Trypanosoma cruzi: predominance of IgG2a in nonspecific humoral response during experimental Chagas' disease.

The kinetic and isotypic pattern of hypergammaglobulinemia has been investigated in C3H/HeJ infected with Trypanosoma cruzi. Hypergammaglobulinemia appeared 14 days postinfection, increased until Day 28 postinfection, and persisted throughout the chronic phase (greater than 60 days of infection). The main isotype secreted was IgG2a, reaching 10-fold the control level. High titers of autoantibodies were found of IgM and IgG subclasses. Isotypic characterization of antibodies against myosin, myelin, and keratin, was performed and determined to be IgG2a subclass in the chronic stage of infection. Specific responses against T. cruzi took place 2 weeks postinfection when the parasitemia was high. Interestingly, parasite-specific response was maximal after 4 weeks of infection and plateaued during the chronic phase when parasites were rare. In contrast to the humoral polyclonal response in the chronic stage, showing a preferential IgG2a pattern, the anti-T. cruzi response consisted of all the different isotypes: IgM, IgG1, IgG3, IgG2a, and IgG2b, throughout the infection. Identical patterns of parasite antigens were recognized by IgG2a and IgG2b antibodies. Few different antigens were identified by the IgG3. Some antigens were recognized by several isotypes, others by only one isotype. With regard to the existence of antigenic cross-reactivities between host and parasite, we designed absorption experiments on parasite-specific immunoadsorbent showing that specific antibodies eluted from the column failed to recognize the natural antigens. These studies suggest that nonspecific and antiparasite-specific responses may be maintained by different regulatory pathways.     

Immunological aspects of infection of 6 inbred strains of mice by 3 different strains of Trypanosoma cruzi

We evaluated humoral and cellular immune responses in 6 inbred mouse strains (BALB/c, B-10, C3H, A/J, AKR and DBA) infected with 3 Trypanosoma cruzi strains (Peruvian, 21 SF and Colombian), which are the standards for the 3 strains Types of Andrade's classification. Negative delayed-type hipersensitivity reactions to parasite antigens were evidence of suppressed cell-mediated immunity. An early drop of IgG1 and rise of IgM levels were observed in almost all mouse strains infected by any T. cruzi strain. Elevation of IgG2a and/or IgG2b levels was higher in resistant mouse strains. Anti-T. cruzi antibody levels (Indirect immunofluorescence and ELISA) did not correlate with survival. Despite some differences among mouse strains there was a definition of an overall pattern of host response and the maintenance of biological standards which characterize the basic types of T. cruzi strains.     

A 38-kDa Antigen of Mycobacterium tuberculosis Predominantly Induces the Secretion of Interleukin-2, Interferon-gamma and IgG2a Antibodies

In the present study, mice of 3 different haplotypes (H-2d, H-2k and H-2b) were sensitized subcutaneously with heat-killed H37Ra or 38-kDa antigen of Mycobacterium tuberculosis. Lymphocytes obtained from immunized animals were challenged in vitro with 38-kDa antigen in both cases. The dominant pattern of Th1-like lymphokines (IL-2 and IFN-gamma) and preferential production of 38-kDa specific IgG2a-type antibody were observed. It was noted that 38-kDa antigen was recognized permissively by all 3 strains of mice used in the present study. It was interesting to note that C3H/HeJ mice, which express BCG-resistant alleles showed a higher level of proliferative as well as cytokine response as compared to BALB/c and C57BL/6 mice, which bear BCG-susceptible alleles. These results suggest that not only in recall responses but also during the induction as well as expression phase of the immune response mediated by 38-kDa antigen of M. tuberculosis the Th1-like immune response predominates.     

融合表达小鼠IgG2b-Fc可增强人类免疫缺陷病毒1型Gag DNA疫苗的免疫原性

为阐明小鼠IgG2b-Fc对DNA疫苗免疫原性的增强作用,首先构建表达人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)CN54Gag基因的DNA疫苗及表达Gag与小鼠IgG2b-Fc融合基因的DNA疫苗,限制性酶切和DNA测序结果表明这两个疫苗均构建成功,蛋白免疫印迹结果也显示其正确表达。然后,利用上述DNA疫苗接种C57BL/6小鼠,比较两个DNA疫苗所诱导的特异性体液免疫反应和特异性细胞免疫反应。结果显示,融合表达小鼠IgG2b-Fc对特异性细胞免疫和体液免疫反应均有增强作用,但只有对特异性体液免疫反应的增强作用有统计学意义。     

Analysis of antibody cross-reactivity in experimental American trypanosomiasis.

Susceptible C3H/He mice were immunized with the avirulent Corpus Christi strain of Trypanosoma cruzi and subsequently infected with virulent Brazil stain organisms. Seventy days after infection sera were isolated and analyzed on western blots of electrophoretically separated T. cruzi antigens prepared from culture-form parasites (primarily epimastigotes). More than 25 bands were identified. The antibodies were fractionated by elution from various regions of western blots corresponding to average molecular weights of approximately greater than 130, 77, 70, 60, 48, or 38 kDa. Each of these antibody preparations was then incubated with strips of nitrocellulose containing all of the electrophoretically separated T. cruzi, and cross-reactivity was determined. Antibodies isolated from the 130-, 77-, and 70-kDa regions all cross-reacted with each other. Antibodies eluted from the 60-kDa region bound antigens in the 60-, 70-, and the 77-kDa regions. More importantly, antibodies eluted from every region bound antigens in the 70-kDa region. Conversely, antibodies eluted from this region bound to antigens in all of the other regions. These data indicate the presence of (a) common antigenic epitope(s) in T. cruzi infections in these mice that is predominantly found in the 70-kDa antigen-antibody complex on western blots.     

Changes in humoral responses to Trypanosoma cruzi during the course of infection in mice held at elevated temperature.

A parasite-specific, enzyme-linked immunosorbent assay and immunoblot analysis were used to examine the development of humoral immunity in Trypanosoma cruzi-infected C3H mice that survive acute infection when held at elevated environmental temperature. Both parasite-specific antibody levels and numbers of antigens identified increased during infection in mice held at 36 C, with the greatest reactivity measured in sera from mice that had resolved parasitemias. Heat shock of culture forms of T. cruzi resulted in production of different antigens, but there was no strong difference in the antigens recognized by sera from mice held at room temperature and those recognized by sera from mice held at 36 C. Immunoblot analysis using blood-form trypomastigote antigens identified a band of approximately 61 kDa produced by trypomastigotes in mice held at 36 C that was strongly detected by sera from mice held at 36 C. Little if any reactivity to this antigen was observed using sera from mice held at room temperature.     

Trypanosoma cruzi: H2 complex and genetic background influence on the humoral immune response against epimastigotes

Using A.SW, A.CA, B10.S and B10.M congenic mouse strains, we measured the IgG specific humoral immune responses against sonicated and live Trypanosoma cruzi epimastigotes. Genes located in the A background (A.SW and A.CA strains) mediate higher IgG responses against the parasite antigenic complexes than those located in the B background (strains B10.S and B10.M), regardless of the H2 haplotypes. Thus, non H2 genetic elements seem to be more important in determining differences in the total IgG immune response against T. cruzi. Whether a detectable H2 effect, in favor of the H2(s) haplotype, occurred in the A or B background, was contingent on the immunisation protocol used. Thus, the H2(s) haplotype mediates a higher IgG response in the A background, if immunised with live epimastigotes, and in the B background against sonicated epimastigotes. Most likely this represents a complex sequence of events, controlled by non-MHC genes, involving antigen handling and processing and depending on the physical form of antigen delivery.     

Trypanosoma cruzi: cross-reactive anti-heart autoantibodies produced during infection in mice

Preimmunization with attenuated Corpus Christi stain Trypanosoma cruzi provides survival to C3H mice and enhances resistance of C57 mice to Brazil strain infection. C3H(He) and C57 B1/6 mice surviving acute infection of T. cruzi are shown to have heart specific autoantibodies through acute and chronic infection. ELISA assays were performed using nondenatured extract of hearts from normal syngeneic mice as target antigen reacted with sera from immunized and/or infected mice. Surviving C3H mice developed a specific anti-heart response as early as Day 21 of infection and this response continued at a high level to Day 300. The response in C57 mice, both immunized-infected and infected only, increased to Day 100 followed by a decline in intensity. The heart specificity of the response in mice was suggested by negligible reaction of sera with smooth muscle preparations and a reduced autoreactivity with skeletal muscle. Laminin, a suggested target of autoimmunity in Chagas' disease, was shown not to be the target of the responses in these mice. Immunoaffinity-purified heart specific antibodies show strong cross-reactivity with parasite antigen and like purified parasite specific antibodies, reacted with heart antigen.     

Heterogeneity in the immune response to serotype b LPS of Actinobacillus actinomycetemcomitans in inbred strains of mice

We investigated the heterogeneity of the humoral immune responses to whole cells and lipopolysaccharide (LPS) of Actinobacillus actinomycetemcomitans serotype b and production of cytokines in inbred strains of mice. Nine such strains were tested: A/J (H-2(a)), C57BL/6 (H-2(b)), BALB/c (H-2(d)), DBA/2 (H-2(d)), B10.BR (H-2(k)), C3H/He (H-2(k)), C3H/HeJ (H-2(k)), DBA/1 (H-2(q)) and B10.S (H-2(s)). Mice were immunized intraperitoneally with whole cells of A. actinomycetemcomitans ATCC 43718 (serotype b) in phosphate buffered saline (PBS; pH 7.2) emulsified with an equal volume of Freund's incomplete adjuvant. Serum immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) levels against A. actinomycetemcomitans were measured by an ELISA system. ELISA analysis, using LPS fractions from serotype a, b or c strains of A. actinomycetemcomitans as the coating antigens, revealed that mice strains C3H/He, C3H/HeJ, B10.BR and B10.S had an extremely high-IgM response against serotype b LPS. High-IgM titer sera contain also elevated levels of IgA antibodies to the antigen. To compare the cytokine production among inbred mice, the amounts of interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-6 (IL-6) released from mouse splenocytes were measured using ELISA systems specific for these cytokines. A. actinomycetemcomitans serotype b LPS stimulation induced IL-6 release from murine splenocytes of all tested strains. However, IL-4 and IL-5 were detected only in high-IgM/IgA responders to A. actinomycetemcomitans serotype b LPS, not in low-IgM/IgA responders. Thus, we found a relationship between the humoral immune response to LPS of A. actinomycetemcomitans serotype b and production of type 2 cytokines by splenocytes.     

Leishmania amazonensis: humoral response to amastigote excreted-antigens in murine leishmaniasis

With the purpose of studying the antigenic role that factors excreted by Leishmania amastigotes might have during murine infection, immunoblots were carried out with sera from C57BL/6 and BALB/c mice infected with two strains of Leishmania (L.) amazonensis, NR and IFLA/BR. Both strains differ widely in virulence in BALB/c mice. BALB/c but not C57BL/6 sera recognized several excretion products. The excreted antigens showed a strong response towards IgG1 and IgG2a isotypes whilst they reacted only weakly against IgG2b and IgG3. A low-molecular weight antigen (about 20 kDa) excreted by both Leishmania strains was strongly recognized by IgG1 from BALB/c mice sera infected with IFLA/BR, the most virulent strain. Sera from NR infected mice were incapable of recognizing this antigen in spite of its presence in NR excreted products. The results indicate that the humoral immune response to excreted antigens of amastigotes depends on both the host genetic background and the parasite strain.     

Screening and characterization of monoclonal antibodies to the surface antigens of Listeria monocytogenes serotype 4b

Aims:  This study aims to develop and characterize monoclonal antibodies (Mabs) with high specificity and affinity for surface antigens of an epidemiologically important serotype 4b of Listeria monocytogenes .
Methods and Results:  Hybridoma clones were derived from B lymphocytes of mice immunized with L. monocytogenes serotype 4b and screened against this strain by an enzyme-linked immunosorbent assay. Twenty-nine clones secreting Mabs reactive with formalin-killed bacteria were obtained; 15, 8, 5 and 1 Mabs were immunoglobulin subclasses IgG2a, IgG2b, IgM and IgG1, respectively. Immuofluoresence or immunogold labelling demonstrated all except five IgM and one IgG2a Mabs bound to the surface of a live L. monocytogenes serotype 4b. The majority of the 23 surface-binding Mabs recognized linear epitopes on a 77-kDa protein. These surface-binding Mabs exhibited little or no cross-reactivity with non-4b serotypes (1/2a, 1/2b, 3a, etc.) of L. monocytogenes , five other Listeria species, Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium.
Conclusions:  The Mabs recognizing a 77-kDa surface protein are novel antibodies with specificity and affinity for L. monocytogenes serotype 4b.
Significance and Impact of the Study:  These anti-77 kDa surface protein Mabs may be explored as reagents for the development of Mabs-based diagnostic immunoassays for L. monocytogenes serotype 4b strains.     

Antibody responses to the fucosylated LacdiNAc glycan antigen in Schistosoma mansoni-infected mice and expression of the glycan among schistosomes

Infections of animals with parasitic worms, such as Schistosoma mansoni, induce humoral immune responses to carbohydrate antigens, raising the possibility that such antigens might be useful targets for the development of vaccines and new diagnostic approaches. Here we describe the identification of fucosylated LacdiNAc (LDNF) [GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc-R] as a new carbohydrate antigen in S. mansoni that induces humoral immune responses in infected mice. The presence of antibodies was determined by ELISA using a neoglycoconjugate synthesized to express LDNF sequences. Sera from S. mansoni-infected, but not uninfected, mice contain IgM, IgG, IgA, and IgE antibodies to LDNF. The IgG antibodies are primarily of the IgG1 and IgG3 subclasses, with no detectable levels of the complement-fixing IgG2a and IgG2b isotypes. An IgM monoclonal antibody, designated SMLDNF1, was generated from the spleens of S. mansoni-infected mice, and the antibody exhibits specific recognition of LDNF sequences, but not other fucosylated glycans tested. Immunocytochemical analysis demonstrates that LDNF antigens are localized on the tegumental surface of adult S. mansoni. Western blot analysis indicates that LDNF sequences are expressed on numerous high-molecular-weight glycoproteins from the three major human schistosome species, as well as the bird schistosome Trichobilharzia ocellata. The identification of LDNF antigen on the tegumental glycoproteins of schistosomes and the ability to synthesize LDNF conjugates should aid in the development of glycan-based vaccines and immunodiagnostic tests for schistosomiasis and in determining the role(s) of the glycans in worm development and pathogenesis.     

In vivo allogeneic effects: shift in the isotype profile of primary TI-2 responses in mice undergoing graft-vs-host reaction

We showed previously that primary responses to T-dependent (TD) and T-independent type 2 (TI-2) antigens were differentially affected by allogeneic effects induced in vivo during a graft-vs-host reaction (GVH). TD responses were greater than or equal to 80% suppressed, whereas the TI-2 responses were greatly enhanced, particularly the IgG component, which normally is very low. We have analyzed the IgG subclass distribution in primary responses of normal and GVH F1 mice in order to determine whether the strong T cell signals that occur during GVH reactions also induce shifts in the isotype profile. The effect of GVH on responses to TI-2 antigens was of particular interest because they are usually dominated by IgM and IgG3 classes in normal mice. We found a threefold to 10-fold increase in the PFC numbers of all four IgG subclasses in the response to TI-2 antigens, with an apparent shift from the usual IgG3 dominance to IgG1 in GVH mice. This IgG1 dominance was not found in serum antibodies where IgG3, IgG1, and IgG2b were equally expressed, although total IgG was increased greater than 20-fold. No isotype shift was found in either the TNP-KLH response, which was greater than or equal to 75% suppressed (IgG1 dominance was retained), or in the TI-1 response to TNP-Ba. The latter response was reduced (25 to 50%) in GVH mice and continued to be dominated by IgG2b/2a and IgG3. Unlike the unique isotype patterns found in primary responses, TNP-KLH primed mice challenged with TD, TI-1, or TI-2 antigens gave memory responses with identical isotype profiles that were dominated by IgG1 PFC. The role of T cells in B cell differentiation and isotype expression is discussed.     

Induction of mouse homocytotropic antibodies to Timothy pollen antigens.

The IgG1 and IgE homocytotropic antibody responses of LAF and C3H mice to timothy pollen antigens are defined. Both mouse strains responded to low doses of crude timothy pollen extract (WST) or a major antigen of timothy pollen coupled to a purified fraction of Ascaris suum (Antigen B-Ascaris). Titers in LAF mice were greater than those in C3H mice. Regardless of the immunogen, antigen B was the major determinant recognized by the homocytotropic antibodies; PCA titers with WST or antigen B for challenge were equivalent and PCA activity could be inhibited by antigen D, a dialyzable fraction of timothy pollen possessing the antigen B determinant in monovalent form. The possible usefulness of antigen D for in vivo and in vitro studies of specific immune suppression of cellular activity is discussed.     

B subunit of Escherichia coli heat-labile enterotoxin as adjuvant of humoral immune response in recombinant BCG vaccination

The B subunit of Escherichia coli heat-labile enterotoxin (LTB), a nontoxic molecule with potent biological properties, is a powerful mucosal and parenteral adjuvant that induces a strong immune response against co-administered or coupled antigens. In this paper, the effect of LTB on the humoral immune response to recombinant BCG (rBCG) vaccination was evaluated. Isogenic mice were immunized with rBCG expressing the R1 repeat region of the P97 adhesin of Mycoplasma hyopneumoniae alone (rBCG/R1) or fused to LTB (rBCG/LTBR1). Anti-R1 systemic antibody levels (IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA) were measured by ELISA using recombinant R1 as antigen. With the exception of IgM, LTB doubled the anti-R1 antibody levels in rBCG vaccination. The IgG1/IgG2a mean ratio showed that both rBCG/LTBR1 and rBCG/R1 induced a mixed Th1/Th2 immune response. Interestingly, anti-R1 serum IgA was induced only by rBCG/LTBR1. These results demonstrate that LTB has an adjuvant effect on the humoral immune response to recombinant antigens expressed in BCG.     

Evaluation of immune responses raised against Tc13 antigens of Trypanosoma cruzi in the outcome of murine experimental infection

We have previously reported that genetic immunization with Tc13Tul antigen of Trypanosoma cruzi, the aetiological agent of Chagas' disease, triggers harmful effects and non-protective immune responses. In order to confirm the role of Tc13 antigens during T. cruzi infection, herein we studied the humoral and cellular immune responses to the Tc13Tul molecule and its EPKSA C-terminal portion in BALB/c T. cruzi-infected mice or mice immunized with recombinant Tc13Tul. Analysis of the antibody response showed that B-cell epitopes that stimulate a sustained IgM production along the infection and high levels of IgG in the acute phase are mainly located at the Tc13 N- and C-terminal domains, respectively. DTH assays showed that T-cell epitopes are mainly at the Tc13 N-terminal segment and that they do not elicit an efficient memory response. Recombinant Tc13Tul did not induce IFN-gamma secretion in either infected or immunized mice. However, a putative CD8+Tc13Tul-derived peptide was found to elicit IFN-gamma production in chronically infected animals. Immunization with recombinant Tc13Tul did not induce pathology in tissues and neither did it protect against the infection. Our results show that in the outcome of T. cruzi infection the Tc13 family protein mainly triggers non-protective immune responses.     

4-Hydroxy-3-nitrophenyl (NP) acetyl-hapten specific lymphocyte proliferation. I. Mice bearing Igh-1b allotype can cross-react with 4-hydroxy-5-iodo-3-nitrophenyl (NIP) acetyl hapten

Hapten specific T cell proliferation was induced in several strains of mice. When lymph node T cells from 4-hydroxy-3-nitrophenyl acetyl-keyhole lympet hemocyanin (NP-KLH)-primed mice were stimulated in vitro with NP-polymer glutamic acid-lysine-phenyl alanine (NP-GL phi) or NP-ovalbumin (NP-OVA), they displayed a good level of proliferative responses. It was observed that NP-GL phi could induce NP-hapten specific proliferation even with NP-KLH lymphocytes from GL phi nonresponder strains. NP-KLH primed lymphocytes from C57BL/6 (H-2b, Igh-1b), CKB (H-2k, Igh-1b), CWB (H-2b, Igh-1b), and B10.BR (H-2k, Igh-1b) mice showed good proliferative responses to both 4-hydroxy-5-iodo-3-nitrophenyl (NIP) acetyl-GL phi and NIP-OVA antigens. However, NP-KLH primed lymphocytes from C3H/He (H-2k, Igh-1j) and C3H. SW (H-2b, Igh-1j) mice displayed poor proliferative responses to NIP-GL phi and NIP-OVA antigen. These results suggested that the gene coding for the NIP-cross-reaction might be mapped in the Ig heavy-chain linked locus.     

IgG Suppresses Antibody Responses in Mice Lacking C1q,C3, Complement Receptors 1 and 2, or IgG Fc-Receptors

Antigen-specific IgG antibodies, passively administered to mice or humans together with large particulate antigens like erythrocytes, can completely suppress the antibody response against the antigen. This is used clinically in Rhesus prophylaxis, where administration of IgG anti-RhD prevents RhD-negative women from becoming immunized against RhD-positive fetal erythrocytes aquired transplacentally. The mechanisms by which IgG suppresses antibody responses are poorly understood. We have here addressed whether complement or Fc-receptors for IgG (FcγRs) are required for IgG-mediated suppression. IgG, specific for sheep red blood cells (SRBC), was administered to mice together with SRBC and the antibody responses analyzed. IgG was able to suppress early IgM- as well as longterm IgG-responses in wildtype mice equally well as in mice lacking FcγRIIB (FcγRIIB knockout mice) or FcγRI, III, and IV (FcRγ knockout mice). Moreover, IgG was able to suppress early IgM responses equally well in mice lacking C1q (C1qA knockout mice), C3 (C3 knockout mice), or complement receptors 1 and 2 (Cr2 knockout mice) as in wildtype mice. Owing to the previously described severely impaired IgG responses in the complement deficient mice, it was difficult to assess whether passively administered IgG further decreased their IgG response. In conclusion, Fc-receptor binding or complement-activation by IgG does not seem to be required for its ability to suppress antibody responses to xenogeneic erythrocytes.     

Genetics of resistance to the African trypanosomes. III. Variant-specific antibody responses of H-2-compatible resistant and susceptible mice

Genetically based differences in variant-specific immunity to the African trypanosomes were examined. H-2-compatible inbred mouse strains that differed in relative resistance were infected with Trypanosoma rhodesiense clone LouTat 1. Antibody responses to exposed epitopes of the LouTat 1 variant-specific surface glycoprotein (VSG) were measured. Relatively resistant B10.BR mice (H-2k) made predictable IgM antibody responses to the VSG of LouTat 1 which were associated with clearance of the LouTat 1 variant antigenic type from blood; IgG responses to LouTat 1 surface antigen appeared after clearance occurred, and were lower than peak titers of IgM. Intermediately susceptible CBA mice (H-2k) also made predictable IgM and IgG responses which followed the same pattern as the more resistant strain. Peak titers were lower for both Ig classes, however, and a delayed appearance of antibody was correlated with delayed clearance of LouTat 1. In contrast to B10.BR and CBA mice, the susceptible C3H mice (H-2k) failed to make detectable antibodies to LouTat 1 surface antigen and also failed to control the first peak of parasitemia. The absence of immunity in infected C3H mice was selective for antibody to exposed epitopes of LouTat 1 VSG because antibody was detectable to invariant VSG or internal trypanosome antigens. Also, the C3H strain was shown not to be a genetic nonresponder to LouTat 1 surface antigen because VSG-specific antibodies appeared within 1 wk after trypanocidal chemotherapy. Finally, we demonstrated that the susceptibility of C3H mice was not associated with an inability of the mononuclear phagocyte system to clear the parasites because drug cure, passive transfer of immune serum, or sensitization of trypanosomes with antibody all led to trypanosome clearance from blood by the liver. In summary, we show for the first time that major differences in variant-specific immunity occur in MHC-compatible animals after infection with the African trypanosomes.