Joseph P. Casazza

HIV preferentially infects HIV-specific CD4+ T cells

HIV infection is associated with the progressive loss of CD4+ T cells through their destruction or decreased production. A central, yet unresolved issue of HIV disease is the mechanism for this loss, and in particular whether HIV-specific CD4+ T cells are preferentially affected. Here we show that HIV-specific memory CD4+ T cells in infected individuals contain more HIV viral DNA than other memory CD4+ T cells, at all stages of HIV disease. Additionally, following viral rebound during interruption of antiretroviral therapy, the frequency of HIV viral DNA in the HIV-specific pool of memory CD4+ T cells increases to a greater extent than in memory CD4+ T cells of other specificities. These findings show that HIV-specific CD4+ T cells are preferentially infected by HIV in vivo. This provides a potential mechanism to explain the loss of HIV-specific CD4+ T-cell responses, and consequently the loss of immunological control of HIV replication. Furthermore, the phenomenon of HIV specifically infecting the very cells that respond to it adds a cautionary note to the practice of structured therapy interruption.

PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection

Here, we report on the expression of programmed death (PD)-1 on human virus-specific CD8+ T cells and the effect of manipulating signaling through PD-1 on the survival, proliferation, and cytokine function of these cells. PD-1 expression was found to be low on naive CD8+ T cells and increased on memory CD8+ T cells according to antigen specificity. Memory CD8+ T cells specific for poorly controlled chronic persistent virus (HIV) more frequently expressed PD-1 than memory CD8+ T cells specific for well-controlled persistent virus (cytomegalovirus) or acute (vaccinia) viruses. PD-1 expression was independent of maturational markers on memory CD8+ T cells and was not directly associated with an inability to produce cytokines. Importantly, the level of PD-1 surface expression was the primary determinant of apoptosis sensitivity of virus-specific CD8+ T cells. Manipulation of PD-1 led to changes in the ability of the cells to survive and expand, which, over several days, affected the number of cells expressing cytokines. Therefore, PD-1 is a major regulator of apoptosis that can impact the frequency of antiviral T cells in chronic infections such as HIV, and could be manipulated to improve HIV-specific CD8+ T cell numbers, but possibly not all functions in vivo.

Analysis of Total Human Immunodeficiency Virus (HIV)-Specific CD4+ and CD8+ T-Cell Responses: Relationship to Viral Load in Untreated HIV Infection

ABSTRACT Human immunodeficiency virus (HIV)-specific T-cell responses are thought to play a key role in viral load decline during primary infection and in determining the subsequent viral load set point. The requirements for this effect are unknown, partly because comprehensive analysis of total HIV-specific CD4+ and CD8+T-cell responses to all HIV-encoded epitopes has not been accomplished. To assess these responses, we used cytokine flow cytometry and overlapping peptide pools encompassing all products of the HIV-1 genome to study total HIV-specific T-cell responses in 23 highly active antiretroviral therapy naı̈ve HIV-infected patients. HIV-specific CD8+ T-cell responses were detectable in all patients, ranging between 1.6 and 18.4% of total CD8+ T cells. HIV-specific CD4+ T-cell responses were present in 21 of 23 patients, although the responses were lower (0.2 to 2.94%). Contrary to previous reports, a positive correlation was identified between the plasma viral load and the total HIV-, Env-, and Nef-specific CD8+ T-cell frequency. No correlation was found either between viral load and total or Gag-specific CD4+ T-cell response or between the frequency of HIV-specific CD4+ and CD8+ T cells. These results suggest that overall frequencies of HIV-specific T cells are not the sole determinant of immune-mediated protection in HIV-infection.

T-Cell Subsets That Harbor Human Immunodeficiency Virus (HIV) In Vivo: Implications for HIV Pathogenesis

ABSTRACT Identification of T-cell subsets that are infected in vivo is essential to understanding the pathogenesis of human immunodeficiency virus (HIV) disease; however, this goal has been beset with technical challenges. Here, we used polychromatic flow cytometry to sort multiple T-cell subsets to 99.8% purity, followed by quantitative PCR to quantify HIV gag DNA directly ex vivo. We show that resting memory CD4+ T cells are the predominantly infected cells but that terminally differentiated memory CD4+ T cells contain 10-fold fewer copies of HIV DNA. Memory CD8+ T cells can also be infected upon upregulation of CD4; however, this is infrequent and HIV-specific CD8+ T cells are not infected preferentially. Naïve CD4+ T-cell infection is rare and principally confined to those peripheral T cells that have proliferated. Furthermore, the virus is essentially absent from naïve CD8+ T cells, suggesting that the thymus is not a major source of HIV-infected T cells in the periphery. These data illuminate the underlying mechanisms that distort T-cell homeostasis in HIV infection.

Acquisition of direct antiviral effector functions by CMV-specific CD4+T lymphocytes with cellular maturation

The role of CD4+ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1β, and surface mobilization of the degranulation marker CD107a by CD4+ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4+ T cell maturation. The functional profile of virus-specific CD4+ T cells in chronic CMV infection was unique compared with that observed in other viral infections. Salient features of this profile were: (a) the simultaneous production of MIP-1β, TNF-α, and IFN-γ in the absence of IL-2; and (b) direct cytolytic activity associated with surface mobilization of CD107a and intracellular expression of perforin and granzymes. This polyfunctional profile was associated with a terminally differentiated phenotype that was not characterized by a distinct clonotypic composition. Thus, mature CMV-specific CD4+ T cells exhibit distinct functional properties reminiscent of antiviral CD8+ T lymphocytes.

Evidence for increased T cell turnover and decreased thymic output in HIV infection.

The effects of HIV infection upon the thymus and peripheral T cell turnover have been implicated in the pathogenesis of AIDS. In this study, we investigated whether decreased thymic output, increased T cell proliferation, or both can occur in HIV infection. We measured peripheral blood levels of TCR rearrangement excision circles (TREC) and parameters of cell proliferation, including Ki67 expression and ex vivo bromodeoxyuridine incorporation in 22 individuals with early untreated HIV disease and in 15 HIV-infected individuals undergoing temporary interruption of therapy. We found an inverse association between increased T cell proliferation with rapid viral recrudescence and a decrease in TREC levels. However, during early HIV infection, we found that CD45RO−CD27high (naive) CD4+ T cell proliferation did not increase, despite a loss of TREC within naive CD4+ T cells. A possible explanation for this is that decreased thymic output occurs in HIV-infected humans. This suggests that the loss of TREC during HIV infection can arise from a combination of increased T cell proliferation and decreased thymic output, and that both mechanisms can contribute to the perturbations in T cell homeostasis that underlie the pathogenesis of AIDS.

Immunisation with BCG and recombinant MVA85A induces long-lasting, polyfunctional mycobacterium tuberculosis-specific CD4+ memory T lymphocyte populations

In the search for effective vaccines against intracellular pathogens such as HIV, tuberculosis and malaria, recombinant viral vectors are increasingly being used to boost previously primed T cell responses. Published data have shown prime‐boost vaccination with BCG‐MVA85A (modified vaccinia virus Ankara expressing antigen 85A) to be highly immunogenic in humans as measured by ex vivo IFN‐γ ELISPOT. Here, we used polychromatic flow cytometry to investigate the phenotypic and functional profile of these vaccine‐induced Mycobacterium tuberculosis (M.tb) antigen 85A‐specific responses in greater detail. Promisingly, antigen 85A‐specific CD4+ T cells were found to be highly polyfunctional, producing IFN‐γ, TNF‐α, IL‐2 and MIP‐1β. Surface staining showed the responding CD4+ T cells to be relatively immature (CD45RO+ CD27intCD57–); this observation was supported by the robust proliferative responses observed following antigenic stimulation. Furthermore, these phenotypic and functional properties were independent of clonotypic composition and epitope specificity, which was maintained through the different phases of the vaccine‐induced immune response. Overall, these data strongly support the use of MVA85A in humans as a boosting agent to expand polyfunctional M.tb‐specific CD4+ T cells capable of significant secondary responses.

A novel approach to the analysis of specificity, clonality, and frequency of HIV-specific T cell responses reveals a potential mechanism for control of viral escape.

Escape from the CD8+ T cell response through epitope mutations can lead to loss of immune control of HIV replication. Theoretically, escape from CD8+ T cell recognition is less likely when multiple TCRs target individual MHC/peptide complexes, thereby increasing the chance that amino acid changes in the epitope could be tolerated. We studied the CD8+ T cell response to six immunodominant epitopes in five HIV-infected subjects using a novel approach combining peptide stimulation, cell surface cytokine capture, flow cytometric sorting, anchored RT-PCR, and real-time quantitative clonotypic TCR tracking. We found marked variability in the number of clonotypes targeting individual epitopes. One subject recognized a single epitope with six clonotypes, most of which were able to recognize and lyse cells expressing a major epitope variant that arose. Additionally, multiple clonotypes remained expanded during the course of infection, irrespective of epitope variant frequency. Thus, CD8+ T cells comprising multiple TCR clonotypes may expand in vivo in response to individual epitopes, and may increase the ability of the response to recognize virus escape mutants.

Putative Immunodominant Human Immunodeficiency Virus-Specific CD8+ T-Cell Responses Cannot Be Predicted by Major Histocompatibility Complex Class I Haplotype

ABSTRACT Recent studies of human immunodeficiency virus (HIV)-specific CD8+ T cells have focused on responses to single, usually HLA-A2-restricted epitopes as surrogate measures of the overall response to HIV. However, the assumption that a response to one epitope is representative of the total response is unconfirmed. Here we assess epitope immunodominance and HIV-specific CD8+ T-cell response complexity using cytokine flow cytometry to examine CD8+ T-cell responses in 11 HLA-A2+HIV+ individuals. Initial studies demonstrated that only 4 of 11 patients recognized the putative immunodominant HLA-A2-restricted p17 epitope SLYNTVATL, suggesting that the remaining subjects might lack significant HIV-specific CD8+ T-cell responses. However, five of six SLYNTVATL nonresponders recognized other HIV epitopes, and two of four SLYNTVATL responders had greater responses to HIV peptides restricted by other class I alleles. In several individuals, no HLA-A2-restricted epitopes were recognized, but CD8+ T-cell responses were detected to epitopes restricted by other HLA class I alleles. These data indicate that an individuals overall CD8+ T-cell response to HIV is not adequately represented by the response to a single epitope and that individual major histocompatibility complex class I alleles do not predict an immunodominant response restricted by that allele. Accurate quantification of total HIV-specific CD8+ T-cell responses will require assessment of the response to all possible epitopes.

Virologic effects of broadly neutralizing antibody VRC01 administration during chronic HIV-1 infection

A single infusion with broadly neutralizing antibody VRC01 resulted in lowered plasma virus load in HIV-1–infected subjects. Passive aggression for HIV Antibodies that neutralize HIV could add to the therapeutic arsenal to prevent and treat disease. Lynch et al. have now tested one such antibody—VRC01—in HIV-infected individuals. Although little difference was observed in viral reservoir in individuals on antiretroviral therapy, plasma viremia was reduced in untreated subjects with a single infusion of VRC01, preferentially suppressing neutralization-sensitive strains. Passive immunization with neutralizing antibodies could therefore aid in viral suppression in HIV-infected individuals. Passive immunization with HIV-1–neutralizing monoclonal antibodies (mAbs) is being considered for prevention and treatment of HIV-1 infection. As therapeutic agents, mAbs could be used to suppress active virus replication, maintain suppression induced by antiretroviral therapy (ART), and/or decrease the size of the persistent virus reservoir. We assessed the impact of VRC01, a potent human mAb targeting the HIV-1 CD4 binding site, on ART-treated and untreated HIV-1–infected subjects. Among six ART-treated individuals with undetectable plasma viremia, two infusions of VRC01 did not reduce the peripheral blood cell–associated virus reservoir measured 4 weeks after the second infusion. In contrast, six of eight ART-untreated, viremic subjects infused with a single dose of VRC01 experienced a 1.1 to 1.8 log10 reduction in plasma viremia. The two subjects with minimal responses to VRC01 were found to have predominantly VRC01-resistant virus before treatment. Notably, two subjects with plasma virus load <1000 copies/ml demonstrated virus suppression to undetectable levels for over 20 days until VRC01 levels declined. Among the remaining four subjects with baseline virus loads between 3000 and 30,000 copies, viremia was only partially suppressed by mAb infusion, and we observed strong selection pressure for the outgrowth of less neutralization-sensitive viruses. In summary, a single infusion of mAb VRC01 significantly decreased plasma viremia and preferentially suppressed neutralization-sensitive virus strains. These data demonstrate the virological effect of this neutralizing antibody and highlight the need for combination strategies to maintain virus suppression.