Non-immunized Donors Research Articles

Accelerated hepatitis B vaccination: Efficacy in young, regular blood donors

Background and Objectives: Hepatitis B (HB) – a vaccine-preventable, transfusion-transmitted infection – has been estimated to have high residual risk, especially in medium- and high-endemic areas. This potent immunogenic vaccine is routinely injected at 0, 1, and 6 months. Accelerated schedule has been recommended in adults to increase the completion rate. We studied the response rate of accelerated schedule in young, regular blood donors in a high-prevalence area for HB virus (HBV) in Iran. Methods: Young, regular nonimmunized blood donors were vaccinated with recombinant DNA HB surface antigen vaccine (Pasteur Institute of Iran) containing 20 μg of material in 1-ml solution injected intramuscularly to the deltoid muscle at 0, 1, and 2 months. HB surface antibody (HBsAb) was titered 1 month after the completion of doses. Nonresponders took one additional dose and tested a month later. Results: A total of 357 donors with a mean age of 26 ± 3.5 years were enrolled, of whom 287 donors completed three doses of vaccination (80% adherence rate). HBsAb titer was >10 IU/ml in 274 donors (95.5% response rate), whereas after one booster dose injection, the titer in 13 nonresponder persons (4.5%) increased to >10 IU/ml. Conclusion: Accelerated HBV vaccination with near 100% response rate and 80% adherence rate in young, regular blood donors has been successful in the immunization of young blood donors. This strategy improves not only blood safety but also donors' health.

Design and construction of a new human naïve single-chain fragment variable antibody library, IORISS1

Human monoclonal antibodies are a powerful tool with increasingly successful exploitations and the single chain fragment variable format can be considered the building block for the implementation of more complex and effective antibody-based constructs. Phage display is one of the best and most efficient methods to isolate human antibodies selected from an efficient and variable phage display library. We report a method for the construction of a human naïve single-chain variable fragment library, termed IORISS1. Many different sets of oligonucleotide primers as well as optimized electroporation and ligation reactions were used to generate this library of 1.2×109 individual clones. The key difference is the diversity of variable gene templates, which was derived from only 15 non-immunized human donors. The method described here, was used to make a new human naïve single-chain fragment variable phage display library that represents a valuable source of diverse antibodies that can be used as research reagents or as a starting point for the development of therapeutics. Using biopanning, we determined the ability of IORISS1 to yield antibodies. The results we obtained suggest that, by using an optimized protocol, an efficient phage antibody library can be generated.

Risk Factors of Changes in Hepatitis B Antibody Status after Allogeneic Hematopoietic Stem Cell Transplantation in Chinese Children with Thalassemia Major

Objective: This retrospective study evaluated the risk factors involved in the changes in HBsAb status in patients with thalassemia major at a single center in China.Methods: A total of 104 children who underwent allo-HSCT, using NF-08-TM transplant protocol in our center, between January 2010 and June 2012 were recruited.Hepatitis B markers, including HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc were examined by TRFIA (time-resolved fluoroimmunoassay) or ELISA (Enzyme-Linked Immunosorbent Assay) for recipients before and after allo-HSCT (at least up to 6 months) and for donors prior to transplantation. HBsAg positive recipients and donors received lamivudine antiviral therapy before allo-HSCT and the treatment was continued in recipients up to 6 months post transplantation.The demographic and clinical characteristics of the patients and their donors were summarized by descriptive statistics. For identification of risk factors that influenced the post-transplant anti-HBs loss and HBV reactivation, both univariate and multivariate logistic regression was used, and odds ratio (OR) and 95% confidence interval (CI) were determined for the covariates that were shown to be statistically significant. All tests were 2-sided, with the type I error rate fixed at 0.05. Statistical analyses were performed using IBM SPSS 20 (SPSS Statistics V20, IBM Corporation, Somers, New York).Results: Of the 104 patients, 2（1.9%） recipients were positive for HBsAg and 102（98.1%） recipients were negative for HBsAg. Of the 102 patients negative for HBsAg before transplantation, the proportion of positive anti-HBs was 69.6% (71 of 102 patients). Of the 104 donors, 99 (95.2%)were negative for HBsAg and 5 (4.8%)were positive for HBsAg. Of the 99 donors negative for HBsAg before transplantation, 72 donors (72.7%) had anti-HBs. After transplantation, of the 69 patients, 27 (39.1%) patients lost their HBV immunity in a median follow-up period of 30 months (range: 21–45); the remaining 42 (60.9 %) patients maintained the immunity against HBV after a median follow-up period of 28.5 months (range: 19–46). 33 patients were anti-HBs negative before the allo-HSCT. The 33 patients included 11 patients with donors who had no anti-HBs and 22 patients with donors who had anti-HBs. After the allo-HSCT, 15 of the 33 patients were found to have newly gained HBV immunity, as represented by the presence of anti-HBs. While 14 of them who developed adoptive immunity had immunized donors (63.6%; 14 out of 22), 1 of them (9.1%; 1 out of 11) with a non-immunized donor (donors without anti-HBs) also had developed HBV immunity.Multivariate logistic regression analysis of 104 patients who underwent allo-HSCT revealed that, patients with pre-HSCT titer of HBsAb < 257.47mIU/mL (adjusted odds ratio, 10.5, 95% CI, 2.1–53.3) and HBsAb-immunized donors (51.3, 2.8–938.6) were significant risk factors for post allo-HSCT HBV loss and acquisition, respectively. In addition, the post-transplant HBV reactivation rate was 11.1%.Conclusions: Current results indicate that pre-transplant HBsAb titer is a key determinant in the loss of HBV immunity after allo-HSCT and HBsAb negative patients with immunized donors are more likely to gain HBV immunity after allo-HSCT than those with non-immunized donors. Further, preemptive antiviral treatment with lamivudine significantly reduces HBV reactivation. This is the first study to have indicated the significant predictors of changes in HBsAg status in children with thalassemia major. DisclosuresNo relevant conflicts of interest to declare.

Construction of a human scFv antibody library with VH regions randomized and its application

A non-immunized human single chain variable fragment (scFv) library containing 2.5 × 10(7) individual clones was constructed from antibody variable region genes of 200 non-immunized donors. ScFv gene repertories were generated by randomly combining rearranged variable regions of heavy chain (VH) and natural occurring light chain (VL) using overlapping extension PCR (OE-PCR). Five recombinant protein antigens from different species were successfully used to select specific binders. Phage ELISA showed that the recombinant phage particle could specifically bind to non-structural protein 1 of Avian influenza virus. This method can therefore efficiently generate a phage antibody library.

A compact phage display human scFv library for selection of antibodies to a wide variety of antigens

BackgroundPhage display technology is a powerful new tool for making antibodies outside the immune system, thus avoiding the use of experimental animals. In the early days, it was postulated that this technique would eventually replace hybridoma technology and animal immunisations. However, since this technology emerged more than 20 years ago, there have only been a handful reports on the construction and application of phage display antibody libraries world-wide.ResultsHere we report the simplest and highly efficient method for the construction of a highly useful human single chain variable fragment (scFv) library. The least number of oligonucleotide primers, electroporations and ligation reactions were used to generate a library of 1.5 × 108 individual clones, without generation of sub-libraries. All possible combinations of heavy and light chains, among all immunoglobulin isotypes, were included by using a mixture of primers and overlapping extension PCR. The key difference from other similar libraries was the highest diversity of variable gene repertoires, which was derived from 140 non-immunized human donors. A wide variety of antigens were successfully used to affinity select specific binders. These included pure recombinant proteins, a hapten and complex antigens such as viral coat proteins, crude snake venom and cancer cell surface antigens. In particular, we were able to use standard bio-panning method to isolate antibody that can bind to soluble Aflatoxin B1, when using BSA-conjugated toxin as a target, as demonstrated by inhibition ELISA.ConclusionThese results suggested that by using an optimized protocol and very high repertoire diversity, a compact and efficient phage antibody library can be generated. This advanced method could be adopted by any molecular biology laboratory to generate both naïve or immunized libraries for particular targets as well as for high-throughput applications.

Human monoclonal ScFv neutralize lethal Thai cobra, Naja kaouthia, neurotoxin

Animal derived anti-Naja. kaouthia (Thai cobra) venom is used for specific treatment of the snake bitten victims. Many recipients develop allergic reaction or anti-isotype response which causes serum sickness. A better therapeutic antibody is needed. In this study, long alpha-neurotoxin was purified from the N. kaouthia holovenom and verified by 2D-LC/MS-MS. The toxin was used as antigen in a phage bio-panning to select phage clones displaying human single chain variable antibody fragments (HuScFv) from a phage display antibody library constructed from immunoglobulin genes of non-immunized Thai blood donors. HuScFv that specifically bound to the neurotoxin were produced from huscfv-phagemid transformed E. coli clones and affinity purified. The HuScFv could neutralize toxicity of the N. kaouthia neurotoxin and rescued the envenomized mice from the neurotoxin mediated lethality. Peptide mimotope of the neutralizing HuScFv matched with an amino acid sequence (epitope) located in the loop-3 of the N. kaouthia long alpha-neurotoxin which functions in acetylcholine receptor binding. The mimotope is also similar to peptide sequences found on other snake venom neurotoxins implying a possibility of the HuScFv to exert pan-neutralizing activity against multiple snake neurotoxins.

Rapid detection of JMH antibodies with recombinant Sema7A (CD108) protein and the particle gel immunoassay

At present, identification of antibodies against the high-prevalence JMH antigen is difficult and limited to reference laboratories having panels of rare red blood cell (RBC) specimens in stock. Here, a novel method is described for detection of anti-JMH with particles coated with recombinant semaphorin 7A (Sema7A, CD108), the protein that carries the JMH blood group antigens. Recombinant Sema7A protein was generated and coupled onto superparamagnetic particles coated with streptavidin. The coated particles were tested in the presence of different serum and plasma samples (11 anti-JMH, 20 other antibodies, and 50 samples from nonimmunized blood donors) with the particle gel immunoassay and flow cytometry. Sema7A-coated particles reacted with all 11 samples containing anti-JMH, but not with samples lacking anti-JMH. In addition, the anti-JMH agglutination scores were higher with Sema7A-coated particles than with JMH-positive RBCs in all cases. Recombinant blood group proteins have the potential to replace RBCs as antigen carriers for identification of certain RBC alloantibodies.

In the Presence of Pre-Existing Factor VIII (FVIII) Immunity, Hematopoietic Stem Cells (HSC) That Are Genetically Modified To Express FVIII in Platelets Were Successfully Transplanted into Hemophilic Mice under Myeloablative and Various Non-Myeloablative Conditions.

While genetic induction of FVIII expression in platelets can restore hemostasis in hemophilia A mice, this approach has not been studied in the clinical setting of pre-existing FVIII inhibitory antibodies, and whether such antibodies would affect therapeutic engraftment. The development of inhibitors to exogenous FVIII is considered a severe and important complication of FVIII infusion in hemophilia A patients. The clinical hallmark of inhibitor development in hemophilia A patients is failure to response to routine replacement therapy for bleeding episodes. In this study, we wanted to explore whether syngeneic transplantation of HSC that are genetically modified to express FVIII in platelets would restore hemostasis to hemophilic mice with pre-existing FVIII immunity. We generated a line of transgenic mice that express FVIII only in platelets using the platelet-specific αIIb promoter (2bF8) and bred this 2bF8 transgene into a FVIIInull background. Bone marrow (BM) from immunized or non-immunized heterozygous 2bF8 transgenic mice was transplanted into immunized FVIIInull mice following lethal or sub-lethal irradiation. The recipients were analyzed by PCR, quantitative real-time PCR, platelet-lysate FVIII activity assay, inhibitor assay, and tail clip survival test to assess engraftment and phenotypic correction. Our results demonstrated that pre-existing anti-FVIII inhibitory antibodies in recipients did not inhibit successful engraftment of BM cells with 2bF8 genetic modification into hemophilia A mice. When we looked at the effect of inhibitor titer, following one month BM reconstitution, all recipients with inhibitor titers of 50 – 400 BU/ml (n = 9) survived tail clipping and 80% of recipients with a titer of 401 – 20,000 BU/ml (n = 25) also survived tail clipping when 1100 R (myeloablative) or 660 R (non-myeloablative) regimens were employed. When comparing conditioning regimens, in immunized recipients conditioned with 1100 R that received BM from 2bF8 donors the tail clip survival rate was 85% (n = 20). In the group of recipients in which 660R was employed 85.7% (n = 14) recipients survived tail clipping. When immunized recipients were conditioned with 440 R, 60% (n = 5) survived. In the group of recipients in which 220 R was employed, none (n = 4) survived tail clipping. The tail clip survival rate was not significantly different between the groups that received BM from immunized 2bF8 donors and from non-immunized donors. Different proportions of mixed BM from 2bF8 transgenic mice and FVIIInull mice were transplanted into lethally irradiated FVIIInull recipients to address what fraction of platelets would need to express FVIII in order to restore hemostasis to hemophilic mice with inhibitors. Our results showed that transplantation with only 5% 2bF8 BM cells still improved hemostasis in hemophilia A mice with inhibitors. These results demonstrate that the presence of FVIII-specific immunity in recipients does not alter engraftment of 2bF8 genetically modified HSC and transplantation of these HSC can efficiently restore hemostasis to hemophilic mice with pre-existing inhibitory antibodies under either myeloablative or non-myeloablative regimens.

Syndrome d'œdème pulmonaire lésionnel aigu post-transfusionnel (Trali) après perfusion de plasma frais congelé au cours d'une microangiopathie thrombotique congénitale

Introduction Transfusion-related acute lung injury is a post-transfusion interstitial lung injury. Case report We reported a post-transfusion acute lung injury in a 23-years old woman having a chronic thrombotic microangiopathy related to an ADAMTS 13 constitutional deficiency receiving monthly plasma infusion for six years. The temporal relationship between the lung injury and the infusion of fresh frozen plasma led to the diagnosis of transfusion-related acute lung injury. The finding in the donor of the transfused plasma of an anti-HLA class II antibody recognizing HLA-DR52 present on leucocytes of the recipient suggests a causal relationship between this antigen-antibody conflict and the triggering of the TRALI. This chronic pathologic state requiring monthly plasma transfusions for thrombotic accident prevention raises the question of the selection of plasma obtained from non-immunized donors. Conclusion The occurrence of a post transfusion pulmonary edema without cardio-vascular overload, must lead to consider a TRALI especially in predisposing clinical situations. In the case reported the role of constitutional ADAMTS 13 deficiency in genesis of TRALI is considered.

Peritransplantation Vaccination with Chaperone-Rich Cell Lysate Induces Antileukemia Immunity

We have reported that chaperone-rich cell lysate (CRCL) is an effective anticancer vaccine in immunocompetent mice. In this study, we explored the therapeutic applicability of CRCL in the context of syngeneic hematopoietic cell transplantation (HCT) to treat preexisting leukemia. Our results demonstrate that tumor growth is significantly delayed in mice receiving syngeneic HCT from 12B1 tumor CRCL-immunized donors compared with animals receiving HCT from nonimmunized donors. CRCL immunization after immune HCT further hindered tumor growth when compared with immune HCT without posttransplantation vaccination. The magnitude of the immune response was consistent with the antitumor effects observed in vivo. Rechallenge of surviving mice with 12B1 or A20 cells in opposite groins confirmed that mice had developed long-term tumor-specific immunity against 12B1 tumor cells. In addition, we documented that both T cells and natural killer cells contributed to the antitumor effect of CRCL vaccination, because depletion of either subset hampered tumor growth delay. Thus, our results indicate that CRCL is a promising vaccine capable of generating specific immune responses. This antitumor immunity can be effectively transferred to a host via HCT and further enhanced after HCT with additional tumor CRCL immunizations.

Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity

Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.

Adoptive transfer of immunity: a new strategy to interfere with severe hepatitis virus reinfection after woodchuck liver transplantation.

The effective transfer of humoral immunity to hepatitis B virus was first demonstrated after clinical and experimental bone marrow transplantation. This strategy is now evaluated in the woodchuck transplantation model, in which protection from reinfection can be tested. Animals negative for woodchuck hepatitis virus (WHV) (n = 3) were vaccinated using plasmids expressing woodchuck hepatitis virus surface (WHs), woodchuck hepatitis virus core (WHc), and woodchuck interferon-gamma in combination with a protein vaccine (WHs antigen [Ag]) three times before liver donation. Control animals (n= 4) received the liver from non-immunized donors. Chronic WHV carriers served as recipients. The viral load in serum and liver tissue was monitored pretransplant and posttransplant for up to 11 weeks by dot blot, Northern blot, Southern blot, and immunohistochemistry for WHc and WhsAg. Donor vaccination was effective, as indicated by the development of anti-WHc and anti-WHs antibodies. Transplanting the livers of these donors resulted in a reduction of viral load in two of three animals. No viral DNA was detected in repeated serum samples by dot-blot hybridization technique. However, polymerase chain reaction for viral DNA extracted from serum was always positive. WHV replication intermediates and WHV RNA were absent in repeated liver biopsies. Only few hepatocytes stained weakly positive for WHc protein and frequency, and the intensity of WHsAg positive hepatocytes was low. The third animal developed severe reinfection within 3 weeks, similar to the woodchucks in the control group. Liver transplantation from immunized donors to chronic carriers seems to be a promising strategy to reduce and delay severe reinfection, which may be applicable in clinical liver transplantation.

Function of C3 in a humoral response: iC3b/C3dg bound to an immune complex generated with natural antibody and a primary antigen promotes antigen uptake and the expression of co-stimulatory molecules by all B cells, but only stimulates immunoglobulin synthesis by antigen-specific B cells.

Previous studies have shown that an optimal humoral response to a primary protein antigen requires C3 and CR2 (CD21). Sera from non-immunized donors contain natural IgM and IgG antibodies to the primary antigen keyhole limpet haemocyanin (KLH), and these have been previously shown to form immune complexes (IC) that activate the classical pathway of C, fixing iC3b/C3dg onto the KLH antigen. Such KLH IC bind to CR2 on KLH-non-specific B lymphocytes, resulting in antigen processing and MHC class II-dependent presentation to KLH-specific helper T cells. KLH IC also induce B lymphocytes to express the CD80 costimulatory molecule via simultaneous CR2 ligation with C3 and Fc gammaRII (CD32) stimulation by IgG natural antibody. The current study demonstrated that KLH IC ligation to either CR2 or Fc gammaRII resulted in activation of a second co-stimulatory molecule, LFA-1 (CD11a, CD18). The possibility of polyclonal B cell stimulation by the presentation of KLH-iC3b/C3dg by antigen-non-specific B cells was excluded by demonstration that in vitro cultivation of peripheral blood mononuclear cells (PBMC) with KLH-iC3b/C3dg elicited only anti-KLH, and did not stimulate synthesis of antibodies to hepatitis C virus (HCV) or tetanus toxoid (TT). Of greatest significance, a specific anti-KLH response was only detectable in cultures stimulated with KLH-iC3b/C3dg and not in cultures stimulated with KLH alone or KLH-IgG. Thus, iC3b/C3dg that was bound to a primary protein antigen enhanced recognition and specific immunoglobulin synthesis by antigen-specific B cells, even though the antigen was taken up and processed via CR2 by both antigen-specific and non-specific B cells.

Construction of large naïve Fab libraries.

In recent years, a number of single-pot antibody (Ab) libraries have been described, which permit the rapid isolation of high-affinity Abs against large panels of antigens (Ags). Naïve libraries have been generated by tapping the natural primary (unselected) immune repertoire via cloning of Abs that recognize a variety of Ags (,). The rearranged V genes were amplified with the polymerase chain reaction (PCR) from B-cell mRNAs encoding immunoglobulin M (IgM) taken from nonimmunized donors. By using this procedure, Abs were recovered prior to encounter with Ag and unscreened for tolerance by the immune system. Indeed, a naïve library represents a good source of Abs against self, nonimmunogenic, and toxic Ags if the library is sufficiently large and diverse.

Selection of high affinity human neutralizing antibodies to VEGFR2 from a large antibody phage display library for antiangiogenesis therapy.

Compelling evidence suggests that vascular endothelial growth factor (VEGF) and its receptors play an important role in angiogenesis associated with tumor growth and metastasis. VEGF exerts its biologic activities through 2 transmembrane tyrosine kinase receptors: the fms-like tyrosine kinase receptor (Flt-1, or VEGFR1) and kinase insert domain-containing receptor (KDR or VEGFR2). We have previously produced a panel of antibodies directed against KDR from mice immunized with the recombinant form receptor. These antibodies efficiently neutralized VEGF-induced KDR activation and mitogenesis of human umbilical vascular endothelial cells (HUVEC). Murine antibodies, however, may not be suitable candidates for human therapy because of their propensity to elicit human anti-mouse antibody response. Here we isolated several high-affinity human Fab antibody fragments directed against KDR from an antibody phage display library constructed from the pooled B lymphocytes of nonimmunized healthy human donors. These human Fab fragments bind specifically to KDR with nanomolar affinity and block KDR/VEGF interaction with IC(50) of approximately 2-20 nM. Further, they effectively inhibit VEGF-stimulated mitogenesis of HUVEC and migration of human leukemia cells. Epitope mapping studies demonstrated that all neutralizing human antibodies bound the epitope(s) located within the first 3 N-terminal immunoglobulin-like domains of KDR, the same region that encompasses the binding site of VEGF. Our results suggest that these human anti-KDR antibodies may have potential application in the treatment of cancer and other diseases in which pathologic angiogenesis occurs.

Application of IL-5 ELISPOT assays to quantification of antigen-specific T helper responses

To be able to determine the frequencies of helper-type T lymphocyte (Th) precursors specific for viral or tumor-associated antigens, an ELISPOT assay for IL-5 production was developed. The assay reproducibility was first determined using fresh or cryopreserved peripheral blood mononuclear cells (PBMC) obtained from six normal donors and tested for IL-5 production after 48 h stimulation with phytohemagglutinin (PHA). Both inter-assay (within-subject CV=1.6%) and intra-assay (within-subject CV=0.8%) variabilities were found to be acceptable, and the frequencies of IL-5 secreting cells were comparable in cryopreserved and fresh PBMC of the same donors. The presence and frequency of Th precursor cells to viral capsid L1 protein (viral-like particles, VLP-L1) of human papillomavirus type 16 (HPV-16) in PBMC obtained from seven non-immunized donors and two volunteers immunized with VLP-L1 were then evaluated. Using autologous dendritic cells as antigen-presenting cells (APC) for VLP-L1 in direct 48-h ELISPOT assays, the mean frequency of IL-5 secreting CD4+ T cells was found to be <1/10 5 (negative) in normal donors but was 1/2, 436 and 1/3678 in the two volunteers immunized with VLP-L1. The assay is applicable to monitoring of the frequency of antigen-specific T cells in the peripheral circulation of individuals immunized with HPV-derived antigens. To test the assay utility in the assessment of Th tumor-reactive lymphocytes, we also used it to determine the frequency of the wild-type sequence (wt) p53 22–36 peptide-specific, HLA-DR4-restricted T cells in the bulk CD4+ T cell line. This frequency was 1/33. The ELISPOT assay for IL-5 production can be reliably used to measure Th-type responses in a variety of experimental settings.

Migratory patterns of thoracic duct lymphocytes into bronchus-associated lymphoid tissue of immunized rats.

Lymphocytes continuously circulate between the bloodstream and lymphoid organs, and their migration into lymphatic tissues presumably occurs through selective mechanisms. Although bronchus-associated lymphoid tissue (BALT) is known as an inductive tissue of the common mucosal immune system, little is known about how effectively the lymphocytes in the blood vessels migrate into the BALT, thereby enabling the BALT to act as an effector tissue in the immunologic responses of the lungs. To analyze whether or not thoracic duct lymphocytes (TDL) from immunized and nonimmunized rats possess different migratory patterns to the BALT, 5-(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled TDL were injected into rats with BALT hyperplasia that was produced by intratracheal administration of TNP-KLH, and then the number of labeled cells in the BALT were examined by immunohistochemical methods. We studied the following three groups at 12 h after the injection: group A, intraintestinally immunized donors and intratracheally immunized recipients; group B, nonimmunized donors and intratracheally immunized recipients; group C (control group), nonimmunized donors and nonimmunized recipients. Time course studies 0.5, 4, 12, and 24 h after the injection were done in groups A and C. In a cytokinetic study, larger numbers of CFSE-labeled lymphocytes were found at 12 h and 24 h in group A than in group C. At 12 h after the injection, the absolute number of CFSE-labeled lymphocytes per BALT was significantly higher in group A than in group B (p < 0.05), and was lowest in group C. Histologically, there was a marked proliferation of high endothelial venules (HEV), with findings of adhesion and influx of lymphocytes inside the HEV in group A. These observations indicate that the immunized BALT actively recruits immunized TDL through a specific mechanism of lymphocyte-endothelium recognition in HEV, which partially explains the process of BALT development as an effector tissue for local immunity.

Affinity Maturation of Natural Antibody Using a Chain Shuffling Technique and the Expression of Recombinant Antibodies in Escherichia coli

The affinity of natural antibody (Ka = 8 × 106 M−1) recognizing preS1 of hepatitis B virus (HBV) was improved by replacing the heavy (H) chain gene with repertoires of VH genes, obtained from two nonimmunized donors. Two separate clones, 1C2 and 1E4, showed affinities of 2.3 × 107 and 5.2 × 107 M−1, which were increased by factors of 2.8 and 6.5, respectively, compared to the parental clone. Recombinant scFvs (rscFvs) were expressed as fusion protein with minor coat protein, pIII, and secreted into medium after 3 h of induction with 1 mM IPTG. The expression level of functional rscFv capable of binding to preS1 reached a peak after 6–10 h (1C2) and 8–10 h (1E4) of IPTG induction, and afterwards decreased gradually. In order to achieve the overexpression of rscFv in E. coli, gene encoding scFv of 1C2 or 1E4 was inserted into pRSET vector. RscFvs were overexpressed as cytoplasmic inclusion bodies in E. coli BL 21 strain, which were denatured and carefully refolded using a continuous dialysis system. The purified recombinant fragments were pure when analyzed by SDS–PAGE and had the predicted size of 34 kDa. Clone 1E4 used the heavy chain gene belonging to family VII and subgroup III. Chain shuffling offers an alternative to random point mutation for affinity maturation of human antibody in vitro.

Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library.

To generate a stable resource from which high affinity human antibodies to any given antigen can be rapidly isolated, functional V-gene segments from 43 non-immunized human donors were used to construct a repertoire of 1.4 x 10(10) single-chain Fv (scFv) fragments displayed on the surface of phage. Fragments were cloned in a phagemid vector, enabling both phage displayed and soluble scFv to be produced without subcloning. A hexahistidine tag has been incorporated to allow rapid purification of scFv by nickel chelate chromatography. This library format reduces the time needed to isolate monoclonal antibody fragments to under two weeks. All of the measured binding affinities show a Kd < 10 nM and off-rates of 10(-3) to 10(-4) s-1, properties usually associated with antibodies from a secondary immune response. The best of these scFvs, an anti-fluorescein antibody (0.3 nM) and an antibody directed against the hapten DTPA (0.8 nM), are the first antibodies with subnanomolar binding affinities to be isolated from a naive library. Antibodies to doxorubicin, which is both immunosuppressive and toxic, as well as a high affinity and high specificity antibody to the steroid hormone oestradiol have been isolated. This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.

Characterization of human blood group scFv antibodies derived from a V gene phage‐display library

We previously reported the initial characterization of five human single-chain Fv (scFv) antibody fragments specific for the blood group antigens B, D(Rh), E(Rh), Kpb and HI. The scFvs were isolated from a phage-antibody library constructed from the variable region genes of two non-immunized donors. In this paper we report the specificity, affinity and kinetics of antigen binding of these scFv fragments. All five scFvs agglutinated the appropriate red cell phenotype following the addition of a monoclonal antibody which recognizes a peptide tag incorporated into the scFv. The anti-B and anti-HI scFv molecules, which recognize high density carbohydrate antigens, spontaneously polymerized and agglutinated red cells directly. None of the antibody fragments showed cross-reactivity with other red cell antigens, with the exception of the anti-E which reacted weakly with E-negative cells. Specific scFv binding was confirmed by ELISA, flow cytometry and radioactive labelling. The anti-D scFv recognized 17,600 sites on cDE/cDE red cells with an association constant (Ka), of 5.2 x 10(7) M-1 and a rate constant for dissociation (koff) of 1.9 x 10(-2) s-1. The anti-E scFv recognized 29,800 and 39,800 sites on cDE/cDE red cells in two experiments with Kas of 8.4 x 10(6) and 4.4 x 10(7) M-1. The koff for this antibody was 2.7 x 10(-2) s-1. The results demonstrate that scFv antibody fragments specific for cell surface antigens and possessing affinities typical of the primary immune response can be obtained from a phage-display library.