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

• Published in: Virology (New York, N.Y.) N.Y.), 2004-06, Vol.323 (2), p.220-232
• Main Authors: Chikhlikar, Priya, Barros de Arruda, Luciana, Agrawal, Shikha, Byrne, Barry, Guggino, William, August, J.Thomas, Marques, Ernesto T.A
• Format: Article
• Language: English
• Subjects: AAV; Animals; Antibodies, Viral - blood; CD4; CD4-Positive T-Lymphocytes - immunology; CD8; CD8-Positive T-Lymphocytes - immunology; Cell Line; Dependovirus - genetics; DNA vaccine; Female; Gene Products, gag - genetics; Gene Products, gag - immunology; Genetic Vectors; HIV-1 Gag; Humans; Immunization Schedule; Inverted terminal repeat; LAMP; Lysosomal Membrane Proteins; Membrane Glycoproteins - genetics; Membrane Glycoproteins - immunology; MHC II; Mice; Mice, Inbred BALB C; Plasmids; Protein Precursors - genetics; Protein Precursors - immunology; rAAV; Recombinant Fusion Proteins - immunology; Terminal Repeat Sequences - immunology; Vaccines, DNA - administration & dosage; Vaccines, DNA - immunology
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1016/j.virol.2004.02.025

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.