Studies on the potency test of HB vaccine by the parallel line assay method in guinea pigs

The parallel line assay method was applied on the potency test of HB vaccine in guinea pigs. The applicability of the method was investigated to obtain information on the possible effect of inactivation process of HBs antigen, on the magnitude of adjuvant effect of aluminum compound and so on. The precision of the method was found to be comparable to those of diphtheria and tetanus toxoids.     

Quantitative estimation of diphtheria and tetanus toxoids. 5. Comparative assays in mice and in guinea-pigs of two diphtheria toxoid preparations

Two freeze-dried international reference diphtheria toxoids of different origin were compared in biological assays in guinea-pigs and mice under different adjuvant conditions. When the antigenic content in the two toxoids was used as denominator for determination of relative potency, that is to say quantitation of immunogenic power per unit amount of antigen, the design of the animal assay proved to have a major influence. Similar observations have been made previously also for tetanus vaccines. It is concluded that diphtheria vaccines as well as tetanus vaccines can hardly be quantitated unambiguously using the currently recommended potency assays in animals. A new scheme for control of toxoid vaccine production is suggested, with more emphasis on the control of the bulk purified toxoid, which would make the release of final products more simple and rapid.     

Antibody responses elicited by a polyvalent vaccine containing synthetic diphtheric, streptococcal and hepatitis peptides coupled to the same carrier

Three synthetic peptides copying fragments of the diphtheria toxin, the M protein of the streptococcus type 24 and the hepatitis B virus surface antigen (HBs) have been conjugated together to the tetanus toxoid. This polyvalent vaccine has been administered to mice. High antibody titers were obtained against the three antigens. No cross-reactivity could be observed between them as demonstrated by the ability of each peptide to inhibit only the antibodies against the natural M protein and the synthetic M protein peptide indicated that the avidity of the antibodies raised against a monovalent streptococcal vaccine were identical to those raised following injection of the polyvalent vaccine. Antibodies raised against the polyvalent streptococcal vaccine were also protective as shown by opsonophagocytic assays.     

Comparative study of the immunoglobulins and specific antibodies in the blood of rabbits immunized with ADTP vaccine and ADT-anatoxin with normal and reduced antigen contents

Adsorbed DPT vaccine and adsorbed DT toxoids with normal and reduced antigen content were used for the immunization of rabbits. The levels of IgM and IgG and the dynamics of antibodies to diphtheria and tetanus toxins and to Bordetella pertussis in the blood sera of the animals were studied in the postvaccinal period (on days 15 and 34). This study revealed that the reduction of the antigen content of adsorbed DT toxoid to 5 Lf of diphtheria toxoid and 5 binding units of tetanus toxoid did not decrease the capacity of the preparation for increasing the levels of IgG and IgM, antibodies to diphtheria and tetanus toxins in the sera of the rabbits. The reduced content of these toxoids in adsorbed DPT vaccine did not affect its capacity for inducing the enhanced synthesis of IgG, antibodies to diphtheria and tetanus toxins, while the production of IgM and IgA remained unchanged. At the same time an increase in the titers of antibodies to B. pertussis in the animals was less pronounced than that observed after the injection of commercial adsorbed DPT vaccine. Additional investigations are necessary in order to establish the protective potency of the pertussis component in adsorbed DPT vaccine with the reduced content of toxoids and to find out the optimum antigenic composition for this preparation.     

Use of adsorbed diphtheria-tetanus (DT) toxoid containing different doses of antigens in booster immunizations of children

To decrease the antigenic load in booster immunizations of children against diphtheria and tetanus, the immunological effectiveness and reactogenicity of different doses of both antigens (i. e 1 Lf and 1 BU; 2.5 Lf and 2.5 BU; 5 Lf and 5 BU) were studied. The study revealed that all these doses of the preparation were practically nonreactogenic: the total of systemic reactions was 0.9%. The study of the immunological effectiveness of adsorbed DT toxoid with reduced antigen content revealed the advantage of the commercial dose of the preparation (5 Lf of diphtheria antigen and 5 BU of tetanus antigen) over such doses of diphtheria and tetanus antigens as, respectively, 1 Lf and 1 BU; 2.5 Lf and 2.5 BU. Still, this advantage disappeared as early as 6 months after booster immunization. The results of these investigations indicate that booster immunization of children aged 6 years and over may be made with lower doses of adsorbed DT toxoid with reduced antigen content.     

Comparative immunologic effectiveness of variants of the DTP vaccine

The immunological effectiveness of two batches of adsorbed DPT vaccine, the batch with the normal content of antigens (control) and the batch with the content of diphtheria and tetanus toxoids reduced to 20 Lf/ml and 5 BU/ml respectively (test batch), has been studied under the conditions of controlled trial. As a result, the reduction of the antigenic content of adsorbed DPT vaccine has been found to exert no negative influence on the immunological effectiveness of the diphtheria, tetanus, and pertussis components of this preparation under the conditions of the new immunization schedule.     

Reactogenic properties of a DTP vaccine with a reduced antigen dosage

The reactogenic properties of batches of adsorbed DPT vaccine with the normal content of antigens and with the content of diphtheria and tetanus toxoids reduced, respectively, to 10 Lf and 5 BU per immunization dose have been studied under the conditions of a controlled epidemiological trial. The reduced antigenic content of adsorbed DPT vaccine decreased the number of vaccinal reactions 1.8 times, as well as the intensity of their manifestations.     

Simultaneous Immunization with B.C.G., Diphtheria-tetanus,and Oral Poliomyelitis Vaccines in Children Aged 13-14

The simultaneous administration of B.C.G. vaccine, diphtheria-tetanus toxoid aluminium hydroxide adsorbed vaccine, and oral poliovaccine was studied in 628 children aged 13-14 years between 1966 and 1969 in Newham, London. The efficacy of these vaccines was unaffected by administering them at the same time; routine simultaneous administration is considered justified when organizational difficulties prevent the attainment of high immunization rates with the vaccines given separately. No adverse reactions to B.C.G. or oral poliomyelitis vaccines took place, but 8% of children had moderately severe local reactions after diphtheria-tetanus aluminium hydroxide adsorbed vaccine, which were attributed to diphtheria toxoid.Serological studies showed the need for immunization against diphtheria, tetanus, and poliomyelitis at 13-14 years of age. Because of the adverse reactions to diphtheria toxoid, however, simultaneous administration of tetanus toxoid aluminium hydroxide adsorbed, oral poliomyelitis, and B.C.G. vaccines only is recommended at present.An “adult type” diphtheria-tetanus toxoid might overcome the problem of reactions, though in two to three years'' time most children aged 13-14 years will have received diphtheria-tetanus-pertussis vaccine in infancy and reinforcement might then be accomplished by a small intradermal dose of the currently available fluid diphtheria-tetanus vaccine.Continued serological studies of diphtheria and tetanus antitoxins and polio antibody are necessary to determine the future need for reinforcement of immunity; such studies should become an essential part of the surveillance of the community immunization programme.     

The level of postvaccinal immunity to diphtheria, tetanus and poliomyelitis in relation to the number of inoculations]

The work was aimed at the comparative study of the intensity of immunity to diphtheria, tetanus and poliomyelitis, depending on the number of injections of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine and poliomyelitis vaccine, for the purpose of finding out the possibility of reducing the antigenic load given to children without diminishing the intensity of immunity. To determine the level of immunity to diphtheria and tetanus, 1900 children under school age were serologically studied by the micromethod in the passive hemagglutination test. The intensity of immunity to poliomyelitis virus was studied in 333 children by means of the neutralization test. The immunogenic properties of the diphtheria component of adsorbed DPT vaccine were found to be less pronounced than those of the tetanus component of the vaccine, which made it impossible to reduce the antigenic load by decreasing the number of vaccinations against diphtheria. The results of the study of postvaccinal immunity to poliomyelitis suggest that during the first and second year of life the course of vaccination against poliomyelitis may be reduced to 3 injections.     

The role of the O antigen in adjuvant activity of lipopolysaccharide

Adjuvant activities of isogenic Salmonella enterica, serovar Typhimurium, O-6,7 and O-4,5,12 lipopolysaccharide (LPS), lipid A and Bordetella pertussis LPS were compared by immunizing groups of mice subcutaneously with diphtheria and tetanus toxoid vaccine alone or mixed with one of the LPS derivatives. Five weeks later the mice were bled and the tetanus and diphtheria antibodies in the sera were measured. All the LPS derivatives efficiently increased the antibody responses when compared to the vaccine alone, but the mannose-rich O-6,7 LPS and lipid A were significantly more potent than O-4,5,12 LPS and B. pertussis LPS. We conclude that the quality of the O antigen influences the adjuvant activity of LPS.     

Comparative quantitation for the protein content of diphtheria and tetanus toxoids by DC protein assay and Kjeldahl method.

DPT, a combination vaccine against diphtheria, tetanus and pertussis is available since many years and still continued in the national immunisation schedule of many countries. Although highly potent, reactions to DPT vaccine are well known, mainly attributed to the factors like Pertussis component, aluminum adjuvant and lower purity of tetanus and diphtheria toxoids. The latter most important aspect has become a matter of concern, specially for the preparation of next generation combination vaccines with more number of antigens in combination with DPT.Purity of toxoid is expressed as Lf (Limes flocculation) per mg of protein nitrogen. The Kjeldahl method (KM) of protein nitrogen estimation suggested by WHO and British Pharmacopoeia is time consuming and less specific. Need has been felt to explore an alternative method which is quick and more specific for toxoid protein determination. DC (detergent compatible) protein assay, an improved Lowry's method, has been found to be much more advantageous than Kjeldahl method.     

Antibody status to poliomyelitis,measles, rubella,diphtheria and tetanus,Ontario, 1969-70: deficiencies discovered and remedies required.

A serologic survey was made in 15 health unit areas, testing some 5000 individuals in the age groups 4 to 6, 11 to 13, 15 to 17 and 23 to 45 years. Two types of serious deficiency were found. Only 65% of children 4 to 6 years old had antibodies to all three types of poliovirus, the antibodies being due almost entirely to immunization with Salk vaccine. Even in children who had had six or more doses only 74% had antibodies to the three types. The high percentage of students 11 to 13 and 15 to 17 years old with poliovirus antibodies can be attributed largely to natural infection and to Sabin vaccine in the mass campaign of 1962, as well as to Salk vaccine. In children who had received Sabin vaccine as well as Salk vaccine a very high level of immunity was found. The immunity of the school-age population will decline to an insufficient level unless Sabin vaccine is used after immunization with Salk vaccine. Of children 4 to 6 years old 18% had no diphtheria antitoxin and 6% had no tetanus antitoxin. Even in those who had had six or more doses of the antigens 5% had no diphtheria antitoxin and 1 to 2% had no tetanus antitoxin. This apparently refractory state is probably due to the use of unadsorbed toxoids, and it is clear that adsorbed toxoids should be used. In the adults, diphtheria antitoxin was found in only 55% and tetanus antitoxin in only 38%.     

The partially degraded lipopolysaccharide of Burkholderia cepacia and ornithine-containing lipids derived from some Gram-negative bacteria are useful complex lipid adjuvants

The partially degraded lipopolysaccharide of Burkholderia cepacia (LPSdegr) and the ornithine-containing lipids were purified from some bacteria. The substances were developed as complex lipid adjuvants, because they have weak toxicity and are able to activate the immune systems of the living body. After various toxoid antigens such as pertussis toxoid, diphtheria toxoid and tetanus toxoid were mixed with the complex lipid adjuvants, the mixtures were administered to mice subcutaneously. Antitoxoid IgG antibody titers in the serum were measured several times over 3 months. The efficacy of the LPSdegr as adjuvant was almost as high as that of the ornithine-containing lipids, and it was almost equal to that of the aluminum hydroxide adjuvant (Alum), which is generally used as a vaccine adjuvant.     

Reactions to and immunogenic properties of the combined vaccine Bubo-M for immunization of children against diphtheria, tetanus and viral hepatitis B

Bubo-M, the first Russian combined vaccine, was found to have low reactogenicity. The difference between the number of postvaccinal reactions in the group of children immunized with Bubo-M (25.9%) and those in the group of children who had been simultaneously injected into different sites of the body with ADS-M toxoid (adsorbed DT toxoid with reduced antigen content) and hepatitis B vaccine (26.7%) was not statistically significant. Following immunization a considerable increase in the level of diphtheria and tetanus antibodies (p < 0.005) was observed in all children (100%), the level of HBs antibodies in the group of children immunized with Bubo-M (the geometric mean titer: 13,721 IU/l) essentially exceeding that observed in the control group injected with ADS-M toxoid and hepatitis B vaccine (the geometric mean of the titer: 2,441 IU/l). Bubo-M was duly registered and allowed for industrial production and medical use.     

Alternative diphtheria, tetanus and whooping cough immunization schedule to evoke a Th2 tetanus and a Th1 pertussis immune response

In a previous study, using BALB/c mice, we found that while diphtheria (D), tetanus (T) and whooping cough (Pw, whole-cell Bordetella pertussis) immunization induces a Th1/Th2 tetanus response and memory T cells able to proliferate in response to in vitro stimulation with B. pertussis, DTPa immunization induces a Th2 tetanus immune response and no memory T cells that recognize B. pertussis as stimulus. Considering that a pro-inflammatory cytokine production is not necessary for protection against tetanus and therefore should be avoided, an alternative DTP immunization schedule with minimal Pw exposure was assessed in order to obtain a Th2 tetanus response and a Th1 pertussis response. BALB/c mice were primed with DT vaccine at day 0, with Pw vaccine at day 14 and boosted with DTPa vaccine at days 21 and 28. A control group was inoculated with saline. Antibodies against B. pertussis surface antigens, tetanus and diphtheria toxoids were produced by mice. Spleen cells stimulated in vitro with B. pertussis produced IL-6 and IFNgamma. Only IL-5 was produced by cells in response to tetanus toxoid stimulation. These results are in line with the low IgG1/IgG2a ratio for pertussis antibodies compared with those corresponding to tetanus and diphtheria. The immunization protocol presented herein succeeded in producing tetanus and pertussis immune responses of Th2 and Th1 type, respectively. In contrast to previous results obtained with DTPw immunization, no IL-12 production was observed. Our findings provide direct evidence that an immunization protocol with an interval of 14 days between DT and Pw primings, followed by DTPa boosters, can induce appropriate immune responses against DTP vaccine antigens.     

Liposomes in immunology: Evidence that their adjuvant effect results from surface exposition of the antigens

The immune responses against human serum albumin (HSA) and bovine gamma globulin (BGG) were studied in rabbits after intravenous injections of various preparations of these antigens. Antigens were injected free in saline, coated on “empty” liposomes or both coated on liposomes, and entrapped in their inner compartments. The earlier established adjuvant effect of the liposomes was confirmed for both antigens. Although the amount of antigen entrapped in the liposomes was much higher than the amount coated on their outer surfaces, liposomes containing the antigen both in their inner compartments and on their outer surface showed no stronger adjuvant effect than “empty” liposomes coated with the antigen only. The results support the hypothesis that the adjuvant effect of liposomes is mediated by antigens exposed on the outer surfaces of the liposomes. Suggestions are made for the use of liposomes as a practical immunoadjuvant with definite advantages over many other adjuvants.     

Bacterial genome variability and its impact on vaccine design

The majority of currently available successful vaccines induce host responses against antigens that are highly conserved in the targeted pathogens. The diphtheria, tetanus, and pertussis vaccines confer protection by inducing neutralizing antibodies to the conserved bacterial toxins that are the major virulence factors. The Hemophilus influenzae B vaccine induces responses to conserved epitopes in the sugar structure of the bacterial capsular polysaccharide. However, the efficacy of more recently developed vaccines is limited by antigen variation, which also presents a challenge for future vaccine development. This review will explore bacterial genome variability and its impact on vaccine development.     

Evaluation of a guinea pig model to assess interference in the immunogenicity of different components of a combination vaccine comprising diphtheria, tetanus and acellular pertussis (DTaP) vaccine and haemophilus influenzae type b capsular polysaccharide conjugate vaccine.

A guinea pig model to assess the immunogenicity of a combination vaccine containing diphtheria, tetanus and acellular pertussis (DTaP) vaccine and Haemophilus influenzae type b (Hib) capsular polysaccharide conjugated to tetanus toxoid (HibT) was evaluated comparatively with the mouse immunogenicity test to study the effect of combining these antigens on the immunogenicity of various components. The immunogenicity test in mice was performed by subcutaneous injection of groups of 10 animals twice at an interval of four weeks with 1/10 of a single human dose of various formulations of combination vaccines, DTaP or HibT vaccine. The animals were bled at 4 and 6 weeks and IgG or total antibodies to various components were determined by ELISA or RIA. The guinea pig immunogenicity model included groups of animals injected subcutaneously twice at an interval of six weeks with 1.5 times the single human dose of various formulations. The animals were bled at 4, 6 and 8 weeks and serum samples were tested for antibodies to various components by ELISA, RIA and/or neutralization tests. Additionally, potency of tetanus and diphtheria components was assessed as per the US Food and Drug Administration's regulations. Aluminium phosphate (AIPO(4)) adsorbed HibT vaccine or HibT as a combination with AIPO(4)adsorbed DTaP vaccine showed significant increases in IgG antibodies to tetanus toxin in mice as well increased tetanus antitoxin levels in guinea pigs as compared to soluble HibT vaccine. In general, combining DTaP and HibT vaccines did not affect the antibody levels to tetanus and diphtheria toxoids whereas DTaP-HibT combination vaccine elicited significantly lower IgG antibodies to pertussis toxin and filamentous haemagglutinin than DTaP vaccine alone, particularly after first injection. Mice showed similar Hib antibody responses for the combination and HibT alone whereas guinea pigs consistently showed lower anamnestic responses to Hib for combination formulations than for HibT alone. Reducing the amount of HibT and/or tetanus toxoid in the combination formulations reduced this suppression of Hib antibody response in guinea pigs. Suppression of Hib antibody response in combination vaccines has also been reported from recent clinical trials. Based on the results from this study, it appears that the guinea pig model may be able to predict the human response to various components of combination vaccines.     

Oral immunogenicity of tomato-derived sDPT polypeptide containing Corynebacterium diphtheriae, Bordetella pertussis and Clostridium tetani exotoxin epitopes

DPT vaccine, designed to immunize against diphtheria, pertussis, and tetanus, has been shown to be effective in humans. Nevertheless, dissatisfaction with the whole-cell preparations is due to the reactogenicity, which has to lead to the development of new safer formulations. Previously, we described the expression in tomato of a plant-optimized synthetic gene encoding the recombinant polypeptide sDPT, containing mainly immunoprotective epitopes of the diphtheria, pertussis and tetanus exotoxins and two adjuvants. In this study, we examined whether the ingestion of tomato-derived sDPT protein induces specific antibodies in mice after three weekly doses scheme. A positive group immunized with DPT toxoids was included. Specific antibody levels were assessed in serum, gut and lung. Sera tested for IgG antibody response to pertussis, tetanus and diphtheria toxin showed responses to the foreign antigens; interestingly, the response to diphtheria epitope was similar to those observed in the positive group. We found higher IgG1 than IgG2a responses in serum. A modest IgG response was observed in the tracheopulmonary fluid. High response of IgA against tetanus toxin was evident in gut, which was statistically comparable to that obtained in the positive group. The levels of response in these groups were higher than those in mice that received wild-type tomato. These findings support the concept of using transgenic tomatoes expressing sDPT polypeptide as model for edible vaccine against diphtheria, pertussis, and tetanus.     

Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.