Abraham Eappen - Academia.edu (original) (raw)

Papers by Abraham Eappen

BACKGROUND Vaccines and new drugs are needed to prevent nearly one million deaths and hundreds of... more BACKGROUND Vaccines and new drugs are needed to prevent nearly one million deaths and hundreds of millions of cases caused by Plasmodium falciparum (Pf) malaria annually. To accelerate development of these vaccines and drugs, volunteers are infected by the bites of Pf sporozoite (SPZ)-infected mosquitoes under controlled conditions. Such infections are limited to the few centers with production of PfSPZ-infected mosquitoes. We assessed the capacity to infect volunteers by intradermal (ID) injection of aseptic, purified, vialed, cryopreserved PfSPZ (PfSPZ Challenge), produced by Sanaria Inc.

Nature Biotechnology, 2000

Figure 2. (A) Activity of plasmin in a P1-Lys positional-scanning synthetic combinatorial library... more Figure 2. (A) Activity of plasmin in a P1-Lys positional-scanning synthetic combinatorial library. (B) Activity of thrombin in a P1-Lys positional-scanning synthetic combinatorial library. y-axis indicates picomoles of fluorophore released per second. x-axis indicates the amino acid held constant at each position, designated by the one-letter code (with n representing norleucine). On p. 188 of the February 2000 issue, "Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin" by Bradley J. Backes, Jennifer L. Harris, Francesco Leonetti, Charles S. Craik, and Jonathan A. Ellman, Figure 2 was incorrectly displayed. Here we reprint the figure in its entirety: On p. 1000 of the October 1999 issue, "Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides" by

蚕糸 昆虫農業技術研究所研究資料, Mar 1, 1999

Scientific Reports, 2022

The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human i... more The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human infectious diseases by conserving stocks, new isolates, lab-bred strains, and transgenic lines that currently require continuous life cycle maintenance. Efforts over several decades to develop a method for cryopreservation have, until now, been fruitless: we describe here a method for the cryopreservation of Anopheles stephensi embryos yielding hatch rates of ~ 25%, stable for > 5 years. Hatched larvae developed into fertile, fecund adults and blood-fed females, produced fully viable second generation eggs, that could be infected with Plasmodium falciparum at high intensities. The key components of the cryopreservation method are: embryos at 15–30 min post oviposition, two incubation steps in 100% deuterated methanol at − 7 °C and − 14.5 °C, and rapid cooling. Eggs are recovered by rapid warming with concomitant dilution of cryoprotectant. Eggs of genetically modified A. stephensi and ...

PLOS Pathogens, 2021

PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufac... more PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line, Δaslrim1, by embryonic genome editing using CRISPR-Cas9. Δaslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of Δaslrim1 mosquitoes and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to basel...

The Journal of Infectious Diseases, 2019

Direct venous inoculation of 3.2 × 103 aseptic, purified, cryopreserved, vialed Plasmodium falcip... more Direct venous inoculation of 3.2 × 103 aseptic, purified, cryopreserved, vialed Plasmodium falciparum (Pf) strain NF54 sporozoites, PfSPZ Challenge (NF54), has been used for controlled human malaria infection (CHMI) in the United States, 4 European countries, and 6 African countries. In nonimmune adults, this results in 100% infection rates. We conducted a double-blind, randomized, dose-escalation study to assess the infectivity of the 7G8 clone of Pf (PfSPZ Challenge [7G8]). Results showed dose-dependent infectivity from 43% for 8 × 102 PfSPZ to 100% for 4.8 × 103 PfSPZ. PfSPZ Challenge (7G8) will allow for more complete assessment by CHMI of antimalarial vaccines and drugs.

Malaria Journal, 2019

Background: Saglin, a 100 kDa protein composed of two 50 kDa homodimers, is present in the saliva... more Background: Saglin, a 100 kDa protein composed of two 50 kDa homodimers, is present in the salivary glands of Anopheles gambiae and has been considered an essential receptor for sporozoites (SPZ) of Plasmodium berghei and Plasmodium falciparum (Pf), allowing SPZ to recognize, bind to, and infect mosquito salivary glands. Spatial and temporal patterns of Saglin expression reported here, however, suggest that this model does not fully describe the Saglin-SPZ interaction. Results: Saglin protein was detected by indirect immunofluorescence microscopy only in the medial and proximallateral lobes, but not in the distal-lateral lobes, of the salivary glands of An. gambiae; the pattern of expression was independent of mosquito age or physiological state. These results were confirmed by steady-state Saglin transcript and protein expression using qRT-PCR and Western-blot analysis, respectively. Saglin was localized to the basal surface of the cells of the medial lobes and was undetectable elsewhere (intracellularly, on the lateral or apical membranes, the cells' secretory vacuoles, or in the salivary duct). In the cells of the proximal lateral lobes of the salivary glands, Saglin was distinctly intracellular and was not localized to any of the cell surfaces. Transgenic Anopheles stephensi were produced that expressed An. gambiae Saglin in the distal lateral lobes of the salivary gland. Additional Saglin expression did not enhance infection by PfSPZ compared to non-transgenic siblings fed on the same gametocyte-containing blood meal. Conclusions: The absence of Saglin in the distal lateral lobes of the salivary glands, a primary destination for SPZ, suggests Saglin is not an essential receptor for Plasmodium SPZ. The lack of any correlation between increased Saglin expression in the distal lateral lobes of the salivary glands of transgenic An. stephensi and PfSPZ infection is also consistent with Saglin not being an essential salivary gland receptor for Plasmodium SPZ.

Proceedings of the National Academy of Sciences of the United States of America, Mar 21, 2017

A live-attenuated malaria vaccine, Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine), conf... more A live-attenuated malaria vaccine, Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine), confers sterile protection against controlled human malaria infection (CHMI) with Plasmodium falciparum (Pf) parasites homologous to the vaccine strain up to 14 mo after final vaccination. No injectable malaria vaccine has demonstrated long-term protection against CHMI using Pf parasites heterologous to the vaccine strain. Here, we conducted an open-label trial with PfSPZ Vaccine at a dose of 9.0 × 10(5) PfSPZ administered i.v. three times at 8-wk intervals to 15 malaria-naive adults. After CHMI with homologous Pf parasites 19 wk after final immunization, nine (64%) of 14 (95% CI, 35-87%) vaccinated volunteers remained without parasitemia compared with none of six nonvaccinated controls (P = 0.012). Of the nine nonparasitemic subjects, six underwent repeat CHMI with heterologous Pf7G8 parasites 33 wk after final immunization. Five (83%) of six (95% CI, 36-99%) remained without parasitemia co...

Nature, Feb 15, 2017

A highly protective malaria vaccine would greatly facilitate the prevention and elimination of ma... more A highly protective malaria vaccine would greatly facilitate the prevention and elimination of malaria and containment of drug-resistant parasites. A high level (more than 90%) of protection against malaria in humans has previously been achieved only by immunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (PfSPZ) inoculated by mosquitoes; by intravenous injection of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ ('PfSPZ Vaccine'); or by infectious PfSPZ inoculated by mosquitoes to volunteers taking chloroquine or mefloquine (chemoprophylaxis with sporozoites). We assessed immunization by direct venous inoculation of aseptic, purified, cryopreserved, non-irradiated PfSPZ ('PfSPZ Challenge') to malaria-naive, healthy adult volunteers taking chloroquine for antimalarial chemoprophylaxis (vaccine approach denoted as PfSPZ-CVac). Three doses of 5.12 × 10(4) PfSPZ of PfSPZ Challenge at 28-day intervals were well tolerated and safe, an...

Nature Medicine, 2016

for the VRC 312 and VRC 314 Study Teams An attenuated Plasmodium falciparum (Pf) sporozoite (SPZ)... more for the VRC 312 and VRC 314 Study Teams An attenuated Plasmodium falciparum (Pf) sporozoite (SPZ) vaccine, PfSPZ Vaccine, is highly protective against controlled human malaria infection (CHMI) 3 weeks after immunization, but the durability of protection is unknown. We assessed how vaccine dosage, regimen, and route of administration affected durable protection in malaria-naive adults. After four intravenous immunizations with 2.7 × 10 5 PfSPZ, 6/11 (55%) vaccinated subjects remained without parasitemia following CHMI 21 weeks after immunization. Five non-parasitemic subjects from this dosage group underwent repeat CHMI at 59 weeks, and none developed parasitemia. Although Pf-specific serum antibody levels correlated with protection up to 21-25 weeks after immunization, antibody levels waned substantially by 59 weeks. Pf-specific T cell responses also declined in blood by 59 weeks. To determine whether T cell responses in blood reflected responses in liver, we vaccinated nonhuman primates with PfSPZ Vaccine. Pf-specific interferon-g-producing CD8 T cells were present at ~100-fold higher frequencies in liver than in blood. Our findings suggest that PfSPZ Vaccine conferred durable protection to malaria through long-lived tissue-resident T cells and that administration of higher doses may further enhance protection.

Entomological Research, 2007

Insects are responsible for the transmission of major infectious diseases. Recent advances in ins... more Insects are responsible for the transmission of major infectious diseases. Recent advances in insect genomics and transformation technology provide new strategies for the control of insect borne pathogen transmission and insect pest management. One such strategy is the genetic modification of insects with genes that block pathogen development. Another is to suppress insect populations by releasing either sterile males or males carrying female-specific dominant lethal genes into the environment. Although significant progress has been made in the laboratory, further research is needed to extend these approaches to the field. These insect control strategies offer several advantages over conventional insecticide-based strategies. However, the release of genetically modified insects into the environment should proceed with great caution, after ensuring its safety, and acceptance by the target populations.

This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or c... more This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles

PLoS pathogens, 2012

The mosquito is the obligate vector for malaria transmission. To complete its development within ... more The mosquito is the obligate vector for malaria transmission. To complete its development within the mosquito, the malaria parasite Plasmodium must overcome the protective action of the mosquito innate immune system. Here we report on the involvement of the Anopheles gambiae orthologue of a conserved component of the vertebrate immune system, LPS-induced TNFα transcription factor (LITAF), and its role in mosquito anti-Plasmodium immunity. An. gambiae LITAF-like 3 (LL3) expression is up-regulated in response to midgut invasion by both rodent and human malaria parasites. Silencing of LL3 expression greatly increases parasite survival, indicating that LL3 is part of an anti-Plasmodium defense mechanism. Electrophoretic mobility shift assays identified specific LL3 DNA-binding motifs within the promoter of SRPN6, a gene that also mediates mosquito defense against Plasmodium. Further experiments indicated that these motifs play a direct role in LL3 regulation of SRPN6 expression. We conc...

Proceedings of the National Academy of Sciences, 2005

We have functionally analyzed the orthologous SRPN6 genes from Anopheles stephensi and Anopheles ... more We have functionally analyzed the orthologous SRPN6 genes from Anopheles stephensi and Anopheles gambiae using phylogenetic, molecular, reverse genetic, and cell biological tools. The results strongly implicate SRPN6 in the innate immune response against Plasmodium . This gene belongs to a mosquito-specific gene cluster including three additional Anopheles serpins. SRPN6 expression is induced by Escherichia coli and both rodent and human malaria parasites. The gene is specifically expressed in midgut cells invaded by Plasmodium ookinetes and in circulating and attached hemocytes. Knockdown of SRPN6 expression by RNA interference in susceptible An. stephensi leads to substantially increased parasite numbers, whereas depletion in susceptible An. gambiae delays progression of parasite lysis without affecting the number of developing parasites. However, the An. gambiae SRPN6 knockdown increases the number of melanized parasites in the L3-5 refractory strain and in susceptible G3 mosquit...

Proceedings of the National Academy of Sciences, 2011

Clinical studies and mathematical models predict that, to achieve malaria elimination, combinatio... more Clinical studies and mathematical models predict that, to achieve malaria elimination, combination therapies will need to incorporate drugs that block the transmission of Plasmodium falciparum sexual stage parasites to mosquito vectors. Efforts to measure the activity of existing antimalarials on intraerythrocytic sexual stage gametocytes and identify transmission-blocking agents have, until now, been hindered by a lack of quantitative assays. Here, we report an experimental system using P. falciparum lines that stably express gametocyte-specific GFP-luciferase reporters, which enable the assessment of dose- and time-dependent drug action on gametocyte maturation and transmission. These studies reveal activity of the first-line antimalarial dihydroartemisinin and the partner drugs lumefantrine and pyronaridine against early gametocyte stages, along with moderate inhibition of mature gametocyte transmission to Anopheles mosquitoes. The other partner agents monodesethyl-amodiaquine an...

PLoS ONE, 2013

Controlled human malaria infection (CHMI) is a powerful method for assessing the efficacy of anti... more Controlled human malaria infection (CHMI) is a powerful method for assessing the efficacy of anti-malaria vaccines and drugs targeting pre-erythrocytic and erythrocytic stages of the parasite. CHMI has heretofore required the bites of 5 Plasmodium falciparum (Pf) sporozoite (SPZ)-infected mosquitoes to reliably induce Pf malaria. We reported that CHMI using the bites of 3 PfSPZ-infected mosquitoes reared aseptically in compliance with current good manufacturing practices (cGMP) was successful in 6 participants. Here, we report results from a subsequent CHMI study using 3 PfSPZ-infected mosquitoes reared aseptically to validate the initial clinical trial. We also compare results of safety, tolerability, and transmission dynamics in participants undergoing CHMI using 3 PfSPZ-infected mosquitoes reared aseptically to published studies of CHMI using 5 mosquitoes. Nineteen adults aged 18-40 years were bitten by 3 Anopheles stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of Pf. All 19 participants developed malaria (100%); 12 of 19 (63%) on Day 11. The mean pre-patent period was 258.3 hours (range 210.5-333.8). The geometric mean parasitemia at first diagnosis by microscopy was 9.5 parasites/mL (range 2-44). Quantitative polymerase chain reaction (qPCR) detected parasites an average of 79.8 hours (range 43.8-116.7) before microscopy. The mosquitoes had a geometric mean of 37,894 PfSPZ/ mosquito (range 3,500-152,200). Exposure to the bites of 3 aseptically-raised, PfSPZ-infected mosquitoes is a safe, effective procedure for CHMI in malaria-naïve adults. The aseptic model should be considered as a new standard for CHMI trials in non-endemic areas. Microscopy is the gold standard used for the diagnosis of Pf malaria after CHMI, but qPCR identifies parasites earlier. If qPCR continues to be shown to be highly specific, and can be made to be practical, rapid, and standardized, it should be considered as an alternative for diagnosis.

PLoS ONE, 2013

Successful development of Plasmodium in the mosquito is essential for the transmission of malaria... more Successful development of Plasmodium in the mosquito is essential for the transmission of malaria. A major bottleneck in parasite numbers occurs during midgut invasion, partly as a consequence of the complex interactions between the endogenous microbiota and the mosquito immune response. We previously identified SRPN6 as an immune component which restricts Plasmodium berghei development in the mosquito. Here we demonstrate that SRPN6 is differentially activated by bacteria in Anopheles stephensi, but only when bacteria exposure occurs on the lumenal surface of the midgut epithelium. Our data indicate that AsSRPN6 is strongly induced following exposure to Enterobacter cloacae, a common component of the mosquito midgut microbiota. We conclude that AsSRPN6 is a vital component of the E. cloacae-mediated immune response that restricts Plasmodium development in the mosquito An. stephensi.

Molecular and General Genetics MGG, 2000

In the silkworm, Bombyx mori, many eye-and egg-colour mutations aecting the synthesis and accumul... more In the silkworm, Bombyx mori, many eye-and egg-colour mutations aecting the synthesis and accumulation of ommochrome pigments have been described. In order to understand the pigment precursor transporters involved, ABC transporter genes homologous to the Drosophila white gene were isolated from the silkworm. Reverse transcriptase-polymerase chain reactions (RT-PCR) using embryonic mRNA ampli®ed three cDNA fragments, named Bmwh1, Bmwh2 and Bmwh3 that showed homology to the white gene. Since Bmwh3 shows the highest degree of sequence identity and a similar expression pattern to the Drosophila homologue, we characterised this gene further. A 2667-bp Bmwh3 cDNA isolated from an embryonic library has one ORF encoding a polypeptide of 687 amino acids. The predicted protein has one ATP-binding domain, six transmembrane-spanning segments and high similarity to the Drosophila WHITE protein. Southern analysis indicates that Bmwh3 is a single-copy gene. Polyadenylated Bmwh3 transcripts about 2.7 kb long were detected in eggs, Malpighian tubules and pupal heads, but not in testes, posterior silk glands or fat body cells. The level of Bmwh3 mRNA was reduced in w3 and w3 ol mutants but normal in other egg-and eye-colour mutants, suggesting that Bmwh3 correspond to the w3 locus. Genetic analysis was used to map the cloned gene to chromosome 10.

BACKGROUND Vaccines and new drugs are needed to prevent nearly one million deaths and hundreds of... more BACKGROUND Vaccines and new drugs are needed to prevent nearly one million deaths and hundreds of millions of cases caused by Plasmodium falciparum (Pf) malaria annually. To accelerate development of these vaccines and drugs, volunteers are infected by the bites of Pf sporozoite (SPZ)-infected mosquitoes under controlled conditions. Such infections are limited to the few centers with production of PfSPZ-infected mosquitoes. We assessed the capacity to infect volunteers by intradermal (ID) injection of aseptic, purified, vialed, cryopreserved PfSPZ (PfSPZ Challenge), produced by Sanaria Inc.

Nature Biotechnology, 2000

Figure 2. (A) Activity of plasmin in a P1-Lys positional-scanning synthetic combinatorial library... more Figure 2. (A) Activity of plasmin in a P1-Lys positional-scanning synthetic combinatorial library. (B) Activity of thrombin in a P1-Lys positional-scanning synthetic combinatorial library. y-axis indicates picomoles of fluorophore released per second. x-axis indicates the amino acid held constant at each position, designated by the one-letter code (with n representing norleucine). On p. 188 of the February 2000 issue, "Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin" by Bradley J. Backes, Jennifer L. Harris, Francesco Leonetti, Charles S. Craik, and Jonathan A. Ellman, Figure 2 was incorrectly displayed. Here we reprint the figure in its entirety: On p. 1000 of the October 1999 issue, "Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides" by

蚕糸 昆虫農業技術研究所研究資料, Mar 1, 1999

Scientific Reports, 2022

The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human i... more The ability to cryopreserve mosquitoes would revolutionize work on these vectors of major human infectious diseases by conserving stocks, new isolates, lab-bred strains, and transgenic lines that currently require continuous life cycle maintenance. Efforts over several decades to develop a method for cryopreservation have, until now, been fruitless: we describe here a method for the cryopreservation of Anopheles stephensi embryos yielding hatch rates of ~ 25%, stable for > 5 years. Hatched larvae developed into fertile, fecund adults and blood-fed females, produced fully viable second generation eggs, that could be infected with Plasmodium falciparum at high intensities. The key components of the cryopreservation method are: embryos at 15–30 min post oviposition, two incubation steps in 100% deuterated methanol at − 7 °C and − 14.5 °C, and rapid cooling. Eggs are recovered by rapid warming with concomitant dilution of cryoprotectant. Eggs of genetically modified A. stephensi and ...

PLOS Pathogens, 2021

PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufac... more PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line, Δaslrim1, by embryonic genome editing using CRISPR-Cas9. Δaslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of Δaslrim1 mosquitoes and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to basel...

The Journal of Infectious Diseases, 2019

Direct venous inoculation of 3.2 × 103 aseptic, purified, cryopreserved, vialed Plasmodium falcip... more Direct venous inoculation of 3.2 × 103 aseptic, purified, cryopreserved, vialed Plasmodium falciparum (Pf) strain NF54 sporozoites, PfSPZ Challenge (NF54), has been used for controlled human malaria infection (CHMI) in the United States, 4 European countries, and 6 African countries. In nonimmune adults, this results in 100% infection rates. We conducted a double-blind, randomized, dose-escalation study to assess the infectivity of the 7G8 clone of Pf (PfSPZ Challenge [7G8]). Results showed dose-dependent infectivity from 43% for 8 × 102 PfSPZ to 100% for 4.8 × 103 PfSPZ. PfSPZ Challenge (7G8) will allow for more complete assessment by CHMI of antimalarial vaccines and drugs.

Malaria Journal, 2019

Background: Saglin, a 100 kDa protein composed of two 50 kDa homodimers, is present in the saliva... more Background: Saglin, a 100 kDa protein composed of two 50 kDa homodimers, is present in the salivary glands of Anopheles gambiae and has been considered an essential receptor for sporozoites (SPZ) of Plasmodium berghei and Plasmodium falciparum (Pf), allowing SPZ to recognize, bind to, and infect mosquito salivary glands. Spatial and temporal patterns of Saglin expression reported here, however, suggest that this model does not fully describe the Saglin-SPZ interaction. Results: Saglin protein was detected by indirect immunofluorescence microscopy only in the medial and proximallateral lobes, but not in the distal-lateral lobes, of the salivary glands of An. gambiae; the pattern of expression was independent of mosquito age or physiological state. These results were confirmed by steady-state Saglin transcript and protein expression using qRT-PCR and Western-blot analysis, respectively. Saglin was localized to the basal surface of the cells of the medial lobes and was undetectable elsewhere (intracellularly, on the lateral or apical membranes, the cells' secretory vacuoles, or in the salivary duct). In the cells of the proximal lateral lobes of the salivary glands, Saglin was distinctly intracellular and was not localized to any of the cell surfaces. Transgenic Anopheles stephensi were produced that expressed An. gambiae Saglin in the distal lateral lobes of the salivary gland. Additional Saglin expression did not enhance infection by PfSPZ compared to non-transgenic siblings fed on the same gametocyte-containing blood meal. Conclusions: The absence of Saglin in the distal lateral lobes of the salivary glands, a primary destination for SPZ, suggests Saglin is not an essential receptor for Plasmodium SPZ. The lack of any correlation between increased Saglin expression in the distal lateral lobes of the salivary glands of transgenic An. stephensi and PfSPZ infection is also consistent with Saglin not being an essential salivary gland receptor for Plasmodium SPZ.

Proceedings of the National Academy of Sciences of the United States of America, Mar 21, 2017

A live-attenuated malaria vaccine, Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine), conf... more A live-attenuated malaria vaccine, Plasmodium falciparum sporozoite vaccine (PfSPZ Vaccine), confers sterile protection against controlled human malaria infection (CHMI) with Plasmodium falciparum (Pf) parasites homologous to the vaccine strain up to 14 mo after final vaccination. No injectable malaria vaccine has demonstrated long-term protection against CHMI using Pf parasites heterologous to the vaccine strain. Here, we conducted an open-label trial with PfSPZ Vaccine at a dose of 9.0 × 10(5) PfSPZ administered i.v. three times at 8-wk intervals to 15 malaria-naive adults. After CHMI with homologous Pf parasites 19 wk after final immunization, nine (64%) of 14 (95% CI, 35-87%) vaccinated volunteers remained without parasitemia compared with none of six nonvaccinated controls (P = 0.012). Of the nine nonparasitemic subjects, six underwent repeat CHMI with heterologous Pf7G8 parasites 33 wk after final immunization. Five (83%) of six (95% CI, 36-99%) remained without parasitemia co...

Nature, Feb 15, 2017

A highly protective malaria vaccine would greatly facilitate the prevention and elimination of ma... more A highly protective malaria vaccine would greatly facilitate the prevention and elimination of malaria and containment of drug-resistant parasites. A high level (more than 90%) of protection against malaria in humans has previously been achieved only by immunization with radiation-attenuated Plasmodium falciparum (Pf) sporozoites (PfSPZ) inoculated by mosquitoes; by intravenous injection of aseptic, purified, radiation-attenuated, cryopreserved PfSPZ ('PfSPZ Vaccine'); or by infectious PfSPZ inoculated by mosquitoes to volunteers taking chloroquine or mefloquine (chemoprophylaxis with sporozoites). We assessed immunization by direct venous inoculation of aseptic, purified, cryopreserved, non-irradiated PfSPZ ('PfSPZ Challenge') to malaria-naive, healthy adult volunteers taking chloroquine for antimalarial chemoprophylaxis (vaccine approach denoted as PfSPZ-CVac). Three doses of 5.12 × 10(4) PfSPZ of PfSPZ Challenge at 28-day intervals were well tolerated and safe, an...

Nature Medicine, 2016

for the VRC 312 and VRC 314 Study Teams An attenuated Plasmodium falciparum (Pf) sporozoite (SPZ)... more for the VRC 312 and VRC 314 Study Teams An attenuated Plasmodium falciparum (Pf) sporozoite (SPZ) vaccine, PfSPZ Vaccine, is highly protective against controlled human malaria infection (CHMI) 3 weeks after immunization, but the durability of protection is unknown. We assessed how vaccine dosage, regimen, and route of administration affected durable protection in malaria-naive adults. After four intravenous immunizations with 2.7 × 10 5 PfSPZ, 6/11 (55%) vaccinated subjects remained without parasitemia following CHMI 21 weeks after immunization. Five non-parasitemic subjects from this dosage group underwent repeat CHMI at 59 weeks, and none developed parasitemia. Although Pf-specific serum antibody levels correlated with protection up to 21-25 weeks after immunization, antibody levels waned substantially by 59 weeks. Pf-specific T cell responses also declined in blood by 59 weeks. To determine whether T cell responses in blood reflected responses in liver, we vaccinated nonhuman primates with PfSPZ Vaccine. Pf-specific interferon-g-producing CD8 T cells were present at ~100-fold higher frequencies in liver than in blood. Our findings suggest that PfSPZ Vaccine conferred durable protection to malaria through long-lived tissue-resident T cells and that administration of higher doses may further enhance protection.

Entomological Research, 2007

Insects are responsible for the transmission of major infectious diseases. Recent advances in ins... more Insects are responsible for the transmission of major infectious diseases. Recent advances in insect genomics and transformation technology provide new strategies for the control of insect borne pathogen transmission and insect pest management. One such strategy is the genetic modification of insects with genes that block pathogen development. Another is to suppress insect populations by releasing either sterile males or males carrying female-specific dominant lethal genes into the environment. Although significant progress has been made in the laboratory, further research is needed to extend these approaches to the field. These insect control strategies offer several advantages over conventional insecticide-based strategies. However, the release of genetically modified insects into the environment should proceed with great caution, after ensuring its safety, and acceptance by the target populations.

This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or c... more This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles

PLoS pathogens, 2012

The mosquito is the obligate vector for malaria transmission. To complete its development within ... more The mosquito is the obligate vector for malaria transmission. To complete its development within the mosquito, the malaria parasite Plasmodium must overcome the protective action of the mosquito innate immune system. Here we report on the involvement of the Anopheles gambiae orthologue of a conserved component of the vertebrate immune system, LPS-induced TNFα transcription factor (LITAF), and its role in mosquito anti-Plasmodium immunity. An. gambiae LITAF-like 3 (LL3) expression is up-regulated in response to midgut invasion by both rodent and human malaria parasites. Silencing of LL3 expression greatly increases parasite survival, indicating that LL3 is part of an anti-Plasmodium defense mechanism. Electrophoretic mobility shift assays identified specific LL3 DNA-binding motifs within the promoter of SRPN6, a gene that also mediates mosquito defense against Plasmodium. Further experiments indicated that these motifs play a direct role in LL3 regulation of SRPN6 expression. We conc...

Proceedings of the National Academy of Sciences, 2005

We have functionally analyzed the orthologous SRPN6 genes from Anopheles stephensi and Anopheles ... more We have functionally analyzed the orthologous SRPN6 genes from Anopheles stephensi and Anopheles gambiae using phylogenetic, molecular, reverse genetic, and cell biological tools. The results strongly implicate SRPN6 in the innate immune response against Plasmodium . This gene belongs to a mosquito-specific gene cluster including three additional Anopheles serpins. SRPN6 expression is induced by Escherichia coli and both rodent and human malaria parasites. The gene is specifically expressed in midgut cells invaded by Plasmodium ookinetes and in circulating and attached hemocytes. Knockdown of SRPN6 expression by RNA interference in susceptible An. stephensi leads to substantially increased parasite numbers, whereas depletion in susceptible An. gambiae delays progression of parasite lysis without affecting the number of developing parasites. However, the An. gambiae SRPN6 knockdown increases the number of melanized parasites in the L3-5 refractory strain and in susceptible G3 mosquit...

Proceedings of the National Academy of Sciences, 2011

Clinical studies and mathematical models predict that, to achieve malaria elimination, combinatio... more Clinical studies and mathematical models predict that, to achieve malaria elimination, combination therapies will need to incorporate drugs that block the transmission of Plasmodium falciparum sexual stage parasites to mosquito vectors. Efforts to measure the activity of existing antimalarials on intraerythrocytic sexual stage gametocytes and identify transmission-blocking agents have, until now, been hindered by a lack of quantitative assays. Here, we report an experimental system using P. falciparum lines that stably express gametocyte-specific GFP-luciferase reporters, which enable the assessment of dose- and time-dependent drug action on gametocyte maturation and transmission. These studies reveal activity of the first-line antimalarial dihydroartemisinin and the partner drugs lumefantrine and pyronaridine against early gametocyte stages, along with moderate inhibition of mature gametocyte transmission to Anopheles mosquitoes. The other partner agents monodesethyl-amodiaquine an...

PLoS ONE, 2013

Controlled human malaria infection (CHMI) is a powerful method for assessing the efficacy of anti... more Controlled human malaria infection (CHMI) is a powerful method for assessing the efficacy of anti-malaria vaccines and drugs targeting pre-erythrocytic and erythrocytic stages of the parasite. CHMI has heretofore required the bites of 5 Plasmodium falciparum (Pf) sporozoite (SPZ)-infected mosquitoes to reliably induce Pf malaria. We reported that CHMI using the bites of 3 PfSPZ-infected mosquitoes reared aseptically in compliance with current good manufacturing practices (cGMP) was successful in 6 participants. Here, we report results from a subsequent CHMI study using 3 PfSPZ-infected mosquitoes reared aseptically to validate the initial clinical trial. We also compare results of safety, tolerability, and transmission dynamics in participants undergoing CHMI using 3 PfSPZ-infected mosquitoes reared aseptically to published studies of CHMI using 5 mosquitoes. Nineteen adults aged 18-40 years were bitten by 3 Anopheles stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of Pf. All 19 participants developed malaria (100%); 12 of 19 (63%) on Day 11. The mean pre-patent period was 258.3 hours (range 210.5-333.8). The geometric mean parasitemia at first diagnosis by microscopy was 9.5 parasites/mL (range 2-44). Quantitative polymerase chain reaction (qPCR) detected parasites an average of 79.8 hours (range 43.8-116.7) before microscopy. The mosquitoes had a geometric mean of 37,894 PfSPZ/ mosquito (range 3,500-152,200). Exposure to the bites of 3 aseptically-raised, PfSPZ-infected mosquitoes is a safe, effective procedure for CHMI in malaria-naïve adults. The aseptic model should be considered as a new standard for CHMI trials in non-endemic areas. Microscopy is the gold standard used for the diagnosis of Pf malaria after CHMI, but qPCR identifies parasites earlier. If qPCR continues to be shown to be highly specific, and can be made to be practical, rapid, and standardized, it should be considered as an alternative for diagnosis.

PLoS ONE, 2013

Successful development of Plasmodium in the mosquito is essential for the transmission of malaria... more Successful development of Plasmodium in the mosquito is essential for the transmission of malaria. A major bottleneck in parasite numbers occurs during midgut invasion, partly as a consequence of the complex interactions between the endogenous microbiota and the mosquito immune response. We previously identified SRPN6 as an immune component which restricts Plasmodium berghei development in the mosquito. Here we demonstrate that SRPN6 is differentially activated by bacteria in Anopheles stephensi, but only when bacteria exposure occurs on the lumenal surface of the midgut epithelium. Our data indicate that AsSRPN6 is strongly induced following exposure to Enterobacter cloacae, a common component of the mosquito midgut microbiota. We conclude that AsSRPN6 is a vital component of the E. cloacae-mediated immune response that restricts Plasmodium development in the mosquito An. stephensi.

Molecular and General Genetics MGG, 2000

In the silkworm, Bombyx mori, many eye-and egg-colour mutations aecting the synthesis and accumul... more In the silkworm, Bombyx mori, many eye-and egg-colour mutations aecting the synthesis and accumulation of ommochrome pigments have been described. In order to understand the pigment precursor transporters involved, ABC transporter genes homologous to the Drosophila white gene were isolated from the silkworm. Reverse transcriptase-polymerase chain reactions (RT-PCR) using embryonic mRNA ampli®ed three cDNA fragments, named Bmwh1, Bmwh2 and Bmwh3 that showed homology to the white gene. Since Bmwh3 shows the highest degree of sequence identity and a similar expression pattern to the Drosophila homologue, we characterised this gene further. A 2667-bp Bmwh3 cDNA isolated from an embryonic library has one ORF encoding a polypeptide of 687 amino acids. The predicted protein has one ATP-binding domain, six transmembrane-spanning segments and high similarity to the Drosophila WHITE protein. Southern analysis indicates that Bmwh3 is a single-copy gene. Polyadenylated Bmwh3 transcripts about 2.7 kb long were detected in eggs, Malpighian tubules and pupal heads, but not in testes, posterior silk glands or fat body cells. The level of Bmwh3 mRNA was reduced in w3 and w3 ol mutants but normal in other egg-and eye-colour mutants, suggesting that Bmwh3 correspond to the w3 locus. Genetic analysis was used to map the cloned gene to chromosome 10.