Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative pathogen of Coronavirus Disease 2019 (COVID-19). Successful vaccination aims to elicit neutralizing antibodies (NAbs) which inhibit viral infection. Traditional NAb quantification methods (neutralization assays) are labor-intensive and expensive, with limited practicality for routine use (e.g. monitoring vaccination response). Thus, a rapid (10-minute) lateral flow assay (LFA) for quantification of SARS-CoV-2 NAbs was developed. Using the NAb LFA, an 18-month longitudinal study assessing monthly NAb titers was conducted in a cohort of over 500 COVID-19 mRNA vaccine recipients. Three NAb response groups were identified: vaccine strong responders (VSRs), moderate responders (VMRs), and poor responders (VPRs). VSRs generated high and durable NAb titers. VMRs initially generated high NAb titers but showed more rapid waning with time post-vaccination. Finally, VPRs rarely generated NAb titers ≥1:160, even after 3rd dose. Although strong humoral responses correlate with vaccine effectiveness, viral-specific CD4+ and CD8+ T cells are critical for long-term protection. Discordant phenotypes of viral-specific CD8+ and CD4+CXCR5+ T follicular helper (cTfh) cells have recently been associated with differential NAb responses. The second portion of this dissertation was to investigate whether/how SARS-CoV-2 T cell responses differ in individuals with impaired NAb titers following mRNA vaccination. Thus, phenotypic and functional characterization of T cell activation across NAb response groups was conducted. It was hypothesized that VPRs would exhibit discordant SARS-CoV-2 T cell activation and altered cTfh phenotypes. Peripheral blood mononuclear cells were isolated from VPRs, VMRs, VSRs, naturally infected, and normal donors. SARS-CoV-2 responsive T cells were characterized using in vitro activation induced marker assays, multicolor flow cytometry, and multiplex cytokine analysis. Further, CXCR5+ cTfh were examined for chemokine receptor expression (CCR6 and CXCR3). Results demonstrated that despite differential NAb responses, activation of SARS-CoV-2 responsive CD4+ and CD8+ T cells was comparable across NAb groups. However, double-positive CD4+CD8+, CD8low, and activated CD4+CXCR5+CCR6-CXCR3+ (Tfh1-like) T cells were expanded in VPRs compared to VMR and VSRs. Interestingly, a unique population of CD8+CXCR5+ T cells was also expanded in VPRs. These novel findings may aid in identification of individuals with impaired or altered immune responses to COVID-19 mRNA vaccination.

Coccidioidomycosis or Valley Fever (VF) is an emerging fungal respiratory infection endemic to the southwest region of the United States, and parts of Mexico, Central and South America. Satellite cases have also been reported in Washington and Oregon. It is estimated that in Maricopa County alone, VF accounts for 10-30% of community-acquired pneumonia. Difficulty in diagnosis is largely attributed to lack of antibody reactivity to antigens used in diagnosis, especially early in disease. Serological detection of VF employs mycelial-phase culture filtrates as antigen. While culture filtrates are thought to provide the most specific diagnostic antigen, preparation includes the growth of large volume Coccidioides cultures which require employment of extensive safety precautions in a BSL3 setting. An additional concern with use of culture filtrates as an antigen source is batch variability, as expression of immunogenic proteins within each lot are variable. To address safety and batch variability concerns, this thesis proposes the use of recombinant Coccidioides proteins as a consistent and reliable antigen source. For the purpose of this study, I expressed known antigenic Coccidioides proteins in a eukaryotic, recombinant protein expression system. Recombinant endochitinase-1 (rCTS1) and recombinant heat-labile antigen (rHL-Ag) were evaluated for serologic reactivity by ELISA, using a sample set of 55 known serologically positive and 55 known negative human sera specimens, previously tested in Mayo Clinic Arizona (MCA) serologic laboratories. Evaluation by ELISA demonstrated 94.55% sensitivity and 92.72% specificity using combined rCTS1 and rHL-Ag as an antigen source, indicating promising diagnostic utility.