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1.
Vaccines (Basel) ; 9(8)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34451973

RESUMO

Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which has reached pandemic proportions. A number of effective vaccines have been produced, including mRNA vaccines and viral vector vaccines, which are now being implemented on a large scale in order to control the pandemic. The mRNA vaccines are composed of viral Spike S1 protein encoding mRNA incorporated in a lipid nanoparticle and stabilized by polyethylene glycol (PEG). The mRNA vaccines are novel in many respects, including cellular uptake and the intracellular routing, processing, and secretion of the viral protein. Viral vector vaccines have incorporated DNA sequences, encoding the SARS-CoV-2 Spike protein into (attenuated) adenoviruses. The antigen presentation routes in MHC class I and class II, in relation to the induction of virus-neutralizing antibodies and cytotoxic T-lymphocytes, will be reviewed. In rare cases, mRNA vaccines induce unwanted immune mediated side effects. The mRNA-based vaccines may lead to an anaphylactic reaction. This reaction may be triggered by PEG. The intracellular routing of PEG and potential presentation in the context of CD1 will be discussed. Adenovirus vector-based vaccines have been associated with thrombocytopenic thrombosis events. The anti-platelet factor 4 antibodies found in these patients could be generated due to conformational changes of relevant epitopes presented to the immune system.

2.
BMC Infect Dis ; 21(1): 677, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34256735

RESUMO

BACKGROUND: SARS-CoV-2 has swept across the globe, causing millions of deaths worldwide. Though most survive, many experience symptoms of COVID-19 for months after acute infection. Successful prevention and treatment of acute COVID-19 infection and its associated sequelae is dependent on in-depth knowledge of viral pathology across the spectrum of patient phenotypes and physiologic responses. Longitudinal biobanking provides a valuable resource of clinically integrated, easily accessed, and quality-controlled samples for researchers to study differential multi-organ system responses to SARS-CoV-2 infection, post-acute sequelae of COVID-19 (PASC), and vaccination. METHODS: Adults with a history of a positive SARS-CoV-2 nasopharyngeal PCR are actively recruited from the community or hospital settings to enroll in the Northern Colorado SARS-CoV-2 Biorepository (NoCo-COBIO). Blood, saliva, stool, nasopharyngeal specimens, and extensive clinical and demographic data are collected at 4 time points over 6 months. Patients are assessed for PASC during longitudinal follow-up by physician led symptom questionnaires and physical exams. This clinical trial registration is NCT04603677 . RESULTS: We have enrolled and collected samples from 119 adults since July 2020, with 66% follow-up rate. Forty-nine percent of participants assessed with a symptom surveillance questionnaire (N = 37 of 75) had PASC at any time during follow-up (up to 8 months post infection). Ninety-three percent of hospitalized participants developed PASC, while 23% of those not requiring hospitalization developed PASC. At 90-174 days post SARS-CoV-2 diagnosis, 67% of all participants had persistent symptoms (N = 37 of 55), and 85% percent of participants who required hospitalization during initial infection (N = 20) still had symptoms. The most common symptoms reported after 15 days of infection were fatigue, loss of smell, loss of taste, exercise intolerance, and cognitive dysfunction. CONCLUSIONS: Patients who were hospitalized for COVID-19 were significantly more likely to have PASC than those not requiring hospitalization, however 23% of patients who were not hospitalized also developed PASC. This patient-matched, multi-matrix, longitudinal biorepository from COVID-19 survivors with and without PASC will allow for current and future research to better understand the pathophysiology of disease and to identify targeted interventions to reduce risk for PASC. Registered 27 October 2020 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT04603677 .


Assuntos
Bancos de Espécimes Biológicos , Teste para COVID-19/métodos , COVID-19/complicações , SARS-CoV-2/genética , Sobreviventes , Adulto , Idoso , COVID-19/sangue , COVID-19/epidemiologia , COVID-19/patologia , COVID-19/virologia , Colorado/epidemiologia , Progressão da Doença , Feminino , Seguimentos , Hospitalização , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Manejo de Espécimes , Adulto Jovem
3.
Vaccines (Basel) ; 9(4)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916180

RESUMO

The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of vaccine candidates for this disease and other emerging diseases. Several novel methods for preparing vaccine candidates are currently undergoing clinical evaluation in response to the urgent need to prevent the spread of COVID-19. In many cases, these methods rely on new approaches for vaccine production and immune stimulation. We report on the use of a novel method (SolaVAX) for production of an inactivated vaccine candidate and the testing of that candidate in a hamster animal model for its ability to prevent infection upon challenge with SARS-CoV-2 virus. The studies employed in this work included an evaluation of the levels of neutralizing antibody produced post-vaccination, levels of specific antibody sub-types to RBD and spike protein that were generated, evaluation of viral shedding post-challenge, flow cytometric and single cell sequencing data on cellular fractions and histopathological evaluation of tissues post-challenge. The results from this preliminary evaluation provide insight into the immunological responses occurring as a result of vaccination with the proposed vaccine candidate and the impact that adjuvant formulations, specifically developed to promote Th1 type immune responses, have on vaccine efficacy and protection against infection following challenge with live SARS-CoV-2. This data may have utility in the development of effective vaccine candidates broadly. Furthermore, the results of this preliminary evaluation suggest that preparation of a whole virion vaccine for COVID-19 using this specific photochemical method may have potential utility in the preparation of one such vaccine candidate.

4.
Vox Sang ; 116(10): 1076-1083, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33835489

RESUMO

BACKGROUND AND OBJECTIVES: Convalescent plasma (CP) has been embraced as a safe therapeutic option for coronavirus disease 2019 (COVID-19), while other treatments are developed. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not transmissible by transfusion, but bloodborne pathogens remain a risk in regions with high endemic prevalence of disease. Pathogen reduction can mitigate this risk; thus, the objective of this study was to evaluate the effect of riboflavin and ultraviolet light (R + UV) pathogen reduction technology on the functional properties of COVID-19 CP (CCP). MATERIALS AND METHODS: COVID-19 convalescent plasma units (n = 6) from recovered COVID-19 research donors were treated with R + UV. Pre- and post-treatment samples were tested for coagulation factor and immunoglobulin retention. Antibody binding to spike protein receptor-binding domain (RBD), S1 and S2 epitopes of SARS-CoV-2 was assessed by ELISA. Neutralizing antibody (nAb) function was assessed by pseudovirus reporter viral particle neutralization (RVPN) assay and plaque reduction neutralization test (PRNT). RESULTS: Mean retention of coagulation factors was ≥70%, while retention of immunoglobulins was 100%. Starting nAb titres were low, but PRNT50 titres did not differ between pre- and post-treatment samples. No statistically significant differences were detected in levels of IgG (P ≥ 0·3665) and IgM (P ≥ 0·1208) antibodies to RBD, S1 and S2 proteins before and after treatment. CONCLUSION: R + UV PRT effects on coagulation factors were similar to previous reports, but no significant effects were observed on immunoglobulin concentration and antibody function. SARS-CoV-2 nAb function in CCP is conserved following R + UV PRT treatment.


Assuntos
Anticorpos Neutralizantes , COVID-19 , Anticorpos Antivirais , COVID-19/terapia , Humanos , Imunização Passiva , Riboflavina , SARS-CoV-2 , Tecnologia , Raios Ultravioleta
5.
Vaccine ; 38(45): 7156-7165, 2020 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-32978002

RESUMO

Although vaccination with BCG prevents disseminated forms of childhood tuberculosis (TB), it does not protect against pulmonary infection or Mycobacterium tuberculosis (Mtb) transmission. In this study, we generated a complete deletion mutant of the Mtb Esx-5 type VII secretion system (Mtb Δesx-5). Mtb Δesx-5 was highly attenuated and safe in immunocompromised mice. When tested as a vaccine candidate to boost BCG-primed immunity, Mtb Δesx-5 improved protection against highly virulent Mtb strains in the murine and guinea pig models of TB. Enhanced protection provided by heterologous BCG-prime plus Mtb Δesx-5 boost regimen was associated with increased pulmonary influx of central memory T cells (TCM), follicular helper T cells (TFH) and activated monocytes. Conversely, lower numbers of T cells expressing exhaustion markers were observed in vaccinated animals. Our results suggest that boosting BCG-primed immunity with Mtb Δesx-5 is a potential approach to improve protective immunity against Mtb. Further insight into the mechanism of action of this novel prime-boost approach is warranted.


Assuntos
Mycobacterium bovis , Mycobacterium tuberculosis , Tuberculose , Sistemas de Secreção Tipo VII , Animais , Antígenos de Bactérias , Vacina BCG , Cobaias , Imunização Secundária , Camundongos , Mycobacterium tuberculosis/genética , Tuberculose/prevenção & controle , Vacinação
6.
Sci Rep ; 10(1): 7651, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32377001

RESUMO

Flow cytometers can now analyze up to 50 parameters per cell and millions of cells per sample; however, conventional methods to analyze data are subjective and time-consuming. To address these issues, we have developed a novel flow cytometry analysis pipeline to identify a plethora of cell populations efficiently. Coupled with feature engineering and immunological context, researchers can immediately extrapolate novel discoveries through easy-to-understand plots. The R-based pipeline uses Fluorescence Minus One (FMO) controls or distinct population differences to develop thresholds for positive/negative marker expression. The continuous data is transformed into binary data, capturing a positive/negative biological dichotomy often of interest in characterizing cells. Next, a filtering step refines the data from all identified cell phenotypes to populations of interest. The data can be partitioned by immune lineages and statistically correlated to other experimental measurements. The pipeline's modularity allows customization of statistical testing, adoption of alternative initial gating steps, and incorporation of other datasets. Validation of this pipeline through manual gating of two datasets (murine splenocytes and human whole blood) confirmed its accuracy in identifying even rare subsets. Lastly, this pipeline can be applied in all disciplines utilizing flow cytometry regardless of cytometer or panel design. The code is available at https://github.com/aef1004/cyto-feature_engineering.


Assuntos
Citodiagnóstico/métodos , Suscetibilidade a Doenças/imunologia , Citometria de Fluxo , Animais , Biomarcadores , Células Sanguíneas/metabolismo , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Camundongos , Mycobacterium tuberculosis/imunologia , Fenótipo , Tuberculose/diagnóstico , Tuberculose/imunologia , Tuberculose/microbiologia
7.
Curr Protoc Cytom ; 93(1): e74, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32421215

RESUMO

Flow cytometry allows the visualization of physical, functional, and/or biological properties of cells including antigens, cytokines, size, and complexity. With increasingly large flow cytometry panels able to analyze up to 50 parameters, there is a need to standardize flow cytometry protocols to achieve high-quality data that can be input into analysis algorithms. Without this clean data, algorithms may incorrectly categorize the cell populations present in the samples. In this protocol, we outline a comprehensive methodology to prepare samples for polychromatic flow cytometry. The use of multiple washing steps and rigorous controls creates high-quality data with good separation between cell populations. Experimental data acquired using this protocol can be analyzed via computational algorithms that perform end-to-end analysis. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of single-cell suspension for flow cytometry Support Protocol 1: Lung preparation Support Protocol 2: Counting cells on a flow cytometer Basic Protocol 2: Surface and intracellular flow cytometry staining Support Protocol 3: Single-color bead controls.


Assuntos
Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Animais , Contagem de Células , Espaço Intracelular/metabolismo , Pulmão/citologia , Camundongos Endogâmicos C57BL , Análise de Célula Única , Baço/citologia , Linfócitos T/imunologia
8.
Immunol Lett ; 217: 77-83, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31726189

RESUMO

Activated mouse B cells as compared to the resting B cells are known to internalize substantially more acid-functionalized single walled carbon nanotubes (AF-SWCNTs). It was hypothesized that the antigens coupled to AF-SWCNTs would also be taken up more efficiently by B cells. Further the enhanced uptake of the antigen by B cells may facilitate antigen presentation by B cells resulting in a better antibody response. Aim of this study was to test this hypothesis. Ovalbumin was chemically coupled to AF-SWCNTs that yielded a coupled product that had 0.08% of all carbon atoms in AF-SWCNTs occupied by ovalbumin. Coupling of ovalbumin to AF-SWCNTs was confirmed by staining the product with anti-ovalbumin antibodies. B cells incubated with ovalbumin-AF-SWCNT internalized more ovalbumin than the B cells incubated with free ovalbumin. Groups of mice were immunized subcutaneously with (a) free ovalbumin, (b) free ovalbumin and AF-SWCNTs at two different subcutaneous sites respectively on mice, and (c) ovalbumin-AF-SWCNT coupled product. In each case a primary immunization was followed by three weekly booster doses. It was found that the anti-ovalbumin antibody response assessed by ELISA, was highest in the group where ovalbumin coupled to AF-SWCNTs was used for immunization (p < 0.001). Antibody response in ovalbumin-AF-SWCNT group was comparable to the group where ovalbumin was used for immunization using complete and incomplete Freund's adjuvant (primary and secondary immunizations respectively). We propose that AF-SWCNTs could be explored as an adjuvant to improve the antibody response especially in vaccine development.


Assuntos
Linfócitos B/imunologia , Macrófagos/imunologia , Nanotubos de Carbono/química , Ovalbumina/imunologia , Adjuvantes Imunológicos , Animais , Formação de Anticorpos , Apresentação do Antígeno , Linfócitos B/metabolismo , Imunização/métodos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
9.
Nanotoxicology ; 13(6): 849-860, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31232140

RESUMO

Uptake of polydispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) in resting and LPS-activated B cells was studied using fluorescence-tagged AF-SWCNTs (FAF-SWCNTs). Activated B cells internalized substantially higher amounts of FAF-SWCNTs [76.5% AF-SWCNT+ B cells, mean fluorescence intensity (MFI) 720.6] as compared to the resting B cells [39.5% AF-SWCNT+ B cells, MFI 198.5]. B cells in S and G2/M phases were found to have significantly higher uptake of FAF-SWCNTs as compared to cells in G0/G1 phase. Confocal microscopy indicated that AF-SWCNTs were essentially localized on cell membrane in resting B cells, whereas in activated B cells, AF-SWCNTs were distributed throughout the cytoplasm. Targeting of AF-SWCNTs specifically to activated B cells in vivo was examined by first administering intravenously LPS-activated B cells tagged with fluorescence tracer (CFSE) in mice, followed by FAF-SWCNTs through the same route. It was found that FAF-SWCNTs were specifically taken up by CFSE+CD19+-activated B cells (95% FAF-SWCNT+ B cells, MFI 3725) as compared to CFSE- CD19+ resting B cells (31.1% FAF-SWCNT+ B cells, MFI 428). Administration (i.v.) of LPS resulted in a significant increase in the proportion of B cell in mouse spleen that was reduced by 68% by administering AF-SWCNTs. In control mice, the corresponding decrease in B cell proportion was 49%, which was significantly lower (p < 0.005) than the decline in LPS-treated mice. These results indicate that AF-SWCNTs may have the potential as an agent for depleting activated B cells in vivo.


Assuntos
Linfócitos B/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Animais , Linfócitos B/imunologia , Linfócitos B/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citoplasma/efeitos dos fármacos , Citoplasma/imunologia , Citoplasma/ultraestrutura , Fluorescência , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia
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