Identification of Promiscuous Epitopes from the Mycobacterial 65-Kilodalton Heat Shock Protein Recognized by Human CD4⁺T Cells of theMycobacterium lepraeMemory Repertoire

Abstract

By using a synthetic peptide approach, we mapped epitopes from the mycobacterial 65-kDa heat shock protein (HSP65) recognized by human T cells belonging to the Mycobacterium leprae memory repertoire. A panel of HSP65 reactive CD4+ T-cell lines and clones were established from healthy donors 8 years after immunization with heat-killed M. leprae and then tested for proliferative reactivity against overlapping peptides comprising both the M. leprae and Mycobacterium tuberculosisHSP65 sequences. The results showed that the antigen-specific T-cell lines and clones established responded to 12 mycobacterial HSP65 peptides, of which 9 peptides represented epitopes crossreactive between the M. tuberculosis and M. leprae HSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 445, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) representedM. leprae HSP65-specific epitopes. Major histocompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from self HLA-DRB1 genes, whereas 3 peptides with sequences completely identical between the M. leprae andM. tuberculosis HSP65 were presented to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was presented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition, the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epitopes from the mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4+ T cells which belong to the human memory T-cell repertoire against M. leprae. The results suggest that such epitopes might be used in the peptide-based design of subunit vaccines against mycobacterial diseases.

Identification of Immune Responses to Japanese Encephalitis Virus Specific T Cell Epitopes

“…Epitopes presented by MHC class II alleles are shown to be highly promiscuous. The same epitope has shown to be presented by multiple T cell alleles in breast cancer (HER2) (43), Ag85B T-cell epitope in Mycobacterium tuberculosis (44), T cell epitopes in Mycobacterium leprae (45) and in many other instances.…”

Th1 Cell Reactivity and HLA‐DR Binding Prediction for Promiscuous Recognition of MPT63 (Rv1926c), a Major Secreted Protein of Mycobacterium tuberculosis

“…The above results suggest that MPT63 could be useful in vaccine formulations against TB. However, it is well known that the protective Th1 cells recognize protein antigens in association with highly polymorphic HLA class II molecules, and particularly HLA‐DR molecules which are usually involved in the presentation of mycobacterial antigens/epitopes to Th1 cells [28, 31, 34, 46, 51, 52]. Therefore, amongst the requirements for any antigen to qualify as a vaccine candidate is its recognition by HLA heterogeneous group of donors.…”

Construção de uma vacina de DNA bivalente para tuberculose expressando a proteína gD do HSV-1 e os epítopos da Hsp65 micobacteriana

“…Mustafá e colaboradores (1999) identificaram três epitopos da Hsp65 (P7-aa 61 ao 75, P13-aa 141 ao 155 e P47-aa 501 ao 515) reconhecidos pelas células T CD4 + de indivíduos imunizados com M. leprae (Mustafa, Lundin et al, 1999) que se associam a várias moléculas de HLA, que não são reconhecidos pelas células T de pacientes com doenças auto-imunes e que não estão na região de grande homologia dessa proteína (Quayle, Wilson et al, 1992). Ao contrário, Charo (2001) e colaboradores definiram o primeiro epitopo reconhecido por células T CD8 + na seqüência da Hsp65 micobacteriana (Mhsp65(9 369 )-aa 369 a 377) e demonstraram que existem células T específicas a esse peptídeo no repertório humano, que ele é gerado naturalmente a partir da proteína Hsp65 e da BCG e que não apresenta reação cruzada com seu correspondente humano.…”

“…Apesar dos epítopos citados acima serem bons alvos para as células T e gerarem uma resposta imunológica como no caso do peptídeo Mhsp65(9 369 ) (Charo, Geluk et al, 2001) uma característica inerente a eles é a fraca imunogenicidade na ausência de potentes adjuvantes (Mustafa, Lundin et al, 1999). Entretanto, avanços nas tecnologias de clonagens e expressão têm mostrado que peptídeos podem ser "entregues" de forma altamente imunogênica ao sistema imunológico na ausência de adjuvantes clássicos (Hetzel, Janssen et al, 1998;Lasaro, Alves et al, 2003;Lasaro, Diniz et al, 2005).…”

“…As seqüências dos epitopos da proteína Hsp65 (Mustafa, Lundin et al, 1999;Charo, Geluk et al, 2001) (Tabela 1) estão distribuídas dentro da seqüência do gene hsp65 em diferentes sítios. Como não tínhamos um molde de DNA para ser usado na amplificação da seqüência Vac1 (5 epitopos da proteína Hsp65), foi necessário primeiro a construção desse molde (Tabela 2) (Figura 12) por meio de uma síntese gênica utilizando a PCR.…”