(~) INSTITUTPASTEUR/ELSEVIER Paris 1992

Res. Virol. 1992, 143, 321-328

In vitro infection of human placental trophoblast

by wild-type vaccinia virus and recombinant virus expressing HIV envelope glycoprotein N. NCrskov-Lauritsen O), V. Zachar O)(2), P . M . Petersen (l), H. Hager 0), G. Aboagye-Mathiesen (i) and P. Ebbesen o) (') tl) Department o f Virus & Cancer, Danish Cancer Society, Gustav Wieds Vej 10, DK-8000 ,~rhus C (Denmark), and f2) Center f o r Molecular Biology, Wayne State University, Detroit, MI (USA).

SUMMARY Short-time ( ~< 7 days) cultures of trophoblast mononuclear cells isolated from term placentae were challenged with vaccinia virus. Cytopathic effects were induced in crude placental cell preparations as well as in cultures established after negative immunosorting of major histocompatibility complex class I epitope-expressing cells, i.e. cultures exclusively derived from villous cytotrophoblast according to our present state of knowledge. The trophoblast in vitro supported a full replicative cycle of both wild-type viruses and a recombinant clone serving as a vector for the human immunodeficiency virus type 1 envelope gene. Results may shed light on mechanisms involved in the rarely observed foetal damage caused by smallpox vaccination during pregnanqy.

Key-words: Vaccinia, Foetus, Vaccination, AIDS; Transplacental virus transmission, Recombinant vaccinia virus.

INTRODUCTION The term variolae vaccinae (latin vacca, cow) was first used to describe a smallpox-related disease o f cows by Edward Jenner in 1798, and the deliberate inoculation of material from such lesions was soon to be designated vaccination. The vaccinia virus was removed from the armamentarium of preventive medicine when smallpox virus became eradicated in 1977. The virus now serves as a useful vector for expressing genes within the cytoplasm of eukaryotic cells (Moss,

Submitted January 9, 1992, accepted August 28, 1992. (*) Correspondingauthor.

1991). Recombinant poxviruses may be candidates for new live vaccines, and a phase I trial o f recombinant live vaccinia virus expressing HIV envelope glycoprotein has recently been reported (Cooney et al., 1991). However, if we are to embark on a new encounter with vaccinia virus, the risk associated with the infection will have to be reconsidered. One of the complications associated with smallpox vaccination was the possibility o f foetal involvement during pregnancy. The platen-

322

N. N O R S K O V-LA U R I T S E N E T A L .

tal trophoblast layer constitutes a physical and biochemical barrier between mother and foetus, and infection o f the trophoblast is a likely pathogenetic step in the transmission o f virus to the foetus. The present investigation was undertaken to determine the in vitro susceptibility o f h u m a n term trophoblast to infection with wildtype and recombinant vaccinia virus. Results may have bearing on the general pathogenesis o f foetal infections, and serve as a reminder o f the small but not negligible risk o f vaccination with live virus in pregnancy (Levine et al., 1974).

MATERIALS

AND METHODS

Trophoblast Placentae were immediately placed in closed plastic bags at 4°C and processed within 8 h. The isolation of villous cytotrophoblast from biopsies is based on trypsinization (Thiede, 1960). "Percoil" centrifugation (Kliman et al., 1986) and negative immunosorting of MHC-expressing cells (Douglas and King, 1989), with minor modifications. Due to the efficiency of immunosorting, we simplified the gradient to two layers (70 and 25 070 Percoll in PBS). Cells were underlaid and centrifuged at 1,200 g for 20 min to separate the erythrocytes (bottom) from the mononuclear placental cells (interphase) and the villous tissue remnants (top). Antibodies to MHC class II, originally included in the purification procedure (Douglas and King, 1989), were omitted. Trophoblast was seeded at 5 x 105 cells in l-cm 2 wells and was cultured as previously described (NCrskov-Lauritsen et al., 1992). Not all cells adhered to the surface. Employed as a control target cell was a continuous human cervix carcinoma cell line (MS-751 from the ATCC, Rockville~ MD, USA). The cells were seeded at 2-7 x 104 cells in 1-cm 2 wells and infected when the cultures were subconfluent. The lymphoblastoid, CD4-expressing cell line CEM-SS used for coculture experiments was obtained from Dr. Nara (Nara et al., 1987).

vPE16, a recombinant virus expressing HIV gpl60 (Earl et aL, 1990), and vaccinia WR, the control wildtype strain, were obtained from the NIAID and NIH AIDS Reference Reagent Program (catalog Nos. 362 and 353, respectively). The viruses were propagated and plaque-titred on African green monkey kidney (Vero) cells. For trophoblast infection, the inl~ut infectivity dose/well varied between 3 x 1 0 ° and 8 x 105 plaque-forming units (PFU), or 0.006-1.6 PFU per trophoblast cell seeded. The input multiplicity of infection (moi) of the separate experiments is given according to this calculation. The dose thus calculated is arbitrary due to the unknown number of trophoblast mononuclear cells and syncytia present at the time of infection. If not otherwise stated, viruses were added to cultures in serum-free medium and were allowed to adsorb for 1 h at 37°C before withdrawal, wash and replenishment with culture medium (RPMI-1640 supplemented with 10 °70 foetal calf serum and antibiotics). In some experiments, the viruses were left in contact with trophoblast for 24 h in culture medium. Viral content of cultures was assayed after withdrawal of medium, cell lysis with 10 mM Tris-HCl pH 8.0, and pooling of media plus lysate. In coculture experiments, trophoblast was infected at unit moi after 24 h in culture. Viruses were removed after 1-h contact and the cultures were washed 3 times with serum-free medium and incubated with culture medium for 24 h. The cultures were then incubated for 6 h with CEM-SS cells at a' ratio of 1:1. Other methods Assay for hCG, as well as immunofluorescence and immunocytochemistry procedures are described elsewhere (Nerskov-Lauritsen et al., 1992). HIV antigen-capture ELISA was performed using IgG isolated from high-titred human sera according to the method of Nielsen et al. (1987). The pH of the cultivation medium during incubation was determined with a "Radiometer ABL-30" analyser (Ebbesen et al., I991).

RESULTS

Vaccinia wild-type (strain Elstree) was obtained from the ATCC (catalog No. VR-862). Vaccinia-

Listed in rations used preparations trypsin from

ATCC HIV MHC

moi PBS PFU

Viruses and infection

= = =

American Type Culture Collection. h u m a n immunodeficiency virus. major histocompatihi|ity complex.

= = =

table I are the t r o p h o b l a s t prepain the present investigation. C r u d e o f m o n o n u c l e a r cells released by villous biopsies (in the range o f 106

mu|tiplicity o f infection. phosphate-buffered saline. plaque-forming unit.

INFECTION OF PLACENTAL

TROPHOBLAST

cells per gram wet weight) from term placentae were composed of 60-90 °70 epithelial cells, but MHC-directed negative immunosorting (Douglas and King, 1989) reproducibly yielded an "essentially pure" human term trophoblast cell suspension (table I). The efficient removal of fibroblast cells was also demonstrated by prolonged cultivation, where 500,000 cells did not give rise to fibroblast cultures after 3 weeks of cultivation, whereas crude placental cell preparations were overgrown within 7-10 days (data not shown).

323

BY VACCINIA VIRUSES

cytopathic effect was induced on the trophoblast at a similar rate (micrographs not shown). When crude placental cell preparations were challenged with vaccinia, a cytopathic alteration was observed on all cells (fig. 1D). We have demonstrated the deterioration o f trophoblast hCG secretion following cytopathic herpes simplex virus infection (NerskovLauritsen et al., 1992) and have used the same trophoblast marker following challenge with vaccinia virus (fig. 2). The hCG synthesis rate varied between preparations from separate placentae, but the kinetics of synthesis in relation to cultivation time was preserved. All additions of virus to cultures during the first days of cultivation were clearly deleterious to subsequent hormone production (fig. 2). Infection at low multiplicities required 2-3 days to influence hormone synthesis, and no significant difference was observed between the inhibition induced by a wild-type virus and the recombinant virus. Mock infection of trophoblast with an inoculum prepared from uninfected Vero cell cultures did not inhibit the release of hCG, excluding the possibility that an

Addition of vaccinia virus to 24-h cultures of villous trophoblast induced a cytopathic alteration, with condensation o f cytoplasm and cell rounding (fig. 1B). The condensation o f trophoblast revealed cytoplasmic bridges between neighbouring trophoblast aggregates, indicating that some irreversible in vitro formation of syncytiotrophoblast had already taken place after 24 h in culture. When trophoblast cultures were infected after 5 days in culture, at which time syncytia formation was maximal, as evaluated by the homogenous cytoplasmic extension and rearrangement of nuclei into groups, a

Table I. Term trophoblast mononuclear cell preparations used for study. Placenta no.

CK

VIM %

MHC I

1.3 (< 1) (0) 0.1 (< 1) 0.2 (0) ND 2.6 (0)

1.3 (0) (0) ND

196 79 60 460

ND

200

P6 P7 P8 t2)

98.9 ND ND 96.5 (94) 94.4 (100) 99.1 94.6 (100)

0.1 3.8 ND

150 1,840 132

P1 t') P3 (') P7 ~*~

86.3 (82) 83.5

4.5 (18) 8.0

5.1 (33) ND

31 9 64

P1 P2 P3 P4 P5

hCG it) nU/cell/24 h

Mononuclearcellsuspensionswerecharacterizedby reactionwithmonocionalantibodiesagainstepithelial(cytokeratin)and connective tissue(vimentin)markers,and MHC class l surfaceepitopes'; 104cellswereanalysedby flowcytometryor -"100cellswerecounted by fluorescencemicroscopy(numbersin brackets).The maximalreleaseof hCG after in vitro differentiationoccurredduringday 4 or 5 of culture. o~Maximal24-h hCG secretionduring in vitro cultivation(see fig. 1). ~2~Mononuclearcells from 3 separate placentaepooled beforethe immunosortingprocedure. ~'~Without immunosoningpurification. ND= not done.

324

N. N t ~ R S K O V - L A U R I T S E N E T A L .

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Fig. 1. Susceptibility of trophoblast to infection with vaccinia virus (strain Elstree). Immunosorted (A, B) or crude (C, D) trophoblast preparation PI was cultivated for 24 h before addition of virus at unit moi (B, D) (A, C : uninfected control). Twenty-four hours after infection, cultures were f'Lxedand processed for immunocytochemical staining directed against cytokeratin (A, B) or vimentin (C, D). The vimentin-stained cultures were photographed by phase-contrast microscopy in order to also reveal the unstained trophoblast. Original magnification x 120 (A, B) or x 240 (C, D).

adventitious agent present in the Vero cells could be responsible for the cytopathic effect (data not shown). Synthesis of crude trophoblast preparation P3* was 7 times less than that obtained after the immunosorting purification, but the kinetics of synthesis and the inhibition induced by addition of virus were virtually identical (fig. 2, P3 vs P3*). The replication of recombinant vaccinia virus in human trophoblast relative to a carcinoma cell line is shown in figure 3. Forty-eight hours were required for a 100-fold increase in total culture content of infectious virus, which is in contrast with the 5 lOgl0-fold increase obtained in 24 h with MS-751 cells (fig. 3A). The viral content in trophoblast cultures did not in-

crease further after the 48 h depicted (data not shown). Vaccinia vPEI6 carries the HIV e n v gene controlled by an early vaccinia virus promoter (Earl et al., 1990), and synthesis of gpl60 was demonstrated in MS-751 cells by HIV antigen-capture ELISA 6 h before progeny virus could be detected. In the trophoblast preparation, total culture content of HIV protein peaked around 30 h after addition of recombinant virus at high input moi (fig. 3B). To examine whether HIV gp120 was presented on the trophoblast surface in a state and an amount necessary for interaction with other cell types, we performed coculture experiments using the CD4-positive CEM-SS cell line. When CD4 + cells were cocultivated with wild-type

I N F E C T I O N OF P L A C E N T A L T R O P H O B L A S T B Y VACCINIA VIRUSES

10 9

10 P2

200

o

P~

0 150

P8

0 0

-

w,,,,,. =

-

P3

~ ~oo

,-

Q.

Z

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~,

325

-

iv

v

v

/

1

0 (days)

5

-t 8

Cultivation time

0

Fig. 2. In vitro production of hCG by 6 separate trophoblast preparations infected with vaccinia virus. Medium was changed daily and examined for hCG content. Note the wide variation in maximal release between different preparations and the invariable end result from

addition of virus. Solid line, uninfected control. Filled symbols, vaccinia wild type (strain Elstree); open symbols, vaccinia vPE16. Viruses were added to the cultures after 0 (circle), 1 (square), 2 (diamond), 3 (triangle) or 5 (inverse triangle) days of incubation. The input moi (see "Materials and Methods" for calculation) for wild type and vPEl6 was 1.4 and 0.03 (P2, P3, P3*), 0.03 and 0.006 (P4, PS), 0.6 and 0.7 (PS), respectively. Infection was carried out for I h (P4, PS) or 24 h (P2, P3, P3*, P8).

vaccinia-virus-infected trophoblast, the appearance after fixation and cytokeratin staining resembled that of a string of pearls (fig. 4A), with the unstained CEM-SS cells adhering to the condensed and stained trophoblast. In marked contrast, addition of CEM-SS to vPE 16-infected trophoblast elicited a pronounced, hybrid cell fusion between cytokeratin-positive and -negative cells (fig. 4B). The p H of the culture m e d i u m decreased less that 0.1 p H unit during the 6-h cocultivation period, excluding the possibility of an acid-dependent vaccinia-virus-induced cell fusion (Doms et al., 1990). DISCUSSION

Based on immunohistochemical examinations, the only placental m o n o n u c l e a r cell not

0.2

~

0

21.

1.8

Time after virus addition (h} Fig. 3. Kinetics of recombinant vaccinia virus replication. Vaccinia vPEl6 (8x 105 PFU) were added to l-cm 2 wells with l-day cultured human term trophoblast (PS, P6) (e) or subconfluent MS-751 carcinoma cells ( • ). At indicated intervals, separate wells were processed for plaque titration (a) or HIV-antigen capture ELISA (b). ELISA values represent the absorption at 492 nm of 8-fold dilutions of cells combined with medium. Wild-type vaccinia (strain Elstree) (&) served as control in the ELISA.

expressing M H C class I epitopes is confined to villous cytotrophoblast (Sasagawa et al., 1987). Thus, the i m m u n o s o r t e d cell suspension can be considered to be villous cytotrophoblast, and results obtained with such cultures may have limited bearing for other types of trophoblast. We therefore examined the in vitro interaction of vaccinia virus with human trophoblast in both crude placental cell cultures and in essentially homogeneous cultures of villous trophoblast. All trophoblast cells were susceptible to cytopathic infection with vaccinia virus, as revealed by immunostaining directed against cytoskeleton components in fixed cells. Viral infection also strongly inhibited the synthesis of hCG, although the low h o r m o n e output o f older cultures m a d e the virus impact on synthesis less easy to evaluate than the m o r p h o -

326

N. NORSKOV-LA URITSEN E T AL.

Fig. 4. Syncytia formation between purified villous trophoblast (P7) and CEM-SS cells induced by recombinant vaccinia virus expressing HIV gp160. Cocultivation was initiated 24 h after removal of virus (see "Materials and Methods"). After a further 6 h, cultureswerewashed, fixedand processedfor immunostainingdirectedagainst cytokeratin. A = wild-typevacciniavirus (strain WR); B = recombinantvacciniavirus (vPE16). Original magnification x 240.

logic cytopathic effect. The kinetics of trophoblast differentiation in vitro, as measured by the daily production of hCG, was similar to that described from other laboratories (Kliman et aL, 1987; Bax et al., 1989; Kato and Braunstein, 1990). However, we found wide"variation between individual placental cell preparations with respect to the hormone synthesis rate. We were not able to relate subsequent in vitro hormone production to any macroscopic characteristics of the placenta, nor could it solely reflect the proportion of seeded cells which attached to the culture wells, since low-producing preparations could appear as dense and viable as the cultures characterized by a high hormone output. Removal of cells during the immunosorting procedure was generally in the range of 0.3-0.5 of the total number subjected to the procedure, but the purification resulted in a 6- to 30-fold increase in hCG production after cultivation

(table I). A likely explanation is that other cell types present within the villous trophoblast may disturb the differentiation into hCG-producing tissue by direct cell contacts or secreted compounds. Vaccinia virus replicated slowly and to low titres in trophoblast, compared to replication in a continuous cell line (fig. 3). A similar difference was observed with herpes simplex virus replication in trophoblast relative to placental fibroblasts and malignant transformed trophoblast (NCrskov-Lauritsen et al., 1992). In contrast, normal term trophoblast was found to be second to none when compared to the other cell types tested with respect to production of interferon in response to synthetic RNA (Toth et al., 1990a), Sendal virus (Toth et al., 1990b) and Newcastle disease virus (Aboagye-Mathiesen, unpublished observation). Trophoblast susceptibility to virus should, however, be compared

INFECTION OF P L A C E N T A L T R O P H O B L A S T B Y VACCINIA VIRUSES

with that of other primary explant cultures not replicating in vitro. Many different viruses can infect the foetus in utero (Isada and Grossman, 1986). Foetal or congenital vaccinia was first described in 1932 (Lynch, 1932), and a total of 25 cases have been characterized (Levine et al., 1974; Hanshaw et al., 1985 ; Fenner et al., 1988). Most cases were fatal, the foetus being stillborn or dying a few days after birth. There is no convincing evidence of a teratogenic effect of foetal vaccinia; the foetus usually died, but if it survived, it recovered completely. Two women acquired vaccinia infection by contact with their vaccinated offspring; of the pregnant women vaccinated, 3 had already been vaccinated previously, but 2 of the 3 women responded to revaccination during pregnancy with primary-like reactions and fever. Most of the women were infected in the second trimester, but the stage of pregnancy ranged from the third week of gestation to near-term. After a mean delay of 8 weeks, pregnancy terminated. A sharp increase in foetal wastage as a consequence of vaccination in the second or third month of pregnancy was demonstrated after an outbreak of smallpox in Scotland 1950 (MacArthur, 1952). Other studies did not find an increased abortion rate associated with vaccination during pregnancy, and the possible adverse effect of the vaccine in the first 12 weeks of pregnancy has been subject to controversy (Levine et al., 1974; Hanshaw et aL, 1985). Levine et aL (1974) judged that cumulative evidence attested to the relative safety of vaccination after the 12th week of pregnancy, but considered the procedure mandatory for any pregnant woman travelling to a smallpox endemic area. If immunization with a new recombinant vaccinia virus was considered for approval, the possible risk of foetal vaccinia would be minimized by careful selection of vaccination subjects, which is also the case with progressive vaccinia (occurring in immunoincompetent individuals) and eczema vaccinatum (in eczematous individuals); however, transmission of virus to susceptible individuals by contact with vaccinated subjects cannot be entirely controlled. Of greater concern is postvaccinal encephalitis, an unpredictable

327

complication taking place at a rate o f 10-5-10 -4 vaccinations (Fenner et al., 1988; Fenner, 1990). A major paediatric health problem is the growing number of patients with congenital HIV infection. Recent findings suggest that the trophoblast may play a role in transplacental transmission of HIV (Lewis et al., 1990; Maury et al., 1989). Elimination of virus-infected trophoblast would pose a special problem to the immune system due to lack of expression of M H C on certain types of trophoblast, as well as other immunologic suppressive mechanisms associated with the state of pregnancy. Trophoblast has been infected with HIV in vitro (Douglas et al., 1991; Zachar et al., 1991), but permissiveness was low. The use of recombinant vaccinia virus to induce expression of HIV glycoprotein on trophoblast might eliminate this obstacle. We are presently using this as a convenient experimental system for investigation of specific immune responses against trophoblast in vitro. Acknowledgements

The work was supported in part by Danish Medical Research Council Grant no. 512-967#-2. The recombinant vaccinia virus vPEI6 was contributed to the NIAID and NIH AIDS Research and Reference Reagent Program by Dr. Patricia Earl and Dr. Bernard Moss.

Infection/!1 vitro du trophoblaste placentaire humain par un virus sauvage de la vaccine et par un recombinant exprimant la glycoprot~ine d'enveloppe du VIH

Des cultures de courte dur~e ( 6 7 jours) de cellules mononucl~6es trophoblastiques isol~s de placenta ~ terme ont ~t6 inocuh~esavec du virus vaccinal. Des actions cytopathog~nes ont 6t~ induites darts des preparations de cellules placentaires fra~ches et dans des cultures effectu~es apr~s ~ immunotriage n~gat i f . de ceUules exprimant I'~pitope de classe I du complexe majeur d'histocompatibilit~, c'est-~-dire des cultures d~riv~es exclusivement du cytotrophoblaste villeux, selon nos actuelles connaissances. In vitro, le trophoblaste assure un cycle r~plicatif complet du virus sauvage ainsi que d'un clone recombinant utilis6 COlmne vecteur du g~ne codant pour l'enveloppe du virus de l'immunod~ficience humaine

328

N. N O R S K O V - L A U R I T S E N E T A L .

(VIH) de type 1. Les r~sultats peuvent 6clairer un m~canisme impliqu6 dans les l~sions foetales caus~es, bien que rarement, par une vaccination antivariolique pendant la grossesse. Mots-cl~s: Vaccine, Foetus, Vaccination, SIDA; Transmission virale transplacentaire, Virus vaccinal recombinant.

References Bax, C.M.R., Ryder, T.A., Mobberley, M.A., Tyros, A.S., Taylor, D.L. & Bloxam, D.L. (1989), Ultrastructural changes and immunocytochemical analysis of human placental trophoblast during short time culture. Placenta, 10, 179-194. Cooney, E.L., Collier, A.C., Greenberg, P.D., Coombs, R.W., Zarling, J., Arditti, D.E., Hoffman, M.C., Hu, S.-L. & Corey, L. (1991), Safety and immunological response to a recombinant vaccinia virus expressing HIV envelope glycoprotein. Lancet, 337, 567-572. Doms, R.W., Blumenthal, R. & Moss, B. (1990), Fusion of intra- and extracellular forms of vaccinia virus with the cell membrane. J. Virol., 64, 4884-4892. Douglas, G.C. & King, B.F. (1989), Isolation of pure villous cytotrophoblast from human term placenta using immunomagnetic microspheres. J. Immunol. Methods, 119, 259-268. Douglas, G.C., Fry, G.N., Thirkill, T., Holmes, E., Hakim, H., Jennings, M. & King, B.F. (1991), Cellmediated infection of human placental trophoblast with HIV in vitro. AIDS Res. Hum. Retroviruses, 7, 735-740. Earl, P.L., Hugin, A.W. & Moss, B. (1990), Removal of cryptic poxvirus transcription termination signals from the human immunodeficiency virus type I envelope gene enhances expression and immunogenicety of a recombinant vaccinia virus. J. Virol., 64, 2448-2451. Ebbesen, P., Toth, F.D., Villadsen, J.A. & NerskovLauritsen, N. (1991), In vitro interferon and virus production at in vivo physiologic oxygen tensions. In Vivo, 5, 355-358. Fenner, F., Henderson, D.A., Arita, I., Jezek, Z. & Ladnyi, I.D. (1988), Developments in vaccination and control 1900-1966, in "Smallpox and its eradication" (same authors) (pp. 277-314). WHO, Genava. Fermer, F. (1990), Poxviruses, in "Virology" (B.N. Fields) (pp. 2113-2136). Raven Press, New York. Hanshaw, J.B., Dudgeon, J.A. & Marshall, W.C. (1985), Smallpox and vaccinia, in "Viral diseases of the fetus and newborn" (same authors) (175-181). W.B. Saunders, Philadelphia. Isada, N.B. & Grossman, J.H. (1986), Perinatal infections, in "Ostetrics" (S.G. Gabbe, J.R. Niebyl & J.L. Simpson) (pp. 979-1050). Churchill Livingstone, New York. Kato, Y. & Braunstein, G.D. (1990), Purified first and third trimester placental trophoblasts differ in in vitro hor-

mone secretion. 3". Clin. Endocrin. Metab., 70, 1187-1192. Kliman, H.J., Nestler, J.E., Sermasi, E., Sanger, J.M. & Straus, J.F. (1986), Purification, characterization and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology, 118, 1567-1582. Kliman, H.J., Feinman, M.A. & Straus, J.F. (1987), Differentiation of human cytotrophoblast into syncytiotrophoblast in culture. Trophoblast Res., 2, 407-421. Levine, M.M., Edsall, J. & Bruce-Chwatt, L.J. (1974), Live-virus vaccines in pregnancy. Risk and recommendations. Lancet, II, 34-39. Lewis, S.H., Reynolds-Kohler, C., Fox, H.E. & Nelson, J.A. (1990), HIV-1 in trophoblastic and villous Hofbauer cells, and haematological precursors in eightweek foetuses. Lancet, I, 565-568. Lynch, F.W. (1932), Dermatologie conditions of the fetus. Arch. Dermatol. SyphiloL, 26, 997-1019. MacArthur, P. (1952), Congenital vaccinia and vaccinia gravidarum. Lancet, II, 1104-1106. Maury, W., Potts, B.J. & Rabson, A.B. (1989), HIV-I infection of first-trimester and term human placental tissue: a possible mode of maternal-fetal transmission. J. infect. Dis., 160, 583-588. Moss, B. (1991), Vaccinia virus: a tool for research and vaccine development. Science, 252, 1662-1667. Nielsen, C.M., Bygbjerg, I.C. & Vestergaard, B.F. (1987), Detection of HIV antigens in eluates from whole blood collected on filter paper. Lancet, I, 566-7. Nara, P.L., Hatch, W.C., Dunlop, N.M., Robey, W.G., Arthur, L.O., Gonda, M.A. & Fischinger, P.J. (1987), Simple, rapid, quantitative, syncytiumforming microassay for the detection of human immunodeficiency virus neutralizing antibody. AIDS Res. Hum. Retroviruses, 3,283-302. Nerskov-Lauritsen, N., Aboagye-Mathiesen, G., Juhl, C.B., Petersen, P.M., Zachar, V. & Ebbesen, P. (1992), Herpes simplex virus infection of cultured human term trophoblast. J. Med. ViroL, 36, 162-166. Sasagawa, M., Yamazaki, T., Endo, M., Kanarawa, K. & Takeuchi, S. (1987), Immunohistochemical localiration of HLA antigens and placental proteins (ahCG, [3hCG, CTP, hPL, and SPI) in villous and extravilIous trophoblast in normal human pregnancy : a distinctive pathway of differentiation of extravillous trophoblast. Placenta, 8, 515-528. Thiede, H.A. (1960), Studies on the human trophoblast in tissue culture. - - 1. Cultural methods and histochemical staining. Amer. J. Obst. Gynecol., 79, 636-647. Toth, F.D., Nerskov-Lauritsen, N., Juhl, C.B. & Ebbesen, P. (1990a), Interferon production by cultured human trophoblast induced with double-stranded polyribonucleotide. J. Reprod. Immun., 17, 217-227. Toth, F.D., Nerskov-Lauritsen, N., Juhl, C.B., AboagyeMathiesen, G. & Ebbesen, P. (1990b), Interferon production by cultured human trophoblasts and choriocarcinoma cell lines induced by Sendal virus. J. gen. ViroL, 71, 3067-3069. Zachar, V., Nerskov-Lauritsen, N., Juhl, C., Spire, B., Chermann, J.C. & Ebbesen, P. (1991), Susceptibility of cultured human trophoblasts to infection with human immunodeficiency virus type 1. J. gen. ViroL, 72, 1253-1260.

In vitro infection of human placental trophoblast by wild-type vaccinia virus and recombinant virus expressing HIV envelope glycoprotein.

Short-time (< or = 7 days) cultures of trophoblast mononuclear cells isolated from term placentae were challenged with vaccinia virus. Cytopathic effe...
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