Bruce B. Reinhold

Induction of anti-tumor cytotoxic T cell responses through PLGA-nanoparticle mediated antigen delivery

Nanotechnology-based antigen delivery has been developing as a vaccine strategy due to its dose-sparing and prolonged antigen presentation features. In the current study, we examined the feasibility of nanoparticle (NP)-mediated delivery of antigenic peptides to efficiently induce cytotoxic T lymphocyte responses against tumor-associated self-antigens in C57BL/6 mouse models. The biodegradable poly(D,L-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying murine melanoma antigenic peptides, hgp100(25-33) and TRP2(180-188), were prepared by double emulsion method. Efficient uptake of PLGA-NP by murine dendritic cells was shown in vitro and in vivo, using NP labeled with the fluorescent dye DiD. Intradermal injection of peptide-loaded PLGA-NP into mice induced antigen-specific T cell responses more strongly than the peptides mixed with Freunds adjuvant. More importantly, vaccination with PLGA-NP carrying both TRP2(180-188) and a toll-like receptor 4 agonist, monophosphoryl lipid A, significantly delayed growth of subcutaneously inoculated B16 melanoma cells in a prophylactic setting. Furthermore, the anti-tumor activity of NP-mediated peptide vaccination was significantly augmented by combined treatment with interferon-γ, which might prevent tumor escape through up-regulation of MHC class I expression on tumor cells. Our findings demonstrate the feasibility of NP-mediated antigen delivery for cancer immunotherapy, in particular when immune escape mechanisms of tumor cells are blocked simultaneously.

Electrospray Ionization Mass Spectrometry: Deconvolution by an Entropy-Based Algorithm

Summary The more demanding complication of the entropy processing of ES1 spectra is the presence of large, unresolved backgrounds on which many individual peaks are superimposed. In working with these spec- tra, we generally employ a combination of Fourier deconvolution and baseline subtraction. Such steps can be managed as a preftlter to the entropy deconvo- lution since the optimization (Fourier cutoff, exponen- tial factor) does not require knowledge of the entropy fit parameters. Figure 4 illustrates the application of these strategies to an ES1 mass spectrum of a gly- copeptide with two glycosolation sites. ties of the data. rates which are modeled as multiply charged Gauss- ian ion profiles. The other noise is shot noise corre- The computer spectra were generated by a numeri- cal simulation of a Poisson process in which a param- sponding to the finite ion counts. Of course, back- eter representing the expected number of counts for each mass value mi is defined. Independent scans are ground peaks or chemical “noise” are, from the obtained by sampling the Poisson distribution corre- sponding to this parameter [7]. In these simulations standpoint of the simulation, just additional real noise can arise in two ways. The ftrst is the relative fraction of counts that are real ions to counts that are peaks. representing background. From the standpoint of the simulation program they are distinguished only be- cause the background has a mass-independent arrival rate while the signal corresponds to mass-dependent This report has discussed a novel algorithm for ex- tracting parent masses from spectra containing multi- ply charged ions, a common feature of ESI. The algo- rithm works with raw data and does not require the

CARBOHYDRATE SEQUENCE ANALYSIS BY ELECTROSPRAY IONIZATION-MASS SPECTROMETRY

Abstract This chapter summarizes several strategies for a more complete understanding of carbohydrate structure with a focus on N-linked glycans. The techniques include periodate oxidation to impart greater molecular specificity, functional group blocking by methylation, electrospray for “soft” and efficient ionization, collision-induced dissociation to obtain detailed fragmentation, and tandem mass spectrometry for mass separation and analysis. The lipophilic products following derivatization contribute to product cleanup by solvent extraction; they also improve sensitivity during ES and, when combined with CID, yield detailed sequence, linkage, and branching information.

Uptake and processing of glycosylated mycolates for presentation to CD1b-restricted T cells.

Antigen presenting cells (APCs) expressing CD1b mediate the specific T cell recognition of mycobacterial lipid antigens. These lipid antigens require internalization by APCs prior to presentation, but the detailed mechanisms of uptake and intracellular processing are not known. Here we have examined several steps in the presentation of two related classes of CD1b-presented antigens, free and glycosylated mycolates. T cell recognition of glucose monomycolate (GMM) was blocked by agents that fix APC membranes or neutralize the pH of endosomes, indicating a requirement for GMM uptake into an acidic compartment prior to recognition. Different T cell lines responded to free mycolate or GMM without crossreactivity, yet both antigens were taken up by APCs at the same rate. This demonstrated that differential recognition of these antigens resulted from T cell specificity for their hydrophilic caps and that APCs were unable to interconvert these antigens by enzymatic or chemical deglycosylation or glycosylation. APCs were also unable to cleave mycobacterial trehalose dimycolate (TDM) at its most chemically labile linkages to yield antigenic free mycolates or GMM. Our results indicate that these mycolate-containing antigens are resistant to chemical or enzymatic cleavage by APCs, suggesting that molecular trimming is not a universal feature of lipid antigen processing.

A Conserved E7-derived Cytotoxic T Lymphocyte Epitope Expressed on Human Papillomavirus 16-transformed HLA-A2+ Epithelial Cancers

Human Papillomavirus 16 (HPV-16) has been identified as the causative agent of 50% of cervical cancers and many other HPV-associated tumors. The transforming potential/tumor maintenance capacity of this high risk HPV is mediated by two viral oncoproteins, E6 and E7, making them attractive targets for therapeutic vaccines. Of 21 E6 and E7 peptides computed to bind HLA-A*0201, 10 were confirmed through TAP-deficient T2 cell HLA stabilization assay. Those scoring positive were investigated to ascertain which were naturally processed and presented by surface HLA molecules for CTL recognition. Because IFNγ ELISpot frequencies from healthy HPV-exposed blood donors against HLA-A*0201-binding peptides were unable to identify specificities for tumor targeting, their physical presence among peptides eluted from HPV-16-transformed epithelial tumor HLA-A*0201 immunoprecipitates was analyzed by MS3 Poisson detection mass spectrometry. Only one epitope (E711–19) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E711–20), previously used as a vaccine candidate, was neither detected by MS3 on HPV-transformed tumor cells nor effectively recognized by 9-mer specific CTL. These data underscore the importance of precisely defining CTL epitopes on tumor cells and offer a paradigm for T cell-based vaccine design.

Design, Expression, and Immunogenicity of a Soluble HIV Trimeric Envelope Fragment Adopting a Prefusion gp41 Configuration

The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) is comprised of non-covalently associated gp120/gp41 subunits that form trimeric spikes on the virion surface. Upon binding to host cells, Env undergoes a series of structural transitions, leading to gp41 rearrangement necessary for fusion of viral and host membranes. Until now, the prefusion state of gp41 ectodomain (e-gp41) has eluded molecular and structural analysis, and thus assessment of the potential of such an e-gp41 conformer to elicit neutralizing antibodies has not been possible. Considering the importance of gp120 amino (C1) and carboxyl (C5) segments in the association with e-gp41, we hypothesize that these regions are sufficient to maintain e-gp41 in a prefusion state. Based on the available gp120 atomic structure, we designed several truncated gp140 variants by including the C1 and C5 regions of gp120 in a gp41 ectodomain fragment. After iterative cycles of protein design, expression and characterization, we obtained a variant truncated at Lys665 that stably folds as an elongated trimer under physiologic conditions. Several independent biochemical/biophysical analyses strongly suggest that this mini-Env adopts a prefusion e-gp41 configuration that is strikingly distinct from the postfusion trimer-of-hairpin structure. Interestingly, this prefusion mini-Env, lacking the fragment containing the 2F5/4E10 neutralizing monoclonal antibody binding sites, displays no detectable HIV-neutralizing epitopes when employed as an immunogen in rabbits. The result of this immunogenicity study has important implications for HIV-1 vaccine design efforts. Moreover, this engineered mini-Env protein should facilitate three-dimensional structural studies of the prefusion e-gp41 and serve to guide future attempts at pharmacologic and immunologic intervention of HIV-1.

TRANSFER OF MAN9GLCNAC TO L-FUCOSE BY ENDO-BETA -N-ACETYLGLUCOSAMINIDASE FROM ARTHROBACTER PROTOPHORMIAE

We have reported that transglycosylation activity of endo-β-N-acetylglucosaminidase fromArthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995)J Biol Chem270: 17723–29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man9GlcNAc, tol-Fuc using Man9GlcNAc2Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2m l-Fuc was used as acceptor. The transglycosylation product was purified by high performance liquid chromatography on a graphitized carbon column and the presence ofl-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man9GlcNAc-l-Fuc-PA, revealed that a β-anomeric configuration linkage was formed between GlcNAc andl-Fuc. The GlcNAc was found to be 1,2-linked tol-Fuc by two methods; i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man9GlcNAc-l-Fuc; and ii) preparation of Man9GlcNAc-l-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man9GlcNAcβ(1,2)-l-Fuc. Methyl β-l-fucopyranoside,l-Gal are also acceptors for the enzymic transglycosylation. However, transglycosylation failed when methyl α-l-fucopyranoside,d-Fuc andd-Gal were used. These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a4C1-conformation or equivalent conformation of the acceptor to perform transglycosylation.

Physical detection of influenza A epitopes identifies a stealth subset on human lung epithelium evading natural CD8 immunity.

Significance Influenza A viruses (IAVs) are a cause of major morbidity in the human population. Being RNA viruses, replication is error prone, and proteins such as viral envelope hemagglutinin rapidly mutate. Current vaccines stimulate antibodies targeting exposed virion proteins but require annual reformation due to constant sequence variation. In contrast, vaccines that stimulate CD8 T cells directed at conserved peptides from internal proteins would offer stable immunity if these peptides are displayed by HLA proteins on infected cells. Currently, functional readouts infer the IAV peptides displayed. Using new MS technology, epitopes on infected human HLA-A2+ lung epithelium are identified and abundances characterized. The data show interconnections between viral evasion, immunodominance, and stealth responses that will aid in developing cellular vaccines against influenza. Vaccines eliciting immunity against influenza A viruses (IAVs) are currently antibody-based with hemagglutinin-directed antibody titer the only universally accepted immune correlate of protection. To investigate the disconnection between observed CD8 T-cell responses and immunity to IAV, we used a Poisson liquid chromatography data-independent acquisition MS method to physically detect PR8/34 (H1N1), X31 (H3N2), and Victoria/75 (H3N2) epitopes bound to HLA-A*02:01 on human epithelial cells following in vitro infection. Among 32 PR8 peptides (8–10mers) with predicted IC50 < 60 nM, 9 were present, whereas 23 were absent. At 18 h postinfection, epitope copies per cell varied from a low of 0.5 for M13–11 to a high of >500 for M158–66 with PA, HA, PB1, PB2, and NA epitopes also detected. However, aside from M158–66, natural CD8 memory responses against conserved presented epitopes were either absent or only weakly observed by blood Elispot. Moreover, the functional avidities of the immunodominant M158–66/HLA-A*02:01-specific T cells were so poor as to be unable to effectively recognize infected human epithelium. Analysis of T-cell responses to primary PR8 infection in HLA-A*02:01 transgenic B6 mice underscores the poor avidity of T cells recognizing M158–66. By maintaining high levels of surface expression of this epitope on epithelial and dendritic cells, the virus exploits the combination of immunodominance and functional inadequacy to evade HLA-A*02:01-restricted T-cell immunity. A rational approach to CD8 vaccines must characterize processing and presentation of pathogen-derived epitopes as well as resultant immune responses. Correspondingly, vaccines may be directed against “stealth” epitopes, overriding viral chicanery.

Molecular Detection of Targeted Major Histocompatibility Complex I-Bound Peptides Using a Probabilistic Measure and Nanospray MS3 on a Hybrid Quadrupole-Linear Ion Trap

A nanospray MS3 method deployed on a quadrupole linear ion trap hybrid can detect targeted peptides with high dynamic range and high sensitivity from complex mixtures without separations. The method uses a recognition algorithm that is a modification of the relative (Kullback−Leibler, KL) entropy characterization of probabilistic distance to detect if reference MS3 fragmentation patterns are components of acquired MS3 spectra. The recognition reflects the probabilistic structure of physical MS measurements unlike the Euclidean or inner product metrics widely used for comparing spectra. It capably handles spectra with a significant chemical ion background in contrast to the Euclidean metric or the direct relative entropy. The full nanospray MS3 method allows both the detection and quantitation of targets without the need to obtain isotopically labeled standards. By avoiding chromatographic separations and its associated surface losses, the detection can be applied to complex samples on a very limited material scale. The methodology is illustrated by applications to the medically important problem of detecting targeted major histocompatibility complex (MHC) I associated peptides extracted from limited cell numbers.

Direct identification of an HPV-16 tumor antigen from cervical cancer biopsy specimens.

Persistent infection with high-risk human papilloma viruses (HPV) is the worldwide cause of many cancers, including cervical, anal, vulval, vaginal, penile, and oropharyngeal. Since T cells naturally eliminate the majority of chronic HPV infections by recognizing epitopes displayed on virally altered epithelium, we exploited Poisson detection mass spectrometry (MS3) to identify those epitopes and inform future T cell-based vaccine design. Nine cervical cancer biopsies from HPV-16 positive HLA-A*02 patients were obtained, histopathology determined, and E7 oncogene PCR-amplified from tumor DNA and sequenced. Conservation of E7 oncogene coding segments was found in all tumors. MS3 analysis of HLA-A*02 immunoprecipitates detected E711–19 peptide (YMLDLQPET) in seven of the nine tumor biopsies. The remaining two samples were E711–19 negative and lacked the HLA-A*02 binding GILT thioreductase peptide despite possessing binding-competent HLA-A*02 alleles. Thus, the conserved E711–19 peptide is a dominant HLA-A*02 binding tumor antigen in HPV-16 transformed cervical squamous and adenocarcinomas. Findings that a minority of HLA-A*02:01 tumors lack expression of both E711–19 and a peptide from a thioreductase important in processing of cysteine-rich proteins like E7 underscore the value of physical detection, define a potential additional tumor escape mechanism and have implications for therapeutic cancer vaccine development.