Immunization of the female reproductive tract is important for protection against sexually transmitted diseases and other pathogens of the reproductive tract. However, intravaginal immunization with soluble antigens generally does not induce high levels of secretory immunoglobulin A (IgA). We recently developed safe mucosal adjuvants by genetically detoxifying Escherichia coli heat-labile enterotoxin, a molecule with a strong mucosal adjuvant activity, and here we describe the use of the nontoxic mutant LTK63 to induce a response in the mouse vagina against ovalbumin (Ova). We compared intravaginal and intranasal routes of immunization for induction of systemic and vaginal responses against LTK63 and Ova. We found that LTK63 is a potent mucosal immunogen when given by either the intravaginal or intranasal route. It induces a strong systemic antibody response and IgG and long-lasting IgA in the vagina. The appearance of vaginal IgA is delayed in the intranasally immunized mice, but the levels of vaginal anti-LTK63 IgA after repeated immunizations are higher in the intranasally immunized mice than in the intravaginally immunized mice. LTK63 also acts as a mucosal adjuvant, inducing a serum response against Ova, when given by both the intravaginal and intranasal routes. However, vaginal IgA against Ova is stimulated more efficiently when LTK63 and antigen are given intranasally. In conclusion, our results demonstrate that LTK63 can be used as a mucosal adjuvant to induce antigen-specific antibodies in vaginal secretions and show that the intranasal route of immunization is the most effective for this purpose.

The introduction of two amino acid substitutions within the enzymatically active subunit Si of pertussis toxin (PT) abolishes its ADP-ribosyltransferase activity and toxicity on CHO cells (Pizza et al., Science 246:497-500, 1989). These genetically inactivated molecules are also devoid of other in vivo adverse reactions typical of PT, such as induction of leukocytosis, potentiation of anaphylaxis, stimulation of insulin secretion, and histamine sensitivity. However, the mutant PT molecules are indistinguishable from wild-type PT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and maintain all the physical and chemical properties of PT, including affinity for toxin-neutralizing poly-and monoclonal antibodies. Either alone or stabilized with formaldehyde, PT mutants are able to induce high levels of neutralizing antibodies and to protect mice in a dose-dependent fashion against intracerebral challenge with virulent B. pertussis. These results clearly show that these genetically inactivated PT molecules are nontoxic but still immunogenic and justify their development as a component of a new, safer aceliular vaccine against whooping cough.

Chronic infection of the gastroduodenal mucosae by the gram-negative spiral bacterium Helicobacter pylori is responsible for chronic active gastritis, peptic ulcers, and gastric cancers such as adenocarcinoma and low-grade gastric B-cell lymphoma. The success of eradication by antibiotic therapy is being rapidly hampered by the increasing occurrence of antibiotic-resistant strains. An attractive alternative approach to combat this infection is represented by the therapeutic use of vaccines. In the present work, we have exploited the mouse model of persistent infection by mouse-adapted H. pylori strains that we have developed to assess the feasibility of the therapeutic use of vaccines against infection. We report that an otherwise chronic H. pylori infection in mice can be successfully eradicated by intragastric vaccination with H. pylori antigens such as recombinant VacA and CagA, which were administered together with a genetically detoxified mutant of the heat-labile enterotoxin of Escherichia coli (referred to as LTK63), in which the serine in position 63 was replaced by a lysine. Moreover, we show that therapeutic vaccination confers efficacious protection against reinfection. These results represent strong evidence of the feasibility of therapeutic use of VacA-or CagA-based vaccine formulations against H. pylori infection in an animal model and give substantial preclinical support to the application of this kind of approach in human clinical trials.

A dedicated 3D numerical model based on coupled mode theory and solving the rate equations has been developed to analyse, design and optimize an optical amplifier obtained by using a tapered fiber and a Er³⁺-doped chalcogenide microsphere. The simulation model takes into account the main transitions among the erbium energy levels, the amplified spontaneous emission and the most important secondary transitions pertaining to the ion-ion interactions. The taper angle of the optical fiber and the fiber-microsphere gap have been designed to efficiently inject into the microsphere both the pump and the signal beams and to improve their spatial overlapping with the rare earth doped region. In order to reduce the computational time, a detailed investigation of the amplifier performance has been carried out by changing the number of sectors in which the doped area is partitioned. The simulation results highlight that this scheme could be useful to develop high efficiency and compact mid-infrared amplifiers.

The age‐related and T cell‐independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long‐lasting T helper cell immunity. Herewe describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4+ T cell epitopes from various pathogen‐derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti‐Hib antibody response in mice. In the case of the N19‐Hib conjugate, this response is at least as good as that observed with CRM197‐Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

SummaryPT9K/129G, a nontoxic mutant of pertussis toxin (PT) obtained by genetic manipulation, has been shown in animal models to be a promising candidate for new vaccines against whooping cough. To assess the safety and the immunogenicity of PT-9K/129G in humans, a pilot study has been performed in adult volunteers. The protein was found to be safe, capable of inducing high titers of toxin-neutralizing antibodies, and capable of generating immunological memory. In fact, vaccination caused an increase of cell-mediated response to PT, PT9K/129G, S1 subunit, and B oligomer, indicating that memory T cells are induced by the vaccine. Since PT-9K/129G is mitogenic for T lymphocytes in vitro, it was investigated whether this activity is also present in vivo. No variation was observed in the proportion of T cells (CD3'), T helper cells (CD4+), and cytotoxic T cells (CD8+), as well as in that of other lymphoid populations, by FACS analysis. Interestingly, no thorough correlation was found between humoral and cellular responses. In one case, a very high cellular response was present in absence of detectable antibodies, suggesting that the antibody response, which is the only parameter measured in most clinical trials, may not give a complete picture of the response induced by a vaccine.

Proteins to be used as vaccines are frequently treated with formaldehyde, although little is known about the effects of this treatment on protein antigenicity. To investigate the effect of formaldehyde treatment on antigen recognition by T cells, we compared the in vitro T-cell response to proteins that have been formaldehyde treated with the response to untreated proteins. We found that peripheral blood mononuclear cells from individuals vaccinated with three formaldehyde-treated proteins (pertussis toxin, filamentous hemagglutinin, pertactin) of Bordetella pertussis showed little or no response to the formaldehyde-treated proteins but proliferated very well in response to the corresponding untreated protein. These findings were further confirmed with CD4+ T-cell clones specific for defined epitopes of the bacterial proteins. We found that some epitopes are presented poorly or not at all when formaldehyde-treated proteins are used, whereas other epitopes are equally presented to T-cell clones when either formaldehyde-treated or untreated antigens are used. However, T-cell recognition could be restored by either antigen degradation before formaldehyde treatment or heat denaturation after such treatment. Parallel digestion with trypsin of both formaldehyde-treated and untreated proteins showed that fragments generated from the two forms of the same antigen were different in size. These results demonstrate that formaldehyde treatment can constrain antigen presentation to T cells and that this may be due to an altered proteolytic processing of formaldehyde-treated proteins.

Colonization of human gastric mucosa with cytotoxic strains of the bacterium Helicobacter pylori is associated with peptic ulcer and with chronic gastritis. Since little is known about the T-cell response to H. pylori, we investigated the CD4 ؉ T-cell response both in peripheral blood mononuclear cells (PBMCs) and at the site of infection. First, we compared the bulk PBMC proliferative response to the bacterium in individuals with and without symptoms of gastroduodenal disease. We found that the PBMCs from virtually all individuals proliferate in response to heat-inactivated bacteria. Second, we cloned H. pylori-specific CD4 ؉ T lymphocytes from the PBMCs of three patients and from both the gastric mucosa and PBMCs of a fourth patient. We have found that CD4 ؉ T-cell clones specific for H. pylori from peripheral blood samples and gastric mucosae of infected patients are major histocompatibility complex class II restricted and discriminate between several cytotoxic and noncytotoxic bacterial strains. Moreover, they are polyclonal in terms of T-cell receptor usage and major histocompatibility complex restriction. Our results demonstrate that the T-cell response to the whole bacterium in PBMCs does not correlate with antibody response, infection, or disease. However, H. pylori-specific CD4 ؉ T cells are detectable, at the clonal level, in both the periphery and gastric mucosa of infected patients. Localization of these cells at the site of disease suggests they are effectors of the immune response to the bacteria.