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Folding of the human coxsackie and adenovirus receptor immunoglobulin (Ig) variable-type domain (CAR D1) during overexpression in the Escherichia coli cytoplasm was shown previously to be partially rescued by fusion to a 22-residue C-terminal peptide. Here, peptide sequence features required for solubilization and folding of CAR D1 and similar Ig variable-type domains from two other human membrane proteins were investigated. Peptide extensions with net negative charge > -6 fully solubilized CAR D1, and approximately half of the peptide-solubilized protein was correctly folded. The Ig variable-type domains from human A33 antigen and myelin P-zero proteins were only partially solubilized by peptide extensions with net charge of -12, however, and only the solubilized P-zero domain appeared to fold correctly whereas the A33 domain formed soluble microaggregates of misfolded protein. Our results suggest a model where the large net charge of peptide extensions increases electrostatic repulsion between nascent polypeptides. The resulting decrease in aggregation rate can enable some polypeptides to fold spontaneously into their native protein conformations. Analysis of the solubility and folding status of sets of structurally homologous proteins, such as the Ig variable-type domains described here, during overexpression could provide insights into how amino acid and gene sequences influence the efficiency of spontaneous protein folding.

Signaling through the B cell antigen receptor (BCR) induces activation and proliferation of B cells, a response that requires the adaptor protein BASH (also known as BLNK/SLP-65). Although BASH and other molecules, such as Btk, PLCgamma2 and PKCbeta, are known to be essential for T cell-independent immune responses in vivo, their requirement during T cell-dependent immune responses, especially their role in antibody affinity-maturation and memory B cell generation remains unclear. In this study, we examined primary and memory immune responses to the T cell-dependent hapten antigen, (4-hydroxy-3-nitrophenyl)acetyl (NP) conjugated to chicken gammaglobulin (CGG), in BASH-deficient mice on a C57BL/6 background. In the primary response, NP-specific IgM was barely produced and the typical anti-NP IgG1/lambda production was markedly attenuated, but kappa chain was unexpectedly over-represented in the anti-NP antibodies. In contrast, CGG-specific IgG1 was normally produced. In the memory response, IgG1/lambda antibody with high affinity to NP was produced at normal level in the mutant mice. The frequency and distribution of somatic mutations in the V(H)186.2 genes of the anti-NP IgG1/lambda antibody were also normal. These results indicate that BASH-mediated BCR signaling is dispensable for somatic hypermutation and affinity selection, as well as generation and response of memory B cells. Interestingly, mutated V(H) genes with the same clonal origin were prominent in the anti-NP antibodies of BASH-deficient mice, indicating that a limited number of original clones had been recruited into the memory compartment. Thus, the scarcity of specific clones in the primary repertoire and an impaired primary response is not detrimental to the quality and quantity of a memory response.

Recently, the existence of "heavy-chain" antibody in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to peptides. In addition, there was not any report of production of single-domain antibodies in two-humped camels (Camelus bactrianus). In the present study, these questions are addressed. We showed the feasibility of immunizing old world camels, cloning the repertoire of the variable domain of their heavy-chain antibodies, panning and selection, leading to the successful identification of minimum-sized antigen binders. Antigen-specific fragments of the heavy-chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable, highly soluble, and react specifically and with high affinity to the antigens. In this study, we immunized two camels (Camelus dromedarius and Camelus bactrianus) with homogenized cancerous tissues, synthetic peptide, and human milk fat globule membrane (HMFG), and generated two V(HH) libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the tandem repeat region of MUC1. The camels' single-domain V(HH) harbor the original, intact antigen binding site and reacted specifically and with high affinity to the tandem repeat region of MUC1. Indeed soluble, specific antigen binders and good affinities (in the range of 0.2 x 10(9) M(-1) to 0.6 x 10(9) M(-1)) were identified from these libraries. This is the first example of the isolation of camel anti-peptide V(HH) domains.

Single chain antibodies (scFv) exhibiting specific binding to Listeria monocytogenes strains were isolated from a pool of random scFvs expressed on the surface of filamentous bacteriophages. Positive selection (panning) using L. monocytogenes was used to enrich for phage clones with the desired binding affinity, and negative selection using L. innocua and L. ivanovii was used to remove phages expressing cross-reactive antibody fragments. A single phage clone, P4:A8, was selected using two independent panning schemes. A rapid assay was devised to determine phage antibody binding specificity and was used to develop a selectivity profile for individual phage clones. The P4:A8 clone was screened against a panel of bacteria consisting of eight strains of L. monocytogenes, one each of the other six species of Listeria and nine other relevant bacterial species. A collection of individual clones from the penultimate panning was also screened against a subset of the panel of bacteria. The selectivity profiles indicate that multiple clones, including P4:A8, exhibit binding to one or more strains of L. monocytogenes without cross-reactivity toward any other species in the panel. This is the first report of a species-specific antibody for viable cells of L. monocytogenes (i.e., the ability to bind to L. monocytogenes without cross-reactivity toward any other species of Listeria).

N-Linked carbohydrates are frequently found in the V region of Ig H chains and can have a positive or negative effect on Ag binding affinity. We have studied a murine anti-alpha(1-->6) dextran V(H) that contains a carbohydrate in complementarity-determining region 2 (CDR2). This carbohydrate remains high mannose rather than being processed to a complex form, as would be expected for glycans on exposed protein loops. We have shown that the glycan remained high mannose when murine-human chimeric Abs were produced in a variety of cell types. Also, when another carbohydrate was present in CDR1, CDR2, or CDR3 of the L chain, the V(H) CDR2 glycan remained high mannose. Importantly, we found that when the anti-dextran V(H) CDR2 replaced CDR2 of an anti-dansyl V(H), the glycosylation site was used, but H chains were withheld in the endoplasmic reticulum and did not traffic to the Golgi apparatus. These results suggest that inappropriate V region glycosylation could contribute to ineffective Ab production from expressed Ig genes. In some cases, a carbohydrate addition sequence generated by either V region rearrangement or somatic hypermutation may result in an Ab that cannot be properly folded and secreted.

Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.

Most mAbs to the capsular polysaccharide glucuronoxylomannan (GXM) of Cryptococcus neoformans are generated from the same VH and VL gene families. Prior Ab studies have assessed protective efficacy, Id structure and binding to capsular polysaccharides, and peptide mimetics. These data have been interpreted as indicating that most mAbs to GXM have the same specificity. A new approach to Ab specificity analysis was investigated that uses genetic manipulation to generate C. neoformans variants with structurally different capsules. C. neoformans mutants expressing GXM with defective O-acetylation were isolated and complemented by the C. neoformans gene CAS1, which is necessary for the O-acetylation of GXM. The mAbs exhibited differences in their binding to the GXM from these mutant strains, indicating previously unsuspected differences in specificity. Analysis of three closely related IgMs revealed that one (mAb 12A1) bound to an epitope that did not require O-acetylation, another (mAb 21D2) was inhibited by O-acetylation, and the third (mAb 13F1) recognized an O-acetylation-dependent conformational epitope. Furthermore, an IgG Ab (mAb 18B7) in clinical development retained binding to de-O-acetylated polysaccharide; however, greater binding was observed to O-acetylated GXM. Our findings suggest that microbial genetic techniques can provide a new approach for epitope mapping of polysaccharide-binding Abs and suggest that this method may applicable for studying the antigenic complexity of polysaccharide Ags in other capsulated microorganisms.

The bone marrow is an important source of Abs involved in long-term protection from recurrence of infections. Allogenic bone marrow transplantation (BMT) fails to restore this working memory. Attempts to overcome this immunodeficiency by immunization of the donor have not been very successful. More needs to be known about transfer of B cell memory by BMT. We tracked memory B cells from the donor to the recipient during BMT of a girl with leukocyte adhesion deficiency. Vaccination of her HLA-identical sibling donor 7 days before harvest induced Haemophilus influenzae type b (Hib) capsular polysaccharide (HibCP)-specific B cells readily detectable in marrow and blood. BMT did not lead to spontaneous production of HibCP Abs, but the recipient responded well to booster immunizations 9 and 11 mo after BMT. HibCP-specific B cells were obtained 7 days after the vaccinations, and their V(H) genes were sequenced and analyzed for rearrangements and unique patterns of somatic hypermutations identifying clonally related cells. Ninety (74%) of 121 sequences were derived from only 16 precursors. Twelve clones were identified in the donor, and representatives from all of them were detected in the recipient where they constituted 61 and 68% of the responding B cells after the first and second vaccinations, respectively. No evidence for re-entry of memory clones into the process of somatic hypermutation was seen in the recipient. Thus, memory B cells were transferred from the donor, persisted for at least 9 mo in the recipient, and constituted the major part of the HibCP-specific repertoire.

Aim: To construct a human phage displayed single chain Fv antibody (scFv) library and screen specific scFv against rabies virus., Methods: Using a phage-display technique, IgVH and IgVL genes were cloned from peripheral blood lymphocytes from three donors immunized with the WISTAR PM strain vaccine. Genes encoding scFv fragments were constructed by random linkage of VH gene and VL gene by SOE PCR, and then the constructed scFv genes were cloned into phagemid vector and transfected into E. coli TG1. The recombinant phages were screened by four rounds of panning with rabies virus vaccine. The positive recombinant phages were sequenced and antigen-binding activity of recombinant scFv was detected by competition ELISA., Results: A human phage-displayed scFv library with about 7 x 10(8) sink size was constructed. A new human scFv was selected, which bound specifically to rabies virus and had higher affinity., Conclusion: The results lay a solid foundation for preparation of human engineering antibody to rabies virus reported herein with higher affinity.

Use of the serogroup B meningococcal capsular polysaccharide (MenB CP) as a vaccine is hampered by the presence of epitopes that cross-react with human polysialic acid. As non-cross-reactive, protective capsular epitopes have also been described, we set out to develop protein mimics of one of such epitopes using as a template a highly protective mAb (mAb Seam 3) raised against a chemically modified form of the MenB CP (N-Pr MenB CP). Using phage display, anti-idiotypic single-chain Ab fragments (scFvs) were obtained from spleen cells of mice immunized with the Seam 3 mAb. Two Seam 3-specific scFvs competed with N-Pr MenB CP for binding to either mAb Seam 3 or rabbit Abs present in typing sera. Moreover, in mice and rabbits the scFvs elicited the production of Abs reacting with both N-Pr MenB CP and whole meningococci, but not with human polysialic acid. These scFv-induced Ab responses were boostable and of the Th1 type, as shown by a predominance of IgG2a. In addition, passive immunization with sera from scFv-immunized animals partially protected neonatal mice from experimental infection with group B meningococci. In conclusion, we have produced anti-idiotypic scFvs that mimic a protective MenB CP epitope and may be useful in the development of an alternative group B meningococcal vaccine.

Avian coccidiosis is due to a protozoan intracellular parasite belonging to the genus Eimeria which multiplies in the intestine of the host. In order to identify Eimeria antigens which reflect the natural avian humoral immune response, chicken hybridomas were produced by fusion of myeloma MuH1 with B lymphocytes from Eimeria tenella infected chicken. B lymphocytes used for fusions were isolated from tonsils at the basis of caeca where the parasite develops. One of the clones (G1F5) recognised oocyst antigens and the macrogamont stage of the parasite in ELISA and immunofluorescence assay. A single-chain variable fragment (scFv) antibody was cloned from the light chain variant region (VL) and heavy chain variant region (VH) genes of the hybridoma. This recombinant antibody (scFv G1F5) exhibited antigen binding specificity to oocysts and macrogamonts of E. tenella equivalent to the mAb produced by the clone G1F5. Nucleotide sequence analysis of VL genes from scFv G1F5 compared to the germ-line revealed vestiges of gene conversion. scFv derived from chicken B lymphocytes isolated from the gut-associated lymphoid tissue following experimental infection can reveal specific antigens recognised by the avian immune response.

Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance. In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT. We studied the mechanism of bacteria-induced GALT development. Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits). We then identified specific members of the intestinal flora that promote GALT development. The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-C micro genes in appendix B cells. Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis. B. fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response. To identify bacterial pathways required for GALT development, we introduced B. fragilis along with stress-response mutants of B. subtilis into germfree-appendix rabbits. We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development. We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses.

The large interallelic distances between the three rabbit Ig V(H)a lineages, a1, a2 and a3, suggest that the persistence time of the V(H)a polymorphism could amount to 50 million years, which is much longer than that of MHC polymorphisms. Rabbit originated in the Iberian Peninsula where two subspecies coexist, one of which is confined to Southwestern Iberia (Oryctolagus cuniculus algirus). We studied the V(H) loci in the original species range to obtain a better understanding of the evolutionary history of this unusual polymorphism. Serological surveys revealed that sera from the subspecies algirus, when tested with V(H)a locus-specific alloantisera, showed either cross-reactivity ("a-positive" variants) or no reaction at all ("a-blank"). Using RT-PCR, we determined 120 sequences of rearranged V(H) genes expressed in seven algirus rabbits that were typed as either a-positive or a-blank. The data show that the V(H) genes transcribed in a-positive rabbits are closely related to the V(H)1 alleles of domestic rabbits. In contrast, a-blank rabbits were found to preferentially use V(H) genes that, although clearly related to the known V(H)a genes, define a new major allotypic lineage, designated a4. The a4 sequences have hallmark rabbit V(H)a residues together with a number of unprecedented amino acid changes in framework region 2 and 3. The net protein distances between the V(H)a4 and the V(H)a1, a2, and a3 lineages were 20, 29, and 21% respectively. We conclude that at least four distantly related lineages of the rabbit V(H)a locus exist, one of which seems to be endemic in the Iberian range.

Previous studies have suggested that B cell Ag receptor (BCR) down-regulation by potentially pathological autoreactive B cells is associated with pathways leading to developmental arrest and receptor editing, or anergy. In this study we compare the primary development of B cells in two strains of mice expressing transgenic BCRs that differ by a single amino acid substitution that substantially increases reactivity for nuclear autoantigens such as DNA. Surprisingly, we find that both BCRs promote efficient development to mature follicular phenotype, but the strongly autoreactive BCR fails to promote marginal zone B cell development. The follicular B cells expressing the strongly autoreactive BCR do not appear to be anergic, as they robustly respond to polyclonal stimuli in vitro, are not short-lived, and can participate in germinal center reactions. Strikingly however, substantial and progressive down-modulation of surface IgM and IgD takes place throughout their primary development in the BM and periphery. We propose that BCR-autoantigen interactions regulate this pathway, resulting in reduced cellular avidity for autoantigens. This process of "learned ignorance" could allow autoreactive B cells access to the foreign Ag-driven memory B cell response, during which their self-reactivity would be attenuated by somatic hypermutation and selection in the germinal center.

The accurate partitioning of Ig H chain V(H)DJ(H) junctions and L chain V(L)J(L) junctions is problematic. We have developed a statistical approach for the partitioning of such sequences, by analyzing the distribution of point mutations between a determined V gene segment and putative Ig regions. The establishment of objective criteria for the partitioning of sequences between V(H), D, and J(H) gene segments has allowed us to more carefully analyze intervening putative nontemplated (N) nucleotides. An analysis of 225 IgM H chain sequences, with five or fewer V mutations, led to the alignment of 199 sequences. Only 5.0% of sequences lacked N nucleotides at the V(H)D junction (N1), and 10.6% at the DJ(H) junction (N2). Long N regions (>9 nt) were seen in 20.6% of N1 regions and 17.1% of N2 regions. Using a statistical analysis based upon known features of N addition, and mutation analysis, two of these N regions aligned with D gene segments, and a third aligned with an inverted D gene segment. Nine additional sequences included possible alignments with a second D segment. Four of the remaining 40 long N1 regions included 5' sequences having six or more matches to V gene end motifs, which may be the result of V gene replacement. Such sequences were not seen in long N2 regions. The long N regions frequently seen in the expressed repertoire of human Ig gene rearrangements can therefore only partly be explained by V gene replacement and D-D fusion.

ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs. In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv. The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain. Selected scFvs derived from an anti-MUC-1 scFv phage library were expressed in pCANTAB 5E and its modified version pCANTAB 5E Cys vectors, and compared for key characteristics. Production yields of scFv and scFv-Cys in shaker flask and biofermentor were compared. In the absence of a reducing agent, stable dimers (covalent scFv homodimers (scFv-Cys)2) were the major form of scFv-Cys. These diabodies provided substantial signal enhancement for immunohistochemical staining of tissues. In the presence of a reducing agent, scFv-Cys molecules remained monomeric, with the free SH available for conjugation to a PEG(maleimide)2 scaffold to form immunoreactive PEG(scFv)2 bioconjugates. ScFv expression from pCANTAB 5E Cys allowed for the production of soluble scFv-Cys protein from E.coli, either as stable scFv-Cys or (scFv-Cys)2. ScFv-Cys can be used for conjugation to PEG to form bivalent PEG (scFv-Cys)2 molecules or used as (scFv-Cys)2 for increased sensitivity in IHC.

Oligonucleotide primers were designed for recovery of Ig H, kappa and lambda transcripts from porcine splenic cDNA. The products were cloned and scFvs constructed in a phage display vector. E. coli HB2151 was transformed with the constructs and upon induction, scFvs of the predicted molecular weight could be detected in culture supernatants by Western blotting and immunoprecipitation with anti-pig IgG. Bacteriophage displaying a swine scFv were diluted into an excess of phage carrying a human anti-thyroglobulin scFv and then panned on plastic coated with anti-pig IgG. Rapid enrichment of phage carrying the porcine scFv through three rounds of selection demonstrated the successful display of an authentically folded pig Ig at the viral surface. The data demonstrate that phage display techniques can be applied successfully to porcine Igs and that expression of recombinant pig scFvs in bacteria can be achieved. These techniques offer the opportunity to generate authentic porcine monoclonal reagents for basic and applied studies.

The methods described in this article are relative to the use of a positive cloning/screening recombinant system for the generation in Escherichia coli of foreign proteins fused to a highly active bacterial alkaline phosphatase (PhoA) variant as reporter enzyme. Appropriate insertion of the DNA encoding the foreign peptides, proteic domains, or proteins between codons +6 and +7 of the phoa gene restores the initial frame of the phoa gene in the vector. Consequently, only recombinant clones appear as blue colonies when plating onto an agar medium containing a chromogenic substrate for PhoA. The presence of an intact PhoA signal peptide yields to a systematic secretion of the fusion proteins into the periplasm where the PhoA dimerises to its active form, and disulfides can be formed if necessary. The resultant PhoA-tagged proteins are particularly convenient novel tools that can be used in a wide range of applications, including expression, epitope mapping, histochemistry, immunoblotting, mutant analysis, and competition or sandwich ELISAs. Expression of an scFv antibody fragment derived from an IgG2a/kappa immunoglobulin specific for curaremimetic toxins from snake (named M-alpha2-3), will be used to illustrate the methods utilized for its cloning, expression in E.coli, extraction, and functional characterization.

Deoxyribonucleic acid double-stranded breaks act as intermediates in Ig V(D)J recombination and probably perform a similar function in class switch recombination between IgH C genes. In SCID mice, V(D)J recombination is blocked because the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein is defective. We show in this study that switching to all isotypes examined was detectable when the SCID mutation was introduced into anti-hen egg lysozyme transgenic B cells capable of undergoing class switch recombination, but switching was significantly reduced in comparison with control B cells of the same specificity lacking the RAG1 gene. Thus, DNA-PKcs is involved in switching to all isotypes, but plays a lesser role in the switching process than it does in V(D)J-coding joint formation. The higher level of switching observed by us in SCID B cells compared with that observed by others in DNA-PKcs(null) cells raises the possibility that kinase-deficient DNA-PKcs can function in switching. Point mutation of G:C base pairs with cytidines on the sense strand was greatly reduced in recombined switch regions from SCID cells compared with control RAG1(-/-) B cells. The preferential loss of sense strand cytidine mutations from hybrid S regions in SCID cells suggests the possibility that nicks might form in S regions of activated B cells on the template strand independently of activation-induced cytidine deaminase and are converted to double-strand breaks when activation-induced cytidine deaminase deaminates the non-template strand.

In mammals that use gut-associated lymphoid tissues for expansion and somatic diversification of the B cell repertoire, B lymphopoiesis occurs early in ontogeny and does not appear to continue throughout life. In these species, including sheep, rabbit, and cattle, little is known about the pathway of B cell development and the time at which B lymphopoiesis wanes. We examined rabbit bone marrow by immunofluorescence with anti-CD79a and anti-mu and identified both proB and preB cells. The proB cells represent the vast majority of B-lineage cells in the bone marrow at birth and by incorporation of 5-bromo-2'-deoxyuridine, they appear to be a dynamic population. PreB cells reach maximum levels in the bone marrow at 3 wk of age, and B cells begin to accumulate at 7 wk of age. We cloned two VpreB and one lambda5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and lambda5 coimmunoprecipitated with the mu-chain in lysates of 293T cells transfected with VpreB, lambda5, and mu, indicating that VpreB, lambda5, and mu-chains associate in a preB cell receptor-like complex. By 16 wk of age, essentially no proB or preB cells are found in bone marrow and by PCR amplification, B cell recombination excision circles were reduced 200-fold. By 18 mo of age, B cell recombination excision circles were reduced 500- to 1000-fold. We suggest that B cell development in the rabbit occurs primarily through the classical, or ordered, pathway and show that B lymphopoiesis is reduced over 99% by 16 wk of age.

A single-chain fragment variable (scFv) antibody library displayed on phage was constructed using spleen cells from mice immunized with human amyloid-beta peptide (Abeta42). This first anti-Abeta42 scFv immune antibody library was selected against human Abeta42. A number of positive clones were obtained, and sequences of VH and Vkappa genes were analyzed using ExPASy and BLAST computer tools. This analysis revealed that only two unique clones with identical VH and Vkappa complementarity determining region (CDR) (except HCDR2) and identical germline genes were selected, indicating that oligoclonal immune response was occurring in Abeta42-immunized mice. Abeta42-specific scFv antibodies selected from this first immune anti-Abeta42 phage antibody library may be an important tool for the development of therapeutic molecules for Alzheimer's disease (AD).

Immunoglobulin E is produced by nasal B cells in response to allergen. We have analyzed IgE V(H) region sequences expressed in the nasal mucosa of patients suffering from allergic rhinitis. V(H) region sequences were amplified by RT-PCR from IgE(+) B cells from nasal biopsies. In two of six patients, sequence analysis clearly demonstrated the presence of closely related IgE(+) B cell clones: cells displaying identical signature regions across CDR3/FWR4, indicating a common clonal ancestry, but a mixture of shared and diverse somatic mutations across the V(H) region. Furthermore, in one of the two patients exhibiting related IgE(+) B cell clones, five IgA(+) B cell clones, related to the IgE(+) B cell family, were also isolated from the patient's nasal mucosa. This evidence, combined with the local expression of mRNA transcripts encoding activation-induced cytidine deaminase, suggests that local somatic hypermutation, clonal expansion, and class switch recombination occur within the nasal mucosa of allergic rhinitics. The presence of related B cells in the nasal mucosa does not appear to result from the random migration of IgE(+) cells from the systemic pool, as analysis of a nonatopic subject with highly elevated serum IgE did not exhibit any detectable V(H)-Cepsilon transcripts in the nasal mucosa. We have provided evidence that suggests for the first time that the nasal mucosa of allergic rhinitics is an active site for local somatic hypermutation, clonal expansion, and class switch recombination, making it of major significance for the targeting of future therapies.

Ab repertoires exhibit marked restrictions during fetal life characterized by biases of variable gene usage and lack of junctional diversity. We tested the hypothesis that Ab repertoire restriction contributes to the observed poor quality of specific Ab responses made by infants to viral infections. We analyzed the molecular determinants of B cell responses in humans to two Ags of rotavirus (RV), a common and clinically important infection of human infants. We sequenced Ab H and L chain V region genes (V(H) and V(L)) of clones expanded from single B cells responding to RV virus protein 6 or virus protein 7. We found that adults exhibited a distinct bias in use of gene segments in the V(H)1 and V(H)4 families, for example, V(H)1-46, V(H)4-31, and V(H)4-61. This gene segment bias differed markedly from the V(H)3 dominant bias seen in randomly selected adult B cells. Recombinant Abs incorporating any of those three immunodominant V(H) segments bound to RV-infected cells and also to purified RV particles. The RV-specific B cell repertoires of infants aged 2-11 mo and those of adults were highly related when compared by V(H), D, J(H), V(L), and J(L) segment selection, extent of junctional diversity, and mean H chain complementarity determining region 3 length. These data suggest that residual fetal bias of the B cell repertoire is not a limiting determinant of the quality of Ab responses to viruses of infants beyond the neonatal period.

Although the Ig H chains of anti-nuclear Abs (ANA) have been described to possess certain shared molecular signatures, it remains unclear whether the L chains of these Abs also possess distinctive molecular features. The present study examines this by generating and analyzing two comprehensive murine Ig L chain databases, one consisting of 264 monoclonal ANAs and the other consisting of 145 non-ANAs, drawn from previously published work. Importantly, clonal replicates were represented only once each, so as to minimize bias. ANAs and non-ANAs did not differ in Vkappa family or Jkappa gene usage, nor in their mutation frequencies. Interestingly, the L chains of ANAs exhibited differential usage of certain complementarity-determining region residues, arising almost entirely from the increased usage of certain Vkappa germline genes, notably, Vkappa ai4 among anti-dsDNA ANAs, Vkappa23-45 among anti-ssDNA ANAs, and Vkappa21-12 among non-ANAs. Finally, prompted by the increased prevalence of a particular Vkappa1 family sequence among ANAs, we proceeded to clone a novel New Zealand Black Vkappa1 germline gene, named bb1.1, which appears to be frequently used to encoded anti-ssDNA Abs. Collectively, these studies underline the potential contribution of particular Vkappa germline genes in promoting or thwarting DNA binding.

The scFv antibody towards the Burkholderia pseudomallei exotoxin was previously constructed by phage display and exhibited good specificity towards the exotoxin. We report here the optimization of the scFv expression in an E. coli expression system. Four different E. coli strains (ER2537, TG1, HB2151, and XL1-Blue) were examined for optimal expression of the scFv protein. Two types of carbon source (i.e. 0.2% glucose and 0.2% glycerol) were also tested for their ability to induce the scFv expression. Cells that carried the scFv construct were grown at 30 degrees C and induced with 0.05 mM IPTG. The expression was then monitored by SDS-PAGE, Western blotting, and indirect ELISA. The Western blot profile showed different levels of the scFv expression among the host strains; XL1-Blue exhibited the highest level of the scFv protein expression. Glycerol at a concentration of 0.2% (v/v) significantly increased the scFv protein expression level when compared to 0.2% (w/v) glucose. Further optimization demonstrated that the scFv protein expression in XL1-Blue was the most optimal with a glycerol concentration as low as 0.05%. However, by indirect ELISA, only the scFv protein that was expressed in 0.2% (v/v) glycerol exhibited high specificity towards the Burkholderia pseudomallei exotoxin.

Affinity maturation of the Ab repertoire in germinal centers leads to the selection of high affinity Abs with selected heavy chain constant regions. Ab maturation involves two modifications of the Ig genes, i.e., somatic hypermutation and class switch recombination. The mechanisms of these two processes are not fully understood. As shown by the somatic hypermutation and class switch recombination-deficient phenotype of activation-induced cytidine deaminase (AID)-deficient patients (hyperIgM type 2 syndrome) and mice, both processes require the AID molecule. Somatic DNA modifications require DNA breaks, which, at least for class switch recombination, lead to dsDNA breaks. By using a ligation-mediated PCR, it was found that class switch recombination-induced dsDNA breaks in S mu switch regions were less frequent in AID-deficient B cells than in AID-proficient B cells, thus indicating that AID acts upstream of DNA break induction.

In L2 mice, a high expression level of the transgenic lambda2(315) L chain results in nearly complete exclusion of endogenous L chains and a predominance of B-1a cells. In this study, we show that splenic and peritoneal B-1a cells differ considerably in their Ab repertoire and gene expression profile. Splenic B-1a cells exhibit a more diversified repertoire under L chain limitation. Despite oligoclonal overlaps between both B-1a compartments, some B cell receptor specificities are clearly restricted to the peritoneum. The capacity of peritoneal B-1a cells to enter the splenic B-1a compartment was found to be very limited. Gene expression profiling revealed genes up-regulated in splenic B-1a cells that are involved in mediating specialized first-line-of-defense effector functions and interaction with T cells. Thus, splenic and peritoneal B-1a cells differ not only in their developmental program but also in functional properties.

Conventional drug development for treatment of cocaine addiction is greatly hindered by the extreme difficulty in designing a selective cocaine antagonist. We employed anti-idiotypic (anti-Id) antibodies to generate cocaine antagonists. The purpose of this study was to investigate the feasibility of this alternative approach. Herein, we describe the molecular cloning, bacterial expression, and functional properties of an anti-Id monoclonal antibody (mAb), designated K2-3f, which possesses an internal image of cocaine within its variable regions. The heavy and light chain variable domains of K2-3f were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and a single chain antibody variable fragment (scFv) was assembled for expression in Escherichia coli. The scFv bound to the human dopamine transporter (hDAT) with moderate affinity (K(a)=5.3 x 10(6) M(-1)) and excellent mimicry of the cocaine molecule completely inhibited cocaine binding at a molar concentration closely resembling in vivo conditions while allowing approximately 90% of equimolar dopamine uptake. Our data suggest that the use of anti-Id antibody as a template for generation of a cocaine antagonist is a promising approach well worth pursuing. If this strategy is successful, it could be applied to potential ligand-receptor interactions in the treatment of other diseases.

The Fv is the smallest antigen binding fragment of the antibody and is made of the variable domains of the light and heavy chains, V(L) and V(H), respectively. The 26-kDa Fv is amenable for structure determination in solution using multi-dimensional hetero-nuclear NMR spectroscopy. The human monoclonal antibody 447-52D neutralizes a broad spectrum of HIV-1 isolates. This anti-HIV-1 antibody elicited in an infected patient is directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. The V3 loop is an immunodominant neutralizing epitope of HIV-1. To obtain the 447-52D Fv for NMR studies, an Escherichia coli bicistronic expression vector for the heterodimeric 447-52D Fv and vectors for single chain Fv and individually expressed V(H) and V(L) were constructed. A pelB signal peptide was linked to the antibody genes to enable secretion of the expressed polypeptides into the periplasm. For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pelB sequence and following the termination codon. A set of oligonucleotides that prime the leader peptide genes of all potential antibody human antibodies were designed as backward primers. The forward primers for the V(L) and V(H) were based on constant region sequences. The 447-52D Fv could not be expressed either by a bicistronic vector or as single chain Fv, probably due to its toxicity to Escherichia coli. High level of expression was obtained by individual expression of the V(H) and the V(L) chains, which were then purified and recombined to generate a soluble and active 447-52D Fv fragment. The V(L) of mAb 447-52D was uniformly labeled with 13C and 15N nuclei (U-13C/15N). Preliminary NMR spectra demonstrate that structure determination of the recombinant 447-52D Fv and its complex with V3 peptides is feasible.

Phosphocholine (PC) is a naturally occurring Ag common to many pathogenic microorganisms. Early in the primary response to PC conjugated to keyhole limpet hemocyanin (KLH), T15 Id(+) Abs constitute >90% of the serum Ig in BALB/c mice. During the late primary and memory response to PC-protein, a shift in the repertoire occurs and T15 Id(+) Abs lose dominance. In this study, we use immunohistochemistry and single germinal center microdissection to locate T15 Id(+) cells in the spleen in a primary response to PC-KLH. We demonstrate T15 Id(+) B cells and V(H)1-DFL16.1-JH1 and V kappa 22-J kappa 5 rearrangements in germinal centers early in the immune response; thus loss of T15 dominance is not due to lack of T15 cells within germinal centers. One-hundred thirty one V(H)1 and 57 V kappa 22 rearrangements were cloned and sequenced. Thirty four percent of the V(H)1 clones and 37% of the V kappa 22 clones contained somatic mutations indicating participation in the germinal center response. Six variant T15 H clones were expressed with wild-type T15 L chain in vitro. Two of these Abs were defective in secretion providing the first evidence that mutation occurring in vivo can disrupt Ig assembly and secretion. Of the four secretion-competent Abs, two failed to display binding to PC-protein, while the other two displayed altered carrier recognition. These results indicate that somatic mutation of T15 in vivo can result in the loss of binding and secretion, potentially leading to B cell wastage. The failure of T15 to gain affinity enhancing mutations in the face of these detrimental changes may contribute to repertoire shift.

Staphylococcal enterotoxin H (SEH) is a bacterial superantigen secreted by Staphylococcus aureus. Superantigens are presented on the MHC class II and activate large amounts of T cells by cross-linking APC and T cells. In this study, RT-PCR was used to show that SEH stimulates human T cells via the Valpha domain of TCR, in particular Valpha10 (TRAV27), while no TCR Vbeta-specific expansion was seen. This is in sharp contrast to all other studied bacterial superantigens, which are highly specific for TCR Vbeta. It was further confirmed by flow cytometry that SEH stimulation does not alter the levels of certain TCR Vbeta. In a functional assay addressing cross-reactivity, Vbeta binding superantigens were found to form one group, whereas SEH has different properties that fit well with Valpha reactivity. As SEH binds on top of MHC class II, an interaction between MHC and TCR upon SEH binding is not likely. This concludes that the specific expansion of TCR Valpha is not due to contacts between MHC and TCR, instead we suggest that SEH directly interacts with the TCR Valpha domain.

The gradual removal of the denaturing reagent guanidine HCl (GdnHCl) using stepwise dialysis with the introduction of an oxidizing reagent and l-arginine resulted in the highly efficient refolding of various denatured single-chain Fv fragments (scFvs) from inclusion bodies expressed in Escherichia coli. In this study, the influence of the additives on the intermediates in scFv refolding was carefully analyzed on the basis of the stepwise dialysis, and it was revealed that the additive effect critically changes the pathway of scFv refolding. Circular dichroism and tryptophan fluorescence emission spectroscopies demonstrated that distinct secondary and tertiary structures were formed upon dialysis from 2 m GdnHCl to 1 m GdnHCl, and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt binding analysis indicated that the addition of l-arginine to the stepwise dialysis system effectively stabilized the exposed hydrophobic area on the scFv. Quantification of the free thiol groups in the scFv by means of Ellman's assay revealed that there was a particular stage in which most of the free thiol groups were oxidized and that adding an oxidizing reagent (the oxidized form of glutathione, GSSG) at that stage was important for complete refolding of the scFv. The particular stage depended on the nature of the refolding solution, especially on whether l-arginine was present. Spontaneous folding at the 1 m GdnHCl stage resulted in a structure in which a free thiol group accessed to the proper one for correct disulfide linkage; however, the addition of l-arginine resulted in the formation of a partially folded intermediate without disulfide linkages. Mass spectrometry experiments on alkylated scFv were carried out at each stage to determine the effects of l-arginine. The spectroscopic studies revealed two different pathways for scFv refolding in the stepwise dialysis system, pathways that depended on whether l-arginine was present. Controlled coupling of the effects of GSSG and l-arginine led to the complete refolding of scFv in the stepwise dialysis.

B cell lymphogenesis in mammals occurs in various tissues during development but it is generally accepted that it operates by the same mechanism in all tissues. We show that in swine, the frequency of in-frame (IF) VDJ rearrangements differs among yolk sac, fetal liver, spleen, early thymus, bone marrow, and late thymus. All VDJ rearrangements recovered and analyzed on the 20th day of gestation (DG20) from the yolk sac were 100% IF. Those recovered at DG30 in the fetal liver were >90% IF, and this predominance of cells with apparently a single IF rearrangement continued in all organs until approximately DG45, which corresponds to the time when lymphopoiesis begins in the bone marrow. Thereafter, the proportion of IF rearrangements drops to approximately 71%, i.e., the value predicted whether VDJ rearrangement is random and both chromosomes were involved. Unlike other tissues, VDJs recovered from thymus after DG50 display a pattern suggesting no selection for IF rearrangements. Regardless of differences in the proportion of IF rearrangements, we observed no significant age- or tissue-dependent changes in CDR3 diversity, N region additions, or other characteristics of fetal VDJs during ontogeny. These findings indicate there are multiple sites of B cell lymphogenesis in fetal piglets and differences in the frequency of productive VDJ rearrangements at various sites. We propose the latter to result from differential selection or a developmentally dependent change in the intrinsic mechanism of VDJ rearrangement.

We previously described T15H chain mutants that were impaired in assembly with L chain and in ability to be secreted from the cell. The unmutated T15L chain is unusual in that it is secretion-impaired in the absence of assembly with H chain. The T15L chain preferentially pairs with T15H in vivo, suggesting that if we introduced mutations that would allow secretion of free T15L chain, they might also lead to the secretion of the complex with the defective H chain. We mutated four positions in the germline T15L that had amino acids infrequently found in other kappa-chains. Mutation to the most frequently occurring amino acid at three of the four positions allowed secretion of free L chain, while the combination of two secretion-restoring mutations was synergistic. Coexpression of secretion-restored mutant L chains with the secretion-defective mutant H chains rescued secretion of the assembled H(2)L(2) complex, suggesting that during somatic hypermutation in vivo, deleterious mutations at the H chain may be compensated by mutations on the L chain. To our knowledge, this is the first example of mutations in IgL chains that are able to restore secretion-defective H chains to secretion competence in mammalian cells.

Ribonucleic acid expression profiles of seven consecutive stages of mouse thymocyte development were generated on high density oligonucleotide arrays. Previously known expression patterns of several genes were confirmed. Ten percent (1,304 of more than 13,000) of the monitored genes were found with 99% confidence to be differentially expressed across all T cell developmental stages. When compared with 1,204 genes differentially expressed in five consecutive B lineage developmental stages of bone marrow, >40% (546 genes) appeared to be shared by both lineages. However, when four pools of functionally distinct cell stages were compared between B and T cell development, DJ-rearranged precursor cells and resting immature precursor cells before and after surface Ag receptor expression shared less than 10%, mature resting lymphocytes between 15 and 20%, and only cycling precursors responding to precursor lymphocyte receptor deposition shared >50% of these differentially expressed genes. Three general rules emerge from these results: 1) proliferation of cells at comparable stages is in majority executed by the same genes; 2) intracellular signaling and intercellular communication are effected largely by different genes; and 3) most genes are not used strictly at comparable, but rather at several, stages, possibly in different functional contexts.

Interfering with increased levels of plasminogen activator inhibitor-1 (PAI-1) might offer new therapeutic strategies for a variety of cardiovascular diseases. Inactivation of PAI-1 can be accomplished by a number of monoclonal antibodies (MA), including MA-8H9D4. In a previous study, a single-chain variable fragment (scFv-8H9D4) was cloned and found to have the same properties as the parental MA-8H9D4. In the present study, we identified the residues of scFv-8H9D4 that contribute significantly to the paratope. The complementarity determining region 3 from the heavy (H3) and the light (L3) chain were analysed through site-directed mutagenesis. Out of twelve mutations, only four residues appeared to contribute to the paratope. The affinity of scFv-8H9D4-H3-L97D for PAI-1 was 38-fold decreased (K(A) = 4.8 +/- 0.2 x 10(7) M(-1) vs. 1.8 +/- 0.7 x 10(9) M(-1) for scFv-8H9D4) whereas scFv-8H9D4-H3-R98Y did not bind to PAI-1. The affinities of scFv-8H9D4-L3-Y91S and scFv-8H9D4-L3-F94D for PAI-1 were 9- and 5-fold reduced, respectively, whereas the combined mutation resulted in an 86-fold decreased affinity (K(A) = 2.1 +/- 0.2 x 10(7) M(-1)). In accordance with the affinity data, these mutants had no, or a reduced, PAI-1 inhibitory capacity, confirming that these four particular residues form the major interaction site of scFv-8H9D4 with PAI-1. In combination with the three-dimensional structure, these data contribute to the rational design of PAI-1 neutralizing compounds.

Assembly of TCRbeta variable region genes is ordered during thymocyte development with Dbeta to Jbeta rearrangement preceding Vbeta to DJbeta rearrangement. The 5'Dbeta 12-RSS is required to precisely and efficiently target Vbeta rearrangement beyond simply enforcing the 12/23 rule. By prohibiting direct Vbeta to Jbeta rearrangement, this restriction ensures Dbeta gene segment use in the assembly of essentially all TCRbeta variable region genes. In this study, we show that rearrangement of Vbeta 23-RSSs is significantly biased to the Dbeta 12-RSS over Jbeta 12-RSSs on extrachromosomal recombination substrates in nonlymphoid cells that express the recombinase-activating gene-1/2 proteins. These findings demonstrate that targeting of Vbeta to Dbeta rearrangement can be enforced by the V(D)J recombinase in the absence of lymphoid-specific factors other than the recombinase-activating gene-1/2 proteins.

Antibodies represent an interesting protein framework on which catalytic functions can be grafted. In previous studies, we have reported the characterization of the catalytic antibody 4B2 obtained on the basis of the "bait and switch" strategy which catalyzes two different chemical reactions: the allylic isomerization of beta,gamma-unsaturated ketones and the Kemp elimination. We have cloned the antibody 4B2 and expressed it as a single-chain Fv (scFv) fragment in different expression systems, Escherichia coli and two yeasts species, in order to elicit the most suitable system to study its catalytic activity. The scFv4B2 was secreted as an active form in the culture medium of Pichia pastoris and Kluyveromyces lactis, which led respectively to 4 and 1.3mg/l after purification. In E. coli, different strategies were investigated to increase the cytoplasmic soluble fraction, which resulted, in all cases, in the expression of a low amount of functional antibodies. By contrast, substantial amount of scFv4B2 could be purified when it was expressed as inclusion bodies (12mg/l) and submitted to an in vitro refolding process. Its catalytic activity was measured and proved to be comparable to that of the whole IgG. However, the instability of the scFv4B2 in solution prevented from an exhaustive characterization of its activity and stabilization of this protein appears to be essential before designing strategies to improve its catalytic activity.

The quasi-monoclonal mouse has limited B cell diversity, whose major (approximately 80%) B cell Ag receptors are comprised of the knockin V(H) 17.2.25 (V(H)T)-encoded H chain and the lambda1 or lambda2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although V(H)T/lambda1 and V(H)T/lambda2 IgM were equally produced, V(H)T/lambda2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 of V(H)T (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated V(H)T-encoded gamma-chains could form lambda1-bearing IgG in Chinese hamster ovary cells, apparent absence of V(H)T/lambda1 anti-pNP IgG may not be due to the incompatibility between the gamma-chains and the lambda1-chain, but may be explained by the fact that V(H)T/lambda1 B cells showed 50- to 100-fold lower affinity for pNP than V(H)T/lambda2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an affinity maturation process.

Epitopes differing among isoenzymes of creatine kinase (CK) are apparently limited in number and poorly immunogenic in vivo. Especially for the BB-CK isoenzyme, very few monoclonal antibodies (mAb) are available. Here, we use in vitro selection with a synthetic human phage display antibody library and develop isoenzyme competition and peptide panning strategies to obtain human single chain Fv (scFv) antibodies against specific CK isoenzymes. We isolated and characterized seven scFv clones that recognize native as well as denatured cytosolic BB-CK in ELISA, immunoblot, immunofluorescence histochemistry and surface plasmon resonance (SPR) spectroscopy. To a variable but minor degree, they also react with cytosolic MM-CK, but not with mitochondrial CK isoenzymes. Epitope mapping revealed that the scFv antibodies recognize different BB-CK epitopes, including the N-terminus and the isoenzyme-specific box, a highly conserved sequence of unknown function for which no mAb were available so far. With a K(D) of 3.5-9.6 x 10(-7) M, the isolated scFv compare favorably with mouse mAb and may overcome certain of their limitations. Our results demonstrate the advantages of in vitro antibody selection for the generation of isoenzyme-specific antibodies.

Objective: To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti-Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer., Methods: Balb/c mice were immunized i.p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TG1. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA., Results: The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed beta or gamma type anti-Id ScFv., Conclusion: The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.

Although mice transgenic (Tg) for human MHC (HLA) class I alleles could provide an important model for characterizing HLA-restricted viral and tumor Ag CTL epitopes, the extent to which Tg mouse T cells become HLA restricted in the presence of endogenous H2 class I and recognize the same peptides as in HLA allele-matched humans is not clear. We previously described Tg mice carrying the HLA-B27, HLA-B7, or HLA-A2 alleles expressed as fully native (HLA(nat)) (with human beta(2)-microglobulin) and as hybrid human/mouse (HLA(hyb)) molecules on the H2(b) background. To eliminate the influence of H2(b) class I, each HLA Tg strain was bred with a H2-K(b)/H2-D(b)-double knockout (DKO) strain to generate mice in which the only classical class I expression was the human molecule. Expression of each HLA(hyb) molecule and HLA-B27(nat)/human beta(2)-microglobulin led to peripheral CD8(+) T cell levels comparable with that for mice expressing a single H2-K(b) or H2-D(b) gene. Influenza A infection of Tg HLA-B27(hyb)/DKO generated a strong CD8(+) T cell response directed at the same peptide (flu nucleoprotein NP383-391) recognized by CTLs from flu-infected B27(+) humans. As HLA-B7/flu epitopes were not known from human studies, we used flu-infected Tg HLA-B7(hyb)/DKO mice to examine the CTL response to candidate peptides identified based on the B7 binding motif. We have identified flu NP418-426 as a major HLA-B7-restricted flu CTL epitope. In summary, the HLA class I Tg/H2-K/H2-D DKO mouse model described in this study provides a sensitive and specific approach for identifying and characterizing HLA-restricted CTL epitopes for a variety of human disease-associated Ags.

Immunization with recombinant heat shock protein 60 (rHsp60) from Histoplasma capsulatum or a region of the protein designated fragment 3 (F3) confers protection from a subsequent challenge in mice. To determine the T cell repertoire involved in the response to Hsp60, T cell clones from C57BL/6 mice immunized with rHsp60 were generated and examined for Vbeta usage by flow cytometry and RT-PCR. Vbeta8.1/8.2(+) T cells were preferentially expanded; other clones bore Vbeta4, -6, or -11. When Vbeta8.1/8.2(+) cells were depleted in mice, Vbeta4(+) T cell clones were almost exclusively isolated. Measurement of cytokine production demonstrated that nine of 16 Vbeta8.1/8.2(+) clones were Th1, while only three of 13 non-Vbeta8.1/8.2(+) clones were Th1. In mice immunized with rHsp60, depletion of Vbeta8.1/8.2(+), but not Vbeta6(+) plus Vbeta7(+), T cells completely abolished the protective efficacy of Hsp60 to lethal and sublethal challenges. Examination of the TCR revealed that a subset of Vbeta8.1/2(+) clones that produced IFN-gamma and were reactive to F3 shared a common CDR3 sequence, DGGQG. Transfer of these T cell clones into TCR alpha/beta(-/-) or IFN-gamma(-/-) mice significantly improved survival, while transfer of other Vbeta8.1/8.2(+) clones that were F3 reactive but were Th2 or clones that were not reactive to F3 but were Th1 did not confer protection. These data indicate that a distinct subset of Vbeta8.1/8.2(+) T cells is crucial for the generation of a protective response to rHsp60.

The primary burst of Ab and germinal center (GC) formation in response to T-dependent Ag is compromised in aging mice. Here we examine the effects of aging on the post-GC phase of memory B cell differentiation and the late Ab repertoire maturation in bone marrow (BM) in mice immunized with a hapten nitrophenyl coupled to chicken gamma-globulin. Specific Ab-forming cells (AFC) with mutated V(H) genes accumulated preferentially in the BM of aged mice, although the AFC numbers and average number of mutations per V(H) were lower, and the D gene usage was less restricted compared with those in the young animals. However, the repertoire of AFC after an Ag boost demonstrated the hallmarks of Ag selection, including the recurrent mutations and canonical VD rearrangements, similar to the late primary response in young animals. It is postulated that the Ab repertoire maturation in aged mice is delayed and may be notably improved by repeated immunizations.

Impaired development of local Ab responses may predispose HIV-1-infected patients to an increased rate, severity, and duration of mucosal infections. We characterized the repertoire of Ig-producing cells in the intestinal effector compartment (the lamina propria) of HIV-1-infected (n = 29) and seronegative control (n = 27) subjects. The density of Ig-producing cells per area was similar in both groups. However, the proportions of IgA-producing cells were lower in both the duodenum and colon from HIV-1-infected patients compared with those of control subjects (p < 0.05), with compensatory increases in IgG-producing cells in the colon and IgM-producing cells in the duodenum. Similarly, among Abs in the lumen the proportions of IgA were also decreased and the proportions of IgG were increased among HIV-1-infected patients. On a molecular level, V(H) gene repertoire analyses by RT-PCR revealed comparable proportions of the V(H)3 family among duodenal IgA transcripts (50-53%) from both groups. V(H)3 expression was decreased only for IgM among patients with advanced HIV-1 disease (n = 6) compared with that of control subjects (n = 8) (48 +/- 8 vs 62 +/- 13%; p < 0.01). Moreover, the frequencies of individual IgM and IgA V(H)3 genes were comparable in each group, including rates of putative HIV-1 gp120-binding V(H)3 genes (V3-23, V3-30, V3-30/3-30.5). We conclude that, despite a decrement in local IgA producing cells, the density and molecular V(H) repertoire of mucosal plasma cells are relatively intact among patients with HIV-1 infection. These data suggest that HIV-1-infected patients use functional regulatory mechanisms to provide sufficient V(H) diversity and effective induction and differentiation of mucosal B cells.

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.

Primary amyloid L chain (AL) amyloidosis is a plasma cell disorder in which depositions of AL cause progressive organ failure. The lack of effective therapies for this fatal disease prompts exploration of newer treatment avenues. We have investigated the application of antisense oligonucleotides (AS) for the inhibition of monoclonal Ig production. The monoclonal L chain was identified by using primers designed for amplifying the human lambda Ig V (Vlambda) region. We demonstrated that AS against L chain complementarity-determining regions inhibited the production of L chain in vitro. RPMI 8226 myeloma cells injected in SCID mice developed s.c. tumors. RT-PCR analysis showed Vlambda mRNA expression in the tumors. In addition, the presence of human Ig in the sera of mice given injection of RPMI 8226 cells was confirmed by ELISA. Administration of AS inhibited the expression of Vlambda mRNA in the s.c. tumors and decreased the concentration of L chain in serum. Therefore, we have shown that it is possible to determine the sequence of Vlambda mRNA and design specific complementary oligonucleotides, suggesting that treatment with Vlambda antisense could represent a rational novel approach to improve treatment outcome in AL amyloidosis.

Cells are inherently robust to environmental perturbations and have evolved to recover readily from short-term exposure to heat, pH changes, and nutrient deprivation during times of stress. The stress of unfolded protein accumulation has been implicated previously in low protein yields during heterologous protein expression. Here we describe the dynamics of the response to this stress, termed the unfolded protein response (UPR), during the expression of the single chain antibody 4-4-20 (scFv) in Saccharomyces cerevisiae. Expression of scFv decreased the growth rate of yeast cells whether the scFv was expressed from single-copy plasmids or integrated into the chromosome. However, the growth rates recovered at longer expression times, and surprisingly, the recovery occurred more quickly in the high-copy integration strains. The presence of a functional UPR pathway was necessary for a recovery of normal growth rates. During the growth inhibition, the UPR pathway appeared to be activated, resulting in decreased intracellular scFv levels and intermittent recovery of the chaperone BiP within the endoplasmic reticulum. Intracellular scFv was observed primarily in the endoplasmic reticulum, consistent with activation of the UPR pathway. Although the intracellular scFv levels dropped over the course of the expression, this was not a result of scFv secretion. A functional UPR pathway was necessary for the drop in intracellular scFv, suggesting that the decrease was a direct response of UPR activation. Taken together, these results suggest that control of heterologous gene expression to avoid UPR activation will result in higher production levels.

Intracellular expression of Ab fragments has been efficiently used to inactivate therapeutic targets, oncogene products, and to induce viral resistance in plants. Ab fragments expressed in the appropriate cell compartment may also help to elucidate the functions of a protein of interest. We report in this study the successful targeting of the protein tyrosine kinase Syk in the RBL-2H3 rat basophilic leukemia cell line. We isolated from a phage display library human single-chain variable fragments (scFv) directed against the portion of Syk containing the Src homology 2 domains and the linker region that separates them. Among them, two scFv named G4G11 and G4E4 exhibited the best binding to Syk in vivo in a yeast two-hybrid selection system. Stable transfectants of RBL-2H3 cells expressing cytosolic G4G11 and G4E4 were established. Immunoprecipitation experiments showed that intracellular G4G11 and G4E4 bind to Syk, but do not inhibit the activation of Syk following FcepsilonRI aggregation, suggesting that the scFv do not affect the recruitment of Syk to the receptor. Nevertheless, FcepsilonRI-mediated calcium mobilization and the release of inflammatory mediators are inhibited, and are consistent with a defect in Bruton's tyrosine kinase and phospholipase C-gamma2 tyrosine phosphorylation and activation. Interestingly, FcepsilonRI-induced mitogen-activated protein kinase phosphorylation is not altered, suggesting that intracellular G4G11 and G4E4 do not prevent the coupling of Syk to the Ras pathway, but they selectively inhibit the pathway involving phospholipase C-gamma2 activation.

Purpose: To clone the variable region gene of light chain of monoclonal antibody against human retinoblastoma and to analyze the characterization of its nucleotide sequence as well as amino acid sequence., Methods: Total RNA was extracted from 3C6 hybridoma cells secreting specific monoclonal antibody (McAb) against human retinoblastoma (RB), then transcripted reversely into cDNA with olig-dT primers. The variable region of the light chain (VL) gene fragments was amplified using polymerase chain reaction(PCR) and further cloned into pGEM -T Easy vector. Then, 3C6 VL cDNA was sequenced by Sanger's method. Homologous analysis was done by NCBI BLAST., Results: The complete nucleotide sequence of 3C6 VL cDNA consisted of 321 bp encoding 107 amino acid residues, containing four workframe regions (FRs) and three complementarity-determining regions(CDRs) as well as the typical structure of two cys residues. The sequence is most homological to a member of the Vk9 gene family, and its chain utilizes the Jkl gene segment., Conclusion: The light chain variable region gene of the McAb against human RB was amplified successfully, which belongs to the Vk9 gene family and utilizes Vk-Jkl gene rearrangement. This study lays a good basis for constructing a recombinant antibody and for making a new targeted therapeutic agents against retinoblastoma.