RESUMO
RATIONALE: Biomarkers that can be used to evaluate new interventions against latent tuberculosis infection (LTBI) and predict reactivation TB disease are urgently required. OBJECTIVES: To evaluate ESAT-6 and CFP-10 (EC) IFN-γ ELISPOT as a biomarker for treatment efficacy in LTBI. METHODS: This was a randomized, blinded, and placebo-controlled trial of INH in EC ELISPOT and Mantoux test positive participants. MEASUREMENTS AND MAIN RESULTS: Participants received a 6-month course of 900 mg INH twice weekly or a matching placebo. INH acetylator genotypes were determined and urine tested for INH metabolites to confirm adherence. The proportion of positive responders for CFP-10 and ESAT-6 between treatment arms was compared using mixed effects logistic regression models. A Tweedie (compound Poisson) model was fitted to allow for zero inflation and overdispersion of quantitative response. The proportions of EC ELISPOT-positive subjects reduced over time (P < 0.001) but did not differ by study arm (P = 0.36). Median spot-forming units for ESAT-6 and CFP-10 also declined significantly with time (P < 0.001) but did not differ by study arm (P = 0.74 and 0.71, respectively). There was no evidence of an interaction between acetylator status and INH treatment with respect to ELISPOT results over time. CONCLUSIONS: In contacts with LTBI, INH therapy plays no role in observed decreases in Mycobacterium tuberculosis antigen-specific T-cell responses over time. IFN-γ ELISPOT is probably not a useful biomarker of treatment efficacy in LTBI. Clinical trial registered with www.clinicaltrials.gov (NCT 00130325).
Assuntos
Antituberculosos/uso terapêutico , ELISPOT/métodos , Interferon gama/sangue , Isoniazida/uso terapêutico , Tuberculose Latente/sangue , Tuberculose Latente/tratamento farmacológico , Adulto , Biomarcadores/sangue , Método Duplo-Cego , ELISPOT/normas , Feminino , Gâmbia , Humanos , Interferon gama/efeitos dos fármacos , Masculino , Mycobacterium tuberculosis/efeitos dos fármacos , Resultado do Tratamento , Adulto JovemRESUMO
Current influenza vaccines could be augmented by including recombinant neuraminidase (rNA) protein antigen to broaden protective immunity and improve efficacy. Toward this goal, we investigated formulation conditions to optimize rNA physicochemical stability. When rNA in sodium phosphate saline buffer (NaPBS) was frozen and thawed (F/T), the tetrameric structure transitioned from a "closed" to an "open" conformation, negatively impacting functional activity. Hydrogen deuterium exchange experiments identified differences in anchorage binding sites at the base of the open tetramer, offering a structural mechanistic explanation for the change in conformation and decreased functional activity. Change to the open configuration was triggered by the combined stresses of acidic pH and F/T. The desired closed conformation was preserved in a potassium phosphate buffer (KP), minimizing pH drop upon freezing and including 10% sucrose to control F/T stress. Stability was further evaluated in thermal stress studies where changes in conformation were readily detected by ELISA and size exclusion chromatography (SEC). Both tests were suitable indicators of stability and antigenicity and considered potential critical quality attributes (pCQAs). To understand longer-term stability, the pCQA profiles from thermally stressed rNA at 6 months were modeled to predict stability of at least 24-months at 5°C storage. In summary, a desired rNA closed tetramer was maintained by formulation selection and monitoring of pCQAs to produce a stable rNA vaccine candidate. The study highlights the importance of understanding and controlling vaccine protein structural and functional integrity.
Assuntos
Vacinas contra Influenza , Influenza Humana , Humanos , Influenza Humana/prevenção & controle , Neuraminidase/genética , Vacinas Sintéticas/genética , RNARESUMO
The ability to measure immunomodulatory effects of a vaccine is crucial for novel vaccine design. While traditional animal models have been effective, a better understanding of the response in humans to new vaccines in pre-clinical development is critical for advancement to clinical trials. A translational methodology that can capture the complexity of a vaccine-driven response in a human model, which does not require human exposure, is needed. Here we have designed a platform that uses fresh human whole blood as a key component to study the adaptive immune memory response to vaccine formulations. The response is monitored by high-parameter single cell analysis using mass cytometry (Helios, CyTOF System), allowing for a rapid, in-depth characterization of antigen specific proliferation and expansion of preexisting memory T cells in concert with an innate adjuvant-driven response. In this work we demonstrate the capability of this platform to characterize biologically relevant changes in the cellular response across memory T-cells, B cells, monocytes, and NK cells, at an unprecedented level of detail. This approach that we call Immunocartography has the potential to transform the way new vaccines can be assessed before and throughout clinical development.
Assuntos
Linfócitos B/efeitos dos fármacos , Vacinas contra Difteria, Tétano e Coqueluche Acelular/farmacologia , Imunogenicidade da Vacina , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Monitorização Imunológica , Proteômica , Análise de Célula Única , Linfócitos T/efeitos dos fármacos , Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos/farmacologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Memória Imunológica/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Valor Preditivo dos Testes , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fluxo de TrabalhoRESUMO
The mutant gdPT R9K/E129G is a genetically detoxified variant of the pertussis toxin (PTx) and represents an attractive candidate for the development of improved pertussis vaccines. The impact of the mutations on the overall protein structure and its immunogenicity has remained elusive. Here we present the crystal structure of gdPT and show that it is nearly identical to that of PTx. Hydrogen-deuterium exchange mass spectrometry revealed dynamic changes in the catalytic domain that directly impacted NAD+ binding which was confirmed by biolayer interferometry. Distal changes in dynamics were also detected in S2-S5 subunit interactions resulting in tighter packing of B-oligomer corresponding to increased thermal stability. Finally, antigen stimulation of human whole blood, analyzed by a previously unreported mass cytometry assay, indicated broader immunogenicity of gdPT compared to pertussis toxoid. These findings establish a direct link between the conserved structure of gdPT and its ability to generate a robust immune response.
Assuntos
Toxina Pertussis/química , Vacina contra Coqueluche/genética , Conformação Proteica , Toxoides/genética , Animais , Bordetella pertussis/genética , Bordetella pertussis/patogenicidade , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Medição da Troca de Deutério , Humanos , Toxina Pertussis/genética , Vacina contra Coqueluche/química , Coqueluche/microbiologia , Coqueluche/prevenção & controleRESUMO
BACKGROUND: Very little longitudinal information is available regarding the performance of T cell-based tests for Mycobacterium tuberculosis infection. To address this deficiency, we conducted a longitudinal assessment of the enzyme-linked immunosorbent spot test (ELISPOT) test in comparison to the standard tuberculin skin test (TST). METHODS AND FINDINGS: In tuberculosis (TB) contacts we repeated ELISPOT tests 3 mo (n = 341) and 18 mo (n = 210) after recruitment and TSTs at 18 mo (n = 130). We evaluated factors for association with conversion and reversion and investigated suspected cases of TB. Of 207 ELISPOT-negative contacts, 51 (24.6%) had 3-mo ELISPOT conversion, which was associated with a positive recruitment TST (odds ratio [OR] 2.2, 95% confidence interval [CI] 1.0-5.0, p = 0.048) and negatively associated with bacillus Calmette-Guérin (BCG) vaccination (OR 0.5, 95% CI 0.2-1.0, p = 0.06). Of 134 contacts, 54 (40.2%) underwent 3-mo ELISPOT reversion, which was less likely in those with a positive recruitment TST (OR 0.3, 95% CI 0.1-0.8, p = 0.014). Between 3 and 18 mo, 35/132 (26.5%) contacts underwent ELISPOT conversion and 28/78 (35.9%) underwent ELISPOT reversion. Of the 210 contacts with complete results, 73 (34.8%) were ELISPOT negative at all three time points; 36 (17.1%) were positive at all three time points. Between recruitment and 18 mo, 20 (27%) contacts had ELISPOT conversion; 37 (50%) had TST conversion, which was associated with a positive recruitment ELISPOT (OR 7.2, 95% CI 1.4-37.1, p = 0.019); 18 (32.7%) underwent ELISPOT reversion; and five (8.9%) underwent TST reversion. Results in 13 contacts diagnosed as having TB were mixed, but suggested higher TST sensitivity. CONCLUSIONS: Both ELISPOT conversion and reversion occur after M. tuberculosis exposure. Rapid ELISPOT reversion may reflect M. tuberculosis clearance or transition into dormancy and may contribute to the relatively low reported ELISPOT conversion rate. Therefore, a negative ELISPOT test for M. tuberculosis infection should be interpreted with caution.
Assuntos
Ensaio de Imunoadsorção Enzimática , Interferon gama/análise , Mycobacterium tuberculosis/isolamento & purificação , Linfócitos T/metabolismo , Tuberculose/diagnóstico , Adolescente , Adulto , Idoso , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Estudos de Coortes , Busca de Comunicante , DNA Bacteriano/genética , Bases de Dados Factuais , Reações Falso-Negativas , Feminino , Seguimentos , Gâmbia/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Escarro/microbiologia , Linfócitos T/imunologia , Fatores de Tempo , Teste Tuberculínico , Tuberculose/epidemiologia , Tuberculose/imunologia , Tuberculose/microbiologiaRESUMO
Commercial tests measuring IFN-gamma responses to ESAT-6 and CFP-10 are available for diagnosing Mycobacterium tuberculosis infection. Measures that minimize cost and complexity will facilitate their application in less-developed countries. We investigated whether overlapping peptides representing both ESAT-6 and CFP-10 are required to detect M. tuberculosis infection in a high TB-burden country, and whether they can be combined in a single pool. ESAT-6 and CFP-10 peptides were compared in IFN-gamma enzyme-linked immunospot (ELISPOT) in 183 HIV-negative smear-positive TB cases and 1673 HIV-negative household contacts. Separate peptide pools for each antigen were compared with a combined pool in 498 contacts. Forty per cent of responsive contacts recognized both antigens, 51% only ESAT-6 and 10% only CFP-10, whereas 56% of responsive cases recognized both antigens, 30% only ESAT-6 and 13% only CFP-10. Accordingly, CFP-10 response rates were higher for TB cases (odds ratio 2.409, P<0.001). Low purified protein derivative response rates indicated that responses to CFP-10 only were non-specific in contacts. Agreement between peptides in separate versus combined pools was good (kappa=0.758, r=0.840). Therefore a combined ESAT-6/CFP-10 peptide pool provided maximum sensitivity and efficiency, but CFP-10 was mainly required to detect active disease.
Assuntos
Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Ensaio de Imunoadsorção Enzimática/métodos , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/diagnóstico , Adolescente , Adulto , Criança , Feminino , Gâmbia , Humanos , Masculino , Sensibilidade e Especificidade , Teste Tuberculínico , Tuberculose Pulmonar/transmissãoRESUMO
Contact investigation is a key component of tuberculosis (TB) control in developed, but not developing, countries. We aimed to measure the prevalence of TB among household contacts of sputum-smear-positive TB cases in The Gambia and to assess the sensitivity of an enzyme-linked immunospot (ELISPOT) assay in this regard. Household contacts of adult smear-positive TB patients were assessed by questionnaire, purified protein derivative (PPD) skin test, ELISPOT assay, physical examination, chest X-ray and sputum/gastric aspirate. Thirty-three TB cases were identified from 2174 of 2381 contacts of 317 adult smear-positive pulmonary TB patients, giving a prevalence of 1518/100000. The cases identified tended to have milder disease than those passively detected. The sensitivity of ESAT-6/CFP-10 ELISPOT test as a screening test for TB disease was estimated as 71%. Fifty-six per cent of contacts with a PPD skin test result >or=10mm induration had detectable responses to ESAT-6/CFP-10 by ELISPOT; 11% with a negative PPD skin test (<10mm) had a positive ESAT-6/CFP-10 response. Active screening for TB among contacts of TB patients may have a role in TB control in The Gambia. These individuals are a high-risk group, and the disease identified is less advanced than that found through passive case detection. An ELISPOT assay was relatively insensitive as a screening test for TB.
Assuntos
Busca de Comunicante/métodos , Tuberculose Pulmonar/epidemiologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Características da Família , Feminino , Gâmbia/epidemiologia , Humanos , Lactente , Masculino , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Prevalência , Sensibilidade e Especificidade , Testes Cutâneos/métodos , Escarro/microbiologia , Teste Tuberculínico , Tuberculose Pulmonar/diagnósticoRESUMO
The restimulation of an immune memory response by in vitro culture of blood cells with a specific antigen has been used as a way to gauge immunity to vaccines for decades. In this commentary we discuss a less appreciated application to support vaccine process development. We report that human whole blood from pre-primed subjects can generate a profound adjuvant-modulated, antigen-specific response to several different vaccine formulations. The response is able to differentiate subtle changes in the quality of an immune memory response to vaccine formulations and can be used to select optimal conditions relating to a particular manufacture process step. While questions relating to closeness to in vivo vaccination remain, the approach is another big step nearer to the more relevant human response. It has special importance for new adjuvant development, complementing other preclinical in vivo and in vitro approaches to considerably de-risk progression of novel vaccines before and throughout early clinical development. Broader implications of the approach are discussed.
Assuntos
Adjuvantes Imunológicos , Sangue/imunologia , Memória Imunológica , Vacinas/imunologia , Imunidade Adaptativa , Ensaios de Triagem em Larga Escala , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Linfócitos T/imunologia , VacinaçãoRESUMO
Monitoring the immunological functionality of vaccine formulations is critical for vaccine development. While the traditional approach using established animal models has been relatively effective, the use of animals is costly and cumbersome, and animal models are not always reflective of a human response. The development of a human-based approach would be a major step forward in understanding how vaccine formulations might behave in humans. Here, we describe a platform methodology using fresh human whole blood (hWB) to monitor adjuvant-modulated, antigen-specific responses to vaccine formulations, which is amenable to analysis by standard immunoassays as well as a variety of other analytical techniques.
Assuntos
Sangue , Composição de Medicamentos , Vacinas , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Vacinas/química , Vacinas/farmacologiaRESUMO
Tobacco use is a major risk factor for tuberculosis (TB). Secondhand smoke (SHS) is also a risk factor for TB and to a lesser extent, Mycobacterium tuberculosis infection without disease. We investigated the added risk of M. tuberculosis infection due to SHS exposure in childhood contacts of TB cases in The Gambia. Participants were childhood household contacts aged ≤ 14 years of newly diagnosed pulmonary TB (PTB) cases. The intensity of exposure to the case was categorized according to whether contacts slept in the same room, same house, or a different house as the case. Contacts were tested with an enzyme-linked immunospot interferon gamma release assay. In multivariate regression models, M. tuberculosis infection was associated with increasing exposure to a case (odds ratios [OR]: 3.9, 95% confidence interval [CI]: 2.11-71.4, P < 0.001]) and with male gender (OR: 1.5 [95% CI: 1.12-2.11], P = 0.008). Tobacco use caused a 3-fold increase in the odds of M. tuberculosis infection in children who slept closest to a case who smoked within the same home compared with a nonsmoking case (OR: 8.0 [95% CI: 2.74-23.29] versus 2.4 [95% CI: 1.17-4.92], P < 0.001). SHS exposure as an effect modifier appears to greatly increase the risk of M. tuberculosis infection in children exposed to PTB cases. Smoking cessation campaigns may be important for reducing transmission of M. tuberculosis to children within households.
Assuntos
Mycobacterium tuberculosis/patogenicidade , Nicotiana/efeitos adversos , Poluição por Fumaça de Tabaco/efeitos adversos , Tuberculose/etiologia , Tuberculose/fisiopatologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Gâmbia , Humanos , Lactente , Relações Interpessoais , Masculino , Pessoa de Meia-Idade , Razão de Chances , Relações Pais-Filho , Fatores de Risco , Adulto JovemRESUMO
Tuberculosis remains a substantial global health problem despite effective drug treatments. The efficacy of BCG, the only available vaccine, is variable, especially in tuberculosis-endemic regions. Recent advances in the development of new vaccines against tuberculosis mean that the first of these are now entering into early clinical trials. A recombinant modified vaccinia virus Ankara expressing a major secreted antigen from Mycobacterium tuberculosis, antigen 85A, was the first new tuberculosis vaccine to enter into clinical trials in September 2002. This vaccine is known as MVA85A. In a series of phase I clinical trials in the UK, MVA85A had an excellent safety profile and was highly immunogenic. MVA85A was subsequently evaluated in a series of phase I trials in The Gambia, a tuberculosis-endemic area in west Africa. This vaccine is the only new subunit tuberculosis vaccine to enter into clinical trials in Africa to date. Here, we discuss some of the issues that were considered in the protocol design of these studies including recruitment, inclusion and exclusion criteria, reimbursement of study participants, and HIV testing. These issues are highly relevant to early clinical trials with all new tuberculosis vaccines in the developing world.
Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Ensaios Clínicos como Assunto/métodos , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose , Tuberculose/prevenção & controle , África , Ensaios Clínicos como Assunto/ética , Ensaios Clínicos como Assunto/normas , Infecções por HIV/diagnóstico , Infecções por HIV/epidemiologia , Humanos , Imunidade Celular , Hospedeiro Imunocomprometido , Vacinas contra a Tuberculose/imunologia , Vacinas contra a Tuberculose/uso terapêutico , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêutico , Vaccinia virus/imunologiaRESUMO
BACKGROUND: New tools are required to improve tuberculosis (TB) diagnosis and treatment, including enhanced ability to compare new treatment strategies. The ELISPOT assay uses Mycobacterium tuberculosis-specific antigens to produce a precise quantitative readout of the immune response to pathogen. We hypothesized that TB patients in The Gambia would have reduced ELISPOT counts after successful treatment. METHODS: We recruited Gambian adults with sputum smear and culture positive tuberculosis for ELISPOT assay and HIV test, and followed them up one year later to repeat testing and document treatment outcome. We used ESAT-6, CFP-10 and Purified Protein Derivative (PPD) as stimulatory antigens. We confirmed the reliability of our assay in 23 volunteers through 2 tests one week apart, comparing within and between subject variation. RESULTS: We performed an ELISPOT test at diagnosis and 12 months later in 89 patients. At recruitment, 70/85 HIV-negative patients (82%) were ESAT-6 or CFP-10 (EC) ELISPOT positive, 77 (90%) were PPD ELISPOT positive. Eighty-two cases (96%) successfully completed treatment: 44 (55%; p < 0.001) were EC ELISPOT negative at 12 months, 17 (21%; p = 0.051) were PPD ELISPOT negative. Sixty (73%) cured cases had a CFP-10 ELISPOT count decrease, 64 (78%) had an ESAT-6 ELISPOT count decrease, 58 (70%) had a PPD ELISPOT count decrease. There was a mean decline of 25, 44 and 47 SFU/2 x 105 cells for CFP-10, ESAT-6 and PPD respectively (p < 0.001 for all). Three of 4 HIV positive patients were cured, all 3 underwent ELISPOT reversion; all 4 not cured subjects (3 HIV-negative, 1 HIV positive) were ESAT-6, CFP-10 and PPD ELISPOT positive at 12 months. CONCLUSION: Successful tuberculosis treatment is accompanied by a significant reduction in the M. tuberculosis-specific antigen ELISPOT count. The ELISPOT has potential as a proxy measure of TB treatment outcome. Further investigation into the decay kinetics of T-cells with treatment is warranted.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Mycobacterium tuberculosis/imunologia , Tuberculose/tratamento farmacológico , Tuberculose/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Bactérias/imunologia , Feminino , Gâmbia/epidemiologia , Humanos , Interferon gama/imunologia , Masculino , Pessoa de Meia-Idade , Escarro/microbiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Tuberculose/sangue , Tuberculose/microbiologiaRESUMO
Vaccines based on conserved pneumococcal proteins are being investigated because serotype coverage by pneumococcal polysaccharide and polysaccharide conjugate vaccines is incomplete and may eventually decrease due to serotype replacement. Here, we examined the functionality of human antibodies induced by a candidate bivalent choline-binding protein A- pneumococcal histidine triad protein D (PcpA-PhtD) vaccine. Pre- and post-immune sera from subjects who had been vaccinated with the PcpA-PhtD candidate vaccine were tested in an established passive protection model in which mice were challenged by intravenous injection with Streptococcus pneumoniae serotype 3 strain A66.1. Serum antibody concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Bacterial surface binding by serum antibodies was determined by a flow cytometry-based assay. Sera from 20 subjects were selected based on low activity of pre-immune samples in the passive protection model. Bacterial surface binding correlated more strongly with anti-PcpA (0.87; p < 0.0001) than with anti-PhtD (0.71; p < 0.0001). The odds ratio for predicting survival in the passive protection assay was higher for the anti-PcpA concentration (470 [95% confidence interval (CI), 46.8 to >999.9]) than for the anti-PhtD concentration (3.4 [95% CI, 1.9 to 5.6]) or bacterial surface binding (9.4 [95% CI, 3.6 to 24.3]). Pooled post-immune serum also protected mice against a challenge with S. pneumoniae serotype 3 strain WU2. Both anti-PcpA and anti-PhtD antibodies induced by the bivalent candidate vaccine mediate protection against S. pneumoniae. The results also showed that the ELISA titer might be useful as a surrogate for estimating the functional activity of antibodies induced by pneumococcal protein vaccines.
Assuntos
Anticorpos Antibacterianos/administração & dosagem , Proteínas de Bactérias/imunologia , Hidrolases/imunologia , Imunização Passiva , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Adolescente , Adulto , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos Endogâmicos CBA , Pessoa de Meia-Idade , Vacinas Pneumocócicas/administração & dosagem , Análise de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Currently, reliable efficacy markers for assessment of new interventions against tuberculosis (TB) are limited to disease and death. More precise measurement of the human immune response to Mycobacterium tuberculosis infection may be important. A qualitative enzyme-linked immunospot assay (ELISPOT) result for early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) offers improved specificity over the purified protein derivative (PPD) skin test reaction in the detection of M. tuberculosis infection. We evaluated the quantitative ELISPOT and PPD skin test responses to recent M. tuberculosis exposure. METHODS: We studied quantitative PPD skin test and PPD ELISPOT results in 1052 healthy household contacts of index patients with cases of sputum smear-positive and culture-positive TB in The Gambia, according to a positive or negative ex vivo interferon gamma ELISPOT response to M. tuberculosis-specific antigens (ESAT-6/CFP-10). We then studied the quantitative PPD skin test and PPD ELISPOT results in patient contacts who had positive ESAT-6/CFP-10 results against a natural exposure gradient according to sleeping proximity to a patient with TB. RESULTS: The number of positive results was significantly greater for both PPD skin test and PPD ELISPOT in ESAT-6/CFP-10-positive subjects, compared with others (P<.0001). However, when quantitative PPD skin test and PPD ELISPOT results were compared in ESAT-6/CFP-10-positive subjects, only the ELISPOT count was sensitive to the exposure gradient, increasing significantly according to exposure (P=.009). CONCLUSIONS: The quantitative ELISPOT response to PPD in specific-antigen-positive contacts of patients with TB reflects the infectious load of M. tuberculosis as a result of recent exposure. This finding offers new possibilities for assessment of the efficacy of new interventions, and adjustment should be made for it when relating the early immune response to progression to disease.
Assuntos
Busca de Comunicante , Mycobacterium tuberculosis/isolamento & purificação , Linfócitos T/imunologia , Tuberculose/microbiologia , Tuberculose/transmissão , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Gâmbia/epidemiologia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Teste Tuberculínico , Tuberculose/epidemiologia , Tuberculose/imunologiaRESUMO
Currently marketed Streptococcus pneumoniae vaccines are based on polysaccharide capsular antigens from the most common strains. Pneumococcal histidine triad protein D (PhtD) is a conserved surface protein that is being evaluated as a candidate for a vaccine with improved serotype coverage. Here, we measured the functional activity of human anti-PhtD antibodies in a passive protection model wherein mice were challenged with a lethal dose of S. pneumoniae by intravenous injection. This functional activity was compared with anti-PhtD antibody concentrations measured by enzyme-linked immunosorbent assay (ELISA) to estimate the 50% protective dose (ED50). Anti-PhtD antibodies affinity purified from pooled normal human sera passively protected mice with an ED50 of 1679 ELISA units/ml (95% confidence interval, 1420-1946). Sera from subjects injected with aluminum-adjuvanted PhtD in a phase I trial had similar activity per unit of antibody (ED50 = 1331 ELISA units/ml [95% confidence interval, 762-2038]). Vaccine-induced activity in the passive protection model was blocked by pre-incubation with recombinant PhtD but not by a control S. pneumoniae antigen (LytB). These results show that human anti-PhtD antibodies, whether naturally acquired or induced by the PhtD candidate vaccine, are functional. This supports the development of the PhtD candidate as part of a broadly protective pneumococcal vaccine.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Imunização Passiva/métodos , Proteínas de Membrana/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/uso terapêutico , Streptococcus pneumoniae/imunologia , Adulto , Idoso , Animais , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/uso terapêutico , Especificidade de Anticorpos , Antígenos de Bactérias/imunologia , Ensaios Clínicos Fase I como Assunto , Humanos , Camundongos , Pessoa de Meia-Idade , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/imunologia , Análise de SobrevidaRESUMO
A tuberculosis (TB) vaccine consisting of a recombinant fusion protein (H4) and a novel TLR9 adjuvant (IC31) is in clinical development. To better understand the H4-IC31 ratio, we measured the binding capacity of IC31 for H4 protein and immunized mice with formulations that contained limiting to excess ratios of IC31 to H4. An immunomodulated H4-specific IFNγ response was only observed when IC31 was present in excess of H4. Since TLR expression is species-specific and the vaccine is intended to boost BCG-primed immunity, we questioned whether data in mice would translate to humans. To address this question, we used the fresh human Whole Blood (hWB) recovered from BCG-vaccinated subjects to screen H4-IC31 formulations. We found IC31 modulation in hWB to be quite distinct from the TLR4-Adjuvant. Unlike TLR4-Adjuvant, IC31 formulations did not induce the pro-inflammatory cytokine TNFα, but modulated a robust H4-specific IFNγ response after 12 d of culture. We then re-stimulated the fresh hWB of 5 BCG-primed subjects with formulations that had excess or limiting IC31 binding for H4 protein and again found that an immunomodulated H4-specific IFNγ response needed an excess of IC31. Finally, we monitored the zeta (ζ) potential of H4-IC31 formulations and found that the overall charge of H4-IC31 particles changes from negative to positive once IC31 is in greater than 9-fold excess. Using two diverse yet mutually supportive approaches, we confirm the need for an excess of IC31 adjuvant in H4 TB vaccine formulations and suggest surface potential may be an important factor.
Assuntos
Antígenos de Bactérias/imunologia , Imunomodulação , Oligodesoxirribonucleotídeos/farmacologia , Oligopeptídeos/farmacologia , Vacinas contra a Tuberculose/imunologia , Animais , Células Cultivadas , Química Farmacêutica , Combinação de Medicamentos , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Camundongos , Vacinação/métodosRESUMO
The purified protein derivative (PPD) skin test for Mycobacterium tuberculosis infection lacks specificity. We assessed 2 more specific M. tuberculosis antigens (ESAT-6 and CFP-10) by enzyme-linked immunospot assay (ELISPOT) compared with PPD by ELISPOT and skin test in The Gambia. Of 735 household contacts of 130 sputum smear-positive tuberculosis cases, 476 (65%) tested positive by PPD ELISPOT, 300 (41%) tested positive by PPD skin test, and 218 (30%) tested positive by ESAT-6/CFP-10 ELISPOT. Only 15 (2%) had positive ESAT-6/CFP-10 results and negative PPD results by ELISPOT. With increasing M. tuberculosis exposure, the percentage of subjects who were PPD skin test positive/ESAT-6/CFP-10 ELISPOT negative increased (P<.001), whereas the percentage of subjects who were PPD skin test negative/PPD ELISPOT positive decreased (P=.011). Eighteen (31%) ESAT-6/CFP-10 ELISPOT-positive subjects in the lowest exposure category had negative PPD skin test results. ESAT-6/CFP-10 ELISPOT probably offers increased specificity in the diagnosis of M. tuberculosis infection in this tropical setting of endemicity, at the cost of some sensitivity.
Assuntos
Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Ensaio de Imunoadsorção Enzimática/métodos , Mycobacterium tuberculosis/isolamento & purificação , Testes Cutâneos/métodos , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Gâmbia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Tuberculina/análiseRESUMO
Understanding the relevant biological activity of any pharmaceutical formulation destined for human use is crucial. For vaccine-based formulations, activity must reflect the expected immune response, while for non-vaccine therapeutic agents, such as monoclonal antibodies, a lack of immune response to the formulation is desired. During early formulation development, various biochemical and biophysical characteristics can be monitored in a high-throughput screening (HTS) format. However, it remains impractical and arguably unethical to screen samples in this way for immunological functionality in animal models. Furthermore, data for immunological functionality lag formulation design by months, making it cumbersome to relate back to formulations in real-time. It is also likely that animal testing may not accurately reflect the response in humans. For a more effective formulation screen, a human whole blood (hWB) approach can be used to assess immunological functionality. The functional activity relates directly to the human immune response to a complete formulation (adjuvant/antigen) and includes adjuvant response, antigen response, adjuvant-modulated antigen response, stability, and potentially safety. The following commentary discusses the hWB approach as a valuable new tool to de-risk manufacture, formulation design, and clinical progression.
Assuntos
Sangue/imunologia , Química Farmacêutica/métodos , Vacinas/imunologia , HumanosRESUMO
BACKGROUND: Our objective was to evaluate procalcitonin (PCT) and C-reactive protein (CRP) as predictors of a pneumococcal etiology in community-acquired pneumonia (CAP) in hospitalized children. METHODS: Children requiring hospitalization for CAP were prospectively enrolled. The following indices were determined: antibodies against pneumococcal surface proteins (anti-PLY, pneumococcal histidine triad D, pneumococcal histidine triad E, LytB and pneumococcal choline-binding protein A), viral serology, nasopharyngeal cultures and polymerase chain reaction for 13 respiratory viruses, blood pneumococcal polymerase chain reaction, pneumococcal urinary antigen, PCT and CRP. Presumed pneumococcal CAP (P-CAP) was defined as a positive blood culture or polymerase chain reaction for Streptococcus pneumoniae or as a pneumococcal surface protein seroresponse (≥2-fold increase). RESULTS: Seventy-five patients were included from which 37 (49%) met the criteria of P-CAP. Elevated PCT and CRP values were strongly associated with P-CAP with odds ratios of 23 (95% confidence interval: 5-117) for PCT and 19 (95% confidence interval: 5-75) for CRP in multivariate analysis. The sensitivity was 94.4% for PCT (cutoff: 1.5 ng/mL) and 91.9% for CRP (cutoff: 100 mg/L). A value≤0.5 ng/mL of PCT ruled out P-CAP in >90% of cases (negative likelihood ratio: 0.08). Conversely, a PCT value≥1.5 ng/mL associated with a positive pneumococcal urinary antigen had a diagnostic probability for P-CAP of almost 80% (positive likelihood ratio: 4.59). CONCLUSIONS: PCT and CRP are reliable predictors of P-CAP. Low cutoff values of PCT allow identification of children at low risk of P-CAP. The association of elevated PCT or CRP with a positive pneumococcal urinary antigen is a strong predictor of P-CAP.