Laura B. Martin

Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum

BACKGROUNDnThe ability of T cells, acting independently of antibodies, to control malaria parasite growth in people has not been defined. If such was shown to be effective, an additional vaccine strategy could be pursued. Our aim was to ascertain whether or not development of cell-mediated immunity to Plasmodium falciparum blood-stage infection could be induced in human beings by exposure to malaria parasites in very low density.nnnMETHODSnWe enrolled five volunteers from the staff at our research institute who had never had malaria. We used a cryopreserved inoculum of red cells infected with P falciparum strain 3D7 to give them repeated subclinical infections of malaria that we then cured early with drugs, to induce cell-mediated immune responses. We tested for development of immunity by measurement of parasite concentrations in the blood of volunteers by PCR of the multicopy gene STEVOR and by following up the volunteers clinically, and by measuring antibody and cellular immune responses to the parasite.nnnFINDINGSnAfter challenge and a extended period without drug cure, volunteers were protected against malaria as indicated by absence of parasites or parasite DNA in the blood, and absence of clinical symptoms. Immunity was characterised by absence of detectable antibodies that bind the parasite or infected red cells, but by the presence of a proliferative T-cell response, involving CD4+ and CD8+ T cells, a cytokine response, consisting of interferon gamma but not interleukin 4 or interleukin 10, induction of high concentrations of nitric oxide synthase activity in peripheral blood mononuclear cells, and a drop in the number of peripheral natural killer T cells.nnnINTERPRETATIONnPeople can be protected against the erythrocytic stage of malaria by a strong cell-mediated immune response, in the absence of detectable parasite-specific antibodies, suggesting an additional strategy for development of a malaria vaccine

Vesicle-associated Membrane Protein 2 Plays a Specific Role in the Insulin-dependent Trafficking of the Facilitative Glucose Transporter GLUT4 in 3T3-L1 Adipocytes

Vesicle-associated membrane protein 2 (VAMP2) has been implicated in the insulin-regulated trafficking of GLUT4 in adipocytes. It has been proposed that VAMP2 co-localizes with GLUT4 in a postendocytic storage compartment (Martin, S., Tellam, J., Livingstone, C., Slot, J. W., Gould, G. W., and James, D. E. (1996) J. Cell Biol. 134, 625–635), suggesting that it may play a role distinct from endosomal v-SNAREs (solubleN-ethylmaleimide-sensitive factor attachment protein receptors) such as cellubrevin that are also expressed in adipocytes. The present study examines the effects of recombinant glutathioneS-transferase (GST) fusion proteins encompassing the entire cytoplasmic tails of VAMP1, VAMP2, and cellubrevin on insulin-stimulated GLUT4 translocation in streptolysin O permeabilized 3T3-L1 adipocytes. GST-VAMP2 inhibited insulin-stimulated GLUT4 translocation by ∼35%, whereas GST-VAMP1 and GST-cellubrevin were without effect. A synthetic peptide corresponding to the unique N terminus of VAMP2 also inhibited insulin-stimulated GLUT4 translocation in a dose-dependent manner. This peptide had no effect on either guanosine 5′-3-O-(thio)triphosphate-stimulated GLUT4 translocation or on insulin-stimulated GLUT1 translocation. These results imply that GLUT4 and GLUT1 may undergo insulin-stimulated translocation to the cell surface from separate intracellular compartments. To confirm this, adipocytes were incubated with a transferrin-horseradish peroxidase conjugate to fill the itinerant endocytic system after which cells were incubated with H2O2 and diaminobenzidine. This treatment completely blocked insulin-stimulated movement of GLUT1, whereas in the case of GLUT4, movement to the surface was delayed but still reached similar levels to that observed in insulin-stimulated control cells after 30 min. These results suggest that the N terminus of VAMP2 plays a unique role in the insulin-dependent recruitment of GLUT4 from its intracellular storage compartment to the cell surface.

Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Substantial evidence exists to suggest that in the basal state GLUT4 resides in discrete storage vesicles. A direct interaction of GLUT4 storage vesicles with the plasma membrane has been implicated because the v-SNARE, vesicle-associated membrane protein-2 (VAMP2), appears to be a specific component of these vesicles. In the present study we sought to identify the cognate target SNAREs for VAMP2 in mouse 3T3-L1 adipocytes. Membrane fractions were isolated from adipocytes and probed by far Western blotting with the cytosolic portion of VAMP2 fused to glutathione S-transferase. Two plasma membrane-enriched proteins, p25 and p35, were specifically labeled with this probe. By using a combination of immunoblotting, detergent extraction, and anion exchange chromatography, we identified p35 as Syntaxin-4 and p25 as the recently identified murine SNAP-25 homologue, Syndet (mSNAP-23). By using surface plasmon resonance we show that VAMP2, Syntaxin-4, and Syndet form a ternary SDS-resistant SNARE complex. Microinjection of anti-Syndet antibodies into 3T3-L1 adipocytes, or incubation of permeabilized adipocytes with a synthetic peptide comprising the C-terminal 24 amino acids of Syndet, inhibited insulin-stimulated GLUT4 translocation to the cell surface by ∼40%. GLUT1 trafficking remained unaffected by the presence of the peptide. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin.

Impact on Malaria Parasite Multiplication Rates in Infected Volunteers of the Protein-in-Adjuvant Vaccine AMA1-C1/Alhydrogel+CPG 7909

Background Inhibition of parasite growth is a major objective of blood-stage malaria vaccines. The in vitro assay of parasite growth inhibitory activity (GIA) is widely used as a surrogate marker for malaria vaccine efficacy in the down-selection of candidate blood-stage vaccines. Here we report the first study to examine the relationship between in vivo Plasmodium falciparum growth rates and in vitro GIA in humans experimentally infected with blood-stage malaria. Methods In this phase I/IIa open-label clinical trial five healthy malaria-naive volunteers were immunised with AMA1/C1-Alhydrogel+CPG 7909, and together with three unvaccinated controls were challenged by intravenous inoculation of P. falciparum infected erythrocytes. Results A significant correlation was observed between parasite multiplication rate in 48 hours (PMR) and both vaccine-induced growth-inhibitory activity (Pearson ru200a=u200a−0.93 [95% CI: −1.0, −0.27] Pu200a=u200a0.02) and AMA1 antibody titres in the vaccine group (Pearson ru200a=u200a−0.93 [95% CI: −0.99, −0.25] Pu200a=u200a0.02). However immunisation failed to reduce overall mean PMR in the vaccine group in comparison to the controls (vaccinee 16 fold [95% CI: 12, 22], control 17 fold [CI: 0, 65] Pu200a=u200a0.70). Therefore no impact on pre-patent period was observed (vaccine group median 8.5 days [range 7.5–9], control group median 9 days [range 7–9]). Conclusions Despite the first observation in human experimental malaria infection of a significant association between vaccine-induced in vitro growth inhibitory activity and in vivo parasite multiplication rate, this did not translate into any observable clinically relevant vaccine effect in this small group of volunteers. Trial Registration ClinicalTrials.gov [NCT00984763]

Anti-Apical-Membrane-Antigen-1 Antibody Is More Effective than Anti-42-Kilodalton-Merozoite-Surface-Protein-1 Antibody in Inhibiting Plasmodium falciparum Growth, as Determined by the In Vitro Growth Inhibition Assay

ABSTRACT Apical membrane antigen 1 (AMA1) and the 42-kDa merozoite surface protein 1 (MSP142) are leading malaria vaccine candidates. Several preclinical and clinical trials have been conducted, and an in vitro parasite growth inhibition assay has been used to evaluate the biological activities of the resulting antibodies. In a U.S. phase 1 trial with AMA1-C1/Alhydrogel plus CPG 7909, the vaccination elicited anti-AMA1 immunoglobulin G (IgG) which showed up to 96% inhibition. However, antibodies induced by MSP142-C1/Alhydrogel plus CPG 7909 vaccine showed less than 32% inhibition in vitro. To determine whether anti-MSP142 IgG had less growth-inhibitory activity than anti-AMA1 IgG in vitro, the amounts of IgG that produced 50% inhibition of parasite growth (Ab50) were compared for rabbit and human antibodies. The Ab50s of rabbit and human anti-MSP142 IgGs were significantly higher (0.21 and 0.62 mg/ml, respectively) than those of anti-AMA1 IgGs (0.07 and 0.10 mg/ml, respectively) against 3D7 parasites. Ab50 data against FVO parasites also demonstrated significant differences. We further investigated the Ab50s of mouse and monkey anti-AMA1 IgGs and showed that there were significant differences between the species (mouse, 0.28 mg/ml, and monkey, 0.14 mg/ml, against 3D7 parasites). Although it is unknown whether growth-inhibitory activity in vitro reflects protective immunity in vivo, this study showed that the Ab50 varies with both antigen and species. Our data provide a benchmark for antibody levels for future AMA1- or MSP142-based vaccine development efforts in preclinical and clinical trials.

Evolution of Resistance to Sulfadoxine-Pyrimethamine in Plasmodium falciparum

ABSTRACT The development of resistance to sulfadoxine-pyrimethamine by Plasmodium parasites is a major problem for the effective treatment of malaria, especially P. falciparum malaria. Although the molecular basis for parasite resistance is known, the factors promoting the development and transmission of these resistant parasites are less clear. This paper reports the results of a quantitative comparison of factors previously hypothesized as important for the development of drug resistance, drug dosage, time of treatment, and drug elimination half-life, with an in-host dynamics model of P. falciparum malaria in a malaria-naïve host. The results indicate that the development of drug resistance can be categorized into three stages. The first is the selection of existing parasites with genetic mutations in the dihydrofolate reductase or dihydropteroate synthetase gene. This selection is driven by the long half-life of the sulfadoxine-pyrimethamine combination. The second stage involves the selection of parasites with allelic types of higher resistance within the host during an infection. The timing of treatment relative to initiation of a specific anti-P. falciparum EMP1 immune response is an important factor during this stage, as is the treatment dosage. During the third stage, clinical treatment failure becomes prevalent as the parasites develop sufficient resistance mutations to survive therapeutic doses of the drug combination. Therefore, the model output reaffirms the importance of correct treatment of confirmed malaria cases in slowing the development of parasite resistance to sulfadoxine-pyrimethamine.

Origin and Dissemination of Chloroquine-Resistant Plasmodium falciparum with Mutant pfcrt Alleles in the Philippines

ABSTRACT The pfcrt allelic type and adjacent microsatellite marker type were determined for 82 Plasmodium falciparum isolates from the Philippines. Mutant pfcrt allelic types P1a and P2a/P2b were dominant in different locations. Microsatellite analysis revealed that P2a/P2b evolved independently in the Philippines, while P1a shared common ancestry with Papua New Guinea chloroquine-resistant parasites.

Analysis of immunological nonresponsiveness to the 19-kilodalton fragment of merozoite surface Protein 1 of Plasmodium yoelii: rescue by chemical conjugation to diphtheria toxoid (DT) and enhancement of immunogenicity by prior DT vaccination.

ABSTRACT The Plasmodium merozoite surface protein 1 (MSP1) is a leading vaccine candidate for protecting against the blood stage of malaria. Previous studies have shown that the 19-kDa carboxyl terminus of this protein is able to induce protective immunity in some monkey and mouse strains. We show that immunization with the recombinant Plasmodium yoelii 19-kDa fragment of MSP1 (MSP119) expressed in Saccharomyces cerevisiae (yMSP119) can induce protective antibodies in several inbred mouse strains and one outbred mouse strain. However, mice expressing the H-2s major histocompatibility complex haplotype are unable to generate yMSP119-specific antibodies. While synthetic peptides derived from MSP119 are immunogenic in B10.S mice, they cannot function as helper epitopes, and immunization with yMSP119 does not induce T cells that recognize the recombinant protein or synthetic peptides corresponding to its sequence. Nonresponsiveness could be overcome by using chemical linkers to conjugate yMSP119 to diphtheria toxoid (DT), resulting in immunogens capable of inducing protective yMSP119-specific antibodies in both MSP119-responsive and otherwise nonresponsive mouse strains. The ability of sera from mice immunized with the conjugate to inhibit binding of a protective monoclonal antibody (MAb 302) to yMSP119 correlated strongly with a delay in the prepatent period. Chemical conjugation of yMSP119 to DT may be a preferred method to enhance immunogenicity, as carrier priming experiments demonstrated that an existing immune response to DT enhanced a subsequent antibody response to yMSP119 after vaccination with yMSP119-DT. These results have important implications for the development of a malaria vaccine to protect a population with diverse HLAs.

Mutational analysis of the carboxy-terminal phosphorylation site of GLUT-4 in 3T3-L1 adipocytes

The carboxy terminus of GLUT-4 contains a functional internalization motif (Leu-489Leu-490) that helps maintain its intracellular distribution in basal adipocytes. This motif is flanked by the major phosphorylation site in this protein (Ser-488), which may play a role in regulating GLUT-4 trafficking in adipocytes. In the present study, the targeting of GLUT-4 in which Ser-488 has been mutated to alanine (SAG) has been examined in stably transfected 3T3-L1 adipocytes. The trafficking of SAG was not significantly different from that of GLUT-4 in several respects. First, in the absence of insulin, the distribution of SAG was similar to GLUT-4 in that it was largely excluded from the cell surface and was enriched in small intracellular vesicles. Second, SAG exhibited insulin-dependent movement to the plasma membrane (4- to 5-fold) comparable to GLUT-4 (4- to 5-fold). Finally, okadaic acid, which has previously been shown to stimulate both GLUT-4 translocation and its phosphorylation at Ser-488, also stimulated the movement of SAG to the cell surface similarly to GLUT-4. Using immunoelectron microscopy, we have shown that GLUT-4 is localized to intracellular vesicles containing the Golgi-derived gamma-adaptin subunit of AP-1 and that this localization is enhanced when Ser-488 is mutated to alanine. We conclude that the carboxy-terminal phosphorylation site in GLUT-4 (Ser-488) may play a role in intracellular sorting at the trans-Golgi network but does not play a major role in the regulated movement of GLUT-4 to the plasma membrane in 3T3-L1 adipocytes.The carboxy terminus of GLUT-4 contains a functional internalization motif (Leu-489Leu-490) that helps maintain its intracellular distribution in basal adipocytes. This motif is flanked by the major phosphorylation site in this protein (Ser-488), which may play a role in regulating GLUT-4 trafficking in adipocytes. In the present study, the targeting of GLUT-4 in which Ser-488 has been mutated to alanine (SAG) has been examined in stably transfected 3T3-L1 adipocytes. The trafficking of SAG was not significantly different from that of GLUT-4 in several respects. First, in the absence of insulin, the distribution of SAG was similar to GLUT-4 in that it was largely excluded from the cell surface and was enriched in small intracellular vesicles. Second, SAG exhibited insulin-dependent movement to the plasma membrane (4- to 5-fold) comparable to GLUT-4 (4- to 5-fold). Finally, okadaic acid, which has previously been shown to stimulate both GLUT-4 translocation and its phosphorylation at Ser-488, also stimulated the movement of SAG to the cell surface similarly to GLUT-4. Using immunoelectron microscopy, we have shown that GLUT-4 is localized to intracellular vesicles containing the Golgi-derived γ-adaptin subunit of AP-1 and that this localization is enhanced when Ser-488 is mutated to alanine. We conclude that the carboxy-terminal phosphorylation site in GLUT-4 (Ser-488) may play a role in intracellular sorting at the trans-Golgi network but does not play a major role in the regulated movement of GLUT-4 to the plasma membrane in 3T3-L1 adipocytes.

Nontyphoidal salmonella disease: Current status of vaccine research and development.

Among more than 2500 nontyphoidal Salmonella enterica (NTS) serovars, S. enterica serovar Typhimurium and S. enterica serovar Enteritidis account for approximately fifty percent of all human isolates of NTS reported globally. The global incidence of NTS gastroenteritis in 2010 was estimated to be 93 million cases, approximately 80 million of which were contracted via food-borne transmission. It is estimated that 155,000 deaths resulted from NTS in 2010. NTS also causes severe, extra-intestinal, invasive bacteremia, referred to as invasive nontyphoidal Salmonella (iNTS) disease. iNTS disease usually presents as a febrile illness, frequently without gastrointestinal symptoms, in both adults and children. Symptoms of iNTS are similar to malaria, often including fever (>90%) and splenomegaly (>40%). The underlying reasons for the high rates of iNTS disease in Africa are still being elucidated. Evidence from animal and human studies supports the feasibility of developing a safe and effective vaccine against iNTS. Both antibodies and complement can kill Salmonella species in vitro. Proof-of-principle studies in animal models have demonstrated efficacy for live attenuated and subunit vaccines that target the O-antigens, flagellin proteins, and other outer membrane proteins of serovars Typhimurium and Enteritidis. More recently, a novel delivery strategy for NTS vaccines has been developed: the Generalized Modules for Membrane Antigens (GMMA) technology which presents surface polysaccharides and outer membrane proteins in their native conformation. GMMA technology is self-adjuvanting, as it delivers multiple pathogen-associated molecular pattern molecules. GMMA may be particularly relevant for low- and middle-income countries as it has the potential for high immunologic potency at a low cost and involves a relatively simple production process without the need for complex conjugation. Several vaccines for the predominant NTS serovars Typhimurium and Enteritidis, are currently under development.