Toxins

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Submit to this Journal Review for this Journal Edit a Special Issue ► ▼ Article Menu Article Menu Subscribe SciFeed Recommended Articles Related Info Link Google Scholar More by Authors Links on DOAJ De-Simone, S. G. Napoleão-Pêgo, P. Lechuga, G. C. Carvalho, J. P. R. S. Gomes, L. R. Cardozo, S. V. Morel, C. M. Provance, D. W. Silva, F. R. da on Google Scholar De-Simone, S. G. Napoleão-Pêgo, P. Lechuga, G. C. Carvalho, J. P. R. S. Gomes, L. R. Cardozo, S. V. Morel, C. M. Provance, D. W. Silva, F. R. da on PubMed De-Simone, S. G. Napoleão-Pêgo, P. Lechuga, G. C. Carvalho, J. P. R. S. Gomes, L. R. Cardozo, S. V. Morel, C. M. Provance, D. W. Silva, F. R. da /ajax/scifeed/subscribe Article Views Citations - Table of Contents Altmetric share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up ... Endorse textsms ... Comment Need Help? Support

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Get Information clear JSmol Viewer clear first_page settings Order Article Reprints Font Type: Arial Georgia Verdana Font Size: Aa Aa Aa Line Spacing:    Column Width:    Background: Open AccessArticle High-Throughput IgG Epitope Mapping of Tetanus Neurotoxin: Implications for Immunotherapy and Vaccine Design by

Salvatore G. De-Simone 1,2,3,* ORCID

Paloma Napoleão-Pêgo 1,2,

Guilherme C. Lechuga 1,2,

João P. R. S. Carvalho 1,2,3,

Larissa R. Gomes 1,2 ORCID

Sergian V. Cardozo 4,

Carlos M. Morel 1 ORCID

David W. Provance, Jr. 1,2 ORCID

and

Flavio R. da Silva 1,2 1 Center for Technological Development in Health (CDTS)/National Institute of Science and Technology for Innovation in Diseases of Neglected Populations (INCT-IDPN), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro 21040-900, RJ, Brazil 2 Laboratory of Epidemiology and Molecular Systematics (LESM), Oswaldo Cruz Institute, Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro 21040-900, RJ, Brazil 3 Post-Graduation Program in Science and Biotechnology, Department of Molecular and Cellular Biology, Biology Institute, Federal Fluminense University, Niterói 22040-036, RJ, Brazil 4 Department of Health, Graduate Program in Translational Biomedicine (BIOTRANS), University of Grande Rio (UNIGRANRIO), Caxias 25071-202, RJ, Brazil * Author to whom correspondence should be addressed. Toxins 2023, 15(4), 239; https://doi.org/10.3390/toxins15040239 Received: 11 February 2023 / Revised: 3 March 2023 / Accepted: 8 March 2023 / Published: 24 March 2023 (This article belongs to the Special Issue Immunotoxins: Current Status and Future Perspectives) Download Download PDF Download PDF with Cover Download XML Download Epub Download Supplementary Material Browse Figures Versions Notes

Abstract: 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. Keywords: tetanus neurotoxin; B-cell linear epitopes; immunological diagnostic; peptide ELISA Key Contribution: Three epitopes critical for the production of vaccine-induced neutralizing antibodies were identified through a fine epitope map that provides a deeper understanding of the protection mechanisms against toxin activity, neuronal cell receptor binding, and the presentation of tetanus.

Graphical Abstract

1. IntroductionTetanus is a highly fatal, noncommunicable, and preventable disease caused by infections with Clostridium tetani [1,2,3]. Children, women, and newborns are at the highest risk of acquiring tetanus due to low immunization rates and unhygienic deliveries [4]. Consequently, tetanus is a public health problem with a recorded worldwide yearly incidence of a million neonatal cases and 34,000 deaths in 2015, according to WHO estimates [4]. The child mortality rate ranges from 20 to 50% [5,6,7,8]. The vaccination schedule of the Brazilian national immunization program advocates for an initial dose of the tetanus vaccine during the first two months of life with two more doses at 4 months and at 6 months of age. In addition, two boosters are administered: one at 15 months of age and the other at 4 years old. Children who complete the vaccination cycle show a high level of protection, avoiding death. Even the immunization of pregnant women, or women of childbearing age, reduces neonatal tetanus mortality by about 94% [4,7].Virulence is associated with tetanus neurotoxin (TeNT), a 150 kDa AB toxin excreted by the bacteria consisting of a light chain (Lc) of 50 kDa and a heavy chain (Hc) of 100 kDa that are joined by a disulfide bond [9]. Lethality is related to a block in neurotransmitter release caused by the N-terminal zinc-dependent metalloendopeptidase in the Lc and its cleavage of VAMP/synatobrevin, a membrane-associated protein involved in synaptic vesicle fusion [10].Residues 233 to 237 in the N-terminus of the Lc present a His-Glu-XX-His Zn2+ motif that forms a primary sphere of residues in the enzymatic active site for coordinating the binding of a zinc ion [11]. A secondary sphere of residues determines the proteolytic specificity of the peptidase activity [10]. The Hc is responsible for entry of the toxin into the cytoplasm of neurons and consists of two domains [12]. A translocation domain is located in the N-terminal region, which is responsible for internalization and translocation into the neuronal cytosol through a clathrin-mediated pathway involving dynamin and AP-2 [13]. The receptor-binding domain resides in the C-terminal region that forms a double bond to membrane gangliosides [1,14,15]. The C-terminal domain, often referred to as the C-fragment, can be further divided into two subdomains of 25 kDa, the N-proximal subdomain (HcRN) and the carboxy-terminal subdomain (HcRC) [16]. In the HcRC subdomain, there is a crystalline structure that is primarily responsible for binding gangliosides [17]. Gangliosides are a type of glycosphingolipid in the plasma membrane composed of sialic acid residues with an acidic nature that can serve as signaling receptors between cells and the extracellular environment [18]. Although gangliosides are present in all tissues, they are most abundant in nerve cell membranes [19]. With respect to TeNT, gangliosides have a high affinity for both C-fragment binding sites and can be considered as dual-function receptors [14]. Despite extensive vaccination, tetanus remains an significant cause of death worldwide, particularly in developing countries [20,21]. Vaccines against tetanus are clinically effective [22,23], rarely present side effects [24], and are composed of a chemically inactivated tetanus toxoid (CITTo). However, CITTo is a crude preparation that can contain hundreds of Clostridium tetani proteins, and the biomedically relevant component is present at variable levels that sometimes only represent a minor percentage of vaccine mass [25]. Furthermore, the toxoids do not resemble the native toxins after formaldehyde treatment in vitro and apparently lack some important neutralizing epitopes. As a result, these modified toxoids may only elicit mild or moderate protection in vivo [26], which necessitates multiple injections of a vaccine to reach a long-lasting immunization capable of protecting against tetanus. This can explain the ineffectiveness of vaccination programs in some developing countries, which can be exacerbated by low vaccination coverage [27,28] https://www.sciencedirect.com/science/article/pii/S004101012030341X?casatoken=T1AsuIB7MZEAAAAA:74MXzK3Yx3vzXdItFo5FstsRB-SI5r3PvSa3W3BuSKtUkAwplrW20Bbp331rUELR8ezUh0XLCA (accessed on 15 January 2023).Together with other issues, such as the use of formaldehyde, which complicates manufacturing procedures through production risks and pollution, many countries are actively researching for the development of a new generation of safer and more effective tetanus vaccines through biotechnological approaches that are capable of lifelong immunization against tetanus [29]. A good protective immune response against TeNT after parenteral immunization has been demonstrated using the HcR domain, which encompasses the host receptor-binding motif [13,26,30]. Recently, a recombinant, non-tagged isoform of the Hc domain of the TeNT alone was successfully evaluated in mice [31]. A different recombinant tetanus vaccine was engineered with eight individual amino acid mutations (8MTT) in TeNT to genetically inactivate catalysis, translocation, and host receptor-binding functions. This protein retained 99.4% amino acid identity to native tetanus toxin (TT) and elicited a potent immune response in mice, which displayed an effective vaccine potency against a TT challenge [32]. Another approach is to generate a highly directed vaccine focused on sites within the TTo antigen that is directly involved with the protective humoral immune response observed with vaccination. However, a structurally resolved analysis of the epitopes in TeNT underlying the molecular mechanisms of antibody neutralization is unavailable. Eleven different epitopes have been deposited in the IEDB (Immune Epitope Database and Analysis Resource; https://www.iedb.org/home_v3.php (accessed on 15 January 2023)) database, and several works have described the isolation of several monoclonal antibodies without defining their cognate binding site [33,34,35,36,37,38,39,40,41]. However, the structure of these epitopes is not known and many of the epitopes still needed to be identified. To reveal the full range of epitopes recognized in TeNT that are elicited by current tetanus vaccines, complete and careful immunological mapping was performed using the high-resolution methodology of printable SPOT synthesis. The epitopes identified along with their implication in immunotherapy and vaccination are discussed. In addition, a set of four epitopes were employed in peptide ELISAs to evaluate the coverage of vaccinations post-pandemic in a group of children living in Rio de Janeiro, Brazil. 2. Results 2.1. Identification of the Immunodominant IgG Epitopes in TeNT Linear epitopes often encompass 4 to 9 amino acid residues in size, which suggests that a detectable signal of high confidence can be observed across a library of 15-mer peptides overlapping by 10 amino acids and containing epitope motifs. Applying this principle, signal intensities for each of the peptides were mapped back to the respective TeNT sequence from which the peptides were derived. By plotting signal intensity as a function of peptide position in the toxin sequence, the binding profile of the TeNT pair was obtained. In Figure 1, panels A and B present the role of each peptide and the measured reactivity intensity, respectively, from the chemiluminescent detection of IgG antibodies in sera pooled from vaccinated children (n = 15). The absolute signals were normalized to percentages using 100% as defined by the positive control. The list of the synthetic peptides’ positions on the membranes can be found in Table S1. Fourth-three IgG epitopes were identified in TeNT with 14 epitopes in the L-chain and 29 epitopes in the H-chain (labeled in gray in Table 1). 2.2. Epitope Reactivity by ELISA-MAP4The method of synthesizing multiple antigenic peptides (MAP) was applied to improve the immunoreactivity of synthetic peptides that represent distinct regions of TeNT. Four selected tetrameric MAP with multiple incorporated overlapping B-cell epitopes were combined with a particular TeNT sequence representing the peptide in the C-terminus of the TeNT and evaluated with a panel of sera from children who sought vaccination in 2021 at a clinic of the Brazilian Ministry of Health. Sera were distributed by age, and the diagnostic performance of peptides [215 (MAP4-VPERYEFGTKPEDFN), 216 (MAP4-EYVPTFDNVIENTTS), 217 (MAP4-EKTLNDYKFQFDSNG) and 218 (MAP4-GTVNTQFQ YEYKIYS)] was evaluated with sera from ninety-three DTP vaccinated children (1–4 years, 5–9 years, 10–13 years) who were tested on an ELISA peptide-based assay (Figure 2).All ChVS were collected at the same time. Children aged 1–4 years old had received the three doses of the vaccine according to the vaccination schedule of the Brazilian Ministry of Health. Children aged 5–9 years old had received the two booster applications at 15 months and 4 years in addition to the three doses of vaccines. Children aged 10–13 years had completed the full vaccination cycle and were also vaccinated with the childhood DT (diphtheria and tetanus) vaccine administered at over 7 years of age. The main observed difference in the age groups was that the sera from 10–13-year-olds showed the lowest title, which may have been due to not having completed the vaccination cycle for childhood DT vaccine (booster) necessary to maintain the level of circulating IgG antibodies. Even all adults are advised to receive the adult DT vaccine every 10 years to support the degree of protection.The performances of the peptides were compared to a commercial kit for classic tetanus IgG (Serion Brazil, Pinhais, PR, Brazil). A significant number of the 92 DTP-positive ChVS reacted with the highly conserved 215–218 peptides. Standard negative human sera from a commercial kit were used in peptide ELISA for peptides 215–218 but did not react. Negative controls were used to determine the cut-off (0.109). Peptide ELISAs using the 215–218 antigens demonstrated that no significant statistical difference was found between ChVS from 1–4- and 5–9-year-olds. However, the group of 10–13-year-olds presented a statistically significant decline in reactivity with a mean optic density reduction that varied from 66 to 79%. The tests presented different performances. Peptide TT-215 showed a greater number of negative reactions (n = 32), followed by peptide TT-216 and 217 (n = 5) and TT-218 (n = 3). The maximum accuracy value is one, which represents a perfect test. Our peptide in-house ELISA immunoassay presented an area under the curve that varied from 0.95 to 0.98 (Figure 2B). The same samples of ChVS were also analyzed using a commercial kit. According to the manufacturer, the qualitative ELISA has >99% sensibility and 97% specificity to monitor the immunological state of vaccinated individuals. Applying the cut-off (0.1 UI/mL), the tests were nearly equivalent to that of the SERION ELISA classic tetanus IgG. Of 93 ChVS, 88 were confirmed as positive and 5 were negative (

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