Intrapatient sequence variation of the gag gene of human immunodeficiency virus type 1 plasma virions.

Because certain regions of the gag gene, such as p24, are highly conserved among human immunodeficiency virus (HIV) isolates, many therapeutic strategies have been directed at gag gene targets. Although intrapatient variation of segments of gag have been determined, little is known about the variability of the full-length gag gene for HIV isolated from a single individual. To evaluate intrapatient full-length gag variability, we derived the nucleotide sequences of at least 10 cDNA gag clones of virion RNA isolated from plasma for each of four asymptomatic HIV type 1-infected patients with relatively high CD4+ T-cell counts (300 to 450 cells per mm3). Mean values of intrapatient gag nucleotide variation obtained by pairwise comparisons ranged from 0.55 to 2.86%. For three subjects, this value was equivalent to that reported for intrapatient full-length env variation. The greatest range of intrapatient mean nucleotide variation for individual protein-coding regions was observed for p7. We did not detect any G-to-A hypermutation, as A-to-G and G-to-A transitions occurred at similar frequencies, accounting for 29 and 25%, respectively, of the changes. Mean variation values and phylogenetic analysis suggested that the extent of nucleotide variation correlated with the length of viral infection. Furthermore, no distinct subpopulations of quasispecies were detectable within an individual. The predicted amino acid sequences indicated that there were no regions within a gag protein that were comprised of clustered changes.     

HLA-B*57 Micropolymorphism shapes HLA allele-specific epitope immunogenicity, selection pressure, and HIV immune control

The genetic polymorphism that has the greatest impact on immune control of human immunodeficiency virus (HIV) infection is expression of HLA-B*57. Understanding of the mechanism for this strong effect remains incomplete. HLA-B*57 alleles and the closely related HLA-B*5801 are often grouped together because of their similar peptide-binding motifs and HIV disease outcome associations. However, we show here that the apparently small differences between HLA-B*57 alleles, termed HLA-B*57 micropolymorphisms, have a significant impact on immune control of HIV. In a study cohort of >2,000 HIV C-clade-infected subjects from southern Africa, HLA-B*5703 is associated with a lower viral-load set point than HLA-B*5702 and HLA-B*5801 (medians, 5,980, 15,190, and 19,000 HIV copies/ml plasma; P = 0.24 and P = 0.0005). In order to better understand these observed differences in HLA-B*57/5801-mediated immune control of HIV, we undertook, in a study of >1,000 C-clade-infected subjects, a comprehensive analysis of the epitopes presented by these 3 alleles and of the selection pressure imposed on HIV by each response. In contrast to previous studies, we show that each of these three HLA alleles is characterized both by unique CD8(+) T-cell specificities and by clear-cut differences in selection pressure imposed on the virus by those responses. These studies comprehensively define for the first time the CD8(+) T-cell responses and immune selection pressures for which these protective alleles are responsible. These findings are consistent with HLA class I alleles mediating effective immune control of HIV through the number of p24 Gag-specific CD8(+) T-cell responses generated that can drive significant selection pressure on the virus.     

Synthesis and structure–activity relationship of a novel series of heterocyclic sulfonamide γ-secretase inhibitors

γ-Secretase inhibitors have been shown to reduce the production of β-amyloid, a component of the plaques that are found in brains of patients with Alzheimer’s disease. A novel series of heterocyclic sulfonamide γ-secretase inhibitors that reduce β-amyloid levels in cells is reported. Several examples of compounds within this series demonstrate a higher propensity to inhibit the processing of amyloid precursor protein compared to Notch, an alternative γ-secretase substrate.     

(S)-N-(5-Chlorothiophene-2-sulfonyl)-β,β-diethylalaninol a Notch-1-sparing γ-secretase inhibitor

Accumulation of beta-amyloid (Aβ), produced by the proteolytic cleavage of amyloid precursor protein (APP) by β- and γ-secretase, is widely believed to be associated with Alzheimer’s disease (AD). Research around the high-throughput screening hit (S)-4-chlorophenylsulfonyl isoleucinol led to the identification of the Notch-1-sparing (9.5-fold) γ-secretase inhibitor (S)-N-(5-chlorothiophene-2-sulfonyl)-β,β-diethylalaninol 7.b.2 (Aβ _40/42 EC₅₀ = 28 nM), which is efficacious in reduction of Aβ production in vivo.     

Bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced preimplantation bovine embryos following artificial exposure

The objective was to determine the average amount of bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced bovine embryos following recommended processing procedures for embryos. In vivo-derived and in vitro-produced bovine embryos at 7 d post-fertilization were exposed (for 2 h) to 2 × 10^5-7 cell culture infective dose (CCID₅₀)/mL of SD-1 (a noncytopathic, Type 1a strain of BVDV), and then washed according to International Embryo Transfer Society (IETS) guidelines prior to testing. Of the 87 in vivo-derived embryos tested, 27% were positive for virus by quantitative polymerase chain reaction (qPCR). The range in amount of virus associated with 99% of the contaminated embryos was ≤6.62 ± 1.57 copies/5 μL; 90% of the contaminated embryos had ≤4.64 ± 1.57 viral copies/5 μL of embryo-associated virus, using tolerance intervals (P < 0.05). The SEM was 0.33 and the mean of averages was 1.12/5 μL. Of the 87 in vitro-produced embryos, 42% were positive for virus. The range in amount of virus associated with 99% of the contaminated embryos was ≤3.44 ± 0.89 copies/5 μL; 90% of the contaminated embryos had ≤2.40 ± 0.89 viral copies/5 μL of embryo-associated virus using tolerance intervals (P < 0.05; S.E.M. was 0.14 and the mean of averages was 0.55/5 μL). Therefore, although many embryos were positive for virus, there were limited numbers of copies, thereby posing doubt regarding their potential for contamination following embryo transfer.