Construction of HBsAg–MPER variants
The synthetic S-gene HBsAg (Berkower et al., 2004
) amino acid 2 to 226 was used as a scaffold to implant the membrane proximal regions of HIV-1 gp41 at the N-terminus, C-terminus or the extra-cellular loop of the HBsAg. S1-gene was PCR-amplified from the vector pGEM using the forward primer 5′ GGAGCTCGTCGACAGCAA3′ and reverse primer 5′GCTCTA GACCCGATGTAGACCCA 3′ to introduce Sal I site at the 5′ and Xba I site at the 3′ end of the gene. The PCR-amplified product was cloned in to pCMV/R vector at the Sal I and Xba I sites. Variants of gp41 sequences containing the MPER were PCR-amplified using codon-optimized HIV-1 YU2 gp160 or JRFLgp160 constructs as the template. Primer pairs used are listed in . The MPER was positioned at the C-terminus of S1 gene (C-MPER) and at the N-terminus after the 2nd
amino acids (E and F, respectively), of the HBsAg S1 sequence (N-MPER). An Age I site was created in the extra cellular loop of the S1 gene by replacing amino acids P126
with T and G substitutions. Following the G residue, the MPER codons (with a 3 amino acid linker GTG at the C-terminus of the MPER codons) were cloned at the Age I site to place it in the extra cellular loop of S1 sequences (ECL-MPER). The second sets of constructs were generated to introduce different lengths of HIV-1 transmembrane region after the lysine 683 of the MPER, 1
for C-MPER-5, 1
for C-MPER-10, 1
for C-MPER-15 and 1
TETSQVAPA – C9 tag for C-MPER-22-C9 in order to further stabilize and orient the 4E10 epitope. The final set of constructs was generated with HIV-1 gp41 region, C-heptad and or the MPER at the C-terminus of HBsAg. Between the HBsAg and the gp41 region, a two amino acids S and R were introduced and following the K at residue 683, a glycine was placed immediately before the stop codon. The T4 fibritin trimerization domain, foldon, was also introduced in two of the constructs to see the effect of trimerization on recombinant HBsAg particle production and recognition of 2F5 and 4E10.
Oligonucleotides used for generating HBsAg-MPER constructs
Transient transfections, particle production and analysis
All the constructs were transfected in HEK293T cells. One day prior to transfection, 8 million HEK 293T cells in DMEM, 10 % FBS, 1 % penicillin-streptomycin (pen-strep) were seeded in a 150 mm tissue culture dish. The cells were transfected with the plasmids encoding recombinant HBsAg-MPER and MPER variants or wild-type HBsAg using Fugene6 (Roche) at a ratio of DNA:Fugene6 1:3 and 10 ug total DNA plate. Four days after transfection, cells and supernatant were collected. Supernatant was concentrated using Centricon Plus-80 100 kDa Biomax membrane (Millipore, Billerica, MA) to 25 mls. The cells were lysed by resuspending it in 10 ml of 1 X PBS and sonication for 1 min at 20 Hz every 10 sec using a probe sonicator and the cell lysate was clarified by centrifugation at 15,000 rpm in an Eppendorf centrifuge for 15 mins. Cell lysates were loaded on a 20% sucrose cushion (20% sucrose in PBS) and centrifuged at 23,000 rpm for 16 hr (Surespin rotor, Sorvall). The partially purified VLPs were resuspended in PBS and analyzed by ELISA or Western blotting. To further purify the VLPs, the particles were loaded onto a 10–40% (wt/wt) CsCl step gradient (in PBS) and centrifuged at 22h at 36000 rpm (TV-860 rotor; Sorvall), and a 500μl fractions were taken from the bottom of the tube. ELISA identified the fractions containing VLPs. The positive fractions were, desalted, concentrated, washed with PBS using Amicon YM-100 filter (Millipore).
EM analysis of the particles
The HEK293 cells transfected with C-terminus HBsAG-MPER DNA were collected 24 hrs after transfection and washed with PBS. Approximately 3 × 106 cells were fixed in 2% paraformaldehyde-2% glutaraldehyde in 0.10M cacodylate buffer. Thin sections were negatively stained with 2% methylamine tungstate and analyzed by electron microscopy at a magnification of 100,000X. For cell-free particles, the fractions from the CsCl gradient were selected by optical density and the particles contained in those fractions were negatively stained with 2% methylamine tungstate and analyzed by electron microscopy.
Recombinant baculovirus particle production
The HBsAg-MPER sequence was PCR-amplified from pCMV/R HBsAg-MPER construct and ligated into the pFastbac plasmid between the Sal I and Spe I restriction sites. The pFastbac sAg-MPER plasmid was transformed into competent cells containing full-length bacmid DNA (Invitrogen), to generate recombinant bacmid. Two positive bacmids were sent to ATG Laboratories, Inc., in Eden Prairie, MN for titer and production of recombinant baculovirus in Sf9 cells. For particle production, the Hi5 or Sf8 cells were infected at a multiplicity of infection of four and. the cell pellets were harvested at about 48 hours following infection. Cells were lysed by sonication and the lysate was layered onto sucrose gradients. After sedimentation for 2 hrs at 27,000 rpm in an SW28 rotor, fractions were collected from the bottom. Each fraction was assayed for MPER content, using monoclonal antibody 2F5. Particles were further purified either by CsCl gradient centrifugation for immunogenicity as described above or on a Macro-prep methyl HIC column (from Bio Rad) for SDS gel analysis. For purification by the hydrophobic methyl Macro-prep method, particles in the pooled sucrose fractions were suspended in 0.01 M sodium phosphate buffer, pH 6.8 with 0.6 M ammonium sulfate and 0.05% CHAPSO (Anagrade), washed in the same buffer and eluted with 0.2 M ammonium sulfate.
ELISA analysis of the HBsAg-MPER particles
To determine the presence of HBsAg, HBsAg-MPER and MPER variants VLPs in the preparations, ELISAs were performed as follows. For direct ELISA, the particles were adsorbed onto a high-protein-binding microwell plate (Corning) for 2hrs, then incubated with blocking buffer (bb; PBS with 2% dry milk). After one wash with PBS/0.2% Tween-20, anti-HBsAg antibody NE3 or NF5 (Aldevron) was added to each well in serial dilution and incubated at 37C for 1hr. After three washes with PBS/0.2% Tween-20, a secondary Anti-Mouse-IgG-HRP antibody (Sigma) was added in washing buffer at a 1:5000 dilution for 1 h at 37C. Following three washes, the ELISAs were developed with 100 μl TMB Peroxidase substrate (KPL). The reaction was stopped by adding 100 μl 1 M HCl to each well. The optical density at 450 nm was read on a microplate reader (Molecular Devices).
For sandwich ELISA, 500 nanograms of the HBsAg-specific mouse monoclonal antibody NE3 (Aldevron) was adsorbed onto each well overnight at 4°C. The next day, following incubation of bb, the particles were resuspended in PBS and 100 ul of the suspension was added to each well and incubated 37°C for 2 hr. After one wash with PBS/0.2% Tween-20, either the antibody 2F5 or 4E10 (kindly provided by H Katinger) or HIVIgG (NIH AIDS Reagent Repository Program) was added to each well as a serial dilution and incubated at 37°C for 1hr. After three washes with PBS/0.2% Tween-20, a secondary Anti-human-IgG-HRP antibody (Jackson Immuno Research labs) was added in washing buffer at a 1:5000 dilution for 1 h at 37°C. Following three washes, the ELISAs were developed as described above.
For competition ELISA, all the steps similar to sandwich ELISA were performed except that the peptide NEQELLELDKWASLWN was mixed along with 2F5 diluted and incubated at 37C for 1hr. To determine the effect of lipid on antibody binding, the Baculovirus expressed C-terminus MPER particles were treated with high and low pH and in PBS containing 1% CHAPSO and then diafiltered to remove the lipids and concentrate the particles. To a portion of the delipidated particles, the lipids DOPC:DOPS (7:3) (Avanti Polar lipids, Inc) were added, the samples was dialyzed against PBS and all particle preparations were analyzed by sandwich ELISA.
BalB/c mice were inoculated by the intramuscular route at intervals of 3 weeks with 5 μg of HBsAg or HBsAg-MPER particles in the presence of alum or 50 μg of HBsAg, HBsAg-MPER, HBsAg-MPER+15 DNA followed by HBsAg or HBsAg-MPER particle/alum boost. Animals were inoculated up to a total of five times. Sera was collected 7–10 days after each inoculation and analyzed either by ELISA, cell surface staining by FACS or in selected instances by a neutralization assay.
For the prime:boost immunization strategy, rabbits were immunized by two priming immunizations at weeks 0 and 4, followed by two boosting immunizations at weeks 8 and 12. The dose of inoculation of the ADA Env-PLs was approximately 20 to 30 ug of envelope glycoproteins as estimated by SDS gels using a defined volume of Env-PL bead suspension. Inoculation was by the intradermal route and CpG plus alum was used as adjuvant for all the immunogens.
Fluorescence-activated cell sorting (FACS) staining at the cell surface
FACS staining was performed as previously described (Koch et al., 2003
; Pancera and Wyatt, 2005
). Forty-eight hours following transfection, the cells were harvested and washed in FACS buffer (PBS, 5% fetal bovine serum, 0.02% azide) and stained with sera from mice immunized with HBsAg or HBsAg-MPER. The polyclonal antibodies were washed in FACS buffer and anti-mouse-FITC (SIGMA) at a 1:200 dilution was added for 30 minutes and then washed to remove unbound secondary antibody. The stained cells were analyzed by FACS on a Beckman Coulter Calibur Instrument.
Virus Neutralization Assay
Single round virus neutralization was performed as described earlier (Li et al., 2006
; Shu et al., 2007
). In brief, HIV-1 was pseudotyped with selected envelope glycoproteins by the cotransfection of an env
expressor and viral genomic DNA with a deletion of Env into 293T cells. The MN Env plasmid was provided by David Montefiori, MW965 was obtained for the NIH AIDS Research and Regent Program. Following the production of pseudotyped virus, a luciferase-based neutralization assay was performed as previously described. Briefly, TZM-bl cells expressing CD4, CXCR4, and CCR5 were used for HIV-1 infection. These target cells contain Tat-responsive reporter genes for firefly luciferase and the Escherichia coli
β-galactosidase gene under the regulatory control of the HIV-1 long terminal repeat. The level of HIV-1 infection was quantified by measuring relative light units (RLU) of luminescence, which is directly proportional to the amount of virus input. The assays were performed using a 96-well microtiter plate format with 10,000 TZM-bl cells per well. For neutralization assays, each pseudotyped virus stock was diluted to a level that produced approximately 100,000 to 500,000 RLU. The percentage of virus neutralization by each immune serum sample was derived by calculating the reduction in RLUs in the test wells compared to the RLUs in the wells containing preimmune serum from the corresponding animal. To control for nonspecific neutralization in protein-immunized rabbits, sera from two animals immunized with BSA were analyzed. All serum samples were also assayed for neutralizing activity against a pseudovirus expressing the amphotropic murine leukemia virus envelope to test for non-HIV-1-specific plasma effects (Mascola et al., 2002
). To obtain IC50 and IC80 data, fivefold serial dilution of immune sera were incubated with viruses before infection of target cells. Antiserum dose-response curves were fit with a nonlinear function, and the IC50 and IC80 for the corresponding virus was calculated by a least-squares regression analysis.