R. Rappuoli

Mucosal vaccines: non toxic derivatives of LT and CT as mucosal adjuvants.

Most vaccines are still delivered by injection. Mucosal vaccination would increase compliance and decrease the risk of spread of infectious diseases due to contaminated syringes. However, most vaccines are unable to induce immune responses when administered mucosally, and require the use of strong adjuvant on effective delivery systems. Cholera toxin (CT) and Escherichia coli enterotoxin (LT) are powerful mucosal adjuvants when co-administered with soluble antigens. However, their use in humans is hampered by their extremely high toxicity. During the past few years, site-directed mutagenesis has permitted the generation of LT and CT mutants fully non toxic or with dramatically reduced toxicity, which still retain their strong adjuvanticity at the mucosal level. Among these mutants, are LTK63 (serine-to-lysine substitution at position 63 in the A subunit) and LTR72 (alanine-to-arginine substitution at position 72 in the A subunit). The first is fully non toxic, whereas the latter retains some residual enzymatic activity. Both of them are extremely active as mucosal adjuvants, being able to induce very high titers of antibodies specific for the antigen with which they are co-administered. Both mutants have now been tested as mucosal adjuvants in different animal species using a wide variety of antigens. Interestingly, mucosal delivery (nasal or oral) of antigens together with LTK63 or LTR72 mutants also conferred protection against challenge in appropriate animal models (e.g. tetanus, Helicobacter pylori, pertussis, pneumococci, influenza, etc). In conclusion, these LTK63 and LTR72 mutants are safe adjuvants to enhance the immunogenicity of vaccines at the mucosal level, and will be tested soon in humans.

Intranasal Immunization with SAG1 and Nontoxic Mutant Heat-Labile Enterotoxins Protects Mice against Toxoplasma gondii

ABSTRACT Effective protection against intestinal pathogens requires both mucosal and systemic immune responses. Intranasal administration of antigens induces these responses but generally fails to trigger a strong protective immunity. Mucosal adjuvants can significantly enhance the immunogenicities of intranasally administered antigens. Cholera toxin (CT) and heat-labile enterotoxin (LT) are strong mucosal adjuvants with a variety of antigens. Moreover, the toxicities of CT and LT do not permit their use in humans. Two nontoxic mutant LTs, LTR72 and LTK63, were tested with Toxoplasma gondii SAG1 protein in intranasal vaccination of CBA/J mice. Vaccination with SAG1 plus LTR72 or LTK63 induced strong systemic (immunoglobulin G [IgG]) and mucosal (IgA) humoral responses. Splenocytes and mesenteric lymph node cells from mice immunized with LTR72 plus SAG1, but not those from mice immunized with LTK63 plus SAG1, responded to restimulation with a T. gondii lysate antigen in vitro. Gamma interferon and interleukin 2 (IL-2) production by splenocytes and IL-2 production by mesenteric lymph node cells were observed in vitro after antigen restimulation, underlying a Th1-like response. High-level protection as assessed by the decreased load of cerebral cysts after a challenge with the 76K strain of T. gondiiwas obtained in the group immunized with LTR72 plus SAG1 and LTK63 plus SAG1. They were as well protected as the mice immunized with the antigen plus native toxins. This is the first report showing protection against a parasite by using combinations of nontoxic mutant LTs and SAG1 antigen. These nontoxic mutant LTs are now attractive candidates for the development of mucosally delivered vaccines.

Enhancement of Protective Efficacy following Intranasal Immunization with Vaccine Plus a Nontoxic LTK63 Mutant Delivered with Nanoparticles

ABSTRACT Most vaccines are still given parenterally. Mucosal vaccination would offer different advantages over parenteral immunization, including blocking of the pathogens at the portal of entry. In this paper, nontoxic Escherichia coli heat-labile enterotoxin (LT) mutants and Supramolecular Biovector systems (SMBV) were evaluated in mice as mucosal adjuvants and delivery systems, respectively, for intranasal immunization with the conjugated group C meningococcal vaccine. The conjugated vaccine formulated together with the LT mutants and the SMBV induced very high titers of serum and mucosal antibodies specific for the group C meningococcal polysaccharide. This vaccination strategy also induced high titers of antibodies with bactericidal activity, which is known to correlate with efficacy. Importantly, the mucosal vaccination, but not the conventional parenteral vaccination, induced bactericidal antibodies at the mucosal level. These data strongly support the feasibility of development of intranasal vaccines with an enhanced protective efficacy against meningococci and possibly against other encapsulated bacteria.

Genome sequencing of disease and carriage isolates of nontypeable Haemophilus influenzae identifies discrete population structure

Significance Several human pathogens exploit genomic variability to adapt to the host environment. Genome sequencing of collections of isolates and classification of strains according to their genomic content are pivotal to the formulation of vaccines able to elicit broad protection. We sequenced a collection of carriage and disease isolates of nontypeable Haemophilus influenzae, a component of the microbial flora of the upper respiratory tract that can cause a spectrum of diseases, including otitis media and meningitis. We identified distinct evolutionary clades that correlate with the presence of selected surface-associated proteins and virulence determinants. The high-resolution definition of the population structure of nontypeable Haemophilus influenzae allowed by whole-genome sequencing will be key for the development of efficacious containment strategies for this important human pathogen. One of the main hurdles for the development of an effective and broadly protective vaccine against nonencapsulated isolates of Haemophilus influenzae (NTHi) lies in the genetic diversity of the species, which renders extremely difficult the identification of cross-protective candidate antigens. To assess whether a population structure of NTHi could be defined, we performed genome sequencing of a collection of diverse clinical isolates representative of both carriage and disease and of the diversity of the natural population. Analysis of the distribution of polymorphic sites in the core genome and of the composition of the accessory genome defined distinct evolutionary clades and supported a predominantly clonal evolution of NTHi, with the majority of genetic information transmitted vertically within lineages. A correlation between the population structure and the presence of selected surface-associated proteins and lipooligosaccharide structure, known to contribute to virulence, was found. This high-resolution, genome-based population structure of NTHi provides the foundation to obtain a better understanding, of NTHi adaptation to the host as well as its commensal and virulence behavior, that could facilitate intervention strategies against disease caused by this important human pathogen.

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.

The response of primary articular chondrocytes to micrometric surface topography and sulphated hyaluronic acid-based matrices

Understanding the response of chondrocytes to topographical cues and chemical patterns could provide invaluable information to advance the repair of chondral lesions. We studied the response of primary chondrocytes to nano‐ and micro‐grooved surfaces, and sulphated hyaluronic acid (HyalS). Cells were grown on grooves ranging from 80 nm to 9 μm in depth, and from 2 μm to 20 μm in width. Observations showed that the cells did not spread appreciably on any groove size, or alter morphology or F‐actin organization, although cells showed accelerated movement on 750 nm deep grooves in comparison to flat surfaces. On chemical patterns, the cells migrated onto, and preferentially attached to, HyalS and showed a greater degree of spreading and F‐actin re‐arrangement. This study shows that 750 nm deep grooves and sulphated hyaluronic acid elicit responses from primary chondrocytes, and this could have implications for the future direction of cartilage reconstruction and orthopaedic treatments in general.

Novel polysaccharide hydrogels: characterization and properties

New chemical hydrogels, potentially suitable for biomedical applications, have been synthesized and characterized by 13C NMR, and FT-IR spectroscopy. The polysaccharide components of these hydrogels are hyaluronane, alginate and carboxymethylcellulose, while the novel cross-linking procedure consists of activating the carboxylate moieties by 2-chloro-1-methylpyridinium iodide CMPJ and using 1,3-diaminopropane as a chemical bridge. Varying the amount of CMPJ three series of hydrogels were obtained with different cross-linking degrees (5, 50, 100%). Their percentages were determined by 13C NMR and FT-IR analysis. The morphology of the gels was studied by scanning electron-microscopy and the pore sizes were determined in order to find a relationship with the swelling properties. Cell adhesion, using human hepatocytes, and platelet adhesion studies on the different series of cross-linked compounds allowed us to envisage their utilization as extracellular matrix materials and cardiovascular biomaterials. Copyright

Sulphated hyaluronic acids: a chemical and biological characterisation

This review focuses on the possibility of modifying hyaluronic acid by the insertion of sulphate groups in order to obtain heparin-like compounds with controllable properties. In particular, the physicochemical and biological behaviour of sulphated hyaluronic acid (HyalSx) with various degrees of sulphation and different molecular weights has been particularly investigated. The mobilization of endothelial cells was investigated by the use of copper(II) complexes with HyalSx and the results were compared with those from the corresponding complex with heparin. New materials containing HyalSxare proposed for biomedical applications. ©1998 SCI.

Nuclear relaxation studies in ligand-macromolecule affinity index determinations

Abstract An ‘affinity index’, representing the global affinity between a ligand and a biomacromolecular receptor, is proposed. It is determined as the slope of the linear relations between ΔR1SE and the receptor concentration and has the advantage of providing a measure of the ligand-biomacromolecule global affinity which is independent of the number of interaction sites. The method was applied to the calculation of the lamotrigine-albumin affinity index.

Pathogenic Mechanisms of Helicobacter pylori: Production of Cytotoxin

Bacteria associated with mucosal infections of the digestive system generally produce toxins, especially when they cause inflammatory lesions. Illnesses due to thermotolerant campylobacters, to enterohemorrhagic Escherichia coli, and to Clostridium difficileare only some examples. It would be surprising if Helicobacter pylori (HP) did not produce any toxic substances. The difficulty consists in attributing a pathogenic meaning to the toxin, since the range is quite wide of clinical and histological presentation of gastroduodenal inflammatory diseases linked to the presence in the stomach of H. pylori organisms [1]. Johnson and Lior [2] firstly reported the production of heat-labile cytotoxin by 80.6% of 36 HP strains they tested. However, most of our knowledge of the cytotoxigenicity of HP is from Leunk et al. [3] whose work has inspired us in part. They found that about 55% of 201 HP strains isolated in four different parts of the world produced a substance which caused intracellular vacuolization in cells of several lines in vitro, not only in lines generally employed in toxigenicity tests, like Chinese hamster ovary (CHO) cells, Vero cells, and Y-1 cells, but also in human tumoral cells like HeLa, KATO III, and HEp-2, as well as in human embryonic intestinal cells which were the most responsive. They also inferred that the toxin was proteinaceous in nature being heat labile (destroyed at 70 °C for 30 min), protease sensitive, and ammonium-sulfate precipitable. Its molecular weight ought to be higher than 100 kDa since cytotoxic activity could be found only in the retentate of a concentrated broth culture filtrate (CBCF) passed through 100 kDa molecular weight limit ultrafiltration membrane.