RESUMEN
We previously described PASD1 as a new cancer testis antigen in multiple myeloma (MM) that is retained post-therapy, suggesting the use of vaccination strategies to induce anti-PASD1 immunity in a setting of minimal residual disease. We have focused on DNA fusion gene vaccines, coupling fragment C domain (DOM) of tetanus toxin with PASD1 sequence, and examined efficacy in Human Leukocyte Antigen (HLA)-A2 (HHD) transgenic mice using a human MM cell line expressing PASD1 protein and chimeric HLA-A2 class I molecules as target. DNA vaccines encoded two HLA-A2-restricted epitopes (p.DOM-PASD1(1), p.DOM-PASD1(2)) and full-length PASD1 (p.DOM-PASD1FL). p.DOM-PASD1(1) proved superior to p.DOM-PASD1(2) in generating T-cell responses in HHD mice, able to lyse the chimeric murine RMA-HHD cells. Boosting by electroporation significantly enhanced p.DOM-PASD1(1). Only p.DOM-PASD1(1) induced cytotoxic T-lymphocytes (CTLs) were able to lyse human MM target cells expressing endogenous antigen. The p.DOM-PASD1FL vaccine predominantly induced strong PASD1(1) over PASD1(2) T-cell immune responses, indicative of immunodominance. Importantly, p.DOM-PASD1FL generated immune-mediating killing of native chimeric MM cells, in the absence of exogenous added peptide, implicating PASD1(1) specific CTLs. These data demonstrate that PASD1-derived epitopes are both efficiently and selectively processed and presented by native human MM cells. Notably, they permit the use of PASD1-encoding DNA vaccine therapy in a clinical setting.
Asunto(s)
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos Nucleares/genética , Antígenos Nucleares/inmunología , Mieloma Múltiple/inmunología , Vacunas de ADN/uso terapéutico , Animales , Antígenos de Neoplasias/farmacología , Antígenos Nucleares/farmacología , Epítopos/inmunología , Antígenos HLA-A/inmunología , Antígeno HLA-A2/inmunología , Humanos , Linfoma/inmunología , Ratones , Ratones Transgénicos , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/inmunologíaAsunto(s)
Formación de Anticuerpos , Linfoma Anaplásico de Células Grandes/inmunología , Neoplasia Residual/diagnóstico , Proteínas Tirosina Quinasas/inmunología , Adolescente , Quinasa de Linfoma Anaplásico , Anticuerpos/sangre , Niño , Preescolar , Humanos , Lactante , Cinética , Linfoma Anaplásico de Células Grandes/diagnóstico , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Neoplasia Residual/inmunología , Proteínas Nucleares/inmunología , Nucleofosmina , Proteínas de Fusión Oncogénica/inmunología , Proteínas Tirosina Quinasas ReceptorasAsunto(s)
Antígenos de Neoplasias/análisis , Antígenos Nucleares/análisis , Linfoma/química , Proteínas Nucleares/análisis , Anticuerpos/uso terapéutico , Antígenos de Neoplasias/inmunología , Antígenos Nucleares/inmunología , Línea Celular Tumoral , Humanos , Linfoma/terapia , Proteínas Nucleares/inmunologíaRESUMEN
A meningococcal genomic expression library was screened for potent CD4+ T-cell antigens, using patients' peripheral blood lymphocytes (PBLs). One of the most promising positive clones was fully characterized. The recombinant meningococcal DNA contained a single, incomplete, open reading frame (ORF), which was fully reconstructed with reference to available genomic sequence data. The gene was designated autA (auto-transporter A) as its peptide sequence shares molecular characteristics of the auto-transporter family of proteins. Only a single copy of this gene was detected in the meningococcal, and none in the gonococcal, genomic sequence databases. The complete autA gene, when cloned into an expression vector, expressed a protein of approximately 68 kDa. Purified rAutA recalled strong secondary T-cell responses in PBLs of patients and some healthy donors, and induced strong primary T-cell responses in healthy donors. The human B-cell immunogenicity and cross-reactivity of AutA, purified under native conditions, was confirmed in dot immunoblot experiments. Immunoblots with rabbit polyclonal antibodies to rAutA demonstrated the conserved nature, antigenicity and cross-reactivity of AutA amongst meningococci of different serogroups and strains representing different hypervirulent lineages. AutA showed homology with another meningococcal and gonococcal ORF (designated AutB). AutB was cloned and expressed and used to raise an autB-specific antiserum. Immunoblot experiments indicated that AutB is not expressed in meningococci and does not cross-react with AutA. Thus, AutA, being a potent CD4+ T-cell and B-cell-stimulating antigen, which is highly conserved, deserves further investigation as a potential vaccine candidate.
Asunto(s)
Antígenos Bacterianos/genética , Linfocitos B/inmunología , Proteínas Bacterianas/inmunología , Linfocitos T CD4-Positivos/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Meningitis Meningocócica/microbiología , Neisseria meningitidis/inmunología , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/clasificación , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Proteínas Portadoras/clasificación , Proteínas Portadoras/aislamiento & purificación , Clonación Molecular , ADN Bacteriano , Femenino , Expresión Génica , Humanos , Memoria Inmunológica/inmunología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Neisseria meningitidis/genética , Conejos , Sepsis/microbiologíaRESUMEN
In the 1997-98 academic year, we conducted a longitudinal study of meningococcal carriage and acquisition among first-year students at Nottingham University, Nottingham, United Kingdom. We examined the dynamics of long-term meningococcal carriage with detailed characterization of the isolates. Pharyngeal swabs were obtained from 2,453 first-year students at the start of the academic year (October), later on during the autumn term, and again in March. Swabs were immediately cultured on selective media, and meningococci were identified and serologically characterized. Nongroupable strains were genetically grouped using a PCR-based assay. Pulsed-field gel electrophoresis was used to determine the link between sequential isolates. Of the carriers initially identified in October, 44.1% (98 of 222) were still positive later on in the autumn (November or December); 57.1% of these remained persistent carriers at 6 months. Of the index carriers who lost carriage during the autumn, 16% were recolonized at 6 months. Of 344 index noncarriers followed up, 22.1% acquired carriage during the autumn term and another 13.7% acquired carriage by March. Overall, 43.9% (397 of 904) of the isolates were noncapsulated (serologically nongroupable); by PCR-based genogrouping, a quarter of these belonged to the capsular groups B and C. The ratio of capsulated to noncapsulated forms for group B and C strains was 2.9 and 0.95, respectively. Sequential isolates of persistent carriers revealed that individuals may carry the same or entirely different organisms at different times. We identified three strains that clearly switched their capsular expression on and off at different times in vivo. One student developed invasive meningococcal disease after carrying the same organism for over 7 weeks. The study revealed a high rate of turnover of meningococcal carriage among students. Noncapsulated organisms are capable of switching their capsular expression on and off (both ways) in the nasopharynx, and group C strains are more likely to be noncapsulated than group B strains. Carriage of a particular meningococcal strain does not necessarily protect against colonization or invasion by a homologous or heterologous strain.
Asunto(s)
Portador Sano , Infecciones Meningocócicas/epidemiología , Vacunación , Susceptibilidad a Enfermedades , Estudios de Seguimiento , Humanos , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/prevención & control , Nasofaringe/microbiología , Neisseria meningitidis/clasificación , Neisseria meningitidis/aislamiento & purificación , Estudiantes , Reino Unido/epidemiología , UniversidadesRESUMEN
OBJECTIVE: To determine the rates of, and risk factors for, meningococcal carriage and acquisition among university students. DESIGN: Repeated cross sectional study. PARTICIPANTS: 2,507 students in their first year at university. MAIN OUTCOME MEASURES: Prevalence of carriage of meningococci and risk factors for carriage and acquisition of meningococci. RESULTS: Carriage rates for meningoccoci increased rapidly in the first week of term from 6.9% on day 1, to 11.2% on day 2, to 19.0% on day 3, and to 23.1% on day 4. The average carriage rate during the first week of term in October among students living in catered halls was 13.9%. By November this had risen to 31.0% and in December it had reached 34. 2%. Independent associations for acquisition of meningococci in the autumn term were frequency of visits to a hall bar (5-7 visits: odds ratio 2.7, 95% confidence interval 1.5 to 4.8), active smoking (1.6, 1.0 to 2.6), being male (1.6, 1.2 to 2.2), visits to night clubs (1. 3, 1.0 to 1.6), and intimate kissing (1.4, 1.0 to 1.8). Lower rates of acquisition were found in female only halls (0.5, 0.3 to 0.9). The most commonly acquired meningococcal strain was C2a P1.5 (P1.2), which has been implicated in clusters of invasive meningococcal disease at other UK universities. CONCLUSIONS: Carriage rates of meningococci among university students increase rapidly in the first week of term, with further increases during the term. The rapid rate of acquisition may explain the increased risk of invasive meningococcal disease and the timing of cases and outbreaks in university students.
Asunto(s)
Portador Sano/microbiología , Infecciones Meningocócicas/microbiología , Estudiantes , Actividades Cotidianas , Adulto , Portador Sano/diagnóstico , Estudios Transversales , Femenino , Humanos , Estilo de Vida , Modelos Logísticos , Masculino , Infecciones Meningocócicas/diagnóstico , Infecciones Meningocócicas/transmisión , Faringe/microbiología , Factores de Riesgo , Factores de TiempoRESUMEN
In search for novel T-cell immunogens involved in protection against invasive meningococcal disease, we screened fractionated proteins of Neisseria meningitidis (strain SD, B:15:P1.16) by using peripheral blood mononuclear cells (PBMCs) and specific T-cell lines obtained from normal individuals and patients convalescing from N. meningitidis infection. Proteins of iron-depleted meningococci produced higher PBMC proliferation indices than proteins of iron-replete organisms, indicating that iron-regulated proteins are T-cell immunogens. Insoluble proteins of the iron-depleted cells, which produced better T-cell stimulation than soluble ones, were fractionated by using sodium dodecyl sulfate-polyacrylamide gels and recovered as five fractions (F1 to F5) corresponding to decreasing molecular weight ranges. The proteins were purified (by elution and precipitation) or electroblotted onto nitrocellulose membranes (dissolved and precipitated) before use in further T-cell proliferation assays. One of the fractions (F1), containing high-molecular-mass proteins (>130 kDa), consistently showed the strongest T-cell proliferation responses in all of the T-cell lines examined. F1 proteins were subdivided into four smaller fractions (F1A to F1D) which were reexamined in T-cell proliferation assays, and F1C induced the strongest responses in patients' T-cell lines. Rabbit polyclonal antibodies to F1C components were used to screen a genomic expression library of N. meningitidis. Two major clones (C1 and C24) of recombinant meningococcal DNA were identified and fully sequenced. Sequence analysis showed that C24 (1,874 bp) consisted of a single open reading frame (ORF), which was included in clone C1 (2, 778 bp). The strong CD4(+) T-cell-stimulating effect of the polypeptide product of this ORF (named TspA) was confirmed, using a patient T-cell line. Immunogenicity for B cells was confirmed by showing that convalescent patients' serum antibodies recognized TspA on Western blots. Additional genetic sequence downstream of C24 was obtained from the meningococcal genomic sequence database (Sanger Centre), enabling the whole gene of 2,761 bp to be reconstructed. The DNA and deduced amino acid sequence data for tspA failed to show significant homology to any known gene, except for a corresponding (uncharacterized) gene in Neisseria gonorrhoeae genome sequences, suggesting that tspA is unique to the genus Neisseria. The DNA and deduced amino acid sequence of the second ORF of clone C1 showed significant homology to gloA, encoding glyoxalase I enzyme, of Salmonella typhimurium and Escherichia coli. Thus, we have identified a novel neisserial protein (TspA) which proved to be a strong CD4(+) T-cell- and B-cell-stimulating immunogen with potential as a possible vaccine candidate.