Immunization against yellow fever

TRANSACTIONS OF THE

ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. VOL. XXVII.

No. 5.

MARCH.

Proceedings of an Ordinary Meeting ot the Society, held at Manson House, 26, Portland Place, London, W.1, at 8.15 p.m., on Thursday, 18th January, 1934. Major-General SIR LEONARD ROGERS, K.C.S.I., M.D., F.R.S., President, in the Chair.

PAPER. I M M U N I Z A T I O N A G A I N S T YELLOW FEVER. BY

G. M. FINDLA¥. Wellcome Bureau of Scientific Research, London.

Introduction. Neurotropic Yellow Fever Virus. Immunization with Neurotropic Virus. The Preparation of Virus and Immune Serum.

Inoculations against Yellow Fever in Man. Reactions to Inoculation. Development and Duration of Immune Bodies. Circulation of Virus in the Blood. Discussion.

INTRODUCTION. Five centuries ago improvements in the science of navigation and in the art of building ships firstmade it possible for sailorsto put forth upon uncharted seas and thus to discover first the West Coast of Africa and then the N e w World (cf.PRESAGE, 1933)' Only a few years later,and certainlyas early as 1503, B

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Tropical America and West Africa were already firmly linked together by the slave trade, while shortly afterwards accounts began to appear of epidemics of a disease in many respects resembling yellow fever. Wherever the place of origin of yellow fever, and, as CANTER (1931) points out, the biological and historical evidence is entirely consistent with an African origin, it is certain that it was the slave trade which carried yellow fever backwards and forwards between Africa and America till each accused the other, and with truth, of being the immediate source of its infection. T o d a y the application of the internal combustion engine to transport on land and in the air has so increased the rapidity of travel that a further extension of yellow fever is within the bounds of possibility, since any country can now be infected if it already harbours the mosquito vector of yellow fever and can be reached by a traveller from an endemic focus within a period of six or possibly even ten days. Some method not merel~ of controlling but of eradicating yellow fever is thus urgently necessary. Such eradication can be brought about only by destruction of either the vector or the virus of the disease. Unfortunately the vast areas in Africa and in South America now known to be infected with yellow fever render efforts to annihilate the mosquito vector a practical impossibility, more especially as recent work has shown that in addition to A~des ~egypti, other mosquitoes may possibly play a part in the genesis of certain epidemics, as in that recently recorded in the Chanaan Valley, State of Espirito Santo, Brazil (SoPER, PENNA, CASDOSO, S~AFIM, FROBISHER and PINHEIRO, 1933). If, on the other hand, a successful method of immunization against yellow fever could be introduced then, in the absence either of any other known mammalian reservoir of the virus, or of transmission of virus either directly from mosquito to mosquito or through the eggs, it would be sufficient to maintain immunity in the h u m a n population for a period only slightly exceeding the m a x i m u m life span of female tildes cegypti: yellow fever would thus be automatically eliminated. It is of interest to note that the first suggestion of the possibility of immunizing against yellow fever was made by CARLOSJUAN FINLAY(1894), while as early as 1903, MARCHOUX, SALIMBENI and SIMMOND (1903), had produced immunity in a volunteer by means of human serum from a convalescent case of yellow fever. No serious advances in immunization against yellow fever were possible, however, till STOKES,BAtma and HUDSON (1928), had shown that rhesus monkeys were susceptible to yellow fever, a discovery which first clearly demonstrated that the cause of yellow fever is an ultramicroscopic virus. In common with many other virus diseases, the following methods of immunization then suggested themselves: (I) Immune serum. (II) Virus, killed or attenuated by physical or chemical agents. (III) Virus and immune serum. (IV) Virus modified biologically. The use of immune serum, even in considerable doses, was soon shown to be of little use owing to the short duration of the resultant passive immunity. BAUER(1931), for instance, showed that monkeys inoculated with considerable doses of immune serum had lost their passive immunity within 14 days. HINDLE(1928) was the first to prepare a vaccine by the action either of phenol or formol on an emulsion of liver obtained from rhesus monkeys dead of yellow fever. Such a

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vaccine, while the virus is still alive, may be capable of producing immunity without obvious signs of disease. Vaccines of similar type were prepared also by PETTIT and STm~ANOPOULO (1928), BEAUREPAIRE-ARAGXO(1928) and MONTEmO (1930): the results of vaccinating more than 25,000 persons in Brazil during the epidemic in Rio de Janeiro were, however, irregular (CHAGAS, 1931). DAVIS(1930), employed a chloroformed vaccine which in laboratory tests either infected monkeys or failed to immunize. Apart from difficulties in preparing such vaccines, their antigenic capacity, as pointed out by OKELL (1930), is usually quickly lost, probably as a result of death of the virus. Thus BURKE and DAvis (1930), record the death of a person in Brazil from yellow fever 5 months after vaccination with monkeys' liver treated with formalin and phenol. In addition, so long as the vaccine contains living virus there is always a possibility that virus may appear in the peripheral circulation, an occurrence that may lead to subsequent infection of the mosquito vector. In 1928, PETTIT,STEFANOPOULOand FRASE¥(1928), showed that anti-yellow fever serum protects rhesus monkeys against subsequent injection of virus when given up to as long as 3 days after injection of the virus. Such monkeys were still resistant 14 days after inoculation to a second injection of living virus. THEILERand SELLAaDS(1928), also obtained immunity by the use of serum and virus for as long as 2½ months after inoculation. These results were confirmed by ARAGJtO(1931), and by FINDL^Y and HINDLE(1931), who found that monkeys vaccinated with virus and immune serum were still immune 14 months later. No serious attempts to vaccinate human beings by the combined use of serum and virus were, however, made till after the discovery of a biologically-modified strain of yellow fever virus ; while in March, 1931, a Commission of the French Academy of Medicine concluded that no method of vaccination against yellow fever had been sufficiently studied for use in practice (PETTIT, 1931). NEUROTROPIC YELLOW FEVER VIRUS. In 1930 MAX THEILER made the important discovery that yellow fever can be transmitted to mice b y intracerebral inoculation. Mice thus inoculated did not develop viscerotropic lesions similar to those f o u n d in rhesus monkeys and man, but died from an encephalitis. Intraperitoneal inoculation was only fatal in young mice in which the virus rapidly became localized in the central and peripheral nervous systems and in the adrenal glands. After serial passage the virus lost its power to produce viscerotropic lesions in monkeys, although it could still be neutralized b y i m m u n e yellow fever serum. T h e s e results have since been amply confirmed. In addition to mice it has been found that all species of monkey so far tested develop an encephalitis after intracerebral inoculation (LLOYD and PENNA, 1933) while some other species of rodents, such as the guineapig, agouti (Dasyprocta agutO, field vole (Microtus agrestis) and red squirrel (Sciurus vulgaris), are also susceptible to the neurotropic virus. Rats, rabbits, wood voles, bank voles and golden hamsters fail to develop encephalitis after intracerebral inoculation as also do cats, ferrets, pigeons, hens and canaries (FINDLAY,1934). 8AWYER and LLOYD (1931) showed that intraperitoneal inoculation of the neurotropic virus if accompanied by damage to the central nervous system, as b y the intracerebral injection of some foreign substance such as broth or starch, invariably leads to the development of yellow fever encephalitis--a finding which forms the basis of their test for detecting the presence of protective i m m u n e bodies in the sera of persons recovered from yellow fever.

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IMMUNIZATION AGAINST YELLOW FEVER.

Intracutaneous, subcutaneous or intraperitoneal inoculation of the neurotropic virus in animals on the other hand, u n a c c o m p a n i e d b y cerebral t r a u m a , m a y or m a y not be followed b y a febrile r e a c t i o n ; while the virus does not reach the central nervous system it m a y be present for a longer or shorter period in the peripheral blood stream. L a t e r there appear in the blood i m m u n e bodies which are capable of neutralising b o t h the neuro- and viscero-tropic strains of yellow fever virus. Inoculation into the sciatic nerve of m o n k e y s has also been shown b y LLOYD and PENNA (1933) to lead to the d e v e l o p m e n t of i m m u n e bodies in the blood, while THmLER (1930) has f o u n d that direct inoculation into the eye-ball of mice m a y lead to a fatal encephalitis. Intranasal instillation of the neurotropic virus m a y also lead to the developm e n t of encephalitis b o t h in mice and monkeys, as shown b y the following experiments : - A 20 per cent. suspension of mouse brain infected with neurotropic yellow fever virus (French strain--passage 202) in 1 in 10 serum saline was centrifuged at 3,000 R.P.M. for 10 minutes to throw down gross particles. The supematant liquid was taken up in a fine capillary pipette and one drop was instilled into both nostrils of twelve mice. Seven mice died with encephalitic symptoms 4, 5, 8, 8, 9, 9 and 10 days after inoculation. The histological changes in the brains were similar to those produced by intracerebral inoculation of yellow fever virus, while inoculation of a suspension of one of the brains into a further series of six mice caused death of all the mice with encephalitis on the fourth day after inoculation. After an interval of 3 weeks the five mice which had survived intranasal instillation were inoculated intraperitoneally with a suspension of infected mouse brain, while starch was inoculated intracerebrally. All the mice died with symptoms of encephalitis. Of a total of thirty-six mice" inoculated intranasally, seventeen have developed encephalitic symptoms. Possibly preliminary instillation of phosphate buffer at pH 5.0, as recommended by SCHULTZand G~HARDT (1933), in the case of poliomyelitis in monkeys, might possibly have increased the percentage of positive results. Three rhesus monkeys had 0.5 c.c. of a 20 per cent. suspension of infected mouse brain, prepared as before, instilled into the nostrils. Symptoms of encephalitis developed 8, 8 and 9 days later, with twitching of the facial muscles and general muscular weakness, most marked in the lower limbs. The monkeys were killed 24 hours later and the organs tested for the presence of virus by intracerebral inoculation into mice. Virus was invariably present in the brain, but absent from the liver, kidneys and adrenals. In two monkeys virus was also entirely absent from the blood : in the case of the third monkey one out of six mice inoculated with blood died from encephalitis 6 days after inoculation. In two cases virus was demonstrated in the sciatic nerve. N e u r o t r o p i c yellow fever virus is thus capable of producing infection after intranasal inoculation, behaving in this respect in the same way as the viruses o f anterior poliomyelitis, louping ill, vaccinia and Rift Valley fever. Preliminary observations suggest that as in the case of poliomyelitis, neurotropic yellow fever virus reaches the central nervous system b y the axis cylinders of the olfactory nerves rather t h a n b y the perineural lymphatics and the subarachnoid space. I t will be r e m e m b e r e d that the olfactory nerve terminations lie exposed in the nasal mucosa so that a direct contact between virus and nervous tissue is here possible. So far as their pathogenicity to animals is concerned the neurotropic and viscerotropic viruses thus differ greatly. I n their immunological properties

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and in certain of their physical properties they are more akin. Thus, as shown by FROBISHER (1933), the resistance to heat is similar while the size, 18 to 27 #/~, is approximately the same in both strains (FINDLAY and BROOM, 1933). The question whether the neurotropic strain of yellow fever can ever be reconverted into the viscerotropic strain is obviously one of great practical and theoretical importance. Although viscerotropic lesions may be produced in monkeys during the earlier stages of the adaptation of the virus to the nervous tissue of mice, after the 100th passage in mouse brain such a reconversion has, up to the present, proved impossible so far as monkeys and mice are concerned. Thus LLOYDand PENNA(1933) state that many and various attempts to cause mouse-brain-adapted yellow fever virus of the French strain, after the 146th passage, to reacquire the general viscerotropic properties of the natural yellow virus in Macacus rhesus have met with failure regardless of the site of inoculation employed, the time of the subinoculation of the virus, the kind of tissue used as a virus-containing inoculum and the amount of virus injected. Repeated passage of the neurotropic virus in mice is also unaccompanied by any tendency to produce viscerotropic lesions in any way similar to those produced by the ordinary strain of yellow fever virus in rhesus monkeys or man. In fact repeated passage in the brains of mice has apparently been attended with increased neurotropism for, as LLOYD, PENNA and MAHAFFY (1933) point out, at the 250th passage a considerable proportion of mice are dead 72 hours after intracerebral inoculation, while in the earlier passages most of the mice died 7 to 8 days after inoculation. The length of the incubation period diminished more rapidly in the earlier passages than in the later. In these laboratories the French strain of neurotropic yellow fever virus has now been carried to its 420th passage. The majority of the mice still die on the third or fourth day after inoculation but in addition there is an occasional tendency for adult mice to develop encephalitis after intraperitoneal inoculation unaccompanied by any form of cerebral trauma. DAVIS, LLOYD and FROBISHER (1932) have also noted that occasionally in monkeys the virus may localize in the brain in the absence of any known trauma. Other factors such as anoxmmia which may apparently have an influence in localizing the virus in the central nervous system will be discussed in a forthcoming paper. After more than one hundred passages in tissue cultures of chick embryo cells the neurotropic virus still produced encephalitis in mice (HAAGEN,1932) while, after passage through A~des aegypti, DAVIS, LLOYD and FRO/3ISHER(1932) could still cause encephalitis in monkeys and mice, no marked lesions being produced in liver or kidneys. In guineapigs dying with encephalitis following intracerebral inoculation of the neurotropic yellow fever virus, specific viscerotropic lesions are also absent while the virus is found only in the central nervous system and adrenals. As noted by LLOYD, PENNA and MAHAFFY(1933) and FINDLAY (1934), a con-

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IMMUNIZATIONAGAINSTYELLOW FEVER.

siderable degree of fatty infiltration of the liver may, however, occasionally be encountered t h o u g h actual necrosis of the liver p a r e n c h y m a , infiltration with m o n o n u c l e a r cells or p o l y m o r p h o n u c l e a r leucocytes and acidophilic intranuclear inclusions are not seen. Field voles and red squirrels dying f r o m yellow fever encephalitis also fail to exhibit specific lesions in the abdominal organs. So far, therefore, as monkeys and rodents are concerned there is no evidence in favour of any return of viscerotropic activity on the part of the neurotropic strain of yellow fever virus. Recently, however, experiments have been carried out on the susceptibility of the hedgehog, Erinaceus europceus, to neurotropic yellow fever virus. I n this animal species intracerebral inoculation of the neurotropic virus m a y produce necrotic changes in the liver. T h e s e experiments are being continued. IMMUNIZATION WITH NEUROTROPIC VIRUS. T h e ideal m e t h o d of i m m u n i z a t i o n against yellow fever should c o n f o r m to the following conditions : - (I) An active i m m u n i t y of long duration should be produced. ( I I ) N o severe reaction should follow and there should be no risk of any serious sequela. ( I I I ) T h e possibility of virus circulating in the peripheral blood s t r e a m should be reduced to a m i n i m u m . (IV) T h e m e t h o d should be easily applicable to a large n u m b e r of persons. I f these conditions are to be fulfilled it is obvious that the neurotropic strain of yellow fever is far m o r e suitable than the viscerotropic for purposes of immunization. In 1931, SAWY-~, KITCHEN and LLOYD published a preliminary account of the immunization of ten persons with a mixture of neurotropic yellow fever virus and immune serum, while in 1932, they gave a more complete account of the immunization of fifteen persons. After preliminary experiments in monkeys, fifteen persons were immunized by a single injection of a dried mixture of living yellow fever virus, fixed for mice, and human immune serum, with separate injections of enough additional serum to prevent the circulation of virus in the peripheral blood stream. One person was similarly immunized by injecting immune serum and dried virus separately. These experiments involved the injection of from 0.0008 to 0.003 gramme of virus-containing brain tissue per 161ogram of body weight and from 0.3 to 0.66 c.c. of immune serum per kilogram of body weight for an adult male, or from 35 to 45 c.c. of immune serum. S~LLARDS and LAXOR~r (1932), dispensed entirely with the use of immune serum, vaccinating six persons with dilutions of mouse fixed living virus. Reactions were slight or entirely insignificant. In seven other cases subsequently vaccinated in this manner, there were in three cases severe reactions after incubation periods of from 13 to 15 days (LAIGRET, 1933). In one, there was fever of 4 days duration with slight albuminuria, in the others the illness lasted for from 8 to 10 days, with albuminuria, icterus, well marked nervous symptoms and hsemorrhagie sputum. Later LAICRET (1933), made use of three injections of virus, the first injection of supposedly non-virulent material, the second injection of virus lethal to mice in doses of from 0.01 to 0.1 c.c. but not in higher dilutions, the third lethal to mice in doses of from 0.001 to 0.01 c.c. The infected mouse brains were attenuated by preservation in glycerine at room temperature for from

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FINDLAY.

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1 to 4 days and then either dried or kept in the refrigerator at approximately -15 ° C., when the virus retains its activity for prolonged periods. The three injections were given at intervals of 20 days. A~cXo (1933), in Brazil, has also made a small number of experiments on five human subjects, of whom two were inoculated subcutaneously with a mixture of 4 c.cm. of human immune yellow fever serum and 0-001 gran'mae of fresh virus-containing mouse brain. Both subjects experienced a fairly definite febrile reaction a week later and in the blood o f o n e of them virus was shown to be present by the mouse test. Three more human subjects w e r e given a preliminary dose of 3 c.cm. of immune serum followed 6 hours later by the serum-virus mixture. The subsequent reactions were less severe and virus could not be demonstrated in the blood. The sera of all the five patients were subsequently shown by tests on monkeys to contain protective antibodies against the viscerotropic strain of yellow fever. While the use of suitably-graded doses of neurotropic yellow fever virus and human yellow fever immune serum thus seems to be the most suitable method of immunization against yellow fever, there is a real practical difficulty in obtaining large quantifies of high titre human yellow fever immune serum when a single inoculation alone requires from 35 tO 45 C.C. PETTITand STEFANOPOULO(1933) have, therefore, proposed the use of horse o r baboon anti-yellow fever serum. Such sera are as a rule of a higher titre than human yellow fever immune serum and may thus be injected in correspondingly smaller doses. M y own work on immunization against yellow fever began in N o v e m b e r , 1932, after returning f r o m New York, whither, at the invitation of the International Health Division, Rockefeller Foundation, to the British Colonial Office, I had proceeded to study the mode of preparation of the yellow fever vaccine and its practical application. I should like to take this opportunity of expressing m y gratitude, not only to the Medical Authorities of the Colonial Office for extending to me the o p p o r t u n i t y of going to New York, but also to Dr. F. F. RUSSELL, Director of the International Health Division, Rockefeller Foundation, Dr. W. A. SAWYER, Director of the Yellow Fever Laboratories, New York, and all their staff who r e n d e r e d m y visit as profitable as it was pleasant. Preliminary accounts of the immunization experiments undertaken in this c o u n t r y have already appeared (FINDLAY, 1933 and 1934). I n the present communication an account is given of the results obtained up to 31st D e c e m b e r , 1933, in the immunization of 200 persons against yellow fever b y the c o m b i n e d use of h u m a n i m m u n e yellow fever serum and mouse fixed virus. THE PREPARATION OF VIRUS AND IMMUNE SERUM FOR IMMUNIZATION. T h e technique employed in preparing the virus and i m m u n e serum is similar to that of SAWYER, KITCHEN and LLOYD (1932) and need not, therefore, be described in detail. Nevertheless, there are certain points of importance which require emphasis. Virus-containing cornponent.--For the preparation of the virus it is essential to employ a strain of mice all of which are completely susceptible to the virus while the danger of all intercurrent infections, more especially those due to ultramicroscopic viruses, must be carefully guarded against.

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IMMUNIZATIONAGAINSTYELLOWFEVER.

The mice used for the preparation of virus in these laboratories are bred in the country and, on arrival, are carefully isolated from all other strains of mice. They are kept under observation for at least a fortnight before being used in the preparation of virus and from time to time brains are removed from apparently healthy mice. Some of the brains are submitted to histological examination, others are emulsified in serum saline and are injected intracerebrally into mice of another strain. That such precautions are not too great is shown by the fact that recently, in the brains of mice of another strain used in these laboratories, there has been found by my colleague, Dr. R. D. MACKENZIEand myself a pathogenic organism with some similarities to a Rickettsia. Possibly in the future it may be possible to employ virus grown in tissue cultures of chick embryo in place of infected mouse brains. For the preparation of virus the French strain of neurotropic virus has invariably been used after from 110 to 120 passages in mouse brains. It would seem to be undesirable to employ virus after about the 150th passage in mice, not only because of the increased neurotropism previously described but also in view of the fact that in the case of other viruses, e.g. anterior poliomyelitis, repeated animal passage may cause antigenic modification with the result that the passage strain is incapable of protecting completely against a freshly isolated strain (cf. BUaNETT and MACNAM~, 1931). In preparing the vaccine~ mice are inoculated intracerebrally with 0.03 c.c. of a 20 per cent. suspension of infected mouse brain in 1 in 10 serum saline. They are killed 4 days after inoculation whether or not symptoms of encephalitis are present. The brains are removed aseptically, weighed and ground up in normal human serum to form a 1 in 10 suspension. The normal human serum should be Wassermann negative and must have been tested for sterility after filtration through a Seitz filter. The suspension of mouse brain is lightly centrifuged at 3,000 R.P.M. for 10 minutes to throw down gross particles, the supernatant liquid is pipetted off and passed through a Seitz filter. Both before and after filtration aerobic and anaerobic cultures of the mouse brain suspension are made in glucose serum broth. After filtration the virus serum mixture is placed in 0.5 c.c. amounts in small glass ampoules lightly plugged with absorbent wool. The ampoules are then placed in a dish containing absolute alcohol to which is added solid carbon dioxide.* In from 3 to 4 minutes the serum in the ampoules is completely frozen. The ampoules are then rapidly transferred to a previously prepared Hempel " improved " desiccator which is exhausted by motor pump and replaced in the ice chest. Complete desiccation occurs after from 36 to 48 hours. The ampoules are then sealed in the blast lamp and stored in the ice-chest. Before use each batch of vaccine is tested for sterility under aerobic and *A simple and inexpensive apparatus for making solid COs may be obtained from Sparklers, Ltd., 93, Regent Street, London, W.1.

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anaerobic conditions and is titrated out for virus-content by redissolving the dried serum-virus mixture in 0"5 c.c. of sterile distilled water. Dilutions are then inoculated intracerebrally into mice. The minimum lethal dose for mice is generally found to be 0.03 c.c. of a dilution 10-8 or 10"r. Virus kept at a temperature of -]- 4 ° C. for a year has shown only very slight diminution in titre. The Hempel " improved " desiccator is prepared some 2 to 3 hours before it is actually required. Anhydrous sulphuric acid is poured into the upper compartment, while in the bottom is placed a light metal container holding at least 20 ampoules. In order to ensure complete contact between the bottom of the desiccator and the ampoules a thin layer of anhydrous glycerine may be poured into the bottom of the desiccator. The prepared desiccator is then put into a wooden bucket and, completely surrounded by freezing mixture, is kept in the ice chest till required. Preparation of immune serum.--Serum is obtained from Wassermann negative donors who have previously suffered from yellow fever. The immune body titre of each donor is tested at intervals and if any marked diminution in titre is noted mouse brain virus is injected subcutaneously. This usually has the effect of raising the titre. Blood is removed from a vein in the arm and collected in the flask devised by OKELL and HART (1932). These flasks have the advantage of dispensing with all rubber tubing and can easily be manipulated by one person. The blood is allowed to clot in the flask and the serum decanted off. The serum is then passed through a Seitz filter and 0.2 c.c. of ether and 0.2 c.c. of tricresol are added to each 100 c.c. of serum. Aerobic and anaerobic cultures are made before and after filtration. The serum is stored in convenient amounts in sealed glass ampoules in the ice chest. LLOYD and PENNA (1933) have found that yellow fever immune serum frozen and dried in vacuo may be kept for long periods without appreciable loss of immune body titre. Before being employed in vaccinations sera are tested by the method of SAWYER and LLOYD (1931) for their protective power. Satisfactory sera should give protection in dilutions of from 1 in 256 to 1 in 1,024. INOCULATIONS AGAINST YELLOW FEVER IN MAN.

Of the 200 persons immunized up to the end of December, 1933, 152 were men, 48 women. With the exception of one Indian and two negroes all were of European descent. The age of the persons immunized varied from 16 to 63 years. Those immunized included civil and military officials, medical officers and nursing sisters of the West African Medical Service, missionaries, traders and the wives of officials proceeding to the West Coast as well as laboratory workers: fifty-one had never visited West Africa, while one hundred and forty-nine had lived there for longer or shorter periods.

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IMMUNIZATION AGAINST YELLOW FEVER.

The persons immunized may be divided into three groups, according to the relative amounts of immune serum and virus injected : Group I--forty persons were inoculated subcutaneously in the abdomen with from 0.3 to 0.S c.c. of human immune yellow fever serum per kilogram of body weight followed 4 to 6 hours later by from 0-25 to 0.S c.c. of 10 per cent. virus injected subcutaneously in the arm. Group I I - - o n e hundred and ten persons were inoculated intradermally in the abdomen with 1 c.c. of human immune yellow fever serum followed 2 hours later 'by from 0.1 to 0.5 c.c. of 10 per cent. virus injected into the same area. Group III--fifty persons were inoculated intradermally with 1 c.c. of human immune yellow fever serum followed 2 hours later by the intradermal injection of from 0-00625 to 0.05 c.c. of 10 per cent. virus together with the subcutaneous injection of 5 c.c. of human immune yellow fever serum. REACTIONS TO INOCULATION.

Group I. The method of inoculation applied to this group was that latterly used by SAWY~, KITCHEN and LLOYD (1932). Of the forty persons thus immunized, of whom thirty-two were men and eight women, fifteen had comparatively slight reactions unaccompanied by fever, twenty-five reacted with a rise in temperature above 99 ° F. One of the persons who had no reaction beyond the slight local tenderness inseparable from the injections had suffered seven months previously while resident on the Gold Coast, from an attack of jaundice, which, though not diagnosed at the time as yellow fever, had nevertheless been of sufficient severity to necessitate his remaining in bed for 10 days. A test of his blood serum, removed before immunization, showed that immune bodies were present in a titre of 1 in 256. With one exception the reactions were comparatively mild. In three cases there was a feeling of faintness coming on immediately after the injections of serum, while in almost every case there was tenderness in the abdominal wall for some hours at the sites of the serum !njections. In one instance 50 c.c. of immune serum were required in order to give 0.5 c.c. per kilogram of body weight. As a rule the abdominal tenderness had greatly diminished after 24 hours though in one instance the pain at the sites of injection persisted for as long as 4 days. In no case was there pain, redness or any form of local reaction at the site of injection of the virus component. Of the fourteen non-immune persons who exhibited no appreciable rise of temperature, four made no complaint of any unusual subjective feelings, while the other ten had vague aching in the bones and a feeling of tiredness or lethargy

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which came on about 30 hours after inoculation of the virus and continued for from 24 to 48 hours. One subject complained of very persistent constipation for 3 days following the inoculations. Of the 25 persons whose temperature rose above 99 ° F. the reaction in all but three instances began on the day after the inoculations, usually in from 25 to 30 hours after injection of the virus c o m p o n e n t . I n the exceptions there was no rise of temperature till f r o m 48 to 54 hours after injection. I n the majority of cases the reaction began rather suddenly, sometimes with a definite rigor. I n eight fever lasted only for 24 hours ; in the six others for 48 hours or more. All persons who had had a febrile attack complained that for a day or two after the cessation of fever they felt a little lethargic with a tendency to pain in the extrinsic muscles of the eyes. I n seven cases the temperature was a b o v e 102-0 ° F., the highest temperature recorded being 103.4 ° F. Apart from the fever the most c o m m o n s y m p t o m was headache of a frontal and post-orbital character, in some cases associated with photophobia. Other complaints were of aching in the muscles of the back and shoulders and in the calves of the legs. M a n y patients felt restless and one complained of general hypermsthesia. T h r e e complained of nausea, and two actually vomited, while isolated complaints were ear-ache and tenderness of all the superficial l y m p h glands. Bradycardia and some degree of leucopenia were also usually present. T h e two most severe cases in this group were : Case No. 2. J.C.B., doctor of medicine, engaged in medical research, aged 30. Weight, 64 kilogrammes. Patient had been for a short time in the tropics but had never suffered from malaria and was in good health at the time of the inoculation. 26th October, 1932, at 11 a.m., 82 c.c. (0.5 c.c. per kilogram of body weight) of human yellow fever immune serum were injected in three places under the skin of the abdomen. At 5 p.m. the virus component, dissolved in 0.5 c.c. of freshly distilled water, was injected subcutaneously into the right arm. Next day there was no complaint of ill-health till the evening when there was a slight feeling of chilliness and the temperature, taken in the mouth, was found to be 99.1 ° F. Patient passed a good night and the next morning (28th October, 1932), the temperature was only 97"1° F. He continued to work in the laboratory though feeling a little cold during the day. On returning home he became definitely unwell, began to shiver and about 8 p.m. had a severe rigor. The temperature was now 102.6 ° F. He was unable to eat any solid food for dinner and on going to bed spent an extremely restless night. Although there was no pain at the sites of the injections, there was very considerable tenderness in the bones as a result of which it was impossible to rest for long in any one position. There was also severe frontal headache, and pain in the extrinsic eye muscles. 29th October, 1932. At about 3 a.m. the patient vomited. When examined, some 7 hours later the temperature had fallen to 100"2° F., pulse 78. There was complaint of severe frontal headache, pains in all the bones and photophobia. The conjunctiwe were not congested, but the uvula and back of the pharynx were injected. The tongue was covered with a thick, whitish fur. In the evening the temperature was again 101.8° F., pulse 80, though the subjective symptoms had decreased in severity. The bowels were constipated. 30th October, 1932. Patient slept comfortably, sweating a good deal, the temperature in the morning was 101"1° F., pulse .78 ; the pains in the bones had decreased, but there was still a dull post-orbital ache, pain in the extrinsic muscles of the eyes and photophobia. The tongue was still furred and the back of the pharynx injected. There was no evidence of any skin rash, neither the spleen nor liver was enlarged or tender and the lungs and heart appeared normal. There was no head retraction but the reflexes all showed increased

448

IMMUNIZATION AGAINST YELLOW FEVER.

activity. T h e urine contained a trace of albumin and in the sediment there were red blood corpuscles and a few leucocytes. During the day the general condition improved. At 3 p.m., the temperature had fallen to 99"4 ° F., pulse 78, while at 9 p.m., it was only 98"8 ° F., pulse 74. T h e patient slept well and next morning (31st October, 1932) the headache had gone and the patient felt well though weak. There was still some photophobia however. This symptom continued for a further 5 or 6 days. Urine examined on 31st October, 1932, still contained a few red blood corpuscles, though by 5th November, 1982, these had disappeared. During the period of convalescence there occurred an outbreak of lingual herpes on the left side of the face, and during this period the deep reflexes were somewhat reduced. It was about 10 days from the time that the temperature became normal till convalescence was complete. It may be added that at the same time as J.C.B., there was also injected a second laboratory worker, A.E.G., who received some of the same immune serum and virus. This patient had only a slight reaction, the temperature rising to 100"2 ° F., 30 hours after the injection of the vaccine and returning to normal after 24 hours. T h e explanation of the symptoms in the case of J.C.B. is somewhat difficult. T h e absence of any urticarial rash, arthritis or a~dema does not suggest any sensitivity to the human serum, while the appearance of red blood corpuscles in the urine is not a symptom associated either with encephalitic syndromes or with ordinary yellow fever, though KLIGLm~ (1928) has noted the transitory occurrence of red cells in the urine in sandfly fever. On the other hand, the headache, pains in the body, photophobia and subsequent appearance of lingual herpes is suggestive of an infection. It may be added that yellow fever virus could not be obtained from the blood stream during the illness, though subsequently the serum gave a satisfactory immune body titre against yellow fever. Case No. 8. A.M.M., medical officer, West African Medical Service, aged 38. Weight, 79.5 kilograms. Patient was in good health at the time of inoculation. 7th December, 1932, at noon, 40 c.c. of human yellow fever immune serum were injected in four places under the skin of the abdomen. At 4.30 p.m., 0"25 c.c. of the virus component was injected subcutaneously into the left arm. Therg was no reaction on the day of inoculation or the following day. 9th December, 1932. About 10 p.m., patient began to feel unwell with slight general malaise : later post-orbital and occipital headache developed with shivering. Temperature 101-0°F. Patient felt very restless and could not get to sleep : therewas some sweating. 10th December, 1932. A t 3 a.m., the temperature was 103-4 ° F : by 8 a.m. it was 99 ° F. and by noon 98"4 ° F. T h e headache had disappeared and patient felt better although tired and listless for the next 24 hours. 19th December, 1932 (12 days after inoculation) patient felt out of sorts and rather nauseated, with lack of appetite. These symptoms continued for 3 days. There was no temperature but one or two roseolar spots were present on the trunk. A p a r t f r o m t h e r e m o t e s y m p t o m s d e s c r i b e d i n t h e a b o v e case t h e r e was also i n o n e case definite p a i n a n d t e n d e r n e s s i n t h e left a n k l e j o i n t 9 d a y s a f t e r i n o c u l a t i o n . T h e s y m p t o m s d i s a p p e a r e d a f t e r 48 h o u r s .

Group H. I n t h i s g r o u p an a t t e m p t was m a d e to t e s t t h e r e s u l t s o f r e d u c i n g t h e a m o u n t o f i m m u n e s e r u m . W i t h t h i s e n d i n v i e w o n l y 1 c.c. o f h u m a n i m m u n e y e l l o w f e v e r s e r u m ( i m m u n e b o d y t i t r e 1 i n 512 o r 1 i n 1,024) was i n j e c t e d i n t r a d e r m a l l y i n t o a n a r e a o f s k i n o n t h e a b d o m e n , 2 h o u r s l a t e r t h e s a m e a r e a o f s k i n was injected intradermally with the virus-containing component. While the amount o f s e r u m r e m a i n e d c o n s t a n t t h e d o s e o f t h e v i r u s was p r o g r e s s i v e l y r e d u c e d f r o m 0 . 5 c.c. t o 0.1 c.c. o f 10 p e r c e n t . v i r u s .

G. M. ~INDLAY.

449

Of the one h u n d r e d and ten persons thus immunized, eighty-three were men, twenty-seven women. Of these s i x t y - t h r e e - - f o r t y - t h r e e men and t w e n t y w o m e n - - h a d no significant rise of temperature. T h e majority of those who had no significant rise of temperature made no complaints, others, as in G r o u p I, mentioned slight headache, some feeling of lassitude and occasionally shivering often accompanied b y a subnormal temperature. T h e s e symptoms generally began about 30 hours after inoculation of the virus and continued for from 24 to 48 hours. Local reactions were extremely slight while the tenderness due to the large injections of serum was absent. T w o persons only complained of stiffness of the abdominal muscles near the site of the injection, while two others noted desquamation of the skin r o u n d the point of the injection. In one case in which fever occurred on the fifth day after inoculation there appeared at the same time a fine urticarial rash, limited to the area r o u n d the site of injection. T h i s rash faded after 5 days. In three instances, all women, the only complaint was of tenderness in the superficial inguinal glands on the same side as the injection. T h e tenderness began some 24 hours after the inoculation and lasted for f r o m 24 to 48 hours. T h e forty-seven cases with temperatures above 99 ° F. may be divided into three classes, according to the time of incidence of the reaction : (I) Reaction within 48 hours of inoculation of the v i r u s ; (II) Reaction within 48 hours followed b y a later exacerbation ; (III) Reaction delayed till four or more days after inoculation. Fifteen cases fall into the first class ; six into the second ; and twenty-six into the third. In the first class the highest temperature recorded was 102-6°F., and the fever had fallen at the end of 48 hours. T h e symptoms were as before, headache, backache and aching in the limbs. In three cases there were complaints of nausea. F o u r persons in this group remarked on the similarity of the attack to one of malaria from which they had all suffered. T h e histories of the second group are briefly as follows : Case No. 37. Male, schoolmaster. 2 years on Gold Coast : no previous illnesses ; 5th July, 1938, immunized by intradermal inoculation with 0.3 c.c. of 1 in 10 suspension of filtered mouse brain 2 hours after the intradermal inoculation of 1 c.c. of human yellow fever immune serum. 6th July, 1933, in the evening sweated heavily. Temperature, 99'6 ° F. 7th July, 1933, felt better; 8th July, 1933, travelled to Austria ; 10th July, 1933, typical malarial attack--parasites found in the blood film. 12th July, 1933. Temperarare normal. 31st July, 1933. Immunity test for yellow fever--protection 1 in 128. Case No. 39. Male, veterinary officer. 6th July, 1933, immunized by intradermal inoculation with 0.5 c.c. of 1 in 10 suspension of filtered mouse brain, 2 hours after the intradermal inoculation of 1 c.c. of human yellow fever immune serum. 7th July, 1933, 33 hours after inoculation of virus--Temperature, 99"6° F. 8th July, 1933, Temperature normal all day. 9th July, 1933, and 10th July, 1933, and l l t h July, 1933, very active during day. Temperature in the evenings, 99-0 ° F. to 99"4° F. 12th July, 1933, felt tired during da~. Temperature, morning, 99"2° F. ; evening, 100° F. 13th July, 1933. About 6 a.m., woke with headache and pain in the back, slight photophobia and a feeling of nausea, temperature, 101° F. At 11 a.m., temperature had fallen to 99"0° F., and at 4 p.m.

450

IMMUNIZATION AGAINST YELLOW FEVER.

was 98"4 ° F. : there was no albuminuria. 29th August, 1933, immunity test for yellow fever--protection 1 in 256. Case No. 62. Wife of political officer. 25th July, 1933, immunized by intradermal inoculation with 0-5 c.c. of 1 in 10 suspension of filtered mouse brain 2 hours after the intradermal inoculation of 1 c.c. of human immune serum. 26th July, 1933, about 12 hours after inoculation suffered from nausea. Temperature, 100 ° F., a.m. : 99 ° F., p.m. 27th July, 1933, felt quite well till the evening of 29th July, 1933, when she again suffered nausea and vomited once. Temperature, 99 ° F. 30th July, 1933, completely recovered. Immunity test for yellow fever, 3rd August, 1933, 9 days after immunization--protection 1 in8. Case No. 74. Male, medical officer in West African Medical Service, had had attacks of pyrexia of unknown origin at intervals of 3 to 4 months. 2nd August, 1933, immunized by intradermal inoculation with 0.5 c.c. of 1 in 10 suspension of filtered mouse brain 2 hours after the intradermal inoculation of 1 c.c. of human immune serum. 4th August, 1933, slight feeling of malaise in the evening. Temperature, 99"2 ° F. Well till 8th Augugt, 1933, when in the evening he suddenly felt ill and experienced a severe dull headache over the vertex. I n a short time the pain shifted and became located in the post-orbital and supraorbital regions. Movement of the eyeballs produced a good deal of discomfort and there was definite objection to light. Accompanying the headache there was a feeling of lassitude with aching in the long bones, ankles and wrists and lumbar region. Temperature, 101 ° F. Patient slept well. 9th August, 1933. Temperature, 100.4 ° F., aching less but increased in the evening when the temperature was 100.5 ° F. 10th August, 1933. Temperature, 99"6 ° F. In the morning felt much improved, by the evening the temperature had become normal. Immunity test for yellow fever : protection 1 in 256. Case No. 121. Male, medical officer in West African Medical Service. 27th September, 1933, immunized by intradermal inoculation with 0"15 c.c. of 1 in 10 suspension of filtered mouse brain 2 hours after intradermal inoculation of 1 c.c. of human immune yellow fever serum : felt faint immediately after the inoculation and in the evening of the same day felt r a t h e r " muzzy " i n the head but without fever. 30th September, 1933. In the afternoon again felt thick in the head. Temperature, 99"0 ° F. : no pain or discomfort. 29th September, 1933, felt well. 3rd October, 1933. In the evening, temperature 100 ° F. : felt as if developing an attack of influenza, diarrhoea : symptoms lasted for about 24 hours though the bowels continued loose for about a week. Case No. 141. Male, Government official, Nigeria. 12th October, 1933, immunized by intradermal inoculation with 0-15 c.c. of 1 in 10 suspension of filtered mouse brain 2 hours after intradermal inoculation of 1 c.c. of human immune yellow fever serum. 13th October, 1933, in the evening aching in the back and limbs. Temperature, 99 ° F. 14th October, 1933, aching in the limbs continued till the evening then disappeared and patient felt quite well till the evening o f 18th October, 1933, when there was a return of the aching in the back and limbs. Temperature, 100 "° F. : symptoms lasted 24 hours. I n o n e case, t h e r e f o r e , t h e s e c o n d a r y r e a c t i o n was a s s o c i a t e d w i t h m a l a r i a , i n t h e o t h e r s it l a s t e d f r o m 24 to 48 h o u r s . O f t h e t w e n t y - s i x p e r s o n s i n t h e t h i r d class i n w h i c h t h e r e a c t i o n was d e l a y e d till a t l e a s t 4 d a y s a f t e r i n o c u l a t i o n , o n e h a d a r e a c t i o n , b e g i n n i n g 5 d a y s a f t e r i n o c u l a t i o n , d u e to a n i n t e r c u r r e n t i n f e c t i o n w i t h B . paratyphosus B. T h i s p a t i e n t h a d n o n e o f t h e s y m p t o m s a s s o c i a t e d w i t h r e a c t i o n to t h e y e l l o w f e v e r virus but had a furred tongue, distended abdomen, constipation and a febrile r e a c t i o n l a s t i n g 14 d a y s . Y e l l o w f e v e r v i r u s c o u l d n o t b e o b t a i n e d f r o m his b l o o d . H i s wife, w h o was i m m u n i z e d at t h e s a m e t i m e as h e r h u s b a n d , h a d a r e a c t i o n c o m i n g o n 30 h o u r s a f t e r i n o c u l a t i o n a n d l a s t i n g f o r less t h a n 48 h o u r s . P a r t i c u l a r s o f t h e t w e n t y - f i v e d e l a y e d r e a c t i o n s a p p a r e n t l y d u e to t h e i n o c u l a t i o n s a r e s h o w n in T a b l e I.

G. M. FINDLAY.

451

TABLE I. DELAYED REACTIONS FOLLOWING

YELLOW FEVER

(Group II.)

IMMUNIZATION. i

Virus Inoculum (1 in 10 suspension of Filtered Mouse Brain in Normal Human

Number of Case.

Number of Days between Inoculation and Reaction.

Maximum Temperature °F.

Serum).

0.50 c.c.

0.30 c.e.

0.25 c.c.

0.20 c.c.

0.15 c.c.

0.10 c.c. 0.05 c.c.

In two for In cases is

44 50 68 70 47 57 77 51 80 82 88 95 97 98 100 102 118 121 131 134 135 137 143 144 147

99"0 99'0 100"4 102 "8 99"0 101"0 101"2 101"0 99 "6 100"8 99"0 103"0 101"0 102"0 103'0 lO1 "4 100"2 100"6 100"4 101'0 100"8 100"2 100"0 101"0 102 "8

t e n cases t h e f e v e r l a s t e d f o r 24 h o u r s ; in e l e v e n f o r 48 h o u r s a n d i n 72 h o u r s . t w o cases t h e r e was f e v e r f o r 15 a n d 11 days. T h e h i s t o r y o f t h e s e t w o as f o l l o w s .

Case No. 97, nursing sister, West African Medical Service : had been on the Gold Coast for 5 years and had had attacks of malaria and dysentery though on this leave she had been quite well : inoculated 6th September, 1933, with 0.2 c.c. 1 in 10 filtered mouse brain virus. 7th September, 1933, in the evening felt nauseated. Temperature, 99°F. 8th September, 1933, felt better in the morning, but in the evening the temperature again rose to 99 .0 F. This evening rise and morning fall of temperature continued for 5 days. 12th September, 1933, the temperature rose about 3 a.m. to 100.8 ° 17. : patient felt ill with severe headache and shooting pains in the legs : 6 pxn., temperature, 101"4 ° F. 13th October, 1933, patient felt better and the fever gradually abated till at 2 a.m., 14th October, 1933, it had reached normal. An evening rise of temperature tO 99 ° F. continued, however,

452

IMMUNIZATION AGAINST YELLOW

FEVER.

for 6 days, though the patient did not feel ill. By 22nd October, 1933, there was complete recovery. Case No. 95. Male, Agricultural Department, Gold Coast, had suffered from malaria. 1st September, 1933, inoculated with 0.25 c.c. of a 1 in 10 suspension of filtered mouse brain. 3rd September, 1933, felt a little heavy, but had no rise of temperature. 6th September, 1933, joined the boat on returning from leave. 7th September, 1933, he fell ill with a temperature of 103 ° F. T h e temperature remained between 100 ° and 101 ° F. till 18th September, 1933, when it fell to normal. He was treated as a case of malaria and given quinine by mouth ; on the seventh day of his illness, he received an intramuscular injection of quinine. On 20th October, 1933, he was examined and found to have no enlargement of spleen or liver. Urine, sp.g. 1020, acid, no albumin. Blood examination : No malarial parasites seen. R.B.C., 5,765,000 ; leucocytes, 12,800 ; differential count ; polymorphonuclears 77.2 per cent., lymphocytes 19.6 per cent., large mononuclears 2 per cent., eosinophils 1.2 per cent. For particulars of this case, I am indebted to Dr. D. Du~F, Director of Medical and

Sanitary Services,Gold Coast. The clinical symptoms in the remaining twenty-three cases wcre very similar to those previously described and consisted of headache of varying intensity, aching in the back and limbs, and occasionally photophobia. Four persons complained of nausea, while two, a husband and wife, both remarked on hyper~esthcsia of the skin. In onc case a labial hcrpcs dcvclopcd 10 days aftcr inoculation. With the fall in temperature thc symptoms, as a rule, rapidly disappeared. In regard to the hypcr~esthcsia of the skin it is of interest that this symptom has also bccn recorded by DOOLE£ (1932) as occurring in a patient who had received an injection of h u m a n serum. Case No. 44. An American educational expert en route for Liberia, presented certain abnormal features. 4 days after inoculation he had a slightheadache and a rise in temperature to 99 ° F. This lastedfor 24 hours, and hc fcltcompletely recovered until 10 days later (15 days after inoculation). Hc then again developed slightfever, unaccompanied by any of the other symptoms suggestive of a reaction to yellow fever. On arrival at Monrovia, he was found to be sufferingfrom high blood pressure and was diagnosed as a case of interstitialnephritis as the urine contained fine granular casts. Complete recovery followed restand dieteticmeasurcs. The random distribution of delayed reactions among the 110 cases of Group II makes any explanation, other than personal idiosyncrasy, a matter of considerable difficulty. Thc only predisposing causes which may possibly have played some part in the development of the reactions arc physical exercise and prolongcd residence in the tropics, associated cspccially with malaria. Thus Case No. 47 walked 20 milcs, Case No. I00 rowed, an unaccustomed cxcrtion, before the onsct of their reactions. In regard to tropical residence, twcnty-thrcc out of the twcnty-fivc pcrsons who suffered from dclaycd reactions had bccn residcnt in West Africa and fifteen at least of the West African residents had a malarial history. One of thc most frequent dcscriptions of thc reaction was, in fact, that it was vcry similar to an attack of malaria. Group I I I . I n this g r o u p , c o m p r i s i n g t h i r t y - e i g h t m e n a n d t w e l v e w o m e n , t h e i n t r a d e r m a l i n o c u l a t i o n s of v i r u s a n d s e r u m were g i v e n as before, b u t i n a d d i t i o n

G. M. FINDLAY.

458

5 c.c. of human yellow fever immune serum were injected subcutaneously at the same time as the injection of the virus-containing component. This additional immune serum was given with the object of (1) reducing the number of delayed reactions ; (2) decreasing the risk of virus circulating in the blood. At the same time the amount of virus injected was progressively decreased from 0.025 c.c. to 0.00625 c.c. of a 1 in 10 suspension of filtered mouse brain. Of the fifty persons thus inoculated fifteen--twelve men and three women-had a rise of temperature above 99 ° F., while thirty-five--twenty-six men and nine women--either made no complaint of any sort or suffered only from slight headache and muscular aching, coming on 30 to 36 hours after injection of the virus and lasting not more than 24 hours. Four other cases suffered from an immediate reaction with rises of temperature varying from 99.2 ° F. to 101 ° F., again lasting for not more than 24 hours. In two cases, a husband and wife, there were both immediate and delayed reactions. The history of these cases is briefly as follows : Case No. 168. M e d i c a l officer, W e s t A f r i c a n M e d i c a l Service. 14th N o v e m b e r , 1933, i n o c u l a t e d w i t h 0.05 c.c. of 1 i n 10 s u s p e n s i o n of filtered m o u s e b r a i n a n d 5 c.c. of h u m a n yellow fever i m m u n e s e r u m . 15th N o v e m b e r , in the e v e n i n g h a d a rise of t e m p e r a t u r e to 99 ° F . - - n o malaise. 20th ,, pains in t h e back a n d joints, t e m p e r a t u r e 101-102 ° F . 21st ,, pains c o n t i n u e d , w i t h a c h i n g in t h e groins a n d b e h i n d t h e e y e s - n o t unlike d e n g u e . 22nd recovered. Case No. 469. Wife of above, similar i n o c u l a t i o n , 14th N o v e m b e r , 1933. 15th N o v e m b e r , in t h e e v e n i n g h a d a rise in t e m p e r a t u r e to 100 ° F . - - n o malaise. 19th in t h e evening, pains in t h e b a c k a n d j o i n t s , t e m p e r a t u r e 100-2 ° F. 20th ,, recovered.

Particulars of the nine delayed reactions are shown in Table II. TABLE II. DELAYED REACTIONS FOLLOWING YELLOW FEVER IMMUNIZATION (GROUP

III).

i

Number of Case.

152 153 157 162 171 174 175 176 178

Virus Inoculum (1 in l0 suspension of Filtered Mouse Brain in ]Normal H u m a n Serum). 0.025

N u m b e r of Days between Inoculation and Reaction.

Maximum Temperature °F.

5 6 5 6 6 6

102"2 102.0 100.4 102"6 100"8 100"0 99.2 101.2 100"2

c.c.

0.0125 c.c.

Duration of Fever. Hours. 48

48 24 48 24 48 48 48 48

C

454

IMMUNIZATION AGAINST YELLOW FEVER.

The symptoms in these nine cases were similar to those previously described, Case No. 178 complained of hyper~esthesia of the skin, while Case No. 157 developed labial herpes after the temperature had fallen to normal. Eight of the nine cases exhibiting delayed reactions were resident in West Africa. It will be noted that the last delayed reaction was Case No. 178. Among the last twenty-one cases no delayed reaction has occurred, possibly owing to the fact that the dose of virus has been reduced to 0.00625 c.c. of a 1 in 10 suspension of filtered mouse brain. Though this dose of virus is so much smaller than the 0.5 c.c. of a 1 in 10 suspension of mouse brain employed in the early cases, nevertheless it still represents approximately 2,500,000 minimal lethal doses for mice, since the minimal lethal dose for mice of the virus injected was 0.03 c.c. of a dilution 10"r. Among these last twenty-one cases there has been no rise of temperature except in one instance where, on the evening of the day after inoculation, the temperature rose to 101 ° F. 12 hours later the temperature had fallen to normal. Of the other twenty persons six had no reaction of any sort, the others complained of headache or heaviness, coming on some 30 hours after inoculation and lasting from 24 to 48 hours. In one instance 7 days after inoculation the face was said to be puffy. This puffiness lasted 3 days and coincided with a cold in the head, so that its relationship to the inoculations is somewhat uncertain. DEVELOPMENT AND DURATION OF IMMUNE BODIES. The presence of protective immune bodies has been demonstrated in persons immunized against yellow fever by the intraperitoneal protection test in mice, described by SAWYER and LLOYD (1931). In this test 0.03 c.c. of sterile 2 per cent. starch solution is injected intracerebrally into each of six mice to localize the virus in the brain. Immediately thereafter the mice are given an intraperitoneal injection of a mixture of 0.2 c.c. of 20 per cent. suspension of virus-containing mouse brain in 1 in 10 normal human serum and saline, and 0.4 c.c. of the serum to be tested. (In the original test the viruscontaining mouse brain was made up as a 10 per cent. suspension.) Control groups of mice are given injections in the same way, except that known normal and immune sera are substituted for the serum to be tested. Mice dying in from 5 to 10 days after inoculation are considered to have died of yellow fever encephalitis if the results in the control groups are satisfactory. If three out of the six mice survive the result is considered" protection." The factors necessary for the correct application of the test have been fully considered by HUGHES and SAWYER (1932) and MAHAFFY,LLOYD and PENNA (1933). They comprise : - (I) A strain of mice fully susceptible to yellow fever virus. (II) A strain of mice in which there are no deaths due to intercurrent infections.

G. M. FINDLAY.

455

( I I I ) Virus which is fully active when inoculated intracerebrally in doses of 0.03 c.c. in a dilution of at least 10 "e. In order to determine the specific character of the test, HUGHES and SAWYER(1932), examined sera from persons resident in China and Canada where yellow fever has never been known to occur. One hundred and three sera from China were all negative, while of one hundred and twenty-two sera from Canada, all except three gave no protection. Of these three, one gave protection in two of seven tests, each of the others in one of five or six successive tests (of. JAMES, 1933). On the other hand, the sera of twenty-one persons who had either been vaccinated against yellow fever or had suffered from attacks of yellow fever all gave protection, as did fourteen out of ninety-seven sera from Panama. In this group of sera the positives were all 30 or more years of age, a finding in elose agreement with the fact that the last known case of yellow fever occurred on the Isthmus of Panama 27 years before the sera were examined. The results of the examination of 3,274 sera from Brazil and West Africa by the mouse protection test have recently been analysed by MAH~-~Y, LLOYD and PE~q-NA (1933). Conclusive results were obtained in 96.6 per cent. of sera on initial test. In order to determine to what extent the results of the test are in agreement with known epidemiological data, the incidence of protective immune bodies in the area of susceptible persons in certain districts in Brazil and West Africa was ascertained. Two areas in Brazil, in which the high altitude and the absence of any history of yellow fever were believed to eliminate the possibility of past infection were chosen for study. Two hundred and twenty-one sera were collected from these areas and in none could the presence of yellow fever protective antibodies be demonstrated. In two other areas in, Brazil and one in West Africa, where yellow fever epidemics were known to have occurred recently, the sera yielding positive results were 61, 72 and 72 per cent. SAWYERand LLOYD (1931), in their original observations were able to demonstrate a close agreement between the immunological reactions in the protection test in rhesus monkeys and those in the intraperitoneal protection test in mice. These findings have been confirmed by MAHAm~Y,LLOYD and PENNA(1933). Thus in one area in Brazil in which 72 per cent. of sera were positive by the mouse protection test, 55.3 per cent. were positive by the monkey protection test (SoPER, FRO~ISn~R, K ~ R and DAVIS, 1932), while in an area in West Africa in which 72 per cent. of sera were positive by the mouse protection test, 68 per cent. were positive by the monkey protection test. Before describing the results obtained with the sera of persons i m m u n i z e d against yellow fever, it may be of interest to discuss briefly the results obtained with sera from persons resident in Great Britain and from Europeans in West Africa, together with sera f r o m a few natives in Barbados. (Table I l l . ) Of the first group resident in Great Britain, none had lived in West Africa or South America t h o u g h two had been exposed to yellow fever in the course of laboratory work. F o u r of the sera in this g r o u p contained protective antibodies against Rift Valley fever, a disease which, despite certain clinical and pathological similarities to yellow fever, has already been shown not to confer any i m m u n i t y to yellow fever (FINDLAY, 1932). T w o had also recovered from dengue. STErANOPOULO and CALLmICOS (1932) have previously shown that sera f r o m recovered cases of dengue do not give protection by the mouse intraperitoneal test. BEEUWK~S (1933) has also reported lack of protection in t w e n t y - f o u r sera from persons who had suffered from dengue during the recent epidemic in Athens. Of the twenty-six Europeans who had been resident in West Africa one only (Case No. 26) gave a positive protection and he gave a definite history of

456

IMMUNIZATION AGAINST YELLOW FEVER.

a previous attack of jaundice. The majority of these residents had had various attacks of malaria. The seven sera from Barbados were obtained through the kindness of Dr. J. F. C. HASLAM, Principal Medical Officer, Barbados; all had suffered TABLE III.

Class.

(1) Residents in Great Britain (21 British : 1 Negro) (2) European residents in West Africa (3) Natives of Barbados (4) Accidental laboratory infections (5) Known attacks of yellow fever

Total

N u m b e r of Sera N u m b e r of Sera giving giving no Protection. Protection.

Number of Sera. 22

I

0

22

26 7

25 7

5

0

4

0

64

10

Range of Titres.

54

1/256 1/128 to 1/1024 1/128 to 1/1024

t

within the past year from a form of infective jaundice which has previously been reported from this island (GuITERAS, 1919). All failed to give protection. On the other hand, the sera from five cases of laboratory infection and from four persons who were known to have had previous attacks of yellow fever all gave a positive protection. The conclusion may, therefore, reasonably be drawn that the mouse protection test for immune bodies to yellow fever possesses a high degree of specificity. 115 persons have now been examined at varying intervals of time after immunization for the presence of protective immune bodies to yellow fever. The results are shown in Table IV from which it will be seen that immune bodies become demonstrable in the blood about the eighth or ninth day after inoculation, irrespective of the method of immunization employed. The immune body titre reaches its maximum in from 4 to 5 weeks after inoculation. No correlation appears to exist between the degree of immunity and the severity of the reaction to inoculation. In Table V are shown observations on the sera of eleven persons from 5 to 16 months after immunization. Seven of these persons were exposed to yellow fever infection during the course of laboratory work. Although immune bodies could be detected in all the sera, the titre, nevertheless, had in certain cases shown a considerable fall. The evidence at present available therefore shows that immune bodies against yellow fever may still be detected in the serum for at least 12 months after immunization.

G. M. FINDLAY.

457

TABLE IV. PROTECTIVE IMMUNE BODIES IN THE SERA OF PERSONS IMMUNIZED AGAINST YELLOW" FEVER.

Number of Persons

Inoculation

Group

Interval between Inoculation and Observation. Days.

Examined.

I

II

III

18

1-- 7 8--15 16--21 22--42 1-- 7 8--15 16--21 22--42 1-- 7 8--15 16--21 22--42

61

36

I m m u n e Body Titre.

Number I

of

i

Observa- i tions.

3 7 7 6 2 29 21 9 6 16 9 6

i/4

3 B

--

--

1/32

4

3 2

1

1/64 1/128

1/256

3 4

1 1

1

--

3

2

6

1/16

-__

2

I/8

10

8

1

2

5 9

1

7 2

2 3

3 2

1 3

2

-2

3

3

6

1

--

2 ,

TABLE V. DURATION OF IMMUNE BODIES AFTER IMMUNIZATION AGAINST YELLOW FEVER.

N u m b e r of M o n t h s since i Immunization.

Immune Body Titre.

i J

1 1 1 1

!

G.M.F.

J

J.C.B. A.E.G.

iL

I I

i

7 16 6 14

5 14

R.D.M.

!

5

i i

13

in 256 in 64 in 512 in 32 1 in 64 1 in 64 1 in 128 1 in 8

Immune Body Titre.

N u m b e r of M o n t h s since I Immunization. R.J.R. *P.A.B. *D.J.L. *A.J.R.O'B. J.H. L.S.S.

i !

5 13 12 11

i

10

i

9

!!

2 6

*B.E.B.

5

1 i 1 1 1 1 1 1 1 1

in in in in in in in in in

512 128 32 16 16 128 256 128 256

[ * N o t exposed to laboratory infection with yellow fever.

CIRCULATION OF VIRUS IN THE BLOOD. The question of whether living yellow fever virus can circulate in the blood of man after combined immunization with virus and immune body is

458

IMMUNIZATION AGAINST YELLOW FEVER.

one of considerable importance since both DAVIS, LLOYD and FROBISHER (1932) and ROUBAUDand STEFANOPOULO(1933) have shown that the fully fixed neurotropic virus can survive in A$des eegypti and after mosquito passage can produce encephalitis both in monkeys and white mice. Viscerotropic lesions, however, are not produced nor can the fixed neurotropic virus be maintained in the mosquito host as easily as the viscerotropic. In an attempt to discover whether neurotropic virus circulates in the blood after the administration of virus and immune serum, a series of experiments was carried out in monkeys. T ~ t . ~ VI. OCCURRENCE OF NEUROTROPIC VIRUS IN THE BLOOD OF RHESUS MONKEYS AFTER IMMUNIZATION WITH IMMUNE SERUM AND NEUROTROPIC VIRUS.

Presence of I m m u n e B o d y i R e a c t i o n t o Virus i n Titre 3 Injection th e Blood a t M o n t h s after of M o n k e y I n t e r v a l s of I m m u n i z a t i o n VisceroD a y s after (by M o u s e tropic Inoculation. Protection Virus 1 3 5 Te s t ). (French strain).

Amount I A m o u n t in c.c. Number in c.c. Body Filtered Virus-conof of S e r u m W e i g h t tanning M o u s e M o n k e y . Injected. in kiloBrain d i l u t e d ( T i t r e 1 grams, in Serum. in 512). t

I I

C.C.

1

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 I7 18 19 20

1

,,

,, ,, ,,

, ,,

,

2.5 2.9 2.8 2.5 3.1 2.6 2.7 2.6 3.0 2.8 2.6 2.8 2.4 2.6 2.2 2.5 2.7 2.6 2.8

c.c. 0.40 0.25

Diluted. 1 in 10

+ +

+ +

+ +

--

+

+

+

+

+

1 in 128

256 1 ,, 256 1 ,, 128 1 ,, 64 1 ,, 128 1 ,, 64 1

,,

Protection J~

J~

J,

0.10

i

,,

,,

1 ,, 1 0 0

"

1 ,, 1,000

,,

l

10+000

1

100,000

1 ,, 64 1 ,, 128 1 ,, 128 1 ,, 64 1 ,, 256 1 ,, 128 1 ,, 32 1 ,, 64 1 ,, 64 1 ,, 128 0

1 ,, 1 , 0 0 0 , 0 0 0

3.0 !

I

* K i l l e d - - B r a i n a n d s e r u m d i d n o t c o n t a i n virus.

Protection

i,

JJ

N o prot e c t i on

G. M. FINDLAY.

459

The results are shown in Table VI from which it will be seen that when from 0.4 to 0.25 c.c. of a 1 in 10 filtered suspension of virus-containing mouse brain was injected into rhesus monkeys, together with 1 c.c. of human immune yellow fever serum, virus could at some period be detected in the blood stream. With smaller doses of virus of from 0"1 c.c. of a 1 in 10 suspension to 0.1 c.c. of a 1 in 1,000,000 filtered suspension of virus-containing mouse brain no virus could be detected in the blood stream. Incidentally, Table VI demonstrates the fact already emphasized by SAWYER and LLOYD (1.931) and others that the detection of immune bodies by the mouse protection test is also evidence of immunity to the viscerotropic strain of yellow fever virus. The examination of the sera of persons from 1 to 6 days after inoculation is shown in Table VII. In two of the sixteen cases virus was present in the blood serum on the sixth and fifth days respectively after inoculation : in all the others virus could not be demonstrated in the serum. While the absence of virus at all times and in all cases cannot, of course, be entirely excluded it is obvious that the circulation

TABLE V I I . EXAMINATION OF BLOOD FOR NEUROTROPIC YELLOW FEWR VIRUS ~OLLOWINO IMMUNIZATION,

Case Number.

Immune Serum.

C.C.

2 lO 14 29 30 57 75 83 133 134 150 159 160

32 33 30 26 35 1 1 1 1 1 6 6 6

Virus I n o c u l u m (1 in 10 suspension of filtered m o u s e brain in normal human serum).

Day of Examination after Inoculation.

Presence of Virus.

C.C.

0.5

3 2 1"

[

0.25 0.15 0.025 0.050 0.025

179

0.0125

195 196

0.00625 0.00625

2 2 5 6 2 5 5 5 1 6 1 5 2 2

+ +

460

IMMUNIZATIONAGAINSTYELLOWFEVER.

of virus is by no means a common occurrence, and when it does occur is probably of comparatively short duration. DISCUSSION.

The experiments here recorded confirm the original observations of SAWYER, KITCHEN and LLOYD (1932) in regard to the practical application of immunization against yellow fever by the combined injection of fixed neurotropic yellow fever virus and human immune yellow fever serum. Of the 200 persons immunized in this country only eighty-six, or 43-0 per cent. developed febrile reactions to the inoculations while the reactions with but a few exceptions were of a comparatively mild character. No case of encephalitis has yet occurred nor any " accident paralytique " similar to those occasionally recorded after immunization against rabies. Although the immune body titre shows a tendency to fall some months after inoculation immune bodies can still be detected at least 12 months after immunization. The possibility that after this form of inoculation neurotropic yellow fever virus may sometimes circulate in the blood stream is one which cannot be entirely excluded though, with careful adjustment of the dose of virus and serum, such an occurrence is likely to be rare. Even if mosquitoes do actually become infected in this way, passage of the neurotropic virus through mosquitoes does not cause a return of viscerotropic properties, while the experiments of DAvis, LLOYD and FROBISHER (1932) show that the neurotropic strain of virus is not easily maintained in Stegomyia mosquitoes. The one draw-back to the method of immunization proposed by SAWYER, KITCHEN and LLOYD is the large amount of human immune yellow fever serum required. To obviate this difficulty various modifications have been proposed. SELLARDS and LAIGRET (1932) have injected neurotropic virus without immune serum. Certain severe reactions occurred and virus almost certainly circulates in the blood. The use by LAIGRET (1933) of three increasing doses of virus at intervals of 20 days fails to remove the danger of circulating virus and, though reactions are slight, the practical difficulty of giving three inoculations at long intervals is obvious. Possibly also the danger o f " accidents paralytiques " may be increased by three inoculations of nervous tissue since the evidence at present available suggests that these accidents are due to the inoculation of some toxic substance present in nervous tissues whether of heterologous or homologous origin. Although a method of dispensing with the use of immune yellow fever serum is highly desirable, it has not yet been found possible to destroy the pathogenicity of yellow lever virus without at the same time destroying its antigenic power. In the case of dog distemper virus, PERDRAUand TODD (1933)

G. M. FINDLAY.

461

have recently shown that exposure of the virus to a pointolite in the presence of 1 in 100,000 methylene blue inactivates the virus but does not destroy its antigenic capacity. Both the viscerotropic and neurotropic strains of yellow fever virus can be readily inactivated by exposure to a pointolite for 20 minutes in the presence of I in 100,000 methylene blue solution, but all attempts to demonstrate antigenic capacity in such inactivated virus have so far failed. The substitution for human immune serum of anti-yellow fever serum from the horse or baboon, suggested by PETTIT and STEFANOPOULO(1933), has much to recommend it, more especially where the immunization of large native populations is concerned. In the case of Europeans, however, many have already received injections of horse serum, while if it became necessary to repeat the inoculations against yellow fever difficulties would almost certainly arise. In addition, the combined effects of reactions to heterologous serum and to the yellow fever virus are as yet unknown. In the meantime, therefore, it seems desirable at any rate in the case of Europeans to continue with homologous immune serum where such can be obtained in sufficient quantities and of sufficiently high titre. The experiments here brought forward show that it is possible to reduce very considerably the dose of both virus and human immune serum from that originally recommended by SAWYER, KITCHEN and LLOYD (1932) without preventing the subsequent development of immune bodies. So far the complete success of immunization against yellow fever has only been demonstrated in the case of laboratory workers, for among these, since the introduction of prophylactic immunization, there has been a complete absence of the accidental infections which were at one period so common. Immunization is now being extended to those Europeans living in endemic areas whose work unavoidably brings them into contact with possible sources of infection. In the future, in view of increased traffic by aeroplane and by car it may be desirable to immunize all persons who wish to travel from an endemic to a non-endemic area if the non-endemic area can be reached within the incubation period of yellow fever and in addition harbours the mosquito vector. Immunization may also be of considerable value in limiting the spread of infection when once an epidemic has broken out, since under natural conditions epidemics apparently cease when the available number of non-immunes is reduced to a low level. Finally, a practical method of immunization against yellow fever renders the total eradication of the disease at least possible if not immediately practicable. Since there is no known animal reservoir of yellow fever virus, and since the virus is not transmitted from mosquito to mosquito either hereditarily or by direct contact it follows that the virus cannot be maintained indefinitely among mosquitoes in the absence of susceptible human beings. The life-span of female A~des ~egypti has, under the best laboratory conditions, been shown by BEEUWKES, KERR, WEATHERBEEand TAYLOR (1933) not to exceed 225 days.

462

I M M U N I Z A T I O N AGAINST Y E L L O W FEVER.

If, therefore, the whole population of the endemic areas in Africa and South America could ever be successfully immunized and could be kept immune for as short a period as 1 year, then the chain of infection would be broken, and yellow fever would be exterminated once and for all. CONCLUSIONS. (1) The immunization of 20() persons against yellow fever with varying doses of mouse-fixed neurotropic yellow fever virus and human immune )ellow fever serum is described. (2) Details are given of the preparation of the vaccine and immune serum. (3) Eighty-six, or 43 per cent., of the persons immunized reacted with a temperature above 99 ° F. : the clinical symptoms of the reactions are described. (4) Virus has only been detected in the blood in two out of sixteen cases. (5) The specificity of the mouse protection test for immune bodies to yellow fever is discussed. (6) The sera of 115 persons were examined for immune bodies after immunization. Immune bodies begin to appear in the blood stream 8 to 9 days after immunization : they attain their maximum titre in from 4 to 5 weeks. (7) 10 to 11 months after immunization the immune body titre decreases but protective immune bodies can still be detected up to at least 16 months after inoculation. (8) The sera of twenty-two residents in Great Britain all failed to show the presence of immune bodies to yellow fever as did the sera of seven natives of Barbados who had suffered from a form of infective jaundice : of twenty-six Europeans resident in West Africa only one serum gave positive protection : in nine persons with a history of yellow fever the sera all gave positive protection. (9) The neurotropic fixity of yellow fever virus is discussed with reference to its behaviour in monkeys, mice and other rodents. (10) Neurotropic yellow fever virus can infect mice and monkeys when introduced into the nostrils, producing an encephalitis. (l l) Both the neurotropic and viscerotropic strains of yellow fever virus can be inactivated by exposure to a pointolite in the presence of 1 in 100,000 methylene b|ue : antigenic activity is also destroyed. REFERENCES. A~AGAO, H. DE B. (1928)• Relatorio a respeito de algumas pesquizas sobre a. febre amarella. Mere. Inst. OstoaldoCruz., Suppl. 2, 23. --~. (1931). S6ro-virusvaccina~onafebreamarella. Ibid.xxv, 213. • (1933). Emploi de virus vivant dans la vaccination contre la fibvre jaune. C.R. Soc. Biol., cxii, 1,471. BAUER, J . H . (1931). Duration of passive immunity in yellow fever. Amer. J. Trop. Med., xi, 451. BZSUWKm,H., KERR,J. A., W,V.ATHEaBEE,A.A. & TAYLOa, A.W. (1933), Observations on the bionomics and comparative prevalence of the vectors of yellow fever and other domestic mosquitoes of West Africa and the epidemiological significance of seasonal variations. Trans. Roy. Soc. Trop. Med. ~ Hyg., xxvi, 425.

C. M. FINDLAY.

463

BURKE, A. W. & DAVIS, N . C . (1930). Notes on laboratory infections with yellow fever. Amer. y. Trop. Med., x, 419. BURNETT, F. M. & MACNAMARA,J. (1931). Immunological differences between strains of poliomyelitis virus. Brit..~. Experim.Path., xii, 57. CHAGAS, C. (1931). Quoted by PETTIT, A. Bull. Acad. Mdd., cv, 522. CARTER, H . R . (1931). Yellow Fever---an Epiderniological and Historical Study of its Place of Or/g/n. [Edited by L. A. CARTERand W. H. FROST]. Baltimore : The Williams & Wilkins Company. DAVIS, N. C. (1931). Uso experimental de urea vaccina cloroformada contre a febre amarella. Brazil-Medico, xlv, 268. DAVIS, N. C., LLOYD, W. & FROmSHER, M. JR. (1932). The transmission of neurotropic yellow fever virus by Stegomyia. ~. Experim. Med., lvi, 853. DOOLEY, P. (1932). Serum disease : report of case following injection of homogeneous serum. ~. Amer. Med. Assoc., xcix, 1,778. FINDLAY, G . M . (1932). Rift Valley fever or enzootic hepatitis. Trans. Roy. Soc. Trop. Med. (~ Hyg., xxv, 229. • (1933). Sur l'immunisation contre la fi&vre jaune. Bull. Off. internat, d'Hyg. publ., xxv, 1,009. • (19341). Nouvelles expdriences concernant l'immunisation contre la fi~vre jaune. Ibid., xxvi, 43. . (1934~). T h e infectivity of neurotropic yellow fever virus for animals. ~. Path. Bact., xxxviii, 1. FINDLAY, G. h/I. & BROOM, J . C . (1933). Experiments on the fltration of yellow fever virus t h r o u g h " gradocol "membranes. Brit.y. Experim. Path., x iv, 391. FINDLAY, G. M. & HINDLE, E. (1931). Combined use of living virus and immune serum for immunization against virus infections. Brit. Med. ~., i, 740. FINLAY, J . C . (1894). La fiebre amarilla. Cr6n. Med. Quir. de la Habana, xxi, 143. FROBISHER, NI., JR. (1933). A comparison of certain properties of the neurotropic virus of yellow fever with those of the corresponding viscerotropic virus. Amer. ~. Hyg., xviii, 354. FROBISHER, M. JR., DAVIS, N. C. & SHANNON,R . C . (1931). On the failure of yellow fever virus to persist in a colony of A~des cegypti. Ibid., xiv, 142. GUITERAS, J. (1919). Epidemic of febrile jaundice in Barbados in 1919. New Orleans Med. ~ Surg. J., lxxii, 360. HAAGEN, E. (1933). Weitere Untersuchungen i~ber das Verhalten des Geldfiebervirus in der Gewebekultur. Zentralbl./. Baht., I. Abt. Orig., cxxviii, 13. HINDLE, E. (1928). A yellow fever vaccine. Brit. Med.j%, i, 976. HU~HES, T . P~ & SAWYER,W . A . (1932). Significance of immunity tests in epidemiology as illustrated in yellow fever. ~. Amer. Med. Assoc., xcix, 978. JAMES, S. P. (1933). Recent knowledge concerning yellow fever. A report prepared by the Yellow Fever Commission of the Office international d'Hygi~ne publique. Trop. Dis. Bull., xxx, 265. KLIOLER, I . J . (1928). Studies on the etiology of Phlebotomus and dengue fever. Ann. Trop. Med. ~ Parasit., xxii, 143. LAIGRET,J. (1932). Recherches exp6rimentales sur la fi~vre jaune. Arch. Inst. Pasteur de Tunis, xxi, 412. ---. (19331). Recherches exp6rimentales sur la fi6vrejaune (3 e m memoire). Technique pour la pr6paration des virus amarils : Vaccins glyc6rin6s et vaccins sec. Ibid., xxii. (19332). La vaccination contre la fi~vre jaune. Bull. Soc. Path. Exot., xxvi, 806 LLOYD, W. & PENNA, H . A . (19331). Studies on the pathogenesis of neurotropic yellow fever virus in Macacus rhesus. Amer. ~. Trop. Med., xiii, 1. (19332). Yellow fever encephalitis in South American monkeys. Ibid., xiii, 243. (1933a). The preservation of yellow fever immune sera. Ibid., xiii, 291. LLOYD,W., PENNA,H. A. & ]VIAHAFFY,A. (1933)• Yellow fever encephalitis in rodents, Amer. ~. Hyg., xviii, 323.

464

IMMUNIZATION AGAINST YELLOW FEVER.

MAHAFFY, A., LLOYD, W. & PENNA, H. A. (1933). Two years' experience with the intraperitoneal protection test in mice in epidemiological studies of yellow fever. Ibid., xviii, 618. MARCHOUX,E., SALIMBENI,A. & SIMMOND,P• L. (1903). La figure jaune. Rapport de la mission fran¢aise. Ann. Inst. Pasteur, xvii, 665. MONTEIRO, J. L. (1930). Nouvelle technique pour la pr6paration du vaccin contre la fi~vre jaune. C.R. Soc. Biol., civ, 695. OKELL, C . C . (1930). Experiments with yellow fever vaccine in monkeys• Trans. Roy. Soc. Trop. Med. ~# Hyg., xxlv, 251. OKELL, C. C. ~ HART, P. D'A. (1932), A simple apparatus for venesection. Lancet, ii, 514. PERDRAU, J. R. & TODD, C. (1933). Canine distemper. The high antigenic value of the virus after photodynamic inactivation by methylene blue• ft. Comp. Path. Therap., xlvi, 78. PETTIT, A. (1931). Rapport sur la valeur irnmunisante des vaccins employ6s contre la fi~vre jaune et la valeur th6rapeutique du serum antiamari!. Bull. Acad. Mdd., cv, 522. PETTIT, A. & STEFANOPOULO,G . J . (1928). Le virus de la fi~vrejaune. Ibid., c, 921. (1933). Utilisation du sdrum antiamaril d'origine animale pour la vaccination de l'homme. Ibid., cx, 67. PETTIT, A., STEFANOPOULO,G. J. ~; FRASEy, V. (1928). Pouvoir prdventif et curatif du sdrum antiamaryllique. C . R . Soc. Biol., xcix, 1,114. PRESAGE, E. (1933). The Portugese Pioneers. The Pioneer Histories. London : Black. ROUBAUD, E. & STEFANOPOULO,G. J. (1933). Recherches sur la transmission par la vole stdgomyienne du virus neurotrope murin de la fi~vre jaune. Bull. Soc. Path. Exot., xxvi, 305. SAWYER, W. A., KITCHEN, S. F. & LLOYD, W. (1931). Vaccination of humans against yellow fever with immune serum and virus fixed for mice. Proc. Soc. Experim. Biol. Med., xxix, 62. (1932). Vaccination against yellow fever with immune serum and virus fixed for mice. ft. Experim. Med., lv, 945. SAWYER, W. A. & LLOYD, W. (1931). The use of mice in tests of immunity against yellow fever. Ibid., liv, 533. SELLARDS,A . W . & LAIGRET,J. (19321). Vaccination de l'homme contre la fi~vre jaune. C. R. Acad. Sc., cxciv, 1,609. . (19322). ContrSle, par dpreuve sur Macacus rhesus, du pouvoir protecteur du sdrum des hommes vaccin6s contre la fi~vre jaune par l'inoculation du virus de souris. Ibid., cxciv, 2,175. . (19323). Immunisation de l'homme contre la fi~vre jaune par l'inoculation du virus de souris. Arch. Inst. Pasteur de Tunis, xxi, 229. SCHULTZ, E. W. & GEBHARDT, L. P. (1933). Observations on the intranasal route of infection in experimental poliomyelitis. Proc. Soc. Experim. Biol. ¢# Med., xxx, 110. SOPER,F. L., FROBISHER,M. JR., KERR, J . A . & DAVIS,N . C . (1932). Studies of distribution of immunity to yellow fever in Brazil : postepidemic survey of Mag6, Rio de Janeiro by complement fixation and monkey protection tests, ft. Preventive Med., vi, 341. SOPER,F. L., PENNA, H., CARDOSO,E., SERAFIM,J., FROBISHER,M. JR. & PINHEIRO, J. L. (1933). Yellow fever without A~des cegypti. Study of a rural epidemic in the Valle do Chanaan, Espirito Santo, Brazil. Amer.ff. Hyg., xviii, 555. STEFANOPOULO, G. J. & CALLINICOS,G. (1932). Absence d'anticorps pour le virus amaril dans le sang des sujets atteints de dengue. C.R. Soc. Biol., cx, 1,230. STOKES,A., BAUER,J . H . & HUDSON, N . P . (1928). Experimental transmission of yellow fever to laboratory animals. Amer. ft. Trop. Med., viii, 103. THEILER, M. (1930). Studies on the action of yellow fever virus in mice. Ann. Trop. Med. ~# Parasit., xxiv, 249. THEILER, M. & SELLARDS, A. W. (]928). The immunological relationship of yellow fever as it occurs in West Africa and in South America. Ibid., xxii, 449.

465 DISCUSSION.

Colonel $. P. James in opening the discussion said : I am very glad to have this opportunity of congratulating Dr. FINDLAY on his admirable paper and particularly on the success that has attended his highly important work. When we reflect on the extent to which the possession of a practicable and harmless method of immunization against yellow fever aids in making our tropical colonies safe for the white man, we are justified in being proud of the important share that English workers have taken in the steps leading to the production of a suitable vaccine. Some of these workers, as we know, lost their lives while engaged in the task, others contracted the disease but happily recovered. As Dr. FINDLAY has said, the complete cessation of these laboratory accidents since prophylactic inoculation became available is clear proof of the efficacy of the measure. This being so, we are on safe ground in recommending it not only as a sure method of personal protection applicable to all colonial officers and others whose work takes them to yellow fever areas, but also as a means of preventing the spread of yellow fever from one country to another. For example, it is obviously the method of choice for preventing the spread of yellow fever by aeroplanes. In this traffic the real practical risk is concerned with the human case in the incubation period of the disease, and it is clear that this risk can be entirely eliminated by requiring the effective immunization of the personnel of aircraft and all intending passengers. As this is so I am sure it will be agreed that the spread of yellow fever from West to East Africa and to India may be prevented by less drastic measures than that of placing an embargo on all air traffic between those countries. So many other points are brought out in Dr. FINDLAY'S paper that it is not easy to decide which to select for brief comment, but I was very interested in his view that theoretically, at any rate, immunization might be employed as a means of eradicating yellow fever from an area. In this connection it is important that, according to results obtained by the protection test in Africa and America, immunization against yellow fever is already occurring under natural conditions in many regions without the appearance of any disease resembling yellow fever. If, on a map of the world, one were to spot all the localities where positive results by the protection test had been found, and all the localities where clinical cases of yellow fever had occurred, it would be seen that there are large areas where a virus is present which immunizes against yellow fever but does not cause the clinical disease. Dr. SAWYERcalls these areas " silent regions." The situation is analogous to that of major and minor smallpox in thc world generally, and it has been suggested that perhaps in yellow fever, as in smallpox, the existence of a mild strain as well as of a virulent strain must be recognised. On this suggestion it is clearly very important to endeavour to isolate the strain of virus which occurs in a country where people become immune without having yellow fever and to ascertain if its low virulence persists over a series of years, as is the case with

466

DISCUSSION.

the virus of variola minor (alastrim). If the mild strain which immunizes without causing art appreciable illness is found to breed true, it would be a virus that could be used for prophylactic vaccination without the necessity of using human immune serum. We should then be reproducing artificially what is apparently happening naturally over very large areas of the tropical world. Of course, the idea that in yellow fever, as in smallpox, two strains of virus are concerned, introduces into the problem many other questions of interest. The mild strain is obviously advantageous to the populations infected with it and the question arises whether it would be wise to endeavour to eradicate it or to reduce its prevalence. This question cannot be answered until a supply of this strain of virus has been obtained and studied intensively with regard to its virulence and other characters. Thus it seems that, as yet, we are only in the early stage of knowledge on various questions which have an important bearing on the prevention and control of yellow fever.

Dr. N. Hamilton Fairley said that he spoke with some feeling on the subject because two of his laboratory technicians had contracted yellow fever some two years ago, and one of them had died, an accident which would hard been prevented if the present method of immunization had been available then. These men were doing ordinary routine biochemical investigations on yellow fever cases and developed the disease though, as far as was known, they had no skin lesions whatever. ; one of them had never been in contact with the patient at all but had simply handled specimens sent down to the laboratory. It was now obvious from monkey experiments that an almost infinitesimal quantity of blood in contact with the skin could produce the disease. The fact that these cases occurred out of all possible range of infection by mosquito transmission showed the care that had to be taken by medical officers and nursing staffs in countries where the disease was endemic. There was no doubt that many doctors and nurses must have contracted the disease in the past when taking blood specimens in the incubation stage or during the first day or two of fever, before diagnosis was possible. These accidents showed how important it was to use gloves, and how necessary it was for those dealing with yellow fever, or potential yellow fever cases in endemic areas, to be immunized as soon as possible. The second point that interested him was the type of reaction following immunization. Dr. FrNDLAY had asked him to see Dr. BROOM during the peculiar reaction which he had experienced. From a clinical point of view it certainly appeared to be a modified attack of yellow fever. He would like to know whether those who had febrile types of reaction showed bradycardia, for this was a feature of Dr. BROOM'S reaction. The only other experience he had had of the reaction to yellow fever inoculation was a much more pleasant one. Recently he had been working on some of Dr. FINDLAY'S monkeys and had thought it advisable to be immunized. This had been carried out by Dr. FINDLAY and had been followed by no reaction whatever, though a very

DISCUSSION.

467

satisfactory grade of immunity, as measured by protection tests, had developed. Dr. G. Carmichael Low said that Dr. FINDLAY'S remark that serological tests carried out in Santa Cruz showed during an epidemic there a greater number of people with immunity to yellow fever than had actually had the disease, reminded him of his experiences in the West Indies many years ago. There it was recognized that, during an outbreak of yellow fever, there were many cases of slight illness, some with traces of albumin in the urine, and the question arose whether there were really mild cases of yellow fever. It was also known that the locally born population, not necessarily black, did not, as a rule, get yellow fever in the epidemics in Barbados, St. Lucia and other West Indian Islands. It was supposed that they had suffered from mild yellow fever when they were young, and so had gained immunity to the disease. The application of the serological tests might throw light on the immunity of these people. Dr. FINDLAY had suggested that it was STOKES, BAUER and HUDSON who had shown that the virus of yellow fever was filterable, whereas in his (Dr. Low's) opinion, this had been clearly demonstrated by the members of the original American Commission who had investigated the disease in 1901. Dr. FINDLAY said that Dr. FAIRLEY had not given a reaction to immunization though afterwards his blood became highly protective. Was it not possible that Dr. FAIRLEYhad had a slight attack of the disease and so developed an immunity while treating the cases of accidental infection in London ? It would have been interesting if his blood had been tested for immunity first. Dr. A. J. R. O'Brien said that in a long experience in West Africa he had seen many cases of yellow fever, most of which had been in Europeans who had died. He and other medical officers with experience of yellow fever considered that the natives of West Africa had some natural immunity to the disease, because, as a rule, in them it was very difficult to diagnose. Though in many cases yellow fever was suspected it had not been possible to prove the diagnosis till the monkey inoculation test had been discovered. Most of the Europeans who contracted the disease died, but now it was possible to look forward to the time when all Europeans would be protected. Though, as a rule, the cases in natives were mild, he could remember that in the Assamankessi outbreak, the virulence and mortality was as high in natives as in Europeans. Examination of the blood of Europeans, who had been resident for several years in West Africa, had shown by the presence of immune bodies, that several must have contracted yellow fever at some time, although in such a mild form that the disease was not diagnosed or even suspected. This and the Assamankessi outbreak rather supported the theory of Colonel JAMES that there might be two strains of the virus. He would like to have some information as to the duration of the immunity. When the protective titre of the serum got low he supposed it would be possible to raise it again easily by giving further doses of mouse brain virus.

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DISCUSSION.

Dr. A. Felix said that the position in regard to yellow fever at present was like that of typhus fever some 8 or 10 years ago, when it was suggested that immunization should be carried out by the simultaneous injection of immune serum and virus. The two viruses were similar in that the form which existed in the tissues of the vertebrate failed to produce immunity if injected after being killed. In the case of the typhus group of diseases, however, the virus in the bloodsucking insects was different, for killed suspensions of virus from the louse, tick or rat-flea were capable of producing active immunity and were being used at present on a small scale. In spite of the dangers of the experiment he thought it might be worth while testing the immunizing power of the killed yellow fever virus as it existed in the mosquito.

Dr. H. $. Stannus : The change in character of the virus in the hedgehog mentioned by Dr. FINDLAY,led him to ask if there were any variations in the reactions in these animals according to the time of the year, and if hibernation had anything to do with it. Hibernation was well-known to have an effect upon infectability and there was the interesting observation also that if the adrenals of a hibernating hedgehog were removed symptoms of deficiency did not develop until after the period of hibernation was over. Dr. Findlay (in reply) : It must be remembered that immunization against yellow fever is as yet only in its infancy : further improvements in technique will almost certainly be developed, for even in the case of active immunization against smallpox, initiated in the East more than 250 years ago, there is as yet no definite agreement as to the best and most satisfactory procedure. Colonel JAMrS has raised the important point as to whether strains of yellow fever virus of low virulence may not be responsible for yellow fever infections in certain areas, aptly termed " silent areas," where outbreaks of yellow fever have not been recorded though sera containing protective antibodies against yellow fever have been obtained. The study of yellow fever in laboratory animals has shown that different strains of yellow fever virus undoubtedly do exist, some such as the F.W. strain being much less pathogenic than others for monkeys. This difference in virulence persists after repeated passage. On the other hand so little is as yet known about these silent areas that no dogmatic statement is at present possible. In one silent area, sparsely populated by Europeans, epidemics of jaundice are known to have occurred; certain of these epidemics may have been yellow fever. There is in addition the question of personal idiosyneracy both to infection with the virus of yellow fever and to immunization. The most severe reaction which we have had occurred in my colleague Dr. BROOM, who was immunized by the original method involving the use of 0"5 c.c. of immune serum per kilogram of body weight. The symptoms resembled a mild case of yellow

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fever, yet my laboratory assistant who was immunized with the same material on the same day had a very mild reaction of less than 24 hours duration. While no one today can gainsay the importance of the fundamental work of REED and the American Commission on Yellow Fever which showed that the virus of yellow fever was filterable it must be remembered that there is a great difference between the terms " filterable " and " ultramicroscopic." Many organisms such as the Leptospira are filterable but can hardly be looked upon as ultramicroscopic. Due credit should therefore be given to the fundamental discovery of STOKES, BAUr~R and HUDSON whose work definitely placed the mtiological agent of yellow fever among the group of ultramicroscopic viruses. The suggestion of Dr. FELIX in regard to the use of virus derived from the insect host as antigen is of considerable interest ; Rickettsia, however, undergo active multiplication in their insect hosts while the evidence at present available does not suggest that the yellow fever virus increases in amount in the mosquito vector. With regard to the question of Dr. FAIRLEY'S immunity it may be stated that in the absence of any illness resembling yellow fever the development of immunity is so rare as to be almost unknown. Dr. FAIRLEY had no illness when in contact with actual yellow fever cases.