Jacob J. Schlesinger

Type- and Subcomplex-Specific Neutralizing Antibodies against Domain III of Dengue Virus Type 2 Envelope Protein Recognize Adjacent Epitopes

ABSTRACT 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.

Protection of Mice Against Dengue 2 Virus Encephalitis by Immunization with the Dengue 2 Virus Non-structural Glycoprotein NS1

Immunization of mice with the dengue 2 virus (DEN 2)-specified non-structural protein NS1 provided significant protection against intracerebral challenge with the virus in the absence of detectable neutralizing or other anti-virion antibody. NS1, purified from lysates of infected Vero cells by immunoaffinity chromatography, expressed an antigenic site(s) common to each of the four DEN serotypes, and hyperimmunization of rabbits with NS1 stimulated production of complement-fixing (CF) antibody with broad DEN serotype specificity. However, cross-protection was not observed: mice immunized with DEN 2 NS1 developed little or no heterologous CF antibody and were not protected against challenge with neurovirulent DEN 1. Induction of a protective immune response by NS1 suggests that it be considered for incorporation into possible synthetic or recombinant DNA DEN vaccines.

Synergistic Interactions of Anti-NS1 Monoclonal Antibodies Protect Passively Immunized Mice from Lethal Challenge with Dengue 2 Virus

Non-neutralizing, serotype-specific anti-NS1 monoclonal antibodies partially protected passively immunized mice from lethal dengue 2 virus intracerebral challenge. There was no apparent correlation between complement-fixing activity and protective capacity among individual anti-NS1 monoclonal antibodies. Immunization with specific combinations of non-protective or partially protective antibodies resulted in prolonged survival or reduced mortality. Solid protection, equal to that achieved after immunization with neutralizing polyclonal antibody, was achieved only with an antibody pair which individually fixed complement to high titre with homologous virus. Some groups of mice had increased morbidity after immunization with combinations of protective monoclonal antibodies that bind to overlapping epitopes. These results may affect the design of recombinant dengue vaccines which may require the inclusion of serotype-specific antigenic domains.

Purification and Characterization of the Respiratory Syncytial Virus Fusion Protein

The fusion protein of respiratory syncytial virus was purified by affinity chromatography using a monoclonal antibody. Under various conditions the protein was recovered as a 145K dimer or a 70K monomer. The 70K monomer was composed of disulphide-linked fragments of 48K and 23K. Polyclonal rabbit serum produced to the dimerized fusion protein neutralized virus but did not inhibit fusion, while rabbit serum to the 2-mercaptoethanol-treated dimerized protein neutralized virus and inhibited fusion of infected cells. Only the latter serum strongly recognized the 23K fragment when studied by Western blot analysis.

Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein.

Sixteen overlapping fragments of the dengue-2 virus envelope (E) protein, expressed as trpE-E fusion products in Escherichia coli, were used to map the epitopes defined by a panel of 20 monoclonal antibodies (MAbs) by immunoblotting. Using this technique, the amino acid sequence of six antigenic domains on the E protein was characterized. Nonneutralizing MAbs were found to define either linear-specific, subcomplex-specific (amino acids 22-58), and complex-specific (amino acids 304-332) epitopes or a subcomplex conformational-dependent epitope requiring the presence of two closely linked amino acid sequences from the E protein, 60-97 and 298-397. Neutralizing MAbs, however, defined either group-reactive epitopes present on two overlapping domains (amino acids 60-135; amino acids 60-205) or type-, subcomplex-, complex-, subgroup-, and group-specific determinants (amino acids 298-397). These neutralizing epitopes were all found to be dependent upon disulfide bridges. Our results suggest that the maintenance of a topographical arrangement of discontinuous antigenic domains in the flavivirus E-protein is necessary to induce neutralizing and protective antibodies.

Monoclonal antibodies distinguish between wild and vaccine strains of yellow fever virus by neutralization, hemagglutination inhibition, and immune precipitation of the virus envelope protein

Nineteen monoclonal antibodies were produced to the 17D strain of yellow fever virus (17D YF). Virus-specific structural and nonstructural proteins were identified for 17D YF and for the parent wild Asibi YF by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fourteen of the monoclonal antibodies were directed against the envelope glycoprotein, E, and five against the nonstructural protein gp 48. The E protein of 17D YF was resolved as a double complex whereas the E protein of Asibi YF appeared as a single band of slightly lower molecular weight. The only IgM anti-E antibody obtained precipitated and neutralized 17D YF specifically with no activity against Asibi YF. This antibody also distinguished clearly by neutralization (N) between the 17D-204 derived vaccine strain to which the animal had been immunized and 17D YF strains of different origin. All 13 IgG anti-E monoclonal antibodies had hemagglutination-inhibition (HI) activity to 17D YF and all but one neutralized Asibi YF; however, only 3 of the 13 neutralized 17D YF. Four anti-E antibodies cross-reacted with other flaviviruses by HI or HI and N. Three of the five anti-gp 48 antibodies had complement-fixation (CF) titers against 17D YF and Asibi YF but none had N or HI activity.

Differential Enhancement of Dengue Virus Immune Complex Infectivity Mediated by Signaling-Competent and Signaling-Incompetent Human FcγRIA (CD64) or FcγRIIA (CD32)

ABSTRACT Fcγ receptor (FcγR)-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. FcγRIA (CD64) and FcγRIIA (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 γ-chain subunit that associates with FcγRIA and constitutively in FcγRIIA is required for phagocytosis mediated by these receptors. To determine whether FcγRIA and FcγRIIA 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 FcγRIIA appeared to do so far more effectively. Abrogation of FcγRIA signaling competency, either by expression without γ-chain or by coexpression with γ-chain mutants, was associated with significant impairment of phagocytosis and of dengue virus immune complex infectivity. Abrogation of FcγRIIA 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 FcγRIA and FcγRIIA with respect to their immune-enhancing capabilities and suggest that different mechanisms of dengue virus immune complex internalization may operate between these FcγRs.

Lethal 17D yellow fever encephalitis in mice. I. Passive protection by monoclonal antibodies to the envelope proteins of 17D yellow fever and dengue 2 viruses.

Monoclonal antibodies to the envelope proteins (E) of the 17D vaccine strain of yellow fever virus (17D YF) and to dengue 2 virus were examined for their ability to confer passive protection against lethal 17D YF encephalitis in mice. All 13 IgG anti-17D YF antibodies, regardless of neutralizing capacity, conferred solid protection when given in a relatively high dose prior to intracerebral inoculation of virus. Three antibodies with high in vitro neutralizing titres were all protective at a low dose as were several non-neutralizing antibodies. One flavivirus group-reactive antibody to dengue 2 virus conferred similar protection at low dose. Protection was also observed when antibodies were given several days after virus inoculation when peak infectious virus titres and histopathological evidence of infection were present in brains. The ability of a non-neutralizing antibody to protect could not be attributed to complement-dependent lysis of virus-infected cells and did not correlate with avidity or with proximity of its binding site to a critical neutralizing epitope of the E protein. Some antibodies, characterized as non-neutralizing by plaque reduction assay on Vero cells, inhibited the growth of virus in a mouse neuroblastoma cell line, suggesting one possible mechanism of protection. These results may be relevant to the design of prospective flavivirus vaccines and support the possibility of conferring broadened protection among flaviviruses by stimulating the antibody response to appropriate epitopes of the E protein.

Cell surface expression of yellow fever virus non-structural glycoprotein NS1: consequences of interaction with antibody.

Among antibodies to flaviviral proteins only those directed at the virion envelope protein (E) or the non-structural glycoprotein NS1 are known to confer protection. To investigate the possible role of complement-mediated cytolysis (CMC) in protection we measured the capacity of anti-NS1, or E monospecific serum or monoclonal antibodies to bind to yellow fever virus (YFV)-infected cells and of anti-NS1 or E serum to sensitize them to CMC. Although both anti-NS1 and anti-E antibody bound to YFV-infected cells, CMC was observed only with anti-NS1 antibody. Greater binding by anti-NS1 antibody suggested the presence of larger amounts of NS1 than E associated with the cell membrane. Using the cell membrane-impermeable, cross-linking reagent BS3, cell surface NS1, but not E, was detected as a homopolymer, a form in which bound antibody might be expected to activate complement more efficiently. Peak titres of progeny virus were reduced 10- to 100-fold when infected cells were treated with complement-fixing, anti-NS1 monoclonal antibody or monospecific, anti-NS1 rabbit serum and complement. Taken together these results are consistent with the hypothesis that CMC subserved by anti-NS1 antibody provides an alternative to direct neutralization of virus in the protective immune response to flaviviral infection.

Primary Human Splenic Macrophages, but Not T or B Cells, Are the Principal Target Cells for Dengue Virus Infection In Vitro

ABSTRACT 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.