Rhona L. Glickman

Functional and neutralization profile of seven overlapping antigenic sites on the HN glycoprotein of Newcastle disease virus: monoclonal antibodies to some sites prevent viral attachment

We have previously identified five antigenic sites on the hemagglutinin-neuraminidase (HN) glycoprotein of the Australia-Victoria isolate of Newcastle disease virus (Iorio and Bratt, J. Virol. 48, 440-450; Iorio et al., J. Gen. Virol. 67, 1393-1403). Two additional sites (designated 12 and 23) are now described, bringing to a total of seven the number of antigenic sites defined by our panel of neutralizing anti-HN antibodies. Competition antibody binding and additive neutralization assays reveal that each of these newly-identified sites overlaps two previously-defined ones. The seven HN antigenic sites thus form a continuum in the three-dimensional conformation of the molecule. Studies on the inhibition of hemagglutination (HA), neuraminidase (NA) and the attachment of virus to chick cell monolayers have been used to construct a functional profile of each antigenic site. Monoclonal antibodies (mAbs) to three overlapping sites (12, 2 and 23) inhibit HA and NA and prevent viral attachment to chick cell monolayers. These findings are consistent with the domains recognized by these mAbs being close to the NA and receptor-binding sites. MAbs to two other overlapping sites, 14 and 1 (which in turn, overlap site 12), inhibit HA quite effectively, and attachment to a lesser extent. Sites 14 and 1 probably identify a second domain involved in receptor recognition. MAbs to the two remaining sites (3 and 4), though neutralizing, are negative in all three assays, thus recognizing domains not involved in HA or NA or attachment to chick cells.

Effective Induction of Simian Immunodeficiency Virus-Specific Systemic and Mucosal Immune Responses in Primates by Vaccination with Proviral DNA Producing Intact but Noninfectious Virions

ABSTRACT We report a pilot evaluation of a DNA vaccine producing genetically inactivated simian immunodeficiency virus (SIV) particles in primates, with a focus on eliciting mucosal immunity. Our results demonstrate that DNA vaccines can be used to stimulate strong virus-specific mucosal immune responses in primates. The levels of immunoglobulin A (IgA) detected in rectal secretions of macaques that received the DNA vaccine intradermally and at the rectal mucosa were the most striking of all measured immune responses and were higher than usually achieved through natural infection. However, cytotoxic T lymphocyte responses were generally low and sporadically present in different animals. Upon rectal challenge with cloned SIVmac239, resistance to infection was observed, but some animals with high SIV-specific IgA levels in rectal secretions became infected. Our results suggest that high levels of IgA alone are not sufficient to prevent the establishment of chronic infection, although mucosal IgA responses may have a role in reducing the infectivity of the initial viral inoculum.

Induction of Mucosal Homing Virus-Specific CD8+ T Lymphocytes by Attenuated Simian Immunodeficiency Virus

ABSTRACT Induction of virus-specific T-cell responses in mucosal as well as systemic compartments of the immune system is likely to be a critical feature of an effective AIDS vaccine. We investigated whether virus-specific CD8+ lymphocytes induced in rhesus macaques by immunization with attenuated simian immunodeficiency virus (SIV), an approach that is highly effective in eliciting protection against mucosal challenge, express the mucosa-homing receptor α4β7 and traffic to the intestinal mucosa. SIV-specific CD8+ T cells expressing α4β7 were detected in peripheral blood and intestine of macaques infected with attenuated SIV. In contrast, virus-specific T cells in blood of animals immunized cutaneously by a combined DNA-modified vaccinia virus Ankara regimen did not express α4β7. These results demonstrate the selective induction of SIV-specific CD8+ T lymphocytes expressing α4β7 by a vaccine approach that replicates in mucosal tissue and suggest that induction of virus-specific lymphocytes that are able to home to mucosal sites may be an important characteristic of a successful AIDS vaccine.

Genetic variation within a neutralizing domain on the haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus

Previously, a panel of monoclonal antibodies recognizing epitopes in four antigenic sites on the haemagglutinin-neuraminidase (HN) glycoprotein of the Australia-Victoria strain of Newcastle disease virus were used in strain comparisons. Epitopes in three sites were found to be conserved while the epitope recognized by the single antibody to site 3 was not. A new panel of antibodies is described, two of which bind to epitopes in site 3 and six of which bind to a site (site 1,4) that overlaps with sites 1 and 4 as determined by analyses of variants, temperature-sensitive mutants, and strains by assays of neutralization of infectivity and binding in a radioimmunoassay. Neutralization of heterologous strains with the panel of antibodies revealed that both new site 3 epitopes are also highly divergent, while three additional epitopes outside site 3 (those in site 1,4) are highly conserved. The new site 3 antibodies can bind to virions of several heterologous strains without neutralizing infectivity. Thus, of the 10 epitopes we have now examined, all of three in site 3 are specific with respect to neutralization of infectivity for the homologous strain, while all of seven in other sites are conserved in heterologous strains. This suggests that the strain specificity originally described for a single site 3 epitope is, instead, a property of a much more extensive, poorly conserved domain on the HN molecule.

Reducing agent-sensitive dimerization of the hemagglutinin-neuraminidase glycoprotein of newcastle disease virus correlates with the presence of cysteine at residue 123

Viruses within the Newcastle disease virus (NDV) serotype induce a wide array of disease manifestations ranging from an almost apathogenic pattern to the high mortality caused by avirulent or virulent isolates, respectively. A disulfide-linked dimer form of the NDV hemagglutinin-neuraminidase (HN) glycoprotein can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions for only some of these isolates. For others, indeed the majority of those we have studied, no such reducing agent-sensitive dimeric form of HN is demonstrable. Apparently, there is no causal relationship between disulfide-linked dimeric HN and virulence. Using the deduced amino acid sequence of the dimeric HN of isolate AV as a basis for selection of oligonucleotide primers, we sequenced three additional reducing agent-sensitive dimeric HN glycoproteins and eight for which a disulfide-linked dimer has not been identified, using primer extension and dideoxy sequencing. The deduced amino acid sequences reveal a strict correlation between the presence of cysteine at residue 123 and reducing agent-sensitive dimerization of HN.

Inhibition of fusion by neutralizing monoclonal antibodies to the haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus

The majority of neutralizing monoclonal antibodies (MAbs) to the haemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus prevent attachment of the virus to cellular receptors and inhibits virion-induced fusion from without (FFWO) and fusion from within (FFWI) mediated by the virus glycoprotein-laden infected cell surface. For these antibodies, the inhibition of fusion is presumed to be the result of the prevention of HN-mediated bridging of potential fusion partners. MAbs against antigenic sites 3 and 4 neutralize virus infectivity, but by a mechanism other than the prevention of attachment, the exact nature of which remains to be established. Antibodies to both of these sites effectively inhibit virion-induced FFWO, even when the inducing virus is not infectious. This is consistent with the mechanism of neutralization of these MAbs involving the inhibition of an early, post-attachment step in infection. MAbs to site 3 also inhibit FFWI, but those to site 4 do not, even when added at high concentrations. This suggests that the requirement for HN may be different in the two modes of fusion. The epitopes recognized by MAbs to sites 3 and 4 have been delineated by the identification of individual nucleotide substitutions in the HN genes of neutralization escape variants. Some of the deduced amino acid substitutions result in additional N-linked glycosylation sites in HN, which are utilized and presumably account for the escape from neutralization.

Identification of amino acid residues important to the neuraminidase activity of the HN glycoprotein of Newcastle disease virus

Monoclonal antibodies (MAbs) to three overlapping antigenic sites (designated 12, 2, and 23) on the hemagglutinin-neuraminidase glycoprotein (HN) of Newcastle disease virus (NDV) were previously shown to inhibit neuraminidase activity (NA) on neuraminlactose (R. M. Iorio and M. A. Bratt, 1984a, J. Immunol. 133, 2215-2219; R. M. Iorio et al., 1989, Virus Res. 13, 245-262). However, a competitive inhibitor of NA blocks the binding of only MAbs to site 23, suggesting that the domain they recognize may be closely related to the NA site. Antigenic variants selected with site 23 MAbs have single amino acid substitutions at HN residues 192, 193, or 200. Virions of variants, which have a substitution at residue 193 or 200, have alterations in NA which are not attributable to a commensurate change in HN content. A revertant of a temperature-sensitive mutant, which has markedly diminished NA relative to the wild type, has an amino acid substitution at residue 175. A second step revertant having partially restored NA has an additional substitution at residue 192 identical to that in one of the site 23 variants, which, in turn, also makes the revertant resistant to neutralization by site 23 MAbs. Thus, an amino acid substitution at residue 175, 193, or 200 of the HN of NDV can have marked effects on the NA of the protein. The amino acids in the region around residue 175 are highly conserved between the HNs of NDV and other paramyxoviruses, suggesting that this domain is important to the integrity of the NA site in this group of viruses.

Characterization of SIV-specific CD4+ T-helper proliferative responses in macaques immunized with live-attenuated SIV

Abstract: Analysis of immune responses generated by live‐attenuated simian immunodeficiency virus (SIV) strains may provide clues to the mechanisms of protective immunity induced by this approach. We examined SIV‐specific T‐helper responses in macaques immunized with the live‐attenuated SIV strains SIVmac239Δnef and SIVmac239Δ3. Optimization of the concentration and duration of antigenic stimulation resulted in the detection of relatively strong SIV‐specific proliferative responses, with peak stimulation indices of up to 84. SIV‐specific proliferative responses were mediated by CD4 + T cells and were major histocompatibility (MHC) class II restricted. Limiting dilution analysis revealed SIV‐specific T‐helper precursor frequencies of up to 96 per 106 peripheral blood mononuclear cells (PBMC). Intracellular flow‐cytometric analysis demonstrated the production of interleukin (IL)‐2, interferon (IFN)‐γ, RANTES and macrophage inhibitory protein‐1α (MIP‐1α) by T lymphocytes from SIVmac239Δnef‐vaccinated animals following SIV p55 stimulation. Induction of strong SIV‐specific T‐helper responses by live‐attenuated SIV vaccines may play a role in their ability to induce protective immunity.

Temperature-sensitive mutants of Newcastle disease virus altered in HN glycoprotein size, stability, or antigenic maturity

It has been suggested that the 11 group B, C, and BC temperature-sensitive (ts) mutants of Newcastle disease virus (NDV), strain Australia-Victoria (AV-WT), have lesions in the gene for the hemagglutinin/neuraminidase glycoprotein (HN), and that complementation between groups B and C is intracistronic. Virions produced by these mutants even at permissive temperature contain greatly reduced amounts of HN, and the accompanying hemagglutinating and neuraminidase functions. To explore the basis for decreased HN incorporation into virions and the temperature sensitivity of these mutants, infected chick embryo cells were examined for changes in HN characteristics. The HN of two of the mutants was clearly altered in electrophoretic migration rates in both virions and infected cells. The migrational differences were not due to differences in glycosylation because altered migration rates were also observed in the presence of tunicamycin. In all cases, cells infected by these mutants produced as much HN as did AV-WT-infected cells, but the HN of six of these mutants was metabolically unstable. All of the mutants, including those with metabolically stable HN, exhibited greatly restricted ability to convert HN to an antigenically reactive form, indicating an early block in processing. For most of these mutants, the neuraminidase activities of infected cells were somewhat temperature sensitive, but the production of hemadsorbing activities on cell surfaces was not temperature sensitive. In contrast, the hemadsorbing and neuraminidase activities of cells infected by one mutant, BC2, were temperature sensitive, probably a reflection of the previously described extreme thermolability of the HN of this mutant. The relationship between these mutant characteristics, their temperature sensitivity and the virion phenotypes, is discussed. The data presented here confirm the assignment of these 11 group B, C, and BC mutants to defects in HN and begin to separate them into groups with different characteristics.