Observations on the therapeutic value of specific immune serum in experimental poliomyelitis

O B S E I ~ V A T I O N S ON T H E T H E R A P E U T I C V A L U E OF S P E C I F I C IMMUNE SERU~

IN EXPERIMENTAL

POLIOMYELITIS

E. W. SC~VLTZ, M.D., AND I~. P. GEBHAR1)T STANFORD UNIVERSITY, CALIF.

established t h a t the serum f r o m m o n k e y s h a v i n g r e c o v e r e d I Tf rISo mwellpoliomyelitis is capable o2 neutralizing the virus in v i t r o (Levaditi and L a n d s t e i n e r , 1 Leiner and yon Weisner, 2 RSmer and Joseph, ~ F1exner and Lewis, 4 and o t h e r s ) : This p r o p e r t y is also exhibited b y the s e r u m of h u m a n beings following r e c o v e r y f r o m acute poliomyelitis ( N e t t e r and Levaditi, 5 F ] e x n e r a n d Lewis, ~ and others). N o t only h u m a n poliomyelitis convalescent serum, but also the s e r u m of a considerable p e r c e n t a g e (about 75 per cent) of the adult population who have to t h e i r k n o w l e d g e not h a d poliomyelitis m a y exhibit this p r o p e r t y ( A y c o e k and K r a m e r ; ~ Shaughnessy, H a r m o n , a n d Gordan ;7 Schultz a n d G e b h a r d t ;s and others). Besides m a n and monkeys, individual animals in the poliomyelitis r e f r a c t o r y group (e.g., sheep, goats, and horses) m a y respond to r e p e a t e d injections of poliomyelitis virus with the p r o d u c t i o n of a virueidal sernm of more or less high a n t i b o d y content ( N e u s t a e d t e r and B a n z h a f ; 9 P e t t i t ; 1~ Weyer, P a r k , and B a n z h a f ; ~ F a i r b r o t h e r ; ~ F a i r b r o t h e r and M o r g a n ; ~3 H o w i t t ; ~4 and Schultz and G e b h a r d t ~ ) . This p a p e r deals with the t h e r a p e u t i c value of poliomyelitis i m m u n e serum in the experimental disease. R]~VIEW OF TI-IE LITER&TUR.E

Comparatively little work on the therapeutic ~ value of immune serum in e x p e r i m e n t a l poliomyelitis has thus f a r been reported. L e v a d i t i and L a n d s t e i n e r 1G in 1910 were a p p a r e n t l y the first to r e p o r t on the value of s e r u m in the e x p e r i m e n t a l disease. T h e y w e r e unable to a l t e r the course of the disease in m o n k e y s b y i n t r a p e r i t o n e a l injections of m o n k e y convalescent serum a d m i n i s t e r e d on the same d a y a n d one a n d five days a f t e r inoculation with virus. The same y e a r F l e x n e r a n d Lewis, 17 however, observed that, if the i n t r a c e r e b r a l injection of virus is sufficiently small (0.1 c.e. of virus filtrate) and fo.llowed within t w e n t y - f o u r hours b y conva.leseent serum i n t r a s p i n a l l y (2 e.e.), one :From t h e D e p a r t m e n t of ] ~ a e t e r i o l o g y a n d E x p e r i m e n t a l P a t h o l o g y , S t a n f o r d U n i v'ersity, C a l i f o r n i a . T h i s i n v e s t i g a t i o n w a s s u p p o r t e d b y t h e iYirs. M a r y I-Iooper S'omers M v d i c a l :P~es e a r c h :Fund, S t a n f o r d U n i v e r s i t y . *The t e r m " t h e r a p e u t i c , " a s u s e d t h r o u g h o u t t h i s p a p e r , r e f e r s to t h e a d m i n i s t r a tion of s e r u m a n y t i m e a f t e r e x p e r i m e n t a l i n o c u l a t i o n of a n i m a l s w i t h v i r u s . In o t h e r wor.ds, t h e t e r m is n o t u s e d in t h e m o r e r e s t r i c t e d sense, s o m e t i m e s e m p l o y e d , of t r e a t m e n t a p p l i e d a f t e r t h e o.nset of c l i n i c a l s y m p t o m s . 615

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may in some cases prevent the development of symptoms. Similar results were reported later by Flexner and Amoss} s Neustaedter and BanzhaP in limited experimental observations noted that intramuscular injection of immune horse serum twenty-four hours aft er intraspinal injection of virus served to prevent the development of symptoms. However, serum administered forty-eight hours aft er inoculation with the virus failed to modify the experimental disease. Weyer, Park, and Banzhaf 11 have reported a high percentage of survivors following the use of a concentrated globulin fraction of poliomyelitis immune horse serum administered at varying intervals of time after three daily intranasal instillations of virus. The same year Rhoads, TM however, using the Pa r k serum in an experiment with three monkeys, obtained inconclusive results. Howitt ~~ in an extensive experimental study in which convalescent monkey serum was administered by different routes and in varying amounts also obtained what may be regarded as highly inconclusive results. EXPI~R,I1V~E I ~ T A L W O R K

A. Procedures Macacus rhesus monkeys were employed in the studies here reported. The viruses used were the Aycoek and the so-called " M V " strains. Both were originally obtained from Dr. Simon Flexner. In the early part of these studies the virus was inoculated by the intracraniai route. This route, however, was never regarded by us as altogether satisfactory in a study of this kind. Therefore, before proceeding with the major part of these studies, we carried out an investigation to determine whether the usual incidence of infection by the intranasal route could in some way be appreciably increased. The results of this preliminary work have already been reported (Schultz and Gebhardt21). The procedure described has in our hands yielded infection in 90 to 95 per cent of the animals inoculated. The essential features of the procedure are: (1) the use of a mixed virus suspension of high potency (pooled cords from several animals); (2) thorough irrigation of the nasal passages of the animal with a phosphate buffer solution (pH 5) immediately before each instillation of virus; (3) deep ether anesthesia to facilitate carrying out, not only the in~ranasal irriga*ions, but also the instillations of virus; (4) three intranasal irrigations and three virus ~ instillations (at about threehour intervals) on the same day; (5) virus instillations made high into the nasal vault, at least 0.5 c.c, of a 10 per cent pooled virus suspension being introduced into each nasal passage with each instillation. Um less otherwise stated, this procedure was followed in all inoculations indicated as having been made by the " i n t r a n a s a l r o u t e . "

The serum employed in these studies consisted for the most part of poliomyelitis immune horse serum exhibiting an unusually high neu-

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617

tralizing titer in i~ vitro tests. The history of this serum has been described in a n o t h e r paper. 1~ It is i m p o r t a n t to note t h a t this serum in r e p e a t e d t i t r a t i o n studies has exhibited what seems to be the highest virucidal titer of a n y poliomyelitis immune serum, either of h u m a n or animal origin, hitherto reported, a f a c t which should be k e p t in mind in reviewing the observations r e p o r t e d f u r t h e r on. Poliomyelitis m o n k e y convalescent serum was employed in several of the experiments. The q u a n t i t y of serum used and the route b y which it was administered arc indicated in describing the individual experiments.

B. Observc~tions Experiment 1.--Six monkeys were injected intraerania]ly with 1.5 c.e. of poliomyelitis convalescent monkey serum 1, 6, 20, 30, 42, and 68 hours, respectively, a f t e r intracranial inoculation (Apr. 10, 1930') with 0.5 c.c. of a 5 per cent suspension of virus-cord. All the animals developed poliomyelitis in six to ten days following the inocula.t~on of virus. Neither the length of the incubation period nor the severity of the disease bore any relationship to the time i n t e r v a l which elapsed before the serum was administered. Experiment 2.--Two animals were inoculated intracranially (Apr. 25, 1930) with 0.5 e.e. of a 5 per cent suspension of virus-cord. On the first appearance of symptoms, such as tremors, five days later, spinal fluid was removed b y l u m b a r p u n c t u r e and 10 c.c. of m o n k e y convalescent serum was injected slowly into the lateral ventricles, the lumbar p u n c t u r e needle being left in place to c a r r y off excess fluid. Both animals, nevertheless, developed extensive paralysis. Experiment 3 . - - S i x monkeys, were injected intracranially (Mar. 17, 1931) with 1 e.e. each of a 1:1,000 virus-cord suspension (aqueous fraction a f t e r ether extraction). F o u r of these animals (398, 399, 400, 401) were given 20 c.e. each of poliomyelitis immune horse serum (Jan. 22, 1931, bleeding 15) by the intramuscular route on the appearance of tremors a n d excitability (five days a f t e r inoculation). All of the t r e a t e d animals developed as extensive paralysis as the controls (396, 397). Experiment 4 . - - T w o monkeys (796, 797) were inoculated with virus by the intranasa] route (Oct. 21, 1932), Six days later, and before the appearance of symptoms one of the monkeys (797) was injected with 10 e.e. of imnmne horse serum (May 3, 1932, bleeding t~) by the intraeardia] route. Both the control and serum-treated animal began to show symptoms of the disease o.n the eighth day and developed essentially the same d e g r e e of paralysis. Experiment 5.--Fo.ur monkeys were inoculated with v i r u s b y the intranasal route (Nov. 9, 1932). One m o n k e y (830) received no serum, while three Were given 10 c.c. each of immune horse serum (May 3, 1932,

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bleeding ~) by the intracardial route. One monkey (831) received the serum twenty-four hours, one (832) forty-eight hours, and one (833) seventy-two hours after inoculation with virus. The contro.l animal developed the disease on the ninth day; the three treas animals on the seventh, eighth, and ninth days. No difference in the extent of the paralysis of the control and the serum-treated animals was noted.

Experiment 6.--Three monkeys were inoculated with virus by the intranasal route (Dec. 14, 1932). Twenty-four hours later two of the monkeys (878, 879) were injected subcutaneously with 20 e.c. each of immune horse serum (May 3, 1932, bleedingl~). One of the animals (879) developed extensive poliomyelitis ten days later and lived for twelve days thereafter; the other (878) remained well. The control (869) developed moderately extensive paralysis on the fifteenth day. Experiment 7.--Six monkeys were inoculated with virus by the intranasal route (Jan. 10, 1933). Two monkeys served as controls (900, 901). Each of the remaining animals (902, 903, 904, 90,5) were given three injections of 20 e.c: of immune horse serum (1Kay 3, 1932, bleeding 15) subcutaneously two, four, and six days after the intranasal instillations of virus, each monkey receiving a total of 60 c.c. of serum. All animals in the series developed the disease eight days after inoculation with virus, and no important difference was noted in the extent of the paralysis developed by the serum-treated and the control animals. Experiment 8.--Convalescent monkey serum (pooled serum from eleven monkeys) was used in this experiment. Six monkeys were inoculated with virus by the intranasal route (Jan. 23, 1933). Two monkeys (914, 915) served as controls while the remainder received immune serum by the subcutaneous route. Two o f the latter (910, 911) received single injections of 20 c.c. of serum twenty-four hours after virus instillation. Both of these developed the disease six days later. Two monkeys (912, 913) received three injections of 20 c.c. each twenty-four, forty-eight, and seventy-two hours after inoculation with virus. These developed the disease on the tenth and twelfth days, respectively. Both controls (914, 915) came down on the eighth day. The two (912, 913), which had received three injections of serum, were not only slower in developing the disease, but also suffered less extensive paralysis and died nine days (912) and fourteen days (913), respectively, after the remainder had succumbed (which was about three days after the appearance of symptoms). Experiment 9.--Three monkeys (988, 989, 990) received pooled monkey convalescent serum (from fourteen monkeys); three monkeys (991, 992, 993) received immune horse serum (July 7, 1933, bleedi n g ~ ) ; and three monkeys (985, 986, 987) served as controls. The serum-treated animals received 15 c.c. of serum by the intracardial

SCHULTZ AND GEBHARDT:

I M M U N E SERUM I N POLIOMYELITIS

619

route twenty-four, forty-eight, and seventy-two hours after intranasal inoculation with virus (July 12, 1933), each monkey receiving a total of 45 c.c. of serum. All of the serum-treated animals developed poliomyelitis eight days after inoculation with virus and presented extensive paralysis within two days after the onset of clinical symptoms. One of the controls (985) remained well; two (986, 987) developed poliomyelitis on the seventh day.

Experiment 10.--Eight monkeys were given the usual three intranasal instillations of virus on May 13, 1934. Twenty-four hours after the last instillation four of the monkeys were injected intramuscularly with immune horse serum (pooled bleedings. J u l y 7, ]933 and Jan. 29, 1934 ~5) in doses of 2 c.c. per kilogram of body weight. These injections were repeated approximately 24, 48, 72, 96, 120, and 144 hours after the intranasal instillations of virus. The results of this experiment are given in Table I. TABLE

MONKEY

B - 97 B - 98 B - 99 B-100 B-]Ol B-102 B-103

~VEIGIIT IN KG.

2.8 3.0 3.0 3.1

I

AMOUNT 0t~ TOTAL SE~U1Vs PEP~ AMOUNT OlV DOSE, IN- C'.C. SERUM IN" 0.0. 5.6 6.0 6.0 6.2 nolle ]loll@ ]lo]le

33.6 36.0 36.0 37.2 ]lone ]lo]le ]lone

RESULTS Poliomyelitis Poliomyelitis Po]iorayelitis Poliomyelitis Poliomyelitis Poliomyelitis Poliomyelitis

on 8 t h on 9 t h on 9 t h on 8 t h on 7 t h on l O t h on 7 t h

day day day day day day day

In view of the fact that the serum employed in this experiment was definitely known from previous tests to possess an exceptionally high virucidal titer and was administered in repeated and in relatively large doses, the first dose having been administered twenty-four hours after inoculation of the animals with virus; it would appear that the results of this experiment should be regarded as highly significant. Not only did the serum-treated animals develop the disease after approximately the same period of incubation as the controls, but little, if any, difference was observed in the extent of the paralysis presented by the two groups--treated and untreated. SUMMARY

The results of ten sets of experiments designed to test the therapeutic value os i m m u n e serum in experimental poliomyelitis are summarized in Table II. It will be noted that the observations are surprisingly uniform, despite the fact that the serum w a s administered in v a r y i n g quantities by different routes and at v a r y i n g intervals of time after inoculation of the animals w i t h virus. While several of the

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earlier e x p e r i m e n t s (:l, 2, 3) cannot be r e g a r d e d as crucial, l a t e r ones, in which the v i r u s was inoculated by the i n t r a n a s a l route, seem to leave little to s u p p o r t the possibiiity t h a t immune serum does a p p r e ciably m o d i f y the eonrse of the disease once the virus has become imp l a n t e d in the n e r v o u s tissue. The results o f E x p e r i m e n t 8 suggest t h a t the pooled m o n k e y convalescent s e r u m m a y possibly ha~e exercised some t h e r a p e u t i c effect. It, however% is also quite possible t h a t the difference n o t e d between the t r e a t e d and control animals r e s t s on some f a c t o r o t h e r t h a n on the use of serum. I t seems p a r t i c u l a r l y n o t e w o r t h y t h a t the a d m i n i s t r a t i o n of serum as e a r l y as t w e n t y - f o u r hours a f t e r inoculation of the animals w i t h virus, w i t h one exception only, failed to p r e v e n t or m o d i f y in a n y significant degree the course of the disease. A n e x p l a n a t i o n of the inability of immune s e r u m to p r e v e n t or m o d i f y the p r o p a g a t i o n of the virus, once it is established in the host, is p r e s e n t e d in the following discussion. DISCUSSION

Recent studies on poliomyelitis indicate t h a t f r o m the v e r y b e g i n n i n g of tile infection the virus is intimately associated with neurons and t h a t once it is established in the nervous, system it is p r o p a g a t e d largely, if not entirely, along axonal routes. The evidence for' this is varied. I n 1930 F a i r b r o t h e r and H u r s t , 22 for example, r e p o r t e d experiments in which a f t e r inoculation of virus into the brain or oil the nasal mucosa the infection a p p a r e n t l y advanced along nerve p a t h s f r o m the eerebral cortex to the thalamus, f r o m t h e r e to the medulla, f r o m whence it spreads with almost explosive suddenness to the spinal cord. T h e y noted, moreover, t h a t nerve, eell d e g e n e r a tion not only preceded but was. often quite independent of inflammat o r y reactions. V i r u s was d e m o n s t r a b l e in the spinal fluid on r a r e oeeas.ions only. g u n g e b l u t and S p r i n g 2a l a t e r in the same y e a r rep o r t e d the results of an e x p e r i m e n t in which the cord of a m o n k e y was t r a n s e a t e d at the level of the first l u m b a r v e r t e b r a p r i o r to an i n t r a c r a n i a l inoculation with virus. Nine days following inoculation with vir~s, t y p i c a l lesions of poliomyelitis were f o u n d in the cervical a n d thoracic cord while the l u m b a r cord, which is ordinarily most m a r k e d l y i n v o l v e d in the e x p e r i m e n t a l disease, presented a n o r m a l appearance. F a b e r and G e b h a r d t 24 h a v e r e c e n t l y f u r t h e r established the axonal route of dissemination. On s a m p l i n g different p a r t s of the central n e r v o u s system, t h e y f o u n d t h a t for' periods of less t h a n f o u r days following i n t r a n a s a l inoculation the virus is d e m o n s t r a b l e in the o l f a c t o r y bulb b u t not elsewhere. B y the fifth and sixth days it h a d spread, a p p a r e n t l y entirely b y the o l f a c t o r y t r a c t s and t h e i r communications to the h y p o t h a l a m u s where a s e c o n d a r y focus was seemingly established ; f r o m whence it s p r e a d to the m e d u l l a and to the t h a l a m u s

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and midbrain. It was first detected in the spinal cord on the seventh d a y and was present at this time in l a r g e r concentrations in the eervical t h a n in the l u m b a r segments. Certain portions of the central nervous system (e.g., cortex, cerebellum) were n e v e r f o u n d to contain demonstrable quantities of virus, and some evidence was obtained t h a t it m a y largely or entirely d i s a p p e a r in areas once. involved. In an excellent review of this general subject F:aber 25 pointed out that clinical observations on man h a r m o n i z e well with the experimental observations which indicate t h a t the infection is largely limited to individual groups of neurons in the central nervous system. F u r t h e r evidence ;hat the virus f r o m the time of the initial invasion propagates itself in v e r y intimate association with nerve cells is affm'ded b y observations r e c e n t l y a n d simultaneously r e p o r t e d in 1934 b y Schultz and Gebhardt ~6 and Brodie and Elvidge. ~7 This evidence rests on an easily demonstrable f a c t t h a t a f t e r the o l f a c t o r y tracts have been cut it is no longer possible to infect monkeys with poliomyelitis virus administered b y the intranasal route. This simple, exp e r i m e n t seems to p r o v e clearly t h a t from the v e r y beginning of the infection the virus is v e r y i n t i m a t e l y associated with nerve cells, the initial portal of e n t r y being possibly the terminal hairs of the o l f a c t o r y nerve. A d d e d to the evidence a l r e a d y p r e s e n t e d is the fact t h a t degenerative changes in neurons precede inflammatory reactions in the n e a r b y tissues; furthermore, t h a t i n t r a n u c l e a r inclusions and oxychromatic degeneration of the nucleus m a y be demonstrated in at least a few of the p y r a m i d a l cells e a r l y in the disease (Covell, 2s tturst, ~ Schultz~~ Finally, the results of ultrafiltration studies, w h i c h point to virus bodies of a magnitude less than 50 mu. (Clifton, Schultz, and GebhardP~), harmonize wit]~ the evidence which points to an i~traceIlular infection of individual groups of neurons r a t h e r than to a more generalized intercellular infection. In the light of this knowledge it is easy to u n d e r s t a n d the results obtained in the therapeutic studies here reported. I f it is correct that in poliomyelitis the virus is confined largely to neurons and is freed from the infected cells only when t h e y are caused to disintegrate, then it is a p p a r e n t enough w h y a ~omplex proteinaceous agent such as. immune serum, which although it m a y bathe the e x t e r i o r of the cell does not reach the virus established within it. This m a y also explain the results of serum t h e r a p y in other filtrable virus diseases--diseases in which a state of intracelIular r a t h e r than intercellular parasitism exists. W h e t h e r or not an immune serum m a y i n t e r r u p t the passage of poliomyelitis virus from one n e u r o n to a n o t h e r at their synapses, as has been suggested by Faber, 2~ is a possibility which will command consideration in the presence of a n y proof that serum is actually of

SCHULTZ AND GEBHARDT: 1MNUNE SERUM IN POLIOMYELITIS 623 some t h e r a p e u t i c

value.

Our results

do n o t i n d i c a t e t h a t

immune

s e r u m i m p e d e s t h e p r o g r e s s of t h e v i r u s a t a n y p o i n t a l o n g i t s n o r m a l path.

I t is, o f c o u r s e , p o s s i b l e t h a t in t h e n a t u r a l d i s e a s e in m a n , in

w h i c h a s m a l l e r q u a n t i t y of v i r u s m a y be i n v o l v e d a n d in w h i c h t h e d i s e a s e t e n d s to p r o g r e s s m o r e s l o w l y , s o m e b l o c k i n g a c t i o n m a y b e e x e r c i s e d b y s e r u m at t h e s y n a p s i s of n e u r o n s .

H o w e v e r , the r e s u l t s

of r e c e n t c l i n i c a l s t u d i e s , in w h i c h t r e a t e d a n d u n t r e a t e d cases h a v e b e e n c a r e f u l l y c o m p a r e d , do n o t s u p p o r t t h e p o s s i b i l i t y t h a t a signific a n t d i f f e r e n c e e x i s t s b e t w e e n t h e r e s u l t s of s e r u m t r e a t m e n t in t h e n a t u r a l a n d t h e e x p e r i m e n t a l d i sease ( P a r k , 82 L a n d o n ~ ) . CONCLUSIONS S p e c i f i c i m m u n e s e r u m is w i t h o u t d e m o n s t r a b l e v a l u e in t h e t r e a t m e n t of e x p e r i m e n t a l p o l i o m y e l i t i s . REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. rl0. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

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