John T. Roehrig

Monoclonal Antibodies That Bind to Domain III of Dengue Virus E Glycoprotein Are the Most Efficient Blockers of Virus Adsorption to Vero Cells

ABSTRACT The specific mechanisms by which antibodies neutralize flavivirus infectivity are not completely understood. To study these mechanisms in more detail, we analyzed the ability of a well-defined set of anti-dengue (DEN) virus E-glycoprotein-specific monoclonal antibodies (MAbs) to block virus adsorption to Vero cells. In contrast to previous studies, the binding sites of these MAbs were localized to one of three structural domains (I, II, and III) in the E glycoprotein. The results indicate that most MAbs that neutralize virus infectivity do so, at least in part, by the blocking of virus adsorption. However, MAbs specific for domain III were the strongest blockers of virus adsorption. These results extend our understanding of the structure-function relationships in the E glycoprotein of DEN virus and provide the first direct evidence that domain III encodes the primary flavivirus receptor-binding motif.

West Nile Virus Recombinant DNA Vaccine Protects Mouse and Horse from Virus Challenge and Expresses In Vitro a Noninfectious Recombinant Antigen That Can Be Used in Enzyme-Linked Immunosorbent Assays

ABSTRACT Introduction of West Nile (WN) virus into the United States in 1999 created major human and animal health concerns. Currently, no human or veterinary vaccine is available to prevent WN viral infection, and mosquito control is the only practical strategy to combat the spread of disease. Starting with a previously designed eukaryotic expression vector, we constructed a recombinant plasmid (pCBWN) that expressed the WN virus prM and E proteins. A single intramuscular injection of pCBWN DNA induced protective immunity, preventing WN virus infection in mice and horses. Recombinant plasmid-transformed COS-1 cells expressed and secreted high levels of WN virus prM and E proteins into the culture medium. The medium was treated with polyethylene glycol to concentrate proteins. The resultant, containing high-titered recombinant WN virus antigen, proved to be an excellent alternative to the more traditional suckling-mouse brain WN virus antigen used in the immunoglobulin M (IgM) antibody-capture and indirect IgG enzyme-linked immunosorbent assays. This recombinant antigen has great potential to become the antigen of choice and will facilitate the standardization of reagents and implementation of WN virus surveillance in the United States and elsewhere.

Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins.

The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 °C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

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.

Guidelines for Plaque-Reduction Neutralization Testing of Human Antibodies to Dengue Viruses

Through the Advisory Committee on Dengue and other Flavivirus Vaccines, the World Health Organization(WHO) has had a long-standing commitment to facilitate and to guide research and development of vaccines for medically important flaviviruses. Recently, the Paediatric Dengue Vaccine Initiative (PDVI) was formed to accelerate the development, testing, and introduction of dengue (DEN)vaccines worldwide, partnering with WHO in this important public health effort. There are now a variety of DEN vaccines in various stages of the developmental pipeline. In an attempt to make interlaboratory information more directly comparable, WHO with the support of PDVI initiated a program to coordinate the procedures used for the plaque-reduction neutralization test (PRNT). ThePRNT is the most common assay used to measure neutralizing antibody. The presence of antibody is believed to be most relevant means of determining protective anti-DEN virus (DENV) immunity. While other neutralizing antibody assays are being considered for use in large-scale vaccine field trials, the PRNT is still considered to be the laboratory standard against which other neutralizing antibody assays should be compared. The need for PRNT coordination has been identified at several consultations between the WHO and PDVI. A more complete version of these guidelines is available on the WHO website: http://www.who.int/immunization/documents/date/en/index.html.

Antigenic structure of flavivirus proteins.

The increased activity of Dengue virus in the tropical regions of the world and the recent movement of West Nile virus from the eastern to the western hemisphere emphasize the fact that vector-borne flaviviruses are medically important emerging infectious diseases. These facts warrant continued efforts to decode all facets of flavivirus immunology. This chapter reviews current understanding of the antigenic fine structure of flaviviral structural and nonstructural (NS) proteins and their involvement in B- an T-cell host responses. The virion structural glycoprotein E elicits both virus-neutralizing antibodies and antiviral Th-cell responses. Consistent with the current hypothesis of the MHC class I pathway of protein processing, immunodominant flaviviral Tc-cell epitopes mainly reside on the NS proteins. To prepare effective and inexpensive subunit vaccines, we will need to continue to better understand these structure-function relationships of flavivirus proteins.

The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein

Abstract To study the role of the precursor to the membrane protein (prM) in flavivirus maturation, we inhibited the proteolytic processing of the Murray Valley encephalitis (MVE) virus prM to membrane protein in infected cells by adding the acidotropic agent ammonium chloride late in the virus replication cycle. Viruses purified from supernatants of ammonium chloride-treated cells contained prM protein and were unable to fuse C6/36 mosquito cells from without. When ammonium chloride was removed from the cells, both the processing of prM and the fusion activity of the purified viruses were partially restored. By using monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of MVE virus, we found that at least three epitopes were less accessible to their corresponding antibodies in the prM-containing MVE virus particles. Amino-terminal sequencing of proteolytic fragments of the E protein which were reactive with sequence-specific peptide antisera or MAb enabled us to estimate the site of the E protein interacting with the prM to be within amino acids 200 to 327. Since prM-containing viruses were up to 400-fold more resistant to a low pH environment, we conclude that the E-prM interaction might be necessary to protect the E protein from irreversible conformational changes caused by maturation into the acidic vesicles of the exocytic pathway.

Use of Immunoglobulin M Cross-Reactions in Differential Diagnosis of Human Flaviviral Encephalitis Infections in the United States

ABSTRACT To define the virus specificity of the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) among the medically important members of the Japanese encephalitis (JE) virus serocomplex of flaviviruses, 103 IgM-positive human serum samples from patients with confirmed West Nile (WN) virus, St. Louis encephalitis (SLE) virus, or JE virus infections were assembled and simultaneously tested against all three viral antigens in a standardized MAC-ELISA. Of the serum samples tested, 96 (93%) showed higher positive-to-negative absorbance ratios (P/Ns) with the infecting virus antigen compared to those obtained with the other two virus antigens. Of the seven specimens with higher P/Ns with heterologous virus antigens, six were from patients with SLE virus infections (the serum samples had higher levels of reactivity with WN virus antigen) and one was from a patient with a JE virus infection (this serum sample also had a higher level of reactivity with WN virus antigen). Not surprisingly, similar virus specificity was observed with WN virus-elicited IgM in cerebrospinal fluid. As shown in previous studies, a subset of these specimens was even less reactive in the MAC-ELISA with dengue virus, a member of a different flavivirus serocomplex. The degree of virus cross-reactivity did not appear to be related to days postonset, at least during the first 40 days of infection. Infections with WN virus could be correctly distinguished from infections with SLE virus on the basis of the observed anti-viral IgM cross-reactivities alone 92% of the time. Infections with SLE virus resulted in antibody that was more cross-reactive, so identification of SLE virus as the infecting agent by use of MAC-ELISA cross-reactivity alone was more problematic.

Epitope-Blocking Enzyme-Linked Immunosorbent Assays for the Detection of Serum Antibodies to West Nile Virus in Multiple Avian Species

ABSTRACT We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WNV or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.1112G potentially discriminated between WNV and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2B2 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.67G, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.

Identification of epitopes on the E glycoprotein of Saint Louis encephalitis virus using monoclonal antibodies.

Twenty-one hybridomas producing monoclonal antibodies specific for the E glycoprotein of St. Louis encephalitis (SLE) virus, strain MSI-7, have been isolated. Serologic reactivities were initially determined by cross-reactivity indirect immunofluorescence assays using 22 strains of SLE virus and 8 other related flaviviruses. Four groups demonstrating type-, subcomplex-, supercomplex-, and group-specific reactivity patterns were identified. Analysis of hemagglutination-inhibition (HI) and virus neutralization (N) subdivided the cross-reactivity groups into eight epitopes (E-1a,b,c,d, E-2, E-3, and E-4a,b). The antibodies could detect strain differences between SLE viruses isolated from various geographic areas. Analysis of the spatial arrangements of these epitopes using competitive binding assays with representative antibodies possessing similar binding avidities, indicated that the protein was a continuum of six overlapping domains.