Introduction The rapid expansion of the novel SARS-CoV-2 virus has raised serious public health concerns due to the possibility of misdiagnosis in regions where arboviral diseases are endemic. We performed the first study in northern Peru to describe the detection of SARSCoV-2 IgM antibodies in febrile patients with a suspected diagnosis of dengue and chikungunya fever. Materials and methods A consecutive cross-sectional study was performed in febrile patients attending primary healthcare centers from April 2020 through March 2021. Patients enrolled underwent serum sample collection for the molecular and serological detection of DENV and CHIKV. Also, serological detection of IgM antibodies against SARS-CoV-2 was performed. Results 464 patients were included during the study period, of which (40.51%) were positive for one pathogen, meanwhile (6.90%) presented co-infections between 2 or more pathogens. The majority of patients with monoinfections were positive for SARS-CoV-2 IgM with (73.40%), followed by DENV 18.09% and CHIKV (8.51%). The most frequent co-infection was DENV + SARS-CoV-2 with (65.63%), followed by DENV + CHIKV and DENV + CHIKV + SARSCoV-2, both with (12.50%). The presence of polyarthralgias in hands (43.75%, p<0.01) and feet (31.25%, p = 0.05) were more frequently reported in patients with CHIKV monoinfection. Also, conjunctivitis was more common in patients positive for SARS-CoV-2 IgM (11.45%, p<0.01). The rest of the symptoms were similar among all the study groups. Conclusion SARS-CoV-2 IgM antibodies were frequently detected in acute sera from febrile patients with a clinical suspicion of arboviral disease. The presence of polyarthralgias in hands and feet may be suggestive of CHIKV infection. These results reaffirm the need to consider SARS-CoV-2 infection as a main differential diagnosis of acute febrile illness in arboviruses endemic areas, as well as to consider co-infections between these pathogens. Copyright: / Revisión por pares

Background: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, which enter the host cells through interactions between the receptor-binding domain (RBD) on the S-protein and the ACE-2 receptor on the host cell. A novel type of vaccine strategy is peptide vaccines, with great potential as a faster and more selective approach to conventional vaccine development. This study focuses on the possibility of generating an antibody response through synthetic peptides harboring B cell epitopes.  Aim: This project aims to investigate the potential of immunogenic peptides to generate an antibody response when used as synthetically produced peptides. As proof-of-concept, the project studies the interactions between previously identified monoclonal antibodies with defined B cell epitopes and the corresponding peptide sequences.  Method: The interactions are evaluated by different ELISA experiments. The candidate peptides are additionally investigated on their binding to polyclonal serum with established S reactive antibodies. Furthermore, the project includes synthesis of one peptide by solid phase peptide synthesis. Results: The ELISA experiments presented no interaction between the synthetic peptides and the monoclonal antibodies or human sera.  Conclusion: The project fulfilled its aim to study the interaction between the B cell epitopes and the monoclonal antibodies. However, no binding was observed. Despite the many advantages in production and stability, development of B cell epitope vaccines come with many challenges. Future will entail if synthetic peptides harboring B cell epitopes can be used as vaccines, or if peptide vaccines will be a focus when a T cell response is to be induced.

No description available.

<p>  </p> <p>Designing and developing a diagnostic technology that is capable of highly sensitive and specific, multiplexed, high-throughput, and quantitative biomarker assays for disease diagnosis and progression is of the upmost importance in modern medicine and patient care. Current diagnostic assays capable of multiplexed and high-throughput analysis include mass spectrometry, electrochemistry, polymerase chain reaction (PCR), and fluorescence-based techniques, however, these techniques suffer from a lack in sensitivity, false responses, or extensive sample processing that are detrimental to clinical diagnostics. To overcome these sensitivity challenges, the field of nanoplasmonics has become utilized when developing diagnostic assays. Plasmonic-based diagnostic tests utilize the unique optical, chemical, and physical property of nanoparticles to increase the sensitivity of the assay. In this dissertation, novel diagnostic platforms that utilize nanoparticles and their localized surface plasmon resonance (LSPR) property will be introduced. LSPR is an optical property in noble metallic nanoparticles that is referred to as the collective oscillation of free electrons upon light irradiation. It is highly dependent on the shape, size, and dielectric constant (refractive index) of the surrounding medium of the nanoparticle and LSPR sensing is based on a change in these properties. In this dissertation, the LSPR property is utilized to fabricate nanoplasmonic-based diagnostic platforms that are capable of multiplexed and high-throughput biomarker assays, with high sensitivity and specificity. The work presented in this dissertation is presented as six chapters, (1) Introduction. (2) Methods, (3) Fabrication of a LSPR-based multiplexed and high-throughput biosensor platform and its application in performing microRNA assays for the diagnosis of bladder cancer. In this chapter, the advancement of single-plex solid state LSPR-based biosensors into a multiplexed and high-throughput diagnostic biosensor platform is reported for the first time. The diagnostic biosensor platform is first fabricated utilizing different gold nanoparticles (spherical nanoparticles, nanorods, and triangular nanoprisms), and then with the gold triangular nanoprisms as the nanoparticle of choice, microRNA assays were performed. The developed biosensor platform is capable of assaying five different types of microRNAs simultaneously at an attomolar limit of detection. Additionally, five microRNA were assayed in 20-bladder cancer patient plasma samples. (4) Development/optimization of the biosensor platform presented in Chapter 3 for the detection of COVID-19 biomarkers. In this chapter, the biosensor platform utilized in Chapter 3 was designed to assay 10 different COVID-19 specific biomarkers from three classes (six viral nucleic acid gene sequences, two spike protein subunits, and two antibodies) with limit of detections in the attomolar range and with high specificity. The high-throughput capability of the biosensor platform was advanced, with the platform performing analysis of a single biomarker in 92 patient samples simultaneously. Additionally, the biomarker platform was utilized to assay all 10 biomarkers in a total of 80 COVID-19 patient samples.  (5) Further optimization of the biosensor platform for the development of a highly specific antibody detection test for COVID-19. During the COVID-19 pandemic, knowledge was gained on the specificity of antibodies produced against COVID-19. In this chapter, that knowledge was applied towards the optimization of the biosensor platform presented in Chapter 4 in order to assay SARS-CoV-2 neutralizing antibody IgG. The optimization of the biosensor platform included the size of the gold triangular nanoprisms and the receptor molecule of choice. The biosensor platform assayed this highly specific COVID-19 IgG antibody with a limit of detection as low as 30.0 attomolar with high specificity and no cross reactivity. Additionally, as a proof of concept, the biosensor platform was utilized in a high-throughput format to assay SARS-CoV-2 IgG in a large cohort of 121 COVID-19 patient samples simultaneously. (6) Advancement of the biosensor platform from a 96-well plate to a 384-well plate and its application in assaying microRNA for early diagnosis of pancreatic cancer. In this chapter, the high-throughput capabilities of the biosensor platform presented in Chapters 3-5 was expanded by increasing the sensor amount in one platform from 92 to 359. The 384-well plate biosensor platform was designed, optimized, and utilized to perform microRNA assays for early-stage pancreatic cancer diagnosis. The optimization of the biosensor platform included the manipulation of LSPR-based parameters and the -ssDNA receptor molecule in order to obtain low limit of detections (high sensitivity). Additionally, the biosensor platform assayed two microRNA in a large cohort (n=110) of pancreatic cancer and chronic pancreatitis patient samples. </p>

Coronaviruses have caused three large outbreaks in the past century. The most recent one, also still ongoing, is represented by the SARS-CoV-2/Covid-19 pandemic. Efforts have been taken to develop efficient vaccines and antivirals and one of the major virus-based targets in drug development is represented by the main protease of these viruses. Main proteases are proteins (cysteine hydrolases) with high level of conservation among different coronaviruses and have an important role in the virus life cycle. Due to the need of developing broad-spectrum antivirals against Coronaviruses, this study aimed to set up a CPE-based assay for testing compounds against the main protease of human coronavirus 229E. An optimized TCID50 protocol was established by using MRC-5 cells, at a density of 1x104 cells/ml with a 3h incubation prior infection with a concentration of 10-1 of HCoV-229E. The cell viability was assessed through MTT assay. Using reference compounds, with previously demonstrated antiviral potency against the main protease of different coronaviruses (GC-376, Nirmatrelvir), the efficiency of the conceived assay was validated (GC-376 EC50 = 1.24 μM; Nirmatrelvir Ec50= 0.72 μM). Compound 19 was proved to also be active against the main protease of HCoV-229E (EC50 = 0.22 μM), and together with previous findings, it was concluded that this compound has a broad-spectrum activity. Newly developed compounds MP17 and MP19 were also demonstrated to be efficient against HCoV-229E. As a future perspective, further investigations of these compounds should take place for the identification of EC50 values.

Time-resolved cryo-EM has already provided ground-breaking discoveries in various fields, including structural biology, biochemistry, and drug development. Compared to traditional structural biology methods where mostly stabilized conformations are reconstructed, the main advantage of time-resolved cryo-EM is its ability to capture dynamic processes in biological samples at near-atomic resolution, which allows for studying biological structures as they change and interact in real-time. In this project, I focused on the expression and purification of the individual proteins of two dynamic molecular complexes – Staphylococcus aureus (S. aureus) DNA gyrase and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) replication-transcription complex – and attempted to assemble them into their functional forms for cryo-EM imaging.  Both of these complexes are interesting drug targets as they play an essential role in nucleic acid replication. The function of DNA gyrase is to modulate DNA supercoiling, facilitate DNA replication, and resolve intertwined DNA molecules. The replication-transcription complex of SARS-CoV-2 comprises, among other proteins, the RNA-dependent RNA polymerase, which, together with non-structural proteins 7 and 8, is responsible for the replication of the viral genome. There are still many questions about the underlying mechanisms of these key processes, and time-resolved cryo-EM studies will provide valuable information to advance our understanding of them. Here I present expression and purification protocols for S. aureus DNA gyrase subunits A and B and SARS-CoV-2 non-structural proteins 7, 8 and 12. DNA gyrase subunits A and B were expressed in Escherichia coli (E. coli) and purified in several steps, including affinity chromatography (His-Trap), ion exchange chromatography (IEX) and size exclusion chromatography (SEC). Despite many challenges with gyrase A precipitation, I obtained enough of both subunits for the intended cryo-EM. Different strategies to assemble them into a functional tetramer were tested but did not result in the expected outcome. The gained knowledge about the behaviour of the subunits in solution will serve as a basis for further optimization of the protocols before the assembly of the complex can be attempted again. Non-structural proteins 7 and 8 were expressed in E. coli as a polyprotein and successfully purified using His-Trap and SEC. I obtained a great amount of the polyprotein and established a protocol for its cleavage. Nsp12 was expressed using the baculovirus-insect cell expression system. The immunofluorescence assay data showed that the tested lipofection protocol works, and nsp12 is being produced in sufficient quantities. This result provides a solid base for further experiments to establish a purification method and assemble the nsp12-nsp7-nsp8 complex for cryo-EM imaging.

No description available.

The pandemic that struck the entire world 2020 caused by the SARS-CoV-2 (COVID-19) virus, will have an enormous interest for statistical and economical analytics for a long time. While the pandemic of 2020 is not the first that struck the entire world, it is the first pandemic in history where the data were gathered to this extent. Most countries have collected and shared its numbers of cases, tests and deaths related to the COVID-19 virus using different storage methods and different data types. Gaining quality data from the COVID-19 pandemic is a problem most countries had during the pandemic, since it is constantly changing not only for the current situation but also because past values have been altered when additional information has surfaced. The importance of having the latest data available for government officials to make an informed decision, leads to the usage of Business Intelligence tools and techniques for data gathering and aggregation being one way of solving the problem. One of the mostly used software to perform Business Intelligence is the Microsoft develop Power BI, designed to be a powerful visualizing and analysing tool, that could gather all data related to the COVID-19 pandemic into one application. The pandemic caused not only millions of deaths, but it also caused one of the largest drops on the stock market since the Great Recession of 2007. To determine if the deaths or other reasons directly caused the drop, the study modelled the volatility from index funds using Generalized Autoregressive Conditional Heteroscedasticity. One question often asked when talking of the COVID-19 virus, is how deadly the virus is. Analysing the effect the pandemic had on the mortality rate is one way of determining how the pandemic not only affected the mortality rate but also how deadly the virus is. The analysis of the mortality rate was preformed using Seasonal Artificial Neural Network. Forecasting deaths from the pandemic using the Seasonal Artificial Neural Network on the COVID-19 daily deaths data.

Effectively treating infectious diseases often requires a multi-step approach to target different components involved in disease pathogenesis. Similarly, the COVID-19 pandemic has become a global health crisis that requires a comprehensive understanding of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection to develop effective therapeutics. One potential strategy to instill greater immune protection against COVID-19 is boosting the innate immune system. This boosting, termed trained immunity, employs immune system modulators to train innate immune cells to produce an enhanced, non-specific immune response upon reactivation following exposure to pathogens, a process that has been studied in the context of and clinical studies prior to the COVID-19 pandemic. Evaluation of the underlying pathways that are essential to inducing protective trained immunity will provide insight into identifying potential therapeutic targets that may alleviate the COVID-19 crisis. Here we review multiple immune training agents, including Bacillus Calmette-Guérin (BCG), β-glucan, and lipopolysaccharide (LPS), and the two most popular cell types involved in trained immunity, monocytes and natural killer (NK) cells, and compare the signaling pathways involved in innate immunity. Additionally, we discuss COVID-19 trained immunity clinical trials, emphasizing the potential of trained immunity to fight SARS-CoV-2 infection. Understanding the mechanisms by which training agents activate innate immune cells to reprogram immune responses may prove beneficial in developing preventive and therapeutic targets against COVID-19.

This study compared the immunogenicity of inactivated SARS-CoV-2 vaccines between people living with HIV (PLWH) and HIV-negative individuals. We recruited 120 PLWH and 53 HIV-negative individuals aged 18-59 years who had received an inactivated SARS-CoV-2 vaccine in two Chinese cities between April and June 2021. Blood samples were tested for immunogenicity of the inactivated SARS-CoV-2 vaccines. The prevalence and severity of adverse events associated with SARS-CoV-2 vaccines were similar between PLWH and HIV-negative individuals. The seropositivity of neutralizing activity against authentic SARS-CoV-2, of the total amount of antibody (total antibody) and of S-IgG were 71.3%, 81.9%, and 92.6%, respectively, among fully vaccinated PLWH. Among all participants, PLWH had lower neutralizing activity, total antibody, S-IgG, and T-cell-specific immune response levels, compared to HIV-negative individuals, after controlling for types of vaccine, time interval between first and second dose, time after receiving the second dose, and sociodemographic factors. PLWH with a longer interval since HIV diagnosis, who received their second dose 15-28 days prior to study commencement, and who had an interval of ≥21 days between first and second dose had higher neutralizing activity levels. The immunogenicity of the inactivated SARS-CoV-2 vaccines was lower among PLWH as compared to HIV-negative individuals. Vaccination guideline specific for PLWH should be developed.