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The Value of Tissue Culture Vaccine*
CHALAZIONLIKE NEOPLASMS
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7. Spaeth, Ε. B . : Ocular tumors. Α Μ Α A r c h . O p h t h , 4 6 : 4 2 1 - 4 2 3 , 1951. 8. H a g e d o o r n . Α . : Adenocarcinoma o f a meibomian gland. A r c h . O p h t h , 12:850-866, 1934. 9. O'Brien, C. S , and Braley, A . E . : C o m m o n tumors of the eyelids. J . A . M . A , 107:933-938, 1936. 10. Magnus, J. Α . : Adenocarcinoma o f meibomian gland with secondaries in liver. T r . Ophth. S o c . U . Kingdom, 6 7 : 4 3 2 - 4 3 5 , 1947. 11. Rice, M. L , and Lundeke, H . J.: Adenocarcinoma o f the meibomian gland. A m . J. O p h t h , 3 3 : 14-34, 1950. 12. Straatsma, B. R.: Meibomian gland tumors. Α Μ Α A r c h . O p h t h , 5 6 : 7 1 - 9 3 ( J u l y ) 1956.
THE IN H.
THE
BEECHER
VALUE
PROPHYLAXIS CHAPIN,
M.D.,
OF OF
TISSUE RECURRENT
SAM
C.
The history of vaccine therapy in herpes simplex infection in man, employing both dead and live virus, has been disappointing. Even in the presence of circulating antibody attacks occur. Thus any new immunizing procedure admittedly must be viewed with considerable skepticism. Yet in the case of herpetic keratitis in which repeated attacks may lead to blindness, new ideas should be explored. Recent studies have shown that potent tissue culture vaccines, with excellent immunizing properties in experimental animals, deserve more than passing interest. Apart from this, past failures in attempted prophylaxis of the infection in man may be attributed to the failure to recognize the allergic phase of the disease. Thus it is not surprising that routine immunizing procedures consisting of three or four injections of vaccine material are ineffective. 1
It is the object of this paper to explore the usefulness of tissue culture vaccine in preventing recurrent attacks of herpetic keratitis. Concomitantly the use of the antigen in the diagnosis of herpetic infections will be presented. AND
ATTACKS
WONG,
INTRODUCTION
MATERIALS
CULTURE
METHODS
VIRUSES
Three strains of herpes simplex virus were used. Strain " H F " was obtained from * F r o m the Department of A l l e r g y , T h e M a n h a t tan Eye, E a r and T h r o a t Hospital.
OF
PH.D.,
VACCINE* HERPETIC
AND
JANE
KERATITIS REAPSOME,
B.S.
Dr. L. W. Chu of the Wyeth Institute. It was passed four times in the chorioallantoic membrane of 10- to 11-day-old embryonated hens' eggs before the virus was stored as 10-percent chorioallantoic membrane suspension at — 65°C. This virus was used for the production of the egg antigen. The "A" strain was originally isolated by Dr. W. Anderson of the New York Hospital from a vesicle of a case of herpes labialis. This virus was found to give the most consistent results in the serum-virus neutralization test for the detection of antibody. The 1266 strain was originally isolated by Dr. Kilbourne in suckling mice from a case of aphthous stomatitis. It was obtained as a frozen 10-percent mouse brain suspension. There was no history of cell line passage of this virus. It was passed five times in chick fibroblast and twice in rabbit kidney cultures before it was stored at — 65°C. The seed virus has a TCID50 of 10 per ml. 6
s 5
PREPARATION
OF
ANTIGEN*
The term antigen or vaccine will be used interchangeably in this paper. The egg antigen was prepared from the infected allantoic fluid of embryonated eggs employing the method of Jawetz, et al. T w o changes, however, were introduced: ( a ) the virus titer of the infected allantoic fluid was determined by serial 10-fold dilutions in rabbit kidney 5
* W e are indebted to Dr. George H . W a r r e n of the W y e t h Institute for supplying part of the antig e n s used in the investigation.
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REAPSOME
tissue cultures and had a TCID50 of 10 per ml. prior to ultracentrifugation at 15,000 rpm for 60 minutes ; (b) formalin in the final concentration of 1:4,000 was used to inactivate residual virus. Control uninfected allantoic fluid was prepared from embryos of the same age and treated in the same manner as the infected material.
tests were carried out. A total of 100 ml. of the vaccine was obtained. The control TC antigen was prepared in identical manner except no virus was added. All antigens were stored at 4°C. It is pertinent to point out that the T C antigen contained no penicillin or animal serum.
The tissue culture antigen was prepared according to the method of Chu and Warren employing rabbit kidney cultures. Essentially the method consisted of the following: Trypsinized rabbit kidney cells in concentration of 10 X 10 cells contained in 10 ml. of growth medium were grown in 200 ml. serum dilution bottles. The growth medium, however, was modified by adding an equivalent volume of medium 199 to the lactalbumin hydrolyzate medium.
SAFETY
7
1
e
When a complete sheet of cells was formed (usually in five to seven days), the tissues were washed with three changes of 30 ml. each of warm medium 199. Phosphate buffered saline, 0.01 molar, pH 7.3 could not be used for the same purpose for it produced extensive sloughing of the cell sheet; 10 · TCID50 of herpes simplex virus, strain 1266, contained in 4.0 ml. of medium 199 was inoculated per bottle. After three hours of adsorption, the old medium was discarded and 10 ml. of fresh medium 199 containing 100 μg. of streptomycin and 50 units of polymyxin per ml. were added. s
6
5
At the end of 40 hours, tissue destruction was usually complete. Aliquots were taken from each bottle and tested in blood agar plates and thioglycolate medium to rule out bacterial contamination. The bottles were stored at — 20°C. overnight. The following day the bottles were thawed in running tap water, the contents pooled, and centrifuged at 2,000 rpm for 20 minutes. The clear pink supernatant fluid was removed and a portion stored in a C 0 chest in sealed glass ampoules for later virus titration. Formalin was added to the antigen to effect a final concentration of 1:4,000. The mixture was incubated at 37°C. for 24 hours and then stored at 4°C. for two weeks before safety 2
TESTS
Preliminary tests had indicated that rabbit kidney cultures were equal to infant mice in sensitivity in the detection of herpes simplex virus. In addition, the former possessed the advantage of permitting the testing of larger volumes of vaccine with concomitant increase in sensitivity. In view of this finding it was decided to place greater reliance on rabbit kidney cultures in determining residual infective virus. Twenty-five ml. each of egg and TC vaccines were dialyzed in cellophane bags in 0.01 molar phosphate buffered saline, p H 7.0, at 4°C. for 72 hours to remove formalin. Five ml. of each dialyzate was inoculated into each of four bottles of rabbit kidney cells, incubated at 37°C. and observed daily for 10 days for evidence of cytopathic effect. The pH of the medium was adjusted with N a H C 0 solution when necessary and complete change of medium was made every four days. 3
Six mice per group were inoculated with 0.03 and 1.0 ml. of the undialyzed vaccines via the intracerebral and intraperitoneal routes respectively. The animals were observed for three weeks. In addition blood agar plates and thioglycolate medium were inoculated with the antigen and observed for one week. There was no evidence of viable virus in all the tests. The frozen virus representing the antigens prior to addition of formalin was titrated in rabbit kidney cultures employing four tubes per serial 10fold dilutions. The titers were calculated by the method of Reed and Muench. 7
SERUM-VTRUS
NEUTRALIZATION
The neutralization test for the detection of serum antibody was carried out in tissue
PROPHYLAXIS OF HERPETIC
culture employing Chang's conjunctival cells. The method of propagation and maintenance is that described by W o n g and Kilbourne. Human sera which were not used within three days were stored at — 20°C. Before use, the serum was inactivated at 56°C. for 30 minutes. Serial two-fold dilutions of sera in phosphate buffered saline pH 7.3, beginning at 1:2.5 were mixed with equal volumes of herpes simplex virus. The "A" strain was diluted in 0.1-percent casein in 0.01 M phosphate buffer, pH 7.0, to contain 2,000 TDIC50 per ml. The control virus consisted of phosphate buffered saline instead of the serum. For each run a positive and a negative serum were included. 1
8
After thorough mixing, the serum-virus mixtures and the virus controls remained at 22°C. for 60 minutes. Then 0.1 ml of the mixture was added to each of two to four tubes of conjunctival cells. Tubes were read daily, the p H was adjusted with one drop of 0.5 percent N a H C 0 solution when needed and the medium was changed every three or four days. The cytopathic effect was graded from 0 to 4 according to the degree of destruction of tissues. The test was terminated when the control tubes showed 3 to 4-plus cytopathic effect. The serum neutralization titer was the highest dilution of serum showing 2-plus or less cytopathic effect than the control tubes. 3
COMPLEMENT
FIXATION
TESTS
The technique used was the California State Department Public Health procedure. The most satisfactory antigen was prepared from human conjunctival-D cells infected with the "A" strain of herpes simplex virus. Briefly the method consisted of propagating the virus in the same manner as that used for the rabbit kidney antigen employing two-percent horse serum in medium 199 instead of 199 alone. When about 75 percent of the tissues were destroyed (usually in about 72 hours), the remaining cells were scraped off with a rubber policeman. Each batch, consisting of about 50 ml. of tissue fragments and fluid, 9
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KERATITIS
was treated in a Raytheon sonic oscillator, Model 10KC, for 60 seconds. Formalin was added to the homogenate to effect a final concentration of 1:2,000. The antigen was stored at 4°C. Such preparations have yielded consistently complement fixation titers of 1:32 or greater. T w o units were used in the test. The control antigen was prepared from uninfected conjunctival-D cells of similar age in an identical manner. Positive and negative control sera were selected from those giving similar results in the serumvirus neutralization test. Human serum specimens to be tested were frozen immediately after separation of the serum until used. Anticomplementary sera were spun at 10,000 rpm for 10 minutes in a Lourdes centrifuge, Model A T , prior to a treatment described by Lennette and Schmidt for such sera. About 80 to 90 percent of such sera were successfully treated by this technique. The complement and hemolysin were purchased from Cappel Laboratories and the sheep cells from Carworth Farms. 10
INTRACUTANEOUS
TEST
The cutaneous tests were done on the volar surface of the forearm in the following manner: After cleansing the skin with 70 percent ethanol, 0.1 ml. of a 1:10 dilution of the herpes simplex virus antigen and also a comparable volume and dilution of the control material were given intradermally so that the injected bleb of fluid was visibly raised above the normal surface of the skin. The sites were observed at the end of 24 hours for erythema, edema and induration. PATIENTS
The subjects upon whom the cutaneous tests were done were drawn from the staff and patients of the Corneal Clinic of the Ophthalmology Department, the Manhattan Eye, Ear and Throat Hospital. The tests were done upon male and female subjects immediately following the taking of blood serum specimens for the determination of
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antibody titer for herpes simplex virus. Diagnosis in those infected cases included herpetic keratitis either in the chronic quiescent phase or with ulcer formation in the cornea and herpes simplex lesions of the lips. Patients chosen for vaccine therapy. Twenty-three females and 27 males ranging in age from four to 81 years clinically diagnosed as having chronic recurring herpetic keratitis were selected for vaccine therapy. A physical examination, blood count and urinalysis were done routinely on all patients. Five patients with recurrent herpes simplex virus lesions at sites other than the conjunctiva received the control antigen. The recurring lesions of the cornea in all the patients had been present for periods varying from one week to 41 years. In some of the patients the cornea of one eye was involved and in others, both eyes. The interval of recurrences varied widely from patient to patient. In some the attacks occurred yearly, whereas others recurred from two to five years. The youngest patient treated had a bilateral infection which recurred about every three months. One patient was treated who did not have the corneal involvement, the lesions occurring in the labial and genital areas and on the buttocks. Method of administration of vaccine. Vaccine was given to patients in a series of weekly injections starting with 0.1 ml. of 1:100 dilution and increasing the dose until the concentrate was reached. Thereupon the concentrate was given in a fixed dose at varying intervals of from two weeks to three months. A second method consisted of intradermal injections of the concentrate in 0.1 to 0.3 ml. doses. These intradermal injections were given either by themselves or in conjunction with the subcutaneous injections. The material used for control consisted of tissue culture fluid which had not been exposed to the herpes simplex virus. A. Delayed type allergy cutaneous tests to tissue culture herpes simplex virus antigen. Delayed type allergy tests have been done
REAPSOME
upon human subjects for the purpose of comparing the results of using a new herpes simplex virus antigen material prepared in rabbit kidney cell tissue culture with the usual type of herpes simplex virus antigen prepared from egg embryo herpes inoculated material. Nagler was the first to describe a specific cutaneous reaction using an 11-day-old chorioallantoic membrane material inoculated with herpes virus. Later using a virus skin test reagent from amniotic fluid, Nagler stated that the amniotic fluid material was more useful for the test than that prepared from chorioallantoic membrane, as the latter gave too many nonspecific reactions probably because of a higher protein content. 2
3
The following year Rose and Molloy described a cutaneous reaction using antigenic material prepared from herpes-infected chorioallantoic fluid, concluding that the test could be used to advantage as an aid in diagnosing primary herpetic infection. Jawetz used a soluble antigen of herpes simplex virus prepared from infected allantoic fluid to obtain the cutaneous reactions. H e noted a maximum reaction in the skin between 18 and 24 hours. The soluble skin reacting material was heat stable, contained one to five percent of the total active virus and did not deteriorate on storage at 4°C. Its skin reactivity was abolished by 10-fold dilution. 4
5
With the development of tissue culture techniques which can produce potent herpes simplex virus antigens in a relatively pure form, it was decided that the specificity of cutaneous reaction to herpetic infection merited re-examination. The cutaneous tests were performed upon human subjects believed to have been infected with herpes simplex virus. A few tests were done upon individuals believed not to have been so infected. Neutralization and complement fixation tests for antibody to the herpes simplex virus were done upon the sera of these subjects. The same herpes virus antigen material used for the cutaneous tests was given
PROPHYLAXIS OF HERPETIC
as a vaccine to certain of the subjects in an attempt to alter the course of their chronic recurring attacks of acute herpetic infection. The result of this study is summarized in Table 1. A n examination of the results of the measurements in cm. of the herpes sim-
259
KERATITIS
plex cutaneous tests in 50 humans reveals that the average measurements of the egg antigen was 0.113 cm., the average of the egg control material being 0.042 cm. The average of herpes simplex virus T C antigen was 0.187 cm., whereas that of the control
TABLE 1 H U M A N SKIN REACTIONS OF THE D E L A Y E D ALLERGY TYPE TO H E R P E S SIMPLEX A N T I G E N P R E P A R E D I N EGG A N D T I S S U E C U L T U R E
Case N o .
Sex
Age
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
M F M F M M M M F F F F F F M F F F M F M M F F M F M M M F M F F F M M M M F M M M M F M F M M M F
31 70 30 37 70 18 66 40 50 38 40 38 60 30 60 44 61 45 56 53 51 36 37 40 64 47 52 55 51 71 62 31 48 44 51 52 71 54 50 81 67 63 51 50 70 45 35 51 45 58
Egg Antigen Herpes Simplex Control Virus (cm.) (cm.) 0 0.3 0 0 1.5 0 0.5 0 0.8 0.7 1.0 0 0 0 2.2 0.7 0 0.2 0.7 0.6 0.5 0 0.8 0.6 0.5 0 0.8 0.6 0 0.8 0 0 1.1 0 1.8 0.8 0.9 0.6 0 0.5 0 1.5 0 0 0 0 0 0.6 0 0
1.9 1.5 0.8 0 1.5 1.1 1.1 1.7 2.0 1.6 1.4 1.6 1.8 0 2.3 1.3 1.7 0 1.2 2.3 1.5 0 1.4 1.1 1.0 1.7 1.0 2.3 1.9 1.2 0.6 0.6 1.2 1.4 1.0 1.1 1.8 1.1 0 1.1 0.7 2.1 0.7 0 0.7 0.1 0.6 1.0 0 0.8
T i s s u e Culture A n t i g e n Herpes Simplex Control Virus (cm.) (cm.) 0 1.5 0 0 1.5 0 0 0 0.8 1.3 0 2.3 2.7 0 2.2 0 0 0 0.9 0 0.8 0 0.5 0 0 0 0.8 0 0.7 0 0 0 1.2 0 0 0.8 0.8 0.6 0.9 0.8 0 1.6 0 0.5 0 0.1 0 0 0 0
2.3 2.0 1.5 1.0 1.1 1.8 1.7 1.9 2.9 2.2 4.3 2.4 2.9 0.5 2.4 2.2 1.7 2.0 2.9 2.8 2.8 2.0 2.1 2.2 2.9 3.4 1.5 2.6 1.4 1.4 0.9 2.2 1.2 1.5 1.6 1.2 1.3 1.7 2.2 0.8 1.3 3.0 1.2 1.8 0.7 0.1 1.8 1.0 1.0 1.4
Neutralizing Antibody
1:20 1:80 0 2.0 1:80 1:160 1:320 1:80 1:80 0 1:640 1:80 1:80 1:40 1:80 1:40 1:80 1:80 1:160 1:110 1:160 1:160 1:160
1:160
1:80 0 1:20 1:80 1:50 1:5 1:20
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TC fluid was 0.044 cm. It would appear from this study that the herpes simplex virus antigen prepared from rabbit kidney tissue was more active than that of chick embryo allantoic fluid material. It is believed that this is the first time that there has been such a study of the cutaneous delayed allergy tests in humans with tissue cell material. The findings of this study do not warrant conclusions as to the specificity of cutaneous reactions to herpetic infection in humans. Further investigation must be made to determine the veracity of the widespread opinion that a cutaneous reaction is specific for herpes infection. Perhaps a method of performing the cutaneous reaction can be devised that will prove specific but it does not appear that the usefulness of such a test for herpes-infected humans would parallel that of the well-known delayed type allergy test to tuberculin in tuberculosis-infected humans. Only further studies will clarify the presumption. In the present study it is of interest to demonstrate that in six cases (Cases 12, 13, 15, 33, 40 and 4 6 ) , the delayed cutaneous reaction to herpes simplex virus vaccine was equal to or scarcely greater than the control reaction. In three cases (Cases 4, 14 and 4 4 ) , there was no or scarcely measurable neutralizing antibody present in the sera of the patients tested. B. Antigenic activity of herpes simplex virus vaccine fractions. An attempt was made to determine the nature of the skin reacting antigen present in the tissue culture vaccine. Jawetz, et al. showed that the skin reacting principal in herpes simplex virusinfected allantoic fluid is a soluble antigen rather than the intact virus particles. Whether a similar substance is present in the T C antigen remains to be determined. The following experiment was performed with this object in view. Tissue culture material which had a TCID50 of 1 0 herpes simplex virus per ml. was centrifuged at 35,000 rpm for 90 5
8,3
minutes in the Spinco, Model L. The supernatant fluid was carefully removed and subjected to two more such ultracentrifugations. On the last run, only 70 percent of the fluid was removed from the stainless steel tubes. The sediment was resuspended to the original volume in medium 199 and washed three more times at the same speed and time as given. Under these conditions it was found, in the average of two experiments employing frozen tissue culture fluids, that the supernate had a TCID50 of 1 0 per ml., the precipitate 10 · per ml. The ratio of viable virus was 1:60, respectively. Using the formalintreated supernate and sediment materials separately, intradermal tests were done as previously described in herpes simplex keratitis cases, with results shown in Table 2. It is clear from the results that the original antigen contains the most active material. However, it is equally clear that the supernate which contained 60 times less viable virus than the sediment fraction elicited 5 7
7
5
TABLE 2 COMPARATIVE SKIN REACTIONS OF VARIOUS FRACTIONS OF TISSUE CULTURE ANTIGEN IN HUMANS*
Case N o .
Tissue Culture Fluid Control (cm.)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Mean
1 .0 0.4 0 0.8 0.3 0.4 0.3 0.2 0 0.4 0.3 0.2 0.5 0.2 0.6 0 0.35
Original Herpes Herpes Simplex Simplex Virus Virus Sediment Vaccine (cm.) (cm.) 3.7 1.9 1.5 1.8 2.2 3.1 2.2 1.0 0.4 2.0 2.6 1.6 2.4 1.6 5.5 2.0 2.2
1.7 0.9 0.7 0.7 0 2.0 1.2 0.8 0.4 0 1.1 0.5 1.7 0.9 1.0 0.6 0.89
Herpes Simplex Virus Supernate (cm.) 2.0 1.3 1.7 0.8 2.9 2.5 2.6 0.3 0 1.3 2.0 0.5 2.0 2.0 0.7 0.5 1.4
* O n e - t e n t h ml. of each c o n c e n t r a t e material inj e c t e d intradermally o n t h e volar surface of t h e forearm. T e s t s read a t 24 hours.
PROPHYLAXIS OF HERPETIC
a more pronounced skin reaction than did the sediment. Thus the nature of the dermal reacting principal in T C antigen remains to be elucidated. It would appear from these results that a soluble antigen of unequh'ocal validity cannot be demonstrated. Further studies are warranted. C. Neutralising and complement fixation antibody. Neutralizing antibody measured in the sera specimens before treatment in 15 patients varied from 1:20 to more than 1:160. One patient's neutralizing antibody titer, reported elsewhere, was persistently negative at 1:5 both before and after vaccine treatment. Complement fixation tests in 25 patients before treatment varied from zero in one patient to 1:5 to 1:80 in the remaining patients. 11
After vaccine treatment was started, neutralizing antibody in the group of 15 patients rose in a range from 1:160 to 1:1280. Higher titers tended to fall as treatment was continued. In the group of 25 patients, after treatment was started, complement fixation tests rose in a range from 1:80 to 1:160 but tended to vary irregularly as treatment was continued. In the patient previously mentioned whose serum contained no neutralizing antibody, complement fixation antibody determination remained at 1:5 in tests performed at monthly intervals for six months. D. Clinical use of herpes simplex virus antigens. The herpes simplex virus infection is one of the commonest afflictions of m a n . - Recurrent attacks in general are mild and self-limiting. However, when the infection occurs in the cornea, it commonly leads to diminished vision of the infected eye, especially if the infection recurs at periodic intervals. Because of the frequency with which patients suffering from herpetic keratitis present themselves at the Manhattan Eye, Ear and Throat Hospital, it was felt worth while to give a clinical trial of vaccine therapy with the herpes simplex virus antigen. 12
13
There have been reports in the literature
KERATITIS
261
of the use of herpes simplex virus vaccine in treatment of herpes simplex virus infections in humans. That such a vaccine has not been in widespread use suggests the difficulties inherent in its practicability as a therapeutic or prophylactic agent. These difficulties may be divided into two main categories: 14
1. The first involves the problem in the successful manufacturing of a potent high titer herpes simplex virus vaccine with concomitant reduction in extraneous proteins. 2. The second is related to the pathogenesis of the herpes simplex virus infection. The virus in most instances in human infection invades the outer layer of the skin, the conjunctival or corneal tissue or mucous membranes. After the acute inflammatory process has subsided and the involved tissues healed, the virus particles are said to be latent. An unknown mechanism may reactivate the virus periodically throughout life with attendant inflammatory reaction. Unlike most of the commonly used vaccine therapies in which vaccination is done to prevent the primary attack of the disease, therapy with herpes simplex virus vaccine is used to prevent recurrences of the disease. Because of the nature of herpes simplex virus infection in humans, it has been suspected that the standard method of vaccination consisting of two or three injections separated by a suitable interval to allow antibody production, followed by booster doses at yearly or longer intervals, would prove of little therapeutic benefit. The few clinical trials of vaccine using these methods have not proved encouraging. The fact that it is believed there is an allergic reaction to the virus in infected corneal tissue at certain phases of the infection, coupled with the well-known herpes simplex virus allergic cutaneous reaction in humans, suggested a new therapeutic approach based upon standard desensitizing-immunizing procedures used against allergy diseases. Because of the greater activity of the herpes simplex virus antigen prepared from rabbit kidney tissue
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REAPSOME
TABLE 3 I N T E R V A L S B E T W E E N R E C U R R E N T ATTACKS O F A C U T E H E R P E T I C K E R A T I T I S
N o . of Cases
Less t h a n One Y e a r
One Year
One t o F i v e Years
Over Five Years
Irregular Intervals
Primary Attack*
12
6
14
3
S
10
* T h e s e patients h a v e had n o recurrence following the subsidence of o n e initial a c u t e a t t a c k .
culture, it was decided to use this antigen material for the clinical trials. The whole vaccine material which contains both the sediment and supernate materials was used in these patients. Because of the varying intervals of recurrence of herpetic lesions in the eye, the value of any treatment can be assessed only on a long-term basis. The present study was initiated January, 1959, and the observations are continuing. In order to assess the results of vaccine therapy, it is necessary to examine in detail the conditions of the course of infection in individual patients. Although no correlation has been observed as yet between therapeutic result and duration of infection, this factor may have a bearing on the end-result of therapy. In this series of cases, five patients have had the infection for less than one year, 32 patients from one year to nine years, and 13 for 10 or more years. More important, seemingly, than the duration of infection, is the interval between the acute attacks in each patient. For purposes of analysis, the cases have been divided into groups relating to these intervals, as shown in Table 3.
TABLE 4 R E S U L T S OF V A C C I N E T H E R A P Y
Number of Cases
Percent
G o o d , no recurrence Fair, mild recurrence Nonconclusive Poor
23 13 13 1
46.0 26.0 26.0 2.0
TOTAL
50
100.0
Category a. b. c. d.
The therapeutic effectiveness of the vaccine in these patients is summarized in Table 4. Of the 50 cases in Table 4, 23 may be classified as ( a ) , 13 as ( b ) , 13 as ( c ) , and one as ( d ) . In contrast the five control patients, receiving the placebo or nonherpetic tissue culture fluid, had recurrent attacks at the usual intervals with undiminished severity. In summary then it may be stated that herpes simplex virus T C vaccine, given by the method described, exerts a protective effect against recrudescence of herpetic lesions in the cornea. A more detailed demonstration of the value of vaccine treatment is shown in Table 5. In this series of 50 cases treated with tissue culture vaccine, it is seen that 36 showed either nonrecurrence of herpetic keratitis or a lessening of the severity of the lesion. Of those 10 cases in which only the primary attack was observed, there is, of course, no information of the expected interval before recurrence. Either a therapeutic effect was achieved by the use of herpes simplex virus vaccine, or the interval before recurrence exceeded the two years covered by this report on these patients. Of the group of 10 cases shown in Table 5, eight had no recurrences, one in the ( b ) category had labial lesions of diminished severity twice during the period of observation and the one case in the (c) category had no recurrence but, as therapy had been given for only 10 months, the result was considered inconclusive. Of the group in which attacks occurred at irregular intervals, the one case in the ( b )
PROPHYLAXIS OF HERPETIC
KERATITIS
263
TABLE 5 I N T E R V A L S B E T W E E N RECURRENT ATTACKS OF A C U T E HERPETIC KERATITIS
Categories
Less t h a n One Year
a b c
4 6 1
A
U
Controls
1 1
3*
One Year 3 1 2
O n e to F i v e Years 7 4 3
Over F i v e Years
— 3
Irregular Intervals 1 1 3
Primary Attack 8 1 1
Total N o . Cases 23 13 13 1 1
2*
5
* In t h e control cases, the a t t a c k s occurred a t the e x p e c t e d t i m e s with no d i m i n u t i o n in t h e size or duration of t h e lesion.
category had no eye recurrence for two years. This patient, however, during an upper respiratory infection with fever, had labial lesions 50-percent smaller than those observed previously during fevers. The three patients with an interval of over five years between attacks fall into ( c ) category because of the shorter period of observation of this study. Of the patients whose attack interval fell between one to five years in the ( b ) category, two exceeded the period of this study. One of these patients had one attack of labial lesions 50 percent smaller than before therapy. Of the other two, one had a subsiding acute attack at time of onset of therapy ; the other had a mild attack after stopping treatment for four months. The two patients with an attack interval of one year in the ( c ) category have not been observed long enough to give information. One with severe bilateral corneal damage from previous attacks which caused marked thinning of the cornea discontinued treatment before dosage could be stabilized. Of those patients with attack intervals of less than one year, the one patient in the ( c ) category discontinued therapy after three months. No acute attack recurred in this patient while on vaccine therapy. Of the six patients in ( b ) category, the interval between attacks lengthened and the lesions became less severe in one patient with severe bilateral infection; one had labial attacks at the expected interval but of diminished severity. It was noted in one patient with
moderately severe allergic dermatitis involving the antecubital and popliteal spaces, the legs, trunk, arms and neck, that there was an exacerbation of the acute corneal condition concomitant with acute allergic dermatitis which promptly responded to steroid therapy. The one patient in ( d ) category with severe corneal damage from previous attacks, before vaccine dosage was stabilized had no apparent lessening of severity of the infection. Jawetz, et al. suggested that psychosomatic factors may play an important role in the benefits derived from vaccine therapy of herpetic infection in man. This opinion is shared by McNair Scott. T o this may be added personal bias. T o minimize the latter, the patients were examined independently and the clinical status evaluated periodically by an ophthalmologist. Admittedly these two factors may have contributed, at least in part, to the favorable results obtained. Nevertheless, we believe that the majority of the patients were benefited by the desensitizing treatments employing tissue culture antigen. The seriousness of herpetic keratitis in man and the possibility that blindness may result justify the trial of any rational therapy. The potent vaccine, the absence of extraneous proteins, the innocuousness of the antigen fulfill these requirements and therefore justify further studies. 5
15
Side-reactions to herpes simplex virus vaccine. There were no general or systemic reactions of any type noted in patients receiving the vaccine. Leukocyte and erythrocyte counts, blood-cell percentages, and urin-
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alysis findings remained within normal limits during vaccine therapy. One of the patients, aged 63 years, with advanced chronic rheumatoid arthritis resulting in joint deformities, had no adverse reactions and the rheumatoid process was quiescent during the period of vaccine administration. There were no severe local reactions to the vaccine at the site of injection. A mild transient erythematous area varying in size from 0.5 to 2.0 cm. and lasting 24 hours or less was the only noteworthy mild local reaction to subcutaneous injections of the vaccine. It is the consensus of those observing allergy patients that, depending upon the sensitivity state, bacterial vaccine injection may cause exacerbation of attacks of asthma, dermatitis or rhinitis if the dosage is raised beyond a certain level. This type of reactivity may have occurred in three patients receiving herpes simplex virus vaccine; however, the evidence is inconclusive. These patients felt that eye symptoms increased during the 48-hour period following vaccine injection. When the dosage level was lowered in one case, however, the complaint ceased. In this patient, dosage was maintained at a lower level for six months, then gradually in-
creased. N o noted.
REAPSOME
further eye symptoms
SUMMARY
AND
were
CONCLUSIONS
1. Herpes simplex virus vaccine prepared from rabbit kidney tissue culture was used in the study of 50 cases of herpetic keratitis. 2. The dermal reaction to the TC antigen in general was more pronounced than the soluble antigen derived from infected allantoic fluid. 3. The whole TC antigen was more effective than either the supernate or sedimented fractions. 4. There were no recurrent herpetic attacks in 46 percent of the patients receiving T C antigen therapy, during a two-year observation period. Attacks were diminished in severity in 26 percent. The findings were inconclusive in 26 percent. 5. TC vaccine was well tolerated by the patients, giving few or no untoward reactions on a long-term desensitizing basis. 136 East 64th Street (21). ACKNOWLEDGMENT W e w i s h to a c k n o w l e d g e the aid of R. T o w n l e y P a t o n , M . D . , and the members of the Department Ophthalmology, T h e Manhattan E y e , E a r and T h r o a t Hospital, without w h o s e interest this study could not easily have been undertaken.
REFERENCES
1. Chu, L. W., and Warren, G. H . : Pathogenicity and immunogenicity of herpes simplex virus strains propagated in rabbit kidney tissue. P r o c . Soc. E x p e r . Biol. & Med., 105:396, 1960. 2. N a g l e r , F . P . O. : A specific cutaneous reaction in persons infected with the virus of herpes simplex. J. Immunol., 4 8 : 2 1 3 , 1944. 3. : A licrpe» skin test reagent from amniotic fluid. Australian T. Exper. Biol. & Med. Sei., 2 4 : 103, 1946. 4. Rose, H . M., and Molloy, E . : Cutaneous reactions with the virus o f herpes simplex. J. Immunol., 5 6 : 2 8 7 , 1947. 5. Jawetz, E., Coleman, V., and Allende, M. F . : Studies on herpes simplex virus: II. A soluble antigen of herpes virus possessing skin reactive properties. J. Immunol., 6 7 : 1 9 7 , 1951. 6. Kilbourne, E. D . , and H o r s f a l l , F. L., Jr. : P r i m a r y herpes s i m p l e x virus infection o f adult with note on relation of herpes simplex virus to recurrent aphthous stomatitis. Α Μ Α A r c h . Int. Med., 8 8 : 495,1951. 7. Reed, L. J., and Muench, Η . Α . : Simple method of estimating 50 percent end-points. A m . J. H y g . , 2 7 : 4 9 3 , 1938. 8. W o n g , S. C , and Kilbourne, E . D . : Changing viral susceptibility of a human cell line in continuous cultivation: I. Production of infective virus in a variant of the Chang conjunctival cell following i n f e c tion with s w i n e o r N - W S influenza viruses. J. E x p e r . Med., 1 1 3 : 9 5 , 1961. 9. D i a g n o s t i c procedures f o r virus and rickettsial diseases. A m . Public H e a l t h Α., N e w York, 1956, ed. 2, pp. 254-259. 10. Lennette, Ε . H., and Schmidt, M. J.: Studies on the development and persistence of C F neutralizing antibodies in human poliomyelitis. A m . J. H y g . , 6 5 : 2 1 0 , 1957.
PROPHYLAXIS OF HERPETIC KERATITIS
265
11. Chapin, H . B , Doctor, D , and W o n g , S.: Peculiar deficiency o f antibody production in a patient with keratitis receiving herpes s i m p l e x v i r u s vaccine. A m . J . O p h t h , 5 2 : 5 6 9 , 1961. 12. Buddingh, G. J , Schrum, D . I , Lanier, J. C , and Guidry, D . J.: Studies o f natural history of herpes simplex infections. Pediatrics, 1 1 : 5 9 5 , 1953. 13. Rake, G. W . : T h e etiologic role of the virus of herpes simplex in ophthalmic disease. A m . J. O p h t h , 4 3 : 1 1 3 , 1957. 14. Kubelka, V , and V a s s e r m a n n o v a , V . : Antiherpetic vaccine in ophthalmology. Cesk. O f t a l , 1 5 : 1 , 1959. 15. Scott, T . F . M . : E p i d e m i o l o g y of herpetic infections. A m . J. O p h t h , 4 3 : 1 3 4 , 1957.
LYOPHILIZED JUN
TSUTSUI,
M.D.,
CORNEA SAEKO
IN
EXPERIMENTAL
WATANABE,
Okayama,
Transplantation of the lyophilized cornea has been attempted since the work of Weiss and Taylor in 1944. They investigated the possibility of using stored frozen-dried corneal grafts in rats and got a promising result. Subsequently Katzin, who carried out essentially similar experiments with frozen corneal grafts in rabbits, reported failure to obtain transparent grafts. Leopold and Adler, who also worked with frozen-dried corneal grafts in rabbits, have reported similar findings. 1
2
3
In 1955, McNair and King reported an effective method of lyophilization of the cornea in animal experiments and they concluded that grafts dehydrated in glycerine and stored in a vacuum without refrigeration were suitable for lamellar keratoplasty. Bonhoure applied this method for the preservation of the human cornea and a favorable result was obtained in a case of lamellar keratoplasty. 4
5
In 1957, King reported as successful use of lyophilized cornea in both animal and human cases. Corneas preserved and stored as long as seven months resulted in transparent grafts in all of nine animal eyes. In six human patients, the results of the lamellar grafts were considered equal to those obtained when fresh donor material was used 8
* F r o m the E y e Department and E y e - B a n k Laboratory, O k a y a m a Rosai Hospital. T h i s w o r k w a s done under the B o b H o p e F i g h t F o r S i g h t Fund ( G - N o . 2 3 3 - C 2 ) of the National Council to Combat Blindness, Inc., N e w York.
B.SC,
AND
HETEROGRAFTS*
NOBUKO
MURAKAMI,
B.SC.
Japan
but penetrating keratoplasty, utilizing these preserved grafts, has not offered the same degree of the success. Payrau reported seven cases of successful lyophilized lamellar homograft and seven cases of heterografts using pig and dog corneas. U p to the present studies, lyophilization of the cornea was mainly done with the aim of the preservation of the graft. In one of our serial studies on corneal heterografts, lyophilization was employed to reduce the antigenicity of the heterogeneous donor material and this method was effective in reducing the donor-recipient reaction as well as in preserving the graft. 7
8,9
MATERIALS
AND
METHODS
Experimental corneal heterografts were attempted on 16 rabbits' eyes using fresh ox cornea and on 24 eyes using lyophilized o x cornea. In both groups, the corneal disc, with both epithelial layer and stroma, was transplanted into the right eye while stroma only was transplanted into the left eyes. In the group of lyophilized donor material, there were three varieties of rehydration: 1. Six lyophilized corneas were rehydrated with the recipients' serum for three days in a refrigerator. 2. Another six corneas were rehydrated for 10 minutes. 3 . The remaining 12 lyophilized discs were rehydrated with normal saline for 10 minutes. Clinical and histologic results were compared.
255
7. Spaeth, Ε. B . : Ocular tumors. Α Μ Α A r c h . O p h t h , 4 6 : 4 2 1 - 4 2 3 , 1951. 8. H a g e d o o r n . Α . : Adenocarcinoma o f a meibomian gland. A r c h . O p h t h , 12:850-866, 1934. 9. O'Brien, C. S , and Braley, A . E . : C o m m o n tumors of the eyelids. J . A . M . A , 107:933-938, 1936. 10. Magnus, J. Α . : Adenocarcinoma o f meibomian gland with secondaries in liver. T r . Ophth. S o c . U . Kingdom, 6 7 : 4 3 2 - 4 3 5 , 1947. 11. Rice, M. L , and Lundeke, H . J.: Adenocarcinoma o f the meibomian gland. A m . J. O p h t h , 3 3 : 14-34, 1950. 12. Straatsma, B. R.: Meibomian gland tumors. Α Μ Α A r c h . O p h t h , 5 6 : 7 1 - 9 3 ( J u l y ) 1956.
THE IN H.
THE
BEECHER
VALUE
PROPHYLAXIS CHAPIN,
M.D.,
OF OF
TISSUE RECURRENT
SAM
C.
The history of vaccine therapy in herpes simplex infection in man, employing both dead and live virus, has been disappointing. Even in the presence of circulating antibody attacks occur. Thus any new immunizing procedure admittedly must be viewed with considerable skepticism. Yet in the case of herpetic keratitis in which repeated attacks may lead to blindness, new ideas should be explored. Recent studies have shown that potent tissue culture vaccines, with excellent immunizing properties in experimental animals, deserve more than passing interest. Apart from this, past failures in attempted prophylaxis of the infection in man may be attributed to the failure to recognize the allergic phase of the disease. Thus it is not surprising that routine immunizing procedures consisting of three or four injections of vaccine material are ineffective. 1
It is the object of this paper to explore the usefulness of tissue culture vaccine in preventing recurrent attacks of herpetic keratitis. Concomitantly the use of the antigen in the diagnosis of herpetic infections will be presented. AND
ATTACKS
WONG,
INTRODUCTION
MATERIALS
CULTURE
METHODS
VIRUSES
Three strains of herpes simplex virus were used. Strain " H F " was obtained from * F r o m the Department of A l l e r g y , T h e M a n h a t tan Eye, E a r and T h r o a t Hospital.
OF
PH.D.,
VACCINE* HERPETIC
AND
JANE
KERATITIS REAPSOME,
B.S.
Dr. L. W. Chu of the Wyeth Institute. It was passed four times in the chorioallantoic membrane of 10- to 11-day-old embryonated hens' eggs before the virus was stored as 10-percent chorioallantoic membrane suspension at — 65°C. This virus was used for the production of the egg antigen. The "A" strain was originally isolated by Dr. W. Anderson of the New York Hospital from a vesicle of a case of herpes labialis. This virus was found to give the most consistent results in the serum-virus neutralization test for the detection of antibody. The 1266 strain was originally isolated by Dr. Kilbourne in suckling mice from a case of aphthous stomatitis. It was obtained as a frozen 10-percent mouse brain suspension. There was no history of cell line passage of this virus. It was passed five times in chick fibroblast and twice in rabbit kidney cultures before it was stored at — 65°C. The seed virus has a TCID50 of 10 per ml. 6
s 5
PREPARATION
OF
ANTIGEN*
The term antigen or vaccine will be used interchangeably in this paper. The egg antigen was prepared from the infected allantoic fluid of embryonated eggs employing the method of Jawetz, et al. T w o changes, however, were introduced: ( a ) the virus titer of the infected allantoic fluid was determined by serial 10-fold dilutions in rabbit kidney 5
* W e are indebted to Dr. George H . W a r r e n of the W y e t h Institute for supplying part of the antig e n s used in the investigation.
H . B E E C H E R C H A P I N , S A M C. W O N G A N D J A N E
256
REAPSOME
tissue cultures and had a TCID50 of 10 per ml. prior to ultracentrifugation at 15,000 rpm for 60 minutes ; (b) formalin in the final concentration of 1:4,000 was used to inactivate residual virus. Control uninfected allantoic fluid was prepared from embryos of the same age and treated in the same manner as the infected material.
tests were carried out. A total of 100 ml. of the vaccine was obtained. The control TC antigen was prepared in identical manner except no virus was added. All antigens were stored at 4°C. It is pertinent to point out that the T C antigen contained no penicillin or animal serum.
The tissue culture antigen was prepared according to the method of Chu and Warren employing rabbit kidney cultures. Essentially the method consisted of the following: Trypsinized rabbit kidney cells in concentration of 10 X 10 cells contained in 10 ml. of growth medium were grown in 200 ml. serum dilution bottles. The growth medium, however, was modified by adding an equivalent volume of medium 199 to the lactalbumin hydrolyzate medium.
SAFETY
7
1
e
When a complete sheet of cells was formed (usually in five to seven days), the tissues were washed with three changes of 30 ml. each of warm medium 199. Phosphate buffered saline, 0.01 molar, pH 7.3 could not be used for the same purpose for it produced extensive sloughing of the cell sheet; 10 · TCID50 of herpes simplex virus, strain 1266, contained in 4.0 ml. of medium 199 was inoculated per bottle. After three hours of adsorption, the old medium was discarded and 10 ml. of fresh medium 199 containing 100 μg. of streptomycin and 50 units of polymyxin per ml. were added. s
6
5
At the end of 40 hours, tissue destruction was usually complete. Aliquots were taken from each bottle and tested in blood agar plates and thioglycolate medium to rule out bacterial contamination. The bottles were stored at — 20°C. overnight. The following day the bottles were thawed in running tap water, the contents pooled, and centrifuged at 2,000 rpm for 20 minutes. The clear pink supernatant fluid was removed and a portion stored in a C 0 chest in sealed glass ampoules for later virus titration. Formalin was added to the antigen to effect a final concentration of 1:4,000. The mixture was incubated at 37°C. for 24 hours and then stored at 4°C. for two weeks before safety 2
TESTS
Preliminary tests had indicated that rabbit kidney cultures were equal to infant mice in sensitivity in the detection of herpes simplex virus. In addition, the former possessed the advantage of permitting the testing of larger volumes of vaccine with concomitant increase in sensitivity. In view of this finding it was decided to place greater reliance on rabbit kidney cultures in determining residual infective virus. Twenty-five ml. each of egg and TC vaccines were dialyzed in cellophane bags in 0.01 molar phosphate buffered saline, p H 7.0, at 4°C. for 72 hours to remove formalin. Five ml. of each dialyzate was inoculated into each of four bottles of rabbit kidney cells, incubated at 37°C. and observed daily for 10 days for evidence of cytopathic effect. The pH of the medium was adjusted with N a H C 0 solution when necessary and complete change of medium was made every four days. 3
Six mice per group were inoculated with 0.03 and 1.0 ml. of the undialyzed vaccines via the intracerebral and intraperitoneal routes respectively. The animals were observed for three weeks. In addition blood agar plates and thioglycolate medium were inoculated with the antigen and observed for one week. There was no evidence of viable virus in all the tests. The frozen virus representing the antigens prior to addition of formalin was titrated in rabbit kidney cultures employing four tubes per serial 10fold dilutions. The titers were calculated by the method of Reed and Muench. 7
SERUM-VTRUS
NEUTRALIZATION
The neutralization test for the detection of serum antibody was carried out in tissue
PROPHYLAXIS OF HERPETIC
culture employing Chang's conjunctival cells. The method of propagation and maintenance is that described by W o n g and Kilbourne. Human sera which were not used within three days were stored at — 20°C. Before use, the serum was inactivated at 56°C. for 30 minutes. Serial two-fold dilutions of sera in phosphate buffered saline pH 7.3, beginning at 1:2.5 were mixed with equal volumes of herpes simplex virus. The "A" strain was diluted in 0.1-percent casein in 0.01 M phosphate buffer, pH 7.0, to contain 2,000 TDIC50 per ml. The control virus consisted of phosphate buffered saline instead of the serum. For each run a positive and a negative serum were included. 1
8
After thorough mixing, the serum-virus mixtures and the virus controls remained at 22°C. for 60 minutes. Then 0.1 ml of the mixture was added to each of two to four tubes of conjunctival cells. Tubes were read daily, the p H was adjusted with one drop of 0.5 percent N a H C 0 solution when needed and the medium was changed every three or four days. The cytopathic effect was graded from 0 to 4 according to the degree of destruction of tissues. The test was terminated when the control tubes showed 3 to 4-plus cytopathic effect. The serum neutralization titer was the highest dilution of serum showing 2-plus or less cytopathic effect than the control tubes. 3
COMPLEMENT
FIXATION
TESTS
The technique used was the California State Department Public Health procedure. The most satisfactory antigen was prepared from human conjunctival-D cells infected with the "A" strain of herpes simplex virus. Briefly the method consisted of propagating the virus in the same manner as that used for the rabbit kidney antigen employing two-percent horse serum in medium 199 instead of 199 alone. When about 75 percent of the tissues were destroyed (usually in about 72 hours), the remaining cells were scraped off with a rubber policeman. Each batch, consisting of about 50 ml. of tissue fragments and fluid, 9
257
KERATITIS
was treated in a Raytheon sonic oscillator, Model 10KC, for 60 seconds. Formalin was added to the homogenate to effect a final concentration of 1:2,000. The antigen was stored at 4°C. Such preparations have yielded consistently complement fixation titers of 1:32 or greater. T w o units were used in the test. The control antigen was prepared from uninfected conjunctival-D cells of similar age in an identical manner. Positive and negative control sera were selected from those giving similar results in the serumvirus neutralization test. Human serum specimens to be tested were frozen immediately after separation of the serum until used. Anticomplementary sera were spun at 10,000 rpm for 10 minutes in a Lourdes centrifuge, Model A T , prior to a treatment described by Lennette and Schmidt for such sera. About 80 to 90 percent of such sera were successfully treated by this technique. The complement and hemolysin were purchased from Cappel Laboratories and the sheep cells from Carworth Farms. 10
INTRACUTANEOUS
TEST
The cutaneous tests were done on the volar surface of the forearm in the following manner: After cleansing the skin with 70 percent ethanol, 0.1 ml. of a 1:10 dilution of the herpes simplex virus antigen and also a comparable volume and dilution of the control material were given intradermally so that the injected bleb of fluid was visibly raised above the normal surface of the skin. The sites were observed at the end of 24 hours for erythema, edema and induration. PATIENTS
The subjects upon whom the cutaneous tests were done were drawn from the staff and patients of the Corneal Clinic of the Ophthalmology Department, the Manhattan Eye, Ear and Throat Hospital. The tests were done upon male and female subjects immediately following the taking of blood serum specimens for the determination of
258
H . B E E C H E R C H A P I N , S A M C. W O N G A N D J A N E
antibody titer for herpes simplex virus. Diagnosis in those infected cases included herpetic keratitis either in the chronic quiescent phase or with ulcer formation in the cornea and herpes simplex lesions of the lips. Patients chosen for vaccine therapy. Twenty-three females and 27 males ranging in age from four to 81 years clinically diagnosed as having chronic recurring herpetic keratitis were selected for vaccine therapy. A physical examination, blood count and urinalysis were done routinely on all patients. Five patients with recurrent herpes simplex virus lesions at sites other than the conjunctiva received the control antigen. The recurring lesions of the cornea in all the patients had been present for periods varying from one week to 41 years. In some of the patients the cornea of one eye was involved and in others, both eyes. The interval of recurrences varied widely from patient to patient. In some the attacks occurred yearly, whereas others recurred from two to five years. The youngest patient treated had a bilateral infection which recurred about every three months. One patient was treated who did not have the corneal involvement, the lesions occurring in the labial and genital areas and on the buttocks. Method of administration of vaccine. Vaccine was given to patients in a series of weekly injections starting with 0.1 ml. of 1:100 dilution and increasing the dose until the concentrate was reached. Thereupon the concentrate was given in a fixed dose at varying intervals of from two weeks to three months. A second method consisted of intradermal injections of the concentrate in 0.1 to 0.3 ml. doses. These intradermal injections were given either by themselves or in conjunction with the subcutaneous injections. The material used for control consisted of tissue culture fluid which had not been exposed to the herpes simplex virus. A. Delayed type allergy cutaneous tests to tissue culture herpes simplex virus antigen. Delayed type allergy tests have been done
REAPSOME
upon human subjects for the purpose of comparing the results of using a new herpes simplex virus antigen material prepared in rabbit kidney cell tissue culture with the usual type of herpes simplex virus antigen prepared from egg embryo herpes inoculated material. Nagler was the first to describe a specific cutaneous reaction using an 11-day-old chorioallantoic membrane material inoculated with herpes virus. Later using a virus skin test reagent from amniotic fluid, Nagler stated that the amniotic fluid material was more useful for the test than that prepared from chorioallantoic membrane, as the latter gave too many nonspecific reactions probably because of a higher protein content. 2
3
The following year Rose and Molloy described a cutaneous reaction using antigenic material prepared from herpes-infected chorioallantoic fluid, concluding that the test could be used to advantage as an aid in diagnosing primary herpetic infection. Jawetz used a soluble antigen of herpes simplex virus prepared from infected allantoic fluid to obtain the cutaneous reactions. H e noted a maximum reaction in the skin between 18 and 24 hours. The soluble skin reacting material was heat stable, contained one to five percent of the total active virus and did not deteriorate on storage at 4°C. Its skin reactivity was abolished by 10-fold dilution. 4
5
With the development of tissue culture techniques which can produce potent herpes simplex virus antigens in a relatively pure form, it was decided that the specificity of cutaneous reaction to herpetic infection merited re-examination. The cutaneous tests were performed upon human subjects believed to have been infected with herpes simplex virus. A few tests were done upon individuals believed not to have been so infected. Neutralization and complement fixation tests for antibody to the herpes simplex virus were done upon the sera of these subjects. The same herpes virus antigen material used for the cutaneous tests was given
PROPHYLAXIS OF HERPETIC
as a vaccine to certain of the subjects in an attempt to alter the course of their chronic recurring attacks of acute herpetic infection. The result of this study is summarized in Table 1. A n examination of the results of the measurements in cm. of the herpes sim-
259
KERATITIS
plex cutaneous tests in 50 humans reveals that the average measurements of the egg antigen was 0.113 cm., the average of the egg control material being 0.042 cm. The average of herpes simplex virus T C antigen was 0.187 cm., whereas that of the control
TABLE 1 H U M A N SKIN REACTIONS OF THE D E L A Y E D ALLERGY TYPE TO H E R P E S SIMPLEX A N T I G E N P R E P A R E D I N EGG A N D T I S S U E C U L T U R E
Case N o .
Sex
Age
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
M F M F M M M M F F F F F F M F F F M F M M F F M F M M M F M F F F M M M M F M M M M F M F M M M F
31 70 30 37 70 18 66 40 50 38 40 38 60 30 60 44 61 45 56 53 51 36 37 40 64 47 52 55 51 71 62 31 48 44 51 52 71 54 50 81 67 63 51 50 70 45 35 51 45 58
Egg Antigen Herpes Simplex Control Virus (cm.) (cm.) 0 0.3 0 0 1.5 0 0.5 0 0.8 0.7 1.0 0 0 0 2.2 0.7 0 0.2 0.7 0.6 0.5 0 0.8 0.6 0.5 0 0.8 0.6 0 0.8 0 0 1.1 0 1.8 0.8 0.9 0.6 0 0.5 0 1.5 0 0 0 0 0 0.6 0 0
1.9 1.5 0.8 0 1.5 1.1 1.1 1.7 2.0 1.6 1.4 1.6 1.8 0 2.3 1.3 1.7 0 1.2 2.3 1.5 0 1.4 1.1 1.0 1.7 1.0 2.3 1.9 1.2 0.6 0.6 1.2 1.4 1.0 1.1 1.8 1.1 0 1.1 0.7 2.1 0.7 0 0.7 0.1 0.6 1.0 0 0.8
T i s s u e Culture A n t i g e n Herpes Simplex Control Virus (cm.) (cm.) 0 1.5 0 0 1.5 0 0 0 0.8 1.3 0 2.3 2.7 0 2.2 0 0 0 0.9 0 0.8 0 0.5 0 0 0 0.8 0 0.7 0 0 0 1.2 0 0 0.8 0.8 0.6 0.9 0.8 0 1.6 0 0.5 0 0.1 0 0 0 0
2.3 2.0 1.5 1.0 1.1 1.8 1.7 1.9 2.9 2.2 4.3 2.4 2.9 0.5 2.4 2.2 1.7 2.0 2.9 2.8 2.8 2.0 2.1 2.2 2.9 3.4 1.5 2.6 1.4 1.4 0.9 2.2 1.2 1.5 1.6 1.2 1.3 1.7 2.2 0.8 1.3 3.0 1.2 1.8 0.7 0.1 1.8 1.0 1.0 1.4
Neutralizing Antibody
1:20 1:80 0 2.0 1:80 1:160 1:320 1:80 1:80 0 1:640 1:80 1:80 1:40 1:80 1:40 1:80 1:80 1:160 1:110 1:160 1:160 1:160
1:160
1:80 0 1:20 1:80 1:50 1:5 1:20
260
H . B E E C H E R C H A P I N , S A M C. W O N G A N D J A N E R E A P S O M E
TC fluid was 0.044 cm. It would appear from this study that the herpes simplex virus antigen prepared from rabbit kidney tissue was more active than that of chick embryo allantoic fluid material. It is believed that this is the first time that there has been such a study of the cutaneous delayed allergy tests in humans with tissue cell material. The findings of this study do not warrant conclusions as to the specificity of cutaneous reactions to herpetic infection in humans. Further investigation must be made to determine the veracity of the widespread opinion that a cutaneous reaction is specific for herpes infection. Perhaps a method of performing the cutaneous reaction can be devised that will prove specific but it does not appear that the usefulness of such a test for herpes-infected humans would parallel that of the well-known delayed type allergy test to tuberculin in tuberculosis-infected humans. Only further studies will clarify the presumption. In the present study it is of interest to demonstrate that in six cases (Cases 12, 13, 15, 33, 40 and 4 6 ) , the delayed cutaneous reaction to herpes simplex virus vaccine was equal to or scarcely greater than the control reaction. In three cases (Cases 4, 14 and 4 4 ) , there was no or scarcely measurable neutralizing antibody present in the sera of the patients tested. B. Antigenic activity of herpes simplex virus vaccine fractions. An attempt was made to determine the nature of the skin reacting antigen present in the tissue culture vaccine. Jawetz, et al. showed that the skin reacting principal in herpes simplex virusinfected allantoic fluid is a soluble antigen rather than the intact virus particles. Whether a similar substance is present in the T C antigen remains to be determined. The following experiment was performed with this object in view. Tissue culture material which had a TCID50 of 1 0 herpes simplex virus per ml. was centrifuged at 35,000 rpm for 90 5
8,3
minutes in the Spinco, Model L. The supernatant fluid was carefully removed and subjected to two more such ultracentrifugations. On the last run, only 70 percent of the fluid was removed from the stainless steel tubes. The sediment was resuspended to the original volume in medium 199 and washed three more times at the same speed and time as given. Under these conditions it was found, in the average of two experiments employing frozen tissue culture fluids, that the supernate had a TCID50 of 1 0 per ml., the precipitate 10 · per ml. The ratio of viable virus was 1:60, respectively. Using the formalintreated supernate and sediment materials separately, intradermal tests were done as previously described in herpes simplex keratitis cases, with results shown in Table 2. It is clear from the results that the original antigen contains the most active material. However, it is equally clear that the supernate which contained 60 times less viable virus than the sediment fraction elicited 5 7
7
5
TABLE 2 COMPARATIVE SKIN REACTIONS OF VARIOUS FRACTIONS OF TISSUE CULTURE ANTIGEN IN HUMANS*
Case N o .
Tissue Culture Fluid Control (cm.)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Mean
1 .0 0.4 0 0.8 0.3 0.4 0.3 0.2 0 0.4 0.3 0.2 0.5 0.2 0.6 0 0.35
Original Herpes Herpes Simplex Simplex Virus Virus Sediment Vaccine (cm.) (cm.) 3.7 1.9 1.5 1.8 2.2 3.1 2.2 1.0 0.4 2.0 2.6 1.6 2.4 1.6 5.5 2.0 2.2
1.7 0.9 0.7 0.7 0 2.0 1.2 0.8 0.4 0 1.1 0.5 1.7 0.9 1.0 0.6 0.89
Herpes Simplex Virus Supernate (cm.) 2.0 1.3 1.7 0.8 2.9 2.5 2.6 0.3 0 1.3 2.0 0.5 2.0 2.0 0.7 0.5 1.4
* O n e - t e n t h ml. of each c o n c e n t r a t e material inj e c t e d intradermally o n t h e volar surface of t h e forearm. T e s t s read a t 24 hours.
PROPHYLAXIS OF HERPETIC
a more pronounced skin reaction than did the sediment. Thus the nature of the dermal reacting principal in T C antigen remains to be elucidated. It would appear from these results that a soluble antigen of unequh'ocal validity cannot be demonstrated. Further studies are warranted. C. Neutralising and complement fixation antibody. Neutralizing antibody measured in the sera specimens before treatment in 15 patients varied from 1:20 to more than 1:160. One patient's neutralizing antibody titer, reported elsewhere, was persistently negative at 1:5 both before and after vaccine treatment. Complement fixation tests in 25 patients before treatment varied from zero in one patient to 1:5 to 1:80 in the remaining patients. 11
After vaccine treatment was started, neutralizing antibody in the group of 15 patients rose in a range from 1:160 to 1:1280. Higher titers tended to fall as treatment was continued. In the group of 25 patients, after treatment was started, complement fixation tests rose in a range from 1:80 to 1:160 but tended to vary irregularly as treatment was continued. In the patient previously mentioned whose serum contained no neutralizing antibody, complement fixation antibody determination remained at 1:5 in tests performed at monthly intervals for six months. D. Clinical use of herpes simplex virus antigens. The herpes simplex virus infection is one of the commonest afflictions of m a n . - Recurrent attacks in general are mild and self-limiting. However, when the infection occurs in the cornea, it commonly leads to diminished vision of the infected eye, especially if the infection recurs at periodic intervals. Because of the frequency with which patients suffering from herpetic keratitis present themselves at the Manhattan Eye, Ear and Throat Hospital, it was felt worth while to give a clinical trial of vaccine therapy with the herpes simplex virus antigen. 12
13
There have been reports in the literature
KERATITIS
261
of the use of herpes simplex virus vaccine in treatment of herpes simplex virus infections in humans. That such a vaccine has not been in widespread use suggests the difficulties inherent in its practicability as a therapeutic or prophylactic agent. These difficulties may be divided into two main categories: 14
1. The first involves the problem in the successful manufacturing of a potent high titer herpes simplex virus vaccine with concomitant reduction in extraneous proteins. 2. The second is related to the pathogenesis of the herpes simplex virus infection. The virus in most instances in human infection invades the outer layer of the skin, the conjunctival or corneal tissue or mucous membranes. After the acute inflammatory process has subsided and the involved tissues healed, the virus particles are said to be latent. An unknown mechanism may reactivate the virus periodically throughout life with attendant inflammatory reaction. Unlike most of the commonly used vaccine therapies in which vaccination is done to prevent the primary attack of the disease, therapy with herpes simplex virus vaccine is used to prevent recurrences of the disease. Because of the nature of herpes simplex virus infection in humans, it has been suspected that the standard method of vaccination consisting of two or three injections separated by a suitable interval to allow antibody production, followed by booster doses at yearly or longer intervals, would prove of little therapeutic benefit. The few clinical trials of vaccine using these methods have not proved encouraging. The fact that it is believed there is an allergic reaction to the virus in infected corneal tissue at certain phases of the infection, coupled with the well-known herpes simplex virus allergic cutaneous reaction in humans, suggested a new therapeutic approach based upon standard desensitizing-immunizing procedures used against allergy diseases. Because of the greater activity of the herpes simplex virus antigen prepared from rabbit kidney tissue
262
H . B E E C H E R C H A P I N , S A M C. W O N G A N D J A N E
REAPSOME
TABLE 3 I N T E R V A L S B E T W E E N R E C U R R E N T ATTACKS O F A C U T E H E R P E T I C K E R A T I T I S
N o . of Cases
Less t h a n One Y e a r
One Year
One t o F i v e Years
Over Five Years
Irregular Intervals
Primary Attack*
12
6
14
3
S
10
* T h e s e patients h a v e had n o recurrence following the subsidence of o n e initial a c u t e a t t a c k .
culture, it was decided to use this antigen material for the clinical trials. The whole vaccine material which contains both the sediment and supernate materials was used in these patients. Because of the varying intervals of recurrence of herpetic lesions in the eye, the value of any treatment can be assessed only on a long-term basis. The present study was initiated January, 1959, and the observations are continuing. In order to assess the results of vaccine therapy, it is necessary to examine in detail the conditions of the course of infection in individual patients. Although no correlation has been observed as yet between therapeutic result and duration of infection, this factor may have a bearing on the end-result of therapy. In this series of cases, five patients have had the infection for less than one year, 32 patients from one year to nine years, and 13 for 10 or more years. More important, seemingly, than the duration of infection, is the interval between the acute attacks in each patient. For purposes of analysis, the cases have been divided into groups relating to these intervals, as shown in Table 3.
TABLE 4 R E S U L T S OF V A C C I N E T H E R A P Y
Number of Cases
Percent
G o o d , no recurrence Fair, mild recurrence Nonconclusive Poor
23 13 13 1
46.0 26.0 26.0 2.0
TOTAL
50
100.0
Category a. b. c. d.
The therapeutic effectiveness of the vaccine in these patients is summarized in Table 4. Of the 50 cases in Table 4, 23 may be classified as ( a ) , 13 as ( b ) , 13 as ( c ) , and one as ( d ) . In contrast the five control patients, receiving the placebo or nonherpetic tissue culture fluid, had recurrent attacks at the usual intervals with undiminished severity. In summary then it may be stated that herpes simplex virus T C vaccine, given by the method described, exerts a protective effect against recrudescence of herpetic lesions in the cornea. A more detailed demonstration of the value of vaccine treatment is shown in Table 5. In this series of 50 cases treated with tissue culture vaccine, it is seen that 36 showed either nonrecurrence of herpetic keratitis or a lessening of the severity of the lesion. Of those 10 cases in which only the primary attack was observed, there is, of course, no information of the expected interval before recurrence. Either a therapeutic effect was achieved by the use of herpes simplex virus vaccine, or the interval before recurrence exceeded the two years covered by this report on these patients. Of the group of 10 cases shown in Table 5, eight had no recurrences, one in the ( b ) category had labial lesions of diminished severity twice during the period of observation and the one case in the (c) category had no recurrence but, as therapy had been given for only 10 months, the result was considered inconclusive. Of the group in which attacks occurred at irregular intervals, the one case in the ( b )
PROPHYLAXIS OF HERPETIC
KERATITIS
263
TABLE 5 I N T E R V A L S B E T W E E N RECURRENT ATTACKS OF A C U T E HERPETIC KERATITIS
Categories
Less t h a n One Year
a b c
4 6 1
A
U
Controls
1 1
3*
One Year 3 1 2
O n e to F i v e Years 7 4 3
Over F i v e Years
— 3
Irregular Intervals 1 1 3
Primary Attack 8 1 1
Total N o . Cases 23 13 13 1 1
2*
5
* In t h e control cases, the a t t a c k s occurred a t the e x p e c t e d t i m e s with no d i m i n u t i o n in t h e size or duration of t h e lesion.
category had no eye recurrence for two years. This patient, however, during an upper respiratory infection with fever, had labial lesions 50-percent smaller than those observed previously during fevers. The three patients with an interval of over five years between attacks fall into ( c ) category because of the shorter period of observation of this study. Of the patients whose attack interval fell between one to five years in the ( b ) category, two exceeded the period of this study. One of these patients had one attack of labial lesions 50 percent smaller than before therapy. Of the other two, one had a subsiding acute attack at time of onset of therapy ; the other had a mild attack after stopping treatment for four months. The two patients with an attack interval of one year in the ( c ) category have not been observed long enough to give information. One with severe bilateral corneal damage from previous attacks which caused marked thinning of the cornea discontinued treatment before dosage could be stabilized. Of those patients with attack intervals of less than one year, the one patient in the ( c ) category discontinued therapy after three months. No acute attack recurred in this patient while on vaccine therapy. Of the six patients in ( b ) category, the interval between attacks lengthened and the lesions became less severe in one patient with severe bilateral infection; one had labial attacks at the expected interval but of diminished severity. It was noted in one patient with
moderately severe allergic dermatitis involving the antecubital and popliteal spaces, the legs, trunk, arms and neck, that there was an exacerbation of the acute corneal condition concomitant with acute allergic dermatitis which promptly responded to steroid therapy. The one patient in ( d ) category with severe corneal damage from previous attacks, before vaccine dosage was stabilized had no apparent lessening of severity of the infection. Jawetz, et al. suggested that psychosomatic factors may play an important role in the benefits derived from vaccine therapy of herpetic infection in man. This opinion is shared by McNair Scott. T o this may be added personal bias. T o minimize the latter, the patients were examined independently and the clinical status evaluated periodically by an ophthalmologist. Admittedly these two factors may have contributed, at least in part, to the favorable results obtained. Nevertheless, we believe that the majority of the patients were benefited by the desensitizing treatments employing tissue culture antigen. The seriousness of herpetic keratitis in man and the possibility that blindness may result justify the trial of any rational therapy. The potent vaccine, the absence of extraneous proteins, the innocuousness of the antigen fulfill these requirements and therefore justify further studies. 5
15
Side-reactions to herpes simplex virus vaccine. There were no general or systemic reactions of any type noted in patients receiving the vaccine. Leukocyte and erythrocyte counts, blood-cell percentages, and urin-
264
H . B E E C H E R C H A P I N , S A M C. W O N G A N D J A N E
alysis findings remained within normal limits during vaccine therapy. One of the patients, aged 63 years, with advanced chronic rheumatoid arthritis resulting in joint deformities, had no adverse reactions and the rheumatoid process was quiescent during the period of vaccine administration. There were no severe local reactions to the vaccine at the site of injection. A mild transient erythematous area varying in size from 0.5 to 2.0 cm. and lasting 24 hours or less was the only noteworthy mild local reaction to subcutaneous injections of the vaccine. It is the consensus of those observing allergy patients that, depending upon the sensitivity state, bacterial vaccine injection may cause exacerbation of attacks of asthma, dermatitis or rhinitis if the dosage is raised beyond a certain level. This type of reactivity may have occurred in three patients receiving herpes simplex virus vaccine; however, the evidence is inconclusive. These patients felt that eye symptoms increased during the 48-hour period following vaccine injection. When the dosage level was lowered in one case, however, the complaint ceased. In this patient, dosage was maintained at a lower level for six months, then gradually in-
creased. N o noted.
REAPSOME
further eye symptoms
SUMMARY
AND
were
CONCLUSIONS
1. Herpes simplex virus vaccine prepared from rabbit kidney tissue culture was used in the study of 50 cases of herpetic keratitis. 2. The dermal reaction to the TC antigen in general was more pronounced than the soluble antigen derived from infected allantoic fluid. 3. The whole TC antigen was more effective than either the supernate or sedimented fractions. 4. There were no recurrent herpetic attacks in 46 percent of the patients receiving T C antigen therapy, during a two-year observation period. Attacks were diminished in severity in 26 percent. The findings were inconclusive in 26 percent. 5. TC vaccine was well tolerated by the patients, giving few or no untoward reactions on a long-term desensitizing basis. 136 East 64th Street (21). ACKNOWLEDGMENT W e w i s h to a c k n o w l e d g e the aid of R. T o w n l e y P a t o n , M . D . , and the members of the Department Ophthalmology, T h e Manhattan E y e , E a r and T h r o a t Hospital, without w h o s e interest this study could not easily have been undertaken.
REFERENCES
1. Chu, L. W., and Warren, G. H . : Pathogenicity and immunogenicity of herpes simplex virus strains propagated in rabbit kidney tissue. P r o c . Soc. E x p e r . Biol. & Med., 105:396, 1960. 2. N a g l e r , F . P . O. : A specific cutaneous reaction in persons infected with the virus of herpes simplex. J. Immunol., 4 8 : 2 1 3 , 1944. 3. : A licrpe» skin test reagent from amniotic fluid. Australian T. Exper. Biol. & Med. Sei., 2 4 : 103, 1946. 4. Rose, H . M., and Molloy, E . : Cutaneous reactions with the virus o f herpes simplex. J. Immunol., 5 6 : 2 8 7 , 1947. 5. Jawetz, E., Coleman, V., and Allende, M. F . : Studies on herpes simplex virus: II. A soluble antigen of herpes virus possessing skin reactive properties. J. Immunol., 6 7 : 1 9 7 , 1951. 6. Kilbourne, E. D . , and H o r s f a l l , F. L., Jr. : P r i m a r y herpes s i m p l e x virus infection o f adult with note on relation of herpes simplex virus to recurrent aphthous stomatitis. Α Μ Α A r c h . Int. Med., 8 8 : 495,1951. 7. Reed, L. J., and Muench, Η . Α . : Simple method of estimating 50 percent end-points. A m . J. H y g . , 2 7 : 4 9 3 , 1938. 8. W o n g , S. C , and Kilbourne, E . D . : Changing viral susceptibility of a human cell line in continuous cultivation: I. Production of infective virus in a variant of the Chang conjunctival cell following i n f e c tion with s w i n e o r N - W S influenza viruses. J. E x p e r . Med., 1 1 3 : 9 5 , 1961. 9. D i a g n o s t i c procedures f o r virus and rickettsial diseases. A m . Public H e a l t h Α., N e w York, 1956, ed. 2, pp. 254-259. 10. Lennette, Ε . H., and Schmidt, M. J.: Studies on the development and persistence of C F neutralizing antibodies in human poliomyelitis. A m . J. H y g . , 6 5 : 2 1 0 , 1957.
PROPHYLAXIS OF HERPETIC KERATITIS
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11. Chapin, H . B , Doctor, D , and W o n g , S.: Peculiar deficiency o f antibody production in a patient with keratitis receiving herpes s i m p l e x v i r u s vaccine. A m . J . O p h t h , 5 2 : 5 6 9 , 1961. 12. Buddingh, G. J , Schrum, D . I , Lanier, J. C , and Guidry, D . J.: Studies o f natural history of herpes simplex infections. Pediatrics, 1 1 : 5 9 5 , 1953. 13. Rake, G. W . : T h e etiologic role of the virus of herpes simplex in ophthalmic disease. A m . J. O p h t h , 4 3 : 1 1 3 , 1957. 14. Kubelka, V , and V a s s e r m a n n o v a , V . : Antiherpetic vaccine in ophthalmology. Cesk. O f t a l , 1 5 : 1 , 1959. 15. Scott, T . F . M . : E p i d e m i o l o g y of herpetic infections. A m . J. O p h t h , 4 3 : 1 3 4 , 1957.
LYOPHILIZED JUN
TSUTSUI,
M.D.,
CORNEA SAEKO
IN
EXPERIMENTAL
WATANABE,
Okayama,
Transplantation of the lyophilized cornea has been attempted since the work of Weiss and Taylor in 1944. They investigated the possibility of using stored frozen-dried corneal grafts in rats and got a promising result. Subsequently Katzin, who carried out essentially similar experiments with frozen corneal grafts in rabbits, reported failure to obtain transparent grafts. Leopold and Adler, who also worked with frozen-dried corneal grafts in rabbits, have reported similar findings. 1
2
3
In 1955, McNair and King reported an effective method of lyophilization of the cornea in animal experiments and they concluded that grafts dehydrated in glycerine and stored in a vacuum without refrigeration were suitable for lamellar keratoplasty. Bonhoure applied this method for the preservation of the human cornea and a favorable result was obtained in a case of lamellar keratoplasty. 4
5
In 1957, King reported as successful use of lyophilized cornea in both animal and human cases. Corneas preserved and stored as long as seven months resulted in transparent grafts in all of nine animal eyes. In six human patients, the results of the lamellar grafts were considered equal to those obtained when fresh donor material was used 8
* F r o m the E y e Department and E y e - B a n k Laboratory, O k a y a m a Rosai Hospital. T h i s w o r k w a s done under the B o b H o p e F i g h t F o r S i g h t Fund ( G - N o . 2 3 3 - C 2 ) of the National Council to Combat Blindness, Inc., N e w York.
B.SC,
AND
HETEROGRAFTS*
NOBUKO
MURAKAMI,
B.SC.
Japan
but penetrating keratoplasty, utilizing these preserved grafts, has not offered the same degree of the success. Payrau reported seven cases of successful lyophilized lamellar homograft and seven cases of heterografts using pig and dog corneas. U p to the present studies, lyophilization of the cornea was mainly done with the aim of the preservation of the graft. In one of our serial studies on corneal heterografts, lyophilization was employed to reduce the antigenicity of the heterogeneous donor material and this method was effective in reducing the donor-recipient reaction as well as in preserving the graft. 7
8,9
MATERIALS
AND
METHODS
Experimental corneal heterografts were attempted on 16 rabbits' eyes using fresh ox cornea and on 24 eyes using lyophilized o x cornea. In both groups, the corneal disc, with both epithelial layer and stroma, was transplanted into the right eye while stroma only was transplanted into the left eyes. In the group of lyophilized donor material, there were three varieties of rehydration: 1. Six lyophilized corneas were rehydrated with the recipients' serum for three days in a refrigerator. 2. Another six corneas were rehydrated for 10 minutes. 3 . The remaining 12 lyophilized discs were rehydrated with normal saline for 10 minutes. Clinical and histologic results were compared.