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The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice Ana Ruth Pastor a , William A. Rodríguez-Limas a , Martha A. Contreras a , Ernesto Esquivel a,b , Fernando Esquivel-Guadarrama b , Octavio T. Ramírez a , Laura A. Palomares a,∗ a Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ave. Universidad 2001, Cuernavaca, Morelos 62210, Mexico b Facultad de Medicina, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
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Article history: Available online xxx Keywords: Rotavirus Protein assembly VLP VP6 Vaccine Challenge
a b s t r a c t Viral protein assemblies have shown to be superior immunogens used in commercial vaccines. However, little is known about the effect of protein assembly structure in immunogenicity and the protection conferred by a vaccine. In this work, rotavirus VP6, a polymorphic protein that assembles into nanotubes, icosahedra (dlRLP) or trimers was used to compare the immune response elicited by three different assemblies. VP6 is the most antigenic and abundant rotavirus structural protein. It has been demonstrated that antibodies against VP6 interfere with the replication cycle of rotavirus, making it a vaccine candidate. Groups of mice were immunized with either nanotubes, dlRLP or trimers and the humoral response (IgG and IgA titers) was measured. Immunized mice were challenged with EDIM rotavirus and protection against rotavirus infection, measured as viral shedding, was evaluated. Immunization with nanotubes resulted in the highest IgG titers, followed by immunization with dlRLP. While immunization with one dose of nanotubes was sufficient to reduce viral shedding by 70%, two doses of dlRLP or trimers were required to obtain a similar protection. The results show that the type of assembly of VP6 results in different humoral responses and protection efficacies against challenge with live virus. This information is important for the design of recombinant vaccines in general. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Several viral proteins are polymorphic and can assemble into sheets, nanotubes, trimers, icosahedra, etc. [1]. The property of assembling into different types of structures allows the use of viral proteins for a wide variety of applications [2,3]. An important advantage of protein assemblies is their superior immunogenicity that allows the design of highly effective recombinant vaccines [4]. So far, recombinant viral proteins assembled into icosahedra (VLP) have been used as commercial vaccines against hepatitis B and human papilloma virus [5]. Furthermore, it has been shown that other types of assemblies may elicit high protective immune responses. That is the case of viral protein nanotubes [6,7]. The main
∗ Corresponding author. Tel.: +52 777 3291646; fax: +52 777 3138811. E-mail addresses: [email protected] (A.R. Pastor), [email protected] (W.A. Rodríguez-Limas), [email protected] (M.A. Contreras), [email protected] (E. Esquivel), [email protected] (F. Esquivel-Guadarrama), [email protected] (O.T. Ramírez), [email protected] (L.A. Palomares).
advantage of VLP as vaccines is that their size and the epitope distribution in their surface are optimal for recognition by the immune system to generate a humoral response [8]. Both properties depend on the type of assembly of the viral protein. Icosahedra and nanotubes have different spatial arrangement of the protein subunits, which results in different interepitope spaces [9]. In contrast, soluble trimers lack such arrangements. Even when the structure of viral protein assemblies can determine their immunogenicity, to our knowledge, little research has been done investigating the effect of such different structures in the immunogenicity of a recombinant protein. In order to investigate the effect of protein assembly in immunogenicity and protection against virus infection, we used rotavirus VP6, a polymorphic protein, to immunize mice. Rotavirus is the main cause of severe gastroenteritis in infants and provokes diarrhea in neonates from several mammalian species. Traditional live virus vaccines against rotavirus infection are available, but they have several disadvantages, such as possible contamination with other viruses and potential side effects. VP6 is the most antigenic and abundant rotavirus structural protein. It has been
http://dx.doi.org/10.1016/j.vaccine.2014.02.018 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Pastor AR, et al. The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.018
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Table 1 Groups of 4–5 mice were immunized subcutaneously as described below. The second dose was administered 14 dpi. Each group was kept in one cage. Immunogen
Group
VP6, g/mice
Doses
Challenge
Nanotubes
1 2 3 4 5
10 20 40 40 40
1 1 1 1 2
No No No Yes Yes
dlRLP
6 7 8 9 10
10 20 40 40 40
1 1 1 1 2
No No No Yes Yes
Trimers
11 12
40 40
1 2
Yes Yes
None
Control 1 Control 2 Control 3
0 0 0
1 1 2
No Yes Yes
demonstrated that antibodies against VP6 interfere with the replication cycle of rotavirus, making it an interesting vaccine candidate [10–12]. VP6 constitutes the middle layer of the rotavirus and can assemble into trimers, icosahedral particles or nanotubes depending on pH, ionic strength and the presence of VP2, which forms the inner layer of the rotavirus [9,13]. When VP6 is recombinantly expressed with VP2, it assembles over it to conform doublelayered rotavirus-like particles (dlRLP). In this work, we compared the humoral immune response and protective action of different recombinant VP6 assemblies, trimers, dlRLP and nanotubes. Mice were immunized with VP6 and challenged with live virus. VP6 in its nanotube form protected mice more efficiently from challenge than trimers or dlRLP, confirming that different viral protein assemblies confer different protection against viral infection. 2. Materials and methods VP6 nanotubes were produced and purified as previously described [14]. VP6 trimers were obtained by adding a 1 M CaCl2 solution to nanotubes to obtain a calcium concentration of 167 mM. dlRLP (containing rotavirus VP2 and VP6) were produced as previously reported [15]. VP6 concentration was measured using the ProSpecT ELISA kit (Oxoid, UK). Purified VP6 was used as standard. Various VP6 amounts and assemblies were used to immunize subcutaneously groups of six- to eight-week old female CD-1 mice as described in Table 1. Each group contained four or five mice. The amount of immunogen administered to mice was referred to VP6 without considering the VP2 content in the dlRLP preparation. VP6 buffer (30 mM Tris, 10 mM EDTA pH 8.0) was administered to control groups. To measure the anti-VP6 humoral response, individual blood samples were collected through the tail vein at 0, 14, 21, 28, 35, 42, 56, 121 and 142 days after the first immunization (dpi). IgG or IgA VP6 specific antibodies in mouse serum samples were measured by ELISA. Briefly, EIA plates (Corning, Costar) were coated with 5 g/mL of purified VP6 in carbonate buffer pH 9.5. Plates were blocked with 5 mg/mL of gelatin solution (Bio-Rad). After washing with Tris buffer with 0.05% Tween 20, serial 3-fold dilutions of serum samples were added to each well. This was followed by the addition of alkaline phosphatase-conjugated goat anti-mouse IgG or IgA antibody (Jackson Immunoresearch, USA). Titers were calculated as the reciprocal of the serum dilution at which the response was 50%. After 142 days of the first immunization, some groups of mice (Table 1) were challenged orally with 1 × 104 focus forming units (FFU) of the murine rotavirus strain EDIMwt (100-fold the 50% diarrhea dose, DD50 ). Feces were collected 0–8 days post-challenge
Fig. 1. Serum IgG titers in mice immunized once with different VP6 assemblies. (A) Mice immunized with 10 g of VP6 nanotubes (crosses) or dlRLP (circles). (B) Mice immunized with 20 g of VP6 nanotubes (crosses) or dlRLP (circles). (C) Mice immunized with 40 g of VP6 nanotubes (crosses), dlRLP (circles) or trimers (triangles). The geometric mean and standard deviation within groups of 4 or 5 mice are shown.
(dpc) and stored at −20 ◦ C for rotavirus detection. Rotavirus shedding was measured as previously reported [16]. Virus shedding curves (OD versus dpc) for each animal were plotted and the reduction of the viral load was calculated as the difference between the average area under the curve of the control group and the immunized groups. 3. Results To determine the humoral response toward different VP6 assemblies, mice were immunized subcutaneously with several VP6 concentrations (10, 20 and 40 g), as described in Table 1 (groups 1–3, 6–8 and 11 with control 1). VP6-specific IgG and IgA titers in serum were measured. No VP6-specific IgG or IgA titers were detected in preimmune sera or in sera from control mice. Moreover, no VP6 specific IgA titers were detected in the serum samples from any of the groups (data not shown), as expected when immunizing subcutaneously. VP6 specific IgG were measurable at 15 dpi (Fig. 1), when the titers obtained with the three VP6 assemblies and concentrations were similar. Immunization with
Please cite this article in press as: Pastor AR, et al. The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.018
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Fig. 2. Serum IgG titers (geometric means) in mice immunized once (white) or twice (black, second immunization at 14 dpi) with 40 g of (A) VP6 nanotubes, (B) dlRLP or (C) VP6 trimers.
10 g of VP6 resulted in IgG titers around 100. The IgG maximum titer was obtained at 28 dpi with nanotubes, and titers decreased after that time (Fig. 1A). Increasing VP6 concentration to 20 g resulted in higher IgG titers (Fig. 1B). At this VP6 concentration, the maximum IgG titer was obtained at 35 dpi with dlRLP. A slight decrease in titer was observed at 42 dpi. The highest VP6-specific IgG titers were obtained when immunizing with 40 g of dlRLP (Fig. 1C). While in mice immunized with nanotubes and dlRLP, titers consistently increased until 42 dpi, IgG titers did not significantly change over time in mice immunized with trimers. As sustained and higher IgG titers were obtained when immunizing with 40 g of VP6, this amount was selected for the following experiments. Groups 4, 5 and 9–12 were immunized once or twice as described in Table 1. Control groups 2 and 3 were tested simultaneously. No VP6-specific IgA was detected in sera, while IgG titers increased as time progressed (Fig. 2). IgG titers in mice immunized with VP6 nanotubes were the highest, almost doubling the titers obtained when immunizing with dlRLP. A second immunization with VP6 nanotubes did not further increase IgG titers (Fig. 2A). In contrast, IgG titers in mice immunized with dlRLP or trimers increased significantly when a second immunization was performed (Fig. 2B and C). The IgG titers in mice immunized with trimers were the lowest, reaching only about one third of the titers obtained with dlRLP. For nanotubes and dlRLP, the maximum IgG titers were obtained at 121 dpi, and decreased afterwards. Mice in groups 4, 5, 9 and 10 and controls 2 and 3 were challenged with EDIM rotavirus at 142 dpi. Immunization with all VP6 formats significantly reduced viral shedding (Fig. 3A). While a single immunization with VP6 nanotubes was sufficient to obtain a high level of protection against rotavirus infection, two doses of either dlRLP or trimers were needed for a similar protection. No significant difference in virus shedding was found between mice immunized with nanotubes, two doses of dlRLP or two doses of VP6 trimers, while a significantly lower protection from rotavirus challenge was observed in mice immunized only once with dlRLP or trimers. Even when not significantly different, a higher protection by two doses of dlRLP than by two doses of trimers was observed. Higher IgG titers were associated with a higher level of protection against rotavirus infection (Fig. 3B). It can be seen that IgG titers of 1500 were sufficient to protect mice against infection.
been previously observed by Thönes et al. [17] when comparing the IgG response toward capsomeres or VLP. Moreover, Jegerleher et al. [8] constructed structures with different epitope densities and observed higher IgG titers as density increased. Changes in IgG titers are a result of differences in the extent of cross-linking of B-cell receptors, where highly organized antigens increase cross-linking [17]. In accordance with such results, we observed lower titers in mice immunized with trimers than in mice immunized with dlRLP or nanotubes. The difference between the IgG titers induced by nanotubes or dlRLP can also be explained by
4. Discussion The type of VP6 assembly used to immunize mice determined the serum IgG titers and conferred different protection against rotavirus infection. While for the first 42 dpi, dlRLP induced the highest IgG titers (300), as time progressed mice immunized with nanotubes had serum IgG titers above 8000. The lowest IgG titers were obtained in mice immunized with trimers. The effect of the protein quaternary structure on the induction of IgG titers has
Fig. 3. (A) Protection from EDIM rotavirus challenge in mice immunized once or twice with 40 g of VP6 nanotubes, dlRLP or trimers. Mean and standard deviation within groups of 4 mice are shown. Significantly different values are indicated with: *p < 0.05; **p < 0.005. (B) Correlation between IgG titers at 142 dpi and the reduction in virus shedding. Standard deviations within groups are shown.
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changes in B-cell receptor crosslinking, in this case most likely provoked by the different curvature of dlRLP and nanotubes, as well as differences in epitope geometry that can be inferred from the structures of such assemblies [9]. The high immunogenicity of tubular assemblies has been previously reported, even when no formal comparison with other types of assemblies had been performed. Ghosh et al. [6] found that the nonstructural NS1 protein of bluetongue virus can be used as a scaffold that efficiently induces humoral responses toward inserted peptides. Moreover, Blazevic et al. [7] have shown that VP6 nanotubes induce a strong systemic antibody response in mice. High IgG titers correlated with high protection from rotavirus infection. Reductions of viral shedding above 70% were observed when IgG titers were above 1500. No significant increase in protection occurred when IgG titers increased over 1500, probably because the reduction of virus shedding was already very high. Reductions of viral shedding above 90% were observed in some mice immunized with nanotubes. Two doses of trimers or dlRLP were necessary to achieve protection similar to that conferred by nanotubes. The correlation between high IgG titers and protection is in agreement with the work by Westerman et al. [18], who observed protection from rotavirus infection by serum IgG. The absence of serum IgA discard their role in the protection observed here, at least during the first hours after infection. Differences in protection between assemblies most probably did not involve CD8+ T-cell responses, as it has been shown that differences in protein quaternary structure do not affect them, as they are not directly dependent on repetitive antigen patterns [17]. It should also be considered that VP6 is an internal protein in the rotavirus capsid. Therefore, immunization with it does not induce rotavirus neutralizing antibodies. Protection from rotavirus infection by VP6specific antibodies requires their transcytosis into the rotavirus infected cells [10,19], which has been previously shown to occur for IgG [18,20]. Differences in the assembly of VP6 have other possible important effects on their immunogenicity. First, epitopes may be differentially exposed in each assembly, provoking differences in the IgG repertoire that is generated. Antibodies against some of the epitopes may be more efficient on neutralizing rotavirus replication than others. Second, the assemblies tested here have very different sizes. Trimers have a size below 20 nm, dlRLP have a diameter of 70 nm and the length of nanotubes is in the micrometer range. Such different sizes can result in differences in the efficiency of entry into lymph vessels or in the efficiency of uptake by antigen presenting cells [4]. This is the first time that the immunogenicity of three different protein assemblies is compared along with protection against challenge with live virus. It was demonstrated that VP6 assemblies induce different protection against rotavirus infection in mice. Such protection correlated with the IgG antibody titers in serum. Future studies to understand the molecular mechanisms in immune response against VP6 are necessary. The obtained information is important for the design of novel recombinant viral vaccines. Acknowledgements ´ and the Animal Technical support by V. Hernández, Rosa Roman Facility of the Instituto de Biotecnología, UNAM. We dedicate this
work to the memory of Ernesto Esquivel, who passed away while the manuscript was under review. Source of funding: SEP-Conacyt 101847. The funding source did not participate in study design, data analysis or manuscript writing. References [1] Lavelle L, Gingery M, Phillips M, Gelbart WM, Knobler CM, Cadena-Nava RD, et al. Phase diagram of self-assembled viral capsid protein polymorphs. J Phys Chem B 2009;113:3813–9. [2] Palomares LA, Ramírez OT. Challenges for the production of virus-like particles in insect cells: the case of rotavirus-like particles. Biochem Eng J 2009;45:158–67. [3] Plascencia-Villa G, Saniger JM, Ascencio JA, Palomares LA, Ramírez OT. Use of recombinant rotavirus VP6 nanotubes as a multifunctional template for the synthesis of nanobiomaterials functionalized with metals. Biotechnol Bioeng 2009;104:871–81. [4] Bachmann MF, Jennings GT. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat Rev 2010;10:787–96. [5] Roldao A, Mellado MCM, Castilho LR, Carrondo MJT, Alves PM. Virus-like particles in vaccine development. Expert Rev Vaccines 2010;9:1149–76. [6] Ghosh MK, Borca MV, Roy P. Virus-derived tubular structure displaying foreign sequences on the surface elicit CD4+ Th cell and protective humoral responses. Virology 2002;302:383–92. [7] Blazevic V, Lappalainen S, Nurminen K, Huhti L, Vesikari T. Norovirus VLP and rotavirus VP6 as combined vaccine for childhood gastroenteritis. Vaccine 2011;29:8126–33. [8] Jegerlehner A, Storni T, Lipowsky G, Schmid M, Pumpens P, Bachmann MF. Regulation of IgG antibody responses by epitope density and CD21-mediated coestimulation. Eur J Immunol 2002;32:3305–14. [9] Lepault J, Petitpas I, Erk I, Navaza J, Bigot D, Dona M, et al. Structural polymorphism of the major capsid protein of rotavirus. EMBO J 2001;20:1498– 507. [10] Corthésy B, Benureau Y, Perrier C, Fourgeux C, Parez N, Greenberg H, et al. Rotavirus anti-VP6 secretory immunoglobulin A contributes to protection via intracellular neutralization but not via immune exclusion. J Virol 2006;80:10692–1699. [11] McNeal M, Rae MN, Conner ME, Ward RL. Stimulation of local immunity and protection in mice by intramuscular immunization with tripleor double-layered rotavirus particles and QS-21. Virology 1998;243: 158–66. [12] Bertolotti-Ciarlet A, Ciarlet M, Crawford SE, Conner ME, Estes MK. Immunogenicity and protective efficacy of rotavirus 2/6-virus-like particles produced by a dual baculovirus expression vector and administered intramuscaularly, intranasally or orally to mice. Vaccine 2003;21:3885–900. [13] Mena JA, Ramírez OT, Palomares LA. Intracellular distribution of rotavirus structural proteins and virus-like particles expressed in the insect cell-baculovirus system. J Biotechnol 2006;122:443–52. [14] Plascencia-Villa G, Mena JA, Castro-Acosta RM, Fabián JC, Ramírez OT, Palomares LA. Strategies for the purification and characterization of protein scaffolds for the production of hybrid nanobiomaterials. J Chromatogr B 2011;879:1105–11. [15] Castro-Acosta RM, Revilla AL, Ramírez OT, Palomares LA. Separation and quantification of double- and triple-layered rotavirus-like particles by CZE. Electrophoresis 2010;31:1376–81. [16] Esquivel FR, Lopez S, Guitierrez-X L, Arias C. The internal rotavirus protein VP6 primes for an enhanced neutralizing antibody response. Arch Virol 2000;145:813–25. [17] Thönes N, Herreiner A, Schädlich L, Piuko K, Müller M. A direct comparison of human papillomavirus type 16 L1 particles reveals a lower immunogenicity of capsomeres than viruslike particles with respect to the induced antibody response. J Virol 2008;82:5472–85. [18] Westerman L, McClure HM, Jiang B, Almond JW, Glass RI. Serum IgG mediates mucosal immunity against rotavirus infection. Proc Natl Acad Sci 2005;102:7268–73. [19] Schwartz-Cornil I, Benureau Y, Greenberg H, Hendrickson BA, Cohen J. Heterologous protection induced by the inner capsid proteins of rotavirus requires transcytosis of mucosal immunoglobulins. J Virol 2002;76: 8110–7. [20] Rojas R, Apodaca G. Immunoglobulin transport across polarized epithelial cells. Nat Rev Mol Cell Biol 2002;3:944–55.
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The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice Ana Ruth Pastor a , William A. Rodríguez-Limas a , Martha A. Contreras a , Ernesto Esquivel a,b , Fernando Esquivel-Guadarrama b , Octavio T. Ramírez a , Laura A. Palomares a,∗ a Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Ave. Universidad 2001, Cuernavaca, Morelos 62210, Mexico b Facultad de Medicina, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, Mexico
a r t i c l e
i n f o
Article history: Available online xxx Keywords: Rotavirus Protein assembly VLP VP6 Vaccine Challenge
a b s t r a c t Viral protein assemblies have shown to be superior immunogens used in commercial vaccines. However, little is known about the effect of protein assembly structure in immunogenicity and the protection conferred by a vaccine. In this work, rotavirus VP6, a polymorphic protein that assembles into nanotubes, icosahedra (dlRLP) or trimers was used to compare the immune response elicited by three different assemblies. VP6 is the most antigenic and abundant rotavirus structural protein. It has been demonstrated that antibodies against VP6 interfere with the replication cycle of rotavirus, making it a vaccine candidate. Groups of mice were immunized with either nanotubes, dlRLP or trimers and the humoral response (IgG and IgA titers) was measured. Immunized mice were challenged with EDIM rotavirus and protection against rotavirus infection, measured as viral shedding, was evaluated. Immunization with nanotubes resulted in the highest IgG titers, followed by immunization with dlRLP. While immunization with one dose of nanotubes was sufficient to reduce viral shedding by 70%, two doses of dlRLP or trimers were required to obtain a similar protection. The results show that the type of assembly of VP6 results in different humoral responses and protection efficacies against challenge with live virus. This information is important for the design of recombinant vaccines in general. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Several viral proteins are polymorphic and can assemble into sheets, nanotubes, trimers, icosahedra, etc. [1]. The property of assembling into different types of structures allows the use of viral proteins for a wide variety of applications [2,3]. An important advantage of protein assemblies is their superior immunogenicity that allows the design of highly effective recombinant vaccines [4]. So far, recombinant viral proteins assembled into icosahedra (VLP) have been used as commercial vaccines against hepatitis B and human papilloma virus [5]. Furthermore, it has been shown that other types of assemblies may elicit high protective immune responses. That is the case of viral protein nanotubes [6,7]. The main
∗ Corresponding author. Tel.: +52 777 3291646; fax: +52 777 3138811. E-mail addresses: [email protected] (A.R. Pastor), [email protected] (W.A. Rodríguez-Limas), [email protected] (M.A. Contreras), [email protected] (E. Esquivel), [email protected] (F. Esquivel-Guadarrama), [email protected] (O.T. Ramírez), [email protected] (L.A. Palomares).
advantage of VLP as vaccines is that their size and the epitope distribution in their surface are optimal for recognition by the immune system to generate a humoral response [8]. Both properties depend on the type of assembly of the viral protein. Icosahedra and nanotubes have different spatial arrangement of the protein subunits, which results in different interepitope spaces [9]. In contrast, soluble trimers lack such arrangements. Even when the structure of viral protein assemblies can determine their immunogenicity, to our knowledge, little research has been done investigating the effect of such different structures in the immunogenicity of a recombinant protein. In order to investigate the effect of protein assembly in immunogenicity and protection against virus infection, we used rotavirus VP6, a polymorphic protein, to immunize mice. Rotavirus is the main cause of severe gastroenteritis in infants and provokes diarrhea in neonates from several mammalian species. Traditional live virus vaccines against rotavirus infection are available, but they have several disadvantages, such as possible contamination with other viruses and potential side effects. VP6 is the most antigenic and abundant rotavirus structural protein. It has been
http://dx.doi.org/10.1016/j.vaccine.2014.02.018 0264-410X/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Pastor AR, et al. The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.018
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Table 1 Groups of 4–5 mice were immunized subcutaneously as described below. The second dose was administered 14 dpi. Each group was kept in one cage. Immunogen
Group
VP6, g/mice
Doses
Challenge
Nanotubes
1 2 3 4 5
10 20 40 40 40
1 1 1 1 2
No No No Yes Yes
dlRLP
6 7 8 9 10
10 20 40 40 40
1 1 1 1 2
No No No Yes Yes
Trimers
11 12
40 40
1 2
Yes Yes
None
Control 1 Control 2 Control 3
0 0 0
1 1 2
No Yes Yes
demonstrated that antibodies against VP6 interfere with the replication cycle of rotavirus, making it an interesting vaccine candidate [10–12]. VP6 constitutes the middle layer of the rotavirus and can assemble into trimers, icosahedral particles or nanotubes depending on pH, ionic strength and the presence of VP2, which forms the inner layer of the rotavirus [9,13]. When VP6 is recombinantly expressed with VP2, it assembles over it to conform doublelayered rotavirus-like particles (dlRLP). In this work, we compared the humoral immune response and protective action of different recombinant VP6 assemblies, trimers, dlRLP and nanotubes. Mice were immunized with VP6 and challenged with live virus. VP6 in its nanotube form protected mice more efficiently from challenge than trimers or dlRLP, confirming that different viral protein assemblies confer different protection against viral infection. 2. Materials and methods VP6 nanotubes were produced and purified as previously described [14]. VP6 trimers were obtained by adding a 1 M CaCl2 solution to nanotubes to obtain a calcium concentration of 167 mM. dlRLP (containing rotavirus VP2 and VP6) were produced as previously reported [15]. VP6 concentration was measured using the ProSpecT ELISA kit (Oxoid, UK). Purified VP6 was used as standard. Various VP6 amounts and assemblies were used to immunize subcutaneously groups of six- to eight-week old female CD-1 mice as described in Table 1. Each group contained four or five mice. The amount of immunogen administered to mice was referred to VP6 without considering the VP2 content in the dlRLP preparation. VP6 buffer (30 mM Tris, 10 mM EDTA pH 8.0) was administered to control groups. To measure the anti-VP6 humoral response, individual blood samples were collected through the tail vein at 0, 14, 21, 28, 35, 42, 56, 121 and 142 days after the first immunization (dpi). IgG or IgA VP6 specific antibodies in mouse serum samples were measured by ELISA. Briefly, EIA plates (Corning, Costar) were coated with 5 g/mL of purified VP6 in carbonate buffer pH 9.5. Plates were blocked with 5 mg/mL of gelatin solution (Bio-Rad). After washing with Tris buffer with 0.05% Tween 20, serial 3-fold dilutions of serum samples were added to each well. This was followed by the addition of alkaline phosphatase-conjugated goat anti-mouse IgG or IgA antibody (Jackson Immunoresearch, USA). Titers were calculated as the reciprocal of the serum dilution at which the response was 50%. After 142 days of the first immunization, some groups of mice (Table 1) were challenged orally with 1 × 104 focus forming units (FFU) of the murine rotavirus strain EDIMwt (100-fold the 50% diarrhea dose, DD50 ). Feces were collected 0–8 days post-challenge
Fig. 1. Serum IgG titers in mice immunized once with different VP6 assemblies. (A) Mice immunized with 10 g of VP6 nanotubes (crosses) or dlRLP (circles). (B) Mice immunized with 20 g of VP6 nanotubes (crosses) or dlRLP (circles). (C) Mice immunized with 40 g of VP6 nanotubes (crosses), dlRLP (circles) or trimers (triangles). The geometric mean and standard deviation within groups of 4 or 5 mice are shown.
(dpc) and stored at −20 ◦ C for rotavirus detection. Rotavirus shedding was measured as previously reported [16]. Virus shedding curves (OD versus dpc) for each animal were plotted and the reduction of the viral load was calculated as the difference between the average area under the curve of the control group and the immunized groups. 3. Results To determine the humoral response toward different VP6 assemblies, mice were immunized subcutaneously with several VP6 concentrations (10, 20 and 40 g), as described in Table 1 (groups 1–3, 6–8 and 11 with control 1). VP6-specific IgG and IgA titers in serum were measured. No VP6-specific IgG or IgA titers were detected in preimmune sera or in sera from control mice. Moreover, no VP6 specific IgA titers were detected in the serum samples from any of the groups (data not shown), as expected when immunizing subcutaneously. VP6 specific IgG were measurable at 15 dpi (Fig. 1), when the titers obtained with the three VP6 assemblies and concentrations were similar. Immunization with
Please cite this article in press as: Pastor AR, et al. The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.018
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Fig. 2. Serum IgG titers (geometric means) in mice immunized once (white) or twice (black, second immunization at 14 dpi) with 40 g of (A) VP6 nanotubes, (B) dlRLP or (C) VP6 trimers.
10 g of VP6 resulted in IgG titers around 100. The IgG maximum titer was obtained at 28 dpi with nanotubes, and titers decreased after that time (Fig. 1A). Increasing VP6 concentration to 20 g resulted in higher IgG titers (Fig. 1B). At this VP6 concentration, the maximum IgG titer was obtained at 35 dpi with dlRLP. A slight decrease in titer was observed at 42 dpi. The highest VP6-specific IgG titers were obtained when immunizing with 40 g of dlRLP (Fig. 1C). While in mice immunized with nanotubes and dlRLP, titers consistently increased until 42 dpi, IgG titers did not significantly change over time in mice immunized with trimers. As sustained and higher IgG titers were obtained when immunizing with 40 g of VP6, this amount was selected for the following experiments. Groups 4, 5 and 9–12 were immunized once or twice as described in Table 1. Control groups 2 and 3 were tested simultaneously. No VP6-specific IgA was detected in sera, while IgG titers increased as time progressed (Fig. 2). IgG titers in mice immunized with VP6 nanotubes were the highest, almost doubling the titers obtained when immunizing with dlRLP. A second immunization with VP6 nanotubes did not further increase IgG titers (Fig. 2A). In contrast, IgG titers in mice immunized with dlRLP or trimers increased significantly when a second immunization was performed (Fig. 2B and C). The IgG titers in mice immunized with trimers were the lowest, reaching only about one third of the titers obtained with dlRLP. For nanotubes and dlRLP, the maximum IgG titers were obtained at 121 dpi, and decreased afterwards. Mice in groups 4, 5, 9 and 10 and controls 2 and 3 were challenged with EDIM rotavirus at 142 dpi. Immunization with all VP6 formats significantly reduced viral shedding (Fig. 3A). While a single immunization with VP6 nanotubes was sufficient to obtain a high level of protection against rotavirus infection, two doses of either dlRLP or trimers were needed for a similar protection. No significant difference in virus shedding was found between mice immunized with nanotubes, two doses of dlRLP or two doses of VP6 trimers, while a significantly lower protection from rotavirus challenge was observed in mice immunized only once with dlRLP or trimers. Even when not significantly different, a higher protection by two doses of dlRLP than by two doses of trimers was observed. Higher IgG titers were associated with a higher level of protection against rotavirus infection (Fig. 3B). It can be seen that IgG titers of 1500 were sufficient to protect mice against infection.
been previously observed by Thönes et al. [17] when comparing the IgG response toward capsomeres or VLP. Moreover, Jegerleher et al. [8] constructed structures with different epitope densities and observed higher IgG titers as density increased. Changes in IgG titers are a result of differences in the extent of cross-linking of B-cell receptors, where highly organized antigens increase cross-linking [17]. In accordance with such results, we observed lower titers in mice immunized with trimers than in mice immunized with dlRLP or nanotubes. The difference between the IgG titers induced by nanotubes or dlRLP can also be explained by
4. Discussion The type of VP6 assembly used to immunize mice determined the serum IgG titers and conferred different protection against rotavirus infection. While for the first 42 dpi, dlRLP induced the highest IgG titers (300), as time progressed mice immunized with nanotubes had serum IgG titers above 8000. The lowest IgG titers were obtained in mice immunized with trimers. The effect of the protein quaternary structure on the induction of IgG titers has
Fig. 3. (A) Protection from EDIM rotavirus challenge in mice immunized once or twice with 40 g of VP6 nanotubes, dlRLP or trimers. Mean and standard deviation within groups of 4 mice are shown. Significantly different values are indicated with: *p < 0.05; **p < 0.005. (B) Correlation between IgG titers at 142 dpi and the reduction in virus shedding. Standard deviations within groups are shown.
Please cite this article in press as: Pastor AR, et al. The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.018
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changes in B-cell receptor crosslinking, in this case most likely provoked by the different curvature of dlRLP and nanotubes, as well as differences in epitope geometry that can be inferred from the structures of such assemblies [9]. The high immunogenicity of tubular assemblies has been previously reported, even when no formal comparison with other types of assemblies had been performed. Ghosh et al. [6] found that the nonstructural NS1 protein of bluetongue virus can be used as a scaffold that efficiently induces humoral responses toward inserted peptides. Moreover, Blazevic et al. [7] have shown that VP6 nanotubes induce a strong systemic antibody response in mice. High IgG titers correlated with high protection from rotavirus infection. Reductions of viral shedding above 70% were observed when IgG titers were above 1500. No significant increase in protection occurred when IgG titers increased over 1500, probably because the reduction of virus shedding was already very high. Reductions of viral shedding above 90% were observed in some mice immunized with nanotubes. Two doses of trimers or dlRLP were necessary to achieve protection similar to that conferred by nanotubes. The correlation between high IgG titers and protection is in agreement with the work by Westerman et al. [18], who observed protection from rotavirus infection by serum IgG. The absence of serum IgA discard their role in the protection observed here, at least during the first hours after infection. Differences in protection between assemblies most probably did not involve CD8+ T-cell responses, as it has been shown that differences in protein quaternary structure do not affect them, as they are not directly dependent on repetitive antigen patterns [17]. It should also be considered that VP6 is an internal protein in the rotavirus capsid. Therefore, immunization with it does not induce rotavirus neutralizing antibodies. Protection from rotavirus infection by VP6specific antibodies requires their transcytosis into the rotavirus infected cells [10,19], which has been previously shown to occur for IgG [18,20]. Differences in the assembly of VP6 have other possible important effects on their immunogenicity. First, epitopes may be differentially exposed in each assembly, provoking differences in the IgG repertoire that is generated. Antibodies against some of the epitopes may be more efficient on neutralizing rotavirus replication than others. Second, the assemblies tested here have very different sizes. Trimers have a size below 20 nm, dlRLP have a diameter of 70 nm and the length of nanotubes is in the micrometer range. Such different sizes can result in differences in the efficiency of entry into lymph vessels or in the efficiency of uptake by antigen presenting cells [4]. This is the first time that the immunogenicity of three different protein assemblies is compared along with protection against challenge with live virus. It was demonstrated that VP6 assemblies induce different protection against rotavirus infection in mice. Such protection correlated with the IgG antibody titers in serum. Future studies to understand the molecular mechanisms in immune response against VP6 are necessary. The obtained information is important for the design of novel recombinant viral vaccines. Acknowledgements ´ and the Animal Technical support by V. Hernández, Rosa Roman Facility of the Instituto de Biotecnología, UNAM. We dedicate this
work to the memory of Ernesto Esquivel, who passed away while the manuscript was under review. Source of funding: SEP-Conacyt 101847. The funding source did not participate in study design, data analysis or manuscript writing. References [1] Lavelle L, Gingery M, Phillips M, Gelbart WM, Knobler CM, Cadena-Nava RD, et al. Phase diagram of self-assembled viral capsid protein polymorphs. J Phys Chem B 2009;113:3813–9. [2] Palomares LA, Ramírez OT. Challenges for the production of virus-like particles in insect cells: the case of rotavirus-like particles. Biochem Eng J 2009;45:158–67. [3] Plascencia-Villa G, Saniger JM, Ascencio JA, Palomares LA, Ramírez OT. Use of recombinant rotavirus VP6 nanotubes as a multifunctional template for the synthesis of nanobiomaterials functionalized with metals. Biotechnol Bioeng 2009;104:871–81. [4] Bachmann MF, Jennings GT. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat Rev 2010;10:787–96. [5] Roldao A, Mellado MCM, Castilho LR, Carrondo MJT, Alves PM. Virus-like particles in vaccine development. Expert Rev Vaccines 2010;9:1149–76. [6] Ghosh MK, Borca MV, Roy P. Virus-derived tubular structure displaying foreign sequences on the surface elicit CD4+ Th cell and protective humoral responses. Virology 2002;302:383–92. [7] Blazevic V, Lappalainen S, Nurminen K, Huhti L, Vesikari T. Norovirus VLP and rotavirus VP6 as combined vaccine for childhood gastroenteritis. Vaccine 2011;29:8126–33. [8] Jegerlehner A, Storni T, Lipowsky G, Schmid M, Pumpens P, Bachmann MF. Regulation of IgG antibody responses by epitope density and CD21-mediated coestimulation. Eur J Immunol 2002;32:3305–14. [9] Lepault J, Petitpas I, Erk I, Navaza J, Bigot D, Dona M, et al. Structural polymorphism of the major capsid protein of rotavirus. EMBO J 2001;20:1498– 507. [10] Corthésy B, Benureau Y, Perrier C, Fourgeux C, Parez N, Greenberg H, et al. Rotavirus anti-VP6 secretory immunoglobulin A contributes to protection via intracellular neutralization but not via immune exclusion. J Virol 2006;80:10692–1699. [11] McNeal M, Rae MN, Conner ME, Ward RL. Stimulation of local immunity and protection in mice by intramuscular immunization with tripleor double-layered rotavirus particles and QS-21. Virology 1998;243: 158–66. [12] Bertolotti-Ciarlet A, Ciarlet M, Crawford SE, Conner ME, Estes MK. Immunogenicity and protective efficacy of rotavirus 2/6-virus-like particles produced by a dual baculovirus expression vector and administered intramuscaularly, intranasally or orally to mice. Vaccine 2003;21:3885–900. [13] Mena JA, Ramírez OT, Palomares LA. Intracellular distribution of rotavirus structural proteins and virus-like particles expressed in the insect cell-baculovirus system. J Biotechnol 2006;122:443–52. [14] Plascencia-Villa G, Mena JA, Castro-Acosta RM, Fabián JC, Ramírez OT, Palomares LA. Strategies for the purification and characterization of protein scaffolds for the production of hybrid nanobiomaterials. J Chromatogr B 2011;879:1105–11. [15] Castro-Acosta RM, Revilla AL, Ramírez OT, Palomares LA. Separation and quantification of double- and triple-layered rotavirus-like particles by CZE. Electrophoresis 2010;31:1376–81. [16] Esquivel FR, Lopez S, Guitierrez-X L, Arias C. The internal rotavirus protein VP6 primes for an enhanced neutralizing antibody response. Arch Virol 2000;145:813–25. [17] Thönes N, Herreiner A, Schädlich L, Piuko K, Müller M. A direct comparison of human papillomavirus type 16 L1 particles reveals a lower immunogenicity of capsomeres than viruslike particles with respect to the induced antibody response. J Virol 2008;82:5472–85. [18] Westerman L, McClure HM, Jiang B, Almond JW, Glass RI. Serum IgG mediates mucosal immunity against rotavirus infection. Proc Natl Acad Sci 2005;102:7268–73. [19] Schwartz-Cornil I, Benureau Y, Greenberg H, Hendrickson BA, Cohen J. Heterologous protection induced by the inner capsid proteins of rotavirus requires transcytosis of mucosal immunoglobulins. J Virol 2002;76: 8110–7. [20] Rojas R, Apodaca G. Immunoglobulin transport across polarized epithelial cells. Nat Rev Mol Cell Biol 2002;3:944–55.
Please cite this article in press as: Pastor AR, et al. The assembly conformation of rotavirus VP6 determines its protective efficacy against rotavirus challenge in mice. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.02.018
