新型疟疾传播阻断疫苗候选抗原PbPSOP7基因截短片段的克隆及表达特点的研究

传播阻断疫苗(transmission-blocking vaccines,TBVs)可以有效地阻断疟原虫从蚊媒向人的传播,是控制疟疾流行的关键,但目前的TBVs候选抗原十分有限,迫切需要寻找有效的候选抗原。动合子分泌蛋白7(putative secreted ookinete protein 7,PSOP7)在疟原虫有性生殖阶段发挥着至关重要的作用,本研究对伯氏疟原虫抗原PSOP7(Pb PSOP7)进行简要的生物信息学分析,并应用原核表达系统高效表达纯化了截短的重组Pb PSOP7蛋白(r Pb PSOP7),免疫BALB/c小鼠后,获得小鼠高滴度多克隆抗体。经Western Blot方法证实该多克隆抗体可识别疟原虫抗原。间接免疫荧光实验显示,Pb PSOP7主要表达于疟原虫的合子与动合子表面。这些特点符合TBVs的基本设计理念,为确认和证实Pb PSOP7蛋白具有疟疾TBVs候选抗原的潜能奠定基础。     

Uses of mosquito-stage transmission-blocking vaccines against Plasmodium falciparum

A quantitative framework is used to explore the potential applications and probable effects of sexual stage or mosquito stage transmission blocking vaccines (TBVs) against malaria. The combination of TBVs with biocides or other malaria vaccines will increase chances of interrupting transmission, whereas the value of TBVs for morbidity control will be limited. Vaccine combination will also protect against selection of insensitive parasites. Simulations indicate that TBVs will reduce risks of reestablishment of transmission when vector control is withdrawn. Simple mathematical analysis shows that efficacy and coverage are equally important, implying that a vaccine that requires a small number of doses (ideally one) is preferable to one that is difficult to deliver, even if this entails accepting a lower efficacy.     

Tick-Borne Viruses

Ticks are important vectors for the transmission of pathogens including viruses. The viruses carried by ticks also known as tick-borne viruses (TBVs), contain a large group of viruses with diverse genetic properties and are concluded in two orders, nine families, and at least 12 genera. Some members of the TBVs are notorious agents causing severe diseases with high mortality rates in humans and livestock, while some others may pose risks to public health that are still unclear to us. Herein, we review the current knowledge of TBVs with emphases on the history of virus isolation and identification, tick vectors, and potential pathogenicity to humans and animals, including assigned species as well as the recently discovered and unassigned species. All these will promote our understanding of the diversity of TBVs, and will facilitate the further investigation of TBVs in association with both ticks and vertebrate hosts.     

Heterologous expression of the C-terminal antigenic domain of the malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonas reinhardtii

Malaria is a widespread and infectious disease that is a leading cause of death in many parts of the world. Eradication of malaria has been a major world health goal for decades, but one that still remains elusive. Other diseases have been eradicated using vaccination, but traditional vaccination methods have thus far been unsuccessful for malaria. Infection by Plasmodium species, the causative agent of malaria, is currently treated with drug-based therapies, but an increase in drug resistance has led to the need for new methods of treatment. A promising strategy for malaria treatment is to combine transmission blocking vaccines (TBVs) that prevent spread of disease with drug-based therapies to treat infected individuals. TBVs can be developed against surface protein antigens that are expressed during parasite reproduction in the mosquito. When the mosquito ingests blood from a vaccinated individual harboring the Plasmodium parasite, the antibodies generated by vaccination prevent completion of the parasites life-cycle. Animal studies have shown that immunization with Pfs48/45 results in the production of malaria transmission blocking antibodies; however, the development of this vaccine candidate has been hindered by poor expression in both prokaryotic and eukaryotic hosts. Recently, the chloroplast of Chlamydomonas reinhardtii has been used to express complex recombinant proteins. In this study, we show that the C-terminal antigenic region of the Pfs48/45 antigen can be expressed in the chloroplast of the green algae C. reinhardtii and that this recombinant protein has a conformation recognized by known transmission blocking antibodies. Production of this protein in algae has the potential to scale to the very large volumes required to meet the needs of millions at risk for contracting malaria.     

Molecular characterization of calreticulin from Anopheles stephensi midgut cells and functional assay of the recombinant calreticulin with Plasmodium berghei ookinetes

Transmission blocking vaccines (TBVs) that target the antigens on the midgut epithelium of Anopheles mosquitoes are among the promising tools for the elimination of the malaria parasite. Characterization and analysis of effective antigens is the first step to design TBVs. Calreticulin (CRT), a lectin-like protein, from Anopheles albimanus midgut, has shown antigenic features, suggesting a promising and novel TBV target. CRT is a highly conserved protein with similar features in vertebrates and invertebrates including anopheline. We cloned the full-length crt gene from malaria vector, Anopheles stephensi (AsCrt) and explored the interaction of recombinant AsCrt protein, expressed in a prokaryotic system (pGEX-6p-1), with surface proteins of Plasmodium berghei ookinetes by immunofluorescence assay. The cellular localization of AsCrt was determined using the baculovirus expression system. Sequence analysis of the whole cDNA of AsCrt revealed that AsCrt contains an ORF of 1221 bp. The amino acid sequence of AsCrt protein obtained in this study showed 64% homology with similar protein in human. The AsCrt shares the most common features of CRTs from other species. This gene encodes a 406 amino-acid protein with a molecular mass of 46 kDa, which contains a predicted 16 amino-acid signal peptides, conserved cysteine residues, a proline-rich region, and highly acidic C-terminal domain with endoplasmic reticulum retrieval sequence HDEL. The production of GST-AsCrt recombinant protein was confirmed by Western blot analysis using an antibody against the GST protein. The FITC-labeled GST-AsCrt exhibited a significant interaction with P. berghei ookinete surface proteins. Purified recombinant GST-AsCrt, labeled with FITC, displayed specific binding to the surface of P. berghei ookinetes in comparison with control. Moreover, the expression of AsCrt in baculovirus expression system indicated that AsCrt was localized on the surface of Sf9 cells. Our results suggest that AsCrt could be utilized as a potential target for future studies in TBV area for malaria control.     

A whole parasite vaccine to control the blood stages of Plasmodium: the case for lateral thinking

Now, 27 years following the cloning of malaria antigens with the promise of the rapid development of a malaria vaccine, we face significant obstacles that are belatedly being addressed. Poor immunogenicity of subunit vaccine antigens and significant antigenic diversity of target epitopes represent major hurdles for which there are no clear strategies for a way forward within the current paradigm. Thus, a different paradigm - a vaccine that uses the whole organism - is now being examined. Although most advances in this approach relate to a vaccine for the pre-erythrocytic stages (sporozoites, liver stages), this opinion paper will outline the possibilities of developing a whole parasite vaccine for the blood stage and address some of the challenges for this strategy, which are entirely different to the challenges for a subunit vaccine. It is the view of the author that both vaccine paradigms should be pursued, but that success will come more quickly using the paranormal approach of exposing individuals to ultra-low doses of whole attenuated or killed parasites.     

Sugar epitopes as potential universal disease transmission blocking targets

One promising method to prevent vector-borne diseases is through the use of transmission blocking vaccines (TBVs). However, developing several anti-pathogen TBVs may be impractical. In this study, we have identified a conserved candidate carbohydrate target in the midguts of several Arthropod vectors. A screen of the novel GlycoChip glycan array found that the anti-carbohydrate malaria transmission blocking monoclonal antibody (MG96) preferentially recognized D-mannose (alpha) and the type II lactosamine disaccharide. The specificity for D-mannose was confirmed by competition ELISA using alpha-methyl mannoside as inhibitor. Con A, which identifies terminal mannose residues, did not inhibit MG96 reactivity with mosquito midgut lysates, suggesting that Con A has differential recognition of this monosaccharide. However, the jack bean lectin, Jacalin, which recognizes D-mannose (alpha), d-galactose (alpha/beta) and the T antigen, not only displays a similar banding profile to that recognized by MG96 on immunoblot but was also shown to effectively inhibit MG96. Wheat-germ agglutinin, which recognizes N-acetyllactosamine units, only partially inhibited MG96 reactivity. This highlights the contribution of both glycan moieties to the MG96 epitope or glycotope. Enzyme deglycosylation results suggest that MG96 recognizes a mannose alpha1-6 substitution on an O-linked oligosaccharide. Taken together, the data suggest that MG96 recognizes a discontinuous glycotope composed of Manalpha1-6 proximal to Galbeta1-4GlcNAc-alpha-O-R glycans on arthropod vector midguts. As such, these glycotopes may represent potential transmission blocking vaccine targets for a wide range of vector-borne pathogens.     

Microorganisms and autoimmunity: making the barren field fertile?

Microorganisms induce strong immune responses, most of which are specific for their encoded antigens. However, microbial infections can also trigger responses against self antigens (autoimmunity), and it has been proposed that this phenomenon could underlie several chronic human diseases, such as type 1 diabetes and multiple sclerosis. Nevertheless, despite intensive efforts, it has proven difficult to identify any single microorganism as the cause of a human autoimmune disease, indicating that the 'one organism-one disease' paradigm that is central to Koch's postulates might not invariably apply to microbially induced autoimmune disease. Here, we review the mechanisms by which microorganisms might induce autoimmunity, and we outline a hypothesis that we call the fertile-field hypothesis to explain how a single autoimmune disease could be induced and exacerbated by many different microbial infections.     

Lymphoid organ dendritic cells: beyond the Langerhans cells paradigm

The immune system has developed mechanisms to detect and initiate responses to a continual barrage of immunological challenges. Dendritic cells (DC), a heterogeneous population of leucocytes, play a major role as immunosurveillance agents. To accomplish this function, DC are equipped with highly efficient mechanisms to detect pathogens, to capture, process and present antigens, and to initiate T-cell responses. These mechanisms are developmentally regulated during the DC life cycle in a process termed 'maturation', which was originally defined using Langerhans cells (LC), a DC type of the epidermis. LC exist in the skin in an immature state dedicated to capturing antigens, and in the subcutaneous lymph nodes in a mature state dedicated to presenting those antigens to T cells. The phenotypic changes undergone by LC during maturation, and the correlation of these changes with tissue localization, have been generally considered a paradigm for all DC. However, studies of the multiple DC types found in the lymphoid organs of mice and humans have revealed that most DC subsets do not follow the life cycle typified by LC. In this review we discuss the limitations of the 'LC paradigm' and suggest that this model should be revised to accommodate the heterogeneity of the DC system. We also discuss the implications of the maturational status of the DC subsets contained in the lymphoid organs for their putative roles in the induction of immune responses and the maintenance of peripheral tolerance.     

Model of T-Cell Nuclear Deformation by the Cortical Actin Layer

Deformations of cell nuclei accompany a number of essential intracellular processes. Although evidence is being accumulated on the primary role actin structures play in controlling the shape of the nucleus, the mechanisms behind this phenomenon remain unknown. Here, we consider theoretically a specific paradigm of nuclear deformation, and a related actin rearrangement, in T cells stimulated by contact with a bead covered by surrogate antigens. We suggest that the nucleus is deformed by the elastic forces developed within a cylindrical layer of polymerized actin, which is generated as a result of the receptor-mediated T-cell activation. We substantiate this proposal with a theoretical analysis of mutual deformations in the actin layer and the nucleus, which recovers the experimentally observed nuclear shapes. Furthermore, we evaluate the forces developed by the actin polymerization that drives the nuclear deformation. The model predicts the mode of actin polymerization generated by the surrogate antigens covering a bead and the values of the elastic moduli of the nuclear shell. We provide a qualitative experimental support for the model assumptions by visualizing the stages of nuclear shape change and the corresponding evolution of the cortical actin.     

Model of T-Cell Nuclear Deformation by the Cortical Actin Layer

Deformations of cell nuclei accompany a number of essential intracellular processes. Although evidence is being accumulated on the primary role actin structures play in controlling the shape of the nucleus, the mechanisms behind this phenomenon remain unknown. Here, we consider theoretically a specific paradigm of nuclear deformation, and a related actin rearrangement, in T cells stimulated by contact with a bead covered by surrogate antigens. We suggest that the nucleus is deformed by the elastic forces developed within a cylindrical layer of polymerized actin, which is generated as a result of the receptor-mediated T-cell activation. We substantiate this proposal with a theoretical analysis of mutual deformations in the actin layer and the nucleus, which recovers the experimentally observed nuclear shapes. Furthermore, we evaluate the forces developed by the actin polymerization that drives the nuclear deformation. The model predicts the mode of actin polymerization generated by the surrogate antigens covering a bead and the values of the elastic moduli of the nuclear shell. We provide a qualitative experimental support for the model assumptions by visualizing the stages of nuclear shape change and the corresponding evolution of the cortical actin.     

Plasmodium vivax transmission-blocking vaccines: Progress,challenges and innovation

Existing control measures have significantly reduced malaria morbidity and mortality in the last two decades, although these reductions are now stalling. Significant efforts have been undertaken to develop malaria vaccines. Recently, extensive progress in malaria vaccine development has been made for Plasmodium falciparum. To date, only the RTS,S/AS01 vaccine has been tested in Phase 3 clinical trials and is now under implementation, despite modest efficacy. Therefore, the development of a malaria transmission-blocking vaccine (TBV) will be essential for malaria elimination. Only a limited number of TBVs have reached pre-clinical or clinical development with several major challenges impeding their development, including low immunogenicity in humans. TBV development efforts against P. vivax, the second major cause of malaria morbidity, lag far behind those for P. falciparum. In this review we summarize the latest progress, challenges and innovations in P. vivax TBV research and discuss how to accelerate its development.     

Broad antigenic coverage induced by vaccination with virus-based cDNA libraries cures established tumors

Effective cancer immunotherapy requires the release of a broad spectrum of tumor antigens in the context of potent immune activation. We show here that a cDNA library of normal tissue, expressed from a highly immunogenic viral platform, cures established tumors of the same histological type from which the cDNA library was derived. Immune escape occurred with suboptimal vaccination, but tumor cells that escaped the immune pressure were readily treated by second-line virus-based immunotherapy. This approach has several major advantages. Use of the cDNA library leads to presentation of a broad repertoire of (undefined) tumor-associated antigens, which reduces emergence of treatment-resistant variants and also permits rational, combined-modality approaches in the clinic. Finally, the viral vectors can be delivered systemically, without the need for tumor targeting, and are amenable to clinical-grade production. Therefore, virus-expressed cDNA libraries represent a novel paradigm for cancer treatment addressing many of the key issues that have undermined the efficacy of immuno- and virotherapy to date.     

Advances in computational structure-based antibody design

Antibodies are currently the most important class of biotherapeutics and are used to treat numerous diseases. Recent advances in computational methods are ushering in a new era of antibody design, driven in part by accurate structure prediction. Previously, structure-based antibody design has been limited to a relatively small number of cases where accurate structures or models of both the target antigen and antibody were available. As we move towards a time where it is possible to accurately model most antibodies and antigens, and to reliably predict their binding site, there is vast potential for true computational antibody design. In this review, we describe the latest methods that promise to launch a paradigm shift towards entirely in silico structure-based antibody design.     

Ookinete-interacting proteins on the microvillar surface are partitioned into detergent resistant membranes of Anopheles gambiae midguts

Lipid raft microdomains, a component of detergent resistant membranes (DRMs), are routinely exploited by pathogens during host-cell entry. Multiple membrane-surface proteins mediate Plasmodium ookinete invasion of the Anopheles midgut, a critical step in the parasite life cycle that is successfully targeted by transmission-blocking vaccines (TBV). Given that lipid rafts are a common feature of host-pathogen interactions, we hypothesized that they promote the partitioning of midgut surface proteins and thus facilitate ookinete invasion. In support of this hypothesis, we found that five of the characterized Anopheles TBV candidates, including the leading Anopheles TBV candidate, AgAPN1, are present in Anopheles gambiae DRMs. Therefore, to extend the repertoire of putative midgut ligands that can be targeted by TBVs, we analyzed midgut DRMs by tandem mass spectrometry. We identified 1452 proteins including several markers of DRMs. Since glycosylphosphotidyl inositol (GPI)-anchored proteins partition to DRMs, we characterized the GPI subproteome of An. gambiae midgut brush-border microvilli and found that 96.9% of the proteins identified in the GPI-anchored fractions were also present in DRMs. Our study vastly expands the number of candidate malarial TBV targets for subsequent analysis by the broader community and provides an inferred role for midgut plasmalemma microdomains in ookinete cell invasion.     

Molecular Mimics of the Tumour Antigen MUC1

A key requirement for the development of cancer immunotherapy is the identification of tumour-associated antigens that are differentially or exclusively expressed on the tumour and recognized by the host immune system. However, immune responses to such antigens are often muted or lacking due to the antigens being recognized as “self”, and further complicated by the tumour environment and regulation of immune cells within. In an effort to circumvent the lack of immune responses to tumour antigens, we have devised a strategy to develop potential synthetic immunogens. The strategy, termed mirror image phage display, is based on the concept of molecular mimicry as demonstrated by the idiotype/anti-idiotype paradigm in the immune system. Here as ‘proof of principle’ we have selected molecular mimics of the well-characterised tumour associated antigen, the human mucin1 protein (MUC1) from two different peptide phage display libraries. The putative mimics were compared in structure and function to that of the native antigen. Our results demonstrate that several of the mimic peptides display T-cell stimulation activity in vitro when presented by matured dendritic cells. The mimic peptides and the native MUC1 antigenic epitopes can cross-stimulate T-cells. The data also indicate that sequence homology and/or chemical properties to the original epitope are not the sole determining factors for the observed immunostimulatory activity of the mimic peptides.     

Monoclonal antibodies for the structural analysis of the Na+/H+ antiporter NhaA from Escherichia coli

Since their advent some 25 years ago, monoclonal antibodies have developed into powerful tools for structural and functional analysis of their cognate antigens. Together with the respective antigen binding fragments, antibodies offer exclusive capacities in detection, characterization, purification and functional assays for every given ligand. Antibody-fragment mediated crystallization represents a major advance in determining the three-dimensional structure of membrane-bound protein complexes. In this review, we focus on the methods used to generate monoclonal antibodies against the NhaA antiporter from Escherichia coli as a paradigm of secondary transporters. We describe examples on how antibodies are helpful in understanding structure and function relationships for this important class of integral membrane proteins. The generated conformation-specific antibody fragments are highly valuable reagents for co-crystallization attempts and structure determination of the antiporter.     

Continuum electrostatic methods applied to pH-dependent properties of antibody-antigen association

Protein association events are a critical component of the functioning of biological systems. Antibody/antigen association, which involves extraordinarily specific interactions, has been a paradigm for the study of structural factors and intermolecular forces controlling protein-protein association. As new experimental approaches to the study of antibody/antigen affinity have become routine, and as more structures of complexes of antibodies and their antigens have become available, it has become possible to use computational approaches to study these interactions. Electrostatic interactions are known to play an important role in protein complex formation. In this review, we focus on the use of continuum electrostatic methods to compute pH-dependent properties of proteins and discuss the use of these methods in the study of antibody/antigen complexes.     

Gut-associated lymphoid tissue, T cell trafficking, and chronic intestinal inflammation

The etiologies of the inflammatory bowel diseases (IBD; Crohn's disease, ulcerative colitis) have not been fully elucidated. However, there is very good evidence implicating T cell and T cell trafficking to the gut and its associated lymphoid tissue as important components in disease pathogenesis. The objective of this review is to provide an overview of the mechanisms involved in naive and effector T cell trafficking to the gut-associated lymphoid tissue (GALT; Peyer's patches, isolated lymphoid follicles), mesenteric lymph nodes and intestine in response to commensal enteric antigens under physiological conditions as well as during the induction of chronic gut inflammation. In addition, recent data suggests that the GALT may not be required for enteric antigen-driven intestinal inflammation in certain mouse models of IBD. These new data suggest a possible paradigm shift in our understanding of how and where naive T cells become activated to yield disease-producing effector cells.     

Monoclonal antibodies for the structural analysis of the Na/H antiporter NhaA from Escherichia coli

Since their advent some 25 years ago, monoclonal antibodies have developed into powerful tools for structural and functional analysis of their cognate antigens. Together with the respective antigen binding fragments, antibodies offer exclusive capacities in detection, characterization, purification and functional assays for every given ligand.Antibody-fragment mediated crystallization represents a major advance in determining the three-dimensional structure of membrane-bound protein complexes. In this review, we focus on the methods used to generate monoclonal antibodies against the NhaA antiporter from Escherichia coli as a paradigm of secondary transporters. We describe examples on how antibodies are helpful in understanding structure and function relationships for this important class of integral membrane proteins.The generated conformation-specific antibody fragments are highly valuable reagents for co-crystallization attempts and structure determination of the antiporter.