RESUMO
Herpes zoster (shingles) causes significant morbidity in immune compromised hosts and older adults. Whereas a vaccine is available for prevention of shingles, its efficacy declines with age. To help to understand the mechanisms driving vaccinal responses, we constructed a multiscale, multifactorial response network (MMRN) of immunity in healthy young and older adults immunized with the live attenuated shingles vaccine Zostavax. Vaccination induces robust antigen-specific antibody, plasmablasts, and CD4+ T cells yet limited CD8+ T cell and antiviral responses. The MMRN reveals striking associations between orthogonal datasets, such as transcriptomic and metabolomics signatures, cell populations, and cytokine levels, and identifies immune and metabolic correlates of vaccine immunity. Networks associated with inositol phosphate, glycerophospholipids, and sterol metabolism are tightly coupled with immunity. Critically, the sterol regulatory binding protein 1 and its targets are key integrators of antibody and T follicular cell responses. Our approach is broadly applicable to study human immunity and can help to identify predictors of efficacy as well as mechanisms controlling immunity to vaccination.
Assuntos
Vacina contra Herpes Zoster/imunologia , Imunidade Adaptativa , Adulto , Idoso , Envelhecimento , Formação de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Fosfatos de Inositol/imunologia , Estudos Longitudinais , Masculino , Metabolômica , Pessoa de Meia-Idade , Caracteres Sexuais , Esteróis/metabolismo , Carga ViralRESUMO
Here we have used a systems biology approach to study innate and adaptive responses to vaccination against influenza in humans during three consecutive influenza seasons. We studied healthy adults vaccinated with trivalent inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV). TIV induced higher antibody titers and more plasmablasts than LAIV did. In subjects vaccinated with TIV, early molecular signatures correlated with and could be used to accurately predict later antibody titers in two independent trials. Notably, expression of the kinase CaMKIV at day 3 was inversely correlated with later antibody titers. Vaccination of CaMKIV-deficient mice with TIV induced enhanced antigen-specific antibody titers, which demonstrated an unappreciated role for CaMKIV in the regulation of antibody responses. Thus, systems approaches can be used to predict immunogenicity and provide new mechanistic insights about vaccines.
Assuntos
Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Orthomyxoviridae/imunologia , Imunidade Adaptativa/imunologia , Adolescente , Adulto , Animais , Anticorpos Antivirais/sangue , Perfilação da Expressão Gênica , Testes de Inibição da Hemaglutinação , Humanos , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Estações do Ano , Biologia de Sistemas/métodos , Vacinação/métodos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Adulto JovemRESUMO
The differentiation of human memory CD8 T cells is not well understood. Here we address this issue using the live yellow fever virus (YFV) vaccine, which induces long-term immunity in humans. We used in vivo deuterium labelling to mark CD8 T cells that proliferated in response to the virus and then assessed cellular turnover and longevity by quantifying deuterium dilution kinetics in YFV-specific CD8 T cells using mass spectrometry. This longitudinal analysis showed that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is maintained by quiescent cells that divide less than once every year (doubling time of over 450 days). Although these long-lived YFV-specific memory CD8 T cells did not express effector molecules, their epigenetic landscape resembled that of effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Epigênese Genética , Memória Imunológica/imunologia , Vacina contra Febre Amarela/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA , Deutério , Perfilação da Expressão Gênica , Meia-Vida , Humanos , Memória Imunológica/genética , Contagem de Linfócitos , Camundongos , Técnica de Diluição de Radioisótopos , Transcrição Gênica , Febre Amarela/imunologia , Febre Amarela/virologia , Vírus da Febre Amarela/imunologiaRESUMO
There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.
Assuntos
Adjuvantes Imunológicos , Linfócitos B/imunologia , Reações Cruzadas/imunologia , Memória Imunológica , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Linfócitos B/metabolismo , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Imunização Secundária , Masculino , Plasmócitos/imunologia , Plasmócitos/metabolismoRESUMO
Exhausted T cells in chronic infections and cancer have sustained expression of the inhibitory receptor programmed cell death 1 (PD-1). Therapies that block the PD-1 pathway have shown promising clinical results in a significant number of advanced-stage cancer patients. Nonetheless, a better understanding of the immunological responses induced by PD-1 blockade in cancer patients is lacking. Identification of predictive biomarkers is a priority in the field, but whether peripheral blood analysis can provide biomarkers to monitor or predict patients' responses to treatment remains to be resolved. In this study, we analyzed longitudinal blood samples from advanced stage non-small cell lung cancer (NSCLC) patients (n = 29) receiving PD-1-targeted therapies. We detected an increase in Ki-67+ PD-1+ CD8 T cells following therapy in â¼70% of patients, and most responses were induced after the first or second treatment cycle. This T-cell activation was not indiscriminate because we observed only minimal effects on EBV-specific CD8 T cells, suggesting that responding cells may be tumor specific. These proliferating CD8 T cells had an effector-like phenotype (HLA-DR+, CD38+, Bcl-2lo), expressed costimulatory molecules (CD28, CD27, ICOS), and had high levels of PD-1 and coexpression of CTLA-4. We found that 70% of patients with disease progression had either a delayed or absent PD-1+ CD8 T-cell response, whereas 80% of patients with clinical benefit exhibited PD-1+ CD8 T-cell responses within 4 wk of treatment initiation. Our results suggest that peripheral blood analysis may provide valuable insights into NSCLC patients' responses to PD-1-targeted therapies.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares , Ativação Linfocitária/efeitos dos fármacos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T CD8-Positivos/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , NivolumabeRESUMO
Herpes zoster (HZ) (shingles) is the clinical manifestation of varicella-zoster virus (VZV) reactivation. HZ typically develops as people age, due to decreased cell-mediated immunity. However, the importance of antibodies for immunity against HZ prevention remains to be understood. The goal of this study was to examine the breadth and functionality of VZV-specific antibodies after vaccination with a live attenuated HZ vaccine (Zostavax). Direct enumeration of VZV-specific antibody-secreting cells (ASCs) via enzyme-linked immunosorbent spot assay (ELISPOT assay) showed that Zostavax can induce both IgG and IgA ASCs 7 days after vaccination but not IgM ASCs. The VZV-specific ASCs range from 33 to 55% of the total IgG ASCs. Twenty-five human VZV-specific monoclonal antibodies (MAbs) were cloned and characterized from single-cell-sorted ASCs of five subjects (>60 years old) who received Zostavax. These MAbs had an average of â¼20 somatic hypermutations per VH gene, similar to those seen after seasonal influenza vaccination. Fifteen of the 25 MAbs were gE specific, whereas the remaining MAbs were gB, gH, or gI specific. The most potent neutralizing antibodies were gH specific and were also able to inhibit cell-to-cell spread of the virus in vitro Most gE-specific MAbs were able to neutralize VZV, but they required the presence of complement and were unable to block cell-to-cell spread. These data indicate that Zostavax induces a memory B cell recall response characterized by anti-gE > anti-gI > anti-gB > anti-gH antibodies. While antibodies to gH could be involved in limiting the spread of VZV upon reactivation, the contribution of anti-gE antibodies toward protective immunity after Zostavax needs further evaluation.IMPORTANCE Varicella-zoster virus (VZV) is the causative agent of chickenpox and shingles. Following infection with VZV, the virus becomes latent and resides in nerve cells. Age-related declines in immunity/immunosuppression can result in reactivation of this latent virus, causing shingles. It has been shown that waning T cell immunity correlates with an increased incidence of VZV reactivation. Interestingly, serum with high levels of VZV-specific antibodies (VariZIG; IV immunoglobulin) has been administered to high-risk populations, e.g., immunocompromised children, newborns, and pregnant women, after exposure to VZV and has shown some protection against chickenpox. However, the relative contribution of antibodies against individual surface glycoproteins toward protection from shingles in elderly/immunocompromised individuals has not been established. Here, we examined the breadth and functionality of VZV-specific antibodies after vaccination with the live attenuated VZV vaccine Zostavax in humans. This study will add to our understanding of the role of antibodies in protection against shingles.
Assuntos
Anticorpos Antivirais/imunologia , Glicoproteínas/imunologia , Vacina contra Herpes Zoster/administração & dosagem , Herpes Zoster/imunologia , Herpesvirus Humano 3/imunologia , Imunidade Celular/imunologia , Vacinas Atenuadas/imunologia , Idoso , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Herpes Zoster/prevenção & controle , Herpes Zoster/virologia , Humanos , Hospedeiro Imunocomprometido , Pessoa de Meia-Idade , VacinaçãoRESUMO
Antibody responses are critical components of protective immune responses to many pathogens, but parameters determining which proteins are targeted remain unclear. Vaccination with individual MHC-II-restricted vaccinia virus (VACV, smallpox vaccine) epitopes revealed that CD4(+) T cell help to B cells was surprisingly nontransferable to other virion protein specificities. Many VACV CD4(+) T cell responses identified in an unbiased screen targeted antibody virion protein targets, consistent with deterministic linkage between specificities. We tested the deterministic linkage model by efficiently predicting new vaccinia MHC II epitopes (830% improved efficiency). Finally, we showed CD4(+) T cell help was limiting for neutralizing antibody development and protective immunity in vivo. In contrast to the standard model, these data indicate individual proteins are the unit of B cell-T cell recognition for a large virus. Therefore, MHC restriction is a key selective event for the antiviral antibody response and is probably important for vaccine development to large pathogens.
Assuntos
Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacina Antivariólica/imunologia , Vaccinia virus/imunologia , Transferência Adotiva , Animais , Especificidade de Anticorpos , Antígenos Virais/metabolismo , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/metabolismo , Epitopos/imunologia , Epitopos/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Testes de Neutralização , Vacina Antivariólica/metabolismo , Vacínia/imunologia , Vacínia/prevenção & controle , Vacínia/virologiaRESUMO
The emergence of pandemic influenza viruses poses a major public health threat. Therefore, there is a need for a vaccine that can induce broadly cross-reactive antibodies that protect against seasonal as well as pandemic influenza strains. Human broadly neutralizing antibodies directed against highly conserved epitopes in the stem region of influenza virus HA have been recently characterized. However, it remains unknown what the baseline levels are of antibodies and memory B cells that are directed against these conserved epitopes. More importantly, it is also not known to what extent anti-HA stem B-cell responses get boosted in humans after seasonal influenza vaccination. In this study, we have addressed these two outstanding questions. Our data show that: (i) antibodies and memory B cells directed against the conserved HA stem region are prevalent in humans, but their levels are much lower than B-cell responses directed to variable epitopes in the HA head; (ii) current seasonal influenza vaccines are efficient in inducing B-cell responses to the variable HA head region but they fail to boost responses to the conserved HA stem region; and (iii) in striking contrast, immunization of humans with the avian influenza virus H5N1 induced broadly cross-reactive HA stem-specific antibodies. Taken together, our findings provide a potential vaccination strategy where heterologous influenza immunization could be used for increasing the levels of broadly neutralizing antibodies and for priming the human population to respond quickly to emerging pandemic influenza threats.
Assuntos
Formação de Anticorpos/imunologia , Reações Cruzadas/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Vacinação , Adulto , Especificidade de Anticorpos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Imunoglobulina G/sangue , Memória Imunológica , Influenza Humana/sangue , Influenza Humana/imunologia , Influenza Humana/virologia , Plasmócitos/imunologiaRESUMO
Human herpesviruses are important causes of potentially severe chronic infections for which T cells are believed to be necessary for control. In order to examine the role of virus-specific CD8 T cells against Varicella Zoster Virus (VZV), we generated a comprehensive panel of potential epitopes predicted in silico and screened for T cell responses in healthy VZV seropositive donors. We identified a dominant HLA-A*0201-restricted epitope in the VZV ribonucleotide reductase subunit 2 and used a tetramer to analyze the phenotype and function of epitope-specific CD8 T cells. Interestingly, CD8 T cells responding to this VZV epitope also recognized homologous epitopes, not only in the other α-herpesviruses, HSV-1 and HSV-2, but also the γ-herpesvirus, EBV. Responses against these epitopes did not depend on previous infection with the originating virus, thus indicating the cross-reactive nature of this T cell population. Between individuals, the cells demonstrated marked phenotypic heterogeneity. This was associated with differences in functional capacity related to increased inhibitory receptor expression (including PD-1) along with decreased expression of co-stimulatory molecules that potentially reflected their stimulation history. Vaccination with the live attenuated Zostavax vaccine did not efficiently stimulate a proliferative response in this epitope-specific population. Thus, we identified a human CD8 T cell epitope that is conserved in four clinically important herpesviruses but that was poorly boosted by the current adult VZV vaccine. We discuss the concept of a "pan-herpesvirus" vaccine that this discovery raises and the hurdles that may need to be overcome in order to achieve this.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Herpesvirus Humano 3/imunologia , Herpesvirus Humano 4/imunologia , Simplexvirus/imunologia , Adulto , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Antígeno HLA-A2/imunologia , Vacina contra Herpes Zoster/imunologia , Humanos , Vacinas Virais/imunologiaRESUMO
We have previously shown that broadly neutralizing antibodies reactive to the conserved stem region of the influenza virus hemagglutinin (HA) were generated in people infected with the 2009 pandemic H1N1 strain. Such antibodies are rarely seen in humans following infection or vaccination with seasonal influenza virus strains. However, the important question remained whether the inactivated 2009 pandemic H1N1 vaccine, like the infection, could also induce these broadly neutralizing antibodies. To address this question, we analyzed B-cell responses in 24 healthy adults immunized with the pandemic vaccine in 2009. In all cases, we found a rapid, predominantly IgG-producing vaccine-specific plasmablast response. Strikingly, the majority (25 of 28) of HA-specific monoclonal antibodies generated from the vaccine-specific plasmablasts neutralized more than one influenza strain and exhibited high levels of somatic hypermutation, suggesting they were derived from recall of B-cell memory. Indeed, memory B cells that recognized the 2009 pandemic H1N1 HA were detectable before vaccination not only in this cohort but also in samples obtained before the emergence of the pandemic strain. Three antibodies demonstrated extremely broad cross-reactivity and were found to bind the HA stem. Furthermore, one stem-reactive antibody recognized not only H1 and H5, but also H3 influenza viruses. This exceptional cross-reactivity indicates that antibodies capable of neutralizing most influenza subtypes might indeed be elicited by vaccination. The challenge now is to improve upon this result and design influenza vaccines that can elicit these broadly cross-reactive antibodies at sufficiently high levels to provide heterosubtypic protection.
Assuntos
Linfócitos B/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Memória Imunológica/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Adulto , Anticorpos Monoclonais/imunologia , Sequência de Bases , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , ELISPOT , Citometria de Fluxo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Imunoglobulina G/imunologia , Região Variável de Imunoglobulina/genética , Vacinas contra Influenza/administração & dosagem , Dados de Sequência Molecular , Testes de Neutralização , Filogenia , Análise de Sequência de DNARESUMO
CD4(+) T follicular helper (TFH) cells are central for generation of long-term B-cell immunity. A defining phenotypic attribute of TFH cells is the expression of the chemokine R CXCR5, and TFH cells are typically identified by co-expression of CXCR5 together with other markers such as PD-1, ICOS, and Bcl-6. Herein, we report high-level expression of the nutrient transporter folate R 4 (FR4) on TFH cells in acute viral infection. Distinct from the expression profile of conventional TFH markers, FR4 was highly expressed by naive CD4(+) T cells, was downregulated after activation and subsequently re-expressed on TFH cells. Furthermore, FR4 expression was maintained, albeit at lower levels, on memory TFH cells. Comparative gene expression profiling of FR4(hi) versus FR4(lo) Ag-specific CD4(+) effector T cells revealed a molecular signature consistent with TFH and TH1 subsets, respectively. Interestingly, genes involved in the purine metabolic pathway, including the ecto-enzyme CD73, were enriched in TFH cells compared with TH1 cells, and phenotypic analysis confirmed expression of CD73 on TFH cells. As there is now considerable interest in developing vaccines that would induce optimal TFH cell responses, the identification of two novel cell surface markers should be useful in characterization and identification of TFH cells following vaccination and infection.
Assuntos
Regulação da Expressão Gênica/imunologia , Receptores de Superfície Celular/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , 5'-Nucleotidase/biossíntese , 5'-Nucleotidase/genética , 5'-Nucleotidase/imunologia , Doença Aguda , Animais , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/biossíntese , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/biossíntese , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Proteínas Proto-Oncogênicas c-bcl-6 , Receptores CXCR5/biossíntese , Receptores CXCR5/genética , Receptores CXCR5/imunologia , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/metabolismo , Viroses/genética , Viroses/imunologia , Viroses/metabolismoRESUMO
The Clinical and Laboratory Standards Institute (CLSI) H62-Validation of Assays Performed by Flow Cytometry guideline, released in 2021, provides recommendations for platform workflow and quality system essentials, instrument setup and standardization, assay development and optimization and fit-for-purpose analytical method validation. In addition, CLSI H62 includes some recommendations for the validation strategies after a validated flow cytometric method has been modified. This manuscript builds on those recommendations and discusses the impact of different types of assay modifications on assay performance. Recommendations regarding which validation parameters to evaluate depending on the type of modification are provided. The impact of assay modification on the assay's intended use is discussed. When recommending minor deviations from the CLSI H62 process for a laboratory-initiated assay revision (e.g., specimen numbers for sensitivity, specificity, or precision studies), a rationale based on expert opinion is provided with the understanding that not every laboratory, assay type, and circumstance can be comprehensively addressed in this paper. These recommendations are meant as a practical recommendation and are not intended to be restrictive, prescriptive, or understood as necessarily sufficient to meet every specific requirement from regulatory bodies (e.g., FDA or New York State Department of Health).
RESUMO
The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on 19-23 June 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2023 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 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 2) covers the recommendations on Biomarkers, IVD/CDx, LBA and Cell-Based Assays. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 9 and 7 (2024), respectively.
Assuntos
Biomarcadores , Terapia Baseada em Transplante de Células e Tecidos , Vacinas , Humanos , Biomarcadores/análise , Vacinas/imunologia , Citometria de Fluxo , Bioensaio/métodos , União Europeia , BrancosRESUMO
Flow cytometry is a powerful technology used in research, drug development and clinical sample analysis for cell identification and characterization, allowing for the simultaneous interrogation of multiple targets on various cell subsets from limited samples. Recent advancements in instrumentation and fluorochrome availability have resulted in significant increases in the complexity and dimensionality of flow cytometry panels. Though this increase in panel size allows for detection of a broader range of markers and sub-populations, even in restricted biological samples, it also comes with many challenges in panel design, optimization, and downstream data analysis and interpretation. In the current paper we describe the practices we established for development of high-dimensional panels on the Aurora spectral flow cytometer to aid clinical sample analysis.
Assuntos
Citometria de Fluxo , Ensaios Clínicos como Assunto , HumanosRESUMO
The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th 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 14th 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.
Assuntos
Bioensaio , Biotecnologia , Terapia Baseada em Transplante de Células e Tecidos , Terapia Genética , Relatório de Pesquisa , Biomarcadores/análise , HumanosRESUMO
Antibodies against the extracellular virion (EV or EEV) form of vaccinia virus are an important component of protective immunity in animal models and likely contribute to the protection of immunized humans against poxviruses. Using fully human monoclonal antibodies (MAbs), we now have shown that the protective attributes of the human anti-B5 antibody response to the smallpox vaccine (vaccinia virus) are heavily dependent on effector functions. By switching Fc domains of a single MAb, we have definitively shown that neutralization in vitro--and protection in vivo in a mouse model--by the human anti-B5 immunoglobulin G MAbs is isotype dependent, thereby demonstrating that efficient protection by these antibodies is not simply dependent on binding an appropriate vaccinia virion antigen with high affinity but in fact requires antibody effector function. The complement components C3 and C1q, but not C5, were required for neutralization. We also have demonstrated that human MAbs against B5 can potently direct complement-dependent cytotoxicity of vaccinia virus-infected cells. Each of these results was then extended to the polyclonal human antibody response to the smallpox vaccine. A model is proposed to explain the mechanism of EV neutralization. Altogether these findings enhance our understanding of the central protective activities of smallpox vaccine-elicited antibodies in immunized humans.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Isotipos de Imunoglobulinas/imunologia , Varíola/prevenção & controle , Vaccinia virus/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Peso Corporal , Complemento C1q/imunologia , Complemento C3/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Testes de Neutralização , Análise de SobrevidaRESUMO
Antibody neutralization is an important component of protective immunity against vaccinia virus (VACV). Two distinct virion forms, mature virion and enveloped virion (MV and EV, respectively), possess separate functions and nonoverlapping immunological properties. In this study we examined the mechanics of EV neutralization, focusing on EV protein B5 (also called B5R). We show that neutralization of EV is predominantly complement dependent. From a panel of high-affinity anti-B5 monoclonal antibodies (MAbs), the only potent neutralizer in vitro (90% at 535 ng/ml) was an immunoglobulin G2a (IgG2a), and neutralization was complement mediated. This MAb was the most protective in vivo against lethal intranasal VACV challenge. Further studies demonstrated that in vivo depletion of complement caused a >50% loss of anti-B5 IgG2a protection, directly establishing the importance of complement for protection against the EV form. However, the mechanism of protection is not sterilizing immunity via elimination of the inoculum as the viral inoculum consisted of a purified MV form. The prevention of illness in vivo indicated rapid control of infection. We further demonstrate that antibody-mediated killing of VACV-infected cells expressing surface B5 is a second protective mechanism provided by complement-fixing anti-B5 IgG. Cell killing was very efficient, and this effector function was highly isotype specific. These results indicate that anti-B5 antibody-directed cell lysis via complement is a powerful mechanism for clearance of infected cells, keeping poxvirus-infected cells from being invisible to humoral immune responses. These findings highlight the importance of multiple mechanisms of antibody-mediated protection against VACV and point to key immunobiological differences between MVs and EVs that impact the outcome of infection.
Assuntos
Proteínas do Sistema Complemento/fisiologia , Vaccinia virus/imunologia , Vírion/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Feminino , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células VeroRESUMO
A universal vaccine against influenza would ideally generate protective immune responses that are not only broadly reactive against multiple influenza strains but also long-lasting. Because long-term serum antibody levels are maintained by bone marrow plasma cells (BMPCs), we investigated the production and maintenance of these cells after influenza vaccination. We found increased numbers of influenza-specific BMPCs 4 weeks after immunization with the seasonal inactivated influenza vaccine, but numbers returned to near their prevaccination levels after 1 year. This decline was driven by the loss of BMPCs induced by the vaccine, whereas preexisting BMPCs were maintained. Our results suggest that most BMPCs generated by influenza vaccination in adults are short-lived. Designing strategies to enhance their persistence will be a key challenge for the next generation of influenza vaccines.
Assuntos
Células da Medula Óssea/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Plasmócitos/imunologia , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Humanos , Imunoglobulina G/sangue , Influenza Humana/sangue , Influenza Humana/imunologia , VacinaçãoRESUMO
The smallpox vaccine is widely considered the gold standard for human vaccines, yet the key antibody targets in humans remain unclear. We endeavored to identify a stereotypic, dominant, mature virion (MV) neutralizing antibody target in humans which could be used as a diagnostic serological marker of protective humoral immunity induced by the smallpox vaccine (vaccinia virus [VACV]). We have instead found that diversity is a defining characteristic of the human antibody response to the smallpox vaccine. We show that H3 is the most immunodominant VACV neutralizing antibody target, as determined by correlation analysis of immunoglobulin G (IgG) specificities to MV neutralizing antibody titers. It was determined that purified human anti-H3 IgG is sufficient for neutralization of VACV; however, depletion or blockade of anti-H3 antibodies revealed no significant reduction in neutralization activity, showing anti-H3 IgG is not required in vaccinated humans (or mice) for neutralization of MV. Comparable results were obtained for human (and mouse) anti-L1 IgG and even for anti-H3 and anti-L1 IgG in combination. In addition to H3 and L1, human antibody responses to D8, A27, D13, and A14 exhibited statistically significant correlations with virus neutralization. Altogether, these data indicate the smallpox vaccine succeeds in generating strong neutralizing antibody responses not by eliciting a stereotypic response to a single key antigen but instead by driving development of neutralizing antibodies to multiple viral proteins, resulting in a "safety net" of highly redundant neutralizing antibody responses, the specificities of which can vary from individual to individual. We propose that this is a fundamental attribute of the smallpox vaccine.
Assuntos
Anticorpos Antivirais/sangue , Vacina Antivariólica/imunologia , Vaccinia virus/imunologia , Proteínas de Transporte/imunologia , Humanos , Imunoglobulina G/sangue , Testes de Neutralização , Proteínas do Envelope Viral/imunologia , Proteínas Virais/imunologia , Vírion/imunologiaRESUMO
Quantitative PCR (QPCR, or real time PCR (rtPCR)) has emerged as a powerful virologic technique for measuring viral replication and viral loads in humans and animal models. We have developed a QPCR assay to accurately quantify lymphocytic choriomeningitis virus (LCMV) in infected mice. We first validated this assay using plasmid DNA and LCMV viral stocks. We then demonstrated that the LCMV QPCR assay can detect LCMV in serum and tissues of chronically infected mice (LCMV-clone 13), with greater sensitivity than conventional plaque assay. Subsequently, we demonstrated that the QPCR assay can detect LCMV in tissues of CD40L(-/-) mice during a low grade chronic infection with LCMV Armstrong. Finally, we improved the assay further such that it was approximate 1000-fold more sensitive than plaque assay for detection of the presence of LCMV in tissue.