RESUMEN
Organ transplantation is a modern medical success story. However, since its inception it has been limited by the need for pharmacological immunosuppression. Regulatory cellular therapies offer an attractive solution to these challenges by controlling transplant alloresponses through multiple parallel suppressive mechanisms. A number of cell types have seen an accelerated development into human trials and are now on the threshold of a long-awaited breakthrough in personalized transplant therapeutics. Here we assess recent developments with a focus on the most likely candidates, some of which have already facilitated successful immunosuppression withdrawal in early clinical trials. We propose that this may constitute a promising approach in clinical transplantation but also evaluate outstanding issues in the field, providing cause for cautious optimism.
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Trasplante de Órganos , Tolerancia al Trasplante , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Tolerancia Inmunológica , Terapia de InmunosupresiónRESUMEN
Neisseria meningitidis serogroup B (MenB) is the leading cause of meningococcal meningitis and sepsis in industrialized countries, with the highest incidence in infants and adolescents. Two recombinant protein vaccines that protect against MenB are now available (i.e. 4CMenB and MenB-fHbp). Both vaccines contain the Factor H Binding Protein (fHbp) antigen, which can bind the Human Factor H (fH), the main negative regulator of the alternative complement pathway, thus enabling bacterial survival in the blood. fHbp is present in meningococcal strains as three main variants which are immunologically distinct. Here we sought to obtain detailed information about the epitopes targeted by anti-fHbp antibodies induced by immunization with the 4CMenB multicomponent vaccine. Thirteen anti-fHbp human monoclonal antibodies (mAbs) were identified in a library of over 100 antibody fragments (Fabs) obtained from three healthy adult volunteers immunized with 4CMenB. Herein, the key cross-reactive mAbs were further characterized for antigen binding affinity, complement-mediated serum bactericidal activity (SBA) and the ability to inhibit binding of fH to live bacteria. For the first time, we identified a subset of anti-fHbp mAbs able to elicit human SBA against strains with all three variants and able to compete with human fH for fHbp binding. We present the crystal structure of fHbp v1.1 complexed with human antibody 4B3. The structure, combined with mutagenesis and binding studies, revealed the critical cross-reactive epitope. The structure also provided the molecular basis of competition for fH binding. These data suggest that the fH binding site on fHbp v1.1 can be accessible to the human immune system upon immunization, enabling elicitation of human mAbs broadly protective against MenB. The novel structural, biochemical and functional data are of great significance because the human vaccine-elicited mAbs are the first reported to inhibit the binding of fH to fHbp, and are bactericidal with human complement. Our studies provide molecular insights into the human immune response to the 4CMenB meningococcal vaccine and fuel the rationale for combined structural, immunological and functional studies when seeking deeper understanding of the mechanisms of action of human vaccines.
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Anticuerpos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Meningitis Meningocócica/inmunología , Vacunas Meningococicas/administración & dosificación , Neisseria meningitidis/inmunología , Adulto , Anticuerpos/sangre , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Humanos , Meningitis Meningocócica/metabolismo , Meningitis Meningocócica/microbiología , Meningitis Meningocócica/prevención & controlRESUMEN
Cutaneous squamous cell carcinoma (CSCC) is a major cause of morbidity and mortality after organ transplant. Many patients subsequently develop multiple CSCC following a first CSCC, and the risk of metastasis and death is significantly increased compared to the general population. Post-transplant CSCC represents a disease at the interface of dermatology and transplant medicine. Both systemic chemoprevention and modulation of immunosuppression are frequently employed in patients with multiple CSCC, yet there is little consensus on their use after first CSCC to reduce risk of subsequent tumors. While relatively few controlled trials have been undertaken, extrapolation of observational data suggests the most effective interventions may be at the time of first CSCC. We review the need for intervention after a first post-transplant CSCC and evidence for use of various approaches as secondary prevention, before discussing barriers preventing engagement with this approach and finally highlight areas for future research. Close collaboration between specialties to ensure prompt deployment of these interventions after a first CSCC may improve patient outcomes.
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Carcinoma de Células Escamosas , Neoplasias Cutáneas , Humanos , Carcinoma de Células Escamosas/etiología , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/prevención & controlRESUMEN
The 4 component meningococcus B vaccine (4CMenB) vaccine is the first vaccine containing recombinant proteins licensed for the prevention of invasive meningococcal disease caused by meningococcal serogroup B strains. 4CMenB contains 3 main recombinant proteins, including the Neisseria meningitidis factor H binding protein (fHbp), a lipoprotein able to bind the human factor H. To date, over 1000 aa sequences of fHbp have been identified, and they can be divided into variant groups 1, 2, and 3, which are usually not crossprotective. Nevertheless, previous characterizations of a small set (n = 10) of mAbs generated in humans after 4CMenB immunization revealed 2 human Fabs (huFabs) (1A12, 1G3) with some crossreactivity for variants 1, 2, and 3. This unexpected result prompted us to examine a much larger set of human mAbs (n = 110), with the aim of better understanding the extent and nature of crossreactive anti-fHbp antibodies. In this study, we report an analysis of the human antibody response to fHbp, by the characterization of 110 huFabs collected from 3 adult vaccinees during a 6-mo study. Although the 4CMenB vaccine contains fHbp variant 1, 13 huFabs were also found to be crossreactive with variants 2 and 3. The crystal structure of the crossreactive huFab 1E6 in complex with fHbp variant 3 was determined, revealing a novel, highly conserved epitope distinct from the epitopes recognized by 1A12 or 1G3. Further, functional characterization shows that human mAb 1E6 is able to elicit rabbit, but not human, complement-mediated bactericidal activity against meningococci displaying fHbp from any of the 3 different variant groups. This functional and structural information about the human antibody response upon 4CMenB immunization contributes to further unraveling the immunogenic properties of fHbp. Knowledge gained about the epitope profile recognized by the human antibody repertoire could guide future vaccine design.-Bianchi, F., Veggi, D., Santini, L., Buricchi, F., Bartolini, E., Lo Surdo, P., Martinelli, M., Finco, O., Masignani, V., Bottomley, M. J., Maione, D., Cozzi, R. Cocrystal structure of meningococcal factor H binding protein variant 3 reveals a new crossprotective epitope recognized by human mAb 1E6.
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Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Factor H de Complemento/inmunología , Epítopos/inmunología , Vacunas Meningococicas/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factor H de Complemento/genética , Factor H de Complemento/metabolismo , Cristalografía por Rayos X , Epítopos/genética , Epítopos/metabolismo , Variación Genética , Humanos , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/prevención & control , Vacunas Meningococicas/administración & dosificación , Modelos Moleculares , Neisseria meningitidis/efectos de los fármacos , Neisseria meningitidis/inmunología , Neisseria meningitidis/fisiología , Unión Proteica , Conformación ProteicaRESUMEN
Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. In immunosuppressed populations it is a source of considerable morbidity and mortality due to its enhanced recurrence and metastatic potential. In common with many malignancies, leucocyte populations are both protective against cancer development and also play a role in 'sculpting' the nascent tumor, leading to loss of immunogenicity and tumor progression. UV radiation and chronic viral carriage may represent unique risk factors for cSCC development, and the immune system plays a key role in modulating the response to both. In this review, we discuss the lessons learned from animal and ex vivo human studies of the role of individual leucocyte subpopulations in the development of cutaneous SCC. We then discuss the insights into cSCC immunity gleaned from studies in humans, particularly in populations receiving pharmacological immunosuppression such as transplant recipients. Similar insights in other malignancies have led to exciting and novel immune therapies, which are beginning to emerge into the cSCC clinical arena.
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Inmunidad Adaptativa , Carcinoma de Células Escamosas/inmunología , Inmunidad Innata , Neoplasias Cutáneas/inmunología , Piel/inmunología , Animales , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/terapia , Humanos , Inmunoterapia , Piel/patología , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapiaRESUMEN
Stability is one of the critical attributes of a protein-based therapeutic or vaccine product, which is directly linked to product quality and efficacy. Elucidating protein degradation pathways is required to obtain thorough understanding of the product and ensure degradation products are properly monitored. We observed a unique protein degradation involving nonenzyme catalyzed loss of a complete N-linked glycan under stress condition from an engineered respiratory syncytial virus (RSV) prefusion F protein (RSVPreF3). Investigations involving mass spectrometry, molecular modeling, and mutagenesis revealed that the glycan shedding was site-specific, dependent on structural elements, and required a glycine residue immediately following the site of glycosylation. The glycan loss did not negatively affect the binding between the main immunogenic epitope Site Ø and the neutralizing antibody D25. Further study indicated that the glycan shedding followed a similar but different mechanism than that of conventional deamidation. Since glycosylation is an important attribute for many recombinant therapeutic proteins or vaccine antigens, the finding from this study suggests the need to monitor this new type of degradation, especially when glycosylation has an impact on efficacy or safety.
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Polisacáridos/análisis , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/química , Proteínas Virales/química , Calor , Humanos , Modelos Moleculares , Estabilidad Proteica , ProteolisisRESUMEN
Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of 'conformational selection' by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in many MarR family proteins, and (iv) four residues (His7, Ser9, Asn11 and Phe25), which are involved in binding 4-HPA, and were confirmed in vitro to have key roles in the regulatory mechanism in bacteria. Overall, this study deepens our molecular understanding of the sophisticated regulatory mechanisms of the expression of nadA and other genes governed by NadR, dependent on interactions with niche-specific signal molecules that may play important roles during meningococcal pathogenesis.
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Proteínas Bacterianas/química , Meningitis Meningocócica/inmunología , Proteínas Represoras/química , Factores de Virulencia/química , Adhesinas Bacterianas/biosíntesis , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Western Blotting , Rastreo Diferencial de Calorimetría , Cromatografía Líquida de Alta Presión , Regulación Bacteriana de la Expresión Génica , Humanos , Espectroscopía de Resonancia Magnética , Mutagénesis Sitio-Dirigida , Neisseria meningitidis Serogrupo B/química , Neisseria meningitidis Serogrupo B/inmunología , Conformación Proteica , Proteínas Represoras/inmunología , Proteínas Represoras/metabolismo , Resonancia por Plasmón de Superficie , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismo , Difracción de Rayos XRESUMEN
Factor H-binding protein (fHbp) is an important antigen of Neisseria meningitidis that is capable of eliciting a robust protective immune response in humans. Previous studies on the interactions of fHbp with antibodies revealed that some anti-fHbp monoclonal antibodies that are unable to trigger complement-mediated bacterial killing in vitro are highly co-operative and become bactericidal if used in combination. Several factors have been shown to influence such co-operativity, including IgG subclass and antigen density. To investigate the structural basis of the anti-fHbp antibody synergy, we determined the crystal structure of the complex between fHbp and the Fab (fragment antigen-binding) fragment of JAR5, a specific anti-fHbp murine monoclonal antibody known to be highly co-operative with other monoclonal antibodies. We show that JAR5 is highly synergic with monoclonal antibody (mAb) 12C1, whose structure in complex with fHbp has been previously solved. Structural analyses of the epitopes recognized by JAR5 and 12C1, and computational modeling of full-length IgG mAbs of JAR5 and 12C1 bound to the same fHbp molecule, provide insights into the spatial orientation of Fc (fragment crystallizable) regions and into the possible implications for the susceptibility of meningococci to complement-mediated killing.
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Anticuerpos Monoclonales/metabolismo , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Neisseria meningitidis/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/química , Proteínas Bacterianas/química , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Epítopos/inmunología , Epítopos/metabolismo , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
Most morbidity associated with malignancy in long-term renal transplant recipients is due to cutaneous squamous cell carcinoma (SCC). Previously identified measures to stratify SCC risk have limited use, however. We hypothesized that an increased proportion of senescent, terminally differentiated CD8(+) T cells would identify renal transplant recipients at elevated SCC risk. Peripheral blood lymphocytes were isolated from 117 stable transplant recipients at high risk of SCC and analyzed phenotypically by flow cytometry. Participants were followed up prospectively for SCC development. The predictive value of variables was assessed using Cox regression. Age at transplant and enrollment, dialysis duration, and previous disease were predictive of SCC development during follow-up. Previously published clinical phenotype-based risk scores lost predictive value with the removal of age as a covariate. The percentage of CD57-expressing CD8(+) T cells was the strongest immunologic predictor of future SCC and correlated with increasing CD8(+) T cell differentiation. We dichotomized participants into those with a majority (CD57hi) and a minority (CD57lo) of CD8(+) T cells expressing CD57; CD57hi participants were more likely to develop SCC during follow-up (hazard ratio, 2.9; 95% confidence interval, 1.0 to 8.0), independent of potential confounders, and tended to develop earlier recurrence. The CD57hi phenotype was stable with time and associated with increasing age and cytomegalovirus seropositivity. Our results show that the CD57hi phenotype is a strong predictor of SCC development and recurrence in this cohort of long-term, high-risk renal transplant recipients. This information may allow identification of recipients who may benefit from intensive dermatologic screening and immunosuppression reduction.
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Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Escamosas/epidemiología , Inmunosenescencia , Trasplante de Riñón , Complicaciones Posoperatorias/epidemiología , Neoplasias Cutáneas/epidemiología , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Medición de RiesgoRESUMEN
Mapping of epitopes recognized by functional monoclonal antibodies (mAbs) is essential for understanding the nature of immune responses and designing improved vaccines, therapeutics, and diagnostics. In recent years, identification of B-cell epitopes targeted by neutralizing antibodies has facilitated the design of peptide-based vaccines against highly variable pathogens like HIV, respiratory syncytial virus, and Helicobacter pylori; however, none of these products has yet progressed into clinical stages. Linear epitopes identified by conventional mapping techniques only partially reflect the immunogenic properties of the epitope in its natural conformation, thus limiting the success of this approach. To investigate antigen-antibody interactions and assess the potential of the most common epitope mapping techniques, we generated a series of mAbs against factor H binding protein (fHbp), a key virulence factor and vaccine antigen of Neisseria meningitidis. The interaction of fHbp with the bactericidal mAb 12C1 was studied by various epitope mapping methods. Although a 12-residue epitope in the C terminus of fHbp was identified by both Peptide Scanning and Phage Display Library screening, other approaches, such as hydrogen/deuterium exchange mass spectrometry (MS) and X-ray crystallography, showed that mAb 12C1 occupies an area of â¼1,000 Å(2) on fHbp, including >20 fHbp residues distributed on both N- and C-terminal domains. Collectively, these data show that linear epitope mapping techniques provide useful but incomplete descriptions of B-cell epitopes, indicating that increased efforts to fully characterize antigen-antibody interfaces are required to understand and design effective immunogens.
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Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Epítopos/inmunología , Vacunas Meningococicas/inmunología , Neisseria meningitidis/inmunología , Neisseria meningitidis/patogenicidad , Factores de Virulencia/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Antígenos Bacterianos/química , Proteínas Bacterianas/química , Técnicas de Visualización de Superficie Celular , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Mapeo Epitopo , Epítopos/química , Espectrometría de Masas , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/prevención & control , Modelos Moleculares , Péptidos/química , Péptidos/inmunología , Unión Proteica/inmunología , Resonancia por Plasmón de Superficie , Factores de Virulencia/químicaRESUMEN
The use of protein X-ray crystallography for structure-based design of small-molecule drugs is well-documented and includes several notable success stories. However, it is less well-known that structural biology has emerged as a major tool for the design of novel vaccine antigens. Here, we review the important contributions that protein crystallography has made so far to vaccine research and development. We discuss several examples of the crystallographic characterization of vaccine antigen structures, alone or in complexes with ligands or receptors. We cover the critical role of high-resolution epitope mapping by reviewing structures of complexes between antigens and their cognate neutralizing, or protective, antibody fragments. Most importantly, we provide recent examples where structural insights obtained via protein crystallography have been used to design novel optimized vaccine antigens. This review aims to illustrate the value of protein crystallography in the emerging discipline of structural vaccinology and its impact on the rational design of vaccines.
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Antígenos Bacterianos/química , Antígenos Virales/química , Epítopos/química , Vacunas Sintéticas/química , Secuencia de Aminoácidos , Antígenos Bacterianos/inmunología , Antígenos Virales/inmunología , Sitios de Unión de Anticuerpos , Cristalografía , Epítopos/inmunología , Datos de Secuencia Molecular , Vacunas Sintéticas/inmunologíaRESUMEN
Staphylococcus aureus is a human pathogen causing globally significant morbidity and mortality. The development of antibiotic resistance in S. aureus highlights the need for a preventive vaccine. In the present paper we explore the structure and function of FhuD2 (ferric-hydroxamate uptake D2), a staphylococcal surface lipoprotein mediating iron uptake during invasive infection, recently described as a promising vaccine candidate. Differential scanning fluorimetry and calorimetry studies revealed that FhuD2 is stabilized by hydroxamate siderophores. The FhuD2-ferrichrome interaction was of nanomolar affinity in surface plasmon resonance experiments and fully iron(III)-dependent. We determined the X-ray crystallographic structure of ligand-bound FhuD2 at 1.9 Å (1 Å=0.1 nm) resolution, revealing the bilobate fold of class III SBPs (solute-binding proteins). The ligand, ferrichrome, occupies a cleft between the FhuD2 N- and C-terminal lobes. Many FhuD2-siderophore interactions enable the specific recognition of ferrichrome. Biochemical data suggest that FhuD2 does not undergo significant conformational changes upon siderophore binding, supporting the hypothesis that the ligand-bound complex is essential for receptor engagement and uptake. Finally, immunizations with FhuD2 alone or FhuD2 formulated with hydroxamate siderophores were equally protective in a murine staphylococcal infection model, confirming the suitability and efficacy of apo-FhuD2 as a protective antigen, and suggesting that other class III SBPs might also be exploited as vaccine candidates.
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Proteínas Bacterianas/química , Proteínas de Transporte de Membrana/química , Proteínas de Unión Periplasmáticas/química , Staphylococcus aureus/metabolismo , Factores de Virulencia/química , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Compuestos Férricos/metabolismo , Ferricromo/metabolismo , Genes Bacterianos , Humanos , Ácidos Hidroxámicos/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/inmunología , Proteínas de Transporte de Membrana/metabolismo , Ratones , Modelos Moleculares , Proteínas de Unión Periplasmáticas/genética , Proteínas de Unión Periplasmáticas/inmunología , Proteínas de Unión Periplasmáticas/metabolismo , Estabilidad Proteica , Sideróforos/metabolismo , Vacunas Estafilocócicas/química , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Staphylococcus aureus/patogenicidad , Electricidad Estática , Transferrina/metabolismo , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismoRESUMEN
In the human pathogen Staphylococcus aureus, there exists an enormous diversity of proteins containing DUFs (domains of unknown function). In the present study, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to Staphylococci. The 18 Csa paralogues in S. aureus Newman are highly similar at the sequence level, yet were found to be expressed in multiple cellular locations. Extracellular Csa1A was shown to be post-translationally processed and released. Molecular interaction studies revealed that Csa1A interacts with other Csa paralogues, suggesting that these proteins are involved in the same cellular process. The structures of Csa1A and Csa1B were determined by X-ray crystallography, unveiling a peculiar structure with limited structural similarity to other known proteins. Our results provide the first detailed biological characterization of this family and confirm the uniqueness of this family also at the structural level. We also provide evidence that Csa family members elicit protective immunity in in vivo animal models of staphylococcal infections, indicating a possible important role for these proteins in S. aureus biology and pathogenesis. These findings identify the Csa family as new potential vaccine candidates, and underline the importance of mining the bacterial unknown proteome to identify new targets for preventive vaccines.
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Antígenos Bacterianos/química , Proteínas Bacterianas/química , Proteoma/química , Staphylococcus aureus/metabolismo , Animales , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Minería de Datos , Ratones , Ratones Endogámicos , Proteoma/genética , Proteoma/metabolismo , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/inmunologíaRESUMEN
Spatial transcriptomic (ST) profiling is the mapping of gene expression within cell populations with preservation of positional context and represents an exciting new approach to develop our understanding of local and regional influences upon skin biology in health and disease. With the ability to probe from a few hundred transcripts to the entire transcriptome, multiple ST approaches are now widely available. In this paper, we review the ST field and discuss its application to dermatology. Its potential to advance our understanding of skin biology in health and disease is highlighted through the illustrative examples of 3 research areas: cutaneous aging, tumorigenesis, and psoriasis.
RESUMEN
Thousands of disease cases and hundreds of deaths occur globally each year due to invasive meningococcal disease. Neisseria meningitidis serogroup B (MenB) is the leading cause of such disease in developed countries. Two vaccines, 4CMenB and MenB-fHbp, that protect against MenB are available and include one or two forms respectively of factor H binding protein (fHbp), a key protective antigen. Studies of circulating meningococci have identified over 1380 different fHbp amino acid sequences, which form three immunologically distinct clusters, termed variants 1, 2, and 3. Neither of the current vaccines contains a variant 2 antigen, which is less well characterized than fHbp variants 1 and 3. We characterized the interaction of fHbp variant 2 with humAb 1B1 using biochemical methods and live meningococcal assays. Further, we determined the crystal structure of the complex at 2.4 Å resolution, clearly revealing the epitope and providing the first detailed report of an antibody with distinct specificity for fHbp variant 2. Extensive mutagenesis and binding studies elucidated key hotspots in the interface. This combination of structural and functional studies provides a molecular explanation for the bactericidal potency and specificity of humAb 1B1 for fHbp variant 2. Our studies, focused on fHbp variant 2, expand the understanding of this previously under characterized group of the vast family of variants of fHbp, a virulence factor present on all meningococci. Moreover, the definition of a protective conformational epitope on fHbp variant 2 may support the design and development of novel variant 2-containing MenB vaccines affording greater breadth of protection.
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INTRODUCTION: Powerful immunosuppressive regimens have reduced rejection risk, leading to an expanding cohort of long-term kidney transplant recipients who are likely to encounter practitioners in other specialties. SOURCES OF DATA: Key review papers and primary literature identified through searches of PubMed, Google Scholar and Medline. AREAS OF AGREEMENT: Death from cardiovascular disease and malignancy remain the chief causes of transplant loss. Risk factors and phenotypes for these differ from the general population. AREAS OF CONTROVERSY: Many guidelines for renal transplant recipients are based on extrapolation from studies on non-transplant cohorts and may not be appropriate. Emerging studies demonstrate that established interventions in the general population are less efficacious in transplant recipients. GROWING POINTS: The influence of immunosuppression on the development of complications. AREAS TIMELY FOR DEVELOPING RESEARCH: Markers to guide individualized optimal immunosuppression and predict the development of complications would allow for targeted early intervention.
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Trasplante de Riñón/efectos adversos , Enfermedades Cardiovasculares/etiología , Humanos , Huésped Inmunocomprometido , Terapia de Inmunosupresión/efectos adversos , Terapia de Inmunosupresión/métodos , Neoplasias/etiología , Neoplasias/inmunología , Cuidados Posoperatorios/efectos adversos , Cuidados Posoperatorios/métodos , Factores de RiesgoRESUMEN
The protein PCSK9 (proprotein convertase subtilisin/kexin type 9) is a key regulator of low-density lipoprotein receptor (LDLR) levels and cardiovascular health. We have determined the crystal structure of LDLR bound to PCSK9 at neutral pH. The structure shows LDLR in a new extended conformation. The PCSK9 C-terminal domain is solvent exposed, enabling cofactor binding, whereas the catalytic domain and prodomain interact with LDLR epidermal growth factor(A) and ß-propeller domains, respectively. Thus, PCSK9 seems to hold LDLR in an extended conformation and to interfere with conformational rearrangements required for LDLR recycling.
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Proproteína Convertasas/química , Receptores de LDL/química , Receptores de LDL/metabolismo , Serina Endopeptidasas/química , Regulación hacia Abajo , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Proproteína Convertasa 9 , Proproteína Convertasas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteolisis , Serina Endopeptidasas/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Staphylococcus aureus is a clinically important bacterial pathogen that has become resistant to treatment with most routinely used antibiotics. Alternative strategies, such as vaccination and phage therapy, are therefore actively being investigated to prevent or combat staphylococcal infections. Vaccination requires that vaccine targets are expressed at sufficient quantities during infection so that they can be targeted by the host's immune system. While our knowledge of in vitro expression levels of putative vaccine candidates is comprehensive, crucial in vivo expression data are scarce and promising vaccine candidates during in vitro assessment often prove ineffective in preventing S. aureus infection. Here, we show how a newly developed high-throughput quantitative reverse transcription-PCR (qRT-PCR) assay monitoring the expression of 84 staphylococcal genes encoding mostly virulence factors can inform the selection and design of effective vaccine candidates against staphylococcal infections. We show that this assay can accurately quantify mRNA expression levels of these genes in several host organs relying only on very limited amounts of bacterial mRNA in each sample. We selected two highly expressed genes, lukE and lukD, encoding pore-forming leukotoxins, to inform the design of detoxified recombinant proteins and showed that immunization with recombinant genetically detoxified LukED antigens conferred protection against staphylococcal skin infection in mice. Consequently, knowledge of in vivo-expressed virulence determinants can be successfully deployed to identify and select promising candidates for optimized design of effective vaccine antigens against S. aureus. Notably, this approach should be broadly applicable to numerous other pathogens. IMPORTANCE Vaccination is an attractive strategy for preventing bacterial infections in an age of increased antimicrobial resistance. However, vaccine development frequently suffers significant setbacks when candidate antigens that show promising results in in vitro experimentation fail to protect from disease. An alluring strategy is to focus resources on developing bacterial virulence factors that are expressed during disease establishment or maintenance and are critical for bacterial in-host survival as vaccine targets. While expression profiles of many virulence factors have been characterized in detail in vitro, our knowledge of their in vivo expression profiles is still scarce. Here, using a high-throughput qRT-PCR approach, we identified two highly expressed leukotoxins in a murine infection model and showed that genetically detoxified derivatives of these elicited a protective immune response in a murine skin infection model. Therefore, in vivo gene expression can inform the selection of promising candidates for the design of effective vaccine antigens.
Asunto(s)
Infecciones Estafilocócicas , Vacunas , Animales , Ratones , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/metabolismo , Leucocidinas/genética , Leucocidinas/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Vacunas/metabolismo , Infecciones Estafilocócicas/microbiología , Perfilación de la Expresión GénicaRESUMEN
Some people remain healthier throughout life than others but the underlying reasons are poorly understood. Here we hypothesize this advantage is attributable in part to optimal immune resilience (IR), defined as the capacity to preserve and/or rapidly restore immune functions that promote disease resistance (immunocompetence) and control inflammation in infectious diseases as well as other causes of inflammatory stress. We gauge IR levels with two distinct peripheral blood metrics that quantify the balance between (i) CD8+ and CD4+ T-cell levels and (ii) gene expression signatures tracking longevity-associated immunocompetence and mortality-associated inflammation. Profiles of IR metrics in ~48,500 individuals collectively indicate that some persons resist degradation of IR both during aging and when challenged with varied inflammatory stressors. With this resistance, preservation of optimal IR tracked (i) a lower risk of HIV acquisition, AIDS development, symptomatic influenza infection, and recurrent skin cancer; (ii) survival during COVID-19 and sepsis; and (iii) longevity. IR degradation is potentially reversible by decreasing inflammatory stress. Overall, we show that optimal IR is a trait observed across the age spectrum, more common in females, and aligned with a specific immunocompetence-inflammation balance linked to favorable immunity-dependent health outcomes. IR metrics and mechanisms have utility both as biomarkers for measuring immune health and for improving health outcomes.
Asunto(s)
COVID-19 , Longevidad , Femenino , Humanos , Envejecimiento , Inflamación , Evaluación de Resultado en la Atención de SaludRESUMEN
Background: Malignancy is a major cause of morbidity and mortality in transplant recipients. Identification of those at highest risk could facilitate pre-emptive intervention such as reduction of immunosuppression. Reduced circulating monocytic HLA-DR density is a marker of immune depression in the general population and associates with poorer outcome in critical illness. It has recently been used as a safety marker in adoptive cell therapy trials in renal transplantation. Despite its potential as a marker of dampened immune responses, factors that impact upon monocytic HLA-DR density and the long-term clinical sequelae of this have not been assessed in transplant recipients. Methods: A cohort study of stable long-term renal transplant recipients was undertaken. Serial circulating monocytic HLA-DR density and other leucocyte populations were quantified by flow cytometry. Gene expression of monocytes was performed using the Nanostring nCounter platform, and 13-plex cytokine bead array used to quantify serum concentrations. The primary outcome was malignancy development during one-year follow-up. Risk of malignancy was calculated by univariate and multivariate proportionate hazards modelling with and without adjustment for competing risks. Results: Monocytic HLA-DR density was stable in long-term renal transplant recipients (n=135) and similar to non-immunosuppressed controls (n=29), though was suppressed in recipients receiving prednisolone. Decreased mHLA-DRd was associated with accumulation of CD14+CD11b+CD33+HLA-DRlo monocytic myeloid-derived suppressor-like cells. Pathway analysis revealed downregulation of pathways relating to cytokine and chemokine signalling in monocytes with low HLA-DR density; however serum concentrations of major cytokines did not differ between these groups. There was an independent increase in malignancy risk during follow-up with decreased HLA-DR density. Conclusions: Dampened chemokine and cytokine signalling drives a stable reduction in monocytic HLA-DR density in long-term transplant recipients and associates with subsequent malignancy risk. This may function as a novel marker of excess immunosuppression. Further study is needed to understand the mechanism behind this association.