INFECTION AND IMMUNITY, Apr. 1979, p. 160-166 0019-9567/79/04-0160/07$02.00
Vol.24, No. 1
Adjuvant Effects of Low Doses of a Nuclease-Resistant Derivative of Polyinosinic Acid. Polycytidylic Acid on Antibody Responses of Monkeys to Inactivated Venezuelan Equine Encephalomyelitis Virus Vaccine D. G. HARRINGTON,'` C. L. CRABBS,' D. E. HILMAS,'t J. R. BROWN,' G. A. HIGBEE,' F. E. COLE, JR.,' AND H. B. LEVY2 United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21701,' and Laboratory of Viral Diseases, National Institute ofAllergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014' Received for publication 15 January 1979
Polyriboinosinic -polyribocytidylic acid [poly(I) poly(C)] stabilized with polyI-lysine and carboxymethylcellulose [poly(ICLC)] has been previously shown to be a compound with marked adjuvant activity when given in high doses with inactivated Venezuelan equine encephalomyelitis (VEE) virus vaccine. This study investigated the effects of much lower doses of poly(ICLC) on the magnitude and kinetics of the primary and secondary humoral antibody responses of rhesus monkeys to inactivated VEE virus vaccine. Monkeys given a single injection of vaccine developed very low neutralizing antibody titers, whereas those given adjuvant plus vaccine had 30- to 100-fold-higher titers which remained elevated for longer than 6 months. Low doses of poly(ICLC) given with VEE virus vaccine resulted in a profound but transient increase in priming of secondary antibody responses to the antigen. In contrast, the administration of poly-l-lysine and carboxymethylcellulose alone without the poly(I) -poly(C) component of the complex had no adjuvant effect on antibody responses of monkeys to VEE virus vaccine. The temporal development of antibody by class (immunoglobulin Mimmunoglobulin G) in monkeys given two injections of adjuvant-vaccine was not different from that with vaccine alone. Serial hematological and clinical chemistry determinations on monkeys given single or multiple doses of poly(ICLC) with vaccine were not different from values in monkeys given vaccine alone. Double-stranded ribonucleic acid complexes have numerous biological activities, including the ability to induce interferon both in vivo (11) and in vitro (12) and to enhance resistance to infectious agents (10, 15, 22, 31, 33, 40) and tumors (13, 14, 27). Recent attention has focused on synthetic double-stranded polynucleotide complexes, such as polyriboinosinic-polyribocytidylic acid [poly(I) - poly(C)] and polyriboadenylic-polyribouridylic acid [poly(A).poly(U)] (see reference 3 for reviews). The adjuvant action of poly(I) .poly(C) and poly(A) - poly(U) on both cell-mediated and humoral immune responses has been reviewed by Johnson (24). It has been shown that poly(I) . poly(C) is not an effective interferon inducer in humans and nonhuman primates, apparently because of rapid enzymatic hydrolysis of the compound by primate serum (29; V. DeVita, G. Canellos, P. t Present address: U.S. Army Medical Research and Development Command, Fort Detrick, MD 21701.
Garbone, S. Baron, H. Levy, and H. Gralnick, Proc. Am. Assoc. Cancer Res. 11:21, 1970). However, stabilization of the poly(I) . poly(C) complex with poly-l-lysine and carboxymethylcellulose [poly(ICLC)] resulted in a complex that is 5- to 10- times more resistant to hydrolysis tban nonstabilized poly(I) .poly(C). Poly(ICLC) has the ability to induce moderate to high leveLs of interferon in nonhuman primates (26, 35) andv humans (H. Champney, H. B. Levy, and A. M. Lerner, Clin. Res. 24:-415A, 1976). Poly(ICLC8) was used successfully in nonhuman primates to alter the pathogenesis of several experimental virus infections, including yellow fever (39), simian hemorrhagic fever (28), Japanese encephalitis (16), rabies (2), hepatitis (32), Venezuelan equine encephalomyelitis (VEE) (17!, Russian spring-summer encephalitis (5), and vaccinia keratitis (1). In comparison with live attenuated VEE virus vaccine, inactivated vaccine is weak-C aantigenic in rhesus monkeys (21). However, when 160
VOL. 24, 1979
ADJUVANTICITY OF POLY(ICLC) IN MONKEYS
poly(ICLC) was used in high doses (1 to 3 mg/ kg) as an adjuvant with the inactivated antigen, anibody titers in monkeys were comparable to those elicited to live attenuated VEE vaccine (19). Also, when given in low doses (10 to 300 ,Ag/kg) with A/NJ/76 (New Jersey; swine) influenza virus subunit vaccine (38), poly(ICLC) has been shown to enhance humoral antibody responses in rhesus monkeys. The objectives of this study were to (i) assess the adjuvant action of lower doses of poly(ICLC) combined with inactivated VEE virus vaccine, (ii) determine what component of the complex is responsible for adjuvant activity by studying poly-l-lysine and carboxymethylcellulose (PIL-CMC) without poly(I) * poly(C), (iii) measure both primary and secondary humoral antibody responses of monkeys to various vaccineadjuvant combinations, (iv) follow the kinetics of production of immunoglobulin M (IgM) and IgG classes of neutralizing antibody, and (v) determine the effect of administration of the complex on hematological and clinical chemistry values in rhesus monkeys. (This study was presented in part at the 77th Annual Meeting of the American Society for Microbiology, New Orleans, La., 8-13 May 1977.)
161
TABLE 1. Vaccination schedule for rhesus monkeys inoculated with inactivated VEE virus vaccine with or withoutpoly(ICLC) or PlL-CMC as an adjuvant (n = 4) Inoculation'
Group
1 2 3 4 5 6 7
Primary, day 0 VEE + poly(ICLC) VEE + poly(ICLC) VEE + poly(ICLC) VEE + poly(ICLC)
Secondary, day 28 VEE + poly(ICLC)
VEE Poly(ICLC) Saline VEE VEE Saline VEE VEE + PIL-CMC VEE + PlL-CMC aAll injections were given s.c. VEE virus vaccine (0.5 ml) was combined just before inoculation with 200 lg of poly(ICLC) per kg or an equivalent (0.1 ml/kg) volume of PIL-CMC. n = 4.
combined with poly(ICLC) or PIL-CMC, vaccine alone, poly(ICLC), or saline (Table 1). After vaccination, blood samples were taken at selected intervals for hematology, serum chemistry, serology, and interferon determinations. Serum-neutralizing test. Titrations for VEE virus serum-neutralizing (SN) antibody were done in triplicate by a modification of the plaque reduction neutralizing test of Earley et al. (9), using Vero cells grown in six-well plastic trays (9.6 cm2/well, Linbro Scientific Co., Inc., New Haven, Conn.). Serial fourfold dilutions, starting with an initial dilution of 1:8, were MATERIALS AND METHODS prepared in phosphate-buffered saline, pH 7.2. The Animals. All animals were healthy, conditioned, 3- greatest serum dilution giving 80% plaque reduction to 5-kg rhesus monkeys (Macaca mulatta) of either was selected as the end point. End points below the initial dilution of 1:8 were assigned a value of 1:4 in sex, seronegative for VEE virus-neutralizing antibody. Complexes. Poly(ICLC) and PIL-CMC were pre- order to perform calculations of geometric mean titers. pared as previously described (26). The final solution Differentiation of antibody class. Immune sera of poly(ICLC) used in this study contained 2 mg of collected from monkeys (groups 1 and 5) on days 10, poly(I) .poly(C) per ml, bound as a complex with poly- 15, 22, 29, 45, and 78 postinoculation were separated l-lysine (1.5 mg/ml) and carboxymethylcellulose (5 into specific IgG- and IgM-containing fractions by the mg/ml). The PIL-CMC complex was the same con- method of Houston et al. (20). centration as that used to prepare the stabilized poly(I) Interferon assays. Serum interferon was measpoly(C). The complexes were stored at 40C and mixed ured by reduction of cytopathic effects induced by just before injection with either vaccine or an equiva- vesicular stomatitis virus in human foreskin fibroblast lent volume of pyrogen-free 0.85% saline solution. (HFS-1) tissue culture as previously described (30). Vaccine. The Formalin-inactivated VEE (TC-83) The titers are expressed as reference units, using as strain virus vaccine used in all experiments has been reference interferon National Institute of Allergy and described previously (6). VEE virus vaccine (0.5 ml) Infectious Diseases reagent catalog no. G023901537. was combined just before inoculation with 0.1 ml of Interferon titers were determined at 8 and 24 h after either poly(ICLC), PIL-CMC, or saline per kg of body primary and booster inoculations and were expressed weight. The drug contents are indicated in the descrip- as reference units per milliliter of serum. Interferon tion of the individual experiments. All injections were titers
Vol.24, No. 1
Adjuvant Effects of Low Doses of a Nuclease-Resistant Derivative of Polyinosinic Acid. Polycytidylic Acid on Antibody Responses of Monkeys to Inactivated Venezuelan Equine Encephalomyelitis Virus Vaccine D. G. HARRINGTON,'` C. L. CRABBS,' D. E. HILMAS,'t J. R. BROWN,' G. A. HIGBEE,' F. E. COLE, JR.,' AND H. B. LEVY2 United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21701,' and Laboratory of Viral Diseases, National Institute ofAllergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014' Received for publication 15 January 1979
Polyriboinosinic -polyribocytidylic acid [poly(I) poly(C)] stabilized with polyI-lysine and carboxymethylcellulose [poly(ICLC)] has been previously shown to be a compound with marked adjuvant activity when given in high doses with inactivated Venezuelan equine encephalomyelitis (VEE) virus vaccine. This study investigated the effects of much lower doses of poly(ICLC) on the magnitude and kinetics of the primary and secondary humoral antibody responses of rhesus monkeys to inactivated VEE virus vaccine. Monkeys given a single injection of vaccine developed very low neutralizing antibody titers, whereas those given adjuvant plus vaccine had 30- to 100-fold-higher titers which remained elevated for longer than 6 months. Low doses of poly(ICLC) given with VEE virus vaccine resulted in a profound but transient increase in priming of secondary antibody responses to the antigen. In contrast, the administration of poly-l-lysine and carboxymethylcellulose alone without the poly(I) -poly(C) component of the complex had no adjuvant effect on antibody responses of monkeys to VEE virus vaccine. The temporal development of antibody by class (immunoglobulin Mimmunoglobulin G) in monkeys given two injections of adjuvant-vaccine was not different from that with vaccine alone. Serial hematological and clinical chemistry determinations on monkeys given single or multiple doses of poly(ICLC) with vaccine were not different from values in monkeys given vaccine alone. Double-stranded ribonucleic acid complexes have numerous biological activities, including the ability to induce interferon both in vivo (11) and in vitro (12) and to enhance resistance to infectious agents (10, 15, 22, 31, 33, 40) and tumors (13, 14, 27). Recent attention has focused on synthetic double-stranded polynucleotide complexes, such as polyriboinosinic-polyribocytidylic acid [poly(I) - poly(C)] and polyriboadenylic-polyribouridylic acid [poly(A).poly(U)] (see reference 3 for reviews). The adjuvant action of poly(I) .poly(C) and poly(A) - poly(U) on both cell-mediated and humoral immune responses has been reviewed by Johnson (24). It has been shown that poly(I) . poly(C) is not an effective interferon inducer in humans and nonhuman primates, apparently because of rapid enzymatic hydrolysis of the compound by primate serum (29; V. DeVita, G. Canellos, P. t Present address: U.S. Army Medical Research and Development Command, Fort Detrick, MD 21701.
Garbone, S. Baron, H. Levy, and H. Gralnick, Proc. Am. Assoc. Cancer Res. 11:21, 1970). However, stabilization of the poly(I) . poly(C) complex with poly-l-lysine and carboxymethylcellulose [poly(ICLC)] resulted in a complex that is 5- to 10- times more resistant to hydrolysis tban nonstabilized poly(I) .poly(C). Poly(ICLC) has the ability to induce moderate to high leveLs of interferon in nonhuman primates (26, 35) andv humans (H. Champney, H. B. Levy, and A. M. Lerner, Clin. Res. 24:-415A, 1976). Poly(ICLC8) was used successfully in nonhuman primates to alter the pathogenesis of several experimental virus infections, including yellow fever (39), simian hemorrhagic fever (28), Japanese encephalitis (16), rabies (2), hepatitis (32), Venezuelan equine encephalomyelitis (VEE) (17!, Russian spring-summer encephalitis (5), and vaccinia keratitis (1). In comparison with live attenuated VEE virus vaccine, inactivated vaccine is weak-C aantigenic in rhesus monkeys (21). However, when 160
VOL. 24, 1979
ADJUVANTICITY OF POLY(ICLC) IN MONKEYS
poly(ICLC) was used in high doses (1 to 3 mg/ kg) as an adjuvant with the inactivated antigen, anibody titers in monkeys were comparable to those elicited to live attenuated VEE vaccine (19). Also, when given in low doses (10 to 300 ,Ag/kg) with A/NJ/76 (New Jersey; swine) influenza virus subunit vaccine (38), poly(ICLC) has been shown to enhance humoral antibody responses in rhesus monkeys. The objectives of this study were to (i) assess the adjuvant action of lower doses of poly(ICLC) combined with inactivated VEE virus vaccine, (ii) determine what component of the complex is responsible for adjuvant activity by studying poly-l-lysine and carboxymethylcellulose (PIL-CMC) without poly(I) * poly(C), (iii) measure both primary and secondary humoral antibody responses of monkeys to various vaccineadjuvant combinations, (iv) follow the kinetics of production of immunoglobulin M (IgM) and IgG classes of neutralizing antibody, and (v) determine the effect of administration of the complex on hematological and clinical chemistry values in rhesus monkeys. (This study was presented in part at the 77th Annual Meeting of the American Society for Microbiology, New Orleans, La., 8-13 May 1977.)
161
TABLE 1. Vaccination schedule for rhesus monkeys inoculated with inactivated VEE virus vaccine with or withoutpoly(ICLC) or PlL-CMC as an adjuvant (n = 4) Inoculation'
Group
1 2 3 4 5 6 7
Primary, day 0 VEE + poly(ICLC) VEE + poly(ICLC) VEE + poly(ICLC) VEE + poly(ICLC)
Secondary, day 28 VEE + poly(ICLC)
VEE Poly(ICLC) Saline VEE VEE Saline VEE VEE + PIL-CMC VEE + PlL-CMC aAll injections were given s.c. VEE virus vaccine (0.5 ml) was combined just before inoculation with 200 lg of poly(ICLC) per kg or an equivalent (0.1 ml/kg) volume of PIL-CMC. n = 4.
combined with poly(ICLC) or PIL-CMC, vaccine alone, poly(ICLC), or saline (Table 1). After vaccination, blood samples were taken at selected intervals for hematology, serum chemistry, serology, and interferon determinations. Serum-neutralizing test. Titrations for VEE virus serum-neutralizing (SN) antibody were done in triplicate by a modification of the plaque reduction neutralizing test of Earley et al. (9), using Vero cells grown in six-well plastic trays (9.6 cm2/well, Linbro Scientific Co., Inc., New Haven, Conn.). Serial fourfold dilutions, starting with an initial dilution of 1:8, were MATERIALS AND METHODS prepared in phosphate-buffered saline, pH 7.2. The Animals. All animals were healthy, conditioned, 3- greatest serum dilution giving 80% plaque reduction to 5-kg rhesus monkeys (Macaca mulatta) of either was selected as the end point. End points below the initial dilution of 1:8 were assigned a value of 1:4 in sex, seronegative for VEE virus-neutralizing antibody. Complexes. Poly(ICLC) and PIL-CMC were pre- order to perform calculations of geometric mean titers. pared as previously described (26). The final solution Differentiation of antibody class. Immune sera of poly(ICLC) used in this study contained 2 mg of collected from monkeys (groups 1 and 5) on days 10, poly(I) .poly(C) per ml, bound as a complex with poly- 15, 22, 29, 45, and 78 postinoculation were separated l-lysine (1.5 mg/ml) and carboxymethylcellulose (5 into specific IgG- and IgM-containing fractions by the mg/ml). The PIL-CMC complex was the same con- method of Houston et al. (20). centration as that used to prepare the stabilized poly(I) Interferon assays. Serum interferon was measpoly(C). The complexes were stored at 40C and mixed ured by reduction of cytopathic effects induced by just before injection with either vaccine or an equiva- vesicular stomatitis virus in human foreskin fibroblast lent volume of pyrogen-free 0.85% saline solution. (HFS-1) tissue culture as previously described (30). Vaccine. The Formalin-inactivated VEE (TC-83) The titers are expressed as reference units, using as strain virus vaccine used in all experiments has been reference interferon National Institute of Allergy and described previously (6). VEE virus vaccine (0.5 ml) Infectious Diseases reagent catalog no. G023901537. was combined just before inoculation with 0.1 ml of Interferon titers were determined at 8 and 24 h after either poly(ICLC), PIL-CMC, or saline per kg of body primary and booster inoculations and were expressed weight. The drug contents are indicated in the descrip- as reference units per milliliter of serum. Interferon tion of the individual experiments. All injections were titers
