Defective Parvoviruses Acquired via the Transplacental Route

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1 INFECTION AND IMMUNITY, July 1982, p /82/ $02.00/0 Vol. 37, No. 1 Defective Parvoviruses Acquired via the Transplacental Route Protect Mice Against Lethal Adenovirus Infection BINIE V. LIPPS AND HEATHER D. MAYOR* Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas Received 20 November 1981/Accepted 18 March 1982 Adeno-associated virus type 1 (AAV-1) interfered with the replication of its murine adenovirus (MAV) helper in primary mouse kidney cells and in 1-day-old ICR mice. Mice carrying AAV-1 acquired via the transplacental route were protected against lethal infection with MAV. The replication of AAV-1 in these mice could be triggered by multiple challenges with MAV, and antibodies to AAV-1 were subsequently detected. Although all viruses are dependent upon suitable cells for their replication, adeno-associated viruses (AAVs) can only grow completely in cells coinfected with a helper adenovirus (1, 7, 20). Adenoviruses have been classified into different subgroups depending upon their natural hosts, such as human, simian, murine, avian, and bovine. It appears that the replication of defective parvoviruses such as AAV can take place in almost any cells which support the replication of the respective helper virus. For example, the replication of AAV type 1 (AAV-1) takes place in chicken embryo cells when CELO virus is used as a helper (9). Recently, we have demonstrated the replication of AAV-1 in primary mouse kidney (PMK) and continuous mouse 929 L cells by using murine adenovirus (MAV) as a helper virus (15). It has been shown that AAVs interfere with the replication of their helper viruses in coinfections in tissue cultures (5, 20, 22). To our knowledge such interference has not been studied in the in vivo system, although the effect of coinfection with oncogenic adenoviruses has been reported. Injection of oncogenic viruses into neonatal Syrian hamsters leads to tumor formation. Injection of AAV together with adenovirus greatly reduces the frequency of these tumors (12). Appreciable reduction in tumor formation or complete abrogation of tumors in newborn hamsters infected with AAV-1 and challenged with adenovirus type 31 (Ad3l) has also been demonstrated (19). Various autonomous parvoviruses have been shown by a number of investigators to cause fetal infections in animals, resulting in stillbirths, malformations, and embryonic diseases (3, 4, 11, 17, 23). To date there are no definitive data that defective parvoviruses such as AAV cause disease in any host. Recently, we have accomplished the transplacental infection of mice with AAV-1 by using 200 MAV as a helper virus (16). In this paper we report the interference of AAV-1 with the infectivity of its helper virus, MAV, in 1-day-old ICR mice. In newborn mice, MAV can produce a fatal infection (6). We report here the protection afforded in mice carrying AAV, acquired via the transplacental route, against challenge with MAV. We also report that mice carrying AAV acquired via the transplacental route produce AAV antibodies after challenge with MAV. MATERIALS AND METHODS Tissue cultures. Human epidermoid carcinoma of the larynx (HEp-2) cells were purchased from American Type Culture Collection, Rockville, Md. PMK cells were prepared from the kidneys of 8- to 10-day suckling ICR Swiss white mice as described elsewhere (15). Growth media. Cells were grown in Eagle minimal essential medium (Flow Laboratories, Inc., McLean, Va.) containing 5% fetal calf serum, 0.1% sodium bicarbonate, and 20 U of penicillin per ml and 20,ug of streptomycin per ml. The same growth medium containing only 2% fetal calf serum was used to maintain infected cultures. Viruses. Human Ad2 and MAV were grown in HEp- 2 and PMK cells, respectively. The cells were each infected with 1 tissue culture infective dose of virus, which was adsorbed for 1 h at 37 C. The maintenance medium was added, and the cultures were incubated further until advanced cytopathic effects were observed. Virus was prepared by freezing and thawing the cultures three times, sonicating, and removing cell debris by centrifuging at 2,000 rpm for 15 min. AAV-1 was grown in HEp-2 cells by coinfecting each cell with 2 immunofluorescent (IF) units of AAV and 1 tissue culture infective dose of Ad2. The viruses were allowed to adsorb for 1 h at 37 C, and then the maintenance medium was added. The cultures were further incubated until advanced cytopathic effects were observed due to adenovirus and then processed as described above. Ad2 was heat inactivated at 560C for 20 min, and the remaining AAV-containing fluid was retained as stock virus. Concentration and purification of AAV. Production of AAV for concentration and purification was similar

2 VOL. 37, 1982 PROTECTION BY ADENO-ASSOCIATED VIRUS 201 to what was described above. The virus stock was centrifuged at 100,000 x g for 3h. Virus pellets were reconstituted into 1/10 or 1/100 volume of the maintenance medium of the original volume of the virus. For the purification of virus, the pellets were suspended 1x SSC (0.15 M NaCl plus M sodium citrate), concentrating it to 100x. Virus pellets were sonicated to break up virus aggregates and applied to to 1.50-g/cm3 preformed CsCl discontinuous gradients. Gradients were spun with a Spinco SW50.1 rotor at 35,000 rpm at 4 C for 18 h. Fractions were collected by bottom puncture, and those containing infectious AAV (1.38 to 1.43 g/cm3) were dialyzed against 1x SSC for 48 h to remove CsCl. Electron microscopic particle counts of purified AAv were done by the method of Smith et al. (22). AAV infectivity of the concentrated and purified preparations was assayed by IF antigen titration in HEp-2 cells with Ad2 as a helper virus by the method of Ito et al. (10). Mice. ICR Swiss white mice of various age groups were used for these studies. To have 1-day-old mice, pregnant mice were ordered and cared for in the laboratory, and the newborns were kept either with the same mothers or with foster mothers. Interference of AAV-1 with the infectivity of MAV in PMK cells. Monolayer cultures of PMK cells grown in Costar wells (microtissue culture dishes) were used. Six wells were used per dilution of the virus, and 0.1 ml of inoculum per well was used. The cultures were inoculated with serially diluted MAV and mixed with an equal volume of the maintenance medium to study the interference. Sets of serially diluted MAV were mixed with an equal volume of AAV-1 untreated or concentrated 10 and 100 times before infecting the cultures. The infected cultures were incubated at 37 C for 1 h for adsorption of the viruses. The maintenance medium (1 ml per well) was added, and the trays were incubated for 10 days. At the end of the incubation period, the cytopathic effects in each well were determined microscopically. The tissue culture infective dose per milliliter was calculated by the method of Reed and Muench (21). Interference of AAV-1 with the infectivity of MAV in 1-day-old mice. MAV serially diluted 10-fold was mixed with an equal volume of maintenance medium for virus titration. It was also mixed with times concentrated AAV-1 preparations. Newborn mice from several litters were mixed and then randomly distributed. The mice were injected subcutaneously in the back with 0.1 ml per mouse. From 10 to 13 mice were used for each dilution. The injected mice were kept with their respective mothers and were observed for 30 days. At the end of the incubation period, the 50% lethal dose per milliliter was calculated by the method of Reed and Muench (21). The interference afforded by AAV-1 was determined by subtracting the virus titer obtained using the mixture of AAV-1 and MAV from that of MAV alone. Interference with infectivity of MAV in 1-day-old mice carrying AAV-1 acquired via the transplacental route. Mice pregnant for 13 to 15 days were injected subcutaneously with a mixture of purified AAV-1 and MAV. Each pregnant mouse received a dose of 0.5 ml. After the mice delivered the newborns, the infected litters were divided into two groups. The first group of newborns were kept with their own infected mothers. The newborns of the second group were transferred immediately to uninfected foster mothers. The newborns in both these groups carry AAV virus, acquired via the transplacental route (16). The third group consisted of mice born to and kept with their uninfected mothers. The 1-day-old mice of all three groups were injected subcutaneously with various serial dilutions of MAV. Antibody production in mice carrying AAV-1 acquired via the transplacental route after multiple challenges with MAV. The surviving mice from the experiment described above were rechallenged with MAV at l0o3 dilutions 4 weeks after the first challenge. The mice were bled before the second challenge and thereafter at weekly intervals. IF antibody titers against AAV-1 were assayed in the mouse sera. Detection of antibodies against AAV-1. Antibodies were detected in HEp-2 cells grown on 12-mm cover slips placed in the wells of Costar trays. The monolayers on the cover slips were coinfected with AAV-1 and Ad2. The cover slips were air dried 24 h postinfection, fixed with acetone, and reacted with serial dilutions of mouse serum at 37 C for 30 min. After several rinsings with phosphate-buffered saline, the cover slips were stained with fluorescein-conjugated goat anti-mouse gamma globulin heavy chains (Cappel Laboratories, Downington, Pa.) for 30 min at 37 C. After this the cover slips were washed three times with phosphatebuffered saline and then mounted on microscope slides in glycerol-phosphate-buffered saline (9:1). The fluorescence-positive cell nuclei were detected with the aid of an Ortholux microscope (E. Leitz Inc., Rackleigh, N.J.). RESULTS Interference with the infectivity of MAV in PMK cells was directly proportional to the amount of AAV-1 present in the inoculating mixture (Table 1). This finding is in agreement with results obtained with AAV-4 and SV15 as a helper virus in tissue culture (20). Furthermore, our results show that the interference with infectivity of MAV in 1-day-old mice was dependent upon the concentration of AAV. We also observed that the incubation period in mice for fatal infection due to MAV was prolonged in the presence of AAV-1 by 4 to 6 days. We have previously demonstrated (16) that when pregnant mice are injected with a mixture of AAV and MAV, only AAV passes through the placenta. Infectious AAV was detected in the homogenates of kidneys and lungs of fetuses as well as in newborn mice. The presence of AAV acquired via the transplacental route in newborn mice interfered with the infectivity of MAV (Table 2). Mice born and kept with the mothers infected with a mixture of AAV and MAV were protected against a very high dose of MAV (Table 2, group III). This enhanced protection appears partly to be passive and was no doubt due to antibodies excreted in milk. The mice born to mothers infected with a mixture of AAV and MAV, but transferred to

3 202 LIPPS AND MAYOR INFECT. IMMUN. TABLE 1. Interference with infectivity of MAV by AAV-1 in PMK cells and 1-day-old micea PMK cells 1-day-old mice IF titer of LDs5ml titer AAV-1 stockb of MAV MAV per ml in Interference with LD5Jml titer of MAV in Interference with perpe ml with coinfection AAV-1 infectivity of MAV of MAV with coinfection AAV-1 infectivity of MAV Unconcentratedd NI' 1Ox concentrated x concentrated a All numbers are b log1o. AAV-1 was concentrated by centrifuging the virus stock at 100,000 x g for 3 h. It was then suspended into 1/ 10 and 1/100 volume of the maintenance medium. cld50, fifty percent lethal dose of MAV. d The IF titer of unconcentrated AAV-1 was loglo 6.2 IF units/ml in HEp-2 cells when Ad2 was used as a helper. e NI, No interference. normal foster mothers immediately after birth, also showed appreciable protection against MAV. The protection afforded by AAV-1 which had crossed the placenta was almost three logs. This is quite significant (Table 2, group IV). In the process of AAV purification, it is almost impossible to obtain pure AAV without any structurally derived components from the helper virus. To rule out any doubt that the protection afforded was due to antibodies to Ad2 components which could neutralize MAV, pregnant mice were injected with Ad2 at a 10-2 dilution which was heated at 56 C for 20 min in a manner similar to the AAV-1 preparations. The results of group II show that there was no protection against MAV when their newborns were injected with MAV. The virus titers in control mice (group I) and group II mice were almost identical. Surviving mice from experiments shown in Table 2 were challenged at 4 weeks of age with MAV at a 10-3 dilution. Each mouse received 0.1 ml of virus subcutaneously. Sera were tested for AAV IF antibodies before the challenge and at weekly intervals thereafter. Results showed a tremendous increase in IF titers in sera of mice in groups III and IV. This clearly indicates that the mice in these groups had acquired AAV via the transplacental route after the challenge with MAV and then replicated and evoked the antibody response observed. Mice born to the mothers injected with heated Ad2 did not show any protection against MAV and did not develop significant antibodies to AAV. TABLE 2. Protection afforded against MAV in 1-day-old mice carrying AAV-1 acquired transplacentally and a subsequent production of antibodies to AAV-1 after challenging with MAVa Protection AAV-1 IF antibody titer in mouse sera after challenge with Group Description of mice LD5OIOg,n afforded MAV (log10) (log10) Base line' l wk 2 wk 3 wk I Control mice born to and kept with normal 6.3 1:10 1:10 1:10 1:20 mothers II Mice born to and kept with mothers injected 6.6 NP3d 1:10 1:20 1:20 1:40 with heated Ad2 at a 10-2 dilution III Mice born to and kept with mothers injected :20 1:80 1:160 1:320 with a mixture of AAV-1 and MAV IV Mice born to mothers injected with a mixture of :10 1:40 1:80 1:320 AAV-1 and MAV, transferred to foster normal mothers immediately after birth a Pregnant mice were injected subcutaneously with a mixture of density gradient-purified AAV-1 and crude MAV. The IF titer of purified AAV-1 was log IF units/ml in HEp-2 cells when Ad2 was used as a helper virus. The particle count was 4.5 x 109/ml. b LD50, fifty percent lethal dose of MAV. c Sera of surviving mice 4 weeks after injection with MAV. At this point the mice were challenged with MAV and then bled at weekly intervals. d NP, No protection.

4 VOL. 37, 1982 DISCUSSION AAV are unconditionally defective members of the parvovirus group and cannot replicate completely unless host cells are coinfected with helper adenoviruses (1, 7, 20). Adenoviruses are host specific. For example, CELO adenovirus will not replicate in human or mammalian cells, but it grows well in chicken cells. Similarly, MAV fails to replicate in human cells, but it grows well in certain mouse cells. In attempting to develop a high-titer stock of MAV, we have observed that this adenovirus is organ specific. It replicates well in the PMK cells of various species such as the ICR Swiss, C3H, and BALB/c breeds, but it fails to replicate either in mouse fibroblast cells or in rat kidney cells. It is clear that only those cells which support the replication of this adenovirus can also support the replication of AAV. Thus, MAV complements AAV IF antigen as well as infectious virus production in PMK cultures from various breeds of mice (15). Several investigators have studied transplacental infection with various viruses. A murine strain of poliomyelitis produces abortion rates from 72 to 88% when injected into mice on day 1 of gestation (13). Likewise, the Lansing murine strain has been found in fetuses, suggesting that it passes through the placenta in mice (2). These authors have also observed high abortion rates in the infected animals. Howell et al. (8) demonstrated an increased abortion rate in mice infected during gestation with swine influenza virus. Komrower (14) has presented evidence that lymphocytic choriomeningitis virus crosses the placenta in humans, although this virus has not been shown to be a major fetal pathogen in humans. Recently, we have demonstrated that when pregnant mice are coinfected with a mixture of AAV and MAV, only AAV crosses the placenta (16). Infectious AAV was isolated from the homogenates of pooled kidneys and lungs of fetuses 7 to 8 days postinfection. The virus persisted for 3 weeks in the kidneys of those mice which were born to infected mothers and transferred to normal foster mothers immediately after birth. The results presented in Table 2 clearly indicate that when pregnant mice were coinfected with AAV and MAV, replication of both viruses took place. Only AAV passed through the placenta, persisted in newborns, and then interfered with the replication of MAV after challenge. Thus, the challenged mice were protected against the lethal effect of MAV. We have previously demonstrated that mice born to mothers infected with a mixture of AAV and MAV develop an AAV IF titer of 1:20 at 3 weeks of age. There is an increase in titer after these mice are challenged with MAV (IF titer, 1:160) (16). In the studies reported here, we PROTECTION BY ADENO-ASSOCIATED VIRUS 203 have demonstrated that when newborn mice carrying AAV acquired via the transplacental route were infected with MAV at birth and challenged with MAV after 4 weeks, they developed much higher AAV IF antibody titers (1:320) (Table 2, groups III and IV). This indicates that multiple infections with MAV in mice enhance antibody response. Mice in group II showed a slight increase in antibody to AAV. This could be a reaction to small amounts of AAV surviving in the laboratory environment. To date there are no definitive data that defective parvoviruses such as AAV cause disease in any host. In fact, they interfere with the growth and potential oncogenesis of those adenoviruses on whose functions they depend (12, 19). Furthermore, Mayor et al. (18) have demonstrated markedly lower titers of antibodies to AAV in sera of patients suffering from either cervical or prostatic carcinoma. These data suggest that exposure to AAV may afford protection against certain kinds of cancers. We plan to test whether AAV acquired transplacentally can protect newborn mice from developing tumors following inoculation shortly after birth with an oncogenic adnovirus such as Ad3l. Given the similarity between human and murine placentation, the mouse model of fetal infection described here holds great promise for future studies in human systems. ACKNOWLEDGMENTS This work was supported by grants from the West Foundation and the Cockrell Foundation, Houston, Tex. LITERATURE CITED 1. Atchison, R. W., B. C. Casto, and W. McD. Hammon Adeno-associated virus particles. Science 149: Byrd, C. L., Jr Influence of injection with Lansing strain poliomyelitis virus on pregnant mice. Neuropathol. Exp. Neurol. 9: Cartwright, S. S., and R. A. Huck Viruses isolated in association with herd infertility abortions and stillbirths in pigs. Vet. Rec. 81: Carwrlight, S. F., M. Lucas, and R. A. 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5 204 LIPPS AND MAYOR tional-lethal mutants of CELO virus at the restrictive temperature. Virology 45: Ito, M., J. L. Melnick, and H. D. Mayor An immunofluorescence assay for studying replication of adenosatellite virus. J. Gen. Virol. 1: Johnson, R. H A search for parvoviridiae (Picodnaviridae). Vet. Rec. 84: Kirchstein, R. L., K. 0. Smith, and E. A. Peters Inhibition of adenovirus 12 oncogenicity by adeno-associated virus. Proc. Soc. Exp. Biol. Med. 128: Knox, A. W Infection and immunity in offspring of mice inoculated during gestation with murine poliomyelitis virus. Proc. Soc. Exp. Biol. Med. 74:792-7%. 14. Komrower, G Lymphocytic choriomeningitis in newborns: probable transplacental infection. Lancet 1: Lipps, B. V., and H. D. Mayor Properties of adenoassociated virus type 1 replicated in rodent cells by murine adenovirus. J. Gen. Virol. 51: Lipps, B. V., and H. D. Mayor Transplacental infection with adeno-associated virus type 1 in mice. Intervirology 14: Marimoto, T., H. Kurogi, T. Muria, T. Sagimori, and Y. INFECT. IMMUN. FeyisakL Isolation of Japanese encephalitis virus and hemagglutinating: DNA virus from brains of stillborn piglets. Natl. Inst. of Anim. Health Q. 12: Mayor, H. D., S. Drake, J. Stahmann, and D. M. Mum. ford Antibodies to adeno-associated satellite virus and herpes simplex in sera from cancer patients and normal adults. Am. J. Obstet. Gynecol. 126: Mayor, H. D., G. S. Houlditch, and D. M. Mumford Influence of adeno-associated satellite virus on adenovirus induced tumors in hamsters. Nature (London) New Biol. 241: Parks, W. O., A. M. Casazza, J. Alcott, and J. L. Melnick Adeno-associated satellite virus interference with the replication of its helper adenovirus. J. Exp. Med. 127: Reed, W., and H. Muench A simple method of estimating fifty percent end points. Am. J. Hyg. 27: Smith, K. O., W. P. Gehle, and J. F. Thiel Properties of a small virus associated with adenovirus type 4. J. Imunol. 97: Toolan, H. W Experimental production of mongoloid hamsters. Science 31: