Host Humoral Immune Response Research Articles

Serological Analysis of Autoantigens of Kawasaki Disease by cDNA Expression Cloning

Kawasaki Disease (KD) is an acute vasculitis of young childfood. However, little is known about the pathogenesis and immune response of KD. In this study, autoantigens of KD recognized by the host humoral immune response were investigated by serological analysis of antigens by recombinant expression cloning(SEREX). Immmunoscreening of a cDNA expression library from human umbilical vascular endotherial cells, which have been activated by IL-1 and TNF-α, with serum from a KD patient identified more than 20 autoantigens, including several antigen processing related proteins. To analyze whether immunerecognition of these autoantigens was KD related, allogeneic sera samples obtained from normal blood donors, patients with KD and patients with other diseases were tested for reactivity against the autoantigens defined in the study.

Generation and characterization of human monoclonal scFv antibodies against Helicobacter pylori antigens.

Infection with Helicobacter pylori is chronic despite a vigorous cellular and humoral immune response and causes severe pathology in some patients. In this study, phage display was used as a new approach in order to investigate the role of the host's humoral immune response in the pathogenesis of H. pylori gastritis. Human monoclonal single-chain Fv (scFv) antibody fragments against H. pylori cell lysate and the H. pylori urease were isolated from an immune phage display library, constructed from peripheral blood lymphocytes of an H. pylori-infected patient. After affinity selection, 23% of the clones tested showed binding activity against a lysate of the H. pylori Sydney strain in enzyme-linked immunosorbent assay (ELISA) and 9% bound the H. pylori urease. Further characterization by PCR-fingerprint analysis and sequencing revealed that two closely related H. pylori binders and one antiurease scFv could be isolated. The selected scFvs were highly specific as analyzed by ELISA and immunoblots using various bacterial lysates and recombinant proteins. Analysis of the humoral immune response following H. pylori infection using human monoclonal antibodies might contribute to a better understanding of the pathogenesis of the disease. Moreover, using immune phage display libraries, it might be possible for relevant epitopes of H. pylori antigens to be determined, which might be of use for vaccine development.

Influence of alcohol consumption on immunological status: a review.

The aim of this review is to present and discuss the effect of different levels of alcohol consumption on the immune system. Not only the amount consumed but also the type of alcoholic beverage have to be taken into account in order to determine the consequences on activity, number, distribution, balance, interaction and response of immunocompetent cells. The association between alcohol exposure and the risk of developing an alcohol-related disease is multifactorial. In fact, age, gender, smoking habits, dietary intake and exercise are involved among other factors. The evaluation of the host cellular and humoral immune responses has shown that alcohol may induce some benefits when consumption is moderate. Moreover, those alcoholic beverages that contain antioxidants, such as red wine, could be protectors against immune cell damage. According to the literature consulted, the daily consumption of 10-12 g and 20-24 g of alcohol for women and men, respectively, is considered to be a moderate intake; the type of beverage has been established not to be important when defining moderation. Particular attention is often focused on the U- or J-shaped curve which also suggests that light to moderate drinking produces a protective effect. Such an inverse relationship indicates a reduction of risk for both light and moderate consumers and a higher risk not only for hard drinkers, but also for non-consumers.

Generation of infectious and transmissible virions from a GB virus B full-length consensus clone in tamarins.

The strong similarity between GB virus B (GBV-B) and hepatitis C virus (HCV) makes tamarins infected by GBV-B an acceptable surrogate animal model for HCV infection. Even more attractive, for drug discovery purposes, is the idea of constructing chimeric viruses by inserting HCV genes of interest into a GBV-B genome frame. To accomplish this, infectious cDNA clones of both viruses must be available. The characterization of several HCV molecular clones capable of infecting chimpanzees has been published, whereas only one infectious GBV-B clone inducing hepatitis in tamarins has been reported so far. Here we describe the infection of tamarins by intrahepatic injection of RNA transcribed from a genomic GBV-B clone (FL-3) and transmission of the disease from infected to naive tamarins via serum inoculation. The disease resulting from both direct and secondary infection was characterized for viral RNA titre and hepatitis parameters as well as for viral RNA distribution in the hepatic tissue. Host humoral immune response to GBV-B antigens was also monitored. The progression of the disease was compared to that induced by intravenous injection of different amounts of the non-recombinant virus.

Feline immunodeficiency virus-infected cat sera associated with the development of broad neutralization resistance in vivo drive similar reversions in vitro.

We previously reported that, upon reinoculation into cats, a neutralization-sensitive, tissue culture-adapted strain of feline immunodeficiency virus constantly reverted to the broad neutralization resistance typical of primary virus isolates and identified residue 481 in the V4 region of the surface glycoprotein as a key determinant of the reversion. Here, we found that well-characterized immune sera, obtained from cats in which such reversion had occurred, selected in tissue culture in favor of virus variants that also had a neutralization-resistant phenotype and had amino acid 481 changed, thus indicating that the host's humoral immune response is capable of driving the reversion in the absence of other intervening factors. In contrast, a second group of immune sera, elicited by a virus variant that had already reverted to neutralization resistance in independent cats, induced the emergence of escape mutants lacking broad neutralization resistance and neutralized fewer virus variants. It is proposed that the viral variants used to produce the two sets of sera may have generated different antibody repertoires.

Recombinant adenovirus vectors for gene therapy and clinical trials.

In the last decade adenovirus (AdV) vectors have emerged as promising technology in gene therapy. They have been used for genetic modification of a variety of somatic cells in vitro and in vivo. They have been widely used as gene delivery vectors in experiments both with curative and preventive purposes. AdV vectors have been used in the experimental and in some extent in the clinical gene therapy of a variety of cancers. The combination of recombinant AdV technology with chemotherapy (pro drug system) seems to be promising, too. AdV vectors offer several advantages over other vectors. Replication defective vectors can be produced in very high titers (10(11) pfu/ml) thus allowing a substantially greater efficiency of direct gene transfer; they have the capacity to infect both replicating and nonreplicating (quiescent) cells from a variety of tissues and species. Several important limitations of adenovirus mediated gene transfer are also known, such as the relatively short-term (transient) expression of foreign genes, induction of the host humoral and cellular immune response to viral proteins and viral infected cells, which may substantially inhibit the effect of repeated treatment with AdV vectors, the limited cloning capacity and the lack of target cell specificity. However, the well-understood structure, molecular biology and host cell interactions of AdV-s offer some potential solutions to these limitations.

Localization of a new neutralizing epitope on the mumps virus hemagglutinin–neuraminidase protein

Four protein fragments which span the entire hemagglutinin–neuraminidase protein (HN) of mumps virus were expressed in HeLa cells and cell extracts were tested for their capability to induce neutralizing antibodies in mice. Fragment HN3 (aa 213–372) was able to induce the production of hemagglutination-inhibiting and neutralizing antibodies. When a subfragment of HN3, the synthetic peptide NSTLGVKSAREF (aa 329–340 of HN) was used for immunization, hemagglutination-inhibiting and neutralizing antibodies against mumps wild type virus but not against the Urabe Am9 vaccine virus were raised. The peptide could, therefore, contain a new epitope, which may be critical for protective host humoral immune response.

Human C-Reactive Protein Is Protective against FatalSalmonella entericaSerovar Typhimurium Infection in Transgenic Mice

C-reactive protein (CRP) is an acute-phase protein with a well-known association with infection and other inflammatory conditions. We have shown that expression of human CRP by CRP transgenic (CRPtg) mice is protective against lethal infection by Streptococcus pneumoniae, an effect likely mediated by CRP's ability to bind to this gram-positive pathogen. In the present study we tested whether CRPtg mice are resistant to infection with Salmonella enterica serovar Typhimurium, a gram-negative pathogen that causes the murine equivalent of typhoid fever. CRPtg mice experimentally infected with a virulent Typhimurium strain lived longer and had significantly lower mortality than their non-tg littermates. The greater resistance of CRPtg mice could be attributed to significantly increased early (0 to 4 h) blood clearance of salmonellae and significantly decreased numbers of bacteria in the liver and spleen on day 7 postinfection. In addition, 14 days after infection with an avirulent Salmonella strain, the serum titer of anti-Salmonella immunoglobulin G antibodies was higher in CRPtg than non-tg mice. This study provides unequivocal evidence that CRP plays an important role in vivo in host defense against salmonellae during the early stages of infection. In addition, as the beneficial effect of CRP includes enhancement of the host's humoral immune response, CRP may also contribute indirectly to host defense during later stages of infection.

Serological analysis of BALB/C methylcholanthrene sarcoma Meth A by SEREX: identification of a cancer/testis antigen.

Antigens of BALB/c methylcholanthrene-induced fibrosarcoma Meth A recognized by the host humoral immune response were investigated by serological analysis of antigens by recombinant expression cloning (SEREX). Immunoscreening a cDNA library from Meth A (Kgamma) cells (Meth A retrovirally transfected with murine IFN-gamma cDNA) with sera from BALB/c mice growing parental Meth A transplants identified 10 antigens. One of them, OY-MS-4, showed characteristics of a cancer/testis (CT) antigen. Nucleotide sequence analysis revealed that OY-MS-4 was identical to a mouse placenta and embryonic expression gene (pem) known to be selectively expressed during embryogenesis and in transformed cell lines. In adult mice, expression of OY-MS-4 was restricted to testis and placenta. Four of 6 methylcholanthrene-induced fibrosarcomas in BALB/c mice showed strong expression of OY-MS-4. In 6 T-cell leukemias, only a dimethylbenzanthracene-induced leukemia, EL4 (C57BL), showed strong expression. Two other tumors, A20.2J and P815, induced by ethylnitrosourea and methylcholanthrene, respectively, also strongly expressed OY-MS-4. The other 9 gene products identified in Meth A by SEREX were expressed in all 15 tumors tested and in a range of normal tissues. Sequence analysis of cDNA inserts coding for the SEREX-defined antigens showed no evidence of mutation. Despite the expression of OY-MS-1-10 antigens in methylcholanthrene sarcomas other than Meth A, no antibody was detected in the sera of mice bearing these other sarcomas. The basis for the unique immunogenicity of OY-MS-1-10 presented by Meth A, but not by other syngeneic tumors expressing these gene products, is unknown.

Antigenic characterization and cytolocalization of P35, the major Mycoplasma penetrans antigen.

Mycoplasma penetrans is a mycoplasma with unique morphology, recently identified in urine samples collected from HIV-infected patients. This mycoplasma has been found to be statistically associated with HIV infection, and to be cytopathic in vitro. The dominant antigen recognized during natural and experimental infections is an abundant lipoprotein, P35, which, upon extraction, segregates in the Triton X-114 detergent phase. It is used as the basis of M. penetrans-specific serological assays. Although mycoplasma lipoproteins, including M. penetrans P35, are the main antigens recognized by the host humoral immune response, very little is known about the nature of the epitopes involved. Immunoelectron microscopy revealed that all P35 is exposed at the cell surface and is distributed all over the membrane. P35 linear B-epitopes were mapped by an ELISA approach based on a set of overlapping peptides covering the entire mature polypeptide. The immunoreactivity of the peptides was first tested with sera from immunized animals. The dominant B-epitopes were found at the C- and N-terminal regions, in partial agreement with algorithmic predictions. Patient sera were evaluated with the same assay. Only some reacted with linear epitopes whereas others did not, indicating the importance of P35 nonsequential epitopes. Statistical analysis of the results allowed the definition of a set of peptides which were clearly immunodominant. Finally, the P35-encoding gene was modified by in vitro mutagenesis to allow the production and purification of a recombinant protein (rP35delta0) in Escherichia coil. The antigenicity of rP35delta0 was tested by Western blotting and compared to that of another recombinant product, rP35delta3, a truncated P35 polypeptide. Although rP35delta0 reacted with the M. penetrans-seropositive patient sera tested, rP35delta3 was only immunoreactive with one of six sera. This result confirmed that P35-nonsequential epitopes dominate during M. penetrans infection. Our results have important implications for the understanding of lipoprotein antigenicity during mycoplasma infections. In addition, the P35-derived immunodominant synthetic peptides defined in this study, as well as the purified rP35delta0, provide the antigenic material for the necessary improvement of M. penetrans serological assays.

Host response and vaccine development to Helicobacter pylori infection.

Studies in both humans and animals demonstrate that H. pylori is capable of illiciting an innate response that in part is regulated by the genetic makeup of the host. These innate responses includes stimulating immune effector mechanisms at the cellular and biochemical level resulting in the influx of neutrophils into the lamina propria and have even been shown to modify gastric acid secretion. The availability of good animal models of chronic Helicobacter infection has also allowed investigators to begin to examine how the adaptive host immune response prevents and/or exacerbates Helicobacter-induced gastroduodenal disease. The experimental H. felis/mouse model has been utilized by a number of laboratories to investigate mechanisms of host defense against chronic Helicobacter infection. This model and the more recently developed H. pylori rodent model has not only allowed investigators to confirm the feasibility of immunotherapy to prevent and/or cure Helicobacter infection but also to begin to examine how the host immune response prevents and/or exacerbates Helicobacter-induced gastroduodenal disease. Based on these studies a hypothesis is emerging that suggests that protection and/or cure from Helicobacter infection is mediated primarily by an upregulated cellular immune response which may act via an antibody independent mechanism. Paradoxically, following natural infection with H. pylori, a component of the cellular immune response also promotes chronic gastric inflammation without clearance of the organism. The recent development of reliable and reproducible H. pylori/rodent models of disease and the availability of numerous inbred strains, transgenic and knockout animals, will allow investigators to continue to explore the role the host cellular and humoral immune response plays in promoting or preventing this infection.

The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein.

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N-glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.

Nucleic acid-based antiviral and gene therapy of chronic hepatitis B infection.

Persistent hepatitis B virus (HBV) infection often leads to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma. There is a need to develop new antiviral approaches for the treatment of this disease. We have explored various nucleic acid-based strategies designed to inhibit HBV replication including: the use of antisense RNA and DNA constructs, DNA-based immunization techniques to stimulate broad-based cellular immune responses with particular emphasis on the generation of cytotoxic lymphocyte (CTL) activity to viral structural proteins, hammerhead ribozymes to cleave HBV pregenomic RNA in vitro and dominant negative HBV core mutant proteins as inhibitors of nucleocapsid formation within cells. In order to optimize these antiviral effects, various novel expression vectors have been developed to deliver such DNA constructs to cells. For example, adenoviral vectors carrying genes that encode for dominant negative proteins have been employed to transfect hepatocytes in vitro and in vivo. In addition, plasmid vectors have been produced to promote expression of HBV structural genes following injection into muscle cells as a means to stimulate the host's cellular and humoral immune response in the context of histocompatibility antigen (HLA) class I and II antigen presentation. These experimental approaches may have important implications for the generation of efficient antiviral effects during chronic HBV infection.

Long Term Correction of Bilirubin-UDP-glucuronosyltransferase Deficiency in Gunn Rats by Administration of a Recombinant Adenovirus during the Neonatal Period

Injection of a recombinant adenovirus expressing human bilirubin-UGT1 (Ad-hBUGT1) (3 x 10(9) plaque-forming units (pfu) intravenously) in adult bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient Gunn rats resulted in biliary excretion of bilirubin glucuronides and a 70% reduction of serum bilirubin levels. However, the effect was transient, and host humoral and cellular immune response prevented transgene expression after subsequent injections. To determine whether injection during the neonatal period would tolerize the host to the recombinant virus, we injected 1 x 10(8) pfu of Ad-hBUGT1 or Ad-LacZ (a recombinant adenovirus expressing Escherichia coli beta-galactosidase) into 1-3-day-old Gunn rats. Two subsequent injections (3 x 10(9) pfu) were given 56 and 112 days after the initial injection. Injection of Ad-BUGT1, but not Ad-LacZ, reduced serum bilirubin by 70-76% of the levels in untreated pups (9 +/- 1.3 mg/dl), followed by a gradual increase to 3.25 +/- 0.3 mg/dl in 56 days; similar or greater reductions occurred after the second and third injection. Serum neutralizing antibody titer and cytotoxic lymphocyte activity against adenovirus-infected hepatocytes were low or undetectable. Thus, tolerization by injection of the virus during the neonatal period permits long term gene therapy by repeated injection of the virus.

Antigenic analysis of type O foot-and-mouth disease virus in the persistently infected bovine

The antigenic profiles of serotype O strains of FMDV collected from the oropharynx of persistently infected cattle were defined with a panel of monoclonal antibodies (mAb's) in an indirect antigen-trapping ELISA. The mAb profiling showed no significant loss of reactivity in two neutralising antigenic sites of persistent FMDV isolates collected over a period of eight months. Early and late serum taken from a carrier animal showed similar neutralising activity against early and late carrier isolates. The antigenic stability of serotype O strains of FMDV collected during the carrier state suggests that antigenic variation facilitated by pressure from the host humoral immune response is not critical to the establishment or maintenance of a persistent infection with FMDV.

Modulation of cardiac physiology by an anti‐Trypanosoma cruzi monoclonal antibody after interaction with myocardium

Circulating antibodies from human and murine chagasic sera are able to interact with myocardium, activating neurotransmitter receptors. Here, we studied the effects of a monoclonal antibody (MAb CAK20.12), which recognizes a 150 kilodalton antigen of Trypanosoma cruzi and reacts with normal human and murine striated muscles and with cardiac tissue. The MAb CAK20.12 binds to purified cardiac membranes and interferes with the binding of beta-adrenergic receptor radioligand ([125I]CYP) and muscarinic cholinergic receptor (mAChR) radioligand ([3H]QNB) in a noncompetitive way. As a consequence of this interaction, beta-adrenergic receptor and mAChR were activated, leading to increased intracellular levels of cyclic AMP as a result of beta-adrenergic receptor-coupled adenylate cyclase triggering. When its sympathetic action was abrogated, it also induced an mAChR-mediated increase in cyclic GMP. Furthermore, cardiac physiology was modified by MAb CAK20.12, as it was able to increase cardiac contractility through beta-adrenoceptor activation and to decrease atrial frequency as a result of mAChR activation. The fact that this MAb modulates and modifies the mechanical and biochemical activity of normal murine heart established an important basis for future research and understanding of how the host's humoral immune response acts on the course and development of the chronic chagasic myocardiopathy.

Antibodies that block virus attachment to vero cells are a major component of the human neutralizing antibody response against dengue virus type 2

Epidemiological data strongly implicate a role for the host humoral immune response in both protection against and exacerbation of dengue virus-caused disease. In an effort to characterize elements of the normal human immune response against dengue virus we have addressed the issue of antibody-mediated neutralization of dengue virus. We show here the ability of both mouse monoclonal antibody 3H5 and human anti-dengue neutralizing sera to block binding of dengue-2 virus to monkey kidney (Vero) cells. Since Vero cells possess virus receptors but not Fc receptors we conclude that the major effect of host neutralizing antibodies is to block virus attachment to Vero cell dengue virus receptors. Analysis of 61 patient antisera yielded good correlation (Pearson's coefficient = 0.90; P < 0.001) between neutralizing activity and ability to block virus-cell attachment suggesting that antibody-mediated neutralization of dengue virus occurs primarily extracellularly and less by a postattachment mechanism as has been described for certain other viruses.

A brief history of the pneumococcus in biomedical research: a panoply of scientific discovery.

Because of its prominence as a cause of disease in humans, Streptococcus pneumoniae has been the subject of intensive investigation at both the clinical level and the basic scientific level during the past century. In a number of instances, these studies have resulted in important progress toward the comprehension of basic biological principles. The areas advanced by studies of the pneumococcus include an understanding of the concept of pathogenesis of infectious disease; the development of Gram's stain for identification of bacteria in specimens from patients; the elucidation of the role of the bacterial capsule in resistance to phagocytosis by cells of the host's immune system; the demonstration that molecules other than proteins are capable of eliciting the host's humoral immune responses and later, by extension, that isolated bacterial exopolysaccharides can be used safely and effectively as vaccines in humans; the documentation of the efficacy of penicillin; the collection of conclusive evidence that DNA encodes genetic information; and the investigation of putative proteinaceous virulence factors.

An antibody- and synthetic peptide-defined rubella virus E1 glycoprotein neutralization domain.

We previously described a monoclonal antibody (MAb) library generated by infecting BALB/c mice with rubella virus (RV) and selected by an enzyme-linked immunosorbent assay (ELISA) using purified virion targets. Plasmid pARV02-01, which expresses the fusion protein RecA1-35-GIGDLGSP-E1(202)-E1(283)-GDP-LacZ9-1015 in Escherichia coli, was shown to be a ligand for MAbs E1-18 and E1-20 (J. S. Wolinsky, M. McCarthy, O. Allen-Cannady, W. T. Moore, R. Jin, S. N. Cao, A. Lovett, and D. Simmons, J. Virol. 65:3986-3994, 1991). Both of these MAbs neutralize RV infectivity. A series of five overlapping synthetic peptides was made to further explore the requirements of this MAb binding domain. One of these peptides (SP15; E1(208) to E1(239)) proved an effective ligand for both MAbs in the ELISA. Stepwise synthesis of SP15 defined the minimal amino-terminal requirement for binding MAb E1-18 as E1(221) and that of MAb E1-20 as E1(223); the minimal carboxyl-terminal requirement is uncertain but does not exceed E1(239). Immunization of mice and rabbits with SP15 induced polyvalent antibody reactive with SP15, with other overlapped and related but not unrelated synthetic peptides, and with RV. The rabbit anti-SP15 antibody showed neutralization activity to RV similar to that of MAbs E1-18 and E1-20 but lacked hemagglutination inhibition activity. These data define a neutralization domain on E1 and suggest that the RV epitopes conserved by SP15 may be critical for protective host humoral immune responses.

Lipopolysaccharide-independent radioimmunoprecipitation and identification of structural and in vivo induced immunogenic surface proteins of Salmonella typhi in typhoid fever

The humoral response to Salmonella typhi is important for protective immunity against typhoid fever, as indicated by the protection obtained with killed cell vaccines and component vaccines (outer membrane proteins, Vi antigen) in animals and human beings. Nonetheless, analysis and interpretation of host humoral immune response to S. typhi surface antigens have been difficult because of the complex structure of the S. typhi envelope and the lack of purified reagents for detection of immune response to individual surface components. Normal and convalescent human sera from typhoid fever patients were absorbed with S. typhi lipopolysaccharide. These sera were used in radioimmunoprecipitation assays of whole S. typhi cells and S. typhi membranes labelled with either 125I or 35S-methionine. This strategy has permitted the unequivocal identification of a humoral immune response to structural and in vivo induced outer membrane proteins of S. typhi. In this manner, we have identified the porins, lipoprotein, the iron-starvation-induced proteins, and three proteins of 30, 18.5 and 15 kDa as surface-exposed immunogens of S. typhi in patients with typhoid fever. These studies suggest that further experimental work is needed to characterize the relevance of both anti- S. typhi outer membrane protein and anti-lipopolysaccharide antibodies in recovery from S. typhi infections and protective immunity.