Temperature enhancement of syncytium formation by HIV and Sendai virus.

Syncytium formation, the characteristic cytopathic effect (CPE) of the human immunodeficiency virus (HIV) and cell fusion by Sendai virus, is accelerated by increasing the ambient temperature to values at which normal metabolic activity is inhibited. Uninfected C8166, CEM, and H9 cells were absorbed at 4 degrees C onto monolayers of H9 cells chronically infected with HIV and incubated subsequently at either 37 degrees C or 45 degrees C. Similarly chick and human erythrocytes and Hela cells were agglutinated with Sendai virus at 4 degrees C before incubation at temperatures of up to 50 degrees C. With both viruses the rate of cell fusion was directly related to temperature. Since membrane fluidity is dependent on the phase-transition temperature points of the membrane lipids it is proposed that sufficient membrane fluidity is essential for cell fusion to occur. The implication of these observations on the cytopathology of HIV is discussed.     

Adhesion molecule monoclonal antibodies inhibit experimental autoimmune thyroiditis.

To examine the role played by adhesion molecules in thyroid autoimmunity, we have assessed the effect of administering monoclonal antibodies (mAb) against intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) in experimental autoimmune thyroiditis, induced by immunizing rats with thyroglobulin in complete Freund's adjuvant. The antibody against LFA-1, but not against ICAM-1, reduced thyroglobulin antibody production (P < 0.01) and both antibodies caused a significant reduction (P < 0.002) in the severity of the thyroidal lymphocytic infiltration. In vitro, both mAb impaired the proliferative response of splenic and lymph node T cells to thyroglobulin, but only the antibody against LFA-1 reduced thyroid cell killing assessed using splenic lymphocytes as effectors. Monoclonal antibodies against both these adhesion molecules appear to inhibit cell-mediated autoimmunity in vivo, but only the LFA-1 mAb reduced the autoantibody response.     

E-rosette formation using heteropolymeric monoclonal antibodies.

We have used bispecific, cross-linked monoclonal antibodies (heteropolymers, HP) to facilitate rosette formation between human erythrocytes (EH) and dinitrophenylated sheep erythrocytes (DNP-ES) in the absence of complement. The HP contain monoclonal antibodies (mAbs) specific for both the EH C3b receptor (CR1), and the DNP group, and control experiments with homologous competing non-cross-linked mAbs and naive EH and ES confirm the specificity of the rosetting reaction. These results extend our previous studies, of HP-mediated binding of simple protein antigens to EH CR1, to complex particulate antigens and may eventually allow for the targeting and clearance from the circulation of a variety of pathogens associated with infectious disease.     

Anti-idiotypic antibodies against Puumala virus glycoprotein-specific monoclonal antibodies inhibit virus infection in cell cultures

Summary Rabbit anti-idiotypic antibodies (anti-ids) were generated against three bank vole and one human monoclonal antibody (MAb) specific for the two envelope glycoproteins of Puumala virus (G 1 and G 2). The anti-ids were purified by sequential immunoaffinity chromatography. Each anti-id inhibited the antigen binding of its respective MAb in a competitive ELISA. This inhibition, and the absence of cross-reactivity among the anti-ids for heterologous MAbs, showed that they all were specific for unique determinants on the antigen binding site of the homologous MAb. The anti-ids reacted with non-infected Vero E 6 cells when examined by immunofluorescence and ELISA, indicating the presence of antibodies that mimic epitopes on the virus. Preincubation of Vero E 6 cells with two of the anti-ids produced against neutralizing MAbs inhibited Puumala virus infection, suggesting that these two anti-ids blocked a cellular component involved in virus infection.     

多种单抗联合检测HIV抗原

目的 建立多种单抗联合早期检测HIV抗原的夹心ELISA方法.方法 以SAS盐析沉淀法和亲和层析法纯化抗HIV-1 p24、gp41、gp120及抗HIV-2 gp36的腹水型单克隆抗体(McAb),用高碘酸钠法将纯化的McAb以HRP进行标记.建立针对单个抗原的双抗体夹心ELISA法,对其灵敏度及特异性进行检测.将筛选得到的4株捕获McAb按比例混合作为捕获抗体,4株酶标McAb按比例混合作为检测抗体,建立多种单抗联合检测HIV抗原的夹心ELISA方法,检测混合HIV抗原.结果 按确定的最优反应条件建立的多种McAb联合夹心ELISA方法,检测到的最高稀释度的HIV混合抗原中各抗原的终浓度分别为:重组HIV-1 p24:0.625 pg/ml,gp41:6.25 ng/ml,gp120:6.25 ng/ml;HIV-2 gp36:9.25 ng/ml.结论 建立了具有高度敏感性的鸡尾酒式多种单抗联合检测HIV抗原的夹心ELISA法,为早期榆测HIV抗原提供了新的思路,为后续的研究奠定了一定基础.     

多种单抗联合检测HIV抗原

目的 建立多种单抗联合早期检测HIV抗原的夹心ELISA方法.方法 以SAS盐析沉淀法和亲和层析法纯化抗HIV-1 p24、gp41、gp120及抗HIV-2 gp36的腹水型单克隆抗体(McAb),用高碘酸钠法将纯化的McAb以HRP进行标记.建立针对单个抗原的双抗体夹心ELISA法,对其灵敏度及特异性进行检测.将筛选得到的4株捕获McAb按比例混合作为捕获抗体,4株酶标McAb按比例混合作为检测抗体,建立多种单抗联合检测HIV抗原的夹心ELISA方法,检测混合HIV抗原.结果 按确定的最优反应条件建立的多种McAb联合夹心ELISA方法,检测到的最高稀释度的HIV混合抗原中各抗原的终浓度分别为:重组HIV-1 p24:0.625 pg/ml,gp41:6.25 ng/ml,gp120:6.25 ng/ml;HIV-2 gp36:9.25 ng/ml.结论 建立了具有高度敏感性的鸡尾酒式多种单抗联合检测HIV抗原的夹心ELISA法,为早期榆测HIV抗原提供了新的思路,为后续的研究奠定了一定基础.     

Rat monoclonal antibodies which inhibit the in vitro multiplication of plasmodium knowlesi

A total of 28 double cloned monoclonal antibodies specific for Plasmodium knowlesi were raised by fusion of Y3 rat myeloma cells with spleen cells of A0 rats immunized with W1 variant isolated merozoites. Four of these antibodies reacted positively in a solid phase radioimmunoassay against glutaraldehyde-fixed schizonts but gave no detectable reaction on indirect immunofluorescence against methanol-fixed schizonts or merozoites. The remaining 24 antibodies could be divided into 13 distinctive immunofluorescent categories on the basis of their patterns of binding to schizonts and merozoites and reactivity with Plasmodium falciparum. Eight antibodies were studied for their ability to inhibit the in vitro multiplication of W1 P. knowlesi as assessed by parasite incorporation of ³H-amino acids and parasite counts. Partially purified antibody preparations from ascitic fluids were all inhibitory for parasite growth; however, when fully purified antibodies were tested, six of the eight proved to be non-inhibitory. Two of the purified antibodies, both IgG2a isotype, inhibited the in vitro multiplication of P. knowlesi in a dose-dependent manner. Inhibition was not associated with detectable damage to intracellular parasites, suggesting that the inhibitory monoclonal antibodies act by blocking the reinfection of red cells by newly released merozoites. On immunofluorescent analysis both inhibitory antibodies bound to methanol-fixed schizonts, with the intensity increasing for progressively more mature parasites; both reacted diffusely with isolated merozoites, and neither cross-reacted with P. falciparum. Both bound specifically to a single metabolically labelled polypeptide which appears to be a minor parasite component and has an approximate molecular weight of 66,000 when analysed by SDS-PAGE fluorography. The putative protective antigen of P. knowlesi has potential interest as a vaccine against P. knowlesi malaria.     

Characterization of antibodies that inhibit HIV gp120 antigen processing and presentation

Antibodies to the CD4-binding site (CD4bs) of HIV-1 envelope gp120 have been shown to inhibit MHC class II presentation of this antigen, but the mechanism is not fully understood. To define the key determinants contributing to the inhibitory activity of these antibodies, a panel of anti-CD4bs monoclonal antibodies with different affinities was studied and compared to antibodies specific for the chemokine receptor-binding site or other gp120 regions. Anti-CD4bs antibodies that completely obstruct gp120 presentation exhibit three common properties: relatively high affinity for gp120, acid-stable interaction with gp120, and the capacity to slow the kinetics of gp120 proteolytic processing. None of these antibodies prevents gp120 internalization into APC. Notably, the broadly virus-neutralizing anti-CD4bs IgG1b12 does not block gp120 presentation as strongly, because although IgG1b12 has a relatively high affinity, it dissociates from gp120 more readily at acidic pH and only moderately retards gp120 proteolysis. Other anti-gp120 antibodies, regardless of their affinities, do not affect gp120 presentation. Hence, high-affinity anti-CD4bs antibodies that do not dissociate from gp120 at endolysosomal pH obstruct gp120 processing and prevent MHC class II presentation of this antigen. The presence of such antibodies could contribute to the dearth of anti-gp120 T helper responses in chronically HIV-1-infected patients.     

Anti-gD monoclonal antibodies inhibit cell fusion induced by herpes simplex virus type 1

Monoclonal antibodies directed against glycoprotein D of herpes simplex virus completely inhibited fusion of Vero cells infected with type 1 virus. In contrast, several monoclonal antibodies directed against other viral glycoproteins, including B, were ineffective or were only minimally inhibitory at the highest concentrations tested.     

Differences in affinity of anti-CD4 monoclonal antibodies predict their effects on syncytium induction by human immunodeficiency virus.

A panel of 20 anti-CD4 monoclonal antibodies (mAb) was ranked in terms of affinity, using an inhibition radioimmunoassay. The ability of these antibodies to inhibit the induction of syncytia by human immunodeficiency virus (HIV) and to prevent binding of the HIV envelope glycoprotein 120 (gp120) to CD4 was also measured. Syncytium inhibition correlated strongly with affinity (P less than 0.001) but only weakly with inhibition of gp120 binding (P = 0.038). Some antibodies partially blocked binding of gp120 to CD4 but did not inhibit syncytia, and some antibodies inhibited syncytia but only weakly blocked binding of gp120. These results suggest that the syncytium inhibition assay is highly affinity-dependent, and that epitopes on CD4 concerned with virus binding are distinct from those involved in syncytium formation.     

The properties of monoclonal antibodies to HIV gag gene proteins]

Immunization of BALB/c mice with natural purified HIV antigen, fusion of spleen cells with myeloma cells and subsequent selection of hybrid clones using recombinant gag antigen of HIV gave hybridomas producing monoclonal antibodies (MCA) to HIV. The immune blotting method demonstrated that 3 clones interacted with protein p24 and 4 clones with protein p17 of HIV. Competitive EIA led to a conclusion that the resulting MCA detected at least 3 antigenic determinants in proteins, products of gag gene of HIV. The potentials of using these MCA for the detection of viral antigen in HIV-infected continuous cell lines were demonstrated.     

Modified HIV envelope proteins with enhanced binding to neutralizing monoclonal antibodies

The target for neutralizing antibodies against human immunodeficiency virus (HIV) is the trimeric Env protein on the native virion. Conserved neutralizing epitopes of receptor binding sites are located in the recessed core of the Env protein, partially masked by glycosylations and variable loops. In this study, we have investigated the effects of modifications of the HIV Env protein by glycosylation site mutations, deletions of variable loops, or combinations of both types of mutations on their protein functions and reactivities with neutralizing antibodies. Modified Env proteins were expressed in insect or mammalian cells, and their reactivity with epitope-specific broadly neutralizing monoclonal antibodies (Mabs) was determined by flow cytometry. A unique mutant designated 3G with mutations in three glycosylation motifs within the V3/C3 domains surrounding the CD4 binding site showed higher levels of binding to most broadly neutralizing Mabs (b12 and 2F5) in both insect and mammalian expression systems. Mutants with a deletion of both V1 and V2 loop domains or with a unique combination of both types of mutations also bound to most neutralizing Mabs at higher levels compared to the wild-type control. Most mutants maintained the ability to bind CD4 and to induce syncytium formation at similar or higher levels as compared to that of the wild-type Env protein, except for a mutant with a combination of variable loop deletions and deglycosylation mutations. Our study suggests that modified HIV Env proteins with reduced glycosylation in domains surrounding the CD4 binding site or variable loop-deleted mutants expose important neutralizing epitopes at higher levels than wild type and may provide novel vaccine immunogens.     

Cytoskeletal inhibitors, anti-adhesion molecule antibodies, and lectins inhibit hepatocyte spheroid formation

We investigated the role of cytoskeletons, adhesion molecules, membrane-glycosylations, and proteoglycans in forming the shape of adult rat hepatocyte spheroids. Isolated hepatocytes were cultured on dishes coated with chondroitin sulfate phosphatidyl ethanolamine (CS-PE). Spheroid-forming ability was observed after adding cytoskeletal inhibitors (cytochalasin D, colchicine, okadaic acid, mycalolide B), anti-adhesion molecule antibodies (anti-E-cadherin, anti-connexin 32, anti-zo-1), a glycosphingolipid synthetic inhibitor (N-butyldeoxynojirimycin), a proteoglycan synthetic inhibitor (p-nitrophenyl-beta-D-xylopyranoside), and several lectins. Localization of actin was studied using confocal microscopy after rhodamine-phalloidin staining. Adding cytoskeletal inhibitors on the initial day resulted in weakly clustered cell aggregates rather than smoothly formed spheroids. These effects disappeared at lower reagent concentrations. When reagents were added on day 3, after the formation of spheroids, only mycalolide B was associated with an irregular spheroid surface; the others had no effect. Adding the anti-E-cadherin, anti-connexin 32 on the initial day showed inhibition of spheroid formation, but anti-zo-1 and proteoglycan synthetic inhibitor had no effects. Among the several lectins, only Wheat Germ Agglutinin (WGA), Ricinus communis Agglutinin I (RCA-I), and Concanavalin A (ConA) showed inhibition. These results suggest that cytoskeletal conformation and some adhesion molecules are necessary to form spheroids. Based on the interactions between lectins and hepatocytes in the present study, hepatocytes appear to contain an N-linked complex or N-linked hybrid glycosylated chains.     

Multispecific monoclonal antibodies

Monoclonal antibodies are frequently shown to participate in unexpected cross reactions involving two apparently dissimilar antigens. This can be attributed either to partial epitope identity or to irrelevant interactions involving additional binding capacity of the antibody. The majority of such interactions appear to fall into the latter category. Such cross reactions are most commonly detected when one of the antigens has a high epitope density and the antibody is multivalent so that a spurious interaction of low intrinsic affinity is amplified by local concentration effects. In this review Souravi Ghosh and Ailsa Campbell discuss the selection of a monoclonal antibody for maximum affinity for the antigens it is designed to study and minimum cross-reactivity.     

Red plaque formation of Coxiella burnetii and reduction assay by monoclonal antibodies

A red plaque technique for C. burnetii which utilizes primary chicken embryo cells, is described. Red plaques could be consistently detected as early as 6 days, usually 8 days post inoculation (p.i.), reflecting that C. burnetii proliferated within the phagolysosomes of host cells. Incubation with phase II monoclonal antibodies or inactivated immune sera containing phase I and phase II antibodies or phase II antibodies only, markedly reduced phase II C. burnetii red plaques. On the other hand, red plaques from phase I organisms increased several times when phase I cells were mixed with phase I monoclonal antibodies or inactivated immune sera containing phase I and phase II antibodies. By indirect red plaque reduction assay red plaque production by phase II cells could be reduced as well.     

Purification of immunoreactive radiolabeled monoclonal antibodies with anti-idiotypic monoclonal antibodies

A method is described to purify immunoreactive monoclonal antibodies from radiolabeled monoclonal antibody preparations. The method is based on incubation of radiolabeled monoclonal antibodies with insolubilized anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of monoclonal antibodies to be purified and elution of bound monoclonal antibodies with a low pH buffer. The immunoreactive fraction of the purified monoclonal antibodies was at least 82%; the yield was at least 73%. The purification procedure did not cause any detectable change in the affinity constant of the eluted monoclonal antibodies. The method is simple and rapid; the requirement for anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of the antibodies to be purified is not likely to represent a major limitation in the broad application of the present method, since the hybridoma technology has greatly facilitated the development of anti-idiotypic monoclonal antibodies.     

B cell responses to HIV and the development of human monoclonal antibodies.

In this review B cell responses in HIV-infected individuals are summarized together with the techniques used to date to produce human monoclonals to HIV and the properties of these antibodies. Profound disturbances in B cell responses are apparent both in vivo and in vitro. While there is evidence in vivo of marked polyclonal B cell activation, primary and secondary antibody responses are impaired. Similarly these cells exhibit spontaneous immunoglobulin secretion upon in vitro culture but do not readily respond to B cell mitogens and recall antigens including HIV. Furthermore, certain of these defects can be reproduced in normal B cells in vitro by incubation with HIV or HIV coded peptides. Individuals infected with HIV develop antibodies to HIV structural proteins (e.g. p17, p24, gp41 and gp120) and regulatory proteins (e.g. vif, nef, RT). Autoantibodies against a number of immunologically important molecules are also frequently observed. The anti-HIV antibodies are predominantly of the IgG1 isotype and exhibit a variety of effects on the virus in vitro. To date, using conventional immortalization strategies, an appreciable number of human monoclonals to HIV have been developed. These have been specific for gp41, gp120 and gag with antibodies of the former specificity predominating. The majority of these antibodies have been of the IgG1 isotype. Only a small number of the antibodies neutralize virus in vitro and most of these react with gp120. The neutralizing antibodies recognize conformational and carbohydrate epitopes or epitopes in amino acid positions 306-322. The predominant epitopes recognized by the anti-gp41 antibodies were in amino acid positions 579-620 and 644-662. A high percentage (congruent to 25%) of these antibodies enhance viral growth in vitro. The problems relating to the production of human monoclonals to HIV are discussed together with strategies that could be used in the future.