Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection.

Plasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections.

Plasmodium vivax VIR Proteins Are Targets of Naturally-Acquired Antibody and T Cell Immune Responses to Malaria in Pregnant Women.

P. vivax infection during pregnancy has been associated with poor outcomes such as anemia, low birth weight and congenital malaria, thus representing an important global health problem. However, no vaccine is currently available for its prevention. Vir genes were the first putative virulent factors associated with P. vivax infections, yet very few studies have examined their potential role as targets of immunity. We investigated the immunogenic properties of five VIR proteins and two long synthetic peptides containing conserved VIR sequences (PvLP1 and PvLP2) in the context of the PregVax cohort study including women from five malaria endemic countries: Brazil, Colombia, Guatemala, India and Papua New Guinea (PNG) at different timepoints during and after pregnancy. Antibody responses against all antigens were detected in all populations, with PNG women presenting the highest levels overall. P. vivax infection at sample collection time was positively associated with antibody levels against PvLP1 (fold-increase: 1.60 at recruitment -first antenatal visit-) and PvLP2 (fold-increase: 1.63 at delivery), and P. falciparum co-infection was found to increase those responses (for PvLP1 at recruitment, fold-increase: 2.25). Levels of IgG against two VIR proteins at delivery were associated with higher birth weight (27 g increase per duplicating antibody levels, p<0.05). Peripheral blood mononuclear cells from PNG uninfected pregnant women had significantly higher antigen-specific IFN-γ TH1 responses (p=0.006) and secreted less pro-inflammatory cytokines TNF and IL-6 after PvLP2 stimulation than P. vivax-infected women (p<0.05). These data demonstrate that VIR antigens induce the natural acquisition of antibody and T cell memory responses that might be important in immunity to P. vivax during pregnancy in very diverse geographical settings.

Plasmodium vivax: comparison of immunogenicity among proteins expressed in the cell-free systems of Escherichia coli and wheat germ by suspension array assays.

BACKGROUND: In vitro cell-free systems for protein expression with extracts from prokaryotic (Escherichia coli) or eukaryotic (wheat germ) cells coupled to solid matrices have offered a valid approach for antigen discovery in malaria research. However, no comparative analysis of both systems is presently available nor the usage of suspension array technologies, which offer nearly solution phase kinetics. METHODS: Five Plasmodium vivax antigens representing leading vaccine candidates were expressed in the E. coli and wheat germ cell-free systems at a 50 µl scale. Products were affinity purified in a single-step and coupled to luminex beads to measure antibody reactivity of human immune sera. RESULTS: Both systems readily produced detectable proteins; proteins produced in wheat germ, however, were mostly soluble and intact as opposed to proteins produced in E. coli, which remained mostly insoluble and highly degraded. Noticeably, wheat germ proteins were recognized in significantly higher numbers by sera of P. vivax patients than identical proteins produced in E. coli. CONCLUSIONS: The wheat germ cell-free system offers the possibility of expressing soluble P. vivax proteins in a small-scale for antigen discovery and immuno-epidemiological studies using suspension array technology.

Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins.

Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could be confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro.

Interaction of the hepatitis B virus protein HBx with the human transcription regulatory protein p120E4F in vitro.

Infection with the hepatitis B virus has been identified as one of the major causes of liver cancer. A large body of experimental work points to a central role for the virally encoded protein HBx in this form of carcinogenesis. HBx is expressed in HBV-infected liver cells and interacts with a wide range of cellular proteins, thereby interfering in cellular processes including cell signaling, cycle regulation and apoptosis. In order to identify possible new targets of the HBx protein, we performed a yeast two-hybrid screen using a truncated protein mini-HBx(18-142) as the bait. In addition to known interacting partners, such as RXR and UVDDB1, we identified several new candidates including the human transcriptional regulatory protein p120E4F, which has been implicated in the regulation of mitosis and the cell cycle. In vitro pull down experiments confirmed the interaction and transcription activation assays in the yeast demonstrated that HBx protein was able to repress GAL4AD-p120E4F-dependent activation of a reporter gene under the control of E4F binding sites found in the adenovirus E4 promoter and the HBV enhancer II region. We also showed that the cysteine residues in HBx are necessary for its interaction with UVDDB1 but not for the interaction with RXR or p120E4F. The possible functional relevance of the interaction between HBx and E4F proteins is discussed in the contexts of cellular transformation and host-virus co-evolution.

Expression and spectroscopic analysis of a mutant hepatitis B virus onco-protein HBx without cysteine residues.

Chronic infection of the hepatitis B virus (HBV) is one of the causes leading to liver cancer. The 3.2kb genome of HBV encodes four proteins: core antigen, surface antigen, a DNA polymerase and the X protein (HBx). The biological functions of HBx are not fully understood. It has been shown that HBx is a potent trans-activator, which activates transcription of many cellular and viral promoters indirectly via protein-protein interactions. These transactivating activities of HBx may contribute to the development of hepatocellular carcinoma. In this paper a truncated mini-HBx(-Cys) (18-142) protein, where the cysteines had been either deleted or substituted by serines, was constructed by site-directed mutagenesis and overexpressed as a 6xHis fusion protein in Escherichia coli. The 6xHis-mini-HBx(-Cys) protein was isolated from inclusion bodies, purified by Ni-affinity chromatography under denaturing conditions and refolded by sequential dialysis. The structure of the 6xHis-mini-HBx(-Cys) protein was analyzed by circular dichroism, fluorescence and one-dimensional NMR spectroscopic assays. The data presented here suggest that HBx is unstructured but has a propensity to gain secondary structure under specific experimental conditions. Its conformational flexibility might partially explain its functional complexity, namely its capacity to interact with a wide array of signaling proteins, transcriptional regulators and nucleic acids.

The cysteine residues of the hepatitis B virus onco-protein HBx are not required for its interaction with RNA or with human p53.

The hepatitis B virus (HBV) protein HBx has been implicated to induce liver cancer in transgenic mice and transactivates a variety of viral and cellular promoters. The 17 kDa protein HBx consists of 154 amino acids, contains 10 cysteine residues and is translated during the viral infection. It has been shown previously that the HBx protein is able to bind to singlestranded DNA and RNA. This nucleic acid binding activity might be relevant for HBx oncogenic character. Furthermore, HBx has been reported to interact with a series of cellular proteins, especially with transcription factors, including the tumor suppressor protein p53. To evaluate the importance of the cysteine residues in HBx for its interaction with RNA and p53 we expressed full-length HBx-wt as well as several truncated mini-HBx(18-142) proteins with multiple cysteine to serine point mutations as 6xHis fusion proteins in Escherichia coli. Using UV cross-linking assays we demonstrate that all truncated mini-HBx proteins with cysteine/serine point mutations maintained the ability to bind to an AU-38 RNA oligonucleotide. Furthermore, we performed in vitro binding assays of selected HBx mutants with GST-p53, circular dichroism spectroscopic analysis of the mutant HBx protein secondary structure and a p53 based transcription activation assay in yeast cells. In summary, our data suggest that the cysteine residues in the HBx protein are of minor importance for its interaction with both RNA and the p53 protein.