Application and comparison of lyophilisation protocols to enhance stable long-term storage of filovirus pseudotypes for use in antibody neutralisation tests.

AIMS: Filoviruses encompass highly pathogenic viruses placing significant public health burden on countries affected. Efforts for improved diagnostics and surveillance are needed. The requirement for high-containment can be circumvented by using pseudotype viruses (PV), which can be handled safely, in tropism, drug screening, vaccine evaluation, and serosurveillance studies. We assessed the stability and functionality after long-term storage of lyophilised filovirus pseudotypes for use in neutralisation assays. METHODS AND RESULTS: We generated a panel of filovirus lentiviral pseudotypes followed by lyophilisation and storage in different conditions. Next, we reconstituted and tested PVs in infection experiments and pseudotype neutralisation assays where possible. Lyophilised Ebola and Marburg PVs retained production titres for at least two years when stored at +4˚C or less. Lyophilised Ebola PVs performed similarly to non-lyophilised PVs in neutralisation assays after reconstitution. When stored at high temperatures (+37˚C), lyophilised PVs did not retain titres after 1-month storage, however, when lyophilised using pilot-scale facilities EBOV PVs retained titres and performed as standard in neutralisation assays after on 1-month storage at 37˚C. CONCLUSIONS: Filovirus PVs are amenable to lyophilisation and can be stored for at least 2 years in a household fridge to be used in antibody assays. Lyophilisation performed in the right conditions would allow transportation at room temperature, even in warmer climates.

Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites.

Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug-sensitivity profiles as wild type parasites. We show that MRP1-deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2-deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development.

Microneedle-based drug and vaccine delivery via nanoporous microneedle arrays.

In the literature, several types of microneedles have been extensively described. However, porous microneedle arrays only received minimal attention. Hence, only little is known about drug delivery via these microneedles. However, porous microneedle arrays may have potential for future microneedle-based drug and vaccine delivery and could be a valuable addition to the other microneedle-based drug delivery approaches. To gain more insight into porous microneedle technologies, the scientific and patent literature is reviewed, and we focus on the possibilities and constraints of porous microneedle technologies for dermal drug delivery. Furthermore, we show preliminary data with commercially available porous microneedles and describe future directions in this field of research.

Alternative delivery of a thermostable inactivated polio vaccine.

In the near future oral polio vaccine (OPV) will be replaced by inactivated polio vaccine (IPV) as part of the eradication program of polio. For that reason, there is a need for substantial amount of safe and more affordable IPV for low-income countries. Bioneedles, which are biodegradable mini-implants, have the potential to deliver vaccines outside the cold-chain and administer them without the use of needles and syringes. In the current study, Bioneedles were filled with IPV, subsequently lyophilized, and antigenic recoveries were determined both directly after IPV-Bioneedle preparation as well as after elevated stability testing. Further, we assessed the immunogenicity of IPV-Bioneedles in rats and the residence time at the site of administration. Trivalent IPV was formulated in Bioneedles with recoveries of 101±10%, 113±18%, and 92±15%, respectively for serotypes 1, 2 and 3. IPV in Bioneedles is more resistant to elevated temperatures than liquid IPV: liquid IPV retained less than half of its antigenicity after 1 day at 45°C and IPV in Bioneedles showed remaining recoveries of 80±10%, 85±4% and 63±4% for the three serotypes. In vivo imaging revealed that IPV administered via Bioneedles as well as subcutaneously injected liquid IPV showed a retention time of 3 days at the site of administration. Finally, an immunogenicity study showed that IPV-filled Bioneedles are able to induce virus-neutralizing antibody titers similar to those obtained by liquid intramuscular injection when administered in a booster regime. The addition of LPS-derivate PagL in IPV-filled Bioneedles did not increase immunogenicity compared to IPV-Bioneedles without adjuvant. The current study demonstrates the pre-clinical proof of concept of IPV-filled Bioneedles as a syringe-free alternative delivery system. Further pre-clinical and clinical studies will be required to assess the feasibility whether IPV-Bioneedles show sufficient safety and efficacy, and may contribute to the efforts to eradicate and prevent polio in the future.

A genetically attenuated malaria vaccine candidate based on P. falciparum b9/slarp gene-deficient sporozoites.

A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of malaria but is currently unavailable. High-level protection in humans can be achieved by experimental immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug coverage. Immunization with genetically attenuated parasites (GAP) would be an attractive alternative approach. In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development. In the rodent malaria model, PbΔb9ΔslarpGAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection. The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection. These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine.

Minipigs as an animal model for dermal vaccine delivery.

Appropriate animal models for intradermal vaccine delivery are scarce. Given the high similarity of their skin anatomy to that of humans, minipigs may be a suitable model for dermal vaccine delivery. Here we describe the immunization of Göttingen minipigs by using intradermal and intramuscular delivery of hepatitis B surface antigen (HBsAg). Intradermal vaccine delivery by needle and syringe and by needle-free jet injection induced humoral antiHBsAg responses. Priming immunization by using the disposable syringe jet injector (DSJI) resulted in a higher antibody titer than did conventional intradermal immunization and a titer comparable to that after intramuscular vaccination with HBsAg and Al(OH)3 adjuvant. This study highlights the utility of the minipig model in vaccine studies assessing the efficacy of conventional and novel methods of dermal delivery. Moreover, we include suggestions regarding working with minipigs during dermal vaccine delivery studies, thereby fostering future work in this area of vaccinology.

Two Plasmodium 6-Cys family-related proteins have distinct and critical roles in liver-stage development.

The 10 Plasmodium 6-Cys proteins have critical roles throughout parasite development and are targets for antimalaria vaccination strategies. We analyzed the conserved 6-cysteine domain of this family and show that only the last 4 positionally conserved cysteine residues are diagnostic for this domain and identified 4 additional "6-Cys family-related" proteins. Two of these, sequestrin and B9, are critical to Plasmodium liver-stage development. RT-PCR and immunofluorescence assays show that B9 is translationally repressed in sporozoites and is expressed after hepatocyte invasion where it localizes to the parasite plasma membrane. Mutants lacking B9 expression in the rodent malaria parasites P. berghei and P. yoelii and the human parasite P. falciparum developmentally arrest in hepatocytes. P. berghei mutants arrest in the livers of BALB/c (100%) and C57BL6 mice (>99.9%), and in cultures of Huh7 human-hepatoma cell line. Similarly, P. falciparum mutants while fully infectious to primary human hepatocytes abort development 3 d after infection. This growth arrest is associated with a compromised parasitophorous vacuole membrane a phenotype similar to, but distinct from, mutants lacking the 6-Cys sporozoite proteins P52 and P36. Our results show that 6-Cys proteins have critical but distinct roles in establishment and maintenance of a parasitophorous vacuole and subsequent liver-stage development.

Combined DNA extraction and antibody elution from filter papers for the assessment of malaria transmission intensity in epidemiological studies.

BACKGROUND: Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution. METHODS: Filter paper blood spots were collected as part of a large cross-sectional survey in the Kenyan highlands. DNA was extracted using a saponin/chelex method. The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures. Antibody elution efficiency was assessed by total IgG ELISA for malaria antigens apical membrane antigen-1 (AMA-1) and merozoite-surface protein-1 (MSP-142). The sensitivity of nested 18S rRNA and cytochrome b PCR assays and the impact of doubling filter paper material for PCR sensitivity were determined. The distribution of cell material and antibodies throughout filter paper blood spots were examined using luminescent and fluorescent reporter assays. RESULTS: Antibody levels measured after the combined antibody/DNA extraction technique were strongly correlated to those measured after standard antibody elution (p < 0.0001). Antibody levels for both AMA-1 and MSP-142 were generally slightly lower (11.3-21.4%) but age-seroprevalence patterns were indistinguishable. The proportion of parasite positive samples ranged from 12.9% to 19.2% in the different PCR assays. Despite strong agreement between outcomes of different PCR assays, none of the assays detected all parasite-positive individuals. For all assays doubling filter paper material for DNA extraction increased sensitivity. The concentration of cell and antibody material was not homogenously distributed throughout blood spots. CONCLUSION: Combined DNA extraction and antibody elution is an operationally attractive approach for high throughput assessment of cumulative malaria exposure and current infection prevalence in endemic settings. Estimates of antibody prevalence are unaffected by the combined extraction and elution procedure. The choice of target gene and the amount and source of filter paper material for DNA extraction can have a marked impact on PCR sensitivity.

Plasmodium liver load following parenteral sporozoite administration in rodents.

One of the bottlenecks in the development of a whole sporozoite malaria vaccine is the route and method of sporozoite administration. Immunization and challenge of human volunteers by mosquito bites is effective, but cannot be used as a vaccine. Intravenous immunization with sporozoites is effective in rodents and non-human primates, and being studied in humans, but is not yet used for licensed vaccines for infectious diseases. Intradermal and subcutaneous immunization regimens show a strong decrease in protective efficacy, which in rodents, is associated with a decreased degree of parasite liver infection during immunization. The objective of this study was to explore alternative routes of sporozoite administration to increase efficiency of liver infection. Using in vivo imaging, we found that IM injection of sporozoites resulted in a greater parasite liver load compared to ID and SC injection. The use of small inoculation volumes and multiple injections further increased the subsequent liver load. These observations were corroborated in a Plasmodium yoelii model using cryopreserved sporozoites administered ID. Our findings provide a rationale for the design of clinical trials to optimize needle and syringe administration of Plasmodium falciparum sporozoites.

Plasmodium berghei Δp52&p36 parasites develop independent of a parasitophorous vacuole membrane in Huh-7 liver cells.

The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Δp52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Δp52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Δp52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Δp52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Δp52&p36 parasites were able to fully mature and produced infectious merozoites. These Δp52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Δp52&p36 liver stages were infectious for C57BL/6 mice.

Assessing the adequacy of attenuation of genetically modified malaria parasite vaccine candidates.

The critical first step in the clinical development of a malaria vaccine, based on live-attenuated Plasmodium falciparum sporozoites, is the guarantee of complete arrest in the liver. We report on an approach for assessing adequacy of attenuation of genetically attenuated sporozoites in vivo using the Plasmodium berghei model of malaria and P. falciparum sporozoites cultured in primary human hepatocytes. We show that two genetically attenuated sporozoite vaccine candidates, Δp52+p36 and Δfabb/f, are not adequately attenuated. Sporozoites infection of mice with both P. berghei candidates can result in blood infections. We also provide evidence that P. falciparum sporozoites of the leading vaccine candidate that is similarly attenuated through the deletion of the genes encoding the proteins P52 and P36, can develop into replicating liver stages. Therefore, we propose a minimal set of screening criteria to assess adequacy of sporozoite attenuation necessary before advancing into further clinical development and studies in humans.

Evaluation of immunity against malaria using luciferase-expressing Plasmodium berghei parasites.

BACKGROUND: Measurement of liver stage development is of key interest in malaria biology and vaccine studies. Parasite development in liver cells can be visualized in real-time, both in culture and in live mice, using a transgenic Plasmodium berghei parasite, PbGFP-Luccon, expressing the bioluminescent reporter luciferase. This study explores the benefit of using these parasites for the evaluation of immunity against malaria, compared to qRT-PCR techniques in vivo and in vitro. METHODS: Mice were immunized with either radiation attenuated sporozoites (RAS) or wildtype sporozoites under chloroquine prophylaxis (CPS) and challenged with PbGFP-Luccon. The in vitro transgenic sporozoites neutralization assay (TSNA) was adapted by replacing PbCS(Pf) parasites for PbGFP-Luccon parasites. RESULTS: Application of PbGFP-Luccon transgenic parasites provides live quantitative visual information about the relation between parasite liver load and protection. Moreover, fast and reproducible results are obtained by using these parasites in the transgenic sporozoites neutralization assay, measuring functional antibody-mediated immune responses. CONCLUSIONS: PbGFP-Luccon parasites are a straightforward and valuable tool for comprehension of the biological and immunological principles underlying protection against malaria.

Aging attenuates the vasodilator response to relaxin.

Relaxin, an insulin-like growth factor peptide, increases endothelium-dependent vasodilation and vascular compliance and decreases myogenic reactivity. These vascular effects significantly contribute to the physiological circulatory adaptations in pregnancy, particularly in the mesentery and kidney. Aging predisposes to vascular maladaptation and gestational hypertensive disease. We hypothesized that mild aging reduces the vascular responses to relaxin. In 20 young (10-12 wk) and 20 middle-aged (40-46 wk) female Wistar Hannover rats, vascular responses to chronic exposure of relaxin vs. placebo (5 days) were quantified in isolated mesenteric arteries and kidney. Vascular responses were evaluated using pressure-perfusion myograph, wire myograph, and an isolated perfused rat kidney model. Rxfp1 (relaxin family peptide) gene expression was determined by quantitative polymerase chain reaction. In young rats, relaxin stimulated nitric oxide (NO)-dependent flow-mediated vasodilation (2.67-fold, from 48 ± 9 to 18 ± 4 µl/min), reduced myogenic reactivity (from -1 ± 2 to 7 ± 3 µm/10 mmHg), and decreased mesenteric sensitivity to (28%, from 1.39 ± 0.08 to 1.78 ± 0.10 µM) but did not change compliance and renal perfusion flow (RPFF). In aged rats, relaxin did not affect any of the analyzed mesenteric or renal parameters. In aged compared with young placebo-treated rats, all mesenteric characteristics were comparable, while RPFF was lower (17%, from 6.9 ± 0.2 to 5.7 ± 0.1 ml·min⁻¹·100 g⁻¹) even though NO availability was comparable. Rxfp1 expression was not different among young and aged rats. Our findings suggest that moderate aging involves normal endothelial function but blunts the physiological endothelium-dependent and -independent vasodilator response to relaxin.

Memory-like IFN-γ response by NK cells following malaria infection reveals the crucial role of T cells in NK cell activation by P. falciparum.

NK cells are rapid IFN-γ responders to Plasmodium falciparum-infected erythrocytes (PfRBC) in vitro and are involved in controlling early parasitaemia in murine models, yet little is known about their contribution to immune responses following malaria infection in humans. Here, we studied the dynamics of and requirements for in vitro NK responses to PfRBC in malaria-naïve volunteers undergoing a single experimental malaria infection under highly controlled circumstances, and in naturally exposed individuals. NK-specific IFN-γ responses to PfRBC following exposure resembled an immunological recall pattern and were tightly correlated with T-cell responses. However, although PBMC depleted of CD56(+) cells retained 20-55% of their total IFN-γ response to PfRBC, depletion of CD3(+) cells completely abrogated the ability of remaining PBMC, including NK cells, to produce IFN-γ. Although NK responses to PfRBC were partially dependent on endogenous IL-2 signaling and could be augmented by exogenous IL-2 in whole PBMC populations, this factor alone was insufficient to rescue NK responses in the absence of T cells. Thus, NK cells make a significant contribution to total IFN-γ production in response to PfRBC as a consequence of their dependency on (memory) T-cell help, with likely positive implications for malaria vaccine development.

Persistence of full-length caspase-12 and its relation to malaria in West and Central African populations.

BACKGROUND: The full-length (L-) variant of caspase-12 is believed to predispose to sepsis. It has been replaced in the genome of most human populations by the (S-) variant, which leads to premature termination of translation. Strikingly, the L-allele is still widely prevalent in African populations, presumably due to a counterbalancing selective force specific to this continent, for which malaria is a prime candidate. METHODS: We investigated associations between caspase-12 genotype and malarial parameters in three West-African populations, in studies encompassing immunological, clinical and obstetric data. RESULTS: The caspase-12 L-allele was found at frequencies of 11-34%. Plasmodium falciparum-stimulated mononuclear cells from S/L heterozygote donors produced stronger interferon-gamma and interleukin-10 responses than S/S homozygotes (p = 0.011 and p = 0.023 in uninfected and infected donors respectively). Nevertheless, we found no association between caspase-12 genotype and either the presentation of severe malaria or individual clinical parameters in sick children. Amongst pregnant women, the caspase-12 genotype did not influence peripheral or placental malaria infection, or basic obstetric parameters. Interestingly, perinatal mortality was more frequent in children of both S/S and L/L than S/L mothers, independent of placental P. falciparum-infection. CONCLUSION: We find little clinical or epidemiological evidence that malaria has contributed to the persistence of functional caspase-12 in Africa, suggesting either that alternative selective forces are at work or that genetic drift underlies its current global distribution.

Early interferon-gamma response against Plasmodium falciparum correlates with interethnic differences in susceptibility to parasitemia between sympatric Fulani and Dogon in Mali.

INTRODUCTION: Interethnic differences in susceptibility to malaria provide a unique opportunity to explore immunological correlates of protection. The Fulani of Sahelian Africa are known for their reduced susceptibility to Plasmodium falciparum, compared with surrounding tribes, yet the immunology underlying this is still poorly understood. METHODS AND RESULTS: Here, we show that mononuclear cells from Fulani elicit >10-fold stronger interferon (IFN)-gamma production following a 24-h in vitro coincubation with asexual parasites than cells from sympatric Dogon. This response appears to be specific for P. falciparum among a panel of other human pathogens and is independent of the lower number of regulatory T cell counts present in Fulani. IFN-gamma responses in both tribes were inversely correlated with peripheral parasite density as quantified by nucleic acid sequenced-based amplification, but responses of Fulani remained significantly stronger than those of Dogon after adjustment for concurrent parasitemia, suggesting that hard-wired immunological differences underlie the observed protection. CONCLUSIONS: These results underscore the value of early IFN-gamma responses to P. falciparum as a correlate of anti-parasite immunity, not only in this setting but also in the wider context of malaria, and support the development of malaria vaccines aimed at inducing such responses.

Visualisation and quantitative analysis of the rodent malaria liver stage by real time imaging.

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc(con), expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1-5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.