M.S. - Biomedical Sciences (Tropical Medicine)

Permanent URI for this collectionhttps://hdl.handle.net/10125/1938

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COVID-19-related hospitalization and mortality among Native Hawaiians and other Pacific Islanders in Hawaii
(University of Hawai'i at Manoa, 2025) Nicholas, Chelsea; Sy, Angela; Biomed Science (Tropical Medicine)
OBJECTIVE. To examine associations between pre-existing health conditions and clinical outcomes among racial and ethnic groups of individuals hospitalized for COVID-19. METHODS. De-identified clinical data from the Queen’s Healthcare System (QHS) on COVID-19 patients admitted from March 15, 2020, to April 2, 2023, were reviewed (N=7,365). Selection criteria included all patients with a positive test for SARS-CoV-2 on qualitative polymerase chain reaction assay. The outcome measures of interest were ICU with mechanical ventilation (MV) and in-hospital death. Descriptive results on demographics, type of health insurance, vaccination status, clinical care outcomes, and comorbidities were analyzed. Comorbidities included asthma, atrial fibrillation, chronic kidney disease (CKD), diabetes, heart failure, hypertension, obesity III or body-mass index (BMI) of 40+ lbs/in2, pneumonia, and smoking history. Bivariate analysis was used to measure associations between clinical outcomes, comorbidities, and race/ethnicity. Odds ratios with 95% confidence intervals of comorbidities by clinical outcome among race and ethnic groups were calculated and adjusted for race, age, sex, insurance, and vaccination status. Statistical analysis was performed using R, version 4.2.2. Values of p≤0.05 were considered statistically significant. RESULTS. Of the 7,365 COVID-19 patients in this study, 18% self-reported as NH White, 13% as NH, 16% as OPI, 24% as Filipino, 11% as Japanese, 10% as Other Asian, and 8% as Others. Overall, NH patients were 51% female, aged 55 years, 39% relied on government insurance, 57% were unvaccinated, 7.5% died in hospital, 55% had a history of smoking, and had a BMI of 32 lbs/in2. OPI patients were 54% male, aged 53 years, 50% relied on Medicaid insurance, 71% were unvaccinated, 6.9% died, 47% had diabetes, and had a BMI of 35 lbs/in2. Of all study patients, 11% utilized MV in the ICU and 8% died. NH (aOR=1.7, CIs=1.3-2.2, p<0.001), and OPI (aORs=1.4, CIs=1.0-1.8, p=0.04) had higher odds of ICU ventilation. For in-hospital mortality, NH (aORs=1.6, CIs=1.1-2.3, p=0.01) and OPI (aORs=1.4, CIs=1.0-2.1, p=0.05) had higher odds. CONCLUSION. NHOPI COVID-19 patients hospitalized in Queens Healthcare System were significantly younger, had higher BMIs, and exhibited higher rates of hypertension, diabetes, CKD, and smoking history compared to other racial groups. Unvaccinated status, age 50 years and older, male sex, NH or OPI ethnicity, and the presence of pneumonia or heart failure were each independently associated with increased odds of severe COVID-19 clinical outcomes. The findings confirmed persistent health disparities and patient predictors of severe COVID-19. Culturally and linguistically tailored prevention and outreach strategies and efforts addressing the underlying causes of morbidity and mortality are critical for NHOPI communities in Hawaii.
Novel antiviral drug targets for screening potential FDA-approved compounds to counter Flavivirus infection
(University of Hawai'i at Manoa, 2025) Bilog-Mina, Jaymie D.; Kaufusi, Pakieli H.; Biomed Science (Tropical Medicine)
Dengue virus (DENV) and West Nile virus (WNV) are major global health concerns, with WNV prevalent in the U.S. and DENV cases increasing in the U.S. with cases detected in Hawaii, Florida and Texas. Native Hawaiians, Other Pacific Islanders (NHOPIs), and Filipinos have an elevated risk of severe WNV and DENV disease due to high rates of chronic conditions. Currently, no antiviral treatments exist for flavivirus infections, highlighting the need for new drug targets. NS3, a key enzyme in flavivirus replication, must localize to the replication organelle (RO), composed of convoluted membranes (CMs) and vesicle packets (VPs). This study explores the role of NS4A, NS4B, and NS1 in facilitating NS3's movement and stabilization to the rough endoplasmic reticulum (ER), and in the RO, respectively. HEK293 cells were transfected with NS3 in combination with NS4A, NS4B, or NS4AB, with Sec61β-RFP (an ER marker), under WNV-infected and uninfected conditions. High resolution confocal imaging and Pearson Correlation Coefficient (PCC) analysis demonstrated that neither NS4A, NS4B, or NS4AB is involved in the ER localization of NS3. However, NS3 localizes to the NS4A-, NS4B-, and NS4AB-induced RO (which appears as fluorescent aggregates), indicating a random recruitment event, where cytoplasmic NS3 was enwrapped during RO formation. In infected cells, NS3 in combination with NS4A, NS4B, and NS4AB was additionally recruited to the ER before reaching the RO, as expected due to the presence of other native viral proteins. This observation also suggests that other viral proteins besides NS4A and NS4B play a role in NS3 ER localization and stabilization in the RO. NS1 in combination with NS3 and NS4A, NS4B, NS4AB, or Sec61β-RFP, were assayed in non-permeabilized and permeabilized conditions. NS1 is partially colocalized with NS4A, NS4B, or NS4AB in both the ER and the foci in non-permeabilized conditions. Additionally, NS1 is visually evident on the plasma membrane (PM) as expected for hexameric NS1. NS1 association with these proteins increased in permeabilized conditions, as expected, since this enables lumen-localized NS1 to be more accessible to the antibody staining, but the PM associated hexameric NS1 is visually decreasing. When NS3 was added to these combinations, it was partially visualized in the ER and the RO in both non-permeabilized and permeabilized conditions. These findings suggest two possible mechanisms for NS3 recruitment: (1) random encapsulation during RO formation and (2) a sequential process requiring additional viral factors. NS3 reaches the ER either through random encapsulation by the RO or via a stepwise mechanism involving NS2B. NS3 is possibly retained in the RO with mechanism (1), but it may be partially stabilized in the ER and the RO by NS1, but not NS4A, NS4B, or NS4AB with mechanism (2). Understanding these interactions may help identify new antiviral drug targets for further development of safe and effective anti-flaviviral therapies.
Ingenuity pathway analysis of biomarkers and immune mediators of Kawasaki disease
(University of Hawai'i at Manoa, 2025) Aguilar, Tatiana; Nerurkar, Vivek R.; Biomed Science (Tropical Medicine)
Kawasaki Disease (KD) is an acute systemic vasculitis of unknown etiology primarily affecting children younger than five. As the etiology of KD remains unknown, diagnosis relies on a trained clinician identifying the sequential appearance of a constellation of clinical signs, and treatment consists of non-specific suppression of inflammation. A multitude of biomarkers associated with acute KD have been studied to understand the underlying mechanisms that may lead to a distinguishable diagnosis. Ingenuity Pathway Analysis (IPA) software is an essential tool for visualizing canonical pathways, upstream and downstream regulators and signaling networks. This study employed IPA to analyze the protein expression profiles of select markers for inflammation, tissue degradation, and vascular remodeling in clinically characterized KD patient serum samples to understand the pathophysiological pathways in acute KD, the development of coronary artery abnormalities (CAA) in KD patients, and intravenous immunoglobulin (IVIG) treatment response. While studies have highlighted the identification of potential biomarkers related to the immune response behind KD, subgroup analyses associated with KD publicly available within the IPA platform did not include all three clinical phases of KD, the acute, subacute and convalescent, and did not include analyses based on the presence or absence of CAA. Comparative analysis not only between the clinical phases of KD, but also between various clinical outcomes in KD patients (i.e., presentation of CAA and IVIG resistance), will allow for a better understanding of KD immunopathogenesis, identify prognostic biomarkers for diagnosis of acute KD and risks of CAA formation, and differentiate between IVIG resistant and responsive patients.
ASSESSING THE IMPEDIMENTS ASSOCIATED WITH SARS-COV-2 WHOLE GENOME SEQUENCING
(University of Hawai'i at Manoa, 2024) Salomon, Renn Silve Chua; Nerurkar, Vivek R.; Biomed Science (Tropical Medicine)
Genomic surveillance can help track the emergence of SARS-COV-2 variants with novel mutations that can alert scientists and policymakers to ensure a proper response is implemented, such as updating vaccine formulations and public health policies. Due to logistics and costs, whole genome sequencing (WGS) cannot be conducted on all nasopharyngeal swabs (NPS). Further, there are regions of ambiguity in each sequence, which needs to be resolved. Therefore, the objective of this research is to enhance the WGS workflow for the submission of high-coverage SARS-CoV-2 sequences to global genomic databases. COVID-19-positive NPS were collected from clinical laboratories in Oahu and underwent a WGS workflow. During the workflow, different measurements that ascertain viral genomic levels were assessed to enhance the WGS workflow using statistical analysis to predict if a nucleotide sequence meets the criteria for submission to GenBank. Furthermore, if dropout regions were observed in the sequences, primers were designed to flank these regions for Sanger sequencing. This Sanger sequence was compared to an updated in-house developed novel bioinformatic workflow that fills in the ambiguous bases using an updated consensus reference sequence. These results demonstrate that utilizing a DNA cut-off of 1.096 ng/ µL increases the submission rate of SARS-CoV-2 sequences to the GenBank database by 15%. Additionally, the proposed updated bioinformatic workflow decreased ambiguous bases within a sequence and had a high coverage accuracy of 99.5-100% when compared to the Sanger sequencing. These data suggest that the updated bioinformatic workflow described in this thesis will enhance the quality of the WGS sequences submitted to various global genomic databases. These findings have the potential to be applied to other pathogens currently being monitored such as the influenza virus or antimicrobial-resistant bacteria.
Dysregulation of Complement Components Associated with Inflammation and Coagulation in Virally Suppressed People Living with HIV (PLWH)
(University of Hawai'i at Manoa, 2024) Subia, Natalie Tejero; Park, Juwon; Biomed Science (Tropical Medicine)
Although HIV has become a manageable chronic disease with the introduction of antiretroviral therapy (ART), people living with HIV (PLWH) experience a higher prevalence of age-related non-AIDS comorbidities (NACMs), such as cardiovascular disease (CVD). PLWH often presents chronic inflammation and higher thrombogenicity, which are key contributing factors for NACM’s CVD development. The interplay between the complement system and platelets with neutrophil activation and neutrophil extracellular traps (NETs) has been a major contribution to chronic inflammation and higher thrombogenicity, but little attention has been directed to its association with virally suppressed PLWH in the context of NACM CVD. We used 40 virally suppressed HIV-seropositive plasma samples and 39 HIV-seronegative plasma samples to quantify and correlate important complement components (C2, C3a, C5a, C9) and platelet activation (von-Willebrand factor-A2 (vWF-A2), ADAMTS13, tissue factor (TF), protein C, and fibrinogen) analytes. We then used ex-vivo NET assays to see further correlations with plasma proteins in inducing NET formation. Results showed that participants had similar demographics, blood parameters, and co-morbidities, with significant differences in hepatitis B and C between groups. PLWH plasma displayed significant altered differences in complement components C2 and C5a, with both of these markers significantly strongly correlated with platelet and neutrophil NET activation markers. C2 also significantly positively correlated with chronic inflammatory markers (serum amyloid A (SAA), serum amyloid P (SAP), IL-1beta, and vascular endothelial growth factor VEGF)). Furthermore, HIV status was a predictive value of C2 and C5a concentration levels in PLWH’s plasma. PLWH’s plasma was also able to induce NETosis compared to HIV-negative plasma, showing that these soluble factors can contribute to both NETosis and non-lytic NET formation as well, and platelets specifically can facilitate NETosis. Our findings highlight the association between complement dysregulation, inflammation, and coagulation in virally suppressed PLWH’s plasma.
USING AND OPTIMIZING DROSOPHILA S2 EXPRESSION SYSTEMS AND AFFINITY CHROMATOGRAPHY FOR RECOMBINANT ANTIGEN PRODUCTION
(University of Hawai'i at Manoa, 2024) Eiser, Isabelle Elaine Yazel; Lehrer, Axel T.; Biomed Science (Tropical Medicine)
Development of recombinant proteins that are key antigens of priority pathogens, such as SARS-CoV-2, Nipah Virus (NiV), and Crimean-Congo Hemorrhagic Fever Virus (CCHFV), are critical for public and global health research. Recombinant proteins have numerous research and biopharmaceutical applications including the use in surveillance of potential outbreaks, studying immunological responses to infection, and the development of serological assays and vaccines. Biopharmaceutical use of recombinant proteins requires reproducible, standardized, and consistent protein production which can be achieved using the Drosophila S2 expression system and affinity purification methods. Here we present a potential platform for the production of SARS-CoV-2 Spike (S) protein, Nipah Virus (pre)fusion (pf/F), attachment (G), and nucleocapsid (N) proteins as well as CCHFV receptor recognition glycoprotein (Gc and Gn) and nucleocapsid (N) proteins using the Drosophila S2 expression platform. We demonstrate a universal, tagless immunoaffinity purification method applicable to all SARS-CoV-2 variant Spike proteins by restoring the CR3022-binding epitope in our engineered BA.1 and BA.5 Spike variant constructs. NiV and CCHFV protein genes were purified using immobilized metal ion affinity chromatography. This research allows for production of antigens for serological surveillance of disease, monitoring of immunogenicity and efficacy of newly developed vaccines, and recombinant subunit protein vaccine production to combat new and evolving viral diseases and could be used for the generation of other, purpose-engineered vaccine antigens.
Investigating Antibody Avidity In SARS-CoV-2 Naïve And Convalescent Vaccinees
(University of Hawaii at Manoa, 2022) Odo, Troy Kazuma; Lehrer, Axel T.; Biomed Science (Tropical Medicine)
Over the past two years, SARS-CoV-2 has had a devastating impact across the world. Despite the historic success of the multiple SARS-CoV-2 vaccines, additional research is needed to further understand the underlying mechanisms of protection. While many studies focus primarily on neutralizing antibody titers, another aspect of humoral immunity that is often overlooked is antibody avidity. Avidity, the total binding strength of all interactions between an antibody and antigen, is important to consider in the context of vaccination because it provides an important measure of antibody maturation and development. To date, there is no “gold standard” approach to measure antibody avidity. Solid-state immunoassays such as ELISAs are commonly modified with a chaotropic agent capable of disrupting antigen-antibody interactions. This overall focus of this study is to measure and compare antibody avidity in SARS-CoV-2 naïve and convalescent vaccinees. To do so we optimized an ammonium thiocyanate-modified multiplex immunoassay that can be used to measure SARS-CoV-2 antibody avidity. We found that standardizing antibody concentrations is an important step when measuring antibody avidity. This helps to prevent oversaturation of the assay and ensures that avidity can be measured and compared between sera with different antibody populations. Additionally, we used this assay to measure longitudinal sera collected from SARS-CoV-2 naïve and convalescent vaccinees to investigate the relationship between natural infection and vaccination. Sera were collected prior to vaccination, >14 days post-dose 1, and >14 days post-dose 2 and individuals were grouped based on whether they had SARS-CoV-2 infection prior to vaccination. Additionally, in both the naïve and convalescent groups, antibody avidity increased following each vaccine dose. A single antigen exposure in the convalescent group (natural infection), generated higher antibody avidity than a single vaccine dose in the naïve group. Avidity was also higher following two antigen exposures in the convalescent group (natural infection + a single vaccine dose) compared to two vaccine doses in the naïve group. Our assay demonstrates differences in the antibody response following natural infection and vaccination which opens the door for further investigation into these differences.
Development of Microfluidics for Use in a Novel Way to Characterize Latently HIV-Infected Cells
(University of Hawaii at Manoa, 2022) Mak, Bradley; MacPherson, Iain; Biomed Science (Tropical Medicine)
Antiretroviral therapy (ART) has been transformative in helping people living with HIV (PLWH) in the last 30 years. Despite the many advances in ART, it cannot eradicate HIV due to its inability to target the HIV latent reservoir. Therapeutic approaches have been considered for eradicating latent HIV such as “shock and kill” and “block and lock”but are still in development and have shown poor results in clinical trials thus far. Laboratory techniques such as STIP-Seq and HIV-Flow have been developed to quantify and characterize the HIV reservoir but do not aim to specifically identify and characterize genuine latently infected cells. Such techniques fall short due to the requirement to activate latently infected cells for identification and analysis, since activation changes cellular gene expression globally. Therefore, we introduce HIV-Seek, a novel approach to characterizing latently HIV infected cells in their pre-activated state. HIV-Seek aims to combine microfluidics, single cell RNA-sequencing, and molecular biology to identify biomarkers for immunological or pharmacological control of the viral reservoir. The focus of this thesis is to establish the development of microfluidics for both its fabrication and function in HIV-Seek alongside the integration of microfluidics and molecular biology. Designs and fabrication conditions were developed for the custom microfluidics for use in HIV-Seek. Encapsulation of beads and cells was performed using microfluidics and a coencapsulation rate of 4.38 ± 0.31% was achieved. Further experimentation with the integration of the molecular biology with the microfluidics is required but shows promise as shown with the dPCR.
Elucidating The Antimicrobial & Antiviral Activity Of An Underexplored Class Of Organobismuth Compounds
(University of Hawaii at Manoa, 2022) Faundo, Michael; Lehrer, Axel T.; Biomed Science (Tropical Medicine)
Infections of antibiotic-resistant pathogens pose an ever-increasing threat to mankind. The investigation of novel approaches for tackling the issue of antimicrobial resistance (AMR) must be part of any global response to this problem if an unwanted reversion to the pre-penicillin era of medicine is to be avoided 1. One such promising avenue of research is looking into the potential of metallodrugs as a solution. Given the historical usage of bismuth compounds in the treatment of certain types of bacterial infections before the treatment of modern antibiotics and its interesting safety profile despite its heavy-metal grouping has made it a driving interest in its basic and applied research 2. Specific research efforts are focused on the development of novel Bi-based compounds, nanoparticles and composites with applications in treating cancer and microbial infections, imaging, theranostics and biosensing 3. Moreover, while metal agents have demonstrated their value as potential antimicrobial and anticancer agents, their antiviral activities have been rarely explored. Despite its growing interests in the medicinal chemistry landscape, organobismuth chemistry is far less developed in comparison to other main-group organometallic chemistry due to two obstacles. First, the C–Bi bond is weak (bond disassociation energy = 46 kcal/mol), making it prone to dismutation, a substituent scrambling process, which complicates the synthesis of unsymmetrical triarylbismuthanes of the general formula Ar21Ar2Bi (Ar1 ≠= Ar2) 4. However, the Hyvl lab at the University of Hawaii at Manoa has designed an efficient two-step synthetic protocol that affords the synthesis of a electronically diverse set of heteroleptic triarylbismuthanes without the formation of dismutated contaminants. The long-term goal of this study is to use the lead structures identified in this work to facilitate the development of novel antimicrobials and antivirals. The immediate objective of the study is to elucidate the antimicrobial and antiviral activities of this group of organobismuth compounds. Given bismuth’s historical use in treating microbial infections before the advent of modern antibiotics, such as syphilis and Helicobacter pylori, we hypothesize that these organobismuth compounds may exhibit a comparable level of antimicrobial activity. Moreover, while metal compounds have demonstrated their applications as antimicrobial and anticancer agents, their antiviral activities have rarely been explored. Due to the novelty of the structure of this underexplored class of compounds, we also hypothesize that these compounds may exert their activity through other modes of action rather than the better understood mechanisms of Bi(III). In the first aim, we screened thirty-one unique organobismuth compounds for their various levels of antimicrobial activities in the context of multiple Streptococcus spp., Staphylococcus aureus strains, and several species of Gram-negative organisms. Additionally, we determined the minimum inhibitory concentration (MIC) for each individual compound that passed initial antimicrobial activity screenings. Additionally, we also determined a range of concentrations at which each compound displays to be cytotoxic to Vero and human embryonic kidney (HEK293T) cells. We found that of the thirty-one unique compounds, twenty-eight exhibited some level of antimicrobial activity in a dose-dependent manner. While the observed activity showed variability between different species and strains of Gram-positive organisms, there did appear to be stronger levels of inhibition skewed towards strains of S. aureus, more so towards strains of methicillin-resistant S. aureus (MRSA) in certain compounds; however, we do not see any inhibitory activity against any Gram-negative bacteria. These data suggest that the targets of for some of these compounds may be specific to S. aureus and even more specifically to MRSA strains in certain contexts. Moreover, we found that twenty-one compounds demonstrate very low levels of cytotoxicity, being well tolerated even at the highest concentration tested (30 µM) displaying no noticeable toxicity when compared to healthy untreated controls measured by MTT assay. In the second aim, we investigated the potential antiviral activity for each of the thirty-one unique compounds. Using recombinant vesicular stomatitis viruses (rVSV), we identified a total of twenty positive hits. Additionally, we found seventeen compounds that demonstrate significant levels of antiviral activity while simultaneously showing no noticeable levels of cytotoxicity even at the highest assayed concentration. Of these positive hits, we did see some overlap with lead compounds described in the previous aim assessing antimicrobial activity. However, we were also able to identify nine hit compounds that solely demonstrated antiviral activity to rVSV pseudotyped viruses. In these structures we are able to observe preliminary structure activity relationships (SARs) and how substitution at certain positions affects compound activity. Preliminary work has qualitatively shown that some compounds are capable of also inhibiting Dengue virus serotype 2 (DENV-2). These results suggest that the activity conferred by these compounds may have the potential to affect additional virus families beyond what has been investigated here. Moreover, when examining compounds under pre-treatment conditions versus virucidal conditions, we found that the compounds are more likely to act directly on the cell to mediate inhibition of viral replication and not directly on the virus itself; however, the specific mechanisms of how they do so remain unclear. If this is indeed the case, these findings may implicate their capacity to act as broad-spectrum antiviral agents.
Role of WNV NS4A and NS4B Proteins in Reducing Type I IFN Response via Interactions with Host IKKe and TBK1 Proteins
(University of Hawaii at Manoa, 2022) Awamura, Thomas Ken; Kaufusi, Pakieli H.; Biomed Science (Tropical Medicine)
West Nile virus (WNV) is a zoonotic flavivirus, with the potential to cause severe virally induced meningitis and encephalitis in humans. Currently, there are no specific antivirals or vaccine options for treatment or prevention of West Nile virus infection. Research on the closely related Dengue virus (DENV) has shown that both nonstructural proteins 4A and 4B (NS4A and NS4B) play key roles in the inhibition of type I interferon (IFN) by infected cells, aiding in the virus’ ability to evade the innate immune system. Despite this, the mechanism by which NS4A and NS4B inhibits the production of IFN-β is inadequately understood, especially for WNV. Recent findings for DENV suggest that the virus may target TANK-binding kinase 1 (TBK1) and inhibitor of nuclear factor kappa B kinase subunit epsilon (IKKe), both potent activators of the type I IFN pathway. The expression of IKKe or TBK1 is required for full expression of IFN-β in cell models, and are a potential target for many viruses. To elucidate if WNV NS4A and/or NS4B proteins interacted with TBK1 and/or IKKe, we examined potential interactions between viral and host cell proteins and determined the degree of IFN inhibition in TBK1 and IKKe overexpressing cells transfected with WNV protein. Human endothelial kidney 293T (HEK293T) cells were transfected with WNV NS4A or NS4B, and IKKe or TBK1 plasmids (n=10-15). Pearson correlation coefficient (PCC) values were used to determine degree of colocalization of WNV proteins to host cell proteins shown in immunofluorescence assay (IFA) images. Calculations of PCC were done using FIJI (version 2.1.0/1.53c), an application within ImageJ, utilizing the coloc2 plugin. To corroborate the findings from the IFA imaging, Western blots of sucrose gradients from transfected cell lysate were performed to visualize recruitment of IKKe and TBK1 away from its intended location in the cytoplasm towards fractions containing WNV protein. To determine whether NS4A and NS4B were able to inhibit type I IFN production in cells overexpressing IKKe and/or TBK1, a dual-reporter luciferase assay was conducted to determine the degree to which these proteins could inhibit IKKe and TBK1 activation of IFN-β production. IFA imaging data indicated that cells expressing WNV proteins saw a significant degree of colocalization with IKKe and TBK1 (PCC ≥0.5) with NS4B seeing the highest degree of colocalization. Additional wWestern blot imaging taken from sucrose gradients conducted on transfected cells lysates showed a clear recruitment of IKKe and TBK1 proteins from the cytoplasm in control cells, towards the rough endoplasmic reticulum (RER) in cells expressing WNV protein. Luciferase assay data showed that in the presence of NS4A or NS4B, in comparison to other WNV NS proteins, IFN-β production was remarkably reduced (p<0.0001). Our study has identified interactions between NS4A and NS4B with both IKKe and TBK1 proteins and potential sequestration of these cytoplasmic host cell proteins towards the RER where the WNV proteins are located. Additionally, we have demonstrated that NS4A and NS4B significantly inhibit IFN-β production in cells expressing high levels of TBK1 and/or IKKe, likely via the targeting of said proteins. These findings provide further evidence that NS4A and NS4B may be optimal candidates for further antiviral development to combat WNV infection.
Characterization Of Circulating Fibrocytes In People Living With HIV
(University of Hawaii at Manoa, 2022) Dean, Logan Skyler; Shikuma, Cecilia; Biomed Science (Tropical Medicine)
Fibrosis is estimated to account for 45% of all-cause mortality in the developed world. Immune dysregulation and chronic inflammation are potent contributors to fibrotic disease, increasingly so in people living with HIV (PLWH), independent of viral suppression. Fibrocytes are bone marrow-derived fibroblast-like cells whose increased numbers have been correlated with incidence and severity of fibrotic diseases. Despite their relevance to fibrosis, studies characterizing the fibrocyte in PLWH are scarce. Using 34 HIV+ individuals on suppressive antiretroviral therapy and 34 healthy controls (HC) we identified and quantified circulating fibrocytes in peripheral blood mononuclear cell (PBMC) samples. Participants were similar in age with a median age of 59.96 (56-63) years in HIV+ and 59.23 (56-64) years in HC and both groups were 88.2% male. No significant difference in non-infectious comorbidity prevalence was noted between the groups. HIV+ participantsʻ median CD4 count was within normal limits at 747.5 (601-898) cells/μL. Fibrocyte and activated fibrocyte numbers were not different between HIV+ and HC (p=0.257 and p=0.866, respectively). Similarly, the percentage of fibrocytes and activated fibrocytes from total CD45+ cells did not significantly differ (p=0.235 and p=0.710, respectively). However, fibrocyte numbers and the percentage of fibrocytes from CD45+ cells were significantly associated with increasing age in both HIV+ and HC groups (r=0.558, p=<0.0001 and r=0.575, p=<0.0001, respectively). Cultured fibrocytes isolated from HIV+ PBMC exhibited an increasing trend of mRNA expression of collagen-1 (COL-1), α-smooth muscle actin (α-SMA), vimentin, CCR5, and CCR7 with a trend towards decreased mRNA expression of CXCR4 and CCR3 compared to HC fibrocytes. Cultured HIV+ and HC fibrocytes treated with TGF-β1 at 5ng/mL and 20ng/mL enhanced fold change expression in COL-1, α-SMA, and chemokine receptor genes. Interestingly, fibrocytes from HIV+ PBMC exhibited decreased susceptibility to TGF- β1-induced COL-1 and CCR7 expression compared to HC. Our study demonstrates that circulating fibrocyte and activated fibrocyte populations are comparable between and strongly associated with age in virally suppressed PLWH and HC. In addition, cultured fibrocytes from PLWH demonstrated different responses to TGF- β1 stimulation compared to HC.
Development of the Molecular Biology of HIV-Seek: A Novel Approach to Characterizing Latently Infected CD4+ T Cells
(University of Hawaii at Manoa, 2022) Gamez, Eduardo Rafael; MacPherson, Iain; Biomed Science (Tropical Medicine)
Although antiretroviral therapy (ART) is effective at managing HIV infection and extending the lives of people living with HIV (PLWH), it is limited by the inability to target the HIV viral reservoir, specifically in latently infected CD4+ T cells. Strategies for targeting the viral reservoir, including “shock and kill” and “block and lock,” while still in development, require more supportive clinical data to legitimize this approach. These strategies may benefit from an accurate characterization of latently infected cells. However, current techniques for characterization are limited due to the scarcity of latency and the stipulation to activate latently infected cells. Cell reprogramming in the activated state of CD4+ T cells obscures the biology of the cell in its latent state. Therefore, we introduce HIV-Seek, a novel approach to characterize the pre-activated transcriptome of latently infected resting memory CD4+ T cells. This characterization will reveal more information on an infected cell in its latent state and may then enable the identification of a biomarker unique to latently infected cells. In turn, a successful biomarker candidate could lead to immunological or pharmacological control of the viral reservoir. HIV-Seek utilizes a variety of technologies including microfluidics and single-cell transcriptomics to isolate and analyze the pre-activated transcriptome of latently infected cells. In this thesis, we establish bead-based molecular techniques that demonstrate the use of oligo-dT-conjugated beads to facilitate reactions involved in HIV-Seek.
Scyllo-inositol And Other Brain Metabolites In Relation To Regional Brain Volumes, Cognition, And Inflammatory Markers In HIV
(University of Hawaii at Manoa, 2021) Lee, Awapuhi; Kallianpur Tata, Kalpana J.; Biomed Science (Tropical Medicine)
HIV-associated neurocognitive disorders (HAND) are a manifestation of neuropsychological (NP) impairment. Brain atrophy that exceeds normal age-related volumetric loss has been observed in HIV patients with NP impairment, and cerebral metabolites have been associated with reduced regional brain volumes and cognitive impairment. Low levels of scyllo-inositol (sI) were recently found to be related to NP impairment in a cohort of HIV patients with mild-to-moderate cognitive impairment. sI has not been extensively studied in the HIV population, so its role in HIV infection and cognitive impairment is unknown. To elucidate the potential roles of sI and other cerebral metabolites in cognitive impairment, we examined associations between metabolites, regional brain volumes, NP performance, and inflammatory markers in HIV+ individuals on combination antiretroviral therapy (cART) who had mild-to-moderate cognitive impairment. We also examined changes in metabolites among HIV patients before and after initiation of Cenicriviroc (CVC) or Maraviroc (MVC).Magnetic resonance imaging (MRI) was used to obtain T1-weighted scans, which were processed using FreeSurfer (version 6.0) to obtain regional brain volumes and total intracranial volume (ICV) of HIV+ participants (N=30) from the CVC and Maraviroc MVC studies from the Hawaiʻi Center for AIDS. Single-voxel magnetic resonance spectroscopy (MRS) and magnetic resonance spectroscopy imaging (MRSI) at 3T was used to measure brain metabolites. Participants underwent NP tests in multiple domains. Immune and inflammatory markers were measured by multi-parametric flow cytometry. Multivariable regression models, Pearson and Spearman correlations, Wilcoxon signed-ranked tests, and Mann-Whitney tests were performed using SPSS (versions 26 and 27). There were many significant associations and correlations between cerebral metabolites, regional brain volumes, NP domains, and inflammatory markers. Choline concentrations increased among patients treated with CVC. HIV patients in the MVC treatment group vs. the placebo group had higher mean differences in GABA and choline. Our study identified multiple associations between cerebral metabolites and regional brain volumes, including sI being positively associated with the putamen, cerebellar cortex and white matter, and the brain stem. This metabolite should be studied further as it could play a role in improving brain health and neurocognitive performance among HIV+ individuals.
Comparison of Antibody Responses in Mice Generated by Different Multivalent Filovirus Vaccines Based on Recombinant Glycoprotein Subunits
(University of Hawaii at Manoa, 2020) Tashiro, Taylor Emi; Lehrer, Axel T.; Biomed Science (Tropical Medicine)
Filoviruses cause fulminant hemorrhagic fevers with case-fatality rates up to 90%. The objective of this research is to achieve a multivalent filovirus vaccine that can be thermostabilized and used to prevent the spread of lethal filovirus disease. The central hypothesis is that since each immunogen was found to elicit a strong antibody response on its own, it should be possible to optimize the formulation dosages of each immunogen to generate a combination vaccine that induces a balanced, robust antibody response and neutralizing antibody titers against the most pathogenic filovirus species, EBOV. Recombinant glycoprotein (GP) derived from Ebola virus (EBOV), Sudan virus (SUDV) and Marburg virus (MARV) have been expressed from stably transformed Drosophila S2 cells and used to formulate recombinant subunit vaccine candidates showing efficacy in rodents and non-human primates. Immunogenicity of 12 different formulations of our trivalent subunit vaccine with adjuvant was tested in Swiss Webster mice, and serum antibody concentration and neutralization of rVSV-EBOV GP was measured and compared to responses from monovalent formulations. The 12 formulations were categorized into three sets of four groups of increasing dosage – The first set being formulations of equal antigen, the second being equal EBOV and MARV antigen with less SUDV antigen, and the third being equal amounts of Ebolavirus to Marburgvirus antigen. These formulations were determined in order to set up assays for potency testing of the vaccine and to understand antibody responses to different formulations. When quantifying antibody concentration, the results were as expected as trivalent formulations induced high antibody concentrations for all antigens at higher dosages. Also, the trivalent vaccine is able to produce comparable antibody concentrations to being vaccinated with monovalent formulations which suggests that trivalent formulations can still elicit antibodies that are specific for each antigen. It was observed that higher dosages of antigen elicit higher antibody concentrations against EBOV and SUDV GP, but a dose-response was not detected for antibody concentrations against MARV GP suggesting that it may be the most immunogenic antigen. Ultimately, the formulation that was able to elicit the highest, and balanced antibody concentration against EBOV, SUDV, and MARV GP was the formulation of equal antigens at the highest dosage. Results from the neutralization tests against rVSV-EBOV GP show that higher neutralization titers are achieved with increased dosages. Neutralization titers were also increased with inclusion of SUDV GP antigen as compared to monovalent vaccination and further increases when there is less SUDV GP present compared to the EBOV and MARV antigens, suggesting SUDV GP interference. This led to highest neutralization observed from groups containing less SUDV GP as compared to EBOV and MARV GP. From these findings, we have shown that trivalent formulations can be balanced to both optimize total antigen-binding IgG and neutralizing responses. This data will be used to formulate the next vaccines for testing in NHP’s and to develop potency tests for antigen- and batch-release testing.
Congenital Zika Syndrome in Guinea Pigs
(University of Hawaii at Manoa, 2019) Stone, Shannon Blair; Kumar, Mukesh; Biomed Science (Tropical Medicine)
Zika virus (ZIKV) infection during pregnancy may cause diverse and serious congenital defects in the developing fetus. In this study, we utilized pregnant guinea pigs to study congenital Zika syndrome. Female guinea pigs early in pregnancy (weeks 3–4 of gestation) were inoculated with Asian ZIKV strain (PRVABC59) or PBS (mock) via subcutaneous route. Dams were weighed daily, and blood and urine were collected at regular intervals to assess the presence of virus. Weight loss was observed in ZIKV-infected dams during first week of the infection. ZIKV-infected animals seroconverted with significant viremia and viral secretion in the urine. During the period between infection and delivery of the pups, significant viral RNA and NS1 protein were detected in all animals from 2 to 5 days after infection, with peak viral replication at day 3. We also detected robust viral RNA shedding in urine, with a prolonged duration relative to that of viremia. Dams developed remarkably robust ZIKV-specific neutralizing antibody response, and anti-ZIKV antibodies were also recovered from pups. Notably, ZIKV was efficiently transmitted from infected guinea pigs to their naïve co-caged mates. ZIKV infection of pregnant guinea pigs caused fetal damage. Sixty percent of ZIKV-infected dams showed abnormal pregnancies in that they all delivered at least one or more abnormal pup. Pups from ZIKV-infected animals exhibited significant intrauterine growth retardation. ZIKV was detected in the brain of pups from ZIKV-infected animals. ZIKV RNA and anti-ZIKV antibody levels in the dams reliably predicted abnormalities in pups. ZIKV detection in the brain tissues correlated with the pup abnormalities. In conclusion, the pregnant guinea pig model provides quantifiable congenital abnormality readouts to assess pregnancy outcomes; and may serve as a good model to test therapeutics, and to elucidate the mechanisms of ZIKV congenital pathogenesis.
Histone deacetylase inhibitors modulate human polyomavirus JC replication
(University of Hawaii at Manoa, 2019) Fisher, Michellei Chiemi; Nerurkar, Vivek R.; Biomed Science (Tropical Medicine)
The human polyomavirus JC (JCPyV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the brain. Although archetype JCPyV exists as an asymptomatic infection in the healthy population, PML occurs almost exclusively in individuals with immunodeficiencies or on immunomodulatory medication. When immune function is perturbed, the mechanisms which maintain viral latency are disrupted and can lead to the development of PML. Therefore, an important question in understanding PML pathogenesis are the molecular mechanisms which maintain JCPyV latency. The JCPyV genome incorporates host-derived histones and closely resembles host chromatin structure, therefore we hypothesize it is subject to epigenetic regulation. Histone deacetylase inhibitors (HDACi) have been demonstrated to increase rearranged JCPyV replication in a transfection model and are candidates for latency reversal agents in HIV treatment. The potential for these HDACi to reactivate HIV suggests the possibility of reactivating other viruses as well. The objective of this study is to characterize the effects of histone deacetylase inhibitors (HDACis) on archetype JCPyV infection, replication, and rearrangement in vitro and in vivo. Here, we demonstrate that HDACi, Trichostatin A, treatment of primary human brain cortical astrocytes and renal proximal tubule epithelial cells increases both early and late archetype JCPyV replication in a cell-specific manner. Further, we demonstrate in patients treated with HDACi, panobinostat, statistically higher increase in archetype JCPyV genome copy number in urine, which was abrogated after treatment. To our knowledge, this is the first study to show the effect of HDACi on archetype JCPyV replication in an in vitro infection model and the first to investigate JCPyV viruria during HDACi treatment. Taken together, these findings suggest that HDACi modulate archetype JCPyV replication. This study emphasizes the need to understand the effects of these global HDACi on other viruses to improve risk stratification for latency reactivation agent treatments. These findings will open new therapeutic strategies for treatment of PML aimed at preventing viral replication and maintaining JCPyV in a latent state.
Developing Novel Serological Tests to Distinguish Flaviviral Infections.
(University of Hawaii at Manoa, 2018-08) Tyson, Jasmine M.; Biomedical Sciences (Tropical Medicine)
Our study developed serological tests to distinguish dengue (DENV) and Zika (ZIKV) virus infections. We expressed the precursor peptide of DENV1, yellow fever virus, West Nile virus (WNV), and ZIKV using two expression systems. Using the pr protein of DENV1, we were able to distinguish DENV and ZIKV infections with high specificity and sensitivity. Furthermore, we improved upon our previous work that a combination of three enzyme-linked immunosorbent assays (ELISAs) based on non-structural protein 1 (NS1) can distinguish DENV and ZIKV infections by replacing the DENV1-NS1 ELISA with a pooled DENV1-4-NS1 ELISA. This increased the overall sensitivity and specificity of our assays. Lastly, we developed a WNV-NS1 ELISA that can be used in combination with DENV- and ZIKV-NS1 ELISAs to distinguish secondary flaviviral infections. With the current limitations of serodiagnosis of DENV and ZIKV due to cross-reactivity, our findings provide a promising addition to the existing CDC recommendations.
Defining the Role of Alpha-Macroglobulins in the Pathogenesis of Flavivirus Encephalitis
(University of Hawaii at Manoa, 2018-05) Krause, Keeton K.; Biomedical Sciences (Tropical Medicine)
West Nile Virus (WNV) and Japanese Encephalitis Virus (JEV) are positive sense, singlestranded RNA viruses belonging to the family Flaviviridae, where the primary mode of transmission occurs through arthropod vectors such as mosquitos. WNV and JEV are the leading etiological agents for arboviral encephalitis in humans, and sporadic outbreaks continue to occur over time resulting in symptomatic infections that account for thousands of deaths each year. Although a number of vaccines have been developed to prevent JEV infections in highly endemic regions, issues with efficacy exist and the vaccine often fails to protect at risk populations from the development of encephalitis, which can be fatal. For both WNV and JEV, no clinically approved therapies currently exist for treatment of the central nervous system (CNS) involvement, which can lead to encephalitis, the most severe, and highly fatal form of the disease. Alpha-macroglobulins, which are physiological proteinase inhibitors have been shown to bind to viral proteins and enhance viral infections in vitro. Moreover, in humans alphamacroglobulins such as pregnancy zone protein (PZP) and alpha-2-macroglobulin (A2M) also serve as immune-modulatory proteins where their normal functions within the body include binding and shuttling of protease inhibitors, growth factors, cytokines, hormones, disease factors, and various small molecule nucleophilic ligands. Previous reports from our laboratory have shown the up-regulation of alpha-macroglobulins in wild type (WT) mice after a lethal subcutaneous WNV infection. Therefore, to define the role of a-macroglobulins during flavivirus infection in vivo, we investigated the susceptibility of mice deficient in amacroglobulins (PZP and MUG-1 double knockout; DKO) against lethal subcutaneous infection with either WNV or JEV. Results of our study show that DKO mice are completely resilient to lethal flaviviral infections of WNV and JEV. Likewise, DKO mice had a significantly milder clinical disease through the course of the study, and this outcome can be coupled with the finding of significantly reduced viral burden in the blood, peripheral organs (kidney and spleen), and brains of the DKO mice when compared to WT mice. The DKO mice had a significantly reduced inflammatory response which was characterized by lower concentrations of pro-inflammatory cytokines and chemokines in the blood, spleen, and brain of DKO mice when compared to WT. Consistent with the multiplex immunoassay data, DKO mice also displayed a significantly decreased level of mRNA corresponding to immune genes in response to WNV infection when compared to WT. Overall, the data from this study demonstrates the significant impact that alpha-macroglobulins have in the pathogenesis of flavivirus-associated encephalitis in mice.
DNA Vaccine Expressing Mature Dengue Virus-Like Particles and Serological Tests to Distinguish Dengue and Zika.
(University of Hawaii at Manoa, 2017-08) Youn, Han Ha; Biomedical Sciences (Tropical Medicine)
Our study investigated DNA vaccines expressing mature dengue virus (DENV) viruslike particles (VLPs) and developed serological tests to distinguish dengue and Zika. We showed that human-codon optimized DNA vaccines improved the expression and production of VLPs in vitro compared with non-codon optimized DNA vaccines. Human-codon optimized DNA vaccine expressing mature VLPs induced similar level of anti-DENV and neutralizing antibodies compared with that expressing mixed VLPs. Secondly, we found that DNA-prime and inactivated virion-boost regimens improved the immunogenicity in mice compared with DNA vaccine alone. Future challenge experiments are needed to show the protective effect of these vaccine candidates. Thirdly, we demonstrated that combination of three enzyme-linked immunosorbent assays based on non-structural protein 1 can distinguish DENV and Zika virus infections with high specificity and sensitivity. In light of the cross-reactivity of current serological tests, our findings have implications for serodiagnosis between DENV and Zika virus or other flaviviral infections.
Zika Virus Infects Human Sertoli Cells And Trespasses The Blood-Testes Barrier To Gain Entry Into The Seminiferous Epithelium
(University of Hawaii at Manoa, 2017-08) Siemann, David; Biomedical Sciences (Tropical Medicine)
Over the past decade, Zika virus (ZIKV) has re-emerged as a pathogen of major health concern in the Western Hemisphere. Specifically, since 2007, 76 countries have announced new outbreaks of ZIKV, 29 of which also report increased incidence of CNS malformations such as microcephaly and also Guillain-Barre Syndrome [1]. These alarming statistics combined with the unique ability of ZIKV to be transmitted both sexually and in utero highlight the urgent need to study the mechanisms of ZIKV pathogenesis in order to ultimately develop vaccines, effective anti-viral therapies, and policies to control the spread of ZIKV disease. While ZIKV can be detected in human seminal fluid for months after the clearance of viremia, the cellular targets and mechanisms associated with persistent infection in the testes remains unclear. Mouse and NHP studies have recently shown that ZIKV can infect and damage the seminiferous tubules within the testes [2-5]. The seminiferous tubules are an immune privileged organ with a tight blood-testes barrier also known as the Sertoli-cell barrier (SCB), which protects developing spermatozoa from peripheral pathogens and environmental toxins. However, increased inflammatory mediators such as TNF-α and IL-1β, matrix metalloproteinases, and cell-adhesion molecules (CAM) can disrupt the integrity of the SCB leading to pathologic outcomes [6, 7]. Therefore, the objective of this study was to characterize ZIKV replication kinetics and immune responses in primary human Sertoli cells (SC) and develop an in vitro SCB model to understand mechanisms of ZIKV transmigration across the barrier. We demonstrate that primary human SC are highly susceptible to ZIKV as compared to the closely related dengue virus and induced expression of IFN-α, key cytokines and celladhesion molecules (VCAM-1 and ICAM-1). Further, using an in vitro SCB model, we show that ZIKV was released on the adluminal side of the SCB model with higher efficiency when compared to the blood-brain barrier model. ZIKV-infected SC also exhibited enhanced adhesion of leukocytes that correlated with decrease in the SCB integrity. While ZIKV infection did not affect the expression of tight and adherens junction proteins such as ZO-1, claudin and JAM-A, exposure of SC to inflammatory mediators derived from ZIKV-infected macrophages led to the degradation of ZO-1 protein that correlated with increased SCB permeability. Collectively, our data suggest that infection of SC may be one of the crucial steps by which ZIKV gains access to the site of spermatozoa development and identifies SC as a therapeutic target. Finally, the SCB model opens up opportunities to assess interactions of SC with other testicular cells and lays the platform for future studies to test the ability of anti-ZIKV drugs to cross the barrier and clear testicular infection.

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