Complement-fixing Antibody Response Research Articles

Antibody Targets and Properties for Complement-Fixation Against the Circumsporozoite Protein in Malaria Immunity.

The Plasmodium falciparum circumsporozoite protein (CSP) forms the basis of leading subunit malaria vaccine candidates. However, the mechanisms and specific targets of immunity are poorly defined. Recent findings suggest that antibody-mediated complement-fixation and activation play an important role in immunity. Here, we investigated the regions of CSP targeted by functional complement-fixing antibodies and the antibody properties associated with this activity. We quantified IgG, IgM, and functional complement-fixing antibody responses to different regions of CSP among Kenyan adults naturally exposed to malaria (n=102) and using a series of rabbit vaccination studies. Individuals who acquired functional complement-fixing antibodies had higher IgG, IgM and IgG1 and IgG3 to CSP. Acquired complement-fixing antibodies targeted the N-terminal, central-repeat, and C-terminal regions of CSP, and positive responders had greater antibody breadth compared to those who were negative for complement-fixing antibodies (p<0.05). Using rabbit vaccinations as a model, we confirmed that IgG specific to the central-repeat and non-repeat regions of CSP could effectively fix complement. However, vaccination with near full length CSP in rabbits poorly induced antibodies to the N-terminal region compared to naturally-acquired immunity in humans. Poor induction of N-terminal antibodies was also observed in a vaccination study performed in mice. IgG and IgM to all three regions of CSP play a role in mediating complement-fixation, which has important implications for malaria vaccine development.

T-Cell Stimulation and Regulation: With Complements from CD46

Crosslinking of CD46 and CD3 on naïve human CD4+ T-lymphocytes induces interleukin-10 secretion and granzyme B expression. These highly proliferative T-regulatory type 1-like T-regulatory T-cells (Tregs) can suppress an immune response. We propose that this process is important in the prevention of chronic inflammation such as at epithelial borders and in deactivation of a successful immune response. Relative to the latter, once a complement-fixing polyclonal antibody response has been mounted, in most cases, the pathogen will be rapidly destroyed. At this time, the C3b/C4b-bearing immune complexes could initiate the deactivation arm of an immune response by shutting down immunocompetent cells through CD46-generated T-cells. Herein, we review this pathway for the induction of Tregs, focusing on a role for the complement system and especially signaling through CD46 on human T-cells.

Antibody-mediated CNS demyelination: focal spinal cord lesions induced by implantation of an IgM anti-galactocerebroside-secreting hybridoma.

O1 hybridoma cells, which secrete an IgM antigalactocerebroside, were implanted into the spinal cord of cyclosporine-treated juvenile or adult rats, and the animals were sacrificed approximately 2-3 wk later. About half the recipient animals developed myelin lesions. In some, sharply circumscribed foci of demyelination formed within the dorsal columns. Cellular reaction consisted of macrophages containing refractile globules in the parenchyma and within enlarged perivascular spaces as well as thickened endothelial cells. "Shadow plaques" also developed, i.e. regions in which axons were surrounded by thin myelin sheaths, compatible with remyelination. In addition, we found damaged axons, some of which were swollen with organelles, comparable to the enlarged axon profiles seen at sites of constriction or interruption. Compromise of the blood-brain barrier at sites of hybridoma growth was demonstrated by extravasation of Evans blue dye. Discontinuation of cyclosporine was followed by an anti-hybridoma, complement-fixing antibody response within 2-3 d. This model of focal CNS demyelination and remyelination, with evidence of some axon damage, is mediated by a defined IgM antiglycolipid monoclonal antibody secreted within the spinal cord parenchyma. The lesions, which are similar to those of multiple sclerosis, probably result from the interaction between the intrathecally secreted IgM antibody and complement entering from the circulation at foci of compromised blood-brain barrier plus activation of endogenous or hematogenous macrophages via their complement receptors.

Molecular cloning and characterization of the Coccidioides immitis complement fixation/chitinase antigen.

Detection of anti-Coccidioides complement-fixing (CF) antibody is a valuable diagnostic and prognostic aid in coccidioidomycosis. The CF antibody response is directed against a heat-labile antigen that has chitinase activity, hereafter referred to as the CF/chitinase protein. To identify and clone this immunoreactive enzyme, we constructed a Coccidioides immitis cDNA lambda ZAP expression library from spherule RNA and detected fusion peptides expressing CF epitopes by immunoscreening. A cDNA clone consisting of 1,623 bp was identified, sequenced, and found to contain a single open reading frame that encodes a protein of 47 kDa with 427 amino acids. Deduced amino acid sequence analyses showed that the cloned CF/chitinase cDNA contains a 35-amino-acid region, beginning at Ser-18 and ending at and ending at Arg-52 which has 92% homology with the reported N-terminal amino acid sequence of authentic CF/chitinase protein. The first 17 amino acids in the deduced sequence of the cloned cDNA are not present on the mature CF/chitinase protein, suggesting that it may be a signal peptide. Expression of the CF/chitinase cDNA insert by using the pGEX-4T-3 vector yields a fusion peptide that bears CF-specific epitopes and shows chitinase activity. The CF/chitinase clone will enable large-scale production of the recombinant CF antigen for use in immunoassays and facilitate studies on the role of chitinase in the morphogenesis of C. immitis.

Vaccination of Lewis rats against Mycoplasma arthritidis- induced arthritis

Summary The nature of Mycoplasma arthritidis antigens responsible for eliciting protective immunity in rats was studied by inoculation of rats with mycoplasmal components that had been subjected to a variety of physical and chemical treatments. All inocula tested induced good protection against development of clinical illness, as assessed by changes in body weight and appearance of joint swelling and/or temporary hind limb paralysis. Although all preparations stimulated development in inoculated rats of high titer of antimycoplasmal antibodies measured by elisa, the complement-fixation antibody response was poor and, in some cases, lacking altogether. This indicated that completion-fixation antibodies may not be involved in protecting rats against M arthritidis-indaced illness. Protective antigens were stable to heat (100 C for 10 minutes), formalin, and denaturation by sodium dodecyl sulfate (sds). Inoculation with membrane and soluble cytoplasmic fractions was protective, as was inoculation with 5 M arthritidis fractions separated according to molecular weight by sds-polyacrylamide gel electrophoresis (sds-page). For this latter experiment, rat antisera obtained after vaccination, but prior to challenge exposure, were tested by immunoblot analysis against electrophoretically separated M arthritidis membrane proteins. Interestingly, all antisera from these rats recognized antigens migrating far outside the molecular weight range of the cell fractions with which rats were inoculated. This indicated either that the protective antigens may be composed of numerous antigenically related subunits that separated by sds-page into a variety of molecular weight ranges or that a few major antigens may exist in several forms or phases within a given population of M arthritidis.

Cytomegalovirus infection after bone marrow transplantation: Relation of pneumonia to postgrafting immunosuppressive treatment

Five fatal cases of CMV associated interstitial pneumonia occurred among 20 patients who had received allogenic bone marrow transplantation for acute leukemia or aplastic anaemia. This outcome was analysed in relation to prospectively obtained data on complement fixing (CF) and IgM anti-CMV titres and excretion of CMV in urine or pharynx. Altogether 16 patients had primary or reactivated CMV infection. Five of these patients receiving corticosteroids at the time of infection, at a dose of more than 1 mg/kg/day, failed to mount a significant CF antibody response, excreted large amounts of virus, and died from pneumonia, while the remaining 11 infected patients obtained high CF antibody titres and showed no severe symptoms related to CMV infection. IgM antibodies occurred simultaneously with CF antibodies in most cases of primary and reactivated infection. The results of the study suggest a protective role of the humoral immune response to CMV infection in BMT recipients, and passive immunisation with CMV hyperimmune globulin should be attempted especially in patients given additional immunosuppression with high dose corticosteroids.

Observations on anaplasmosis following field exposure to heavy and light infestations with Boophilus microplus

Two groups of Normandy calves were exposed to heavy ( X half body count = > 300 ) or light ( X half body count = 9 ) natural infestations with Boophilus microplus. All of the calves became infected with Anaplasma marginale. Despite the difference in tick challenge level, there was no significant difference in the incubation period, increase in body temperature, level or duration of Anaplasma parasitemia, decrease in packed cell volume, or complement-fixing antibody response, between the heavily and lightly infected calves. Neither the incubation period nor the clinical severity of anaplasmosis was significantly influenced by the number of infested tick vectors.

Experimental St. Louis encephalitis virus infection of sloths and cormorants.

Experimental infection of 11 Bradypus variegatus and Choloepus hoffmanni sloths with St. Louis encephalitis (SLE) virus produced detectable viremias of seven to 27 (median 13) days duration and maximum titers of 2.7 to 6.5 (median 5.1) log10 median suckling mouse intracranial lethal doses (SMicLD50) per ml. Experimental SLE viremia onset was delayed and maximum titer depressed in two sloths concurrently infected with naturally acquired viruses. SLE viremias in four experimentally inoculated cormorants Phalacrocorax olivaceus were shorter, and of equal or lower titer, than in sloths. Colonized Culex pipiens quinquefasciatus mosquitoes were infected by feeding on sloths circulating at least 4.8 log10 SMicLD50 of SLE virus per ml, and subsequently transmitted the infection to mice and chicks. An uninoculated baby Bradypus became infected by contact transmission from its mother. The antibody response of sloths to SLE virus was slow, being undetectable until several weeks post-inoculation. However, both sloth species developed high and long-lasting neutralizing and hemagglutination-inhibition antibody titers. The complement-fixation antibody response in Bradypus was lower and slower to develop than in Choloepus. Sloths with naturally acquired SLE virus antibody did not become detectably viremic after experimental inoculation. Neither sloths nor cormorants become overly ill from SLE virus infection.

Alteration of immune function by staphylococcal pyrogenic exotoxin type C: possible role in toxic-shock syndrome.

Staphylococcal pyrogenic exotoxin type C (PE-C) suppressed synthesis of IgM antibodies to sheep erythrocytes by in vitro cultures of murine and rabbit splenocytes. Maximal suppression was observed on day 4 of culture and at PE-C doses of 0.1 and 0.01 microgram per 10(7) splenocytes. Endotoxin, added to cell cultures containing PE-C, caused further suppression of IgM synthesis. Endotoxin alone added to cultures was not immunosuppressive. Sublethal doses of PE-C plus endotoxin also suppressed complement-fixing antibody responses in rabbits to sheep erythrocytes; neither agent alone was suppressive. Further, the combination of PE-C and endotoxin blocked reticuloendothelial clearance of colloidal carbon by 50% during a 20-min period. PE-C enhanced the skin reactivity of rabbits previously sensitized to purified protein derivative. One-half of rabbits immunized with PE-C emulsified in adjuvant failed to make antibody to PE-C during a 112-day period. The animals that were nonresponsive to PE-C were able to make antibodies to sheep erythrocytes.

Virological studies in amyotrophic lateral sclerosis.

Complement-fixing antibody response to eleven different viruses were measured in amyotrophic lateral sclerosis (ALS) patients, contacts of ALS patients, neurological controls, and normal controls. The normal controls showed a decreased response to adeno-associated virus and an increased response to adenovirus when compared to the other groups. Levels of interferon-like substances also were investigated in sera and cerebrospinal fluids of ALS patients and neurological controls. Responses were of a low titer and were not increased in the ALS group. Explant cultures were established from tissues of 24 ALS autopsy cases. Cultures were investigated directly or following fusion to various indicator cell lines for viral-like agents. Techniques such as interference assays, 5-bromodeoxyuridine (BudR) induction, hemadsorption, and fluorescent antibody staining failed to detect virus. By addition of helper adenovirus to primary explant cultures, adeno-associated virus was isolated from 2 of 11 ALS cases.

Patterns of shedding of hepatitis A virus antigen in feces and of antibody responses in patients with naturally acquired type A hepatitis.

Serial stools and sera from 13 patients with hepatitis A were collected during an epidemic in 1978 in a rural village near Hangzhou, China, and were studied to determine the patterns of shedding of hepatitis A antigen (HAAg) and of antibody response in some cases. Of 44 stool specimens, 31 were HAAg-positive by a radioimmunoprecipitation-polyethylene glycol assay. The highest percentage of HAAg positivity was in stools collected one week before and one week after the peak elevation of serum glutamic pyruvic transaminase (SGPT) levels, and peak HAAg shedding in each patient usually occurred in the early stools. Fecal shedding of HAAg was detected as early as day 19 before peak elevation in SGPT level and as late as day 11 afterward and continued for at least 25 days. The pattern of HAAg shedding in feces and of complement-fixation and immune adherence antibody response in relation to the change in SGPT activity and jaundice in four patients with hepatitis A was demonstrated.

Complement fixing antibody responses to virus infection in children with acute lymphoblastic leukaemia.

Thirty children with acute lymphoblastic leukaemia (ALL) were studied and had a virus isolated. Only 50% produced a significant rise in complement fixing (CF) antibody titre compared to 100% of normal children. The failure to produce antibodies was unpredictable. CF antibodies are not a reliable guide to virus infections in children with ALL.

Limitations of the complement-fixation test for distinguishing naturally acquired from vaccine-induced yellow fever infection in flavivirus-hyperendemic areas.

On the basis of previous studies, it has long been stated that 17D yellow fever (YF) vaccine generally does not induce complement-fixing (CF) antibodies, and that the presence of CF antibodies could be used in epidemiological studies to distinguish individuals infected with wild YF virus from vaccinated persons. In January 1979, seroepidemiological investigations were conducted during a YF epidemic in The Gambia, West Africa. Since a mass vaccination campaign was also in progress, it was important to confirm that the CF test could be used for serodiagnosis and determination of the incidence of natural YF infections. The serological responses of 58 individuals who received 17D YF vaccine were studied. The vaccinees fell into three gorups: 1) those with prevaccination YF neutralizing (N) antibodies; 2) immunological virgins without prevaccination YF-N antibody or hemagglutination-inhibiting (HI) antibodies to heterologous flaviviruses (Zika, West Nile, dengue 1, Uganda S, Spondweni, or Ntaya; and 3) those without prevaccination YF-N antibodies but with heterologous flaviviral HI antibodies. Vaccination of persons without prior flaviviral immunological experience resulted in monotypic YF HI and/or N antibody seroconversions, but no CF antibody response. The presence of prevaccination YF N antibodies blocked serological response to the vaccine in a high proportion of the cases; however, 24% of vaccinees in this group had a marked rise in log2 YF CF antibody titer (mean increase of 3.9). Thirteen (46%) of 28 persons without prevaccination YF N, but with heterologous flaviviral HI antibodies demonstrated YF CF antibody seroconversion or increase in titer following vaccination; in this group the mean increase in log2/ YF CF antibody titer was 2.1. The CF antibody response was generally broadly cross-reactive; but in a few individuals, the YF CF antibody response was homotypic. Nine different patterns of HI and CF homologous and heterologous antibody responses were defined and are discussed. The practical significance of these studies is that they demonstrate that in a high percentage of persons with prior flavivirus exposure, anamnestic serological responses to YF vaccine result in CF antibodies similar to those induced by natural YF virus infection. In Africa and tropical America, where the background of flaviviral immunity is high, it is imperative that seroepidemiologic investigations during or after YF outbreaks be conducted prior to vaccination.

INFLUENZA A AND B VIRUS INFECTION IN INFANTS AND YOUNG CHILDREN DURING THE YEARS 1957–19761

Influenza A virus activity was demonstrated in infants and young children from metropolitan Washington, DC during each of 19 successive August-July respiratory disease years, and during 17 of these years at least 2% of hospitalized respiratory disease patients yielded an influenza A or B virus and/or showed an influenza A or B serum complement-fixing (CF) antibody response. Between October 1957 and July 1976, 14.3% of 860 croup patients and 5.3% of a total of 5655 hospitalized respiratory patients, including croup patients, showed evidence of influenza A or B infection. The mean period of hospitalization with either virus was about 8 days, though serious infection with influenza A virus was 4.5 times more common than with influenza B virus. Both influenza viruses were detected more frequently in respiratory disease outpatients than in respiratory disease inpatients. Patients with serious influenza A virus infections were especially likely to have croup (particularly during the H3N2 era), to be seen during December through February, and to be black male infants. During the peak month of a composite of 13 consecutive influenza A virus outbreaks, influenza A virus infection was demonstrated in 67.6% of croup patients and in 35.6% of all hospitalized respiratory patients including croup patients. During the peak month of a composite of 6 consecutive influenza B virus outbreaks, influenza B virus infection was demonstrated in 36.0% of croup patients and in 10.8% of all hospitalized respiratory disease patients including croup patients.

Antibody responses to influenza vaccines containing A/New Jersey/8/76.

Studies were undertaken with whole virus, subunit and adsorbed subunit influenza vaccines containing the A/New Jersey/8/76 strain to compare the antibody response induced by immunization with these vaccines. There were no significant differences in the haemagglutination-inhibiton (HI) antibody responses to A/New Jersey/8/76 induced by these vaccines in the adult groups under study. Vaccinees over 23 years of age produced good HI antibody responses to one dose of vaccine. Neuraminidase antibody and complement-fixing (CF) antibody responses were determined for the A/Victoria/3/75 component of bivalent vaccines. There were significant increases in neuraminidase inhibiting (NI) antibody titres but no increase in the complement-fixing antibody titres after vaccination.

360 INFLUENZA A AND B VIRUS INFECTION IN INFANTS AND YOUNG CHILDREN DURING THE YEARS 1957–1976

Influenza A virus activity was demonstrated in infants and young children from metropolitan Washington, D. C. during each of 19 successive August-July respiratory disease years, and during 17 of these years at least two per cent of hospitalized respiratory disease patients yielded an influenza A or B virus and/or showed an influenza A or B serum complement fixing (CF) antibody response. Between October 1957 and July 1976, 14.3 per cent of 860 croup patients and 5.3 per cent of 5,655 hospitalized respiratory patients showed evidence of influenza A or B virus infection. Serious illness with influenza A virus was 4.5 times more common than with influenza B virus. Influenza A virus infections were seen more than twice as frequently during the era of the H3N2 virus subtype as during the H2N2 era. Patients with serious influenza A virus infections were especially likely to have croup (particularly during the H3N2 era), to be seen during December through February, and to be black male infants. During the peak month of a composite of 13 consecutive influenza A virus outbreaks, influenza A virus infection was demonstrated in 67.6 per cent of croup patients and 35.6 per cent of all hospitalized respiratory patients. During the peak month of a composite of 6 consecutive influenza B virus outbreaks, influenza B virus infection was demonstrated in 36.0 per cent of croup patients and in 10.8 per cent of all hospitalized respiratory disease patients

Complement-fixing antibody response in pneumococcal pneumonia.

Previous studies of complement-fixing antibodies to pneumococcal capsular polysaccharides in humans have yielded conflicting results. We studied 65 sera from 25 patients with pneumococcal pneumonia, using both fresh sera and heat-inactivated sera with added human complement. Only 4 of the 25 patients developed detectable levels of complement-fixing anticapsular antibody. Of the 25 patients, 22 developed detectable levels of hemagglutinating anticapsular antibody, indicating that they were able to develop an immunological response during the infection. Most of the antibody detected by hemagglutination was sensitive to 2-mercaptoethanol, but some 2-mercaptoethanol-resistant antibody was also detected. In studies with rabbit antiserum, the complement fixation test was found to be as sensitive as the hemagglutination test for detection of anticapsular antibody. It is not clear why detectable levels of complement-fixing antibody do not develop more often in patients with pneumococcal pneumonia. Studies of purified anticapsular antibody would be of interest to determine whether or not these antibodies are restricted to immunoglobulin subclasses having a limited capacity to fix complement.

Relative Importance of Viruses and Bacteria in the Etiology of Pediatric Diarrhea in Taiwan

Of 80 children with acute gastroenteritis in Taiwan, 75 were tested for evidence of infection with a reovirus-like agent (RVLA) by electron microscopic examination of stools and/or by assays of complement-fixing and immunofluorescent antibody responses to the serologically related Nebraska calf diarrhea virus; 42 (56%) were positive. Of children whose stools contained the agent, 90% showed a fourfold or greater rise in titer of immunofluorescent antibodies, and 85% developed a fourfold or greater rise in titer of complement-fixing antibodies to the agent. Strains of Escherichia coli producing only heat-labile toxin (LT) were isolated from 7% of 57 ill children who had not received previous antibiotics and from 4% of 131 healthy control children (P = 0.49). Strains of E. coli producing only heat-stable toxin (ST) were isolated from five (9%) of 57 age-matched children in the group of 131 healthy controls tested. Invasive E. coli were not found. In addition, three of six children with bacterial and! or serological evidence of an infection with LT-producing E. coli and four of five children infected with ST-producing E. coli had infections with RVLA. RVLA appeared to be the major cause of diarrheal illness in Taiwan during the summer of 1975, whereas toxigenic E. coli appeared to be responsible for a smaller proportion of pediatric enteritis.

Antigenic relationships among several simian varicella-like viruses and varicella-zoster virus

Cross-neutralization and complement fixation tests demonstrated the immunological identity of the Delta herpesvirus, the 592S virus, the Liverpool vervet monkey virus, the herpesvirus of patas monkeys, and the Medical Lake macaque virus. These viruses were isolated from diverse outbreaks of varicella-like disease in simians and from various simian species. All of the simian viruses were shown to be related to human varicella-zoster (V-Z) virus, as evidenced by the fact that immunization of monkeys with each of the simian viruses elicited the production of both neutralizing and complement-fixing antibodies to V-Z virus. However, cross-complement fixation tests indicated that the simian viruses are not so closely related to V-Z virus as they are to one another. Varicella or zoster infections in humans produced neutralizing and complement-fixing antibody responses to each of the simian viruses; the responses were more marked in zoster infections than in varicella infections but, in most patients, antibody levels produced to the simian viruses were much lower than those to the homologous V-Z virus.

Complement-fixing reactivity of Varicella-Zoster virus subunit antigens with sera from homotypic infections and heterotypic Herpes simplex virus infections

Various subunit antigens of varicella-zoster (V-Z) virus were examined for complement-fixing (CF) activity with sera from homotypic infections and from herpes simplex virus (HSV) infections in which a CF antibody titer rise was demonstrated with crude V-Z antigen. The subunit antigens included nucleocapsids, envelopes, a soluble antigen produced from infected culture fluids by sucrose density gradient centrifugation, a soluble antigen produced by reducing the volume of clarified infected culture fluids, a soluble antigen derived from infected cell lysates, a "viral" antigen consisting largely of enveloped particles with a few nucleocapsids, and a cell membrane-associated antigen. None was more suitable than crude V-Z antigen for serodifferentiation of V-Z virus and HSV infections. The envelope antigen, cell membrane antigen, and the soluble antigen prepared by density gradient centrifugation showed little reactivity with sera from varicella and HSV infections, but gave high antibody titers with sera from zoster infections, suggesting that a secondary V-Z virus infection is required to produce an antibody response to these subunit antigens. Patients with varicella and zoster infections and the selected patients with HSV infections all showed significant CF antibody responses to the other V-Z subunit antigens.