RESUMO
INTRODUCTION: Neisseria meningitidis serogroup B (NmB) antigens are inherently diverse with variable expression among strains. Prediction of meningococcal B (MenB) vaccine effectiveness therefore requires an assay suitable for use against large panels of epidemiologically representative disease-causing NmB strains. Traditional serum bactericidal antibody assay using exogenous human complement (hSBA) is limited to the quantification of MenB vaccine immunogenicity on a small number of indicator strains. AREAS COVERED: Additional and complementary methods for assessing strain coverage developed previously include the Meningococcal Antigen Typing System (MATS), Meningococcal Antigen Surface Expression (MEASURE) assay, and genotyping approaches, but these do not estimate vaccine effectiveness. We provide a narrative review of these methods, highlighting a more recent approach involving the hSBA assay in conjunction with expanded NmB strain panels: hSBA assay using endogenous complement in each vaccinated person's serum (enc-hSBA) against a 110-strain NmB panel and the traditional hSBA assay against 14 (4 + 10) NmB strains. EXPERT OPINION: The enc-hSBA is a highly standardized, robust method that can be used in clinical trials to measure the immunological effectiveness of MenB vaccines under conditions that mimic real-world settings as closely as possible, through the use of endogenous complement and a diverse, epidemiologically representative panel of NmB strains.
Meningococcal disease refers to illnesses caused by the bacterium Neisseria meningitidis (meningococcus), including infections of the brain lining and spinal cord (meningitis) and bloodstream (septicemia). It is rare but often severe and can be deadly. Invasive meningococcal disease can be prevented through vaccination. Nearly all cases are caused by six serogroups (types) of meningococci, including meningococcal serogroup B. Vaccines are available against meningococcal serogroup B but, because of the uncommonness of the disease, standard clinical trials could not be performed to prove these vaccines are effective. Instead, an indirect measure, called the 'hSBA assay' (serum bactericidal antibody assay using human complement), is used to measure the ability of vaccines to provide protection against specific N. meningitidis strains that have antigens (substances that cause the immune system to react) sharing characteristics with components of the vaccines. However, meningococcal serogroup B strains are diverse in the genetic composition and expression of vaccine antigens. Hence, a large number of N. meningitidis serogroup B strains would have to be tested to make sure that the vaccine is effective against these strains. This is not feasible using the traditional hSBA assay, which requires a human complement (a protein system, which is part of the immune system) that has not come from the vaccinated person and is difficult and time-consuming to source. Recently, an alternative hSBA assay was developed that uses the complement present in each vaccinated person's blood (endogenous complement) and which overcomes these challenges. By allowing testing against a broad panel of N. meningitidis serogroup B strains, this new assay may enable a more accurate estimation of the effectiveness of vaccines against serogroup B meningococci.
Assuntos
Infecções Meningocócicas , Vacinas Meningocócicas , Neisseria meningitidis Sorogrupo B , Neisseria meningitidis , Humanos , Ensaios de Anticorpos Bactericidas Séricos/métodos , Sorogrupo , Eficácia de Vacinas , Anticorpos Antibacterianos , Antígenos de Bactérias/genética , Neisseria meningitidis Sorogrupo B/genética , Proteínas do Sistema Complemento , Infecções Meningocócicas/prevenção & controleRESUMO
Cholera remains a significant public health burden worldwide, and better methods for monitoring cholera incidence would enhance the effectiveness of public health interventions. The serum bactericidal assay (SBA) has been used extensively for Vibrio cholerae vaccine assessments and serosurveillance. Current SBA approaches for V. cholerae rely on colony enumeration or optical density (OD600nm) readings to measure viable bacteria following complement-mediated lysis. These methods provide titer values that are constrained to discrete dilution values and rely on bacterial outgrowth, which is time consuming and prone to variation. Detection of bacterial proteins following complement-mediated lysis presents a faster and potentially less variable alternative approach independent of bacterial outgrowth. Here, we present an SBA that measures luciferase luminescence driven by lysis-released adenylate kinase. This approach is faster and less variable than growth-dependent SBAs and directly measures continuous titer values. This novel SBA method can potentially be applied to other bacteria of interest.
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Anticorpos Antibacterianos/imunologia , Cólera/epidemiologia , Ensaios de Anticorpos Bactericidas Séricos/métodos , Vibrio cholerae/imunologia , Cólera/imunologia , Cólera/prevenção & controle , Vacinas contra Cólera/uso terapêutico , Análise Custo-Benefício , Monitoramento Epidemiológico , Humanos , Imunogenicidade da Vacina , Medições Luminescentes , Reprodutibilidade dos Testes , Estudos Soroepidemiológicos , Fatores de TempoRESUMO
The classical complement pathway is triggered when antigen-bound immunoglobulins bind to C1q through their Fc region. While C1q binds to a single Fc with low affinity, a higher avidity stable binding of two or more of C1q globular heads initiates the downstream reactions of the complement cascade ultimately resulting in bacteriolysis. Synergistic bactericidal activity has been demonstrated when monoclonal antibodies recognize nonoverlapping epitopes of the same antigen. The aim of the present work was to investigate the synergistic effect between antibodies directed toward different antigens. To this purpose, we investigated the bactericidal activity induced by combinations of monoclonal antibodies (mAbs) raised against factor H-binding protein (fHbp) and Neisserial Heparin-Binding Antigen (NHBA), two major antigens included in Bexsero, the vaccine against Meningococcus B, for prevention from this devastating disease in infants and adolescents. Collectively, our results show that mAbs recognizing different antigens can synergistically activate complement even when each single Mab is not bactericidal, reinforcing the evidence that cooperative immunity induced by antigen combinations can represent a remarkable added value of multicomponent vaccines. Our study also shows that the synergistic effect of antibodies is modulated by the nature of the respective epitopes, as well as by the antigen density on the bacterial cell surface.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas do Sistema Complemento/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Fator H do Complemento/imunologia , Epitopos/imunologia , Neisseria meningitidis/imunologia , Ensaios de Anticorpos Bactericidas Séricos/métodosRESUMO
The capsular group B meningococcal (MenB) four component vaccine (4CMenB) has been licensed for the prevention of invasive disease caused by MenB. The vaccine causes fever in infants, particularly when given in combination (concomitant) with other routinely-administered vaccines (routine), such as the standard diphtheria, tetanus, pertussis (DTP)-containing vaccine. To assess the suitability of a mouse immunisation model to study this phenomenon, we monitored temperature in mice after a second dose of routine vaccines, with or without 4CMenB, and compared the results with those in humans. Using this mouse model, we explored the reactogenicity of 4CMenB components by measuring changes in temperature, cytokines, and gene expression induced by 4CMenB, one of its components, wild-type or attenuated endotoxin outer membrane vesicles (OMVs), or lipopolysaccharide (LPS). A significant rise (p < 0.01) in temperature was observed in mice immunised with 4CMenB, wild-type OMVs, and LPS. RNA-sequencing of mouse whole blood revealed a gene signature shared by the 4CMenB, OMV, and LPS groups consisting of bacterial pattern recognition receptors and neutrophil activation marker genes. Sequencing of neutrophils isolated after concomitant 4CMenB identified cells expressing the OMV-associated genes Plek and Lcp1. Immunisation with 4CMenB or OMVs led to increased IL-6 in serum and significant upregulation (p < 0.0001) of prostaglandin-synthesising enzymes on brain tissue. These data demonstrate the suitability of a mouse model for assessing vaccine reactogenicity and strongly indicate that the fever following vaccination with 4CMenB in human infants is induced by endotoxin contained in the OMV component of the vaccine.
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Membrana Externa Bacteriana/imunologia , Endotoxinas/imunologia , Infecções Meningocócicas/imunologia , Vacinas Meningocócicas/imunologia , Transcriptoma/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Feminino , Imunização/métodos , Esquemas de Imunização , Interleucina-6/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Ensaios de Anticorpos Bactericidas Séricos/métodos , Vacinação/métodosRESUMO
The global spread of multidrug-resistant gonorrhea has spurred efforts to develop a safe and effective vaccine against gonorrhea. Complement plays an important role in host defenses against Neisseria infections. Complement-dependent bactericidal activity of antibodies (either natural antibodies or those elicited by immunization) is a well-established correlate of protection against meningococcal infections. Although correlates of protection against gonococcal infection have not been defined, there is evidence to suggest that complement-mediated killing may also predict vaccine efficacy against this disease. This chapter describes methods to prepare human complement sources and perform bactericidal assays against Neisseria gonorrhoeae.
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Anticorpos Antibacterianos/imunologia , Proteínas do Sistema Complemento/imunologia , Gonorreia/imunologia , Neisseria gonorrhoeae/imunologia , Ensaios de Anticorpos Bactericidas Séricos/métodos , Proteínas da Membrana Bacteriana Externa/imunologia , Gonorreia/microbiologia , Humanos , Imunidade HumoralRESUMO
Invasive meningococcal disease is rare and potentially devastating but often vaccine-preventable. Evaluation of meningococcal vaccine effectiveness is impractical owing to relatively low disease incidence; protection is therefore estimated using serum bactericidal antibody (SBA) assays. Original experiments on natural immunity established a titer of ≥4 as the correlate of protection for SBA assays using human complement (hSBA), but human complement is relatively difficult to obtain and standardize. Use of baby rabbit complement (rSBA assays), per standard guidelines for serogroups A and C, generally results in comparatively higher titers. Postlicensure effectiveness data for serogroup C conjugate vaccines support acceptance of rSBA titers ≥8 as the correlate of protection for this serogroup, but no thresholds have been formally established for serogroups A, W, and Y. Studies evaluating MenACWY-TT (Nimenrix®; Pfizer Inc, Sandwich, UK) immunogenicity have used both hSBA and rSBA assays, and ultimately suggest that rSBA may be more appropriate for these measurements.
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Proteínas do Sistema Complemento/análise , Proteínas do Sistema Complemento/imunologia , Vacinas Meningocócicas/imunologia , Ensaios de Anticorpos Bactericidas Séricos/normas , Animais , Anticorpos Antibacterianos/sangue , Humanos , Infecções Meningocócicas/diagnóstico , Infecções Meningocócicas/prevenção & controle , Coelhos/imunologia , Sorogrupo , Ensaios de Anticorpos Bactericidas Séricos/métodos , Fatores de Tempo , Vacinas Conjugadas/imunologiaRESUMO
Serum bactericidal antibody (SBA) assays measure functional antibody titers against Neisseria meningitidis in sera. Induction of complement-dependent SBA after vaccination with meningococcal polysaccharide or conjugate or protein based vaccines is regarded as the surrogate of protection and thus acceptable evidence of the potential efficacy of these vaccines. This chapter discusses and details SBA assay protocols for measuring the complement-mediated lysis of serogroup A, B, C, W, X, and Y meningococci by human sera, for example, following vaccination or disease.
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Anticorpos Antibacterianos/imunologia , Proteínas do Sistema Complemento/imunologia , Infecções Meningocócicas/imunologia , Neisseria meningitidis/imunologia , Sorogrupo , Ensaios de Anticorpos Bactericidas Séricos/métodos , Humanos , Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/administração & dosagem , Neisseria meningitidis/classificação , VacinaçãoRESUMO
BACKGROUND: Current serological tests cannot discriminate between bactericidal Borrelia burgdorferi antibodies from others that are merely a response to Borrelia antigenic stimulation. OBJECTIVE: To develop a sensitive and convenient luminescence-based serum bactericidal assay (L-SBA) to identify serum borreliacidal activity. STUDY DESIGN: Prospective validation study and method comparison. METHODS: Serum samples were obtained either from archives of the Animal Health Diagnostic Center at Cornell University (N = 7) or from a vaccination trial (N = 238). Endogenous complement-inactivated serum sample was incubated with exogenic complement and B. burgdorferi ML23 pBBE22luc, which is able to process luciferin with luciferase and produce luminescence in viable Borrelia. After incubation, a light signal can be detected by using a luminometer to calculate the borreliacidal antibody titre. RESULTS: Components of the reaction mixture including spirochetes and complement from various sources and concentrations were tested to identify a reliable recipe for our complement-mediated L-SBA. We also applied this L-SBA on measuring bactericidal antibody activities and calculated the half inhibitory concentration (IC50 ) of serum samples from clinical collections. Furthermore, we analysed the L-SBA titres and anti-outer surface protein A (OspA) antibody levels from vaccinated horses using the multiplex assays and found that there is a relationship between results generated using these two different assays. The increases of L-SBA titres correlated with increases of anti-OspA antibody titre in sera (r = 0.423). MAIN LIMITATIONS: Immunoreactivity of commercial complement may differ from different batches. Clinical protection of borreliacidal antibody levels has not been determined. CONCLUSIONS: The L-SBA provided a sensitive and easy-operating platform for the evaluation of bactericidal antibody to B. burgdorferi, and we anticipated L-SBA would function well as an evaluation tool of vaccine efficiency in the future.
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Anticorpos Antibacterianos/sangue , Borrelia burgdorferi/imunologia , Doenças dos Cavalos/prevenção & controle , Medições Luminescentes/veterinária , Vacinas contra Doença de Lyme/imunologia , Ensaios de Anticorpos Bactericidas Séricos/veterinária , Animais , Doenças dos Cavalos/sangue , Cavalos , Medições Luminescentes/métodos , Ensaios de Anticorpos Bactericidas Séricos/métodosRESUMO
Better and more diverse biomarkers for the development of simple point-of-care tests for active tuberculosis (TB), a clinically heterogeneous disease, are urgently needed. We generated a proteomic Mycobacterium tuberculosis (Mtb) High-Density Nucleic Acid Programmable Protein Array (HD-NAPPA) that used a novel multiplexed strategy for expedited high-throughput screening for antibody responses to the Mtb proteome. We screened sera from HIV uninfected and coinfected TB patients and controls (n = 120) from the US and South Africa (SA) using the multiplex HD-NAPPA for discovery, followed by deconvolution and validation through single protein HD-NAPPA with biologically independent samples (n = 124). We verified the top proteins with enzyme-linked immunosorbent assays (ELISA) using the original screening and validation samples (n = 244) and heretofore untested samples (n = 41). We identified 8 proteins with TB biomarker value; four (Rv0054, Rv0831c, Rv2031c and Rv0222) of these were previously identified in serology studies, and four (Rv0948c, Rv2853, Rv3405c, Rv3544c) were not known to elicit antibody responses. Using ELISA data, we created classifiers that could discriminate patients' TB status according to geography (US or SA) and HIV (HIV- or HIV+) status. With ROC curve analysis under cross validation, the classifiers performed with an AUC for US/HIV- at 0.807; US/HIV+ at 0.782; SA/HIV- at 0.868; and SA/HIV+ at 0.723. With this study we demonstrate a new platform for biomarker/antibody screening and delineate its utility to identify previously unknown immunoreactive proteins.
Assuntos
Proteínas de Bactérias/imunologia , Infecções por HIV/sangue , Mycobacterium tuberculosis/metabolismo , Análise Serial de Proteínas/métodos , Proteômica/métodos , Ensaios de Anticorpos Bactericidas Séricos/métodos , Tuberculose/imunologia , Adulto , Idoso , Biomarcadores/sangue , Coinfecção , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Curva ROC , África do Sul , Estados Unidos , Adulto JovemRESUMO
Scrub typhus, caused by Orientia tsutsugamushi, is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targeting O. tsutsugamushi 47-kDa, groEL, and human interferon beta (IFN-ß gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness.
Assuntos
Proteínas de Bactérias/genética , Chaperonina 60/genética , Orientia tsutsugamushi/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Tifo por Ácaros/diagnóstico , Ensaios de Anticorpos Bactericidas Séricos/métodos , Adulto , Anticorpos Antibacterianos/sangue , Carga Bacteriana/métodos , Proteínas de Bactérias/análise , Chaperonina 60/análise , Febre/diagnóstico , Febre/microbiologia , Amplificação de Genes , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Interferon beta/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Orientia tsutsugamushi/isolamento & purificação , Tifo por Ácaros/microbiologia , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Bivalent rLP2086 is a recombinant factor H binding protein-based vaccine approved in the USA for prevention of meningococcal serogroup B disease in 10-25-year-olds. We aimed to assess the persistence of bactericidal antibodies up to 4 years after a three-dose schedule of bivalent rLP2086. METHODS: We did this randomised, single-blind, placebo-controlled, phase 2 trial at 25 sites in Australia, Poland, and Spain. In stage 1 of the study (February, 2009-May, 2010), healthy adolescents (aged 11-18 years) were randomly assigned, via an interactive voice and web-response system with computer-generated sequential random numbers, to receive either ascending doses of vaccine (60 µg, 120 µg, and 200 µg) or placebo at months 0, 2, and 6. Dispensing staff were not masked to group allocation, but allocation was concealed from principal investigators, participants and their guardians, and laboratory personnel. In stage 2 of the study (reported here), we enrolled healthy adolescents who had received three doses of 120 µg bivalent rLP2086 (the optimum dose level identified in stage 1) or saline. Immunogenicity was determined in serum bactericidal antibody assay using human complement (hSBA) by use of four meningococcal serogroup B test strains expressing vaccine-heterologous factor H binding protein variants: PMB80 (A22), PMB2001 (A56), PMB2948 (B24), and PMB2707 (B44). Immunogenicity in stage 2 was assessed at months 6, 12, 24, and 48 post-vaccination. We did analysis by intention to treat. This trial is registered as ClinicalTrials.gov number NCT00808028. FINDINGS: Between March 17, 2010, and Feb 8, 2011, 170 participants who received 120 µg of bivalent rLP2086 and 80 participants who received placebo in stage 1 of the study were entered into stage 2; 210 participants completed stage 2 up to 48 months. 1 month after the third vaccination, 93% (n=139/149) to 100% (n=48/48) of vaccine recipients achieved protective hSBA titres equal to or greater than the lower limit of quantification to each test strain, compared with 0% (n=0/25) to 35% (n=8/23) of control recipients. Despite initial declines in seroprotective hSBA titres for all four test strains, for three test strains (A22, A56, and B24), more than 50% of bivalent rLP2086 recipients continued to achieve titres equal to or greater than the lower limit of quantification at months 6 (57% [n=93/163] to 89% [n=42/47]), 12 (54% [n=84/155] to 69% [n=33/48]), 24 (53% [n=26/49] to 54% [n=82/152]), and 48 (51% [n=24/47] to 59% [n=79/134]); corresponding values in the control group were 14% (n=11/80) to 22% (n=5/23) at month 6, 13% (n=10/78) to 29% (n=22/76) at month 12, 16% (n=12/74) to 36% (n=8/22) at month 24, and 24% (n=16/68) to 35% (n=8/23) at month 48. For test strain B44, hSBA titres equal to or greater than the lower limit of quantification were shown in 37% (n=18/49) of vaccine recipients at 6 months, in 29% (n=14/48) at 12 months, in 22% (n=11/49) at 24 months, and in 20% (n=10/49) at 48 months, compared with 0% (n=0/25) of control recipients at month 6, 4% (n=1/25) at months 12 and 24, and 12% (n=3/25) at month 48. Adverse events were reported in seven (4%) of 170 participants in the bivalent rLP2086 group and two (3%) of 80 participants in the control group; no event was deemed related to vaccine. INTERPRETATION: After three doses of bivalent rLP2086, protective hSBA titres above the correlate of protection (≥1/4) were elicited up to 4 years in more than 50% of participants for three of four meningococcal serogroup B test strains representative of disease-causing meningococci expressing vaccine-heterologous antigens. Further studies will be needed to assess possible herd immunity effects with meningococcal serogroup B vaccines and the need for a booster dose to sustain individual protection against invasive meningococcal disease. FUNDING: Pfizer.
Assuntos
Antígenos de Bactérias/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/administração & dosagem , Vacinas Sintéticas/imunologia , Adolescente , Anticorpos Antibacterianos/sangue , Austrália , Criança , Feminino , Seguimentos , Humanos , Masculino , Neisseria meningitidis/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Neisseria meningitidis Sorogrupo B/isolamento & purificação , Polônia , Ensaios de Anticorpos Bactericidas Séricos/métodos , Espanha , Adulto JovemRESUMO
BACKGROUND: In the majority of pediatric patients, the hemolytic-uremic syndrome (HUS) is caused by an infection with Shiga toxin-producing Escherichia coli (STEC), mostly serotype O157. It is important to discriminate between HUS caused by STEC and complement-mediated HUS (atypical HUS) due to differences in treatment and outcome. As STEC and its toxins can only be detected in the patient's stool for a short period of time after disease onset, the infectious agent may go undetected using only fecal diagnostic tests. Serum antibodies to lipopolysaccharide (LPS) of STEC persist for several weeks and may therefore be of added value in the diagnosis of STEC. METHODS: All patients with clinical STEC-HUS who were treated at Radboud University Medical Center between 1990 and 2014 were included in this retrospective single-center study. Clinical and diagnostic microbiological data were collected. Immunoglobulin M (IgM) antibodies against LPS of STEC serotype O157 were detected by a serological assay (ELISA). RESULTS: Data from 65 patients weres available for analysis. Fecal diagnostic testing found evidence of an STEC infection in 34/63 patients (54 %). Serological evidence of STEC O157 was obtained in an additional 16 patients. This is an added value of 23 % (p < 0.0001) when the serological antibody assay is used in addition to standard fecal diagnostic tests to confirm the diagnosis STEC-HUS. This added value becomes especially apparent when the tests are performed more than 7 days after the initial manifestation of the gastrointestinal symptoms. CONCLUSIONS: The serological anti-O157 LPS assay clearly makes a positive contribution when used in combination with standard fecal diagnostic tests to diagnose STEC-HUS and should be incorporated in clinical practice.
Assuntos
Anticorpos Antibacterianos/sangue , Infecções por Escherichia coli/diagnóstico , Escherichia coli O157/imunologia , Escherichia coli O157/isolamento & purificação , Fezes/microbiologia , Síndrome Hemolítico-Urêmica/diagnóstico , Lipopolissacarídeos/imunologia , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Infecções por Escherichia coli/sangue , Infecções por Escherichia coli/microbiologia , Feminino , Síndrome Hemolítico-Urêmica/sangue , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Imunoglobulina M/sangue , Lactente , Masculino , Estudos Retrospectivos , Ensaios de Anticorpos Bactericidas Séricos/métodos , Fatores de TempoRESUMO
Determinar el estadio de fibrosis hepática es esencial en el manejo de la enfermedad hepática en pacientes con hepatitis crónica por virus C. La biopsia hepática ha sido considerada el gold standard para diagnosticar la enfermedad, la actividad necroinflamatoria y el estadio de fibrosis, pero tiene algunas limitaciones técnicas y riesgos. Teniendo en cuenta estas limitaciones, existe cierta exigencia en introducir biomarcadores séricos no invasivos para el diagnóstico de fibrosis que podrían ser capaces de reemplazar parcialmente a la biopsia hepática. El biomarcador sérico ideal debería ser específico para el hígado y fácil de determinar. Los marcadores séricos se dividen en directos e indirectos. Los directos reflejan el recambio de la matriz extracelular, mientras que los indirectos reflejan alteraciones en la función hepática. Aunque todavía está limitado el grado de implementación de los test no invasivos de fibrosis hepática, esta revisión es una visión de conjunto de los biomarcadores, índices y algoritmos diagnósticos de fibrosis hepática estudiados en hepatitis crónica C pero con un interés potencial en otras hepatopatías crónicas (AU)
Precise staging of liver fibrosis is essential in the management of liver disease activity in chronic hepatitis C patients. Liver biopsy has been considered the reference method for diagnosing disease, grading necroinflammatory activity, and staging fibrosis, but it has some technical limitations and risks. Taking into account these limitations, there is a need to introduce non-invasive serum markers for fibrosis that would be able to partially replace liver biopsy. Ideal serum markers should be specific for the liver and easy to perform. Serum markers of hepatic fibrosis are divided into direct and indirect. Direct markers reflect extracellular matrix turnover, whereas indirect markers reflect alterations in hepatic function. The degree of implementation of non-invasive diagnostic tests for liver fibrosis still remains limited. This review provides a current overview of biomarkers, indexes and algorithms for hepatic fibrosis diagnosis in chronic hepatitis C patients (AU)
Assuntos
Feminino , Humanos , Masculino , Biomarcadores/análise , Biomarcadores/metabolismo , Cirrose Hepática/diagnóstico , Hepatite Crônica/diagnóstico , Hepatite C/diagnóstico , Metaloproteases , Fator de Crescimento Transformador beta1 , Fibrose/diagnóstico , Algoritmos , Biomarcadores/sangue , Ensaios de Anticorpos Bactericidas Séricos/métodos , Pró-Colágeno , Ácido Hialurônico , Receptores de Fibrinogênio/análiseRESUMO
Complement is an essential component of the immune system and human pathogenic organisms have developed various mechanisms for evading complement mediated serum killing. The "gold standard" for measuring the ability of vaccine-induced antibody to kill Neisseria meningitidis is the serum bactericidal antibody (SBA) assay which measures complement mediated killing via antibody. This assay requires active complement, either intrinsic from the serum being tested or the addition of exogenous complement, either from a human or from another species such as rabbit. For serogroup C, an SBA titre of ≥4 was established as the correlate of protection when using human complement and ≥8 as the threshold when using rabbit complement, based on comparative assay results. Licensure of meningococcal vaccines, including polysaccharide protein conjugate vaccines and serogroup B vaccines has been based on the immune responses measured with the SBA assay, thus on a surrogate of vaccine efficacy. This review examines the use of complement and the SBA assay to assess immunity to meningococcal infection, and provides examples of vaccine trials in different age groups where various assays have been used.
Assuntos
Anticorpos Antibacterianos/imunologia , Atividade Bactericida do Sangue , Proteínas do Sistema Complemento/imunologia , Neisseria meningitidis/imunologia , Ensaios de Anticorpos Bactericidas Séricos/métodos , Animais , Humanos , Infecções Meningocócicas/imunologia , Vacinas Meningocócicas/administração & dosagem , Vacinas Meningocócicas/imunologia , CoelhosRESUMO
Prior to routine immunisation, Haemophilus influenzae serotype b (Hib) was a major cause of serious bacterial infections, particularly in young children. In the United Kingdom, introduction of the Hib conjugate vaccine into the national childhood immunisation schedule has led to a sustained decline in invasive Hib disease across all age-groups. Evaluation of the immune response to Hib conjugate vaccines involves measurement of serum IgG antibodies against the capsular polyribosyl-ribitol-phosphate (PRP) polysaccharide by enzyme-linked immunosorbent assay (ELISA), with accepted short-term and long-term protective thresholds of ≥0.15µg/mL and ≥1.0µg/mL, respectively. These levels were derived by passive immunisation or immunisation with pure polysaccharide, and their relevance for protection following immunisation with conjugate vaccines remains unclear. This study aimed to modify and optimise a serum bactericidal antibody (SBA) assay to evaluate the functional activity of Hib antibodies generated following Hib conjugate vaccination. Validation of the Hib SBA assay was deemed acceptable for all assay parameters tested. A strong correlation between anti-PRP IgG concentrations and SBA titres was observed in vaccinated adults (r=0.81), as well as infants after primary immunisation at 2, 3, and 4 months (r=0.635) and after the 12-month booster (r=0.746). The assay identified some children with high anti-PRP IgG but low SBA activity and vice versa. The predictive protective SBA titre corresponding to a post-booster anti-PRP IgG of 1.0µg/mL was 8. Thus, the optimised Hib SBA assay was specific and reproducible and correlated with anti-PRP IgG. Such assays may have a role in evaluating immune responses to conjugate vaccines in addition to measuring capsular antibodies.
Assuntos
Anticorpos Antibacterianos/sangue , Vacinas Anti-Haemophilus/uso terapêutico , Ensaios de Anticorpos Bactericidas Séricos/métodos , Adulto , Cápsulas Bacterianas , Infecções por Haemophilus/prevenção & controle , Haemophilus influenzae tipo b , Humanos , Imunização Secundária , Imunoglobulina G/sangue , Lactente , Sensibilidade e Especificidade , Ensaios de Anticorpos Bactericidas Séricos/normasRESUMO
BACKGROUND: 4CMenB (Bexsero), a vaccine developed against invasive meningococcal disease caused by capsular group B strains (MenB), was recently licensed for use by the European Medicines Agency. Assessment of 4CMenB strain coverage in specific epidemiologic settings is of primary importance to predict vaccination impact on the burden of disease. The Meningococcal Antigen Typing System (MATS) was developed to predict 4CMenB strain coverage, using serum bactericidal antibody assay with human complement (hSBA) data from a diverse panel of strains not representative of any specific epidemiology. OBJECTIVE: To experimentally validate the accuracy of MATS-based predictions against strains representative of a specific epidemiologic setting. METHODS AND RESULTS: We used a stratified sampling method to identify a representative sample from all MenB disease isolates collected from England and Wales in 2007-2008, tested the strains in the hSBA assay with pooled sera from infant and adolescent vaccinees, and compared these results with MATS. MATS predictions and hSBA results were significantly associated (P=0.022). MATS predicted coverage of 70% (95% CI, 55-85%) was largely confirmed by 88% killing in the hSBA (95% CI, 72-95%). MATS had 78% accuracy and 96% positive predictive value against hSBA. CONCLUSION: MATS is a conservative predictor of strain coverage by the 4CMenB vaccine in infants and adolescents.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Vacinas Meningocócicas/administração & dosagem , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Ensaios de Anticorpos Bactericidas Séricos/métodos , Vacinação/estatística & dados numéricos , Adolescente , Criança , Pré-Escolar , Inglaterra/epidemiologia , Feminino , Voluntários Saudáveis , Humanos , Lactente , Masculino , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/prevenção & controle , Valor Preditivo dos Testes , País de Gales/epidemiologiaRESUMO
The serum bactericidal assay is the correlate of protection for meningococcal disease but the use and comparison of functional immunological assays for the assessment of meningococcal vaccines is complicated by the sourcing of human complement. This is due to high levels of immunity in the population acquired through natural meningococcal carriage and means that many individuals must be screened to find donors with suitably low bactericidal titres against the target strain. The use of different donors for each meningococcal strain means that comparisons of assay responses between strains and between laboratories is difficult. We have developed a method for IgG-depletion of 300 ml batches of pooled human lepirudin-derived plasma using Protein G sepharose affinity chromatography that retains complement activity. However, IgG-depletion also removed C1q. This was also eluted from the affinity matrix, concentrated and added to the complement source. The final complement source retained mean alternative pathway activity of 96.8% and total haemolytic activity of 84.2% in four batches. Complement components C3, C5, properdin and factor H were retained following the process and the IgG-depleted complement was shown to be suitable for use in antibody-mediated complement deposition and serum bactericidal activity assays against serogroup B meningococci. The generation of large IgG-depleted batches of pooled human plasma allows for the comparison of immunological responses to diverse meningococcal strain panels in large clinical trials.
Assuntos
Proteínas do Sistema Complemento/análise , Neisseria meningitidis/imunologia , Ensaios de Anticorpos Bactericidas Séricos/métodos , Animais , Anticorpos Antibacterianos/isolamento & purificação , Cromatografia de Afinidade , Complemento C1q/análise , Complemento C3/análise , Complemento C5/análise , Fator H do Complemento/análise , Via Alternativa do Complemento , Ensaio de Imunoadsorção Enzimática , Hemólise , Humanos , Imunoglobulina G/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Reprodutibilidade dos TestesRESUMO
Nontyphoidal isolates of Salmonella (NTS), particularly Salmonella Typhimurium, are a major cause of invasive bacteremia in Africa. Despite this, no vaccine against NTS is currently available for use in humans. If a NTS vaccine is to be developed in a timely manner, there is a need to develop assays to assess its in vivo efficacy. Assessment of potential efficacy of candidate vaccines in preclinical models is important for proof-of-concept and reduces attrition of vaccines in clinical trials. Serum bactericidal assays (SBA) are often used to assess the functional activity of vaccine-induced antibody responses targeted against Gram-negative bacteria with results given as the maximum dilution of serum that can effect bacterial killing. Previously we have found evidence for a protective role for antibody-induced complement-mediated killing of NTS in African children using an undiluted whole serum SBA. However, endogenous complement in diluted human sera is limiting and insufficient to effect bactericidal activity against S. Typhimurium beyond two two-fold dilutions. In the current study, we examined the requirements for SBA against NTS using baby rabbit serum (BRS) as an exogenous source of complement. We found that the amount of complement required for antibody-mediated bactericidal activity is much higher for the invasive African S. Typhimurium isolate D23580, compared with the laboratory S. Typhimurium LT2 and Salmonella Paratyphi A CVD1901. While 20% BRS was sufficient to kill LT2 and CVD1901, 75% BRS was needed to kill D23580. Our findings demonstrate that one concentration of exogenous complement is not suitable for SBA against all Salmonella isolates. To develop SBA to assess the in vivo efficacy of Salmonella vaccines, it is necessary to optimize the assay for the Salmonella isolates against which the vaccine is targeted.
Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas do Sistema Complemento/imunologia , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Adulto , África , Animais , Proteínas do Sistema Complemento/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Viabilidade Microbiana/imunologia , Coelhos , Salmonella/classificação , Salmonella/imunologia , Salmonella/metabolismo , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Vacinas contra Salmonella/imunologia , Salmonella typhimurium/metabolismo , Ensaios de Anticorpos Bactericidas Séricos/métodos , Especificidade da EspécieAssuntos
Antibacterianos/administração & dosagem , Reservatórios de Doenças , Imunoglobulina G/sangue , Rickettsia prowazekii , Tifo Epidêmico Transmitido por Piolhos , Zoonoses , Animais , Distribuição de Qui-Quadrado , Cloranfenicol/administração & dosagem , Reservatórios de Doenças/microbiologia , Reservatórios de Doenças/estatística & dados numéricos , Ensaio de Imunoadsorção Enzimática , Humanos , Rickettsia prowazekii/efeitos dos fármacos , Rickettsia prowazekii/isolamento & purificação , Fatores de Risco , Sciuridae/microbiologia , Ensaios de Anticorpos Bactericidas Séricos/métodos , Ensaios de Anticorpos Bactericidas Séricos/estatística & dados numéricos , Tetraciclina/administração & dosagem , Tifo Epidêmico Transmitido por Piolhos/diagnóstico , Tifo Epidêmico Transmitido por Piolhos/epidemiologia , Tifo Epidêmico Transmitido por Piolhos/microbiologia , Tifo Epidêmico Transmitido por Piolhos/fisiopatologia , Tifo Epidêmico Transmitido por Piolhos/transmissão , Estados Unidos/epidemiologia , Zoonoses/epidemiologia , Zoonoses/microbiologia , Zoonoses/transmissãoRESUMO
The vibriocidal titer assay can be used to detect antibodies against Vibrio cholerae in serum samples, serving as an indicator of prior infection and potential protection against cholera. The assay can be utilized in research and clinical settings to test the effectiveness of vaccines, and also in epidemiological studies relevant to cholera transmission and surveillance. This unit outlines the steps involved in conducting an easily interpreted colorimetric vibriocidal titer assay with a relatively short turnaround time for results of around 8 hr, with final result observations in 24 hr. The assay can also be easily scaled up or down to accommodate as many or as few serum samples available and is not V. cholerae strain specific.