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A new lipoid adjuvant. I. Preparation and observation of its effectivenes
Abstract
Authors prepared an experimental lipoid adjuvant (ELA) of the oil-in-water type, based on metabolisable isopropylester of palmitic acid (IPP) intended for veterinary use. Poloxamer 105 and Arlacel A were used as detergents to prepare a high quality stable emulsion. The observation of quality and stability of emulsion enabled to determine the optimum conditions of preparation and ratio of individual components of the adjuvant: IPP - 20%; detergent - 10% (7% Poloxamer 105 + 3% Arlacel A); water - 70%. Authors tested the potency-increasing effect of ELA on immunogenic and antigenic activity of inactivated, concentrated and purified rabies vaccine. The immunogenic activity of this vaccine, when used with ELA (in model experiments on mice), increased approximately twofold. The potentiating effect of ELA on the antigenic activity of rabies vaccine was observed by the authors on laboratory animals (guinea pigs) and on target animal species (domestic dogs and cattle). The level of rabies antibodies, determined by the ELISA method, served as a criterion. The results obtained indicated sufficient potency-increasing capability of ELA which was minimally comparable with that of commercial products.
... In the experiments the following vaccines were used: 1. Experimental -inactivated concentrated purified rabies vaccine (S ü li ová et al. 1997;Bení‰ek et al. 1998), potentiated with lipoid adjuvant ( B e ní‰ek et al. 1999;Süliová et al. 1999); 2. Commercial inactivated rabies vaccine Lyscelin, op. No. 260594, produced by Bioveta Ltd. ...
... For the complex efficacy evaluation of experimental inactivated concentrated purified rabies vaccine potentiated with lipoid adjuvant, intended for domestic animals ( B e ní‰ek et al. 1999;Süliová et al. 1999) it was necessary to perform comparative experiments using an important target animal species -cattle. Comparative experiments for the verification of the efficacy of this experimental vaccine on the most important species, domestic dogs, especially with different methods of vaccine administration, were the subjects of our previous work (Bení‰ek et al. 1998). ...
... Significantly higher efficacy of the experimental vaccine (especially from the point of view of humoral immunity indicators -day 180) is not only due to concentrated and purified antigen in the vaccine, but also due to the use of areactogenic metabolisable as well as highly effective lipoid adjuvant ( B e ní‰ek et al. 1999;Süliová et al. 1999) for the support of the vaccine effect. The possibility of adequate rabies immunity induction in cattle administration of a dose of only 1 cm 3 of experimental vaccine is not a negligible fact. ...
Efficacy of an experimental inactivated concentrated and purified rabies vaccine, potentiated with lipoid adjuvant was evaluated in comparative experiments performed in cattle by testing of specific cell-mediated immune response (CMI). Commercial inactivated rabies vaccines -Lyscelin (Czech Republic) and Rabisin (France) were used for comparison. Together with CMI evaluation by leukocytes migration inhibition test (LMI), postvaccination rabies antibodies were quantified by the ELISA method and by the rapid fluorescent focus inhibition test (RFFIT) on days 14, 28, 60 and 180 after immunization of the animals. The results of antigenic activity of tested vaccines show the best efficacy of the experimental vaccine from the point of view of humoral immunity, as well as cell-mediated immunity, even though all tested vaccines induced sufficient level of rabies antibodies (> 0.5 IU/cm3) at all evaluated intervals.
... They are cheap and safe (areactogenic) if a suitable oil component is used and are easy to apply [13]. One adjuvant developed in our laboratory is of the O/W type [14], based on fully metabolisable [15] isopropyl palmitate (IPP)—isopropylester of palmitic acid. This adjuvant has excellent properties [16]; however, its disadvantage is that can be used only for resuscitation of lyophilised vaccines and not for potentiation of liquid inactivated vaccines. ...
... coconut, peanut, olive, soya, lanolin). Metabolisable esters of fatty acids appear to be another suitable oil component of effective adjuvants141516. Very good adjuvant activity was reached by using squalene or squalane [19]. ...
... Besides detergent properties it has also some adjuvant ones, it is a block co-polymer. This emulsifier was used successfully in preparation of adjuvant based on isopropyl-palmitate which exhibits excellent adjuvant prop- erties [14,16]. However, one of his disadvantages is that can be used only for dilution or resuscitation of lyophilised vac- cines. ...
The authors prepared experimental lipoid adjuvant of the oil-in-water (O/W) type, based on squalene (ESA: experimental squalene adjuvant), intended for liquid inactivated veterinary vaccines. The final concentration of squalene in the vaccine was 5% (w/v). Detergents Poloxamer 105 in the concentration of 4% (w/v) and Abil-Care in the concentration of 2% were used as emulsifiers. The water component was inactivated virus suspension. Emulsification at decreased temperatures (up to 37 degrees C) fails to denaturate the immunization antigen. The effectiveness of experimental squalene adjuvant was tested using a liquid inactivated adjuvant rabies vaccine and a liquid porcine parvovirus vaccine in comparison with adjuvant-free vaccines or vaccines potentiated with aluminium hydroxide. The squalene adjuvant increased the immunogenic activity of non-potentiated rabies vaccine approximately 1.8-fold, while aluminium hydroxide increased the effectiveness of rabies vaccine only 1.08-fold. The antigenic activity, i.e. the level of specific antibodies after vaccination was significantly increased by using both adjuvants; ESA was more efficient than aluminium hydroxide.
... The development of new vaccine adjuvants (combined adjuvants) has been hampered by their unacceptable reactogenicity [6]. We prepared in our laboratory an emulsion of oil-in-water type based on esters of fatty acids, namely izopropylester of palmitic acid with the use of block co-polymer Poloxamer 105 (pluronic polyol) as an emulsifier with marked adjuvant activity [7,8]. Pluronic polyols are a family of non-ionic surfactants currently used as drug carriers for antibiotic , anti-inflammatory and anti-neoplastic agents [9]. ...
Model experiments on laboratory animals (guinea pigs) were carried out to test the possible allergic reaction (possibility of sensitisation) to the repeated administration of an experimental lipoid adjuvant prepared on the basis of squalene (experimental squalene adjuvant--ESA). No significant differences were observed between the animals sensitised-provoked with ESA and control animals. In order to evaluate the local tissue reactivity (local reactogenity), also with regard to the process dynamics to the administration of ESA, comparative patho-anatomical and patho-histological examinations of tissues were carried out in the location of adjuvant administration. The examinations indicated very low local reactogenity of the experimental lipoid adjuvant prepared in our laboratory. The test of pyrogenicity also confirmed the safety of ESA, the labelled lysate sensitivity lambda was under 0.25 IU/cm3.
Model experiments on laboratory animals (guinea pigs) were carried out 10 test the possible allergy reaction (possibility of senzitization) to the repeated administration of an experimental lipoid adjuvant (ELA) prepared on the basis of isopropylpalmitate. No significant differences were observed between the animals senzitized-provoked with ELA and the control animals. In order to evaluate the local tissue reactivity (local reactogenicity), also with regard to the process dynamics, to the administration of ELA and to carry out comparisons with other types of lipoid adjuvants, as well as aluminium hydroxide, comparative patho-anatomical and patho-histological examinations of tissues were carried out in the location of adjuvant administration. The examinations indicated very low local reactogenicity of the experimental lipoid adjuvant prepared in our laboratory.
The authors had developed a new experimental adjuvant based on squalene and its efficacy was tested on target domestic animals - cats and dogs by means of different rabies vaccines. In this experiment the authors compared the immune response on the rabies vaccine without adjuvant and with aluminium adjuvant or experimental squalene adjuvant, respectively. The level of rabies antibodies was determined by rapid fluorescence focus inhibition test on days 14, 30, 90 and 450 (dogs), and on days 14, and 30 (cats), respectively. The rabies antibodies level on day 14 still did not reache the protection level determined by WHO. On day 30 each of the vaccines induced an adequate response, while the most effective seemed to be the vaccine with experimental squalene adjuvant. On day 450 the antibodies level decreased, but the average level was over the protection value, when the experimental squalene-adjuvanted vaccine was used. The level of rabies antibodies did not achieve the protection value in 2 from 10 dogs in this group. There is an open question, if it is sufficient to use only one dose of rabies vaccine for safe primovaccination of young animals.
Model experiments on laboratory animals (guinea pigs) were carried out 10 test the possible allergy reaction (possibility of senzitization) to the repeated administration of an experimental lipoid adjuvant (ELA) prepared on the basis of isopropylpalmitate. No significant differences were observed between the animals senzitized-provoked with ELA and the control animals. In order to evaluate the local tissue reactivity (local reactogenicity), also with regard to the process dynamics, to the administration of ELA and to carry out comparisons with other types of lipoid adjuvants, as well as aluminium hydroxide, comparative patho-anatomical and patho-histological examinations of tissues were carried out in the location of adjuvant administration. The examinations indicated very low local reactogenicity of the experimental lipoid adjuvant prepared in our laboratory.
Non-ionic block copolymers and lipopolysaccharides are both effective immunological adjuvants which are thought to act via distinct mechanisms. We hypothesized that they might produce synergistic effects when used together. We prepared a series of lipopolysaccharide (LPS) preparations ranging from the smallest precursor, lipid X through complete LPS with O-polysaccharide chains. Three preparations with reduced toxicity, monophosphoryl lipid A, partially hydrolysed Ra-LPS and LPS of Rhodopseudomonas sphaeroides were also utilized. All LPS preparations except the smallest were effective adjuvants for inducing early antibody responses to trinitrophenyl-conjugated hen egg albumin (TNP-HEA) when injected in squalane-in-water emulsions with copolymer L141. Only the larger LPS preparations induced sustained antibody responses. By itself, emulsions of copolymer L141 induced a predominant IgG1 antibody isotype response with lesser amounts of IgG2a and IgG2b. Surprisingly, all of the LPS preparations tested increased the proportion of IgG2 isotypes even though some had little effect on overall titres. The detoxified Ra-LPS (Ra-detox) was the most effective preparation for both increasing antibody titres and inducing the desirable IgG2a and IgG2b isotypes. These results demonstrate that the combination of LPS and block polymer adjuvants can produce synergistic effects without unacceptable toxicities.
Water-in-oil-in-water (w/o/w) emulsion developed in our laboratory is as effective as water-in-oil (w/o) emulsion of Freund's incomplete adjuvant (FIA) in the stimulation of antibody formation. The emulsion is prepared by redispersion of water-in-sesame oil emulsion of an antigen solution in phosphate buffered saline with emulsifier, Tween 80. The emulsion can be stored at 4 degrees C for at least 3 months without any evidence of change in the adjuvanticity and in the w/o/w state. Even a single injection of bovine serum albumin (BSA) in the w/o/w emulsion elicited a high antibody response in mice over the period of almost whole lifespan. 10 microgram BSA in w/o/w could stimulate antibody formation up to 2(12) in hemagglutination titer, while the same dose in free solution did not elicit any detectable antibody. The tissue reactions caused by the w/o/w emulsion at the injected site and in the regional lymph nodes were much less prominent than those by FIA.
In 1986-1988, adverse anaphylactoid reactions (AR) were observed in animals in Czechoslovakia after the administration of oil adjuvant-containing vaccines or other lipoid drugs. Treated animals showed signs resembling the classic anaphylactic reaction, i.e. restlessness, salivation, pruritus, oedema and cyanosis of udder and vulva, and eyelid oedema, developing within a few minutes. The reactions were not elicited by the antigen alone, but by the oil adjuvant. The aim of our experiments was to identify substances eliciting the reaction in susceptible animals and to investigate possible induction mechanisms. The emulsifier Tween 80 has been demonstrated to be an AR inducing component of vaccines and drugs (Tab. I and III). Weak or moderate reactions were observed in 33% of animals treated with 5% Tween and 66% of those treated with 10% Tween showed strong reactions. On the other hand, no reactions were elicited by treatment with several paraffin oils of different quality (Tab. I) nor with an oil-in-water emulsion containing Montanid as an emulsifier (Tab. II). The role of the vegetative nervous system in the rise of AR has been confirmed. AR were suppressed in animals pretreated with parasympatholytic atropine and enhanced in a part of those pretreated with parasympathomimetic pilocarpine (Tab. III). The percentage of animals affected and the intensity of AR were also lower in animals pretreated with complement inhibitor epsilon-aminocapronic acid (Tab. IV). A major role of complement activation is suggested in the discussion of possible mechanisms of AR induction. It is possible to draw a conclusion on the basis of the results presented here and of the analysis of individual cases that a certain degree of animal susceptibility, depending on the phase of reproductive cycle, metabolism level and neurovegetative balance is necessary besides the administration of an AR inducer (Tween 80 in our case). Hence it seems that the adverse anaphylactoid reactions results from interactions of the two factors, i.e. administration of an AR inducer to susceptible animals.
We evaluated an adjuvant, TiterMax, as an alternative to complete Freund's adjuvant (CFA) for producing antisera in animals. TiterMax, consists of a microparticulate stabilized water-in-oil emulsion of a metabolizable oil, squalene, with the adjuvant block copolymer CRL89-41. This paper reports two evaluations of TiterMax versus CFA and other commercially available adjuvants. In the first study, mice were immunized with a hapten, trinitrophenol, conjugated to hen egg albumin (TNP-HEA) in one of several adjuvants: TiterMax, CFA, Adjuvax, Ribi adjuvant system (RAS), Alhydrogel or Lipovant. TiterMax induced higher longer lasting titers with fewer injections than any of the other adjuvants. The magnitude of the response to TNP varied with species and route of immunization. In the second study, CFA, TiterMax, Adjuvax and RAS were compared in rabbits, mice and goats. Animals were immunized with luteinizing hormone-releasing hormone (LHRH) conjugated to BSA in each adjuvant using comparable protocols. TiterMax induced titers against the peptide equivalent to CFA in all three species. The inflammatory responses induced by TiterMax were mild and transient compared with those induced by CFA. These data suggest that TiterMax is an effective alternative to CFA in many situations.
The adjuvant activity of block copolymers varies with the lengths of the chains of polyoxypropylene (POP) and polyoxyethylene (POE). This project evaluated the adjuvant activity of new copolymers with long polyoxypropylene chains in mice immunized with TNP-hen egg albumin in 2% squalone-in-water emulsions. Two of the new copolymers, L141 and L180.5, not only stimulated higher antibody titres to TNP than older preparations, but also induced a higher percentage of the IgG2 isotypes. The smaller older copolymers, L101 and L121, induced higher absolute levels of IgG3 antibody and relative increases in IgG1. Incorporation of immunomodulators (cell wall skeletons, monophosphoryl lipid A or threonyl muramyl dipeptide) increased mean IgG titres but also increased variability of responsiveness among individuals.
Mice were vaccinated against lethal blood stage P. yoelii malaria with lysates of parasitised blood prepared in various detergents. Two injections, containing 25 micrograms of protein, of a Triton X-100 lysate gave optimal protection when injected intraperitoneally with saponin as adjuvant. Such lysates provide a starting point for the isolation of potential antigens for vaccination, selected for their protective activity in vivo.
Many of the most potent immunoadjuvants for inclusion in vaccines are extremely hydrophobic surfactants. Lipophilic vehicles are needed as carriers for these water-insoluble adjuvants and to provide the hydrophilic-hydrophobic interfacial surface at which they act. We used emulsifiers comprised of fatty acid esters of polyoxyethylene sorbitan (Tweens) or sorbitan (Spans) to prepare oil-in-water (O/W) emulsions of hexadecane. Emulsion stability could be predicted from a ratio of spectrophotometric absorbance readings at 800 and 400 nm but not from published hydrophilelipophile balance (HLB) values. Emulsions that were stable even after heating or freezing resulted when equal volumes of hexadecane and a 70:30 blend of Tween 80/Span 80(T80/S80) were mixed and then diluted with normal saline solution to the desired hexadecane concentration. This blend of monooleate esters has an HLB value of 11.8. Other Tween-Span formulations were mixed to yield emulsifiers with the same HLB value, but only those that contained either T80 or S80 were effective stabilizers. Instability resulted when both esters were derived from saturated fatty acids. Addition of bovine serum albumin (BSA) antigen to the oil phase of 5% hexadecane emulsions tended to destabilize the emulsions, especially at higher protein concentrations. The surface active adjuvant, hexadecylamine, increased emulsion stability. Highest antibody responses in mice were seen when BSA was added to the internal phase of emulsions, i.e., the oil phase of O/W emulsions and the aqueous phase of W/O (Freund's) emulsions. Addition of the hydrophilic T80 to the aqueous phase of O/W emulsions was detrimental to antibody production. In general, stability, oil concentration, and T80/S80 concentration of emulsions had little effect on IgG levels.(ABSTRACT TRUNCATED AT 250 WORDS)
Studies were conducted in mice, hamsters, sheep, and two species of nonhuman primates which demonstrate the adjuvant activity of a new metabolizable lipid emulsion with marginally immunogenic doses of Formalin-inactivated viral vaccines. The lipid base consists of highly refined peanut oil emulsified in aqueous vaccines with glycerol and lecithin. Hamsters and mice inoculated with lipid emulsion plus western or Venezuelan equine encephalitis vaccine were significantly more resistant than vaccinated controls to lethal homologous virus challenge. Sheep given one dose of lipid emulsion plus Rift Valley fever vaccine developed significantly higher antibody titers than control sheep receiving only vaccine. Cynomolgous monkeys inoculated with lipid emulsion plus Rift Valley fever vaccine developed 16-fold greater peak primary and 20-fold greater secondary antibody titers than those of vaccine controls. Similar lipid emulsion-Rift Valley fever studies in rhesus monkeys resulted in 37- and 300-fold increases in primary and secondary titers, respectively, compared with monkeys given vaccine alone. Neither the sequence of combining antigen with lipid nor the exact ratio of aqueous phase to lipid phase affected the survival of Venezuelan equine encephalitis-vaccinated mice challenged with homologous lethal virus. This lipid formulation has several advantages over other water-in-oil adjuvants for potential use in humans. The components are metabolizable or normal host constituents, it is easily emulsified with aqueous vaccines by gentle agitation, and it is relatively nonreactogenic in recipients.
Water-in-oil emulsions of metabolizable fatty acid esters, with the non-toxic surfactant Pluronic L122 as emulsifying agent, potentiated the humoral response to bovine serum albumin and staphylococcal toxoid in the mouse. Adjuvant activity was increased by changing the chemical nature of the esters as follows: (i) using a series of ethyl esters, adjuvant activity appeared when the acyl chain length of the fatty acid component was 16 or greater; (ii) isobutyl and isopropyl esters of palmitic acid (C16:0) were superior to ethyl; (iii) the ethyl esters of oleic (C18:1) and linoleic (C18:2) acids were better than stearic (C18:0). Since emulsions prepared with longer chain saturated esters are very viscous or solid at room temperature, and unsaturated esters are chemically reactive, emulsions were prepared with differing proportions of ethyl caprate (C10:0) and butyl stearate. At a ratio of 9:1 the emulsions possessed the low viscosity of ethyl caprate, but gained the adjuvant activity of butyl stearate. 125I-labelled BSA was retained in the footpad to a significantly greater extent than with a caprate emulsion, but reasons are given for believing that slow release of antigen is not the only mechanism of adjuvant activity. The ester emulsions caused more acute but less chronic local inflammation (footpad swelling) than Freund's incomplete adjuvant.
The authors developed a kit for the purpose of assessment of anti-rabies antibodies by the ELISA immunoenzymatic method in human immunized sera. The results of the detection and quantification of anti-rabies antibodies acquired by the ELISA method were compared with those originating from classical procedures (virusneutralizing test on mice, indirect hemagglutination test), and a sufficient correlation and sensitivity of the immunoenzymatic method were detected. By means of the developed test it is possible to detect the particular level of anti-rabies virusneutralizing IgG antibodies. (Tab. 2, Fig. 1, Ref. 25).
The authors prepared an experimental, inactivated, concentrated and highly purified rabies vaccine from the strain Vnukovo-32/107. The purification and concentration (simultaneous--in one operation) was carried out by affinity chromatography. The content of rabies antigen in the vaccine was 4.256 UE/cm3 (determined by ELISA method). The authors used their own lipoid adjuvant--type oil in water--to resolve lyophilized vaccine to original volume. It is based on esters of fatty the acids. The effectiveness--immunogenic activity--of the experimental preparation was compared with that of commercial rabies vaccines and the reference vaccine by means of the NIH and NRLR methods. The results obtained (2.826 units/cm3 by the NIH and 3.085 units/cm3 by the NRLR method) confirmed that the method of affinity chromatography could be used to prepare a rabies vaccine, exhibiting effectiveness superior to commercial preparations available, and complying with the WHO criteria.
Since early this century, various substances have been added to vaccines and certain formulations have been devised in an attempt to render vaccines more effective. Despite a plethora of options, only aluminium salts have gained acceptance as human vaccine adjuvants and even veterinary vaccines are largely dependent upon the use of aluminium salts. Currently, many new vaccines are under development and there is a desire to simplify vaccination schedules both by increasing the number of components per vaccine and decreasing the number of doses required for a vaccine course. New, more effective adjuvants will be required to achieve this.