Effect of adjuvants on antibody responses of sheep immunised with recombinant pili from Dichelobacter nodosus

Objective To compare the effects of two oil emulsion adjuvants (incomplete Freunds adjuvant and a proprietary oil adjuvant), DEAE-dextran, L-tyrosine particles and Quil A on the humoral immune responses of sheep immunised with recombinant pili of Dichelobacter nodosus (strain A).
Procedure Antibody titres were studied for up to 32 weeks and were measured by bacterial agglutination and ELISA. The relative avidity of antibodies for pili was determined and the incidence and severity of adverse reactions at the site of injection of vaccines were recorded.
Results The oil emulsion adjuvants and Quil A were more effective than either DEAE-dextran or L-tyrosine at stimulating antibodies in sheep. The incidence and severity of adverse reactions was lower in sheep which received vaccines containing either Quil A or DEAE-dextran than in sheep which received vaccines containing oil emulsion adjuvants. L-tyrosine had no adverse effects.
Conclusion Quil A was as effective as oil adjuvants at stimulating high levels of antibodies against recombinant pili in sheep and had the significant advantage of being less irritant after subcutaneous injection.     

Immune response and reactions to various dose regimens for raising hyperimmune antisera in sheep

To determine the optimum procedure for raising hyperimmune sera to tetanus toxin, three adjuvants, four antigen preparations and two routes of administration in various combinations were investigated in sheep. Oil-in-water adjuvants alone or in combination with aluminum gels were superior to aluminium gels on their own. This disadvantage of aluminium gels was partially but not completely abrogated when the frequency of doses was increased to three per week. Intensity of local reaction was strongly correlated with immune response; the more immunogenic a dose, the more reactive. Reactivity of oily adjuvants could be lessened by use of a more suitable route of administration, thus oily adjuvants appeared suitable for use when administered by the intraperitoneal route even though moderate to severe reactions resulted from subcutaneous injections. Of other variables investigated, toxin did not confer any advantage over toxoid as an immunogen, purified toxoid was a significantly better immunogen than unpurified toxoid and two large bleeds (30% of total blood volume each) every six weeks rather than 20 ml test bleeds did not affect the titre of the hyperimmune serum produced.     

Adjuvant regulation of cytokine profile and antibody isotype of immune responses to Mycoplasma agalactiae in mice

Traditionally, adjuvants have been administered with antigens to enhance immunity. We studied the effect of several adjuvants such as Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS), homopolymers of polyinosinic-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U), lithium chloride (LiCl), saponin Quil A and calcium phosphate gel (CaHPO(4)) on the immune response of mice to formalin-inactivated Mycoplasma agalactiae. The specific antibody or cytokine producing splenocytes were detected by ELISAspot and immunocytochemistry, respectively. Depending on the adjuvant given, the number of M. agalactiae-specific antibody producing cells was increased 2.5-6-fold. IgG was the major class of M. agalactiae-specific antibodies followed by IgM, IgA and IgE. Among IgG isotypes, FCA, FIA, Quil A and CaHPO(4) induced an IgG1 response with substantial increase of the IgG2a, IgG2b and IgG3 isotypes while poly I:C shifted the response toward an IgG2a/IgG3 production. Finally, poly A:U induced an IgG2b response while LPS and LiCl augmented the IgG3/IgG1/IgG2a secretion. FCA augmented IL-4, IL-5 and IL-10 production suggesting a strong Th2 response, while IFN-gamma and IL-12 remained low; poly I:C enhanced IFN-gamma, IL-12 and TNF-alpha eliciting a Th1 response; poly A:U resulted in a IL-10, IL-5, IL-6 and IL-12 secretion; and LPS enhanced the IL-10, IL-6 and TNF-alpha production. Our data show that adjuvants augment M. agalactiae-specific antibody production and lead to B cell isotype-switching via the appropriate cytokine milieu. Certain adjuvants, such as poly I:C, therefore, appear as promising immune enhancers for vaccination against M. agalactiae infections.     

Assessment of the immunogenicity of the leptospiral LipL32, LigAni,and LigBrep recombinant proteins in the sheep model

Veterinary leptospirosis vaccines are composed of bacterins and present limitations, for example, the need for bacteriological culture and serovar-dependent immunity. Recombinant antigens represent a promising alternative. LigAni, LigBrep, and LipL32 proteins have been shown to promote a protective immune response against the homologous challenge in hamsters. Therefore, the next step is to evaluate the immunological properties of these immunogens in the actual hosts, as ruminants, which has never been performed before. The objective of this study was to evaluate the immunogenicity and potential adverse effects of the recombinant proteins LigAni, LigBrep, and LipL32 in the ovine model. For this, 16 Santa Inês sheep were allocated into three groups: two experimental (Groups A and B) and one control group (Group C). Group A was inoculated with a formulation containing the recombinant proteins in combination with the aluminum hydroxide adjuvant; Group B was inoculated with a formulation containing the recombinant proteins in combination with the Montanide adjuvant; and Group C was inoculated with adjuvants only. The results revealed that formulations containing the recombinant proteins induced total IgG seroconversion and led to a significant increase in antibody titers in the sheep model. Besides, there were no clinical changes or adverse effects. Thus, LigAni, LigBrep, and LipL32 proteins elicited a significant humoral immune response with elevated serum IgG levels, demonstrating that they possess the immunogenic and safety characteristics necessary to sustain their potential use as leptospirosis vaccines in the ruminant model.     

Adjuvant enhancement of humoral immune response to chemically inactivated bovine viral diarrhea virus.

Potentiation of the antibody response to inactivated bovine viral diarrhea virus by immunological adjuvants was studied in guinea pigs and cattle. The inactivated bovine viral diarrhea virus alone was demonstrated to be a weak immunogen. Addition of either 2 mg per mL diethylaminoethyl-dextran or 5% alhydrogel to inactivated bovine viral diarrhea virus did not or only slightly stimulated the antibody response; the combined adjuvants induced a significantly higher titer. A higher concentration (20 mg per mL) of diethylaminoethyl-dextran, on the contrary, suppressed the immune potentiation by the dual adjuvants. Combination of Bordetella bronchiseptica and alhydrogel adjuvants stimulated a high titer of antibody. The titer was further elevated upon revaccination and was significantly higher than that induced by alhydrogel alone. In cattle, alhydrogel enhanced the immune response and the additional inclusion of diethylaminoethyl-dextran did not cause a significant potentiation of the immunity. However, the antibody decay rate was significantly slower when stimulated by the combined adjuvants.     

Effects of adjuvants on the immune response of staphylococcal alpha toxin and capsular polysaccharide (CPS) in rabbit

This study was performed to isolate a vaccine strain of S. aureus from clinical or subclinical mastitis and to choose the most optimal adjuvant for immune response of alpha toxin and capsular polysaccharide (CPS) of field strain. Of thirty strains of S. aureus isolated from milk of clinical or subclinical mastitis, V112 strain isolated from milk of gangrenous mastitis was used in this vaccine. Twenty one of rabbits were allocated into 5 groups based on adjuvants and immunized twice every 2 weeks for 8 weeks. This vaccine was composed of alpha toxin (10 hemolytic units) and formalinized whole cells (1 x 10(11) cells/ml. Five rabbits received PBS solution as a control group. The highest antibody titers against alpha toxin and CPS were observed in dextran sulfate- and aluminium hydroxide-adjuvant group at 8 weeks after immunization, respectively. These results of the study showed that one adjuvant could not induce strong and long-term immune response of alpha toxin and CPS antigens. Therefore, the use of combined adjuvants in subunit vaccine may be useful and feasible.     

Sheep erythrocyte and bluetongue virus antibody responses of spleen cell cultures from mice.

The optimum conditions for the culture of cells from dissociated spleens were determined. Routinely, 10(7) cells were seeded per ml of RPMI 1640 medium supplemented with 20% pre-tested foetal calf serum. For the assay of the immune response, cultures were supplemented with 30 muMolar mercaptoethanol. The immune responses to sheep erythrocyte and bluetongue virus antigens were determined by the haemolytic plaque-forming cell assays described by Oellermann (1974) and Oellermann, Carter & Marx (1976a). The optimum sheep erythrocyte antigen concentration was 2 X 10(6) erythrocytes per 10(7) spleen cells and maximum IgM plaque-forming cells were detected after 4 days in culture. Successful stimulation of the immune response to bluetongue virus was achieved in spleen cell cultures from mice previously primed with bluetongue virus. The optimum antigen concentration was 30-40 ng bluetongue virus per 10(7) spleen cells and the maximum plaque-forming cell response was observed after 4 days in culture.     

抑制素主动免疫对大鼠生殖激素及卵巢发育的影响:Montanide和Freund’s佐剂效应比较

本研究采用新疆细毛羊抑制素成熟区序列重组融合蛋白(roINH)作为免疫原,以Montanide (MON)和Freund's (FA)作为佐剂,主动免疫SD大鼠,测定生殖激素及卵巢发育情况,评价佐剂效应.选用72只9～10周龄性成熟雌性SD大鼠,随机分为3组,分别皮下注射生理盐水(对照组)、roINH+ MON(MON组)和roINH+FA (FA组).各组大鼠每20 d免疫1次,连续免疫3次.结果,MON组产生的抗体滴度显著高于FA组(P＜0.05);与对照组相比,注射roINH+MON和roINH+FA均可极显著提高大鼠血清FSH平均含量(P＜0.01),显著提高P峰值(P＜0.05),而FSH及P平均含量及峰值在2个免疫组间无显著差异(P＞0.05);LH含量和峰值在2个试验组间无显著差异(P＞0.05); MON和FA组成熟卵泡数无显著差异(P＞0.05),但均显著高于对照组(P＜0.05);MON组脓包直径及炎症反应小于FA组.结果表明,应用roINH作为免疫原并配以MON或FA佐剂免疫大鼠均能取得较好的免疫效果,且MON佐剂炎症反应小于FA佐剂.     

猪圆环病毒2型灭活疫苗免疫佐剂的比较试验

以猪圆环病毒2型(PCV2)遗传标记毒株为毒种,经细胞培养传代,优化了病毒增殖条件,获得较高滴度的病毒培养物用于PCV2灭活疫苗的研制。为了比较不同免疫佐剂的效果,本试验对国产矿物质白油和铝胶两种佐剂以及赛比克公司提供的MONTANIDETMISA206、ISA15VG、IMS1315VG3种佐剂配制的灭活疫苗进行了猪体免疫试验。通过临床观察、血清抗体效价测定,确认ISA15VG佐剂配制的疫苗在增强免疫效果方面优于其它佐剂。按5种佐剂免疫效果排序为ISA15VG、IMS1315VG、铝胶、ISA206、国产白油。ISA15VG佐剂属于水包油剂型,可刺激接种动物产生快速免疫应答反应,抗体产生效价高、持续时间长,有可能成为新型PCV2灭活疫苗佐剂的候选。     

Evaluation of three experimental bovine viral diarrhea virus killed vaccines adjuvanted with combinations of Quil A cholesterol and dimethyldioctadecylammonium (DDA) bromide

Bovine viral diarrhea virus (BVDV) infections cause respiratory, reproductive, and enteric disease in cattle. Vaccination raises herd resistance and limits the spread of BVDV among cattle. Both killed and modified live vaccines against BVDV are available. While modified live vaccines elicit an immune response with a broader range and a longer duration of immunity, killed vaccines are considered to be safer. One way to improve the performance of killed vaccines is to develop new adjuvants. The goal of this research was evaluate new adjuvants, consisting of combinations of Quil A cholesterol and dimethyldioctadecylammonium (DDA) bromide, for use in killed vaccines. Responses to three novel killed vaccines, using combinations of Quil A and DDA as adjuvants, were compared to responses to a commercial modified live and a commercial killed vaccine. Vaccination response was monitored by measuring viral neutralizing antibodies (VN) levels and by response to challenge. All three novel vaccines were efficacious based on reduction in virus isolation, pyrexia, and depression. Compared to a commercial killed vaccine, the three novel vaccines elicited higher VN levels and reduced injection site inflammation.     

Experimental maedi virus infection in sheep: cellular and humoral immune response during three years following intranasal inoculation

A long-term experiment in sheep inoculated intranasally with 2 strains of Norwegian maedi virus was carried out in 2 groups of Norwegian Dala sheep (7 sheep/group). Virus-specific cellular immune response was assayed in the lymphocyte transformation test sequentially during 3 years after sheep were inoculated in group 1 and 4 times in the 3rd year in group 2. Humoral immune response was assayed by immunodiffusion, complement-fixation, and neutralization tests on sequential serum samples collected from the 2 groups. Attempts to isolate virus were made. All group 1 sheep showed transient and irregularly recurring cellular immune responses. In group 2, 6 of the 7 sheep gave similar responses. The frequency of virus isolations was low compared with that reported by various research workers using other breeds for studying experimental maedi-visna infection. Precipitating antibodies were detected earlier, and in more animals, than were complement-fixing antibodies. Both were, however, detected later and less frequently than were reported by other research workers. There was a marked difference in the capability of the 2 maedi virus strains to induce neutralizing antibodies. The sequential sera usually showed distinct differences in neutralizing capacity of the virus strains, indicating that they are antigenically different.     

Effect of four adjuvants on immune response to F4 fimbriae in chickens

IgG antibody response in chickens immunized with F4 fimbriae extracted from local enterotoxigenic Escherichia coli (ETEC) strain was studied during a 98-day immunization period for comparing the efficacy of four adjuvants: Freund' adjuvant, Quil A (QA), propolis and extract from Cochinchina momordica seed (ECMS). For this purpose, chickens were immunized with F4 fimbriae alone or combined with one of the above adjuvants on days 1 and 21. IgG antibody levels in serum and egg yolk (by ELISA) were measured on days 0, 7, 14, 21, 28, 35, 42, 49, 56, 70, 84 and 98. The egg production of each group was also determined during days 1-7 and the following four weeks. The results showed that QA could enhance antibody titre, as good or almost as good as Freund's adjuvant, whereas the titres of ECMS and propolis groups were relatively lower, with the overall order: Freund's adjuvant>QA>ECMS>propolis both in serum and egg yolk. However, the significant decrease of egg production was merely observed in the Freund's adjuvant group. It is concluded that the four adjuvants tested can stimulate immune response to F4 fimbriae in chickens, with Freund's adjuvant giving the best results, followed by QA.     

Haemonchus contortus infection in sheep: parasite fecundity correlates with worm size and host lymphocyte counts

Two experiments were conducted to elucidate the timing and nature of the sheep immune response to Haemonchus contortus (Barber's pole worm). The first experiment examined the establishment of H. contortus populations and the immune response by comparing a bolus infection of third-stage larvae in na?ve sheep with a group previously primed by a trickle infection. The second experiment used staggered doses of ivermectin-resistant larvae to compare the development of adult worms during different durations of trickle infection with ivermectin-sensitive larvae. Infections successfully generated pathological signs of haemonchosis such as anaemia. Image analysis software was used to measure the area and perimeter of worms collected at post-mortem, and the number of eggs present in individual adult females (fecundity) was significantly correlated with worm size. A significant inverse correlation was found between blood lymphocyte counts and worm fecundity. The absence of correlation between worm fecundity and other leukocyte and erythrocyte counts highlighted the specificity of the lymphocyte response. This is the first report of a link between haematology profiles and worm fecundity in haemonchosis. The correlation observed between adult worm size and egg content leads to the hypothesis that egg production in H. contortus is limited by immune regulation of worm size and presumably growth. Mean worm size and fecundity declined as sheep received more prolonged trickle infections before necropsy, confirming previous reports that immune responses to adult worms are enhanced by ongoing larval challenge. Immunohistochemical results showed trends consistent with a Th2 (humoral) immune response which has been implicated in reducing nematode burdens in several species.     

Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccine formulated with saponin or selected adjuvants.

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.     

Enzyme-linked immunosorbent assay for detection of bluetongue virus antibodies

The immune response to bluetongue virus in sheep and cattle was studied by applying a newly developed indirect enzyme-linked immunosorbent assay (ELISA). Purified virus obtained by sucrose gradient centrifugation was used at a concentration of 0.01 optical density units (formula: see text) to coat individual wells (200 microliter) of a microtitration plate. Dilution of antigen was performed in 0.05 M carbonate buffer, pH 9.6, and adsorption lasted for at least 16 hours at 4 C. Coated plates retained their activity for 10 weeks when stored at 4 C. Sera recovered from experimentally infected sheep and cattle were tested together with known negative sera. A good correlation between results was obtained with the modified complement-fixation test and the ELISA; however, the ELISA proved to be more sensitive. The group specificity of the ELISA was proven by testing various type-specific sheep and cattle immune sera. The ELISA has potential for the detection of group-specific antibodies to bluetongue virus infection.     

Immunity in neonates

Passively derived maternal immunity hampers active immunization of newborns. Further, an immature immune system contributes to a weak and Th2 polarized immunity. This state of immunity in early life sustains endemic infections in man and continuous reinfections in animal herds. The endemic infections of the young occur preferentially when the immune system is still functionally immature and when the low levels of maternal antibodies are no longer protective but yet blocks protective immune responses. Vaccines overcoming these problems would have strong positive effects on the herd health and environmental benefits. The Th2 bias of the newborn is mediated by high levels of progesterone and Th2 cytokines produced in the maternal-fetal interface. The activity of the innate system is enhanced in the mother during the prepartus period, certainly having effects on the offspring. Newborn, 2-days-old, mice can be primed with Sendai virus envelope proteins as model antigens to induce Th1 or Th2 responses, dependent on the supplementation of the virus antigen formulation with Th1 or Th2 adjuvants. This priming has a strong life-long effect when complemented with subsequent boosts. However and importantly this priming effect can be modulated by adjuvants focusing for Th1 and Th2 when applied to the mice at 6 weeks of age, i.e. when they are immunologically adult. It has been shown in various species, besides mice, i.e. dog, sheep, horse and seal, that a strong Th1 driving adjuvant can induce immune response and protection in newborns when conventional vaccines fail. In conclusion, the Th2 bias prevailing around partus can be overcome by appropriate immunological treatments, permitting effective vaccination and protective immunity in the newborn.     

Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccine formulated with saponin or selected adjuvants

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.     

Border disease: virus persistence, antibody response and transmission studies

Three groups of five and one group of four oestrus-synchronised sheep were inoculated with Border disease (BD) virus at 52 +/- 2 days after their first service. Transmission of virus to offspring as demonstrated by virus isolation, detection of viral antigen and, or antibody response occurred in 12 of 19 sheep and probably in four others which aborted or produced stillborn lambs. Both apparently normal and clinically affected animals excreted virus in saliva, urine and faeces, and excretion and contact transmission to sheep and pigs persisted for up to two and a half years. Most of the tissues of infected sheep contained virus titres between 10(3.5) and 10(5.5) TCID50 per g. The immune response in the lambs varied, in some it began before birth, in others a transient or low level response was observed in the first or second year, while others remained serologically negative for two and a half years.     

Assessing humoral and cell-mediated immune response in Hawaiian green turtles, Chelonia mydas

Seven immature green turtles, Chelonia mydas, captured from Kaneohe Bay on the island of Oahu were used to evaluate methods for assessing their immune response. Two turtles each were immunized intramuscularly with egg white lysozyme (EWL) in Freund's complete adjuvant, Gerbu, or ISA-70; a seventh turtle was immunized with saline only and served as a control. Humoral immune response was measured with an indirect enzyme linked immunosorbent assay (ELISA). Cell-mediated immune response was measured using in vitro cell proliferation assays (CPA) using whole blood or peripheral blood mononuclear cells (PBM) cultured with concanavalin A (ConA), phytohaemagglutinin (PHA), or soluble egg EWL antigen. All turtles, except for one immunized with Gerbu and the control, produced a detectable humoral immune response by 6 weeks which persisted for at least 14 weeks after a single immunization. All turtles produced an anamnestic humoral immune response after secondary immunization. Antigen specific cell-mediated immune response in PBM was seen in all turtles either after primary or secondary immunization, but it was not as consistent as humoral immune response; antigen specific cell-mediated immune response in whole blood was rarely seen. Mononuclear cells had significantly higher stimulation indices than whole blood regardless of adjuvant, however, results with whole blood had lower variability. Both Gerbu and ISA-70 appeared to potentiate the cell-mediated immune response when PBM or whole blood were cultured with PHA. This is the first time cell proliferation assays have been compared between whole blood and PBM for reptiles. This is also the first demonstration of antigen specific cell-mediated response in reptiles. Cell proliferation assays allowed us to evaluate the cell-mediated immune response of green turtles. However, CPA may be less reliable than ELISA for detecting antigen specific immune response. Either of the three adjuvants appears suitable to safely elicit a detectable immune response in green turtles.     

Influence of adjuvant formulation on the induced protection of mice immunized with total soluble antigen of Trichinella spiralis

Vaccination of pigs against the helminth nematode Trichinella could be a good alternative to prevent the risk of human infection. In order to develop an efficient and safe vaccine, the choice of the adjuvant is an important issue. In this study, two adjuvants were selected to prepare vaccines based on total soluble Trichinella spiralis muscle larvae (ML) antigen: Montanide ISA 70 water in oil emulsion and Montanide IMS nanoparticles. Aluminium hydroxide was used as a reference adjuvant. The immune response was checked by ELISA of parasite antigen specific IgG1 and IgE. Finally, protection induced in vaccinated mice was measured after a T. spiralis challenge by counting ML burdens. The results clearly showed an impact of adjuvants on the specific IgG1 and IgE antibody responses against T. spiralis. Differences were observed between the rates of protection induced according to the type of formulation, although the three adjuvants tested were able to enhance the humoral immune response. This work demonstrated the need to use an adjuvant to obtain a specific IgG1 and IgE responses directed against the total soluble extract of T. spiralis.