INFEcTION AND IMMUNITY, June 1976, p. 1559-1562 Copyright 0 1976 American Society for Microbiology
Vol. 13, No. 6 Printed in USA.
Adjuvant Effects of Diethylaminoethyl-Dextran WILLIAM E. HOUSTON,* CARROLL L. CRABBS, ROBERT J. KREMER,' AND JOHN W. SPRINGER United States Army Medical Research Institute ofInfectious Diseases, Fort Detrick, Frederick, Maryland 21701 Received for publication 12 January 1976
Diethylaminoethyl-dextran exhibited a significant adjuvant effect on the immune response of rhesus monkeys to formalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (IVEE). Antibody formed to IVEE and adjuvant followed a classic immunoglobulin M-immunoglobulin G pattern; however, as compared with vaccine alone, use of this adjuvant with IVEE reduced the time required for onset of immunoglobulin G synthesis.
primary
Formalin-inactivation of virus vaccines significantly reduces the toxicity to a host that is often associated with administration of live, attenuated vaccines. Unfortunately, many times this process also decreases the antigenicity and protective effects of the vaccine. To compensate for this various methods including the use of adjuvants have been used to enhance immunogenicity. The identification of new adjuvants that are safe and effective would be valuable to improve the immunogenicity of weak, but potentially useful antigens. Diethylaminoethyl-dextran (DEAE-D) an anion-exchange resin, has been established as an efficient adjuvant for immunization of guinea pigs and swine with inactivated foot-and-mouth disease virus (15, 16). We now describe experiments which show that DEAE-D also exerts adjuvant activity in rhesus monkeys immunized with fonnalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (IVEE).
were administered per kg of body weight. Antibody class differentiation. Sera from rhesus monkeys with specific neutralizing antibody for VEE virus were diluted with an equal volume of PBS. Two-tenths milliliter of the diluted serum was layered over a linear 10 to 40% sucrose gradient; after centrifugation at 40,000 rpm (195,400 x g) for 16 h (L5-75 Ultracentrifuge, SW41 rotor, Beckman Instruments Co., Baltimore, Md.), the gradient was fractionated in 0.5-ml volumes with an Isco fraction collector (model 1200 PUP, Instrumentations Specialties Co., Lincoln, Neb.). Immunoglobulin G (IgG) was confined to fractions 5 to 9 and IgM to fractions 18 to 21 as indicated by radial immunodiffusion with class-specific anti-human serum (Immuno-Plate III Products, Hyland Division, Travenol Laboratories, Inc., Costa Mesa, Calif.). For neutralization tests, fractions 5 to 8 were pooled to determine IgG antibody, and fractions 18 to 21 for IgM antibody. Titrations with selected fraction pools were corrected for dilution in the gradient, i.e., if the volumes for the serum placed on the gradient and for the combined fraction were 0.2 and 2.0 ml, respectively, the dilution factor was 1:10. Serological tests. The presence of neutralizing antibody was determined as previously described (8). Monkeys were bled sequentially; the titers of individual animals from each group were used to calculate a geometric mean titer for each point in time.
MATERIALS AND METHODS Animals. Twenty healthy, young-adult, 2- to 4-kg rhesus monkeys (Macaca mulatta) of both sexes, housed in individual cages, were utilized. Preimmunization plaque reduction neutralizing titers to Venezuelan equine encephalomyelitis (VEE) virus RESULTS were less than 1:4. Immunization was accomplished by a single subcutaneous inoculation. A significant adjuvant effect was produced in DEAE-D. DEAE-D (Sigma Chemical Co., St. rhesus monkeys immunized with IVEE virus Louis, Mo.), molecular weight approximately 2 x vaccine when vaccine was combined with 106, was used as the adjuvant in this study. A stock DEAE-D (Fig. 1). Antibody titers from monthe solution was prepared by suspending polycation in phosphate-buffered saline, pH 7.3, to a concentra- keys receiving antigen plus 5 mg of DEAE-D per kg peaked at levels greater than 1:400 and tion of 20 mg/ml (wt/vol). Virus and virus-adjuvant mixtures. IVEE (TC-83 persisted for at least 72 days after a single strain) virus vaccine was used as the antigen in all immunization. In contrast, monkeys immustudies (4). Vaccine (0.5 ml) was combined just prior nized with vaccine alone responded with signifto immunization with an appropriate dilution of icantly lower titers. Without DEAE-D, the stock DEAE-D solution so that doses of 1 or 5 mg maximum response was observed on day 10 and I Present address: Route 1, Bonnots Mill, Mo. 65016. decreased thereafter until relatively little re1559
1560
INFECT. IMMUN.
HOUSTON E'1 AL.
1000
w F 2
CL
z 100
4
-A*
0
10
I
10
I
I
L230 0 5 7i I I I I I I
20
I
30
40
50
72
DAYS AFTER IMMUNIZATION FIG. 1. Serum plaque reduction neutralizing (PRN) titers of rhesus monkeys immunized with inactivated VEE virus and DEAE-D. Symbols: *, IVEE control N = 8; A, KVEE + 1 mg of DEAE-D per kg (N = 4); *, KVEE + 5 mg of DEAE-D per kg (N = 8), *, P
Vol. 13, No. 6 Printed in USA.
Adjuvant Effects of Diethylaminoethyl-Dextran WILLIAM E. HOUSTON,* CARROLL L. CRABBS, ROBERT J. KREMER,' AND JOHN W. SPRINGER United States Army Medical Research Institute ofInfectious Diseases, Fort Detrick, Frederick, Maryland 21701 Received for publication 12 January 1976
Diethylaminoethyl-dextran exhibited a significant adjuvant effect on the immune response of rhesus monkeys to formalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (IVEE). Antibody formed to IVEE and adjuvant followed a classic immunoglobulin M-immunoglobulin G pattern; however, as compared with vaccine alone, use of this adjuvant with IVEE reduced the time required for onset of immunoglobulin G synthesis.
primary
Formalin-inactivation of virus vaccines significantly reduces the toxicity to a host that is often associated with administration of live, attenuated vaccines. Unfortunately, many times this process also decreases the antigenicity and protective effects of the vaccine. To compensate for this various methods including the use of adjuvants have been used to enhance immunogenicity. The identification of new adjuvants that are safe and effective would be valuable to improve the immunogenicity of weak, but potentially useful antigens. Diethylaminoethyl-dextran (DEAE-D) an anion-exchange resin, has been established as an efficient adjuvant for immunization of guinea pigs and swine with inactivated foot-and-mouth disease virus (15, 16). We now describe experiments which show that DEAE-D also exerts adjuvant activity in rhesus monkeys immunized with fonnalin-inactivated Venezuelan equine encephalomyelitis virus vaccine (IVEE).
were administered per kg of body weight. Antibody class differentiation. Sera from rhesus monkeys with specific neutralizing antibody for VEE virus were diluted with an equal volume of PBS. Two-tenths milliliter of the diluted serum was layered over a linear 10 to 40% sucrose gradient; after centrifugation at 40,000 rpm (195,400 x g) for 16 h (L5-75 Ultracentrifuge, SW41 rotor, Beckman Instruments Co., Baltimore, Md.), the gradient was fractionated in 0.5-ml volumes with an Isco fraction collector (model 1200 PUP, Instrumentations Specialties Co., Lincoln, Neb.). Immunoglobulin G (IgG) was confined to fractions 5 to 9 and IgM to fractions 18 to 21 as indicated by radial immunodiffusion with class-specific anti-human serum (Immuno-Plate III Products, Hyland Division, Travenol Laboratories, Inc., Costa Mesa, Calif.). For neutralization tests, fractions 5 to 8 were pooled to determine IgG antibody, and fractions 18 to 21 for IgM antibody. Titrations with selected fraction pools were corrected for dilution in the gradient, i.e., if the volumes for the serum placed on the gradient and for the combined fraction were 0.2 and 2.0 ml, respectively, the dilution factor was 1:10. Serological tests. The presence of neutralizing antibody was determined as previously described (8). Monkeys were bled sequentially; the titers of individual animals from each group were used to calculate a geometric mean titer for each point in time.
MATERIALS AND METHODS Animals. Twenty healthy, young-adult, 2- to 4-kg rhesus monkeys (Macaca mulatta) of both sexes, housed in individual cages, were utilized. Preimmunization plaque reduction neutralizing titers to Venezuelan equine encephalomyelitis (VEE) virus RESULTS were less than 1:4. Immunization was accomplished by a single subcutaneous inoculation. A significant adjuvant effect was produced in DEAE-D. DEAE-D (Sigma Chemical Co., St. rhesus monkeys immunized with IVEE virus Louis, Mo.), molecular weight approximately 2 x vaccine when vaccine was combined with 106, was used as the adjuvant in this study. A stock DEAE-D (Fig. 1). Antibody titers from monthe solution was prepared by suspending polycation in phosphate-buffered saline, pH 7.3, to a concentra- keys receiving antigen plus 5 mg of DEAE-D per kg peaked at levels greater than 1:400 and tion of 20 mg/ml (wt/vol). Virus and virus-adjuvant mixtures. IVEE (TC-83 persisted for at least 72 days after a single strain) virus vaccine was used as the antigen in all immunization. In contrast, monkeys immustudies (4). Vaccine (0.5 ml) was combined just prior nized with vaccine alone responded with signifto immunization with an appropriate dilution of icantly lower titers. Without DEAE-D, the stock DEAE-D solution so that doses of 1 or 5 mg maximum response was observed on day 10 and I Present address: Route 1, Bonnots Mill, Mo. 65016. decreased thereafter until relatively little re1559
1560
INFECT. IMMUN.
HOUSTON E'1 AL.
1000
w F 2
CL
z 100
4
-A*
0
10
I
10
I
I
L230 0 5 7i I I I I I I
20
I
30
40
50
72
DAYS AFTER IMMUNIZATION FIG. 1. Serum plaque reduction neutralizing (PRN) titers of rhesus monkeys immunized with inactivated VEE virus and DEAE-D. Symbols: *, IVEE control N = 8; A, KVEE + 1 mg of DEAE-D per kg (N = 4); *, KVEE + 5 mg of DEAE-D per kg (N = 8), *, P
