Fine specificity of antibodies against AQP4: epitope mapping reveals intracellular epitopes

The autoantibody to aquaporin-4 (AQP4) is a marker and a pathogenetic factor in Neuromyelitis Optica (NMO) (Devic’s syndrome). Our aim was to identify B-cell antigenic linear epitopes of the AQP4 protein and investigate similarities with other molecules. To this end, we screened sera from 21 patients positive for anti-AQP4 antibodies (study group), from 23 SLE and 23 pSS patients without neurologic involvement (disease controls) and from 28 healthy individuals (normal controls). Eleven peptides, spanning the entire intracellular and extracellular domains of the AQP4 molecule, were synthesized, and all sera were screened for anti-peptide antibodies by ELISA. Specificity was evaluated by homologous inhibition assays. NMO positive sera exhibited reactivity against 3 different peptides spanning the sequences aa1–22 (AQPpep1) (42.9% of patients), aa88–113 (AQPpep4) (33%) and aa252–275 (AQPpep8) (23.8%). All epitopes were localized in the intracellular domains of AQP4. Homologous inhibition rates were ranging from 71.1% to 84.3%. A 73% sequence homology was observed between AQPpep8′ aa257–271, a 15-mer peptide part of the AQPpep8 aa252–275, and the aa219–233 domain of the Tax1-HTLV-1 binding protein (TAX1BP1), a host protein associated with replication of the Human T-Lymphotropic Virus 1 (HTLV-1). Antibodies against the AQP4 and the TAX1BP1 15-mer peptides were detected in 26.3% (N = 5) and 31.6% (N = 6) of NMO positive sera (r_s = 0.81, P < 0.0001). Healthy controls did not react with these peptides, while homologous and cross-inhibition assays confirmed binding specificity. This first epitope mapping for AQP4 reveals that a significant proportion of anti-AQP4 antibodies target linear epitopes localized in the intracellular domains of the channel. One of the epitopes displays high similarity with a portion of TAX1BP1 protein.     

T cell help is required to induce idiotypic-anti-idiotypic autoantibody network after immunization with complementary epitope 289-308aa of La/SSB autoantigen in non-autoimmune mice

Immunotherapies against autoimmune diseases have been of limited success. Preventive vaccines could be developed on the basis to abrogate unwanted immune responses to defined autodeterminants. In this study it is shown that immunization of BALB/c mice with two linear T and B cell epitopes of the human La/SSB autoantigen (spanning the regions 289-308aa and 349-364aa) and their complementary forms specified by the complementary mRNA, results in characteristic B and T cell responses. Mice immunized with the 289-308aa epitope or its complementary peptide elicited specific antibodies against both epitopes. In contrast, mice immunized with the 349-364aa epitope or its complementary peptide mounted antibody titres against the immunizing peptide only. According to these data, the 289-308aa epitope and its complementary form were capable to generate an idiotypic-anti-idiotypic response, which were cross-regulated. Peptide-specific T cell proliferation and cytokine production in vitro revealed the induction of a two-stage T helper response (Th1-->Th2 type) after immunization with either the epitope 289-308 or its complementary peptide. IgG1 was the predominant subclass after immunization with the two forms of epitopes 289-308 and 349-364, while a response of the IgG2b > IgG2a was obtained after the immunization with the complementary form of 349-364 epitope reflecting the TH2/TH1 polarization, respectively. Our data suggest that the complementary peptides of two immunodominant epitopes of human LaSSB can mimic the autoantibodies against these epitopes and establish an active idiotypic-anti-idiotypic network.     

In vivo alteration of humoral responses to HIV-1 envelope glycoprotein gp120 by antibodies to the CD4-binding site of gp120

The binding of antibodies to the CD4-binding site (CD4bs) of the HIV-1 envelope glycoprotein gp120 has been shown to induce gp120 to undergo conformational changes that can expose and/or shield specific epitopes on gp120. Here, we study alterations in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4bs of gp120. The data showed that gp120 bound by anti-CD4bs mAbs had enhanced reactivity with mAbs to the V3 and N-terminal regions, but not with mAb to the C terminus. Moreover, mice immunized with the gp120/anti-CD4bs mAb complexes produced higher titers of gp120-specific serum IgG and IgA than mice immunized with uncomplexed gp120 or other gp120/mAb complexes. Notably, the enhanced antibody production was directed against V3 and correlated with better exposure of V3 on the gp120/anti-CD4bs mAb complexes. The higher antibody reactivity was evident against the homologous V3(LAI) peptide, but not against heterologous V3 peptides. Potent neutralization activity against HIV-1(LAI) was also observed in the sera from mice immunized with gp120/anti-CD4bs mAb complexes, although the sera exhibited poor neutralizing activities against other viruses tested. These results indicate that the anti-CD4bs antibodies alter the antigenicity and immunogenicity of gp120, leading to enhanced production of anti-gp120 antibodies directed particularly against the V3 region.     

Linear epitopes of two different autoantigens-La/SSB and myelin basic protein--with a high degree of molecular similarity,cause different humoral immune responses

The region 147-154aa of La/SSB presents 83% sequence similarity with the 139-146aa region of the human myelin basic protein (MBP). The aim of this study was to investigate the prevalence and significance of antibodies against both epitopes in sera from patients with systemic autoimmune diseases, and to compare the humoral responses produced after rabbit immunization. Peptides 147-154aa of La/SSB and 139-146aa of the MBP were attached on tetrameric sequential oligopeptide carriers and used for immunizations of New Zealand White rabbits. Antibodies to immunizing peptides, as well as to the peptides corresponding to other previously defined La/SSB epitopes (289-308aa, 349-364aa), to the intact human MBP (hMBP) and to the recombinant human La/SSB (rechLa) proteins, were identified using specific ELISA assays. Sera from 45 patients with Sjogren's syndrome (pSS), 49 with Systemic Lupus Erythematosus (SLE), and 18 with Rheumatoid Arthritis (RA) were tested against the two peptides and the hMBP. Twenty-two per cent of sera from pSS patients, 27% of SLE patients, and none from RA sera reacted with the La epitope; 27% from pSS sera, 22% of SLE sera, and 17% of RA sera gave a positive reaction against the MBP peptide. Finally, 19% of pSS, 30% of SLE, and 38% of RA sera reacted with the hMBP. Thirty-five days after immunization of rabbits with the La epitope, antibodies were produced against all three La/SSB peptides, the MBP peptide, and the hMBP and rechLa proteins. Rabbits immunized with the MBP peptide produced antibodies against the immunizing peptide and the mimicking peptide of La shortly after immunization, whilst antibodies against the other La epitopes and the two intact proteins were produced later. Inhibition experiments in rabbit sera with high reactivity against hMBP, using the MBP peptide as inhibitor, revealed that 80% of serum reactivity was abolished. In conclusion, a significant proportion of human autoimmune sera reacted with both La and MBP derived peptides, as well as with hMBP. La 147-154aa peptide, when used for animal immunizations, induces a fast epitope spreading involving both La and MBP. In contrast, the mimicking MBP epitope induces a delayed response against the other La epitopes. Thus, despite the fact that these peptides present molecular similarity, they induce different immune responses.     

Analysis of the Neutralizing Antibody Response to the Measles Virus Using Synthetic Peptides of the Haemagglutinin Protein

Infection or immunization with measles virus induces a protective immune reaction including neutralizing antibodies against the haemagglutinin and fusion protein. The reactivity of the polyclonal IgG response of sera obtained from late convalescent donors was studied, using overlapping 15mer peptides covering the complete sequence of the measles virus haemagglutinin. Most sera reacted with a similar set of peptides generating a characteristic binding pattern. The reactive peptides correspond to a region mediating cell hemolysis (aa310–325), to regions which serve as targets to neutralizing antibodies and to a putative transmembrane region (aa35–58). The latter region contains also a human T-cell epitope providing evidence of a non-random association of T- and B-cell epitopes. We also immunized different strains of mice and rabbits with measles virus. In contrast to the human sera, animal sera with strong neutralizing activities did not react with any of the H-protein peptides. The mostly weak reactivities with the linear sequences contrast with the strong neutralizing activities of the human or animal antibodies, suggesting that these primarily recognize the fusion protein or conformational epitopes of the haemagglutinin protein.     

Mapping of Epitopes Recognized by Antibodies Induced by Immunization of Mice with PspA and PspC

Pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) are important candidates for an alternative vaccine against pneumococcal infections. Since these antigens show variability, the use of variants that do not afford broad protection may lead to the selection of vaccine escape bacteria. Epitopes capable of inducing antibodies with broad cross-reactivities should thus be the preferred antigens. In this work, experiments using peptide arrays show that most linear epitopes recognized by antibodies induced in mice against different PspAs were located at the initial 44 amino acids of the mature protein and that antibodies against these linear epitopes did not confer protection against a lethal challenge. Conversely, linear epitopes recognized by antibodies to PspC included the consensus sequences involved in the interaction with human factor H and secretory immunoglobulin A (sIgA). Since linear epitopes of PspA were not protective, larger overlapping fragments containing 100 amino acids of PspA of strain Rx1 were constructed (fragments 1 to 7, numbered from the N terminus) to permit the mapping of antibodies with conformational epitopes not represented in the peptide arrays. Antibodies from mice immunized with fragments 1, 2, 4, and 5 were capable of binding onto the surface of pneumococci and mediating protection against a lethal challenge. The fact that immunization of mice with 100-amino-acid fragments located at the more conserved N-terminal region of PspA (fragments 1 and 2) induced protection against a pneumococcal challenge indicates that the induction of antibodies against conformational epitopes present at this region may be important in strategies for inducing broad protection against pneumococci.     

Deletional tolerance prevents AQP4‐directed autoimmunity in mice

Neuromyelitis optica (NMO) is an autoimmune disorder of the central nervous system (CNS) mediated by antibodies to the water channel protein AQP4 expressed in astrocytes. The contribution of AQP4‐specific T cells to the class switch recombination of pathogenic AQP4‐specific antibodies and the inflammation of the blood–brain barrier is incompletely understood, as immunogenic naturally processed T‐cell epitopes of AQP4 are unknown. By immunizing Aqp4 ^?/? mice with full‐length murine AQP4 protein followed by recall with overlapping peptides, we here identify AQP4(201‐220) as the major immunogenic IA^b‐restricted epitope of AQP4. We show that WT mice do not harbor AQP4(201–220)‐specific T‐cell clones in their natural repertoire due to deletional tolerance. However, immunization with AQP4(201–220) of Rag1 ^?/? mice reconstituted with the mature T‐cell repertoire of Aqp4 ^?/? mice elicits an encephalomyelitic syndrome. Similarly to the T‐cell repertoire, the B‐cell repertoire of WT mice is “purged” of AQP4‐specific B cells, and robust serum responses to AQP4 are only mounted in Aqp4 ^?/? mice. While AQP4(201–220)‐specific T cells alone induce encephalomyelitis, NMO‐specific lesional patterns in the CNS and the retina only occur in the additional presence of anti‐AQP4 antibodies. Thus, failure of deletional T‐cell and B‐cell tolerance against AQP4 is a prerequisite for clinically manifest NMO.     

Linear and multiple antigen peptides containing defined T and B epitopes of the Plasmodium yoelii circumsporozoite protein: antibody- mediated protection and boosting by sporozoite infection

We previously reported the identification of a T cell epitope in the N- terminal part of the circumsporozoite protein (CSP) of Plasmodium yoelii yoelii (Pyy). CD4+ T cell clones derived from mice immunized with a 21-mer peptide (amino acids 59-79, referred to as Py1) containing this epitope confer complete protection after passive transfer in mice. These clones proliferate in vitro in the presence of a 13-mer peptide (amino acids 59-71, referred to as Py1T). This shorter peptide was found to behave as a Th epitope in vivo, allowing overcoming of the genetic restriction for production of anti-repeat antibodies in BALB/c mice, when cross-linked to three (QGPGAP) repeats of the Pyy CSP. In this study, we report protection in BALB/c mice, against a challenge with Pyy sporozoites after immunization with linear and multiple antigen peptides containing Py1T as T epitope and three repeats QGPGAP (Py3) as B epitope. Multiple antigen peptide (MAP4-Py1T- Py3)-induced immunity was shown to be more effective than immunity induced by the linear form of the conjugate (Py1T-Py3), protecting against challenges with higher numbers of sporozoites. In both cases, levels of anti-repeat antibodies were strongly correlated with anti- parasite antibodies and protection. When tested in vitro, sera from mice immunized with the protective constructs strongly inhibited Pyy liver stages, while lymph node T cells displayed no cytotoxicity. In vivo, depletion of CD4+ or CD8+ T cells did not affect protection. Furthermore, MAP4-Py1T-Py3-immunized mice were not protected against a challenge with P. yoelii nigeriensis sporozoites, a parasite which has the same Py1T sequence but differs from Pyy in its repeated sequence. These results demonstrate that anti-repeat antibodies raised by immunization with the linear or the MAP form are exclusively responsible for the protection. Furthermore, this antibody response is boosted by a sporozoite challenge, allowing protection against a second challenge.      

Production of anti-dengue NS1 monoclonal antibodies by DNA immunization

Monoclonal antibodies against dengue NS1 protein were generated following immunization of mice with plasmid DNA encoding the transmembrane form of NS1 from dengue serotype 2 virus. A mammalian expression vector, pDisplay, was engineered to direct cell surface expression of dengue NS1 and tested for transient expression in COS cells. Two mice were immunized intramuscularly with six doses of 100 microg of plasmid at 2-week intervals; one mouse received a booster of live virus prior to the last plasmid injection. Both mice showed antibody responses against dengue antigens in dot enzyme immunoassay. Following fusion, hybridomas were screened with dot enzyme immunoassay against all four dengue serotypes. Specificity to the NS1 protein was confirmed by western blot analysis. Among five anti-dengue NS1 monoclonal antibodies generated, two clones were serotype 2 specific, two clones reacted with all four serotypes and the last also reacted with Japanese encephalitis virus. Reactivity against native or denatured forms of NS1 revealed three clones with reactivity to linear epitopes and two clones recognizing conformational epitopes. Such diverse specificity of anti-dengue NS1 monoclonal antibodies indicates that DNA immunization, especially with the combination of virus boosting, is an efficient way of producing monoclonal antibodies against viral protein. This has opened up a possibility of producing monoclonal antibodies to rare viral proteins that are difficult to isolate or purify.     

Induction of immune responses against human papillomaviruses by hypervariable epitope constructs

An ideal prophylactic vaccine against human papillomaviruses (HPV) would be one that can induce broadly reactive antibody titres to at least the major oncogenic strains of HPV. It has been previously shown that HPV structural proteins are highly immunogenic but fail to elicit cross-reactive immune responses against heterologous strains of HPV. Recent studies have demonstrated that the immunity induced by virus-like particles is mostly type specific. In the present study, we determined the breadth of reactivity of antibodies induced in mice immunized with hypervariable epitope constructs (HECs), which represent sequence variants of immunodominant B-cell epitopes of the major capsid protein L1 of HPV. In order to test the breadth of reactivity, sera from immunized mice were tested against peptides representing analogous sequences of HPV types 16, 18, 31 and 45. Mice immunized with HECs based on two epitopes mounted antibody responses that cross-reacted with two different analogues, 16 and 18. Significantly, antibodies from mice immunized with HECs also inhibited haemagglutination mediated by HPV-16 L1 VLPs, suggesting that immunization resulted in the development of antibodies that could bind to viral capsid proteins in their native conformation. Our observations suggest that HECs may overcome the restriction of type specific immunity against HPV.     

汉滩病毒部分核蛋白基因原核表达产物 的免疫及其保护作用

目的 探讨汉滩病毒（HTNV）部分核蛋白（NP）基因（37－294bp)原核表达产物NP AA1－86多肽的免疫原性及其免疫保护作用。方法 大肠杆菌表达的HTNV部分核蛋白多肽NP AA1－86混合福氏佐剂,腹腔注射免疫BALB／c小鼠,IFA检测抗体应答;^3H-TdR掺入及4h ^51Cr释放试验检测免疫小鼠脾细胞对HTNV的特异淋巴细胞增殖转化指数及细胞毒T淋巴细胞（CTL）杀伤功能;免疫小鼠脾细胞转输感染HTNV A9株的乳鼠,观察对乳鼠致死性的免疫保护作用。结果 免疫后1周小鼠即出现抗体应答反应,抗体滴度最高达1：12800,与重组完整NP免疫组差异无显著性（P＞0．05）;免疫小鼠的淋巴细胞增殖转化指数、CTL杀伤率较对照组明显增高（P＜0．01）,与完整NP免疫组差异无显著性（P＞0．05）,CTL杀伤率随效：靶比例的增高呈逐渐上升趋势;免疫小鼠脾细胞转输可部分保护病毒致死性感染的乳鼠,保护率约25％。结论 大肠杆菌表达的汉滩病毒部分核蛋白多肽NP AA1－86不仅包含NP几乎所有的体液免疫表位,同时含有部分细胞免疫表位,对汉滩病毒感染可产生部分免疫保护作用。     

Antibodies neutralizing feline leukaemia virus (FeLV) in cats immunized with the transmembrane envelope protein p15E

The feline leukaemia virus (FeLV) vaccines that are currently in wide use are generally poor inducers of virus-neutralizing antibodies, although such antibodies appear after recovering from challenge. However, the presence of neutralizing antibodies in cats recovering from natural FeLV infection clearly correlates with resistance to subsequent infection and passive transfer of antibodies can protect other animals. After demonstrating the induction of neutralizing antibodies in rats and goats immunized with the transmembrane envelope protein p15E of FeLV, cats were immunized with the same antigen. High titres of neutralizing antibodies specific for FeLV were induced and epitope mapping revealed a pattern of recognition similar to that seen following immunization of rats and goats. These epitopes are highly related to epitopes recognized after immunization with porcine endogenous retrovirus (PERV) p15E and to epitopes recognized by neutralizing antibodies in patients infected with human immunodeficiency virus type 1. The ability of p15E to induce neutralizing antibodies in cats suggests that it should be included in the next generation of vaccines. In contrast, sera from FeLV-infected animals usually fail to recognize the neutralization-relevant epitopes in p15E. Since homologous epitope sequences are present in feline endogenous retroviruses, it appears that tolerance against these sequences is not induced.     

A mimotope peptide-based vaccine against Schistosoma mansoni: synthesis and characterization

A panel of four mimotopes of the epitope recognized by the highly protective monoclonal antibody against Schistosoma mansoni (152-66-9B) was obtained by screening a solid-phase 8mer random peptide library. Three of the four mimotopes (p28, p29 and p30) were efficiently recognized in an in vitro radioimmunoassay by the monoclonal antibody and by sera from infected mice and one (p30) induced in vitro proliferation of primed lymphocytes. When the mimotopes were conjugated to bovine serum albumin (BSA) and the conjugates used to immunize C57BL/6J mice, only the p30-BSA-induced antibodies which were effective at complement-mediated killing of schistosomula. The level of complement-mediated killing obtained with the anti-p30 antibodies was comparable to that seen with serum from mice immunized with the protective 9B-antigen. Furthermore, following challenge infection of mimotope-BSA-immunized mice, a greater than 40% reduction in worm burden was observed in p30-BSA-immunized mice, a level comparable to that seen following immunization with the intact 9B-antigen. These results show that a simple synthetic peptide immunogen comprising an eight-amino acid mimotope of a conformational epitope on the 9B-antigen can induce protective immune responses against S. mansoni that are comparable to those obtained following immunization with the far more complex intact antigen. This mimotope may well represent a potential component of a synthetic peptide vaccine against S. mansoni. The inclusion of other B-cell- and T-cell-stimulating synthetic epitopes in such a vaccine, together with a more appropriate carrier, adjuvant and delivery systems may well result in a level of protection even greater than that seen with the single mimotope.     

Immunogenicity of a non-repetitive sequence of Plasmodium falciparum circumsporozoite protein in man and mice

In the present work, the hypothesis that individuals naturally exposed to Plasmodium falciparum malaria infection in endemic areas produce antibodies directed against non-repetitive epitopes of the circumsporozoite protein was investigated. Using a synthetic peptide reproducing the non-repetitive group-conserved region I sequence, we have shown that specific anti-region I antibodies are detectable in sera from endemic countries. Of these sera, 87% also had antibodies against the immunodominant repetitive epitope (Asn-Ala-Asn-Pro, NANP) of P. falciparum. In order to study the immunogenicity of this non-repetitive epitope, a synthetic peptide consisting of both region I and three (NANP) repeats [RI-(NANP)3] was used to immunize inbred strains of mice. H-2b mice produced antibodies against both the repetitive and the non-repetitive epitope. These antibodies were specific for each epitope, recognized P. falciparum sporozoites in immunofluorescence, and inhibited sporozoite penetration into human liver cells in vitro. Non-H-2b mice were completely unresponsive. Lymph node cells from H-2b mice immunized with RI-(NANP)3 peptide proliferated in the presence of RI-(NANP)3 and of (NANP)4 peptide, but never in the presence of RI peptide alone. These findings demonstrate that in the configuration used (i) the non-repetitive epitope region I does not carry T-helper epitopes; (ii) the (NANP) repetitive epitope may act as a carrier for the immune response to region I in mice; and (iii) therefore, immune response to region I in man probably depends on the recognition of T-cell epitopes similar to those involved in the anti-NANP response: i.e. such a T epitope may be NANP itself in responding individuals or another, not yet recognized, sporozoite T-cell epitope.     

Identification of a major antibody binding epitope in the non-structural protein 3D of foot-and-mouth disease virus in cattle and the development of a monoclonal antibody with diagnostic applications

Detection of FMDV non-structural protein 3D antibodies has been used as a complementary method for sero-epidemiological studies as an indirect indicator of FMDV infection. In order to develop a sensitive cELISA to detect FMDV antibodies, immune dominant epitopes in FMDV-3D protein were identified by peptide array analysis. Monoclonal antibodies were then raised to a selected epitope and used in cELISA. Ninety two peptides corresponding to the complete amino acid sequence of FMDV-3D were synthesized. The sera from 15 FMDV infected cows were tested for binding to the peptides in an indirect ELISA. One major peptide (3D-4) was recognized by antisera in 12 of the 15 infected cows (80%). The sequence was formed by amino acid residues 16-30 of FMDV-3D. The mAbs produced from the mice immunized with native 3D showed neither reactivity to this epitope nor competition with sera from FMDV infected cattle. However, the mAbs produced from the mice immunized with native 3D and boosted with the peptide 3D-4 showed reactivity with native 3D, recombinant 3D as well as competition with sera of FMDV infected cattle and sheep in ELISA assays. Immune response to FMDV-3D was determined using a cELISA. All cattle and sheep tested were positive at 9 dpi and remained positive until the end of the experiment on days 28-31 (>50% inhibition). This demonstrated that mAbs directed to the peptide 3D-4 were effective competitors to the polyclonal antibodies against 3D in infected sera. The approach described here provides a useful tool for specific mAb production in the development of new diagnostic tests.     

Characterization of humoral and cellular immune responses in mice induced by immunization with HIV-1 Nef regulatory protein encapsulated in poly(DL-lactide-co-glycolide) microparticles.

We have characterized the humoral and cellular immune responses of BALB/c mice immunized with HIV-1 Nef regulatory protein encapsulated in poly(DL-lactide-co-glycolide) PLG particles. Three groups of mice were immunized with Nef PLG, Nef in the presence of complete Freund's adjuvant (CFA) or Nef alone in PBS. When titers were compared 7 months after the last injection, anti-Nef titers in mice immunized with Nef PLG were still close to the maximum, whereas a significant decrease was observed in mice immunized with Nef alone (five times lower) or with Nef in CFA (three times lower). These results indicate that Nef PLG is at least a similar or better vector/adjuvant than Nef in CFA concerning the duration of the humoral immune response. The analysis of cytokine profiles (IL-5 and IL-10) and the isotypic patterns of anti-Nef antibodies (predominantly IgG1), in the three groups of mice, indicated a predominant Th2 immune response. Using synthetic peptides covering the entire sequence of Nef, we identified at least three linear epitopes within sequences 32-64, 118-167 and 185-205 in the sera of mice immunized with Nef PLG or Nef CFA. In contrast, anti-Nef antibodies against Nef alone failed to recognize synthetic peptides, indicating that the majority of anti-Nef antibodies were primarily directed against conformational epitopes. We then examined the ability of Nef PLG to prime for the antigen-specific proliferative responses in vitro. The data obtained indicate the presence of both B-cell and T-cell epitopes in the C-terminal fragment of the protein after immunization of mice with Nef encapsulated in PLG particles.     

Idiotype-anti-idiotype circuit in non-autoimmune mice after immunization with the epitope and complementary epitope 289-308aa of La/SSB: implications for the maintenance and perpetuation of the anti-La/SSB response

BACKGROUND: Antibodies to La/SSB are usually found in sera of patients with Sjogren's Syndrome (SS) and Systemic Lupus Erythematosus (SLE). Recent work from our laboratory (Mol Med 2002;8:293-305) revealed that an active idiotypic network involving antibodies to epitopes of La/SSB and their anti-idiotypes exist in human sera. The anti-idiotypic antibodies were detected using complementary peptides to B-cell epitopes of the autoantigen. The principle of the complementary peptides is based on the 'molecular recognition' theory. According to this theory, translation of two complementary RNA strands (coding and non-coding strand) into protein, generate a pair of peptides, which bind each other with specificity and high affinity. AIM: To investigate antibody production and T-cell responses in non-autoimmune-susceptible animal strains which were immunized with the epitope 289-308aa of La/SSB as well as its complementary epitope. MATERIALS AND METHODS: Balb/c mice were immunized with a peptide corresponding to epitope 289-308aa (pep) or its complementary (cpep) peptide (5 animals/group). The sera were tested for the presence of antibodies to pep and cpep as well as for epitope spreading to recombinant human La/SSB and a major B-cell epitope of La/SSB spanning the region 349-364aa. Another group of animals was sacrificed on day 10 and T-cell responses against pep and cpep were evaluated in cells from lymph nodes and spleen. RESULTS: Immunizations with either pep or cpep led to the appearance of antibodies against the immunogen peptide by day 31 which subsequently was followed by antibody production to its complementary peptide by day 55. In two out of five animals immunized with the epitope 289-308aa, a spreading of the immune response to epitope 349-364aa was observed. In the remaining three animals, negative for antibodies to pep349-364, a specific treatment of sera, using cpep349-364 revealed that anti-idiotypic antibodies masked antibodies to pep349-364. In all immunization experiments high T-cell proliferative responses to both pep and cpep peptides were detected. CONCLUSIONS: Complementary peptides to epitopes of La/SSB can be utilized as probes to study the development of an idiotypic-anti-idiotypic network towards the major autoantigen. The ability of pep and cpep peptides to induce both B-cell and T-cell responses may provide useful insights into understanding further the initiation and maintenance of autoimmune response and create new tools for therapeutic intervention.     

可用于短肽基因免疫的真核表达载体的构建及应用

目的: 构建可高效表达表位短肽的基因免疫载体, 并研究其在表位为基础的基因免疫中的免疫效应。方法:将基因表达调控序列插入pUC19载体中, 加入KOZAK增强序列、基因克隆位点及转录起始和终止码, 构建可表达短肽的表达载体, 进一步将表位基因克隆到构建的真核载体中,并研究其在体外的表达和体内基因免疫特性。结果: 成功地构建了高效表达短肽的真核表达载体, 并能在小鼠体内诱导表位特异性的免疫应答。结论: 本实验中构建的含PEC的载体以表位为基础的DNA疫苗研究提供了新的设计策略和工具。     

用噬菌体表面显示肽库技术筛选乙酰胆碱酯酶有效抗原表位的研究

目的：筛选和鉴定乙酰胆碱酯酶的有效抗原表位。方法：收集乙酰胆碱受体抗体阴性而乙酰胆碱酯酶抗体阳性的重症肌无力病人血清，采用溴化氰活化的琼脂糖柱（CNBr-actived sepharose^TM4B）纯化抗乙酰胆碱酯酶的多克隆抗体并定量；用纯化的抗乙酰胆碱酯酶的抗体对随机的12肽噬菌体表面显示肽库进行5轮免疫学筛选，随机挑取克隆；采用Western blot免疫识别，识别为阳性的克隆进行核苷酸序列的测定，并与乙酰胆碱酯酶的氨基酸序列进行同源性比较。将获得的不同表位的阳性克隆分别免疫小鼠，采用Western blot筛选能刺激小鼠产生抗乙酰胆碱酯酶抗体的阳性克隆，进行生物学活性的鉴定。结果：经5轮免疫学筛选后挑取的49个克隆中，经Westem blot识别15个克隆能被抗乙酰胆碱酯酶抗体识别。核苷酸序列分析发现共有7种不同的表位，其中1种表位与乙酰胆碱酯酶有较高的同源性，其余6种表位与其无一级结构的同源性。经动物免疫初步实验筛选，共有5个免疫原性较强的阳性克隆，其免疫的鼠血清均可识别人乙酰胆碱酯酶。结论：获得了5种乙酰胆碱酯酶的有效抗原表位，1种可能为结构表位，4种为模拟表位。     

Characterization of autoantibodies generated in mice by immunization with the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins

The R13 peptide sequence (EEEDDDMGFGLFD) that corresponds to the C-terminal region of Trypanosoma cruzi ribosomal P1 and P2 proteins differs from the eukariotic P consensus sequence EESDDDMGFGLFD (H13) only in a nonconservative amino acid substitution. Since the immunization with R13 peptide coupled to a carrier protein like OVA would break the tolerance to a self-sequence and generate autoantibodies, we characterized the antibodies induced in mice by R13 immunization, analyzing by ELISA their capacity to bind to R13 and the self-sequence H13. Besides, we studied the course of these reactivities a long time after immunization. It was found that all R13-immunized mice had antibodies against H13 and this reactivity was always lower than R13 reactivity. The anti-H13 reactivity evaluated by competitive ELISA demonstrated that the H13 peptide is able to inhibit the binding of immune sera to R13 at high doses. When the levels and the avidity of anti-R13 and anti-H13 were evaluated at 10 and 80 days post third immunization, it was observed that anti-R13 levels were higher than anti-H13 levels in all sera from 10 days after the third immunization. However, avidity of both antibodies was high. In sera from 80 days post third immunization, anti-R13 and anti-H13 levels and avidity either remained elevated or showed a rise, whereas anti-OVA levels declined. Moreover, when ECG traces were obtained from immunized mice, the heart functional alterations observed at 10 days continued at 80 days after the third immunization, showing an association with the levels of the antibodies. In addition, the isotype pattern that recognizes R13 and the self-sequence H13 is different. For anti-R13 response, IgG1 reactivity was higher than IgG2; meanwhile, for anti-H13 response IgG2 reactivity was higher than IgG1. These results indicate that sera from R13-immunized mice bind the H13 sequence and this autoreactivity may be self-perpetuating.