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Despite the fact that the majority of people in tuberculosis (TB)-endemic areas are vaccinated with the Bacillus Calmette-Guérin (BCG) vaccine, TB remains the leading infectious cause of death. Data from both animal models and humans show that BCG and subunit vaccines induce T cells of different phenotypes, and little is known about how BCG priming influences subsequent booster vaccines. To test this, we designed a novel Mycobacterium tuberculosis-specific (or "non-BCG") subunit vaccine with protective efficacy in both mice and guinea pigs and compared it to a known BCG boosting vaccine. In naive mice, this M. tuberculosis-specific vaccine induced similar protection compared with the BCG boosting vaccine. However, in BCG-primed animals, only the M. tuberculosis-specific vaccine added significantly to the BCG-induced protection. This correlated with the priming of T cells with a lower degree of differentiation and improved lung-homing capacity. These results have implications for TB vaccine design.
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Antígenos de Bactérias/imunologia , Diferenciação Celular/imunologia , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Linfócitos T , Tuberculose , Animais , Feminino , Cobaias , Camundongos , Linfócitos T/imunologia , Linfócitos T/patologia , Tuberculose/imunologia , Tuberculose/patologia , Tuberculose/prevenção & controle , VacinaçãoRESUMO
Globally, an estimated 131 million new cases of chlamydial infection occur annually. Chlamydia trachomatis infection can cause permanent damage to the fallopian tubes in woman, resulting in infertility and a risk of ectopic pregnancy. There is a great need for a vaccine against Chlamydia trachomatis and as a result there is a need for assays to evaluate functional immune responses for use in future clinical trials and epidemiological studies. Antibodies play a crucial role in the defense against infection and can be protective by several functions, including phagocytosis and neutralization. Vaccine development could greatly benefit from a method to measure functional C. trachomatis-specific antibodies in a large number of samples. In the current in vitro antibody protection assays, which measure the capacity of antibodies to facilitate phagocytic uptake of C. trachomatis, the phagocytosed bacteria have to be counted manually. This is both labor demanding, time consuming, and it prevents high-throughput usage of this method. In this study, we, therefore, developed a simple and rapid flow cytometry based assay to measure the capacity of antibodies to mediate Fc-receptor dependent phagocytosis. This method is highly reproducible and suitable to analyze large numbers of clinical and nonclinical samples. © 2018 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.
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Anticorpos Antibacterianos/análise , Infecções por Chlamydia , Citometria de Fluxo/métodos , Fagocitose , Linhagem Celular , Chlamydia trachomatis , HumanosRESUMO
Chlamydia trachomatis is one of the most common sexually transmitted bacterial pathogens in humans. The infection is often asymptomatic and can lead to chronic manifestations. The infectious elementary body and the replicating reticulate body are the two growth forms in the normal developmental cycle. Under the influence of interferon-γ, the normal cycle is disrupted because of tryptophan degradation, leading to a third persistent form, the aberrant reticulate body.For the genital strain C. trachomatis D/UW-3/CX we established a quantitative, label-free proteomic approach, and identified in total 655 out of 903 (73%) predicted proteins, allowing the first quantitative comparison of all three growth forms. Inclusion membrane proteins and proteins involved in translation were more abundant in the reticulate body (RB)1 and aberrant reticulate body (ARB) forms, whereas proteins of the type III Secretion System and the cell envelope were more abundant in the elementary body (EB) form, reflecting the need for these proteins to establish infection and for host interactions.In the interferon-γ induced ARB proteome, the tryptophan synthase subunits were identified as biomarkers with a strong increase from less than 0.05% to 9% of the total protein content, reflecting an inherent defense strategy for the pathogen to escape interferon-γ mediated immune pressure. Furthermore, the total tryptophan content in the ARB form was 1.9-fold lower compared with the EB form, and we demonstrate that modulation of the protein repertoire toward lower abundance of proteins with high tryptophan content, is a mechanism which contributes to establish and maintain chlamydial persistence. Thus, quantitative proteomics provides insights in the Chlamydia defense mechanisms to escape interferon-γ mediated immune pressure.
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Proteínas de Bactérias/metabolismo , Chlamydia trachomatis/fisiologia , Interferon gama/farmacologia , Proteômica/métodos , Triptofano/metabolismo , Chlamydia trachomatis/efeitos dos fármacos , Cromatografia Líquida , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Development of rapid diagnostic tests for tuberculosis is a global priority. A whole proteome screen identified Mycobacterium tuberculosis antigens associated with serological responses in tuberculosis patients. We used World Health Organization (WHO) target product profile (TPP) criteria for a detection test and triage test to evaluate these antigens. METHODS: Consecutive patients presenting to microscopy centers and district hospitals in Peru and to outpatient clinics at a tuberculosis reference center in Vietnam were recruited. We tested blood samples from 755 HIV-uninfected adults with presumptive pulmonary tuberculosis to measure IgG antibody responses to 57 M. tuberculosis antigens using a field-based multiplexed serological assay and a 132-antigen bead-based reference assay. We evaluated single antigen performance and models of all possible 3-antigen combinations and multiantigen combinations. RESULTS: Three-antigen and multiantigen models performed similarly and were superior to single antigens. With specificity set at 90% for a detection test, the best sensitivity of a 3-antigen model was 35% (95% confidence interval [CI], 31-40). With sensitivity set at 85% for a triage test, the specificity of the best 3-antigen model was 34% (95% CI, 29-40). The reference assay also did not meet study targets. Antigen performance differed significantly between the study sites for 7/22 of the best-performing antigens. CONCLUSIONS: Although M. tuberculosis antigens were recognized by the IgG response during tuberculosis, no single antigen or multiantigen set performance approached WHO TPP criteria for clinical utility among HIV-uninfected adults with presumed tuberculosis in high-volume, urban settings in tuberculosis-endemic countries.
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Antígenos de Bactérias/imunologia , Imunoglobulina G/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Peru , Reprodutibilidade dos Testes , Testes Sorológicos/métodos , Testes Sorológicos/normas , Tuberculose Pulmonar/epidemiologia , Adulto JovemRESUMO
BACKGROUND: User-friendly, rapid, inexpensive yet accurate TB diagnostic tools are urgently needed at points of care in resource-limited settings. We investigated host biomarkers detected in serum samples obtained from adults with signs and symptoms suggestive of TB at primary healthcare clinics in five African countries (Malawi, Namibia, South Africa, The Gambia and Uganda), for the diagnosis of TB disease. METHODS: We prospectively enrolled individuals presenting with symptoms warranting investigation for pulmonary TB, prior to assessment for TB disease. We evaluated 22 host protein biomarkers in stored serum samples using a multiplex cytokine platform. Using a pre-established diagnostic algorithm comprising of laboratory, clinical and radiological findings, participants were classified as either definite TB, probable TB, questionable TB status or non-pulmonary TB. RESULTS: Of the 716 participants enrolled, 185 were definite and 29 were probable TB cases, 6 had questionable TB disease status, whereas 487 had no evidence of TB. A seven-marker biosignature of C reactive protein, transthyretin, IFN-γ, complement factor H, apolipoprotein-A1, inducible protein 10 and serum amyloid A identified on a training sample set (n=491), diagnosed TB disease in the test set (n=210) with sensitivity of 93.8% (95% CI 84.0% to 98.0%), specificity of 73.3% (95% CI 65.2% to 80.1%), and positive and negative predictive values of 60.6% (95% CI 50.3% to 70.1%) and 96.4% (95% CI 90.5% to 98.8%), respectively, regardless of HIV infection status or study site. CONCLUSIONS: We have identified a seven-marker host serum protein biosignature for the diagnosis of TB disease irrespective of HIV infection status or ethnicity in Africa. These results hold promise for the development of a field-friendly point-of-care screening test for pulmonary TB.
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Proteínas Sanguíneas/análise , Tuberculose Pulmonar/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Adulto , África , Algoritmos , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sistemas Automatizados de Assistência Junto ao Leito , Valor Preditivo dos Testes , Atenção Primária à Saúde , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
The VD4 region from the Chlamydia trachomatis major outer membrane protein contains important neutralizing B-cell epitopes of relevance for antibody-mediated protection against genital tract infection. We developed a multivalent vaccine construct based on VD4s and their surrounding constant segments from serovars D, E, and F. Adjuvanted with cationic liposomes, this construct promoted strong immune responses to serovar-specific epitopes, the conserved LNPTIAG epitope and neutralized serovars D, E, and F. Vaccinated mice were protected against challenge, with protection defined as reduced bacterial numbers in vagina and prevention of pathological changes in the upper genital tract. Adoptive transfer of serum and T-cell depletion experiments demonstrated a dominant role for antibodies and CD4(+) T cells in the protective immune response. Integrating a multivalent VD4 construct into the sequence of the major outer membrane protein resulted in a protective and broadly neutralizing vaccine. Our findings emphasize the important role of antibodies in protection against Chlamydia trachomatis.
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Anticorpos Antibacterianos/fisiologia , Anticorpos Neutralizantes/fisiologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Infecções por Chlamydia/prevenção & controle , Chlamydia trachomatis/metabolismo , Animais , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/química , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Infecções por Chlamydia/patologia , Chlamydia trachomatis/imunologia , Feminino , CamundongosRESUMO
Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.
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Imãs , Fagossomos/microbiologia , Coloração e Rotulagem/métodos , Humanos , Lipídeos/química , Listeria monocytogenes/isolamento & purificação , Macrófagos/microbiologia , Macrófagos/ultraestrutura , Imãs/química , Microscopia Eletrônica de Transmissão e Varredura , Microscopia de Fluorescência , Mycobacterium/isolamento & purificação , Fagossomos/ultraestruturaRESUMO
When infected with Mycobacterium tuberculosis, most individuals will remain clinically healthy but latently infected. Latent infection has been proposed to partially involve M. tuberculosis in a nonreplicating stage, which therefore represents an M. tuberculosis phenotype that the immune system most likely will encounter during latency. It is therefore relevant to examine how this particular nonreplicating form of M. tuberculosis interacts with the host immune system. To study this, we first induced a state of nonreplication through prolonged nutrient starvation of M. tuberculosis in vitro. This resulted in nonreplicating persistence even after prolonged culture in phosphate-buffered saline. Infection with either exponentially growing M. tuberculosis or nutrient-starved M. tuberculosis resulted in similar lung CFU levels in the first phase of the infection. However, between week 3 and 6 postinfection, there was a very pronounced increase in bacterial levels and associated lung pathology in nutrient-starved-M. tuberculosis-infected mice. This was associated with a shift from CD4 T cells that coexpressed gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) or IFN-γ, TNF-α, and interleukin-2 to T cells that only expressed IFN-γ. Thus, nonreplicating M. tuberculosis induced through nutrient starvation promotes a bacterial form that is genetically identical to exponentially growing M. tuberculosis yet characterized by a differential impact on the immune system that may be involved in undermining host antimycobacterial immunity and facilitate increased pathology and transmission.
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Imunidade Adaptativa , Antígenos de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Peptídeos/imunologia , Linfócitos T/imunologia , Tuberculose Pulmonar/imunologia , Animais , Antígenos de Bactérias/administração & dosagem , Quimera , Contagem de Colônia Microbiana , Meios de Cultura/química , Meios de Cultura/farmacologia , Modelos Animais de Doenças , Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Imunofenotipagem , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-2/biossíntese , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Peptídeos/administração & dosagem , Peptídeos/síntese química , Inanição , Linfócitos T/microbiologia , Linfócitos T/patologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Tuberculose Pulmonar/prevenção & controle , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia , VacinaçãoRESUMO
In order to successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models that mimic latent infection are valuable tools for describing the changes in metabolism that occur when the bacterium exists in a non-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional difference gel electrophoresis, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. Through the label-free LC-MS/MS analysis of fractionated samples, 1176 proteins were identified from culture filtrates of log phase and nutrient-starved cultures, and the protein levels of 230 proteins were increased in nutrient-starved culture filtrates, whereas those of 208 proteins were decreased. By means of Gene Ontology clustering analysis, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin-antitoxin systems were present in larger quantities in nutrient-starved cultures, supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy. Decreased abundance of proteins involved in amino acid and protein synthesis was apparent, as well as changes in the lipid metabolism. Further analysis of the dataset identified increased abundance of lipoproteins and decreased abundance of ESAT-6 family proteins. Results from the two-dimensional difference gel electrophoresis proteomics demonstrated overall agreement with the LC-MS/MS data and added complementary insights about protein degradation and modification.
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Proteínas de Bactérias/metabolismo , ATPases Bacterianas Próton-Translocadoras/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteoma/metabolismo , Adaptação Fisiológica , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Vias Biossintéticas , Análise por Conglomerados , Lipoproteínas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/crescimento & desenvolvimento , Proteômica , Estresse Fisiológico , Espectrometria de Massas em Tandem , Eletroforese em Gel Diferencial BidimensionalRESUMO
BACKGROUND: Chlamydia trachomatis (CT) Major Outer Membrane Protein (MOMP) holds a neutralising epitope in the Variable Domain 4 (VD4), and this region's immune dominance during infection is well known. This study aimed to assess the antibody response induced after infection and compare it for specificity and functionality to the response following vaccination with the vaccine CTH522, which contains VD4's from serovars D, E, F, and G. METHODS: We assessed the antibody epitopes in MOMP by a high density peptide array. Furthermore, the role of the VD4 epitope in neutralisation was explored by competitive inhibition experiments with a fusion protein holding the neutralising VD4 linear epitope. This was done in two independent groups: 1) MOMP seropositive individuals infected with CT (n = 10, from case-control study) and 2) CTH522/CAF®01-vaccinated females (n = 14) from the CHLM-01 clinical trial. FINDINGS: We identified the major antigenic regions in MOMP as VD4 and the conserved region just before VD3 in individuals infected with CT. The same regions, with the addition of VD1, were identified in vaccine recipients. Overall, the VD4 peptide responses were uniform in vaccinated individuals and led to inhibition of infection in vitro in all tested samples, whereas the VD4 responses were more heterogenous in individuals infected with CT, and only 2 out of 10 samples had VD4-mediated neutralising antibody responses. INTERPRETATION: These data provide insights into the role of antibodies against MOMP VD4 induced after infection and vaccination, and show that their functionality differs. The induction of functional VD4-specific antibodies in vaccine recipients mimics previous results from animal models. FUNDING: This work was supported by the European Commission through contract FP7-HEALTH-2011.1.4-4-280873 (ADITEC) and Fonden til Lægevidenskabens Fremme.
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Anticorpos Antibacterianos , Vacinas Bacterianas , Infecções por Chlamydia , Chlamydia trachomatis , Epitopos , Vacinação , Humanos , Chlamydia trachomatis/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/sangue , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Feminino , Vacinas Bacterianas/imunologia , Epitopos/imunologia , Anticorpos Neutralizantes/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Adulto , Masculino , Estudos de Casos e Controles , Adulto JovemRESUMO
The clinical development of an effective Chlamydia vaccine requires in-depth understanding of how well protective pre-clinical immune signatures translate to humans. Here, we report a comparative immunological characterization of CTH522/CAF®01 in female mice and humans. We find a range of immune signatures that translate from mouse to human, including a Th1/Th17 cytokine profile and antibody functionality. We identify vaccine-induced T cell epitopes, conserved among Chlamydia serovars, and previously found in infected individuals. Using the mouse model, we show that the common immune signature protected against ascending infection in mice, and vaccine induced antibodies could delay bacterial ascension to the oviduct, as well as development of pathology, in a T cell depleted mouse model. Finally, we demonstrate long-lasting immunity and protection of mice one year after vaccination. Based on the results obtained in the present study, we propose to further investigate CTH522/CAF®01 in a phase IIb study.
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Infecções por Chlamydia , Chlamydia , Vacinas , Humanos , Feminino , Animais , Camundongos , Infecções por Chlamydia/microbiologia , Anticorpos , Vacinação , Vacinas BacterianasRESUMO
BACKGROUND: Trachoma is a leading cause of infection-related blindness worldwide. This disease is caused by recurrent Chlamydia trachomatis (Ct) infections of the conjunctiva and develops in two phases: i) active (acute trachoma, characterized by follicular conjunctivitis), then long-term: ii) scarring (chronic trachoma, characterized by conjunctival fibrosis, corneal opacification and eyelid malposition). Scarring trachoma is driven by the number and severity of reinfections. The immune system plays a pivotal role in trachoma including exacerbation of the disease. Hence the immune system may also be key to developing a trachoma vaccine. Therefore, we characterized clinical and local immune response kinetics in a non-human primate model of acute conjunctival Ct infection and disease. METHODOLOGY/PRINCIPAL FINDINGS: The conjunctiva of non-human primate (NHP, Cynomolgus monkeys-Macaca fascicularis-) were inoculated with Ct (B/Tunis-864 strain, B serovar). Clinical ocular monitoring was performed using a standardized photographic grading system, and local immune responses were assessed using multi-parameter flow cytometry of conjunctival cells, tear fluid cytokines, immunoglobulins, and Ct quantification. Clinical findings were similar to those observed during acute trachoma in humans, with the development of typical follicular conjunctivitis from the 4th week post-exposure to the 11th week. Immunologic analysis indicated an early phase influx of T cells in the conjunctiva and elevated interleukins 4, 8, and 5, followed by a late phase monocytic influx accompanied with a decrease in other immune cells, and tear fluid cytokines returning to initial levels. CONCLUSION/SIGNIFICANCE: Our NHP model accurately reproduces the clinical signs of acute trachoma, allowing for an accurate assessment of the local immune responses in infected eyes. A progressive immune response occurred for weeks after exposure to Ct, which subsided into a persistent innate immune response. An understanding of these local responses is the first step towards using the model to assess new vaccine and therapeutic strategies for disease prevention.
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BACKGROUND: There is no vaccine against the major global pathogen Chlamydia trachomatis; its different serovars cause trachoma in the eye or chlamydia in the genital tract. We did a clinical trial administering CTH522, a recombinant version of the C trachomatis major outer membrane molecule, in different dose concentrations with and without adjuvant, to establish its safety and immunogenicity when administered intramuscularly, intradermally, and topically into the eye, in prime-boost regimens. METHODS: CHLM-02 was a phase 1, double-blind, randomised, placebo-controlled trial at the National Institute for Health Research Imperial Clinical Research Facility, London, UK. Participants were healthy men and non-pregnant women aged 18-45 years, without pre-existing C trachomatis genital infection. Participants were assigned into six groups by the electronic database in a pre-prepared randomisation list (A-F). Participants were randomly assigned (1:1:1:1:1) to each of the groups A-E (12 participants each) and 6 were randomly assigned to group F. Investigators were masked to treatment allocation. Groups A-E received investigational medicinal product and group F received placebo only. Two liposomal adjuvants were compared, CAF01 and CAF09b. The groups were intramuscular 85 µg CTH522-CAF01, or placebo on day 0 and two boosters or placebo at day 28 and 112, and a mucosal recall with either placebo or CTH522 topical ocularly at day 140 (A); intramuscular 85 µg CTH522-CAF01, two boosters at day 28 and 112 with additional topical ocular administration of CTH522, and a mucosal recall with either placebo or CTH522 topical ocularly at day 140 (B); intramuscular 85 µg CTH522-CAF01, two boosters at day 28 and 112 with additional intradermal administration of CTH522, and a mucosal recall with either placebo or CTH522 topical ocularly at day 140 (C); intramuscular 15 µg CTH522-CAF01, two boosters at day 28 and 112, and a mucosal recall with either placebo or CTH522 topical ocularly at day 140 (D); intramuscular 85 µg CTH522-CAF09b, two boosters at day 28 and 112, and a mucosal recall with either placebo or CTH522 topical ocularly at day 140 (E); intramuscular placebo (F). The primary outcome was safety; the secondary outcome (humoral immunogenicity) was the percentage of trial participants achieving anti-CTH522 IgG seroconversion, defined as four-fold and ten-fold increase over baseline concentrations. Analyses were done as intention to treat and as per protocol. The trial is registered with ClinicalTrials.gov, NCT03926728, and is complete. FINDINGS: Between Feb 17, 2020 and Feb 22, 2022, of 154 participants screened, 65 were randomly assigned, and 60 completed the trial (34 [52%] of 65 women, 46 [71%] of 65 White, mean age 26·8 years). No serious adverse events occurred but one participant in group A2 discontinued dosing after having self-limiting adverse events after both placebo and investigational medicinal product doses. Study procedures were otherwise well tolerated; the majority of adverse events were mild to moderate, with only seven (1%) of 865 reported as grade 3 (severe). There was 100% four-fold seroconversion rate by day 42 in the active groups (A-E) and no seroconversion in the placebo group. Serum IgG anti-CTH522 titres were higher after 85 µg CTH522-CAF01 than 15 µg, although not significantly (intention-to-treat median IgG titre ratio groups A-C:D=5·6; p=0·062), with no difference after three injections of 85 µg CTH522-CAF01 compared with CTH522-CAF09b (group E). Intradermal CTH522 (group C) induced high titres of serum IgG anti-CTH522 neutralising antibodies against serovars B (trachoma) and D (urogenital). Topical ocular CTH522 (group B) at day 28 and 112 induced higher total ocular IgA compared with baseline (p<0·001). Participants in all active vaccine groups, particularly groups B and E, developed cell mediated immune responses against CTH522. INTERPRETATION: CTH522, adjuvanted with CAF01 or CAF09b, is safe and immunogenic, with 85 µg CTH522-CAF01 inducing robust serum IgG binding titres. Intradermal vaccination conferred systemic IgG neutralisation breadth, and topical ocular administration increased ocular IgA formation. These findings indicate CTH522 vaccine regimens against ocular trachoma and urogenital chlamydia for testing in phase 2, clinical trials. FUNDING: The EU Horizon Program TRACVAC.
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Vacinas Bacterianas , Chlamydia trachomatis , Lipossomos , Tracoma , Humanos , Adulto , Método Duplo-Cego , Feminino , Masculino , Adulto Jovem , Chlamydia trachomatis/imunologia , Tracoma/prevenção & controle , Adolescente , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/efeitos adversos , Pessoa de Meia-Idade , Injeções Intramusculares , Anticorpos Antibacterianos/sangue , Adjuvantes Imunológicos/administração & dosagem , Voluntários SaudáveisRESUMO
The only licensed tuberculosis (TB) vaccine, Bacillus Calmette Guerin (BCG), fails to reliably protect adolescents and adults from pulmonary TB, resulting in ~1.6 million deaths annually. Protein subunit vaccines have shown promise against TB in clinical studies. Unfortunately, most subunit vaccines require multiple administrations, which increases the risk of loss to follow-up and necessitates more complex and costly logistics. Given the well-documented adjuvant effect of BCG, we hypothesized that BCG co-administration could compensate for a reduced number of subunit vaccinations. To explore this, we developed an expression-optimized version of our H107 vaccine candidate (H107e), which does not cross-react with BCG. In the CAF®01 adjuvant, a single dose of H107e induced inferior protection compared to three H107e/CAF®01 administrations. However, co-administering a single dose of H107e/CAF®01 with BCG significantly improved protection, which was equal to BCG co-administered with three H107e/CAF®01 doses. Importantly, combining BCG with a single H107e/CAF®01 dose also increased protection in previously BCG-primed animals. Overall, a single dose of H107e/CAF®01 with BCG induced long-lived immunity and triggered BCG-specific Th17 responses. These data support co-administration of BCG and subunit vaccines in both BCG naïve and BCG-primed individuals as an improved TB vaccine strategy with reduced number of vaccination visits.
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Background: Ocular infections with Chlamydia trachomatis serovars A-C cause the neglected tropical disease trachoma. As infection does not confer complete immunity, repeated infections are common, leading to long-term sequelae such as scarring and blindness. Here, we apply a systems serology approach to investigate whether systemic antibody features are associated with susceptibility to infection. Methods: Sera from children in five trachoma endemic villages in the Gambia were assayed for 23 antibody features: IgG responses towards two C. trachomatis antigens and three serovars [elementary bodies and major outer membrane protein (MOMP), serovars A-C], IgG responses towards five MOMP peptides (serovars A-C), neutralization, and antibody-dependent phagocytosis. Participants were considered resistant if they subsequently developed infection only when over 70% of other children in the same compound were infected. Results: The antibody features assayed were not associated with resistance to infection (false discovery rate < 0.05). Anti-MOMP SvA IgG and neutralization titer were higher in susceptible individuals (p < 0.05 before multiple testing adjustment). Classification using partial least squares performed only slightly better than chance in distinguishing between susceptible and resistant participants based on systemic antibody profile (specificity 71%, sensitivity 36%). Conclusions: Systemic infection-induced IgG and functional antibody responses do not appear to be protective against subsequent infection. Ocular responses, IgA, avidity, or cell-mediated responses may play a greater role in protective immunity than systemic IgG.
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Tracoma , Criança , Humanos , Tracoma/diagnóstico , Tracoma/epidemiologia , Chlamydia trachomatis , Formação de Anticorpos , Olho/metabolismo , Imunoglobulina GRESUMO
After clean drinking water, vaccination is the most impactful global health intervention. However, development of new vaccines against difficult-to-target diseases is hampered by the lack of diverse adjuvants for human use. Of particular interest, none of the currently available adjuvants induce Th17 cells. Here, we develop and test an improved liposomal adjuvant, termed CAF®10b, that incorporates a TLR-9 agonist. In a head-to-head study in non-human primates (NHPs), immunization with antigen adjuvanted with CAF®10b induced significantly increased antibody and cellular immune responses compared to previous CAF® adjuvants, already in clinical trials. This was not seen in the mouse model, demonstrating that adjuvant effects can be highly species specific. Importantly, intramuscular immunization of NHPs with CAF®10b induced robust Th17 responses that were observed in circulation half a year after vaccination. Furthermore, subsequent instillation of unadjuvanted antigen into the skin and lungs of these memory animals led to significant recall responses including transient local lung inflammation observed by Positron Emission Tomography-Computed Tomography (PET-CT), elevated antibody titers, and expanded systemic and local Th1 and Th17 responses, including >20% antigen-specific T cells in the bronchoalveolar lavage. Overall, CAF®10b demonstrated an adjuvant able to drive true memory antibody, Th1 and Th17 vaccine-responses across rodent and primate species, supporting its translational potential.
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ESAT-6 system 1 (ESX-1)-mediated secretion in Mycobacterium tuberculosis is dependent on proteins encoded by the cotranscribed espA-espC-espD gene cluster. While the roles of EspA and EspC with respect to the ESX-1 secretion system have been actively investigated, the function of EspD remains unknown. We show that EspD is secreted by M. tuberculosis, but unlike EspA and EsxA, its export does not exclusively require the ESX-1 system. Evidence for stabilization of cellular levels of EspA and EspC by EspD is presented, and depletion of EspD results in loss of EsxA secretion. Site-directed mutagenesis of EspD reveals that its role in the maintenance of cellular levels of EspA in M. tuberculosis is distinct from its facilitation of EsxA secretion. The same mutagenesis experiments have also shown that secretion of EspD is not required for the secretion of EsxA. Our findings highlight a critical and complex role for EspD in modulating the ESX-1 secretion system in M. tuberculosis.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Transdução de SinaisRESUMO
Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.
Assuntos
Antígenos de Bactérias/química , Proteínas de Bactérias/química , Mycobacterium tuberculosis/química , Proteoma/química , Tuberculina/química , Animais , Antígenos de Bactérias/análise , Antígenos de Bactérias/classificação , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/classificação , Proteínas de Bactérias/imunologia , Cobaias , Mycobacterium tuberculosis/imunologia , Proteoma/análise , Tuberculina/análise , Tuberculina/imunologia , Tuberculose/imunologia , Tuberculose/microbiologiaRESUMO
BACKGROUND: The pathogenesis of Mycobacterium tuberculosis largely depends on the secretion of the 6-kD early secreted antigenic target ESAT-6 (EsxA) and the 10-kD culture filtrate protein CFP-10 (EsxB) via the ESX-1/typeVII secretion system. Although gene products from the core RD1 region have been shown to be deeply implicated in this process, less is known about proteins encoded further upstream in the 5' region of the ESX-1 cluster, such as the ESX-1 secretion-associated proteins (Esps) EspF or EspG(1). METHODS: To elucidate the role of EspF/G(1), whose orthologs in Mycobacterium marinum and Mycobacterium smegmatis are reportedly involved in EsxA/B secretion, we constructed 3 M. tuberculosis knockout strains deleted for espF, espG(1) or the segment corresponding to the combined RD1(bcg)-RD1(mic) region of bacille Calmette-Guérin (BCG) and Mycobacterium microti, which also contains espF and espG(1). RESULTS: Analysis of these strains revealed that, unlike observations with the model organisms M. smegmatis or M. marinum, disruption of espF and espG(1) in M. tuberculosis did not impact the secretion and T cell recognition of EsxA/B but still caused severe attenuation. CONCLUSIONS: The separation of the 2 ESX-1-connected phenotypes (ie, EsxA/B secretion and virulence) indicates that EsxA/B secretion is not the only readout for a functional ESX-1 system and suggests that other processes involving EspF/G(1) also play important roles in ESX-1-mediated pathogenicity.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Deleção de Genes , Técnicas de Inativação de Genes , Teste de Complementação Genética , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica , Mycobacterium tuberculosis/genética , Processamento de Proteína Pós-Traducional , VirulênciaRESUMO
It is of international priority to develop a vaccine against sexually transmitted Chlamydia trachomatis infections to combat the continued global spread of the infection. The optimal immunization strategy still remains to be fully elucidated. The aim of this study was to evaluate immunization strategies in a nonhuman primate (NHP) model. Cynomolgus macaques (Macaqua fascicularis) were immunized following different multi-component prime-boost immunization-schedules and subsequently challenged with C. trachomatis SvD in the lower genital tract. The immunization antigens included the recombinant protein antigen CTH522 adjuvanted with CAF01 or aluminium hydroxide, MOMP DNA antigen and MOMP vector antigens (HuAd5 MOMP and MVA MOMP). All antigen constructs were highly immunogenic raising significant systemic C. trachomatis-specific IgG responses. In particularly the CTH522 protein vaccinated groups raised a fast and strong pecificsIgG in serum. The mapping of specific B cell epitopes within the MOMP showed that all vaccinated groups, recognized epitopes near or within the variable domains (VD) of MOMP, with a consistent VD4 response in all animals. Furthermore, serum from all vaccinated groups were able to in vitro neutralize both SvD, SvE and SvF. Antibody responses were reflected on the vaginal and ocular mucosa, which showed detectable levels of IgG. Vaccines also induced C. trachomatis-specific cell mediated responses, as shown by in vitro stimulation and intracellular cytokine staining of peripheral blood mononuclear cells (PBMCs). In general, the protein (CTH522) vaccinated groups established a multifunctional CD4 T cell response, whereas the DNA and Vector vaccinated groups also established a CD8 T cells response. Following vaginal challenge with C. trachomatis SvD, several of the vaccinated groups showed accelerated clearance of the infection, but especially the DNA group, boosted with CAF01 adjuvanted CTH522 to achieve a balanced CD4/CD8 T cell response combined with an IgG response, showed accelerated clearance of the infection.