FUTIs: an In-Person or Online Graphing, Bioinformatics, and Scientific Literacy Exercise That Explores the Presence of Antibiotic Resistance in Foodborne Urinary Tract Infections.

We developed a course-based undergraduate research experience (CURE) that gives students an opportunity to practice the process of science in a context that intersects with their everyday lives: purchasing grocery store chicken. Student mastery of concepts was assessed by pre- and postassessment questions and lab report worksheets that guided them through the process of writing a scientific paper. Learning to produce graphs from large data sets and comparing the results with published data emphasized quantitative reasoning, while working as a group and writing helped students practice scientific communication. Most students (>90%) met the learning objectives, and students in both groups reported feeling more confident producing graphs and figures; they also showed large gains in confidence and interest in bioinformatics. Lab protocols require biosafety level 2 safety guidelines; however, students in an online or dry lab setting can use the compiled data sets and whole-genome sequences to complete the objectives. Group discussions and essay prompts at the end encourage students to use evidence-based arguments to make decisions that impact the global issue of antimicrobial resistance.

Primer Design for Reverse Transcription-Quantitative PCR Simulation: an Online Lab Exercise Using a SARS-CoV-2 Model.

Since the start of the 2019 coronavirus disease (COVID-19) pandemic, many microbiology lab activities have been conducted online. We produced a simple PCR primer design and virtual PCR activity for introductory students to use as part of an online laboratory course or as an independent activity in a traditional laboratory setting. Most students are aware of basic PCR concepts but struggle with important details, such as how PCR is specific and how false positives and negatives can be generated in a diagnostic test that is not well designed. This exercise helps students review molecular biology concepts within the context of a test that was commonplace during the COVID-19 pandemic. We found that nursing students and Biology and non-Biology majors were able to complete the worksheet as a group with minimal instructor input. This could be used as a stand-alone activity, as a warm-up for other bioinformatics exercises, or as a prelab activity for actual in-lab quantitative PCR experiments, such as the one offered by miniPCR bio. With minor modifications, it could also be used with more advanced students.

Beyond the Traditional Classroom: Increased Course Structure and Cooperative Learning Remove Differences in Achievement between Students in an In-Person versus Hybrid Microbiology Course.

The increase in online learning brought on by the COVID-19 pandemic will likely result in a greater availability of online and hybrid course offerings. In this study, students enrolled in parallel sections of a microbiology lab course with in-person labs and either face-to-face (F2F) or all-online lectures (hybrid, H). Course material and method of assessment in the two sections were identical; student demographics were similar. In the first year, F2F students scored significantly higher on two out of four exams. In the second year, two interventions were introduced: team-building activities (in both sections) and online group discussions (H only). Students in both the F2F and H sections reported similar positive teamwork reviews based on Comprehensive Assessment of Team Member Effectiveness (catme.org) and survey data. Although the COVID-19 pandemic eventually forced all learning online, exam scores from the two sections in the first half of the semester were similar, suggesting that the interventions were effective. In both sections, exam scores were positively correlated with entering grade point averages. This study adds to the body of literature supporting the effectiveness of hybrid learning.

Production and Characterization of SIV-Specific CAR/CXCR5 T Cells.

HIV-specific chimeric antigen receptor (CAR) T cells that target lymphoid follicles have the potential to functionally cure HIV infection. CD8+ T cells, NK cells, or peripheral blood mononuclear cells (PBMC) may be modified to express HIV-specific CARs as well as follicular homing molecules such as CXCR5 to target the virally infected T follicular helper cells that concentrate within B cell follicles during HIV infection. This chapter outlines methods utilizing a simian immunodeficiency virus (SIV) rhesus macaque model of HIV to produce transduced T cells from primary PBMCs. Methods are presented for production of an SIV-specific CAR/CXCR5-encoding retrovirus used to transduce primary rhesus macaque PBMCs. Procedures to evaluate the functionality of the expanded CAR/CXCR5 T cells in vitro and ex vivo are also presented. An in vitro migration assay determines the ability of the T cells expressing CAR/CXCR5 to migrate to the CXCR5 ligand CXCL13, while an ex vivo migration assay allows measurement of the transduced T cell migration into the B cell follicle. Antiviral activity of the CAR/CXCR5 transduced T cells is determined using a viral suppression assay. These methods can be used to produce T cells for immunotherapy in SIV-infected rhesus macaques and to evaluate the functionality of the cells prior to infusion. Similar procedures can be used to produce HIV-specific CAR/CXCR5 T cells.

Low levels of SIV-specific CD8+ T cells in germinal centers characterizes acute SIV infection.

CD8+ T cells play an important role in controlling of HIV and SIV infections. However, these cells are largely excluded from B cell follicles where HIV and SIV producing cells concentrate during chronic infection. It is not known, however, if antigen-specific CD8+ T cells are excluded gradually as pathogenesis progresses from early to chronic phase, or this phenomenon occurs from the beginning infection. In this study we determined that SIV-specific CD8+ T cells were largely excluded from follicles during early infection, we also found that within follicles, they were entirely absent in 60% of the germinal centers (GCs) examined. Furthermore, levels of SIV-specific CD8+ T cells in follicular but not extrafollicular areas significantly correlated inversely with levels of viral RNA+ cells. In addition, subsets of follicular SIV-specific CD8+ T cells were activated and proliferating and expressed the cytolytic protein perforin. These studies suggest that a paucity of SIV-specific CD8+ T cells in follicles and complete absence within GCs during early infection may set the stage for the establishment of persistent chronic infection.

Simian Immunodeficiency Virus (SIV)-Specific Chimeric Antigen Receptor-T Cells Engineered to Target B Cell Follicles and Suppress SIV Replication.

There is a need to develop improved methods to treat and potentially cure HIV infection. During chronic HIV infection, replication is concentrated within T follicular helper cells (Tfh) located within B cell follicles, where low levels of virus-specific CTL permit ongoing viral replication. We previously showed that elevated levels of simian immunodeficiency virus (SIV)-specific CTL in B cell follicles are linked to both decreased levels of viral replication in follicles and decreased plasma viral loads. These findings provide the rationale to develop a strategy for targeting follicular viral-producing (Tfh) cells using antiviral chimeric antigen receptor (CAR) T cells co-expressing the follicular homing chemokine receptor CXCR5. We hypothesize that antiviral CAR/CXCR5-expressing T cells, when infused into an SIV-infected animal or an HIV-infected individual, will home to B cell follicles, suppress viral replication, and lead to long-term durable remission of SIV and HIV. To begin to test this hypothesis, we engineered gammaretroviral transduction vectors for co-expression of a bispecific anti-SIV CAR and rhesus macaque CXCR5. Viral suppression by CAR/CXCR5-transduced T cells was measured in vitro, and CXCR5-mediated migration was evaluated using both an in vitro transwell migration assay, as well as a novel ex vivo tissue migration assay. The functionality of the CAR/CXCR5 T cells was demonstrated through their potent suppression of SIVmac239 and SIVE660 replication in in vitro and migration to the ligand CXCL13 in vitro, and concentration in B cell follicles in tissues ex vivo. These novel antiviral immunotherapy products have the potential to provide long-term durable remission (functional cure) of HIV and SIV infections.

The human IL-15 superagonist ALT-803 directs SIV-specific CD8+ T cells into B-cell follicles.

Sequestering of latent HIV in follicular helper T cells within B-cell follicles that largely exclude cytotoxic T cells is a major barrier to cellular immune-based approaches to eradicate HIV. Here, we show that the clinical-grade human interleukin-15 (IL-15) superagonist ALT-803 activates and redirects simian immunodeficiency virus (SIV)-specific CD8+ T cells from the peripheral blood into B-cell follicles. In agreement with the increased trafficking of SIV-specific cytotoxic T cells to sites of cryptic viral replication, lymph nodes of elite controlling macaques contained fewer cells expressing SIV RNA or harboring SIV DNA post-ALT-803 treatment. These data establish ALT-803 as an immunotherapeutic for HIV and other chronic viral pathogens that evade host immunity by persisting in B-cell follicles.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues.

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled "Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo." The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.