Prediction by genetic MATS of 4CMenB vaccine strain coverage of invasive meningococcal serogroup B isolates circulating in Taiwan between 2003 and 2020.

Neisseria meningitidis serogroup B (NmB) strains have diverse antigens, necessitating methods for predicting meningococcal serogroup B (MenB) vaccine strain coverage. The genetic Meningococcal Antigen Typing System (gMATS), a correlate of MATS estimates, predicts strain coverage by the 4-component MenB (4CMenB) vaccine in cultivable and non-cultivable NmB isolates. In Taiwan, 134 invasive, disease-causing NmB isolates were collected in 2003-2020 (23.1%, 4.5%, 5.2%, 29.8%, and 37.3% from individuals aged ≤11 months, 12-23 months, 2-4 years, 5-29 years, and ≥30 years, respectively). NmB isolates were characterized by whole-genome sequencing and vaccine antigen genotyping, and 4CMenB strain coverage was predicted using gMATS. Analysis of phylogenetic relationships with 502 global NmB genomes showed that most isolates belonged to three global hyperinvasive clonal complexes: ST-4821 (27.6%), ST-32 (23.9%), and ST-41/44 (14.9%). Predicted strain coverage by gMATS was 62.7%, with 27.6% isolates covered, 2.2% not covered, and 66.4% unpredictable by gMATS. Age group coverage point estimates ranged from 42.9% (2-4 years) to 66.1% (≤11 months). Antigen coverage estimates and percentages predicted as covered/not covered were highly variable, with higher estimates for isolates with one or more gMATS-positive antigens than for isolates positive for one 4CMenB antigen. In conclusion, this first study on NmB strain coverage by 4CMenB in Taiwan shows 62.7% coverage by gMATS, with predictable coverage for 29.8% of isolates. These could be underestimated since the gMATS calculation does not consider synergistic mechanisms associated with simultaneous antibody binding to multiple targets elicited by multicomponent vaccines or the contributions of minor outer membrane vesicle vaccine components.IMPORTANCEMeningococcal diseases, caused by the bacterium Neisseria meningitidis (meningococcus), include meningitis and septicemia. Although rare, invasive meningococcal disease is often severe and can be fatal. Nearly all cases are caused by six meningococcal serogroups (types), including meningococcal serogroup B. Vaccines are available against meningococcal serogroup B, but the antigens targeted by these vaccines have highly variable genetic features and expression levels, so the effectiveness of vaccination may vary depending on the strains circulating in particular countries. It is therefore important to test meningococcal serogroup B strains isolated from specific populations to estimate the percentage of bacterial strains that a vaccine can protect against (vaccine strain coverage). Meningococcal isolates were collected in Taiwan between 2003 and 2020, of which 134 were identified as serogroup B. We did further investigations on these isolates, including using a method (called gMATS) to predict vaccine strain coverage by the 4-component meningococcal serogroup B vaccine (4CMenB).

Pemigatinib for patients with previously treated, locally advanced or metastatic cholangiocarcinoma harboring FGFR2 fusions or rearrangements: A joint analysis of the French PEMI-BIL and Italian PEMI-REAL cohort studies.

BACKGROUND: Pemigatinib is approved for patients with pretreated, locally advanced or metastatic CCA harboring FGFR2 rearrangements or fusions. We aim to assess the effectiveness and safety of pemigatinib in real-world setting. MATERIAL AND METHODS: A joint analysis of two multicentre observational retrospective cohort studies independently conducted in France and Italy was performed. All consecutive FGFR2-positive patients affected by CCA and treated with pemigatinib as second- or further line of systemic treatment in clinical practice, within or outside the European Expanded Access Program, were included. RESULTS: Between July 2020 and September 2022, 72 patients were treated with pemigatinib in 14 Italian and 25 French Centres. Patients had a median age of 57 years, 76% were female, 81% had ECOG-PS 0-1, 99% had intrahepatic CCA, 74% had ≥ 2 metastatic sites, 67% had metastatic disease at diagnosis, while 38.8% received ≥ 2 previous lines of systemic treatment. At data cut-off analysis (April 2023), ORR and DCR were 45.8% and 84.7%, respectively. Median DoR was 7 months (IQR: 5.8-9.3). Over a median follow-up time of 19.5 months, median PFS and 1-year PFS rate were 8.7 months and 32.8%. Median OS and 1-year OS rate were 17.1 months and 60.6%. Fatigue (69.4%), ocular toxicity (68%), nail toxicities (61.1%), dermatologic toxicity (41.6%) hyperphosphataemia (55.6%), stomatitis (48.6%), and diarrhea (36.1%) were the most frequent, mainly G1-G2 AEs. Overall incidence of G3 AEs was 22.2%, while no patient experienced G4 AE. Dose reduction and temporary discontinuation were needed in 33.3% and 40.3% of cases, with 1 permanent discontinuation due to AEs. CONCLUSIONS: These results confirm the effectiveness and safety of pemigatinib in a real-world setting.

Using the World Apheresis Association Registry Helps to Improve the Treatment Quality of Therapeutic Apheresis.

Therapeutic apheresis (TA) is prescribed to patients that suffer from a severe progressive disease that is not sufficiently treated by conventional medications. A way to gain more knowledge about this treatment is usually by the local analysis of data. However, the use of large quality assessment registries enables analyses of even rare findings. Here, we report some of the recent data from the World Apheresis Association (WAA) registry. Data from >104,000 procedures were documented, and TA was performed on >15,000 patients. The main indication for TA was the collection of autologous stem cells (45% of patients) as part of therapy for therapy. Collection of stem cells from donors for allogeneic transplantation was performed in 11% of patients. Patients with indications such as neurological diseases underwent plasma exchange (28%). Extracorporeal photochemotherapy, lipid apheresis, and antibody removal were other indications. Side effects recorded in the registry have decreased significantly over the years, with approximately only 10/10,000 procedures being interrupted for medical reasons. CONCLUSION: Collection of data from TA procedures within a multinational and multicenter concept facilitates the improvement of treatment by enabling the analysis of and feedback on indications, procedures, effects, and side effects.

A Serum Metabolomics Classifier Derived from Elderly Patients with Metastatic Colorectal Cancer Predicts Relapse in the Adjuvant Setting.

Adjuvant treatment for patients with early stage colorectal cancer (eCRC) is currently based on suboptimal risk stratification, especially for elderly patients. Metabolomics may improve the identification of patients with residual micrometastases after surgery. In this retrospective study, we hypothesized that metabolomic fingerprinting could improve risk stratification in patients with eCRC. Serum samples obtained after surgery from 94 elderly patients with eCRC (65 relapse free and 29 relapsed, after 5-years median follow up), and from 75 elderly patients with metastatic colorectal cancer (mCRC) obtained before a new line of chemotherapy, were retrospectively analyzed via proton nuclear magnetic resonance spectroscopy. The prognostic role of metabolomics in patients with eCRC was assessed using Kaplan-Meier curves. PCA-CA-kNN could discriminate the metabolomic fingerprint of patients with relapse-free eCRC and mCRC (70.0% accuracy using NOESY spectra). This model was used to classify the samples of patients with relapsed eCRC: 69% of eCRC patients with relapse were predicted as metastatic. The metabolomic classification was strongly associated with prognosis (p-value 0.0005, HR 3.64), independently of tumor stage. In conclusion, metabolomics could be an innovative tool to refine risk stratification in elderly patients with eCRC. Based on these results, a prospective trial aimed at improving risk stratification by metabolomic fingerprinting (LIBIMET) is ongoing.

Cross-reactivity of 4CMenB vaccine-induced antibodies against meningococci belonging to non-B serogroups in Italy.

The four-component meningococcal serogroup B vaccine (4CMenB) contains antigens present in the majority of meningococci causing invasive meningococcal disease (IMD) and may potentially offer protection against strains belonging to non-B serogroups.This study aimed to evaluate the ability of 4CMenB-induced antibodies to kill, in a human serum bactericidal assay (hSBA), non-B meningococci belonging to the main genotypes responsible for IMD in Italy.Meningococci, collected between 2015 and 2017, was characterized for PorA, FetA and sequence type, and for clonal complex. Twenty non-B isolates, representative of the most frequent genotypes, were molecularly characterized for 4CMenB antigens and tested in hSBA with sera from 4CMenB-vaccinated infants and adolescents.Among twenty isolates, eleven were serogroup C, five were Y, two W and two X. All isolates contained genes encoding for fHbp and NHBA antigens and four harbored the NadA full-length encoding gene. Positive hSBA titers were obtained against all serogroup W, X and Y isolates and against five serogroup C isolates.These data show that the 4CMenB vaccine can induce bactericidal antibodies against genetically representative meningococcal W, Y and X strains from Italy. For serogroup C, different susceptibilities to killing were observed for strains with similar antigenic repertoires.

Bactericidal antibodies against hypervirulent Neisseria meningitidis C field strains following MenC-CRM or MenACWY-CRM priming and MenACWY-CRM booster in children.

An increase in invasive meningococcal disease (IMD) incidence was observed in Tuscany in 2015/2016, mainly due to hypervirulent clonal complex (cc) 11 strains. In a post-hoc analysis, we assessed bactericidal activity of antibodies in sera from children primed with MenACWY-CRM or MenC-CRM conjugate vaccines and receiving a MenACWY-CRM booster dose against 5 meningococcal C (MenC) strains isolated from IMD cases. Sera collected from 90 infants/toddlers who participated in a phase III, open-label study (NCT00667602) and its extension (NCT01345721) were tested by serum bactericidal activity assay with human complement (hSBA). Children were primed with either MenACWY-CRM at 6-8 and 12 months of age (group 2_MenACWY; N = 30), MenACWY-CRM (group 1_MenACWY; N = 30), or MenC-CRM at 12 months of age (group 1_MenC; N = 30); all received MenACWY-CRM booster dose at 22-45 months of age. Four tested strains (FI001-FI004) were C:P1.5-1,10-8:F3-6:ST-11 (cc11) and 1 (FI005) was C:P1.7-4,14-6:F3-9:ST-1031 (cc334). Overall, immune responses tended to be higher against Fl002-FI004 than Fl001 and Fl005. Geometric mean titers were high in group 2_MenACWY (range: 94.8 [FI005]-588.1 [FI004]) and very high post-boosting with MenACWY-CRM in all groups (176.9 [FI005]-3911.0 [FI004]). Seroresponse rates tended to be higher in group 1_MenC (33.3% [FI005]-93.3% [FI004]) than in group 1_MenACWY (16.7% [FI005]-73.3% [FI004]). Irrespective of strains tested or the identity/number of priming doses, ≥96.7% of children had hSBA titers ≥1:8 post-MenACWY-CRM booster dose. MenACWY-CRM and MenC-CRM elicited bactericidal antibodies and immunological memory against hypervirulent cc11 and cc334 MenC strains responsible for IMD outbreaks.

Four-component Meningococcal Serogroup B Vaccine Induces Antibodies With Bactericidal Activity Against Diverse Outbreak Strains in Adolescents.

BACKGROUND: Neisseria meningitidis serogroup B (MenB) causes most meningitis outbreaks worldwide. We evaluated the ability of the 4-component MenB vaccine (4CMenB) to induce bactericidal activity against outbreak strains in adolescents. METHODS: Individual sera from 20 United States and 23 Chilean adolescents who received 2 doses of 4CMenB 2 months apart were assayed at prevaccination and 1 month after second dose using a human complement serum bactericidal antibody assay (hSBA) against a full or subset strain panel consisting of 14 MenB outbreak strains and 1 MenW hyperendemic strain collected between 2001 and 2017 in the United States, United Kingdom, and France. Bactericidal activity was determined as the percentage of adolescents with hSBA titer ≥1:4 or ≥1:8. RESULTS: One month after the second 4CMenB dose, antibodies from 65% to 100% of the US adolescents were able to kill 12 of 15 strains at 1:4 dilution. The remaining 3 strains were killed by 45%, 25%, and 15% of US adolescent sera. Similar percentages exhibited hSBA titers of ≥1:8. Across a subset of 4 strains, point estimates for the percentages of Chilean and US adolescents with hSBA titers of ≥1:4 after the second 4CMenB dose were similar (100% for strain M27703, 74% vs. 80% for M26312, 52% vs. 45% for M08 0240745), except for strain M39090 (91% vs. 65%). CONCLUSIONS: This study was the first to evaluate bactericidal activity elicited by a MenB vaccine against 15 outbreak strains. Two doses of 4CMenB elicited bactericidal activity against MenB outbreak strains and a hyperendemic MenW strain.

Does vaccination with 4CMenB convey protection against meningococcal serogroup B strains not predicted to be covered by MATS? A study of the UK clonal complex cc269.

The Meningococcal Antigen Typing System (MATS) has been developed as an hSBA surrogate to evaluate potential coverage afforded by the 4-component meningococcal serogroup B vaccine (4CMenB: Bexsero, GSK). We investigated whether the lower value of MATS coverage among invasive Meningococcus serogroup B clonal complex 269 strains from the United Kingdom (53% in 2014-2015 versus 73% in 2007-2008) reflected the lower bactericidal activity of the vaccine against these isolates. A total of 34 MATS-negative strains (31 were cc269 or closely related) were tested against pooled sera from 32 or 72 4CMenB-vaccinated infants in a serum bactericidal antibody assay in presence of human complement (hSBA). All infants had received four 4CMenB doses in the first 2 y of life. Baseline sera comprised 180 pooled samples from healthy-unvaccinated 2-month-old infants. Twenty of the 34 (59%) MATS-negative strains were killed in hSBA with titers ≥4 by pooled sera from vaccinated infants. There were 13/34 strains with hSBA titers ≥4 and at least a 4-fold rise in titer with respect to pooled baseline sera, and 10/34 with hSBA titers ≥8 and at least a 4-fold rise in titer with respect to baseline. These data confirm MATS as a conservative estimate for predicting strain coverage by 4CMenB.

Novel Multiplex Immunoassays for Quantification of IgG against Group B Streptococcus Capsular Polysaccharides in Human Sera.

Group B Streptococcus (GBS) infections constitute a major cause of invasive disease during the first three months of life and an unmet medical need that could be addressed by maternal vaccination. The GBS capsular polysaccharides (CPSs) have shown promise as vaccine targets in clinical studies. A highly specific serological assay to quantify maternal and neonatal anti-CPS antibody levels will be instrumental for GBS vaccine licensure. Here, we describe the development and comparison of two novel multiplex immunoassays (MIAs) based on the Luminex technology for the quantification of IgG antibodies recognizing the five most frequent GBS capsular variants (Ia, Ib, II, III, and V) out of the ten types identified. The first assay is based on the use of biotinylated CPSs coupled to streptavidin-derivatized magnetic microspheres (Biotin-CPS MIA), while the second is a sandwich assay with plain CPSs coupled to magnetic microspheres coated with polysaccharide-specific mouse monoclonal antibodies (Sandwich MIA). Both assays showed good specificity, linearity, and precision, although the Biotin-CPS MIA presented higher sensitivity and lower complexity than the Sandwich MIA. A panel of human sera representing a wide range of anti-CPS IgG concentrations was tested in parallel by the two assays, which resulted in comparable titers. Our data support the preservation of antigenic epitopes in the biotinylated polysaccharides and the suitability of the Biotin-CPS MIA for the precise determination of GBS anti-CPS IgG concentrations in human sera.IMPORTANCE Group B streptococcal infections can cause death in neonates up to 3 months of age. Intrapartum antibiotic prophylaxis in GBS-colonized mothers has limited early infections but has no impact after the first week of life. The development of a maternal vaccine to address this unmet medical need has been identified as a priority by the World Health Organization, and the GBS CPSs are considered the best antigen targets. However, to date there are no accepted standardized assays to measure immune responses to the investigational vaccines and for establishment of serocorrelates of protection. Here, we describe the performance of two microsphere-based pentaplex immunoassays for the determination of antibodies recognizing the five most frequent GBS serotypes. Our data confirm that an assay based on biotinylated polysaccharides coupled to streptavidin microspheres would be suitable for the intended purpose.

Meningococcal Antigen Typing System (MATS): A Tool to Estimate Global Coverage for 4CMenB, a Multicomponent Meningococcal B Vaccine.

Meningococcal Antigen Typing System (MATS) is the combination of a sandwich ELISA (Enzyme Linked Immunosorbent Assay) developed to estimate the level of expression and immunoreactivity of the antigen components (fHbp, NHBA, and NadA) of the 4CMenB vaccine (Bexsero, GSK Vaccines) in circulating, serogroup B meningococcal (MenB) strains, with the molecular typing of PorA, the main antigenic component in the outer membrane vesicles (OMV). MATS has been proven to be a good surrogate of the accepted correlate of protection for meningococcus (hSBA), thus providing a quick, conservative and reproducible method to assess vaccine coverage. The method has been successfully transferred and standardized in several public health laboratories across Europe, North America, and Australia and used to screen thousands of isolates all over the world. Here we describe the sandwich ELISA method applied to assess the expression and cross-reactivity of fHbp, NHBA, and NadA in MenB isolates.

Evaluation of strain coverage of the multicomponent meningococcal serogroup B vaccine (4CMenB) administered in infants according to different immunisation schedules.

The 4-component vaccine 4CMenB, developed against invasive disease caused by meningococcal serogroup B, is approved for use in infants in several countries worldwide. 4CMenB is mostly used as 3 + 1 schedule, except for the UK, where a 2 + 1 schedule is used, and where the vaccine showed an effectiveness of 82.9%. Here we compared the coverage of two 4CMenB vaccination schedules (3 + 1 [2.5, 3.5, 5, 11 months] versus 2 + 1 [3.5, 5, 11 months of age]) against 40 serogroup B strains, representative of epidemiologically-relevant isolates circulating in England and Wales in 2007-2008, using sera from a previous phase 3b clinical trial. The strains were tested using hSBA on pooled sera of infants, collected at one month post-primary and booster vaccination. 4CMenB coverage was defined as the percentage of strains with positive killing (hSBA titres ≥ 4 after immunisation and negative baseline hSBA titres < 2). Coverage of 4CMenB was 40.0% (95% confidence interval [CI]: 24.9-56.7) and 87.5% (95%CI: 73.2-95.8) at one month post-primary and booster vaccination, respectively, regardless of immunisation schedule. Using a more conservative threshold (post-immunisation hSBA titres ≥ 8; baseline ≤ 2), at one month post-booster dose, strain coverages were 80% (3 + 1) and 70% (2 + 1). We used a linear regression model to assess correlation between post-immunisation hSBA data for each strain in the two groups; Pearson's correlation coefficients were 0.93 and 0.99 at one month post-primary and booster vaccination. Overall, there is no evidence for a difference in strain coverage when 4CMenB is administered according to a 3 + 1 or 2 + 1 infant vaccination schedule.

Protective effect of Group B Streptococcus type-III polysaccharide conjugates against maternal colonization, ascending infection and neonatal transmission in rodent models.

Group B Streptococcus (GBS) is a normal inhabitant of recto-vaginal mucosae in up to 30% of healthy women. Colonization is a major risk factor for perinatal infection which can lead to severe complications such as stillbirth and neonatal invasive disease. Intra-partum antibiotic prophylaxis in colonized women is a safe and cost-effective preventive measure against early-onset disease in the first days of life, but has no effect on late-onset manifestations or on early maternal infection. Maternal immunization with capsular polysaccharide-based vaccines shows promise for the prevention of both early-onset and late-onset neonatal infections, although ability to prevent maternal colonization and ascending infection has been less studied. Here we investigated the effect of a GBS glycoconjugate vaccine since the very early stage of maternal GBS acquisition to neonatal outcome by rodent models of vaginal colonization and ascending infection. Immunization of female mice and rats with a type III glycoconjugate reduced vaginal colonization, infection of chorioamniotic/ placental membranes and bacterial transmission to fetuses and pups. Type III specific antibodies were detected in the blood and vagina of vaccinated mothers and their offspring. The obtained data support a potential preventive effect of GBS glycoconjugate vaccines during the different stages of pregnancy.

Screening for Frailty in Older Patients With Early-Stage Solid Tumors: A Prospective Longitudinal Evaluation of Three Different Geriatric Tools.

BACKGROUND: Frailty increases the risk of adverse health outcomes and/or dying when exposed to a stressor, and routine frailty assessment is recommended to guide treatment decision. The Balducci frailty criteria (BFC) and Fried frailty criteria (FFC) are commonly used, but these are time consuming. Vulnerable Elders Survey-13 (VES-13) score of ≥7, a simple and resource conserving function-based scoring system, may be used instead. This prospective study evaluates the performance of VES-13 in parallel with BFC and FFC, to identify frailty in elderly patients with early-stage cancer. METHODS: Patients aged ≥70 years with early-stage solid tumors were classified as frail/nonfrail based on BFC (≥1 criteria), FFC (≥3 criteria), and VES-13 (score ≥ 7). All patients were assessed for functional decline and death. RESULTS: We evaluated 185 patients. FFC had a 17% frailty rate, whereas BFC and VES-13 both had 25%, with poor concordance seen between the three geriatric tools. FFC (hazard ratio = 1.99, p = .003) and VES-13 (hazard ratio = 2.81, p < .001) strongly discriminated for functional decline, whereas BFC (hazard ratio = 3.29, p < .001) had the highest discriminatory rate for deaths. BFC and VES-13 remained prognostic for overall survival in multivariate analysis correcting for age, tumor type, stage, and systemic treatment. CONCLUSIONS: A VES-13 score of ≥7 is a valuable discriminating tool for predicting functional decline or death and can be used as a frailty-screening tool among older cancer patients in centers with limited resources to conduct a comprehensive geriatric assessment.

Auto-Assembling Detoxified Staphylococcus aureus Alpha-Hemolysin Mimicking the Wild-Type Cytolytic Toxin.

Staphylococcus aureus alpha-hemolysin (Hla) assembles into heptameric pores on the host cell membrane, causing lysis, apoptosis, and junction disruption. Herein, we present the design of a newly engineered S. aureus alpha-toxin, HlaPSGS, which lacks the predicted membrane-spanning stem domain. This protein is able to form heptamers in aqueous solution in the absence of lipophilic substrata, and its structure, obtained by transmission electron microscopy and single-particle reconstruction analysis, resembles the cap of the wild-type cytolytic Hla pore. HlaPSGS was found to be impaired in binding to host cells and to its receptor ADAM10 and to lack hemolytic and cytotoxic activity. Immunological studies using human sera as well as sera from mice convalescent from S. aureus infection suggested that the heptameric conformation of HlaPSGS mimics epitopes exposed by the cytolytic Hla pore during infection. Finally, immunization with this newly engineered Hla generated high protective immunity against staphylococcal infection in mice. Overall, this study provides unprecedented data on the natural immune response against Hla and suggests that the heptameric HlaPSGS is a highly valuable vaccine candidate against S. aureus.

Multiplex immunoassay for in vitro characterization of acellular pertussis antigens in combination vaccines.

Vaccines characterization is required to ensure physical, chemical, and biological integrity of antigens and adjuvants. Current analytical methods mostly require complete antigen desorption from aluminum-based adjuvants and are not always suitable to distinguish individual antigens in multivalent formulations. Here, Luminex technology is proposed to improve the analytics of vaccine characterization. As proof of concept, TdaP (tetanus, diphtheria and acellular pertussis) combination, adjuvanted with aluminum hydroxide, was chosen as model formulation to quantify and determine the level of adsorption of acellular pertussis (aP) antigens onto adjuvant surface at the same time. The assay used specific antibodies bound to magnetic microspheres presenting unique digital signatures for each pertussis antigen, allowing the simultaneous recognition of respective antigens in the whole vaccine, avoiding laborious procedures for adjuvant separation. Accurate and reproducible quantification of aP antigens in TdaP vaccine has been achieved in the range 0.78-50 ng/mL, providing simultaneously information on antigen identity, quantity, and degree of adsorption to aluminum hydroxide. The current study could further be considered as a model to set up in vitro potency assays thus supporting the replacement of animal tests accordingly to the 3Rs concept.

Vaccine composition formulated with a novel TLR7-dependent adjuvant induces high and broad protection against Staphylococcus aureus.

Both active and passive immunization strategies against Staphylococcus aureus have thus far failed to show efficacy in humans. With the attempt to develop an effective S. aureus vaccine, we selected five conserved antigens known to have different roles in S. aureus pathogenesis. They include the secreted factors α-hemolysin (Hla), ess extracellular A (EsxA), and ess extracellular B (EsxB) and the two surface proteins ferric hydroxamate uptake D2 and conserved staphylococcal antigen 1A. The combined vaccine antigens formulated with aluminum hydroxide induced antibodies with opsonophagocytic and functional activities and provided consistent protection in four mouse models when challenged with a panel of epidemiologically relevant S. aureus strains. The importance of antibodies in protection was demonstrated by passive transfer experiments. Furthermore, when formulated with a toll-like receptor 7-dependent (TLR7) agonist recently designed and developed in our laboratories (SMIP.7-10) adsorbed to alum, the five antigens provided close to 100% protection against four different staphylococcal strains. The new formulation induced not only high antibody titers but also a Th1 skewed immune response as judged by antibody isotype and cytokine profiles. In addition, low frequencies of IL-17-secreting T cells were also observed. Altogether, our data demonstrate that the rational selection of mixtures of conserved antigens combined with Th1/Th17 adjuvants can lead to promising vaccine formulations against S. aureus.

Investigating the immunodominance of carbohydrate antigens in a bivalent unimolecular glycoconjugate vaccine against serogroup A and C meningococcal disease.

Multicomponent constructs, obtained by coupling different glycans to the carrier protein, have been proposed as a way to co-deliver multiple surface carbohydrates targeting different strains of one pathogen and reduce the number of biomolecules in the formulation of multivalent vaccines. To assess the feasibility of this approach for anti-microbial vaccines and investigate the potential immunodominance of one carbohydrate antigen over the others in these constructs, we designed a bivalent unimolecular vaccine against serogroup A (MenA) and C (MenC) meningococci, with the two different oligomers conjugated to same molecule of carrier protein (CRM197). The immune response elicited in mice by the bivalent MenAC construct was compared with the ones induced by the monovalent MenA and MenC vaccines and their combinations. After the second dose, the bivalent construct induced good levels of anti-MenA and anti-MenC antibodies with respect to the controls. However, the murine sera from the MenAC construct exhibited good anti-MenC bactericidal activity, and very low anti-MenA functionality when compared to the monovalent controls. This result was explained with the diverse relative avidities against MenA and MenC polysaccharides, which were measured in the generated sera. The immunodominant effect of the MenC antigen was fully overcome following the third immunization, when sera endowed with higher avidity and excellent bactericidal activity against both MenA and MenC expressing strains were elicited. Construction of multicomponent glycoconjugate vaccines against microbial pathogens is a feasible approach, but particular attention should be devoted to study and overcome possible occurrence of immune interference among the carbohydrates.

A new flow-cytometry-based opsonophagocytosis assay for the rapid measurement of functional antibody levels against Group B Streptococcus.

Opsonophagocytosis is the primary mechanism for the clearance of Group B Streptococcus (GBS) by the host, and levels of opsonic antibodies may correlate with protection in preclinical models. A killing-based opsonophagocytosis assay (OPA), can be used to determine the functional activity of vaccine-induced GBS-specific antibodies. The assay, which measures the number of bacterial colonies surviving phagocytic killing in the presence of specific antibodies and complement, is rather expensive, time-consuming and poorly standardized. Here we describe a rapid, sensitive and reproducible fluorescent OPA assay (fOPA) based on flow cytometry analysis (FACS), which allows internalized bacteria to be distinguished from those associated to the plasma membrane of phagocytic cells. Fixed GBS were labeled with pHrodo™, a fluorescent dye which dramatically increases the emitted fluorescence at the acidic conditions present in the phagocytic endosomal compartment. Labeled bacteria were incubated with HL-60 cells differentiated to phagocytes, antibodies and complement, and then analyzed by FACS. A further improvement to our method, allowing to reduce assay variability, consisted on a step of selection of effector cells among the HL-60 population. Analysis of sera from mice immunized with different GBS vaccines revealed comparable sensitivity and specificity with the traditional killing OPA assay (kOPA), and a good correlation between the fluorescent signal of bacteria internalized by HL-60 phagocytes and killing. Remarkably, the pHrodo-based approach reduced the variability observed with other fOPA assays. The obtained data indicate the proposed fOPA as a reliable and useful tool for functional antibody assessment.

Immunization with the RrgB321 fusion protein protects mice against both high and low pilus-expressing Streptococcus pneumoniae populations.

RrgB321, a fusion protein of the three Streptococcus pneumoniae pilus-1 backbone RrgB variants, is protective in vivo against pilus islet 1 (PI-1) positive pneumococci. In addition, antibodies to RrgB321 mediate a complement-dependent opsonophagocytosis of PI-1 positive strains at levels comparable to those obtained with antisera against glycoconjugate vaccines. In the pneumococcus, pilus-1 displays a biphasic expression pattern, with different proportions of two bacterial phenotypes, one expressing and one not expressing the pilus-1. These two populations can be stably separated in vitro giving rise to the enriched high (H) and low (L) pilus expressing populations. In this work we demonstrate that: (i) the opsonophagocytic killing mediated in vitro by RrgB321 antisera is strictly dependent on the pilus expression ratio of the strain used; (ii) during the opsonophagocytosis assay pilus-expressing pneumococci are selectively killed, and (iii) no switch towards the pilus non-expressing phenotype can be observed. Furthermore, in sepsis and pneumonia models, mice immunized with RrgB321 are significantly protected against challenge with either the H or the L pilus-expressing population of strains representative of the three RrgB variants. This suggests that the pilus-1 expression is not down-regulated, and also that the expression of the pilus-1 could be up-regulated in vivo. In conclusion, these data provide evidence that RrgB321 is protective against PI-1 positive strains regardless of their pilus expression level, and support the rationale for the inclusion of this fusion protein into a multi-component protein-based pneumococcal vaccine.

Hyporesponsiveness following booster immunization with bacterial polysaccharides is caused by apoptosis of memory B cells.

BACKGROUND: Repeated immunizations with polysaccharide (PS) vaccines cause hyporesponsiveness through undefined mechanisms. We assessed the effects of a PS booster on immune responses, frequency, and survival of PS-specific B-cell subpopulations in spleen and bone marrow. METHODS: Neonatal mice were primed with meningococcus serotype C (MenC) conjugate MenC-CRM(197)+CpG1826, boosted with MenC-CRM(197), MenC-PS, or saline; subsequently, bromodeoxyuridine (BrdU) was injected daily intraperitoneally. MenC-PS-specific cells were labeled with fluorescent MenC-PS and phenotyped by flow cytometry. RESULTS: After MenC-PS booster, proliferating (BrdU(+)) MenC-PS-specific naive B cells (CD138(-)/B220(+); P = .0003) and plasma cells (CD138(+)/B220(-); P = .0002) in spleen were fewer than after saline booster. BrdU(+) MenC-PS-specific plasma cells were also reduced in bone marrow (P = .0308). Compared to saline, MenC-PS booster reduced BrdU(+) IgG(+) MenC-PS-specific B cells in spleen (P = .0002). Twelve hours after the MenC-PS booster, an increased frequency of apoptotic (AnnexinV(+)) MenC-PS-specific B cells in spleen was observed compared with MenC-CRM(197) (P = .0286) or saline (P = .001) boosters. CONCLUSIONS: We demonstrated that the MenC-PS booster significantly reduced the frequency of newly activated MenC-PS-specific B cells-mostly switched IgG(+) memory cells-by driving them into apoptosis. It shows directly that apoptosis of PS-specific memory cells is the cause of PS-induced hyporesponsiveness. These results should be taken into account prior to consideration of the use of PS vaccines.