Broad neutralization and complement-mediated lysis of HIV-1 by PEHRG214, a novel caprine anti-HIV-1 polyclonal antibody
Verity, Erin Ea,b,d,e; Williams, Lisa Ac; Haddad, Da'ed Nf; Choy, Vernonc; O'Loughlin, Chrisc; Chatfield, Catherineb,e; Saksena, Nitin Kf; Cunningham, Anthonyg; Gelder, Frankc; McPhee, Dale Aa,b,d,e
From the aDepartment of Microbiology, Monash University, Clayton, Victoria, Australia
bMacfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, Australia
cVirionyx Corporation, Auckland, New Zealand
dNational Serology Reference Laboratory, Australia, Fitzroy, Victoria, Australia
eNational Centre in HIV Virology Research, Melbourne, Victoria, Australia
fRetroviral Genetics Division, Westmead Millenium Institute, Sydney, New South Wales, Australia
gWestmead Millenium Institute, Sydney, New South Wales, Australia.
Received 4 November, 2004
Revised 27 October, 2005
Accepted 1 December, 2005
Correspondence to D. McPhee, National Serology Reference Laboratory, at St. Vincent's Institute for Medical Research, 4th floor, Healy Building, 41 Victoria Parade, Fitzroy, Victoria, Australia, 3065. Tel: +61 3 9418 1121; fax: +61 3 9418 1155; e-mail: email@example.com
These data were presented in part at the Keystone Symposium ‘HIV Vaccine Development: Immunological and Biological Challenges’, March 2003, Banff, Alberta, Canada and also at the Australian Society for HIV Medicine Conference ‘Global Crisis: Local Action’, October 2003, Cairns, Queensland, Australia.
Objectives: To assess the potency, breadth of action, and mechanism of action of the polyclonal goat anti-HIV antibody, PEHRG214.
Design: Typical human antibody responses to HIV-1 infection are unable to neutralize virus efficiently, clear the infection, or prevent disease progression. However, more potent neutralizing antibodies may be capable of playing a pivotal role in controlling HIV replication in vivo. PEHRG214 is a polyclonal caprine antibody raised against purified HIV-associated proteins, such that epitopes that are immunologically silent in humans may potentially be recognized in another species. It has been administered safely to HIV-infected individuals in Phase I clinical trials.
Methods: The anti-HIV activity of PEHRG214 was assessed using neutralization and virion lysis assays. The target proteins for PEHRG214 activity were investigated using flow cytometry and by adsorption of anti-cell antibodies from the antibody cocktail.
Results: PEHRG214 strongly neutralized a diverse range of primary HIV-1 isolates, encompassing subtypes A to E and both CCR5 and CXCR4 phenotypes. Neutralization was enhanced by the presence of complement. PEHRG214 also induced complement-mediated lysis of all HIV-1 isolates tested, and recognized or cross-reacted with a number of host cell proteins. Lysis was abrogated by adsorption with T and/or B cells expressing GPI-linked proteins, but not by GPI-deficient B cells or red blood cells.
Conclusions: PEHRG214 was found to potently neutralize and lyse HIV-1 particles. By targeting host cell proteins present in the viral envelope, which are conserved among all strains tested, PEHRG214 potentially opens up a highly novel means of eliminating circulating virus in infected individuals.
HIV-1 infection results in robust immune responses incapable of clearing infection or preventing disease progression. Antibodies recognize all viral proteins, but those associated with virus neutralization target the envelope glycoproteins gp120 and gp41 [1–5]. Although antibodies may neutralize autologous virus they rarely inhibit heterologous virus [5–7]. This can drive virus evolution and allow escape mutants to overcome inhibition [5,8,9].
In addition, virions incorporate cellular proteins in the viral envelope that aid replication and evasion of immune responses. These include the complement control proteins CD46, CD55, and CD59 [10–13]; adhesion molecules [14,15]; MHC class I and II proteins [16,17]; and other lipid raft-associated proteins.
Glycosylphosphatidylinositol (GPI)-linked proteins in particular associate with the viral envelope, including CD14, CD48, CD55, CD58, CD59, and CDw108 [18,19]. Polyclonal antibodies targeting cell-derived proteins, including CD55, CD59, and MHC-II, can neutralize virus and induce complement-mediated lysis (CML) of virus particles [10–12,20].
Despite this, a handful of broadly neutralizing monoclonal antibodies have been identified [21–24]. Passive immunization of primates demonstrated that these antibodies can prevent infection or delay disease progression [25–29]. Interestingly, at least three of these antibodies possess unique structural characteristics [30–32], highlighting the requirement for novel approaches to HIV-1 neutralization.
One such approach is to use antibodies raised in a species other than man. Potentially neutralizing viral epitopes may remain immunologically silent in humans due to mimicry of cellular protein epitopes and the cellular epitopes themselves present on the virion surface. Exposure of these epitopes in animal hosts could induce production of such antibodies.
Here we examine PEHRG214, a novel polyclonal caprine antibody raised against purified virus. Based on preliminary data showing potent neutralization of multiple HIV-1 strains, PEHRG214 was used in Phase I clinical trials in Sydney, Australia and Boston, USA [33,34], where it was generally well tolerated at doses lower than 48 000 U/kg. We show that PEHRG214 is strongly neutralizing in cell culture, that it causes CML of virus particles, and examine reactivity against virion-associated proteins.
Materials and methods
Antibodies and complement
The polyclonal caprine antibody PEHRG214 (Virionyx, Auckland, New Zealand) was raised against purified lysates of pooled HIV-1MN (H9 cells), HIV-1BaL (macrophage), HIV-2NIHZ (HUT-78), and boosted with synthetic peptides mapping to continuous epitopes of HIV-1SF2 Env (aa 4–27, 54–76, and 502–541) and Gag (aa 2–23, 69–94, 89–116, 166–181, 390–410, and 438–443). Lysates were treated to remove lipids and carbohydrates, and immunoaffinity purified against HLA class I and II monoclonal antibodies. The resulting antibodies were IgG purified by immunoaffinity chromatography. PEHRG214 was tested for total protein and IgG concentrations, and immunoblot against viral proteins. Goat pre-immune sera which had undergone the same purification processes (01001) was used as a control antibody.
Human complement was purchased in lyophilized form (Sigma-Aldrich Co., St Louis, Misouri, USA) and was reconstituted immediately before use.
Cells and cell lines
Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation from buffy coats obtained from the Red Cross Blood Bank, taken from healthy, seronegative donors. Seventy-two hours prior to use PBMC were activated using 2.5–10 μg/ml phytohemagglutinin (PHA). The human T cell line PM-1 and pro-myelocytic leukaemia cell line THP-1 were gifts from A. Cunningham (Westmead Millennium Institute, Sydney, New South Wales, Australia). The Epstein–Barr virus (EBV)-transformed, B-lymphoblastoid JY cell line and the derivative, GPI-deficient JY33 cell line were gifts from G. Spear (Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL, USA). The absence of GPI-linked proteins on JY33 cells was confirmed by FACS analysis. PBMC and cell lines were maintained in RF10 medium (RPMI-1640 medium supplemented with 10% foetal bovine serum, 25 μg/ml L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin).
The HIV-1 isolates HIV-1NL43 (X4, subtype B), HIV-1AD8 (R5, subtype B), HIV-1BCB93 (subtype D) and HIV-192TH024 (R5, subtype CRF01_AE) were from the NIH AIDS Reference and Reagent Program (NIAID, Bethesda, MD, USA). HIV-1BaL (R5, subtype B) was from S. Sonza (Burnet Institute, Melbourne, Australia). HIV-1ROK39 (R5, subtype A) and HIV-1SE364 (R5, subtype C) were from P. Cameron (University of Melbourne, Victoria, Australia). The Australian isolates HIV-1MBC200 (X4, subtype B) and HIV-1MBC925 (R5, subtype B) were isolated from infected PBMC . Virus stocks were produced by infection and culture of 3-day PHA-activated PBMC in interleukin (IL)-2 media (RF10 containing 12 mM HEPES, 10 U/ml IL-2, 0.2% polybrene, and 0.1% hydrocortisone). For use in virion lysis assays, infection supernatant was ultracentrifuged at 100 000 × g through a 20% sucrose cushion in a Beckman L-90 ultracentrifuge using an SW41 rotor. Virus was pelleted and resuspended in a tenfold smaller volume of RF10 medium, and analysed for p24 content by ELISA (Beckman Coulter, NSW, Australia). For transfections, 7.5 × 106 JY or JY33 cells were transfected with 20 μg pNL4-3 or pAD8 by electroporation using a Gene Pulser II (Bio-Rad Laboratories, Regents Park, NSW, Australia). Virus harvested on day 5 post-transfection was concentrated and purified as described above.
PEHRG214 (25 μl, to give final concentrations of 750, 500, 250, 100, 50 or 5 μg/ml PEHRG214) was added to 500 tissue culture 50% infectious doses (TCID50) of HIV-1 (25 μl) and 25 μl of 40% v/v human complement each in quadruplicate wells of a 96-well tissue culture plate. This mixture was incubated for 1 h at 37°C and 5% CO2 before the addition of 2 × 105 3-day PHA-activated PBMC. After incubation for 2 h, 100 μl of IL-2 medium was added to each well. PBMC were washed on day 1 post-infection. Neutralization was measured during the logarithmic growth phase of virus replication by analysis for cell-free reverse transcriptase activity and/or p24 antigen content, compared with a control containing no antibody.
Virion lysis assay
Aliquots of 200 pg of p24 associated with purified virus were incubated with PEHRG214 (generally 750 μg/ml) in RF10 for 30 min at 4°C, in a total volume of 500 μl. The virus–antibody mixture was centrifuged through a 20% sucrose cushion at 45 000 × g for 1 h at 4°C in a Heraeus Biofuge Stratos centrifuge. The viral pellet was resuspended in 500 μl of either human complement (1: 4 dilution in RF10) or RF10. After incubation at 37°C for 30 min, any virus remaining intact was separated from the viral debris in the supernatant by centrifugation and was resuspended in 600 μl 0.5% Triton-X-100 in RF10. The p24 concentration in both the pellet and supernatant was measured using p24 ELISA. The percentage of virion lysis was calculated as the percentage of p24 in the supernatant.
Flow cytometric analysis
PEHRG214 was titrated against human non-HIV-1 infected PBMC to determine the concentration required for saturated binding, as opposed to discrimination between virus-infected and non-infected cells. For flow cytometric analysis, 5 × 105 cells were resuspended in 100 ul of PEHRG214 at this saturating concentration of 1 mg/ml in PBS in calcium and magnesium free (PBS−) and incubated for 30 mins on ice. Cells were washed with PBS−, resuspended in 100ul of saturating concentrations of FITC-conjugated rabbit anti-goat antibody (Sigma-Aldrich, St Louis, MO, USA) in PBS− before incubating for 30 min on ice. Cells were washed in PBS−, resuspended in 0.1% paraformaldehyde in PBS− and stored at 4°C until flow cytometric analysis using a FACSCalibur/Cellquest operating system.
For dual labelling studies, single-labelled cells were washed in PBS− and resuspended in 50 μl of 10% v/v phycoerythrin (PE)-conjugated antibody specific for leukocyte subset markers CD4, CD8 or CD14 (Sigma). Labelling reactions were incubated 30 min on ice, washed once and resuspended in 0.1% paraformaldehyde in PBS− before flow cytometric analysis.
For intracellular HIV p24 antigen labelling, paraformaldehyde-fixed single-labelled cells were washed and resuspended in 0.1% Triton X-100 in PBS− for 15 min on ice. The PE-conjugated anti-p24 antibody (KC57, Beckman-Coulter) was added and incubated in permeabilization buffer for 30 min on ice before washing in PBS−. Cells were fixed in 1% paraformaldehyde in PBS− and analysed using flow cytometry.
Adsorption and elution of PEHRG214 and western blot analysis
Anti-cell antibodies were removed by incubation of PEHRG214 (10.5 mg/ml) with an equal volume of packed cells for 30 min on ice, repeated once using fresh cells. The removal of anti-cell antibodies was confirmed by flow cytometry (PBMC, JY, JY33 cells) or haemagglutination testing (red blood cells; RBC). Adsorbed samples were analysed by Western blot using a Genelabs Diagnostics HIV BLOT 2.2 test kit, according to the manufacturer's instructions.
Neutralization of HIV-1 by a caprine polyclonal anti-HIV antibody, PEHRG214
The novel antibody preparation PEHRG214 was initially tested for neutralization of the subtype B and C, X4 and R5 primary isolates HIV-1MBC200 and HIV-1SE364, measured by a reduction in virus replication relative to controls containing no antibody (Fig. 1a and b). Neutralization was dose-dependent, and was stronger and more sustained in the presence of human complement (75 ± 35% or 92 ± 4% neutralization of HIV-1MBC200, or 85 ± 9% or 98 ± 0% neutralization of HIV-1SE364 at 250 μg/ml PEHRG214 in the absence and presence of complement, respectively). Neutralization was variable in the absence of complement. Equivalent goat pre-immune sera (01001) did not inhibit virus replication (data not shown). Cells treated with PEHRG214 were observed to aggregate, but cell toxicity was minimal and cells were able to be productively infected post-antibody exposure (data not shown).
Complement mediated lysis of HIV-1 by PEHRG214
Due to the stronger neutralization observed in the presence of complement, the ability of PEHRG214 to cause CML was assessed. CML was measured as the percentage of virion-associated p24 released into the supernatant upon CML. Treatment of virus with antibody and complement resulted in almost complete virion lysis (Fig. 1c). Lysis did not occur for the control caprine antibody preparation 01001, the monoclonal antibody b12 (40 μg/ml, data not shown) or polyclonal human HIVIG (450 μg/ml, data not shown). The degree of virion lysis was very similar for both HIV-1MBC200 and HIV-1SE364. Some spontaneous lysis (17.9 ± 8.4%) was observed in the absence of antibody or complement, or in the presence of either antibody or complement alone. This background of non-specific lysis was taken into account in each experiment, and differed between different virus preparations.
PEHRG214 neutralizes a broad spectrum of HIV-1 isolates
Neutralizing antibodies elicited by HIV-1 infection are normally low titre and strain specific [5–7]. However, PEHRG214 consistently neutralized a broad spectrum of HIV-1 isolates in the presence of human complement, including viruses from subtypes A to E and viruses utilizing either CCR5 or CXCR4 coreceptors for entry into target cells (Fig. 2a, data not shown for subtype E). All HIV-1 isolates were neutralized by more than 70% at 250 μg/ml PEHRG214. Neutralization was significantly greater in the presence of complement at concentrations of 150 μg/ml PEHRG214 or greater (P ≤ 0.000765), using an unpaired, two-tailed t test. Enhancement of infection was occasionally observed at limiting antibody concentrations with particular isolates (HIV-1MBC925, Fig. 2a).
Complement-mediated lysis of a broad spectrum of HIV-1 isolates
PEHRG214 induced CML of all viruses tested (Fig. 2b). The effective concentration for each virus was very similar, with maximum virion lysis (≥ 95%) occurring at 300 μg/ml PEHRG214 and above (98.8 ± 0.9% at 750 μg/ml PEHRG214; 95.1 ± 2.1% at 300 μg/ml PEHRG214). Lysis was reduced to 84.0 ± 0.7% at 150 μg/ml PEHRG214, and 15.1 ± 6.9% at 30 μg/ml PEHRG214, which was indistinguishable from background levels. The dose–response curve was similar to that observed for the virus neutralization assay (Fig. 1). In the absence of PEHRG214, and to a lesser extent at limiting concentrations of the antibody, there was some variation observed among the viruses tested. To assess breadth of activity, we tested HIV-1BaL derived from macrophages and found that CML of this virus by PEHRG214 was almost identical to that of PBMC-derived viruses (Fig. 2b).
PEHRG214 recognizes cell surface proteins on human T and B cells
To assess reactivity with host cell proteins, FACS analysis was performed. Dual-staining of PBMC populations showed that PEHRG214 bound to both HIV-1-infected and uninfected cells, as measured by intracellular p24 staining (Fig. 3a). Human cells from monocytic (THP-1), T-lymphocytic (PM-1) or B-lymphocytic (JY and JY33) lineages were also labelled by PEHRG214 (Fig. 3b). Further, PEHRG214 binding was detected on the surface of all major subpopulations of uninfected leukocytes: CD4-, CD8-, and CD14-positive cells (Fig. 3c).
PEHRG214 recognition of HIV-1 and cellular proteins by immunoblot analysis
Immunoblot analysis was used to examine the overall recognition of HIV-1 and host cell proteins by PEHRG214 and PEHRG214 adsorbed with PBMC, JY, or JY33 cells (Fig. 4). PEHRG214 recognized the same HIV-1 proteins that were detected by serum from an infected individual, as well as numerous other proteins (Fig. 4). Adsorption with uninfected JY or JY33 cells did not fully remove specificities for the proteins overall. Interestingly, no obvious difference was observed between the proteins recognized by PEHRG214 adsorbed by any of the cell types. This may simply have been due to a low concentration of functionally active antibodies in the PEHRG214 antibody cocktail. Eluates from adsorption with lymphocytes showed low reactivity with p24 and gp41, and several other unidentified bands were observed (not shown).
Functional significance of the PEHRG214 fraction binding human cells
Antibodies binding PHA-activated PBMC, RBC, JY, or GPI-deficient JY33 cells were removed by mixing PEHRG214 with packed viable cells. When tested for neutralizing activity, those samples in which the antibodies binding PBMC, JY, or JY33 cells had been removed demonstrated little or no inhibition of virus replication (Fig. 5a). The removal of antibodies binding RBC had minimal effect on PEHRG214 neutralization activity. To rule out gross removal of anti-HIV antibodies by PBMC adsorption, PEHRG214 was spiked prior to adsorption with a neutralizing serum . The anti-HIV-1 activity of the HIV-positive serum was not removed, demonstrating that PBMC adsorption removed the antibodies responsible for the inhibitory activity of PEHRG214, but not antibodies targeting known neutralizing viral epitopes.
The importance of GPI-anchored cell surface molecules in CML of HIV-1 by PEHRG214
PEHRG214 adsorbed with PBMC, RBC, JY, or JY33 cells was then tested for activity in CML assays. Adsorption with PBMC or JY cells abrogated the ability of PEHRG214 to induce CML of HIV-1 particles (Fig. 5b). However, adsorption with RBC or JY33 cells only slightly reduced the maximum level of virolysis to 85.0 ± 0.8% (RBC) or 82.8 ± 7.7% (JY33 cells). Lysis was maximal because increasing the concentration of antibody did not increase the level of CML (data not shown), indicating only partial removal of functionally active antibodies. In addition, virions derived from transfected JY33 cells (HIV-1NL43 and HIV-1AD8) were not lysed by PEHRG214 that had been adsorbed by either JY or JY33 cells (Fig. 5c). Together, these data support the proposal that GPI-linked proteins are the major targets for the lytic activity of PEHRG214. Surprisingly, virus particles derived from transfection of JY33 cells were lysed (Fig. 5c) and neutralized (data not shown) by unadsorbed PEHRG214, despite the absence of GPI-linked target proteins.
Naturally generated polyclonal antibodies are generally unable to neutralize a broad spectrum of HIV-1 isolates and do not lyse virions. Here, the caprine polyclonal antibody PEHRG214 neutralized diverse HIV-1 isolates in a dose-dependent manner enhanced by the presence of complement. The major mechanism of action was CML of virus particles.
All viruses tested were potently neutralized and lysed by PEHRG214, including strains representing subtypes A–E (clade E data not shown), primary and laboratory isolates, and R5 and X4 phenotypes. PEHRG214 induced ≥95% CML of virus particles at 300 μg/ml, and neutralization was potent at or above 250 μg/ml PEHRG214, a similar functional concentration range to the polyclonal HIVIG [23,26]. Some neutralization was detected in the absence of complement, suggesting that other complement-independent mechanisms were possible in addition to CML. Although a number of monoclonal antibodies neutralize HIV-1 at much lower concentrations [21–24], functionally active antibodies present in PEHRG214 are likely to constitute a small proportion of the total IgG. To our knowledge, no other polyclonal antibody is capable of such broad and potent complement-dependent neutralization of HIV-1.
PEHRG214 recognized both HIV-1-infected and uninfected, resting and activated PBMC, as well as cells from T, B, and myeloid lineages. It is possible that antibody binding was greater in infected cell populations, but the high fluorescence intensities in PBMC populations prevented detection of differences in the presence or absence of HIV-1 infection.
Removal of leukocyte-binding antibodies by adsorption with uninfected PBMC, T, B, or myeloid cells abrogated virus neutralization and lysis by PEHRG214. Neutralizing sera retained activity post-adsorption, and adsorbed PEHRG214 preparations retained anti-HIV immunoblot reactivity, suggesting that functionally active PEHRG214 antibodies react or cross-react with PBMC surface molecules.
Interestingly, removal of antibodies binding RBC or GPI-deficient JY33 cells did not abrogate PEHRG214 lytic activity, or neutralizing activity for RBC. This suggested that CML was mediated by antibodies recognizing molecules absent on these cells. However, protein targets responsible for neutralization were present on JY33 cells. Although CML activity was not removed by RBC or JY33 adsorption, a reduction in the maximum level of CML suggested that lysis was targeted to more than one specificity within PEHRG214. Together, these data suggested that CML was targeted primarily to host cell GPI-linked proteins acquired by HIV-1, with possible contributions from other cell surface proteins. GPI linked proteins present on virions includes the complement control proteins CD55 and CD59, as well as CD14, CD48, CD58, CD73, CD90, and CDw108 , and possibly other unidentified proteins. It is likely that significant activity of PEHRG214 is targeted to one or more of these proteins. RBC, which reduced PEHRG214 lytic activity similar to JY33 cells, express CD55, CD59, and CDw108, suggesting that these proteins are not primary targets for PEHRG214 activity. The results also illustrate that the neutralization assay is not completely reliant on virion lysis for its inhibitory activity, as complement-independent activity was observed in this assay system, albeit variably, and also because JY33 adsorption did not abrogate neutralization.
HIV-1 particles derived from JY33 cells were not lysed by PEHRG214 adsorbed against JY or JY33 cells, suggesting that reactivity against HIV-1-encoded proteins was not able to induce CML, even in the absence of the GPI-linked complement control proteins. Unexpectedly, unadsorbed PEHRG214 lysed virions derived from JY33 cells, suggesting that additional reactivity against non-GPI-linked host cell protein(s) can induce virion lysis in the absence of protective CD55 and CD59. Such antibody targets may also be responsible for CML activity lost upon adsorption with JY33 cells or RBC.
Results from clinical trials suggest that low doses of PEHRG214 are acceptable for patients with high viral loads and low CD4 cell counts [33,34]. Here we show that PEHRG214 mediates neutralization and CML of multiple HIV-1 isolates at concentrations readily achieved in vivo. The antibody cocktail targets both virus- and host-encoded antigens, potentially inhibiting infection through multiple mechanisms of action. With antibody targets that utilize both complement-dependent and -independent mechanisms of action, it may be possible to both harness and bypass the chronically activated complement system of infected individuals to eradicate virus more effectively. Despite this, the action of PEHRG214 in clinical trials showed an indirect and delayed effect in some patients [33,34].
This study also highlights cell-derived proteins as potential targets for antiviral therapeutics, which is not unprecedented. Monoclonal antibodies targeting numerous cellular antigens have been approved for use, or are in clinical trials, for the treatment of cancer and autoimmune disease [37,38]. Indeed, the gp41 monoclonal antibodies 2F5 and 4E10, which have been tested in clinical trials [39–41], autoreact with the phospholipid cardiolipin . Furthermore, an antibody targeting cellular proteins would not allow virus evolution to escape neutralization. This study suggests that this plethora of host and viral antibody targets, some cross-reactive, are together potentially capable of playing a significant therapeutic role in controlling HIV-1 infection, opening up a highly novel means of eliminating circulating virus in infected individuals.
We thank Dr D. Montefiori (Duke University Medical Center, Durham, North Carolina, USA) for his valuable insight that has lead to the characterisation of this antibody. The authors would also like to thank Dr G. Spear and Dr A. Cunningham for provision of cell lines, and Dr P. Cameron, Dr S. Sonza, and the AIDS Reference and Reagent Program for provision of viruses. We would also like to thank G. Paukovics and J. Sanford for technical assistance.
EEV was supported by an Australian Postgraduate Award (Monash University, Victoria, Australia). This study was supported by the Burnet Institute (Melbourne, Victoria, Australia), the National Centre for HIV Virology Research (Melbourne, Victoria, Australia), Virionyx Corporation (Auckland, New Zealand), and the National Serology Reference Laboratory, Australia (Melbourne, Victoria, Australia).
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