Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever
© Branco et al; licensee BioMed Central Ltd. 2010
Received: 24 September 2010
Accepted: 20 October 2010
Published: 20 October 2010
Lassa fever is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. Treatment of acute Lassa fever infections has successfully utilized intravenous administration of ribavirin, a nucleotide analogue drug, but this is not an approved use; efficacy of oral administration has not been demonstrated. To date, several potential new vaccine platforms have been explored, but none have progressed toward clinical trials and commercialization. Therefore, the development of a robust vaccine platform that could be generated in sufficient quantities and at a low cost per dose could herald a subcontinent-wide vaccination program. This would move Lassa endemic areas toward the control and reduction of major outbreaks and endemic infections. To this end, we have employed efficient mammalian expression systems to generate a Lassa virus (LASV)-like particle (VLP)-based modular vaccine platform.
A mammalian expression system that generated large quantities of LASV VLP in human cells at small scale settings was developed. These VLP contained the major immunological determinants of the virus: glycoprotein complex, nucleoprotein, and Z matrix protein, with known post-translational modifications. The viral proteins packaged into LASV VLP were characterized, including glycosylation profiles of glycoprotein subunits GP1 and GP2, and structural compartmentalization of each polypeptide. The host cell protein component of LASV VLP was also partially analyzed, namely glycoprotein incorporation, though the identity of these proteins remain unknown. All combinations of LASV Z, GPC, and NP proteins that generated VLP did not incorporate host cell ribosomes, a known component of native arenaviral particles, despite detection of small RNA species packaged into pseudoparticles. Although VLP did not contain the same host cell components as the native virion, electron microscopy analysis demonstrated that LASV VLP appeared structurally similar to native virions, with pleiomorphic distribution in size and shape. LASV VLP that displayed GPC or GPC+NP were immunogenic in mice, and generated a significant IgG response to individual viral proteins over the course of three immunizations, in the absence of adjuvants. Furthermore, sera from convalescent Lassa fever patients recognized VLP in ELISA format, thus affirming the presence of native epitopes displayed by the recombinant pseudoparticles.
These results established that modular LASV VLP can be generated displaying high levels of immunogenic viral proteins, and that small laboratory scale mammalian expression systems are capable of producing multi-milligram quantities of pseudoparticles. These VLP are structurally and morphologically similar to native LASV virions, but lack replicative functions, and thus can be safely generated in low biosafety level settings. LASV VLP were immunogenic in mice in the absence of adjuvants, with mature IgG responses developing within a few weeks after the first immunization. These studies highlight the relevance of a VLP platform for designing an optimal vaccine candidate against Lassa hemorrhagic fever, and warrant further investigation in lethal challenge animal models to establish their protective potential.
Lassa virus, a member of the Arenaviridae family, is the etiologic agent of Lassa fever, which is an acute and often fatal illness endemic to West Africa. There are an estimated 300,000 - 500,000 cases of Lassa fever each year [1–3], with a mortality rate of 15%-20% for hospitalized patients and as high as 50% during epidemics [4, 5]. Presently, there is no licensed vaccine or immunotherapy available for preventing or treating this disease. Although the antiviral drug Ribavirin is somewhat beneficial, it must be administered at an early stage of infection to successfully alter disease outcome, thereby limiting its utility . Furthermore, there is no commercially available Lassa fever diagnostic assay, which hampers early detection and rapid implementation of existing treatment regimens (e.g. Ribavirin administration). The severity of the disease, ability to be transmitted by aerosol, and lack of a vaccine or therapeutic drug led to its classification as a National Institutes of Allergy and Infectious Diseases (NIAID) Category A pathogen and biosafety level-4 (BSL-4) agent.
The LASV genome is comprised of two ambisense, single-stranded RNA molecules designated small (S) and large (L) . Two genes on the S segment encode the nucleoprotein (NP) and two envelope glycoproteins (GP1 and GP2); whereas, the L segment encodes the viral polymerase (L protein) and RING finger Z matrix protein. GP1 and GP2 subunits result from post-translational cleavage of a precursor glycoprotein (GPC) by the protease SKI-1/S1P . GP1 serves a putative role in receptor binding, while the structure of GP2 is consistent with viral transmembrane fusion proteins . NP is an abundant virion protein that binds and protects the viral RNA. The Z matrix protein associates with GP2 and NP during viral biogenesis, but alone is sufficient to mediate formation and release of viral particles from infected/transfected cells .
LASV gene expression and incorporation in VLP
To determine if tagged arenaviral gene sequences benefitted overall expression levels and incorporation into VLP a series of matrix experiments were performed that combined native and/or 6X-HIS or FLAG epitope tags. Only the addition of a 6X-HIS tag to the C-terminus of the Z gene did not affect its expression and incorporation into VLP (Additional file 2: Figure S2). The addition of C-terminal tags to GPC or NP resulted in lower expression levels and resulting incorporation into VLP. In some cases these tags led to unexpected and untoward proteolytic processing (Additional file 2: Figure S2, lane 6).
Large scale generation of LASV VLP
Generation of LASV VLP from 6 well plates through 15 cm cell culture dishes resulted in linear volumetric increase in particle yields (~100 μg/35 mm well; ~2 mg/15 cm dish). Production of VLP for biochemical characterization and in vivo studies was performed in multiple 15 cm culture dishes, which routinely yielded an average of 2 mg of total VLP protein per dish, as determined by Micro BCA and SDS-PAGE. VLP generated from expression of LASV Z, GPC, and NP gene constructs resulted in particles with higher densities than those produced by expression of Z and GPC alone, as assessed by relative levels of each viral protein throughout the sucrose density spectrum (Figure 1A,B, lanes 2 - 9). The majority of Z+GPC+NP VLP sedimented between 30 and 60% sucrose (Figure 1Ai - iv, lanes 4 - 8), whereas Z+GPC VLP were present in ~25 - 40% sucrose fractions (Figure 1Bi, ii, lanes 3 - 5). Surprisingly, Z+GPC VLP sedimenting through 30 - 60% sucrose contained progressively lower levels of Z matrix protein (Figure 1Bii, lanes 6 - 8) than counterparts containing both NP (Figure 1Aiv, lanes 6 - 8) and Z. In both Z+GPC and Z+GPC+NP VLP preparations a considerable insoluble fraction pelleted through 60% sucrose, and could only be dissolved in reducing SDS-PAGE buffer (Figure 1Ai - iv, 1B i - ii, lane 9 [P]).
Effects of LASV gene expression on mammalian cell morphology - cytotoxicity
LASV VLP contain a multitude of cellular proteins in addition to viral polypeptides
LASV VLP glycoproteins display heterogeneous glycosylation
LASV VLP do not package cellular ribosomes
LASV VLP are morphologically similar to native virions
LASV VLP display glycoprotein resistance to proteolysis by trypsin
LASV VLP are immunogenic in mice and induce a mature IgG response after prime + two boosts intra-peritoneal immunizations
Increasing IgG titers to Lassa virus antigens through the vaccination schedule
pre- 2nd boost
LASV patient sera specifically recognize VLP antigens in conformational and individual recombinant viral proteins
Lassa virus-like particles were generated to contain the major immunological determinants of the virus, resembled native virions structurally, and were immunogenic in mice. Plasmid vectors well suited for high level expression of recombinant proteins in mammalian cells through combination of rational design and proven genetic elements have resulted in high yields of LASV VLP. These vectors afford the possibility of developing a VLP-based vaccine candidate in mammalian cell systems at low cost per dose, using transient expression technologies. Despite incorporation of all LASV proteins into VLP, both glycoproteins were present at significantly higher levels in most sucrose density fractions than either NP or Z (Figure 1). Incorporation of high levels of both glycoproteins in VLP may be beneficial in a vaccine platform, as these viral components alone have been shown to confer full protection against challenge with lethal doses of live LASV in non-human primates [17–21]. Yet, despite the high levels of glycoprotein incorporation into LASV VLP, addition of the nucleoprotein may be of critical importance in establishing more robust and long lived immunity against Lassa virus [19, 22]. Previous studies have demonstrated physical interaction between the glycoprotein complex, the Z matrix, and nucleoproteins during viral biogenesis [23–25]. Thus, these natural interactions are greatly beneficial since they result in the generation of VLP that package all viral immunogenic and protective determinants from a single set of transiently transfected recombinant LASV genes. In these studies we employed the human endothelial kidney cell line HEK-293T/17 for its high levels of transfectability, expression of recombinant proteins from human cytomegalovirus (hCMV) promoter driven gene constructs, and resulting yields of LASV VLP. During the course of this work, we have also established the value of using HEK-293T/17 as an indicator cell line. The profound morphological changes manifested by the cell line upon expression of LASV Z matrix protein is a good indicator of transfection efficiency and overall production levels of resulting VLP (Figure 2). Despite significant adverse metabolic effects on cells expressing LASV proteins and generating budding VLP, culture viability remained high (mean = 70%) at the time of harvest. This desirable aspect of mammalian cell culture-based production is beneficial in downstream purification processes, by reducing host cell components that must be eliminated from the final purified product, namely the cellular proteins, DNA, RNA, and lipids. Other expression platforms cannot be easily employed in the generation of LASV VLP where the glycoprotein complex precursor is used to incorporate processed GP1 and GP2. Truncated versions of the GPC precursor lacking the transmembrane domain have been generated in E. coli (unpublished data from the Viral Hemorrhagic Fever Research Consortium) and in baculovirus expression systems . In E. coli, the protein is neither glycosylated nor cleaved into GP1 and GP2 subunits. In insect cells, the protein is glycosylated but is not cleaved . Both expression systems lack the critical SKI-1/S1P subtilase responsible for co-translational processing of the LASV GPC precursor in mammalian cells . Despite the possibility of co-expressing the subtilase in heterologous systems to facilitate processing of GPC precursor, the glycosylation profile of GP1 and GP2 subunits may play a critical role in the structure and function of each protein in vivo. Thus, a mammalian expression system remains a highly attractive platform for the development of an arenaviral VLP-based vaccine.
We have determined in these studies that LASV VLP contain, in addition to the intended viral polypeptides, a plethora of host cell membrane proteins, presumably acquired during budding from the cell membrane or other intracellular lipid bilayer containing structures, such as the Golgi apparatus. A significant portion of the viral envelope protein content is made up of host cell glycoproteins, as determined by a broad glycan binding analysis performed on sucrose sedimented fractions (Figure 3B,C). The host cell glycoprotein composition varies along the gradient spectrum (Figure 3B). A similar pattern of cellular glycoproteins incorporated into LASV VLP was detected in purified particles generated from expression of Z alone or in combination with GPC and NP (Figure 3C). In Z+GPC or Z+GPC+NP VLP, a diffuse lectin binding pattern could be detected between 38 and 42 kDa which was absent from VLP that did not express the glycoprotein complex. This pattern was detected in addition to a prominent ~ 48 kDa cellular glycoprotein of unknown identity present in all VLP formats (Figure 3 C). The majority of detected cellular glycoproteins incorporated into LASV VLP ranged from 30 to greater than 220 kDa in mass. Recently, Moerdyk-Schauwecker et al. 2009  characterized the spectrum of mammalian host cell proteins incorporated into vesicular stomatitis virus (VSV), an enveloped virus, during viral biogenesis. In total, 64 proteins of host cell origin were identified via a proteomics approach coupled with mass spectrometry (MS). Of the 64 host cell proteins identified in these studies, 10 were glycoproteins . Although a similar study has not been performed for any member of the arenaviridae, it is likely that some common host cell proteins are packaged among a wide array of viral classes, and some of these proteins may even play functional roles during viral infection and replication. Characterization of the host cell protein profile in LASV VLP will be paramount in gaining regulatory clearance of an arenaviral pseudoparticle-based vaccine. The immunological and functional role of such proteins must be known in order to avert untoward side effects, such as autoimmunity and physiological disregulations.
We had previously characterized the gross glycosylation profile of LASV GP1 in the context of a soluble isoform (sGP1) of this viral protein . In the present studies, we characterized LASV VLP-associated GP1 and GP2 glycosylation patterns. Glycoprotein 1 associated with VLP generated essentially the same glycosylation pattern as sGP1, with only partial deglycosylation by Endo H, and insignificant processing by neuraminidase (Figure 4A). These results point to a heterogeneous array of glycans on the surface of GP1 that include some high mannose and branched oligosaccharides. Glycoprotein 2 displayed a more heterogeneous glycan array with a highly homogeneous high mannose and hybrid oligosaccharide content that accounted for approximately 8 kDa of the fully processed mass of the protein, based on the detection of a relatively sharp 30 kDa species upon treatment with Endo H (Figure 4B, lane 3). The remaining 7 kDa of glycan content could be removed by treatment of the protein with PNGase F, but not with neuraminidase (Figure 4B, lanes 2 and 4). A similar micro- and macroheterogeneity in both GP1 and GP2 N-linked glycosylation has not been characterized in native Lassa virions.
Through these studies, we have established that GP1 incorporated into LASV VLP is highly resistant to proteolytic digestion by trypsin (Figure 7A, lanes 4 and 5), despite 13 predicted trypsin recognition sites on the polypeptide backbone (ExPASy proteomics server tools, PeptideCutter ). Similarly, GP2 is resistant to digestion with trypsin, albeit to a lesser extent than GP1, even after solubilization of the pseudoparticle envelope with Triton X-100 (Figure 7B, lanes 4 and 5). The PeptideCutter tool in ExPASy predicted 25 recognition sites with high confidence in the GP2 polypeptide backbone. However, since the glycoprotein complex spike is the viral antigen most readily accessible to the innate immune system and to circulating serum proteases, it is likely that this molecule evolutionarily developed a significant level of proteolytic resistance in the structure-function relationship. It is of paramount importance to the virus that the critical components required for binding and fusion to permissive host cells be preserved. The specific glycosylation patterns on GP1 and GP2 may play a functional role in the observed resistance to proteolytic degradation. In the studies by Schlie et al. 2010 , Proteinase K protection assays performed on GP VLP also revealed partial resistance of the GP2 component against degradation by the protease, although solubilization with Triton X-100 in conjunction with protease resulted in complete digestion of the protein. Glycosylation of arenaviral glycoproteins is critical for protein stability, as unglycosylated GP1 and GP2 generated in E. coli are insoluble and require detergents, zwitterions, and reducing agents to remain in solution [11, 30], and deglycosylating mammalian cell generated GP1 generally produces similar results (unpublished data).
To characterize the structural compartmentalization of viral proteins in LASV we performed trypsin protection assays in the absence or presence of the anionic detergent Triton X-100 (Figure 7). In the absence of detergent, trypsin completely digested non-reduceable GP1 trimer, partially degraded unprocessed GPC, but had no effect of monomeric GP1 (Figure 7A, lane 4). A similar digestion pattern was obtained for GP2 (Figure 7B, lane 4). The addition of detergent to the reaction enhanced digestion of unprocessed GPC and had a minor effect on sensitivity of GP1 to the protease (Figure 7A, lane 5). Dissolution of the envelope by detergent resulted in more pronounced degradation of GP2 by trypsin, although a significant portion of the monomer could be detected (Figure 7B, lane 5). Only treatment of LASV VLP with Triton X-100 resulted in proteolytic degradation of both Z matrix and NP proteins. These results strongly support the model of a LASV VLP containing glycoprotein spikes on the surface of a lipid envelope with an internal matrix of Z protein containing the nucleoprotein component. We have shown that the viral proteins NP, Z, GP1 and GP2 can be co-expressed in VLP. Protein-protein associations appear to be an important aspect to the formation of VLP. Schlie et al. 2010  reported that a co-localization of NP, Z, and GP occurs near the nucleus. Similarly, Eichler et at. 2004  demonstrated that NP and Z co-localize in the cell. They also demonstrated that NP could be precipitated using an antiserum against Z and vice versa. Furthermore, Schlie et al. 2010  determined that NP did not influence the interaction of GP and Z, nor could an interaction between NP and GP be detected in the absence of Z in co-localization and immunoprecipitation experiments. However, pull down experiments performed by Schlie et al. 2010  demonstrated an association between Z and GP and Z and NP. Strecker et al. 2006  reported that Z myristoylation is important for binding to lipid membranes. Flotation experiments using wild-type Z protein and a mutant of Z at the myristoylation site showed that the mutant remains localized in the cytosol, whereas the wild-type associated with the membrane. Thus, the interactions between Z and the membrane and with GP and NP result in VLP formation with relevant proteins incorporated in virions.
Another structural component of native LASV virions are host cell ribosomes that are packaged during virion assembly, presumably for enhanced viral mRNA translation in the early stages of cellular infection. To determine whether LASV VLP containing any combination of Z matrix, GPC, and NP proteins mediated the ability to package cellular ribosomes, total RNA was isolated from pseudoparticles and analyzed by denaturing RNA gel electrophoresis (Figure 5). RNA was also isolated from the corresponding transfected cells and analyzed alongside VLP RNA. All VLP formats analyzed in these studies did not contain significant levels of the 28S and 18S ribosomal RNA species known to be critical components of mammalian ribosomes (Figure 5, lanes 2, 4, 6, 8, 10). In some analyses, RNA was purified from 1 mg of total purified VLP, and the entire purified nucleic acid fraction was analyzed by gel electrophoresis without distinct ribosomal RNA bands visible (data not shown). Despite the lack of rRNA detection in LASV VLP, all pseudoparticle formats analyzed in these studies contained significant levels of low molecular weight RNA species ~ 75 - 200 nt, that co-migrated with cellular 5S (120 nt) and 5.8S (160 nt) rRNA, and transfer RNAs (75 - 95 nt). It is reasonable to assume that in native VLP the incorporation of host cell ribosomes would result in the co-packaging of critical tRNAs for translation of viral mRNAs. Although in these studies the exact nature of the packaged RNA species was not characterized in detail, the results suggest that multiple RNA species of ribosomal origin are incorporated into VLP. To confirm that ribonucleoproteins were not incorporated into virions, we performed western blot analysis on VLP proteins using antibodies raised against U1 snRNP 70, La/SSB, and Ro/SSA. No ribonucleoproteins could be detected in pseudoparticles (data not shown). These studies also point to a critical presence of viral RNA polymerase and genomic RNA segments during replication for subsequent incorporation of host cell ribosomes into nascent viral particles. The lack of detectable ribosomes in LASV VLP represents a regulatory advantage for this platform as a vaccine candidate. Administration of pseudoparticles containing autologous ribosomes to vaccinees has potential to result in untoward immunological affects.
Despite the lack of detectable 28S and 18S rRNA in LASV VLP comprised of any combination of LASV proteins analyzed in these studies, pseudoparticles that contained GPC and/or NP in addition to Z matrix protein were morphologically similar to native virions (Figure 6B,C,D). These VLP were electron dense particles with punctuate inclusions and appeared to associate with highly electron dense subcellular organelles in the cytoplasm, possibly ribosomes despite their lack of incorporation into the pseudoparticle (Figure 6C,D). The size of mammalian ribosomes is approximately 20 nm, in line with the size of the particles associated with nascent LASV VLP imaged in these studies (Figure 6D). However, these subcellular structures could not be detected in VLP budding from the surface of cells transfected with Z matrix protein alone (Figure 6A), which appeared empty and containing only an envelope structure, as shown here and reported by others .
For immunizations, LASV VLP comprised of Z+GPC or Z+GPC+NP were formulated in PBS and used to immunize BALB/c mice, in a prime + 2 boosts schedule, 3 weeks apart, in the absence of an adjuvant, and administered by i.p. injection. After a single immunization some animals showed a low level IgG response to individual LASV antigens, with increasing mean antibody titers after each subsequent boost (Table 1). ELISA analysis of terminal IgG titers showed a clear difference in the response levels against GP1, and whole VLP between Z+GPC and Z+GPC+NP pseudoparticles (p = 0.004 and 0.026, respectively) (Figure 8A,B). VLP containing all three proteins induced a significantly higher response to the glycoprotein components compared to Z+GPC VLP, with a 15 fold overall increase in titer against both GP1 and GP2, despite a not quite significant statistical difference in the GP2 titers (p = 0.092). Likewise, the titers against whole Z+GPC+NP VLP were nearly 3 fold higher than to Z+GPC pseudoparticles (Figure 8A,B).
Lastly, we attempted to use LASV VLP as a diagnostic tool for the detection of viral protein-specific IgM and IgG in the serum of convalescent subjects, patients from the Lassa ward, contacts from patients who succumbed to Lassa fever, and individuals not known to have had the febrile illness at any given time in their lives. The LASV antigen binding profile of these sera was extensively characterized using highly sensitive and specific recombinant protein-based diagnostics under development by the Viral Hemorrhagic Fever Research Consortium. The overall poor level of correlation observed in human serum IgM (r = 0.3297; r2 = 0.1087) and IgG (r = 0.6284; r2 = 0.3949) binding profiles between LASV VLP and recombinant proteins in these studies was not surprising. Recombinant LASV proteins currently employed in diagnostic assays are generated in bacterial or mammalian cell systems, as outlined in Branco et al., 2008 , and Illick et al., 2008 . Individually produced, purified, and characterized proteins are used alone or in combination to coat high protein binding ELISA plates for determination of serum IgM and IgG binding profiles. Thus, it would be expected that protein-protein interactions known to play a role during viral biogenesis and in the formation of LASV VLP result in presentation of different epitopes and conformations than in counterparts generated as individual polypeptides. The known interactions between Z, GPC, and NP proteins in a VLP format likely mask the presentation of relevant epitopes to which a given individual may have generated IgM and IgG. As a result, native presentation of antigens in the context of a VLP, even in the presence of low levels of the membrane solubilizing detergent Tween 20, will likely not result in disruption of protein interactions necessary for the detection of epitope-specific serum antibodies. This is supported by the fact that all five NP-specific mAbs used in this analysis detected and captured recombinantly expressed NP in solution (Figure 9F), albeit at different levels. In combination, these results strongly suggest that LASV proteins in the context of a VLP display epitopes that possibly mimic native conformation and presentation. These observations further support the use of LASV VLP as a vaccine platform by supplying a quasi-native antigen, thus allowing the innate and adaptive immune systems to preferentially target epitopes relevant for immune protection against the virus. In addition, the use of pseudoparticles in clinical assays may offer advantages over the use of recombinantly expressed individual proteins. Immune responses to LASV VLP may be directed against epitopes that are best or exclusively displayed in the context of a quasi native particle containing proteins assembled in a manner similar to functional viral biogenesis.
VLP have gained significant momentum in the past decade as premier vaccine platforms. The approval of Merck & Co., Inc.'s Gardasil(r) (Human Papillomavirus Quadrivalent [Types 6, 11, 16, and 18] Vaccine, Recombinant) by regulatory agencies heralded a new era in vaccinology, demonstrating that VLP are immunogenic, safe, and well tolerated in humans, and confer nearly complete protective immunity against homologous viral strains in canine models [32–38]. ENGERIX-B [Hepatitis B Vaccine (Recombinant)] is a recombinant VLP-like hepatitis B vaccine developed and manufactured by GlaxoSmithKline plc. These "Dane" particles, generated in yeast strains, are comprised of HbsAg and yeast phospholipids, and are subsequently harvested by gradient centrifugation and properly disulfide-linked in vitro. These particles are highly immunogenic, safe, well tolerated, and very efficacious.
VLP-based vaccine candidates have also been developed and tested for their efficacy in preventing a wide array of viral conditions, such as Influenza [40–44], Ebola [45, 46], Marburg [45, 47], West Nile virus , Dengue , Respiratory Syncytial Virus (RSV) , HIV [51–56], and Hepatitis C virus [57–59], and the most recently reported case of Chikungunya . VLP platforms currently being evaluated toward clinical licensure include Novavax's trivalent seasonal influenza vaccine. In recent Phase II clinical trials the vaccine was well tolerated and safe in adults age 60 and older and in healthy volunteers 18 to 48 years of age [61, 62]. Thus, it is reasonable to employ similar strategies to develop a vaccine platform based on VLP that contain all the relevant immunological determinants that are known to confer protective immunity against this viral hemorrhagic fever. Studies are currently ongoing to determine the in vivo efficacy of LASV VLP in relevant in vivo models.
The generation and characterization of a LASV VLP platform displaying all major immunological and protective determinants of the virus, with quasi-native morphological and protein association properties, that induced significant IgG titers in mice potentiate further development as a viable human vaccine platform.
Presently, there is no licensed vaccine or anti-viral therapy available for the prevention or treatment of this disease, and there is no commercially available Lassa fever diagnostic assay. The threat posed by LASV is heightened further by the potential use of the virus as a biological weapon, which is substantiated by the stability of the virion, demonstrated person-to-person transmission, the severity of disease, lack of therapeutic and prophylactic reagents, and the capacity for aerosolization. Collectively, these factors underscore the need for effective diagnostics, vaccines, and therapies against Lassa fever. The work performed in these studies is a first step toward resolving a public health crisis in Africa and bioterrorism concerns elsewhere.
Cells, plasmids, antibodies
HEK-293T/17 cells (ATCC CRL11268) were maintained in complete high glucose Dulbecco's Modified Eagle Medium (cDMEM) supplemented with non-essential amino acids (NEAA) and 10% heat-inactivated fetal bovine serum (ΔFBS).
Plasmid constructs expressing LASV GPC and the backbone vector pcDNA3.1+zeo:int A were described elsewhere . Optimized Z and NP genes for expression were amplified from LASV Josiah infected VERO cell RNA, as previously outlined . For immunoassays, Dr. Randal J. Schoepp kindly provided the LASV-specific GP1 mAb L52-74-7A and GP2 mAb L52-216-7, which were generated against purified gamma-irradiated LASV, as previously described . Monoclonal antibody to poly-histidine (6X-HIS) was purchased from Invitrogen, Inc. LASV NP-specific polyclonal sera were generated in goats by immunizing animals with 100 μg of E.coli generated protein per injection, using a prime + 3 boosts strategy, followed by terminal bleeds (Bethyl Laboratories, Inc.). The LASV NP-specific goat IgG fraction was subsequently purified by affinity column chromatography with agarose beads coupled to NP immobilized by AminoLink chemistry (Thermo Fisher Scientific, Inc., Rockford, IL). Horseradish peroxidase (HRP)-conjugated secondary antibodies specific for goat and mouse IgG-gamma were purchased from Kirkegaard and Perry Laboratories (KPL, Gaithersburg, MD). The NP-specific hybridomas NP 33LN, NP 100LN, NP 61SP, NP 692SP, and NP 1474SP were generated by fusion of the SP2/0-Ag14 myeloma cell line with splenocytes and mesenteric lymph node lymphocytes from BALB/c mice immunized with E. coli-expressed NP, essentially as outlined by Köhler and Milstein [63–65]. Monoclonal antibodies were produced in serum free medium (PFHM II, Invitrogen), purified via Protein-G chromatography, quantitated by A280, BCA, and SDS-PAGE.
Transient expression of LASV gene constructs
Recombinant LASV protein expression was analyzed in HEK-293T/17 cells transiently transfected with mammalian expression vector DNAs, which were prepared using the Endo-Free PureLink HiPure plasmid filter maxiprep kit (Invitrogen, Carlsbad, CA). Transfections and preparation of cell extracts for protein analysis have been described elsewhere . The negative control vector pcDNA3.1(+):intA was included in all transfections. Protein concentration was determined for each sample by A280 with A260 subtraction, and verified using a Micro BCA(tm) Protein Assay Kit, as outlined by the manufacturer (Thermo Scientific).
Generation and purification of LASV VLP
LASV VLP were generated by transfecting HEK-293T/17 cells in 6-well plates (for small scale analysis) or in 15 cm plates (for purification of multi-milligram quantities of VLP) using Lipofectamine 2000 (Invitrogen). Cells were seeded on plates coated with 50 μg/mL Poly-D-Lysine hydrobromide, and were transfected only at ≥90% confluence. Monolayers were transfected with equimolar amounts of vector DNAs, and when required reactions were normalized for DNA content with empty pcDNA3.1(+):intA. Cell supernatants were harvested 4 days post transfection and were clarified by centrifugation at 4000 ×g for 20 minutes at room temperature. Clarified supernatants were transferred to Beckman polyallomer ultratubes and gently mixed with polyethylene glycol-6000 (Sigma/Fluka) and sodium chloride to final concentrations of 5% and 0.25 M, respectively. Reactions were incubated at +4°C overnight, followed by centrifugation for one hour at 15,000 ×g, +4°C, in an SW28 rotor, to pellet the precipitated VLP. Pellets were gently resuspended in 20 mM Tris, pH7.4, 0.1 M NaCl, 0.1 mM EDTA (TNE), or in 1X PBS, pH 7.4, overlayed on 20% sucrose cushions, and centrifuged for 2 hours at 55,000 rpm, +4°C, in an SW60Ti rotor. Pellets were resuspended in TNE or PBS and VLP were further purified on 20 - 60% discontinuous sucrose gradients, as described above for sucrose cushions. VLP were removed from visible bands throughout the gradient, combined, diluted in TNE or PBS, and centrifuged for one hour at 15,000 ×g, +4°C, in an SW28 rotor, to pellet the purified VLP and to remove sucrose. Pellets were resuspended in TNE or PBS and allowed to dissolve fully at 4°C overnight. VLP used for immunizations were filtered through 0.45 μm syringe filters before being assayed for protein content by Micro BCA. VLP preparations were stored at 4°C in TNE or PBS at concentrations ranging from 200 - 3000 μg/mL. VLP for immunizations were tested for endotoxin levels with a high sensitivity Limulus Amebocyte Lysate (LAL) test (Sigma-Aldrich).
Western blot and densitometry analyses
Expression of LASV GP1, GP2, NP, and Z-3'HIS in VLP were confirmed by Western blot analysis using anti-LASV mAbs L52-74-7A, L52-216-7, goat polyclonal antibody (PAb) to E. coli generated nucleoprotein and α-6X-HIS mAb, respectively. Secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) or rabbit anti goat IgG (H+L). Five to ten μg of total VLP protein were denatured, reduced, and resolved on 10% NuPAGE Novex Bis-Tris gels, according to the manufacturer's specifications (Novex, San Diego, CA). Proteins were transferred to 0.45-μm nitrocellulose membranes, blocked, and probed in 1X PBS, pH 7.4, 5% non-fat dry milk, 1% heat inactivated fetal bovine serum, 0.05% Tween-20, and 0.1% thymerosal. Membranes were then incubated in LumiGlo chemiluminescent substrate (KPL) and exposed to Kodak BioMax MS Film. Developed films were subjected to high resolution scanning for densitometry analysis. Quantification of band intensity was performed using National Institutes of Health ImageJ 1.41o software http://rsb.info.nih.gov/ij, and following the procedure outlined in http://www.lukemiller.org/journal/2007/08/quantifying-western-blots-without.html, using TIFF files.
Cell proliferation assays
HEK-293T/17 cell cytotoxicity induced by LASV Z, GPC, and NP expression was monitored with a TACS(tm) MTT Cell Proliferation Assay (R&D Systems, Minneapolis, MN), according to manufacturer's instructions. The transfection procedure was scaled down to a 96-well format, with each condition analyzed in triplicate. Data was plotted as mean absorbance at 562 nm, with standard deviation, and background correction at 650 nm.
Protease protection assays
Pseudovirus-specific protein composition and VLP structure were characterized by trypsin protection assays. Ten μg of purified VLP were treated with 100 μg/mL trypsin in the presence or absence of 1% Triton X-100, for 30 minutes, at room temperature. Reactions were stopped by the addition of soybean trypsin inhibitor to a final concentration of 3 mg/mL, addition of SDS-PAGE buffer and reducing agent (DTT), and heating to 70°C for ten minutes. Proteins were resolved on 10% NuPage gels and detected by western blot, as described above.
PNGase F, Endo H, and neuraminidase assays
The glycosylation patterns of LASV VLP GP1 and GP2 generated from expression of LASV Z+GPC+NP were resolved by treatment with the deglycosidases PNGase F, Endo H, and neuraminidase, as previously described , on sucrose cushion purified VLP. Reactions were performed on heat denatured VLP to conform to manufacturer's recommendations for PNGase F and Endo H digestion conditions, and on non-denatured VLP. Control reactions were similarly processed except that enzymes were not added. Specificity of deglycosidases was assessed by monitoring the effects of all three enzymes on LASV NP and Z proteins packaged into VLP. Proteins were subsequently resolved by reducing SDS-PAGE, blotted, probed with α-LASV GP1, GP2, α-6X-HIS mAbs, or goat PAb α-NP, and developed as described above.
Lectin-based Glycan differentiation assays
Glycosylation patterns of VLP associated proteins were characterized via binding of glycan-specific lectins using a DIG Glycan Differentiation Kit (Roche Applied Science, Mannheim, Germany), according to the manufacturer's instructions. LASV VLP proteins were resolved by reducing SDS-PAGE, blotted onto nitrocellulose, and subjected to lectin binding assays.
RNA extraction from purified VLP
RNA was extracted from VLP with Trizol(tm) reagent/chloroform and isopropanol precipitation, essentially as outlined in the product insert (Invitrogen). RNA pellets were washed with 75% ethanol, air dried, resuspended in DEPC-treated water, and quantitated by A280. RNA was glyoxal-denatured and analyzed on 1.5% agarose gels containing ethidium bromide, essentially as described in Sambrook et al. . Gels were photographed on a Kodak EDAS 120 system and images were saved as TIFF files for densitometry analysis. Total RNA was extracted from corresponding transfected HEK293T/17 cells using the same procedure.
Genomic DNA fragmentation analysis
Genomic DNA was isolated from HEK-293T/17 cells using a Qiagen DNeasy kit, according to the manufacturer's instructions. Purified DNAs were quantitated by A260/A280. Two μgs of each DNA sample were resolved per lane of a 1.8% TAE/agarose gel containing 1 μg/mL ethidium bromide. High resolution gel images were converted to TIFF format for analysis.
Six to eight week-old female BALB/c mice were purchased from Charles River Laboratories and housed according to Tulane University's IACUC guidelines. Research was conducted in compliance with the Animal Welfare Act and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of
Laboratory Animal Care International. For immunizations, mice were randomly divided into groups of 10 and injected intraperitoneally with 10 μg of LASV VLP (Z+GPC or Z+GPC+NP) in 100 μL of sterile TNE. Ten mice were similarly injected with 100 μL TNE as vector control. One prime and two boosts were performed, three weeks apart, each with 10 μg of homologous LASV VLP. Mice were sacrificed by CO2 asphyxiation three weeks after the last boost and whole blood was collected by cardiac puncture. The plasma fraction was isolated and frozen at -80°C until analysis.
IgG and IgM ELISA on recombinant LASV proteins and VLP
Murine immunoglobulin-γ endpoint titers to whole VLP, and IgG-γ to GP1 and GP2 were determined in serially diluted sera samples. Nunc MaxiSorp ELISA plates were coated with 2 μg/mL total VLP protein in carbonate buffer. Recombinant mammalian cell expressed LASV GP1 and GP2, produced by Vybion, Inc., Ithaca, NY, were coated on Nunc PolySorp ELISA strips, pre-blocked, and lyophilized by Corgenix Medical Corp., Broomfield, CO. Plates coated with VLP were blocked in 1X PBS, pH 7.4, 5% NFDM, 1% FBSΔ, 0.05% Tween-20, 0.01% thymerosal. The same buffer was used for all sera and secondary antibody dilutions. Mouse IgG was detected with a Horseradish Peroxidase (HRP)-labeled goat F(ab')2 anti-mouse IgG γ-specific reagent at 1:2500 dilution (KPL). Reactions were developed with TMB for 15 minutes at room temperature, stopped with 0.5 N H2SO4, and plates were read at 450 nm in a BioTek 808 ELISA reader. Viral antigen-specific IgG and IgM analysis in the sera of convalescent patients was similarly performed, with serum samples diluted 1:100 in NFDM blocking reagent, and detected with HRP labelled goat F(ab')2 anti-human IgG, γ or μ-specific reagents, respectively. Monoclonal antibodies to GP2 and NP were used as positive controls on antigen coated plates to verify presence of relevant epitopes on viral proteins. Total IgG fraction from naive mice was used as negative control antibody (ms IgG). Sera collected from North American volunteer blood donors that had never travelled to LHF endemic regions, and that were confirmed naive to LASV antigens by ELISA were used as negative controls. Serum from a patient that tested positive for NP-specific IgM and IgG antibodies in a recombinant NP ELISA was used as a positive control in these assays (G652-3).
HEK-293T/17 cells were harvested at 72 hours post transfection with LASV gene constructs. Cells were pelleted by centrifugation at 200 ×g, washed once in cold (4°C) PBS, and fixed with 2.5% glutaraldehyde in phosphate buffer. Fixed cell pellets were embedded in 1% agarose prepared in phosphate buffer and allowed to solidify at 4°C. Cell pellets in agarose were post fixed with 1% osmium tetroxide, dehydrated in a graded series of ethanol, and embedded in epoxy resin. Thin sections were cut on a Leica UC6 ultramicrotome, stained with uranyl acetate and lead citrate, followed by examination on a Hitachi H-7100 transmission electron microscope.
Statistical analysis and in silico tools
Statistical analysis of data was performed with GraphPad InStat, V3.06 (GraphPad Software, Inc., San Diego, CA), using Analysis of Variance (ANOVA), paired or unpaired Student's t test, and Pearson's correlation. The PeptideCutter analysis tool from the Swiss Institute of Bioinformatics ExPASy Proteomics Server was employed in the in silico analysis of predicted trypsin cleavage sites on LASV GP1 and GP2.
This work was supported by Department of Health and Human Services/National Institutes of Health/National Institute of Allergy and Infectious Diseases Challenge and Partnership Grant Numbers AI067188 and AI082119, and RC-0013-07 from the Louisiana Board of Regents. The research described herein was sponsored in part by the Division of GEIS Operations at the Armed Forces Health Surveillance Center, Research Plan C0169_10_RD. We thank the members of the Hemorrhagic Fever Diagnostic Consortium, and Lassa Fever - Mano River Union for ongoing support. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.
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