Theodore C. Pierson, Ph.D.

Flaviviruses are small RNA viruses responsible for considerable morbidity and mortality worldwide. Flaviviruses are endemic to and cause disease in many regions of the globe that support their arthropod vectors. Each year, dengue virus (DENV) alone results in an estimated 390 million infections. Flaviviruses also have considerable potential for emergence and explosive transmission, as observed with West Nile virus (WNV) and Zika virus (ZIKV) in the Americas during the past twenty-five years. The re-emergence of yellow fever virus (YFV) is also alarming. Fortunately, vaccines have proven effective at controlling flaviviruses. Human vaccines are available for YFV, Japanese encephalitis virus, tick-borne encephalitis virus, and DENV. Neutralizing antibodies (Nabs), which bind to the surface of the virus to directly inhibit infectivity, are a correlate of vaccine-elicited protection for many of these viruses. Complicating an understanding of humoral immunity to flaviviruses is the potential for virus-reactive antibodies to augment infection of Fc-receptor-expressing cells. This mechanism is called antibody-dependent enhancement of infection (ADE) and has been linked mechanistically to severe DENV disease. A DENV vaccine must simultaneously elicit protection against four groups of antigenically-related viruses that may share only some of the epitopes that contribute to protection. Critically, a vaccine cannot sensitize the recipient to severe infection outcomes. Studies of the first licensed tetravalent DENV vaccine raised important questions about the role of Nabs in protection from infection and how to measure protective responses in clinical trials. A goal of the Viral Pathogenesis Section (VPS) is to understand the mechanisms of action of anti-flavivirus antibodies, the complexity of antibody responses elicited by flavivirus infection or vaccination, and the antigenic features of protective vaccine antigens. 

Mechanisms of antibody-mediated neutralization

The VPS explores the characteristics and mechanisms that define neutralizing or protective antibodies. While the importance of antibodies in flavivirus pathogenesis is widely accepted, our understanding of the principles and mechanisms that govern their activity in vivo is insufficient for the rapid rational design of protective vaccines in response to emerging public health threats. The existence of high-resolution structural information for isolated viral envelope proteins and intact virions, coupled with the availability of large numbers of monoclonal antibodies, makes flaviviruses a promising and important system in which to investigate basic principles of humoral immunity that enable findings of translational significance. Many of the concepts explored in our basic and translational research projects are highly relevant to an understanding of humoral immunity to other viruses. The high-throughput quantitative tools used to pursue these studies have proven invaluable for analyzing vaccine-elicited humoral immunity as part of vaccine development campaigns.  

Viral structural heterogeneity and dynamics

The positive-sense flavivirus genomic RNA encodes at least ten functional proteins in a single open reading frame. Three structural proteins (capsid (C), premembrane (prM), and envelope (E)) comprise the infectious virion. The E protein is an elongated three-domain structure connected to the viral membrane by a helical stem and antiparallel transmembrane domains (Figure 1). The three domains of the E protein (E-DI, E-DII, and E-DIII) are connected by flexible linkers that enable structural transitions during the virus assembly, maturation, and entry steps of the virus replication cycle. Newly formed virions bud into the lumen of the ER as immature virions that incorporate E proteins in heterotrimeric complexes with prM. Virion maturation is an essential step in the flavivirus replication cycle defined by the cleavage of prM by the host protease furin. Exposure of immature virions to an acidic environment during egress triggers the reorganization of prM-E trimers that exposes a sequence on prM recognized by furin. Furin cleavage results in a virion-associated small M peptide with unknown function and a cleaved “pr” portion that disassociates from mature virions upon exit from the cell. E proteins are arranged in a herringbone pattern of antiparallel E protein dimers on mature virions. 

Figure 1. Flavivirus maturation. The morphogenesis and trafficking of flaviviruses through the secretory pathway is illustrated. Virion maturation occurs in the trans-Golgi network and depends on the host protease furin and low pH-triggered conformational changes in the arrangement of E proteins on the virion (left inset). prM cleavage may be inefficient, resulting in the release of partially mature or immature virions that retain uncleaved prM. The three domains of E proteins and their arrangement as antiparallel dimers is depicted (top inset). Artwork by Ethan Tyler, NIH.

Credit: NIAID

The stoichiometric framework for understanding flavivirus neutralization and ADE developed in our laboratory provides a reductionist functional perspective to investigate the structural states of infectious viruses. NAbs frequently bind epitopes predicted by structural models to be inaccessible among the E proteins that comprise the virion surface. How antibodies bind these cryptic epitopes in sufficient numbers to inhibit virus infection is an incompletely understood problem with considerable translational importance. We have discovered two mechanisms that impact the structure and biology of virions. First, the inefficient cleavage of prM during virion biogenesis results in considerable structural heterogeneity. Inefficient virion maturation can significantly influence virion neutralization sensitivity. Second, flaviviruses are not static structures (Figure 2). The structural dynamics of flaviviruses, or viral “breathing,” contribute to epitope accessibility and creates a moving target for antibody recognition. Our lab investigates how viral variation shapes the conformational ensemble of virus particles to define virion antigenic structure. These characteristics raise important questions about the structure of vaccine antigens, the structures of infectious virions in vivo, and how to measure the most protective antibody responses. The rational design of vaccine antigens requires a deeper understanding of these complexities and their contribution to immunogenicity and protection.

Figure 2. Flaviviruses are dynamic structures that sample multiple states at equilibrium, called “viral breathing”. Changes in the arrangement of E proteins among the structural ensemble are shown schematically and may alter the presentation or accessibility of epitopes recognized by NAbs. Sequence variation may impact the structural ensemble in ways that are not understood, yet contribute to antibody recognition by NAbs. Artwork by Ethan Tyler, NIH.

Credit: NIAID

The complexity of flavivirus antibody responses

The antibody response to flaviviruses consists of type-specific antibodies that recognize only a single virus type and cross-reactive antibodies capable of binding to more distantly related flaviviruses. Antibodies vary significantly with respect to their capacity to neutralize virus infection and confer protection in vivo. The goal of our studies is to identify functionally significant components of convalescent or vaccine-elicited immune sera. This work is critical for determining correlates of immune protection following vaccination, identifying immune signatures with diagnostic utility, and guiding the selection of antibody classes for which detailed studies will have the most significant translational impact. A detailed understanding of the interplay between antibody repertoires, immunogenicity, and viral antigen structures will accelerate vaccine development.

Development of candidate vaccines to protect against Zika virus infection

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as a considerable threat to public health. While the existence of ZIKV has been known for more than sixty years, until recently, this virus was not a source of significant human disease. Recent ZIKV outbreaks have been associated with neurological complications, including Guillain-Barré syndrome in adults, and devastating neurodevelopmental defects in the infants of women infected while pregnant, including microcephaly. There is an urgent need for interventions to reduce ZIKV transmission and disease. Our laboratory seeks a detailed understanding of the antibody response to ZIKV and to apply these insights into the development of ZIKV vaccines. To this end, we have developed high-throughput quantitative measures of antibody-mediated neutralization of ZIKV, established that ZIKV circulates as a single viral serotype, and collaborated with multiple groups to develop ZIKV vaccine candidates using multiple vaccine platforms. In particular, we have worked closely with intramural colleagues at the Vaccine Research Center to design and test candidate DNA vaccines encoding ZIKV structural proteins and study their immunogenicity in clinical trials. Current efforts are focused on establishing correlates of protection following vaccination and the development of approaches to dissect the functional components of the humoral response to ZIKV vaccination and infection (Figure 3).

Figure 3: Flavivirus vaccine platforms (blue box) may elicit antibody responses that vary with respect to magnitude and qualitative features with the potential to impact protection. Artwork by Ethan Tyler, NIH. Credit: NIAID