诱导免疫应答的肿瘤抗原

肿瘤抗原可以诱导机体的免疫应答,是肿瘤的免疫治疗中多肽疫苗的分子基础,近十年来发展起来的肿瘤疫苗筛选方法,利用肿瘤抗原特异性T细胞或抗体识别肿瘤抗原,为临床肿瘤免疫治疗提供了大量备选抗原分子。文中总结了肿瘤抗原的种类,及迄今几乎所有被证明的含有T细胞识别表位的抗原分子及其血清学反应性,为临床肿瘤疫苗的选择提供了依据。     

基于蛋白质基因组学方法的新抗原鉴定流程

肿瘤新抗原是免疫治疗的重要靶点,但基因组数据产生的候选新抗原数量庞大,预测假阳性肽段过多,实验验证费时费力,影响肿瘤新抗原的临床应用.本研究以乳腺癌为例,使用比转录组水平筛选更严格、比细胞学实验更省时的蛋白质基因组学方法来预测和筛选新抗原.研究发现,C2 (IFN-γdominant)免疫表型的新抗原数量最多. C2免疫表型显示出最高的M1/M2巨噬细胞极化,较强的CD8信号和最大的T细胞受体多样性,这可能导致产生更多具有良好免疫原性的新抗原.另外,我们还观察到乳腺癌肿瘤突变负荷与新抗原数目之间呈正相关.通过同批样本的质谱数据进一步筛选发现,可将两万级别的预测新抗原肽候选降至几十条表达肽段,进而可分析其对应的特异或广谱突变基因.最后,我们进一步分析了新抗原的免疫原性,即被T细胞受体识别的可能性.本文利用蛋白质组学数据对基因组数据计算预测得到的候选新抗原进一步筛选,提高了新抗原预测准确性,大大缩小后续实验验证范围.该流程可为肿瘤新抗原预测与筛选研究提供参考.     

个性化癌症疫苗及其佐剂

个性化肿瘤(癌症)疫苗(personalized cancer vaccines,PCVs)包括新抗原癌症(肿瘤)疫苗(neoantigen cancer vaccines,NCVs)和肿瘤裂解物疫苗(tumor lysate vaccines,TLVs)。NCVs以新表位为抗原。新表位是新抗原中可以激活肿瘤特异性T细胞的免疫活性肽。新抗原是根据肿瘤细胞全基因组测序数据确定的肿瘤细胞特有的突变蛋白。TLVs以肿瘤患者的肿瘤裂解物为抗原。PCVs能激活肿瘤特异性CD4+T细胞和CD8+T细胞,这些T细胞能在肿瘤患者体内抑制、杀伤肿瘤细胞,因而延长肿瘤患者的生存期。为了提高疫苗效力,PCVs必须和佐剂组成一定的剂型。可用于PCVs的佐剂有运载体、纳米颗粒、乳化剂、模式识别受体激动剂、免疫卡点抑制剂和能改变免疫抑制肿瘤微环境的制剂等。     

生物信息学方法预测病原体抗原蛋白序列中多肽疫苗候选表位

根据病原体抗原基因中具有免疫原性的表位氨基酸序列,通过化学合成技术可制备该病原体的多肽疫苗。相较于灭活和减毒疫苗,多肽疫苗因成分简单、特异性强、安全性高、易于保存等优点成为疫苗研发的重点。然而,特异有效的疫苗候选表位的预测和筛选,仍是限制多肽疫苗设计与研发的一个重要因素。本研究综合多个生物信息学工具,构建了一个可用于预测和筛选病原体多肽疫苗候选表位的操作流程。并且,基于严重急性呼吸综合征冠状病毒2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)的结构蛋白序列,该流程被成功地应用于其候选抗原表位的预测和筛选。最终,我们共筛选到34条关于SARS-CoV-2的T细胞候选表位,其中20条候选表位与数据库中经过验证的表位高度同源,且能够被T细胞受体(T cell receptor, TCR)识别。综上所述,本研究不仅为当下广泛蔓延的SARS-CoV-2的多肽疫苗设计提供了候选表位,还建立了一个广泛适用于多肽疫苗候选表位预测和筛选的工作流程。     

新型冠状病毒T细胞表位多肽不同免疫途径及佐剂对免疫应答的影响及长效免疫研究

很多研究表明T细胞免疫在抗新型冠状病毒（SARS-CoV-2）感染和预防重症死亡中起到关键作用。T细胞识别由抗原递呈细胞（Antigen-Presenting Cell,APC）递呈的与主要组织相容性复合体（Major Histocompatibility Complex,MHC）结合的短肽,因此新冠T细胞多肽疫苗成为研究重点之一。本研究利用三条C57BL/6J小鼠MHC II类分子限制性的新冠T细胞表位多肽分别结合目前已经在临床上市使用的佐剂：铝佐剂,5’cytosine-phosphateguanine 3’oligonucleotide(CpG-OND,以下简称CpG)佐剂以及铝加CpG佐剂制备成小鼠新冠T细胞多肽疫苗,通过皮下、肌肉、滴鼻（CpG佐剂组别）三种给药方式两次免疫C57BL/6J小鼠,在二免后14 d和6个月（CpG佐剂组别）后利用荧光免疫斑点实验（FluoroSpot）评价疫苗刺激小鼠脾脏淋巴细胞产生的辅助型T细胞1(T helper 1 cell,Th1)应答细胞因子—干扰素γ(Interferon-γ, IFN-γ)、肿瘤坏死因子α(Tumor Necrosi...     

HPV16 E7抗原CTL表位拟肽设计及活性评价

人乳头瘤病毒(human papillomavirus,HPV)早期基因E7是致癌的关键基因,其表达在宫颈癌细胞癌变进程及维持癌细胞恶性表型方面发挥重要作用,已成为宫颈癌治疗的理想靶标.目前,基于HPV16 E7抗原细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL)表位设计多肽疫苗是抗宫颈癌治疗发展的重要方向,但天然CTL表位肽普遍存在体内半衰期短、激发CTL反应效果不佳等缺点.因此,本研究基于前期HPV16 E7抗原CTL表位鉴定的基础,结合多肽酶解实验结果,进行分子动力学模拟及结合自由能计算,初步筛选了3条表位模拟肽.人工合成相关待测表位肽,并利用T2细胞株测定各肽与HLA-A2分子的结合力.研究结果表明,3条表位模拟肽体外抗酶解能力较天然HPV16 E7抗原CTL表位肽均有提高,以(d)RAHYNIVTF表位模拟肽的效果最为明显.此外,(d)RAHYNIVTF表位模拟肽与HLA-A2分子的结合力也有所提高(荧光系数为2.06).以上结果表明,基于HPV16 E7抗原CTL表位模拟肽进行结构修饰有望为宫颈癌治疗性疫苗的设计奠定基础.     

烟曲霉抗原Asp f16HLA-A*0201限制性的CD8+CTL抗原表位生物信息学预测与实验室鉴定

目的 预测与鉴定烟曲霉抗原Asp f16的HLA-A *0201限制性CD8+细胞毒性T细胞(CTL)抗原表位.方法 以国人常见的HLA-A*0201位点为靶点,依据生物信息学软件扫描烟曲霉特异性抗原Asp f16的全部427个氨基酸序列.使用HLA-A *0201转基因小鼠制备骨髓来源的树突状细胞(DC)和CTL.流式细胞仪技术检测DC表面MHC Ⅱ类抗原,CD80,CD86和CD11c的表达来验证其是否成熟.ELISPOT试验检测烟曲霉抗原多肽特异性CTL产生的细胞因子IFN-γ.四聚体(Tetramer)试验证实烟曲霉特异性CTL与抗原肽,HLA-A*0201分子复合体的亲和性.结果 根据与MHC I类分子结合的半衰期评分,选择了3个HLA-A*0201限制性抗原表位.流式细胞仪分析示成熟DC高表达HLA Ⅱ类抗原,CD80,CD86和CD11c.Tetramer试验证实烟曲霉特异性T细胞受体与抗原肽,HLA-A*0201分子复合体的高亲和性.ELISPOT实验结果 表明烟曲霉抗原肽体外可以活化CD8+CTL,被负载了抗原肽的DC刺激活化后可以产生IFN-γ.结论 本研究成功鉴定烟曲霉抗原Asp f16的HLA-A*0201限制性CD8+CTL表位,可作为疫苗设计的候选表位,为进一步研发新型抗烟曲霉疫苗提供参考.     

Nature:肿瘤特异性抗原表位检测,推动个体化肿瘤免疫治疗

<正>近日,来自德国的科学家在国际学术期刊nature发表文章,提出了一种靶向癌症病人全谱肿瘤特异性突变的个体化肿瘤免疫治疗方案。肿瘤特异性突变是癌症免疫治疗的理想靶向目标,因为它们在健康组织中不表达,因此能够作为新抗原被成熟T细胞所识别。但根据肿瘤特异性突变开发疫苗进行系统性癌症免疫治疗仍存在很大障碍,每个癌症病人都有其独特的肿瘤特异性突变存在,因此要先对其肿瘤特异性突变     

主要组织相容性复合体I类分子抗原肽

被主要组织相容性复合体(MHC)I类分子呈递在细胞表面的抗原肽大部分来源于细胞内新合成蛋白质的降解产物,抗原肽直接体现细胞内功能蛋白质的部分变化,蛋白酶体、氨肽酶和抗原转运体(TAP)参与调控抗原肽的生成。在MHC的组装、折叠过程中,抗原肽促进各亚基的结合和折叠进程;而在起始细胞的免疫应答过程中,抗原肽不仅诱导T细胞抗原受体的特异结合,更为重要的是延长MHC同T细胞抗原受体特异结合的作用时间。     

Mathematical model of a personalized neoantigen cancer vaccine and the human immune system

Cancer vaccines are an important component of the cancer immunotherapy toolkit enhancing immune response to malignant cells by activating CD4⁺ and CD8⁺ T cells. Multiple successful clinical applications of cancer vaccines have shown good safety and efficacy. Despite the notable progress, significant challenges remain in obtaining consistent immune responses across heterogeneous patient populations, as well as various cancers. We present a mechanistic mathematical model describing key interactions of a personalized neoantigen cancer vaccine with an individual patient’s immune system. Specifically, the model considers the vaccine concentration of tumor-specific antigen peptides and adjuvant, the patient’s major histocompatibility complexes I and II copy numbers, tumor size, T cells, and antigen presenting cells. We parametrized the model using patient-specific data from a clinical study in which individualized cancer vaccines were used to treat six melanoma patients. Model simulations predicted both immune responses, represented by T cell counts, to the vaccine as well as clinical outcome (determined as change of tumor size). This model, although complex, can be used to describe, simulate, and predict the behavior of the human immune system to a personalized cancer vaccine.     

Prioritizing candidate peptides for cancer vaccines through predicting peptide presentation by HLA-I proteins

Cancer (treatment) vaccines that are made of neoantigens, or peptides unique to tumor cells due to somatic mutations, have emerged as a promising method to reinvigorate the immune response against cancer. A key step to prioritizing neoantigens for cancer vaccines is computationally predicting which neoantigens are presented on the cell surface by a human leukocyte antigen (HLA). We propose to address this challenge by training a neural network using mass spectrometry (MS) data composed of peptides presented by at least one of several HLAs of a subject. We embed the neural network within a mixture model and train the neural network by maximizing the likelihood of the mixture model. After evaluating our method using data sets where the peptide presentation status was known, we applied it to analyze somatic mutations of 60 melanoma patients and identified a group of neoantigens more immunogenic in tumor cells than in normal cells. Moreover, neoantigen burden estimated by our method was significantly associated with a measurement of the immune system activity, suggesting these neoantigens could induce an immune response.     

Identification of CD4+ T cell epitopes from NY-ESO-1 presented by HLA-DR molecules

In previous studies, the shared cancer-testis Ag, NY-ESO-1, was demonstrated to be recognized by both Abs and CD8+ T cells. Gene expression of NY-ESO-1 was detected in many tumor types, including melanoma, breast, and lung cancers, but was not found in normal tissues, with the exception of testis. In this study, we describe the identification of MHC class II-restricted T cell epitopes from NY-ESO-1. Candidate CD4+ T cell peptides were first identified using HLA-DR4 transgenic mice immunized with the NY-ESO-1 protein. NY-ESO-1-specific CD4+ T cells were then generated from PBMC of a patient with melanoma stimulated with the candidate peptides in vitro. These CD4+ T cells recognized NY-ESO-1 peptides or protein pulsed on HLA-DR4+ EBV B cells, and also recognized tumor cells expressing HLA-DR4 and NY-ESO-1. A 10-mer peptide (VLLKEFTVSG) was recognized by CD4+ T cells. These studies provide new opportunities for developing more effective vaccine strategies by using tumor-specific CD4+ T cells. This approach may be applicable to the identification of CD4+ T cell epitopes from many known tumor Ags recognized by CD8+ T cells.     

Immunoproteomic discovery of novel T cell antigens from the obligate intracellular pathogen Chlamydia

Chlamydia infections cause substantial morbidity worldwide and effective prevention will depend on a vaccine. Since Chlamydia immunity is T cell-mediated, a major impediment to developing a molecular vaccine has been the difficulty in identifying relevant T cell Ags. In this study, we used a combination of affinity chromatography and tandem mass spectrometry to identify 13 Chlamydia peptides among 331 self-peptides presented by MHC class II (I-A(b)) molecules from bone marrow-derived murine dendritic cells infected with Chlamydia muridarum. These MHC class II-bound peptides were recognized by Chlamydia-specific CD4 T cells harvested from immune mice and adoptive transfer of dendritic cells pulsed ex vivo with the peptides partially protected mice against intranasal and genital tract Chlamydia infection. The results provide evidence for lead vaccine candidates for a T cell-based subunit molecular vaccine against Chlamydia infection suitable for human study.     

Poor binding of a HER-2/neu epitope (GP2) to HLA-A2.1 is due to a lack of interactions with the center of the peptide

Class I major histocompatibility complex (MHC) molecules bind short peptides derived from proteins synthesized within the cell. These complexes of peptide and class I MHC (pMHC) are transported from the endoplasmic reticulum to the cell surface. If a clonotypic T cell receptor expressed on a circulating T cell binds to the pMHC complex, the cell presenting the pMHC is killed. In this manner, some tumor cells expressing aberrant proteins are recognized and removed by the immune system. However, not all tumors are recognized efficiently. One reason hypothesized for poor T cell recognition of tumor-associated peptides is poor binding of those peptides to class I MHC molecules. Many peptides, derived from the proto-oncogene HER-2/neu have been shown to be recognized by cytotoxic T cells derived from HLA-A2(+) patients with breast cancer and other adenocarcinomas. Seven of these peptides were found to bind with intermediate to poor affinity. In particular, GP2 (HER-2/neu residues 654-662) binds very poorly even though it is predicted to bind well based upon the presence of the correct HLA-A2.1 peptide-binding motif. Altering the anchor residues to those most favored by HLA-A2.1 did not significantly improve binding affinity. The crystallographic structure shows that unlike other class I-peptide structures, the center of the peptide does not assume one specific conformation and does not make stabilizing contacts with the peptide-binding cleft.     

Identification of MHC class II-restricted tumor antigens recognized by CD4+ T cells

CD4+ T cells play a central role in orchestrating host immune responses against cancer as well as autoimmune and infectious diseases. Identification of major histocompatibility complex (MHC) class II-restricted helper T peptides is important for development of effective vaccines. The lack of effective methods to identify such T-cell peptides is a major hurdle in the use of antigen-specific CD4+ T cells in cancer vaccines. Here we describe a genetic targeting expression system for cloning genes encoding for MHC class II-restricted tumor antigens recognized by tumor-reactive CD4+ T cells. Helper T peptides are subsequently identified by using synthetic peptides to test their ability to stimulate CD4+ T cells.     

Finding neoepitopes in mouse models of personalized cancer immunotherapy

Background

Cancer immunotherapy uses one’s own immune system to fight cancerous cells. As immune system is hard-wired to distinguish self and non-self, cancer immunotherapy is predicted to target cancerous cells specifically, therefore is less toxic than chemotherapy and radiation therapy, two major treatments for cancer. Cancer immunologists have spent decades to search for the specific targets in cancerous cells.

Methods

Due to the recent advances in high throughput sequencing and bioinformatics, evidence has merged that the neoantigens in cancerous cells are probably the cancer-specific targets that lead to the destruction of cancer.We will review the transplantable murine tumor models for cancer immunotherapy and the bioinformatics tools used to navigate mouse genome to identify tumor-rejecting neoantigens.

Results

Several groups have independently identified point mutations that can be recognized by T cells of host immune system. It is consistent with the note that the formation of peptide-MHC I-TCR complex is critical to activate T cells. Both anchor residue and TCR-facing residue mutations have been reported. While TCR-facing residue mutations may directly activate specific T cells, anchor residue mutations improve the binding of peptides to MHC I molecules, which increases the presentation of peptides and the T cell activation indirectly.

Conclusions

Our work indicates that the affinity of neoepitopes for MHC I is not a predictor for anti-tumor immune responses in mice. Instead differential agretopic index (DAI), the numerical difference of epitope-MHC I affinities between the mutated and un-mutated sequences is a significant predictor. A similar bioinformatics pipeline has been developed to generate personalized vaccines to treat human ovarian cancer in a Phase I clinical trial.

     

Vaccines targeting tumor blood vessel antigens promote CD8(+) T cell-dependent tumor eradication or dormancy in HLA-A2 transgenic mice

We have recently shown that effective cytokine gene therapy of solid tumors in HLA-A2 transgenic (HHD) mice lacking murine MHC class I molecule expression results in the generation of HLA-A2-restricted CD8(+) T effector cells selectively recognizing tumor blood vessel-associated pericytes and/or vascular endothelial cells. Using an HHD model in which HLA-A2(neg) tumor (MC38 colon carcinoma or B16 melanoma) cells are not recognized by the CD8(+) T cell repertoire, we now show that vaccines on the basis of tumor-associated blood vessel Ags (TBVA) elicit protective Tc1-dependent immunity capable of mediating tumor regression or extending overall survival. Vaccine efficacy was not observed if (HLA-A2(neg)) wild-type C57BL/6 mice were instead used as recipient animals. In the HHD model, effective vaccination resulted in profound infiltration of tumor lesions by CD8(+) (but not CD4(+)) T cells, in a coordinate reduction of CD31(+) blood vessels in the tumor microenvironment, and in the "spreading" of CD8(+) T cell responses to alternate TBVA that were not intrinsic to the vaccine. Protective Tc1-mediated immunity was durable and directly recognized pericytes and/or vascular endothelial cells flow-sorted from tumor tissue but not from tumor-uninvolved normal kidneys harvested from these same animals. Strikingly, the depletion of CD8(+), but not CD4(+), T cells at late time points after effective therapy frequently resulted in the recurrence of disease at the site of the regressed primary lesion. This suggests that the vaccine-induced anti-TBVA T cell repertoire can mediate the clinically preferred outcomes of either effectively eradicating tumors or policing a state of (occult) tumor dormancy.     

In vivo evidence that peptide vaccination can induce HLA-DR-restricted CD4+ T cells reactive to a class I tumor peptide

Vaccination with class I tumor peptides has been performed to induce tumor-reactive CD8(+) T cells in vivo. However, the kinds of immune responses that vaccination might elicit in patients are not fully understood. In this study we tried to elucidate the mechanisms by which vaccination of class I binding tumor peptides into an HLA-A2(+) lung cancer patient elicited dramatic amounts of IgG1 and IgG2 specific to a nonamer peptide, ubiquitin-conjugated enzyme variant Kua (UBE2V)(43-51). The UBE2V(43-51) peptide contains cysteine at the sixth position. HLA-DR-restricted and UBE2V(43-51) peptide-recognizing CD4(+) T cells were induced from postvaccination, but not from prevaccination, PBMCs of the cancer patient. In addition, a CD4(+) T cell line (UB-2) and its clone (UB-2.3), both of which recognize the UBE2V(43-51) peptide in the context of HLA-DRB1*0403 molecules, were successfully established from postvaccination PBMCs. The peptide vaccination increased the frequency of peptide-specific T cells, especially CD4(+) T cells. In contrast, mass spectrometric analysis revealed that the vaccinated UBE2V(43-51) peptide contained both monomeric and dimeric forms. Both forms, fractionated by reverse phase HPLC, were recognized by UB-2 and UB-2.3 cells. Recognition by these CD4(+) T cells was observed despite the addition of a reduction reagent or the fixation of APC. Overall, these results indicate that vaccination with class I tumor peptides can induce HLA-DR-restricted CD4(+) T cells in vivo and elicit humoral immune responses, and that a cysteine-containing peptide can be recognized by CD4(+) T cells not only as a monomer, but also as a dimer.     

Human CD4+ T cell epitopes from vaccinia virus induced by vaccination or infection

Despite the importance of vaccinia virus in basic and applied immunology, our knowledge of the human immune response directed against this virus is very limited. CD4(+) T cell responses are an important component of immunity induced by current vaccinia-based vaccines, and likely will be required for new subunit vaccine approaches, but to date vaccinia-specific CD4(+) T cell responses have been poorly characterized, and CD4(+) T cell epitopes have been reported only recently. Classical approaches used to identify T cell epitopes are not practical for large genomes like vaccinia. We developed and validated a highly efficient computational approach that combines prediction of class II MHC-peptide binding activity with prediction of antigen processing and presentation. Using this approach and screening only 36 peptides, we identified 25 epitopes recognized by T cells from vaccinia-immune individuals. Although the predictions were made for HLA-DR1, eight of the peptides were recognized by donors of multiple haplotypes. T cell responses were observed in samples of peripheral blood obtained many years after primary vaccination, and were amplified after booster immunization. Peptides recognized by multiple donors are highly conserved across the poxvirus family, including variola, the causative agent of smallpox, and may be useful in development of a new generation of smallpox vaccines and in the analysis of the immune response elicited to vaccinia virus. Moreover, the epitope identification approach developed here should find application to other large-genome pathogens.