Interleukin-11 up-regulates endoplasmic reticulum stress induced target, PDIA4 in human first trimester placenta and in vivo in mice.

Interleukin (IL)11 is a crucial factor for human trophoblast function and placentation. Elevated levels are associated with pregnancy complications including preeclampsia, intrauterine growth restriction (IUGR) and preterm birth. However, the regulation of IL11 in the placenta has not been investigated. We examined the effect of pro-inflammatory cytokines IL1ß and TNFα, as well as low oxygen tension (2%) on IL11 levels in first trimester placental villous explants. IL1ß upregulated IL11 mRNA and protein, while TNFα and low oxygen had no effect. Using mass spectrometry, we identified protein disulfide isomerase 4 (PDIA4) in IL11-treated first trimester human placental explants (100 ng/ml, 24 h, n = 3), but not PBS control tissues. PDIA4 is a member of the PDI family, also known as endoplasmic reticulum (ER) stress protein (ERP)72. We previously identified GRP78 (a master regulator for ER stress) in human placenta for the first time and demonstrated that IL11 up-regulates GRP78 in the placenta. In this report, we demonstrated that IL11 upregulates PDIA4 protein in human placental villous tissue, HTR8-SVneo trophoblasts (cell line) and in vivo in IL11-treated mouse placenta. We aimed to determine whether IL11 upregulates other ER stress proteins in human first trimester placental villous. IL11 stimulated ERP44, but not GRP94, or PDI. Placental endoplasmic reticulum stress has been postulated in the pathophysiology of preeclampsia and IUGR, but its activation remains elusive. Together, these data suggest that IL11 could trigger an ER stress response in the placenta, which may contribute to obstetric complications such as preeclampsia.

Induction of strain-transcending immunity against Plasmodium chabaudi adami malaria with a multiepitope DNA vaccine.

A major goal of current malaria vaccine programs is to develop multivalent vaccines that will protect humans against the many heterologous malaria strains that circulate in endemic areas. We describe a multiepitope DNA vaccine, derived from a genomic Plasmodium chabaudi adami DS DNA expression library of 30,000 plasmids, which induces strain-transcending immunity in mice against challenge with P. c. adami DK. Segregation of this library and DNA sequence analysis identified vaccine subpools encoding open reading frames (ORFs)/peptides of >9 amino acids [aa] (the V9+ pool, 303 plasmids) and >50 aa (V50+ pool, 56 plasmids), respectively. The V9+ and V50+ plasmid vaccine subpools significantly cross-protected mice against heterologous P. c. adami DK challenge, and protection correlated with the induction of both specific gamma interferon production by splenic cells and opsonizing antibodies. Bioinformatic analysis showed that 22 of the V50+ ORFs were polypeptides conserved among three or more Plasmodium spp., 13 of which are predicted hypothetical proteins. Twenty-nine of these ORFs are orthologues of predicted Plasmodium falciparum sequences known to be expressed in the blood stage, suggesting that this vaccine pool encodes multiple blood-stage antigens. The results have implications for malaria vaccine design by providing proof-of-principle that significant strain-transcending immunity can be induced using multiepitope blood-stage DNA vaccines and suggest that both cellular responses and opsonizing antibodies are necessary for optimal protection against P. c. adami.

A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria.

The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in production and secretion of both AMA-1 and MSP4/5 in vitro. Vaccination of BALB/c mice via intraepidermal gene gun and intramuscular routes against AMA-1 and MSP4/5 resulted in antibody production and significant in vitro proliferation of splenocytes stimulated by both AMA-1 and MSP4/5. Survival of BALB/c mice vaccinated with bicistronic constructs after lethal Plasmodium chabaudi adami DS erythrocytic-stage challenge was variable, although significant increases in survival and reductions in peak parasitemia were observed in several challenge trials when the vaccine was delivered by the intramuscular route. This study using a murine model demonstrates that the delivery of malarial antigens via bicistronic vectors is feasible. Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against malaria.

Induction of specific T-cell responses, opsonizing antibodies, and protection against Plasmodium chabaudi adami infection in mice vaccinated with genomic expression libraries expressed in targeted and secretory DNA vectors.

It has been proposed that a multivalent malaria vaccine is necessary to mimic the naturally acquired resistance to this disease observed in humans. A major experimental challenge is to identify the optimal components to be used in such a multivalent vaccine. Expression library immunization (ELI) is a method for screening genomes of a pathogen to identify novel combinations of vaccine sequences. Here we describe immune responses associated with, and the protective efficacy of, genomic Plasmodium chabaudi adami DS expression libraries constructed in VR1020 (secretory), monocyte chemotactic protein-3 (chemoattractant), and cytotoxic T lymphocyte antigen 4 (lymph node-targeting) DNA vaccine vectors. With splenocytes from vaccinated mice, specific T-cell responses, as well as gamma interferon and interleukin-4 production, were observed after stimulation with P. chabaudi adami-infected erythrocytes, demonstrating the specificity of genomic library vaccination for two of the three libraries constructed. Sera obtained from mice vaccinated with genomic libraries promoted the opsonization of P. chabaudi adami-infected erythrocytes by murine macrophages in vitro, further demonstrating the induction of malaria-specific immune responses following ELI. Over three vaccine trials using biolistic delivery of the three libraries, protection after lethal challenge with P. chabaudi adami DS ranged from 33 to 50%. These results show that protective epitopes or antigens are expressed within the libraries and that ELI induces responses specific to P. chabaudi adami malaria. This study further demonstrates that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.

The protective efficacy of MSP4/5 against lethal Plasmodium chabaudi adami challenge is dependent on the type of DNA vaccine vector and vaccination protocol.

The enhancement of immunogenicity of malarial DNA vaccines is important if they are to have practical application in protecting against blood-stage malaria. Here we describe three different DNA vaccine vector types used in conjunction with the blood-stage merozoite surface protein 4/5 (MSP4/5), the murine homologue of Plasmodium falciparum MSP4 and MSP5, in an attempt to enhance survival against lethal Plasmodium chabaudi adami DS blood-stage challenge. MSP4/5 was inserted into VR1020 (secretory), monocyte-chemotactic protein-3 (MCP-3) (chemoattractant), and cytotoxic T-lymphocyte antigen 4 (CTLA4) (lymph node targeting) vectors. Mice were immunized intradermally via gene-gun, IM injection, or boosting with recombinant MSP4/5 protein. Antibody responses after boosting were predominantly of the IgG1 and IgE isotypes, with low avidity antibodies produced in DNA primed groups. Despite antibody responses comparable to recombinant protein immunization, boosting mice primed with antigens encoded by MCP-3 and CTLA4 vectors did not enhance survival compared to vector control groups. Gene-gun vaccination using VR1020/MSP4/5 followed by recombinant MSP4/5 boosting, or gene-gun DNA vaccination alone using MCP-3/MSP4/5, resulted in enhanced survival compared to empty vector control mice. The results suggest that the enhancement of survival against lethal blood-stage malaria challenge after utilizing MSP4/5 DNA vaccination is therefore highly dependent on the route and type of vaccine vector employed.

Expression library immunization protects mice against a challenge with virulent rodent malaria.

Although several candidate vaccine antigens have been developed for malaria, there is as yet no effective single vaccine available. There is a growing consensus that the ultimate malaria vaccine will be multivalent, requiring the identification of a suitable cocktail of antigens. However, evaluation of the multitude of potential malaria vaccine antigens in suitable combinations is a daunting task. Here we describe the validation of expression library immunization (ELI) as a tool for the discovery of sequences protective against malaria infection. A genomic Plasmodium chabaudi expression library was constructed comprising ten separate pools of 3000 plasmids. Over three vaccine trials using biolistic delivery of pools composed of 616 to 30,000 plasmids we report up to 63% protection of mice from a challenge with P. chabaudi adami DS, a highly virulent strain. Overall, ELI protected 36% of vaccinated mice against virulent challenge compared with only 3.2% survival of control mice. These results demonstrate that ELI is a suitable approach for screening the malaria genome to identify the components of multivalent vaccines.