日本血吸虫23 kDa膜蛋白DNA疫苗和蛋白质疫苗联合应用免疫保护性作用的研究

目的　研究 Sj C2 3DNA疫苗和 Sj C2 3蛋白疫苗联合免疫对 C5 7BL/ 6感染小鼠的免疫保护作用。方法　分别构建和制备 Sj C2 3DNA疫苗 (pc DNA3.1- Sj C2 3)和蛋白疫苗 (Sj C2 3大亲水片段融合蛋白 GST- HD)。 4 8只 C5 7BL / 6小鼠随机分为 A、B、C、D 4组 ,每组 12只。 A组 (Sj C2 3DNA+GST- HD联合应用组 )每只小鼠分别在第 0、2周经股四头肌注射 10 0μg Sj C2 3质粒 DNA,在第 4周经背部皮下多点注射 5 0 μg GST- HD+5 0 μg CFA;B组 (Sj C2 3DNA组 )每只小鼠分别在第 0、2、4周经股四头肌注射 10 0 μg Sj C2 3质粒 DNA;C组 (蛋白疫苗组 )于第 4周每鼠经背部皮下多点注射5 0μg GST- HD+5 0μg CFA 1次 ;D组 (对照组 )每鼠分别在第 0、2、4周肌注 10 0μg pc DNA 3.1。各组在末次免疫后 30 d每鼠以 4 5± 2条 /只日本血吸虫尾蚴攻击感染 ,攻击后 4 5 d剖杀小鼠 ,计数成虫及肝内虫卵数。在免疫前、末次免疫后和攻击后从小鼠尾静脉采血 ,分离血清 ,用 Western- blot法检测抗体反应。结果　联合组与质粒对照组相比 ,获得了 36 .9%的减虫率和 30 .7%减卵率 ;而单独Sj C2 3DNA疫苗组的减虫率为 2 6 .9% ,显著低于联合组 (P<0 .0 5 )和 2 2 .2 %的减卵率 ,单独蛋白疫苗组 (GST- HD)为 15 .2 %和 12 .2 %     

Epitope-specific TCRbeta repertoire diversity imparts no functional advantage on the CD8+ T cell response to cognate viral peptides

TCR repertoire diversity has been convincingly shown to facilitate responsiveness of CD8+ T cell populations to mutant virus peptides, thereby safeguarding against viral escape. However, the impact of repertoire diversity on the functionality of the CD8+ T cell response to cognate peptide-MHC class I complex (pMHC) recognition remains unclear. Here, we have compared TCRbeta chain repertoires of three influenza A epitope-specific CD8+ T cell responses in C57BL/6 (B6) mice: D(b)NP(366-374), D(b)PA(224-233), and a recently described epitope derived from the +1 reading frame of the influenza viral polymerase B subunit (residues 62-70) (D(b)PB1-F2(62)). Corresponding to the relative antigenicity of the respective pMHCs, and irrespective of the location of prominent residues, the D(b)PA(224)- and D(b)PB1-F2(62)-specific repertoires were similarly diverse, whereas the D(b)NP(366) population was substantially narrower. Importantly, parallel analysis of response magnitude, cytotoxicity, TCR avidity, and cytokine production for the three epitope-specific responses revealed no obvious functional advantage conferred by increased T cell repertoire diversity. Thus, whereas a diverse repertoire may be important for recognition of epitope variants, its effect on the response to cognate pMHC recognition appears minimal.     

Design of immunogens to elicit broadly neutralizing antibodies against HIV targeting the CD4 binding site

A vaccine which is effective against the HIV virus is considered to be the best solution to the ongoing global HIV/AIDS epidemic. In the past thirty years, numerous attempts to develop an effective vaccine have been made with little or no success, due, in large part, to the high mutability of the virus. More recent studies showed that a vaccine able to elicit broadly neutralizing antibodies (bnAbs), that is, antibodies that can neutralize a high fraction of global virus variants, has promise to protect against HIV. Such a vaccine has been proposed to involve at least three separate stages: First, activate the appropriate precursor B cells; second, shepherd affinity maturation along pathways toward bnAbs; and, third, polish the Ab response to bind with high affinity to diverse HIV envelopes (Env). This final stage may require immunization with a mixture of Envs. In this paper, we set up a framework based on theory and modeling to design optimal panels of antigens to use in such a mixture. The designed antigens are characterized experimentally and are shown to be stable and to be recognized by known HIV antibodies.

Vaccines are the most important medical countermeasure for protecting entire populations against viruses, of which smallpox and measles vaccines are successful examples. In fact, a safe and effective HIV vaccine is considered to be the best way to end the global AIDS epidemic (1). However, how to produce a universal vaccine for highly antigenically variable viruses like HIV is a daunting and yet unsolved problem. The high variability of this virus allows it to elude the immune system, making the produced antibodies ineffective; that is, they are generally specific for a given strain of the virus but not for other strains resulting from mutation. In some cases, HIV-infected patients can elicit antibodies that can recognize and neutralize a broad range of different viral strains (2, 3). These broadly neutralizing antibodies (bnAbs) usually take a long time to appear naturally in infected patients and then only in a subset of such individuals.The reason that bnAbs can arise is that even highly variable pathogens have regions with a well-defined, relatively conserved structure, which is required for their function. In HIV, entry depends on the trimeric spike exposed on the external lipid membrane of the virion, a heterotrimer formed by the gp120 and gp41 glycoproteins produced by posttranslational cleavage of a gp160 precursor. This protein binds to the CD4 coreceptor on CD4 T lymphocytes during HIV infection, and it has some relatively conserved regions that can be used as a target for bnAbs. Indeed, many bnAbs target the CD4 binding site (CD4bs) (4–8). If naive B cells that can bind to one of these relatively conserved regions can be expanded upon exposure to different variants of the virus, antibodies could evolve to better recognize the conserved portions, while avoiding the variable ones. The resulting antibodies can acquire breadth in this way, thereby becoming bnAbs. A successful vaccine would contain immunogens that can guide the immune system to produce bnAbs, rather than strain-specific antibodies.In the past, numerous approaches for the development of an effective HIV vaccine have been tried. They include the use of cleverly chosen natural HIV proteins, the design of a consensus (9) or “center-of-tree” (10) antigens, and the creation of a mosaic protein from different HIV strains (11). All these methods used a single optimized antigen in the vaccine and were shown to be ineffective at eliciting bnAbs (12, 13). One possible reason for this is that, when exposed to a single antigen, the immune system will produce antibodies specific for that particular antigen, and neutralization escape variants can easily develop. A possible solution is to use more than one antigen in a vaccination protocol. This raises a number of questions: How many antigens are necessary? How different should they be from each other? And in what temporal order should they be administered? Answering such questions is far from trivial, in particular due to the limited mechanistic understanding of affinity maturation (AM) in vivo. Another problem is that bnAbs have an unusually high number of somatic mutations, not only in the complementarity-determining regions (CDRs) but also in the immunoglobulin framework regions (7, 14). Recent computational data on the flexibility of the antibody and the need for framework mutations in the simulated AM showed how important it is for a vaccination protocol to have a specific antigen that can prime a good antibody precursor B cell receptor (BCR) (15). Moreover, it has been shown that putative precursors of known classes of bnAbs are generally not able to neutralize HIV or recognize envelope (Env), often due to clashes of the antibody with the glycosylation shield that protects the HIV Env protein (8, 16–18). For example, VRC01-class bnAbs are known to introduce a deletion or a mutation to a flexible glycine in the CDRL1 loop to avoid the glycan at N276 (19, 20).The above discussion led to the proposal of a vaccination strategy consisting of three steps. First, a special purpose antigen is used to activate the correct naïve or precursor B cell (17, 21). Since this precursor will generally not bind to native HIV, as a second step, one or more antigens are used as intermediates to induce somatic mutations and to allow recognition of the native virus. In the third step, one or more antigens are used in a mixture or in sequence to increase the breadth of the antibody population (19, 22, 23). Implementations of the first and second steps have already been shown to be promising in experiments (16, 21, 24–27). However, much less is known about the third step. Some insights into this question can be obtained by in silico simulations of AM. Using coarse-grained models, it has been shown that, while administering a single mixture containing multiple antigens may induce too much frustration to lead to bnAbs formation, a sequential approach, in which antigens are administered one after another, seems to be more effective (23). It was also observed that the number of antigens required in a mixture is correlated with their sequence dissimilarity, and optimal breadth is obtained at an optimal number of antigens and dissimilarity (28). Given the coarse-grained nature of these studies, the actual antigen sequences to use in experiments cannot be obtained from them.In this work, we focus on the third step of the proposed vaccination protocol. In particular, we derive a set of empirical rules and protocols to select an optimal panel of antigens to maximize the breadth of the produced antibodies upon AM. To be able to do so, it is essential to understand, at an atomistic level of detail, the role of each antigen amino acid in the antibody/antigen interaction. This aspect will be presented in the next section based on an analysis of the available crystallographic structures of bnAbs bound to the gp160 Env glycoprotein. However, the structures do not provide information concerning HIV stability and function. For example, generating antigen sequences by introducing purely random mutations will likely lead to sequences that are lethal for the virus and/or are not representative of HIV in vivo. To overcome this problem, it is useful to consider the structural data together with a model of the gp160 fitness landscape (29), which is a measure of the ability of HIV to tolerate mutations in its gp160 sequence to escape immune pressure. Structural and fitness information together provide a classification of the antibody/antigen interface and indicate the residues to mutate and the amino acids that are more probable at those positions.While this analysis helps to reduce the number of antigen sequences to consider by highlighting the “hot spots” of antibody/antigen binding, it leaves open the question of how to select a combination of antigen sequences for use in a vaccine. Given rules of optimal sequence dissimilarity and optimal fitness according to the HIV landscape, a Pareto frontier approach will be described. It is able to select, from all possible panels of antigen sequences, the few that are predicted to best elicit antibodies with a broad activity spectrum. Experimental evidence of the viability of the designed antigens and of their immunogenic properties is presented in the final section.     

Epitope specificities and antibody responses to the EG95 hydatid vaccine

Antibody isotype and epitope specificities were examined in sheep immunized with EG95, a protective recombinant vaccine against hydatid disease. All sheep immunized with EG95 as a fusion protein with glutathione S-transferase (GST) produced prominent IgG antibodies against the EG95 portion of the protein. Linear, antibody-binding epitope specificities of EG95 were mapped using a series of 25 overlapping synthetic peptides. Three immunodominant regions were identified which generated specific IgG₁ and IgG₂ antibodies in the majority of vaccinated sheep. These regions corresponded to the EG95-derived sequences SLKAVNPSDPLVYKRQTAKF, DIETPRAGKKESTVMTSGSA and SALTSAIAGFVFSC. An additional immunogenic region was identified which induced almost exclusively IgG₂ antibody. This epitope was located within the sequence TETPLRKHFNLTPV. The anti-parasitic, protective effects of the EG95 vaccine correlated with the detection of specific antibody to two or more of the four linear immunogenic regions. The identification of these immunogenic peptides of EG95 maybe useful in the development of a synthetic peptide vaccine as a derivative of the EG95 recombinant.     

Intranasal immunization with yeast-expressed 19 kD carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (yMSP119) induces protective immunity to blood stage malaria infection in mice

Variable protection against malaria blood-stage infection has been demonstrated in mice following parenteral immunization with the highly conserved 19 kD carboxylterminal fragment of the merozoite surface protein-1 (MSP1₁₉) using CFA/IFA and other adjuvants. Here we show that intranasal immunization of BALB/C mice with yeast expressed Plasmodium yoelii MSP1₁₉ plus a mixture of native and recombinant cholera toxin B subunit, could induce serum MSP1₁₉-specific antibodies at titres ranging from 20 000 to 2 560 000. The Ig subclass responses were predominantly G1 and G2b. Intranasal immunization led to protection following challenge (peak parasitaemia < 1%) in mice with the highest MSP1₁₉-specific titre (≥ 640 000). In two of the three protected mice, a peak parasitaemia of 0.1%–1% was followed by a boost of the antibody response whereas one of the three protected mice did not boost its antibody response after a peak parasitaemia of 0.02%. In unprotected mice, antibody levels rose, then fell, following the detection of parasites in the peripheral blood. CD4⁺ T cell-depletion abrogated the ability of the mice to boost their antibody response following challenge. These data demonstrate the potential for intranasal immunization with MSP1₁₉ to protect against malaria .     

Safety and immunogenicity of a fully liquid vaccine containing five-component pertussis-diphtheria-tetanus-inactivated poliomyelitis-Haemophilus influenzae type b conjugate vaccines administered at two, four, six and 18 months of age

OBJECTIVE: The safety, immunogenicity and lot consistency of a fully liquid, five-component acellular pertussis combination vaccine, comprised of diphteria, tetanus and acellular pertussis, inactivated polio vaccine, Haemophilus influenzae type b (DTaP-IPV-Hib [Pediacel, sanofi pasteur, Canada]) were assessed and compared with that of Hib vaccine reconstituted with the five-component acellular pertussis combination vaccine (DTaP-IPV//Hib, Pentacel [sanofi pasteur, Canada]). METHODS: Infants were recruited at vaccine study centres in Montreal, Quebec; Simon Fraser Health Region, British Columbia, and southern Alberta after the protocol had been approved by the relevant institutional ethics committees. Written informed consent was obtained from the parents or guardians of all subjects. At two months of age, the infants were randomly assigned to receive one of three consecutive production lots of DTaP-IPV-Hib by intramuscular injection. Reactions to vaccinations were assessed by parental observation and through telephone interviews conducted by study nurses. Blood samples were obtained at two, six, seven, 18 and 19 months of age for measurement of antibodies to vaccine antigens. RESULTS: Most injection site and systemic reactions were mild or moderate, and of brief duration. All infants were protected against tetanus, diphtheria and all three polio serotypes after both primary and booster vaccinations. Antibody responses to pertussis antigens were similar to those observed in Swedish infants, in whom the five-component vaccine was shown to be 85% effective. Proportions of infants with antipolyribosylribitol phosphate antibody of 0.15 mug/mL or greater and 1.0 mug/mL or greater, were 97.9% and 88.9%, respectively, following primary immunization, and 100% and 99% following booster vaccination. Safety and immunogenicity results with both reconstituted and fully liquid combination vaccines were comparable. CONCLUSIONS: The fully liquid combination vaccine was comparable in terms of safety and immunogenicity with the reconstituted combination vaccine.     

Effects of hepatitis A vaccination on atherogenesis in a murine model

Summary. Our laboratory demonstrated that seropositivity to hepatitis A virus (HAV) independently predicts risk for coronary artery disease (CAD). As these findings are based only on the presence of HAV-specific antibodies, and not infectious virus, this prompted questions regarding possible effects of HAV vaccines on CAD development. If seropositivity to HAV alone, resulting from HAV vaccination, leads to increased atherogenesis, this raises important issues regarding the benefit of protection against HAV infection vs the risk of developing CAD. This study examines the effect of HAV vaccination on atherosclerosis development in a cholesterol-fed mouse model. Animals either received HAV vaccine, adjuvant, or saline. After 15 weeks, no significant differences were found in lesion area between the groups: HAV vaccine, 13 470 μm²; adjuvant, 16 332 μm² and saline, 14 356 μm². Only animals receiving HAV vaccination developed HAV-specific IgG. Thus, in this mouse model, vaccination against HAV does not contribute to the development of atherosclerosis.     

Safety and immunogenicity of live attenuated influenza reassortant H5 vaccine (phase I–II clinical trials)

Objective Our studies aimed to evaluate in clinical trials the safety and immunogenicity of an H5 live influenza vaccine candidate obtained using classical reassortment techniques from a low pathogenicity avian influenza (LPAI) A/Duck/Potsdam/1402‐6/86(H5N2) virus and the cold‐adapted (ca) donor strain A/Leningrad/134/17/57(H2N2). Methods During Phase I–II clinical trials, volunteers received intranasally two doses of reassortant influenza vaccine strain A/17/Duck/Potsdam/86/92 (H5N2) 21 days apart. Clinical examination of all vaccinees was conducted 7 days post‐vaccination. Serum antibody responses were measured by hemagglutination‐inhibition and microneutralization and local antibodies were estimated using an enzyme‐linked immunosorbent assay test. Results The vaccine was safe and of low reactogenicity with no febrile reactions. After revaccination 47·1–54·8% of subjects showed ≥fourfold seroconversions of Hamagglutination inhibition (HAI) antibodies to the hemagglutinin (HA) antigen of the A/17/Duck/Potsdam/86/92 (H5N2) virus and 29·4–30·8% were seroconverted to the HA antigen of the reverse genetics reassortant A/Indonesia/05/2005 × PR8 IBCDC‐RG (H5N1). Virus‐neutralizing antibody levels in sera of volunteers were similar to those shown in HAI test. The virus‐specific nasal IgA antibody response after two vaccine doses demonstrated significant increases of ≥fourfold rise SIgA antibodies (65%) geometrical mean titers (16·0) and a rise in SIgA antibodies (2·8) compared with one dose. Conclusion The live attenuated influenza vaccine candidate prepared using the LPAI A(H5N2) strain was well tolerated and elicited serum and local immune responses. There was evident cross‐reactivity to the A(H5N1) strain in the HAI test.     

Protection in lambs vaccinated with Haemonchus contortus antigens is age related, and correlates with IgE rather than IgG1 antibody

The involvement of mucosal mast cells (MMC) in protection against infection with the murine nematode parasite Trichuris muris was studied in genetically mast cell-deficient WBB6F1-W/Wv mice and their normal littermates WBB6F1-+/+ mice. Expulsion of T. muris worms occurred in infected +/+ mice, whereas no worm expulsion was observed in infected W/Wv mice where the infection persisted until at least day 46 postinfection. No MMC responses were induced in either infected W/Wv or +/+ mice. Specific IgG1and IgG2a antibodies to T. muris excretory/secretory antigens were observed in infected W/Wv and +/+ mice, and antibody production showed similar kinetics. Interleukin 4 production by concanavalin A (Con A)-stimulated mesenteric lymph node cells (MLNC) was induced preferentially in infected +/+ mice. T. muris infection increased the levels of IFN-gamma produced by Con A-stimulated MLNC of infected W/Wv and +/+ mice, with the levels of IFN-gamma in infected W/Wv mice being higher than those in infected +/+ mice. Taken together, these results indicate that W/Wv and +/+ mice are susceptible and resistant to T. muris infection, respectively, and that MMC responses are not required for protective immunity.