Ph.D. - Biomedical Sciences (Tropical Medicine)

Permanent URI for this collectionhttps://hdl.handle.net/10125/1939

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Kawasaki disease: Diagnostic and prognostic markers, and treatment of coronary artery lesions
(University of Hawai'i at Manoa, 2025) Ching, Lauren L.; Nerurkar, Vivek R.; Biomed Science (Tropical Medicine)
Background: The acute vasculitis, Kawasaki disease (KD) is the most common pediatric acquired heart disease in young children (less than 5 years old) in the developed world. Long-term sequelae of coronary artery aneurysms are reported in 25% of untreated/delayed treatment and 5% of treated cases. Early diagnosis and treatment are key to avoiding the most severe and long-term complications. Currently there are no laboratory-based tests for the diagnosis and prognosis of KD, or treatments that specifically target the tissue degradation and vascular remodeling involved in the development of coronary artery (CA) aneurysms. Standard KD treatment includes broad spectrum anti-inflammatory drugs, none of which specifically targets the tissue degradation and vascular remodeling that leads to KD-associated CA aneurysms. Materials and Methods: We characterized the inflammatory and vascular markers associated with KD, CA aneurysms, and intravenous gamma globulin (IVIG) resistance in serum samples longitudinally collected from 116 KD patients. KD patients who presented with CA aneurysms were offered enrollment to a phase II prospective, randomized, double-blinded placebo-controlled study of doxycycline for the treatment of KD associated CA aneurysms. Doxycycline was selected for evaluation as a novel treatment for KD-associated CA aneurysms due to its known anti-inflammatory properties that specifically target proteins associated with KD inflammation. Results: Using the patient's convalescent serum samples as self-controls, we identified panel of 10 biomarkers – five immune factors (granulocyte colony stimulating factor/ colony stimulating factor 3 [G-CSF/CSF3], interleukin [IL]-6, C-C motif chemokine ligand 2/monocyte chemotactic protein 1 [CCL2/MCP-1], C-X-C motif chemokine ligand 10/10kDa interferon gamma-induced protein [CXCL10/IP-10], C-X-C motif chemokine ligand 11/interferon-inducible T-cell alpha chemoattractant [CXCL11/ITAC]) and five vascular markers (neutrophil elastase [ELANE], matrix metalloproteinase [MMP]-3, MMP-9, pentraxin 3 [PTX3], soluble E-selectin [sELAM-1]), which demonstrated significant upregulation in the acute phase of KD, with strong performance in identifying acute phase KD samples. Stratifying KD serum samples by IVIG treatment response revealed a panel of four biomarkers - three immune factors (IL-1a, IL-25/IL-17F, tumor necrosis factor beta/lymphotoxin alpha [TNF-b/LTA]) and one vascular marker (pappalysin-1 [dPAPP-A]), which demonstrated significantly elevated levels among those resistant to IVIG treatment, at all clinical phases of KD. Comparing acute phase KD samples among those with and without CA aneurysms identified acute phase PTX3 significantly elevated in KD patients with CA as compared to those with normal CA, suggesting this protein as a novel factor in the development of KD-associated CA aneurysms. Comparisons of absolute and changes in CA diameter Z-scores among KD patients with CA aneurysms treated with doxycycline as compared to those who receive standard KD treatment did not demonstrate statistically significant differences. Conclusions: These findings provide a platform for laboratory-based diagnostic and prognostic markers of KD, CA aneurysms, and IVIG resistance. Further, the findings of the doxycycline treatment clinical trial did not reveal an advantage in using doxycycline treatment for the development or progression of CA aneurysms in KD over current standard of care.
ADVANCING FILOVIRUS VACCINE DEVELOPMENT: DEVELOPING HIGH-YIELD PLATFORMS TO EXPRESS AND PURIFY MARBURG VIRUS GLYCOPROTEIN AND OTHER VACCINE TARGETS
(University of Hawai'i at Manoa, 2024) Mayerlen, Ludwig Immanuel; Lehrer, Axel T.; Biomed Science (Tropical Medicine)
This dissertation focuses on the advancement of recombinant subunit vaccine platforms through the optimized production, purification, and stability testing of a filovirus vaccine candidate. Filoviruses, including Ebola and Marburg virus, represent substantial global health threats due to their high lethality and almost annual recurrence of outbreaks. Since our world has become more and more connected, these outbreaks are not only limited to filovirus-endemic countries but have been occurring on a global scale, reaching countries at a far distance from endemicity, including Germany and the U.S. Although approved vaccines for Ebola virus are available, no approved vaccines exist for other filoviruses, including Marburg virus. Additionally, current vaccines are limited by cold-chain requirements, emphasizing the need for new vaccine platforms for the prevention of filovirus disease. This research aimed to (1) Increase the production of Marburg virus glycoprotein by testing novel expression vectors together with transposases and integrases, (2) develop cost-effective production of antibodies, including MARV GP-specific monoclonal antibody 9A11, for subsequent immunoaffinity purification. (3) evaluate the stability and immunogenicity of different filovirus vaccine formulations under extended storage at elevated temperatures. Our findings demonstrate significant improvements in protein production yields for MARV GP using our newly developed expression vectors, achieving MARV GP production levels suitable for large-scale vaccine manufacturing. We demonstrate the in-house production of multiple antibodies, including monoclonal 9A11, and confirm their ability to purify our vaccine antigens. Furthermore, our thermostability study demonstrates that lyophilized vaccine formulations retain immunogenicity after extended storage at elevated temperatures of 40°C for at least 2 years. Our findings show that our expression platform can produce robust and stable antigens. Together, our findings support a vaccine platform that can be formulated as thermostable, single-vial vaccines conducive to global distribution, including low- and middle-income countries in the Global South.
ONE HEALTH APPROACH FOR STUDYING EMERGING AND RE-EMERGING VIRUSES IN LIBERIA
(University of Hawai'i at Manoa, 2024) Kamara, Varney M.; Lehrer, Axel; Biomed Science (Tropical Medicine)
The production and use of recombinant antigens for integrated serosurveillance for Lassa Virus (LASV) and other emerging and re-emerging epidemic-prone viruses are critical to global health efforts to effectively prevent future disease outbreaks. LASV causes millions of infections and over 10,000 deaths annually and remains endemic in West African countries, including Nigeria, Liberia, Sierra Leone, Guinea, etc. Additionally, the 2014- 2015 Ebola Virus Disease (EVD) outbreak in West Africa resulted in over 28,000 infected individuals and over 11,000 deaths, and the 2022 EVD outbreak in the Democratic Republic of Congo (DRC) led to more than 2,200 lives being lost. With the circulation of these emerging and re-emerging viruses in Africa, there are still challenges in conducting surveillance. The current surveillance system uses traditional enzyme-linked immunosorbent assay (ELISA), which is time-consuming, requires more reagents, and requires copious amounts of viral antigens. Most of these viruses that have epidemic or pandemic potential are category A pathogens. These viruses need to be handled in a Biosafety Level 4 (BSL-4) containment facility, which is lacking in most African countries. The issue of using a live virus is of major concern. Developing a serological assay using recombinant antigens for integrated surveillance is key to solving these challenges. The production of recombinant proteins eliminates the need for BSL-4 containment facilities, which are essential for handling highly infectious pathogens in traditional production platforms. The Multiplex Immuno-assay (MIA) can detect multiple analytes in the same well and is very cost-effective as compared to ELISA. This dissertation centers on the development of recombinant Dengue virus-2 (DENV-2) and West Nile Virus (WNV) NS1 antigens. The rationale for developing DENV-2 and WNV) NS1 antigens is multifaceted, such as developing improved diagnostics and supporting vaccine development and research. We also evaluated the performance of LASV GP and NP, as well as other recombinant antigens from viruses to screen for viral exposure across various species, including humans, rodents, and dogs. We adopted the multiplex immune assay (MIA) platform to conduct population-based seroprevalence studies for several coronaviruses in Liberia using an MIA. We used the Drosophila S2 expression platform and an immobilized metal affinity chromatography (IMAC) purification system to produce DENV and WNV NS1 proteins. Using MIA, we evaluated the performance of LASV and other viral antigens across humans, rodents, and dogs. We collected 200 serum samples from healthy human individuals living in LASV-endemic areas of Liberia. We received an additional 200 rodent and 200 dog serum samples from veterinarians from the Ministry of Agriculture (MoA) in Liberia, covering the same four regions/counties: Bong, Grand Bassa, Lofa, and Nimba. For our nationwide seroprevalence studies, we collected 3500 serum samples, a representative national sample from Bong, Grand Cape Mount, Sinoe, Maryland, and Montserrado counties. Our results showed the successful production of DENV and WNV NS1 proteins, which were validated using immune sera as positive controls and naïve sera as negative controls. These proteins demonstrated stability and strong immunogenicity, paving the way for their successful use in developing multiplex serological assays using these antigens. Results from our seroprevalence studies of LASV and other antigens showed a high prevalence of LASV antibodies in humans, followed by rodents and low seroprevalence in dogs. The low seroprevalence rate observed in dogs suggests that they might not be ideal sentinels for LASV infection. Among the rodent species trapped, the most abundant rodent species was R. rattus, followed by Hylomyscus pamfi, Mastomys species, Graphiurus murinus (African pygmy dormice), and Apodemus sylvaticus. While not considered a reservoir for LASV, R. rattus had the highest seroprevalence for LASV antigens at 27%. Finally, our Coronavirus population-based survey results showed high reactivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variant antigens, followed by less reactivity to SARS-1 spike antigen and low reactivity to the other coronavirus antigens, including Human coronavirus NL63 (HCoV-NL63) spike and Middle East respiratory syndrome coronavirus (MERS-CoV) spike and NP antigens. Overall, we found that our MIA based on coronavirus antigens is an effective means to measure the seroprevalence of different coronaviruses. This study shows the utility of recombinant antigens and MIA in conducting seroprevalence studies in humans and animals in resource-limited countries like Liberia. Findings from this study may, in the future, help public health professionals, including veterinarians, prevent future outbreaks.
Investigating the Use of Ultra-Small Iron Oxide Nanoparticles as a Subunit Vaccine Delivery Platform with Adjuvant-Like Properties
(University of Hawai'i at Manoa, 2024) Golshan, Aneesa; Hui, George; Biomed Science (Tropical Medicine)
Highly engineered vaccines such as subunit vaccines, nucleic acid vaccines, and self-assembling virus-like particles often display improved safety profiles in comparison to their whole-cell vaccine counterparts. While these vaccines have reduced reactogenicity and are safer for immunosuppressed populations such as the elderly, pregnant women, or the immunocompromised, they often lack in immunogenicity and long-term effectiveness. In order to improve upon these vital qualities, adjuvants are frequently added to subunit vaccine formulations. Inorganic nanoparticle delivery systems with built-in adjuvant properties have the potential increase subunit vaccine immunogenicity without the need for additional adjuvants to be added to vaccine formulations. Previous research in our lab indicates that functionalized iron-oxide nanoparticles possess adjuvant-like properties and are internalized by bone marrow-derived dendritic cells (BMDCs). Iron oxide nanoparticles (IO NPs) have been repeatedly demonstrated to be safe in current clinical applications, and are inexpensive, highly reproducible and can be lyophilized and multiplexed – qualities that are vital for the development of effective vaccines that are practical for global distribution. This project aimed to further elucidate, in vitro and in vivo, the extent of adjuvant-like activities of IO NPs, with a focus on dendritic cells (DCs) at the innate-adaptive immune interface. We evaluated 10nm, 20nm and 30nm IO NPs for their ability to activate bone marrow-derived dendritic cells (BMDCs) in vitro, the uptake efficiency and intracellular localization of IO NPs in vitro and in vivo dissemination following subcutaneous (SC) injection, and the ability of IO NPs to enhance vaccine immunogenicity of a recombinant spike protein (SP) SARS-CoV-2 vaccine. We also aimed to identify if correlations existed in IO NP-induced innate immune modulation and performance of IO NPs as a subunit vaccine delivery system. We found that 10nm IO NPs were superior to 20nm and 30nm IO NPs in their ability to induce activation of CD11c+ BMDC maturation and cytokine and chemokine production. 10nm IO NPs were also internalized more efficiently by BMDCs. IO NPs were largely found to utilize clathrin-mediated endocytosis and macropinosytosis for entry into BMDCs. Following SC injection, IO NPs drained to local lymph nodes for uptake by CD11c+ DCs within 24 hours, followed by presence in CD11c+ DCs in the spleen at 48 and 96 hours post injection. SP-IO vaccine was able to induce vaccine titers similar to SP administered with ISA51 following a three-dose regimen. 10nm IO NPs induced the more uniform antibody titers, leading us to conclude that the innate immune signature induced by 10nm IO NPS on DCs impacts adaptive immune responses when used as a subunit vaccine delivery platform. These studies add to the library of information available to researchers regarding potential delivery systems and adjuvants for the rational design of subunit vaccines.
Understanding The Myeloid HIV Reservoir In HIV-Associated Neurological Disorder During Antiretroviral-Treated HIV Infection
(University of Hawai'i at Manoa, 2023) Mitchell, Brooks I.; Shikuma, Cecilia M.; Biomed Science (Tropical Medicine)
Background: Despite effective antiretroviral therapy (ART), the persistence of latent HIV that produce replication-competent virus remains a major barrier to a cure. Since the development of the quantitative viral outgrowth assay (QVOA) over 20 years ago, there has been significant advancements in the detection and quantification of the replication-competent reservoir. However, the original development and ongoing modifications of the QVOA remains mostly T cell-centric. Recent studies have shown compelling evidence that the myeloid compartment harbor HIV in the setting of ART in tissues, yet only a few studies were tailored to measure myeloid-derived replication-competent HIV in circulation in humans. In addition, the myeloid compartment is reported to play an important mechanistic role in the neuropathogenesis of HIV-associated neurological dysfunction, though it remains unclear whether HIV reservoirs that may be persistent in myeloid cells in the setting of suppressive ART are related to neurocognitive impairment. Methods: We obtained cryopreserved peripheral blood mononuclear cells from chronic HIV-infected individuals on long term ART (>5 years) (n=16) and those who initiated ART early during acute infection and remained aviremic for 2 years (n=8), all of whom had available age and education-adjusted neuropsychological testing scores. Cells were sorted to ultra-high purity by flow-cytometry for total monocytes and CD4 T cells and validated. Using a modified novel Monocyte TZM-bl Assay (TZA), (MoCo-TZA), the frequencies of cells producing replication-competent HIV (IUPM) were calculated in both total monocytes and CD4 T cells. Total p24 associated with replication-competent HIV produced by these cell types were quantified using a relative light unit to replication-competent HIV-associated p24 standard curve in the MoCo TZA. Ratios of total replication-competent HIV-associated p24 and IUPMs, termed infectious potential, were calculated for monocytes and CD4 T cells. Results: Among the 16 ART-suppressed chronic HIV-infected individuals evaluated, 11 had detectable peripheral blood monocytes with inducible replication-competent HIV. Although monocyte IUPMs were significantly lower as compared to CD4 T cells, the infectious potential in the monocyte compartment was slightly higher as compared to the CD4 T cell compartment. Higher surface expression of CD16 on intermediate monocytes correlated with elevated monocyte reservoir measurements. In individuals who were treated early during acute infection, all 8 had detectable circulating monocytes with inducible replication-competent HIV after 2 years of ART. Individuals with cognitive impairment had higher reservoir measurements in total monocytes as compared to those with normal cognition. In addition, higher expression of CD16 on intermediate monocytes correlated with lower executive function and global NP z-scores. No differences in CD4 T cell reservoir measurements were observed between cognitive status groups. Conclusion: Utilizing the MoCo-TZA, we show that inducible replication-competent HIV in circulating monocytes is detectable during long-term chronic infection, as well as in individuals who initiated ART early during acute infection and were on treatment for 2 years. Our results highlight the need to further understand the monocyte-derived replication-competent HIV reservoir during ART-suppression and to further investigate this reservoir compartment in HIV-associated neurocognitive disease treatments and in HIV cure efforts.
ASSESSING VACCINE EFFICACY AND SEROPREVALENCE USING MULTIPLEX IMMUNOASSAYS: DEVELOPMENT, VALIDATION, AND APPLICATIONS IN DIVERSE POPULATIONS
(University of Hawai'i at Manoa, 2023) Smith, Olivia Anne; Lehrer, Axel T.; Biomed Science (Tropical Medicine)
This dissertation addresses the critical need for advanced tools in infectious disease prevention by focusing on the development, optimization, validation, and performance of a Multiplex Bead-Based Immunoassay (MIA) as a tool to assess vaccine efficacy and seroprevalence. The study's multifaceted approach encompasses three key objectives: optimization of the MIA using SARS-CoV-2 Spike subunits produced in Drosophila S2 insect cell lines, validation of the MIA for robust performance across diverse geographical and resource settings, and assessment of immunogenicity associated with altered metabolic states post-immunization, specifically in diabetic and obese murine models. The MIA is a high-throughput platform with high analytical accuracy that offers distinct advantages over established assays such as enzyme-linked immunosorbent assays (ELISAs). It simultaneously measures multiple analytes, providing a larger dynamic range and reducing the overall analysis cost. To demonstrate the feasibility of this approach, an MIA for anti-EBOV GP responses was compared to the well-established Filovirus Animal Non-Clinical Group Assay (FANG assay). The assays gave comparable results, but the MIA is more cost-effective and efficient, a particularly important consideration for monitoring efforts in low-resourced areas. An MIA targeting SARS-CoV-2 was then developed, optimized, validated, and applied experimentally in mouse models to shed light on differences in immune responses to SARS-CoV-2 vaccination that can arise as a result of altered metabolic states. By illustrating the systematic steps of assay development and validation, this work helps to overcome the lack of publicly available, formatted MIA protocols that have hindered the adoption of this approach thus far. The study suggests that MIAs have the potential to become useful, routine tools for identifying humoral immune responses associated with vaccine efficacy and antigen exposure. Ongoing refinement and adoption of these assays should improve our capacity to prevent and manage viral diseases, including the persistent challenge of COVID-19. Further research is also needed to attain a more holistic understanding of immune responses to vaccines and natural infections, especially with larger and more diverse populations. This endeavor may be facilitated by tools developed and described as part of this study.
Analysis of Dengue and Zika Antibodies Among a Cohort of Pregnant Women in Salvador, Brazil
(University of Hawaii at Manoa, 2023) Driesse-Keegan, Kaitlin; Chang, Sandra P.; Biomed Science (Tropical Medicine)
Dengue virus has been circulating in a hyperendemic pattern in Brazil for decades and the introduction of Zika virus, a closely related member of the flavivirus genus, into the Americas has complicated this situation. Zika virus has only recently begun to be viewed as a pathogen of significant concern. A dramatic increase in the incidence of microcephaly in Northeastern Brazil was reported in late 2015, coinciding with a large increase in ZIKV infection. This unique pattern of microcephaly and other disabilities linked to infection with the Zika virus during pregnancy is known as Congenital Zika Syndrome (CZS). The pathogenesis driving this phenomenon is unknown, however, due to the similarities between Zika and dengue viruses, it is theorized that dengue virus-mediated immune enhancement in mothers may be a risk factor for the development of CZS in infants. As such, it is important to further study the immune profiles of pregnant women infected with dengue and Zika viruses. Our research analyzes the antibody response to dengue and Zika viruses among pregnant women during the 2015-16 Zika outbreak in Salvador, Brazil, and characterizes cross-reactive antibodies between dengue (DENV) and Zika (ZIKV). This study provides the unique ability to study the background levels of dengue virus immunity in pregnant women in Northeast Brazil at the time of the ZIKV epidemic. Much is unknown about the effect of prior dengue infection and its ability to confer protection or risk of enhancement of Zika virus infection, especially during pregnancy. We aim to better understand this relationship and how it correlates to protection, with the long-term goal of facilitating the development of safe and effective DENV and ZIKV vaccines.
Characterizing Protective Antibody Responses to Recombinant Ebola Virus Subunit Vaccines in Non-Human Primates
(University of Hawaii at Manoa, 2023) Ball, Aquena Hana; Lehrer, Axel T.; Biomed Science (Tropical Medicine)
Ebola virus (EBOV) causes lethal hemorrhagic fevers with case fatality rates of up to 90%. Outbreaks are sporadic and unpredictable, and vaccination has been key to controlling disease. The establishment of correlates of protection is a critical step in vaccine development and assessment and are an asset to outbreak control and robust public health strategies. Although approved vaccines for EBOV are available, limited stock, low durability, and requirement of ultra-cold-chain storage highlight the need for improved vaccine technology. We have developed a recombinant subunit vaccine that shows improved safety and thermostability profiles, allowing easier deployment in endemic regions. Our vaccine has shown high efficacy in the gold standard non-human primate model of cynomolgus macaques; However, we have yet to fully characterize the immune responses that are elicited. This dissertation aims to establish humoral correlates of protection for EBOV by determining antibody characteristics associated with protection as well as important glycoprotein targets and the timing at which various antibody functions control infection. We find that a combination of protective antibody qualities including GP targets, neutralization, and effector function can be used to predict vaccine efficacy. Ability of antibodies to bind virus despite the presence of the mucin-like domain was also correlated with higher vaccine induced protection against EBOV with the majority of neutralizing and Fc effector functioning antibodies binding regions beneath the mucin-like domain. Post-challenge kinetics are also a relatively unexplored aspect of vaccine induced protection. Here we find that rapid antibody recall responses are needed to confer protection in sensitive NHP models and increases in antibody function later after infection, likely stemming from IgM, are not sufficient for protection in non-survivors. Our research highlights the importance of EBOV GP epitopes beneath the mucin-like domain as well as rapid memory recall responses. These findings can be used in the assessment and development of next generation vaccine candidates.
Virologic And Bioinformatic Analysis Of SARS-COV-2 To Facilitate Evidence-based Public-policy And Next-generation Vaccine Design
(University of Hawaii at Manoa, 2023) Maison, David Patrick; Deng, Youping; Biomed Science (Tropical Medicine)
Severe acute respiratory coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), has been the scourge of the world for the past three years. Worldwide, over 619 million cases of COVID-19 have resulted in over 6.5 million deaths. Hawaiʻi has not been spared from this pandemic, with more than 349,000 cases of COVID-19 and 1,691 deaths, with considerable caseloads among ethnic populations. The work herein combines virology, bioinformatics, and algorithms to discover new mutations, evaluate COVID-19 public policy, and facilitate the standardization of next-generation vaccine design. A better understanding of the responses to the COVID-19 pandemic, SARS-CoV-2 evolution, and the future of SARS-CoV-2 in endemicity is needed. First, we sequence the S gene of SARS-CoV-2 positive nasal and oropharyngeal swabs acquired from Hawaiʻi. These sequences were then phylogenetically evaluated against sequences from around the world to determine if multiple introductions of SARS-CoV-2 were evident in Hawaiʻi. After finding that multiple introductions were present, we evaluated the S gene of the sequences found in Hawaiʻi and discovered the P681H amino acid substitution. We then evaluated the P681H substitution against all worldwide sequences and demonstrated a worldwide emergence pattern. Second, we furthered our studies on SARS-CoV-2 in Hawaiʻi by isolating the virus. Following isolation, we developed a whole genome sequencing workflow to identify the viral genome. Following whole genome sequencing, we evolved our statistical evaluation of emergence for the assigned lineages and individual amino acid substitutions. We compared this to in-silico predicted B and T cell epitopes to demonstrate the need for vaccine redesign. We then answer how to redesign vaccines by evolving the emergence statistical analysis into an algorithm to predict emergence according to vaccine manufacturing timelines. Third, we used bioinformatic approaches to evaluate the effectiveness of quarantine early in the pandemic and to apply precision public-health genomics to Hawaiʻi. With the former, we developed a method demonstrating the quarantine delayed the spread of variants. With the latter, we identified the origin of all SARS-CoV-2 variants of concern found in Hawaiʻi. With this, we demonstrated that 76% of all sequences found in Hawaiʻi originate from California. Scientists and public-health officials can use the techniques we developed herein for use in future pandemic preparedness. The studies herein provide a foundation for understanding the COVID-19 pandemic worldwide and in Hawaiʻi. Such an understanding will allow the world to respond more adequately to future pandemics and SARS-CoV-2 as it becomes endemic. This work provides sequencing data from Hawaiʻi and uses that, combined with the worldwide data, to show that quarantine was effective, to develop a rule-based algorithm to monitor and determine virus evolution and emergence worldwide for vaccine redesign, and to track viral entry into Hawaiʻi from around the world.
Advancing Methodologies to Determine Protective Humoral Responses Generated by Recombinant Protein Subunit Filovirus Vaccines
(University of Hawaii at Manoa, 2021) Williams, Caitlin; Lehrer, Axel T.; Biomed Science (Tropical Medicine)
Zaire ebolavirus (EBOV) and other related viruses, Sudan ebolavirus (SUDV) and Marburg marburgvirus (MARV) are endemic in have caused sporadic and deadly outbreaks in Central and West Africa with case fatality rates of up to 90%. The largest Ebola Virus Disease (EVD) outbreak on record caused over 28,000 cases. There is a clear need for comprehensive disease intervention to prevent future outbreaks. Licensed vaccines, Ervebo® and Zabdeno + Mvabea, utilize virally vectored platforms and are contraindicated in pregnant and nursing women. To address the needs of these and of immunocompromised individuals, our lab has developed protein subunit vaccines that have shown protection against all three filoviruses in non-human primate (NHP) models. There are no known correlates of protection for any filovirus-induced disease and therefore no defined goals for what a protective immune response of a successful vaccine should be comprised of. The goal of this project is to study protective antibody responses in murine and NHP models in order to understand what aspects of immunity are important for protection in both the general population as well as during pregnancy and nursing to potentially induce maternal immunization. We developed methods for analyzing the breadth of antibody responses to our vaccine by utilizing a Peptide-Array based ImmunoSignature (IS) approach, as well as conducting a more thorough characterization of vaccine induced antibody function through avidity and surrogate neutralization assays. The IS in conjunction with avidity assays will elucidate both the antibody specificity and the strength of antibody binding that comprises a protected humoral response. Immunotherapies have provided insight to the importance of antibody binding strength, however there are conflicting findings in regard to the role of antibody avidity as a correlate of protection, our studies contribute to the overall ongoing research into EBOV vaccine correlates of protection. These experiments utilized pre challenge NHP sera, which is key for developing tools to determine vaccine efficacy in humans where challenge is not a possibility. Here we demonstrate that an analysis at the repertoire level as well as at the paratope-epitope binding level are capable of differentiating survivors from non survivors. While the avidity assays did not correlate with survival day they informed our understanding of eliciting antigen-specific maturation with three doses of vaccine as well as with increasing valency of vaccine formulations. To assess maternal immunization, we utilized a mouse model to analyze antibody transfer from immunized mothers to nurslings. The maternal immunization studies demonstrated that vaccine induced maternal antibodies are not only transferred in high concentration to suckling pups but are functional in virus neutralization. These experiments provide specific knowledge on vaccine induced immunity to EBOV. We demonstrate that vaccine failure can be detected in a genetically diverse animal model, that antibody avidity is not a correlate of protection but may provide clues to defining a vaccine correlate of protection, and that vaccine specific IgG can be passed mother to child and function to neutralize virus in pups. To our knowledge this is the first instance in defining vaccine failure and partial protection using pre challenge serum samples as well as the first instance of mother to child transfer of neutralizing EBOV vaccine antibody in milk.
Insect Cell-expressed Recombinant Viral Glycoproteins Are Effective Immunogens
(University of Hawaii at Manoa, 2020) To, Albert; Lehrer, Axel T.; Biomed Science (Tropical Medicine)
The ability to rapidly produce industrial quantities of antigen, for use in vaccinations, diagnostic tests and serosurveillance etc., is an essential component of an effective public health strategy. Properly-folded and post-translationally modified recombinant viral glycoproteins are important immunogens, especially used for the containment of highly virulent pathogens, however large scale production for preventative and therapuetic use are bottle-necked by the low protein yields and high cost associated with mammlian cell culture. The Drosophila S2 insect cell expression system is an ideal, alternative platform for expressing native-like proteins as it is capable of eukaryotic post-translational modification, requires lower maintenance than mammalian cells and is easily scalable. Furthermore purification using immunochromatography eliminates the need for peptide tags and yields protein at a high purity. To generate our vaccine antigens, we have developed a production and purification method for the surface glycoproteins of the Sudan Ebolavirus (SUDV), Zika Virus (ZIKV), and Lassa Virus (LASV), expressed in stably transformed Drosophila S2 cell lines and purified using immunochromatography with an appropriate monoclonal antibody. Each of these glycoproteins was recognized by well-characterized antibodies, or convalescent sera from infected animals. When formulated with pre-clinical and clinical grade adjuvants, each of these surface glycoproteins generated maximal antibody titers in vaccinated mice after the second dose that only increased slightly after a third dose. We demonstrate that these glycoproteins can cross-react with glycoproteins from closely related viruses but do not exert any immunointerference. Protection from viral challenge was observed in mice receiving two doses of our ZIKV vaccine candidate, or a passive transfer of high ZIKV E-specific IgG titered sera from vaccine-immunized mice. Formulation of LASV GP with several adjuvants generated a diversified IgG response and a robust cell-mediated response with one of the tested formulations. Together these results suggest that S2-cell expressed vaccine antigens, purified using immunoaffinity chromatography, are native-like in their conformational folding and can be used as effective immunogens when formulated with the appropriate adjuvant, capable of protecting mice from the targeted viral infection. Additionally, the work presented in this dissertation validates the use of insect cell-expressed viral glycoproteins in vaccines and will provide a cheaper and safer alternative than traditional vaccine platforms and can provide greater coverage, even to the vulnerable populations. This is especially important when rapid antigen production is required in response to a public health emergency such as the ongoing COVID-19 pandemic.
Emerging Challenges In HIV And Aging: Role Of A Glycan-binding Immunomodulatory Lectin
(University of Hawaii at Manoa, 2020) Premeaux, Thomas Alan; Ndhlovu, Lishomwa C.; Biomed Science (Tropical Medicine)
Despite antiretroviral therapy, people living with HIV have an increased risk for and earlier onset of age-related comorbidities including cardiovascular, kidney, liver, bone, and neurologic disease. About half of the population living with HIV in the U.S. is older than fifty years of age and this trend to an older demographic is expected to continue. Although multifactorial, evidence suggests chronic inflammation and immune activation, thought to stem from occult viral replication and the senescence, exhaustion, and premature aging of the immune system, are key drivers of early development of these comorbidities. Identifying reliable biomarkers to predict disease progression, determining key pathways in comorbidity development, and developing novel interventions to mitigate these complications are warranted as people with HIV age. One key regulator of immunological functions is interactions between cell-surface glycans and glycan-binding proteins, particularly galectins, a family of soluble β-galactoside-binding lectins. In particular, galectin-9 (Gal-9), a pleiotropic mediator of innate and adaptive immune responses, is elevated in the plasma during early HIV infection and remains high after ART suppression compared to people without HIV. Gal-9 is associated with HIV disease progression, the severity of age-related comorbidities, and can induce HIV transcription and reactivate latent virus. Investigating the significance of Gal-9 in the older HIV+ population could lead to the development of novel therapies to improve immune functionality, and reduce inflammation-associated co-morbidities, during ART-suppressed HIV infection. Our in vitro and ex vivo data show that Gal-9 mediates both HIV transcription and T cell activation through Lck-dependent mechanisms, indicative of a TCR activation pathway. We determined the potential of Gal-9 to serve as a biomarker of morbidity and mortality risk in HIV. Our data illustrate a link between cerebrospinal levels of Gal-9 with poor cognitive performance central nervous system immune activation, as well as plasma Gal-9 levels and mortality risk and the burden of comorbid disease among older people living with HIV. Finally, given that the functionality of endogenous Gal-9 during the course of HIV infection is not fully known and targeting Gal-9-induced pathways in vivo are limited, we generated anti-Gal-9 monoclonal antibodies with high specificity and limited off-target effects that can be used to target Gal-9 pathways in vivo in various animal models of HIV infection. Our work provides a framework in guiding the understanding of glycan-galectin interactions in the pathogenic mechanisms of age-related disorders in older HIV-infected individuals on suppressive ART and the relationship between aging, HIV, and inflammation.
Investigating the testicular tropism of Zika virus using 2D and 3D human tissue culture systems
(University of Hawaii at Manoa, 2020) Strange, Daniel P.; Verma, Saguna; Biomed Science (Tropical Medicine)
Zika virus (ZIKV) is a mosquito-borne flavivirus traditionally associated with mild febrile illness. However, following the 2015–2016 epidemic in the Americas, new severe manifestations of ZIKV disease emerged, including microcephaly in neonates and Guillain-Barre syndrome in adults. Sexual transmission of ZIKV, which is atypical for flaviviruses, also became a common occurrence, predominantly male-to-female, during the epidemic. Cohort and case studies have since shown that ZIKV can be shed in semen regardless of symptoms for several months to more than a year following infection. This indicates a potentially long infectious period for the virus and highlights a risk of disease spread to non-endemic regions. Furthermore, prolonged seminal shedding of ZIKV in humans has been associated with reduction in motile sperm and altered serum levels of testosterone, providing evidence that ZIKV likely infects the testes and impacts testicular function. However, due to the lack of relevant small animal models, little is known regarding ZIKV tropism and infection in the testes, including the cell targets, immune responses, and pathophysiology. These gaps ultimately hamper efforts to properly assess the risks of ZIKV persistence for male reproductive health and also hinders the development of appropriate countermeasures. To begin to address these gaps, here we investigate various aspects of testicular ZIKV tropism and infection using different in vitro systems, including human 2D testis cell cultures and recently established 3D multicellular human testicular organoids (HTO). The testis is composed of two compartments, the interstitium and seminiferous tubules. Here, we show that seminiferous tubule cells (STC), including peritubular myoid cells, spermatogonial stem cells, and Sertoli cells (SC), were all susceptible to ZIKV; however, SC were clearly the most permissive to infection. In contrast, Leydig cells (LC), the major cell type of the interstitium that produces testosterone, were highly resistant to ZIKV. Basal protein levels of the TAM (Tyro3, Axl, Mer) receptor Axl and basal transcript levels of antiviral effectors correlated with the differential permissibility of SC and LC to ZIKV infection. We further document that HTO supported productive ZIKV infection, resulting in reduced HTO viability and function. Temporal transcriptomics analysis of infected SC showed that the most prominent response to ZIKV at the early stage of infection was the suppression of genes involved in cell growth and proliferation, while peak virus replication was associated with the induction of genes involved in innate antiviral defense. Similarly, our proteomics analysis demonstrated that ZIKV infection in SC and STC predominantly impacted pathways and functional networks associated with innate antiviral defense. Increased levels of the interferon (IFN)-I/III-stimulated protein MX1 at the later time points of infection coincided with diminished propagation of ZIKV in SC, and silencing of MX1 and the IFN-stimulated gene (ISG) IFIT1 enhanced peak ZIKV replication in SC. Although IFN-I signaling was found to restrict ZIKV replication in SC, further analysis demonstrated that SC exhibited delayed/dampened IFN response following ZIKV infection. Moreover, neutralization of Axl receptor and its ligand Gas6 strongly attenuated ZIKV entry in SC. However, inhibition of Axl kinase did not affect ZIKV entry but instead led to decreased protein levels of suppressor of cytokine signaling (SOCS)1 and SOCS3, increased expression of ISGs, and reduced ZIKV replication. Similarly, treatment with an Axl kinase inhibitor attenuated ZIKV replication and increased ISG expression in HTO. Collectively, our findings provide new insights into the testicular tropism of ZIKV. The data implicate SC as the principal cell target of robust ZIKV infection in the testes and demonstrate the utility of HTO for studying various aspects of testicular ZIKV infection and pathogenesis in lieu of animal models. Moreover, this study highlights the IFN-I/III response as a driver of the antiviral state that eventually limits ZIKV propagation in SC. Importantly, the data also suggest that delayed/dampened antiviral defense in SC may contribute to the ability of ZIKV to establish persistence in the testes. Furthermore, this study defines a novel role of Axl in modulating ZIKV infection in SC and as a determinant of ZIKV tropism in the testes.
Regulation Of CD8 T-cell Function By Multiple Negative Immune Checkpoint Molecules During HTLV-1 Infection
(University of Hawaii at Manoa, 2020) Clements, Danielle M.; Ndhlovu, Lishomwa C.; Biomedical Sciences
Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive and debilitating inflammatory syndrome of the central nervous system, affects 1–5% of HTLV-1-infected individuals. There is no vaccine for HTLV-1, and treatment options are limited. Cytotoxic T lymphocytes (CTL) play an important role in antiviral immunity and previous studies have shown that CTL dysfunction is associated with increased expression of negative checkpoint receptors (NCRs). NCR expression, particularly the expression of multiple co-expressed NCRs, and whether or not they impact CTL activity has not been fully explored in the context of HTLV-1 infection. The purpose of this research was to determine the extent to which multiple NCRs are expressed on CD8 T cells in HTLV-1 infection and whether NCR blockade effectively enhances the functionality of CTLs in HAM/TSP. We hypothesized that patients with HAM/TSP co-express multiple NCRs on CD8 T cells, and that blocking more than one NCR would enhance CTL function to a greater degree than single-blockade strategies. Results from flow-cytometric analyses demonstrated that different individual NCRs are expressed on T cells from HTLV-1-infected asymptomatic carriers (AC) and HAM/TSP patients. Furthermore, CD8 T cells from HAM/TSP patients co-express multiple NCRs at higher frequencies than AC, including programmed cell death protein 1 (PD-1), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) and lymphocyte-activation gene 3 (LAG-3). Higher frequencies of dendritic cells expressing ligands for PD-1 and TIGIT were also observed in HAM/TSP patients. Upon NCR blockade, expression of interferon-gamma (IFN-), cluster of differentiation 107a (CD107a), and tumor necrosis factor- (TNF-a) increased in CD8 T cells in response to HTLV-1 virus to a greater extent when blocked with multiple (>3) blocking monoclonal antibodies (against PD-1, TIGIT, TIM-3 and LAG-3) compared to either single or dual combination blockade. Most interestingly, blockade of both single and multiple NCRs resulted in decreased frequency of IL-2+ CD8 T cells in HAM/TSP patients, but not in AC. The observed reduction in IL-2 expression after blockade may indicate that NCR blockade not only enhances anti-HTLV-1 CTL activity but decreases IL-2 production by anti-HTLV-1-specific CD8 T cells, perhaps leading to a reduction in infected CD4 T-cell proliferation. These results advance our understanding of NCR expression in HTLV-1 infection and supports future exploration of blocking multiple NCRs as a novel immunotherapeutic strategy for HAM/TSP.
Effect of antiretroviral therapy on blood-brain barrier integrity and central nervous system inflammation
(University of Hawaii at Manoa, 2020) Kettlewell, Joanna; Shiramizu, Bruce; Biomed Science (Tropical Medicine)
Human immunodeficiency virus type 1 (HIV) enters the central nervous system (CNS) as early as eight days after infection and crosses the blood-brain barrier (BBB) primarily via infected monocytes. Even with suppressive antiretroviral therapy (ART), HIV infection of the CNS causes persistent inflammation, neuronal injury, and BBB breakdown leading to neurocognitive impairment, categorized as HIV-associated neurocognitive disorders (HAND). HAND is a common, debilitating complication of HIV infection. Diagnosing and treating HAND remains challenging. Multiple biomarkers have been proposed to aid in the management of patients with HAND, however, their clinical relevance is undetermined. To address this gap, this study analyzed changes in neuroinflammatory mediators in serum and cerebrospinal fluid (CSF) of HIV-infected study participants with HAND on suppressive ART receiving CCR5-inhibitor maraviroc for 48 weeks. Due to inhibition of the CCR5 receptor, which is important for HIV entry into monocytes, the central hypothesis was maraviroc would reduce neuroinflammation and improve BBB integrity in vivo and in vitro, corresponding to improved neuropsychological performance. The effect of maraviroc on BBB integrity was assessed using both in vivo and in vitro functional assays. The study demonstrated a reduction in some neuroinflammatory mediators, but none that corresponded to improved neuropsychological performance. Decreased in vivo BBB integrity corresponded to increased CSF tumor necrosis factor α and serum calcium-binding protein B of the S-100 protein family. This study also assessed maraviroc as an addition to current pre-exposure prophylaxis (PrEP) therapy [tenofovir disoproxil fumarate, emtricitabine] due to concerns of drug resistance. PrEP with and without in vitro addition of maraviroc showed a reduction in monocyte trafficking across the BBB in two out of three study participants. In vitro PrEP exposure of BBB endothelial cells with and without maraviroc showed an increased presence of tight junction protein occludin. These findings indicate CCR5 inhibition with maraviroc may reduce some neuroinflammation and current PrEP drugs tenofovir and emtricitabine, with and without maraviroc, may be neuroprotective. This study contributes to the field on potential treatment and prevention strategies for HIV infection and HAND. Future research will increase the clinical and translational impact of these findings.
The Immune Response To Recombinant Subunit Vaccines
(University of Hawaii at Manoa, 2019) Medina, Liana Odette; Lehrer, Axel T.; Biomed Science (Tropical Medicine)
Zika Virus (ZIKV) is a positive sense, single stranded RNA virus that for many years was not associated with severe clinical symptoms. However, it became a public health threat following an epidemic in French Polynesia 2013–2014 that resulted in neurological complications associated with infection. Ebola virus (EBOV) on the other hand is a negative sense, single stranded RNA virus with case fatality rates in outbreaks reaching 90%. Between 2013 and 2016 an outbreak of an unprecedented scale occurred in West Africa with almost 30,000 people infected and over 11,000 fatalities were reported. There are currently no vaccines or therapeutics approved for ZIKV while the first EBOV vaccine, Ervebo (rVSVG-ZEBOV-GP), was approved in November 2019 by the European Commission. Another candidate vaccine against EBOV combining two different virally vectored approaches is in advanced clinical trials. Several different vaccine platforms are being used to develop additional strategies to prevent infections with these viruses, mostly based on recombinant viral vectors. However, virally vectored vaccines have shown significant safety risks, particularly in immunocompromised populations. Recombinant subunit vaccines have recently been put into use against several viral infections, such as Engerix-B and Recombivax HB against Hepatitis B virus, FluBlok against seasonal influenza, and GARDASIL and CERVARIX against human papilloma virus. Vaccines made using this versatile platform have high safety profiles and are relatively easy to manufacture and scale up. Using a recombinant subunit platform consisting of antigens produced in Drosophila melanogaster S2 cells, we have developed vaccine candidates for ZIKV and EBOV. The efficacy of our recombinant subunits against EBOV has been evaluated in a guinea pig and non-human primate (NHP) model, while the efficacy of our ZIKV vaccine has been evaluated in immunocompetent mice and NHPs. In our ZIKV NHP model, high neutralizing antibody titers were seen in all protected cynomolgus macaques, and passive transfer demonstrated that plasma from these NHPs was sufficient to protect against viremia in mice subsequently infected with ZIKV. Taken together these data demonstrate the immunogenicity and protective efficacy of the recombinant subunit vaccine candidate against ZIKV in NHPs and highlights the importance of neutralizing antibodies in protection against ZIKV infection, validating their potential to serve as a correlate of protection. Vaccine candidates containing the EBOV glycoprotein with or without matrix proteins Viral Protein 24 and Viral Protein 40, formulated with several different adjuvants were tested in mice, guinea pigs and NHPs for immunogenicity and efficacy against lethal EBOV challenge. We also evaluated bi- and trivalent formulations in guinea pigs and NHPs in an effort to develop multivalent filovirus vaccines. The results demonstrated that the monovalent vaccine candidates engendered high titers of antigen-specific antibodies in immunized animals, and two of these vaccine candidates afforded complete or nearly complete protection against lethal challenge. All vaccine candidates were able to elicit virus-specific anti-GP IgG titers in all species tested, but high antibody titers were also seen in animals not protected from viral challenge. Cell-mediated immunity was analyzed in samples taken from vaccinated NHPs with the goal of discovering responses that correlated with protection, and exploring cross-reactive responses to direct development of future formulations. While we were able to find vaccine specific cell-mediated immune responses, we so far were unable to correlate presence or absence or magnitude of these responses with vaccine efficacy.
The Exocyst Complex is Essential for Insulin Stimulated Glucose Uptake in Skeletal Muscle and Whole-Body Glucose Homeostasis
(University of Hawaii at Manoa, 2019) Fujimoto, Brent; Fogelgren, Benjamin C.; Polgar, Noemi; Biomedical Sciences
The earliest detectable sign of Type 2 Diabetes Mellitus is reduced insulin stimulated glucose uptake in skeletal muscle. Skeletal muscle is responsible for 80-90% of insulin stimulated glucose uptake. In the basal state, the membrane bound GLUT4 is sequestered in regions of the cytosol as GLUT4 storage vesicles. When stimulated by insulin, GLUT4 is incorporated into plasma membrane by the process of exocytosis. Conserved from yeast to humans, the Exocyst complex is important for the targeted delivery and docking of cytoplasmic vesicles to the plasma membrane. It has been demonstrated that the Exocyst complex is essential for insulin stimulated GLUT4 exocytosis in adipocytes. Our studies have demonstrated that the Exocyst complex is also critical for GLUT4 exocytosis and glucose uptake in L6-GLUT4myc myoblasts. The Exocyst inhibitor endosidin-2 and a heterozygous knockout of EXOC5 in skeletal myoblast cells both lead to impaired GLUT4 trafficking to the plasma membrane and also hindered glucose uptake. A tamoxifen-inducible skeletal muscle-specific knockout mouse strain of EXOC5 (EXOC5-SMKO) to assess the role of the Exocyst in glucose homeostasis in vivo was generated. EXOC5-SMKO mice had elevated overnight fasting glucose levels and increase body weight after a high fat diet challenge. Male EXOC5-SMKO mice had impaired glucose tolerance, enlarged Islets of Langerhans, and lower serum insulin. The research presented here confirms the central role of the Exocyst complex during insulin stimulated glucose uptake in muscle. This work will help to expand the range of potential targets for therapeutic interventions of Type2 Diabetes Mellitus.
Defining molecular adjuvant effects on human B cell subsets
(University of Hawaii at Manoa, 2019) Posner, Jourdan Kamakanamakamae; Chang, Sandra P.; Biomed Science (Tropical Medicine)
Recent advances in vaccine development include the incorporation of novel adjuvants to increase vaccine immunogenicity and efficacy. Pattern recognition receptor (PRR) ligands are of particular interest as vaccine adjuvants. During early childhood, the B cell compartment contains a high frequency of immature, transitional B cells. This prominent transitional B cell population may encounter PRR ligands during immunization; however, the response of human transitional B cells to these stimuli remains largely unknown. The goal of this dissertation work was to evaluate the capacity of PRR ligands to drive transitional B cells to mature into follicular or marginal zone B cells by assessing transitional B cell maturation and evaluating the transcriptome of mature B cell subsets. This work determined that PRR ligands can drive the phenotypic maturation of human transitional B cells as measured by CD23 expression and Rhodamine 123 retention. In vitro transitional B cell maturation resulted in the generation of both Fo-like and MZ-like B cells as determined by surface phenotype. Additionally, select PRR ligands induced gene expression changes in transitional B cells similar to mature B cells. We analyzed human tonsil follicular and marginal zone-like B cell transcriptomes to identify several genes uniquely associated with either cell fate decision and assessed the expression of some of these genes in our in vitro transitional B cell maturation model. To the best of our knowledge, this is the first study to examine the differential effect of various PRR ligands on the maturation of human transitional B cells into mature, naïve B cells and to examine the follicular versus marginal zone B cell fate decision. The rational design of vaccine adjuvants that take into consideration the effect of PRR ligands on B cell maturation and differentiation may lead to new strategies to improve the immunogenicity and efficacy of childhood vaccines.
Investigation of Archetype Human Polyomavirus JC Cellular Tropism and Genomic Alterations in JC Virus Pathogenesis
(University of Hawaii at Manoa, 2017-08) Lazaga, Nelson; Biomedical Sciences (Tropical Medicine)
The human polyomavirus JC (JCPyV) is the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). While the archetypal form of the virus is ubiquitous in the healthy human population, it is the rearranged form that is responsible for PML. The archetype form of JCPyV has a conserved noncoding control region (NCCR) that is defined by six designated blocks, A-F. However, the rearranged form has deletions and/or duplications in its NCCR. Although it has been established that the rearranged form of JCPyV is pathogenic, the events leading to the reactivation and/or rearrangement in its NCCR have yet to be determined. Thus, the lack of in vitro and in vivo archetype JCPyV replication models have hindered the understanding of mechanisms underlying the development of PML pathogenesis. In this report, we demonstrate in vitro infection and efficient replication of archetype JCPyV in renal proximal tubule epithelial (RPTE) and human brain microvascular endothelial (HBMVE) cells, limited or no replication in human brain cortical astrocytes (HBCA) and primary human fetal glial (PHFG) cells, and in vitro rearrangement of archetype JCPyV at day 645 in COS-7 cells. In addition, we demonstrate that archetype JCPyV (CY) and rearranged JCPyV (Mad1) can replicate in HBMVE cells, while limited replication was observed when HBMVE cells were transfected with the hybrid JCPyV (CYrM1c). Lastly, we demonstrate in vivo infection of JCPyV in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. To our knowledge, this is the first study demonstrating the ability for urine-derived archetype JCPyV to rearrange in vitro, to be infectious in naïve primary cells, and to demonstrate JCPyV infection in humanized NSG mice. This study will therefore give insight on cellular conditions involved in urine-derived archetype JCPyV infection, reactivation, and rearrangement, which will impact the development of much-needed therapeutics for PML
A Tale of Two Viruses: HPV Infection and Associated Anal Dysplasia among Hawai‘i HIV-Seropositive Patients and Related HIV-Seroconversion Risk in a Thai Population
(University of Hawaii at Manoa, 2017-08) Chuang, Eleanore J.; Biomedical Sciences (Tropical Medicine)
Human immunodeficiency virus (HIV) and human papillomavirus (HPV) are both sexually transmitted pathogens. As the most prevalent sexually transmitted infection in the world, HPV affects both men and women across many demographic categories. Although HPV often clears without treatment, persistent infection can cause dysplasia which can progress to cancer. HPV vaccines, licensed in the US since 2006, hold promise for reducing not only infection rates but also incidence of HPV-associated cancers. Albeit less prevalent than HPV, HIV still represents a considerable health burden worldwide even in an era of effective combination anti-retroviral therapy (cART). In the US, the majority of new HIV cases arise among gay and bisexual men. Despite billions of dollars and years devoted to research, an effective HIV vaccine remains elusive. Since HIV-positive individuals are more susceptible to other infections even while on anti-retroviral therapy, they are also prone to HPV infection and associated anal dysplasia. Additionally, a growing body of evidence suggests that HPV also impacts the acquisition of HIV. The axes of interactions between these two viral infections are not yet fully understood. The overall objective of this study was to further elucidate the relationship between HIV and HPV infection in anal dysplasia/cancer by assessing HPV infection in the context of HIV and vice versa. The central hypotheses were that HIV-positive individuals present more frequently with HPV infection and associated anal dysplasia and that HIV seroconversion may occur subsequent to HPV infection. First, the study demonstrated that HPV at non-anal sites may be associated with anal dysplasia/cancer among HIV-positive males. The presence of HPV and the number of HPV genotypes at anatomical sites other than the anus trended toward higher odds of ASIL. Presence of HPV and the number of HPV genotypes at the penile shaft conferred the highest, statistically significant odds of ASIL – greater odds than conferred by presence of HPV and the number of HPV genotypes at the anus itself. Second, the study showed the potential of HPV quantitation for enhanced screening and diagnosis of anal dysplasia/cancer in the context of HIV infection. All participants with HGAIN had HPV-16 E6 DNA levels above 10 copies per cell. Participants with LGAIN or Negative biopsy results and low HPV-16 E6 copy could potentially have avoided invasive HRA if HPV quantitation were used as a supplemental diagnostic marker for anal dysplasia. Third, the study explored the effect of prior HPV infection on acquisition of HIV. Among MSM and TG women in the Thai Test & Treat cohort, HPV acquisition appeared to increase the risk of subsequent HIV seroconversion. Despite remaining ambiguity over HPV’s role in HIV acquisition, some researchers are advocating for study of HPV vaccination as a means for reducing HIV incidence. This study has contributed to the body of knowledge in the field by identifying new diagnostic indicators of HPV-associated anal dysplasia and by providing additional evidence for HPV’s influence on HIV acquisition. Further investigation will permit validation of these novel diagnostic markers and innovative strategy for reducing the global incidence of HIV.

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