Analysis Of Humoral Immune Response Research Articles

Cellular immunotherapy: antigen recognition is just the beginning

Advances in molecular and cellular biology have illustrated both the flexibility and complexity involved in host immune responses. Understanding this response is vital to the further development of therapeutic strategies that involve manipulation of the cellular immune response to target tumors. Mobilized, tumor antigen-specific T cells, the core for most immunotherapeutic strategies, are highly regulated, and capable of a wide spectrum of functional responses. Due to differences in murine and human immunity, broad-scale immune monitoring, particularly high-throughput ex vivo analysis of human immune responses, promises to determine what comprises an effective immunotherapy. Such understanding will lead to more sophisticated clinical trials, earlier determination of efficacy and individualized protocols.

Evaluation of protein allergenic potential in mice: dose-response analyses.

With the increasing interest in novel foods derived from transgenic crop plants, there is a growing need for the development of approaches for the characterization of the allergenic potential of proteins. Whereas immunogenicity is a common property of foreign proteins, including food proteins, relatively few are significant dietary allergens with the inherent capacity to provoke IgE antibody production and immediate-type hypersensitivity responses. In order to evaluate an approach for the measurement of the allergenic potential of proteins, detailed dose-response analyses of humoral immune responses induced following systemic exposure of BALB/c strain mice to proteins known to differ in terms of sensitizing activity have been conducted. Mice were exposed to a range of concentrations of ovalbumin, a major allergenic constituent of hen's egg, a purified peanut allergen, Arachis hypogea agglutinin, or to the milk allergen bovine serum albumin, and to materials considered to lack significant allergenicity: a crude potato protein extract and a purified potato protein, potato agglutinin. The specific IgE antibody was measured by homologous passive cutaneous anaphylaxis assay, and the specific IgG antibody was measured by enzyme-linked immunosorbent assay. Each of the five proteins was immunogenic in mice, inducing IgG antibody responses at all doses tested, although there was some variation with respect to the vigour of IgG responses. Marked differences in the capacity of these proteins to induce IgE responses were observed, however, with relatively high-titre IgE antibody provoked by all three allergens over the dose ranges examined, whereas the potato proteins stimulated low-titre IgE antibody at the highest dose (10%) only. Importantly, differences in IgE antibody production have been observed against a background of equivalent immunogenicity (IgG antibody responses). The data presented here suggest that the measurement of antibody (IgE) responses in BALB/c mice appears to identify allergens accurately and to distinguish them from those materials that apparently lack allergenicity.

Enhancement of immune responses to an HIV env DNA vaccine by a C-terminal segment of listeriolysin O.

An effective vaccine against AIDS should induce both cellular and humoral immune responses. Here we report that immunization of mice with a DNA plasmid encoding a chimeric protein consisting of HIV89.6 Env gp140 and the listeriolysin O (LLO) C-terminal segment (59 amino acids) significantly enhanced both humoral and cellular immune responses against the HIV89.6 Env protein. Plasmid DNA expression vectors with genes codon-optimized for mammalian expression were synthesized for HIV89.6 gp140 as well as for chimeric protein gp140-LLO, in which the coding sequence for the C-terminal 59 amino acids of LLO were fused in frame to the 3' end of the codon-optimized gene for gp140. All plasmid vectors produced high levels of protein expression, and the gp140-LLO chimeric protein was cleaved and secreted as efficiently as gp140. Analysis of humoral immune responses by ELISA showed that the chimeric gp140-LLO construct induced higher antibody responses than the gp140 construct in immunized mice, more notably in the IgG2a antibody subtype. Intracellular cytokine staining and flow cytometry analysis showed that the gp140-LLO construct induced significantly higher levels of cytotoxic T lymphocyte immune responses against the HIV 89.6 Env protein than those observed with the gp140 construct. Our results thus demonstrate that the C-terminal segment of LLO can be effectively employed to enhance both cellular and humoral immune responses against the HIV89.6 Env antigen in the context of a DNA vaccine.

Cutaneous transfection and immune responses to intradermal nucleic acid vaccination are significantly enhanced by in vivo electropermeabilization.

Naked DNA injection with electropermeabilization (EP) is a promising method for nucleic acid vaccination (NAV) and in vivo gene therapy. Skin is an ideal target for NAV due to ease of administration and the accessibility of large numbers of antigen-presenting cells within the tissue. This study demonstrates that in vivo skin EP may be used to increase transgene expression up to an average of 83-fold relative to naked DNA injection (50 microg DNA per dose, P < 0.005). Transfected cells were principally located in dermis and included adipocytes, fibroblasts, endothelial cells, and numerous mononuclear cells with dendritic processes in a porcine model. Transfected cells were also observed in lymph nodes draining electropermeabilized sites. A HBV sAg-coding plasmid was used to test skin EP-mediated NAV in a murine model. Analysis of humoral immune responses including immunoglobulin subclass profiles revealed strong enhancement of EP-mediated NAV relative to naked DNA injection, with a Th1-dominant, mixed-response pattern compared to immunization with HBV sAg protein that was exclusively Th2 (P = 0.02). Applications for these findings include NAV-based modulation of immune responses to pathogens, allergens, and tumor-associated antigens and the modification of tolerance.

Targeted inactivation of the tetraspanin CD37 impairs T-cell-dependent B-cell response under suboptimal costimulatory conditions.

CD37 is a membrane protein of the tetraspanin superfamily, which includes CD9, CD53, CD63, CD81, and CD82. Many of these molecules are expressed on leukocytes and have been implicated in signal transduction, cell-cell interactions, and cellular activation and development. We generated and analyzed mice deficient for CD37. Despite the high expression of CD37 on cells of the immune system, no changes in development and cellular composition of lymphoid organs were observed in mice lacking CD37. Analyses of humoral immune responses revealed a reduced level of immunoglobulin G1 (IgG1) in the sera of nonimmunized mice and an alteration of responses to T-cell-dependent antigens. Antibody responses to model antigen administered in the absence of adjuvant and to viral infections were generally poor in CD37-deficient mice. These poor antibody responses could be overcome by the immunization of antigen together with adjuvant. These results suggest a role for CD37 in T-cell-B-cell interactions which manifests itself under suboptimal costimulatory conditions.

Immunoblot analysis of humoral immune response to Helicobacter pylori in children with and without duodenal ulcer.

Several studies have demonstrated that enzyme-linked immunosorbent assay is not a sensitive and specific method to diagnose Helicobacter pylori infection in children, especially in the younger ones. Since serum immune response can also be determined by immunoblotting and it permits the detection of antibodies to virulence factors such as CagA and VacA, we evaluated the accuracy of a commercial immunoblotting test to diagnose H. pylori infection and to assess the humoral immune response to different H. pylori antigens in 122 children who underwent upper gastrointestinal endoscopy. The presence of H. pylori was determined in antral biopsy specimens by culture, preformed urease test, and histological analysis. H. pylori was identified by microbiological and histopathological methods in 66 children (including all of the 21 who had duodenal ulcer). Antibodies to H. pylori were detected in 63 infected children and in 8 noninfected ones. The sensitivity, specificity, and positive and negative predictive values of the immunoblotting test were 95.5, 85.7, 88.7, and 94.1%, respectively. The number of immunoreactive bands increased with age (P = 0.003), and the bands of 35 kDa (P = 0.013); 89 kDa, the VacA antigen (P = 0.001); and 116 kDa, the CagA antigen (P = 0.00004) were more frequently observed in older children. The frequency of the bands of 89 kDa (P = 0.001) and 116 kDa (P = 0.03) was higher in children with duodenal ulcer than in H. pylori-positive children without the disease. In conclusion, the immunoblotting test appears to be useful for the diagnosis of H. pylori infection in children, even in the younger ones.

Rapidity of specific antibody-antigen interactions.

Analysis of humoral immune responses against viruses has concentrated on studies with serum dilutions, which reflect characteristics pertaining to the diluent buffer but not the serum environment. The majority of virus-specific antibody in serum from foot-and-mouth disease virus (FMDV)-vaccinated cattle bound to antigen within 10-60 s, whereas aspecific reactions evolved more slowly. Upon dilution of sera, the reaction characteristics no longer related to those obtained with the serum, particularly when individual animals were compared. Diagnostic enzyme-linked immunosorbent assay (ELISA) - employing diluted serum - identified little variation in the reactivity of serum samples from different vaccinated cattle. This related to previous analyses showing similar specific antibody titres. In contrast, analyses of serum reactivities over a 10- and 60-s incubation period demonstrated high variation between individual animals. Furthermore, when a challenge infection was performed on vaccinated animals, only those with the higher serum reactivities over a 10- and 60-s incubation were protected. These results demonstrate the importance of the specific serum antibody reactions, which will occur within seconds. Moreover, such qualitative characteristics would be overlooked when employing conventional assays with diluted sera and long incubation periods.

Western blot analysis of humoral immune response to Leishmania donovani antigens in patients with post-kala-azar dermal leishmaniasis

Sera from 32 Indian patients with post-kala-azar dermal leishmaniasis (PKDL) were examined for antibodies by immunoblot analysis using an antigen extract of Leishmania donovani. The study revealed that the humoral immune response in PKDL patients was quite distinct compared to that in kala-azar patients. Antibodies to 3 antigens of L. donovani (molecular sizes 110, 65 and 38–42 kDa) were predominant in a majority (78%) of PKDL patients. The most important finding was the consistent recognition of 2 parasite antigens (of 110 and 65 kDa) by PKDL sera; antibodies to the 110-kDa antigen were detectable in 97% of cases, while antibodies to the 65-kDa antigen were detectable in 100% of cases that were examined. None of the 18 cases of leprosy, 10 of vitiligo, or the 30 healthy persons included in the study showed antibodies to these 2 antigens. Thus Western blot analysis provided a highly sensitive test for PKDL patients. Further, it led to the identification of 2 parasite antigens (110 and 65 kDa) that elicit an antibody response in 97–100% of PKDL patients. Purified or recombinant versions of these proteins deserve consideration as potential target antigens in development of simpler, highly specific and sensitive serodiagnostic tests for PKDL.

Analysis of Humoral Immune Response in Chickens after Inoculation with Cryptosporidium baileyi or Cryptosporidium parvum

White leghorn chickens aged 14 days were orally inoculated with 1 x 10(6) oocysts of Cryptosporidium baileyi or C. parvum to compare the specific immune responses. Cross-reactions were evaluated using homologous or heterologous antigens in enzyme-linked immunosorbent assay (ELISA) and Western blot to determine the occurrence of an antigenic homology between these two species. Blood, bile, whole intestine, bursa of Fabricius, and feces were collected daily from the day of inoculation (day 0) to day 22 postinoculation (PI). Eight control chickens remained negative up to day 22 PI. Chickens inoculated with C. baileyi did not express clinical symptoms but shed oocysts from days 6 to 21 PI. Chickens inoculated with C. parvum exhibited no clinical signs, no oocysts in feces, and no developmental stages of the parasite. However, a specific immune response to both antigens appeared on day 9 PI. ELISA using homologous or heterologous antigens showed that anti-C. baileyi and anti-C. parvum antibodies in serum or bile were detectable using C. baileyi or C. parvum oocysts as antigen, but the intensity of the response was significantly higher when C. baileyi was used. Cross-reactions in immunoblot analysis confirmed ELISA results, revealing a greater number of bands using C. baileyi as antigen but showing that epitopes recognized on the protein with a molecular weight of 15,000-17,000 were different.

Production and characterization of murine monoclonal antibodies specific for baboon IgG heavy and light chain epitopes

We have generated a panel of murine monoclonal antibodies (MAbs) that recognize baboon IgG epitopes. The reactivity of the MAbs with IgG from other primate species was also examined. Specificity for IgG heavy (H) or light (L) chain epitopes was determined by Western blot analysis. The H chain-specific MAbs were analyzed for IgG subclass specificity and the L chain-specific MAbs for reactivity with baboon IgM and polymeric sIgA. Finally, an ELISA was developed to demonstrate the utility of the MAbs in analysis of humoral immune responses in baboons.

Immunoblot analysis of humoral immune responses to Mycobacterium bovis in experimentally infected cattle: early recognition of a 26-kilodalton antigen

Development of a serodiagnostic test for bovine tuberculosis necessitates an understanding of the humoral immune responses of animals following infection with Mycobacterium bovis. The antibody responses in groups of calves challenged intranasally with different doses of M. bovis (approximately 10(2), 10(4), and 10(6) CFU) or placed in contact with the infected animals were analyzed by immunoelectrophoretic blotting in which a whole-cell sonicate of M. bovis was utilized as an antigen. Antibody responses were evident early in infections in which calves were exposed to high doses of M. bovis, while in groups exposed to lower doses, the time until antibody was detected increased as the challenge dose decreased. In cattle exposed to M. bovis, immunoblot analysis showed antibody responses to three main antigens of 26, 22, and 16 kDa. It was further demonstrated that antibody responses to the 26-kDa antigen appeared earliest in the course of infection. Preliminary investigations in this study have identified a 26-kDa antigen for potential use in improved serodiagnosis by enzyme-linked immunosorbent assays.

Isotypic Analysis of Humoral Immune Responses in Rhesus Monkeys to an Adult Microsomal Antigen of Schistosoma Mansoni: an Indicator of Successful Treatment

A study was undertaken to examine the potential role of immunodiagnostic methods in determining successful chemotherapy in schistosomiasis. Fifteen rhesus monkeys were infected with 1,500 Schistosoma mansoni (Puerto Rico strain) cercariae, and 10 of the monkeys were then treated with a curative dose of praziquantel 13 weeks after infection. Five monkeys remained untreated. One monkey was not successfully cured, as confirmed by the presence of both male and female worms at the time of perfusion. Serum samples were longitudinally collected and specific Ig isotypes were quantified with an adult microsomal antigen of S. mansoni using the FAST-ELISA. Specific isotypes were detected with monoclonal antibodies specific for each human Ig isotype, followed by a peroxidase-conjugated anti-mouse Ig. Longitudinally, all monkeys showed similar isotype patterns. Isotypes increased for the first nine weeks following infection, and then began to decrease. Ten to 14 days following treatment, all isotypes increased. The Ig isotype responses of all monkeys followed classic patterns of isotype expression. A ratio of pretreatment (week 13) IgG1 absorbance values to post-treatment IgG1 absorbance values was generated for each monkey. All successfully treated monkeys, determined to be worm-free by perfusion, had IgG1 ratios at week 53 greater than 2.4 (range 2.4-181). The untreated monkeys and the single monkey that was a treatment failure had IgG1 ratios less than 2.1 (range 0.09-2.05) for the same time period.

Immunoblot analysis of humoral immune responses following infection with Bordetella pertussis or immunization with diphtheria-tetanus-pertussis vaccine.

To help develop better diagnostic tests for pertussis, we examined the serologic response to whole-cell proteins of Bordetella pertussis after natural infection or vaccination with diphtheria-tetanus-pertussis vaccine. Serum specimens collected during a pertussis outbreak investigation and from uninfected persons were used in Western blot (immunoblot) analyses to determine the presence of immunoglobulin G (IgG) and IgA antibodies to specific B. pertussis proteins. IgG antibodies to proteins of molecular masses 220 and 210 kilodaltons (kDa) were detected in 14 of 18 serum samples obtained from patients with culture-confirmed pertussis greater than or equal to 40 days after the onset of coughing. IgA antibodies were detected in 15 of the 18 samples. Of 19 serum samples obtained from patients who had not been ill with pertussis, 6 contained IgG antibodies to these proteins and 1 contained IgA antibodies. The two proteins bound antiserum specific for filamentous hemagglutinin and comigrated with purified filamentous hemagglutinin. IgG antibodies to two additional protein bands of molecular masses 84 and 75 kDa were associated with previous vaccination. Antibody to the 84-kDa protein was detected in 15 of 17 vaccinated, never-infected persons, and antibody to the 75-kDa protein was detected in 16 of the 17. None of 11 nonvaccinated, never-infected persons tested had antibodies to either protein. All seven fully vaccinated persons with culture-documented infection had antibodies to both proteins. Antibodies to the 84-kDa protein were detected in 6 of 22 nonvaccinated and infected persons, and antibodies to the 75-kDa protein were detected in 8 of the 22. Use of Western blot analysis in this study allowed us to distinguish antibody responses to infection and immunization.