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The ability to induce a protective response against Helicobacter pylori infection has been investigated by systemic immunization of mice with urease formulated with the cationic lipid DC Chol. This compound acts both as a formulating agent and as an adjuvant and induces a balanced Th1/Th2 response shown to be more effective for protection in our previous studies. Urease-DC Chol induced a significant protection in prophylaxis but not in therapeutic immunization. The protection level was between 1.5 and 2 log reduction of bacterial density measured by quantitative culture compared to unimmunized-infected mice. In parallel, the protective efficacy of other H. pylori antigens formulated in a similar way and administered with DC Chol was tested. These antigens were tested alone or in combination in prophylactic and therapeutic regimens. Some combinations of antigens induced a better prophylactic or therapeutic activity than urease alone (0.5-1.5 log further reduction in prophylaxis and therapy respectively, P<0.05). The combinations that induced the best protection were different in prophylaxis and therapy. In conclusion, DC Chol provides a convenient and efficient method to formulate different antigens even when they are present in non-compatible buffers initially. Moreover, the results obtained in protection against H. pylori with such formulations should lead the way to future clinical trials.

Polymorphonuclear cells (PMNs) from healthy donors and differentiated HL-60 cells were compared in an opsonophagocytic assay using fluorescent latex beads coated with Streptococcus pneumoniae polysaccharide conjugates. Serum-specific phagocytosis was efficiently mediated by both sources of cells, as measured by flow cytometry, but the mean number of beads ingested per cell was three-to fivefold higher when PMNs were used than when HL-60 cells were used. Nevertheless, differentiated HL-60 cells could be a convenient and standardized source of cells to evaluate the functionality of specific antibodies to vaccine candidates as a coating on fluorescent beads.In the search for new vaccines, investigators often have to select a few candidates among a large number of antigens and formulations. A good way to evaluate the potential of such candidates is to measure the specific immune responses against the different antigens in convalescent-phase or resistant hosts after natural infection with the corresponding pathogen. Besides classical enzyme-linked immunosorbent assay techniques, functional assays, such as opsonophagocytic assays, have been demonstrated to be more relevant to analyze the role of antibodies in protection, in particular in the case of Streptococcus pneumoniae (pneumococcal) or Neisseria meningitidis (meningococcal) infections (1-5). An opsonophagocytic assay using fluorescent beads coated with different antigenic structures has been developed by A. Lehmann and coworkers (1), with polymorphonuclear cells (PMNs) from healthy donors as effector cells; opsonophagocytic activity is measured by flow cytometric analysis, the end points being the percentage of fluorescent cells, the mean number of beads per each phagocyte (designated by M in this study), and the product of these two values, the phagocytosis product (PP) (2, 3). In parallel, the group of G. M. Carlone has developed opsonophagocytic assays using differentiated HL-60 cells. In this assay, live bacteria and, more recently, fluorescently labeled and fixed bacteria were used (4, 5). Opsonophagocytic activity is measured in the former case by viable count, while in the latter case, flow cytometry is used.In the present study, we evaluated the combined use of antigen-coated fluorescent beads as targets and HL-60 cells as phagocytes compared to PMNs from healthy donors. Streptococcus pneumoniae polysaccharides from serotypes 4 and 14 conjugated to tetanus toxoid (TT [Pn4-TT and Pn14-TT, respectively]) were used as antigens. The assay was set up with rabbit positive sera (demonstrated in the department to be opsonic with human cells in a viable opsonophagocytic assay) and negative sera (directed against an irrelevant conjugate) in order to have well-identified negative controls, because it was difficult to obtain and select human sera without specific antipneumococcal antibodies. Nevertheless, several human sera, including a reference serum from Sandoz (Sandoglobuline; Sandoz, Rueil Malmaison, France), were tested in a second step. Rabbit sera heated at ...