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Two multiplex immunoassays are routinely used to assess antibody responses in clinical trials of the 9-valent human papillomavirus (9vHPV) vaccine. The HPV6/11/16/18/31/33/45/52/58 competitive Luminex immunoassay (HPV-9 cLIA) and HPV6/11/16/18/31/33/45/52/58 total immunoglobulin G Luminex immunoassay are used for measurements of immunogenicity. Following their initial validation in 2010, both assays were redeveloped, and several parameters were optimized, including the coating concentration of virus-like particles, type of Luminex microspheres, serum sample and reference standard diluent, reference standard starting dilution and titration series, and vendor and concentration of the phycoerythrin-labeled antibodies. Validation studies evaluated the assay performance parameters, including the intra-assay precision (repeatability), intermediate precision, linearity, relative accuracy, and limits of quantitation. In addition, since maintaining a link to the original assays that were used in trials supporting vaccine licensure is critical, the assays were formally bridged to the previous assay versions by using individual patient sera from a 9vHPV vaccine clinical trial (n = 150 day 1 [prevaccination] samples; n = 100 month 7 [1 month post-last vaccine dose] and n = 100 month 36 [30 months post-last vaccine dose; antibody persistence] samples). The results of the validation studies indicate that both optimized assays are accurate, specific, and precise over their respective quantifiable ranges. There was a strong linear association between the new and previous versions of both assays. Assay serostatus cutoffs for the redeveloped assays were established based on the bridging studies and, for the HPV-9 cLIA, further refined, based on additional data from HPV vaccine clinical studies so as to align the seropositivity rates between assay versions. IMPORTANCE Assay modernization is a key aspect of vaccine life cycle management. Thus, new, reoptimized versions of two 9vHPV immunoassays have been developed and validated for use in ongoing and future HPV vaccine clinical trials. These assays are suitable for use in high-throughput testing for HPV antibodies in serum samples. Bridging to previous versions of the assays allows for the continuous monitoring of immune responses across assay versions, including in immunogenicity studies that involve new populations as well as long-term follow-up studies.
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Infecciones por Papillomavirus , Vacunas contra Papillomavirus , Humanos , Virus del Papiloma Humano , Infecciones por Papillomavirus/diagnóstico , Infecciones por Papillomavirus/prevención & control , Anticuerpos Antivirales , Vacunación , PapillomaviridaeRESUMEN
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.
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Infecciones Neumocócicas , Streptococcus pneumoniae , Niño , Humanos , Streptococcus pneumoniae/genética , Serogrupo , Infecciones Neumocócicas/diagnóstico , Infecciones Neumocócicas/prevención & control , Serotipificación/métodos , Vacunas Neumococicas , Antígenos Bacterianos/genética , Oído MedioRESUMEN
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.
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Virus Sincitiales Respiratorios , Vacunas , Anticuerpos Neutralizantes , Calibración , Pruebas de Neutralización , Organización Mundial de la SaludRESUMEN
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.
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Bacteriemia , Infecciones Comunitarias Adquiridas , Infecciones Neumocócicas , Neumonía , Anciano , Niño , Preescolar , Humanos , Infecciones Neumocócicas/epidemiología , Vacunas Neumococicas , Polisacáridos , Serogrupo , Streptococcus pneumoniaeRESUMEN
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.
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Biopelículas , Infecciones Relacionadas con Catéteres/prevención & control , Cateterismo Venoso Central/efectos adversos , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Staphylococcus epidermidis/inmunología , Animales , Femenino , Modelos Animales , Ratas , Ratas Sprague-DawleyRESUMEN
Gamma irradiation (GI) is included in the CDC guidance on inactivation procedures to render a group of select agents and toxins nonviable. The Ebola virus falls within this group because it potentially poses a severe threat to public health and safety. To evaluate the impact of GI at a target dose of 50 kGy on neutralizing antibody titers induced by the rVSVΔG-ZEBOV-GP vaccine (V920), we constructed a panel of 48 paired human serum samples (GI-treated versus non-GI-treated) from healthy participants selected from a phase 3 study of V920 (study V920-012; NCT02503202). Neutralizing antibody titers were determined using a validated plaque-reduction neutralization test. GI of sera from V920 recipients was associated with approximately 20% reduction in postvaccination neutralizing antibody titers. GI was not associated with any change in pre-vaccination neutralizing antibody titers.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra el Virus del Ébola/administración & dosificación , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Sueros Inmunes/efectos de la radiación , Anticuerpos Neutralizantes/análisis , Vacunas contra el Virus del Ébola/síntesis química , Ebolavirus/patogenicidad , Voluntarios Sanos , Fiebre Hemorrágica Ebola/sangre , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/virología , Humanos , Sueros Inmunes/química , Inmunogenicidad Vacunal , Pruebas de Neutralización , Estudios Prospectivos , Vacunación/métodos , Vesiculovirus/química , Vesiculovirus/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
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.
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Pruebas de Neutralización/métodos , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Vacunas contra Virus Sincitial Respiratorio/inmunología , Virus Sincitiales Respiratorios/fisiología , Anciano de 80 o más Años , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Chlorocebus aethiops , Diagnóstico por Imagen , Ensayos Analíticos de Alto Rendimiento , Humanos , Lactante , Recién Nacido , Miniaturización , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de Tiempo , Células Vero , Ensayo de Placa ViralRESUMEN
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.
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Bioensayo/métodos , Vacunas Neumococicas/química , Streptococcus pneumoniae/química , Humanos , SerogrupoRESUMEN
The recombinant vesicular stomatitis virus - Zaire Ebola virus envelope glycoprotein (rVSVΔG-ZEBOV-GP) vaccine is a live recombinant vesicular stomatitis virus (VSV) where the VSV G protein is replaced with ZEBOV-GP. To better understand the immune response after receiving the rVSVΔG-ZEBOV-GP vaccine, the current analyses evaluated different definitions of seroresponse that differentiate vaccine and placebo recipients enrolled in a placebo-controlled clinical trial (PREVAIL; NCT02344407) in which a subset of the study participants had elevated baseline titers. Alternative values for serostatus cutoff (SSCO; 200-500 EU/mL) and/or fold rise (two- to five-fold) were applied to compare their ability to distinguish between participants receiving rVSVΔG-ZEBOV-GP or placebo. The results indicate that an SSCO of 200 EU/mL can be used to define seropositivity at baseline (i.e. pre-vaccination). The use of dual criteria of the same SSCO (200 EU/mL) together with a two-fold rise in antibody level from baseline provided the definition of seroresponse that maximized the statistical significance between vaccine recipients and placebo recipients post-vaccination. Clinical trial registration: NCT02344407.
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Vacunas contra el Virus del Ébola , Ebolavirus , Fiebre Hemorrágica Ebola , Estomatitis Vesicular , Animales , Anticuerpos Antivirales , República Democrática del Congo , Fiebre Hemorrágica Ebola/prevención & control , Humanos , Envoltura ViralRESUMEN
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.
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Bioensayo/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Vacunas Neumococicas/inmunología , Organización Mundial de la Salud/organización & administración , HumanosRESUMEN
The RSV Fusion (F) protein is a target for neutralizing antibody responses and is a focus for vaccine discovery; however, the process of RSV entry requires F to adopt a metastable prefusion form and transition to a more stable postfusion form, which displays less potent neutralizing epitopes. mRNA vaccines encode antigens that are translated by host cells following vaccination, which may allow conformational transitions similar to those observed during natural infection to occur. Here we evaluate a panel of chemically modified mRNA vaccines expressing different forms of the RSV F protein, including secreted, membrane associated, prefusion-stabilized, and non-stabilized structures, for conformation, immunogenicity, protection, and safety in rodent models. Vaccination with mRNA encoding native RSV F elicited antibody responses to both prefusion- and postfusion-specific epitopes, suggesting that this antigen may adopt both conformations in vivo. Incorporating prefusion stabilizing mutations further shifts the immune response toward prefusion-specific epitopes, but does not impact neutralizing antibody titer. mRNA vaccine candidates expressing either prefusion stabilized or native forms of RSV F protein elicit robust neutralizing antibody responses in both mice and cotton rats, similar to levels observed with a comparable dose of adjuvanted prefusion stabilized RSV F protein. In contrast to the protein subunit vaccine, mRNA-based vaccines elicited robust CD4+ and CD8+ T-cell responses in mice, highlighting a potential advantage of the technology for vaccines requiring a cellular immune response for efficacy.
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Human papillomavirus (HPV) DNA genotyping is an essential test to establish efficacy in HPV vaccine clinical trials and HPV prevalence in natural history studies. A number of HPV DNA genotyping methods have been cited in the literature, but the comparability of the outcomes from the different methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons were evaluated for 15 HPV types common in both assays. A slightly higher proportion of samples tested positive by the HPV multiplex PCR than by the HCII-LiPA v2 assay. The sensitivities of the multiplex PCR assay relative to those of the HCII-LiPA v2 assay for HPV types 6, 11, 16, and 18, for example, were 0.806, 0.646, 0.920, and 0.860, respectively; the specificities were 0.986, 0.998, 0.960, and 0.986, respectively. The overall comparability of detection of the 15 HPV types was quite high. Analyses of DNA genotype testing compared to cytology results demonstrated a significant discordance between cytology-negative (normal) and HPV DNA-positive results. This demonstrates the challenges of cytological diagnosis and the possibility that a significant number of HPV-infected cells may appear cytologically normal.
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Hibridación de Ácido Nucleico/métodos , Papillomaviridae/clasificación , Papillomaviridae/genética , Reacción en Cadena de la Polimerasa/métodos , Adolescente , Adulto , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/métodos , Papillomaviridae/aislamiento & purificación , Sensibilidad y Especificidad , Adulto JovenRESUMEN
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.
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Vacuna contra la Varicela , Vacunas contra el Virus del Herpes Simple , Herpesvirus Humano 3/clasificación , Herpesvirus Humano 3/genética , Reacción en Cadena de la Polimerasa/métodos , Simplexvirus/clasificación , Simplexvirus/genética , Cartilla de ADN , Diagnóstico Diferencial , Herpes Simple/diagnóstico , Herpes Zóster/diagnóstico , Herpesvirus Humano 3/aislamiento & purificación , Humanos , Reacción en Cadena de la Polimerasa/normas , Polimorfismo de Nucleótido Simple , Estándares de Referencia , Sensibilidad y Especificidad , Simplexvirus/aislamiento & purificación , Vacunas , Globinas beta/genéticaRESUMEN
rVSVΔG-ZEBOV-GP vaccine is a live recombinant (r) vesicular stomatitis virus (VSV), where the VSV G protein is replaced with the Zaire Ebola virus (ZEBOV) glycoprotein (GP). For vaccine immunogenicity testing, clinical trial sera collected during an active ZEBOV outbreak underwent gamma irradiation (GI) before testing in biosafety level 2 laboratories to inactivate possible wild-type ZEBOV. Before irradiating pivotal trial samples, two independent studies evaluated the impact of GI (50 kGy) on binding ZEBOV-GP (ELISA) antibodies against rVSVΔG-ZEBOV-GP, using sera from a North American phase 1 study. Gamma irradiation was associated with slightly higher antibody concentrations in pre-vaccination samples and slightly lower concentrations postvaccination. Results indicate that GI is a viable method for treating samples from regions where filoviruses are endemic, with minor effects on antibody titers. The impact of GI on immunogenicity analyses should be considered when interpreting data from irradiated specimens.
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Anticuerpos Antivirales/efectos de la radiación , Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/metabolismo , Rayos gamma , Suero/efectos de la radiación , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/fisiología , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Humanos , Glicoproteínas de Membrana , Vacunación , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
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.
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Anticuerpos Antivirales/sangre , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Inmunoensayo/métodos , Virus de la Influenza A/inmunología , Virus de la Influenza B/inmunología , Vacunas contra la Influenza/inmunología , Proteínas de la Matriz Viral/inmunología , Adolescente , Adulto , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Femenino , Humanos , Lactante , Macaca mulatta , Masculino , Péptidos/inmunología , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
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.
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Anticuerpos Neutralizantes/análisis , Anticuerpos Antivirales/análisis , Virus del Dengue/inmunología , Ensayos Analíticos de Alto Rendimiento/normas , Imagen Molecular/métodos , Pruebas de Neutralización/normas , Análisis de la Célula Individual/métodos , Animales , Antraquinonas/química , Chlorocebus aethiops , Dengue/inmunología , Dengue/prevención & control , Dengue/virología , Vacunas contra el Dengue/análisis , Virus del Dengue/aislamiento & purificación , Colorantes Fluorescentes/química , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Células Vero , Carga Viral , Ensayo de Placa ViralRESUMEN
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.
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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.
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Adenoviridae/genética , Vectores Genéticos/análisis , Reacción en Cadena de la Polimerasa/métodos , Vacunas de ADN/genética , Esparcimiento de Virus/fisiología , Adenoviridae/aislamiento & purificación , ADN Viral/aislamiento & purificación , Productos del Gen gag/análisis , Productos del Gen gag/genética , Terapia Genética/métodos , Vectores Genéticos/orina , Humanos , Mucosa Respiratoria/virología , Sensibilidad y Especificidad , Manejo de Especímenes/métodos , Vacunas de ADN/análisisRESUMEN
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).
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Adenoviridae/genética , Adenoviridae/inmunología , Fosfatasa Alcalina/genética , Vectores Genéticos/inmunología , Pruebas de Neutralización/métodos , Vacunas de ADN/inmunología , Adenoviridae/clasificación , Adulto , Anciano , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/inmunología , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/inmunología , Línea Celular , Estudios Epidemiológicos , Femenino , Técnicas de Transferencia de Gen , Genes Reporteros , Humanos , Macaca mulatta , Masculino , Persona de Mediana EdadRESUMEN
In the clinical trials of the quadrivalent human papillomavirus (qHPV) vaccine, antibodies were measured by a competitive Luminex immunoassay (HPV-4 cLIA). A nine-valent HPV (9vHPV) vaccine targeting the types in the qHPV vaccine (HPV6/11/16/18), as well as 5 of the next most frequent HPV types found in cervical cancers worldwide (HPV31/33/45/52/58) is under development. To support the 9vHPV vaccine program, a nine-multiplexed cLIA (HPV-9 cLIA) was developed. Antibody titers were determined in a competitive format, where type-specific phycoerythrin (PE)-labeled, neutralizing mAbs (mAbs-PE) compete with an individual's serum antibodies for binding to conformationally sensitive, neutralizing epitopes on the VLPs. Neutralizing antibody levels were quantitated against a reference standard - a pool of sera from 6 Rhesus macaques that were immunized with the 9vHPV vaccine. Specificity of the mAbs was assessed by measuring their individual binding capacities to the type-specific and non-type-specific VLPs at alternative concentrations of the mAbs. Antibody assignments to the HPV-9 cLIA reference standard for HPV6/11/16/18 were determined to provide for a measure of consistency in serostatus assignment between the HPV-4 and HPV-9 cLIAs. Antibody assignments to the HPV-9 reference standard for HPV31/33/45/52/58 were obtained by calibration to HPV11 using a direct binding IgG assay. For each HPV VLP type, the cross-reactivity of the mAb-PEs in the HPV-9 cLIA was <1% (i.e., the mAb-PEs result in <1% non-specific binding). The antibody concentrations assigned to the HPV-9 cLIA reference standard for types 6/11/16/18/31/33/45/52/58 were 3,817, 2,889, 23,061, 5,271, 3,942, 2,672, 1,489, 1274, and 2263 mMU/mL, respectively.