Lucia Ometto

Viral phenotype and host-cell susceptibility to HIV-1 infection as risk factors for mother-to-child HIV-1 transmission

Objective: To investigate the role of maternal HIV‐1 isolate phenotype and a childs cell susceptibility/resistance to viral infection in mother‐to‐child HIV‐1 transmission. Patients and methods: Forty‐nine women were studied at the time of delivery. Primary isolates, obtained by culturing patient peripheral blood mononuclear cells (PBMC) with PBMC from healthy donors, were characterized for tropism and syncytium‐inducing capability in monocyte‐derived macrophages (MDM), peripheral blood lymphocytes (PBL), and in the MT‐2 and MOLT‐3 T‐cell lines. Results: Seven women transmitted HIV‐1 to their children. Primary isolates were obtained from six and 28 transmitting and non‐transmitting mothers, respectively. All primary isolates from transmitting mothers and their infants but only 50% of those from non‐transmitting mothers replicated in MDM, regardless of their replication capacity in T‐cell lines. PBL and MDM cells from six uninfected children were exposed to the corresponding maternal isolates. Polymerase chain reaction analysis of HIV‐1 DNA in cells and p24 antigen assay in culture supernatants disclosed that two PBL and five MDM cultures were resistant to viral infection; two other PBL cultures, although HIV‐1‐infected, were negative for p24 production. Depletion of CD8+ cells only partially restored productive infection in CD4+ cell cultures. Moreover, all six PBL but only one MDM cultures were productively infected by an isolate obtained from a transmitting mother, thus suggesting that MDM resistance to HIV‐1 infection is not viral isolate‐restricted. Conclusions: Our findings strongly suggest that mother‐to‐child HIV‐1 transmission is influenced by both monocyte‐macrophage tropism of the maternal isolate and susceptibility of the childs target cells, in particular monocyte‐macrophages, to HIV‐1 infection. AIDS 1995, 9:427‐434

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.

Vertical transmission of HIV-1 : lack of detectable virus in peripheral blood cells of infected children at birth

ObjectiveTo evaluate the time-course of HIV-1 detection in peripheral blood mononuclear cells (PBMC) from newborns at risk of vertically acquired infection. Design and methodForty-six infants born to HIV-1-infected mothers were enrolled at birth and examined virologically and clinically in the perinatal period and every 30 days for the first 3 months of life. Follow-up was conducted at intervals of 2–3 months. HIV-1 detection in PBMC was performed using virus culture and polymerase chain reaction (PCR) assay. ResultsOnly one out of 24 newborns tested within 48 h of delivery and two out of 22 infants tested between 3 and 15 days of age were found to be HIV-1-positive by both PCR and virus culture. Further testing performed between 30 and 60 days of life identified an additional eight HIV-1-positive children. Subsequent viral, immunological and clinical follow-up confirmed PCR and virus culture results obtained in 30–60-day-old children. ConclusionsInfected infants had detectable levels of HIV-1 in their PBMC at 1 month of age. The negative PCR and virus culture findings in PBMC of newborns indicate strongly that HIV-1 cannot be diagnosed at birth in the majority of cases, and suggests that viral transmission could occur during late pregnancy and/or delivery.

Immune reconstitution in HIV-1-infected children on antiretroviral therapy: role of thymic output and viral fitness

ObjectiveTo investigate the role of thymic output and viral fitness in immune reconstitution in HIV-1-infected children on antiretroviral therapy. MethodsThymic output was studied by measuring levels of T-cell receptor rearrangement excision circles (TREC) in peripheral blood lymphocytes, using a real-time quantitative PCR assay. Recombinant viruses containing pre-therapy or post-therapy HIV-1 protease domains were evaluated for viral infectivity in a quantitative single-cycle assay. ResultsEighteen HIV-1-infected children who showed a significant increase in CD4 T-cell count after therapy were studied; HIV-1 plasma viraemia was substantially suppressed in 12 children (virological responders), but not in the other six (virological non-responders). TREC were quantified at baseline, and sequentially during the first 12 months of therapy. Both virological responders and non-responders showed an increase in TREC levels that was inversely correlated with baseline TREC and CD4 T cell counts. Changes in TREC positively correlated with CD4 T-cell count increases in virological responders, but not in non-responders; moreover, the ratios between TREC and CD4 T-cell count increases were higher in non-responders than in responders, suggesting a persistence of peripheral CD4 T-cell loss in the former. Drug-resistant viruses with reduced replicative capacity were documented in three out of six non-responders. ConclusionsThese findings indicate that recovery of thymic function is a pivotal event in immune reconstitution, and suggest that CD4 T-cell increase despite persistent viraemia is sustained by a continuous thymic output that compensates peripheral CD4 T-cell depletion which might be slowed down by emerging viruses with reduced fitness.

Telomerase activity in chronic lymphoproliferative disorders of B-cell lineage

The progressive shortening of telomeres at each cell division is a key mechanism in controlling cell proliferative capacity. The activation of telomerase, a reverse transcriptase that extends telomere length, potentially leads to unlimited cell proliferation, and is believed to play a critical role in the neoplastic process. High levels of telomerase activity have been demonstrated in almost all solid tumours; however, little data is available concerning its expression in chronic B‐cell neoplasms. By using a quantitative polymerase chain reaction‐based method we quantified telomerase activity in normal B lymphocytes, and in various B‐cell malignancies, including chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL) and hairy cell leukaemia (HCL). Compared to normal B cells, which expressed very low levels of telomerase activity, malignant cells from most of the patients showed a significant increase in telomerase activity, with highest values observed in HCL samples. Moreover, among the CLL and HCL cases, significantly higher levels of telomerase activity were found in patients with progressive disease at 1 year follow‐up versus patients with stable disease. These data suggest that telomerase activity might correlate with disease progression.

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.

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.

B cell activation in peripheral blood and lymph nodes during HIV infection

Background The spontaneous in-vitro antibody synthesis observed in unstimulated lymphocyte cultures from HIV-infected patients closely reflects the in-vivo activation of the B cell compartment; however, the mechanisms underlying this phenomenon are far from clear. Methods We compared the ability of peripheral blood mononuclear cells (PBMC) and lymph-node cells (LNC) from 10 HIV-infected patients to produce in vitro HIV-specific and total Ig spontaneously, and we correlated these parameters with tumour necrosis factor alpha (TNF-α) expression by CD4 T cells, viral dissemination in the organism, and the extent of HIV spread into lymph-node germinal centres, measured by in-situ hybridization (ISH). Results In-vitro spontaneous synthesis of both HIV-specific and total antibody was significantly higher in PBMC than in LNC; the two variables showed a good correlation in LNC, but not in PBMC. In both compartments, no correlation was found between B cell activation and the percentage of CD4 T cells expressing TNF-α, which was increased compared with seronegative donors. Furthermore, no correlation was found between in-vitro spontaneous antibody synthesis and the number of T cells containing proviral HIV in PBMC and LNC, or the plasma levels of HIV RNA. On the contrary, a good correlation was found between HIV-specific B cell activation and the extent of viral spread into lymph-node germinal centres, evaluated by ISH. Conclusion These data suggest that the adhesion of HIV virions to the follicular dendritic cell network in lymph-node germinal centres may primarily contribute to sustaining the steady B cell activation observed in HIV-infected patients.