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1.
J Immunol Methods ; 519: 113516, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37348647

RESUMO

Streptococcus pneumoniae is one of the most common microorganisms causing acute otitis media (AOM) in children. While bacterial culture of middle ear fluid (MEF) is the gold standard to detect the etiological organisms, several host and pathogen factors impact the survival of the organisms resulting in false negatives. To overcome this limitation, we have developed and validated an innovative multiplex immuno-molecular assay to screen and detect the S. pneumoniae 15-valent pneumococcal conjugate vaccine (PCV15; STs 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) vaccine serotypes in MEF. This novel in vitro approach involves two-step testing. First, the MEF specimens were tested for highly conserved pneumococcal genes, autolysin, lytA, and pneumolysin, ply using direct PCR to identify pneumococcus positive specimens. The pneumococcus positive specimens were screened for the presence of vaccine serotype specific pneumococcal polysaccharides using a 15-plex Pneumococcal Antigen Detection (PAD) assay, with specific capture and detection monoclonal antibodies. Due to the lack of availability of MEF samples, cerebrospinal fluid (CSF) was used as the surrogate matrix for the development and validation of the PCR-PAD assays. The PCR and PAD assays were separately evaluated for sensitivity and specificity. Subsequently, the PCR-PAD assays were cross-validated with human MEF samples (n = 39) which were culture confirmed to contain relevant bacterial strains. The combined PCR-PAD assays demonstrated high rate of agreement 94.9% (95% CI; 82.7, 99.4%) with historical Quellung serotype data of these MEF samples. This novel PCR-PAD assay demonstrates the feasibility of combining molecular and immunological assays to screen and identify PCV15 pneumococcal vaccine serotypes in AOM clinical samples.


Assuntos
Infecções Pneumocócicas , Streptococcus pneumoniae , Criança , Humanos , Streptococcus pneumoniae/genética , Sorogrupo , Infecções Pneumocócicas/diagnóstico , Infecções Pneumocócicas/prevenção & controle , Sorotipagem/métodos , Vacinas Pneumocócicas , Antígenos de Bactérias/genética , Orelha Média
2.
Bioanalysis ; 14(15): 1067-1079, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36125040

RESUMO

Background: Respiratory syncytial virus (RSV) vaccine is an unmet medical need. The virus reduction neutralization test (VRNT) was developed to replace the LI-COR microneutralization assay to measure RSV neutralization titers. Methods: A bridging study using selected V171 phase I samples and calibration studies using the WHO international standard antiserum to RSV were performed to compare VRNT and LI-COR. Results: From the bridging study, we showed good concordance between VRNT and LI-COR titers, and similar post-/prevaccination titer ratios. From the calibration studies, we can convert VRNT and LI-COR titers into similar IU/ml. Conclusion: The VRNT and LI-COR microneutralization assay correlate well and the titers can be standardized as similar IU/ml, enabling direct comparison of titers from different assays.


Assuntos
Vírus Sinciciais Respiratórios , Vacinas , Anticorpos Neutralizantes , Calibragem , Testes de Neutralização , Organização Mundial da Saúde
3.
mSphere ; 7(4): e0011422, 2022 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-35913133

RESUMO

Streptococcus pneumoniae is a major cause of community-acquired pneumonia (CAP) in young children, older adults, and those with immunocompromised status. Since the introduction of pneumococcal vaccines, the burden of invasive pneumococcal disease caused by vaccine serotypes (STs) has decreased; however, the effect on the burden of CAP is unclear, potentially due to the lack of testing for pneumococcal STs. We describe the development, qualification, and clinical validation of a high-throughput and multiplex ST-specific urine antigen detection (SSUAD) assay to address the unmet need in CAP pneumococcal epidemiology. The SSUAD assay is sensitive and specific to the 15 STs in the licensed pneumococcal conjugate vaccine V114 (STs 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) and uses ST-specific monoclonal antibodies for rapid and simultaneous quantification of the 15 STs using a Luminex microfluidics system. The SSUAD assay was optimized and qualified using healthy adult urine spiked with pneumococcal polysaccharides and validated using culture-positive clinical urine samples (n = 34). Key parameters measured were accuracy, precision, sensitivity, specificity, selectivity, and parallelism. The SSUAD assay met all prespecified validation acceptance criteria and is suitable for assessments of disease burden associated with the 15 pneumococcal STs included in V114. IMPORTANCE Streptococcus pneumoniae has more than 90 serotypes capable of causing a range of disease manifestations, including otitis media, pneumonia, and invasive diseases, such as bacteremia or meningitis. Only a minority (<10%) of pneumococcal diseases are bacteremic with known serotype distribution. Culture and serotyping of respiratory specimens are neither routine nor reliable. Hence, the serotype-specific disease burden of the remaining (>90%) noninvasive conditions is largely unknown without reliable laboratory techniques. To address this need, a 15-plex urine antigen detection assay was developed and validated to quantify pneumococcal serotype-specific capsular polysaccharides in urine. This assay will support surveillance to estimate the pneumococcal disease burden and serotype distribution in nonbacteremic conditions. Data obtained from this assay will be critical for understanding the impact of pneumococcal vaccines on noninvasive pneumococcal diseases and to inform the choice of pneumococcal serotypes for next-generation vaccines.


Assuntos
Bacteriemia , Infecções Comunitárias Adquiridas , Infecções Pneumocócicas , Pneumonia , Idoso , Criança , Pré-Escolar , Humanos , Infecções Pneumocócicas/epidemiologia , Vacinas Pneumocócicas , Polissacarídeos , Sorogrupo , Streptococcus pneumoniae
4.
Hum Vaccin ; 7(6): 630-8, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21508677

RESUMO

Indwelling central venous catheters are a common and important source of nosocomial Staphylococcus epidermidis and S. aureus infections, causing increased morbidity and mortality during hospitalization. A model was developed to reflect the clinical situation of catheter colonization by transient hematogeneously spread staphylococci, in order to investigate potential vaccine candidates. Rats were cannulated in the right jugular vein, followed by challenge through the tail vein with either S. epidermidis RP62a, or S. aureus Becker. At 24 hr post challenge, colonizing bacteria were found to be present on the catheter in an early biofilm, as evidenced by the presence of polysaccharide intercellular adhesin (PIA). For vaccination studies, rats were first immunized, surgically cannulated, and then challenged via the tail vein. At 24 hr post challenge, the catheters were harvested and cultured on mannitol salt agar plates. The catheters were scored as positive if there was outgrowth of bacterial colonies, and negative if no colonies were observed. A S. epidermidis antigen (SERP0630, MenD), and a S. aureus antigen (SACOL1138, iron regulated surface determinant B, IsdB) were found to have significant protective activity in this model, compared to mock immunized controls. Using SERP0630 as the test immunogen, it was also determined that a single vaccination of rats after cannulation was sufficient for significant catheter protection. This model may be used to evaluate antigens for protective activity against transient hematogenous spread of staphylococci resulting in catheter colonization and early biofilm formation.


Assuntos
Biofilmes , Infecções Relacionadas a Cateter/prevenção & controle , Cateterismo Venoso Central/efeitos adversos , Infecções Estafilocócicas/prevenção & controle , Vacinas Antiestafilocócicas/imunologia , Staphylococcus aureus/imunologia , Staphylococcus epidermidis/imunologia , Animais , Feminino , Modelos Animais , Ratos , Ratos Sprague-Dawley
5.
J Immunol Methods ; 494: 113054, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33845088

RESUMO

Respiratory syncytial virus (RSV) is a common pathogen causing severe respiratory illness in infants and elder adults. The development of an effective RSV vaccine is an important unmet medical need and an area of active research. The traditional method for testing neutralizing antibodies against RSV in clinical trials is the plaque reduction neutralization test (PRNT), which uses 24-well plates and needs several days post infection to develop viral plaques. In this study, we have developed a virus reduction neutralization test (VRNT), which allows the number of RSV infected cells to be automatically counted by an imaging cytometer at one day post infection in 96-well plates. VRNT was found robust to cell seeding density, detection antibody concentration, virus input and infection time. By testing twenty human sera, we have shown good correlation between VRNT50 and PRNT50 titers for multiple RSV strains: A2, Long and 18537 (serotype B). To understand the VRNT performance, eight human serum samples with high, medium and low neutralization titers were selected for VRNT qualification. We have demonstrated that VRNT had good specificity, precision, linearity and relative accuracy. In conclusion, VRNT is a better alternative to PRNT in serum neutralization test for RSV vaccine candidates.


Assuntos
Testes de Neutralização/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sinciciais Respiratórios/fisiologia , Idoso de 80 Anos ou mais , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Diagnóstico por Imagem , Ensaios de Triagem em Larga Escala , Humanos , Lactente , Recém-Nascido , Miniaturização , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Células Vero , Ensaio de Placa Viral
6.
Bioanalysis ; 12(14): 1003-1020, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32686954

RESUMO

Background: To streamline and improve throughput, the agar-based multiplexed opsonophagocytic killing assay (MOPA) was optimized and validated on a microcolony platform for use in the Phase III clinical trial program for V114, an MSD 15-valent pneumococcal conjugate vaccine candidate. Results & methodology: The precision, dilutional linearity and specificity of the microcolony MOPA (mMOPA) were assessed for each serotype in validation experiments. All prespecified acceptance criteria on assay performance were satisfied. Accuracy was assessed by testing 007sp and the US FDA reference panel and comparing to consensus values. The mMOPA produced comparable results to other opsonophagocytic killing assays/MOPAs. Conclusion: The mMOPA is suitable for measuring functional antibodies in adult and pediatric samples. Benefits include throughput, reduced analyst-to-analyst variability and automation potential.


Assuntos
Bioensaio/métodos , Vacinas Pneumocócicas/química , Streptococcus pneumoniae/química , Humanos , Sorogrupo
7.
Bioanalysis ; 12(19): 1363-1375, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32975436

RESUMO

Aim: To re-optimize the pneumococcal (Pn) electrochemiluminescence (ECL) assay and to validate and bridge the enhanced assay to the WHO ELISA, to support the Phase III clinical trial program for V114, a 15-valent Pn conjugate vaccine. Materials & methods: The Pn ECL assay was re-optimized, validated and formally bridged to the WHO ELISA. Results: The enhanced Pn ECL assay met all prespecified validation acceptance criteria and demonstrated concordance with the WHO ELISA. The corresponding threshold value remains at 0.35 µg/ml for all 15 serotypes. Conclusion: The enhanced Pn ECL assay has been validated for the measurement of antibodies to 15 Pn capsular polysaccharides and is concordant with the WHO ELISA, supporting its use in clinical trials.


Assuntos
Bioensaio/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Vacinas Pneumocócicas/imunologia , Organização Mundial da Saúde/organização & administração , Humanos
8.
J Med Virol ; 81(7): 1310-22, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19475609

RESUMO

A real-time PCR assay was developed to identify varicella-zoster virus (VZV) and herpes simplex virus (HSV) DNA in clinical specimens from subjects with suspected herpes zoster (HZ; shingles). Three sets of primers and probes were used in separate PCR reactions to detect and discriminate among wild-type VZV (VZV-WT), Oka vaccine strain VZV (VZV-Oka), and HSV DNA, and the reaction for each virus DNA was multiplexed with primers and probe specific for the human beta-globin gene to assess specimen adequacy. Discrimination of all VZV-WT strains, including Japanese isolates and the Oka parent strain, from VZV-Oka was based upon a single nucleotide polymorphism at position 106262 in ORF 62, resulting in preferential amplification by the homologous primer pair. The assay was highly sensitive and specific for the target virus DNA, and no cross-reactions were detected with any other infectious agent. With the PCR assay as the gold standard, the sensitivity of virus culture was 53% for VZV and 77% for HSV. There was 92% agreement between the clinical diagnosis of HZ by the Clinical Evaluation Committee and the PCR assay results.


Assuntos
Vacina contra Varicela , Vacinas contra o Vírus do Herpes Simples , Herpesvirus Humano 3/classificação , Herpesvirus Humano 3/genética , Reação em Cadeia da Polimerase/métodos , Simplexvirus/classificação , Simplexvirus/genética , Primers do DNA , Diagnóstico Diferencial , Herpes Simples/diagnóstico , Herpes Zoster/diagnóstico , Herpesvirus Humano 3/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase/normas , Polimorfismo de Nucleotídeo Único , Padrões de Referência , Sensibilidade e Especificidade , Simplexvirus/isolamento & purificação , Vacinas , Globinas beta/genética
9.
J Immunol Methods ; 334(1-2): 11-20, 2008 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-18329663

RESUMO

Current flu vaccines are based on killed or attenuated virus vaccines that must be altered each year to include the hemagglutinin and neuraminidase genes from a strain of virus predicted to predominate in the coming year. A vaccine that could protect against multiple strains of influenza A and B would be a major asset in the fight against flu-related mortality and morbidity. To support development of such a vaccine, we have developed a Flu Multiplex Assay based on a Luminex platform to assess serum antibody levels to two conserved peptides derived from influenza A (M2 protein) and influenza B (hemagglutinin protein). The peptides were synthesized with a biotin label and subsequently coupled to two different LumAvidin microspheres. We then tested various sera against both types of peptide in the multiplex assay format. The data show that sera from Rhesus macaques immunized with a single peptide react only with the homologous peptide while Rhesus macaques immunized with both peptides respond well to both peptides. Additionally, we were able to specifically compete reactivity to both peptides. We have tested serial bleeds from 100 pediatric patients at ages ranging from 16 to 56 weeks as well as single bleeds from over 100 healthy adults. No overall trend in titer relative to pediatric age was detected. Both demographics exhibited a minimal response to either the A/M2 or B/HA0 peptides. However, the average titer for the pediatric serum samples was significantly lower than that found in the adult population. The adult population exhibited a higher prevalence of low reactive samples. Assay reagents and parameters have been optimized and the assay is shown to be repeatable and robust. The assay will be used to support clinical vaccine trials of a bivalent peptide vaccine.


Assuntos
Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoensaio/métodos , Vírus da Influenza A/imunologia , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Proteínas da Matriz Viral/imunologia , Adolescente , Adulto , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Feminino , Humanos , Lactente , Macaca mulatta , Masculino , Peptídeos/imunologia , Padrões de Referência , Sensibilidade e Especificidade
10.
Am J Trop Med Hyg ; 99(6): 1430-1439, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30350775

RESUMO

Vaccine immunogenicity and clinical efficacy are often assessed by the measure of serum-neutralizing antibodies. The present gold standard for detecting neutralizing antibodies against many viruses, including dengue, is the plaque/focus reduction neutralization test (P/FRNT). The FRNT is a cell-based assay that inherits high variability, resulting in poor precision and has lengthy turnaround times. The virus reduction neutralization test (VRNT) is a high-throughput alternative to the standard low-throughput and laborious FRNT. The VRNT is similar to FRNT using unaltered wild-type virus and immunostaining, yet uses imaging cytometry to count virus-infected cells 1 day post-infection, reducing assay time and increasing overall throughput 15-fold. In addition, the VRNT has lowered variability relative to FRNT, which may be explained in part by the observation that foci overlap alters foci count and titer over time, in the FRNT. The ability to count one infected cell, rather than waiting for overlapping foci to form, ensures accuracy and contributes to the precision (7-25% coefficient of variation) and sensitivity of the VRNT. Results from 81 clinical samples tested in the VRNT and FRNT show a clear positive relationship. During sample testing, a 96-well plate edge effect was noted and the elimination of this edge effect was achieved by a simple plate seeding technique. The VRNT is an improvement to the current neutralization assays for its shortened assay time, increased precision and throughput, and an alternative to the P/FRNT.


Assuntos
Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Vírus da Dengue/imunologia , Ensaios de Triagem em Larga Escala/normas , Imagem Molecular/métodos , Testes de Neutralização/normas , Análise de Célula Única/métodos , Animais , Antraquinonas/química , Chlorocebus aethiops , Dengue/imunologia , Dengue/prevenção & controle , Dengue/virologia , Vacinas contra Dengue/análise , Vírus da Dengue/isolamento & purificação , Corantes Fluorescentes/química , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Células Vero , Carga Viral , Ensaio de Placa Viral
11.
Front Immunol ; 6: 553, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26579128

RESUMO

ZOSTAVAX(®) is a live attenuated varicella-zoster virus (VZV) vaccine that is licensed for the protection of individuals ≥50 years against shingles and its most common complication, postherpetic neuralgia. While IFNγ responses increase upon vaccination, the quality of the T cell response has not been elucidated. By using polychromatic flow cytometry, we characterized the breadth, magnitude, and quality of ex vivo CD4(+) and CD8(+) T cell responses induced 3-4 weeks after ZOSTAVAX vaccination of healthy adults. We show, for the first time that the highest frequencies of VZV-specific CD4(+) T cells were poly-functional CD154(+)IFNγ(+)IL-2(+)TNFα(+) cells, which were boosted upon vaccination. The CD4(+) T cells were broadly reactive to several VZV proteins, with immediate early (IE) 63 ranking the highest among them in the fold rise of poly-functional cells, followed by IE62, gB, open reading frame (ORF) 9, and gE. We identified a novel poly-functional ORF9-specific CD8(+) T cell population in 62% of the subjects, and these were boosted upon vaccination. Poly-functional CD4(+) and CD8(+) T cells produced significantly higher levels of IFNγ, IL-2, and TNFα compared to mono-functional cells. After vaccination, a boost in the expression of IFNγ by poly-functional IE63- and ORF9-specific CD4(+) T cells and IFNγ, IL-2, and TNFα by ORF9-specific poly-functional CD8(+) T cells was observed. Responding poly-functional T cells exhibited both effector (CCR7(-)CD45RA(-)CD45RO(+)), and central (CCR7(+)CD45RA(-)CD45RO(+)) memory phenotypes, which expressed comparable levels of cytokines. Altogether, our studies demonstrate that a boost in memory poly-functional CD4(+) T cells and ORF9-specific CD8(+) T cells may contribute toward ZOSTAVAX efficacy.

12.
Hum Gene Ther ; 14(1): 25-36, 2003 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-12573056

RESUMO

Adenoviral vectors are used widely as gene therapy and vaccine delivery systems. An adenovirus-shedding assay may be performed in clinical trials to monitor the safety of the vector and to investigate the potential relation between clinical symptoms and shed vector virus. This report describes the development and statistical performance of the shedding assay. Live adenovirus was recovered from throat swab and urine samples spiked with E1-deleted adenovirus type 5 vector expressing HIV-1 gag [Ad5HIVgag], in the presence or absence of wild-type adenovirus (WT Ad5). Samples were cultured in 293 and A549 cells, and the DNA extracted from virus culture was tested by polymerase chain reaction (PCR) for sequence identity. The results showed that the frequency of Ad5HIVgag infectivity in 293 cells by cytopathic effect (CPE) or an immunofluorescence assay (IFA) was concentration-dependent (53% for 10(2), 94% for 10(4), and 100% for 10(6) viral particles). WT Ad5 virus did not interfere with Ad5HIVgag. PCR amplisets could specifically amplify target sequences in the background of nonspecific DNA matrices and could distinguish Ad5HIVgag from wild-type adenoviruses. This assay may be used for clinical trials using adenovirus vectors as vehicles for vaccines.


Assuntos
Adenoviridae/genética , Vetores Genéticos/análise , Reação em Cadeia da Polimerase/métodos , Vacinas de DNA/genética , Eliminação de Partículas Virais/fisiologia , Adenoviridae/isolamento & purificação , DNA Viral/isolamento & purificação , Produtos do Gene gag/análise , Produtos do Gene gag/genética , Terapia Genética/métodos , Vetores Genéticos/urina , Humanos , Mucosa Respiratória/virologia , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Vacinas de DNA/análise
13.
Hum Gene Ther ; 15(3): 293-304, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15018738

RESUMO

Replication-defective recombinant adenoviruses (rAd) are used as vectors for vaccines as well as for gene therapy. To determine type-specific antibodies to adenovirus (Ad) serotypes 2, 5, 24, 34, and 35, we developed quantitative neutralization assays using recombinant adenoviruses with the secreted alkaline phosphatase (SEAP) reporter gene. Among the standardized parameters, the concentration of infectious and noninfectious adenoviral particles used in the assay is critical for a reliable comparison of data from different studies. The usefulness of this assay was demonstrated in a pilot epidemiologic study of 40 healthy individuals. In this study, the highest prevalence of antiadenovirus antibodies was found for the Ad2 serotype (82.5%), followed by Ad5 (35%). The prevalence of antiadenovirus antibodies for the serotypes 24, 34, and 35 was low (7.5%, 2.5%, and 0%, respectively). In addition, epidemiologic parameters such as gender and age were statistically evaluated. A positive association was found between age and the presence of anti-Ad5 antibodies. The assay was also useful for evaluating the presence of antiadenovirus antibodies in the design of vaccines using a rhesus monkey model. In this animal model, it was possible to determine differential dose and time responses, and the specificity for the detection of neutralizing antibodies was assessed. The evaluation of serotype-specific neutralizing antibodies can be of both clinical and epidemiologic importance as a means of selecting the appropriate serotype adenovector(s).


Assuntos
Adenoviridae/genética , Adenoviridae/imunologia , Fosfatase Alcalina/genética , Vetores Genéticos/imunologia , Testes de Neutralização/métodos , Vacinas de DNA/imunologia , Adenoviridae/classificação , Adulto , Idoso , Fosfatase Alcalina/análise , Fosfatase Alcalina/imunologia , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Linhagem Celular , Estudos Epidemiológicos , Feminino , Técnicas de Transferência de Genes , Genes Reporter , Humanos , Macaca mulatta , Masculino , Pessoa de Meia-Idade
14.
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
15.
Vaccine ; 31(42): 4888-93, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-23941913

RESUMO

There is a need for novel rabies vaccines suitable for short course, pre- and post-exposure prophylactic regimens which require reduced doses of antigen to address the current worldwide supply issue. We evaluated in rhesus macaques the immunogenicity of a quarter-dose of a standard rabies vaccine formulated with Merck's amorphous aluminum hydroxylphosphate sulfate adjuvant, the saponin-based ISCOMATRIX™ adjuvant, or a synthetic TLR9 agonist. All adjuvants significantly increased the magnitude and durability of the humoral immune response as measured by rapid fluorescent focus inhibition test (RFFIT). Several three-dose vaccine regimens resulted in adequate neutralizing antibody of ≥ 0.5 IU/ml earlier than the critical day seven post the first dose. Rabies vaccine with ISCOMATRIX™ adjuvant given at days 0 and 3 resulted in neutralizing antibody titers which developed faster and were up to one log10 higher compared to WHO-recommended intramuscular and intradermal regimens and furthermore, passive administration of human rabies immunoglobulin did not interfere with immunogenicity of this reduced dose, short course vaccine regimen. Adjuvantation of whole-killed rabies vaccine for intramuscular injection may therefore be a viable alternative to intradermal application of non-adjuvanted vaccine for both pre- and post-exposure regimens.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Compostos de Alumínio/administração & dosagem , Colesterol/administração & dosagem , Fosfolipídeos/administração & dosagem , Vacina Antirrábica/administração & dosagem , Vacina Antirrábica/imunologia , Saponinas/administração & dosagem , Receptor Toll-Like 9/agonistas , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Combinação de Medicamentos , Feminino , Injeções Intramusculares , Macaca mulatta , Masculino , Testes de Neutralização , Fosfatos/administração & dosagem , Raiva/prevenção & controle , Sulfatos/administração & dosagem , Receptor Toll-Like 9/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia
16.
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
17.
Clin Diagn Lab Immunol ; 12(8): 959-69, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16085914

RESUMO

A human papillomavirus (HPV) multiplexed competitive Luminex immunoassay first described by Opalka et al. (D. Opalka, C. E. Lachman, S. A. MacMullen, K. U. Jansen, J. F. Smith, N. Chirmule, and M. T. Esser, Clin. Diagn. Lab. Immunol. 10:108--15, 2003) was optimized and validated for use in epidemiology studies and vaccine clinical trials. Optimization increased both the analytical sensitivity and the clinical specificity of the assay to more effectively discriminate the low-titer antibody response of HPV-infected persons from noninfected individuals. The characteristics of the assay that were optimized included monoclonal antibody (MAb) specificity, scaling up the conjugation of virus-like particles (VLPs) to microspheres, VLP concentration, MAb concentration, sample matrix, sample dilution, incubation time, heat inactivation of sample sera, and detergent effects on assay buffer. The assay was automated by use of a TECAN Genesis Workstation, thus improving assay throughput, reproducibility, and operator safety. Following optimization, the assay was validated using several distinct serum panels from individuals determined to be at low and high risk for HPV infection. The validated assay was then used to determine the clinical serostatus cutoff. This high-throughput assay has proven useful for performing epidemiology studies and evaluating the efficacy of prophylactic HPV vaccines.


Assuntos
Anticorpos Monoclonais/sangue , Anticorpos Antivirais/sangue , Imunoensaio/métodos , Papillomaviridae/imunologia , Infecções por Papillomavirus/imunologia , Infecções Tumorais por Vírus/imunologia , Adolescente , Adulto , Criança , Epitopos/imunologia , Feminino , Humanos , Masculino , Microesferas , Papillomaviridae/classificação , Infecções por Papillomavirus/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Infecções Tumorais por Vírus/diagnóstico
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