Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Virology. Author manuscript; available in PMC 2012 March 15.
Published in final edited form as:
PMCID: PMC3100196

Antibody-mediated neutralization of flaviviruses: A reductionist view


Flaviviruses are a group of ~70 small RNA viruses responsible for significant morbidity and mortality across the globe. Efforts to develop effective vaccines for several clinically important flaviviruses are underway. Antibodies are a significant component of the host’s protective response against flavivirus infection with the potential to contribute to immunity via several distinct mechanisms, including an ability to directly neutralize virus infection. Conversely, virus-reactive antibodies have been implicated in the increased risk of severe clinical manifestations following secondary dengue virus infection. In this review, we will discuss recent progress toward understanding the molecular basis of antibody-mediated neutralization of flaviviruses. Neutralization requires engagement of the virion with a stoichiometry that exceeds a required threshold. From this perspective, we will discuss viral and host factors that impact the number of antibody molecules bound to the virus particle and significantly modulate the potency of neutralizing antibodies.

Antibodies are glycoproteins that equip the adaptive humoral immune system with the capacity to recognize a constantly evolving spectrum of pathogens. A significant role for antibodies in protection of the host from virus infection has been established (reviewed in (Parren and Burton, 2001)). Antibodies exert their antiviral effects by virtue of a capacity to bind viruses and directly neutralize infectivity, as well as via effector functions coordinated by the crystalizable fragment (Fc) region of the antibody heavy chain (Burton, 2002; Nimmerjahn and Ravetch, 2008). Passive transfer of virus-reactive antibody or immune sera has been shown to confer protection from infection by many virus types (reviewed in (Parren and Burton, 2001; Roehrig et al., 2001; Sawyer, 2000)). These findings highlight the general importance of humoral immunity to virus infection, and may enable the identification of desirable functional properties of the antibody response to be targeted by vaccination.

Flaviviruses are a group of positive-stranded RNA viruses capable of significant morbidity and mortality that are transmitted to humans primarily by arthropod vectors (Lindenbach, Thiel, and Rice, 2007). Highly-effective vaccines for several flaviviruses are in use, including yellow fever virus (YFV) (Barrett and Teuwen, 2009), tick-borne encephalitis virus (TBEV) (Heinz et al., 2007), and Japanese encephalitis virus (JEV) (Halstead and Thomas, 2010). A vaccine to protect against dengue virus (DENV) is not yet available despite an urgent need (>50 million human infections annually) and considerable effort by the public and private sectors (Guy et al., 2010; Whitehead et al., 2007). DENV vaccine development is complicated considerably by a perceived need to simultaneously protect against the four serotypes of DENV that circulate in nature, and the possibility that a non-protective humoral response may contribute to more severe clinical outcomes in vaccinated populations (Whitehead et al., 2007). Flavivirus-reactive antibodies may be protective or pathogenic depending on circumstances and context (Halstead, 2003). In this regard, understanding the breadth, specificity, and functional properties of the polyclonal antibody response to infection and vaccination will contribute significantly to the development of safe vaccines against flaviviruses.

A reductionist approach toward understanding how antibodies contribute to protection from flaviviruses considers how their neutralizing activity is controlled at the level of binding to individual virions and in what numbers (Burton, Saphire, and Parren, 2001; Della-Porta and Westaway, 1978). This stoichiometric viewpoint serves as an interesting reference from which to consider the impact of cellular factors and viral dynamics on antibody-mediated neutralization.

Flaviviruses are complex antigens

Flaviviruses are small spherical virus particles composed of three structural proteins (capsid, envelope, and pre-membrane/membrane), an RNA genome, and a lipid envelope (Mukhopadhyay, Kuhn, and Rossmann, 2005). The envelope (E) protein is a type II viral fusion protein composed of three distinct domains connected to the viral membrane by a helical structure called the stem anchor (Fig. 1a) (reviewed in (Harrison, 2008)). The E protein orchestrates the processes of virus assembly, budding, and entry into target cells. This protein is also the primary target of neutralizing antibodies; all three E protein domains contain epitopes recognized by antibodies capable of neutralizing infection under some circumstances (reviewed in (Roehrig, 2003)). Flaviviruses bud into the endoplasmic reticulum as immature virions on which the E proteins interact with the pre-membrane (prM) protein as heterotrimeric spikes arranged with icosahedral symmetry (Fig. 1b) (Zhang et al., 2003; Zhang et al., 2007). Interactions between the prM and E proteins prevent fusion of the virus with membranes of the producer cell during egress through acidic compartments of the secretory pathway (Guirakhoo et al., 1991; Heinz et al., 1994). Cleavage of prM by the cellular protease furin during transit through the Golgi network is a required step in the viral lifecycle that defines the transition from an immature non-infectious virus particle into an infectious form (Elshuber et al., 2003). The products of cleavage are a ~8 kDa M protein that remains associated with the virion, and a “pr” portion that disassociates from the virus particle upon release from the cell (Li et al., 2008; Yu et al., 2008), although as discussed below in greater detail, this cleavage event is often inefficient and results in the release of infectious virions that retain some uncleaved prM molecules. While prM-specific antibodies display limited neutralizing activity (Aaskov et al., 1988; Beltramello et al., 2010; Kaufman et al., 1989; Vazquez et al., 2002), it has been suggested that they contribute to the pathogenesis of DENV virus infection (Dejnirattisai et al., 2010; Rodenhuis-Zybert et al., 2010).

Figure 1
Structure of the flavivirus E protein and its arrangement on the virion

Mature flaviviruses incorporate 180 copies of the E protein arranged as anti-parallel dimers in an unusual herringbone pseudo-icosahedral symmetry pattern (Kuhn et al., 2002; Mukhopadhyay et al., 2003). In this configuration the E protein exists in three distinct chemical environments defined by proximity to the three different symmetry axes of the virion (Fig. 1c). This dense arrangement of E proteins adds considerably to the complexity of the antigenic surface of the virion as epitopes may be differentially accessible for antibody binding depending on their position on the virus particle (Kaufmann et al., 2006; Kaufmann et al., 2010; Nybakken et al., 2005).

Models of the stoichiometric requirements for antibody-mediated neutralization

Considering how the number of antibody molecules bound to the virion determines whether or not virus neutralization occurs is a useful framework to explore factors that govern antibody potency. Two models to explain the behavior of neutralizing antibodies have been proposed and debated extensively. An excellent historical perspective of both schools of thought is provided by Parren and Burton (Parren and Burton, 2001).

“Single-hit” concepts of neutralization postulate that neutralization occurs when the virion is bound by antibody at a “critical site” (Dulbecco, Vogt, and Strickland, 1956). This model posits that antibody binding to functionally important locations on the virion is sufficient to disrupt required steps of the virus entry process and irreversibly inactivate virions (Mandel, 1976). In this regard, not all binding events are equivalent and antibodies bound to non-critical sites on the virion do not contribute to neutralization. Experiments demonstrating that neutralization proceeds via first order kinetics have been used to support this perspective, and suggest neutralization begins immediately after exposure to antibody (Dulbecco, Vogt, and Strickland, 1956). However, interpretation of these results is somewhat controversial. The inability to observe a lag phase in kinetic neutralization experiments, corresponding to the time required to load virions with multiple antibody molecules, may simply reflect limitations of experimental conditions (discussed in (Della-Porta and Westaway, 1978)). Furthermore, the implied requirement that coordinated events across the virus particle during virus entry may be inhibited by a single binding event is difficult to envision in light of the diversity of virus structures and methods for penetrating host cells.

A “multiple-hit” requirement suggests that neutralization occurs when an individual virion is bound by antibody with a stoichiometry that exceeds a required threshold, and is reversible (Burnet, Keogh, and Lush, 1937) (reviewed by (Parren and Burton, 2001)). Neutralization can therefore be considered from a reductionist perspective focused on the number of antibodies bound to the virion during critical steps of the virus entry process. A review of data supporting a multiple-hit mechanism of neutralization of flaviviruses is provided by Della-Porta and Westaway (Della-Porta and Westaway, 1978). Evidence consistent with this perspective discussed within includes: i.) the epitope occupancy requirements for neutralization of flaviviruses, ii.) an estimate of the stoichiometric requirements for neutralization, iii.) the existence of a fraction of viruses resistant to neutralization at any concentration of antibody, iv.) the phenomenon of antibody-dependent enhancement of infection, and v.) the augmented neutralizing activity of anti-flavivirus antibodies in the presence of complement.

Antibody affinity controls the percentage of viral epitopes bound by antibody

At least two factors define conditions that allow engagement of virions by antibody with a stoichiometry that exceeds the threshold requirements for neutralization. One of these factors, antibody affinity, defines the fraction of epitopes bound by antibody molecules at any particular concentration (Klasse and Sattentau, 2002). For example, incubation of a virion that displays 180 epitopes with antibody at a concentration equal to its Kd will result in occupancy of virus particles with an average of 90 antibody molecules (Fig. 2a). Thus, it is not surprising that in many instances, antibody affinity correlates with neutralizing activity in vitro. Under non-saturating conditions, antibodies that bind viral antigens via high affinity interactions will dock onto the virion in greater numbers than those that bind with low-affinity to the same epitope. In this regard, vaccination strategies that elicit high-affinity antibodies are desirable, as this translates into increased neutralization at lower concentrations of antibody. Conversely, antigenic variants that reduce the strength of antibody binding are less sensitive to neutralization as a function of reduced occupancy at any non-saturating concentration of antibody. For example, the recent demonstration that genotypic variation of DENV reduces the neutralizing activity of antibodies against heterologous strains of the same DENV serotype can be explained by differences in antibody affinity (Brien et al., 2010; Sukupolvi-Petty et al., 2010; Wahala et al., 2010).

Figure 2
Antibody affinity and epitope accessibility govern the neutralization potency of anti-flavivirus antibodies

Epitope accessibility is a critical and dynamic factor that governs antibody-mediated neutralization of viruses

Not all antibodies that bind virions with high affinity are characterized by significant neutralizing activity. For example, monoclonal antibodies (mAbs) E16 and E53, described in detail below, bind structurally distinct epitopes on the West Nile virus (WNV) E protein with high affinity, yet differ in their capacity to neutralize infection by several orders of magnitude (Nelson et al., 2008; Oliphant et al., 2005; Oliphant et al., 2006; Pierson et al., 2007). Antibody affinity determines the fractional occupancy of epitopes on the surface of the virion, not the number of antibody molecules bound to the virion under any condition. Thus, insight into the affinity of antibody-virion interactions is not sufficient to predict whether a particular antibody can exceed the stoichiometric requirements for neutralization. The stoichiometry of antibody binding is determined both by antibody affinity and by the total number of epitopes on the virion that are accessible for binding. Epitope accessibility defines the occupancy requirements for neutralization (Fig. 2b), which can vary considerably for antibodies that bind flaviviruses. For example, the occupancy requirements for neutralization by mAbs specific for a type-specific epitope on E-DIII of DENV serotype 2 (DENV-2) were markedly lower than the fractional occupancy required for neutralization via adjacent, partially overlapping, group- and sub-complex-reactive epitopes (Gromowski and Barrett, 2007; Gromowski, Barrett, and Barrett, 2008; Gromowski et al., 2010). In agreement, the occupancy requirements for a panel of WNV-reactive mAbs were determined and varied considerably as a function of the predicted accessibility of the epitope on the mature virus particle (Pierson et al., 2007). The widely varying occupancy requirements for neutralization of flaviviruses directly support the multiple-hit model. Factors that govern epitope accessibility and the occupancy requirements of neutralization are discussed below:

Steric constraints arising from virion structure

The dense herringbone arrangement of E proteins on the mature flavivirus imposes steric constraints on antibody recognition. In fact, none of the mAbs characterized to date using structural methods are capable of binding all 180 E proteins incorporated into the virion (Cherrier et al., 2009; Kaufmann et al., 2006; Kaufmann et al., 2010; Lok et al., 2008; Nybakken et al., 2005). For example, the neutralizing mAb E16 binds a discontinuous epitope on the upper lateral ridge of domain III of the E protein (DIII-LR) (Nybakken et al., 2005; Oliphant et al., 2005). Cryoelectron microscopic (cryo-EM) reconstructions of Fab fragments of E16 bound to WNV demonstrate that E16 binds a maximum of 120 epitopes on the mature virion (Kaufmann et al., 2006). E16 cannot bind E proteins proximal to the five-fold symmetry axis due to steric constraints imposed by the very close proximity of neighboring DIII molecules (Fig. 1c). Antibody occupancy of a relatively small fraction (~25%) of the 120 accessible DIII-LR epitopes is required for neutralization (Pierson et al., 2007).

In contrast, many WNV- and DENV-reactive antibodies neutralize infection only at maximal occupancy, in the presence of saturating concentrations of antibody. Residues that contribute significantly to antibody binding have been mapped for large panels of flavivirus mAbs (Beltramello et al., 2010; Brien et al., 2010; Gromowski and Barrett, 2007; Gromowski, Barrett, and Barrett, 2008; Gromowski et al., 2010; Kiermayr, Stiasny, and Heinz, 2009; Mandl et al., 1989; Oliphant et al., 2005; Oliphant et al., 2006; Shrestha et al., 2010; Sukupolvi-Petty et al., 2010; Sukupolvi-Petty et al., 2007). In many cases, plotting the location of these residues on the reconstruction of the intact mature virus particle does not provide a clear structural basis for antibody recognition due to accessibility considerations. For example, the highly conserved fusion loop at the top of E domain II (DII-FL) recognized by mAb E53 is buried between DI and DIII of the opposing E protein of the dimers arranged on the mature virion (Cherrier et al., 2009; Mukhopadhyay, Kuhn, and Rossmann, 2005). Antibodies specific for these poorly accessible determinants have to bind essentially all of them in order to exceed the stoichiometric threshold of neutralization (Nelson et al., 2008; Stiasny et al., 2006). Importantly, many antibodies that can bind virions display very little in vitro neutralizing activity because exceeding this threshold is not possible at any concentration of antibody (Nelson et al., 2008; Oliphant et al., 2006; Pierson et al., 2007). The limited neutralizing activity of antibodies specific for the prM protein may also reflect an inability to exceed the threshold requirements for neutralization.

The impact of structural dynamics

The utility of molecular models of viral proteins and their organization on virions are powerful tools that reveal the average state of proteins on the virion. Estimates of epitope accessibility based on these structural models may be complicated by the possibility that virions are in constant motion. It is well established that proteins sample many conformations at equilibrium (reviewed by (Boehr and Wright, 2008)). This is also undoubtedly true of proteins incorporated into virus particles (reviewed in (Johnson, 2003; Witz and Brown, 2001)). For example, limited proteolysis studies of picornaviruses suggest the capsids of these viruses “breathe” (Bothner et al., 1998). Interestingly, a small molecule inhibitor that blocks capsid dynamics neutralizes infectivity (Lewis et al., 1998). Poliovirus VP4 is recognized by neutralizing antibodies even though this protein is not predicted to be accessible on the exterior of the particle (Li et al., 1994), implying that epitopes transiently and reversibly exposed on virions via structural dynamics may be stabilized by antibody recognition.

Viral dynamics may also have a role in the accessibility of epitopes on flaviviruses. The mAb 1A1D-2 is a DENV sub-complex specific mAb that recognizes an epitope on the A-strand of E-DIII and is capable of neutralizing DENV serotypes 1, 2, and 3 (Lok et al., 2008; Roehrig, Bolin, and Kelly, 1998). Analysis of the accessibility of residues involved in 1A1D-2 recognition identified steric conflicts that should prevent binding to DIII in all three symmetry environments of the mature virus particle, consistent with an inability to dock Fab fragments of this antibody onto virions at 4°C (Lok et al., 2008). Interestingly, antibody binding was possible when incubations were carried out at 37°C. Cryo-EM reconstructions of 1A1D-2 in complex with DENV revealed a marked rotation of the E proteins away from the surface of the virion as compared to the arrangement of E proteins in the average state of the mature virion. The structure of 1A1D-2 bound to DENV provides insight into one of many potential conformations sampled by DENV at equilibrium, and demonstrates how such structural changes can modulate epitope accessibility. Whether viral dynamics play a more widespread role in neutralization via other epitopes remains to be determined.

Changes in oligomeric state occurring during virion maturation

Populations of flaviviruses released from infected cells contain not only immature and mature virions, but also virus particles that share structural features of both these forms. While cleavage of prM is required for the production of an infectious particle (Elshuber et al., 2003), it may be inefficient. In fact, recent studies suggest that as many as 92% of DENV virions incorporate prM proteins that are not cleaved during virus egress (Junjhon et al., 2010). At least some of these partially mature virions remain infectious, although whether there is a limit to the number of uncleaved prM molecules that an infectious virion may retain is unknown (Davis et al., 2006; Guirakhoo, Bolin, and Roehrig, 1992). While the structure of partially mature virions is not yet known, the E proteins on these heterogeneous virus particles likely exist as both anti-parallel dimers that lie flat against the viral membrane (characteristic of mature virions), as well as spikes that project off the surface of the virion (characteristic of immature virions). E proteins in these distinct conformations and oligomeric states present very different surfaces for antibody recognition (Guirakhoo, Bolin, and Roehrig, 1992; Heinz et al., 1994).

Analysis of the neutralization profiles of several mAbs specific for epitopes predicted to be poorly accessible on the mature virion revealed a subset of virions resistant to neutralization by saturating concentrations of antibody (Nelson et al., 2008; Pierson et al., 2007). A similar pattern was observed when the number of epitopes on the virion recognized by the potently neutralizing mAb E16 was reduced using genetic methods, as discussed below (Mehlhop et al., 2009; Pierson et al., 2007). Increasing the efficiency of virus maturation yielded a homogenous population of mature WNV resistant to neutralization by mAb E53 and other mAbs specific for cryptic determinants (Nelson et al., 2008). Conversely, inhibiting maturation increased sensitivity to neutralization by these mAbs, and significantly reduced the size of the resistant fraction described above. These results suggest the process of virion maturation modulates the sensitivity of WNV to neutralization by mAbs that recognize several distinct classes of epitopes through changes in epitope accessibility (Fig. 2c). The structural basis of this phenomenon has been confirmed. The mAb E53 binds the highly conserved fusion loop at the terminus of E-DII (DII-FL) and neutralizes WNV in a maturation state-dependent fashion. Cryo-EM reconstruction studies of Fab fragments of E53 bound to WNV and DENV demonstrated binding to E proteins only when complexed with prM in the heterotrimeric spikes of immature virions (Cherrier et al., 2009). In this context, binding to two of three fusion loop epitopes was possible; binding to the third E protein was prevented by steric conflicts between the DII-FL and prM. Due to the inaccessibility of the DII-FL epitope on the mature form of the virion (discussed above), significant binding or neutralization of E53 to mature virions was not detected.

Limits on epitope accessibility imposed by the steric bulk of intact antibody molecules

Antibodies are relatively large molecules that occupy considerably more space than simply the surface area of the virion in contact with the paratope. This steric bulk is thought to contribute to neutralization by sterically preventing required interactions with cellular factors during the virus entry process or by interfering with conformational changes in viral proteins required for fusion (Klasse and Sattentau, 2002). In contrast, the size of antibody molecules may in some instances be detrimental. For example, the broadly neutralizing mAb 4E10 binds the conserved membrane proximal external region (MPER) of HIV-1 gp41. Fabs and a smaller engineered fragment of mAb 4E10 displayed greater neutralizing activity than the intact antibody molecule, presumably due to a reduction in the degree of steric occlusion (Klein et al., 2009). Antibody size also may influence the maximal number of antibody molecules that can simultaneous bind to virions (Kiermayr, Stiasny, and Heinz, 2009).

Estimates of the stoichiometry of antibody-mediated neutralization of West Nile virus

The number of antibodies required for the neutralization of several virus types has been investigated (reviewed in (Burton, Saphire, and Parren, 2001)). The stoichiometric requirement for neutralization varies considerably among viruses. For example, neutralization of poliovirus occurs following engagement by 4–5 mAbs, whereas inhibition of rabies virus requires more than 200 IgG molecules (Flamand et al., 1993; Icenogle et al., 1983). One unifying principle of antibody-mediated neutralization is that the number of antibodies required to neutralize infection is determined by the surface area of the virus particle (Burton, Saphire, and Parren, 2001); increased virion size requires a larger number of antibody molecules to sterically interfere with required steps in the virus entry process. The stoichiometric requirement for neutralization of WNV has been studied using two antibodies specific for the DIII-LR and was estimated as ~30 antibody molecules (Mehlhop et al., 2009; Pierson et al., 2007). Estimates for the number of antibodies required to neutralize WNV are in agreement with the predictions of this “coating theory” (Klasse and Burton, 2007). However, the functional significance of this number is not presently clear, as the number of antibodies required for neutralization does not in itself provide insight into the mechanism of neutralization. Antibodies have the potential to block multiple steps in the virus entry process. Antibodies that block flavivirus attachment (Crill and Roehrig, 2001; He et al., 1995; Nybakken et al., 2005) and fusion (Gollins and Porterfield, 1986; Roehrig, Bolin, and Kelly, 1998; Stiasny et al., 2007; Thompson et al., 2009; Vogt et al., 2009) have been described, although it is quite possible that antibodies have the potential to neutralize via multiple mechanisms depending on epitope occupancy. For example, mAb E16 blocks viral fusion by preventing the conformational changes in the E protein required to drive fusion (Kaufmann et al., 2009; Thompson et al., 2009) and has a modest effect on virus attachment at high concentrations. These observations suggest that the threshold requirements for neutralization may differ depending on the step in the virus entry/fusion pathway blocked by the antibody. Likewise, it remains to be determined whether the threshold requirements for neutralization will differ for antibodies specific for structurally distinct epitopes, as suggested by studies of mAbs that neutralize bovine papillomavirus (Booy et al., 1998; Roden et al., 1994). To date, the analysis of the stoichiometric requirements for antibodies that bind outside the DIII-LR has been complicated by the significant impact of virion maturation and/or structural dynamics on epitope accessibility.

Antibody-dependent enhancement of infection

Antibody-dependent enhancement of infection (ADE) describes a marked increase in the efficiency of virus infection in the presence of non-neutralizing concentrations of virus-reactive antibody (Halstead and O’Rourke, 1977; Hawkes, 1964). ADE is most commonly observed using cells expressing Fc- or complement- receptors, and has been observed in vitro for several viruses (reviewed in (Porterfield, 1986)). The most clearly established role for ADE in vivo exists for DENV (reviewed in (Halstead, 2003)). Infection by any of the four serotypes of DENV results in a febrile illness called dengue fever from which that individual will typically recover and thereafter be immune to reinfection by the same serotype of DENV. However, re-infection of that same individual with a heterologous serotype of DENV may result in a more severe and potentially fatal clinical course (reviewed in (Halstead, 2002)). While multiple host and viral factors likely contribute to circumstances that lead to severe manifestations of disease (Green and Rothman, 2006; Halstead, 2003), one possibility is that antibodies raised during primary infection not only fail to protect from secondary infection by a heterologous DENV serotype, but also have the potential to enhance virus infection in vivo. The increased risk of severe disease following primary infection of children of DENV-immune mothers provides direct support for a role for antibody in DENV pathogenesis (Kliks et al., 1988; Kliks et al., 1989). In addition, enhanced viremia has been observed following passive transfer of antibody in primates (Goncalvez et al., 2007; Halstead, 1979), and two recent reports describe the characterization of a murine model that recapitulates some aspects of severe DENV, including the role of antibody in augmenting infection in vivo (Balsitis et al., 2010; Zellweger, Prestwood, and Shresta, 2010).

Virus neutralization and ADE are two phenomena related by the number of antibodies bound to the virion (Morens, Halstead, and Marchette, 1987; Pierson et al., 2007). ADE occurs at concentrations of antibody bounded at an upper limit by the stoichiometric threshold for neutralization, and at lower concentrations by the minimal number of antibodies required to support stable attachment of the virion to cells. Analysis of the stoichiometric requirements for ADE of K562 cells that express the Fcγ-receptor CD32 suggest that ~15 antibodies are required for enhanced infection of WNV (Pierson et al., 2007). This provides a useful perspective in which to evaluate the likelihood that antibodies of known specificity will enhance infection. Potently neutralizing antibodies support ADE when diluted past concentrations sufficient to neutralize infection. For antibodies that recognize poorly accessible determinants on the virion, which typically require full occupancy to exceed the stoichiometric threshold for neutralization, even small changes in the number of antibodies bound to the virion result in ADE. Furthermore, antibodies that recognize infrequently displayed epitopes that do not support neutralization may enhance infection even at saturation. Indeed, the enhancing properties of poorly neutralizing prM-reactive antibodies can be observed over a wide range of antibody concentrations (Beltramello et al., 2010; Dejnirattisai et al., 2010).

The cell biology of ADE remains incompletely understood. ADE is most readily observed on cell types that do not efficiently bind virus particles. Antibodies complexed to virions may significantly increase the efficiency of virus attachment to cells through multivalent interactions with Fc-receptors. ADE can be inhibited by antibodies that block Fc-receptor engagement (Peiris et al., 1981), removal of the antibody heavy chain (Zellweger, Prestwood, and Shresta, 2010), or deletion of the N-linked sugar on IgG molecules required for interactions with Fcγ-receptors (Balsitis et al., 2010; Goncalvez et al., 2007; Pierson et al., 2007). The expression of cellular attachment factors on Fcγ-receptor expressing cells markedly reduces the extent to which virus infection may be enhanced in the presence of antibody (Boonnak et al., 2008; Pierson et al., 2007). For example, immature dendritic cells (DCs) do not appear to support ADE of DENV despite expressing readily detectable Fcγ-receptors presumably because they express the DENV attachment factor CD209 (DC-SIGN). In contrast, mature DCs express reduced levels of CD209 and support ADE through Fcγ-RIIa (Boonnak et al., 2008). In some instances, ADE may occur when antibody molecules cross-react with both viral and cellular antigens. DENV prM-specific antibodies were identified that were capable of binding simultaneously to the virion and target cell, providing a Fcγ-receptor-independent mechanism for internalization and infection (Huang et al., 2006). A recent study demonstrated that 75% of mAbs cloned from HIV-1-infected patients demonstrated polyreactive specificities (Mouquet et al., 2010), however, whether flavivirus-specific antibodies with these properties are common remains unclear.

While Fc-receptor-mediated uptake of DENV immune complexes results in increased infection simply by increasing the number of susceptible cells, recent studies have suggested signaling events that occur following the ligation of Fc-receptors may be important for ADE (Moi et al., 2010; Rodrigo et al., 2006), resulting in the inhibition of the antiviral response and increased viral replication (intrinsic ADE). Specifically, intrinsic ADE is thought to occur by two separate mechanisms: 1) disruption of the RIG-I/MDA5 signaling cascade, which in turn abrogates the type I IFN response, and 2) induction of anti-inflammatory cytokines such as IL-10 coupled with suppression of pro-inflammatory cytokines such as IL-12 and TNF-α (Boonnak et al., 2008; Chareonsirisuthigul, Kalayanarooj, and Ubol, 2007; Ubol et al., 2010).

Serum complement proteins increase the neutralizing activity of antibodies via multiple mechanism

While the engagement of virions by antibody has the potential to directly neutralize infectivity, in the host these interactions occur in the presence of serum proteins capable of modulating antibody function. The complement system is a component of the innate immune response capable of pathogen recognition and clearance that is regulated by a complex cascade of cleavage events. The importance of the complement system in viral immunity is highlighted by the number and diversity of strategies evolved by viruses to antagonize it (reviewed by (Lambris, Ricklin, and Geisbrecht, 2008)). Activation of the complement system occurs via three convergent pathways that ultimately lead to the release of anaphylatoxins and chemotactic factors, deposition of the opsonic complement protein C3b on the surface of the pathogen, and the assembly of the membrane attack complex (MAC) capable of pathogen lysis. The classical pathway of complement activation is triggered by interactions between the complement protein C1q and the Fc-portion of antibody molecules. C1q is a ~460kDa multimeric protein composed of six globular heads connected at the amino-terminus by collagenous flexible stalks (reviewed by (Kishore and Reid, 2000)). While C1q can bind efficiently to a single pentameric IgM molecule bound to the surface of a pathogen, the affinity of monomeric interactions with IgG is quite low. Efficient interactions with IgG molecules require multivalent interactions with antibody molecules arrayed on the surface of the pathogen, and occur in an isotype-dependent manner. In humans, IgG3 and IgG1 efficiently interact with C1q (IgG3 > IgG1), whereas C1q binding to IgG2 and IgG4 subclasses is very weak (the hierarchy of C1q binding to murine antibodies is IgG2a > IgG2b > IgG1) (Leatherbarrow and Dwek, 1984; Schumaker et al., 1976).

An understanding of the impact of complement on the potency of antibodies has advanced considerably since the first reports that heat-labile factors in serum augment the neutralizing activity of antibodies. Complement has been shown to increase the neutralization activity of antibodies against many viruses, including flaviviruses (Della-Porta and Westaway, 1978). Several lines of evidence suggest that C1q is sufficient for enhancing the neutralizing activity of antibodies that bind flaviviruses (Mehlhop et al., 2009). Incubation of virion-antibody complexes with fresh, but not heat-inactivated sera, results in more efficient neutralization in vitro; this effect is lost when serum obtained from C1q- but not C3- or C5-deficient mice is added to neutralization experiments. The addition of recombinant C1q to neutralization tests also results in increased neutralization in an antibody isotype-dependent fashion. Together, these data suggest that molecules in the complement cascade downstream of C1q activation, including the formation of the MAC on virions, are not required for enhanced neutralization of flaviviruses. Studies of the mechanism of complement-enhanced neutralization of influenza virus reached similar conclusions (Feng, Mozdzanowska, and Gerhard, 2002). Neutralization of the extracellular enveloped form of vaccinia virus (EV) by B5R-specific antibodies was antibody isotype- and C1q-dependent, but also required deposition of C3 (but not C5 activity) (Benhnia et al., 2009).

C1q has the potential to directly increase the neutralizing activity of antibodies through one or more mechanisms. Because C1q only binds efficiently to multiple antibody molecules arrayed on the surface of the virus particle (Kishore and Reid, 2000), C1q engagement may increase the functional avidity of antibodies for the virion. This would translate into enhanced neutralization via increased antibody occupancy at any non-saturating concentration of antibody. Multivalent C1q interactions also cross-link antibodies (and viral proteins) on the surface of the virus particle together, which may have significant implications for the viral fusion machinery (Kaufmann et al., 2009; Whiteman et al., 2010). Finally, the relatively large size of C1q adds substantially to the bulk of antibody molecules bound to the virion, which may increase neutralizing activity due to enhanced steric effects.

Our studies with WNV suggest that C1q enhances antibody-mediated neutralization by significantly reducing the stoichiometric requirements for neutralization (Mehlhop et al., 2009). Neutralization of WNV can be controlled by manipulating the number of epitopes displayed on the average virion using genetic approaches (Pierson et al., 2007) or via changes in the maturation state of the virus particle (Nelson et al., 2008), as discussed above. Reducing the number of epitopes on the virion below the stoichiometric threshold required for neutralization results in the appearance of a fraction of viruses resistant to neutralization, even when incubated with saturating concentrations of antibody. The addition of C1q to neutralization studies dramatically reduced the size of this resistant fraction in an isotype-dependent manner. In this context, C1q-mediated increases in avidity are not sufficient to explain this increase in neutralization because all of the accessible epitopes on the virion are already bound by saturating concentrations of antibody. Instead these results suggest that C1q increases neutralizing activity through significant changes in the stoichiometric threshold for neutralization. This effect could be mimicked by incubation of virion-antibody complexes with Fab2 molecules specific for IgG, but not Fab fragments of the same specificity incapable of bivalent interactions with antibodies bound to the virion. These findings rule out a requirement for the very large size of the C1q molecule, and suggest that cross-linking is mechanistically important. A reduced stoichiometric requirement for neutralization in the presence of C1q also provides a mechanism to explain complement modulation of ADE (Mehlhop et al., 2007; Yamanaka, Kosugi, and Konishi, 2008).

Toward understanding the complexities of the polyclonal antibody response

Experiments with mAbs have yielded an increasingly sophisticated understanding of the molecular mechanisms of flavivirus neutralization. A quantitative view of neutralization provides a framework to define the functional and biochemical features of the most potently neutralizing antibodies, as well as those with the greatest potential to enhance infection. The identification of antibodies with therapeutic value is an exciting practical product of these efforts (Morrey et al., 2006). However, relatively little is known about the composition and functional properties of antibodies elicited by flavivirus infection. The antibody response to infection is likely a polyclonal ensemble of antibodies specific for structurally distinct epitopes on the virus particle. While many of the most potent neutralizing antibodies bind epitopes in E-DIII (Beasley and Barrett, 2002; Gromowski and Barrett, 2007; Oliphant et al., 2005), recent studies of human mAbs isolated from immortalized B cells or the analysis of combinatorial libraries of B cell donors suggest the repertoire of antibodies produced in vivo is focused elsewhere (Beltramello et al., 2010; Dejnirattisai et al., 2010; Throsby et al., 2006). Cross-reactive E-DII-reactive antibodies characterized by limited neutralization activity are common. This conclusion is supported by biochemical and serologic analysis of antibody reactivity (Crill et al., 2009; Crill, Trainor, and Chang, 2007; Lai et al., 2008; Oliphant et al., 2007). While it is possible that low concentrations of potently neutralizing E-DIII-reactive antibodies could be functionally significant (Crill et al., 2009), two approaches suggest this is not the case. Ablation of the DIII-LR epitope on WNV had little to no effect on the neutralization titers of antibody in the sera of infected humans or horses (Nelson et al., 2008; Oliphant et al., 2007; Sanchez et al., 2007). In agreement, a recent study demonstrated that the depletion of the DIII-reactive antibodies present in DENV-immune sera did not have a significant impact on neutralization when compared to non-depleted sera (Wahala et al., 2009). While the analysis of the functional interplay between components of the polyclonal antibody response is at a relatively early stage, it holds great promise for the identification of epitopes associated with protective responses, insight into circumstances that may contribute to augmented virus infection in vivo, and more focused targets for immunization.

Figure 3
Factors that govern the neutralizing activity of anti-flavivirus antibodies


The authors thank Drs. Michael Diamond, Heather Hickman, Thomas Kristie and members of our laboratory for helpful discussions and critical comments on this manuscript. This work was supported by the intramural program of the National Institute of Allergy and Infectious Disease.


Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.


  • Aaskov JG, Williams L, Fletcher J, Hay R. Failure of a dengue 1 subunit vaccine to protect mice against a lethal dengue virus infection. Am J Trop Med Hyg. 1988;39(5):511–8. [PubMed]
  • Balsitis SJ, Williams KL, Lachica R, Flores D, Kyle JL, Mehlhop E, Johnson S, Diamond MS, Beatty PR, Harris E. Lethal antibody enhancement of dengue disease in mice is prevented by Fc modification. PLoS Pathog. 2010;6(2):e1000790. [PMC free article] [PubMed]
  • Barrett AD, Teuwen DE. Yellow fever vaccine - how does it work and why do rare cases of serious adverse events take place? Curr Opin Immunol. 2009;21(3):308–13. [PubMed]
  • Beasley DW, Barrett AD. Identification of neutralizing epitopes within structural domain III of the West Nile virus envelope protein. J Virol. 2002;76(24):13097–13100. [PMC free article] [PubMed]
  • Beltramello M, Williams KL, Simmons CP, Macagno A, Simonelli L, Quyen NT, Sukupolvi-Petty S, Navarro-Sanchez E, Young PR, de Silva AM, Rey FA, Varani L, Whitehead SS, Diamond MS, Harris E, Lanzavecchia A, Sallusto F. The human immune response to Dengue virus is dominated by highly cross-reactive antibodies endowed with neutralizing and enhancing activity. Cell Host Microbe. 2010;8(3):271–83. [PMC free article] [PubMed]
  • Benhnia MR, McCausland MM, Laudenslager J, Granger SW, Rickert S, Koriazova L, Tahara T, Kubo RT, Kato S, Crotty S. Heavily isotype-dependent protective activities of human antibodies against vaccinia virus extracellular virion antigen B5. J Virol. 2009;83(23):12355–67. [PMC free article] [PubMed]
  • Boehr DD, Wright PE. Biochemistry. How do proteins interact? Science. 2008;320(5882):1429–30. [PubMed]
  • Boonnak K, Slike BM, Burgess TH, Mason RM, Wu SJ, Sun P, Porter K, Rudiman IF, Yuwono D, Puthavathana P, Marovich MA. Role of dendritic cells in antibody-dependent enhancement of dengue virus infection. J Virol. 2008;82(8):3939–51. [PMC free article] [PubMed]
  • Booy FP, Roden RB, Greenstone HL, Schiller JT, Trus BL. Two antibodies that neutralize papillomavirus by different mechanisms show distinct binding patterns at 13 A resolution. J Mol Biol. 1998;281(1):95–106. [PubMed]
  • Bothner B, Dong XF, Bibbs L, Johnson JE, Siuzdak G. Evidence of viral capsid dynamics using limited proteolysis and mass spectrometry. J Biol Chem. 1998;273(2):673–6. [PubMed]
  • Brien JD, Austin SK, Sukupolvi-Petty S, O’Brien KM, Johnson S, Fremont DH, Diamond MS. Genotype Specific Neutralization and Protection by Antibodies against Dengue Virus Type 3. J Virol 2010 [PMC free article] [PubMed]
  • Burnet FM, Keogh EV, Lush D. The immunological reactions of the filterable viruses. The Australian Journal of Experimental Biology and Medical Science. 1937;15:227–368.
  • Burton DR. Antibodies, viruses and vaccines. Nat Rev Immunol. 2002;2(9):706–13. [PubMed]
  • Burton DR, Saphire EO, Parren PW. A model for neutralization of viruses based on antibody coating of the virion surface. Curr Top Microbiol Immunol. 2001;260:109–43. [PubMed]
  • Chareonsirisuthigul T, Kalayanarooj S, Ubol S. Dengue virus (DENV) antibody-dependent enhancement of infection upregulates the production of anti-inflammatory cytokines, but suppresses anti-DENV free radical and pro-inflammatory cytokine production, in THP-1 cells. J Gen Virol. 2007;88(Pt 2):365–75. [PubMed]
  • Cherrier MV, Kaufmann B, Nybakken GE, Lok SM, Warren JT, Chen BR, Nelson CA, Kostyuchenko VA, Holdaway HA, Chipman PR, Kuhn RJ, Diamond MS, Rossmann MG, Fremont DH. Structural basis for the preferential recognition of immature flaviviruses by a fusion-loop antibody. EMBO J. 2009;28(20):3269–76. [PubMed]
  • Crill WD, Hughes HR, Delorey MJ, Chang GJ. Humoral immune responses of dengue fever patients using epitope-specific serotype-2 virus-like particle antigens. PLoS ONE. 2009;4(4):e4991. [PMC free article] [PubMed]
  • Crill WD, Roehrig JT. Monoclonal antibodies that bind to domain III of dengue virus E glycoprotein are the most efficient blockers of virus adsorption to Vero cells. J Virol. 2001;75(16):7769–73. [PMC free article] [PubMed]
  • Crill WD, Trainor NB, Chang GJ. A detailed mutagenesis study of flavivirus cross-reactive epitopes using West Nile virus-like particles. J Gen Virol. 2007;88(Pt 4):1169–74. [PubMed]
  • Davis CW, Nguyen HY, Hanna SL, Sanchez MD, Doms RW, Pierson TC. West Nile virus discriminates between DC-SIGN and DC-SIGNR for cellular attachment and infection. J Virol. 2006;80(3):1290–301. [PMC free article] [PubMed]
  • Dejnirattisai W, Jumnainsong A, Onsirisakul N, Fitton P, Vasanawathana S, Limpitikul W, Puttikhunt C, Edwards C, Duangchinda T, Supasa S, Chawansuntati K, Malasit P, Mongkolsapaya J, Screaton G. Cross-reacting antibodies enhance dengue virus infection in humans. Science. 2010;328(5979):745–8. [PMC free article] [PubMed]
  • Della-Porta AJ, Westaway EG. A multi-hit model for the neutralization of animal viruses. J Gen Virol. 1978;38(1):1–19. [PubMed]
  • Dulbecco R, Vogt M, Strickland AG. A study of the basic aspects of neutralization of two animal viruses, western equine encephalitis virus and poliomyelitis virus. Virology. 1956;2(2):162–205. [PubMed]
  • Elshuber S, Allison SL, Heinz FX, Mandl CW. Cleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virus. J Gen Virol. 2003;84(Pt 1):183–91. [PubMed]
  • Feng JQ, Mozdzanowska K, Gerhard W. Complement component C1q enhances the biological activity of influenza virus hemagglutinin-specific antibodies depending on their fine antigen specificity and heavy-chain isotype. J Virol. 2002;76(3):1369–78. [PMC free article] [PubMed]
  • Flamand A, Raux H, Gaudin Y, Ruigrok RW. Mechanisms of rabies virus neutralization. Virology. 1993;194(1):302–13. [PubMed]
  • Gollins SW, Porterfield JS. A new mechanism for the neutralization of enveloped viruses by antiviral antibody. Nature. 1986;321(6067):244–6. [PubMed]
  • Goncalvez AP, Engle RE, St Claire M, Purcell RH, Lai CJ. Monoclonal antibody-mediated enhancement of dengue virus infection in vitro and in vivo and strategies for prevention. Proc Natl Acad Sci U S A. 2007;104(22):9422–7. [PubMed]
  • Green S, Rothman A. Immunopathological mechanisms in dengue and dengue hemorrhagic fever. Curr Opin Infect Dis. 2006;19(5):429–36. [PubMed]
  • Gromowski GD, Barrett AD. Characterization of an antigenic site that contains a dominant, type-specific neutralization determinant on the envelope protein domain III (ED3) of dengue 2 virus. Virology. 2007;366(2):349–60. [PubMed]
  • Gromowski GD, Barrett ND, Barrett AD. Characterization of Dengue Complex-specific Neutralizing Epitopes on the Envelope Protein Domain III of Dengue 2 Virus. J Virol 2008 [PMC free article] [PubMed]
  • Gromowski GD, Roehrig JT, Diamond MS, Lee JC, Pitcher TJ, Barrett AD. Mutations of an antibody binding energy hot spot on domain III of the dengue 2 envelope glycoprotein exploited for neutralization escape. Virology. 2010;407(2):237–46. [PubMed]
  • Guirakhoo F, Bolin RA, Roehrig JT. The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. Virology. 1992;191(2):921–31. [PubMed]
  • Guirakhoo F, Heinz FX, Mandl CW, Holzmann H, Kunz C. Fusion activity of flaviviruses: comparison of mature and immature (prM-containing) tick-borne encephalitis virions. J Gen Virol. 1991;72 (Pt 6):1323–9. [PubMed]
  • Guy B, Guirakhoo F, Barban V, Higgs S, Monath TP, Lang J. Preclinical and clinical development of YFV 17D-based chimeric vaccines against dengue, West Nile and Japanese encephalitis viruses. Vaccine. 2010;28(3):632–49. [PubMed]
  • Halstead SB. In vivo enhancement of dengue virus infection in rhesus monkeys by passively transferred antibody. J Infect Dis. 1979;140(4):527–33. [PubMed]
  • Halstead SB. Dengue. Curr Opin Infect Dis. 2002;15(5):471–6. [PubMed]
  • Halstead SB. Neutralization and antibody-dependent enhancement of dengue viruses. Adv Virus Res. 2003;60:421–67. [PubMed]
  • Halstead SB, O’Rourke EJ. Antibody-enhanced dengue virus infection in primate leukocytes. Nature. 1977;265(5596):739–41. [PubMed]
  • Halstead SB, Thomas SJ. Japanese encephalitis: new options for active immunization. Clin Infect Dis. 2010;50(8):1155–64. [PubMed]
  • Harrison SC. Viral membrane fusion. Nat Struct Mol Biol. 2008;15(7):690–8. [PMC free article] [PubMed]
  • Hawkes RA. Enhancement of the Infectivity of Arboviruses by Specific Antisera Produced in Domestic Fowls. Aust J Exp Biol Med Sci. 1964;42:465–82. [PubMed]
  • He RT, Innis BL, Nisalak A, Usawattanakul W, Wang S, Kalayanarooj S, Anderson R. Antibodies that block virus attachment to Vero cells are a major component of the human neutralizing antibody response against dengue virus type 2. J Med Virol. 1995;45(4):451–61. [PubMed]
  • Heinz FX, Holzmann H, Essl A, Kundi M. Field effectiveness of vaccination against tick-borne encephalitis. Vaccine. 2007;25(43):7559–67. [PubMed]
  • Heinz FX, Stiasny K, Puschner-Auer G, Holzmann H, Allison SL, Mandl CW, Kunz C. Structural changes and functional control of the tick-borne encephalitis virus glycoprotein E by the heterodimeric association with protein prM. Virology. 1994;198(1):109–17. [PubMed]
  • Huang KJ, Yang YC, Lin YS, Huang JH, Liu HS, Yeh TM, Chen SH, Liu CC, Lei HY. The dual-specific binding of dengue virus and target cells for the antibody-dependent enhancement of dengue virus infection. J Immunol. 2006;176(5):2825–32. [PubMed]
  • Icenogle J, Shiwen H, Duke G, Gilbert S, Rueckert R, Anderegg J. Neutralization of poliovirus by a monoclonal antibody: kinetics and stoichiometry. Virology. 1983;127(2):412–25. [PubMed]
  • Johnson JE. Virus particle dynamics. Adv Protein Chem. 2003;64:197–218. [PubMed]
  • Junjhon J, Edwards TJ, Utaipat U, Bowman VD, Holdaway HA, Zhang W, Keelapang P, Puttikhunt C, Perera R, Chipman PR, Kasinrerk W, Malasit P, Kuhn RJ, Sittisombut N. Influence of pr-M cleavage on the heterogeneity of extracellular dengue virus particles. J Virol. 2010;84(16):8353–8. [PMC free article] [PubMed]
  • Kaufman BM, Summers PL, Dubois DR, Cohen WH, Gentry MK, Timchak RL, Burke DS, Eckels KH. Monoclonal antibodies for dengue virus prM glycoprotein protect mice against lethal dengue infection. Am J Trop Med Hyg. 1989;41(5):576–80. [PubMed]
  • Kaufmann B, Chipman PR, Holdaway HA, Johnson S, Fremont DH, Kuhn RJ, Diamond MS, Rossmann MG. Capturing a flavivirus pre-fusion intermediate. PLoS Pathog. 2009;5(11):e1000672. [PMC free article] [PubMed]
  • Kaufmann B, Nybakken GE, Chipman PR, Zhang W, Diamond MS, Fremont DH, Kuhn RJ, Rossmann MG. West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody. Proc Natl Acad Sci U S A. 2006;103(33):12400–4. [PubMed]
  • Kaufmann B, Vogt MR, Goudsmit J, Holdaway HA, Aksyuk AA, Chipman PR, Kuhn RJ, Diamond MS, Rossmann MG. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354. Proc Natl Acad Sci U S A. 2010;107(44):18950–5. [PubMed]
  • Kiermayr S, Stiasny K, Heinz FX. Impact of quaternary organization on the antigenic structure of the tick-borne encephalitis virus envelope glycoprotein E. J Virol. 2009;83(17):8482–91. [PMC free article] [PubMed]
  • Kishore U, Reid KB. C1q: structure, function, and receptors. Immunopharmacology. 2000;49(1–2):159–70. [PubMed]
  • Klasse PJ, Burton DR. Antibodies to West Nile virus: a double-edged sword. Cell Host Microbe. 2007;1(2):87–9. [PubMed]
  • Klasse PJ, Sattentau QJ. Occupancy and mechanism in antibody-mediated neutralization of animal viruses. J Gen Virol. 2002;83(Pt 9):2091–108. [PubMed]
  • Klein JS, Gnanapragasam PN, Galimidi RP, Foglesong CP, West AP, Jr, Bjorkman PJ. Examination of the contributions of size and avidity to the neutralization mechanisms of the anti-HIV antibodies b12 and 4E10. Proc Natl Acad Sci U S A. 2009;106(18):7385–90. [PubMed]
  • Kliks SC, Nimmanitya S, Nisalak A, Burke DS. Evidence that maternal dengue antibodies are important in the development of dengue hemorrhagic fever in infants. Am J Trop Med Hyg. 1988;38(2):411–9. [PubMed]
  • Kliks SC, Nisalak A, Brandt WE, Wahl L, Burke DS. Antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever. Am J Trop Med Hyg. 1989;40(4):444–51. [PubMed]
  • Kuhn RJ, Zhang W, Rossmann MG, Pletnev SV, Corver J, Lenches E, Jones CT, Mukhopadhyay S, Chipman PR, Strauss EG, Baker TS, Strauss JH. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell. 2002;108(5):717–25. [PMC free article] [PubMed]
  • Lai CY, Tsai WY, Lin SR, Kao CL, Hu HP, King CC, Wu HC, Chang GJ, Wang WK. Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II. J Virol. 2008;82(13):6631–43. [PMC free article] [PubMed]
  • Lambris JD, Ricklin D, Geisbrecht BV. Complement evasion by human pathogens. Nat Rev Microbiol. 2008;6(2):132–42. [PMC free article] [PubMed]
  • Leatherbarrow RJ, Dwek RA. Binding of complement subcomponent C1q to mouse IgG1, IgG2a and IgG2b: a novel C1q binding assay. Mol Immunol. 1984;21(4):321–7. [PubMed]
  • Lewis JK, Bothner B, Smith TJ, Siuzdak G. Antiviral agent blocks breathing of the common cold virus. Proc Natl Acad Sci U S A. 1998;95(12):6774–8. [PubMed]
  • Li L, Lok SM, Yu IM, Zhang Y, Kuhn RJ, Chen J, Rossmann MG. The flavivirus precursor membrane-envelope protein complex: structure and maturation. Science. 2008;319(5871):1830–4. [PubMed]
  • Li Q, Yafal AG, Lee YM, Hogle J, Chow M. Poliovirus neutralization by antibodies to internal epitopes of VP4 and VP1 results from reversible exposure of these sequences at physiological temperature. J Virol. 1994;68(6):3965–70. [PMC free article] [PubMed]
  • Lindenbach BD, Thiel HJ, Rice CM. Flaviviridae: The Viruses and Their Replication. In: Knipe DM, Howley PM, editors. Fields Virology. 5. Vol. 1. Lippincott-Williams & Wilkins; Philadelphia: 2007. pp. 1101–1152. 2 vols.
  • Lok SM, Kostyuchenko V, Nybakken GE, Holdaway HA, Battisti AJ, Sukupolvi-Petty S, Sedlak D, Fremont DH, Chipman PR, Roehrig JT, Diamond MS, Kuhn RJ, Rossmann MG. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins. Nat Struct Mol Biol. 2008;15(3):312–7. [PubMed]
  • Mandel B. Neutralization of poliovirus: a hypothesis to explain the mechanism and the one-hit character of the neutralization reaction. Virology. 1976;69(2):500–10. [PubMed]
  • Mandl CW, Guirakhoo F, Holzmann H, Heinz FX, Kunz C. Antigenic structure of the flavivirus envelope protein E at the molecular level, using tick-borne encephalitis virus as a model. J Virol. 1989;63(2):564–71. [PMC free article] [PubMed]
  • Mehlhop E, Ansarah-Sobrinho C, Johnson S, Engle M, Fremont DH, Pierson TC, Diamond MS. Complement protein C1q inhibits antibody-dependent enhancement of flavivirus infection in an IgG subclass-specific manner. Cell Host Microbe. 2007;2(6):417–26. [PMC free article] [PubMed]
  • Mehlhop E, Nelson S, Jost CA, Gorlatov S, Johnson S, Fremont DH, Diamond MS, Pierson TC. Complement protein C1q reduces the stoichiometric threshold for antibody-mediated neutralization of West Nile virus. Cell Host Microbe. 2009;6(4):381–91. [PMC free article] [PubMed]
  • Moi ML, Lim CK, Takasaki T, Kurane I. Involvement of the Fc gamma receptor IIA cytoplasmic domain in antibody-dependent enhancement of dengue virus infection. J Gen Virol. 2010;91(Pt 1):103–11. [PubMed]
  • Morens DM, Halstead SB, Marchette NJ. Profiles of antibody-dependent enhancement of dengue virus type 2 infection. Microb Pathog. 1987;3(4):231–7. [PubMed]
  • Morrey JD, Siddharthan V, Olsen AL, Roper GY, Wang H, Baldwin TJ, Koenig S, Johnson S, Nordstrom JL, Diamond MS. Humanized Monoclonal Antibody against West Nile Virus Envelope Protein Administered after Neuronal Infection Protects against Lethal Encephalitis in Hamsters. J Infect Dis. 2006;194(9):1300–8. [PubMed]
  • Mouquet H, Scheid JF, Zoller MJ, Krogsgaard M, Ott RG, Shukair S, Artyomov MN, Pietzsch J, Connors M, Pereyra F, Walker BD, Ho DD, Wilson PC, Seaman MS, Eisen HN, Chakraborty AK, Hope TJ, Ravetch JV, Wardemann H, Nussenzweig MC. Polyreactivity increases the apparent affinity of anti-HIV antibodies by heteroligation. Nature. 2010;467(7315):591–5. [PMC free article] [PubMed]
  • Mukhopadhyay S, Kim BS, Chipman PR, Rossmann MG, Kuhn RJ. Structure of West Nile virus. Science. 2003;302(5643):248. [PubMed]
  • Mukhopadhyay S, Kuhn RJ, Rossmann MG. A structural perspective of the flavivirus life cycle. Nat Rev Microbiol. 2005;3(1):13–22. [PubMed]
  • Nelson S, Jost CA, Xu Q, Ess J, Martin JE, Oliphant T, Whitehead SS, Durbin AP, Graham BS, Diamond MS, Pierson TC. Maturation of West Nile virus modulates sensitivity to antibody-mediated neutralization. PLoS Pathog. 2008;4(5):e1000060. [PMC free article] [PubMed]
  • Nimmerjahn F, Ravetch JV. Fcgamma receptors as regulators of immune responses. Nat Rev Immunol. 2008;8(1):34–47. [PubMed]
  • Nybakken GE, Oliphant T, Johnson S, Burke S, Diamond MS, Fremont DH. Structural basis of West Nile virus neutralization by a therapeutic antibody. Nature. 2005;437(7059):764–9. [PubMed]
  • Oliphant T, Engle M, Nybakken GE, Doane C, Johnson S, Huang L, Gorlatov S, Mehlhop E, Marri A, Chung KM, Ebel GD, Kramer LD, Fremont DH, Diamond MS. Development of a humanized monoclonal antibody with therapeutic potential against West Nile virus. Nat Med. 2005;11(6):522–30. [PMC free article] [PubMed]
  • Oliphant T, Nybakken GE, Austin SK, Xu Q, Bramson J, Loeb M, Throsby M, Fremont DH, Pierson TC, Diamond MS. Induction of epitope-specific neutralizing antibodies against West Nile virus. J Virol. 2007;81(21):11828–39. [PMC free article] [PubMed]
  • Oliphant T, Nybakken GE, Engle M, Xu Q, Nelson CA, Sukupolvi-Petty S, Marri A, Lachmi BE, Olshevsky U, Fremont DH, Pierson TC, Diamond MS. Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein. J Virol. 2006;80(24):12149–59. [PMC free article] [PubMed]
  • Parren PW, Burton DR. The antiviral activity of antibodies in vitro and in vivo. Adv Immunol. 2001;77:195–262. [PubMed]
  • Peiris JS, Gordon S, Unkeless JC, Porterfield JS. Monoclonal anti-Fc receptor IgG blocks antibody enhancement of viral replication in macrophages. Nature. 1981;289(5794):189–91. [PubMed]
  • Pierson TC, Fremont DH, Kuhn RJ, Diamond MS. Structural insights into the mechanisms of antibody-mediated neutralization of flavivirus infection: implications for vaccine development. Cell Host Microbe. 2008;4(3):229–38. [PMC free article] [PubMed]
  • Pierson TC, Xu Q, Nelson S, Oliphant T, Nybakken GE, Fremont DH, Diamond MS. The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection. Cell Host Microbe. 2007;1(2):135–45. [PMC free article] [PubMed]
  • Porterfield JS. Antibody-dependent enhancement of viral infectivity. Adv Virus Res. 1986;31:335–55. [PubMed]
  • Roden RB, Weissinger EM, Henderson DW, Booy F, Kirnbauer R, Mushinski JF, Lowy DR, Schiller JT. Neutralization of bovine papillomavirus by antibodies to L1 and L2 capsid proteins. J Virol. 1994;68(11):7570–4. [PMC free article] [PubMed]
  • Rodenhuis-Zybert IA, van der Schaar HM, da Silva Voorham JM, van der Ende-Metselaar H, Lei HY, Wilschut J, Smit JM. Immature dengue virus: a veiled pathogen? PLoS Pathog. 2010;6(1):e1000718. [PMC free article] [PubMed]
  • Rodrigo WW, Jin X, Blackley SD, Rose RC, Schlesinger JJ. Differential enhancement of dengue virus immune complex infectivity mediated by signaling-competent and signaling-incompetent human Fcgamma RIA (CD64) or FcgammaRIIA (CD32) J Virol. 2006;80(20):10128–38. [PMC free article] [PubMed]
  • Roehrig JT. Antigenic structure of flavivirus proteins. Adv Virus Res. 2003;59:141–75. [PubMed]
  • Roehrig JT, Bolin RA, Kelly RG. Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica. Virology. 1998;246(2):317–28. [PubMed]
  • Roehrig JT, Staudinger LA, Hunt AR, Mathews JH, Blair CD. Antibody prophylaxis and therapy for flavivirus encephalitis infections. Ann N Y Acad Sci. 2001;951:286–97. [PubMed]
  • Sanchez MD, Pierson TC, Degrace MM, Mattei LM, Hanna SL, Del Piero F, Doms RW. The neutralizing antibody response against West Nile virus in naturally infected horses. Virology. 2007;359(2):336–48. [PubMed]
  • Sawyer LA. Antibodies for the prevention and treatment of viral diseases. Antiviral Res. 2000;47(2):57–77. [PubMed]
  • Schumaker VN, Calcott MA, Spiegelberg HL, Muller-Eberhard HJ. Ultracentifuge studies of the binding of IgG of different subclasses to the Clq subunit of the first component of complement. Biochemistry. 1976;15(23):5175–81. [PubMed]
  • Shepherd CM, Borelli IA, Lander G, Natarajan P, Siddavanahalli V, Bajaj C, Johnson JE, Brooks CL, 3rd, Reddy VS. VIPERdb: a relational database for structural virology. Nucleic Acids Res. 2006;34(Database issue):D386–9. [PMC free article] [PubMed]
  • Shrestha B, Brien JD, Sukupolvi-Petty S, Austin SK, Edeling MA, Kim T, O’Brien KM, Nelson CA, Johnson S, Fremont DH, Diamond MS. The development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1. PLoS Pathog. 2010;6(4):e1000823. [PMC free article] [PubMed]
  • Stiasny K, Brandler S, Kossl C, Heinz FX. Probing the flavivirus membrane fusion mechanism by using monoclonal antibodies. J Virol. 2007;81(20):11526–31. [PMC free article] [PubMed]
  • Stiasny K, Kiermayr S, Holzmann H, Heinz FX. Cryptic properties of a cluster of dominant flavivirus cross-reactive antigenic sites. J Virol. 2006;80(19):9557–68. [PMC free article] [PubMed]
  • Sukupolvi-Petty S, Austin SK, Engle M, Brien JD, Dowd KA, Williams KL, Johnson S, Rico-Hesse R, Harris E, Pierson TC, Fremont DH, Diamond MS. Structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2. J Virol. 2010;84(18):9227–39. [PMC free article] [PubMed]
  • Sukupolvi-Petty S, Austin SK, Purtha WE, Oliphant T, Nybakken GE, Schlesinger JJ, Roehrig JT, Gromowski GD, Barrett AD, Fremont DH, Diamond MS. Type- and subcomplex-specific neutralizing antibodies against domain III of dengue virus type 2 envelope protein recognize adjacent epitopes. J Virol. 2007;81(23):12816–26. [PMC free article] [PubMed]
  • Thompson BS, Moesker B, Smit JM, Wilschut J, Diamond MS, Fremont DH. A therapeutic antibody against west nile virus neutralizes infection by blocking fusion within endosomes. PLoS Pathog. 2009;5(5):e1000453. [PMC free article] [PubMed]
  • Throsby M, Geuijen C, Goudsmit J, Bakker AQ, Korimbocus J, Kramer RA, Clijsters-van der Horst M, de Jong M, Jongeneelen M, Thijsse S, Smit R, Visser TJ, Bijl N, Marissen WE, Loeb M, Kelvin DJ, Preiser W, ter Meulen J, de Kruif J. Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus. J Virol. 2006;80(14):6982–92. [PMC free article] [PubMed]
  • Ubol S, Phuklia W, Kalayanarooj S, Modhiran N. Mechanisms of immune evasion induced by a complex of dengue virus and preexisting enhancing antibodies. J Infect Dis. 2010;201(6):923–35. [PubMed]
  • Vazquez S, Guzman MG, Guillen G, Chinea G, Perez AB, Pupo M, Rodriguez R, Reyes O, Garay HE, Delgado I, Garcia G, Alvarez M. Immune response to synthetic peptides of dengue prM protein. Vaccine. 2002;20(13–14):1823–30. [PubMed]
  • Vogt MR, Moesker B, Goudsmit J, Jongeneelen M, Austin SK, Oliphant T, Nelson S, Pierson TC, Wilschut J, Throsby M, Diamond MS. Human Monoclonal Antibodies Induced by Natural Infection Against West Nile Virus Neutralize at a Post-Attachment Step. J Virol 2009 [PMC free article] [PubMed]
  • Wahala WM, Donaldson EF, de Alwis R, Accavitti-Loper MA, Baric RS, de Silva AM. Natural strain variation and antibody neutralization of dengue serotype 3 viruses. PLoS Pathog. 2010;6(3):e1000821. [PMC free article] [PubMed]
  • Wahala WM, Kraus AA, Haymore LB, Accavitti-Loper MA, de Silva AM. Dengue virus neutralization by human immune sera: role of envelope protein domain III-reactive antibody. Virology. 2009;392(1):103–13. [PMC free article] [PubMed]
  • Whitehead SS, Blaney JE, Durbin AP, Murphy BR. Prospects for a dengue virus vaccine. Nat Rev Microbiol. 2007;5(7):518–28. [PubMed]
  • Whiteman MC, Li L, Wicker JA, Kinney RM, Huang C, Beasley DW, Chung KM, Diamond MS, Solomon T, Barrett AD. Development and characterization of non-glycosylated E and NS1 mutant viruses as a potential candidate vaccine for West Nile virus. Vaccine. 2010;28(4):1075–83. [PubMed]
  • Witz J, Brown F. Structural dynamics, an intrinsic property of viral capsids. Arch Virol. 2001;146(12):2263–74. [PubMed]
  • Yamanaka A, Kosugi S, Konishi E. Infection-enhancing and -neutralizing activities of mouse monoclonal antibodies against dengue type 2 and 4 viruses are controlled by complement levels. J Virol. 2008;82(2):927–37. [PMC free article] [PubMed]
  • Yu IM, Zhang W, Holdaway HA, Li L, Kostyuchenko VA, Chipman PR, Kuhn RJ, Rossmann MG, Chen J. Structure of the immature dengue virus at low pH primes proteolytic maturation. Science. 2008;319(5871):1834–7. [PubMed]
  • Zellweger RM, Prestwood TR, Shresta S. Enhanced infection of liver sinusoidal endothelial cells in a mouse model of antibody-induced severe dengue disease. Cell Host Microbe. 2010;7(2):128–39. [PMC free article] [PubMed]
  • Zhang Y, Corver J, Chipman PR, Zhang W, Pletnev SV, Sedlak D, Baker TS, Strauss JH, Kuhn RJ, Rossmann MG. Structures of immature flavivirus particles. Embo J. 2003;22(11):2604–13. [PubMed]
  • Zhang Y, Kaufmann B, Chipman PR, Kuhn RJ, Rossmann MG. Structure of immature West Nile virus. J Virol. 2007;81(11):6141–5. [PMC free article] [PubMed]