Adjuvant effects of nonionic block polymer surfactants on liposome-induced humoral immune response

The ability of several surface-active agents to stimulate the humoral immune response in mice against haptenated liposomes was tested. The surfactants were block copolymers of hydrophilic polyoxyethylene (POE) and hydrophobic polyoxypropylene (POP) that differed in m.w., percentage of POE, and mode of linkage of POP to POE. The liposomes were haptenated with tripeptide-enlarged dinitrophenyl coupled to phosphatidylethanolamine, which was incorporated into the liposomal membrane. Additional injection of mice with surfactant stimulated serum hemagglutination titers and splenic plaque-forming cell (PFC) numbers to varying extents. Block polymers with POP chains flanking a POE center, as well as polymers with POE chains flanking a POP center, displayed high adjuvant activity. These block polymers stimulated the antibody response in a dose-dependent manner. They stimulated the antibody response with both high and low antigen doses. Furthermore, the addition of one of these adjuvants (25R1) reduced the amount of carrier lipid required in the liposome in order to obtain an optimal antibody response. The surfactants, which displayed high adjuvant activity, did not interfere with liposome stability as measured with a liposome lysis assay. Moreover, in vitro preincubation of liposomes with a block polymer did not affect their immunogenicity. Optimal adjuvant activity was observed when both adjuvant and liposomes were administered by the same route. Simultaneous injection of both components, however, is not a prerequisite. Conclusively, it can be stated that nonionic block polymer surfactants are potent adjuvants for stimulation of the antibody response against haptenated liposomes.     

The choice of adjuvants in Mycoplasma vaccines

The use of adjuvants in vaccine production is an important aspect of potent vaccines. This investigation was concerned with finding the most efficient adjuvants for use in Mycoplasma vaccines produced in Nigeria. Four different vaccines were produced from the Gladysdale strain of Mycoplasma mycoides subspecies mycoides. They differed depending on the type of adjuvants used. Each vaccine was used to vaccinate eight cattle using a dose of 1 ml. Two other groups of eight cattle were used as controls. One of the two groups received 1 ml dose of inactivated Gladysdale vaccine without adjuvant while the second group received 1 ml dose of saline. The number of cattle that had the peak complement fixing (CF) antibody titres of 1/80 in each group of cattle was four for vaccine containing aluminium hydroxide gel, eight for vaccine containing liquid paraffin, one for vaccine containing sodium alginate and one for vaccine without adjuvant. Seven cattle from the group vaccinated with vaccine containing Freund's incomplete adjuvant had peak CF antibody titres of 1/80 or higher. The two groups vaccinated with vaccine containing liquid paraffin and Freund's incomplete adjuvant survived challenge at 6 months post vaccination. Freund's incomplete adjuvant and liquid paraffin containing 10% Arlacel A are the most efficient adjuvants.     

Mixed adjuvant formulations reveal a new combination that elicit antibody response comparable to Freund's adjuvants

Adjuvant formulations capable of inducing high titer and high affinity antibody responses would provide a major advance in the development of vaccines to viral infections such as HIV-1. Although oil-in-water emulsions, such as Freund's adjuvant (FCA/FIA), are known to be potent, their toxicity and reactogenicity make them unacceptable for human use. Here, we explored different adjuvants and compared their ability to elicit antibody responses to FCA/FIA. Recombinant soluble trimeric HIV-1 gp140 antigen was formulated in different adjuvants, including FCA/FIA, Carbopol-971P, Carbopol-974P and the licensed adjuvant MF59, or combinations of MF59 and Carbopol. The antigen-adjuvant formulation was administered in a prime-boost regimen into rabbits, and elicitation of antigen binding and neutralizing antibodies (nAbs) was evaluated. When used individually, only FCA/FIA elicited significantly higher titer of nAbs than the control group (gp140 in PBS (p<0.05)). Sequential prime-boost immunizations with different adjuvants did not offer improvements over the use of FCA/FIA or MF59. Remarkably however, the concurrent use of the combination of Carbopol-971P and MF59 induced potent adjuvant activity with significantly higher titer nAbs than FCA/FIA (p<0.05). This combination was not associated with any obvious local or systemic adverse effects. Antibody competition indicated that the majority of the neutralizing activities were directed to the CD4 binding site (CD4bs). Increased antibody titers to the gp41 membrane proximal external region (MPER) and gp120 V3 were detected when the more potent adjuvants were used. These data reveal that the combination of Carbopol-971P and MF59 is unusually potent for eliciting nAbs to a variety of HIV-1 nAb epitopes.     

Vaccines against blowfly strike: the effect of adjuvant type on vaccine effectiveness.

Vaccination of sheep with a partially purified extract of Lucilia cuprina larvae in some cases resulted in marked reduction of growth in larvae which fed on the sheep. Twelve adjuvants were assessed, in vitro and in vivo, to determine which induced the largest inhibitory effect on larval growth. The Freund's complete adjuvant and Quil A groups produced ELISA antibody levels significantly higher (P less than 0.05) than other groups. Seven adjuvants mediated an immune response which caused significant inhibition of larval growth (P less than 0.05). When the sheep were assessed by in vivo larval culture, only larvae feeding on sheep vaccinated with the antigen presented in Freund's complete adjuvant or dextran sulphate or a dextran sulphate/Freund's incomplete adjuvant mixture weighed significantly less (P less than 0.05) than larvae feeding on control sheep. The effect on larvae was monitored in vitro for 70 days after vaccination, by which time significant reduction in larval weight was no longer observed. The loss of larval growth inhibition was not associated with a corresponding reduction in overall antibody levels.     

An evaluation of several adjuvant emulsion regimens for the production of polyclonal antisera in rabbits.

Emulsion adjuvants have been used for production of polyclonal antisera in rabbits (Oryctolagus cunniculi) for decades. Complete Freund's adjuvant has a reputation as a very effective immunoenhancer, but adverse physiological effects, including fever, inflammation and sterile abscess formation, have prompted a search for alternatives to complete Freund's. In this study, we quantitatively compared five adjuvant regimens: (a) a primary inoculation with complete Freund's followed by three boosts with incomplete Freund's; (b) four serial inoculations of incomplete Freund's adjuvant augmented with 6-bromoguanisine; (c) four serial inoculations with RIBI's MPL + TDM + CWS adjuvant emulsion; (d) four serial inoculations with Montanide ISA 50 emulsion; and (e) four serial inoculations with Montanide ISA 70 emulsion. We chose a small (12 amino acid) chain polypeptide coupled to bovine serum albumin as our test antigen. When compared, no system could be seen to be significantly better than a regimen of a primary immunization with complete Freund's adjuvant followed by serial reimmunization with incomplete Freund's adjuvant. The commercially available RIBI adjuvant produced significantly lower antibody levels, while other systems produced essentially equivalent levels. With all five adjuvants, antibody quantities plateaued after the second injection and further immunization did not increase titers significantly. Boost injections did yield greater intradermal tissue reaction than primary inoculations, and intramuscular inoculum volumes of 0.4 cc caused chronic lesions still detectable by the gross necropsy 2 weeks after the final injection.     

Evaluation of ethylene-vinyl acetate copolymer as a non-inflammatory alternative to Freund's complete adjuvant in rabbits

Ethylene-vinyl acetate copolymer (EVAc) was evaluated as an antigen delivery device in laboratory rabbits. Bovine serum albumin (BSA) was incorporated with EVAc in a pellet, which was implanted subcutaneously. Serum antibody titers to BSA in four implanted rabbits were equal to titers in four rabbits injected twice with BSA in complete Freund's adjuvant. Three of four rabbits implanted with EVAc displayed no inflammation or systemic illness in response to the pellet. The fourth rabbit repeatedly developed a small abscess at the implantation site, but the lesions were less severe than complete Freund's adjuvant injection sites. The EVAc pellet is recommended as a non-inflammatory alternative method to Freund's adjuvants for producing serum antibody in rabbits.     

免疫佐剂对幽门螺杆菌免疫原性的影响

幽门螺杆菌是一类高传染性的致病细菌,能导致人类多种疾病。为比较不同佐剂对该致病菌的免疫原性的影响,实验中利用简单的布氏肉汤添加环糊精液体培养基体外培养幽门螺杆菌,分别与福氏佐剂、自制油佐剂、氢氧化铝佐剂混合免疫昆明种小鼠,经间接ELISA法分析抗血清效价证实,三种免疫佐剂都能有效地刺激小鼠对幽门螺杆菌产生明显的体液应答,其中福氏佐剂的效果最好,自制油佐剂略强于氢氧化铝佐剂的免疫活化作用。三者免疫的抗血清效价分别为,福氏佐剂1∶25600,自制油佐剂1∶12800,氢氧化铝佐剂1∶12800。     

Immunogenic Properties of Colloidal Gold

We studied the capacity of colloidal gold for enhancing specific and nonspecific immune response in laboratory animals (rabbits, rats, and mice) immunized with antigens of various nature. The antibody titers obtained with colloidal gold as a carrier were higher as compared to the standard immunization techniques (free antigen or its combination with Freund's adjuvant). Application of colloidal gold also enhanced nonspecific immune responses, such as lysozyme concentration in the blood, activity of the complement system proteins, as well as phagocytic and bactericidal activities. The antibodies were tested by immunodot assay using gold markers. Immunization of the animals with colloidal gold conjugates with haptens or complete antigens (without other adjuvants) was shown to induce the production of highly active antibodies. In addition, the amount of antigen used for animal immunization with colloidal gold was an order of magnitude lower, compared to immunization with complete Freund's adjuvant. This fact can be evidence for adjuvant properties of colloidal gold proper.     

不同佐剂条件下Aβ多肽B细胞表位疫苗诱导产生抗体的免疫反应特性分析

目的:研究阿尔茨海默病β淀粉样肽(Aβ)B细胞表位疫苗2Aβ1-15-PADRE(Aβ-T)诱导产生抗体的免疫反应特性,并探讨不同佐剂对该疫苗免疫反应效果的影响。方法:合成了含2个Aβ42的 B细胞表位—Aβ1-15及1个辅助T细胞表位—PADRE的多肽2Aβ1-15-PADRE。采用Al(OH)₃佐剂,弗氏佐剂,Abisco佐剂,MF59佐剂分别与多肽疫苗联合免疫小鼠,并另设3个对照组:无佐剂多肽免疫组(Mock),PBS免疫组(PBS),未免疫组(Native)。结果:5组多肽免疫组小鼠均产生了针对Aβ的特异性抗体,无佐剂多肽免疫组的IgG抗体滴度最低,Al(OH)₃佐剂组,MF59佐剂组,Abisco佐剂组小鼠IgG抗体滴度较高,弗氏佐剂组IgG抗体滴度最高。斑点杂交实验结果显示5组小鼠免疫后血清与Aβ42单体反应较弱,与寡聚体反应最明显,与纤维状Aβ42几乎不反应。结论:4种佐剂均能提高多肽疫苗的免疫反应,产生高水平抗Aβ的特异性抗体。5组免疫小鼠产生的抗体均与Aβ寡聚体反应较强,与纤维状Aβ42反应较弱,表明该多肽疫苗具有良好的应用前景。     

The generation of monoclonal antibodies in mice: influence of adjuvants on the immune response, fusion efficiency and distress

The objective of this study was to find a reliable alternative to Freund's adjuvant in order to reduce the distress imposed on the animals without impairing the fusion efficiency for immune-positive clones. For this purpose several commercially available adjuvants and adjuvant formulations representing different classes of molecules were compared. Humoral responses and animals' distress evaluated by clinical assessment and histopathological examinations were investigated and compared to fusion efficiencies. In a first set of experiments seven adjuvants were tested essentially to determine their potential to induce distress. Poly(A).poly(U) and GERBU were selected for further investigations due to their low overall toxicity. They were combined with five different antigens and compared to the classic Freund's adjuvant system (CFA/IFA) and to control immunizations without adjuvant. The results showed that adjuvants of very low toxicity could induce a high fusion efficiency. According to a standardized immunization protocol, GERBU induced polyclonal titres similar to Freund's whereas animals treated with poly(A).poly(U) did not attain titres higher than mice immunized with antigen in saline. Poly(A).poly(U) however, exhibited the best fusion efficiency, Freund and GERBU were slightly less efficient. Therefore poly(A).poly(U) and GERBU may serve as valuable alternatives to Freund's adjuvant for generating monoclonal antibodies. Furthermore, these two adjuvants are very easy to use.     

Immunological adjuvants efficiently induce antigen-specific T cell responses in old mice: implications for vaccine adjuvant development in aged individuals

The morbidity and mortality of infectious diseases are significantly increased in aged humans. Hence, vaccination has been suggested as a means to reduce or prevent the impact of infections on old individuals. However, it has remained unresolved whether or not standard vaccine adjuvants such as aluminum hydroxide (Alum) are similarly efficacious in old individuals, as compared to young adults. Here, we have investigated the effects of prototypic immunological adjuvants, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), or Alum on HEL-specific T cell responses in young adult and old mice. We report that independent of the adjuvant used, the induced T cell responses to the prototypic protein antigen hen eggwhite lysozyme (HEL) were similar in young adult and old mice in terms of cytokine production, T cell frequencies, determinant specificity, and T cell repertoire. The results suggest that vaccine adjuvants developed in young adults should be equally effective in inducing T cell immunity in old individuals.     

Adjuvants and antibody production: dispelling the myths associated with Freund's complete and other adjuvants

Adjuvants have been used for more than 70 yr to enhance the immune response of the host animal to an antigen. Among the mechanisms that adjuvants use to enhance the immune response are the "depot" effect, antigen presentation, antigen targeting, immune activation/modulation, and cytotoxic lymphocyte induction. The immunostimulatory properties of adjuvants result in inflammation, tissue destruction, and the potential for resulting pain and distress in the host animal. The inflammatory lesions produced by adjuvants such as Freund's complete adjuvant (FCA) have led some to conclude that pain and distress are present, even in cases where the scientific evidence fails to support this conclusion. Recommendations and regulations in the literature, based on available scientific evidence, provide guidance on total adjuvant volumes, volumes per site, routes of injection, booster injections, and adjuvants used for antibody production. Among the numerous adjuvants that are used for experimental antibody production reviewed in this article, many claim to be less inflammatory, tissue destructive, and painful than FCA while producing equal or superior antibody responses. Although no adjuvant surpasses FCA for experimental antibody production against a wide range of antigenic molecules, many produce excellent antibody responses with less inflammation and tissue destruction. Balancing the requisite degree of immuno-stimulation and the extent of inflammation, necrosis, and potential pain and distress requires consideration of the nature of the antigen, the host immune responsiveness, the adjuvant's mechanisms of action, and the desired end-product. In cases where the antigen is a weak immunogen or has a very limited availability, the type and role of adjuvant becomes a critical component in producing an acceptable immune response and humoral antibody response.     

Stable biocompatible adjuvants--a new type of adjuvant based on solid lipid nanoparticles: a study on cytotoxicity,compatibility and efficacy in chicken

A new type of adjuvant was tested for its ability to initiate antibody production in chickens, and its cellular and tissue compatibility were assessed. The stable biocompatible adjuvants tested are based on surface-modified solid lipid nanoparticles (SLNs), made from paraffin or biodegradable glycerides, and are simply admixed to the antigens before administration. The tissue-damaging potency of four formulations of the new adjuvants (H1, H2, H3 and H4) were first tested in vitro by using human foreskin fibroblasts and RAW 264.7 macrophages. The adjuvants were well tolerated by both cell types. Immunisation studies in chickens were performed by using a Mycoplasma bovis antigen and mouse immunoglobulin G (IgG). The resulting antibodies were non-invasively extracted from egg yolk. The use of the various adjuvant formulations resulted in a significant production of specific antibodies after the first and second booster immunisations. Freund's complete adjuvant (FCA), considered until now to be the "gold standard" among the adjuvants, revealed the highest antibody titre against mouse IgG. SLNs with a particle size of more than 100 nm exhibited a clear adjuvant activity, whereas SLNs with a particle size below 100 nm, in various concentrations, revealed a lower adjuvant activity. Immunisation of chickens with the mouse IgG alone, dissolved in phosphate-buffered saline, resulted in a slow antibody titre development. At the end of the experiment, the chickens were examined for vaccination-associated tissue damage. In contrast to FCA, the SLN formulations caused only minor tissue irritation at the injection sites. In conclusion, SLNs seem to be a promising alternative to FCA for antibody production in chickens, and potentially in other animals.     

Induction of protective immunity against S. mansoni in mice vaccinated with Sm10-DLC

Sm10-DLC, a 10-kDa dynein light chain protein identified as a strong T cell immunogen in a large number of subjects sensitized by natural infections, may be of interest for vaccination. To assess the vaccine potential of Sm10 we carried out immunization trials in CBA/J mice using recombinant Sm10 (rSm10) expressed in E. coli and tested its capacity to induce protection against a challenge infection. With one rSm10 preparation injected intramuscularly in Freund's adjuvant, a significant reduction in worm burden (27% reduction) was obtained in two independent experiments. We have not obtained protection with other adjuvants, in particular with alum. In addition, a negative correlation was observed between the antibody response and the worm burden reduction. These results suggest that rSm10 injected with Freund's adjuvant was able to induce a protective immunity against Schistosoma mansoni.     

Learning impairment in honey bees caused by agricultural spray adjuvants

Background

Spray adjuvants are often applied to crops in conjunction with agricultural pesticides in order to boost the efficacy of the active ingredient(s). The adjuvants themselves are largely assumed to be biologically inert and are therefore subject to minimal scrutiny and toxicological testing by regulatory agencies. Honey bees are exposed to a wide array of pesticides as they conduct normal foraging operations, meaning that they are likely exposed to spray adjuvants as well. It was previously unknown whether these agrochemicals have any deleterious effects on honey bee behavior.

Methodology/Principal Findings

An improved, automated version of the proboscis extension reflex (PER) assay with a high degree of trial-to-trial reproducibility was used to measure the olfactory learning ability of honey bees treated orally with sublethal doses of the most widely used spray adjuvants on almonds in the Central Valley of California. Three different adjuvant classes (nonionic surfactants, crop oil concentrates, and organosilicone surfactants) were investigated in this study. Learning was impaired after ingestion of 20 µg organosilicone surfactant, indicating harmful effects on honey bees caused by agrochemicals previously believed to be innocuous. Organosilicones were more active than the nonionic adjuvants, while the crop oil concentrates were inactive. Ingestion was required for the tested adjuvant to have an effect on learning, as exposure via antennal contact only induced no level of impairment.

Conclusions/Significance

A decrease in percent conditioned response after ingestion of organosilicone surfactants has been demonstrated here for the first time. Olfactory learning is important for foraging honey bees because it allows them to exploit the most productive floral resources in an area at any given time. Impairment of this learning ability may have serious implications for foraging efficiency at the colony level, as well as potentially many social interactions. Organosilicone spray adjuvants may therefore contribute to the ongoing global decline in honey bee health.     

Role of adjuvants in inhibitory influence of immunization with porcine zona pellucida antigen (ZP-3) on ovarian folliculogenesis in bonnet monkeys: a morphological study

Female bonnet monkeys were immunized with 55 kDa porcine zona antigen (ZP-3), with either complete Freund's adjuvant (CFA) or sodium phthalylated lipopolysaccharide (SPLPS) as adjuvant. Anti-ZP-3 antibody titers were monitored and the effect of immunization on the ovarian morphology was assessed by high-resolution light microscopy. The study demonstrated that both adjuvants used were equally potent in eliciting antibody response against ZP-3. Although no morphological damage to ovarian components was observed in animals immunized with SPLPS as adjuvant, immunization using CFA resulted in profound ovarian follicular atrophy, sparing only the primordial follicles. The atrophic phenomenon involved those follicles that either already had or were in the process of forming zona pellucida. The results of this study indicate that choice of adjuvant may be an important consideration for immunization against zona antigens. These findings encourage further investigations for developing better immunization regimen aimed at using zona antigens for immunocontraception.     

The immune response of the brown trout, Salmo trutta, L. to MS2 bacteriophage: immunogen concentration and adjuvants

The humoral antibody response to single intraperitoneal inoculations of MS2 bacteriophage was followed in Salmo trutta using a 50% bacteriophage neutralisation litre ( sd ₅₀) method. Immune memory was observed with enhancement of both the time and level of antibody production, though an initial suppression was observed 7 days after secondary inoculation. Antibody titres were related to MS2 concentration, and adjuvants were found to enhance the response. An increased enhancement of antibody production by Freund's complete adjuvant over that of incomplete adjuvant was observed.     

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.     

Comparison of immune and adverse effects induced by AdjuVac and Freund's complete adjuvant in New Zealand white rabbits (Oryctolagus cuniculus)

Though Freund's complete adjuvant effectively increases immune response to vaccines in various species, its potentially severe inflammatory effects have led many animal researchers to seek alternative immunological adjuvants. In a study of New Zealand white rabbits, the authors compared the immune and adverse effects of Freund's complete adjuvant with the effects of two formulations of AdjuVac, an immunological adjuvant previously developed by their group. All three adjuvants improved humoral immune response but also caused inflammation. Inflammatory reactions caused by AdjuVac, however, tended to be less severe than those caused by Freund's complete adjuvant.     

Does Freund's adjuvant denature protein antigens? EPR studies of emulsified hemoglobin.

Use of complete Freund's adjuvant for production of antibodies to study protein conformation is valid only if emulsification in adjuvant does not denature protein antigens. Using electron paramagnetic resonance to observe directly the protein in situ in the opaque emulsions, we demonstrate that hemoglobin is not denatured by emulsification or storage in adjuvant for 24 hr at 4 degrees C, conditions comparable to the usual handling of antigens before immunization.