Immune responses to H particles of poliovirus

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Immune

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Responses to H Particles of Poliovirus

TABLE PRODUCTION IMMUNIZED

Poliovirus of any given type consists of two distinct antigens. The N or D antigen represents complete particles, which may or may not be infectious, whereas the H or C antigen appears morphologically as empty shells which are deficient. of nucleic acid. The K antigen can be converted to H in vitro by chemical or physical treatment (l-3) and immunization of animals with this artificially produced H antigen has produced sera showing high homologous titers, but no anti-N nor neutralizing antibodies (4, 5). During studies on the structure of poliovirus particles we found that H antigenic particles prepared from highly purified poliovirus induced not only homologous antibody but also anti-N as well as neutralizing antibodies. We have evaluated these preliminary results and excluded the possibility of contamination of the H-reactive immunogen by N antigenic particles. This communication reports these observations, and, on the basis of the results, a new concept of the antigenic structure of poliovirus is proposed. Poliovirus type I strain Mahoney was grown in HeLa S3 cells. The virus progeny was concentrated and purified according to procedures described previously (6). CF-N and CF-H antigen units were assayed against monoreactive anti-N and anti-H guinea pig sera (4-6). Plaque-forming units (PFU) were determined on monolayers of HeLa S3 cells according to standard procedures. H virus particles were prepared by ultraviolet (UV) irradiation (4,000 erg/square cm/second for 120 minutes) or by alkali treatment (heating at 40” in 0.01 M borate buffer, pH 10.5, for 30 minutes) of the purified virus preparation, according to the procedures described in previous reports (7, 8). In the first series of experiments three groups of adult albino rabbits were immunized with either the purified virus preparation in its native state or the same material converted to H-reactive particles by UV. The production of antibodies in these three groups of rabbits is summarized in Table 1. The three rabbits in the first group injected with the native purified virus prep-

1

OF ANTIBODIES WITH

INFECTIOUS

H

UV-INDUCED

I II III

A”tip&itY-

200 N <20 Nb 200H

RABBITS VIRUS

OR

PARTICLES Antibodies

Jmmunogen %ixSOf

IN

PFU

lo9 105 7

CF-N

CF-H

Neutralizing

3/3”

3/3

3/3

O/6 6/6

O/6 6/6

o/6 w3

LiNumber of antibody-positive rabbits/number of rabbits immunized. b It can be calculated to be 0.02 N on the basis of lO,OOO-fold dilution of 200 N.

aration containing log PFU and 200 CFU-N produced CF-N, CF-H, as well as neutralizing antibodies. When this material was diluted lO,OOO-fold and injected into six rabbits (group 2) no antibodies were evident after 31 days. This confirms previous observations (9, 10) that less than lo6 PFU of poliovirus are insufficient to produce antibodies in rabbits. The original material was UV irradiated, subsequently exhibiting 200 CFU-H. When injected into six rabbits (group 3) CF-H, CF-N, as well as neutralizing antibodies were detectable. The antibody curves in each rabbit of the first and third groups are shown in Fig. 1. The differences between the two curves were as follows : 1. Initiation of CF-H antibody responses in group 3 receiving UV produced H-reactive material became apparent only after a second injection in three of six rabbits, while a single injection elicited this response in all rabbits of group 1. Eventual maximum titers of CF-H in both groups were similar, thus the purified infectious virus preparation containing only N antigen with no detectable H induced CF-H responses sooner than the purely H-reactive preparation. 2. In the first group, curves of CF-N and CF-H were similar in each individual rabbit,. In the third group, however, CF-N antibodies reached lower titers and appeared later than in group 1. 3. Production of neutralizing antibodies in the third group was different, from that in the first. The neutralizing antibodies ap-

774

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COMMUNICATIONS Group 1-3

Group I - 3 I H

I H

Group I1

1 L .

DAYS AFTER

THE FIRST

0 INJECTION

6

I

p--$-A ’ : : ,d 0’ 3’ : : ::

(i) -I

IO

20

30

FIG. 1. Kinetics of the formation of antibodies in rabbits immunized with infectious virus or UVinduced H particles. Animals were injected intravenously with indicated dose (see Table 1) of immunogens suspended in phosphate-buffered saline, pH 7.2, twice at a-week intervals. Panels a-c and d-i show the antibody-formation curves in rabbits in Group I (109PFU) and Group III (lo9 PFU converted to H by UV), respectively. A-----A, CF-N antibody; A-----A, CF-H antibody; O---O, neutralizing antibody.

very late and the maximum titers were low as compared to those for group 1. These results suggested that H antigen produced by UV irradiation elicits both H and N antibodies as well as neutralizing antibodies. This may have been due to the fact that small amounts of noninfectious N particles were present in the UV-irradiated material (7). To exclude this possibility, the following experiments were carried out,. By CF test it was found that UV treatment of the original preparation reduced N reactivity by more than 99%. In order to eliminate all possible N reactivity, the UV-irradiated preparation was treated with 10 pg/ml of Deared

RNase for 30 minutes at, 37”, because we have shown previously (7,11) that this treatment, affects a conversion of RNase-sensitive, noninfectious N particles induced by UV irradiation to H-reactive particles. Subsequently the H particles were purified twice in a 15-30% sucrose gradient, which also separates N from H efficiently. The final H-reactive product contained 1,000 CFU-H. It was diluted to a level of 200 CFUH and used for immunization. It was calculated that the successive treatments and the final dilution resulted in reduction of original PFU by a factor of 5 X 105. Since it is known that viral preparation containing

SHORT Group 4l

V- I

Group 4l -

I

I

b-

L -B

P-

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COMMUNICATIONS

t

I-I

I H

DAYS

AFTER

THE

FIRST

INJECTION

2. Kinetics of the formation of antibodies in rabbits immunized with 200 CFU-H purified particles in the same manner as in Fig. 1. Panels a-c and d-f show the antibody-formation curves in rabbits in Groun IV WV-irradiated preparation) and Group V (alkali-treated preparation), respectively. A-----A, CF-N antibody; A-----A,-CF-H antibody. FIG.

less than 106 PFU are nonimmunogenic, we have to assume that N reactivity was reduced to a point where it would not have played a role in eliciting homologous antibodies. The second immunogen was an alkalitreated preparation. This treatment reduced N antigenicity by more than 99 %. This preparation was further purified by two cycles of sucrose-gradient centrifugation, and the final product was diluted to contain also 200 CFU-H. The reduction factor based on the original PFU was more than 1 X 105, reducing it, below the level of PFU necessary to induce anti-N. Two groups of rabbits were immunized with both preparations, and the results are shown in Fig. 2. The rabbits in both groups produced both CF-N and CF-H antibodies. The antibody-production curves were essentially similar to those in the rabbits immunized with UV-irradiated virus preparations in the first series of experiments. Neutralizing antibodies were not determined in the latter two groups. From the foregoing results, it is evident that production of CF-N, and neutralizing antibodies, as well as homologous CF-H

antibodies were elicited by H-antigenic particles in these preparations. The results imply that the N-antigenic determinant is masked in the H-particle in a state which cannot, be detected by in vitro serologic reaction, but it is, nevertheless, immunogenic. The structural relationships of the N- and H-antigenic determinant on the viral capsids are unknown. The following possibility may be considered. A change in primary or secondary structure of the capsid polypeptides may determine the qualitative difference between the two antigenic determinants, or, alternatively, either N- or H-antigenic. particles, assuming that they contain either only N or H determinants, can induce not only monoreactive antibody molecules but also antibody molecules which are able to react with both N- and H-antigen in vitro. This may be possible if the two antigens are closely related to each other in their primary structure as is known in the case of 2,4 dinitrophenyl peptides (12). The antigenic structure of other picornaviruses may be reevaluated by the experimental approach presented here.

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COMMUNICATIONS

ACKNOWLEDGMENTS The authors thank Dr. Klaus Hummeler for his valuable discussions and revising the manuscript. The study was aided partly by grants from the Waksman Foundation of Japan and from the Ministry of Education of the Japanese Government. REFERENCES 1. ROIZMAN, B., MAYER, M. M., and RAPP, J. J., J. Zmmunol. 81,419425 (1958). 2. HUMMELER, K., and HAMPARIAN, V. V., J. Zmmunol. 81,499-505 (1958). 3. LE BOUVIER, G. L., Brit. J. Exp. Pathol. 40, 605-620 (1959). 4. HUMMELER, K., and TUMILOWICZ, J. J., J. Zmmunol. 84, 630-634 (1960). 6. KATAGIRI, S., and HINUMA, Y., Tohoku J. Exp. Med. 88,153-160 (1966). 6. HINUMA, Y., KATAGIRI, S., FUKUDA, M., FUKUSHI, K., and WATANABE, Y., Biken J. 8, 143-153 (1965). 7. KATAGIRI, S., HINUMA, Y., and ISHIDA, N., Virology 32, 337-343 (1967). 8. MAIZEL, J. V., JR., PHILLIPS, B. A., and SUMMERS, D. F., Virology 32,692-699 (1967). 9. SVEHAG, S. E., and MANDEL, B., J. Exp. Med. 119, 1-19 (1964). 10. HINUMA, Y., Uirusugaku-no-Shinten (5th Virus Symp. Inst. Virus Res. Kyoto Univ.) 5,141-153 (1965) (in Japanese). 11. KATAGIRI, S., HINUMA, Y., and ISHIDA, N., Virology 34,797-799 (1968). 18. PARKER, C. W., GOTT, S. M., and JOHNSON, M. C., Biochemistry 5,2314-2326 (1966). YORIO HIMUMA SUSUMU KATAGIRI SHOGO AII~AWA Deparlment of Microbiology Tohoku University School Sendai, Japan Accepted

Partial

December

of Dentistry

18, 1969

Purification

of the Epstein-Barr

Virus and Some Properties of its DNA’ The Epstein-Barr virus (EBV) has been incriminated as the etiologic agent of infectious mononucleosis (1, 2). Various lymphoblastoid cell lines, originating from 1 After this paper had been cation, a study on EB virus

submitted of Burkitt

for publilympho-

patients with infectious mononucleosis, other diseases of the hematopoietic system, as well as from healthy donors, were, at least temporarily, positive for EBV (S-5). Especially, a number of lymphoblastoid cell lines derived from patients with Burkitt lymphoma have been shown to be persistently infected with EBV (6-8). Electron microscopic studies identified EBV as a herpes-like particle (8-9). The antigenic properties were distinct from well-known representatives of the herpes virus group, e.g., herpes simplex, varicella, cytomegalovirus, as revealed by immunofluorescence (10, 11). Owing to the small percentage of virus-producing cells, concentration of the virus was difficult to achieve, and the nature of the viral nucleic acid has not yet been clearly established. Some preliminary reports, however, indicated that the viral core contains deoxyribonucleic acid (DNA) (12, 13). The following is a brief account of a concentration procedure for EBV which selectively concentrates enveloped particles not penetrable by phosphotungstic acid (PTA). In addition, it shows that EBV contains double-stranded DNA with an estimated G + C content of .!i9 %. The P3HR-1 clone of Burkitt lymphoma cells, isolated by Hinuma et al. (14), was used for the concentration procedures. Raji cells (15), derived also from Burkitt lymphoma and found to be EBV-negative by immunofluorescence and electron microscopy (16’), served as negative controls. Both lines were repeatedly controlled for mycoplasma contamination and found to be negative. The cells were kept at least for 11 days in the same medium (RPM1 1629, Grand Island Biological Co., Grand Island, N. Y., containing 10% calf serum, 100 U/ml of penicillin, and 100 pg/ml streptomycin). 3H-Thymidine (1 &X/ml, sp act 21.3 Ci/mmole, The Radiochemical Centre, Amersham) was added 2 days aft.er seeding the cells and remained until harvest 9 days later. For the harvest of the virus the supernatant fluid was clarified by low-speed centrifblasts was published by A. Weinberg Becker (Virology 39,312-321, 1969). Upon gation of intracellular virus these authors a buoyant density for EBV-DNA of 1.720

and Y. investireported g/cm3.