Nucleic Acid-Induced Resistance to Viral Infection

JOURNAL OF BACTERIOLOGY Dec. 1965 Copyright 1965 American Society for Microbiology Vol. 9, No. 6 Printed in U.S.A. Nucleic Acid-Induced Resistance to Viral Infection KOUICHI TAKANO, JOEL WARREN, KEITH E. JENSEN, AND ALAN L. NEAL Department of Biologics Research, Chas. Pfizer & Co., Inc., Terre Haute, Indiana Received for publication 6 July 1965 ABSTRACT TAKANO, KOUICHI (Chas. Pfizer & Co., Inc., Terre Haute, Ind.), JOEL WARREN, KEITH E. JENSEN, AND ALAN L. NEAL. Nucleic acid resistance to viral infection. J. Bacteriol. 9:1542-1547. 1965.-Administration of nonviral nucleic acids to mice increased their resistance to a subsequent infection with influenza or encephalomyocarditis viruses. Injection of ribonucleic acid or deoxyribonucleic acid by peripheral routes did not modify susceptibility to intranasal infection. Lung tissue extracts from animals previously treated with yeast nucleic acid inhibited the growth of vaccinia and influenza viruses. The protective effect of exogenous nucleic acids persisted in mice for several days, but gradually diminished to undetectable levels. Although there have been at least two reports of the ability of parenterally injected nucleic acids to modify viral infection in vivo (O'Dell, Wright, and Bieter, 1953; Thely et al., 1963), it was not until recently that these could be interpreted on a rational basis. The observations of Rotem, Cox, and Isaacs (1963) and Jensen et al. (1963) that nucleic acids from various nonviral sources could induce interferonlike activity in chick fibroblast cultures, and the finding by Gifford (1965) that mononucleotides and yeast ribonucleic acid (RNA) would inhibit vaccinia plaque formation, suggested that contact of a cell with a foreign nucleic acid could induce interferon or an interferonlike inhibitor. Takano, Warren, and Jensen (1964) and Warren et al. (Arch. Ges. Virusforsch., in press) presented preliminary data on the ability of nonviral nucleic acids to increase pulmonary resistance to several viruses; the present report describes these studies in more detail. MATERIALS AND METHODS Viruses. Influenza virus was used as 1% suspensions of emulsified mouse lung containing the B/GL/1739/54 strain (21st to 28th serial mouse passage). A neurotropic strain of encephalomyocarditis virus (infected mouse brain) was inoculated into mice for five passages. A 1% suspension of infected brain or lung tissues was prepared from infected mice and stored at -7 C until required. Preparation of nucleic acids. Yeast nucleic acid (YNA) from Calbiochem and herring sperm deoxyribonucleic acid (DNA) obtained from Nutritional Biochemicals Corp., Cleveland, Ohio, were dissolved in.85% NaCl with addition of 2 N NaOH to bring the ph to 7.. An equal volume of redistilled 9% phenol was then added, and the mixture was stirred vigorously for 1 hr at room temperature. After refrigeration overnight, the phases were separated by addition of an equal volume of.85% NaCl and centrifugation. A second phenol extraction was performed in a similar manner, and the nucleic acid was finally precipitated from the aqueous solution by the addition of 2 volumes of 95% ethyl alcohol. This was collected by centrifugation, and the sediment was washed twice with 66% ethyl alcohol. The NA was then dissolved in.85% NaCl and extracted twice with two-thirds volume ethyl ether, after which the latter was removed by aeration and the product was dried. Yeast RNA and sperm DNA concentrations were determined spectrophotometrically on solutions in phosphate-buffered saline (ph 7. to 7.2). Mice. Female Swiss albino mice weighing 15 to 17 g were caged in groups of 1 to 15, and not fewer than 15 animals were used for challenge with a given virus concentration. Procedures. A nucleic acid solution containing 2 to 6 mg/ml was administered by one or more daily, intranasal instillations of.5 ml while mice were under light ether anesthesia. After 24 hr, these and control mice, which received a placebo of buffered saline solution, were inoculated intranasally with.5 ml of virus administered at several different concentrations. Animals were observed daily, and those dying within 72 hr after challenge were discarded from the experiment. RESULTS Protective effect of nucleic acids administered prior to infection with influenza and encephalomyocarditis (EMC) viruses in mice. Instillation of solutions containing 2 to 6 mg/ml of YNA into the nostrils of mice prior to infection increased the resistance to a subsequent intranasal 1542

X/- VOIJ. 9, 1965 RESISTANCE TO VIRAL INFECTION 1543 TABLE 1. Effect of intranasal administration of yeast nucleic acid* on influenza infection in the mouse Mortality at LD5o Time of Interval before Group Treatment challenge 32 lo 32 1 3 days hr 1 4 24 5/18 (28%) 2/18 (11%) /17 (%) 2 1 48 11/2 (55%) 9/2 (45%) 2/2 (1%) 3 1 72 18/2 (9%) 14/2 (7%) 5/2 (25%) 4 None Controls 15/15 (1%) 15/15 (1%) 15/15 (1%) * Dosage of 3 mg per.5 ml. x~ 5 \!a~ \ \x c' G- I-. 4t : J2 ( LD 5 2 6 CHALLENGE FIG. 1. Effect of nucleic acids on respiratory infection with EMC virus. Symbols: X, untreated control;, yeast nucleic acid, 3 mg per dose;, yeast nucleic acid,.5 mg per dose; A, sperm DNA, 3 mg per dose; A, sperm DNA,.5mg per dose. challenge with influenza or EMC viruses. Shown in Table 1 are typical mortality rates for treated and control animals when challenged with various doses of B/GL influenza virus. Mice in group 1 were treated with YNA on four successive days, and were infected 24 hr later. Groups 2 and 3 were treated only once and then were challenged 2 and 3 days later, respectively. All 45 control animals receiving 3 to 32 LD5o of virus succumbed within 8 days. Reduction of mortality in each of the other groups was related to the extent of treatment before challenge. YNA had no direct inactivating effect on virus when mixtures containing as much as 1 mg/ml were incubated with several concentrations of influenza for 1 hr and then titrated in mice or embryonated eggs. The protective effect of YNA against EMC virus was determined in basically the same fashion. Figure 1 contains data from an exper-.6 8 5 8 LD5 CHALLENGE FIG. 2. Relation of yeast nucleic acid dosage to protection in mouse influenza. Dosages are given in milligrams per mouse per day, administered four times intranasally. iment in which groups of 15 mice received four daily treatments with purified YNA or herring sperm DNA at concentrations of.5 and 3. mg/dose. On the following day, these and control animals were challenged by intranasal instillation of varying LDuo doses of lung-adapted EMC virus. Protection induced by the 3-mg dose of either nucleic acid was superior to that resulting from.5 mg. Relation of dosage to protection. To obtain information on the dose-response relationship, we performed several experiments with influenza virus in which the concentration of YNA was varied. Figure 2 illustrates the decrease in mor-

1544 TAKANO ET AL. J. BACTERIOL. S~~~~~~~~ I 5 I-- O 3 2 1 L D5 CHALLENGE FIG. 3. Comparison of single and repeated treatment with yeast nucleic acid. tality as the amount of YNA was increased from.25 to 2 mg and given daily for 4 days. A test of the effectiveness of a single treatment and four daily treatments with YNA indicated that it was possible to achieve complete protection against 1 LD5o of B/GL virus when a dose of 3. mg per mouse was used (Fig. 3). It was of interest that approximately 4% of the group survived a virus inoculation of 1 LDo when infected 24 hr after a single treatment with 3 mg. Duration of protection. To determine the duration of prophylaxis, animals were treated intranasally for four successive days with YNA at 3 mg per dose and were challenged with three different levels of influenza virus after various intervals. The results of two separate experiments are shown in Fig. 4. The level of protection remained essentially unchanged for approximately 4 days but had diminished considerable after I week. In another experiment of similar type (not shown), mice were not resistant when challenged 1 days after the cessation of treatment. Absence of curative effects and requirements of a post-treatment incubation period. There was no significant reduction in mortality when YNA was given simultaneously or 1 hr before virus; a period of approximately 24 hr had to elapse before challenge to obtain maximal protection. When mice were first infected with influenza and high concentrations of YNA were administered, subsequently, there was no reduction in the mortality. 2 3.211 3 2 LOD CHALLENGE LD5 CHALLENGE EXP A EXP B FIG. 4. Persistence of yeast nucleic acid-induced resistance in mice. Dosage was 8 mg per mouse per day, administered four times intranasally. Concentration of influenza virus in the lungs of nucleic acid-treated mice. In the studies described above, protection was estimated in terms of reduced mortality after virus challenge. In earlier work (Takano, Jensen, and Warren, 1963), it was found that the lungs of mice pretreated intranasally with an exogenous viral interferon contained less influenza virus than did control animals at the same stage of infection. It was therefore decided to determine the concentration of virus in the lungs of animals pretreated with nucleic acid. Accordingly, a group of mice was given 3 mg of YNA intranasally for 4 successive days. After a 24-hr rest period, they were divided into three groups and inoculated intranasally with 3, 1, or 3 LD5 of B/GL virus. Similar numbers of untreated control mice were also inoculated with these doses of virus. After 3 days, 2 of the treated and 24 control mice were killed, their lungs were removed, and their content of virus was determined individually by inoculation of lung tissue suspensions into embryonated eggs. The distribution of titers is shown in Fig. 5. In each instance, there was less virus in the pulmonary tissues of the YNAtreated mice, and the difference between the average EID5 in control and treated mice was approximately 3 logs. Also shown is the mortality in a similar group of 45 controls and 53 treated animals which were not killed, to permit an estimation of mortality at the three levels of challenge virus employed. If one assumes that the death rate in the animals killed to obtain lung tissue would have been essentially the same as in those which were permitted to survive, the data indicate that mice having a virus

VOL. 9, 1965 RESISTANCE TO VIRAL INFECTION 15D45 MORTALITY - DAYS CONTROL 15/15 15/15 15/15 TREATED 5/18 2/18 /IT 8. _ LUNG VIRUS CONTENT - 3 DAYS T. E 6. - 5.- * CONTROL 4.- 8 3. O 2.- IC YNA O TREATED 3 io CHALLENGE L D 5 FIG. 5. Influenza virus concentration in lungs of treated and control mice. concentration of 14- EID5o per g or less on the third postinfection day would have recovered. A similar virustatic effect has been found in the lungs of mice treated with YNA and then infected with EMC virus. Relation of route of nucleic acid administration to prctection. To test the effects of aerosol inhalation, a DeVilbis ultrasonic nebulizer attached to a stainless-steel chamber was used. Mice were treated with nucleic acid aerosols for various periods and then were challenged with influenza virus. Results of a typical experiment are shown in Fig. 6 in which groups of 1 mice were exposed for 15 min daily for 7 days to a solution of herring sperm DNA (6 mg/ml) at a flow rate of 4 ml/ min. Control and treated groups were infected with influenza virus 1 day after cessation of treatment. Significant protection was obtained but, in general, the direct intranasal deposition of drug has been superior to an aerosol; however, optimal methods for aerosolization and delivery have not yet been determined. In contrast to the findings obtained when a nucleic acid was placed in direct contact with the respiratory tract of mice, injections by intraperitoneal, subcutaneous, or intravenous routes with even larger amounts of YNA in 3. 25 8 25 8 2.5 LD5 CHALLENGE FIG. 6. Influenza protection by instilled or nebulized yeast nucleic acid. aqueous solution or subcutaneously in a mineral oil adjuvant failed to induce resistance to intranasally administered influenza or EMC virus. These findings support the conclusion that the prophylactic effects of YNA were related to its direct contact with the respiratory epithelium. Induced resistance in relation to route of infection. When it was found that peripherally administered nucleic acid failed to protect the respiratory tract, a set of converse experiments was performed to determine whether intranasal instillation of drug would protect against virus infection through other portals of entry. Groups of animals were given YNA and 1 day after the last of four treatments were challenged with various concentrations of EMC virus; one group was infected intracerebrally, one group intraperitoneally, and one was infected intranasally. EMC virus was used because it would produce a lethal infection after parenteral injection. The results of such an experiment are shown in Fig. 7. Although resistance to respiratory infection was increased by intranasal medication, such treated animals were fully susceptible when the virus was introduced into the peritoneal cavity or the central nervous system. Stimulation of interferonlike activity by YNA. The need for direct contact between nucleic acid and the target zone of infection, together with the delay required between its adminis-

1546 TAKANO ET AL. J. BACTERIOL. ;.n. INFECTION i.c. INFECTION i.p. INFECTION 2. *HYNA-IN DUCE1-ASSAYED I IN VITRO ICETCI B/GL-INDUCED-- ASSAYED IN VVO (M IICE) O 5 X, - 5- O O 5- o 1.5 -id C- IT a.5 H'~~~@ H o II, F 16 16 6,16 63 6 3 6 32 3 2 3 L5 CHALLENGE LD5 CHALLENGE LD5 CHALLENGE FIG. 7. Induced resistance and route of infection with EMC virus (neurotropic). tration and virus challenge, suggested that formation of an intermediate material might be involved in protection. We considered formation of an interferon in view of the earlier demonstrations that nonviral YNA added to cell monolayers caused the appearance of an interferonlike factor in the cell-free supernatant culture fluids (Rotem et al., 1963; Jensen et al., 1963). Accordingly, experiments were performed in which groups of mice were treated for 4 days with YNA, (3 mg per dose), their lungs removed 1 day later, and tissue extracts were treated at low ph as described earlier (Takano et al., 1963). For purposes of comparison with a known interferon induced by virus alone, a similar group of animals was inoculated intranasally with approximately 1 LD5Q of B/GL influenza virus, and their lung tissues were processed 3 days after infection. These lung extracts were then compared for their capacity to interfere with vaccinia virlus in chick embryo tissue culture monolayers. The extracts of YNA-treated or virus-infected mouse lungs significantly inhibited the growth of vaccinia virus and were also capable of protecting mice against an intranasal challenge with influenza virus (Fig. 8). This effect was abolished if either extract was treated with trypsin or heated at 8 C for 1 hr. These findings suggest that exposure of the mouse lung to a foreign nucleic acid caused the formation of a substance resembling virus-induced interferon. Comparative activity of certain antiviral compounds in stimulating respiratory resistance in mice. To determine whether several known antiviral substances would also modify respiratory susceptibility when administered intranasally, mice were treated daily for 4 days with the following compounds and were challenged with influenza virus: 2-hydroxybenzimidazole, cycloleucine, 5-iodo-2-deoxyuridine, dimethyl-(p-carboxyphenyl azo)-hydroxybenzene, isatin B thio- A II 2 4 8 t16 132 64 DILUTION- INTERFERON t1% LUNG EXTRGCT) FIG. 8. Dose response in vitro and in vivo to virus and nucleic acid-induced interferons. semicarbazone (IBT), and N-methyl IBT at.5 mg per dose; tetramethylene benzimidazole hydrochloride and streptonigrin at.5 mg; and 1-adamantanamine at.13 mg. None of these compounds significantly reduced the mortality in several experiments wherein a reference lot of YNA was found to be effective. This is not to imply that certain of these, as adamantanamine, will not either directly inhibit growth of influenza virus or alter the course of infection. Rather, we found them to be ineffective when operating through the same mechanism whereby intranasal foreign nucleic acids modified respiratory susceptibility in the mouse. Because of the possibility that mechanical factors, perhaps related to the viscosity of YNA, might account for the reduction in influenzal mortality, we inoculated mice intranasally with 5% hydrolyzed gelatin in buffered saline and challenged them after four such treatments in the usual fashion. There was no difference in the influenza mortality in these and in a group of untreated controls. DISCUSSION These studies demonstrate that susceptibility to virus infection of the mouse respiratory tract can be modified by the administration of certain nucleic acids prior to infection. The highest levels of protection were obtained after intranasal instillation; peripherally injected nucleic acids were ineffective against influenza at the concentrations used. Conversely, significant protection was not achieved when EMC virus was inoculated into the peritoneal cavity or the central nervous system of treated animals. These findings suggest that contact of nucleic acid with the target zone of infection may be essential to successful prophylaxis. This is not in accord with earlier work of O'Dell et al. (1953), who found that some protection against MM in-

VOL. V91RESISTANCE 9, 1965 TO VIRAL INFECTION 1547 fection of the central nervous system resulted from intraperitoneal injection of nucleic acid into mice. On the other hand, it is possible that peripheral administration of large amounts of nucleic acids might influence the course of those virus infections in which viremia plays a greater role than it does in the case of murine influenza. It has been repeatedly observed in our studies of influenza infection in mice that to obtain maximal enhancement of resistance a latent period is required between the time of nucleic acid administration and virus challenge. Since yeast nucleic acid did not inactivate influenza virus in vitro, it is possible that the latent period is required for uptake of nucleic acid through the cell membrane, synthesis of an inhibitor (interferon?), and perhaps release of the inhibitor into the extracellular fluids. Repeated demonstration of the superiority of herring sperm DNA over the other nucleic acids studied suggests that the source of nucleic acid may be of importance and related to variation in the ability of nucleic acids to penetrate cells as suggested by Gifford (1964). Nucleic acid treatment of the mouse respiratory tract suppressed influenza infection but did not completely block it. This was shown by direct titration of the virus content in the lungs of individual mice. Such suppression without sterilization may be a useful aspect of nucleic acid prophylaxis in that it would enable host antibody mechanisms to curtail the infection and insure a level of humoral immunity. Available information does not permit us to define more specifically the mode or site of action of foreign nucleic acids in enhancing resistance. Although it is attractive to postulate that local stimulation of interferon follows the intranasal instillation of nucleic acid in the respiratory tract, the lack of species specificity we have repeatedly seen in lung extracts argues against this. Further, we have recently learned from S. Baron (personal communication) that the virus inhibitory factor extractable from nucleic acidtreated mouse lung does not firmly attach to cells of the test system, in contrast to "classical" interferon. On the other hand, we have recently obtained evidence that significantly higher levels of influenza virus-induced interferon develop more rapidly in the lungs of mice pretreated with YNA as compared with the levels in control mice which received virus only. This suiggests that a combination of factors may result from nucleic acid treatment, including a nonspecific antiviral response of the pulmonary epithelium and a metabolic change which results in the accelerated production of interferon in response to the challenge virus. Experiments are now in progress to compare the virus and interferon concentration in the lung, brain, and blood of nucleic acid-treated and control animals. These promise to shed additional light on this problem. LITERATURE CITED GIFFORD, G. E. 1964. Studies on the induction of interferon production. Federation Proc. 23:54. GIFFORD, G. E. 1965. Inhibitory effect of mononucleotides on virus plaque formation. Proc. Soc. Exptl. Biol. Med. 119:9-12. JENSEN, K. E., A. L. NEAL, R. E. OWENS, AND J. WARREN. 1963. Interferon responses of chick embryo fibroblasts to nucleic acids and related compounds. Nature 2:433-434. O'DELL, T. B., H. N. WRIGHT, AND R. N. BIETER. 1953. Chemotherapeutic activity of nucleic acids and high protein diets against the infection caused by the MM virus in mice. J. Pharmacol. 17 :232-24. ROTEM, Z., R. A. Cox, AND A. ISAACS. 1963. Inhibition of virus multiplication by foreign nucleic acid. Nature 197:564-566. TAKANO, K., K. E. JENSEN, AND J. WARREN. 1963. Passive interferon protection in mouse influenza. Proc. Soc. Exptl. Biol. Med. 114:472-475. TAKANO, K., J. WARREN, AND K. E. JENSEN. 1964. Resistance conferred by interferons stimulated with non-viral nucleic acids. Federation Proc. 23:57. THELY, M. J. CHOAY, L. DHENNIN, AND L. DHENNIN. 1963. Virostase induite in vivo par des acides ribonucleiques non infectieux. Compt. Reiid. 14:148-15.