RESUMO
In this paper, we describe development of a high-throughput, highly sensitive method based on Lab Chip CGE-SDS platform for purity determination and characterization of virus-like particle (VLP) vaccines. A capillary gel electrophoresis approach requiring about 41 s per sample for analysis and demonstrating sensitivity to protein initial concentrations as low as 20 µg/mL, this method has been used previously to evaluate monoclonal antibodies, but this application for lot release assay of VLPs using this platform is unique. The method was qualified and shown to be accurate for the quantitation of VLP purity. Assay repeatability was confirmed to be less than 2% relative standard deviation of the mean (% RSD) with interday precision less than 2% RSD. The assay can evaluate purified VLPs in a concentration range of 20-249 µg/mL for VEE and 20-250 µg/mL for EEE and WEE VLPs.
Assuntos
Eletroforese Capilar/métodos , Vírus da Encefalite Equina do Leste/isolamento & purificação , Vírus da Encefalite Equina Venezuelana/isolamento & purificação , Vírus da Encefalite Equina do Oeste/isolamento & purificação , Ensaios de Triagem em Larga Escala/métodos , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Fluorescência , Corantes Fluorescentes/química , Humanos , Sensibilidade e Especificidade , Vacinas de Partículas Semelhantes a Vírus/químicaRESUMO
The relative conservation of the influenza hemagglutinin (HA) stem compared to that of the immunodominant HA head makes the HA stem an attractive target for broadly protective influenza vaccines. Here we report the first-in-human, dose-escalation, open-label trial (NCT04579250) evaluating an unadjuvanted group 2 stabilized stem ferritin nanoparticle vaccine based on the H10 A/Jiangxi-Donghu/346/2013 influenza HA, H10ssF, in healthy adults. Participants received a single 20 mcg dose (n = 3) or two 60 mcg doses 16 weeks apart (n = 22). Vaccination with H10ssF was safe and well tolerated with only mild systemic and local reactogenicity reported. No serious adverse events occurred. Vaccination significantly increased homologous H10 HA stem binding and neutralizing antibodies at 2 weeks after both first and second vaccinations, and these responses remained above baseline at 40 weeks. Heterologous H3 and H7 binding antibodies also significantly increased after each vaccination and remained elevated throughout the study. These data indicate that the group 2 HA stem nanoparticle vaccine is safe and induces stem-directed binding and neutralizing antibodies.
RESUMO
Cross-reactive neutralizing antibodies (NAbs) are found in the sera of many HIV-1-infected individuals, but the virologic basis of their neutralization remains poorly understood. We used knowledge of HIV-1 envelope structure to develop antigenically resurfaced glycoproteins specific for the structurally conserved site of initial CD4 receptor binding. These probes were used to identify sera with NAbs to the CD4-binding site (CD4bs) and to isolate individual B cells from such an HIV-1-infected donor. By expressing immunoglobulin genes from individual cells, we identified three monoclonal antibodies, including a pair of somatic variants that neutralized over 90% of circulating HIV-1 isolates. Exceptionally broad HIV-1 neutralization can be achieved with individual antibodies targeted to the functionally conserved CD4bs of glycoprotein 120, an important insight for future HIV-1 vaccine design.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Especificidade de Anticorpos , Linfócitos B/imunologia , Sítios de Ligação de Anticorpos , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Reações Cruzadas , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Genes de Cadeia Pesada de Imunoglobulina , Genes de Cadeia Leve de Imunoglobulina , Anticorpos Anti-HIV/isolamento & purificação , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
During Caenorhabditis elegans ovulation, the somatic gonad integrates signals from germ cells and propels a mature oocyte into the spermatheca for fertilization. Previous work suggests that phosphoinositide signaling plays important roles in C. elegans fertility. To fully understand inositol-1,4,5-trisphosphate (IP(3)) signaling in ovulation, we have examined the function of phosphatidylinositol-4-phosphate 5' kinase (PIP5K) in C. elegans. Our results show that the C. elegans PIP5K homolog, ppk-1, is essential for ovulation in C. elegans; ppk-1 is mainly expressed in somatic gonad, and depletion of ppk-1 expression causes defective ovulation, reduced gonad sheath contractility, and sterility. Increased IP(3) signaling compensates for ppk-1 (RNAi)-induced sterility, suggesting that ppk-1 is linked to IP(3) signaling. These results demonstrate that ppk-1 plays an essential role in IP(3) signaling and cytoskeleton organization in somatic gonad.