MRKAd5 HIV-1 Gag/Pol/Nef Vaccine-Induced T-Cell Responses Inadequately Predict Distance of Breakthrough HIV-1 Sequences to the Vaccine or Viral Load

Background

The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect.

Methods

Ninety-one male participants (37 placebo and 54 vaccine recipients) were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits.

Findings

Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0·04) and greater breadth of post-infection response (median 4.5 vs 2; p = 0·06). Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0·04; Pol p = 0·13; Gag p = 0·89). Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p>0·50). Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4·7 vs 5·1) but the difference was not significant (p = 0·27). Neither was acute viral load associated with distance of the viral sequence to the insert (p>0·30).

Interpretation

Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect suppression.     

The Conserved Bud20 Zinc Finger Protein Is a New Component of the Ribosomal 60S Subunit Export Machinery

The nuclear export of the preribosomal 60S (pre-60S) subunit is coordinated with late steps in ribosome assembly. Here, we show that Bud20, a conserved C₂H₂-type zinc finger protein, is an unrecognized shuttling factor required for the efficient export of pre-60S subunits. Bud20 associates with late pre-60S particles in the nucleoplasm and accompanies them into the cytoplasm, where it is released through the action of the Drg1 AAA-ATPase. Cytoplasmic Bud20 is then reimported via a Kap123-dependent pathway. The deletion of Bud20 induces a strong pre-60S export defect and causes synthetic lethality when combined with mutant alleles of known pre-60S subunit export factors. The function of Bud20 in ribosome export depends on a short conserved N-terminal sequence, as we observed that mutations or the deletion of this motif impaired 60S subunit export and generated the genetic link to other pre-60S export factors. We suggest that the shuttling Bud20 is recruited to the nascent 60S subunit via its central zinc finger rRNA binding domain to facilitate the subsequent nuclear export of the preribosome employing its N-terminal extension.     

Development of HIV fusion inhibitors.

In the past 25 years, the worldwide AIDS epidemic has grown such that roughly 38 million people were estimated to be living with the disease worldwide at the end of 2003. The introduction of antiretroviral-based therapies, beginning in 1987, has enabled many to live with HIV as a chronic, rather than terminal, disease. However, the emergence and spread of drug-resistant strains highlights the continued need for new therapies with novel modes of action. In 2003, the FDA and EMEA approved enfuvirtide (Fuzeon), a 36 amino acid peptide derived from the natural gp41 HR2 sequence, as the first HIV fusion inhibitor. T-1249, a 39 amino acid fusion inhibitor, is active against viruses that develop resistance to enfuvirtide. The development of FIs and the processes to manufacture enfuvirtide and T-1249 on an unprecedented scale for peptide therapeutics are presented. Synthetic routes based on a combination of solid phase peptide synthesis and solution phase fragment condensation as well as the analytical controls necessary to insure a robust process are discussed.     

Changes in anthropometrical characteristics during two years in 6 year old children

The purpose of this study was to investigate changes in anthropometrical characteristics during the last year (autumn-spring) in preschool and first year (autumn-spring) in school. Changes in anthropometrical characteristics during summer months were also studied (spring-autumn). The subjects were 130 boys and 122 girls, 6 years of age at the beginning of the investigation. The body height and mass were measured and the BMI (kg/m2) was calculated. In total, eight skinfolds, 10 girths and two bone widths were measured. In boys, the body height and mass were higher than in girls, while the skinfold thicknesses were thicker in girls. There were only very few significant differences between sexes in girth parameters. The width parameters were higher in boys. Our results indicated that there were more significant changes in skinfold thickness, girths and width parameters during winter in preschool in comparison with first year in school in boys, while there were only a very few significant changes during summer months in boys. In contrast, there were more significant changes in school in girls. The Spearman correlations between baseline and after 6, 12 and 18 months of study indicated that the tracking of body height was high in boys and girls. Body mass tracking was high in girls and moderate in boys, while BMI tracking was moderate in both groups. The tracking coefficients of skinfold thicknesses were relatively stable during the two years of study and mostly at the moderate level (r = 0.3-0.6). Tracking coefficients of girths measures were at moderate level, but lower in boys in comparison with girls. It was concluded that social changes and probably changes in the physical activity during transition from preschool to school slightly influenced the measured anthropometrical parameters.     

Electron transfer from the Rieske iron-sulfur protein (ISP) to cytochrome f in vitro. Is a guided trajectory of the ISP necessary for competent docking?

The time course of electron transfer in vitro between soluble domains of the Rieske iron-sulfur protein (ISP) and cytochrome f subunits of the cytochrome b(6)f complex of oxygenic photosynthesis was measured by stopped-flow mixing. The domains were derived from Chlamydomonas reinhardtii and expressed in Escherichia coli. The expressed 142-residue soluble ISP apoprotein was reconstituted with the [2Fe-2S] cluster. The second-order rate constant, k(2)((ISP-f)) = 1.5 x 10(6) m(-1) s(-1), for ISP to cytochrome f electron transfer was <10(-2) of the rate constant at low ionic strength, k(2)((f-PC))(> 200 x 10(6) m(-1) s(-1)), for the reduction of plastocyanin by cytochrome f, and approximately 1/30 of k(2)((f-PC)) at the ionic strength estimated for the thylakoid interior. In contrast to k(2)((f-PC)), k(2)((ISP-f)) was independent of pH and ionic strength, implying no significant role of electrostatic interactions. Effective pK values of 6.2 and 8.3, respectively, of oxidized and reduced ISP were derived from the pH dependence of the amplitude of cytochrome f reduction. The first-order rate constant, k(1)((ISP-f)), predicted from k(2)((ISP-f)) is approximately 10 and approximately 150 times smaller than the millisecond and microsecond phases of cytochrome f reduction observed in vivo. It is proposed that in the absence of electrostatic guidance, a productive docking geometry for fast electron transfer is imposed by the guided trajectory of the ISP extrinsic domain. The requirement of a specific electrically neutral docking configuration for ISP electron transfer is consistent with structure data for the related cytochrome bc(1) complex.     

Effect of heavy increase in training stress on the plasma leptin concentration in highly trained male rowers

AIM: To characterize the effects of a heavy increase in training stress followed by a reduced stress on fasting plasma leptin levels in highly trained male rowers. METHODS: 12 rowers underwent a 3-week period of maximally increased training stress followed by a 2-week tapering period. RESULTS: A mean 22% increase in training stress caused a significant decrease (by 8%) in the leptin concentrations. A further increase in training stress by 25% significantly reduced leptin further by 35%. A 1st tapering week, during which the training stress was rapidly reduced by approximately 50%, significantly increased the plasma leptin concentrations by 29%. Plasma leptin was significantly increased further (by 4%) during the 2nd tapering week. CONCLUSION: Leptin is sensitive to pronounced changes in training stress in highly trained male rowers.     

Changes in Laminin Expression Pattern during Early Differentiation of Human Embryonic Stem Cells

Laminin isoforms laminin-511 and -521 are expressed by human embryonic stem cells (hESC) and can be used as a growth matrix to culture these cells under pluripotent conditions. However, the expression of these laminins during the induction of hESC differentiation has not been studied in detail. Furthermore, the data regarding the expression pattern of laminin chains in differentiating hESC is scarce. In the current study we aimed to fill this gap and investigated the potential changes in laminin expression during early hESC differentiation induced by retinoic acid (RA). We found that laminin-511 but not -521 accumulates in the committed cells during early steps of hESC differentiation. We also performed a comprehensive analysis of the laminin chain repertoire and found that pluripotent hESC express a more diverse range of laminin chains than shown previously. In particular, we provide the evidence that in addition to α1, α5, β1, β2 and γ1 chains, hESC express α2, α3, β3, γ2 and γ3 chain proteins and mRNA. Additionally, we found that a variant of laminin α3 chain—145 kDa—accumulated in RA-treated hESC showing that these cells produce prevalently specifically modified version of α3 chain in early phase of differentiation.