The amino acid residues at 102 and 104 in GP5 of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody

Two neutralizing monoclonal antibodies (MAbs) against canine calicivirus (CaCV), which has a distinct antigenicity from feline calicivirus (FCV), were obtained. Both MAbs recognized conformational epitopes on the capsid protein of CaCV and were used to identify these epitopes. Neutralization-resistant variants of CaCV were selected in the presence of individual MAbs in a cell culture. Cross-neutralization tests using the variants indicated that the MAbs recognized functionally independent epitopes on the capsid protein. Recombinantly expressed ORF2 products (capsid precursors) of the variants showed no reactivity to the MAbs used for the selection, suggesting that the resistance was induced by a failing in binding of the MAbs to the variant capsid proteins. Several nucleotide changes resulting in amino acid substitutions in the capsid protein were found by sequence analysis. Reactivities of the MAbs to the revertant ORF2 products produced from each variant ORF2 by site-directed mutagenesis identified a single amino acid substitution in each variant capsid protein responsible for the failure of MAb binding. The amino acid residues related to forming the conformational neutralizing epitopes were located in regions equivalent to the 5′ and 3′ hypervariable regions of the FCV capsid protein, where antigenic sites were demonstrated in previous studies. The recombinant ORF2 products expressed in bacteria failed to induce neutralizing antibody, suggesting that neutralizing antibodies were only generated when properly folded capsid protein was used as an antigen. In CaCV, the conformational epitopes may play a more important role in neutralization than do linear epitopes.

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Influence of N-Linked Glycosylation of Porcine Reproductive and Respiratory Syndrome Virus GP5 on Virus Infectivity, Antigenicity, and Ability To Induce Neutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.80.8.3994-4004.2006 ◽

2006 ◽

Vol 80 (8) ◽

pp. 3994-4004 ◽

Cited By ~ 183

Author(s):

Israrul H. Ansari ◽

Byungjoon Kwon ◽

Fernando A. Osorio ◽

Asit K. Pattnaik

Keyword(s):

Amino Acid ◽

Neutralizing Antibodies ◽

Protective Efficacy ◽

Neutralizing Antibody ◽

Respiratory Syndrome Virus ◽

Virus Infectivity ◽

Neutralization Epitope ◽

Enhanced Sensitivity

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 5 (GP5) is the most abundant envelope glycoprotein and a major inducer of neutralizing antibodies in vivo. Three putative N-linked glycosylation sites (N34, N44, and N51) are located on the GP5 ectodomain, where a major neutralization epitope also exists. To determine which of these putative sites are used for glycosylation and the role of the glycan moieties in the neutralizing antibody response, we generated a panel of GP5 mutants containing amino acid substitutions at these sites. Biochemical studies with expressed wild-type (wt) and mutant proteins revealed that the mature GP5 contains high-mannose-type sugar moieties at all three sites. These mutations were subsequently incorporated into a full-length cDNA clone. Our data demonstrate that mutations involving residue N44 did not result in infectious progeny production, indicating that N44 is the most critical amino acid residue for infectivity. Viruses carrying mutations at N34, N51, and N34/51 grew to lower titers than the wt PRRSV. In serum neutralization assays, the mutant viruses exhibited enhanced sensitivity to neutralization by wt PRRSV-specific antibodies. Furthermore, inoculation of pigs with the mutant viruses induced significantly higher levels of neutralizing antibodies against the mutant as well as the wt PRRSV, suggesting that the loss of glycan residues in the ectodomain of GP5 enhances both the sensitivity of these viruses to in vitro neutralization and the immunogenicity of the nearby neutralization epitope. These results should have great significance for development of PRRSV vaccines of enhanced protective efficacy.

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Localization of the binding site for a factor VIII activity neutralizing antibody to amino acid residues Asp1663-Ser1669.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)81497-7 ◽

1988 ◽

Vol 263 (21) ◽

pp. 10198-10203

Author(s):

M Shima ◽

C A Fulcher ◽

S de Graaf Mahoney ◽

R A Houghten ◽

T S Zimmerman

Keyword(s):

Amino Acid ◽

Factor Viii ◽

Binding Site ◽

Neutralizing Antibody ◽

Amino Acid Residues ◽

Factor Viii Activity

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Identification of an Antigenic Epitope inHelicobacter pylori Urease That Induces Neutralizing Antibody Production

Infection and Immunity ◽

10.1128/iai.69.11.6597-6603.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 6597-6603 ◽

Cited By ~ 41

Author(s):

Kaoru Hirota ◽

Kumiko Nagata ◽

Yoshihiko Norose ◽

Seiji Futagami ◽

Yohko Nakagawa ◽

...

Keyword(s):

Amino Acid ◽

Enzymatic Activity ◽

Neutralizing Antibody ◽

Antigenic Peptides ◽

Amino Acid Residues ◽

Antigenic Epitope ◽

Amino Acid Deletion ◽

Entire Sequence ◽

H Pylori ◽

Insight Into

ABSTRACT We previously reported a mouse monoclonal antibody (MAb), termed L2, specific for Helicobacter pylori urease strongly inhibited its enzymatic activity. Here, to gain insight into how this antibody affects urease activity, the epitope that was recognized by the antibody was determined. By screening a panel of overlapping synthetic peptides covering the entire sequence of the two subunits (UreA and UreB), we identified a stretch of UreB-derived 19 amino acid (aa) residues (UB-33; aa 321 to 339, CHHLDKSIKEDVQFADSRI) that was specifically recognized by the L2 antibody. Further sequential amino acid deletion of the 19-mer peptide from either end allowed us to determine the minimal epitope as 8 amino acid residues (F8; SIKEDVQF) for L2 reactivity. This epitope appears to lie exactly on a short sequence which formed a flap over the active site of urease, suggesting that binding of the L2 antibody sterically inhibits access of urea, the substrate of urease. Finally, immunization of rabbits with either the 19-mer peptide or the 8-mer minimal epitope resulted in generation of antiurease antibodies that were capable of inhibiting the enzymatic activity. Since urease is critical for virulence of H. pylori, antigenic peptides that induce production of antibodies to inhibit its enzymatic activity may potentially be a useful tool as a vaccine for prevention and treatment of H. pyloriinfection.

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