The development of a vaccine against malaria has public health priority. In a controlled setting, preliminary data on the efficacy of Plasmodium falciparum vaccine candidates can be obtained by ...exposing immunized human volunteers to the bites of laboratory-reared P. falciparum—infected mosquitoes. Using empirical data, we show that these trials, with small numbers of volunteers, are sufficiently powered to detect protective biological effects induced by preerythrocytic and/or blood-stage candidate vaccines if parasitemia is measured daily by quantitative polymerase chain reaction. Sporozoite challenge trials are thus a powerful tool for early selection of candidates that warrant efficacy of trials in the field.
Aim
: Pre-targeting is a proven strategy for
in vivo
delivery of a diagnostic or therapeutic payload. The pre-targeting concept can be realized through various conjugation strategies, one of which is ...based on copper-free "click" chemistry. Copper-free click reactions have shown
in vivo
potential for imaging and radionuclide therapy, but this conjugation strategy has not yet been explored in combination with microspheres or unicellular organisms. This study aims to evaluate the
in vivo
efficacy of strain-promoted azide-alkyne cycloaddition (SPAAC) reactions to achieve imaging and targeting of azide-functionalized macro-aggregated albumin (MAA) microspheres and
Staphylococcus aureus
bacteria.
Methods
: MAA microspheres (diameter 10-90 μm) were functionalized with a biorthogonal Cy5 fluorophore, bearing an azide functionality (N
3
), to generate MAA-Cy5-N
3
.
S. aureus
(diameter ∼1 μm) were functionalized with
99m
Tc-UBI
29-41
-Cy5-N
3
, generating
S. aureus
-
99m
Tc-UBI
29-41
-Cy5-N
3
.
In situ
and
in vitro
click conjugation on the -N
3
moieties was studied for 20 h using a radioactivity-based assay and fluorescence microscopy. For
in vivo
validation, both primary entities, radiolabeled with
99m
Tc, were deposited into the microvasculature of the liver
via
intrasplenic injections. Secondary targeting was realized following the intravenous administration of indium-111-radiolabeled diethylenetriaminepentaacetic acid-dibenzocyclooctyne (
111
In-DTPA-DBCO). To assess click reaction efficiency
in vivo
,
99m
Tc and
111
In-biodistributions were measured (SPECT and %ID g
−1
). Use of
111
In-DTPA-DBCO in mice without MAA deposits or mice infected with non-functionalized
S. aureus
served as controls.
Ex vivo
confocal fluorescence imaging was carried out in excised tissues to confirm the presence of functionalized MAA and bacteria.
Results: In vitro
data confirmed effective click reactions on both the MAA particles and the bacterial membrane. SPECT imaging and biodistribution studies revealed significantly (
p
< 0.05) increased accumulation of
111
In-DTPA-DBCO at the sites where MAA-Cy5-N
3
(7.5 ± 1.5%ID g
−1
vs
. 3.5 ± 0.5%ID g
−1
in control mice) and
S. aureus
-
99m
Tc-UBI
29-41
-Cy5-N
3
(9.3 ± 1.3%ID g
−1
vs.
6.0 ± 0.5%ID g
−1
in control mice) resided.
Ex vivo
fluorescence imaging confirmed the presence of either functionalized MAA or
S. aureus
in excised spleens and livers of mice.
Conclusion
: Copper-free click chemistry between a DBCO moiety and Cy5-N
3
-functionalized microspheres or bacterial entities in the liver can be used to realize
in vivo
imaging and targeting.
Pre-targeting is a proven strategy for
in vivo
delivery of a diagnostic or therapeutic payload.
Background. Through 2 international traveler-focused surveillance networks (GeoSentinel and TropNet), we identified and investigated a large outbreak of acute muscular sarcocystosis (AMS), a rarely ...reported zoonosis caused by a protozoan parasite of the genus Sarcocystis, associated with travel to Tioman Island, Malaysia, during 2011–2012. Methods. Clinicians reporting patients with suspected AMS to GeoSentinel submitted demographic, clinical, itinerary, and exposure data. We defined a probable case as travel to Tioman Island after 1 March 2011, eosinophilia (>5%), clinical or laboratory-supported myositis, and negative trichinellosis serology. Case confirmation required histologic observation of sarcocysts or isolation of Sarcocystis species DNA from muscle biopsy. Results. Sixty-eight patients met the case definition (62 probable and 6 confirmed). All but 2 resided in Europe; all were tourists and traveled mostly during the summer months. The most frequent symptoms reported were myalgia (100%), fatigue (91%), fever (82%), headache (59%), and arthralgia (29%); onset clustered during 2 distinct periods: "early" during the second and "late" during the sixth week after departure from the island. Blood eosinophilia and elevated serum creatinine phosphokinase (CPK) levels were observed beginning during the fifth week after departure. Sarcocystis nesbitti DNA was recovered from 1 muscle biopsy. Conclusions. Clinicians evaluating travelers returning ill from Malaysia with myalgia, with or without fever, should consider AMS, noting the apparent biphasic aspect of the disease, the later onset of elevated CPK and eosinophilia, and
Summary Background We have shown that immunity to infection with Plasmodium falciparum can be induced experimentally in malaria-naive volunteers through immunisation by bites of infected mosquitoes ...while simultaneously preventing disease with chloroquine prophylaxis. This immunity was associated with parasite-specific production of interferon γ and interleukin 2 by pluripotent effector memory cells in vitro. We aim to explore the persistence of protection and immune responses in the same volunteers. Methods In an open-label study at the Radboud University Nijmegen Medical Centre (Nijmegen, Netherlands), from November to December, 2009, we rechallenged previously immune volunteers (28 months after immunisation) with the bites of five mosquitoes infected with P falciparum . Newly recruited malaria-naive volunteers served as infection controls. Our primary outcome was the detection of blood-stage parasitaemia by microscopy. We assessed the kinetics of parasitaemia with real-time quantitative PCR (rtPCR) and recorded clinical signs and symptoms. In-vitro production of interferon γ and interleukin 2 by effector memory T cells was studied after stimulation with sporozoites and red blood cells infected with P falciparum . Differences in cellular immune responses between the study groups were assessed with the Mann-Whitney test. This study is registered with ClinicalTrials.gov , number NCT00757887. Findings Four of six immune volunteers were microscopically negative after rechallenge. rtPCR-based detection of blood-stage parasites in these individuals was negative throughout follow-up. Patent parasitaemia was delayed in the remaining two immunised volunteers. In-vitro assays showed the long-term persistence of parasite-specific pluripotent effector memory T-cell responses in protected volunteers. The four protected volunteers reported several mild to moderate adverse events, of which the most commonly reported symptom was headache (one to three episodes per volunteer). The two patients with delayed patency had adverse events similar to those in the control group. Interpretation Artificially induced immunity lasts longer than generally recorded after natural exposure; providing a new avenue of research into the mechanisms of malaria immunity. Funding Dioraphte Foundation.
To assess the feasibility of using freehand Single Photon Emission Computed Tomography (freehandSPECT) for the identification of technetium-99m-hydroxydiphosphonate (99mTc-HDP) positive bone lesions ...and to evaluate the possibility of using these imaging data-sets for augmented- and virtual-reality based navigation approaches.
In 20 consecutive patients referred for scintigraphy with 99mTc-HDP, 21 three-dimensional freehandSPECT-images were generated using a handheld gamma camera. Concordance of the two different data sets was ranked. Furthermore, feasibility of segmenting the hotspot of tracer accumulation for navigation purposes was assessed.
In 86% of the cases freehandSPECT images showed good concordance with the corresponding part of the scintigraphic images. In lesions with a signal to background ratio (SBR) >1.36, freehandSPECT provided an automatically segmented reference point for navigation purposes. In 14% of the cases (average SBR 1.82, range 1.0–3.4) freehandSPECT images showed intermediate concordance due to difficult anatomical area or negative bone scintigraphy and could not be used as navigation targets.
In this pilot study, in 86% of the cases freehandSPECT demonstrated good concordance with traditional scintigraphy. A lesion with a SBR of 1.36 or more was suitable for navigation. These high-quality freehandSPECT images supported the future exploration navigation strategies, e.g. guided needle biopsies.
Determinar la factibilidad de la freehandSPECT en la identificación de lesiones óseas con captación de 99mTc-HDP evaluando los datos generados mediante la utilización de sistemas de navegación con realidad aumentada y virtual.
Se generaron 21 imágenes 3D utilizando freehandSPECT con una gammacámara manual en 20 pacientes referidos para gammagrafía ósea con 99mTc-HDP. Las imágenes freehandSPECT y las gammagrafías fueron comparadas y se analizóel grado de concordancia. Además, se evaluó la factibilidad de navegación hacia las lesiones óseas hipercaptantes.
En el 86% de los casos freehandSPECT mostró una buena concordancia con las imágenes correspondientes de la gammagrafía ósea. En lesiones con una señal lesión/fondo de >1,36 freehandSPECT pudo automáticamente proporcionar puntos de referencia segmentados con finalidad de navegación. En el 14% de los casos (índice lesión/fondo: valor promedio 1,82, rango 1,0-3,4) las imágenes freehandSPECT mostraron concordancia intermedia debido a que estaban localizadas en regiones anatómicas difíciles o asociadas con una gammagrafía ósea negativa y fueron consideradas como no apropiadas para la navegación dirigida.
En este estudio piloto, se encontró un 86% de los casos apropiados para propósitos de navegación con una buena concordancia entre freehandSPECT y la gammagrafía ósea. Un índice lesión/fondo de 1.36 o más facilitó la navegación con freehandSPECT. La alta calidad de las imágenes generadas con freehandSPECT potencialmente asegura una exitosa estrategia de navegación para biopsias óseas guiadas.
Abstract Purpose To assess the feasibility of using freehand Single Photon Emission Computed Tomography (freehandSPECT) for the identification of technetium-99m-hydroxydiphosphonate (99m Tc-HDP) ...positive bone lesions and to evaluate the possibility of using these imaging data-sets for augmented- and virtual-reality based navigation approaches. Material and methods In 20 consecutive patients referred for scintigraphy with99m Tc-HDP, 21 three-dimensional freehandSPECT-images were generated using a handheld gamma camera. Concordance of the two different data sets was ranked. Furthermore, feasibility of segmenting the hotspot of tracer accumulation for navigation purposes was assessed. Results In 86% of the cases freehandSPECT images showed good concordance with the corresponding part of the scintigraphic images. In lesions with a signal to background ratio (SBR) >1.36, freehandSPECT provided an automatically segmented reference point for navigation purposes. In 14% of the cases (average SBR 1.82, range 1.0–3.4) freehandSPECT images showed intermediate concordance due to difficult anatomical area or negative bone scintigraphy and could not be used as navigation targets. Conclusion In this pilot study, in 86% of the cases freehandSPECT demonstrated good concordance with traditional scintigraphy. A lesion with a SBR of 1.36 or more was suitable for navigation. These high-quality freehandSPECT images supported the future exploration navigation strategies, e.g. guided needle biopsies.
Studies in animal models suggest that protection against malaria induced by intradermal (ID) administration of sporozoites is less effective compared to intravenous injection (IV). We investigated in ...a murine model the protective efficacy and immune responses after ID or IV immunization of sporozoites. Mice were immunized via either IV or ID route with Plasmodium berghei sporozoites in combination with chloroquine treatment (CPS) (allowing full liver stage development) or by γ-radiation-attenuated sporozoites (RAS) (early liver stage arrest). While IV immunization with both RAS and CPS generated 90-100% protection, ID immunization resulted in reduced levels of protection with either immunization strategy in both Balb/cByJ (50%) and C57BL/6j mice (7-13%). Lower protection by ID routing associated with a 30-fold lower parasite liver load P < 0.001 (χ(2) = 49.08, d.f. = 1) assessed by real-time in vivo imaging of bioluminescent P. berghei parasites. Unlike IV, ID immunization did not result in expansion of CD8+ T cells with effector memory phenotype and showed lower IFNγ responses irrespective of the immunization regime. In conclusion, protection against sporozoite infection is likely dependent on parasite liver infection and subsequently generated cellular immune responses.
Controlled human malaria infection with sporozoites is a standardized and powerful tool for evaluation of malaria vaccine and drug efficacy but so far only applied by exposure to bites of Plasmodium ...falciparum (Pf)-infected mosquitoes. We assessed in an open label Phase 1 trial, infection after intradermal injection of respectively 2,500, 10,000, or 25,000 aseptic, purified, vialed, cryopreserved Pf sporozoites (PfSPZ) in three groups (N = 6/group) of healthy Dutch volunteers. Infection was safe and parasitemia developed in 15 of 18 volunteers (84%), 5 of 6 volunteers in each group. There were no differences between groups in time until parasitemia by microscopy or quantitative polymerase chain reaction, parasite kinetics, clinical symptoms, or laboratory values. This is the first successful infection by needle and syringe with PfSPZ manufactured in compliance with regulatory standards. After further optimization, the use of such PfSPZ may facilitate and accelerate clinical development of novel malaria drugs and vaccines.
PDF
