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1.
J Infect ; : 106257, 2024 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-39216830

RESUMO

Children with Hb AC or AS have decreased susceptibility to clinical malaria. Parasite variant surface antigen (VSA) presentation on the surface of infected erythrocytes is altered in erythrocytes with hemoglobin C (Hb AC) or sickle trait (Hb AS) mutations in vitro. The protective role of incomplete or altered VSA presentation against clinical malaria in individuals with Hb AC or AS is unclear. Using a high-throughput protein microarray, we sought to use serological responses to VSAs as a measure of host exposure to VSAs among Malian children with Hb AC, Hb AS, or wildtype hemoglobin (Hb AA). In uncomplicated malaria, when compared to Hb AA children, Hb AC children had significantly lower serological responses to extracellular Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) domains but did not differ in responses to intracellular PfEMP1 domains and other VSAs, including members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family. Healthy children with Hb AC and Hb AS genotypes recognized fewer extracellular PfEMP1s compared to children with Hb AA, especially CD36-binding PfEMP1s. These reduced serologic responses may reflect reduced VSA presentation or lower parasite exposure in children with Hb AC or AS and provide insights into mechanisms of protection.

2.
Front Immunol ; 14: 1166261, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37266444

RESUMO

Introduction: In the context of recurrent surges of SARS-CoV-2 infections, a detailed characterization of antibody persistence over a 6-month period following vaccine booster dose is necessary to crafting effective public health policies on repeat vaccination. Methods: To characterize the SARS-CoV-2 antibody profile of a healthcare worker population over a 6-month period following mRNA vaccination and booster dose. 323 healthcare workers at an academic medical center in Orange County, California who had completed primary vaccination and booster dose against SARS-CoV-2 were recruited for the study. A total of 690 blood specimens over a 6-month period were collected via finger-stick blood and analyzed for the presence of antibodies against 9 SARS-CoV-2 antigens using a coronavirus antigen microarray. Results: The primary outcome of this study was the average SARS-CoV-2 antibody level as measured using a novel coronavirus antigen microarray. Additional outcomes measured include levels of antibodies specific to SARS-CoV-2 variants including Delta, Omicron BA.1, and BA.2. We also measured SARS-CoV-2 neutralization capacity for a subset of the population to confirm correlation with antibody levels. Although antibodies against SARS-CoV-2 wane throughout the 6-month period following a booster dose, antibody levels remain higher than pre-boost levels. However, a booster dose of vaccine based on the original Wuhan strain generates approximately 3-fold lower antibody reactivity against Omicron variants BA.1 and BA.2 as compared to the vaccine strain. Despite waning antibody levels, neutralization activity against the vaccine strain is maintained throughout the 6-month period. Discussion: In the context of recurrent surges of SARS-CoV-2 infections, our data indicate that breakthrough infections are likely driven by novel variants with different antibody specificity and not by time since last dose of vaccination, indicating that development of vaccinations specific to these novel variants is necessary to prevent future surges of SARS-CoV-2 infections.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/prevenção & controle , Anticorpos Antivirais , Pessoal de Saúde , Vacinas de mRNA
3.
Res Sq ; 2022 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-36561177

RESUMO

In the context of recurrent surges of SARS-CoV-2 infections, a detailed characterization of antibody persistence over a 6-month period following vaccine booster dose is necessary to crafting effective public health policies on repeat vaccination. To characterize the SARS-CoV-2 antibody profile of a healthcare worker population over a 6-month period following mRNA vaccination and booster dose. 323 healthcare workers at an academic medical center in Orange County, California who had completed primary vaccination and booster dose against SARS-CoV-2 were recruited for the study. A total of 690 blood specimens over a 6-month period were collected via finger-stick blood and analyzed for the presence of antibodies against 9 SARS-CoV-2 antigens using a coronavirus antigen microarray. The primary outcome of this study was the average SARS-CoV-2 antibody level as measured using a novel coronavirus antigen microarray. Additional outcomes measured include levels of antibodies specific to SARS-CoV-2 variants including Delta, Omicron BA.1, and BA.2. We also measured SARS-CoV-2 neutralization capacity for a subset of the population to confirm correlation with antibody levels. Although antibodies against SARS-CoV-2 wane throughout the 6-month period following a booster dose, antibody levels remain higher than pre-boost levels. However, a booster dose of vaccine generates approximately 3-fold lower antibody reactivity against Omicron variants BA.1 and BA.2 as compared to the original Wuhan strain. Despite waning antibody levels, neutralization activity against the original Wuhan strain is maintained throughout the 6-month period. In the context of recurrent surges of SARS-CoV-2 infections despite vaccination with booster doses, our data indicate that breakthrough infections are likely driven by novel variants with different antibody specificity and not by time since last dose of vaccination, indicating that development of vaccinations specific to these novel variants is necessary to prevent future surges of SARS-CoV-2 infections.

4.
Biomedicines ; 10(9)2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-36140339

RESUMO

High-throughput and rapid screening testing is highly desirable to effectively combat the rapidly evolving COVID-19 pandemic co-presents with influenza and seasonal common cold epidemics. Here, we present a general workflow for iterative development and validation of an antibody-based microarray assay for the detection of a respiratory viral panel: (a) antibody screening to quickly identify optimal reagents and assay conditions, (b) immunofluorescence assay design including signal amplification for low viral titers, (c) assay characterization with recombinant proteins, inactivated viral samples and clinical samples, and (d) multiplexing to detect a panel of common respiratory viruses. Using RT-PCR-confirmed SARS-CoV-2 positive and negative pharyngeal swab samples, we demonstrated that the antibody microarray assay exhibited a clinical sensitivity and specificity of 77.2% and 100%, respectively, which are comparable to existing FDA-authorized antigen tests. Moreover, the microarray assay is correlated with RT-PCR cycle threshold (Ct) values and is particularly effective in identifying high viral titers. The multiplexed assay can selectively detect SARS-CoV-2 and influenza virus, which can be used to discriminate these viral infections that share similar symptoms. Such protein microarray technology is amenable for scale-up and automation and can be broadly applied as a both diagnostic and research tool.

5.
NPJ Vaccines ; 7(1): 103, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-36042229

RESUMO

Current seasonal and pre-pandemic influenza vaccines induce short-lived predominantly strain-specific and limited heterosubtypic responses. To better understand how vaccine adjuvants AS03 and MF59 may provide improved antibody responses to vaccination, we interrogated serum from subjects who received 2 doses of inactivated monovalent influenza A/Indonesia/05/2005 vaccine with or without AS03 or MF59 using hemagglutinin (HA) microarrays (NCT01317758 and NCT01317745). The arrays were designed to reflect both full-length and globular head HA derived from 17 influenza A subtypes (H1 to H16 and H18) and influenza B strains. We observed significantly increased strain-specific and broad homo- and heterosubtypic antibody responses with both AS03 and MF59 adjuvanted vaccination with AS03 achieving a higher titer and breadth of IgG responses relative to MF59. The adjuvanted vaccine was also associated with the elicitation of stalk-directed antibody. We established good correlation of the array antibody responses to H5 antigens with standard HA inhibition and microneutralization titers.

6.
Lab Chip ; 22(14): 2695-2706, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35737382

RESUMO

Heterogeneous immunoassays (HI) are an invaluable tool for biomarker detection and remain an ideal candidate for microfluidic point-of-care diagnostics. However, automating and controlling sustained fluid flow from benchtop to microfluidics for the HI reaction during the extended sample incubation step, remains difficult to implement; this leads to challenges for assay integration and assay result interpretation. To address these issues, we investigated the liquid reciprocation process on a microfluidic centrifugal disc (CD) to generate continuous, bidirectional fluid flow using only a rotating motor. Large volumetric flow rates (µL s-1) through the HI reaction chamber were sustained for extended durations (up to 1 h). The CD liquid reciprocation operating behavior was characterized experimentally and simulated to determine fluid flow shear rates through our HI reaction chamber. We demonstrated the continuous CD liquid reciprocation for target molecule incubation for a microarray HI and that higher fluid shear rates negatively influenced our fluorescence intensity. We highlight the importance of proper fluid flow considerations when integrating HIs with microfluidics.


Assuntos
COVID-19 , Técnicas Analíticas Microfluídicas , Bioensaio , Humanos , Imunoensaio , Microfluídica
7.
Front Immunol ; 13: 817345, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35493473

RESUMO

Recent studies provide conflicting evidence on the persistence of SARS-CoV-2 immunity induced by mRNA vaccines. Here, we aim to quantify the persistence of humoral immunity following vaccination using a coronavirus antigen microarray that includes 10 SARS-CoV-2 antigens. In a prospective longitudinal cohort of 240 healthcare workers, composite SARS-CoV-2 IgG antibody levels did not wane significantly over a 6-month study period. In the subset of the study population previously exposed to SARS-CoV-2 based on seropositivity for nucleocapsid antibodies, higher composite anti-spike IgG levels were measured before the vaccine but no significant difference from unexposed individuals was observed at 6 months. Age, vaccine type, or worker role did not significantly impact composite IgG levels, although non-significant trends towards lower antibody levels in older participants and higher antibody levels with Moderna vaccine were observed at 6 months. A small subset of our cohort were classified as having waning antibody titers at 6 months, and these individuals were less likely to work in patient care roles and more likely to have prior exposure to SARS-CoV-2.


Assuntos
COVID-19 , SARS-CoV-2 , Idoso , Anticorpos Antivirais , COVID-19/prevenção & controle , Pessoal de Saúde , Humanos , Imunoglobulina G , Lactente , Estudos Prospectivos
8.
NPJ Vaccines ; 6(1): 132, 2021 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-34737318

RESUMO

We analyzed data from two ongoing COVID-19 longitudinal serological surveys in Orange County, CA., between April 2020 and March 2021. A total of 8476 finger stick blood specimens were collected before and after a vaccination campaign. IgG levels were determined using a multiplex antigen microarray containing antigens from SARS-CoV-2, SARS, MERS, Common CoV, and Influenza. Twenty-six percent of specimens from unvaccinated Orange County residents in December 2020 were SARS-CoV-2 seropositive; out of 852 seropositive individuals 77 had symptoms and 9 sought medical care. The antibody response was predominantly against nucleocapsid (NP), full length, and S2 domain of spike. Anti-receptor binding domain (RBD) reactivity was low and not cross-reactive against SARS S1 or SARS RBD. A vaccination campaign at the University of California Irvine Medical Center (UCIMC) started on December, 2020 and 6724 healthcare workers were vaccinated within 3 weeks. Seroprevalence increased from 13% pre-vaccination to 79% post-vaccination in January, 93% in February, and 99% in March. mRNA vaccination induced higher antibody levels than natural exposure, especially against the RBD domain and cross-reactivity against SARS RBD and S1 was observed. Nucleocapsid protein antibodies can be used to distinguish vaccinees to classify pre-exposure to SARS-CoV-2 Previously infected individuals developed higher antibody titers to the vaccine than non pre-exposed individuals. Hospitalized patients in intensive care with severe disease reach significantly higher antibody levels than mild cases, but lower antibody levels compared to the vaccine. These results indicate that mRNA vaccination rapidly induces a much stronger and broader antibody response than SARS-CoV-2 infection.

9.
NPJ Vaccines ; 6(1): 115, 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34518543

RESUMO

Knowledge of the Plasmodium falciparum antigens that comprise the human liver stage immunoproteome is important for pre-erythrocytic vaccine development, but, compared with the erythrocytic stage immunoproteome, more challenging to classify. Previous studies of P. falciparum antibody responses report IgG and rarely IgA responses. We assessed IgG and IgA antibody responses in adult sera collected during two controlled human malaria infection (CHMI) studies in malaria-naïve volunteers and in 1- to 6-year-old malaria-exposed Malian children on a 251 P. falciparum antigen protein microarray. IgG profiles in the two CHMI groups were equivalent and differed from Malian children. IgA profiles were robust in the CHMI groups and a subset of Malian children. We describe immunoproteome differences in naïve vs. exposed individuals and report pre-erythrocytic proteins recognized by the immune system. IgA responses detected in this study expand the list of pre-erythrocytic antigens for further characterization as potential vaccine candidates.

10.
Sci Rep ; 11(1): 7554, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824382

RESUMO

A coronavirus antigen microarray (COVAM) was constructed containing 11 SARS-CoV-2, 5 SARS-1, 5 MERS, and 12 seasonal coronavirus recombinant proteins. The array is designed to measure immunoglobulin isotype and subtype levels in serum or plasma samples against each of the individual antigens printed on the array. We probed the COVAM with COVID-19 convalescent plasma (CCP) collected from 99 donors who recovered from a PCR+ confirmed SARS-CoV-2 infection. The results were analyzed using two computational approaches, a generalized linear model (glm) and random forest (RF) prediction model, to classify individual specimens as either Reactive or non-reactive against the SARS-CoV-2 antigens. A training set of 88 pre-COVID-19 specimens (PreCoV) collected in August 2019 and102 positive specimens from SARS-CoV-2 PCR+ confirmed COVID-19 cases was used for these analyses. Results compared with an FDA emergency use authorized (EUA) SARS-CoV2 S1-based total Ig chemiluminescence immunoassay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and with a SARS-CoV-2 S1-S2 spike-based pseudovirus micro neutralization assay (SARS-CoV-2 reporter viral particle neutralization titration (RVPNT) showed high concordance between the three assays. Three CCP specimens that were negative by the VITROS CoV2T immunoassay were also negative by both COVAM and the RVPNT assay. Concordance between VITROS CoV2T and COVAM was 96%, VITROS CoV2T and RVPNT 93%, and RVPNT and COVAM 91%. The discordances were all weakly reactive samples near the cutoff threshold of the VITROS CoV2T immunoassay. The multiplex COVAM allows CCP to be grouped according to antibody reactivity patterns against 11 SARS-CoV-2 antigens. Unsupervised K-means analysis, via the gap statistics, as well as hierarchical clustering analysis revealed three main clusters with distinct reactivity intensities and patterns. These patterns were not recapitulated by adjusting the VITROS CoV2T or RVPNT assay thresholds. Plasma classified by COVAM reactivity patterns offers potential to improve CCP therapeutic efficacy CoV2T alone. The use of a SARS-CoV-2 antigen array can qualify CCP for administration as a treatment for acute COVID-19, and interrogate vaccine immunogenicity and performance in preclinical, clinical studies, and routine vaccination to identify antibody responses predictive of protection from infection and disease.


Assuntos
Anticorpos Antivirais/imunologia , COVID-19/imunologia , COVID-19/terapia , SARS-CoV-2/imunologia , Imunidade Adaptativa , Coronavirus/imunologia , Humanos , Imunidade Humoral , Imunização Passiva , Soroterapia para COVID-19
11.
Front Immunol ; 12: 653092, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815413

RESUMO

Q fever is caused by the obligate intracellular bacterium, Coxiella burnetii, a designated potential agent of bioterrorism because of its route of transmission, resistance to disinfectants, and low infectious dose. The only vaccine licensed for human use is Q-VAX® (Seqirus, licensed in Australia), a formalin-inactivated whole-cell vaccine, which produces severe local and systemic reactogenic responses in previously sensitized individuals. Accordingly, the U.S. Food and Drug Administration and other regulatory bodies around the world, have been reluctant to approve Q-VAX for widespread use. To obviate these adverse reactions, we prepared recombinant protein subunit vaccine candidates containing purified CBU1910, CBU0307, CBU0545, CBU0612, CBU0891, and CBU1398 proteins and TLR triagonist adjuvants. TLR triagonist adjuvants combine different TLR agonists to enhance immune responses to vaccine antigens. We tested both the protective efficacy and reactogenicity of our vaccine candidates in Hartley guinea pigs using intratracheal infection with live C. burnetii. While all of our candidates showed varying degrees of protection during challenge, local reactogenic responses were significantly reduced for one of our vaccine candidates when compared with a formalin-inactivated whole-cell vaccine. Our findings show that subunit vaccines combined with novel TLR triagonist adjuvants can generate protective immunity to C. burnetii infection while reducing reactogenic responses.


Assuntos
Adjuvantes Imunológicos/farmacologia , Vacinas Bacterianas/farmacologia , Coxiella burnetii/imunologia , Febre Q/prevenção & controle , Receptores Toll-Like/antagonistas & inibidores , Adjuvantes Imunológicos/uso terapêutico , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/farmacologia , Antígenos de Bactérias/uso terapêutico , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/uso terapêutico , Modelos Animais de Doenças , Cobaias , Humanos , Imunogenicidade da Vacina , Febre Q/imunologia , Febre Q/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/farmacologia , Vacinas de Subunidades Antigênicas/uso terapêutico , Vacinas Sintéticas/genética , Vacinas Sintéticas/farmacologia , Vacinas Sintéticas/uso terapêutico
12.
Nat Commun ; 12(1): 6, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397903

RESUMO

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/sangue , COVID-19/imunologia , SARS-CoV-2/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , COVID-19/sangue , COVID-19/diagnóstico , Teste para COVID-19 , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Análise em Microsséries/métodos , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Testes de Neutralização , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
13.
Lab Chip ; 20(18): 3302-3309, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32743622

RESUMO

To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100× more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Diagnóstico por Imagem/instrumentação , Diagnóstico por Imagem/métodos , Análise em Microsséries/métodos , Pneumonia Viral/diagnóstico , COVID-19 , Teste para COVID-19 , Infecções por Coronavirus/imunologia , Humanos , Microscopia , Pandemias , Pneumonia Viral/imunologia , Impressão Tridimensional , Vigilância em Saúde Pública , Pontos Quânticos
14.
bioRxiv ; 2020 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-32511302

RESUMO

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

15.
bioRxiv ; 2020 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-32511369

RESUMO

To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100x more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months and years to come to implement efficient containment and mitigation measures, as well as help develop therapeutics and vaccines to treat and prevent the spread of COVID-19.

16.
Sci Rep ; 10(1): 3952, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127565

RESUMO

Vaccines based on Plasmodium falciparum apical membrane antigen 1 (AMA1) have failed due to extensive polymorphism in AMA1. To assess the strain-specificity of antibody responses to malaria infection and AMA1 vaccination, we designed protein and peptide microarrays representing hundreds of unique AMA1 variants. Following clinical malaria episodes, children had short-lived, sequence-independent increases in average whole-protein seroreactivity, as well as strain-specific responses to peptides representing diverse epitopes. Vaccination resulted in dramatically increased seroreactivity to all 263 AMA1 whole-protein variants. High-density peptide analysis revealed that vaccinated children had increases in seroreactivity to four distinct epitopes that exceeded responses to natural infection. A single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy. Antibody measurements using whole antigens may be biased towards conserved, immunodominant epitopes. Peptide microarrays may help to identify immunogenic epitopes, define correlates of vaccine protection, and measure strain-specific vaccine-induced antibodies.


Assuntos
Anticorpos Antiprotozoários/imunologia , Formação de Anticorpos/fisiologia , Antígenos de Protozoários/imunologia , Proteínas de Membrana/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Formação de Anticorpos/imunologia , Vacinas Antimaláricas/imunologia , Vacinas Antimaláricas/uso terapêutico , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/patogenicidade
17.
Front Immunol ; 11: 611661, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33584691

RESUMO

The anti-CD20 antibody Rituximab to deplete CD20+ B cells is an effective treatment for rheumatoid arthritis and B cell malignancies, but is associated with an increased incidence of respiratory infections. Using mouse models we have investigated the consequences of B cell depletion on natural and acquired humoral immunity to Streptococcus pneumoniae. B cell depletion of naïve C57Bl/6 mice reduced natural IgM recognition of S. pneumoniae, but did not increase susceptibility to S. pneumoniae pneumonia. ELISA and flow cytometry assays demonstrated significantly reduced IgG and IgM recognition of S. pneumoniae in sera from mice treated with B cell depletion prior to S. pneumoniae nasopharyngeal colonization compared to untreated mice. Colonization induced antibody responses to protein rather than capsular antigen, and when measured using a protein array B cell depletion prior to colonization reduced serum levels of IgG to several protein antigens. However, B cell depleted S. pneumoniae colonized mice were still partially protected against both lung infection and septicemia when challenged with S. pneumoniae after reconstitution of their B cells. These data indicate that although B cell depletion markedly impairs antibody recognition of S. pneumoniae in colonized mice, some protective immunity is maintained, perhaps mediated by cellular immunity.


Assuntos
Linfócitos B/efeitos dos fármacos , Imunidade Humoral , Imunidade Inata , Fatores Imunológicos/farmacologia , Depleção Linfocítica , Pneumonia Pneumocócica/prevenção & controle , Rituximab/farmacologia , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Antibacterianos/sangue , Linfócitos B/imunologia , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Imunidade Celular , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Camundongos Endogâmicos C57BL , Pneumonia Pneumocócica/sangue , Pneumonia Pneumocócica/imunologia , Pneumonia Pneumocócica/microbiologia , Streptococcus pneumoniae/patogenicidade
18.
Cell Host Microbe ; 26(5): 579-590.e5, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31726028

RESUMO

Extreme diversity of the major Plasmodium falciparum antigen, PfEMP1, poses a barrier to identifying targets of immunity to malaria. Here, we used protein microarrays containing hundreds of variants of the DBLα domain of PfEMP1 to cover the diversity of Papua New Guinean (PNG) parasites. Probing the plasma of a longitudinal cohort of malaria-exposed PNG children showed that group 2 DBLα antibodies were moderately associated with a lower risk of uncomplicated malaria, whereas individual variants were only weakly associated with clinical immunity. In contrast, antibodies to 85 individual group 1 and 2 DBLα variants were associated with a 70%-100% reduction in severe malaria. Of these, 17 variants were strong predictors of severe malaria. Analysis of full-length PfEMP1 sequences from PNG samples shows that these 17 variants are linked to pathogenic CIDR domains. This suggests that whereas immunity to uncomplicated malaria requires a broad repertoire of antibodies, immunity to severe malaria targets a subset of conserved variants. These findings provide insights into antimalarial immunity and potential antibody biomarkers for disease risk.


Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Plasmodium falciparum/imunologia , Domínios Proteicos/imunologia , Proteínas de Protozoários/imunologia , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Pré-Escolar , Feminino , Humanos , Lactente , Estudos Longitudinais , Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Masculino , Papua Nova Guiné , Análise Serial de Proteínas , Domínios Proteicos/genética , Proteínas de Protozoários/genética
19.
ACS Cent Sci ; 5(7): 1137-1145, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31403067

RESUMO

Traditional vaccination strategies have failed to generate effective vaccines for many infections like tuberculosis and HIV. New approaches are needed for each type of disease. The protective immunity and distinct responses of many successful vaccines come from activating multiple Toll-like receptors (TLRs). Vaccines with multiple TLRs as adjuvants have proven effective in preclinical studies, but current research has not explored two important elements. First, few multi-TLR systems explore spatial organization-a critical feature of whole-cell vaccines. Second, no multi-TLR systems to date provide systematic analysis of the combinatorial space of three TLR agonists. Here, we present the first examination of the combinatorial space of several spatially defined triple-TLR adjuvants, by synthesizing a series of five triple-TLR agonists and testing their innate activity both in vitro and in vivo. The combinations were evaluated by measuring activation of immune stimulatory genes (Nf-κB, ISGs), cytokine profiles (IL12-p70, TNF-α, IL-6, IL-10, CCL2, IFN-α, IFN-ß, IFN-γ), and in vivo cytokine serum levels (IL-6, TNF-α, IL12-p40, IFN-α, IFN-ß). We demonstrate that linking TLR agonists substantially alters the resulting immune response compared to their unlinked counterparts and that each combination results in a distinct immune response, particularly between linked combinations. We show that combinations containing a TLR9 agonist produce more Th1 biasing immune response profiles, and that the effect is amplified upon conjugation. However, combinations containing TLR2/6 agonist are skewed toward TH2 biasing profiles despite the presence of TLR9. These results demonstrate the profound effects that conjugation and combinatorial administration of TLR agonists can have on immune responses, a critical element of vaccine development.

20.
J Vis Exp ; (149)2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31403629

RESUMO

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens. This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera. Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Influenza Humana/imunologia , Análise Serial de Proteínas/métodos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Estudos de Coortes , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vacinas contra Influenza/imunologia , Neuraminidase/imunologia , Estudos Prospectivos , Reprodutibilidade dos Testes , Proteínas Virais/imunologia
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