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Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever.

Branco LM, Grove JN, Geske FJ, Boisen ML, Muncy IJ, Magliato SA, Henderson LA, Schoepp RJ, Cashman KA, Hensley LE, Garry RF - Virol. J. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tulane University Health Sciences Center, New Orleans, LA, USA.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

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Purification of HEK-293T/17 generated LASV VLP by sucrose gradient sedimentation and detection of GP1, GP2, NP, and Z proteins in fractions by western blot analysis. LASV VLP were precipitated with PEG-6000/NaCl and concentrated by ultracentrifugation. Pellets were resuspended in 500 μL of TNE or PBS, overlayed on discontinuous 20 - 60% sucrose gradients, and sedimented by ultracentrifugation. Eight fractions of 500 μL each were collected from sucrose gradients. Ten μL from each fraction were separated on denaturing 10% NuPAGE gels, blotted and probed with LASV protein-specific mAbs. LASV VLP packaging Z+GPC+NP (A) and Z+GPC (B) were analyzed for distribution of GP1 (Ai, Bi), GP2 (Aii), NP (Aiii), and Z (Aiv, Bii) throughout the gradient spectrum. Fraction 1 contained input supernatant (S) loaded onto gradients. Fractions 2 through 8 were from 20 - 60% sucrose gradients. Lane 9 contained insoluble material that pelleted through 60% sucrose (P). The size of each protein in kDa is indicated to the right of each blot (unprocessed GPC: 75 kDa, GP1: 42 kDa, GP2: 38 kDa, NP: 60 kDa, and Z: 12 kDa).
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Figure 1: Purification of HEK-293T/17 generated LASV VLP by sucrose gradient sedimentation and detection of GP1, GP2, NP, and Z proteins in fractions by western blot analysis. LASV VLP were precipitated with PEG-6000/NaCl and concentrated by ultracentrifugation. Pellets were resuspended in 500 μL of TNE or PBS, overlayed on discontinuous 20 - 60% sucrose gradients, and sedimented by ultracentrifugation. Eight fractions of 500 μL each were collected from sucrose gradients. Ten μL from each fraction were separated on denaturing 10% NuPAGE gels, blotted and probed with LASV protein-specific mAbs. LASV VLP packaging Z+GPC+NP (A) and Z+GPC (B) were analyzed for distribution of GP1 (Ai, Bi), GP2 (Aii), NP (Aiii), and Z (Aiv, Bii) throughout the gradient spectrum. Fraction 1 contained input supernatant (S) loaded onto gradients. Fractions 2 through 8 were from 20 - 60% sucrose gradients. Lane 9 contained insoluble material that pelleted through 60% sucrose (P). The size of each protein in kDa is indicated to the right of each blot (unprocessed GPC: 75 kDa, GP1: 42 kDa, GP2: 38 kDa, NP: 60 kDa, and Z: 12 kDa).

Mentions: Transient transfection of HEK-293T/17 cells with LASV GPC, NP, and Z gene constructs resulted in high level expression of all proteins, including their known post-translational processing. The glycoprotein complex (GPC) was detected as a 75 kDa polyprotein precursor in transfected cell extracts, and in VLP preparations (Figure 1 Ai, Aii, Bi lanes 2 - 9; Additional file 1: Figure S1 Ci lane 4). Similarly, the proteolytically processed GP1 and GP2 subunits were detected in cell extracts (Additional file 1: Figure S1 Ci lane 4) and in purified VLP (Figure 1 Ai, Aii, Bi lanes 2 - 9) as 42 and 38 kDa glycosylated species, respectively. In VLP cell culture supernatants cleared by ultracentrifugation, the soluble LASV GP1 isoform previously described in this expression system was also detected at high levels (Figure 1 Ai, lane 1) [11,12]. Nucleoprotein (NP) was mainly detected as a 60 kDa species with smaller fragments identified, namely a 24 kDa protein corresponding to a previously described proteolysis product generated during LASV infection in vitro (Figure 1 Aiii lanes 2 - 9; Additional file 1: Figure S1 Ci, lane 1), [13-16]. The nucleoprotein was largely absent from the extracellular milieu (Additional file 1: Figure S1 Cii, lane 1) unless the Z matrix protein was co-expressed (Figure 1 Aiii, Aiv, lanes 2 - 9). Nucleoprotein that was not associated with VLP was present in the input fraction, as assessed by corresponding lack of GP2 and Z matrix protein detection (Figure 1 Aiii, lane 1). The Z matrix protein was detected in cell extracts (Additional file 1: Figure S1 Ci, lane 2) and in VLP preparations, as a 12 kDa protein (Figure 1 Aiv, Bii, lanes 2 - 9). An N-terminal 6X-HIS tagged Z protein gene variant starting at amino acid position +3 that disrupted the known mirystoylation domain also expressed at high levels, but failed to generate VLPs, as determined by lack of detection of the protein in cell culture supernatants (Additional file 1: Figure S1 Ci, ii, lane 3).


Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever.

Branco LM, Grove JN, Geske FJ, Boisen ML, Muncy IJ, Magliato SA, Henderson LA, Schoepp RJ, Cashman KA, Hensley LE, Garry RF - Virol. J. (2010)

Purification of HEK-293T/17 generated LASV VLP by sucrose gradient sedimentation and detection of GP1, GP2, NP, and Z proteins in fractions by western blot analysis. LASV VLP were precipitated with PEG-6000/NaCl and concentrated by ultracentrifugation. Pellets were resuspended in 500 μL of TNE or PBS, overlayed on discontinuous 20 - 60% sucrose gradients, and sedimented by ultracentrifugation. Eight fractions of 500 μL each were collected from sucrose gradients. Ten μL from each fraction were separated on denaturing 10% NuPAGE gels, blotted and probed with LASV protein-specific mAbs. LASV VLP packaging Z+GPC+NP (A) and Z+GPC (B) were analyzed for distribution of GP1 (Ai, Bi), GP2 (Aii), NP (Aiii), and Z (Aiv, Bii) throughout the gradient spectrum. Fraction 1 contained input supernatant (S) loaded onto gradients. Fractions 2 through 8 were from 20 - 60% sucrose gradients. Lane 9 contained insoluble material that pelleted through 60% sucrose (P). The size of each protein in kDa is indicated to the right of each blot (unprocessed GPC: 75 kDa, GP1: 42 kDa, GP2: 38 kDa, NP: 60 kDa, and Z: 12 kDa).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984592&req=5

Figure 1: Purification of HEK-293T/17 generated LASV VLP by sucrose gradient sedimentation and detection of GP1, GP2, NP, and Z proteins in fractions by western blot analysis. LASV VLP were precipitated with PEG-6000/NaCl and concentrated by ultracentrifugation. Pellets were resuspended in 500 μL of TNE or PBS, overlayed on discontinuous 20 - 60% sucrose gradients, and sedimented by ultracentrifugation. Eight fractions of 500 μL each were collected from sucrose gradients. Ten μL from each fraction were separated on denaturing 10% NuPAGE gels, blotted and probed with LASV protein-specific mAbs. LASV VLP packaging Z+GPC+NP (A) and Z+GPC (B) were analyzed for distribution of GP1 (Ai, Bi), GP2 (Aii), NP (Aiii), and Z (Aiv, Bii) throughout the gradient spectrum. Fraction 1 contained input supernatant (S) loaded onto gradients. Fractions 2 through 8 were from 20 - 60% sucrose gradients. Lane 9 contained insoluble material that pelleted through 60% sucrose (P). The size of each protein in kDa is indicated to the right of each blot (unprocessed GPC: 75 kDa, GP1: 42 kDa, GP2: 38 kDa, NP: 60 kDa, and Z: 12 kDa).
Mentions: Transient transfection of HEK-293T/17 cells with LASV GPC, NP, and Z gene constructs resulted in high level expression of all proteins, including their known post-translational processing. The glycoprotein complex (GPC) was detected as a 75 kDa polyprotein precursor in transfected cell extracts, and in VLP preparations (Figure 1 Ai, Aii, Bi lanes 2 - 9; Additional file 1: Figure S1 Ci lane 4). Similarly, the proteolytically processed GP1 and GP2 subunits were detected in cell extracts (Additional file 1: Figure S1 Ci lane 4) and in purified VLP (Figure 1 Ai, Aii, Bi lanes 2 - 9) as 42 and 38 kDa glycosylated species, respectively. In VLP cell culture supernatants cleared by ultracentrifugation, the soluble LASV GP1 isoform previously described in this expression system was also detected at high levels (Figure 1 Ai, lane 1) [11,12]. Nucleoprotein (NP) was mainly detected as a 60 kDa species with smaller fragments identified, namely a 24 kDa protein corresponding to a previously described proteolysis product generated during LASV infection in vitro (Figure 1 Aiii lanes 2 - 9; Additional file 1: Figure S1 Ci, lane 1), [13-16]. The nucleoprotein was largely absent from the extracellular milieu (Additional file 1: Figure S1 Cii, lane 1) unless the Z matrix protein was co-expressed (Figure 1 Aiii, Aiv, lanes 2 - 9). Nucleoprotein that was not associated with VLP was present in the input fraction, as assessed by corresponding lack of GP2 and Z matrix protein detection (Figure 1 Aiii, lane 1). The Z matrix protein was detected in cell extracts (Additional file 1: Figure S1 Ci, lane 2) and in VLP preparations, as a 12 kDa protein (Figure 1 Aiv, Bii, lanes 2 - 9). An N-terminal 6X-HIS tagged Z protein gene variant starting at amino acid position +3 that disrupted the known mirystoylation domain also expressed at high levels, but failed to generate VLPs, as determined by lack of detection of the protein in cell culture supernatants (Additional file 1: Figure S1 Ci, ii, lane 3).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tulane University Health Sciences Center, New Orleans, LA, USA.

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus