(Pathogenic mechanisms of "Streptococcus pneumoniae" involved in invasive disease and chronic obstructive pulmonary disease)

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Microbiología III, leída el 16-06-2017Streptococcus pneumoniae, también conocido como neumococo, es uno de los patógenos humanos más importantes responsable de infecciones severas tales como neumonía bacteriémica y meningitis, así como de enfermedades no tan graves como neumonía adquirida en la comunidad, otitis media aguda, sinusitis y conjuntivitis (Bogaert et al., 2004), que afectan principalmente a niños, personas mayores de 65 años y pacientes inmunocomprometidos (Koedel et al., 2002; van der Poll y Opal, 2009). La enfermedad neumocócica es precedida por la colonización de la nasofaringe, que es particularmente común en niños con más de un serotipo colonizando simultáneamente (Bogaert et al., 2004). Se calcula que la tercera parte de las muertes anuales que se producen en el mundo son debidas a enfermedades infecciosas y, entre todas ellas, las enfermedades respiratorias son las responsables de la muerte de 4 millones de personas cada año (Morens et al., 2004). Según la Organización Mundial de la Salud (OMS), la sepsis, la neumonía neonatal y la neumonía comunitaria en niños más mayores fueron responsables del 15% de las casi un millón de muertes anuales en niños menores de 5 años ocurridas en el 2015, siendo S. pneumoniae el causante más frecuente de neumonía infantil severa, tanto en países desarrollados como en aquellos en vías de desarrollo (OMS, 2016; UNICEF, 2015; UNICEF y WHO, 2006). La capsula polisacarídica está considerada como uno de los principales factores de virulencia de la bacteria (López y García, 2004), de la cual se han descrito más de 96 polisacáridos capsulares (CPS) diferentes (Jauneikaite et al., 2015). Las vacunas conjugadas existentes, como son la PCV7, PCV10 y PCV13, confieren protección frente a la enfermedad neumocócica invasiva (ENI) (Feikin et al., 2013; O'Brien y Levine, 2006). La activación de la cascada del complemento por anticuerpos específicos conduce a la formación del componente clave C3b, el cual juega un papel crucial en la defensa del hospedador frente a neumococo, recubriendo los microorganismos y estimulando la fagocitosis. Sin embargo, uno de los inconvenientes de las vacunas conjugadas es el limitado número de serotipos frente a los que protegen y, como consecuencia de la introducción de las mismas, el fenómeno conocido como reemplazo de serotipos o “capsular switching” (Coffey et al., 1991), que permite a los clones preexistentes de S. pneumoniae que expresan serotipos vacunales escapar de la inmunidad inducida por la vacunación mediante la adquisición de genes capsulares de neumococo no incluidos en las vacunas (Brueggemann et al., 2007; Golubchik et al., 2012)...Streptococcus pneumoniae, the pneumococcus, is an important human pathogen that usually colonizes asymptomatically the mucosal surfaces of the upper respiratory tract in early childhood with more than one serotype colonizing the nasopharynx of the same individual at the same time (Bogaert et al., 2004). Once carriage is established, the microorganism may invade several sterile sites, leading to what is known as invasive pneumococcal disease (IPD). Indeed, the pneumococcus is one of the major etiologic agents causing episodes of communityacquired pneumonia, bacteremia and meningitis. However, S. pneumoniae is also a major cause of non-invasive diseases such as non-bacteraemic pneumonia, acute otitis media, sinusitis and conjunctivitis, mainly in children, the elderly, and immunocompromised patients (Henriques- Normark and Tuomanen, 2013). More than 25% of the 57 million annual deaths worldwide are estimated to be directly related to infectious diseases. Particularly, respiratory infections are responsible of the death of 4 million people every year. According to the World Health Organization (WHO), estimates of sepsis or pneumonia in neonates and also pneumonia in older children accounted for 15% of the nearly 1 million deaths of children younger than 5 years old reported in 2015, being Streptococcus pneumoniae the most common cause of severe pneumonia among children in more and less developed countries (OMS, 2016; UNICEF, 2015; UNICEF and WHO, 2006). The capsule is considered the main pneumococcal virulence factor and up to 96% distinct capsular polysaccharides (CPS) have been described (Jauneikaite et al., 2015). Current prophylactic measures against pneumococcal infections such as the introduction of PCV7, PCV10 and PCV13, are a highly efficient approach for preventing IPD. Activation of complement cascades by specific antibodies leads to the formation of C3b, the key complement component that is crucial in host defense against pneumococcus by coating the microorganism and stimulating phagocytosis (Markieswski and Lambris, 2007). However, among the main disadvantages of conjugated vaccines are the limited number of serotypes covered and the phenomenon known as “capsular switching” (Coffey et al., 1991) which allows pre-existing clones of S. pneumoniae expressing vaccine serotypes to escape vaccine-induced immunity by acquisition of capsular genes from non-vaccine serotypes (Brueggemann et al., 2007; Golubchik et al., 2012)...Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEunpu

Emergence of Amoxicillin-Resistant Variants of Spain(9V)-ST156 Pneumococci Expressing Serotype 11A Correlates with Their Ability to Evade the Host Immune Response

Capsular switching allows pre-existing clones of Streptococcus pneumoniae expressing vaccine serotypes to escape the vaccine-induced immunity by acquisition of capsular genes from pneumococci of a non-vaccine serotype. Here, we have analysed the clonal composition of 492 clinical isolates of serotype 11A causing invasive disease in Spain (2000-2012), and their ability to evade the host immune response. Antibiograms, serotyping and molecular typing were performed. The restriction profiles of pbp2x, pbp1a and pbp2b genes were also analysed. Interaction with the complement components C1q, C3b, C4BP, and factor H was explored whereas opsonophagocytosis assays were performed using a human cell line differentiated to neutrophils. Biofilm formation and the polymorphisms of the major autolysin LytA were evaluated. The main genotypes of the 11A pneumococci were: ST62 (447 isolates, 90.6%), followed by ST6521 (35 isolates, 7.3%) and ST838 (10 isolates, 2.1%). Beta lactam resistant serotype 11A variants of genotypes ST838 and ST6521 closely related to the Spain9V-ST156 clone were first detected in 2005. A different pattern of evasion of complement immunity and phagocytosis was observed between genotypes. The emergence of one vaccine escape variant of Spain9V-ST156 (ST652111A), showing a high potential to avoid the host immune response, was observed. In addition, isolates of ST652111A showed higher ability to produce biofilms than ST83811A or ST6211A, which may have contributed to the emergence of this PEN-resistant ST652111A genotype in the last few years. The emergence of penicillin-resistant 11A invasive variants of the highly successful ST156 clonal complex merits close monitoring

Promoting Fc-Fc interactions between anti-capsular antibodies provides strong immune protection against Streptococcus pneumoniae.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and an important cause of childhood mortality. Despite the introduction of successful vaccines, the global spread of both non-vaccine serotypes and antibiotic-resistant strains reinforces the development of alternative therapies against this pathogen. One possible route is the development of monoclonal antibodies (mAbs) that induce killing of bacteria via the immune system. Here, we investigate whether mAbs can be used to induce killing of pneumococcal serotypes for which the current vaccines show unsuccessful protection. Our study demonstrates that when human mAbs against pneumococcal capsule polysaccharides (CPS) have a poor capacity to induce complement activation, a critical process for immune protection against pneumococci, their activity can be strongly improved by hexamerization-enhancing mutations. Our data indicate that anti-capsular antibodies may have a low capacity to form higher-order oligomers (IgG hexamers) that are needed to recruit complement component C1. Indeed, specific point mutations in the IgG-Fc domain that strengthen hexamerization strongly enhance C1 recruitment and downstream complement activation on encapsulated pneumococci. Specifically, hexamerization-enhancing mutations E430G or E345K in CPS6-IgG strongly potentiate complement activation on S. pneumoniae strains that express capsular serotype 6 (CPS6), and the highly invasive serotype 19A strain. Furthermore, these mutations improve complement activation via mAbs recognizing CPS3 and CPS8 strains. Importantly, hexamer-enhancing mutations enable mAbs to induce strong opsonophagocytic killing by human neutrophils. Finally, passive immunization with CPS6-IgG1-E345K protected mice from developing severe pneumonia. Altogether, this work provides an important proof of concept for future optimization of antibody therapies against encapsulated bacteria

Promoting Fc-Fc interactions between anti-capsular antibodies provides strong immune protection against Streptococcus pneumoniae.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and an important cause of childhood mortality. Despite the introduction of successful vaccines, the global spread of both non-vaccine serotypes and antibiotic-resistant strains reinforces the development of alternative therapies against this pathogen. One possible route is the development of monoclonal antibodies (mAbs) that induce killing of bacteria via the immune system. Here, we investigate whether mAbs can be used to induce killing of pneumococcal serotypes for which the current vaccines show unsuccessful protection. Our study demonstrates that when human mAbs against pneumococcal capsule polysaccharides (CPS) have a poor capacity to induce complement activation, a critical process for immune protection against pneumococci, their activity can be strongly improved by hexamerization-enhancing mutations. Our data indicate that anti-capsular antibodies may have a low capacity to form higher-order oligomers (IgG hexamers) that are needed to recruit complement component C1. Indeed, specific point mutations in the IgG-Fc domain that strengthen hexamerization strongly enhance C1 recruitment and downstream complement activation on encapsulated pneumococci. Specifically, hexamerization-enhancing mutations E430G or E345K in CPS6-IgG strongly potentiate complement activation on S. pneumoniae strains that express capsular serotype 6 (CPS6), and the highly invasive serotype 19A strain. Furthermore, these mutations improve complement activation via mAbs recognizing CPS3 and CPS8 strains. Importantly, hexamer-enhancing mutations enable mAbs to induce strong opsonophagocytic killing by human neutrophils. Finally, passive immunization with CPS6-IgG1-E345K protected mice from developing severe pneumonia. Altogether, this work provides an important proof of concept for future optimization of antibody therapies against encapsulated bacteria

Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria

Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice. In addition, the Fc-engineered variant improves on-target complement-mediated killing of cancer cells as well as both gram-positive and gram-negative bacteria. Hence, this versatile Fc technology should be broadly applicable in antibody design aiming for long-acting prophylactic or therapeutic interventions

Vaccination with LytA, LytC, or Pce of Streptococcus pneumoniae Protects against Sepsis by Inducing IgGs That Activate the Complement System.

The emergence of non-vaccine serotypes of Streptococcus pneumoniae after the use of vaccines based in capsular polysaccharides demonstrates the need of a broader protection vaccine based in protein antigens and widely conserved. In this study, we characterized three important virulence factors of S. pneumoniae namely LytA, LytC, and Pce as vaccine candidates. These proteins are choline-binding proteins that belong to the cell wall hydrolases' family. Immunization of mice with LytA, LytC, or Pce induced high titers of immunoglobulins G (IgGs) of different subclasses, with IgG1, IgG2a, and IgG2b as the predominant immunoglobulins raised. These antibodies activated the classical pathway of the complement system by increasing the recognition of C1q on the surface of pneumococcal strains of different serotypes. Consequently, the key complement component C3 recognized more efficiently these strains in the presence of specific antibodies elicited by these proteins, activating, therefore, the phagocytosis. Finally, a mouse sepsis model of infection was established, confirming that vaccination with these proteins controlled bacterial replication in the bloodstream, increasing the survival rate. Overall, these results demonstrate that LytA, LytC, and Pce can be protein antigens to be contained in a future universal vaccine against S. pneumoniae.This research was funded by Ministerio de Economía, Industria y Competitividad (grant number SAF2017-83388). B.C. and L.A. were supported, respectively, by a fellowship from the Brazilian Program Ciencia Sem Fronteiras (CsF) from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and by an FPI fellowship from MINECO.S

Immunization with LytB protein of Streptococcus pneumoniae activates complement-mediated phagocytosis and induces protection against pneumonia and sepsis.

The cell wall glucosaminidase LytB of Streptococcus pneumoniae is a surface exposed protein involved in daughter cell separation, biofilm formation and contributes to different aspects of the pathogenesis process. In this study we have characterized the antibody responses after immunization of mice with LytB in the presence of alhydrogel as an adjuvant. Enzyme-linked immunosorbent assays measuring different subclasses of immunoglobulin G, demonstrated that the antibody responses to LytB were predominantly IgG1 and IgG2b, followed by IgG3 and IgG2a subclasses. Complement-mediated immunity against two different pneumococcal serotypes was investigated using sera from immunized mice. Immunization with LytB increased the recognition of S. pneumoniae by complement components C1q and C3b demonstrating that anti-LytB antibodies trigger activation of the classical pathway. Phagocytosis assays showed that serum containing antibodies to LytB stimulates neutrophil-mediated phagocytosis against S. pneumoniae. Animal models of infection including invasive pneumonia and sepsis were performed with two different clinical isolates. Vaccination with LytB increased bacterial clearance and induced protection demonstrating that LytB might be a good candidate to be considered in a future protein-based vaccine against S. pneumoniae.This work was supported by grants SAF2012-39444-C01/02 from Ministerio de Economía y Competitividad (MINECO). Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES) is an initiative of ISCIII. BC and LA were supported, respectively, by a fellowship from the Brazilian Program Ciencia Sem Fronteiras (CsF) from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and by an FPI fellowship from MINECO. The authors wish to thank Sandra Martín for technical assistanceS

Validation of new therapeutic targets in antibacterials throughout an academic Drug Discovery Platform

2 p.The relentless increase in bacterial resistance to our current armamentarium of antibiotics could shortly take Europe and other global communities into a post-antibiotic era in which many common infections would be unable to be effectively treated. To avert this danger requires a fundamental change in antibacterial drug discovery that will involve the identification of new targets, the development of new chemical compounds. We present the MHit initiative, a Drug Discovery Platform that provides the framework for blending the academia innovative approaches together with their translation in real practical solutions, where the industry plays a fundamental role.Peer reviewe

Auranofin efficacy against MDR Streptococcus pneumoniae and Staphylococcus aureus infections

Aguinagalde, Leire et al.[Background] Auranofin is an FDA-approved, gold-containing compound in clinical use for the oral treatment of rheumatoid arthritis and has been recently granted by the regulatory authorities due to its antiprotozoal properties.[Methods] A reprofiling strategy was performed with a Streptococcus pneumoniae phenotypic screen and a proprietary library of compounds, consisting of both FDA-approved and unapproved bioactive compounds. Two different multiresistant S. pneumoniae strains were employed in a sepsis mouse model of infection. In addition, an MRSA strain was tested using both the thigh model and a mesh-associated biofilm infection in mice.[Results] The repurposing approach showed the high potency of auranofin against multiresistant clinical isolates of S. pneumoniae and Staphylococcus aureus in vitro and in vivo. Efficacy in the S. pneumoniae sepsis model was obtained using auranofin by the oral route in the dose ranges used for the treatment of rheumatoid arthritis. Thioglucose replacement by alkyl chains showed that this moiety was not essential for the antibacterial activity and led to the discovery of a new gold derivative (MH05) with remarkable activity in vitro and in vivo.[Conclusions] Auranofin and the new gold derivative MH05 showed encouraging in vivo activity against multiresistant clinical isolates of S. pneumoniae and S. aureus. The clinical management of auranofin, alone or in combination with other antibiotics, deserves further exploration before use in patients presenting therapeutic failure caused by infections with multiresistant Gram-positive pathogens. Decades of clinical use mean that this compound is safe to use and may accelerate its evaluation in humans.This work was supported by grants SAF2009-10824, SAF2012-39444-C01/02, SAF2013-48271, BIO2011-30503, AES/PI12/01628 (Ministerio de Economía y Competitividad; MINECO), CM S2011/BMED-2353 (Comunidad de Madrid) and FEDER Funds. Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES) is an initiative of the Instituto de Salud Carlos III (ISCIII). L. A. was supported by an FPI fellowship from MINECO.Peer Reviewe