Éva Rajnavölgyi

Evaluation of monoclonal antibodies having specificity for human IgG sub-classes: results of an IUIS/WHO collaborative study.

Seventy-four monoclonal antibodies (McAb) of putative specificity for human IgG (11), the IgG sub-classes (59) or Gm allotypes (4) have been evaluated for reactivity and specificity in eight laboratories employing different assay techniques or protocols. For the IgG, IgG3, IgG4, G1m(f) and G3m(u) specificities McAb have been produced that can be satisfactorily applied in most methodologies employed and have potential as reference reagents. The IgG1 and particularly IgG2 specificities proved problematical with all McAb evaluated demonstrating apparent assay restriction and whilst performing well in some assays proved to be poor or inactive reagents in others. However, the study identifies McAb individually suited to application within most commonly employed methodologies. Epitope display is the probable variability rather than capricious behaviour by the McAb. IgG1 and IgG2 were the least immunogenic of the sub-class proteins and there is evidence that epitope display is influenced by the physical and chemical procedures used to immobilize or fix antigen - a common requirement in the assay systems studied.

A hemagglutinin-based multipeptide construct elicits enhanced protective immune response in mice against influenza A virus infection

Multipeptide constructs, comprising adjacent sequences of the 317-341 intersubunit region of immature influenza A hemagglutinin (H1N1), were designed and the functional properties of these branched peptides were compared to that of the corresponding linear peptides. In vivo studies revealed that the immunogenicity of the peptides was dependent on the presence of the hydrophobic fusion peptide (comprised in FP3), encompassing the N-terminal 1-13 sequence of the HA2 subunit. Antibody and T cell recognition, however, was directed against the 317-329 HA1 sequence, comprised in the P4 peptide. Multiple copies of P4, covalently linked by branched lysine residues, significantly enhanced the efficiency of antibody binding and the capacity of peptides to elicit B- and T-cell responses. A fraction of peptide induced antibodies reacted with immature or with proteolitically cleaved hemagglutinin (HA) molecules pretreated at low pH. Immunization with a multipeptide construct, (P4)4-FP3, not only resulted in elevated antibody and T cell responses but conferred enhanced protection against lethal A/PR/8/34 (H1N1) infection as compared to its subunit peptides. The beneficial functional properties of this artificial peptide antigen may be acquired by multiple properties including: (i) stabilized peptide conformation which promotes strong, polyvalent binding to both antibodies and MHC class II molecules; (ii) appropriate P4 conformation for antibody recognition stabilized by the covalently coupled fusion peptide, resulting in the production of virus cross reactive antibodies which inhibit the fusion activity of the virus; (iii) activation of peptide specific B cells which potentiate antigen presentation and peptide specific T cell responses; and (iv) generation of helper T cells which secrete lymphokines active in the resolution of infection.

Interaction of C3 and C3b with immunoglobulin G

Human C3b as well as native C3 were found to bind to solid phase human and rabbit IgG. Haemolytically active C3 had significantly higher binding capacity to IgG than the C3b fragment. Inhibition experiments proved that C3 and C3b have common binding sites on the Fab and Fc part of the IgG molecule but the character of these binding sites was different. As a functional consequence of C3-IgG interaction, C3 binding was found to inhibit the specific precipitation of an IgG antibody preparation.

Targeting of influenza epitopes to murine CR1/CR2 using single-chain antibodies

Single-chain variable fragment (scFv) antibodies are genetically engineered molecules comprising the variable regions responsible for specific binding. scFv that recognize certain surface molecules on professional antigen presenting cells could therefore be suitable for targeting Ag to these cells. We have produced an scFv that recognizes murine complement receptors 1 and 2 (CR1/CR2) and genetically fused it with different numbers of influenza hemagglutinin peptides which contain both B and T cell epitopes. The CR1/CR2 specific hybridoma 7G6 was used for RT-PCR to obtain the variable regions, which were then combined to create an scFv fragment. The influenza hemagglutinin intersubunit peptide HA317-41 (IP) was engineered to the N terminus of the scFv in one, two or three copies. The so obtained IP(1-3)7G6scFv still bound the complement receptors; the peptides in the construct were recognized by the peptide specific monoclonal IP2-11-1 on Western blots and ELISAs. The CR1/CR2 positive B lymphomas A20 and 2PK3 presented the peptide to an I-Ed restricted IP specific T cell hybridoma more efficiently when incubated with the IP(1)7G6 constructs as compared to the free peptide. The results suggest that scFv could work as targeting devices in subunit vaccines.

T cell recognition of the posttranslationally cleaved intersubunit region of influenza virus hemagglutinin

The influenza virus hemagglutinin is synthesized as a single polypeptide chain, but upon maturation it will posttranslationally be modified by a host cell related trypsin-like enzyme. The enzymatic cleavage attacks the so-called intersubunit region of the molecule giving rise to covalently linked HA1 and HA2 subunits. An I-Ed-restricted T cell epitope was identified in the highly conserved intact intersubunit region of the influenza virus hemagglutinin. T cell recognition of a 25-mer synthetic peptide comprising the intact intersubunit region does not require further processing and the elimination of the intervening Arg residue coupling the fusion peptide to the C-terminal segment of HA1 does not abolish the T cell activating capacity. The fine specificity pattern of a T cell hybridoma similar to that of the polyclonal T cell response demonstrates that a single T cell receptor is able to recognize peptides of different sizes representing not only the uncleaved but also the cleaved form of this hemagglutinin region. Based on specificity studies the epitope was localized to the C-terminal 11 amino acids of the HA1 subunit. The cross-reactivity of peptide-primed T cells with influenza virus infected antigen-presenting cells shows that fragments comprising the identified epitope of the intersubunit region can be generated as a result of natural processing of the hemagglutinin molecule. As antigen-presenting cells are lacking the enzyme which is responsible for the posttranslational modification of newly synthesized hemagglutinin molecules, the role of immature viral proteins in immune recognition is discussed.

Anti-cholesterol antibodies (ACHA) in patients with different atherosclerotic vascular diseases and healthy individuals. Characterization of human ACHA

In animal experiments the protective role of anti-cholesterol antibodies (ACHA) in the development of atherosclerosis has been demonstrated. Despite the fact that ACHA are present in the serum of healthy humans, no data on the occurrence of these antibodies in human diseases are available. We determined serum concentrations of IgG type ACHA by an enzyme immunosorbent assay in 600 patients with atherosclerotic vascular diseases (86 patients with peripheral occlusive atherosclerosis, 146 patients with cerebrovascular diseases, 341 patients with severe coronary heart disease (CHD) who received aorto-coronary by-pass, 27 patients with myocardial infarction who did not undergo by-pass operation), in 57 patient controls (complaints of CHD, without coronarographic alterations) and in 218 healthy individuals. ACHA were present in the sera of all persons tested. No serum cofactor is needed for the binding of human ACHA to solid phase cholesterol, binding can be inhibited dose-dependently by LDL and even more strongly with LDL/VLDL preparations purified from human serum. ACHA levels were found to be considerably lower in patients with peripheral occlusive atherosclerosis and cerebrovascular diseases compared with the levels in healthy individuals. By contrast, the ACHA levels of patients with CHD were considerably higher. No differences in the IgG subclass distribution and binding efficiency of ACHA in the sera of CHD patients and controls were found. Thus, our present findings indicate that both low and high ACHA production may be associated with different atherosclerotic vascular diseases.

The Influence of Branched Polypeptide Carriers on the Immunogenicity of Predicted Epitopes of HSV-1 Glycoprotein D

To investigate the role of synthetic polypeptide carriers in inducing an epitope‐specific immune response relevant for vaccine design, peptides comprising two distinct regions of herpes simplex virus type I glycoprotein D (1–23 and 273–284) have been conjugated to the branched polypeptides with polylysine backbone, poly[L‐Lys‐(DL‐Alam)] (AK), or poly[L‐Lys‐(LeUi‐DL‐Alam)] (LAK) and to keyhole limpet haemocyanin (KLH). The magnitude, fine specificity and isotype distribution of the conjugate‐, peptide and carrier‐specific antibody responses were characterized in immunized BALB/c and CBA mice. Conjugates containing the polypeptide carrier AK were the most effective in inducing HSV gD‐peptide specific antibody responses while KLH peptide conjugates resulted in conjugate‐specific antibody responses without measurable peptide specificity. The efficacy of AK‐peptide conjugates was verified by the dominant appearance of peptide‐specific antibodies belonging to functionally efficient IgG isotopes, accompanied by low levels of carrier specific antibody responses. Preimmunization of BALB/ or CBA mice with AK conjugates comprising the 1–23 or 276–284 HSV peptides resulted in prolonged survival of animals infected with a lethal dose of infectious HSV‐1. The potency of these conjugates in eliciting a protective immune response shows a close correlation with the relative levels of conjugate‐induced virus specific antibodies and the neutralizing activity of sera as measured in preimmunized survivors.

The Intersubunit Region of the Influenza Virus Haemagglutinin is Recognized by Antibodies During Infection

The influenza virus haemagglutinin has an important role in the infectious cycle of the virus and carries multiple B and T cell epitopes. It is synthesized as a single polypeptide chain but viral infectivity depends on its post‐translational enzymatic cleavage. The cleavage site of a trypsin‐like enzyme responsible for this modification is found in the most conserved intersubunit region of the molecule. In this study the role of this region in antibody recognition was investigated. Synthetic peptides comprising the intact and cleaved forms of the intersubunit segment were used to examine the specificity of virus‐ or peptide‐ induced antibodies. The immune response elicited by viral infection resulted in the appearance of antibodies capable of neutralizing the virus without interfering with its binding to the receptor. A monoclonal antibody (MoAb) of such functional properties was shown to recognize the intact intersubunit region both in the uncleaved haemagglutinin molecule and in a 25‐mer synthetic peptide comprising the intact intersubunit region. Specificity and functional studies revealed the conformation dependent recognition of the C‐terminal segment of the haemagglutinin 1 subunit by this MoAb. The binding of the antibody was shown to inhibit the trypsin‐mediated cleavage of the haemagglutinin molecule and the membrane fusion event. The enzymatic cleavage of the haemagglutinin was demonstrated to abolish antibody recognition of the infective virus suggesting an escape mechanism mediated by the functional destruction of this highly conserved region. The synthetic peptide corresponding to the intact intersubunit region is characterized by an ordered structure and is able to elicit an antibody response in BALB/c mice while its subfragments are nonimmunogenic. Furthermore, this peptide elicited a protective immune response demonstrated by in vivo experiments.

Synthetic peptides in the search for T- and B-cell epitopes

In this article, Eva Rajnavölgyi describes two aspects of the rigorous application of organic chemistry to the task of synthesizing peptides that induce immune responses. First, the development of viral peptides that activate T and B cells without the need for carrier molecules and secondly, the production of a new generation of immunologically inert carrier molecules.

Isotype distribution and fine specificity of the antibody response of inbred mouse strains to four compounds belonging to a new group of synthetic branched polypeptides

A new group of synthetic branched polypeptides was developed to initiate a systematic study of the relationships between the chemical structure (charge, size, primary structure, configuration and conformation), the carrier potential and the antigenic properties of these biodegradable and biocompatible macromolecules. This model system has two main advantages over the previously used ones: (i) the side chains grafted to the poly(L-lysine) backbone are composed of about three DL-Ala and a single chain-terminating amino acid with different absolute configuration and/or identity, and (ii) the conformation of these polypeptides is characterized in solution. The size, charge and inside area of the four molecules selected for this study were identical; however, the identity, the absolute configuration of the chain-terminating amino acids (D-Leu, Leu, Phe or D-Phe) and, in consequence, the conformation of the macromolecules were different. The qualitative and quantitative features of the antibody response induced by the four polypeptides were characterized in inbred mouse strains by IgM and IgG type antibody levels, as well as by isotype distribution and fine specificity of antibodies produced during the primary and memory response. The intensity of the memory response and the characteristics of subclass distribution were dependent on the conformation of the branched polypeptides. These molecules carry at least two types of antigenic determinants. One is ordered to the tetrapeptide side chain, the expression of which proved to be inversely correlated with the backbone-originated helix content of the molecules. The other antigenic determinant corresponds to the common inside area of the polypeptides which is less conformation-dependent and therefore common to all four polypeptides.