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1 EVALUATION OF IMMUNOMODULATIO N BY MONOCLONAL ANTIBODIES AGAINST Streptococcus mutans ADHESIN P1 By REBEKAH ANN ROBINETTE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008
2 2008 Rebekah Ann Robinette
3 To Dr. Ann Bragg and Ms. Barbara Briscoe
4 ACKNOWLEDGMENTS I would lik e to thank Dr. Brady for providing me a place in her laboratory in which to complete my graduate studies and for her excel lent mentorship. I am also thankful to the members of the Brady laboratory for their tech nical advice and moral support throughout my time as a graduate student. Additionally, I thank the people who graciously served on my committee, Dr. William McArthur, Dr. David Os trov, Dr. Art Edison, and Dr. Sally Litherland for their ideas and suggestions. I wish to express my deepest appreciation to Dr. Ann Bragg and Ms. Barbara Briscoe for being the support upon which I depended during my time as a gradua te student. I thank them for helping me achieve success by not only pushi ng me to acknowledge my full potential, but by standing beside me through each step of my academic career. Lastly, I express my gratitude to Catherine Steen for her support throughout the last three years of my graduate studies.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES.........................................................................................................................7 LIST OF ABBREVIATIONS.......................................................................................................... 9 ABSTRACT...................................................................................................................................10 CHAP TER 1 INTRODUCTION..................................................................................................................12 Dental Caries and Streptococcus mutans ................................................................................12 Virulence Factors and Vaccine Targ ets of S. mutans .............................................................13 P1 Structure and Function......................................................................................................14 The Natural Immune Response against S. mutans ..................................................................16 Immunization against Dental Caries and S. mutans Colonization ..........................................17 Passive Immunotherapy against S. mutans Colonization ....................................................... 18 Immunomodulation by Antibody........................................................................................... 20 Mechanisms of Immunomodulation by Antibodies............................................................... 22 Antigen Structure and Immunogenicity.................................................................................. 23 Immunomodulation by Anti-P1 Monoclonal Antibodies .......................................................24 Summary and Goals................................................................................................................26 2 MATERIALS AND METHODS........................................................................................... 30 Bacterial Strains, Plasmids and Growth Conditions...............................................................30 Construction of P1 Sub-clones...............................................................................................30 Construction of P1 Sub-clone RR2................................................................................. 30 Construction of P1 Sub-clones LT1, LT2, and LT3........................................................ 31 Purification of His-Ta gged Fusion Proteins ...........................................................................31 Purification of Maltose Bindi ng Protein Fusion Proteins ....................................................... 32 Mice........................................................................................................................................33 Source of Antibodies..............................................................................................................33 Immunizations and Sample Collections.................................................................................34 Inhibition of S. mutans Adherence .........................................................................................35 Pepsin Digestion of MAbs and Purification of F(ab)2 Fragments.......................................... 36 Biotin Labeling of Monoclonal Antibodies............................................................................36 Biotin-Labeled MAb 1-6F Competition ELISA.....................................................................37 RR2 and MAb 3-10E Competition ELISA............................................................................. 38 Measurement of the AntiS. mutans Immune Res ponse by Quantitative ELISA...................38 Measurement of the Anti-NR21 Imm une Response by Quantitative ELISA .........................39 Measurement of the Anti-NR21 and Anti-P1 Reactivity by Western Blot............................ 40 Anti-P1 Polypeptide ELISA................................................................................................... 40
6 Measurement of in vitro Immune Ce ll Proliferation..............................................................41 Cytokine Measurement from Culture Supernatants............................................................... 41 3 RESULTS...............................................................................................................................43 Evaluation of Immunomodulator y Properties of MAb 5-5D .................................................43 Further Definition of P1 Amino Acid Se quences Required for MAb 3-10E Binding ........... 44 Recombinant Polypeptide RR2 Achiev es a Native-like P1 Structure .................................... 45 The Genetic Background of the Host Aff ects Immunom odulation by Anti-P1 MAbs..........45 Kinetics and Isotype Composition of the AntiS. mutans Response ...............................47 MAb 1-6F Competition by Serum of MAb 6-11A and 5-5D IC-Imm unized Mice........ 47 Measurement of Competition against MAb 1-6F by Serum from IC-immunized Mice........ 48 Isotype Determination of MAb 1-6F-Like Abs in the Serum of IC-immunized Mice........... 49 Role of Activating Fc Receptors in Immunomodulation by Anti-P1 MAbs .......................... 51 Role of the Fc Portion of Anti-P1 MAbs on Immunomodulation.......................................... 52 Re-evaluation of the Immunomodulatory Properties of MAb 4-10A .................................... 53 P1 Binding by MAb 4-10A Enhances the Binding of MAb 1-6F..........................................55 Evaluation of Cell-Level Immunomo dulatory Changes Induced by MAb ............................56 Serum Reactivity of Proliferat ion Assay Mice against A3VP1 ...................................... 57 Splenocyte Proliferation Assa ys and Cytokine Analysis ................................................ 58 Evaluation of Guyss 13 Plantibody f or Immunomodulatory Properties............................... 61 4 DISCUSSION AND CONCLUSIONS.................................................................................. 91 Overview....................................................................................................................... ..........91 Anti-P1 MAb 3-10E Recognizes a Complex Native-like P1 Structure.................................. 94 Immunomodulation by Anti-P1 MAbs................................................................................... 95 Beneficial Immunomodulati on is Dependent on the Genetic Background of the Host ......... 96 Beneficial Immunomodula tion is Fc-Independent ................................................................. 99 Enhanced Epitope Exposure Explai ns Beneficial I mmunom odulation................................ 101 Immunomodulatory MAbs Affect the Kinetics of the AntiS. mutans Immune Response.. 103 Detection of Immunomodulatory Ac tivity at the Cellular Level .......................................... 105 Guys 13 Plantibody Exhibits Immu nom odulatory Characteristics..................................... 108 Summary and Significance...................................................................................................110 LIST OF REFERENCES.............................................................................................................113 BIOGRAPHICAL SKETCH.......................................................................................................132
7 LIST OF FIGURES Figure page 1-1 Schematic representation of P1 and relevant dom ains; primary sequence or combinations thereof known to achieve epitopes recognized by anti-P1 MAbs............... 29 3-1 Adherence inhibition by serum fr om MAb 5-5D IC-immunized m ice............................. 63 3-2 Evaluation of the minimal P1 sequences required for MAb 3-10E recognition................ 64 3-3 P1 sequence requirements for achievement of the MAb 3-10E epitope on the surface of S. mutans .......................................................................................................................65 3-4 Adherence inhibition by sera of MAb 6-11A and 5-5D IC-immunized BALB/c and C57/BL6 m ice................................................................................................................... .66 3-5 Anti-S. mutans whole cell IgG sub-class reactivity in the sera from immunized BALB/c and C57/BL6 mice............................................................................................... 67 3-6 MAb 1-6F competition by the sera f rom MAb 6-11A and 5-5D IC-immunized BALB/c and C57/BL6 mice............................................................................................... 69 3-7 MAb 1-6F Competition by serum from anti-P1 MAb IC-immunized BALB/c m ice....... 70 3-8 Schematic of recombinant P1 polype ptide encoded by sub-clone NR21 and MAb reactivity against full-length P1 and P1 polypeptide NR21.. .............................................71 3-9 Anti-NR21 IgG subclass reac tivity in the ser a from S. mutans and IC-immunized BALB/c mice.................................................................................................................... .72 3-10 Western blot analysis of anti-NR21 IgG subclass reac tivity in the sera from S. mutans and MAb 5-5D IC-immunized mice..................................................................... 75 3-11 Evaluation of the role of Fc receptors in beneficial imm unomodulation by anti-P1 MAbs..................................................................................................................................76 3-12 Evaluation of the role of the Fc portion of anti-P1 MAbs on beneficial immunom odulation............................................................................................................ 77 3-13 Re-evaluation of the immunom odul atory properties of MAb 4-10A................................ 79 3-14 MAb 4-10A enhances the expos ure of the MAb 1-6F epitope. .........................................80 3-15 Schematic representation of recombinan t polypeptides encoded by P1 subclones used in proliferation assays. .......................................................................................................81 3-16 Serum reactivity of immunized mice used in splenocyte pro liferation assays against recombinant P1 polypeptide A3VP1................................................................................. 82
8 3-17 Tritiated thymidine uptake and cytokine production by splenocytes from immunized mice stimulated in vitro with recombinant P1 polypeptide A3VP1.................................. 84 3-18 Evaluation of cytokine production in the culture supernatants of splenocytes from immunized mice stimulated in vitro with a panel of recombinant P1 polypeptides.......... 87 3-19 Total antiS. mutans IgG and anti-NR21 IgG subclass reac tivity in the serum from mice immunized with S. mutans and Guys 13 plantibody IC.......................................... 89 3-20 Anti-A3VP1 IgG subclass reactivity in the serum from mice immunized with S. mutans and Guys 13 plantibody IC.................................................................................. 90 4-1 Structural models of P1.................................................................................................... 112
9 LIST OF ABBREVIATIONS Ab Antibody Ag Antigen APCs Antigen presenting cells Fc Fragment crystalizable portion of an antibody FcRs Fc receptors GBPs Glucan binding proteins GTFs Glucosyltransferases IC Immune complex Ig Immunoglobulin MAb Monoclonal antibody SAG Salivary agglutinin
10 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EVALUATION OF IMMUNOMODULATIO N BY MONOCLONAL ANTIBODIES AGAINST Streptococcus mutans ADHESIN P1 By Rebekah Ann Robinette August 2008 Chair: L. Jeannine Brady Major: Medical Sciences--Immunology and Microbiology Streptococcus mutans is the primary causative agent of dental caries. One important vaccine candidate against S. mutans is a multifunctional cell surface localized adhesin, P1. Previous studies identified five anti-P1 IgG1 MAbs that alter the anti-P1 response when administered intraperitoneally to BALB/c mice as part of an immune complex (IC) bound to the surface of S. mutans whole cells. Differences in the imm une response include changes in the ability of sera from IC-immunized mice to inhibit S. mutans adherence to immobilized salivary agglutinin (SAG), as measured using a whol e cell BiaCore surface pl asmon resonance (SPR) assay, as well as changes in the specificity and isotype composition of anti-P1 antibodies elicited in mice receiving IC compared to S. mutans alone. Anti-P1 MAbs are not equal in their ability to directly inhib it adherence of S. mutans to SAG or in their ability to modulate the immune response against P1, and the regi ons of P1 required for forma tion of their cognate epitopes varies. Results of the current study revealed that beneficial immunomodulation is produced by four MAbs whose epitopes are contributed to by an interac tion between the discontinuous alanine richand proline rich-repeat regions of P1 and sequences flanking them. Furthermore, it
11 was demonstrated that the mechanism of their action is Fc-independent but dependent upon the genetic background of the immunized host and the concentration of the MA b used to coat the bacterial cells. Additionally, changes in Ab specif icity, isotype distribution, and biological activity in the serum of IC-immunized mice were defined. The data confirmed that epitope specificity and the nature of the antibody-antigen interaction is seminal to the mechanism of action of beneficial anti-P1 MAbs. Their binding enhanced the immunogenicity and antigenicity of epitopes that are better targets of efficaci ous Abs. Immunomodulator y changes on a cellular level and cytokine level were also assessed and were consistent w ith serological data. This study provides valuable information about the m echanism by which anti-P1 MAbs modulate the immune response towards a desirable outcome a nd furthers our understanding of the correlates of protection against S. mutans adherence.
12 CHAPTER 1 INTRODUCTION Dental Caries and Strep tococcus mutans Dental Caries is one of the most common in fectious diseases in the United States and worldwide. It is most common in children; however, it is still a major concern for people of all ages. It is estimated by the American Dental As sociation that, in the United States alone, $40 billion is spent annually for th e treatment and preven tion of dental caries. However, despite prophylactic measures such as to pical or systemic use of fluor ides and dietary control, the incidence of caries remains high. In fact, nearly one-half of 5-year-old children have caries and 95% of adults have experienced caries (51). Caries occurs when the enamel of the tooth is de-mineralized by a localized acidic environment on the tooth surface. This acidic envi ronment is caused by bacterial agents present in the oral cavity that utilize dietar y carbohydrates and produce acid as a by-product. Streptococcus mutans ( S. mutans ) was shown over four decades ago to be the bacterial species that is the primary etiologic ag ent of dental caries in humans ( 71, 111). Initially isolated from a human carious lesion by Clarke in the early 20th century, S. mutans was named based on its mutant appearance by gram stain in that it appeared more oval than round (41). Based on differences in cell wall carbohydrates (24, 146) and DNA hybridization studies (43, 44), Streptococcus mutans isolates have been divided into eight serotypes, designated a through h, and four genetic groups. These include S. sobrinus (serotypes d, g, h), S. cricetus (serotype a), S. rattus (serotype b) and S. mutans (serotypes c, e, f). These gr oups are known collectively as mutans streptococci (45). The mo st prevalent mutans streptococci isolated from human dental plaque is Streptococcus mutans serotype c and S. mutans and S. sobrinus are the two predominant species found in humans (71, 111).
13 Virulence Factors and Vaccine Targets of S. mutans In order for any organism to ex ert its pathogenic effect (s) it must first be able to occupy its desired niche, wherever that m ay be, or be ab le to produce its pathogenic factor(s). Generally speaking, a virulence factor is referred to as anything that aids an organism in habitation, infection, and/or disease progres sion of the host. While numerous definitions of a virulence factor exist, Casadevall and Piro fski suggest that virulence cannot exist in the absence of a host and that host damage cannot occur without microbi al virulence factors (34) Simply defined as a microbial characteristic, a virulence factor can be anything from secreted factors to adhesive proteins. The primary virulence factors of S. mutans that have been studie d as vaccine candidates are glucosyltransferases (GTFs) glucan binding proteins (GBPs), and antigen P1 (69, 131, 181, 182, 222). These virulence factors of S. mutans are all involved with helping this cariogenic organism with colonizing the oral cavity. Gl ucosyltransferases are extracellular enzymes produced by S. mutans which catalyze the synthesis of wate r-soluble and waterinsoluble glucans from sucrose and these enzymes are considered im portant vaccine targets due to their ability to both synthesize and bind to glucan s which have been implicated in the plaque-forming potential of S. mutans (182, 183). Due to the role of glucans in plaque formation, the proteins which bind these molecules are also important targets of vaccine studies. Glucan binding proteins aid S. mutans in colonization of the oral cavity by helping it adhere to the glucan components of the dental plaque. S. mutans synthesizes a minimum of two GBPs which lack GTF enzymatic activity that have been studied as vaccine candidates (162, 178). The virulence factor and vaccine candidate th at is the focus of the current study is a Mr~185,000 surface protein of S. mutans serotype c called P1 (56). P 1, originally identified as Antigen I/II (Ag I/II) (160), and also called An tigen B (163) and PAc (142) is a member of a
14 family of structurally complex cell-surface anchored multi-functional adhesins. P1-like polypeptides are produced by almost all species of oral streptococci indigenous to the human oral cavity and mediate interactions with salivary co nstituents, host cell matrix proteins such as fibronectin, fibrinogen, collagen, an d other oral bacteria. They ar e comprised of multiple ligandbinding sites and regions within these polypeptides are reported to bind human salivary glycoproteins, other microbial cells, and calcium (7, 86, 100). P1 Structure and Function The structure and m olecular biology of P1 has been extensively studied (6, 13, 19-22, 46, 47, 91, 92, 105, 106, 156, 166, 202). The gene encoding P1, called spaP or pac was cloned (105, 141) and sequenced (92, 141) and the c onstruction of isogenic mutants of spaP allowed characterization of P1 as an adhesive component of the bacterial surf ace (46, 106). P1 is a multidomain adhesin that contains an amino-terminal signal sequence, a series of three 82 residue alanine-rich repeats (A-region) within the ami no-terminal third of the molecule, a 150 residue variable region where most sequence variations between P1 from different bacterial strains are clustered, a series of three 39 resi due proline-rich repeats (P-region) in the central portion of the molecule, and a carboxy-terminal sequence characteristic of wall and membrane spanning domains of streptococcal surface proteins wh ich includes the LPXTG motif conserved among sortase substrates (F igure 1-1) (104). Based on the sequence of spaP secondary structural predic tions suggested that the Aregion would most likely form an alpha-helical coiled-coil structure and the P-region of the molecule would form a beta-sheet structure (91) The crystal structure data available on the intervening region between the Aand P-regions of P1 from S. mutans serotype f indicates that this region forms a flexible beta sandwich and th at the Aand Pregions of the molecule most likely are in close proximity in the native form of the protein (197). The tertiary structure of the
15 molecule has not been fully elucidated; however, it is known that the Aand P-regions interact in the native structure and that this interac tion contributes to the formation of complex discontinuous epitopes (127, 166, 200). In collaboration with Dr. Champion Deivanayagam at the University of Alabama at Birmingham, a partial crystal structure of a P1 polypeptide spanning the third A-region through the first P-region repeat (A3VP1) has been obtained (103). This novel protein structure revealed the association of the proline-rich domain forming an unusually long single polyproline type II helix wrapped with th e alanine-rich domain as an extended alpha-helix. The interaction between the alpha-helix and the polyproline helix is primarily mediated through hydrophobic interactions and the region intervening the Aand Psegments achieved a structure similar to that prev iously reported by Troffe r-Carlier, et al. (197). P1 presumably functions on the ce ll surface by mediating adherence of S. mutans to the surface of the tooth via a large molecular weight glycoprotein called salivary agglutinin (SAG) (22, 96, 106, 136), now known to represent the huma n salivary scavenger protein glycoprotein 340 (gp 340) (151). The interaction of P1 with salivary components has been shown to be complex (65) and different regions of P1 have be en shown to be involved in its interaction with human SAG depending on whether it is in fluid-phase or immobilized on a surface (22). While P1 interacts with both fluid-phase and immobilized SAG, it is its intera ction with immobilized SAG that is suspected to allow it to better exer t its cariogenic effects. Additionally, it has been suggested that members of the Ag I/II fa mily of proteins, including Ag I/II of S. mutans and SspA and SspB of S. gordonii may also function as invasins of dental tubules by recognition of collagen (112, 113). Consistent with this idea, one study in which germ-free rats infected with S. mutans or a P1-deficient strain reported lower cari es in the mutant infected rats despite there being no difference in cultivatable numbers of bacteria between the groups of rats (46).
16 The Natural Immune Response against S. muta ns The oral cavity and systemic circulation of humans has been shown to contain antibodies (Abs) to numerous oral microorganisms, including S. mutans (129, 131). The class of Abs found in the oral cavity are not only IgA1 and Ig A2. Varying amounts of IgG and IgM are also detectable in human saliva, most likely via tr ansudation through the gi ngival crevice (52). Humoral immunity against human dental caries ha s been reported for many years. However, a definitive correlate of protection has yet to be identified. Numerous studies have reported that salivary and serum antibodies can be both prot ective or non-protective depending on the specific study (25, 40, 62, 63, 80, 93, 109, 157, 193). One study reported that low caries inciden ce correlated with increased serum IgG antibodies specific for Ag I/II ( 40). Another study reported that high affinity serum IgG against S. mutans whole cells correlated with de ntal plaque absent in mutans streptococci (193).Gregory et al. reported the importance of the specificity of the response with regard to caries sensitivity by showing that caries-susceptible individuals have significantly higher levels of antibodies against S. mutans native antigens (63). Consis tent with the success of S. mutans in colonizing the oral cavity of caries sensitive individuals, Kelly et al. reported a limited antibody response against adhesion epitopes of Ag I/II (93). In contrast, another study reported that caries susceptible subjects had high levels of anti-Ag I/II salivary IgA (80). However, Bratthall et al. showed that the relationship betw een salivary IgA and dental caries is complex in that low caries children had less diverse reactivity to S. mutans and S. sobrinus antigens (Ags) and that high caries children mounted a more dive rse salivary IgA response (25). Studies have also looked at the humoral im mune response in naturally sensitized humans against P1. The results of these studies highlight the importance of differences in T cell help and lymphocyte proliferative responses against certain T cell epitopes ra ther than total Ab responses
17 against full-length Ag I/II in determining the caries susceptibility of the host (93, 126). In one such study of naturally sensiti zed individuals (93), serum antibody responses were shown to be predominantly against N-terminal residues (39 to 481) and central residue s (816 to 1213) of P1 which include the Aand P-region of the protein. T-cell responses were also found to be directed predominantly towards the central region. Furtherm ore, this study demonstrated that there is a limited B-cell response to adhesion epitopes, an obse rvation that is consistent with the success of S. mutans in colonizing the oral cavity in the pres ence of an immune response. As a whole, previous studies have focused on the evaluation of the immune response against linear epitopes. However, the current study poi nts toward the importance of evaluating the response towards complex epitopes in order to elucidate relevant target epitopes that ar e involved in protection from or susceptibility to S. mutans colonization. Immunization against Dental Caries and S. mutans Coloniz ation Previously, immunization strategies utilizing P1 or P1-derived Ags and a variety of other S. mutans vaccine targets have been employed with varying degrees of success (90, 116, 117, 119, 132, 133, 164, 170, 176, 177, 179, 192). It was reported by Lehner et al. that immunization of rhesus monkeys with Ag I or Ag I/II was pr otective against dental car ies and that protection was associated with IgG in their serum and gi ngival crevicular fluid (108). Studies have evaluated the immunogenicity of the entire P1 molecule or fr agments of the antigen using a variety of adjuvants, bacterial vector delivery systems, immuni zation sites, and even genetic immunization with recombinant plasmid DNA, all with reported success (14, 27, 54, 66-68, 73, 81, 82, 85, 87, 89, 95, 154, 159, 161, 164, 167, 168, 192, 195, 196, 215, 216, 218, 222). However, immunization with subunits of P1 have not achieved the same level of protection as immunization with the full-lengt h protein (1, 68, 169, 191). Also, when different routes of immunization are employed, the specific ity of the immune response against S. mutans antigens
18 have been reported to vary (23, 57, 58, 161). Ho wever, one study did report protection from S. mutans colonization in mice immunized with a ch imeric protein made of the saliva-binding region (SBR) of P1 and the glucan-binding domain (GLU) of a GTF (222). Mice were immunized with a chimeric prot ein, SBR-GLU, contai ning the SBR of P1, which corresponds to the A-region (47, 65), and GLU of GTF-I from S. mutans. Immunization with the chimeric protein resulted in significantly enhanced le vels of serum anti-SBR IgG1 and IgG2a, a significant increase in salivary and vaginal Ig A antibody responses to SBR and GLU, and a significant reduction in S. mutans colonization was also observed. Passive Immunotherapy against S. mutans Coloniz ation The term passive immunotherapy can simp ly be defined as the application or administration of a preformed A b. A number of studies that focus on and suggest the use of passive immunotherapies to protect against S. mutans colonization and dental caries have been published (55, 107, 116, 130, 145). Previously it was reported that local administration of an anti-Ag I/II MAb, Guys 13, to the tooth surface of human vol unteers after treatment with chlorohexidine prevented re-colonization of S. mutans for up to two years while placebo control subjects had detectable levels of S. mutans as early as 10 days afte r chlorohexidine treatment (117). Further studies using Guy's 13 demonstrated prevention of S. mutans re-colonization by administration of F(ab)2 fragments of the MAb but the Fab fr agments of the molecule failed to prevent re-colonization. In f act, subjects receiving MAb Fab fragments experienced S. mutans levels in dental plaque and saliva simila r to that found in sham-immunized subjects. Interestingly, protecti on against colonization by S. mutans lasting up to 2 years was observed in immunized subjects even though MAb was only a pplied over a period of 3 weeks and was only detectable up to 3 days after its application to the teeth (115). A subse quent study demonstrated that MAb epitope specificity appeared to be an important factor in efficacy since MAbs Guy's 11
19 and 13 are the same isotype and both recogni ze a protein determinant, but only Guy's 13 prevented S. mutans colonization when used as a passive immunotherapy (116). With the observed success of passive admini stration of Guys 13, the MAb was later reengineered in tobacco plants as a chimeric IgA/IgG with a rabbit secretory component (118) and is now generally referred to as Guys 13 plan tibody. Subsequent human trials using plantibody demonstrated its ability to prevent S. mutans re-colonization for up to four months (114). However, repeated human clinical trials in the United States failed to demonstrate the efficacy of plantibody at the concentrations tested ( 209). The mechanism(s) by which passively administered Guys 13 and Guys 13 plantibody prevents S. mutans re-colonization for such a long time has never been satisfactorily explained. It was speculated that S. mutans recolonization was inhibited by other species of oral bacteria being ab le to compete with S. mutans and occupy its niche within the oral cavity. In addition to the use of Guys 13 and Guys 13 plantibody for S. mutans /P1-related therapies, application of a MAb speci fic for Antigen B (P1) and PAg of S. sobrinus has also been shown to decrease the colonization of S. sobrinus in a rat model (201, 221). Passive immunotherapy directed against non -P1 antigens have been reported as well. It has been reported that passive administration of polyclonal hen-egg-yolk antibodies (IgY) prepared against whole S. mutans cells (145), S. mutans glucosyltransf erase (70), and S. mutans glucan binding protein B (GBP-B) (180) protected rats ag ainst dental caries. Furthermor e, human subjects passively immunized with anti-S. mutans IgY demonstrated a decrease in the percentage of S. mutans in plaque of treated subjects, but the length of the effect was much shorter than that reported after application of anti-P1 MAb Guys 13 (74).
20 Immunomodulation by Antibody There is long-standing and recent evidence th at passively administered Ab may not be acting passively, but may have immuno modulatory effects (23, 26, 79, 98, 99, 125, 128, 148, 175, 194, 198, 204). The idea of passively admini stered antibody-containing substances improving host protection via an immunomodulator y mechanism was suggested in a study using human plasma derived immunoglobulin (IVIG) in conjunction with Streptococcus pneumoniae challenge in mice (153). Furthermore, therapeutic effects of IVIG have been demonstrated in a multitude of autoimmune and inflammatory diseases (171, 172). Immunization of Ag bound by specific Ab has been shown to exert a number of effects including suppression, enhancement and differen ces in the elicited immune response (15, 173, 213) and it has been suggested that exogenously applied Ab may act as a therapeutic agent by redirecting the host immune response (153, 219). Changes in the immune response toward an antigen by immunization with Ag complexed w ith MAbs has been documented (17, 75-78, 97, 138, 217). IC consisting of HBsAg vaccine bound to different MAbs resulted in enhancement, indifference, and/or decreases in the immune response depending on MAb used within the IC (16, 128). It has also been demonstrated that immunization of chickens with IC containing Ag subunits of Newcastle Disease virus and specifi c polyclonal Abs protects against live viral challenge (150). Alterations in both the cellular and humoral immune response have been reported as a result of immunization with Ag-A b IC. In fact, enhanced proliferation and activa tion of antigenspecific T cell lines has been shown to occur wh en specific Ag is administered in an immune complex with either affinity-purified polyclona l antibodies or monoclonal antibodies compared to antigen administered alone (39, 121, 165). Presentation of human serum albumin (HSA) to HSA primed T cells has been shown to be more efficient when it is bound by specific Ab versus
21 Ag alone (124) and HSA IC elicits a stronger in vivo cellular immune response (125). Accelerated development of memory B cells, form ation of germinal centers, and maturation of antibody affinity has been shown to occur by immunization with preformed Ag-Ab complexes when compared with immunizati on of Ag alone (97). Furthermor e, B cells from mice immunized with the Ag-Ab complexes have been shown to demonstrate more heterogeneous VH gene expression versus B cells from mice receiving Ag alone (138). The authors of one study suggested that isotype switching and a memory response can be induced without the need for second priming of the immune response. In this pa rticular study they repo rted that immunization of mice with immune complexes consisting of IgE and 2,4,6-trinitrophenylated bovine serum albumin as the Ag resulted in a multiple-hundr ed-fold higher Ag-specifi c humoral response and the mice that received the IC showed a 500-fold increase in specific IgGsecreting splenic cells (212). Administration of a MAb-containing IC has al so been used to increase the number of MAb-producing hybrids and to induce the formati on of antibodies against subdominant antigenic epitopes (16, 17). Immunization wi th antigen-antibody complexes has also been used to generate MAbs that recognize novel epitopes of an an tigen (214). Investigators have also used immunization with IC to cha nge the immune response against specific pathogens such as Hepatitis B virus (210), Simian Immunodeficiency Virus (72), and infectious bursal disease virus (64). Additionally, another study reported intranasal immunization with Hepatitis B surface antigen complexed with Ag-specific antibodie s induced both systemic and mucosal immune responses in BALB/c mice (128). Immunomodulation by a MAb represents a strate gy to enhance the protective immunity of vaccine antigens by eliciting the formation of antibodies against sub-dominant but protective
22 epitopes, suppressing th e immune response against nonprotective epitopes or changing the subclass distribution of immunoglobulins to more effective isotypes (15, 120, 122, 219, 220). Immunomodulatory effects of Abs have been impli cated in a variety of situations which range from regulation of inflammation and cell-mediated immunity to the classic effects of opsonization and complement fixation (30-33, 35-38) In addition, our laboratory has shown that immunization of mice with anti-P1 MAbs as part of an IC with S. mutans whole cells modulates the antiS. mutans and P1 response in various specific ways (23, 84, 144, 155, 156). In these studies, mice were immunized orally, in tranasally, or intraperitoneally with S. mutans alone and S. mutans coated with a MAb directed against the P1 major surface protein. Mice immunized with the MAb-coated bacteria showed changes in the intensity and specificity of the immune response as well as the subclass distribution of serum antibodies compared to mice immunized with bacteria alone. Mechanisms of Immunomodulation by Antibodies There are several known m echanisms by which antibody (Ab) can modulate the host immune response when it is bound to its specific Ag as part of an IC. While not mutually exclusive and likely to overlap, these alterati ons of the host immune response can be broadly grouped into Fc-dependent or Fc-independent mechanisms. Fc-dependent mechanisms can involve increased uptake via Fc receptors (FcRs) and/or differential engagement of stimulatory versus inhibitory FcRs on antigen presenting cells (59, 75, 78, 102, 122, 174, 211). Additionally, Ab-mediated complement activation with subseq uent uptake of Ag via complement receptors can be considered an Fc-dependent mechanism (78, 83, 102, 120, 190). Fc-independent mechanisms involve, for example, Ab masking of dominant antigenic epitopes, exposure of cryptic epitopes revealed upon Ab binding, and/or changes in proteolysis and Ag presentation (2, 4, 5, 8, 9, 12, 97, 110, 120, 122, 173, 205).
23 Whether initiated by an Fc-depe ndent or independent mechanism, presence of Ab with an Ag can influence proteolysis and antigen pro cessing (5, 120, 122, 173, 205), leading to a shift in presentation of class II-restricted T-cell epitopes (3, 4, 110), cha nges in cytokine expression by antigen-presenting cells and/or T cells (2, 3, 10, 11), enhanced germinal center formation and generation of strong memory responses (78, 88, 94, 97, 99, 152, 188), changes in usage of germline-encoded VH genes (138, 189), and induction of somatic hypermutation (138, 188, 189). Exogenous antibody can therefore affect the spectrum of T lymphocytes induced to proliferate in response to presentation by MHC mol ecules by altering proteo lytic suscep tibility of the Ag which results in changes in the peptides generated for display. Proteases perform two key functions in the class II MHC antigen-pro cessing pathway, initiation and removal of the invariant chain chaperone for MHC class II and generation of peptides from foreign and self peptides for capture and display to T cells (207, 208). Presentation of particular antigen-specific T-cell determinants can be enhanced or suppressed as a direct c onsequence of antibody modulation of antigen processing (4, 5, 122, 173, 205). Indeed in our laboratory, an anti-P1 MAb has been shown to increase the rate and change the proteoly tic digestion pattern of bacterial cell-surfacelocalized P1 in vitro (156). Antigen Structure and Immunogenicity The three-dimensional structure of an antigen impacts its antigenicity and alterations in native structure can lead to ch anges in the immune response to ward that Ag. It has been documented that destabilization of structure and shifts in proteoly tic susceptibility are associated with exposure of cryptic epitope s which leads to a more robust helper T cell response (28, 29, 49, 137, 186, 187). Furthermore, it has been shown that Ag-Ab complexes, rather than Ag alone, may be a common way in which Ag is encoun tered by APCs. Since Ab can influence the processing and presentation by MHC Class II of the processed Ag with a subsequent alteration in
24 the adaptive T helper cell res ponse (134, 173, 205, 206), this would be expected to also influence the nature and specificity of the resultant antibody response against the Ag (101, 123, 135). Studies have demonstrated that protease-sensitive mobile loops of heat shock protein 10 (Hsp10) from mycobacteria, Escherichia coli and bacteriophage T4 (T4Hsp10) are associated with adjacent immunodominant he lper T-cell epitopes and that mobile-loop deletion in T4Hsp10 eliminates the protease sensitivity of Hsp10 and resolves any associated epitope immunodominance (28). In a furt her study, protease-sensitivity and epitope presentation was analyzed in a group of T4Hsp10 vari ant proteins and the results de monstrated that a proteolytic nick in the mobile-loop of T4Hs p10 unlocks three-dimensional st ructure allowing the epitope to become available for binding of MHC (29). Thes e studies support the id ea that even minor changes in antigen structure can have dramatic effects upon the immunogeni city and antigenicity of the protein. Immunomodulation by Anti-P1 Monoclonal Antibodies Previous studies by our laboratory identified five anti-P1 Ig G1 MAbs that alte r the anti-P1 response when administered intraperitoneally to BALB/c mice as part of an IC bound to the surface of S. mutans whole cells. Differences in the imm une response include changes in the ability of sera from IC-immunized mice to inhibit S. mutans adherence to SAG, as measured using a whole cell BiaCore surface plasmon resona nce (SPR) assay, as well as changes in the specificity and isotype compositi on of anti-P1 antibodies elicited in mice receiving IC compared to S. mutans alone (84, 143, 144). Initial experiment s identified similar changes in antibody specificity in mucosally and parenterally immuni zed animals and demonstrated that effects can vary depending on the concentration of MAb in the IC. Anti-P1 MAbs are not equal in their ability to inhibit adherence of S. mutans to SAG or in their abili ty to modulate the immune response against P1, and the regions of P1 required for formation of their cognate epitopes varies
25 (Figure 1-1). In brief, the epitopes of MAbs 6-11A, 3-10E, 5-5D, and 4-10A are contributed to by an interaction between the di scontinuous Aand P-regions of P1 and differences among the MAbs exist in regards to their requiremen t for the presence of other P1 segments. Anti-P1 MAbs 6-11A, 3-10E, and 5-5D have be en shown to be beneficial modulators of the immune response in that they promote the fo rmation of a polyclonal response more inhibitory of bacterial adherence, but these MAbs themselv es do not inhibit adherence. In contrast, MAbs 1-6F and 4-9D both promote the formation of a pol yclonal response less inhibitory of adherence when they are administered as part of an IC while they themselves inhibit adherence. MAb 410A, which also inhibits adherence, was found to be neutral in its affect (144). The beneficial effects of MAbs 6-11A, 3-10E, and 5-5D appear to relate to an epitope requirement that depends upon an interaction of the discon tinuous alanine-rich (A) and pr oline-rich (P) regions of P1. Previous data which demonstrated that immuni zation with IC containi ng A/P-dependent anti-P1 MAbs results in a change in the specificity of the anti-P1 serum response (84, 143, 144) suggests that MAb binding may alter the antig en in some way as to expose different epitopes that provide better targets of protective antibod ies. Such an alteration of P1 structure would be expected to lead to changes in the T helper cell repertoire and the B cell re sponse. Studies evaluating T and B cell epitopes in human subjects that were naturally sensitized to S. mutans Ag I/II revealed a correlation of a T cell epitope with caries resist ance (93). Recognition of certain T cell epitopes would imply the role of antigen processing and peptide presentation and suggest that host MHC make up could contribute to th e immunomodulatory outcome. In fact, the immune responses to P1 of S. mutans was evaluated in various strains of mice using peptides spanning the entire protein with attention being given to the haplotype of MHC II genes (191). This study revealed that subcutaneous immunizat ion of mice carrying the MHC
26 class II I-Ad gene (BALB/c, B10.D2, B10.GD, and (B10.D2 x B10.G)F1 mice) with a 301 to 319 peptide induced strong serum IgG responses to recombinant P1 (rP1) and the peptide. Immunization of mice carrying th e haplotype k or b of the H2 I-A gene (C3H/HeN, C57BL/6, B10.BR, B10.A, or B10 mice) with the same pep tide induced intermediate serum IgG responses to rP1 and the peptide and immunization of mice carrying the haplotype s or q of the H-2 I-A gene (DBA/1, B10.S, or B10.G mice) induced w eak serum IgG responses to rP1 and the 301-319 peptide. The authors mapped antigenic epitopes in the P1 301-319 peptide and P1 in mice bearing different H-2 haplotype s by using 10 overlapping decape ptides covering P1 residues 301-319 and 153 decapeptides covering the entire ma ture P1 protein. Epitope-scanning analysis of the mature P1 molecule showed that an tigenic epitopes were scattered throughout the molecule and that antigenic epitope patterns differed in mice with different H-2 haplotypes. Summary and Goals Previous studies have demons trated that certain anti-P1 MAbs exert immunom odulatory effects on the resulting immune response when they are administered as part of an IC with S. mutans whole cells. Characteristics among the MAbs that exert beneficial immunomodulatory effects versus those MAbs that result in unde sirable outcomes include differences in their epitopes and their ability to inhibit bacterial a dherence to immobilized SAG. The overall goals of the current study were to better understand the P1 sequences required to form the complex epitopes recognized by our anti-P1 MAbs, to fu rther characterize the changes in the immune response following immunization with IC comprised of S. mutans and beneficial immunomodulatory anti-P1 MAbs, and to gain an understanding of the mechanism(s) by which our anti-P1 MAbs modulate the re sultant immune response when they are administered as part of an IC in a BALB/c host.
27 Previous experiments began to demonstrate a change in the specificity and isotype composition of the resultant immune response toward S. mutans P1, N-chlorosuccinimide (NCS)-digested P1 fragments, and partial P1 polypeptides encoded by spaP sub-clones in the serum of mice immunized with IC versus S. mutans alone. As such, an objective of this study was to further define and evaluate the changes in specificity and isotype distribution of Abs contained within the polyclonal immune serum from IC-immunized mice compared to that of mice receiving bacteria alone. Additionally, in light of previous reports that the response against P1 epitopes varies between mice of differing genetic backgrounds (191), the importance of host genetic background on immunomodulatory ch anges and outcomes was examined. As described previously, immuno modulatory effects by Ab can be classified in one of two categories: Fc-dependent and Fc -independent. Another major purpos e of this study was to first determine if beneficial immunomodulation is Fc-dependent. To accomplish this, experiments were performed to evaluate the role of FcRs and the Fc portion of anti-P1 MAbs on their beneficial immunomodulatory effects. Given that changes in the specificity, isotype distribution, and prot ective efficacy of the immune response had been demonstrated in IC -immunized mice, it would be expected that changes at a cell and/or cytokine level might occur as a result of immunization with IC. The last aspiration of this study was to evaluate changes that might occur on a cellu lar level in order to begin to understand the molecular mechanisms involved in beneficial immunomodulation by anti-P1 MAbs that may be linked to observe d changes in serology of immunized mice. Immunomodulation by Ab represents an often over-looked and underutilized strategy to redirect immunity toward increased efficac y. Defining specific changes will help predict correlates of protection, thus far poorly unders tood for P1. Understanding the molecular
28 mechanism of Ab-mediated immunomodulation may also suggest potential ways to shift the balance of the antiS. mutans response in the hosts favor wit hout the need for immunization with immune complexes.
29 Figure 1-1. Schematic represen tation of P1 and relevant domains; primary sequence or combinations thereof known to achieve epitopes recognized by anti-P1 MAbs.
30 CHAPTER 2 MATERIALS AND METHODS Bacterial Strains, Plasmids and Growth Conditions Serotype c S. mutans strain NG8 was kindly provided by K. W. Knox, Institute for Dental Research, Sydney, Australia. Escherichia coli host strains used to express recombinant P1 polypeptides included DH5 INV F (InVitrogen Corp., San Diego, CA) and M15 (pREP4) (Qiagen, Santa Clarita, CA). Escherichia coli strains were grown ae robically at 37C with vigorous shaking in Luria-Bertani broth (1% [wt/ vol] tryptone, 0.5% [wt/vol] yeast extract, 1% [wt/vol] NaCl) supplemented with ampicillin (50-100 g/mL) or kanamycin (25-50 g/mL) as appropriate. Plasmids pCR2.1 (InVitrogen Corp), pQE30 Xa (Qiagen), pQE30 UA (Qiagen) and pMal-p2x (New England Biolabs, Inc. [NEB], Beverly, MA) were used as cloning and expression vectors. Construction of P1 Sub-clones All P1 sub-clones were derived by P CR a mplification from serotype c S. mutans strain NG8 chromosomal DNA or from plasmid DNA derived from NG8 chromosomal DNA. P1 constructs encoding recombinant polypept ides NR21, CK1, CK2, and A3VP1 were made previously in our laboratory (127) or by collaborators (unpublished). Construction of P1 Sub-clone RR2. P1 construct, RR2, was cloned by PCR amplifi cation of base pairs 250-3669 of the P1 gene from S. mutans (NG8) chromosomal DNA using FWD primer 5-CCC GGG ACA AAT GGT TCA ATA CCA GTT-3 which included a Sma I restriction site and REV primer, 5-AAG CTT TCA GTC AGT CAA TCC TGA CGC AAT TCA-3 which included a Hind III restriction site. The resulting 3.4 kb PCR product was then cloned into the PQE-30 Xa Stu I and Hind III restriction sites and transformed into M15 [pREP4] E.coli for expression.
31 Construction of P1 Sub -clones LT1, LT2, and LT3 P1 sub-clone, LT1, was constructed by PCR amplification of the base pairs correlating to amino acids 465-825 from NG8 chromosomal D NA. Primers used were LT1-FWD 5-AAA GAT TTA GCA GAC TAT CCA G-3 and LT1-REV 5CGC ACG GAT TTT ACC ATT TAA A-3. LT2 was constructed by PCR amplifi cation of the base pair s correlating to amino acids 185-679 from NG8 chromosomal DNA. Primers used were LT2-FWD 5CAT AAA GCC GAG GTT GAA CG -3 and LT2-REV 5-ATA GAA AGT GAA TTC ATT TTT AAT AA -3. LT3 was constructed by PCR amplification of the base pairs correlating to amino acids 185-825 from NG8 chromosomal DNA. Primers used were LT2-FWD 5CAT AAA GCC GAG GTT GAA CG -3 and LT1-REV 5CGC ACG GA T TTT ACC ATT TAA A-3. All PCR products were then cloned into the PQE-30 UA cloning vector and propagated in M15 E. coli harboring the pREP4 plasmid. Purification of His-Tagged Fusion Proteins M15 pREP4 E. coli containing the plasm id harboring th e P1 constructs were grown as described in the Bacterial Strains, Plasmids and Growth Conditions sect ion. Overnight cultures were added 1:100 into fresh medi a containing ampicillin (50-100 g/mL) and kanamycin (25-50 g/mL) and grown with shaking until an OD600 of 0.45-0.6 was reached. The cultures were induced for 3-5 hours at 37C w ith 0.1-0.5 mM IPTG (Fisher). The cells were centrifuged to pellet the cells and purificati on steps were then followed. All P1 constructs produced in the PQE-fam ily of vectors were purified by affinity chromatography using nickel resin. Induced cell pellets were lysed using bugbuster (Novagen) at 20 mL per liter of cells and shaking at room temperature for 20 minutes. The lysed cells were centrifuged and the protein containing lysate was removed. Approximately 5 mL of washed
32 nickel resin was added to the cl eared lysate and it was incubated for 2 hours to overnight with rotation at 4C. The resin and ly sate mixture was poured into a reusable column and washed 2x with 1X tris-buffered saline (T BS), 15 mM imidazole. The His-tagged proteins were then eluted with 1X TBS, 300 mM imidazole and 2 mL fractions were collected for analysis by SDS PAGE and Western blot. Pure fractions were pooled for use in experi ments. Impure fractions were subjected to another passage over the nickel column and/or were further purified by gel filtration or anion exchange chromatography. The presence of full-length protein was assessed by Western blot using appropriate conformation dependent anti-P1 MAbs. Duplicate membranes were also stained with colloidal gold to determine the presence or absence of protein breakdown products and/or contaminating E. coli proteins. Purification of Maltose Bindi ng Protein Fusion Proteins E. coli containing the plasm ids harboring the P1 c onstructs were grown as described in the Bacterial Strains, Plasmids and Growth Conditions section. Overnight cultures were added 1:100 into fresh media containing ampicillin (50-100 g/mL) and grown with shaking until an OD600 of 0.45-0.6 was reached. The cultures were then indu ced for 3-5 hours at 37C with 0.1-0.5 mM IPTG (Fisher). The cells were centrifuged to pelle t the cells and purificat ions steps were then followed. Induced cell pellets were lysed by an osmotic shock method. Briefly, cells were resuspended in 200 ml of 30 mM Tris-HCl, 20% sucrose (pH 8.0) and EDTA (0.5 M, pH 8.0) was added to 1 mM and the mixture was shaken at room temperature for 10 minutes. The cells were centrifuged at 10,000 x g for 20 minutes at 4 C and decanted. They were resuspended in 200 ml of ice-cold 5 mM MgSO4 and shaken in an ice bath for 10 minutes followed by centrifugation at 8000 x g for 20 minutes at 4 C. The resulting supernat ant containing the fusion
33 protein was then collected and 4 ml of 0.5 M sodium phosphate buffer (pH 7.2) was added to the supernatant. The fusion protein was affinity purified from the E. coli lysate by column chromatography using amylose resin (New England Biolabs (NEB)) according to the manufacturers protocol. The column buffer used was 0.5 M sodium phosphate buffer, 200 mM NaCl, 1mM sodium azide, 1mM EDTA, pH 7.0. Elution buffer was column buffer containing 10 mM maltose. Fractions (2 ml) were collected and anal yzed by SDS PAGE and Western blot. Pure fractions were pooled for use in experiments. Impu re fractions were subjected to another passage over the amylose column and/or were further purified by gel filtration or anion exchange chromatography. The presence of full-length protein was assessed by Western blot using appropriate conformation-dependent anti-P1 MAbs (48, 127, 155). Duplicate membranes were also stained with colloidal gold to determine the presence or absen ce of protein breakdown products and/or contaminating E. coli proteins. Mice Six-eight week old fem ale BALB/c and/or C57/BL6 mice were purchased from Charles River Laboratories, Wilmington, MA. Fou r-twelve week old male and female Fcer1g (FcR ) transgenic mice were purchased from Taconic Laboratories, Hudson, NY. All mice were housed in biosafety level 2 facilities und er infectious disease conditions and were fed a standard diet. Source of Antibodies Guys 13 plantibody was a gift obtained as a reagent known as caro Rx from Planet Biotech. All anti-P1 MAbs were obtained from previously established hybridomas by our laboratory. Polyclonal Abs were also previous ly obtained via rabbit immunizations by our laboratory. Peroxidase-labeled MAbs were obtained from Southern Biotech at a concentration of 1 mg/ml. All anti-P1 MAbs used in this st udy were purified by column chromatography from
34 ascites fluid using an ImmunoPure (A Plus) IgG Purification Kit (Pierce, Rockford, IL) according to the manufacturers protocol. Briefly, ascites fluid was diluted 1:1 in Binding Buffer and the sample was applied to the equilibrated protein A column and the flow through was saved for further evaluation. The column was then wash ed twice with binding buffer prior to eluting the bound antibody. The antibody was eluted using ~10 ml of elution buffer and 1.0 mL fractions were collected with each tube containing 50-100 l of neutralization buffer. The Ab containing fractions were then determined by measuring th eir absorbance at 280 nm and analysis by SDS PAGE and Western blot. Immunizations and Sample Collections Groups of six m ice were immunized in traperitoneally (IP) with ~1.5 109 CFUs of S. mutans in 150 l of PBS or the same amount of S. mutans coated with either a saturating or subsaturating concentrations of each MAb. The dilution of MAb n ecessary to saturate the bacterial cells was predetermined by serial titration and do t blot analysis. In each experiment, a negative control group received PBS only. To exclude the possibility of anti-idiotype effects and to control for other potentia l effects of MAbs occurring independ ently of antigen, additional control groups received MAb alone at the concentration required to saturate th e immunizing dose of bacteria. Mice were pre-bled one week before the first inoculation, i mmunized on days 0 and14 and exsanguinated on day 30-40. Interim bleeds were also taken on days 3-7 in some cases. For proliferation assays mice were immunized on day 0 and spleens were removed on day 7-12 in most cases. However, some mice were immunized on day 0 and/or day 13 and spleens were removed on day 28-33. These mice were also bled on days 0, 13, and 26 in addition to terminal bleeds upon sacrifice.
35 Inhibition of S. mutans Adherence Inhibition of S. mutans adherence by serum from imm unized mice was measured by a Biacore assay as previously described (143). Salivary agglutinin was prepared by a modification of the technique of Rundegren and Arnold (22, 158). Inhibition of adherence of S. mutans whole cells to salivary agglutinin immobilized on an F1 sensor chip was assayed using the BIAcore 3000 machine (BIAcore AB, Upps ala, Sweden). Salivary agglutinin was immobilized on the BIAcore F1 sensor chip surface in flow cell 2 by amine coupling, and the dextran matrix was activated with 35 l of an equal mixture of N-hydrosuccinimide (11.5 mg/ml) and N-ethyl-N'(dimethylaminopropyl) carbodiimide (75 mg/ml) as suggested by the manufacturer. Agglutinin (100 ng/ml) was diluted 1:5 in acetate buffer (pH 5), and 2 to 20 l was injected manually until the change in refractive units was >1,000. The re maining activated dextran was inactivated by injection of two aliquots of 35 l of 1 M ethanolamine Flow cell 1 was treated in the same way but the addition of agglutinin was omitted, and it served as a reference surface. A flow rate of 10l/min was used throughout the experiment Adherence buffer (AB; 0.78 mM KH2PO4, 1.22 mM K2HPO4, 50 mM KCl, 1 mM CaCl2*6H2O; pH 7.2) was used as running buffer. Whole S. mutans NG8 cells (10^9 CFU/ml) in AB were sonicat ed to dechain the cells and injected at a flow rate of 10 l/min for 60 s, totaling 1 x 107 cells per injection. The surface was regenerated with 5 to 25 l of PBS containing 0.03% Tween, 10 mM EDTA, 100 mM NaCl, and 100 mM NaOH. A spaP -negative isogenic mutant of S. mutans devoid of P1 (46) that does not adhere above background levels to the control surface was used as a negative control in this assay (143). This indicates that the detected change in resona nce signal is entirely P1 mediated and is not due to an interaction of non-P1 cell surface component s with low levels of potential contaminants, such as sIgA in the agglutinin preparation. Adhe rence inhibition was perf ormed with murine sera pooled from each treatment group or with a protein A-purified anti-P1 MAb. One hundred
36 microliters of a suspension with a 1.010-CFU/ml density was incubated with pooled sera or with protein A-purified anti-P1 MAb as previously desc ribed (143), and the mixtures were rotated end over end at room temperature for 1 h. The cells were harvested by centr ifugation, washed twice with AB, and then used for BIAcore analysis. Pepsin Digestion of MAbs and Purification of F(ab)2 Fragments MAbs 5-5D and 6-11A were buffer exchanged into 0.2 M Sodium Acetate adjusted to pH 4.0, using glacial acetic acid, overnight at 4C. Once buffer exchanged, the MAbs were diluted or concentrated to ~ 2.0 mg/ml and 0.1 mg/ml pepsin (Sigma) dissolved in 0.2 M Sodium Acetate, pH 4.0 was added until an enzyme:Ab ratio of 1:20 was obtained. The samples were placed at 37C for 24-72 hours. After incubation at 37C, 2 M Tris base was added to each sample until the pH reached 7.0-8.0 and the sa mples were dialyzed into 1X PBS, pH 8.0 overnight. The F(ab)2 fragments were first depleted of any Fc-containing fragments by purification on protein A using an ImmunoPure (A Plus) IgG Purification Kit (Pierce, Rockford, IL) according to the manufacturers instructions. Briefly, the protein A column was equilibrated with binding buffer and the digested sample was diluted 1:1 in binding buffer and applied to the column. The flow through was saved along with th e first wash in order to obtain Fc-free and F(ab)2 containing samples. Further purification of F(ab)2 fragments was then performed on a Bio-silect 250 gel filtration column (Bio Rad, Hercules, CA). Purity of F(ab)2 was verified by Western blot using anti-Fc, antichain, antichain, and antichain specific secondary antibodies (Southern Biotech). Biotin Labeling of Monoclonal Antibodies MAb 1-6F was purified from ascites fluid usi ng an ImmunoPure (A Plus) IgG Purification Kit (Pierce, Rockford, IL) according to the manuf acturers protocol. Briefly, ascites fluid was diluted 1:1 in Binding Buffer. The antibody sample was then applied to the equilibrated protein
37 A column and the flow through was saved for fu rther evaluation. The column was then washed twice with binding buffer prior to eluting th e bound antibody. The antibody was then eluted using ~10 ml of elution buffer and 1.0 mL fractions were collected with each tube containing 50100 l of neutralization buffer. The Ab containing fractions were then determined by measuring their absorbance at 280 nm and analys is by SDS PAGE and Western blot. Approximately 1 mg of purified MAb 1-6F was biotin labeled using EZ-Link BiotinLC-Hydrazide (Pierce, Rockford, IL) according to the manufacturers protocol. 1 mL of purified Ab was dialyzed overnight into coupling buffer (0.1 M sodium acetate buffer, pH 5.5) and 1 mL of cold 20 mM sodium meta -periodate solution was added. Th e oxidation reaction was allowed to proceed for 30 minutes on ice or at 4C in th e dark and then glycerol was added to a final concentration of 15 mM and inc ubated for 5 minutes on ice or at 4C to stop the oxidation. The oxidized sample was then dialy zed overnight into coupling buffe r and biotin hydrazide solution was added to a final concentration of 5 mM and the sample was agitated for two hours at room temperature. Finally, the biotin reacted sample was dialyzed overnight into 1X PBS to separate any excess biotin from the mixture. The presence of biotin labeled MAb 1-6F was verified using an avidin-horseradish peroxida se conjugate reagent (Pierce, Rockford, IL) by enzyme-linked immunosorbent assay (ELISA) and Western blot. Biotin-Labeled MAb 1-6F Competition ELISA The ability of antibodies contained within im m une serum of immunized mice to compete for the MAb 1-6F epitope was evaluated by a competition ELISA. ELISA plate wells were coated with Streptococcus mutans serotype c strain NG8 whole cells (6) in carbonate-bicarbonate buffer, pH 9.6. Immune sera from S. mutans and S. mutans -MAb ICs immunized mice were serially diluted two-fold beginning at 1:50 and 100l of each dilution was added to the wells followed by 100l of diluted biotin-labeled MAb 1-6F. The plates were then incubated at 37C
38 for two hours. Avidin-HRP conjugate (Pierce, IL) was used at a 1:15,000 dilution for 30 minutes at room temperature. Plates were developed with 0.1 M o-phenyl enediamine dihydrochloride and 0.012% hydrogen peroxide in 0.01 M phosphate citrate buffer. Plates were incubated for 30 min at room temperature in the dark, and the ab sorbance at 450 nm was recorded using an MPM Titertek model 550 ELISA plate reader (Bio-Rad, CA ). Percent inhibition of Biotin-labeled MAb 1-6F binding was calcula ted as %Inhibition=[OD450 direct binding of bio tin-labeled 1-6F OD450 experimental well / OD450 direct binding of biotin-label ed 1-6F] X 100. Controls included nonlabeled MAb 1-6F and AV-HRP only wells. RR2 and MAb 3-10E Competition ELISA E. coli cell lysates con taining recombinant P1 polypeptides RR2, CK1, and CK2 and vector only controls were adjusted to comparable OD280 and 100 l of serial two-fold dilutions were added to S. mutans coated ELISA plates, blocked with 5% skim milk in 1X PBS/0.03% Tween 20, immediately prior to the addition of 100l of a 1:1000 dilution of MAb 3-10E (1.8 mg/ml). The plates were then incubated for 2 hours at 37C, washed 3X, and 100 l of a 1:2000 dilution of goat anti-mouse IgG-HRP conjugate was added to each well and incubated for 2 hours at 37C. Plates were developed with 0.1 M o-phenylenediamine dihydrochloride and 0.012% hydrogen peroxide in 0.01 M phosphate citrate buffer. Plates were incubated for 30 min at room temperature in the dark, and the ab sorbance at 450 nm was recorded using an MPM Titertek model 550 ELISA plate reader (Bio-R ad, CA). The positive control for MAb 3-10E binding were no inhibitor wells and negative controls included use of polypeptides CK1 and CK2 along with vector only lysate wells. Measurement of the AntiS. mu tans Immune Response by Quantitative ELISA Serum samples were assayed for antiS. mutans IgG1, IgG2a, and IgG2b isotype antibodies by ELISA. ELISA plate wells were coated with NG8 whole cells in carbonate-bicarbonate
39 buffer, pH 9.6. Mouse immune sera were serially diluted two-fold beginning at 1:50 and added to the wells. Antibody reactivity was detected using affinity-purified peroxidase-labeled goat antimouse peroxidase conjugated, IgG subclass sp ecific antibodies (Sout hern Biotech) at the following dilutions: anti-IgG1, 1:1000; anti-IgG2 a, 1:1000; anti-IgG2b, 1:1000; and anti-IgG3, 1:1000. Plates were developed with 0.1 M ophenylenediamine dihydrochloride and 0.012% hydrogen peroxide in 0.01M phosphate citrate buffer. Plates were incubated for 30 min at room temperature in the dark, and the absorbance at 450 nm was recorded using an MPM Titertek model 550 ELISA plate reader (Bio-Rad, CA). The concentrations of antiS. mutans IgG subclass antibodies were calculate d by interpolation on standard curves generated using purified mouse subclass reagents (Southern Biotech, AL). Measurement of the Anti-NR21 Immune Response by Quantitative ELISA Serum samples were assayed for anti-NR21 Ig G1, IgG2a, and IgG2b isotype antibodies by ELISA. Plate wells were coated with appr oximately 200 ng/well purified NR21 in carbonatebicarbonate buffer, pH 9.6. Mouse immune sera were serially diluted two-fold beginning at 1:50 and added to the NR21-coated wells. Antibody re activity was detected us ing affinity-purified peroxidase-labeled goat anti-m ouse peroxidase conjugated, Ig G subclass specific antibodies (Southern Biotech) at the followi ng dilutions: anti-IgG1, 1:1000; anti-IgG2a, 1:1000; anti-IgG2b, 1:1000; and anti-IgG3, 1:1000. Plates were developed with 0.1 M o-phenylenediamine dihydrochloride and 0.012% hydrogen peroxide in 0.01 M phosphate citrate buffer. Plates were incubated for 30 min at room temperature in the dark, and the absorbance at 450 nm was recorded using an MPM Titertek model 550 ELISA plate reader (Bio-Rad, CA). Concentrations of anti-NR21 antibodies were cal culated by interpolation on sta ndard curves generated using purified mouse subclass reagents (Southern Biot ech, AL). All control sera were non-reactive with NR21.
40 Measurement of the Anti-NR21 and Anti -P1 Reactivity by Western Blot Recom binant full-length P1 and recombinan t polypeptide NR21 were electrophoresed on three replicate 7% SDS-polyacrylamide prepar atory slab gels and electroblotted onto nitrocellulose filters. Each nitrocellulose filter was cut into strips and replicate strips were reacted with our panel of 11 MAbs or appropriate mouse serum. Blot stri ps were then reacted with affinity-purified peroxidase-labeled goat anti-mouse peroxidase conjugated, IgG specific antibody (Southern Biotech) at 1:1000 followed by washing and development with 4-chloro-1naphthol solution (7 ml of PBS, 1 ml of 4-ch loro-1-naphthol [Sigma; 3 mg/ml in ice-cold methanol], and 8 l of 30% hydrogen peroxide). Controls included a polyclonal anti-P1 Ab as well as secondary Ab only-reacted blot strips. Anti-P1 Polypeptide ELISA ELISA plates were coated with appro ximately 200 ng/ well of each respective P1-derived polypeptide overnight in carbonate-b icarbonate buffer, pH 9.6. Mouse immune sera were serially diluted two-fold beginning at 1:50 and added to the coated wells. Antibody reactivity was detected using affinity-purifie d peroxidase-labeled goat anti-m ouse peroxidase conjugated, IgG subclass specific antibodies (Southern Biotech ) at the following dilutions: anti-IgG1, 1:1000; anti-IgG2a, 1:1000; anti-IgG2b, 1: 1000; and anti-IgG3, 1:1000. Plat es were developed with 0.1 M o-phenylenediamine dihydrochloride and 0.0 12% hydrogen peroxide in 0.01 M phosphate citrate buffer. Plates were incubated for 30 mi n at room temperature in the dark, and the absorbance at 450 nm was recorded using an MP M Titertek model 550 ELISA plate reader (BioRad, CA). Concentrations of anti-P1 polypeptide antibodies were calculated by interpolation on standard curves generated using purified mouse subclass reagents (Southe rn Biotech, AL). All control sera were non-reactive with each respective P1 sub-clone.
41 Measurement of in vitr o Immune Cell Proliferation BALB/c mice were immunized with 1X PBS, S. mutans whole cells, or IC containing MAb 4-10A at a 1:6000 or 1:8000 dilution. In so me cases mice were immunized on day 0 and spleens were harvested on day 7. Some mice were immunized on day 7 and spleens were harvested on day 33. Other mice were immunized on day 7 and day 13 and spleens were harvested on day 33. All spleens were excised and placed in media (RPMI, 10% Hyclone FBS, 100 U/ml penicillin, 100ug/ml streptomycin, HEPES (1.87 g/500 mL), and 50 uM 2-ME, pH 7.4) on ice. They were then washed 2-3 times with me dia and placed in a tissue sieve with 5 mL fresh media. The spleens were then pushed through th e nylon membrane to single cell suspension and the cells and media were colle cted and centrifuged for 5 minutes at 4C, 300xg. RBCs were lysed with 1 mL 0.84%NH4Cl for 5 min on ice and 10 mL media was added to stop lysis. The cells were then washed 2 times with media and resuspended in media. Cells were counted using trypan blue on a hemacytometer and 1 million ce lls were plated per well. Approximately 50 g/mL of each antigen (recombinant P1 polypeptid e) was used per well (experimental wells) and Con A was used at 5 g/mL (positive controls). The total volume of each well was 200 l and wells containing cells alone served as negative controls. The plat e was then incubated at 37C, 5% CO2 for 5 days. Con A cells were incubated for four days. On Day 6, or Day 4 in the case of Con A treated wells, cells were pulsed with 4.0E-4 mC i thymidine (Perkin Elmer cat # NET027005MC) per well and were harvested 16 hours later using a Micro 96 Cell Harvester (Molecular Devices part # 0200-3923) Proliferation was then m easured using a 1450 Microbeta Trilux liquid scintillation and luminescence counter (Wallac). Cytokine Measurement from Culture Supernatants Culture supernatants were rem oved from the wells containing stimulated splenocytes prior to the addition of thymidine and placed in eppe ndorf tubes for storage at minus 20C or minus
42 80C. Supernatants were thawed and replicat e samples were pooled immediately prior to analysis. Sample preparation and analysis was performed ex actly as recommended in the manufacturers protocol. Concentrations of INF-gamma, IL-2, IL -10, IL-4, and TNF-alpha were measured using a Beadlyte mous e multi-cytokine detec tion system 1 (Millipore cat# 48-005, lot # 1467271) and concentrations of TGF-beta isoforms 1, 2, and 3 were measured using a multiplex Beadlyte TGF-beta 1, beta 2, beta 3 detection system (Mill ipore cat # 48-015, lot # 1460119). All samples were treated exactly as described in the protocols for the kits. Supernatants were acid treated as described in the TGF-beta protocol as well. Analytes were measured on a luminex 100 system using the settings recommended in each respective manufacturers protocol.
43 CHAPTER 3 RESULTS Evaluation of Immunomodulat ory Properties of MAb 5-5D Results of experim ents undert aken prior to this study ai med at defining the minimal primary amino acid sequence required for recogn ition of P1 by our panel of 11 anti-P1 MAbs and results of prior immunization experiments with IC containing MAbs 6-11A, 3-10E, 1-6F, 49D, and 4-10A suggested that MAb epitope might play a role in benefi cial immunomodulation observed when MAbs 6-11A and 3-10E were used as part of an IC to immunize a murine host. The core P1 sequences required for binding of be neficial immunomodulatory MAbs indicated an importance of an interaction of the discontinuous Aand P-regions of the adhesin. Additionally, an inability of the MAb to inhi bit bacterial adheren ce to immobilized SAG was suggested as a key feature of beneficial imm unomodulatory MAbs. However, some disparity existed in that A/P-dependent MAb 4-10A is a dir ect inhibitor of adherence and wa s shown to be neutral at the concentrations tested within the IC. Also, a nother Aand P-region dependent MAb, 5-5D, was not tested for immunomodulatory properties in th e earlier studies. To further evaluate the beneficial immuno modulatory properties of our anti-P1 MAbs, murine immunization experiments were undertak en using MAb 5-5D. This MAb was tested because its epitope, like MAbs 6-11A and 3-10E, is contributed to by an interaction of the discontinuous A-and P-regions of the P1 molecule as well as sequences upstream of the Aregion. BALB/c mice were immunized as describe d in Chapter 2. Briefly, mice were immunized intraperitoneally (IP) with S. mutans whole cells or whole cells co ated with a 1:200 [saturating] or a 1:2000 [0.1x sub-saturating] dilution of MAb 5-5D. All Imm unizations in the current study were done with whole S. mutans cells because evaluation of the dominant response against P1 as it exists in its functional form on the cell surface and how to change that response for the better
44 was the objective. Parenteral rather than muco sal immunizations were performed because of practicality of obtaining sufficien t sample volumes (larger volume of serum compared to volume of saliva) in order to conduct multiple experime ntal evaluations and Rhodin, et al. had already demonstrated similar MAb-mediated alterations in specificity of mucosal and systemic responses (156). As can be seen in Figure 3-1, serum from mice immunized with both a [saturating] and a [0.1x sub-saturating] was increase d in its ability to inhibit S. mutans adherence to immobilized SAG over the serum from S. mutans -immunized mice. Furthermore, MAb 5-5D is unable to directly inhibit S. mutans adherence to immobilized SAG (dat a not shown). These results provide further support that MAb epitope an d a lack in the ability to dire ctly inhibit bacterial adherence are key features of benefi cial immunom odulatory MAbs. Further Definition of P1 Amino Acid Sequences Required for MAb 3-10E Binding Previous MAb epitope m apping ex periments revealed that anti-P1 MAb 3-10E recognizes the most complex, discontinuous epitope of all our anti-P1 MAbs. In order to gain a better understanding of the P1 sequen ces contributing to recognitio n by MAb 3-10E, the sequences required for MAb recognition were further define d. Based on its reactivity with full-length P1 and lack of reactivity with P1 sub-clones CK 1 and CK2 (see schematic in Figure 3-2A), it was determined that MAb 3-10E requires the presence of not only the Aand P-regions, but that sequence N-terminal of the A-region and C-terminal of the P-region are an absolute requirement for its binding to P1 (Figure 32B). In order to further define the minimal sequence required Cterminal to the P-region, P1 sub-clone RR2 was made. The RR2 construct encodes for a polypeptide which includes amino acids 84-1218 of the P1 protein sequence. When tested by Western blot, it was shown that recombinant polypeptide RR2 is capable of restoring P1 recognition by MAb 3-10E (Figure 3-2B). While RR2 most likely does not re present the absolute
45 minimum sequence required for MAb 3-10E re cognition, it does provide information that suggests important components contributing to epitope formation are contained within the sequences C-terminal to the P-region and that an interaction between not only the Aand Pregions of the molecule but an interaction between pr e-A and post-P region sequence is required for MAb 3-10E recognition. Recombinant Polypeptide RR2 Achiev es a Native-like P1 Structure Given that recom binant polypeptide RR2 is capable of restoring binding of MAb 3-10E, it seemed possible that this polype ptide might also contain all the requisite elements of the P1 sequence required for folding of the protein into a native-like state. A competition ELISA was used to determine if recombinant RR2 achieves a folded structure that closely resembles that of native P1 on the cell surface. Recombinant P1 po lypeptides CK1 and CK2 were also included in the assay to confirm their lack of recognition by MAb 3-10E vi a ELISA. Indeed, it was shown that RR2 is capable of competing with native P1 localized on the surface of S. mutans whole cells for MAb 3-10E binding at a level of nearly 90% (Figure 3-3). In addition the data shows that there is a complete lack of competition for S. mutans binding with both recombinant polypeptides CK1 and CK2 and the vector only ly sates which served as negative controls. These data suggest that folding of P1 into its native three-dimensional structur e not only requires an interaction between the Aand P -regions of the protein but also an interaction of sequence Nterminal of the A-region and C-terminal of the P-region. Furthermore, it is suggested that MAb 3-10E may be a useful tool for the detection of P1 which is in a native-like folded state. The Genetic Background of the Host Affe cts Immunomodulation by Anti-P1 MAbs W ith the discovery that MAb 5-5D is a be neficial immunomodulator, it became apparent that the ability of MAbs 6-11A, 3-10E, and 5-5D to beneficially modulate the antiS. mutans immune response is likely linked to a commonality in their epitopes which potentially results by
46 an influence upon P1 structure leading to the exposure of more efficacious targets for Ab formation. If, indeed, some common feature in MAb epitope results in a structural perturbation, this could lead to changes in Ag processing and presentation by antigen presenting cells (APCs). Any change in Ag processing and presentation would be expected to be refl ected in that different results would be predicted in mice with differing MHC II haplotypes. In order to determine what effect(s), if any, the genetic background (MHC II haplotype ) of the host might have on immunomodulation by anti-P1 MAbs, immunizati on experiments were performed in BALB/c and C57BL/6 mice. Groups of six female BALB/c and C57BL/6 mi ce were immunized in parallel with IC containing MAbs 6-11A and 5-5D at [saturating] and [0.1x subsaturating] and with uncoated S. mutans whole cells. The immunizations were carried out as described in the methods. However, test bleeds were taken from all mice on day 7 in order to assess the kinetics of the antiS. mutans immune response. SPR results demonstrated that serum from BALB/c mice immunized with ICs containing MAbs 6-11A and 5-5D wa s increased in its ability to inhibit bacterial adherence to immobilized SAG and that differing results we re observed in C57BL/6 mice treated in an identical manner. The serum from BALB/c mice immunized with a [saturating] of MAb 6-11A and both a [saturating] and [0.1x sub-saturating] of MAb 5-5D wa s increased in its ability to inhibit S. mutans adherence to immobilized SAG over the serum from S. mutans -immunized mice. However, the serum from C57BL/6 mi ce immunized with ICs containing either MAb showed no measurable increase in ability to i nhibit bacterial adherenc e (Figure 3-4). These results suggest that the genetic background of the host does affect the immunomodulatory outcome when IC containing anti-P1 MAbs 6-11A and 5-5D are administered.
47 Kinetics and Isotype Composition of the AntiS. mutans Response In order to better evaluate other immunom odulatory propertie s of MAbs 6-11A and 5-5D, the serum from immunized BALB/c and C57BL/6 mice were also examined for changes in the kinetics and isotype composition of the antiS. mutans response. As described in the previous section, both mouse strains we re immunized with IC and S. mutans alone. In order to evaluate the immune response in these mice early in th e immunization regimen, the mice were subjected to blood collection 7 days post primary im munization and the serum was tested for S. mutans reactivity and the isotype of these Abs we re also determined by quantitative ELISA. Interestingly, the serum from BALB/c mice receiving ICs containing MAb 6-11A showed an early rise (7 days pos t-primary immunizat ion) in anti-S. mutans IgG1, IgG2a, and IgG2b versus the serum from mice receiving S. mutans alone and the seru m from C57BL/6 mice exhibited different changes in the immune res ponse (Figure 3-5A). Add itionally, the serum from BALB/c mice immunized with IC s containing MAb 5-5D also c ontained early and increased levels of antiS. mutans Abs of the IgG2a and IgG2b isotypes over the serum from S. mutans immunized BALB/c mice while th e serum from C57BL/6-immuni zed mice exhibited different outcomes (Figure 3-5B). The data demonstrate that im munization with IC versus bacteria alone boosts the resultant immune response towards S. mutans and that the Ab isotype composition of the polyclonal response is increased in specific IgG subclass Abs. These results further support the observed effect of host genetic background on the beneficial immunomodulatory properties of anti-P1 MAbs 6-11A, 3-10E, and 5-5D. MAb 1-6F Competition by Serum of MAb 6-11A and 5-5D IC-Immuni zed Mice In order to further define the changes in spec ificity of the resultant immune response in ICimmunized BALB/c and C57BL/6 mice, the polyc lonal serum from these mice were evaluated
48 for the presence of Abs that recognized the same or similar epitopes as a MAb that is a direct inhibitor of adherence such as MAb 1-6F. MAb 1-6F directly inhibits the binding of S. mutans to immobilized SAG and as such one would expect that the serum of IC-immunized mice which have been shown to inhibit adhe rence at an increased level might contain Abs that recognize the same or similar epitopes as MAb 1-6F. In order to explore this possibility, a competition ELISA was used. The ability of the serum from S. mutans and IC-immunized BALB/c and C57BL/6 mice to compete for binding of native P1 present on the surface of S. mutans whole cells with biotinlabeled MAb 1-6F was measured. Consistent wi th previous data, the serum from MAb 6-11A and 5-5D IC-immunized BALB/c, but not C57BL/6 mice, did exhibit an increase in levels of Abs capable of competing for S. mutans binding over the serum from mice receiving bacteria alone (Figure 3-6). These data suggest that beneficial immunomodulatory MAb binding acts in some way to enhance the exposure of the P1 se gments which contain the MAb 1-6F epitope. Measurement of Competition against MAb 16F by Serum from IC-immuniz ed Mice With the discovery that immunization with IC containing MAbs 6-11A and 5-5D results in a polyclonal immune response that contains increased levels of Abs capable of competing for S. mutans binding with biotin labeled 1-6F, further competition experiments were undertaken to evaluate the presence of MAb 1-6F-like Abs in the serum of BALB/c mice immunized with IC containing all the anti-P1 MAbs tested for immunomodulatory activit y. Previously, MAbs 611A, 3-10E, 5-5D, 1-6F, 4-9D, and 4-10A were tested for immunomodulatory activity when administered to BALB/c mice as part of an IC (84, 144). Sera stored from these previous experiments were tested for their ability to compete for S. mutans binding with biotin-labeled MAb 1-6F.
49 Interestingly, the serum from the mice im munized with IC containing beneficial immunomodulatory MAbs 6-11A, 3-10E, and 5-5D a ll contained Abs increased in their ability to compete with MAb 1-6F for S. mutans binding when compared to serum from S. mutansimmunized mice. Consistent with previous data serum from mice immunized with IC containing those MAbs shown to be non-bene ficial or neutral in their i mmunomodulatory effects did not exhibit any such increase in 1-6F-like Abs (Fi gure 3-7). These data de monstrate a link between beneficial immunomodulatory properties and enhanced exposure of more relevant targets of efficacious antibodies. Isotype Determination of MAb 1-6F-Like Ab s in the Seru m of IC-immunized Mice Prior studies conducted in our laboratory demonstr ated a statistically si gnificant correlation between the ability of serum fr om IC-immunized mice to inhibit S. mutans adherence to immobilized SAG and the presence of antiS. mutans Abs of the IgG2a and IgG2b subclass (144). Given that the sera from mice i mmunized with IC containing beneficial immunomodulatory MAbs contains MAb 1-6F-l ike Abs that can be related to enhanced adherence inhibition, experiments were performed to assess the isotypes of the MAb 1-6F-like Abs within those sera. In order to determine the isotype of the MAb 1-6F-like Abs contained within the serum of IC-immunized mice, a quantitative subclass ELISA was employed. The Ag chosen for use in these ELISA experiments was recombinant P1 polypeptide NR21. Polypeptide NR21 was chosen as the Ag for these experiments because it is r ecognized by MAb 1-6F but none of the other antiP1 MAbs (Figure 3-8). Therefore, it stands to reason that any IgG subcla ss Ab reactivity against this protein fragment can be considered as MAb 1-6F-like in nature or to at least recognize a similar epitope as MAb 1-6F. Again the sera from previous studies were used to evaluate the isotype composition of 1-6F-like Abs.
50 As can be seen in Figure 3-9A, immunizati on with IC containing MAbs 6-11A, 3-10E, and 4-10A results in increased serum levels of NR21-specific IgG1. Figure 3-9B shows that immunization with MAb 6-11A, 5-5D, and 4-10A cont aining ICs results in increased levels of anti-NR21 IgG2a in the serum of immunized mice. Anti-NR21 IgG2b levels were also increased in the serum of mice receiving ICs of MAbs 5-5D and 4-10A (Figure 3-9C). Any differences seen between the levels of an ti-NR21 Abs in the serum from S. mutans only-immunized mice are a result of experimental varia tion in that each immunization e xperiment with each individual MAb was performed at different times with diffe rent shipments of mice. Therefore, it is important to only compare the differences be tween levels of anti-NR21 Abs in IC vs. S. mutans immunized mice within a given experiment. To further examine the NR21 reactivity of the serum from IC-immunized mice, Western blot analysis was also performed using the se rum from individual mice immunized with IC containing MAb 5-5D. Only the seru m from a few mice immunized with S. mutans alone reacted with NR21 despite the Ab isotype measured. Howeve r, there was an increas e in reactivity of the IgG1 and IgG2a anti-NR21 isotypes in the sera of mice receiving IC containing MAb 5-5D (Figure 3-10). Taken together, these data suggest that the pr eviously observed correla tion with specific antiS. mutans IgG subclass Abs and the ability of se rum from immunized mice to inhibit bacterial adherence may potentially be due to the presence of MAb 1-6F-like Abs within those sera. The data demonstrate an increased level of anti-NR21 Abs (MAb 1-6F-like) of certain IgG subclasses in the serum from mice immunized with IC containing beneficial immunomodulatory MAbs.
51 Role of Activating Fc Receptors in Immunomodulation by Anti-P1 MAbs Immunization with ICs containing MAbs 6-11A 3-10E, and 5-5D not only results in an immune response that is increase d in its ability to inhibit bacterial adherence to SAG but the response is altered specif ically towards less immunodom inant but more relevant targets such as the MAb 1-6F epitope containing regions of P1. Despite evidence that the MAb epitope requirements play a significant role in the immunomodulatory properties of beneficial MAbs, other mechanisms by which Ab can modulate th e immune response toward an Ag must be evaluated in our system. As described previous ly, there are two major categories by which Ab can modulate the resultant immune response: Fc -dependent and Fc-independent. In order to determine Fc-dependence on immunomodulation in our system, the mechanistic role that activating Fc receptors (FcRs) present on the surf ace of antigen presenting cells (APCs) have in immunomodulatory changes by our beneficial MAbs was evaluated. Immunization experiments were carried out in a BALB/c mouse strain lacking activating Fc receptors. Wild-type BALB/c and BALB/c activating Fc R targeted mutation transgenic mice, Fcer1g, were immunized with IC containing MA b 5-5D at a [saturating] and a [0.1x subsaturating] or S. mutans alone as described in the methods section. The resulting serum responses were then evaluated for their immunomodulatory pr operties by testing thei r ability to inhibit bacterial adherence, compete for S. mutans binding with biotin-l abeled MAb 1-6F, and measuring the levels of NR21 specific IgG subclass Abs. These experiments were performed using activating Fc R targeted mutation transgenic mice because the three receptors known to bind IgG and activate an immune response, Fc RI, Fc RIII, and Fc RIV, all depend upon a common chain for signaling through an ITAM motif and this mouse lacks a functional chain (139, 140).
52 Results of adherence inhibition experiments are illustrated in Figure 3-11A. The data demonstrate a decrease in the ability of S. mutans whole cells to bind immobilized SAG in the presence of serum from both wild-type and FcR deficient (Fcer1g) BALB/c mice immunized with IC containing MAb 5-5D when compared to S. mutans binding in the presence of serum from mice receiving S. mutans alone. Furthermore, as can be seen in Figure 3-11B, the serum from Fcer1g mice immunized with IC containing MAb 5-5D is able to compete with biotinlabeled MAb 1-6F for S. mutans binding over the serum from S. mutans -immunized mice. Figure 3-11C demonstrates increased levels of anti-NR 21 Abs of all three IgG isotypes tested in the serum from MAb 5-5D IC-immunized versus the serum from bacter ia only immunized Fcer1g mice. Taken together these results suggest immunomodulation by beneficial MAbs 6-11A, 310E, and 5-5D is independent of activating FcRs on the surface of APCs. The data suggest a mechanism of action that is Fc-independent, but does not exclude Fc-dependence altogether. Role of the Fc Portion of An ti-P1 MAbs on Immunomo dulation Despite ruling out the role of activating Fc receptors in the mechanism underlying beneficial immunomodulation, the role of the Fc portion of the MAb on immunomodulation still required evaluation to determine if Ab mediated complement fixation and subsequent uptake of the IC via complement receptors or uptake via i nhibitory FcRs are involved in our system. In order to evaluate the role that the Fc portion of the anti-P1 MAbs might have in their beneficial immunomodulatory effects, murine immunizatio n experiments were carried out using IC containing F(ab)2 fragments of MAbs 6-11A and 55D in place of intact MAb. MAbs 6-11A and 5-5D were subjecte d to pepsin digestion and F(ab)2 fragments were purified by passage over a protein A column an d by use of size exclusion chromatography. The [saturating] and [0.1x sub-satura ting] concentrations of F(ab)2 fragments and intact MAb were determined by dot blot. Groups of six fe male BALB/c mice were immunized with S. mutans
53 alone, F(ab)2-containing IC or MAb-containing IC (positive control) exactly as previous immunizations were carried out. The sera fr om each group of mice was then evaluated for immunomodulatory changes in adherence inhibi tion, MAb 1-6F competition, and levels of antiNR21 IgG subclass Abs as was previously test ed in the sera from IC-immunized mice. Consistent with previous data the sera from the mice immuni zed with a [saturating] of MAb 6-11A F(ab)2 IC and a [0.1x sub-satura ting] of MAb 5-5D F(ab)2 IC demonstrated an increase in adherence inhibiti on by SPR (Figure 3-12A). The sera from the mice receiving both [F(ab)2] of MAb 6-11A exhibited a st atistically significant increase in biotin-labeled MAb 1-6F competition while the sera from mice receiving IC of [0.1x sub-saturating] of MAb 5-5D F(ab)2 was also increased in MAb 1-6F competition for S. mutans binding (Figure 3-12B). As can be seen in Figure 3-12C, immunization of mice with both [MAb 6-11A F(ab)2] IC resulted in increased levels of anti-NR21 IgG1, IgG2a, and IgG2b and immunizatio n of mice with a [0.1x sub-saturating] of MAb 5-5D F(ab)2 IC resulted in increased levels of anti-NR21 IgG1 and IgG2a and, to some extent, IgG2b. These data exclude any involvement of th e Fc portion of the MAb in beneficial immunomodulation by our anti-P1 MAbs. Taken together with the activating Fcer1g transgenic mice data and previous data which demonstrated that the beneficial MA bs were not opsonic and that complement fixation did not correlate with immunomodulatory characteristics (84), the data exclude Fc-dependence from the mechanism of immunomodulation by our beneficial MAbs. Re-evaluation of the Immunomodul atory Properties of MAb 4-10A W ith Fc receptors and the Fc portion of MAbs ruled out as being involved in the mechanism(s) of beneficial immunomodulation by MAbs 6-11A, 3-10E, and 5-5D, it became even more likely that their mechanism of action is linked to their common epitope features. All three MAbs epitopes are significantl y contributed to by an interaction of the Aand P-regions of
54 P1. However, a discrepancy in th is concept existed in that anot her Aand P-region-dependent MAb had been shown to be neutral in its imm unomodulatory effects. Ho wever, prozone-like effects have been previously observed with our anti-P1 MAbs in that the immunomodulatory effects of anti-P1 MAbs appear to be dependent upon the concentration of MAbs within the IC. To determine if Aand P-region dependent MAb 4-10A does exhibit beneficial immunomodulatory properties, it was re-evaluated in murine immunization experiments over a broader concentration ra nge within the IC. Groups of six female BALB/c mice were immunized with S. mutans alone and S. mutans coated with MAb 4-10A (3.0 mg/ml) at a 1:2000, 1:4000, 1:8000, 1:16000, or 1:32000 dilution. The mice were immunized and sacrificed accord ing to previous experimental schedules. In addition, small test bleeds were also taken from the mice 7 days post-primary immunization and these sera were also evaluated fo r early immunomodulatory changes. As can be seen in Figure 3-13A, immunization with IC of MAb 4-10A at lower concentrations indeed results in a serum response that is increa sed in its ability to inhibit bacterial adherence to immobilized SAG, as measured by SPR. It is also evident that a prozonelike effect exists with MAb 4-10A in that too much or too little Ab re sults in non-beneficial outcomes. The data also demonstr ate that immunization of mice w ith IC containing intermediate concentrations of MAb 4-10A also results in a response that is incr eased in ability to compete for S. mutans binding with biotin-labeled MAb 1-6F as early as seven days post-primary immunization (Figure 3-13B). Lastly, consistent with other results, the presence of MAb 4-10A at intermediate concentrations wi thin the IC also results in an anti-NR21 response in the sera of immunized mice that is increased in all three Ig G isotypes, again only se ven days after primary immunization, but with IgG1 and IgG2a be ing the most pronounced (Figure 3-13C).
55 These data demonstrate that MAb 4-10A is immunomodulatory a nd that prozone-like effects do exist with this MAb. These result s further support a mechanism of beneficial immunomodulation that is de pendent upon commonalities among MA b epitope requirements and that is Fc-independent. Again, a correlation with MAb 1-6F-like Abs of certain IgG isotypes and increased inhibition of S. mutans adherence to immobilized SAG is suggested. Furthermore, the results demonstrate an early increase in serum Abs capable of competing with MAb 1-6F binding of S. mutans and in serum IgG subclass Abs directed towards NR21 in that these effects were observed only 7 days after primary immunization. P1 Binding by MAb 4-10A Enhances the Binding of MAb 1-6F W ith a final confirmation that all four MA bs whose epitopes involve an interaction between the Aand P-regions of P1 were ind eed beneficial immunomodu lators and that the serum from the mice immunized with IC c ontaining MAbs 6-11A, 3-10E, 5-5D, and 4-10A contains MAb 1-6F-like Abs, it seemed appropria te to examine whether any of these MAbs are actually enhancing the exposure of the MAb 1-6F epitope by binding of cell surface-localized P1. As can be seen in Figure 3-8, the MAb 16F epitope is normally not well exposed in the context of purified full-length P1. Therefore, it stands to reason that bi nding of P1 by beneficial immunomodulatory MAbs might influence the st ructure of the protein in some way as to enhance the exposure of the MAb 1-6F epitope. To address this question, a modification of a competition ELISA was employed. In this assay, S. mutans whole cells were coated onto the ELISA plate wells and two-fold dilutions of MAb 4-10A were added to the plate. This wa s followed immediately by addition of several dilutions of biotin-labeled MAb 1-6F and the plate was incubated at 37C for 1.5-2.0 hours. After washing, addition of Avidin-HRP, and addition of s ubstrate, the plate was read at 450 nm and the
56 percent increase or d ecrease of biotin-labeled MAb 1-6F bound to S. mutans on the plate was determined against reference wells cont aining only biotin-labeled MAb 1-6F. As can be seen in Figure 3-14, the presence of MAb 4-10A at dilutions similar to those which exhibited beneficial eff ects in murine immunization expe riments increased the binding of MAb 1-6F up to 150%. These data suggest that binding of an A/P-dependent anti-P1 MAb may alter the structure of P1 in so me way as to better expose the MA b 1-6F epitope. This speculation is supported by previous studies wh ich demonstrated that binding of P1 by MAb 6-11A alters the proteolytic digestion of cell surface-localized P1 in vitro including both elimination of as well as appearance of certain proteolytic fragments (156). This is sugges tive of a structural perturbation that masks or exposes sites within the molecule. Evaluation of Cell-Level Immunomo dulatory Changes Induced by MAb Previous data suggested that immunization with IC contai ning MAbs 6-11A, 3-10E, 5-5D, and 4-10A could potentially result in alterations of the antigen processing and/or presentation pathway by causing a structural perturbation in P1 which might alter the availability of proteolytic sites within the molecule. The data also excluded any role of the Fc portion of the MAb and FcRs in the observed immunomodulatory changes. Furthermore, the ability of MAb 410A to enhance the exposure of the MAb 1-6F epitope also s uggests that MAb binding effects the native structure of P1. To determ ine if immunization with IC versus S. mutans alone results in detectable changes on a cellular level, a sple nocyte proliferation assa y (measured by tritiated thymidine incorporation) and multi-plex cytokine analysis was used. These experiments were undertaken in an attempt to begin to understa nd what is a complex cascade of downstream changes involving the complexities of cell-cell interactions, antigen processing and presentation, and cytokine effects.
57 As was discussed in the methods section of this document, BALB/c mice were immunized with 1X PBS, S. mutans or IC containing MAb 4-10A (3.0 mg/ml) at a 1:8000 dilution and the spleens of the immunized mice were used in proliferation assays and their response to recombinant P1 polypeptides was measured. Se rum responses of the immunized mice were evaluated over time prior to the mice receiving a splenectomy. Culture supernatants were also saved prior to addition of tritiated thymidine for cytokine analysis. The P1 polypeptides used as antigens in these assays are schematically represented in Figure 3-15. Serum Reactivity of Proliferation Assay Mice against A3VP1 In order to determ ine which P1-derived polype ptide might be the most appropriate antigen to use in the splenocyte proliferation assa ys, the serum IgG reactivity of immunized mice towards a panel of recombinant P1 polype ptides was measured on day 26 post primary immunization. Serum reactivity against recombinant P1 polypeptide encoded by A3VP1 was found to be the most appropriate antigen based on a differential between the serum response of mice immunized with S. mutans and those receiving MAb 4-10A-S. mutans IC. As is shown in Figure 3-16A, the total IgG response towards A3VP1 in the serum of mice receiving one injection of IC was increased in comparison to the serum of mice receivi ng only one injection of S. mutans Additionally, while not as notable of an increase, a similar effect was observed in the sera of mice receiving a second booster injection. To evaluate the isotype of anti-AV3P1 antibod ies contained within th e polyclonal serum of immunized mice at the time the spleens were harv ested for use in splenocyt e proliferation assays (33 days post primary immunization), serum was obtained from each mouse via cardiac puncture prior to removal of their spleens. The termin al serum samples were then analyzed by IgG subclass quantitative ELISA against recombin ant P1 polypeptide AV3P1. Consistent with previous ELISA data, an increase in anti-AV3 P1 IgG1, IgG2a, and IgG2b was observed in the
58 serum of mice receiving one inje ction of MAb 4-10A IC when co mpared to the serum from mice receiving one injection of S. mutans alone (Figure 3-16B). Howeve r, similar levels of IgG subclass antibodies were observed in the serum of mice receiving a sec ondary booster injection (Figure 3-16B). Splenocyte Proliferation Ass ays and Cytokine Analysis In order to evaluate potential imm unomodulat ory changes on a cellular level, tritiated thymidine uptake assays utilizing crude splenoc ytes from immunized mice and recombinant P1 polypeptides were employed. Groups of two fema le BALB/c mice were immunized with PBS, S. mutans whole cells, or S. mutans coated with MAb 4-10A. PBS-immunized mice were injected once on day 0 and received a splenectomy on day 33. S. mutans and MAb 4-10A IC-immunized mice were either immunized on day 0 and rested until day 33 or were immunized on day 0 and boosted on day 13 and rested until day 33. On day 33 all spleens were harvested and duplicate spleens were pooled together for splenocyte ex traction. One million cells were plated per well and recombinant A3VP1 was added at 50 g/ml to the appropriate experimental wells. Con A was added at 5 g/ml to positive control wells. Negative control wells received cells alone. The stimulation index values of nave (PBS) sple nocytes, splenocytes from mice receiving one injection of S. mutans or IC, and splenocytes r eceiving two injections of S. mutans or IC in response to AV3P1 are shown in Figure 3-17A. Cons istent with the serological data from these mice, the splenocytes from mice receiving one immunization with IC exhibited a higher proliferative response to A3VP1 than those from mice receiving one immunization with S. mutans alone. However, it appears that boosting with IC actually decreased or eliminated the proliferative response towards A3 VP1. At least in this specific experiment, it appears that boosting with IC may result in an anergic-li ke state as measured by this particular in vitro assay.
59 In addition to serological and proliferation da ta, the culture supernatants from the tritiated thymidine uptake assays were also analyzed for cytokine content using a multi-plex approach on a luminex 100 system. The supernatants were tested for interferon gamma (INF-gamma), interleukin 10 (IL-10), interleukin 2 (IL-2), interleukin 4 (IL-4), tumor necrosis factor alpha (TNF-alpha), and tumor growth factor beta (TGF-beta) isoforms 1, 2, and 3. These cytokines were chosen due to their known association with Ab class switching to IgG1, IgG2a, and IgG2b as well as their involvement in other areas of the immune response (42, 50, 61, 184). After analysis of the cytokine assay results it appears that IL-2, IL -4, and TNF-alpha were not altered with IC-immunization compared to S. mutans -immunization (data not shown). Interestingly, INF-gamma levels were increased in the supernatants of splenocytes from mice receiving one injection of MAb 4-10A IC versus splenocytes fr om mice receiving one injection of S. mutans alone (Figure 3-17B) and this is consis tent with the observed increase in antiA3VP1 IgG2a in the serum of those same mice which received one immunization with MAb 410A IC. Figure 3-17B also shows that a decrease in IL-10 was observed in the supernatants from splenocytes of mice receiving two injections of MAb 4-10A IC when compared to the supernatants from A3VP1 stimulated splenocytes from mice receiving two injections of uncoated S. mutans whole cells. These data appear to agree w ith the stimulation index values from those same splenocytes. The exact role of TGF-beta isoforms in the immune response and Ab isotype class switching to IgG2b is sti ll unclear but it is known that this cytokine works closely with other cytokines in many situations. While no definite role(s) of TGF-beta 1,2,3 in immunomodulatory outcomes against A3VP1 can be defined by the cy tokine analysis resu lts, some noteworthy changes were observed (Figure 3-17C). TGF-beta isoform 1 s howed a disconnect with the
60 serological data which showed an increase in anti-A3VP1 IgG2b in the serum of mice immunized with one injection of MAb 4-10A IC versus those receiving one immunization of S. mutans in that there was no observed in crease in this cytokine in th e supernatants of splenocytes from these mice stimulated with A3VP1. However, TGF-beta 2 appeared to be slightly increased in the supernatants of splenocytes from MA b 4-10A IC immunized mice stimulated with recombinant A3VP1. However, there is an absence of TGF-beta 3 isoform in the supernatants of splenocytes from IC-immunized mice stimulated w ith A3VP1 while it is present in those from S. mutans -immunized mice. In order to better evaluate the cytokine re sponse in the supernatants of splenocytes stimulated in vitro with recombinant P1 polypept ides, another set of culture supernatants from splenocytes stimulated with P1 polypeptides we re analyzed for cytokine content. In these experiments mice were immunized with PBS (nave), S. mutans or S. mutans coated with MAb 4-10A (4-10A IC) once on day 0 and the spleen s were harvested on day 7. Splenocytes were plated at one million cells per well and 50 g /ml of recombinant P1 polypeptides LT1, LT2, LT3, or NR21 were added to their representative wells. Positive control wells received 5 g/ml Con A and negative wells received cells alone. Th e cells were incubated with antigen for six days and culture supernatants were collected on day 6. Consistent with previous serological data from MAb 4-10A IC-immunized mice against NR21 which showed an increase in anti-NR21 IgG2a, INF-gamma was increased in the culture supernatants of splenocytes derived from MAb 4-10A IC-immunized mice stimulated in vitro with NR21 (Figure 3-18A). However, there was no observed increase or differences in the supernatants of splenocytes stimulated with LT1, LT2, or LT3. Taken together with the results of the cytokine analysis of supe rnatants from A3VP1 stimulated cells, it appears there is a
61 connection with INF-gamma produc tion and the stimulating polype ptides in the assay which vary in their structural complex ity resulting in part from the in teraction of the discontinuous Aand P-regions. Figure 3-18A demonstrates that IL-10 production in response to LT3 stimulation is decreased in splenocytes from IC-immunized versus S. mutans -immunized mice. TGF-beta 1 in the supernatants of splenocytes from MAb 4-10A IC-immunized mice was increased in LT2 and LT3 stimulated cells and decreased in LT1 and NR21 stimulated cell s versus splenocytes from S. mutans -immunized mice (Figure 3-18 B). Furthermor e, the results of TGF-beta 3 (Figure 3-18B) analysis further suppor t the observation that TGF-be ta production in splenocytes stimulated with recombinant polypeptides depend s on protein structure in that it appears as the complexity of the polypeptide increases that the level of TGF-beta 3 decreases in the culture supernatants. Evaluation of Guyss 13 Plantibo dy for Immunomodulatory Properties As discussed in the introducti on, anti-P1 MAbs have been used by others as passive immunotherapies to decrease S. mutans colonization. In particular Guys 13 and its tobacco plant-derived counterpart, Guys 13 plantibody, were s hown to protect against S. mutans recolonization in hum an subjects for up to two years af ter their passive application to the surface of the teeth of study volunteers. However, the m echanism by which these passively administered reagents produced such long-term effects was never satisfactorily e xplained. One would not expect the effects of a passive approach such as that used in those studies to have such lasting effects on S. mutans re-colonization. Indeed, the results suggest that an active immunologic mechanism may be involved. Based on the results of the passive immunothera py experiments and the fact that Guys 13 and Guys 13 plantibody also require an interaction between the Aand P-regions for P1 recognition, it seemed plausible that they might exhibit benefici al immunomodulatory
62 characteristics similar to those observed with our MAbs. To examine this possibility, murine immunization experiments using S. mutans and S. mutans coated with various concentrations (saturating and multiple non-satura ting) of Guys 13 plantibody were performed. Groups of six female BALB/c mice were immunized in the same manner as previous experiments with PBS, Ab only, S. mutans only, and S. mutans coated with Guys 13 pl antibody (2.0 mg/ml) at a dilution of 1:100, 1:1000, and 1:10,000. The resulta nt polyclonal serum response in those mice was then evaluated for immunomodulatory characteristics. The serum from mice immunized with IC contai ning intermediate concentrations of Guys 13 plantibody versus S. mutans alone contained increased levels of antiS. mutans total IgG and, as was observed in MAb 4-10A IC mice, increased levels of anti-NR21 IgG1, IgG2a, and IgG2b subclass Abs (Figure 3-19). Furthermore, the sa me results were observed when anti-A3VP1 IgG subclass Abs was measured in the serum of imm unized mice (Figure 3-20). These results suggest that Guys 13 plantibody does exhibit i mmunomodulatory characteristics and these characteristics may explain the long-term e ffects observed in human clinical trials.
63 Figure 3-1. Adherence inhibiti on by serum from MAb 5-5D IC -immunized mice. Sera from mice receiving S. mutans alone or IC of S. mutans coated with MAb 5-5D were tested for their ability to inhibit adherence of S. mutans to immobilized SAG by SPR. The percent inhibition of adherence was calcul ated and is represented by bar graph and High and Low Ab indicate that [saturating] and [0.1x sub-saturating] of MAb were used to coat S. mutans prior to immunization. The standard deviations between replicate experiments are denoted by error bars. The data are representative of at least three independent experiments.
64 Figure 3-2. Evaluation of the minimal P1 se quences required for MAb 3-10E recognition. (A) Schematic representation of full-length P1 and P1 sub-clones encoding recombinant polypeptides CK1, CK2, and RR2. (B) Western blot reactivity of anti-P1 MAbs with recombinant full-length P1, CK1, CK2, and RR2.
65 Figure 3-3. P1 sequence requirements for achieve ment of the MAb 3-10E epitope on the surface of S. mutans Inhibition of MAb 3-10E binding to S. mutans whole cells was measured by competition ELISA following incubation with threefold serial dilutions of E. coli lysates containing P1 polypeptides RR2, CK1, CK2, or the vector-only controls (VC) (RR2 VC and CK1 & 2 VC). The percent inhibition was calculated as follows: 100[(mean OD450 of MAb 3-10E + P1 polypeptide/mean OD450 of MAb 3-10E alone) X100]. Standard deviations from the means are represented by error bars. The data are representative of a mi nimum of duplicate experimental wells from each of two independent experiments.
66 Figure 3-4. Adherence inhibiti on by sera of MAb 6-11A and 55D IC-immunized BALB/c and C57/BL6 mice. BiaCore anal ysis of inhibition of S. mutans adherence to immobilized salivary agglutinin by serum from BALB/c and C57/BL6 mice immunized with ICs of MAbs 6-11A and 5-5D versus S. mutans alone. Binding of S. mutans to immobilized SAG in the presence of serum from S. mutans and IC-immunized mice is shown by sensor grams. High and Low Ab indicate that [saturating] and [0.1x subsaturating] of MAb were used to coat S. mutans prior to immunization. Positive and negative controls included m easurement of the binding of S. mutans and a P1 deficient S. mutans strain in the absence of serum. The data ar e representative of a minimum of three independent experiments.
67 Figure 3-5. AntiS. mutans whole cell IgG sub-class reactivity in the sera from immunized BALB/c and C57/BL6 mice. Sera from S. mutans MAb 6-11A IC (A), and MAb 55D IC (B) immunized BALB/c and C57/ BL6 mice taken 7 days after primary immunization were evaluated for anti-S. mutans IgG sub-class reactivity by quantitative ELISA. High and Low Ab indi cate that [saturating] and [0.1x subsaturating] of MAb were used to coat S. mutans prior to immunization. AntiS. mutans IgG subclass concentrations were extrap olated from standard curves and are represented by line graphs. Standard deviat ions from the means are represented by error bars. Positive controls included binding of appropriate anti-P1 MAbs and negative controls included appropriate sec ondary only reagent wells All assays were performed in duplicate wells in multiple independent experiments.
68 Figure 3-5. Continued.
69 Figure 3-6. MAb 1-6F competition by the se ra from MAb 6-11A and 5-5D IC-immunized BALB/c and C57/BL6 mice. Competition ELISA was used to evaluate the presence of MAb 1-6F-like Abs in the serum of BALB/c and C57/BL6 mice immunized with ICs of MAb 6-11A and 5-5D versus S. mutans alone. ELISA plate wells were coated with S. mutans strain NG8 whole cells and inhibi tion of binding of biotin-labeled anti-P1 MAb 1-6F was measured. High and Low Ab indicate that [saturating] and [0.1x sub-saturating] of MAb were used to coat S. mutans prior to immunization. Positive and negative controls included inhibition by unlabeled MAbs 1-6F and 410A (not shown). The percent competition is depicted by bar graph and the standard error of the means observe d between replicate wells are shown as error bars. Statistical significance is indicated by P values The data are represen tative of at least three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova.
70 Figure 3-7. MAb 1-6F Competition by serum fr om anti-P1 MAb IC-immunized BALB/c mice. Competition ELISA was used to evaluate th e presence of MAb 1-6F-like Abs in the serum of BALB/c mice imm unized with ICs of MAb 6-11A, 3-10E, 5-5D, 1-6F, 49D, and 4-10A versus S. mutans alone. ELISA plate wells were coated with S. mutans strain NG8 whole cells and inhibition of binding of biotin-labeled anti-P1 MAb 1-6F was measured. High and Low Ab indicate that [saturating] and [0.1x sub-saturating] of MAb were used to coat S. mutans prior to immunization. Positive and negative controls included inhibition by unlabeled MAbs 1-6F a nd 4-10A (not shown). The percent competition is depicted by bar gr aph and the standard error of the means observed between replicate wells are shown as error bars. Statistical significance is indicated by P values. The data are repres entative of at least three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova.
71 Figure 3-8. Schematic of recombinant P1 polypeptide encoded by sub-clone NR21 and MAb reactivity against full-length P1 a nd P1 polypeptide NR21. (A) schematic representation showing the regions of P1 contained within P1 sub-clone encoding recombinant polypeptide NR21. (B) Western bl ot analysis of anti-P1 MAb reactivity with recombinant full-length P1 and pol ypeptide NR21. Controls included reactivity with appropriate polyclonal anti-P 1 Abs and secondary reagents only.
72 Figure 3-9. Anti-NR21 IgG subclass reactivity in the sera from S. mutans and IC-immunized BALB/c mice. Sera from the S. mutans MAb 6-11A IC, 3-10E IC, 5-5D IC, 1-6F IC, 4-19D IC, and 4-10A IC-immunized mice were evaluated for the presence of NR21specific IgG1 (A) IgG2a (B), and IgG 2b (C) subclass Abs by quantitative ELISA. Anti-NR21 isotype concentrations were ex trapolated from standard curves. The means of duplicate wells at each serum dilution are represented by line graph and standard deviations are depicted by e rror bars. High and Low Ab indicate that [saturating] and [0.1x sub-saturati ng] of MAb were used to coat S. mutans prior to immunization. Positive controls included binding of appropriate anti-P1 MAbs and negative controls included appropriate s econdary only reagent wells. The data are representative of at least th ree independent experiments.
73 Figure 3-9. Continued.
74 Figure 3-9. Continued.
75 Figure 3-10. Western blot analysis of antiNR21 IgG subclass reactivity in the sera from S. mutans and MAb 5-5D IC-immunized mice. R eactivity of serum from individual mice immunized with S. mutans (n=5) [lanes 1-5], IC containing a [saturating] of MAb 5-5D (n=6) [lanes 6-11] and a [0.1x s ub-saturating] of MAb 5-5D (n=6) [lanes 12-17] was measured by Western blot ag ainst purified recombinant polypeptide NR21. Ab subclass was measured by anti-IgG1, IgG2a, and IgG2b secondary reagents. Negative controls included use of appropriate secondary reagent only strips (lane 18) and positive controls (not shown) included reactivity with appropriate antiP1 MAbs.
76 Figure 3-11. Evaluation of the role of Fc recep tors in beneficial immunomodulation by anti-P1 MAbs. Adherence inhibition, MAb 1-6F competition, and Anti-NR21 sub-class reactivity by the sera of IC-immunized Fcer1g transgenic mice. High and Low Ab indicate that [saturating] and [0.1x sub-saturating] of MAb were used to coat S. mutans prior to immunization. (A) BiaC ore analysis of inhibition of S. mutans adherence to SAG by serum fr om wild-type BALB/c and Fcer1g transgenic mice immunized with ICs of MAb 5-5D versus S. mutans alone. (B) MAb 1-6F competition ELISA using the serum from S. mutans and MAb 5-5D IC-immunized Fcer1g transgenic mice. (C) Quantitative an ti-NR21 IgG subclass ELISA using the serum from S. mutans and MAb 5-5D IC-immunized Fcer1g transgenic mice. Sera from S. mutans and 5-5D IC-immunized Fcer1g knock-out mice were evaluated for the presence of NR21-specific IgG1, IgG2 a, and IgG2b subclass Abs. Anti-NR21 isotype concentrations were extrapolated from standard curves. Positive controls included binding of appropriate anti-P1 MAbs and negative controls included appropriate secondary only reagent wells. Th e means of duplicate wells at each serum dilution are represented by lin e graph and standard deviations are depicted by error bars. The data are representative of at least three independent experiments.
77 Figure 3-12. Evaluation of the role of th e Fc portion of anti-P1 MAbs on beneficial immunomodulation. Adherence inhib ition, MAb 1-6F competition, and NR21 reactivity by sera of F(ab)2 IC-immunized mice. High and Low Ab indicate that [saturating] and [0.1x sub-saturating] of MAb or F(ab)2 were used to coat S. mutans prior to immunization. (A) BiaCor e analysis of Inhibition of S. mutans adherence to salivary agglutinin via the serum of BA LB/c mice immunized with ICs of MAb 611A F(ab)2 and MAb 5-5D F(ab)2 versus S. mutans alone. (B) MAb 1-6F competition ELISA using the serum of BALB/c mice im munized with ICs of MAb 6-11A F(ab)2 and MAb 5-5D F(ab)2 versus S. mutans alone. ELISA plate wells were coated with S. mutans strain NG8 whole cells and inhibition of binding of biotin -labeled anti-P1 MAb 1-6F was measured. Positive and negative controls included inhibition by unlabeled MAbs 1-6F and 4-10A (not show n). The percent competition is depicted by bar graph and the standard error of the means observed between replicate wells are shown as error bars. Statistical significan ce is indicated by P values. The data are representative of at least three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova (C) Quantitative anti-NR21 IgG sub-class ELISA. Sera from S. mutans MAb 6-11A F(ab)2 and MAb 5-5D F(ab)2 immunized mice were evaluated for the presence of NR21-specific IgG1, IgG2a, and IgG2b subclass Abs. Anti-NR21 isotype concentrations were extrapolated from st andard curves. Positive controls included binding of appropriate anti-P1 MAbs and negative controls included appropriate secondary only reagent wells. The means of duplicate wells at ea ch serum dilution are represented by line graph and standard devia tions are depicted by error bars. The data are representative of at least three independent experiments.
78 Figure 3-12. Continued.
79 Figure 3-13. Re-evaluation of the immunomodulatory properties of MAb 4-10A. Adherence inhibition, MAb 1-6F competition, and anti-N R21 IgG quantitative ELISA using sera from MAb 4-10A IC-immunized mice. The x-ax is of all graphs are labeled with the dilutions of MAb 4-10A present within th e IC used to immunize mice. (A) BiaCore analysis of Inhibition of S. mutans adherence to salivary agglutinin via the terminal serum of BALB/c mice imm unized with ICs of MAb 4-10A at a 1:2000 to 1:32000 dilution and S. mutans alone. (B) MAb 1-6F competition ELISA using the serum (7day bleeds) of BALB/c mice immunized w ith ICs of MAb 4-10A dilutions versus S. mutans alone. ELISA plate wells were coated with S. mutans strain NG8 whole cells and inhibition of binding of biotin-labeled anti-P1 MAb 1-6F was measured. Positive and negative controls included inhibition by unlabeled MAbs 1-6F and 4-10A (not shown). The percent competition is depicted by bar graph and the standard error of the means observed between replicate well s are shown as error bars. Statistical significance is indicated by P values. (C ) Quantitative anti-NR21 IgG subclass ELISA. Sera (7-day bleeds) from S. mutans and MAb 4-10A IC-immunized mice were evaluated for the presence of NR21-specific IgG1, IgG2a, and IgG2b subclass Abs. Anti-NR21 isotype concentrations were extrapolated from standard curves. Positive controls included binding of appropria te anti-P1 MAbs and negative controls included appropriate secondary only reagent wells. The means of duplicate wells are represented by bar graph and standard de viations are depict ed by error bars. Statistical significance is indicated by P values The data are represen tative of at least three independent experiments. Statistical analysis was performed using graph pad prism 4.0 and analysis included one-way anova.
80 Figure 3-14. MAb 4-10A enhances the expos ure of the MAb 1-6F epitope. MAb 4-10A enhancement of MAb 1-6F binding to S. mutans whole cells by ELISA. Two-fold serial dilutions of MAb 4-10A were added to ELISA wells coated with S. mutans whole cells immediately followed by the addi tion of biotin labeled MAb 1-6F. The resulting level of MAb 1-6F bound to P1 on the surface of S. mutans whole cells in the presence of MAb 4-10A was then measured. The percent increase or decrease of biotin labeled MAb 1-6F binding was cal culated and the percent change in S. mutans binding by biotin labeled MAb 1-6F at each dilution of MAb 4-10A is represented by bar graph. Positive and negative controls included direct MAb 1-6F binding, unlabeled MAb 1-6F, and secondary reagen t only wells. The data are representative of multiple independent experiments.
81 Figure 3-15. Schematic represen tation of recombinant polypep tides encoded by P1 subclones used in proliferation assays.
82 Figure 3-16. Serum reactivity of immunized mice used in splenocyte prolif eration assays against recombinant P1 polypeptide A3VP1. (A) To tal anti-A3VP1 IgG in the serum from S. mutans immunized mice and 4-10A IC-imm unized mice and comparison between mice receiving one versus two injections. The OD 450 nm at each serum dilution is represented by line graph and the standard deviations between replicate wells are depicted by error bars. (B) Anti-A3VP1 quantitative subclass ELISA on the serum from S. mutans and MAb 4-10A IC-immunized mice receiving either one or two injections. Serum was taken the same day th e spleens were excised for proliferation assay and evaluated for the presence of A3VP1-specific IgG1, IgG2a, and IgG2b subclass Abs. Anti-A3VP1 isotype concentrations were extrapolated from standard curves. Positive controls included binding of appropriate anti-P1 MAbs and negative controls included appropriate secondary onl y reagent wells. The means of duplicate wells at each serum dilution are represented by line graph and sta ndard deviations are depicted by error bars. The data are representative of multiple independent experiments.
83 Figure 3-16. Continued.
84 Figure 3-17. Tritiated thym idine uptake and cytokine production by splenocytes from immunized mice stimulated in vitro with recombinant P1 polypeptide A3VP1. (A) Splenocytes from mice immunized with PBS (nave), S. mutans or MAb 4-10A IC either once (1X) or twice (2X) were stimulated in vitro with polypeptide A3VP1 and tritiated thymidine uptake was measured. S timulation index values were determined by dividing the cpm of stimulated cells by the cpm of unstimulated cells and are represented by bar graph. Con A stimulated cells (not shown) were included as a positive control in the assay. The data are repr esentative of triplicate experiments. (B) Measurement of INF-gamma and IL-10 produc tion in the culture supernatants of splenocytes from S. mutans and MAb 4-10A IC immunized mice stimulated in vitro with recombinant P1 polypeptide A3VP1. (C) Measurement of acid activated TGFbeta isoforms 1, 2, and 3 in the culture supernatants of splenocytes from S. mutans and MAb 4-10A IC immunized mice stimulated in vitro with recombinant P1 polypeptide A3VP1. The x-axis of the graphs are labeled with the immunizing source of the splenocytes and the P1 polypeptide used to stimulate the cells in vitro. Black bars represent cytokine levels from unstimul ated cells and rust colored bars represent cytokine levels from stimulated cells. Cytokine analysis was performed using multiplex bead assays on a luminex 100 system. Each measured value is a result of the average of a minimum of 50 data points from duplicate experiments (100 total) extrapolated from internal standard curves.
85 Figure 3-17. Continued.
86 Figure 3-17. Continued.
87 Figure 3-18. Evaluation of cytoki ne production in the culture supe rnatants of splenocytes from immunized mice stimulated in vitro with a panel of recomb inant P1 polypeptides. (A) Measurement of INF-gamma and IL-2 produc tion in the culture supernatants of splenocytes S. mutans and MAb 4-10A IC immunized mice stimulated in vitro with recombinant P1 polypeptides LT1, LT2, LT3, and NR21. (B) Measurement of acid activated TGF-beta isoforms 1, 2, and 3 in the culture supernatants of splenocytes from PBS, S. mutans and MAb 4-10A IC immunized mice stimulated in vitro with recombinant P1 polypeptides LT1, LT2, LT3, and NR21. The x-axis of the graphs are labeled with the immunizing source of the splenocytes and the P1 polypeptide used to stimulate the cells in vitro. Black bars represent cytoki ne levels from unstimulated cells and colored bars represent cytokine levels from stimulated cells. Cytokine analysis was performed using multi-plex bead assays on a luminex 100 system. Each measured value is a result of the aver age of a minimum of 50 data points from duplicate experiments (100 to tal) extrapolated from in ternal standard curves.
88 Figure 3-18. Continued.
89 Figure 3-19. Total antiS. mutans IgG and anti-NR21 IgG subclass reactivity in the serum from mice immunized with S. mutans and Guys 13 plantibody IC. Mice were immunized as described in the methods section and terminal serum samples from each group were tested for total antiS. mutans IgG content by standard ELISA and anti-NR21 IgG1, IgG2a, and IgG2b subclass Abs by quantitative ELISA. The numbers next to IC in the legends of the graphs represent the dilution of Guys 13 plantibody used to coat the bacterial ce lls prior to immunization. Anti-NR 21 isotype concentrations were extrapolated from standard curves. The means of duplicate wells at each serum dilution are represented by lin e graph and standard deviations are depicted by error bars. Positive controls incl uded binding of appropriate anti-P1 MAbs and negative controls included appropriate secondar y only reagent wells. The data are representative of at least th ree independent experiments.
90 Figure 3-20. Anti-A3VP1 IgG s ubclass reactivity in the serum from mice immunized with S. mutans and Guys 13 plantibody IC. Mice were immunized as described in the methods section and terminal serum sample s from each group were tested for antiA3VP1 IgG1, IgG2a, and IgG2b subclass Abs by quantitative ELISA. Anti-A3VP1 isotype concentrations were extrapolated fr om standard curves. The numbers next to IC in the legends of the graphs represent the dilution of Guys 13 plantibody used to coat the bacterial cells prio r to immunization. Means of duplicate wells at each serum dilution are represented by lin e graph and standard deviations are depicted by error bars. Positive controls incl uded binding of appropriate anti-P1 MAbs and negative controls included appropriate secondar y only reagent wells. The data are representative of at least th ree independent experiments.
91 CHAPTER 4 DISCUSSION AND CONCLUSIONS Overview Advances in dental care and caries preventi on have reduced the incidence of caries in developed countries but the incidence of caries worldwide has remained unchanged for the past two plus decades (147). This widespread infectio us disease is still prev alent in the U.S. and elsewhere, especially among people of low soci oeconomic status and education levels who do not have access to dental care ( 53, 149, 203). In developing countries the situation is even worse with an almost complete lack of dental care. Due to the persistence of caries in the human population, despite currently available prophylactic measures and treatments, an increased understandi ng of the correlates of protection and/or targets of prot ective immunity against S. mutans is needed. A better understanding of these factors would lead to more effective prev entative measures and may overcome the current lack of resources that is hinderi ng caries prevention and treatment. It was shown over forty years a go that the bacterial species that is the primary etiologic agent of dental caries in humans is S. mutans (71, 111). Since that time, a large number of studies have been undertaken in order to understand the factors involv ed in the virulence of this organism. The majority of these studies have focused on glucosyltransferases (GTFs), glucan binding proteins (GBPs), and antigen P1 (69, 131, 181, 182, 222). The virulence factor and vaccine candidate that was the focus of this study is the surface protein of S. mutans called Ag I/II (160) or P1 (56). P1 is a member of a family of structural ly complex cell-surface anchored multi-functional adhesins. Almost all species of oral streptococci indigenous to the human oral cavity produce P1-like polypeptides. P1 and P1-l ike polypeptides mediate interactions with
92 salivary constituents, host cell matrix proteins such as fibronectin, fibrinogen, collagen, and other oral bacteria (7, 86, 100). It is widely accepted that P1 functions on the cell surface in me diating adherence of S. mutans to the acquired pellicle of the tooth via a large molecular weight glycoprotein found in saliva, gp340 (22, 96, 106, 136). The in teraction of P1 with salivar y components has been shown to be complex (65) and different regions of P1 have been shown to be in volved in its interaction with human salivary agglutinin depending on whether it is in fl uid-phase or immobilized on a surface (22). Binding to immobilized agglutinin has been demonstrated by Scatchard plot analysis to involve two binding si tes (65). P1 interacts with both fluid-phase and immobilized SAG, but it is its interaction with immobilized SAG that is believed to contribute to pathogenesis and cariogenicity. The oral cavity and systemic circulation of humans has been shown to contain Abs against numerous oral microorganisms, including S. mutans (129, 131). Not only are IgA1 and IgA2 found in the oral cavity, but IgG and IgM are also detectable in human saliva, most likely via transudation through the gingival crevice (52). In one study serum antibodies to Ag I/II and S. mutans whole cells were examined in 108 subjects and the relationship to dental caries of isotype-specific antibodies to both antigens was determined (40). The results of this study demonstrated that serum IgG antibodies to Ag I/II and S. mutans were significantly greater in subjects with a low caries history than in subject s with a history of high caries. Furthermore, it was shown that serum IgM antibodies to Ag I/II and not S. mutans whole cells were correlated with a low incidence of caries while IgA antibodie s against either antigen were not related to caries incidence. Additionally Tenovuo, et. al re ported that high affinity serum IgG against S. mutans whole cells correlated with an absence of mutans streptococ ci in dental plaque (193).
93 Gregory et al. reported the importance of the specificity of the response with regard to caries sensitivity by showing that caries-susceptible individuals have significantly higher levels of salivary antibodies against S. mutans native antigens (63). In a subsequent study, Gregory et al. demonstrated that caries susceptibility correlated with higher Ab responses to S. mutans coated with saliva or altered by acid rather than to native S. mutans antigens (62). Another study reported that caries susceptible subjects had high levels of anti-Ag I/II salivary IgA against S. sanguis and S. sobrinus (80). However, Bratthall et al. s howed that the relationship between salivary IgA and dental caries is complex in th at low caries children had a less diverse reactivity to S. mutans and S. sobrinus antigens Ags while high caries children mounted a more diverse salivary IgA response (25). Taken together, the results of multiple studies indicate that much remains to be learned about the most relevant fe atures of protective anti bodies against dental caries, but specificity and isotype are clearly important factors. Even though humoral immunity against human dental caries has been reported for many years, S. mutans and many other pathogens are often able to persist in the presence of an immune response. As stated above, a definitive correlat e of protection against car ies at the epitope level has yet to be fully identified. Immunomodul ation by exogenously added MAb represents an often overlooked method of improving the effica cy of the immune response against a pathogen by shifting the response towards more protectiv e and potentially subdominant targets of protection. The overall goal of this study was to further defi ne MAb-mediated changes in antibody specificity and isotype and to gain an un derstanding of the mechanism(s) by which antiP1 MAbs modulate and improve the efficacy of the resultant immune response when they are administered as part of an IC in a BALB/c host.
94 Anti-P1 MAb 3-10E Recognizes a Co mplex Native-like P 1 Structure Previously, the binding sites of 11 anti-P1 MA bs were approximated based on reactivity with full-length and truncated P1 polypeptides (21, 47, 127, 155). MAbs 6-11A, 4-10A, and 55D all recognize epitopes depende nt on the presence of both the Aand P-regions, while 3-10E requires the P-region with binding substantially diminished in th e absence of the A-region (20, 166). The Aand P-regions of P1 interact a nd contribute to the formation of complex discontinuous and/or conformational epitopes (20, 155, 166, 199) and binding of this family of MAbs is reconstituted to varying degrees upon inte raction of the isolated Aand P-regions or longer P1 polypeptides including these domains The segment immediately upstream of the Aregion (a.a. 84-186) also contributes to the epitopes recognized by MAbs 6-11A and 5-5D and is requisite for recognition by MAb 3-10E (127, 155). All three of th ese MAbs promote beneficial immune responses (23, 84, 144, 156). In contrast, MAbs 4-9D and 1-6F promote the formation of antibodies less inhibitory of adherence when administered as part of an IC (144) and their cognate epitopes are contributed to by sequences contained in the intervening region between the Aand P-regions (127). Based on reactivity with full-length recomb inant P1 and lack of reactivity with recombinant P1 polypeptides CK1 and CK2, it was discovered that sequence C-terminal to the Pregion of P1 also contributes to the epitope recognized by MAb 3-10E (127). In the current study, a P1 sub-clone RR2 which encodes a re combinant polypeptide spanning amino acids 841218 was constructed in order to identify the pr imary amino acid sequence of P1 necessary to achieve the epitope recognized by MAb 3-10E. Polypeptide RR2 but no other P1 fragments tested was recognized by MAb 3-10E. Indeed, MAb 3-10E demonstrates the most complex epitope requirements in terms of intramolecular interactions between discontinuous segments of P1 of all the beneficial immunomodulatory MA bs studied to date. Results of a MAb 3-10E
95 competition ELISA using RR2, CK1, and CK2 suggest that P1 polypeptide RR2 achieves a more complete and native-like structure than shorter polypeptides lacking sequence amino-terminal to the A-region and C-terminal to the P-region. Immunomodulation by Anti-P1 MAbs Previous s tudies revealed that mucosal immunization of BALB/c mice with S. mutans coated with MAb 6-11A results in changes in the amount, isotype distribution, and specificity of serum antibodies compared with mice immunized with bacteria alone (23). Prior work in our laboratory demonstrated that changes in antibod y specificity in the se rum of MAb 6-11A ICimmunized mice were associated with changes in biological activity a nd suggested that the targets of biologically relevant antibodies involve complex epit opes not reconstituted by linear sequences of P1 (156). More recent studies de monstrated that parenteral immunization of BALB/c mice with IC consisting of S. mutans coated with other anti-P1 MAbs also results in alterations of the polyclonal imm une response in the serum of t hose mice when compared to the serum of mice immunized with S. mutans alone. When used as part of an IC to immunize mice, MAbs 6-11A, 3-10E, and 5-5D were shown to redirect the serum re sponse toward one of increased ability to inhibit S. mutans adherence to immobilized SAG, as measured by SPR, while MAbs 1-6F and 4-9D redirected the serum response toward a decreased ability to inhibit adherence (84, 144). Oli, et al. also demonstrat ed that MAbs 6-11A, 3-10E, 1-6F, 4-9D, and 410A influenced murine anti-P1 serum antibody responses in terms of subclass distribution and/or specificity but that the effects differed based on which MAb was used an d the concentration of the MAb used within the IC (144) Furthermore, this same study re vealed an inverse relationship between the ability of the MAbs to inhibit adherence and the abil ity of the MAbs to promote the formation of a serum Ab response better able to inhibit adherence. In addition, statistically
96 significant correlations were demo nstrated between the levels of anti-P1 serum IgG2a and IgG2b and the ability of sera from immunized animals to inhibit bacterial adherence. Results of the current study revealed that not only are MAbs 6-11A, 5-5D, and 3-10E beneficial immunomodulators but that MAb 4-10A also exhibits beneficial rather than neutral immunomodulatory characteristics. However, the ability of MAb 4-10A to modulate the immune response toward one of increased efficacy is strongly dependent upon the concentration of MAb used to coat the bacterial cells. The sera from MAb 6-11A, 5-5D, and 3-10E IC-immunized mice and, subsequently MAb 4-10A IC-immunized mice were shown to exhibit a ch ange in specificity towards MAb 1-6F-like epitopes an d those sera were increased in IgG2a and/or IgG2b subclass Abs, the isotypes previously linked with enha nced adherence inhib ition (144). Overall the current data are consistent with earlier studies by our laboratory with the exception that MAb 410A is itself an inhibitor of adherence. The ability of MAb 4-10A to inhibit S. mutans adherence to immobilized SAG is speculated to be due to steric effects as this MAb does not bind to the region of P1 that is now suspect ed to contribute to binding of S AG or that is recognized by the adherence inhibiting MAbs 4-9D and 1-6F, altho ugh these epitopes likely lie in close proximity in the folded structure. Beneficial Immunomodulation is Dependent on the Geneti c Background of the Host An epitope scanning approach evaluating T cell a nd linear B cell epitopes revealed that the response to P1 varied depending on the MHC II ha plotype of the murine host (191). This study revealed that subcutaneous immunization of mi ce carrying the MHC class II I-Ad gene with a peptide corresponding to amino acids 301 to 319 induced strong serum IgG responses to recombinant P1 (rP1) and the peptide itself. I mmunization of mice carrying the haplotype k or b of the H-2 I-A gene with the same peptide induced intermediate serum IgG responses to rP1 and the immunizing peptide whereas, mice carrying th e haplotype s or q of the H-2 I-A gene
97 mounted a weak serum IgG respons es to rP1 and the 301-319 peptid e. Furthermore, the authors mapped antigenic epitopes in the P1 301-319 pep tide and P1 in mice bearing different H-2 haplotypes by using 10 overlapping decapep tides covering P1 residues 301-319 and 153 decapeptides covering the entire mature P1 prot ein and demonstrated that antigenic epitope patterns differed in mice with different H-2 haplotypes. The beneficial effects of MAbs 6-11A, 3-10E 5-5D, and 4-10A appear to relate to a common epitope requirement that depends upon an interaction of the discontinuous Aand Pregions of P1. If, as suggested by changes in the specificity of the subsequent immune response, these MAbs act in some way as to expose diffe rent epitopes that prov ide better targets of protective antibodies, one would predict the immunomodulatory effect to vary depending on the MHC II haplotype and genetic background of the hos t. Any perturbation of P1 structure would be expected to lead to change s in antigen processing and presen tation, the subset of stimulated T helper cells, and the ultimate B cell response. A study evaluating naturall y sensitized cariesresistant compared to caries-sensitive human subjects revealed a correlation to an S. mutans Ag I/II T cell epitope, despite prot ection against caries being antibody rather than cell-mediated. (93). In this study no correlati on was found between caries levels and B cell epitopes; however, only antibody responses against simple linear epitopes were evaluated. Presumably, in cariesresistant individuals a more effective antibody response against more complex and relevant epitopes was facilitated by the pa rticular helper T cell specificity iden tified in the study. The stimulation of particular T cell subsets is remarkably dependent on minor alterations in an Ag. Alteration of a single amino acid residue w ithin tetanus toxin C fragment has been shown to substantially alter antigen processing and lead to changes in presentation of numerous T cell epitopes (4). Ab can also influence the processing and presentation by MHC Class II of the
98 processed Ag with a subsequent alteration in the adaptive T helper cell response (134, 173, 205, 206). In fact, it has been shown th at binding of tetanus toxin by antibodies dramatically affects the proteolytic processing of the Ag and protects or footprints specific domains of the antigen during processing which results in modulation of loading of class II MHC-restricted T cell epitopes (205). Furthermore, processing and clas s II MHC loading of some T cell epitopes within the footprinted region was hindered but presenta tion of other T cell epitopes were boosted. Our immunomodulatory MAb 6-11A has been shown to alter the proteolytic se nsitivity of P1 on the bacterial cell surface in vitro with more complete and rapid digestion upon treatment with a variety of proteases suggesting destabilizat ion of P1 structure upon Ab binding (156). The current study tested and confirmed th e importance of host genetic background in beneficial immunomodulation by an ti-P1 MAbs. Serum from BALB/c mice immunized with IC of MAbs 6-11A and 5-5D both demonstrated an in creased ability to inhibit bacterial adherence compared to the serum from S. mutans -immunized BALB/c, but the serum from IC-immunized C57BL/6 mice was not increased in its ability to disrupt bacterial adhe rence to immobilized SAG. The kinetics and isotype distribution of the anti-S. mutans response was evaluated in both strains of mice. Results rev ealed that IC-immunization in BALB/c, but not C57BL/6, mice results in an early rise (7 days post-primary immunization) in antiS. mutans IgG1, IgG2a, and/or IgG2b. Lastly, changes in the specificity of the anti-P1 response were observed in the serum from IC-immunized BALB/c mice in that these sera demonstrated an increased ability to compete for S. mutans binding with a MAb known to be an inhibitor of adherence, 1-6F. However, biotin labeled MAb 1-6F competiti on was not observed in the serum from ICimmunized C57BL/6 mice. These results dem onstrate the genetic background of the host contributes in large part to the beneficial outcome defined in BALB/c mice resulting from
99 immunization with IC containing immunomodulatory anti-P1 MAbs and is consistent with the hypothesis that serological changes stem from changes in antigen processing and subsequent presentation of different peptides by MHC II molecules. Beneficial Immunomodulat ion is Fc-Independent The immune response toward an antigen varies when it is encountered in the context of an IC versus the antigen alone (18). There ar e several known m echanis ms by which antibody can modulate the host immune response when it is b ound to its specific Ag as part of an IC. Alterations of the host immune response can stem broadly from Fc-dependent and Fcindependent mechanisms. However, these mechan isms are not necessarily mutually exclusive and could well overlap. Fc-dependent mechan isms include increased uptake by antigen presenting cells via FcRs and/or differential engagement of stimulatory versus inhibitory FcR on antigen presenting cells (59, 75, 78, 102, 122, 174, 211). Additionally, Ab-mediated complement activation with subsequent uptake of Ag via co mplement receptors can be considered an Fcdependent mechanism (78, 83, 102, 120, 190). Fc-independent mechanisms include, for example, Ab masking of dominant antigen ic epitopes, exposure of crypt ic epitopes revealed upon Ab binding, and/or changes in proteolysis which lead s to changes in Ag presentation (2, 4, 5, 8, 9, 12, 97, 110, 120, 122, 173, 205). Recent studies in our laboratory showed th at neither opsonophagocytosis nor complement activation is directly involved in the immunomodu latory mechanism of th ose anti-P1 MAbs that redirect the response toward one of increased efficacy (84). Even though IgG1 has been reported to be a non-complement activating isotype in mice, exceptions can be found and binding of MAbs 5-5D and 1-6F to S. mutans cells in the presence of comple ment resulted in C3 deposition on the bacteria. However, there was no obvious para llel of complement ac tivation with observed immunomodulatory effects. Inte restingly, an inverse correlation between adherence inhibition
100 and opsonization was observed with beneficial immunomodulato ry MAbs 6-11A, 5-5D, and 310E being non-opsonic and adhe rence inhibiting MAbs 4-9D 1-6F, and 4-10A promoting bacterial uptake and killing by a macrophage cell line. Results of the current study demonstrate th at beneficial immuno modulation by anti-P1 MAbs is independent of activ ating FcRs. Immunization of Fcer1g transgenic BALB/c mice with IC containing MAb 5-5D resulted in the same immunomodulatory outcome s as those observed in IC-immunized wild-type BALB/c. Demonstrated changes in the serum of IC-immunized mice versus S. mutans -immunized mice included an increased abil ity to inhibit bact erial adherence, increased competition with MAb 1-6F, and increased levels anti-NR21 IgG subclass Abs. Numerous studies have suggested that Fc-mediated mechanisms may be the predominant way that Ab modulates the immune response (60, 75, 78). Although Fc-dependent pathways have been presumed to represent the major mech anism of antibody-mediat ed modulation of the immune response, the results of studies by our laborat ory suggest that this is not always the case. Results of the current study fu rther substantiate that complement activation and/or Fc receptors are not the primary mechanism of be neficial immunomodulation by anti-P1 MAbs in that the Fc region of the MAb is not required to mediate the e ffects. Immunization experiments carried out using pepsin-digested F(ab)2 fragments of MAbs 6-11A a nd 5-5D in place of intact IgG in the IC revealed that the serum from F(ab)2-immunized mice exhibited parallel immunomodulatory changes as t hose observed with immunization of IC containing whole IgG. Sera of F(ab)2 IC-immunized mice versus S. mutans -immunized mice demonstrated an increased ability to inhibit bacterial adherence, increased competition with MAb 1-6F, and increased levels anti-NR21 IgG subclass Abs. Taken together with the results of the st udy by Isoda, et al. (84),
101 the findings of the current study demonstrate indepe ndence of direct effect s of the Fc portion of the MAb and FcRs on beneficial im munomodulation by anti-P1 MAbs. Enhanced Epitope Exposure Explains Beneficial I mmunomodulation With Fc-dependence ruled out as being involved in beneficial i mmunomodulation, it is apparent that a common denominator among th e anti-P1 MAbs that promote a desirable adherence inhibiting antibody response is shared features of their cognate epitopes. MAbs 611A, 5-5D, 3-10E, and 4-10A recognize comple x conformational determinants involving an interaction between the discontinuous Aand P-regions and/or segments flanking these domains. This implies that the mechanism responsible for their redirection of the immune response involves the nature of the Ag-Ab interaction itself and requires binding that is dependent on structural features of the adhe sin. P1 binding by three of the f our A/P-dependent MAbs (6-11A, 5-5D, and 3-10E) also involves a contribution of pr e-A region sequence (amino acids 84-190). Elimination of this region substantially increas es the binding of the a dherence inhibiting MAb 16F even though MAb 1-6F maps to the interven ing region between the Aand P-regions (amino acids 465-679) (127). Crystallographic information suggests this segment forms a domain that is spatially separated by at least 155 angstroms from sequence N-term inal to the A-region (103). A schematic representation of the A3VP1 pol ypeptide (Figure 3-15) based on its solved crystal structure is shown in Figure 4-1, panel A. The protein structure rev ealed an association of the first tandem repeat of th e proline-rich domain forming an unusually long single polyproline type II helix wrapped with the third tandem repeat of the alan ine-rich domain as an extended alpha-helix. The interaction was shown to be mediated primarily through hydrophobic interactions and isothermal titration calorimetri c studies showed both high binding affinity and high enthalpy changes and a stable 1:1 complex. This is consistent with previous results from our laboratory (166) that demonstrat ed a 1:1 stoichiometry of the A-region and P-region interaction.
102 It is speculated but currently unclear whether the interaction of the complete A-region with the complete P-region would form a single exte nded helix. It appears likely from epitope mapping experiments using additivity ELISA (127) and experiments in the current study with RR2 that pre-A-region sequence interacts with post-P-region sequence to form the cognate epitope of MAb 3-10E. Based on this informati on and binding characteristics of other anti-P1 MAbs a model of the structure of cell surface localized P1 is proposed (Figure 4-1, panel B). In this model the adherence inhibiting MAbs 4-9D a nd 1-6F bind on one side of the extended helix while MAbs 5-5D, 6-11A, and 3-10E that do not inhibit adherence bind on the other. Guys 13 and MAb 4-10A are both known to be fully reconstitu ted by an interaction of the isolated Aand P-regions without a contribution of flanking sequence (127, 199). The consensus sequence of residues reported to contribute to the Guys 13 epitope is repeated multiple times throughout the P-region (200). Work in our laboratory has identified that MAb 4-10A binding is reconstituted when a single proline-rich repeat is reintroduced into a P-region-deletion construct of P1 (48). Therefore, it is believed that MAb 4-10A also recognizes a repeated epitope. Hence, Guys 13 and 4-10A are illustrated as binding at multiple sites along the helical structure formed by the interacting Aand P-regions. Binding of immunomodulatory MAbs 6-11A, 5-5D, and 3-10E may act to influence P1 structure and expose the 1-6F epitope in a manne r similar to removal of pre-A-region sequence (127). A relationship exists between the abil ity of serum from mi ce immunized with ICs containing MAbs 6-11A, 5-5D, 3-10E, and 4-10A to compete with biotin-l abeled MAb 1-6F for binding to S. mutans with the presence of anti-NR21 specifi c IgG1, IgG2a and/or IgG2b, and to inhibit bacterial adherence to immobilized SAG. The induction of anti-NR21 Abs of the IgG2a
103 and IgG2b subclass is consistent with the previously observed stat istical correlation of adherence inhibition and anti-P1 serum IgG2a and IgG2b (144). Collectively, these data suggest that bindi ng of beneficial imm unomodulatory MAbs, or structural disruption of key elements of th eir epitopes, increases the antigenicity and immunogenicity of other epitopes that are more re levant targets of protec tive antibodies. Western blot data (Figure 3-8) demonstr ates that certain epitopes may be more or less accessible within the context of the whole molecule. The most convincing evidence that immunomodulation is linked to a structural perturba tion of P1 on the cell surface is that MAb 4-10A increases the binding of biotin-lab eled MAb 1-6F to S. mutans whole cells at the sa me dilutions shown to promote MAb 1-6F-like responses in the serum of IC-immunized mice. Immunomodulatory MAbs Affect the Kinetics of the AntiS. mutans Immune Response For m ost of the immunomodulation experiment s with anti-P1 MAbs, serum samples were collected only at the termination of the expe riments. Based on the in teresting findings of alterations in the relative levels of different IgG subclasses in terminal bleeds reactive with S. mutans and later with particular P1-derived polype ptides which was suggestive of an influence on Ab class switching, as well as published reports that immunization with an IC can promote germinal center formation (78, 88, 94, 99, 152, 188) an interest developed in whether the presence of anti-P1 MAbs in the IC used for immunization affected the kinetics of the subsequent humoral immune respons e. Therefore, collection of interim serum samples at 7 days post-primary immunization was begun in order to asses the kinetics of the response in immunized mice, although volumes of these sa mples were limited and a full battery of experiments was not possible. Primary immunization (one inje ction) with IC containing MA b 6-11A resulted in a 7-day response that was increased in antiS. mutans IgG2a, IgG2b, and IgG1. In the case of MAb 5-5D,
104 antiS. mutans IgG2a and IgG2b were increased in IC -immunized mice while IgG1 was similar in all groups (Figure 3-5). Similar to the resu lts with MAb 6-11A, primary immunization with MAb 4-10A IC resulted in increased levels of all three isotypes, IgG1, IgG2a, and IgG2b when P1 polypeptide NR21 was used as the antigen in ELISA assays (Figure 3-13). Taken together, these results demonstrate that there is not only an early appearance of antibodies against S. mutans in general, but also of an tibodies reactive with specific P1 polypeptides now known to achieve epitopes recognized by anti-P1 MAbs capable of inhibiting bacterial adherence to SAG. In further support of this conclusion, the sa me 7-day serum samples from MAb 4-10A ICimmunized mice were increased in their ability to compete with the adherence inhibiting MAb 16F for S. mutans binding over those from S. mutans -immunized mice (Figure 3-13). These results are of interest because it is often pr esumed that because the gamma 1 constant region gene segment is most upstream of all the gamma gene segments, that IgG1 is the first IgG isotype expressed following exposure to Ag. While IgG1 has been shown to be the predominant isotype of antiS. mutans IgG in the terminal serum samples of S. mutans and IC-immunized mice, the results of the current study suggest th at other more biologically efficacious isotypes may be induced early in the response even before a disproportionate rise in the level of serum IgG1 is obvious. Immunization with IC has been shown previ ously by others to a lter the magnitude and kinetics of the immune response and it has been suggested that immunization with IC can mimic a secondary response. A study by Gustavsson, et al. demonstrated that mice immunized with IgE/2,4,6-trinitrophenylated bovine serum albumin IC (IgE/TNPBSA) exhibited an antigen specific serum immune response that was seve ral hundred-fold higher in magnitude than was observed in mice immunized with Ag alone (64) A subsequent study measured the up-regulated
105 anti-TNP-BSA response as the number of Ab-secreting B cells in the spleens of IgE/Ag ICimmunized mice and demonstrated at least a 500-fo ld increase of specific IgG-secreting spleen cells which peaked only 6 days after primary i mmunization (212). The aut hors suggest that such a rapid increase in the number of IgG-secreting cells in the spleens of th e mice was a result of IgE/Ag IC induction of a seconda ry-like antibody response in th e absence of and without the requirement for conventional priming. Consistent with this speculation, re sults of the current study suggest that immunization with IC containing beneficial immunom odulatory anti-P1 MAbs may induce a primary immune response that is similar in nature to a prim ed secondary response. Additionally, the resu lts suggest a MAb-mediated influence on cellular interactions and resultant cytokine profiles that affect th e serological response as is ev idenced by the early and continued presence of specific IgG2a and IgG2b in the serum of IC-immunized mice. Detection of Immunomodulatory Ac tivity a t the Cellular Level Whether initiated by an Fc-depe ndent or independent mechanism, presence of Ab with an Ag can lead to alterations in proteolysis and antigen processing (5, 120, 122, 173, 205), a shift in presentation of class II-restricted T-cell epitopes (3, 4, 110), cha nges in cytokine expression by antigen-presenting cells and/or T cells (2, 3, 10, 11), enhanced germinal center formation and generation of strong memory responses (78, 88, 94, 97, 99, 152, 188), changes in usage of germline-encoded VH genes (138, 189), and induction of somatic hypermutation (138, 188, 189). Immunization with ICs contai ning anti-P1 MAbs 6-11A, 3-10E 5-5D, and 4-10A leads to an increased response against at l east one P1 epitope that is not r eadily exposed in the context of the purified full-length protein. Also, MAb 6-11A was previously shown to alter the proteolytic digestion of P1 (155). These data sugges t that Ag binding by the four beneficial immunomodulatory MAbs induces a structural perturbation in cell surface localized P1 which could lead to changes in proteo lytic digestion of the molecule within APCs and, hence, an
106 alteration in peptides available for display by MHC II molecules to CD4+ helper T cells. Consistent with this speculat ion it was found that IC immuni zation in mouse strains with a different MHC II haplotype results in different immunomodulatory outcomes. One would expect that cha nges in epitope display by AP Cs would be reflected in proliferation assays of immune cells ha rvested from IC-immunized compared to S. mutans immunized mice (3, 4, 110). As expected, results of splenocyte proliferation assays against P1 polypeptide A3VP1 demonstrated that the prolif erative response differed between splenocytes derived from mice immunized with MAb 4-10A IC and splenocytes obtained from S. mutans immunized mice. The response to A3VP1 is relevant in that this polypeptid e is recognized by all three MAbs that directly inhibit bacterial a dherence to immobilized SAG. Consistent with serological reactivity against A3VP1 from the same mice, primary immunization with IC enhanced the in vitro proliferative response of splenoc ytes against A3VP1 compared to splenocytes from S. mutans -immunized mice. However, when the proliferative response against A3VP1 was assessed in splenocytes derived from mice immunized with two doses of immunogen, an anergic-like response was obser ved in the splenocytes from mice immunized with MAb 4-10A IC compared to those from S. mutans -immunized mice. These results are not understood at this time, but it is clear there are differences among groups of mice receiving different numbers of imm unizing doses of immunogens. Other immunomodulatory changes that can occur by immunization with immune complexes include alterations in the cytokine response of APCs and/or T cells (2, 3, 10, 11). Analysis of cytokine levels in the culture supernatants of splenocytes stimulated in vitro with a panel of recombinant P1 polypeptides revealed a number of parallels with observed proliferation data and serological responses of immunized mice. While the effects of cytokines and the
107 interplay of the ratios of cytokines relative to one another are complex and not fully understood, the published literature provides so me insight into certain functi ons that influence the humoral immune response. Interferon gamma (IFN-gamma) has been implicated in Ab class switching to IgG2a, interleukin 4 (IL-4) has been implicated in class switching to IgG1 and IgE, and tumor growth factor beta isoform 1 has been implicat ed in class switching to IgG2b and IgA (50, 184, 185) Levels of IFN-gamma in the culture supern atants of splenocytes from MAb 4-10A ICimmunized mice stimulated with A3VP1 and NR21 were increased over the levels observed in stimulated splenocytes from mice immunized with S. mutans only. These data are consistent with the observed increase in anti-A3VP1 IgG2a a nd anti-NR21 IgG2a in the serum of MAb 4-10A IC-immunized mice. Also, in parallel with the aner gy-like state of splenocyt es derived from mice that had been immunized and also boosted with IC against A3VP1, the levels of IL-10 in the supernatants from these splenocyte cultures were decreased. Deenick, et al. (50) have reported a role fo r TGF-beta 1 in cla ss switching to IgG2b although there was no observable difference in the level of TGF-beta 1 in the culture supernatants from splenocytes derived from S. mutans compared to MAb 4-10A IC-immunized mice. In the current study the data suggest that as the complexity of the stimulating polypeptide antigen increases (Figures 3-17C an d 3-18B) that there is a decrease in TGF-beta 3 in the culture supernatants of splenocytes from the IC immuni zed mice. This suggests that the ratio of TGFbeta isoforms may be critical as a decrease in TGFbeta 3 appears to correlate with an increase in desirable outcomes. It is possible, therefore, that TGF-beta 3 coul d act as a negative regulator of TGF-beta 1 in that in the absence of TG F-beta 3 a desirable outcome is enabled.
108 The results of the current study begin to provide a foundation for a better understanding and design of future experiments to measure effect s that occur at the cellu lar level as a result of immunization with IC containing anti-P1 MAbs. Furt hermore, they demonstrated that differences in cytokine expression which result from anti-P1 MAb-mediated immunomodulation are consistent with the published literature a nd observed changes in serum Ab isotypes. Guys 13 Plantibody Exhibits Immunomodulatory Characteristics Previously it was reported that local adm inistration of an anti-Ag I/II MAb, Guys 13, to the tooth surface of human volunteers after treatment with chlorohexidine prevented recolonization of S. mutans for up to two years (117). Further studies using Guy's 13 demonstrated prevention of S. mutans re-colonization by administration of F(ab)2 fragments of the MAb but the Fab fragments of the molecule failed to pr event re-colonization. In terestingly, protection against colonization by S. mutans lasting up to 2 years was observ ed in immunized subjects even though MAb was only applied over a period of 3 w eeks and was only detectable up to 3 days after its application to the teet h (115). A subsequent study demons trated that favorable results were linked to MAb epitope specificity (116). MA b Guys 13 was later re-engineered in tobacco plants as a chimeric IgA/IgG with a rabbit se cretory component (118) and is now generally referred to as Guys 13 plantibody. Subsequent human trials usi ng Guys 13 plantibody demonstrated its ability to prevent S. mutans re-colonization for up to four months (114). However, a human clinical trial repeated at a different institution fa iled to demonstrate the efficacy of Guys 13 plantibody at the concentrations tested (209). The mechanism(s) by which passively admini stered Guys 13 and Guys 13 plantibody prevented S. mutans re-colonization in some studies fo r such a long time has never been satisfactorily explained. It was speculated that S. mutans re-colonization was inhibited by other
109 species of oral bacteria displacing S. mutans and occupying its niche within the oral cavity but no differences in numbers of other bact erial species were demonstrated. It has been shown that, like our anti-P1 MAbs Guys 13 recognizes a complex epitope that is dependent upon an interaction between the disc ontinuous Aand P-regions of P1 (199). Also, it has been previously documented that immunomodulation by benefici al anti-P1 MAbs is dependent on the concentration of MAb used to coat the bacteria. The effects of MAb 4-10A were notably concentration dependent and a prozone-like phenomenon was evident. Guys 13 plantibody is similar to MAb 4-10A in that they both require only an interaction of the discontinuous Aand P-regions of P1 for MAb binding, but they are not identical. While MAb 410A is a direct inhibitor of b acterial adherence, interestingl y, Guys 13 plan tibody, like MAbs 611A, 5-5D, and 3-10E, does not inhibit adherence. In fact, it enhances adherence as measured by our BiaCore assay (Brady lab unpublished data). Again this suggests a MAb-mediated perturbation of P1 on the cell surface resulting in a measurab le alteration of P1 function, antigenicity, and immunogenicity. Based on similarities with our anti-P1 MAbs and the lack of a satisfactory explanation for its long-term effects when used as a passive immunotherapy, it was theo rized that Guys 13 may possess immunomodulatory propertie s. Indeed, results of muri ne immunization experiments using IC containing Guys 13 plantibody reveal ed similar immunomodulatory changes in the serum of Guys 13 plantibody IC-i mmunized mice as were observed with other anti-P1 MAbs and a prozone-like effect was observed. Sera from mice immunized with Guys 13 plantibody IC containing intermediate concentrations of MAb de monstrated increased levels of anti-NR21 and anti-A3VP1 IgG1, IgG2a, and/or IgG2b versus the sera from S. mutans -immunized mice.
110 If Guys 13 plantibody was able to confer long-term protection from S. mutans recolonization by an immunomodulat ory mechanism, a source of S. mutans would have to have been present in order for IC to be formed in the oral cavity of MAb treated subjects. Indeed, prior reports indicated that S. mutans began to appear in sham-treat ed patients during the window of time when MAb was still being applied to the teet h of treated subjects. Of potential consequence is that human subjects in the successful clinical trials were dental stude nts who would have been exposed to a potential source of S. mutans via patient contact whereas subjects in the unsuccessful repeated trial were predominantly middle-aged women and we re not recruited from a population of dental practitioners (Wycoff, Planet Biotech Inc., personal communication). The results of the current study support previous findings in ou r laboratory that beneficial immunomodulation by anti-P1 MAbs is linked to commonalities in their c ognate epitopes. Also the current study suggests that the results of previous studies usi ng Guys 13 or Guys 13 plantibody may be difficult to repr oduce in that its eff ects appear to be concentration dependent. The results of the current study provide a longsought explanation for the observed long-term effects of passively administered Guys 13 a nd Guys 13 plantibody and may also provide an explanation for the observed negative results in the repeated clinical trials in that when desired outcomes were not observed more MAb was app lied (Wycoff, Planet Biotech Inc., personal communication). Summary and Significance The goal of this study was to gain a better understanding of the m echanism(s) involved in beneficial immunomodulation by an ti-P1 MAbs. Also, a greater insight into the epitopes and characteristics of adherenceinhibiting antibodies against S. mutans was sought and gained. This study demonstrated that certain MAbs mediate a desirable affect upon the subsequent immune response towards their cognate Ag. The data support a mechanism of action by beneficial
111 immunomodulatory anti-P1 MAbs that is Fc-independent, influenced by the genetic background of the host, results in alterations in cellular inte ractions and cytokine prof iles, and is likely linked to a structural perturbation of the P1 Ag on the bacterial surface le ading changes in its immunogenicity and antigenicity as manifested by measurable differences in the kinetics, specificity, isotype and adherence-inhibi ting capability of antibodies against it. Numerous studies have demonstr ated immune protection against S. mutans colonization and dental caries in animal models and naturally-sensitized human subjects; however, an established correlate(s) of protection that has been reduced to pr actice in the form of an effective vaccine remains elusive. The current study provi des information on a method to manipulate the immune response toward the induction of anti-P1 an tibodies with a more de sirable characteristic, i.e. inhibition of S. mutans adherence to its known physiologi cal ligand, and may represent a therapeutic modality that can improve our ability to combat the widespread infectious disease of human dental caries. In addi tion, the information gained regarding the interactions of immunomodulatory as well as elicited antibodie s with components of the P1 antigen that contribute to a complex higher order struct ure suggest that immuni zation with immune complexes in an animal model can also be used as screening tool to learn qualities of an effective immune response and to guide re-engineering of th e antigen itself in such a way as to improve the response without th e need for immunization with immu ne complexes. The approach of antibody-mediated immunomodulation e ither as a direct therapy or guide to an effective immune response may be broadly applicab le to numerous pathogens beyond S. mutans for which vaccines are not yet available and that share the common charac teristic of being able to persist in the face of an ineffective immune response.
112 Figure 4-1. Structural models of P1. (A) Sc hematic representation of P1 polypeptide A3VP1 based on its solved crystal structure (103) (B) Predicted model of full-length P1 based on the crystal structure of A3VP1 a nd regions within the primary amino acid sequence known to interact and/or cont ribute to epitopes recognized by anti-P1 MAbs. NT (amino-terminal region), CT (carboxy-terminal region), and CW (cell wall).
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BIOGRAPHICAL SKETCH Rebekah Ann Robinette was born o n Decem ber 12, 1976 at Baptist Hospital in Little Rock, Arkansas. She grew up in Benton, Arkansas and remained in the state until she was 22 years old. Rebekah was a graduate of a home school program in February 1994. In the fall of 1996, she began her undergraduate studies at Garland County Community College (now known as National Park Community College) in Hot Sp rings National Park, Arkansas and received an A.S. degree in Medical Laborat ory Technology in May 1999. Rebekah received a scholarship to attend High Point University and moved to High Point, North Carolina in August 1999. She earned her B.S. in biology in May, 2001 and remain ed in the area to continue her career at MWG Biotech. In August 2002, Rebekah moved to Gainesvill e, Fl to join the Interdisciplinary Program in Biomedical Sciences at UF. During this time, she joined the Immunology and Microbiology concentration and ultimately joined the laborat ory of Dr. Brady in the Department of Oral Biology, UF College of Dentistry. Rebekah is curre ntly continuing her trai ning as a post-doctoral fellow at UF. Her future plans are to pursue a research-based career in academia or industry.