VariceUa-Zoster Virus Immunizes Patas Monkeys against Simian Varicella-like Disease

Transcription

1 J. gen Virol. 0979), 4z, Printed in Great Britain I7I VariceUa-Zoster Virus Immunizes Patas Monkeys against Simian Varicella-like Disease By AMBHAN D. FELSENFELD* AND NATHALIE J. SCHMIDTt * Delta Regional Primate Research Center, Tulane University, Covington, Louisiana, 7o433, U.S.A. and Viral and Rickettsial Disease Laboratory, State of California Department of Health, Berkeley, California 94704, U.S.A. (Accepted 3I July I978) SUMMARY To define further the antigenic relationship between human varicella-zoster virus and herpesviruses which produce varicella-like disease in certain simian species, patas monkeys were inoculated with varicella-zoster virus and then challenged with Delta herpesvirus, which uniformly produces severe, clinically apparent disease in susceptible animals. Protection against Delta herpesvirus was conferred both by hyperimmunization with varicella-zoster virus and by a single immunization with a cell-free preparation of varicella-zoster virus. Although the immunological relationship between the human and simian varicella viruses is not completely reciprocal, these studies confirmed that antigens which induce immunity are shared by the human and simian viruses. No clinical symptoms were seen in monkeys inoculated with varicella-zoster virus, but the rapid and marked antibody responses to the virus suggested that subclinical infection had occurred. In contrast, a chimpanzee inoculated with one of the same varicella-zoster virus preparations produced only low levels of antibody. INTRODUCTION By cross-neutralization and cross-complement fixation tests we have shown that Delta herpesvirus (Ayres, I97I; Riopelle et al. r97i), 592S virus (Allen et al. I974), Liverpool vervet monkey virus (Clarkson et al. I967), the herpesvirus of patas monkeys (McCarthy et al. I968), and the Medical Lake macaque virus (Blakely et al. I973) are each immunologically related to varicella-zoster (V-Z) virus of man (Felsenfeld & Schmidt, i975, ~977). These simian herpesviruses, isolated from diverse outbreaks of varicella-like disease and from various simian species, were immunologically indistinguishable from one another, but cross-complement fixation tests and cross-neutralization tests with sera from natural infections indicated that the simian varicella viruses are not so closely related to V-Z virus as they are to one another (Felsenfeld & Schmidt, 1977). Thus, although the simian and human viruses clearly share major common antigens, they appear to have additional specific antigens as well. For the present studies, patas monkeys (Erythrocebus patas) were inoculated with V-Z virus preparations and then challenged with the 592S strain of Delta herpesvirus (DHV), which in unprotected animals uniformly produces severe, clinically apparent infection with a high mortality rate (Allen et al. I974). o /79/oooo-3296 $02.00 ~ 1979 SGM

2 I72 A. D. FELSENFELD AND N. J. SCHMIDT METHODS Viruses. The CaQu strain of V-Z virus was isolated in human foetal diploid kidney cells from a lesion specimen of a patient with a vesicular exanthema clinically resembling herpes zoster. Specific identification of the virus was made by immunofluorescence staining with immune rhesus monkey serum (Schmidt et al. 1965). For the present studies the virus strain had been passaged twice in human foetal diploid kidney cells and then eight times in human foetal diploid lung (HFDL) cells. Cell-free virus, prepared as described previously (Schmidt & Lennette, I976), and having a titre of 3"7 x IO p.f.u./ml was used for inoculation. Uninfected control cell culture material was prepared in the same manner from the same batch of HFDL cells. The SPu strain of V-Z virus was recovered in HFDL cells from a clinical case of varicella and was also identified by immunofluorescence staining. The virus was at passage levels of 2 to 6 when used in these studies. Virus-infected cells suspended in growth medium with 35 ~ sorbitol at a concentration of 6.8 x lo 5 cells/ml was stored at -8o C. This preparation, representing the second virus passage, was used for the initial inoculation of monkeys, and fresh trypsin-dispersed, infected HFDL cells representing virus passage levels of 3 to 6 were used at a concentration of approx. 4"5 x Io 5 cells/ml for booster immunizations. Uninfected control inocula were prepared by the same procedures from HFDL cells of the same batches. The 592S strain of DHV was isolated in Veto cells from the blood of moribund patas monkey involved in the 1973 outbreak of vadcella-like disease at the Delta Primate Center (Allen et al. 1974). The virus was used in the present studies at the fifth and seventh passage levels in Veto cells. Stock virus was prepared as described previously (Felsenfeld & Schmidt, 1977). The preparations had infectivity titres of 4"5 to 6.0 lo 5 p.f.u./ml, and consistently produced varicella-like disease in susceptible patas monkeys. Antibody assays. Neutralizing antibody to V-Z virus was assayed by a plaque reduction method (Schmidt & Lennette, 1975) both with and without fresh guinea pig serum in the reaction mixture, since it has been shown that complement enhances the neutralization of V-Z virus (Schmidt & Lennette, 1975). Neutralizing antibody to DHV was also determined by a plaque reduction technique (Felsenfeld & Schmidt, 1975). Complement-fixing (CF) and indirect fluorescent antibody (FA) assays were performed by the standard procedures of the State of California Department of Health Virus Laboratory (Schmidt et al. 1965; Lennette, 1969). Virus isolation attempts. Efforts were made to isolate V-Z virus from washed lymphocytes and from throat swabs of inoculated animals by inoculation into HFDL cells (Schmidt, I974), and to recover DHV from the same types of specimens by inoculation into Vero cells (Allen et al. 1974). These specimens were collected at 2 to 3 day intervals over a period of Io to 14 days after inoculation with virus. The DHV isolates were identified by neutralization with specific antiserum. Inoculation of animals. Animals were handled in strict accordance with the rules and regulations of the Animal Resources Board and the National Research Council. Ketamine HC1 at t ms/ks of body weight was given for anaesthesia. Intratracheal inoculation was performed by inserting a 22 gauge needle between the cartilaginous rings I to 1"5 cm above the suprasternal notch. Before the inoculum was injected, air was aspirated to ensure that the needle was in the proper position. Patas monkeys 2 to 3 years of age weighing 4 to 5 ks, which had no demonstrable neutralizing antibodies for V-Z virus or DHV, received I"5 ml of virus preparation or

3 Simian and human varicella viruses I73 Table I. Clinical and virological findings in patas monkeys hyperimmunized with V-Z virus and challenged with DHV Monkey Absolute lymphocyte Virus Inocula Day Rash SGOT SGPT count isolation Cell control+dhv o-i4 o --* o o.... o 3 o ~ + L ~ 5 o L 7 o t 26o~ t 259~ + 66~ +L,T 9 + 1' 64o~ ~ 6o4~ -- +L,T I [ -Jr- 1' 340 ~ ~" 376 % -- o V-Z virus + DHV o--i 4 o o 3 o o 5 o L 7 o o 9 o o I[ V-Z virus + DHV o--i 4 o ~ o O--I I * -, Values within baseline range. Baseline ranges at day o for cell control or V-Z virus: SGOT = 28 to 39, SGPT = 21 to 42, absolute lymphocyte count = Io66 to Ranges at day o for DHV: SGOT = x8 to 34, SGPT = 2I to 26, absolute lymphocyte count = 2769 to t L, Virus isolation from lymphocytes; T, isolation from throat. :~ Per cent increase or decrease from baseline level. Mild leucocytopenia seen at day 2 (~, 40 ~) and day 4 (4. 33 ~). corresponding uninfected cell culture material by the intratracheal route, followed immediately by an inoculation of I-5 ml of the same material by the subcutaneous route. The monkeys inoculated with the SPu strain of V-Z virus, or with corresponding uninfected cell culture material, were hyperimmunized by five, weekly subcutaneous injections of I'5 ml of freshly trypsinized infected or uninfected cells (approx. 3 to 4 x Io 5 cells/ml) beginning at zz days after the initial intratracheal-subcutaneous inoculations. At 63 days after the initial injections, these animals were challenged with I-8 x io 5 p.f.u, of DHV administered by the intratracheal-subcutaneous routes. Monkeys inoculated with the CaQu strain of V-Z virus received only an initial inoculation of 1.5 ml of cell-free virus by the intratracheal route and I-5 ml by the subcutaneous route (a total of i.i x lo T p.f.u.) and were challenged with DHV 35 days later. Control animals were inoculated in the same manner with uninfected cell culture material and then challenged with DHV. An effort was made to infect a chimpanzee (Pan troglodytes) with V-Z virus by giving a juvenile animal approximately 8 to 9 years of age, without demonstrable neutralizing antibodies to V-Z virus or DHV, the same preparation of CaQu virus which was given to the patas monkeys, in the same vol. and by the same routes. A control chimpanzee of approximately the same age, also lacking V-Z and DHV neutralizing antibodies, was inoculated in the same manner with the corresponding uninfected cell culture material.

4 174 A. D. FELSENFELD AND N. J. SCHMIDT Table 2. Antibody responses in patas monkeys hyperimmunized with V-Z virus and challenged with DHV V-Z virus antibody titres neutralization* DHV c ~ ~ neutralization Monkey Inocula Day - C + C CF titre 5583 Cell control+dhv o < 4 < 4 < 8 < 4 22 <4 <4 -- <4 36 <4 <4 -- <4 48 <4 <4 -- <4 63(o)t < 4 < 4 < 8 < 4 85 (22) 4 I V-Z virus+dhv o < 4 < 4 < 8 < o (o) I6 85 (22) I I2 559O V-Z virus+dhv o < 4 < 4 < 8 < Io IO I6384 Io (o) IO (22) * - C, complement absent in serum-virus mixture; + C, complement t Number in parentheses is day with respect to DHV inoculation. present. RESULTS Experiments in paras monkeys hyperimmunized with V-Z virus Clinical and virological findings in patas monkeys hyperimmunized with the SPu strain of V-Z virus and then challenged with DHV are shown in Table I, and antibody responses are shown in Table 2. The control animal immunized with uninfected cell culture material developed a typical simian varicella-like disease in response to inoculation with DHV, with rash, elevated serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) levels, and a depressed lymphocyte count, and DHV was isolated over a period of 6 days. Neither of the monkeys showed clinical symptoms or abnormal liver function in response to V-Z virus inoculation. One animal developed a mild leucocytopenia, but the significance of this is questionable since the animal had a very high initial lymphocyte count. V-Z virus was not recovered from lymphocyte or throat specimens. However, as shown in Table 2, a rapid and marked antibody response occurred to V-Z virus and antibody levels, even those produced by the initial inoculation of V-Z virus, were as high as or higher than those commonly seen in primary V-Z infections (varicella) in humans. Detectable neutralizing antibody was produced to DHV, even by the initial V-Z virus inoculation, but titres remained low until after challenge with DHV. Challenge with DHV produced no clinical symptoms nor abnormal liver function and haematologieal findings in the monkeys hyperimmunized with V-Z virus. DHV was isolated from only a single specimen, a 5-day lymphocyte specimen from animal No

5 Simian and human varicella viruses 175 Monkey Table 3. Clinical and virological findings in patas monkeys receiving a single immunization with cell-free V-Z virus before challenge with DHV Inocula 5785 Cell control + DHV 579I Cell control+ DHV 5587 V-Z virus + DHV 5779 V-Z virus + DHV Absolute lymphocyte Virus Day Rash SGOT SGPT count isolation o to t3 o --* o 3 o L, Tt ,~ 62 %~ +L 8 + I" 89% '~ 249/oo ~ 26% +L,T Io + I' 80% I" 84% -- 0 OtO 13 O O 3 o L, T 6 o ~, 53 % +L 8 + ~" 80% # 235% + 30% +L,T IO + f 69~ J' 119o~ -- o o to 13 o o 3 o L 6to ]o o o 3 0 ~ 281 ~oo ~" 13o ~ ~ 31% o 6 o I" 789~ t" 79 I~ ~ 43~ o 8 0 I' 157% I' 319~ -- 0 Io o ~' 98 ~ I' 314 ~ -- O 13 0 I' 40 ~ t 220 ~oo -~ 41% 0 3 o o 6 o -- f 22% ~ 28% I * --, Values within baseline range. Baseline ranges at day o for cell control or V-Z virus: SGOT = 23 to42,sgpt = 18 to t 29, absolute lymphocyte count = 2542 to372o. RangesatdayoforDHV: SGOT = 21 to 28, SGPT = 2o to 44, absolute lymphocyte count = 2491 to 5o4o. I" L, Virus isolation from lymphocytes; T, isolation from throat. Per cent increase or decrease from baseline level. Experiments in patas monkeys receiving a single immunization with cell free V-Z virus After the previous experiments showed that multiple inoculations of V-Z virus preparations of undetermined infectivity titre could induce immunity to DHV, the protective capacity of a single administration of cell-free V-Z virus of known titre was examined. Two patas monkeys were inoculated with the CaQu virus strain, and two with corresponding uninfected cell culture material, as described in the Methods. The animals were challenged with DHV 35 days later. Clinical and virological findings are shown in Table 3, and antibody responses in Table 4. Both control animals showed typical varicella-like disease in response to DHV inoculation, and DHV was isolated over a 5 day period. Again, inoculation with V-Z virus failed to produce vesicular lesions in the monkeys, and one animal showed normal liver function and haematological findings. One of the animals (No. 5779) showed elevated SGOT and SGPT levels and depressed lymphocyte counts. Efforts to recover V-Z virus from lymphocyte or throat specimens were unsuccessful. As shown in Table 4, high levels of V-Z neutralizing and CF antibody were present 35 days I2 VIR 42

6 176 A. D. FELSENFELD AND N. J. SCHMIDT Table 4. Antibody responses in patas monkeys receiving a single immunization with cell-free V-Z virus before challenge with DHV V-Z virus antibody titres neutralization* DHV r ~ ~ neutralization Monkey Inocula Day - C + C CF titre 5785 Cell control+dhv o < 4 < 4 < 8 < 4 35(o)t < 4 < 4 < 8 < 4 55 (2I) I (30) (49) Cell control+dhv o < 4 < 4 < 8 < 4 35 (o) < 4 < 4 < 8 < 4 55 (2I) (30) I I (49) 32 I28 I6 5~ V-Z virus+dhv o < 4 < 4 < 8 < 4 35 (O) IO (2I) (30) 256 I (49) 256 IO V-Z virus + DHV o < 4 < 4 < 8 < 4 35 (o) Io24 < 4 55 (2I) (30) I (49) * -C, complement absent in serum-virus mixture; + C, complement present. t Number in parentheses is day with respect to DHV inoculation. after inoculation. In one animal a DHV neutralizing antibody titre of t : 8 was demonstrable at the time of challenge; the other animal had a titre of < I : 4. However, both animals were protected against DHV disease. DHV was isolated only from a 3 day lymphocyte specimen from monkey No Inoculation of a chimpanzee with V-Z virus An attempt was made to infect a chimpanzee with the same cell-free V-Z preparation used to immunize the patas monkeys against DHV infection. Although this material produced high levels of neutralizing and CF antibody in the monkeys, the chimpanzee responded with very low levels of antibody. V-Z virus antibody titres at 3o and 44 days were I : 64 by neutralization and indirect immunofluorescence staining and I : 4 by CF, and titres of DHV were < I : 4. Clinical evidence of V-Z infection was not seen in the animal, and V-Z virus isolation attempts on leucocyte and throat specimens taken on days o, 2, 6, 9, Ii and 13 were negative. DISCUSSION Previous results from our laboratories (Felsenfeld & Schmidt, I975, 1977) showing that heterologous neutralizing antibodies were produced in seronegative individuals by infection with either V-Z virus or DHV furnished the first evidence that antigens which elicit the production of protective antibodies are shared by the simian and human varicella viruses. The present studies extended these findings to show that immunization with V-Z virus stimulates humoral, and perhaps cellular, immunity against DHV disease in patas monkeys.

7 Simian and human varicella viruses ~77 In addition to providing additional information on the antigenic relationship between simian and human varicella viruses, these findings may have practical implications. Immunizing paras monkeys with V-Z virus and challenging with DHV may prove to be a useful method for evaluating experimental vaccines against V-Z virus. Also, infection of paras monkeys with V-Z virus appears to be a very effective method for producing hightitred antiserum; this is a simpler procedure than others which have been described for preparing experimental antisera to V-Z virus (Schmidt et al. I965; Kissling et al. I968; Martos et al. I97o). Although vesicular exanthemas were not produced, it appears likely that the patas monkeys inoculated with V-Z virus experienced subclinical infections. The high levels of V-Z antibody attained early after inoculation suggest that virus replication occurred, increasing the antigenic stimulus. In addition to the animals used in the experiments reported here, a monkey inoculated with HFDL cells infected with another strain of V-Z virus (MTh), and two monkeys inoculated with another cell-free preparation of the CaQu strain of V-Z virus produced similar high titres of V-Z antibodies at z2 days after a single inoculation. The failure to isolate V-Z virus from lymphocyte or throat specimens does not rule out the possibility of subclinical infection, since these are not optimal specimens for recovery of V-Z virus in human infections, but had to be resorted to in the absence of vesicular lesion materials. It is also noteworthy that one monkey responded to V-Z virus inoculation with elevated SGOT and SGPT levels and depressed lymphocyte levels. In contrast to the high titres of antibody elicited in patas monkeys by the V-Z virus preparation, the chimpanzee inoculated in the same manner with the same virus produced only low levels of V-Z antibody and no detectable DHV antibody, suggesting that virus replication may not have occurred, and that the weak serological response was to the initial inoculum of virus. The chimpanzee was inoculated with the V-Z virus preparation before the patas monkeys were, ruling out the possibility that the poor serological response in the former was due to deterioration of infectivity or antigenicity of the inoculum upon storage. None of the CaQu virus material used for inoculation had previously been subjected to freezing and thawing. Since chimpanzees are more closely related phylogenetically to humans than are patas monkeys, it might be expected that they would be more susceptible to infection with the human varicella virus. Varicella-like disease has been reported in infant chimpanzees, and a herpesvirus similar to V-Z virus was isolated from their vesicular lesions (McClure & Keeling, I97I). However, the method used for identifying these agents was not stated, so it is uncertain how closely they were related to V-Z virus. Certainly chimpanzees experience natural infection with an agent immunologically related to V-Z virus, since, of I9 juvenile, wild-caught chimpanzees which we tested, I3 had neutralizing antibody titres of /> ~ :4 to V-Z virus. Our experiment in a single chimpanzee is clearly not definitive insofar as determining whether human V-Z virus can produce disease in chimpanzees. This study was supported in part by a grant to the Delta Regional Primate Research Center by the Division of Research Resources, Public Health Research Grant No. RRoo 164, and by Public Health Service Grant AI-oi475 from the National Institute of Allergy and Infectious Diseases to the State of California Department of Health. Z2-2

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