Ontogeny of Yellow Fever 17D Vaccine: RNA Oligonucleotide Fingerprint and Monocional Antibody Analyses of Vaccines Produced World-Wide

J. gen. Viral. (1983), 64, 627-637. Printed in Great Britain 627

Key words: yellowfever 17D vaccine/oligonucleotidefingerprint/Asibi virus/monoclonal antibody

Ontogeny of Yellow Fever 17D Vaccine: RNA Oligonucleotide Fingerprint and Monocional Antibody Analyses of Vaccines Produced World-wide

By T. P. MONATH, 1. g. M. KINNEY, 1 J. J. SCHLESINGER, 2 M. W. BRANDRISS 2 AND PAUL BR]~S 3 1Division of Vector-Borne Viral Diseases, Center for Infectious Diseases, Centers for Disease Control, Public Health Service, U.S. Department of Health and Human Services, Fort Collins, Colorado 80522, U.S.A., 2Department of Medicine, The Rochester General Hospital and University of Rochester, School of Medicine and Dentistry, Rochester, New York 14621, U.S.A. and 3 Virus Diseases Unit, World Health Organization, Geneva, Switzerland

(Accepted 23 September 1982)

SUMMARY Yellow fever 17D vaccines are currently manufactured with approval of the World Health Organization (WHO) in 11 countries. These vaccines have proven highly efficacious and safe. Nevertheless, they have not been fully characterized genetically, a problem for future standardization and modernization of vaccine manufacture now being proposed by WHO. Vaccines in use are derived from two distinct substrains (17D-204 and 17DD) which represent independently maintained passage series from original 17D. In this study, all 17D vaccines produced world-wide were characterized by RNA oligonucleotide fingerprinting. Forty-two large oligonucleotides were compared, and differences from an arbitrarily selected reference strain (produced by Connaught Laboratories in the U.S.A.) were determined. With one exception (vaccine produced in South Africa), fingerprints of vaccines derived from substrain 17D-204 were identical. The South African primary seed differed in position of one oligonucleotide, reflecting a charge shift due to a single base change. This difference occurred within one egg passage; a further change in the South African vaccine occurred within one or two passages from primary seed. No antigenic differences between 17D-204-derived vaccines (including South Africa) were demonstrated by neutralization tests using monoclonal antibody. Vaccines derived from the 17DD substrain consistently differed from 17D-204 vaccines in the absence of one oligonucleotide (No. 37). This change probably occurred during 40 additional egg passages in development of the 17DD vaccines. A clear antigenic difference was shown between 17D-204 and 17DD substrain vaccines using monoclonal antibody. 17DD vaccines showed minor genotypic differences, suggesting a higher degree of genetic instability than 17D-204 vaccines. No oligonucleotide fingerprint differences were found between avian leukosis virus (ALV)-free and ALV-contaminated vaccines. No definite genomic correlate of neurovirulence was defined by fingerprinting strains with a history of encephalitic complications in man or of failure to pass monkey neurovirulence tests. Parent Asibi virus showed several oligonucleotide differences and was serologically distinct from 17D vaccine.

INTRODUCTION Yellow fever (YF) was the first disease of man shown to be caused by a virus and the third virus infection to be subjugated by the development of a vaccine (after smallpox and rabies). Theiler & Smith (1937a) described the attenuation of yellow fever virus (Asibi strain) by in vitro passage in chick embryo tissue and reported the first trials with this vaccine strain (17D) in man (Theiler & Smith, 1937 b). Mass vaccination was conducted in the late 1930's and early 1940's in Brazil, various countries in Africa, and in the armed forces of the United States and its allies.

0022-1317/83/0000-5348 628 T. P. MONATH AND OTHERS

Vaccine production in this era was not standardized; vaccine lots were prepared by inoculation of chicken eggs with the current in vitro subculture of the virus. Certain vaccine lots that had been subcultured more than 300 times were found to have become over-attenuated, resulting in loss of immunogenicity for man (Theiler, 1951), and, conversely, others demonstrated increased neurovirulence, with clinical encephalitis in a number of human vaccinees and an unusually high incidence of encephalitis in monkeys (Fox et al., 1941). These problems were addressed by the acceptance in 1945 of standards for YF vaccine production, including the adoption of a seed lot system (WHO, 1945, 1956). Primary and secondary seed lots must pass satisfactory monkey safety and immunity tests; vaccine batches prepared from the same secondary seed lot are thereby stabilized as to passage level and biological characteristics. Yellow fever 17D vaccines are currently manufactured with approval of the World Health Organization (WHO) in 11 countries (the United States, England, Federal Republic of Germany, France, the Netherlands, India, Senegal, South Africa, Australia, Brazil and Colombia). Many millions of doses have been administered, and these vaccines have proven both highly efficacious and safe. Despite this admirable record, several problems remain. First, there is a disparity between vaccines in their substrain origin. Vaccines presently in use are derived from two distinct substrains which represent independently maintained series of passages from the original 17D virus developed by Theiler & Smith (1937a). Some manufacturers use seed lots derived from substrain 17DD at the 284th to 286th passage level, whereas others use lots derived from substrain 17D-204 at the 233rd to 237th passage level. Second, certain producers have used various techniques to free vaccine seed lots from the avian leukosis virus (ALV) contaminant. Both ALV-free and ALV-contaminated vaccines are currently used. Third, full characterization of 17D yellow fever vaccines has not been accomplished, despite clear indications for heterogeneity and the demonstrated presence of plaque mixtures with variable mouse neurotropism (Liprandi, 1981). Finally, there are difficulties in large-scale vaccine production, resulting from shortages of suitable embryonated eggs, short shelf-life of vaccines, and antiquated production methods; also, periodic urgent demands for millions of doses must be met during epidemics. At a recent conference on yellow fever (WoodaU, 1981) these problems led a group of experts to recommend that the Pan American and World Health Organizations "urgently undertake a program directed at modernizing yellow fever vaccine .... evaluate cell cultures as virus production substrates .... and determine virus heterogeneity (by) molecular.., methods." The present study, using RNA oligonucleotide mapping, was designed to investigate the genetic heterogeneity of YF 17D vaccines produced world-wide. Monoclonal antibodies were used to investigate antigenic differences between 17D vaccines. YF 17D vaccines were also compared to the parent virus (Asibi).

METHODS Vaccine viruses. Primary and secondaryseed lots and representative vaccinebatches were kindly supplied to the Division of Vector-Borne Viral Diseases, Centers for Disease Control, Fort Collins, Co., U.S.A. by the directors of the 11 vaccineproduction institutes (see Acknowledgements)(Table l). Vaccines were maintained in a mechanical freezer (-70 °C) for up to 9 months until tested. The history of development of 17D viral substrains has been described elsewhere (Sawyer et al., 1944; Fox & Penna, 1943). A surveyof producers was conducted recently by WHO to define the pedigrees and passage levels of current seed lots and vaccines (WHO, 1980). Other viruses. Yellow fever virus (Asibi strain) was kindly supplied by Drs R. Shope and R. Tesh at the Yale Arbovirus Research Unit, New Haven, Conn., U.S.A. This virus (the parent of YF 17D) had been passed six times in monkeys and once in Aedes albopictus C6/36 cells. An additional passage in C6/36 cells was made to produce a seed pool. A seed lot (6766) produced by Connaught Laboratories and found to be unsatisfactory in the monkey neurovirnlence test was kindly sent by Dr B. Elisberg, Bureau of Biologics, Food and Drug Administration, Washington, D.C., U.S.A. Two other vaccine batches, NY-310 and NY-318 produced in the Rockefeller Foundation Yellow Fever Laboratoryin New York were also supplied by Dr Shope. One of these lots (NY-310) had been associated with encephalitic reactions in Brazil in 1941 (Fox et al., 1941). Growth and purification. Growth curves of a single vaccine lot (2654 LE, Connaught Laboratories) were performed in four different continuous cell lines (LLC-MK2,-Vero,gW2i3 and A~ albopictus C6/36)to determine Yellow fever 17D vaccines 629

Table 1. Yellow fever seed 17D and vaccine lots studied by RNA oligonucleotide fingerprinting Country Manufacturer* Material tested Lot no. U.S.A. Connaught Laboratories, Inc. Vaccine 2654 LE U.S.A. Connaught Laboratories, Inc. Vaccine 2834 U.S.A. Connaught Laboratories, Inc. Vaccine 2090 England Wellcome Research Laboratories Vaccine YF/1/188 F.R.G. Robert Koch-Institut Vaccine 248-80 France Institut Pasteur Vaccine 0910 Netherlands Institute of Tropical Hygiene Vaccine 189.2 India Central Research Institute Vaccine E-76 Senegal Institut Pasteur Primary seed 18-A Senegal Institut Pasteur Vaccine 659 Senegal Institut Pasteur Vaccine 672 S. Africa National Institute for Virology Primary seed 1530/1534 S. Africa National Institute for Virology Vaccine 10802 Australia Commonwealth Serum Laboratories Vaccine 905-13-002.1 Brazil Fundacao Oswaldo Cruz Secondary seed S-101 Brazil Fundacao Oswaldo Cruz Vaccine 997 Colombia Instituto Nacional de Salud Primary seed ICF-374 Colombia lnstituto Nacional de Salud Secondary seed INDES-915 Colombia Instituto Nacional de Salud Vaccine 284 Colombia Instituto Nacional de Salud Vaccine 292 * Use of names of commercial sources is for identification only and does not constitute endorsement by the Public Health Service or the U.S. Department of Health and Human Services. the most suitable substrate for virus growth. Infectivity titrations of supernatant fluid samples were performed by plaque assay in Vero cells. Peak titres of 107° p.f.u./ml were obtained at 60 to 70 h in LLC-MK2 and Vero cells, of 107.9 p.f.u./ml at 54 to 58 h in SW-13 cells (37 °C), and of 1063 p.f.u./ml at 7 days in C6/36 cells (28 °C). SW-13 (human adrenocortical carcinoma) cells were selected for growth of the vaccines, since our laboratories have used these cells extensively in similar studies of other flaviviruses. Asibi virus proved difficult to grow to sufficient titre in these cells, and was therefore propagated in LLC-MK2 cells. Vaccines were reconstituted in 0.9 ~ NaC1; working seeds were prepared from infected culture fluids of two to three monolayer cultures of SW-13 cells grown in 25 cm 2 plastic flasks. For RNA studie:, 15 to 20 monolayer cultures grown in 150 or 175 cm z plastic flasks were inoculated at a ratio of approximately 1 p.f.u./cell. After 1 h adsorption at 37 °C, 30 ml of maintenance medium (Dulbecco's minimal essential medium containing 5 ~ foetal bovine serum heated at 56 °C for 1 h) was added to each flask. Supernatant fluids were harvested 60 to 72 h after infection, and the cell debris was removed by centrifugation (10000 g for 30 rain). Virus was precipitated with polyethylene glycol, resuspended in TNE buffer (10 mM-Tris-HCl pH 8.5, 150 mM-NaC1, 2 mM-EDTA), and subjected to rate-zonal (40000 rev/min for 2 h) and then isopycnic centrifugation in combination gradients of potassium tartrate and glycerol (Obijeski et al., 1976). The virus band recovered was pelleted, resuspended in TNE, and used for RNA extraction. RNA extraction from virus. Purified virus in 2 ml TNE was treated with 1 mg proteinase K (Beckman Instruments) for 30 min at 37 °C, solubilized with 1 ~ (w/v) SDS for 15 min at 37 °C, and extracted twice with an equal volume mixture of phenol : chloroform : isoamyl alcohol : 8-hydroxyquinoline at 25 : 24 : 1:0-5 (by wt.). RNA was precipitated for at least 14 h at - 20 °C by adding 0.2 ml of a 5 M-LiCI solution and 2-5 vol. ethanol (Trent & Grant, 1980). Five to 10 ltg RNA measured by u.v. absorption at 260 nm was used for digestion and labelling. Ribonuclease TI digestion and 32p labelling of oligonucleotides. RNA extracts were digested with 5 units ribonuclease T~ (Calbiochem-Behring Corp., San Diego, Ca., U.S.A.) in TE buffer (20 mM-Tris-HCl pH 8.0, 2 mM-EDTA) for 1 h at 37 °C. Digested RNA was incubated (3 min, 50 °C) with 2 litre 10 mM-spermine. Oligonucleotide fragments were then labelled at the 5' end with 32p using [~,.32p]AT P (New England Nuclear) and 15 units of T4 polynucleotide kinase, as described by Pedersen & Haseltine (1980). Oligonucleotide fingerprints. Oligonucleotides were separated by two-dimensional polyacrylamide gel electrophoresis, as described by DeWachter & Fiers (1972) and modified by Clewley et al. (1977a). Two reference dye markers, bromophenol blue and xylene cyanol, were included in the electrophoreses. After electrophoresis, gels were frozen, autoradiographed (Trent & Grant, 1980), and the autoradiographs were used for comparative analyses. Monoclonat antibody. Methods for hybridoma construction and characterization wilt be described elsewhere (Schlesinger et al., 1983). Briefly, 6-week-old BALB/c mice were immunized by intraperitoneal (i.p.) injection of 6 x l0 s p.f.u, of ALV-free 17D YF vaccine (substrain 17D-204, lot no. 2091, Connaught Laboratories). Thirty days 630 T. P. MONATH AND OTHERS later, 1 × 107 p.f.u, of this vaccine, passaged once in BHK-21 cells, was injected intravenously, and the same dose was given i.p. the next day. Three days later, the spleens were removed, and spleen cells were fused with non- secretor P3X653/Ag8.653 mouse myeloma cells. Hybrid cultures were screened for YF 17D antibodies by a microneutralization test. An IgM monoclonal antibody preparation (8A3) with high RIA and neutralizing activity was selected for study. Hybridomas were cloned twice by limiting dilution and immune ascitic fluids produced in pristane-treated BALB/c mice. Neutralization (N) tests, plaque-reduction N tests were performed in Vero cell monolayers grown in six-well plastic plates (Linbro). Monoclonal antibody 8A3 diluted 1 : 10 or 1 : 15 was mixed with an equal volume (0.1 ml) of virus containing approximately 200 p.f.u, and the mixture incubated for 1 h at 37 °C before inoculation of cell cultures. A double agar overlay technique was used; the second overlay containing 1:25000 neutral red, was applied after 6 days incubation. N antibody titre was expressed as the reciprocal of the highest dilution reducing plaque titre by >90%.

RESULTS

Pedigrees and passage levels of vaccines Figures 1 and 2 show the origins of vaccines derived from substrains 17D-204 and 17DD respectively. Vaccines derived from substrain 17D-204 are at passages 233 to 238, whereas 17DD-derived vaccines are at a higher level (286 to 287) due principally to a series of 40 additional passages in embryonated eggs in Brazil (Fig. 2). In Senegal and Colombia, vaccines have been produced from both substrains. The ALV-contaminated vaccine manufactured at the Institut Pasteur, Dakar, Senegal is prepared from primary and secondary seed lots originally derived from Brazilian 17DD primary seed (lot 458). The Senegal secondary seed (SLII-75-1) had been used directly to prepare vaccine in Colombia (Fig. 2). ALV-free vaccine manufactured in Senegal has also recently been prepared from 17D-204 substrain seed provided by France (Fig. 1). Colombian seed lots and vaccines have also been produced from the 17D-204 substrain (Fig. 1). Figure 3 shows the passage history of the NY-310 and NY-318 vaccines produced in 1940 by the RockefeUer Founoatlon.

RNA oligonucleotide fingerprint analyses of YF 17D vaccines One lot of vaccine (2654 LE) produced in the United States was arbitrarily chosen as a reference for comparative studies. Five replicate fingerprints of this virus grown in SW-13, and two replicates of virus grown in LLC-MK2 cells were shown to be identical. Forty-two of the larger oligonucleotides were assigned numbers, as shown in Fig. 4. Autoradiographs of other viruses were visually compared with that of 2654 LE, using the internal dye markers as reference points, and differences in these 42 oligonucleotides were defined. The results were expressed in terms of (i) the number (percentage) of spots shared with the reference strain, (ii) the assigned number of any missing oligonucleotide(s), and (iii) the presence of new oligonucleotide(s). With one exception, fingerprints of all current vaccines and seed lots derived from substrain 17D-204 were identical with the reference strain (Table 2). The fingerprint of NY-318 vaccine (Fig. 3) was also identical to the reference strain. Vaccine produced in South Africa showed minor differences: oligonucleotide no. 11 was missing and a new spot appeared to the left of the missing position (Fig. 5). This may be explained by a single base change and charge difference in oligonucleotide no. 11. In addition, a new spot appeared between oligonucleotides no. 3 and 4 (Fig. 5). The primary seed (1530/1534) from which the South Africa vaccine originated was also fingerprinted. Oligonucleotide no. 11 was again missing, and the new spot to the left of the no. 11 position was noted; therefore, these changes occurred before or during growth of the primary seed lot. Since the Colombian primary and secondary seeds and the vaccines produced in India and England originated from the same stock (RF-555, Fig. 1) as the South African vaccine, we conclude that the change in oligonucleotide no. 11 occurred during a single egg passage in the preparation of the South African primary seed from RF-555. The appearance of a new spot between oligonucleotides no. 3 and 4 in the South African vaccine (lot 10802) reflects a further change, which occurred within two egg passages from the primary seed. Yellow fever 17D vaccines 631

(ORIGINAL lTD]sc 180

Subculture 200 [

210 I

220 F 228,17 [ I r i t 229 RF 1~45-3 RF ~ 505 I I 230 RML YF-I RF ! 555 I I I I I I 231 RMLYF 10 RH>o R~I~60 RH',815 R~!847 I COLOMBIAIIS. AFRICAI ENGLANDIIINDIAI I w I i 232 ~. ,AB237~ SI* SI* C/80/C $2 (ICF374) (1530/1534) II (R-20) I I 233 SI~_ ALIA]'(5~231)'~k AB616 $2 ~1 Vaccine* (YFS/P/3) (E 76) 234 s'~ ~, s2' Vaccine". Sl ~ , H (771-2) (IP/F2) (002-2) (2654, 2834)~ AB617A(AB617) ] Vaccine Vaccine* $2 (YFS/10-12) II II t ~ I 8366 (10802) II 235 Vaccine* VacCine* Vaccine* S I ~1 $2 , (672) (0910) (905-13-002.1) C1600 (AB619)(AB618)IFED- REP~GERMANY Ii Vaccine* II | I (YF/I/188) 236 $2 $2 SI ALV-free (1613) (AB620) Vaccine (112 69) 237 Vaccine* Vaccine* (2090) (189.3) (160-72) ALV&ee ALV-free II 238 Vaccine* (248 80) 239 ALV-free Fig. 1. Passage history of vaccines derived from the 17D-204 substrain. --, Passage in eggs; - - - leukosis-free passage; *, tested by RNA oligonucleotide fingerprinting; S1, primary seed; $2', secondary seed.

Subculture 180 190 ~sc 195 200 sc 204 210 220 (see Fig. l) 230 240 sc 243 250 Subculture 260 I 180 I ORIGINAL 17D ] 270 17DD high 280 283 sc 283 200 284 SI/"~SI (458) (PI) 285 SI* $2" (18 A) (S-!01) 286 s'z 220 I Vaccine* ! (SLII 75-1) (997) 287 Vaccine* Vaccine* I COLOMBIA 88 I (292,284) (659) NY-~'I0* NY'-312 NY 3"18" !t [ COLOMBIA [ SENEGAL ] BRAZIL [ 240 Current vaccines Fig. 2 Fig. 3 Fig. 2. Passage history of vaccines derived from the 17DD substrain. , Subculture in tissue culture; = = =, subculture in eggs; *, tested by RNA oligonucleotide fingerprinting; S1, primary seed; $2, secondary seed. Fig. 3. Pedigree of NY-310 and NY-318 vaccines produced by the Rockefeller Foundation, New York, 1940. *, Tested by RNA oligonucleotide fingerprinting. 632 T. P. MONATH AND OTHERS

: (b)

Fig. 4. RNA oligonucleotide fingerprint (a) and diagram (b) of yellow fever 17D vaccine (17D-204 substrain) produced in U.S.A. by Connaught Laboratories, lot 2654 LE, used as the reference strain in these studies.

Table 2. RNA oligonucleotide homologies, 17D yellow fever vaccines derived from substrain 17D-204 Material tested Subculture Homology with Missing No. of new Producer (lot no.) level ALV reference strain oligonucleotide oligonucleotides U.S.A. Vaccine (2654 LE) 234 + (reference strain) U.S.A. Vaccine (2834) 234 + 42/42 (100%) - - U.S.A. Vaccine (2090) 237 - 42/42 (100%) - - India Vaccine (E-76) 233 + 42/42 (100~) - - Colombia S1 (ICF-374) 232 + 42/42 (100~) - - Colombia $2 (INDES-915) 233 + 42/42 (100~) - - Senegal Vaccine (672) 235 42/42 (100%) - - France Vaccine (0910) 235 42/42 (100%) - - Australia Vaccine (905-13- 235 + 42/42 (100~) - - 002.1) England Vaccine (YF/1/188) 237 - 42/42 (100~) - - Netherlands Vaccine (189.2) 237 - 42/42 (100%) - - F.R.G. Vaccine (248-80) 238 42/42 (100~) - - S. Africa S1 (1530/1534) 232 + 41/42 (98%) no. 11 1 S. Africa Vaccine (10802) 234 + 41/42 (98~) no. 11 2

Vaccines derived from substrain 17DD differed from those originating from substrain 17D- 204 in the absence of oligonucleotide no. 37. The loss of oligonucleotide no. 37 must have occurred between the divergence of the 17DD substrain line at subculture 195 and the preparation of EP-774, from which Brazilian primary seed lots were made (Fig. 2). Both vaccine (lot 997) and secondary seed (S-101) produced in Brazil showed an additional change (missing oligonucleotide no. 25 and appearance of a new spot to the right of the no. 25 position, possibly due to a base change and charge difference in this oligonucleotide; Fig. 6). Since the change in oligonucleotide no. 25 was not reflected in the primary seed, lot 18-A, made in Senegal or in the vaccines produced in Senegal or Colombia (Table 3), it occurred during the two passages between EP-774 and the Brazilian secondary seed S-101 (Fig. 2). Two lots of vaccine produced in Colombia showed a further minor difference: the appearance of a new oligonucleotide (Fig. 7). This spot was not present in the primary seed from which the vaccines originated (Senegal lot 18-A, Fig. 2). Yellow fever 17D vaccines 633

Fig. 5. RNA oligonucleotide fingerprint (a) and diagram (b) of yellow fever 17D vaccine (17D-204 substrain) produced in South Africa, lot 10802. This vaccine differs from the reference strain in the absence of oligo no. 11 (*) and the presence of two new oligonucleotides (a and b).

• •••• "••••~~•~• ':~i: iiii!/i~i~i,~!~ ~i?•

~LY ® ® ® L

• "~(~)

Fig. 6. RNA oligonucleotide fingerprint (a) and diagram (b) of yellow fever 17D vaccine (17DD substrain) produced in Brazil (Fundacao Oswaldo Cruz, lot 997), Showing the absence of oligo no. 25 and 37 (*) and the appearance of a new spot (c) to the fight of no. 25.

Table 3. RNA oligonucleotide homologies of 17D yellow fever vaccines derived from substrain 17DD Material tested Subculture Homology with Missing No. of new Producer (lot no.) level ALV reference strain oligonucleotide oligonucleotides Brazil $2 (S-101) 285 + 40/42 (95%) no. 25, 37 1 Brazil Vaccine (997) 286 + 40/42 (95%) no. 25, 37 1 Senegal S1 (18-A) 285 + 41/42 (98%) no. 37 0 Senegal Vaccine (659) 287 + 41/42 (98%) no. 37 0 Colombia Vaccine (292) 287 + 41/42 (98%) no. 37 1 Colombia Vaccine (284) 287 + 41/42 (98%) no. 37 1 634 T. P. MONATH AND OTHERS

(b) _

Fig. 7. RNA oligonucleotide fingerprint (a) and diagram (b) of yellow fever 17D vaccine (17DD substrain) produced in Colombia, showing the absence of oligo no. 37 (*) and the appearance of a new oligonucleotide (d).

Fig. 8. RNA oligonucleotide fingerprint of parent Asibi virus, showing appearance of three new spots (marked e, f, and g, in positions above no. 2 and 5 and below no. 36 reference strain) and absence of oligo no. 29 (*).

Table 4. Plaque-reduction neutralization (N) test results, monoclonal antibody D vaccines and parent Asibi viruses N titre Virus Substrain origin monoclone 8A3" YF 17D Connaught 2848 17D-204 81920 YF 17D Wellcome YF/1/189 17D-204 81920 YF 17D R. Koch 213/A/80 17D-204 81920 YF 17D S. Africa primary 17D-204 40960 YF 17D S. Africa 10802 17D-204 40960 YF 17D Osw. Cruz S-101 17DD <20 YF 17D Bogota 284 17DD 40 YF 17D Dakar 18A 17DD 20 YF Asibi <20 * Monoclonal antibody produced to YF 17D Connaught, IgM isotype. Yellow fever 17D vaccines 635

Neurovirulent vaccines The NY-310 vaccine, implicated in encephalitic reactions in Brazil (Fox et al., 1941), differed from the reference strain in the presence of a new oligonucleotide. By direct visual inspection, this oligonucleotide is very close to the new spot in the fingerprint of the Brazilian secondary seed and vaccine (Fig. 6). The fingerprint of virus 6766 (the seed lot which failed monkey neurovirulence tests) was identical to the reference strain.

Avian leukosis virus contamination No differences were found between the reference strain (ALV-contaminated) and any of the vaccines which had been freed of ALV at six different institutes (Table 2). Development of ALV-free vaccines involved up to five passages; all cleaned vaccines were from 17D-204 substrain stock (Fig. 2).

Parent Asibi virus Several differences between the parent virus and the 17D-204 reference strains were found, none of which correspond to oligonucleotide variations seen among 17D vaccines (Fig. 8). In the Asibi fingerprint, oligonucleotide no. 29 is missing and three additional spots are present (above spots no. 2 and 5 and below spot no. 36 of the reference strain).

Antigenic correlations Monoclonal antibody 8A3 was tested by plaque-reduction N test against representative vaccines and seeds derived from the 17D-204 and 17DD substrains as well as parent Asibi virus (Table 4). This antibody clearly distinguished vaccines of different substrain origin, consistent with the oligonucleotide differences. The South African seed and vaccine, which differed slightly from the reference strain by RNA fingerprinting, was not antigenically distinct. 17D and parent Asibi viruses differed serologically. A number of additional IgG monoclonai antibodies were also examined but none distinguished substrains of 17D virus; these results will be published elsewhere (Schlesinger et al., 1983).

DISCUSSION In the early phase of YF 17D vaccine development and production, a number of different substrains were deployed in pathogenicity studies and human trials. Some of these were found to be unsatisfactory. Vaccine produced in Brazil from the 17DD substrain which had been passed in tissue culture more than 300 times ('17DD-high') was found to be overattenuated (Theiler, 1951). Other vaccines, produced from both the 17DD and the 17D-204 substrains, were associated with an increased incidence of central nervous system signs in man and in intracerebrally inoculated monkeys (Theiler, 1951 ; Fox et al., 1941 ; Sawyer et al., 1944). These difficulties attested to the potential genetic instability of 17D virus and were resolved by establishment of the seed lot system in 1945. However, the disparate substrain origin of early vaccines continues to be reflected in contemporary vaccine manufacture; both the 17DD and 17D-204 substrains are currently used, and vaccines vary with respect to passage level. Despite the disparate origins and passage levels, a high degree of genetic similarity was found between vaccines produced world-wide. At the level of sensitivity of the RNA oligonucleotide fingerprinting technique (examination of approx. 10 to 15 ~ of the genome), all vaccines shared 95 to 100~ of their oligonucleotides, reflecting an estimated sequence homology of 98 to 100~ (Aaronson et al., 1982). This was an encouraging reaffirmation of the seed lot system as a means of assuring genetic stability and of the widely held concept that YF 17D vaccines are stable and extremely safe products for human use. Nevertheless, several observations illustrate the potential for genetic changes during vaccine production. The South African vaccine derived from substrain 17D-204 and the Colombian vaccines derived from substrain 17DD had minor oligonucleotide map changes which probably arose within a single passage in embryonated eggs. Similarly, the viruses prepared in Brazil and Senegal (17DD substrain) differed in one oligonucleotide (no. 25), a difference which must have occurred within one or two passages from 636 T. P. MONATH AND OTHERS their common ancestor (EP-774, Fig. 2). These changes confirm the view that RNA viral genomes can rapidly evolve due to high mutation frequencies (Holland et al., 1982). Future work to develop a new generation of YF 17D vaccines in cell culture substrates will have to consider the possibility that such rapid genetic changes can occur. The vaccines derived from the 17DD substrain demonstrated a consistent difference from those derived from substrain 17D-204: absence of oligonucleotide no. 37. Loss of this oligonucleotide occurred at some point in the independent lineage of 17DD, probably during 40 additional egg passages made before establishment of a seed lot system. Among 17DD-derived vaccines there appeared to be somewhat more genetic variability than for those developed from 17D-204 substrains. This observation, plus the fact that to date no 17DD-derived vaccine has been freed from ALV, suggest certain advantages of the 17D-204 lineage for future standardized vaccine development. In fact, WHO has recently contracted for the manufacture of an ALV-free official primary seed lot prepared from 17D-204 derived primary seed at the Robert Koch- Institut, F.R.G. Recently, Liprandi (1981) showed that both the Wellcome and South African vaccines contained plaque-size variants which, when cloned, significantly differed in neurovirulence for intracerebrally inoculated adult mice. These studies underscore the genetic variability of current vaccines. We are unable to extend these observations, since RNA oligonucleotide fingerprints were performed on viruses passed in SW-13 cell cultures without cloning or biological characterization. WHO requirements specify that seed lots be safety tested in rhesus monkeys, an increasingly expensive and scarce laboratory host. It was thus of interest to examine YF 17D viruses with undesirable neurotropism by a molecular technique. No definite marker of neurovirulence was defined by oligonucleotide fingerprints of the NY-310 and 6766 strains. Fox et al. (1941) reported a 0-53~ incidence of severe neurological reactions in persons given NY-310 vaccine in Brazil in 1941 ; this incidence was much lower than that associated with another batch of vaccine (E718), which was not available for study by us. The single oligonucleotide difference between the reference strain and NY-310 could reflect a genomic correlate of neurovirulence; however, minor differences of this type were found among several 17D vaccines without increased neurovirulence properties. The 6766 virus is also considered to be only mildly encephalitogenic in monkey neurovirulence tests (B. Elisberg, personal communication, 1981). The fingerprint of 6766 virus was identical to the reference strain. In future studies on the genetics of YF neurovirulence, it would be interesting to compare the French neurotropic vaccine strain with its viscerotropic parent. Several oligonucleotide differences were found between parent Asibi virus and 17D vaccines. The relationship between these changes and phenotypic expression of virulence and serological reactivity remains obscure. However, it is of interest that 98 ~ of the Asibi virus oligonucleotides (probably reflecting less than a 1 ~ difference in RNA sequence; Aaronson et al., 1982) was conserved in the long series of passages made in the development of 17D vaccine. Similar stability of RNA genomes with passage have been reported for vesicular stomatitis virus and other viruses (Holland et al., 1982; Clewley et al., 1977b) probably reflecting the "strong selective fitness (of certain RNA genomic species) that under appropriate conditions ... dominate the equilibrium population" (Holland et al., 1982). The consistent genotypic difference between substrain 17D-204- and 17DD-derived vaccines (in oligonucleotide no. 37) was also reflected by different reactivities in N tests using monoclonal antibody produced against 17D vaccine (Connaught Laboratories). This antigenic difference between 17D-204 and 17DD vaccines is probably of no practical significance, since polyclonal antibodies in the vaccinated host have broad neutralizing reactivity against yellow fever viruses. The antigenic difference between 17D and parent Asibi virus noted in our study confirms the earlier observation by Clarke (1960), who showed by antibody absorption that 17D virus possessed an antigen not present in Asibi virus.

We are deeply indebted to the following individuals at YF 17D vaccine manufacturing institutes for their assistance in providingvaccines and seed lots: Drs Walter E. Woods (U.S.A.), J. Prydie (England), J. L'age-Stehr Yellow fever 17D vaccines 637

(F.R.G.), C. Hannoun (France), C. Lucasse (The Netherlands), S. N. Saxena (India), J.-J. Salaun (Senegal), O. W. Prozesky (South Africa), W. D. Collins (Australia), A. Homma (Brazil), and C. E. Bernal Cubides (Colombia). As mentioned in the text, Drs R. E. Shope and R. B. Tesh at the Yale Arbovirus Research Unit, and Dr B. Elisberg, Bureau of Biologics, Food and Drug Administration, also kindly sent YF virus strains. We also wish to thank Messrs C. B. Cropp, J. Brubaker, and G. Wiggett, for considerable technical assistance in the study.

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(Received 21 July 1982)