SPOT Synthesis Research Articles

Enhanced Assessment of Cross-Reactive Antigenic Determinants within the Spike Protein.

Despite successful vaccination efforts, the emergence of new SARS-CoV-2 variants poses ongoing challenges to control COVID-19. Understanding humoral responses regarding SARS-CoV-2 infections and their impact is crucial for developing future vaccines that are effective worldwide. Here, we identified 41 immunodominant linear B-cell epitopes in its spike glycoprotein with an SPOT synthesis peptide array probed with a pool of serum from hospitalized COVID-19 patients. The bioinformatics showed a restricted set of epitopes unique to SARS-CoV-2 compared to other coronavirus family members. Potential crosstalk was also detected with Dengue virus (DENV), which was confirmed by screening individuals infected with DENV before the COVID-19 pandemic in a commercial ELISA for anti-SARS-CoV-2 antibodies. A high-resolution evaluation of antibody reactivity against peptides representing epitopes in the spike protein identified ten sequences in the NTD, RBD, and S2 domains. Functionally, antibody-dependent enhancement (ADE) in SARS-CoV-2 infections of monocytes was observed in vitro with pre-pandemic Dengue-positive sera. A significant increase in viral load was measured compared to that of the controls, with no detectable neutralization or considerable cell death, suggesting its role in viral entry. Cross-reactivity against peptides from spike proteins was observed for the pre-pandemic sera. This study highlights the importance of identifying specific epitopes generated during the humoral response to a pathogenic infection to understand the potential interplay of previous and future infections on diseases and their impact on vaccinations and immunodiagnostics.

Patterns of Diversity and Humoral Immunogenicity for HIV-1 Antisense Protein (ASP).

HIV-1 has an antisense gene overlapping env that encodes the ASP protein. ASP functions are still unknown, but it has been associated with gp120 in the viral envelope and membrane of infected cells, making it a potential target for immune response. Despite this, immune response patterns against ASP are poorly described and can be influenced by the high genetic variability of the env gene. To explore this, we analyzed 100k HIV-1 ASP sequences from the Los Alamos HIV sequence database using phylogenetic, Shannon entropy (Hs), and logo tools to study ASP variability in worldwide and Brazilian sequences from the most prevalent HIV-1 subtypes in Brazil (B, C, and F1). Data obtained in silico guided the design and synthesis of 15-mer overlapping peptides through spot synthesis on cellulose membranes. Peptide arrays were screened to assess IgG and IgM responses in pooled plasma samples from HIV controllers and individuals with acute or recent HIV infection. Excluding regions with low alignment accuracy, several sites with higher variability (Hs > 1.5) were identified among the datasets (25 for worldwide sequences, 20 for Brazilian sequences). Among sites with Hs < 1.5, sequence logos allowed the identification of 23 other sites with subtype-specific signatures. Altogether, amino acid variations with frequencies > 20% in the 48 variable sites identified were included in 92 peptides, divided into 15 sets, representing near full-length ASP. During the immune screening, the strongest responses were observed in three sets, one in the middle and one at the C-terminus of the protein. While some sets presented variations potentially associated with epitope displacement between IgG and IgM targets and subtype-specific signatures appeared to impact the level of response for some peptides, signals of cross-reactivity were observed for some sets despite the presence of B/C/F1 signatures. Our data provides a map of ASP regions preferentially targeted by IgG and IgM responses. Despite B/C/F1 subtype signatures in ASP, the amino acid variation in some areas preferentially targeted by IgM and IgG did not negatively impact the response against regions with higher immunogenicity.

Redefining Peptide 14D: Substitutional Analysis for Accelerated TB Diagnosis and Enhanced Activity against Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a predominant cause of mortality, especially in low- and middle-income nations. Recently, antimicrobial peptides have been discovered that at low concentrations could stimulate the growth of M. tuberculosis (hormetic response). In this study, such a peptide was used to investigate the effects on the time to positivity (TTP). A systematic substitution analysis of peptide 14D was synthesized using Spot synthesis technology, resulting in 171 novel peptides. Our findings revealed a spectrum of interactions, with some peptides accelerating M. tuberculosis growth, potentially aiding in faster diagnostics, while others exhibited inhibitory effects. Notably, peptide NH2-wkivfiwrr-CONH2 significantly reduced the TTP by 25 h compared to the wild-type peptide 14D, highlighting its potential in improving TB diagnostics by culture. Several peptides demonstrated potent antimycobacterial activity, with a minimum inhibitory concentration (MIC) of 20 µg/mL against H37Rv and a multidrug-resistant M. tuberculosis strain. Additionally, for two peptides, a strongly diminished formation of cord-like structures was observed, which is indicative of reduced virulence and transmission potential. This study underscores the multifaceted roles of antimicrobial peptides in TB management, from enhancing diagnostic efficiency to offering therapeutic avenues against M. tuberculosis.

Multilingual sound sopt synthesis systems

Localized sound zone synthesis, which can generate acoustically bright and dark zones using loudspeakers, is gaining attention as one of important acoustic communication techniques. By superposing multiple localized sound zones, multiple sound spot synthesis can simultaneously deriver different sound signals at different zones using a loudspeaker array. Additionally, it can be implemented as a user interface for real-time speech translation by combining multilingual speech synthesis technology. This paper introduces multilingual sound spot synthesis systems based on spatial Fourier transform implemented by using compact circular array of 16 loudspeakers and linear array of 64 loudspeakers combined with multilingual neural speech synthesis.

Mapping IgA Epitope and Cross-Reactivity between Severe Acute Respiratory Syndrome-Associated Coronavirus 2 and DENV.

The newly introduced COVID-19 vaccines have reduced disease severity and hospitalizations. However, they do not significantly prevent infection or transmission. In the same context, measuring IgM and IgG antibody levels is important, but it does not provide information about the status of the mucosal immune response. This article describes a comprehensive mapping of IgA epitopes of the S protein, its cross-reactivity, and the development of an ELISA-peptide assay. IgA epitope mapping was conducted using SPOT synthesis and sera from RT-qPCR COVID-19-positive patients. Specific and cross-reacting epitopes were identified, and an evolutionary analysis from the early Wuhan strain to the Omicron variant was performed using bioinformatics tools and a microarray of peptides. The selected epitopes were chemically synthesized and evaluated using ELISA-IgA. A total of 40 IgA epitopes were identified with 23 in S1 and 17 in the S2 subunit. Among these, at least 23 epitopes showed cross-reactivity with DENV and other organisms and 24 showed cross-reactivity with other associated coronaviruses. Three MAP4 polypeptides were validated by ELISA, demonstrating a sensitivity of 90-99.96% and a specificity of 100%. Among the six IgA-RBD epitopes, only the SC/18 epitope of the Omicron variants (BA.2 and BA.2.12.1) presented a single IgA epitope. This research unveiled the IgA epitome of the S protein and identified many epitopes that exhibit cross-reactivity with DENV and other coronaviruses. The S protein of variants from Wuhan to Omicron retains many conserved IgA epitopes except for one epitope (#SCov/18). The cross-reactivity with DENV suggests limitations in using the whole S protein or the S1/S2/RBD segment for IgA serological diagnostic tests for COVID-19. The expression of these identified specific epitopes as diagnostic biomarkers could facilitate monitoring mucosal immunity to COVID-19, potentially leading to more accurate diagnoses and alternative mucosal vaccines.

Mapping possible interaction sites for crotoxin in CNF, a gamma PLA2 inhibitor from Crotalus durissus terrificus rattle snake, using SPOT synthesis

Phospholipases A2 (PLA2s) are main components of snake venoms. Several snake species possess endogenous PLA2 inhibitors in their circulating blood, which are generally known as sbPLIs (an acronym for snake blood phospholipase A2inhibitors). The sbPLIs are categorized in three classes (alpha, beta or gamma) depending on the existence of distinguishing protein domains in their structure. The Crotalus durrissus terrificus venom has a highly neurotoxic PLA2 - crotoxin (CTX) - in its composition and the self-protection of the snake is mainly ensured by a sbγPLI named CNF (standing for Crotalusneutralizing factor). In an attempt to find smaller molecules able to inhibit the catalytic activity of CTX, in the present study we used linear peptide arrays to identify CNF segments possibly involved in the interaction with the toxin. Five reacting segments were identified as possible interacting regions. The target peptides were synthesized and located in the in silico CNF structure. Although all of them are exposed to the solvent, high concentrations were needed to inhibit the PLA2 activity of the whole venom or CTX. Limitations of the methodology employed and particular characteristics of CTX inhibition by CNF are discussed.

High-Throughput IgG Epitope Mapping of Tetanus Neurotoxin: Implications for Immunotherapy and Vaccine Design

Tetanus is an acute, fatal disease caused by exotoxins released from Clostridium tetani during infections. A protective humoral immune response can be induced by vaccinations with pediatric and booster combinatorial vaccines that contain inactivated tetanus neurotoxin (TeNT) as a major antigen. Although some epitopes in TeNT have been described using various approaches, a comprehensive list of its antigenic determinants that are involved with immunity has not been elucidated. To this end, a high-resolution analysis of the linear B-cell epitopes in TeNT was performed using antibodies generated in vaccinated children. Two hundred sixty-four peptides that cover the entire coding sequence of the TeNT protein were prepared in situ on a cellulose membrane through SPOT synthesis and probed with sera from children vaccinated (ChVS) with a triple DTP-vaccine to map continuous B-cell epitopes, which were further characterized and validated using immunoassays. Forty-four IgG epitopes were identified. Four (TT-215-218) were chemically synthesized as multiple antigen peptides (MAPs) and used in peptide ELISAs to screen post-pandemic DTP vaccinations. The assay displayed a high performance with high sensitivity (99.99%) and specificity (100%). The complete map of linear IgG epitopes induced by vaccination with inactivated TeNT highlights three key epitopes involved in the efficacy of the vaccine. Antibodies against epitope TT-8/G can block enzymatic activity, and those against epitopes TT-41/G and TT-43/G can interfere with TeNT binding to neuronal cell receptors. We further show that four of the epitopes identified can be employed in peptide ELISAs to assess vaccine coverage. Overall, the data suggest a set of select epitopes to engineer new, directed vaccines.

B-Cell Epitope Mapping of the Vibrio cholera Toxins A, B, and P and an ELISA Assay.

Oral immunization with the choleric toxin (CT) elicits a high level of protection against its enterotoxin activities and can control cholera in endemic settings. However, the complete B-cell epitope map of the CT that is responsible for protection remains to be clarified. A library of one-hundred, twenty-two 15-mer peptides covering the entire sequence of the three chains of the CT protein (CTP) was prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice vaccinated with an oral inactivated vaccine (Schankol™) allowed the mapping of continuous B-cell epitopes, topological studies, multi-antigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Eighteen IgG epitopes were identified; eight in the CTA, three in the CTB, and seven in the protein P. Three V. cholera specific epitopes, Vc/TxA-3, Vc/TxB-11, and Vc/TxP-16, were synthesized as MAP4 and used to coat ELISA plates in order to screen immunized mouse sera. Sensitivities and specificities of 100% were obtained with the MAP4s of Vc/TxA-3 and Vc/TxB-11. The results revealed a set of peptides whose immunoreactivity reflects the immune response to vaccination. The array of peptide data can be applied to develop improved serological tests in order to detect cholera toxin exposure, as well as next generation vaccines to induce more specific antibodies against the cholera toxin.

Design, synthesis, and structural activity relationship of antimicrobial peptides against multi-drug resistant organisms

Antimicrobial peptides (AMPs) are a family of tiny peptides found throughout nature that play a vital role in various organisms' innate immune systems. Antimicrobial peptides and proteins (AMPs) are a varied family of naturally occurring chemicals produced by all multicellular organisms as a first line of defense. The rising prevalence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) diseases is the most evident cause for concern. Traditional antimicrobial drugs work by targeting certain metabolic pathways or microbiological structures to kill or hinder bacterial growth. The development of new lead structures to combat multidrug-resistant bacteria is critical, and cationic antimicrobial peptides (AMPs) have the ability to do so. Based on the SPOT synthesis, we present an outline of a strategy for screening peptides for antimicrobial activity. Multidrug-resistant bacteria have grown fast in recent decades, leading to a rise in nosocomial infections and in-hospital mortality, as well as posing a threat to world health. We can say that he discovery of antibiotics signaled the start of a golden period in human medicine.

Multiepitope Proteins for the Differential Detection of IgG Antibodies against RBD of the Spike Protein and Non-RBD Regions of SARS-CoV-2.

The COVID-19 pandemic has exposed the extent of global connectivity and collective vulnerability to emerging diseases. From its suspected origins in Wuhan, China, it spread to all corners of the world in a matter of months. The absence of high-performance, rapid diagnostic methods that could identify asymptomatic carriers contributed to its worldwide transmission. Serological tests offer numerous benefits compared to other assay platforms to screen large populations. First-generation assays contain targets that represent proteins from SARS-CoV-2. While they could be quickly produced, each actually has a mixture of specific and non-specific epitopes that vary in their reactivity for antibodies. To generate the next generation of the assay, epitopes were identified in three SARS-Cov-2 proteins (S, N, and Orf3a) by SPOT synthesis analysis. After their similarity to other pathogen sequences was analyzed, 11 epitopes outside of the receptor-binding domain (RBD) of the spike protein that showed high reactivity and uniqueness to the virus. These were incorporated into a ß-barrel protein core to create a highly chimeric protein. Another de novo protein was designed that contained only epitopes in the RBD. In-house ELISAs suggest that both multiepitope proteins can serve as targets for high-performance diagnostic tests. Our approach to bioengineer chimeric proteins is highly amenable to other pathogens and immunological uses.

Identification of a Neutralizing Epitope on TOSV Gn Glycoprotein.

Emerging and re-emerging viral infections have been an important public health problem in recent years. We focused our attention on Toscana virus (TOSV), an emergent neurotropic negative-strand RNA virus of the Phenuiviridae family. The mechanisms of protection against phlebovirus natural infection are not known; however, it is supposed that a virus-neutralizing antibody response against viral glycoproteins would be useful to block the first stages of infection. By using an improved memory B cell immortalization method, we obtained a panel of human mAbs which reacted with TOSV antigens. We identified three epitopes of TOSV Gn glycoproteins by neutralizing mAbs using synthetic peptide arrays on membrane support (SPOT synthesis). These epitopes, separated in primary structure, might be exposed near one another as a conformational epitope in their native structure. In vivo studies were conducted to evaluate the humoral response elicited in mice immunized with the identified peptides. The results underlined the hypothesis that the first two peptides located in the NH2 terminus could form a conformational epitope, while the third, located near the transmembrane sequence in the carboxyl terminus, was necessary to strengthen neutralizing activity. Our results emphasize the importance of identifying neutralizing epitopes shared among the various phleboviruses, which could be exploited for the development of a potential epitope-based diagnostic assay or a polyvalent protective vaccine against different phleboviruses.

Immunoinformatics and Pepscan strategies on the path of a peptide-based serological diagnosis of COVID19

Several diagnostic tools have been developed for clinical and epidemiological assays. RT-PCR and antigen detection tests are more useful for diagnosis of acute disease, while antibody tests allow the estimation of exposure in the population. Currently, there is an urgent need for the development of diagnostic tests for COVID-19 that can be used for large-scale epidemiological sampling. Through a comprehensive strategy, potential 16 mer antigenic peptides suited for antibody-based SARS-CoV-2 diagnosis were identified. A systematic scan of the three structural proteins (S,N and M) and the non-structural proteins (ORFs) present in the SARS-CoV-2 virus was conducted through the combination of immunoinformatic methods, peptide SPOT synthesis and an immunoassay with cellulose-bound peptides (Pepscan). The Pepscan filter paper sheets with synthetic peptides were tested against pools of sera of COVID-19 patients. Antibody recognition showed a strong signal for peptides corresponding to the S, N and M proteins of SARS-CoV-2 virus, but not for the ORFs proteins. The peptides exhibiting higher signal intensity were found in the C-terminal region of the N protein. Several peptides of this region showed strong recognition with all three immunoglobulins in the pools of sera. The differential reactivity observed between the different immunoglobulin isotypes (IgA, IgM and IgG) within different regions of the S and N proteins, can be advantageous for ensuring accurate diagnosis of all infected patients, with different times of exposure to infection. Few peptides of the M protein showed antibody recognition and no recognition was observed for peptides of the ORFs proteins.

Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay.

Background: The diphtheria toxoid antigen is a major component in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. Although antibodies are considered critical for diphtheria protection, little is known about the antigenic determinants that maintain humoral immunity. Methods: One-hundred and twelve 15 mer peptides covering the entire sequence of diphtheria toxin (DTx) protein were prepared by SPOT synthesis. The immunoreactivity of membrane-bound peptides with sera from mice immunized with a triple DTP vaccine allowed mapping of continuous B-cell epitopes, topological studies, multiantigen peptide (MAP) synthesis, and Enzyme-Linked Immunosorbent Assay (ELISA) development. Results: Twenty epitopes were identified, with two being in the signal peptide, five in the catalytic domain (CD), seven in the HBFT domain, and five in the receptor-binding domain (RBD). Two 17 mer (CB/Tx-2/12 and CB/DTx-4–13) derived biepitope peptides linked by a Gly-Gly spacer were chemically synthesized. The peptides were used as antigens to coat ELISA plates and assayed with human (huVS) and mice vaccinated sera (miVS) for in vitro diagnosis of diphtheria. The assay proved to be highly sensitive (99.96%) and specific (100%) for huVS and miVS and, when compared with a commercial ELISA test, demonstrated a high performance. Conclusions: Our work displayed the complete picture of the linear B cell IgG response epitope of the DTx responsible for the protective effect and demonstrated sufficient specificity and eligibility for phase IIB studies of some epitopes to develop new and fast diagnostic assays.

In silico and in vitro Evaluation of Mimetic Peptides as Potential Antigen Candidates for Prophylaxis of Leishmaniosis.

Antigen formulation is the main feature for the success of leishmaniosis diagnosis and vaccination, since the disease is caused by different parasite species that display particularities which determine their pathogenicity and virulence. It is desirable that the antigens are recognized by different antibodies and are immunogenic for almost all Leishmania species. To overcome this problem, we selected six potentially immunogenic peptides derived from Leishmania histones and parasite membrane molecules obtained by phage display or spot synthesis and entrapped in liposome structures. We used these peptides to immunize New Zealand rabbits and determine the immunogenic capacity of the chimeric antigen. The peptides induced the production of antibodies as a humoral immune response against L. braziliensis or L. infantum. Next, to evaluate the innate response to induce cellular activation, macrophages from the peptide mix-immunized rabbits were infected in vitro with L. braziliensis or L. infantum. The peptide mix generated the IFN-γ, IL-12, IL-4 and TGF-β that led to Th1 and Th2 cellular immune responses. Interestingly, this mix of peptides also induced high expression of iNOS. These results suggest that the mix of peptides derived from histone and parasites membrane molecules was able to mimic parasites proteins and induce cytokines important to CD4+ T cell Th1 and Th2 differentiation and effector molecule to control the parasite infection. Finally, this peptide induced an immune balance that is important to prevent immunopathological disorders, inflammatory reactions, and control the parasite infection.

Immunoprotection against lethal effects of Crotalus durissus snake venom elicited by synthetic epitopes trapped in liposomes

Snakebites caused by Crotalus genus are the second most frequent in Brazil. Crotoxin is a beta-neurotoxin responsible for the main envenomation effects of Crotalus biting, while crotamine immobilizes the animal hind limbs, contributing to prey immobilization and to envenoming symptoms. As crotoxin and crotamine represent about 90% of Crotalus venom dry weight, these toxins are of great importance for antivenom therapy. In this sense, knowledge regarding the antigenicity/immunogenicity at the molecular level of these toxins can provide valuable information for the improvement of specific antivenoms. Therefore, the aims of this study are the identification of the B-cell epitopes from crotoxin and crotamine; and the characterization of the neutralizing potency of antibodies directed against the corresponding synthetic epitopes defined in the current study. Linear B-cell epitopes were identified using the Spot Synthesis technique probed with specific anti-C. d. terrificus venom horse IgG. One epitope of crotamine (F12PKEKICLPPSSDFGKMDCRW32) and three of crotoxin (L10LVGVEGHLLQFNKMIKFETR30; Y43CGWGGRGRPKDATDRCCFVH63 and T118YKYGYMFYPDSRCRGPSETC138) were identified. After synthesis in their soluble form, the peptides mixture correspondent to the mapped epitopes was entrapped in liposomes and used as immunogens for antibody production in rabbits. Anti-synthetic peptide antibodies were able to protect mice from the lethal activity of C. d. terrificus venom.

Identification Of A Novel Histone Derived Antimicrobial Peptide In Airway Surface Liquid.

The airway surface liquid (ASL), a protective layer secreted by the airway epithelium, represents the first line of defence against inhaled infectious material. It contains a complex array of secreted proteins and peptides that aid the neutralisation and removal of inhaled microbes and toxicants. The ASL also contains many proteases that can cleave proteins to generate further bioactive peptides. We therefore hypothesised that the airway ASL sustained an extensive peptidome containing novel bioactive peptides. We examined the ASL peptide profile of normal human bronchial epithelial cells transformed with BMI‐1 (NHBE‐BMI1) and cell‐lines Calu3 (submucosal adenocarcinoma) and H441 (Clara cell like adenocarcinoma). ASL was acquired by washing the apical surface of epithelial monolayers grown at air‐liquid interface with PBS (100μl) at 0, 24 and 120 hours after exposure to hyperglycaemic (25mM glucose) or normoglycaemic (5mM glucose and 20mM mannitol) basolateral medium. The ASL peptides &lt; 10kDa, were subsequently isolated and analysed using the Q Exactive™ HF‐X Hybrid Quadrupole‐Orbitrap™ Mass Spectrometer. The resulting spectra of native peptides and their subsequent fragments were analysed using the pNovo De novo sequencing tool. Our preliminary results identified 4765 unique peptides (NHBE‐BMI1: 646, Calu3: 3150, H441: 1197). Of these, 37 peptides were common to all samples regardless of glycaemic state. A number of histone derived peptides were identified in all samples and cell lines. These peptides had a proline‐alanine rich N‐terminal region and a cationic C‐terminal, similar to antimicrobial peptides from histone H1 found in Atlantic salmon skin mucous. Synthetic peptides were produced by SPOT synthesis and the antimicrobial activity tested against luminescent Pseudomonas aeruginosa over 18 hours at 37°C in (100mM Tris (pH 7) and 20mM glucose). One of the crude peptides elicited a dose dependent inhibition of bacterial luminescence at 4.63±1.34 μM (n=3), indicating no bacteria survived. Truncated variants of this peptide containing the alanine‐proline rich N‐terminal sequence or the cationic C‐terminal sequence were produced to determine if a specific region of the peptide was required for antimicrobial activity. The N and the C terminal peptides had their antimicrobial activity significantly decreased, only reducing bacterial luminescence by 28.06±2.27% and 35.28±9.45% respectively compared to the full‐length peptide; 99.99±0.001% (all normalised to untreated bacterial luminescence, n=4, p&lt;0.0001, ±SD). This indicates that both regions are required for antimicrobial activity. An immunoblot of concentrated native ASL peptides and cell lysates showed the presence of known histone protein epitopes in our peptide of interest. Furthermore, we identified this histone‐derived peptide in sputum samples from both Cystic Fibrosis and normal healthy patients. Our preliminary data indicates that this novel histone derived peptide may serve an antimicrobial function in the ASL.Support or Funding InformationMedical Research Council London Intercollegiate Doctoral Training Programme

Engineered protein containing crotoxin epitopes induces neutralizing antibodies in immunized rabbits

Crotoxin (Ctx) is the main lethal component of Crotalus durissus terrificus venom. It is a neurotoxin, composed of two subunits associated by noncovalent interactions, the non-toxic acid subunit (CA), named Crotapotin, and the basic subunit (CB), with phospholipase A2 (PLA2) activity. Employing the SPOT synthesis technique, we determined two epitopes located in the C-terminal of each Ctx subunit. In addition, 3 other epitopes were mapped in different regions of Ctx using subcutaneous spot implants surgically inserted in mice. All epitopes mapped here were expressed together as recombinant multi-epitopic protein (rMEPCtx), which was used to immunize New Zealand rabbits. Anti-rMEPCtx rabbit serum cross-reacted with Ctx and crude venoms from C. d. terrificus, Crotalus durissus ruruima, Peruvian C. durissus and Bothrops jararaca (with lower intensity). Furthermore, anti-rMEPCtx serum was able to neutralize Ctx lethal activity. As the recombinant multiepitopic protein is not toxic, it can be administered in larger doses without causing adverse effects on the immunized animals health. Therefore, our work evidences the identification of neutralizing epitopes of Ctx and support the use of recombinant multiepitopic proteins as an innovation to immunotherapeutics production.

SPOT Synthesis: The Solid-Phase Peptide Synthesis on Planar Surfaces.

Peptide libraries are a highly useful tool for drug development. While most preparations of peptide libraries are laborious during either the synthesis or its screening, the SPOT synthesis offers the possibility of directly synthesizing large numbers of peptides on a planar surface. As a positionally addressable, multiple solid-phase synthesis technique, the synthesis allows a very convenient handling during the screening of that peptide library in a form of an array. This publication will provide protocols for the basic procedures of the SPOT synthesis and references to some important literature regarding that technique and its application.

Chemical Modification of Cellulose Membranes for SPOT Synthesis

Since the development of solid-phase peptide synthesis in the 1960s, many laboratories have modified the technology for the production of peptide arrays to facilitate the discovery of novel peptide mimetics and therapeutics. One of these, known as SPOT synthesis, enables parallel peptide synthesis on cellulose paper sheets and has several advantages over other peptide arrays methods. Today, the SPOT technique remains one of the most frequently used methods for synthesis and screening of peptides on arrays. Although polypropylene and glass can be used for the preparation of peptide arrays, the most commonly used material for SPOT membranes is cellulose. Critical to the success of the SPOT synthesis is the ability to modify a cellulose membrane to make it more suitable for solid-phase peptide synthesis of peptides and their analogues. In this review, we highlight the current range of chemical modifications of cellulose that have been developed to enable SPOT synthesis and further enhance its impact on peptide drug discovery. This will contribute to further chemical modifications and applications of SPOT synthesis for peptide arrays and peptide therapeutic screening.

Porcine parvovirus VP1/VP2 on a time series epitope mapping: exploring the effects of high hydrostatic pressure on the immune recognition of antigens

Porcine parvovirus (PPV) is a DNA virus that causes reproductive failure in gilts and sows, resulting in embryonic and fetal losses worldwide. Epitope mapping of PPV is important for developing new vaccines. In this study, we used spot synthesis analysis for epitope mapping of the capsid proteins of PPV (NADL-2 strain) and correlated the findings with predictive data from immunoinformatics. The virus was exposed to three conditions prior to inoculation in pigs: native (untreated), high hydrostatic pressure (350 MPa for 1 h) at room temperature and high hydrostatic pressure (350 MPa for 1 h) at − 18 °C, and was compared with a commercial vaccine produced using inactivated PPV. The screening of serum samples detected 44 positive spots corresponding to 20 antigenic sites. Each type of inoculated antigen elicited a distinct epitope set. In silico prediction located linear and discontinuous epitopes in B cells that coincided with several epitopes detected in spot synthesis of sera from pigs that received different preparations of inoculum. The conditions tested elicited antibodies against the VP1/VP2 antigen that differed in relation to the response time and the profile of structurally available regions that were recognized.