RESUMEN
BACKGROUND: This article describes the lessons learned from an international pilot assessment using the first version of the HTA Core Model® and Guidelines for rapid Relative Effectiveness Assessment (REA) of pharmaceuticals based on input from three different perspectives: the assessors, the users (health technology assessment organisations) and the marketing authorisation holder. METHODS: A pilot assessment was performed of pazopanib for the treatment of advanced or metastatic renal cell carcinoma for which 54 individuals from 22 EUnetHTA member organisations from 16 European countries gave their contribution. The work was divided in eight domain teams. Subsequently, results of these domain teams were synthesised in one pilot report. Feedback on the outcomes of the pilot was gathered throughout the project and through structured surveys. RESULTS: The first version of the assessment was produced in six months and consisted of 55 question and answer pairs, 8 domain reports and a synthesis section that combined the results from the different domains. The organisation of the pilot required intense coordination. Main points of criticism on the assessment were the lengthiness of the document and overlap of information throughout the assessment. CONCLUSIONS: A reduction in the number of authoring organisations and individuals participating is necessary to avoid information overlap and increase efficiency in undertaking the assessment. Involving several organisations (e.g. five) in an in-depth review could still ensure the benefit of broad participation from various countries. The focus of a rapid REA should be on the first four domains of the Model.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Cooperación Internacional , Pirimidinas/farmacología , Sulfonamidas/farmacología , Evaluación de la Tecnología Biomédica/organización & administración , Carcinoma de Células Renales/tratamiento farmacológico , Investigación sobre la Eficacia Comparativa , Europa (Continente) , Humanos , Indazoles , Neoplasias Renales/tratamiento farmacológico , Proyectos Piloto , Evaluación de Programas y Proyectos de Salud , Prohibitinas , Encuestas y CuestionariosRESUMEN
In animals, effective immune responses against malignancies and against several infectious pathogens, including malaria, are mediated by T cells. Here we show that a heterologous prime-boost vaccination regime of DNA either intramuscularly or epidermally, followed by intradermal recombinant modified vaccinia virus Ankara (MVA), induces high frequencies of interferon (IFN)-gamma-secreting, antigen-specific T-cell responses in humans to a pre-erythrocytic malaria antigen, thrombospondin-related adhesion protein (TRAP). These responses are five- to tenfold higher than the T-cell responses induced by the DNA vaccine or recombinant MVA vaccine alone, and produce partial protection manifest as delayed parasitemia after sporozoite challenge with a different strain of Plasmodium falciparum. Such heterologous prime-boost immunization approaches may provide a basis for preventative and therapeutic vaccination in humans.
Asunto(s)
Inmunización Secundaria , Vacunas contra la Malaria/inmunología , Linfocitos T/inmunología , Vacunas de ADN/inmunología , Vacunas Sintéticas/inmunología , Virus Vaccinia/inmunología , Animales , Antígenos de Protozoos/inmunología , Humanos , Esquemas de Inmunización , Interferón gamma/inmunología , Interferón gamma/metabolismo , Activación de Linfocitos , Malaria Falciparum/prevención & control , Malaria Falciparum/terapia , Péptidos/inmunología , Péptidos/metabolismo , Plásmidos , Plasmodium falciparum/genética , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Linfocitos T/metabolismo , Virus Vaccinia/genéticaRESUMEN
BACKGROUND: The cellular immune responses that protect against tuberculosis have not been identified. METHODS: We assessed baseline interferon γ (IFNγ) and lymphocyte proliferation assay (LPA) responses to antigen 85 (Ag85), early secretory antigenic target 6 (ESAT6), and Mycobacterium tuberculosis whole cell lysate (WCL) in human immunodeficiency virus (HIV)-infected and bacille CalmetteGuérin (BCG)-immunized adults with CD4 cell counts of >or= 200 cells/µL who received placebo in the DarDar tuberculosis vaccine trial in Tanzania. Subjects were followed prospectively to diagnose definite or probable tuberculosis. RESULTS: Tuberculosis was diagnosed in 92 of 979 subjects during a mean followup of 3.2 years. The relative risk of tuberculosis among subjects with positive IFNγ responses to Ag85 was 0.51 (95% confidence interval [CI], 0.26-0.99; P = .049), to ESAT6 was 0.44 (95% CI, 0.23-0.85; P = .004), and to WCL was 0.67 (95% CI, 0.49-0.88; P = .002). The relative risk of tuberculosis was not significantly associated with baseline LPA responses. In a multivariate Cox regression model, subjects with IFNγ responses to ESAT6 and WCL had a lower hazard of developing tuberculosis, with a hazard ratio for ESAT6 of 0.35 (95% CI, 0.160.77; P = .009) and a hazard ratio for WCL of 0.30 (95% CI, 0.16-0.56; P < .001). CONCLUSIONS: Baseline IFNγ responses to ESAT-6 and WCL were associated with protection from subsequent tuberculosis among HIV-infected subjects with childhood BCG immunization in a region of high tuberculosis prevalence. Trial registration. ClinicalTrials.gov identifier: NCT00052195.
Asunto(s)
Antígenos Bacterianos/inmunología , Infecciones por VIH/complicaciones , Infecciones por VIH/inmunología , Interferón gamma/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/complicaciones , Tuberculosis/inmunología , Adulto , Vacuna BCG/inmunología , Recuento de Linfocito CD4 , Femenino , Humanos , Interferón gamma/sangre , Masculino , Factores de Riesgo , TanzaníaRESUMEN
In this study experimental mouse model for Chlamydia pneumoniae infection was used to elucidate the nature of immune response developing during primary and secondary infection. First we examined the mononuclear cells from different lymphoid organs in BALB/c mice during C. pneumoniae infection and detected a strong lymphocyte influx into mediastinal lymph nodes (MLN). To further characterize the C. pneumoniae induced immune response the gene expression profiles of MLN derived lymphocytes was studied. To identify genes characteristic for reinfection we compared gene expression profiles during reinfection and primary infection and found 148 genes to be differentially regulated in CD19+ cells, 7 in CD4+ cells and 12 in CD8+ cells. A panel of these genes was selected to be confirmed by real-time RT-PCR. Genes related to interferon signaling like Ifit1, Ifit3, Gbp2, Irf7 and Usp18 were found to be upregulated when reinfection was compared to primary infection. In our study we were able to identify 8 genes that were differentially expressed between reinfection and primary infection in lymphocytes. These novel gene expression signatures provide new insights and clues to the nature of protective immunity established during experimental C. pneumoniae immunity.
Asunto(s)
Infecciones por Chlamydia/genética , Infecciones por Chlamydia/inmunología , Chlamydophila pneumoniae/inmunología , Perfilación de la Expresión Génica , Linfocitos/inmunología , Animales , Células Cultivadas , Infecciones por Chlamydia/microbiología , Chlamydophila pneumoniae/fisiología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Chlamydia pneumoniae is a common human respiratory pathogen, and sera from infected individuals recognize several proteins of C. pneumoniae. We produced C. pneumoniae-specific proteins in a Bacillus subtilis expression system. We then used these recombinant C. pneumoniae proteins and purified C. pneumoniae elementary bodies as antigens in enzyme immunoassays to assess the kinetics and protein specificity of the systemic and mucosal antibody responses induced by C. pneumoniae intranasal infection in BALB/c mice. The systemic antibodies in mice recognized strong 'key' immunogens of Chlamydia, Omp2 and Hsp60, but weakly targeted the MOMP protein, the major immunogen in chlamydial species other than C. pneumoniae. The IgA antibodies in bronchial secretions specifically recognized the putative surface protein of C. pneumoniae, Omp4. Our preliminary observations point to the necessity of further characterization of the mucosal antibody response during C. pneumoniae infection.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Infecciones por Chlamydia/inmunología , Chlamydophila pneumoniae/inmunología , Mucosa Intestinal/inmunología , Animales , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/inmunología , Chaperonina 60/biosíntesis , Chaperonina 60/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunidad Mucosa , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Organismos Libres de Patógenos EspecíficosRESUMEN
BACKGROUND: We sought to determine the prevalence of active tuberculosis among ambulatory HIV-infected persons in Tanzania with CD4 cell counts of > or =200 cells/mm3 and a bacille Calmette-Guerin vaccination scar. METHODS: Subjects who volunteered for a tuberculosis booster vaccine trial were screened for active tuberculosis by obtainment of a history, physical examination, chest radiography, sputum culture and acid fast bacillus (AFB) stain, and blood culture. All subjects underwent a tuberculin skin test (TST) and lymphocyte proliferation assays (LPAs) for detection of responses to mycobacterial antigens. RESULTS: Active tuberculosis was identified at baseline in 14 (15%) of the first 93 subjects who were enrolled: 10 (71%) had clinical tuberculosis (symptoms or chest radiograph findings), and 4 (29%) had subclinical tuberculosis (positive sputum AFB stain or culture results but no symptoms or chest radiograph findings). An additional 6 subjects with subclinical tuberculosis were identified subsequently. The 10 subjects with subclinical tuberculosis included 3 with positive sputum AFB stains results and 7 who were only identified by a positive sputum culture result. Compared with subjects who did not have tuberculosis, the 10 subjects with subclinical tuberculosis were more likely to have peripheral lymphadenopathy, positive TST results, and elevated LPA responses to early secreted antigenic target-6 (ESAT). Eight of 10 patients had received isoniazid because of a positive TST result before active tuberculosis was recognized. CONCLUSIONS: Clinical and subclinical tuberculosis are common among ambulatory HIV-infected persons, and some cases can only be identified by sputum culture. World Health Organization guidelines for screening for latent tuberculosis before treatment do not recommend sputum culture and, therefore, may fail to identify a substantial number of HIV-infected persons with subclinical, active tuberculosis.
Asunto(s)
Infecciones por VIH/complicaciones , Tuberculosis/complicaciones , Tuberculosis/epidemiología , Adulto , Recuento de Linfocito CD4 , Femenino , Infecciones por VIH/microbiología , Humanos , Masculino , Tanzanía/epidemiología , Tuberculosis/diagnósticoRESUMEN
OBJECTIVE: Prior to the widespread use of Mycobacterium bovis, Bacille Calmette-Guerin (BCG), inactivated whole cell mycobacterial vaccines had been shown effective in the prevention of tuberculosis. The present study was conducted to determine the safety and immunogenicity of an inactivated whole cell mycobacterial vaccine in persons with HIV infection.DESIGN Randomized, controlled trial. METHODS: A total of 39 HIV-positive patients with prior BCG immunization and CD4 cell counts >/= 200 x 10(6) cells/l were randomized to five doses of inactivated Mycobacterium vaccae (MV) vaccine or control vaccine (CV). Lymphocyte proliferation (LPA) and interferon gamma (IFN-gamma) responses to mycobacterial antigens were assayed at baseline, after three and five doses of vaccine and > 1 year later. Parallel studies were conducted in 10 HIV-negative subjects with prior BCG immunization. RESULTS: Among HIV-positive patients, 19 MV recipients had higher LPA and IFN-gamma responses to MV sonicate than 20 CV recipients after three and five doses of vaccine and > 1 year later. LPA responses to Mycobacterium tuberculosis whole cell lysate increased over time in both groups consistent with prior BCG immunization and current antiretroviral therapy; after three doses, responses were boosted to higher levels in MV subjects than CV subjects. LPA responses to WCL were also boosted in HIV-negative MV recipients. Immunization was safe and had no adverse effects on HIV viral load or CD4 cell count. CONCLUSIONS: In BCG-primed, HIV-positive and HIV-negative subjects, MV induces durable cellular immune responses to a new mycobacterial antigen and boosts pre-existing responses to WCL. MV is a candidate for clinical trials for the prevention of HIV-associated tuberculosis.
Asunto(s)
Infecciones por VIH/complicaciones , Infecciones Oportunistas/prevención & control , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Adulto , Vacuna BCG , Recuento de Linfocito CD4 , Relación Dosis-Respuesta Inmunológica , Femenino , Infecciones por VIH/inmunología , Humanos , Inmunidad Celular , Interferón gamma/biosíntesis , Activación de Linfocitos/inmunología , Masculino , Mycobacterium/inmunología , Infecciones Oportunistas/complicaciones , Tuberculosis/complicaciones , Vacunas de Productos Inactivados/inmunologíaRESUMEN
Mycobacterium bovis, bacille Calmette-Guérin (BCG) is administered widely to newborns throughout the world and has been shown to be effective in preventing childhood tuberculosis but not reactivation pulmonary disease or human immunodeficiency virus-associated tuberculosis. Development of a more effective, better standardized, affordable vaccine with durable activity and fewer side effects is a major priority. Contemporary molecular techniques have identified promising immunodominant antigens and novel immunization strategies. Vaccine development has also been informed by an improved understanding of the role of nontuberculous mycobacteria in the efficacy of BCG and in the prevention of tuberculosis. Vaccines under investigation include attenuated or enhanced whole-cell live, whole-cell inactivated, subunit, DNA, and prime-boost vaccines. Several candidate vaccines have demonstrated activity in animal models that is equal to or superior to that of BCG, and human trials are under way. Because there is no identified surrogate marker for protection, identification of an improved vaccine will require long-term efficacy trials in humans.
Asunto(s)
Vacuna BCG/uso terapéutico , Tuberculosis/prevención & control , Vacuna BCG/efectos adversos , Ensayos Clínicos como Asunto , Humanos , Mycobacterium bovis/inmunología , Subunidades de Proteína , Tuberculosis/inmunología , Vacunas de ADN , Vacunas de Productos InactivadosRESUMEN
BACKGROUND: Surrogate immunologic markers for natural and vaccine-mediated protection against tuberculosis (TB) have not been identified. METHODS: HIV-infected adults with childhood BCG immunization entering the placebo arm of the DarDar TB vaccine trial in Dar es Salaam, Tanzania, were assessed for interferon gamma (IFN-γ) responses to three mycobacterial antigen preparations--secreted Mycobacterium tuberculosis antigens 85 (Ag85), early secretory antigenic target 6 (ESAT-6) and polyantigenic whole cell lysate (WCL). We investigated the association between the number of detectable IFN-γ responses at baseline and the subsequent risk of HIV-associated TB. RESULTS: During a median follow-up of 3.3 years, 92 (9.4%) of 979 placebo recipients developed TB. The incidence of TB was 14% in subjects with no detectable baseline IFN-γ responses vs. 8% in subjects with response to polyantigenic WCL (Pâ=â0.028). Concomitant responses to secreted antigens were associated with further reduction in the incidence of HIV-associated TB. Overall the percentage of subjects with 0, 1, 2 and 3 baseline IFN-γ responses to mycobacterial preparations who developed HIV-associated TB was 14%, 8%, 7% and 4%, respectively (Pâ=â0.004). In a multivariate Cox regression model, the hazard of developing HIV-associated TB was 46% lower with each increment in the number of detectable baseline IFN-γ responses (P<0.001). CONCLUSIONS: Among HIV-infected adults who received BCG in childhood and live in a TB-endemic country, polyantigenic IFN-γ responses are associated with decreased risk of subsequent HIV-associated TB. TRIAL REGISTRATION: ClinicalTrials.gov NCT0052195.
Asunto(s)
Infecciones por VIH/complicaciones , Inmunización , Interferón gamma/inmunología , Mycobacterium bovis/inmunología , Tuberculosis Pulmonar/complicaciones , Tuberculosis Pulmonar/prevención & control , Adulto , Antígenos Bacterianos/inmunología , Niño , Bases de Datos Factuales , Femenino , Humanos , Masculino , Análisis Multivariante , Medición de RiesgoRESUMEN
Preventive immunization with whole inactivated Mycobacterium vaccae (MV) confers protection against HIV-associated tuberculosis (TB) in BCG-immunized adults with CD4 counts ≥200 cells/µl. We evaluated the immunogenicity of MV in the 2013 subjects of the phase III DarDarTrial using an interferon gamma (IFN-γ) enzyme linked immunosorbent assay (ELISA), tritiated thymidine lymphocyte proliferation assay (LPA) and an ELISA for antibodies to the TB glycolipid lipoarabinomannan (LAM). MV immunization boosts IFN-γ and LPA responses to MV sonicate, and antibody responses to LAM. Post-immunization immune responses to MV correlated with baseline clinical factors, but the responses did not predict protection from HIV-associated TB.
Asunto(s)
Infecciones por VIH/complicaciones , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/prevención & control , Adulto , Anticuerpos Antibacterianos/sangre , Formación de Anticuerpos , Antígenos Bacterianos/inmunología , Antituberculosos/administración & dosificación , Vacuna BCG/inmunología , Método Doble Ciego , Ensayo de Inmunoadsorción Enzimática , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/microbiología , Humanos , Interferón gamma/inmunología , Isoniazida/administración & dosificación , Lipopolisacáridos/inmunología , Masculino , Tanzanía , Prueba de Tuberculina , Tuberculosis/complicaciones , Tuberculosis/inmunología , Carga ViralRESUMEN
In interleukin-10 knock out (IL-10 KO) mice, accelerated clearance of pulmonary Chlamydia pneumoniae infection was observed. On the other hand, the histopathological changes in lung tissue were more pronounced in IL-10 KO mice at all time points after infection and repeated infection than in the wild type mice. Both ex vivo induced antigen-specific proliferation as well as production of proinflammatory cytokines by splenocytes were higher in IL-10 KO mice than in WT mice. Also, intrapulmonary proinflammatory cytokine levels were higher in IL-10 KO mice than in the WT mice. The lack of anti-inflammatory action of IL-10 is likely to contribute to the enhanced clearance but severe inflammation in this experimental model.
Asunto(s)
Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/patología , Chlamydophila pneumoniae/inmunología , Interleucina-10/inmunología , Neumonía Bacteriana/inmunología , Animales , Proliferación Celular , Infecciones por Chlamydia/microbiología , Chlamydophila pneumoniae/patogenicidad , Chlamydophila pneumoniae/fisiología , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Interleucina-10/genética , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neumonía Bacteriana/microbiología , Neumonía Bacteriana/patologíaRESUMEN
As a step toward understanding the role played by host gene expression in the development and pathogenesis of persistent Chlamydia pneumoniae infection, modulation of the host-cell transcriptional response during interferon (IFN)- gamma -induced persistent C. pneumoniae infection of HL cells was examined by a cDNA array and then selectively by a real-time quantitative reverse transcription-polymerase chain reaction. We identified 9 host cell genes whose transcription was consistently altered during IFN- gamma -induced persistent C. pneumoniae infection. The strongest up-regulation of persistent infection, compared with controls (active infection and IFN- gamma ) was identified for insulin-like growth factor-binding protein 6, IFN-stimulated protein 15 kDa, cyclin D1, and interleukin-7 receptor genes. These results suggest that, during persistent infection, C. pneumoniae reprograms the host transcriptional machinery that regulates a variety of cellular processes, including adhesion, regulation of the cell cycle, growth, and inflammatory response, all of which might play important roles in the pathogenesis of persistent C. pneumoniae infection.
Asunto(s)
Infecciones por Chlamydia/inmunología , Chlamydophila pneumoniae/patogenicidad , Células Epiteliales/microbiología , Interferón gamma/farmacología , Proteínas/metabolismo , Regulación hacia Arriba , Células Cultivadas , Infecciones por Chlamydia/microbiología , Chlamydophila pneumoniae/crecimiento & desarrollo , Regulación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Most new tuberculosis vaccines will be administered as a booster to subjects primed with bacille Calmette-Guérin (BCG) during childhood. METHODS: We investigated in vivo and in vitro immune responses to mycobacteria in human immunodeficiency virus (HIV)-positive subjects in Tanzania primed with BCG during childhood and entering a tuberculosis booster vaccine trial. Tests included intradermal skin testing for Mycobacterium tuberculosis purified protein derivative (PPD) and Mycobacterium avium sensitin (MAS); lymphocyte proliferation assays and interferon (IFN)-gamma levels after stimulation with Mycobacterium vaccae sonicate (MVS), M. tuberculosis early secreted antigen (ESAT)-6, M. tuberculosis antigen 85 (Ag85), or M. tuberculosis whole-cell lysate (WCL); and determination of serum antibody to lipoarabinomannin (LAM). RESULTS: A total of 888 subjects with CD4 cell counts > or = 200 cells/mm3 were enrolled. PPD and MAS test results were positive in 34% and 30% of the subjects, respectively. Proliferative responses were detected as follows: MVS, 6%; Ag85, 24%; ESAT-6, 21%; and WCL, 59%. IFN-gamma responses were 2%, 6%, 12%, and 38%, respectively. LAM antibody was detected in 28% of the subjects. Subjects were more likely to have detectable proliferative and IFN-gamma responses if they had positive PPD test results or CD4 cell counts > or = 500 cells/mm3. Overall, 94% of the subjects had evidence of primed mycobacterial immune responses. CONCLUSION: Of HIV-positive BCG-immunized adults with CD4 cell counts > or = 200 cells/mm3 in Tanzania, 94% are primed for booster mycobacterial immunization.
Asunto(s)
Vacuna BCG/administración & dosificación , Vacuna BCG/inmunología , Infecciones por VIH/inmunología , Inmunidad Celular/inmunología , Inmunización Secundaria , Tuberculosis/prevención & control , Adulto , Recuento de Linfocito CD4 , Infecciones por VIH/complicaciones , Humanos , Activación de Linfocitos/inmunología , Mycobacterium/inmunología , Prueba de Tuberculina , Tuberculosis/complicacionesRESUMEN
T-cell-mediated responses against the liver-stage of Plasmodium falciparum are critical for protection in the human irradiated sporozoite model and several animal models. Heterologous prime-boost approaches, employing plasmid DNA and viral vector delivery of malarial DNA sequences, have proved particularly promising for maximising T-cell-mediated protection in animal models. The T-cell responses induced by this prime-boost regime, in animals and humans, are substantially greater than the sum of the responses induced by DNA or MVA vaccines used alone, leading to the term introduced here of "synergistic" prime-boost immunisation. The insert in our first generation clinical constructs is known as multiple epitope-thrombospondin-related adhesion protein (ME-TRAP). We have performed an extensive series of phase I/II trials evaluating various prime-boost combination regimens for delivery of ME-TRAP in over 500 malaria-naïve and malaria-exposed individuals. The three delivery vectors are DNA, modified vaccinia virus Ankara (MVA) and, more recently, fowlpox strain 9 (FP9). Administration was intra-epidermal and intramuscular for DNA and intradermal for MVA and FP9. Doses of DNA ranged from 4 microg to 2mg. Doses of MVA were up to 1.5 x 10(8) plaque forming units (pfu) and of FP9, up to 1.0 x 10(8)pfu. Further trials employing bacille Calmette-Guérin (BCG) as the priming agent and MVA expressing antigen 85A of Mycobacterium tuberculosis as the boosting agent has extended the scope of synergistic prime-boost vaccination. In this review we summarise the safety, immunogenicity and efficacy results from these malaria and tuberculosis vaccine clinical trials.
Asunto(s)
Malaria/prevención & control , Tuberculosis/prevención & control , Vacunas de ADN/administración & dosificación , Virus Vaccinia/inmunología , Adulto , Animales , Vacuna BCG/administración & dosificación , Humanos , Plasmodium falciparum/inmunologíaRESUMEN
Heterologous prime-boost immunisation with RTS,S/AS02A and the poxvirus MVA-CS was evaluated in 18 healthy malaria-naïve subjects in Oxford. Both priming with RTS,S and boosting MVA-CS, and the reverse, were found to be safe and well tolerated. T cell responses as measured by IFN-gamma ex vivo ELISPOT were induced, but the responses were low to moderate in both groups, with heterologous boosting yielding only small increments in T cell immunogenicity and no increased antibody response. Protection against 3D7 Plasmodium falciparum sporozoite challenge 4 weeks after the final vaccination was equal for both regimens at 33% (95% C.I. 4.3-77.7%), with one subject remaining fully protected on rechallenge at 5 months.
Asunto(s)
Inmunización Secundaria , Vacunas contra la Malaria/inmunología , Malaria/prevención & control , Virus Vaccinia/inmunología , Adolescente , Adulto , Anticuerpos Antiprotozoarios/sangre , Femenino , Humanos , Interferón gamma/biosíntesis , Malaria/inmunología , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/efectos adversos , Masculino , Persona de Mediana Edad , Linfocitos T/inmunologíaRESUMEN
Heterologous prime-boost vaccination has been shown to be an efficient way of inducing T cell responses in animals and in humans. We have used three vaccine vectors, naked DNA, modified vaccinia virus Ankara (MVA), and attenuated fowlpox strain, FP9, for prime-boost vaccination approaches against Plasmodium falciparum malaria in humans. In this study, we characterize, using two types of ELISPOT assays and FACS analysis, cell-mediated immune responses induced by different prime-boost combinations where all vectors encode a multiepitope string fused to the pre-erythrocytic Ag thrombospondin-related adhesion protein. We show that these different vectors need to be used in a specific order for an optimal ex vivo IFN-gamma response. From the different combinations, DNA priming followed by MVA boosting and FP9 priming followed by MVA boosting were most immunogenic and in both cases the IFN-gamma response was of broad specificity and cross-reactive against two P. falciparum strains (3D7 and T9/96). Immunization with all three vectors showed no improvement over optimal two vector regimes. Strong ex vivo IFN-gamma responses peaked 1 wk after the booster dose, but cultured ELISPOT assays revealed longer-lasting T cell memory responses for at least 6 mo. In the DNA-primed vaccinees the IFN-gamma response was mainly due to CD4(+) T cells, whereas in the FP9-primed vaccinees it was mainly due to CD4-dependent CD8(+) T cells. This difference may be of importance for the protective efficacy of these vaccination approaches against various diseases.
Asunto(s)
Vectores Genéticos/inmunología , Interferón gamma/biosíntesis , Malaria Falciparum/prevención & control , Vacunas de ADN/inmunología , Vacunas Virales/inmunología , Animales , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Citometría de Flujo , Virus de la Viruela de las Aves de Corral/inmunología , Humanos , Interferón gamma/inmunología , Subgrupos Linfocitarios/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Linfocitos T/inmunología , Vacunas Atenuadas/inmunología , Virus Vaccinia/inmunologíaRESUMEN
Immunological memory is a required component of protective antimalarial responses raised by T cell-inducing vaccines. The magnitude of ex vivo IFN-gamma T cell responses is widely used to identify immunogenic vaccines although this response usually wanes and may disappear within weeks. However, protection in the field is likely to depend on durable central memory T cells that are not detected by this assay. To identify longer-lived memory T cells, PBMC from malaria-naive vaccinated volunteers who had received prime boost vaccinations with a combination of DNA and/or viral vectors encoding the multiepitope string-thrombospondin-related adhesion protein Ag were cultured in vitro with Ag for 10 days before the ELISPOT assay. Ex vivo T cell responses peaked at 7 days after the final immunization and declined substantially over 6 mo, but responses identified after T cell culture increased over the 6-mo period after the final immunization. Moreover, individual cultured ELISPOT responses at the day of challenge time point correlated significantly with degree of protection against malaria sporozoite challenge, whereas ex vivo responses did not, despite a correlation between the peak ex vivo response and magnitude of memory responses 6 mo later. This cultured assay identifies long-lasting protective T cell responses and therefore offers an attractive option for assessments of vaccine immunogenicity.
Asunto(s)
Memoria Inmunológica , Vacunas contra la Malaria/inmunología , Malaria/prevención & control , Linfocitos T/inmunología , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunización Secundaria , Interferón gamma/biosíntesis , Activación de LinfocitosRESUMEN
ICC-1132, a recombinant virus-like particle comprising of a modified hepatitis B core protein with a B cell (NANP) and two T cell epitopes of Plasmodium falciparum circumsporozoite protein (CSP), was administered i.m. as a single 50 microg dose in Seppic ISA 720 to 11 volunteers. Local reactogenicity and systemic side effects were acceptable with the predominant finding being mild pain at the injection site. This regimen induced anti-NANP antibodies in 10/11 and modest T cell responses. There was no evidence of protection from experimental challenge with P. falciparum sporozoites. Other formulations and/or multi-dose regimens will be required to enhance the immunogenicity and efficacy of ICC-1132.
Asunto(s)
Eritrocitos/inmunología , Eritrocitos/parasitología , Vacunas contra la Malaria/efectos adversos , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Plasmodium falciparum/inmunología , Adulto , Secuencia de Aminoácidos , Animales , Epítopos de Linfocito T/administración & dosificación , Epítopos de Linfocito T/inmunología , Femenino , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Humanos , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/genética , Malaria Falciparum/parasitología , Malaria Falciparum/prevención & control , Masculino , Esporozoítos/inmunologíaRESUMEN
Malaria is a major global health problem for which an effective vaccine is required urgently. Prime-boost vaccination regimes involving plasmid DNA and recombinant modified vaccinia virus Ankara-encoding liver-stage malaria antigens have been shown to be powerfully immunogenic for T cells and capable of inducing partial protection against experimental malaria challenge in humans, manifested as a delay in time to patent parasitemia. Here, we report that substitution of plasmid DNA as the priming vector with a specific attenuated recombinant fowlpox virus, FP9, vaccine in such prime-boost regimes can elicit complete sterile protection that can last for 20 months. Protection at 20 months was associated with persisting memory but not effector T cell responses. The protective efficacy of various immunization regimes correlated with the magnitude of induced immune responses, supporting the strategy of maximizing durable T cell immunogenicity to develop more effective liver-stage vaccines against Plasmodium falciparum malaria.
Asunto(s)
Inmunización Secundaria/métodos , Vacunas contra la Malaria/inmunología , Malaria Falciparum/inmunología , Malaria Falciparum/prevención & control , Plasmodium falciparum/inmunología , Linfocitos T/inmunología , Vacunas de ADN/inmunología , Adulto , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Virus de la Viruela de las Aves de Corral/genética , Virus de la Viruela de las Aves de Corral/inmunología , Humanos , Inmunidad Celular/inmunología , Inmunoglobulina G/inmunología , Memoria Inmunológica/inmunología , Interferón gamma/sangre , Vacunas contra la Malaria/genética , Masculino , Persona de Mediana Edad , Plásmidos/genética , Plásmidos/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Virus Vaccinia/genética , Virus Vaccinia/inmunologíaRESUMEN
Due to intracellular growth requirements, large-scale cultures of chlamydiae and purification of its proteins are difficult and laborious. To overcome these problems we produced chlamydial proteins in a heterologous host, Bacillus subtilis, a gram-positive nonpathogenic bacterium. The genes of Chlamydia pneumoniae major outer membrane protein (MOMP), the cysteine-rich outer membrane protein (Omp2), and the heat shock protein (Hsp60) were amplified by PCR, and the PCR products were cloned into expression vectors containing a promoter, a ribosome binding site, and a truncated signal sequence of the alpha-amylase gene from Bacillus amyloliquefaciens. C. pneumoniae genes were readily expressed in B. subtilis under the control of the alpha-amylase promoter. The recombinant proteins MOMP and Hsp60 were purified from the bacterial lysate with the aid of the carboxy-terminal histidine hexamer tag by affinity chromatography. The Omp2 was separated as an insoluble fraction after 8 M urea treatment. The purified proteins were successfully used as immunogens and as antigens in serological assays and in a lymphoproliferation test. The Omp2 and Hsp60 antigens were readily recognized by the antibodies appearing after pulmonary infection following intranasal inoculation of C. pneumoniae in mice. Also, splenocytes collected from mice immunized with MOMP or Hsp60 proteins proliferated in response to in vitro stimulation with the corresponding proteins.