RESEARCH Open Access Antibody dependent infection of human macrophages by severe acute respiratory syndrome coronavirus Ming Shum Yip 1 Nancy Hiu Lan Leung 1 Chung Yan Cheung 2 Ping Hung Li 1 Horace Hok Yeung Lee 1 Marc Da ron 3 4 Joseph Sriyal Malik Peiris 1 Roberto Bruzzone 1 5 and Martial Jaume 1 Abstract Background Public health risks associated to infection by human coronaviruses remain considerable and vaccination is a key option for preventing the resurgence of severe acute respiratory syndrome coronavirus SARS CoV We have previously reported that antibodies elicited by a SARS CoV vaccine candidate based on recombinant full length SARS CoV Spike protein trimers trigger infection of immune cell lines These observations prompted us to investigate the molecular mechanisms and responses to antibody mediated infection in human macrophages Methods We have used primary human immune cells to evaluate their susceptibility to infection by SARS CoV in the presence of anti Spike antibodies Fluorescence microscopy and real time quantitative reverse transcriptase polymerase chain reaction RT PCR were utilized to assess occurrence and consequences of infection To gain insight into the underlying molecular mechanism we performed mutational analysis with a series of truncated and chimeric constructs of fragment crystallizable receptors Fc R which bind antibody coated pathogens Results We show here that anti Spike immune serum increased infection of human monocyte derived macrophages by replication competent SARS CoV as well as Spike pseudotyped lentiviral particles SARS CoVpp Macrophages infected with SARS CoV however did not support productive replication of the virus Purified anti viral IgGs but not other soluble factor s from heat inactivated mouse immune serum were sufficient to enhance infection Antibody mediated infection was dependent on signaling competent members of the human Fc RII family which were shown to confer susceptibility to otherwise na ve ST486 cells as binding of immune complexes to cell surface Fc RII was necessary but not sufficient to trigger antibody dependent enhancement ADE of infection Furthermore only Fc RII with intact cytoplasmic signaling domains were competent to sustain ADE of SARS CoVpp infection thus providing additional information on the role of downstream signaling by Fc RII Conclusions These results demonstrate that human macrophages can be infected by SARS CoV as a result of IgG mediated ADE and indicate that this infection route requires signaling pathways activated downstream of binding to Fc RII receptors Keywords SARS CoV Spike Antibody dependent enhancement Macrophage Fc receptor Antibodies Pseudotypes Correspondence bruzzone hku hk breizh hku hk Equal contributors 1 HKU Pasteur Research Pole and Center of Influenza Research School of Public Health LKS Faculty of Medicine The University of Hong Kong Hong Kong Hong Kong SAR 5 Department of Cell Biology and Infection Institut Pasteur Paris France Full list of author information is available at the end of the article 2014 Yip et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License http creativecommons org licenses by 2 0 which permits unrestricted use distribution and reproduction in any medium provided the original work is properly credited The Creative Commons Public Domain Dedication waiver http creativecommons org publicdomain zero 1 0 applies to the data made available in this article unless otherwise stated Yip et al Virology Journal 2014 11 82 Background The continuous threat of respiratory viruses to public health was exemplified by the global impact of the SARS CoV outbreak in 2003 1 by the occurrence since 2003 of confirmed human cases of H5N1 avian influenza in many countries particularly across Asia 2 and the 2009 H1N1 influenza pandemic 3 The recent emergence in the Arab peninsula of a novel coronavirus responsible for theMiddleEastrespiratorysyndrome MERS CoV 4 5 and the new H7N9 strain of avian influenza that has jumped into humans 6 7 in China underscore the need to continue work in this direction It is now agreed that SARS CoV can infect not only the respiratory tract but can also affect other organ sys tems and several reports have demonstrated infection of hematopoietic cells 8 10 however the mechanism by which SARS CoV enters into immune cells which do not express the SARS CoV receptor angiotensin converting enzyme 2 ACE2 11 12 has remained poorly under stood Both C type lectin receptors such as liver lymph node specific intercellular adhesion molecule 3 grabbing integrin L SIGN or dendritic cell specific intercellular adhesion molecule 3 grabbing non integrin DC SIGN 13 14 as well as antibody mediated infection may provide SARS CoV with an opportunity to modify its tropism Because of the lack of effective antiviral strategies to control coronaviruses infections vaccination is still regarded as a major option for preventing resurgence of SARS and related diseases We previously showed that a SARS CoV vaccine candidate based on recombinant full length SARS CoV Spike protein trimers triggered infection of human B cell lines despite eliciting in vivo a neutralizing and protective immune response in rodents 15 More recently we demonstrated that anti Spike antibody potentiates infection of both monocytic and lymphoid immune cell lines not only by SARS CoVpp but also by replication competent SARS coronavirus 16 thus providing evidence for a novel and versatile mechan ism by which SARS CoV can enter into target cells that do not express the conventional ACE2 virus receptor and are otherwise refractory to the virus Such infection path way may have implications for understanding the tropism and pathogenesis of the virus and therefore we further investigated the molecular and cellular mechanisms under lying ADE of SARS CoV infection By monitoring the susceptibility of human bulk primary immune cells i e peripheral blood mononuclear cells we have established the occurrence of ADE of SARS CoVpp infection in different circulating immune cell types among which the monocytic lineage CD68 cells was the pri mary target In addition to monocytes human macro phages were also infected by SARS CoV in presence of anti Spike antibodies only ADE mediated infection of macrophages however did not support productive rep lication of the virus Finally we have provided evidence that the intracellular signaling motif but not the IgG binding motif of the Fc R is the key molecular deter minant for triggering ADE of SARS CoVpp Our findings conclusively demonstrate that anti spike serum promotes internalization of SARS CoV by human macrophages Results Primary human macrophages are susceptible to SARS CoV infection through antibody mediated pathway Because our previous observations revealed that a human monocytic cell line THP 1 was susceptible to ADE of infection 16 we investigated the occurrence of ADE of infection in primary human macrophages in vitro firstly by taking advantage of SARS CoVpp that can be safely used to mimic the viral entry process 15 17 We found that SARS CoVpp opsonized with a 1 1000 dilution of anti Spike serum readily infected over 80 of primary human macrophages as determined by immunofluores cence staining of firefly luciferase at 72 hours post infec tion h p i By contrast cells exposed to SARS CoVpp opsonized with control serum did not show any positive staining of the luciferase reporter protein Figure 1 These experiments extend our previous observations and indi cate that anti Spike antibodies facilitate infection of SARS CoVpp into human macrophages We next tested whether this altered tropism was also displayed by replication competent SARS CoV Human macrophages were in fected at a multiplicity of infection MOI of 1 with the same dilutions of anti Spike or control immune serum and the infection pattern was examined by both im munofluorescence staining of SARS CoV nucleocapsid protein and real time quantitative PCR measurement of viral RNAs Positive immunofluorescence signals were detected only at 6 h p i when SARS CoV was opsonized with both control and anti Spike serum Figure 2 Inter estingly whereas presence of control serum led to only modest infection by SARS CoV 5 of cells a 4 fold in crease in the percentage of positive cells was noted in the presence of anti Spike serum for two out of three donors Figure 2 Such enhanced infection pattern was paralleled by the number of viral gene copies measured Figure 3 Thus compared to inoculums containing control serum there was a 2 to 3 fold increase in the detection of both positive and negative strands of viral genes in macro phages infected in presence of anti Spike serum which was more pronounced at 1 and 6 h p i There was a rapid decline in viral RNA from 6 to 24 h p i for both conditions and no further changes were detected at later time points Figure 3 However it should be noted that the primer set used for the nucleocapsid gene could also detect the negative strand of the viral genomic RNA and could not distinguish it from sub genomic material In addition Yip et al Virology Journal 2014 11 82 Page 2 of 11 we measured by real time quantitative PCR copies of both viral nucleocapsid and ORF1b genes in culture su pernatants to determine the release of SARS CoV parti cles from the infected macrophages Copy numbers of both viral RNAs however remained unchanged at all the selected time points during the course of the experi ment less than 200 copies l regardless of whether macrophages had been infected in the presence of either control or anti Spike serum data not shown ADE of SARS CoVpp infection is dependent on the dose of IgG fraction of heat inactivated immune serum We have previously demonstrated that mouse anti Spike serum could trigger infection of Raji cells 16 In the context of ADE antibodies against viral proteins are considered as the major players of enhancement 18 19 see for review 20 21 To eliminate the possibility of the participation of other soluble factors during ADE of SARS in this section we investigated the capability of anti Spike antibodies alone in enhancing infection of immune cells We purified IgG by protein G sepharose chromatography from mouse anti Spike and control serum and used 2 fold serial dilutions from 10 to 0 125 g mL of the purified portion to form immune complex with SARS CoVpp and then infected Raji cells Our results show that puri fied IgG from mouse anti Spike serum triggered infec tion in Raji cells which was more pronounced with increasing immunoglobulin concentrations Figure 4 The flow through FT from the same serum which was diluted by the appropriate factor to be comparable to the final IgG concentration used elicited no detectable infection at all concentrations data not shown Signifi cant differences between ADE of infection with purified mouse anti Spike IgG and flow through were observed at concentrations 10 and 2 5 g mL and marginal differ ence was observed at 0 625 g mL As expected neither purified IgG Figure 4 nor flow through from mouse control serum triggered significant ADE of infection at all concentrations Molecular determinants of Fc RII underlying ADE of SARS CoVpp Our recent work had revealed the predominant role of human Fc RII CD32 in mediating ADE of SARS CoV 16 To gain further insight into the underlying molecular mechanism we have investigated the involvement of differ ent domains of Fc RII in mediating ADE To this end we produced a series of truncated constructs that only carried ectodomain and transmembrane domains of Fc RII and chimeric constructs with the ectodomain of one re ceptor and transmembrane and endodomain of another Figure 5A We then verified the expression of these constructs on ST486 cells by flow cytometry using a specific monoclonal antibody clone FLI8 26 that binds to the second Ig like domain D2 of Fc RII Of note theFcbindingportionoftheFc RII group is very simi lar among Fc RIIA H131 Fc RIIA R131 Fc RIIB1 22 and also Fc RIIB2 as it harbors identical extracellular Figure 1 Anti Spike but not control serum triggered infection of human macrophages by SARS CoVpp A Monocyte derived macrophages were incubated for 1 hour with SARS CoVpp carrying a luciferase reporter gene in the presence of a 1 1000 dilution of either anti Spike or control serum and infected cells were detected by fluorescence staining of firefly luciferase green as described under Materials and methods Cell nuclei were labeled with DAPI blue SARS CoVpp infection of macrophages occurred only in the presence of anti Spike serum Images are representatives of five independent experiments using macrophages from five donors B Infection was quantified by counting the percentage of immunofluorescence positive cells in randomly chosen fields using Metamorph software Results are shown as means SEM Scale bar 20 m Yip et al Virology Journal 2014 11 82 Page 3 of 11 structures as Fc RIIB1 Our findings indicate that all Fc RII constructs exhibited detectable expression of Fc RII darkgrey inST486cellsincomparisontoiso type control light grey Figure 5B We then tested the ability of the various constructs to bind purified anti Spike IgG SARS CoVpp immune complexes and ob served that all ST486 transfectants were able to bind immune complexes Figure 5C Finally we investigated whether any difference in susceptibility to SARS CoVpp ADE of infection was conferred by different Fc RII con structs When immune complexes formed by 5 g mL of purified mouse anti Spike IgG the concentration at which the highest infection level was observed in Raji cells and SARS CoVpp were added to Fc RII expressing ST486 cells all four transfectants expressing wild type Fc RII forms cf Fc RIIA H vs Fc RIIA R and Fc RIIB1 vs Fc RIIB2 corresponding to constructs 1 5 9 10 respectively were infected Figure 5D with Fc RIIA expressing ST486 being more prone to infec tion than Fc RIIB cf constructs 1 and 5 with 9 All the endodomain truncated constructs Fc RIIA H IC Fc RIIA R IC and Fc RIIB IC corresponding to con structs 2 6 11 respectively were not susceptible to ADE of infection indicating that binding of anti Spike IgG SARS CoVpp immune complexes was not suffi cient to mediate entry and that the signaling competent endodomain was required However not all chimeric constructs were able to sustain ADE of infection sug gesting that domain swapping may have partially inter fered with signal transduction Thus only chimeras with Fc RIIA H ectodomain and Fc RIIB1 endomain or with Fc RIIB ectodomain and Fc RIIA endomain exhibited a statistically significant ADE of infection Figure 5D This cannot be explained by differences in surface expression or binding of opsonized pseudoparticles as Fc RIIB EC IIA IC viz construct 12 showed robust ADE despite one of the lowest binding ability of immune complexes Discussion The possibility that immune response to pathogens may have also deleterious effects on the host homeostasis has been the focus of several studies For example the hyper induction of cytokines following avian influenza infection has been implicated in the severity of the dis ease 23 and infection of cells by ADE has been known to occur for several viral diseases 20 21 Here we dem onstrate that anti Spike antibody potentiates infection Figure 2 Anti Spike serum enhanced SARS CoV infection in human monocyte derived macrophages A Monocyte derived macrophages were infected with SARS CoV strain HK39849 at an MOI of 1 in the presence of a 1 1000 dilution of either anti Spike black bars or control white bars serum for 1 hour and then cultured with fresh medium for 6 hours as described under Materials and methods After fixation cells were permeabilized for intracellular staining of SARS CoV nucleocapsid protein which was revealed by TRITC conjugated goat anti mouse antibody orange while cell nuclei were stained with DAPI blue Images shown are representatives of the results obtained with macrophages from three donors at 6 hours post infection Scale bar 20 m B Infectivity of SARS CoV was determined by calculating the percentage of nucleocapsid positive cells in five randomly selected fields using Metamorph software Presence of anti Spike serum increased infectivity of SARS CoV by 3 4 fold in macrophages of two out of three donors Note that the ordinate s scale for Donor 3 is different for ease of visualization Results are shown as means SEM Yip et al Virology Journal 2014 11 82 Page 4 of 11 of human primary immune cells by SARS CoVpp and replication competent SARS coronavirus Although we unambiguously obtained evidence of on going infection e g de novo synthesis of the structural viral protein N ADE infected macrophages did not support productive replication of SARS CoV and after initiation of viral gene transcription and viral protein synthesis the replication process stalled ultimately end ing in an abortive viral cycle without detectable release of progeny virus Abortive replication of SARS CoV into macrophages has already been documented 24 but at variance with this previous report in which 90 of the macrophages were infected by SARS CoV in the absence of immune serum MOI 1 2 we observed a much lower infection rate about 5 7 One possibility is that such discrepancy may be due to difference of time of sampling 6 hours in our study versus 15 hours in Cheung s study and the protocol used for in vitro differentiation viz macrophages were differentiated in the presence of fetal calf serum in our study and au tologous plasma was removed two days prior to infec tion in ref 8 leading to difference in infectivity of the cells observed in the studies Alternatively we should also consider that the readout of the pseudo particle ex periments was the expression of the luciferase reporter gene which is under the control of the HIV backbone This may lead to higher level of protein expression when compared to the abortive replication that follows SARS CoV infection of macrophages Thus the difference may be in part due to the inability to detect low amounts of Spike protein by immunofluorescence and the differ ence in sensitivity of the two methods Of note the anti Spike mediated entry is specific for Spike pseudotyped particles as shown in Figure 1 of 16 Because clinical observations have reported poor disease outcomes in early seroconverted SARS patients 25 27 it would be of interest to test SARS patient sera collected at different time points after SARS onset However we have been unable thus far to conduct conclusive assays on a well characterized serum library moreover we have to be cognizant of the possibility that ADE may only occur within a narrow window during the course of an infection and only in a subset of infected patients The alternative possibility that internalization by macrophages may in fact represent an additional mechanism to control viral spread requires further investigation In general studies aiming at better understanding antibody dependent enhancement of viral infections are focusing on either identifying the immune receptor s and or serum component s allowing penetration of the pathogen into the target cell also known as extrinsic ADE or the outcome response s of the target cell 1 6 24 72 0 3000 6000 9000 162472 0 100 200 1 6 24 72 0 2000 4000 6000 162472 0 30 60 90 1 6 24 72 0 100 200 162472 0 10 20 1 6 24 72 0 1000 2000 162472 0 3 6 9 1 6 24 72 0 1000 2000 162472 0 10 20 30 1 6 24 72 0 500 1000 1500 162472 0 3 6 9 Do n o r 1 Do n o r 2 Do n o r 3 Nucleocapsid ORF1b Positive strand Negative strand Positive strand Negative strand Control Anti Spike AB ge ne c o pi e s 1 0 8 18S rRN A co p ies hour s post infection hp i Figure 3 Nucleocapsid and ORF1b gene expression in SARS CoV infected human macrophages Monocyte derived macrophages from three independent donors were infected with SARS CoV as described in the legend to Figure 2 in the presence of anti Spike black bars or control serum white bars Pos