Marcela A. Manghi

Partial characterization of enterocin MR99 from a corn silage isolate of Enterococcus faecalis

Aims:  To assess the inhibitory activity on Gram‐positive and Gram‐negative bacteria of several species of enterococci recovered from a natural corn silage.

A possible explanation for the discrepancy between ELISA and neutralising antibodies to tetanus toxin

The structure and protective activity of tetanus antibodies elicited in rabbits after whole-cell pertussis diphtheria-tetanus vaccine (DTPw) vaccination was studied. ELISA antibody levels and toxin neutralisation activity (TNT) were measured in individual serum samples. The ratio of symmetric and asymmetric (functionally monovalent) IgG molecules was determined by concanavalin A (Con A) chromatography. This test is based on the fact that the carbohydrate group responsible for the molecular asymmetry has high affinity for the lectin Con A. Asymmetric molecule ratio was observed to increase with immunisation time, as well as differences between TNT and ELISA levels. All serum samples were overestimated by ELISA as compared to TNT assay, in line with the markedly higher proportion of asymmetric molecules which have lower toxin neutralising activity. Protective levels could not be predicted reasonably from ELISA results below 0. 222 IU/ml, because this methodology fails to discriminate between both types of antibodies and only an in vivo serum neutralisation procedure (TNT) reflects the true neutralising serum activity.

Development of an ELISA for measuring the activity of tetanus toxoid in vaccines and comparison with the toxin neutralization test in mice

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure anti-tetanus toxoid antibody levels in immunized guinea-pig sera as a useful alternative to the currently used toxin neutralization test (TNT) in determining the activity of the tetanus toxoid in vaccines. The ELISA was found to measure antibody levels as low as 5.8 x 10(-5) IU/ml. Furthermore, a comparison of the results from ELISA and TNT involving 132 different commercial vaccines showed a very good correlation (r = 0.94, p < 0.001) between antibody levels measured by both methods. The results suggest that the proposed ELISA is a reliable, simple and economical alternative to the TNT in mice for assessing the activity of tetanus toxoids in vaccines.

Binding of immunoglobulins and immune complexes to erythrocytes of vertebrates

Abstract Previous investigations which have referred to red cell Fc receptor in rabbit, guinea pig. sheep and man, have been extended to other vertebrates, such as reptiles, amphibia, birds and mammalians. In these investigations, red cells from different animal species have been analyzed. As ligand DNP-BSA anti-DNP non-precipitating antibody, aggregated IgG by the bis-diazotized benzidine method. IgM 7S and non-modified IgG, IgA and IgM were used. The ligand binding to red cells was detected by Coombs test with specific anti-immunoglobulin serum. The analyzed red cells showed exposed Fc receptor. In the case of human red cells, to render such receptor evident it is necessary to submit the cells to trypsinization. All the analyzed erythrocytes bind IgM 7S, Ab-Ag complex and aggregated IgG, and some of these cells bind non-modified immunoglobulins, although none of them bind IgM 19S. These bindings were inhibited by Fcγ, Fcμ and Ag-Ab complex, but they were not inhibited by Fabγ. When specific anti-immunoglobulin serum was added to red cells with bound IgM 7S or Ag-Ab, the clusters which formed by nucleated erythrocytes were bigger than those obtained with non-nucleated red-cells. In the case of chicken (nucleated) and sheep (non-nucleated) erythrocytes. the interaction between 131 I-IgM s -red cells at equilibrium showed a similar K 0 for both red cells (chicken K 0 = 1.8 × 10 8 L/M; sheep K 0 = 1.1 × 10 8 L/M). The number of Fc receptors by red cell were 1.23 × 10 6 and 1.21 × 10 5 for chicken and sheep erythrocytes, respectively. Taking into account these results, the different behaviour of nucleated and non-nucleated red cells with bound IgM 7S, when they react with anti-IgM antibody, would be the consequence of different avidity for both equilibrated systems and not a particular characteristic of their Fc receptors.

Whole-cell Bordetella pertussis vaccine component modulates the mouse immune response to an unrelated soluble antigen.

Several factors are involved in the selective activation of Th1 or Th2 cells, such as different physical characteristics of antigens and the type of antigen-presenting cells involved in the immune response, among others. To study the influence of a particulate antigen on Th1/Th2 cell differentiation during the immune response to another antigen, we analysed the immune response to tetanus toxoid (soluble antigen) in BALB/c mice immunized with one of the three following vaccines: tetanus and diphtheria toxoids (DT), or DT associated with whole-cell Bordetella pertussis or its soluble antigens (DTPw and DTPa, respectively). Similar total antibody levels were observed for all vaccines. DT vaccine showed a higher IgG1/IgG2a ratio than the similar values observed for DTPw and DTPa vaccines. DT- and DTPa-primed spleen cells showed a Th2 (IL-5) profile while a Th1/Th2 (IFN gamma, IL-5) profile was observed for DTPw. IL-6 was only produced by DTPw-primed cells. Besides, IL-12 levels induced by DTPw were three times higher than the ones induced by both DT and DTPa. Our findings indicate that whole-cell B. pertussis priming modifies the tetanus immune response from Th2 to Th1/Th2 type probably via inflammatory mechanisms. In addition, in the light of conflicting reports regarding the mechanisms of protection induced by DTP vaccines, we studied the pertussis immune response. Only DTPw immunization generated memory T cells capable of proliferating with B. pertussis as an in vitro stimulus. Results might indicate that these cells may not play a key role in protecting against B. pertussis when the host is vaccinated with DTPa.

Non-precipitating antibodies isolated by immunoadsorption

Abstract In previous papers we have eliminated several possibilities regarding the lack of formation of insoluble complexes when antigen was mixed with purified non-precipitating antibody from different mammalian species by immunoadsorption. From the results of this investigation it seems reasonable to assume that in the two antigen binding arms of non-precipitating antibody there exist combining sites which behave differently, expecially with large antigens. This assumption is based on the following: (1) the difference in K 0 values between anti-DNP sheep precipitating and non-precipitating IgG1 is no greater than 1 × 10 1 M −1 ; (2) the bi-modal characteristics of the Scatchard plots when different populations of non-precipitating antibody (isolated by affinity chromatography from the same serum sample) are analyzed; and (3) the formation of precipitating antibody upon random hybridization of non-precipitating antibody half-molecules. The foregoing should enable us to understand why non-precipitating antibody, which does not give rise to insoluble complexes with antigen, can become firmly bound and incorporated into the complexes formed by precipitating antibody.

Serum antibodies to diphtheria-tetanus-pertussis vaccine components in Argentine children.

The Argentine vaccination schedule against diphtheria, tetanus and pertussis (DTP) recommends three doses of DTP vaccine at 2, 4 and 6 months of age, two boosters at 18 months and 6 years, a booster dose of tetanus vaccine every 10 years and two doses during pregnancy. To evaluate the effect of this schedule, antibodies against pertussis toxin (PT) and filamentous hemagglutinin (FHA) and against tetanus and diphtheria toxoids were determined by ELISA in serum samples from children (1 month to 6 years) who received different doses of DPT vaccine: 0 dose (n=50), 1 dose (n=25), 2 doses (n=25), 3 doses (n=55), first and second booster (n=25); 25 pregnant women and their offspring, and 45 adults. High antibody levels against PT (>140 EU/ml) and FHA (>80 EU/ml) were recorded in mothers and in the newborn. Antibody titers against PT increased with the number of doses given and decreased with time. Full protection against tetanus (titers >0.1 IU/ml) was observed in the group of adults (0.37 IU/ml), in mothers (4.4 IU/ml) and their newborn offspring (5.5 IU/ml), and in children after receiving the second dose of DTP vaccine (1.86 IU/ml). The immune status for diphtheria was far lower, as most of the groups lacked adequate protection. After the third dose of DTP vaccine, only 78% of the children had antibody titers above the protective level (0.1 IU/ml). Since antibody levels considered to provide full protection were only achieved after the first booster dose of DTP vaccine, the primary three-dose schedule seems to be insufficient to confer adequate immunity in all vaccinees. Because of the high proportion of non-protected adults, a booster dose of Td vaccine should be considered for this group.SummaryThe Argentine vaccination schedule against diphtheria, tetanus and pertussis (DTP) recommends three doses of DTP vaccine at 2, 4 and 6 months of age, two boosters at 18 months and 6 years, a booster dose of tetanus vaccine every 10 years and two doses during pregnancy. To evaluate the effect of this schedule, antibodies against pertussis toxin (PT) and filamentous hemagglutinin (FHA) and against tetanus and diphtheria toxoids were determined by ELISA in serum samples from children (1 month to 6 years) who received different doses of DPT vaccine: 0 dose (n=50), 1 dose (n=25), 2 doses (n=25), 3 doses (n=55), first and second booster (n=25); 25 pregnant women and their offspring, and 45 adults. High antibody levels against PT (>140 EU/ml) and FHA (>80 EU/ml) were recorded in mothers and in the newborn. Antibody titers against PT increased with the number of doses given and decreased with time. Full protection against tetanus (titers >0.1 IU/ml) was observed in the group of adults (0.37 IU/ml), in mothers (4.4 IU/ml) and their newborn offspring (5.5 IU/ml), and in children after receiving the second dose of DTP vaccine (1.86 IU/ml). The immune status for diphtheria was far lower, as most of the groups lacked adequate protection. After the third dose of DTP vaccine, only 78% of the children had antibody titers above the protective level (0.1 IU/ml). Since antibody levels considered to provide full protection were only achieved after the first booster dose of DTP vaccine, the primary three-dose schedule seems to be insufficient to confer adequate immunity in all vaccinees. Because of the high proportion of non-protected adults, a booster dose of Td vaccine should be considered for this group.

Structure and Protective Capacity of Tetanus and Diphtheria Antibodies Produced During Human Pregnancy and Transferred to New-Born

PROBLEM: The structure and protective activity of antibodies against tetanus (anti‐T) and diphtheria (anti‐D), produced during human pregnancy and transferred to new‐born, was studied.

Development of an alternative method for testing the immunogenicity of diphtheria vaccines

The immunogenicity of the diphtheria component of 73 commercial vaccines from five different manufacturers was tested by the toxin neutralization test (TNT) and the enzyme-linked immunosorbent assay (ELISA) developed in our laboratory. A comparison of the antibody levels measured by both assays showed a very good correlation (r = 0.95, p < 0.001). The results suggest that the proposed ELISA is a reliable, simple and economical alternative to the TNT in guinea pigs. Also, the ELISA was found to measure IgG antibody levels as low as 5.5 x 10(-5) IU ml-1. To evaluate the possibility of accelerating the active immunization during the activity test of vaccines, an alternative schedule using one single human dose was assayed. A very good correlation was observed between the IgG antibody response obtained with this schedule and with the traditional programme. Therefore, the cost and the time required to perform the activity test may be considerably reduced when both the rapid immunization schedule and the ELISA are used.

Pertussis Whole Cell Vaccine: Relation Between Intracerebral Protection in Mice and Antibody Response to Pertussis Toxin, Filamentous Hemagglutinin and Adenylate Cyclase

N:NIH mice were vaccinated according to the WHO recommendations for the potency test with the Second International Standard for Pertussis Vaccine (ISPV). Blood for serological investigation was taken from the animals on day 14 post immunization before intracerebral challenge with Bordetella pertussis 18323 was done. The relationship between anti-pertussis toxin, anti-filamentous hemagglutinin and anti-adenylate cyclase antibody levels as measured by ELISA and protection from intracerebral challenge was studied. The proportion of surviving mice increased in correlation with increasing anti-PT titres; a protective level of 4 ELISA units/ml was found. Such relationship between protection against intracerebral challenge and antibody titres was not found for anti-FHA nor for anti-AC antibodies, thus suggesting that these antibodies do not play an important role in protection in this model. The excellent correlation between anti-PT antibody titres and protection suggests that the measure of anti-PT response could be a useful tool for estimating the potency of whole-cell vaccines. The development of an alternative method for testing the potency of pertussis whole-cell vaccines based on the anti-PT response should be considered.