Gabriella Scarlatti

Determination of Coreceptor Usage of Human Immunodeficiency Virus Type 1 from Patient Plasma Samples by Using a Recombinant Phenotypic Assay

ABSTRACT We developed a recombinant virus technique to determine the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from plasma samples, the source expected to represent the most actively replicating virus population in infected subjects. This method is not subject to selective bias associated with virus isolation in culture, a step required for conventional tropism determination procedures. The addition of a simple subcloning step allowed semiquantitative evaluation of virus populations with a different coreceptor (CCR5 or CXCR4) usage specificity present in each plasma sample. This procedure detected mixtures of CCR5- and CXCR4-exclusive virus populations as well as dualtropic viral variants, in variable proportions. Sequence analysis of dualtropic clones indicated that changes in the V3 loop are necessary for the use of CXCR4 as a coreceptor, but the overall context of the V1-V3 region is important to preserve the capacity to use CCR5. This convenient technique can greatly assist the study of virus evolution and compartmentalization in infected individuals.

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report

Background Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). Methods In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. Findings Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. Conclusions Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend parallel use of PSV and VIA for vaccine evaluation.

Cell-to-Cell Contact Results in a Selective Translocation of Maternal Human Immunodeficiency Virus Type 1 Quasispecies across a Trophoblastic Barrier by both Transcytosis and Infection

ABSTRACT Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant childs virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.

Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains.

ABSTRACT The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on coreceptor usage phenotype. These results provide the first evidence of a correlation between HIV-1 biological phenotype and neutralization sensitivity, raising the possibility that the in vivo evolution of HIV-1 coreceptor usage may be influenced by the selective pressure of specific host antibodies.

A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load

Quantification of human immunodeficiency virus type-1 (HIV-1) proviral DNA is increasingly used to measure the HIV-1 cellular reservoirs, a helpful marker to evaluate the efficacy of antiretroviral therapeutic regimens in HIV-1–infected individuals. Furthermore, the proviral DNA load represents a specific marker for the early diagnosis of perinatal HIV-1 infection and might be predictive of HIV-1 disease progression independently of plasma HIV-1 RNA levels and CD4+ T-cell counts. The high degree of genetic variability of HIV-1 poses a serious challenge for the design of a universal quantitative assay capable of detecting all the genetic subtypes within the main (M) HIV-1 group with similar efficiency. Here, we describe a highly sensitive real-time PCR protocol that allows for the correct quantification of virtually all group-M HIV-1 strains with a higher degree of accuracy compared with other methods. The protocol involves three stages, namely DNA extraction/lysis, cellular DNA quantification and HIV-1 proviral load assessment. Owing to the robustness of the PCR design, this assay can be performed on crude cellular extracts, and therefore it may be suitable for the routine analysis of clinical samples even in developing countries. An accurate quantification of the HIV-1 proviral load can be achieved within 1 d from blood withdrawal.

Polymerase chain reaction, virus isolation and antigen assay in HIV-1-antibody-positive mothers and their children

Diagnosis of perinatal HIV-1 infection is complicated by the persistence of maternal antibodies and the conflicting reports on polymerase chain reaction (PCR) reactivity in children born to HIV-1-seropositive mothers. We have compared PCR with other diagnostic methods for perinatal HIV-1 infection and have attempted also to identify maternal markers which correlate with risk of transmission. PCR was the most sensitive method for early diagnosis of perinatal transmission of HIV-1, but the PCR-positive children (n = 11) developed at least one additional sign of infection. The PCR-negative children (n = 76) were clinically healthy, virus isolation negative, and their serum was HIV-1-antigen-negative. All children who had become seronegative (n = 36) were both PCR- and isolation-negative. Antigenaemia in the mothers correlated significantly with higher risk of perinatal transmission of HIV-1, while no other parameters (clinical stage, lymphocyte subsets, PCR and isolation) showed such a correlation. This indicates that the level of virus expression may be of key importance for the risk of vertical transmission of HIV-1 infection.

Prognostic significance of immunologic changes in 675 infants perinatally exposed to human immunodeficiency virus

Neutrophil, lymphocyte, and T-cell subset numbers and immunoglobulin levels were evaluated at birth to age 2 years in 675 children born to mothers infected with the human immunodeficiency virus type 1 (58 infected symptom-free subjects (P-1), 203 infected subjects with symptoms (P-2), and 414 uninfected subjects). The P-2 patients had (even at birth to age 1 month) lower CD4+ lymphocyte and higher IgA and IgM values than P-1 and uninfected children had. Increased IgG values (from 1 to 6 months of age) and increased CD8+ lymphocyte numbers (at 13 to 24 months of age) were also observed. The P-1 children differed from uninfected children only at 13 to 24 months of age (decreased CD4+ and increased CD8+ lymphocytes). Progressive immunologic changes were found in P-2 patients who had severe clinical conditions and in those who died. To evaluate the predictive meaning of the immunologic changes, we selected 164 children (25 P-2, 15 P-1, and 124 uninfected children) because they had been examined sequentially from birth and they were classified as in the indeterminate state of infection (P-0) at immunologic evaluations at birth to age 1 and at 1 to 6 months of age. During the 1- to 6-month period, P-2 patients had higher immunoglobulin and lower CD4+ lymphocyte values than P-1 and uninfected children had; no difference was found between P-1 and uninfected subjects. These results indicate that in infants with perinatal human immunodeficiency virus type 1 infection, immunologic abnormalities correlate with the clinical condition and are predictive of the clinical outcome rather than the infection status.

Polymorphisms in the MBL2 promoter correlated with risk of HIV-1 vertical transmission and AIDS progression

We investigated the polymorphisms of the promoter region of the MBL2 gene, which codifies for the Mannose-binding protein (MBP). The study population included 90 children with vertically acquired HIV-infection, further divided on the basis of the disease rate, 27 HIV exposed-uninfected children, and 74 healthy control subjects matched for ethnic origin to evaluate the MBP involvement in the risk of HIV-1 infection and to assess the role of the MBP promoter in AIDS progression. A region of 380 bp in the promoter of the MBL2 gene was analysed by PCR and direct sequencing of both DNA strands. We found that the polymorphism at position −550 influences the risk of HIV-infection and AIDS progression. Also a 6 bp deletion at position −328 was correlated with HIV-1 infection. This study indicates that the promoter of the MBL2 gene influences vertical transmission of HIV and the course of perinatal infection.

Low Rate of Mother-to-Child Transmission of HIV-1 After Nevirapine Intervention in a Pilot Public Health Program in Yaounde, Cameroon

Objective: To determine the percentage of infected children for whom nevirapine (NVP) was used to prevent peripartum mother‐tochild transmission (MTCT) of HIV in Yaoundé, Cameroon. Design: The study was a prospective Public Health Pilot Program covering a 3‐year period (January 2000‐December 2002). Methods: Counseled and consenting HIV‐1‐positive pregnant women were given a single dose of NVP at the onset of labor. Babies were given 2 mg/kg NVP syrup within the first 72 hours of life. NVPtreated children were regularly followed up and examined for HIV‐1 infection at 6‐8 weeks and 5‐6 months through plasma viral load (VL) quantification with the bDNA system. Results: One hundred twenty‐three children were diagnosed with perinatal HIV‐1 infection at 6‐8 weeks and 5‐6 months. Thirteen children (10.6% [13/123]; 95% confidence interval, 5.1‐16) were infected and presented with high VLs, in general >500,000 copies/mL. Two children had intermediate VLs (between 50 and 3500 copies/mL) at both time points. One hundred seven children (87%) were considered not infected at 6‐8 weeks of age. Conclusions: Our results indicate that the HIV‐1 MTCT rate 6‐8 weeks after NVP administration was not >13% (16/123), thus demonstrating the effectiveness of NVP for lowering the risk of HIV‐1 MTCT in real‐life settings.

Selection of Maternal Human Immunodeficiency Virus Type 1 Variants in Human Placenta

To determine the mechanisms by which human immunodeficiency virus type 1 (HIV-1) crosses the placenta into the fetal blood, 12 matched samples of serial maternal blood, term placentas, and infant blood obtained from a cohort of pregnant women in Cameroon identified as predominantly infected by subtype A viruses were studied. HIV-1 env sequences were detected by polymerase chain reaction (PCR) in both chorionic villi and enriched trophoblastic cells of all 12 placentas but at variable rates of detection. Heteroduplex mobility assay analysis showed the presence of multiple HIV-1 env quasispecies in sequential maternal peripheral blood mononuclear cell samples, but only a small number of env variants were found in chorionic villi and enriched trophoblastic cells. These data indicate that HIV-1 env sequences are always present in term placentas of seropositive women, contrasting with the low frequency at which infection is diagnosed by PCR in neonates with tat, gag, and env primers. Maternal HIV-1 variants appear to undergo a strong negative selection by different cell populations within the placental villi.