Vaccine 29 (2011) 49134922

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Vaccine
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HIV-Gag VLPs presenting trimeric HIV-1 gp140 spikes constitutively expressed in stable double transfected insect cell line
M. Tagliamonte a , M.L. Visciano a , M.L. Tornesello a , A. De Stradis b , F.M. Buonaguro a , L. Buonaguro a,c,
a b c

Lab. of Molecular Biology and Viral Oncogenesis, Istituto Nazionale Tumori Fond. G. Pascale, Naples, Italy Natl. Res. Council Inst. Plant Virology, Bari, Italy Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, USA

a r t i c l e

i n f o

a b s t r a c t
We have previously described the establishment and characterization of a stably transfected insect cell line for the constitutive and efcient expression of Pr55 HIV Gag proteins, which auto-assemble into enveloped Virus-Like Particles (VLPs) released into the cell culture supernatant. Such HIV-Gag VLPs have been shown to elicit a specic systemic humoral response in vivo, proving the appropriate antigenic presentation of the HIV Gag protein to the immune system. Here we describe the establishment of a stable double transfected insect cell line for the constitutive and reproducible production of Pr55GagVLPs expressing on their surface trimeric forms of HIV-1 envelope glycoproteins. The persistence of HIV coding genes has been veried in clonal resistant insect cells, the protein expression and conformation has been veried by Western blot analysis. The resulting HIV-VLPs have been visualized by standard transmission electron microscopy and their immunogenicity has been evaluated in vivo. This represents, to our knowledge, the rst example of stable double transfected insect cell line for the constitutive production of enveloped HIV-Gag VLPs presenting trimeric HIV-gp140 on their surface. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 11 January 2011 Received in revised form 7 April 2011 Accepted 1 May 2011 Available online 17 May 2011 Keywords: Virus-Like Particles Delivery system HIV Stable expression Insect cells

1. Introduction Virus-Like Particles (VLPs) are an effective form of subunit vaccine based on viral capsid proteins self-assembling into structures closely resembling immature forms of the natural virus from which they are derived. Moreover, lacking regulatory proteins as well as genetic material, they are unable to induce productive viral infection [14]. VLPs can be considered nanoparticles and represent a highly attractive presenting and delivery system for antigenic structures as well as foreign DNA and drug molecules [47]. Moreover, the ease of engineering and purication makes VLPs key candidates for vaccine as well as gene therapy strategies for a wide variety of diseases. Indeed, as vaccine model, VLPs efciently stimulate the immune system, being taken up by key immune cells including dendritic cells (DCs), macrophages, mast cells and B cells. In particular, in the absence of infection or intracellular replication, VLPs have been shown to enter both the extracellular and the endogenous processing pathway within antigen presenting cells (APCs), activating antigen-specic CD8+ cytotoxic T lymphocytes (CTL),

Corresponding author at: Lab. Molecular Biology and Viral Oncogenesis, Istituto Nazionale Tumori Fond. G. Pascale, Via Mariano Semmola, 1, 80131 Naples, Italy, Tel.: +39 081 5903 273; fax: +39 081 5451276. E-mail address: irccsvir@unina.it (L. Buonaguro). 0264-410X/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.05.004

a phenomenon known as cross-priming [811]. This characteristic has been exploited by modifying VLPs to act as vehicles for pathogen as well as tumor-associated antigens [8,12]. HIV-1 Pr55Gag-based VLP model has been employed to deliver additional antigenic structures, such as whole HIV envelope protein or specic individual epitopes, with induction of both arms of the immune response. In particular, HIV-1 envelope V3 epitopes have been introduced into dispensable Pr55Gag domains [13,14] or fused to the 3 -end of the gag ORF, taking advantage of the ribosomal frameshifting signals [15]. Both strategies have resulted in the induction of an efcient immune response against the HIV-1 epitopes, although the antibody response against the Gag protein appeared much stronger than against the V3 envelope epitopes [15,16]. In order to improve the Env antigenicity and to present conformational epitopes, the entire gp120 molecule has been expressed on the Pr55Gag-VLPs and anchored through the transmembrane (TM) portion of the Epstein-Barr virus (EBV) gp220/350 (EnvGaghybrid VLPs). Such approach increases the expression and stability of the gp120 glycoprotein on the VLPs surface, without affecting its oligomerization [17,18]. Nevertheless, the substitution of the autologous signal sequence (ASS) with the one of Honeybee Mellitin signal sequence (HMSS) as well as the fusion of HIV-1 gp120 envelope protein with the trans-membrane domain of the baculovirus major glycoprotein gp64 (gp120gp64 fusion protein) have been shown to increase HIV-1 gp120 expression, glycosyla-

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tion, secretion from the SF9 insect cells and incorporation on the VLPs surface [19,20]. Additional strategies have been more recently described to express trimeric forms of whole HIV-1 envelopes on the surface of Pr55Gag-VLPs [21,22] (Visciano et al., submitted for publication). All such approaches result in induction of a relevant Env-specic humoral and cellular immune response. HIV-VLPs have been produced in different expression systems, such as Vaccinia virus, yeast spheroplasts, mammalian cells, plant cells (i.e. tobacco) [4,21,2325]. However, the Baculovirus-based transient protein expression vector system (BEVS) is the most widely used, considering the many advantages of the insect-cellbased protein production: (1) the large production of correctly folded recombinant proteins obtained in eukaryotic cells at highdensity cell culture conditions; (2) the absence of mammalian-cell derived supplements in culture media, drastically reducing the risk of culturing opportunistic pathogens; (3) the narrow host range of the baculovirus, limited to few species of Lepidoptera, without any threat to vaccinated individuals [26]; and (4) the effective scale-up of baculovirus system for large-scale vaccine production [27]. VLPs based on HIV-1 precursor 55 kDa (Pr55) Gag protein have been produced in baculovirus expression system by many groups, including ours [13,16,18,2830]. In particular, the VLPs developed in our laboratory display an entire gp120 molecule from an Ugandan HIV-1 isolate of the A clade [31,32] anchored through the trans-membrane (TM) portion of the Epstein-Barr virus (EBV) gp220/350 [18]. These HIV-VLPA s induce HIV-1-specic CD4+ and CD8+ T cell responses as well as cross-clade neutralizing antibodies in immunized BALB/C mice [33]. Moreover, the intraperitoneal and intranasal administration of HIV-VLPA s in mice induce antibody responses at systemic as well as mucosal (vaginal and intestinal) level [34,35]. More recently, we have developed Pr55Gag-VLPs expressing chimeric HIV gp140 envelope proteins (gp120 + gp41 ectodomain) linked to the trans-membrane domain from the heterologous baculovirus major glycoprotein gp64 (gp64TM), showing a trimeric conformation and induction of humoral response with neutralization activity (Visciano et al., submitted for publication). In parallel to the BEVS strategy, we recently described the establishment and characterization of a stably transfected insect cell line for the constitutive expression of the Pr55 HIV Gag protein which auto-assemble into enveloped Virus-Like Particles (VLPs). Such VLPs are released into the cell culture supernatant and have shown to elicit specic anti-Gag systemic humoral response in vivo [36]. This new approach seems to improve the expression and extracellular secretion of VLPs particles circumventing many difculties associated with the conventional baculovirus-infected cell approach [3739]. In this report, we describe the establishment of a stable double transfected insect cell line for the constitutive and reproducible production of Pr55Gag-VLPs expressing on their surface trimeric forms of whole HIV-1 envelopes. Such VLPs have been characterized for both protein expression and conformation and their in vivo immunogenicity has been evaluated in a mouse model.

to replace at NH2 -terminus the MRVMGTQTSWQNLWRWGTMILGMIIIC signal sequence of the HIV envelope. On the other hand, the sequence codifying for the MAEGELAAKLTSFMFGHVVNFVIILIVILFLYCMIRNRNRQY trans-membrane domain of the baculovirus major glycoprotein gp64 (gp64TM) was introduced at C-terminus of the chimeric gene. The chimeric HIV gp140 envelope gene was sub-cloned into EcoRV site of the pUC57 vector (GenScript Corporation), and positive clones were screened for the correct orientation relative to the driving promoter by nucleotide sequence analysis.

2.2. Construction of gp140 expression plasmid The chimeric codon-optimized HIV-1 gp140 coding region (2232 bp) has been PCR amplied using the primer pair 5 BNG (5 -TATCGCATGAAGTTCCTCGTGAACG-3 ) and 3 BNG (5 GCGGCGTTAGTACTGTCTATTTCTG-3 ), where nucleotides in bold indicate the 5-prime and 3-prime sequence of the amplied chimeric envelope gene. The full-length PCR product was subcloned into NcoI site of the pBiEx-3 dual-host expression vector (Novagen, Darmstadt, Germany), which allows protein expression in both bacteria and insect cells. The insertion of the full-length HIV-1 gp140 fragment in the vector was veried by PCR analysis; moreover, the correct in-frame integration with respect to the upstream T7 promoter was conrmed by nucleotide sequence analysis.

2.3. Transient expression of gp140 protein in gag-transgenic High-Five insect cell line Gag-transgenic High-Five insect cell line [36], derived from Trichoplusia ni egg cell homogenates (Life Technologies, Carlsbad, CA), was propagated in suspension in serum-free SF900 medium (Life Technologies, Carlsbad, CA) in presence of 1 mg/ml G-418 at 28 C, shaking at 150 rpm. 1 107 cells, in the logarithmic growth phase, were seeded in 8 ml of serum-free medium in a 125-ml Erlenmeyer ask and transfected with 20 g of gp140-recombinant pBiEx3, according to the protocol provided with the Insect GeneJuce Transfection Reagent (Novagen, Darmstadt, Germany). Cells were incubated and collected 2 days post-transfection for evaluation of HIV gag and env gene and protein expression.

2.4. Constitutive expression of gp140 protein in gag-transgenic High-Five insect cell line Gag-transgenic High-Five insect cell line [36] were seeded at a concentration of 1 106 cells per well in 6-well plates. A mixture of 2 g of gp140-recombinant pBiex-3 and 0.2 g of pIE1-Hygro plasmids were combined with Insect GeneJuce Transfection Reagent (Novagen, Darmstadt, Germany) (dened as transfection mixture). After 15 min of incubation at room temperature, the transfection mixture was added to the seeded cells and incubated at 28 C for 24 h. Cells were detached from the wells by gentle squirting, to generate multiple sparsely seeded culture dishes at several densities, by two-fold serial dilutions starting from 5 104 cells per 60-mm Petri plate. SF900 medium (Life Technologies, Carlsbad, CA), supplemented with 10% FCS, 1 mg/ml of G418 and 200 g/ml of hygromycin, was added to each plate and cells were incubated at 28 C until single resistant colonies were visible to the naked eye (approximately 2 weeks). Individual colonies were identied and transferred to a new plate for a stepwise amplication until a sufcient number of cells were available for gene and protein expression analysis.

2. Materials and methods 2.1. Generation of chimeric HIV envelope genes The chimeric HIV gp140 envelope genes containing the Honeybee Mellitin signal sequence (HMSS) at the 5 -end and the transmembrane domain of the baculovirus major glycoprotein gp64 (gp64TM) at the 3 -end, were synthesized and codon-optimized for expression in eukaryotic cells (GenScript Co., Piscataway, NJ, USA) (Fig. 1). In particular, the sequence codifying for the MKFLVNVALVFMVVYISYIYA Honeybee Mellitin signal sequence was used

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Fig. 1. Schematic representation of chimeric HIV envelope proteins. (A) Full-length gp140 has been generated including coding sequences for gp120 and gp41 ectodomain. It has been fused to Honeybee Mellitin signal sequence (HMSS), at the 5 -end, and to the trans-membrane domain of baculovirus major glycoprotein gp64 (gp64TM), at the 3 -end. (B) Modications to generate SOSIP gp140 are indicated.

2.5. Molecular characterization of High-Five transfected cells 2.5.1. PCR analysis Genomic DNA was extracted by Maxwell DNA purication kit (Promega, Madison, WI) from transient or G418 and hygromycinresistant stable double transfected High-Five insect cells, as well as from control cells, according to the manufacturers instructions. The fragments spanning the p24 and p7 region of the HIV gag gene (460 bp) and the entire gp140 env gene (2232 bp) were amplied by PCR from approximately 1 g of puried DNA (corresponding to 1.5 105 cells), using the primer pairs H1GAG158 (5 -AAAGATGGATAATCCTGGG-3 ; position 9911009 in HIVHXB2 clone) and G17 (5 -TCCACATTTCCAACAGCCCTTTTT3 ; position 14341457 in HIVHXB2 clone), and 5 BNG (5 -TATCGCATGAAGTTCCTCGTGAACG-3 ) and 3 BNG (5 -GCGGCGTTAGTACTGTCTATTTCTG-3 ), respectively, for gag and gp140 env genes as previously described [40]. 2.5.2. RT-PCR analysis Total RNA was extract from transient or G418 and hygromycinresistant stable double transfected High-Five insect cells, as well as from control cells, by TRIzol solution (Life Technologies, Rockville, MD). Purity of the RNA preparation was veried by spectrophotometric reading and integrity of extracted RNA was evaluated by separation on agarose gel. One hundred nanogram of each total RNA sample was analyzed by RT-PCR using the same primer pairs indicated in the previous section with rTth RNA PCR kit (Applied Biosystems, Foster City, CA). 2.5.3. SDS-PAGE, BN-PAGE and Western blot analysis Total protein concentration in cytoplasmic fraction as well as concentrated culture supernatant was assessed by the Bradford method (Bio-Rad Laboratories, Hercules CA), using bovine serum albumine as a standard. Equivalent amount of total proteins were mixed with same volume of 2 Laemmli sample buffer [41], boiled for 5 min and electrophoresed on a 10% denaturing SDS-PAGE. Recombinant HIV p24 (ARP689, NIBSC Centralised Facility for AIDS Reagents, Hertfordshire, UK) as well as recombinant HIV gp140 (ARP698, NIBSC Centralised Facility for AIDS Reagents, Hertfordshire, UK) were run in parallel as controls. Electrophoresed proteins were visualized by staining with Comassie Brilliant Blue R-250 (SigmaAldrich, St. Louis, MO). For native BN-PAGE analysis, VLPs were incubated in an equal volume of solubilization buffer (0.12% Triton X-100 in 1 mM EDTA/1.5 M aminocaproic acid) to liberate envelope proteins [21,42] and separated on a 416% Bis-Tris NuPAGE gel (Invitrogen). Ferritin (Amersham) was used as a size standard. The gel was transferred onto polyvinylidene diuoride blotting membrane. Excess Coomassie blue dye was removed after blotting by washing with 30% methanol/10% acetic acid and subsequently with 100% methanol.

Gag and gp140 Env proteins expression, after SDS- or BN-PAGE, was evaluated by WB analysis incubating blotted proteins with a 1:400 dilution of rabbit polyclonal anti-p24 anti-serum (ARP432, NIBSC Centralised Facility for AIDS Reagents, Hertfordshire, UK) or mouse monoclonal anti-gp140 (ARP3119, NIBSC Centralised Facility for AIDS Reagents, Hertfordshire, UK). After an overnight incubation, secondary peroxidase-conjugated goat anti-rabbit and anti-mouse IgG, respectively (Santa Cruz Biotechnology Inc., Santa Cruz, CA) (1:1000 dilution) were added for 2 h. After extensive washes with 1 TBS, bound antibodies were visualized adding 4chloro-1-naphtol as substrate. 2.6. Characterization of env glycoprotein conformation The conformation of Env glycoproteins presented on the VLP surface has been evaluated by an enzyme-linked immunosorbent assay (ELISA). 96-well MICROTEST assay plates (Becton Dickinson) were coated with 2.5 g of VLPs, native or heat-inactivated (1 h at 56 C), alternatively with 200 ng of either trimeric or monomeric Ugandan clade A gp140 envelope protein. Plates were incubated with different anti-gp120 MAbs: CA13 (NIBSC-CFAR, Catalog # ARP3119), 2G12 (NIBSC-CFAR, Catalog # EVA3064), b12 (NIBSCCFAR, Catalog # ARP3065). After washes with PBS-Tween (0.1%), wells were incubated 90 min at 37 C with HRP-conjugated rabbit anti-mouse (CA13) or anti-human (2G12 and b12) IgG antibodies (1:1000 dilution). Positive reactions were visualized with TMB Ultra 1-step solution (Thermo Scientic) and stopped with 2 N sulfuric acid. 2.7. Electron microscopy Supernatants from G418 and hygromycin-resistant stable double transfected and Mock-transfected High-Five insect cells were collected for electron microscopy analysis. Supernatants were claried by centrifugation at 2000 g for 15 min at 4 C, VLPs were pelleted by ultra-centrifugation at 100,000 g for 75 min through a 25% sucrose cushion and resuspended in 1 PBS, as previously described [18,43]. The presence of VLPs expressing the HIV1 gp140 released into extracellular medium or budding from the stable double transfected High-Five insect cells were determined by standard analytical methods for transmission electron microscope [44]. In particular, VLPs were adsorbed on microscope carbon-coated grids by submersion for 2 min at room temperature, washed with distilled water and negative-stained with 2% uranyl acetate aqueous solution. For the immunogold analysis, adsorbed VLPs were incubated for 5 min at 38 C with a mix of 1:10 dilution of anti-gp41 (2F5 and 4E10) and anti-gp120 (2G12 and b12) MAbs (all from NIBSC). Final concentration of each MAb was: 100 g/ml (2F5), 50 g/ml

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(4E10), 200 g/ml (2G12) and 100 g/ml (b12). Specic secondary anti-human IgG gold conjugated 10 nm (SigmaAldrich Chemie, Germany) were used at 1:20 dilution. Observations were performed with FEI Morgagni 282D electron microscope operating at 60 kV. In parallel, stable double transfected High-Five insect cells were prepared for ultrathin sectioning according to standard procedures [45]. In particular, cells were xed in ice-bath for 1 h in 2.5% glutaraldehyde in 0.05 M phosphate buffer, post-xed in 1% osmium tetroxide, stained overnight in 0.5% aqueous uranyl acetate, dehydrated in graded ethanol dilutions and embedded in TAAB SPURR RESIN with ERL 4221D-medium grade (Agar Scientic, UK). Thin sections were stained with lead citrate prior to viewing under the FEI MORGAGNI 282D electron microscope using an accelerating voltage of 60 kV. 2.8. Immunization experiments BALB/C mice were injected subcutaneously (s.c.) with 20 g of puried VLPs resuspended in 200 l of PBS, with or without 20 g of TLR9 ligand ODN1826 (Invivogen, San Diego, CA). The immunization schedule was based on a 2-dose regimen, where the booster inoculation was administered at 3 weeks after the primary injection; serum samples were collected 1 week after each of the two immunizations by blood drawing from the retro-orbital vein. All animals were sacriced at week 4 by cervical dislocation, with collection of serum samples. 2.9. Serological assays The presence and the titer of specic antibodies was evaluated in serum samples obtained from immunized mice by an enzymelinked immunosorbent assay (ELISA). 96-well MICROTEST assay plates (Becton Dickinson) were coated with 100 ng of puried gp140 Env protein (NIBSC-CFAR, Catalog # ARP698). Plates were incubated with dilutions of heat-inactivated mouse sera ranging from 1:100 to 1:100,000. After washes with PBS-Tween (0.1%), wells were incubated 90 min at 37 C with HRP-conjugated rabbit anti-mouse IgG (BioRad) antibodies (1:1000 dilution). Positive reactions were visualized with TMB Ultra 1-step solution (Thermo Scientic) and stopped with 2 N sulfuric acid. Absorbance was determined at O.D.450 and reactions were considered positive when exceeding the mean absorbance 2 standard deviations of equal dilutions of pre-immunization sera collected from animals. The antibody levels were evaluated as the geometric mean titer of the last positive dilution of sera from the animals of each set. 3. Results 3.1. Design of chimeric Env proteins The chimeric gp140 sequence to be displayed on the VLP surface was based upon the gp120 derived from the A-clade Ugandan HIV-1 isolate previously characterized in our Laboratory [31,32] (GenBank Accession No. AF062521) and used to generate the HIVVLPA s developed by our group [18,3335]. As previously dened, the gp140 envelope was obtained including at the carboxyl terminus of the gp120 sequence the gp41 ectodomain, which retains the oligomerization domain [46,47]. Such gp140 has been further modied, given that (1) the autologous signal sequence has been substituted with the heterologous Honeybee Mellitin signal sequence (HMSS) and (2) the trans-membrane domain from the baculovirus major glycoprotein gp64 (gp64TM) has been linked to the carboxyl terminus of the gp41 sequence (Fig. 1A). Furthermore, a disulde bond has been introduced between the C-terminal region of gp120 (G517C) and the immunodominant segment of the gp41 ectodomain (P621C), to stabilize the association between the two subunits, as previously described (SOS gp140) [48]. Moreover, an I575P substitution into the N-terminal heptad repeat region of gp41 has been introduced to stabilize the gp41-gp41 interactions (Fig. 1B). This modied gp140 (designated SOSIP gp140) has been previously shown to be properly folded, proteolytically cleaved, substantially trimeric, with appropriate receptor binding and antigenic properties [49]. 3.2. Transient expression of gp140 protein in High-Five cells The full-length chimeric gp140 sequence (2232 bp) was PCR amplied and cloned in the NcoI restriction site downstream of T7 RNA polymerase promoter of the dual-host expression vector pBiEx-3 (Novagen, Darmstadt, Germany). The expected length of the sub-cloned fragment was veried by PCR analysis; moreover, the correct in-frame integration with respect to the upstream T7 promoter was conrmed by nucleotide sequence analysis (data not shown). Gag-transgenic High-Five insect cells [36] were transfected with gp140-recombinant pBiex-3 and transient intracellular expression was investigated at 2 days post-transfection. As expected, the gp140 sequence was identied by PCR only in cells transfected with the pBiex-3 plasmid carrying the gp140 envelope DNA sequence (Fig. 2, lane 4). In parallel, the gag sequence was identied in all transfected cells, regardless the transfection mixture, conrming the retention of DNA sequences in the Gagtransgenic High-Five insect cells used in this experiment (Fig. 2, lanes 57). Protein expression was veried in such transiently transfected cells and, as previously reported for the transiently gag transfected High-Five cells [36], the expected chimeric gp140 Env protein was not detectable at Comassie staining in the cytoplasmic fraction (data not show). On the contrary, Western blot analysis, performed using mouse monoclonal anti-gp140, revealed a band compatible with the full-length gp140 protein only in cells transfected with the pBiex-3 plasmid carrying the gp140 DNA sequence (Fig. 3A, lane 4 pBiexRec). In parallel, rabbit polyclonal anti-p24 antibody

Fig. 2. Transient transfection of gp140 env gene in gag-transgenic High-Five insect cells. Amplication of gag and gp140 genes was performed by PCR on transfected gag-transgenic High-Five cells. The expected fragments of 474 bp and 2232 bp, respectively for gag and env genes, were identied by electrophoretic separation on a 1% agarose gel and are indicated by the arrows. Neg = reactions negative control; Mock = gag-transgenic High-Five cells treated only with Transfection Reagent; pBiex = gag-transgenic High-Five cells transfected with pBiex vehicle DNA; pBiexRec = gag-transgenic High-Five cells transfected with gp140recombinant pBiex-3 plasmid. M = Lambda Hind III DNA Marker.

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showed the constitutive expression of Pr55Gag protein in the gagtransgenic High-Five insect cells (Fig. 3B). 3.3. Stable expression of gp140 protein in Gag-transgenic High-Five cells The observed expression in the transient transfection assay, led our efforts to establish a stable double transfected High-Five insect cell line constitutively producing Pr55Gag-VLPs presenting the HIV-gp140 Env protein. Neomicin-resistant gag-transgenic HighFive insect cells were co-transfected with the gp140-recombinant pBiex-3 and the pIE1-Hygro plasmids, and cultured in the presence of G418 and hygromycin antibiotics, to select double-transfected cell clones. Individual double-antibiotic resistant colonies were progressively expanded and investigated for intracellular HIV Gag and gp140 Env protein expression. The persistence of gag and gp140 env genes in neomycinhygromycin resistant and stable double transfected HighFive clones, was conrmed in two clones by PCR analysis using gag and env-specic primers pairs (Fig. 4A, lanes 2, 3, 5, and 6). As expected, only the gag, and not the gp140 env fragment was amplied in the Mock-transfected cells (Fig. 4A, lanes 1 and 4). Furthermore, in the same stable double transfected clones, the presence of specic gag and gp140 env transcripts was veried by RT-PCR analysis (Fig. 4B, lanes 2, 3, 5, and 6). The expression of HIV-1 Gag and gp140 Env proteins in stable double transfected HighFive clones was investigated by Western blot analysis, using anti-Gag rabbit polyclonal and anti-gp140 Env mouse monoclonal antibodies. Fig. 5 shows the presence of a specic band corresponding to Gag and gp140 Env proteins in the cytoplasmic fraction of stable double transfected HighFive clones 1 and 2 (Fig. 5A and B, lanes 1 and 2) as well as in their culture supernatant, after ultra-centrifugation through a 25% sucrose cushion, suggesting the release of Pr55Gag/gp140Env-VLPs (Fig. 5A and B, lanes 3 and 4). The expression of Pr55 Gag protein was monitored by Western blot analysis on cytoplasmic as well as supernatant fractions and reproducibly observed over 15 cell culture sub-passages in presence of G418. In particular, the continuous VLP expression is routinely veried by WB analysis every ve cell culture passages. The expression of released VLPs ranges about 3 mg/L, at a cell density of 1.52 106 cell/ml, with a less than 10% variation in production and purication yield overtime (data not shown).

Fig. 3. Transient expression of gp140 Env protein in gag-transgenic High-Five insect cells. Total protein extract from cytoplasmic fraction was separated on 10% SDS-PAGE. Western blot analysis performed using mouse monoclonal antigp120 antibody (A) or rabbit polyclonal anti-p24 antibody (B). The expected gp140 Env protein or Pr55 kDa Gag protein are indicated by arrows in the two panels. Mock = gag-transgenic High-Five cells treated only with Transfection Reagent; pBiex = gag-transgenic High-Five cells transfected with pBiex vehicle DNA; pBiexRec = gag-transgenic High-Five cells transfected with gp140-recombinant pBiex-3 plasmid.

Fig. 4. Stable transfection of gp140 env gene in gag-transgenic High-Five insect cells. (A) Amplication of gag and gp140 genes was performed by PCR on stable double transfected High-Five cells. The expected fragments of 474 bp and 2232 bp, respectively for gag and env genes, were identied by electrophoretic separation on a 1% agarose gel and is indicated by the arrows. (B) RT-PCR analysis performed on the same cells. Neg = reactions negative control; Mock = gag-transgenic High-Five cells treated only with Transfection Reagent; Clones 12 = two independent clones of gag-transgenic High-Five cells transfected with gp140-recombinant pBiex-3 plasmid. M = Lambda Hind III DNA Marker.

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Fig. 7. Evaluation of conformational epitopes on gp140 Envelope proteins expressed on Pr55Gag/gp140 Env-VLPs. VLPs have been plated as a native or heat-denaturated form, to disrupt the trimeric structure of presented envelope glycoproteins, and probed with the three MAbs CA13, 2G12 and b12. Results are shown as percentage of binding reduction to plated antigen. VLP (denatured/native) and gp140 (monomeric/trimeric). Fig. 5. gp140 Env protein expression in double stable transfected High-Five cells. Western blot analysis was performed using rabbit polyclonal anti-p24 antibody (A), and mouse monoclonal anti-gp120 antibodies (B). The expected Pr55 kDa Gag and gp140 Env proteins are indicated by arrows in the two panels. Cytoplasmic fraction (lanes 1 and 2) and cell culture supernatant (lanes 3 and 4) from double stable transfected High-Five cells are shown.

3.4. Characterization of gp140 Env protein conformation on VLPs In order to verify the conformation of gp140 Env proteins expressed on the surface of the released VLPs, proteins were electrophoresed in native conditions by BN-PAGE. The subsequent WB analysis showed, besides the gp120/gp41 monomer band, an additional and prevalent band of approximately 400 kDa corresponding to authentic trimers, conrming that expression in membranes provides stability to Env trimers [21,42] (Fig. 6A). Furthermore, in order to verify whether the gp120 and gp41 subunits in the trimers were covalently or noncovalently associated, VLPs were analyzed in denaturing SDS-PAGE under reducing conditions. The results showed that increasing concentrations of reducing agent (dithiothreitol, DTT) were able to completely disrupt the gp120-gp41 intra-molecule disulde bonds, leading to identication of a single band compatible with gp41 protein (Fig. 6B, lane 2) and conrming that the gp120/gp41 monomers are non-covalently associated. Furthermore, the conformation of env glycoproteins presented on the VLP surface has been evaluated also by an enzyme-linked immunosorbent assay (ELISA). VLPs have been plated as a native or heat-denaturated form, to disrupt the trimeric structure of pre-

sented envelope glycoproteins. The results showed a 50% reduction in the binding to denatured VLPs by MAbs b12 and 2G12, which are known to recognize conformational epitopes, compared to the preservation of the binding by the MAb CA13 (Fig. 7). Such results conrm that, when the trimeric conformation of the env glycoprotein is disrupted, the binding of antibodies to conformational epitopes is severely affected. In parallel, binding to the monomeric soluble protein is much less affected compared to the trimeric, conrming the preservation of the target conformational epitopes. 3.5. Characterization of Pr55Gag/gp140Env-VLPs by electron microscopy analysis The formation of Pr55Gag-VLPs expressing the gp140 Env protein on the surface was investigated by electron microscopy analysis performed on stable double transfected HighFive clones or on their culture supernatant concentrated by ultra-centrifugation through a 25% sucrose cushion. HighFive clones were found to produce immature Virus-Like Particles with size and morphology appropriate for HIV-VLPs; the diameter is of approximately 100 nm and VLPs show a characteristic high density Gag layer underneath the cell membrane (Fig. 8A and B). The same cells, showing VLPs budding from the membrane, contains also long hollow cylinders (Fig. 8C) which closely recall the structure formed by helical arrays of Gag-p24 hexamers [5053]. When released Pr55Gag/gp140Env-VLPs were immunostained with a mix of MAbs targeting the HIV envelope glycoprotein

Fig. 6. Characterization of gp140 Envelope proteins expressed on Pr55Gag/gp140 Env-VLPs. Full-length gp140 were analyzed by native BN-PAGE, to verify the trimeric structure of the gp120/gp41 expressed on VLPs (A). Alternatively, proteins were analyzed in denaturing SDS-PAGE under reducing conditions (B). Proteins were detected with mixture of anti-gp120 b12 and 2G12 MAbs (A) or a mixture of anti-gp41 2F5 and 4E10 MAbs (B).

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Fig. 8. Standard transmission electron microscopy (EM) analysis of Pr55Gag/gp140 Env-VLPs. Stable double transfected High-Five cells showing budding VLPs from the cell membrane (A and B) and intracellular long hollow cylinders (C). Gp140 spikes on released Pr55Gag/gp140 Env-VLPs were immunostained with gold particles (D). The bars represent 100 nm; MC: mitochondria.

before electron microscopy observation. In particular, the mix contained MAbs 2F5 and 4E10, specic for gp41 epitopes [54,55]; 2G12 and b12, specic for gp120 epitopes [5557]. Gold particles (10 nm diameter) were found surrounding the Gag particles, conrming the HIV gp140 Env spikes anchored on the surface of particles (Fig. 8D), with a density similar to the one previously observed by cryoelectron microscopy tomography on wild-type HIV-1 virions [58]. Gold particles not associated to VLPs may possibly result from binding to shedded gp140 Env molecules. 3.6. In vivo humoral immune response induced by Pr55Gag/gp140Env-VLPs In order to evaluate the induction of immune response targeting gp140 Env proteins displayed by Pr55Gag/gp140Env-VLPs produced by stable double transfected HighFive clones, VLPs were administered subcutaneously (s.c.) in BALB/C mice in duplicated experiments with a prime and single homologous boost immunization schedule, with or without 20 g of the TLR9 ligand ODN1826. Blood samples were drawn from the retro-orbital vein a week before the priming dose (pre-immunization) and a week after each administration. Each immunization group contained 3 animals which did not show signs of toxicity and remained healthy up to the end of the vaccination protocol. ELISA tests were performed on microwell plates coated with 100 ng of recombinant gp140 protein and data show that the immunization protocol effectively elicited a progressive increase of serum anti-Env titers, up to a nal 1:105 dilution (Fig. 9). In particular, the adjuvanted formulation induced a 1-log increase in the

specic serum anti-Env titers (two-tailed P = 0.0131), with an overall intra-group variability lower than 5%. These results are in line with previous data obtained with VLPs produced with Baculovirusbased transient protein expression vector system (BEVS) [33,34]

Fig. 9. Levels of anti-Env humoral response induced in mice by Pr55Gag/gp140 Env-VLPs. Geometric mean titers of serum antibodies induced in BALB/C mice in duplicated experiments by administration of VLPs via subcutaneous route. Serum samples were obtained at the end of the immunization protocol and analyzed for specic mouse IgG antibodies by ELISA, performed in triplicate on 96-well plates coated with rgp140. Results are expressed as the individual average reciprocal last dilution exceeding the mean absorbance 2 standard deviations of equal dilutions of pre-immunization sera. Bars represent the geometric mean.

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and show the effective immunogenicity of Pr55Gag/gp140EnvVLPs generated by the stable double transfected High-Five cells.

4. Discussion Gag-transgenic High-Five insect cells, constitutively expressing the HIV Pr55 Gag protein autoassembling into enveloped Virus-Like Particles (VLPs) [36], were transfected with HIV gp140 Env expressing vector to obtain a double transfected insect cell clones for the constitutive, efcient and reproducible production of Pr55Gag -VLPs presenting trimeric HIV gp140 Env proteins (Pr55Gag/gp140Env-VLPs). In particular, the HIV gp140 env gene, derived from an Ugandan HIV-1 isolate of the A-clade and used to generate the HIV-VLPA s developed by our group [18,3135], has been modied to stabilize the association between the gp120 and gp41 subunits as well as the gp41gp41 interactions (SOSIP gp140) [48,49]. These modications are required, given that the cleavage and post-translational maturation of the gp140 into gp120 and gp41 occurs in insect cells driven by the endogenous furin [22,59]. Constitutive expression of HIV Pr55 Gag and gp140 Env proteins was conrmed by Western blot analysis in several independent resistant clones, which showed different levels of protein expression. Individual transfected clones, indeed, may show a wide range of protein expression levels due to possible different reasons, including copy numbers of transfected plasmids, percentage of transduced cells and genomic integration sites. In fact, antibiotic selection does not fully guarantee retention and/or co-expression of the unselected marker(s) present on co-transfected plasmid [37]. Furthermore, the trimerization of gp120/gp41 complex presented on the surface of the Pr55Gag/gp140Env-VLPs was demonstrated by non-denaturing BN-PAGE and the non-covalent association between the two subunits of each gp120/gp41 monomer has been conrmed by SDS-PAGE analysis performed in different reducing conditions. Furthermore, the presentation of conformational epitopes on trimeric env glycoproteins was demonstrated by severe reduction of binding by MAbs 2G12 and b12 to heat-denatured VLPs. Considering, indeed, that envelope proteins on the virus surface are assembled into trimers, it is assumed that the induction of potent and broad anti-HIV neutralizing antibodies can be achieved only through HIV envelope trimers closely mimicking the structure of native functional Env complexes [6062]. The stability of both HIV Gag and Env protein expression was monitored and reproducibly observed over at least 15 cell culture passages and is routinely checked to conrm the genetic stability of double transfected cell line. Constitutively expressed Pr55Gag/gp140Env-VLPs show size and morphology appropriate for HIV-VLPs and are released by cell membrane budding into the cell culture supernatant, as demonstrated by standard transmission electron microscopy. Moreover, the expression of gp140 Env protein on the VLPs surface has been conrmed by immunogold staining with a mix of MAbs targeting the HIV envelope gp41 and gp120 glycoproteins. Such observation conrm that, in the stable double transfected insect cells, the intra-cellular expression level of HIV Pr55 Gag protein reaches the required threshold necessary for the auto-assembling of the protein, formation and release into extracellular medium of Virus-Like Particles presenting the gp140 Env protein with a density similar to the one observed on wild-type HIV-1 virions [58]. The Pr55Gag/gp140Env-VLPs generated by the stable double transfected High-Five cells have been shown to elicit systemic antiEnv humoral response in vivo, conrming the appropriate antigenic presentation of the HIV gp140 Env proteins to the immune system. This represent, to our knowledge, the rst report on production of enveloped Pr55Gag-VLPs presenting trimeric HIV-gp140

on their surface, using stable transfection instead of the transient baculovirus expression vector system (BEVS). The choice of such approach is supported by data reporting signicantly higher production of extracellular or membrane-bound proteins compared to conventional transient baculovirus infection strategy [3739]. It has been shown, indeed, that the overall yield of foreign proteins may be affected by a cysteine protease produced during baculovirus infection cycle [39,63,64] and that expression of extracellular and membrane-bound proteins is perturbed by adverse effects of baculovirus infection on the host insect cell secretory pathway [65]. Moreover, production of VLPs from stable transfected insect cells overcome the contamination by co-puried baculovirus particles, produced during baculovirus infection cycle and representing a potential concern for regulatory Agencies. The here described strategy to generate stable double transfected insect cell clones for the constitutive expression of enveloped Pr55Gag-VLPs, presenting trimeric gp140 Env proteins on the surface, will allow the consistent as well as reproducible production of VLPs, increasing the opportunities of expressing additional antigenic and/or adjuvanting proteins within VLPs as well as facilitating the scale up step for GLP/GMP production.

Acknowledgements The study was supported by the European Communitys Seventh Framework Programme NGIN (FP7/2007-2013) under grant Agreement No. 201433. M.T. and M.L.V. are funded by the NGIN Programme. Recombinant HIV p24 (ARP689), gp140 (ARP698), gp41 (ARP680), rabbit polyclonal anti-p24 anti-serum (ARP432), mouse monoclonal anti-gp140 (ARP3119), anti-gp41 2F5 and 4E10 MAbs (EVA3063 and ARP3239), anti-gp120 2G12 and b12 MAbs (EVA3064 and EVA3065) were obtained through the NIBSC Centralised Facility for AIDS Reagents, Hertfordshire, UK. We thank Don Jarvis, University of Wyoming, for kindly providing us pIE1-Hygro plasmid. Contributors: M.T. performed the studies and contributed to writing the paper; M.L.V. contributed to immunological evaluations; M.L.T. contributed to the data analysis; A.D.S. performed the electron microscopy analysis; F.M.B. supervised the whole project; L.B. was responsible for the overall planning and coordination of the study as well as writing the paper. All authors read and approved the nal manuscript. Conict of interest statement: The authors declare no competing nancial or other interests.

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