Vitamin E adjuvant formulations in
mice
R.P. Tengerdy* and N.G. Lacetera t
Water-in-oil-type adjuvants prepared with natural or synthetic vitamin E as the oil phase,
Arlacel A or Montanide 103 as emulsifier and 1.0% aqueous solution o f bovine serum
albumin ( B S A ) as antigen were tested in mice. An emulsion o f 0.42 ml vitamin E, 0.42 ml
special mineral oil, O.15 ml Arlacel A and 1.0 ml BSA 9ave maximal humoral response, but
0.85 ml special mineral oil, 0.15 ml Arlacel A and 1.0 ml BSA (standard Freund's
incomplete adjuvant formulation) 9ave the 9reatest delayed-type hypersensitivity response.
Keywords:Vitamin E; adjuvants; humoral and cell-mediated immunity
INTRODUCTION
Water-in-oil (w/o)-type adjuvants may enhance the
immunogenicity of antigens, may result in lasting
immunity and may modulate humoral and cell-mediated
immune responses ~. The classic w/o adjuvant is the
Freund's incomplete adjuvant (FIA), but recently there
has been a renewed interest in novel-type w/o adjuvants I .
Among these novel-type w/o adjuvants are fat-soluble
vitamin E and vitamin A, replacing the mineral oil in FIA
formulations2'3. These antioxidant vitamins by them-
selves are good immunopotentiators4'5, because they
protect the sensitive, rapidly proliferating cells of the
immune system from oxidation damage and increase
cell cell interaction by membrane alteration. In the w/o
emulsion the vitamins are present in a high local
concentration, trapped in a droplet together with antigen,
normally deposited in a subcutaneous tissue. The
adjuvant emulsion amplifies local inflammatory reac-
tions, attracting polymorphonucleocytes, dendritic cells,
macrophages and lymphocytes to the site of injection,
allowing optimal interaction between antigen, antigen-
processing cells and the vitamin 6. The vitamin acts as a
physical constituent of the emulsion as well as a potent
immunoenhancer.
Vitamin E adjuvant vaccines have been tested earlier in
this laboratory against enterotoxaemia of sheep v, ram
epididymitiss'9, and E. coli infection of chickens 3. The
vitamin E adjuvant was more effective than FIA or
alum-precipitated aqueous antigen suspensions. Interest-
ingly, in contrast to FIA, which causes a strong
inflammatory reaction manifested by abscesses at the site
of injection that prevents its widespread use in veterinary
medicine and prohibits its use in human medicine, the
Department of Microbiology, Colorado State University, Fort
Collins, CO 80523, USA. tPresent address: Istituto di
Produzioni Animali, Universita di Perugia, Via San Costanzo
4, 061100, Perugia, Italy. *To whom correspondence should be
addressed. (Received 2 February 1990; accepted 4 September
1990)
0264~410X/91 j/ 030204-03
:~: 1991 Butterworth°Heinernann Ltd
204 Vaccine, Vol. 9, March 1991
vitamin E adjuvants did not cause abscesses in sheep and
chicken.
The purpose of the present study was to optimize
vitamin E adjuvant formulations by the following criteria:
maximum stimulation of immune responses, maximum
stability, storability and ease of delivery of emulsions
without side effects in the animal. The physical properties
of the emulsion were changed systematically by mixing
natural or synthetic vitamin E with mineral oil and by
using different emulsifiers. The inflammatory stimulation
was modified by using different grades of mineral oil and
dimethylsulphoxide. Bovine serum albumin was selected
as antigen, because of the well-known humoral and
cellular responses of this antigen to adjuvant immuniz-
ation. The study was performed in mice to allow a large
number of variations in adjuvant formulation.
MATERIALS AND METHODS
Adjuvant formulations
The following ingredients were used: bovine serum
albumin (BSA), Cohn fraction 5 (Sigma) prepared as
1.0% (w/v) solution in PBS; synthetic vitamin E,
DL-~-tocopheryl acetate (Roche Chem., Nutley, NJ);
natural vitamin E, RRR-D-~-tocopherol (Henkel Corp.,
La Grange, IL); regular mineral oil (USP grade), used in
FIA; special mineral oil (Exxon, NJ) light fraction,
purified; Arlacel A (mannide monoleate) (Sigma); emulsi-
fier; Montanide-103 (anhydro-mannitol-oleic esters),
(Seppic Corp., Paris, France): emulsifier: dimethyl
sulphoxide (DMSO) (Sigma).
Emulsions were prepared with a high-speed (20 000 rev
rain -1) homogenizer (Virtis Co., Gardiner, NJ, USA),
first mixing the oil phase with the emulsifier, then adding
dropwise the aqueous phase with continuous stirring.
Arlacel A was used in most experiments, as this is the
emulsifier in FIA, but Montanide-103 gave less viscous
emulsions, easier to inject. Because of the high viscosity of
tocopherol and its esters, this may be a practical
consideration in field applications.
 Vitamin E adjuvant formulations: R.P. Tengerdy and N.G. Lacetera

Immunization (DTH) was measured 24 and 48h after antigen

Swiss outbred mice, 3 months old, male and female,
obtained from the Communicable Disease Center (Fort
Collins, CO, USA) colony were distributed randomly,
five per cage, four replicates per group, into treatment
groups. The mice were fed and watered ad libitum. The
mice in each group were vaccinated with 0.2 ml each of the
respective adjuvant formulations or physiological buf-
fered saline (PBS) pH 7.4 for the control, subcutaneously.
In the preliminary experiment, the mice were bled at
21 days postvaccination by intracardiac puncture and
serum antibody level was measured by enzyme-linked
immunosorbent assay (ELISA), using goat anti-mouse
IgG peroxidase conjugate (Sigma Chem. Co., St Louis,
MO, USA) in U-bottomed polyvinyl microtitre plates
(Dynatec Labs, Alexandria, VA, USA) at 1/200 serum
dilution in PBS, in triplicate 1°. After addition of
substrate, 2,2'-azino di-(3-ethyl benzthiazolin sulphonic
acid) (Sigma), the absorbance (A4os/45Onm) of the
coloured reaction product was read in a dual beam
Dynatec reader. The absorbancy values are useful for
comparing relative antibody levels in different sera at a
single dilution.
In the main experiment, 21 days postvaccination, the
mice were challenged each with 250/~g BSA in 0.02 ml
PBS in the left hind footpad, and 0.02 ml PBS (sham
injection for control) in the right hind footpad. Footpad
thickness as a measure of delayed-type hypersensitivity
Table I Primary humoral immune response to BSA in mice immunized
with vitamin E adjuvant
Adjuvant~ Relative antibody level ~
PBS control 0.05_+ 0.01ac
BSA control 0.23 + 0.02b
E placebo 0.10_0.01a
Vitamin E 0.70_+ 0.10c
FIA 0.51 _+0.10d
aPBS, physiological buffered saline; BSA, 1.0% bovine serum albumin in
PBS; E, nL-c(-tocopheryl acetate; FIA, Freund's incomplete adjuvant.
Adjuvants were prepared by emulsifying 0.85 ml E (or mineral oil in FIA),
0.15 ml Arlacel A and 1.0 ml BSA. The E placebo contained PBS without
BSA. bMean OD40e~0~,,values in ELISA at 1/200 serum dilution, 21 days
postimmunization, n = 2 0 in four replicates of five mice each, triplicate
tests for each serum. CDifferent letters indicate significant differences
(p ~<0.05)
sensitization. The amount of swelling was measured with
a dial gauge caliper reading to 0.01 mm (Oditest, HC
Kropin, Hessen, Germany). Blood was collected 7 days
after challenge by intracardiac puncture and serum
antibody level was measured as described above.
Statistical evaluation was performed by one-way analysis
of variance.
RESULTS A N D D I S C U S S I O N
In a preliminary experiment, Freund's incomplete
adjuvant (FIA) was compared with vitamin E by
replacing the mineral oil with vitamin E in the FIA
formulation (Table 1). The relative antibody level to BSA
was significantly higher at 21 days with vitamin E-BSA
than with FIA BSA, or BSA in PBS. These results
represent a primary response to adjuvant immunization.
Similar results were obtained in guinea-pigs earlier 2.
In the main experiment, FIA was compared with a
variety of adjuvant formulations, comparing the
effectiveness of synthetic versus natural vitamin E, special
versus regular mineral oil, Arlacel A versus Montanide-
103, and DMSO. The adjuvant formulations shown in
Table 2 were used in primary immunization. The
secondary humoral and cell-mediated immune response
(delayed-type hypersensitivity) was observed after
challenge with BSA. All adjuvant formulations gave
significantly higher levels of antibody and more
pronounced delayed-type hypersensitivity reactions than
aqueous BSA. An equal mixture of special mineral oil and
vitamin E gave the best adjuvant formulation for humoral
immune response (Table 3). This corroborated our earlier
findings in chicken with a vitamin E-E. coli adjuvant 3,
and also that of Franchini and others in chicken with
a vitamin E-Newcastle disease virus adjuvant 11. The
mineral oil apparently increases the inflammatory
reaction, attracting cells to the site of injection, and the
vitamin E enhances cell-cell interaction and lymphocyte
proliferation. There was no significant difference between
special and regular mineral oil in these experiments,
although in other experiments the lighter special oil gave
better results 3. The strongest delayed-type hypersensitiv-
ity reaction was observed with FIA, probably because
mineral oil caused a stronger inflammatory reaction.

Table 2 Adjuvant formulations

Amount (ml)

Adjuvant Oil phase Aqueous phase Emulsifier

PBS control -- 1.0 PBS --

BSA control -- 1.0 BSA --
E placebo 0.85 E 1.0 PBS 0.15 A
RRR placebo 0.85 RRR 1.0 PBS 0.15 A
EA 0.85 E 1.0 BSA 0.15 A
EM 0.85 E 1.0 BSA 0.15 M
RRR 0.85 RRR 1.0 BSA 0.15 A
MO (FIA) 0.85 MO 1.0 BSA 0.15 A
SO 0.85 SO 1.0 BSA 0.15 A
E/MO 0.425 E+0.425 MO 1.0 BSA 0.15 A
E/SO 0.425 E+0.425 SO 1.0 BSA 0.15 A
E/SO, DMSO 0.425 E+0.425 SO 1.0 BSA with 25% DMSO 0.15 A

PBS, physiologic buffered saline, pH 7.4; BSA, 1.0% bovine serum albumin in PBS; E, synthetic OL-C~-tocopherylacetate; EA, E adjuvant prepared with Arlacel
A; EM, E adjuvant prepared with Montanide 103; RRR, natural c--~-tocopherol; MO, regular mineral oil in Freund's incomplete adjuvant (FIA);
SO: special mineral oil; DMSO: dimethyl sulphoxide; A: Arlacel A; M: Montanide-103

Vaccine, Vol. 9, M a r c h 1991 205

V i t a m i n E a d j u v a n t f o r m u l a t i o n s : R.P. T e n g e r d y a n d N.G. L a c e t e r a

Table 3 Humoral and cell-mediated immune response to BSA in gave a stable, easily injectable formulation, and
adjuvant-immunized and BSA--challenged mice
stimulated both humoral and cell-mediated immune
Relative Skin test (mm) ~ responses in mice.
antibody
Adjuvant level a 24 h 48 h
ACKNOWLEDGEMENTS
PBS control 0.05i0.01a 0.0±0.1a 0.0_+ 0.1a
BSA control 0.48_+ 0.02c 2.0 ± 0.5b 2.0 ± 0.5b This research was supported by a grant from F. Hoffmann
E placebo 0.25±0.02b 1.1 ±0.3b 1.2_+0.3b LaRoche & Co. AG, Basel, Switzerland. N.G.L. was
RRR placebo 0.20_+ 0.02b 1.1 +,0.2b 1.1 ±0.2b supported by a fellowship from the Consiglio Nacionale
EA 0.80 ± 0.05e 12.3 ±0.7c 13.1 +, 0.9c delle Ricerche, Italy.
EM 0.85 ± 0.06e 11.8 ± 0.8c 12.6 _+0.9c
RRR 0.85±0.07e 12.0±0.7c 12.8±0.9c
MO (FIA) 0.65 +_0.03d 21.0& 1.0e 22.3_+ 1.3e REFERENCES
SO 0.70 ± 0.05d 19.0__+1.2e 20.1 ± 1.2e
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206 Vaccine, Vol. 9, M a r c h 1991