Andrea R. Stacey

Induction of a Striking Systemic Cytokine Cascade prior to Peak Viremia in Acute Human Immunodeficiency Virus Type 1 Infection, in Contrast to More Modest and Delayed Responses in Acute Hepatitis B and C Virus Infections

ABSTRACT Characterization of the immune responses induced in the initial stages of human immunodeficiency virus type 1 (HIV-1) infection is of critical importance for an understanding of early viral pathogenesis and prophylactic vaccine design. Here, we used sequential plasma samples collected during the eclipse and exponential viral expansion phases from subjects acquiring HIV-1 (or, for comparison, hepatitis B virus [HBV]or hepatitis C virus [HCV]) to determine the nature and kinetics of the earliest systemic elevations in cytokine and chemokine levels in each infection. Plasma viremia was quantitated over time, and levels of 30 cytokines and chemokines were measured using Luminex-based multiplex assays and enzyme-linked immunosorbent assays. The increase in plasma viremia in acute HIV-1 infection was found to be associated with elevations in plasma levels of multiple cytokines and chemokines, including rapid and transient elevations in alpha interferon (IFN-α) and interleukin-15 (IL-15) levels; a large increase in inducible protein 10 (IP-10) levels; rapid and more-sustained increases in tumor necrosis factor alpha and monocyte chemotactic protein 1 levels; more slowly initiated elevations in levels of additional proinflammatory factors including IL-6, IL-8, IL-18, and IFN-γ; and a late-peaking increase in levels of the immunoregulatory cytokine IL-10. Notably, there was comparatively little perturbation in plasma cytokine levels during the same phase of HBV infection and a delayed response of more intermediate magnitude in acute HCV infection, indicating that the rapid activation of a striking systemic cytokine cascade is not a prerequisite for viral clearance (which occurs in a majority of HBV-infected individuals). The intense early cytokine storm in acute HIV-1 infection may have immunopathological consequences, promoting immune activation, viral replication, and CD4+ T-cell loss.

Evidence of dysregulation of dendritic cells in primary HIV infection

Myeloid and plasmacytoid dendritic cells (DCs) are important mediators of both innate and adaptive immunity against pathogens such as HIV. During the course of HIV infection, blood DC numbers fall substantially. In the present study, we sought to determine how early in HIV infection the reduction occurs and whether the remaining DC subsets maintain functional capacity. We find that both myeloid DC and plasmacytoid DC levels decline very early during acute HIV infection. Despite the initial reduction in numbers, those DCs that remain in circulation retain their function and are able to stimulate allogeneic T-cell responses, and up-regulate maturation markers plus produce cytokines/chemokines in response to stimulation with TLR7/8 agonists. Notably, DCs from HIV-infected subjects produced significantly higher levels of cytokines/chemokines in response to stimulation with TLR7/8 agonists than DCs from uninfected controls. Further examination of gene expression profiles indicated in vivo activation, either directly or indirectly, of DCs during HIV infection. Taken together, our data demonstrate that despite the reduction in circulating DC numbers, those that remain in the blood display hyperfunctionality and implicates a possible role for DCs in promoting chronic immune activation.

Elevation of Intact and Proteolytic Fragments of Acute Phase Proteins Constitutes the Earliest Systemic Antiviral Response in HIV-1 Infection

The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, β-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5–7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha–1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.

Cross-Sectional Detection of Acute HIV Infection: Timing of Transmission, Inflammation and Antiretroviral Therapy

Background Acute HIV infection (AHI) is a critical phase of infection when irreparable damage to the immune system occurs and subjects are very infectious. We studied subjects with AHI prospectively to develop better treatment and public health interventions. Methods Cross-sectional screening was employed to detect HIV RNA positive, antibody negative subjects. Date of HIV acquisition was estimated from clinical history and correlated with sequence diversity assessed by single genome amplification (SGA). Twenty-two cytokines/chemokines were measured from enrollment through week 24. Results Thirty-seven AHI subjects were studied. In 7 participants with limited exposure windows, the median exposure to HIV occurred 14 days before symptom onset. Lack of viral sequence diversification confirmed the short duration of infection. Transmission dates estimated by SGA/sequencing using molecular clock models correlated with transmission dates estimated by symptom onset in individuals infected with single HIV variants (mean of 28 versus 33 days). Only 10 of 22 cytokines/chemokines were significantly elevated among AHI participants at enrollment compared to uninfected controls, and only 4 participants remained seronegative at enrollment. Discussion The results emphasize the difficulty in recruiting subjects early in AHI. Viral sequence diversity proved accurate in estimating time of infection. Regardless of aggressive screening, peak viremia and inflammation occurred before enrollment and potential intervention. Given the personal and public health importance, improved AHI detection is urgently needed.

Origin and Evolution of HIV-1 in Breast Milk Determined by Single-Genome Amplification and Sequencing

ABSTRACT HIV transmission via breastfeeding accounts for a considerable proportion of infant HIV acquisition. However, the origin and evolution of the virus population in breast milk, the likely reservoir of transmitted virus variants, are not well characterized. In this study, HIV envelope (env) genes were sequenced from virus variants amplified by single-genome amplification from plasmas and milk of 12 chronically HIV-infected, lactating Malawian women. Maximum likelihood trees and statistical tests of compartmentalization revealed interspersion of plasma and milk HIV env sequences in the majority of subjects, indicating limited or no compartmentalization of milk virus variants. However, phylogenetic tree analysis further revealed monotypic virus variants that were significantly more frequent in milk (median proportion of identical viruses, 29.5%; range, 0 to 61%) than in plasma (median proportion of identical viruses, 0%; range, 0 to 26%) (P = 0.002), suggesting local virus replication in the breast milk compartment. Moreover, clonally amplified virus env genes in milk produced functional virus Envs that were all CCR5 tropic. Milk and plasma virus Envs had similar predicted phenotypes and neutralization sensitivities to broadly neutralizing antibodies in both transmitting and nontransmitting mothers. Finally, phylogenetic comparison of longitudinal milk and plasma virus env sequences revealed synchronous virus evolution and new clonal amplification of evolved virus env genes in milk. The limited compartmentalization and the clonal amplification of evolving, functional viruses in milk indicate continual seeding of the mammary gland by blood virus variants, followed by transient local replication of these variants in the breast milk compartment.

HIV-1 gp41 envelope IgA is frequently elicited after transmission but has an initial short response half-life

Prevention of HIV-1 transmission at mucosal surfaces will likely require durable pre-existing mucosal anti-HIV-1 antibodies (Abs). Defining the ontogeny, specificities and potentially protective nature of the initial mucosal virus-specific B-cell response will be critical for understanding how to induce protective Ab responses by vaccination. Genital fluids from patients within the earliest stages of acute HIV-1 infection (Fiebig I–VI) were examined for multiple anti-HIV specificities. Gp41 (but not gp120) Env immunoglobulin (Ig)A Abs were frequently elicited in both plasma and mucosal fluids within the first weeks of transmission. However, shortly after induction, these initial mucosal gp41 Env IgA Abs rapidly declined with a t1/2 of ∼2.7 days. B-cell-activating factor belonging to the TNF family (BAFF) was elevated immediately preceding the appearance of gp41 Abs, likely contributing to an initial T-independent Ab response. HIV-1 transmission frequently elicits mucosal HIV-1 envelope-specific IgA responses targeted to gp41 that have a short half-life.

HIV-1 infection–induced apoptotic microparticles inhibit human DCs via CD44

Acute HIV-1 infection results in dysregulated immunity, which contributes to poor control of viral infection. DCs are key regulators of both adaptive and innate immune responses needed for controlling HIV-1, and we surmised that factors elicited during acute HIV-1 infection might impede DC function. We derived immature DCs from healthy donor peripheral blood monocytes and treated them with plasma from uninfected control donors and donors with acute HIV-1 infections. We found that the plasma from patients with HIV specifically inhibited DC function. This suppression was mediated by elevated apoptotic microparticles derived from dying cells during acute HIV-1 infection. Apoptotic microparticles bound to and inhibited DCs through the hyaluronate receptor CD44. These data suggest that targeting this CD44-mediated inhibition by apoptotic microparticles could be a novel strategy to potentiate DC activation of HIV-specific immunity.

Distinct patterns of hepcidin and iron regulation during HIV-1, HBV, and HCV infections

Significance Altered iron levels correlate with disease progression in HIV type-1 (HIV-1) infection, and cellular iron promotes HIV-1 replication. In chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, increased liver iron levels contribute to disease. The peptide hormone hepcidin controls iron distribution. We find that hepcidin increases during the acute phase of HIV-1 infection, early hepcidin predicts later plasma viral set-point, and hepcidin remains high even in chronically infected individuals receiving antiretroviral therapy. Conversely hepcidin is not induced, and blood iron is not decreased, during the acute response to HBV and HCV. Therefore, the nature of iron redistribution during the response to infections is a pathogen-specific phenomenon; furthermore, the deleterious effects of chronic infection on hepcidin and iron appear to be established early in infection. During HIV type-1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections, altered iron balance correlates with morbidity. The liver-produced hormone hepcidin dictates systemic iron homeostasis. We measured hepcidin, iron parameters, cytokines, and inflammatory markers in three cohorts: plasma donors who developed acute HIV-1, HBV, or HCV viremia during the course of donations; HIV-1–positive individuals progressing from early to chronic infection; and chronically HIV-1–infected individuals (receiving antiretroviral therapy or untreated). Hepcidin increased and plasma iron decreased during acute HIV-1 infection, as viremia was initially detected. In patients transitioning from early to chronic HIV-1 infection, hepcidin in the first 60 d of infection positively correlated with the later plasma viral load set-point. Hepcidin remained elevated in individuals with untreated chronic HIV-1 infection and in subjects on ART. In contrast to HIV-1, there was no evidence of hepcidin up-regulation or hypoferremia during the primary viremic phases of HCV or HBV infection; serum iron marginally increased during acute HBV infection. In conclusion, hepcidin induction is part of the pathogenically important systemic inflammatory cascade triggered during HIV-1 infection and may contribute to the establishment and maintenance of viral set-point, which is a strong predictor of progression to AIDS and death. However, distinct patterns of hepcidin and iron regulation occur during different viral infections that have particular tissue tropisms and elicit different systemic inflammatory responses. The hypoferremia of acute infection is therefore a pathogen-specific, not universal, phenomenon.

Interleukin-10 responses to therapeutic vaccination during highly active antiretroviral therapy and after analytical therapy interruption.

We investigated whether therapeutic vaccination in highly active antiretroviral therapy (HAART)-treated patients with a modified vaccinia virus Ankara-vectored HIV-1 vaccine, with or without therapy interruption, induced the production of interleukin (IL)-10. Plasma IL-10 levels were not significantly increased postvaccination, but increased in parallel with viraemia in patients who interrupted therapy. Surprisingly, IL-10 blockade augmented HIV-specific T cell proliferative responses in HAART-suppressed patients but had no effect once virological control was lost. Modulation of IL-10 might enhance vaccine-induced immune responses.

Innate immune responses in acute HIV-1 infection: protective or pathogenic?

Background The importance of events in acute HIV-1 infection (AHI) in determining the subsequent disease course prompts a need to understand the virus-immune system interactions in this phase of infection that impact on concurrent and ensuing viral replication and pathogenesis. The speed with which innate responses can be mobilized following pathogen exposure suggests they may play critical roles in AHI. We sought to characterise the innate responses activated during AHI and identify components of the response that contribute to control of viremia or conversely promote immune activation and virus replication.