Development of a glycoconjugate vaccine to prevent meningitis in Africa caused by meningococcal serogroup X.

Neisseria meningitidis is a major cause of bacterial meningitis worldwide, especially in the African meningitis belt, and has a high associated mortality. The meningococcal serogroups A, W, and X have been responsible for epidemics and almost all cases of meningococcal meningitis in the meningitis belt over the past 12 y. Currently no vaccine is available against meningococcal X (MenX). Because the development of a new vaccine through to licensure takes many years, this leaves Africa vulnerable to new epidemics of MenX meningitis at a time when the epidemiology of meningococcal meningitis on the continent is changing rapidly, following the recent introduction of a glycoconjugate vaccine against serogroup A. Here, we report the development of candidate glycoconjugate vaccines against MenX and preclinical data from their use in animal studies. Following optimization of growth conditions of our seed MenX strain for polysaccharide (PS) production, a scalable purification process was developed yielding high amounts of pure MenX PS. Different glycoconjugates were synthesized by coupling MenX oligosaccharides of varying chain length to CRM197 as carrier protein. Analytical methods were developed for in-process control and determination of purity and consistency of the vaccines. All conjugates induced high anti-MenX PS IgG titers in mice. Antibodies were strongly bactericidal against African MenX isolates. These findings support the further development of glycoconjugate vaccines against MenX and their assessment in clinical trials to produce a vaccine against the one cause of epidemic meningococcal meningitis that currently cannot be prevented by available vaccines.

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.

Synthesis and immunological evaluation of protein conjugates of Neisseria meningitidis X capsular polysaccharide fragments.

A vaccine to prevent infections from the emerging Neisseria meningitidis X (MenX) is becoming an urgent issue. Recently MenX capsular polysaccharide (CPS) fragments conjugated to CRM197 as carrier protein have been confirmed at preclinical stage as promising candidates for vaccine development. However, more insights about the minimal epitope required for the immunological activity of MenX CPS are needed. We report herein the chemical conjugation of fully synthetic MenX CPS oligomers (monomer, dimer, and trimer) to CRM197. Moreover, improvements in some crucial steps leading to the synthesis of MenX CPS fragments are described. Following immunization with the obtained neoglycoconjugates, the conjugated trimer was demonstrated as the minimal fragment possessing immunogenic activity, even though significantly lower than a pentadecamer obtained from the native polymer and conjugated to the same protein. This finding suggests that oligomers longer than three repeating units are possibly needed to mimic the activity of the native polysaccharide.

RrgB321, a fusion protein of the three variants of the pneumococcal pilus backbone RrgB, is protective in vivo and elicits opsonic antibodies.

Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.

CD81 is a central regulator of cellular events required for hepatitis C virus infection of human hepatocytes.

Infection with hepatitis C virus (HCV) is still a major public health problem, and the events leading to hepatocyte infection are not yet fully understood. Combining confocal microscopy with biochemical analysis and studies of infection requirements using pharmacological inhibitors and small interfering RNAs, we show here that engagement of CD81 activates the Rho GTPase family members Rac, Rho, and Cdc42 and that the block of these signaling pathways drastically reduces HCV infectivity. Activation of Rho GTPases mediates actin-dependent relocalization of the HCV E2/CD81 complex to cell-cell contact areas where CD81 comes into contact with the tight-junction proteins occludin, ZO-1, and claudin-1, which was recently described as an HCV coreceptor. Finally, we show that CD81 engagement activates the Raf/MEK/ERK signaling cascade and that this pathway affects postentry events of the virus life cycle. In conclusion, we describe a range of cellular events that are manipulated by HCV to coordinate interactions with its multiple coreceptors and to establish productive infections and find that CD81 is a central regulator of these events.

Functional activity of maternal and cord antibodies elicited by an investigational group B Streptococcus trivalent glycoconjugate vaccine in pregnant women.

OBJECTIVES: The main aim of this exploratory study was to evaluate functional activity of antibodies elicited by a maternal Group B Streptococcus (GBS) investigational vaccine composed of capsular polysaccharides Ia, Ib, and III conjugated to genetically detoxified Diphtheria toxin CRM197. The second objective was to investigate the relationship between serotype-specific IgG concentrations and functional activity in maternal and cord sera. METHODS: Maternal and cord sera collected at baseline and at delivery from vaccine and placebo recipients during a double-blind placebo-controlled Phase II study (www.clinicaltrials.gov, NCT01446289) were tested in an opsono-phagocytic bacterial killing assay. Cord sera from vaccine recipients were also passively transferred to newborn mice to investigate conferred protection against bacterial challenge. RESULTS: Antibody-mediated GBS phagocytic killing was significantly increased in maternal serum at delivery and in cord sera from the investigational vaccine group as compared to the placebo group. Anti-capsular IgG concentrations above 1 µg/mL mediated in vitro killing against GBS strains belonging to all three serotypes and IgG levels correlated with functional titers. Passively administered cord sera elicited a dose-dependent protective response against all GBS serotypes in the in vivo model. CONCLUSIONS: The maternal vaccine elicited functional antibodies that were placentally transferred. Anti-capsular IgG concentrations in maternal and cord sera were predictive of functional activity and in vivo protection in the mouse model.

Synergy between stents and extracorporeal membrane oxygenation in multitrauma patients with inferior vena cava injury.

Despite the lack of evidence in the literature, we report the case of a 25-year-old man involved in a road traffic accident, who had an inferior vena cava (IVC) injury and severe lung contusion with parenchymal bleeding requiring an extracorporeal membrane oxygenation (ECMO). An emergency procedure to implant a stent graft was successful in repairing the IVC injury. Moreover, we think that ECMO, in addition to providing oxygenation, reduced bleeding by creating a negative pressure along the injured IVC. The patient was decannulated on the eighth day and discharged 31 days after the accident.

Immunoactivity of protein conjugates of carba analogues from Neisseria meningitidis a capsular polysaccharide.

Neisseria meningitidis type A (MenA) is a Gram-negative encapsulated bacterium that is a major cause of epidemic meningitis, especially in the sub-Saharan region of Africa. The development and manufacture of a liquid glycoconjugate vaccine against MenA are hampered by the poor hydrolytic stability of its capsular polysaccharide (CPS), consisting of (1→6)-linked 2-acetamido-2-deoxy-α-d-mannopyranosyl phosphate repeating units. The replacement of the ring oxygen with a methylene group to generate a carbocyclic analogue leads to enhancement of its chemical stability. Herein, we report conjugation of carbocyclic analogue monomer, dimer, and trimer to the protein carrier CRM197. After immunization in mice, only the conjugated trimer was able to induce specific anti-MenA polysaccharide IgG antibodies with in vitro bactericidal activity, although to a lesser extent than pentadecamer and hexamer oligomers obtained from mild acid hydrolysis of the native polysaccharide conjugated to the same protein carrier. This study represents the first proof-of-concept that hydrolytically stable structural analogues of saccharide antigens can be used for the development of efficacious antimicrobial preventative therapies. Conjugates with longer carbocyclic oligomers and/or precise acetylation patterns could further increase the induced immune response to a level comparable with those of commercially available anti-meningococcal glycoconjugate vaccines.

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.

Non infective severe aortic paravalvular leakage 7 years after surgery: the role of suture technique.

We report a case of redo aortic prosthesis replacement for a severe paravalvular leak (PVL) in a man operated with continuous suture technique 7 years earlier. The severe aortic regurgitation was due to the rupture of the suture. In spite of operations to replace malfunctioning heart valves are common procedures and performed all over the world from more than 50 years, there is still an open debate about the most suitable suture technique. In this case report, we'll discuss if the suture technique has a role in preventing or leading complications as severe PVL.

Determinants of pericardial drainage for cardiac tamponade following cardiac surgery.

OBJECTIVE: We aimed to identify independent risk factors predisposing toward postoperative surgical or percutaneous pericardial drainage following cardiac surgery, and to assess late survival. METHODS: A retrospective review of preoperative, intra-operative and postoperative variables was conducted in 5818 patients, who underwent adult heart surgery in a 7-year time span (2002-2009). Pericardial drainage was performed in 117 patients (2%), of whom 52 (44%) were evacuated by surgical drainage and 65 (56%) by echocardiographic-guided pericardiocentesis. Patients were divided in two groups: patients with two-dimensional (2D) echocardiographic evidence of cardiac tamponade, who underwent pericardial surgical or percutaneous drainage (group I: 117 patients); and patients without cardiac tamponade (group II: 5701 patients), who served as the control group. RESULTS: The two groups were compared with univariate analysis, and variables significantly (p ≤ 0.05) or possibly (p ≤ 0.2) associated with pericardial drainage were entered into multivariable logistic regression analysis models assessing the role of pre-, intra- and postoperative variables together or separately. Pericardial drainage was more likely to occur in patients undergoing combined procedures such as double/triple valves or surgery on ascending aorta, in patients with higher EuroSCORE (European System for Cardiac Operative Risk Evaluation) levels, whereas patients receiving aspirin treatment before surgery had a lower risk of this complication. In addition, postoperative blood product transfusion and the occurrence of renal failure after surgery increased the risk of this complication. CONCLUSIONS: Postoperative pericardial drainage is an uncommon complication after heart surgery, mainly managed percutaneously. Our study has identified different independent causative factors for cardiac tamponade requiring pericardiocentesis. The identification of preoperative and postoperative risk factors may be useful to adopt strategies to further reduce the incidence of pericardial tamponade requiring drainage.