Observation of Collider Muon Neutrinos with the SND@LHC Experiment.

We report the direct observation of muon neutrino interactions with the SND@LHC detector at the Large Hadron Collider. A dataset of proton-proton collisions at sqrt[s]=13.6 TeV collected by SND@LHC in 2022 is used, corresponding to an integrated luminosity of 36.8 fb^{-1}. The search is based on information from the active electronic components of the SND@LHC detector, which covers the pseudorapidity region of 7.2<η<8.4, inaccessible to the other experiments at the collider. Muon neutrino candidates are identified through their charged-current interaction topology, with a track propagating through the entire length of the muon detector. After selection cuts, 8 ν_{µ} interaction candidate events remain with an estimated background of 0.086 events, yielding a significance of about 7 standard deviations for the observed ν_{µ} signal.

Meal patterns across ten European countries - results from the European Prospective Investigation into Cancer and Nutrition (EPIC) calibration study.

OBJECTIVE: To characterize meal patterns across ten European countries participating in the European Prospective Investigation into Cancer and Nutrition (EPIC) calibration study. DESIGN: Cross-sectional study utilizing dietary data collected through a standardized 24 h diet recall during 1995-2000. Eleven predefined intake occasions across a 24 h period were assessed during the interview. In the present descriptive report, meal patterns were analysed in terms of daily number of intake occasions, the proportion reporting each intake occasion and the energy contributions from each intake occasion. SETTING: Twenty-seven centres across ten European countries. SUBJECTS: Women (64 %) and men (36 %) aged 35-74 years (n 36 020). RESULTS: Pronounced differences in meal patterns emerged both across centres within the same country and across different countries, with a trend for fewer intake occasions per day in Mediterranean countries compared with central and northern Europe. Differences were also found for daily energy intake provided by lunch, with 38-43 % for women and 41-45 % for men within Mediterranean countries compared with 16-27 % for women and 20-26 % for men in central and northern European countries. Likewise, a south-north gradient was found for daily energy intake from snacks, with 13-20 % (women) and 10-17 % (men) in Mediterranean countries compared with 24-34 % (women) and 23-35 % (men) in central/northern Europe. CONCLUSIONS: We found distinct differences in meal patterns with marked diversity for intake frequency and lunch and snack consumption between Mediterranean and central/northern European countries. Monitoring of meal patterns across various cultures and populations could provide critical context to the research efforts to characterize relationships between dietary intake and health.

Impact of thearubigins on the estimation of total dietary flavonoids in the European Prospective Investigation into Cancer and Nutrition (EPIC) study.

Thearubigins (TR) are polymeric flavanol-derived compounds formed during the fermentation of tea leaves. Comprising ∼70% of total polyphenols in black tea, TR may contribute majorly to its beneficial effects on health. To date, there is no appropriate food composition data on TR, although several studies have used data from the US Department of Agriculture (USDA) database to estimate TR intakes. We aimed to estimate dietary TR in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort and assess the impact of including TR or not in the calculation of the total dietary flavonoid intake. Dietary data were collected using a single standardized 24-h dietary recall interviewer-administered to 36 037 subjects aged 35-74 years. TR intakes were calculated using the USDA database. TR intakes ranged from 0.9 mg/day in men from Navarra and San Sebastian in Spain to 532.5 mg/day in men from UK general population. TR contributed <5% to the total flavonoid intake in Greece, Spain and Italy, whereas in the UK general population, TR comprised 48% of the total flavonoids. High heterogeneity in TR intake across the EPIC countries was observed. This study shows that total flavonoid intake may be greatly influenced by TR, particularly in high black tea-consuming countries. Further research on identification and quantification of TR is needed to get more accurate dietary TR estimations.

Dietary intakes and food sources of phytoestrogens in the European Prospective Investigation into Cancer and Nutrition (EPIC) 24-hour dietary recall cohort.

BACKGROUND/OBJECTIVES: Phytoestrogens are estradiol-like natural compounds found in plants that have been associated with protective effects against chronic diseases, including some cancers, cardiovascular diseases and osteoporosis. The purpose of this study was to estimate the dietary intake of phytoestrogens, identify their food sources and their association with lifestyle factors in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. SUBJECTS/METHODS: Single 24-hour dietary recalls were collected from 36,037 individuals from 10 European countries, aged 35-74 years using a standardized computerized interview programe (EPIC-Soft). An ad hoc food composition database on phytoestrogens (isoflavones, lignans, coumestans, enterolignans and equol) was compiled using data from available databases, in order to obtain and describe phytoestrogen intakes and their food sources across 27 redefined EPIC centres. RESULTS: Mean total phytoestrogen intake was the highest in the UK health-conscious group (24.9 mg/day in men and 21.1 mg/day in women) whereas lowest in Greece (1.3 mg/day) in men and Spain-Granada (1.0 mg/day) in women. Northern European countries had higher intakes than southern countries. The main phytoestrogen contributors were isoflavones in both UK centres and lignans in the other EPIC cohorts. Age, body mass index, educational level, smoking status and physical activity were related to increased intakes of lignans, enterolignans and equol, but not to total phytoestrogen, isoflavone or coumestan intakes. In the UK cohorts, the major food sources of phytoestrogens were soy products. In the other EPIC cohorts the dietary sources were more distributed, among fruits, vegetables, soy products, cereal products, non-alcoholic and alcoholic beverages. CONCLUSIONS: There was a high variability in the dietary intake of total and phytoestrogen subclasses and their food sources across European regions.

A bivariate measurement error model for nitrogen and potassium intakes to evaluate the performance of regression calibration in the European Prospective Investigation into Cancer and Nutrition study.

OBJECTIVES: Within the European Prospective Investigation into Cancer and Nutrition (EPIC) study, the performance of 24-h dietary recall (24-HDR) measurements as reference measurements in a linear regression calibration model is evaluated critically at the individual (within-centre) and aggregate (between-centre) levels by using unbiased estimates of urinary measurements of nitrogen and potassium intakes. METHODS: Between 1995 and 1999, 1072 study subjects (59% women) from 12 EPIC centres volunteered to collect 24-h urine samples. Log-transformed questionnaire, 24-HDR and urinary measurements of nitrogen and potassium intakes were analysed in a multivariate measurement error model to estimate the validity of coefficients and error correlations in self-reported dietary measurements. In parallel, correlations between means of 24-HDR and urinary measurements were computed. Linear regression calibration models were used to estimate the regression dilution (attenuation) factors. RESULTS: After adjustment for sex, centre, age, body mass index and height, the validity coefficients for 24-HDRs were 0.285 (95% confidence interval: 0.194, 0.367) and 0.371 (0.291, 0.446) for nitrogen and potassium intakes, respectively. The attenuation factors estimated in a linear regression calibration model were 0.368 (0.228, 0.508) for nitrogen and 0.500 (0.361, 0.639) for potassium intakes; only the former was different from the estimate obtained using urinary measurements in the measurement error model. The aggregate-level correlation coefficients between means of urinary and 24-HDR measurements were 0.838 (0.637, 0.932) and 0.756 (0.481, 0.895) for nitrogen and potassium intakes, respectively. CONCLUSIONS: This study suggests that 24-HDRs can be used as reference measurements at the individual and aggregate levels for potassium intake, whereas, for nitrogen intake, good performance is observed for between-centre calibration, but some limitations are apparent at the individual level.

The EPIC nutrient database project (ENDB): a first attempt to standardize nutrient databases across the 10 European countries participating in the EPIC study.

OBJECTIVE: This paper describes the ad hoc methodological concepts and procedures developed to improve the comparability of Nutrient databases (NDBs) across the 10 European countries participating in the European Prospective Investigation into Cancer and Nutrition (EPIC). This was required because there is currently no European reference NDB available. DESIGN: A large network involving national compilers, nutritionists and experts on food chemistry and computer science was set up for the 'EPIC Nutrient DataBase' (ENDB) project. A total of 550-1500 foods derived from about 37,000 standardized EPIC 24-h dietary recalls (24-HDRS) were matched as closely as possible to foods available in the 10 national NDBs. The resulting national data sets (NDS) were then successively documented, standardized and evaluated according to common guidelines and using a DataBase Management System specifically designed for this project. The nutrient values of foods unavailable or not readily available in NDSs were approximated by recipe calculation, weighted averaging or adjustment for weight changes and vitamin/mineral losses, using common algorithms. RESULTS: The final ENDB contains about 550-1500 foods depending on the country and 26 common components. Each component value was documented and standardized for unit, mode of expression, definition and chemical method of analysis, as far as possible. Furthermore, the overall completeness of NDSs was improved (>or=99%), particularly for beta-carotene and vitamin E. CONCLUSION: The ENDB constitutes a first real attempt to improve the comparability of NDBs across European countries. This methodological work will provide a useful tool for nutritional research as well as end-user recommendations to improve NDBs in the future.

Effects of the adjuvant cholera toxin on dendritic cells: stimulatory and inhibitory signals that result in the amplification of immune responses.

Cholera toxin (CT) is a potent mucosal adjuvant. When administered through the mucosal route CT amplifies B and T lymphocyte responses to co-administered antigens. Since the discovery of CT as a mucosal adjuvant, other bacterial enterotoxins have been found to have this property. These molecules or their detoxified derivatives are all important for the development of mucosal vaccines for human use, and it is thus necessary to understand their mechanism of action. CT has immunomodulatory effects on different cell types, however, the interaction of CT with dendritic cells (DCs), which have a primary role in the priming of immune responses, may be crucial for its adjuvant activity.

Rationally designed strings of promiscuous CD4(+) T cell epitopes provide help to Haemophilus influenzae type b oligosaccharide: a model for new conjugate vaccines.

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. Here we 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.

Cholera toxin induces maturation of human dendritic cells and licences them for Th2 priming.

Cholera toxin (CT) is a potent mucosal adjuvant that amplifies B and T cell responses to mucosally co-administered antigens, stimulating predominant Th2-type responses. However, little is known about the mechanism of adjuvanticity of CT and on the influence this toxin may have on Th2 cell development during the priming of an immune response. We analyzed the effect of CT on dendritic cells (DC), which are responsible for the priming of immune responses at the systemic as well as at the mucosal level. We found that CT induces phenotypic and functional maturation of blood monocyte-derived DC. Indeed, CT-treated DC up-regulate expression of HLA-DR molecules, B7. 1 and B7.2 co-stimulatory molecules, and are able to prime naive CD4(+)CD45RA(+) T cells in vitro, driving their polarization towards the Th2 phenotype. Furthermore, CT-matured DC express functional chemokine receptors CCR7 and CXCR4 which may render them responsive to migratory stimuli towards secondary lymphoid organs. Interestingly, the maturation program induced by CT is unique since CT does not induce but rather inhibits cytokine (IL-12p70 and TNF-alpha) and chemokine (RANTES, MIP-1alpha and MIP-1beta) secretion by lipopolysaccharide- or CD40 ligand-activated DC. Our results help to elucidate the mechanism of action of CT as an adjuvant and highlight a new stimulus of bacterial origin that promotes maturation of DC.

The comparison of the effect of LTR72 and MF59 adjuvants on mouse humoral response to intranasal immunisation with human papillomavirus type 6b (HPV-6b) virus-like particles.

Infections with genital human papillomaviruses (HPV) are likely to be neutralised more efficiently if a mucosal immune response can be elicited at the viral entry site. Local IgA antibodies are highly induced when antigens are co-administered with mucosal adjuvants, such as cholera toxin (CT) and Escherichia coli heat labile enterotoxin (LT) which, however, are not expected to have wide application because of their pronounced toxicity. We have immunised mice intranasally with HPV-6b virus-like particles (VLPs) and a genetically modified LT-derived molecule with only residual toxicity, LTR72, and compared the humoral responses with those obtained following systemic immunisation with VLPs and the MF59 adjuvant. Titration of anti-HPV antibodies in sera and vaginal secretions established that LTR72 was able to elicit higher serum and mucosal IgA titers, in addition to IgG serum levels, comparable to those obtained by parenteral immunisation. These results confirm the potential of toxin-derived adjuvants and extend their use in combination with HPV antigens.

Zonula occludens toxin is a powerful mucosal adjuvant for intranasally delivered antigens.

Zonula occludens toxin (Zot) is produced by toxigenic strains of Vibrio cholerae and has the ability to reversibly alter intestinal epithelial tight junctions, allowing the passage of macromolecules through the mucosal barrier. In the present study, we investigated whether Zot could be exploited to deliver soluble antigens through the nasal mucosa for the induction of antigen-specific systemic and mucosal immune responses. Intranasal immunization of mice with ovalbumin (Ova) and recombinant Zot, either fused to the maltose-binding protein (MBP-Zot) or with a hexahistidine tag (His-Zot), induced anti-Ova serum immunoglobulin G (IgG) titers that were approximately 40-fold higher than those induced by immunization with antigen alone. Interestingly, Zot also stimulated high anti-Ova IgA titers in serum, as well as in vaginal and intestinal secretions. A comparison with Escherichia coli heat-labile enterotoxin (LT) revealed that the adjuvant activity of Zot was only sevenfold lower than that of LT. Moreover, Zot and LT induced similar patterns of Ova-specific IgG subclasses. The subtypes IgG1, IgG2a, and IgG2b were all stimulated, with a predominance of IgG1 and IgG2b. In conclusion, our results highlight Zot as a novel potent mucosal adjuvant of microbial origin.

NMR studies on the structure/function correlations of T-cell-epitope analogs from pertussis toxin.

A synthetic tridecapeptide, corresponding to the 30-42 fragment of the S1 subunit of pertussis toxin, has been structurally characterised by using NMR spectroscopy. The molecule corresponds to a T-cell epitope of the bacterial toxin which has been extensively analysed with the alanine scanning approach to check the relevance of each residue for the biological activity of the peptide. Five of these Ala-substituted analogs have also been spectroscopically studied. In the experimental conditions used, different extents of helicity were found for the six peptides in a way which cannot be related to their capabilities of of binding to major histocompatibility complex (MHC) class II and inducing T-cell proliferation. Backbone flexibility around helical transient conformations seems to constitute the structural intermediate step between the structure of the corresponding sequence within the parental protein and in the MHC class II complex. A model of the latter complex, which accounts for the different biological activities of the analogs, is proposed.

Adjuvant effect of non-toxic mutants of E. coli heat-labile enterotoxin following intranasal, oral and intravaginal immunization.

Cholera toxin and Escherichia coli heat-labile enterotoxin (LT) are known to be very effective mucosal adjuvants, but their toxicity limits their use in humans. We genetically detoxified LT by substituting single residues in the active site of the enzymatic A subunit and obtained mutant molecules that retain mucosal adjuvant activity but are devoid of toxicity. These mutant LT molecules induce mucosal and systemic responses to antigens delivered intranasally, orally and intravaginally in mice. Furthermore, mucosal immunization with these molecules confers protection against systemic challenge with tetanus toxin (TT) and mucosal challenge with Helicobacter pylori.

Therapeutic intragastric vaccination against Helicobacter pylori in mice eradicates an otherwise chronic infection and confers protection against reinfection.

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.

Acellular pertussis vaccines containing genetically detoxified pertussis toxin induce long-lasting humoral and cellular responses in adults.

New generation pertussis vaccines, containing only purified Bordetella pertussis antigens, have been proven safe, immunogenic and efficacious. They have, however, raised new questions regarding the mechanism of protection from whooping cough and the duration of the immune response following vaccination. In addition to the antibody (Ab) titer, the level of pertussis toxin (PT) neutralizing antibodies may be very important in protection and the role of cell-ediated immunity needs to be defined. We have previously reported the safety and immunogenicity results of two phase I trials in adult volunteers with two acellular pertussis vaccines containing genetically detoxified PT alone or in combination with filamentous hemagglutinin (FHA) and 69K protein. In this work, we present the results of a long term follow-up study of the immune response in the same vaccinees. We evaluated the Ab response, the PT neutralizing titer and the peripheral blood T cell response up to 4 years following vaccination. Our results show that in adults the level of antibodies to PT, FHA and 69K and the PT neutralizing titers slightly decline between 2.5 and 12 months after the last vaccine dose, but they remain high in the following 2-4 years, showing levels 10-100 times higher than pre-vaccination values. The T cell responses were more heterogeneous among vaccinees but they did not show any significant decline throughout the period monitored.

Induction of CD4+ T cell depletion in mice doubly transgenic for HIV gp120 and human CD4.

It has been suggested that loss of uninfected T cells in HIV infection occurs because of lymphocyte activation resulting in cell death by apoptosis. To address the question of whether cross-linking of CD4/HIV gp120 complexes by antibodies were sufficient to induce T cell depletion in vivo, we developed an animal model of continuous interaction between human CD4 (hCD4), gp120 and anti-gp120 antibodies in the absence of other viral factors. Double-transgenic mice have been generated in which T cells express on their membrane hCD4 and secrete HIV gp120. Although these mice have hCD4/gp120 complexes present on the surface of T cells, they do not show gross immunological abnormalities, and they are able to produce anti-gp120 antibodies following immunization with denaturated gp120. However, double-transgenic mice with antibodies to gp120, when immunized with tetanus toxoid, mount an IgG response that is significantly lower than that of double-transgenic mice without antibodies to gp120. Furthermore, the presence of anti-gp120 antibodies leads to CD4+ T cell depletion and immunodeficiency in the absence of HIV infection. Thus, the antibody response to gp120 can lead to CD4+ T cell attrition in vivo.

Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant.

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 reaction of bacterial toxins with formaldehyde and its use for antigen stabilization.

Since the discovery of diphtheria toxin inactivation in the early 1920s, formaldehyde has been used to inactivate bacterial toxins and viruses used as vaccine antigens. More recently, formaldehyde was used to inactivate pertussis toxin (PT), a component of the newly developed diphtheria-tetanus-acellular pertussis (DTaP) vaccine. This application however illustrated the complexity of the reaction. To eliminate the need for inactivation, the mutant PT-9K/129G was developed. This toxin analogue is irreversibly devoid of toxicity and is a more immunogenic antigen than chemically detoxified PT. Native antigens however proved less stable than detoxified antigens upon storage or heating. We investigated the use of low concentrations of formaldehyde as a stabilizing agent for PT-9K/129G. Under the conditions selected, its antigenic characteristics were retained. Enhanced immunogenicity compared to detoxified preparations was demonstrated in clinical trials in infants where DTaP vaccines containing formalin-stabilized PT-9K/129G were compared to other DTaP vaccines containing detoxified wild type PT. Additional studies with filamentous haemagglutinin (FHA), another component of acellular pertussis vaccines, showed how high formaldehyde concentrations could depress the presentation of epitopes to T-cells by limiting the antigen processing. In conclusion, mild formaldehyde treatment can be applied to stabilize vaccine antigens while retaining optimum antigenic activity.

Helicobacter pylori-specific CD4+ T-cell clones from peripheral blood and gastric biopsies.

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.

Acellular pertussis vaccine composed of genetically inactivated pertussis toxin.

Whooping cough, an acute respiratory disease affecting over sixty million infants, can be prevented by vaccination. The vaccine currently used, composed of killed bacterial cells, however, has been associated with many side effects. An improved vaccine against the disease should contain pertussis toxin (PT), a major virulent factor of Bordetella pertussis (B. pertussis). In order to be included in the vaccine, PT needs to be detoxified and the chemical methods used so far are not completely satisfactory, since they give a product with reduced immunogenicity and possible residual toxicity. To avoid this problem, we have used recombinant DNA technologies to clone the PT gene, express it in bacteria, map the B and T cell epitopes of the molecule and identify the amino acids that are important for the enzymatic activity and toxicity. Based on this information, the gene coding for PT was mutated to produce an inactive protein. This genetically modified PT was non toxic, highly immunogenic and able to protect mice from intracerebral challenge with virulent B. pertussis. The mutant was included as a main component of an acellular pertussis vaccine which has been shown in numerous clinical trials to be more safe and immunogenic than the old cellular vaccine.