Quantitative Approach to Supramolecular Assembly Engineering for Isolating and Activating Antigen-Specific T Cells

T cell immunotherapy is a novel therapeutic strategy that aims to leverage the antigen-specific nature of a T cell immune response to treat a variety of immunological conditions. Over the past twenty years, T cell immunotherapy has been applied to treat several types of cancer, autoimmune conditions, and chronic infections, culminating in the FDA approval of two highly effective chimeric antigen receptor (CAR) T cell therapies targeting hematological cancers in 2017. While the initial success of T cell immunotherapy has been encouraging, identifying appropriate antigenic targets and optimizing T cell activation to promote effective responses in vivo remain significant challenges. In this dissertation, we discuss the development and application of new molecular tools for identifying, isolating, and activating antigen-specific T cells, which are directly relevant to the current challenges facing T cell immunotherapy. One of the greatest obstacles to developing a successful T cell immunotherapy is the selection of appropriate antigenic targets. T cells naturally recognize antigen-derived peptides presented on polymorphic major histocompatibility complex (MHC) proteins, and different MHC alleles exhibit different peptide binding specificities. Therefore, peptides that promiscuously bind multiple MHC alleles representing a diverse population have significant potential in the development of broadly protective peptide-based therapeutics and vaccines. A number of high-throughput in silico strategies have been developed to predict peptide-MHC binding; however, the accuracy of these approaches is generally inadequate for the reliable prediction of class II peptide-MHC (MHCII) interactions. In contrast, most experimental systems designed to measure peptide-MHCII binding emphasize quantitative detail over throughput. In this dissertation, we develop and validate a high-throughput screening strategy to evaluate peptide binding to four common MHCII alleles. Using this strategy, which we have termed microsphere-assisted peptide screening (MAPs), we screened overlapping peptide libraries of antigenic viral proteins and identified 12 promiscuously MHCII-binding peptides. Subsequent structural analysis indicated that nearly half of these peptides overlapped with antibody neutralization sites on the respective viral protein. Together, these results indicate that the MAPS strategy can be used to rapidly identify promiscuously binding and immunodominant peptides that have therapeutic relevance. Another significant challenge limiting the successful application of T cell immunotherapy is expanding a clinically relevant number of therapeutically effective T cells. The effectiveness of a T cell response is largely determined by the spatial and stoichiometric organization of signals delivered to the T cell during T cell activation. One strategy for promoting an effective T cell response is to tune the presentation of stimulatory and costimulatory signals through artificial antigen presentation. However, existing technologies have a limited ability to control the spatial and stoichiometric organization of T cell ligands on 3D surfaces. In this dissertation, we introduce a novel strategy for presenting highly organized clusters of stimulatory and costimulatory ligands to T cells using protein-scaffold directed assembly. Using this approach, we systematically investigated how the global surface density, local valency, and stoichiometric ratio of T cell ligands on a 3D cellular (yeast) surface can be manipulated to tune T cell activation. After validating this approach, we further develop more complex scaffold-assembly schemes to enhance the controllability of isolating and activating antigen-specific T cells. We believe that MAPS and artificial antigen presentation using protein-scaffold directed assembly provide a robust toolset for identifying, isolating, and activating antigen-specific T cells for T cell immunotherapy.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/147510/1/masonrsm_1.pd

Development of Novel Zika and Anthrax Viral Nanoparticle Vaccines

Vaccines protect against numerous infectious diseases and prevent millions of deaths annually, but there are still many infectious diseases for which no licensed vaccine exists. Developing a new vaccine requires balancing safety and efficacy, and viral nanoparticle (VNP) vaccines possess both of these characteristics. The work herein demonstrates how tobacco mosaic virus (TMV) nanoparticles can serve as a platform to create candidate vaccines for Zika virus (ZIKV) and anthrax. In the first study, a ZIKV-specific epitope was genetically fused to TMV to create a safe and inexpensive vaccine that proved highly immunogenic in mice and led to the discovery of ZIKV-specific neutralizing antibodies that may have applications in therapeutics and diagnostics. In the second study, anthrax toxin domains were expressed, purified, and conjugated to the outer surface of modified TMV nanoparticles. These VNPs were readily recognized by anthrax immune serum, but further studies will be necessary to ascertain their ability to induce a protective immune response. As demonstrated in these studies, genetic fusions and chemical conjugations to TMV each have distinct benefits and limitations. However, both methods result in the production of TMV-based VNPs, in which the TMV virion acts as both a scaffold and delivery mechanism, ensuring that the foreign antigens are taken up by DCs, transported to lymph nodes, and stimulate robust, antigen-specific B and T cell responses. In summation, this work shows how TMV VNPs displaying exogenous antigens can be used to create novel vaccines against both viral and bacterial pathogens

Enhancement of plant expression vectors using replication and silencing suppressor elements

Molecular farming is gaining traction as a cost-effective platform to produce recombinant proteins. Further improvements can be made, however, to increase overall yield especially for difficult to express proteins. In this study virus-derived silencing suppressors and replication elements were used with the aim of increasing expression and yield of enhanced green fluorescent protein (EGFP) and the Zika PrME polyprotein in Nicotiana benthamiana. A comparison of four viral silencing suppressor proteins was performed: these were tomato spotted wilt virus non-structural protein, NSs, tomato aspermy virus (TAV) 2b, tomato bushy stunt virus P19 and begomovirus alphasatellite Rep. Differences in EGFP expression in N. benthamiana due to the silencing suppression were determined using immunoblotting and fluorescence of EGFP. In addition, replication elements from three viruses (bean yellow dwarf virus [BeYDV], beak and feather disease virus [BFDV] and begomovirus alphasatellite) were assembled into novel plant expression vectors using GoldenBraid (GB) cloning technology and assessed using EGFP. Finally, the two approaches were combined in an attempt to express the Zika PrME polyprotein, which was assessed using immunoblotting. EGFP expression was found to be greatest in the presence of the TAV 2b protein and no difference in fluorescence intensity between the original BeYDV replicating plant expression vector and that constructed using GB could be detected; however, the GB assembly of the BFDV and alphasatellite plant expression vectors was unsuccessful. The TAV 2b combined with the BeYDV replicating elements were used for the expression of Zika PrME. The gene was successfully cloned into the replicating BeYDV vector and a vector that does not replicate (negative control). The PrME was not detected using anti-His tag immunoblotting despite optimisation for Agrobacterium infiltration density, harvest day post infiltration, signal peptides and buffers during extraction. In this study I demonstrated the following: that the TAV 2b protein out-performed all other silencing suppressors; that the GB cloning technology can be successfully applied in the development of novel plant expression vectors, although further optimisation is required for these and for Zika PrME expression. Further work in characterising the effect of silencing suppression on recombinant protein expression can be assessed using RT-qPCR to measure the effect on mRNA levels. In summary, these improvements in plant recombinant protein expression can be readily applied to large scale production of novel therapeutics and vaccines

Antiviral activities and applications of ribosomally synthesized and post-translationally modified peptides (RiPPs)

The emergence and re-emergence of viral epidemics and the risks of antiviral drug resistance are a serious threat to global public health. New options to supplement or replace currently used drugs for antiviral therapy are urgently needed. The research in the field of ribosomally synthesized and post-translationally modified peptides (RiPPs) has been booming in the last few decades, in particular in view of their strong antimicrobial activities and high stability. The RiPPs with antiviral activity, especially those against enveloped viruses, are now also gaining more interest. RiPPs have a number of advantages over small molecule drugs in terms of specificity and affinity for targets, and over protein-based drugs in terms of cellular penetrability, stability and size. Moreover, the great engineering potential of RiPPs provides an efficient way to optimize them as potent antiviral drugs candidates. These intrinsic advantages underscore the good therapeutic prospects of RiPPs in viral treatment. With the aim to highlight the underrated antiviral potential of RiPPs and explore their development as antiviral drugs, we review the current literature describing the antiviral activities and mechanisms of action of RiPPs, discussing the ongoing efforts to improve their antiviral potential and demonstrate their suitability as antiviral therapeutics. We propose that antiviral RiPPs may overcome the limits of peptide-based antiviral therapy, providing an innovative option for the treatment of viral disease

Improving Vaccine Design For Viral Diseases Using Modified Antigens And Vectors

Two of the principal challenges facing vaccine design today are how to generate protective antibody responses against viruses that have evolved sophisticated strategies to evade the humoral immune system and how to more rapidly and effectively produce vaccines to address emerging epidemics. In this regard, we explored multiple strategies to improve vaccine design for HIV-1 and Zika virus. In one approach, we derived CD4-independent variants of HIV-1 envelope (Env) with the hypothesis that such Envs would expose conserved epitopes that may be targets of protective, non-neutralizing antibodies. We characterized the biological and structural properties of two CD4-independent Env clones and found that they exhibited significantly greater exposure of a relatively conserved, linear epitope in the second variable loop (V2) that had previously been associated with decreased risk of infection in a clinical HIV-1 vaccine trial. This epitope was significantly more immunogenic in mice and nonhuman primates and, intriguingly, was associated with more rapid development of antibody-dependent cell-mediated cytotoxicity. In another approach, we designed mutations in the cytoplasmic tail of HIV-1 Env that were predicted to increase its cell surface expression and thus its immunogenicity in a vaccinia prime-protein boost vaccine protocol. We found that the highest level of surface expression was mediated by Envs with truncated cytoplasmic tails, and this was associated with higher levels of binding and neutralizing antibodies after vaccinia primes and protein boosts, respectively. These two studies revealed that modifications to HIV-1 Env immunogens are able to influence both the quality and magnitude of desirable antibody responses. Finally, we used a newly developed vaccine platform based on nucleoside-modified mRNA to design a vaccine against Zika virus. This vaccine, encoding the surface prM and E proteins, was potently immunogenic and elicited high and sustained titers of neutralizing antibodies in mice and nonhuman primates following a single intradermal immunization. We observed rapid and durable protection from Zika virus infection in mice and a high level of protection in monkeys challenged five weeks after vaccination. This vaccine thus represents a promising candidate for clinical use in controlling the spread of Zika virus

Structural and functional analysis of Zika Virus NS5 protein

[eng] Zika virus (ZIKV) belongs to the Flaviviridae family and constitute an important public health concern since ZIKV infection produced devastating effects in new born infants. Flaviviruses present a positive sense single stranded RNA genome flanked by highly structured untranslated regions (UTR) carrying one open reading frame that codifies for three structural proteins (C, prM, E) and five nonstructural proteins (NS1-5). At the most C-terminal end, NS5 protein carries a RNA dependent RNA polymerase (RdRP) and a methyl transferase domain (MTase) for genome copying and 5’ capping activities of the newly synthesized RNA, respectively. Given the crucial role of this enzyme for viral replication, NS5 constitutes an attractive antiviral target to inhibit viral replication. In this study, we determined the structure of the ZIKV NS5 protein using X-Ray crystallography combined with several structural biology approaches to characterize the supramolecular arrangement of the ZIKV NS5 protein. We identified the monomer-monomer and dimer-diner interactions to form fibril-like structures, and evaluated the role of oligomer formation, using in-vitro polymerization assays. We also evaluated the in-vivo effect of NS5-oligomerisation in chicken embryos, stablishing a connection between this protein and microcephaly. One of the most important RNA structures present at the 5’UTR of flavivirus genomes is the 5SLA. This structure was identified previously to bind the NS5 protein, acting as a promoter and being essential for viral replication. We assayed and optimized the NS5-5SLA complex stability using biophysical and biochemical techniques and determined the structure of the complex by single particle cryo-EM. Comparisons between the NS5-5SLA complex and the NS5 crystallographic structure revealed for the first time in flavivirus, important conformational changes in the NS5 RdRP. We identified the residues involved in complex formation and characterized the effect of this binding on NS5 polymerization, shedding new light on the understanding of replication mechanisms in flaviviruses.[spa] El virus Zika (ZIKV) pertenece a la familia Flaviviridae y constituye una amenaza para la salud pública, especialmente debido a las malformaciones provocadas en neonatos. Los flavivirus presentan un genoma RNA de simple cadena con polaridad positiva, flanqueado por regiones no traducidas (UTR) que presentan una elevada estructura secundaria, seguido de una región codificante para una única poliproteína que por proteólisis dará lugar a tres proteínas estructurales (C, prM, E) y cinco proteinas no estructurales (NS1-5). En el extremo C-terminal se encuentra la proteina NS5 que presenta actividad ARN polimerasa dependiente de ARN (RdRP) y un dominio metil-transferasa (MTase) para copiar el genoma y añadir una caperuza al extremo 5’ del nuevo ARN sintetizado, respectivamente. Dado el papel crucial de este enzima en la replicación viral, la proteina NS5 constituye una diana antiviral muy atractiva para inhibir la replicación del virus. En este estudio, determinamos la estructura de la proteína NS5 de ZIKV, usando cristalografía de Rayos-X combinada con diferentes técnicas biofísicas para caracterizar la organización supramolecular de la proteína. Identificamos las interacciones monomero-monomero y dimero-dimero para caracterizar las estructuras fibrilares de la proteína y evaluamos los efectos de la dimerización en la actividad polimerasa in-vitro. También evaluamos los efectos de la oligomerización de NS5 in-vivo en embriones de pollo, estableciendo una conexión entre esta proteína y la aparición de microcefalia en fetos infectados. Una de las estructuras de ARN más importantes presentes en el 5’UTR del genoma de los flavivirus es el 5SLA. Previamente se describió que esta estructura se unía a NS5 y actuaba como un promotor, siendo ademas esencial para la replicación viral. Medimos y optimizamos la estabilidad del complejo NS5-5SLA mediante técnicas biofísicas y bioquímicas y determinamos la estructura del complejo mediante cryo-EM. Las comparaciones entre la estructura cristalográfica y cryo-EM de NS5 revelaron, por primera vez en flavivirus, cambios conformacionales importantes en el dominio RdRP. Identificamos los residuos implicados en la formación del complejo y caracterizamos el efecto de la unión de NS5 a 5SLA sobre su actividad polimerasa. Estos resultados arrojan nueva luz para entender los mecanismos de replicación en los flavivirus

Has Molecular Docking Ever Brought us a Medicine?

Molecular docking has been developed and improving for many years, but its ability to bring a medicine to the drug market effectively is still generally questioned. In this chapter, we introduce several successful cases including drugs for treatment of HIV, cancers, and other prevalent diseases. The technical details such as docking software, protein data bank (PDB) structures, and other computational methods employed are also collected and displayed. In most of the cases, the structures of drugs or drug candidates and the interacting residues on the target proteins are also presented. In addition, a few successful examples of drug repurposing using molecular docking are mentioned in this chapter. It should provide us with confidence that the docking will be extensively employed in the industry and basic research. Moreover, we should actively apply molecular docking and related technology to create new therapies for diseases

유전체 비교분석을 통한 포유류 감염성 바이러스의 진화에 대한 통찰

학위논문(석사)--서울대학교 대학원 :농업생명과학대학 농생명공학부,2019. 8. 김희발.감염성 바이러스는 인간을 비롯한 많은 종의 동물을 감염시켜 돌이킬 수 없는 결과를 초래하기도 합니다. 수많은 사람을 죽음에 이르게 하는 것은 물론, 매 해마다 대규모 가축 감염사례로 인하여 축산업에 커다란 경제적 피해를 끼치고 있습니다. 그렇기 때문에 감염성 바이러스에 대한 충분한 연구가 필요합니다. 바이러스는 다른 미생물이나 생명체에 비하여 유전자 변형이 보다 빠르고 무작위로 이루어지는 특징이 있습니다. 대부분의 바이러스는 숙주의 종에 따라 감염 여부가 달라지지만, 뉴클레오타이드와 아미노산 서열 하나의 변형으로도 새로운 종의 숙주를 감염시키거나 그 독성이 달라지기도 하기 때문에 그들의 유전체 차원에서의 특징을 발견하고 분석하는 것은 상업적 및 과학적 주요한 가치를 제공합니다. 이러한 유전체 특징 중에서 단일 유전자 변이체(Single Nucleotide and Amino acid variant)는 많은 연구에서 연구 대상으로 사용되고 있습니다. 실제적으로 바이러스 연구에서 바이러스의 종을 동정하거나 백신 개발 등 다양한 분야에 사용되고 있습니다. 챕터 2지카바이러스는 일반적인 성인이 감염되었을 시에는 지카열, 두통 및 관절통 등의 증상을 유발하지만 임산부가 감염되었을 시에는 태아의 소두증을 일으키는 것과 연관이 있다고 알려져 있습니다. 지난 10년간 전 세계에 폭발적으로 퍼져 나갔으며 많은 학자들이 지카바이러스의 분자 메커니즘에 대한 연구를 수행했습니다. 그러나 치료와 예방을 위한 의약품 및 백신 개발은 아직까지 진행 중이며 보다 많은 유전체 수준에서의 연구가 필요합니다. 이 연구에서 공개데이터베이스로부터 이용 가능한 지카바이러스의 NGS 유전체 데이터를 수집하고 분석을 통하여 지리적, 시기적 관점을 고려한 지역 특이적 유전체 변이(Single Nucleotide and Amino Acid variants)를 유전자 마커로써 제시하였습니다. 진화적 연관분석과 자율학습 k-means 클러스터링 알고리즘을 이용하여 4개의 대표그룹을 선정하였습니다. 대표 4그룹에 초점을 맞추어 통계적으로 유의미한 유전체 변이들을 찾아내고 dN/dS 진화 분석으로 진화적으로 가속화된 단백질 암호화 영역을 확인했습니다. 이후 그룹 기능성 단백질 영역과 B-cell, T-cell 특이적 항원결정기 후보를 예측하여 찾아낸 유전체 변이들이 단백질 및 항원결정기 형성의 결정적인 역할을 확인하여 그룹별 주요 유전자 마커로써 제안하였습니다. 챕터 3인플루엔자의 새로운 타입으로 분류된 인플루엔자 D 바이러스는 소를 비롯한 반추동물을 감염시키는 호흡기성 바이러스입니다. 감염 증상은 경미하지만 다른 치명적인 호흡기성 바이러스 감염을 유발하고 인간에게도 감염될 수 있는 잠재성이 있기 때문에 유전체 차원에서의 연구를 수행하였습니다. 인플루엔자 D 바이러스의 모든 유전자 단편 NGS데이터를 이용한 유전체 특성 및 진화적 상관관계 분석으로 하나의 유전자 단편을 통한 분석의 결과와의 차이점을 밝혀냈습니다. 그 결과를 토대로 선정한 대표 그룹을 초점으로, 통계적으로 유의미한 특이적 유전체 변이를 찾아냈습니다. 이후 dN/dS 진화 분석과 단백질 코딩영역, B-cell 특이적 항원결정기 예측 분석 결과와 비교하여 그룹 특이적 유전자 마커로써 제안하였습니다. 이 연구를 통하여 감염성 바이러스의 그룹별 특이적 유전자 마커를 제시하고 이 마커가 새로운 바이러스 종의 동정과 병독성 진화에 대한 통찰, 그리고 백신 개발에 도움을 줄 수 있을 것입니다.Infectious viruses infect many species of animal, including human, and cause irreversible consequence. They bring fetal death to human and cause massive economic losses to livestock industry due to the large-scale infection. Therefore, we need more research on infectious viruses. Viruses have faster and random genetic variable features than other organisms. Most viruses are susceptible to infection depending on the host species. However, since a single nucleotide and amino acid sequence variation leads infection to a new species or alter its toxicity, genomic level of virus research provides major commercial and scientific value. Therefore, many researchers focus on the single genetic variation for identification of a new virus species or vaccine study. Chapter 1Zika virus (ZIKV) is known to be associated with a serious brain disease, fetal microcephaly in pregnant women, and has been explosively spread throughout the world over the last decade. Virologists of most countries attempted investigations of ZIKV molecular mechanisms to prevent the worldwide proliferation. However, only few genetic variants in several regions were anticipated as targets of vaccines and medicines. Here, I analyzed all of available ZIKV complete genomes from the Virus Pathogen Resource (ViPR) database to identify novel genetic markers by considering geographical and temporal perspectives. By principal component and phylogenetic analysis, ZIKV strains formed four clusters according to collected continent. Focusing on the major groups in African, Asian, Central America and Caribbean, I found single nucleotide variants (SNVs) supported by statistical significance. From the dN/dS analysis, I identified the protein coding regions that were evolutionary accelerated in each group. Out of the intercontinental SNVs, non-synonymous and synonymous variants on functional protein domains and predicted B-cell and T-cell epitopes were suggested as regional markers. I believe these local genetic markers can improve medical strategies for ZIKV prevention, diagnosis, and treatment. Chapter 2Influenza D virus (IDV), a new type of influenza, is a respiratory virus that infects ruminants, including cattle. Because the infection symptoms of IDV are mild, but, causes fatal infection of other respiratory viruses and have potential for infection in human, I conducted researches at the genomic level. Using the results of phylogeny and principal coordinate analysis (PCoA), we compared concatenated all of coding sequence dataset and each of genes coding sequence dataset. I confirmed that concatenated dataset results were more appropriately clustered into four groups with isolated region, and I selected the main three groups. Focusing on the main three groups, I found statistically significant genetic markers in comparison with dN/dS analysis, searching protein coding region, and B-cell epitope prediction analysis. Through this study, I suggest local-specific genetic markers of infectious virus, and these markers will give a deep insight for further studies.ABSTRACT IV CONTENTS VII LIST OF TABLES VIII LIST OF FIGURES IX CHAPTER 1. LITERATURE REVIEW 1 CHAPTER 2. IDENTIFICATION OF LOCAL-SPECIFIC GENETIC MARKERS OF ZIKA VIRUS ACROSS THE ENTIRE GLOBE 7 2.1 ABSTRACT 8 2.2 INTRODUCTION 9 2.3 MATERIALS AND METHODS 12 2.4 RESULTS 18 2.5 DISCUSSION 26 CHAPTER 3. LOCAL GENETIC MARKERS CLUSTERED BY CODING SEQUENCES OF INFLUENZA D VIRUS 56 3.1 ABSTRACT 57 3.2 INTRODUCTION 59 3.3 MATERIALS AND METHODS 61 3.4 RESULTS 66 3.5 DISCUSSION 72 REFERENCES 93 요약(국문초록) 100Maste

In Silico Modeling and Immunoinformatics Probing Disclose the Epitope Based PeptideVaccine Against Zika Virus Envelope Glycoprotein

Zika virus (ZIKV) is an aedes mosquito borne pathogen belonging to the member of flaviviridae subgroup is the causative agent of an emerging disease called Zika fever, known as a benign infection usually presenting as influenza like illness with cutaneous rash. Due to recent epidemic outbreaks it is realized as a major health risk which need enhanced surveillance, but no attempt has been made to design an epitope based peptide vaccine against Zika virus. Viral envelope proteins are derived from host cell membrane proteins with some viral glycoproteins and are used to cover their protective protein capsid, help the viruses to enter host cells and help them to avoid the host immune response. In this study, amino acid sequence of ZIKV envelope glycoprotein was obtained from a protein database and examined with in silico approaches to determine the most immunogenic epitopes for B cell and T cell which could induce humoral as well as cell mediated immune response. Both the linear and conformational epitopes for B cell were predicted by immunoinformatics tools housed in IEDB resources. The peptide sequence DAHAKRQTVVVLGSQEGAV from position 121 and peptide sequence from 117-137 amino acids were predicted as most potential B cell linear and conformational epitopes respectively. Epitopes for CD4+ and CD8+ T cell were also predicted by using tools within IEDB resource and peptide sequence MMLELDPPF from position 250-258 amino acids was predicted as most immunogenic CD8+ T cell epitope with immune response evoking ability prediction score (I pMHC) of 0.09139 and conservancy of 52.17%. The innate immune response for ZIKV envelope glycoprotein was determined by interferon (IFN)-gamma effectuation and mimicking capacity by immunoinformatics and molecular docking study respectively. However, this is an introductory approach to design an epitope based peptide vaccine against Zika virus; we hope this model will be very much helpful in designing and predicting novel vaccine candidate

Characterizing the Humoral Response to Flavivirus Infection

Flaviviruses are positive (+) sense, single-stranded RNA viruses of the Flaviviridae family that are transmitted by mosquitoes. For our studies, we focused on Zika virus (ZIKV) and Japanese encephalitis virus (JEV). Most human infections with ZIKV historically resulted in a mild self-limiting febrile illness. However, since 2013, a worldwide spread and increase in ZIKV infections has been observed. Notably, ZIKV has been associated with autoimmune ascending paralysis (Guillain-Barré Syndrome) and ophthalmologic effects in adults and intrauterine growth restriction and microcephaly in developing fetuses. Current vaccine efforts utilize technologies implemented for related flaviviruses (yellow fever virus (YFV), Dengue virus (DENV), and JEV) including subunit-based, chemically inactivated, and live-attenuated vaccines. Furthermore, co-circulation of flaviviruses, such as DENV and ZIKV in regions of South America, make it desirable to generate a vaccine that protects against both. JEV infections are usually clinically asymptomatic or result in a mild self-limiting febrile illness. However, disseminated infection and viral penetration of the blood-brain barrier into the central nervous system results in meningitis and encephalitis, which are associated with high morbidity and mortality. Children are especially vulnerable to neuroinvasion due to lack of prior immunity and the relative immaturity of their immune responses. Although vaccination programs in endemic countries have decreased the incidence of disease, existing vaccines have limitations including multiple dose requirements and reactogenicity. Finally, a major issue in vaccine efficacy is the derivation from genotype III (GIII) strains, the concurrent diversity of JEV worldwide, and the scarcity of efficacy testing across multiple genotypes. Currently, there are five genotypes of JEV that encompass approximately 100 unique strains. In addition, the dominant genotypes vary by country and are not static over time. We are interested in understanding the immunologic restriction of flavivirus infection by characterizing the interaction between viruses and the humoral response. We identified a panel of mouse and human derived anti-ZIKV monoclonal mAbs and found that ZIKV specific mAbs strongly neutralize multiple strains of ZIKV of Asian and African lineages compared to mAbs that recognize a cross-reactive determinant. Additionally, we identified a novel conformational inter-dimer epitope that when bound, results in significant reduction in in vitro infection and in vivo protection. We tested the prophylactic and therapeutic efficacy of the strongest neutralizing mAbs in adult male mice for lethality and pregnant female mice for transplacental protection of fetuses. We also tested a panel of anti-DENV mAbs derived from naturally infected patients. We confirmed that EDE1 mAbs, which have stronger virus binding in the absence of glycosylation compared to EDE2 mAbs, are more potent neutralizers of multiple ZIKV strains. We demonstrated that viral seeding of immune privileged sites, such as testis and fetus, occurs by the second day post-infection and mAb administration after this may reduce but not eliminate viral burden and effects in the acute and persistent stages of infection. For JEV, we generated a panel of mouse and human anti-JEV mAbs. We identified a subset of domain I and domain III specific mAbs that can neutralize JEV strains representative of four different genotypes. Subsequent in vivo testing demonstrated a broad range of effective doses that protected prior to and following infection with highly virulent strains of JEV representative of multiple genotypes. We anticipate that further understanding of epitope specificity for neutralization and protection is essential for understanding the efficacy of current (for JEV) and future (for ZIKV) vaccines to multiple strains and genotypes. Moreover, this will improve our understanding of correlates of protection of flavivirus vaccines which remain poorly understood, apart from YFV