ORFDB: an information resource linking scientific content to a high-quality Open Reading Frame (ORF) collection.

The ORFDB (http://orf.invitrogen.com/) represents an ongoing effort at Invitrogen Corporation to integrate relevant scientific data with an evolving collection of human and mouse Open Reading Frame (ORF) clones (Ultimate ORF Clones). The ORFDB serves as a central data warehouse enabling researchers to search the ORF collection through its web portal ORFBrowser, allowing researchers to find the Ultimate ORF clones by blast, keyword, GenBank accession, gene symbol, clone ID, Unigene ID, LocusLink ID or through functional relationships by browsing the collection via the Gene Ontology (GO) Browser. As of October 2003, the ORFDB contains 6200 human and 2870 mouse Ultimate ORF clones. All Ultimate ORF clones have been fully sequenced with high quality, and are matched to public reference protein sequences. In addition, the cloned ORFs have been extensively annotated across six categories: Gene, ORF, Clone Format, Protein, SNP and Genomic links, with the information assembled in a format termed the ORFCard. The ORFCard represents an information repository that documents the sequence quality, alignment with respect to public protein sequences, and the latest publicly available information associated with each human and mouse gene represented in the collection.

Immunogenicity of HIV-1 IIIB and SHIV 89.6P Tat and Tat toxoids in rhesus macaques: induction of humoral and cellular immune responses.

This study compared immune responses in rhesus macaques immunized with unmodified HIV-1 IIIB Tat, SHIV89.6P Tat, and carboxymethylated IIIB and 89.6P Tat toxoids. Immunization with either IIIB or 89.6P preparation induced high titer and broadly crossreactive serum anti-Tat IgG that recognized HIV-1 subtype-E and SIVmac251 Tat. However, the response was delayed, and titers were lower in 89.6P vaccination groups. Serum anti-Tat IgG recognized peptides corresponding to the amino-terminus, basic domain, and carboxy-terminal region. Cellular proliferative responses to Tat toxoids corresponding to the immunogen were evident in vitro in both IIIB and 89.6P groups. Crossreactive proliferative responses were observed in IIIB groups in response to stimulation with 89.6P or SIVmac251 Tat toxoids, but were much less prevalent in 89.6P groups. The truncated 86 amino acid IIIB Tat appears to be more immunogenic than the 102 amino acid 89.6P Tat with respect to both humoral and cellular immune responses, and may be a better vaccine component. Despite induction of robust humoral and cellular immune responses (including both CD4+ and CD8+ T-cell responses) to Tat, all animals were infected upon intravenous challenge with 30 MID(50) of SHIV89.6P and outcome of vaccine groups was not different from controls. Sequencing both Tat exons from serum viral RNA revealed no evidence of escape mutants. These results suggest that with intravenous SHIV89.6P challenge in rhesus macaques, precipitous CD4+ T-cell decline overwhelms potentially protective immune responses. Alternatively, Tat specific CD8+ T-cell responses may not appropriately recognize infected cells in vivo in this model. In view of evidence demonstrating Tat specific CTLs in the SIV model and in humans infected with HIV-1, results in this pathogenic SHIV model may not apparently predict the efficacy of this approach in human studies. The potency and cross-reactivity of these immune responses confirm Tat toxoid as an excellent candidate vaccine component.

Antibodies to Tat and Vpr in the GRIV cohort: differential association with maintenance of long-term non-progression status in HIV-1 infection.

The HIV-1 regulatory protein Tat and the accessory protein Vpr are thought to stimulate viral replication and contribute to viral pathogenesis as extracellular proteins. Humoral immune responses to these early viral proteins may therefore be beneficial. We examined serum anti-Tat and anti-Vpr IgG by ELISA in the GRIV cohort of HIV-1 seropositive slow/non-progressors (NP) and fast-progressors (FP), and in seronegative controls. Based on information obtained during a brief follow-up period (median = 20 months), NPs were sub-grouped as those maintaining non-progression status and therefore stable (NP-S), and those showing signs of disease progression (NP-P). As the primary comparison, initial serum anti-Tat and anti-Vpr IgG (prior to follow-up) were analyzed in the NP sub-groups and in FPs. Anti-Tat IgG was significantly higher in stable NP-S compared to unstable NP-P (P = 0.047) and FPs (P < 0.0005); the predictive value of higher anti-Tat IgG for maintenance of non-progression status was 92% (P = 0.029). In contrast, no-difference was observed in anti-Vpr IgG between NP-S and NP-P, although both were significantly higher than FPs (P

Outcome of simian-human immunodeficiency virus strain 89.6p challenge following vaccination of rhesus macaques with human immunodeficiency virus Tat protein.

The regulatory proteins Nef, Rev, and Tat of human immunodeficiency virus type 1 (HIV-1) are attractive targets for vaccine development, since induction of effective immune responses targeting these early proteins may best control virus replication. Here we investigated whether vaccination with biologically active Tat or inactive Tat toxoid derived from HIV-1(IIIB) and simian-human immunodeficiency virus (SHIV) strain 89.6p would induce protective immunity in rhesus macaques. Vaccination induced high titers of anti-Tat immunoglobulin G in all immunized animals by week 7, but titers were somewhat lower in the 89.6p Tat group. Dominant B-cell epitopes mapped to the amino terminus, the basic domain, and the carboxy-terminal region. Tat-specific T-helper responses were detected in 50% of immunized animals. T-cell epitopes appeared to map within amino acids (aa) 1 to 24 and aa 37 to 66. In addition, Tat-specific gamma interferon responses were detected in CD4+ and/or CD8+ T lymphocytes in 11 of 16 immunized animals on the day of challenge. However, all animals became infected upon intravenous challenge with 30 50% minimal infective doses of SHIV 89.6p, and there were no significant differences in viral loads or CD4+ T-cell counts between immunized and control animals. Thus, vaccination with HIV-1(IIIB) or SHIV 89.6p Tat or with Tat toxoid preparations failed to confer protection against SHIV 89.6p infection despite robust Tat-specific humoral and cellular immune responses in some animals. Given its apparent immunogenicity, Tat may be more effective as a component of a cocktail vaccine in combination with other regulatory and/or structural proteins of HIV-1.