Françoise Seillier-Moiseiwitsch

Resistance of Hiv-1 to antiretroviral agents in blood and seminal plasma: implications for transmission

Objectives:To evaluate blood and genital secretions from HIV-infected men for HIV-1 resistant to antiretroviral agents. Design:A longitudinal study of 11 men with HIV infection and persistent detectable HIV RNA levels in blood and semen on antiretroviral therapy. Methods:HIV-1 from the blood and seminal plasma, obtained before the initiation of a new therapeutic regimen and on therapy, were evaluated by population-based sequencing of reverse transcriptase (RT) and protease RNA for the development of resistance to antiretroviral therapy. The genetic relatedness of sequences over time was compared. Results:RT genotypic resistance markers were present in seminal plasma at baseline in three out of six individuals with previous RT inhibitor experience. Eight out of 10 men, from whom the viral sequence was available on new therapy, demonstrated the evolution of new resistance mutations in the blood or seminal plasma, or both. The evolution of resistance mutations in blood and semen were frequently discordant, although over time similar patterns were seen. In two individuals, protease inhibitor resistance mutations evolved in the blood but not in the major variant in seminal plasma. Comparisons of the viral sequences between blood and seminal plasma from six men revealed two patterns. Three men showed a clustering of sequences from blood and semen. Three had sequences that appeared to evolve separately in the two compartments. Conclusions:HIV-1 variants with genotypic resistance markers are present in the male genital tract and evolve over time on incompletely suppressive antiretroviral therapy. The absence of genotypic changes consistent with protease inhibitor resistance in the semen, despite their presence in blood plasma, suggests the possibility of limited penetration of these agents into the male genital tract. Sexual transmission of resistant variants may have a negative impact on treatment outcome in newly infected individuals and on the spread of the diseases within a population. Therapeutic strategies that fully suppress HIV-1 in the genital tract should be a public health priority.

Variability in the Human Immunodeficiency Virus Type 1 gp120 Env Protein Linked to Phenotype-Associated Changes in the V3 Loop

ABSTRACT Isolates of human immunodeficiency virus type 1 (HIV-1) are classified according to the chemokine receptor (coreceptor) used in conjunction with CD4 to target and enter cells: viruses using CCR5 and CXCR4 are classified as R5 and X4, respectively. The major determinant of entry-related HIV-1 phenotypes is known to reside in the third variable region of gp120 (V3). It is clear, however, that positions outside of V3 play some role in influencing phenotype, although marked context dependence and extensive variability among HIV-1 isolates have made the identification of these positions difficult. We used the presence of previously described substitutions in V3 to classify a large set of HIV-1 subtype B gp120 sequences available in public databases as X4-like or R5-like. Using these classifications, we searched for positions outside of V3 where either amino acid composition or variability differed significantly among sequences of different inferred phenotypes. Our approach took the epidemiological relationships among sequences into account. A cluster of positions linked to changes in V3 was identified between amino acids 190 and 204 of gp120, immediately C-terminal of V2; changes at position 440 in C4 were also linked to inferred phenotype. Structural data place these positions at the coreceptor-binding face of gp120 in a surface-exposed location. We also noted a significant increase in net positive charge in a highly variable region of V2. This study both confirms previous observations and predicts specific positions that contribute to a functional relationship between V3, V2, and C4.

Effects of genital tract inflammation on human immunodeficiency virus type 1 V3 populations in blood and semen.

ABSTRACT We have examined cell-free viral populations in the blood plasma and seminal plasma compartments of men infected with subtype C human immunodeficiency virus type 1 (HIV-1) using the V3-specific heteroduplex tracking assay (V3-HTA). We studied two cohorts of subjects who had visited either a sexually transmitted disease (STD) clinic for genital tract inflammation in the form of urethritis (n = 43) or a dermatology clinic (controls,n = 14) in Malawi. We have previously shown that the presence of urethritis is associated with an eightfold increase in virus load in the seminal plasma compartment (M. S. Cohen et al., Lancet 349:1868–1873, 1997). The purpose of this study was to determine whether genital tract inflammation and its treatment caused genetic instability in cell-free HIV-1 populations. In a cross-sectional analysis at study entry, three-fourths of the STD and control subjects had multiple V3 populations in their blood while 60% of the STD subjects and 79% of the control subjects had multiple V3 populations in their semen. Overall, one-fourth of all of the subjects showed discordance between results with blood and semen specimens when samples were compared for the presence and absence of subpopulations. When differences in the relative levels of abundance of bands were also taken into account, two-fifths of all of the subjects showed discordance between the compartments. Among the subset of subjects in whom multiple virus populations could be detected, half showed discordance between the compartments. There were no differences between STD and control cohorts for these comparisons of the compartments in this cross-sectional analysis at study entry. Longitudinal analysis of the viral populations from two separate clinic visits over 1 to 4 weeks showed that the complexity of each V3 population as measured by Shannon entropy was different in blood and semen at the two time points, indicating that the blood and semen constitute different compartments for HIV-1. The seminal plasma compartment was more dynamic than the blood plasma compartment for the STD subjects who were treated for urethritis, with changes being noted in the presence or absence of V3-HTA bands in the semen of 29% of these subjects but in the blood of only 9% of these subjects. However, the changes were generally small. Overall, our results suggest that 40% of male subjects show discordance between seminal and blood viral populations and that the complexity of each V3 population was different between the two compartments. Both of these results point to the partial independence of the seminal compartment as a viral niche within the body.

Epstein-Barr virus strain variation in nasopharyngeal carcinoma from the endemic and non-endemic regions of China

Nasopharyngeal carcinoma (NPC) occurs with a striking geographic incidence and is endemic in parts of southern China, where it is the major cause of cancer death. Epstein–Barr virus (EBV) is detected in all cells of the majority of NPC cases regardless of geographic origin. A small subset of EBV genes is expressed in NPC, including the latent membrane protein (LMP‐1). LMP‐1 is essential for transformation of B lymphocytes and is considered to be the EBV oncogene. This analysis of the DNA sequence variation within the LMP‐1 gene reveals a consensus sequence for a strain, denoted China1, which predominates in East Asia where NPC is endemic. The China1 strain is characterized by nucleotide changes at 13 loci in the amino terminal portion of the LMP‐1 gene when compared with the B95‐8 prototype, including a point mutation resulting in the loss of an Xho1 restriction site. This strain was present in 9 of 15 NPC biopsy specimens from the endemic region and in 7 of 13 from northern China, where NPC is non‐endemic. A second strain, China2, was detected in 4 of 15 endemic isolates and in 2 of 13 non‐endemic isolates; this strain was characterized by a cluster of 5 nucleotide changes in the amino terminal portion of LMP‐1 in addition to those seen in China1. It was also marked by distinct changes in the carboxy terminal region of LMP‐1 including the retention of amino acids 343–352. All China1 isolates were EBV type 1, whereas the China2 isolates did not correlate with EBV type. Phylogenetic relationships between these 2 strains were determined, as were signature amino acid alterations that discriminate between them. Int. J. Cancer 76:207–215, 1998.© 1998 Wiley‐Liss, Inc.

PREDICTIVE DIAGNOSTICS FOR LOGISTIC MODELS

Novel methodology is implemented to assess the predictive power of covariate information associated with sequential binary events. Logistic models are first fitted on the basis of a subset of the observations and then evaluated sequentially on the rest. The probabilistic forecasts are compared to the outcomes via a scoring function, but as most validation samples are small, the usual reference distribution for the test statistics is inadequate. However, bootstrap-based distributions can easily be constructed. The first example pertains to the evaluation of screening tests for major depression. It illustrates that goodness-of-fit and predictive assessments lead to the selection of very different models. The second example deals with the prediction of a major event in the natural history of HIV-induced disease. It shows that this type of analysis can reveal features missed by other approaches.

Identifying genetic markers to assess the presence of gene-environment interactions

We analyzed a randomly chosen replicate with the goals of locating the closest markers to the genes involved in the discrete trait and utilizing these as surrogates for the genes in assessing the presence of gene‐environment interactions. We screened the markers with an association test prior to using the transmission‐disequilibrium test. We performed a segregation analysis, with regressive models and including the selected markers, to understand the underlying genetic mechanism and the role of the environmental factor. We were unsuccessful in locating the relevant markers due to the absence of linkage disequilibrium. Nevertheless, some insights were gained from the methods used.