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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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Related in: MedlinePlus

Trypsin protection assay on LASV Z+GPC+NP VLP. LASV VLP expressing Z, GPC, and NP proteins were subjected to trypsin protection assays to assess the enveloped nature of pseudoparticles and compartmentalization of viral proteins. LASV VLP incorporated unprocessed 75 kDa GPC precursor (7A, 7B, lane 1), and monomeric 42 kDa GP1 (7A, lane 1), and 38 kDa GP2 (7B, lane 1). LASV VLP also incorporated trimerized, non-reduceable 126 kDa GP1 isoforms (7A, lane 1), and 114 kDa GP2 trimers to a lesser extent (7B, lane 1). For trypsin protection assays ten μg of LASV VLP were either left untreated (lane 1), treated with 3 mg/mL soybean trypsin inhibitor (lane 2), 1% Triton X-100 (lane 3), 100 μg/mL trypsin (lane 4), 1% Triton X-100 and 100 μg/mL trypsin (lane 5), or 100 μg/mL trypsin in the presence of 3 mg/mL soybean trypsin inhibitor (lane 6). Trypsin alone completely digested trimerized GP1 (7A, lane 4) and GP2 (7B, lane 4), while partially degrading GPC precursor, but having little effect on monomeric glycoproteins. Trypsin treatment of intact VLP did not significantly affect the levels of NP (7C, lane 4), and Z (7D, lane 4) proteins. Treatment of VLP with Triton X-100 in the presence of trypsin resulted in the complete digestion of NP (7C, lane 5) and Z (7D, lane 5), while only partially degrading monomeric GP1 (7A, lane 5) and GP2 (7B, lane 5) proteins. Treatment of VLP with trypsin in the presence of soybean trypsin inhibitor completely prevented digestion of any form of all viral proteins (7A - 7D, lane 6).
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Figure 7: Trypsin protection assay on LASV Z+GPC+NP VLP. LASV VLP expressing Z, GPC, and NP proteins were subjected to trypsin protection assays to assess the enveloped nature of pseudoparticles and compartmentalization of viral proteins. LASV VLP incorporated unprocessed 75 kDa GPC precursor (7A, 7B, lane 1), and monomeric 42 kDa GP1 (7A, lane 1), and 38 kDa GP2 (7B, lane 1). LASV VLP also incorporated trimerized, non-reduceable 126 kDa GP1 isoforms (7A, lane 1), and 114 kDa GP2 trimers to a lesser extent (7B, lane 1). For trypsin protection assays ten μg of LASV VLP were either left untreated (lane 1), treated with 3 mg/mL soybean trypsin inhibitor (lane 2), 1% Triton X-100 (lane 3), 100 μg/mL trypsin (lane 4), 1% Triton X-100 and 100 μg/mL trypsin (lane 5), or 100 μg/mL trypsin in the presence of 3 mg/mL soybean trypsin inhibitor (lane 6). Trypsin alone completely digested trimerized GP1 (7A, lane 4) and GP2 (7B, lane 4), while partially degrading GPC precursor, but having little effect on monomeric glycoproteins. Trypsin treatment of intact VLP did not significantly affect the levels of NP (7C, lane 4), and Z (7D, lane 4) proteins. Treatment of VLP with Triton X-100 in the presence of trypsin resulted in the complete digestion of NP (7C, lane 5) and Z (7D, lane 5), while only partially degrading monomeric GP1 (7A, lane 5) and GP2 (7B, lane 5) proteins. Treatment of VLP with trypsin in the presence of soybean trypsin inhibitor completely prevented digestion of any form of all viral proteins (7A - 7D, lane 6).

Mentions: Trypsin protection assays were employed to characterize protein content and structural compartmentalization of LASV antigens. Treatment of VLP with soybean trypsin inhibitor alone, with 1% Triton X-100 alone, or with soybean trypsin inhibitor and trypsin had no effect on the integrity of GP1, GP2, Z, and NP proteins when compared to untreated controls (Figure 7A - 7D, lanes 2, 3, 6 versus lane 1). Treatment of VLP with trypsin alone completely digested the approximately 120 kDa trimerized GP1 species and partially digested unprocessed GPC, while monomeric GP1 remained largely resistant to the protease (Figure 7A, lane 4). Similarly, trypsin completely digested the approximately 120 kDa trimerized GP2 species, but only partially digested monomeric GP2 (Figure 7B, lane 4). Trypsin treatment of intact LASV VLP did not significantly affect detection of NP and Z proteins (Figure 7C,D, lane 4). Whereas, treatment of LASV VLP with Triton X-100 and trypsin resulted in increased digestion of both glycoproteins, but significant levels of GP1 and GP2 could still be detected (Figure 7A,B, lane 5). Under these conditions, both NP and Z proteins were completely digested by trypsin (Figure 7C,D, lane 5). Digestion of intact VLP in the presence of soybean trypsin inhibitor completely prevented digestion of any form of the exposed glycoprotein complex (Figure 7A,B, lane 6).


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)

Trypsin protection assay on LASV Z+GPC+NP VLP. LASV VLP expressing Z, GPC, and NP proteins were subjected to trypsin protection assays to assess the enveloped nature of pseudoparticles and compartmentalization of viral proteins. LASV VLP incorporated unprocessed 75 kDa GPC precursor (7A, 7B, lane 1), and monomeric 42 kDa GP1 (7A, lane 1), and 38 kDa GP2 (7B, lane 1). LASV VLP also incorporated trimerized, non-reduceable 126 kDa GP1 isoforms (7A, lane 1), and 114 kDa GP2 trimers to a lesser extent (7B, lane 1). For trypsin protection assays ten μg of LASV VLP were either left untreated (lane 1), treated with 3 mg/mL soybean trypsin inhibitor (lane 2), 1% Triton X-100 (lane 3), 100 μg/mL trypsin (lane 4), 1% Triton X-100 and 100 μg/mL trypsin (lane 5), or 100 μg/mL trypsin in the presence of 3 mg/mL soybean trypsin inhibitor (lane 6). Trypsin alone completely digested trimerized GP1 (7A, lane 4) and GP2 (7B, lane 4), while partially degrading GPC precursor, but having little effect on monomeric glycoproteins. Trypsin treatment of intact VLP did not significantly affect the levels of NP (7C, lane 4), and Z (7D, lane 4) proteins. Treatment of VLP with Triton X-100 in the presence of trypsin resulted in the complete digestion of NP (7C, lane 5) and Z (7D, lane 5), while only partially degrading monomeric GP1 (7A, lane 5) and GP2 (7B, lane 5) proteins. Treatment of VLP with trypsin in the presence of soybean trypsin inhibitor completely prevented digestion of any form of all viral proteins (7A - 7D, lane 6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Trypsin protection assay on LASV Z+GPC+NP VLP. LASV VLP expressing Z, GPC, and NP proteins were subjected to trypsin protection assays to assess the enveloped nature of pseudoparticles and compartmentalization of viral proteins. LASV VLP incorporated unprocessed 75 kDa GPC precursor (7A, 7B, lane 1), and monomeric 42 kDa GP1 (7A, lane 1), and 38 kDa GP2 (7B, lane 1). LASV VLP also incorporated trimerized, non-reduceable 126 kDa GP1 isoforms (7A, lane 1), and 114 kDa GP2 trimers to a lesser extent (7B, lane 1). For trypsin protection assays ten μg of LASV VLP were either left untreated (lane 1), treated with 3 mg/mL soybean trypsin inhibitor (lane 2), 1% Triton X-100 (lane 3), 100 μg/mL trypsin (lane 4), 1% Triton X-100 and 100 μg/mL trypsin (lane 5), or 100 μg/mL trypsin in the presence of 3 mg/mL soybean trypsin inhibitor (lane 6). Trypsin alone completely digested trimerized GP1 (7A, lane 4) and GP2 (7B, lane 4), while partially degrading GPC precursor, but having little effect on monomeric glycoproteins. Trypsin treatment of intact VLP did not significantly affect the levels of NP (7C, lane 4), and Z (7D, lane 4) proteins. Treatment of VLP with Triton X-100 in the presence of trypsin resulted in the complete digestion of NP (7C, lane 5) and Z (7D, lane 5), while only partially degrading monomeric GP1 (7A, lane 5) and GP2 (7B, lane 5) proteins. Treatment of VLP with trypsin in the presence of soybean trypsin inhibitor completely prevented digestion of any form of all viral proteins (7A - 7D, lane 6).
Mentions: Trypsin protection assays were employed to characterize protein content and structural compartmentalization of LASV antigens. Treatment of VLP with soybean trypsin inhibitor alone, with 1% Triton X-100 alone, or with soybean trypsin inhibitor and trypsin had no effect on the integrity of GP1, GP2, Z, and NP proteins when compared to untreated controls (Figure 7A - 7D, lanes 2, 3, 6 versus lane 1). Treatment of VLP with trypsin alone completely digested the approximately 120 kDa trimerized GP1 species and partially digested unprocessed GPC, while monomeric GP1 remained largely resistant to the protease (Figure 7A, lane 4). Similarly, trypsin completely digested the approximately 120 kDa trimerized GP2 species, but only partially digested monomeric GP2 (Figure 7B, lane 4). Trypsin treatment of intact LASV VLP did not significantly affect detection of NP and Z proteins (Figure 7C,D, lane 4). Whereas, treatment of LASV VLP with Triton X-100 and trypsin resulted in increased digestion of both glycoproteins, but significant levels of GP1 and GP2 could still be detected (Figure 7A,B, lane 5). Under these conditions, both NP and Z proteins were completely digested by trypsin (Figure 7C,D, lane 5). Digestion of intact VLP in the presence of soybean trypsin inhibitor completely prevented digestion of any form of the exposed glycoprotein complex (Figure 7A,B, lane 6).

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