Vol. 15, No. 4

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1979, p. 547-553

0066-4804/79/04-0647/07$02.00/0

Effect of Pyran on Latency After Herpes Simplex Virus Infections PAGE S. MORAHAN, PAUL F. CLINE, MARY C. BREINIG,t AND BYRON K. MURRAY Department of Microbiology, Medical College of Virginia, Commonwealth University, Richmond, Virginia 23298

Received for publication 25 January 1979

The immunomodulator pyran protected mice against both herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections. In infections of the lip with HSV1, prophylactic administration of pyran reduced the severity of the herpetic lesions and enhanced their resolution, but did not decrease the high incidence of development of latent HSV-1 infection of the trigeminal ganglia. In vaginal infections with HSV-2, prophylactic administration of pyran either systemically or locally reduced mortality, reduced the incidence of mice with vaginal HSV-2 infection, and did not alter the low incidence of latent infection of the spinal dorsal root ganglia. Pyran treatinent before systemic herpetic infection after intravenous inoculation of HSV-2 also reduced mortality and virus replication, as evidenced by a decreased antibody response in the survivors, and it either reduced latent infection in the spinal dorsal root ganglia or did not predispose mice to latent infection. Treatment with the immunomodulator appeared to inhibit or reduce HSV infection early in viral pathogenesis in all three model systems, producing protection from clinical disease and resulting in less virus to induce a systemic antibody response, with either a reduction in latent virus infection or no enhancement of development of latency. In all of the HSV models, the development of latent herpetic infection was closely correlated with sufficient virus replication early in the infection to induce a systemic neutralizing-antibody response. A variety of drugs have exhibited antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections in animals and humans. Included are compounds with direct antiviral properties (1, 2, 11, 18, 19) and compounds with immunomodulatory activity (3-5, 8, 12, 17). In addition, various herpes simplex virus vaccines are being tested (10, 13). Currently, the major index for effectiveness is reduction in clinical signs or virus replication. However, herpes simplex virus has the potential for establishing latent infection in the regional sensory or autonomic ganglia (6) and of then causing recurrent herpetic infection, which has been hypothesized to be involved in the development of oral or cervical carcinoma or central nervous system disorders (7, 21). Effective antiherpetic therapy, thus, should be directed toward inhibiting, or at least not increasing, viral latency. Surprisingly little attention has been given to evaluating the effects of potential antiherpetic drugs or vaccines on viral latency. Treatment of t Present address: Graduate School of Public Health, Department of Microbiology, Pittsburgh, PA 15261.

animals with phosphonoacetic acid prevented latency after intradermal inoculation of mice with HSV-1 (14, 25), whereas adenine arabinoside or adenine arabinoside monophosphate protected mice from lethality but did not prevent latency (14). Intraperitoneal immunization of mice with live HSV-1 reduced latency after vaginal HSV-1 infection, but similar immunization with live HSV-2 did not reduce latency, and in some instances predisposed mice to development of latency after vaginal HSV-2 infection (20, 23). The present work was directed at establishing the effect that treatment with an immunomodulator, pyran, has on clinical signs, virus replication, and latency after lip, vaginal, or systemic HSV infection. MATERLALS AND METHODS

Animals. Male and female BALB/c mice,

5 to 8

weeks old, were obtained from Laboratory Supply Company, Inc., Indianapolis, Ind., or Simonsen Laboratories, Gilroy, Calif. Mice were used in experiments 1 to 2 weeks after arrival. Virus. The HSV-2 strain was obtained through the courtesy of R. W. Tankersly, A. H. Robins Co., Richmond, Va. (16), and the HSV-1 Patton strain was from 547

548

MORAHAN ET AL.

the Virology Department, Baylor College of Medicine, Houston, Tex. (13). Virus pools were prepared in cell culture as described previously (13, 16). Virus pool titers ranged from 106 to 107 plaque-forming units (PFU) per ml on Vero (African green monkey kidney) cells. Drugs. Pyran copolymer, a copolymer of maleic anhydride and divinyl ether, was obtained from D. S. Breslow, Hercules, Inc., Wilmington, Del. The sample preparation used for all studies was number XA 124177, which has an average molecular weight of 32,200. Pyran was dissolved at 56°C in 0.15 M NaCl to which a small quantity of 1 N NaOH was added, and the final pH was adjusted to 6.8 with 1 N NaOH. Solutions were prepared immediately before use. Doses of 25 mg/kg were administered intravenously (i.v.), and doses of either 25 or 250 mg/kg were administered intravaginally 24 h before challenge of animals with HSV. When administered i.v., the drug volume was equivalent to 0.1 ml/10 g of body weight. For intravaginal instillation, the drug volume delivered with a 0.5-ml syringe fitted with a plastic catheter needle was approximately 0.025 ml/10 g of body weight. Toxicity controls at these doses showed no weight loss or other apparent toxicity. Phosphonoacetic acid, lot 52,701-Al, obtained through the courtesy of L. R. Overby, Abbott Laboratories, North Chicago, Ill., was dissolved in phosphate-buffered saline (pH 7.4), in a concentration of 200 mg/ml, for vaginal inoculation. Virus titrations. HSV was assayed for infectivity by plaque formation on Vero cells as described previously (15, 16). Swabs of vaginal secretions were placed in 1 ml of GLB (Hanks balanced salt solution with 0.5% gelatin, 0.25% lactalbumin hydrolysate, and antibiotics). When quantitating the recovered virus, 0.1ml portions of tenfold dilutions were adsorbed onto Vero monolayers of 90 to 100% confluency, virus was adsorbed for 1 h at 36°C, 1% methyl cellulose medium was added, and cells were incubated at 36°C and stained with crystal violet 3 days later (5). The titer was expressed as PFU per milliliter of the GLB-vaginal secretion samples. Serum neutralization. In most instances, serum for antibody was obtained by cardiac puncture when mice were sacrificed. For mice not to be immediately sacrificed, blood was obtained from the retroorbital plexus. The neutralizing ability of dilutions of heatinactivated sera was determined in the presence of 10 hemolytic units of guinea pig complement (Colorado Serum Company, Denver, Colo.). The antibody titers, expressed in units, were interpolated graphically as the reciprocal of the dilution causing 50% plaque reduction. A standard rabbit antiserum against HSV-2 was titrated with each assay; the titer averaged 1:1,800 against HSV-2 and 1:240 against HSV-1. Vaginal HSV-2 infection and latency studies. In the vaginal model, female 6- to 8-week-old mice (14 to 18 g) were inoculated with approximately 4 x 104 PFU of HSV-2. The 50% lethal dose (LD50) for BALB/ c mice of this age was approximately 1.7 x 103 PFU. Infection was consistently achieved in young adult BALB/c mice by instilling approximately 0.04 ml of virus suspension, without the need for preparation of the vagina with alkaline washes or preswabbing as is

ANTIMICROB. AGENTS CHEMOTHER. often necessary with older or outbred mice (2, 5, 11). Swabs of vaginal secretions were taken on individual mice 2 to 3 days after inoculation. Mice were observed daily for clinical signs; two observers did not differ significantly in scoring. Vaginitis was scored as follows: 0 = none; 1 = erythema; 2 = erythema and edema or vaginal discharge; 3 = erythema, edema, vaginal discharge, and/or paralysis. Rarely was paralysis observed without the signs of vaginitis. To insure that the acute phase of the infection had run its course, mice were held for at least 30 days after inoculation before tests for latency were performed. Usually very few mice died after 21 days post-inoculation (16). To remove ganglia, the spinal column was dissected free of surrounding tissue and cut along the ventral side with fine-point angular scissors. The vertebrae were then cracked open to expose the spinal cord. With the use of a dissecting microscope (10x), the dorsal root ganglia (DRG) were removed aseptically and placed in media. Approximately 20 lumbar and sacral ganglia (10 from each side of the spinal cord) were removed from each mouse. The first ganglion past the last rib of the mouse was designated lumbar ganglion number one. After ganglia from three to five mice were removed, the DRG from individual mice were immediately placed on Vero or secondary rabbit kidney cells. Cells with explanted ganglia were observed daily for typical cytopathic effect. The ganglia were transferred to new indicator cells once a week. Cytopathic effect usually occurred between days 8 and 16 in cultures of ganglia that were positive for latent HSV infection. Any cytopathic effect observed before day 4 was considered indicative of acute rather than latent infection. This standard procedure was shortened recently by explanting ganglia alone in media, homogenizing the ganglia 3 days later, infecting indicator cells (Vero or rabbit kidney) with supernatant fluids from the homogenates, and observing the cytopathic effect which occurs within 3 days (24). The efficiency of the shortened procedure for detecting latency was comparable to that of the longer procedure, and was used to obtain the data in Table 4, experiment 2. HSV-1 infection on the lip and latency studies. In this model, which used 8- to 10-week-old mice (18 to 20 g), an abraded area on the upper right lip was swabbed with a cotton swab wetted with 0.1 ml of HSV-1 (approximately 106 PFU). The abrasion was produced by using gentle operation of a steel brush burr on a dental drill on mice that were anesthetized by methoxyfluorane (Pittman-Moore, Washington Crossing, N.J.) inhalation. In such infections, mortality was usually less than 30%. Clinical signs and mortality were recorded daily; two observers did not differ significantly in scoring. The severity of the lip lesion was scored as follows: 0 = no lesions; 1 = erythema with or without a small (1- to 2-mm) lesion present; and 2 = erythema and the presence of a large (>2-mm) lesion. All lesions had healed, and no mice died after approximately 21 days post-inoculation; thus, mice alive on day 30 were considered to be survivors of the acute infection. Trigeminal ganglia were obtained from mice between 30 and 40 days after virus inoculation and tested for latency as described above. Removal of the trigeminal ganglia involved retracting the scalp to

VOL. 15, 1979

PYRAN AFTER HSV INFECTIONS

expose the cranium, opening the cranial vault to expose the brain, removing the cerebrum and cerebellum, and then removing aseptically the trigeminal ganglia lying under the cerebrum and cerebellum. HSV-2 infection i.v. and latency studies. In this model, which used 6- to 10-week-old mice, approximately 104 PFU of HSV-2 were inoculated i.v. via the lateral tail vein. The 50% lethal dose for 6-week-old BALB/c mice was approximately 1.2 x 103 PFU, with most mice dying by day 18 after infection (16). Cervical and thoracic ganglia were removed from mice 40 to 45 days after virus inoculation and explanted as in the vaginal model described above. Up to 30 ganglia were explanted. In some experiments, lumbar as well as cervical and thoracic ganglia were explanted. Statistical procedures. Significant differences in protection of pyran, incidences of infection, or severity of lesions were analyzed by either the chi-square test using Yate's correction factor or by the Student's t test.

549

z 3.0 0

0

v 2.5-

A CONTROL O PYRAN

o 2.0-

a-.2

1.5-

,

1.0-

-J

0 0.5z J 0.0- a

100

80

- 60-

RESULTS

Effect of systemic administration of pyran on intravaginal HSV-2 infection. Administration of pyran i.v. 24 h before virus inoculation significantly reduced vaginitis from a peak clinical score of 2.5 to 1.0 (P < 0.05) and decreased mortality (P < 0.05) (Fig. 1). Pyran also reduced the incidence of virus isolations from the vaginal secretions (Table 1) and vaginal-cervical and uterine tissues (data not shown). The incidences of latent infection were 8% in recipients of pyran and 0% in the controls (Table 1). The presence of HSV-2 in vaginal secretions on days 2 to 3 was almost always predictive of a fatal outcome, whether in control mice or mice receiving pyran. Thus, less than 1% of animals with a vaginal swab positive for virus survived. The survivors, from either control or pyran treatment groups were, with two exceptions, mice that showed vaginal swabs negative for virus. Neutralizing antibody was not detected in any of the surviving controls, and rarely at low titers (1:10, 1:20) in the pyran-treated group. The incidence of latent HSV-2 infection in the DRG was identical to the incidence of neutralizing antibody. Effect of local administration of pyran on intravaginal HSV-2 infection. In most studies, local administration of chemotherapeutic drugs has been much more effective than systemic treatment against intravaginal HSV-2 infections in mice (2, 11). Thus, experiments were carried out to determine whether local administration of the immunomodulator pyran would produce an enhanced antiviral effect (Table 2). In one experiment, groups of mice were therapeutically treated by intravaginal instillations of

-j

cr 0 M 40-

20 -

14 2 6 10 18 DAYS AFTER I VAG INFECTION FIG. 1. Effect of pyran on clinical symptoms (a) and mortality (b) after vaginal HSV-2 infection. BALB/c mice were inoculated intravaginally with HSV-2 24 h after i.v. administration of 0.15 M NaCI or pyran (25 mg/kg). Clinical symptoms of vaginitis were scored as follows: 0 = none; 1 = erythema; 2 = erythema and edema or vaginal discharge; and 3 = erythema, edema, vaginal discharge and/or paralysis.

saline, pyran (250 mg/kg), or phosphonoacetic acid (500 mg/kg), beginning 2 h after intravaginal HSV-2 inoculation and continuing once daily for an additional 3 days. Only phosphonoacetic acid produced significant protection (P < 0.05) against virus replication and mortality. The survivor from the pyran-treated group showed antibody (1:50 titer) and was also positive for latency. To determine whether prophylactic or combined prophylactic-therapeutic regimens of pyran would be effective, pyran or saline was instilled intravaginally 24 h before intravaginal HSV-2 challenge. In some groups, treatment was continued daily on the day of virus inoculation and on 3 additional days. Mortality was reduced

550

MORAHAN ET AL.

ANTIMICROB. AGENTS CHFJ40THER.

TABLE 1. Effect of systemic administration ofpyran on intravaginal infections with HSV-2 Survivors

Vaginal infection

Group'

Vaginal

Controls

MtatydVaginal swabs`C

swabs1b

Mortality,""

121/133

162/180

(90)

(90)

Antibody

Latency

1/16

0/18

(6)

(0)

0/18 (0) 2/26 (8)

2/26 1/17 50/78 30/50 (8) (6) (64)' (60)' a Six-week-old BALB/c mice were inoculated intravaginally with HSV-2 24 h after i.v. admnistation of pyran (25 mg/kg). Controls received 0.2 ml of 0.15 M NaCl i.v. or no treatment; there was no difference between the two groups, and they were combined. The table is a composite of several experiments to obtain sufficient survivors. b'Values indicate number positive per total tested; those in parentheses indicate percent. 'Not all mice were tested for virus in vaginal swabs. All survivors in the control group (18/180) and 26 of the surviving mice in the pyran-treated group (28/78) were tested for antibody and latency. d Values indicate number dead per total tested; those in parentheses indicate percent. P < 0.05 as compared with the appropriate control. Pyran

TABLE 2. Effect of vaginal instiUation ofpyran on intravaginal infections with HSV-2 Survivorsb Vaginal infection Treatmenta Expt no.

Drugrg None Saline

1

Pyran PAA

D(oe (mg/kg) -

Schedule

-

DO, qd X 4 DO, qd x 4 DO, qd x 4

250 500

-

Vaginal

swabse 14/15 (93) 15/15 (100) 10/10 (100) 0/20 (0)d

Mortalityc

Antibody

Latency

14/15 (93) 15/15 (100) 9/10 (90) 0/20 (0)d

0/1

0/1

-

1/1 0/13

1/1 0/13

15/15 (100) 15/15 (100) D-1, qd x 5 Saline 0/6 1/3 5/15 (33)d 250 7/15 (47)d D-1, qd x 5 Pyran NT NTt D-1 250 5/10 (50)d 4/10 (40)d Pyran NT NT 9/10 (90) 25 9/10 (90) D-1, qd x 5 Pyran a BALB/c mice were treated by instilling drugs into the vagina. DO, begun 2 h after intravaginal HSV-2 virus inoculation; D-1, either single or multiple treatment beginning 24 h before virus inoculation; qd, multiple daily treatment over a series of days as designated by x and given number. PAA, phosphonoacetic acid; -, no survivors. b Values indicate number positive per total tested; those in parentheses indicate percent. c Values indicate number dead per total tested; those in parentheses indicate percent.

2

d

p< 0.05.

'

NT, Not tested.

significantly to 50% by the prophylactic regimen tralizing-antibody determination when mice and to 33% with the combined prophylactic- were sacrificed for trigeminal ganglia explantatherapeutic regimen with 250 mg of pyran per tion (Table 3). Although there were no deaths kg (P 0.05). The number of mice with local in either control or treated groups, mice receivvirus replication in the vaginal area was also ing pyran showed a significant decrease (P < decreased, and an estimation of the PFU/mouse 0.05) in severity and an earlier resolution of the obtained from the vaginal swabs indicated herpetic lip lesions. Mean antibody titers in the greater than 100 PFU/mouse in the saline group group receiving pyran were also significantly as compared with 3 PFU/mouse in mice receivlower (P < 0.05) than those in the control group, ing pyran. One of three survivors after pyran although the incidence of mice showing an imtreatment had an antibody titer of 1:25, but no mune response to the virus was similar. There latent infection was detected. was, however, no significant difference in inciEffect of systemic administration of dence of latent HSV-1 infection between the two pyran on HSV-1 lip infections. In this study groups. Effect of systemic administration of either pyran or saline was administered i.v. 24 h before mice were inoculated on the lip with pyran on i.v. HSV-2 infection. AdministraHSV-1. Mice were examined daily for lip lesions tion of pyran i.v. 24 h before virus inoculation and mortality, and blood was collected for neu- decreased mortality and the neutralizing-anti

Effect of pyran on latency after herpes simplex virus infections.

Vol. 15, No. 4 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1979, p. 547-553 0066-4804/79/04-0647/07$02.00/0 Effect of Pyran on Latency After Herpes...
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