The effect of Trim5 polymorphisms on the clinical course of HIV-1 infection.

The antiviral factor tripartite interaction motif 5alpha (Trim5alpha) restricts a broad range of retroviruses in a species-specific manner. Although human Trim5alpha is unable to block HIV-1 infection in human cells, a modest inhibition of HIV-1 replication has been reported. Recently two polymorphisms in the Trim5 gene (H43Y and R136Q) were shown to affect the antiviral activity of Trim5alpha in vitro.

Tight regulation of the Epstein-Barr virus setpoint: interindividual differences in Epstein-Barr virus DNA load are conserved after HIV infection.

Healthy individuals carry a constant number of Epstein-Barr virus-infected B cells in the peripheral blood over time. Here, we show that interindividual differences in Epstein-Barr virus DNA levels are maintained after HIV infection, providing evidence for the existence of an individual Epstein-Barr virus setpoint. Immune activation may contribute to the increase in this setpoint after human immunodeficiency virus seroconversion.

The presence of the Trim5alpha escape mutation H87Q in the capsid of late stage HIV-1 variants is preceded by a prolonged asymptomatic infection phase.

Background: Recently, the tripartite interaction motif 5alpha (Trim5alpha) has been identified as an inhibitory factor blocking infection of a broad range of retroviruses in a species-specific manner. In particular, HIV-1 replication can be efficiently blocked by Trim5alpha from Old World monkeys. The cyclophilin A binding region in the HIV-1 capsid is believed to be the viral determinant for Trim5alpha, and mutations in this region lift the restriction in simian cells.

Shift of CMV-specific CD4+ T-cells to the highly differentiated CD45RO-CD27- phenotype parallels loss of proliferative capacity and precedes progression to HIV-related CMV end-organ disease.

To identify factors related to progression to CMV end-organ disease, cytokine production, proliferative capacity and phenotype of CMV-specific CD4(+) T-cells were analysed longitudinally. Numbers of IFNgamma(+)CD4(+) and IFNgamma(+)IL-2(+)CD4(+) T-cells tended to decrease in individuals progressing to AIDS with CMV end-organ disease (AIDS-CMV), whereas they remained detectable in long-term asymptomatics (LTAs) and progressors to AIDS with opportunistic infections (AIDS-OI). In parallel, CMV-specific proliferative capacity was lost in AIDS-CMV.

Susceptibility of recently transmitted subtype B human immunodeficiency virus type 1 variants to broadly neutralizing antibodies.

The ability of the broadly neutralizing human immunodeficiency virus type 1 (HIV-1) specific human monoclonal antibodies (MAbs) b12, 2G12, 2F5, and 4E10 to neutralize recently transmitted viruses has not yet been explored in detail. We investigated the neutralization sensitivity of subtype B HIV-1 variants obtained from four primary HIV infection cases and six transmission couples (four homosexual and two parenteral) to these MAbs. Sexually transmitted HIV-1 variants isolated within the first 2 months after seroconversion were generally sensitive to 2F5, moderately resistant to 4E10 and b12, and initially resistant but later more sensitive to 2G12 neutralization.

Escape of human immunodeficiency virus type 1 from broadly neutralizing antibodies is not associated with a reduction of viral replicative capacity in vitro.

Although the majority of primary HIV-1 variants can be neutralized by broadly neutralizing antibodies such as b12, 2G12, 2F5 and 4E10, resistance to these antibodies has been reported as well. The ability of the broadly neutralizing antibodies to inhibit a variety of viruses suggests that their epitopes are conserved and escape from these antibodies may thus come at a cost to viral fitness. Here we demonstrate that resistance to broadly neutralizing antibodies was in general not associated with a reduced replicative capacity of the virus in vitro.

Efficient transduction of simian cells by HIV-1-based lentiviral vectors that contain mutations in the capsid protein.

Recently, the cyclophilin A (CyPA)-binding region of the HIV-1 capsid protein was identified as a viral determinant involved in the post-entry restriction in Old World monkey cells. Here, we constructed a panel of HIV-1-based lentiviral vectors (LVs) that contain either mutations in the CyPA-binding region or the CyPA-binding region of the related viruses HIV-1 group O and HIV-2. We demonstrated that amino-acid changes in the CyPA-binding region of the capsid can alter the phenotype of the virus resulting in CyPA-independent infection in human cells and non-restricted infection in simian cells.

Increased neutralization sensitivity of recently emerged CXCR4-using human immunodeficiency virus type 1 strains compared to coexisting CCR5-using variants from the same patient.

CXCR4-using (X4) human immunodeficiency virus type 1 (HIV-1) variants evolve from CCR5-using (R5) variants relatively late in the natural course of infection in 50% of HIV-1 subtype B-infected individuals and subsequently coexist with R5 HIV-1 variants. This relatively late appearance of X4 HIV-1 variants is poorly understood. Here we tested the neutralization sensitivity for soluble CD4 (sCD4) and the broadly neutralizing antibodies IgG1b12, 2F5, 4E10, and 2G12 of multiple coexisting clonal R5 and (R5)X4 (combined term for monotropic X4 and dualtropic R5X4 viruses) HIV-1 variants that were obtained at two time points after the first appearance of X4 variants in five participants of the Amsterdam Cohort Studies on HIV-1 infection and AIDS.

T cell line passage can select for pre-existing neutralization-sensitive variants from the quasispecies of primary human immunodeficiency virus type-1 isolates.

Primary human immunodeficiency type 1 viruses (HIV-1) resist antibody neutralization but become sensitive after passage through T cell lines. We and others previously reported an increased neutralization sensitivity of HIV-1 after prolonged culture on primary peripheral blood mononuclear cells (PBMC). Hence we hypothesized that adaptation to growth in T cell lines is in fact selection of a pre-existing neutralization-sensitive HIV-1 variant from the quasispecies in the PBMC culture.

Long-term highly active antiretroviral therapy in chronic HIV-1 infection: evidence for reconstitution of antiviral immunity.

In this study we investigated the long-term effect of highly active antiretroviral therapy (HAART) on HIV-specific CD4+ T-cell responses in comparison with virus-specific CD4+ T-cell responses against the persistent herpes viruses cytomegalovirus (CMV) and Epstein-Barr virus (EBV). To this end, HIV- and herpes virus-specific cellular immune responses were measured longitudinally in 10 seroconverters with long-term follow-up including 55 months of successful suppression of viral load by HAART. HIV- and CMV-specific CD4+ T cells producing interferon-gamma (IFNgamma) or interleukin-2 (IL-2) were analysed as well as proliferative capacity.

HIV-specific CD4+ T cells and viremia: who's in control?

It has been proposed that HIV-specific CD4+ T cells with a central memory phenotype might be involved in controlling HIV replication. Based on recent data (lack of protective effects of HIV-specific CD4+ T-cell responses in acutely infected patients undergoing treatment interruptions; loss of initially strong T-helper cell responses in progressors to AIDS; and lack of prognostic value of HIV-specific CD4+ T cells in a prospective study) we argue that the level of persistent viremia determines the fate of HIV-specific CD4+ T cells. We postulate that, rather than the absence of HIV-specific T cells, it is the viral and immune activation set points that are major determinants of progression to AIDS.

Detailed kinetics of EBV-specific CD4+ and CD8+ T cells during primary EBV infection in a kidney transplant patient.

The etiology of infectious mononucleosis is poorly understood and usually detected many weeks after infection. Here, we present a unique case of primary symptomatic EBV infection after kidney transplantation, in whom we analyzed both EBV-specific CD4+ and CD8+ T cells in detail from the moment of infection up to latency. We show that EBV-specific T-cell responses in peripheral blood during primary EBV infection after kidney transplantation peaked early after the appearance of viral load, but well before onset of IM symptoms, suggesting that IM in this case is not caused by high numbers of CD8+ T cells per se but may be caused by lack of homing to lymph nodes or tonsils.

Short communication: No detrimental immunological effects of mycophenolate mofetil and HAART in treatment-naive acute and chronic HIV-1-infected patients.

Mycophenolate mofetil has been proposed for HIV-1 therapy because of its guanine-depleting effect, which is expected to interfere with HIV-1 replication directly by hampering reverse transcription and indirectly via inhibition of CD4+ T cell proliferation. However, treatment with mycophenolate mofetil might also compromise lymphocyte reconstitution and HIV-specific immunity. Therefore we longitudinally studied the effects of mycophenolate mofetil in combination with HAART on T cell proliferation, lymphocyte reconstitution, and HIV-specific CD4+ and CD8+ T cell responses in six therapy-naive, acute or chronic HIV-1-infected patients, as compared to eight patients treated with HAART alone.

Direct ex vivo detection of HLA-DR3-restricted cytomegalovirus- and Mycobacterium tuberculosis-specific CD4+ T cells.

In order to detect epitope-specific CD4+ T cells in mycobacterial or viral infections in the context of human class II major histocompatibility complex protein human leukocyte antigen (HLA)-DR3, two HLA-DR3 tetrameric molecules were successfully produced. One contained an immunodominant HLA-DR3-restricted T-cell epitope derived from the 65-kDa heat-shock protein of Mycobacterium tuberculosis, peptide 1-13. For the other tetramer, we used an HLA-DR3-restricted T-cell epitope derived from cytomegalovirus (CMV) pp65 lower matrix protein, peptide 510-522, which induced high levels of interferon (IFN)-gamma-producing CD4+ T cells in three of four HLA-DR3-positive CMV-seropositive individuals up to 0.

Low-level CD4+ T cell activation is associated with low susceptibility to HIV-1 infection.

Different features have been associated with low susceptibility to HIV type 1 (HIV-1) infection in exposed seronegative individuals. These include genetic make-up such as homozygosity for the CCR5-Delta32 allele and the presence of HIV-specific CTLs. We studied immune activation and immune responsiveness in relation to HIV-1 susceptibility in 42 high-risk seronegative (HRSN) participants of the Amsterdam Cohort Studies and 54 men from the same cohort who were seronegative at the moment of analysis but later became HIV seropositive.

Determination of co-receptor usage of HIV-1.

In addition to CD4, HIV-1 uses chemokine receptors for entry in their target cells. The most important chemokine receptors in this respect are beta-chemokine receptor 5 (CCR5) and alpha-chemokine receptor 4 (CXCR4). Coreceptor usage is an important feature of the biological phenotype of HIV-1 variants.

Determination of cell tropism of HIV-1.

With the discovery that changes in the biological properties of HIV-1 correlate with the progression to disease, it became more and more important to develop assays to distinguish between the viral phenotypes. In this chapter, it is described how the biological phenotype of HIV-1 with regard to cellular tropism can be determined on primary monocyte-derived macrophages, established T-cell lines: MT2, SupT1, and H9, and promonocytic cell lines: U937, HL-60, and THP-1.

Reconstitution of EBV latent but not lytic antigen-specific CD4+ and CD8+ T cells after HIV treatment with highly active antiretroviral therapy.

The incidence of (EBV-related) malignancies in HIV-infected subjects has declined since the introduction of highly active antiretroviral therapy (HAART). To investigate the effect of HAART on EBV infection, we performed a longitudinal analysis of the T cell response to both a latent and a lytic Ag and EBV viral load in 10 subjects from early in HIV infection up to 5 years after HAART. All individuals responded to HAART by a decline in HIV viral load, a restoration of total CD4+ T cell numbers, and a decline in T cell immune activation.

Loss of EBNA1-specific memory CD4+ and CD8+ T cells in HIV-infected patients progressing to AIDS-related non-Hodgkin lymphoma.

We previously observed a loss of Epstein-Barr virus (EBV)-specific CD8+ T cells in subjects progressing to EBV-related non-Hodgkin lymphoma (NHL), correlating with loss of CD4+ T cells. The aim of the present study was to determine the role of EBV-specific CD4+ T cells in the development of NHL during chronic HIV infection. To this end, CD4+ and CD8+ memory T cells, capable of both proliferation and subsequent interferon gamma (IFNgamma) production, directed against a latent (Epstein-Barr virus nuclear antigen 1 [EBNA1]) and a lytic (BamH fragment Z left frame 1 [BZLF1]) EBV antigen were studied longitudinally in 9 progressors to NHL, 4 progressors to non-EBV-related AIDS, and 4 slow progressors to AIDS.

Analysis of the effect of highly active antiretroviral therapy during acute HIV-1 infection on HIV-specific CD4 T cell functions.

Background: It has been reported that antiretroviral therapy (HAART) during acute HIV-1 infection may rescue HIV-1-specific CD4 T cell responses.

Objective: To determine the duration of this preserved response by investigating the long-term effects of HAART during acute infection on HIV-specific CD4 T cell function related to possible immune control during subsequent therapy interruption.

Methods: A longitudinal analysis followed HIV-specific CD4 T cell reactivity in 17 individuals with well-documented acute HIV-1 infection where five out of 11 HAART-treated patients stopped therapy and six were untreated.

Natural controlled HIV infection: preserved HIV-specific immunity despite undetectable replication competent virus.

Long-term non-progressive HIV infection, characterized by low but detectable viral load and stable CD4 counts in the absence of antiviral therapy, is observed in about 5% of HIV-infected patients. Here we identified four therapy naïve individuals who are strongly seropositive for HIV-1 but who lack evidence of detectable HIV p24 antigen, plasma RNA, and proviral DNA in routine diagnostic testing. With an ultrasensitive PCR, we established that frequencies of pol proviral DNA sequences were as low as 0.

Sensitivity of primary R5 HTV-1 to inhibition by RANTES correlates with sensitivity to small-molecule R5 inhibitors.

In approximately 50% of HIV-1 subtype B-infected individuals, progression to AIDS is preceded by the emergence of CXCR4-using (X4) variants, whereas the rest progress to AIDS in the presence of CCR5-using (R5) variants only. In a previous study, we showed that during disease progression in the presence of R5 variants only, HIV-1 variants emerge with a decreased sensitivity to inhibition by RANTES, a natural ligand of CCR5 that inhibits cellular entry of R5 variants. This observation was of potential clinical relevance as HIV-1 small-molecule R5 entry inhibitors are a new class of drugs that, in analogy to RANTES, target the binding and subsequent entry of HIV into the target cell.

Progression to CMV end-organ disease in HIV-1-infected individuals despite abundance of highly differentiated CMV-specific CD8+ T-cells.

Since CMV-specific T-cells have been shown to generally express an advanced state of differentiation, we investigated whether these mature CMV-specific T-cells are sustained in HIV-infected patients, who are not treated with HAART, receive no CMV medication, but do progress to AIDS with CMV end-organ disease (AIDS-CMV). CD8+ and CD4+ T-cell phenotype was studied in these patients in comparison with long-term asymptomatic HIV-infected individuals, progressors to AIDS without CMV end-organ disease as well as CMV-seropositive HIV-negative controls. CMV-specific CD8+ T-cells from progressors to AIDS-CMV expressed markers typical of highly differentiated effector T-cells, being CCR7-, CD27- CD45RO+/-, with high CD57 expression and increased Ki67 expression, compatible with functional effector cell capabilities.

Novel method for detection of virus-specific CD41+ T cells indicates a decreased EBV-specific CD4+ T cell response in untreated HIV-infected subjects.

A lower function of EBV-specific CD8(+) T cells in HIV-infected subjects could be related to a lack of specific CD4(+) T cell help. Therefore, we studied EBV-specific CD4(+) T cells in both healthy donors and untreated or highly active antiretroviral therapy (HAART)-treated HIV-seropositive homosexual men. To this end, PBMC were stimulated with overlapping peptide pools from a latent and a lytic EBV protein, EBV nuclear antigen (EBNA)1 and EBV lytic-switch protein ZEBRA (BZLF1), respectively.