Effect of CpG methylation on isotype and magnitude of antibody responses to influenza hemagglutinin-expressing plasmid

We previously showed that intramuscular saline DNA immunizations favor the development of an IgG2a-dominant Th1 immune response, whereas gene gun DNA immunizations stimulate the production of an IgG1-dominant Th2 immune respo nse. Several studies have implicated immunostimulatory CpG sequences as the causative factor in the development of Th1 immune responses to saline DNA immunization. To determine whether the Th1 cytokine-inducing properties of CpG sequences in plasmid DNA (pDNA) were responsible for the induction of a Th1 immune response, in vitro methylated and untreated (nonmethylated) hem agglutinin-expressing pDNA were compared for immunogenicity. Methylation ab rogated the immunostimulatory activity of pDNA for cultured splenocytes and significantly reduced antigen expression. However, methylation of pDNA was not associated with a change from the induction of IgG2a to IgG1, After im munization with the methylated plasmid, the magnitude of the immune respons e was reduced. However, the decline in the total antibody response matched the decline in antigen expression. The dose of DNA or the presence of lipop olysaccharide in pDNA likewise did not affect the preferential development of an IgG2a antibody response, Our findings reveal that high levels of CpG sequences are not required for raising IgG2a-predominant, Th1-biased immune responses to intramuscular injections of hemagglutinin-expressing DNA.