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.     

Liposomes as adjuvant for combination vaccines

In the present study tetanus toxoid (TT) loaded liposomes and diphtheria toxoid (DT) loaded liposomes were prepared by reverse phase evaporation method and after combining these two vaccines the potential advantages were investigated. Prepared systems were characterized for the size, shape and entrapment efficiency. SDS-PAGE analysis of TT and DT was also performed. The selected liposomal formulations were administered subcutaneously to Balb/c mice and their immune responses were determined using ELISA after 15, 30, 45 days. After boosting the maximum immune response was observed after 45 days and was found to be 0.831 and 0.749 for TT loaded liposome and DT loaded liposomes respectively. When the mice were immunized subcutaneously with the physical mixture of TT loaded liposomes and DT loaded liposomes the immune response for the combination vaccine was found to be 1.44 and 0.741 for the TT and DT respectively. The result showed that the immune response of TT increased when it was combined with DT in liposomes. This confirms adjuvantcity of DT vis-a-vis immunogenicity. Thus, carrier mediated cocktail vaccination holds promise for clinical applications.     

b型流感嗜血杆菌结合疫苗中载体蛋白质的效力研究

考察b型流感嗜血杆菌(Hib)结合疫苗的载体蛋白质—破伤风类毒素(TT)的免疫原性,为百白破与Hib四联疫苗中TT的使用提供参考。方法:将小鼠随机分为四组,分别注射Hib结合疫苗、Hib与百白混合疫苗、Hib与百白破混合疫苗、破伤风类毒素,比较它们在NIH小鼠体内所诱导产生的特异性抗体水平,并对这四组疫苗进行破伤风效力保护试验。结果表明,破伤风类毒素组,Hib与百白破混合疫苗组刺激的TT抗体水平远大于Hib结合疫苗组和Hib与百白混合疫苗组。效力试验虽四组间没有差异,只说明一定的抗体量就可以对小鼠提供保护。认为Hib结合疫苗中的载体蛋白并不能替代百白破疫苗中的破伤风类毒素。     

In vitro induction of a diphtheria toxoid specific antibody response in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid

In comparison with the presently used potency test for diphtheria vaccine, in vitro examination of the immunogenicity of the vaccine would have great advantages. For this reason in vitro induction of diphtheria toxoid specific antibody synthesis in human peripheral blood lymphocytes cultivated in the presence of diphtheria toxoid was investigated. The results showed that a dose dependent synthesis of diphtheria antibody was induced by adsorbed diphtheria toxoid and combined vaccines containing the diphtheria toxoid component. Plain diphtheria toxoid appeared to be less immunogenic in comparison with adsorbed toxoid. There is some indication that the pertussis component had a stimulating effect on the diphtheria antibody synthesis. In conclusion, these results are promising for in vitro examination of the immunogenicity of diphtheria vaccines. The model will be validated for the routine control of diphtheria vaccine.     

不同蛋白载体的痢疾多糖结合疫苗小鼠免疫原性对比试验

试验中以小鼠为动物模型,对不同蛋白载体的痢疾多糖结合疫苗进行免疫效果观察。3种福氏2a痢疾结合疫苗和3种宋内氏痢疾结合疫苗分别皮下免疫NIH小鼠,同时设置O-SP(O-特异性多糖)对照组,免疫3针,在不同免疫针次间采血,用ELISA测定抗体滴度。单独使用福氏2aO-SP和宋内氏O-SP免疫后,小鼠血清中几乎没有抗LPS IgG抗体产生,而用结合疫苗免疫后,小鼠血清中产生了抗LPS IgG抗体,且第二次、第三次免疫后,小鼠血清中抗LPS IgG抗体水平有显著升高,表明结合疫苗具有加强免疫应答效应。三种不同的痢疾结合疫苗相比较,F2a-O-SP-rEPA结合疫苗较F2a-O-SP-TT结合疫苗和F2a-0-SP—DT结合疫苗的小鼠抗LPS IgG抗体水平高,S-O-SP-rEPA结合疫苗较S-O-SP-TT结合疫苗和S-O-SP—CRM9,结合疫苗的小鼠抗LPS IgG抗体水平高。以rEPA作为载体的痢疾结合疫苗比DT,TT作为载体的痢疾结合疫苗的免疫原性要强。     

A novel Enzyme-Linked Immuno-Sorbent Assay (ELISA) for the quantification of total and free polysaccharide in Haemophilus influenzae b–Tetanus toxoid conjugate vaccines in monovalent and combined vaccine formulations

Current Haemophilus influenzae b conjugate vaccines (Hib), which are made of purified capsular polysaccharide (poly-ribosyl-ribitol-phosphate; PRP) conjugated to a carrier protein, are almost completely evaluated by physico-chemical methods to ensure the integrity and stability of the vaccine and consistency of manufacture of batches. The absence of a potency assay makes the quantification of total PRP content (in SI units) and of % free polysaccharide in final fills or bulk components of Hib vaccines critical release tests for both manufacturers and national control authorities. Here we describe a simple and sensitive Enzyme-Linked Immuno-sorbent Assay (ELISA) which has been developed to quantify total and free PRP content in Hib–TT vaccine alone or when in combination with other vaccines. The assay is robust, specific and highly sensitive.     

Haemophilus influenzae type b-outer membrane protein complex glycoconjugate vaccine induces cytokine production by engaging human toll-like receptor 2 (TLR2) and requires the presence of TLR2 for optimal immunogenicity

Conjugate vaccines consisting of the capsular polysaccharide (PS) of Haemophilus influenzae type b (Hib) covalently linked to carrier proteins, unlike pure PS, are immunogenic in infants and have significantly reduced Hib infections in the United States, but require multiple doses to induce protective anti-PS Ab titers. Hib-meningococcal outer membrane protein complex (OMPC) conjugate vaccine, however, elicits protective anti-PS Ab titers after one dose. We found that OMPC and Hib-OMPC engaged human Toll-like receptor 2 (TLR2) expressed in human embryonic kidney (HEK) cells, inducing IL-8 production, and engaged mouse TLR2 on bone marrow-derived dendritic cells, inducing TNF release. Hib conjugated to the carrier proteins CRM(197) and tetanus toxoid did not engage TLR2 on HEK or dendritic cells. Engagement of TLR2 by Hib-OMPC was MyD88 dependent, as Hib-OMPC-induced TNF production was ablated in MyD88 knockout (KO) mice. Hib-OMPC was significantly less immunogenic in TLR2 KO mice, inducing lower Hib PS IgG and IgM titers compared with those in wild-type mice. Splenocytes from OMPC-immunized TLR2 KO mice also produced significantly less IL-6 and TNF-alpha than those from wild-type mice. Hib-OMPC is unique among glycoconjugate vaccines by engaging TLR2, and the ability of Hib-OMPC to elicit protective levels of Abs after one dose may be related to TLR2-mediated induction and regulation of cytokines produced by T cells and macrophages in addition to the peptide/MHC II-dependent recruitment of T cell help commonly afforded by carrier proteins. TLR2 engagement by an adjuvant or carrier protein may be a useful strategy for augmentation of the anti-PS Ab response induced by glycoconjugate vaccines.     

Influence of carrier proteins on the immunologic response to haptenic antitetrodotoxin vaccine

Tetrodotoxin (TTX) is a haptenic, highly toxic neurotoxin with no specific antidote available yet. Anti-TTX vaccine is being studied for antitoxin development. The effectiveness of the carrier protein in eliciting TTX-specific antibody response was comparatively studied. TTX was conjugated to Tachypleus tridentatus hemocyanin (TTH), Limulus polyphemus hemocyanin (LPH), tetanus toxoid (TT), diphtheria toxoid (DT), and bovine serum albumin (BSA) chemically to form artificial antigens TTH-TTX, LPH-TTX, TT-TTX, DT-TTX, and BSA-TTX, respectively, with which BALB/c mice were immunized, and the antibody response and antitoxic efficacy were detected. The serum anti-TTX antibody response and antitoxic efficacy varied markedly with adopted carrier protein. TTH-TTX elicited the best and BSA-TTX the worst TTX-specific antibody response. The proportion of the immunized mice surviving a 3x lethal dose (LD) dose of TTX challenge was 92%, 75%, 42%, 8%, and 0% for TTH-, TT-, LPH-, DT-, and BSA-TTX conjugates, respectively. The rank order of total efficacy of carrier protein for both anti-TTX antibody response and antitoxic effect was TTH > TT > LPH > DT > BSA. As a result of formaldehyde treatment in coupling of TTX carriers, the relative immunogenicity of TTX vs carrier, that is, the ratio of TTX- to carrier-specific antibody response, evidently varied with respective carrier adopted, in a rank order of TT > BSA > TTH > DT > LPH. The results suggest that the carrier protein used in haptenic TTX vaccine is greatly important in eliciting potent anti-TTX antibody, and both TTH and TT are the preferred carriers for development of excellent experimental TTX vaccine.     

Modulation of carrier-induced epitopic suppression by Bordetella pertussis components and muramyl peptide

Synthetic antigens employed in experimental synthetic vaccines are generally small haptenic peptides. Therefore, effective immunization with these antigens usually requires the use of an immunogenic carrier. Tetanus toxoid has been proposed for use as a carrier in future synthetic vaccines due to its high immunogenicity and acceptance for human use. Previous studies employing standard hapten/carrier systems such as DNP/KLH have demonstrated, however, that an epitope-specific suppression occurs when mice previously primed with carrier are subsequently immunized with an haptenic epitope conjugated to the same carrier. These same studies have shown that Bordetella pertussis vaccine administered at the time of carrier priming abrogates epitopic suppression. In the present investigation, epitopic suppression was studied in a synthetic vaccine model employing tetanus toxoid as a carrier. Results from these studies indicated that mice primed with tetanus toxoid 1 month before immunization with a peptide-tetanus toxoid conjugate exhibited enhanced secondary anti-tetanus toxin responses but decreased anti-peptide responses. Furthermore, injection of pertussis vaccine or purified B. pertussis toxin or endotoxin at the time of carrier priming could block the establishment of epitopic suppression. Administration of B. pertussis components enhanced antibody responses to both the carrier and the synthetic peptides as compared with responses of control animals. In addition, administration of an adjuvant-active nonpyrogenic derivative of muramyl dipeptide. Murabutide, with carrier priming reduced epitopic suppression of anti-peptide responses. B. pertussis toxin or endotoxin administered to mice previously suppressed by carrier priming with the first injection of carrier-peptide conjugate overcame epitopic suppression with resultant titers of anti-peptide antibody equal to or greater than nonsuppressed controls. These results suggest that the use of adjuvants with future synthetic vaccines may contribute the additional advantage of overcoming epitopic suppression, thus permitting the use of common, well-tolerated carrier systems such as tetanus toxoid in synthetic vaccine preparations.     

Correlation of Clostridium botulinum type C antitoxin titers in mink and guinea pigs to protection against type C intoxication in mink

In USA, the potency of commercial vaccines containing Clostridium botulinum type C toxoid is determined by a mink vaccination-challenge assay outlined in the Code of Federal Regulations, Title 9, Part 113.110. A more humane potency test is desired, and this study provides preliminary data in support of a serological assay that correlates post-vaccination antitoxin titers of guinea pigs to vaccine efficacy in mink. Mink and guinea pigs were injected with varying dilutions of a vaccine containing C. botulinum type C toxoid. Blood samples were collected from each animal prior to challenging the mink with type C toxin. Serum antitoxin titers of mink and guinea pigs were measured by a mouse protection test, and the results were compared to the outcome of the toxin challenge in mink. A dose-dependent antitoxin response was observed in guinea pigs vaccinated with the critical dilutions of vaccine bracketing the minimum protective dose in mink. These preliminary data suggest that it may be possible to correlate post-vaccination antitoxin titers in guinea pigs to vaccine efficacy in mink. This correlation could be used as the basis for a more humane potency test for C. botulinum type C toxoids.     

Level of bacterial endotoxins in Haemophilus influenzae type b vaccines conjugated with toxoids (td, Di)

Quantitative evaluation of bacterial endotoxin was performed in the following vaccines: Act-Hib, Hiberix, Hib-Titer. The aim of this study was to assess the accuracy and precision of chromogenic LAL test with S-2423 substrate for this particular biopreparations and after that to determine the amounts of endotoxin as a factor of vaccine safety. Because of the lack of information concerning the presence of endotoxin in Act-Hib vaccine, we also tried to establish the limits for the presence of endotoxin in this type of vaccine. The estimated level of endotoxin was as follows: 110 EU/ml in Act-Hib, 1.64 EU/ml in Hiberix and 2.4 EU/ml in Hib-Titer. The results of this study showed that the amounts of endotoxin was dependent on the molecular size of polysaccharide PRP and on the presence of protein component. The limit of endotoxin presence in Act-Hib vaccine recommended by us is max. 150 EU/ml.     

Carrier-induced epitopic suppression, a major issue for future synthetic vaccines

Synthetic antigens have been shown, in experimental models, to induce protective immunity against a variety of pathogens. These studies have demonstrated that, due to their low immunogenicity, these synthetic antigens required conjugation to carrier molecules. Therefore, the choice of appropriate carriers for human immunization by future synthetic vaccines is a major issue. Tetanus toxoid is generally considered to be an effective potential carrier devoid of side-effects. However, the present study performed in mice with two synthetic vaccine models demonstrates that the immune response against the synthetic epitopes conjugated to tetanus toxoid can be suppressed by pre-existing immunity against this same carrier. Because most humans have been exposed to this antigen, this effect may have important implications for the development of synthetic vaccines.     

Development of a mouse model to estimate the potency of the diphtheria toxoid component of diphtheria-tetanus and diphtheria-tetanus-pertussis vaccines

A mouse model to estimate the potency of the diphtheria toxoid component in diphtheria-tetanus vaccines and diphtheria-tetanus-pertussis vaccines has been developed as an alternative to the conventional method of testing in guinea-pigs. Optimal conditions with regard to dose, route and period of immunization have been standardized. The maximum levels of antitoxin were detected five weeks after vaccination and the s.c. route was found to be optimal. Potency data have been compared with other studies in mouse models and with those obtained by the conventional method in guinea-pigs.     

Importance of the degree of antigen polymerization by detoxification in modulating the immunogenicity of acellular pertussis vaccine

For the acellular pertussis vaccine with a high immunogenicity, the concentration, composition and characteristics of acellular pertussis antigens are the crucial points to be considered. Nevertheless, it has not been proved yet whether or not the polymerization degree, one of the characteristics of formalin-detoxified acellular pertussis antigens, has an influence on vaccine potency. Thus, in the present study, the correlations among detoxification conditions of acellular pertussis bulks, their polymerization degrees and their immunogenicities were examined. In addition, the relative importance of pertussis toxoid in vaccine immunogenicity was also investigated. Results show that a lower lysine concentration during detoxification induces highly-polymerized antigens, the immunogenicity has a great dependency on the polymerization degree of antigens, and also pertussis toxoid has a relatively stronger influence on the immunogenicity than other antigens. Accordingly, in the aspect of the potency of detoxified acellular pertussis vaccine, it can be demonstrated that the polymerization of antigens and its degree are the major factors affecting the immunogenicity along with a relatively high content of pertussis toxoid.     

MF59 and Pam3CSK4 boost adaptive responses to influenza subunit vaccine through an IFN type I-independent mechanism of action

The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSK4) adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in mouse. Although all adjuvants boosted the Ab responses to TT, only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine, we investigated the gene signatures induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is required for adjuvanticity to influenza vaccines, we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate immunity pathways. By contrast, resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb, we confirmed that the adjuvanticity of MF59 and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent.     

不同纯度、剂量白喉类毒素对迟发型超敏反应的影响

研究不同剂量及不同白喉类毒素纯度引起的迟发型超敏反应的状况,用于指导疫苗的生产,提高疫苗的质量。以豚鼠为动物模型,采用迟发型超敏试验法,对原制白喉类毒素、精制白喉类毒素、纯化精制白喉类毒素、高纯度的精制白喉类毒素的剂量与超敏反应试验。试验结果表明,注射白喉类毒素剂量的大小与超敏反应成正相关,与纯度成负相关。剂量越大,超敏反应越强;纯度越高,超敏反应愈弱。     

聚合破伤风类毒素抗原特性的研究

由戊二醛脱毒的聚合破伤风类毒素,经高压液相层析及聚丙烯酰胺凝胶电泳分析,类毒素中多聚体含量占８１．９％以上,多聚体分子量为８００ｋＤ,常规破类多聚体仅占有２．２４％。聚合破类免疫豚鼠后,平均心血抗体单位达２ＩＵ／ｍｌ,常规破类仅为０．７５ＩＵ／ｍｌ（Ｔ＝１３．１５,Ｐ＜０．００１）,聚合破类免疫马匹后所诱发的抗体水平较常规抗原的高。     

Evaluation of reactogenicity and immunogenicity of Bubo-M, the Russian combined vaccine for immunization of adults against diphtheria, tetanus and viral hepatitis B

Bubo-M, the first Russian associated vaccine, was found to have low reactogenicity and high immunogenic potency. The frequency of postvaccinal reactions in the group of persons immunized with Bubo-M (20%) appeared to be considerably lower than among persons who received the combined injection of adsorbed DT toxoid with reduced antigen content and vaccine against hepatitis B (47.7%). Following the course of vaccination the level of anti-HBs considerably exceeded the protective level. Immune response to the diphtheria and tetanus components of Bubo-M exceeded that observed after immunization with absorbed DT toxoid with reduced antigen content (p < 0.05).     

Enhanced potency of individual and bivalent DNA replicon vaccines or conventional DNA vaccines by formulation with aluminum phosphate

DNA vaccines against botulinum neurotoxin (BoNTs) induce protective humoral immune responses in mouse model, but when compared with conventional vaccines such as toxoid and protein vaccines, DNA vaccines often induce lower antibody level and protective efficacy and are still necessary to increase their potency. In this study we evaluated the potency of aluminum phosphate as an adjuvant of DNA vaccines to enhance antibody responses and protective efficacy against botulinum neurotoxin serotypes A and B in Balb/c mice. The administration of these individual and bivalent plasmid DNA replicon vaccines against botulinum neurotoxin serotypes A and B in the presence of aluminum phosphate improved both antibody responses and protective efficacy. Furthermore, formulation of conventional plasmid DNA vaccines encoding the same Hc domains of botulinum neurotoxin serotypes A and B with aluminum phosphate adjuvant increased both antibody responses and protective efficacy. These results indicate aluminum phosphate is an effective adjuvant for these two types of DNA vaccines (i.e., plasmid DNA replicon vaccines and conventional plasmid DNA vaccines), and the vaccine formulation described here may be an excellent candidate for further vaccine development against botulinum neurotoxins.