Vibriolytic IgG immunocyte response of mice after primary and secondary immunization with cholera somatic antigens.

Antibody plaque-forming cells (FC) to the somatic antigens of Vibrio cholerae were enumerated in the spleen of mice after primary and secondary immunization with a heat-killed vaccine prepared from the vibrios. Immunocytes releasing both high efficiency IgM and low efficiency IgG antibody were readily detected using a direct and facilitated plaque procedure in agar gel. Whereas the peak numbers of IgM-PFC after primary immunization occurred on days 12 to 14, the peak IgG-PFC response developed somewhat later (16-18 days). After a second injection of vaccine larger numbers of both IgM- and IgG-PFE appeared in the mouse spleens, with peak responses for both occurring between days 5 and 8. The largest number of IgG-PFC developed in spleens of mice given a second injection of vaccine 6-8 weeks after primary immunization. The dose of killed vibrios used for priming markedly affected both the magnitude and the class of antibody-forming cells appearing during the secondary response; 1--10 mug vaccine was more effective than higher or lower doses for priming the mice to a heightened secondary response. Furthermore, the antigenic specificity of both the IgM- and IgG-PFC appearing after secondary immunization was directly related to the strain of cholera bacilli used for priming. When mice were immunized with the Ogawa strains of cholera most of the secondary PFC after booster immunization with the serologically distinct Inaba strain was directed towards the common antigen shared by both strains and not to the type specific antigen of the Inaba vibrios. The specificity of the anti-vibrio PFC during both the primary and secondary responses was readily demonstrable by inhibition experiments using sonicated or soluble cholera antigens. Prior incubation of these antigens with test spleen cells in the agar gel effictively inhibited development of the vibriolytic plaques, regardless of antibody class. Similar antigen extracts from toher bacteria had no effect. The immunoglobulin nature of the plaques was also demonstrable by inhibition with low dilutions of rabbit anti-mouse globulin serum incorporated into the agar plates prior to testing; both IgM and IgG plaues were inhibited.     

Immunoglobulin M, A, and G antibody response to lipopolysaccharide O antigen in symptomatic and asymptomatic Shigella infections.

The antilipopolysaccharide antibody response in sera obtained from subjects involved in 10 outbreaks of shigellosis occurring in Israeli military field units was determined by an enzyme-linked immunosorbent assay and a passive hemagglutination test. Both tests were found to be sensitive and specific for the diagnosis of shigellosis. A significant antibody response was detected in 73 to 82% of the symptomatic and 48 to 60% of the asymptomatic subjects during the Shigella sonnei and Shigella flexneri outbreaks. Fifty percent of the symptomatic and none of the asymptomatic subjects showed a significant antibody response in the Shigella boydii outbreaks. An examination of the kinetics of the antibody levels over a 10-week period after the onset of disease revealed that immunoglobulin A (IgA) levels were highest 2 weeks after infection and had declined to initial levels within 2.5 months. In contrast, IgG levels at the late convalescent stage were half those measured at early convalescence, still being about twice as high as the initial titers. Although the IgM levels showed a pattern similar to that of IgA, their elevation at the early convalescent stage was less pronounced. We conclude that the detection of an increase in the level of the IgA fraction appeared to be the best indicator for recent symptomatic, as well as symptomatic, infections due to Shigella organisms.     

Immunoglobulin class-specific antibody response in serum, spleen, lungs, and bronchoalveolar washings after primary and secondary sendai virus infection of germfree mice.

Immunoglobulin class-specific antibodies were measured by a solid-phase radioimmunoassay in serum, bronchoalveolar washings (BAW), lung cell lysates, and spleen cell lysates in germfree mice after intranasal (i.n.) and intraperitoneal (i.p.) primary and secondary 10(5), 10(4), and 10(3) mean tissue culture infective doses (TCID(50)) of live parainfluenza 1 (Sendai) virus. The earliest antibody detected in lungs after i.n. virus challenge was immunoglobulin G (IgG), followed by IgM and, lastly, IgA. The local IgA response after both primary and secondary i.n. virus challenge was lowest after the severest infection. It is suggested that the delayed appearance of IgA antibody and the lower response after severe lung damage may be related to a temporary local secretory component-producing cell deficiency. The lungs were a major source of serum IgG antibody after both primary and secondary i.n. virus challenge. Only IgG and IgM antibodies were detectable in lung cell lysates after the i.n. 10(3) TCID(50) secondary response. A secondary response was detected in IgG, IgA, and IgM after secondary i.n. challenge with the other two doses. The lung response to all of primary and secondary i.p. doses of virus was exclusively IgG and IgM. Calculation of radioimmunoassay antibody per microgram of IgG, IgA, and IgM in serum and BAW after both i.n. and i.p. virus challenges showed that, when BAW antibody was present, the ratio in BAW was always higher than that in serum. This finding in the i.n. mice, together with the presence of IgA antibody-containing cells in the lungs, strongly indicates local manufacture and secretion of IgA antibodies in these animals and suggests that the same conclusion could apply to local IgG and IgM antibodies after both i.n. and i.p. challenges.     

HL-A antigens and antibody response after influenza A vaccination. Decreased response associated with HL-A type W16.

We investigated possible associations of HL-A types and antibody-response patterns during clinical trials with a live, attenuated intranasal influenza A vaccine. After vaccination, subjects with HL-A type W16 had, as a group, a mean convalescent-phase hemagglutination-inhibiting antibody titer of 14, which was significantly lower (P less than 0.001) than the mean titer of 36 in subjects without Type W16. Of 25 subjects with a poor antibody response, 32 per cent had HL-A type W16, whereas only 5 per cent with a good response had Type W16. The mean titers in nasal secretions of five W16 subjects at 13 and 30 days were less than 3; in contrast, similar titers of 22 subjects without W16 were 8 and 9 respectively. The results suggest that the lower antibody response in W16 subjects is due to increased cellular resistance to infection rather than to a suppressed immune response because other subjects with W16 had normal antibody responses after vaccination with killed influenza vaccine.     

Development of subtype-specific and heterosubtypic antibodies to the influenza A virus hemagglutinin after primary infection in children

Children undergoing primary infection with an H1N1 or H3N2 influenza A virus developed subtype-specific hemagglutination inhibition antibodies and enzyme-linked immunosorbent assay antibodies to purified hemagglutinin (HA) of the infecting virus subtype. They also developed lower titered ELISA antibodies to the noninfecting H1 or H3 HA and to H8 (an avian strain) HA. Thus, after primary infection with an influenza A virus, children develop enzyme-linked immunosorbent assay, but not hemagglutination inhibition, antibodies reactive with heterosubtypic HAs. These heterosubtypic antibodies could influence the response to infection with other wild-type or attenuated vaccine strains of influenza A virus.     

IgE antibody response to mite antigens in mite infested mice.

Mice infested at birth with the mouse mite Myocoptes musculinus developed positive skin tests to mite antigens at the age of 5 weeks. Serum IgE antibodies directed against mite antigens were first detected at 6 weeks of age and high levels of IgE were present as long as 1 year later. Similar kinetics of IgE formation were observed in mice infected as adults. Mast cell degranulation by mite extract was demonstrated in connective tissue obtained from the skin of mite infested mice.     

Immunoglobulin G and M composition of naturally occurring antibody to type III group B streptococci.

Human sera were examined by an enzyme-linked immunosorbent assay for immunoglobulin G (IgG) and IgM antibodies to purified type III polysaccharide of group B streptococci. The antigen-binding capacity of a reference human serum was determined by a radioimmunoassay, and the total antibody content was determined by quantitative precipitation. The serum was then depleted of IgM and IgA to determine the effect on the antigen-binding capacity. Duplicate samples of 81 sera were tested by the enzyme-linked assay in comparison with reference standard serum. Although levels of IgG antibody were greater in subjects who had carried type III streptococci during pregnancy, concentrations of this antibody were generally low. Only 2 of 28 sera (7%) from parturient subjects and 7 of 25 sera (28%) from adult volunteers contained greater than or equal to 1 microgram of IgG antibody per ml; the mean levels were 0.13 and 0.53 micrograms/ml, respectively. In contrast, 19 of 28 maternal sera (68%) and 22 of 25 (88%) volunteer adult sera contained greater than or equal to 1 microgram/ml of IgM antibody; mean levels were 1.33 and 1.54 micrograms/ml, respectively. The cord serum levels of IgG antibody were almost identical to maternal serum concentrations, whereas IgM antibody was essentially undetected.     

Immunoglobulin G subclass antibody responses in influenza A and parainfluenza type 1 virus infections

Antibody responses in immunoglobulin G1, G2, G3, G4, A (IgA1) and M isotypes were studied in 10 patients with an acute influenza A and in another 10 patients with a parainfluenza type 1 virus infection using radioimmunoassay with standardized monoclonal anti-immunoglobulins. A four-fold or greater increase of antibody in patients have an acute influenza A virus infection, were found in IgG1 (all 10 cases), IgG3 (seven cases), IgG4 (eight cases) and in IgA1 (six cases) whereas IgG2 and IgM responses were observed only in one and three cases, respectively. The antibody titre values were converted to immunoglobulin units by multiplying the titre by a pre-determined correction coefficient compensating for the varying affinity of the individual monoclonal anti-immunoglobulins. These units were then used to calculate the actual proportions of each isotype. In the convalescent phase, 78% of total anti-influenza A antibodies were estimated to be of IgG1 isotype and other immunoglobulin isotypes varied from 3 to 7% of total. Similar results in parainfluenza virus antibodies were obtained with serum pairs from patients with an acute parainfluenza virus infection.     

Immunoglobulin G antibody response to infection with coccoid forms of Helicobacter pylori

An increasing number of studies support a potential role for coccoid forms in Helicobacter pylori infection. Evidence for this was obtained through scanning microscopy, genetic analysis for virulence traits, examination of the presence and activity of key enzymes, and other methods. We studied the serum immunoglobulin G responses to coccoid H. pylori forms by enzyme-linked immunosorbent assay (ELISA) and immunoblotting and compared them with those of bacillary cells. Sera from a total of 295 infected individuals were studied; these included sera from 100 patients with duodenal ulcers, 98 patients with nonulcer dyspepsia, 11 patients with gastroduodenal cancer, and 86 asymptomatic individuals. Initially, we characterized and selected coccoid and bacillary antigenic preparations by one-dimensional (1-D) and 2-D gel electrophoresis and immunoblotting. Data showed that coccoid and bacillary preparations with comparable protein contents have similar patterns in 1-D and 2-D electrophoresis gels and antigenic recognition at blotting. These results revealed that coccoid and spiral antigens in ELISA can equally recognize specific antibodies to H. pylori in sera from infected individuals. The analysis of the spiral and coccoid preparations by Western blotting showed no major differences in antigen recognition. No specific bands or profiles associated with a single gastric condition were identified.     

Comparative antibody response to Salmonella antigens in genetically resistant and susceptible mice.

The ELISA was used to titrate the antibody response in mice inoculated with salmonella antigens. The genetically resistant A/J and susceptible C57BL/6J mice were either infected with the virulent or the avirulent Salmonella typhimurium. Alternatively, they were inoculated either once or twice with the heat-killed salmonella vaccine. No appreciable difference could be detected in the relative ability of these two strains of mice to produce antibodies against the lipopolysaccharide antigens of this pathogen under these four conditions.     

Immunoglobulin G to virus-specific early antigens in congenital, primary, and reactivated human cytomegalovirus infections.

Immunoglobulin G antibody to human cytomegalovirus (CMV)-specific early antigens (EA-Ab) was determined by the immunoperoxidase antibody technique in several cases of congenital, primary, and reactivated CMV infections. Mothers of congenitally infected infants and a group of leukemic children and pregnant women were also studied. In 11 cases of congenital infection, CMV EA-Ab was always associated with CMV excretion whether immunoglobulin M antibody was present or not. Nine mothers of congenitally infected infants had CMV EA-Ab for several months after delivery, but association with CMV elimination was not established when urine and/or saliva were tested for virus isolation. In all nine cases of primary CMV infection, CMV EA-Ab was present, and in five its detection was associated with CMV isolation. In one case, disappearance of EA-Ab occurred when virus excretion ceased. In five cases of reactivated CMV infections, a consistent association between CMV EA-Ab and virus isolation was found. Six of 31 leukemia children had CMV EA-Ab, and virus was isolated from 3 of these. Four of 28 pregnant women showed EA-Ab in their serum, but tests for isolation were not done. These data suggest that CMV EA-Ab is not a marker of a current primary CMV infection, as previously reported, but a marker of an active CMV replication which can take place in primary as well as in congenital and reactivated CMV infections.     

Idiotypic modulation of the antibody response of mice to Echinococcus granulosus antigens.

In this work we analysed the modulation of the antibody response to Echinococcus granulosus antigens via anti-idiotype (Ab2) administration. In a first set of experiments, we determined the antibody response to hydatid cyst fluid antigen (HCFA) of mice immunized with Ab2. Results showed that Ab2 elicited anti-HCFA antibodies in mice that had never been exposed to HCFA before. Moreover, that response was characterized by booster effect, avidity maturation and an inverse correlation between avidity and Ab2 dose. We infected these mice and measured the titre and avidity of anti-HCFA antibodies during 250 days of infection. Lack of avidity maturation was the most important feature observed, suggesting that the parasite (either protoscolex or cyst) could modulate the antibody response of its host. In a second set of experiments, we investigated the presence of regulatory anti-idiotypes in the Ab2 preparation. In this context we treated neonates with different doses of Ab2, immunized them with HCFA 12 weeks later and determined their anti-HCFA titres. A specific and Ab2 dose-dependent suppression of the response to HCFA was observed, suggesting the existence of regulatory anti-idiotypes in the Ab2 preparation. Although this Ab2 could be involved in the regulation of the anti-HCFA response, other anti-idiotypes could also be involved. Our results show that it may be possible to improve the avidity of the anti-HCFA response via the administration of the anti-idiotype Ab. However, the live parasite could successfully revert this effect by mechanisms not yet characterized.     

Immunoglobulin allotypes and IgG subclass antibody response to Pseudomonas aeruginosa antigens in chronically infected cystic fibrosis patients.

Chronic Pseudomonas aeruginosa lung infection is the leading cause of death in patients with cystic fibrosis (CF). Poor prognosis correlates with a high number of anti-pseudomonas precipitins and with high levels of IgG2 and IgG3 anti-pseudomonas antibodies. Reports of several highly significant associations between certain Gm (genetic markers of IgG on human chromosome 14) and Km (k-type light chain determinants on chromosome 2) phenotypes and immune responsiveness to various antigens suggest that allotype-linked immune response genes do exist in man. Furthermore correlation between Gm types and IgG subclass levels has been reported. A group of 143 CF patients were investigated (31 non-infected and 112 chronic infected). The IgG subclass antibodies to three different P. aeruginosa antigens (P. aeruginosa standard antigen (St-Ag), alginate and LPS) were determined. Immunoglobulin allotypes were determined by haemagglutination inhibition. Samples were typed for G1m(1,2,3, and 17), G2m(23), G3m(5,21), and Km(1,3). Statistical analysis of our data demonstrate that IgG3 anti-pseudomonas antibody levels and Gm markers are related. IgG3 antibody levels to all investigated P. aeruginosa antigens are significantly higher in sera homozygous for Gm(3;5), somewhat lower in heterozygous sera, and significantly lower in sera homozygous for Gm(1,2,17;21). We suggest that genetic differences between the patients may explain the present differences in subclass patterns.     

Type- and subtype-specific detection of influenza viruses in clinical specimens by rapid culture assay.

A rapid culture assay which allows for the simultaneous typing and subtyping of currently circulating influenza A(H1N1), A(H3N2), and B viruses in clinical specimens was developed. Pools of monoclonal antibodies (MAbs) against influenza A and B viruses and MAbs HA1-71 and HA2-76, obtained by immunizing mice with the denatured hemagglutinin subfragments HA1 and HA2 of influenza virus A/Victoria/3/75, were used for immunoperoxidase staining of antigens in infected MDCK cells. MAb HA1-71 reacted exclusively with influenza A viruses of the H3 subtype, while MAb HA2-76 reacted with subtypes H1, H3, H4, H6, H8, H9, H10, H11, and H12, as determined with 78 human, 4 swine, and 10 avian influenza virus reference strains subtyped by the hemagglutination inhibition test. To determine if the technique can be used as a rapid diagnostic test, 263 known influenza virus-positive frozen nasal or throat swabs were inoculated into MDCK cells. After an overnight incubation, the cells were fixed and viral antigens were detected by immunoperoxidase staining. Influenza A viruses of the H1 and H3 subtypes were detected in 31 and 113 specimens, respectively. The subtypes of 10 influenza A virus-positive specimens could not be determined because they contained too little virus. Influenza B viruses were detected in 84 specimens, and 25 specimens were negative. We conclude that this assay is a rapid, convenient, non-labor-intensive, and relatively inexpensive test for detecting, typing, and subtyping influenza viruses in clinical specimens.     

Detection of cells producing surface antigen-specific antibody to influenza viruses.

B cells producing antibodies to influenza virus antigens were detected and quantitated by a hemolytic plaque assay. Responses of mice after primary infection and immunization with influenza viruses were measured and compared with responses after secondary immunization. The B-cell responses were specific and differentiated between A and B influenza viruses and between different subtypes of A influenza viruses. Responses to closely related influenza A virus strains of the H3N2 subtype cross-reacted but could also be differentiated. Cells secreting antibody to either of the virus surface antigens (hemagglutinin and neuraminidase) could be separately enumerated. Evidence that immunoglobulin G- secreting cells are detected in the assay without the use of facilitating anti-immunoglobulin G sera is presented.     

Biological Activities of Rabbit Immunoglobulin M and Immunoglobulin G Antibodies to Pseudomonas aeruginosa

Immunoglobulin (Ig)M and IgG antibodies, prepared in the rabbit against the protective antigen of Pseudomonas aeruginosa P4, were compared as to their biological activities in vitro and in vivo. In vitro biological activities of these antibodies were determined by passive hemagglutination, bactericidal, and opsonophagocytic tests. Increased effectiveness of IgM over IgG on a molar basis was demonstrated in all of these tests. However, in mouse protection tests, in which the purified globulins were injected intraperitoneally 4 hr prior to challenge with P. aeruginosa suspended in hog gastric mucin, IgM anticapsular antibody was found to be less effective than IgG antibody. The exact mechanism whereby IgG antibody exerts more protective ability than IgM antibody is still unknown. We present evidence to suggest that the difference in activity between the two classes of antibody is due to the ability of the IgG antibody to enter the bloodstream more rapidly than the IgM antibody and also to the ability of IgG to diffuse rapidly through the tissues of the organs.     

HLA antigens and the antibody response after vaccination to hepatitis B

The aim of our work was the search for immunogenetic factors that influence the antibody response to HBs antigen. We analyzed the HLA-A, -B, and -DR antigen frequencies in 19 seropositive to HBs and 28 seronegative to HBs healthy persons finding an elevated frequency of B5 in the seropositive group (p value 0.037). After vaccination with Hevac B Pasteur vaccine of the seronegative persons, the low antibody response was associated with B13 (p value 0.041). An association between local side reactions to the vaccine and HLA A3 and B35 were also found (p values 0.042 and 0.022 respectively). The presented p values are not significant after correction for the number of antigens tested and for this reason our findings require confirmation in an independent study.     

Natural influenza A virus infection of mice elicits strong antibody response to HA2 glycopolypeptide

Two influenza viruses, A/Dunedin/4/73 (H3N2) and A/Mississippi/1/85 (H3N2) were adapted to BALB/c mice. Groups of BALB/c mice were intranasally (i.n.) infected with either single dose of particular virus strain or successively with both virus strains and titers of serum antibodies against influenza virus antigens ("influenza virus antibodies") and those just against the HA2 part of hemagglutinin (HA) ("HA2 antibodies") were determined. Successive infection with virus strains Dunedin and Mississippi in interval of 21 days led to the strong increase of the proportion of anti-HA2 antibodies in sera, though whole antiviral titres remained in general unchanged. These observations confirmed that the HA2 glycopolypeptide (gp) part of influenza virus HA is very strong immunogen in natural infection.