HUMAN VACCINES & IMMUNOTHERAPEUTICS 2016, VOL. 12, NO. 4, 988–989 http://dx.doi.org/10.1080/21645515.2015.1116655
COMMENTARY
Recombinant immune complexes as versatile and potent vaccines Hugh S. Mason Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ, USA
ABSTRACT
ARTICLE HISTORY
Immune complexes (IC) used as vaccines have the potential to enhance both antibody and cell-mediated immune responses over those obtained with antigen alone. However, difficulty of manufacture represents a significant hurdle to the widespread use of IC as vaccines. Recombinant IC (RIC) and their expression in plants enable manufacturing by the coordinate expression of immunoglobulin and antigen as a fusion protein. The use of a modular RIC system facilitates insertion of antigen genes and provides a broadly applicable platform that can be adapted for a wide variety of antigens.
Received 15 October 2015 Accepted 31 October 2015
Immune complexes (IC) comprising immunoglobulin molecules bound to their cognate antigen represent a potentially powerful yet relatively unexploited strategy for vaccine delivery for humans and agricultural animals. One of the earliest observations showed enhanced immune responses in mice when antigen and antiserum were co-delivered.1 Another early paper involved establishment of B memory cells via stimulation with preformed antigen-antibody complexes.2 Many more recent studies have demonstrated enhanced immune responses in animals that were vaccinated with IC, as compared to the antigens alone.3-7 The data strongly suggest that the mechanism of immune enhancement involves the enabling of binding of the antigen-antibody complex to dendritic cells (DC) or other antigen presenting cells via interaction with Fc domains of the immunoglobulin component, or by binding to components of complement that enhance dendritic cell interaction.2-4,8-10 These interactions would enable efficient uptake and processing of foreign antigens to provide more effective T-cell help to activated B cells, and thus more robust production of antibodies. Moreover, evidence indicates that IC-induced maturation of DC enables them to prime CD8C cytotoxic T cells, as well as CD4C T-helper cells.8-10 Why has the IC strategy not yet been further developed for application in clinical studies? An important reason is the difficulty of manufacture, which can require the separate production of antigen and antibody, often in different expression host cells or organisms in order to optimize yield or quality of the biologic products. A good solution to this problem is the use of “recombinant immune complexes” (RIC), which could be produced in various hosts, but very conveniently in plants using either stably integrated3,7 or transient expression11-13 constructs. This strategy entails the fusion of a vaccine protein to the C-terminus of the heavy chain of an IgG that binds specifically to the antigen. Co-expression of this fusion protein with the light chain of the antibody produces a fully formed IgG that is self-reactive, and results in the creation of IC due to the
CONTACT Hugh S. Mason © 2016 Taylor & Francis
[email protected]
KEYWORDS
antibody fusion; immune complex; recombinant plant; transient expression; vaccine
bivalent binding capacity of the IgG (see Fig. 1 in ref. 3 and Fig. 9 in ref. 12). Chargelegue and coworkers showed that a plant-produced RIC comprising tetanus toxin fragment C (TTFC) displayed the expected secondary effector functions that enable binding to complement C1q and Fcg receptor, as well as uptake by antigen-presenting cells.3 Interestingly, although Fc effector functions can depend on specific Asnlinked glycan patterns,14 the plant-specific glycans of the tobacco host cells15 did not impede the immune functions of TTFC-RIC as measured by in vitro binding or in vivo antibody response. One potential problem (but also an opportunity) with IC vaccines is that the choice of antibody (either monoclonal or polyclonal) may affect the outcome. Hioe and colleagues have used this phenomenon to select monoclonal antibodies (mAb) to combine with the gp120 antigen of HIV in order to obtain optimized broadly reactive antibody responses against the V3 domain.6 Thus, substantial empirical study may be required in order to choose the best combination of antibody and antigen. The author and colleagues have recently developed11 a modular system using a mAb directed at a linear epitope of Ebola GP1,12 which enables convenient epitope tagging of a protein antigen and fusion to the mAb heavy chain (Fig. 1). In the event that the antigen module requires a free C-terminus for optimal immunogenicity, the epitope tag may be moved to the N-terminal side of the antigen, with appropriate linkers flanking it. Thus, the incorporated antigen is not directly bound by the mAb and is relatively free to interact with B cell receptors in order to stimulate the B cells for further development. The model antigen studied was a dengue virus envelope glycoprotein domain III, which is a small and compact globular domain of 103 amino acids.11 The resulting RIC, produced by rapid and robust transient expression in Nicotiana benthamiana leaves and conveniently purified by protein A-affinity, displayed complement C1q binding and provoked strong, virus-neutralizing
P.O. Box 874501, Arizona State University, Tempe, AZ 85287-4501, USA.
HUMAN VACCINES & IMMUNOTHERAPEUTICS
Figure 1. Structure of the modular mAb fusion protein for production of RIC. The heavy chain of anti-Ebola GP1 mAb 6D8 is extended from its C-terminus by a linker (G4S)3, an antigen module whose sequence is flanked by unique BamHI and SpeI sites, and the Ebola GP1 6D8 epitope including the amino acids “HNTPVYKLDISEATQ”.
antibody responses in mice without an adjuvant. The fact that a much larger antigen, Ebola GP1, was also amenable to the RIC strategy12,13 suggests that a wide range of antigen sizes can be accommodated by the modular platform. However, it is likely that some antigens will perform less well, depending on their particular characteristics and folding patterns. Further, since the mAb fusion constructs are targeted to the ER in the host cells, the structure and folding of the fused antigen must be compatible with the ER environment, with the realization that Asn residues that occur in the context “N-X-S/T” are likely to be glycosylated.15 Nonetheless, many protein antigens will be reasonably good targets for the modular RIC strategy. An issue that needs further work is the degree of size heterogeneity of RIC and its effect on induction of favorable immune responses, which will be important for manufacturing, purification, and application in clinical trials. The number of IgGfusion molecules involved in an average RIC is unclear, but measurements on Ebola GP1 RIC suggest that it is in the range of 4-6 molecules per complex.12 However, it is likely that the average size of a particular RIC will be affected by characteristics of the antibody and antigen used, as well as methods of purification and storage. In some cases, the antigen itself could drive oligomerization,7 which could complicate matters further. In this regard, the use of a defined antibody and antigen as a platform for insertion of antigen modules11 can simplify and systematize the requirements for purification. We conclude that the use of IC as vaccines represent a viable and in many cases preferable alternative to soluble antigens, as the benefit of effective targeting to dendritic cells via Fc receptor interaction can enable strong antibody responses in without the use of adjuvants. Moreover, the potential of IC to stimulate CD8C CTL responses is a strong advantage. Finally, the use of a modular RIC system provides a broadly applicable platform that can be adapted for a wide variety of antigens and greatly facilitate the development of vaccines.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References [1] Morrison SL, Terres G. Enhanced immunologic sensitization of mice by the simultaneous injection of antigen and specific antiserum. II. Effect of varying the antigen-antibody ratio and the amount of immune complex injected. J Immunol 1966; 96:901-5; PMID:5913760
989
[2] Klaus GG. The generation of memory cells. II. Generation of B memory cells with preformed antigen-antibody complexes. Immunology 1978; 34:643-52; PMID:309848. [3] Chargelegue D, Drake PM, Obregon P, Prada A, Fairweather N, Ma JK. Highly immunogenic and protective recombinant vaccine candidate expressed in transgenic plants. Infect Immun 2005; 73:5915-22; PMID:16113311; http://dx.doi.org/10.1128/ IAI.73.9.5915-5922.2005 [4] Herrada AA, Contreras FJ, Tobar JA, Pacheco R, Kalergis AM. Immune complex-induced enhancement of bacterial antigen presentation requires Fcgamma receptor III expression on dendritic cells. Proc Natl Acad Sci U S A 2007; 104:13402-7; PMID:17679697; http://dx.doi.org/10.1073/pnas.0700999104 [5] Hioe CE, Visciano ML, Kumar R, Liu J, Mack EA, Simon RE, Levy DN, Tuen M. The use of immune complex vaccines to enhance antibody responses against neutralizing epitopes on HIV-1 envelope gp120. Vaccine 2009; 28:352-60; PMID:19879224; http://dx.doi.org/ 10.1016/j.vaccine.2009.10.040 [6] Kumar R, Tuen M, Liu J, Nadas A, Pan R, Kong X, Hioe CE. Elicitation of broadly reactive antibodies against glycan-modulated neutralizing V3 epitopes of HIV-1 by immune complex vaccines. Vaccine 2013; 31:5413-21; PMID:24051158; http://dx.doi.org/10.1016/j. vaccine.2013.09.010 [7] Pepponi I, Diogo GR, Stylianou E, van Dolleweerd CJ, Drake PM, Paul MJ, Sibley L, Ma JK, Reljic R. Plant-derived recombinant immune complexes as self-adjuvanting TB immunogens for mucosal boosting of BCG. Plant Biotechnol J 2014; 12:840-50; PMID:24629003; http://dx.doi.org/10.1111/pbi.12185 [8] Regnault A, Lankar D, Lacabanne V, Rodriguez A, Thery C, Rescigno M, Saito T, Verbeek S, Bonnerot C, Ricciardi-Castagnoli P, Amigorena S. Fcgamma receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J Exp Med 1999; 189:371-80; PMID:9892619; http://dx.doi.org/10.1084/jem.189.2.371 [9] Schuurhuis DH, Ioan-Facsinay A, Nagelkerken B, van Schip JJ, Sedlik C, Melief CJ, Verbeek JS, Ossendorp F. Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8C CTL responses in vivo. J Immunol 2002; 168:2240-6; PMID:11859111; http://dx.doi.org/10.4049/jimmunol.168.5.2240 [10] van Montfoort N, t Hoen PA, Mangsbo SM, Camps MG, Boross P, Melief CJ, Ossendorp F, Verbeek JS. Fcgamma receptor IIb strongly regulates fcgamma receptor-facilitated T cell activation by dendritic cells. J Immunol 2012; 189:92-101; PMID:22649202; http://dx.doi. org/10.4049/jimmunol.1103703 [11] Kim MY, Reljic R, Kilbourne J, Ceballos-Olvera I, Yang MS, Reyesdel Valle J, Mason HS. Novel vaccination approach for dengue infection based on recombinant immune complex universal platform. Vaccine 2015; 33:1830-8; PMID:25728317; http://dx.doi.org/ 10.1016/j.vaccine.2015.02.036 [12] Phoolcharoen W, Bhoo SH, Lai H, Ma J, Arntzen CJ, Chen Q, Mason HS. Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana. Plant Biotechnol J 2011a; 9:807-16; http://dx.doi. org/10.1111/j.1467-7652.2011.00593.x [13] Phoolcharoen W, Dye JM, Kilbourne J, Piensook K, Pratt WD, Arntzen CJ, Chen Q, Mason HS, Herbst-Kralovetz MM. A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge. Proc Natl Acad Sci U S A 2011b; 108:20695-20700; http://dx.doi.org/ 10.1073/pnas.1117715108 [14] Jefferis R Isotype and glycoform selection for antibody therapeutics. Arch Biochem Biophys 2012; 526:159-66; PMID:22465822; http://dx. doi.org/10.1016/j.abb.2012.03.021 [15] Strasser R, Stadlmann J, Schahs M, Stiegler G, Quendler H, Mach L, Glossl J, Weterings K, Pabst M, Steinkellner H (2008) Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnol J 6:392-402; PMID:18346095; http://dx. doi.org/10.1111/j.1467-7652.2008.00330.x
COMMENTARY
Recombinant immune complexes as versatile and potent vaccines Hugh S. Mason Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ, USA
ABSTRACT
ARTICLE HISTORY
Immune complexes (IC) used as vaccines have the potential to enhance both antibody and cell-mediated immune responses over those obtained with antigen alone. However, difficulty of manufacture represents a significant hurdle to the widespread use of IC as vaccines. Recombinant IC (RIC) and their expression in plants enable manufacturing by the coordinate expression of immunoglobulin and antigen as a fusion protein. The use of a modular RIC system facilitates insertion of antigen genes and provides a broadly applicable platform that can be adapted for a wide variety of antigens.
Received 15 October 2015 Accepted 31 October 2015
Immune complexes (IC) comprising immunoglobulin molecules bound to their cognate antigen represent a potentially powerful yet relatively unexploited strategy for vaccine delivery for humans and agricultural animals. One of the earliest observations showed enhanced immune responses in mice when antigen and antiserum were co-delivered.1 Another early paper involved establishment of B memory cells via stimulation with preformed antigen-antibody complexes.2 Many more recent studies have demonstrated enhanced immune responses in animals that were vaccinated with IC, as compared to the antigens alone.3-7 The data strongly suggest that the mechanism of immune enhancement involves the enabling of binding of the antigen-antibody complex to dendritic cells (DC) or other antigen presenting cells via interaction with Fc domains of the immunoglobulin component, or by binding to components of complement that enhance dendritic cell interaction.2-4,8-10 These interactions would enable efficient uptake and processing of foreign antigens to provide more effective T-cell help to activated B cells, and thus more robust production of antibodies. Moreover, evidence indicates that IC-induced maturation of DC enables them to prime CD8C cytotoxic T cells, as well as CD4C T-helper cells.8-10 Why has the IC strategy not yet been further developed for application in clinical studies? An important reason is the difficulty of manufacture, which can require the separate production of antigen and antibody, often in different expression host cells or organisms in order to optimize yield or quality of the biologic products. A good solution to this problem is the use of “recombinant immune complexes” (RIC), which could be produced in various hosts, but very conveniently in plants using either stably integrated3,7 or transient expression11-13 constructs. This strategy entails the fusion of a vaccine protein to the C-terminus of the heavy chain of an IgG that binds specifically to the antigen. Co-expression of this fusion protein with the light chain of the antibody produces a fully formed IgG that is self-reactive, and results in the creation of IC due to the
CONTACT Hugh S. Mason © 2016 Taylor & Francis
[email protected]
KEYWORDS
antibody fusion; immune complex; recombinant plant; transient expression; vaccine
bivalent binding capacity of the IgG (see Fig. 1 in ref. 3 and Fig. 9 in ref. 12). Chargelegue and coworkers showed that a plant-produced RIC comprising tetanus toxin fragment C (TTFC) displayed the expected secondary effector functions that enable binding to complement C1q and Fcg receptor, as well as uptake by antigen-presenting cells.3 Interestingly, although Fc effector functions can depend on specific Asnlinked glycan patterns,14 the plant-specific glycans of the tobacco host cells15 did not impede the immune functions of TTFC-RIC as measured by in vitro binding or in vivo antibody response. One potential problem (but also an opportunity) with IC vaccines is that the choice of antibody (either monoclonal or polyclonal) may affect the outcome. Hioe and colleagues have used this phenomenon to select monoclonal antibodies (mAb) to combine with the gp120 antigen of HIV in order to obtain optimized broadly reactive antibody responses against the V3 domain.6 Thus, substantial empirical study may be required in order to choose the best combination of antibody and antigen. The author and colleagues have recently developed11 a modular system using a mAb directed at a linear epitope of Ebola GP1,12 which enables convenient epitope tagging of a protein antigen and fusion to the mAb heavy chain (Fig. 1). In the event that the antigen module requires a free C-terminus for optimal immunogenicity, the epitope tag may be moved to the N-terminal side of the antigen, with appropriate linkers flanking it. Thus, the incorporated antigen is not directly bound by the mAb and is relatively free to interact with B cell receptors in order to stimulate the B cells for further development. The model antigen studied was a dengue virus envelope glycoprotein domain III, which is a small and compact globular domain of 103 amino acids.11 The resulting RIC, produced by rapid and robust transient expression in Nicotiana benthamiana leaves and conveniently purified by protein A-affinity, displayed complement C1q binding and provoked strong, virus-neutralizing
P.O. Box 874501, Arizona State University, Tempe, AZ 85287-4501, USA.
HUMAN VACCINES & IMMUNOTHERAPEUTICS
Figure 1. Structure of the modular mAb fusion protein for production of RIC. The heavy chain of anti-Ebola GP1 mAb 6D8 is extended from its C-terminus by a linker (G4S)3, an antigen module whose sequence is flanked by unique BamHI and SpeI sites, and the Ebola GP1 6D8 epitope including the amino acids “HNTPVYKLDISEATQ”.
antibody responses in mice without an adjuvant. The fact that a much larger antigen, Ebola GP1, was also amenable to the RIC strategy12,13 suggests that a wide range of antigen sizes can be accommodated by the modular platform. However, it is likely that some antigens will perform less well, depending on their particular characteristics and folding patterns. Further, since the mAb fusion constructs are targeted to the ER in the host cells, the structure and folding of the fused antigen must be compatible with the ER environment, with the realization that Asn residues that occur in the context “N-X-S/T” are likely to be glycosylated.15 Nonetheless, many protein antigens will be reasonably good targets for the modular RIC strategy. An issue that needs further work is the degree of size heterogeneity of RIC and its effect on induction of favorable immune responses, which will be important for manufacturing, purification, and application in clinical trials. The number of IgGfusion molecules involved in an average RIC is unclear, but measurements on Ebola GP1 RIC suggest that it is in the range of 4-6 molecules per complex.12 However, it is likely that the average size of a particular RIC will be affected by characteristics of the antibody and antigen used, as well as methods of purification and storage. In some cases, the antigen itself could drive oligomerization,7 which could complicate matters further. In this regard, the use of a defined antibody and antigen as a platform for insertion of antigen modules11 can simplify and systematize the requirements for purification. We conclude that the use of IC as vaccines represent a viable and in many cases preferable alternative to soluble antigens, as the benefit of effective targeting to dendritic cells via Fc receptor interaction can enable strong antibody responses in without the use of adjuvants. Moreover, the potential of IC to stimulate CD8C CTL responses is a strong advantage. Finally, the use of a modular RIC system provides a broadly applicable platform that can be adapted for a wide variety of antigens and greatly facilitate the development of vaccines.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
References [1] Morrison SL, Terres G. Enhanced immunologic sensitization of mice by the simultaneous injection of antigen and specific antiserum. II. Effect of varying the antigen-antibody ratio and the amount of immune complex injected. J Immunol 1966; 96:901-5; PMID:5913760
989
[2] Klaus GG. The generation of memory cells. II. Generation of B memory cells with preformed antigen-antibody complexes. Immunology 1978; 34:643-52; PMID:309848. [3] Chargelegue D, Drake PM, Obregon P, Prada A, Fairweather N, Ma JK. Highly immunogenic and protective recombinant vaccine candidate expressed in transgenic plants. Infect Immun 2005; 73:5915-22; PMID:16113311; http://dx.doi.org/10.1128/ IAI.73.9.5915-5922.2005 [4] Herrada AA, Contreras FJ, Tobar JA, Pacheco R, Kalergis AM. Immune complex-induced enhancement of bacterial antigen presentation requires Fcgamma receptor III expression on dendritic cells. Proc Natl Acad Sci U S A 2007; 104:13402-7; PMID:17679697; http://dx.doi.org/10.1073/pnas.0700999104 [5] Hioe CE, Visciano ML, Kumar R, Liu J, Mack EA, Simon RE, Levy DN, Tuen M. The use of immune complex vaccines to enhance antibody responses against neutralizing epitopes on HIV-1 envelope gp120. Vaccine 2009; 28:352-60; PMID:19879224; http://dx.doi.org/ 10.1016/j.vaccine.2009.10.040 [6] Kumar R, Tuen M, Liu J, Nadas A, Pan R, Kong X, Hioe CE. Elicitation of broadly reactive antibodies against glycan-modulated neutralizing V3 epitopes of HIV-1 by immune complex vaccines. Vaccine 2013; 31:5413-21; PMID:24051158; http://dx.doi.org/10.1016/j. vaccine.2013.09.010 [7] Pepponi I, Diogo GR, Stylianou E, van Dolleweerd CJ, Drake PM, Paul MJ, Sibley L, Ma JK, Reljic R. Plant-derived recombinant immune complexes as self-adjuvanting TB immunogens for mucosal boosting of BCG. Plant Biotechnol J 2014; 12:840-50; PMID:24629003; http://dx.doi.org/10.1111/pbi.12185 [8] Regnault A, Lankar D, Lacabanne V, Rodriguez A, Thery C, Rescigno M, Saito T, Verbeek S, Bonnerot C, Ricciardi-Castagnoli P, Amigorena S. Fcgamma receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J Exp Med 1999; 189:371-80; PMID:9892619; http://dx.doi.org/10.1084/jem.189.2.371 [9] Schuurhuis DH, Ioan-Facsinay A, Nagelkerken B, van Schip JJ, Sedlik C, Melief CJ, Verbeek JS, Ossendorp F. Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8C CTL responses in vivo. J Immunol 2002; 168:2240-6; PMID:11859111; http://dx.doi.org/10.4049/jimmunol.168.5.2240 [10] van Montfoort N, t Hoen PA, Mangsbo SM, Camps MG, Boross P, Melief CJ, Ossendorp F, Verbeek JS. Fcgamma receptor IIb strongly regulates fcgamma receptor-facilitated T cell activation by dendritic cells. J Immunol 2012; 189:92-101; PMID:22649202; http://dx.doi. org/10.4049/jimmunol.1103703 [11] Kim MY, Reljic R, Kilbourne J, Ceballos-Olvera I, Yang MS, Reyesdel Valle J, Mason HS. Novel vaccination approach for dengue infection based on recombinant immune complex universal platform. Vaccine 2015; 33:1830-8; PMID:25728317; http://dx.doi.org/ 10.1016/j.vaccine.2015.02.036 [12] Phoolcharoen W, Bhoo SH, Lai H, Ma J, Arntzen CJ, Chen Q, Mason HS. Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana. Plant Biotechnol J 2011a; 9:807-16; http://dx.doi. org/10.1111/j.1467-7652.2011.00593.x [13] Phoolcharoen W, Dye JM, Kilbourne J, Piensook K, Pratt WD, Arntzen CJ, Chen Q, Mason HS, Herbst-Kralovetz MM. A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge. Proc Natl Acad Sci U S A 2011b; 108:20695-20700; http://dx.doi.org/ 10.1073/pnas.1117715108 [14] Jefferis R Isotype and glycoform selection for antibody therapeutics. Arch Biochem Biophys 2012; 526:159-66; PMID:22465822; http://dx. doi.org/10.1016/j.abb.2012.03.021 [15] Strasser R, Stadlmann J, Schahs M, Stiegler G, Quendler H, Mach L, Glossl J, Weterings K, Pabst M, Steinkellner H (2008) Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnol J 6:392-402; PMID:18346095; http://dx. doi.org/10.1111/j.1467-7652.2008.00330.x
