Immune responses in baboons vaccinated with HIV-2 genetic expression libraries.

Immunization using genetic expression libraries may be an improvement over conventional DNA immunization using a single gene because more epitopes are simultaneously presented to the immune system. In this study, we evaluated the effectiveness of an HIV-2 vaccine made from a genomic expression library in baboons. We found that HIV-2 expression library immunization induced HIV-2-specific memory responses but low levels of CD8+ cell anti-viral responses and neutralizing antibodies. After intravenous virus challenge using a homologous pathogenic variant, HIV-2UC2/9429, viral loads were similar in the HIV-2-immunized and control baboons. We conclude that although immunization using HIV-2 expression libraries induces immune responses, this approach does not provide protection in baboons against intravenous challenge with HIV-2.

Genetic live vaccines mimic the antigenicity but not pathogenicity of live viruses.

The development of an effective HIV vaccine is both a pressing and a formidable problem. The most encouraging results to date have been achieved using live-attenuated immunodeficiency viruses. However, the frequency of pathogenic breakthroughs has been a deterrent to their development. We suggest that expression libraries generated from viral DNA can produce the immunologic advantages of live vaccines without risk of reversion to pathogenic viruses. The plasmid libraries could be deconvoluted into useful components or administered as complex mixtures. To explore this approach, we designed and tested several of these genetic live vaccines (GLVs) for HIV. We constructed libraries by cloning overlapping fragments of the proviral genome into mammalian expression plasmids, then used them to immunize mice. We found that inserting library fragments into a vector downstream of a secretory gene sequence led to augmented antibody responses, and insertion downstream of a ubiquitin sequence enhanced cytotoxic lymphocyte responses. Also, fragmentation of gag into subgenes broadened T-cell epitope recognition. We have fragmented the genome by sequence-directed and random methods to create libraries with different features. We propose that the characteristics of GLVs support their further investigation as an approach to protection against HIV and other viral pathogens.

Linear expression elements: a rapid, in vivo, method to screen for gene functions.

The increasing accumulation of genomic sequence information has accentuated the need for new methods to efficiently assess gene function and to prepare reagents to study these functions. Toward solving this general problem in functional genomics, we report a method by which any PCR-amplified open-reading frame (ORF) can be noncovalently linked to a eukaryotic promoter and terminator, and directly injected into animals to produce local gene expression. We also demonstrate that ORFs can be delivered into mice to produce antibodies specific for the encoded foreign protein by simply attaching mammalian promoter and terminator sequences. This technology makes it possible to screen large numbers of genes rapidly for their functions in vivo or to produce immune responses without the necessity of cloning, bacterial propagation, or protein purification.