A Vaccine Encoding Conserved Promiscuous HIV CD4 Epitopes Induces Broad T Cell Responses in Mice Transgenic to Multiple Common HLA Class II Molecules

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, “promiscuous” (multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II- transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.     

Broad and Cross-Clade CD4+ T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4⁺ T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4⁺ T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4⁺ T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA^b and IA^d. Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-γ secretion against 11 out of the 27 peptides in BALB/c mice; CD4⁺ and CD8⁺ T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4⁺ and CD8⁺ T cells, able to simultaneously proliferate and produce IFN-γ and TNF-α, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.     

Immune Activation Promotes Evolutionary Conservation of T-Cell Epitopes in HIV-1

The immune system should constitute a strong selective pressure promoting viral genetic diversity and evolution. However, HIV shows lower sequence variability at T-cell epitopes than elsewhere in the genome, in contrast with other human RNA viruses. Here, we propose that epitope conservation is a consequence of the particular interactions established between HIV and the immune system. On one hand, epitope recognition triggers an anti-HIV response mediated by cytotoxic T-lymphocytes (CTLs), but on the other hand, activation of CD4⁺ helper T lymphocytes (T_H cells) promotes HIV replication. Mathematical modeling of these opposite selective forces revealed that selection at the intrapatient level can promote either T-cell epitope conservation or escape. We predict greater conservation for epitopes contributing significantly to total immune activation levels (immunodominance), and when T_H cell infection is concomitant to epitope recognition (trans-infection). We suggest that HIV-driven immune activation in the lymph nodes during the chronic stage of the disease may offer a favorable scenario for epitope conservation. Our results also support the view that some pathogens draw benefits from the immune response and suggest that vaccination strategies based on conserved T_H epitopes may be counterproductive.     

Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope ¹²³¹PTGSGKSTK¹²³⁹ (EP05) or core motif ¹³⁷³IPFYGKAI¹³⁸⁰ (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.     

用人MHC转基因小鼠模型鉴定高致病禽流感病毒核蛋白CTL表位

目的:筛选高致病禽流感病毒核蛋白(NP)中可用于高致病禽流感病毒感染检测或疫苗设计的CTL表位,为评价疫苗接种效果和开发新型疫苗奠定基础。方法:根据NCBI公布的NP的核苷酸序列设计特异性引物,以2006年深圳株高致病禽流感H5N1病人分离的病毒cDNA为模板扩增NP全长基因(1500 bp)并测序。通过生物信息学方法,预测NP氨基酸序列中潜在的HLA-A觹0201限制性表位。构建重组pJW4303-NP核酸疫苗并肌肉免疫HLA-A2/DP4转基因小鼠,利用ELISPOT法筛选特异性CTL表位。结果:克隆了2006年深圳株高致病禽流感NP基因,构建的重组pJW4303-NP核酸疫苗能在体外COS-7细胞中表达,免疫小鼠后能引起小鼠产生特异性的体液免疫和细胞免疫。结论:生物信息学和转基因小鼠模型筛选相结合的方法,能用于高致病性禽流感核蛋白CTL表位的筛选。     

Species-Specific and Conserved Epitopes on Mouse and Human E-Selectin Important for Leukocyte Adhesion

Selectins are C-type, cell surface lectins that are key players in leukocyte adhesion to the blood vessel wall endothelium. We describe here epitopes for a series of novel monoclonal antibodies (moAbs), UZ4-UZ7, directed against mouse E-selectin. All four antibodies specifically bind to mouse E-selectin, but not to P- or L-selectin, and all inhibit the adhesion of granulocytes, peripheral blood lymphocytes, and promyelocytic HL-60 cells to cytokine-activated mouse endothelium. Three moAbs, UZ5, UZ7, and UZ6, specifically inhibit mouse E-selectin-mediated adhesion by binding to epitopes in domains CR1 or CR2. moAb UZ4 inhibits leukocyte adhesion to both human and murine endothelium activated with IL-1 or other proinflammatory stimuli. UZ4 is the first described moAb that detects an epitope in the lectin domain which is conserved in both murine and human E-selectin (CXKKKL), but is not present in the other members of the selectin family, P- and L-selectin. Interestingly, UZ5, UZ6, and UZ7 more efficiently interfere with lymphocyte than with granulocyte adhesion to cytokine-activated endothelium, while UZ4 completely blocks adhesion of PMN, lymphocytes, and HL-60 and U937 cell lines. The data suggest that E-selectin–ligand engagement differs between lymphocytes and PMN, and that these differences may be accentuated by the CR1 and CR2 domains in the E-selectin cell adhesion molecule.     

Analysis of Promiscuous T cell Epitopes for Vaccine Development Against West Nile Virus Using Bioinformatics Approaches

West Nile virus (WNV) is a major pathogenic flavivirus which causes human neuro-invasive disease, worldwide. Still successful vaccine and therapeutic treatment against WNV infection is not available, which demands the development of more potential WNV vaccines. The present study used immunoinformatics methods viz. Matrix and Artificial Neural Network (ANN) based algorithm to identify the promiscuous and conserve T cell epitopes from entire WNV proteome followed by structure based analysis of identified epitopes. The epitopes were also taken for TAP binding analysis and epitope conservancy analysis. Among 89 identified epitopes, eight epitopes showed high potential and conserve nature but two epitopes viz. capsid ⁴⁰FVLALLAFF⁴⁸ and NS2B ⁹LMFAIVGGL¹⁷ were found most promiscuous and having high population coverage in comparison of other identified epitopes and known antigenic positive control epitopes. Further, Autodock 4.2 and NAMD–VMD molecular dynamics simulation were used for docking and molecular dynamics simulation respectively, to validate epitope and allele complex stability. The 3D structure models were generated for epitopes and corresponding HLA allele by Pepstr and Modeller 9.10, respectively. Epitope FVLALLAFF-B*3501 allele and epitope LMFAIVGGL-B*5101 HLA allele complexes have shown best energy minimization and stable complexes during simulation. The study also showed the optimum binding epitopes FVLALLAFF and LMFAIVGGL with cTAP1 (PDB ID: 1JJ7) cavity, as revealed by Autodock 4.2, concluding favored passage through the ER membrane from cytosol to the ER lumen during cytosolic processing. The docking experiment of epitopes FVLALLAFF, LMFAIVGGL with cTAP1 very well show 1 H-bond state with a binding energy of ?1.62 and ?0.23 kcal/mol, respectively. These results show a smooth pass through of the epitope across the channel of cTAP1 via being weakly bonded and released into the ER lumen through the cavity of cTAP1. Overall, identified peptides have potential application in the development of short peptide based vaccines and diagnostic agents for West Nile virus.     

Immunogenicity of a Human Immunodeficiency Virus (HIV) Polytope Vaccine Containing Multiple HLA A2 HIV CD8+ Cytotoxic T-Cell Epitopes

Compelling evidence now suggests that alphabeta CD8 cytotoxic T lymphocytes (CTL) have an important role in preventing human immunodeficiency virus (HIV) infection and/or slowing progression to AIDS. Here, we describe an HIV type 1 CTL polyepitope, or polytope, vaccine comprising seven contiguous minimal HLA A2-restricted CD8 CTL epitopes conjoined in a single artificial construct. Epitope-specific CTL lines derived from HIV-infected individuals were able to recognize every epitope within the construct, and HLA A2-transgenic mice immunized with a recombinant virus vaccine coding for the HIV polytope also generated CTL specific for different epitopes. Each epitope in the polytope construct was therefore processed and presented, illustrating the feasibility of the polytope approach for HIV vaccine design. By simultaneously inducing CTL specific for different epitopes, an HIV polytope vaccine might generate activity against multiple challenge isolates and/or preempt the formation of CTL escape mutants.     

SARS-CoVN蛋白抗原表位的筛选和鉴定

利用噬菌体展示的线性12肽库从马抗SARS-CoVIgG筛选SARS-CoV的抗原表位。经生物淘洗富集的噬菌体克隆被测序。获得两个共有序列:DXXDP和TXTLL。它们分别与SARS-CoVN蛋白341-345和392-396位氨基酸序列高度同源。含共有序列的克隆在ELISA竞争抑制试验中与SARS-CoVN蛋白竞争结合马抗SARS-CoVIgG。将两个共有序列肽通过基因重组技术成功展示到大肠杆菌鞭毛,获得重组菌F1和F2。用重组菌F1和F2免疫接种试验Balb/c小鼠产生的血清均能与SARS-CoVN蛋白特异结合。说明DXXDP和TXTLL是SARS-CoVN蛋白的两个连续抗原表位。     

SARS-CoV N蛋白抗原表位的筛选和鉴定

利用噬菌体展示的线性12肽库从马抗SARS-CoV IgG筛选SARS-CoV的抗原表位.经生物淘洗富集的噬菌体克隆被测序.获得两个共有序列DXXDP和TXTLL.它们分别与SARS-CoV N蛋白341-345和392-396位氨基酸序列高度同源.含共有序列的克隆在ELISA竞争抑制试验中与SARS-CoV N蛋白竞争结合马抗SARS-CoVIgG.将两个共有序列肽通过基因重组技术成功展示到大肠杆菌鞭毛,获得重组菌F1和F2.用重组菌F1和F2免疫接种试验Balb/c小鼠产生的血清均能与SARS-CoVN蛋白特异结合.说明DXXDP和TXTLL是SARS-CoVN蛋白的两个连续抗原表位.     

Identification of Multiple Protective Epitopes (Protectopes) in the Central Conserved Domain of a Prototype Human Respiratory Syncytial Virus G Protein

A recombinant fusion protein (BBG2Na) comprising the central conserved domain of the respiratory syncytial virus subgroup A (RSV-A) (Long) G protein (residues 130 to 230) and an albumin binding domain of streptococcal protein G was shown previously to protect mouse upper (URT) and lower (LRT) respiratory tracts against intranasal RSV challenge (U. F. Power, H. Plotnicky-Gilquin, T. Huss, A. Robert, M. Trudel, S. Stahl, M. Uhlén, T. N. Nguyen, and H. Binz, Virology 230:155-166, 1997). Panels of monoclonal antibodies (MAbs) and synthetic peptides were generated to facilitate dissection of the structural elements of this domain implicated in protective efficacy. All MAbs recognized native RSV-A antigens, and five linear B-cell epitopes were identified; these mapped to residues 152 to 163, 165 to 172, 171 to 187 (two overlapping epitopes), and 196 to 204, thereby covering the highly conserved cysteine noose domain. Antibody passive-transfer and peptide immunization studies revealed that all epitopes were implicated in protection of the LRT, but not likely the URT, against RSV-A challenge. Pepscan analyses of anti-RSV-A and anti-BBG2Na murine polyclonal sera revealed lower-level epitope usage within the central conserved region in the former, suggesting diminished immunogenicity of the implicated epitopes in the context of the whole virus. However, Pepscan analyses of RSV-seropositive human sera revealed that all of the murine B-cell protective epitopes (protectopes) that mapped to the central conserved domain were recognized in man. Should these murine protectopes also be implicated in human LRT protection, their clustering around the highly conserved cysteine noose region will have important implications for the development of RSV vaccines.     

Accurate Prediction of Immunogenic T-Cell Epitopes from Epitope Sequences Using the Genetic Algorithm-Based Ensemble Learning

BackgroundT-cell epitopes play the important role in T-cell immune response, and they are critical components in the epitope-based vaccine design. Immunogenicity is the ability to trigger an immune response. The accurate prediction of immunogenic T-cell epitopes is significant for designing useful vaccines and understanding the immune system.MethodsIn this paper, we attempt to differentiate immunogenic epitopes from non-immunogenic epitopes based on their primary structures. First of all, we explore a variety of sequence-derived features, and analyze their relationship with epitope immunogenicity. To effectively utilize various features, a genetic algorithm (GA)-based ensemble method is proposed to determine the optimal feature subset and develop the high-accuracy ensemble model. In the GA optimization, a chromosome is to represent a feature subset in the search space. For each feature subset, the selected features are utilized to construct the base predictors, and an ensemble model is developed by taking the average of outputs from base predictors. The objective of GA is to search for the optimal feature subset, which leads to the ensemble model with the best cross validation AUC (area under ROC curve) on the training set.ResultsTwo datasets named ‘IMMA2’ and ‘PAAQD’ are adopted as the benchmark datasets. Compared with the state-of-the-art methods POPI, POPISK, PAAQD and our previous method, the GA-based ensemble method produces much better performances, achieving the AUC score of 0.846 on IMMA2 dataset and the AUC score of 0.829 on PAAQD dataset. The statistical analysis demonstrates the performance improvements of GA-based ensemble method are statistically significant.ConclusionsThe proposed method is a promising tool for predicting the immunogenic epitopes. The source codes and datasets are available in S1 File.     

The Capsid Gene of Feline Calicivirus Contains Linear B-Cell Epitopes in both Variable and Conserved Regions

In order to map linear B-cell (LBC) epitopes in the major capsid protein of feline calicivirus (FCV), an expression library containing random, short (100- to 200-bp) fragments of the FCV F9 capsid gene was constructed. Analysis of this library showed it to be representative of the region of the capsid gene that encodes the mature capsid protein. The library was screened by using polyclonal antisera from a cat that had been challenged experimentally with F9 to identify immunoreactive clones containing LBC epitopes. Twenty-six clones that reacted positively to feline antisera in immunoblots were identified. FCV-derived sequence from these clones mapped to a region of the capsid that spanned 126 amino acids and included variable regions C and E. An overlapping set of biotinylated peptides corresponding to this region was used to further map LBC epitopes by using F9 antisera. Four principal regions of reactivity were identified. Two fell within the hypervariable region at the 5' end of region E (amino acids [aa] 445 to 451 [antigenic site (ags) 2] and aa 451 to 457 [ags 3]). However, the other two were in conserved regions (aa 415 to 421 [ags 1; region D] and aa 475 to 479 [ags 4; central region E]). The reactivity of the peptide set with antisera from 11 other cats infected with a range of FCV isolates was also determined. Ten of 11 antisera reacted to conserved ags 4, suggesting that this region may be useful for future recombinant vaccine design.     

Increased Sequence Coverage through Combined Targeting of Variant and Conserved Epitopes Correlates with Control of HIV Replication

A major challenge in the development of an HIV vaccine is that of contending with the extensive sequence variability found in circulating viruses. Induction of HIV-specific T-cell responses targeting conserved regions and induction of HIV-specific T-cell responses recognizing a high number of epitope variants have both been proposed as strategies to overcome this challenge. We addressed the ability of cytotoxic T lymphocytes from 30 untreated HIV-infected subjects with and without control of virus replication to recognize all clade B Gag sequence variants encoded by at least 5% of the sequences in the Los Alamos National Laboratory HIV database (1,300 peptides) using gamma interferon and interleukin-2 (IFN-γ/IL-2) FluoroSpot analysis. While targeting of conserved regions was similar in the two groups (P = 0.47), we found that subjects with control of virus replication demonstrated marginally lower recognition of Gag epitope variants than subjects with normal progression (P = 0.05). In viremic controllers and progressors, we found variant recognition to be associated with viral load (r = 0.62, P = 0.001). Interestingly, we show that increased overall sequence coverage, defined as the overall proportion of HIV database sequences targeted through the Gag-specific repertoire, is inversely associated with viral load (r = −0.38, P = 0.03). Furthermore, we found that sequence coverage, but not variant recognition, correlated with increased recognition of a panel of clade B HIV founder viruses (r = 0.50, P = 0.004). We propose sequence coverage by HIV Gag-specific immune responses as a possible correlate of protection that may contribute to control of virus replication. Additionally, sequence coverage serves as a valuable measure by which to evaluate the protective potential of future vaccination strategies.     

Somatic Variation of T-Cell Receptor Genes Strongly Associate with HLA Class Restriction

Every person carries a vast repertoire of CD4⁺ T-helper cells and CD8⁺ cytotoxic T cells for a healthy immune system. Somatic VDJ recombination at genomic loci that encode the T-cell receptor (TCR) is a key step during T-cell development, but how a single T cell commits to become either CD4⁺ or CD8⁺ is poorly understood. To evaluate the influence of TCR sequence variation on CD4⁺/CD8⁺ lineage commitment, we sequenced rearranged TCRs for both α and β chains in naïve T cells isolated from healthy donors and investigated gene segment usage and recombination patterns in CD4⁺ and CD8⁺ T-cell subsets. Our data demonstrate that most V and J gene segments are strongly biased in the naïve CD4⁺ and CD8⁺ subsets with some segments increasing the odds of being CD4⁺ (or CD8⁺) up to five-fold. These V and J gene associations are highly reproducible across individuals and independent of classical HLA genotype, explaining ~11% of the observed variance in the CD4⁺ vs. CD8⁺ propensity. In addition, we identified a strong independent association of the electrostatic charge of the complementarity determining region 3 (CDR3) in both α and β chains, where a positively charged CDR3 is associated with CD4⁺ lineage and a negatively charged CDR3 with CD8⁺ lineage. Our findings suggest that somatic variation in different parts of the TCR influences T-cell lineage commitment in a predominantly additive fashion. This notion can help delineate how certain structural features of the TCR-peptide-HLA complex influence thymic selection.     

基于纳米抗体CDR3区抗原表位展示制备生存素抗体及其鉴定

目的:通过纳米抗体CDR3区展示生存素N端表位的方式,探索纳米抗体在抗原表位展示中的作用。方法:通过基因合成方法将生存素N端起始表位(氨基酸序列1~15)插入纳米抗体CDR3区,再构建到原核表达载体pET24a中,IPTG诱导表达,用带His标签的填料纯化,获得高纯度的目的蛋白,免疫雌性BALB/c小鼠,间接ELISA检测5次免疫后的效价,用抗原偶联纯化介质纯化免疫多抗,Western印迹检测多抗特异性。结果:IPTG诱导后,目的蛋白主要以包涵体形式存在,亲和层析获得纯度大于96%的目的蛋白,包涵体经复性后免疫小鼠,效价可达1∶512000,West?ern印迹特异性检测显示免疫多抗能够特异性结合生存素。结论:纳米抗体CDR3区生存素抗原N端表位展示的方法可用于抗生存素抗体的制备,并为今后纳米抗体表位展示相关研究奠定基础。     

Identification of Continuous Human B-Cell Epitopes in the Envelope Glycoprotein of Dengue Virus Type 3 (DENV-3)

Background

Dengue virus infection is a growing global public health concern in tropical and subtropical regions of the world. Dengue vaccine development has been hampered by concerns that cross-reactive immunological memory elicited by a candidate vaccine could increase the risk of development of more severe clinical forms. One possible strategy to reduce risks associated with a dengue vaccine is the development of a vaccine composed of selected critical epitopes of each of the serotypes.

Methodology/Principal Findings

Synthetic peptides were used to identify B-cell epitopes in the envelope (E) glycoprotein of dengue virus type 3 (DENV-3). Eleven linear, immunodominant epitopes distributed in five regions at amino acid (aa) positions: 51–65, 71–90, 131–170, 196–210 and 246–260 were identified by employing an enzyme- linked immunosorbent assay (ELISA), using a pool of human sera from dengue type 3 infected individuals. Peptides 11 (aa51–65), 27 and 28 (aa131–150) also reacted with dengue 1 (DENV-1) and dengue 2 (DENV-2) patient sera as analyzed through the ROC curves generated for each peptide by ELISA and might have serotype specific diagnostic potential. Mice immunized against each one of the five immunogenic regions showed epitopes 51–65, 131–170, 196–210 and 246–260 elicited the highest antibody response and epitopes131–170, 196–210 and 246–260, elicited IFN-γ production and T CD4+ cell response, as evaluated by ELISA and ELISPOT assays respectively.

Conclusions/Significance

Our study identified several useful immunodominant IgG-specific epitopes on the envelope of DENV-3. They are important tools for understanding the mechanisms involved in antibody dependent enhancement and immunity. If proven protective and safe, in conjunction with others well-documented epitopes, they might be included into a candidate epitope-based vaccine.