First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity.

Immediate passive immune prophylaxis as part of rabies post-exposure prophylaxis (PEP) often cannot be provided due to limited availability of human or equine rabies immunoglobulin (HRIG and ERIG, respectively). We report first clinical data from two phase I studies evaluating a monoclonal antibody cocktail CL184 against rabies. The studies included healthy adult subjects in the USA and India and involved two parts. First, subjects received a single intramuscular dose of CL184 or placebo in a double blind, randomized, dose-escalation trial. Second, open-label CL184 (20IU/kg) was co-administered with rabies vaccine. Safety was the primary objective and rabies virus neutralizing activity (RVNA) was investigated as efficacy parameter. Pain at the CL184 injection site was reported by less than 40% of subjects; no fever or local induration, redness or swelling was observed. RVNA was detectable from day 1 to day 21 after a single dose of CL184 20 or 40IU/kg. All subjects had adequate (>0.5IU/mL) RVNA levels from day 14 onwards when combined with rabies vaccine. CL184 appears promising as an alternative to RIG in PEP.

A sensitive cell-based assay for the detection of residual infectious West Nile virus.

Ensuring complete viral inactivation is critical for the safety of vaccines based on an inactivated virus. Detection of residual infectious virus is dependent on sensitivity of the assay, sample volume analyzed and the absence of interference with viral infection. Here we describe the development and qualification of a sensitive cell-based assay for the detection of residual infectious West Nile Virus (WNV). The results of the assay are in good agreement with the assumption that at low concentrations the number of infectious units in relatively small samples follows a Poisson distribution. The assay can detect 1 infectious unit with a confidence of 99%, provides statistical controls for interference and can easily be scaled up to test large amounts of vaccine material. Furthermore, we show equivalence in sensitivity between the cell-based assay and an in vivo assay for detection of infectious WNV. Finally, the assay has been used for successful release testing of clinical lots of inactivated WNV vaccine. Given the principle and generic setup of the method we envision broad applicability to the detection of very low concentrations of infectious virus.

A rapid method for immunotitration of influenza viruses using flow cytometry.

Reliable assays for accurate titration of influenza virus in infectious samples are pivotal to both influenza research and vaccine development. A titration assay adopted commonly for this purpose is the plaque assay on Madin-Darby canine kidney (MDCK) cells, despite it being time and labour consuming. A novel assay is described for titration of influenza viruses based on the detection of intracellular viral nucleoprotein (NP) by fluorescence-activated cell sorting (FACS). By using a panel of viruses of different type, subtype and origin, it is demonstrated that there is a mathematical correlation between titres measured by immunotitration and by classical plaque assay on MDCK cells. Moreover, the availability of NP antibodies specific for type A or type B influenza virus ensures the specificity of the assay. Based on speed, accuracy and specificity, it is concluded that the FACS-based immunotitration of influenza virus represents a valid and efficient alternative to the classical plaque assay.

Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease.

Since targeting of recombinant adenovirus vectors to defined cell types in vivo is a major challenge in gene therapy and vaccinology, we explored the natural diversity in human adenovirus tissue tropism. Hereto, we constructed a library of Ad5 vectors carrying fibers from other human serotypes. From this library, we identified vectors that efficiently infect human cells that are important for diverse gene therapy approaches and for induction of immunity. For several medical applications (prenatal diagnosis, artificial bone, vaccination, and cardiovascular disease), we demonstrate the applicability of these novel vectors. In addition, screening cell types derived from different species revealed that cellular receptors for human subgroup B adenoviruses are not conserved between rodents and primates. These results provide a rationale for utilizing elements of human adenovirus serotypes to generate chimeric vectors that improve our knowledge concerning adenovirus biology and widen the therapeutic window for vaccination and many different gene transfer applications.

The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines.

Influenza viruses for vaccine production are currently grown on embryonated eggs. This manufacturing system conveys many major drawbacks such as inflexibility, cumbersome down stream processing, inability of some strains to replicate on eggs to high enough yields, and selection of receptor-binding variants with reduced antigenicity. These limitations emphasize the need for a cell line-based production system that could replace eggs in the production of influenza virus vaccines in a pandemic proof fashion. Here we present the efficient propagation of influenza A and B viruses on the fully characterized and standardized human cell line PER.C6.

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.

Suppression of hepatitis B virus replication mediated by hepatitis A-induced cytokine production.

BACKGROUND: Acute hepatitis A virus (HAV) infection can cause severe hepatitis especially in patients with underlying chronic liver disease. In patients with pre-existing chronic hepatitis B (HBV) acute HAV infection can suppress HBV replication. The exact mechanism of HBV suppression during acute HAV infection is still a subject of debate. One mechanism may be the production of HAV infection-induced cytokines leading to suppression of HBV replication and viral clearance. AIM: To evaluate cytokine production and HBV-specific lympho-proliferative responses (LPR) during acute HAV infection in a patient with chronic HBV infection-clearing markers of active HBV replication. DESIGN: Early detection of a case of acute HAV infection in an HBeAg-positive, HBV DNA-positive chronic HBV patient treated with lamivudine. RESULTS: At the time of HAV infection a sharp peak in the gamma-interferon (IFN-gamma) level occurred just before the rise in serum transaminase activity. This was subsequently followed by a decrease in HBV DNA and HBeAg below the limit of detection of the assay. However the HBV-specific T-cell response was not modified. After resolution of the acute HAV infection and withdrawal of antiviral therapy HBV replication relapsed. CONCLUSION: The sharp rise in IFN-gamma production mediated by the acute HAV infection may be pivotal in the suppression of HBV replication in chronic hepatitis B.

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.

Human peptide transporter deficiency: importance of HLA-B in the presentation of TAP-independent EBV antigens.

Two siblings with a peptide TAP deficiency were recently described. Despite poor cell surface expression of HLA class I molecules, these patients were not unusually susceptible to viral infections. The majority of the cell surface-expressed class I molecules were HLA-B products as assessed by cytofluorometry and biochemical analysis. Analysis of two peptides eluted from the class I molecules expressed by TAP-deficient EBV B lymphoblastoid cell lines indicated that both were derived from cytosolic proteins and presented by HLA-B molecules. Peripheral alphabeta CD8+ T cells were present and their TCR repertoire was polyclonal. Most of the alphabeta CD8+ T cell clones studied (21 of 22) were nonreactive against cells expressing normal levels of the same HLA alleles as those of the TAP-deficient patients. However, it was possible to isolate one cytotoxic CD8+ alphabeta T cell clone recognizing the EBV protein LMP2 presented by HLA-B molecules on TAP-deficient cells. These observations suggest that in the TAP-deficient patients, CD8+ alphabeta T cells could mature and be recruited in immune responses to mediate HLA class I-restricted cytotoxic defense against viral infections. They also strengthen the physiologic importance of a TAP-independent processing pathway of the LMP2 protein, which was previously shown to contain several other TAP-independent epitopes.

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.

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.

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 single injection of Staphylococcus aureus enterotoxin B reduces autoimmunity in MRL/lpr mice.

MRL/Mp-lpr/lpr (MRL/lpr) mice carry a mutation in the Fas gene whose product is involved in the regulation of lymphocyte apoptosis. This mutation is associated with the lpr phenomenon, i.e., a massive expansion of phenotypically abnormal CD4-CD8- cells ("double negative," DN) alpha/beta T cells (lpr cells) that becomes manifest at 3-4 months of age. As in normal mice, intravenous SEB injection into 2- or 6-month-old female MRL/lpr mice causes a transient expansion of SEB-reactive V beta 8+ T cells, followed by a deletion of this subset. In contrast, in the same animals, the frequency of abnormal V beta 8+CD4-CD8- cells is not modulated by SEB. Whereas DN T cells are completely resistant to SEB-mediated deletion in vivo, their precursors appear susceptible to SEB-induced deletion. Thus, a single injection of SEB prior to the surge of DN T cells in peripheral lymphoid organs, at 2 months of age, is sufficient to cause a stable long-term (6 months) deletion of DN cells. This is accompanied by a significant amelioration of autoimmune parameters (autoantibody titers, incidence of arthritis and nephritis), thus pointing to the feasibility of employing superantigens for simple manipulations of the immune repertoire that result in the long-term prophylaxis of autoimmune diseases.

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.

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.

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.

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.