Glycan mediated immune responses to tumor cells

8 downloads 120 Views 499KB Size Report
Oct 8, 2010 - immune responses as a survival strategy.6 Cancer cells might also use ... metastatic models.23,24 Theoretically, cancer vaccines treatment.
review

Human Vaccines 7: Supplement, 156-165; January/February 2011; © 2011 Landes Bioscience

Glycan-mediated immune responses to tumor cells Anastas Pashov, Behjatolah Monzavi-Karbassi and Thomas Kieber-Emmons* Winthrop P. Rockefeller Cancer Institute; Department of Pathology; University of Arkansas for Medical Sciences; Little Rock, AR USA

Key words: cancer, mimotopes, carbohydrate, cytotoxic T lymphocytes, antigen presentation

Preclinical animal studies convincingly demonstrate that tumor immunity to self-antigens can be actively induced and can translate into effective anti-tumor responses. Among the most challenging of clinical targets for cancer immunotherapy is Tumor Associated Carbohydrate Antigens (TACA). The molecular characterization of TACA suggest that these glycans are both altered and self-antigens. A new appreciation of the interaction of glycans with immune effector cells that will benefit immunotherapy strategies is emerging as more information on the nature of molecular interactions of glycan recognition molecules is obtained. Carbohydrate recognition affects more or less every aspect of the innate and adaptive immune response and their role in immunotherapy of cancer should be considered beyond the existing paradigm of traditional TACA based-vaccines.

as more information on the nature of molecular interactions of glycan recognition molecules is obtained.8 For example, parasitic helminths have co-evolved with vertebrate immune systems to enable long-term survival of worms in infected hosts. Some worm glycan antigens share structural features with host-like glycans, including Le(X) (Galbeta1-4[Fucalpha1-3] GlcNAc-), LDNF (GalNAcbeta1-4[Fucalpha1-3]GlcNAc-), LDN (GalNAcbeta1-4GlcNAc-) and Tn (GalNAcalpha1-OThr/Ser), some of which are Tumor Associated Carbohydrate Antigens (TACA) displayed as altered or embryonic forms. In worms, these glycan epitopes regulate and suppress host immune responses as a survival strategy.6 Cancer cells might also use these shifts in glycan code to evade immune surveillance, but they might also suggest strategies for treatments that accompany neoplastic transformation.9,10 TACA affect a myriad of processes that correlate with poor prognosis of cancer affecting cell signaling and communication, cell motility and adhesion, angiogenesis and organ tropism.11,12 Changes in glycosylation are often a hallmark of the transition from normal to inflamed or neoplastic tissue.13-15 TACA, like pathogen glycan antigens, are targeted by innate immune responses suggesting that innate immune surveillance is operative against TACA.16,17 Many host cell types, including endothelial and epithelial cells, neutrophils, monocytes, Natural Killer (NK) cells, Dendritic Cells (DC) and macrophages, initiate the first line of defense against infection by sensing conserved microbial structures through Toll-like receptors (TLRs).18 Consequently, glycans may carry danger signals for the immune system, often playing a role in immune recognition but not necessarily immune response.16,19 The rationale for targeting TACA was elegantly laid out years ago in terms of tissue distribution and therapeutic importance.20-22 Preclinical studies support the hypothesis that vaccine-induced responses against TACAs might have their greatest impact in the adjuvant setting as such responses inhibit tumor outgrowth in metastatic models.23,24 Theoretically, cancer vaccines treatment is akin to the induction of autoimmunity caused tissue rejection.25 Carbohydrate-targeting tissue rejection is best typified by the natural antibody reactivity directed against the α-Gal antigen, a major barrier in porcine-to-human xenotransplantation.19 This antibody-mediated tissue rejection model therefore also supports a rationale for targeting TACAs as tumor-induced antibody responses resemble autoimmune responses.26 Such observations suggest that sustained immunity against TACAs might be

©201 1L andesBi os c i enc e. Donotdi s t r i but e. Introduction

Glycosylation is among the most common post-translational modifications proteins undergo that affects many of their activities including protein folding, cellular adhesion and cell differentiation and growth.1-4 Glycan residues, synthesized by the repertoire of glycosyltransferases and glycosidases, provide the mammalian glycome - a large array of glycan structures that adds to the diversity already created by the proteome.5 At the same time, many glycan structures are ubiquitous in nature.6 Pathogen glycans can mimic native mammalian glycosphingolipids that lend to their pathophysiology, teaching us along the way about pathogenic mechanisms that include recognition and signaling, as well as how the immune system is high-jacked by glycan processing.7 Their multifaceted intersection with variable and invariant receptors of the immune system renders carbohydrates the functional property of bridging innate and adaptive immune responses. Lessons learned from immune responses associated with pathogen glycans might be of benefit in understanding tumor biology and tumor immunology. A new appreciation of the interaction of glycans with immune effector cells is emerging *Correspondence to: Thomas Kieber-Emmons; Email: [email protected] Submitted: 10/08/10; Accepted: 10/17/10 DOI: 10.4161/hv.7.0.14578

156

Human Vaccines Volume 7 Supplement

REVIEW

review

beneficial to prevent the recurrence of disease much like the way the immune system works in the context of surveillance. In this review, focus is placed upon the role carbohydrate reactive receptors and cells play in mediating immune responses as part of the immune surveillance machinery that could provide further rationale in developing immunotherapies for cancer. A unique advantage in targeting TACAs is that multiple proteins and lipids on the cancer cell can be modified with the same carbohydrate structure. Since tissue rejection is the goal of cancer immunotherapies broad-spectrum tumor associated antigens like TACA could become plausible targets once the problem of their low immunogenicity is solved. Thus, targeting TACAs has the potential to broaden the spectrum of antigens recognized by the immune response, thereby lowering the risk of developing resistant tumors due to the loss of a given protein antigen. Reading the Glycan Signatures A variety of protein-glycan interactions control the innate and adaptive immune responses to both pathogens and tumor cells alike.8,27 N and O-linked glycans are associated with TACA. O-glycosylation starts with the addition of a single monosaccharide, such as GalNAc in the conventional mucin-type O-glycosylation, attached to serine or threonine. This single GalNAc residue can be extended to form different O-glycan core structures, which can be further elongated to generate more complex glycosylation. N-glycosylation biosynthesis starts with attachment of GlcNAc to the asparagine residue that can be further expanded with mannose residues to produce high mannose or complex oligosaccharides, followed by trimming and branching of glycans and the sequential addition of a variety of monosaccharides, depending on the glycosyltransferases present, thus creating a wide diversity of N-glycan structures. Collectively, glycosylation presents a glycan signature that needs to be decoded. The innate immune system recognizes microorganisms through a series of pattern recognition receptors (PRR) that are highly conserved in evolution.28 Molecular interactions of endogenous glycan-binding proteins represented by C-type lectins, galectins and siglecs function as the decoding agents of glycan signatures.29-31 Glycan decoding receptors are strategically placed at the interface of the immune system with potential antigens, most notably on Antigen Presenting Cells (APC). APCs interact with antigens through an array of PRR as well as through complexes with antibodies and complement utilizing Fcγ and complement receptors to facilitate the cross-presentation of exogenous glycan containing antigens. Many C-type lectins and several siglecs (Siglecs 1, 5, 7 and H) are PRRs that interact with specific glycan structures on pathogens, such as Lewis X,Y, GlcNAc, high-mannose structures, sialic acids and GalNAc, that mediate internalization and enhanced antigen processing and presentation by APCs. DCs are specialized in MHC class I/II cross-presentation of exogenous antigens. Cross-presentation may be the final outcome of antigen internalization in DC by most pathways. This includes most of the known C-type lectin receptors (CLR) but in this case the result, as a rule, is tolerogenic DC phenotype.32 Hardwired to distinguish dangerous non-self, PRR recognize also out of place

“self.” CD205 and dectin-1 are examples pointing to the possible coincidence of PRRs for pathogen associated molecular patterns (PAMPs) and receptors recognizing apoptotic cells, necrotic cells and may be danger associated molecular patterns (DAMPs) therein.33,34 Many cross-presenting receptors bind carbohydrate epitopes on PAMPs and exposed hydrophobic determinants on PAMPs and DAMPs. Furthermore, these interactions are characterized by a broad polyspecificity for “patterns”. Here the quasispecific recognition is seen as a one step generalization the system uses to respond to signals of common biological meaning.35 The immature mouse DC involved in cross-presentation express a wide range of type II and IV CLRs36 CLRs of type IV are represented by type I transmembrane proteins with several carbohydrate recognition domains (CRD). These include CD205 (DEC-205—specificity still unknown) and CD206 (mannose receptor),37 CD205 recognizes dying cells, and may be important for the presentation of self-antigens in the peripheral tolerance.33 It is expressed mostly on CD8 + DC. CD206 is involved mainly in the internalization of soluble antigens38 and this explains to a great extent the successful use of mannose/mannan for delivery of antigens39,40 as well as its possible role in undesirable immunogenicity.41 Under certain circumstances, CD206 transmits signals that cause DCs expressing it to switch on an anti-inflammatory, immunosuppressive cytokine pattern.42 Among the type II CLR (the asialoglycoprotein receptor family), CD207 and CD209 (DC-DSIGN) have been implicated in cross-presentation.43,44 They recognize a large set of sugars including fucose, mannose, N-acetylglucosamine, sulfated sugars, etc.45-48 Dectin-1 also belongs to this family and its primary ligand is β-glucan.49 It participates in the uptake of cellular antigens by human monocyte-derived DC, which leads to crosstolerization to the associated antigens.34 DNGR-1 (CLEC9A) is also a member of this family. Structurally related to dectin-1, it is used as a novel, highly specific, marker of mouse and human DC subsets cross-priming CTL.50 Similar to dectin-1 and CD205, it is expressed on CD8 + mouse DC and mediates loading on class I antigens from apoptotic cell.51 Also like CD205, its intracellular ligand is protease sensitive.33,51 There is a considerable body of evidence now in favor of a role for glycan reactive antibodies as mediators of cellular responses. In this context antibodies function as decoding agents as they are reactive with glycans, but then they can function as bridge molecules to facilitate cellular responses to the captured antigen. Glycan-binding antibodies are mostly IgM and their functions depend to a large extent on complement and complement receptors.52 Serum IgMs are produced mostly by B1 B cells and contain most of the natural auto-antibodies. The latter are polyreactive, low-affinity immunoglobulins involved in the first-line of defense against infectious agents. IgG reactive antiglycan antibodies exist too. IgG2 in humans and IgG3 in mice are produced after isotype switch from IgM in a T-cell independent process. Furthermore, anti-carbohydrate antibodies of other IgG isotypes can be produced under certain circumstances (e.g., a strong protein antigen as a carrier).53,54 Antibodies affect T-cell responses in all types of immune activity by interaction with antigen-presenting cells through

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

www.landesbioscience.com

Human Vaccines

157

their Fc and complement receptors. There is a point in the infectious cycle of almost any pathogen when it is found outside of cells and is, thus, vulnerable to antibody action.55 Immune complexes are formed and those containing IgG target the antigens to Fc receptors (FcγRs). FcR cross-linking can have profound effects both as signal transduction in the APC as well as presentation pathway of antigens.56,57 This mechanism, for instance, is the fundamental basis for using the Gal epitope in vaccine design.19,58 It is expected that antibody mediated internalization of antigens with Gal epitope coupled to them will facilitate epitope spreading mostly through cross-presentation of antigens by DCs. DCs, as professional APCs, are the principal cells capable to cross-present antigens. CD8 + DCs cross-present exogenous antigens on MHC class I molecules constitutively while in CD8- DCs this function is switched on after activation via FcγRs cross-linking.59 In humans the equivalent of the CD8 + DCs are the BDCA3 + DC and have a restricted expression of DNGR1.60,61 In the process of cross-presentation, the APCs acquire proteins from other cells also through endocytosis, mostly phagocytosis or macropinocytosis. Endoplasmic reticulum (ER) contribution to phagosomal membranes is thought to provide antigen access to the ER-associated degradation (ERAD) machinery, allowing cytosolic dislocation.62 Cross-presentation is the only way for the immune system to detect and respond by CTLs to intracellular parasites or mutations that do not affect professional APCs. Cross-presentation of antigen aided by immune complexes63 seems to depend on FcgRIIA. Unlike CLRs, this receptor is not downregulated during maturation.64 The inhibitory FcγRIIB counterbalances this signal in immature DC but its level diminish in the process of maturation. Thus, unlike other internalization pathways, mature DC cross-present through the FcγR better than iDC.64 The role of immune complexes in CTL priming has implications both for tumor vaccine design as well as for understanding the action of monoclonal antibodies used in immunotherapy. The hierarchy of the efficiency of antitumor antibodies of different IgG isotypes in mice was found to be IgG2a≥IgG2b>IgG1>>IgG3. This followed the values of their affinity with the activating FcRs (FcγRIII-IgG1, FcγRIV-IgG2a and IgG2b) and related to the affinity with the inhibitory FcγRIIB.65 In this context it is suggested that the mechanism underlying the long-standing observation of subclass dominance in function is provided by the differential affinities of IgG subclasses for specific activating IgG Fc receptors compared with their affinities for the inhibitory IgG Fc receptor.65 FcRg1 is specific for IgG2a/b. FcRg1 is suggested to play a particularly important role in immune clearance of pathogens and tumor cells present in the circulating blood, as well as in tissues, linking the tumor targeting Th1 response with the IgG2a response. In humans IgG1 and IgG3 show higher affinity than IgG2 for most FcRs. As pointed out above, IgG2 is often the major carbohydrate reactive isotype in humans. The induction of IgG2 antibodies might lend to deficiencies in antibody mediated crosspresentation. The corresponding Th1 linked antibody subclass in humans is hard to discern. However, because of the nature of antibody mediated cross-presentation we suggest that anticancer strategies using Th2 immune response have been underestimated

and warrant further investigations in the context of epitope spreading.66 The Case for Glycan-Mediated Cellular Responses As T-cell-dependent (TD) antigens, proteins have long been seen as the primary target of adaptive immune responses. In contrast, carbohydrates are characterized as T-cell-independent (TI) (either Type 1 or Type 2) antigens.67 Nevertheless, CD4 T cells are involved in functional responses to polysaccharides.68 AntiTACA immune responses induced by glycan conjugates that confer a high protection rate in animals depends on the induction of anti-TACA antibodies requiring T-cell help69 but how B cells and T cells communicate to affect this synergy is unclear. Early studies demonstrated that T cells could recognize carbohydrate antigen vaccines.70 For example, T-cell clones from rabbits immunized with a group C streptococcus vaccine showed strong proliferation in the presence of soluble group C carbohydrate, in addition to the particulate vaccine.70 Post-translationally modified T-cell epitopes constitute a small fraction of both Major Histocompatability Complex Class I and II (MHC-I and MHC-II)-bound peptides, and a number of modifications are identified as natural MHC ligands in vivo.71 Antigen processing and presentation to CD4 + T cells by the MHC-II endocytic pathway has been considered strictly limited to protein antigens. However, the demonstration that T cells can recognize non-protein antigens has modified ideas on the breadth of antigens capable of interacting with T cells.72 T cells are demonstrated to react with processed glycopeptides and glycolipids often representing TACA.73 Some types of carbohydrates seem to be processed and presented to T cells by MHC-II.74,75 Specific T-cell clones have been generated from mice immunized with a meningococcal group C (MCP) (alpha2→9-sialic acid) polysaccharide-tetanus toxoid (TT) (MCPS-TT) conjugate.76 These clones were MHC independent but still needed contact with antigen presenting cells for optimal activation.76 Structures of MHC Class II/peptide complexes suggest analogies with helical carbohydrate structures that could fit the MHC class II antigen-binding groove.75 The resemblance with peptide complexes is even stronger for zwitterionic carbohydrate structures. Existence of MHC/carbohydrate complexes implies recognition by and oligoclonal expansion of specific CD4 + T cells. Crystal structure analysis of TCR-glycopeptide interactions validate that TCR can recognize glycans presented on a peptide backbone.77,78 Existing structures display the key interaction of the core of the peptide ligand with the TCR the CDR3 region shaping a “cavity” often accommodating aromatic amino acid residues. The latter are successfully mimicked in size and conformation by short glycans like TF or the monomer Tn. The helical structure of peptides in the groove of MHC-II actually is classified as type II polyproline helix, which has also been considered a structural basis for carbohydrate mimicry.79,80 Nevertheless, both MHC Class I groove fitting peptides as well as carbohydrate structures and the corresponding carbohydrate mimetic peptides (CMP) with extended secondary structure have been reported in reference 81. A prototypic CMP with a central

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

158

Human Vaccines Volume 7 Supplement

have long-lasting effects on the development of tumor-specific immune responses. An active role of T regulatory cells (Treg) and tolerogenic DC (Tol-DC) is believed important for the induction and maintenance of transplantation tolerance. Toll-like receptor (TLR) triggering on Tol-DC results in TLR2 upregulation and a reduced proinflammatory immune program.87 TLRs are PRRs that act as primary sensors of microbial products. There are now some 10 TLRs identified in humans, found in both the innate and the adaptive immune system. They initiate and modulate the response against distinct, structurally conserved components of pathogens. After co-engagement, TLR receptors also switch on the cross-presenting function for some of the CLRs, as well as modify the immune context of the ensuing T-cell stimulation.88 Vaccine Approaches to Facilitate Anti-Glycan Responses Figure 1. Model of CMP p106 (GVV IYW RYD IYW RYD IYW RYD) in HLA A*0201 based on the structure of a complex with Wilms tumor protein p126–134 (3MYJ.pdb). An A*0201 epitope—VVIYWRDYI, was predicted in p106 using the Immune Epitope Data Base. This epitope was threaded through the Wilms tumor protein epitope, the structure was improved and the energy of the complex minimized using SwissProt pdb viewer. The resulting structure had free energy not worse than the template model. The YW motif is seen protruding from the cleft and possibly interacting with a recognizing TCR. This motif is structurally similar to small carbohydrate haptens like Tn and TF antigens. The secondary structure of the HLA molecule is color coded. The red regions represent the helix forming the peptide binding cleft while the yellow regions represent beta sheets.

Bridging innate and adaptive immunity relies on notions of immune surveillance17 whereby the immune system can recognize nascent transformed cells, leading to suppression of the formation of a primary tumor. Immune surveillance, as a model and rationale for immunotherapy, requires that cancer cells are recognized as “non-self” or may be “perturbation of self.” Often the ability of inducing a rejection strength response has been associated with appearance of xeno-antigenic determinants but now the other dimension of immunogenicity—the danger signal is recognized as even more important. TACA are associated with the transformation process, arising as under- or overexpressed altered self-antigens or as embryonic antigens. Consequently, much like pathogen glycans they can present a danger signature for surveillance mechanisms to act against. Arguably, the concepts and strategies employed in the development of bacterial targeting carbohydrate-based vaccines serve as a paradigm for the development of TACA-based vaccines for cancer.89,90 Being structurally distinct from their mammalian counterparts, most pathogen glycans are valid vaccine targets. In this aspect, tumor vaccines are a challenge since TACA are often self-antigens and there is a different requirement in the type of response. Most importantly, carbohydrates play a controversial role in cancer immunity and any vaccine, designed on their basis should be considered potentially a source of opposing signals to the immune system. There are four principle vaccine approaches being evaluated in clinical trials or close to clinical trials to augment responses to TACA. The first is TACA-based vaccines to induce tumor cell reactive antibodies.89,90 Typically, TACA-based conjugate vaccines in clinical development are directed toward gangliosides,91-94 polysialic acid, Globo-H,95 Lewis Y (LeY),96 and the sialylated Tn (STn) antigen.97 The potential impact of vaccines that induce antibodies to TACAs is demonstrated in clinical trials where patient survival significantly correlates with carbohydrate-reactive IgM levels.98 However, the implementation of TACA-based vaccines is to induce predominately IgG1 antibodies much like that for carbohydrate based vaccines for pathogens. Conjugated carbohydrate-based vaccines typically do not induce a cytokine profile reflective of anti-tumor immunity as conjugation is often

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

sequence motif WYPY displays an extended beta structure that superimposes with the pentasaccharide (β-GlcNAc-(1–2)-αMan-(1–3)-[β-GlcNAc-(1–2)-α-Man-(1–6)]-Man) associated with Concavalin A (Con A); resulting in a root mean square deviation of 0.18 A between the carbohydrate and peptide suggesting very close structural similarities of their respective backbones. The WYPY motif and a sister sequence motif YWRY are projected to be structurally similar and shown to induce CTLs against tumor cells.82 Figure 1 illustrates the positioning of the YWRY motif within MHC-I. Immune cell activation, differentiation and homing can be affected by glycan signatures, while immune responses themselves lead to glycan remodeling of tumor cells as they escape surveillance. The skewing of immune responses mediated by glycan-recognition events is evident in several ways. Siglec activation may downregulate NK cells if triggered by gangliosides shed from tumor cells.83 The same process can occur for tumorassociated glycoproteins. Internalization pathways that favor cross-presentation are understood. Proteins and particulate antigens are cross-presented more efficiently than peptides.84 The cross-presentation of peptides may be greatly improved if they are attached to liposomes.85 The stability of the antigen rather than the size may be the reason for the inefficient cross-presentation of peptides since another small molecule, ganglioside GD3, is efficiently cross-presented albeit in the context of CD1.86 Glycans expressed on DCs or DCs exposed to glycans might themselves promote regulatory T-cell activity that may

www.landesbioscience.com

Human Vaccines

159

associated with a Th2 type immune response with generation of IgG1 antibodies as the primary endpoint. Theoretically, anticarbohydrate IgG1 antibodies contribute to cross-presentation of antigens and thus, to epitope spreading.66 Cross-presentation can however lead either to tolerance or to immunity. This pathway can, therefore, be further exploited in strategies for antibody mediated induction of both CD4(+) and CD8(+) T-cell immunity. Three issues need consideration in the development of TACA based conjugate vaccines. The first is functionality of the induced antibodies. Induced antibodies could mediate cell killing through complement or by engaging NK cells as well as opsonization, receptor triggered apoptosis, adhesion inhibition or immune regulation through feedback and cross-presentation. In this context, the immunogen chosen defines to a great extent the desired effect, which might be offset by the nature of the isotype induced. Glycoprotein targeting might mediate effector responses like ADCC, while glycolipid targeting might be better at mediating signal transduction mechanisms lending to apoptosis. Targeting shed glycans by antibodies might benefit feedback or regulatory mechanisms. Shed glycan often provide negative signaling to T cells and NK cells shutting them down. The formation of immune complexes with shed glycans might disrupt such signaling. As glycans are targets of natural immunity, it seems the instruments used by nature, like natural antibodies, are obviously excellent templates to correlate target with functionality useful in implementing vaccine design strategies. The second is the multivalency of TACAs expressed on the tumor cell surface. This has two forms; one targeting multiple TACA expressed on the cell surface and the second better emulating the clustered presentation of TACA on the cell surface. In this context multivalent vaccines are being developed representing multiple antigens.69,99,100 In one clinical study of a hexavalent vaccine in prostate cancer antibody titers against several of the antigens were lower than those seen in individual monovalent trials.101 The hexavalent vaccine included GM2, Globo H, Lewis(y), glycosylated MUC-1-32mer and Tn and TF in a clustered formation, conjugated to KLH and mixed with QS-21. The third issue is the potential for low efficiency of conjugation of TACAs since classical conjugation strategies fail to enhance uniformly carbohydrate immunogenicity.102,103 Due to the lack of cellular immune responses to TACA themselves, continuous boosting is still necessary to maintain titers. The lack of typical immune memory for carbohydrate antigens is believed to be secondary to the inability of carbohydrates to associate with MHC class II molecules and thus a failure to recruit cognate CD4 + T-cell help. Thus, the relative roles of B cell processing of carbohydrate antigens and cognate B- and T-cell interactions are emphasized.104 The second vaccine approach focuses on the generation of tumor cell reactive T cells because T cells are thought to play an important role in cancer immune therapy. As discussed in a previous section this component of the immune system is not specifically recruited by carbohydrate antigens alone, or by carbohydrate-conjugate vaccines without the help of cross-presentation pathways. Consequently, efforts are directed toward defining and

developing glycoproteins and glycopeptides that facilitate cellular responses. In the last decade a number of researchers report that T-cell receptors can recognize glycopeptides, albeit the anticipated low degree of expressed glycopeptides, carrying mono- and disaccharides in a MHC-restricted manner.72 Recognition of peptide-MHC complexes by T-cells initiates a cascade of signals in T-cells and activated cells either destroy or help to destroy the APC and an understanding of these processes can lend to design of molecular vaccines in general.105 The size of the carbohydrate chains as well as O- versus N-glycosylation varies depending on tumor histotypes. Computer based sequence analysis suggests that only a minimal portion of experimentally verified T-cell epitopes are potentially N- or O-glycosylated (2.26% and 1.22%, respectively).106 These observations are very important in understanding the complexity of the antitumor response especially in terms of abnormal glycan expression patterns. The recognition that T-cell receptors can interact with glycopeptides has facilitated concepts for new antigens being developed to activate anti-tumor responses. The feasibility of T-cell antigens design based on carbohydrate structures is strongly supported by crystallography of several HLA/ peptide complexes. These include designer glycopeptides to facilitate CTL activation,107 glycan modification of antigens to target to APC to enhance both CD4 and CD8 T-cell responses,44,108,109 addition of the Gal epitope to both cells and to molecular antigens58 and other bio-engineering approaches to facilitate antigen uptake to improve immunogenicity.3,110,111 It is not surprising that sometimes glycopeptides offer no significant benefit as targets for cytotoxic immune response. The reason could be also that (a) the elicited CTLs are cross-reactive with both the glycosylated and non-glycosylated forms of the same peptide and (b) the glycopeptides were of low abundance of on tumor target cells.112 A third approach is using PRR to augment or deliver vaccines. TLR ligands are now viewed as critical activators of innate immunity and are being developed as vaccine adjuvants.113-116 Mannan conjugated to vaccine preparations are in the clinic.117 Interestingly, depending on whether mannan is in an oxidized or reduced state, in vivo immune responses can be steered towards a Th1 or Th2 response118 and applicable to protein119 and DNA120 formulations. Oxidized mannan is able to mature DCs and enhance antigen presentation in a TLR4-dependent manner120,121 suggesting that targeting TLR4 might be efficient in tailoring Th1 responses.122 In contrast, tailoring to TLR2 might promote Th2 responses.122-124 Targeting CLRs may not only provide the DC-specific target but also may simultaneously facilitate antigen internalization for MHC- or CD1-mediated antigen presentation.125 The substitution of carbohydrate epitopes with protein or peptide surrogates is the fourth approach to overcome the TI nature of the “anti-carbohydrate” response.126 The molecular mimicry concept is well received in the context of mechanisms associated with linking autoimmune diseases and immune responses caused by pathogens but not well-viewed as a means to harness this concept for immune therapy of cancer. Clinical characterization of anti-idiotypic antibodies that mimic the GD3 ganglioside antigen127 and GD2,128 have been described.

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

160

Human Vaccines Volume 7 Supplement

There are several rationales underlying vaccination strategies that employ surrogates of TACA. First, surrogates function as xenoantigens and consequently, provide an advantage to overcome tolerance to TACA self-antigens. Antibodies induced by surrogates are thought to have low affinities for TACA, which might be of benefit in not inducing tissue damage to normal cells. Specific targeting of tumor cells is due in part to overexpression of the carbohydrate antigen on tumor cells, which compensates for the low affinity of the carbohydrate cross-reactive antibodies. Consequently, tumor cells and not normal tissue will be reactive with surrogate induced TACA reactive antibodies. Second, surrogate antigens of TACA have the potential to overcome immune deficiencies that prevent vaccine-induced carbohydrate-directed responses.81 Unlike carbohydrate antigens and carbohydrate-conjugate vaccines, mimotopes also prime B and T cells for subsequent memory of carbohydrate antigens, facilitating long-term surveillance through recall of carbohydrate immune responses.129 This effect may minimize the need for constant boosting. In addition, they can functionally emulate conserved structures of TACA, inducing antibodies that recognize multiple TACA, and therefore function like a TACA multivalent vaccine.130-133 Third, surrogate antigens can be manipulated in ways that TACA cannot. Protein and peptide surrogates of TACA can be engineered to induce CD8 + T cells cross-reactive with tumorassociated glycopeptides and/or to induce CD4 + T cells that benefit the further expansion of CD8 + T cells and B cells.82,131,134,135 They can also be encoded into platforms not available to glycans typified by DNA immunization. Our group has defined many of the paradigms associated with immunization with Carbohydrate Mimetic Peptides (CMPs), pioneering the concept that CMPs hold the potential to generate a multifaceted TACA-reactive immune response. CMPs can induce antibodies with anti-tumor properties.24,136 As T-cell antigens, immunization with peptide mimotopes can contribute to cellular responses, taking the form of CD4 + T-cell help135 in antibody production or facilitating communication between B cells and T cells,137 facilitating carbohydrate specific Delay Type Hypersensitivity (DTH) reactions,138 and activating tumor specific CTLs.82,135 Starting with the idea to convert TI-2 antigens to TD antigens, now carbohydrate mimotope based cancer vaccines should be tuned rather to promoting only the anti-tumor capacity from the plethora of controversial signals carbohydrate trigger. The characterization of CMPs is at present limited to preclinical studies but we are anticipating moving a CMP into Phase I trial this coming year. Unlike unconjugated TACA antigens, we have shown that unconjugated multivalent antigen peptide forms (MAPs)-CMPs prime for subsequent memory of unconjugated TACA antigens, facilitating long-term surveillance through recall of TACA immune responses.139 CMPs are shown to promote B/T-cell crosstalk (including cognate interactions) as CMPs induce antibodies in IgM and anti-carbohydrate response deficient animals.137 Immunization with CMPs recruits peptide specific, IFNγ producing Th1 cells and cytotoxic T cells.82,135 The deviation to

Th1 responses could be due to a possible interaction of the glycan mimic with polyreactive B1 cells. Peritoneal B1 cells preferentially promoted Th1 and Th17 cell differentiation140 and we have shown that CMPs immune responses are dependent on B1 B cells.137 The targeting of different B cell populations further may expand the potential to control the outcome of immunization and may be a mechanism contributing to the observed diverse effects of carriers on the immunogenicity of haptens. Interestingly, CMP may trigger carbohydrate specific T cells dependent on cell contact similar to that observed for MCP induced antigen proliferation of T cells.76 Despite the fact that an optimal tumor antigen-specific vaccine will ideally incorporate a panel of dominant tumor antigens recognized by tumor-specific CD8 + T cells, our vaccination strategy may augment anti-tumor cell responses for select metastatic tumor types by inducing both a tumor-associated carbohydrate reactive humoral response and a Th1/Tc1 biased cellular response that goes beyond general inflammatory responses as characterized by delayed type hypersensitivity reactions that are carbohydrate associated.141 MUC-1 as a Special Case Glycoprotein and CMP

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

www.landesbioscience.com

The immune system differentially recognizes various epitopes of tumor-associated antigens either as self or as foreign, and this controls the strength of antitumor immunity.142 Among glycoprotein tumor associated antigen targets is the human milk mucin MUC1. MUC1 is a type I transmembrane protein where the extracellular domain contains a variable number tandem repeat (VNTR) region comprising 20 amino acids of sequence GSTAPPAHGVTSAPDTRPAP, each of which contains five sites for potential O-glycosylation.143-146 The overexpression of MUC1 on tumor cells coupled with an altered glycosylation of the extracellular VNTR domain leading to the generation of TACAs makes it an ideal vaccine candidate. However, there are aspects of MUC-1 that are largely ignored in the literature that might impact on its utility as an immunogen. Recently it was found that several of the tumor-related glycoforms of carcinoembryonic antigen and MUC-1 might affect CLR signaling and DC differentiation. These are specific ligands for the pattern recognition receptors DC-SIGN147 and macrophage galactose-type C-type lectin (MGL),148 expressed on DCs. MGL1/2-positive cells are interesting as they represent a distinct sub-population of macrophages, having unique functions in the generation and maintenance of granulation tissue induced by antigenic stimuli.149 MGL1 is postulated to be actively involved in the inflammatory processes.150 Consequently, Tn glycans on MUC-1 that bind MGL might instruct DC to drive Th2mediated responses, which, unlike those of Th1 effector cells, are thought not to contribute to tumor cell eradication. This has several ramifications. Cancer patients with MUC-1 expression profiles may exhibit a Th2-skewed cytokine profile within blood and tumor-infiltrating lymphocytes. This Th1/Th2 imbalance would coincide with disease progression and immunotherapy response. Various lines of evidence suggest that in vivo skewing of T-cell responses toward a Th2 type is an important mechanism of

Human Vaccines

161

immune evasion in cancer patients.151-153 Terminal glycan structures shared by both host and parasitic helminths include Le X, LDN and LDNF, and the truncated O-glycans known as the T (Galβ13GalNAcα1-O-Thr/Ser) and Tn antigens, all glycan antigens that may interact with host lectins that skew the immune response to Th2 profiles.6 This skewing may limit the efficacy of immunotherapeutic approaches.154 Immunization with formulations that reflect a Th2 bias of the native antigen might only exacerbate the Th2 response. Ensuring induction of a strong type 1 response may be critical to the development of effective cancer vaccines. Another issue that might affect MUC-1 strategies is that peptide sequences of MUC-1 function as CMPs. In a series of manuscripts McKenzie’s group showed that anti-α-Gal antibodies reacted with MUC-1 antigens and that anti-MUC1 antibodies reacted with the α-Gal sugar.155-157 In mice, MUC-1 peptide immunization results in cellular responses with reported little humoral response. In this context it can be rationalized that peptide immunization resulted in activating cellular responses typical of peptide loading pathways. In contrast, the MUC-1 induced strong cellular response in mice transformed to a strong antibody response in human immunization.155 It was argued that preexisting anti-Gal antibodies caused a deviation of the immune response in humans compared with mice that do not have anti-Gal antibodies attributed to cross-reactivity of natural anti-Gal antibodies to MUC-1. In mainstream immunology terms, probably MUC-1 peptide was seen as a strong nonself T-cell antigen for the mice while in humans the T-cell tolerance to it was circumvented by the abundance of memory B cells specific for the cross-reactive α-Gal epitope, exacerbating the humoral response to MUC-1 at the expense of the cellular response. The cross-reactivity between natural anti-Gal antibodies and MUC-1 would also suggest that it might be difficult to ascertain the contribution of a de novo antibody immune response to MUC-1 versus a response by anti-Gal memory B cells stimulated in a thymus independent manner. Furthermore, an important consideration is the nature of the anti-Gal antibody isotype in individuals—e.g., IgG2 or IgG1. This simple concept lends to a personalized medicine approach to vaccination with MUC-1 formulations, which is not presently performed but will affect immunization out-come. Another interesting aspect is a possible explanation of the observed protection against breast cancer induced by pregnancy.158 It is tempting to speculate that TF, Tn and other sugars found in milk159 react with natural antibodies to facilitate protective responses. All humans normally possess antibodies that react with the TF and the Tn antigens which are predominately IgM.160 However humans do have natural IgG antibodies to TF and Tn antigens which relate to survival.161,162 The presence of natural IgG antibodies, would lead to the formation of immune complexes taken up by macrophages and DCs, if the isotype of the complex lends to efficient uptake. In this context, uptake would facilitate cellular responses when tolerance allows for them. TF and Tn antigen show some structural similarity to the blood group A

antigen. The TF and Tn antigens, and blood type A antigens are immunologically considered to be quite similar because of their shared terminal sugar (N-acetylgalactosamine), and so might be confused by the immune system of blood type A individuals. Not surprisingly, blood type A individuals have the least aggressive antibody immune response against the TF and probably Tn antigens. Consequently, blood type A individuals would be at an immunologic disadvantage in attacking any cell bearing TF and Tn antigenic markers. Blood-group-A cancer patients have the greatest and uniform suppression of the level of TF antibody agglutinins, irrespective of age, cancer stage or tumor morphology, and lower levels of anti-B isohemagglutinins. This is probably at least a part of the explanation for the poorer outcomes in many cancers among blood type A individuals. This has been demonstrated in breast cancer, where a substantially greater amount of cancer patients (as compared with healthy non-cancer controls) have depressed levels of anti-TF antibody.163 Early studies suggested that breast cancer patients displayed DTH responses to TF no matter what the stage or invasive nature of the cancer.164 Summary

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

162

Intrinsic to exploiting immune surveillance we need to understand that there are myths in tumor immunology. These include that cancer cells are ignored by the immune system, the immune responses are directed to only “unique antigen,” that strong immune responses are necessary, that cancers are weakly immunogenic or are tolorogenic because of immunologic ignorance, that tumor-specific T cells alone are sufficient for tumor regression and that tumor cells are passive targets for anti-tumor responses. A prevailing view is that carbohydrates, generally, are immunologically inert or ligands for receptors downregulating immune functions. In contrast to this viewpoint glycans do function as a bridge between innate and adaptive immune responses. Because of the biological importance of TACA in cancer and because the immune system itself has evolved in a way to search out glycans, optimizing sustained immunity against TACA is important.89,90 A variety of findings illustrate the relevance of altered glycosylation in the tumor microenvironment in the impaired function of DCs. Of particular importance is how cellular responses might be bridged by carbohydrate reactive lymphocytes, innate immune cells and the role glycopeptides and carbohydrate mimetic peptides might play in further mediating innate and adaptive tumor reactive responses. Studies in vitro and in vivo in both mice and human have demonstrated that high-affinity binding structures, such as antibodies and glycans, may be used to ‘direct’ vaccine vehicles to CLRs on DC and thus modulate antigen-specific T-cell responses. However, a better understanding of the natural humoral response to TACA might also contribute to the development of anti-tumor cell strategies.

Human Vaccines Volume 7 Supplement

References 1.

Apweiler R, Hermjakob H, Sharon N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta 1999; 1473:4-8. 2. Shental-Bechor D, Levy Y. Folding of glycoproteins: toward understanding the biophysics of the glycosylation code. Curr Opin Struct Biol 2009; 19:524-33. 3. Bertozzi CR, Kiessling LL. Chemical glycobiology. Science 2001; 291:2357-64. 4. Varki A. Biological roles of oligosaccharides: all of the theories are correct. Glycobiology 1993; 3:97-130. 5. Cattaruzza S, Perris R. Approaching the proteoglycome: molecular interactions of proteoglycans and their functional output. Macromol Biosci 2006; 6:667-80. 6. van Die I, Cummings RD. Glycan gimmickry by parasitic helminths: a strategy for modulating the host immune response? Glycobiology 2009; 20:2-12. 7. Tsai CM. Molecular mimicry of host structures by lipooligosaccharides of Neisseria meningitidis: characterization of sialylated and nonsialylated lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structures in lipooligosaccharides using monoclonal antibodies and specific lectins. Adv Exp Med Biol 2001; 491:525-42. 8. van Kooyk Y, Rabinovich GA. Protein-glycan interactions in the control of innate and adaptive immune responses. Nat Immunol 2008; 9:593-601. 9. Dube DH, Bertozzi CR. Glycans in cancer and inflammation—potential for therapeutics and diagnostics. Nat Rev Drug Discov 2005; 4:477-88. 10. Ravindranath MH, Yesowitch P, Sumobay C, Morton DL. Glycoimmunomics of human cancer: current concepts and future perspectives. Future Oncol 2007; 3:201-14. 11. Ono M, Hakomori S. Glycosylation defining cancer cell motility and invasiveness. Glycoconj J 2004; 20:71-8. 12. Hakomori S. Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines. Adv Exp Med Biol 2001; 491:369-402. 13. Meezan E, Wu HC, Black PH, Robbins PW. Comparative studies on the carbohydrate-containing membrane components of normal and virus-transformed mouse fibroblasts. II. Separation of glycoproteins and glycopeptides by sephadex chromatography. Biochemistry 1969; 8:2518-24. 14. Magnani JL. Carbohydrate differentiation and cancerassociated antigens detected by monoclonal antibodies. Biochem Soc Trans 1984; 12:543-5. 15. Singhal A, Hakomori S. Molecular changes in carbohydrate antigens associated with cancer. Bioessays 1990; 12:223-30. 16. Vollmers HP, Brandlein S. Natural antibodies and cancer. N Biotechnol 2009; 25:294-8. 17. Pashov A, Monzavi-Karbassi B, Raghava GP, KieberEmmons T. Bridging innate and adaptive antitumor immunity targeting glycans. J Biomed Biotechnol 2010; 354-68. 18. Medvedev AE, Sabroe I, Hasday JD, Vogel SN. Tolerance to microbial TLR ligands: molecular mechanisms and relevance to disease. J Endotoxin Res 2006; 12:133-50. 19. Galili U. The alpha-gal epitope and the anti-Gal antibody in xenotransplantation and in cancer immunotherapy. Immunol Cell Biol 2005; 83:674-86. 20. Livingston PO, Zhang S, Lloyd KO. Carbohydrate vaccines that induce antibodies against cancer. 1. Rationale. Cancer Immunol Immunother 1997; 45:1-9. 21. Ragupathi G. Carbohydrate antigens as targets for active specific immunotherapy. Cancer Immunol Immunother 1996; 43:152-7. 22. Hakomori S. Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines. Adv Exp Med Biol 2001; 491:369-402.

23. Zhang S, Zhang HS, Reuter VE, Slovin SF, Scher HI, Livingston PO. Expression of potential target antigens for immunotherapy on primary and metastatic prostate cancers. Clin Cancer Res 1998; 4:295-302. 24. Monzavi-Karbassi B, Artaud C, Jousheghany F, Hennings L, Carcel-Trullols J, Shaaf S, et al. Reduction of spontaneous metastases through induction of carbohydrate cross-reactive apoptotic antibodies. J Immunol 2005; 174:7057-65. 25. Hurwitz AA, Ji Q. Autoimmune depigmentation following sensitization to melanoma antigens. Methods Mol Med 2004; 102:421-7. 26. Preiss S, Kammertoens T, Lampert C, Willimsky G, Blankenstein T. Tumor-induced antibodies resemble the response to tissue damage. Int J Cancer 2005; 115:456-62. 27. Avci FY, Kasper DL. How bacterial carbohydrates influence the adaptive immune system. Annu 2010; 28:107-30. 28. Dziarski R, Gupta D. Peptidoglycan recognition in innate immunity. J Endotoxin Res 2005; 11:304-10. 29. Rabinovich GA, Toscano MA. Turning ‘sweet’ on immunity: galectin-glycan interactions in immune tolerance and inflammation. Nat Rev Immunol 2009; 9:338-52. 30. Paulson JC, Blixt O, Collins BE. Sweet spots in functional glycomics. Nat Chem Biol 2006; 2:238-48. 31. Nonaka M, Ma BY, Murai R, Nakamura N, Baba M, Kawasaki N, et al. Glycosylation-dependent interactions of C-type lectin DC-SIGN with colorectal tumor-associated Lewis glycans impair the function and differentiation of monocyte-derived dendritic cells. J Immunol 2008; 180:3347-56. 32. Mahnke K, Qian Y, Knop J, Enk AH. Induction of CD4+/CD25+ regulatory T cells by targeting of antigens to immature dendritic cells. Blood 2003; 101:4862-9. 33. Shrimpton RE, Butler M, Morel AS, Eren E, Hue SS, Ritter MA. CD205 (DEC-205): a recognition receptor for apoptotic and necrotic self. Mol Immunol 2009; 46:1229-39. 34. Weck MM, Appel S, Werth D, Sinzger C, Bringmann A, Grunebach F, et al. hDectin-1 is involved in uptake and cross-presentation of cellular antigens. Blood 2008; 111:4264-72. 35. Cohn M. A rationalized set of default postulates that permit a coherent description of the immune system amenable to computer modeling. Scand J Immunol 2008; 68:371-80. 36. Kilpatrick DC. Animal lectins: a historical introduction and overview. Biochimica et Biophysica Acta (BBA)— General Subjects 2002; 1572:187-97. 37. East L, Isacke CM. The mannose receptor family. Biochim Biophys Acta 2002; 1572:364-86. 38. Burgdorf S, Lukacs-Kornek V, Kurts C. The mannose receptor mediates uptake of soluble but not of cellassociated antigen for cross-presentation. J Immunol 2006; 176:6770-6. 39. He LZ, Crocker A, Lee J, Mendoza-Ramirez J, Wang XT, Vitale LA, et al. Antigenic targeting of the human mannose receptor induces tumor immunity. J Immunol 2007; 178:6259-67. 40. Ramakrishna V, Treml JF, Vitale L, Connolly JE, O’Neill T, Smith PA, et al. Mannose receptor targeting of tumor antigen pmel17 to human dendritic cells directs anti-melanoma T cell responses via multiple HLA molecules. J Immunol 2004; 172:2845-52. 41. Dasgupta S, Navarrete AM, Bayry J, Delignat S, Wootla B, Andre S, et al. A role for exposed mannosylations in presentation of human therapeutic selfproteins to CD4+ T lymphocytes. Proc Natl Acad Sci USA 2007; 104:8965-70. 42. Chieppa M, Bianchi G, Doni A, Del Prete A, Sironi M, Laskarin G, et al. Cross-Linking of the mannose receptor on monocyte-derived dendritic cells activates an anti-inflammatory immunosuppressive program. J Immunol 2003; 171:4552-60.

43. Idoyaga J, Cheong C, Suda K, Suda N, Kim JY, Lee H, et al. Cutting edge: langerin/CD207 receptor on dendritic cells mediates efficient antigen presentation on MHC I and II products in vivo. J Immunol 2008; 180:3647-50. 44. Singh SK, Stephani J, Schaefer M, Kalay H, GarciaVallejo JJ, den Haan J, et al. Targeting glycan modified OVA to murine DC-SIGN transgenic dendritic cells enhances MHC class I and II presentation. Mol Immunol 2009; 47:164-74. 45. Galustian C, Park CG, Chai W, Kiso M, Bruening SA, Kang YS, et al. High and low affinity carbohydrate ligands revealed for murine SIGN-R1 by carbohydrate array and cell binding approaches and differing specificities for SIGN-R3 and langerin. Int Immunol 2004; 16:853-66. 46. Monzavi-Karbassi B, Luo P, Cunto-Amesty G, Jousheghany F, Pashov A, Weissman D, et al. Fucosylated lactosamines participate in adhesion of HIV-1 envelope glycoprotein to dendritic cells. Arch Virol 2004; 149:75-91. 47. Takahara K, Omatsu Y, Yashima Y, Maeda Y, Tanaka S, Iyoda T, et al. Identification and expression of mouse Langerin (CD207) in dendritic cells. Int Immunol 2002; 14:433-44. 48. van Die I, van Vliet SJ, Nyame AK, Cummings RD, Bank CM, Appelmelk B, et al. The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x. Glycobiology 2003; 13:471-8. 49. Ujita M, Nagayama H, Kanie S, Koike S, Ikeyama Y, Ozaki T, et al. Carbohydrate binding specificity of recombinant human macrophage beta-glucan receptor dectin-1. Biosci Biotechnol Biochem 2009; 73:237-40. 50. Sancho D, Mourao-Sa D, Joffre OP, Schulz O, Rogers NC, Pennington DJ, et al. Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type lectin. J Clin Invest 2008; 118:2098-110. 51. Sancho D, Joffre OP, Keller AM, Rogers NC, Martinez D, Hernanz-Falcon P, et al. Identification of a dendritic cell receptor that couples sensing of necrosis to immunity. Nature 2009; 458:899-903. 52. van Montfoort N, de Jong JM, Schuurhuis DH, van der Voort EI, Camps MG, Huizinga TW, et al. A novel role of complement factor C1q in augmenting the presentation of antigen captured in immune complexes to CD8+ T lymphocytes. J Immunol 2007; 178:7581-6. 53. Soininen A, Seppala I, Nieminen T, Eskola J, Kayhty H. IgG subclass distribution of antibodies after vaccination of adults with pneumococcal conjugate vaccines. Vaccine 1999; 17:1889-97. 54. van de Wijgert JH, Verheul AF, Snippe H, Check IJ, Hunter RL. Immunogenicity of Streptococcus pneumoniae type 14 capsular polysaccharide: influence of carriers and adjuvants on isotype distribution. Infect Immun 1991; 59:2750-7. 55. Casadevall A. Antibody-mediated immunity against intracellular pathogens: two-dimensional thinking comes full circle. Infect Immun 2003; 71:4225-8. 56. Casadevall A, Pirofski L. Insights into mechanisms of antibody-mediated immunity from studies with Cryptococcus neoformans. Curr Mol Med 2005; 5:421-33. 57. Moore T, Ekworomadu CO, Eko FO, MacMillan L, Ramey K, Ananaba GA, et al. Fc receptor-mediated antibody regulation of T cell immunity against intracellular pathogens. J Infect Dis 2003; 188:617-24. 58. Galili U. Autologous tumor vaccines processed to express alpha-gal epitopes: a practical approach to immunotherapy in cancer. Cancer Immunol Immunother 2004; 53:935-45. 59. den Haan JM, Bevan MJ. Constitutive versus activation-dependent cross-presentation of immune complexes by CD8(+) and CD8(-) dendritic cells in vivo. J Exp Med 2002; 196:817-27.

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

www.landesbioscience.com

Human Vaccines

163

60. Bachem A, Guttler S, Hartung E, Ebstein F, Schaefer M, Tannert A, et al. Superior antigen cross-presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells. J Exp Med 2010; 207:1273-81. 61. Poulin LF, Salio M, Griessinger E, Anjos-Afonso F, Craciun L, Chen JL, et al. Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8alpha+ dendritic cells. J Exp Med 2010; 207:1261-71. 62. Rock KL, Shen L. Cross-presentation: underlying mechanisms and role in immune surveillance. Immunol Rev 2005; 207:166-83. 63. Rafiq K, Bergtold A, Clynes R. Immune complex-mediated antigen presentation induces tumor immunity. J Clin Invest 2002; 110:71-9. 64. Liu Y, Gao X, Masuda E, Redecha PB, Blank MC, Pricop L. Regulated expression of FcgammaR in human dendritic cells controls cross-presentation of antigenantibody complexes. J Immunol 2006; 177:8440-7. 65. Nimmerjahn F, Ravetch JV. Divergent immunoglobulin γsubclass activity through selective Fc receptor binding. Science 2005; 310:1510-2. 66. Dhodapkar KM, Krasovsky J, Williamson B, Dhodapkar MV. Antitumor monoclonal antibodies enhance crosspresentation of Cellular antigens and the generation of myeloma-specific killer T cells by dendritic cells. J Exp Med 2002; 195:125-33. 67. Cobb BA, Kasper DL. Coming of age: carbohydrates and immunity. Eur J Immunol 2005; 35:352-6. 68. Stephen TL, Fabri M, Groneck L, Rohn TA, Hafke H, Robinson N, et al. Transport of Streptococcus pneumoniae capsular polysaccharide in MHC Class II tubules. PLoS Pathog 2007; 3:32. 69. Lo-Man R, Vichier-Guerre S, Bay S, Deriaud E, Cantacuzene D, Leclerc C. Anti-tumor immunity provided by a synthetic multiple antigenic glycopeptide displaying a tri-Tn glycotope. J Immunol 2001; 166:2849-54. 70. Jackson S, Folks TM, Wetterskog DL, Kindt TJ. A rabbit helper T cell clone reactive against groupspecific streptococcal carbohydrate. J Immunol 1984; 133:1553-7. 71. Petersen J, Purcell AW, Rossjohn J. Post-translationally modified T cell epitopes: immune recognition and immunotherapy. J Mol Med 2009; 87:1045-51. 72. Carbone FR, Gleeson PA. Carbohydrates and antigen recognition by T cells. Glycobiology 1997; 7:725-30. 73. Zhao XJ, Cheung NK. GD2 oligosaccharide: target for cytotoxic T lymphocytes. J Exp Med 1995; 182:67-74. 74. Tzianabos AO, Finberg RW, Wang Y, Chan M, Onderdonk AB, Jennings HJ, et al. T cells activated by zwitterionic molecules prevent abscesses induced by pathogenic bacteria. J Biol Chem 2000; 275:6733-40. 75. Cobb BA, Kasper DL. Characteristics of carbohydrate antigen binding to the presentation protein HLA-DR. Glycobiology 2008; 18:707-18. 76. Muthukkumar S, Stein KE. Immunization with meningococcal polysaccharide-tetanus toxoid conjugate induces polysaccharide-reactive T cells in mice. Vaccine 2004; 22:1290-9. 77. Glithero A, Tormo J, Haurum JS, Arsequell G, Valencia G, Edwards J, et al. Crystal structures of two H-2Db/ glycopeptide complexes suggest a molecular basis for CTL cross-reactivity. Immunity 1999; 10:63-74. 78. Speir JA, Abdel-Motal UM, Jondal M, Wilson IA. Crystal structure of an MHC class I presented glycopeptide that generates carbohydrate-specific CTL. Immunity 1999; 10:51-61. 79. Anderson MW, Gorski J. Cooperativity during the formation of peptide/MHC class II complexes. Biochemistry 2005; 44:5617-24. 80. Kaur KJ, Khurana S, Salunke DM. Topological analysis of the functional mimicry between a peptide and a carbohydrate moiety. J Biol Chem 1997; 272:5539-43.

81. Cunto-Amesty G, Dam TK, Luo P, Monzavi-Karbassi B, Brewer CF, Van Cott TC, et al. Directing the immune response to carbohydrate antigens. J Biol Chem 2001; 276:30490-8. 82. Monzavi-Karbassi B, Luo P, Jousheghany F, TorresQuinones M, Cunto-Amesty G, Artaud C, et al. A mimic of tumor rejection antigen-associated carbohydrates mediates an antitumor cellular response. Cancer Res 2004; 64:2162-6. 83. Nicoll G, Avril T, Lock K, Furukawa K, Bovin N, Crocker PR. Ganglioside GD3 expression on target cells can modulate NK cell cytotoxicity via siglec7-dependent and -independent mechanisms. Eur J Immunol 2003; 33:1642-8. 84. Zehn D, Cohen CJ, Reiter Y, Walden P. Efficiency of peptide presentation by dendritic cells compared with other cell types: implications for cross-priming. Int Immunol 2006; 18:1647-54. 85. Taneichi M, Ishida H, Kajino K, Ogasawara K, Tanaka Y, Kasai M, et al. Antigen chemically coupled to the surface of liposomes are cross-presented to CD8+ T cells and induce potent antitumor immunity. J Immunol 2006; 177:2324-30. 86. Wu DY, Segal NH, Sidobre S, Kronenberg M, Chapman PB. Cross-presentation of disialoganglioside GD3 to natural killer T cells. J Exp Med 2003; 198:173-81. 87. Chamorro S, Garcia-Vallejo JJ, Unger WW, Fernandes RJ, Bruijns SC, Laban S, et al. TLR triggering on tolerogenic dendritic cells results in TLR2 upregulation and a reduced proinflammatory immune program. J Immunol 2009; 183:2984-94. 88. van Vliet SJ, Garcia-Vallejo JJ, van Kooyk Y. Dendritic cells and C-type lectin receptors: coupling innate to adaptive immune responses. Immunol Cell Biol 2008; 86:580-7. 89. Guo Z, Wang Q. Recent development in carbohydratebased cancer vaccines. Curr Opin Chem Biol 2009; 13:608-17. 90. Buskas T, Thompson P, Boons GJ. Immunotherapy for cancer: synthetic carbohydrate-based vaccines. Chem Commun (Camb) 2009; 5335-49. 91. Guthmann MD, Bitton RJ, Carnero AJ, Gabri MR, Cinat G, Koliren L, et al. Active specific immunotherapy of melanoma with a GM3 ganglioside-based vaccine: a report on safety and immunogenicity. J Immunother 2004; 27:442-51. 92. Krug LM, Ragupathi G, Hood C, Kris MG, Miller VA, Allen JR, et al. Vaccination of patients with small-cell lung cancer with synthetic fucosyl GM-1 conjugated to keyhole limpet hemocyanin. Clin Cancer Res 2004; 10:6094-100. 93. Ragupathi G, Livingston PO, Hood C, Gathuru J, Krown SE, Chapman PB, et al. Consistent antibody response against ganglioside GD2 induced in patients with melanoma by a GD2 lactone-keyhole limpet hemocyanin conjugate vaccine plus immunological adjuvant QS-21. Clin Cancer Res 2003; 9:5214-20. 94. Ragupathi G, Meyers M, Adluri S, Howard L, Musselli C, Livingston PO. Induction of antibodies against GD3 ganglioside in melanoma patients by vaccination with GD3-lactone-KLH conjugate plus immunological adjuvant QS-21. Int J Cancer 2000; 85:659-66. 95. Slovin SF, Ragupathi G, Adluri S, Ungers G, Terry K, Kim S, et al. Carbohydrate vaccines in cancer: immunogenicity of a fully synthetic globo H hexasaccharide conjugate in man. Proc Natl Acad Sci USA 1999; 96:5710-5. 96. Sabbatini PJ, Kudryashov V, Ragupathi G, Danishefsky SJ, Livingston PO, Bornmann W, et al. Immunization of ovarian cancer patients with a synthetic Lewis(y)protein conjugate vaccine: a phase 1 trial. Int J Cancer 2000; 87:79-85. 97. Holmberg LA, Sandmaier BM. Vaccination with Theratope (STn-KLH) as treatment for breast cancer. Expert Rev Vaccines 2004; 3:655-63.

98. Takahashi T, Johnson TD, Nishinaka Y, Morton DL, Irie RF. IgM anti-ganglioside antibodies induced by melanoma cell vaccine correlate with survival of melanoma patients. J Invest Dermatol 1999; 112:205-9. 99. Keding SJ, Danishefsky SJ. Prospects for total synthesis: a vision for a totally synthetic vaccine targeting epithelial tumors. Proc Natl Acad Sci USA 2004; 101:11937-42. 100. Ragupathi G, Gathuru J, Livingston P. Antibody inducing polyvalent cancer vaccines. Cancer Treat Res 2005; 123:157-80. 101. Slovin SF, Ragupathi G, Fernandez C, Diani M, Jefferson MP, Wilton A, et al. A polyvalent vaccine for high-risk prostate patients: “are more antigens better?". Cancer Immunol Immunother 2007; 56:1921-30. 102. Gilewski T, Ragupathi G, Bhuta S, Williams LJ, Musselli C, Zhang XF, et al. Immunization of metastatic breast cancer patients with a fully synthetic globo H conjugate: a phase I trial. Proc Natl Acad Sci USA 2001; 98:3270-5. 103. McCool TL, Harding CV, Greenspan NS, Schreiber JR. B- and T-cell immune responses to pneumococcal conjugate vaccines: divergence between carrier- and polysaccharide-specific immunogenicity. Infect Immun 1999; 67:4862-9. 104. Khan AQ, Lees A, Snapper CM. Differential regulation of IgG anti-capsular polysaccharide and antiprotein responses to intact Streptococcus pneumoniae in the presence of cognate CD4+ T cell help. J Immunol 2004; 172:532-9. 105. Apostolopoulos V, Lazoura E, Yu M. MHC and MHC-like molecules: structural perspectives on the design of molecular vaccines. Adv Exp Med Biol 2008; 640:252-67. 106. Szabo TG, Palotai R, Antal P, Tokatly I, Tothfalusi L, Lund O, et al. Critical role of glycosylation in determining the length and structure of T cell epitopes. Immunome Res 2009; 5:4. 107. Xu Y, Gendler SJ, Franco A. Designer glycopeptides for cytotoxic T cell-based elimination of carcinomas. J Exp Med 2004; 199:707-16. 108. Singh SK, Streng-Ouwehand I, Litjens M, Kalay H, Saeland E, van Kooyk Y. Tumour-associated glycan modifications of antigen enhance MGL2 dependent uptake and MHC class I restricted CD8 T cell responses. Int J Cancer 2010; 128:1371-83 109. Denda-Nagai K, Aida S, Saba K, Suzuki K, Moriyama S, Oo-Puthinan S, et al. Distribution and function of macrophage galactose-type C-type lectin 2 (MGL2/ CD301b): efficient uptake and presentation of glycosylated antigens by dendritic cells. J 285:19193-204. 110. Champion E, Andre I, Moulis C, Boutet J, Descroix K, Morel S, et al. Design of alpha-transglucosidases of controlled specificity for programmed chemoenzymatic synthesis of antigenic oligosaccharides. J Am Chem Soc 2009; 131:7379-89. 111. Pon RA, Biggs NJ, Jennings HJ. Polysialic acid bioengineering of neuronal cells by N-acyl sialic acid precursor treatment. Glycobiology 2007; 17:249-60. 112. Stepensky D, Tzehoval E, Vadai E, Eisenbach L. O-glycosylated versus non-glycosylated MUC1-derived peptides as potential targets for cytotoxic immunotherapy of carcinoma. Clin Exp Immunol 2006; 143:139-49. 113. Adams S. Toll-like receptor agonists in cancer therapy. Immunotherapy 2009; 1:949-64. 114. Smits EL, Ponsaerts P, Berneman ZN, Van Tendeloo VF. The use of TLR7 and TLR8 ligands for the enhancement of cancer immunotherapy. Oncologist 2008; 13:859-75. 115. Dubensky TW Jr, Reed SG. Adjuvants for cancer vaccines. Semin 22:155-61. 116. Lahiri A, Das P, Chakravortty D. Engagement of TLR signaling as adjuvant: towards smarter vaccine and beyond. Vaccine 2008; 26:6777-83.

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

164

Human Vaccines Volume 7 Supplement

117. Apostolopoulos V, Pietersz GA, Tsibanis A, Tsikkinis A, Drakaki H, Loveland BE, et al. Pilot phase III immunotherapy study in early-stage breast cancer patients using oxidized mannan-MUC1 [ISRCTN71711835]. Breast Cancer Res 2006; 8:27. 118. Apostolopoulos V, Pietersz GA, Loveland BE, Sandrin MS, McKenzie IF. Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses. Proc Natl Acad Sci 1995; 92:10128-32. 119. Apostolopoulos V, Pietersz GA, McKenzie IF. Cellmediated immune responses to MUC1 fusion protein coupled to mannan. Vaccine 1996; 14:930-8. 120. Tang CK, Sheng KC, Esparon SE, Proudfoot O, Apostolopoulos V, Pietersz GA. Molecular basis of improved immunogenicity in DNA vaccination mediated by a mannan based carrier. Biomaterials 2009; 30:1389-400. 121. Sheng KC, Pouniotis DS, Wright MD, Tang CK, Lazoura E, Pietersz GA, et al. Mannan derivatives induce phenotypic and functional maturation of mouse dendritic cells. Immunology 2006; 118:372-83. 122. Agrawal S, Agrawal A, Doughty B, Gerwitz A, Blenis J, Van Dyke T, et al. Cutting edge: different Toll-like receptor agonists instruct dendritic cells to induce distinct Th responses via differential modulation of extracellular signal-regulated kinase-mitogen-activated protein kinase and c-Fos. J Immunol 2003; 171:4984-9. 123. Wenink MH, Santegoets KC, Broen JC, van Bon L, Abdollahi-Roodsaz S, Popa C, et al. TLR2 promotes Th2/Th17 responses via TLR4 and TLR7/8 by abrogating the type I IFN amplification loop. J Immunol 2009; 183:6960-70. 124. Moyle PM, Toth I. Self-adjuvanting lipopeptide vaccines. Curr Med Chem 2008; 15:506-16. 125. Young DC, Moody DB. T-cell recognition of glycolipids presented by CD1 proteins. Glycobiology 2006; 16:103-12. 126. Pashov A, Monzavi-Karbassi B, Kieber-Emmons T. Immune surveillance and immunotherapy: lessons from carbohydrate mimotopes. Vaccine 2009; 27:3405-15. 127. Chapman PB. Vaccinating against GD3 ganglioside using BEC2 anti-idiotypic monoclonal antibody. Curr Opin Investig Drugs 2003; 4:710-5. 128. Foon KA, Lutzky J, Baral RN, Yannelli JR, Hutchins L, Teitelbaum A, et al. Clinical and immune responses in advanced melanoma patients immunized with an antiidiotype antibody mimicking disialoganglioside GD2. J Clin Oncol 2000; 18:376-84. 129. Monzavi-Karbassi B, Shamloo S, Kieber-Emmons M, Jousheghany F, Luo P, Lin KY, et al. Priming characteristics of peptide mimotopes of carbohydrate antigens. Vaccine 2003; 21:753-60. 130. Cunto-Amesty G, Luo P, Monzavi-Karbassi B, Lees A, Alexander J, del Guercio MF, et al. Peptide mimotopes as prototypic templates of broad-spectrum surrogates of carbohydrate antigens. Cell Mol Biol (Noisy-le-grand) 2003; 49:245-54. 131. Cunto-Amesty G, Dam TK, Luo P, Monzavi-Karbassi B, Brewer CF, Van Cott TC, et al. Directing the immune response to carbohydrate antigens. J Biol Chem 2001; 276:30490-8. 132. Pashov A, Monzavi-Karbassi B, Raghava G, KieberEmmons T. Peptide mimotopes as prototypic templates of broad-spectrum surrogates of carbohydrate antigens for cancer vaccination. Crit Rev Immunol 2007; 27:247-70. 133. Pashov AD, Plaxco J, Kaveri SV, Monzavi-Karbassi B, Harn D, Kieber-Emmons T. Multiple antigenic mimotopes of HIV carbohydrate antigens: relating structure and antigenicity. J Biol Chem 2006; 281:29675-83. 134. Cunto-Amesty G, Luo P, Monzavi-Karbassi B, KieberEmmons T. Exploiting molecular mimicry: defining rules of the game. Int Rev Immunol 2001; 20:157-80.

135. Monzavi-Karbassi B, Cunto-Amesty G, Luo P, Shamloo S, Blaszcyk-Thurin M, Kieber-Emmons T. Immunization with a carbohydrate mimicking peptide augments tumor-specific cellular responses. Int Immunol 2001; 13:1361-71. 136. Kieber-Emmons T, Luo P, Qiu J, Chang TYOI, Blaszczyk-Thurin M, et al. Vaccination with carbohydrate peptide mimotopes promotes anti-tumor responses. Nature Biotechnology 1999; 17:660-5. 137. Cunto-Amesty G, Luo P, Monzavi-Karbassi B, Lees A, Kieber-Emmons T. Exploiting molecular mimicry to broaden the immune response to carbohydrate antigens for vaccine development. Vaccine 2001; 19:2361-8. 138. Wondimu A, Zhang T, Kieber-Emmons T, Gimotty P, Sproesser K, Somasundaram R, et al. Peptides mimicking GD2 ganglioside elicit cellular, humoral and tumor-protective immune responses in mice. Cancer Immunol Immunother 2008; 57:1079-89. 139. Monzavi-Karbassi B, Shamloo S, Kieber-Emmons M, Jousheghany F, Luo P, Lin KY, et al. Priming characteristics of peptide mimotopes of carbohydrate antigens. Vaccine 2003; 21:753-60. 140. Zhong X, Gao W, Degauque N, Bai C, Lu Y, Kenny J, et al. Reciprocal generation of Th1/Th17 and T(reg) cells by B1 and B2 B cells. Eur J Immunol 2007; 37:2400-4. 141. Ravindranath MH, Bauer PM, Amiri AA, Miri SM, Kelley MC, Jones RC, et al. Cellular cancer vaccine induces delayed-type hypersensitivity reaction and augments antibody response to tumor-associated carbohydrate antigens (sialyl Le(a), sialyl Le(x), GD3 and GM2) better than soluble lysate cancer vaccine. Anti Cancer Drugs 1997; 8:217-24. 142. Ryan SO, Turner MS, Gariepy J, Finn OJ. Tumor antigen epitopes interpreted by the immune system as self or abnormal-self differentially affect cancer vaccine responses. Cancer 2010; 70:5788-96. 143. Spicer AP, Parry G, Patton S, Gendler SJ. Molecular cloning and analysis of the mouse homologue of the tumor-associated mucin, MUC1, reveals conservation of potential O-glycosylation sites, transmembrane and cytoplasmic domains and a loss of minisatellite-like polymorphism. J Biol Chem 1991; 266:15099-109. 144. Hanisch FG, Muller S. MUC1: the polymorphic appearance of a human mucin. Glycobiology 2000; 10:439-49. 145. Patton S, Gendler SJ, Spicer AP. The epithelial mucin, MUC1, of milk, mammary gland and other tissues. Biochim Biophys Acta 1995; 1241:407-23. 146. Irimura T, Denda K, Iida S, Takeuchi H, Kato K. Diverse glycosylation of MUC1 and MUC2: potential significance in tumor immunity. J Biochem 1999; 126:975-85. 147. Samsen A, Bogoevska V, Klampe B, Bamberger AM, Lucka L, Horst AK, et al. DC-SIGN and SRCL bind glycans of carcinoembryonic antigen (CEA) and CEArelated cell adhesion molecule 1 (CEACAM1): recombinant human glycan-binding receptors as analytical tools. Eur Cell Biol 2010; 89:87-94. 148. Saeland E, van Vliet SJ, Backstrom M, van den Berg VC, Geijtenbeek TB, Meijer GA, et al. The C-type lectin MGL expressed by dendritic cells detects glycan changes on MUC1 in colon carcinoma. Cancer Immunol Immunother 2007; 56:1225-36. 149. Sato K, Imai Y, Higashi N, Kumamoto Y, Mukaida N, Irimura T. Redistributions of macrophages expressing the macrophage galactose-type C-type lectin (MGL) during antigen-induced chronic granulation tissue formation. Int Immunol 2005; 17:559-68.

150. Chun KH, Imai Y, Higashi N, Irimura T. Migration of dermal cells expressing a macrophage C-type lectin during the sensitization phase of delayed-type hypersensitivity. J Leukoc Biol 2000; 68:471-8. 151. Becker Y. Molecular immunological approaches to biotherapy of human cancers—a review, hypothesis and implications. Anticancer Res 2006; 26:1113-34. 152. Botella-Estrada R, Escudero M, O’Connor JE, Nagore E, Fenollosa B, Sanmartin O, et al. Cytokine production by peripheral lymphocytes in melanoma. Eur Cytokine Netw 2005; 16:47-55. 153. Knutson KL, Disis ML. Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer Immunol Immunother 2005; 54:721-8. 154. Minkis K, Kavanagh DG, Alter G, Bogunovic D, O’Neill D, Adams S, et al. Type 2 Bias of T cells expanded from the blood of melanoma patients switched to type 1 by IL-12p70 mRNA-transfected dendritic cells. Cancer Res 2008; 68:9441-50. 155. Apostolopoulos V, Osinski C, McKenzie IF. MUC1 cross-reactive Gal alpha(1,3)Gal antibodies in humans switch immune responses from cellular to humoral. Nat Med 1998; 4:315-20. 156. Apostolopoulos V, Sandrin MS, McKenzie IF. Carbohydrate/peptide mimics: effect on MUC1 cancer immunotherapy. J Mol Med 1999; 77:427-36. 157. Sandrin MS, Vaughan HA, Xing PX, McKenzie IFC. Natural human anti-Galα(1,3)Gal antibodies react with human mucin peptides. Glycoconjugate Journal 1997; 14:97-105. 158. Croce MV, Isla-Larrain MT, Capafons A, Price MR, Segal-Eiras A. Humoral immune response induced by the protein core of MUC1 mucin in pregnant and healthy women. Breast cancer research and treatment 2001; 69:1-11. 159. Hanisch FG, Stadie TR, Deutzmann F, Peter-Katalinic J. MUC1 glycoforms in breast cancer—cell line T47D as a model for carcinoma-associated alterations of 0-glycosylation. Eur J Biochem 1996; 236:318-27. 160. Zanetti M, Lenert G, Springer GF. Idiotypes of preexisting human anti-carcinoma anti-T and anti-Tn antibodies. Int Immunol 1993; 5:113-9. 161. Kurtenkov O, Klaamas K, Rittenhouse-Olson K, Vahter L, Sergejev B, Miljukhina L, et al. IgG immune response to tumor-associated carbohydrate antigens (TF, Tn, alphaGal) in patients with breast cancer: impact of neoadjuvant chemotherapy and relation to the survival. Exp Oncol 2005; 27:136-40. 162. Kurtenkov O, Klaamas K, Mensdorff-Pouilly S, Miljukhina L, Shljapnikova L, Chuzmarov V. Humoral immune response to MUC1 and to the ThomsenFriedenreich (TF) glycotope in patients with gastric cancer: relation to survival. Acta Oncol 2007; 46:316-23. 163. Springer GF, Desai PR, Scanlon EF. Blood group MN precursors as human breast carcinoma-associated antigens and “naturally” occurring human cytotoxins against them. Cancer 1976; 37:169-76. 164. Springer GF, Desai PR, Murthy MS, Scanlon EF. Delayed-type skin hypersensitivity reaction (DTH) to Thomsen-Friedenreich (T) antigen as diagnostic test for human breast adenocarcinoma. Klin Wochenschr 1979; 57:961-3.

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

www.landesbioscience.com

Human Vaccines

165