Immunology, 1975, 29, 1001.

Naturally Occurring Double-Stranded RNA and Immune Responses III. IMMUNOGENICITY AND ANTIGENICITY IN ANIMALS P. G. CUNNINGTON AND J. D. NAYSMITH* Beecham Research Laboratories, Chemotherapeutic Research Centre, Brockham Park, Betchworth,

Surrey (Received 23rd May 1975; acceptedfor publication 19th June 1975)

Summary. Naturally occurring double-stranded RNA (ds-RNA) was immunogenic when injected into mice, rats, guinea-pigs, rabbits, dogs and baboons. The response to native material administered intravenously (i.v.) was strongest in rabbits and mice, and weakest in baboons. Mice, guinea-pigs and baboons injected with ds-RNA complexed with methylated BSA emulsified in Freund's complete adjuvant all gave high antibody responses. When ds-RNA was given in aerosol form to mice and guinea-pigs the response was weaker than that following i.v. injection, and baboons did not respond to antigen given as an aerosol. In most species the immune response obtained was predominantly IgM in nature, and there was no evidence for cell-mediated immunity in any species. The only evidence of an adverse reaction associated with repeated administration of ds-RNA was a systemic anaphylactic-type response in a small group of mice given ds-RNA repeatedly in aerosol form and challenged with ds-RNA i.v. INTRODUCTION Double-stranded polyribonucleotides produce resistance to virus infections (Lampson, Tytell, Field, Nemes and Hilleman, 1967; Field, Tytell, Lampson and Hilleman, 1967; Vilcek, Ng, Friedman-Kein and Krawciw, 1968) and have enhancing effects on various immune responses (Braun and Nakano, 1967; Johnson, Schmidtke, Merritt and Han, 1968; Turner, Chan and Chirigos, 1970). Recently one of these nucleotides, poly I-poly C, has been found to be itself immunogenic (Steinberg, Baron, Uhlendorf and Talal, 1971a; Field, Tytell, Lampson and Hilleman, 1972; Dianzani, Forni, Ponzi, Pugliese and Cavallo, 1972), and this raises the important question (both from a theoretical viewpoint and because of its implications for the therapeutic potential of double-stranded polyribonucleotides) of the extent to which the biological effects of such molecules may be altered by prior immunization. There is a need to define the conditions under which double-stranded polyribonucleotides induce immune responses, and to ascertain the likelihood of adverse effects occurring in immunized individuals on subsequent exposure. * Present address: Department of Pathology, University of Bristol, The Medical School, Bristol BS8 lTD. Correspondence: Mr P. G. Cunnington, Beecham Research Laboratories, Chemotherapeutic Research Centre,

Brockham

Park, Betchworth, Surrey.

1001

1002 P. G. Cunnington and J. D. ;Nysmith This laboratory has been studying a naturally occurring fungal, double-stranded RNA (ds-RNA) (BRL 5907) which is a potent antiviral agent and interferon inducer (Sharpe, Birch and Planterose, 1971), an immunological adjuvant (Cunnington and Naysmith, 1975; Gough, Allen, Knight and Leiper, 1974) and an anti-tumour agent (Heyes and Catherall, 1974). It has already been established that ds-RNA is immunogenic in mice, and that the presence of antibody to ds-RNA does not interfere with the induction of anti-viral protection, although it may be associated with reduced serum interferon levels (Naysmith, Sharpe and Planterose, 1974). There is also evidence that anti-ds-RNA antibody does not interfere with the adjuvant activity of ds-RNA in mice (Cunnington and Butlin, to be published). In this paper attempts to immunize animals of six species to ds-RNA using different routes and forms of antigen presentation are described. In those species which responded the type of immune response obtained has been studied, and adverse effects in immunized animals subsequently exposed to ds-RNA challenge have been sought.

MATERIALS AND METHODS

Double-stranded RNA The derivation and properties of this material, BRL 5907, have previously been described (Cunnington and Naysmith, 1975). Tritium-labelled ds-RNA ([3H]ds-RNA) was prepared from fermentations containing tritiated adenosine. Other nucleic acids Polyinosinic: polycytidylic acid (poly I-poly C) was purchased from P-L Biochemicals Incorporated, Milwaukee, Wisconsin (double-stranded duplex, lyophilized sodium salt, lot number 047231). Tritium-labelled poly I-poly C ([3H]poly I-poly C) of specific activity 33 mCi/mMP was obtained from Miles Laboratories Incorporated, Elkhart, Indiana. Polyadenylic and polyuridylic acid single-stranded polymers were from P-L Biochemicals. The double-stranded duplex, poly A-poly U, was prepared for use by mixing equimolar solutions of the single-stranded polymers and incubating at 370 for 30 minutes. Calf thymus DNA and single-stranded yeast RNA were purchased from the Sigma Chemical Company, Kingston, Surrey.

Animals- source and exposure to ds-RNA Animals of six species: mice, rats, rabbits, guinea-pigs, dogs and baboons were used in these studies. Two strains of mice were used: one the Fl hybrid between parents of the inbred strains CBA and C57B1/6 obtained from the L.A.C., Carshalton and the other the outbred CFLP strain obtained from Carworth (Europe). The remaining animals were outbred, though usually members of closed colonies. (Guinea-pigs were obtained from Olac Ltd, Oxford; rats from Carworth Europe Ltd; rabbits from Ranch Rabbits Ltd, Crawley; dogs and twenty baboons from Huntingdon Research Centre, Huntingdon; and the remaining baboons from Animal Suppliers (London) Ltd, Welwyn.) In the basic plan for this study animals were given soluble ds-RNA once a week by intravenous (i.v.) injection for 8 weeks, and antibody levels were assayed in serum samples taken regularly over this period. Prior to the first injection of ds-RNA all animals were skin-tested with small quantities of ds-RNA, and on completion of dosing this procedure

Antibody Response to ds-RNA 1003 was repeated. A few days later the animals were challenged with a further (usually larger) dose and observed for signs of adverse effects. A second group of animals were dosed with ds-RNA in aerosol form instead of i.v., and a third group was given ds-RNA coupled to methylated BSA (MeBSA) in adjuvant followed by booster injections of ds-RNA in saline. Important points in designing these three dosage schedules are appended below, but the details of each experiment (source, sex and weight of animal, actual doses given, and timings of doses) are listed in Tables 1, 2 and 3. Groups of mice were also given poly I-poly C to compare the immunogenicity of this material with ds-RNA in this species. (i) Intravenous route. Animals of all six species were given once a week for 8 weeks, a dose of ds-RNA which approximated to one-tenth of the LD50 for ds-RNA in that species. The actual dose used reflects our previous experience with each species and the accuracy with which the LD50 is known. Prior to commencement of treatment the animals were bled and skin tested. The skin test involved the intradermal injection of 0-1 ml saline containing 50 jig of ds-RNA; control sites on the same animal were injected with 0 1 ml of saline. At periods of 30 minutes, 4 hours and 24 hours after inoculation the skin sites were observed for signs of inflammation. The blood samples taken before inoculation, and regularly throughout the study, were obtained in the following ways: by cardiac puncture in rats and guinea-pigs, from the retro-orbital sinus in mice, from the marginal ear vein in rabbits, and by venepuncture of a forelimb in dogs and baboons. The blood was allowed to clot and the separated serum stored at -200. Nine weeks after the first i.v. injection of ds-RNA all animals were skin tested as before. Two days later a 'challenge' injection of three times the regular dose was administered i.v., and the animals were observed closely for 24 hours. (This dose level, which is still well below the LD50, would not be expected to produce acute toxic effects in the majority of animals.) TABLE 1 DETAILS OF ANIMALS DOSED REGULARLY WITH ds-RNA BY THE INTRAVENOUS ROUTE

Animal Mouse Dog Baboon Rabbit Rat Guinea-pig *

Species or strain CFLP (CBA x C57B1)Fl Beagle Papio cynocephalus NZW Wistar Dunkin-Hartley

Number

40**t 20t 8t 8$ 6t 18t 18t

Weight

Weekly dose

Final challenge dose (i.v.)

18-22 g 20-25 g

50 pg/animal 50 pg/animal 50 ,g/kg 2 mg/kg 500 pg/animal 500 pg/animal 250 ,g/animal

150 pg 150 ug 150 pg/kg 6 mg/kg 1 mg 1-5 mg 750 pg

9-11 kg 3-4-5 kg 3-4 kg 150-200 g 250-300 g

Twenty of these mice were dosed and challenged with poly I-poly C instead of ds-RNA.

t All male. t Equal numbers of males and females.

Further experimental details concerning this group of animals are recorded in Table 1. (ii) Aerosol route. Further groups of mice, guinea-pigs and baboons were exposed weekly to ds-RNA delivered in aerosol form. The mice and guinea-pigs were placed in inhalation chambers of suitable size, into which a 1 mg/ml solution of ds-RNA was nebulized via a Wright's nebulizer for a period of 6 hours. The average dose delivered to the lungs of

1004 P. G. Cunnington and J. D. Naysmith each animal was approximately 14 yug per mouse and 150 jug per guinea-pig. The baboons were seated in primate restraining chairs, fitted with face masks connected to a Dautreband generator, and exposed to an aerosol generated from a 10 mg/ml solution of ds-RNA. With this apparatus an exposure time of 5-10 minutes sufficed to deliver a dose of approximately 1 mg/kg. Pre-inoculation and post-inoculation skin tests, regular blood sampling, and i.v. challenge with ds-RNA were carried out in these aerosol-dosed animals as already described. The experimental details for this group of animals are recorded in Table 2. TABLE 2 DETAILS OF ANIMALS DOSED REGULARLY WITH ds-RNA IN AEROSOL FORM

Animal Mouse Guinea-pig Baboon

Species or strain CFLP Dunkin-Hartley Papio cynoccphalus and P. anubis

Number 40*t 12t

lot

Weight

Weekly dose

Final challenge dose (i.v.)

18-22 g 250-300 g 4-5-6-1 kg

14 pg/animal 150 pg/animal 1 mg/kg

150 pg 750 pg 6 mg/kg

* Twenty of these mice were dosed and challenged with poly I-poly C instead of ds-RNA. t All male. + Equal numbers of males and females.

(iii) Dosing in adjuvant. Groups of mice, guinea-pigs and baboons were injected with ds-RNA complexed with MeBSA by the method of Plescia, Braun and Palczuk (1964) and Wold, Young, Tan and Farr (1968). Injections were intraperitoneal or intramuscular; the initial dose being emulsified in Freund's complete adjuvant whilst the second and third doses consisted of the complex suspended in saline. Additional i.v. injections of ds-RNA in saline were also given in some cases. Preinoculation and post-inoculation skin tests and regular blood sampling were carried out in these immunized animals as already described. These animals were 'challenged' twice: first with ds-RNA in aerosol form then, after an interval of 5 or 6 days, with ds-RNA in saline given intravenously. The experimental details for this section are recorded in Table 3.

Antibody determination Serum samples were assayed for the presence of anti-ds-RNA antibody by a modification of the Farr technique using [3H]ds-RNA or [3H]poly I-poly C, similar to that described by Wold et al. (1968) with DNA. In each duplicated test, 0 1-ml volumes of serum (diluted in borate buffer, pH 8.3) and 0.1-ml volumes of [3H]ds-RNA were mixed and incubated, with shaking, at 370 for 1 hour. Following overnight incubation at 4°, bound antigen was precipitated by adding 0-2 ml of 70 per cent saturated ammonium sulphate. After a further hour at 40 the precipitate was spun down, washed with 1 0 ml of 35 per cent saturated ammonium sulphate, reprecipitated, and finally solubilized in Soluene (Nuclear Chicago Enterprises, Ltd). After dispersion in 12 ml of scintillation fluid (2-ethyoxyamnifluor containing 0-2 ml of glacial acetic acid) radioactivity of the precipitate was counted.

1005

Antibody Response to ds-RNA TABLE 3

DETAILS OF

ANIMALS IMMUNIZED WITH

ds-RNA+MeBSA IN ADJUVANT Treatment

Final challenge dose

Animal

Species or strain

Number

Weight

Day

Mouse

CFLP

20*

18-22 g

(CBAx C57Bl)FI

20t

21-25 g

P. cynocephalus and P. anubis

lot

3-1-5-2 kg

Dunkin-Hartley

18*

250-300 g

150 jug 14 pg/animal 0 50 pg+MeBSA in on day 35 on day 41 adjuvant (i.p.) 7 50,pg+MeBSA in saline (i.p.) 14 50 pg+MeBSA in saline (i.p.) 21 50 pg in saline (i.v.) 2 mg/kg 6 mg/kg 0 5 mg/kg+MeBSA in on day 37 on day 42 adjuvant (i.m.) 7 5 mg/kg+MeBSA in saline (i.m.) 14 5 mg/kg+MeBSA in saline (i.m.) 21 2 mg/kg in saline (i.v.) 0 250 pg+MeBSA in 150 pg/animal 750 jpg on day 69 on day 75 adjuvant (i.p.) 7 250 ug+MeBSA in saline (i.p.) 14 250 pg+MeBSA in saline (i.p.) 21 250 pig in saline (i.v.) 48 250 pg in saline (i.v.) 56 250 pg in saline (i.v.)

Baboon

Guinea-pig

Dosage

Aerosol

Intravenous

* All male.

t Equal numbers of males and females.

After conversion of counts to d/minute, the percentage added antigen bound by a given quantity of serum was calculated from the radioactivity precipitated by an immune serum less that precipitated by the corresponding pre-inoculation (normal) serum. Normally in this study the antibody content of a serum sample will be reported in terms of the percentage of added antigen bound by a specified dilution of that serum. However, in those species where antibody activity was high enough, it is reported as the antigenbinding capacity (ABC33)-which is calculated from that serum dilution capable of binding 33 per cent of added antigen (determined graphically from a semi-log plot of percentage binding).

Class-specific antibody determination The immunoglobulin classes containing antibody activity in selected whole sera were determined using standard gel filtration and 2-mercaptoethanol inactivation techniques. Gel filtration was carried out using a Sephadex G-200 column equilibrated with TrisHCl buffer in 0-2 M sodium chloride, pH 8-0. The column measured 3-2 x 91 cm and was calibrated with blue dextran 2000 (V0 = 237 ml) at a flow rate of 25 ml/hour. Samples of 5-6 ml of normal or immune serum were applied to the column in a 1: 1 mixture with buffer. The optical densities of individual tubes from the fraction collector were measured at 280 nm and samples pooled within each of the three major peaks obtained. Binding activity for [3H]ds-RNA of the pooled samples was determined as for whole serum. Inactivation of the 19S (IgM) component of selected immune sera was achieved by incubating equal volumes of serum and 0-2 M 2-mercaptoethanol solution at 370 for 30

1 006 P. G. Cunnington and J3. D. Naysmith minutes. After addition of sufficient buffered saline to give the required final dilution of serum, residual antibody activity was determined as before.

Degradation of ds-RNA by plasma in vitro Equal volumes of ds-RNA solution, fresh blood plasma (diluted 1:5 in saline), and 0 06 M Tris-HCl buffer, pH 7 6, were mixed in stoppered tubes and incubated at 37°. At intervals between 0 and 15 minutes the reaction was stopped by adding phenol containing 8-hydroxyquinoline. Undegraded ds-RNA was precipitated from the aqueous phase by 2 volumes of chilled ethanol, separated by centrifugation in the cold, and redissolved in 0415 M NaCl solution. (This extraction procedure has an efficiency of approximately 70 per cent for ds-RNA, with complete removal of protein.) Electrophoresis of samples involved the application of 50 yl in EDTA-sucrose buffer to 2 75 per cent acrylamide gels using a micro-syringe, running at 5 mA for 3 hours, followed by brief washing in water and fixation in 7 per cent acetic acid. Fixed gels were scanned at 260 nm in a Gilford spectrophotometer and the ds-RNA peaks integrated. Results for plasma-degraded samples are presented as percentage loss of intact ds-RNA with time.

Statistics Statistical evaluation of results, where applicable, was performed by means of Student's t-test, or by the paired t-test for sequential samples. RESULTS ANTIBODY FORMATION

(i) Intravenous ds-RNA Antibody formation was consistently found in mice, rats and rabbits when ds-RNA was administered i.v. Table 4 gives the ABC33 values of serum samples taken from these species after six injections of ds-RNA. The mean ABC33 of CFLP mice at this time was 105 jg/ml, and that of (CBA x C57B1)Fl hybrid mice was 1 64 pg/ml. In nine rats sampled at this time the mean ABC33 was lower (0 56 pg/ml) but there was a marked variation between individual animals. A markedly stronger antibody response was found in rabbits. After six injections of ds-RNA, four out of five rabbits had an ABC33 between 9 5 and 15 0 ug/ml. The fifth animal (not included in the table) responded poorly, its ABC33 was 0-21 Pg/ml. In these three species antigen-binding activity was found at all time intervals studied, beginning with the first bleed taken 14 days after the first injection of antigen. TABLE 4 ANTIGEN-BINDING ACTIVITY FOR ds-RNA IN THE SERA OF MICE RATS AND RABBITS BLED 1 WEEK AFTER SIX i.\v. INJECTIONS OF ds-RNA

Species

Strain

of animals

Mean ABC33 Pg/mi (± 1 s.d.)

Mouse

CFLP (CBA x C57B1)F1

15 14 9 4

1-05+0 90 1*64+ 0-96 0-56 + 0 37 12-60 + 2-20

Number

Rat Rabbit

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1008 P. G. Cunnington and J. D. 1N/aysmith In guinea-pigs, dogs and baboons antigen-binding activity was more difficult to detect; and when present was at a much lower level. In guinea-pigs, tested after six injections of ds-RNA, only two animals showed a significant level of antigen-binding activity using serum at a dilution of 1:5. A full time-course study using undiluted serum, however, revealed that all the guinea-pigs produced antibody at some time during immunization. There was great variation between individual animals in both the pattern and magnitude of the response (Table 5). Similarly in dogs, tested after six injections of ds-RNA, only two animals possessed antigen-binding activity and this was at a very low level. From the time course study, however, the interesting finding emerged that antigen-binding activity was present in most sera 14 days after the first injection of antigen, and declined steadily thereafter (Table 5). Higher levels of binding were found in male than in female dogs. In baboons, after six injections of ds-RNA, binding activity was barely detectable. When all the serum samples were assayed, six out of eight animals showed very low binding at some time during immunization. But unlike the situation in dogs there was no consistent pattern in the development of this activity (Table 5). The i.v. administration of poly Ipoly C to mice resulted in low levels of antigen-binding activity (for [3H]poly I-poly C) in the CFLP strain, and in (CBA x C57B1)Fl hybrid females (Table 5). TABLE 6 COMPARISON OF ANTIGEN-BINDING ACTIVITY FOR POLY I-POLY C AND ds-RNA IN THE POOLED CFLP MICE DOSED REGULARLY WITH POLY I-POLY C OR ds-RNA IN AEROSOL FORM

Immunizing antigen

Assaying antigen*

Percentage added antigen bound at successive bleedst

i+

Poly I-poly C ds-RNA

SERA OF

2

3

4

5

6

(PolyI-polyC

01

06

02

1-2

06

0-1

07

ds-RNA tPoly I-poly C

0-3 11-6

0-5 22-1

06 45-2

07 50 7

05 46-1

0-3 53-1

1-9 49-8

Lds-RNA

34-9

51-7

63-6

69-3

66.5

7

67-0

* [3H]Poly I-poly C = 20,646 d/min (97 ng/tube); [3H]ds-RNA = 82,223 d/min (98 ng/tube). t Equal volumes of twelve individual serum samples were pooled for each time interval, and used undiluted. t First bleed sera obtained 2 weeks after first dose of antigen. Successive bleeds at weekly intervals

thereafter-except for the seventh bleed which was obtained

17

days after the sixth bleed.

(ii) Aerosol ds-RNA The antigen-binding activity in serum samples of animals exposed to ds-RNA by aerosol was much lower than that detected following systemic injection of antigen. Fourteen out of fifteen mice sampled after six exposures had clearly demonstrable antibody, and the time course study shows that nineteen out of twenty mice produced antibody at some time during dosing (Tables 5 and 6). The mean ABC33 for the nine mouse sera in which activity was high enough to make this calculation possible was 0A46 yug/ml (range 0-24-0-8 pg/ml). Antigen-binding activity was observed in the sera of several mice after only one or two exposures to antigen. All guinea-pigs sampled after seven doses of ds-RNA contained antigen-binding activity, and a few animals showed such activity at earlier stages (Table 5). However, it was only possible to detect this activity using undiluted serum, which indicates the weakness of the response induced in guinea-pigs by aerosol exposure.

Antibody Response to ds-RN#A

1009

Using our standard assay we were unable to detect any specific binding activity in the sera of baboons dosed by the aerosol route. In an attempt to detect very low levels of activity we used 40 per cent saturated ammonium sulphate in the assay (to increase precipitation, both specific and non-specific) and when this was done eight out of the ten baboons proved to have very low levels of activity at some point in the dosing schedule (Table 5). However at no point in the study did the mean d/minute values show a statistically significant difference from that found with pre-dosing sera. Poly I-poly C administered to CFLP mice by aerosol resulted in only very low levels of activity which were barely detectable (Table 6). (iii) ds-RNA in adjuvant The sera of all three animal species injected with ds-RNA complexed with MeBSA in Freund's complete adjuvant contained high levels of antigen-binding activity when assayed 28 days after the first injection (Table 7). Of thirty mice assayed twelve had an TABLE 7 ANTIGEN-BINDING ACTIVITY FOR ds-RNA IN THE SERA OF MICE, GUINEA-PIGS AND BABOONS 28 AND 63 DAYS AFTER STARTING IMMUNIZATION WITH ds-RNA+MeBSA IN ADJUVANT

Mean ABC33 pUg/ml

Species Mouse

Guinea-pig Baboon

Strain

(±1 S.d.)

Number of animals

CFLP (CBAX C57BI)F1 -

18

12 18 12

Day 28

Day 63

18-39+ 13-4 15*87+8-7 1[37+ 069 7.22 + 6-3

0-71+04

ABC33 greater than 50 jig/ml, eleven had an ABC33 between 10 and 50 ug/ml, and the remaining seven had an ABC33 between 2-8 and 10 jg/ml. The mean ABC33 for eighteen guinea-pigs was 1 37 yug/ml (range 0-32-2-50 ,ug/ml). An attempt was made to further boost the antibody levels in guinea-pigs by giving two additional injections of 250 ,ug ds-RNA in saline. When these animals were subsequently bled, on day 63, their sera contained less antibody than before (mean ABC33 = 0-71 yg/ml). Somewhat surprisingly (in view of the weak response obtained following i.v. and aerosol exposures in this species) the sera from all ten baboons contained high antigen-binding activity. The mean ABC33 was 7-22 yug/ml (range 0-40-18 10 pg/ml). This level of anti-ds-RNA binding activity was only exceeded in this study among rabbits injected repeatedly by the i.v. route and mice injected with ds-RNA+ MeBSA in adjuvant.

(iv) Characterization and specificity of anti-ds-R.NA antibodies Individual terminal bleed sera containing moderate to high binding activity were selected for each species and fractionated on Sephadex G-200. Three protein peaks were obtained: fraction A (IgM); fraction B (IgG); fraction C (albumin). Table 8 shows the antigen-binding activities of pooled samples from within each of these peaks compared with normal pre-inoculation sera fractionated in the same way. In every case antigen-

P. G. Cunnington and J. D. Naysmith 1010 binding activity was higher in fraction A than in fraction B. Only in the guinea-pig was there no detectable activity in fraction B. TABLE 8 ANTIGEN-BINDING ACTIVITY FOR ds-RNA IN SERUM FRACTIONS OBTAINED BY SEPHADEX G-200 CHROMATOGRAPHY OF NORMAL AND IMMUNE SERA FROM FIVE ANIMAL SPECIES

Percentage added antigen bound*

Species

Immunizing treatment

Mouse

ds-RNA i.v.

Baboon

ds-RNA+MeBSA in adjuvant ds-RNA i.v.

Rabbit

ds-RNA i.v.

Rat

Guinea-pig

ds-RNA+MeBSA in adjuvant

Serum

Fraction A (Kav -0 03 to 0.03)

Fraction B (Kav 0-13 to 0-28)

Fraction C (Kav 0-34 to 0-51)

Normal Immune Normal Immune Normal Immune Normal Immune Normal Immune

1-4 14-6 06 43-7 1-2 41-6 1-3 16-8 1.0 4-2

1-5 6-9 0-7 10-2 1-0 21-2 1-5 4-8 09 09

1-2 1-1 0-7 08 0-7 06 0-7 1-0 0-6 08

Immune complexes were precipitated using 40 per cent saturated ammonium sulphate. * [3H]ds-RNA added: baboon, rabbit and guinea-pig, 17,184 d/min (20 ng/tube); mouse and rat, 13,072 (15-4 ng/tube).

dlmin

The antigen-binding activity in serial serum samples from three rabbits was assayed before and after incubation of the sera with 2-mercaptoethanol (Table 9). Mercaptoethanol-sensitive antibody' (19S, IgM) was present throughout the immunization procedure, and in two out of three rabbits was the predominant antibody type even in the sera from later bleeds. A similar distribution of antibody activity was found in the sera of mice immunized with ds-RNA (results not presented). TABLE 9 ANTIGEN-BINDING ACTIVITY FOR ds-RNA IN THE SERA OF THREE RABBITS, DOSED REGULARLY WITH ds-RNA i.v. AFTER INCUBATION OF THE SERUM SAMPLES WITH 2-MERCAPTOETHANOL (2-ME) OR SALINE

Percentage added antigen bound* at successive bleedst Rabbit number

Treatment of serum

1

Saline 2-ME Saline 2-ME Saline 2-ME

2 3

1

2

3

4

2-1

2-4 02 3-9 0 13-4 0-2

36-7 05 26 0 0 45-4 43-8

46-2 20 6 39-1 05 33-2 46-3

0-1 06 0 01 0-1

5

6

7

50 3

45-7 42-4 39-1 16-6 44-2 45-8

50 8 17-3

38-1 34-2 4-9 44-1 43-8

449 46-8

* [3H]ds-RNA added; 25,075 d/min (29 ng/tube). t First bleed sera obtained 2 weeks after first dose of antigen. Successive bleeds at weekly intervals

thereafter, except for the seventh bleed which was obtained 17 days after the sixth bleed.

The specificity of the antibodies produced in response to ds-RNA administration was tested by incubating antibody-containing serum samples with a 10-15-fold excess of

Antibody Response to ds-RNA 1011 various unlabelled nucleic acid antigens prior to the addition of [3H]ds-RNA. Such inhibition experiments were carried out only with antisera which we knew gave adequate binding with known amounts of [3H]ds-RNA. As can be seen from Table 10, in all species only ds-RNA antigens inhibited binding of ds-RNA. Except in the rabbit, the synthetic polyribonucleotides poly I-poly C and poly Apoly U gave a similar degree of inhibition as fungal ds-RNA. Since under the conditions of this experiment there was little inhibition of binding in rat sera, and in the sera of baboons given ds-RNA+MeBSA in adjuvant, the binding of these sera for [14C]DNA was tested directly, with uniformly negative results (details not included). In addition the sera from baboons given ds-RNA by all three routes were tested for anti-nuclear antibodies by means of an indirect fluorescent antibody technique. Frozen sections of normal rat liver were used as the target tissue, and a positive serum from a case of human SLE was used for control purposes. No anti-nuclear antibodies were found in the sera of any of the baboons immunized with ds-RNA. TABLE 1 0 INHIBITION OF [3H]ds-RNA BINDING BY ANTI-ds-RNA SERA FOLLOWING INCUBATION WITH ds-RNA, POLY I-POLY C, POLY A-POLY U, DNA OR ss-RNA

Species

Mouse Mouse Dog Rabbit Guinea-pig Guinea-pig Rat Baboon

Route of

Mean percentage inhibition of binding (relative to saline)*

immunization

ds-RNA

i.v. Aerosol i.v. i.v. i.v. +MeBSA in adjuvant i.v. +MeBSA in adjuvant

46-3 57-7 22-6 53-1 89-4 50-0 25-4 28-5

*

Poly I-poly C Poly A-poly U

DNA

ss-RNA

20-7 0-9 0-1 0-7 2-0 2-1

2-3 0 0 6-0

33-0

47-7 78-6 83-7 0 70-0 10-2

0 4-2

0 11-0

13-5 1-4

58-8 75-3 22-6 0

60-7

0-8 0-9 0

1-2

Mean of values for two or three individual sera.

BIOLOGICAL EFFECTS OF REPEATED DOSING WITH ds-RNA

In this study 248 animals, belonging to six different species, were regularly given large doses of ds-RNA or poly I-poly C. In only two situations was there any indication of adverse effects associated with this ds-RNA administration. The first situation involves the intradermal injection of 50 ug of ds-RNA which was given to all animals prior to the dosing regimen, and again when dosing was completed. In mice, rats and baboons, no inflammation was observed at the site of injection either before or after dosing. In rabbits, dogs and guinea-pigs the intradermal injection of dsRNA provoked the development of an inflammatory reaction at the injection site. In all three species the gross appearance of the reaction was similar, and at 24 48 hours consisted of a pale central area surrounded by erythema. The reactions were slow to developerythema being apparent in dogs and rabbits at 7 hours, and in guinea-pigs at 4 hours. The maximum reaction size was found in guinea-pigs, and had a diameter of 20-30 mm. The reactions resolved over the ensuing 1-2 days leaving no signs of damage. In guinea-pigs and rabbits, the post-dosing intradermal inflammation may have been more E

1012 P. G. Cunnington and J. D. Naysmith severe, and in dogs less severe than that seen in animals prior to dosing. However, when these lesions were quantified by measuring the diameter of reactions and the incidence of positive reactions with time after challenge, there was no statistically significant difference between pre-dosed and post-dosed animals. Neither was any correlation found between antibody level and the severity of intradermal inflammation when individual animals were considered. The second situation involved seven CFLP mice which had been given seven exposures to ds-RNA by aerosol (estimated dose = 14 pg per mouse per exposure) over a period of 8 weeks. One week after the final aerosol dose and terminal skin tests, these animals were given an i.v. injection of 150 pg of ds-RNA. Within 1 hour of the injection, five of these animals became obviously ill: they exhibited convulsions, panting, ruffled fur, hunched posture, lack of co-ordination and eventually lethargy. (Difficulty was experienced in obtaining blood samples from these animals, probably due to a fall in blood pressure.) Two of the five affected mice became moribund and were killed, the other three gradually recovered and appeared normal 24 hours later. The five surviving mice were killed, and compared with the two which had become moribund and control mice in terms of gross anatomy and detailed histology. Particular attention was paid to liver, kidney and lung tissues, and kidney sections were examined by electron microscopy. No significant differences emerged between any of these animals which might explain the onset of distress in the affected animals. Although the immediate signs of distress suggested the onset of some form of anaphylactic or allergic reaction, it was not possible to confirm this by any recognized criterion. The affected mice had antibody to ds-RNA, but the levels were quite low (ABC33 values were less than 1 jg/ml). DEGRADATION OF

ds-RNA

BY FRESH PLASMA

The ability of fresh plasma to degrade ds-RNA in vitro is shown, for seven species, in Table 1 1. Over a 15-minute period more than 80 per cent of intact ds-RNA was destroyed in human and baboon plasma whereas there was only 10-25 per cent breakdown in rabbits, rats and guinea-pigs. Over the same period there was no significant breakdown in dog mouse plasma. As can be seen from the table there is no obvious correlation between slow plasma breakdown and immunogenicity of ds-RNA on i.v. injection. TABLE 11 COMPARISON OF ds-RNA IMMUNOGENICITY AND DEGRADATION OF ds-RNA BY FRESH PLASMA IN VARIOUS ANIMAL SPECIES

Species Mouse Rabbit

Dog Rat

Guinea-pig Baboon Human

Percentage intact ds-RNA Immunogenicity* after 15 minutes in plasma

High High Medium Medium Low Low Not known

100 88 100 85 77 2

12

* Based on antigen-binding activity induced in serum during i.v. dosing study.

1013 Antibody Response to ds-RNA These results were extended in mice, dogs, rabbits and guinea-pigs by injecting radiolabelled ds-RNA into animals and measuring the radioactivity in the acid-insoluble fraction of serum at various time intervals thereafter. In the rabbit and the guinea-pig the half-life of ds-RNA was relatively short (13-27 minutes and 15-33 minutes respectively). A similar result was obtained in the dog, but in mice the half-life of ds-RNA was considerably longer (65-75 minutes) and there was virtually no loss during the first 50 minutes following injection. Again there is no obvious correlation between in vivo half-life and

immunogenicity. DISCUSSION The results presented in this paper indicate that fungal ds-RNA is immunogenic in a wide variety of mammalian species. When injected i.v. in native form to mice and rabbits high levels of antibody were consistently obtained. Rats and dogs also produced moderate to good antibody responses when immunized by this route, but there was more variation between individual animals in these species and there was a tendency for antibody formation to decrease in dogs as ds-RNA administration continued. Guinea-pigs and baboons responded only weakly to i.v. administration, and there was much variation between individual animals. When ds-RNA was delivered to the respiratory tracts of mice, guineapigs and baboons in aerosol form an antibody response was only obtained in mice and guinea-pigs. In both cases the response was markedly less than that obtained following i.v. injection of ds-RNA. A similar result was found when i.v. administration of poly Ipoly C to mice was compared with aerosol administration, and the antibody level induced by poly I-poly C was significantly lower than that induced by equivalent doses of ds-RNA. When ds-RNA was complexed with MeBSA and given to mice, guinea-pigs and baboons together with Freund's complete adjuvant, very high levels of anti-ds-RNA antibody were obtained in each case. Although antigen was administered repeatedly and sometimes injected in adjuvant (both conditions which favour IgG antibody formation) in most species in this study IgM antibody tended to predominate throughout immunization. Only in the rabbit was this not clearly observed, and the rabbit was also an exception when the specificity of the anti-ds-RNA antibodies was studied. In most species antigenbinding activity was inhibited by poly I-poly C or poly A-poly U, suggesting that antibody specificity was directed against the double-stranded configuration common to the naturally occurring and synthetic polyribonucleotides. No inhibition was observed with DNA or ss-RNA, and when high titre rat, mouse and baboon anti-ds-RNA antisera were tested against ['4C]DNA directly, no binding was detected. Only rabbit antisera could not be inhibited by synthetic polyribonucleotides suggesting that this species can recognize additional antigenic structures (possibly particular sequences of bases) present in ds-RNA

preparations. Although all the animals in this study received repeated high doses of ds-RNA (very much higher than doses required to demonstrate antiviral or adjuvant activity) the incidence of adverse effects attributable to ds-RNA was remarkably low. This was true of animals with high anti-ds-RNA antibody, and in animals lacking such activity. Intradermal injection of ds-RNA to non-immune rabbits, guinea-pigs and dogs was followed by the development of erythematous inflammatory lesions at the sites of injection. Similar lesions also developed on injection of ds-RNA in these three species following immunization-there was however, no evidence to suggest any change in the underlying mechanism

1014 P. G. Cunnington and J. D. Naysmith neither was there any correlation between antibody level and severity of the lesion. In mice, rats and baboons intradermal injection of ds-RNA provoked no discernible lesions either before or after immunization. In one group of seven mice, which had been immunized by exposure to ds-RNA in aerosol form, i.v. challenge with ds-RNA produced an immediate systemic reaction in five animals. Although the reaction appeared superficially to resemble classical systemic anaphylaxis, we have no firm evidence to confirm or deny this possibility. The antibody levels present in the affected animals were lower than in mice which had been immunized i.v. or by ds-RNA given in adjuvant, none of which showed any adverse effects on challenge with ds-RNA given i.v. No untoward reaction was observed in guinea-pigs and baboons exposed to ds-RNA repeatedly by aerosol and challenged i.v. In this study, which is the first extensive analysis of the immunogenicity of a naturally occurring ds-RNA and the first to report antibody formation against any kind of doublestranded RNA in rats and dogs, the results agree broadly with findings of others who have used synthetic polyribonucleotides such as poly I-poly C and poly A-poly U. The differences which do exist can be satisfactorily explained on two grounds: (a) the greater inherent immunogenicity of ds-RNA compared with poly I-poly C which was demonstrated directly in the present work, and (b) our use of a primary-binding technique to demonstrate antibody rather than secondary techniques such as complement fixation and passive haemagglutination which have been used in some other studies. There seems to be general agreement that double-stranded polyribonucleotides are highly immunogenic in rabbits (Field et al., 1972; Parker and Steinberg, 1973) and this may explain the recent finding in this laboratory that the difference in immunogenicity between poly I-poly C and ds-RNA seen in mice is much less marked in rabbits (Cunnington, to be published). Double-stranded polyribonucleotides of many types are also immunogenic in mice (Steinberg, Chused, Jacobs and Talal, 1970; Stollar, Fuchs and Mozes, 1973) but to obtain high antibody titres most previous investigators have had to use materials incorporated in adjuvant or complexed with MeBSA (Lambert and Dixon, 1970; Talal and Steinberg, 1971; Steinberg, Pincus and Talal, 197 1b), and there appear to be marked differences between the responses obtained in different mouse strains. Field et al. (1972) using a complement-fixation technique, failed to obtain antibody formation against poly I-poly C in guinea-pigs following its repeated i.v. injection; but, as with ds-RNA in the present study, Van Boxel, Steinberg and Green (1972) obtained a weak response when poly I-poly C was injected into guinea-pigs in its native form, and a much more marked response when it was given in adjuvant or coupled to MeBSA. There are few reports bearing on the immunogenicity of polyribonucleotides in primates, but Field et al. (1972) and Field, Young, Krakoff, Tytell, Lampson, Nemes and Hilleman (1971) failed to detect antibodies in the sera of grivet monkeys and humans respectively following i.v. injection of poly I-poly C. However, these two studies are subject to significant limitations: in both, complement fixation was used in an attempt to detect antibody, and in the latter study the subjects were terminal cancer patients given relatively low doses of polyribonucleotide and only a few received repeated injections. In the present study very weak responses were obtained to native ds-RNA in baboons, but the fact that ds-RNA was highly immunogenic in this species when given as a complex with MeBSA in adjuvant suggests that double-stranded polyribonucleotides may be immunogenic in primates as they are in all other groups of animals which have been adequately tested. There is however, clear evidence of differences in the immunogenicity of native material which

1015 Antibody Response to ds-RNA largely disappears when polyribonucleotides are coupled to carrier molecules or given in adjuvant. This differential immunogenicity of native material might be explained if there was a species-specific rate of breakdown in plasma, thus allowing different effective doses of immunogenic material to reach the lymphoid tissues. A serum nuclease, specific for double-stranded polyribonucleotides, has been described in a number of species (Stern, 1970), and Hadjiyannaki and Lachmann (1972) have described an inhibitor of nuclease activity in the serum of NZB mice which they argue could be associated with the high levels of anti-DNA antibody found in that strain. In the present study the rate of breakdown of ds-RNA in fresh mouse plasma was found to be relatively low, while in baboon plasma the rate of breakdown was high. A consideration of only these two species would suggest a good correlation between rate of breakdown and immunogenicity. However, the rabbit, which is a high responder to ds-RNA, had an appreciable plasma breakdown rate; and the dog, which had a plasma breakdown rate similar to that of the mouse, gave a much lower and more variable level of antibody formation than the mouse. So although it is clear that no absolute correlation exists, it seems likely that in species such as the baboon very rapid breakdown of ds-RNA may preclude the development of marked immune responses. In species with slow breakdown rates, genetic factors such as those demonstrated for different mouse strains (Lambert and Dixon, 1970; Steinberg et al., 1971b; Stoller et al., 1973; Scher, Frantz and Steinberg, 1973), and perhaps operating through differential uptake of ds-RNA from the circulation (present study) may be of importance. The enhancing effect of complexing polyribonucleotides with carrier molecules and injecting them in adjuvant presumably acts, at least in part, by protecting material from enzymic degradation, but it has been shown that the interferon response of mice to poly I-poly C can be enhanced by injecting Freund's complete adjuvant separately up to 2 days later (DeClercq, Nuwer and Merigan, 1970), so there remains a possibility that the biological effects of double-stranded polyribonucleotides (including immunogenicity) may be influenced by adjuvant in as yet unrecognized ways. One of the areas of most concern when considering the therapeutic use of doublestranded polyribonucleotides in man is the specificity of the antibodies which might be induced, and the possibility that they might cross-react with autologous nuclear antigens. In the present study only rabbits produced anti-ds-RNA antibodies whose specificity was not restricted to double-stranded polyribonucleotide molecules, a result which agrees with the findings of Steinberg et al. (1971b) who used poly I-poly C and poly A-poly U in mice. The situation in the rabbit appears to be more complex, and various studies have produced conflicting results. Nahon, Michelson and Lacour (1967) and Nahon-Merlin, Michelson, Verger and Lacour (1971) found that antibody from rabbits immunized with poly I-poly C or poly A-poly U complexed with MeBSA could recognize various singlestranded RNAs and also DNA, whereas similar antisera raised by Schwartz and Stollar (1969), Field et al. (1972), and Francki andJackson (1972) did not cross-react with singlestranded RNAs or native DNA. These discrepancies are probably due partly to methodological differences since a variety of immunizing schedules and antibody detection systems have been used, but it also seems likely that since rabbits are high responders to this material they may be able to recognize determinant groups on double-stranded polyribonucleotides which are not recognized in most other species. We think it is important that in the present study no DNA-binding activity was found in the sera of baboons immunized with ds-RNA, neither was there any evidence of LE cells or antinuclear antibodies in these immunized animals.

P. G. Cunnington and J. D. Naysmith 1016 We found no evidence in the present study to suggest that immunization with ds-RNA induced cell-mediated immune responses in general or delayed-type hypersensitivity reactions in particular in any species. This agrees closely with the findings of Van Boxel et al. (1972) using synthetic ribonucleic acids in guinea-pigs, who also showed that immunization with RNA alone did not stimulate anamnestic responses. In the present study and that of Stollar et al. (1973) there was evidence to suggest that immunization with polyribonucleotides results mainly in IgM antibody responses, a finding which supports the suggestion of Van Boxel et al. (1972) that double-stranded polyribonucleotides lack carrier function and thus would not be expected to stimulate T cells or thymus-dependent immune mechanisms. There was further evidence for this idea in the finding of the present study that in a number of species, but particularly dogs, the continued administration of ds-RNA resulted in an eventual decrease of antibody formation-a feature that is reminiscent of the properties of thymus-independent antigens in mice (Baker, Stashak, Amsbaugh and Prescott, 1971). Of the two adverse effects of ds-RNA found in the present study, it seems likely that the inflammatory response induced following intradermal injection of ds-RNA in guineapigs, rabbits and dogs is another example of the well-known non-specific toxicity of doublestranded polyribonucleotides in many species (e.g. Talal, 1971). Such inflammatory reactions did not increase in severity in immunized animals, and there was no evidence that similar reactions were induced in originally non-reactive species following exposure to ds-RNA. The occurrence of an anaphylactic-like response in five out of seven mice dosed with ds-RNA and challenged i.v. is more serious, since there is already one report indicating that acute systemic anaphylaxis may accompany immunization of guineapigs with poly I-poly C or poly A-poly U (Van Boxel et al., 1972). We were unable to detect anaphylaxis in guinea-pigs in the present study, and only mice exposed to ds-RNA by aerosol prior to i.v. challenge displayed evidence of a systemic reaction. If this reacsion was antibody-mediated then it seems likely that aerosol immunization favoured the production of a particular type of antibody which was not stimulated by other routes of immunization, or in other species.

ACKNOWLEDGMENTS We thank our colleagues Dr R. C. Imrie and Dr R. A. Edmondson for giving permission to quote their unpublished data on the half-life of ds-RNA in vivo and rates of degradation of ds-RNA in plasma samples, respectively; and Mr B. Hatt for preparing the [3H]dsRNA. Dr G. Johnson, Canadian Red Cross Hospital, Taplow kindly provided serum from a patient with SLE. We are also indebted to Mrs J. L. Feaver, Miss J. Boden and Miss M. Asher for their skilled technical assistance. REFERENCES

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