Mahamoud Sama Cherif

Immunogenicity of novel nanoparticle-coated MSP-1 C-terminus malaria DNA vaccine using different routes of administration.

An important aspect in optimizing DNA vaccination is antigen delivery to the site of action. In this way, any alternative delivery system having higher transfection efficiency and eventual superior antibody production needs to be further explored. The novel nanoparticle, pDNA/PEI/γ-PGA complex, is one of a promising delivery system, which is taken up by cells and is shown to have high transfection efficiency. The immunostimulatory effect of this novel nanoparticle (NP) coated plasmid encoding Plasmodium yoelii MSP1-C-terminus was examined. Groups of C57BL/6 mice were immunized either with NP-coated MSP-1 plasmid, naked plasmid or NP-coated blank plasmid, by three different routes of administration; intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c). Mice were primed and boosted twice at 3-week intervals, then challenged 2 weeks after; and 100%, 100% and 50% mean of survival was observed in immunized mice with coated DNA vaccine by i.p., i.v. and s.c., respectively. Coated DNA vaccine showed significant immunogenicity and elicited protective levels of antigen specific IgG and its subclass antibody, an increased proportion of CD4(+) and CD8(+) T cells and INF-γ and IL-12 levels in the serum and cultured splenocyte supernatant, as well as INF-γ producing cells in the spleen. We demonstrate that, NP-coated MSP-1 DNA-based vaccine confers protection against lethal P. yoelii challenge in murine model across the various route of administration and may therefore, be considered a promising delivery system for vaccination.

Development and Evaluation of Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) Assay Coupled with a Portable Device for Rapid Diagnosis of Ebola Virus Disease in Guinea

Given the current absence of specific drugs or vaccines for Ebola virus disease (EVD), rapid, sensitive, and reliable diagnostic methods are required to stem the transmission chain of the disease. We have developed a rapid detection assay for Zaire ebolavirus based on reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and coupled with a novel portable isothermal amplification and detection platform. The RT-LAMP assay is based on primer sets that target the untranscribed trailer region or nucleoprotein coding region of the viral RNA. The test could specifically detect viral RNAs of Central and West African Ebola virus strains within 15 minutes with no cross-reactivity to other hemorrhagic fever viruses and arboviruses, which cause febrile disease. The assay was evaluated using a total of 100 clinical specimens (serum, n = 44; oral swab, n = 56) collected from suspected EVD cases in Guinea. The specificity of this diagnostic test was 100% for both primer sets, while the sensitivity was 100% and 97.9% for the trailer and nucleoprotein primer sets, respectively, compared with a reference standard RT-PCR test. These observations suggest that our diagnostic assay is useful for identifying EVD cases, especially in the field or in settings with insufficient infrastructure.

Effect of nanoparticle coating on the immunogenicity of plasmid DNA vaccine encoding P. yoelii MSP-1 C-terminal

In order to assess a new strategy for DNA vaccine formulation and delivery, plasmid encoding Plasmodium yoelii MSP-1 C-terminal was formulated with newly designed nanoparticle-an anionic ternary complex of polyethylenimine and γ-polyglutamic acid (pVAX-MSP-1/PEI/γ-PGA), and intravenously administered to C57BL/6 mice in four different doses, three times at 3-week interval. Antibody response as determined by ELISA, IFA and Western blot, was dose-dependent and subsequent challenge with 10(5)P. yoelii-infected red blood cells revealed 33-60% survival in repeated experiments at a dose of 80 μg pDNA/mouse. IgG subtypes and cytokine levels in the serum and culture supernatants of stimulated spleen cells were also measured. Antigen-specific IgG response provoked by the DNA vaccination was dominated by IgG1 and IgG2b. Although the elevation of IL-12p40 and IFN-γ was marginal (P≥0.354) in the coated group, interleukin-4 levels were significantly higher (P≥0.013) in the coated group than in the naked or control group, suggesting a predominant Th2-type CD4(+) T cell response. These results therefore, overall indicate the possibility of selection and optimization of DNA vaccine formulation for intravenous delivery and may be useful in designing a nanoparticle-coated DNA vaccine that could optimally elicit a desired antibody response for various disease conditions.

Immunogenicity and anti-fecundity effect of nanoparticle coated glutathione S-transferase (SjGST) DNA vaccine against murine Schistosoma japonicum infection.

There is still urgent need for a vaccine against schistosomiasis, especially in Schistosoma japonicum endemic areas where even a vaccine that will interrupt zoonotic transmission will be potentially effective as an intervention tool. We had developed a novel nanoparticle gene delivery system, which has proven efficacious in gene transfection to target immune cells with complementary adjuvant effect and high protective efficacy in several diseases. Here, we applied this nanoparticle system in combination with S. japonicum glutathione S-transferase (SjGST) DNA vaccine to show the immunogenicity and anti-fecundity effect of the nanoparticle coated vaccine formulation against murine schistosomiasis. The nanoparticle-coated DNA vaccine formulation induced desired immune responses. In comparison with the nanoparticle coated empty vector, it produced significantly increased antigen-specific humoral response, T-helper 1 polarized cytokine environment, higher proportion of IFN-γ producing CD4(+) T-cells and the concomitant decrease in IL-4 producing CD4(+) T-cells. Although there was no effect on worm burden, we recorded a marked reduction in tissue egg burden. There was up to 71.3% decrease in tissue egg burden and 55% reduction in the fecundity of female adult worms. Our data showed that SjGST DNA vaccine, delivered using the nanoparticle gene delivery system, produced anti-fecundity effect on female adult schistosomes as previously described by using conventional subunit vaccine with adjuvant, proving this DNA vaccine formulation as a promising candidate for anti-pathology and transmission blocking application.

Nanoparticle formulation enhanced protective immunity provoked by PYGPI8p-transamidase related protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model

We have previously reported the new formulation of polyethylimine (PEI) with gamma polyglutamic acid (γ-PGA) nanoparticle (NP) to have provided Plasmodium yoelii merozoite surface protein-1 (PyMSP-1) plasmid DNA vaccine with enhanced protective cellular and humoral immunity in the lethal mouse malaria model. PyGPI8p-transamidase-related protein (PyTAM) was selected as a possible candidate vaccine antigen by using DNA vaccination screening from 29 GPI anchor and signal sequence motif positive genes picked up using web-based bioinformatics tools; though the observed protection was not complete. Here, we observed augmented protective effect of PyTAM DNA vaccine by using PEI and γ-PGA complex as delivery system. NP-coated PyTAM plasmid DNA immunized mice showed a significant survival rate from lethal P. yoelii challenge infection compared with naked PyTAM plasmid or with NP-coated empty plasmid DNA group. Antigen-specific IgG1 and IgG2b subclass antibody levels, proportion of CD4 and CD8T cells producing IFN-γ in the splenocytes and IL-4, IFN-γ, IL-12 and TNF-α levels in the sera and in the supernatants from ex vivo splenocytes culture were all enhanced by the NP-coated PyTAM DNA vaccine. These data indicates that NP augments PyTAM protective immune response, and this enhancement was associated with increased DC activation and concomitant IL-12 production.

Selection and identification of malaria vaccine target molecule using bioinformatics and DNA vaccination

Following a genome-wide search for a blood stage malaria DNA-based vaccine using web-based bioinformatic tools, 29 genes from the annotated Plasmodium yoelii genome sequence (www.PlasmoDB.org and www.tigr.org) were identified as encoding GPI-anchored proteins. Target genes were those with orthologues in P. falciparum, containing an N-terminal signal sequence containing hydrophobic amino acid stretch and signal P criteria, a transmembrane-like domain and GPI anchor motif. Focusing on the blood stage, we extracted mRNA from pRBCs, PCR-amplified 22 out of the 29 selected genes, and eventually cloned nine of these into a DNA vaccine plasmid, pVAX 200-DEST. Biojector-mediated delivery of the nine DNA vaccines was conducted using ShimaJET to C57BL/6 mice at a dose of 4 μg/mouse three times at an interval of 3 weeks. Two weeks after the second booster, immunized mice were challenged with P. y. yoelii 17XL-parasitized RBCs and the level of parasitaemia, protection and survival was assessed. Immunization with one gene (PY03470) resulted in 2-4 days of delayed onset and level of parasitaemia and was associated with increased survival compared to non-immunized mice. Antibody production was, however, low following DNA vaccination, as determined by immunofluorescence assay. Recombinant protein from this gene, GPI8p transamidase-related protein (rPyTAM) in PBS or emulsified with GERBU adjuvant was also used to immunize another set of C57BL/6 mice with 10-20 μg/mouse three times at 3-week interval. Higher antibody response was obtained as determined by ELISA with similar protective effects as observed after DNA vaccination.

Human-applicable dendrigraft poly-l-lysine-based nanoparticle-coated Plasmodium yoelii-transamidase DNA vaccine is immunogenic and protective as the polyethylenimine-based formulation

The objective was to assess the immunoequivalence and protective efficacy of the novel, relatively safe dendrigraft poly-l-lysine-based nanoparticle formulation in comparison to the non-degradable polyethylenimine-based system. Groups of 6-week-old female C57BL/6 mice were immunized three times biweekly. Each mouse received 100 µg of the Plasmodium yoelii GPI8p-transamidase PyTAM formulated with nanoball that consisted of PyTAM/PEI/γ-PGA or PyTAM/DGL/γ-PGA and their respective nanoparticle-coated blank vector controls. Two weeks after the last immunization, the humoral responses and cellular immune response were assessed. The survival and parasitemia were evaluated in each group challenged intraperitoneally with 106 of a lethal strain of P. yoelii 17XL-parasitized red blood cells. Mice immunized with PyTAM/PEI/γ-PGA or PyTAM/DGL/γ-PGA showed similar survival rates, humoral responses and T helper 1 pro-inflammatory cellular immune responses in vivo and ex vivo. In particular, the PyTAM/DGL/γ-PGA formulation showed a significant increase in conventional dendritic cells in the spleen, which were consistently associated with high interleukin-12 production, the driver of the T helper 1 response. We show that the substitution of non-degradable polyethylenimine with the biodegradable dendrigraft poly-l-lysine in the nanoparticle formulation is immunoequivalent and elicits protective immunity against the lethal strain of P. yoelii. Therefore, this new gene-delivery vehicle with a good safety profile presents an exciting prospect for application in vaccination strategies.

Elevated IL-17 levels in semi-immune anaemic mice infected with Plasmodium berghei ANKA

BackgroundAlterations in inflammatory cytokines and genetic background of the host contribute to the outcome of malaria infection. Despite the promising protective role of IL-17 in infections, little attention is given to further understand its importance in the pathogenesis of severe malaria anaemia in chronic/endemic situations. The objective of this study, therefore, was to evaluate IL-17 levels in anaemic condition and its association with host genetic factors.MethodsTwo mice strains (Balb/c and CBA) were crossed to get the F1 progeny, and were (F1, Balb/c, CBA) taken through 6 cycles of Plasmodium berghei (ANKA strain) infection and chloroquine/pyrimethamine treatment to generate semi-immune status. Cytokine levels and kinetics of antibody production, CD4+CD25+T regulatory cells were evaluated by bead-based multiplex assay kit, ELISA and FACs, respectively.ResultsHigh survival with high Hb loss at significantly low parasitaemia was observed in Balb/c and F1. Furthermore, IgG levels were two times higher in Balb/c, F1 than CBA. While CD4+CD25+ Treg cells were lower in CBA; IL-4, IFN-γ, IL-12α and IL-17 were significantly higher (p < 0.05) in Balb/c, F1.ConclusionsIn conclusion, elevated IL-17 levels together with high IL-4, IL-12α and IFN-γ levels may be a marker of protection, and the mechanism may be controlled by host factor (s). Further studies of F2 between the F1 and Balb/c will be informative in evaluating if these genes are segregated or further apart.

The predictor of mortality outcome in adult patients with Ebola virus disease during the 2014–2015 outbreak in Guinea

The purpose of this study was to examine the association of any demographic and clinical factors with mortality outcome among adult patients with Ebola virus disease (EVD) in Guinea. This retrospective observational study analyzed medical records of laboratory confirmed EVD adult patients during the 2014–2015 EVD outbreak in Guinea. The associations between any demographic or clinical variables and mortality outcome of EVD were assessed using univariate and multivariate logistic regression analyses. Of 2,310 EVD adult patients included for analysis, the overall case fatality rate was 68.1%. Univariate analyses identified factors possibly associated with mortality outcome, including patient age (p < 0.001), history of visiting or close contact with a suspected or confirmed EVD patient (p = 0.035), and seven clinical symptoms on admission, i.e., fever (p = 0.003), hiccups (p < 0.001), vomiting (p = 0.003), diarrhea (p < 0.001), cough (p = 0.001), sore throat (p = 0.016), and unexplained bleeding (p = 0.021). The multivariate analysis showed that patient age was independently associated with mortality outcome of EVD (OR = 1.06; 95%CI = 1.03–1.09; p < 0.001), while none the of clinical symptoms on admission were significantly associated with the mortality outcome. Our analysis indicates that older age was the only independent factor associated with death among EVD adult patients in Guinea. This suggests that older EVD patients should receive intensive medical care and be carefully monitored.

Prognostic and Predictive Factors of Ebola Virus Disease Outcome in Elderly People during the 2014 Outbreak in Guinea

Elderly people occupy a prominent position in African societies; however, their potential linkage to high case fatality rate (CFR) in Ebola virus disease (EVD) was often overlooked. We describe the predictive factors for EVD lethality in the elderly. A total of 2,004 adults and 309 elderly patients with confirmed EVD were included in the analysis. The median age (interquartile range) was 35 years (23-44) in adults and 65 years (60-70) in the elderly. The proportion of funeral participation was significantly higher in the elderly group than in the adult group. Duration (in days) between the onset of symptoms and admission was significantly longer in elderly. CFR in the elderly people was also significantly higher (80.6%) than in the adult group (66.2%). Funeral participation constituted a risk factor for the transmission of EVD in elderly people.