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1.
BMC Infect Dis ; 18(1): 613, 2018 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-30509199

RESUMO

BACKGROUND: Community-acquired pneumonia is a leading infectious cause of hospitalization. A few vaccines exist to prevent pneumococcal disease in adults, including a pneumococcal polysaccharide unconjugated vaccine and a protein conjugated polysaccharide vaccine. Previous studies on the human immune response to the unconjugated vaccine showed that the vaccine boosted the existing memory B cells. In the present study, we investigated the human B cell immune response following pneumococcal polysaccharide conjugate vaccination. METHODS: Plasmablast B cells from a pneumococcal polysaccharide conjugate vaccinee were isolated and cloned for analysis. In response to primary vaccination, identical sequences from the plasmablast-derived antibodies were identified from multiple B cells, demonstrating evidence of clonal expansion. We evaluated the binding specificity of these human monoclonal antibodies in immunoassays, and tested there in vitro function in a multiplexed opsonophagocytic assay (MOPA). To characterize the plasmablast B cell response to the pneumococcal conjugated vaccine, the germline usage and the variable region somatic hypermutations on these antibodies were analyzed. Furthermore, a serotype 4 polysaccharide-specific antibody was tested in an animal challenge study to explore the in vivo functional activity. RESULTS: The data suggests that the pneumococcal polysaccharide conjugate vaccine boosted memory B cell responses, likely derived from previous pneumococcal exposure. The majority of the plasmablast-derived antibodies contained higher numbers of variable region somatic hypermutations and evidence for selection, as demonstrated by replacement to silent ratio's (R/S) greater than 2.9 in the complementarity-determining regions (CDRs). In addition, we found that VH3/JH4 was the predominant germline sequence used in these polysaccharide-specific B cells. All of the tested antibodies demonstrated narrow polysaccharide specificity in ELISA binding, and demonstrated functional opsonophagocytic killing (OPK) activity in the MOPA assay. The in-vivo animal challenge study showed that the tested serotype 4 polysaccharide-specific antibody demonstrated a potent protective effect when administered prior to bacterial challenge. CONCLUSIONS: The findings on the pneumococcal polysaccharide conjugate vaccine responses from a vaccinated subject reported in this study are similar to previously published data on the pneumococcal polysaccharide unconjugated vaccine responses. In both vaccine regimens, the pre-existing human memory B cells were expanded after vaccination with preferential use of the germline VH3/JH4 genes.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Linfócitos B/imunologia , Memória Imunológica , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/uso terapêutico , Hipermutação Somática de Imunoglobulina , Adulto , Animais , Anticorpos Antibacterianos/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Feminino , Rearranjo Gênico do Linfócito B/genética , Rearranjo Gênico do Linfócito B/imunologia , Humanos , Memória Imunológica/genética , Memória Imunológica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções Pneumocócicas/genética , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas/imunologia , Sorogrupo , Hipermutação Somática de Imunoglobulina/genética , Hipermutação Somática de Imunoglobulina/imunologia , Streptococcus pneumoniae/imunologia , Vacinação , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/uso terapêutico
2.
BMC Microbiol ; 16(1): 165, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27464881

RESUMO

BACKGROUND: Chlamydia trachomatis is a human pathogen which causes a number of pathologies, including genital tract infections in women that can result in tubal infertility. Prevention of infection and disease control might be achieved through vaccination; however, a safe, efficacious and cost-effective vaccine against C. trachomatis infection remains an unmet medical need. C. trachomatis major outer membrane protein (MOMP), a ß-barrel integral outer membrane protein, is the most abundant antigen in the outer membrane of the bacterium and has been evaluated as a subunit vaccine candidate. Recombinant MOMP (rMOMP) expressed in E. coli cytoplasm forms inclusion bodies and rMOMP extracted from inclusion bodies results in a reduced level of protection compared to the native MOMP in a mouse challenge model. RESULTS: We sought to target the recombinant expression of MOMP to the E. coli outer membrane (OM). Successful surface expression was achieved with codon harmonization, utilization of low copy number vectors and promoters with moderate strength, suitable leader sequences and optimization of cell culture conditions. rMOMP was extracted from E. coli outer membrane, purified, and characterized biophysically. The OM expressed and purified rMOMP is immunogenic in mice and elicits antibodies that react to the native antigen, Chlamydia elementary body (EB). CONCLUSIONS: C. trachomatis MOMP was functionally expressed on the surface of E. coli outer membrane. The OM expressed and purified rMOMP elicits antibodies that react to the native antigen, Chlamydia EB, in a mouse immunogenicity model. Surface expression of MOMP could provide useful reagents for vaccine research, and the methodology could serve as a platform to produce other outer membrane proteins recombinantly.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Chlamydia trachomatis/genética , Escherichia coli/genética , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/biossíntese , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/química , Células Cultivadas , Infecções por Chlamydia/prevenção & controle , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/metabolismo , Feminino , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
3.
Vaccine ; 41(11): 1774-1777, 2023 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-36781335

RESUMO

Pneumococcal conjugate vaccines (PCVs) have reduced vaccine-type pneumococcal disease but in turn have also resulted in replacement with non-vaccine serotypes. One such serotype, 35B, a multidrug resistant type, has been associated with an increase in disease. Mice were immunized intramuscularly with monovalent pneumococcal polysaccharide 35B conjugated to CRM197 containing aluminum phosphate adjuvant on days 0, 14, and 28. Pneumococcal enzyme-linked immunosorbent assay, opsonophagocytic killing assays, and competition OPA were performed for STs 35B and 29 to measure serotype-specific binding and functional antibodies. On day 52, mice were intratracheally challenged with S. pneumoniae ST29 to evaluate cross-protection. 35B-CRM197 immunized mice had binding and functional antibodies to both PnPs 35B and 29. 35B-CRM197 immunized mice were 100% protected from IT challenge with S. pneumoniae ST29 as compared to 30% survival in the naïve group. Future vaccines containing polysaccharide 35B, such as the investigational 21-valent PCV, V116, may provide cross protection against the non-vaccine serotype 29 due to structural similarity.


Assuntos
Infecções Pneumocócicas , Pneumonia , Animais , Camundongos , Sorogrupo , Proteção Cruzada , Streptococcus pneumoniae , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas , Vacinas Conjugadas , Anticorpos Antibacterianos
4.
Vaccine ; 41(35): 5113-5125, 2023 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-37321893

RESUMO

Development of a vaccine drug product requires formulation optimization to ensure that the vaccine's effectiveness is preserved upon storage throughout the shelf-life of the product. Although aluminum adjuvants have been widely used in vaccine formulations to safely and effectively potentiate an immune response, careful attention must be directed towards ensuring that the type of aluminum adjuvant does not impact the stability of the antigenic composition. PCV15 is a polysaccharide-protein conjugate vaccine comprising the pneumococcal polysaccharide (PnPs) serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F), each individually conjugated to the protein carrier CRM197. PCV15 was formulated with either amorphous aluminum hydroxyphosphate sulfate adjuvant (AAHS) or aluminum phosphate adjuvant (AP) and examined for both stability and immunogenicity. Using a collection of methods to evaluate vaccine stability, it was discovered that certain PCV15 serotypes (e.g., 6A, 19A, 19F) formulated with AAHS resulted in a reduction of immunogenicity in vivo and a reduction in recoverable dose as tested by an in vitro potency assay. The same polysaccharide-protein conjugates formulated with AP were stable regarding all measures tested. Moreover, the reduction in potency of certain serotypes correlated with chemical degradation of the polysaccharide antigen caused by the aluminum adjuvant as measured by reducing polyacrylamide gel electrophoresis (SDS-PAGE), High-Pressure Size Exclusion Chromatography coupled with UV detection (HPSEC-UV) and ELISA immunoassay. This study suggests a formulation, which includes AAHS, may negatively impact the stability of a pneumococcal polysaccharide-protein conjugate vaccine that contains phosphodiester groups. This decrease in stability would likely result in a decrease in the "active" concentration of antigen dose, and herein, it is shown that such instability directly compromised vaccine immunogenicity in an animal model. The results presented in this study help to explain critical degradation mechanisms of pneumococcal polysaccharide-protein conjugate vaccines.


Assuntos
Alumínio , Infecções Pneumocócicas , Animais , Vacinas Conjugadas , Vacinas Pneumocócicas , Sorogrupo , Adjuvantes Imunológicos , Infecções Pneumocócicas/prevenção & controle , Anticorpos Antibacterianos
5.
Vaccine ; 41(4): 903-913, 2023 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-36566163

RESUMO

Despite the widespread effectiveness of pneumococcal conjugate vaccines on the overall incidence of invasive pneumococcal disease, the global epidemiological landscape continues to be transformed by residual disease from non-vaccine serotypes, thus highlighting the need for vaccines with expanded disease coverage. To address these needs, we have developed V116,an investigational 21-valent non-adjuvanted pneumococcal conjugate vaccine (PCV),containingpneumococcal polysaccharides (PnPs) 3, 6A, 7F, 8, 9N, 10A, 11A,12F, 15A, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F, 35B, anda de-O-acetylated 15B(deOAc15B) individually conjugated to the nontoxic diphtheria toxoid CRM197 carrier protein. Preclinical studies evaluated the immunogenicity of V116 inadult monkeys, rabbits, and mice. Following one dose, V116 was found to be immunogenic in preclinical animal species and induced functional antibodies for all serotypes included in the vaccine, in addition to cross-reactive functional antibodies to serotypes 6C and 15B. In these preclinical animal studies, the increased valency of V116 did not result in serotype-specific antibody suppression when compared to lower valent vaccines V114 or PCV13. In addition, when compared with naïve controls, splenocytes from V116 to immunized animals demonstrated significant induction of CRM197-specific T cells in both IFN-γ and IL-4 ELISPOT assays, as well as Th1 and Th2 cytokine induction through in vitro stimulation assays, thus suggesting the ability of V116 to engage T cell dependent immune response pathways to aid in development of memory B cells. V116 also demonstrated significant protection in mice from intratracheal challenge with serotype 24F, a novel serotype not contained in any currently licensed vaccine.


Assuntos
Infecções Pneumocócicas , Streptococcus pneumoniae , Coelhos , Camundongos , Animais , Vacinas Pneumocócicas , Vacinas Conjugadas , Macaca mulatta , Anticorpos Antibacterianos , Infecções Pneumocócicas/prevenção & controle , Sorogrupo , Modelos Animais de Doenças
6.
Microb Pathog ; 50(1): 39-47, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21035535

RESUMO

Animal models predictive of human disease are generally difficult to establish and reproduce. In the case of the Group A Streptococcus (GAS) bacterium, which is predominantly a human pathogen, virulence assessment in animal models is problematic. We compared a monkey colonization and pharyngitis model of infection in two macaque species to determine the optimal model for vaccine candidate evaluation. Rhesus and cynomolgus macaques were intranasally infected with a streptomycin resistant (Str(r)) GAS strain. Monkeys were monitored for body weight and temperature changes, throat swabs and sera were collected, and clinical observations were noted throughout the study. Both species exhibited oropharyngeal colonization by GAS, with rhesus macaques demonstrating a more sustained colonization through day 28 post-challenge. Veterinary observations revealed no significant differences between GAS-infected rhesus and cynomolgus macaques. Mock-infected monkeys did not exhibit clinical symptoms or GAS colonization throughout the study. ELISA results demonstrated that both rhesus and cynomolgus macaques developed anti-streptolysin-O antibody titers, with cynomolgus generating higher titers. Sera from infected monkeys produced opsonophagocytic killing and bound to the bacterium in an immunofluorescence assay. Both rhesus and cynomolgus macaques can be used for colonization studies with this GAS M3 strain, yet only mild clinical signs of pharyngitis and tonsillitis were observed.


Assuntos
Macaca fascicularis/imunologia , Macaca mulatta/imunologia , Modelos Animais , Infecções Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas , Farmacorresistência Bacteriana/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Fagocitose , Infecções Estreptocócicas/microbiologia
7.
Vaccine ; 39(30): 4231-4237, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34074546

RESUMO

Invasive pneumococcal disease (IPD) is responsible for serious illnesses such as bacteremia, sepsis, meningitis, and pneumonia in young children, older adults, and persons with immunocompromising conditions and often leads to death. Although the most recent pneumococcal conjugate vaccines (PCVs) have been designed to target serotypes identified as the primary causative agents of IPD, the epidemiological landscape continues to change stressing the need to develop new PCVs. We have developed an investigational 24-valent PCV (PCV24) including serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F all conjugated to CRM197 and evaluated this vaccine in adult monkeys. PCV24 was shown to be immunogenic and induced functional antibody for all vaccine serotypes. Of the serotypes common to PCV13 and V114 (PCV15), PCV24 had a similar immunogenic response with the exceptions of 23F which had higher IgG GMCs for PCV13 and V114, and 7F which had higher GMCs for PCV13. Functional antibody responses were similar for the serotypes in common between PCV24, PCV13 and V114 vaccines, with the exception of serotype 7F which was greater for PCV13. Overall, this study shows that PCV24 provided similar immunogenicity as the lower valent vaccines in adult monkeys with no apparent serotype interference. In addition, PCV24 also provided protection against pneumococcal infection in a mouse challenge model.


Assuntos
Infecções Pneumocócicas , Vacinas Pneumocócicas , Idoso , Animais , Anticorpos Antibacterianos , Pré-Escolar , Haplorrinos , Humanos , Lactente , Camundongos , Infecções Pneumocócicas/prevenção & controle , Vacinas Conjugadas
8.
Pediatr Infect Dis J ; 39(1): 70-77, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31725555

RESUMO

BACKGROUND: Evaluation of a pneumococcal conjugate vaccine (PCV) in an animal model provides an initial assessment of the performance of the vaccine prior to evaluation in humans. Cost, availability, study duration, cross-reactivity and applicability to humans are several factors which contribute to animal model selection. PCV15 is an investigational 15-valent PCV which includes capsular polysaccharides from pneumococcal serotypes (ST) 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F all individually conjugated to cross-reactive material 197 (CRM197). METHODS: Immunogenicity of PCV15 was evaluated in infant rhesus macaques (IRM), adult New Zealand white rabbits (NZWR) and CD1 mice using multiplexed pneumococcal electrochemiluminescent (Pn ECL) assay to measure serotype-specific IgG antibodies, multiplexed opsonophagocytosis assay (MOPA) to measure serotype-specific functional antibody responses and bacterial challenge in mice to evaluate protection against a lethal dose of S. pneumoniae. RESULTS: PCV15 was immunogenic and induced both IgG and functional antibodies to all 15 vaccine serotypes in all animal species evaluated. PCV15 also protected mice from S. pneumoniae serotype 14 intraperitoneal challenge. Opsonophagocytosis assay (OPA) titers measured from sera of human infants vaccinated with PCV15 in a Phase 2 clinical trial showed a good correlation with that observed in IRM (rs=0.69, P=0.006), a medium correlation with that of rabbits (rs=0.49, P=0.06), and no correlation with that of mice (rs=0.04, P=0.89). In contrast, there was no correlation in serum IgG levels between human infants and animal models. CONCLUSIONS: These results demonstrate that PCV15 is immunogenic across multiple animal species, with IRM and human infants showing the best correlation for OPA responses.


Assuntos
Imunogenicidade da Vacina , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Vacinas Conjugadas/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Vacina Pneumocócica Conjugada Heptavalente/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos
9.
Vaccine ; 38(51): 8145-8153, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33162203

RESUMO

Pneumococcal conjugate vaccines (PCVs) have been effective in reducing the disease burden caused by Streptococcus pneumoniae. The first licensed PCV (PCV7) was composed of capsular polysaccharides from seven serotypes. This was followed by PCV10, then PCV13, and currently there are a number of higher valency vaccines in development. As part of licensure, new vaccine iterations require assessment of immunogenicity. Since some antibodies can be non-functional, measuring functional antibodies is desirable. To meet this need, opsonophagocytic assays (OPAs) have been developed. Previous studies have shown there can be significant variations in OPA results from different laboratories. We have previously shown that standardizing OPA data using reference serum 007sp can decrease this variation. To extend this approach to additional serotypes, a panel of sera was tested by five laboratories using a multiplexed OPA for serotypes 2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20B, 22F, and 33F. Each sample was tested in five runs with 007sp tested three times in each run. Results were analyzed using a mixed effects ANOVA model. Standardization of the results significantly decreased the inter-laboratory variation for some serotypes. For serotypes 2, 8, and 11A, the variability was reduced by 40%, 45%, and 40%, respectively. For serotypes 12F, 17F, and 20B, the reductions were more modest (14%, 19%, and 24%, respectively). Standardization had little effect for the remaining serotypes. In many cases, the impact of normalization was blunted by the results from five sera that were collected after an extended post-vaccination interval. We have previously reported consensus values for 007sp for 13 serotypes, as well as the creation of a calibration serum panel ("Ewha Panel A"). Here, we report consensus values for 11 additional serotypes for 007sp and the creation of a second serum panel ("Ewha Panel B"). These consensus values will facilitate the development of next-generation PCVs.


Assuntos
Infecções Pneumocócicas , Streptococcus pneumoniae , Anticorpos Antibacterianos , Calibragem , Humanos , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas , Sorogrupo , Vacinas Conjugadas
10.
Vaccine ; 35(6): 865-872, 2017 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-28087148

RESUMO

Pneumococcal disease continues to be a medical need even with very effective vaccines on the market. Globally, there are extensive research efforts to improve serotype coverage with novel vaccines; therefore, conducting preclinical studies in different animal models becomes essential. The work presented herein focuses on evaluating a 15-valent pneumococcal conjugate vaccine (PCV15) in mice. Initially we evaluated several doses of PCV15 in Balb/c mice. The optimal vaccine dose was determined to be 0.4µg per pneumococcal polysaccharide (PS) (0.8µg of 6B) for subsequent studies. This PS dose was chosen for PCV evaluation in mice based on antibody levels determined by multiplexed electrochemiluminescent (ECL) assays, T-cell responses following in vitro stimulation with CRM197 peptides and protection from pneumococcal challenge. We then selected four mouse strains for evaluation: Balb/c, C3H/HeN, CD1 and Swiss Webster (SW), immunized with PCV15 by either intraperitoneal (IP) or intramuscular (IM) routes. We assessed IgG responses by ECL assays and functional antibody activity by multiplexed opsonophagocytic assays (MOPA). Every mouse strain evaluated responded to all 15 serotypes contained in the vaccine. Mice tended to have lower responses to serotypes 6B, 23F and 33F. The IP route of immunization resulted in higher antibody titers for most serotypes in Balb/c, C3H and SW. CD1 mice tended to respond similarly for most serotypes, regardless of route of immunization. Similar trends were observed with the four mouse strains when evaluating functional antibody activity. Given the differences in antibody responses based on mouse strain and route of immunization, it is critical to evaluate pneumococcal vaccines in multiple animal models to determine the optimal formulation before moving to clinical trials.


Assuntos
Anticorpos Antibacterianos/biossíntese , Imunoglobulina G/biossíntese , Vacinas Pneumocócicas/administração & dosagem , Pneumonia Pneumocócica/prevenção & controle , Streptococcus pneumoniae/efeitos dos fármacos , Vacinação , Animais , Proteínas de Bactérias/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Injeções Intramusculares , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos , Vacinas Pneumocócicas/síntese química , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/microbiologia , Polissacarídeos Bacterianos/administração & dosagem , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/imunologia , Sorogrupo , Especificidade da Espécie , Streptococcus pneumoniae/química , Streptococcus pneumoniae/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Vacinas Conjugadas
11.
PLoS One ; 11(7): e0160055, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27467585

RESUMO

Chlamydia trachomatis is among the most prevalent of sexually transmitted diseases. While Chlamydia infection is a reportable event and screening has increased over time, enhanced surveillance has not resulted in a reduction in the rate of infections, and Chlamydia infections frequently recur. The development of a preventative vaccine for Chlamydia may be the only effective approach for reducing infection and the frequency of pathological outcomes. Current vaccine research efforts involve time consuming and/or invasive approaches for assessment of disease state, and MRI presents a clinically translatable method for assessing infection and related pathology both quickly and non-invasively. Longitudinal T2-weighted MRI was performed over 63 days on both control or Chlamydia muridarum challenged mice, either with or without elementary body (EB) immunization, and gross necropsy was performed on day 65. A scoring system was developed to assess the number of regions affected by Chlamydia pathology and was used to document pathology over time and at necropsy. The scoring system documented increasing incidence of pathology in the unimmunized and challenged mice (significantly greater compared to the control and EB immunized-challenged groups) by 21 days post-challenge. No differences between the unchallenged and EB immunized-challenged mice were observed. MRI scores at Day 63 were consistently higher than gross necropsy scores at Day 65, although two of the three groups of mice showed no significant differences between the two techniques. In this work we describe the application of MRI in mice for the potential evaluation of disease pathology and sequelae caused by C. muridarum infection and this technique's potential for evaluation of vaccines for Chlamydia.


Assuntos
Infecções por Chlamydia/diagnóstico por imagem , Modelos Animais de Doenças , Animais , Infecções por Chlamydia/microbiologia , Células HeLa , Humanos , Imageamento por Ressonância Magnética , Camundongos
12.
Clin Vaccine Immunol ; 21(5): 689-97, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24623624

RESUMO

Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates.


Assuntos
ADP Ribose Transferases/imunologia , Anticorpos Neutralizantes/sangue , Proteínas de Bactérias/imunologia , Ensaios de Triagem em Larga Escala/métodos , Animais , Chlorocebus aethiops , Cricetinae , Macaca mulatta , Células Vero
13.
Hum Vaccin Immunother ; 9(3): 488-96, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23249976

RESUMO

Vaccine development for Group A streptococcal (GAS) infection has been extensively focused on the N-terminal hypervariable or the C-terminal conserved regions of the M protein, a major virulence factor of GAS. We evaluated the immunogenicity and functional activity of the conserved C-terminal peptide vaccine candidate, J8, conjugated to CRM197, in two mouse strains: C3H (H2(k)) and Balb/c (H2(d)), and in rhesus macaques. Mice were immunized with J8-CRM197 formulated with Amorphous Aluminum Hydroxyphosphate Sulfate Adjuvant (AAHSA), and non-human primates were immunized with J8-CRM197 formulated with AAHSA, ISCOMATRIX (TM) adjuvant, or AAHSA/ISCOMATRIX adjuvant. J8-CRM197 was immunogenic in mice from both H2(k) and H2(d) backgrounds, and the antibodies generated bound to the surface of four different GAS serotypes and had functional bacterial opsonic activity. Mice immunized with J8-CRM197/AAHSA demonstrated varying degrees of protection from lethal challenge. We also demonstrated that J8-CRM197 is immunogenic in non-human primates. Our data confirm the utility of J8 as a potential GAS vaccine candidate and demonstrate that CRM197 is an acceptable protein carrier for this peptide.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Proteínas de Bactérias/administração & dosagem , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/imunologia , Streptococcus pyogenes/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Adjuvantes Imunológicos/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Feminino , Macaca mulatta , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Infecções Estreptocócicas/imunologia , Vacinas Estreptocócicas/administração & dosagem , Vacinas Estreptocócicas/genética , Vacinas Estreptocócicas/metabolismo , Streptococcus pyogenes/genética , Análise de Sobrevida , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/genética , Vacinas Conjugadas/imunologia , Vacinas Conjugadas/metabolismo , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/metabolismo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/metabolismo
14.
Clin Vaccine Immunol ; 20(4): 517-25, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23389929

RESUMO

Clostridium difficile produces two major virulence toxins, toxin A (TcdA) and toxin B (TcdB). Antitoxin antibodies, especially neutralizing antibodies, have been shown to be associated with a lower incidence of C. difficile infection (CDI) recurrence, and antibody levels are predictive of asymptomatic colonization. The development of an assay to detect the presence of neutralizing antibodies in animal and human sera for the evaluation of vaccine efficacy is highly desired. We have developed such an assay, which allows for the quantification of the effect of toxins on eukaryotic cells in an automated manner. We describe here the optimization of this assay to measure toxin potency as well as neutralizing antibody (NAb) activity against C. difficile toxins using a design-of-experiment (DOE) methodology. Toxin concentration and source, cell seeding density, and serum-toxin preincubation time were optimized in the assay using Vero cells. The assay was shown to be robust and to produce linear results across a range of antibody concentrations. It can be used to quantify neutralizing antibodies in sera of monkeys and hamsters immunized with C. difficile toxoid vaccines. This assay was shown to correlate strongly with traditional assays which rely on labor-intensive methods of determining neutralizing antibody titers by visual microscopic inspection of intoxicated-cell monolayers. This assay has utility for the selection and optimization of C. difficile vaccine candidates.


Assuntos
Anticorpos Neutralizantes/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Clostridioides difficile/imunologia , Técnicas Citológicas/métodos , Enterotoxinas/imunologia , Testes de Neutralização/métodos , Proteínas Repressoras/imunologia , Animais , Automação Laboratorial/métodos , Chlorocebus aethiops , Cricetinae , Masculino , Mesocricetus , Células Vero
15.
Vaccine ; 29(48): 8870-6, 2011 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-21964055

RESUMO

The incidence of invasive pneumococcal disease (IPD), caused by the approximately 91 serotypes of Streptococcus pneumoniae (PN), varies geographically and temporally as a result of changing epidemiology and vaccination patterns as well as due to regional measurement differences. Prevnar(®) (Pfizer), the first licensed pneumococcal conjugate vaccine (PCV), comprises polysaccharides (PS) from 7 serotypes conjugated to the mutant diphtheria toxin carrier protein, CRM197. In the United States and elsewhere, this vaccine has been highly efficacious in reducing the incidence of IPD caused by vaccine serotypes, however, the incidence of non-vaccine serotypes (e.g., 19A, 22F, and 33F) has increased, resulting in the need for vaccines with higher valencies. In response, 10- and 13-valent PCVs have recently been licensed. To further increase serotype coverage, we have developed a 15-valent PCV containing PS from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197 and formulated on aluminum phosphate adjuvant. Vaccine immunogenicity was evaluated in infant rhesus monkeys since they, like human infants, respond poorly to unconjugated PN PS. Infant (2-3 month old) rhesus monkeys were vaccinated three times with PCV-15 or Prevnar(®) at 2 month intervals, and serotype-specific IgG antibodies were measured using a multiarray electrochemiluminescence (ECL) assay. The results indicate that antibody responses to PCV-15 and Prevnar(®) were comparable for the 7 common serotypes and that post-vaccination responses to PCV-15 were >10-fold higher than baseline for the 8 additional serotypes.


Assuntos
Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/imunologia , Polissacarídeos Bacterianos/imunologia , Animais , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Vacina Pneumocócica Conjugada Heptavalente , Imunoglobulina G/sangue , Macaca mulatta , Vacinas Pneumocócicas/administração & dosagem , Sorotipagem , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologia
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