Antibodies elicited by influenza virus hemagglutinin fail to bind to synthetic peptides representing putative antigenic sites

doi:10.1016/S0161-5890(85)80008-0

Molecular Immunology

Volume 22, Issue 2, 1985, Pages 145-154

https://doi.org/10.1016/S0161-5890(85)80008-0 Get rights and content

Abstract

A number of peptides of the hemagglutinin (HA) of X-31 influenza virus have been synthesised. The amino acid sequences of some of these peptides represent regions of HA which have been postulated [Wiley et al., Nature, Lond. 289, 373–378 (1981)] to form the antigenic sites of this molecule. Animals were immunized with free peptide or peptide conjugated to a carrier and the resulting antisera examined for their capacities to bind to homologous peptide, whole HA, reduced and alkylated HA, and intact virus. Not all peptides examined in this way were immunogenic. Only antibodies raised against the C-terminus of HA₁ peptide displayed binding to virus. This antiserum bound to the intact HA but not to the reduced and alkylated form of the molecule. These results raise questions as to the feasibility of using synthetic peptides of the influenza HA in short linear sequences to elicit neutralising antibody.

References (60)

AltschuhD. et al.
Immunochemical studies of tobacco mosaic virus—V. Localization of four epitopes in the protein subunit by inhibition tests with synthetic peptides and cleavage peptides from three strains
Molec. Immun.
(1983)
BreschkinA.M. et al.
Antigenic determinants of influenza virus hemagglutinin VIII. Topography of the antigenic regions of influenza virus hemagglutinin determined by competitive radio-immunoassay with monoclonal antibodies
Virology
(1981)
GillT.J. et al.
Studies on synthetic polypeptide antigens II. The immunochemical properties of a group of linear synthetic polypeptides
J. biol. Chem.
(1961)
GrandgenettD.P. et al.
Antibodies against a synthetic peptide of the avian retrovirus pp32 protein and the β DNA polymerase subunit
Virology
(1983)
GreenN. et al.
Immunogenic structure of the influenza virus hemagglutinin
Cell
(1982)
HuangR.T.C. et al.
Influenza viruses cause hemolysis and fusion of cells
Virology
(1981)
JacksonD.C.
Some effects of chloramine-T induced radioiodination on the physico-chemical properties of oligomeric proteins
J. immun. Meth.
(1980)
JacksonD.C. et al.
Antigenic determinants of influenza virus hemagglutinin. II. Antigenic reactivity of the isolated N-terminal cyanogen bromide peptide of A/Memphis/72 hemagglutinin heavy chain
Virology
(1979)
JacksonD.C. et al.
Antigenic activity of a synthetic peptide comprising the “loop” region of influenza virus hemagglutinin
Virology
(1982)
JacksonD.C. et al.
Antigenic determinants of influenza virus hemagglutinin. I. Cyanogen bromide peptides derived from A/Memphis/72 hemagglutinin possess antigenic activity
Virology
(1978)

LaverW.G.

Separation of two polypeptide chains from the hemagglutinin subunit of influenza virus

Virology

(1971)

LaverW.G. et al.

Antigenic drift in type A influenza virus: sequence differences in the hemagglutinin of Hong Kong (H3N2) variants selected with monoclonal hybridoma antibodies

Virology

(1979)

RichardsonC.D. et al.

Specific inhibition of paramyxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F₁ or HA₂ viral polypeptides

Virology

(1980)

Shi. et al.

The antigencity of myoglobin-related peptides synthesized on polyacrylamide and polystyrene resin supports

Molec. Immun.

(1984)

TamuraT. et al.

Antibodies against synthetic peptides as a tool for functional analysis of the transforming protein pp60^src

Cell

(1983)

WebsterR.G. et al.

Determination of the number of nonoverlapping antigenic areas on Hong Kong (H3N2) influenza virus hemagglutinin with monoclonal antibodies and the selection of variants with potential epidemiological significance

Virology

(1980)

AbueloJ.G. et al.

Dinitrophenylated bacitracin as an antigen in the guinea pig

J. Immun.

(1965)

ArnonR.

Antibodies to enzymes—a tool in the study of antigenic specificity determinants

Curr. Topics Microbiol. Immun.

(1971)

ArnonR. et al.

Antibodies to a unique region in lysozyme provoked by a synthetic antigen conjugate

AtassiM.Z. et al.

Localization, synthesis and activity of an antigenic site on influenza virus hemagglutinin

BaranyG. et al.

BaronM.H. et al.

Antibodies against a synthetic peptide of the poliovirus replicase protein: reaction with native, virus encoded proteins and inhibition of virus-specific polymerase activities in vitro

J. Virol.

(1982)

BerglundG.

Preparation of antiserum to an antigen of low molecular weight

Nature, Lond.

(1965)

BittleJ.L. et al.

Protection against foot and mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence

Nature, Lond.

(1982)

BoltonA.E. et al.

The labeling of proteins to high specific radioactivity by conjugation to a ¹²⁵I-containing acylating agent. Application to the radio-immunoassay

Biochem. J.

(1973)

BrandC.M. et al.

Crystalline antigen from the influenza virus envelope

Nature New Biol.

(1972)

BrownL.E. et al.

Antigenic determinants of influenza virus hemagglutinin V. Antigenicity of the HA₂ chain

J. Immun.

(1980)

CrumptonM.J.

Protein antigens: the molecular bases of antigenicity and immunogenicity

DietrichF.M.

Immunogenicity of synthetic angiotensin II

Int. Archs Allergy appl. Immun.

(1966)

EminiE.A. et al.

Priming for and induction of anti-poliovirus neutralizing antibodies by synthetic peptides

Nature, Lond.

(1983)

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Citation Excerpt :
The HA1/HA2 joint region is the most broadly conserved, with the HA2 N-terminal 11 amino acid (a.a.) conserved among all influenza A subtypes [11–13]. Several groups have reported generation of antibodies against the HA1/HA2 joint region with the use of branched peptides or peptide-carrier conjugate [11–15]. Yet, attempts to generate antibodies against the more universally conserved N-terminus of the HA2 (the fusion peptide) have not been successful [11–16].
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Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines. In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization. Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different. The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses. In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis. Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma. These data suggest that antibody response to P. aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides.
Liposome-induced conformational changes of an epitopic peptide and its palmitoylated derivative of influenza virus hemagglutinin
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The conformation of synthetic HA_317-329-NH₂representing the major B- and T-cell epitopic region of influenza virus hemagglutinin, its palmitoylated derivative (HA_317-329-Thr(Pal)-NH₂), and the intersubunit peptide (HA_317-341-NH₂) comprising also the fusion peptide, were studied in aqueous buffer and in the presence of neutral and negatively charged liposomes. The free peptide is unordered in aqueous solution, even in the presence of liposomes. However, grafting the palmitic acid or the fusion peptide onto the C-terminus of the peptide enables the hydrophilic HA_317-329to adopt folded (turn) and β-strand structure on the surface of neutral and negatively charged liposomes, respectively. The results emphasize the importance of some kind of anchor for achieving a specific conformation of epitopic peptide HA_317-329-NH₂on the surface of liposomes.
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1996, Molecular Immunology
The interaction between two monoclonal antibodies and their corresponding Fab′ fragments with a synthetic peptide, corresponding to the C-terminal 23 residues of the HA₁ chain of influenza virus hemagglutinin against which they were generated, has been examined using an optical biosensor employing the detection principal of surface plasmon resonance (Pharmacia BIAcore™). The data obtained has been analysed in detail by linear transformation of the primary data and non-linear regression analysis, as well as by analysis of equilibrium binding data. The $21$ antibodies and their Fab′ fragments displayed higher affinity than the corresponding $11$ proteins. The IgGs were found to have equilibrium association constants (K_A) 10–20-fold higher than the corresponding Fab′ fragments. This appears largely to be due to differences in the dissociation rate constant (k_d) and probably reflects increased avidity due to bivalent binding.
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The immunogenicity of a soluble, non-self protein or peptide can be greatly enhanced by injecting this antigen coupled to an antibody specific for class II MHC molecules in the recipient. This adjuvant-independent immunization strategy is known as immunotargeting. We have investigated the ability of a mouse anti-class II MHC antibody to provide the three-dimensional framework for the reconstitution of a heterologous conformational B-cell epitope, specifically the A loop epitope from the influenza virus hemagglutinin (HA). From a panel of three anti-class II MHC immunoglobulin (Ig)-A loop constructs, we found that one of these, an insertion into the FR3 region of the Ig molecule, retained its specificity for mouse I-A^k. Although mouse monoclonal antibodies specific for the A loop region in the HA molecule were unable to react with the Ig-A loop variants, we did find that the heavy chain CDR3 insertion construct was able to elicit an A loop-specific, HA-reactive antibody response when used as an immunogen in rabbits. These results demonstrate the potential for the Ig molecule to function successfully as a structural framework for the reconstitution and presentation of heterologous conformational B-cell epitopes.
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Previously, we reported 12 synthetic T- and B-cell recognition regions representing surface areas of the hemagglutinin (HA) of X31 influenza virus. In the present study, four of these peptides were examined in Balb/c mice for their ability to produce protective immunity against lethal infection with a dose equivalent to 10 LD₅₀ of influenza virus. These peptides corresponded to the following sequences: 23–36 (HA1-1); 138–152 (HA1-3); 183–199 (HA1-6) and 1–11 (HA2-10). Each of the selected peptides, in their free form, evoked anti-peptide antibodies that cross-react with intact X31 virus. Two of the peptides, HA1-1 and HA1-3, also elicited virus-specific delayed type hypersensitivity (DTH) responses. These two peptides, when injected into mice, not only failed to protect the immunized mice against challenge with influenza virus, but in fact caused greater susceptibility to viral infection as compared to control animals that had been injected with saline. In contrast, peptides HA1-6 and HA2-10, which were unable to induce adequate virus-specific DTH responses, conferred 42–46% and 54–73% protection, respectively, compared to the control group that received only saline (P<0.03 to P<0.01).

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^*: This work was supported by a grant from the National Health and Medical Research Council of Australia.

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Antibodies elicited by influenza virus hemagglutinin fail to bind to synthetic peptides representing putative antigenic sites*

Abstract

Molec. Immun.

Virology

J. biol. Chem.

Virology

Cell

Virology

J. immun. Meth.

Virology

Virology

Virology

Virology

Virology

Virology

Molec. Immun.

Cell

Virology

Dinitrophenylated bacitracin as an antigen in the guinea pig

J. Immun.

Antibodies to enzymes—a tool in the study of antigenic specificity determinants

Curr. Topics Microbiol. Immun.

Antibodies to a unique region in lysozyme provoked by a synthetic antigen conjugate

Localization, synthesis and activity of an antigenic site on influenza virus hemagglutinin

Antibodies against a synthetic peptide of the poliovirus replicase protein: reaction with native, virus encoded proteins and inhibition of virus-specific polymerase activities in vitro

J. Virol.

Preparation of antiserum to an antigen of low molecular weight

Nature, Lond.

Protection against foot and mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence

Nature, Lond.

The labeling of proteins to high specific radioactivity by conjugation to a 125I-containing acylating agent. Application to the radio-immunoassay

Biochem. J.

Crystalline antigen from the influenza virus envelope

Nature New Biol.

Antigenic determinants of influenza virus hemagglutinin V. Antigenicity of the HA2 chain

J. Immun.

Protein antigens: the molecular bases of antigenicity and immunogenicity

Immunogenicity of synthetic angiotensin II

Int. Archs Allergy appl. Immun.

Priming for and induction of anti-poliovirus neutralizing antibodies by synthetic peptides

Nature, Lond.

The labeling of proteins to high specific radioactivity by conjugation to a ¹²⁵I-containing acylating agent. Application to the radio-immunoassay

Antigenic determinants of influenza virus hemagglutinin V. Antigenicity of the HA₂ chain