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A Potency Test for Trachoma Vaccine Utilizing the Mouse Toxicity Prevention Test
A POTENCY T E S T F O R TRACHOMA VACCINE U T I L I Z I N G T H E M O U S E TOXICITY P R E V E N T I O N T E S T SAN-PIN W A N G , M.D.,
AND J. THOMAS GRAYSTON,
After it became possible to cultivate the agent of trachoma and inclusion conjunctiv itis (TRIC) in series and quantity, investi gators have explored the use of vaccine in prevention of the disease.1 Results of vac cine studies among human volunteers,2 field trials in preschool children3·4 and a series of monkey experiments5-7 indicate that vac cines can either modify the disease or pre vent infections. Similar findings also have been obtained by other study groups.8'12 If vaccines are to become practical for disease prevention, development of better vaccines is necessary. For this goal, an appropriate biologic assay for potency of vaccine is a requisite. Unfortunately, few animal species are known to be susceptible to trachoma. Vaccine tests in monkeys, enabling us to evaluate protection by experimental eye in fection, would be ideal.6·7 However, ex pense, labor, and time consumption make the use of primates impractical. One possi ble potency assay is the mouse toxicity pre vention ( M T P T ) test. Briefly, this system involves active immunization of mice with TRIC antigens and subsequent challenge with toxin to assay protection. This system is applicable to most TRIC strains, and is the most specific immunologie test we know
M.D.
at this time for differentiating antigenic variations among TRIC strains.13·15 A most important finding is that antigenic differences demonstrated by M T P T also have been dem onstrated in vaccine protection of monkeys against experimental eye infections.6·7 In our previous report,14 we classified TRIC strains by cross protection tests in mice after active immunization. Intravenous injections of sublethal doses (one-third or one-half minimal 100% lethal doses) were used for immunization and the minimal 100% lethal dose was the intravenous chal lenge. Further revision of this test leads to a method for quantitative assay of immuniz ing antigens. This paper presents the results of the application of the M T P T to the de velopment of a potency assay of trachoma vaccine. MATERIALS AND METHODS
TRIC strains. TW-1, TW-3, TW-5, and TW-12 are Taiwan trachoma strains. The first two represent Taiwan immunologie prototypes, while TW-5 and TW-12 are antigenically similar to TW-1. 1 4 Strain ND-3 from New Delhi, India, is antigenically simi lar to TW-3. 1 4 The 10th to 35th yolk-sac pas sage level of these strains was used. The Bour strain 16 was isolated in California, and was From the Department of Preventive Medicine, obtained from Drs. P. Thygeson and E. JaUniversity of Washington School of Medicine, wetz. The 20th—30th yolk sac passages were Seattle, and the United States Naval Medical Re search Unit No. 2, Taipei, Taiwan. This study used. was supported in part by a United States Public Mice. Mice used in this study were the Health Service research grant NB-03144 from N I H White Swiss strain originally obtained the National Institute of Neurological Diseases and Blindness and by the Bureau of Medicine and from the Naval Medical Research Institute Surgery, United States Navy. The opinions or in Bethesda, Maryland, and maintained in assertions contained herein are the private ones the laboratory of NAMRU-2. A commercial of the authors, and are not to be construed as official or reflecting the views of the Navy Depart source of White Swiss Webster mice also ment or the Naval Service at large. was employed. Both strains were equally Reprint requests to: Dr. San-pin Wang, Depart susceptible to trachoma toxin. Normal mice ment of Preventive Medicine, University of Wash ington School of Medicine, Seattle, Washington of homogenous age and weight were used. 98105. As a routine, five-week-old mice (15-17 gm) 1443/417
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of random sex were used for toxicity titration and three-week-old mice (9-11 gm) for initiation of immunization. Preparation of toxic pools. Toxicity pools to be used for toxic challenge were prepared as follows. Seven-day-old embryonated eggs were inoculated by the yolk-sac route with 0.2 ml of highly infectious ( l % - 2 % ) yolk sac propagated agent. When one-third to one-half of the inoculated eggs had died, the remaining eggs were harvested and yolk sacs of normal appearance were excluded. A 40% suspension, in terms of original yolk sac (1.0 gm per ml = 100%) was made in sucrose-potassium-glutamate (SPG) 1 4 by grinding and emulsifying with sterile mor tar, sand, and pestle. Precautions were taken to keep materials cold during pro cessing. After a light centrifugation at 4°C (500 X G for 10 minutes), fat material floating on the surface of the supernate and large particles in the pellet were discarded. The supernatant emulsion was distributed in appropriate quantities in screw-capped vials and stored at —65° C in a mechanical freezer. Toxicity titration in mice. A sample of the frozen 40% yolk sac pool was thawed, and a series of 3:4 serial dilutions was made in SPG. The dilutions were kept in an iced container during mouse injection. Each of five or six mice in a group was injected in travenously with 0.5 ml of each dilution of agent and observed for 24 hours. The num ber of hours to death were recorded and a 50% end-point calculated according to the method of Reed and Muench.17 The lethal dose (LD50) was expressed as the percent of crude yolk sac agent in 0.5 ml. Immunizing antigens. Both crude yolk sac agent suspensions and purified preparations were used in this study. For formalinized vaccine, formalin was added in a final con centration of 0.02%. Methods of purifica tions included one or a combination of the following procedures described by Lai 18 with some modifications. They are briefly as fol lows: TRYPSIN DIGESTION. Trypsin, twice recrystalinized product from National Bio
MAY, 1967
chemical Corporation, Cleveland, Ohio, or crude product (1:250) of Difco Co., was added at a final concentration of 0.2% to a yolk-sac homogenate in buffered saline (pH 7.2) ; p H was adjusted to 7.6 with sodium bicarbonate solution, and incubated at 37°C for 15 minutes. This was followed by one cycle of differential centrifugation (see be low) and resuspended to original concentra tion. ULTRASONIC DISINTEGRATION. A volume of 50-70 ml of infected yolk-sac homogenate (10%-20%) was subjected to ultrasonic disintegration for five minutes. The sonicator used was an MSE (Measuring and Sci entific Equipment, Ltd.) ultrasonic disinte grator with a 4J4-inch probe. An electric signal generator was tuned between 18,000 to 20,000 cycles per second. DIFFERENTIAL CENTRIFUGATION. The
su
pernate from a low-speed centrifugation at 500 X G for 10 minutes in an International Refrigerated Centrifuge, was centrifuged at high speed at 30,000 X G for 30 minutes in a Servali RC-2 ss-34 angled rotator to sedi ment elementary bodies. DEAE SEPHADEX COLUMN CHROMATOGRAPHIC SEPARATION. D E A E Sephadex A-50 was obtained from Pharmacia, Ltd., Rochester, Minnesota. It was allowed to swell in distilled water, washed with 1.0 molar NaCl, and then several times by water. After washing, 18 to 20 gm of Sephadex were packed into a column (40 mm in diam eter and 600 mm in length) and washed with 0.02 M phosphate buffer (pH 7.2). A 500% yolk sac TRIC agent suspension, which had been partially purified through three cycles of differential centrifugation, was then developed in the column, and eluted with 0.35-0.4 M NaCl containing 0.4 M su crose-phosphate buffer. Usually a ratio of 1.0 gm Sephadex to 0.6 ml of 500% partially purified organisms was used for purification. The eluate was subjected to one cycle of dif ferential centrifugation in SPG or phosphate buffered saline (PBS, pH 7.2). Infectivity is retained in this procedure. GENETRON TREATMENT. Genetron 113 or
TRACHOMA AND ALLIED DISEASES Genesolv-D (trifluorotrichloroethane) from Allied Chemical Corporation, New York, was used. One-half volume of genetron was added to one volume of a partially purified 20% yolk-sac agent suspension and mixed in a blender for two minutes. After centrifu, gation at 500 X G for 10 minutes, the top aqueous layer was collected, and the pro cedure was repeated. Infectivity was lost by genetron treatment. The aqueous layer was subjected to three cycles of washing and high speed centrifugation and the pellet resus, pended to an appropriate concentration in PBS. SEDIMENTATION
IN
SUCROSE-GRADIENT.
The procedure employed was that of Ribi and Hoyer 19 with some modification. A con tinuous linear gradient from 0%-40% su?crose against 1.0 M KCl was prepared in 50 ml lusteroid tubes. One ml of 500% partially purified yolk sac agent suspension was layered on the top of a gradient tube and was centrifuged at 1500 X G for two hours with a No. 269 head of an Interna tional refrigerated centrifuge. The resulting dense zone in the gradient was collected and subjected to a cycle of differential centrifu gation in SPG. Infectivity is retained after this procedure. Particle count. Particle count was carried out by electron microscopy. At least two 10-fold dilutions (usually 10-3-10-5) of each sample were tested. One ml of each di lution was placed in a particle chamber,20 and sedimented at 30,000 X G (Servali su rotator) for 20 minutes on two 400 mesh grids which were coated with parlodium and attached to the base of the chamber.21 This base area of the chamber was 81 sq mm and one grid opening was 1084 sq micron. A count was made with the electron microscope for each of 20 openings per grid, a total of 40 openings in two grids. The dilution which gave an average count of more than five but less than 50 per grid opening was used for calculation of the total number of particles. The total number of particles was obtained by multiplying the average number per grid opening by factors
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of 8.1 X 104 and the dilution employed. Mouse toxicity prevention test .(MTPT). Unless otherwise noted, immunization of mice was carried out by the intravenous route. A series of 10-fold dilutions of test antigen was made in PBS or SPG (in case of live preparations of the organisms), and 0.5 ml of each dilution was injected intrave nously into each of a group of 10 to 15 three-week-old mice. The same dosage was again injected into the same mouse one week later. One week after the second im munization, the mice were intravenously challenged by injecting 0.5 ml of approxi mately 1.5 LD 5 0 of crude yolk sac TRIC agent. Control mice (at least 15) were in jected with two doses of 0.5 ml PBS or SPG at the same time as the immunized mice. The challenge material was retitrated in the control mice by dividing them into three groups and challenging with three di lutions of agent 1:1, 1:1.5, and 1:2.25. Mice dying within one hour after challenge were assumed to have died from anaphylaxis and they were excluded from the calculations. In the latter part of the study, epinephrine solu tion (0.05 mg adrenalin chloride/ml) was given intrapèritoneally to each mouse (0.2 ml per mouse) one to five minutes prior to chal lenge in order to prevent anaphylactic death.22 The 50% end-point was calculated according to the method of Reed and Muench.17 For the immunized group, a 50% effective dose (ED 5 0 ) was obtained. It was expressed by percentage concentration of yolk sac and/or particle count of the immun izing antigen. The LD 5 0 obtained from the control mice measured the challenge dose which was actually used. RESULTS
Mouse toxicity titration. It was learned from our previous experiments 14 that the challenge dose in the M T P T was a critical factor in obtaining satisfactory results, since protection was easily overwhelmed by an increased challenge dose. A precise and re producible titration was required for a toxic pool before it was used for challenge. In
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TABLE 1 SAMPLE OF MOUSE TOXICITY TITRATION WITH TAIWAN TRACHOMA STRAIN TW-1
Dilution of 40% Yolk Sac - 2 (22.S%) -'(16.9%) ■54(12.7%) " e ( 9.5%) " ( 7.1%)
Death (hr)
No. of Mice 4i 4 41 18
4i
4
4i 3
4i 4J 18
5 Si 18
18 18
D/T
LD6i
5/5 5/5 4/5 2/5 0/5
f-4.68
Calculation for f -4 · 68 (f-4/3)
Logf- 4 - 6 8 =-4.68XLog 1.333 = -4.68X0.12483 = -0.5742 So {-*·>* =1/3.75 (LD50 in terms of dilution from f°) 40%(f»)Xl/3.75 = 10.6% (LD50 in terms of yolk sac suspension)
order to obtain an LD 50 in a titration and also to give precision in the range from 5% to 30% (in terms of original yolk sac weight), a dilution factor of 3:4 was used for making serial dilutions of test material. A graded pattern of deaths usually was obtained with this increment of dilution. An example of toxicity titration for a TW-1 pool is given in Table 1. The LD 50 for this pool was cal culated to be the dilution of f"4·68 (f :4/3) or 1:3.75 of the original 40% yolk-sac agent material. The LD 50 could be computed as 10.6% yolk-sac suspension. For the chal lenge, 1.5 LD 50 s or 16.4% yolk-sac suspen sion was used, which was close to the mimimal 100% lethal dose we used in previous studies.14 A satisfactory toxic pool of TRIC strain was found to have an LD 50 in the range of 5% to 20% when tested in five-week-old mice. Effect of age and weight of mice on toxici ty titration. In order to test the influence of age and body weight of mice on the result
of toxicity titration, a toxic pool of TW-5 40% crude infected yolk-sac suspension was simultaneously tested in three (9-10 gm), four (12-13 gm), and five (16-17 gm) week-old mice. Each age group was selected for homogenous body weight. The results of titration are shown in Table 2. LD 50 s of 11.7%, 14.1%, and 14.5% were obtained. The younger mice were more sensitive than the older mice, indicating that mice of ho mogenous age and body weight are neces sary for reproducible titrations. Stability of toxin to the influence of tem perature. It is known that the toxicity of TRIC strains is stable when the toxic mate rial is stored at —65°C.14 In order to test the stability of toxin to the influence of different temperatures, a frozen Bour 40% yolk-sac suspension was thawed, distributed in an appropriate number of samples and each sample then exposed to different tem peratures and time periods. Conditions tested were 56°C for five and 10 minutes,
TABLE 2 INFLUENCE OF AGE AND WEIGHT OF MICE ON MOUSE TOXICITY TITRATION OF TRACHOMA STRAIN TW-5 Dilution of 4 0 % Infected Yolk Sac f-»(22.5%) f-'(16.9%) f-*(12.7%) f- 5 ( 9 . 5 % ) f-«( 7 . 1 % )
3-week-old (9-10 gm)
4-week-old (12-13 gm)
5-week-old (16-17 gm)
9/9* 9/10 5/9 3/10 0/9
9/9 9/10 1/9 1/10 1/10
7/8 7/8 2/7 0/10 0/10
14.1%
14.5%
LD60 11.7% f=4/3 * No. of toxic death/No. of mice tested
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TRACHOMA AND ALLIED DISEASES 5-1 -65'C
10-
Fig. 1 (Wang and Grayston). Stability of mouse toxicity of Bow 40% yolk sac at different tempera tures.
20
\23'-24"C
25-
30-
35 >40
03612
48
72
188
Time exposure in Hours
37°C for 1, 3, 6, 15 and 24 hours, 23-24°C room temperature for one and two days, and 2°C-5°C for 1, 2, 3 and 7 days. After these exposures, each sample was titrated for toxicity in mice. The results are shown in Figure 1. The initial titer of the pool was 7.5%. Toxicity was rapidly destroyed at 56° C, since the agent exposed to this tem perature for five and 10 minutes did not kill mice at 40%. A relatively rapid inactivation of toxin also was found at 37°C. This ex plains the poor results with yolk sac agent harvested from dead eggs, since the agent has been exposed to the incubation tempera ture (35°C) after release from cells. At room temperature, toxin was inactivated more slowly than at 37°C. Only a slight di minution of toxicity titers was observed with toxic material stored at 2°C-5°C. These results indicate that the M T P T can be performed without worry of toxicity loss within several hours of thawing the chal lenge inoculum if it is kept in an iced con tainer. Very similar stability results were obtained in another experiment with TW-12 trachoma strain. The LD 50 fell only from 5.0% to 7.5% after six days at 2°C-5°C, and to more than 30% after two days at room temperature, 15 hours at 37°C, or one minute at 56° C.
Specific protection of purified trachoma vaccines. Intraperitoneal immunization of mice with an inactivated and concentrated trachoma agent has been shown to give specific protection against mouse toxicity.13 In order to test whether intravenous injec tion, as the method of immunization with purified inactivated TRIC agent, would also give specific protection, the following exper iment was attempted. Highly purified vac cines of TW-1 and TW-3, two antigenically different Taiwan prototype strains, were prepared by either DEAE-Sephadex or trypsin-genetron-sucrose gradient (TGG) methods. In the Sephadex preparations, the organisms were still infectious to eggs with EID 50 s of 10-2 to IO"3 per 0.2 ml of 100% concentration, while in the TGG prep arations, the organisms were inactivated following genetron treatment. Two 0.5 ml doses (one week apart) of the 100% con centrations were used for intravenous im munization. The immunized mice were cross-challenged one week after the last dose of vaccine with an estimated minimal 100% lethal doses14 of TW-1 and TW-3 toxic materials. The results are shown in Table 3. Both the Sephadex and TGG prep arations showed a clear-cut homologous pro tection. These results were gratifying in
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AMERICAN JOURNAL OF OPHTHALMOLOGY TABLE 3
CROSS IMMUNIZATION WITH PURIFIED ANTIGENS IN THE MOUSE TOXICITY PREVENTION TEST
Immunizing Antigen (given i.v.) TW-1, TGG, 100%, TW-1, DSP, 100%,
Challenge Strains TW-1 0/13* 0/15
TW-3 10/12 11/13
TW-3, TGG, 100% TW-3, DSP, 100%
11/12 10/10
0/12 3/14
Control
16/19
17/17
* No. of toxic death / No. of mice tested TGG: purified by trypsin, genetron, and sucrosegradient (dead vaccine) DSP: purified by DEAE-Sephadex (live vaccine)
that vaccines with highly purified orga nisms, even though killed, were able to con fer a specific protection by intravenous im munization. Quantitative assay for protection of tra choma vaccine. In order to carry out a quan titative evaluation of the protection of trachoma vaccine, trials of mouse immuniza tion with a series of 2-fold, 4-fold, S-fold, or 10-fold dilutions of vaccine were made. After a series of preliminary experiments, we came to the conclusion that immunization with a series of 10-fold dilutions of vaccine gave the most useful pattern of protection in the MTPT. A 50% end-point of effective dose (ED 5 o) was usually obtainable within
MAY, 1967
the range of 10% to 0.001% of vaccine. A typical sample of a protection test is illus trated by the test of TW-3 TGG formalinized vaccine in Table 4. The original prep aration of this vaccine was in 2000% concentration with a particle count of 1.3 X 10 9 /ml. Five serial 10-fold dilutions ranging from 10% through 0.001% were tested for intravenous immunization. A graded pattern of protection according to dilution was ob tained with a challenge dose of 1.35 LD 50 of TW-3 crude yolk-sac suspension. The ED 5 0 was calculated as 0.246% of the vaccine, or 1.6 X 105 in terms of particle count. If a protection unit is defined for one ED 50 dose of vaccine, the original vaccine (2000%, 1.3 X 10 9 /ml) is estimated to have approxi mately 8,000 mouse protection units by this test. Two preparations of 100% TW-1 vaccine were made from the same partially purified material of TW-1 infected yolk sac; one was further purified with genetron and the other by sucrose-gradient. The genetron vaccine was inactivated with a particle count of 1.5 X 1010/ml while the gradient vaccine was live with an elementary body particle count of 8.5 X 10 9 /ml. Both vaccines were potency tested against 1.5 LD 5 0 challenge dose of TW-5. The results are shown in Table 5. A graded pattern of protection was
TABLE 4 POTENCY TEST OF PURIFIED TW-3 TRACHOMA VACCINE IN THE MOUSE TOXICITY PREVENTION TEST
Dilution of Vaccine
No. of Mice
10% 1% 0.1% 0.01% 0.001%
10 10 10 10 10
Death (hr) after Challenge ON ON 3 3 3
ON 3 4 4 4 4 5 3 4 4
5i ON 5 5 5 ON ON ON 4§ 5 5 7 7 ON
Toxicity titration of challenge dose used, TW-3:20% y.s. 3 5 ON ON ON 6 TW-3 y.s. 20% ON ON TW-3 13.3% 6 ON TW-3 8.9% 6
D/T
ED 60
1/10 2/10 6/10 10/10 10/10
0.246% (1.6X105)
5/6 2/6 1/6
(LD 50 : 14.8%)
Challenge dose = 20%/14.8% = 1.35 LD50 Note: Vaccine tested: TW-3, TGG-formalinized, 2000% (1.3X10 9 particles/ml) Immunization is given intravenously two 0.5 ml doses of vaccine dilution one week apart, and challenge made 0.5 ml intravenously one week after the last dose of vaccine ON: Mouse died overnight
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TRACHOMA AND ALLIED DISEASES TABLE 5 POTENCY TEST FOR TWO TW-1 TRACHOMA VACCINES PURIFIED BY DIFFERENT METHODS
Dilution of Vaccine 10% 1% 0.1% 0.01% 0.001%
Genetron-Purified D/T
ED6,
Gradient-Purified D/T
ED 50
0/10 0/10 1/10 3/9 8/10
0.0046%6 (6.9X10 )
0/10 0/10 1/10 2/10 8/10
0.0042%5 (3.6X10 )
Toxicity titration for challenge dose used, TW-5:23% y.s. 23% 8/10 15.3% 6/10 LD 60 : 15.3% 10.2% 0/9 Challenge dose = 23%/15.3% = 1.50 LD50 Genetron-purified—100% (1.5X1010 particles/ml) Gradient-purified—100% (8.5X10» particles/ml) D/T: No. of toxic death / No. of mice tested
seen in both vaccines with an ED 5 0 of 0.0046% or 6.9 X 105 particles for the gene tron vaccine and 0.0042% or 3.6 X 105 particles for the gradient vaccine. It is esti mated that the original TW-1 genetron vac cine has 22,000 mouse protection units/ml and the TW-1 gradient vaccine 24,000 units/ml. The genetron vaccine (killed prep aration) was nearly as potent as the gra dient vaccine (live preparation). Route of immunization in the mouse tox icity prevention test. The quantitative dif ference of immunizing efficiency between two injection routes is presented in the fol lowing experiment. An ND-3 gradient-puri fied vaccine (100%, 1.5 X 1010 particles/ml) was tested in the range of 10% through 0.001%. One group of mice received intra
venous and the other intraperitoneal immun ization with the same schedule and the same dose of vaccine. Both groups were challenged simultaneously by intravenous route with a toxic dose of ND-3. The results are shown in Table 6. An ED5„ of 0.021% or 3.2 X 10e particles was obtained with intravenous im munization, whereas 1.76% or 2.6 X 108 par ticles was the ED 5 0 with intraperitoneal im munization. There was nearly a two-log dif ference of immunizing efficiency between the two routes. A more irregular pattern of pro tection was apt to follow intraperitoneal than intravenous immunization. Time of challenge after immunization in the mouse toxicity prevention test. The best time for challenge after immunization and the duration of immunity were evaluated in
TABLE 6 COMPARISON OF IMMUNIZATION ROUTES IN THE MOUSE TOXICITY PREVENTION TEST
Dilution of Vaccine* 10% 1% 0.1% 0.01% 0.001%
Immunized Intraperitoneally 2/llt 8/9 6/7 5/8 9/11
ED 60 : 1.76% 8 (2.6X10 )
Immunized Intravenously 2/11 1/9 1/8 4/7 7/9
Toxicity t itration of challenge dose used, ND-3:2 2.5% 22.5% 7/10 15.0% 0/9 10.0% 0/9 Challenge dose = 22.5%/22.0% = 1.01 LD 50 * ND-3 sucrose-gradient purified vaccine: 100%—1.5X1010 particles/ml f No. of toxic death / No. of mice tested
ED5„: 0.021% (3.2X10·)
LD60: 22.0%
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TABLE 7 COMPARISON OF TIME INTERVALS BETWEEN IMMUNIZATION AND CHALLENGE IN THE MOUSE TOXICITY PREVENTION TEST
Intervals Immunization*— Challenge 1 weekf 2 weeks 3 weeks
Retitration of Challenge Doset
ED 60 0.025% (7.0X10«) 0.059% (1.7X10») 0.054% (1.5X10«)
Age of Mice
LD60
5 weeks 6 weeks 7 weeks
14.9% 16.8% 16.3%
Test vaccine: TW-5 genetron-purified 100%—2.8X10»/ml * Two doses of each of the 10-fold dilutions (10%—0.001%) of vaccine given intravenously one week apart t Intravenous challenge made after the last dose of immunization t Challenge dose: TW-5 yolk sac suspension—18% the following experiments. Three groups of mice each received a similar schedule of intravenous immunization (two 0.5 ml doses at weekly intervals) with serial 10-fold dilutions of TW-5 genetron-purified vaccine (100%, 2.8 X 109 particles/ml). The time of immunization was adjusted for each of the three groups so that the immunization was initiated with three-week-old mice and com-
pleted one, two and three weeks respectively prior to the simultaneous challenge. Control mice were set aside for each immunized group and they were used for titrations of the challenge dose. The results are shown in Table 7. There was no significant difference in protection among the three groups although the group challenged one week after immunization appeared to be better protect-
TABLE 8 REPEAT OF POTENCY TEST FOR TRACHOMA VACCINES
%
ED 60 (Particles
Challenge Dose: LDJO (Date)
0.006%(1.4X10«] 0.147% (3.2X10« 0.033% (7.2X10* 0.013% (2.9X10«:
1.2(1-29-64) 1.3(3-31-64)
Bour, TGG, 2000% (8.0X10 9 /ml)
0.087%(3.5X10«] 0.055%(2.2X10 6
1.2(1-29-64) 1.3(3-31-64)
TW-5, genetron, 2000%, formalinized (5.0X10 10 /ml)
0.008% (4.2X10^ 0.007%(7.2X10« 0.048% (2.4X10«)
1.3(6-22-65) 1.3(9-8-65) 1.3(2-24-66)
TW-5, gradient, 100% (l.lXlO^/ml)*
0.017%(1.9X10« 0.010%(1.1X10» 0.023% (2.5X10« 0.060% (6.6X10«) 0.010%(1.1X10« 0.045% (5.OX 10« 0.025% (2.8X10« 0.032% (3.5X10« 0.012%(1.3X10« 0.047%(5.2X10« 0.047%(5.2X10« 0.050%(5.5X10« 0.032%(3.5X10» 0.017%(1·9Χ10«
1.1(9-2-65) 1.2(2-16-66) 1.6(3-2-66) 1.3(3-23-66) 1.2(3-30-66) 1.4(4-28-66) 1.1(5-5-66) 1.3(5-25-66) 1.2(6-15-66) 1.4(6-22-66) 1.6(6-29-66) 1.3(7-6-66) 1.2(7-13-66) 1.2(7-13-66)
Bour, Sephadex, 2000% (4.3X10»/ml) Bour, Sephadex, 2000%, formalinized (4.3X10»/ml)
* Vaccine kept at -20°C The rest vaccines were kept at 2°C-5°C in the ordinary refrigerator temperature
1.2(1-29-64) 1.3(3-31-64)
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TRACHOMA AND ALLIED DISEASES TABLE 9 EFFECT OF PARTIAL PURIFICATION ON THE MOUSE TOXICITY PROTECTING ANTIGEN
Material After Process
Egg Infectivity Log EID 50 /ELD 60
Mouse Potency Test* ED5o
7.0/6.8 6.7/6.0 6.8/6.5 7.0/6.3
0.0046% >1% 0.0008% 0.0225% 0.0153% 0.0075% 0.0228%
Original ( 1 % Bour y.s.) Supernate after 20,000XG, 1 hr Pellet after 20,000XG, 1 hr Original (10% Bour y.s.) Pellet after: Washing (4X, high spin) : Trypsin & washing : Sonication, trypsin & wash * Tested against 1.8 LD50s of Bour toxin
ed than the others. The six- and sevenweek-old mice challenged two and three weeks after the last immunization were slightly more resistant than the five-weekold mice challenged one week after immuni zation. Nevertheless, two intravenous im munizations with dead vaccine provided good protection against toxic challenge three weeks after the immunization. Reproducibility of potency test. The re sults of repeated potency tests with Bour and TW-S vaccines are shown in Table 8. These vaccines, except TW-5 gradient vaccine, were stored at 2°C-5°C during the period of repeat tests. The ED 50 s obtained for each sample were usually reprodicible. The last sample, TW-5 gradient vaccine, was stored at — 20°C and tested 14 times over a period of approximately 10 months. The results in dicate that this sample did not change in potency during this period. The coefficient of variation for the test results was S/χ = 54%, indicating good reproducibility. Application of MTPT in evaluation of purification of TRIC agent. Crude yolk-sac materials harvested from trachoma infected eggs contain a variety of antigenic sub stances besides whole TRIC organisms. In order to determine whether any significant amount of soluble substances might be immunogenic in mouse toxicity prevention, 1% of crude Bour yolk sac suspension was centrifuged at 20,000 X G for one hour. The original material and the resultant su pernatant fluid and pellet (resuspended to
the original 1% by volume) were simulta neously tested in the MTPT. The results are shown in the upper part of Table 9. It is obvious that the active immunizing antigen was associated with the sedimented pellet. In another series of experiments, Bour crude yolk sac, 10% in SPG suspension, was divided in four parts. The first part served as control. The second part was washed four times with SPG and centrifuged at 20,000 X G for one hour. The third was di gested with trypsin and washed four times, and the fourth sonicated, digested with trypsin, and then washed four times. The final pellet of each was resuspended to the original 10% concentration in SPG. The results of egg infectivity titration 23 and M T P T are shown in the lower part of Table 9. In these partial purification proce dures, there was neither appreciable loss of &gg infectivity nor the active immunizing power of the Bour antigen. It is also worthy to note that the M T P T was fairly reproduc ible and that a small amount of antigen was enough to immunize mice against toxic chal lenge. DISCUSSION
In our previous report,14 immunization of mice against toxic challenge was made by intravenous injection of sublethal doses of living trachoma agents. It can be argued that the protection afforded in that case might be due to toxin tolerance rather than to immunologie response. This seems un-
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likely since the protection afforded by in travenous immunization has been highly specific, and in this report protection was obtained by immunization with nontoxic and highly diluted antigen. Protection was demonstrated without much loss three weeks after intravenous immunization with a nontoxic antigen. Unequivocal demonstration of immune mechanism by neutralizing of toxin with serum antibody has been accomplished in recent experiments (unreported). Using M T P T as a tool, we have demon strated that the potency of a vaccine may be evaluated in terms of its immunizing capacity against toxic death of mice. Whether the antitoxic immunity is an adequate measure of trachoma immunity is not yet definite. A concept that the pathologic changes of tra choma are due to the toxic effect of the agent has been presented.24 Furthermore, good correlation between the results of mouse toxicity prevention and the preven tion of monkey eye infection can be demon strated. In our successful vaccine trials in monkeys, protection was observed only with homologous vaccine but not with heterologous vaccine, the specificity of which paral leled the antigenic difference determined by the mouse toxicity prevention test.7 The po tency of vaccine assessed in the prevention of monkey eye infection has been shown to reflect the results of potency tests in the MTPT. 7 This would seem to indicate a close association between antitoxic immuni ty and protection from trachoma. In this sense, it is believed that the M T P T is use ful for potency assay of trachoma vaccine and for investigation of protective antigens. Characterization of trachoma antigens which are responsible for prevention of mouse tox icity is being studied, and the results will be reported separately.25 A minimum antigenic mass was found to be necessary for immunization of mice against toxic challenge, irrespective of prep arations of vaccine used. ED 50 was estimat ed to be about 105-106 particles of the agent.
MAY, 1967
We have also found that the critical anti genic mass is 108-109 particles for protection against experimental monkey eye infections when similar vaccines are administered with an oil adjuvant.7 This study showed that a small amount of antigen is sufficient for immunizing mice against toxic challenge. Antigenic typing of new TRIC strains can be simplified by em ploying 1% of crude yolk-sac suspension for immunization, and 1.5 LD 50 dose of crude infected yolk-sac suspension for chal lenge.15 SUMMARY
A quantitative modification of the mouse toxicity prevention test was described for potency assay of trachoma vaccine. The method of toxicity titration of toxin was re vised. Closely spaced dilutions (3:4) of in fected yolk sac suspension (ranging from 5% to 30%) were intravenously titrated in mice. The 50% end-point of lethality (LD 50 ) was obtained and the results of titrations were reproducible. The age and weight of mice were critical to reproducible titration. Using this titration method, thermostability of toxin was investigated. Toxin was rapidly inactivated within five minutes at 56°C, but was stable for days at the refrig erator temperature of 2°C-5°C, and appar ently stable for several months at — 65°C. Although toxin was found to be labile, ei ther live (DEAE-Sephadex purified) or killed (trypsin-genetron-sucrose gradient purified) organisms were effective immuniz ing antigens. A quantitative measurement of protecting antigen was devised as follows: two doses of serial 10-fold dilutions of vaccine ranging from 10% to 0.001% concentration were injected intravenously into groups of 10-15 mice at weekly intervals. They were intra venously challenged one week after the last immunization with 1.5 LD 50 of toxin. A graded pattern of protection was usually ob tained by dilution of antigen and a 50% end-
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point of effective dose (ED50) of vaccine and Alexander, E. R. : Prevention of trachoma with Arch. Environ. Hlth. 8:518, 1964. can be obtained. The results were reproduc vaccine. 7. Wang, S. P., Grayston, J. T. and Alexander, ible in repeated testings of a given vaccine. E. R. : Trachoma vaccine studies in monkeys. Much better results were obtained with Am. J. Ophth. 63:1615, 1967. 8. Dawson, C, Jawetz, E., Thygeson, P. and intravenous immunization than with intraHanna, L. : Trachoma viruses isolated in the peritoneal. Protection can be demonstrated United States. 4. Infectivity and immunogenicity without much loss three weeks after the for monkeys. Proc. Soc. Exper. Biol. & Med. intravenous immunization. In a parallel test, 106:898, 1961. 9. Collier, L. H. : Experiments with trachoma killed (genetron purified) and live (sucrose- vaccines. 1. Experimental system using inclusion gradient purified) preparations of vaccine blenorrhea virus. Lancet, 1:795, 1961. 10. Snyder, J. C, Bell, S. D., Jr., Murray, E. derived from the same infected yolk sac S., Thygeson, P. and Haddad, N. A. : Attempt to materials were shown to be equally protec immunize a volunteer (with formalin-inactivated tive in the quantitative test. virus) against experimental trachoma induced by This potency test has been used for evalu Saudi Arabian strain 2. Ann. N.Y. Acad. Sci. 98:368, 1962. ation of purification methods for vaccine. 11. Sampaio, A. A., Ayres, L., Haddad, N. A., The results indicate that the immunizing an Bell, S. D., Jr., Murray, E. S. and Snyder, J. tigen is closely associated with TRIC agent C. : Studies on trachoma. IV. Investigation in Portugal on formalin-killed trachoma vaccine elementary body particles, and that treat with special reference to serologie response. Am. ment of the infected crude yolk sacs with J. Trop. Med. & Hyg. 12:909, 1963. 12. Snyder, J. C, Nichols, R. L., Bell, S. D., 0.2% trypsin at 37°C for 15 minutes, ultra Haddad, N. O.. Murray, E. S. and McComb, D. sonic disintegration at 18,000-20,000 cycles E. : Vaccination against trachoma in Saudi Ara per second for five minutes, and repeated bia: Design of field trials and initial results. Inwashings with SPG do not remove infectivi dust & Trop. Health, 5:65, 1963. 13. Bell, S. D., Jr. and Theobald, B. : Differ ty or protective antigens. ACKNOWLEDGMENTS
We wish to acknowledge the technical assistance of Mr. Chia-ho Liu, Mr. Ju-chun Ho, Miss Jean C. A. Lin and Mrs. Judy Tesmer. REFERENCES
1. 'Collier, L. H. : The present status of tracho ma vaccination studies. Bull. World Health Org. 34:233, 1966. 2. Grayston J. T., Wang, S. P., Yang, Y. F. and Woolridge, R. L. : The effect of trachoma virus vaccine on the course of experimental infection in blind human volunteers. J. Exp. Med. 115:1009, 1962. 3. Grayston J. T., Woolridge, R. L , Wang, S. P., Yen, C. H., Cheng, K. S. and Chang, I. S.: Field studies of protection from infection by ex perimental trachoma virus vaccine in preschoolage children in Taiwan. Proc. Soc. Exper. Biol. &Med. 112:589,1963. 4. Dhir, S. P., Agarwal, L. P., Detels, R., Wang, S. P. and Grayston, J. T.: Field trial of two bi valent trachoma vaccines in children of a Punjabi Indian village. Am. J. Ophth. 63:1639, 1967. 5. Grayston, J. T., Woolridge, R. L. and Wang, S. P. : Trachoma vaccine studies on Taiwan. Ann. N.Y. Acad. Sci. 98:352, 1962. 6. Grayston, J. T., Wang, S. P., Woolridge, R. L.
entiation of trachoma strains on the basis of im munization against toxic death of mice. Ann. N.Y. Acad. Sci. 98:337, 1962. 14. Wang, S. P. and Grayston, J. T.: Clas sification of trachoma virus strains by protection of mice from toxic death. J. Immunol. 90:849, 1963. 15. Alexander, E. R., Wang, S. P. and Grayston, J. T.: Further classification of TRIC agents from ocular trachoma and other sources by the mouse toxicity prevention test. Am. J. Ophth. 63: 1469,1967. 16. Hanna, L., Thygeson, P., Jawetz, E. and Dawson, C. : Elementary body virus isolated from clinical trachoma in California. Science, 130:1339, 1959. 17. Reed, L. J. and Muench, H. : A simple method of estimating fifty per cent end point. Am. J. Hyg. 27 :493, 1938. 18. Lai, J. S. : Purification of trachoma agent. Thesis for M.S. degree, University of Washington Department of Microbiology, 1962. 19. Ribi, E. and Hoyer, B. H.: Purification of Q fever rickettsiae by density-gradient sedimen tation. J. Immunol. 85:314, 1960. 20. Sharp., D. G. : Enumeration of virus particles by electron micrography. Proc. Soc. Exper. Biol. & Med. 70 :S4, 1949. 21. Litwin, J. : The growth cycle of the psitta cosis group of micro-organisms. J. Infect. Dis. 105:129, 1959.
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22. Alexander, E. R. and Fresh, J. W. : Preven 24. Thygeson, P. and Nataf, R.: Etiologic tion of anaphylactic death in the mouse toxicity problems in trachoma. Rev. Int. Trach., 35:177, prevention test for TRIC agents. Am. J. Ophth. 63 : 1958. 1462, 1967. 25. Wang, S. P., Kenny, G. E. and Grayston, J. 23. Wang, S. P. and Grayston, J. T. : Egg infec- T. : Characterization of trachoma antigens protec tivity assay of trachoma virus. Proc. Soc. Exper. tive against mouse toxicity. Am. J. Ophth. 63 : Biol. &Med. 115:587, 1964. 1454, 1967.
CHARACTERIZATION O F TRACHOMA ANTIGENS PROTECTIVE AGAINST MOUSE TOXICITY SAN-PIN WANG, M.D.,
GEORGE E. K E N N Y ,
J. THOMAS GRAYSTON,
Although active immunization with tra choma vaccine may prevent or modify dis ease in man1-5 and primates,6"9 the nature of the protective antigens is unknown. Vaccine potency can be evaluated in monkeys9 but this procedure is difficult and expensive. A more convenient model is the mouse toxicity prevention test which not only permits differentiation of TRIC strains10"12 but also has permitted the development of a quanti tative method for assay of protective antigens.13 The results of vaccine potency assay in the mouse toxicity system correlate well with the results of prevention of mon key eye infection.0 This potency assay was employed for the evaluation of methods of vaccine preparation and also to characterize the protective antigens. This report presents preliminary findings concerning the physicochemical stability of the protective antigens of trachoma. MATERIAL AND METHOD
TRIC strains. Strains of TRIC agents used in this study were three ocular trachoFrom the Department of Preventive Medicine, University of Washington, School of Medicine, Seattle. This work was supported by a United States Public Health Service research grant NB03144 from the National Institute of Neurologi cal Diseases and Blindness, National Institutes of Health. Reprint requests to : Dr. San-pin Wang, Depart ment of Preventive Medicine, University of Wash ington School of Medicine, Seattle, Washington 98105.
P H . D . AND
M.D
ma strains isolated by us in Taiwan (TW-3, TW-5 and ND-3). 1 1 The 10th to 38th yolk sac passages of these organisms were em ployed. Purification of TRIC agent. Infected yolk sacs were disrupted by one of the following techniques: (1) shaking with glass beads for 10 minutes, (2) homogenization with an electric blender (Servali "Omnimix") and (3) sonic disruption with an ultrasonic de vice (Branson) at 18,000-20,000 cycles/ second for one hour in an icebath. The organisms were partially purified from the yolk sac suspension by cycles of washing at low (500 X G) and high (30,000 X G) speed centrifugation with phosphate buf fered saline ( P B S ) . The pellets from lowspeed sedimentations were discarded, the supernatant was centrifuged at high speed and this pellet saved and the process repeated. These partially purified organisms were then purified by the following methods: Sucrose-KCl gradient sedimentation. De tails are described separately.13 Briefly, a 100% concentration* of partially purified organisms was layered on the top of the centrifuge bottles containing 250 ml of a continuous 0%-40% sucrose 1.0 M KC1 grad ient, centrifuged at 1500 X G for two hours, and the dense zones in the gradient were collected. After cycles of washing with PBS, the final pellets were suspended at * Concentration values are in %. The original yolk sac is considered 100%.
AND J. THOMAS GRAYSTON,
After it became possible to cultivate the agent of trachoma and inclusion conjunctiv itis (TRIC) in series and quantity, investi gators have explored the use of vaccine in prevention of the disease.1 Results of vac cine studies among human volunteers,2 field trials in preschool children3·4 and a series of monkey experiments5-7 indicate that vac cines can either modify the disease or pre vent infections. Similar findings also have been obtained by other study groups.8'12 If vaccines are to become practical for disease prevention, development of better vaccines is necessary. For this goal, an appropriate biologic assay for potency of vaccine is a requisite. Unfortunately, few animal species are known to be susceptible to trachoma. Vaccine tests in monkeys, enabling us to evaluate protection by experimental eye in fection, would be ideal.6·7 However, ex pense, labor, and time consumption make the use of primates impractical. One possi ble potency assay is the mouse toxicity pre vention ( M T P T ) test. Briefly, this system involves active immunization of mice with TRIC antigens and subsequent challenge with toxin to assay protection. This system is applicable to most TRIC strains, and is the most specific immunologie test we know
M.D.
at this time for differentiating antigenic variations among TRIC strains.13·15 A most important finding is that antigenic differences demonstrated by M T P T also have been dem onstrated in vaccine protection of monkeys against experimental eye infections.6·7 In our previous report,14 we classified TRIC strains by cross protection tests in mice after active immunization. Intravenous injections of sublethal doses (one-third or one-half minimal 100% lethal doses) were used for immunization and the minimal 100% lethal dose was the intravenous chal lenge. Further revision of this test leads to a method for quantitative assay of immuniz ing antigens. This paper presents the results of the application of the M T P T to the de velopment of a potency assay of trachoma vaccine. MATERIALS AND METHODS
TRIC strains. TW-1, TW-3, TW-5, and TW-12 are Taiwan trachoma strains. The first two represent Taiwan immunologie prototypes, while TW-5 and TW-12 are antigenically similar to TW-1. 1 4 Strain ND-3 from New Delhi, India, is antigenically simi lar to TW-3. 1 4 The 10th to 35th yolk-sac pas sage level of these strains was used. The Bour strain 16 was isolated in California, and was From the Department of Preventive Medicine, obtained from Drs. P. Thygeson and E. JaUniversity of Washington School of Medicine, wetz. The 20th—30th yolk sac passages were Seattle, and the United States Naval Medical Re search Unit No. 2, Taipei, Taiwan. This study used. was supported in part by a United States Public Mice. Mice used in this study were the Health Service research grant NB-03144 from N I H White Swiss strain originally obtained the National Institute of Neurological Diseases and Blindness and by the Bureau of Medicine and from the Naval Medical Research Institute Surgery, United States Navy. The opinions or in Bethesda, Maryland, and maintained in assertions contained herein are the private ones the laboratory of NAMRU-2. A commercial of the authors, and are not to be construed as official or reflecting the views of the Navy Depart source of White Swiss Webster mice also ment or the Naval Service at large. was employed. Both strains were equally Reprint requests to: Dr. San-pin Wang, Depart susceptible to trachoma toxin. Normal mice ment of Preventive Medicine, University of Wash ington School of Medicine, Seattle, Washington of homogenous age and weight were used. 98105. As a routine, five-week-old mice (15-17 gm) 1443/417
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of random sex were used for toxicity titration and three-week-old mice (9-11 gm) for initiation of immunization. Preparation of toxic pools. Toxicity pools to be used for toxic challenge were prepared as follows. Seven-day-old embryonated eggs were inoculated by the yolk-sac route with 0.2 ml of highly infectious ( l % - 2 % ) yolk sac propagated agent. When one-third to one-half of the inoculated eggs had died, the remaining eggs were harvested and yolk sacs of normal appearance were excluded. A 40% suspension, in terms of original yolk sac (1.0 gm per ml = 100%) was made in sucrose-potassium-glutamate (SPG) 1 4 by grinding and emulsifying with sterile mor tar, sand, and pestle. Precautions were taken to keep materials cold during pro cessing. After a light centrifugation at 4°C (500 X G for 10 minutes), fat material floating on the surface of the supernate and large particles in the pellet were discarded. The supernatant emulsion was distributed in appropriate quantities in screw-capped vials and stored at —65° C in a mechanical freezer. Toxicity titration in mice. A sample of the frozen 40% yolk sac pool was thawed, and a series of 3:4 serial dilutions was made in SPG. The dilutions were kept in an iced container during mouse injection. Each of five or six mice in a group was injected in travenously with 0.5 ml of each dilution of agent and observed for 24 hours. The num ber of hours to death were recorded and a 50% end-point calculated according to the method of Reed and Muench.17 The lethal dose (LD50) was expressed as the percent of crude yolk sac agent in 0.5 ml. Immunizing antigens. Both crude yolk sac agent suspensions and purified preparations were used in this study. For formalinized vaccine, formalin was added in a final con centration of 0.02%. Methods of purifica tions included one or a combination of the following procedures described by Lai 18 with some modifications. They are briefly as fol lows: TRYPSIN DIGESTION. Trypsin, twice recrystalinized product from National Bio
MAY, 1967
chemical Corporation, Cleveland, Ohio, or crude product (1:250) of Difco Co., was added at a final concentration of 0.2% to a yolk-sac homogenate in buffered saline (pH 7.2) ; p H was adjusted to 7.6 with sodium bicarbonate solution, and incubated at 37°C for 15 minutes. This was followed by one cycle of differential centrifugation (see be low) and resuspended to original concentra tion. ULTRASONIC DISINTEGRATION. A volume of 50-70 ml of infected yolk-sac homogenate (10%-20%) was subjected to ultrasonic disintegration for five minutes. The sonicator used was an MSE (Measuring and Sci entific Equipment, Ltd.) ultrasonic disinte grator with a 4J4-inch probe. An electric signal generator was tuned between 18,000 to 20,000 cycles per second. DIFFERENTIAL CENTRIFUGATION. The
su
pernate from a low-speed centrifugation at 500 X G for 10 minutes in an International Refrigerated Centrifuge, was centrifuged at high speed at 30,000 X G for 30 minutes in a Servali RC-2 ss-34 angled rotator to sedi ment elementary bodies. DEAE SEPHADEX COLUMN CHROMATOGRAPHIC SEPARATION. D E A E Sephadex A-50 was obtained from Pharmacia, Ltd., Rochester, Minnesota. It was allowed to swell in distilled water, washed with 1.0 molar NaCl, and then several times by water. After washing, 18 to 20 gm of Sephadex were packed into a column (40 mm in diam eter and 600 mm in length) and washed with 0.02 M phosphate buffer (pH 7.2). A 500% yolk sac TRIC agent suspension, which had been partially purified through three cycles of differential centrifugation, was then developed in the column, and eluted with 0.35-0.4 M NaCl containing 0.4 M su crose-phosphate buffer. Usually a ratio of 1.0 gm Sephadex to 0.6 ml of 500% partially purified organisms was used for purification. The eluate was subjected to one cycle of dif ferential centrifugation in SPG or phosphate buffered saline (PBS, pH 7.2). Infectivity is retained in this procedure. GENETRON TREATMENT. Genetron 113 or
TRACHOMA AND ALLIED DISEASES Genesolv-D (trifluorotrichloroethane) from Allied Chemical Corporation, New York, was used. One-half volume of genetron was added to one volume of a partially purified 20% yolk-sac agent suspension and mixed in a blender for two minutes. After centrifu, gation at 500 X G for 10 minutes, the top aqueous layer was collected, and the pro cedure was repeated. Infectivity was lost by genetron treatment. The aqueous layer was subjected to three cycles of washing and high speed centrifugation and the pellet resus, pended to an appropriate concentration in PBS. SEDIMENTATION
IN
SUCROSE-GRADIENT.
The procedure employed was that of Ribi and Hoyer 19 with some modification. A con tinuous linear gradient from 0%-40% su?crose against 1.0 M KCl was prepared in 50 ml lusteroid tubes. One ml of 500% partially purified yolk sac agent suspension was layered on the top of a gradient tube and was centrifuged at 1500 X G for two hours with a No. 269 head of an Interna tional refrigerated centrifuge. The resulting dense zone in the gradient was collected and subjected to a cycle of differential centrifu gation in SPG. Infectivity is retained after this procedure. Particle count. Particle count was carried out by electron microscopy. At least two 10-fold dilutions (usually 10-3-10-5) of each sample were tested. One ml of each di lution was placed in a particle chamber,20 and sedimented at 30,000 X G (Servali su rotator) for 20 minutes on two 400 mesh grids which were coated with parlodium and attached to the base of the chamber.21 This base area of the chamber was 81 sq mm and one grid opening was 1084 sq micron. A count was made with the electron microscope for each of 20 openings per grid, a total of 40 openings in two grids. The dilution which gave an average count of more than five but less than 50 per grid opening was used for calculation of the total number of particles. The total number of particles was obtained by multiplying the average number per grid opening by factors
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of 8.1 X 104 and the dilution employed. Mouse toxicity prevention test .(MTPT). Unless otherwise noted, immunization of mice was carried out by the intravenous route. A series of 10-fold dilutions of test antigen was made in PBS or SPG (in case of live preparations of the organisms), and 0.5 ml of each dilution was injected intrave nously into each of a group of 10 to 15 three-week-old mice. The same dosage was again injected into the same mouse one week later. One week after the second im munization, the mice were intravenously challenged by injecting 0.5 ml of approxi mately 1.5 LD 5 0 of crude yolk sac TRIC agent. Control mice (at least 15) were in jected with two doses of 0.5 ml PBS or SPG at the same time as the immunized mice. The challenge material was retitrated in the control mice by dividing them into three groups and challenging with three di lutions of agent 1:1, 1:1.5, and 1:2.25. Mice dying within one hour after challenge were assumed to have died from anaphylaxis and they were excluded from the calculations. In the latter part of the study, epinephrine solu tion (0.05 mg adrenalin chloride/ml) was given intrapèritoneally to each mouse (0.2 ml per mouse) one to five minutes prior to chal lenge in order to prevent anaphylactic death.22 The 50% end-point was calculated according to the method of Reed and Muench.17 For the immunized group, a 50% effective dose (ED 5 0 ) was obtained. It was expressed by percentage concentration of yolk sac and/or particle count of the immun izing antigen. The LD 5 0 obtained from the control mice measured the challenge dose which was actually used. RESULTS
Mouse toxicity titration. It was learned from our previous experiments 14 that the challenge dose in the M T P T was a critical factor in obtaining satisfactory results, since protection was easily overwhelmed by an increased challenge dose. A precise and re producible titration was required for a toxic pool before it was used for challenge. In
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TABLE 1 SAMPLE OF MOUSE TOXICITY TITRATION WITH TAIWAN TRACHOMA STRAIN TW-1
Dilution of 40% Yolk Sac - 2 (22.S%) -'(16.9%) ■54(12.7%) " e ( 9.5%) " ( 7.1%)
Death (hr)
No. of Mice 4i 4 41 18
4i
4
4i 3
4i 4J 18
5 Si 18
18 18
D/T
LD6i
5/5 5/5 4/5 2/5 0/5
f-4.68
Calculation for f -4 · 68 (f-4/3)
Logf- 4 - 6 8 =-4.68XLog 1.333 = -4.68X0.12483 = -0.5742 So {-*·>* =1/3.75 (LD50 in terms of dilution from f°) 40%(f»)Xl/3.75 = 10.6% (LD50 in terms of yolk sac suspension)
order to obtain an LD 50 in a titration and also to give precision in the range from 5% to 30% (in terms of original yolk sac weight), a dilution factor of 3:4 was used for making serial dilutions of test material. A graded pattern of deaths usually was obtained with this increment of dilution. An example of toxicity titration for a TW-1 pool is given in Table 1. The LD 50 for this pool was cal culated to be the dilution of f"4·68 (f :4/3) or 1:3.75 of the original 40% yolk-sac agent material. The LD 50 could be computed as 10.6% yolk-sac suspension. For the chal lenge, 1.5 LD 50 s or 16.4% yolk-sac suspen sion was used, which was close to the mimimal 100% lethal dose we used in previous studies.14 A satisfactory toxic pool of TRIC strain was found to have an LD 50 in the range of 5% to 20% when tested in five-week-old mice. Effect of age and weight of mice on toxici ty titration. In order to test the influence of age and body weight of mice on the result
of toxicity titration, a toxic pool of TW-5 40% crude infected yolk-sac suspension was simultaneously tested in three (9-10 gm), four (12-13 gm), and five (16-17 gm) week-old mice. Each age group was selected for homogenous body weight. The results of titration are shown in Table 2. LD 50 s of 11.7%, 14.1%, and 14.5% were obtained. The younger mice were more sensitive than the older mice, indicating that mice of ho mogenous age and body weight are neces sary for reproducible titrations. Stability of toxin to the influence of tem perature. It is known that the toxicity of TRIC strains is stable when the toxic mate rial is stored at —65°C.14 In order to test the stability of toxin to the influence of different temperatures, a frozen Bour 40% yolk-sac suspension was thawed, distributed in an appropriate number of samples and each sample then exposed to different tem peratures and time periods. Conditions tested were 56°C for five and 10 minutes,
TABLE 2 INFLUENCE OF AGE AND WEIGHT OF MICE ON MOUSE TOXICITY TITRATION OF TRACHOMA STRAIN TW-5 Dilution of 4 0 % Infected Yolk Sac f-»(22.5%) f-'(16.9%) f-*(12.7%) f- 5 ( 9 . 5 % ) f-«( 7 . 1 % )
3-week-old (9-10 gm)
4-week-old (12-13 gm)
5-week-old (16-17 gm)
9/9* 9/10 5/9 3/10 0/9
9/9 9/10 1/9 1/10 1/10
7/8 7/8 2/7 0/10 0/10
14.1%
14.5%
LD60 11.7% f=4/3 * No. of toxic death/No. of mice tested
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TRACHOMA AND ALLIED DISEASES 5-1 -65'C
10-
Fig. 1 (Wang and Grayston). Stability of mouse toxicity of Bow 40% yolk sac at different tempera tures.
20
\23'-24"C
25-
30-
35 >40
03612
48
72
188
Time exposure in Hours
37°C for 1, 3, 6, 15 and 24 hours, 23-24°C room temperature for one and two days, and 2°C-5°C for 1, 2, 3 and 7 days. After these exposures, each sample was titrated for toxicity in mice. The results are shown in Figure 1. The initial titer of the pool was 7.5%. Toxicity was rapidly destroyed at 56° C, since the agent exposed to this tem perature for five and 10 minutes did not kill mice at 40%. A relatively rapid inactivation of toxin also was found at 37°C. This ex plains the poor results with yolk sac agent harvested from dead eggs, since the agent has been exposed to the incubation tempera ture (35°C) after release from cells. At room temperature, toxin was inactivated more slowly than at 37°C. Only a slight di minution of toxicity titers was observed with toxic material stored at 2°C-5°C. These results indicate that the M T P T can be performed without worry of toxicity loss within several hours of thawing the chal lenge inoculum if it is kept in an iced con tainer. Very similar stability results were obtained in another experiment with TW-12 trachoma strain. The LD 50 fell only from 5.0% to 7.5% after six days at 2°C-5°C, and to more than 30% after two days at room temperature, 15 hours at 37°C, or one minute at 56° C.
Specific protection of purified trachoma vaccines. Intraperitoneal immunization of mice with an inactivated and concentrated trachoma agent has been shown to give specific protection against mouse toxicity.13 In order to test whether intravenous injec tion, as the method of immunization with purified inactivated TRIC agent, would also give specific protection, the following exper iment was attempted. Highly purified vac cines of TW-1 and TW-3, two antigenically different Taiwan prototype strains, were prepared by either DEAE-Sephadex or trypsin-genetron-sucrose gradient (TGG) methods. In the Sephadex preparations, the organisms were still infectious to eggs with EID 50 s of 10-2 to IO"3 per 0.2 ml of 100% concentration, while in the TGG prep arations, the organisms were inactivated following genetron treatment. Two 0.5 ml doses (one week apart) of the 100% con centrations were used for intravenous im munization. The immunized mice were cross-challenged one week after the last dose of vaccine with an estimated minimal 100% lethal doses14 of TW-1 and TW-3 toxic materials. The results are shown in Table 3. Both the Sephadex and TGG prep arations showed a clear-cut homologous pro tection. These results were gratifying in
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AMERICAN JOURNAL OF OPHTHALMOLOGY TABLE 3
CROSS IMMUNIZATION WITH PURIFIED ANTIGENS IN THE MOUSE TOXICITY PREVENTION TEST
Immunizing Antigen (given i.v.) TW-1, TGG, 100%, TW-1, DSP, 100%,
Challenge Strains TW-1 0/13* 0/15
TW-3 10/12 11/13
TW-3, TGG, 100% TW-3, DSP, 100%
11/12 10/10
0/12 3/14
Control
16/19
17/17
* No. of toxic death / No. of mice tested TGG: purified by trypsin, genetron, and sucrosegradient (dead vaccine) DSP: purified by DEAE-Sephadex (live vaccine)
that vaccines with highly purified orga nisms, even though killed, were able to con fer a specific protection by intravenous im munization. Quantitative assay for protection of tra choma vaccine. In order to carry out a quan titative evaluation of the protection of trachoma vaccine, trials of mouse immuniza tion with a series of 2-fold, 4-fold, S-fold, or 10-fold dilutions of vaccine were made. After a series of preliminary experiments, we came to the conclusion that immunization with a series of 10-fold dilutions of vaccine gave the most useful pattern of protection in the MTPT. A 50% end-point of effective dose (ED 5 o) was usually obtainable within
MAY, 1967
the range of 10% to 0.001% of vaccine. A typical sample of a protection test is illus trated by the test of TW-3 TGG formalinized vaccine in Table 4. The original prep aration of this vaccine was in 2000% concentration with a particle count of 1.3 X 10 9 /ml. Five serial 10-fold dilutions ranging from 10% through 0.001% were tested for intravenous immunization. A graded pattern of protection according to dilution was ob tained with a challenge dose of 1.35 LD 50 of TW-3 crude yolk-sac suspension. The ED 5 0 was calculated as 0.246% of the vaccine, or 1.6 X 105 in terms of particle count. If a protection unit is defined for one ED 50 dose of vaccine, the original vaccine (2000%, 1.3 X 10 9 /ml) is estimated to have approxi mately 8,000 mouse protection units by this test. Two preparations of 100% TW-1 vaccine were made from the same partially purified material of TW-1 infected yolk sac; one was further purified with genetron and the other by sucrose-gradient. The genetron vaccine was inactivated with a particle count of 1.5 X 1010/ml while the gradient vaccine was live with an elementary body particle count of 8.5 X 10 9 /ml. Both vaccines were potency tested against 1.5 LD 5 0 challenge dose of TW-5. The results are shown in Table 5. A graded pattern of protection was
TABLE 4 POTENCY TEST OF PURIFIED TW-3 TRACHOMA VACCINE IN THE MOUSE TOXICITY PREVENTION TEST
Dilution of Vaccine
No. of Mice
10% 1% 0.1% 0.01% 0.001%
10 10 10 10 10
Death (hr) after Challenge ON ON 3 3 3
ON 3 4 4 4 4 5 3 4 4
5i ON 5 5 5 ON ON ON 4§ 5 5 7 7 ON
Toxicity titration of challenge dose used, TW-3:20% y.s. 3 5 ON ON ON 6 TW-3 y.s. 20% ON ON TW-3 13.3% 6 ON TW-3 8.9% 6
D/T
ED 60
1/10 2/10 6/10 10/10 10/10
0.246% (1.6X105)
5/6 2/6 1/6
(LD 50 : 14.8%)
Challenge dose = 20%/14.8% = 1.35 LD50 Note: Vaccine tested: TW-3, TGG-formalinized, 2000% (1.3X10 9 particles/ml) Immunization is given intravenously two 0.5 ml doses of vaccine dilution one week apart, and challenge made 0.5 ml intravenously one week after the last dose of vaccine ON: Mouse died overnight
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TRACHOMA AND ALLIED DISEASES TABLE 5 POTENCY TEST FOR TWO TW-1 TRACHOMA VACCINES PURIFIED BY DIFFERENT METHODS
Dilution of Vaccine 10% 1% 0.1% 0.01% 0.001%
Genetron-Purified D/T
ED6,
Gradient-Purified D/T
ED 50
0/10 0/10 1/10 3/9 8/10
0.0046%6 (6.9X10 )
0/10 0/10 1/10 2/10 8/10
0.0042%5 (3.6X10 )
Toxicity titration for challenge dose used, TW-5:23% y.s. 23% 8/10 15.3% 6/10 LD 60 : 15.3% 10.2% 0/9 Challenge dose = 23%/15.3% = 1.50 LD50 Genetron-purified—100% (1.5X1010 particles/ml) Gradient-purified—100% (8.5X10» particles/ml) D/T: No. of toxic death / No. of mice tested
seen in both vaccines with an ED 5 0 of 0.0046% or 6.9 X 105 particles for the gene tron vaccine and 0.0042% or 3.6 X 105 particles for the gradient vaccine. It is esti mated that the original TW-1 genetron vac cine has 22,000 mouse protection units/ml and the TW-1 gradient vaccine 24,000 units/ml. The genetron vaccine (killed prep aration) was nearly as potent as the gra dient vaccine (live preparation). Route of immunization in the mouse tox icity prevention test. The quantitative dif ference of immunizing efficiency between two injection routes is presented in the fol lowing experiment. An ND-3 gradient-puri fied vaccine (100%, 1.5 X 1010 particles/ml) was tested in the range of 10% through 0.001%. One group of mice received intra
venous and the other intraperitoneal immun ization with the same schedule and the same dose of vaccine. Both groups were challenged simultaneously by intravenous route with a toxic dose of ND-3. The results are shown in Table 6. An ED5„ of 0.021% or 3.2 X 10e particles was obtained with intravenous im munization, whereas 1.76% or 2.6 X 108 par ticles was the ED 5 0 with intraperitoneal im munization. There was nearly a two-log dif ference of immunizing efficiency between the two routes. A more irregular pattern of pro tection was apt to follow intraperitoneal than intravenous immunization. Time of challenge after immunization in the mouse toxicity prevention test. The best time for challenge after immunization and the duration of immunity were evaluated in
TABLE 6 COMPARISON OF IMMUNIZATION ROUTES IN THE MOUSE TOXICITY PREVENTION TEST
Dilution of Vaccine* 10% 1% 0.1% 0.01% 0.001%
Immunized Intraperitoneally 2/llt 8/9 6/7 5/8 9/11
ED 60 : 1.76% 8 (2.6X10 )
Immunized Intravenously 2/11 1/9 1/8 4/7 7/9
Toxicity t itration of challenge dose used, ND-3:2 2.5% 22.5% 7/10 15.0% 0/9 10.0% 0/9 Challenge dose = 22.5%/22.0% = 1.01 LD 50 * ND-3 sucrose-gradient purified vaccine: 100%—1.5X1010 particles/ml f No. of toxic death / No. of mice tested
ED5„: 0.021% (3.2X10·)
LD60: 22.0%
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TABLE 7 COMPARISON OF TIME INTERVALS BETWEEN IMMUNIZATION AND CHALLENGE IN THE MOUSE TOXICITY PREVENTION TEST
Intervals Immunization*— Challenge 1 weekf 2 weeks 3 weeks
Retitration of Challenge Doset
ED 60 0.025% (7.0X10«) 0.059% (1.7X10») 0.054% (1.5X10«)
Age of Mice
LD60
5 weeks 6 weeks 7 weeks
14.9% 16.8% 16.3%
Test vaccine: TW-5 genetron-purified 100%—2.8X10»/ml * Two doses of each of the 10-fold dilutions (10%—0.001%) of vaccine given intravenously one week apart t Intravenous challenge made after the last dose of immunization t Challenge dose: TW-5 yolk sac suspension—18% the following experiments. Three groups of mice each received a similar schedule of intravenous immunization (two 0.5 ml doses at weekly intervals) with serial 10-fold dilutions of TW-5 genetron-purified vaccine (100%, 2.8 X 109 particles/ml). The time of immunization was adjusted for each of the three groups so that the immunization was initiated with three-week-old mice and com-
pleted one, two and three weeks respectively prior to the simultaneous challenge. Control mice were set aside for each immunized group and they were used for titrations of the challenge dose. The results are shown in Table 7. There was no significant difference in protection among the three groups although the group challenged one week after immunization appeared to be better protect-
TABLE 8 REPEAT OF POTENCY TEST FOR TRACHOMA VACCINES
%
ED 60 (Particles
Challenge Dose: LDJO (Date)
0.006%(1.4X10«] 0.147% (3.2X10« 0.033% (7.2X10* 0.013% (2.9X10«:
1.2(1-29-64) 1.3(3-31-64)
Bour, TGG, 2000% (8.0X10 9 /ml)
0.087%(3.5X10«] 0.055%(2.2X10 6
1.2(1-29-64) 1.3(3-31-64)
TW-5, genetron, 2000%, formalinized (5.0X10 10 /ml)
0.008% (4.2X10^ 0.007%(7.2X10« 0.048% (2.4X10«)
1.3(6-22-65) 1.3(9-8-65) 1.3(2-24-66)
TW-5, gradient, 100% (l.lXlO^/ml)*
0.017%(1.9X10« 0.010%(1.1X10» 0.023% (2.5X10« 0.060% (6.6X10«) 0.010%(1.1X10« 0.045% (5.OX 10« 0.025% (2.8X10« 0.032% (3.5X10« 0.012%(1.3X10« 0.047%(5.2X10« 0.047%(5.2X10« 0.050%(5.5X10« 0.032%(3.5X10» 0.017%(1·9Χ10«
1.1(9-2-65) 1.2(2-16-66) 1.6(3-2-66) 1.3(3-23-66) 1.2(3-30-66) 1.4(4-28-66) 1.1(5-5-66) 1.3(5-25-66) 1.2(6-15-66) 1.4(6-22-66) 1.6(6-29-66) 1.3(7-6-66) 1.2(7-13-66) 1.2(7-13-66)
Bour, Sephadex, 2000% (4.3X10»/ml) Bour, Sephadex, 2000%, formalinized (4.3X10»/ml)
* Vaccine kept at -20°C The rest vaccines were kept at 2°C-5°C in the ordinary refrigerator temperature
1.2(1-29-64) 1.3(3-31-64)
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TRACHOMA AND ALLIED DISEASES TABLE 9 EFFECT OF PARTIAL PURIFICATION ON THE MOUSE TOXICITY PROTECTING ANTIGEN
Material After Process
Egg Infectivity Log EID 50 /ELD 60
Mouse Potency Test* ED5o
7.0/6.8 6.7/6.0 6.8/6.5 7.0/6.3
0.0046% >1% 0.0008% 0.0225% 0.0153% 0.0075% 0.0228%
Original ( 1 % Bour y.s.) Supernate after 20,000XG, 1 hr Pellet after 20,000XG, 1 hr Original (10% Bour y.s.) Pellet after: Washing (4X, high spin) : Trypsin & washing : Sonication, trypsin & wash * Tested against 1.8 LD50s of Bour toxin
ed than the others. The six- and sevenweek-old mice challenged two and three weeks after the last immunization were slightly more resistant than the five-weekold mice challenged one week after immuni zation. Nevertheless, two intravenous im munizations with dead vaccine provided good protection against toxic challenge three weeks after the immunization. Reproducibility of potency test. The re sults of repeated potency tests with Bour and TW-S vaccines are shown in Table 8. These vaccines, except TW-5 gradient vaccine, were stored at 2°C-5°C during the period of repeat tests. The ED 50 s obtained for each sample were usually reprodicible. The last sample, TW-5 gradient vaccine, was stored at — 20°C and tested 14 times over a period of approximately 10 months. The results in dicate that this sample did not change in potency during this period. The coefficient of variation for the test results was S/χ = 54%, indicating good reproducibility. Application of MTPT in evaluation of purification of TRIC agent. Crude yolk-sac materials harvested from trachoma infected eggs contain a variety of antigenic sub stances besides whole TRIC organisms. In order to determine whether any significant amount of soluble substances might be immunogenic in mouse toxicity prevention, 1% of crude Bour yolk sac suspension was centrifuged at 20,000 X G for one hour. The original material and the resultant su pernatant fluid and pellet (resuspended to
the original 1% by volume) were simulta neously tested in the MTPT. The results are shown in the upper part of Table 9. It is obvious that the active immunizing antigen was associated with the sedimented pellet. In another series of experiments, Bour crude yolk sac, 10% in SPG suspension, was divided in four parts. The first part served as control. The second part was washed four times with SPG and centrifuged at 20,000 X G for one hour. The third was di gested with trypsin and washed four times, and the fourth sonicated, digested with trypsin, and then washed four times. The final pellet of each was resuspended to the original 10% concentration in SPG. The results of egg infectivity titration 23 and M T P T are shown in the lower part of Table 9. In these partial purification proce dures, there was neither appreciable loss of &gg infectivity nor the active immunizing power of the Bour antigen. It is also worthy to note that the M T P T was fairly reproduc ible and that a small amount of antigen was enough to immunize mice against toxic chal lenge. DISCUSSION
In our previous report,14 immunization of mice against toxic challenge was made by intravenous injection of sublethal doses of living trachoma agents. It can be argued that the protection afforded in that case might be due to toxin tolerance rather than to immunologie response. This seems un-
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likely since the protection afforded by in travenous immunization has been highly specific, and in this report protection was obtained by immunization with nontoxic and highly diluted antigen. Protection was demonstrated without much loss three weeks after intravenous immunization with a nontoxic antigen. Unequivocal demonstration of immune mechanism by neutralizing of toxin with serum antibody has been accomplished in recent experiments (unreported). Using M T P T as a tool, we have demon strated that the potency of a vaccine may be evaluated in terms of its immunizing capacity against toxic death of mice. Whether the antitoxic immunity is an adequate measure of trachoma immunity is not yet definite. A concept that the pathologic changes of tra choma are due to the toxic effect of the agent has been presented.24 Furthermore, good correlation between the results of mouse toxicity prevention and the preven tion of monkey eye infection can be demon strated. In our successful vaccine trials in monkeys, protection was observed only with homologous vaccine but not with heterologous vaccine, the specificity of which paral leled the antigenic difference determined by the mouse toxicity prevention test.7 The po tency of vaccine assessed in the prevention of monkey eye infection has been shown to reflect the results of potency tests in the MTPT. 7 This would seem to indicate a close association between antitoxic immuni ty and protection from trachoma. In this sense, it is believed that the M T P T is use ful for potency assay of trachoma vaccine and for investigation of protective antigens. Characterization of trachoma antigens which are responsible for prevention of mouse tox icity is being studied, and the results will be reported separately.25 A minimum antigenic mass was found to be necessary for immunization of mice against toxic challenge, irrespective of prep arations of vaccine used. ED 50 was estimat ed to be about 105-106 particles of the agent.
MAY, 1967
We have also found that the critical anti genic mass is 108-109 particles for protection against experimental monkey eye infections when similar vaccines are administered with an oil adjuvant.7 This study showed that a small amount of antigen is sufficient for immunizing mice against toxic challenge. Antigenic typing of new TRIC strains can be simplified by em ploying 1% of crude yolk-sac suspension for immunization, and 1.5 LD 50 dose of crude infected yolk-sac suspension for chal lenge.15 SUMMARY
A quantitative modification of the mouse toxicity prevention test was described for potency assay of trachoma vaccine. The method of toxicity titration of toxin was re vised. Closely spaced dilutions (3:4) of in fected yolk sac suspension (ranging from 5% to 30%) were intravenously titrated in mice. The 50% end-point of lethality (LD 50 ) was obtained and the results of titrations were reproducible. The age and weight of mice were critical to reproducible titration. Using this titration method, thermostability of toxin was investigated. Toxin was rapidly inactivated within five minutes at 56°C, but was stable for days at the refrig erator temperature of 2°C-5°C, and appar ently stable for several months at — 65°C. Although toxin was found to be labile, ei ther live (DEAE-Sephadex purified) or killed (trypsin-genetron-sucrose gradient purified) organisms were effective immuniz ing antigens. A quantitative measurement of protecting antigen was devised as follows: two doses of serial 10-fold dilutions of vaccine ranging from 10% to 0.001% concentration were injected intravenously into groups of 10-15 mice at weekly intervals. They were intra venously challenged one week after the last immunization with 1.5 LD 50 of toxin. A graded pattern of protection was usually ob tained by dilution of antigen and a 50% end-
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point of effective dose (ED50) of vaccine and Alexander, E. R. : Prevention of trachoma with Arch. Environ. Hlth. 8:518, 1964. can be obtained. The results were reproduc vaccine. 7. Wang, S. P., Grayston, J. T. and Alexander, ible in repeated testings of a given vaccine. E. R. : Trachoma vaccine studies in monkeys. Much better results were obtained with Am. J. Ophth. 63:1615, 1967. 8. Dawson, C, Jawetz, E., Thygeson, P. and intravenous immunization than with intraHanna, L. : Trachoma viruses isolated in the peritoneal. Protection can be demonstrated United States. 4. Infectivity and immunogenicity without much loss three weeks after the for monkeys. Proc. Soc. Exper. Biol. & Med. intravenous immunization. In a parallel test, 106:898, 1961. 9. Collier, L. H. : Experiments with trachoma killed (genetron purified) and live (sucrose- vaccines. 1. Experimental system using inclusion gradient purified) preparations of vaccine blenorrhea virus. Lancet, 1:795, 1961. 10. Snyder, J. C, Bell, S. D., Jr., Murray, E. derived from the same infected yolk sac S., Thygeson, P. and Haddad, N. A. : Attempt to materials were shown to be equally protec immunize a volunteer (with formalin-inactivated tive in the quantitative test. virus) against experimental trachoma induced by This potency test has been used for evalu Saudi Arabian strain 2. Ann. N.Y. Acad. Sci. 98:368, 1962. ation of purification methods for vaccine. 11. Sampaio, A. A., Ayres, L., Haddad, N. A., The results indicate that the immunizing an Bell, S. D., Jr., Murray, E. S. and Snyder, J. tigen is closely associated with TRIC agent C. : Studies on trachoma. IV. Investigation in Portugal on formalin-killed trachoma vaccine elementary body particles, and that treat with special reference to serologie response. Am. ment of the infected crude yolk sacs with J. Trop. Med. & Hyg. 12:909, 1963. 12. Snyder, J. C, Nichols, R. L., Bell, S. D., 0.2% trypsin at 37°C for 15 minutes, ultra Haddad, N. O.. Murray, E. S. and McComb, D. sonic disintegration at 18,000-20,000 cycles E. : Vaccination against trachoma in Saudi Ara per second for five minutes, and repeated bia: Design of field trials and initial results. Inwashings with SPG do not remove infectivi dust & Trop. Health, 5:65, 1963. 13. Bell, S. D., Jr. and Theobald, B. : Differ ty or protective antigens. ACKNOWLEDGMENTS
We wish to acknowledge the technical assistance of Mr. Chia-ho Liu, Mr. Ju-chun Ho, Miss Jean C. A. Lin and Mrs. Judy Tesmer. REFERENCES
1. 'Collier, L. H. : The present status of tracho ma vaccination studies. Bull. World Health Org. 34:233, 1966. 2. Grayston J. T., Wang, S. P., Yang, Y. F. and Woolridge, R. L. : The effect of trachoma virus vaccine on the course of experimental infection in blind human volunteers. J. Exp. Med. 115:1009, 1962. 3. Grayston J. T., Woolridge, R. L , Wang, S. P., Yen, C. H., Cheng, K. S. and Chang, I. S.: Field studies of protection from infection by ex perimental trachoma virus vaccine in preschoolage children in Taiwan. Proc. Soc. Exper. Biol. &Med. 112:589,1963. 4. Dhir, S. P., Agarwal, L. P., Detels, R., Wang, S. P. and Grayston, J. T.: Field trial of two bi valent trachoma vaccines in children of a Punjabi Indian village. Am. J. Ophth. 63:1639, 1967. 5. Grayston, J. T., Woolridge, R. L. and Wang, S. P. : Trachoma vaccine studies on Taiwan. Ann. N.Y. Acad. Sci. 98:352, 1962. 6. Grayston, J. T., Wang, S. P., Woolridge, R. L.
entiation of trachoma strains on the basis of im munization against toxic death of mice. Ann. N.Y. Acad. Sci. 98:337, 1962. 14. Wang, S. P. and Grayston, J. T.: Clas sification of trachoma virus strains by protection of mice from toxic death. J. Immunol. 90:849, 1963. 15. Alexander, E. R., Wang, S. P. and Grayston, J. T.: Further classification of TRIC agents from ocular trachoma and other sources by the mouse toxicity prevention test. Am. J. Ophth. 63: 1469,1967. 16. Hanna, L., Thygeson, P., Jawetz, E. and Dawson, C. : Elementary body virus isolated from clinical trachoma in California. Science, 130:1339, 1959. 17. Reed, L. J. and Muench, H. : A simple method of estimating fifty per cent end point. Am. J. Hyg. 27 :493, 1938. 18. Lai, J. S. : Purification of trachoma agent. Thesis for M.S. degree, University of Washington Department of Microbiology, 1962. 19. Ribi, E. and Hoyer, B. H.: Purification of Q fever rickettsiae by density-gradient sedimen tation. J. Immunol. 85:314, 1960. 20. Sharp., D. G. : Enumeration of virus particles by electron micrography. Proc. Soc. Exper. Biol. & Med. 70 :S4, 1949. 21. Litwin, J. : The growth cycle of the psitta cosis group of micro-organisms. J. Infect. Dis. 105:129, 1959.
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22. Alexander, E. R. and Fresh, J. W. : Preven 24. Thygeson, P. and Nataf, R.: Etiologic tion of anaphylactic death in the mouse toxicity problems in trachoma. Rev. Int. Trach., 35:177, prevention test for TRIC agents. Am. J. Ophth. 63 : 1958. 1462, 1967. 25. Wang, S. P., Kenny, G. E. and Grayston, J. 23. Wang, S. P. and Grayston, J. T. : Egg infec- T. : Characterization of trachoma antigens protec tivity assay of trachoma virus. Proc. Soc. Exper. tive against mouse toxicity. Am. J. Ophth. 63 : Biol. &Med. 115:587, 1964. 1454, 1967.
CHARACTERIZATION O F TRACHOMA ANTIGENS PROTECTIVE AGAINST MOUSE TOXICITY SAN-PIN WANG, M.D.,
GEORGE E. K E N N Y ,
J. THOMAS GRAYSTON,
Although active immunization with tra choma vaccine may prevent or modify dis ease in man1-5 and primates,6"9 the nature of the protective antigens is unknown. Vaccine potency can be evaluated in monkeys9 but this procedure is difficult and expensive. A more convenient model is the mouse toxicity prevention test which not only permits differentiation of TRIC strains10"12 but also has permitted the development of a quanti tative method for assay of protective antigens.13 The results of vaccine potency assay in the mouse toxicity system correlate well with the results of prevention of mon key eye infection.0 This potency assay was employed for the evaluation of methods of vaccine preparation and also to characterize the protective antigens. This report presents preliminary findings concerning the physicochemical stability of the protective antigens of trachoma. MATERIAL AND METHOD
TRIC strains. Strains of TRIC agents used in this study were three ocular trachoFrom the Department of Preventive Medicine, University of Washington, School of Medicine, Seattle. This work was supported by a United States Public Health Service research grant NB03144 from the National Institute of Neurologi cal Diseases and Blindness, National Institutes of Health. Reprint requests to : Dr. San-pin Wang, Depart ment of Preventive Medicine, University of Wash ington School of Medicine, Seattle, Washington 98105.
P H . D . AND
M.D
ma strains isolated by us in Taiwan (TW-3, TW-5 and ND-3). 1 1 The 10th to 38th yolk sac passages of these organisms were em ployed. Purification of TRIC agent. Infected yolk sacs were disrupted by one of the following techniques: (1) shaking with glass beads for 10 minutes, (2) homogenization with an electric blender (Servali "Omnimix") and (3) sonic disruption with an ultrasonic de vice (Branson) at 18,000-20,000 cycles/ second for one hour in an icebath. The organisms were partially purified from the yolk sac suspension by cycles of washing at low (500 X G) and high (30,000 X G) speed centrifugation with phosphate buf fered saline ( P B S ) . The pellets from lowspeed sedimentations were discarded, the supernatant was centrifuged at high speed and this pellet saved and the process repeated. These partially purified organisms were then purified by the following methods: Sucrose-KCl gradient sedimentation. De tails are described separately.13 Briefly, a 100% concentration* of partially purified organisms was layered on the top of the centrifuge bottles containing 250 ml of a continuous 0%-40% sucrose 1.0 M KC1 grad ient, centrifuged at 1500 X G for two hours, and the dense zones in the gradient were collected. After cycles of washing with PBS, the final pellets were suspended at * Concentration values are in %. The original yolk sac is considered 100%.