Sergey S. Seregin

Cutting Edge: Coding Single Nucleotide Polymorphisms of Endoplasmic Reticulum Aminopeptidase 1 Can Affect Antigenic Peptide Generation In Vitro by Influencing Basic Enzymatic Properties of the Enzyme

ER aminopeptidase 1 (ERAP1) customizes antigenic peptide precursors for MHC class I presentation and edits the antigenic peptide repertoire. Coding single nucleotide polymorphisms (SNPs) in ERAP1 were recently linked with predisposition to autoimmune disease, suggesting a link between pathogenesis of autoimmunity and ERAP1-mediated Ag processing. To investigate this possibility, we analyzed the effect that disease-linked SNPs have on Ag processing by ERAP1 in vitro. Michaelis–Menten analysis revealed that the presence of SNPs affects the Michaelis constant and turnover number of the enzyme. Strikingly, specific ERAP1 allele-substrate combinations deviate from standard Michaelis–Menten behavior, demonstrating substrate-inhibition kinetics; to our knowledge, this phenomenon has not been described for this enzyme. Cell-based Ag-presentation analysis was consistent with changes in the substrate inhibition constant Ki, further supporting that ERAP1 allelic composition may affect Ag processing in vivo. We propose that these phenomena should be taken into account when evaluating the possible link between Ag processing and autoimmunity.

Transient Pretreatment With Glucocorticoid Ablates Innate Toxicity of Systemically Delivered Adenoviral Vectors Without Reducing Efficacy

More than 300 human clinical trials utilize recombinant adenoviruses (rAds) as a gene transfer vector, confirming that rAds continue to be of high clinical interest. A primary weakness of rAds is their known propensity to trigger an innate, proinflammatory immune response rapidly after high-dose, systemic administration. In this study, we investigated what affects that pre-emptive treatment with anti-inflammatory glucocorticoids might have upon Ad vector-triggered inflammatory immune responses. We found that a simple pretreatment regimen with Dexamethasone (DEX) can significantly reduce most Ad-induced innate immune responses. DEX prevented rAd induction of systemic cytokine/chemokine releases in a dose-dependent fashion, with higher dosages preventing rAd induction of acute thrombocytopenia, endothelial cell activation, proinflammatory gene induction, and leukocyte infiltration into transduced organs. Transient glucocorticoid pretreatment also significantly reduced rAd-induced adaptive immune responses, including a decreased induction of Ad-neutralizing antibodies (NAbs). Importantly, use of DEX did not reduce the efficacy of rAd-mediated gene transduction nor rAd-derived transgene expression. Our results demonstrate that a simple, pre-emptive and transient glucocorticoid pretreatment is a viable approach to reduce rAd-associated acute toxicities that currently limit the use of Ad vectors in systemic clinical applications.

Overcoming pre-existing adenovirus immunity by genetic engineering of adenovirus-based vectors

Adenovirus (Ad)-based vectors offer several benefits showing their potential for use in a variety of vaccine applications. Recombinant Ad-based vaccines possess potent immunogenic potential, capable of generating humoral and cellular immune responses to a variety of pathogen-specific antigens expressed by the vectors. Ad5 vectors can be readily produced, allowing for usage in thousands of clinical trial subjects. This is now coupled with a history of safe clinical use in the vaccine setting. However, traditional Ad5-based vaccines may not be generating optimal antigen-specific immune responses, and generate diminished antigen-specific immune responses when pre-existing Ad5 immunity is present. These limitations have driven initiation of several approaches to improve the efficacy of Ad-based vaccines, and/or allow modified vaccines to overcome pre-existing Ad immunity. These include: generation of chemically modified Ad5 capsids; generation of chimeric Ads; complete replacement of Ad5-based vaccine platforms with alternative (human and non-human origin) Ad serotypes, and Ad5 genome modification approaches that attempt to retain the native Ad5 capsid, while simultaneously improving the efficacy of the platform as well as minimizing the effect of pre-existing Ad immunity. Here we discuss recent advances in- and limitations of each of these approaches, relative to their abilities to overcome pre-existing Ad immunity.

A Common Single Nucleotide Polymorphism in Endoplasmic Reticulum Aminopeptidase 2 Induces a Specificity Switch That Leads to Altered Antigen Processing

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) cooperate to trim antigenic peptide precursors for loading onto MHC class I molecules and help regulate the adaptive immune response. Common coding single nucleotide polymorphisms in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. It has been hypothesized that altered Ag processing by these enzymes is a causal link to disease etiology, but the molecular mechanisms are obscure. We report in this article that the common ERAP2 single nucleotide polymorphism rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared with the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. These effects are primarily due to changes in the catalytic turnover rate (kcat) and not in the affinity for the substrate. X-ray crystallographic analysis of the ERAP2 392K allele suggests that the polymorphism interferes with the stabilization of the N terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis. This specificity switch allows the 392N allele of ERAP2 to supplement ERAP1 activity for the removal of hydrophobic N-terminal residues. Our results provide mechanistic insight to the association of this ERAP2 polymorphism with disease and support the idea that polymorphic variation in Ag processing enzymes constitutes a component of immune response variability in humans.

Rationally designed inhibitor targeting antigen-trimming aminopeptidases enhances antigen presentation and cytotoxic T-cell responses

Significance The human immune system fights disease by eradicating sick cells after first recognizing that they are infected or cancerous. This is achieved by specialized cells that detect on the surface of other cells small molecules called antigenic peptides. Pathogens and cancer can evade the immune system by stopping the generation of antigenic peptides. We designed, synthesized and evaluated artificial small molecules that can effectively block a group of enzymes that are key for the production or destruction of antigenic peptides. We show that these compounds can enhance the generation of antigenic peptides in cells and enhance the immune system reaction toward cancer. Inhibitors of this kind may provide a new approach to coax the immune system into recognizing and eliminating cancer cells. Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2’ pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway.

Complex interactions with several arms of the complement system dictate innate and humoral immunity to adenoviral vectors

The complement system is known to play critical roles in pathogen identification, initiation of innate immune responses and facilitation of adaptive immune responses. Several studies have suggested that recombinant adenoviruses (rAds) interact with proteins of the complement system within minutes of administration. In this study, we assessed the roles of the alternative (Factor B), classical (C1q and C4) and common (C3) arms of the complement system in the innate and humoral response to systemic rAd administration using mice genetically deficient for each of these functions. Although most plasma cytokines and chemokines induced by Ads appeared to be elicited in a C3-dependent manner, we found that rAd-induced thrombocytopenia was dependent on Factor B and C3, implicating the alternative pathway as responsible for this response. Alteration of the complement-dependent transcriptome response after rAd-induced liver gene expression was also found to be Factor B- and C3-dependent. Ad interactions with the classical and alternative arms of the complement system can also be redundant, as many complement-dependent, Ad-induced innate immune responses appeared to be primarily C3-dependent. We also identified a C3 dependence of Ad-mediated induction of the nuclear factor-κB (NF-κB) activation pathway. Finally, we confirmed that humoral immune responses to the vector capsid, and the transgene it encodes, are also complement-dependent.

Wild-type adenoviruses from groups A–F evoke unique innate immune responses, of which HAd3 and SAd23 are partially complement dependent

Alternative human and non-human Ad serotype vectors are currently studied for gene therapy and/or vaccine applications to capitalize upon their likely ability to avoid pre-existing immunity to HAd5. However, relatively little attention has been given to the nature and scope of innate immune responses generated by alternative Ad serotypes. In this study, we characterized several innate immune responses after intravenous administration of wild-type Ad serotypes HAd31, HAd3, HAd5, HAd37, SAd23 and HAd41, representing groups A–F, respectively. Notably, biodistribution studies revealed significant differences between the serotypes, with high levels of HAd3 genomes found in the liver and lung, and HAd37 genomes found in the spleen after systemic administration. Relative to similar treatments with other Ad serotypes, HAd3 and SAd23 induced altered innate immune responses, illustrated by induction of higher levels of cellular gene transcription in several tissues, and higher plasma levels of cytokines and chemokines. We also investigated whether complement interactions have a role in HAd3- and SAd23-induced responses. We confirmed complement dependent gene transcription, plasma cytokine/chemokine responses, and liver toxicities incurred after administration of HAd3 and SAd23. This study highlights the potential benefits and/or limitations to the proposed use of alternative Ad serotypes for gene therapy or vaccine applications.

Endoplasmic reticulum aminopeptidase-1 alleles associated with increased risk of ankylosing spondylitis reduce HLA-B27 mediated presentation of multiple antigens

Abstract Ankylosing spondylitis (AS) is a chronic systemic arthritic disease that leads to significant disability and loss of quality of life in the ∼0.5% of the worldwide human population it affects. There is currently no cure for AS and mechanisms underlying its pathogenesis remain unclear. AS is highly genetic, with over 70% of the genetic risk being associated with the presence of HLA-B27 and endoplasmic reticulum aminopeptidase-1 (ERAP1) alleles. Furthermore, gene-gene interactions between HLA-B27 and ERAP1 AS risk alleles have recently been confirmed. Here, we demonstrate that various ERAP1 alleles can differentially mediate surface expression of antigens presented by HLA-B27 on human cells. Specifically, for all peptides tested, we found that an ERAP1 variant containing high AS risk SNPs reduced the amount of the peptide presented by HLA-B27, relative to low AS risk ERAP1 variants. These results were further validated using peptide catalysis assays in vitro, suggesting that high AS risk alleles have an enhanced catalytic activity that more rapidly destroys many HLA-B27-destined peptides, a result that correlated with decreased HLA-B27 presentation of the same peptides. These findings suggest that one mechanism underlying AS pathogenesis may involve an altered ability for AS patients harboring both HLA-B27 and high AS risk ERAP1 alleles to correctly display a variety of peptides to the adaptive arm of the immune system, potentially exposing such individuals to higher AS risk due to abnormal display of pathogen or self-derived peptides by the adaptive immune system.

Sublingual Administration of an Adenovirus Serotype 5 (Ad5)-Based Vaccine Confirms Toll-Like Receptor Agonist Activity in the Oral Cavity and Elicits Improved Mucosal and Systemic Cell-Mediated Responses against HIV Antigens despite Preexisting Ad5 Immunity

ABSTRACT HIV/AIDS continue to devastate populations worldwide. Recent studies suggest that vaccines that induce beneficial immune responses in the mucosal compartment may improve the efficacy of HIV vaccines. Adenovirus serotype 5 (Ad5)-based vectors remain a promising platform for the development of effective vaccines. In an effort to improve the efficacy of Ad5-based vaccines, even in the presence of preexisting Ad5 immunity, we evaluated the potential for an Ad5-based HIV vaccine to induce antigen-specific immune responses following sublingual (s.l.) administration, a route not previously tested in regard to Ad-based vaccines. s.l. vaccination with an Ad5-based HIV-Gag vaccine resulted in a significant induction of Gag-specific cytotoxic T-lymphocyte (CTL) responses in both the systemic and the mucosal compartment. We also show that s.l. immunization not only avoided preexisting Ad5 immunity but also elicited a broad repertoire of antigen-specific CTL clones. Additionally, we confirm for the first time that oral delivery of a vaccine expressing a potent Toll-like receptor (TLR) agonist can stimulate innate immune responses through induction of cytokines and chemokines and activation of NK cells, NKT cells, and macrophages in vivo. These results positively correlated with improved antigen-specific CTL responses. These results could be achieved both in Ad5-naïve mice and in mice with preexisting immunity to Ad5. The simplicity of the s.l. vaccination regimen coupled with augmentation of TLR-dependent pathways active in the oral cavity makes s.l. delivery a promising method for HIV vaccine development specifically, as well as for many other vaccine applications in general.

Adenovirus-based vaccination against Clostridium difficile toxin A allows for rapid humoral immunity and complete protection from toxin A lethal challenge in mice.

Clostridium difficile associated diarrhea (CDAD) is a critical public health problem worldwide with over 300,000 cases every year in the United States alone. Clearly, a potent vaccine preventing the morbidity and mortality caused by this detrimental pathogen is urgently required. However, vaccine efforts to combat C. difficile infections have been limited both in scope as well as to efficacy, as such there is not a vaccine approved for use against C. difficile to date. In this study, we have used a highly potent Adenovirus (Ad) based platform to create a vaccine against C. difficile. The Ad-based vaccine was able to generate rapid and robust humoral as well as cellular (T-cell) immune responses in mice that correlated with provision of 100% protection from lethal challenge with C. difficile toxin A. Most relevant to the clinical utility of this vaccine formulation was our result that toxin A specific IgGs were readily detected in plasma of Ad immunized mice as early as 3 days post vaccination. In addition, we found that several major immuno-dominant T cell epitopes were identified in toxin A, suggesting that the role of the cellular arm in protection from C. difficile infections may be more significant than previously appreciated. Therefore, our studies confirm that an Adenovirus based-C. difficile vaccine could be a promising candidate for prophylactic vaccination both for use in high risk patients and in high-risk environments.