- Email: [email protected]
Flaviviruses and their antigenic structure
Journal of Clinical Virology 55 (2012) 289–295
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Review
Flaviviruses and their antigenic structure F.X. Heinz ∗ , Karin Stiasny Department of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria
a r t i c l e
i n f o
Article history: Received 7 August 2012 Accepted 25 August 2012 Keywords: Flaviviruses Molecular antigenic structure Virus neutralization Flavivirus vaccines
a b s t r a c t Flaviviruses comprise important arthropod-transmitted human pathogens, including yellow fever (YF), dengue (Den), Japanese encephalitis (JE), West Nile (WN) and tick-borne encephalitis (TBE) viruses that have the potential of expanding their endemic areas due to global climatic, ecological and socio-economic changes. While effective vaccines against YF, JE and TBE are in widespread use, the development of a dengue vaccine has been hampered for a long time because of concerns of immunopathological consequences of vaccination. Phase III clinical trials with a recombinant chimeric live vaccine are now ongoing and will show whether the enormous problem of dengue can be resolved or at least reduced by vaccination in the future. Unprecedented details of the flavivirus particle structure have become available through the combined use of X-ray crystallography and cryo-electron microscopy that led to novel and surprising insights into the antigenic structure of these viruses. Recent studies provided evidence for an important role of virus maturation as well as particle dynamics in virus neutralization by antibodies and thus added previously unknown layers of complexity to our understanding of flavivirus immune protection. This information is invaluable for interpreting current investigations on the functional activities of polyclonal antibody responses to flavivirus infections and vaccinations and may open new avenues for studies on flavivirus cell biology and vaccine design. © 2012 Elsevier B.V. All rights reserved.
Contents 1.
2. 3.
4.
Human flaviviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Impact of flavivirus diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Laboratory diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Flavivirus vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flavivirus structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular antigenic structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Antigenic relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Characterization of epitopes and mechanism of neutralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Antibody-dependent enhancement (ADE) of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Role of particle dynamics on antibody binding and neutralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
289 289 290 290 290 290 290 292 292 293 293 294 294 294 294 294
1. Human flaviviruses Abbreviations: Den, dengue; JE, Japanese encephalitis; mab, monoclonal antibody; sE, soluble E; TBE, tick-borne encephalitis; WN, West Nile; YF, yellow fever. ∗ Corresponding author. Tel.: +43 1 40160 65510; fax: +43 1 40160 965599. E-mail address: [email protected] (F.X. Heinz). 1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.08.024
1.1. Impact of flavivirus diseases Flaviviruses represent some of the most important humanpathogenic arboviruses worldwide. They form a genus of more
290
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295
than 70 different viruses in the family Flaviviridae and comprise the mosquito-borne yellow fever (YF), dengue (Den), Japanese encephalitis (JE), and West Nile (WN) viruses as well as tick-borne encephalitis (TBE) virus,1 all of which have a significant impact on public health in their respective endemic and/or epidemic regions.2 Because of their dependence on specific natural hosts, vectors and ecosystems in general, flaviviruses are not uniformly distributed but have distinct, sometimes overlapping geographical distributions.2 The dynamic situation of ecological and climatic changes as well as factors associated with urbanization, international travel, trade and the possible adaptation of flaviviruses to new host species increase the potential of flavivirus emergence in previously unaffected regions of the world. This is most dramatically exemplified by the expansion of dengue hyperendemic areas,3 the introduction of WN virus to New York in 1999 and its subsequent expansion in North- and South-America,4 increased WN activity in Mediterranean countries5 and also the detection of new infection sites of TBE virus in Europe.6 With respect to global disease incidence, dengue has by far the highest impact, with an estimated 50–100 million infections per year (resulting in 500,000 cases of hemorrhagic dengue fever (DHF) and/or dengue shock syndrome (DSS) with more than 20,000 deaths) and 2.5 billion people living in dengue-endemic tropical and subtropical regions.3,7,8 In Africa and South-America, dengue areas overlap with those of YF (estimated number of annual cases 200,0009 ) and in South-East Asia with those of JE (estimated number of annual cases 50,000.10 TBE virus, on the other hand, does not occur in the tropics/subtropics but is endemic in large parts of Europe as well as Central and Eastern Asia.6,11 1.2. Laboratory diagnosis Human flavivirus infections are usually diagnosed by serology using various IgM and IgG immunoassay formats which have replaced previously used hemagglutination-inhibition and complement fixation tests (reviewed in Refs. 12,13). Because of the antigenic relationships between different flaviviruses (see Section 3.1), serological cross-reactions can pose a problem in the specific laboratory diagnosis of flavivirus infections. Especially in the case of sequential infections with different Den virus serotypes, the type-specific serodiagnosis is difficult and may require special immunoassay formats,14 virus neutralization tests and the analysis of paired sera. Compared to serology, nucleic acid detection assays are very specific and allow precise identification of the infecting virus by sequence analysis. As a drawback of this technology in routine flavivirus diagnosis, in many instances severe symptoms leading to hospitalization develop only at the end of viremia when the virus has already reached undetectable levels. 1.3. Flavivirus vaccines In principle, flavivirus diseases can be effectively prevented by vaccination, exemplified by the live attenuated YF vaccine,9 both live- and inactivated JE vaccines,10 as well as inactivated TBE vaccines,15 all of which are in widespread use. For dengue, however, – despite its enormous public health impact – no vaccine has yet become available on the market. The major obstacle are long-standing concerns that vaccination may predispose to an exacerbation of infection by immunological enhancement phenomena also observed in sequential infections with different dengue serotypes16,17 (see Section 3.3). This problem of dengue immunopathogenesis has been investigated extensively but is still not completely resolved.16 Nevertheless, great efforts were made in the last decades for the development of dengue vaccines which should ideally induce life-long protection against all four serotypes. The approaches include live-attenuated, inactivated whole virus,
recombinant protein, DNA as well as vectored vaccines.17–20 Currently, the most advanced of these candidate vaccines is a tetravalent recombinant chimeric live vaccine (Chimerivax, Sanofi Pasteur) based on the yellow fever strain 17D backbone combined with the structural proteins of all four dengue serotypes.21 Ongoing phase III clinical trials22 will hopefully provide conclusive evidence for protection in the absence of adverse effects and eventually lead to an effective means for the immunoprophylaxis of dengue. 2. Flavivirus structure Flaviviruses are small enveloped viruses with only three structural proteins, designated E (envelope), prM/M (precursor of membrane or membrane, respectively) and C (capsid). The first assembly products are non-infectious immature virions that contain complexes between E and prM in the viral membrane and are formed by budding into the endoplasmic reticulum (Fig. 1A, left).23 Upon transport of these particles through the exocytotic pathway of the infected cell, prM is cleaved by the cellular protease furin in the trans-Golgi network,24 finally resulting in the release of mature infectious viruses (Fig. 1A, right) into the extracellular fluid. Molecular details of the flavivirus structure were resolved by X-ray crystallography of soluble forms of E and cryo-electron microscopy of immature and mature virus particles (reviewed in Ref. 25). In immature virions, E is associated with prM and forms 60 spikes of trimers of prM-E heterodimers (Fig. 1A, left).26 The processes of virus maturation (prM cleavage) result in a complete rearrangement of E proteins in the viral envelope27 and the formation of smooth-surfaced particles with a herringbonelike icosahedral lattice of antiparallel E dimers (Fig. 1B).28 The ectodomain of the E dimer (soluble E; sE) lacks the trans-membrane anchor and a membrane-associated element called ‘stem’; Fig. 1A, right). It is composed of three distinct domains (DI, DII, DIII), forming an elongated rod that is gently curved to accommodate the shape of the viral surface (Fig. 1C and D). During cell infection, the E protein not only mediates receptorbinding but also fusion of the viral membrane with endosomal membranes after uptake by receptor-mediated endocytosis.25,29,30 In this low pH-triggered process, the E dimer dissociates, exposes the highly conserved fusion peptide at the tip of DII (Fig. 1C), rearranges its domains to form a hairpin-like structure and is converted into a trimer.31,32 Because of its essential functions in virus entry, the E protein is the major target of flavivirus neutralizing antibodies which block infection by inhibiting cell attachment, endocytosis and/or membrane fusion.33 In certain cell types, the cleavage of prM may be quite inefficient – especially in the case of Den viruses – resulting in the release of varying proportions of immature, partially mature, and mature particles.34,35 The finding that partially mature virus particles are infectious36 and that even completely immature particles can infect cells when taken up by antibody- and Fc receptormediated endocytosis37 suggested a possible role of maturation cleavage in the pathogenesis of flaviviruses.34 3. Molecular antigenic structure 3.1. Antigenic relationships Originally, the flaviviruses (former group B arboviruses) were grouped together on the basis of cross-reactions observed in hemagglutination inhibition assays using polyclonal sera.38 Virus neutralization is more specific and allowed the definition of serocomplexes containing more closely related flaviviruses,39 as displayed in Fig. 2A. The E proteins of viruses from different serocomplexes share only about 40% identical amino acids, concentrated in the interior of the protein, so that their exposed
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295
291
Fig. 1. Flavivirus structure. (A) Schematic representation of a flavivirus particle. Left: immature virion; right: mature virion. The unstructured spherical capsid contains the positive-stranded genomic RNA and multiple copies of the capsid protein C. Immature virions are covered by spiky complexes of 60 trimers of prM-E heterodimers. The proteolytic cleavage of prM results in the reorganization of the E proteins and the formation of smooth-surfaced particles covered with 90 E dimers. sE: soluble form of E that lacks the membrane anchor and an adjacent sequence element called ‘stem’. M: Membrane-associated cleavage product of prM. (B) Herringbone-like arrangement of 90 E protein dimers at the virion surface as determined by cryo-electron microscopy (PDB 14KR). The triangle indicates 2-, 3-, and 5-fold symmetry axes. (C) and (D) Ribbon diagrams of the crystal structure of the TBE virus sE protein dimer (PDB 1SVB) in its top view and side view, respectively. Printed black and white version: In all panels, the three E protein domains are displayed in different shades of grey (DIII darkest; DI intermediate; DII lightest). The fusion peptide at the tip of DII interdigitates with a hydrophobic pocket provided by DIII-DI of the second subunit in the dimer. Online color version: In all panels, the three E protein domains are colored as follows: DIII blue; DI red; D II yellow; fusion peptide green. The fusion peptide at the tip of DII (green) interdigitates with a hydrophobic pocket provided by DIII-DI of the second subunit in the dimer.
protein surfaces differ significantly in pairwise comparisons (Fig. 2B) – consistent with the lack of cross-protection between distantly related flaviviruses. Even within serocomplexes the divergence can be quite significant and – as an example – the four dengue serotypes differ by up to ∼37% in their E protein amino
acid sequences (Fig. 2A). Pairwise comparison of E proteins from different dengue serotypes (as displayed for Den 2 and 4 in Fig. 2C) reveals a mosaic of variable but also conserved patches, allowing the induction of antibodies that cross-neutralize at least to a certain extent. In natural infections, however, cross-protection between
Fig. 2. Relationships of flaviviruses. (A) Dendrogram based on the percentages of identical amino acids between the E proteins of the most important human-pathogenic flaviviruses. Printed black and white version: (B) Surface representation of the Den virus 2 sE dimer, with surface-exposed amino acids that differ in comparison to TBE virus highlighted in black. (C) Surface representation of the Den virus 2 sE dimer, with surface-exposed amino acids that differ in comparison to Den virus 4 highlighted in black. Online color version: (B) Surface representation of the Den virus 2 sE dimer, with surface-exposed amino acids that differ in comparison to TBE virus highlighted in blue. (C) Surface representation of the Den virus 2 sE dimer, with surface-exposed amino acids that differ in comparison to Den virus 4 highlighted in blue. Virus strains used for panels B and C: Den virus 2 strain 159 S1 (GenBank M19197; PDB 1OAN); TBE virus strain Neudoerfl (GenBank U27495); Den virus 4 strain H241 (GenBank AY947539).
292
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295
dengue serotypes lasts only for a few months16,40 and a single person can have four Den virus infections in a life time. The waning concentration of poorly neutralizing cross-reactive antibodies with time has been implicated in the higher frequency of severe forms of Den virus infections (DHF/DSS) observed in sequential infections through the phenomenon of antibody-dependent enhancement of infection (ADE; see Section 3.3).16,41 On the other hand, based on the relatively close relationship between JEV and WNV (about 80% sequence identity in E; Fig. 2A) and animal experiments, it was recently proposed that potent new generation JEV vaccines could induce sufficiently high antibody titers to mediate protection against both diseases.42 In the context of such a cross-protective vaccination approach, it will be important to study the kinetics of waning immunity and theoretically possible phenomena of enhanced infection as described for dengue, although these have been considered to be more remote within the JE serocomplex of flaviviruses.42 3.2. Characterization of epitopes and mechanism of neutralization The E protein is the major target of neutralizing antibodies and corresponding epitopes have been mapped for different flaviviruses by a number of approaches, including the analysis of neutralization escape mutants, specifically engineered mutants, peptide mapping, yeast surface display of variant forms of E as well as X-ray crystallography and cryo-electron microscopy of complexes of antibody fragments with sE and whole virions, respectively. These analyses revealed that in principle the whole surface of E exposed in the virion can induce and bind neutralizing antibodies. Importantly, even sites that appear occluded in the cryo-electron microscopy model shown in Fig. 1B can become accessible through temperature-dependent dynamic structural changes of the E protein arrangement at the viral surface (see Section 3.4). Monoclonal antibody (mab)-defined epitopes (Fig. 3A) have been mapped to sites within each of the three domains,43–46 to domain-overlapping sites within the same E monomer,45 to E dimer-specific sites involving residues from both monomers,47 and to sites not represented by soluble forms of E but requiring the herringbone-like quaternary arrangement in virus particles48 (Fig. 3B). The role of such sites in polyclonal responses is currently under intensive investigation and recent studies with human dengue post-infection sera provide evidence that the majority of neutralizing antibodies are directed to quaternary epitopes not represented by soluble forms of E.49 The neutralization of flaviviruses by antibodies is a multi-hit phenomenon, i.e. it requires a number of antibody molecules bound to the virus particle that exceeds a certain stoichiometric threshold.33 This threshold is primarily determined by antibody affinity as well as by the nature and location of the epitope, especially its accessibility on the virus particle. These factors determine the fraction of epitopes on the virion occupied by antibody at any given concentration (occupancy) and the occupancy requirements for neutralization, respectively. For one of the best characterized neutralizing mabs (E16, specific for DIII of West Nile virus), it was shown that only 120 of the potential 180 DIII-binding sites per virion are accessible because of steric constraints at the five-fold symmetry axes50 (Fig. 1B) and that 30 antibodies must have bound to achieve 50% neutralization.51 The analysis of a panel of WNVspecific mabs has revealed that the stoichiometric requirements for neutralization are subject to strong variation and complete occupancy may be necessary for achieving neutralization in the case of poorly accessible determinants.51 Mechanistically, the binding of antibodies to the flavivirus E protein can lead to virus neutralization by inhibiting its principal functions in virus entry, including cell attachment and membrane
Fig. 3. Types of epitopes mapped in E. (A) Domain-specific: restricted to a single protein domain. Domain-overlapping: contains contact amino acid residues from two adjacent domains of the same E monomer. Subunit-overlapping (=E dimerspecific): contains contact amino acid residues of both monomers in the dimer. (B) ‘Herringbone’-specific epitope, dependent on the quaternary arrangement of E dimers in the virus particle, as determined by cryo EM of complexes between WN virus and Fabs of a neutralizing monoclonal antibody. Left: Whole virion. The triangle indicates 2-, 3-, and 5-fold symmetry axes, as in Fig. 1B. Right: ‘Raft’ of three parallel E dimers, with the densities of bound Fabs highlighted in grey. Reproduced from Kaufmann et al.,48 with permission of the authors and the publisher. Color codes are the same as in Fig. 1.
fusion after uptake by receptor-mediated endocytosis. Flavivirus receptors are still incompletely defined and may differ for different viruses, hosts and tissues (reviewed in Ref. 25). Because experimental data suggest that antibodies to DIII neutralize by inhibiting virus binding to cells, this domain has been proposed to interact with putative cellular receptors (reviewed in Ref. 52). As shown by studies with both polyclonal and monoclonal antibodies, virus neutralization can also occur at post-attachment steps, most likely due to interference with the structural rearrangements of E that are necessary for viral membrane fusion in the endosome.53–56 It is plausible that the mechanism of neutralization of many E protein-specific antibodies involves both steps of virus entry and is modulated by the composition of antibody-populations in polyclonal sera. The degree of prM cleavage can vary between different flaviviruses, strains and the cell type in which the virus replicates.34,35 The grade of particle maturation can have a strong influence on the results of neutralization assays.57 Indeed, substantial strain- and genotype-specific variations of neutralization have been observed with Den 1,58 Den 259 and Den 3 viruses.60,61 These variables therefore pose a problem for the standardization of assays measuring vaccine-induced neutralizing antibodies – generally assumed to provide a good correlate of in vivo protection62,63 – and may be responsible for discrepancies observed between these parameters in some instances.59,64 3.3. Antibody-dependent enhancement (ADE) of infection ADE has been described with Fc-receptor-bearing cells for a number of different viruses (reviewed in Refs. 65–67). In the
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295
293
Fig. 4. Antibody-induced rearrangement of E proteins in the viral envelope. Left: Structure of mature Den virus in the absence of antibody. Right: E protein organization in the same virus after complex formation at 37 ◦ C with the Fab fragment of a neutralizing monoclonal antibody recognizing a partially cryptic epitope. Triangles indicate 2-, 3-, and 5-fold symmetry axes, as in Fig. 1B. Reprinted by permission from Lok et al. Nature Structural and Molecular Biology Macmillan Publishers LTd.69 Color codes are the same as in Fig. 1.
case of dengue, however, this phenomenon is believed to contribute – together with other factors16 – to the development of DHF and DSS, which are most frequently observed in secondary infections with different dengue serotypes and in children of seropositive mothers several months after birth, when the originally protective maternal antibodies have declined.41 Den viruses have a tropism for monocytes and macrophages, and complexes of virions with non-neutralizing antibodies can lead to enhanced infection of these cell types through a strongly increased virus uptake by Fc-receptor-mediated endocytosis. Originally, it was believed that neutralizing and enhancing antibodies are induced by different epitopes of the virion.65 Model studies with panels of neutralizing mabs, however, provided conclusive evidence that any antibody that neutralizes at sufficiently high concentration can lead to enhanced infection at sub-neutralizing concentration, and the same also holds true for polyclonal sera.51 ADE has not only been demonstrated with E protein-specific but also with prM-specific antibodies that are poorly neutralizing but seem to represent a significant proportion of the total antibody response in dengue patients.68 Enhanced infection by partially or completely immature dengue virus particles in complex with anti-prM antibodies may thus contribute to the pathogenesis of severe forms of dengue virus infections.37,68 The potential detrimental role of poorly neutralizing dengue cross-reactive and anti prM antibodies, together with the possible decline of strongly neutralizing antibodies to subneutralizing concentrations pose formidable challenges for the development and introduction of dengue vaccines. 3.4. Role of particle dynamics on antibody binding and neutralization There is strong evidence now that the envelopes of flaviviruses are much more dynamic than previously thought and several observations of antibody binding and neutralization cannot be explained by static models of virion structure as determined by cryo-electron microscopy. Specifically, epitopes of neutralizing monoclonal antibodies have been identified that would be at least partly occluded and therefore inaccessible for antibodies in the herringbone model of mature virions shown in Fig. 1B.69–71 Studies with the mouse mab 1A1D-2 – which neutralizes Den virus serotypes 1, 2 and 3 and recognizes an epitope in DIII – have revealed that its interaction with the virion is strongly temperature-dependent and efficient
binding occurs only at 37 ◦ C.69 At this temperature the antibody apparently gains access to an otherwise cryptic site and induces structural changes that result in a complete reorganization of the E proteins in the virus envelope – as revealed by cryo EM of Den 2 virus in complex with the 1A1D-2 Fab fragment (Fig. 4A and B).69 The neutralizing activity of a mab that reacts with a similar epitope in DIII of all dengue serotypes70 was hypothesized to be based on its capacity to disrupt the contacts between the fusion peptide of one monomer and its accommodating pocket in the second monomer (Fig. 1C), resulting in E dimer dissociation and concomitantly the rearrangement of E proteins at the virion surface. Significant conformational changes in the viral envelope must also take place for allowing the binding of the dengue mab 2H2.71 This study further suggests that the extent of envelope ‘breathing’ may differ between the dengue serotypes. In any case, the existing evidence indicates that the viral envelope proteins are in constant motion at 37 ◦ C and that the binding of antibodies to specific sites can shift the equilibrium to E protein arrangements that are strikingly different from those determined in the absence of antibodies. The impact of temperature and incubation time on West Nile virus neutralization by different monoclonal antibodies corroborates the dynamic model of flavivirus structure and helps to explain the contribution of occluded antigenic sites in virus neutralization.72 The new evidence for the flexibility of the viral envelope is fully consistent with original descriptions of antibody-induced conformational changes by E protein-specific mabs, in particular with the finding that certain antibodies (and their Fab fragments) not only increased the avidities but also the number of binding sites for other antibodies.73,74 These data can now be interpreted in the light of the dynamics and antibody-induced rearrangements of E protein lattices in the flavivirus envelope. Such rearrangements are likely to have implications for possible cooperative interactions between antibody populations in polyclonal sera that influence neutralizing activity. As suggested by Cockburn et al.,70 the dynamic nature of the flavivirus envelope may not only allow antibody-induced but also receptor-induced rearrangements that could contribute to cell attachment and facilitate virus entry into cells. 4. Conclusion The combination of X-ray crystallography, cryo-electron microscopy and monoclonal antibody studies have dramatically
294
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295
increased our molecular understanding of flavivirus antigenic structure and allowed the elucidation of diverse mechanisms of virus neutralization by antibodies. Most remarkably, it becomes increasingly clear that the seemingly closed shell of flavivirus particles is not a static structure but a dynamic ensemble of proteins that are in constant temperature-dependent motion, resulting in the exposure of previously believed cryptic antigenic sites that can contribute to virus neutralization by antibodies. The demonstration of particle dynamics in the context of antibody binding has raised the question whether this phenomenon might also play a role in certain stages of the viral life cycle, e.g. allowing receptor-induced rearrangements important for virus entry. Future studies will show in which way the new structural insights can provide leads for elucidating as yet unrecognized aspects of virus-cell interactions and whether they can be translated into a better understanding of polyclonal antibody responses and the development of new concepts in the design of novel vaccines. Funding None. Competing interests None declared. Ethical approval Not required. Acknowledgements We thank Michael G. Rossmann for providing the originals to Figs. 3B and 4. References 1. Simmonds P, Becher P, Collett MS, Gould EA, Heinz FX, Meyers G, et al. Family Flaviviridae. In: King AMQ, Lefkowitz E, Adams MJ, Carstens EB, editors. Virus Taxonomy IXth Report of the International Committee on Taxonomy of Viruses. San Diego: Elsevier Academic Press; 2011. 2. Gubler D, Kuno G, Markhoff L. Flaviviruses. In: Knipe DM, Howley PM, Griffin DE, et al., editors. Fields virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins; 2007. p. 1153–252. 3. Vasilakis N, Cardosa J, Hanley KA, Holmes EC, Weaver SC. Fever from the forest: prospects for the continued emergence of sylvatic dengue virus and its impact on public health. Nat Rev Microbiol 2011;9:532–41. 4. Gubler DJ. The continuing spread of West Nile virus in the western hemisphere. Clin Infect Dis 2007;45:1039–46. 5. Calistri P, Giovannini A, Hubalek Z, Ionescu A, Monaco F, Savini G, et al. Epidemiology of West Nile in Europe and in the Mediterranean basin. Open Virol J 2010;4:29–37. 6. Suss J. Tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in Europe and Asia—an overview. Ticks Tick Borne Dis 2011;2:2–15. 7. Gubler DJ. Emerging vector-borne flavivirus diseases: are vaccines the solution? Expert Rev Vaccines 2011;10:563–5. 8. Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, Gubler DJ, et al. Dengue: a continuing global threat. Nat Rev Microbiol 2010;8:S7–16. 9. Monath TP, Cetron MS, Teuwen DE. Yellow fever vaccine. In: Plotkin SA, Orenstein WA, Offit PA, editors. Vaccines. Saunders Elsevier; 2008. p. 959–1055. 10. Halstead SB, Thomas SJ. New Japanese encephalitis vaccines: alternatives to production in mouse brain. Expert Rev Vaccines 2011;10:355–64. 11. Lindquist L, Vapalahti O. Tick-borne encephalitis. Lancet 2008;371:1861–71. 12. Endy TP, Nisalak A, Vaughn DW. Diagnosis of dengue virus infections. In: Halstead SB, editor. Dengue. London: Imperial College Press; 2008. p. 327–60. 13. Hunsperger E. Flavivirus diagnostics. In: Shi PY, editor. Molecular virology and control of flaviviruses. Norfolk: Caister Academic Press; 2012. p. 271–96. 14. Midgley CM, Bajwa-Joseph M, Vasanawathana S, Limpitikul W, Wills B, Flanagan A, et al. An in-depth analysis of original antigenic sin in dengue virus infection. J Virol 2011;85:410–21. 15. WHO. Vaccines against tick-borne encephalitis: WHO position paper. Wkly Epidemiol Rec 2011;86:241–56. 16. Rothman AL. Immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms. Nat Rev Immunol 2011;11:532–43.
17. Simmons CP, Farrar JJ, Nguyen vV, Wills B. Dengue. N Engl J Med 2012;366:1423–32. 18. Heinz FX, Stiasny K. Flaviviruses and flavivirus vaccines. Vaccine 2012;30:4301–6. 19. Coller BA, Clements DE. Dengue vaccines: progress and challenges. Curr Opin Immunol 2011;23:391–8. 20. Whitehead SS, Durbin AP. Prospects and challenges for dengue virus vaccine development. In: Hanley KA, Weaver SC, editors. Frontiers in dengue virus research. Norfolk, UK: Caister Academic Press; 2010. p. 221–37. 21. 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;28:632–49. 22. Guy B, Barrere B, Malinowski C, Saville M, Teyssou R, Lang J. From research to phase III: preclinical, industrial and clinical development of the Sanofi Pasteur tetravalent dengue vaccine. Vaccine 2011;29:7229–41. 23. Lindenbach BD, Thiel HJ, Rice CM. Flaviviridae: the viruses and their replication. In: Knipe DM, Howley PM, Griffin DE, et al., editors. Fields virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins; 2007. p. 1101–52. 24. Stadler K, Allison SL, Schalich J, Heinz FX. Proteolytic activation of tick-borne encephalitis virus by furin. J Virol 1997;71:8475–81. 25. Kaufmann B, Rossmann MG. Molecular mechanisms involved in the early steps of flavivirus cell entry. Microbes Infect 2011;13:1–9. 26. Zhang Y, Corver J, Chipman PR, Zhang W, Pletnev SV, Sedlak D, et al. Structures of immature flavivirus particles. EMBO J 2003;22:2604–13. 27. Yu IM, Zhang W, Holdaway HA, Li L, Kostyuchenko VA, Chipman PR, et al. Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 2008;319:1834–7. 28. Kuhn RJ, Zhang W, Rossmann MG, Pletnev SV, Corver J, Lenches E, et al. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 2002;108:717–25. 29. Stiasny K, Heinz FX. Flavivirus membrane fusion. J Gen Virol 2006;87:2755–66. 30. Smit JM, Moesker B, Rodenhuis-Zybert I, Wilschut J. Flavivirus cell entry and membrane fusion. Viruses 2011;3:160–71. 31. Harrison SC. Viral membrane fusion. Nat Struct Mol Biol 2008;15:690–8. 32. Stiasny K, Fritz R, Pangerl K, Heinz FX. Molecular mechanisms of flavivirus membrane fusion. Amino Acids 2011;41:1159–63. 33. 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;4:229–38. 34. Rodenhuis-Zybert IA, Wilschut J, Smit JM. Partial maturation: an immuneevasion strategy of dengue virus? Trends Microbiol 2011;19:248–54. 35. Pierson TC, Diamond MS. Degrees of maturity: the complex structure and biology of flaviviruses. Curr Opin Virol 2012;2:168–75. 36. Mukherjee S, Lin TY, Dowd KA, Manhart CJ, Pierson TC. The infectivity of prMcontaining partially mature West Nile virus does not require the activity of cellular furin-like proteases. J Virol 2011;85:12067–72. 37. Rodenhuis-Zybert IA, van der Schaar HM, da Silva Voorham JM, van der EndeMetselaar H, Lei HY, Wilschut J, et al. Immature dengue virus: a veiled pathogen? PLoS Pathog 2010;6:e1000718. 38. Porterfield JS. Antigenic characteristics and classification of Togaviridae. In: Schlesinger RW, editor. The Togaviruses biology, structure, replication. New York: Academic Press; 1980. p. 13–46. 39. Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, Westaway EG, et al. Antigenic relationships between flaviviruses as determined by crossneutralization tests with polyclonal antisera. J Gen Virol 1989;70(Pt 1):37–43. 40. Sabin AB. Research on dengue during World War II. Am J Trop Med Hyg 1952;1:30–50. 41. Halstead SB, Pathogenesis:. Risk factors for infection. In: Halstead SB, editor. Dengue. London: Imperial College Press; 2008. p. 219–56. 42. Lobigs M, Diamond MS. Feasibility of cross-protective vaccination against flaviviruses of the Japanese encephalitis serocomplex. Expert Rev Vaccines 2012;11:177–87. 43. Wahala WM, Silva AM. The human antibody response to dengue virus infection. Viruses 2011;3:2374–95. 44. 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;437:764–9. 45. Oliphant T, Nybakken GE, Engle M, Xu Q, Nelson CA, Sukupolvi-Petty S, et al. Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein. J Virol 2006;80:12149–59. 46. 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;83:8482–91. 47. Throsby M, Geuijen C, Goudsmit J, Bakker AQ, Korimbocus J, Kramer RA, et al. Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus. J Virol 2006;80:6982–92. 48. Kaufmann B, Vogt MR, Goudsmit J, Holdaway HA, Aksyuk AA, Chipman PR, et al. 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 USA 2010;107:18950–5. 49. de Alwis R, Smith SA, Olivarez NP, Messer WB, Huynh JP, Wahala WM, et al. Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions. Proc Natl Acad Sci USA 2012;109:7439–44. 50. Kaufmann B, Nybakken GE, Chipman PR, Zhang W, Diamond MS, Fremont DH, et al. West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody. Proc Natl Acad Sci USA 2006;103:12400–4.
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295 51. Pierson TC, Xu Q, Nelson S, Oliphant T, Nybakken GE, Fremont DH, et al. The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection. Cell Host Microbe 2007;1:135–45. 52. Pierson TC, Diamond MS. Molecular mechanisms of antibody-mediated neutralisation of flavivirus infection. Expert Rev Mol Med 2008;10:e12. 53. Gollins SW, Porterfield JS. A new mechanism for the neutralization of enveloped viruses by antiviral antibody. Nature 1986;321:244–6. 54. Stiasny K, Brandler S, Kossl C, Heinz FX. Probing the flavivirus membrane fusion mechanism by using monoclonal antibodies. J Virol 2007;81:11526–31. 55. 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:e1000453. 56. Vogt MR, Moesker B, Goudsmit J, Jongeneelen M, Austin SK, Oliphant T, et al. Human monoclonal antibodies against West Nile virus induced by natural infection neutralize at a postattachment step. J Virol 2009;83:6494–507. 57. Nelson S, Jost CA, Xu Q, Ess J, Martin JE, Oliphant T, et al. Maturation of West Nile virus modulates sensitivity to antibody-mediated neutralization. PLoS Pathog 2008;4:e1000060. 58. Shrestha B, Brien JD, Sukupolvi-Petty S, Austin SK, Edeling MA, Kim T, et al. The development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1. PLoS Pathog 2010;6:e1000823. 59. Sukupolvi-Petty S, Austin SK, Engle M, Brien JD, Dowd KA, Williams KL, et al. Structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2. J Virol 2010;84:9227–39. 60. Brien JD, Austin SK, Sukupolvi-Petty S, O’Brien KM, Johnson S, Fremont DH, et al. Genotype-specific neutralization and protection by antibodies against dengue virus type 3. J Virol 2010;84:10630–43. 61. 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:e1000821. 62. Roehrig JT, Staudinger LA, Hunt AR, Mathews JH, Blair CD. Antibody prophylaxis and therapy for flavivirus encephalitis infections. Ann NY Acad Sci 2001;951:286–97. 63. Diamond MS, Pierson TC, Fremont DH. The structural immunology of antibody protection against West Nile virus. Immunol Rev 2008;225:212–25.
295
64. Simmons M, Porter KR, Hayes CG, Vaughn DW, Putnak R. Characterization of antibody responses to combinations of a dengue virus type 2 DNA vaccine and two dengue virus type 2 protein vaccines in rhesus macaques. J Virol 2006;80:9577–85. 65. Porterfield JS. Antibody-dependent enhancement of viral infectivity. Adv Virus Res 1986;31:335–55. 66. Takada A, Kawaoka Y. Antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications. Rev Med Virol 2003;13: 387–98. 67. Halstead SB, Mahalingam S, Marovich MA, Ubol S, Mosser DM. Intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes. Lancet Infect Dis 2010;10: 712–22. 68. Dejnirattisai W, Jumnainsong A, Onsirisakul N, Fitton P, Vasanawathana S, Limpitikul W, et al. Cross-reacting antibodies enhance dengue virus infection in humans. Science 2010;328:745–8. 69. Lok SM, Kostyuchenko V, Nybakken GE, Holdaway HA, Battisti AJ, SukupolviPetty S, et al. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins. Nat Struct Mol Biol 2008;15: 312–7. 70. Cockburn JJ, Navarro Sanchez ME, Fretes N, Urvoas A, Staropoli I, Kikuti CM, et al. Mechanism of dengue virus broad cross-neutralization by a monoclonal antibody. Structure 2012;20:303–14. 71. Midgley CM, Flanagan A, Tran HB, Dejnirattisai W, Chawansuntati K, Jumnainsong A, et al. Structural analysis of a dengue cross-reactive antibody complexed with envelope domain III reveals the molecular basis of cross-reactivity. J Immunol 2012;188:4971–9. 72. Dowd KA, Jost CA, Durbin AP, Whitehead SS, Pierson TC. A dynamic landscape for antibody binding modulates antibody-mediated neutralization of West Nile virus. PLoS Pathog 2011;7:e1002111. 73. Heinz FX, Mandl C, Berger R, Tuma W, Kunz C. Antibody-induced conformational changes result in enhanced avidity of antibodies to different antigenic sites on the tick-borne encephalitis virus glycoprotein. Virology 1984;133:25–34. 74. Heinz FX. Epitope mapping of flavivirus glycoproteins. Adv Virus Res 1986;31:103–68.
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Review
Flaviviruses and their antigenic structure F.X. Heinz ∗ , Karin Stiasny Department of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria
a r t i c l e
i n f o
Article history: Received 7 August 2012 Accepted 25 August 2012 Keywords: Flaviviruses Molecular antigenic structure Virus neutralization Flavivirus vaccines
a b s t r a c t Flaviviruses comprise important arthropod-transmitted human pathogens, including yellow fever (YF), dengue (Den), Japanese encephalitis (JE), West Nile (WN) and tick-borne encephalitis (TBE) viruses that have the potential of expanding their endemic areas due to global climatic, ecological and socio-economic changes. While effective vaccines against YF, JE and TBE are in widespread use, the development of a dengue vaccine has been hampered for a long time because of concerns of immunopathological consequences of vaccination. Phase III clinical trials with a recombinant chimeric live vaccine are now ongoing and will show whether the enormous problem of dengue can be resolved or at least reduced by vaccination in the future. Unprecedented details of the flavivirus particle structure have become available through the combined use of X-ray crystallography and cryo-electron microscopy that led to novel and surprising insights into the antigenic structure of these viruses. Recent studies provided evidence for an important role of virus maturation as well as particle dynamics in virus neutralization by antibodies and thus added previously unknown layers of complexity to our understanding of flavivirus immune protection. This information is invaluable for interpreting current investigations on the functional activities of polyclonal antibody responses to flavivirus infections and vaccinations and may open new avenues for studies on flavivirus cell biology and vaccine design. © 2012 Elsevier B.V. All rights reserved.
Contents 1.
2. 3.
4.
Human flaviviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Impact of flavivirus diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Laboratory diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Flavivirus vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flavivirus structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular antigenic structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Antigenic relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Characterization of epitopes and mechanism of neutralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Antibody-dependent enhancement (ADE) of infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Role of particle dynamics on antibody binding and neutralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
289 289 290 290 290 290 290 292 292 293 293 294 294 294 294 294
1. Human flaviviruses Abbreviations: Den, dengue; JE, Japanese encephalitis; mab, monoclonal antibody; sE, soluble E; TBE, tick-borne encephalitis; WN, West Nile; YF, yellow fever. ∗ Corresponding author. Tel.: +43 1 40160 65510; fax: +43 1 40160 965599. E-mail address: [email protected] (F.X. Heinz). 1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.08.024
1.1. Impact of flavivirus diseases Flaviviruses represent some of the most important humanpathogenic arboviruses worldwide. They form a genus of more
290
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295
than 70 different viruses in the family Flaviviridae and comprise the mosquito-borne yellow fever (YF), dengue (Den), Japanese encephalitis (JE), and West Nile (WN) viruses as well as tick-borne encephalitis (TBE) virus,1 all of which have a significant impact on public health in their respective endemic and/or epidemic regions.2 Because of their dependence on specific natural hosts, vectors and ecosystems in general, flaviviruses are not uniformly distributed but have distinct, sometimes overlapping geographical distributions.2 The dynamic situation of ecological and climatic changes as well as factors associated with urbanization, international travel, trade and the possible adaptation of flaviviruses to new host species increase the potential of flavivirus emergence in previously unaffected regions of the world. This is most dramatically exemplified by the expansion of dengue hyperendemic areas,3 the introduction of WN virus to New York in 1999 and its subsequent expansion in North- and South-America,4 increased WN activity in Mediterranean countries5 and also the detection of new infection sites of TBE virus in Europe.6 With respect to global disease incidence, dengue has by far the highest impact, with an estimated 50–100 million infections per year (resulting in 500,000 cases of hemorrhagic dengue fever (DHF) and/or dengue shock syndrome (DSS) with more than 20,000 deaths) and 2.5 billion people living in dengue-endemic tropical and subtropical regions.3,7,8 In Africa and South-America, dengue areas overlap with those of YF (estimated number of annual cases 200,0009 ) and in South-East Asia with those of JE (estimated number of annual cases 50,000.10 TBE virus, on the other hand, does not occur in the tropics/subtropics but is endemic in large parts of Europe as well as Central and Eastern Asia.6,11 1.2. Laboratory diagnosis Human flavivirus infections are usually diagnosed by serology using various IgM and IgG immunoassay formats which have replaced previously used hemagglutination-inhibition and complement fixation tests (reviewed in Refs. 12,13). Because of the antigenic relationships between different flaviviruses (see Section 3.1), serological cross-reactions can pose a problem in the specific laboratory diagnosis of flavivirus infections. Especially in the case of sequential infections with different Den virus serotypes, the type-specific serodiagnosis is difficult and may require special immunoassay formats,14 virus neutralization tests and the analysis of paired sera. Compared to serology, nucleic acid detection assays are very specific and allow precise identification of the infecting virus by sequence analysis. As a drawback of this technology in routine flavivirus diagnosis, in many instances severe symptoms leading to hospitalization develop only at the end of viremia when the virus has already reached undetectable levels. 1.3. Flavivirus vaccines In principle, flavivirus diseases can be effectively prevented by vaccination, exemplified by the live attenuated YF vaccine,9 both live- and inactivated JE vaccines,10 as well as inactivated TBE vaccines,15 all of which are in widespread use. For dengue, however, – despite its enormous public health impact – no vaccine has yet become available on the market. The major obstacle are long-standing concerns that vaccination may predispose to an exacerbation of infection by immunological enhancement phenomena also observed in sequential infections with different dengue serotypes16,17 (see Section 3.3). This problem of dengue immunopathogenesis has been investigated extensively but is still not completely resolved.16 Nevertheless, great efforts were made in the last decades for the development of dengue vaccines which should ideally induce life-long protection against all four serotypes. The approaches include live-attenuated, inactivated whole virus,
recombinant protein, DNA as well as vectored vaccines.17–20 Currently, the most advanced of these candidate vaccines is a tetravalent recombinant chimeric live vaccine (Chimerivax, Sanofi Pasteur) based on the yellow fever strain 17D backbone combined with the structural proteins of all four dengue serotypes.21 Ongoing phase III clinical trials22 will hopefully provide conclusive evidence for protection in the absence of adverse effects and eventually lead to an effective means for the immunoprophylaxis of dengue. 2. Flavivirus structure Flaviviruses are small enveloped viruses with only three structural proteins, designated E (envelope), prM/M (precursor of membrane or membrane, respectively) and C (capsid). The first assembly products are non-infectious immature virions that contain complexes between E and prM in the viral membrane and are formed by budding into the endoplasmic reticulum (Fig. 1A, left).23 Upon transport of these particles through the exocytotic pathway of the infected cell, prM is cleaved by the cellular protease furin in the trans-Golgi network,24 finally resulting in the release of mature infectious viruses (Fig. 1A, right) into the extracellular fluid. Molecular details of the flavivirus structure were resolved by X-ray crystallography of soluble forms of E and cryo-electron microscopy of immature and mature virus particles (reviewed in Ref. 25). In immature virions, E is associated with prM and forms 60 spikes of trimers of prM-E heterodimers (Fig. 1A, left).26 The processes of virus maturation (prM cleavage) result in a complete rearrangement of E proteins in the viral envelope27 and the formation of smooth-surfaced particles with a herringbonelike icosahedral lattice of antiparallel E dimers (Fig. 1B).28 The ectodomain of the E dimer (soluble E; sE) lacks the trans-membrane anchor and a membrane-associated element called ‘stem’; Fig. 1A, right). It is composed of three distinct domains (DI, DII, DIII), forming an elongated rod that is gently curved to accommodate the shape of the viral surface (Fig. 1C and D). During cell infection, the E protein not only mediates receptorbinding but also fusion of the viral membrane with endosomal membranes after uptake by receptor-mediated endocytosis.25,29,30 In this low pH-triggered process, the E dimer dissociates, exposes the highly conserved fusion peptide at the tip of DII (Fig. 1C), rearranges its domains to form a hairpin-like structure and is converted into a trimer.31,32 Because of its essential functions in virus entry, the E protein is the major target of flavivirus neutralizing antibodies which block infection by inhibiting cell attachment, endocytosis and/or membrane fusion.33 In certain cell types, the cleavage of prM may be quite inefficient – especially in the case of Den viruses – resulting in the release of varying proportions of immature, partially mature, and mature particles.34,35 The finding that partially mature virus particles are infectious36 and that even completely immature particles can infect cells when taken up by antibody- and Fc receptormediated endocytosis37 suggested a possible role of maturation cleavage in the pathogenesis of flaviviruses.34 3. Molecular antigenic structure 3.1. Antigenic relationships Originally, the flaviviruses (former group B arboviruses) were grouped together on the basis of cross-reactions observed in hemagglutination inhibition assays using polyclonal sera.38 Virus neutralization is more specific and allowed the definition of serocomplexes containing more closely related flaviviruses,39 as displayed in Fig. 2A. The E proteins of viruses from different serocomplexes share only about 40% identical amino acids, concentrated in the interior of the protein, so that their exposed
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295
291
Fig. 1. Flavivirus structure. (A) Schematic representation of a flavivirus particle. Left: immature virion; right: mature virion. The unstructured spherical capsid contains the positive-stranded genomic RNA and multiple copies of the capsid protein C. Immature virions are covered by spiky complexes of 60 trimers of prM-E heterodimers. The proteolytic cleavage of prM results in the reorganization of the E proteins and the formation of smooth-surfaced particles covered with 90 E dimers. sE: soluble form of E that lacks the membrane anchor and an adjacent sequence element called ‘stem’. M: Membrane-associated cleavage product of prM. (B) Herringbone-like arrangement of 90 E protein dimers at the virion surface as determined by cryo-electron microscopy (PDB 14KR). The triangle indicates 2-, 3-, and 5-fold symmetry axes. (C) and (D) Ribbon diagrams of the crystal structure of the TBE virus sE protein dimer (PDB 1SVB) in its top view and side view, respectively. Printed black and white version: In all panels, the three E protein domains are displayed in different shades of grey (DIII darkest; DI intermediate; DII lightest). The fusion peptide at the tip of DII interdigitates with a hydrophobic pocket provided by DIII-DI of the second subunit in the dimer. Online color version: In all panels, the three E protein domains are colored as follows: DIII blue; DI red; D II yellow; fusion peptide green. The fusion peptide at the tip of DII (green) interdigitates with a hydrophobic pocket provided by DIII-DI of the second subunit in the dimer.
protein surfaces differ significantly in pairwise comparisons (Fig. 2B) – consistent with the lack of cross-protection between distantly related flaviviruses. Even within serocomplexes the divergence can be quite significant and – as an example – the four dengue serotypes differ by up to ∼37% in their E protein amino
acid sequences (Fig. 2A). Pairwise comparison of E proteins from different dengue serotypes (as displayed for Den 2 and 4 in Fig. 2C) reveals a mosaic of variable but also conserved patches, allowing the induction of antibodies that cross-neutralize at least to a certain extent. In natural infections, however, cross-protection between
Fig. 2. Relationships of flaviviruses. (A) Dendrogram based on the percentages of identical amino acids between the E proteins of the most important human-pathogenic flaviviruses. Printed black and white version: (B) Surface representation of the Den virus 2 sE dimer, with surface-exposed amino acids that differ in comparison to TBE virus highlighted in black. (C) Surface representation of the Den virus 2 sE dimer, with surface-exposed amino acids that differ in comparison to Den virus 4 highlighted in black. Online color version: (B) Surface representation of the Den virus 2 sE dimer, with surface-exposed amino acids that differ in comparison to TBE virus highlighted in blue. (C) Surface representation of the Den virus 2 sE dimer, with surface-exposed amino acids that differ in comparison to Den virus 4 highlighted in blue. Virus strains used for panels B and C: Den virus 2 strain 159 S1 (GenBank M19197; PDB 1OAN); TBE virus strain Neudoerfl (GenBank U27495); Den virus 4 strain H241 (GenBank AY947539).
292
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295
dengue serotypes lasts only for a few months16,40 and a single person can have four Den virus infections in a life time. The waning concentration of poorly neutralizing cross-reactive antibodies with time has been implicated in the higher frequency of severe forms of Den virus infections (DHF/DSS) observed in sequential infections through the phenomenon of antibody-dependent enhancement of infection (ADE; see Section 3.3).16,41 On the other hand, based on the relatively close relationship between JEV and WNV (about 80% sequence identity in E; Fig. 2A) and animal experiments, it was recently proposed that potent new generation JEV vaccines could induce sufficiently high antibody titers to mediate protection against both diseases.42 In the context of such a cross-protective vaccination approach, it will be important to study the kinetics of waning immunity and theoretically possible phenomena of enhanced infection as described for dengue, although these have been considered to be more remote within the JE serocomplex of flaviviruses.42 3.2. Characterization of epitopes and mechanism of neutralization The E protein is the major target of neutralizing antibodies and corresponding epitopes have been mapped for different flaviviruses by a number of approaches, including the analysis of neutralization escape mutants, specifically engineered mutants, peptide mapping, yeast surface display of variant forms of E as well as X-ray crystallography and cryo-electron microscopy of complexes of antibody fragments with sE and whole virions, respectively. These analyses revealed that in principle the whole surface of E exposed in the virion can induce and bind neutralizing antibodies. Importantly, even sites that appear occluded in the cryo-electron microscopy model shown in Fig. 1B can become accessible through temperature-dependent dynamic structural changes of the E protein arrangement at the viral surface (see Section 3.4). Monoclonal antibody (mab)-defined epitopes (Fig. 3A) have been mapped to sites within each of the three domains,43–46 to domain-overlapping sites within the same E monomer,45 to E dimer-specific sites involving residues from both monomers,47 and to sites not represented by soluble forms of E but requiring the herringbone-like quaternary arrangement in virus particles48 (Fig. 3B). The role of such sites in polyclonal responses is currently under intensive investigation and recent studies with human dengue post-infection sera provide evidence that the majority of neutralizing antibodies are directed to quaternary epitopes not represented by soluble forms of E.49 The neutralization of flaviviruses by antibodies is a multi-hit phenomenon, i.e. it requires a number of antibody molecules bound to the virus particle that exceeds a certain stoichiometric threshold.33 This threshold is primarily determined by antibody affinity as well as by the nature and location of the epitope, especially its accessibility on the virus particle. These factors determine the fraction of epitopes on the virion occupied by antibody at any given concentration (occupancy) and the occupancy requirements for neutralization, respectively. For one of the best characterized neutralizing mabs (E16, specific for DIII of West Nile virus), it was shown that only 120 of the potential 180 DIII-binding sites per virion are accessible because of steric constraints at the five-fold symmetry axes50 (Fig. 1B) and that 30 antibodies must have bound to achieve 50% neutralization.51 The analysis of a panel of WNVspecific mabs has revealed that the stoichiometric requirements for neutralization are subject to strong variation and complete occupancy may be necessary for achieving neutralization in the case of poorly accessible determinants.51 Mechanistically, the binding of antibodies to the flavivirus E protein can lead to virus neutralization by inhibiting its principal functions in virus entry, including cell attachment and membrane
Fig. 3. Types of epitopes mapped in E. (A) Domain-specific: restricted to a single protein domain. Domain-overlapping: contains contact amino acid residues from two adjacent domains of the same E monomer. Subunit-overlapping (=E dimerspecific): contains contact amino acid residues of both monomers in the dimer. (B) ‘Herringbone’-specific epitope, dependent on the quaternary arrangement of E dimers in the virus particle, as determined by cryo EM of complexes between WN virus and Fabs of a neutralizing monoclonal antibody. Left: Whole virion. The triangle indicates 2-, 3-, and 5-fold symmetry axes, as in Fig. 1B. Right: ‘Raft’ of three parallel E dimers, with the densities of bound Fabs highlighted in grey. Reproduced from Kaufmann et al.,48 with permission of the authors and the publisher. Color codes are the same as in Fig. 1.
fusion after uptake by receptor-mediated endocytosis. Flavivirus receptors are still incompletely defined and may differ for different viruses, hosts and tissues (reviewed in Ref. 25). Because experimental data suggest that antibodies to DIII neutralize by inhibiting virus binding to cells, this domain has been proposed to interact with putative cellular receptors (reviewed in Ref. 52). As shown by studies with both polyclonal and monoclonal antibodies, virus neutralization can also occur at post-attachment steps, most likely due to interference with the structural rearrangements of E that are necessary for viral membrane fusion in the endosome.53–56 It is plausible that the mechanism of neutralization of many E protein-specific antibodies involves both steps of virus entry and is modulated by the composition of antibody-populations in polyclonal sera. The degree of prM cleavage can vary between different flaviviruses, strains and the cell type in which the virus replicates.34,35 The grade of particle maturation can have a strong influence on the results of neutralization assays.57 Indeed, substantial strain- and genotype-specific variations of neutralization have been observed with Den 1,58 Den 259 and Den 3 viruses.60,61 These variables therefore pose a problem for the standardization of assays measuring vaccine-induced neutralizing antibodies – generally assumed to provide a good correlate of in vivo protection62,63 – and may be responsible for discrepancies observed between these parameters in some instances.59,64 3.3. Antibody-dependent enhancement (ADE) of infection ADE has been described with Fc-receptor-bearing cells for a number of different viruses (reviewed in Refs. 65–67). In the
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295
293
Fig. 4. Antibody-induced rearrangement of E proteins in the viral envelope. Left: Structure of mature Den virus in the absence of antibody. Right: E protein organization in the same virus after complex formation at 37 ◦ C with the Fab fragment of a neutralizing monoclonal antibody recognizing a partially cryptic epitope. Triangles indicate 2-, 3-, and 5-fold symmetry axes, as in Fig. 1B. Reprinted by permission from Lok et al. Nature Structural and Molecular Biology Macmillan Publishers LTd.69 Color codes are the same as in Fig. 1.
case of dengue, however, this phenomenon is believed to contribute – together with other factors16 – to the development of DHF and DSS, which are most frequently observed in secondary infections with different dengue serotypes and in children of seropositive mothers several months after birth, when the originally protective maternal antibodies have declined.41 Den viruses have a tropism for monocytes and macrophages, and complexes of virions with non-neutralizing antibodies can lead to enhanced infection of these cell types through a strongly increased virus uptake by Fc-receptor-mediated endocytosis. Originally, it was believed that neutralizing and enhancing antibodies are induced by different epitopes of the virion.65 Model studies with panels of neutralizing mabs, however, provided conclusive evidence that any antibody that neutralizes at sufficiently high concentration can lead to enhanced infection at sub-neutralizing concentration, and the same also holds true for polyclonal sera.51 ADE has not only been demonstrated with E protein-specific but also with prM-specific antibodies that are poorly neutralizing but seem to represent a significant proportion of the total antibody response in dengue patients.68 Enhanced infection by partially or completely immature dengue virus particles in complex with anti-prM antibodies may thus contribute to the pathogenesis of severe forms of dengue virus infections.37,68 The potential detrimental role of poorly neutralizing dengue cross-reactive and anti prM antibodies, together with the possible decline of strongly neutralizing antibodies to subneutralizing concentrations pose formidable challenges for the development and introduction of dengue vaccines. 3.4. Role of particle dynamics on antibody binding and neutralization There is strong evidence now that the envelopes of flaviviruses are much more dynamic than previously thought and several observations of antibody binding and neutralization cannot be explained by static models of virion structure as determined by cryo-electron microscopy. Specifically, epitopes of neutralizing monoclonal antibodies have been identified that would be at least partly occluded and therefore inaccessible for antibodies in the herringbone model of mature virions shown in Fig. 1B.69–71 Studies with the mouse mab 1A1D-2 – which neutralizes Den virus serotypes 1, 2 and 3 and recognizes an epitope in DIII – have revealed that its interaction with the virion is strongly temperature-dependent and efficient
binding occurs only at 37 ◦ C.69 At this temperature the antibody apparently gains access to an otherwise cryptic site and induces structural changes that result in a complete reorganization of the E proteins in the virus envelope – as revealed by cryo EM of Den 2 virus in complex with the 1A1D-2 Fab fragment (Fig. 4A and B).69 The neutralizing activity of a mab that reacts with a similar epitope in DIII of all dengue serotypes70 was hypothesized to be based on its capacity to disrupt the contacts between the fusion peptide of one monomer and its accommodating pocket in the second monomer (Fig. 1C), resulting in E dimer dissociation and concomitantly the rearrangement of E proteins at the virion surface. Significant conformational changes in the viral envelope must also take place for allowing the binding of the dengue mab 2H2.71 This study further suggests that the extent of envelope ‘breathing’ may differ between the dengue serotypes. In any case, the existing evidence indicates that the viral envelope proteins are in constant motion at 37 ◦ C and that the binding of antibodies to specific sites can shift the equilibrium to E protein arrangements that are strikingly different from those determined in the absence of antibodies. The impact of temperature and incubation time on West Nile virus neutralization by different monoclonal antibodies corroborates the dynamic model of flavivirus structure and helps to explain the contribution of occluded antigenic sites in virus neutralization.72 The new evidence for the flexibility of the viral envelope is fully consistent with original descriptions of antibody-induced conformational changes by E protein-specific mabs, in particular with the finding that certain antibodies (and their Fab fragments) not only increased the avidities but also the number of binding sites for other antibodies.73,74 These data can now be interpreted in the light of the dynamics and antibody-induced rearrangements of E protein lattices in the flavivirus envelope. Such rearrangements are likely to have implications for possible cooperative interactions between antibody populations in polyclonal sera that influence neutralizing activity. As suggested by Cockburn et al.,70 the dynamic nature of the flavivirus envelope may not only allow antibody-induced but also receptor-induced rearrangements that could contribute to cell attachment and facilitate virus entry into cells. 4. Conclusion The combination of X-ray crystallography, cryo-electron microscopy and monoclonal antibody studies have dramatically
294
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295
increased our molecular understanding of flavivirus antigenic structure and allowed the elucidation of diverse mechanisms of virus neutralization by antibodies. Most remarkably, it becomes increasingly clear that the seemingly closed shell of flavivirus particles is not a static structure but a dynamic ensemble of proteins that are in constant temperature-dependent motion, resulting in the exposure of previously believed cryptic antigenic sites that can contribute to virus neutralization by antibodies. The demonstration of particle dynamics in the context of antibody binding has raised the question whether this phenomenon might also play a role in certain stages of the viral life cycle, e.g. allowing receptor-induced rearrangements important for virus entry. Future studies will show in which way the new structural insights can provide leads for elucidating as yet unrecognized aspects of virus-cell interactions and whether they can be translated into a better understanding of polyclonal antibody responses and the development of new concepts in the design of novel vaccines. Funding None. Competing interests None declared. Ethical approval Not required. Acknowledgements We thank Michael G. Rossmann for providing the originals to Figs. 3B and 4. References 1. Simmonds P, Becher P, Collett MS, Gould EA, Heinz FX, Meyers G, et al. Family Flaviviridae. In: King AMQ, Lefkowitz E, Adams MJ, Carstens EB, editors. Virus Taxonomy IXth Report of the International Committee on Taxonomy of Viruses. San Diego: Elsevier Academic Press; 2011. 2. Gubler D, Kuno G, Markhoff L. Flaviviruses. In: Knipe DM, Howley PM, Griffin DE, et al., editors. Fields virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins; 2007. p. 1153–252. 3. Vasilakis N, Cardosa J, Hanley KA, Holmes EC, Weaver SC. Fever from the forest: prospects for the continued emergence of sylvatic dengue virus and its impact on public health. Nat Rev Microbiol 2011;9:532–41. 4. Gubler DJ. The continuing spread of West Nile virus in the western hemisphere. Clin Infect Dis 2007;45:1039–46. 5. Calistri P, Giovannini A, Hubalek Z, Ionescu A, Monaco F, Savini G, et al. Epidemiology of West Nile in Europe and in the Mediterranean basin. Open Virol J 2010;4:29–37. 6. Suss J. Tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in Europe and Asia—an overview. Ticks Tick Borne Dis 2011;2:2–15. 7. Gubler DJ. Emerging vector-borne flavivirus diseases: are vaccines the solution? Expert Rev Vaccines 2011;10:563–5. 8. Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, Gubler DJ, et al. Dengue: a continuing global threat. Nat Rev Microbiol 2010;8:S7–16. 9. Monath TP, Cetron MS, Teuwen DE. Yellow fever vaccine. In: Plotkin SA, Orenstein WA, Offit PA, editors. Vaccines. Saunders Elsevier; 2008. p. 959–1055. 10. Halstead SB, Thomas SJ. New Japanese encephalitis vaccines: alternatives to production in mouse brain. Expert Rev Vaccines 2011;10:355–64. 11. Lindquist L, Vapalahti O. Tick-borne encephalitis. Lancet 2008;371:1861–71. 12. Endy TP, Nisalak A, Vaughn DW. Diagnosis of dengue virus infections. In: Halstead SB, editor. Dengue. London: Imperial College Press; 2008. p. 327–60. 13. Hunsperger E. Flavivirus diagnostics. In: Shi PY, editor. Molecular virology and control of flaviviruses. Norfolk: Caister Academic Press; 2012. p. 271–96. 14. Midgley CM, Bajwa-Joseph M, Vasanawathana S, Limpitikul W, Wills B, Flanagan A, et al. An in-depth analysis of original antigenic sin in dengue virus infection. J Virol 2011;85:410–21. 15. WHO. Vaccines against tick-borne encephalitis: WHO position paper. Wkly Epidemiol Rec 2011;86:241–56. 16. Rothman AL. Immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms. Nat Rev Immunol 2011;11:532–43.
17. Simmons CP, Farrar JJ, Nguyen vV, Wills B. Dengue. N Engl J Med 2012;366:1423–32. 18. Heinz FX, Stiasny K. Flaviviruses and flavivirus vaccines. Vaccine 2012;30:4301–6. 19. Coller BA, Clements DE. Dengue vaccines: progress and challenges. Curr Opin Immunol 2011;23:391–8. 20. Whitehead SS, Durbin AP. Prospects and challenges for dengue virus vaccine development. In: Hanley KA, Weaver SC, editors. Frontiers in dengue virus research. Norfolk, UK: Caister Academic Press; 2010. p. 221–37. 21. 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;28:632–49. 22. Guy B, Barrere B, Malinowski C, Saville M, Teyssou R, Lang J. From research to phase III: preclinical, industrial and clinical development of the Sanofi Pasteur tetravalent dengue vaccine. Vaccine 2011;29:7229–41. 23. Lindenbach BD, Thiel HJ, Rice CM. Flaviviridae: the viruses and their replication. In: Knipe DM, Howley PM, Griffin DE, et al., editors. Fields virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins; 2007. p. 1101–52. 24. Stadler K, Allison SL, Schalich J, Heinz FX. Proteolytic activation of tick-borne encephalitis virus by furin. J Virol 1997;71:8475–81. 25. Kaufmann B, Rossmann MG. Molecular mechanisms involved in the early steps of flavivirus cell entry. Microbes Infect 2011;13:1–9. 26. Zhang Y, Corver J, Chipman PR, Zhang W, Pletnev SV, Sedlak D, et al. Structures of immature flavivirus particles. EMBO J 2003;22:2604–13. 27. Yu IM, Zhang W, Holdaway HA, Li L, Kostyuchenko VA, Chipman PR, et al. Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 2008;319:1834–7. 28. Kuhn RJ, Zhang W, Rossmann MG, Pletnev SV, Corver J, Lenches E, et al. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 2002;108:717–25. 29. Stiasny K, Heinz FX. Flavivirus membrane fusion. J Gen Virol 2006;87:2755–66. 30. Smit JM, Moesker B, Rodenhuis-Zybert I, Wilschut J. Flavivirus cell entry and membrane fusion. Viruses 2011;3:160–71. 31. Harrison SC. Viral membrane fusion. Nat Struct Mol Biol 2008;15:690–8. 32. Stiasny K, Fritz R, Pangerl K, Heinz FX. Molecular mechanisms of flavivirus membrane fusion. Amino Acids 2011;41:1159–63. 33. 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;4:229–38. 34. Rodenhuis-Zybert IA, Wilschut J, Smit JM. Partial maturation: an immuneevasion strategy of dengue virus? Trends Microbiol 2011;19:248–54. 35. Pierson TC, Diamond MS. Degrees of maturity: the complex structure and biology of flaviviruses. Curr Opin Virol 2012;2:168–75. 36. Mukherjee S, Lin TY, Dowd KA, Manhart CJ, Pierson TC. The infectivity of prMcontaining partially mature West Nile virus does not require the activity of cellular furin-like proteases. J Virol 2011;85:12067–72. 37. Rodenhuis-Zybert IA, van der Schaar HM, da Silva Voorham JM, van der EndeMetselaar H, Lei HY, Wilschut J, et al. Immature dengue virus: a veiled pathogen? PLoS Pathog 2010;6:e1000718. 38. Porterfield JS. Antigenic characteristics and classification of Togaviridae. In: Schlesinger RW, editor. The Togaviruses biology, structure, replication. New York: Academic Press; 1980. p. 13–46. 39. Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, Westaway EG, et al. Antigenic relationships between flaviviruses as determined by crossneutralization tests with polyclonal antisera. J Gen Virol 1989;70(Pt 1):37–43. 40. Sabin AB. Research on dengue during World War II. Am J Trop Med Hyg 1952;1:30–50. 41. Halstead SB, Pathogenesis:. Risk factors for infection. In: Halstead SB, editor. Dengue. London: Imperial College Press; 2008. p. 219–56. 42. Lobigs M, Diamond MS. Feasibility of cross-protective vaccination against flaviviruses of the Japanese encephalitis serocomplex. Expert Rev Vaccines 2012;11:177–87. 43. Wahala WM, Silva AM. The human antibody response to dengue virus infection. Viruses 2011;3:2374–95. 44. 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;437:764–9. 45. Oliphant T, Nybakken GE, Engle M, Xu Q, Nelson CA, Sukupolvi-Petty S, et al. Antibody recognition and neutralization determinants on domains I and II of West Nile Virus envelope protein. J Virol 2006;80:12149–59. 46. 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;83:8482–91. 47. Throsby M, Geuijen C, Goudsmit J, Bakker AQ, Korimbocus J, Kramer RA, et al. Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus. J Virol 2006;80:6982–92. 48. Kaufmann B, Vogt MR, Goudsmit J, Holdaway HA, Aksyuk AA, Chipman PR, et al. 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 USA 2010;107:18950–5. 49. de Alwis R, Smith SA, Olivarez NP, Messer WB, Huynh JP, Wahala WM, et al. Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions. Proc Natl Acad Sci USA 2012;109:7439–44. 50. Kaufmann B, Nybakken GE, Chipman PR, Zhang W, Diamond MS, Fremont DH, et al. West Nile virus in complex with the Fab fragment of a neutralizing monoclonal antibody. Proc Natl Acad Sci USA 2006;103:12400–4.
F.X. Heinz, K. Stiasny / Journal of Clinical Virology 55 (2012) 289–295 51. Pierson TC, Xu Q, Nelson S, Oliphant T, Nybakken GE, Fremont DH, et al. The stoichiometry of antibody-mediated neutralization and enhancement of West Nile virus infection. Cell Host Microbe 2007;1:135–45. 52. Pierson TC, Diamond MS. Molecular mechanisms of antibody-mediated neutralisation of flavivirus infection. Expert Rev Mol Med 2008;10:e12. 53. Gollins SW, Porterfield JS. A new mechanism for the neutralization of enveloped viruses by antiviral antibody. Nature 1986;321:244–6. 54. Stiasny K, Brandler S, Kossl C, Heinz FX. Probing the flavivirus membrane fusion mechanism by using monoclonal antibodies. J Virol 2007;81:11526–31. 55. 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:e1000453. 56. Vogt MR, Moesker B, Goudsmit J, Jongeneelen M, Austin SK, Oliphant T, et al. Human monoclonal antibodies against West Nile virus induced by natural infection neutralize at a postattachment step. J Virol 2009;83:6494–507. 57. Nelson S, Jost CA, Xu Q, Ess J, Martin JE, Oliphant T, et al. Maturation of West Nile virus modulates sensitivity to antibody-mediated neutralization. PLoS Pathog 2008;4:e1000060. 58. Shrestha B, Brien JD, Sukupolvi-Petty S, Austin SK, Edeling MA, Kim T, et al. The development of therapeutic antibodies that neutralize homologous and heterologous genotypes of dengue virus type 1. PLoS Pathog 2010;6:e1000823. 59. Sukupolvi-Petty S, Austin SK, Engle M, Brien JD, Dowd KA, Williams KL, et al. Structure and function analysis of therapeutic monoclonal antibodies against dengue virus type 2. J Virol 2010;84:9227–39. 60. Brien JD, Austin SK, Sukupolvi-Petty S, O’Brien KM, Johnson S, Fremont DH, et al. Genotype-specific neutralization and protection by antibodies against dengue virus type 3. J Virol 2010;84:10630–43. 61. 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:e1000821. 62. Roehrig JT, Staudinger LA, Hunt AR, Mathews JH, Blair CD. Antibody prophylaxis and therapy for flavivirus encephalitis infections. Ann NY Acad Sci 2001;951:286–97. 63. Diamond MS, Pierson TC, Fremont DH. The structural immunology of antibody protection against West Nile virus. Immunol Rev 2008;225:212–25.
295
64. Simmons M, Porter KR, Hayes CG, Vaughn DW, Putnak R. Characterization of antibody responses to combinations of a dengue virus type 2 DNA vaccine and two dengue virus type 2 protein vaccines in rhesus macaques. J Virol 2006;80:9577–85. 65. Porterfield JS. Antibody-dependent enhancement of viral infectivity. Adv Virus Res 1986;31:335–55. 66. Takada A, Kawaoka Y. Antibody-dependent enhancement of viral infection: molecular mechanisms and in vivo implications. Rev Med Virol 2003;13: 387–98. 67. Halstead SB, Mahalingam S, Marovich MA, Ubol S, Mosser DM. Intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes. Lancet Infect Dis 2010;10: 712–22. 68. Dejnirattisai W, Jumnainsong A, Onsirisakul N, Fitton P, Vasanawathana S, Limpitikul W, et al. Cross-reacting antibodies enhance dengue virus infection in humans. Science 2010;328:745–8. 69. Lok SM, Kostyuchenko V, Nybakken GE, Holdaway HA, Battisti AJ, SukupolviPetty S, et al. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins. Nat Struct Mol Biol 2008;15: 312–7. 70. Cockburn JJ, Navarro Sanchez ME, Fretes N, Urvoas A, Staropoli I, Kikuti CM, et al. Mechanism of dengue virus broad cross-neutralization by a monoclonal antibody. Structure 2012;20:303–14. 71. Midgley CM, Flanagan A, Tran HB, Dejnirattisai W, Chawansuntati K, Jumnainsong A, et al. Structural analysis of a dengue cross-reactive antibody complexed with envelope domain III reveals the molecular basis of cross-reactivity. J Immunol 2012;188:4971–9. 72. Dowd KA, Jost CA, Durbin AP, Whitehead SS, Pierson TC. A dynamic landscape for antibody binding modulates antibody-mediated neutralization of West Nile virus. PLoS Pathog 2011;7:e1002111. 73. Heinz FX, Mandl C, Berger R, Tuma W, Kunz C. Antibody-induced conformational changes result in enhanced avidity of antibodies to different antigenic sites on the tick-borne encephalitis virus glycoprotein. Virology 1984;133:25–34. 74. Heinz FX. Epitope mapping of flavivirus glycoproteins. Adv Virus Res 1986;31:103–68.