The opsonizing activity of the sera of hamadryas baboons immunized with a preparation of the outer membranes of Francisella tularensis (based on data from the luminol-dependent luminescence method)]

The opsonizing properties of sera obtained from hamadryas baboons immunized with the preparation of F. tularensis outer membranes (OM) were studied with the use of luminol-dependent chemiluminescence (CL) of whole blood. The immunization of monkeys with the OM preparation was shown to lead to the formation of functionally active antibodies possessing opsonizing properties with respect to virulent F. tularensis. Immune sera obtained from the animals immunized with live vaccine and from those immunized with OM preparation had no essential differences in their opsonizing properties. The level of IgG antibodies in immune sera correlated with the CL parameters of whole blood in the presence of F. tularensis opsonized with these sera. Increased CL of phagocytes observed after addition of bacteria and immune sera under test to whole blood taken from a nonimmune donor made it possible to evaluate the functional activity of antibodies, thus permitting its use as a test for the evaluation of the effectiveness of new vaccine preparations.     

Natural and immune human antibodies reactive with antigens of virulent Neisseria gonorrhoeae: immunoglobulins G, M, And A

Natural and immune human antibodies reactive with heat-labile and heat-stable antigens of virulent Neisseria gonorrhoeae were studied by use of an indirect fluorescent-antibody (IFA) procedure. The immunoglobulin class of the reactive antibodies was identified by using fluorescein-conjugated antisera specific for human IgG, IgA, or IgM in the IFA procedure. The effects of heat and mercaptoethanol on IFA reactivities were also studied. It appeared that antibodies of the IgG, IgM, and IgA classes present in the sera of both infected persons (immune antibodies) and normal persons with no history of gonococcal infection (natural antibodies) react with heat-stable somatic antigens. Immune IgG antibodies, however, were distinguishable from natural IgG antibodies by their ability to recognize heat-labile surface antigens. The distinction between natural and immune IgM antibodies was less obvious. IgM antibodies from both infected and normal persons appeared to react with heat-labile antigens. Some, but not all, infected persons had immune IgA antibodies to heat-labile as well as to heat-stable antigens. Treatment of sera with mercaptoethanol had no effect on IgG antibodies. The IFA activity of IgM antibodies was decreased, but not abolished. The effects of mercaptoethanol on IgA antibodies were variable. Some sera showed a decrease in IgA titer, and others showed an increase in IgA activity to certain antigens. Immune IgG antibodies were more resistant to heating than were natural IgG antibodies. Natural and immune IgM antibodies appeared equally sensitive to heating. IgA activity, on the other hand, was increased by heating sera at 60 C, but was decreased at higher temperatures. Thus, it appears that natural and immune human IgG antibodies to N. gonorrhoeae may be distinguished by their interactions with heat-labile antigens and by their resistance to heating.     

Effect of antibodies to denatured DNA on the human bone marrow cells forming colonies in semi-solid agar]

gamma-globulin from rabbit sera containing antibodies to denatured DNA or cytidine suppressed the efficiency of colony formation in agar cultures of human bone marrow cells. The antibodies to DNA isolated from the immune sera by the immunosorbent produced an analogous effect. gamma-globulin from the sera of intact animals and immune sera after the removal of antibodies to denatured DNA from them produced a different effect. gamma-globulin in a concentration of 0.28 and 5 mg failed to influence the colony-formation of 2.10(5) explanted nucleus-containing cells, by stimulated the colony growth when added in a concentration of 15 mg.     

Specific antibodies bound to secretory IgA in the sera of patients with intestinal infections]

Specific IgA and sIgA antibodies were studied in the sera of patients suffering from various intestinal diseases (dysentery, salmonellosis, typhoid fever, chronic typhoid carrier state) and in the sera of healthy persons immunized by parenteral route with typhoid alcohol vaccine. The nature of antibodies was identified in Coombs' test, using monospecific antisera to alpha-chain and to the secretory component. IgA and sIgA antibodies were revealed most frequently in the sera of dysentery patients and of chronic typhoid carriers. No sIgA antibodies were found in the sera of subcutaneously immunized persons. The presence of specific sIgA antibodies in the serum reflects the participation of local immune mechanisms in the formation of systemic immunity in the intestinal infections.     

Antisperm antibodies in human immunodeficiency virus infection: effects on fertilization and embryonic development

Sera from human immunodeficiency virus (HIV)-infected males (n = 10) and females (n = 5) were analyzed for the presence of antisperm antibodies reacting against sperm-specific antigens. Of the HIV-positive males tested, sera of 40% were positive for human sperm extract (HSE), 70% for protamine, and 70% for fertilization antigen (FA-1) for at least one class of antibodies, compared to sera from HIV-negative males. Of the HIV-positive females tested, sera of 40% were positive for HSE, 30% for protamine, and 30% for FA-1 compared to sera from HIV-negative females. The majority of the sperm antigen-reactive antibodies belonged to the IgG class. The reactions observed with FA-1 were weaker than those with other antigens. Ninety percent of HIV-positive male sera and 80% of the HIV-negative female sera were found to contain immune complexes, 20% of which showed the presence of FA-1. HIV-positive male or female sera did not bind to any specific protein on the Western blot of HSE. The minimal amount of free anti-FA-1 antibodies present in sera did not bind to live sperm in the sperm immobilization technique, sperm agglutination technique, or immunobead binding technique and thus were incapable of affecting human sperm penetration of zona-free hamster ova (SPA). Nor did HIV-positive sera induce any apparent abnormality in the development of 2-cell embryos to blastocysts in vitro in murine bioassay. In conclusion, these results indicate that HIV-infected patients have sperm-specific antibodies in their sera that do not adversely affect SPA and murine embryo bioassay. There was a high incidence of immune complex formation after HIV infection. These data will provide the basis for exploring further the role of sperm antigens in altering the immunoregulatory mechanisms after HIV infection.     

Broad HIV-1 neutralization mediated by CD4-binding site antibodies

We have identified several patient sera showing potent and broad HIV-1 neutralization. Using antibody adsorption and elution from selected gp120 variants, the neutralizing specificities of the two most broadly reactive sera were mapped to the primary receptor CD4-binding region of HIV-1 gp120. Novel antibodies to the CD4-binding site are elicited in some HIV-1-infected individuals, and new approaches to present this conserved region of gp120 to the immune system may result in improved vaccine immunogens.     

Use of I125 labelled poly(I). poly(C) for determination of antibodies to double-stranded RNA by immune complex absorption to nitrocellulose filters]

125I-labeled double-stranded polyribonucleotide complex was used for detection of antibodies to double-stranded RNA in sera from people and immunized animals by the method of immune complex adsorption by the nitrocellulose filters. The technique is simple and sensitive. Antibodies to double-stranded RNA WERE detected in sera from patients with different diseases and from normal individuals. Systemic lupus erythematosus sera contain as a rule higher amounts of antibodies to double-stranded RNA. Often these antibodies were measured together with those directed towards native and denatured DNA. Anti-double-stranded RNA antibodies from sera of immunised animals and patients with systemic lupus erythematosus are highly specific.     

Serological Diagnosis of Classical Swine Fever

Antibody levels in post-infection sera from a pig inoculated with a low virulent strain of classical swine fever virus (Hannover 62) and in sera from two pigs inoculated with another low virulent strain (Spielbach 66) and from an in-contact pig were assayed by complement fixation and immunofluorescence using classical swine fever virus (ALD strain) and bovine virus diarrhoea virus (UG 59 strain) as antigens. The complement fixation test used was modified by addition of a preparation of porcine Glq to the complement and by mercaptoethanol treatment of the immune serum before use. The mercaptoethanol treatment of the immune serum resulted in complete elimination of a haemolytic prozone often seen with porcine immune sera. In the sera from the inoculated animals complement-fixing antibodies appeared earlier than neutralizing antibodies. A few weeks after inoculation there was a correlation between the presence of complement-fixing and neutralizing antibodies. During the entire observation period of 13 weeks it was not possible to demonstrate complement-fixing or neutralizing antibodies in serum from a pig exposed to infection by contact with the two pigs inoculated with the Spièlbach 66 strain of classical swine fever virus.     

Mapping the idiotopes of a monoclonal anti-myoglobin antibody with syngeneic monoclonal anti-idiotypic antibodies: detection of a common idiotope

A panel of syngeneic monoclonal anti-idiotypic antibodies was prepared by immunizing A.SW mice with keyhole limpet hemocyanin-coupled A.SW monoclonal anti-myoglobin (HAL 19, IgG1) and screening the cloned hybridomas for production of IgG2 binding to idiotype but not to certain other anti-myoglobin antibodies of the same subclass in an ELISA. With these antibodies, we identified three nonoverlapping idiotopes, based on three clusters of monoclonal anti-idiotopes that mutually inhibit within each cluster, but not between clusters (Cluster I: S2, S6, S8; Cluster II: S5, S7; Cluster III: S9). Only Cluster II antibodies block the binding of myoglobin to HAL 19 and so identify a binding site-related idiotope(s). Binding of both Cluster II monoclonals (S5 and S7) to Hal 19 is inhibited by a rabbit anti-idiotype that we previously reported detects a common cross-reactive anti-myoglobin idiotope in immune sera. However, only one of these, S7, and not S5, identifies an idiotope that is present on 20 to 30% of A.SW anti-myoglobin antibodies in immune sera and ascites. The panel of syngeneic monoclonal anti-idiotype antibodies also detects new idiotopes not detected by the rabbit anti-idiotype. The development of a panel of syngeneic monoclonal anti-idiotypic antibodies to different clusters of idiotopes on the same antibody molecule, including one that identifies a major common idiotope in immune sera, should allow the analysis of possible idiotype network regulation in vivo and in vitro in a completely syngeneic system.     

The potential protective effect of immunization with outer-membrane protein I from Neisseria gonorrhoeae

Immunization of rabbits with outer membranes (OM) of Neisseria gonorrhoeae produced antibodies directed against outer-membrane proteins PI and PIII. The antibodies directed against PIII reacted equally well on Western blots with all strains tested, but antibodies directed against PI reacted only with the homologous strain. When purified PIB was used for immunization the immune response was quite different: the sera obtained reacted with both homologous and heterologous PIB types and also reacted with strains expressing PIA. Western blotting of peptides produced by sequential cleavage of PIB revealed that the antigenic determinants recognized by anti-OM sera were predominantly located in the central surface-exposed region of PIB, as is the epitope recognized by the protective anti-PIB monoclonal antibody SM24. In contrast antibodies produced by immunization with purified PI reacted with antigenic determinants in the N-terminal portion of PIB. Nevertheless these determinants are accessible to immune attack on the native protein since the anti-PI sera were opsonic and were strongly bactericidal for both PIA- and PIB-expressing strains.     

Isolation of a cDNA clone for a catalytic subunit of Torpedo marmorata acetylcholinesterase

We have constructed a cDNA library from Torpedo marmorata electric organ poly(A+) RNA in the lambda phage expression vector lambda gt11. This library has been screened with polyclonal anti-acetylcholinesterase antibodies. One clone, lambda AChE1, produced a fusion protein which was recognized by the antibodies and which prevented the binding of native acetylcholinesterase in an enzymatic immune assay. These results indicate that lambda AChE1 contains a cDNA insert coding for a part of a catalytic subunit of Torpedo acetylcholinesterase. The 200-base-pair cDNA insert hybridized to three mRNAs (14.5, 10.5 and 5.5 kb) from Torpedo electric organs. These mRNAs were also detected in Torpedo electric lobes.     

Comparing antigenicity and immunogenicity of engineered gp120

We have engineered monomeric gp120 in such a way as to favorably present the conserved epitope for the broadly neutralizing antibody b12 while lowering the exposure of epitopes recognized by some weakly neutralizing and nonneutralizing antibodies. The work presented here describes the immune response in rabbits immunized with two prototype, engineered gp120s to explore the relationship between antigenicity and immunogenicity for these mutants. The GDMR gp120 mutant (residues 473 to 476 on gp120 altered from GDMR to AAAA) has a series of substitutions on the edge of the CD4 binding site (CD4bs), and the mCHO gp120 mutant has seven extra glycans relative to the wild-type protein. Importantly, serum mapping showed that both mutants did not elicit antibodies against a number of epitopes that had been targeted for dampening. The sera from rabbits immunized with the GDMR gp120 mutant neutralized some primary viruses at levels somewhat better than the wild-type gp120 immune sera as a result of an increased elicitation of anti-V3 antibodies. Unlike wild-type gp120 immune sera, GDMR gp120 immune sera failed to neutralize HXBc2, a T-cell line adapted (TCLA) virus. This was associated with loss of CD4bs/CD4-induced antibodies that neutralize TCLA but not primary viruses. The mCHO gp120 immune sera did not neutralize primary viruses to any significant degree, reflecting the masking of epitopes of even weakly neutralizing antibodies without eliciting b12-like antibodies. These results show that antibody responses to multiple epitopes on gp120 can be dampened. More precise focusing to a neutralizing epitope will likely require several iterations comparing antigenicity and immunogenicity of engineered proteins.     

Cholestatic Liver Disease after Rituximab and Adalimumab and the Possible Role of Cross-Reacting Antibodies to Fab 2 Fragments

Background

Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs) for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA)-associated granulomatosis with polyangiitis (GPA) who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group.

Methods

Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin.

Results

Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients’ sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected.

Conclusion

This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects and accelerated drug clearance in patients on Tmab therapy.     

流行性出血热患者皮肤组织内病毒抗原及免疫复合物检测与意义

应用常规病理、免疫病理及超微病理技术,对３３例流行性出血热（ＥＨＦ）患者皮肤活检标本的病理变化及病毒抗原、免疫复合物进行观察,同时与血清病毒抗原、抗体及循环免疫复合物检出情况进行比较。在２３例ＥＨＦ患者皮肤微血管内皮细胞中检出病毒抗原,部分组织中可同时检出免疫球蛋白及Ｃ３,少数组织仅能检出病毒抗原或免疫球蛋白。配对血清小也可检出ＥＨＦ病毒抗原、抗体及循环免疫复合物。组织及血清免疫复合物形成与血清补体Ｃ３水平下降有关,组织内肥大细胞脱颗粒与血清ＩｇＥ水平升高相关,提示多种变态反应参与了流行性出血热的发病机制。     

In vitro response of Ichthyophthirius multifiliis to sera from immune channel catfish

Sera from channel catfish rendered immune to the protozoan pathogen Ichthyophthirius multifiliis were screened for activity against live parasites. Cells in the infective stage (tomites) were incubated in doubling dilutions of immune and pre-immune sera from fish that had been immunized by exposure to sublethal infections. When examined by light microscopy, tomites were found to agglutinate in the presence of immune sera. While the stength of individual sera varied, agglutination of cells occurred at dilutions as high as 1:128. Cells showed little tendency to agglutinate in pre-immune sera, and virtually no effects were seen with dilutions of pre-immune sera greater than 1:16. Agglutination was usually accompanied by release of mucus from cells, and while tomites appeared to be immobilized, their cilia continued to beat. Low dilutions of immune sera appeared to be toxic. Similar effects on tomites were seen with rabbit antisera prepared against Ichthyophthirius cilia. The involvement of humoral antibodies in agglutination and protective immunity is discussed.     

Dynamics of formation of antibodies of different immunochemical types to several L-forms and mycoplasmae]

Both IgM- and IgG-immunoglobulins were revealed in the sera of rabbits immunized with L-forms of hemolytic streptococcus, group A, and Mycoplasma fermentans. In the process of immune response the hemagglutinin titres proved to increase, with IgG antibodies prevalence in the hyperimmune sera.     

Antibodies to meningococcal H.8 (Lip) antigen fail to show bactericidal activity

Purified H.8 (Lip) antigen was coupled to tresyl-activated Sepharose 4B and used in affinity columns to purify anti-Lip antibodies from convalescent patient sera and from immune rabbit sera. Affinity-purified anti-Lip antibodies isolated from two convalescent patient sera contained 1000 and 1280 ELISA units of antibody and included antibodies of IgG, IgA, and IgM isotypes. An anti-Lip mouse monoclonal ascites (2-1-CA2) had 28,400 ELISA units of antibody. Bactericidal assays were performed using three different case strains of Neisseria meningitidis group B, namely 44/76, 8532, and 8047. Neither preparation of purified human anti-Lip antibodies had detectable bactericidal activity against strains 44/76 and 8532, but one of the two had a titer of 1:4 against strain 8047. Anti-Lip antibodies that were purified from immune rabbit serum and contained 1600 ELISA units of anti-Lip antibodies also failed to show detectable bactericidal activity. The rabbits were immunized with purified Lip antigen and showed specific antibody levels of 2000-2200 units by ELISA, but even the unfractionated sera had little or no bactericidal activity against the test strains. The high titer mouse monoclonal ascites had no bactericidal activity against the test strains. The poor bactericidal activity associated with monoclonal and polyclonal antibodies to the Lip antigen suggest that in spite of other attractive properties it may not be useful as a meningococcal vaccine.     

Antibody-dependent cellular cytotoxicity-inducing antibodies against human immunodeficiency virus. Presence at different clinical stages

The presence of antibodies mediating antibody-dependent cellular cytotoxicity (ADCC) against human immunodeficiency virus (HIV)-infected target cells was investigated with 170 sera from patients with varying severity of HIV infection. Approximately 40% of sera from individuals representing all stages of infection were ADCC-positive when tested against HTLV-IIIB infected 0937 clone 2 target cells. The positive sera had higher HIV antibody titers as measured by enzyme-linked immunosorbent assay compared with ADCC-negative sera. ADCC titers were lower in patients with acquired immune deficiency syndrome than in asymptomatic carriers. This decline in ADCC titer was not correlated with a general decrease of HIV antibodies. No correlation between the CD4:CD8 lymphocyte ratio and ADCC activity was found. The possible beneficial effect of ADCC-inducing antibodies early in infection is discussed in relation to the effect of ADCC-inducing antibodies in other retrovirus systems and to the nature of lentivirus infections.     

Detection of poliovirus antigens and antibodies: microindirect haemagglutination and haemagglutination inhibition tests for poliovirus types I, II, and III.

Microtitre indirect haemagglutination (IHA) and IHA-inhibition (IHAI) procedures were adapted to determine the reactivities of type I, II, and III poliovirus antibodies and antigens. Glutaraldehyde-fixed sheep erythrocytes were sensitized for these tests with concentrated, partially purified preparations of type I, II, and III poliovirus. Antibody titres by IHA were generally 10 to 100 times greater than serum microneutralization (SN) titres. The SN and IHA reactivities of three kinds of sera were compared. Of these sera, virus type specific antibodies, in monospecific guinea pig sera one week after immunization and in sera from hyperimmunized horses, could be readily differentiated and measured; antibodies in human diagnostic specimens, however, showed some intertypic cross reactivity. Monovalent one-week immune guinea pig sera reacted specifically in the IHAI test to differentiate viruses, and could be used for virus typing and differentiating strains of poliovirus type III.     

Nippostrongylus brasiliensis: further properties of antibody-damaged worms and induction of comparable damage by maintaining worms in vitro.

When adult Nippostrongylus brasiliensis were maintained in vitro they became damaged. Using the criteria of ultrastructural morphology, acetylcholinesterase isoenzyme pattern and the behaviour of the worms after transfer to a normal rat, this damage appeared to be similar to that produced by the in vivo action of antibodies. Antibodies were shown to be responsible for the anterior migration of adult worms which occurs during primary infections in mature rats and in the prolonged infections seen in lactating and immature rats. Antibody damaged worms and worms unaffected by antibodies were equally able to stimulate the immune response required for worm expulsion. Apparently antibody damage is not required for the initiation of the second immune component necessary for expulsion of this parasite.