Murine antibody response to oral infection with live aroA recombinant Salmonella dublin vaccine strains expressing filamentous hemagglutinin antigen from Bordetella pertussis.

Two plasmids which express either nearly intact or truncated filamentous hemagglutinin (FHA) from Bordetella pertussis and which are marked with a tetracycline resistance (Tcr) gene were transformed into Salmonella dublin SL1438, an aroA deletion mutant intended for use as an attenuated oral vaccine against salmonellosis. These S. dublin recombinants, when fed to mice, induced serum immunoglobulin, immunoglobulin M (IgM), and sometimes IgA antibody responses to FHA and S. dublin. In addition, IgA antibodies against FHA were found in gut wash fluids. S. dublin carrying pDB2300, a multicopy plasmid encoding truncated FHA protein, induced a better antibody response than did S. dublin carrying pDB2000, a low-copy-number plasmid encoding full-sized FHA. Administration of tetracycline to mice enhanced the stability of recombinant plasmids, and tetracycline-treated mice developed higher anti-FHA titers. Although neither strain examined is suitable for use in a human oral vaccine, these data demonstrated that an immune response against B. pertussis FHA could be induced by oral administration of live attenuated recombinant strains of S. dublin and suggested that development of a live oral attenuated vaccine against pertussis may be possible.     

The haemagglitinating activity of different anti-DNP antibody populations when dinitrophenylated sheep and human red cells are used as agglutinogen

By means of serial precipitations, using 1/20th of the antigen (DNP - BSA) dose necessary for optimal antibody precipitation, it is possible to separate precipitating antibodies from anti-DNP sera corresponding to different antibody species. If the remaining serum is passed through a column of immunoadsorbent, a population of non-precipitating antibodies, capable of co-precipitating with the precipitating antibodies is retained. In the serum which has passed through the immunoadsorbent there remains a third population of antibodies lacking in precipitating and co-precipitating activity. Its quantification can only be achieved by saturation at high free hapten concentration. The average Ko and a values are as follows: precipitating antibodies. Ko = 1.14 × 10⁵ L/M, a = 0.72; non-precipitating (co-precipitating) antibodies isolated by immunoadsorption, Ko = 3.16 × 10⁴ L/M, a = 0.51; and non-precipitating antibodies isolated from the serum lacking in precipitating and co-precipitating antibodies, Ko = 1.05 × 10³ L/M, a = 0.69.By means of passive haemagglutination and the use of dinitrophenylated sheep and human red cells as agglutinogen it is possible to differentiate the three populations of antibodies. The precipitating antibodies agglutinate both types of red cells; the non-precipitating antibodies separable by immunoadsorption only agglutinate sheep red cells; while the third population of antibodies is totally lacking in haemagglutinating activity. This suggests that haemagglutination detects only those antibodies capable of firm union with antigen at two combination sites, at least in the case of red cells without exposed Fc receptors (human red cells), while if the red cells possess such receptors (sheep red cells) haemagglutination takes place even with antibodies capable of a firm union to the antigen with at least one of its combination sites (non-precipitating antibodies retained by immunoadsorption or co-precipitating antibodies) as previously demonstrated (Margin et al., 1974). The antibodies which do not precipitate with the antigen and which are not capable of fixation to immunoadsorbents lack haemagglutinating activity.The difference between the titers of haemagglutination obtained with dinitrophenylated sheep and human red cells gives an approximate estimate of the content of precipitating and non-precipitating anti-DNP antibodies separable by immunoadsorption present in a sample.     

Accuracy of single radial hemolysis test for rubella immunity when internal reference standards are used to estimate antibody levels.

The accuracy and reproducibility of antibody levels obtained by single radial hemolysis with six internal reference sera were evaluated. The test was performed in a clinical laboratory for routine assessment of immunity to rubella infection over a period of 1 year. A linear relationship exists between the antibody titer (expressed in log dilution) and zone diameters. In 43 of 44 test runs the correlation coefficient of the standard curve was over 0.990. Prediction limits of 95% around the curve showed that on replication of the test, zone diameters could be found within less than half a doubling-dilution step. The antibody level can thus be determined more accurately by the single radial hemolysis test than in the conventional hemagglutination inhibition test. This is particularly important in assessing immunity when antibody titers are low, since the hemagglutination inhibition test is less reliable then. The use of standard sera calibrated international units would render results of different laboratories comparable and allow standardization at threshold values.     

Time to peak serum antibody response to influenza vaccine in the elderly.

The earliest time at which serum antibody peaks following administration of influenza virus vaccine in elderly persons is not clearly defined. We compared the time intervals of 2 and 4 weeks after vaccination. A commercial trivalent vaccine containing the hemagglutinins of influenza viruses A/Texas/36/91(H1N1), A/Shangdong/9/93(H3N2), and B/Panama/45/90 was used. The hemagglutination inhibition antibody titers at 2 weeks after vaccination were identical to the hemagglutination inhibition antibody titers at 4 weeks for all three vaccine components.     

Time to earliest peak serum antibody response to influenza vaccine in the elderly.

The earliest time at which serum antibody levels peak following administration of an influenza virus vaccine in elderly persons is not clearly defined. We compared the time intervals of 1 and 2 weeks after vaccination. A commercial trivalent vaccine containing the hemagglutinins of influenza viruses A/Texas/36/91 (H1N1), A/Johannesburg/33/94 (H3N2), and B/Harbin/7/94 was used. The hemagglutination inhibition (HAI) antibody titers at 1 week after vaccination were significantly lower than the HAI titers at 2 weeks postvaccination for all three vaccine components.     

Evidence that inhibitor(s) are formed which may interfere with the growth of revertant colonies in the Ames Salmonella and the E. coli tryptophan reverse mutation assays when strictly anaerobic conditions are used.

Spontaneous and chemically induced revertant colonies were not observed on plates when a strictly anaerobic environment and anaerobically prepared media were used to perform the Ames histidine reversion assay with each of eight different Salmonella strains. A similar effect was observed when the E. coli tryptophan reverse mutation assay was performed under strictly anaerobic conditions. We provide evidence here that under anaerobic conditions growth inhibitor(s) are formed by the S. typhimurium and E. coli bacteria when the limited histidine and tryptophan, respectively, are depleted from the medium. The inhibitor(s) are nonspecific and inhibit the growth not only of prototrophic bacteria but also of the inhibitor-producing bacteria as measured by neutralized supernatants of growth-limiting minimal liquid cultures. Inhibitor(s) are also formed in stationary phase cultures of Salmonella and E. coli in minimal liquid medium supplemented with excess histidine and tryptophan, respectively. These results suggest that inhibitor formation under anaerobic conditions is a physiological phenomenon which interferes with at least two reverse mutation assays. Whether or not it also interferes with the reverse mutagenesis process remains to be determined.     

Immunological unresponsiveness to protein antigens in rabbits: II. The nature of the subsequent antibody response

Rabbits made immunologically unresponsive by neonatal administration of HSA, HGG or BSA were given a course of intravenous injections of the respective antigens, adsorbed on alum, after a lapse of 13–27 months since the last administration of antigen. 8/12 responded to HSA, 4/5 to HGG, 9/10 to BSA, as judged by immune elimination of antigen, but this was delayed in onset and slow compared with that in previously untreated rabbits. The antibody formed was small in quantity and usually failed to precipitate with antigen.

The sedimentation coefficients of ¹³¹I-labelled antigens, in the presence of excess antibody, were measured by ultracentrifugation through a sucrose density gradient. These showed that only small complexes were formed in some of the non-precipitating antisera. In one instance the diffusion coefficient of the complex was also measured, by a technique based on diffusion through agar gel. The calculated molecular weight of the complex, 330,000 indicated the presence of only two combining sites on the antigen.

Combination of the anti-HSA sera with an HSA fragment was also measured. Whereas the amount of the fragment bound by ordinary hyperimmune anti-HSA sera was about one-fifth the HSA bound, the amounts bound by the test sera were relatively much less. Some non-precipitating sera failed to bind the fragment, although they bound HSA.

These findings indicate that following neonatally induced immunological unresponsiveness the capacity to respond to antigen returns piecemeal in respect of different parts of the antigenic mosaic, and that it may be severely restricted. The theoretical implications are discussed.

     

Immunoregulation of the in vitro anti-HBs antibody synthesis in chronic HBsAg carriers and in recently boosted anti-hepatitis B vaccine recipients.

We report a study on immunoregulation of in vitro antibody to hepatitis B surface antigen (anti-HBs) synthesis induced by pokeweed mitogen (PWM) from peripheral blood mononuclear cells (PBMC) in chronic hepatitis B surface antigen (HBsAg) carriers and in 'high responders', (anti-HBs RIA ratio greater than or equal to 20 in serum), recently boosted with anti-hepatitis B vaccine. Anti-HBs was detected in 11 days PBMC supernatants (SN) from 24 out of 36 'high responders', but in none from 31 chronic HBsAg carriers, despite detectable amounts of polyclonal IgG and antibody to hepatitis B core antigen (anti-HBc) were produced. The lack of anti-HBs production by chronic HBsAg carriers did not seem to be determined by suppressor influences because T lymphocytes from the majority of chronic HBsAg carriers, co-cultured with 'high responders' PBMC did not suppress anti-HBs production. Co-cultures between HBsAg carriers T4 positive (helper/inducer) cells and allogenic 'high responder' non-T cells produced anti-HBs antibody, indicating that HBsAg carrier T cells are not deficient in this allogenic helper function under PWM stimulation. Allogenic cocultures between HBsAg carrier non-T cells and 'high responder' T4 positive cells failed in anti-HBs production: a specific B lymphocyte defect might be involved in the lacking anti-HBs synthesis in chronic HBV patients. Antigen-induced specific anti-HBs synthesis experiments indicate that B cells themselves seem to be the target for HBsAg-induced suppression of anti-HBs antibody response.     

Analysis of immunity to infection with Salmonella typhimurium in outbred mice. I. Requirement of the antibody to non-O antigen for protection in mice that are not protected by the RNA-rich vaccine.

Two outbred mouse strains, ddY and CF1, were tested for their ability to be protected against infection with Salmonella typhimurium by several types of salmonella vaccines. These strains have the same levels of innate susceptibility to S. typhimurium, and also have the same capacity to develop delayed-type hypersensitivity (DTH) to salmonella antigens. Both the crude ribosomal fraction (CRF) and live-cell vaccines conferred acquired resistance on both strains, characterized by greater responses of T cells to salmonella antigens. Mice of the ddY strain were also protected by the purified transfer RNA (tRNA) vaccine, which was free of O antigens, but CF1 mice were not, despite the presence of T-cell reactivity with salmonella antigens. Neither strain was protected by the phenol-water-extracted lipopolysaccharide (LPS). The tRNA-immunized CF1 mice were protected by transfer of antiserum to CRF, but not by transfer of anti-LPS antibody. This antiserum to CRF, however, did not transfer acquired resistance into non-immune mice of either strain. These observations suggest that CF1 mice may require an antibody to another non-O antigen existing in CRF to develop acquired resistance, and that stimulation of the defence system by tRNA may be essential to the development of acquired resistance in CF1 mice.     

Immunological responses to Salmonella R antigens. The bacterial cell and the protein edestin as carriers for R oligosaccharide determinants.

Responses in rabbits to heat-killed Salmonella minnesota R mutants (chemotypes Ra, Rc and Re) were heterogeneous with respect to the amounts and specific haemagglutinin activities (SHAA) of IgM and IgG antibodies produced to each mutant. Amounts of antibodies in IgM and IgG fractions of sera were determined by quantitative precipitation. For comparison, antibodies were also isolated using an R oligosaccharide-specific immunoadsorbent and quantitated spectrophotometrically. SHAA (haemagglutinating units/mg antibody) of IgG antibodies were similar for all three mutants. In contrast, the Ra mutant induced IgM antibodies with the highest SHAA, while the Re mutant induced IgM antibodies 10-fold lower in activity. The ratio of the amount of IgM/IgG produced was approximately 1/1 for both the Ra and the Rc mutants, while the ratio for the Re mutant was about 1/2. Salmonella R oligosaccharide-protein conjugates (chemotypes Rb2, Rc and Re) were prepared, and the responses to these antigens were compared with those to the heat-killed mutants. The conjugates were specific for the given chemotype, and they were strongly immunogenic when incorporated into Freund's complete adjuvant and administered intramuscularly. Haemagglutinin titres were relatively high, but amounts of antibodies were considerably reduced when the conjugates were administered intravenously without adjuvant. Rabbits immunized with the conjugates in the same manner as with heat-killed R mutants produced predominantly IgM responses in all three cases.     

The effect of antilymphocytic antibody on the humoral immune response in different strains of mice: II. The response to sheep erythrocytes

The effect of an horse anti-mouse thymocyte globulin (ALG) preparation on the primary and secondary immune responses to sheep erythrocytes (RBC) was studied in 2–3-month-old male A/HeJ, C57B1, BALB/c, DBA/1, CBA and C3H mice. A wide variation in the primary and secondary responses of the individual strains was noticed but in all cases these were significantly suppressed by ALG pretreatment. The capacity of the ALG to suppress the primary immune response was not overcome by increasing the antigen dose from 3 × 10⁷ to 3 × 10⁹ sheep RBC.     

The influence of particulate carriers and of mineral oil in adjuvants on the antibody response in rabbits to human gamma globulin.

A single inoculation of rabbits with human gamma globulin, administered with polystyrene latex particles and mineral oil as adjuvant, gave rise to at least the same level and duration of antibody formation as could be achieved with classical Freund's adjuvant made "complete" with killed mycobacteria.     

The effect of anti-lymphocytic antibody on the humoral immune response in different strains of mice: I. The response to bovine serum albumin

The effect of a single batch of horse anti-mouse thymocyte globulin on the humoral immune response has been extensively investigated in 10–12-week-old male mice of six inbred strains. The preparation under test readily suppressed the primary immune response of A/HeJ, C57B1, DBA/1, CBA and C₃H mice to 0.1, 1.0 and 10.0 mg doses of alum precipitated bovine serum albumin (alum BSA). However, at the doses used it generally failed to significantly suppress the primary immune response of BALB/c mice to 0.1 and 1.0 mg doses of this antigen. Furthermore, the effectiveness of the suppression was somewhat diminished by the incorporation of bordetella pertussis organisms into the alum BSA inoculum. The product also suppressed the secondary immune response to alum BSA in C₃H mice and C57B1 mice but, at the same doses, was without significant effect on the secondary responses of A/HeJ and CBA mice.     

表达hCGβ的重组乳酸杆菌经口接种小鼠产生抗hCGβ体液免疫应答

目的：用表达hCGβ的重组乳酸杆菌活疫苗经口接种不同品系小鼠产生抗hCGβ免疫应答。方法：用10^8、10^9、10^10个重组乳酸杆菌Lb．hCGβ灌服6～8周龄雌性BALB／c和C57BL／6小鼠。3周后用相同剂量加强免疫1次。间接ELISA检测初次免疫后2～8周血清和阴道灌洗液抗hCGβIsG和IgA抗体滴度。流式细胞仪分析B和T细胞的增殖情况，ELISpot计数抗hCGβIsG和IsA抗体分泌细胞克隆数。结果：不同品系小鼠经口腔接种10^9、10^10Lb．hCGβ可诱导相近的免疫应答；而10。诱导的抗体滴度较低。在加强免疫后，10^9和10^10Lb．hCGβ诱导的抗血清结合hCGβ抗原的能力〉100ng／n11。Lb．hCGβ口腔接种后，BALB／c小鼠子宫和阴道的B细胞增殖；C57BL／6小鼠子宫及阴道的抗体分泌细胞克隆数较脾脏明显增多（P〈0．05）。结论：重组乳酸杆菌活疫苗Lb．hCGβ可经口接种诱导有效的抗hCGβ免疫应答，产生的应答强度与免疫剂量呈正相关。     

The effect of antilymphocytic antibody on the humoral immune response of different strains of mice. V. Effects on the class and subclass of antibody produced, and the antibody responses of congenitally athymic mice

Antilymphocytic globulin (ALG) depressed the antibody responses of mice to SRBC as measured in terms of splenic PFCs of IgM, IgG1, IgG2a and IgG2b specificity. This effect was apparent over a range of antigen doses. ALG also reduced the antibody responses of congenitally athymic mice to SRBC and SIII. The possible mode of action of ALG is discussed in the light of these, and other, observations.     

Lentogenic field isolates of Newcastle disease virus isolated in Canada and Hungary are identical with the vaccine type used in the region.

Lentogenic field isolates of Newcastle disease virus were examined by restriction enzyme analysis of RT-PCR products generated from the matrix protein gene that discriminates between strains LaSota and B-1, the two most widely used lentogenic vaccine viruses. Isolates were derived from regions where, exclusively or predominantly, only one type of vaccine was employed. Viruses collected in Hungary for two decades were exclusively of LaSota-type while the Canadian collection predominantly included B-1, which corresponded to the vaccine types used in the regions. Isolation of vaccine type lentogenic viruses from unvaccinated flocks supports the occurrence of area spread of these lentogenic viruses.     

Comparison of Newcastle disease vaccine administered as powder or liquid in relation to the serum antibody response and adverse vaccinal reactions in broilers

Liquid spray and aerosol mass vaccination of poultry have several drawbacks, such as uncontrolled deposition of vaccine particles in the respiratory tract and vaccine virus inactivation by formation and evaporation of droplets. These may be addressed by using dry powder vaccines with defined particle size distribution targeting the upper (primary vaccination) or the entire respiratory tract (booster vaccination). Therefore, a coarse Newcastle disease (LZ58 strain) powder vaccine was administered to specified pathogen free (SPF) broiler hens to compare the antibody response and adverse vaccinal reactions with those induced by a coarse liquid spray and a fine liquid aerosol. Groups of 40 broilers each housed in isolators were vaccinated at 4 days of age and intratracheally inoculated with Escherichia coli (strain 506) at 11 days of age. Adverse vaccinal reactions were evaluated by measuring body weight gain and mortality between 4 and 11 days of age and between 11 and 18 days of age, and by recording colibacillosis lesions at 18 days of age. The antibody serum response was measured at 18 days of age by the haemagglutination inhibition test. Despite the relative low initial vaccine virus loss and narrow particle size distribution of the powder vaccines in comparison with their liquid counter parts, no significant differences (P > 0.05) regarding adverse vaccinal reactions and antibody response were observed between broilers vaccinated with the powder vaccines or with their liquid counterparts.     

The effect of antilymphocytic antibody on the humoral immune response in different strains of mice: III. The response to type III pneumococcus polysaccharide

The effect of a single batch of horse anti-mouse thymocyte globulin on the immune response to type III polysaccharide antigen has been investigated in 2–3-month-old male A/HeJ, C₅₇B1, BALB/c, DBA/1, CBA and C₃H mice. In almost all cases the intraperitoneal administration of 5 mg of this material on days –4 and –2 significantly suppressed the immune response to 0.1, 1.0 and 5.0 μg of antigen injected i.v. on day 0. Further studies undertaken in BALB/c mice indicated that effective suppression of the immune response to type III polysaccharide antigen could also be achieved by injecting 5 mg of this product (i.p.) some 15–30 minutes prior to antigenic challenge. Preliminary cell reconstitution studies in antilymphocytic antibody-treated CBA mice indicate that the ability to respond to type III polysaccharide can be partially restored by the injection of syngeneic thymocytes, bone marrow cells or spleen cells.     

Genetic control of the immune response to myoglobin. IX. Overcoming genetic control of antibody response to antigenic sites by increasing the dose of antigen used in immunization

Previously, it was reported that the immune response to myoglobin (Mg) was under genetic control, with the response to each site being under separate Ir-gene control. Here we have investigated the effect of antigen dose on the control of the antibody response to the five antigenic sites of sperm-whale Mb to determine whether or not the overcoming of genetic control by antigen dose has a uniform effect on all five antigenic sites. The antibody response to sperm whale myoglobin (Mb) and its five antigenic was measured in the following inbred strains of mice, C57BL/6J, AKR and SWR/J. These strains of mice are low responders to Mb following immunization with 50 micrograms, responding only to site 4. After immunization with 200 micrograms Mb: C57BL/6J mice are high responders to Mb and respond to antigenic sites 1, 3, 4 and 5; AKR mice are high responders to Mb and respond to antigenic sites 1 and 4; SWR/J mice are high responders to Mb and respond to all five antigenic sites. It was concluded that the genetic control of the immune response to Mb and its synthetic antigenic sites is dependent on antigen dose. Also, these studies have enabled us for the first time to separate the response to site 1 from the response to site 2 and thus have conclusively established that sites 1 and 2 are controlled by separate Ir-genes.