Localization of protective epitopes of the amino terminus of type 5 streptococcal M protein.

We have used a set of overlapping chemically synthesized peptides representing the amino terminus of type 5 streptococcal M protein to localize protective, as opposed to nonprotective and tissue-crossreactive epitopes that might be appropriate for vaccine formulations. Rabbit antisera raised against SM5(1-35) reacted in high titer with pep M5 by ELISA and opsonized type 5 streptococci. None of the antisera crossreacted with human heart tissue or myosin. Antisera against SM5(26-35) reacted with SM5(1-35) and pep M5 but failed to opsonize type 5 streptococci. Particle-phase ELISA indicated that SM5(26-35) antibodies were directed against nonprotective determinants of pep M5 that were not exposed on the surface of viable organisms. Opsonization and ELISA inhibition assays showed that, of the SM5(1-35) antibodies that reacted with M5, all were inhibited by SM5(14-35), whereas none was inhibited by SM5(26-35), suggesting that the protective epitopes of SM5(1-35) resided between residues 14 and 26. This was confirmed by subsequent chemical synthesis of this region; SM5(14-26) totally inhibited SM5(1-35) antibodies that reacted with pep M5 in ELISA, and completely inhibited opsonization of type 5 streptococci by SM5(1-35) antibodies. SM5(14-26) evoked high titers of type-specific, opsonic antibodies against type 5 streptococci, confirming the protective immunogenicity of this 13-residue peptide of type 5 M protein.

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Protective antigenic determinant of streptococcal M protein shared with sarcolemmal membrane protein of human heart.

Journal of Experimental Medicine ◽

10.1084/jem.156.4.1165 ◽

1982 ◽

Vol 156 (4) ◽

pp. 1165-1176 ◽

Cited By ~ 63

Author(s):

J B Dale ◽

E H Beachey

Keyword(s):

Disulfide Bonds ◽

Antigenic Determinant ◽

Human Heart ◽

Heart Tissue ◽

M Protein ◽

Definitive Evidence ◽

Streptococcal M Protein ◽

Complex Protein ◽

The Cross ◽

Human Heart Tissue

We present definitive evidence that at least one protective antigenic determinant on type 5 M protein of group A streptococci evokes antibody that is cross-reactive with human heart tissue. One of nine rabbits immunized with a peptide fragment of type 5 M protein (pep M5) produced antibody that cross-reacted by immunofluorescence with sarcolemmal membranes of human heart. The cross-reactive antibody could be removed by absorbing the antiserum with sarcolemmal membranes, types 5 and 19 streptococci, or their pepsin-extracted M proteins, but with no other serotypes tested. Although each of the pep M5 immune sera was opsonic for type 5 streptococci, only the heart-reactive antiserum opsonized type 19 streptococci. The opsonization of type 19 streptococci was abolished by absorbing the antiserum with sarcolemmal membranes isolated from human heart tissue. Purified heart-reactive antibodies eluted from sarcolemmal membranes opsonized both types 5 and 19 streptococci, indicating that the heart cross-reactive determinant of type 5 M protein is cross-protective. The cross-reactive antigen was purified by affinity chromatography from detergent extracts of sarcolemmal membranes and determined to be a complex protein composed of four subunits apparently linked by disulfide bonds.

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Radioimmunoassay of aspartate aminotransferase isoenzymes in human serum.

Clinical Chemistry ◽

10.1093/clinchem/30.8.1361 ◽

1984 ◽

Vol 30 (8) ◽

pp. 1361-1365 ◽

Cited By ~ 4

Author(s):

F Y Leung ◽

A E Niblock ◽

A R Henderson

Keyword(s):

Human Serum ◽

Column Chromatography ◽

Aspartate Aminotransferase ◽

Human Heart ◽

Solid Phase ◽

High Titer ◽

Heart Tissue ◽

Cross Reactivity ◽

Specific Radioimmunoassay ◽

Human Heart Tissue

Abstract We describe the development of a sensitive, specific radioimmunoassay for the cytoplasmic and mitochondrial isoenzymes of human aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase; EC 2.6.1.1). Isoenzymes from human heart tissue were purified to homogeneity and used to raise high-titer antisera in rabbits. We partly purified the antisera by selective column chromatography. The Bolton-Hunter reagent was used to radioiodinate the isoenzymes. The assay requires 100 microL of serum, includes a solid-phase second-antibody separation, and can be completed in less than 3 h. There was no cross reactivity between the two isoenzymes. As little as 5 micrograms (50 pmol) of each aspartate aminotransferase can be measured per liter of serum.

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A Lipid Core Peptide Construct Containing a Conserved Region Determinant of the Group A Streptococcal M Protein Elicits Heterologous Opsonic Antibodies

Infection and Immunity ◽

10.1128/iai.70.5.2734-2738.2002 ◽

2002 ◽

Vol 70 (5) ◽

pp. 2734-2738 ◽

Cited By ~ 41

Author(s):

Colleen Olive ◽

Michael R. Batzloff ◽

Anikó Horváth ◽

Allan Wong ◽

Timothy Clair ◽

...

Keyword(s):

Immune Responses ◽

High Titer ◽

Heart Tissue ◽

M Protein ◽

Lipid Core ◽

Streptococcal M Protein ◽

Parenteral Immunization ◽

Group A ◽

Core Peptide ◽

Conserved Region

ABSTRACT The study reported here investigated the immunogenicity and protective potential of a lipid core peptide (LCP) construct containing a conserved region determinant of M protein, defined as peptide J8. Parenteral immunization of mice with LCP-J8 led to the induction of high-titer serum immunoglobulin G J8-specific antibodies when the construct was coadministered with complete Freund's adjuvant (CFA) or administered alone. LCP-J8 in CFA had significantly enhanced immunogenicity compared with the monomeric peptide J8 given in CFA. Moreover, LCP-J8/CFA and LCP-J8 antisera opsonized four different group A streptococcal (GAS) strains, and the antisera did not cross-react with human heart tissue proteins. These data indicate the potential of an LCP-based M protein conserved region GAS vaccine in the induction of broadly protective immune responses in the absence of a conventional adjuvant.

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Benefit of microwave-assisted acid hydrolysis of proteins for mass spectrometric profiling of the human heart tissue proteome

Rapid Communications in Mass Spectrometry ◽

10.1002/rcm.3125 ◽

2007 ◽

Vol 21 (16) ◽

pp. 2779-2783 ◽

Cited By ~ 5

Author(s):

Mulu Gebremedhin ◽

Hongying Zhong ◽

Shaohua Wang ◽

Michael Weinfeld ◽

Liang Li

Keyword(s):

Acid Hydrolysis ◽

Human Heart ◽

Heart Tissue ◽

Mass Spectrometric ◽

Microwave Assisted ◽

Hydrolysis Of ◽

Tissue Proteome ◽

Human Heart Tissue

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IMMUNOLOGIC RELATION OF STREPTOCOCCAL AND TISSUE ANTIGENS

Journal of Experimental Medicine ◽

10.1084/jem.119.4.651 ◽

1964 ◽

Vol 119 (4) ◽

pp. 651-666 ◽

Cited By ~ 109

Author(s):

Melvin H. Kaplan ◽

Kathryn H. Svec

Keyword(s):

Human Heart ◽

Streptococcal Infection ◽

Heart Tissue ◽

Antigenic Determinants ◽

Heat Inactivation ◽

Prolonged Storage ◽

Acute Glomerulonephritis ◽

Streptococcal Antigen ◽

Reactive Antibody ◽

Human Heart Tissue

Sera from patients with recent streptococcal infection or non-suppurative sequelae exhibit with variable frequency a precipitin reaction in agar gel with a partially purified streptococcal antigen which has been shown previously to be immunologically related to human heart tissue. This precipitin could be absorbed from sera with normal human heart tissue homogenates but not with homogenates of other organs. Demonstration of this cross-reaction by heart absorption was found dependent both upon the serologic properties of individual sera and the nature or state of purification of the streptococcal product employed as test antigen. Evidence was obtained of a close association of heart-related and non-heart-related antigenic determinants in partially purified preparations of the streptococcal antigen by both gel diffusion and immunoelectrophoresis. On immunoelectrophoretic analysis, cross-reactive antigen exhibited a more rapid mobility toward the anode than M protein. It was destroyed by digestion with trypsin, pepsin, and chymotrypsin. Based on specific absorption tests with a Type 5 and Type 19 strain, the antigen was localized to cell walls and to a lesser extent to cell membranes of these strains. Precipitating activity related to cross-reactive antibody was localized to the immunoglobulin zone in immunoelectrophoresis. Reactive sera showed diminution or loss of serological activity following heat inactivation at 56°C or after prolonged storage at 4°C. Sera containing cross-reactive precipitating antibody exhibited an immunofluorescent reaction with sarcolemma of cardiac myofibers, which was inhibited by streptococcal cross-reactive antigen. By this inhibition test, the immunofluorescent reaction related to cross-reactive antibody could be distinguished from that due to other heart-reactive factors. Antibody to streptococcal cross-reactive antigen defined by precipitation-absorption tests was observed in 24 per cent of patients with recent history of uncomplicated streptococcal infection and in the majority of patients with acute rheumatic fever, rheumatic heart disease, or acute glomerulonephritis. It was observed rarely in patients with non-streptococcal related disease. These data provide evidence that induction of cross-reactive autoantibody to heart in certain individuals is associated with streptococcal infection.

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