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. 2004 Jun 1;323(2):220-32.
doi: 10.1016/j.virol.2004.02.025.

Inverted terminal repeat sequences of adeno-associated virus enhance the antibody and CD8(+) responses to a HIV-1 p55Gag/LAMP DNA vaccine chimera

Priya Chikhlikar  1 , Luciana Barros de Arruda, Shikha Agrawal, Barry Byrne, William Guggino, J Thomas August, Ernesto T A Marques Jr
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Free article

Inverted terminal repeat sequences of adeno-associated virus enhance the antibody and CD8(+) responses to a HIV-1 p55Gag/LAMP DNA vaccine chimera

Priya Chikhlikar et al. Virology. .
Free article

Abstract

The immune responses to an HIV-1 p55Gag vaccine encoded as a DNA chimera with the lysosomal associated membrane protein-1 (LAMP) have been examined for the effect of the addition of the inverted terminal repeat (ITR) sequences of the adeno-associated virus (AAV) to the DNA plasmid construct, and of packaging the LAMP/gag gene as a recombinant AAV vector (rAAV). DNA plasmids encoding Gag and the LAMP/Gag protein chimera were constructed in two vectors, the pcDNA3.1 and a corresponding plasmid containing the ITR sequences (pITR) flanking the expression elements of the plasmid, and the pITR LAMP/gag DNA plasmid was encapsidated in the rAAV vector. Human 293 cells transfected in vitro with LAMP/gag plasmids either in pcDNA3.1 or pITR produced much Gag protein in cell extracts (1.6 and 2.2 ng of Gag/mg of protein, respectively). The immune responses of mice to immunization with these constructs were examined under three protocols: DNA prime/DNA boost, DNA prime/rAAV boost, and a single rAAV immunization. The results demonstrated that under DNA prime/DNA boost protocol, the "naked" DNA vaccines encoding the LAMP/gag chimera, either as pcDNA3.1 or pITR DNA plasmid constructs, elicited strong CD4(+) T cell responses. In contrast, significantly higher levels of CD8(+) and antibody responses were observed with the pITR-DNA constructs. Immunization with the rAAV vector under the DNA prime/rAAV boost protocol resulted in sustained T cell responses and a markedly increased antibody response, predominantly of the IgG(1) isotype resulting from the activation of the Th2 subset of CD4(+) T cells, that was sustained for at least 5 months after immunization.

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