Immunization of Macaca tascicularis (Macaca irus) Monkeys with Streptococcus mutans: Specificity of Antibody Responses in Saliva F. G. EMMINGS,* R. T. EVANS, and R. J. GENCO Department of Oral Biology, School of Dentistry, State University New York at Buffalo, Buffalo, New York 14226, USA
During the 1960s, the groundwork was laid for a resurgence of interest in immunization as a preventive measure against dental caries. The work of Keyes' in 1960 established dental caries as an infectious, transmissable disease and in that same year, Fitzgerald and Keyes2 confirmed Streptococcus mutans as a causative agent of caries. In 1965, Tomasi et a13 characterized the secretory immune system with its unique secretory IgA immunoglobulin molecule and its independence from other immune mechanisms. Bowen,4,5 in a series of reports, characterized the Macaca fascicularis monkey as a model in which to study caries. In 1969, Bowen issued a preliminary report6 stating that in monkeys intravenous immunization with live S mutans resulted in protection against caries. In this experiment, the salivary antibodies were not examined. Genco and Taubman7 then showed that the tenets demonstrated8 to govern the central secretory immune system, that is, "local stimulation-local response," also applied to peripheral secretory organs. In the rabbit, they first showed that direct injection into the mammary gland was followed by colostral IgA antibody and later9 used direct salivary gland injection to induce a salivary IgA response. Building on these findings, Taubman and Smith10 showed that a salivary IgA antibody response to S mutans, stimulated by direct injection in the vicinity of the major glands in rats, could be correlated with a lower mean caries scores and fewer carious lesions. This investigation was supported by USPHS Grants DE04061, DE0167, DE0384, RR05330, RR07006, and DE02814. * Present address: University of Rochester-Strong Memorial Hospital, Rochester, NY 14642.
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We proposed to stimulate a salivary IgA antibody response to S mutans in the M fascicularis monkey and to examine some in vitro and in vivo functions of the induced antibodies. We have already reported"1"2 our success in inducing a salivary IgA response to S mutans in young adult monkeys and offered evidence13 that immunization inhibited the establishment of the immunizing strain in dental plaque. We will summarize this data and then show the results obtained when we attempted to implant a second strain of S mutans belonging to a different serotype than the immunizing strain. We will describe the dynamics of the immune response in a group of young monkeys, having only their primary dentition, and compare the responses of the two groups.
Materials and Methods MONKEYS.-Group 1 was comprised of eight wild-caught, female irus monkeys between 3 and 6 years of age (estimated by analyses of their dentition) with most of their permanent dentition erupted. Group 2 consisted of eight monkeyrs of either sex, about 12 to 14 months of age, weighing about 2 lb, with only their primary dentition erupted. Eight times a day they were fed a high sucrose, low fiber diet augmented with Nutrical,a a vitamin, mineral, and protein supplement. Sucrose was dissolved in their drinking water to a final concentration of 3% (w/v). The water was taken directly from the fluoridated municipal supply. Weights of group 1 monkeys remained stable (5 to 7 lb) throughout the experimental period, whereas group 2 monkeys grew to about 3 lb. Mirror and explorer examinaa
Evsco Pharmaceutical Corp., Oceanside, NJ.
C181 Downloaded from jdr.sagepub.com at University of British Columbia Library on June 28, 2015 For personal use only. No other uses without permission.
EMMINGS, EVANS, AND GENCO
C182
J Dent Res Special Issue C
tions and bitewing radiographs showed no caries, and periodontal tissues showed mild, generalized gingivitis. When necessary, monkeys in group 1 were sedated with a combination of Sernylanb (Phencyclidine HCl; 4 to 5 mg) and Innovar (0.05 to 0. I ml) c This combination of drugs, even in considerably reduced doses, resulted in respiratory depression in the younger monkeys, and Promazine (6.5 mg) d was used with Sernylan instead of Innovar. BACTERIA.-S mutans strain 6715e and S mutans strains GS-5 and FA-If were used. Organisms were grown for 36 hours at 37 C in Trypticase soy broth; the cells were harb Biocentric Laboratories, St. Joseph, Mo. e Pitman Moore, Washington Crossing, NJ. Ft. Dodge Laboratories, Ft. Dodge, Iowa. e Dr. Martin Taubman, Forsyth Dental Center, Boston, Mass. f Dr. Robert Fitzgerald, VA Hospital, Miami, Fla. d
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FIG 1.-Titers of antibody responses to S muas measured by indirect immunofluorescent staining. Titers are expressed as reciprocal of dilution. Each point represents mean of samples taken from four monkeys. V'ertical bars indicate subcutaneous (SC) immunizations. Arrows indicate immunizations via parotid duct (PD). Double vertical lines signify beginning of PD immunization. Letter at each point denotes immunoglobin class of antibody. All points on abscissa (
During the 1960s, the groundwork was laid for a resurgence of interest in immunization as a preventive measure against dental caries. The work of Keyes' in 1960 established dental caries as an infectious, transmissable disease and in that same year, Fitzgerald and Keyes2 confirmed Streptococcus mutans as a causative agent of caries. In 1965, Tomasi et a13 characterized the secretory immune system with its unique secretory IgA immunoglobulin molecule and its independence from other immune mechanisms. Bowen,4,5 in a series of reports, characterized the Macaca fascicularis monkey as a model in which to study caries. In 1969, Bowen issued a preliminary report6 stating that in monkeys intravenous immunization with live S mutans resulted in protection against caries. In this experiment, the salivary antibodies were not examined. Genco and Taubman7 then showed that the tenets demonstrated8 to govern the central secretory immune system, that is, "local stimulation-local response," also applied to peripheral secretory organs. In the rabbit, they first showed that direct injection into the mammary gland was followed by colostral IgA antibody and later9 used direct salivary gland injection to induce a salivary IgA response. Building on these findings, Taubman and Smith10 showed that a salivary IgA antibody response to S mutans, stimulated by direct injection in the vicinity of the major glands in rats, could be correlated with a lower mean caries scores and fewer carious lesions. This investigation was supported by USPHS Grants DE04061, DE0167, DE0384, RR05330, RR07006, and DE02814. * Present address: University of Rochester-Strong Memorial Hospital, Rochester, NY 14642.
of
We proposed to stimulate a salivary IgA antibody response to S mutans in the M fascicularis monkey and to examine some in vitro and in vivo functions of the induced antibodies. We have already reported"1"2 our success in inducing a salivary IgA response to S mutans in young adult monkeys and offered evidence13 that immunization inhibited the establishment of the immunizing strain in dental plaque. We will summarize this data and then show the results obtained when we attempted to implant a second strain of S mutans belonging to a different serotype than the immunizing strain. We will describe the dynamics of the immune response in a group of young monkeys, having only their primary dentition, and compare the responses of the two groups.
Materials and Methods MONKEYS.-Group 1 was comprised of eight wild-caught, female irus monkeys between 3 and 6 years of age (estimated by analyses of their dentition) with most of their permanent dentition erupted. Group 2 consisted of eight monkeyrs of either sex, about 12 to 14 months of age, weighing about 2 lb, with only their primary dentition erupted. Eight times a day they were fed a high sucrose, low fiber diet augmented with Nutrical,a a vitamin, mineral, and protein supplement. Sucrose was dissolved in their drinking water to a final concentration of 3% (w/v). The water was taken directly from the fluoridated municipal supply. Weights of group 1 monkeys remained stable (5 to 7 lb) throughout the experimental period, whereas group 2 monkeys grew to about 3 lb. Mirror and explorer examinaa
Evsco Pharmaceutical Corp., Oceanside, NJ.
C181 Downloaded from jdr.sagepub.com at University of British Columbia Library on June 28, 2015 For personal use only. No other uses without permission.
EMMINGS, EVANS, AND GENCO
C182
J Dent Res Special Issue C
tions and bitewing radiographs showed no caries, and periodontal tissues showed mild, generalized gingivitis. When necessary, monkeys in group 1 were sedated with a combination of Sernylanb (Phencyclidine HCl; 4 to 5 mg) and Innovar (0.05 to 0. I ml) c This combination of drugs, even in considerably reduced doses, resulted in respiratory depression in the younger monkeys, and Promazine (6.5 mg) d was used with Sernylan instead of Innovar. BACTERIA.-S mutans strain 6715e and S mutans strains GS-5 and FA-If were used. Organisms were grown for 36 hours at 37 C in Trypticase soy broth; the cells were harb Biocentric Laboratories, St. Joseph, Mo. e Pitman Moore, Washington Crossing, NJ. Ft. Dodge Laboratories, Ft. Dodge, Iowa. e Dr. Martin Taubman, Forsyth Dental Center, Boston, Mass. f Dr. Robert Fitzgerald, VA Hospital, Miami, Fla. d
SERUM GGG
I800-
G
500250-
'Ml
A- /A
//\\
w i
o10- _gr IMMUIINIZATION
.5
-~
.
z
.
.7-
-W=;L.
.
A.
SCHEDULE
1
'a
4
90
150
4
a
CD
30
210
270
DAYS
FIG 1.-Titers of antibody responses to S muas measured by indirect immunofluorescent staining. Titers are expressed as reciprocal of dilution. Each point represents mean of samples taken from four monkeys. V'ertical bars indicate subcutaneous (SC) immunizations. Arrows indicate immunizations via parotid duct (PD). Double vertical lines signify beginning of PD immunization. Letter at each point denotes immunoglobin class of antibody. All points on abscissa (
