Eric S. Rosenberg

Latent infection of CD4 + T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy

Combination therapy for HIV-1 infection can reduce plasma virus to undetectable levels, indicating that prolonged treatment might eradicate the infection. However, HIV-1 can persist in a latent form in resting CD4+ T cells. We measured the decay rate of this latent reservoir in 34 treated adults whose plasma virus levels were undetectable. The mean half-life of the latent reservoir was very long (43.9 months). If the latent reservoir consists of only 1 × 105 cells, eradication could take as long as 60 years. Thus, latent infection of resting CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective anti-retroviral therapy.

Immune control of HIV-1 after early treatment of acute infection

Virus-specific T-helper cells are considered critical for the control of chronic viral infections. Successful treatment of acute HIV-1 infection leads to augmentation of these responses, but whether this enhances immune control has not been determined. We administered one or two supervised treatment interruptions to eight subjects with treated acute infection, with the plan to restart therapy if viral load exceeded 5,000 copies of HIV-1 RNA per millilitre of plasma (the level at which therapy has been typically recommended) for three consecutive weeks, or 50,000 RNA copies per ml at one time. Here we show that, despite rebound in viraemia, all subjects were able to achieve at least a transient steady state off therapy with viral load below 5,000 RNA copies per ml. At present, five out of eight subjects remain off therapy with viral loads of less than 500 RNA copies per ml plasma after a median 6.5 months (range 5–8.7 months). We observed increased virus-specific cytotoxic T lymphocytes and maintained T-helper-cell responses in all. Our data indicate that functional immune responses can be augmented in a chronic viral infection, and provide rationale for immunotherapy in HIV-1 infection.

Comprehensive Epitope Analysis of Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Responses Directed against the Entire Expressed HIV-1 Genome Demonstrate Broadly Directed Responses, but No Correlation to Viral Load

ABSTRACT Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/106 PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.

Evolution and transmission of stable CTL escape mutations in HIV infection

Increasing evidence indicates that potent anti-HIV-1 activity is mediated by cytotoxic T lymphocytes (CTLs); however, the effects of this immune pressure on viral transmission and evolution have not been determined. Here we investigate mother–child transmission in the setting of human leukocyte antigen (HLA)-B27 expression, selected for analysis because it is associated with prolonged immune containment in adult infection. In adults, mutations in a dominant and highly conserved B27-restricted Gag CTL epitope lead to loss of recognition and disease progression. In mothers expressing HLA-B27 who transmit HIV-1 perinatally, we document transmission of viruses encoding CTL escape variants in this dominant Gag epitope that no longer bind to B27. Their infected infants target an otherwise subdominant B27-restricted epitope and fail to contain HIV replication. These CTL escape variants remain stable without reversion in the absence of the evolutionary pressure that originally selected the mutation. These data suggest that CTL escape mutations in epitopes associated with suppression of viraemia will accumulate as the epidemic progresses, and therefore have important implications for vaccine design.

Genetic and Immunologic Heterogeneity among Persons Who Control HIV Infection in the Absence of Therapy

BACKGROUND Spontaneous control of human immunodeficiency virus (HIV) infection has been documented in a minority of HIV-infected individuals. The mechanisms behind this outcome remain largely unknown, and a better understanding of them will likely influence future vaccine strategies. METHODS HIV-specific T cell and antibody responses as well as host genetics were examined in untreated HIV-infected patients who maintain comparatively low plasma HIV RNA levels (hereafter, controllers), including those with levels of < 50 RNA copies/mL (elite controllers, n = 64), those with levels of 50-2000 copies/mL (viremic controllers, n = 60); we also examined HIV-specific T cell and antibody responses as well as host genetics for patients with levels of >10,000 copies/mL (chronic progressors, n = 30). RESULTS CD8+ T cells from both controller groups preferentially target Gag over other proteins in the context of diverse HLA class I alleles, whereas responses are more broadly distributed in persons with progressive infection. Elite controllers represent a distinct group of individuals who have significantly more CD4 and CD8 T cells that secrete interferon-gamma and interleukin-2 and lower levels of HIV-neutralizing antibodies. Individual responses were quite heterogeneous, and none of the parameters evaluated was uniquely associated with the ability to control viremia. CONCLUSIONS Elite controllers are a distinct group, even when compared to persons with low level viremia, but they exhibit marked genetic and immunologic heterogeneity. Even low-level viremia among HIV controllers was associated with measurable T cell dysfunction, which has implications for current prophylactic vaccine strategies.

Upregulation of CTLA-4 by HIV-specific CD4+ T cells correlates with disease progression and defines a reversible immune dysfunction

In progressive viral infection, antiviral T cell function is impaired by poorly understood mechanisms. Here we report that the inhibitory immunoregulatory receptor CTLA-4 was selectively upregulated in human immunodeficiency virus (HIV)–specific CD4+ T cells but not CD8+ T cells in all categories of HIV-infected subjects evaluated, with the exception of rare people able to control viremia in the absence of antiretroviral therapy. CTLA-4 expression correlated positively with disease progression and negatively with the capacity of CD4+ T cells to produce interleukin 2 in response to viral antigen. Most HIV-specific CD4+ T cells coexpressed CTLA-4 and another inhibitory immunoregulatory receptor, PD-1. In vitro blockade of CTLA-4 augmented HIV-specific CD4+ T cell function. These data, indicating a reversible immunoregulatory pathway selectively associated with CD4+ T cell dysfunction, provide a potential target for immunotherapy in HIV-infected patients.

Loss of HIV-1–specific CD8+ T Cell Proliferation after Acute HIV-1 Infection and Restoration by Vaccine-induced HIV-1–specific CD4+ T Cells

Virus-specific CD8+ T cells are associated with declining viremia in acute human immunodeficiency virus (HIV)1 infection, but do not correlate with control of viremia in chronic infection, suggesting a progressive functional defect not measured by interferon γ assays presently used. Here, we demonstrate that HIV-1–specific CD8+ T cells proliferate rapidly upon encounter with cognate antigen in acute infection, but lose this capacity with ongoing viral replication. This functional defect can be induced in vitro by depletion of CD4+ T cells or addition of interleukin 2–neutralizing antibodies, and can be corrected in chronic infection in vitro by addition of autologous CD4+ T cells isolated during acute infection and in vivo by vaccine-mediated induction of HIV-1–specific CD4+ T helper cell responses. These data demonstrate a loss of HIV-1–specific CD8+ T cell function that not only correlates with progressive infection, but also can be restored in chronic infection by augmentation of HIV-1–specific T helper cell function. This identification of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important implications for immunotherapeutic interventions.

Selective escape from CD8+ T-Cell responses represents a major driving force of Human Immunodeficiency Virus Type 1 (HIV-1) sequence diversity and reveals constraints on HIV-1 evolution

ABSTRACT The sequence diversity of human immunodeficiency virus type 1 (HIV-1) represents a major obstacle to the development of an effective vaccine, yet the forces impacting the evolution of this pathogen remain unclear. To address this issue we assessed the relationship between genome-wide viral evolution and adaptive CD8+ T-cell responses in four clade B virus-infected patients studied longitudinally for as long as 5 years after acute infection. Of the 98 amino acid mutations identified in nonenvelope antigens, 53% were associated with detectable CD8+ T-cell responses, indicative of positive selective immune pressures. An additional 18% of amino acid mutations represented substitutions toward common clade B consensus sequence residues, nine of which were strongly associated with HLA class I alleles not expressed by the subjects and thus indicative of reversions of transmitted CD8 escape mutations. Thus, nearly two-thirds of all mutations were attributable to CD8+ T-cell selective pressures. A closer examination of CD8 escape mutations in additional persons with chronic disease indicated that not only did immune pressures frequently result in selection of identical amino acid substitutions in mutating epitopes, but mutating residues also correlated with highly polymorphic sites in both clade B and C viruses. These data indicate a dominant role for cellular immune selective pressures in driving both individual and global HIV-1 evolution. The stereotypic nature of acquired mutations provides support for biochemical constraints limiting HIV-1 evolution and for the impact of CD8 escape mutations on viral fitness.

HLA Alleles Associated with Delayed Progression to AIDS Contribute Strongly to the Initial CD8+ T Cell Response against HIV-1

Background Very little is known about the immunodominance patterns of HIV-1-specific T cell responses during primary HIV-1 infection and the reasons for human lymphocyte antigen (HLA) modulation of disease progression. Methods and Findings In a cohort of 104 individuals with primary HIV-1 infection, we demonstrate that a subset of CD8+ T cell epitopes within HIV-1 are consistently targeted early after infection, while other epitopes subsequently targeted through the same HLA class I alleles are rarely recognized. Certain HLA alleles consistently contributed more than others to the total virus-specific CD8+ T cell response during primary infection, and also reduced the absolute magnitude of responses restricted by other alleles if coexpressed in the same individual, consistent with immunodomination. Furthermore, individual HLA class I alleles that have been associated with slower HIV-1 disease progression contributed strongly to the total HIV-1-specific CD8+ T cell response during primary infection. Conclusions These data demonstrate consistent immunodominance patterns of HIV-1-specific CD8+ T cell responses during primary infection and provide a mechanistic explanation for the protective effect of specific HLA class I alleles on HIV-1 disease progression.

Whole genome deep sequencing of HIV-1 reveals the impact of early minor variants upon immune recognition during acute infection

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.