Marisa Zanchetta

Regression of AIDS-related Kaposi's sarcoma following antiretroviral therapy with protease inhibitors: biological correlates of clinical outcome

The clinical response of AIDS-related Kaposis sarcoma (KS) to highly active antiretroviral therapy (HAART), a combination of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase inhibitors, was studied in 11 patients, all but one with progressive KS. CD4+ cell counts, plasma HIV-1 RNA levels, and antibody titres to lytic ORF65 and latency-associated human herpes virus type 8 (HHV-8) proteins were determined in sequential samples. Six complete and three partial clinical responses were achieved in a median time of 6 and 3 months, respectively, and confirmed after a median time of 16 months on HAART. 2 patients showed disease progression. A consistent decrease in HIV-1 RNA levels, paralleled by an increase in CD4+ cell counts, was observed in all patients who showed complete or partial clinical response; HIV-1 RNA levels remained persistently high in the two patients who progressed, despite a change in HAART. HHV-8 antibody titres were generally higher in patients with mucosal/visceral involvement compared with patients with limited disease; a decrease in ORF65 antibody titre was significantly associated with a clinical response. These results indicate that HAART is effective for AIDS-related KS; the clinical response correlates with a decrease in plasma HIV-1 RNA levels, an increase in CD4+ lymphocytes, and a decrease in antibodies to ORF65 HHV-8 protein.

Dynamics of Epstein-Barr virus in HIV-1-infected subjects on highly active antiretroviral therapy.

Objective Patients infected with HIV-1 are at high risk of developing Epstein–Barr virus (EBV)-associated lymphoproliferative disorders. This study evaluated the impact of highly active antiretroviral therapy (HAART) on EBV infection. Methods To measure EBV content in peripheral blood lymphocytes (PBL) and in plasma, we set up a quantitative analysis using the real-time PCR. EBV latent membrane protein 1 (LMP1) expression was determined by reverse transcriptase-PCR. Results EBV levels were determined in 33 HIV-1- and EBV-coinfected patients at the start of HAART, and during therapy. At baseline, EBV content in PBL samples ranged from 8 to 14 532 copies/μg DNA. EBV levels transiently increased in nine out of 17 patients in whom HIV-1 plasmaviraemia declined to undetectable levels (virological response) and CD4 cell counts increased (immunological response), while they remained fairly stable or decreased in the other eight virological and immunological responders, and in seven patients who showed a virological response only. Of interest, a significant increase in EBV load was observed in five out of nine patients who showed an increase in CD4 cell counts but lack of HIV-1 suppression during HAART. This EBV increase was accompanied by the detection of both LMP1 transcripts in PBL and EBV DNA in plasma, and was paralleled by an increase in immunoglobulin levels, a marker of B-cell stimulation. Conclusions These findings suggest that peripheral immune reconstitution during HAART without a reduction in HIV-1 replication may increase B-cell stimulation and the number of EBV-infected B cells.

Immune reconstitution in human immunodeficiency virus type 1-infected children with different virological responses to anti-retroviral therapy

Immune repopulation, despite virological failure, often occurs in children under highly active anti‐retroviral therapy (HAART). The aim of this study was to analyse the characteristics of immune repopulation and activation in children with and without virological response to HAART. Fourteen human immunodeficiency virus type 1 (HIV‐1)‐infected children with suppression of HIV‐1 plasma viraemia (virological responders, VR) and 16 virological non‐responders (VNR) to therapy were studied at baseline and after approximately 2 years of HAART. During therapy, CD4+ T cells increased in both groups, but were higher in the VR than in the VNR group. All CD4+ T cell subsets (naive, central memory, effector/memory and CD38+) increased significantly in VR children, while there was a significant increase only in naive cells in VNR children. Naive CD8+ T cells and T cell receptor rearrangement excision circles (TREC), an indicator of thymic output, increased in both VR and VNR children. Activated CD8+CD38+ T cells decreased in VR but remained high in VNR children. Levels of circulating lipopolysaccharide (LPS), an indicator of microbial translocation, further increased in VNR children. In conclusion, HAART induced an increase in naive cells in all children, regardless of their virological response. However, the persistence of viraemia resulted in an impaired expansion of memory CD4+ T cells susceptible to HIV‐1 infection, and together with the microbial translocation sustained the persistence of a high level of immune activation.

Co-receptor usage of HIV-1 primary isolates, viral burden, and CCR5 genotype in mother-to-child HIV-1 transmission

ObjectiveTo investigate the relationship between CC chemokine receptor 5 (CCR5) genotype, viral load and co-receptor usage of maternal HIV-1 isolates in perinatal HIV-1 transmission. Patients and methodsA total of 181 mothers and infants were studied at the time of delivery. Wild-type (wt) and Δ32 CCR5 alleles were determined by means of polymerase chain reaction (PCR). The viral load in maternal plasma samples was determined by a quantitative reverse transcriptase–PCR assay; co-receptor usage of maternal isolates was determined by viral infection in cells stably expressing CCR5 or CXC chemokine receptor 4 (CXCR4) co-receptors. ResultsHIV-1 transmission rates in wt/wt and wt/Δ32 mothers (14.7 versus 15.8%), and in wt/wt and wt/Δ32 infants (14.6 versus 14.3%) were similar. Mothers transmitting infection to wt/Δ32 infants had significantly higher HIV-1-RNA levels than those who transmitted infection to wt/wt infants (5.4 versus 4.1 log10 copies/ml, P = 0.03). In wt/wt children there was a positive relationship between transmission rate and maternal viral load over the entire range of HIV-1 values, whereas in wt/Δ32 children transmission occurred only at viral loads greater than 4.0 log10 copies/ml. Logistic regression analysis confirmed that the relationship between viral load and transmission varied according to the childs CCR5 genotype (P = 0.035; adjusted for zidovudine prophylaxis and mode of delivery, P = 0.090). Moreover, the majority of wt/wt transmitting mothers had R5-type isolates, whereas none of the wt/Δ32 mothers with an R5-type virus transmitted HIV-1 to their wt/Δ32 infants. ConclusionTaken together, these findings suggest that CCR5 Δ32 heterozygosity exerts a protective effect against perinatal transmission in children exposed to a low maternal viral burden of an R5-type isolate.

Polymorphisms in the CCR5 Promoter Region Influence Disease Progression in Perinatally Human Immunodeficiency Virus Type 1–Infected Children

The effect of CC-chemokine receptor 5 (CCR5) promoter polymorphisms on the natural history of human immunodeficiency virus (HIV) disease was studied in 73 HIV-1-infected children. The CCR5(59338-59537) promoter haplotype, CCR5-59029A/G polymorphism, and CCR5Delta32 and CCR2-64I alterations were investigated. After exclusion of carriers of CCR5Delta32 or CCR2-64I, Kaplan-Meier analysis disclosed that children with the P1/P1(59353C,59356C,59402A) genotype progressed faster to disease than did children with other haplotypes (P=.016). When CCR2-64I carriers were included, this effect had borderline significance (P=.065) and was lost when CCR5Delta32 carriers were also considered (P=.387). The P1/P1 effect was strongest early after infection, when progression to disease was mainly associated with CCR5 coreceptor-using viruses. These results indicate that the P1/P1 genotype is predictive of rapid progression in HIV-1-infected children lacking CCR5Delta32 or CCR5-64I alleles. The observation of a linkage disequilibrium between P1 and 59029A might explain the previously reported association between 59029A homozygosity and rapid disease progression.

Human immunodeficiency virus type 1 modulates telomerase activity in peripheral blood lymphocytes.

The effect of human immunodeficiency virus type 1 (HIV-1) on telomerase activity in peripheral blood lymphocytes (PBL) was examined. Telomerase is an enzyme that is involved in mechanisms that control cell life span and replicative potential. HIV-1 reduced telomerase activity in in vitro-infected PBL and impaired enzyme activation upon cell stimulation. Telomerase activity was significantly lower in PBL from 23 HIV-1-infected patients than in PBL from healthy donors and significantly increased during highly active antiretroviral therapy (HAART) in 10 patients who had both a virological and an immunological response and in 5 and 8 patients with a virological or an immunological response, respectively. Further analyses of fractionated cells revealed that telomerase activity increased mainly in CD4(+) lymphocytes. Overall, these findings demonstrate that HIV-1 infection down-modulates telomerase activity and suggest that both the HIV-1 decline and immunorestoration in response to HAART contribute to increased telomerase activity in CD4(+) lymphocytes.

Long-term decay of the HIV-1 reservoir in HIV-1-infected children treated with highly active antiretroviral therapy.

To investigate the decay of the human immunodeficiency virus type 1 (HIV-1) reservoir in children receiving highly active antiretroviral therapy (HAART), we measured HIV-1 DNA in peripheral blood mononuclear cells from 14 children who achieved and maintained suppression of plasma viremia up to 48 months after the initiation of HAART. Levels of intracellular unspliced and multiply spliced HIV-1 RNA were used as markers of residual viral replication. During the first month of HAART, there were significant decays in levels of both plasma HIV-1 RNA and multiply spliced HIV-1 RNA, yet unspliced HIV-1 RNA persisted in most of the children. Greater HIV-1 DNA decay during the first month of HAART correlated with a higher concomitant increase in CD4(+) cell counts (P=.028) and a smaller subsequent HIV-1 DNA decay (P=.0012). Furthermore, HIV-1 DNA decayed faster from 1 to 9 months of HAART (median half-life, 5 months) than during the subsequent follow-up period (median half-life, 30 months). Moreover, after 9 months of HAART, HIV-1 DNA tended to decay more slowly in children with detectable levels of unspliced HIV-1 RNA. These findings suggest that clearance of the viral reservoir in HAART-treated children may be influenced by immune repopulation and residual viral replication and may help in refining long-term treatment strategies.

Role of β-defensin-1 polymorphisms in mother-to-child transmission of hiv-1

Background and Objectives:Mother-to-child transmission (MTCT) of HIV-1, the main source of pediatric AIDS, is multifactorial. Defensins provide microbial barriers and function as effectors of innate immunity. This study investigated the relationship between genetic variants of β-defensin-1 gene and MTCT of HIV-1. Patients and Methods:Three hundred children, 118 HIV-1 infected and 182 HIV-1 uninfected, born to HIV-1-infected mothers who had not undergone antiretroviral therapy during pregnancy, and 84 HIV-1-infected mothers were analyzed. The single nucleotide polymorphisms -44C/G (rs1800972) and -52G/A (rs1799946) were genotyped by TaqMan allelic discrimination assay and sequencing. Statistical analyses were performed using SNPStats and Bonferroni correction for multiple tests. Results:In children, the -52GG genotype and the -44G/-52G haplotype had a protective role against HIV-1 infection [odds ratio (OR) = 0.52, 95% confidence interval (CI) 0.31 to 0.86, P = 0.03 and OR = 0.50, 95% CI 0.31 to 0.83, P = 0.014, respectively]. In mothers, the -52GG genotype and the -44G/-52G haplotype were associated with low levels of HIV-1 plasma viremia (<1000 copies/mL) and a lower risk of maternal HIV-1 transmission (OR = 0.14, 95% CI 0.03 to 0.67, P = 0.009 and OR = 0.23, 95% CI 0.08 to 0.66, P = 0.012, respectively). Conclusions:These results demonstrate a significant relationship between genetic variants of β-defensin-1 gene, viral load, and MTCT of HIV-1, thus supporting a critical role of innate immunity in pediatric HIV-1 infection.

Toll-like receptor 9 polymorphisms influence mother-to-child transmission of human immunodeficiency virus type 1

BackgroundToll-like receptors (TLRs) recognize pathogen-associated molecular patterns and play a crucial role in the hosts innate immune response. Genetic variations in TLR genes may influence host-viral interactions and might impact upon the risk of mother-to-child transmission (MTCT) of Human Immunodeficiency Virus type 1 (HIV-1). The aim of this study was to investigate the influence of genetic variants of TLR 9 gene on MTCT.MethodsThree hundred children (118 HIV-1-infected and 182 HIV-1-uninfected) born to HIV-1-infected mothers were studied. Single nucleotide polymorphisms (SNPs) NM_017442.2: c.4-44G > A (rs352139) and c.1635A > G (rs352140) of the TLR9 gene were genotyped by TaqMan allelic discrimination assay. Statistical analyses were performed using SNPStats program.ResultsWhen considered separately, neither of the two SNPs was significantly associated with risk of HIV-1 infection. However, the [A;A] and [G;G] haplotypes were associated with a higher risk of HIV-1 infection compared to the prevalent [G;A] haplotype [odds ratio (OR) = 3.16, 95% confidence interval (CI) 1.24-8.03, p = 0.016, and OR = 5.54, 95% CI 1.76-17.50, p = 0.004, respectively].ConclusionsOverall, results demonstrate a significant correlation between specific genetic variants of the TLR9 gene and risk of MTCT of HIV-1, thus confirming a critical role of innate immunity in perinatal HIV-1 infection. Strategies aimed at modulating innate immunity might be useful for future treatment of pediatric HIV-1 infection and AIDS.

Restriction of HIV type 1 infection in macrophages heterozygous for a deletion in the CC-chemokine receptor 5 gene.

Homozygosity for a 32-base pair deletion (delta32) within the CC-chemokine receptor 5 (CCR5) gene confers resistance to infection by R5-type HIV-1 isolates. To ascertain how CCR5delta32 heterozygosity influences the susceptibility of lymphocytes and macrophages to HIV-1 infection, peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDMs) from three HIV-1-uninfected CCR5delta32 heterozygous infants and three HIV-1-uninfected CCR5 wild-type homozygous infants were exposed to two R5-type primary isolates. HIV-1 infection was monitored by DNA-PCR and p24 antigen determination; CCR5 and CCR5delta32 transcripts were quantified by competitive reverse transcription-PCR. Wild-type homozygous MDMs and PBLs and heterozygous PBLs were infected by both viral isolates, albeit with different efficiencies, but heterozygous MDMs showed restriction to HIV-1 infection. Lower levels of CCR5 mRNA and protein expression were found in heterozygous versus wild-type homozygous MDMs and PBLs. Interestingly, wild-type homozygous MDMs showed higher levels of CCR5 mRNA expression compared with wild-type homozygous PBLs, while heterozygous MDMs had lower levels of CCR5 wild-type mRNA and a higher CCR5delta32/CCR5 mRNA ratio compared with heterozygous PBLs. These findings suggest that CCR5delta32 heterozygosity confers a different degree of protection against HIV-1 in PBLs and MDMs, depending on the ratio of wild-type and mutant CCR5 mRNA in the two cell types, and may delay virus spread in the host by preventing infection of monocytes and macrophages.