JOURNAL OF VIROLOGY, Oct. 2011, p. 10582–10597
`0022-538X/11/$12.00 doi:10.1128/JVI.00671-11
`Copyright © 2011, American Society for Microbiology. All Rights Reserved.
`
`Vol. 85, No. 20
`
`Anti-Severe Acute Respiratory Syndrome Coronavirus Spike
`Antibodies Trigger Infection of Human Immune Cells
`via a pH- and Cysteine Protease-Independent
`Fc␥R Pathway䌤
`Martial Jaume,1* Ming S. Yip,1 Chung Y. Cheung,2 Hiu L. Leung,1 Ping H. Li,1 Francois Kien,1
`Isabelle Dutry,1,2 Benoıˆt Callendret,3,4‡ Nicolas Escriou,3,4 Ralf Altmeyer,1† Beatrice Nal,1,5
`Marc Dae¨ron,6,7 Roberto Bruzzone,1,8 and J. S. Malik Peiris1,2
`HKU-Pasteur Research Centre, 8 Sassoon Road, Hong Kong SAR, People’s Republic of China1; Department of Microbiology,
`The University of Hong Kong, 21 Sassoon Road, Hong Kong SAR, People’s Republic of China2; Institut Pasteur, Unite´ de
`Ge´ne´tique Mole´culaire des Virus a` ARN, De´partement de Virologie, 25 Rue du Docteur Roux, F-75015 Paris,
`France3; CNRS, URA30I5, F-75015 Paris, France4; Department of Anatomy, The University of Hong Kong,
`21 Sassoon Road, Hong Kong SAR, People’s Republic of China5; Institut Pasteur, De´partement d’lmmunologie,
`Unite´ d’Allergologie Mole´culaire et Cellulaire, 25 Rue du Docteur Roux, F-75015 Paris, France6;
`INSERM, Unité 760, 25 Rue du Docteur Roux, F-75015 Paris, France7; and Institut Pasteur,
`Department of Cell Biology and Infection, 25 Rue du Docteur Roux, F-75015 Paris, France8
`
`Received 4 April 2011/Accepted 5 July 2011
`
`Public health measures successfully contained outbreaks of the severe acute respiratory syndrome coronavirus
`(SARS-CoV) infection. However, the precursor of the SARS-CoV remains in its natural bat reservoir, and reemer-
`gence of a human-adapted SARS-like coronavirus remains a plausible public health concern. Vaccination is a major
`strategy for containing resurgence of SARS in humans, and a number of vaccine candidates have been tested in
`experimental animal models. We previously reported that antibody elicited by a SARS-CoV vaccine candidate based
`on recombinant full-length Spike-protein trimers potentiated infection of human B cell lines despite eliciting in vivo
`a neutralizing and protective immune response in rodents. These observations prompted us to investigate the
`mechanisms underlying antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro. We demonstrate
`here that anti-Spike immune serum, while inhibiting viral entry in a permissive cell line, potentiated infection of
`immune cells by SARS-CoV Spike-pseudotyped lentiviral particles, as well as replication-competent SARS corona-
`virus. Antibody-mediated infection was dependent on Fc␥ receptor II but did not use the endosomal/lysosomal
`pathway utilized by angiotensin I converting enzyme 2 (ACE2), the accepted receptor for SARS-CoV. This suggests
`that ADE of SARS-CoV utilizes a novel cell entry mechanism into immune cells. Different SARS vaccine candidates
`elicit sera that differ in their capacity to induce ADE in immune cells despite their comparable potency to neutralize
`infection in ACE2-bearing cells. Our results suggest a novel mechanism by which SARS-CoV can enter target cells
`and illustrate the potential pitfalls associated with immunization against it. These findings should prompt further
`investigations into SARS pathogenesis.
`
`Although public health measures have successfully con-
`tained human outbreaks of the severe acute respiratory syn-
`drome coronavirus (SARS-CoV) infection, a disease with a
`case-fatality ratio of 10% (13, 32), the precursor SARS-CoV-
`like virus remains endemic in its natural bat reservoir (34), and
`future reemergence of a SARS-like disease remains a credible
`public health threat. Therefore, efforts have continued to de-
`velop safe vaccine strategies against SARS-CoV.
`Neutralizing antibodies are elicited in patients recovering
`from SARS and studies on experimental animal models have
`
`* Corresponding author. Mailing address: HKU-Pasteur Research
`Centre, Dexter H. C. Man Building, 8 Sassoon Road, Pokfulam, Hong
`Kong SAR, China. Phone: (852) 2816 8423. Fax: (852) 2872 5782.
`E-mail: breizh@hku.hk.
`‡ Present address: Center for Vaccines and Immunity, the Research
`Institute at Nationwide Children’s Hospital, 700 Childrens Dr., Co-
`lumbus, OH 43205.
`† Present address: Institut Pasteur of Shanghai, Chinese Academy of
`Sciences, Shanghai, People’s Republic of China.
`䌤 Published ahead of print on 20 July 2011.
`
`shown that antibodies can prevent infection by SARS-CoV (6).
`Among the four major structural SARS-CoV proteins, the
`Spike envelope glycoprotein (S) has been identified as the
`most important antigen inducing neutralizing and protective
`antibodies (1, 3, 72). Furthermore, it has been demonstrated
`that binding of Spike to its receptor angiotensin I converting
`enzyme 2 (ACE2) is a key event in the entry of SARS-CoV into
`cells (40).
`Several vaccine strategies aiming at preventing infection by
`SARS-CoV have therefore targeted the Spike glycoprotein
`(14, 15). Such a strategy, however, poses concern since previ-
`ous attempts at vaccination against coronaviruses have re-
`sulted in markedly different outcomes. Immune protection
`against animal coronaviruses, such as mouse hepatitis and
`transmissible gastroenteritis viruses (11, 46), can be induced by
`immunization with Spike glycoprotein. On the other hand, this
`vaccine approach led to a worsened disease in the feline coro-
`navirus (FCoV) infection due to the induction of infection-
`enhancing antibodies (8, 25, 44, 65). Immune-mediated infec-
`tions and,
`in particular, antibody-dependent enhancement
`
`10582
`
`Moderna 2030
`BioNTech-Pfizer v. Moderna
`IPR2023-01359
`
`

`

`VOL. 85, 2011
`
`IMMUNE-MEDIATED ENHANCEMENT OF SARS-CoV INFECTION
`
`10583
`
`(ADE) have long been known to be exploited by a variety of
`viruses, such as dengue virus, HIV, and FCoV, as an alterna-
`tive way to infect host cells (59, 60). In addition to interaction
`between viral protein and host receptors, these viruses can
`enter into cells through binding of virus/antibody immune
`complexes to Fc receptors (FcR) or complement receptors or,
`alternatively, by inducing a conformational change in envelope
`glycoproteins that are required for virus-cell membrane fusion
`(59, 60). Thus, immunization of cats with recombinant vaccinia
`virus preparations expressing the FCoV Spike protein resulted
`in the induction of Spike-specific antibodies responsible for an
`enhanced susceptibility to challenge infection (65). The en-
`hanced infection of macrophages following antibody-mediated
`entry of the FCoV is responsible for the occurrence of the
`severe disease feline infectious peritonitis (8, 25, 44).
`We previously reported that a SARS-CoV vaccine candidate
`based on recombinant, full-length SARS-CoV Spike-protein
`trimers triggered infection of human B cell lines despite elic-
`iting in vivo a neutralizing and protective immune response in
`rodents (30). These observations prompted us to further in-
`vestigate the molecular and cellular mechanisms underlying
`ADE of SARS-CoV infection in vitro.
`By monitoring the susceptibility of immune cell lines having
`different patterns of Fc␥ receptor (Fc␥R) expression, we have
`demonstrated the predominant role of human Fc␥RII (CD32)
`in mediating ADE of SARS-CoV. Furthermore, we have pro-
`vided evidence that, in contrast to the ACE2-mediated infec-
`tion, ADE pathways are independent of endosomal or lyso-
`somal pH and are minimally affected by the activities of
`cysteine proteases. Finally, we have found marked differences
`between different SARS vaccines in their capacity to elicit a
`humoral immune response that neutralizes or facilitates infec-
`tion by SARS-CoV in vitro, an observation that should prompt
`further studies to develop safe immunization protocols.
`
`MATERIALS AND METHODS
`
`Cell lines. The following cell lines were used in the present study: VeroE6
`(African green monkey kidney epithelial cells), K-562 (human chronic myelog-
`enous leukemia cells), U-937 (human histiocytic lymphoma cells), THP-1 (hu-
`man acute monocytic leukemia cells), SUP-T1 (human lymphoblastic leukemia/T
`lymphoblast), MOLT-3 (human acute lymphoblastic leukemia/T lymphoblast),
`MT4/R5 (human T cell lymphoblast expressing CCR5), Raji (Burkitt’s lympho-
`ma/B lymphoblast), Daudi (Burkitt’s lymphoma/B lymphoblast), parental ST486
`(Burkitt’s lymphoma/B lymphoblast lacking expression of Fc␥R) and Fc␥R/
`ST486, 721.221 (Epstein-Barr virus-transfected human B cells), and P388D1 and
`J774A.1 (murine macrophage-like lymphoblasts). VeroE6 cells were cultured in
`Dulbecco modified Eagle medium (DMEM) supplemented with 10% of heat-
`inactivated fetal bovine serum (FBS), and hematopoietic cells were cultured in
`RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% nones-
`sential amino acids, 4 mM L-glutamine, 1 mM sodium pyruvate, and 20 ␮M
`␤2-mercaptoethanol (all from Invitrogen). All cells were maintained in a humid-
`ified atmosphere at 37°C with a 5% CO2 supply.
`Immunization with recombinant Spike proteins or inactivated SARS-CoV.
`Recombinant SARS-CoV Spike proteins were produced as described elsewhere
`(4, 30). Six to eight-week-old BALB/c mice (n ⫽ 4 to 5 per group) were immu-
`nized intraperitoneally with one of the following antigens: 2 ␮g of recombinant
`codon-optimized SARS-CoV Spike full-length (S, amino acids [aa] 1 to 1255), 2
`␮g of recombinant codon-optimized SARS-CoV Spike ectodomain (S.ECD, aa
`1 to 1184), 2 ␮g of recombinant codon-optimized SARS-CoV Spike subunit 1
`(S1, aa 1 to 757), or 10 ␮g of recombinant, soluble SARS-CoV Spike truncated
`early after the transmembrane domain (Ssol, aa 1 to 1193) (see Fig. 8A for
`details). All recombinant proteins were FLAG tagged according to standard
`molecular biology techniques. An additional group, immunized with 2 ␮g (Spike-
`equivalent) of gamma-irradiated SARS-CoV virion (60Co based, 50 KGy; whole
`
`killed virus [WKV]), was also included, as well as a control group injected with
`saline solution. Mice received two immunizations in the presence of 1 mg of
`Alum at 3-week intervals, and blood samples were collected by bleeding the
`saphenous vein on days ⫺1, 27, and 55 postimmunization in accordance with
`local guidelines for animal handling. For all groups (n ⫽ 4 to 5), serum samples
`were collected at day 55 postimmunization, and an equal volume from each
`animal was pooled, heat inactivated for 30 min at 56°C, and stored at ⫺20°C for
`subsequent use.
`Production and use of lentiviral pseudotyped particles. The pseudotyped viral
`particles expressing a luciferase reporter gene were essentially produced as
`described elsewhere (42). Briefly, SARS-CoV Spike-pseudotyped lentiviral par-
`ticles (SARS-CoVpp), vesicular stomatitis virus glycoprotein (VSV-G)-pseu-
`dotyped lentiviral particles (VSVpp), or lentiviral particles lacking expression of
`any viral envelope protein (⌬env.pp) were obtained by transfection of HEK293T
`cells with an HIV-1 provirus construction (pNL4.3.LucR⫺E⫺pro⫺) and a plas-
`mid encoding the viral envelope protein of interest, i.e., SARS-CoV Spike,
`VSV-G, or empty vector (pcDNA3.1; Invitrogen), respectively. After a purifica-
`tion step on a 20% sucrose cushion, the concentrated viral particles were titrated
`by enzyme-linked immunosorbent assay (ELISA) for lentivirus-associated HIV-1
`p24 protein according to the manufacturer’s instruction (Cell Biolabs, Inc.), and
`the viral stocks were stored at ⫺80°C until use.
`For neutralization and ADE assays, 100 ␮l of serial, 2-fold dilutions of heat-
`inactivated mouse serum were incubated for 1 h at 37°C with 100 ␮l of pseu-
`dotyped viral particles. The same inoculum was then used to infect in parallel
`both VeroE6 (SARS-CoVpp neutralization test) and hematopoietic (ADE as-
`say) cells. Briefly, 104 VeroE6 cells were seeded 24 h before the infection in a
`96-well Opti-plate (Perkin-Elmer). On the day of infection, the cells were washed
`twice, and 25 ␮l of inoculum was added to an equivalent volume of supplemented
`DMEM. Hematopoietic cells were washed and diluted to 2 ⫻ 106 cells/ml in
`supplemented RPMI 1640 medium. Portions (25 ␮l) of inoculum were deposited
`in a 96-well Opti-plate, and an equal volume of the cell suspension was added
`immediately thereafter. After 1 h of incubation at 37°C, the cells were washed
`and incubated for additional 65 to 75 h in 100 ␮l of supplemented culture
`medium. The cells were quenched by adding 100 ␮l of BrightGlow luciferase
`substrate (Promega) directly to each well, and the luciferase activity was mea-
`sured with a MicroBeta Jet counter (Perkin-Elmer). Background values, moni-
`tored from uninfected cells and cells infected with lentiviral particles lacking
`expression of any viral envelope protein (⌬env.pp) were consistently below 200
`relative luminescence units (see Fig. 1 for reference).
`Infection with SARS-CoV. Serial, 2-fold dilutions of heat-inactivated mouse
`sera were incubated for 1 h at 37°C with an equal volume of live SARS-CoV
`(strain HKU-39849) under appropriate containment in a BSL3 laboratory (De-
`partment of Microbiology, The University of Hong Kong). Both VeroE6 and
`Raji cells were infected at a multiplicity of infection (MOI) of 1 for 60 min at
`37°C, washed, and then incubated in supplemented culture medium containing
`appropriate dilutions of mouse serum. At the end of the experiment, the cells
`were either fixed in 4% paraformaldehyde (dissolved in phosphate-buffered
`saline) for immunofluorescence microscopy or resuspended in lysis buffer (RLT
`buffer, RNeasy RNA minikit; Qiagen) for endpoint and real-time quantitative
`PCR and stored appropriately until use. In addition, samples of the cell culture
`supernatants (100 ␮l) harvested at different time points were mixed with 350 ␮l
`of RLT buffer and stored at ⫺80°C until use.
`Immunofluorescence microscopy. To assess SARS-CoV infection, both
`VeroE6 and Raji cells were incubated for 45 min with either a mouse monoclonal
`antibody specific for the viral nucleoprotein (N) (7) or rabbit polyclonal anti-
`bodies recognizing the viral membrane (M) protein (ProSci), which were re-
`vealed by secondary TRITC (tetramethyl rhodamine isothiocyanate)-conjugated
`goat anti-mouse (Zymed Laboratories) and fluorescein isothiocyanate (FITC)-
`conjugated goat anti-rabbit antibody (Jackson Immunoresearch), respectively.
`Slides were assembled with DAPI (4⬘,6⬘-diamidino-2-phenylindole)-containing
`mounting reagent (Southern Biotech) and analyzed with an AxioObserver Z1
`microscope (Zeiss). Pictures from 10 to 30 randomly selected fields were ac-
`quired with an Axiocam MRm camera and processed with MetaMorph software
`(Molecular Devices).
`Endpoint and real-time quantitative reverse transcriptase PCR (RT-PCR) for
`viral gene expression. Total RNAs were extracted with an RNeasy RNA minikit
`(Qiagen), with DNase digestion, according to the manufacturer’s instructions.
`Extracted RNAs were stored at ⫺80°C until use. Superscript III reverse trans-
`criptase (Invitrogen) and random hexamer primers (Invitrogen) or gene-specific
`oligonucleotides were used to convert RNAs to cDNAs. The amounts of viral
`and host RNA were measured either by conventional endpoint PCR or by
`real-time quantitative PCR using TaqMan MGB probe-based technology on a
`LightCycler 480-II instrument (Roche). The primers and conditions for detection
`
`

`

`10584
`10584
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`

`

`VOL. 85, 2011
`
`IMMUNE-MEDIATED ENHANCEMENT OF SARS-CoV INFECTION
`
`10585
`
`of the GAPDH and SARS-CoV genomic and subgenomic species (18), as well as
`the SARS-CoV ORF1b and nucleoprotein genes (7), have been described pre-
`viously. Positive and negative controls were included in each run and, when
`appropriate, the levels of SARS-CoV gene expression were normalized to those
`of the 18S rRNA gene, which were determined using 600 nM concentrations of
`both forward (5⬘-CggAggTTCgAAgACgATCA-3⬘) and reverse (5⬘-ggCgggTCA
`TgggAATAAC-3⬘) primers and a 100 nM concentration of the probe (5⬘HEX-
`ATACCgTCgTAgTTCCgACCA-BHQ3⬘).
`Human Fc␥ receptor profiling by conventional endpoint PCR. Total RNAs
`were extracted with an RNeasy RNA minikit (Qiagen), with DNase digestion,
`according to the manufacturer’s instructions, and stored at ⫺80°C until use.
`Superscript III reverse transcriptase (Invitrogen) and random hexamer primers
`(Invitrogen) were used to convert RNAs to cDNAs. Additional samples consist-
`ing of negative RT controls (RT⫺) were prepared by omitting Superscript III
`during reverse transcription. The amounts of RNA/cDNA encoding FcεR1␥
`chain, Fc␥RIA (CD64a), Fc␥RIIA (CD32a), Fc␥RIIB (CD32b), Fc␥RIIIA
`(CD16a), ACE2, and GAPDH were measured by conventional endpoint PCR
`using the primer pairs described in Table 2. Negative and positive controls (i.e.,
`DNA plasmid containing the full coding sequence of the gene of interest) were
`included in each run. The amplification procedure consisted of an initial dena-
`turation step at 94°C for 5 min, followed by 30 cycles of three steps of 45 s each,
`including denaturation at 94°C, primer annealing at 60°C, and primer extension
`at 72°C. The protocol also included a final extension step at 72°C for 7 min.
`Amplicons were eventually visualized by ethidium bromide staining after elec-
`trophoresis in 2% agarose gel, and negative images were taken.
`Flow cytometry. Cells were harvested at 4°C in phosphate-buffered saline
`containing 1% FBS, 2% normal human serum, 3% normal goat serum, 2 mM
`EDTA, and 0.1% sodium azide and then incubated for 30 to 45 min at 4°C with
`1 ␮g of either isotype-matched control or monoclonal antibody/ml for the indi-
`cated human Fc␥R. The following mouse monoclonal antibodies were used: 3G8
`anti-hCD16, 3D3, FLI8.26 anti-hCD32, or 10.1 anti-hCD64 (all from BD Phar-
`mingen) and MOPC-21 (IgG1, ␬) or MPC-11 (IgG2b, ␬) isotype controls (both
`from BioLegend). The cells were then washed, and the primary antibody binding
`was revealed by staining at 4°C for 30 min with FITC-conjugated goat anti-mouse
`antibodies (Jackson Immunoresearch). Finally, washed cells were further incu-
`bated with an optimal concentration of the fixable viability dye eFluor 660
`(FVD660; eBioscience). The data were collected from ⱖ30,000 singlet living cells
`on a LSRII flow cytometer (BD Biosciences), and postacquisition analyses were
`performed using the FlowJo software (TreeStar).
`Blockade of human Fc␥ receptor in vitro. Cells were washed with cold, sup-
`plemented RPMI 1640 medium and maintained on ice throughout the anti-Fc␥R
`blocking step only. Briefly, 2 ⫻ 105 cells were treated for 45 to 60 min in 200 ␮l
`of supplemented culture medium containing 5 ␮g of either Fc␥R-specific mouse
`monoclonal antibody (3G8, anti-hCD16; FLI8.26, anti-hCD32; and 10.1, anti-
`hCD64 [BD Pharmingen]) or isotype-matched controls (clone MOPC-21 and
`MPC-11 [BioLegend])/ml. An equal volume of inoculum was directly added to
`the tubes, and the samples were incubated at 37°C for 60 min. Cells were washed
`twice and resuspended in supplemented culture medium (5 ⫻ 105 cells/ml), and
`100 ␮l (triplicate) was transferred to a 96-well Opti-plate (Perkin-Elmer). The
`plates were then incubated at 37°C for 65 to 75 h, and the luciferase activity was
`measured as described above.
`Lentiviral transfer vector construction. We constructed the lentiviral plasmids
`encoding human FcεR␥-chain and/or human Fc␥R by substituting enhanced
`green fluorescent protein (eGFP) and/or hygromycin resistance gene from the
`bicistronic vector pCHMWS-eGFP_IRES_hygromycin (kindly provided by Rik
`Gijsbers and Zeger Debyser [Katholieke Universiteit Leuven, Leuven, Bel-
`
`gium]). The FcεR␥-chain coding sequence (GenBank accession no. M33195) was
`obtained by PCR amplification from the huFcR␥/pBJ1 neo plasmid (kindly
`provided by Jean-Pierre Kinet [Harvard Medical School, Boston, MA]) (38). The
`unique restriction sites BamHI and XhoI were added to the 5⬘ and 3⬘ ends,
`respectively. After amplification, the human FcεR␥-chain PCR product was
`digested with BamHI and XhoI and inserted into the transfer plasmid to yield
`pCHMWS-huFcεR␥-chain_IRES_hygromycin. The latter construction was used
`to generate pCHMWS-huFcεR␥-chain_IRES_huFc␥RIA and pCHMWS-
`huFcεR␥-chain_IRES_huFc␥RIIIA.
`The human Fc␥RIA (hCD64a) coding sequence (GenBank accession no.
`NM_000566) was obtained by PCR amplification from the huFc␥RIA/pcDNA-1
`plasmid (kindly provided by Clark L. Anderson, Ohio State University, Colum-
`bus, OH) (41), with BamHI and SpeI linkers at the 5⬘ and 3⬘ ends, respectively.
`After amplification, the human Fc␥RIA PCR product (1,151 bp) was digested
`with BamHI and SpeI and inserted into the transfer plasmid digested by BclI and
`SpeI to yield pCHMWS-huFcεR␥-chain_IRES_huFc␥RIA. A human Fc␥RIIIA
`(hCD16a) fragment (1,417 bp) was removed from the LL649 plasmid (kindly
`provided by Lewis L. Lanier, University of California, San Francisco, CA) (33)
`using BamHI and EcoRI and inserted into the pIRES plasmid (Clontech) using
`the same restriction enzymes. A cDNA fragment (183 bp) was PCR amplified
`from human peripheral blood mononuclear cells using the forward (5⬘-TgACgg
`ATCCAggAAATTggTgggTgACAg-3⬘) and reverse (5⬘-gTCAggATCCTAgCAg
`AgCAgTTgggAggA-3⬘) primers, both flanked by an BamHI site, and then inserted
`into LL649/pIRES plasmid at the BamHI site. The full human Fc␥RIIIA coding
`sequence (GenBank accession no. NM_000569) was generated by excision of the
`undesirable fragment (225 bp) with EcoRV and direct religation, yielding the
`huFc␥RIIIA/pIRES shuttle vector. The human Fc␥RIIIA fragment was subse-
`quently excised using BamHI and NcoI⫹Klenow blunting and cloned into the
`lentiviral vector resulting in pCHMWS-huFcεR␥-chain_IRES_huFc␥RIIIA. Coding
`sequences for human Fc␥RIIA (hCD32a) isoforms (huFc␥RIIA.R131 and
`huFc␥RIIA.H131 GenBank accession no. NM_021642) and human Fc␥RIIB1
`(hCD32b; GenBank accession no. AF543826) flanked by BglII and SalI sites were
`commercially synthesized (GeneArt, Regensburg, Germany). The synthetic se-
`quences were digested by BglII and SalI and inserted into the original transfer
`plasmid to yield pCHMWS-huFc␥RIIA.R131_IRES_hygromycin, pCHMWS-
`huFc␥RIIA.H131_IRES_hygromycin,
`pCHMWS-huFc␥RIIB1_IRES_
`and
`hygromycin. To verify that PCR amplification and cloning procedures had not
`introduced random mutations, all constructs were sequenced by the Genome
`Research Centre (The University of Hong Kong).
`Generation of Fc␥R-expressing cell lines using lentiviral particle-based gene
`transduction. Pseudotyped viral particles were essentially produced as described
`elsewhere (42). Briefly, VSV-G-pseudotyped lentiviral particles were obtained by
`transfecting HEK293T cells with a packaging plasmid (pCMV⌬R8.91), a plasmid
`encoding the envelope of VSV (pCI-VSVg), and a pCHMWS-derived transfer
`plasmid (described above) coding for a human Fc receptor as specified. Stable
`cell lines were generated by the transduction of monoclonal ST486 cells with the
`VSV-G-pseudotyped lentiviral particles. Briefly, target cells were washed and
`diluted to 3 ⫻ 106 cells/ml in supplemented RPMI 1640 medium. One milliliter
`of crude supernatant from pseudoparticle-producing 293T cells was deposited in
`a 24-well plate, and an equal volume of the cell suspension was added immedi-
`ately thereafter. After 3 to 8 h of incubation at 37°C, the cells were washed and
`incubated for additional 40 to 48 h in 2 ml of supplemented culture medium. At
`2 days postinfection, the cell surface expression of the human Fc␥R was moni-
`tored by flow cytometry, and the cells were subsequently cultured in selective
`medium containing 250 ␮g of hygromycin (Invitrogen)/ml when appropriate.
`Finally, several monoclonal cell lines for each construct were isolated by Pois-
`
`FIG. 1. Susceptibility of hematopoietic cell lines to infection by SARS-CoV Spike pseudoparticles (SARS-CoVpp). (A) SARS-CoVpp were incu-
`bated in the presence or absence of different dilutions (1/1,000, 1/2,000, and 1/4,000) of either control (solid bars) or anti-Spike (hatched bars) serum for
`1 h prior to addition to the cells. At 3 days postinfection, luciferase substrate reagent was added to wells, and the luminescence was measured. The data
`were normalized to control conditions, viz., cells incubated with SARS-CoVpp in the absence of any serum (taken as unity), and are expressed as the fold
`changes in luminescence. Because the results for SARS-CoVpp with or without control serum were virtually identical with all cell lines, for the sake of
`clarity only one dilution (1/4,000) is shown. The results are the means ⫾ the SD of six measurements from two independent experiments. Statistical
`significance was assessed by comparing the appropriate dilutions of control and anti-Spike serum (‡, P ⬍ 0.05; *, P ⬍ 0.001 [unpaired Student t test]).
`(B to D) HIV Gag-normalized lentiviral particles (0.1 ng of p24 protein/␮l) pseudotyped with the envelope glycoprotein of SARS-CoV Spike
`(SARS-CoVpp [B]) or vesicular stomatitis virus (VSVpp [C]) or lacking any viral envelope protein (⌬env.pp [D]) were incubated in the presence or
`absence of a 1/1,000 dilution of either control (solid gray bars) or anti-Spike (hatched bars) serum for 1 h prior to addition to the cells. At 3 days
`postinfection, luciferase substrate reagent was added, and the luminescence was measured. The results are the means ⫾ the SD of nine measurements
`from three independent experiments. When not visible, the SD values were contained within the size of the symbols. Anti-Spike serum either significantly
`decreased (VeroE6) or increased (THP-1, Raji, and Daudi) entry of SARS-CoVpp. *, P ⬍ 0.001 (unpaired Student t test).
`
`

`

`10586
`
`JAUME ET AL.
`
`J. VIROL.
`
`son’s limiting dilution procedure, and expression of the transgene was confirmed
`by RT-PCR (data not shown) and flow cytometry (see Fig. 6A).
`Lysosomotropic agent and protease inhibitor treatments. Cells were preincu-
`bated with the indicated amounts of drugs, either ammonium chloride (NH4Cl)
`or E-64d/cathepsin L inhibitor (Cat L Inh; Calbiochem), for 1 h or 3 h prior to
`infection, respectively. SARS-CoVpp, pretreated with immune or control serum,
`were mixed with the same concentrations of drugs in tubes and added to the cells.
`After 5 h (E-64d or Cat L Inh) or 7 h (NH4Cl), the cells were washed and further
`incubated with fresh medium without any drug. Cells were assayed for luciferase
`activity 60 to 65 h after infection, as described above.
`Statistical analysis. Results are shown as means ⫾ the standard deviations
`(SD) of the indicate number of observations. The statistical difference between
`groups was determined by using an unpaired Student t test with a 0.05 signifi-
`cance level.
`
`RESULTS
`
`Anti-Spike immune serum promotes the infection of human
`hematopoietic cells by SARS-CoV. SARS-CoV-induced pa-
`thology is not confined to the respiratory tract but also involves
`other tissues and organs, most importantly cells of the gastro-
`intestinal tract and the immune system (19, 39, 74). Although
`several reports have shown that SARS-CoV can infect hema-
`topoietic cells, it is not known how the virus gets a foothold
`into these immune cells that do not express the specific SARS-
`CoV receptor ACE2 (20, 21).
`In order to investigate the possibility of antibody-mediated
`infection of immune cells during SARS pathogenesis, we have
`taken advantage of SARS-CoV Spike-pseudotyped particles
`(SARS-CoVpp) to compare the effect of anti-Spike immune
`serum on the prototypic permissive VeroE6 cells and a panel
`of immune cell lines (Fig. 1A). These recombinant viruses
`encoding a reporter gene and bearing the SARS-CoV Spike
`protein at the virion surface have been shown to faithfully
`mimic the SARS-CoV entry process (57, 71). As expected,
`SARS-CoVpp efficiently infected VeroE6 cells, whereas the
`luminescence signal detected in any of the immune cell types
`never exceeded values measured in the absence of SARS-
`CoVpp (Fig. 1B). To explore the occurrence of antibody-me-
`diated infection, we preincubated SARS-CoVpp with either
`mouse anti-Spike immune-serum (Fig. 1A and B, hatched
`bars) or control serum (Fig. 1A and B, solid gray bars) prior to
`infection and then compared the resulting luminescence signal
`intensities.
`The outcome of infection with SARS-CoVpp in the pres-
`ence of anti-Spike immune-serum depended on the target cell
`type. Although heat-inactivated serum inhibited SARS-CoVpp
`entry into the permissive VeroE6 cell line in a dose-dependent
`fashion, as demonstrated by a dramatic drop in the intensity of
`luminescence (Fig. 1A and B, hatched bars), it facilitated in-
`fection of the human monocytic cell line THP-1 and of the B
`cell lines Daudi and Raji. In contrast, no infection of these cell
`lines was noticed when SARS-CoVpp were preincubated with
`control serum (Fig. 1A and B, solid bars), and similar back-
`ground levels of luminescence were detected in the presence of
`immune serum only (data not shown). Of note, infection of the
`THP-1, Raji, and Daudi cell lines by recombinant viral parti-
`cles pseudotyped with the glycoprotein of the vesicular stoma-
`titis virus (VSVpp; Fig. 1C) or no viral envelope protein
`(⌬env.pp; Fig. 1D) was never affected by the presence of anti-
`SARS-CoV Spike immune serum. These experiments indicate
`that anti-Spike antibodies
`facilitate infection of SARS-
`
`CoVpp—but not VSVpp or ⌬env.pp—into distinct immune
`cell types.
`Altered tropism of replication-competent SARS-CoV toward
`human immune cells in the presence of anti-Spike immune
`serum. Because Raji cells displayed the greatest susceptibility
`to antibody-mediated infection of SARS-CoVpp (Fig. 1A and
`B), we used this B-cell-derived human cell line to investigate
`whether a change of tropism could also be observed during
`infection with replication-competent SARS-CoV.
`As reported previously (30), infection of permissive VeroE6
`cells remained unchanged in the presence of control serum,
`whereas anti-Spike immune serum fully abrogated it (Fig. 2A).
`In contrast, when Raji cells were infected in the presence of
`anti-Spike immune-serum, detection of intracellular viral pro-
`teins (viz., membrane and nucleocapsid) was markedly differ-
`ent from those infected in the presence of control serum (Fig.
`2B). Such enhanced infection was also demonstrated at the
`molecular level, since the viral gene detection by conventional
`RT-PCR (i.e., endpoint PCR; Fig. 3A) fully paralleled the
`real-time quantitative PCR measurements (Fig. 3B). Com-
`pared to inoculum containing control serum, there was signif-
`icantly enhanced detection of subgenomic viral RNA (sgRNA)
`and nucleocapsid genes observed in Raji cells infected in the
`presence of anti-Spike immune serum (Fig. 3B, P ⬍ 0.001).
`Although we never detected SARS-CoV proteins in cells chal-
`lenged in the presence of control serum, trace amounts of PCR
`products related to SARS-CoV ORF1b (data not shown), nu-
`cleocapsid genes and other viral genomic and subgenomic
`RNA were detectable (Fig. 3). These background levels were
`likely the result of nonspecific binding/uptake of SARS-CoV.
`Taken together, these experiments indicate that anti-Spike se-
`rum can trigger infection of immune cells by live SARS-CoV,
`similarly to that observed with the pseudotyped viral particles.
`Antibody-mediated enhancement of SARS-CoV infection in
`Raji cells leads to abortive infection. To assess the capability of
`SARS-CoV to productively replicate into ADE-infected Raji
`cells, we monitored by PCR the cellular viral load, as well as
`the release of SARS-CoV progeny into cell culture superna-
`tant. As previously seen at 15 hours postinfection (hpi) (Fig.
`3A), all samples were determined to be positive by endpoint
`PCR for the detection of SARS-CoV genomic RNA. At most
`of the time points,
`low but noticeably higher amounts of
`SARS-CoV sgRNA were visible in cells infected in the pres-
`ence of anti-Spike serum (not shown). Such results are in
`accordance with the quantitative measurements of either pos-
`itive (i.e., genomic and mRNA) or negative (i.e., subgenomic
`replicative intermediates) RNA strands, demonstrating an in-
`creased viral load at 6 hpi, followed by a continuing decrease of
`both viral RNA species with time so that no more difference
`between groups was noted later on (Fig.