Recombinant HIV-1 vaccine candidates based on replication-defective flavivirus vector.

Multiple approaches utilizing viral and DNA vectors have shown promise in the development of an effective vaccine against HIV. In this study, an alternative replication-defective flavivirus vector, RepliVax (RV), was evaluated for the delivery of HIV-1 immunogens. Recombinant RV-HIV viruses were engineered to stably express clade C virus Gag and Env (gp120TM) proteins and propagated in Vero helper cells. RV-based vectors enabled efficient expression and correct maturation of Gag and gp120TM proteins, were apathogenic in a sensitive suckling mouse neurovirulence test, and were similar in immunogenicity to recombinant poxvirus NYVAC-HIV vectors in homologous or heterologous prime-boost combinations in mice. In a pilot NHP study, immunogenicity of RV-HIV viruses used as a prime or boost for DNA or NYVAC candidates was compared to a DNA prime/NYVAC boost benchmark scheme when administered together with adjuvanted gp120 protein. Similar neutralizing antibody titers, binding IgG titers measured against a broad panel of Env and Gag antigens, and ADCC responses were observed in the groups throughout the course of the study, and T cell responses were elicited. The entire data demonstrate that RV vectors have the potential as novel HIV-1 vaccine components for use in combination with other promising candidates to develop new effective vaccination strategies.

In men at risk of HIV infection, IgM, IgG1, IgG3, and IgA reach the human foreskin epidermis.

We profiled the humoral response in the penis, an area that has been minimally explored but may be relevant for protecting insertive men against HIV and other sexually acquired infections. Comparing paired tissue samples from 20 men at risk of HIV infection, foreskin contains less immunoglobulin A (IgA) and more IgG2 than colon. Using foreskin dermal and epidermal explants and paired plasma from 17 men, we examined Ig accumulation by normalizing Ig to human serum albumin (HSA) transudation. Dermal IgM, IgG2, IgA, and IgE ratios were greater than that in plasma, suggesting there is local antibody secretion at the dermis. Local Ig transcription was concentrated at the inner rather than the outer foreskin, and inner foreskin Ig ratios did not correlate with blood, indicating that localized production can contribute to the foreskin response. IgM, IgG1, IgG3, and IgA have preferential access to the foreskin epidermis, whereas IgG2, IgG4, and IgE are restricted to the dermis. Lastly, Ad5-specific IgA was selectively present in the colon, whereas foreskin Ad5 IgG was mainly derived from blood, and reached the inner epidermis at higher ratios than the outer (P<0.002). In summary, the foreskin antibody response combines local and systemic sources, and there is selective isotype accumulation in the epidermis.

A mammalian sequence-dependent upstream open reading frame mediates polyamine-regulated translation in yeast.

In mammals, control of S-adenosylmethionine decarboxylase (AdoMetDC) translation is one component of a feedback network that regulates intracellular levels of the polyamines, spermidine, and spermine. AdoMetDC mRNA from mammals contains a highly conserved upstream open reading frame (uORF) within its leader sequence that confers polyamine-regulated suppression of translation on the associated downstream cistron. This regulation is mediated through an interaction that depends on the amino acid sequence of the uORF-encoded hexapeptide. It remains to be shown whether polyamines participate directly in this interaction or indirectly through a specialized signal transduction pathway. We show that Saccharomyces cerevisiae does not have a uORF associated with its AdoMetDC gene (SPE2) and that ribosome loading on the SPE2 mRNA is not positively influenced by polyamine depletion, as it is in mammalian cells. Nevertheless, the mammalian AdoMetDC uORF, when introduced into a polyamine auxotroph of yeast, conferred polyamine regulation of both translational efficiency and ribosome loading on the associated mRNA. This regulatory activity depended on the amino acid sequence encoded by the fourth and fifth codons of the uORF, as in mammalian cells. The fact that the regulatory properties of this mammalian translational control element are quite similar in both mammalian and yeast cells suggests that a specialized signal transduction pathway is not required. Rather, it seems likely that polyamines may be directly participating in an interaction between the uORF-encoded peptide and a constitutive component of the translation machinery, which leads to inhibition of ribosome activity.

In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from the S-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism.

Role of two upstream open reading frames in the translational control of oncogene mdm2.

Overexpression of oncoprotein MDM2 has been found in a significant number of human soft tissue tumors. In a subset of these tumors, overexpression is a result of enhanced translation of mdm2 mRNA. There are two transcripts from the mdm2 gene that differ only in their 5' leaders: a long form (L-mdm2) and a short form (S-mdm2) that arise from the use of different promoters. L-mdm2 mRNA contains two upstream open reading frames (uORFs) and this mRNA was loaded with ribosomes inefficiently in comparison with S-mdm2. The 5' leader of L-mdm2 was sufficient to transfer translational repression to a reporter gene and the two uORFs acted synergistically to achieve full suppression. In contrast, the 5' leader of S-mdm2 allowed efficient translation of an attached reporter gene in the tumor cells. These results are consistent with a model in which overexpression of MDM2 in certain tumors results from a change in mRNA structure due to a switch in promoter usage.

Direct analysis of protein complexes using mass spectrometry.

We describe a rapid, sensitive process for comprehensively identifying proteins in macromolecular complexes that uses multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) to separate and fragment peptides. The SEQUEST algorithm, relying upon translated genomic sequences, infers amino acid sequences from the fragment ions. The method was applied to the Saccharomyces cerevisiae ribosome leading to the identification of a novel protein component of the yeast and human 40S subunit. By offering the ability to identify >100 proteins in a single run, this process enables components in even the largest macromolecular complexes to be analyzed comprehensively.

The inhibitory upstream open reading frame from mammalian S-adenosylmethionine decarboxylase mRNA has a strict sequence specificity in critical positions.

The upstream open reading frame (uORF) in the 5' leader of the mammalian mRNA encoding S-adenosylmethionine decarboxylase (AdoMetDC) serves as a negative regulatory element by suppressing translation of the associated downstream cistron. Certain changes in the amino acid sequence of the hexapeptide (sequence MAGDIS) encoded by the uORF destroy suppressive activity, implying specific interaction with a cellular target. In this paper, we examine the extent of alterations that can be tolerated in this uORF. The mammalian AdoMetDC uORF inhibits downstream translation when placed into the 5' leader of a yeast mRNA with characteristics resembling those in mammalian cells, suggesting that the encoded peptide has a similar target across species. Using yeast for the initial screen, we tested the specificity of the critical three codons at the 3' end of the uORF by saturation mutagenesis. Altered uORFs selected from the primary yeast screen were then retested in mammalian cells. The requirements at codons 4 and 5 were quite stringent; only aspartic acid at codon 4 yielded a fully suppressive peptide, and only valine could substitute productively for isoleucine at codon 5. The specificity at codon 6 was much looser, with many substitutions retaining suppressive activity in both yeast and mammalian cells.

Transcription factor ZBP-89 regulates the activity of the ornithine decarboxylase promoter.

Appropriate cellular levels of polyamines are required for cell growth and differentiation. Ornithine decarboxylase is a key regulatory enzyme in the biosynthesis of polyamines, and precise regulation of the expression of this enzyme is required, according to cellular growth state. A variety of mitogens increase the level of ornithine decarboxylase activity, and, in most cases, this elevation is due to increased levels of mRNA. A GC box in the proximal promoter of the ornithine decarboxylase gene is required for basal and induced transcriptional activity, and two proteins, Sp1 and NF-ODC1, bind to this region in a mutually exclusive manner. Using a yeast one-hybrid screening method, ZBP-89, a DNA-binding protein, was identified as a candidate for the protein responsible for NF-ODC1 binding activity. Three lines of evidence verified this identification; ZBP-89 copurified with NF-ODC1 binding activity, ZBP-89 antibodies specifically abolished NF-ODC1 binding to the GC box, and binding affinities of 12 different double-stranded oligonucleotides were indistinguishable between NF-ODC1, in nuclear extract, and in vitro translated ZBP-89. ZBP-89 inhibited the activation of the ornithine decarboxylase promoter by Sp1 in Schneider's Drosophila line 2, consistent with properties previously attributed to NF-ODC1.

Differential regulation of proto-oncogenes c-jun and c-fos in T lymphocytes activated through CD28.

The T cell surface molecule CD28 binds to ligands on accessory cells and APCs, playing an important costimulatory role in the response of T cells to Ags. Our knowledge of the intracellular signaling pathways coupled to this receptor is incomplete. In addition to activation of phospholipase C gamma 1, ligation of this receptor also seems to activate a calcium-independent, CD28-specific pathway. In this paper, we report that cross-linking of CD28 (but not CD2, CD5, LFA-1, or CD7) leads to an elevation of c-jun mRNA, with only minimal activation of c-fos expression. CD28-dependent induction of c-jun expression requires protein tyrosine kinase activity, but does not depend on activation of a phorbol ester-responsive protein kinase C or elevation of cytosolic calcium. Furthermore, CD28-dependent elevation of c-jun mRNA does not appear to be mediated at the level of mRNA stability. A mechanism is suggested whereby expression of c-jun and junB, in the absence of members of the fos family, can prevent inappropriate activation of T cells caused by ligation of CD28 in the absence of a specific antigenic stimulus.