antiretroviral therapy (natural viral suppressors, NVSs). Design:It is a cross-sectional study. Methods:We compared Vγ2Vδ2 T-cell frequency, T-cell repertoire, and responses to isopentenyl pyrophosphate stimulation between NVSs (n = 21) and HIV-uninfected controls (n = 27) and between NVSs and HIV-infected patients taking HAART with suppressed viral replication (HIV-P; n = 25). Results:NVSs had a mean frequency of 1.06 ± 0.82% CD3+Vδ2+ cells among total lymphocytes, which was significantly higher than both control groups (HIV-negative: 0.50 ± 0.53%, P = 0.042; HIV-P: 0.34 ± 0.37%, P = 0.002). The proportion of Vγ2 chains correlating with the Vγ2-Jγ1.2 rearrangement was reduced among NVSs compared with HIV-negative controls (0.57 ± 0.06 vs. 0.32 ± 0.04; P = 0.016) but was increased compared with HIV-P patients (0.32 ± 0.04 vs. 0.22 ± 0.03; P = 0.03). NVSs had a similar baseline frequency of CD27−/CD45RA− effector cells (19.6 ± 4.2%) compared with HIV-negative controls (20.8 ± 12.9%; P = 0.35). Conclusion:The altered γδ T-cell receptor repertoire among NVS was consistent with the known effect of HIV-1 on these cells. Uniquely among all HIV-infected groups, NVS reconstituted the γδ T-cell population, eventually reaching levels significantly above controls. This capacity to recover γδ T-cell numbers and function distinguishes individuals who control HIV-1 with and without HAART.

Chronic immune activation and decreased CD4 cell counts associated with hepatitis C infection in HIV-1 natural viral suppressors.

We have established a cohort of natural viral suppressors (NVS) who can suppress HIV-1 replication to less than 400 copies/ml in the absence of therapy (similar to Elite Controllers/Elite Suppressors). Of the 59 patients currently in the NVS cohort, 45.8% have chronic hepatitis C virus (HCV) infection, thereby presenting a unique opportunity to study immune activation and the interaction between HCV and HIV. NVS with chronic HCV infection had elevated levels of immune activation (CD38-positive HLA-DR-positive CD8 cells) compared to NVS without chronic HCV (P = 0.004). The increased levels of immune activation were not associated with sex, HLA B57 status, or injection drug use use. NVS patients with chronic HCV had lower mean CD4 cell counts, CD4 percentage, and CD4/CD8 ratios than NVS without chronic HCV infection (P = 0.038, P = 0.008, and P = 0.048, respectively). The difference in CD4 cell count appeared to occur early in HIV infection with no difference observed in CD4 slopes between groups. Among all NVS, there was a direct correlation between mean CD4 cell count, mean CD4 percentage, and mean CD4/CD8 ratio with percentage of CD38+ HLA-DR+ CD8 cells (P = 0.0018; P = 0.0069; and P = 0.0014, respectively). This study suggests a relationship between HCV infection, immune activation, and CD4 cell counts in the NVS, with chronic HCV infection associated with lower CD4 cell counts and higher levels of immune activation. Further studies are needed to determine if successful HCV treatment lowers immune activation levels and/or increases CD4 cell counts in these patients.

Increased expression of suppressor of cytokine signaling-1 (SOCS-1): A mechanism for dysregulated T helper-1 responses in HIV-1 disease.

Maintenance of Th1 responses and dendritic cell (DC) functions are compromised in HIV-1 infected individuals. To better understand these immune abnormalities, we developed an HIV-1 transgenic (Tg) rat. We report that Tg DCs induce elevated levels of SOCS-1 and secrete decreased IL-12p40 and elevated levels of IL-10 following TLR-4 stimulation by LPS. This leads to further induction of SOCS-1 by IL-10 and decreased IFN-gamma-mediated induction of interferon response factor (IRF)-1 and IL-12Rbeta1 expression in CD4+ T cells and to decreased IL-12-induction of IFN-gamma production by Th1 polarized T cells. We also show that SOCS-1 is elevated in CD4+ T cells from HIV-1 infected progressors, and is correlated with defective induction of IRF-1 following IFN-gamma stimulation, compared with healthy controls and HIV-1 natural viral suppressor (NVS) patients. These results suggest a link between high levels of SOCS-1, defects in innate immunity and adaptive Th1 responses that may be reflected in the loss of Th1 immune competence observed with AIDS patients.

Soluble factors from T cells inhibiting X4 strains of HIV are a mixture of β chemokines and RNases

T-cell-derived soluble factors that inhibit both X4 and R5 HIV are recognized as important in controlling HIV. Whereas three β chemokines, regulated-on-activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β, account for the suppression of R5 HIV by blockade of HIV entry, the major components responsible for the inhibition of X4 HIV strains have not been identified previously. We identify these factors primarily as a mixture of three β chemokines [macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), and I-309] and two RNases (angiogenin and RNase 4) of lesser potency and show that in a clade B population, some correlate with clinical status and are produced by both CD4+ and CD8+ T cells (chemokines, angiogenin) or only by CD8+ T cells (RNase 4). The antiviral mechanisms of these HIV X4-suppressive factors differ from those of the previously described HIV R5-suppressive β chemokines.

Correlation between circulating HIV-1 RNA and broad HIV-1 neutralizing antibody activity

Objective:To examine the relationship between HIV-1 antigenic load (plasma RNA copies/mL) and broad HIV-1 neutralizing antibody activity. Methods:Plasma from 120 HIV-1-infected patients, including HIV-1 natural viral suppressors (similar to elite controllers), was tested for neutralization against 15 Tier 1/Tier 2 HIV-1 pseudoviruses. Broad HIV-1 neutralizing antibody activity was confirmed with immunoglobulin G and heterlogous clade testing (18 pseudoviruses from Clades A, C, and CRF02_AG). Statistical analysis was performed to determine factors associated with broad HIV-1 neutralizing antibody activity. Results:Ten individuals with broad HIV-1 neutralizing antibody activity were identified. These individuals had a median CD4 count of 589 cells per microliter (range 202-927), 1611 HIV-1 RNA copies per milliliter (range 110-8964), and 13 years since HIV diagnosis (range 1-22). There was a significant correlation between the presence of broadly neutralizing antibodies in those with HIV-1 RNA between 100 and 10,000 copies per milliliter compared with those <100 or >10,000 copies per milliliter (P = 0.0003 and 0.0245, respectively). Individuals with HIV-1 RNA 100-10,000 copies per milliliter had a higher number of Tier 2 viruses neutralized compared with the <100 or >10,000 copies per milliliter groups (P ≤ 0.0001 and P = 0.076, respectively). Male sex was associated with broad HIV-1 neutralizing antibody activity (P = 0.016). Conclusions:These results indicate that low but persistent HIV antigen expression correlates with broad HIV-1 neutralizing antibody activity. At higher levels of plasma viremia, neutralization titers were diminished. Conversely, at lower levels, there seems to be insufficient antigen stimulation to maintain high neutralization titers. These findings may have important implications in furthering the understanding of the humoral response to HIV infection.