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1.
Front Immunol ; 12: 669357, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34349756

RESUMO

Development of adaptive immunity after COVID-19 and after vaccination against SARS-CoV-2 is predicated on recognition of viral peptides, presented on HLA class II molecules, by CD4+ T-cells. We capitalised on extensive high-resolution HLA data on twenty five human race/ethnic populations to investigate the role of HLA polymorphism on SARS-CoV-2 immunogenicity at the population and individual level. Within populations, we identify wide inter-individual variability in predicted peptide presentation from structural, non-structural and accessory SARS-CoV-2 proteins, according to individual HLA genotype. However, we find similar potential for anti-SARS-CoV-2 cellular immunity at the population level suggesting that HLA polymorphism is unlikely to account for observed disparities in clinical outcomes after COVID-19 among different race/ethnic groups. Our findings provide important insight on the potential role of HLA polymorphism on development of protective immunity after SARS-CoV-2 infection and after vaccination and a firm basis for further experimental studies in this field.


Assuntos
COVID-19/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Imunidade Celular , SARS-CoV-2/imunologia , Apresentação do Antígeno , Linfócitos T CD4-Positivos/imunologia , COVID-19/genética , Genótipo , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Peptídeos/imunologia , Polimorfismo Genético , Proteoma/imunologia , Proteínas Virais/imunologia
2.
Cell Rep ; 36(8): 109570, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34390647

RESUMO

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.


Assuntos
COVID-19/prevenção & controle , Linfócitos T/imunologia , Vacinas Sintéticas/administração & dosagem , Anticorpos Antivirais/sangue , Ligante de CD40/metabolismo , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Interferon gama/metabolismo , Interleucina-2/metabolismo , Peptídeos/imunologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Vacinação , Vacinas Sintéticas/imunologia
3.
J Immunol ; 207(4): 1138-1149, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34341168

RESUMO

Group A streptococcal infections are a significant cause of global morbidity and mortality. A leading vaccine candidate is the surface M protein, a major virulence determinant and protective Ag. One obstacle to the development of M protein-based vaccines is the >200 different M types defined by the N-terminal sequences that contain protective epitopes. Despite sequence variability, M proteins share coiled-coil structural motifs that bind host proteins required for virulence. In this study, we exploit this potential Achilles heel of conserved structure to predict cross-reactive M peptides that could serve as broadly protective vaccine Ags. Combining sequences with structural predictions, six heterologous M peptides in a sequence-related cluster were predicted to elicit cross-reactive Abs with the remaining five nonvaccine M types in the cluster. The six-valent vaccine elicited Abs in rabbits that reacted with all 11 M peptides in the cluster and functional opsonic Abs against vaccine and nonvaccine M types in the cluster. We next immunized mice with four sequence-unrelated M peptides predicted to contain different coiled-coil propensities and tested the antisera for cross-reactivity against 41 heterologous M peptides. Based on these results, we developed an improved algorithm to select cross-reactive peptide pairs using additional parameters of coiled-coil length and propensity. The revised algorithm accurately predicted cross-reactive Ab binding, improving the Matthews correlation coefficient from 0.42 to 0.74. These results form the basis for selecting the minimum number of N-terminal M peptides to include in potentially broadly efficacious multivalent vaccines that could impact the overall global burden of group A streptococcal diseases.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Reações Cruzadas/imunologia , Vacinas Estreptocócicas/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Epitopos/imunologia , Feminino , Humanos , Masculino , Camundongos , Peptídeos/imunologia , Vacinas Sintéticas/imunologia
4.
Microb Pathog ; 158: 105095, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34280501

RESUMO

Short peptide antigens covering conserved T or B cell epitopes have been investigated in influenza vaccines. Bursal pentapeptide V (BPP-V) and bursal peptide IV (BP-IV) are small molecular peptides that were isolated and identified from the bursa of Fabricius (BF) and induce a strong immune response at both the humoural and cellular levels. To explore the molecular adjuvant potential of BPP-V and BP-IV with an epitope vaccine, an epitope peptide (HA284-298, GNCVVQCQTERGGLN) rich in T and B cell epitopes for the H9N2 avian influenza virus (AIV) haemagglutinin (HA) protein was selected. BPP-V and BP-IV were coupled with the epitope peptide sequence to form BPP-V and BP-IV-epitope vaccines, respectively. The immunoefficacy of BPP-V and BP-IV-epitope peptide vaccines was evaluated. The results showed that the epitope peptide had weak immunogenicity. The BPP-V-epitope peptide vaccine promoted only the secretion of anti-HA IgG and IgG1 antibodies. The BP-IV-epitope peptide vaccine not only promoted the production of anti-HA IgG and IgG1 antibodies but also significantly induced the production of the IgG2a antibody. The BP-IV-epitope peptide vaccine significantly promoted the production of interleukin (IL-4) and interferon-γ (IFN-γ) (the BPP-V epitope peptide vaccine promoted only the production of IL-4), enhanced the cytotoxic T lymphocyte (CTL) response, and increased the proportion of CD3+ T lymphocytes. Moreover, the BP-IV-epitope peptide vaccine promoted a cell-mediated immune response similar to that of the AIV vaccine group. Furthermore, BPP-V and BP-IV-epitope peptide vaccines could also accelerate the clearance of pulmonary virus and reduce pathological damage after the challenge with H9N2 AIV. This study demonstrates the potential of BP-IV as an effective adjuvant for the epitope peptide vaccine for the H9N2 AIV.


Assuntos
Adjuvantes Imunológicos , Vírus da Influenza A Subtipo H9N2 , Vacinas contra Influenza , Influenza Aviária , Animais , Anticorpos Antivirais , Galinhas , Epitopos de Linfócito B , Epitopos de Linfócito T , Influenza Aviária/prevenção & controle , Peptídeos/imunologia , Vacinas de Subunidades
5.
J Immunol ; 207(4): 1009-1017, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34321228

RESUMO

The human CD8+ T cell clone 6C5 has previously been shown to recognize the tert-butyl-modified Bax161-170 peptide LLSY(3-tBu)FGTPT presented by HLA-A*02:01. This nonnatural epitope was likely created as a by-product of fluorenylmethoxycarbonyl protecting group peptide synthesis and bound poorly to HLA-A*02:01. In this study, we used a systematic approach to identify and characterize natural ligands for the 6C5 TCR. Functional analyses revealed that 6C5 T cells only recognized the LLSYFGTPT peptide when tBu was added to the tyrosine residue and did not recognize the LLSYFGTPT peptide modified with larger (di-tBu) or smaller chemical groups (Me). Combinatorial peptide library screening further showed that 6C5 T cells recognized a series of self-derived peptides with dissimilar amino acid sequences to LLSY(3-tBu)FGTPT. Structural studies of LLSY(3-tBu)FGTPT and two other activating nonamers (IIGWMWIPV and LLGWVFAQV) in complex with HLA-A*02:01 demonstrated similar overall peptide conformations and highlighted the importance of the position (P) 4 residue for T cell recognition, particularly the capacity of the bulky amino acid tryptophan to substitute for the tBu-modified tyrosine residue in conjunction with other changes at P5 and P6. Collectively, these results indicated that chemical modifications directly altered the immunogenicity of a synthetic peptide via molecular mimicry, leading to the inadvertent activation of a T cell clone with unexpected and potentially autoreactive specificities.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Fragmentos de Peptídeos/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Apresentação do Antígeno/imunologia , Células Cultivadas , Epitopos de Linfócito T/imunologia , Humanos , Ligantes , Biblioteca de Peptídeos
6.
Commun Biol ; 4(1): 825, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34211107

RESUMO

Extracellular vesicles can modulate diverse processes ranging from proliferation and tissue repair, to chemo-resistance and cellular differentiation. With the advent of tissue and immunological targeting, extracellular vesicles are also increasingly viewed as promising vectors to deliver peptide-based cancer antigens to the human immune system. Despite the clinical relevance and therapeutic potential of such 'cell-free' approaches, the natural antigen presentation landscape exported in extracellular vesicles is still largely uncharted, due to the challenging nature of such preparations and analyses. In the context of therapeutic vesicle production, a critical evaluation of the similarity in vesicular antigen presentation is also urgently needed. In this work, we compared the HLA-I peptide ligandomes of extracellular vesicles against that of whole-cells of the same cell line. We found that extracellular vesicles not only over-represent HLA-B complexes and peptide ligands, but also cysteinylated peptides that may modulate immune responses. Collectively, these findings describe the pre-existing provision of vesicular HLA complexes that may be utilized to carry peptide vaccines, as well as the propensity for different peptide and post-translationally modified ligands to be presented, and will outline critical considerations in devising novel EV vaccination strategies.


Assuntos
Apresentação do Antígeno/imunologia , Cisteína/imunologia , Vesículas Extracelulares/imunologia , Antígenos HLA-B/imunologia , Linhagem Celular , Cromatografia Líquida/métodos , Cisteína/metabolismo , Vesículas Extracelulares/metabolismo , Antígenos HLA-B/metabolismo , Humanos , Ligantes , Peptídeos/imunologia , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/imunologia , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
7.
Viruses ; 13(7)2021 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199077

RESUMO

Many viruses, especially RNA viruses, utilize programmed ribosomal frameshifting and/or stop codon readthrough in their expression, and in the decoding of a few a UGA is dynamically redefined to specify selenocysteine. This recoding can effectively increase viral coding capacity and generate a set ratio of products with the same N-terminal domain(s) but different C-terminal domains. Recoding can also be regulatory or generate a product with the non-universal 21st directly encoded amino acid. Selection for translation speed in the expression of many viruses at the expense of fidelity creates host immune defensive opportunities. In contrast to host opportunism, certain viruses, including some persistent viruses, utilize recoding or adventitious frameshifting as part of their strategy to evade an immune response or specific drugs. Several instances of recoding in small intensively studied viruses escaped detection for many years and their identification resolved dilemmas. The fundamental importance of ribosome ratcheting is consistent with the initial strong view of invariant triplet decoding which however did not foresee the possibility of transitory anticodon:codon dissociation. Deep level dynamics and structural understanding of recoding is underway, and a high level structure relevant to the frameshifting required for expression of the SARS CoV-2 genome has just been determined.


Assuntos
Vírus de DNA/genética , Vírus de DNA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Evasão da Resposta Imune , Vírus de RNA/genética , Antivirais/farmacologia , Códon de Terminação , Vírus de DNA/efeitos dos fármacos , Mudança da Fase de Leitura do Gene Ribossômico , Antígenos de Histocompatibilidade Classe I/genética , Conformação de Ácido Nucleico , Peptídeos/imunologia , Biossíntese de Proteínas , Vírus de RNA/efeitos dos fármacos , Vírus de RNA/imunologia
8.
Viruses ; 13(7)2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202029

RESUMO

The current COVID-19 pandemic has highlighted the urgent need to develop effective therapeutic strategies. We evaluated the in vitro antiviral effect against SARS-CoV-2 of a hepatitis B virus (HBV) hexamer peptide, Poly6, which is capable of eliciting an antiviral effect against human immunodeficiency virus -1 (HIV-1), as a novel HIV-1 integrase inhibitor, and a strong anticancer immune response in an IFN-I-dependent manner, as a novel potential adjuvant in anticancer immunotherapy. Here, we report that Poly6 exerts an anti-SARS-CoV-2 effect, with an estimated 50% inhibitory concentration of 2.617 µM, in the human bronchial epithelial cell line, Calu-3 but not in Vero-E6 cells, which are deficient in type 1 interferon (IFN-I) signaling. We proved via assays based on mRNA profiles, inhibitors, or blocking antibodies that Poly6 can exert an anti-SARS-CoV-2 effect in an IFN-I-dependent manner. We also found that Poly6 inhibits IL-6 production enhanced by SARS-CoV-2 in infected Calu-3 cells at both the transcription and the translation levels, mediated via IL-10 induction in an IFN-I-dependent manner. These results indicate the feasibility of Poly6 as an IFN-I-inducing COVID-19 drug with potent antiviral and anti-inflammatory activities.


Assuntos
Antivirais/farmacologia , Células Epiteliais/efeitos dos fármacos , Vírus da Hepatite B/química , Interferon Tipo I/imunologia , Peptídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Brônquios/citologia , Brônquios/virologia , Chlorocebus aethiops , Células Epiteliais/imunologia , Células Epiteliais/virologia , Vírus da Hepatite B/genética , Humanos , Pulmão/citologia , Pulmão/virologia , Peptídeos/imunologia , SARS-CoV-2/imunologia , Células Vero
9.
Front Immunol ; 12: 693054, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34326844

RESUMO

Advanced age is associated with severe symptoms and death upon SARS-CoV-2 infection. Virus-specific CD8+ T-cell responses have shown to be protective toward critical COVID-19 manifestations, suggesting that suboptimal cellular immunity may contribute to the age-pattern of the disease. The induction of a CD8+ T-cell response against an emerging pathogen like SARS-CoV-2 relies on the activation of naive T cells. To investigate whether the primary CD8+ T-cell response against this virus is defective in advanced age, we used an in vitro approach to prime SARS-CoV-2-specific naive CD8+ T cells from healthy, unexposed donors of different age groups. Compared to younger adults, older individuals display a poor SARS-CoV-2-specific T-cell priming capacity in terms of both magnitude and quality of the response. In addition, older subjects recognize a lower number of epitopes. Our results implicate that immune aging is associated with altered primary SARS-CoV-2-specific CD8+ T-cell responses.


Assuntos
Envelhecimento/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Antígenos Virais/imunologia , Células Cultivadas , ELISPOT , Epitopos de Linfócito T/imunologia , Regulação da Expressão Gênica , Voluntários Saudáveis , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Ativação Linfocitária , Pessoa de Meia-Idade , Peptídeos/imunologia , Adulto Jovem
10.
Nat Commun ; 12(1): 4365, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272369

RESUMO

Activating RAS missense mutations are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here we employ a multi-omics approach to characterize HLA class I-restricted mKRAS epitopes. We provide proteomic evidence of mKRAS epitope processing and presentation by high prevalence HLA class I alleles. Select epitopes are immunogenic enabling mKRAS-specific TCRαß isolation. TCR transfer to primary CD8+ T cells confers cytotoxicity against mKRAS tumor cell lines independent of histologic origin, and the kinetics of lytic activity correlates with mKRAS peptide-HLA class I complex abundance. Adoptive transfer of mKRAS-TCR engineered CD8+ T cells leads to tumor eradication in a xenograft model of metastatic lung cancer. This study validates mKRAS peptides as bona fide epitopes facilitating the development of immune therapies targeting this oncoprotein.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinogênese/imunologia , Epitopos de Linfócito T/imunologia , Neoplasias Pulmonares/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transferência Adotiva , Alelos , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Peptídeos/genética , Peptídeos/imunologia , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Front Immunol ; 12: 635942, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34127926

RESUMO

SARS-CoV-2 infection takes a mild or clinically inapparent course in the majority of humans who contract this virus. After such individuals have cleared the virus, only the detection of SARS-CoV-2-specific immunological memory can reveal the exposure, and hopefully the establishment of immune protection. With most viral infections, the presence of specific serum antibodies has provided a reliable biomarker for the exposure to the virus of interest. SARS-CoV-2 infection, however, does not reliably induce a durable antibody response, especially in sub-clinically infected individuals. Consequently, it is plausible for a recently infected individual to yield a false negative result within only a few months after exposure. Immunodiagnostic attention has therefore shifted to studies of specific T cell memory to SARS-CoV-2. Most reports published so far agree that a T cell response is engaged during SARS-CoV-2 infection, but they also state that in 20-81% of SARS-CoV-2-unexposed individuals, T cells respond to SARS-CoV-2 antigens (mega peptide pools), allegedly due to T cell cross-reactivity with Common Cold coronaviruses (CCC), or other antigens. Here we show that, by introducing irrelevant mega peptide pools as negative controls to account for chance cross-reactivity, and by establishing the antigen dose-response characteristic of the T cells, one can clearly discern between cognate T cell memory induced by SARS-CoV-2 infection vs. cross-reactive T cell responses in individuals who have not been infected with SARS-CoV-2.


Assuntos
COVID-19/imunologia , COVID-19/virologia , Interações Hospedeiro-Patógeno/imunologia , SARS-CoV-2/imunologia , Linfócitos T/imunologia , Antígenos Virais/imunologia , Biomarcadores , COVID-19/metabolismo , Reações Cruzadas/imunologia , Citocinas/metabolismo , Epitopos de Linfócito T/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Memória Imunológica , Peptídeos/imunologia , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/metabolismo
12.
Bioconjug Chem ; 32(8): 1606-1616, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34181851

RESUMO

In the near future, the increase in the number of required tests for COVID-19 antibodies is expected to be many hundreds of millions. Obviously, this will be done using a variety of analytical methods and using different antigens, including peptides. In this work, we compare three method variations for detecting specific immunoglobulins directed against peptides of approximately 15-aa of the SARS-CoV-2 spike protein. These linear peptide epitopes were selected using antigenicity algorithms, and were synthesized with an additional terminal cysteine residue for their bioconjugation. In two of the methods, constructs were prepared where the peptide (F, function) is attached to a negatively charged hydrophilic spacer (S) linked to a dioleoylphosphatidyl ethanolamine residue (L, lipid) to create a function-spacer-lipid construct (FSL). These FSLs were easily and controllably incorporated into erythrocytes for serologic testing or in a lipid bilayer deposited on a polystyrene microplate for use in an enzyme immunoassays (EIA). The third method, also an EIA, used polyacrylamide conjugated peptides (peptide-PAA) prepared by controlled condensation of the cysteine residue of the peptide with the maleimide-derived PAA polymer which were immobilized on polystyrene microplates by physisorption of the polymer. In this work, we describe the synthesis of the PAA and FSL peptide bioconjugates, design of test systems, and comparison of the bioassays results, and discuss potential reasons for higher performance of the FSL conjugates, particularly in the erythrocyte-based serologic assay.


Assuntos
Anticorpos Antivirais/análise , Desenho de Fármacos , Peptídeos/química , Peptídeos/imunologia , SARS-CoV-2/imunologia , Anticorpos Antivirais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia
13.
Cell ; 184(15): 3962-3980.e17, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34171305

RESUMO

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.


Assuntos
Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Fases de Leitura Aberta/genética , Peptídeos/imunologia , Proteoma/imunologia , SARS-CoV-2/imunologia , Células A549 , Alelos , Sequência de Aminoácidos , Animais , Apresentação do Antígeno/imunologia , COVID-19/imunologia , COVID-19/virologia , Feminino , Células HEK293 , Humanos , Cinética , Masculino , Camundongos , Peptídeos/química , Linfócitos T/imunologia
14.
Theranostics ; 11(15): 7308-7321, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34158852

RESUMO

Dendritic cells (DCs) can process the antigens of cancer vaccine and thus stimulate the CD8+ T cells to recognize and kill the tumor cells that express these antigens. However, lack of promising carriers for presenting the antigens to DCs is one of the main barriers to the development of clinically effective cancer vaccines. Another limitation is the risk of inflammatory side effects induced by the adjuvants. It is still unclear how we can develop ideal adjuvant-free DC vaccine carriers without adjuvants. Methods: A 12-mer peptide carrier (CBP-12) with high affinity for Clec9a expressed on DCs was developed using an in silico rational optimization method. The therapeutic effects of the adjuvant-free vaccine comprising CBP-12 and exogenous or endogenous antigenic peptides were investigated in terms of antigen cross-presentation efficacy, specific cytotoxic T lymphocyte response, and antitumor activity. We also explored the mechanism involved in the antitumor effects of the adjuvant-free CBP-12 vaccine. Finally, we assessed the effects of the CBP-12 conjugated peptide vaccine combined with radiotherapy. Results: Here, we developed CBP-12 as a vaccine carrier that enhanced the uptake and cross-presentation of the antigens, thus inducing strong CD8+ T cell responses and antitumor effects in both anti-PD-1-responsive (B16-OVA) and -resistant (B16) models, even in adjuvant-free conditions. CBP-12 bound to and activated Clec9a, thereby stimulating Clec9a+ DC to product IL-21, but not IL-12 by activating of Syk. The antitumor effects of the CBP-12 conjugated peptide vaccines could be blocked by an IL-21 neutralizing antibody. We also observed the synergistic antitumor effects of the CBP-12 conjugated peptide vaccine combined with radiotherapy. Conclusions: CBP-12 could serve as an adjuvant-free peptide vaccine carrier for cancer immunotherapy.


Assuntos
Vacinas Anticâncer , Células Dendríticas/imunologia , Sistemas de Liberação de Medicamentos , Interleucinas/imunologia , Lectinas Tipo C/imunologia , Melanoma Experimental/imunologia , Peptídeos , Receptores Imunológicos/imunologia , Transdução de Sinais/efeitos dos fármacos , Quinase Syk/imunologia , Animais , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/farmacologia , Feminino , Interleucinas/genética , Lectinas Tipo C/genética , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Camundongos Knockout , Peptídeos/imunologia , Peptídeos/farmacologia , Receptores Imunológicos/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Quinase Syk/genética , Vacinas de Subunidades/imunologia , Vacinas de Subunidades/farmacologia
15.
Nat Commun ; 12(1): 3346, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099720

RESUMO

Characterizing the human leukocyte antigen (HLA) bound ligandome by mass spectrometry (MS) holds great promise for developing vaccines and drugs for immune-oncology. Still, the identification of non-tryptic peptides presents substantial computational challenges. To address these, we synthesized and analyzed >300,000 peptides by multi-modal LC-MS/MS within the ProteomeTools project representing HLA class I & II ligands and products of the proteases AspN and LysN. The resulting data enabled training of a single model using the deep learning framework Prosit, allowing the accurate prediction of fragment ion spectra for tryptic and non-tryptic peptides. Applying Prosit demonstrates that the identification of HLA peptides can be improved up to 7-fold, that 87% of the proposed proteasomally spliced HLA peptides may be incorrect and that dozens of additional immunogenic neo-epitopes can be identified from patient tumors in published data. Together, the provided peptides, spectra and computational tools substantially expand the analytical depth of immunopeptidomics workflows.


Assuntos
Aprendizado Profundo , Peptídeos/imunologia , Espectrometria de Massas em Tandem/métodos , Linhagem Celular , Epitopos , Proteínas da Matriz Extracelular/metabolismo , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Ligantes , Espectrometria de Massas , Medicina Molecular , Peptídeos/metabolismo , Proteômica
16.
Cell Rep ; 35(13): 109305, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34166618

RESUMO

The human leukocyte antigen (HLA)-bound viral antigens serve as an immunological signature that can be selectively recognized by T cells. As viruses evolve by acquiring mutations, it is essential to identify a range of presented viral antigens. Using HLA peptidomics, we are able to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-derived peptides presented by highly prevalent HLA class I (HLA-I) molecules by using infected cells as well as overexpression of SARS-CoV-2 genes. We find 26 HLA-I peptides and 36 HLA class II (HLA-II) peptides. Among the identified peptides, some are shared between different cells and some are derived from out-of-frame open reading frames (ORFs). Seven of these peptides were previously shown to be immunogenic, and we identify two additional immunoreactive peptides by using HLA multimer staining. These results may aid the development of the next generation of SARS-CoV-2 vaccines based on presented viral-specific antigens that span several of the viral genes.


Assuntos
Antígenos Virais/imunologia , COVID-19/imunologia , COVID-19/virologia , Peptídeos/imunologia , SARS-CoV-2/imunologia , Apresentação do Antígeno , Antígenos Virais/metabolismo , Vacinas contra COVID-19 , Linhagem Celular , Epitopos de Linfócito T/imunologia , Células HEK293 , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Peptidomiméticos , SARS-CoV-2/genética , Linfócitos T
17.
BMC Microbiol ; 21(1): 194, 2021 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-34174835

RESUMO

BACKGROUND: Serological test is helpful in confirming and tracking infectious diseases in large population with the advantage of fast and convenience. Using the specific epitope peptides identified from the whole antigen as the detection antigen is sensitive and relatively economical. The development of epitope peptide-based detection kits for COVID-19 patients requires comprehensive information about epitope peptides. But the data on B cell epitope of SARS-CoV-2 spike protein is still limited. More importantly, there is a lack of serological data on the peptides in the population. In this study, we aimed to identify the B cell epitope peptides of spike protein and detect the reactivity in serum samples, for further providing data support for their subsequent serological applications. RESULTS: Two B cell linear epitopes, P104 and P82, located in non-RBD region of SARS-CoV-2 S protein were identified by indirect ELISA screening of an overlapping peptide library of the S protein with COVID-19 patients' convalescent serum. And the peptides were verified by testing with 165 serum samples. P104 has not been reported previously; P82 is contained in peptide S21P2 reported before. The positive reaction rates of epitope peptides S14P5 and S21P2, the two non-RBD region epitopes identified by Poh et al., and P82 and P104 were 77.0%, 73.9%, 61.2% and 30.3%, respectively, for 165 convalescent sera, including 30 asymptomatic patients. Although P104 had the lowest positive rate for total patients (30.3%), it exhibited slight advantage for detection of asymptomatic infections (36.7%). Combination of epitopes significantly improved the positive reaction rate. Among all combination patterns, (S14P5 + S21P2 + P104) pattern exhibited the highest positive reaction rate for all patients (92.7%), as well as for asymptomatic infections (86.7%), confirming the feasibility of P104 as supplementary antigen for serological detection. In addition, we analyzed the correlation between epitopes with neutralizing antibody, but only S14P5 had a medium positive correlation with neutralizing antibody titre (rs = 0.510, P < 0.01). CONCLUSION: Our research proved that epitopes on non-RBD region are of value in serological detection especially when combination more than one epitope, thus providing serological reaction information about the four epitopes, which has valuable references for their usage.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19 , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos de Linfócito B , Glicoproteína da Espícula de Coronavírus/química , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/diagnóstico , COVID-19/imunologia , COVID-19/virologia , Criança , Pré-Escolar , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Peptídeos/imunologia , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
18.
PLoS One ; 16(6): e0252198, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34077451

RESUMO

Once an obscure pathogen, Zika virus (ZIKV) has emerged as a significant global public health concern. Several studies have linked ZIKV infection in pregnant women with the development of microcephaly and other neurological abnormalities, emphasizing the need for a safe and effective vaccine to combat the spread of this disease. Preclinical studies and vaccine development efforts have largely focused on the role of humoral immunity in disease protection. Consequently, relatively little is known in regard to cellular immunity against ZIKV, although an effective vaccine will likely need to engage both the humoral and cellular arms of the immune system. To that end, we utilized two-dimensional liquid chromatography coupled with tandem mass spectrometry to identify 90 ZIKV peptides that were naturally processed and presented on HLA class I and II molecules (HLA-A*02:01/HLA-DRB1*04:01) of an immortalized B cell line infected with ZIKV (strain PRVABC59). Sequence identity clustering was used to filter the number of candidate peptides prior to evaluating memory T cell recall responses in ZIKV convalescent subjects. Peptides that individually elicited broad (4 of 7 subjects) and narrow (1 of 7 subjects) T cell responses were further analyzed using a suite of predictive algorithms and in silico modeling to evaluate HLA binding and peptide structural properties. A subset of nine broadly reactive peptides was predicted to provide robust global population coverage (97.47% class I; 70.74% class II) and to possess stable structural properties amenable for vaccine formulation, highlighting the potential clinical benefit for including ZIKV T cell epitopes in experimental vaccine formulations.


Assuntos
COVID-19/terapia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Adulto , Linfócitos B/imunologia , COVID-19/imunologia , Linhagem Celular , Epitopos de Linfócito T/imunologia , Feminino , Humanos , Imunidade Celular , Imunidade Humoral , Imunização Passiva/métodos , Memória Imunológica , Masculino , Espectrometria de Massas/métodos , Peptídeos/imunologia , Linfócitos T/imunologia , Zika virus/metabolismo , Zika virus/patogenicidade , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologia
19.
Int J Biol Macromol ; 184: 522-529, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34119553

RESUMO

Sericin, as the main component of silkworm cocoon silk, surrounds and protects the silk fibroin. Sericin is a natural macromolecular protein complex encoded by the genes Ser1, Ser2, and Ser3. At present, there are no available antibodies against sericin that may be used to identify and locate it at the protein level, hindering the study of its secretion mechanism and materials application. Therefore, the development of effective antibodies against sericin is an urgent necessity. To address this problem, we prepared polyclonal antibodies against the Ser1, Ser2 and Ser3 proteins using synthesized peptides for the first time. The specificity of the antibodies was confirmed using dot blot, immunoblotting and mass spectrometry on the hybrid bands of the middle silk gland. The immunoblotting results of anti-sericin antibodies showed that sericin has different molecular weights in different regions of the middle silk gland and strains in the 5th instar. Through immunohistochemistry, anti-sericin antibodies revealed that sericin presented different distributions in the anterior part of the middle silk gland of 872 strain at the 7th day of 5th instar. In addition, the prepared antibodies not only detected intact sericin molecules, but also detected degraded sericin that was dissolved in five different solvents. In summary, this work prepared effective sericin antibodies for silk protein synthesis and secretion research and provides a possible molecular detection method for biological products containing silkworm sericin.


Assuntos
Anticorpos/análise , Bombyx/crescimento & desenvolvimento , Peptídeos/imunologia , Sericinas/química , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Bombyx/imunologia , Bombyx/metabolismo , Peso Molecular , Família Multigênica , Peptídeos/genética , Sericinas/genética , Sericinas/imunologia , Especificidade da Espécie
20.
JCI Insight ; 6(13)2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34081630

RESUMO

BACKGROUNDThe role of humoral immunity in COVID-19 is not fully understood, owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome.METHODSUsing SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients' plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution.RESULTSWe identified linear epitopes from the spike (S) and nucleocapsid (N) proteins and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S-811-825, S-881-895, and N-156-170 epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes.CONCLUSIONEpitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, but it also may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern in both the peptide array and latex agglutination formats.FUNDINGOntario Research Fund (ORF) COVID-19 Rapid Research Fund, Toronto COVID-19 Action Fund, Western University, Lawson Health Research Institute, London Health Sciences Foundation, and Academic Medical Organization of Southwestern Ontario (AMOSO) Innovation Fund.


Assuntos
Testes de Aglutinação/métodos , Formação de Anticorpos/imunologia , Teste Sorológico para COVID-19/métodos , COVID-19/imunologia , Epitopos de Linfócito B/imunologia , SARS-CoV-2/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , COVID-19/sangue , COVID-19/mortalidade , Epitopos/imunologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Humanos , Imunidade Humoral , Análise em Microsséries/métodos , Nucleocapsídeo/química , Nucleocapsídeo/genética , Nucleocapsídeo/imunologia , Peptídeos/imunologia , SARS-CoV-2/genética , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
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