Dengue Reporter Virus Particles for Measuring Neutralizing Antibodies against Each of the Four Dengue Serotypes

The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination

Beyond bNAbs: Uses, Risks, and Opportunities for Therapeutic Application of Non-Neutralising Antibodies in Viral Infection

The vast majority of antibodies generated against a virus will be non-neutralising. However, this does not denote an absence of protective capacity. Yet, within the field, there is typically a large focus on antibodies capable of directly blocking infection (neutralising antibodies, NAbs) of either specific viral strains or multiple viral strains (broadly-neutralising antibodies, bNAbs). More recently, a focus on non-neutralising antibodies (nNAbs), or neutralisation-independent effects of NAbs, has emerged. These can have additive effects on protection or, in some cases, be a major correlate of protection. As their name suggests, nNAbs do not directly neutralise infection but instead, through their Fc domains, may mediate interaction with other immune effectors to induce clearance of viral particles or virally infected cells. nNAbs may also interrupt viral replication within infected cells. Developing technologies of antibody modification and functionalisation may lead to innovative biologics that harness the activities of nNAbs for antiviral prophylaxis and therapeutics. In this review, we discuss specific examples of nNAb actions in viral infections where they have known importance. We also discuss the potential detrimental effects of such responses. Finally, we explore new technologies for nNAb functionalisation to increase efficacy or introduce favourable characteristics for their therapeutic applications

A Structural Perspective of Antibody Neutralization of Dengue Virus

The four dengue viruses: DENV) are mosquito-borne flaviviruses and are considered the world\u27s most significant arboviruses in terms of worldwide disease burden. Symptoms of dengue disease are classified into dengue fever, a mild, febrile illness, and the potentially fatal severe dengue, which can include hemorrhaging and shock. Antibody protection against DENV correlates with the production of neutralizing antibodies against the envelope: E) glycoprotein. To understand the role of antibodies in DENV infection, we sought to dissect the relationship between epitope and function. Virologic studies had identified that the most potently neutralizing antibodies are against domain III: DIII) of the E protein. We have identified five epitopes within DENV DIII. Our data suggests that the most potently neutralizing antibodies are specific for a single serotype, while cross-reactive antibodies are relatively poorly neutralizing. Additionally, we were surprised to define neutralizing epitopes that were shown to be inaccessible on the surface of the virion in cryo-electron microscopy studies. Fine epitope mapping was used to define the epitopes of a panel of existing DENV-2 antibodies. Antibodies against the lateral ridge were the most potently neutralizing antibodies and reacted only with the DENV-2 serotype. The second epitope was centered on the DIII A-strand, and antibodies against this epitope reacted with several serotypes of DENV. Several poorly neutralizing antibodies reacted to all four DENV serotypes, as well as West Nile virus, a related flavivirus, mapped to the highly conserved AB loop of DIII. We expanded our studies of DIII-specific antibodies to the DENV-1 serotype. One antibody, E106, potently neutralized the five DENV-1 strains representing the five genotypes, and bound a composite epitope of the lateral ridge and A-strand epitopes. Despite the potency of E106-mediated neutralization, a combination of structural, biophysical, virologic data suggest that potent DENV-1 neutralization by E106 is coincident with bivalent engagement of the virus. Additionally, we determined the crystal structures of E111 bound to a novel fifth CC\u27 loop epitope on domain III: DIII) of the E protein from two different DENV-1 genotypes. The available atomic models of DENV virions revealed that the E111 epitope was inaccessible, suggesting that it recognizes an uncharacterized virus conformation. While the affinity of binding between E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC\u27 loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development

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

The Molecular Basis of Antibody Mediated Neutralization of Hepatitis C Virus

Hepatitis C virus: HCV) is positive strand, blood-borne, hepatotropic RNA virus that causes chronic infection in ~170 million people worldwide and is the leading cause of liver transplantation in the United States. HCV entry and attachment is mediated by the envelope protein E2 through interaction with several cellular receptors including CD81, scavenger receptor B1: SRB-1), claudin-1, and occludin, although the exact mechanism by which these receptors facilitate infection remains unclear, largely due to the absence of a structural model of E2. The production of neutralizing antibodies against E2 is thought to be important for controlling HCV infection, likely by blocking virus interaction with these receptors. To better understand the structural and molecular basis of antibody neutralization of HCV, which could be used to inform novel therapeutic or vaccine approaches, we generated a panel of 78 monoclonal antibodies: MAbs) against the E2 protein from HCV genotypes 1 and 2 and assessed their neutralizing activity in vitro. Using this approach and by performing mechanistic studies, we identified three neutralizing MAbs, H77.16, H77.39, and J6.36, that inhibit infection at a post-attachment step. Using a yeast display library of E2 protein variants, we mapped the critical binding residues of these MAbs to distinct regions of the E2 protein: H77.16 binds within the HVR1 and to a conserved CD81 binding region ~125 amino acid residues C-terminal to the HVR1; H77.39 binds to conserved residues upstream of the hypervariable region: HVR1); and J6.36 binds to amino acid residues within HVR1 as well a site ~150 amino acids C-terminal to HVR1. Receptor-binding inhibition studies using E2 demonstrated that H77.16 potently inhibits binding to SR-B1, H77.39 potently inhibits binding to SR-B1 and CD81, and J6.36 potently inhibits binding to SR-B1 and modestly inhibits binding to CD81. Further mechanistic studies demonstrated that MAb-mediated neutralization could be enhanced by increases in pre-incubation temperature and time and that these results were likely due to altered epitope exposure on the viral surface. Together, these data provide new insight into the mechanisms by which antibodies neutralize infection of HCV

Chikungunya Virus

The chikungunya virus (CHIKV) is an RNA virus that is transmitted to humans by Aedes mosquitos commonly found in tropical and subtropical countries. In humans, CHIKV causes an infection with symptoms strikingly like those of the dengue virus and Zika disease, both of which are also transmitted to humans by the same genus of mosquitos. This book delves into the history of the disease and the molecular characterization of the virus. It sheds light on modern diagnosis tools that allow unambiguous identification of CHIKV infection. In addition, this book addresses the epidemiology of chikungunya fever, the distribution and spread of the disease, and the promising approaches under consideration for preventing and treating the disease

Antibody Cross-Reactivity Between Zika Virus, Kedougou Virus, and Spondweni Virus

Zika virus (ZIKV), dengue virus (DENV), Kedougou virus (KEDV), and Spondweni virus (SPOV) are closely-related, mosquito-borne flaviviruses. It is well-established that ZIKV and DENV both cause fever, rash, and joint pain, in addition to more severe disease states such as microcephaly and hemorrhagic fever, respectively. However, little is known about KEDV or SPOV infections because they have not yet caused large human outbreaks. Given that cross- reactive antibodies between ZIKV and DENV complicate serodiagnostic tests in regions where both viruses circulate, it is crucial to understand and develop serological applications for other flaviviruses, such as KEDV and SPOV, which are even more closely related to ZIKV than DENV is. In addition, identifying a molecular basis for the cross-neutralization of ZIKV, KEDV, and SPOV can help inform the development of neutralizing antibodies and broadly protective vaccines. The objective of this project was to assess the neutralization of ZIKV (strain H/PF/ 2013), KEDV (D14701), and SPOV (SA Ar 94) by mouse polyclonal immune sera (anti-ZIKV, anti-KEDV, and anti-SPOV) and human convalescent sera (ZIKV-immune and DENV-immune). Another goal was to determine whether a panel of monoclonal antibodies (mAbs), specific to particular epitopes on the flavivirus E protein (domain I, domain II, domain III, fusion loop), broadly neutralized ZIKV, KEDV, and SPOV. Here, we found that anti-ZIKV mouse immune sera cross-neutralized KEDV and SPOV, though anti-KEDV and anti-SPOV sera did not cross- neutralize ZIKV (FRNT≤25). We observed that several mouse and human mAbs targeting the fusion loop bound ZIKV, KEDV, and SPOV. However, these fusion loop mAbs neutralized SPOV but not ZIKV, suggesting that virion maturation state could impact cross-neutralization. In addition, we identified two mAbs—G9E and EDEC8—that cross-neutralized both ZIKV and SPOV by targeting the E protein cross-dimer interface, including domains I and II. These findings allow for subsequent studies on the cross-neutralization of ZIKV, KEDV, and SPOV by an expanded panel of ZIKV-immune and DENV-immune human convalescent sera samples, as well as other mAbs that cross-neutralize these viruses. This would facilitate both vaccine design and serodiagnostics in the event that a new flavivirus emerges in ZIKV/DENV co-endemic areas.Bachelor of Scienc

Innovative and New Approaches to Laboratory Diagnosis of Zika and Dengue: A Meeting Report

Epidemics of dengue, Zika, and other arboviral diseases are increasing in frequency and severity. Current efforts to rapidly identify and manage these epidemics are limited by the short diagnostic window in acute infection, the extensive serologic cross-reactivity among flaviviruses, and the lack of point-of-care diagnostic tools to detect these viral species in primary care settings. The Partnership for Dengue Control organized a workshop to review the current landscape of Flavivirus diagnostic tools, identified current gaps, and developed strategies to accelerate the adoption of promising novel technologies into national programs. The rate-limiting step to bringing new diagnostic tools to the market is access to reference materials and well-characterized clinical samples to facilitate performance evaluation. We suggest the creation of an international laboratory-response consortium for flaviviruses with a decentralized biobank of well-characterized samples to facilitate assay validation. Access to proficiency panels are needed to ensure quality control, in additional to in-country capacity building

Antibodies to combat viral infections: development strategies and progress.

Monoclonal antibodies (mAbs) are appealing as potential therapeutics and prophylactics for viral infections owing to characteristics such as their high specificity and their ability to enhance immune responses. Furthermore, antibody engineering can be used to strengthen effector function and prolong mAb half-life, and advances in structural biology have enabled the selection and optimization of potent neutralizing mAbs through identification of vulnerable regions in viral proteins, which can also be relevant for vaccine design. The COVID-19 pandemic has stimulated extensive efforts to develop neutralizing mAbs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with several mAbs now having received authorization for emergency use, providing not just an important component of strategies to combat COVID-19 but also a boost to efforts to harness mAbs in therapeutic and preventive settings for other infectious diseases. Here, we describe advances in antibody discovery and engineering that have led to the development of mAbs for use against infections caused by viruses including SARS-CoV-2, respiratory syncytial virus (RSV), Ebola virus (EBOV), human cytomegalovirus (HCMV) and influenza. We also discuss the rationale for moving from empirical to structure-guided strategies in vaccine development, based on identifying optimal candidate antigens and vulnerable regions within them that can be targeted by antibodies to result in a strong protective immune response

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