Nonhuman Primate Liver Research Articles

Modulation of AAV9 Galactose Binding Yields Novel Gene Therapy Vectors and Predicts Cross-Species Differences in Glycan Avidity.

Effective use of adeno-associated viruses (AAVs) for clinical gene therapy is limited by their propensity to accumulate in and transduce the liver. This natural liver tropism is associated with severe adverse events at the high doses that can be necessary for achieving therapeutic transgene expression in extrahepatic tissues. To improve the safety and cost of AAV gene therapy, capsid engineering efforts are underway to redirect invivo AAV biodistribution away from the liver toward disease-relevant peripheral organs such as the heart. Building on previous work, we generated a series of AAV libraries containing variations at three residues (Y446, N470, and W503) of the galactose-binding pocket of the AAV9 VP1 protein. Screening of this library in mice identified the XRH family of variants (Y446X, N470R, and W503H), the strongest of which, HRH, exhibited a 6-fold reduction in liver RNA expression and a 10-fold increase in cardiac RNA expression compared with wild-type AAV9 in the mouse. Screening of our library in a nonhuman primate (NHP) revealed reduced performance of AAV9 and two closely related vectors in the NHP liver compared with the mouse liver. Measurement of the galactose-binding capacity of our library further identified those same three vectors as the only strong galactose binders, suggesting an altered galactose presentation between the mouse and NHP liver. N-glycan profiling of these tissues revealed a 9% decrease in exposed galactose in the NHP liver compared with the mouse liver. In this work, we identified a novel family of AAV variants with desirable biodistribution properties that may be suitable for targeting extrahepatic tissues such as the heart. These data also provide important insights regarding species- and tissue-specific differences in glycan presentation that may have implications for the development and translation of AAV gene therapies.

High-dose systemic adeno-associated virus vector administration causes liver and sinusoidal endothelial cell injury

We analyzed retrospective data from toxicology studies involving administration of high doses of adeno-associated virus expressing different therapeutic transgenes to 21 cynomolgus and 15 rhesus macaques. We also conducted prospective studies to investigate acute toxicity following high-dose systemic administration of enhanced green fluorescent protein-expressing adeno-associated virus to 10 rhesus macaques. Toxicity was characterized by transaminitis, thrombocytopenia, and alternative complement pathway activation that peaked on post-administration day 3. Although most animals recovered, some developed ascites, generalized edema, hyperbilirubinemia, and/or coagulopathy that prompted unscheduled euthanasia. Study endpoint livers from animals that recovered and from unscheduled necropsies of those that succumbed to toxicity were analyzed via hypothesis-driven histopathological and unbiased single-nucleus RNA sequencing. All liver cell types expressed high transgene transcript levels at early unscheduled timepoints that subsequently decreased. Thrombocytopenia coincided with sinusoidal platelet microthrombi and sinusoidal endothelial injury identified via immunohistology and single-nucleus RNA sequencing. Acute toxicity, sinusoidal injury, and liver platelet sequestration were similarly observed with therapeutic transgenes and enhanced green fluorescent protein at doses ≥1×1014 GC/kg, suggesting it was the consequence of high-dose systemic adeno-associated virus administration, not green fluorescent protein toxicity. These findings highlight a potential toxic effect of high-dose intravenous adeno-associated virus on nonhuman primate liver microvasculature.

Moderate maternal nutrient reduction in pregnancy alters fatty acid oxidation and RNA splicing in the nonhuman primate fetal liver.

Fetal liver tissue collected from a nonhuman primate (NHP) baboon model of maternal nutrient reduction (MNR) at four gestational time points (90, 120, 140, and 165 days gestation [dG], term in the baboon is ∼185 dG) was used to quantify MNR effects on the fetal liver transcriptome. 28 transcripts demonstrated different expression patterns between MNR and control livers during the second half of gestation, a developmental period when the fetus undergoes rapid weight gain and fat accumulation. Differentially expressed transcripts were enriched for fatty acid oxidation and RNA splicing-related pathways. Increased RNA splicing activity in MNR was reflected in greater abundances of transcript splice variant isoforms in the MNR group. It can be hypothesized that the increase in splice variants is deployed in an effort to adapt to the poor in utero environment and ensure near-normal development and energy metabolism. This study is the first to study developmental programming across four critical gestational stages during primate fetal liver development and reveals a potentially novel cellular response mechanism mediating fetal programming in response to MNR.

Effects of long-term simulated microgravity on liver metabolism in rhesus macaques.

The liver is an essential multifunctional organ and constantly communicates with nearly all the tissues in the body. Spaceflight or simulated microgravity has a significant impact on the livers of rodent models, including lipid accumulation and inflammatory cell infiltration. Whether similar liver lipotoxicity could occur in humans is not known, even though altered circulating cholesterol profile has been reported in astronauts. Using a 42-day head-down bed rest (HDBR) model in rhesus macaques, the present study investigated whether simulated microgravity alters the liver of non-human primates at the transcriptome and metabolome levels. Its association with stress and intestinal changes was also explored. Compared to the controls, the HDBR monkeys showed mild liver injury, elevated ANGPTL3 level in the plasma, and accumulation of fat vacuoles and inflammatory cells in the liver. Altered transcriptome signatures with up-regulation of genes in lipid metabolisms and down-regulation of genes in innate immune defense were also found in HDBR group-derived liver samples. The metabolic profiling of the liver revealed mildly disturbed fatty acid metabolism in the liver of HDBR monkeys. The intestinal dysbiosis, its associated endotoxemia and changes in the composition of bile acids, and elevated stress hormone in HDBR monkeys may contribute to the altered lipid metabolisms in the liver. These data indicate that liver metabolic functions and gut-liver axis should be closely monitored in prolonged spaceflight to facilitate strategy design to improve and maintain metabolic homeostasis.

Endogenous ADAR-mediated RNA editing in non-human primates using stereopure chemically modified oligonucleotides

Technologies that recruit and direct the activity of endogenous RNA-editing enzymes to specific cellular RNAs have therapeutic potential, but translating them from cell culture into animal models has been challenging. Here we describe short, chemically modified oligonucleotides called AIMers that direct efficient and specific A-to-I editing of endogenous transcripts by endogenous adenosine deaminases acting on RNA (ADAR) enzymes, including the ubiquitously and constitutively expressed ADAR1 p110 isoform. We show that fully chemically modified AIMers with chimeric backbones containing stereopure phosphorothioate and nitrogen-containing linkages based on phosphoryl guanidine enhanced potency and editing efficiency 100-fold compared with those with uniformly phosphorothioate-modified backbones in vitro. In vivo, AIMers targeted to hepatocytes with N-acetylgalactosamine achieve up to 50% editing with no bystander editing of the endogenous ACTB transcript in non-human primate liver, with editing persisting for at least one month. These results support further investigation of the therapeutic potential of stereopure AIMers.

Therapeutic application of genome editing in dyslipidemia.

To summarize recent advances with respect to the use of genome editing to modify blood lipid levels in vivo. Genome-editing technologies have been successfully used to target the PCSK9 gene in the livers of nonhuman primates and significantly reduce blood LDL cholesterol levels. Multiple proof-of-concept nonhuman primate studies raise the prospect of genome editing empowering 'one-and-done' therapies for the treatment of dyslipidemic patients.

Long-term stable reduction of low-density lipoprotein in nonhuman primates following in vivo genome editing of PCSK9

Gene disruption via programmable, sequence-specific nucleases represents a promising gene therapy strategy in which the reduction of specific protein levels provides a therapeutic benefit. Proprotein convertase subtilisin/kexin type 9 (PCSK9), an antagonist of the low-density lipoprotein (LDL) receptor, is a suitable target for nuclease-mediated gene disruption as an approach to treat hypercholesterolemia. We sought to determine the long-term durability and safety of PCSK9 knockdown in non-human primate (NHP) liver by adeno-associated virus (AAV)-delivered meganuclease following our initial report on the feasibility of this strategy. Six previously treated NHPs and additional NHPs administered AAV-meganuclease in combination with corticosteroid treatment or an alternative AAV serotype were monitored for a period of up to 3 years. The treated NHPs exhibited a sustained reduction in circulating PCSK9 and LDL cholesterol (LDL-c) through the course of the study concomitant with stable gene editing of the PCSK9 locus. Low-frequency off-target editing remained stable, and no obvious adverse changes in histopathology of the liver were detected. We demonstrate similar on-target nuclease activity in primary human hepatocytes using a chimeric liver-humanized mouse model. These studies demonstrate that targeted invivo gene disruption exerts a lasting therapeutic effect and provide pivotal data for safety considerations, which support clinical translation.

Absence of OATP1B (Organic Anion-Transporting Polypeptide) Induction by Rifampin in Cynomolgus Monkeys: Determination Using the Endogenous OATP1B Marker Coproporphyrin and Tissue Gene Expression.

Organic anion-transporting polypeptide (OATP) 1B induction is an evolving mechanism of drug disposition and interaction. However, there are contradictory reports describing OATP1B expression in hepatocytes and liver biopsies after administration of an inducer. This study investigated the in vivo effects of the common inducer rifampin (RIF) on the activity and expression of cynomolgus monkey OATP1B1 and OATP1B3 transporters, which are structurally and functionally similar their human OATP1B counterparts. Multiple doses of oral RIF (15 mg/kg) resulted in a steady 3.9-fold increase of CYP3A biomarker, 4β-hydroxycholesterol (4βHC), in the plasma samples collected before each RIF dose during the treatment period (i.e., predose). In contrast, the predose plasma levels of OATP1B biomarkers coproporphyrin (CP) I and CPIII did not change when compared with RIF treatment. The trough concentration, area under plasma concentration-time curve (AUC), and half-life of RIF decreased markedly during RIF treatment, suggesting that RIF induced its own clearance. Consequently, RIF treatment increased CPI and CPIII AUCs substantially after a single administration and, to a lesser extent, after multiple administrations compared with preadministration AUCs. In addition, OATP1B1 and OATP1B3 mRNA expressions were not modulated by RIF treatment (0.85-1.3-fold), whereas CYP3A8 expression was increased 3.7-5.0-fold, which correlated well with the predose levels of CP and 4βHC. Rifampin treatment showed 2.0-3.3-fold increases in P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2) expression in the small intestine. Collectively, these findings indicate that monkey OATP1B and OATP1B3 are not induced by RIF, and further investigation of OATP1B induction by RIF and other nuclear receptor activators in humans is warranted. SIGNIFICANCE STATEMENT: In this study, combined endogenous biomarker and gene expression data suggested that RIF did not induce OATP1B in cynomolgus monkeys. For the first time, the study determines transporter gene expression in the nonhuman primate liver, gut, and kidney tissues after administration of RIF for 7 days, leading to a better understanding of the induction of OATP1B and other major drug transporters. Finally, it provides evidence to strengthen the claim that coproporphyrin is a suitable endogenous probe of OATP1B activity.

Atlas-based liver segmentation for nonhuman primate research.

Certain viral infectious diseases cause systemic damage and the liver is an important organ affected directly by the virus and/or the hosts' response to the virus. Medical imaging indicates that the liver damage is heterogenous, and therefore, quantification of these changes requires analysis of the entire organ. Delineating the liver in preclinical imaging studies is a time-consuming and difficult task that would benefit from automated liver segmentation. A nonhuman primate atlas-based liver segmentation method was developed to support quantitative image analysis of preclinical research. A set of 82 computed tomography (CT) scans of nonhuman primates with associated manual contours delineating the liver was generated from normal and abnormal livers. The proposed technique uses rigid and deformable registrations, a majority vote algorithm, and image post-processing operations to automate the liver segmentation process. This technique was evaluated using Dice similarity, Hausdorff distance measures, and Bland-Altman plots. Automated segmentation results compare favorably with manual contouring, achieving a median Dice score of 0.91. Limits of agreement from Bland-Altman plots indicate that liver changes of 3 Hounsfield units (CT) and 0.4 SUVmean (positron emission tomography) are detectable using our automated method of segmentation, which are substantially less than changes observed in the host response to these viral infectious diseases. The proposed atlas-based liver segmentation technique is generalizable to various sizes and species of nonhuman primates and facilitates preclinical infectious disease research studies. While the image analysis software used is commercially available and facilities with funding can access the software to perform similar nonhuman primate liver quantitative analyses, the approach can be implemented in open-source frameworks as there is nothing proprietary about these methods.

A nonhuman primate model of liver fibrosis towards cell therapy for liver cirrhosis

Orthotopic liver transplantation (OLT) is the only curative treatment for refractory chronic liver failure in liver cirrhosis. However, the supply of donated livers does not meet the demand for OLT due to donor organ shortage. Cell therapy using hepatocyte-like cells derived from human induced pluripotent stem cells (hiPSC-HLCs) is expected to mitigate the severity of liver failure, postpone OLT and ameliorate the insufficient liver supply. For the successful clinical translation of hiPSC-based cell therapy against liver cirrhosis, realistic animal models are required. In this study, we created a nonhuman primate (NHP) liver fibrosis model by repeated administrations of thioacetamide (TAA) and evaluated the short-term engraftment of hiPSC-HLCs in the fibrotic liver. The NHP liver fibrosis model reproduced well the pathophysiology of human liver cirrhosis including portal hypertension. Under immunosuppressive treatment, we transplanted ALBUMIN-GFP reporter hiPSC-HLC aggregates into the fibrotic livers of the NHP model via the portal vein. Fourteen days after the transplantation, GFP-expressing hiPSC-HLC clusters were detected in the portal areas of the fibrotic livers. These results will facilitate preclinical studies using the NHP liver fibrosis model and help establish iPSC-based cell therapies against liver cirrhosis.

Primate fetal hepatic responses to maternal obesity: epigenetic signalling pathways and lipid accumulation.

Maternal obesity (MO) and exposure to a high-fat, high-simple-carbohydrate diet during pregnancy predisposes offspring to obesity, metabolic and cardiovascular disorders in later life. Underlying molecular pathways and potential epigenetic factors that are dysregulated in MO were identified using unbiased transcriptomic methods. There was increased lipid accumulation and severe steatosis in the MO baboon fetal liver suggesting that these offspring are on an early trajectory of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. Maternal obesity (MO) increases offspring cardiometabolic disease risk. Altered fetal liver development in response to the challenge of MO has metabolic consequences underlying adverse offspring life-course health outcomes. Little is known about the molecular pathways and potential epigenetic changes regulating primate fetal liver responses to MO. We hypothesized that MO would induce fetal baboon liver epigenetic changes resulting in dysregulation of key metabolic pathways that impact lipid metabolism. MO was induced prior to pregnancy by a high-fat, high-fructose diet. Unbiased gene and microRNA (small RNA Seq) abundance analyses were performed on fetal baboon livers at 0.9 gestation and subjected to pathway analyses to identify fetal liver molecular responses to MO. Fetal baboon liver lipid and glycogen content were quantified by the Computer Assisted Stereology Toolbox. In response to MO, fetal livers revealed dysregulation of TCA cycle, proteasome, oxidative phosphorylation, glycolysis and Wnt/β-catenin signalling pathways together with marked lipid accumulation supporting our hypothesis that multiple pathway dysregulation detrimentally impacts lipid management. This is the first study of MO programming of the non-human primate fetal liver using unbiased transcriptome analysis to detect changes in hepatic gene expression levels and identify potential microRNA epigenetic regulators of metabolic disruption.

The human longevity gene homolog INDY and interleukin-6 interact in hepatic lipid metabolism.

Targeting human mINDY may have therapeutic potential in obese patients with nonalcoholic fatty liver disease. German Clinical Trials Register: DRKS00005450. (Hepatology 2017;66:616-630).

Successful Repeated Hepatic Gene Delivery in Mice and Non-human Primates Achieved by Sequential Administration of AAV5ch and AAV1

In the gene therapy field, re-administration of adeno-associated virus (AAV) is an important topic because a decrease in therapeutic protein expression might occur over time. However, an efficient re-administration with the same AAV serotype is impossible due to serotype-specific, anti-AAV neutralizing antibodies (NABs) that are produced after initial AAV treatment. To address this issue, we explored the feasibility of using chimeric AAV serotype 5 (AAV5ch) and AAV1 for repeated liver-targeted gene delivery. To develop a relevant model, we immunized animals with a high dose of AAV5ch-humansecreted embryonic alkaline phosphatase (hSEAP) that generates high levels of anti-AAV5ch NAB. Secondary liver transduction with the same dose of AAV1-human factor IX (hFIX) in the presence of high levels of anti-AAV5ch NAB proved to be successful because expression/activity of both reporter transgenes was observed. This is the first time that two different transgenes are shown to be produced by non-human primate (NHP) liver after sequential administration of clinically relevant doses of both AAV5ch and AAV1. The levels of transgene proteins achieved after delivery with AAV5ch and AAV1 illustrate the possibility of both serotypes for liver targeting. Furthermore, transgene DNA and RNA biodistribution patterns provided insight into the potential cause of decrease or loss of transgene protein expression over time in NHPs.

Biokinetics of Plutonium in Nonhuman Primates

A major source of data on metabolism, excretion and retention of plutonium comes from experimental animal studies. Although old world monkeys are one of the closest living relatives to humans, certain physiological differences do exist between these nonhuman primates and humans. The objective of this paper was to describe the metabolism of plutonium in nonhuman primates using the bioassay and retention data obtained from macaque monkeys injected with plutonium citrate. A biokinetic model for nonhuman primates was developed by adapting the basic model structure and adapting the transfer rates described for metabolism of plutonium in adult humans. Significant changes to the parameters were necessary to explain the shorter retention of plutonium in liver and skeleton of the nonhuman primates, differences in liver to bone partitioning ratio, and significantly higher excretion of plutonium in feces compared to that in humans.

Inhibition of Glycolate Oxidase With Dicer-substrate siRNA Reduces Calcium Oxalate Deposition in a Mouse Model of Primary Hyperoxaluria Type 1

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive, metabolic disorder caused by mutations of alanine-glyoxylate aminotransferase (AGT), a key hepatic enzyme in the detoxification of glyoxylate arising from multiple normal metabolic pathways to glycine. Accumulation of glyoxylate, a precursor of oxalate, leads to the overproduction of oxalate in the liver, which accumulates to high levels in kidneys and urine. Crystalization of calcium oxalate (CaOx) in the kidney ultimately results in renal failure. Currently, the only treatment effective in reduction of oxalate production in patients who do not respond to high-dose vitamin B6 therapy is a combined liver/kidney transplant. We explored an alternative approach to prevent glyoxylate production using Dicer-substrate small interfering RNAs (DsiRNAs) targeting hydroxyacid oxidase 1 (HAO1) mRNA which encodes glycolate oxidase (GO), to reduce the hepatic conversion of glycolate to glyoxylate. This approach efficiently reduces GO mRNA and protein in the livers of mice and nonhuman primates. Reduction of hepatic GO leads to normalization of urine oxalate levels and reduces CaOx deposition in a preclinical mouse model of PH1. Our results support the use of DsiRNA to reduce liver GO levels as a potential therapeutic approach to treat PH1.

Efficient and Targeted Transduction of Nonhuman Primate Liver With Systemically Delivered Optimized AAV3B Vectors

Recombinant adeno-associated virus serotype 3B (rAAV3B) can transduce cultured human liver cancer cells and primary human hepatocytes efficiently. Serine (S)- and threonine (T)-directed capsid modifications further augment its transduction efficiency. Systemically delivered capsid-optimized rAAV3B vectors can specifically target cancer cells in a human liver cancer xenograft model, suggesting their potential use for human liver-directed gene therapy. Here, we compared transduction efficiencies of AAV3B and AAV8 vectors in cultured primary human hepatocytes and cancer cells as well as in human and mouse hepatocytes in a human liver xenograft NSG-PiZ mouse model. We also examined the safety and transduction efficacy of wild-type (WT) and capsid-optimized rAAV3B in the livers of nonhuman primates (NHPs). Intravenously delivered S663V+T492V (ST)-modified self-complementary (sc) AAV3B-EGFP vectors led to liver-targeted robust enhanced green fluorescence protein (EGFP) expression in NHPs without apparent hepatotoxicity. Intravenous injections of both WT and ST-modified rAAV3B.ST-rhCG vectors also generated stable super-physiological levels of rhesus chorionic gonadotropin (rhCG) in NHPs. The vector genome predominantly targeted the liver. Clinical chemistry and histopathology examinations showed no apparent vector-related toxicity. Our studies should be important and informative for clinical development of optimized AAV3B vectors for human liver-directed gene therapy.

240. Efficient and Targeted Transduction of Nonhuman Primate Liver With Optimized AAV3B Vectors Through Systemic Delivery

240. Efficient and Targeted Transduction of Nonhuman Primate Liver With Optimized AAV3B Vectors Through Systemic Delivery

Post mortem histological artifacts created by poor tissue handling during necropsy

Histological artifacts may be introduced into the tissue of laboratory animals during sample collection at the time of necropsy or during tissue processing. To identify and characterize tissue artifacts, modifications in necropsy/tissue processing standard operating procedures were made in order to reproduce some of these effects in the lungs, thyroid glands, and stomach of rats; the liver, spleen, and kidney of dogs; and the liver and spleen of non-human primates. Induced artifacts included lungs over-infused with formalin, stomachs subjected to mechanical damage, and thyroid glands roughly handled with forceps. Oversized sections of liver, spleen, and kidney tissues were forced into cassettes. All tissues were preserved for 24 hours in formalin before trimming. These tissues were processed, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin (H&E). Tissues were then examined microscopically by a pathologist and compared with control tissues collected at necropsy under standard conditions. The evaluation confirmed that artifacts such as those induced in this study can interfere with accurate histologic examination, ultimately confounding pathology interpretation. These results emphasize the importance of rigorous necropsy and tissue collection practices to ensure a consistently high quality of tissue specimens.

Perinatal Exposure to a High-Fat Diet Is Associated with Reduced Hepatic Sympathetic Innervation in One-Year Old Male Japanese Macaques

Our group recently demonstrated that maternal high-fat diet (HFD) consumption is associated with non-alcoholic fatty liver disease, increased apoptosis, and changes in gluconeogenic gene expression and chromatin structure in fetal nonhuman primate (NHP) liver. However, little is known about the long-term effects that a HFD has on hepatic nervous system development in offspring, a system that plays an important role in regulating hepatic metabolism. Utilizing immunohistochemistry and Real-Time PCR, we quantified sympathetic nerve fiber density, apoptosis, inflammation, and other autonomic components in the livers of fetal and one-year old Japanese macaques chronically exposed to a HFD. We found that HFD exposure in-utero and throughout the postnatal period (HFD/HFD), when compared to animals receiving a CTR diet for the same developmental period (CTR/CTR), is associated with a 1.7 fold decrease in periportal sympathetic innervation, a 5 fold decrease in parenchymal sympathetic innervation, and a 2.5 fold increase in hepatic apoptosis in the livers of one-year old male animals. Additionally, we observed an increase in hepatic inflammation and a decrease in a key component of the cholinergic anti-inflammatory pathway in one-year old HFD/HFD offspring. Taken together, these findings reinforce the impact that continuous exposure to a HFD has in the development of long-term hepatic pathologies in offspring and highlights a potential neuroanatomical basis for hepatic metabolic dysfunction.

Adaptive clinical trials of three PfSPZ products for development of a whole sporozoite vaccine that prevents Plasmodium falciparum infection, disease and transmission

An ideal, single stage vaccine useful for elimination of Plasmodium falciparum (Pf) would prevent infection at the pre-erythrocytic stage of the parasite life cycle, thereby preventing all Pf-caused disease and transmission from humans to mosquitoes. The only approach to immunization shown to consistently induce greater than 90% protection against infection and protection sustained for at least 10-28 months has been immunization by mosquito bite with whole Pf sporozoites (SPZ) of two types. The first type, radiation-attenuated PfSPZ, invade hepatoctyes and expresses new proteins, but cannot replicate. The second type fully develop in hepatocytes, producing tens of thousands of merozoites that invade erythrocytes, but are unable to fully develop within erythrocytes because they are killed by an antimalarial drug. This approach called chemoprophylaxis with sporozoites (CPS) harnesses the infectious agent’s inherent replicative properties to amplify production of protective immunogens spanning multiple developmental stages, and then eliminates the infectious agent with an anti-infective drug before the onset of disease. Sanaria was founded to develop PfSPZ vaccines. The first vaccine developed and tested was the PfSPZ Vaccine. The PfSPZ Vaccine is comprised of aseptic, purified, radiation attenuated, cryopreserved PfSPZ. It was shown to be safe and well-tolerated when administered ID or SC to 80 volunteers in the U.S., but sub-optimally immunogenic. It is now being tested in the U.S. and soon in Tanzania when administered by IV injection, since it induced high levels of PfSPZ-specific CD8+ T cells in the livers of immunized non-human primates when administered IV. A second product, PfSPZ Challenge, is comprised of non-irradiated, fully infectious PfSPZ. PfSPZ Challenge has been shown to infect 100% of volunteers after ID or IM administration by needle and syringe. It has been or will be tested for optimization of adminstration by the ID, IM, and IV routes in 2012 or early 2013 in the Netherlands, UK, Tanzania, U.S., Germany, Spain, and Kenya. A third product, PfSPZ-CVac, is comprised of PfSPZ Challenge administered to volunteers receiving chloroquine chemoprophylaxis. It will be assessed in 2012-2013 in the Netherlands, Mali, Germany and Tanzania. Assessment of these three products in synergistic, interactive and adaptive clinical trials will facilitate progress toward optimizing administration and dosage regimen of all three whole PfSPZ products, as well as those developed in the future from genetically altered parasites, thereby facilitating licensure of one or more PfSPZ-based vaccines. Progress and plans for development will be discussed.