Experimental Infection and Immune Response of Guinea Pigs with Varicella-Zoster Virus

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1 INFECTION AND IMMUNITY, Aug. 1982, p /82/ $02.00/0 Vol. 37, No. 2 Experimental Infection and Immune Response of Guinea Pigs with Varicella-Zoster Virus YOSHINO MATSUNAGA, KOICHI YAMANISHI,* AND MICHIAKI TAKAHASHI Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan Received 17 August 1981/Accepted 8 April 1982 An immune response (fluorescent antibody to membrane antigen) was detected in guinea pigs inoculated with varicella-zoster virus (VZV) adapted to guinea pig embryonic cells, including the Oka vaccine strain, even when inoculation was by an external route, i.e., nasal or corneal. Live or UV-inactivated virus having the same virus titer before irradiation was administered to guinea pigs by the corneal route, and antibody induction was detected only with live virus. The transmission of VZV from infected guinea pigs to noninfected ones was suggested by the appearance of antibody in the serum of the latter, who were kept in the same cage. The time course of the appearance of humoral and cellular immune responses in guinea pigs was examined by the fluorescent antibody to membrane antigen test and the skin reaction, with varicella antigen representing delayed-type hypersensitivity. When VZV was injected subcutaneously, skin reaction appeared as early as 4 days after inoculation, which preceded the appearance of detectable antibody by 2 to 6 days. In in vitro studies, the Oka vaccine showed a higher adsorption rate and better growth in guinea pig embryonic cells than did other wild-type strains when assayed by the infectious center assay. These results suggest that a system of VZV adapted to guinea pig cells and guinea pigs provides a good animal experimental model for immunological study of VZV infection. We have developed a live varicella vaccine which has been passaged in guinea pig embryonic cells 12 times (14, 15). This vaccine has been widely used, tnainly for immunocompromised children, conferring high immunity against varicella without adverse clinical reaction (4, 13). The immunopathological study of varicella-zoster virus (VZV) has long been hampered by the lack of a suitable experimental system using small animals, although monkeys have been used (5, 10, 11). In the investigation of biological markers of the vaccine virus, we found that the vaccine virus induced neutralizing or complement-fixing antibody response in guinea pigs when administered by subcutaneous inoculation, whereas little or no such response was observed with other wild-type strains (13, 18). Recently Myers et al. (8) reported that weanling guinea pigs inoculated intranasally or subcutaneously with VZV grown in fetal guinea pig tissue culture shed virus from the nasopharynx and seroconverted to VZV. Animal-to-animal transmission of VZV was also observed by them. In the present study we confirmed the observation of Myers et al., extending the VZV strains used for inoculation and the routes of inoculation, i.e., via a corneal route. Furthermore, the time course of humoral and cellular immune responses in guinea pigs inoculated with VZV (Oka vaccine strain) was investigated by the fluorescent antibody to membrane antigen (FAMA) test and by the varicella skin test, which represents delayed-type hypersensitivity (DTH). In addition, replication of VZV of various strains in cultured guinea pig cells was investigated in connection with the above in vivo experiments. MATERIALS AND METHODS Cells. Human embryonic fibroblast (HuEF) or lung (HEL) cells were grown in Roux bottles, using a mixture of an equal amount of Eagle minimum essential medium and medium 199 supplemented with 10 or 3% calf serum for growth or maintenance medium, respectively. All media contained 100 U of penicillin and 100 p.g of streptomycin per ml. Cells were subcultured at 6- to 7-day intervals, and cultures from the 5th to the 15th passage were used for the experiments. Cells were not contaminated with mycoplasma. Primary cultures of guinea pig embryonic kidney (GPEK), lung (GPEL), skin (GPES), and fibroblast (GPEF) cells were prepared from White Hartley guinea pig embryos, using the same media as above. The second-passaged cells were used for the virus infection. Virus. Oka, Kawaguchi (14), and Inoue strains were isolated from vesicles of varicella patients and passaged 5, 10, and 5 times, respectively, in HEL cells. Oka vaccine strain was passaged 11 times in HEL cells and 12 times in GPEF cells and then propagated in human diploid cells (14, 15). Inoue strain, passaged 407

2 408 MATSUNAGA, YAMANISHI, AND TAKAHASHI INFECT. IMMUN. nine times in GPEF cells after five passages in HEL cells, was also used. For the passages of virus, infected cells were trypsinized, suspended in maintenance medium, and inoculated to fresh cell sheet at a ratio of approximately 1 infected cell to 5 uninfected cells. Cell-free virus was obtained by the sonication method described previously (2). UV irradiation. Two milliliters of virus preparation was irradiated in 60-mm plastic plates with a UV germicidal lamp at a distance producing a radioactive dose of 50 erg mm-2 s-' for 10 min, resulting in a complete loss of infectivity. Animals and VZV inoculation of animals. Male White Hartley guinea pigs weighing about 200 g were used for VZV infection. Cell-free virus prepared from infected HEL cells or infected GPEF cells were administered to animals by subcutaneous, corneal, or nasal routes. GPEF cells infected with the Oka vaccine strain were harvested when more than 80%o of the cell sheet showed cytopathic effect. The numbers of infected cells were calculated on HuEF cells by an infectious center assay (17) just before the animals were inoculated. In corneal inoculation, the animals were anesthetized with ether, and the cornea of each eye was scarified with a sterile 25-gauge needle. Then 0.1 ml of virus suspension was dropped onto the surface of the eye. In nasal inoculation, the animals were likewise anesthetized, and 0.1 ml of virus suspension was dropped into each nostril. In subcutaneous inoculation, 0.5 ml of virus suspension was injected at the gluteal area. Blood was collected by cardiac puncture before and usually 30 days after inoculation except in cases indicated in the text. Sera were inactivated at 56 C for 30 min for the serological test. FAMA test. For the FAMA test, the method of Williams et al. (16) was essentially followed. HEL cells were planted into each well of flat-bottom microtiter plates (Linbro, Flow Laboratories) and were inoculated with VZV-infected cells at a ratio of 1 infected to 20 uninfected cells. Infected monolayers in each well were used for the FAMA test when cytopathic effect appeared in about 80% of cells. After each well was washed twice with phosphate-buffered saline (PBS), serial dilutions of serum samples prepared in transfer plates were added to each well. The plates were incubated for 30 min at 37 C. Subsequently, each well was washed three times with PBS, and ml offluorescein isothiocyanate-conjugated antiguinea pig immunoglobulin G (IgG) rabbit serum (Miles Laboratories, Inc.), usually at a dilution of 1:20, was poured into each well. After incubation for 30 min at 37 C, cells were washed an additional three times with PBS. Then 0.1% EDTA in PBS was added to each well to detach cells, and then suspended cells were dropped onto a glass slide. A cover slip was placed over the cells and slides were examined with a fluorescence microscope. As a control, uninfected HEL cells were also examined for sample sera to check the reaction against HEL cells. Preparation of skin test antigen and DTH test. The testing antigen was prepared in essentially the same way as that for human (1, 6), except for using guinea pig embryonic cells instead of human diploid cells. Oka vaccine strain was used for infection. After a monolayer of GPEF cells had formed, the medium was decanted, and the cells were inoculated with infected GPEF cells. When extensive cytopathic effect appeared after incubation at 37 C, the cell sheet was washed three times with PBS. Then the cells were collected with a rubber policeman, pelleted by centrifugation at 3,000 rpm, and suspended in PBS. The cell suspension was sonicated (Tomy Ultrasonic Disrupter, model UR-200W output 11, for 30 s), followed by centrifugation at 3,000 rpm for 10 min at 4 C. The supernatant was harvested and, after heating at 56 C for 30 min, stored at -80 C until testing. The test antigen had the antigen titer of 1:2 to 1:4 in the complement-fixing test, using human zoster convalescent serum. Control antigen, having a protein content comparable to that of the testing antigen, was prepared from uninfected GPEF cells in the same way. The backs of the animals were shaved with an electric clipper, and intradermal inoculation of 0.1 ml of testing antigen and control antigen was done with 25-gauge needles. Inoculation sites were examined at 48 h after injection. Since repeated skin tests might result in an enhancement of the immunological reaction of guinea pigs, animals were challenged only once. Inoculation of VZV to guinea pig cell cultures. Monolayers of GPEK, GPEL, and GPES cells in 60-mm plastic plates were inoculated with cell-free virus. After 1 h of adsorption at 37 C, cultures were washed with PBS three times, flooded with the maintenance medium, and kept at 37 C. Cells were trypsinized at 4, 24, 48, 72, %, and 120 h after infection and suspended in 2 ml of maintenance medium per plate, and 0.2 ml of cell suspension was inoculated onto monolayers of HuEF cells in a 60-mm plate. The number of infected guinea pig cells was thus assessed by counting VZV foci that appeared 5 days after inoculation. RESULTS Antibody responses of guinea pigs subcutaneously inoculated with various strains of VZV. Twenty-four guinea pigs were subcutaneously injected with cell-free virus of Oka, Inoue, and Kawaguchi strains, four to five animals for each strain. Viruses differing in their passage history were used for the inoculation. When animals were injected with Oka, Inoue, or Kawaguchi strain that had been passaged only in HEL cells, with a viral dose of 1.5 x 104 to 5.8 x 104 PFU/ dose, no antibody response was observed by FAMA test. In contrast, when Oka vaccine strain or Inoue strain, passaged nine times in GPEF cells, was injected, all guinea pigs showed antibody responses even with a lower dose of virus (2 x 1 0 to 2.5 x 103 PFU/dose) (Table 1). Antibody responses of guinea pigs inoculated with Oka vaccine strain of VZV by various routes. Viral preparation injected into animals may induce an antibody response even if virus does not replicate in them. To confirm the viral replication in them, VZ vaccine virus was externally administered to animals, and antibody responses were compared with those of subcutaneously inoculated animals. Twenty-seven guinea pigs were inoculated with either infected cells or cellfree virus of Oka vaccine strain by the subcuta-

3 VOL. 37, 1982 TABLE 1. Antibody response in guinea pigs inoculated with VZVa irus V(PFru/dite) Antibody titer (FAMA) Oka (vaccine) 2.0 x 103 8, 8, 8, 8, 4 Oka (HEL, 5.8 x 104 <2, <2, <2, <2, <2 7th passage) Inoue (HEL, 5.0 x 104 <2, <2, <2, <2, <2 5th passage) Inoue (GPEF, 2.5 x 103 4, 4, 4, 8, 8 9th passage) Kawaguchi 1.5 x 104 <2, <2, <2, <2 (HEL, 10th passage) a Animals were injected subcutaneously with cellfree virus. neous, corneal, or nasal route, and antibody responses were tested by FAMA technique. All guinea pigs inoculated with either infected cells or cell-free virus by the subcutaneous route showed antibody response (Table 2). The range of their antibody titers was between 1:8 and 1:16. Three of four guinea pigs showed antibody responses when they were inoculated by the corneal route with infected cells, and four of five guinea pigs showed antibody responses when inoculated by the same route with cell-free virus. Two of three guinea pigs showed antibody response when they were inoculated with infected cells by the nasal route, and four of five guinea pigs showed antibody responses when inoculated by the same route with cell-free virus. The level of antibody titer induced by the nasal route was lower than that induced by the subcutaneous route. Clinical symptoms, such as exanthema, were not observed in any guinea pig by any method of inoculation. Antibody responses of guinea pigs inoculated with live or UV-inactivated VZV of Oka vaccine strain by corneal route. Ten guinea pigs were inoculated by the corneal route with cell-free virus of Oka vaccine strain having the titer of 6.0 x 102 PFU/dose or UV-inactivated virus having the same titer before irradiation, with five animals for each group. Antibody response occurred in four of five guinea pigs inoculated with live virus, but no guinea pig showed antibody response with UV-irradiated virus (Table 3). Transmission of VZV from infected guinea pigs to noninfected ones. Five animals were injected subcutaneously with Oka vaccine strain of VZV having the titer 1.5 x 104 infected guinea pig cells per dose and housed together with five noninfected animals. Another five animals were sham-infected and housed separately as control. Animals were bled to test antibody response 30 or 45 days after injection. All guinea pigs inoculated with VZV produced antibodies (Table 4). VZV INFECTION OF GUINEA PIGS 409 TABLE 2. Antibody responses in guinea pigs inoculated with VZV of Oka vaccine strain by various routesa Route of Inoculated Virus titer Antibody titer inoculation material (PFU/dose) by FAMA s.c.b Infected cell' 2.3 x 104 8, 8, 16, 16, 16 Cell-free virus 2.0 x 103 8, 8, 8, 8 Corneal Infected cell 2.0 x 104 <2, 2, 4, 8 Cell-free virus 6.0 x 102 <2, 2, 4, 8, 8 Nasal Infected cell 1.4 x 105 <2, 2, 2 Cell-free virus 1.0 x 103 <2, 2, 2, 2, 2 a Animals were inoculated with either infected cells or cell-free virus by subcutaneous, corneal, or nasal route and bled for test of antibody response 30 days after inoculation. b s.c., Subcutaneous. ' Infected cell number was calculated by infectious center assay on HuEF cells. Seroconversion was observed on the 30th day in two of five uninoculated animals housed together with the inoculated animals and the number of seroconverted animals increased to four out of the five at the 45th day. Increase of antibody titer was also observed at the 45th day, but their antibody titers were generally lower than those of the injected ones. No animals which were housed separately showed any antibody response. Time course of appearance of cellular and humoral immune responses in guinea pigs subcutaneously inoculated with VZV of Oka vaccine strain. FAMA and DTH to VZV were examined to ascertain the humoral and cellular immune response in guinea pigs inoculated with VZV (Oka vaccine strain). For this experiment, Oka vaccine strain virus, passaged once in GPEF cells, was used for immunization. A skin test was used to detect the cellular immune response. Five guinea pigs were subcutaneously injected with VZV-infected GPEF cells (1.5 x 103 infected cells per animal), and tested for DTH by intradermal injection with 0.1 ml of viral and control antigens as described above. Injected sites were examined for erythematous change at 0.5, 3, 6, 16, 24, 48, and 72 h after injection (Table 5). Specific erythema started to TABLE 3. Antibody response of guinea pigs inoculated with live or UV-inactivated VZV of Oka vaccine strain by corneal route Virus Antibody titer (FAMA) Live virusa... <2, 2, 4, 8, 8 UV-inactivated virusb... <2, <2, <2, <2, <2 a Amount was 6 x 102 PFU/dose. b The virus dose before irradiation was the same as that of live virus.

4 410 MATSUNAGA, YAMANISHI, AND TAKAHASHI TABLE 4. Transmission of VZV from inoculated guinea pigs to noninfected guinea pigs housed in the same cage' Group Days infection after Antibody titer (FAMA) Infected 30 8, 16, 16, 64, 64 Cagemates 30 <2, <2, <2, 2, 2, Cagemates 45 <2, 4, 4, 4, 4 Control 45 <2, <2, <2, <2, <2 a Five animals were injected subcutaneously with Oka vaccine strain (1.5 X 104 infected cells per animal) and housed together with five noninfected animals. Another five animals were sham-infected and housed separately as controls. Animals were bled to test antibody response 30 or 45 days after injection. occur at 6 h and remained until 48 h postinjection. Erythema was observed also in the site injected with control antigen at 6 h but it diminished by 16 h postinjection. So it was easy to scale the erythema between 16 and 48 h postinjection in infected guinea pigs. As a control, five uninfected animals were challenged with the skin antigen, and no specific reaction was observed. Thirty-one guinea pigs were subcutaneously infected with VZV-infected GPEF cells (1.5 x 103 infected cells per animal). Three to five animals were injected intradermally with skin antigen at days 2, 4, 8, 14, 20, 29, and 110, and the sites injected were examined after 48 h. When the diameter of erythema was scaled, animals were bled, and FAMA tests were performed to compare the cellular immunity with humoral immunity. No immunological responses were detected on day 2 (Fig. 1). On day 4, two offour guinea pigs responded with skin reaction, and all animals showed the skin reaction after 8 days. On the other hand, antibody responses were not detected until day 6, but all animals TABLE 5. Cellular immune response in infected guinea pigs challenged with VZV antigen or control antigena Erythema (diam in mm) with Time after skin test antigen challenge (h)vzcotl VZV Control antigen antigen 0.5 ob ob a Animals were inoculated with 1.5 x 103 infected GPEF cells per animal and intradermally injected with skin antigens on the 30th day after inoculation. b Average diameter of five animals. 120 av INFECT. IMMUN lio Days after injection FIG. 1. Time course of the appearance of DTH to VZV in guinea pigs. Thirty-one guinea pigs were inoculated subcutaneously with Oka vaccine strain (1.5 x 103 infected guinea pig cells per dose). At days 2, 4, 8, 14, 20, 29, and 110 after inoculation, groups of guinea pigs (each group consisted of three to five guinea pigs) were examined by the skin test with varicella antigen. Skin test antigen (0.1 ml) was injected intradermally, and erythematous change was measured 48 h later. Each dot represents the mean diameter of erythematous change in guinea pigs of each group. showed antibody increases on day 10 (Fig. 2). When the immunological examination was done 4 months after infection, both humoral and cellular immunities were found to persist. Adsorption and growth of VZV of various strains in cultured guinea pig embryonic cells. GPEL, GPES, and GPEK cells were inoculated with cell-free virus of Oka (vaccine), Oka (HEL, 5th passage), Kawaguchi (HEL, 10th passage), and Inoue (HEL, 5th passage) strains to compare the adsorption and growth of viruses in these cells. The conventional method to examine adsorption and growth of virus seemed unsuitable for the purpose of the present study,because the infectivity of VZV is not stable under usual environmental conditions, and the yields of cell-free infectious virus from guinea pig cultures were not large enough to permit depiction of the precise growth curve of the viruses. Therefore, infectious center assay was performed at 4 h after infection (early stage of infection) and at later hours (advanced stage of infection) to assess the adsorption and growth of viruses. GPEL and GPES cells were far more susceptible to VZV, 30 to 50 times higher than GPEK cells, when Oka vaccine strain was used for infection (Table 6). Oka vaccine strain showed a higher adsorption rate to all kinds of cells, particularly to GPEL cells, than did other strains. Next, GPEL cells were inoculated with VZV of Oka vaccine, Oka (HEL, 5th passage), and Kawaguchi (HEL, 10th passage) strains, and

5 VOL. 37, % A ig I ahb I I a 4. 0 VZV INFECTION OF GUINEA PIGS VZV strains passaged in guinea pig embryonic cells. Myers et al. (8) reported that VZV grown in 00 see guinea pig cells infects weanling guinea pigs when they are inoculated intranasally and we * e e * also demonstrated in the present study that VZV of Oka vaccine strain can infect guinea pigs by an external route, i.e., corneal or nasal inocula- 00 * s tion. Furthermore, live or UV-inactivated virus having the same virus titer before irradiation was used to inoculate guinea pigs by the corneal 22~ route, and antibody was induced only with live virus preparation. Also, in our in vitro study after inljection.using various kinds of guinea pig embryonic FIG. 2. Time course of appearance of FAMA anti- cells, Oka vaccine virus showed higher adsorpa pigs. The same guinea pigs tion and growth rate than did other VZV. These body to VZV in guine, inoculated subcutaneoiusly with VZV-infected cells that (in the legend to Fig. 1) were bled by cardiac results suggest that VZV adapted to guinea pig puncture on the days off measurement of erythema for cells were really replicating in guinea pigs. Howgroup consisted of three to five ever, antibody production was generally small the skin reaction. Each guinea pigs. Symbols: I0, FAMA antibody titer of each by nasal inoculation as compared with that by guinea pig; A, the aver, age antibody titer. subcutaneous inoculation, which suggests that the susceptibility of respiratory tract cells of guinea pigs to VZV was relatively low. infectious center assaiy was performed at 24, 48, Myers et al. (8) observed shedding of VZV 72, %, and 120 h posstinfection to examine their from the nasopharynx of weanling guinea pigs growth in GPEL celis. As a result, the highest intranasally or subcutaneously inoculated with growth was observe( d with Oka vaccine strain, VZV grown in fetal guinea pig cells and animal- reaching the maxim infection (Fig. 3). um at 96 to 120 h after to-animal transmission of virus, also detected serologically. We confirmed transmission of VZV from subcutaneously injected guinea pigs to DIS'CUSSION untreated ones housed in the same cage by seroconversion, but not by virus isolation. But We reported previiously that a varicella vac- the antibody titers induced in the untreated cine virus (Oka straiin) induced neutralizing or cagemates were considerably lower than those complement-fixing antibody in guinea pigs by subcutaneously injected. This might be due to subcutaneous inoculation, whereas other wild- the rather poor susceptibility of respiratory tract type virus did not (1 3, 18). This was thought to cells of guinea pigs, as observed in the preceding be due to the differenice of passage history of the nasal inoculation experiments. viruses because the vaccine strain had been DTH to herpes simplex virus has been reportguinea pig embryonic cells ed in guinea pigs (7, 12) and mice (9). Since the passaged 12 times in before propagation irn human diploid cells. This VZV vaccine was developed, the skin reaction was confirmed in the present study using other test has been used to detect induction and per- TABLE 6. Adsorption of various strains of VZV to GPEL, GPES, and GPEK cells assessed by the infectious center assay Virus Input virus No. (PFU) of infectious centers (%) with: dose (PFU) GPEL GPES GPEK Oka (vaccine) 7.0 x x 102 (52.8)" 2.6 x 102 (36.5) 1.3 x 101 (1.8) Oka (HEL, 5th passage) 4.8 x x 102 (4.0) 2.4 x 102 (5.1) 9.6 x 100 (0.2) Kawaguchi (HEL, 10th passage) 1.1 x x 101 (7.9) 1.3 x 102 (12.2) 5.5 x 100 (0.5) Inoue (HEL, 5th passage) 2.1 x x 102 (5.1) 8.8 x 101 (4.2) 2.1 x 100 (0.1) a Number of infectious center at 4 h postinfection (PFU) Input virus (PFU) GPEL, GPES, and GPEK in 60-mm plastic plates were inoculated with the above dose of cell-free virus of Oka (vaccine), Oka (HEL, 5th passage), Kawaguchi (HEL, 10th passage), and Inoue (HEL, 5th passage) strains. At 4 h after inoculation, cells were trypsinized, and an infectious center assay was performed on HuEF cells.

6 412 MATSUNAGA, YAMANISHI, AND TAKAHASHI INFECT. IMMUN. La. 0.- IL cl v) , ' FIG.: 3. Growth of VZV of various strains in cul- Experimental infection of guinea pigs with varicella-zoster tured GIPEL cells. GPEL cells in 60-mm dishes were virus. J. Infect. Dis. 142: inoculateed with VZV of Oka (vaccine), Oka (HEL, 5th 9. Nash, A. A., H. J. Field, and R. Quartey-Papafio passage) I, and Kawaguchi (HEL, 10th passage) strains. Cell-mediated immunity in herpes simplex virus-infected After a: mice: 1-h adsorption period, cultures were washed induction, characterization and antiviral effects of with PB, three times,flooedwiththemaintenadelayed, type hypersensitivity. J. Gen. Virol. 48: S three flooded wlth the maintenance 10. Rivers, T. M Nuclear inclusions in the testicles of medium, and kept at 37 C. Cultures were trypsinized monkeys infected with the tissue of human varicella at 24, 4$8, 72, 96, and 120 h after infection, and lesions. J. Exp. Med. 43: infectiouis center assay was performed on HuEF cells. 11. Rivers, T. M Nuclear inclusions produced by vari- the input doses of cell-free viruses. cella virus in the testicles of monkeys. J. Exp. Med. Arrow iindicates Symbols passage) recover using yuinfromigscinical vreflla experimen 18. Yamanishi, K., Y. Matsunaga, T. Otsuka, and M. Takaha- pigs might be useful to investigate shi Immune response of guinea pigs to varicella gruinea lular immune response to VZV. vaccine strain (Oka) and wild strains. Biken J. 23: the cell LITERATURE CITED 1. Asano, Y., K. Shiraki, M. Takahashi, H. Nagai, T. Ozaki, and T. Yazaki Soluble skin test antigen of varicellazoster virus prepared from the fluid of infected cultures. J. Infect. Dis. 143: Asano, Y., and M. Takahashi Studies on neutralization of varicella-zoster virus and serological follow-up of cases of varicella and zoster. Biken J. 21: Baba, K., H. Yabuuchi, H. Okuni, and M. Takahashi Studies with live varicella vaccine and inactivated skin test antigen: protective effect of the vaccine and clinical application of the skin test. Pediatrics 61: Gershon, A. A Live attenuated varicella vaccine. Rev. Infect. Dis. 2: Heuschele, W. P Varicella (chicken pox) in three young anthropoid apes. J. Am. Vet. Med. Assoc. 15: Kamiya, H., T. Ihara, A. Hattori, T. Iwasa, M. Sakurai, T. Izawa, A. Yamada, and M. Takahashi Diagnostic skin test reactions with varicella virus antigen and clinical, s, application of the test. J. Infect. Dis. 136: Lausch, R., J. Swyers, and H. Kaufman Delayed hypersensitivity to herpes simplex virus in the guinea pig. J. Immunol. 96: Hours after inoculation 8. Myers, M. G., H. L. Duer, and C. K. Hausler , Oka vaccine strain; A, Oka (HEL, 5th 45: , Kawaguchi (HEL, 10th passage). 12. Rogers, H. W., L. V. Scott, and R. A. Patnode Sensitization of guinea pigs to herpes simplex virus. J. Immunol. 109: sistencei of cellular immunity against VZV in 13. Takahashi, M., Y. Asano, H. Kamiya, K. Baba, and K. humans (1, 3, 6). In this paper, the time course Yamanishi Active immunization for varicella-zoster of the aippearances of humoral and cellular im- virus, p In A. Nahmias, W. R. Dowdle, and R. F. in injected guinea mune ri Schinazi (ed.), The human herpes viruses. Elsevier/ esponses to v E v In InJectea gulnea plgs North-Holland Publishing Co., New York. was prezsented. Skin reaction with vaficella anti- 14. Takahashi, M., Y. Okuno, T. Otsuka, J. Osame, A. gen wars first observed on day 4, and all animals Takanizawa, T. Sasada, and T. Kubo Development responcled with skin antigen on day 8. On the of a live attenuated varicella vaccine. Biken J. 18: other hand, no antibody was detected on day Takahashi, M., T. Otsuka, Y. Okuno, Y. Asano, T. Skin re; actin. culdbe Yazaki, and S. Isomura Live vaccine used to action could detected up to 4 months, as coul prevent the spread of varicella in children in hospital. d humoral antibody. When varicella vac- Lancet 1i: cine wi as used for healthy children, a positive 16. Williams, V., A. Gershon, and P. A. Brunell Seron in the skin test was detected as early as logic response to varicella-zoster membrane antigens reactiol 5 days after vaccination, and neutralizing anti- measured by indirect immunofluorescence. J. Infect. Dis. 130: body a ppeared Lppeared 1 week week later laer (3). The Te immune 17. Yamanishi, K., Y. Matsunaga, Y. Ogino, M. Takahashi, responsses in guinea pigs and vaccinated children and A. Takamizawa Virus replication and localizahus very similar. As cellular immune tion of varicella-zoster virus antigens in human embryonic were t] responsse has been presumed important for the fibroblast cells infected with cell-free virus. Infect. Imry from clinical varicella, experiments mun. 28: