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Biomedical subjects

H Zaghouani

Publications and source records attributed to H Zaghouani.

At least 37 records · Page 2Linked to original sources
PubMed1994

Immunogenicity of microbial peptides grafted in self immunoglobulin molecules.

The advent of genetic engineering has allowed for the expression and production of recombinant proteins carrying short immunogenic epitopes of foreign antigens. These antigenized molecules represent valuable tools to investigate the molecular basis of antigen fragmentation, generation and presentation of peptide to lymphocytes, the induction of epitope specific immunity and potentially the development of a new generation of vaccines. Recently, we expressed viral epitopes on immunoglobulin molecules by replacing the D segment of a variable region of the heavy chain (VH) gene with a B cell epitope from the V3-loop of HIV-1 envelope protein, as well as a cytotoxic T lymphocyte (CTL) and a T helper epitope from influenza virus nucleoprotein and hemagglutinin, respectively. The T cell peptides generated from the immunoglobulin molecules produced by cells transfected with chimeric V genes, activated specific T cells as they do when generated from viral proteins. Possible practical applications for the development of prophylactic and immunotherapeutic reagents are envisioned for immunoglobulin molecules bearing foreign epitopes.

Amino Acid Sequence↗
PubMed1993

Efficient loading of identical viral peptide onto class II molecules by antigenized immunoglobulin and influenza virus.

Several prior reports have identified peptides that are naturally associated with major histocompatibility complex (MHC) class II molecules on presenting cells. We have examined the delivery of a peptide from exogenous sources to MHC class II molecules. The peptide derives from the influenza virus hemagglutinin (HA) and activates a CD4+ T cell hybridoma. In functional assays of antigen presentation, this epitope is delivered effectively to T cells either in the context of influenza virus or chimeric immunoglobulin (Ig) molecules (Ig-HA) in which the peptide has replaced the CDR3 loop of the heavy chain. We find that the identical 11-mer peptide can be isolated from mouse MHC class II antigens whether the exogenous source of peptide is free HA peptide, the Ig-HA chimera, or ultraviolet-inactivated PR8 influenza virus. The Ig-HA chimera proves to be the most efficient vehicle for charging class II molecules via the exogenous route. Given the fact that self Igs represent natural long-lived carriers, we suggest that antigenized Igs have considerable potential for peptide delivery to MHC molecules in situ.

Amino Acid Sequence↗
PubMed1993

A sensitive method to detect defined peptide among those eluted from murine MHC class II molecules.

We developed a sensitive competitive inhibition radioimmunoassay able to trace pmoles of a defined peptide eluted from major histocompatibility complex (MHC) class II molecules that were subsequently fractionated by RP-HPLC. In this assay we used a model synthetic peptide corresponding to amino acid residues 110-120 from the hemagglutinin (HA) of PR8 influenza virus, and affinity purified rabbit antibodies specific for this peptide. The HA110-120 peptide binds to I-Ed class II molecules on the surface of APCs and is recognized by specific CD4+ T helper cells. 2PK3 B lymphoma cells (H-2d) were pulsed with HA110-120 peptide or PR8 virus, lysed, the MHC class II molecules extracted, and bound peptides eluted. After separation by RP-HPLC, the fractions were tested for inhibition of the binding of rabbit anti-HA110-120 antibodies to peptide coated microtiter plates. A significant inhibitory activity was observed with one peak when the cells were pulsed with HA110-120 peptide and two peaks when pulsed with PR8 virus. The inhibitory activity was correlated with the presence of HA110-120 peptide as demonstrated by peptide sequencing. The assay is reproducible and sensitive to 1 pmol of antigenic peptide. This assay can be useful to identify microbial peptides with defined structure and antigenicity among the multiple peptides bound to class II molecules.

Amino Acid Sequence↗
PubMed1993

Presentation of a viral T cell epitope expressed in the CDR3 region of a self immunoglobulin molecule.

Synthetic peptides corresponding to microbial epitopes stimulate T cell immunity but their immunogenicity is poor and their half-lives are short. A viral epitope inserted into the complementarity-determining region 3 (CDR3) loop of the heavy chain of a self immunoglobulin (Ig) molecule was generated from the Ig context and was presented by I-Ed class II molecules to virus-specific, CD4+ T cells. Chimeric Ig-peptide was presented 100 to 1000 times more efficiently than free synthetic peptide and was able to prime virus-specific T cells in vivo. These features suggest that antigenized Ig can provide an improved and safe vaccine for the presentation of microbial and other peptides.

Amino Acid Sequence↗
PubMed1993

In vivo priming effect during various stages of ontogeny of an influenza A virus nucleoprotein peptide.

In vivo priming of cytotoxic T lymphocytes (CTL) by an influenza virus nucleoprotein (NP) peptide was studied at various stages of development. Adult mice immunized twice with the NP peptide in complete Freund's adjuvant and incomplete Freund's adjuvant, respectively, produce significant CTL responses. Neonates immunized at birth with large amounts of NP peptide and boosted twice with the peptide during adulthood, also mount a weak but significant CTL response. By contrast, offspring from mothers immunized with the NP peptide at days 15, 17, and 19 of pregnancy showed unresponsiveness to the peptide subsequent to a similar regimen of peptide immunization at the age of 1 month. The data indicate that the contact of T cell precursors with antigen during fetal life induces CTL tolerance, whereas, after birth the precursors are not susceptible to tolerogenic signals.

Animals↗
PubMed1993

Contrasting efficacy of presentation by major histocompatibility complex class I and class II products when peptides are administered within a common protein carrier, self immunoglobulin.

Major histocompatibility complex (MHC) class I and II products are specialized to present antigens via different intracellular processing routes. Peptides originating from proteins in the cytoplasm can gain access to class I peptide-binding grooves, most likely in the rough endoplasmic reticulum. Peptides from proteins in acidic endocytic vacuoles gain access to class II. It has been proposed that MHC class I products also can capture peptides from "exogenous" or noninfectious sources, and this assumption underlies the use of intact proteins as vaccines for CD8+ cytotoxic T lymphocytes. Here we describe quantitative information comparing the efficacy of peptide presentation from exogenous proteins by administering a class I- and II-restricted peptide within the same context, the CDR3 loop of the VH domain of a self immunoglobulin. Antigen-presenting cells (APC), including primary dendritic cells, efficiently present an influenza hemagglutinin peptide from the immunoglobulin (Ig) carrier (50% maximal response at 10 nM Ig-HA) to an MHC class II-restricted T cell. In contrast, these same APC are unable to present an influenza nucleoprotein (NP) peptide from the same context (1 microM Ig-NP) to an MHC class I-restricted T cell. Ig-NP DNA transfectants do present the nucleoprotein viral peptide on class I. Thus, peptides within the complementarity-determining region loops of Ig carriers can be presented on class I or II MHC products, but the endocytic compartment, when offered MHC class I- and II-restricted peptides within the same carrier protein context, favors presentation by class II by at least 1000-fold.

Amino Acid Sequence↗
PubMed1993

Further studies on the adjuvanticity of stearyl tyrosine and ester analogues.

A new family of immunoadjuvants, long-chain stearyl esters of amino acids and peptides, are described and examined with bacterial and viral vaccines. The parent compound, stearyl tyrosine, displayed significant adjuvant activity with these vaccines. Stearyl glycyl glycine displayed superior activity with viral vaccines. A number of analogues of stearyl tyrosine were adjuvant-active. Further, these adjuvants were able to elicit a neutralizing antibody response. Stearyl tyrosine and stearyl ester analogues represent promising adjuvants for human vaccines.

Adjuvants, Immunologic↗
PubMed1993

Priming of cytotoxic T lymphocytes at various stages of ontogeny with transfectoma cells expressing a chimeric Ig heavy chain gene bearing an influenza virus nucleoprotein peptide.

The amino acid residues 147-161 of the nucleoprotein (NP) of influenza virus represent a T cell epitope recognized by cytotoxic T cells (CTLs) in association with Kd class I molecules. When SP2/0 myeloma B cells are transfected with a chimeric heavy chain gene bearing this particular NP(147-161) peptide, they are lysed by CTLs specific for the NP(147-161) peptide. Cells that are transfected with this heavy chain chimera and the parental light chain secreted a soluble Ig-NP chimera and were also lysed by the CTLs. Herein, we present evidence that transfectoma cells are able to induce in vitro proliferation of NP specific CTL, whereas immobilized Ig-NP chimeras do not. Furthermore, the transfectoma cells expressing the chimeric heavy chain prime NP specific CTLs in adult as well as in newborn mice, while SP2/0 cells coated with NP(147-161) synthetic peptide do not. These data indicate that the NP peptide needs to be cleaved from the Ig context in order to be presented to T cells and that only endogenously generated NP peptide is immunogenic.

Amino Acid Sequence↗
PubMed1993

Chimeric influenza virus induces neutralizing antibodies and cytotoxic T cells against human immunodeficiency virus type 1.

Expression vectors based on DNA or plus-stranded RNA viruses are being developed as vaccine carriers directed against various pathogens. Less is known about the use of negative-stranded RNA viruses, whose genomes have been refractory to direct genetic manipulation. Using a recently described reverse genetics method, we investigated whether influenza virus is able to present antigenic structures from other infectious agents. We engineered a chimeric influenza virus which expresses a 12-amino-acid peptide derived from the V3 loop of gp120 of human immunodeficiency virus type 1 (HIV-1) MN. This peptide was inserted into the loop of antigenic site B of the influenza A/WSN/33 virus hemagglutinin (HA). The resulting chimeric virus was recognized by specific anti-V3 peptide antibodies and a human anti-gp120 monoclonal antibody in both hemagglutination inhibition and neutralization assays. Mice immunized with the chimeric influenza virus produced anti-HIV antibodies which were able to bind to synthetic V3 peptide, to precipitate gp120, and to neutralize MN virus in human T-cell culture system. In addition, the chimeric virus was also capable of inducing cytotoxic T cells which specifically recognize the HIV sequence. These results suggest that influenza virus can be used as an expression vector for inducing both B- and T-cell-mediated immunity against other infectious agents.

Amino Acid Sequence↗
PubMed1993

Engineered immunoglobulin molecules as vehicles for T cell epitopes.

The variable regions (V) of immunoglobulins (Ig) bear antigenic determinants that can stimulate both humoral and cellular immune responses subsequent to hetero, allo or iso-immunization. The expression of these determinants by Igs usually correlates with the presence of specific amino acid residues within the CDR loops of the V regions. Since the CDR loops varies in length, we reasoned that they would represent permissive sites to insert foreign peptides and create antigenized Igs expressing selected determinants. Taking advantage of these properties and the fact that Igs are self and long-lived molecules, we expressed a CTL and a helper epitope of influenza virus nucleoprotein and hemagglutinin respectively, within the heavy chain CDR3 loop of an anti-arsonate antibody. We found that foreign peptides comprised of 11 to 15 amino acid residues can be expressed within the V region of the heavy chain without alteration of pairing with the light chain. More striking, the cellular processing machinery is able to generate the peptides from the Ig context which were then recognized by specific T cells. Furthermore, the engineered Igs are able to induce T cell responses specific for the inserted peptide and for influenza virus. The use of engineered Ig molecules as vehicles for T and B cell peptides might represent a valuable strategy to generate safe, long lived reagents able to stimulate strong specific immune responses. This would then overcome the short half life of synthetic peptides based vaccines and the side effects seen in case of recombinant viral proteins or inactivated viruses based vaccines.

Animals↗
PubMed1992

A single amino acid mutation in CDR3 of the 3-14-9 L chain abolished expression of the IDA 10-defined idiotope and antigen binding.

The ABPC 48 myeloma protein and the 3-14-9 mAb derive their V region genes from the same VH and V kappa gene families. They also share a cross-reactive idiotope defined by the anti-Id mAb IDA 10. Whereas ABPC 48 is specific for bacterial levan, 3-14-9 showed a significant Ag-binding activity to aminophenyl-beta-N-acetylglucosaminide (AZO). In order to define the molecular basis of idiotope expression and Ag-binding activity, we have cloned the genes encoding the 3-14-9 H and L chain V region genes, generated antibodies that carry mutations within the L chain genes, by site-directed mutagenesis, and investigated the effects of those mutations with respect to IDA 10 idiotope expression and binding to AZO. Our findings show that, whereas expression of the IDA 10-defined idiotope requires association of both the H and L chains, a single change (glycine to phenylalanine) at position 91 in the third complementarity-determining region of the L chain abolished both idiotope expression and Ag-binding activity. In addition, a L chain change of alanine to threonine at position 25 allowed idiotope expression to some extent but significantly reduced binding activity to AZO. These data suggest that a single amino acid change can play a crucial role in the functional activity and structural integrity of antibodies.

Animals↗
PubMed1992

Cells expressing an H chain Ig gene carrying a viral T cell epitope are lysed by specific cytolytic T cells.

The epitope corresponding to amino acid residues 147-161 of the nucleoprotein (NP) of influenza A virus is recognized by CTL in association with H-2Kd class I Ag. Herein, we engineered an Ig molecule carrying this CTL epitope by replacing the diversity gene segment of the H chain V region of an anti-arsonate antibody with an oligonucleotide that encodes the CTL epitope. The chimeric H chain gene was expressed either alone or together with the parental L chain in the nonsecreting BALB/c myeloma B cell line, SP2/0. The Ig produced by cells transfected with both the chimeric H chain and parental L chains genes expressed the NP epitope but lost the original arsonate binding activity. In addition, SP2/0 cells expressing the chimeric H chain either alone or together with the parental L chain were lysed by class I restricted NP-epitope specific CTL. By contrast, SP2/0 cells pulsed with soluble chimeric Ig molecules were not lysed by the specific CTL. These observations indicate that: 1) this particular CTL epitope can be expressed on Ig molecules without altering the H and L chain pairing; 2) this CTL epitope can be generated from this chimeric Ig in which it is surrounded by flanking regions distinct from those of the viral NP; and 3) the generation of this CTL epitope from the Ig molecule requires the endogenous pathway as do viral proteins.

Amino Acid Sequence↗
PubMed1992

Adjuvanticity of stearyl tyrosine on the antibody response to peptide 503-535 from HIV gp160.

In this present report we compare the humoral immune response induced by immunization with an HIV-1 gp160 peptide corresponding to amino acid sequence 503-535 complexed with different adjuvants. Specifically, the antipeptide, anti-HIV-1 gp160 and neutralizing antibody responses were measured in groups of mice and baboons that received peptide 503-535 conjugated to a carrier protein in either saline, alum, or stearyl tyrosine. The highest antibody responses were induced when mice and baboons were immunized with peptide adsorbed on stearyl tyrosine. These data indicate that stearyl tyrosine represents a potent candidate as a nontoxic adjuvant not only for subunit viral vaccines, but also for HIV peptides.

Adjuvants, Immunologic↗
PubMed1991

Induction of antibodies to the envelope protein of the human immunodeficiency virus by immunization with monoclonal anti-idiotypes.

Anti-idiotypes that possess the internal image of antigen can induce protective humoral immunity toward microbes. Herein we demonstrate antigen mimicry by monoclonal anti-idiotypes of a distinct epitope of the human immunodeficiency virus (HIV) envelope protein that is defined by a synthetic peptide. This peptide, corresponding to amino acid residues 503-535 (peptide 503-535) of HIV-1 IIIB gp160, induced antibodies in three mammalian species that interacted with HIV-1 gp120 and inhibited in vitro syncytium formation caused by HIV-1, IIIB and MN isolates. Three monoclonal anti-idiotypes were generated against rabbit anti-gp120 antibodies specific for peptide 503-535. These anti-idiotypes recognize an interspecies cross-reactive idiotype expressed on mouse, chimpanzee, baboon, rabbit, and human anti-gp120 antibodies specific for peptide 503-535. The interaction with the cross-reactive idiotype is inhibited by synthetic peptide and HIV-1 gp160. Furthermore, rabbits immunized with the monoclonal anti-idiotypes produced antibodies that also bind HIV-1 gp120 and gp160 and recognized the epitope defined by peptide 503-535.

Amino Acid Sequence↗
PubMed1990

V kappa gene usage, idiotype expression, and antigen binding among clones expressing the VHX24 gene family derived from naive and anti-idiotype immune BALB/c mice.

The BALB/c myeloma protein ABPC48 binds beta(2-6)-linked fructosans and expresses genes derived from the VHX24 and V kappa 10 gene families. We have selected 30 hybridomas expressing the VHX24 gene family derived from mitogen-stimulated spleen cells of naive BALB/c mice and mice injected at birth with the syngeneic monoclonal anti-ABPC48Id, IDA10. The majority of mAb with kappa L chains uses V kappa 1. Antibodies reacting with IDA10 use both V kappa 10 and V kappa 1. Most of these VHX24+ mAb reacted with one or more members of a limited panel of predominantly polysaccharide Ag that have been previously observed to interact with antibodies expressing the VHX24 gene family. Nucleotide sequencing of selected VH and V kappa genes shows a very low frequency of somatic mutation. The effect of neonatal anti-Id injection on VHX24-V kappa pairing and Id expression is discussed.

Animals↗