E. A. Gould

Neutralizing (54K) and non-neutralizing (54K and 48K) monoclonal antibodies against structural and non-structural yellow fever virus proteins confer immunity in mice

The capacity of monoclonal antibodies to protect mice passively against yellow fever (YF) virus infection was investigated. Both neutralizing (54K-specific) and non-neutralizing (54K- and 48K-specific) antibodies protected mice against challenge with the RMP substrain of YF virus. Average survival times of mice inoculated intracerebrally with a standard lethal dose of YF virus differed according to the strain used: thus mice inoculated with the most neurovirulent viruses, FNV and Asibi, survived for 6 X 50 and 7 X 65 days respectively, and those with RMP virus survived for 15 X 75 days. The capacity of antibodies to protect mice passively against virus challenge was directly related to virus neurovirulence. Possible mechanisms and the significance of protection by antibodies against non-structural proteins that do not mediate neutralization, are discussed.

Examination of the Immunological Relationships between Flaviviruses Using Yellow Fever Virus Monoclonal Antibodies

Monoclonal antibodies prepared against vaccine strains of yellow fever (YF) virus were initially characterized by fluorescence microscopy of Vero cells infected with YF virus strain 17D. When similarly tested against representatives of all flavivirus subgroups, the antibodies produced a wide spectrum of reactions ranging from the monospecific to the broadly cross-reactive; at least five antigenic domains in the YF virus envelope glycoprotein were identified. Monoclonal antibodies differentiated between YF virus vaccine strains (17D, 17DD, FNV), wild-type viruses and plaque variants selected from a 17D pool. One isolate from a patient with YF was antigenically similar to the Brazilian vaccine strain 17DD. Several of the antibodies reacting with the YF viral envelope glycoprotein in biological tests identified the 54K envelope glycoprotein; 45K and 26K polypeptides in YF 17D virus-infected cells were also identified by radioimmunoprecipitation and polyacrylamide gel electrophoresis. Neither of these polypeptides was found in uninfected cells. They may represent short-lived precursors of the 54K protein, post-translational cleavage or breakdown products. Other antibodies reacted with a 48K polypeptide in virus-infected cell lysates. This may be the non-structural NV3 protein described for YF virus. Its appearance on the surface of unfixed infected cells, but not on released virions, was demonstrated by fluorescence microscopy.

Monoclonal antibodies to Sindbis virus glycoprotein E1 can neutralize, enhance infectivity, and independently inhibit haemagglutination or haemolysis.

Two monoclonal antibodies raised against Sindbis virus were shown to be specific for the envelope glycoprotein E1 by radioimmunoprecipitation (RIP). They had a number of contrasting biological properties. One of them was capable of neutralizing virus infectivity and inhibiting haemagglutination, while the other had no significant neutralizing or haemagglutination-inhibiting capability, but did inhibit virus-mediated haemolysis. Both monoclonal antibodies could enhance virus infectivity of Fc receptor-bearing macrophage-like cells when present at suitable dilutions.

Use of the biotin-streptavidin interaction to improve flavivirus detection by immunofluorescence and ELISA tests

The specificity and sensitivity of immunofluorescence microscopy and ELISA tests were compared both with and without the use of biotinylated anti-species antibody and streptavidin, conjugated with either fluorescein isothiocyanate or horse radish peroxidase. In the biotin-streptavidin system monoclonal and polyclonal antibodies against yellow fever virus had titres between ten- and fifty-fold higher and background readings, particularly in ELISA tests, were noticeably reduced. Plaque variants of yellow fever virus, selected for their apparent loss of antigenic determinants against some monoclonal antibodies in conventional fluorescence tests, were found to possess the antigenic determinants when tested with the more sensitive system.

Antibody-mediated Early Death in vivo after Infection with Yellow Fever Virus

The phenomenon known as antibody-dependent enhancement (ADE) has been demonstrated in vitro but its significance in viral pathogenesis is uncertain even though it has been associated with dengue shock syndrome. Here we report for the first time the enhancement of virus virulence in mice using monoclonal antibodies (MAbs) prepared against yellow fever (YF) viruses. Our results show that the average survival time of mice was reduced by up to 33% (i.e. 6.7 to 4.5 days) and that ADE is both antibody dose-dependent and antibody- and virus strain-specific. A total of 12 YF viruses and 11 MAbs were examined and of these only three YF viruses (FNV, Asibi and B11) could be enhanced in vivo by only two MAbs (427 and 126). A particular combination of virus and antibody is required for ADE to take place.

Neutralization of Yellow Fever Virus Studied Using Monoclonal and Polyclonal Antibodies

Monoclonal and polyclonal antibodies with known specificity for either the 54K envelope glycoprotein or the 48K non-structural glycoprotein of yellow fever (YF) virus-infected cells were studied in plaque reduction neutralization tests. Viruses employed in the tests comprised wild-type and vaccine strains of YF and a selection of other flaviviruses. Of 17 monoclonal antibodies examined, six of the 54K-specific antibodies neutralized at least one YF preparation. Both vaccine and wild-type YF viruses varied in their susceptibility to neutralization and there were also differences between individual 17D vaccine strains. The monoclonal antibodies produced a range of titres with the different viruses, the most potent, 864, leaving no non-neutralizable fraction. Addition of anti-globulin, complement or other antibodies did not affect the results. YF-neutralize antibodies which bound to other flaviviruses did not necessarily neutralize them; hence, neutralization could be defined as either homotypic, heterotypic or both homotypic and heterotypic. A polyclonal antiserum and a broadly reacting monoclonal antibody produced almost identical neutralization results in tests with wild-type YF viruses. In contrast, the polyclonal antiserum produced higher titres with vaccine strains of YF. In mouse passive protection experiments on the other hand, the monoclonal antibody did not differentiate between these viruses.

Comparison of Neurovirulence of Different Strains of Yellow Fever Virus in Mice

The virulence of different vaccine and wild-type yellow fever (YF) viruses for young adult mice was compared using both intracerebral and intranasal routes of administration. Thirty-five different YF viruses killed mice within 12 days following intracerebral inoculation. In contrast, only seven of those examined killed mice following intranasal administration; these were Asibi virus (YF-AS), the French neurotropic vaccine, two out of three 17DD vaccine substrain viruses (Brazil, Colombia but not Dakar) and three out of six wild-type isolates (YF-B7, YF-B12 and YF-B15). None of eight distinct preparations from the 17D-204 vaccine substrain was virulent by the intranasal route. Thus, strains of YF virus can be distinguished on the basis of their virulence for mice if the intranasal route is used. Evidence of heterogeneity in mouse virulence within the populations was obtained using large and small plaques selected from wild-type stocks of virus. Following intranasal inoculation, a YF-AS small plaque variant was more virulent than either its parent virus or a large plaque variant. On the other hand, a large and a small plaque variant from a nonvirulent wild-type strain could not be distinguished in these tests.

Detection of virus-specific antigen in the nuclei or nucleoli of cells infected with Zika or Langat virus.

Two monoclonal antibodies (MAbs) with molecular specificities for either the viral envelope glycoprotein (MAb 541) or the non-structural NS1 glycoprotein (MAb 109) were derived using West Nile and yellow fever (YF) viruses respectively. Their antigenic reactivity with a large number of flaviviruses was tested by indirect immunofluorescence microscopy. Both produced cytoplasmic fluorescent staining patterns with the homologous virus against which they were raised. Additionally, MAb 541 reacted with two substrains of YF virus whereas MAb 109 reacted with Bussuquara, YF and Ntaya viruses. These reactions were exclusively cytoplasmic. Two unexpected patterns of fluorescent labelling were observed when the antibodies were tested with Zika and Langat viruses. MAb 541 produced fluorescent staining of the nuclei, but not the cytoplasm, of cells infected with Zika virus and MAb 109 labelled only the nucleoli of cells infected with Langat virus. Double-labelling experiments showed that the nuclear fluorescent label was confined to virus-infected cells, and antibody absorption experiments with virus-infected cell packs confirmed the virus specificity of the nuclear antigen. The unexpected presence of virus-specific antigen in the nuclei or nucleoli of Zika or Langat virus-infected cells brings into question the role of the nucleus in flavivirus replication.

Reduction of yellow fever virus mouse neurovirulence by immunization with a bacterially synthesized non-structural protein (NS1) fragment

Part of a yellow fever virus-specified non-structural protein (NS1) was expressed in Escherichia coli as a fusion protein with beta-galactosidase. Immunization of mice with this partially purified NS1-beta-galactosidase fusion protein induced yellow fever virus-specific antibodies and provided some protection against intracerebral challenge with the virus.

Immune Enhancement of Yellow Fever Virus Neurovirulence for Mice: Studies of Mechanisms Involved

Enhancement of yellow fever virus neurovirulence for mice by specific antibody was studied with the French neurotropic vaccine strain. Experimental conditions for enhancement required mice between 14 and 40 days old and intraperitoneal administration of a selected monoclonal antibody 24 h before or up to 72 h after intracerebral virus challenge. Virus infectivity titrations were similar in brains of antibody-treated and untreated mice. Virus recovered from brains of mice with enhanced viral infections was neither qualitatively nor quantitatively different from standard virus. Humoral immune responses in enhanced infections were normal, macrophages did not become infected and viraemia was not significant. Both hydrocortisone treatment and complement depletion with cobra venom resulted in prolongation of mouse survival times but virulence enhancement persisted. Antithymocyte serum had no effect on enhancement although it reduced the humoral immune response. It is proposed that virulence enhancement is due to the combined effects of virus-specific antibody on infected cells, complement-mediated cytolysis and resultant host anti-cellular activity. There is no analogy between mechanisms effecting increased arbovirus growth in vitro in the presence of specific antibody and increased yellow fever virus neurovirulence in vivo after parenteral administration of antibody.