Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 pg of purified DNA in saline, 67-95% of test mice and inoculations were undertaken (i) by a route that supports unusually efficient transfection (muscle), (ii) by routes that support less efficient transfection but represent routes frequently used for the administration of an antigen to a test animal (subcutaneous, intraperitoneal), and (iii) by routes that support less efficient transfection but deliver DNA to tissues with high levels of local immune surveillance (skin and respiratory passages).The effect of the route of inoculation on DNA vaccination was evaluated in murine and avian influenza virus models. In both models, the vaccine consisted of purified plasmid DNA that had been designed to express an influenza virus hemagglutinin glycoprotein. This glycoprotein mediates adsorption and penetration of virus and represents a major target for neutralizing antibody (16, 17). A number of antigenically distinct subtypes of hemagglutinin glycoproteins are found in naturally occurring influenza virus infections (18). In the murine model, plasmid DNA expressing the hemagglutinin subtype 1 (Hi) protein was used to protect against a lethal challenge with a mouse-adapted influenza virus with an identical Hi gene. In the chicken model, DNA expressing the hemagglutinin subtype 7 (H7) protein was used to vaccinate against a lethal H7 virus with an antigenically distinct H7 glycoprotein (19 §To whom reprint requests should be addressed.11478
In Bacillus subtilis, competence for transformation develops in 5-10% of the cells in a stationary phase culture. These cells exhibit a prolonged lag in the resumption of growth and cell division during the escape from competence. To better understand the basis of this lag, we have characterized competent cultures microscopically. To distinguish the minority of competent cells, a translational fusion between ComK, the competence transcription factor, and the green fluorescent protein (GFP) was used as a marker. Only 5-10% of the cells in a competent culture were fluorescent, indicating that ComK synthesis is an all or nothing event. To validate the identification of competent cells, we demonstrated the coincident expression of comEA, a late competence gene, and comK-gfp. Competent cells resemble stationary phase cells; the majority are single (not in chains), contain single nucleoids, and rarely contain FtsZ rings. Upon dilution into fresh medium, competent cells maintain this appearance for about 2 h. In contrast, the majority of non-competent cells rapidly resume growth, exhibiting chaining, nuclear division and FtsZ-ring formation. The late competence protein ComGA is required for the competence-related block in chromosome replication and cell division. In the competent cells of a comGA mutant culture, chromosomal replication and FtsZ-ring formation were no longer blocked, although competent comGA mutant cells were abnormal in appearance. It is likely that one role for ComGA is to prevent growth, chromosome replication and cell division until ComK can be eliminated by degradation. A mutation in the ATP-binding site of comGA inactivated the protein for transformation but did not prevent it from inhibiting DNA replication and cell division. The buoyant density difference between competent and non-competent cells depends on the competence-specific growth arrest.
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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