Vol. 16, No. 2 Printed in U.S.A.
INFECTION AND IMMUNITY, May 1977, p. 717-719 Copyright C 1977 American Society for Microbiology
Efficacy of Herpes Simplex Virus Type 1 Immunization in Protecting Against Acute and Latent Infection by Herpes Simplex Virus Type 2 in Mice ROBERT R. McKENDALL Department of Neurology, Veterans Administration Hospital and University of California, San Francisco, California 94121 Received for publication 22 February 1977
ICR mice were immunized with herpes simplex virus type 1 (HSV-1) and later challenged with HSV-2 by footpad inoculation. Both immunized animals and age-matched, nonimmunized controls were observed for ascending neurological disease and latent infection of spinal ganglia resulting from the HSV-2 challenge. Control animals had a 78% incidence of acute and latent infection compared with a 1.7% incidence in immunized mice. The data show immunity to HSV-1 is protective against both acute and latent infection by HSV-2. Increasing evidence has accumulated over the last few years to document the existence of two closely related subtypes of herpes simplex virus (HSV) - HSV-1 and HSV-2 (9-11, 14). Peripheral inoculation of either subtype into mice and other experimental animals results in a neurological disease characterized by progressive paralysis of the limb of the inoculated side and later of the opposite side (15-18). Various studies show that the ocurrence of ascending neurological disease is accomplished by neural transit of virus (1-4, 21). A variety of immunological studies suggest that host factors may moderate both the appearance and severity of disease (5, 7, 8, 12, 13, 19). Although knowledge of cross-neutralizing antibody in vitro (i.e., antibody to one HSV subtype neutralizes the other subtype) has been available for several years, investigation of in vivo cross-immunity has not until now been undertaken. Epidemiological data in man associate HSV1 with supradiagphragmatic sites of infection whereas HSV-2 primarily occurs about genitally related areas (9). Up to 55% of people develop neutralizing HSV-1 antibody prior to the onset of sexual activity (20). In light of recent increases of all human venereal diseases, including HSV-2, a study was undertaken in mice to determine whether prior HSV-1 exposure and immunity was protective against the ascent of HSV-2 along nerves. This design was chosen not only because HSV-2 exposure presumably occurs after attainment of sexual maturity, and hence later than HSV-1 in most people, but also because HSV-2 is more neurovirulent (16) and, therefore, might circumvent the immune system ability to clear virus.
Immunization of 4-week-old ICR mice was accomplished by footpad inoculation of a single dose containing 1,300 50% tissue culture infective doses (TCID50) of HSV-1, a human strain previously described (6). Three weeks later, the HSV-1-immunized mice and 55 age-sexmatched control mice, which had been held concurrently in our animal facility, were inoculated in the opposite footpad with 1,000 TCID50 of HSV-2, a strain isolated from a human penile vesicle. Animal groups were coded and evaluated every day or two for evidence of ascending neurological disease. After the period of acute illness was completed, the code was broken and the results were tabulated. Following a minimum interval of 12 weeks after HSV-2 challenge, study of spinal ganglia for the presence of latent infection was done by explant technique. The lumbosacral dorsal root ganglia from both sides of the illness-free animals were aseptically removed and cultured in petri dishes with medium 199 supplemented with antibiotics, 1% glutamine, and 10% heat-inactivated fetal calf serum. Culture supernatants were taken at 1- to 5-day intervals over the course of 30 days and frozen for later viral assay by observation of a cytopathic effect on primary rabbit kidney cell culture. All isolates were identified by complete neutralization with HSV hyperimmune guinea pig serum. HSV typing by neutralization kinetics (17) was kindly provided by Ron Duff (North Chicago, Ill.). Following immunization with HSV-1, 92 of 250 (36.8%) mice developed neurological disease. Of these mice, 63 recovered monoplegic animals and 58 mice that had remained well were challenged with HSV-2. Table 1 shows
717
718
INFECT. IMMUN.
NOTES
that neurological illness resulting from HSV-2 challenge developed in 36 to 53% of the control mice compared with 0 to 4% of the mice immunized with HSV-1 (P < 0.001). Among the HSV-1-immunized mice, prior HSV-1 disease did not influence the response to HSV-2 challenge. Since animals that remain free of illness may harbor latent virus in the dorsal root ganglia, study of illness-free animals after HSV-2 challenge for possible latent ganglionic infection was undertaken to determine whether the ascent of HSV-2 along nerves had been completely averted, or whether illness alone had been prevented by the HSV-1 immunization. Of 45 immunized mice which remained illness-free upon HSV-2 challenge, 23 had latent virus only on the side of HSV-1 immunization, and three had latent infection on both sides (Table 2). To determine whether latent infection on the side of HSV-2 challenge was the result of the HSV-1 immunization or of the HSV-2 challenge, isolates from these animals were typed and identified as HSV-1 by neutralization kinetics. In fact, all 45 mice were protected against ascent of HSV-2. In comparison, 18 illness-free control animals were studied, and six (33%) were shown to harbor latent virus on the side of the HSV-2 challenge. Therefore, the HSV-1-immune animals had a clearly lower incidence of latent HSV-2 infection compared with nonimmunized controls (P < 0.05). To assess the overall effectiveness of HSV-1 immunization on the prevention of HSV-2 ascent along peripheral nerves, the prevention of combined acute and latent infection must be considered. Table 3 indicates that 1.7% of the HSV-1-immunized mice developed acute or latent infection compared with 78% of the unimmunized mice (P < 0.001). The mechanism of this cross-immunity is unclear. Although the titer of neutralizing antibody in pooled sera from six immunized mice was 1:16 1 day prior to HSV-2 challenge, further studies will be required to determine whether humoral or cell-mediated immunity provides the protection. The data indicate that HSV-1 immunity under some circumstances TABLE 1. Ascending neurological disease in mice challenged with HSV-2 after immunization with HSV-1 Illness after HSV-2 challenge Group Expt 1
0/690 9/25
Expt 2
2/52 16/30 a Number sick/number of mice inoculated with 1,000 TCID50 of HSV-2 by footpad. HSV-1 immune Nonimmune controls
TABLE 2. Asymptomatic latent ganglionic infection in immunized versus nonimmunized mice challenged with HSV-2 Left side (chalRight side (immunized with HSV-1) lenged with HSV-2) Group No. posi- Isolate No. posi- Isolate tive/no. tive/no. subtype subtype tested tested HSV-1 3/45 HSV-1 immu- 26/45a HSV-1 nized 6/18 HSV-2 Controls 0/18 a Number of mice with latent infection by explant technique/number of mice explanted.
TABLE 3. Effect of HSV-1 immunization on the ascent of HSV-2 along nerves in mice Group
Latent HSV-2 infecAcute HSV-2 disease tion in illness-free mice %
HSV-1 immune 1.7 45 Nonimmune controls
No. 121 55
% 0.0 33
No. 45 18
may prevent the ascent of HSV-2 along nerves, resulting in aversion of both acute and latent herpetic infection. Consideration should be given to the concept that prior HSV-1 exposure may be an important influence on the acquisition of acute and latent HSV-2 disease in humans. LITERATURE CITED 1. Cook, M. L., and J. G. Stevens. 1973. Pathogenesis of herpetic neuritis and ganglonitis in mice: evidence for intra-axonal transport of infection. Infect. Immun. 7:272-288. 2. Goodpasture, E. W. 1925. The axis-cylinders of peripheral nerves as portals of entry to the central nervous system for the virus of herpes simplex in experimentally infected rabbits. Am. J. Pathol. 1:11-31. 3. Goodpasture, E. W., and 0. Teague. 1923. Transmission of the virus of herpes fibrilis along nerves in experimentally infected rabbits. J. Med. Res. 44:139188. 4. Johnson, R. T. 1964. The pathogenesis of herpes virus encephalitis. I. Virus pathways to the nervous system of suckling mice demonstrated by fluorescent antibody staining. J. Exp. Med. 119:343-356. 5. Johnson, R. T. 1964. The pathogenesis of herpes virus encephalitis. II. A cellular basis for the development of resistance with age. J. Exp. Med. 120:359-374. 6. McKendall, R. R., N. Vogelzang, and G. G. Jackson. 1974. Herpes virus latency in spinal ganglia of mice without illness. Proc. Soc. Exp. Biol. Med. 146:10931096. 7. Mori, R., T. Tasaki, G. Kimura, and K. Takeya. 1967. Depression of acquired resistance against herpes simplex virus infection in neonatally thymectomized mice. Arch. Gesamte Virusforsch. 21:459-462. 8. Nahmias, A. J., M. S. Hirsch, J. H. Kramer, and J. A. Murphey. 1969. Effect of antithymocyte serum on herpes virus hominis (type 1) infection in adult mice. Proc. Soc. Exp. Biol. Med. 132:696-698. 9. Nahmias, A. J., and B. Roizman. 1973. Infection with
VOL. 16, 1977
10. 11. 12.
13. 14.
15.
herpes-simplex viruses 1 and 2. N. Engl. J. Med. 289:667-674. Nahmias, A. J., and B. Roizman. 1973. Infection with herpes-simplex viruses 1 and 2. N. Engl. J. Med. 289:719-724. Nahmias, A. J., and B. Roizman. 1973. Infection with herpes-simplex viruses 1 and 2. N. Engl. J. Med. 289:781-789. Oakes, J. E. 1975. Invasion of the central nervous system by herpes simplex virus type 1 after subcutaneous inoculation of immunosuppressed mice. J. Infect. Dis. 131:51-57. Oakes, J. E. 1975. Role for cell-mediated immunity in the resistance of mice to subcutaneous herpes simplex virus infection. Infect. Immun. 12:166-172. Plummer, G. 1973. A review of the identification and titration of antibodies to herpes simplex viruses Type 1 and Type 2 in human sera. Cancer Res. 33:14691476. Plummer, G., and S. Hackett. 1965. Herpes simplex virus and paralysis of animals. Br. J. Exp. Pathol.
NOTES
719
47:82-85. 16. Plummer, G., J. L. Waner, and C. P. Bowling. 1968. Comparative studies of type 1 and type 2 "herpes simplex" viruses. Br. J. Exp. Pathol. 49:202-208. 17. Plummer G., J. L. Waner, A. Phuangsab, and C. R. Goodheart. 1970. Type 1 and type 2 herpes simplex viruses: serological and biological differences. J. Virol. 5:51-59. 18. Price, R. W., M. A. Walz, C. Wohlenberg, and A. L. Notkins. 1975. Latent infection of sensory ganglia with herpes simplex virus: efficacy of immunization. Science 188:938-940. 19. Rager-Zisman, B., and A. C. Allison. 1976. Mechanics of immunologic resistance to herpes simplex virus 1 (HSV-1) infection. J. Immunol. 116:35-40. 20. Smith, I. W., J. F. Peutherer, and F. 0. MacCallum. 1967. The incidence of Herpesvirus hominis antibody in the population. J. Hyg. 65:395-408. 21. Wildy, P. 1967. The progression of herpes simplex virus to the central nervous system of the mouse. J. Hyg. 65:173-192.
INFECTION AND IMMUNITY, May 1977, p. 717-719 Copyright C 1977 American Society for Microbiology
Efficacy of Herpes Simplex Virus Type 1 Immunization in Protecting Against Acute and Latent Infection by Herpes Simplex Virus Type 2 in Mice ROBERT R. McKENDALL Department of Neurology, Veterans Administration Hospital and University of California, San Francisco, California 94121 Received for publication 22 February 1977
ICR mice were immunized with herpes simplex virus type 1 (HSV-1) and later challenged with HSV-2 by footpad inoculation. Both immunized animals and age-matched, nonimmunized controls were observed for ascending neurological disease and latent infection of spinal ganglia resulting from the HSV-2 challenge. Control animals had a 78% incidence of acute and latent infection compared with a 1.7% incidence in immunized mice. The data show immunity to HSV-1 is protective against both acute and latent infection by HSV-2. Increasing evidence has accumulated over the last few years to document the existence of two closely related subtypes of herpes simplex virus (HSV) - HSV-1 and HSV-2 (9-11, 14). Peripheral inoculation of either subtype into mice and other experimental animals results in a neurological disease characterized by progressive paralysis of the limb of the inoculated side and later of the opposite side (15-18). Various studies show that the ocurrence of ascending neurological disease is accomplished by neural transit of virus (1-4, 21). A variety of immunological studies suggest that host factors may moderate both the appearance and severity of disease (5, 7, 8, 12, 13, 19). Although knowledge of cross-neutralizing antibody in vitro (i.e., antibody to one HSV subtype neutralizes the other subtype) has been available for several years, investigation of in vivo cross-immunity has not until now been undertaken. Epidemiological data in man associate HSV1 with supradiagphragmatic sites of infection whereas HSV-2 primarily occurs about genitally related areas (9). Up to 55% of people develop neutralizing HSV-1 antibody prior to the onset of sexual activity (20). In light of recent increases of all human venereal diseases, including HSV-2, a study was undertaken in mice to determine whether prior HSV-1 exposure and immunity was protective against the ascent of HSV-2 along nerves. This design was chosen not only because HSV-2 exposure presumably occurs after attainment of sexual maturity, and hence later than HSV-1 in most people, but also because HSV-2 is more neurovirulent (16) and, therefore, might circumvent the immune system ability to clear virus.
Immunization of 4-week-old ICR mice was accomplished by footpad inoculation of a single dose containing 1,300 50% tissue culture infective doses (TCID50) of HSV-1, a human strain previously described (6). Three weeks later, the HSV-1-immunized mice and 55 age-sexmatched control mice, which had been held concurrently in our animal facility, were inoculated in the opposite footpad with 1,000 TCID50 of HSV-2, a strain isolated from a human penile vesicle. Animal groups were coded and evaluated every day or two for evidence of ascending neurological disease. After the period of acute illness was completed, the code was broken and the results were tabulated. Following a minimum interval of 12 weeks after HSV-2 challenge, study of spinal ganglia for the presence of latent infection was done by explant technique. The lumbosacral dorsal root ganglia from both sides of the illness-free animals were aseptically removed and cultured in petri dishes with medium 199 supplemented with antibiotics, 1% glutamine, and 10% heat-inactivated fetal calf serum. Culture supernatants were taken at 1- to 5-day intervals over the course of 30 days and frozen for later viral assay by observation of a cytopathic effect on primary rabbit kidney cell culture. All isolates were identified by complete neutralization with HSV hyperimmune guinea pig serum. HSV typing by neutralization kinetics (17) was kindly provided by Ron Duff (North Chicago, Ill.). Following immunization with HSV-1, 92 of 250 (36.8%) mice developed neurological disease. Of these mice, 63 recovered monoplegic animals and 58 mice that had remained well were challenged with HSV-2. Table 1 shows
717
718
INFECT. IMMUN.
NOTES
that neurological illness resulting from HSV-2 challenge developed in 36 to 53% of the control mice compared with 0 to 4% of the mice immunized with HSV-1 (P < 0.001). Among the HSV-1-immunized mice, prior HSV-1 disease did not influence the response to HSV-2 challenge. Since animals that remain free of illness may harbor latent virus in the dorsal root ganglia, study of illness-free animals after HSV-2 challenge for possible latent ganglionic infection was undertaken to determine whether the ascent of HSV-2 along nerves had been completely averted, or whether illness alone had been prevented by the HSV-1 immunization. Of 45 immunized mice which remained illness-free upon HSV-2 challenge, 23 had latent virus only on the side of HSV-1 immunization, and three had latent infection on both sides (Table 2). To determine whether latent infection on the side of HSV-2 challenge was the result of the HSV-1 immunization or of the HSV-2 challenge, isolates from these animals were typed and identified as HSV-1 by neutralization kinetics. In fact, all 45 mice were protected against ascent of HSV-2. In comparison, 18 illness-free control animals were studied, and six (33%) were shown to harbor latent virus on the side of the HSV-2 challenge. Therefore, the HSV-1-immune animals had a clearly lower incidence of latent HSV-2 infection compared with nonimmunized controls (P < 0.05). To assess the overall effectiveness of HSV-1 immunization on the prevention of HSV-2 ascent along peripheral nerves, the prevention of combined acute and latent infection must be considered. Table 3 indicates that 1.7% of the HSV-1-immunized mice developed acute or latent infection compared with 78% of the unimmunized mice (P < 0.001). The mechanism of this cross-immunity is unclear. Although the titer of neutralizing antibody in pooled sera from six immunized mice was 1:16 1 day prior to HSV-2 challenge, further studies will be required to determine whether humoral or cell-mediated immunity provides the protection. The data indicate that HSV-1 immunity under some circumstances TABLE 1. Ascending neurological disease in mice challenged with HSV-2 after immunization with HSV-1 Illness after HSV-2 challenge Group Expt 1
0/690 9/25
Expt 2
2/52 16/30 a Number sick/number of mice inoculated with 1,000 TCID50 of HSV-2 by footpad. HSV-1 immune Nonimmune controls
TABLE 2. Asymptomatic latent ganglionic infection in immunized versus nonimmunized mice challenged with HSV-2 Left side (chalRight side (immunized with HSV-1) lenged with HSV-2) Group No. posi- Isolate No. posi- Isolate tive/no. tive/no. subtype subtype tested tested HSV-1 3/45 HSV-1 immu- 26/45a HSV-1 nized 6/18 HSV-2 Controls 0/18 a Number of mice with latent infection by explant technique/number of mice explanted.
TABLE 3. Effect of HSV-1 immunization on the ascent of HSV-2 along nerves in mice Group
Latent HSV-2 infecAcute HSV-2 disease tion in illness-free mice %
HSV-1 immune 1.7 45 Nonimmune controls
No. 121 55
% 0.0 33
No. 45 18
may prevent the ascent of HSV-2 along nerves, resulting in aversion of both acute and latent herpetic infection. Consideration should be given to the concept that prior HSV-1 exposure may be an important influence on the acquisition of acute and latent HSV-2 disease in humans. LITERATURE CITED 1. Cook, M. L., and J. G. Stevens. 1973. Pathogenesis of herpetic neuritis and ganglonitis in mice: evidence for intra-axonal transport of infection. Infect. Immun. 7:272-288. 2. Goodpasture, E. W. 1925. The axis-cylinders of peripheral nerves as portals of entry to the central nervous system for the virus of herpes simplex in experimentally infected rabbits. Am. J. Pathol. 1:11-31. 3. Goodpasture, E. W., and 0. Teague. 1923. Transmission of the virus of herpes fibrilis along nerves in experimentally infected rabbits. J. Med. Res. 44:139188. 4. Johnson, R. T. 1964. The pathogenesis of herpes virus encephalitis. I. Virus pathways to the nervous system of suckling mice demonstrated by fluorescent antibody staining. J. Exp. Med. 119:343-356. 5. Johnson, R. T. 1964. The pathogenesis of herpes virus encephalitis. II. A cellular basis for the development of resistance with age. J. Exp. Med. 120:359-374. 6. McKendall, R. R., N. Vogelzang, and G. G. Jackson. 1974. Herpes virus latency in spinal ganglia of mice without illness. Proc. Soc. Exp. Biol. Med. 146:10931096. 7. Mori, R., T. Tasaki, G. Kimura, and K. Takeya. 1967. Depression of acquired resistance against herpes simplex virus infection in neonatally thymectomized mice. Arch. Gesamte Virusforsch. 21:459-462. 8. Nahmias, A. J., M. S. Hirsch, J. H. Kramer, and J. A. Murphey. 1969. Effect of antithymocyte serum on herpes virus hominis (type 1) infection in adult mice. Proc. Soc. Exp. Biol. Med. 132:696-698. 9. Nahmias, A. J., and B. Roizman. 1973. Infection with
VOL. 16, 1977
10. 11. 12.
13. 14.
15.
herpes-simplex viruses 1 and 2. N. Engl. J. Med. 289:667-674. Nahmias, A. J., and B. Roizman. 1973. Infection with herpes-simplex viruses 1 and 2. N. Engl. J. Med. 289:719-724. Nahmias, A. J., and B. Roizman. 1973. Infection with herpes-simplex viruses 1 and 2. N. Engl. J. Med. 289:781-789. Oakes, J. E. 1975. Invasion of the central nervous system by herpes simplex virus type 1 after subcutaneous inoculation of immunosuppressed mice. J. Infect. Dis. 131:51-57. Oakes, J. E. 1975. Role for cell-mediated immunity in the resistance of mice to subcutaneous herpes simplex virus infection. Infect. Immun. 12:166-172. Plummer, G. 1973. A review of the identification and titration of antibodies to herpes simplex viruses Type 1 and Type 2 in human sera. Cancer Res. 33:14691476. Plummer, G., and S. Hackett. 1965. Herpes simplex virus and paralysis of animals. Br. J. Exp. Pathol.
NOTES
719
47:82-85. 16. Plummer, G., J. L. Waner, and C. P. Bowling. 1968. Comparative studies of type 1 and type 2 "herpes simplex" viruses. Br. J. Exp. Pathol. 49:202-208. 17. Plummer G., J. L. Waner, A. Phuangsab, and C. R. Goodheart. 1970. Type 1 and type 2 herpes simplex viruses: serological and biological differences. J. Virol. 5:51-59. 18. Price, R. W., M. A. Walz, C. Wohlenberg, and A. L. Notkins. 1975. Latent infection of sensory ganglia with herpes simplex virus: efficacy of immunization. Science 188:938-940. 19. Rager-Zisman, B., and A. C. Allison. 1976. Mechanics of immunologic resistance to herpes simplex virus 1 (HSV-1) infection. J. Immunol. 116:35-40. 20. Smith, I. W., J. F. Peutherer, and F. 0. MacCallum. 1967. The incidence of Herpesvirus hominis antibody in the population. J. Hyg. 65:395-408. 21. Wildy, P. 1967. The progression of herpes simplex virus to the central nervous system of the mouse. J. Hyg. 65:173-192.
