Abundance Of Epitopes Research Articles

Variable content and distribution of arabinogalactan proteins in banana (Musa spp.) under low temperature stress.

Information on the spatial distribution of arabinogalactan proteins (AGPs) in plant organs and tissues during plant reactions to low temperature (LT) is limited. In this study, the extracellular distribution of AGPs in banana leaves and roots, and their changes under LT stress were investigated in two genotypes differing in chilling tolerance, by immuno-techniques using 17 monoclonal antibodies against different AGP epitopes. Changes in total classical AGPs in banana leaves were also tested. The results showed that AGP epitopes recognized by JIM4, JIM14, JIM16, and CCRC-M32 antibodies were primarily distributed in leaf veins, while those recognized by JIM8, JIM13, JIM15, and PN16.4B4 antibodies exhibited predominant sclerenchymal localization. Epitopes recognized by LM2, LM14, and MAC207 antibodies were distributed in both epidermal and mesophyll cells. Both genotypes accumulated classical AGPs in leaves under LT treatment, and the chilling tolerant genotype contained higher classical AGPs at each temperature treatment. The abundance of JIM4 and JIM16 epitopes in the chilling-sensitive genotype decreased slightly after LT treatment, and this trend was opposite for the tolerant one. LT induced accumulation of LM2- and LM14-immunoreactive AGPs in the tolerant genotype compared to the sensitive one, especially in phloem and mesophyll cells. These epitopes thus might play important roles in banana LT tolerance. Different AGP components also showed differential distribution patterns in banana roots. In general, banana roots started to accumulate AGPs under LT treatment earlier than leaves. The levels of AGPs recognized by MAC207 and JIM13 antibodies in the control roots of the tolerant genotype were higher than in the chilling sensitive one. Furthermore, the chilling tolerant genotype showed high immuno-reactivity against JIM13 antibody. These results indicate that several AGPs are likely involved in banana tolerance to chilling injury.

Pectic-β(1,4)-galactan, extensin and arabinogalactan-protein epitopes differentiate ripening stages in wine and table grape cell walls.

Cell wall changes in ripening grapes (Vitis vinifera) have been shown to involve re-modelling of pectin, xyloglucan and cellulose networks. Newer experimental techniques, such as molecular probes specific for cell wall epitopes, have yet to be extensively used in grape studies. Limited general information is available on the cell wall properties that contribute to texture differences between wine and table grapes. This study evaluates whether profiling tools can detect cell wall changes in ripening grapes from commercial vineyards. Standard sugar analysis and infra-red spectroscopy were used to examine the ripening stages (green, véraison and ripe) in grapes collected from Cabernet Sauvignon and Crimson Seedless vineyards. Comprehensive microarray polymer profiling (CoMPP) analysis was performed on cyclohexanediaminetetraacetic acid (CDTA) and NaOH extracts of alcohol-insoluble residue sourced from each stage using sets of cell wall probes (mAbs and CBMs), and the datasets were analysed using multivariate software. The datasets obtained confirmed previous studies on cell wall changes known to occur during grape ripening. Probes for homogalacturonan (e.g. LM19) were enriched in the CDTA fractions of Crimson Seedless relative to Cabernet Sauvignon grapes. Probes for pectic-β-(1,4)-galactan (mAb LM5), extensin (mAb LM1) and arabinogalactan proteins (AGPs, mAb LM2) were strongly correlated with ripening. From green stage to véraison, a progressive reduction in pectic-β-(1,4)-galactan epitopes, present in both pectin-rich (CDTA) and hemicellulose-rich (NaOH) polymers, was observed. Ripening changes in AGP and extensin epitope abundance also were found during and after véraison. Combinations of cell wall probes are able to define distinct ripening phases in grapes. Pectic-β-(1,4)-galactan epitopes decreased in abundance from green stage to véraison berries. From véraison there was an increase in abundance of significant extensin and AGP epitopes, which correlates with cell expansion events. This study provides new ripening biomarkers and changes that can be placed in the context of grape berry development.

The Influenza Virus–Specific CTL Immunodominance Hierarchy in Mice Is Determined by the Relative Frequency of High-Avidity T Cells

Virus-specific CTL responses typically fall into reproducible hierarchies with particular epitopes eliciting either immunodominant or subdominant responses after viral challenge. The recently acquired capacity to directly enumerate naive CTL precursors (CTLps) in both mice and humans has implicated CTLp frequency as a key predictor of immune response magnitude after Ag challenge. However, recent studies have indicated that naive CTLp frequencies do not necessarily predict the size of the Ag-driven response, indicating an important role for differential CTLp recruitment and/or expansion. This study characterizes the early emergence of various influenza epitope-specific CTL responses at multiple sites in C57BL/6 mice, and probes the role of Ag dose and TCR avidity in dictating immune response hierarchies. Despite large naive CTLp numbers, subdominance was found to arise largely as a consequence of the abrupt and premature cessation of CTL proliferation, at least for one epitope specificity. Investigation into the possible drivers of the poor proliferation observed for subdominant specificities showed that the immunodominance hierarchy endured irrespective of epitope abundance, and correlated with the prevalence of high-avidity T cells in both the naive and immune compartments. Our study strongly indicates that the quality, and not simply the quantity, of antiviral CTLs dictate response magnitude.

HIV-1 Adaptation to Antigen Processing Results in Population-Level Immune Evasion and Affects Subtype Diversification

SummaryThe recent HIV-1 vaccine failures highlight the need to better understand virus-host interactions. One key question is why CD8+ T cell responses to two HIV-Gag regions are uniquely associated with delayed disease progression only in patients expressing a few rare HLA class I variants when these regions encode epitopes presented by ∼30 more common HLA variants. By combining epitope processing and computational analyses of the two HIV subtypes responsible for ∼60% of worldwide infections, we identified a hitherto unrecognized adaptation to the antigen-processing machinery through substitutions at subtype-specific motifs. Multiple HLA variants presenting epitopes situated next to a given subtype-specific motif drive selection at this subtype-specific position, and epitope abundances correlate inversely with the HLA frequency distribution in affected populations. This adaptation reflects the sum of intrapatient adaptations, is predictable, facilitates viral subtype diversification, and increases global HIV diversity. Because low epitope abundance is associated with infrequent and weak T cell responses, this most likely results in both population-level immune evasion and inadequate responses in most people vaccinated with natural HIV-1 sequence constructs. Our results suggest that artificial sequence modifications at subtype-specific positions in vitro could refocus and reverse the poor immunogenicity of HIV proteins.

The mouse transcription factor‐like 5 gene encodes a protein localized in the manchette and centriole of the elongating spermatid

Spermiogenesis is the final phase of spermatogenesis. During this process, haploid round spermatids differentiate into spermatozoa, with dramatic morphological changes, including elongation and condensation of the nuclei, and formation of the flagella. Meig1 is one of many genes involved in the regulation of this process. Male mice deficient in MEIG1 are sterile with a severe defect in spermiogenesis, associated with dramatic disruption of the spermatid manchette and failure of flagellogenesis. A yeast two-hybrid screen using full-length MEIG1 as bait identified transcription factor-like 5 protein (TCFL5) as a putative interacting proteins. Interestingly, this protein was also identified as a potential binding partner of SPAG16, another protein essential for spermatogenesis, and also a binding partner of MEIG1. The interaction between TCFL5 and MEIG1 was confirmed in cultured cells over-expressing the two proteins. The mouse Tcfl5 transcript is present only in the testis, and its expression is significantly increased during spermiogenesis. However, little is known about TCFL5 protein and its role in male germ cells. A rabbit polyclonal antibody was generated against the C-terminal region of TCFL5. Mouse TCFL5 protein was expressed in the testis but not in mature spermatozoa. During the first wave of spermatogenesis, TCFL5 expression was dramatically increased at day 30 after birth. In the testis and a mixture of dispersed testicular cells, the protein co-localized with α-tubulin, a manchette marker in early elongating spermatids. The protein also localized in the centrioles of late elongating spermatids. No obvious differences in TCFL5 epitope abundance and localization were observed between wild type and the Meig1-deficient mice. These findings suggest that TCFL5 may play a role upstream of MEIG1 action, and based on putative binding partners and localization is likely to be involved in spermiogenesis and formation of the sperm flagella.

Kinetics of Antigen Expression and Epitope Presentation during Virus Infection

Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity.

Systematically exploring the contribution of individual immunoproteasome subunits to influenza Cd8+ T cell repertoire and epitope abundance

Systematically exploring the contribution of individual immunoproteasome subunits to influenza Cd8+ T cell repertoire and epitope abundance

Direct quantitation of MHC‐bound peptide epitopes by selected reaction monitoring

We describe a cell-free approach that employs selected reaction monitoring (SRM) in tandem mass spectrometry to identify and quantitate T-cell epitopes. This approach utilises multiple epitope-specific SRM transitions to identify known T-cell epitopes and an absolute quantitation (AQUA) peptide strategy to afford AQUA. The advantage of a mass spectrometry-based approach over more traditional cell-based assays resides in the robustness and transferability of an SRM approach between laboratories and the ability of this strategy to detect multiple peptides simultaneously without the requirement of epitope-specific reagents such as T-cell lines. Thus, the SRM strategy for epitope quantitation will find application in studies of antigen density, the link between epitope abundance and immunogenicity, the dynamic range of epitope presentation and the abundance of T-cell epitopes in disease.

Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance

Although cytotoxic T lymphocytes (CTLs) in people infected with human immunodeficiency virus type 1 can potentially target multiple virus epitopes, the same few are recognized repeatedly. We show here that CTL immunodominance in regions of the human immunodeficiency virus type 1 group-associated antigen proteins p17 and p24 correlated with epitope abundance, which was strongly influenced by proteasomal digestion profiles, affinity for the transporter protein TAP, and trimming mediated by the endoplasmatic reticulum aminopeptidase ERAAP, and was moderately influenced by HLA affinity. Structural and functional analyses demonstrated that proteasomal cleavage 'preferences' modulated the number and length of epitope-containing peptides, thereby affecting the response avidity and clonality of T cells. Cleavage patterns were affected by both flanking and intraepitope CTL-escape mutations. Our analyses show that antigen processing shapes CTL response hierarchies and that viral evolution modifies cleavage patterns and suggest strategies for in vitro vaccine optimization.

The role of innate immunity in atherogenesis

Lipid peroxidation is a common event in health and is greatly accelerated in pro-inflammatory settings such as hypercholesterolemia. Consequently, oxidation-specific epitopes are generated, which are pro-inflammatory and immunogenic, leading to both adaptive and innate responses. Because innate immune mechanisms use conserved germline pattern recognition receptors (PRRs) that are preformed and present at birth, it is not obvious why they should bind to such epitopes. In this review, we put forward the hypothesis that because oxidation-specific epitopes are ubiquitous in both health and disease, and because they in essence represent "danger signals," they constitute a class of pathogen-associated molecular patterns leading to the natural selection of multiple innate PRRs that target such epitopes. We suggest that apoptotic cells, and the blebs and microparticles released from such cells, which are rich in oxidation-specific epitopes and thus pro-inflammatory, constitute an endogenous set of selecting antigens. In turn, natural antibodies, scavenger receptors, and soluble innate proteins, such as pentraxins, all represent PRRs that target such epitopes. We discuss the evidence for this hypothesis and the consequences of such responses in health and disease, such as atherosclerosis.

A virus-specific CD8+T cell immunodominance hierarchy determined by antigen dose and precursor frequencies

Immunodominance hierarchies are a substantial, but poorly understood, characteristic of CD8(+) T cell-mediated immunity. Factors influencing the differential responses to the influenza A virus nucleoprotein (NP(366-374)) and acid polymerase (PA(224-233)) peptides presented by H2D(b) have been analyzed by disabling (N5--> Q substitution) these peptides in their native configuration, then expressing them in the viral neuraminidase protein. This strategy of shifting epitopes within the same viral context resulted in an apparent equalization of D(b)NP(366) [epitope consisting of viral nucleoprotein (NP) amino acid residues 366-374 complexed with the H2D(b) MHC class I glycoprotein] and D(b)PA(224) (H2D(b)+PA(224-233)) epitope abundance after direct infection in vitro and induced reproducible changes in the magnitude of the D(b)NP(366)- and D(b)PA(224)-specific T cell subsets generated after infection of mice. Comparison of D(b)NP(366)- and D(b) PA(224)-specific CD8(+) T cell responses induced from the native configuration and from the viral neuraminidase stalk demonstrated that the size of both primary and secondary responses is influenced by relative epitope levels and that, at least after secondary challenge, the magnitude of responses is also determined by CD8(+) T cell precursor frequency. Thus, this immunodominance hierarchy is a direct function of antigen dose and T cell numbers.

Tolerance induction to a mammalian blood group—like carbohydrate antigen by syngeneic lymphocytes expressing the antigen, II: tolerance induction on memory B cells

AbstractInduction of immune tolerance on memory B cells specific to transplantation carbohydrate antigens was studied in the experimental animal model of α1,3galactosyltransferase knockout (KO) mice, which lack the α-gal epitope (Galα1-3Galβ1-4GlcNAc-R) and can produce the anti-Gal antibody against it. Memory anti-Gal B cells were generated by immunization of KO mice with pig kidney membranes (ie, xenogeneic cell membranes expressing an abundance of α-gal epitopes). Lymphocytes including memory anti-Gal B cells were administered into lethally irradiated KO mice, together with syngeneic wild-type (WT) lymphocytes expressing α-gal epitopes. Memory anti-Gal B cells were completely tolerized after being in vivo for 14 days with WT lymphocytes. This was indicated by the lack of anti-Gal immunoglobulin G (IgG) response following immunization with pig kidney membranes vs the extensive anti-Gal response in mice that did not receive WT lymphocytes. Tolerance induction was prevented if T cells were activated by alloantigens. This tolerance was highly specific to anti-Gal B cells and did not affect memory B cells with closely related specificity, such as B cells with anti–blood group A specificity. Tolerance induction on anti-Gal B cells was found to be time dependent and required more than 10 days of in vivo exposure of these B cells to WT lymphocytes. These observations suggest a novel method for induction of tolerance to transplantation carbohydrate antigens in humans, by in vitro transduction of autologous blood lymphocytes with an adenovirus containing the corresponding glycosyltransferase gene and administration of the transduced cells into the circulation after removal of natural antibodies to the antigen. (Blood. 2003;102:229-236)

Competition between two MHC binding registers in a single peptide processed from myelin basic protein influences tolerance and susceptibility to autoimmunity.

Experimental allergic encephalomyelitis (EAE) is an animal model for multiple sclerosis induced by stimulating myelin basic protein (MBP)-specific T cells. The MBP-specific repertoire in B10.PL mice is shaped by tolerance mechanisms that eliminate MBP121–150–specific T cells. In contrast, MBPAc1–11–specific T cells escape tolerance and constitute the encephalitogenic repertoire. To determine if this differential tolerance is caused by differences in the abundance of MBP epitopes generated by processing, MBP peptides were eluted from I-Au complexes and analyzed by mass spectrometry. Peptides were identified from both the NH2-terminal and MBP121–150 regions. Unexpectedly, MBPAc1–18 and Ac1–17, which contain the MBPAc1–11 epitope, were much more abundant than MBP121–150 peptides. The results demonstrate that competition between two I-Au binding registers, a low affinity register defined by MBPAc1–11 and a high affinity register defined by MBP5–16, prevents most of the NH2-terminal naturally processed peptides from binding in the MBPAc1–11 register. The small fraction of MBPAc1–18 bound in the MBPAc1–11 register is not sufficient to induce tolerance but provides a ligand for MBPAc1–11–specific T cells during disease. These results provide a basis for both the lack of tolerance to MBPAc1–11 and the ability of this epitope to become a target during autoimmunity.

Developmental regulation of pectic epitopes during potato tuberisation

We show, by immunogold labelling, that potato (Solanum tuberosum L. cv Karnico) pectic epitopes are developmentally regulated within regions of the stolon, in addition to showing tissue-specific differences in abundance and localisation. The (1-->4)-beta-D-galactan and (1-->5)-alpha-arabinan epitopes demarcate two distinct zones within stolons; galactans are enriched in primary walls of elongating cells proximal to the stolon hook, whilst arabinans predominate in younger cells distal to the hook. Low-methoxyl homogalacturonan epitopes are concentrated in the middle lamella and show a proximo-distal gradient in stolons similar to that of galactans, whilst high-methoxyl homogalacturonan is uniformly abundant. Calcium pectate is restricted to the middle lamella at cell corners and pit fields. Calcium-binding sites are uniformly present in stolon cell walls, but their total density is reduced and they become localised to a few cell corners in mature tubers, as determined by image-electron energy loss spectroscopy. During the transition from elongation growth to isodiametric expansion during tuberisation of the stolon hook, there were no detectable changes in pectic epitope abundance or localisation. As tubers matured, all epitopes increased in abundance in parenchymal cell walls, except for calcium pectate. We conclude that potentially significant changes in pectic composition occur as young cells distal to the stolon hook move into the zone of cell elongation proximal to the hook.

Immunodominance among EBV-derived epitopes restricted by HLA-B27 does not correlate with epitope abundance in EBV-transformed B-lymphoblastoid cell lines.

Using synthetic peptides, the HLA-B27-restricted CTL response to EBV in asymptomatic virus carriers has been mapped to four epitope regions in EBV latent cycle Ags. One of these peptide-defined epitopes (RRIYDLIEL) tends to be immunodominant and is recognized in the context of all three B27 subtypes studied, B*2702, B*2704, and B*2705. The other peptide-defined epitopes induce responses only in the context of one subtype, the immunogenic combinations being RRARSLSAERY/B*2702, RRRWRRLTV/B*2704, and FRKAQIQGL/B*2705. We used immunoaffinity chromatography to isolate the naturally presented viral peptides associated with these MHC class I molecules on the surface of EBV-transformed B-LCL. Using CTL reconstitution assays in conjunction with mass spectrometry, we established that the naturally processed and presented peptides are identical with the previously identified synthetic sequences. Despite the subtype-specific immunogenicity of three of the four epitopes, all four epitope peptides were found in association with each of the three different HLA-B27 subtypes. Indeed, those peptides that failed to induce a response in the context of a particular HLA-B27 subtype were frequently presented at greater abundance by that subtype than were the immunogenic peptides. Furthermore, among the peptides that did induce a response, immunodominance did not correlate with epitope abundance; in fact the immunodominant RRIYDLIEL epitope was least abundant, being present at less than one copy per cell. The relationship of this unexpected finding to the persistence of EBV is discussed.

Adult and neonatal anti-Gal response in knock-out mice for alpha1,3galactosyltransferase.

The knockout mouse to alpha1,3galactosyltransferase (alpha1,3GT KO) lacks the ability to synthesize alpha-gal epitopes (Galalpha1,3Galbeta1,4GlcNAc-R) and is capable of producing low amounts of the natural anti-Gal antibody. The present study indicates that repeated immunization of these mice with rabbit red blood cell (RRBC) membranes results in production of anti-Gal in titers and specificity similar to those in humans. In contrast, immunized wild-type mice completely lack anti-Gal. Anti-Gal in the alpha1,3GT KO mice is produced in the circulation as the various IgG subclasses and as IgM isotype, but not IgA. In view of previous reports on the possible induction of T cell tolerance by immunization of mice with large amounts of antigen up to 24 days of age, we assayed possible induction of neonatal B cell tolerance toward the alpha-gal epitope. Eight-day-old neonates were subjected to immunization with 1 x 10(8) RRBC membranes, or 30 x 10(6) wild type mouse splenocytes, both of which express an abundance of alpha-gal epitopes. These neonatal exposures to alpha-gal epitopes did not prevent subsequent production of anti-Gal. Thus, the tolerance induction to this carbohydrate epitope is likely to be mediated by mechanisms other than those inducing neonatal T cell tolerance.

Production and structure characterisation of recombinant chromogranin A N-terminal fragments (vasostatins) -- evidence of dimer-monomer equilibria.

Vasostatins (VS) are vasoinhibitory peptides derived from the N-terminal domain of chromogranin A, a secretory protein present in the electron-dense granules of many neuroendocrine cells. In this work we describe a method for the production in Escherichia coli of large amounts of recombinant vasostatins, corresponding to chromogranin A residues 1-78 (VS-1), and 1-115 (VS-2), and the use of these materials for structure characterisation. The masses of both products were close to the expected values, by SDS/PAGE and mass spectrometry analysis. However, their hydrodynamic behaviours in size-exclusion chromatography corresponded to that of proteins with a larger size. SDS/PAGE analysis of VS-1 and VS-2 after cross-linking with disuccinimidyl suberate indicated that both polypeptides form dimers. VS-2 was almost entirely dimeric at > 4 microM, but rapidly converted to monomer after dilution to 70 nM. The rapid dimer-monomer transition of VS-2 after dilution could be part of a mechanism for regulating its activity and localising its action. Immunological studies of VS-1 have shown that residues 37-70 constitute a highly antigenic region characterised by an abundance of linear epitopes efficiently mimicked by synthetic peptides. The recombinant products and the immunological reagents developed in this work could be valuable tools for further investigating the structure and the function of chromogranin A and its fragments.

Structure and distribution of lignin in primary and secondary cell walls of maize coleoptiles analyzed by chemical and immunological probes

Lignin is an integral constituent of the primary cell walls of the dark-grown maize (Zea mays L.) coleoptile, a juvenile organ that is still in the developmental state of rapid cell extension. Coleoptile lignin was characterized by (i) conversion to lignothiolglycolate derivative, (ii) isolation of polymeric fragments after alkaline hydrolysis, (iii) reactivity to antibodies against dehydrogenative polymers prepared from monolignols, and (iv) identification of thioacidolysis products typical of lignins. Substantial amounts of lignin could be solubilized from the coleoptile cell walls by mild alkali treatments. Thioacidolysis analyses of cell walls from coleoptiles and various mesocotyl tissues demonstrated the presence of guaiacyl-, syringyl- and (traces of)p-hydroxyphenyl units besidesp-coumaric and ferulic acids. There are tissue-specific differences in amount and composition of lignins from different parts of the maize seedling. Electron-microscopic immunogold labeling of epitopes recognized by a specific anti-guaiacyl/syringyl antibody demonstrated the presence of lignin in all cell walls of the 4-d-old coleoptile. The primary walls of parenchyma and epidermis were more weakly labeled than the secondary wall thickenings of tracheary elements. No label was found in middle lamellae and cell corners. Lignin epitopes appeared first in the tracheary elements on day 2 and in the parenchyma on day 3 after sowing. Incubation of coleoptile segments in H2O2 increased the amount of extractable lignin and the abundance of lignin epitopes in the parenchyma cell walls. Lignin deposition was temporally and spatially correlated with the appearance of epitopes for prolinerich proteins, but not for hydroxyproline-rich proteins, in the cell walls. The lignin content of coleoptiles was increased by irradiating the seedlings with white or farred light, correlated with the inhibition of elongation growth, while growth promotion by auxin had no effect. It is concluded that wall stiffness, and thus extension growth, of the coleoptile can be controlled by lignification of the primary cell walls. Primary-wall lignin may represent part of an extended polysaccharide-polyphenol network that limits the extensibility of the cell walls.

Parameters that influence the efficiency of processing antigenic epitopes expressed in Salmonella typhimurium

We investigated parameters that affect the efficiency with which antigenic epitopes from Salmonella typhimurium are processed for presentation to T lymphocytes. As a model system, the hen egg white lysozyme 52-61 [HEL(52-61)] epitope, which binds the murine major histocompatibility complex class II (MHC-II) molecule I-Ak, was expressed in soluble fusion proteins in S. typhimurium. Murine peritoneal macrophages mediated phagocytic processing of viable S. typhimurium expressing fusion proteins of the HEL epitope for presentation via I-Ak regardless of the bacterial compartment in which the epitope was contained (i.e., surface exposed, facing the periplasmic space, or in the cytoplasm). Minor differences in processing efficiency observed with different epitope compartmentalizations could be overcome by altering the relative expression level, indicating that epitope abundance is an important factor for efficient processing of epitopes from S. typhimurium. This processing pathway required phagocytosis of bacteria followed by passage through an acidic compartment, suggesting a pathway involving phagolysosomal degradation of the bacteria to liberate epitopes that bind MHC-II. HEL(52-61) was processed more efficiently from heat-killed S. typhimurium than from viable bacteria, and in addition, the HEL epitope was processed more efficiently from a rough lipopolysaccharide (LPS) strain than from its isogenic smooth LPS counterpart, most likely because of enhanced phagocytosis of the rough LPS strain. These data suggest that the efficiency of epitope processing from S. typhimurium for presentation via MHC-II is affected by bacterial viability, epitope abundance, and LPS phenotype, factors which may be important to consider in development of recombinant S. typhimurium vaccine strains.

Compartmentalization of defined epitopes expressed in Escherichia coli has only a minor influence on efficiency of phagocytic processing for presentation by class I and class II major histocompatibility complex molecules to T cells

The effect of abundance and compartmentalization of antigenic epitopes expressed in Escherichia coli on phagocytic processing was studied by expressing fusion proteins containing the epitope from positions 52 to 61 of hen egg white lysozyme [HEL(52-61)], which binds the I-Ak murine major histocompatibility complex class II (MHC-II) molecule or the epitope from positions 257 to 264 of chicken egg ovalbumin [OVA(257-264]), which binds the Kb murine MHC-I molecule. Epitopes expressed as fusion proteins in the outer membrane protein LamB allowed exposure of the epitopes either at the bacterial surface, in the periplasmic space, or in the cytoplasm. Regardless of epitope compartmentalization within the bacterium, MHC-II-restricted or MHC-I-restricted presentation to T hybridoma cells occurred after macrophages phagocytosed bacteria producing the HEL(52-61) epitope or the OVA(257-264) epitope, respectively. Increased epitope abundance within a given microbial compartment resulted in increased processing and presentation to epitope-specific T hybridoma cells. Minor differences in the efficiency of epitope processing between the constructs was observed, and the HEL or OVA epitope exposed in the periplasmic space was processed most efficiently compared with the surface- or cytoplasm-localized epitopes. These differences could be overcome by increasing the amount of epitope per bacterium as little as two to five times. The minor differences in processing efficiency may be due to differing protein contexts of the epitope as well as differing epitope compartmentalizations within the bacteria. Thus, production of abundant epitope is the important parameter influencing processing of epitopes expressed in E. coli to induce T-cell responses rather than targeting of an epitope to a specific bacterial compartment.