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6 Generating Novel HIV-1 Envelope Vaccine Candidates Using DNA Shuffling Technology
L. Xu1, W. Zhang1, X. Du1, D. R. Burton2, P. W. H. I. Parren2, and R. G. Whalen*1
1Maxygen, Inc., Redwood City, CA, USA and 2The Scripps Res. Inst., La Jolla, CA, USA
Background: DNA shuffling captures the power of genetic recombination in a simple test-tube format. Typically, homologous genes are used to create large libraries of highly chimeric sequences encoding proteins that exhibit novel and improved biological properties. This technology has been used to improve enzymes, antibodies, viral antigens, and cytokines. By shuffling the antigen coding sequences in a DNA vaccine for hepatitis B surface antigen, we obtained a 10- to 25-fold increase in immunogenicity in mice. Currently, we are applying DNA shuffling to create novel HIV-1 envelope genes and proteins for testing as HIV vaccines.
Methods: Gp160 genes from HIV-1 clade B primary isolates were used in DNA shuffling reactions to generate recombinant libraries with or without constrained cysteine residues between gp120 and gp41. Protein expression from the shuffled clones was verified using immunostaining of transfected cells with a mouse anti-gp160 polyclonal antibody. Surface expression was assessed using monoclonal antibodies and flow cytometry. The envelope-expressing DNA clones were used to directly immunize mice, and the sera were screened for reactivity with rgp160 in ELISA and for HIV-1 neutralizing activity in vitro.
Results: Partial DNA sequence analyses of shuffled clones indicated that recombination had occurred among the parental gp160 genes. All clones were highly chimeric. A number of the shuffled clones showed improved immunogenicity compared with the parental clones.
Conclusions: DNA shuffling of gp160 genes resulted in enhanced immunogenicity of the corresponding DNA vaccines compared with the parental genes. The application of DNA shuffling to HIV vaccine development provides a new approach to generate novel envelope structures, potentially with the ability to induce broadly cross-reactive neutralizing antibodies against primary isolates of HIV.
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