Antigen loading of MHC class I molecules in the endocytic tract.

Major histocompatibility complex (MHC) class I molecules bind antigenic peptides that are translocated from the cytosol into the endoplasmic reticulum by the transporter associated with antigen processing. MHC class I loading independent of this transporter also exists and involves peptides derived from exogenously acquired antigens. Thus far, a detailed characterization of the intracellular compartments involved in this pathway is lacking. In the present study, we have used the model system in which peptides derived from measles virus protein F are presented to cytotoxic T cells by B-lymphoblastoid cells that lack the peptide transporter. Inhibition of T cell activation by the lysosomotropic drug ammoniumchloride indicated that endocytic compartments were involved in the class I presentation of this antigen. Using immunoelectron microscopy, we demonstrate that class I molecules and virus protein F co-localized in multivesicular endosomes and lysosomes. Surprisingly, these compartments expressed high levels of class II molecules, and further characterization identified them as MHC class II compartments. In addition, we show that class I molecules co-localized with class II molecules on purified exosomes, the internal vesicles of multivesicular endosomes that are secreted upon fusion of these endosomes with the plasma membrane. Finally, dendritic cells, crucial for the induction of primary immune responses, also displayed class I in endosomes and on exosomes.

Recycling MHC class I molecules and endosomal peptide loading.

MHC class I molecules usually present peptides derived from endogenous antigens that are bound in the endoplasmic reticulum. Loading of exogenous antigens on class I molecules, e.g., in cross-priming, sometimes occurs, but the intracellular location where interaction between the antigenic fragment and class I takes place is unclear. Here we show that measles virus F protein can be presented by class I in transporters associated with antigen processing-independent, NH(4)Cl-sensitive manner, suggesting that class I molecules are able to interact and bind antigen in acidic compartments, like class II molecules. Studies on intracellular transport of green fluorescent protein-tagged class I molecules in living cells confirmed that a small fraction of class I molecules indeed enters classical MHC class II compartments (MIICs) and is transported in MIICs back to the plasma membrane. Fractionation studies show that class I complexes in MIICs contain peptides. The pH in MIIC (around 5.0) is such that efficient peptide exchange can occur. We thus present evidence for a pathway for class I loading that is shared with class II molecules.

Measles virus fusion protein- and hemagglutinin-transfected cell lines are a sensitive tool for the detection of specific antibodies by a FACS-measured immunofluorescence assay.

A FACS-measured immunofluorescence assay was developed for the detection of antibodies directed against the hemagglutinin (H) and fusion (F) glycoproteins of measles virus (MV). Human melanoma cell lines transfected with either the MV H or F genes, which showed a high surface expression of the respective proteins in their native conformation, were used as target cells. The cells were incubated with diluted plasma samples, and stained subsequently with FITC-conjugated secondary antibodies. The FACS-measured fluorescence signals correlated directly with the amount of specific immunoglobulins over a wide concentration range. The use of different conjugates enabled the separate detection of MV-specific IgG, IgM, IgA and IgG subclasses, with relatively low backgrounds. Hemagglutinin-specific IgG, IgM and IgA fluorescence signals were shown to correlate well with MV-specific IgG ELISA titers and MV-specific IgM or IgA capture ELISA OD450-values, respectively. The polyclonal conjugates with specificity for human immunoglobulins offered sufficient cross-reactivity to detect MV-specific IgG, IgM and IgA in plasma samples of cynomolgus macaques, making this technique a useful tool for studying serological responses in vaccination and challenge experiments in non-human primate models.

The SCID mouse as a model for multiple myeloma.

The SCID mouse was investigated as a potential animal model for human multiple myeloma (MM). Duplicate samples of bone marrow mononuclear cells (BMMC) and/or peripheral blood mononuclear cells (PBMC) of six MM patients in different clinical phases and one patient with monoclonal gammapathy of undetermined significance (MGUS) were injected intraperitoneally into SCID mice. Human immunoglobulins (Ig) in the SCID sera were quantified with a light-chain isotype-specific ELISA, and their monoclonality biochemically characterized, using a sensitive immunoblotting technique after agar gel electrophoresis. Successful transplantation of bone marrow derived-tumour cells in SCID mice was obtained with BMCC of two MM patients with progressive disease. Human plasma cells were detected in the mesenteric fat tissue around the pancreas and the spleen. This model in SCID mice may facilitate studies on processes involved in tumour progression and provides a new tool for therapeutic approaches in MM.

Intracellular processing and presentation of T cell epitopes, expressed by recombinant Escherichia coli and Salmonella typhimurium, to human T cells.

Vaccines based on recombinant attenuated bacteria represent a potentially safe and effective immunization strategy. A carrier system was developed to analyze in vitro whether foreign T cell epitopes, inserted in the outer membrane protein PhoE of Escherichia coli and expressed by recombinant bacteria, are efficiently processed and presented via human leukocyte antigen (HLA) class I and II molecules by bacterial infected human macrophages. A well-defined HLA-B27-restricted cytotoxic T cell (CTL) epitope and an HLA-DR53 restricted T helper (Th) epitope of the fusion protein of measles virus were genetically inserted in a surface-exposed region of PhoE, and the chimeric proteins were expressed in E. coli and Salmonella typhimurium. Macrophages infected with both recombinant bacteria presented the Th epitope to the specific CD4+ T cell clone, but failed to present the CTL epitope to the specific CD8+ T cell clone. Presentation of the Th epitope by the infected macrophages was inhibited by cytochalasin D, indicating that phagocytic processing of intact bacteria within infected macrophages was essential for antigen presentation via HLA class II. Presentation of the Th epitope to the CD4+ T cell clone by infected macrophages was blocked by brefeldin A and cycloheximide, indicating the requirement of nascent HLA class II molecules for presentation. The efficiency of macrophages to process and present the inserted Th epitope was similar for both the recombinant E. coli and S. typhimurium strains.

Viral replication and development of specific immunity in macaques after infection with different measles virus strains.

Cynomolgus monkeys (Macaca fascicularis) were experimentally infected with a wild type measles virus (MV) strain (MV-BIL). Following intratracheal inoculation with different infectious doses, the virus could be isolated from peripheral blood mononuclear cells (PBMC), lung lavage cells, and pharyngeal cells. The kinetics of the cell-associated viremia was similar in all infected animals. They developed specific serum IgM, IgG, and neutralizing antibody responses as well as MV-specific T cell-mediated immunity. Monkeys infected intratracheally or intramuscularly with the wild type MV-Edmonston or the attenuated MV-Schwartz strain showed a lower level of PBMC-associated viremia and less pronounced specific IgM responses. Nine months after infection with MV strains, all of the monkeys were protected from intratracheal reinfection with MV-BIL. This monkey model is suitable for study of new generations of vaccines and vaccination strategies for measles.

A subset of HLA-B27 molecules contains peptides much longer than nonamers.

An unusual monoclonal antibody (MARB4) directed against HLA-B27 that reacts with only approximately 5-20% of the cell surface HLA-B27 was used for large-scale purification of these molecules. Subsequent mass spectrometry of HLA-B27-bound peptides showed that the minor MARB4-reactive population contained peptides primarily from 900 to 4000 Da in size (approximately 8-33 amino acid residues), whereas the major HLA-B27 population contained peptides in the mass range of 900-1400 Da (approximately 8-12 amino acid residues). Thus, a subset of HLA-B27 molecules binds to peptides much longer than nonamers. Typical HLA-B27-binding peptides contain arginine in position 2. Further analysis by Edman sequencing of the pooled bound peptides revealed that the major population contained substantial amounts of arginine at positions 1 and 9 (40-50%) and exclusively arginine at position 2, as expected. The minor population of peptides also contained detectable amounts of arginine at these positions, but at the level of only approximately 10%; no marked enrichment at any position was observed. These long HLA-B27-bound peptides could represent either intermediates in the formation of nonamers or adventitiously bound peptides. Lastly, in the TAP2 mutant cell line BM36.1 transfected with HLA-B*2705, MARB4-reactive HLA-B27 molecules were absent from the cell surface, indicating that the peptide transporter was required for delivery of the long peptides. Thus, during the folding of class I heavy chains, peptides of diverse lengths are available and participating.

Mitogen and antigen induced B and T cell responses of peripheral blood mononuclear cells from the harbour seal (Phoca vitulina).

In vitro assays were developed for studies concerning the functioning of the immune system of the harbour seal (Phoca vitulina). Proliferative responses of peripheral blood mononuclear cells (PBMC) were measured after stimulation with different concentrations of the mitogens concanavalin A (Con A), pokeweed mitogen (PWM), phytohaemagglutinin (PHA) or lipopolysaccharide from Salmonella typhimurium (LPS). Con A and PWM induced strong proliferative responses, while PHA and LPS induced comparatively low proliferative responses. Responses of mitogen stimulated PBMC to recombinant human interleukin-2 (rhIL-2) and in vitro immunoglobulin production by mitogen stimulated PBMC were measured to discriminate between stimulation of T cells and B cells. It was found that Con A and PHA stimulate phocine T cells, PWM stimulates both T cells and B cells and LPS predominantly stimulates phocine B cells. Antigen-specific immune responses were measured after immunization of seals with an inactivated rabies vaccine and/or with tetanus toxoid. Antigen-specific proliferation of PBMC and the presence of antigen-specific antibody forming cells were demonstrated for both antigens in the PBMC of immunized animals. The responses measured in vitro correlated well with the development of specific serum antibody titers to these antigens.

Structural and functional studies on a unique linear neutralizing antigenic site (G5) of the rabies virus glycoprotein.

The core of a unique linear neutralization epitope (G5) on the glycoprotein of rabies virus, recognized by a virus-neutralizing mouse monoclonal antibody (MAb 6-15C4), was determined by Pepscan analysis. The G5 epitope was defined as an octapeptide (LHDFRSDE). The contribution of the individual amino acids of the G5 epitope to the binding of MAb 6-15C4 was analysed with a set of synthetic peptides in which the individual amino acids had been replaced in turn by each of the other 19 naturally occurring amino acids. Five amino acids of the octapeptide proved to be essential for the binding of MAb 6-15C4. The conservation of the G5 epitope within the glycoprotein of the different rabies virus strains sequenced to date proved to be absolute at the amino acid level. Studies concerning the immunodominance of the G5 epitope were carried out by determining the presence of G5 epitope-specific serum antibodies in vaccinated human and mice, and by determining the frequency of G5 epitope-specific B lymphocytes in the blood of vaccinated humans. These studies indicated that antibodies to the G5 epitope constitute a minor population of the rabies virus-specific serum antibodies induced by rabies vaccination.

Human HLA class I- and HLA class II-restricted cloned cytotoxic T lymphocytes identify a cluster of epitopes on the measles virus fusion protein.

The transmembrane fusion (F) glycoprotein of measles virus is an important target antigen of human HLA class I- and class II-restricted cytotoxic T lymphocytes (CTL). Genetically engineered F proteins and nested sets of synthetic peptides spanning the F protein were used to determine sequences of F recognized by a number of F-specific CTL clones. Combined N- and C-terminal deletions of the respective peptides revealed that human HLA class I and HLA class II-restricted CTL efficiently recognize nonapeptides or decapeptides representing epitopes of F. Three distinct sequences recognized by three different HLA class II (DQw1, DR2, and DR4/w53)-restricted CTL clones appear to cluster between amino acids 379 and 466 of F, thus defining an important T-cell epitope area of F. Within this same region, a nonamer peptide of F was found to be recognized by an HLA-B27-restricted CTL clone, as expected on the basis of the structural homology between this peptide and other known HLA-B27 binding peptides.

Measles virus transmembrane fusion protein synthesized de novo or presented in immunostimulating complexes is endogenously processed for HLA class I- and class II-restricted cytotoxic T cell recognition.

The routes used by antigen-presenting cells (APC) to convert the transmembrane fusion glycoprotein (F) of measles virus (MV) to HLA class I and class II presentable peptides have been examined, using cloned cytotoxic T lymphocytes in functional assays. Presentation by Epstein-Barr virus-transformed B lymphoblastoid cell lines was achieved using live virus, ultraviolet light-inactivated virus, and purified MV-F delivered either as such or incorporated in immunostimulating complexes (MV-F-ISCOM). Only live virus and MV-F-ISCOM allow presentation by class I molecules, while all antigen preparations permit class II-restricted presentation. We observe presentation of MV-F from live virus and as MV-F-ISCOM by class II molecules in a fashion that is not perturbed by chloroquine. Our studies visualize novel presentation pathways of type I transmembrane proteins.

Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

Monoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these anti-Id antibodies as Ab2 gamma or Ab2 beta. By inhibiting experiments it was shown that these anti-Id antibodies did not recognize interspecies cross-reactive idiotopes, but recognized private idiotopes, uniquely associated with the Id of the anti-CPV MoAb used for immunization. This classifies these anti-Id antibodies as non-internal image Ab2 gamma. The potential use of these non-internal image anti-Id antibodies for the induction of antiviral antibodies in the CPV system is discussed.

A human CD5+ B cell clone that secretes an idiotype-specific high affinity IgM monoclonal antibody.

We previously demonstrated the occurrence of a naturally arisen human anti-idiotypic B cell clone, that we transformed with EBV (EBV383). We show evidence that EBV383 not only expresses the CD5 surface Ag, but also contains the 2.7-kb mRNA transcript encoding this protein. In addition, we show the presence of the 3.6-kb mRNA precursor. Most Ig produced by CD5+ B cells are polyreactive natural IgM antibodies encoded by unmutated copies of germline VH genes. However, in this study we present data demonstrating the monoreactive high affinity character of the anti-idiotypic antibody (mAb383) produced by EBV383. These data are in agreement with our previous observations, showing that the VH chain of mAb383 is encoded by an extensive somatically mutated VHV gene in a way that is consistent with an Ag-driven immune response. A possible role for this remarkable anti-idiotypic antibody in the maintenance of B cell memory is discussed.

The use of enzyme-linked immunosorbent assay systems for serology and antigen detection in parvovirus, coronavirus and rotavirus infections in dogs in The Netherlands.

Complex trapping blocking (CTB) enzyme-linked immunosorbent assays (ELISAs) and indirect ELISAs for the detection of antibodies to canine parvovirus (CPV), canine coronavirus (CCV) and rotavirus in sera of dogs were established. Double antibody sandwich ELISAs for the detection of CPV-, CCV- and rotavirus antigens in fecal samples were also developed. Both the serological and antigen-detection ELISAs were used to screen samples from dogs in The Netherlands, with or without a history of acute diarrhea. It was shown that the results of the respective serological ELISAs correlated well and that CPV was the major cause of virus-induced acute diarrhea in dogs in The Netherlands.

Identification of Hantavirus serotypes by testing of post-infection sera in immunofluorescence and enzyme-linked immunosorbent assays.

Serum samples were collected from 27 individuals who had been infected with a member of the genus Hantavirus in the Netherlands or Belgium during the last 15 years. These samples were tested in an immunofluorescence assay (IFA) and two enzyme-linked immunosorbent assay (ELISA) systems, using different virus strains that represented each of the four recently proposed serotypes of this genus. The serum samples from 11 individuals who had been infected through contacts with laboratory rats showed the highest reactivities with Hantaan virus (serotype I) and SR-11 (serotype II) in the IFA and ELISA systems. The samples of 16 individuals who had probably been infected through contacts with wild rodents showed the highest reactivities with Hällnäs virus (serotype III) in the IFA. All except two of these also showed the highest reactivity with Hällnäs virus in the two different ELISA systems.

A synthetic peptide derived from the amino acid sequence of canine parvovirus structural proteins which defines a B cell epitope and elicits antiviral antibody in BALB c mice.

Synthetic peptides, recombinant fusion proteins and mouse monoclonal antibodies were used to delineate a B cell epitope of the VP'2 structural protein of canine parvovirus (CPV). Although this epitope is not preferentially recognized in the normal antibody response to CPV, virus-specific antibodies could be induced in BALB/c mice with a synthetic peptide representing the epitope. The potential of this non-dominant B cell epitope to induce antiviral immunity in the presence of maternal CPV-specific antibodies, is discussed.

Delineation of canine parvovirus T cell epitopes with peripheral blood mononuclear cells and T cell clones from immunized dogs.

Three synthetic peptides derived from the amino acid sequence of VP2 of canine parvovirus (CPV) which were recently shown to represent three distinct T cell epitopes for BALB/c mice could prime BALB/c mice for a CPV-specific proliferative T cell response upon immunization. Proliferative responses of peripheral blood mononuclear cells (PBMC) from CPV-immunized dogs upon stimulation with these and other peptides, covering the major part of the sequence of VP2', identified the presence of T cell epitopes for this species. Most of these epitopes were recognized by PBMC from only a minority of the dogs tested. With three newly generated canine Thyl+ T cell clones, which recognized CPV antigen in association with major histocompatibility complex class II molecules, two distinct T cell epitopes were identified within the unique sequence of VP1.

Mass mortality in seals caused by a newly discovered morbillivirus.

During a recent disease outbreak among harbour seals (Phoca vitulina) in the North and Baltic seas, more than 17,000 animals have died. The clinical symptoms and pathological findings were similar to those of distemper in dogs. Based on a seroepizootiological study, using a canine distemper virus (CDV) neutralization assay, it was shown that CDV or a closely related morbillivirus (phocid distemper virus-PDV) was the primary cause of the disease. The virus was isolated in cell culture from the organs of dead seals and characterized as a morbillivirus by serology (immunofluorescence neutralization and enzyme-linked immunosorbent assays) and by negative contrast electron microscopy. Experimental infection of SPF dogs resulted in the development of mild clinical signs of distemper and CDV-neutralizing antibodies. The disease was reproduced in seals by experimental inoculation of organ material from animals that had died during the outbreak. However, seals that had been vaccinated with experimental inactivated CDV vaccines were protected against this challenge. This fulfilled the last of Koch's postulates, confirming that the morbillivirus isolated from the seal organs, was the primary cause of the disease outbreak. The recent demonstration of the presence of a similar virus in Lake Baikal seals (Phoca sibirica), which infected these Siberian seals 1 year before the northwestern European seals were infected, raises new questions about the origin of this infectious disease in pinnipeds.

Establishment and characterization of canine parvovirus-specific murine CD4+ T cell clones and their use for the delineation of T cell epitopes.

Canine parvovirus (CPV)-specific T cell clones were generated by culturing lymph node cells from CPV-immunized BALB/c mice at limiting dilutions in the presence of CPV antigen and interleukin-2 (IL-2). All isolated T cell clones exhibited the cell surface phenotype Thy1+, CD4+, CD8- and proliferated specifically in response to CPV antigen. After stimulation with CPV antigen in culture the T cell clones produced IL-2 and proliferated in the absence of exogenous IL-2. Naive mice to which CPV-specific T cell clones had been adoptively transferred developed a CPV-specific delayed type hypersensitivity reaction upon simultaneous intracutaneous injection of CPV in their ears. The ability of recombinant viral fusion proteins, representing the VP2 capsid protein of the antigenically closely related feline panleukopenia virus and of synthetic peptides derived from the amino acid sequence of the VP2 of CPV, to stimulate these T cell clones enabled the identification of T cell epitopes.