Publications by authors named "Daniela Verthelyi"

72 Publications

Lessons learned: new insights on the role of cytokines in COVID-19.

Nat Immunol 2021 04;22(4):404-411

Laboratory of Immunology, Division of Biotechnology Review and Research-III, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41590-021-00901-9DOI Listing
April 2021

2020 White Paper on Recent Issues in Bioanalysis: Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation ( - Recommendations on Immunogenicity Assay Strategies, NAb Assays, Biosimilars and FDA/EMA Immunogenicity Guidance/Guideline, Gene & Cell Therapy and Vaccine Assays).

Bioanalysis 2021 Mar 3;13(6):415-463. Epub 2021 Feb 3.

Intellia Therapeutics, Cambridge, MA, USA.

The 14 edition of the Workshop on Recent Issues in Bioanalysis (14 WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14 WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4155/bio-2021-0007DOI Listing
March 2021

2020 White Paper on Recent Issues in Bioanalysis: BAV Guidance, CLSI H62, Biotherapeutics Stability, Parallelism Testing, CyTOF and Regulatory Feedback ( - Recommendations on Biotherapeutics Stability, PK LBA Regulated Bioanalysis, Biomarkers Assays, Cytometry Validation & Innovation - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

Bioanalysis 2021 Mar 29;13(5):295-361. Epub 2021 Jan 29.

Health Canada, Ottawa, ON, Canada.

The 14 edition of the Workshop on Recent Issues in Bioanalysis (14 WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14 WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4155/bio-2021-0005DOI Listing
March 2021

Long-term persistence of infectious Zika virus: Inflammation and behavioral sequela in mice.

PLoS Pathog 2020 12 10;16(12):e1008689. Epub 2020 Dec 10.

US Food and Drug Administration, Office of Biotechnology Products, Silver Spring, Maryland, United States of America.

The neurodevelopmental defects associated with ZIKV infections early in pregnancy are well documented, however the potential defects and long-term consequences associated with milder infections in late pregnancy and perinatal period are less well understood. To model these, we challenged 1 day old (P1) immunocompetent C57BL/6 mice with ZIKV. The animals developed a transient neurological syndrome including unsteady gait, kinetic tremors, severe ataxia and seizures 10-15 days post-infection (dpi) but symptoms subsided after a week, and most animals survived. Despite apparent recovery, MRI of convalescent mice show reduced cerebellar volume that correlates with altered coordination and motor function as well as hyperactivity and impulsivity. Persistent mRNA levels of pro-inflammatory genes including Cd80, Il-1α, and Ifn-γ together with Cd3, Cd8 and perforin (PrfA), suggested persistence of low-grade inflammation. Surprisingly, the brain parenchyma of convalescent mice harbor multiple small discrete foci with viral antigen, active apoptotic processes in neurons, and cellular infiltrates, surrounded by activated astrocytes and microglia as late as 1-year post-infection. Detection of negative-sense strand viral RNA and isolation of infectious virus derived from these convalescent mice by blinded passage in Vero cells confirmed long-term persistence of replicating ZIKV in CNS of convalescent mice. Although the infection appears to persist in defined reservoirs within CNS, the resulting inflammation could increase the risk of neurodegenerative disorders. This raises concern regarding possible long-term effects in asymptomatic children exposed to the virus and suggests that long-term neurological and behavioral monitoring as well as anti-viral treatment to clear virus from the CNS may be useful in patients exposed to ZIKV at an early age.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1008689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728251PMC
December 2020

The dynamic changes in cytokine responses in COVID-19: a snapshot of the current state of knowledge.

Nat Immunol 2020 10;21(10):1146-1151

Laboratory of Immunology, Division of Biotechnology Review and Research-III, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41590-020-0779-1DOI Listing
October 2020

CpG Oligonucleotides Protect Mice From Alphavirus Encephalitis: Role of NK Cells, Interferons, and TNF.

Front Immunol 2020 18;11:237. Epub 2020 Feb 18.

Division of Biotechnology Review and Research-III, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States.

Arboviruses including alphavirus are responsible for most emerging infectious diseases worldwide. Recent outbreaks of chikungunya virus serve as a stark reminder to their pathogenic potential. There are no vaccines or therapeutics currently available to contain alphavirus outbreaks. In this study we evaluated the effect of immunomodulatory CpG ODN on the clinical progression of neurotropic Sindbis virus infection. Neonatal C57Bl-6 mice challenged with Sindbis virus AR339 (25 PFU Subcutaneous) infect neurons in the CNS leading to the development of ataxia, seizures, paralysis, and death. We show that systemic administration of CpG ODN modulates the cytokine and chemokine gene expression levels in the CNS and ultimately protects neonatal mice from lethal neurotropic infection. The protection conferred by CpG ODN is controlled by innate immune response and T and B cells were dispensable. Further, protection required Type I, Type II interferons, and TNF as well as functional NK cells, but did not involve iNOS. This study confirms that administration of innate immune modulators can be used as a strategy to boost host innate immune responses and protect against neurotropic viruses reducing their pathogenic footprint.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.00237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040238PMC
March 2021

CpG ODN D35 improves the response to abbreviated low-dose pentavalent antimonial treatment in non-human primate model of cutaneous leishmaniasis.

PLoS Negl Trop Dis 2020 02 28;14(2):e0008050. Epub 2020 Feb 28.

Laboratory of Immunology, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.

Cutaneous leishmaniasis (CL) affects the lives of 0.7-1 million people every year causing lesions that take months to heal. These lesions can result in disfiguring scars with psychological, social and economic consequences. Antimonials are the first line of therapy for CL, however the treatment is lengthy and linked to significant toxicities; further, its efficacy is variable and resistant parasites are emerging. Shorter or lower dose antimonial treatment regimens, which would decrease the risk of adverse events and improve patient compliance, have shown reduced efficacy and further increase the risk emergence of antimonial-resistant strains. The progression of lesions in CL is partly determined by the immune response it elicits, and previous studies showed that administration of immunomodulatory type D CpG ODNs, magnifies the immune response to Leishmania and reduces lesion severity in nonhuman primates (NHP) challenged with Leishmania major or Leishmania amazonensis. Here we explored whether the addition of a single dose of immunomodulating CpG ODN D35 augments the efficacy of a short-course, low-dose pentavalent antimonial treatment regimen. Results show that macaques treated with D35 plus 5mg/kg sodium stibogluconate (SbV) for 10 days had smaller lesions and reduced time to re-epithelization after infection with Leishmania major. No toxicities were evident during the studies, even at doses of D35 10 times higher than those used in treatment. Critically, pentavalent antimonial treatment did not modify the ability of D35 to induce type I IFNs. The findings support the efficacy of D35 as adjuvant therapy for shorter, low dose pentavalent antimonial treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0008050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075640PMC
February 2020

SampPick: Selection of a Cohort of Subjects Matching a Population HLA Distribution.

Front Immunol 2019 20;10:2894. Epub 2019 Dec 20.

Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, United States.

Immune responses to therapeutic proteins and peptides can adversely affect their safety and efficacy; consequently, immunogenicity risk-assessments are part of the development, licensure and clinical use of these products. In most cases the development of anti-drug antibodies is mediated by T cells which requires antigen presentation by Major Histocompatibility Complex Class II (MHCII) molecules (also called Human Leucocyte Antigen, HLA in humans). Immune responses to many protein therapeutics are thus HLA-restricted and it is important that the distribution of HLA variants used in the immunogenicity assessments provides adequate coverage of the target population. Due to biases inherent to the collection of samples in a blood bank or donor pool, simple random sampling will not achieve a truly representative sample of the population of interest. To help select a donor cohort we introduce SampPick, an implementation of simulated annealing which optimizes cohort selection to closely match the frequency distribution of a target population or subpopulation. With inputs of a target background frequency distribution for a population and a set of available, HLA-typed donors, the algorithm will iteratively create a cohort of donors of a user selected size that will closely match the target population rather than a random sample. In addition to optimizing the HLA types of donor cohorts, the software presented can be used to optimize donor cohorts for any other biallelic or monoallelic trait.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933600PMC
November 2020

2019 White Paper On Recent Issues in Bioanalysis: FDA BMV Guidance, ICH M10 BMV Guideline and Regulatory Inputs ( - Recommendations on 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and Regulatory Agencies' Input on Bioanalysis, Biomarkers and Immunogenicity).

Bioanalysis 2019 Dec 9;11(23):2099-2132. Epub 2019 Dec 9.

US FDA, Silver Spring, MD, USA.

The 2019 13 Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA on 1-5 April 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations on the 2018 FDA BMV guidance, 2019 ICH M10 BMV draft guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy. Part 1 (Innovation in small molecules and oligonucleotides and mass spectrometry method development strategies for large molecules bioanalysis) and Part 3 (New insights in biomarker assay validation, current and effective strategies for critical reagent management, flow cytometry validation in drug discovery and development and CLSI H62, interpretation of the 2019 FDA immunogenicity guidance and gene therapy bioanalytical challenges) are published in volume 10 of , issues 22 and 24 (2019), respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4155/bio-2019-0270DOI Listing
December 2019

2019 White Paper on Recent Issues in Bioanalysis: FDA Immunogenicity Guidance, Gene Therapy, Critical Reagents, Biomarkers and Flow Cytometry Validation (Part 3 - Recommendations on 2019 FDA Immunogenicity Guidance, Gene Therapy Bioanalytical Challenges, Strategies for Critical Reagent Management, Biomarker Assay Validation, Flow Cytometry Validation & CLSI H62).

Bioanalysis 2019 Dec 10;11(24):2207-2244. Epub 2019 Dec 10.

Janssen R&D, Spring House, PA, USA.

The 2019 13 Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches. This 2019 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2019 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers New Insights in Biomarker Assay Validation, Current & Effective Strategies for Critical Reagent Management, Flow Cytometry Validation in Drug Discovery & Development & CLSI H62, Interpretation of the 2019 FDA Immunogenicity Guidance and Gene Therapy Bioanalytical Challenges. Part 1 (Innovation in Small Molecules and Oligonucleotides & Mass Spectrometry Method Development Strategies for Large Molecule Bioanalysis) and Part 2 (Recommendations on the 2018 FDA BMV Guidance, 2019 ICH M10 BMV Draft Guideline and regulatory agencies' input on bioanalysis, biomarkers, immunogenicity and gene therapy) are published in volume 11 of , issues 22 and 23 (2019), respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4155/bio-2019-0271DOI Listing
December 2019

Pseudovirus rVSVΔG-ZEBOV-GP Infects Neurons in Retina and CNS, Causing Apoptosis and Neurodegeneration in Neonatal Mice.

Cell Rep 2019 02;26(7):1718-1726.e4

Division of Biotechnology Review and Research-III, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA. Electronic address:

Zaire Ebola virus (ZEBOV) survivors experience visual and CNS sequelae that suggests the ZEBOV glycoprotein can mediate neurotropism. Replication-competent rVSVΔG-ZEBOV-GP vaccine candidate is generally well tolerated; however, its potential neurotropism requires careful study. Here, we show that a single inoculation of rVSVΔG-ZEBOV-GP virus in neonatal C57BL/6 mice results in transient viremia, neurological symptoms, high viral titers in eyes and brains, and death. rVSVΔG-ZEBOV-GP infects the inner layers of the retina, causing severe retinitis. In the cerebellum, rVSVΔG-ZEBOV-GP infects neurons in the granular and Purkinje layers, resulting in progressive foci of apoptosis and neurodegeneration. The susceptibility to infection is not due to impaired type I IFN responses, although MDA5, IFNβ, and IFNAR1 mice have accelerated mortality. However, boosting interferon levels by co-administering poly(I:C) reduces viral titers in CNS and improves survival. Although these data should not be directly extrapolated to humans, they challenge the hypothesis that VSV-based vaccines are non-neurotropic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.01.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748882PMC
February 2019

Multiplexed Gene Expression as a Characterization of Bioactivity for Interferon Beta (IFN-β) Biosimilar Candidates: Impact of Innate Immune Response Modulating Impurities (IIRMIs).

AAPS J 2019 02 8;21(2):26. Epub 2019 Feb 8.

Laboratory of Immunology, Division of Biotechnology Review and Research III, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Building 52-Room 2112, 10903 New Hampshire Av., Silver Spring, Maryland, 20993, USA.

Recombinant human interferon-β (rhIFN-β) therapy is the first-line treatment in relapsing-remitting forms of multiple sclerosis (MS). The mechanism of action underlying its therapeutic activity is only partially understood as IFN-βs induce the expression of over 1000 genes modifying multiple immune pathways. Currently, assessment of potency for IFN-β products is based on their antiviral effect, which is not linked to its therapeutic effect. Here, we explore the use of a multiplexed gene expression system to more broadly characterize IFN-β bioactivity. We find that MM6 cells stimulated with US-licensed rhIFN-βs induce a dose-dependent and reproducible pattern of gene expression. This pattern of gene expression was used to compare the bioactivity profile of biosimilar candidates with the corresponding US-licensed rhIFN-β products, Rebif and Betaseron. While the biosimilar candidate for Rebif matched the pattern of gene expression, there were differences in the expression of a subset of interferon-inducible genes including CXCL-10, CXCL-11, and GBP1 induced by the biosimilar candidate for Betaseron. Assessment of product impurities in both products suggested that the difference was rooted in the presence of innate immune response modulating impurities (IIRMIs) in the licensed product. These studies indicate that determining the expression levels for an array of reporter genes that monitor different pathways can be informative as part of the demonstration of biosimilarity or comparability for complex immunomodulatory products such as IFN-β, but the sensitivity of each gene to potential impurities in the product should be examined to fully understand the results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1208/s12248-019-0300-7DOI Listing
February 2019

IL-6 Impairs Vaccine Responses in Neonatal Mice.

Front Immunol 2018 20;9:3049. Epub 2018 Dec 20.

Division of Bacterial Allergenic and Parasitic Diseases, Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, United States.

The inability of infants to mount proper follicular helper T (T) cell response renders this age group susceptible to infectious diseases. Initial instruction of T cells by antigen presenting cells and subsequent differentiation into T cells are controlled by T cell receptor signal strength, co-stimulatory molecules and cytokines such as IL-6 and IL-21. In immunized adults, IL-6 promotes T development by increasing the expression of CXCR5 and the T master transcription factor, B cell lymphoma 6. Underscoring the importance of IL-6 in T generation, we found improved antibody responses accompanied by increased T cells and decreased follicular regulatory helper T (T) cells, a Foxp3 expressing inhibitory CD4 T cell occupying the germinal center (GC), when a tetanus toxoid conjugated pneumococcal polysaccharide type 14 vaccine was injected in adult mice together with IL-6. Paradoxically, in neonates IL-6 containing PPS14-TT vaccine suppressed the already impaired T development and antibody responses in addition to increasing T cell population. Supporting the diminished T development, we detected lower frequency of phospho-STAT-3 T in immunized neonatal T cells after IL-6 stimulation than adult cells. Moreover, IL-6 induced more phospho-STAT-3 T in neonatal cells than adult cells. We also measured lower expression of IL-6R on T cells and higher expression on T cells in neonatal cells than adult cells, a possible explanation for the difference in IL-6 induced signaling in different age groups. Supporting the flow cytometry findings, microscopic examination revealed the localization of T cells in the splenic interfollicular niches of immunized adult mice compared to splenic follicles in neonatal mice. In addition to the limitations in the formation of IL-21 producing T cells, neonatal mice GC B cells also expressed lower levels of IL-21R in comparison to the adult mice cells. These findings point to diminished IL-6 activity on neonatal T cells as an underlying mechanism of the increased T T ratio in immunized neonatal mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2018.03049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307459PMC
November 2019

2018 White Paper on Recent Issues in Bioanalysis: focus on immunogenicity assays by hybrid LBA/LCMS and regulatory feedback (Part 2 - PK, PD & ADA assays by hybrid LBA/LCMS & regulatory agencies' inputs on bioanalysis, biomarkers and immunogenicity).

Bioanalysis 2018 Dec 29;10(23):1897-1917. Epub 2018 Nov 29.

F Hoffmann-La Roche, Basel, Switzerland.

The 2018 12 Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies' input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of , issues 22 and 24 (2018), respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4155/bio-2018-0285DOI Listing
December 2018

2018 White Paper on Recent Issues in Bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 - LBA/cell-based assays: immunogenicity, biomarkers and PK assays).

Bioanalysis 2018 Dec 29;10(24):1973-2001. Epub 2018 Nov 29.

Amador Bioscience, Pleasanton, CA, USA (formerly of OncoMed, Redwood City, CA, USA).

The 2018 12 Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of , issues 22 and 23 (2018), respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4155/bio-2018-0287DOI Listing
December 2018

2018 White Paper on Recent Issues in Bioanalysis: 'A global bioanalytical community perspective on last decade of incurred samples reanalysis (ISR)' (Part 1 - small molecule regulated bioanalysis, small molecule biomarkers, peptides & oligonucleotide bioanalysis).

Bioanalysis 2018 Nov 29;10(22):1781-1801. Epub 2018 Nov 29.

UK MHRA, London, UK.

The 2018 12 Workshop on Recent Issues in Bioanalysis (12th WRIB) took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LC-MS, hybrid ligand binding assay (LBA)/LC-MS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers the recommendations for LC-MS for small molecules, peptides, oligonucleotides and small molecule biomarkers. Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) and Part 3 (large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays) are published in volume 10 of Bioanalysis, issues 23 and 24 (2018), respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4155/bio-2018-0268DOI Listing
November 2018

Aggregates of IVIG or Avastin, but not HSA, modify the response to model innate immune response modulating impurities.

Sci Rep 2018 07 31;8(1):11477. Epub 2018 Jul 31.

Division of Biotechnology Review and Research-III, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, 20993, USA.

Therapeutic proteins can induce immune responses that affect their safety and efficacy. Product aggregates and innate immune response modulating impurities (IIRMI) are risk factors of product immunogenicity. In this study, we use Intravenous Immunoglobulin (IVIG), Avastin, and Human Serum Albumin (HSA) to explore whether increased aggregates activate innate immune cells or modify the response to IIRMI. We show that increased aggregates (shaken or stirred) in IVIG and Avastin, but not HSA, induced activation of MAPKs (pp38, pERK and pJNK) and transcription of immune-related genes including IL8, IL6, IL1β, CSF1, CCL2, CCL7, CCL3, CCL24, CXCL2, IRAK1, EGR2, CEBPβ, PPARg and TNFSF15 in human PBMC. The immunomodulatory effect was primarily mediated by FcγR, but not by TLR. Interestingly, increased aggregates in IVIG or Avastin magnified innate immune responses to TLR2/4 agonists, but diminished responses to TLR3/9 agonists. This study shows that IIRMI and aggregates can modify the activity of immune cells potentially modifying the milieu where the products are delivered highlighting the complex interplay of different impurities on product immunogenicity risk. Further, we show that aggregates could modify the sensitivity of PBMC-based assays designed to detect IIRMI. Understanding and managing immunogenicity risk is a critical component of product development and regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-29850-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6068171PMC
July 2018

ZIKA virus infection causes persistent chorioretinal lesions.

Emerg Microbes Infect 2018 May 25;7(1):96. Epub 2018 May 25.

Division of Biotechnology Review and Research-III, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, 20993, USA.

Zika-infected patients can have eye involvement ranging from mild conjunctivitis to severe chorioretinal lesions, however the possible long-term sequelae of infection and timeline to recovery remain unknown. Here we describe the partial recovery of chorioretinal lesions in an immunocompetent patient diagnosed with bilateral posterior uveitis associated with Zika infection and show that some lesions resolved with focal atrophy evident as pigmentary changes on funduscopy. To better understand the progression of the lesions and correlate the changes in fundus imaging with local viral load, immune responses, and retinal damage, we developed a symptomatic mouse model of ocular Zika virus infection. Imaging of the fundus revealed multiple hypopigmentary patches indicative of chorioretinal degeneration as well as thinning of the retina that mirror the lesions in patients. Microscopically, the virus primarily infected the optic nerve, retinal ganglion cells, and inner nuclear layer cells, showing thinning of the outer plexiform layer. During acute infection, the eyes showed retinal layer disorganization, retinitis, vitritis, and focal choroiditis, with mild cellular infiltration and increased expression of tumor necrosis factor, interferon-γ, granzyme B, and perforin. Focal areas of gliosis and retinal degeneration persisted 60 dpi. The model recapitulates features of ZIKA infections in patients and should help elucidate the mechanisms underlying the damage to the eyes and aid in the development of effective therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41426-018-0096-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970181PMC
May 2018

Regulation of the maturation of human monocytes into immunosuppressive macrophages.

Blood Adv 2017 Dec 4;1(26):2510-2519. Epub 2017 Dec 4.

Cancer and Inflammation Program, National Cancer Institute, National Institutes of Health, Frederick MD; and.

Human monocytes differentiate into either proinflammatory or immunosuppressive macrophages in response to distinct stimuli. Results show that the Toll-like receptor 2/1 agonist PAM3 replicates the ability of macrophage colony-stimulating factor (M-CSF) to induce the preferential generation of immunosuppressive macrophages in vitro, an activity confirmed by in vivo studies of rhesus macaques. By comparing the gene expression pattern of monocytes treated with M-CSF vs PAM3, the pathways regulating macrophage maturation were identified. NF-κB and Akt were found to play a central role in the overall process of monocyte into macrophage differentiation. Pathways regulated by p38 MAPK and PTGS2 biased this process toward the generation of immunosuppressive rather than proinflammatory macrophages. ERK and JNK contribute to PAM3- but not M-CSF-driven monocyte maturation. These findings clarify the mechanisms underlying the generation of immunosuppressive macrophages and support the use of PAM3 in the treatment of autoimmune and inflammatory diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2017011221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728638PMC
December 2017

PF4-HIT antibody (KKO) complexes activate broad innate immune and inflammatory responses.

Thromb Res 2017 Nov 21;159:39-47. Epub 2017 Sep 21.

Food and Drug Administration, CDER, Division of Pharmaceutical Analysis, St Louis, MO 63110, United States. Electronic address:

Introduction: Heparin-induced thrombocytopenia (HIT) is an immune-mediated complication of heparin anticoagulation therapy resulting in thrombocytopenia frequently accompanied by thrombosis. Current evidence suggests that HIT is associated with antibodies developed in response to multi-molecular complexes formed by platelet factor 4 (PF4) bound to heparin or cell surface glycosaminoglycans. These antibody complexes activate platelets and monocytes typically through FcγRIIA receptors increasing the production of PF4, inflammatory mediators, tissue factor and thrombin. The influence of underlying events in HIT including complex-induced pro-inflammatory cell activation and structural determinants leading to local inflammatory responses are not fully understood.

Methods: The stoichiometry and complex component requirements were determined by incubating fresh peripheral blood mononuclear cells (PBMC) with different concentrations of unfractionated heparin (H), low molecular weight heparin (LMWH), PF4- and anti-PF4-H complex antibodies (KKO). Cytokine mRNA or protein were measured by qRT-PCR or Meso Scale Discovery technology, respectively. Gene expression profile analysis for 594 genes was performed using Nanostring technology and analyzed using Ingenuity Pathway Analysis software.

Results And Conclusions: The data show that antibodies magnify immune responses induced in PBMCs by PF4 alone or in complex with heparin or LMWH. We propose that following induction of HIT antibodies by heparin-PF4 complexes, binding of the antibodies to PF4 is sufficient to induce a local pro-inflammatory response which may play a role in the progression of HIT. In vitro assays using PBMCs may be useful in characterizing local inflammatory and innate immune responses induced by HIT antibodies in the presence of PF4 and different sources of heparins.

Fda Disclaimer: The findings and conclusions in this article are solely the responsibility of the authors and are not being formally disseminated by the Food and Drug Administration. Thus, they should not be construed to represent any Agency determination or policy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.thromres.2017.09.018DOI Listing
November 2017

Prior Exposure to Zika Virus Significantly Enhances Peak Dengue-2 Viremia in Rhesus Macaques.

Sci Rep 2017 09 5;7(1):10498. Epub 2017 Sep 5.

Uniformed Services University, Bethesda, MD, 20814, USA.

Structural and functional homologies between the Zika and Dengue viruses' envelope proteins raise the possibility that cross-reactive antibodies induced following Zika virus infection might enhance subsequent Dengue infection. Using the rhesus macaque model we show that prior infection with Zika virus leads to a significant enhancement of Dengue-2 viremia that is accompanied by neutropenia, lympocytosis, hyperglycemia, and higher reticulocyte counts, along with the activation of pro-inflammatory monocyte subsets and release of inflammatory mediators. Zika virus infection induced detectable Dengue cross-reactive serum IgG responses that significantly amplified after Dengue-2 virus infection. Serum from Zika virus immune animals collected prior to Dengue-2 infection showed significant capacity for in vitro antibody dependent enhancement of Dengue-1, 2, 3 and 4 serotypes suggesting that pre-existing immunity to Zika virus could potentially enhance infection by heterologous Dengue serotypes. Our results provide first in vivo evidence that prior exposure to Zika virus infection can enhance Dengue infection, which has implications for understanding pathogenesis and the development of vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-10901-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585353PMC
September 2017

Cell based assay identifies TLR2 and TLR4 stimulating impurities in Interferon beta.

Sci Rep 2017 09 5;7(1):10490. Epub 2017 Sep 5.

Laboratory of Immunology, Division of Biotechnology Review and Research III, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.

Immunogenicity can have devastating consequences on the safety and efficacy of therapeutic proteins. Therefore, evaluating and mitigating the risk of product immunogenicity is critical for the development these products. This study, showed that Betaseron and Extavia, which are reported to be more immunogenic among IFNβ products in clinical usage, contain residual innate immune response modulating impurities (IIRMIs) capable of activating NF-κB and induced expression of inflammatory mediators. These IIRMIs were undetectable in Rebif or Avonex. The stimulatory effect was attributed solely to IIRMIs because it was evident in murine cells lacking the interferon receptor (IFNAR). The IIRMIs in Betaseron and Extavia triggered NF-κB activation in HEK-293 cells bearing TLR2 and TLR4 in MyD88 dependent manner. Importantly, the IIRMIs in Betaseron induced up-regulation of IL-6, IL-1β, and ccl5 in the skin of IFNAR knock out mice following subcutaneous administration. This indicates that trace level IIRMIs in Betaseron could contribute to the higher immunogenicity rates seen in clinics. Together these data suggest that cell based assays can reveal subtle but clinically relevant differences in IIRMIs following manufacturing changes or between products with the same active ingredients but different manufacturing processes. Appreciating these differences may inform immunogenicity risk assessments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-09981-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585229PMC
September 2017

In Vivo Effect of Innate Immune Response Modulating Impurities on the Skin Milieu Using a Macaque Model: Impact on Product Immunogenicity.

J Pharm Sci 2017 03 4;106(3):751-760. Epub 2016 Dec 4.

Laboratory of Immunology, Division of Biotechnology Review and Research III, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration (FDA), Silver Spring, Maryland 20993. Electronic address:

Unwanted immune responses to therapeutic proteins can severely impact their safety and efficacy. Studies show that the presence of trace amounts of host cells and process-related impurities that stimulate pattern recognition receptors (PRR) can cause local inflammation and enhance product immunogenicity. Here we used purified PRR agonists as model impurities to assess the minimal level of individual innate immune response modulating impurities (IIRMIs) that could activate a local immune response. We show that levels of endotoxin as low as 10 pg (0.01 EU), 1 ng for polyinosinic:polycytidylic acid (PolyI:C), 100 ng for synthetic diacylated liopprotein, thiazoloquinolone compound, or muramyl dipeptide, 1 μg for flagellin or β-glucan, or 5 μg for CpG-oligodeoxynucleotide increased expression of genes linked to innate immune activation and inflammatory processes in the skin of rhesus macaques. Furthermore, spiking studies using rasburicase as a model therapeutic showed that the levels of PRR agonists that induced detectable gene upregulation in the skin were associated with increased immunogenicity for rasburicase. This study underscores the need for testing multiple IIRMIs in biologics, strengthening the connection between the local mRNA induction in skin, innate immune activation, and antibody development in primates, and provides an indication of the levels of IIRMI in therapeutic products that could impact product immunogenicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.xphs.2016.11.001DOI Listing
March 2017

Zika (PRVABC59) Infection Is Associated with T cell Infiltration and Neurodegeneration in CNS of Immunocompetent Neonatal C57Bl/6 Mice.

PLoS Pathog 2016 Nov 17;12(11):e1006004. Epub 2016 Nov 17.

Division of Biotechnology Review and Research-III, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.

The recent spread of Zika virus (ZIKV) and its association with increased rates of Guillain Barre and other neurological disorders as well as congenital defects that include microcephaly has created an urgent need to develop animal models to examine the pathogenesis of the disease and explore the efficacy of potential therapeutics and vaccines. Recently developed infection models for ZIKV utilize mice defective in interferon responses. In this study we establish and characterize a new model of peripheral ZIKV infection using immunocompetent neonatal C57BL/6 mice and compare its clinical progression, virus distribution, immune response, and neuropathology with that of C57BL/6-IFNAR KO mice. We show that while ZIKV infected IFNAR KO mice develop bilateral hind limb paralysis and die 5-6 days post-infection (dpi), immunocompetent B6 WT mice develop signs of neurological disease including unsteady gait, kinetic tremors, severe ataxia and seizures by 13 dpi that subside gradually over 2 weeks. Immunohistochemistry show viral antigen predominantly in cerebellum at the peak of the disease in both models. However, whereas IFNAR KO mice showed infiltration by neutrophils and macrophages and higher expression of IL-1, IL-6 and Cox2, B6 WT mice show a cellular infiltration in the CNS composed predominantly of T cells, particularly CD8+ T cells, and increased mRNA expression levels of IFNg, GzmB and Prf1 at peak of disease. Lastly, the CNS of B6 WT mice shows evidence of neurodegeneration predominantly in the cerebellum that are less prominent in mice lacking the IFN response possibly due to the difference in cellular infiltrates and rapid progression of the disease in that model. The development of the B6 WT model of ZIKV infection will provide insight into the immunopathology of the virus and facilitate assessments of possible therapeutics and vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1006004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113993PMC
November 2016

Early treatment with reverse transcriptase inhibitors significantly suppresses peak plasma IFNα in vivo during acute simian immunodeficiency virus infection.

Cell Immunol 2016 12 9;310:156-164. Epub 2016 Sep 9.

Uniformed Services University of the Health Sciences, Bethesda, MD, United States. Electronic address:

Innate interferons (IFN) are comprised of multiple Type I and III subtypes. The in vivo kinetics of subtype responses during human immunodeficiency virus (HIV) infection is not well defined. Using the acute simian immunodeficiency virus (SIV) infection model, we show that plasma IFNα levels peak at day 10 post-infection (pi) after which they rapidly declined. The mRNA expression of Type I and III IFN subtypes were significantly elevated in the lymph nodes (LN) at day 10 pi. Though the expression levels of all subtypes declined by day 14-31 pi, numerous subtypes remained elevated suggesting that ongoing viral replication in LN continues to drive induction of these subtypes. Interestingly, treatment with reverse transcriptase (RT) inhibitors at day 7 pi significantly suppressed plasma IFNα responses by day 10 pi that significantly correlated with cell-associated SIV DNA loads suggesting that RT byproducts such as viral DNA likely plays a role in driving IFN responses during acute SIV infection. Quantification of Type I and III subtype transcripts in sorted subsets of LN CD4+ and CD8+ T cells, CD14+/CD14- monocytes/macrophages, and total CD11c/CD123+ dendritic cells (DC) at day 10 pi showed that DC expressed ∼3-4 log more subtype transcripts as compared to the other subsets. Taken together, our results provide new insights into the kinetics of innate interferon responses during early stages of infection, and provide evidence that DC's are a major in vivo source of innate IFN during acute SIV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cellimm.2016.09.003DOI Listing
December 2016

CD4 and CD8 T cells mediate distinct lethal meningoencephalitis in mice challenged with Tacaribe arenavirus.

Cell Mol Immunol 2017 01 29;14(1):90-107. Epub 2016 Aug 29.

Division of Biologics Review and Research-III, Office of Biotechnology, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.

Neonates are at increased risk of viral encephalopathies that can result in neurological dysfunction, seizures, permanent disability and even death. The neurological damage results from the combined effect of the virus and the immune response it elicits, thus finding tools to facilitate viral clearance from central nervous system (CNS) while minimizing neuron damage remains a critical challenge. Neonatal mice inoculated intraperitoneally with Tacaribe virus (TCRV) develop seizures, hindlimb paralysis and death within 15 days of inoculation. TCRV localizes to the CNS within days of challenge, primarily infecting astrocytes in the cerebellum and brain stem. We show that infection leads to inflammation, T cell and monocyte infiltration into the cerebellar parenchyma, apoptosis of astrocytes, neuronal degeneration and loss of Purkinje cells. Infiltrating antigen-specific T cells fail to clear the virus but drive the disease, as T-cell-deficient CD3ɛ KO mice survive TCRV infection with minimal inflammation or clinical manifestations despite no difference in CNS viral loads in comparison with T-cell sufficient mice. CD8+ T cells drive the pathology, which even in the absence of CD4+ T-cell help, infiltrate the parenchyma and mediate the apoptotic loss of cerebellar astrocytes, neurodegeneration and loss of Purkinje cells resulting in loss of balance, paralysis and death. CD4+ T cells are also pathogenic inducing gliosis and inflammation in the cerebellum and cerebrum that are associated with wasting and death several weeks after CD4+ T-cell transfer. These data demonstrate distinct pathogenic effects of CD4+ and CD8+ T cells and identify them as possible therapeutic targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/cmi.2016.41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5214944PMC
January 2017

Detection of innate immune response modulating impurities in therapeutic proteins.

PLoS One 2015 22;10(4):e0125078. Epub 2015 Apr 22.

Laboratory of Immunology, Division of Biotechnology Review and Research III, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.

Therapeutic proteins can contain multiple impurities, some of which are variants of the product, while others are derived from the cell substrate and the manufacturing process. Such impurities, even when present at trace levels, have the potential to activate innate immune cells in peripheral blood or embedded in tissues causing expression of cytokines and chemokines, increasing antigen uptake, facilitating processing and presentation by antigen presenting cells, and fostering product immunogenicity. Currently, while products are tested for host cell protein content, assays to control innate immune response modulating impurities (IIRMIs) in products are focused mainly on endotoxin and nucleic acids, however, depending on the cell substrate and the manufacturing process, numerous other IIRMI could be present. In these studies we assess two approaches that allow for the detection of a broader subset of IIRMIs. In the first, we use commercial cell lines transfected with Toll like receptors (TLR) to detect receptor-specific agonists. This method is sensitive to trace levels of IIRMI and provides information of the type of IIRMIs present but is limited by the availability of stably transfected cell lines and requires pre-existing knowledge of the IIRMIs likely to be present in the product. Alternatively, the use of a combination of macrophage cell lines of human and mouse origin allows for the detection of a broader spectrum of impurities, but does not identify the source of the activation. Importantly, for either system the lower limit of detection (LLOD) of impurities was similar to that of PBMC and it was not modified by the therapeutic protein tested, even in settings where the product had inherent immune modulatory properties. Together these data indicate that a cell-based assay approach could be used to screen products for the presence of IIRMIs and inform immunogenicity risk assessments, particularly in the context of comparability exercises.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125078PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406594PMC
April 2016

High-throughput quantitative real-time RT-PCR assay for determining expression profiles of types I and III interferon subtypes.

J Vis Exp 2015 Mar 24(97). Epub 2015 Mar 24.

Center for Biologics Evaluation and Research, US Food and Drug Administration;

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-α subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-λ2 and -λ3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/52650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4401384PMC
March 2015

Immunology careers at the NIH, FDA and CDC: different paths that focus on advancing public health.

Nat Immunol 2015 Feb;16(2):129-32

Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, Maryland, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ni.3061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830109PMC
February 2015

Glycosylation, hypogammaglobulinemia, and resistance to viral infections.

N Engl J Med 2014 Apr 9;370(17):1615-1625. Epub 2014 Apr 9.

Infectious Diseases Susceptibility Unit, Laboratory of Host Defenses (M.A.S., N.H., E.N., R.M., M.G., S.D.R.), Laboratory of Immunoregulation (S.M., T.-W.C., P.L., L.J.Y.K., A.L., R.C., J.S.J., D.M.), Laboratory of Infectious Diseases (M.J.M., T.B.), Laboratory of Clinical Infectious Diseases (B.E.M.), Laboratory of Immunogenetics (G.H., D.S., P.S.), and Primary Immunodeficiency Clinic (S.D.R.), National Institute of Allergy and Infectious Diseases, the Undiagnosed Diseases Program, National Human Research Genome Institute (L.W., H.V., Y.H. D.A., C.F.B.), the Department of Laboratory Medicine, Clinical Center (K.R.C., J.S.), and Oral and Pharyngeal Cancer Branch (V.P.) and Adeno-Associated Virus Biology Section (J.A.C., G.D.P.), National Institute of Dental and Craniofacial Research - all at the National Institutes of Health, Bethesda, the Center for Biologics Evaluation and Research (G.K., J.J.) and the Center for Drug Evaluation and Research (D.C.I., C.G., D.V.), Food and Drug Administration Clinical Services Program, Silver Spring, and SAIC-Frederick, Frederick National Laboratory for Cancer Research, Frederick (D.A.L.P., D.B.K.) - all in Maryland; the Department of Human Genetics, Emory University School of Medicine, Atlanta (M. He, M. Hegde); and the IAVI (International AIDS Vaccine Initiative) Center for Neutralizing Antibodies at TSRI and the Department of Immunology and Microbial Science, Scripps Research Institute, La Jolla, CA (Y.L.).

Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1056/NEJMoa1302846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066413PMC
April 2014