VACCINATION WITH VIRUS q/D IN THE CONTROL OF JUNGLE YELLOW FEVER IN BRAZIL b) Fred L. Soper and H. H. Smith, International Health Division of the Rockefeller Foundation, Rio de Janeiro 33 Reprinted from the Transactions of the Third International Congress of Tropical Medicine and Malaria, 1938, Vol. I, pp. 295-313 PRINTED IN HOLLAND BY C. A. SPIN & ZOON N.V. VACCINATION WITH VIRUS 17D IN THE CONTROL OF JUNGLE YELLOW FEVER IN BRAZIL1 by Fred L. Soper and H. H. Smith, International Health Division of the Rockefeller Foundation, Rio de Janeiro Vaccination in Jungle Yellow Fever The first attempt to protect an exposed population against jungle yellow fever by vaccination was made in Parani, Brazil, early in r 9 36 (I), using hyperimmune goat and monkey sera and a virus nlodjfied by culture in mouse embryo tissue (I 7E) (2). The difficulties encountered were such as to cause the discontinuation of this method in the field, and during the yellow fever season (January to May) of 1937, no attempt was made to protect exposed populations. Work with another modified virus (I 7D) developed in the laboratories of the International Health Division of The Rockefeller Foundation in New York, began in Brazil in February, I 93 7 (3). By June, these studies had progressed far enough to justify field vaccination, and the county of Varginha, Minas Geraes, in a region where jungle yellow fever had been found a few weeks previously, was chosen for the first field application of the new vaccine virus. During the next three months, 2,746 persons were vaccinated in the field, with satisfactory results, and, in September, routine field vaccination began, which increased the total field vaccinations for the year 1937 to 36,104. The 193 8 yellow fever season in South Brazil began early in January, with an outbreak of jungle yellow fever at Presidente Wenceslau, S3o Paul0 (4), and shortly thereafter the disease was found at Mathias Barboza, Minas Geraes. Vaccination units were moved into both these districts, and an attempt was made throughout the following months to vaccinate threatened populations wherever yellow fever was found. The I y 3 8 yellow fever season was an active one, with outbreaks in some of the richest and most heavily populated agricultural districts of Brazil, in the states of Minas Geraes, Rio de Janeiro, SZo Paulo and Santa Catharina. The need for vaccine greatly exceeded the initial production capacity of the laboratory and the 1 This report is based on work of many colleagues of the Cooperative Yellow Fever Service, iointlv maintained bv the Ministrv of Education and Health of Brazil and tde `Inte&ational Health' Division of' The Rockefeller Foundation. Special credit for the rapid expansion of vaccination in 1938 must go to the Brazilian Government, which furnished the necessary additional funds. 295 FRED L. SOPER AND H. H. SMITH ability of the field service to apply it. The Brazilian Government opened a special credit of 2,000 cantos, or approximately $ 100,000 UScy., to cover the cost of a program for the vaccination of at least one million persons during the year I 93 8. From January first to July 3 ~st, a total of x57,86 I persons were vaccinated, and the final figures for the year will almost certainly exceed the preliminary estimate of one million. Table I gives the number of persons vaccinated per month in Brazil, from September, I 937, to July, I 93 8, by states. Table II gives the distribution of the same persons by population groups. Origin of Vaccine Virus 17D In December, 1933, Lloyd transferred the Asibi strain to tissue culture containing mouse embryo tissue and monkey serum ; after I 8 subcultures, a second transfer was made to a medium containing whole chick embryo tissue, from which, after 56 passages, it was transplanted to tissue culture containing chick embryo, from which the central nervous system had been removed. After 39 passages in this medium, without central nervous system tissue, this strain of virus, now known as I'D, was tested and found to have lost much of its viscera- and neurotropism, while still retaining the property of stimulating the production of antibodies (5). Virus I'D was first used for human inoculation on November 30, I 936, in New York (6), with material transferred 227 times in tissue culture since its last previous passage in an animal host. Subcultures used as source of vaccine in Brazil have ranged from the 205th to the 3 I 7th. Results Obtained with Virus 17D The points on which a method of vaccination for general use as a public health measure should be judged, may be grouped under the following headings : a. Ease of manufacture of standard product. b. Ease of application under field conditions. C. Safety and comfort of persons vaccinated. d. Safety of persons not vaccinated. e. Antibody production. A. Ease of Manufacture of the Standard Product The titer of virus in tissue culture material is much below that obtained by grokth in the' developing chick embryo. The vaccine 296 VACCINATION WITH VIRUS r7D IN JUNGLE YELLOW FEVER virus is maintained in tissue culture free of central nervous system tissues, to avoid any possible reversion to type, but for the preparation of iaccine, tissue culture is inoculated in the allantoic sac of the sir-day old chick embryo. After further incubation for four days, ,u a temperature of 37' C., the embryo is removed, triturated, and suspended (I o %) in inactivated human serum diluted with equal anlounts of distilled water. The filtrate of this suspension, which is the vaccine material, is distributed in ampoules, frozen, dried in v;Icuum, sealed and stored at about 2' C. In addition to the usual bac- teriological controls for sterility, each lot of vaccine is titrated for virus content by intracerebral inoculation in serial dilutions in white mice, and is inoculated intracerebrally into a rhesus monkey, to test for possible increase in either viscera- or neurotropism. Altough laboratory studies indicate (3) that a much smaller dose may be sufficient, between 350 and 800 MLD 1 for mice are now being allowed for each person vaccinated in Brazil. On this basis the Rio de Janeiro Laboratory `is producing some I 20,000 doses of vaccine per month, at a total cost, including overhead, excepting rent, of less than $ 3,odo UScy., or 2;: cents UScy., per dose. B. Ease of Application under Field Conditions Virus I 7D, even when dried and sealed, is susceptible to ordinary temperatures and to direct sunlight ; the vaccine leaves the Rio laboratory, packed with ice and salt, in wide-mouthed thermos flasks, and is thus kept chilled until the moment of rehy- dration. Even after rehydration with distilled water the ampoule is kept on ice, and the vaccine is finally diluted in physiological saline solution in the syringe itself immediately preceding inoculation. To determine the viability of the virus used, mice are inoculated intracerebrally with the remaining vaccine after the last person has been inoculated. Experience shows that a vaccination unit, consisting of three persons, a doctor, a technical assistant and a secretary-chauffeur, can, under optimum conditions, register and inoculate from I ,000 to 2,000 persons a day a. The actual cost of applying vaccine in Brazil I The end point of titration in mice is considered as that dilution which, when injected in 0.03 C.C. amounts intracerebrally in mice, will produce a mortality. of lo% (7). s The use of three Forsbeck needle-racks by each unit is advisable, to avoid unne- cessary delays in waiting for needles to cool after boiling. It is believed that certain irregular results of postvaccination protection tests are due to failure to cool needles after boiling, with consequent inactivation of the vaccine virus. 297 FRED L. SOPER AND H. H. SMITH in I 9 3 8 has not exceeded, including initial cost of automobiles and equipment, 7 cents UScy., per capita. The actual field operating expense has dropped from 5% cents UScy., per capita, in January, to 3 cents UScy., in June. However, the per capita cost of application must increase rapidly in sparsely populated regions and in areas where transportation is difficult. C. Safey and Comfort of Persons Vaccinated Since the beginning of work with virus I'D in February, I 93 7, a conscientious search has been made among vaccinated groups for evidence of: I. Severe reaction at site of inoculation ; 2. Sensitization to foreign protein ; 3. Serum sickness ; 4. Virus reaction, visceral and neural ; 5. Delayed jaundice, and 6. Infection with other viruses. . Special attention should be called to the distribution of vacci- nated persons by population groups (Table II). `Employees of the Yellow Fever Service, of the airlines, the population of large coffee fazendas, inmates of schools, laborers and highway construction gangs and members of military units, all form very useful groups for observation. Even where it has not been possible for physicians of the Yellow Fever Service to make personal observation, fazenda owners, military medical officers, school directors and other respon- sible persons have given information as to the severity of postvacci- nation reactions. The sum total of observations on vaccinated groups may be stated briefly as follows : For the eighteen months' period, during which almost 600,ooo persons were vaccinated, there is no evidence of severe reaction at the site of inoculation, of sensitization to foreign protein 1, of serum sickness, of delayed jaundice (8), (y), nor of infection with other viruses. The type of relatively mild reaction which is observed seems to be a general, not neural, reaction to the virus itself, after an - l A number of cases have received second and third inoculations of r7D, without any evidence of sensitization to chick protein. 298 VACCINATION WITH VIRUS I'D IN JUNGLE YELLOW FEVER incubation period of generally from five to eight days. 1 The symptoms most frequently noted are : headache, backache, body pains, weakness and malaise, lasting from a few hours to a couple of days. The reaction to virus I'D is not severe enough to have any influence against its general acceptance by the people. Fazenda owners, and others responsible for large groups, generally report from 5 to 8 y0 of reactions, with not more than I to 2 yO of reactions severe enough to cause loss of time from work. A person- to-person canvas, however, will result in 20, 40 or even 50% of individuals questioned reporting at least a slight headache, but the number of severe reactions does not increase correspondingly. The most severe reactions reported are those related to each other by members of the foreign colony in the capital city of Rio de Janeiro ! Considering the number vaccinated, it seems truly remarkable that many more conditions occurring after vaccination have not been credited to the inoculation. Experience has failed to reveal any contraindications to the use of virus I 7D, early restrictions have been entirely removed, and children of all ages and women in all stages of pregnancy are routinely inoculated. D. Safe9 of Persons Not Vaccinated In using a living virus for vaccination, the possibility of such living virus being picked up from the blood stream by some insect vector, and sooner or later reverting to its original virulence, must be considered. Such return to virulence of a yellow fever vaccine would have to depend upon the following factors : I. Circulation of virus in the blood stream in quantities sufficient to infect the insect vector ; 2. Ability of the infected vector to transmit the vaccine virus, and 3. Ability of the vaccine virus to revert to a virulent state. Experimental work indicates that sufficient virus does not circulate to infect the traditional vector, AZdes aegypii, and that even when this mosquito has been infected by special methods it does 1 So far, only one case has been reported, in which symptoms of involvement of the central nervous system were attributed by the attending physicians to inoculation with virus 17D. Case E. R. C., observed by Drs. Raul Azevedo and Deolindo Couto, Rio de Janeiro, to whom we owe thanks for details of this case, developed signs of meningeal involvement one month after vaccination with Lot 136 of virus r7D, the estimated virus used beiig zao MLD for mice. Complete recovery occurred, and studies are now in progress to determine, if possible, the nature of the infection.. 299 FRED L. SOPER AND H. H. SMITH not readily transmit the r7D virus, even after prolonged incubation (IO). Attempts to infect AZdes aesypti by postvaccination feeding on humans and on rhesus monkeys, which have been shown to circulate more virus than do humans, were failures, no virus being demon- strated in the mosquito by either feeding'on monkeys or inoculation into mice. The immersion of AZdes aegypti larvae in high concentration of virus did result in the production of infected mosquitoes, as demonstrated by mouse inoculation ; such infected aegypt' failed completely to transmit virus to susceptible animals, even after prolonged incubation periods. The difficulty of getting virus I'D to circulate in appreciable quantities with regularity, has, so far, prevented conclusive experi- ments with the jungle vectors of yellow fever, only a few of which have very recently been definitely incriminated (I I). The same difficulty has prevented the carrying out of a large series of animal passages, to determine the ability of virus I 7D to revert to its original type ; the relative stability of the virus in tissue culture, embryo passage and in mouse brain passage, suggests that such reversion to virulence, if it did occur at all, would be slow in appear- ing. This opinion is strengthened by the results of other workers, who have not been able to transmit a tissue culture virus with A&les aegypti' (I 2), nor to reconvert it to virulence by direct liver-to-liver passage (I 3). E. Anti body Production The rhesus monkey, which is more highly susceptible to yellow fever than is man, becomes fully resistant to virulent strains, such as Asibi, following inoculation with virus I'D. Similar tests on humans have not been made, but the wide use of virus 17D this year, among exposed populations, during active outbreaks of jungle yellow fever, has resulted in a mass of field observation almost as conclusive as laboratory experiments. Local physicians and other observers report a sudden reduction in observed cases in infected districts shortly after mass vaccination, and cite instances in which individuals, who failed to be inoculated, later contracted the disease in infected forests, while vaccinated members of the same labor gangs escaped. Field experience suggests that the protective effect of vaccination begins not later than a week after inoculation, although laboratory tests fail to show demonstrable antibodies at this time (3). While it is probable that a much larger number of cases of yellow VACCINATION WITH VIRUS r7D IN JUNGLE YELLOW FEVER fever must have occurred among persons infected before vaccination, only eight of these have been reported, four in Minas Geraes, three in Santa Catharina and one in SZo Paulo. Onset in two was on the same day as vaccination, in the other four, between the first and fourth days following. Two of the three fatal cases in this group were confirmed by viscerotomy, and a virus, quite different from the vaccine virus, was isolated from one of the non-fatal cases. Two additional cases of postvaccinationyellow fever have been found, one mild case with onset thirty days, and one fatal case with onset six weeks after vaccination. These cases had received virus from lots 95 and I I 7, both of which gave irregular results, as measured by the protection test (Table IV). It is possible that neither received active virus. The mouse protection test (14) has been used since I 93 I, for determining the presence of yellow fever antibodies in the blood serum of persons and animals. It is customary to inoculate six mice with highly neurotropic virus and with the serum to be tested. Results are read as a fraction showing the proportion of mice living on the fourth day (d enominator), which survive to the tenth day (numerator) after inoculation. Seven readings are possible, of which only two, 6/6 and 516, are, in analyzing critical immunity surveys, considered definite evidence of previous infection with yellow fever ; 4/6 and 3/6 results are considered inconclusive, and 216, 116 and o/6 as negatives. It has been noted in immunity surveys that bloods from regions where yellow fever has never been present give remarkably clearcut negative readings, whereas bloods from endemic regions give an appreciable number of inconclusives, as well as positives and nega- tives. The majority of these inconclusives are probably from indivi- duals who have at some time been exposed to yellow fever infection, and are, almost certainly, not apt to ever again develop clinical yellow fever. It seems'reasonable at the present time to read mouse protection test results as indicating full protection, partial protection and no protection, without attempting to interpret too rigidly these readings in terms of reaction to yellow fever infection, further than to assume that those showing full protection are, at the moment tested, adequately protected against fully virulent virus. Postvacci- nation results, when compared with prevaccination results (Table III), suggest that virus I'D does produce some measurable antibody formation in almost IOO y0 of persons receiving 50 MLD or more FRED L. SOPER AND H. H. SMITH of living virus. It has been noted that in many postvaccination pro- tection tests, in which the final reading is : 216, 116, or even o/6, the average length of survival of inoculated animals is from one to two days longer than for similar negative tests in vaccinated groups. This suggests that sufficient antibody is present to definitely delay the action of virus inoculated in animals. Table III gives the results of pre- and postvaccination tests on the same individuals, including both laboratory and field groups 1, during the preliminary phase of observation, before routine field vaccination began. Attention must be called to the fact that on one occasion, the virus was apparently inactive before inoculation began, since all of the persons tested failed to give evidence of antibody development, and the inoculation of the remaining vaccine into mice failed to cause any deaths. Table IV covers a special investigation to determine the results obtained with different dosages of virus, and to evaluate the viability test as an indication of efficiency of the preceding vaccination. The groups bled for this special study were selected as probably represen- tative of the poorest work of the season, and included groups receiving the lowest doses of virus used during the height of the yellow fever outbreak, working with newly trained personnel, far from head- quarters. The results indicate that doses as low as so, 85 and IOO MLD per person are adequate to give satisfactory results. They also indicate that the viability test, in and of itself, is not a safe indication of the efficiency of the vaccination. For example, lot I I 7 of virus I'D was used and tested in five groups, of which only one gave satisfactory results, the viability tests for which, o/s, 115 and 1/4, were poor. Postvaccination mouse protection tests on a number of persons from vaccinated groups are proving a better method of checking the work of field units than is the test for viability of the remaining vaccine. Table V gives a general summary of all postvaccination protection test results for work with virus I'D in Brazil. A study of Tables III, IV and V and other available information suggest that the differences in the results of vaccination depend in great part upon the delivery of relatively small amounts of active virus below the skin of the individual vaccinated. The results show 1 Vaccination of these groups was carried out under the direct supervision of Dr. H. H. Smith, who, with Drs. Henrique Penna and Adhemar PaolieUo (3), has published a report covering observations on the first 60,000 vaccinations in Brazil. 302 VACCINATION WITH VIRUS r7D IN JUNGLE YELLOW FEWER that with standardized methods of vaccine production and with adequate supervision of the administration of virus in the field, highly satisfactory results can be obtained. Anticipated Epidemiological Results of Vaccination Admitting that the individual can be protected by vaccination, the epidemiological results of vaccination must vary with the con- ditions under which infection,occurs. Where man is an essential element in the cycle of infection, responsible for maintaining the virus, as in urban oegypti-transmitted yellow fever, artificial immuni- zation of the bulk of the population should effectively protect the remaining non-immunes. It is probable that occasional mass vacci- nation will be found more economical and practicable in certain regions, for breaking the cycle of infection, man-aetlypti-man, than is the traditional maintenance of antimosquito services for the prevention of aegypti breeding. In considering jungle yellow fever, however, in which man is, apparently, not an important factor in maintaining the virus, vacci- nation should alter the epidemiological picture, mostly by preventing the infection of vaccinated persons, and, only in a very minor degree, by reduction of the source of virus for forest vectors. Vaccination promises to be a great aid in preventing the transfer of yellow fever infection from one place to another by the human host ; the long-distance transfer of virus, by modem methods of rapid transportation, can be prevented by vaccination, as can also the introduction of virus from jungle to urban areas. Since the jungle infection, apparently, exists independent of the human population, and spreads from place to place by other than human carriers, vaccination cannot be expected to completely eradicate yellow fever. Summary. During the period, September I 937 to July I 93 8, over half a million persons were inoculated with the modified yellow fever virus r7D. Vaccination with this virus was widely used throughout the I 93 8 epidemic of jungle yellow fever in South Brazil. Field obser- vations indicate that vaccination becomes effective within a week after inoculation. Reaction to vaccination is relatively mild, and no contraindications have been found. The results of approximately 3,000 mouse protection tests are presented, showing that a high percentage of persons vaccinated develop demonstrable antibodies. 303 I. 2. 3. 4. 5. 6. 7. 8. 9. IO. II. 12. 13. `4. 304 FRED L. SOPER AND H. H. SMITH Bibliography Soper, F. L. Vacina+o contra a febre amarella no Brasil, de 1930 a 1937. Arch. de Hie. Rio de Janeiro. 1937. 7: 379-390. Lloyd, Wray, Theiler, M., and Ricci, N. I. Modification of the virulence of yellow fever virus by cultivation in tissue in vitro. Tran. Roy. Sot. Trop. Med. Hyg. 1936. 29: 481-529. Smith, H. H., Penna, H. A., and Paoliello, A. Yellow fever vaccination with cultured virus (I 7D) without immune serum. Am. Jr. Trop. Med. 1938. In Press. AragZo, H. de B. Observa@es a respeito de urn foco limitado de febre amarella sylvestre no Estado de SZo Paulo. Brasil Medico. Rio de Janeiro. 1938. 52 : 401-412. Theiler, M., and Smith, H. H. The effect of prolonged culti- vation in vitro upon the pathogenicity of yellow fever virus. /I. Exp. Med. 1937. 65: 767-786. Sawyer, W. A. Experience in vaccinating against yellow fever with immune human serum and virus fixed for mice. Am. Jl. Hyg. 1937. 25: 221-231. Reed, L. J., and Muench, H. A simple method of estimating fifty per cent endpoints. Am. Jl. Hyg. 1938. 27: 493-497. Findlay, G. M., and MacCallum, F. 0. Note on acute hepatitis and yellow fever immunization., Trans. Roy. Sot. Trop. Med. Hyg. 1937. 31 : 297-308. Soper, F. L., and Smith, H. H. Yellow fever vaccination with cultivated virus and immune and hyperimmune serum. Am. /I. Trop. Med. 1938. 18 : II r-134. Whitman, L. Failure of ACdes aeavpti to transmit yellow fever cultured virus (I 7D). I 93 8. In Press. Shannon, R. C., Whitman, L., and Franca, M. Yellow fever virus in jungle mosquitoes. Science. 1938. 88 : (No. 2274) I IO-I I I. Roubaud, E., Stefanopoulo, .G. J., and Findlay, G. M. Essais de transmission par les stegomyies du virus amaril de cultures en tissu embryonnaire. Bull. Sot. Path. Exot. 1937. 30 : 581-583. Findlay, G. M., and MacCallum, .F. 0. Vaccination contre la fievre jaune au moyen du virus pantrope att&mk employ6 seul. Bull. O$. lnternat. d'Hyg. Publ. 1937. 29 : 1145-II.+g. Sawyer, W. A., and Lloyd, Wray. The use of mice in tests of immunity against yellow fever. jl. Exp. Med. 1931. 54 : 533-555. VACCINATION WITH VIRUS 17D IN JUNGLE YELLOW FEVER TABLE I Persons in Brazil vaccinated with virus 17D from September 1937 to July 31st 1938, away from the. laboratory Months September ...... October ........ November ...... December ...... January ........ February ....... March .......... April ........... May ........... June ............ July ........... Total ...... Federal district 46 3.337 13.411 IO.313 6.181 7.224 6.444 11.c-J 77.713 24.768 .09.342 34.368 28 i91.219 Rio de S"a0 Minas Santa Mato Janeiro Paul0 Geraes Catarina Grosso State State state State State 1.861 18.234 17.238 12.894 16.760 10.726 8.103 1.406 11.259 3.719 10.580 7.473 11.540 12.701 31.517 64.118 41.084 66.340 72.393 83.317 22.363 12.005 6 II II Total 3.719 10.580 7.473 11.546 20.850 36.766 96.247 94.998 97.431 99.783 r11.786 FRED L. SOPER AND H. H. SMITH TABLE II Distribution by population groups of persons vaccinated in Brazil September 1937 to July 3rst 1938 v-x$7 ) \yz;i ( Total Farms and hamlets ...... hlilitary units ........... Schools ................ Labor gangs ........... Cities and towns ........ Miscellaneous ........... Total . . . . . . . . . . . . . . . . 16.530 397.809 414.339 I.105 23.730 24.831 994 34.348 3i.342 368 39.183 39.11' 14.361 13.337 61.698 - 9.414 9.454 33.318 557.861 19'.==9 306 TABLE III Immunitv to vellow fever followine vaccination with Virus 17D measured hv nwtwc protection tw+t _ _ Where used - 7 1 1 Viability ) Per- test sons inocu- 1 lated results t Post-vaccination 26126 1 71 Pm-vaccination Post-vaccination Pre-22natiok 77 Post-vaccination 23/23 1 Pre-vaccination Post-vaccination Pre-~%ratic!n "I Post-vaccination 23123 I 79 Pre-vaccination Post-vaccination 27/27J/2 / 589 Pre-vaccination Post-vaccination pr$$~/,7,tioi, `72 Post-vaccination TOTAL 1149 Pre-vaccination Post-vaccination :e is calculated on tE Lot Dilu- No. tion hlousc protection test resulta" I Dose rl.L.D' Mouse) ajoto 7.100 a~.000 25.000 2~.000 11.000 25.000 2.500 2.500 314 ;`I' 414 515 I6 of I lice iur- ving I 4 0 11 I II 2 28 IO 89 a 82 0 22 I 20 I 2 z 7; 26 96 2 2 I 42 18 81 0 29 0 14 0 0 I 24 8: I 34 2 2 IO 185 4 76 II `3 I I 114 164 6 78 CC- ion in- iex4 ber ested o/4 015 d6 `14 I/J .I6 45 16 ! 3 7- 214 16 3lr 16 I I L I I ( - 39 I : I) t : 2) [ : I) t : 2) 1:2 Laboratory 18 `4 0 2 0 0 0 0.6 5.4 40 Laboratory 12 43 I 6 0 0 0 41 Field 66 14 0 9 0 I 3 0 7 0 18 41 I : 2) and) 1:z Laboratory IO 4 0 4 0 0 0 42 52 12 12 Field I32 109 0 16 0 3 0 1:1 I : IO 1: IO Field 69 14 0 `3 0 Field `19 122 4 26 I Field 21 I7 15 0 0 0 25 0 II 4: 0 0 527 417 20 13 I 0 0 0 5 0 3 0 0 0 7 I I 2 18 0 0 3 34 r The endpoint for titration of virus in n nortal My w * The fraction indicates the number of mice dyitg of specijic encephalitis (numerator) in comparison with number alive four days after inoculation 0 -4 (denominator). 3 The fraction indicates the number of mice Juryiving lo the tenth uhy (numerator) in comparison with number alive four days after inoculation (denominator). * Average number of mice surviving calculated on basis of six mouse groups. TABLE IV Immunity to yellow fever following vaccination with Virus 17D measured b xotection test be 1 T Num ber testec IO I7 20 21 23 21 20 21 20 I2 IO 21 22 20 20 II 20 20 20 21 21 21 19 21 s II 21 20 20 21 14 42 36 42 39 L Per- sons nocll lated 47 338 I43 329 310 364 241 237 476 72 I08 `95 94 353 III II 399 414 269 313 417 634 `19 109 405 192 356 243 60 384 1201 `307 954 - 1 i Viability test results Mouse protection test results3 - t i ; T 41 $5 2/4 316 311 I/G 1 - 314 4/5 116 Pro- tec- tion in- dex' ::i 5.8 I*' I.0 4.7 3.8 2.0 5.3 5.8 5.4 5.4 5.6 5.3 5.2 5.4 5.4 1.6 4.7 2.4 4.9 1.4 4.7 4.9 5.8 1.1 1.2 3.8 4-I 3.6 4.2 4.3 4.4 3.8 5.0 % of mice sur- riving 88 81 96 84 a2 z: i; 97 89 89 96 88 87 90 89 93 77 41 88 89 78 a2 96 84 86 64 68 60 70 71 ii: 83 Dose M.L.D' Where used Mouse: ISW 85 85 130 130 SO *:: 850 85 87 170 270 210 140 270 100 100 85 85 110 110 110 110 85 85 85 85 170 81 85 200 200 200 280 Farms & Hamlets Cit. & Towns Milit. Units Farms & Hamlets Farms & Hamlets Cit. & Towns Cit. & Towns Farms & Hamlets Farms & Hamlets Schools Farms & Hamlets Farms & Hamlets Cit. & Towns Farms & Hamlets Farms h Hamlets Cit. & Towns Farms & Hamlets Farms & Hamlets Farms & Hamlets Cit. & Towns Farms & Hamlets Schools Farms & Hamlets Farms & Hamlets Farms & Hamlets Farms & Hamlets Cit. & Towns Cit. & Towns Farms & Hamlets Farms & Hamlets Farms & Hamlets Farms & Hamlets Farms & Hamlets Farms & Hamlets Cit. & Towns Lot Dilu- No. tion 4/4 s/s i/6 014 O/S `16 I 0 0 I 0 0 I 3 0 0 0 0 0 I 0 I 0 I I 2 0 0 0 2 0 0 I 0 3 2 0 I 0 0 I I 0 0 0 I 0 0 0 I 0 0 0 S 0 0 0 2 0 0 I 3 0 0 1 3 0 I 0 0 0 0 0 4 0 0 0 I 0 0 0 I I I I 0 0 0 3 I 0 0 0 3 0 0 I I I I I 0 d 0 0 0 I 0 0 0 0 0 0 3 I 0 0 2 2 0 0 2 I 0 I 0 3 f 7 6 I 3 0 I I I 0 3 0 2 0 5 I 0 I 3 - L I 3 4 7 iI S 0 3 0 4 3 4 S I 3 i 7 : 7 7 S I S I i 7 I2 I2 7 IO 17 z 16 II II IO a 5 14 II 5 `3 16 II 14 7 II `3 9 4 IO II a I2 4 I I2 7 2 Ia I8 Ia 14 IS 69 79 a0 85 91 101 102 `03 106 '15 117 126 016 116 616 z;: 216 6/6 116 414 0 2 0 I I t IO I 0 0 0 0 0 I 0 0 0 3 IO I 0 2 I 0 0 I 5 z 3 G7 II I 4s 616 I*)** 316 w 16116 616 2;; 416 4/s 014 014 s/s S/I o/s I/J 616 616 I/S 116 /6,1+2, s/j 4 314 ( 015 I/S J 4/4 The endpoint for titration of virus in mice is calculated on the basis of 10 % mortality. The fraction indicates the number of micz dying of specifi etrcepbalifis (numerator) in comparison with number alive four days after inoculation (denominator). The fraction indicates the number of price suryiving fo the tenth uky (numerator) in comparison with number alive four days after inoculation (denominator). Average number of mice surviving calculated on basis of six mouse groups. 308 TABLE V Immunity to yellow fever following vaccination with Virus 17D measured by mouse protection test Lot NO. 39 40 41 41 42 12 12 I* 14 14 IS s6 17 s* 19 60 61 62 63 64 65 67 68 69 :t 77 78 79 80 83 84 81 88 91 101 102 103 106 112 11s 7'7 123 126 136 137 T Dilu- tion T Dose M.L.D' :Mouse) 810 to 7.100 8 5 .ooo 2J.000 2J.000 I I .ooo 2 5 .ooo 2.500 2.JOO ? 6.800 540 ? 230 ? I .700 760 I.700 ? 140 340 540 170 r.yoo 170 810 8.50 400 85 130 230 8.50 SO *SO *SO 170 210 100 8.5 230 110 170 200 200 220 280 Where used Laboratory 63165 20 I8 0 Laboratory 201/202 71 $2 I Field and Laboratory Field Field Field Field Field and Laboratory Field Field Field Laboratory School & Field Field School & Field Army & Field City Br Field Field City & Field Laboratory Field Field Army & Field Field City & Field Field Army & Field Field City Field City Fiidj & Lab. School & Field City & Field Field City Laboratory School & Field Citv & Field Field City & Field Laboratory Field IS/I8 27129 34134 23123 28129 I/18 79186 1981203 I21/12~ 1341138 49188 *07/*40 68/84 100/107 196/*v 205/218 134/w 78/78 81184 171/188 41171 17170 so/Ios 184/190 3001377 1391149 40/51 **I37 *70/*79 37147 47154 I 861202 145J163 212/232 264/282 ror/106 20123 85194 27142 I 50/208 118/176 59185 *40/*5 3 87189 TOTAL 77. 141' 840 1245' ISSO 1868 co4 126 1680 1943 22 2284 1866 1743 73 1293 714 2152 4414 5933 2807 1486 1781 3'91 41x4 2182 4752 6233 6822 7995 3867 624 21 3614 `0414 10259 6166 9233 7400 3089; 66 IO 132 69 159 21 I43 76 108 80 2 :i 99 129 s= 56 73 24 18 IO 47 93 128 84 37 44 100 3' 43 62 41 46 70 40 41 14 80 113 I 120 3;: 2944 0 0 0 0 4 `I 3 0 0 2 11 I 4 I 5 I 3 I 0 4 t 6 2 3 3 2 2 0 9 I 11 0 I 0 13 0 4 IS 0 23 0 I 170 Viability test results Pef- sons nocu- lated km- her ested I- O/4 I/4 O/S I/S ,I6 16 Mouse protection test results 3 *Is L/G 214 516 0 -- 311 4 314 415 i/6 -- 0 0 0 0 0 0 I 4 4 0 2 0 2 0 0 I 3 I 0 I I 0 0 0 4 I I 0 0 0 4 0 0 I 3 0 0 0 I 0 I 0 3 0 1 48 I t 41. s/s i/6 % of mice sur- riving Pm- tec- tioo in- kx4 0 3 4 II 89 1.4 0 I 7 II 28 82 S.1 3 0 0 0 2 0 4 0 2 0 0 0 0 I I I 2 0 2 0 0 0 0 I I I 0 2 2 0 0 2 2 I I 0 I 0 I I 0 6 0 0 48 3 0 7 I 18 0 20 3 IO 2 iz 2 7 I2 I 2 3 4 0 I 0 I I II 3 I I s 0 I I I 0 2 0 I 1 s 9 0 1 1 1 162 18 0 3-5 `I 46 0 33 8 28 Y I I2 6 13 16 14 6 II 10 7 I I 4 21 22 IO 3 3 7 3 z 2 7 7 I I 2 s `3 0 9 2 3 427 22 2 42 29 14 0 44 28 32 22 4 20 17 31 32 36 21 16 29 7 3 I 9 24 30 29 7 14 3' 12 II `9 3 7 II II II I 2s 41 I 30 3 `7 85'3 I 20 8 s* 24 34 *i 35 37 34 49 I 29 26 24 tt `9 22 26 IO s 7 23 39 :1: 23 22 49 16 20 24 `9 31 48 24 `3 6 39 19 4 48 3 `I 231 78 96 85 84 76 13 ii 78 89 41 83 82 :: 83 81 78 81 :: 88 73 i! 8.5 `35 83 82 90 71 82 59 9= 92 9= 58 8s 83 72 96 69 81 83 80 4.8 S-8 S-1 I.1 4.1 0.8 4-4 1.3 4.7 1.4 2.4 s.0 4.9 4.8 4.6 s-0 I.0 4.8 4.9 ::i I.1 4.4 4.8 4.9 s.0 S-1 s-0 s.0 1.4 4-3 4.6 3.6 1.5 1.4 S*S 3.5 S.1 s-0 4.2 S-8 4.x 4.9 0 ::* 1 The endpoint for titration of virus in mice is calculated on the basis of 50 % mortality. 2 The fraction indicates the number of mice dying of Jpeci'c encepbafitiis (numerator) m comparison with number alive four days after inoculation (denominator). 3 The fraction indicates the number of mice s~ruiving to the tenth day (numerator) in comparison with number alive four days after inoculation (denominator). 4 Average number of mice survivin,? calculated on basis of six-mouse groups. 5 Revaccination immunes. FRED L. SOPER AND H. H. SMITH IMMUNITY TO YELLOW FEVER BEFORE AND AaFTER UACCINATION WITH VIRUS 170. VACCINATION WITH VIRUS x7D IN JUNGLE YELLOW FEVER MONTHLY PROGRESSOF ROUTINE FIELD VACCINATION IN BRAZIL, SEPTEMBER 1937 TO JULY 1938 l10.00~ 100.001 9o.ooc 8O.OOC 7OOOf 6O.OOC 2 s 3 5o.ooc 2 2 4oooc 30.000 20 000 10.000 o- 3- ,, I- I- I- )- I- A FRED L. SOPER AND H. H. SMITH POST VACCINATION MOUSE PROTECTION TEST RES'ULTS ON 2944 PERSONS INOCUL4TED WITH VIRUS 17D 1.300 1.200 1.100 1.000 900 600 700 600 500 400 300 200 100 0 1 I VACCINATION WITH VIRUS r?D IN JUNGLE YELLOW FEVER Disptltutio. W. A. P. Schiiffner (Holland) : Mit besonderer Genugtuung horte ich die Vorschlage Sopers. die er beziigl. des Ablesens des mouse-protection-tests machte. Sie stimmen mit den von uns in Amsterdam gegebenen erfreulich iiberein. Die Anspriiche, die man an den Mause-Versuch stellen muss, haben sich mit der Zeit ge- andert . Urspriinglich von Theiler und von Sawyer ausgearbeitet, wurden die Methoden von franzosischer und portugiesischer Seite (im Office intemationa1 d'hygi&ne) als nicht spezifisch angegriffen. Um diesen Vorwurf zu entkriften, vermehrte Sawyer die Menge des Virus ; statt einer 10% Emulsion nahm er eine 20% ; damit konnten unspezifische Reaktionen (die iibrigens kaum vorkommen) nlit noch grijsserer Sicherheit ausgeschaltet werden. Aber natiirlich gingen damit schwache spezifische Reaktionen verloren. Heute aber, wo an der Spezifitat des Miuse-Versuchs nicht mehr gezweifelt werden kann, verlangt die Erforschung der Epidemiologie des Gelb- Iiebers such das Erfassen einer schwachen Immunitat. Wir haben daher einmal die schwgchere Emulsion (I o yO, und davon 0,`2 cc. mit 0,q zu priifendem Serum intraperitoneal gegeben) beibehalten und zweitens, ebenso wie heute nun such Soper, vorgeschlagen, die Resul- tate, die jetzt noch als zweifelhaft oder gar als negativ gelten, mit zu beriicksichtigen. Ich stimme Soper vollkommen bei, wenn er daran erinnert, dass selbst ein volkommen negativ abgelaufener Mausever- such (6/6) noch nicht eine Rest-ImmunitEt ausschliesst. Zu dieser Auffassung wurden Snijders und ich friiher bereits bei unsern Dengue- Untersuchungen gedrungen, spgter hatten wir sie fiir das Verstindnis der Verhsltnisse in Suriname, wo der Eingeborene auffallend resistent bei Gelbfieberepidemien war, notig. 313