Journal of Immmmlogieal Methods, 153 (1992)31-41J
3I
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$115.1~1
JIM06382
A comparison of commercially available adjuvants for use in research B e t h B e n n e t t , I r e n e J. C h e c k , M a r g a r e t R. O l s e n a n d R o b e r t L. H u n t e r Department of Pathology and Laboratory Medicine, Emory Unirersity, Atlanta, GA 30322, USA
(Received 15 January 1992,revised received 26 March 1992,accepted 27 March 1992)
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 ( L H R H ) 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. Key words: lmmunoadjuvant;TiterMax; Complete Freund's adjuvant; Antibody titer
Correspondence to: B. Bennett, Department of Pathology, Room 760 WMB, 1639 Pierce Drive, Empty University, Atlanta, GA 30322, USA. Tel.: 4{14-727-5933;Fax: 404-727-2991. t Supported by grant #'6-38943 from CytRx Corporation to Empty University. Robert Hunter is Vice President and Director of Research for CytRx Corporation. This study was presented as a poster at the 1991 FASEB Meeting (abstract #1464). Abbreciations: HEA, hen egg albumin: LHRH-BSA, luteinizing hormone-releasing hormone conjugated to BSA, OPD, ortho-phenylenediamine; POE, polyoxyethylene;POP, polyoxypropylene; RAS, Ribi adjuvant system; TNBS, picrylsulfonic acid; TNP-BSA, trinitrophenol conjugated to bovine serum albumin; TNP-HEA, trinitrophenol conjugated to hen egg albumin; F68, poloxamer 188; PBS, phosphate-buffered saline.
Introduction Complete Freund's adjuvant (CFA) has been used for over 50 years for producing antisera in animals (Freund et al., 1937; Freund, 19511. The severity of its toxicity was recognized immediately, but attempts to find a less toxic and equally effective alternative have not been successful. CFA induces both local and systemic reactions. The site of injection frequently develops hypersensitivity granuloma~ which may ulcerate to form draining abscesses. Particles of the emulsion may spread through the body producing metastatic
granulomas. This may be accompanied by wasting disease, adjuvant arthritis, or other autoimmune conditions (Amyx, 1987; Broderson, 1989). The arthritis can be particularly debilitating especially in larger animals. Investigations of pain research have clearly documented that CFA can produce severe chronic pain and suffering in small animals as well (Amyx, 1987; Broderson, 1989). With advances in many areas of the biological sciences and increasing concern for the welfare of experimental animals, there is increased pressure to ban or restrict the use of CFA. In 1981, we described the adjuvant activity of nonionic block eopolymers (Hunter et al., 1981). These are synthetic surface active agents consisting of a chain, or block, of hydrophobic polyoxypropylene (POP) flanked by two chains of hydrophilic polyoxyethylene (POE). These copolymers bind antigen, as well as complement and other mediators in a way that is thought to produce efficient presentation of antigen to cells in lymph nodes (Hunter et al, 1984, 1986). They induced high, long-lasting antibody titers with minimal toxicity when formulated in oil-in-water emulsions (Hunter et al., 1984, 1986, 1991; Takayama et al., 1991). Such preparations, however, were effective with only a limited spectrum of antigens. We undertook development of an adjuvant formulation which would utilize the potent adjuvant activity of block copolymers in a formulation that would be effective with a wide variety of antigens. In general, adjuvants function best if they facilitate the simultaneous delivery, of the antigen and immunomodulating substances to lymphoid tissue. CFA does this by containing virtually any antigen together with killed mycobaeteria as an immunostimulant in a water-inoil emulsion. The toxicity of CFA has been attributed to both the mineral oil and mycobacteria (Amyx, 1987; Broderson, 1989). We found that squalene, combined with copolymer CRL89-41 bonded to micropartieulate silica, could be used to produce water-in-oil emulsions which were more stable and easier to prepare than CFA. Squalene, a metabolizable oil, is a normal body component. This paper reports two comparisons of the new adjuvant formulation, TiterMax, with other com-
mercially available adjuvants. Studies on the dose and route of injection are included. The results indicate that TiterMax is a safe and effective alternative to CFA. Materials and methods
TNP-HEA antigen Hen egg albumin (HEA, Sigma Chemical Co., St. Louis, MO) was conjugated to picrylsulfonie acid (TNBS, Sigma Chemical Co., St. Louis, MO) in borate buffer, pH 8.5 (Garvey et al., 1977). The hapten-to-protein ratio (9.4:1) was calculated using a molar extinction coefficient of 15,400 at 350 nm for TNP-aminocaproate and a molecular weight of 45,000 for HEA. LHRH-BSA antigen 100/zl of BSA (160 mg/mi in distilled water) was mixed with 100 /zl of ethyl-dimethylaminopropyi earbodiimide-HCI (EDC (300 mg/ml in distilled water)). A total of 5 mg of LHRH (dissoZved in 500 ttl distilled water) was added in 100 tzl portions at 5 rain intervals. The reaction mixture was incubated for 1 h (including the 20 min to add the LHRH) at room temperature, then dialyzed for 24 h (Jennes et ai., 1983). The percent conjugation was 48% and the molar ratio of peptide:BSA was 9:1. Adjuvants TiterMax #R-1 was provided by CytRx Corporation, Norcross, GA. CFA used in the TNP-HEA experiments was purchased from GIBCO, Grand Island, NY and that used for the LHRH-BSA experiments was purchased from Difco, Detroit, Michigan. Ribi adjuvant system (RAS #R-700) was purchased from Ribi Immunochem Research Inc., Hamilton, MT, Adjuvax from Alpha-Beta Technology, Worcester, MA, Lipovant from Accurate Chemical and Scientific Corp., Westbury, NY and Alhydrogel (trademark of Superfos, Denmark) from Accurate Chemical and Scientific Corp., Westbury, NY. Animals Mice and rats. 7-week-old, female, outbred ICR mice (virus and pathogen free) or 6-week-old female rats
were purchased from Harlan Sprague-Dawley (Birmingham, AL facility) and were used for the T N P - H E A experiments. Heparinized plasma was collected by retro-orbital plexus bleeding for mice and from tail vein bleeding for rats. Balb/c mice, randomized sexes, were used for the LHRH-BSA experiments (Charles River Laboratories, Wilmington, MA). Serum was collected by tail vein bleeding. All animals were anesthetized prior to blood collection using metofane. Rabbits. Adult New Zealand White female rabbits from Myrtles Rabbitry (Thompson Station, TN) were used for the T N P - H E A experiments and from Hazleton Washington for LHRH-BSA experiments. Heparinized plasma was collected for the T N P - H E A studies by ear vein bleeding. Serum was collected for the LHRH-BSA studies also by ear vein bleeding. Guinea pigs. Hartley 400 g female guinea pigs (Charles River Laboratories, Kingston, NY facility) were used. Heparinized plasma was collected by collecting blood from a clipped toe pad. Animals were anesthetized prior to blood collection using metofane. Goats. Mixed breed male goats (Hazleton Washington) were used. Serum was collected by neck vein bleeding. Experiments using T N P - H E A were performed at Emory University and all animals were maintained in the animal facilities at Emory University. Experiments using LHRH-BSA were performed at Hazleton Washington, Vienna, VA. In both facilities, animals were routinely screened for viral, bacterial and mycoplasma infections. Commercial food and tap water were provided ad libitum. The temperature range of the animal rooms was 22-25°C and the relative humidity was approximately 50%. Subdued lighting, 12 h on and 12 h off cycle, was used to illuminate the rooms.
Immunization protocols Water-in-oil emulsions of TiterMax, CFA, or IFA were prepared as follows: 0.5 ml of aqueous antigen was added to 1.0 ml oil phase, emulsified to a water-in-oil emulsion and further emulsified with an additional 0.5 mi of aqueous antigen. Stability of the water-in-oil emulsions of CFA
and TiterMax were assessed by placing a drop of the emulsion onto the surface of water. Adjuvax, RAS, Lipovant and AIhydrogel were prepared according to the manufacturers' protocols. Briefly, aqueous antigen was mixed with 1 mg Adjuvax and hydrated by drawing the mixture in and out of an 18 gauge needle 8-10 times. An oil-in-water emulsion of RAS was prepared by warming the vial of adjuvant and adding antigen in sterile saline to the vial and vortexing for several minutes. Antigen in water was added to the Lipovant vial and the vial was vigorously shaken for several seconds. Alhydrogei was mixed with aqueous antigen to allow adsorption of the antigen to alum. In experiments with TNP-HEA, groups of five or six mice were immunized with a total of 50-100 #,g T N P - H E A in a total volume of 40-200/.d of adjuvant. Depending on the study, various routes were used as detailed in the figure legends, including s.c. at the base of the tail, s.c. in 2 - 4 sites on the back, i.p., or intraplantar in one or two hind footpads. Adjuvax,. RAS, Li0ovant, and Albydrogel were injected according to manufacturers' protocols. Briefly, 0.2 ml of antigen-Adjuvax was injected s.c. in two sites (0.1 ml each) in the inner thigh on days 0, 21 and 35. All other adjuvants were injected on days 0 and 28. RAS was injected s.c in two sites, 0.1 ml each site. Lipovant was injected s.c. in two sites, 0.1 ml each site. 0.2 ml of Alhydrogel was injected s.c. in two sites (0.1 ml each). No secondary immunization was given to groups whose results are shown in Figs. 5, 6, and 7. To evaluate the effects of boosting with TiterMax, in one experiment, animals were boosted on day 35 with either 25 #,g in 20 gl of T N P - H E A in a water-in-oil emulsion prepared using TiterMax or with 25 #,g of TNP-HEA in 20 /.d saline. Groups were immunized intraplantar in the hind footpads. In a second boosting experiment, one group of animals was boosted on day 28 with 100 ~ g of TNP-HEA in a water-in-oil emulsion orepared using TiterMax in a total volume of 80 #1 split between the hind footpads. The efficacy of TiterMax in various species was evaluated by measuring antibody titers in mice, rats, guinea pigs, and rabbits receiving a single immunization. Mice received 50 p,g of
TNP-HEA emulsified with 20 p-I of TiterMax in a total volume of 40 p-I injected s.c. at the base of the tail. Rats, guinea pigs, and rabbits each received 100 p-g of antigen emulsified with 40 p-I of TiterMax in a total volume of 80 p-I equally distributed between two sites (except for the i.p. route). Rabbits and guinea pigs received i.m. flank injections while rats received intraplantar, s.c. or i.p. injections. In studies using LHRH-BSA, groups of four mice, rabbits, or goats were immunized with a total of 50 p-g of antigen in adjuvant. The total volumes of antigen in adjuvant which were injected varied according to the manufacturers' instructions and were as follows for mice: LHRHBSA with TiterMax, 40 /~1; LHRH-BSA with CFA, 500 p.I; LHRH-BSA with Adjuvax was injected with 400 p.l; RAS, 200 p-I. With TiterMax, CFA, and Adjuvax, the protocols were the same for rabbits and goats. With RAS, the rabbits received a total of 1 mi antigen/adjuvant distributed as follows: 0.3 ml intradermal (50 p-I in each of six injections), 0.4 ml i.m. (0.2 ml in each hind flank), 0.1 ml s.c. (neck region), and 0.2 mi i.p. With RAS, goats received 1.0 ml antigen/adjuvant distributed between the two hind sites. The immunization routes were i.m. except with Adjuvax where they were s.c. All groups were injected on day 1 and boosted on day 28 (TiterMax and CFA groups), day 21 (RAS group), or day 21 and 35 (Adjuvax group). The second injection in the CFA group was with IFA.
lmmunoassays ELISA assay for antibody to TNP. 96 well, flat bottom EIA plates (Flow Labs., McLean, VA) were coated with 100 p-! of TNP-BSA, 0.5 /zg/ml in PBS, pH 8.4, overnight at 4°C. If not used immediately, plates can be stored, covered, at 4°C for 1 month. Before use, TNP-BSA coating solution was replaced with 100 p-I of blocking reagent, 1% BSA in PBS, pH 7.4. Plates were incubated with blocking reagent for 1 h at room temperature and the coated ELISA plates were washed. This and all subsequent washes were performed three times with 0.05% poloxamer 188 (F68) in phosphate-buffered saline (PBS), pH 7.4, using 150 p.I/well each wash. 100 p-i of diluted plasma (from serial 3-fold dilutions ranging from
1/100 to 1/218,700) in antibody diluent was added. Antibody diluent consisted of 0.1% BSA containing 0.1% F68 (BASF Wyandotte, Wyandotte, MI) in PBS, pH 7.4. Plates were incubated for 1 h at room temperature on a shaker table (200 rpm) then washed. 100 p-I horseradish peroxidase conjugated goat anti-mouse lgG (Fisher Scientific, Norcross, GA) (1/2000 dilution in antibody diluent) was added to each well. Plates were incubated for 1.5 h at 37°C then washed. 100 p-I per well colorizing reagent (0.4 mg/mi ortho-phenylenediamine (OPD) and 0.009% hydrogen peroxide in citrate buffer, pH 5.0) was added for 15 rain at room temperature. The reaction was stopped by adding 100 p-i 2.5 M H2SO4. Absorbance at 490 nm was read using the Bio-Rad Model 3550 Microplate reader. Titers were calculated from raw absorbance data within the linear range using a linear regression program. The reciprocal dilution of plasma producing an absorbance of 1.0 was defined as the titer. Subclass specific ELISA. The ELISA plates were set up as described above except that 100 p-I of a horseradish peroxidase-conjugated goat anti-mouse lgG1, IgG2a, IgG2b or IgG3 (Fisher Scientific, Norcross, GA (1/2000 dilution in antibody diluent)) was added to each well in place of anti-mouse lgG. The plates were incubated for 1.5 h at 37°C, washed and processed as described above. ELISA assay for antibody to BSA. Anti-BSA lgG titers were measured in serum from animals immunized with LHRH-BSA using a microplate sandwich ELISA. Microtiter plates were coated with 100 p-I BSA (4 p-g/ml) coating solution in a humidified chamber at 4°C overnight. Plates were then washed with PBS buffer and blocked with 200 p-I of PBS-gelatin blocking solution for 1 h at 37°C followed by three washes with PBS buffer. Dilutions of serum from goats (1/10-1/50,000), rabbits (1/10-1/100,000) or mice ( 1 / 1 0 - 1 / 100,000) were added to the washed plates in 100 p-i aliquots. Plates were incubated at 37°C for 2 h. Plates were washed three times and 100 p-I peroxidase conjugated anti-lgG specific for each species in PBS added for 2 h at room temperature. Plates were washed with PBS and 100 p-I tetramethyibenzidine added for 6 min at room temperature.
100 /zl of HaPO 4 stop solution was then added and absorbance at 450 nm was read using a microplate reader. The reciprocal dilution of serum producing an absorbance of 0.75 was defined as the titer. Anti-LHRH RIA procedure. Antibody activity against the LHRH peptide was measured in serum from animals immunized with LHRH-BSA using a radioimmunoassay with labeled peptide. Rabbit, mouse or goat serum diluted in EDTAPBS buffer was added to tubes containing radioiodinated native LHRH. Serum and reagents were incubated for 12-18 h at 4°C. In rabbits or mouse assays, I00 p l of species specific anti-lgG was added, the tubes vortexed and incubated at room temperature for 1 h. 600 p,i of 6% polyethylene glycol (PEG) was added to all tubes containing mouse or rabbit sera and 1.5 ml 200 Froof ethanol was added to tubes containing goat sera. The tubes were then vortexed and centrifuged at 2-8°C at 3000 rpm for 20 min. The supernatant was aspirated, the tubes were counted for 1 rain in a gamma counter and percent binding was calculated. Data for the percent peptide bound using a serum dilution in the linear portion of the dose-response curves are shown. All ?~ta are expressed as the mean + SEM. Histology / toxicity studies. 7-weet~-,>ld, female, outbred ICR mice were immunized with 100 p,g TNP-HEA in a water-in-oil emulsion of TiterMax. Animals received a total injection volume of 80 /xl split between two hind footpads. Animals were killed at various intervals and the
hind feet were fixed in PBS-buffered formalin, decalcified and processed for staining with hematoxylin and eosin. Some animals were injected with 100/zg TNP-HEA i.m. (40/zl i.m. in each hind flank) and killed at 28 days. Muscle was removed and fixed in PBS-buffered formalin, processed, sectioned and stained.
Results
Groups of 5 - 6 mice were injected with 50-100 /zg of TNP-HEA in one of six commercially available adjuvants. The number of immunizations varied to conform to the manufacturer's instructions for each preparation. However, animals received identical doses of antigen in each experiment. TiterMax was injected in the same protocol as CFA. The antibody titers were measured over a period of 4 months (Fig. 1). TNP-specific antibody increased more rapidly in the animals injected using TiterMax than in animals immunized using any of the other edjuvants. CFA produced the second highest titers, the other adju~,ants produced lower and less persistent antibody responses. The efficacy of TiterMax in various species was evaluated by measuring antibody titers 14 and 28 days following a single immunization. The results are summarized in Table !. All four species produced significant antibody titers, although the magnitude and duration of the response varied with species and route of immunization. The anti-
TABLE I EFFICACY OF TITERMAX IN VARIOUS SPECIES Species a
Injection route b
Anti-TNP titer c
Mice Rats
s.c. Base of tail Intrapiantar
Day 14 23,857 + 3,944 43,998 _+7,168
Rats Rats Guinea pigs Rabbits
i.p. s.c. Two hind quadriceps i.m. Two hind quadriceps i.m. Two hind quadriceps
I 1,866+3,433 4,335_+ 873 45,228+-3,261 26,262 5:9,554
Day 28 130,374+_22,825 48,997 _+~8,227 10,2Ill _+ 4,368 4,584+ 984 388+- 1,385 158,892+ 33,l}16
Numberof animals per group: five mice, four rats, three guinea pigs, three rabbits. b Mice receiveda total volume of 40 #1; all other groups received80/zl. Antibody titers (mean+-SEM) were measured using an ELISA.
a
Local toxicity None
I mm swelling & erythema None None None None
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"0 Fig. !. Comparison of commercially available adjuvants. 7week-old, female, outbred ICR mice (6 per group) were immunized with 100 #g TNP-HEA in one of six adjuvants. Ribi adjuvant system (RAS), Adjuvax, Lipovant and AIhydrogel were injected according to the manufacturer's protocols. 80 p,I of a 50:50 water-in-oil em~;l~ion of TiterMax or CFA was injected in each hind footpad (40 p.I/footpad). All adjuvants were injected on day 0 and boosted on day 28. Adjuvax (with antigen) was also boosted on day 35. The second injection in the CFA group was with IFA. lgG antibody titers against TNP were determined on plasma at intervals using an ELISA and are expressed as the mean + SEM as indicated.
Fig. 2. Histologic reaction to antigen in TiterMax. i-week-old, female, outbred ICR mice were immunized with 100 pg TNpoHEA in a 50:50 water-in-oil emulsion of TiterMax. Animals received a total injection volume of 80 p.I split between two hind footpads. Animals were killed at intervals, the hind feet fixed in PBS-buffered formalin, decalcified and
processed for staining with hemato~lin and eosin. Some animals were injected with 100/zg TNP-HEA i.m. (40 pl i.m. in each hind flank) and killed at 28 days. Muscle was removed and fixed in PBS-buffered formalin, processed, sectioned and stained.
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Fig. 3. Antibody titers with and without boosting with TiterMax #R-I. 7-week-old, female, outbred ICR mice (six per group) were immunized with 100 p,g TNP-HEA in a total volume of 80 /tl of a water-in-oil emulsion of TiterMax. Groups were immunized intraplantar in footpads. On day 28, one group of animals was boosted with 100/~g of TNP-HEA in a water-in-oil emulsion using TiterMax in a total volume of 80 pl split between each hind footpad or received no boost as indicated in the graph, lgG antibody titers against TNP were determined on plasma at intervals using an ELISA and are expressed as the mean + SEM.
body titers of both rabbits and mice increased five-six fold b e t w e e n days 14 and 28, but w e r e essentially u n c h a n g e d in rats and decreased in guinea pigs. In rats, the intraplantar route was s u p e r i o r to either the s.c. or i.p. route. TiterMax was effective in four different species of experimental animals. I m m u n i z a t i o n protocols should be tailored to each experimental system. T h e inflammatory r e s p o n s e at the site of injection was recorded for each g r o u p of animals. C F A p r o d u c e d the most severe inflammation characterized by r e d n e s s and swelling. All of the o t h e r adjuvants p r o d u c e d mild transient reactions. In animals immunized with TiterMax, histoiogic sections p r e p a r e d from representative sites of injection (Fig. 2) d e m o n s t r a t e d a foreign body reaction characterized by m a c r o p h a g e s filled with foamy material with few lymphocytes and o t h e r cells. T h e r e w a s n o evidence of activated m a c r o p h a g e s or the hypersensitivity g r a n u l o m a s characteristic of CFA. G r o s s and histologic examination of o t h e r o r g a n s failed to reveal metastatic g r a n u l o m a s o f the type r e p o r t e d for CFA. In o t h e r experiments, we have failed to induce adjuvant arthritis in Lewis rats u n d e r conditions w h e r e
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Fig+ 4. Effect of boosting with antigen in saline or adjuvant. 7-week-old, female, oatbred ICR mice (6 per group) were immunized with 50 l~g TNP-HEA in a total volume of 40 ~1 of a 50:50 water-in-oil emulsion of TiterMax, Groups were immunized intraplantar in four footpads ( I x 10/xt). On day 35, animals were boosted with either 25/zg of TNP-HEA in a water-in-oil emulsion using TiterMax in a total volume of 20 #1 split between each hind footpad or with 25/~g of TNP-HEA in saline in 2,10/zl injections in each hind footpad, Mean lgG antibody titers against TIqP were determined on plasma at intervals using an ELISA.
all of the control CFA-treated rats succumb (data not shown). The duration of antibody response has been tl.
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4
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Fig. 6, Effect of number of injection sites on antilx~ly titer. 7-week-old, female, outhred ICR mice (six per group) were immunized with 50/zg TNP-HEA it, a total volume of 40/zl of a water-in-oil emulsion of TiterMax, Groups were immunized s.c. in eRher two or four sites. No .secondary immunization was given, lgG antibody titers against TNP were determined on plasma at intervals using an ELISA and are expressed as the mean ± SEM.
related to the persistence o f antigen. O n e would expect that adjuvants which form stable d e p o t s o f water-'.,n-oil emulsion in tissue might p r o d u c e long-lasting antibody responses. T h i s was f o u n d to be the case with TiterMax (Fig. 3). W h e t h e r mice were injected with only a single dose o f T N P - H E A in TiterMax o r boosted at 28 days, the antibody titers reached high levels by 6 weeks.
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Fig, 5. Effect of injection route on antibody titer. 7.week-old, female, oatbred ICR mice (six per group) were immunized with 50 #g TNP-HEA in a total volume of 40 ~1 of a water-in-oil emulsion of TiterMax. Groups were immunized either s,c. at the base of the tail, i.p. or intraplantar in one hind footpad. No secondary immunization was given, lgG antibody triers against TNP were determined on plasma at intervals using an ELISA and are expressed as the mean:k SEM.
IgG1
IOG2B
igG2b
igG3
Fig. 7. Subcla~ of lgG antibody induced by TiterMax. 7-weekold, female, outbred ICR mice (five per group) were immunized intraplantar in one hind footi~ad with 50 t~g TNPoHEA in a total volume of 40 ,gl of a water-in-oil emulsion of TiterMax. No seconda~ immunization was done. lgG antibody subclass triers against TNP were determined on plasma at 28 days using a subclass-specificELISA.
Following a single injection, t h e titers b e g a n to decline after a few m o n t h s , b u t they rose again after 7 m o n t h s a n d at I y e a r were c o m p a r a b l e to levels in b o o s t e d animals. A n o t h e r g r o u p o f a n i m a l s w a s injected with T i t e r M a x a n d b o o s t e d with a n t i g e n either in saline
or in T i t e r M a x at 28 days. T h o s e i m m u n i z e d a n d b o o s t e d w~th T i t e r M a x displayed a slight r e d u c tion in antibody titers for 2 w e e k s followed by a n i n c r e a s e d a n d long persistence, Surprisingly, t h o s e i m m u n i z e d with T i t e r M a x a n d b o o s t e d with soluble a n t i g e n s h o w e d a n i m m e d i a t e increase o f
Anti-LHRH peptlcle
Anti-BSA carrier
,a~biW
TIz,rM.x
C~A.................),d,j,~a,x
TaerMax
R/~ M
TaerlMiix
CFA
Adjuvsx
CFA
AdJuvax
RAS
AdJuvax
R~
:e
RAS
TitelMllx
CFA
Gc ~ts
TaerMllx
CFA
AdJu¥1tx
RAS
TltllrMiix
CFA
AdJuvllx
RA~;
Fig. 8. Assessmentof TiterMax and other commercially available adjuvants. Groups of animals were immunized with a total of 50 /.~gLHRH-BSA (lute;nizing hormone releasing hormone conjugated to bovine serum albumin) in several adjuvants according to the manufacturer's prot~ls. Four New Zealand White rabbits, mixed breed male goats, or Balb/c mice per group were used. All groups were injected o~ day I and boosted on day 28 (TiterMax and CFA groups), day 21 {RAS group), or day 21 and 35 (Adjuvax group). The second injection in the CFA group was with IFA. Results using serum samples from week 8 are shown, lgG antibody liters against the carrier BSA were measured using an EL1SA. Antibody activity against the LHRH peptide was measured using a radioimmunoassay with labeled peptide. Data for the % peptide bound using a serum dilution (final concentration 1:200 for rabbits: I : 100 for mice: 1:5 for goats) in the linear portion of the dose-response curves are shown. All data are expressed as mean ± SEM.
five-fold in antibody titer which persisted for 3 months before beginning a gradual decline (Fig. 4). Studies were done to evaluate the effect of injection route on antibody titers. Animals injected in the footpad, s.c. at the base of the tail, or i.p. all produced similar titers for the first month after a single injection. In those immunized i.p., the titers then plateaued and began to decline after two months. In the groups immunized in the footpad or s.e,, the titers continued to increase for two months and plateaued at a significantly higher level (Fig. 5). CFA is frequently injected in multiple s.c. sites on the back and flank of animals in order to increase titers and reduce local inflammation. In experiments to evaluate such protocols on the efficacy of TiterMax, animals injected at two sites on opposite flanks produced lower titers than those immunized with an identical volume and dose of antigen distributed over four sites (Fig. 6). As shown in Figs. 5 and 6, the magnitude of the response varies with the route of immunization. The subclass of lgG antibody in mice was determined with a specially calibrated ELISA assay. Animals immunized with TiterMax made comparable amounts of lgG 1 and lgG2b anti-TNP with variable amounts of lgG2a and very little IgG3 (Fig. 7). This demonstrates that TiterMax is able to stimulate all of the isotypes of IgG antibody in mice in a pattern comparable to that of CFA. Animals were immunized by Hazleton Washington with a peptide hormone LHRH conjugated to BSA as a carrier and antibody was measured against both the peptide and the cartier. CFA and TiterMax were superior to Adjuvax or RAS in all comparisons with both antigens and all three species. In rabbits, TiterMax produced significantly higher responses to BSA with comparable responses to the peptide. In mice, the results between CFA and TiterMax were not significantly different for either the peptide or the carrier. In goats, CFA produced higher antibody titers against the carrier while TiterMax produced comparable titers against the peptide (Fig. 8).
Discussion
Previous studies have reported that block copolymer adjuvants are able to induce antibody titers at least comparable to those induced by CFA. Nonionic block copolymers are relatively nontoxic surface active agents and are effective immunoadjuvants(Hunter et al., 1981, 1984, 1986, 1991; Takayama et ai., 1991). However, preparation of effective immunogen formulations has been both difficult and unreliable. The water-inoil emulsion with CFA, in contrast, is readily formulated with a wide variety of antigens. TiterMax was designed to combine the activity of an especially efficacious block copolymer with a stable water-in-oil emulsion which could contain nearly any antigen, it does not contain any protein, peptide, pol$,saceharide or mycobacterial component. TiterMax was developed specifically as a replacement for CFA and was not intended for use in vaccines. We have previously described the adjuvant activity of nonionic block copolymers (Hunter et at., 1981, 1984). CRL89-41 is one such pharmaceutical grade tribloek copolymer. It was specifically developed for use with water-in-squalene emulsions and was selected for its effectivene.~s in this formulation. Its fundamental mechanisms of action, however, are undoubtedly similar to those of previously described block copolymers (Hunter et al, 1981, 1984, 1986, 1991; Takayama et al., 1991). Mineral oil is used in CFA and IFA because previous investigators have been unable to produce stable emulsions with vegetable oils. One exception, adjuvant 65, used peanut oil with aluminum monostearate as a stabilizer (Hilleman, 19671. The efficacy of adjuvant 65 is comparable to that of IFA. Both CFA and adjuvant 65 must be prepared with at least 50% oil. TiterMax represents a significant advance in emulsion technology in that stable water-in-oil emulsions can be prepared with less than 20% oil. We have found that TiterMax produces stable emulsions with antigens containing materials which break CFA emulsions. This includes certain polysaccharide-protein conjugates, 1.0% SDS or 0.75 M urea. Both the block copolymer and the copolym~:r-
coated microparticles are necessary for this stability, It is well known that microparticles contribute to emulsion stability if they have surface p r o p e r ties which facilitate their localization at t h e oilwater interface (Becher, 1965). Silica particles coated with copolymer by a proprietary process were found to have ideal surface properties for stabilization o f wateroin-squalene emulsions, Silica particles themselves have adjuvant activity. However, this activity d e p e n d s u p o n exposure o f the crystalline surface. Since the surface o f t h e particles in TiterMax emulsions is covered by copolymer and is contained within oil, it is unlikely that the inherent adjuvant activity o f silica contributes significantly to antibody titers in TiterMax emulsions. T h e r e have b e e n many advances in adjuvant and emulsification technology since Jules F r e u n d introduced his adjuvant over 50 years ago. T h e p r e s e n t experiments were u n d e r t a k e n as an att e m p t to combine a newly synthesized copolymer adjuvant with m o d e r n emulsification reagents to produce a safe and effective alternative to C F A . T h e result is an adjuvant which contains n o protein, peptide o r polysaccharide and no microbial c o m p o n e n t s . In our studies, t h e inflammatory re. sponses induced by TiterMax were mild and transient c o m p a r e d with those induced by CFA. T h e magnitude o f the antibody response varied with the species a n d route o f immunization. T h e s e data suggest that TiterMax a p p e a r s to b e a safe, effective and chemically d e f i n e d alternative to C F A in many situations.
Acknowledgements T h e authors gratefully thank A n g e l a J o h n s o n for h e r technical assistance with these experiments.
References Amyx, H.L. (1087) Control of animal pain and distress in antibody production and infectious disease studies, J. Am.
Vet. MOd.Assoc. ~91, 1287. Becher, P. (1965)Theory of emulsions: stability. In: P. Beeher, (Ed.), Emulsions: Theory and Practice, 2rid edn. Reinhold, New York, pp. 95-149. Broderson, LR. (1989) A retrospective review of lesions associated with the use of Freund's adjuvant. Lab. Animal Sci. 39, 400. Freund, J., Casals, J. and Hismer, E.P. (1937) Sensitization and antibody formation after injection of tubercle bacilli and paraffin oil. Ptoc. Soc. Exp. BioL Med. 37, 509. Freund, J. (1951) The effect of paraffin oil and mycobecteria on antibody formation and sensitization. Am. J. Clin. Pathol. 21,645. Garvcy, J.S., Cremcr, N.E., and Sussdorf, D.H. (1977) DNP. and TNP-conjugated proteins. In: J.S. Garvey, N,E. Cremer and D.H. Sussdorf (Eds.), Methods in Immunology, 3rd edn. W.A. Benjamin, Reading, MA, pp. 153-158. Hilteman, M.R. (1967) Considerations for safety and application of emulsified oil adjuvants to viral vaccines. In: R.H. Regamay, W. Hennessen. D. ikic and J, Unger, (Eds.), Symposia Series in lmmunobiological Standardization, Vol. 6, International Symposium on Adjuvants of Immunity. S. Karger, New York, p. 18. Hunter, R.L., Strickland, F, and Kezdy, F. (1981) Studies on the adjuvant activity of nonionic block polymer surfacrants. 1. The role of hydrophile-lipophile balance. J. lmmunoL 127, 1244. Hunter, R.L. and Bennett, B. (1984) Studies on the adjuvant activity of nonionic block polymer surfactants. 11. Antibody formation and inflammation related to the structure of triblock and octablock copolymers. J. lmmunol. 133, 1367. Hunter, R.L. and Bennett, B. (1986) The adjuvant activity of nonionic block polymer suffactants. IlL Characterization of selected biologicallyactive surfaces. Scand. J. ImmunoL 23, 287. Hunter, R.L., Olsgn, M. and Buynitzky, S. (1991) Adjuvant activity of non-ionic block copolymers. IV. Effect of molecular weight and formulation on titre and isotype of antibody. Vaccine 9, 250. Jenncs` L. ar~d Stumpf, W,E. (1983) Preparation and use of specific antibodies for immunohistochemistry of neuropeptides. In: P.M, Conn (Ed.), Methods in Enzymology, VoL 103. Academic Press, New York, pp. 448-458. Takayama, K., Olsen, M., Dana, P. and Hunter, R.L. (1991) Adjuvant activity of non-ionic block copolymers. V. Modulation of antibody isotype by lipopolysaccharides` lipid A and precursors. Vaccine 9, 257.
3I
© 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$115.1~1
JIM06382
A comparison of commercially available adjuvants for use in research B e t h B e n n e t t , I r e n e J. C h e c k , M a r g a r e t R. O l s e n a n d R o b e r t L. H u n t e r Department of Pathology and Laboratory Medicine, Emory Unirersity, Atlanta, GA 30322, USA
(Received 15 January 1992,revised received 26 March 1992,accepted 27 March 1992)
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 ( L H R H ) 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. Key words: lmmunoadjuvant;TiterMax; Complete Freund's adjuvant; Antibody titer
Correspondence to: B. Bennett, Department of Pathology, Room 760 WMB, 1639 Pierce Drive, Empty University, Atlanta, GA 30322, USA. Tel.: 4{14-727-5933;Fax: 404-727-2991. t Supported by grant #'6-38943 from CytRx Corporation to Empty University. Robert Hunter is Vice President and Director of Research for CytRx Corporation. This study was presented as a poster at the 1991 FASEB Meeting (abstract #1464). Abbreciations: HEA, hen egg albumin: LHRH-BSA, luteinizing hormone-releasing hormone conjugated to BSA, OPD, ortho-phenylenediamine; POE, polyoxyethylene;POP, polyoxypropylene; RAS, Ribi adjuvant system; TNBS, picrylsulfonic acid; TNP-BSA, trinitrophenol conjugated to bovine serum albumin; TNP-HEA, trinitrophenol conjugated to hen egg albumin; F68, poloxamer 188; PBS, phosphate-buffered saline.
Introduction Complete Freund's adjuvant (CFA) has been used for over 50 years for producing antisera in animals (Freund et al., 1937; Freund, 19511. The severity of its toxicity was recognized immediately, but attempts to find a less toxic and equally effective alternative have not been successful. CFA induces both local and systemic reactions. The site of injection frequently develops hypersensitivity granuloma~ which may ulcerate to form draining abscesses. Particles of the emulsion may spread through the body producing metastatic
granulomas. This may be accompanied by wasting disease, adjuvant arthritis, or other autoimmune conditions (Amyx, 1987; Broderson, 1989). The arthritis can be particularly debilitating especially in larger animals. Investigations of pain research have clearly documented that CFA can produce severe chronic pain and suffering in small animals as well (Amyx, 1987; Broderson, 1989). With advances in many areas of the biological sciences and increasing concern for the welfare of experimental animals, there is increased pressure to ban or restrict the use of CFA. In 1981, we described the adjuvant activity of nonionic block eopolymers (Hunter et al., 1981). These are synthetic surface active agents consisting of a chain, or block, of hydrophobic polyoxypropylene (POP) flanked by two chains of hydrophilic polyoxyethylene (POE). These copolymers bind antigen, as well as complement and other mediators in a way that is thought to produce efficient presentation of antigen to cells in lymph nodes (Hunter et al, 1984, 1986). They induced high, long-lasting antibody titers with minimal toxicity when formulated in oil-in-water emulsions (Hunter et al., 1984, 1986, 1991; Takayama et al., 1991). Such preparations, however, were effective with only a limited spectrum of antigens. We undertook development of an adjuvant formulation which would utilize the potent adjuvant activity of block copolymers in a formulation that would be effective with a wide variety of antigens. In general, adjuvants function best if they facilitate the simultaneous delivery, of the antigen and immunomodulating substances to lymphoid tissue. CFA does this by containing virtually any antigen together with killed mycobaeteria as an immunostimulant in a water-inoil emulsion. The toxicity of CFA has been attributed to both the mineral oil and mycobacteria (Amyx, 1987; Broderson, 1989). We found that squalene, combined with copolymer CRL89-41 bonded to micropartieulate silica, could be used to produce water-in-oil emulsions which were more stable and easier to prepare than CFA. Squalene, a metabolizable oil, is a normal body component. This paper reports two comparisons of the new adjuvant formulation, TiterMax, with other com-
mercially available adjuvants. Studies on the dose and route of injection are included. The results indicate that TiterMax is a safe and effective alternative to CFA. Materials and methods
TNP-HEA antigen Hen egg albumin (HEA, Sigma Chemical Co., St. Louis, MO) was conjugated to picrylsulfonie acid (TNBS, Sigma Chemical Co., St. Louis, MO) in borate buffer, pH 8.5 (Garvey et al., 1977). The hapten-to-protein ratio (9.4:1) was calculated using a molar extinction coefficient of 15,400 at 350 nm for TNP-aminocaproate and a molecular weight of 45,000 for HEA. LHRH-BSA antigen 100/zl of BSA (160 mg/mi in distilled water) was mixed with 100 /zl of ethyl-dimethylaminopropyi earbodiimide-HCI (EDC (300 mg/ml in distilled water)). A total of 5 mg of LHRH (dissoZved in 500 ttl distilled water) was added in 100 tzl portions at 5 rain intervals. The reaction mixture was incubated for 1 h (including the 20 min to add the LHRH) at room temperature, then dialyzed for 24 h (Jennes et ai., 1983). The percent conjugation was 48% and the molar ratio of peptide:BSA was 9:1. Adjuvants TiterMax #R-1 was provided by CytRx Corporation, Norcross, GA. CFA used in the TNP-HEA experiments was purchased from GIBCO, Grand Island, NY and that used for the LHRH-BSA experiments was purchased from Difco, Detroit, Michigan. Ribi adjuvant system (RAS #R-700) was purchased from Ribi Immunochem Research Inc., Hamilton, MT, Adjuvax from Alpha-Beta Technology, Worcester, MA, Lipovant from Accurate Chemical and Scientific Corp., Westbury, NY and Alhydrogel (trademark of Superfos, Denmark) from Accurate Chemical and Scientific Corp., Westbury, NY. Animals Mice and rats. 7-week-old, female, outbred ICR mice (virus and pathogen free) or 6-week-old female rats
were purchased from Harlan Sprague-Dawley (Birmingham, AL facility) and were used for the T N P - H E A experiments. Heparinized plasma was collected by retro-orbital plexus bleeding for mice and from tail vein bleeding for rats. Balb/c mice, randomized sexes, were used for the LHRH-BSA experiments (Charles River Laboratories, Wilmington, MA). Serum was collected by tail vein bleeding. All animals were anesthetized prior to blood collection using metofane. Rabbits. Adult New Zealand White female rabbits from Myrtles Rabbitry (Thompson Station, TN) were used for the T N P - H E A experiments and from Hazleton Washington for LHRH-BSA experiments. Heparinized plasma was collected for the T N P - H E A studies by ear vein bleeding. Serum was collected for the LHRH-BSA studies also by ear vein bleeding. Guinea pigs. Hartley 400 g female guinea pigs (Charles River Laboratories, Kingston, NY facility) were used. Heparinized plasma was collected by collecting blood from a clipped toe pad. Animals were anesthetized prior to blood collection using metofane. Goats. Mixed breed male goats (Hazleton Washington) were used. Serum was collected by neck vein bleeding. Experiments using T N P - H E A were performed at Emory University and all animals were maintained in the animal facilities at Emory University. Experiments using LHRH-BSA were performed at Hazleton Washington, Vienna, VA. In both facilities, animals were routinely screened for viral, bacterial and mycoplasma infections. Commercial food and tap water were provided ad libitum. The temperature range of the animal rooms was 22-25°C and the relative humidity was approximately 50%. Subdued lighting, 12 h on and 12 h off cycle, was used to illuminate the rooms.
Immunization protocols Water-in-oil emulsions of TiterMax, CFA, or IFA were prepared as follows: 0.5 ml of aqueous antigen was added to 1.0 ml oil phase, emulsified to a water-in-oil emulsion and further emulsified with an additional 0.5 mi of aqueous antigen. Stability of the water-in-oil emulsions of CFA
and TiterMax were assessed by placing a drop of the emulsion onto the surface of water. Adjuvax, RAS, Lipovant and AIhydrogel were prepared according to the manufacturers' protocols. Briefly, aqueous antigen was mixed with 1 mg Adjuvax and hydrated by drawing the mixture in and out of an 18 gauge needle 8-10 times. An oil-in-water emulsion of RAS was prepared by warming the vial of adjuvant and adding antigen in sterile saline to the vial and vortexing for several minutes. Antigen in water was added to the Lipovant vial and the vial was vigorously shaken for several seconds. Alhydrogei was mixed with aqueous antigen to allow adsorption of the antigen to alum. In experiments with TNP-HEA, groups of five or six mice were immunized with a total of 50-100 #,g T N P - H E A in a total volume of 40-200/.d of adjuvant. Depending on the study, various routes were used as detailed in the figure legends, including s.c. at the base of the tail, s.c. in 2 - 4 sites on the back, i.p., or intraplantar in one or two hind footpads. Adjuvax,. RAS, Li0ovant, and Albydrogel were injected according to manufacturers' protocols. Briefly, 0.2 ml of antigen-Adjuvax was injected s.c. in two sites (0.1 ml each) in the inner thigh on days 0, 21 and 35. All other adjuvants were injected on days 0 and 28. RAS was injected s.c in two sites, 0.1 ml each site. Lipovant was injected s.c. in two sites, 0.1 ml each site. 0.2 ml of Alhydrogel was injected s.c. in two sites (0.1 ml each). No secondary immunization was given to groups whose results are shown in Figs. 5, 6, and 7. To evaluate the effects of boosting with TiterMax, in one experiment, animals were boosted on day 35 with either 25 #,g in 20 gl of T N P - H E A in a water-in-oil emulsion prepared using TiterMax or with 25 #,g of TNP-HEA in 20 /.d saline. Groups were immunized intraplantar in the hind footpads. In a second boosting experiment, one group of animals was boosted on day 28 with 100 ~ g of TNP-HEA in a water-in-oil emulsion orepared using TiterMax in a total volume of 80 #1 split between the hind footpads. The efficacy of TiterMax in various species was evaluated by measuring antibody titers in mice, rats, guinea pigs, and rabbits receiving a single immunization. Mice received 50 p,g of
TNP-HEA emulsified with 20 p-I of TiterMax in a total volume of 40 p-I injected s.c. at the base of the tail. Rats, guinea pigs, and rabbits each received 100 p-g of antigen emulsified with 40 p-I of TiterMax in a total volume of 80 p-I equally distributed between two sites (except for the i.p. route). Rabbits and guinea pigs received i.m. flank injections while rats received intraplantar, s.c. or i.p. injections. In studies using LHRH-BSA, groups of four mice, rabbits, or goats were immunized with a total of 50 p-g of antigen in adjuvant. The total volumes of antigen in adjuvant which were injected varied according to the manufacturers' instructions and were as follows for mice: LHRHBSA with TiterMax, 40 /~1; LHRH-BSA with CFA, 500 p.I; LHRH-BSA with Adjuvax was injected with 400 p.l; RAS, 200 p-I. With TiterMax, CFA, and Adjuvax, the protocols were the same for rabbits and goats. With RAS, the rabbits received a total of 1 mi antigen/adjuvant distributed as follows: 0.3 ml intradermal (50 p-I in each of six injections), 0.4 ml i.m. (0.2 ml in each hind flank), 0.1 ml s.c. (neck region), and 0.2 mi i.p. With RAS, goats received 1.0 ml antigen/adjuvant distributed between the two hind sites. The immunization routes were i.m. except with Adjuvax where they were s.c. All groups were injected on day 1 and boosted on day 28 (TiterMax and CFA groups), day 21 (RAS group), or day 21 and 35 (Adjuvax group). The second injection in the CFA group was with IFA.
lmmunoassays ELISA assay for antibody to TNP. 96 well, flat bottom EIA plates (Flow Labs., McLean, VA) were coated with 100 p-! of TNP-BSA, 0.5 /zg/ml in PBS, pH 8.4, overnight at 4°C. If not used immediately, plates can be stored, covered, at 4°C for 1 month. Before use, TNP-BSA coating solution was replaced with 100 p-I of blocking reagent, 1% BSA in PBS, pH 7.4. Plates were incubated with blocking reagent for 1 h at room temperature and the coated ELISA plates were washed. This and all subsequent washes were performed three times with 0.05% poloxamer 188 (F68) in phosphate-buffered saline (PBS), pH 7.4, using 150 p.I/well each wash. 100 p-i of diluted plasma (from serial 3-fold dilutions ranging from
1/100 to 1/218,700) in antibody diluent was added. Antibody diluent consisted of 0.1% BSA containing 0.1% F68 (BASF Wyandotte, Wyandotte, MI) in PBS, pH 7.4. Plates were incubated for 1 h at room temperature on a shaker table (200 rpm) then washed. 100 p-I horseradish peroxidase conjugated goat anti-mouse lgG (Fisher Scientific, Norcross, GA) (1/2000 dilution in antibody diluent) was added to each well. Plates were incubated for 1.5 h at 37°C then washed. 100 p-I per well colorizing reagent (0.4 mg/mi ortho-phenylenediamine (OPD) and 0.009% hydrogen peroxide in citrate buffer, pH 5.0) was added for 15 rain at room temperature. The reaction was stopped by adding 100 p-i 2.5 M H2SO4. Absorbance at 490 nm was read using the Bio-Rad Model 3550 Microplate reader. Titers were calculated from raw absorbance data within the linear range using a linear regression program. The reciprocal dilution of plasma producing an absorbance of 1.0 was defined as the titer. Subclass specific ELISA. The ELISA plates were set up as described above except that 100 p-I of a horseradish peroxidase-conjugated goat anti-mouse lgG1, IgG2a, IgG2b or IgG3 (Fisher Scientific, Norcross, GA (1/2000 dilution in antibody diluent)) was added to each well in place of anti-mouse lgG. The plates were incubated for 1.5 h at 37°C, washed and processed as described above. ELISA assay for antibody to BSA. Anti-BSA lgG titers were measured in serum from animals immunized with LHRH-BSA using a microplate sandwich ELISA. Microtiter plates were coated with 100 p-I BSA (4 p-g/ml) coating solution in a humidified chamber at 4°C overnight. Plates were then washed with PBS buffer and blocked with 200 p-I of PBS-gelatin blocking solution for 1 h at 37°C followed by three washes with PBS buffer. Dilutions of serum from goats (1/10-1/50,000), rabbits (1/10-1/100,000) or mice ( 1 / 1 0 - 1 / 100,000) were added to the washed plates in 100 p-i aliquots. Plates were incubated at 37°C for 2 h. Plates were washed three times and 100 p-I peroxidase conjugated anti-lgG specific for each species in PBS added for 2 h at room temperature. Plates were washed with PBS and 100 p-I tetramethyibenzidine added for 6 min at room temperature.
100 /zl of HaPO 4 stop solution was then added and absorbance at 450 nm was read using a microplate reader. The reciprocal dilution of serum producing an absorbance of 0.75 was defined as the titer. Anti-LHRH RIA procedure. Antibody activity against the LHRH peptide was measured in serum from animals immunized with LHRH-BSA using a radioimmunoassay with labeled peptide. Rabbit, mouse or goat serum diluted in EDTAPBS buffer was added to tubes containing radioiodinated native LHRH. Serum and reagents were incubated for 12-18 h at 4°C. In rabbits or mouse assays, I00 p l of species specific anti-lgG was added, the tubes vortexed and incubated at room temperature for 1 h. 600 p,i of 6% polyethylene glycol (PEG) was added to all tubes containing mouse or rabbit sera and 1.5 ml 200 Froof ethanol was added to tubes containing goat sera. The tubes were then vortexed and centrifuged at 2-8°C at 3000 rpm for 20 min. The supernatant was aspirated, the tubes were counted for 1 rain in a gamma counter and percent binding was calculated. Data for the percent peptide bound using a serum dilution in the linear portion of the dose-response curves are shown. All ?~ta are expressed as the mean + SEM. Histology / toxicity studies. 7-weet~-,>ld, female, outbred ICR mice were immunized with 100 p,g TNP-HEA in a water-in-oil emulsion of TiterMax. Animals received a total injection volume of 80 /xl split between two hind footpads. Animals were killed at various intervals and the
hind feet were fixed in PBS-buffered formalin, decalcified and processed for staining with hematoxylin and eosin. Some animals were injected with 100/zg TNP-HEA i.m. (40/zl i.m. in each hind flank) and killed at 28 days. Muscle was removed and fixed in PBS-buffered formalin, processed, sectioned and stained.
Results
Groups of 5 - 6 mice were injected with 50-100 /zg of TNP-HEA in one of six commercially available adjuvants. The number of immunizations varied to conform to the manufacturer's instructions for each preparation. However, animals received identical doses of antigen in each experiment. TiterMax was injected in the same protocol as CFA. The antibody titers were measured over a period of 4 months (Fig. 1). TNP-specific antibody increased more rapidly in the animals injected using TiterMax than in animals immunized using any of the other edjuvants. CFA produced the second highest titers, the other adju~,ants produced lower and less persistent antibody responses. The efficacy of TiterMax in various species was evaluated by measuring antibody titers 14 and 28 days following a single immunization. The results are summarized in Table !. All four species produced significant antibody titers, although the magnitude and duration of the response varied with species and route of immunization. The anti-
TABLE I EFFICACY OF TITERMAX IN VARIOUS SPECIES Species a
Injection route b
Anti-TNP titer c
Mice Rats
s.c. Base of tail Intrapiantar
Day 14 23,857 + 3,944 43,998 _+7,168
Rats Rats Guinea pigs Rabbits
i.p. s.c. Two hind quadriceps i.m. Two hind quadriceps i.m. Two hind quadriceps
I 1,866+3,433 4,335_+ 873 45,228+-3,261 26,262 5:9,554
Day 28 130,374+_22,825 48,997 _+~8,227 10,2Ill _+ 4,368 4,584+ 984 388+- 1,385 158,892+ 33,l}16
Numberof animals per group: five mice, four rats, three guinea pigs, three rabbits. b Mice receiveda total volume of 40 #1; all other groups received80/zl. Antibody titers (mean+-SEM) were measured using an ELISA.
a
Local toxicity None
I mm swelling & erythema None None None None
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"0 Fig. !. Comparison of commercially available adjuvants. 7week-old, female, outbred ICR mice (6 per group) were immunized with 100 #g TNP-HEA in one of six adjuvants. Ribi adjuvant system (RAS), Adjuvax, Lipovant and AIhydrogel were injected according to the manufacturer's protocols. 80 p,I of a 50:50 water-in-oil em~;l~ion of TiterMax or CFA was injected in each hind footpad (40 p.I/footpad). All adjuvants were injected on day 0 and boosted on day 28. Adjuvax (with antigen) was also boosted on day 35. The second injection in the CFA group was with IFA. lgG antibody titers against TNP were determined on plasma at intervals using an ELISA and are expressed as the mean + SEM as indicated.
Fig. 2. Histologic reaction to antigen in TiterMax. i-week-old, female, outbred ICR mice were immunized with 100 pg TNpoHEA in a 50:50 water-in-oil emulsion of TiterMax. Animals received a total injection volume of 80 p.I split between two hind footpads. Animals were killed at intervals, the hind feet fixed in PBS-buffered formalin, decalcified and
processed for staining with hemato~lin and eosin. Some animals were injected with 100/zg TNP-HEA i.m. (40 pl i.m. in each hind flank) and killed at 28 days. Muscle was removed and fixed in PBS-buffered formalin, processed, sectioned and stained.
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Fig. 3. Antibody titers with and without boosting with TiterMax #R-I. 7-week-old, female, outbred ICR mice (six per group) were immunized with 100 p,g TNP-HEA in a total volume of 80 /tl of a water-in-oil emulsion of TiterMax. Groups were immunized intraplantar in footpads. On day 28, one group of animals was boosted with 100/~g of TNP-HEA in a water-in-oil emulsion using TiterMax in a total volume of 80 pl split between each hind footpad or received no boost as indicated in the graph, lgG antibody titers against TNP were determined on plasma at intervals using an ELISA and are expressed as the mean + SEM.
body titers of both rabbits and mice increased five-six fold b e t w e e n days 14 and 28, but w e r e essentially u n c h a n g e d in rats and decreased in guinea pigs. In rats, the intraplantar route was s u p e r i o r to either the s.c. or i.p. route. TiterMax was effective in four different species of experimental animals. I m m u n i z a t i o n protocols should be tailored to each experimental system. T h e inflammatory r e s p o n s e at the site of injection was recorded for each g r o u p of animals. C F A p r o d u c e d the most severe inflammation characterized by r e d n e s s and swelling. All of the o t h e r adjuvants p r o d u c e d mild transient reactions. In animals immunized with TiterMax, histoiogic sections p r e p a r e d from representative sites of injection (Fig. 2) d e m o n s t r a t e d a foreign body reaction characterized by m a c r o p h a g e s filled with foamy material with few lymphocytes and o t h e r cells. T h e r e w a s n o evidence of activated m a c r o p h a g e s or the hypersensitivity g r a n u l o m a s characteristic of CFA. G r o s s and histologic examination of o t h e r o r g a n s failed to reveal metastatic g r a n u l o m a s o f the type r e p o r t e d for CFA. In o t h e r experiments, we have failed to induce adjuvant arthritis in Lewis rats u n d e r conditions w h e r e
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Fig+ 4. Effect of boosting with antigen in saline or adjuvant. 7-week-old, female, oatbred ICR mice (6 per group) were immunized with 50 l~g TNP-HEA in a total volume of 40 ~1 of a 50:50 water-in-oil emulsion of TiterMax, Groups were immunized intraplantar in four footpads ( I x 10/xt). On day 35, animals were boosted with either 25/zg of TNP-HEA in a water-in-oil emulsion using TiterMax in a total volume of 20 #1 split between each hind footpad or with 25/~g of TNP-HEA in saline in 2,10/zl injections in each hind footpad, Mean lgG antibody titers against TIqP were determined on plasma at intervals using an ELISA.
all of the control CFA-treated rats succumb (data not shown). The duration of antibody response has been tl.
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Fig. 6, Effect of number of injection sites on antilx~ly titer. 7-week-old, female, outhred ICR mice (six per group) were immunized with 50/zg TNP-HEA it, a total volume of 40/zl of a water-in-oil emulsion of TiterMax, Groups were immunized s.c. in eRher two or four sites. No .secondary immunization was given, lgG antibody titers against TNP were determined on plasma at intervals using an ELISA and are expressed as the mean ± SEM.
related to the persistence o f antigen. O n e would expect that adjuvants which form stable d e p o t s o f water-'.,n-oil emulsion in tissue might p r o d u c e long-lasting antibody responses. T h i s was f o u n d to be the case with TiterMax (Fig. 3). W h e t h e r mice were injected with only a single dose o f T N P - H E A in TiterMax o r boosted at 28 days, the antibody titers reached high levels by 6 weeks.
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Fig, 5. Effect of injection route on antibody titer. 7.week-old, female, oatbred ICR mice (six per group) were immunized with 50 #g TNP-HEA in a total volume of 40 ~1 of a water-in-oil emulsion of TiterMax. Groups were immunized either s,c. at the base of the tail, i.p. or intraplantar in one hind footpad. No secondary immunization was given, lgG antibody triers against TNP were determined on plasma at intervals using an ELISA and are expressed as the mean:k SEM.
IgG1
IOG2B
igG2b
igG3
Fig. 7. Subcla~ of lgG antibody induced by TiterMax. 7-weekold, female, outbred ICR mice (five per group) were immunized intraplantar in one hind footi~ad with 50 t~g TNPoHEA in a total volume of 40 ,gl of a water-in-oil emulsion of TiterMax. No seconda~ immunization was done. lgG antibody subclass triers against TNP were determined on plasma at 28 days using a subclass-specificELISA.
Following a single injection, t h e titers b e g a n to decline after a few m o n t h s , b u t they rose again after 7 m o n t h s a n d at I y e a r were c o m p a r a b l e to levels in b o o s t e d animals. A n o t h e r g r o u p o f a n i m a l s w a s injected with T i t e r M a x a n d b o o s t e d with a n t i g e n either in saline
or in T i t e r M a x at 28 days. T h o s e i m m u n i z e d a n d b o o s t e d w~th T i t e r M a x displayed a slight r e d u c tion in antibody titers for 2 w e e k s followed by a n i n c r e a s e d a n d long persistence, Surprisingly, t h o s e i m m u n i z e d with T i t e r M a x a n d b o o s t e d with soluble a n t i g e n s h o w e d a n i m m e d i a t e increase o f
Anti-LHRH peptlcle
Anti-BSA carrier
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Fig. 8. Assessmentof TiterMax and other commercially available adjuvants. Groups of animals were immunized with a total of 50 /.~gLHRH-BSA (lute;nizing hormone releasing hormone conjugated to bovine serum albumin) in several adjuvants according to the manufacturer's prot~ls. Four New Zealand White rabbits, mixed breed male goats, or Balb/c mice per group were used. All groups were injected o~ day I and boosted on day 28 (TiterMax and CFA groups), day 21 {RAS group), or day 21 and 35 (Adjuvax group). The second injection in the CFA group was with IFA. Results using serum samples from week 8 are shown, lgG antibody liters against the carrier BSA were measured using an EL1SA. Antibody activity against the LHRH peptide was measured using a radioimmunoassay with labeled peptide. Data for the % peptide bound using a serum dilution (final concentration 1:200 for rabbits: I : 100 for mice: 1:5 for goats) in the linear portion of the dose-response curves are shown. All data are expressed as mean ± SEM.
five-fold in antibody titer which persisted for 3 months before beginning a gradual decline (Fig. 4). Studies were done to evaluate the effect of injection route on antibody titers. Animals injected in the footpad, s.c. at the base of the tail, or i.p. all produced similar titers for the first month after a single injection. In those immunized i.p., the titers then plateaued and began to decline after two months. In the groups immunized in the footpad or s.e,, the titers continued to increase for two months and plateaued at a significantly higher level (Fig. 5). CFA is frequently injected in multiple s.c. sites on the back and flank of animals in order to increase titers and reduce local inflammation. In experiments to evaluate such protocols on the efficacy of TiterMax, animals injected at two sites on opposite flanks produced lower titers than those immunized with an identical volume and dose of antigen distributed over four sites (Fig. 6). As shown in Figs. 5 and 6, the magnitude of the response varies with the route of immunization. The subclass of lgG antibody in mice was determined with a specially calibrated ELISA assay. Animals immunized with TiterMax made comparable amounts of lgG 1 and lgG2b anti-TNP with variable amounts of lgG2a and very little IgG3 (Fig. 7). This demonstrates that TiterMax is able to stimulate all of the isotypes of IgG antibody in mice in a pattern comparable to that of CFA. Animals were immunized by Hazleton Washington with a peptide hormone LHRH conjugated to BSA as a carrier and antibody was measured against both the peptide and the cartier. CFA and TiterMax were superior to Adjuvax or RAS in all comparisons with both antigens and all three species. In rabbits, TiterMax produced significantly higher responses to BSA with comparable responses to the peptide. In mice, the results between CFA and TiterMax were not significantly different for either the peptide or the carrier. In goats, CFA produced higher antibody titers against the carrier while TiterMax produced comparable titers against the peptide (Fig. 8).
Discussion
Previous studies have reported that block copolymer adjuvants are able to induce antibody titers at least comparable to those induced by CFA. Nonionic block copolymers are relatively nontoxic surface active agents and are effective immunoadjuvants(Hunter et al., 1981, 1984, 1986, 1991; Takayama et ai., 1991). However, preparation of effective immunogen formulations has been both difficult and unreliable. The water-inoil emulsion with CFA, in contrast, is readily formulated with a wide variety of antigens. TiterMax was designed to combine the activity of an especially efficacious block copolymer with a stable water-in-oil emulsion which could contain nearly any antigen, it does not contain any protein, peptide, pol$,saceharide or mycobacterial component. TiterMax was developed specifically as a replacement for CFA and was not intended for use in vaccines. We have previously described the adjuvant activity of nonionic block copolymers (Hunter et at., 1981, 1984). CRL89-41 is one such pharmaceutical grade tribloek copolymer. It was specifically developed for use with water-in-squalene emulsions and was selected for its effectivene.~s in this formulation. Its fundamental mechanisms of action, however, are undoubtedly similar to those of previously described block copolymers (Hunter et al, 1981, 1984, 1986, 1991; Takayama et al., 1991). Mineral oil is used in CFA and IFA because previous investigators have been unable to produce stable emulsions with vegetable oils. One exception, adjuvant 65, used peanut oil with aluminum monostearate as a stabilizer (Hilleman, 19671. The efficacy of adjuvant 65 is comparable to that of IFA. Both CFA and adjuvant 65 must be prepared with at least 50% oil. TiterMax represents a significant advance in emulsion technology in that stable water-in-oil emulsions can be prepared with less than 20% oil. We have found that TiterMax produces stable emulsions with antigens containing materials which break CFA emulsions. This includes certain polysaccharide-protein conjugates, 1.0% SDS or 0.75 M urea. Both the block copolymer and the copolym~:r-
coated microparticles are necessary for this stability, It is well known that microparticles contribute to emulsion stability if they have surface p r o p e r ties which facilitate their localization at t h e oilwater interface (Becher, 1965). Silica particles coated with copolymer by a proprietary process were found to have ideal surface properties for stabilization o f wateroin-squalene emulsions, Silica particles themselves have adjuvant activity. However, this activity d e p e n d s u p o n exposure o f the crystalline surface. Since the surface o f t h e particles in TiterMax emulsions is covered by copolymer and is contained within oil, it is unlikely that the inherent adjuvant activity o f silica contributes significantly to antibody titers in TiterMax emulsions. T h e r e have b e e n many advances in adjuvant and emulsification technology since Jules F r e u n d introduced his adjuvant over 50 years ago. T h e p r e s e n t experiments were u n d e r t a k e n as an att e m p t to combine a newly synthesized copolymer adjuvant with m o d e r n emulsification reagents to produce a safe and effective alternative to C F A . T h e result is an adjuvant which contains n o protein, peptide o r polysaccharide and no microbial c o m p o n e n t s . In our studies, t h e inflammatory re. sponses induced by TiterMax were mild and transient c o m p a r e d with those induced by CFA. T h e magnitude o f the antibody response varied with the species a n d route o f immunization. T h e s e data suggest that TiterMax a p p e a r s to b e a safe, effective and chemically d e f i n e d alternative to C F A in many situations.
Acknowledgements T h e authors gratefully thank A n g e l a J o h n s o n for h e r technical assistance with these experiments.
References Amyx, H.L. (1087) Control of animal pain and distress in antibody production and infectious disease studies, J. Am.
Vet. MOd.Assoc. ~91, 1287. Becher, P. (1965)Theory of emulsions: stability. In: P. Beeher, (Ed.), Emulsions: Theory and Practice, 2rid edn. Reinhold, New York, pp. 95-149. Broderson, LR. (1989) A retrospective review of lesions associated with the use of Freund's adjuvant. Lab. Animal Sci. 39, 400. Freund, J., Casals, J. and Hismer, E.P. (1937) Sensitization and antibody formation after injection of tubercle bacilli and paraffin oil. Ptoc. Soc. Exp. BioL Med. 37, 509. Freund, J. (1951) The effect of paraffin oil and mycobecteria on antibody formation and sensitization. Am. J. Clin. Pathol. 21,645. Garvcy, J.S., Cremcr, N.E., and Sussdorf, D.H. (1977) DNP. and TNP-conjugated proteins. In: J.S. Garvey, N,E. Cremer and D.H. Sussdorf (Eds.), Methods in Immunology, 3rd edn. W.A. Benjamin, Reading, MA, pp. 153-158. Hilteman, M.R. (1967) Considerations for safety and application of emulsified oil adjuvants to viral vaccines. In: R.H. Regamay, W. Hennessen. D. ikic and J, Unger, (Eds.), Symposia Series in lmmunobiological Standardization, Vol. 6, International Symposium on Adjuvants of Immunity. S. Karger, New York, p. 18. Hunter, R.L., Strickland, F, and Kezdy, F. (1981) Studies on the adjuvant activity of nonionic block polymer surfacrants. 1. The role of hydrophile-lipophile balance. J. lmmunoL 127, 1244. Hunter, R.L. and Bennett, B. (1984) Studies on the adjuvant activity of nonionic block polymer surfactants. 11. Antibody formation and inflammation related to the structure of triblock and octablock copolymers. J. lmmunol. 133, 1367. Hunter, R.L. and Bennett, B. (1986) The adjuvant activity of nonionic block polymer suffactants. IlL Characterization of selected biologicallyactive surfaces. Scand. J. ImmunoL 23, 287. Hunter, R.L., Olsgn, M. and Buynitzky, S. (1991) Adjuvant activity of non-ionic block copolymers. IV. Effect of molecular weight and formulation on titre and isotype of antibody. Vaccine 9, 250. Jenncs` L. ar~d Stumpf, W,E. (1983) Preparation and use of specific antibodies for immunohistochemistry of neuropeptides. In: P.M, Conn (Ed.), Methods in Enzymology, VoL 103. Academic Press, New York, pp. 448-458. Takayama, K., Olsen, M., Dana, P. and Hunter, R.L. (1991) Adjuvant activity of non-ionic block copolymers. V. Modulation of antibody isotype by lipopolysaccharides` lipid A and precursors. Vaccine 9, 257.
