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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

Mus81-Mms4 endonuclease is an Esc2-STUbL-Cullin8 mitotic substrate impacting on genome integrity

Waizenegger, Anja; Urulangodi, Madhusoodanan; Lehmann, Carl P.; Reyes, Teresa Anne Clarisse; Saugar, Irene; Tercero, José Antonio; Szakal, Barnabas; Branzei, Dana
Nov 2020
10.1038/s41467-020-19503-4
The Mus81-Mms4 nuclease is activated in G2/M via Mms4 phosphorylation to allow resolution of persistent recombination structures. However, the fate of the activated phosphorylated Mms4 remains unknown. Here we find that Mms4 is engaged by (poly)SUMOylation and ubiquitylation and targeted for proteasome degradation, a process linked to the previously described Mms4 phosphorylation cycle. Mms4 is a mitotic substrate for the SUMO-Targeted Ubiquitin ligase Slx5/8, the SUMO-like domain-containing protein Esc2, and the Mms1-Cul8 ubiquitin ligase. In the absence of these activities, phosphorylated Mms4 accumulates on chromatin in an active state in the next G1, subsequently causing abnormal processing of replication-associated recombination intermediates and delaying the activation of the DNA damage checkpoint. Mus81-Mms4 mutants that stabilize phosphorylated Mms4 have similar detrimental effects on genome integrity. Overall, our findings highlight a replication protection function for Esc2-STUbL-Cul8 and emphasize the importance for genome stability of resetting phosphorylated Mms4 from one cycle to another.

Rapid response to pandemic threats: immunogenic epitope detection of pandemic pathogens for diagnostics and vaccine development using peptide microarrays

Heiss, Kirsten; Heidepriem, Jasmin; Fischer, Nico; Weber, Laura K; Dahlke, Christine; Jaenisch, Thomas; Loeffler, Felix F
J. Proteome Res..
Sep 2020
10.1021/acs.jproteome.0c00484
Emergence and re-emergence of pathogens bearing the risk of becoming a pandemic threat are on the rise. Increased travel and trade, growing population density, changes in urbanization, and climate have a critical impact on infectious disease spread. Currently, the world is confronted with the emergence of a novel coronavirus SARS-CoV-2, responsible for yet more than 500 000 deaths globally. Outbreaks caused by viruses such as SARS-CoV-2, HIV, Ebola, influenza, and Zika have increased over the last decade, underlining the urgent need for a rapid development of diagnostics and vaccines. Hence, the rational identification of biomarkers for diagnostic measures on the one hand, and antigenic targets for vaccine development on the other, are of utmost importance. Peptide microarrays can display large numbers of putative target proteins translated into overlapping linear (and cyclic) peptides. Using these highly diverse libraries, covering tens of thousands of peptides, allow for the in-depth analysis of antibody signatures in a multiplexed, high-throughput fashion. In this review, we highlight synthesis platforms that facilitate fast and highly flexible generation of high-density peptide microarrays. We further outline the multifaceted applications of these peptide array platforms for the development of serological tests and vaccines, to quickly encounter pandemic threats.

Expression of different L1 isoforms of Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity

Fu, Yingying; Cao, Rui; Schäfer, Miriam; Stephan, Sonja; Braspenning-Wesch, Ilona; Schmitt, Laura; Bischoff, Ralf; Müller, Martin; Schäfer, Kai; Vinzón, Sabrina E; Rösl, Frank; Hasche, Daniel
Aug 2020
10.7554/eLife.57626
Although many high-risk mucosal and cutaneous human papillomaviruses (HPVs) theoretically have the potential to synthesize L1 isoforms differing in length, previous seroepidemiological studies only focused on the short L1 variants, co-assembling with L2 to infectious virions. Using the multimammate mouse Mastomys coucha as preclinical model, this is the first study demonstrating seroconversion against different L1 isoforms during the natural course of papillomavirus infection. Intriguingly, positivity with the cutaneous MnPV was accompanied by a strong seroresponse against a longer L1 isoform, but to our surprise, the raised antibodies were non-neutralizing. Only after a delay of around 4 months, protecting antibodies against the short L1 appeared, enabling the virus to successfully establish an infection. This argues for a novel humoral immune escape mechanism that may also have important implications on the interpretation of epidemiological data in terms of seropositivity and protection of PV infections in general.

Epitopes of Naturally Acquired and Vaccine‐induced Anti‐Ebola Virus Glycoprotein Antibodies in Single Amino Acid Resolution

Heidepriem, Jasmin; Krähling, Verena; Dahlke, Christine; Wolf, Timo; Klein, Florian; Addo, Marylyn M.; Becker, Stephan; Loeffler, Felix F.
Biotechnol. J..
May 2020
10.1002/biot.202000069
The Ebola virus (EBOV) can cause severe infections in humans, leading to a fatal outcome in a high percentage of cases. Neutralizing antibodies against the EBOV surface glycoprotein (GP) can prevent infections, demonstrating a straightforward way for an efficient vaccination strategy. Meanwhile, many different anti-EBOV antibodies have been identified, whereas the exact binding epitopes are often unknown. Here, the analysis of serum samples from an EBOV vaccine trial with the recombinant vesicular stomatitis virus-Zaire ebolavirus (rVSV-ZEBOV) and an Ebola virus disease survivor, using high-density peptide arrays, is presented. In this proof-of-principle study, distinct IgG and IgM antibodies binding to different epitopes of EBOV GP is detected: By mapping the whole GP as overlapping peptide fragments, new epitopes and confirmed epitopes from the literature are found. Furthermore, the highly selective binding epitope of a neutralizing monoclonal anti-EBOV GP antibody could be validated. This shows that peptide arrays can be a valuable tool to study the humoral immune response to vaccines in patients and to support Ebola vaccine development.

Fibrinogen interaction with complement C3: a potential therapeutic target to reduce thrombosis risk

King, Rhodri J.; Schuett, Katharina; Tiede, Christian; Jankowski, Vera; John, Vicky; Trehan, Abhi; Simmons, Katie; Ponnambalam, Sreenivasan; Storey, Robert F.; Fishwick, Colin W.G.; McPherson, Michael J.; Tomlinson, Darren C.; Ajjan, Ramzi A.
Haematologica.
Apr 2020
10.3324/haematol.2019.239558
Complement C3 binds fibrinogen and compromises fibrin clot lysis thereby enhancing thrombosis risk. We investigated the role of fibrinogen-C3 interaction as a novel therapeutic target to reduce thrombosis risk by analysing: i) consistency in the fibrinolytic properties of C3, ii) binding sites between fibrinogen and C3 and iii) modulation of fibrin clot lysis by manipulating fibrinogen-C3 interactions. Purified fibrinogen and C3 from the same individuals (n=24) were used to assess inter-individual variability in the anti-fibrinolytic effects of C3. Microarray screening and molecular modelling evaluated C3 and fibrinogen interaction sites. Novel synthetic conformational proteins, termed Affimers, were used to modulate C3-fibrinogen interaction and fibrinolysis. C3 purified from patients with type 1 diabetes showed enhanced prolongation of fibrinolysis compared with healthy control protein [195±105 and 522±166 seconds, respectively (p=0.04)], with consistent effects but a wider range (5-51% and 5-18% lysis prolongation, respectively). Peptide microarray screening identified 2 potential C3-fibrinogen interactions sites within fibrinogen β chain (residues 424-433, 435-445). One fibrinogen-binding Affimer was isolated that displayed sequence identity with C3 in an exposed area of the protein. This Affimer abolished C3-induced prolongation of fibrinolysis (728±25.1 seconds to 632±23.7 seconds, p=0.005) and showed binding to fibrinogen in the same region that is involved in C3-fibrinogen interactions. Moreover, it shortened plasma clot lysis of patients with diabetes, cardiovascular disease or controls by 7-11%. C3 binds fibrinogen β-chain and disruption of fibrinogen-C3 interaction using Affimer proteins enhances fibrinolysis, which represents a potential novel target tool to reduce thrombosis in high risk individuals.

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