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Discover how PEPperPRINT Peptide Microarray products have been used in different fields of research.

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A Quantum Vaccinomics Approach for the Design and Production of MSP4 Chimeric Antigen for the Control of Anaplasma phagocytophilum Infections

de la Fuente, José; Moraga-Fernández, Alberto; Alberdi, Pilar; Díaz-Sánchez, Sandra; García-Álvarez, Olga; Fernández-Melgar, Rubén; Contreras, Marinela
Vaccines.
Nov 2022
10.3390/vaccines10121995
Anaplasma phagocytophilum Major surface protein 4 (MSP4) plays a role during infection and multiplication in host neutrophils and tick vector cells. Recently, vaccination trials with the A. phagocytophilum antigen MSP4 in sheep showed only partial protection against pathogen infection. However, in rabbits immunized with MSP4, this recombinant antigen was protective. Differences between rabbit and sheep antibody responses are probably associated with the recognition of non-protective epitopes by IgG of immunized lambs. To address this question, we applied quantum vaccinomics to identify and characterize MSP4 protective epitopes by a microarray epitope mapping using sera from vaccinated rabbits and sheep. The identified candidate protective epitopes or immunological quantum were used for the design and production of a chimeric protective antigen. Inhibition assays of A. phagocytophilum infection in human HL60 and Ixodes scapularis tick ISE6 cells evidenced protection by IgG from sheep and rabbits immunized with the chimeric antigen. These results supported that the design of new chimeric candidate protective antigens using quantum vaccinomics to improve the protective capacity of antigens in multiple hosts.

In silico and in vitro arboviral MHC class I-restricted-epitope signatures reveal immunodominance and poor overlapping patterns

Lopes-Ribeiro, Ágata; Araujo, Franklin Pereira; Oliveira, Patrícia de Melo; Teixeira, Lorena de Almeida; Ferreira, Geovane Marques; Lourenço, Alice Aparecida; Dias, Laura Cardoso Corrêa; Teixeira, Caio Wilker; Retes, Henrique Morais; Lopes, Élisson Nogueira; Versiani, Alice Freitas; Barbosa-Stancioli, Edel Figueiredo; da Fonseca, Flávio Guimarães; Martins-Filho, Olindo Assis; Tsuji, Moriya; Peruhype-Magalhães, Vanessa; Coelho-dos-Reis, Jordana Grazziela Alves
Front. Immunol..
Nov 2022
10.3389/fimmu.2022.1035515
Introduction The present work sought to identify MHC-I-restricted peptide signatures for arbovirus using in silico and in vitro peptide microarray tools. Methods First, an in-silico analysis of immunogenic epitopes restricted to four of the most prevalent human MHC class-I was performed by identification of MHC affinity score. For that, more than 10,000 peptide sequences from 5 Arbovirus and 8 different viral serotypes, namely Zika (ZIKV), Dengue (DENV serotypes 1-4), Chikungunya (CHIKV), Mayaro (MAYV) and Oropouche (OROV) viruses, in addition to YFV were analyzed. Haplotype HLA-A*02.01 was the dominant human MHC for all arboviruses. Over one thousand HLA-A2 immunogenic peptides were employed to build a comprehensive identity matrix. Intending to assess HLAA*02:01 reactivity of peptides in vitro, a peptide microarray was designed and generated using a dimeric protein containing HLA-A*02:01. Results The comprehensive identity matrix allowed the identification of only three overlapping peptides between two or more flavivirus sequences, suggesting poor overlapping of virus-specific immunogenic peptides amongst arborviruses. Global analysis of the fluorescence intensity for peptide-HLA-A*02:01 binding indicated a dose-dependent effect in the array. Considering all assessed arboviruses, the number of DENV-derived peptides with HLA-A*02:01 reactivity was the highest. Furthermore, a lower number of YFV-17DD overlapping peptides presented reactivity when compared to non-overlapping peptides. In addition, the assessment of HLA-A*02:01-reactive peptides across virus polyproteins highlighted non-structural proteins as “hot-spots”. Data analysis supported these findings showing the presence of major hydrophobic sites in the final segment of non-structural protein 1 throughout 2a (Ns2a) and in nonstructural proteins 2b (Ns2b), 4a (Ns4a) and 4b (Ns4b). Discussion To our knowledge, these results provide the most comprehensive and detailed snapshot of the immunodominant peptide signature for arbovirus with MHC-class I restriction, which may bring insight into the design of future virus-specific vaccines to arboviruses and for vaccination protocols in highly endemic areas.

Binding epitope for recognition of human TRPM4 channel by monoclonal antibody M4M

Wei, Shunhui; Behn, Julian; Poore, Charlene Priscilla; Low, See Wee; Nilius, Bernd; Fan, Hao; Liao, Ping
Sci Rep.
Nov 2022
10.1038/s41598-022-22077-4
Abstract Mouse monoclonal antibody M4M was recently designed to block human TRPM4 channel. The polypeptide for generating M4M is composed of peptide A1 between the transmembrane segment 5 (S5) and the pore, and a second peptide A2 between the pore and the transmembrane segment 6 (S6). Using peptide microarray, a 4-amino acid sequence EPGF within the A2 was identified to be the binding epitope for M4M. Substitution of EPGF with other amino acids greatly reduced binding affinity. Structural analysis of human TRPM4 structure indicates that EPGF is located externally to the channel pore. A1 is close to the EPGF binding epitope in space, albeit separated by a 37-amino acid peptide. Electrophysiological study reveals that M4M could block human TRPM4, but with no effect on rodent TRPM4 which shares a different amino acid sequence ERGS for the binding motif. Our results demonstrate that M4M is a specific inhibitor for human TRPM4.

Vivaxin genes encode highly immunogenic, non-variant antigens on the Trypanosoma vivax cell-surface

Romero-Ramirez, Alessandra; Casas-Sánchez, Aitor; Autheman, Delphine; Duffy, Craig W.; Brandt, Cordelia; Clare, Simon; Harcourt, Katherine; André, Marcos Rogério; de Almeida Castilho Neto, Kayo José Garcia; Teixeira, Marta M. G.; Machado, Rosangela Zacharias; Coombes, Janine; Flynn, Robin J.; Wright, Gavin J.; Jackson, Andrew P.
PLoS Negl Trop Dis.
Sep 2022
10.1371/journal.pntd.0010791
Trypanosoma vivax is a unicellular hemoparasite, and a principal cause of animal African trypanosomiasis (AAT), a vector-borne and potentially fatal livestock disease across sub-Saharan Africa. Previously, we identified diverse T. vivax-specific genes that were predicted to encode cell surface proteins. Here, we examine the immune responses of naturally and experimentally infected hosts to these unique parasite antigens, to identify immunogens that could become vaccine candidates. Immunoprofiling of host serum shows that one particular family (Fam34) elicits a consistent IgG antibody response. This gene family, which we now call Vivaxin, encodes at least 124 transmembrane glycoproteins that display quite distinct expression profiles and patterns of genetic variation. We focused on one gene (viv-β8) that encodes one particularly immunogenic vivaxin protein and which is highly expressed during infections but displays minimal polymorphism across the parasite population. Vaccination of mice with VIVβ8 adjuvanted with Quil-A elicits a strong, balanced immune response and delays parasite proliferation in some animals but, ultimately, it does not prevent disease. Although VIVβ8 is localized across the cell body and flagellar membrane, live immunostaining indicates that VIVβ8 is largely inaccessible to antibody in vivo. However, our phylogenetic analysis shows that vivaxin includes other antigens shown recently to induce immunity against T. vivax. Thus, the introduction of vivaxin represents an important advance in our understanding of the T. vivax cell surface. Besides being a source of proven and promising vaccine antigens, the gene family is clearly an important component of the parasite glycocalyx, with potential to influence host-parasite interactions.

The Correlation between Subolesin-Reactive Epitopes and Vaccine Efficacy

Contreras, Marinela; Kasaija, Paul D.; Kabi, Fredrick; Mugerwa, Swidiq; De la Fuente, José
Vaccines.
Aug 2022
10.3390/vaccines10081327
Vaccination is an environmentally-friendly alternative for tick control. The tick antigen Subolesin (SUB) has shown protection in vaccines for the control of multiple tick species in cattle. Additionally, recent approaches in quantum vaccinomics have predicted SUB-protective epitopes and the peptide sequences involved in protein–protein interactions in this tick antigen. Therefore, the identification of B-cell–reactive epitopes by epitope mapping using a SUB peptide array could be essential as a novel strategy for vaccine development. Subolesin can be used as a model to evaluate the effectiveness of these approaches for the identification of protective epitopes related to vaccine protection and efficacy. In this study, the mapping of B-cell linear epitopes of SUB from three different tick species common in Uganda (Rhipicephalus appendiculatus, R. decoloratus, and Amblyomma variegatum) was conducted using serum samples from two cattle breeds immunized with SUB-based vaccines. The results showed that in cattle immunized with SUB from R. appendiculatus (SUBra) all the reactive peptides (Z-score > 2) recognized by IgG were also significant (Z-ratio > 1.96) when compared to the control group. Additionally, some of the reactive peptides recognized by IgG from the control group were also recognized in SUB cocktail–immunized groups. As a significant result, cattle groups that showed the highest vaccine efficacy were Bos indicus immunized with a SUB cocktail (92%), and crossbred cattle were immunized with SUBra (90%) against R. appendiculatus ticks; the IgG from these groups recognized overlapping epitopes from the peptide SPTGLSPGLSPVRDQPLFTFRQVGLICERMMKERESQIRDEYDHVLSAKLAEQYDTFVKFTYDQKRFEGATPSYLS (Z-ratio > 1.96), which partially corresponded to a Q38 peptide and the SUB protein interaction domain. These identified epitopes could be related to the protection and efficacy of the SUB-based vaccines, and new chimeras containing these protective epitopes could be designed using this new approach.

A heterotypic assembly mechanism regulates CHIP E3 ligase activity

Das, Aniruddha; Thapa, Pankaj; Santiago, Ulises; Shanmugam, Nilesh; Banasiak, Katarzyna; Dąbrowska, Katarzyna; Nolte, Hendrik; Szulc, Natalia A; Gathungu, Rose M; Cysewski, Dominik; Krüger, Marcus; Dadlez, Michał; Nowotny, Marcin; Camacho, Carlos J; Hoppe, Thorsten; Pokrzywa, Wojciech
The EMBO Journal.
Aug 2022
10.15252/embj.2021109566
CHIP (C‐terminus of Hsc70‐interacting protein) and its worm ortholog CHN‐1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin‐proteasome system (UPS). CHN‐1 can cooperate with UFD‐2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN‐1–UFD‐2 complex in Caenorhabditis elegans. Our data show that UFD‐2 binding promotes the cooperation between CHN‐1 and ubiquitin‐conjugating E2 enzymes by stabilizing the CHN‐1 U‐box dimer. However, HSP70/HSP‐1 chaperone outcompetes UFD‐2 for CHN‐1 binding, thereby promoting a shift to the autoinhibited CHN‐1 state by acting on a conserved residue in its U‐box domain. The interaction with UFD‐2 enables CHN‐1 to efficiently ubiquitylate and regulate S‐adenosylhomocysteinase (AHCY‐1), a key enzyme in the S‐adenosylmethionine (SAM) regeneration cycle, which is essential for SAM‐dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN‐1 and UFD‐2 in substrate ubiquitylation.

Analysis of the Immune Response and Identification of Antibody Epitopes Against the Sigma C Protein of Avian Orthoreovirus Following Immunization with Live or Inactivated Vaccines

Dawe, W. H.; Kapczynski, D. R.; Linnemann, E. G.; Gauthiersloan, V. R.; Sellers, H. S.
Avian Diseases.
Jan 2022
10.1637/aviandiseases-D-22-99992

Immunity to Influenza is dependent on MHC II polymorphism: study with 2 HLA transgenic strains

Luckey, David; Weaver, Eric A.; Osborne, Douglas G.; Billadeau, Daniel D.; Taneja, Veena
Sci Rep.
Dec 2019
10.1038/s41598-019-55503-1
Major histocompatibility complex II (MHC II) molecules are involved in antigen presentation and the development of a functional adaptive immune response. Evolutionary selection for MHC molecules that effectively clear infectious agents provides an advantage to humans. However, certain class II molecules are associated with autoimmune diseases. In this study we infected autoimmune-susceptible DRB1*0401.AEo and non-susceptible *0402.AEo mice with H1N1 influenza and determined clearance and protective immunity to H3N2 virus. *0401 mice generated a robust TLR-triggered immune response and cleared H1N1 influenza virus infection. After vaccination and challenge with H1N1, *0401 mice, when challenged with H3N2, generated cross-protective immunity to heterosubtypic H3N2 influenza strain whereas *0402 mice cleared the H1N1 infection but did not generate cross-protective immunity against the H3N2 influenza strain. The intracellular trafficking route of MHCII revealed that *0401 molecules traffic through the late endosome/lysosomes while *0402 molecules traffic into early endosomes. This suggested that trafficking of MHCII could affect the functional output of the innate immune response and clearance of viral infections. Also, DRB1*0401 mice live longer than HLA-DRB1*0402 mice. The study provides a potential hypothesis for evolutionary selection of *0401 molecule, even though it is associated with autoreactivity, which may be dependent on the availability of peptide repertoire of self-antigens.

Probing peptide sequences on their ability to generate affinity sites in molecularly imprinted polymers

Piletska, Elena V; Guerreiro, Antonio; Mersiyanova, Margarita; Cowen, Todd; Canfarotta, Francesco; Piletsky, Stanislav S.; Karim, Kal; Piletsky, Sergey A.
Langmuir.
Dec 2019
10.1021/acs.langmuir.9b03410
An array of 4000 defined and addressable tripeptides on a polymer-coated glass slide is used to synthesize molecularly imprinted polymer (MIP) nanoparticles. This work is undertaken to systematically probe the impact of the peptide sequence on the ability to generate affinity MIPs. The polymer affinity is assessed by measuring the fluorescence of bound MIP nanoparticles at each peptide spot on the surface after washing the array to remove any low-affinity polymer. The generic composition commonly used in the preparation of MIPs against proteins seems to be equally suitable for imprinting hydrophobic and hydrophilic tripeptides. The amino acids frequently contributing to the formation of high-affinity MIPs include T, F, D, N, Y, W, and P. The amino acids that rarely contribute to the formation of high-affinity interactions with MIPs are G, V, A, L, I, and M. These observations are confirmed by computational modeling. The basic technique proposed here may be applicable in optimizing polymer compositions for the production of high-affinity MIPs or, more specifically, for the selection of appropriate amino acid sequences when peptide epitopes are used instead of whole protein imprinting.

Analysis of humoral immune responses in chikungunya virus (CHIKV) infected patients and individuals vaccinated with a candidate CHIKV vaccine

Henss, Lisa; Yue, Constanze; von Rhein, Christine; Tschismarov, Roland; Lewis-Ximenez, Lia Laura; Dölle, Albert; Baylis, Sally A; Schnierle, Barbara S
Dec 2019
10.1093/infdis/jiz658
Abstract Background Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe flu like symptoms. The acute symptoms disappear after one week, but chronic arthralgia can persist for years. Here, humoral immune responses in CHIKV-infected patients and vaccinees were analyzed. Methods Alphavirus neutralization activity was analyzed with pseudotyped lentiviral vectors and antibody epitope mapping was performed with a peptide array. Results Greatest CHIKV neutralization activity was observed 60-92 days after onset of symptoms. The amount of CHIKV-specific antibodies, their binding avidity and cross-reactivity with other alphaviruses increased over time. CHIKV and o’nyong-nyong virus (ONNV) were both neutralized to a similar extent. Linear antibody binding epitopes were mainly found in E2 domain B and the acid-sensitive regions (ASRs). In addition, serum samples from healthy volunteers vaccinated with a measles-vectored chikungunya vaccine candidate, MV-CHIK, were analyzed. Neutralization activity in the samples from the vaccine cohort was 2–6-fold lower than in samples from CHIKV-infected patients. In contrast to infection, vaccination only induced cross-neutralization with ONNV and the E2 ASR1 was the major antibody target. Conclusion These data could assist vaccine design and enable the identification of correlates of protection necessary for vaccine efficacy.

Diagnostic Profiling of the Human Public IgM Repertoire With Scalable Mimotope Libraries

Pashov, Anastas; Shivarov, Velizar; Hadzhieva, Maya; Kostov, Victor; Ferdinandov, Dilyan; Heintz, Karen-Marie; Pashova, Shina; Todorova, Milena; Vassilev, Tchavdar; Kieber-Emmons, Thomas; Meza-Zepeda, Leonardo A.; Hovig, Eivind
Front. Immunol..
Dec 2019
10.3389/fimmu.2019.02796
Specific antibody reactivities are routinely used as biomarkers, but the antibody repertoire reactivity (igome) profiles are still neglected. Here, we propose rationally designed peptide arrays as efficient probes for these system level biomarkers. Most IgM antibodies are characterized by few somatic mutations, polyspecificity, and physiological autoreactivity with housekeeping function. Previously, probing this repertoire with a set of immunodominant self-proteins provided a coarse analysis of the respective repertoire profiles. In contrast, here, we describe the generation of a peptide mimotope library that reflects the common IgM repertoire of 10,000 healthy donors. In addition, an appropriately sized subset of this quasi-complete mimotope library was further designed as a potential diagnostic tool. A 7-mer random peptide phage display library was panned on pooled human IgM. Next-generation sequencing of the selected phage yielded 224,087 sequences, which clustered in 790 sequence clusters. A set of 594 mimotopes, representative of the most significant sequence clusters, was shown to probe symmetrically the space of IgM reactivities in patients’ sera. This set of mimotopes can be easily scaled including a greater proportion of the mimotope library. The trade-off between the array size and the resolution can be explored while preserving the symmetric sampling of the mimotope sequence and reactivity spaces. BLAST search of the non-redundant protein database with the mimotopes sequences yielded significantly more immunoglobulin J region hits than random peptides, indicating a considerable idiotypic connectivity of the targeted igome. The proof of principle predictors for random diagnoses was represented by profiles of mimotopes. The number of potential reactivity profiles that can be extracted from this library is estimated at more than 1070. Thus, a quasi-complete IgM mimotope library and a scalable representative subset thereof are found to address very efficiently the dynamic diversity of the human public IgM repertoire, providing informationally dense and structurally interpretable IgM reactivity profiles.

A Low‐Cost Laser‐Based Nano‐3D Polymer Printer for Rapid Surface Patterning and Chemical Synthesis of Peptide and Glycan Microarrays

Eickelmann, Stephan; Tsouka, Alexandra; Heidepriem, Jasmin; Paris, Grigori; Zhang, Junfang; Molinari, Valerio; Mende, Marco; Loeffler, Felix F.
Adv. Mater. Technol..
Nov 2019
10.1002/admt.201900503
A low-cost laser-based printing setup is presented, which allows for the spot-wise patterning of surfaces with defined polymer nanolayers. These nanolayer spots serve as a “solid solvent,” embedding different chemicals, chemical building blocks, materials, or precursors and can be stacked on top of each other. By melting the spot pattern, the polymer-embedded molecules are released for chemical reaction. This enables researchers to quickly pattern a surface with different molecules and materials, mixing them directly on the surface for high-throughput chemical synthesis to generate and screen diverse microarray libraries. In contrast to expensive ink-jet or contact printing, this approach does not require premixing of inks, which enables in situ combinatorial mixing. Easy access and versatility of this patterning approach are shown by generating microarrays of various biomolecules, such as glycans for the first time, to screen interactions of antibodies and lectins. In addition, a layer-by-layer solid-phase synthesis of peptides directly on the microarray is presented. Amino acid–containing nanolayers are repeatedly laser-transferred and reacted with the functionalized acceptor surface in defined patterns. This simple system enables a reproducible array production, down to spot-to-spot distances of 100 µm, and offers a flexible and cheap alternative to expensive spotting robot technology.

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