Immunogenicity of a recombinant chimeric antigen using Aβ1-15 epitope fused to a T helper epitope / 军事医学

Objective To explore the immunogenicity of recombinant chimeric 6Aβ15-T including the Aβ1-15 epitope and a T-helper epitope formulated with different adjuvants and to evaluate its feasibility as a candidate vaccine for Alzheimer disease (AD).Methods The recombinant chimeric antigen 6Aβ15-T formulated with Al adjuvant, Freund′s adjuvant or MF59 adjuvant was administered to two strains of mice .The 6Aβ15-T-immunized group without adjuvants ( Mock) and non-immunized group (Control) were included in this study as control groups .The specific antibody and cellular immune response of the chimeric antigen were evaluated .Results In BALB/c strain mice, three types of adjuvants could substan-tially boost the immunogenicity of chimeric antigen 6Aβ15-T and produce a high level of specific-Aβ(β-amyloid) antibod-ies.In C57BL/6 strain mice, the existence of adjuvants enhanced the immune response of 6Aβ15-T antigen, but the mice in Mock group also produced a strong antibody response .In two strains of mice, prevalence of anti-AβIgG1, which was an indicator of Th2 polarization, was observed in the 6Aβ15-T-immunized mice.Additionally, the Al adjuvant induced a high-er level of IgG1 antibody titers, and the ratio of IgG1/IgG2a was the largest.As expected, the 6Aβ15-T antigen formulated with or without adjuvants induced PADRE-specific, but not Aβ42-specific T cellular immune response .Conclusion The 6Aβ15-T antigens formulated with different types of adjuvants could induce strong Th 2-polarized Aβ42-specific antibody re-sponses without activating self-reactive Aβ42-specific T cells in two strains of mice .The results suggested that the recombi-nant chimeric antigen 6Aβ15-T is a good candidate vaccine for AD .

Antigen-specific helper T cells in patients with pemphigus vulgaris / 中华皮肤科杂志

Objective To study the changes of antigen-specific T helper type 1 (Th1) cells and Th2 cells in patients with pemphigus vulgaris (PV) at different stages,so as to better understand the roles of autoreactive T cells in PV.Methods The DG3 (96-112) peptide was synthesized.Twenty-four patients with PV and 10 health checkup examinees were included in this study.Peripheral blood mononuclear cells (PBMCs) were obtained from the health checkup examinees and all the patients before treatment and seven patients about one month after treatment,and stimulated with the DG3 peptide of 10 mg/L for different durations.Then,enzyme-linked immunospot (ELISPOT) assay was performed to count the number of DG3-specific IFN-γ+ Th1 cells,IL-4+ Th2 cells and memory B cells.One-way analysis of variance (ANOVA) and t test were done to compare the number of cells between different groups,and Pearson correlation coefficient was used to evaluate the correlation among Th cells,memory B cells and anti-desmoglein 3 (Dsg3) antibody titers.Results In these patients,the male to female ratio was 1.67 ∶ 1,and the average age was (56.59 ± 14.66) years.Compared with the health check-up examinees,the patients with PV showed a higher absolute number of DG3-specific IFN-γ+ Th1 cells (420.18 ± 350.29 vs.145.12 ± 86.56,t =3.25,P ＜ 0.05),but a similar absolute number of specific IL-4+ Th2 cells (366.76 ± 192.44 vs.335.88 ± 164.96,P＞ 0.05) per 5 × 105 PBMCs.The percentage of DG3-specific IL-4+ Th2 cells in Th2 cells was 37.03％ ± 23.44％,and the percentage of IFN-γ+ Th1 cells was 23.62％ ± 16.77％ in peripheral blood of patients with PV.The number of DG3-specific IL-4+ Th2 cells per 5 × 105 PBMCs significantly decreased from 452.82 ± 199.29 before treatment to 241.68 ± 160.60 after treatment in seven patients (t =2.48,P ＜ 0.05).There was no significant correlation between specific Th cells,memory B cells and anti-Dsg3 antibody titers (all P ＞ 0.05).Conclusions The peptide DG3 (96-112) has pathogenic epitopes which could be recognized by specific Th cells of patients with PV.Both antigen-specific IFN-γ+ Th1 cells and IL-4+ Th2 cells play certain roles in the pathogenesis of pemphigns vulgaris,and IL-4+ Th2 cells appear to be more important.

Prediction and experimental verification of T cell reactive peptides of mycobacterium tuberculosis antigen Rv2608 / 中国医师杂志

Objective To discover T cell reactive peptides of mycobacterium tuberculosis antigen Rv2608 for treatment or adjuvant treatment for tuberculosis.Methods The T cell epitopes of Rv2608 were predicted by bioinformatics.The long-chain peptides were screened with strong affinity with HLA-A0201,HLA-A1101,and HLA-A2402.The physical and chemical characters of the peptides were predicted.The stable and hydrophilic peptides for chemical synthesis were selected.The T cell reactivity of the peptides in patients with active tuberculosis was detected by ELISPOT.The results were compared with the clinical diagnosis kits TSPOT.TB.Results Four peptides of Rv2608 were predicted and screened,and they could be active T cells of patients with active TB.Rv2608p3 was the most efficient to prime the T cell response.Conclusions Software prediction was consistent with the result of ELISPOT.Rv2608p3 might be the candidate for therapeutic vaccine for tuberculosis.

Characteristics of SLA specific T cell response and its clinical significance in autoimmune hepatitis patients / 中华检验医学杂志

Objective To analyze the feature of SLA specific T cell response in AIH. Methods Thirtyone cases of AIH were enrolled by investigating T-cell reactivity against chemically synthesized peptides spanning the full SLA protein using ELISpot. The 31 cases of AIH included 10 anti-SLA/LP positive cases and 21 negative cases. The control groups included 30 PBC patients, 29 chronic viral hepatitis patients and 30 healthy cases. The secretion of IFN-γ after PBMC stimulated by SLA peptides was observed. Results Eighteen of 31 cases with AIH [56. 08 % ( 18/31 ) ] showed the positive response to SLA peptide pools, and only 1 of 30 patients with primary biliary cirrhosis (3.34%) and 1 of 29 patients with virus hepatitis ( 3.44% ), could be elicited responses by SLA peptide pools. There wasn't positive response in healthy eases. The response frequency to SLA peptides in AIH group was significantly higher than those in PBC cases, chronic viral hepatitis cases and healthy cases (X2 = 21. 295, 20. 655, 15.988, P < 0. 01 ). Amongst 18 AIH patients with positive responds to SLA pool, 8 antigen clusters including aa 1-44, aa 57-132, an 129-180,aa 177-196, aa 193-244, aa 241-268, aa 281-308 and aa 321-428 were highlighted. The mean number of recognized peptides was 6 (2-17), indicating the polyclonal feature. Fourteen of 18 AIH patients with positive response to SLA peptides were subjected to live function test simultaneously when PBMC were collected. There was significant correlation between the breadth of recognized poptides and AST ( logarithm, r = 0. 539, P = 0. 045). Conclusions SLA peptides can induce PBMC in peripheral blood of AIH patients to secrete IFN-γ and it is polyclonal response. The breadth of recognized peptides may reflect the degree of liver inflammation.

Identification of T cell epitopes from ovarian cancer associated anti-idiotype antibody / 中华妇产科杂志

Objective To identify the T cell epitopes from ovarian cancer associated anti-idiotypic antibody 6B11in order to explore the molecular basis of 6B11 induced cellular immune responses against ovarian cancer.Methods Potential human leukocyte antigen(HLA)A0201 ligands were predicted by using SYFPEITHI algorithm and tested by the T2 binding assay for screening of HLA-A2 binding peptides from 6B11 complimentary determining region(CDR).Cytotoxic T lymphocytes(CTL)to 6B11 or peptides were generated by 3 rounds of in vitro stimulation with 6B11 or peptide-pulsod dendritic cells(DC),and then tested by 51Cr-release assay to ascertain the CTL epitope of 6B11.Cell proliferation assay was performed by using 6B11 specific CTL as responder cells and peptide-pulsed DC as stimulators to ascertain the helper T lymphocyte(Th)epitope of 6B11.Cytokine assay and interferon-γ ELISPOT assay were performed to verify the CTL and Th epitope of 6B11 further.Results Light chain CDR3 peptide(VL CDR3)of 6B11 induced specific CTL to kill HLA-A2 and target antigen positive ovarian cancer cells,which could be blocked by anti human major histocompatibility complex(MHC)Ⅰ antibody.Heavy chain CDR3 peptide(VH CDR3)of 6B11 stimulated the proliferation of 6B11-primed CTL,which could be blocked mainly by anti human MHC-Ⅱ antibody,and further experiments showed that part of the VH CDR3 peptide-primed CTL killed K562 cells.Peptides in VL CDR3 and VH CDR3 were the CTL and Th epitope mimicking the original antigen of 6B11 respectively.Collaboration of 6B11 CTL and Th epitope,or 6B11 CTL epitope and keyhole limpet hemocyanin(KLH),the former was more powerful in inducing specific cellular immune responses against ovarian cancer.6B11 or corresponding CTL and Th epitope specific CIL secreted high levels of interleukin-2 (1630,1503 ng/L)and interferon-γ(5620,5421 ng/L),while basal level of interleukin-4 was detected (253,274 ng/L).ELISPOT assay confirmed the existence of specific interferon-γ secreting cells in 6811 or epitopes specific CTL(196/1×106 T cell,184/1×106 T cell).Conclusions VL CDR3 and VH CDR3 peptides of 6B11 are the CTL and Th epitopes mimicking original antigen which could duplicate the activity of intact 6B11 to induce the cellular immune responses against ovarian cancer.The results have significant theoretical and practical value in application of anti-idiotypic antibody ag anti tumor vaccine.The acquired CTL and Th epitopes as constituents of future multiple peptides against ovarian cancer could be used in peptide vaccine based ovarian cancer therapy.

Estudo do reconhecimento de epitopos das proteínas Gag e Nef do HIV-1 por linfócitos T em indivíduos cronicamente infectados pelo HIV-1 não progressores por longo tempo / Study of the recognition of HIV-1 Gag and Nef epitopes by T lymphocytes in chronically infected HIV-1 long-term non-progressors

Os linfócitos T têm um papel central no controle da infecção pelo HIV-1. As respostas mediadas por esses linfócitos contra epitopos do HIV-1 restritos a moléculas HLA de classe I podem estar associadas à proteção natural em indivíduos LTNP. Relatos sugerem que determinados alelos HLA apresentam-se mais representados entre os LTNP. Para avaliar esses aspectos na coorte francesa ALT, coletamos células mononucleares de sangue periférico (CMSP) de 24 indivíduos LTNP e verificamos a frequência de respostas específicas para o HIV-1. Para isso, utilizamos pools de peptídeos sobrepostos de Gag e regiões imunodominantes da RT e Nef, e identificamos epitopos do HIV-1 restritos a moléculas HLA de classe I, associados ou não à proteção, através do ensaio de ELISPOT IFN-?. Todos os indivíduos apresentaram respostas específicas aos pools testados, com uma mediana de 5 (2-12). Todas as proteínas do HIV-1 foram reconhecidas, sendo que Gag-p24 e Nef foram as mais frequentemente reconhecidas pelas CMSP dos indivíduos avaliados. A intensidade total de resposta de linfócitos T específicos aos pools de Gag, RT e Nef do HIV-1 em cada indivíduo variou de 160 a 12307 SFC/106 CMSP (mediana: 2025). Observamos o reconhecimento de 22 epitopos já descritos na literatura, contidos nas proteínas Gag-p17, Gag-p24 e Nef do HIV-1, restritos a moléculas HLA de classe I, a maioria descrita como protetoras da progressão para a doença. Quatro novos epitopos ainda não descritos na literatura também foram observados. Concluímos que: respostas específicas mediadas por linfócitos T, eficazes e dirigidas contra um amplo painel de epitopos do HIV-1, estão presentes nos indivíduos LTNP; a presença de moléculas HLA de classe I associadas à proteção favorece o reconhecimento preferencial de epitopos do HIV-1 restritos por elas na maioria dos indivíduos LTNP; esses aspectos devem ser levados em conta na perspectiva do desenvolvimento de uma vacina candidata contra o HIV-1.

T lymphocytes (T-L) have a paramount role in the control of HIV-1 infection. The responses mediated by these cells against HLA class I epitopes may be associated to the natural protection in long-term non-progressors (LTNP). The literature suggests that some HLA alleles relate to the protection against the immune dysfunction. The aim of this research is to study the recognition of HIV-1 Gag, Nef and RT epitopes by T-L through an ELISPOT IFN-? assay in the peripheral blood mononuclear cells (PBMC) of 24 LTNP selected from French ALT study group. We evaluated the frequency of anti-HIV-1 responses and identified HLA class I epitopes. All individuals presented specific responses to the pools of peptides tested with a median of 5 (2-12). Gag-p24 and Nef were the most frequently recognized proteins. The magnitude of the responses varied from 160 to 12307 SFC/106 PBMC (median=2025). We observed the recognition of 22 epitopes already described in HIV-1 Gag-p17, Gag-p24 and Nef, restricted to HLA class I molecules reported as protective. We have also observed four new epitopes not already described in the literature. Our results suggest that: HIV-1 responses by T-L are present in LTNP; the presence of HLA class I molecules associated with protection in the majority of LTNP are related to the recognition of MHC restricted HIV-1 epitopes; these aspects must be taken into account in the development of a candidate vaccine against HIV-1.

Analysis of variation in CTL epitope of HBV popular in China / 解放军医学杂志

Objective To analyze the variation in CTL epitope of hepatitis B virus (HBV) popular in China. Methods Amino acid sequences of 13 cytotoxic T lymphocyte (CTL) epitopes previously reported abroad were taken as reference sequences, and comparison was made draw between these reference sequences and both the 45 CTL-epitopic sequences obtained from the Genbank and also 5 CTL-epitopic sequences from native patients with chronic hepatitis B, using Vector NTI software. Results Sequence variations were observed frequently within several CTL eptitopes of HBV genotype B and C compared with the reference sequences. In the 13 analyzed CTL epitopes, 4 showed variation accounting over 70% in genotype B, and 2 have variance over 70% in occurrence in genotype C. Among these, C18-27 (FLPSDFFPSV), HBcAg-derived CTL epitope which was the most frequently studied previously, is showed in variation more frequently in HBV genotype B and C popular in China, reaching 71% and 97%, respectively. Conlusion Some CTL epitopic sequences of HBV genotype B and C popular in China have their own charactiristic difference from previously reported ones, which should be considered in the study of CTL response of Chinese subjects.

Molecular Modification and Identification of HPV16E749-57 / 中华皮肤科杂志

Objective To introduce the amino acid substitution for HP V16E749-57(HLA-A2-restricted CTL epitope) and to identify the novel epitopes.Me thods Quantitative method was used to evaluate the affinity of the substituted peptides.To determine the peptide candidates to be synthesized and identified,the molecular models of the HLA-A2-peptide complex and CTL epitope candidates b ound to the HLA-A2 molecule were established by computer molecular modeling.Pep tides were synthesized and purified with standard Fmoc assay,lactate dehydrogen ase (LDH) release assay was used to determine their abilities of inducing the ge neration of specific CTLs.Results Modified peptides met the requirements of H LA-A2-restricted CTL epitopes.Peptide RLHYNIVTF had the abilitiy of inducing th e generation of specific CTLs.Conclusions Compared with HPV17E749-57 the mod ified peptide RLHYNIVTF has a higher antigenicity and affinity to HLA-A2.So,pe ptide RLHYNIVTF may be used as one of the HLA-A2-restricted candidate epitopes,instead of HPV17E749-57,for peptide vaccine in the treatment of HPV infection.

Screening and Identification of Predicted Epitopes of HLA-A2-restricted Cytotoxic T Lymphocytes Derived from the HPV16 E7 Antigen / 中华皮肤科杂志

Objective To screen and identify the predicted epitopes of synthesized predicted HLA-A2-restricted cytotoxic T lymphocytes (CTLs) derived from HPV16 E7 antigen. Methods The predicted epitopes of HLA-A2-restricted CTLs derived from HPV16 E7 antigen were synthesized and purified with Standard Fmoc assays, and the standard 51Cr release assay was used to determine their activities to induce specific CTL. Results Two epitopes of HLA-A2-restricted CTLs, namely E711-19 (YMLDLQPET) and E749-57 (RAHYNIVTF) derived from HPV16 E7 antigen were identified. Conclusion E711-19 (YMLDLQPET) and E749-57 (RAHYNIVTF) have antigenicity, and may be the candidates for development of peptide vaccine in the treatment of HPV infections.

Cell-penetrating effects of HPV16E7_(49-57) epitope modified by peptide HIV-Tat_(49-57) / 中华皮肤科杂志

Objective To investigate the effects of the intracellular delivery of HPV16E7_(49-57) epi- tope modified by cell-penetrating peptide HIV-Tat_(49-57) and its influential factors.Methods The unique HLA-A2+/H-2kb+ limited CTL epitope of HPV16E7 fused with a cell-penetrating sequence (HIV-Tat_(49-57)) was designed and a 18-mer peptide was synthesized with aid of polypeptide solid phase synthesis technique. The intracellular transport capabilities of these peptides were tested with indirect immunofluorescence assay and laser confocal microscopy.In addition,the CTL epitope and 18-met peptide were used to stimulate PBMC of healthy C57BL/6 mice,and the E7_(49-57) specific CTL responses were measured by LDH cytotoxicity detection kit in PBMC of those mice.Results The HIV-Tat_(49-57) peptide could efficiently assist HPV 16E7_(49-57) peptide in penetrating into BHK cells in a time and dose-dependent manner (P＜0.05).Further- more,the specific CTL activity induced by the 18-mer peptide was much stronger than that induced by the single epitope.Conclusion The results show that HIV-Tat_(49-57) can efficiently deliver the exogenous anti- genic peptide into the cytoplasm of live cells,and induce specific CTL response.This is a new way to de- sign peptide-based vaccine of HPV.