Stimulating Role of Toxoids-laden Liposomes in Oral Immunization against Diphtheria and Tetanus Infections

Liposomes have been produced by injecting an ether solution of a mixture of lecithin and cholesterol into a diluted solution of prewarmed diphtheria and tetanus toxoids followed by elimination of the stream of ether vapour by vacuum.In a preliminary study, adjuvant effects of liposomes on the systemic and mucosal immune response have been studied. When a mixture of diphtheria toxoid (DT) and tetanus toxoid (TT) entrapped in liposomes were administered parenterally or orally in rabbit, a significant rise of specific antibodies against both toxoids was noticed. In monkeys receiving a mixture of DT and TT entrapped in liposomes orally, the antibody response after two and three ingestions of this product was mild but when liposomes containing toxoids were adsorbed with aluminium hydroxide in a similar experiment, a significant rise in the specific antibody response in monkey against both toxoids was recorded. Adult volunteers, similarly receiving a mixture of DT and TT, entrapped in liposomes and adsorbed with aluminium hydroxide have shown a significant rise in specific circulating antitoxins. In order to compare the efficacy of this technique of human oral immunization with the previous method, whereby a plant medicinal seed (LRS) was used as adjuvant in oral immunization of man, a second group of volunteers were simultaneously and similarly treated as suggested previously. The comparative results are discussed in the present report.     

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.     

Idiotypic determinants on human T cells and modulation of human T cell responses by anti-idiotypic antibodies

The role of idiotypic anti-idiotypic interactions in the regulation of the human T cell response to tetanus toxoid (TT) antigen was examined in three subjects. Rabbit anti-idiotypic (anti-Id) antisera were raised against IgG (Fab')2 anti-TT obtained 7 to 10 days after booster immunization with TT. F(ab')2 fragments of rabbit-anti-Id IgG were used in conjunction with fluorescein-conjugated goat anti-rabbit Ig in an indirect immunofluorescence assay to determine the frequency of Id-positive cells in T cell-enriched preparations. This frequency was 24, 29, and 38 per 10,000, respectively, in the three subjects studied. Significant contribution of contaminating B cell to fluorescence-staining was ruled out by capping experiments using goat anti-human Ig (GAHIG) and by double staining experiments using rhodamine-conjugated GAHIG. Absorption of anti-Id antisera with Epstein Barr virus (EBV)-transformed B cell lines from the IgG (Fab')2 anti-TT donor, but not with EBV-B cell lines from unrelated donors, removed their reactivity with the T cells. Rabbit anti-Id IgG caused minimal proliferation (two-threefold) of T cells and had no effect on T cell proliferation in response to TT antigen when added to the cultures. Preincubation of T cells for 48 hr with rabbit anti-Id IgG (Fab')2, but not with preimmune rabbit IgG (Fab')2, resulted in the generation of antigen-specific suppressor cells that inhibited T cell proliferation in response to TT, but not in response to diphtheria toxoid (DT). These cells also inhibited the synthesis of IgG anti-TT in response to in vitro stimulation with TT antigen, but not the synthesis of IgG anti-DT in response to DT antigen. Adsorption of T cells over plates coated with rabbit anti-Id IgG (Fab')2 enhanced the proliferative response of the T cells to TT, but not to DT antigen, and enhanced the helper activity of the T cells for the in vitro synthesis of IgG anti-TT but not of IgG anti-DT antibodies. These results suggest that idiotypic-anti-idiotypic interactions play a role in the human T cell response to antigen.     

Immune response in adult immunized with adsorbed DT-M anatoxin with reduced antigen content and Imovax-DT-adulte vaccine

The comparative study of immune response after immunization of adults with adsorbed DT toxoid with reduced antigen content and Imovax-DT-adulte vaccine, as well as the safety of these preparations, was made. The study revealed that immunization of adults with adsorbed DT toxoid having reduced antigen content, made in two injections, and the injection of Imovax-DT-dulte vaccine, as well as the successive injection of these preparations, produced the same the levels of antitetanus immunity. Antidiphtheria immunity, evaluated by the number of seroconverted to diphtheria persons following two injections immunization was similar for the two preparations, while the level of antidiphtheria antibodies was higher in persons immunized with adsorbed DT toxoid. The immune stratum index was rather high among persons aged 16-29 years. This age group exhibited the highest number of persons, seropositive to both diphtheria and tetanus. Both vaccine preparations, adsorbed DT toxoid with reduced antigen content and Imovax-DT-adulte vaccine, were found to be equally capable of inducing autoimmune reactions in the vaccinees, detected by laboratory methods.     

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.     

The induction and suppression of in vitro IgG anti-tetanus toxoid antibody synthesis by human lymphocytes stimulated with tetanus toxoid in the absence of in vivo booster immunizations

This report presents a new approach that by-passes booster immunizations with tetanus toxoid (TT) before in vitro studies of antibody (Ab) production. The methodology for optimal TT-induced synthesis of specific IgG anti-tetanus toxoid Ab (IgG anti-TT) by peripheral blood mononuclear cells (PBMC) from randomly selected TT immune individuals without recent booster immunizations is described. PBMC from most normal immune subjects could be repeatedly induced to produce in vitro IgG anti-TT; PBMC from subjects with high TT titers are not required for this new approach. This approach uses high cell concentrations in multiple replicate microcultures and TT washout to obtain optimal IgG anti-TT synthesis. Washed cultures produced more Ab than nonwashed cultures (p less than or equal to 0.005). The readdition of TT (2.5 to 250 ng/ml) to the culture media after washout of TT on day 4 suppressed specific Ab formation, whereas diphtheria toxoid added at comparable doses did not inhibit specific Ab formation. Suppression of antibody synthesis mediated by T cells could be induced by TT per se, and was not due to binding of synthesized Ab to TT in the latter 8 days of culture. In addition, suppression could not be induced in the first 4 days of culture by IgG anti-TT, IgG, or IgM. This approach permits the analysis of antigen-specific regulatory circuits in the steady and activated immune states, and the evaluation of in vivo and in vitro effects of biologic response modifiers on specific Ab production.     

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。     

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.     

The specific prophylaxis of diphtheria in adults with adsorbed DT-m anatoxin]

The dependence of immune response on commonly observed immunity characteristics prior to immunization has been established on the basis of the study of the kinetics of immune response in adults receiving injections of adsorbed diphtheria-tetanus (DT) toxoid with reduced antigen content, both for routine immunization and on epidemiological indications. The necessity of the practical use of immunological screening for the differentiated approach to the choice of a suitable preparation (adsorbed diphtheria toxoid, adsorbed DT toxoid or adsorbed DT toxoid with reduced antigen content) and immunization schedule for adults, especially in epidemic foci, has been substantiated.     

TLR7 imidazoquinoline ligand 3M-019 is a potent adjuvant for pure protein prototype vaccines

Cancer vaccines, while theoretically attractive, present difficult challenges that must be overcome to be effective. Cancer vaccines are often poorly immunogenic and may require augmentation of immunogenicity through the use of adjuvants and/or immune response modifiers. Toll-like receptor (TLR) ligands are a relatively new class of immune response modifiers that may have great potential in inducing and augmenting both cellular and humoral immunity to vaccines. TLR7 ligands produce strong cellular responses and specific IgG2a and IgG2b antibody responses to protein immunogens. This study shows that a new TLR7 ligand, 3M-019, in combination with liposomes produces very strong immune responses to a pure protein prototype vaccine in mice. Female C57BL/6 mice were immunized subcutaneously with ovalbumin (OVA, 0.1 mg/dose) weekly 4x. Some groups were immunized to OVA plus 3M-019 or to OVA plus 3M-019 encapsulated in liposomes. Both antibody and cellular immune responses against OVA were measured after either two or four immunizations. Anti-OVA IgG antibody responses were significantly increased after two immunizations and were substantially higher after four immunizations in mice immunized with OVA combined with 3M-019. Encapsulation in liposomes further augmented antibody responses. IgM responses, on the other hand, were lowered by 3M-019. OVA-specific IgG2a levels were increased 625-fold by 3M-019 in liposomes compared to OVA alone, while anti-OVA IgG2b levels were over 3,000 times higher. In both cases encapsulation of 3M-019 in liposomes was stronger than either liposomes alone or 3M-019 without liposomes. Cellular immune responses were likewise increased by 3M-019 but further enhanced when it was encapsulated in liposomes. The lack of toxicity also indicates that this combination may by safe, effective method to boost immune response to cancer vaccines.     

The role of liposome charge on immune response generated in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63)

Liposomes as a lipid-based system have been shown to be an effective adjuvant formulation. In this study, the role of liposome charge in induction of a Th1 type of immune response and protection against leishmaniasis in BALB/c mice was studied. Liposomes containing rgp63 were prepared by Dehydration-Rehydration Vesicle (DRV) method. Neutral liposomes consisted of dipalmitoylphosphatidylcholine and cholesterol. Positively and negatively charged liposomes were prepared by adding dimethyldioctadecylammonium bromide (DDAB) or dicetyl phosphate (DCP) to the neutral liposome formulation, respectively. Female BALB/c mice were immunized subcutaneously with negatively, positively charged or neutral liposomes encapsulated with rgp63, rgp63 in soluble form or PBS, three times in 3 week intervals. The extent of protection and type of immune response generated were studied in different groups of mice. The group of mice immunized with rgp63 encapsulated in neutral liposomes showed a significantly (P < 0.01) smaller footpad swelling upon challenge with Leishmania major compared with positively or negatively charged liposomes. The mice immunized with neutral liposomes also showed a significantly (P < 0.01) the lowest splenic parasite burden, the highest IgG2a/IgG1 ratio and IFN-γ production and the lowest IL-4 level compared to the other groups. The results indicated that a Th1 type of immune response was induced in mice immunized with neutral liposomes more efficiently than positively charged liposomes and conversely negatively charged liposomes induced a Th2 type of immune response.     

Dys-regulation of effector CD4+ T cell function by the V3 domain of the HIV-1 gp120 during antigen presentation.

It was recently demonstrated that the semiconserved domain of the V3 region of the HIV-1 surface glycoprotein gp120 can induce an activation-apoptosis phenomenon to memory CD4+ cells from healthy individuals. Studying the effects of V3 on the interaction of antigen presentation between monocyte-derived macrophages and resting memory CD4+ T cells, we observed that V3 affects both cell populations. Macrophages exposed to composite liposomes containing V3 on the surface and tetanus toxoid (TT) as the recall antigen entrapped in the aqueous phase (lipoV3/TT liposomes) were able to activate CD4+ T cells during primary stimulation, but not after restimulation nine days later. Unstimulated macrophages or macrophages exposed to soluble TT responded to second stimuli, lipoV3/TT liposomes, and soluble TT in activating CD4+ T cells. Soluble TT-activated CD4+ T cells could be restimulated by soluble TT but not by lipoV3/TT liposomes, whereas lipoV3/TT liposome-activated CD4+ T cells became unresponsive to a second stimulus. These results show that resting memory CD4+ cells activated by macrophages presenting the recall antigen together with V3 become unresponsive to restimulation.     

Diphtheria-tetanus overimmunization in children with no records: can it be prevented?

A pilot study was undertaken to assess the validity of two new tests for predicting the immune response of Toronto schoolchildren with no acceptable evidence of prior administration of diphtheria or tetanus toxoid to a routine booster injection of diphtheria and tetanus (DT) toxoid. The tests, an inexpensive enzyme-linked immunosorbent assay (ELISA) fingerprick test for tetanus antibodies and a modification of the Schick skin test for susceptibility to diphtheria, were administered before the booster injection. One week later the ELISA test was repeated and the result of the modified Schick test read. On both occasions a diphtheria microneutralization assay was done for "gold standard" evidence of prior exposure to diphtheria toxoid or toxin. The results were used to determine the sensitivity and specificity of a single prebooster tetanus ELISA test or a modified Schick test for predicting which children with no records could be safely protected with only one DT booster dose instead of the primary series of three or four doses usually given to such children. Only 6 of the 34 subjects (18%) were totally without prior exposure to tetanus toxoid. Two of the six (6% of 33 subjects) appeared to mount a primary immune response to diphtheria toxoid as well. An initial ELISA titre of 0.01 IU/ml or lower correctly identified all six children needing a full series of tetanus toxoid (sensitivity for a primary immune response 100%) and falsely identified only 3 of 28 immune children as needing the series (specificity for immunity 89.3%). The modified Schick test appeared to have even greater accuracy for identifying children needing a full series of diphtheria toxoid. However, its use, entailing the costs of an extra nurse visit, would have prevented only seven more children from receiving an unnecessary full series of diphtheria toxoid than use of the baseline tetanus ELISA test alone.     

Combined Poly(Lactide-Co-Glycolide) Microspheres Containing Diphtheria Toxoid for a Single-shot Immunization

To develop a single-shot vaccine containing diphtheria toxoid (DT) with a sufficient immune response, poly(lactide-co-glycolide) (PLGA) microspheres were prepared by water-in-oil-in-water double emulsification and solvent extraction techniques using low or high-molecular-weight PLGA (LMW-MS or HMW-MS). Stearic acid (SA) was introduced to HMW-MS (HMW/SA-MS) as a release modulator. Mean particle sizes (dvs, μm) varied between the prepared microspheres, with LMW-MS, HMW-MS, and HMW/SA-MS having the sizes of 29.83, 110.59, and 69.5 μm, respectively; however, the protein entrapment and loading efficiency did not vary, with values of 15.2–16.8 μg/mg and 61–75%, respectively. LMW-MS showed slower initial release (~?2 weeks) but faster and higher release of antigen during weeks 3~7 than did HMW-MS. HMW/SA-MS showed rapid initial release followed by a continuous release over an extended period of time (~?12 weeks). Mixed PLGA microspheres (MIX-MS), a combination of HMW/SA-MS and LMW-MS (1:1), demonstrated a sufficient initial antigen release and a subsequent boost release in a pulsatile manner. Serum antibody levels were measured by ELISA after DT immunization of Balb/c mice, and showed a greater response to MIX-MS than to alum-adsorbed DT (control). A lethal toxin challenge test with MIX-MS (a DT dose of 18 Lf) using Balb/c mice revealed complete protection, indicating a good candidate delivery system for a single-shot immunization.     

Immune response of mice of different inbred lines to Clostridium oedematiens anatoxin

Mice belonging to a number of inbred strains were immunized intradermally with Cl. oedematiens alpha-toxoid. The immunization was repeated 30 days later. On the 20th and the 30th days after the first injection and on the 10th day after the second one the antibody level against the toxoid was determined in the blood of mice by the passive hemagglutination test. The maximum response to the primary immunization was observed in the mice of the C3H strain, and the minimum one--in mice of the DBA/2 strain; the difference was more than 30-fold. The rest of the strains used in the test (A,CBA, BALB/c, AKR, CC57BR) displayed an intermediate level of the immune response. The differences reduced after the repeated immunization. The immune response to this antigen in mice is supposed to be genetically controlled.     

Assessing immune competence in pigs by immunization with tetanus toxoid

Immune competence can be tested by challenging organisms with a set of infectious agents. However, disease control requirements impose restrictions on the infliction of infections upon domestic pigs. Alternatively, vaccinations induce detectable immune responses that reflect immune competence. Here, we tested this approach with tetanus toxoid (TT) in young domestic pigs. To optimize the vaccination protocol, we immunized the pigs with a commercial TT vaccine at the age of 21 or 35 days. Booster immunizations were performed either 14 or 21 days later. TT-specific antibodies in plasma as well as lymphoproliferative responses were determined both 7 and 14 days after booster immunization using ELISA and lymphocyte transformation tests, respectively. In addition, general IgG and IgM plasma concentrations and mitogen-induced proliferation were measured. The highest TT-specific antibody responses were detected when blood samples were collected 1 week after a booster immunization conducted 21 days after primary immunization. The pigs’ age at primary immunization did not have a significant influence on TT-specific antibody responses. Similarly, the TT-specific proliferative responses were highest when blood samples were collected 1 week after booster immunization, while age and time of primary and booster immunization were irrelevant in our setup. While general IgG and IgM plasma levels were highly age dependent, there were no significant age effects for TT-specific immune responses. In addition, mitogen-induced proliferation was independent of immunization as well as blood sampling protocols. In summary, our model of TT vaccination provides an interesting approach for the assessment of immune competence in young pigs. The detected vaccination effects were not biased by age, even though our data were acquired from immune systems that were under development during our tests.     

Mannosylated Niosomes as Adjuvant-Carrier System for Oral Mucosal Immunization

The aim of the present study was to develop mannosylated niosomes as oral vaccine delivery carrier and adjuvant for the induction of humoral, cellular, and mucosal immunity. Tetanus toxoid (TT) loaded niosomes composed of sorbiton monostearate (Span 60), cholesterol, and stearylamine were prepared by the reverse-phase evaporation method. They were coated with a modified polysaccharide o-palmitoyl mannan (OPM) to protect them from bile salts caused dissolution and enzymatic degradation in the gastrointestinal tract and to enhance their affinity toward the antigen presenting cells of Peyer's patches. Prepared niosomes were characterized in vitro for their size, shape, entrapment efficiency, ligand binding specificity, and stability in simulated gastric fluid and simulated intestinal fluid. OPM-coated niosomes were found to more stable in simulated gastrointestinal conditions. The immune stimulating activity was studied by measuring serum IgG titer, IgG2a/IgG1 ratio in serum, and sIgA levels in intestinal and salivary secretions following oral administration of niosomal formulations in albino rats. The results were compared with alum-adsorbed TT following oral and intramuscular administration, and it was observed that OPM-coated niosomes produced better IgG levels as compared to plain uncoated niosomes and alum-adsorbed TT upon oral administration. Oral niosomes also elicited a significant mucosal immune response (sIgA levels in mucosal secretions). The developed formulations also elicited a combined serum IgG2a/IgG1 response, suggesting that they were capable of eliciting both humoral and cellular response. The study signifies the potential of OPM-coated niosomes as an oral vaccine delivery carrier and adjuvant. The proposed system is simple, stable, and cost-effective and may be clinically acceptable.     

Mannosylated niosomes as adjuvant-carrier system for oral mucosal immunization

The aim of the present study was to develop mannosylated niosomes as oral vaccine delivery carrier and adjuvant for the induction of humoral, cellular, and mucosal immunity. Tetanus toxoid (TT) loaded niosomes composed of sorbiton monostearate (Span 60), cholesterol, and stearylamine were prepared by the reverse-phase evaporation method. They were coated with a modified polysaccharide o-palmitoyl mannan (OPM) to protect them from bile salts caused dissolution and enzymatic degradation in the gastrointestinal tract and to enhance their affinity toward the antigen presenting cells of Peyer's patches. Prepared niosomes were characterized in vitro for their size, shape, entrapment efficiency, ligand binding specificity, and stability in simulated gastric fluid and simulated intestinal fluid. OPM-coated niosomes were found to more stable in simulated gastrointestinal conditions. The immune stimulating activity was studied by measuring serum IgG titer, IgG2a/IgG1 ratio in serum, and sIgA levels in intestinal and salivary secretions following oral administration of niosomal formulations in albino rats. The results were compared with alum-adsorbed TT following oral and intramuscular administration, and it was observed that OPM-coated niosomes produced better IgG levels as compared to plain uncoated niosomes and alum-adsorbed TT upon oral administration. Oral niosomes also elicited a significant mucosal immune response (sIgA levels in mucosal secretions). The developed formulations also elicited a combined serum IgG2a/IgG1 response, suggesting that they were capable of eliciting both humoral and cellular response. The study signifies the potential of OPM-coated niosomes as an oral vaccine delivery carrier and adjuvant. The proposed system is simple, stable, and cost-effective and may be clinically acceptable.     

Adjuvant properties of bacterial product cantastim

Aluminum compounds have been used as adjuvants in practical vaccination for more than 60 years to induce an early, an efficient and a long lasting protective immunity. Nowadays they are the most widely used adjuvants in both veterinary and human vaccines. Unfortunately these adjuvants do not only cause undesirable side effects, but often induce T-helper type 2 (Th2)-biased responses. In this study we investigated the ability of the bacterial product CANTASTIM (CS) to augment the immune responses to a model antigen, tetanus toxoid (TT). Immunization of mice with TT+CS elicited higher anti-TT IgG antibody levels as compared to mice that received TT alone. Moreover, treatment with TT+CS resulted in a lower IgG1/IgG2a ratio and a stronger in vitro IFN-gamma synthesis by splenocytes and T cells cocultured with macrophages. These data suggest that CS can be used to enhance the magnitude of the immune response and to skew it towards the Th1 type.     

Marine gastropod hemocyanins as adjuvants of non-conjugated bacterial and viral proteins

Killed viral vaccines and bacterial toxoids are weakly immunogenic. Numerous compounds are under evaluation as immunological adjuvants and peptide-carriers to improve the immune response. The hemocyanins, giant extracellular copper proteins in the blood of many mollusks, are widely used as immune stimulants. In the present study we investigated the adjuvant properties of hemocyanins isolated from marine gastropods Rapana thomasiana and Megathura crenulata. An immunization with Influenza vaccine or tetanus toxoid combined with Rapana thomasiana hemocyanin (RtH) and Keyhole limpet hemocyanin (KLH) in mice induced an anti-influenza cytotoxic response lasting at least 5 months and an antibody response to viral proteins. The IgG antibody response to the tetanus toxoid (TT) combined with RtH or KLH was comparable to the response of the toxoid in complete Freund's adjuvant. The results obtained demonstrate that the both hemocyanins are acceptable as potential bio-adjuvants for subunit vaccines.