Ph.D. - Biomedical Sciences (Tropical Medicine)

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https://hdl.handle.net/10125/1939

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    ONE HEALTH APPROACH FOR STUDYING EMERGING AND RE-EMERGING VIRUSES IN LIBERIA
    (2024) Kamara, Varney M.; Lehrer, Axel; Biomed Science (Tropical Medicine)
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    Understanding The Myeloid HIV Reservoir In HIV-Associated Neurological Disorder During Antiretroviral-Treated HIV Infection
    (2023) Mitchell, Brooks I.; Shikuma, Cecilia M.; Biomed Science (Tropical Medicine)
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    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.
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    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.
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    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.
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    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.
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    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.