Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection.

Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus-Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE.

A humanized IgG but not IgM antibody is effective in prophylaxis and therapy of yellow fever infection in an AG129/17D-204 peripheral challenge mouse model.

Yellow fever virus (YFV), a member of the genus Flavivirus, is a mosquito-borne virus found in tropical regions of Africa and South America that causes severe hepatic disease and death in humans. Despite the availability of effective vaccines, YFV is responsible for an estimated 200,000 cases and 30,000 deaths annually. There are currently no prophylactic or therapeutic strategies approved for use in human YFV infections. Furthermore, implementation of YFV 17D-204 vaccination campaigns has become problematic due to an increase in reported post-vaccinal adverse events. We have created human/murine chimeric MAbs of a YFV-reactive murine monoclonal antibody (mMAb), 2C9, that was previously shown to protect mice from lethal YFV infection and to have therapeutic activity. The new chimeric (cMAbs) were constructed by fusion of the m2C9 IgG gene variable regions with the constant regions of human IgG and IgM and expressed in Sp2 murine myelomas. The 2C9 cMAbs (2C9-cIgG and 2C9-cIgM) reacted with 17D-204 vaccine strain in an enzyme-linked immunosorbent assay and neutralized virus in vitro similarly to the parent m2C9. Both m2C9 and 2C9-cIgG when administered prophylactically 24h prior to infection protected AG129 mice from peripheral 17D-204 challenge at antibody concentrations ≥1.27 µg/mouse; however, the 2C9-cIgM did not protect even at a dose of 127 µg/mouse. The 17D-204 infection of AG129 mice is otherwise uniformly lethal. While the m2C9 was shown previously to be therapeutically effective in YFV-infected BALB/c mice at day 4 post-infection, the m2C9 and 2C9-cIgG demonstrated therapeutic activity only when administered 1 day post-infection in 17D-204-infected AG129 mice.

Human antibodies against dengue enhance dengue viral infectivity without suppressing type I interferon secretion in primary human monocytes.

It remains unclear whether antibody-dependent-enhancement (ADE) of dengue infection merely augments viral attachment and entry through Fcγ receptors or immune complex binding to Fcγ receptors triggers an intrinsic signaling cascade that changes the viral permissiveness of the cell. Using human dengue-immune sera and novel human monoclonal antibodies against dengue in combination with virologic and immunologic techniques, we found that ADE infection increased the proportion of infected primary human monocytes modestly from 0.2% ± 0.1% (no Ab) to 1.7% ± 1.6% (with Ab) but the total virus output markedly from 2 ± 2 (× 10(3)) FFU to 120 ± 153 (× 10(3))FFU. However, this increased virus production was not associated with a reduced secretion of type I interferon or an elevated secretion of anti-inflammatory cytokine, IL-10. These results demonstrate that the regulation of virus production in ADE infection of primary human monocytes is more complex than previously appreciated.

A tetravalent recombinant dengue domain III protein vaccine stimulates neutralizing and enhancing antibodies in mice.

Dengue viruses co-circulate as four serologically distinct viruses (DENV1-4) that commonly infect individuals sequentially. Current DENV candidate vaccines incorporate the entire virion envelope E protein (E) ectodomain thereby stimulating both DENV serotype-specific and cross-reactive antibodies. Because the latter may enhance naturally acquired infection, such vaccine formulations must be tetravalent. We evaluated the neutralizing and enhancing antibody response to E domain III (dIII) proteins, in which serotype-specific neutralizing determinants are concentrated. Mice immunized with insect cell-secreted recombinant DENV-dIII proteins individually, and in tetravalent combination, produced serotype-specific IgG1 neutralizing antibodies that nevertheless exhibited measurable DENV enhancing activity in FcγR-bearing cells. Vaccine strategies directed to DENV-dIII-targeted neutralizing antibody production remain attractive but will likely require further modifications to induce safe, protective immunity.

Dengue virus neutralization is modulated by IgG antibody subclass and Fcgamma receptor subtype.

Severe dengue virus (DENV) infection is epidemiologically linked to pre-existing anti-DENV antibodies acquired by maternal transfer or primary infection. A possible explanation is that DENV immune complexes evade neutralization by engaging Fcgamma receptors (FcgammaR) on monocytes, natural targets for DENV in humans. Using epitope-matched humanized monoclonal antibodies (mAbs) and stable FcgammaR-transfected CV-1 cells, we found that DENV neutralization by IgG1, IgG3, and IgG4 mAbs was enhanced in high-affinity FcgammaRIA transfectants and diminished in low-affinity FcgammaRIIA transfectants, whereas neutralization by IgG2 mAbs (low-affinity ligands for both FcgammaRs) was diminished equally. In FcgammaR-negative Vero cells, IgG3 mAbs exhibited the strongest neutralizing activity and IgG2, the weakest. Our results demonstrate that DENV neutralization is modulated by the Fc region in an IgG subclass manner, likely through effects on virion and FcgammaR binding. Thus, the IgG antibody subclass profile generated by DENV infection or vaccination may independently influence the magnitude of the neutralizing response.

Primary human endothelial cells support direct but not antibody-dependent enhancement of dengue viral infection.

Microvascular plasma leakage is the hallmark of dengue hemorrhagic fever and dengue shock syndrome. The precise molecular mechanisms leading to microvascular leakage are yet to be determined, but dengue virus (DENV) infection and consequent endothelial cell death has been suggested as its major cause. However, the extent of endothelial cell permissiveness to DENV infection and the magnitude of cell death following DENV infection are controversial. To clarify this issue, we analyzed the kinetics and consequences of DENV infection of human umbilical vein endothelial cells (HUVEC) using a novel molecularly cloned DENV2-16681 virus. Viral replication was detected as early as 24 hr post-infection by RT-PCR and plaque assays. However, merely 2% of HUVEC were DENV antigen-positive even after 96 hr of infection as measured by the FACS indirect immunofluorescence assays. Unlike monocytes/macrophages, HUVEC did not support antibody dependent enhancement of dengue viral infection due to a lack of FcgammaRI and FcgammaRII. Furthermore, DENV infection did not increase HUVEC apoptosis as compared to mock-infected cells. Because in vitro only a small percentage of endothelial cells were productively infected in vitro with no significant apoptosis occurring in either infected or bystander cells, it would be important to re-examine whether direct dengue viral infection of endothelium is the major cause of the extensive vascular leakage observed in patients with dengue hemorrhagic fever and dengue shock syndrome.

An automated Dengue virus microneutralization plaque assay performed in human Fc{gamma} receptor-expressing CV-1 cells.

We describe microneutralization assays that used automated 96-well enzyme-linked immunospot (ELI-SPOT) readout instrumentation to measure human anti-dengue virus (DENV) antibodies in CV-1 cells that were stably transfected to express human FcgammaRIIA (CD32) using conventional Vero cells as a comparator. Classic plaque reduction neutralization test (PRNT) end-point titers were determined by probit analysis. Neutralization titers against DENV measured in CV-1 transfectants were expressed in terms of both conventional 50% to 90% PRNT end-point titers and differential infectivity of antibody-treated virus in control and CD32-expressing CV-1 cells. Significantly reduced PRNT titers and strikingly heightened infectivity (up to 100-fold) of antibody-treated DENV was observed in CV-1 CD32 transfectants compared with that observed in control CV-1 or Vero cells. Because DENVs may preferentially replicate in CD32-expressing monocytes/macrophages and dendritic cells, in vivo, it is possible that CD32 introduced into a conventional DENV neutralization assay might provide results that better correlate with protection.

Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells.

Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcgammaRI, but not its associated gamma-chain, and was not supported by other FcgammaR family members (FcgammaRIIA, FcgammaRIIB, and FcgammaRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcgammaRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer.

Monocytes, but not T or B cells, are the principal target cells for dengue virus (DV) infection among human peripheral blood mononuclear cells.

A better understanding of the pathogenesis of dengue hemorrhagic fever and dengue shock syndrome requires the precise identification of dengue virus (DV) permissive target cells. To examine the relative DV permissiveness among cell subsets, we inoculated unfractionated human peripheral blood mononuclear cells with DV2-16681 in the presence or absence of pooled DV-immune human sera (PHS), and assessed infection with fluorescent dye labeled DV-specific monoclonal antibody and cell surface markers using flow cytometry. We found significantly higher levels of DV antigen staining on DV-infected than mock-infected primary monocytes (3.54 +/- 3.42% vs. 0.50 +/- 0.38%; P = 0.001). The magnitude of infection was markedly enhanced in the presence of highly diluted PHS (10.04 +/- 6.10% vs. 3.54 +/- 3.42%; P = 0.015). Under identical experimental conditions, primary T or B cells were not infected either with or without the addition of PHS (0.06 +/- 0.04% and 0.44 +/- 0.22% for T and B cells, respectively). Furthermore, depletion of CD14+ monocytes prior to DV inoculation abrogated the detection of infected cells, and the addition of monoclonal antibodies to either FcgammaRI (CD64) or FcgammaRII (CD32) led to a 50-70% reduction in antibody-dependent enhancement (ADE) of DV infection. Collectively, these results provide further support to the notion that primary monocytes and FcgammaRs expressed on these cells may be important in the initial steps of immune enhancement observed in some patients with natural DV infection. They also demonstrate that using modern experimental technology, DV infection, and neutralization and enhancement of DV infection can be easily assessed simultaneously in multiple cell types.

Primary human splenic macrophages, but not T or B cells, are the principal target cells for dengue virus infection in vitro.

Understanding the pathogenesis of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) requires the precise identification of dengue virus (DV)-permissive target cells. In a previous study using unfractionated human peripheral blood mononuclear cells, we found that monocytes, but not B or T cells, were the principal DV-permissive cells in the absence of DV-immune pooled human sera (PHS) and the major mediators of antibody-dependent enhancement in the presence of PHS. To further identify DV-permissive target cells in other tissues and organs, we isolated human splenic mononuclear cells (MNCs), inoculated them with DV type 2 (strain 16681) in the presence or absence of PHS, and assessed their infection either directly using flow cytometry and reverse transcription-PCR (RT-PCR) assays or indirectly by plaque assay. We found that in the absence of PHS, a small proportion of splenic macrophages appeared to be positive for DV antigens in comparison to staining controls by the flow cytometric assay (0.77% +/- 1.00% versus 0.18% +/- 0.12%; P = 0.07) and that viral RNA was detectable by the RT-PCR assay in MNCs exposed to DV. Additionally, supernatants from cultures of DV-exposed MNCs contained infectious virions that were readily detectable by plaque assay. The magnitude of infection was significantly enhanced in splenic macrophages in the presence of highly diluted PHS (5.41% +/- 3.53% versus 0.77% +/- 1.00%; P = 0.001). In contrast, primary T and B cells were not infected in either the presence or absence of PHS. These results provide evidence, for the first time, that human primary splenic macrophages, rather than B or T cells, are the principal DV-permissive cells in the spleen and that they may be uniquely important in the initial steps of immune enhancement that leads to DHF/DSS in some DV-infected individuals.

Type- and subcomplex-specific neutralizing antibodies against domain III of dengue virus type 2 envelope protein recognize adjacent epitopes.

Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.

Differential enhancement of dengue virus immune complex infectivity mediated by signaling-competent and signaling-incompetent human Fcgamma RIA (CD64) or FcgammaRIIA (CD32).

Fcgamma receptor (FcgammaR)-mediated entry of infectious dengue virus immune complexes into monocytes/macrophages is hypothesized to be a key event in the pathogenesis of complicated dengue fever. FcgammaRIA (CD64) and FcgammaRIIA (CD32), which predominate on the surface of such dengue virus-permissive cells, were compared for their influence on the infectivity of dengue 2 virus immune complexes formed with human dengue virus antibodies. A signaling immunoreceptor tyrosine-based activation motif (ITAM) incorporated into the accessory gamma-chain subunit that associates with FcgammaRIA and constitutively in FcgammaRIIA is required for phagocytosis mediated by these receptors. To determine whether FcgammaRIA and FcgammaRIIA activation functions are also required for internalization of infectious dengue virus immune complexes, we generated native and signaling-incompetent versions of each receptor by site-directed mutagenesis of ITAM tyrosine residues. Plasmids designed to express these receptors were transfected into COS-7 cells, and dengue virus replication was measured by plaque assay and flow cytometry. We found that both receptors mediated enhanced dengue virus immune complex infectivity but that FcgammaRIIA appeared to do so far more effectively. Abrogation of FcgammaRIA signaling competency, either by expression without gamma-chain or by coexpression with gamma-chain mutants, was associated with significant impairment of phagocytosis and of dengue virus immune complex infectivity. Abrogation of FcgammaRIIA signaling competency was also associated with equally impaired phagocytosis but had no discernible effect on dengue virus immune complex infectivity. These findings point to fundamental differences between FcgammaRIA and FcgammaRIIA with respect to their immune-enhancing capabilities and suggest that different mechanisms of dengue virus immune complex internalization may operate between these FcgammaRs.

Yellow fever virus/dengue-2 virus and yellow fever virus/dengue-4 virus chimeras: biological characterization, immunogenicity, and protection against dengue encephalitis in the mouse model.

Two yellow fever virus (YFV)/dengue virus chimeras which encode the prM and E proteins of either dengue virus serotype 2 (dengue-2 virus) or dengue-4 virus within the genome of the YFV 17D strain (YF5.2iv infectious clone) were constructed and characterized for their properties in cell culture and as experimental vaccines in mice. The prM and E proteins appeared to be properly processed and glycosylated, and in plaque reduction neutralization tests and other assays of antigenic specificity, the E proteins exhibited profiles which resembled those of the homologous dengue virus serotypes. Both chimeric viruses replicated in cell lines of vertebrate and mosquito origin to levels comparable to those of homologous dengue viruses but less efficiently than the YF5.2iv parent. YFV/dengue-4 virus, but not YFV/dengue-2 virus, was neurovirulent for 3-week-old mice by intracerebral inoculation; however, both viruses were attenuated when administered by the intraperitoneal route in mice of that age. Single-dose inoculation of either chimeric virus at a dose of 10(5) PFU by the intraperitoneal route induced detectable levels of neutralizing antibodies against the homologous dengue virus strains. Mice which had been immunized in this manner were fully protected from challenge with homologous neurovirulent dengue viruses by intracerebral inoculation compared to unimmunized mice. Protection was associated with significant increases in geometric mean titers of neutralizing antibody compared to those for unimmunized mice. These data indicate that YFV/dengue virus chimeras elicit antibodies which represent protective memory responses in the mouse model of dengue encephalitis. The levels of neurovirulence and immunogenicity of the chimeric viruses in mice correlate with the degree of adaptation of the dengue virus strain to mice. This study supports ongoing investigations concerning the use of this technology for development of a live attenuated viral vaccine against dengue viruses.