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Endpoint immunoglobulin G (IgG) titers and cytotoxic T-lymphocyte (CTL) activities were identical between mice immunized via the intramuscular and epidermal (gene gun) routes with 100 and 1 g, respectively, of an influenza virus nucleoprotein (NP) expression vector. However, examination of the relative levels of two IgG subclasses demonstrated that muscle inoculation resulted in predominantly IgG2a responses, whereas gene gun immunization yielded a preponderance of IgG1 antibodies. Inasmuch as these data suggested that muscle inoculation and gene gun delivery elicited Th1-like and Th2-like responses, respectively, gamma interferon release profiles from antigen-stimulated splenocytes were remarkably similar between these groups. Interleukin-4 (IL-4) production assays, on the other hand, revealed qualitative differences that could be correlated with the divergent IgG subclass data. Waning gamma interferon production in gene gun-immunized animals was countered by a marked increase in IL-4 production following the third immunization, as was the case in control animals immunized with inactivated influenza virus formulated with Freund's adjuvant. In contrast, significant levels of IL-4 production were not observed in the intramuscular DNA inoculation group, despite similar decreases in gamma interferon production with increasing immunizations. These data show that intramuscular inoculation leads to Th1-like responses due to elevated IgG2a levels, production of gamma interferon, CTL activity, and lack of IL-4. However, gene gun responses are more difficult to categorize because of the presence of significant gamma interferon and CTL activity on the one hand and elevated IgG1 antibodies and increasing IL-4 production with successive immunizations on the other. In addition, there was a lack of correlation between IgG isotype ratios and cytokine production in all of the NP DNA-immunized animals, in that IgG subclass ratios remained fixed while cytokine production patterns fluctuated with successive immunizations. These data are consistent with the idea that the types of responses elicited following DNA immunization are dependent on both the identity of the antigen and the route of DNA administration.

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Maternal antibody is the major form of protection from disease in early life when the neonatal immune system is still immature; however, the presence of maternal antibody also interferes with active immunization, placing infants at risk for severe bacterial and viral infection. We tested the ability of intramuscular and gene gun immunization with DNA expressing influenza virus hemagglutinin (HA) and nucleoprotein (NP) to raise protective humoral and cellular responses in the presence or absence of maternal antibody. Neonatal mice born to influenza virus-immune mothers raised full antibody responses to NP but failed to generate antibody responses to HA. In contrast, the presence of maternal antibody did not affect the generation of long-lived CD8 ؉ T-cell responses to both HA and NP. Thus, maternal antibody did not affect cell-mediated responses but did affect humoral responses, with the ability to limit the antibody response correlating with whether the DNA-expressed immunogen was localized in the plasma membrane or within the cell.Neonates are deficient in several components of inflammatory, innate, and specific immune responses. The presence of high-titer maternal antibody in newborns is the major form of protection from disease in early life. Maternal immunoglobulin G (IgG) crosses the placenta from mother to fetus during development (12) and typically exceeds titers of the same antibody in the mother. This passive antibody slowly declines over the first year of life, a period during which the infant's immune system matures, becomes more experienced, and develops its own repertoire of protective memory immune responses. However, maternal antibody can also interfere with active immunization of the offspring (1). Immunization protocols are often delayed several months and/or require multiple booster immunizations to achieve the desired protective immune response. Thus, a window of time exists when maternal antibody levels are too low to reliably protect an infant from infectious disease but are high enough to prevent responses to vaccines.DNA vaccination is an attractive method for immunization in the presence of maternal antibody. Maternal antibody is thought to interfere with traditional vaccine efficacy by reducing the amount of antigen available for processing and presentation by antigen-presenting cells. The ability of DNA vaccines to directly transfect cells bypasses this problem. The maternal antibody will not inhibit the DNA vaccine itself because antigen is not available until de novo synthesis occurs. Both DNA and subsequent antigen expression persists for several weeks (4, 6). Thus, DNA-raised immune responses could occur as maternal antibody titers wane. Some groups have reported success following neonatal DNA immunization in the presence of maternal antibody (14), while others have failed (11,15,21,25).We have previously shown that intramuscular (i.m.) and gene gun (g.g.) immunization of mice as neonates or adults with an influenza hemagglutinin (HA)-expressing DNA generates long-lasting protectiv...

Neonatal mice have immature immune systems with defects in several components of inflammatory, innate, and specific immune responses and develop a preferential T helper type 2 response following immunization with many vaccine antigens. These studies were undertaken to determine whether 1-day-old neonatal mice immunized with plasmid DNA expressing influenza A/PR/8/34 hemagglutinin (H1) by either intramuscular (im) or gene gun (gg) inoculation were capable of generating humoral responses comparable to those in mice immunized as adults. The newborn mice developed stable, long-lived, protective anti-H1-specific IgG responses similar in titer to those of adult DNA-immunized mice. However, unlike the adult im and gg DNA immunizations, which develop polarized IgG2a and IgG1 responses, respectively, mice immunized as neonates developed a variety of IgG1, IgG2a, and mixed IgG1/IgG2a responses regardless of the inoculation method. Boosting increased but did not change these antibody profiles. In contrast to the DNA immunizations, inoculations of newborn mice with an A/PR/8/34 viral protein subunit preparation failed to elicit an antibody response. Temporal studies revealed that both responsiveness to protein vaccination and development of polarized patterns of T help following DNA immunization appeared by 2 weeks of age.