Immuno-rolling circle amplification using a multibinding fusion protein

Ultrasensitive detection of specific, low level proteins in body fluids is particularly challenging. Owing to the extreme sensitivity of the polymerase chain reaction step, the requirements for immuno-rolling circle amplification (immuno-RCA) are much more stringent than for conventional ELISA. Here, we report the development of a rolling circle amplification procedure using multibinding fusion protein to enhance signals of immuno-RCA to detect a cancer biomarker, α-fetoprotein (AFP). We successfully avoid the covalent linkage between antibody and DNA or antibody and biotin/streptavidin by introducing a new genetically engineered fusion protein which contains the C2 domain of protein G and biotin acceptor peptide (BAP) which is intended to maintain the biological activity of the antibody. The purified fusion protein retained its binding affinity with IgG and streptavidin after efficient expression in Escherichia coli. Immuno-RCA in combination with BAP-C2 specifically and sensitively detected AFP in a microplate format. Therefore, the sensitivity and convenient nature of this method should contribute to effective signal enhancement in immunoassays for cancer biomarker detection.     

Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells

MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.     

Identification of key amino acid residues that determine the ability of high risk HPV16-E7 to dysregulate major histocompatibility complex class I expression

High risk human Papillomavirus (HPV) types are the major causative agents of cervical cancer. Reduced expression of major histocompatibility complex class I (MHC I) on HPV-infected cells might be responsible for insufficient T cell response and contribute to HPV-associated malignancy. The viral gene product required for subversion of MHC I synthesis is the E7 oncoprotein. Although it has been suggested that high and low risk HPVs diverge in their ability to dysregulate MHC I expression, it is not known what sequence determinants of HPV-E7 are responsible for this important functional difference. To investigate this, we analyzed the capability to affect MHC I of a set of chimeric E7 variants containing sequence elements from either high risk HPV16 or low risk HPV11. HPV16-E7, but not HPV11-E7, causes significant diminution of mRNA synthesis and surface presentation of MHC I, which depend on histone deacetylase activity. Our experiments demonstrate that the C-terminal region within the zinc finger domain of HPV-E7 is responsible for the contrasting effects of HPV11- and HPV16-E7 on MHC I. By using different loss- and gain-of-function mutants of HPV11- and HPV16-E7, we identify for the first time a residue variation at position 88 that is highly critical for HPV16-E7-mediated suppression of MHC I. Furthermore, our studies suggest that residues at position 78, 80, and 88 build a minimal functional unit within HPV16-E7 required for binding and histone deacetylase recruitment to the MHC I promoter. Taken together, our data provide new insights into how high risk HPV16-E7 dysregulates MHC I for immune evasion.     

Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv): the case of the hEx3 diabody

Diabodies (Dbs) and tandem single-chain variable fragments (taFv) are the most widely used recombinant formats for constructing small bispecific antibodies. However, only a few studies have compared these formats, and none have discussed their binding kinetics and cross-linking ability. We previously reported the usefulness for cancer immunotherapy of a humanized bispecific Db (hEx3-Db) and its single-chain format (hEx3-scDb) that target epidermal growth factor receptor and CD3. Here, we converted hEx3-Db into a taFv format to investigate how format affects the function of a small bispecific antibody; our investigation included a cytotoxicity assay, surface plasmon resonance spectroscopy, thermodynamic analysis, and flow cytometry. The prepared taFv (hEx3-taFv) showed an enhanced cytotoxicity, which may be attributable to a structural superiority to the diabody format in cross-linking target cells but not to differences in the binding affinities of the formats. Comparable cross-linking ability for soluble antigens was observed among hEx3-Db, hEx3-scDb, and hEx3-taFv with surface plasmon resonance spectroscopy. Furthermore, drastic increases in cytotoxicity were found in the dimeric form of hEx3-taFv, especially when the two hEx3-taFv were covalently linked. Our results show that converting the format of small bispecific antibodies can improve their function. In particular, for small bispecific antibodies that target tumor and immune cells, a functional orientation that avoids steric hindrance in cross-linking two target cells may be important in enhancing the growth inhibition effect.     

Comparative structural and molecular characterization of Streptococcus pneumoniae capsular polysaccharide serogroup 10

Streptococcus pneumoniae serogroup 10 includes four cross-reactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). In the present study, the structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype. Both CPS10C and CPS10F had β1-6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent α1-2 or wcrF-dependent α1-4 linkages between Gal and ribitol-5-phosphate. The presence of one linkage or the other also distinguished the repeating units of CPS10B and CPS10A. However, whereas these polysaccharides both had β1-3-linked Galf branches linked to GalNAc, only CPS10A had additional β1-6-linked Galp branches. These Galp branches and the reaction of a CPS10A-specific monoclonal antibody were eliminated by deletion of wcrG from the cps10A locus. In contrast, deletion of this gene from the cps10B locus had no effect on the structure of CPS10B, thereby identifying wcrG as a pseudogene in this serotype. The β1-3-linked Galf branches of CPS10A and CPS10B were eliminated by deletion of wcrD from each corresponding cps locus. Deletion of this gene also eliminated wcrG-dependent β1-6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD. These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes.     

Loss of T cell antigen recognition arising from changes in peptide and major histocompatibility complex protein flexibility: implications for vaccine design

Modification of the primary anchor positions of antigenic peptides to improve binding to major histocompatibility complex (MHC) proteins is a commonly used strategy for engineering peptide-based vaccine candidates. However, such peptide modifications do not always improve antigenicity, complicating efforts to design effective vaccines for cancer and infectious disease. Here we investigated the MART-1(27-35) tumor antigen, for which anchor modification (replacement of the position two alanine with leucine) dramatically reduces or ablates antigenicity with a wide range of T cell clones despite significantly improving peptide binding to MHC. We found that anchor modification in the MART-1(27-35) antigen enhances the flexibility of both the peptide and the HLA-A*0201 molecule. Although the resulting entropic effects contribute to the improved binding of the peptide to MHC, they also negatively impact T cell receptor binding to the peptide·MHC complex. These results help explain how the "anchor-fixing" strategy fails to improve antigenicity in this case, and more generally, may be relevant for understanding the high specificity characteristic of the T cell repertoire. In addition to impacting vaccine design, modulation of peptide and MHC flexibility through changes to antigenic peptides may present an evolutionary strategy for the escape of pathogens from immune destruction.     

Polymeric structure and host Toll-like receptor 4 dictate immunogenicity of NY-ESO-1 antigen in vivo

In search of intrinsic factors that contribute to the distinctively strong immunogenicity of a non-mutated cancer/testis antigen, we found that NY-ESO-1 forms polymeric structures through disulfide bonds. NY-ESO-1 binding to immature dendritic cells was dependent on its polymeric structure and involved Toll-like receptor-4 (TLR4) on the surface of immature dendritic cells in mouse and human. Gene gun-delivered plasmid encoding the wild-type NY-ESO-1 readily induced T cell-dependent antibody (Ab) responses in wild-type C57BL/10 mice but not TLR4-knock-out C57BL/10ScNJ mice. Disrupting polymeric structures of NY-ESO-1 by cysteine-to-serine (Cys-to-Ser) substitutions lead to diminished immunogenicity and altered TLR4-dependence in the induced Ab response. To demonstrate its adjuvant effect, NY-ESO-1 was fused with a major mugwort pollen allergen Art v 1 and a tumor-associated antigen, carbonic anhydrase 9. Plasmid DNA vaccines encoding the fusion genes generated robust immune responses against otherwise non-immunogenic targets in mice. Polymeric structure and TLR4 may play important roles in rendering NY-ESO-1 immunogenic and thus serve as a potent molecular adjuvant. NY-ESO-1 thus represents the first example of a cancer/testis antigen that is a also damage-associated molecular pattern.     

The polypeptide binding conformation of calreticulin facilitates its cell-surface expression under conditions of endoplasmic reticulum stress

We define two classes of calreticulin mutants that retain glycan binding activity; those that display enhanced or reduced polypeptide-specific chaperone activity, due to conformational effects. Under normal conditions, neither set of mutants significantly impacts the ability of calreticulin to mediate assembly and trafficking of major histocompatibility complex class I molecules, which are calreticulin substrates. However, in cells treated with thapsigargin, which depletes endoplasmic reticulum calcium, major histocompatibility complex class I trafficking rates are accelerated coincident with calreticulin secretion, and detection of cell-surface calreticulin is dependent on its polypeptide binding conformations. Together, these findings identify a site on calreticulin that is an important determinant of the induction of its polypeptide binding conformation and demonstrate the relevance of the polypeptide binding conformations of calreticulin to endoplasmic reticulum stress-induced interactions.     

Merkel cell polyomavirus large T antigen disrupts lysosome clustering by translocating human Vam6p from the cytoplasm to the nucleus

Merkel cell polyomavirus (MCV) has been recently described as the cause for most human Merkel cell carcinomas. MCV is similar to simian virus 40 (SV40) and encodes a nuclear large T (LT) oncoprotein that is usually mutated to eliminate viral replication among tumor-derived MCV. We identified the hVam6p cytoplasmic protein involved in lysosomal processing as a novel interactor with MCV LT but not SV40 LT. hVam6p binds through its clathrin heavy chain homology domain to a unique region of MCV LT adjacent to the retinoblastoma binding site. MCV LT translocates hVam6p to the nucleus, sequestering it from involvement in lysosomal trafficking. A naturally occurring, tumor-derived mutant LT (MCV350) lacking a nuclear localization signal binds hVam6p but fails to inhibit hVam6p-induced lysosomal clustering. MCV has evolved a novel mechanism to target hVam6p that may contribute to viral uncoating or egress through lysosomal processing during virus replication.     

Structural and functional characterization of the Streptococcus pneumoniae RrgB pilus backbone D1 domain

Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.