Adapting immunity with subunit vaccines: case studies with group A Streptococcus and malaria

Although vaccines have widely been regarded as the most cost-effective way to improve public health, for some organisms new technological advances in vaccine design and delivery, incurring additional developmental costs, will be essential. These organisms are typically those for which natural immunity is either slow to develop or does not develop at all. Clearly, such organisms have evolved strategies to evade immune responses and innovative approaches will be required to induce a type of immune response which is both different to that which develops naturally and is effective. This article describes some approaches to develop vaccines for two such organisms (malaria parasites and Streptococcus pyogenes (group A Streptococcus)) that are associated with widespread mortality and morbidity, mostly in the poorest countries of the world. At this stage, the challenges are primarily scientific, but if these hurdles are surmounted then the challenges will become financial ones - developing much needed vaccines for people least able to afford them. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Use of Granada medium to detect group B streptococcal colonization in pregnant women.

Direct inoculation onto Granada medium (GM) in plates and tubes was compared to inoculation into a selective Todd-Hewitt broth (with 8 microg of gentamicin per ml and 15 microg of nalidixic acid per ml) for detection of group B streptococci (GBS) in pregnant women with 800 vaginal and 450 vaginoanorectal samples. Comparatively, GM was found to be as sensitive as the selective broth for the detection of GBS in vaginal specimens and more sensitive than selective broth for the detection of GBS in vaginoanorectal samples (96 versus 82%). The use of GM improved the time to reporting of a GBS-positive result by at least 24 h and reduced the direct cost of screening. We have also found that the inconvenience of anaerobic incubation of GM plates can be avoided when a cover slide is placed upon the inoculum, because aerobic incubation in GM plates with cover slides causes GBS to develop the same pigmentation that it develops with incubation under anaerobic conditions. These data support the routine use of GM plates or tubes as a more accurate, easier, and cheaper method of identification of GBS-colonized women compared to the enrichment broth technique.

Spinal epidural abscess from group A Streptococcus after varicella infection: a case report and review of the literature.

INTRODUCTION: Spinal epidural abscess (SEA) is a very rare condition in pediatric patients. Varicella zoster infection could be a predisposing factor, and SEA should be suspected in patients with signs of secondary bacterial infection and even mild neurological signs. CLINICAL CASE: We describe here a case of a 30-month-old girl with a history of remitting varicella infection, diagnosed for a lumbar epidural abscess and sacro-ileitis, secondary to group A Streptococcus (GAS). DISCUSSION: This is the third case of SEA from GAS reported in the literature in a pediatric population with varicella infection. We discuss here the clinical presentation and the diagnostic challenges for SEA in childhood through a review of the literature.

Lógica en la toma de decisiones de inversión en el sector de extracción de petróleo y gas natural

Este documento se centra en la presentación de información y análisis de la misma a la hora de establecer la manera en que empresas del sector de extracción de gas natural y generación de energía a base de dicho recurso, toman decisiones en cuanto a inversión, centrándose en la lógica que usan a la hora de emprender este proceso. Esto debido a la constante necesidad de establecer procesos que permitan tomar decisiones más acertadas, incluyendo todas las herramientas posibles para tal fin. La lógica es una de estas herramientas, pues permite encadenar factores con el fin de obtener resultados positivos. Por tal razón, se hace importante conocer el uso de esta herramienta, teniendo en cuentas de qué manera y en que contextos es usada. Con el fin de tener una mayor orientación, este estudio estará centrado en un sector específico, el cual es el de la extracción de petróleo y gas natural. Lo anterior entendiendo la necesidad existente de fundamentación teórica que permita establecer de manera clara la forma apropiada de tomar decisiones en un sector tan diverso y complejo como lo es el mencionado. El contexto empresarial actual exige una visión global, no basada en la lógica lineal causal que hoy se tiene como referencia. El sector de extracción de petróleo y gas natural es un ejemplo particular en cuanto a la manera en cuanto se toman decisiones en inversión, puesto que en su mayoría son empresas de capital intensivo, las cuales mantienen un flujo elevado de recursos monetarios.

Evaluation of glycoconjugate antigens as vaccine candidates against group A Streptococcus and human immunodeficiency virus infections

This PhD thesis discusses the rationale for design and use of synthetic oligosaccharides for the development of glycoconjugate vaccines and the role of physicochemical methods in the characterization of these vaccines. The study concerns two infectious diseases that represent a serious problem for the national healthcare programs: human immunodeficiency virus (HIV) and Group A Streptococcus (GAS) infections. Both pathogens possess distinctive carbohydrate structures that have been described as suitable targets for the vaccine design. The Group A Streptococcus cell membrane polysaccharide (GAS-PS) is an attractive vaccine antigen candidate based on its conserved, constant expression pattern and the ability to confer immunoprotection in a relevant mouse model. Analysis of the immunogenic response within at-risk populations suggests an inverse correlation between high anti-GAS-PS antibody titres and GAS infection cases. Recent studies show that a chemically synthesized core polysaccharide-based antigen may represent an antigenic structural determinant of the large polysaccharide. Based on GAS-PS structural analysis, the study evaluates the potential to exploit a synthetic design approach to GAS vaccine development and compares the efficiency of synthetic antigens with the long isolated GAS polysaccharide. Synthetic GAS-PS structural analogues were specifically designed and generated to explore the impact of antigen length and terminal residue composition. For the HIV-1 glycoantigens, the dense glycan shield on the surface of the envelope protein gp120 was chosen as a target. This shield masks conserved protein epitopes and facilitates virus spread via binding to glycan receptors on susceptible host cells. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose oligosaccharides on the gp120 subunit of HIV-1 Env protein. This oligomannose epitope has been a subject to the synthetic vaccine development. The cluster nature of the 2G12 epitope suggested that multivalent antigen presentation was important to develop a carbohydrate based vaccine candidate. I describe the development of neoglycoconjugates displaying clustered HIV-1 related oligomannose carbohydrates and their immunogenic properties.

New insight on a Group A Streptococcus protective antigen contributing to bacterial cell division

Bioinformatic analysis of Group A Streptococcus (GAS) genomes aiming at the identification of new vaccine antigens, revealed the presence of a gene coding for a putative surface-associated protein, named GAS40, inducing protective antibodies in an animal model of sepsis. The aim of our study was to unravel the involvement of GAS40 in cell division processes and to identify the putative interactor. Firstly, bioinformatic analysis showed that gas40 shares homology with ezrA, a gene coding for a negative regulator of Z-ring formation during cell division process. Both scanning and transmission electron microscopy indicated morphological differences between wild-type and the GAS40 knock-out mutant strain, with the latter showing an impaired capacity to divide resulting in the formation of very long chains. Moreover, when the localization of the antigen on the bacterial surface was analyzed, we found that in bacteria grown at exponential phase GAS40 specifically localized at septum, indicating a possible role in cell division. Furthermore, by ELISA and co-sedimentation assays, we found that GAS40 is able to interact with FtsZ, a protein involved in Z-ring formation during cell division process. These data together with the co-localization of GAS40/FtsZ at bacterial septum demonstrated by by confocal microscopy, strongly support the hypothesis for a key role of GAS40 in bacterial cell division.

Geochemical and Strontium Isotope Characterization of Produced Waters from Marcellus Shale Natural Gas Extraction

Extraction of natural gas by hydraulic fracturing of the Middle Devonian Marcellus Shale, a major gas-bearing unit in the Appalachian Basin, results in significant quantities of produced water containing high total dissolved solids (TDS). We carried out a strontium (Sr) isotope investigation to determine the utility of Sr isotopes in identifying and quantifying the interaction of Marcellus Formation produced waters with other waters in the Appalachian Basin in the event of an accidental release, and to provide information about the source of the dissolved solids. Strontium isotopic ratios of Marcellus produced waters collected over a geographic range of ∼375 km from southwestern to northeastern Pennsylvania define a relatively narrow set of values (εSr SW = +13.8 to +41.6, where εSr SW is the deviation of the 87Sr/86Sr ratio from that of seawater in parts per 104); this isotopic range falls above that of Middle Devonian seawater, and is distinct from most western Pennsylvania acid mine drainage and Upper Devonian Venango Group oil and gas brines. The uniformity of the isotope ratios suggests a basin-wide source of dissolved solids with a component that is more radiogenic than seawater. Mixing models indicate that Sr isotope ratios can be used to sensitively differentiate between Marcellus Formation produced water and other potential sources of TDS into ground or surface waters.

Group B Streptococcus colonization in pregnancy: prevalence and prevention strategies of neonatal sepsis

Early onset neonatal sepsis due to Group B streptococci (GBS) is responsible for severe morbidity and mortality of newborns. While different preventive strategies to identify women at risk are being recommended, the optimal strategy depends on the incidence of GBS-sepsis and on the prevalence of anogenital GBS colonization. We therefore aimed to assess the Group B streptococci prevalence and its consequences on different prevention strategies. We analyzed 1316 pregnant women between March 2005 and September 2006 at our institution. The prevalence of GBS colonization was determined by selective cultures of anogenital smears. The presence of risk factors was analyzed. In addition, the direct costs of screening and intrapartum antibiotic prophylaxis were estimated for different preventive strategies. The prevalence of GBS colonization was 21%. Any maternal intrapartum risk factor was present in 37%. The direct costs of different prevention strategies have been estimated as follows: risk-based: 18,500 CHF/1000 live births, screening-based: 50,110 CHF/1000 live births, combined screening- and risk-based: 43,495/1000 live births. Strategies to prevent GBS-sepsis in newborn are necessary. With our colonization prevalence of 21%, and the intrapartum risk profile of women, the screening-based approach seems to be superior as compared to a risk-based approach.

THE OCCURRENCE OF GROUP G STREPTOCOCCUS IN THE DOMESTIC CAT: COMPARISON OF THE PHYSIOLOGICAL PROPERTIES OF GROUP G STREPTOCOCCUS ISOLATED FROM HUMANS AND CATS

The occurrence of group G streptococci in cats and evaluation of the recovered organisms as potential human pathogens was investigated. Throat swabs were obtained from 89 cats (47 males and 42 females) and vaginal swabs from 39 female cats. Eighty-three of the examined cats were housed in individual cages at a University Animal Care Facility. Six cats, 2 mature males, 2 mature females and 2 young females were family pets in a rural area. Beta-hemolytic streptococci were recovered from 33 (37%) of the 89 cat throats cultured, and 27 (30.3%) were identified as group G. More males (34%) than females (24%) had throat cultures positive for group G. From the 39 vaginal cultures examined, 24 (61.5%) contained beta-hemolytic streptococci and 23 (58.9%) were identified as group G streptococci. Streptococci were not recovered from the vaginal cultures of the 5 females under 6 months of age.^ Thirty one group G streptococci isolated from cats were compared with 37 isolates of group G obtained from humans (health status or site of origin unknown). More group G cat isolates (81%) produced deoxyribonuclease (DNase) than did the human isolates (36%). The proportion of cat throat and vaginal isolates producing DNase was the same. Production of nicotinamide adenine dinucleotide glycohydrolase (NADase) by group G isolates of human origin was 70%, cat throat isolates 53% and cat vaginal isolates 37%. The Serum Opacity Factor was present in 73% of the cat throat isolates of group G, 43.7% of the cat vaginal isolates and 58.6% of the human isolates. Possession of an anti-phagocytic factor (M protein like substance) demonstrated by the ability to multiply in fresh human blood was greater in the group G from cat throats (46.7%) than from cat vagina (37.5%) or from the human isolates (13.5%). Many of the biochemical characteristics of the group G streptococci of cat origin were more similar to the biochemical characteristics of group A streptococci, than to the characteristics of group G of human origin. The group G streptococci, found in a large number of cats, could be potential human pathogens, as their physiological and biological characteristics are very similar to those of group A, a known human pathogen. ^

Studies of group B streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity.

Group B streptococci (GBS) cause sepsis and meningitis in neonates and serious infections in adults with underlying chronic illnesses. Specific antibodies have been shown to be an important factor in protective immunity for neonates, but the role of serum complement is less well defined. To elucidate the function of the complement system in immunity to this pathogen, we have used the approach of gene targeting in embryonic stem cells to generate mice totally deficient in complement component C3. Comparison of C3-deficient mice with mice deficient in complement component C4 demonstrated that the 50% lethal dose for GBS infection was reduced by approximately 50-fold and 25-fold, respectively, compared to control mice. GBS were effectively killed in vitro by human blood leukocytes in the presence of specific antibody and C4-deficient serum but not C3-deficient serum. The defective opsonization by C3-deficient serum in vitro was corroborated by in vivo studies in which passive immunization of pregnant dams with specific antibodies conferred protection from GBS challenge to normal and C4-deficient pups but not C3-deficient pups. These results indicate that the alternative pathway is sufficient to mediate effective opsonophagocytosis and protective immunity to GBS in the presence of specific antibody. In contrast, the increased susceptibility to infection of non-immune mice deficient in either C3 or C4 implies that the classical pathway plays an essential role in host defense against GBS infection in the absence of specific immunity.

Toward the development of a synthetic group A streptococcal vaccine of high purity and broad protective coverage

Using native chemical ligation, we synthesized a group A streptococcal. (GAS) vaccine that contained three different GAS M protein peptide epitopes in a chemically well-characterized construct in high purity. Two of the peptide epitopes represented variable amino terminal serotype determinants, and the third represented a carboxyl terminal conserved region determinant of the GAS M protein. We also synthesized a lipid core peptide (LCP) construct containing the same three peptides. Upon immunization of mice, the non-LCP construct only elicited antibody responses to all three epitopes with the use of adjuvant. The LCP construct, however, elicited excellent antibody responses to all three epitopes without the need for any additional adjuvant or carrier. We have synthesized the LCP synthetic vaccine system with good reproducibility.

Genes for the majority of group A streptococcal virulence factors and extracellular surface proteins do not confer an increased propensity to cause invasive disease

Background. The factors behind the reemergence of severe, invasive group A streptococcal (GAS) diseases are unclear, but it could be caused by altered genetic endowment in these organisms. However, data from previous studies assessing the association between single genetic factors and invasive disease are often conflicting, suggesting that other, as-yet unidentified factors are necessary for the development of this class of disease. Methods. In this study, we used a targeted GAS virulence microarray containing 226 GAS genes to determine the virulence gene repertoires of 68 GAS isolates (42 associated with invasive disease and 28 associated with noninvasive disease) collected in a defined geographic location during a contiguous time period. We then employed 3 advanced machine learning methods (genetic algorithm neural network, support vector machines, and classification trees) to identify genes with an increased association with invasive disease. Results. Virulence gene profiles of individual GAS isolates varied extensively among these geographically and temporally related strains. Using genetic algorithm neural network analysis, we identified 3 genes with a marginal overrepresentation in invasive disease isolates. Significantly, 2 of these genes, ssa and mf4, encoded superantigens but were only present in a restricted set of GAS M-types. The third gene, spa, was found in variable distributions in all M-types in the study. Conclusions. Our comprehensive analysis of GAS virulence profiles provides strong evidence for the incongruent relationships among any of the 226 genes represented on the array and the overall propensity of GAS to cause invasive disease, underscoring the pathogenic complexity of these diseases, as well as the importance of multiple bacteria and/ or host factors.

Synthesis and immunological evaluation of M protein targeted tetra-valent and tri-valent group A streptococcal vaccine candidates based on the lipid-core peptide system

Group A streptococcus (GAS) is responsible for causing many clinical complications including the relatively benign streptococcal pharyngitis and impetigo. However. if left untreated. these conditions may lead to more severe diseases such as rheumatic fever (RF) and rheumatic heart disease (RHD). These diseases exhibit high morbidity and mortality, Particularly in developing countries and in indigenous populations of affluent countries. Only ever occur following GAS infection, a vaccine offers Promise for their Prevention. As stich, we have investigated the Use of the lipid-core peptide (LCP) system for the development of multi-valent Prophylactic GAS vaccines. The current study has investigated the capacity of this system to adjuvant LIP to four different GAS peptide epitopes. Presented are the synthesis and immunological assessment of tetra-valent and tri-valent GAS LCP systems. We demonstrated their capacity to elicit systemic IgG antibody responses in B10.BR mice to all GAS peptide epitopes. The data also showed that the LCP systems Were self-adjuvanting. These findings are particularly encouraging for the development of multi-valent LCP-based GAS vaccines.

FUNCTIONAL CHARACTERIZATION OF THE LINK BETWEEN CARBOHYDRATE METABOLISM AND THE PATHOGENESIS OF THE INVASIVE M1T1 GROUP A STREPTOCOCCUS

The Group A Streptococcus (GAS), or Streptococcus pyogenes, is a strict human pathogen that colonizes a variety of sites within the host. Infections can vary from minor and easily treatable, to life-threatening, invasive forms of disease. In order to adapt to niches, GAS utilizes environmental cues, such as carbohydrates, to coordinate the expression of virulence factors. Research efforts to date have focused on identifying how either components of the phosphoenolpyruvate-phosphotransferase system (PTS) or global transcriptional networks affect the regulation of virulence factors, but not the synergistic relationship between the two. The present study investigates the role of a putative PTS-fructose operon encoded by fruRBA and its role in virulence in the M1T1 strain 5448. Growth in fructose resulted in induction of fruRBA. RT-PCR showed that fruRBA formed an operon, which was repressed by FruR in the absence of fructose. Growth and carbon utilization profiles revealed that although the entire fruRBA operon was required for growth in fructose, FruA was the main fructose transporter. The ability of both ΔfruR and ΔfruB mutants to survive in whole human blood or neutrophils was impaired. However, the phenotypes were not reproduced in murine whole blood or in a mouse intraperitoneal infection, indicating a human-specific mechanism. While it is known that the PTS can affect activity of the Mga virulence regulator, further characterization of the mechanism by which sugars and its protein domains affect activity have not been studied. Transcriptional studies revealed that the core Mga regulon is activated more in a glucose-rich than a glucose-poor environment. This activation correlates with the differential phosphorylation of Mga at its PTS regulatory domains (PRDs). Using a 5448 mga mutant, transcriptome studies in THY or C media established that the Mga regulon reflects the media used. Interestingly, Mga regulates phage-encoded DNases in a low glucose environment. We also show that Mga activity is dependent on C-terminal amino acid interactions that aid in the formation of homodimers. Overall, the studies presented sought to define how external environmental cues, specifically carbohydrates, control complex regulatory networks used by GAS, contribute to pathogenesis, and aid in adaptation to various nutrient conditions encountered.

PDIA3, HSPA5 and viementin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78 kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHD