Production of human monoclonal antibodies by the epstein-barr virus method.

Epstein-Barr virus (EBV) is a herpes virus which in vitro efficiently immortalizes nearly all human B lymphocytes. The lymphoblastoid diploid cell lines (LCL's) thus generated preserve the characteristics of the cells initially infected by the virus: the cells produce and secrete immunoglobulins and also express these molecules on their surface. A selection of specific antibody-producing cells (i.e., antigen-committed cells) before EBV-infection or when LCL's have already been established, enables isolation of monoclonal cell lines that secrete specific antibodies. If selection of antigen-committed cells is not feasible, secretion of specific antibodies by cloned LCL's in limiting dilution cultures enables isolation of the desired cell lines. The method allows the production of human IgM, IgG, IgA, and IgE monoclonal antibodies from any individual. Monoclonal antibodies produced by the EBV method resemble the antibody repertoire of the donor of the lymphocytes. Human monoclonal antibodies are promising reagents for passive immunization.

Preferential targeting of somatic hypermutation to hotspot motifs and hypermutable sites and generation of mutational clusters in the IgVH alleles of a rheumatoid factor producing lymphoblastoid cell line.

Epstein-Barr virus transforms human peripheral B cells into lymphoblastoid cell lines (LCL) that secrete specific antibodies. Our previous studies showed that a monoclonal LCL that secretes a rheumatoid factor expressed activation-induced cytidine deaminase (AID) and displayed an ongoing process of somatic hypermutation (SHM) at a frequency of 1.7×10⁻³ mut/bp in its productively rearranged IgVH gene. The present work shows that SHM similarly affects the nonproductive IgVH allele of the same culture. Sequencing of multiple cDNA clones derived from cellular subclones of the parental culture, showed that both alleles exhibited an ongoing mutational process with mutation rates of 2-3×10⁻5 mut/bp×generation with a high preference for C/G transition mutations and lack of a significant strand bias. About 50% of the mutations were targeted to the underlined C/G bases in the WRCH/DGYW and RCY/RGY hotspot motifs, indicating that they were due to the initial phase of AID activity. Mutations were targeted to the VH alleles and not to the Cµ or to the GAPDH genes. Genealogical trees showed a stepwise accumulation of only 1-3 mutations per branch of the tree. Unexpectedly, 27% of all the mutations in the two alleles occurred repeatedly and independently within certain sites (not necessarily the canonical hotspot motifs) in cellular clones belonging to different branches of the lineage tree. Furthermore, some of the mutations seem to arise as recurrent mutational clusters, independently generated in different cellular clones. Statistical analysis showed that it is very unlikely that these clusters were due to random targeting of equally accessible hotspots, indicating the presence of 'hypermutable sites' that generate recurring mutational clusters in the IgVH alleles. Intrinsic hypermutable sites may enhance affinity maturation and generation of effective mutated antibody repertoires against invading pathogens.

Amyloid-ß oligomer specificity mediated by the IgM isotype--implications for a specific protective mechanism exerted by endogenous auto-antibodies.

BACKGROUND: Alzheimers disease (AD) has been strongly linked to an anomalous self-assembly of the amyloid-ß peptide (Aß). The correlation between clinical symptoms of AD and Aß depositions is, however, weak. Instead small and soluble Aß oligomers are suggested to exert the major pathological effects. In strong support of this notion, immunological targeting of Aß oligomers in AD mice-models shows that memory impairments can be restored without affecting the total burden of Aß deposits. Consequently a specific immunological targeting of Aß oligomers is of high therapeutic interest. METHODOLOGY/PRINCIPAL FINDINGS: Previously the generation of conformational-dependent oligomer specific anti-Aß antibodies has been described. However, to avoid the difficult task of identifying a molecular architecture only present on oligomers, we have focused on a more general approach based on the hypothesis that all oligomers expose multiple identical epitopes and therefore would have an increased binding to a multivalent receptor. Using the polyvalent IgM immunoglobulin we have developed a monoclonal anti-Aß antibody (OMAB). OMAB only demonstrates a weak interaction with Aß monomers and dimers having fast on and off-rate kinetics. However, as an effect of avidity, its interaction with Aß-oligomers results in a strong complex with an exceptionally slow off-rate. Through this mechanism a selectivity towards Aß oligomers is acquired and OMAB fully inhibits the cytotoxic effect exerted by Aß(1-42) at highly substoichiometric ratios. Anti-Aß auto-antibodies of IgM isotype are frequently present in the sera of humans. Through a screen of endogenous anti-Aß IgM auto-antibodies from a group of healthy individuals we show that all displays a preference for oligomeric Aß. CONCLUSIONS/SIGNIFICANCE: Taken together we provide a simple and general mechanism for targeting of oligomers without the requirement of conformational-dependent epitopes. In addition, our results suggest that IgM anti-Aß auto-antibodies may exert a more specific protective mechanism in vivo than previously anticipated.

Three decades of human monoclonal antibodies: past, present and future developments.

The hybridoma technique has been shown to be a most reproducible method for producing rodent monoclonal antibodies but poor results were obtained when it was used for generating human monoclonal antibodies. For immunotherapy, murine monoclonal antibodies are inadequate, whereas human monoclonal antibodies are virtually indispensable. Cellular, chemical, genetic and molecular methods to generate human monoclonal antibodies have been developed. Most often, the monoclonal antibodies for therapy are selected after deliberate vaccination, according to their high affinity towards an arbitrarily-chosen epitope of a pathogen or cellular antigen and therefore the selection is obviously skewed. A major hindrance of the production of therapeutic human monoclonal antibodies is the lack of an appropriate strategy to define and select the antibodies that would be effective in vivo. In contrast to antibodies induced by vaccination, there has been only a marginal interest in monoclonal antibodies which reflect antibodies of the innate immunity. In the future, human monoclonal antibodies that resemble antibodies that are ubiquitously present in sera of healthy individuals might serve as novel therapies in diseases such as Alzheimer's disease, where no other therapy exists.

Immunotherapy for Alzheimer's disease.

Patients with Alzheimer's disease (AD) express severe cognitive deficiencies with a concurrent increase in brain deposits of aggregated amyloid-beta (Abeta), a catabolic derivative of the ubiquitous amyloid precursor protein (APP). Interference in the homeostasis of Abeta has been suggested as a treatment for AD patients. In AD murine models it has been shown that active and passive immunization against Abeta alters the equilibrium of the different forms of Abeta in brain and serum, leading to a concomitant cognitive improvement. Generally, the clinical trials that followed the study of the murine AD model confirmed the results of the AD models, although safety issues advocate the passive vaccination approach rather than the active one. However, passive vaccination of patients with monoclonal antibodies derived from nonhuman sources is limited. Anti-Abeta IgM and IgG antibodies, which are present in the serum of every healthy individual and probably play a role in the homeostasis of Abeta in healthy subjects, might be beneficial to AD patients, as shown for the effect exerted by the commercial preparation of intravenous immunoglobulin. Human monoclonal anti-Abeta antibodies, which correspond to the ubiquitous anti-Abeta antibodies, are plausible candidates for future immunotherapy of AD patients.

Ongoing somatic hypermutation of the rearranged VH but not of the V-lambda gene in EBV-transformed rheumatoid factor-producing lymphoblastoid cell line.

Epstein-Barr virus (EBV) transforms human peripheral B cells into lymphoblastoid cell lines (LCLs) that secrete specific antibodies. In contrast to peripheral blood B cells, LCLs express the activation-induced cytidine deaminase (AID) gene, a key enzyme in the generation of somatic hypermutation (SHM) in immunoglobulin variable genes. We have previously studied an LCL that secretes a rheumatoid factor (RF: an IgM(lambda) anti-IgG antibody) and identified the accumulation of SHM at a frequency of 1.5 x 10(-3)mut/bp in the rearranged variable region heavy chain gene (VH) of its RF sub-culture (i.e., RF-2004). The aim of the present work was to find out whether SHM was initiated as an early event following EBV transformation. Our results show that already the earliest RF-culture (RF-1983) mutates its VH at a somewhat higher frequency of 1.9 x 10(-3). Overall, we detected 17 point mutations in the RF-2004 culture and in 26 cellular clones derived from the RF-1983 and RF-2004 cultures. Most of the mutations were due to C to T or G to A transitions, with preferential targeting to WRCH/DGYW hotspot motifs, indicating that they were due to the initial phase of AID-directed mutations. A genealogical tree demonstrates that mutations were accumulated in a stepwise manner with 1-2 mutations per cell division. However, no mutations were found in the rearranged V-lambda (Vlambda) gene in the same RF-cultures and their subclones (i.e., <1.2 x 10(-4)mut/bp). To our knowledge this is the first reported clonal cell line that generates SHM in the VH, but not in the Vlambda. It may be due to abrogation of a cis-regulatory element(s) in the Vlambda or to a lack of a specific trans-acting factor which differentially direct the SHM machinery to this gene. Out of the 17 point mutations detected in both cell lines there were, 1 stop codon, 3 mutations which obliterated the binding of the RF antibody to its IgG antigen and 1 or 2 mutations which enhanced antigen-binding affinity. These results show that the evolutionary developed germline encoded antibody combining site is highly sensitive to amino acid replacements. Our combined findings that the RF cells accumulate in a stepwise manner up to 1-2 point mutations/sequence per cell division and the generation of high percentage of functionally deleterious mutations, are in accord with the 'multiphase-recycling model' of SHM, which states that B cells in the germinal center are subjected to multiple rounds of somatic mutations interchanged with periods of antigenic selection.

Antibody response and resistance of Cyprinus carpio immunized with cyprinid herpes virus 3 (CyHV-3).

CyHV-3 (Cyprinid herpesvirus-3) is a large DNA virus that causes a fatal disease in koi and common carp. Infection with wild type or attenuated virus induces an immune response that renders the fish resistant to further virus challenges. The kinetics and affinity of the antibody response in immune fish depend on the temperature of the water. Virus-inoculated fish produce anti-CyHV-3 antibodies, which gradually decrease during 280 days post infection to a level slightly above that of naïve fish. The protection against the virus is proportional to the titer of anti-virus antibodies in recently inoculated fish. Nevertheless, these immunized fish, even with no-longer detectable antibodies, are resistant to virus infection, probably due to the subsequent rapid response of high affinity anti-virus antibodies. The fact that anti-virus antibodies neutralize in vitro the pathogenic effects of the virus emphasizes the central role probably played by the antibodies in anti-CyHV-3 protection in vivo.

Developing injectable immunoglobulins to treat cognitive impairment in Alzheimer's disease.

BACKGROUND: Alzheimer's disease is a devastating disorder, clinically characterized by a comprehensive cognitive decline. The novel strategy of anti-amyloid-beta immunotherapy has been suggested following encouraging results obtained in murine models of Alzheimer's disease, in non-human primates, and in small-scale clinical trials. OBJECTIVE: To examine the choice between active or passive anti-amyloid-beta immunization and the choice of the molecule to which the immune machinery should be targeted, which are central issues in future immune therapy of Alzheimer's disease. METHODS: Research into the new area of Alzheimer's disease immune therapy is primarily based on in vivo and in vitro studies of murine models of Alzheimer's disease. The studies are hence limited to defined genetic deficiencies. RESULTS/CONCLUSIONS: In humans, infusion of anti-amyloid-beta antibodies is considered a safer approach than active anti-amyloid-beta vaccination. Alzheimer's-disease-protective anti-amyloid-beta monoclonal antibodies should target specific epitopes within the amyloid beta(1 42) peptide, avoiding possibly harmful binding to the ubiquitous normal amyloid precursor protein. Since Alzheimer's disease immunotherapy requires repeated infusion of antibodies over a prolonged period of time, Alzheimer's disease patients will tolerate such antibodies provided the latter are exclusively of human origin. Human monoclonal antibodies that correspond to ubiquitous anti-amyloid-beta, present in all healthy humans, might bear important protective characteristics.

Anti-amyloid beta protein antibody passage across the blood-brain barrier in the SAMP8 mouse model of Alzheimer's disease: an age-related selective uptake with reversal of learning impairment.

Amyloid beta protein (Abeta) levels are elevated in the brain of Alzheimer's disease patients. Anti-Abeta antibodies can reverse the histologic and cognitive impairments in mice which overexpress Abeta. Passive immunization appears safer than vaccination and treatment of patients will likely require human rather than xenogenic antibodies. Effective treatment will likely require antibody to cross the blood-brain barrier (BBB). Unfortunately, antibodies typically cross the BBB very poorly and accumulate less well in brain than even albumin, a substance nearly totally excluded from the brain. We compared the ability of two anti-Abeta human monoclonal IgM antibodies, L11.3 and HyL5, to cross the BBB of young CD-1 mice to that of young and aged SAMP8 mice. The SAMP8 mouse has a spontaneous mutation that induces an age-related, Abeta-dependent cognitive deficit. There was preferential uptake of intravenously administered L11.3 in comparison to HyL5, albumin, and a control human monoclonal IgM (RF), especially by hippocampus and olfactory bulb in aged SAMP8 mice. Injection of L11.3 into the brains of aged SAMP8 mice reversed both learning and memory impairments in aged SAMP8 mice, whereas IgG and IgM controls were ineffective. Pharmacokinetic analysis predicted that an intravenous dose 1000 times higher than the brain injection dose would reverse cognitive impairments. This predicted intravenous dose reversed the impairment in learning, but not memory, in aged SAMP8 mice. In conclusion, an IgM antibody was produced that crosses the BBB to reverse cognitive impairment in a murine model of Alzheimer's disease.

Somatic mutations and activation-induced cytidine deaminase (AID) expression in established rheumatoid factor-producing lymphoblastoid cell line.

Epstein-Barr virus (EBV) transforms human peripheral B cells into lymphoblastoid cell lines (LCLs), allowing the production of specific antibody-secreting cell lines. We and others have previously found that in contrast to peripheral blood B cells, EBV-transformed lymphoblastoid cell lines express the activation-induced cytidine deaminase (AID) gene. The opposite is true for the germinal center-specific BCL6 gene: it is expressed in adult peripheral blood B cells and is no longer expressed in LCLs. The present work extends our findings and shows that whereas AID expression is rapidly induced following EBV infection, BCL6 expression is gradually down-regulated and is fully extinguished in already established LCLs. The question of whether AID activation induces the process of somatic hypermutation (SHM) was investigated in adult-derived LCLs. It was found that the VH gene from the rheumatoid factor-producing RF LCL (derived from a rheumatoid arthritis patient), accumulated somatic point mutations in culture. Overall, nine unique mutations have accumulated in the rearranged VH gene since the generation of the RF cell line. Four additional intraclonal mutations were found among 10 cellular clones of the RF cells. One out of the four was in CDR1 and could be correlated with loss of antigen-binding activity in three out of the 10 clones. Altogether, these 13 mutations were preferentially targeted to the DGYW motifs and showed preference for CG nucleotides, indicating that they were AID-mediated. By contrast, mutations were not detected among 3700-4000 nucleotides each of the Vlambda, Cmu and GAPDH genes derived from the same RF cell cultures and the cellular clones. Our results thus show that AID may generate point mutations in the rearranged Ig VH during in vitro cell culture of adult-LCLs and that these mutations may be responsible, at least in part, for the known instability and occasional loss of antigen-binding activity of antibody-secreting LCLs.

Detection of circulating antibodies against tau protein in its unphosphorylated and in its neurofibrillary tangles-related phosphorylated state in Alzheimer's disease and healthy subjects.

While the presence of naturally occurring antibodies (Abs) against amyloid-beta in AD patients and healthy subjects have been repeatedly reported, no data on the presence of naturally occurring Abs against tau protein, in its unphosphorylated as well as its pathologically phosphorylated state, has been reported so far. We describe here the detection of circulating Abs against unphosphorylated and pathologically phosphorylated tau protein in sera of 17 aged subjects: nine Alzheimer's disease (AD) patients and eight healthy individuals. An ongoing autoimmune process may take place, as is suggested by the presence of both IgM class anti-tau Abs, as well as IgG. A preliminary evidence for higher anti-phosphorylated-tau Abs of IgM class in AD patients relative to controls is indicated, but demands further confirmation in a larger sample. Detection of naturally occurring anti-tau antibodies may point to the possibility that some autoimmune process may take place against the tau neuronal protein, including its pathologically phosphorylated state which compose the neurofibrillary tangles. Whether these Abs are neuroprotective or neurotoxic - is unknown, as it is with anti-amyloid-beta Abs.

Immunotherapy of Alzheimer's disease (AD): from murine models to anti-amyloid beta (Abeta) human monoclonal antibodies.

The deposition of amyloid beta (Abeta) protein is a key pathological feature in Alzheimer's disease (AD). In murine models of AD, both active and passive immunization against Abeta induce a marked reduction in amyloid brain burden and an improvement in cognitive functions. Preliminary results of a prematurely terminated clinical trial where AD patients were actively vaccinated with aggregated Abeta bear resemblance to those documented in murine models. Passive immunization of AD patients with anti-Abeta antibodies, in particular human antibodies, is a strategy that provides a more cautious management and control of any undesired side effects. Sera of all healthy adults contain anti-Abeta IgG autoimmune antibodies. Hence antigen-committed human B-cells are easily immortalized by Epstein-Barr virus (EBV) into anti-Abeta secreting cell lines. Two anti-Abeta human monoclonal antibodies which we recently prepared bind to the N-terminus of Abeta peptide and were shown to stain amyloid plaques in non-fixed brain sections from an AD patient. It is anticipated that specifically selected anti-Abeta human monoclonal antibodies could reduce and inhibit deposits of amyloid in brain while avoiding the cognitive decline that characterizes AD. In the future, this type of antibody may prove to be a promising immune therapy for the disease.

Human monoclonal antibodies against amyloid-beta from healthy adults.

Two anti-amyloid-beta human antibody-producing cell lines were established from amyloid-beta (Abeta)-selected lymphocytes from peripheral blood of healthy adults. ELISA and Western blot analysis showed that the monoclonal antibodies bound with high affinity to the 43 amino acid-long amyloid-beta peptide. The antigen epitope of these antibodies encountered within amino acids 1-16 of the amyloid-beta peptide. The antibodies did not bind to several immunoglobulin light chain amyloids (AL) and amylin. One of the monoclonals was tested by immunohistochemistry for the binding to frozen sections of brains derived from patients with Alzheimer's disease. It specifically and intensively stained diffuse and core amyloid-beta plaques; whereas, sections from normal brains were not stained. Concomitant staining with a commercial mouse anti-amyloid-beta monoclonal antibody co-localized with that of the human antibody. Simultaneous staining with the human antibody and Congo red implied that the antibody binds primarily to an early immature form of beta-amyloid. Human monoclonal antibodies, which resemble physiologically normal non-pathogenic and possibly protective antibodies in healthy adults, might be attractive candidates for immune therapy of Alzheimer's disease.

A peptide mimotope of type 8 pneumococcal capsular polysaccharide induces a protective immune response in mice.

Increasing antibiotic resistance and a rising patient population at risk for infection due to impaired immunity underscore the importance of vaccination against pneumococci. However, available capsular polysaccharide vaccines are often poorly immunogenic in patients at risk for pneumococcal disease. The goal of this study was to explore the potential of peptide mimotopes to function as alternative vaccine antigens to elicit a type-specific antibody response to pneumococci. We used a human monoclonal immunoglobulin A (IgA) antibody (NAD) to type 8 Streptococcus pneumoniae capsular polysaccharide (type 8 PS) to screen a phage display library, and the phage PUB1 displaying the peptide FHLPYNHNWFAL was selected after three rounds of biopanning. Inhibition studies with phage-displayed peptide or the peptide PUB1 and type 8 PS showed that PUB1 is a mimetic of type 8 PS. PUB1 conjugated to tetanus toxoid (PUB1-TT) induced a type 8 PS-specific antibody response in BALB/c mice, further defining it as a mimotope of type 8 PS. The administration of immune sera obtained from PUB1-TT-immunized mice earlier (days 14 and 21) and later (days 87 and 100) after primary and reimmunization resulted in a highly significant prolongation of the survival of naive mice after pneumococcal challenge compared to controls. The survival of PUB1-TT-immunized mice was also prolonged after pneumococcal challenge nearly 4 months after primary immunization. The efficacy of PUB1-TT-induced immune sera provides proof of principle that a mimotope-induced antibody response can protect against pneumococci and suggests that peptide mimotopes selected by type-specific human antibodies could hold promise as immunogens for pneumococci.

Abrogation of Vif function by peptide derived from the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) protease.

The human immunodeficiency virus type 1 (HIV-1) auxiliary gene vif is essential for virus propagation in peripheral blood lymphocytes, macrophages, and in some T-cell lines. Previously, it was demonstrated that Vif inhibits the autoprocessing of truncated HIV-1 Gag-Pol polyproteins expressed in bacterial cells, and that purified recombinant Vif and Vif-derived peptides inhibit and bind HIV-1 protease (PR). Here we show that Vif interacts with the N-terminal region of HIV-1 PR, and demonstrate that peptide derived from the N-terminal region of PR abrogates Vif function in non-permissive cells. Specifically, we show that (i) Vif protein binds HIV-1 PR, but not covalently linked tethered PR-PR; (ii) the four amino acids residing at the N terminus of HIV-1 PR are essential for Vif/PR interaction; (iii) synthetic peptide derived from the N terminus of HIV-1 PR inhibits Vif/PR binding; and (iv) this peptide inhibits the propagation of HIV-1 in restrictive cells. Based on these data, we suggest that Vif interacts with the dimerization sites of the viral protease, and that peptide residing at the N terminus of PR abrogates Vif function(s).

Pathogenesis of acute viral disease induced in fish by carp interstitial nephritis and gill necrosis virus.

A lethal disease of koi and common carp (species Cyprinus carpio) has afflicted many fish farms worldwide since 1998, causing severe financial losses. Morbidity and mortality are restricted to common carp and koi and appear in spring and autumn, when water temperatures are 18 to 28 degrees C. We have isolated the virus causing the disease from sick fish, propagated it in koi fin cell culture, and shown that virus from a single clone causes lethal disease in carp and koi upon infection. Intraperitoneal virus injection or bathing the fish in virus-containing water kills 85 to 100% of the fish within 7 to 21 days. This virus is similar to the previously reported koi herpesvirus; however, it has characteristics inconsistent with the herpesvirus family, and thus we have called it carp interstitial nephritis and gill necrosis virus. We examined the pathobiology of this disease in carp by using immunohistochemistry and PCR. We found large amounts of the virus in the kidneys of sick fish and smaller amounts in liver and brain. A rapid increase in the viral load in the kidneys was detected by using both immunofluorescence and semiquantitative PCR. Histological analyses of fish at various times after infection revealed signs of interstitial nephritis as early as 2 days postinfection, which increased in severity up to 10 days postinfection. There was severe gill disease evidenced by loss of villi with accompanying inflammation in the gill rakers. Minimal focal inflammation was noted in livers and brains. This report describes the etiology and pathology of a recently described viral agent in fish.

Vaccination with Ep-CAM protein or anti-idiotypic antibody induces Th1-biased response against MHC class I- and II-restricted Ep-CAM epitopes in colorectal carcinoma patients.

PURPOSE: The tumor-associated antigen Ep-CAM (epithelial cell adhesion molecule) is overexpressed in colorectal carcinoma (CRC). The aim of the present study was to evaluate and compare the safety and immunogenicity of a recombinant Ep-CAM protein and a human anti-idiotypic antibody (anti-Id) mimicking Ep-CAM. EXPERIMENTAL DESIGN: Patients with resected American Joint Committee on Cancer stages II-IV CRC without remaining macroscopic disease received intradermal/subcutaneous injections of Ep-CAM (400 microg/dose; n = 7) or anti-Id (500 microg/dose; n = 6) at weeks 0, 2, and 6 in combination with granulocyte macrophage colony-stimulating factor (75 microg/day, for 4 consecutive days). RESULTS: Adverse reactions were mild (grade I-II). All patients immunized with the Ep-CAM protein produced Ep-CAM-specific IgG antibodies, predominantly IgG1 and IgG3 subclasses, whereas no humoral response was induced by the anti-Id vaccine. All patients, with one exception in each group, mounted an Ep-CAM-specific proliferative T-cell response. The immune response was more rapid, potent, and protracted after Ep-CAM in comparison with anti-Id vaccination. Interferon-gamma-secreting cells (ELISPOT) were detected in both immunization groups against the Ep-CAM protein as well as various Ep-CAM-derived MHC class I- and II-restricted peptides. Flow cytometry analysis showed that Ep-CAM-specific interferon-gamma- and perforin-producing cells predominantly resided within CD8(+)CD56- and CD8(dim)CD56+ T cells. CONCLUSIONS: Ep-CAM protein in combination with granulocyte macrophage colony-stimulating factor induced a long-lasting, Th1-biased humoral and cellular immune response compared with anti-Id. Ep-CAM-specific T cells and natural killer-like T cells responding in a MHC class I- and II-restricted manner were also induced. Vaccination with Ep-CAM protein may warrant further investigation as a novel therapeutic approach to CRC.

Modulation of polymorphonuclear cell interleukin-8 secretion by human monoclonal antibodies to type 8 pneumococcal capsular polysaccharide.

Pneumococcal capsular polysaccharide (PS) vaccines induce type-specific immunoglobulin M (IgM), IgG, and IgA. Type-specific IgG to the PS is sufficient to confer protection against the homologous serotype of the pneumococcus, but the efficacies of type-specific IgM and IgA are less well understood. We examined the in vitro activities and efficacies in mice of two human monoclonal antibodies (MAbs) to type 8 PS, NAD (IgA) and D11 (IgM). MAb-mediated opsonophagocytic killing was evaluated after coculture of type 8 pneumococci with human polymorphonuclear cells (PMNs), type-specific or control MAbs, and human complement sources. The effects of the MAbs on PMN interleukin-8 (IL-8) and IL-6 secretion were determined in supernatants from cocultures containing pneumococci and PMNs by enzyme-linked immunosorbent assay. MAb efficacy was determined in an intratracheal model of type 8 infection in mice with classical complement pathway deficiency. Both MAbs were protective in 100% of infected mice. Neither MAb promoted a significant amount of killing of type 8 pneumococci compared to its isotype control MAb. Both type-specific MAbs mediated complement-dependent modulation of PMN IL-8 secretion, with increased secretion at effector/target (E:T) ratios of 500:1 and 50:1 and reduced secretion at 1:5. Trypan blue staining revealed that PMNs cocultured with D11 were less viable at an E:T ratio of 1:5 than PMNs cocultured with the control MAb. PMN IL-6 secretion was increased by both type-specific and control MAbs. These results suggest that certain type-specific IgM and IgAs might contribute to host defense by modulation of the inflammatory response to pneumococci.

Efficient vaccine against the virus causing a lethal disease in cultured Cyprinus carpio.

We have isolated a virus, which causes a mortal disease in cultured ornamental Koi and Common carps (Cyprinus carpio) in many countries worldwide. This unclassified virus, which causes nephritis and gill necrosis, and so has been given the name carp nephritis and gill necrosis virus (CNGV), has a morphology resembling the herpes virus, but bears a genomic DNA of ca 250-300 kbp. So far, both others and we have been unable to find CNGV-DNA sequences possessing a significant similarity to known DNA viruses. The virus induces a lethal disease when water temperature ranges between 18 and 25 degrees C (permissive temperature). In this report, we demonstrate that carps, exposed to the virus at 23 degrees C for 3-5 days and then transferred to the non-permissive temperature of 30 degrees C, became resistant to a challenged infection and their sera demonstrated a high level of virus-specific antibodies. We have isolated attenuated non-pathogenic viruses that render virus-vaccinated carps resistant to the disease. Furthermore, vaccinated fish developed high levels of antibodies against the virus. We suggest, therefore, that this attenuated virus could be used as a live vaccine for the eradication of the mortal disease afflicting Common and ornamental carp fisheries in many countries.