Immune response to hepatitis B vaccination in HIV-positive adults with isolated antibodies to HBV core antigen.

OBJECTIVE: To investigate the merits of vaccination against hepatitis B virus (HBV) in HIV-positive individuals with isolated antibodies to hepatitis B core antigen (anti-HBc). METHODS: HIV-positive patients with isolated anti-HBc and CD4 counts >200 cells/mm(3) received HBV vaccination. An antibody titre to hepatitis B surface antigen (anti-HBs titres) ≥10 IU/L one month post-vaccination was termed an anamnestic response; a titre <10 IU/L was termed a primary response. Patients with primary responses received a 3-dose vaccine course. Anti-HBs titres in all responders were measured 12 and 24 months post-vaccination. RESULTS: 37 patients were studied: 19 (51%) were co-infected with hepatitis C; median CD4 count was 443 cells/mm(3). 8/37 patients (22%) elicited an anamnestic response. 29/37 patients (78%) elicited a primary response. After a 3-dose vaccine course, 15/25 primary responders (60%) achieved anti-HBs titres ≥10 IU/L. HIV acquisition through injecting drug use was the only independent predictor of an anamnestic response (OR 22.9, CI 1.71-306.74, P=0.018). Median anti-HBs titres for anamnestic and primary responders were 51 IU/L (13-127) and 157 IU/L (25-650) respectively. Of all responders, 12/23 (52%) retained anti-HBs titres ≥10 IU/L at 24 months. Anti-HBs duration was not significantly different between anamnestic and primary responders. CONCLUSIONS: 23/37 HIV-positive patients (62%) with isolated anti-HBc achieved anti-HBs titres ≥10 IU/L after 1-3 vaccine doses. However, duration of this immune response was short-lived (

Penetration and binding of radiolabeled anti-carcinoembryonic antigen monoclonal antibodies and their antigen binding fragments in human colon multicellular tumor spheroids.

The binding and penetration of two 125I-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAb) and their F(ab')2 and Fab fragments were measured in multicellular spheroids of poorly (HT29) and moderately well differentiated (Co112) human colon adenocarcinomas which express different amounts of CEA. Spheroids cultured in vitro model tumor microenvironments where poor vascular supply may modulate antigen expression and accessibility. The two MAb studied, 202 and 35, were shown previously to react with different CEA epitopes and to have high affinities of 1.2 and 5.8 X 10(9) M-1, respectively. MAb 202 has also been shown to cross-react with antigens present on human granulocytes and normal epithelial cells from human lung and pancreas. Specific binding of intact MAb and fragments of both antibodies was demonstrated for both types of human colon carcinoma spheroids compared to mouse colon carcinoma (CL26) and mammary tumor (EMT6/Ro) spheroids. Total binding of MAb and fragments was greater (1.5- to 2.5-fold) after 4 h compared to 1 h of exposure; the amount of binding compared to control IgG1 was 5- to 30-fold greater after 1-h incubation and 15 to 200 times greater after 4 h. This binding was stable as demonstrated by short and long wash experiments at 37 degrees and 4 degrees C. The binding of F(ab')2 and Fab fragments of the anti-CEA MAb 35 to spheroids of human colon Co112 was almost 2-fold greater than that of the intact MAb. However, for MAb 202, the binding of intact MAb and F(ab')2 was greater than that of Fab fragments. In addition the binding of both intact and F(ab')2 fragments of MAb 202 was greater than that obtained with MAb 35. Specific binding of both antibodies to HT29 spheroids, which express less CEA, was decreased for MAb and fragments of both 202 and 35. Autoradiography and immunoperoxidase experiments were performed to determine the penetration of MAb and fragments after incubation with intact spheroids. Comparisons were made with labeled MAb directly applied to frozen sections of spheroids. F(ab')2 and Fab fragments of both antibodies were bound at the surface of intact spheroids and penetrated to eight to ten cells, but the intact MAb were localized mainly at the spheroid surface and the outer one to three cell layers. There was much less binding at the surfaces of HT29 compared to Co112 spheroids. An enzyme immunoassay using MAb 35 and 202 demonstrated that Co112 spheroids produced about 8-fold more CEA/mg of cell protein than did monolayer cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibodies against idiotypic determinant(s) of the T cell receptor from HPB-ALL cells induce IL2 production in Jurkat cells without apparent evidence of binding.

Two monoclonal antibodies (mAb) directed against idiotypic determinants of the T cell receptor (anti-Ti) from HPB-ALL cells induce interleukin 2 (IL2) production in Jurkat T cells without evidence of binding to these cells as judged by fluorescence-activated cell sorter (FACS) analysis, indirect antibody-binding radioimmunoassay and direct binding studies with 125I-labeled mAb. The IL2 response induced by these mAb observed both in the presence and absence of phorbol myristate acetate was in the range of that obtained when Jurkat cells were stimulated with phytohemagglutinin or anti-T3 mAb (Leu 4). The idiotypic specificity of the two anti-HPB-ALL Ti mAb was demonstrated by several criteria. Both mAb bound specifically to HPB-ALL cells as determined by radioimmunoassay or FACS analysis but not with 8 other T cell lines. The anti-HPB-ALL Ti mAb precipitated a disulfide-linked heterodimer of 85 kDa only from 125I-labeled HPB-ALL cells and not from other cell lines tested. Incubation of HPB-ALL cells with anti-T3 abrogated the expression of T3 and induced co-modulation of the idiotypic structures detected by the two anti-HPB-ALL Ti mAb. Conversely, incubation of HPB-ALL cells with either one of the anti-Ti mAb abrogated the expression of T3 and of the idiotypic structures. Our results suggest that mAb with an apparent unique specificity for the receptor of the immunizing T cell line HPB-ALL can activate Jurkat cells by a very weak cross-reaction with these cells, which is not detectable by conventional binding tests.

Radiolabeled fragments of monoclonal antibodies against carcinoembryonic antigen for localization of human colon carcinoma grafted into nude mice.

Four monoclonal antibodies against carcinoembryonic antigen (CEA) have been selected from 32 hybrids that produce antibodies against this antigen, by the criteria of high affinity for CEA and low cross-reactivity with granulocyte glycoprotein(s). The specificity of tumor localization in vivo of the four MAb, and their F(ab')2 and Fab fragments was compared in nude mice bearing grafts of a serially transplanted, CEA-producing, human colon carcinoma. The distribution of radiolabeled MAb and their fragments after intravenous injection was analyzed by direct measurement of radioactivity in tumor and normal organs, as well as by whole-body scanning and by autoradiography of tumor sections. Paired labeling experiments, in which 131I-labeled antibody or fragments and 125I-labeled control IgG are injected simultaneously, were undertaken to determine the relative tumor uptakes of each labeled protein. The tumor antibody uptake divided by that of control IgG defines the specificity index of localization. Tumor antibody uptakes (as compared with the whole mouse), ranging between 7 and 15, and specificity indices ranging between 3.4 and 6.8, were obtained with the four intact MAb at day 4-5 after injection. With F(ab')2 fragments of the four MAb, at day 3, the tumor antibody uptakes ranged between 12 and 24 and the specificity indices between 5.3 and 8.2. With the Fab fragments prepared from the two most promising MAb, the antibody uptakes reached values of 34 and 82 at day 2-3 and the specificity indices were as high as 12 and 19. The scanning results paralleled those obtained by direct measurement of radioactivity. With intact MAb, tumor grafts of 0.5-1 g gave very contrasted positive scans 3 d after injection. Using MAb fragments, tumors of smaller size were detectable earlier. The best results were obtained with Fab fragments of MAb 35, which gave clear detections of tumors weighing only 0.1 g as early as 48 h after injection. Autoradiographs of tumor sections from mice injected with 125I-labeled MAb demonstrated that the radioactivity was localized in the tumor tissues and not in the stromal connective tissue of mouse origin. The highest radioactivity concentration was localized in areas known to contain CEA such as the pseudolumen of glands and the apical side of carcinoma cells. The penetration of radioactivity in the central part of tumor nodules and the pseudolumen appeared to be increased with the use of MAb fragments.

Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments.

Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats.

Liver kidney microsomal type 1 antibodies reduce the CYP2D6 activity in patients with chronic hepatitis C virus infection.

Liver kidney microsomal type 1 (LKM-1) antibodies have been shown to decrease the CYP2D6 activity in vitro and are present in a minority of patients with chronic hepatitis C infection. We investigated whether LKM-1 antibodies might reduce the CYP2D6 activity in vivo. All patients enrolled in the Swiss Hepatitis C Cohort Study and tested for LKM-1 antibodies were assessed (n = 1723): 10 eligible patients were matched with patients without LKM-1 antibodies. Patients were genotyped for CYP2D6 variants to exclude individuals with a poor metabolizer genotype. CYP2D6 activity was measured by a specific substrate using the dextromethorphan/dextrorphan metabolic ratio to classify patients into four activity phenotypes. All patients had a CYP2D6 extensive metabolizer genotype. The observed phenotype was concordant with the CYP2D6 genotype in most LKM-negative patients, whereas only three LKM-1 positive patients had a concordant phenotype (six presented an intermediate and one a poor metabolizer phenotype). The median DEM/DOR ratio was sixfold higher in LKM-1 positive than in LKM-1 negative patients (0.096 vs. 0.016, P = 0.004), indicating that CYP2D6 metabolic function was significantly reduced in the presence of LKM-1 antibodies. In chronic hepatitis C patients with LKM-1 antibodies, the CYP2D6 metabolic activity was on average reduced by 80%. The impact of LKM-1 antibodies on CYP2D6-mediated drug metabolism pathways warrants further translational studies.

Imaging and therapy with monoclonal antibodies in non-hematopoietic tumors.

Although radiolabelled monoclonal antibodies are useful in tumor imaging, in our opinion their most important role is in the evaluation of the capacity of newly developed monoclonal antibodies to localize in tumors specifically. Intravenous injections of monoclonal antibody fragments, labelled with beta-emitting radionuclides, can completely eradicate large human colon carcinoma xenografts in nude mice whereas this is not achieved by unconjugated monoclonal antibodies. New strategies are being developed to make radioimmunotherapy applicable to carcinoma patients.

Mucosal but not parenteral immunization with purified human papillomavirus type 16 virus-like particles induces neutralizing titers of antibodies throughout the estrous cycle of mice.

We have recently shown that nasal immunization of anesthetized mice with human papillomavirus type 16 (HPV16) virus-like particles (VLPs) is highly effective at inducing both neutralizing immunoglobulin A (IgA) and IgG in genital secretions, while parenteral immunization induced only neutralizing IgG. Our data also demonstrated that both isotypes are similarly neutralizing according to an in vitro pseudotyped neutralization assay. However, it is known that various amounts of IgA and IgG are produced in genital secretions along the estrous cycle. Therefore, we have investigated how this variation influences the amount of HPV16 neutralizing antibodies induced after immunization with VLPs. We have compared parenteral and nasal protocols of vaccination with daily samplings of genital secretions of mice. Enzyme-linked immunosorbent assay analysis showed that total IgA and IgG inversely varied along the estrous cycle, with the largest amounts of IgA in proestrus-estrus and the largest amount of IgG in diestrus. This resulted in HPV16 neutralizing titers of IgG only being achieved during diestrus upon parenteral immunization. In contrast, nasal vaccination induced neutralizing titers of IgA plus IgG throughout the estrous cycle, as confirmed by in vitro pseudotyped neutralization assays. Our data suggest that mucosal immunization might be more efficient than parenteral immunization at inducing continuous protection of the female genital tract.

Antigen binding properties of purified immunoglobulin A and reconstituted secretory immunoglobulin A antibodies.

The hybridoma cell line ZAC3 expresses Vibrio cholerae lipopolysaccharide (LPS)-specific mouse IgA molecules as a heterogeneous population of monomeric (IgAm), dimeric (IgAd), and polymeric (IgAp) forms. We describe a gentle method combining ultrafiltration, ion-exchange chromatography, and size exclusion chromatography for the simultaneous and qualitative separation of the three molecular forms. Milligram quantities of purified IgA molecules were recovered allowing for direct comparison of the biological properties of the three forms. LPS binding specificity was tested after purification; IgAd and IgAp were found to bind strongly to LPS whereas IgAm did not. Secretory IgA (sIgA) could be reconstituted in vitro by combining recombinant secretory component (rSC) and purified IgAd or IgAp, but not IgAm. Surface plasmon resonance-based binding experiments using LPS monolayers indicated that purified reconstituted sIgA and IgA molecules recognize LPS with identical affinity (KA 1.0 x 10(8)M-1). Thus, this very sensitive assay provides the first evidence that the function of SC in sIgA complex is not to modify the affinity for the antigen. KA falls to 6.6 x 10(5) M-1 when measured by calorimetry using detergent-solubilized LPS and IgA, suggesting that the LPS environment is critical for recognition by the antibody.

Immunochemical characterization of two antigens recognized by new monoclonal antibodies against human colon carcinoma.

Two different monoclonal antibodies (MAb), called L-D1 and L-C5, were produced after immunization with either intact cells or the methanol phase of glycolipid extracts, respectively, from the same human colon carcinoma line, LoVo. As determined by an antibody-binding radioimmunoassay (RIA) on intact cells, MAb L-D1 and MAb L-C5 were highly reactive with all five colon carcinoma lines tested and with only one out of the 21 cell lines of various tissue origin tested. No reactivity of either MAb was observed with peripheral blood lymphocytes, granulocytes, or erythrocytes from healthy donors of various blood groups. Both MAb were tested in competitive binding experiments with an anti-CEA MAb from our laboratory (CEA 35) and with two previously described anti-colon carcinoma MAb from the Wistar Institute called 1083-17-1A (17-1A) and NS-19.9. In competitive binding experiments, MAb L-D1 was inhibited by MAb 17-1A and reciprocally, whereas MAb L-C5 was not inhibited by any of the other MAb tested. MAb L-D1 precipitated a major protein band with an apparent molecular weight (MW) of 41 kilodaltons (kD); interestingly, MAb 17-1A, which was reported to react with an uncharacterized antigen, precipitated the same protein band of 41 kD. This was confirmed with immunodepletion experiments. Furthermore, after treatment of the colon carcinoma cell line with tunicamycin, both MAb L-D1 and 17-1A precipitated a protein band of 35 kD. This shift of 6 kD suggests that the glycoprotein recognized by these 2 MAb contains two to three N-linked carbohydrate side chains. MAb L-C5 precipitated a group of three to four protein bands ranging from 43 to 53 kD that were not modified by tunicamycin treatment. A preliminary study conducted by using immunoperoxidase labeling on frozen sections of primary colon carcinoma showed that the two new MAb react strongly with these tumors, but also weakly with the normal adjacent mucosa, as did the other anti-colon carcinoma MAb tested.

Use of radiolabelled monoclonal anti-CEA antibodies for the detection of human carcinomas by external photoscanning and tomoscintigraphy

Paul Ehrlich's inspired concept of 'magic bullets' for the cure of diseases has been revitalized by recent advances in immunology1. In particular, the development of cell fusion technology allowing the production of monoclonal antibodies (Mabs) with exquisite specificities2 triggered new hopes that we may now have the perfect carrier molecules with which to deliver cytotoxic drugs3 or toxins4 to the hidden cancer cells. This article reviews data on one aspect of the magic bullet concept, the use of radiolabelled antibodies as tracers for tumour localization. It will also discuss the very recent clinical use of 131I-labelled Mabs against carcinoembryonic antigen (CEA)5 to detect carcinoma either by conventional external photoscanning or by single photon emission computerized tomography (SPELT). This alliance of the most modern tools from immunology (Mabs) and nuclear medicine (SPELT) appears promising as a way to improve the sensitivity of 'immunoscintigraphy'. However, this approach is not yet ready, for widespread clinical use.