Generation of a neutralizing antibody against RD114-pseudotyped viral vectors

The feline endogenous RD114 glycoprotein has proved to be an attractive envelope to pseudotype both retroviral and lentiviral vectors. As a surface protein, its detection on packaging cells as well as viral particles would be useful in different fields of its use. To address this, we generated a monoclonal antibody against RD114 by immunization of rats, termed 22F10. Once seroconversion was confirmed, purified 22F10 was cloned into murine Fc and characterized with a binding affinity of 10nM. The antibody was used to detect RD114 and its variant envelopes on different stable viral packaging cell lines (FLYRD18 and WinPac-RD). 22F10 was also shown to prevent the infections of different strains of RD-pseudotyped vectors but not related envelope glycoproteins by blocking cell surface receptor binding. We are the first to report the neutralization of viral particles by a monoclonal αRD114 antibody

A Recoding Method to Improve the Humoral Immune Response to an HIV DNA Vaccine

This manuscript describes a novel strategy to improve HIV DNA vaccine design. Employing a new information theory based bioinformatic algorithm, we identify a set of nucleotide motifs which are common in the coding region of HIV, but are under-represented in genes that are highly expressed in the human genome. We hypothesize that these motifs contribute to the poor protein expression of gag, pol, and env genes from the c-DNAs of HIV clinical isolates. Using this approach and beginning with a codon optimized consensus gag gene, we recode the nucleotide sequence so as to remove these motifs without modifying the amino acid sequence. Transfecting the recoded DNA sequence into a human kidney cell line results in doubling the gag protein expression level compared to the codon optimized version. We then turn both sequences into DNA vaccines and compare induced antibody response in a murine model. Our sequence, which has the motifs removed, induces a five-fold increase in gag antibody response compared to the codon optimized vaccine

The A-rich RNA sequences of HIV-1 pol are important for the synthesis of viral cDNA

The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich ‘structurally poor’ RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5–100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using ‘structurally poor’ RNA domains in regulating biological process

Differential Trends in the Codon Usage Patterns in HIV-1 Genes

Host-pathogen interactions underlie one of the most complex evolutionary phenomena resulting in continual adaptive genetic changes, where pathogens exploit the host's molecular resources for growth and survival, while hosts try to eliminate the pathogen. Deciphering the molecular basis of host–pathogen interactions is useful in understanding the factors governing pathogen evolution and disease propagation. In host-pathogen context, a balance between mutation, selection, and genetic drift is known to maintain codon bias in both organisms. Studies revealing determinants of the bias and its dynamics are central to the understanding of host-pathogen evolution. We considered the Human Immunodeficiency Virus (HIV) type 1 and its human host to search for evolutionary signatures in the viral genome. Positive selection is known to dominate intra-host evolution of HIV-1, whereas high genetic variability underlies the belief that neutral processes drive inter-host differences. In this study, we analyze the codon usage patterns of HIV-1 genomes across all subtypes and clades sequenced over a period of 23 years. We show presence of unique temporal correlations in the codon bias of three HIV-1 genes illustrating differential adaptation of the HIV-1 genes towards the host preferred codons. Our results point towards gene-specific translational selection to be an important force driving the evolution of HIV-1 at the population level

IFITM proteins inhibit HIV-1 protein synthesis

Interferon induced transmembrane proteins (IFITMs) inhibit the cellular entry of a broad range of viruses, but it has been suspected that for HIV-1 IFITMs may also inhibit a post-integration replicative step. We show that IFITM expression reduces HIV-1 viral protein synthesis by preferentially excluding viral mRNA transcripts from translation and thereby restricts viral production. Codon-optimization of proviral DNA rescues viral translation, implying that IFITM-mediated restriction requires recognition of viral RNA elements. In addition, we find that expression of the viral accessory protein Nef can help overcome the IFITM-mediated inhibition of virus production. Our studies identify a novel role for IFITMs in inhibiting HIV replication at the level of translation, but show that the effects can be overcome by the lentiviral protein Nef.Wellcome Trust-University of Edinburgh Institutional Strategic Support Fun

Effects of early administration of atorvastatin treatment on thrombotic process in normocholesterolemic patients with unstable angina.

BACKGROUND: Although statin-treatment during the acute phase of unstable coronary syndromes improve the outcome their effects on thrombosis/fibrinolysis system in normocholesterolemic patients admitted with unstable angina remain obscure. We assessed the effects of short-term atorvastatin treatment on thrombotic/fibrinolysis markers in normocholesterolemic in patients with unstable angina. METHODS: Forty-five patients with unstable angina were allocated into two groups to receive atorvastatin 10 mg/day (n = 24) or no statin (n = 21) for 6 weeks. Circulating levels of von Willebrand Factor (vWF), factor V (fV), protein C (prC), tissue plasminogen activator (tPA) and antithrombin III (ATIII) were measured by enzyme linked immunosorbent assay, by the patients admission and at the 1st and 6th week of the study. RESULTS: After 1 week of treatment, a significant increase of ATIII (p < 0.05), fV (p < 0.01) and vWF (p < 0.05) was found in the control group, but not in atorvastatin-treated group. Similarly, at 6 weeks after admission, plasma levels of ATIII were still significantly higher than at baseline in controls (p < 0.05), but not in atorvastatin-treated group. Plasma levels of PrtC were significantly increased in both controls (p < 0.01) and atorvastatin-treated patients (p < 0.05) at 1 week, while remained unaffected in atorvastatin-treated group at 6th week. There was no significant difference in the variations of plasma levels of tPA, PrtS and fVII between the two groups at 1 and 6 weeks after admission. CONCLUSIONS: In normocholesterolemic patients admitted with unstable angina the early administration of atorvastatin, significantly affects von Willebrand factor levels and the expression of liver-derived components of both thrombosis and fibrinolysis system

Effects of early administration of atorvastatin treatment on thrombotic process in normocholesterolemic patients with unstable angina

Background: Although statin-treatment during the acute phase of unstable coronary syndromes improve the outcome their effects on thrombosis/fibrinolysis system in normocholesterolemic patients admitted with unstable angina remain obscure. We assessed the effects of short-term atorvastatin treatment on thrombotic/fibrinolysis markers in normocholesterolemic in patients with unstable angina. Methods: Forty-five patients with unstable angina were allocated into two groups to receive atorvastatin 10 mg/day (n=24) or no statin (n =21) for 6 weeks. Circulating levels of von Willebrand Factor (vWF), factor V (W), protein C (prC), tissue plasminogen activator (tPA) and antithrombin III (ATIII) were measured by enzyme linked immunosorbent assay, by the patients admission and at the 1st and 6th week of the study. Results: After I week of treatment, a significant increase of ATIII (p < 0.05), fV (p < 0.01) and vWF (p < 0.05) was found in the control group, but not in atorvastatin-treated group. Similarly, at 6 weeks after admission, plasma levels of ATIII were still significantly higher than at baseline in controls (p < 0.05), but not in atorvastatin-treated group. Plasma levels of PrtC were significantly increased in both controls (p < 0.01) and atorvastatin-treated patients (p < 0.05) at I week, while remained unaffected in atorvastatin-treated group at 6th week. There was no significant difference in the variations of plasma levels of tPA, PrtS and fVlI between the two groups at 1 and 6 weeks after admission. Conclusions: In normocholesterolemic patients admitted with unstable angina the early administration of atonastatin, significantly affects von Willebrand factor levels and the expression of liver-derived components of both thrombosis and fibrinolysis system. (c) 2005 Elsevier Ireland Ltd. All rights reserved