Additional Epitopes Research Articles

Facts and Hopes in Immunotherapy Strategies Targeting Antigens Derived from KRAS Mutations.

In this commentary, we advance the notion that mutant KRAS (mKRAS) is an ideal tumor neoantigen that is amenable for targeting by the adaptive immune system. Recent progress highlights key advances on various fronts that validate mKRAS as a molecular target and support further pursuit as an immunological target. Because mKRAS is an intracellular membrane localized protein and not normally expressed on the cell surface, we surmise that proteasome degradation will generate short peptides that bind to HLA class I (HLA-I) molecules in the endoplasmic reticulum for transport through the Golgi for display on the cell surface. T-cell receptors (TCR)αβ and antibodies have been isolated that specifically recognize mKRAS encoded epitope(s) or haptenated-mKRAS peptides in the context of HLA-I on tumor cells. Case reports using adoptive T-cell therapy provide proof of principle that KRAS G12D can be successfully targeted by the immune system in patients with cancer. Among the challenges facing investigators is the requirement of precision medicine to identify and match patients to available mKRAS peptide/HLA therapeutics and to increase the population coverage by targeting additional mKRAS epitopes. Ultimately, we envision mKRAS-directed immunotherapy as an effective treatment option for selected patients that will complement and perhaps synergize with small-molecule mKRAS inhibitors and targeted mKRAS degraders.

Chimeric antigen receptors enable superior control of HIV replication by rapidly killing infected cells.

Engineered T cells hold great promise to become part of an effective HIV cure strategy, but it is currently unclear how best to redirect T cells to target HIV. To gain insight, we generated engineered T cells using lentiviral vectors encoding one of three distinct HIV-specific T cell receptors (TCRs) or a previously optimized HIV-targeting chimeric antigen receptor (CAR) and compared their functional capabilities. All engineered T cells had robust, antigen-specific polyfunctional cytokine profiles when mixed with artificial antigen-presenting cells. However, only the CAR T cells could potently control HIV replication. TCR affinity enhancement did not augment HIV control but did allow TCR T cells to recognize common HIV escape variants. Interestingly, either altering Nef activity or adding additional target epitopes into the HIV genome bolstered TCR T cell anti-HIV activity, but CAR T cells remained superior in their ability to control HIV replication. To better understand why CAR T cells control HIV replication better than TCR T cells, we performed a time course to determine when HIV-specific T cells were first able to activate Caspase 3 in HIV-infected targets. We demonstrated that CAR T cells recognized and killed HIV-infected targets more rapidly than TCR T cells, which correlates with their ability to control HIV replication. These studies suggest that the speed of target recognition and killing is a key determinant of whether engineered T cell therapies will be effective against infectious diseases.

Design of a Multi-epitope Vaccine against Covid-19: An In silico Approach

Background: The control of the Covid-19 epidemic depends on designing a novel, effec-tive vaccine against it. Currently, available vaccines cannot provide complete protection against various mutants of Covid-19. Objective: The present investigation aimed to design a new multi-epitope vaccine by using in silico tools. Methods: In the present study, the spike-glycoprotein was targeted, desirably stimulating both B and T-cell lymphocytes, providing effective and safe responses in the host immune system. The de-sired vaccine has been found to possess 448 amino acids of spike glycoprotein. The prognosticated epitopes included 10 CTL, 4 linear B-cells, and 14 HTL, including the 128 amino acid sequence of 50S ribosomal protein adjuvant joined by GPGPG and AAY linkers on the N terminus of linear B-cell, HTL, and CTL epitopes, and the C-terminal joined with HHHHHH (6HIS) linker, indicating stability for vaccine structure. Results: The molecular docking has revealed the protein-protein restricting communication between the immunization construct and the TLR-3-resistant receptor. The vaccine has been developed through selected epitopes, an adjuvant, and an additional epitope. Docking assays with toll-like re-ceptor 3 have been run on a three-dimensional structural model of the vaccine to gauge its immuno-logical potency. Our findings support the hypothesis that our vaccination will activate TLR-mediated downstream immune pathways by aggressively interacting with the innate receptor. Conclusion: The results suggest that the proposed chimeric peptide could initiate an efficient and safe immune response against Covid-19. The proposed vaccine has been proven safe in all critical parameters.

In silico epitope prediction and evolutionary analysis reveals capsid mutation patterns for enterovirus B.

Enterovirus B (EVB) is a common species of enterovirus, mainly consisting of Echovirus (Echo) and Coxsackievirus B (CVB). The population is generally susceptible to EVB, especially among children. Since the 21st century, EVB has been widely prevalent worldwide, and can cause serious diseases, such as viral meningitis, myocarditis, and neonatal sepsis. By using cryo-electron microscopy, the three-dimensional (3D) structures of EVB and their uncoating receptors (FcRn and CAR) have been determined, laying the foundation for the study of viral pathogenesis and therapeutic antibodies. A limited number of epitopes bound to neutralizing antibodies have also been determined. It is unclear whether additional epitopes are present or whether epitope mutations play a key role in molecular evolutionary history and epidemics, as in influenza and SARS-CoV-2. In the current study, the conformational epitopes of six representative EVB serotypes (E6, E11, E30, CVB1, CVB3 and CVB5) were systematically predicted by bioinformatics-based epitope prediction algorithm. We found that their epitopes were distributed into three clusters, where the VP1 BC loop, C-terminus and VP2 EF loop were the main regions of EVB epitopes. Among them, the VP1 BC loop and VP2 EF loop may be the key epitope regions that determined the use of the uncoating receptors. Further molecular evolution analysis based on the VP1 and genome sequences showed that the VP1 C-terminus and VP2 EF loop, as well as a potential "breathing epitope" VP1 N-terminus, were common mutation hotspot regions, suggesting that the emergence of evolutionary clades was driven by epitope mutations. Finally, footprints showed mutations were located on or near epitopes, while mutations on the receptor binding sites were rare. This suggested that EVB promotes viral epidemics by breaking the immune barrier through epitope mutations, but the mutations avoided the receptor binding sites. The bioinformatics study of EVB epitopes may provide important information for the monitoring and early warning of EVB epidemics and developing therapeutic antibodies.

Immunity-driven evolution of virulence and diversity in respiratory diseases.

The time-honoured paradigm in the theory of virulence evolution assumes a positive relation between infectivity and harmfulness. However, the aetiology of respiratory diseases yields a negative relation, with diseases of the lower respiratory tract being less infective and more harmful. We explore the evolutionary consequences in a simple model incorporating cross-immunity between disease strains that diminishes with their distance in the respiratory tract, assuming that docking rate follows the match between the local mix of cell surface types and the pathogen's surface, and cross-immunity the similarity of the pathogens' surfaces. The assumed relation between fitness components causes virulent strains infecting the lower airways to evolve to milder, more transmissible variants. Limited cross-immunity generally causes a readiness to diversify that increases with host population density. In respiratory diseases, that diversity will be highest in the upper respiratory tract. More tentatively, emerging respiratory diseases are likely to start low and virulent, to evolve up and become milder. Our results extend to a panoply of realistic generalisations of the disease's ecology and to including additional epitope axes. These extensions allow us to apply our results quantitatively to elucidate the differences in diversification between rhino- and coronavirus caused common colds.

A human antibody epitope map of the malaria vaccine antigen Pfs25

Pfs25 is a leading antigen for a malaria transmission-blocking vaccine and shows moderate transmission-blocking activity and induction of rapidly decreasing antibody titers in clinical trials. A comprehensive definition of all transmission-reducing epitopes of Pfs25 will inform structure-guided design to enhance Pfs25-based vaccines, leading to potent transmission-blocking activity. Here, we compiled a detailed human antibody epitope map comprising epitope binning data and structures of multiple human monoclonal antibodies, including three new crystal structures of Pfs25 in complex with transmission-reducing antibodies from Malian volunteers immunized with Pfs25 conjugated to EPA and adjuvanted with AS01. These structures revealed additional epitopes in Pfs25 capable of reducing transmission and expanded this characterization to malaria-exposed humans. This work informs immunogen design to focus the antibody response to transmission-reducing epitopes of Pfs25, enabling development of more potent transmission-blocking vaccines for malaria.

Immunochemical studies and gene cluster relationships of closely related O-antigens of Aeromonas hydrophila Pt679, Aeromonas popoffii A4, and Aeromonas sobria K928 strains classified into the PGO1 serogroup dominant in Polish aquaculture of carp and rainbow trout

The present study included three Aeromonas sp. strains isolated from fish tissues during Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreaks on commercial farms, i.e.: Aeromonas hydrophila Pt679 obtained from rainbow trout as well as Aeromonas popoffii A4 (formerly Aeromonas encheleia) and Aeromonas sobria K928 both isolated from carp, which were classified into the new provisional PGO1 serogroup prevailing among aeromonads in Polish aquaculture. The structure of the O-specific polysaccharides of A4 and K928 has been previously established. Here, immunochemical studies of the O-specific polysaccharide of A. hydrophila Pt679 were undertaken. The O-specific polysaccharide was obtained from the lipopolysaccharide of A. hydrophila Pt679 after mild acid hydrolysis and separation by gel-permeation chromatography. The high-molecular-mass fraction was studied using chemical methods and 1H and 13C NMR spectroscopy, including 1H,1H NOESY, and 1H,13C HMBC experiments. The following structure of the branched repeating unit of the O-polysaccharide from A. hydrophila Pt679 was determined:→3)[α-d-Fucp3NAc-(1→2)]-α-l-Rhap-(1→3)-β-l-Rhap-(1→4)-α-l-Rhap-(1→3)-β-d-QuipNAc-(1→2)┘The studies indicated that O-polysaccharides from A. hydrophila Pt679, A. popoffii A4 and A. sobria K928 share similarities but they also contain unique characteristics. Western blotting and an enzyme-linked immunosorbent assay revealed that the cross-reactivity of the related O-antigens is caused by the occurrence of common structural elements, whereas additional epitopes define the specificity of the O-serotypes.For genetic relationship studies, the O-antigen gene cluster was characterized in the genome of the A. hydrophila Pt679 strain and compared with the corresponding sequences of A. popoffii A4 and A. sobria K928 and with sequences available in the databases. The composition of the regions was found to be consistent with the O-antigen structures of Aeromonas strains classified into the same PGO1 serogroup.

Immunoglobulin profiling with large high-density peptide microarrays as screening method to detect candidate proteins for future biomarker detection in dogs with steroid-responsive meningitis-arteritis.

Large high-density peptide microarrays are a suitable screening method to detect possible autoantigens which might be involved in the pathogenesis of SRMA. The IgA and IgG profile of pooled serum samples of 5 dogs with SRMA and 5 dogs with neck pain due to intervertebral disc herniation (IVDH) without ataxia or paresis were compared via commercially available high-density peptide microarrays (Discovery Microarray) containing 29,240 random linear peptides. Canine distemper virus nucleoprotein (CDVN) served as positive control as all dogs were vaccinated. Common motifs were compared to amino acid sequences of known proteins via databank search. One suitable protein was manually selected for further analysis with a smaller customized high-density peptide microarray. Pooled serum of dogs with SRMA and IVDH showed different IgA and IgG responses on Discovery Microarray. Only top IgG responses of dogs with SRMA showed a common motif not related to the control protein CDVN. This common motif is part of the interleukin 1 receptor antagonist protein (IL1Ra). On IL1Ra, dogs with SRMA displayed IgA binding to an additional epitope, which dogs with IVDH did not show. IL1Ra is an anti-inflammatory acute phase protein. Different immunoglobulin binding patterns on IL1Ra could be involved in the pathogenesis of SRMA and IL1Ra might be developed as future biomarker for SRMA.

MEK inhibition enhances presentation of targetable MHC-I tumor antigens in mutant melanomas

Combining multiple therapeutic strategies in NRAS/BRAF mutant melanoma-namely MEK/BRAF kinase inhibitors, immune checkpoint inhibitors (ICIs), and targeted immunotherapies-may offer an improved survival benefit by overcoming limitations associated with any individual therapy. Still, optimal combination, order, and timing of administration remains under investigation. Here, we measure how MEK inhibition (MEKi) alters anti-tumor immunity by utilizing quantitative immunopeptidomics to profile changes in the peptide major histocompatibility molecules (pMHC) repertoire. These data reveal a collection of tumor antigens whose presentation levels are selectively augmented following therapy, including several epitopes present at over 1,000 copies per cell. We leveraged the tunable abundance of MEKi-modulated antigens by targeting four epitopes with pMHC-specific T cell engagers and antibody drug conjugates, enhancing cell killing in tumor cells following MEK inhibition. These results highlight drug treatment as a means to enhance immunotherapy efficacy by targeting specific upregulated pMHCs and provide a methodological framework for identifying, quantifying, and therapeutically targeting additional epitopes of interest.

Prediagnostic Appearance of Thrombospondin Type-1 Domain 7A Autoantibodies in Membranous Nephropathy.

Key Points The entire extracellular domain of thrombospondin type-1 domain 7A (THSD7A) in the luciferase immunoprecipitation system immunoassay was required to detect autoantibodies with high sensitivity in membranous nephropathy (MN).In THSD7A-seropositive MN patients, changes in antibody levels precede changes in clinical status.Seropositive THSD7A antibodies were detected in some patients with MN considered to be secondary to autoimmunity or cancer. Background Pathogenic autoantibodies against thrombospondin type-1 domain 7A (THSD7A) are present in approximately 3% of patients with membranous nephropathy (MN). Compared with PLA2R antibodies, less is known about THSD7A autoantibodies (ABs) because of the relative rarity and the lack of a commercially available quantitative immunoassay. Methods In this study, we describe the development and validation of a highly quantitative luciferase immunoprecipitation system (LIPS) assay for detecting THSD7A ABs and used it to study dominant THSD7A epitopes, disease associations, and monitoring disease activity. The Department of Defense Serum Repository (DODSR) was then used to analyze THSD7A AB in 371 longitudinal serum samples collected before clinical diagnosis of MN from 110 PLA2R-negative MN subjects. Results LIPS analysis demonstrated that a near full-length THSD7A (amino acids 1–1656) detected robust autoantibody levels in all known seropositive MN patients with 100% sensitivity and specificity compared with ELISA and/or Western blotting. Most of the THSD7A-seropositive subjects in our pilot cohort had evidence of coexisting autoimmunity or cancer. Moreover, three THSD7A-seropositive patients undergoing immunosuppressive therapy showed longitudinal autoantibody levels that tracked clinical status. Additional epitope analysis of two smaller protein THSD7A fragments spanning amino acids 1-416 and 1-671 demonstrated lower sensitivity of 32% and 44%, respectively. In the DODSR cohort, THSD7A seropositivity was detected in 4.5% of PLA2R-negative MN patients. In one primary and in one secondary MN-associated with cancer, THSD7A ABs were detectable <1 month before biopsy-proven diagnosis. In addition, three patients with lupus membranous nephropathy had detectable THSD7A ABs years before hypoalbuminemia and biopsy-proven diagnosis. Conclusions Although further studies are needed to explore the significance of THSD7A ABs in lupus membranous nephropathy, this study describes a novel, highly sensitive LIPS immunoassay for detecting THSD7A ABs and adds to the existing literature on THSD7A-associated MN. Clinical Trial registry name and registration number: NCT00977977; registration date: September 16, 2009.

BepiPred-3.0: Improved B-cell epitope prediction using protein language models.

B-cell epitope prediction tools are of great medical and commercial interest due to their practical applications in vaccine development and disease diagnostics. The introduction of protein language models (LMs), trained on unprecedented large datasets of protein sequences and structures, tap into a powerful numeric representation that can be exploited to accurately predict local and global protein structural features from amino acid sequences only. In this paper, we present BepiPred-3.0, a sequence-based epitope prediction tool that, by exploiting LM embeddings, greatly improves the prediction accuracy for both linear and conformational epitope prediction on several independent test sets. Furthermore, by carefully selecting additional input variables and epitope residue annotation strategy, performance was further improved, thus achieving unprecedented predictive power. Our tool can predict epitopes across hundreds of sequences in minutes. It is freely available as a web server and a standalone package at https://services.healthtech.dtu.dk/service.php?BepiPred-3.0 with a user-friendly interface to navigate the results.

Towards the development of an epitope-focused vaccine for SARS-CoV-2

The rapid spread of COVID-19 on all continents and the mortality induced by SARS-CoV-2 virus, the cause of the pandemic coronavirus disease 2019 (COVID-19) has motivated an unprecedented effort for vaccine development. Inactivated viruses as well as vaccines focused on the partial or total sequence of the Spike protein using different novel platforms such us RNA, DNA, proteins, and non-replicating viral vectors have been developed. The high global need for vaccines, now and in the future, and the emergence of new variants of concern still requires development of accessible vaccines that can be adapted according to the most prevalent variants in the respective regions.Here, we describe the immunogenic properties of a group of theoretically predicted RBD peptides to be used as the first step towards the development of an effective, safe and low-cost epitope-focused vaccine. One of the tested peptides named P5, proved to be safe and immunogenic. Subcutaneous administration of the peptide, formulated with alumina, induced high levels of specific IgG antibodies in mice and hamsters, as well as an increase of IFN-γ expression by CD8+ T cells in C57 and BALB/c mice upon in vitro stimulation with P5. Neutralizing titers of anti-P5 antibodies, however, were disappointingly low, a deficiency that we will attempt to resolve by the inclusion of additional immunogenic epitopes to P5. The safety and immunogenicity data reported in this study support the use of this peptide as a starting point for the design of an epitope restricted vaccine.

Linear Peptide Epitopes Derived from ErpP, p35, and FlaB in the Serodiagnosis of Lyme Disease.

Lyme disease is the most common vector-borne disease in the northern hemisphere. Current serodiagnostics are insensitive in early infection. Sensitivity in these seroassays is compromised by the necessity to preserve specificity in the presence of cross-reactive epitopes in Borrelia burgdorferi target antigens. We evaluated the efficacy of using synthetic peptides containing epitopes unique to B. burgdorferi as antigen targets in a Lyme disease seroassay. We performed linear B cell epitope mapping of the proteins p35 (BBH32) and ErpP to identify unique epitopes. We generated peptides containing these newly identified linear epitope sequences along with previously identified epitopes from the antigens FlaB and VlsE and evaluated their diagnostic capabilities via ELISA using large serum sets. Single-epitope peptides, while specific, demonstrated insufficient sensitivity. However, when epitopes from FlaB, ErpP, or p35 were combined in tandem with an epitope from VlsE, the sensitivity of the assay was significantly increased without compromising specificity. The identification of additional unique epitopes from other B. burgdorferi antigens and the further development of a combined multi-peptide-based assay for the laboratory diagnosis of Lyme disease offers a way to address the poor specificity associated with the use of whole protein antigen targets and thus significantly improve the laboratory diagnosis of Lyme disease.

Complexity of Viral Epitope Surfaces as Evasive Targets for Vaccines and Therapeutic Antibodies.

The dynamic interplay between virus and host plays out across many interacting surfaces as virus and host evolve continually in response to one another. In particular, epitope-paratope interactions (EPIs) between viral antigen and host antibodies drive much of this evolutionary race. In this review, we describe a series of recent studies examining aspects of epitope complexity that go beyond two interacting protein surfaces as EPIs are typically understood. To structure our discussion, we present a framework for understanding epitope complexity as a spectrum along a series of axes, focusing primarily on 1) epitope biochemical complexity (e.g., epitopes involving N-glycans) and 2) antigen conformational/dynamic complexity (e.g., epitopes with differential properties depending on antigen state or fold-axis). We highlight additional epitope complexity factors including epitope tertiary/quaternary structure, which contribute to epistatic relationships between epitope residues within- or adjacent-to a given epitope, as well as epitope overlap resulting from polyclonal antibody responses, which is relevant when assessing antigenic pressure against a given epitope. Finally, we discuss how these different forms of epitope complexity can limit EPI analyses and therapeutic antibody development, as well as recent efforts to overcome these limitations.

Rheumatoid Factor and Anti-Modified Protein Antibody Reactivities Converge on IgG Epitopes.

Rheumatoid arthritis (RA) patients often develop rheumatoid factors (RFs), antibodies that bind IgG Fc, and anti-modified protein antibodies (AMPAs), multireactive autoantibodies that commonly bind citrullinated, homocitrullinated, and acetylated antigens. Recently, antibodies that bind citrulline-containing IgG epitopes were discovered in RA, suggesting that additional undiscovered IgG epitopes could exist and that IgG could be a shared antigen for RFs and AMPAs. This study was undertaken to reveal new IgG epitopes in rheumatic disease and to determine if multireactive AMPAs bind IgG. Using sera from patients with RA, systemic lupus erythematosus, Sjögren's disease (SjD), or spondyloarthropathy, IgG binding to native, citrulline-containing, and homocitrulline-containing linear epitopes of the IgG constant region was evaluated by peptide array, with highly bound epitopes further evaluated by enzyme-linked immunosorbent assay (ELISA). Binding of monoclonal AMPAs to IgG-derived peptides and IgG Fc was also evaluated by ELISA. Seropositive RA sera showed high IgG binding to multiple citrulline- and homocitrulline-containing IgG-derived peptides, whereas anti-SSA+ sera from SjD patients showed consistent binding to a single linear native epitope of IgG in the hinge region. Monoclonal AMPAs bound citrulline- and homocitrulline-containing IgG peptides and modified IgG Fc. The repertoire of epitopes bound by AMPAs includes modified IgG epitopes, positioning IgG as a common antigen that connects the otherwise divergent reactivities of RFs and AMPAs.

Linear Epitope Binding Patterns of Grass Pollen-Specific Antibodies in Allergy and in Response to Allergen-Specific Immunotherapy.

Allergic diseases affect many individuals world-wide and are dependent on the interaction between allergens and antibodies of the IgE isotype. Allergen-specific immunotherapy (AIT) can alter the development of the disease, e.g., through induction of allergen-specific IgG that block allergen-IgE interactions. The knowledge of epitopes recognized by allergy-causing and protective antibodies are limited. Therefore, we developed an allergome-wide peptide microarray, aiming to track linear epitope binding patterns in allergic diseases and during AIT. Here, we focused on immune responses to grass pollen allergens and found that such epitopes were commonly recognized before initiation of AIT and that AIT commonly resulted in increased antibody production against additional epitopes already after 1 year of treatment. The linear epitope binding patterns were highly individual, both for subjects subjected to and for individuals not subjected to AIT. Still, antibodies against some linear epitopes were commonly developed during AIT. For example, the two rigid domains found in grass pollen group 5 allergens have previously been associated to a diversity of discontinuous epitopes. Here, we present evidence that also the flexible linker, connecting these domains, contains regions of linear epitopes against which antibodies are developed during AIT. We also describe some commonly recognized linear epitopes on Phl p 2 and suggest how antibodies against these epitopes may contribute to or prevent allergy in relation to a well-defined stereotyped/public IgE response against the same allergen. Finally, we identify epitopes that induce cross-reactive antibodies, but also antibodies that exclusively bind one of two highly similar variants of a linear epitope. Our findings highlight the complexity of antibody recognition of linear epitopes, with respect to both the studied individuals and the examined allergens. We expect that many of the findings in this study can be generalized also to discontinuous epitopes and that allergen peptide microarrays provide an important tool for enhancing the understanding of allergen-specific antibodies in allergic disease and during AIT.

Epitope-coated polymer particles elicit neutralising antibodies against Plasmodium falciparum sporozoites

The current Malaria RTS,S vaccine is based on virus-like particles (VLPs) comprising the NANP repetitive epitopes from the cicumsporozoite protein (CSP) of Plasmodium falciparum. This vaccine has limited efficacy, only preventing severe disease in about 30% of vaccinated individuals. A more efficacious vaccine is urgently needed to combat malaria. Here we developed a particulate malaria vaccine based on the same CSP epitopes but using biopolymer particles (BPs) as an antigen carrier system. Specific B- and T-cell epitope-coated BPs were assembled in vivo inside an engineered endotoxin-free mutant of Escherichia coli. A high-yield production process leading to ~27% BP vaccine weight over biomass was established. The epitope-coated BPs were purified and their composition, i.e., the polymer core and epitope identity, was confirmed. Epitope-coated BPs were used alongside soluble peptide epitopes and empty BPs to vaccinate sheep. Epitope-coated BPs showed enhanced immunogenicity by inducing anti-NANP antibody titre of EC50 > 150,000 that were at least 20 times higher than induced by the soluble peptides. We concluded that the additional T-cell epitope was not required as it did not enhance immunogenicity when compared with the B-cell epitope-coated BPs. Antibodies specifically bound to the surface of Plasmodium falciparum sporozoites and efficiently inhibited sporozoite motility and traversal of human hepatocytes. This study demonstrated the utility of biologically self-assembled epitope-coated BPs as an epitope carrier for inclusion in next-generation malaria vaccines.

HER2/neu-Based Peptide Vaccination-Pulsed with B-Cell Epitope Induced Efficient Prophylactic and Therapeutic Antitumor Activities in TUBO Breast Cancer Mice Model.

Simple SummaryRecently, the role of vaccination has been expanded in the management of HER2/neu-positive breast cancer. Data from various clinical trials indicated that an effective breast cancer vaccine must be able to induce both humoral and cellular immune responses. One approach is the use of a multi-epitope peptide vaccine comprising B, T helper and CTL epitopes. In this study, we have successfully developed a vaccine construct based on a chimeric HER2/neu-derived antigenic peptide possessing B- and T-cell epitopes conjugated to the KLH protein as a source of T-helper epitopes. A similar vaccine construct, but without the presence of a B-cell peptide epitope, was also prepared to examine the role of the humoral immune response in antitumor immunity. Multi-epitope peptide vaccine with additional B-cell epitope was found to be superior in inhibiting the tumor growth and prolongation of survival in tumor mice model compared to vaccine construct without the presence of B-cell epitope.Breast cancer is the most common invasive cancer diagnosed among women. A cancer vaccine has been recognized as a form of immunotherapy with a prominent position in the prevention and treatment of breast cancer. The majority of current breast cancer vaccination strategies aim to stimulate antitumor T-cell responses of the HER2/neu oncogene, which is abnormally expressed in breast cancer cells. However, the role of the B-cell humoral response is often underappreciated in the cancer vaccine design. We have advanced this idea by elucidating the role of B-cells in cancer vaccination by designing a chimeric antigenic peptide possessing both cytotoxic T lymphocytes (GP2) and B-cell (P4) peptide epitopes derived from HER2/neu. The chimeric peptide (GP2–P4) was further conjugated to a carrier protein (KLH), forming a KLH–GP2–P4 conjugate. The immunogenicity of KLH–GP2–P4 was compared with KLH–GP2 (lacking the B-cell epitope) in BALB/c mice. Mice immunized with KLH–GP2–P4 elicited more potent antigen-specific neutralizing antibodies against syngeneic TUBO cells (cancer cell line overexpressing HER2/neu) that was governed by a balanced Th1/Th2 polarization in comparison to KLH–GP2. Subsequently, these immune responses led to greater inhibition of tumor growth and longer survival in TUBO tumor-bearing mice in both prophylactic and therapeutic challenge experiments. Overall, our data demonstrated that the B-cell epitope has a profound effect in orchestrating an efficacious antitumor immunity. Thus, a multi-epitope peptide vaccine encompassing cytotoxic T-lymphocytes, T-helper and B-cell epitopes represents a promising strategy in developing cancer vaccines with a preventive and therapeutic modality for the effective management of breast cancer.

Molecular characterization of Anaplasma marginale based on the msp1a and msp1b genes

Anaplasma marginale is an intracellular rickettsial bacterium causing anaplasmosis in ruminants. A. marginale is transmitted biologically by ticks and mechanically by blood-sucking vectors. Anaplasmosis occurs in tropical and subtropical areas of the world. This disease causes huge economic losses due to decreasing meat yield and milk production. The aims of this study were to determine the genetic diversity and antigenicity of A. marginale based on the msp1a and msp1b genes in cattle in Thailand. The A. marginale msp1a and msp1b genes were amplified by the polymerase chain reaction (PCR). There have been four copies of MSP1a tandem repeats among A. marginale Thailand strain, and thirteen different MSP1a tandem repeats were found including repeats B, 25, 27, M, 3, S, C, H, β, 80, 4, TH1 and TH2. Notably, this study showed two copies of the novel conserved tandem sequences namely Thailand Type 1 (TH1) and Type 2 (TH2). The phylogenetic analysis revealed that A. marginale msp1a and msp1b genes were genetically diverse and showed 9 and 5 clades with similarity ranging from 98 to 100% and 79.5 to 100%, respectively, when compared within the isolates of this study. The results of diversity analysis showed 18 and 16 haplotypes of the msp1a and msp1b genes, respectively. The entropy analyses of msp1a and msp1b nucleic acid sequences showed 39 and 900 high entropy peaks with values ranging from 0.35 to 0.85 and from 0.41 to 1.48, respectively, while those of MSP1a and MSP1b amino acid sequences exhibited 75 and 72 high entropy peaks with values ranging from 0.35 to 1.06 and from 0.41 to 1.55, respectively. In addition, B-cell and T-cell epitopes have also been investigated in this study. Hence, our results could be employed to improve the insight input of molecular phylogenetics, genetic diversity and antigenicity of A. marginale Thailand strain.

Methods to Identify Immunogenic Peptides in SARS-CoV-2 Spike and Protective Monoclonal Antibodies in COVID-19 Patients.

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection and the associated COVID‐19 diseases are an emerging threat to global public health. Although considerable scientific research on the immune, especially antibody, responses to SARS‐CoV‐2 infection have been conducted, additional dominant epitopes and protective antibodies are needed for diagnosis and treatment of COVID‐19 patients. Here, two different phage libraries are used to identify immunogenic epitopes across the spike protein and monoclonal antibodies from COVID‐19 patients. Three peptides are further characterized in the receptor‐binding motif (RBM) and measured their antibody levels in COVID‐19 patients, from which one identifies one most immunodominant epitope with the highest antibody response in COVID‐19 patients and in immunized mice. More importantly, monoclonal antibodies specifically binding to this peptide isolated from COVID‐19 patients have therapeutic potential to neutralize SARS‐CoV‐2 infection. Thus, the approaches to systemically identify immunogenic peptides and directly identify human monoclonal antibodies from patients will provide useful diagnostic and therapeutic tools for COVID‐19 and other emerging infectious diseases.