Erythromycin-induced QT prolongation and polymorphic ventricular tachycardia (torsades de pointes): case report and review.

Although uncommon, ventricular arrhythmias associated with erythromycin use have been reported previously, usually in the presence of heart disease and/or situations causing abnormal cardiac electrophysiology (such as bradycardia, hypokalemia, and the administration of other cardioactive drugs). We report a case of QT prolongation and polymorphic ventricular tachycardia (torsades de pointes) that was precipitated by the intravenous administration of erythromycin. In contrast to most other previously described patients, our patient did not demonstrate significant heart disease or other apparent factors contributing to the genesis of the arrhythmia.

Immunogenicity of a purified fragment of 17D yellow fever envelope protein.

Information on the immunogenic properties of purified flavivirus proteins may be useful in the development of recombinant or synthetic peptide vaccines. Using a monoclonal antibody, an attempt was made to purify the envelope (E) protein of 17D yellow fever virus (17D YF) by affinity chromatography. The purified material could not be identified as intact E protein but it did bear antigenic determinants of E as determined by selective reactivity with anti-E monoclonal antibodies. Rabbits immunized with this material produced antibodies that neutralized 17D YF and dengue-2 viruses in comparable titers, indicating that cross-reactive antigenic determinants were preserved. Immunization of mice resulted in protection against intracerebral challenge with 17D YF.

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.

Comparison of antigenic sites of subtype-specific respiratory syncytial virus attachment proteins.

A panel of 19 monoclonal antibodies (MAbs) were used to probe the antigenic relationships between the G (attachment) proteins of A and B respiratory syncytial virus (RSV) subtypes (GA and GB). At least three and two antigenic sites were present on GA and GB, respectively, including a shared neutralizing site. Most of the antibodies had some degree of complement-independent neutralizing capacity, but in common was a large neutralization-resistant fraction of virus (range 13 to 78%). Passive administration of MAbs to the cross-reactive antigenic site reduced pulmonary virus titres of both A and B subtype virus in the cotton rat model. Protection with subtype-specific MAbs, however, did not always correlate with in vitro neutralizing capacity. The cross-reactive antigenic site appears to be stable to denaturation by polyacrylamide gel electrophoresis and is present on the unglycosylated and partially glycosylated forms of GA and GB by Western blot analysis of infected cell lysates.

Immunological differences between the envelope glycoproteins of two strains of human respiratory syncytial virus.

The envelope glycoproteins of two distinct strains of respiratory syncytial virus (RSV) (Long and 18537 strains) were purified by affinity chromatography and characterized by immunological methods. The fusion (F) proteins from the two strains were similar in molecular weight by gel electrophoresis and were very closely related immunologically. Rabbit antisera to either F protein reacted with near equivalent titres with the heterologous F protein by Western blot, enzyme-linked immunoassay (EIA), neutralization and fusion inhibition assays. In contrast to the similarity of the F proteins, the attachment proteins (G) differed significantly. The 18537 G protein had a molecular weight of 78K compared to 84K for the Long G protein. Rabbit antisera to the G proteins clearly reacted preferentially with the homologous protein, although some cross-reactivity was noted by Western blot and EIA. Anti-G serum neutralized the homologous strain of RSV in the presence or absence of complement to much higher titre than the heterologous virus strain. The implications for vaccine development are discussed.

Immunization with glycoprotein subunits of respiratory syncytial virus to protect cotton rats against viral infection.

The cotton rat model of respiratory syncytial virus infection was used to study immunization with viral glycoproteins. Animals immunized with the purified attachment protein (G) or the fusion protein (F) developed complete pulmonary resistance, but only partial nasal resistance, to challenge with respiratory syncytial virus. Antibody produced to the G protein neutralized virus, whereas antibody to the F protein neutralized virus and also inhibited fusion of infected cells. There was no evidence of enhanced pulmonary pathology in any immunized group.

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.

Protection against yellow fever in monkeys by immunization with yellow fever virus nonstructural protein NS1.

Immunization of monkeys with yellow fever virus-specified nonstructural protein NS1 resulted in protection against fatal hepatitis as well as marked reduction in the magnitude of viremia after subcutaneous challenge with yellow fever virus. The results may be relevant to the design of possible subunit or recombinant flavivirus vaccines.

Partial N-terminal amino acid sequences of three nonstructural proteins of two flaviviruses.

Partial N-terminal amino acid sequences for the three largest nonstructural proteins of two flaviviruses, yellow fever virus and St. Louis encephalitis virus, have been obtained. The determined sequences of these proteins exhibit significant amino acid sequence homology, and allow the positioning of these three nonstructural proteins in the polyprotein sequence deduced from the nucleotide sequence of yellow fever virus (C. M. Rice, E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, and J. H. Strauss, 1985, Science 229, 726-733.) The deduced start points support the hypothesis that the N terminus of nonstructural glycoprotein NS1 results from cleavage by signalase, whereas the N termini of NS3 and NS5 result from cleavages following double basic residues that are flanked by amino acids with short side chains.

Analysis of the respiratory syncytial virus fusion protein using monoclonal and polyclonal antibodies.

At least four distinct epitopes are described on the respiratory syncytial virus fusion protein (VP70) using 13 monoclonal antibodies in solid-phase competitive binding studies. Two, and possibly three, fusion-inhibiting epitopes, one non-fusion-inhibiting neutralizing epitope, and one non-neutralizing epitope are described. All but the latter site demonstrated partial overlap, suggesting possible topographical proximity of these epitopes. Polyclonal rabbit sera to VP70, which neutralized virus but did not inhibit fusion of infected cells, blocked the binding of all fusion-inhibiting monoclonal antibodies to VP70 in the solid-phase assay but did not inhibit their effect in vitro. Western blot analysis of these monoclonal antibodies demonstrates that one fusion-inhibiting epitope is localized on the 48K fragment of VP70 and is resistant to denaturation by heat, 2-mercaptoethanol and SDS.

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.

Protection against 17D yellow fever encephalitis in mice by passive transfer of monoclonal antibodies to the nonstructural glycoprotein gp48 and by active immunization with gp48.

The protective capacity of antiviral antibodies has generally been considered to depend on their interactions with structural components of the virion. Here we report protection against lethal 17D yellow fever virus (YF) encephalitis of mice by passive administration of nonneutralizing monoclonal antibodies to a 17D YF-specified nonstructural glycoprotein, gp48, and by active immunization with purified gp48. Among five anti-gp48 monoclonal antibodies tested, two with high titer complement-fixing (CF) activity were protective, whereas three antibodies with little or no CF activity were not. The ability of antibodies to protect correlated with their ability to promote complement-mediated cytolysis (CMC) of 51Cr-labeled 17D YF-infected mouse neuroblastoma (Neuro 2a) cells. Purified gp48, prepared from lysates of 17D YF-infected Vero cells by immunoaffinity chromatography, was shown to bear both YF type-specific and flavivirus group-reactive determinants in a solid phase radioimmunoassay. Immunization of mice with purified gp48 resulted in solid protection in the absence of detectable anti-virion antibody, measured by neutralization and radioimmunoprecipitation assays. The results are consistent with plasma membrane expression of gp48 and susceptibility of 17D YF-infected neural cells to CMC, a possible mechanism of host defense in 17D YF encephalitis. Protection provided by immunization with gp48, which bears a group-reactive determinant and is highly conserved among flaviviruses, may have implications in regard to flavivirus vaccine design.

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.

Analysis of 17D yellow fever virus envelope protein epitopes using monoclonal antibodies.

Sixteen monoclonal antibodies that reacted with the envelope glycoprotein (E) of 17D vaccine strain yellow fever virus (17D YF), including two antibodies produced against dengue 2 virus, were used in a solid phase competitive binding assay (CBA) to define spatial relationships among antigenic determinants on 17D YF E. The antibodies showed YF strain, type or flavivirus group specificities and nine epitopes were identified on 17D YF E by patterns of neutralization, haemagglutination inhibition and competition of antibody binding. Epitopes defined by neutralizing antibodies with strain and type specificities appeared spatially distant but competition between type-specific neutralizing antibodies and some non-neutralizing antibodies against type and group determinants suggested close proximity among epitopes in these regions. Despite competition between some neutralizing and non-neutralizing monoclonal antibodies in CBA, plaque assays revealed no interference with neutralization by non-neutralizing antibody.

Yellow fever monoclonal antibodies: type-specific and cross-reactive determinants identified by immunofluorescence.

Monoclonal antibodies directed against the envelope glycoprotein and the NV3 non-structural viral protein of yellow fever (YF) were tested by the indirect fluorescent antibody technique against a variety of YF virus strains and heterologous flaviviruses. Monoclonal antibodies directed against the envelope glycoprotein exhibited YF strain-specificity, YF type-specificity, broad group cross-reactivity, or limited subgroup reactivity (YF + Banzi or YF + Koutango + Zika + Usutu + Uganda S). Monoclonal antibodies directed against NV3 reacted either with YF + Koutango or with YF + Banzi. These findings generally correlated with the results of biological tests reported previously. Monoclonal antibodies that were type-specific to YF will be useful for the rapid specific identification of YF virus isolates and are available from the Centers for Disease Control on request.

Purification and characterization of GP90, one of the envelope glycoproteins of respiratory syncytial virus.

The large glycoprotein, GP90, of respiratory syncytial virus (RSV) was purified by affinity chromatography using a monoclonal antibody. Hyperimmune rabbit antiserum directed specifically to the purified GP90 neutralized RSV to high titre but did not inhibit fusion of previously infected cells. 125I-labelled GP90 bound rapidly to HEp-2 cells in an unsaturable manner; binding was inhibited by the hyperimmune rabbit antiserum.

Antibody-mediated infection of P388D1 cells with 17D yellow fever virus: effects of chloroquine and cytochalasin B.

Antibody-mediated 17D yellow fever virus infection of a murine macrophage-like cell line, P388D1, was not inhibited by concentrations of cytochalasin B which did inhibit phagocytosis of antibody-coated sheep red blood cells. Chloroquine did not affect antibody-mediated enhancement of viral growth but inhibited viral replication equally whether infection was carried out in the presence or absence of antibody. The results suggest that the route of internalization of virus and subsequent intracellular pathways of replication are similar for both conditions of infection.

Protection from respiratory syncytial virus infection in cotton rats by passive transfer of monoclonal antibodies.

The effects of passive administration of neutralizing monoclonal antibodies (MAB) to respiratory syncytial virus glycoproteins (GP90 and VP70) was evaluated in cotton rats challenged with respiratory syncytial virus. Animals injected with MAB to VP70 had lower mean viral titers in lung tissues than did controls (log10 2.5 versus 5.4 PFU/g; P less than 0.001), as did cotton rats given MAB to GP90 (log10 2.1 versus 5.0 PFU/g; P less than 0.001). Fifty percent of animals given either MAB had no detectable virus in lung tissues, whereas virus was detected in the lungs of all controls. Virus growth in nasal turbinates was decreased but not eliminated in recipients of either MAB.

17D yellow fever virus infection of P388D1 cells mediated by monoclonal antibodies: properties of the macrophage Fc receptor.

Thirteen IgG monoclonal antibodies to the envelope protein of 17D yellow fever virus (17D YF) were produced. All of the antibodies, whether type-specific to 17D YF or flavivirus cross-reactive, mediated antibody-dependent enhancement (ADE) of virus growth in P388D1 cells. There was no consistent relationship between ADE titres and the degree or pattern of neutralizing and/or haemagglutination inhibition activity. Monoclonal antibodies of different isotypes were used to investigate further the properties of P388D1 Fc receptors. The effects of trypsin treatment of P388D1 on ADE were similar to those previously described in experiments measuring direct binding of IgG proteins or rosetting of sheep red blood cells (SRBC) by macrophages, demonstrating sensitivity to digestion by trypsin of the Fc receptor for monomeric IgG2a but not for IgG2b. Aggregated myeloma proteins of IgG2a and IgG2b isotypes competed equally well with either IgG2a or IgG2b monoclonal antibodies to 17D YF in inhibition of ADE. However, selective inhibition by the homologous isotype was observed when rosetting by P388D1 of SRBC coated with IgG2a or IgG2b monoclonal antibodies was examined. These results may help to explain apparent discrepancies previously reported between experiments utilizing direct binding of IgG proteins and those using rosetting of antibody-coated SRBC to examine Fc receptor properties and indicate that immune complexes of virus and antibody resemble aggregated immunoglobulins with respect to macrophage Fc receptor function and differ from antibody-coated SRBCs.

Infection of a macrophage-like cell line, P388D1 with reovirus; effects of immune ascitic fluids and monoclonal antibodies on neutralization and on enhancement of viral growth.

We studied the effect of antibody on the growth of reovirus, serotypes 1 and 3, in P388D1, a continuous mouse macrophage-like cell line. Enhanced growth of virus was observed when cells were infected in the presence of nonneutralizing monoclonal antibodies or subneutralizing concentrations of either immune ascitic fluids or neutralizing monoclonal antibodies. Both enhancement of viral growth and neutralization were accompanied by an antibody-mediated increase in binding of radiolabeled virus to P388D1 cells. Although neutralization was seen only with monoclonal antibodies directed toward the sigma-1 surface protein of the virus, enhancement was observed with two monoclonal antibodies directed toward other surface proteins. Trypsin treatment of P388D1 cells abrogated enhanced growth of virus mediated by a mouse IgG2a antibody; preincubation with P388D1 with human IgG1 but not IgG2 myeloma proteins also abrogated enhancement by immune ascitic fluid or monoclonal antibody. These observations are compatible with known properties of P388D1 Fc receptors and support the role of the Fc receptor in antibody-mediated infection.