Stabilised aluminium phosphate nanoparticles used as vaccine adjuvant.

Aluminium phosphate is a commonly used adjuvant consisting of heterogeneously sized aggregates up to several micrometers. However, aluminium phosphate nanoparticles may exhibit an improved adjuvant effect. In this study, nanoparticles were made by sonication of commercially available aluminium phosphate adjuvant, resulting in particles with a size (Z-average diameter) between 200-300 nm and a point of zero charge of 4.5. To prevent reaggregation, which occurred within 14 days, a screening of excipients was performed to identify stabilisers effective under physiological conditions (pH 7.4, 290 mOsm). The amino acids threonine, asparagine, and L-alanyl-L-1-aminoethylphosphonic acid (LAPA) stabilised sonicated aluminium phosphate. Particle sizes remained stable between 400-600 nm at 37 °C during 106 days. Contrarily, arginine induced strong reaggregation to a particle size larger than 1000 nm. The stability of aluminium phosphate nanoparticles was strongly affected by the pH. Aggregation mainly occurred below pH 7. The adsorption capacity, a potentially relevant parameter for adjuvants, was slightly reduced in the presence of asparagine, when using a model antigen (lysozyme). LAPA, arginine, threonine and aspartic acid reduced protein adsorption significantly. The adjuvant effect of aluminium phosphate nanoparticles was studied by immunisation of mice with diphtheria toxoid adjuvanted with the aluminium phosphate nanoparticles. The presence of LAPA, threonine, aspartic acid or asparagine did not alter diphtheria toxoid-specific antibody or toxin-neutralising antibody titres. Arginine increased diphtheria toxoid-specific antibody titres but not toxin-neutralising antibody titres. In conclusion, aluminium phosphate nanoparticles were stabilised by particular amino acids and induced an adjuvant effect comparable to that of aluminium phosphate microparticles.

Augmentation of humoral and cellular immunity in response to Tetanus and Diphtheria toxoids by supercritical carbon dioxide extracts of Hippophae rhamnoides L. leaves.

Hippophae rhamnoides L. commonly known as Seabuckthorn (SBT), a wild shrub of family Elaegnacea, has extensively used for treating various ailments like skin diseases, jaundice, asthma, lung troubles. SBT leaves have been reported to possess several pharmacological properties including immunomodulatory, antioxidant, anti-inflammatory, antimicrobial and tissue regeneration etc. The present study was undertaken to evaluate the adjuvant property of supercritical carbon dioxide extracts (SCEs 300ET and 350ET) of SBT leaves in balb/c mice immunized with Tetanus and Diphtheria toxoids. The dynamic changes in the immune response were measured in terms of humoral and cell-mediated immune responses. We have seen the effect of SCEs on immunoglobulin subtypes and secondary immune response generation. In addition, the effect of SCEs on antigen specific cellular immunity was evaluated. Our results show that SCEs 300ET and 350ET significantly enhanced antibody titers in response to both TT and DT antigens. The secondary immune response generated was significantly increased in case of TT immunized animals. SCEs also enhanced cytokine levels (IFN-γ, IL-4, TNF-α and IL-1ß) and increased lymphoproliferation. Besides, both SCEs did not show any toxic effects. Therefore, the study suggests that SCEs are safe and have potent immunostimulatory activity and hence, seems to be a promising balanced Th1 and Th2 directing immunological adjuvant for various veterinary as well as human vaccines.

Identification of a novel vaccine adjuvant that stimulates and maintains diphtheria toxoid immunity.

Immunomodulation by plant-derived medicines is well-documented with effects on both innate and adaptive immunity. This study reports potent and long-lasting diphtheria toxoid-specific immunity by the botanical medicinal, Rehmannia Six Formula, using an in vivo mouse model of vaccine immunity. A significant vaccine adjuvant effect was observed with an increase in serum anti-diphtheria toxoid total and IgG antibodies following oral administration of Rehmannia Six Formula to mice. This response was antigen-specific and was still detectable six months following botanical medicinal treatment, suggesting that Rehmannia Six Formula could help maintain protective antibody levels in populations where vaccine coverage is low. Rehmannia Six Formula was well-tolerated with no adverse effects on mouse weight or survival observed in this study and suggests a potential role as a novel vaccine adjuvant preparation.

Enhanced transdermal-immunization with diptheria-toxoid using local hyperthermia.

In this study we have investigated how mild local hyperthermia could be used for transdermal-immunization. Mild hyperthermia is found unique in many ways. It activates the immune system and it also results in higher mass transport of high molecular weight molecules and nanometer size quantum dots labeled antigens across the skin in experimental animals. Hyperthermia increases the expression of co-stimulatory molecules such as CD80 and CD86. Mice transdermally immunized with diphtheria toxoid, without any adjuvant or penetration enhancing reagent but under the conditions of local hyperthermia generate an antibody response. In the memory recall assay, the splenocytes of hyperthermia enhanced transdermal-immunized mice undergo proliferation, when exposed to diphtheria toxoid. Comparable response was generated when mice were immunized with diphtheria toxoid by hyperthermia enhanced transdermal route or by conventional inter-muscular injection. Hyperthermia enhanced transdermal-immunization procedure is likely to have higher compliance as it does not cause any pain or visible damage to the skin.

Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccine (part 2).

The study is a contribution to the EDQM's efforts to meet some of the expectations of the 3 Rs: Replacement, Reduction and Refinement of animal assays as proposed by Russell and Burch in 1959 and adopted by the European Union in 1986, and specifically to validate alternative assays to replace, for batch-release purposes, the European Pharmacopoeia (Ph. Eur.) in vivo direct challenge procedures for the potency determination of diphtheria toxoid vaccines. The study results may be used in support of the replacement of the multi-dilution direct challenge procedures in different animal models by a single dilution serology test, where appropriate, and to use sera from the same animals for potency testing of several components in combined vaccines. With regard to the latter, the present study explores the possibility of testing both diphtheria and tetanus toxoid potencies using serum from the same animals.

A preliminary evaluation of alternative adjuvants to alum using a range of established and new generation vaccine antigens.

Although alum is the most commonly used vaccine adjuvant, it has some limitations for use with the next generation recombinant antigens. We explored the use of alternative adjuvant formulations (poly lactide co-glycolide (PLG) microparticles, MF59 emulsion, CAP and l-tyrosine suspension) in comparison with five different vaccine antigens--namely, diphtheria toxoid (DT), tetanus toxoid (TT), HBsAg, Men C conjugate and MB1. The results indicated that although alum was optimal for bacterial toxoid based vaccines, it was not highly potent for MB1, Men C or HBsAg antigens. MF59 emulsion stood out as a good alternative to alum for TT, HBsAg, MB1 and Men C vaccines. On the other hand l-tyrosine suspension and CAP did not enhance immune responses over alum with most antigens. PLG microparticles were comparable or better than alum with both MB1 and Men C conjugate vaccine. The study indicates that it is possible to replace alum with other adjuvant formulations like MF59 and PLG and maintain and/or improve immune responses with some vaccine antigens.

Effect of vitamin E TPGS on immune response to nasally delivered diphtheria toxoid loaded poly(caprolactone) microparticles.

The nasal mucosa has many advantages as a potential site for drug and vaccine delivery. The present study has sought to exploit this route of delivery using microparticles composed of D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) as a matrix material blended with poly(caprolactone) for nasal immunisation with diphtheria toxoid. Particles were prepared by a double emulsion method, followed by spray drying and the effect of TPGS on size, zeta potential, loading and release of antigen was assessed. Particles composed of TPGS-PCL blends were spherical, smooth and monodisperse, displaying increasing yields after spray drying with increasing concentrations of TPGS. The immune response to diphtheria toxoid loaded PCL-TPGS microspheres after nasal administration was shown to be higher than that achieved using PCL microspheres alone. We conclude that TPGS shows significant potential as a novel adjuvant either alone or in combination with an appropriate delivery system.

Effect of monophosphoryl lipid A on antibody response to diphtheria toxin and its subunits.

Monophosphoryl lipid A (MPL) was evaluated for its ability to enhance the antibody response to diphtheria toxin and its fragment A and fragment B subunits. BALB/c mice were immunized subcutaneously with 1 Lf of diphtheria toxoid in the presence of 25 microg of MPL on days 0 and 14. Two weeks after the second immunization, sera were obtained from the mice and analysed for antibody response to diphtheria toxin and its subunits. A new ELISA method, developed in our laboratory, was used to measure antibody levels against the toxin, fragment A, and fragment B. It was observed that MPL significantly enhanced antibody responses to diphtheria toxin and its subunits. However, there was no statistical difference between anti-A and anti-B responses. The results indicated that MPL seems to be a potential candidate as an adjuvant for future diphtheria vaccine formulation.

Diphtheria epidemic in the Republic of Georgia, 1993-1997.

Epidemic diphtheria reemerged in the republic of Georgia in 1993. From 1993 to 1997, 1405 cases were reported (28 in 1993, 312 in 1994, 429 in 1995, 348 in 1996, and 288 in 1997), with a cumulative incidence of 25.8/100,000 and a case fatality ratio of 9.5%. During 1993-1997, 53% of the diphtheria cases occurred among persons >/=15 years of age. Unvaccinated patients were more likely to have toxic forms (relative risk=2.24; 95% confidence interval=1.69-2.96) or to die of diphtheria (relative risk=2.24; 95% confidence interval=1. 36-3.68) than those who had received at least one dose of diphtheria toxoid. Improvement in routine childhood vaccination coverage and implementation of mass adult vaccination campaigns have been critical to bringing the epidemic under control. By mid-1998, the overall diphtheria situation in Georgia appeared to have been controlled. Only 53 cases were reported from January to June 1998, representing a 64% decrease from the 148 cases during the corresponding period in 1997.

Simultaneous vitamin A administration at routine immunization contact enhances antibody response to diphtheria vaccine in infants younger than six months.

A randomized, double-blind, placebo-controlled trial was conducted to evaluate the effect of simultaneous vitamin A supplementation and diphtheria, pertussis and tetanus (DPT) vaccination on the antibody levels. Infants aged 6-17 wk (n = 56) were randomly given 15 mg oral vitamin A or placebo at the time of their DPT immunization. Three such doses were given at monthly intervals. Immunoglobulin (Ig) G antibodies to diphtheria, pertussis and tetanus were assayed on enrollment and 1 mo after the third dose. Baseline antibody concentrations to diphtheria, pertussis and tetanus did not differ between the vitamin A-supplemented and placebo-treated groups. The postdose antibody to diphtheria level was significantly greater in the vitamin A than in the placebo-treated group. The geometric mean +/- SEM antibody levels (mg/L) were 22.9 +/- 1.2 and 11.0 +/- 1.3 in the vitamin A and placebo groups, respectively (P = 0.029). The postsupplementation concentrations of antibodies to pertussis and tetanus did not differ between the two groups. These results suggest that antibody response to diphtheria vaccination was potentiated by simultaneous vitamin A administration and DPT immunization.

The induction of systemic and mucosal immune responses following the subcutaneous immunization of mature adult mice: characterization of the antibodies in mucosal secretions of animals immunized with antigen formulations containing a vitamin D3 adjuvant.

Systemic and mucosal immune responses were effectively induced following the subcutaneous administration of Haemophilus influenzae type b oligosaccharide conjugated to diphtheria toxoid vaccine in a formulation containing the active form of vitamin D3. IgA and IgG antibodies with specificity for both the protein and oligosaccharide components of the vaccine were detectable in mucosal secretions following immunization. The IgA and IgG mucosal antibodies were produced locally, and were functional as demonstrated by their diphtheria toxin neutralizing activity. Our data suggests that subcutaneous tissues can effectively serve as effective antigen presenting sites for both mucosal and systemic immune responses to antigens administered in combination with vitamin D3.

Nasal and parenteral immunizations with diphtheria toxoid using monoglyceride/fatty acid lipid suspensions as adjuvants.

A novel suspension system was developed where monoglycerides were formulated together with fatty acids and subsequently admixed with antigens. In the present study, diphtheria toxoid was used as a model antigen primarily due to its weak immunological properties as well as to its importance as a future human vaccine for mucosal, particularly nasal immunization. The formulations were administered parenterally and/or nasally to mice whereafter the immune response was determined. In the present study, we have shown that mono-olein/oleic acid vesicles enhance the immunogenicity of admixed diphtheria toxoid in mice to the same level as Alum adsorbed (or Freund's complete adjuvant) when administered parenterally or nasally. It was also shown that the immunogenicity was linked to the length of the acyl chain of the lipids, where shorter acyl chains resulted in reduced titers. Furthermore, shorter acyl chains also gave rise to more pronounced toxic reactions at the injections sites, such as necrosis and alopeci, both of which were lacking when the optimal formulation consisting of mono-olein and oleic acid was used. Thus, this lipid matrix has in our view a great potential as an immunological adjuvant with an exceptionally simple and efficient preparation procedure without organic solvents and with low cost endogenous lipid based raw materials.

[The antidiphtheria activity of the pre-EGF fragment]. / Antidifteriinaia aktivnost' fragmenta pre-EGF.

The DNA fragment, coding a part of the protein molecule--the precursor of the epidermal growth factor (pre-EGF76-208)--and containing the sequence of 133 N-end amino acid residues, was obtained with the use of gene engineering and molecular biological techniques. For this purpose a fraction of poly(A+) = RNA was isolated from the kidney of a newborn infant; on this fraction the "library" of cDNA fragments whose coding capacity corresponded to the required sequence of mRNA in the pre-EGF gene was obtained in the reaction of reverse transcription, conjugated with the polymerase chain reaction (PCR). After the determination of the nucleotide sequences in 11 fragments only 1 fragment which contained practically no mutations appearing in PCR as the result of amplifications was chosen. This fragment was used for the construction of a hybrid plasmid controlling the synthesis of hybrid fusion protein with the sequence of pre-EGF76-208. Fusion protein was synthesized with very low effectiveness, but the use of metallo-affinity chromatography permitted to isolate and purify it, its purity reaching 98%. Then its antitoxic activity was determined in the skin test on guinea pigs and found to be not less then 10(6) I.U./mg of protein.

Quantitation of tetanus and diphtheria antitoxins in mouse sera by indirect haemagglutination.

Serum samples obtained from 75 groups of mice immunized with various doses of adsorbed tetanus vaccine, adsorbed diphtheria-tetanus vaccine and adsorbed diphtheria-tetanus-pertussis vaccine were titrated for tetanus antitoxin content by an in-vitro indirect haemagglutination (IHA) and by toxin neutralization test (TN) in mice. From these serum samples of 49 groups of mice which were immunized with combined vaccine containing diphtheria toxoid were titrated for their diphtheria antitoxin content by IHA and by i.d. toxin neutralization test (TN) in guinea pigs. Good correlations were found between the estimates obtained by in-vitro IHA and in vivo TN tests in both tetanus and diphtheria antitoxin titrations. The minimum level of tetanus or diphtheria antitoxin detectable by IHA was 0.00039 IU/ml. It is concluded that IHA is a simple, sensitive and reproducible alternative test which can replace the animal TN tests for the estimation of tetanus and diphtheria antitoxins and could reliably be used in the potency assay of tetanus and diphtheria toxoids of combined vaccines based on antibody induction in mice.

Investigation into suitable carrier molecules for use in an anti-gonadotrophin releasing hormone vaccine.

Gonadal function can be controlled through immunoneutralisation of gonadotrophin releasing hormone (GnRH), with an analogue, GnRH-glycys, linked to a carrier molecule and an appropriate adjuvant. In this study, four different types of carrier molecule were investigated: (a) single and branched amino acid polymers--[poly-(D-glu, D-lys) and poly-(phe, glu)-poly(DL-ala)-poly(lys)]; (b) bacterial toxoids--diphtheria (DT) and tetanus (TT); (c) synthetic T-helper epitopes--derived from malarial circumsporozite protein (CS) and measles virus fusion protein (MVF); and (d) thyroglobulin (Thy)--a large protein. The effect of non-ionic surfactant vesicles (NISV) and an aluminum hydroxide based adjuvant (alum), was also examined. Although good antibody responses were achieved with GnRH-glycys-DT, GnRH-glycys-TT and GnRH-glycys-Thy, adsorbed onto alum and the dimerised synthetic T-helper epitope constructs, incorporated into NISV, a critical antibody titre was necessary to result in morphological changes in the gonads and complete suppression of spermatogenesis. This was only achieved with tetanus toxoid and the dimerised T-helper epitopes.

Development of an animal model to assess the immunogenicity of single-dose tetanus and diphtheria vaccines based on controlled release from biodegradable polymer microspheres.

We have determined threshold doses of aluminium phosphate (AlPO4) adsorbed tetanus toxoid (TT) and diphtheria toxoid (DT) in mice and guinea pigs with a view to developing an animal model to assess the immunogenicity of controlled release vaccines. A dose was sought (threshold dose) which produces little antibody after primary injection and a moderate response after a booster injection, thus mimicking the adult human response to TT and DT vaccines adsorbed on to aluminium adjuvants. After the first injection, mice and guinea pigs showed a dose response for both tetanus toxin IgG antibodies and tetanus antitoxin over a wide range of doses of AlPO4-adsorbed TT (0.01 to 0.2 Lf). After the second and third injections, there was no clear dose response for doses between 0.05 and 0.2 Lf However, doses between 0.01 and 0.04 Lf still showed a dose response after the second injection. Dilution of AlPO4-adsorbed TT in saline just before injection did not alter immunogenicity after the first injection, but stronger booster responses were seen in mice to diluted versus undiluted vaccine after the second and third injections. The threshold dose of AlPO4-adsorbed TT for both mice and guinea pigs was 0.01 Lf given in 100 microliters. For AlPO4-adsorbed DT, three strains of mice (inbred, Balb/c and C57/B6, and outbred, CD-1) showed a dose response after the first injection for DT IgG antibodies and diphtheria antitoxin at 0.1 and 0.2 Lf doses. Outbred CD-1 mice showed a dose response after the second and third injections also, whereas inbred mice showed inconsistent dose responses after the second injection and none after the third injection. In contrast to AlPO4-adsorbed TT, mice injected with undiluted AlPO4-adsorbed DT elicited significantly higher antibodies than those injected with diluted formulations, particularly after the first injection. The threshold dose of AlPO4-adsorbed DT for mice was 0.1 Lf in a volume of 250 microliters. Lower doses did not produce consistent antibody responses in mice. We propose that a single dose of controlled release formulations at doses not greater than 1/10 of a single human dose when injected into mice and guinea pigs should produce an antibody response similar or higher than two to three threshold doses of AlPO4-adsorbed TT or DT.

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.

Booster vaccination against diphtheria and tetanus in man. Comparison of three different vaccine formulations--III.

Adverse reactions and antibody levels were compared following a booster vaccination of 177 Danish military recruits with a plain, an aluminium hydroxide (0.5 mg Al per human dose, HD) and a calcium phosphate (0.25 mg Ca per HD) adsorbed diphtheria-tetanus (D-T) vaccine. The calcium phosphate adsorbed vaccine was given in a HD of 3 Lf of D and T toxoids and proved to be of equal efficacy as the aluminium hydroxide adsorbed vaccine which was injected in a dose containing twice the antigen amount. The calcium phosphate vaccine caused fewer adverse reactions than the one adsorbed to aluminium hydroxide. The plain vaccine (6 Lf per HD of D and T toxoid) had the highest efficacy with a similar low occurrence of adverse reactions as the calcium phosphate adsorbed vaccine. Potency assays in mice were in accordance with these immunogenicity results in man if a two dose immunization schedule was followed, but not if the vaccines were compared after a single immunization as requested by the procedure for potency testing according to current WHO and European Pharmacopoeia requirements. Both of the adsorbed vaccines primed mice for specific IgE antibody formation. This could be detected after a second immunization with either of the adsorbed vaccines or with the plain D-T vaccine. Also in humans, immunization with the plain vaccine boosted specific IgE formation to a detectable level. This may be ascribed to adjuvant priming during the primary vaccination series some 20 years previously.

Cynomolgus monkeys (Macaca fascicularis) in preclinical immune function safety testing: development of a delayed-type hypersensitivity procedure.

A delayed-type hypersensitivity (DTH) test commonly used for humans was adapted for use with cynomolgus monkeys (Macaca fascicularis). Pilot experiments showed naive animals had poor response rates and inconsistent reactivity to the antigens. In an exploratory phase, it was determined that monkeys could be experimentally sensitized by immunization with commercially available antigens. Animals were then sensitized with various concentrations of diphtheria and tetanus toxoids, Candida, and Trichophyton in the dose-response phase. Antigens were injected intradermally (i.d.) 3 times over a 7-day period and monkeys were tested 14 days after the last injection. Responses were measured 24, 48, and 72 h post-challenge, with skin biopsies taken from two animals per group at the 24 h interval. Optimal concentrations were 1.2 Lf diphtheria, 6 Lf tetanus, 1000 PNU Candida, and 1000 PNU Trichophyton. These concentrations produced the best balance between DTH responses, homogeneity of dermal mononuclear cell infiltrate and lowest frequency of undesirable skin reactions. Positive responses were seen at 24 and 48 h post-challenge and were waning by 72 h. DTH responses were inhibited by topical corticosteroids. The final phase of these studies assessed whether sensitization of naive animals could be achieved using subcutaneous (s.c.) administration of the optimal antigen concentrations. Comparable responses to i.d. sensitization were obtained and skin sores did not develop at injection sites. These studies show that the DTH test adapted to monkeys was reproducible, minimally invasive, did not require sacrifice of the test animal, allowed repeated measurements and paralleled the reactions observed in humans.

Oral immunization against diphtheria and tetanus infections by fluid diphtheria and tetanus toxoids.

Purified diphtheria toxoid incorporated in egg yolk and mixed with a medicinal plant seed was used to orally immunize rabbits against diphtheria infection. Animals were partially immunized against a lethal diphtheria toxin challenge. The immunity was complete when gastric enzyme juices were inhibited before oral vaccination by aprotinin, a natural protease inhibitor. Rabbits and monkeys were orally immunized against both diphtheria and tetanus in the same way by pre-treatment with aprotinin. Adult volunteers receiving protease inhibitor before administration of oral toxoids have shown a significant rise in specific circulating antitoxins.