Satish Mishra

Self-assembled peptide nanofibers raising durable antibody responses against a malaria epitope.

Biomaterials that modulate innate and adaptive immune responses are receiving increasing interest as adjuvants for eliciting protective immunity against a variety of diseases. Previous results have indicated that self-assembling β-sheet peptides, when fused with short peptide epitopes, can act as effective adjuvants and elicit robust and long-lived antibody responses. Here we investigated the mechanism of immunogenicity and the quality of antibody responses raised by a peptide epitope from Plasmodium falciparum circumsporozoite (CS) protein, (NANP)(3),conjugated to the self-assembling peptide domain Q11. The mechanism of adjuvant action was investigated in knockout mice with impaired MyD88, NALP3, TLR-2, or TLR-5 function, and the quality of antibodies raised against (NANP)(3)-Q11 was assessed using a transgenic sporozoite neutralizing (TSN) assay for malaria infection. (NANP)(3)-Q11 self-assembled into nanofibers, and antibody responses lasted up to 40 weeks in C57BL/6 mice. The antibody responses were T cell- and MyD88-dependent. Sera from mice primed with either irradiated sporozoites or a synthetic peptide, (T1BT*)(4)-P3C, and boosted with (NANP)(3)-Q11 showed significant increases in antibody titers and significant inhibition of sporozoite infection in TSN assays. In addition, two different epitopes could be self-assembled together without compromising the strength or duration of the antibody responses raised against either of them, making these materials promising platforms for self-adjuvanting multi-antigenic immunotherapies.

PK4, a eukaryotic initiation factor 2α(eIF2α) kinase, is essential for the development of the erythrocytic cycle of Plasmodium

In response to environmental stresses, the mammalian serine threonine kinases PERK, GCN2, HRI, and PKR phosphorylate the regulatory serine 51 of the eukaryotic translation initiation factor 2α (eIF2α) to inhibit global protein synthesis. Plasmodium, the protozoan that causes malaria, expresses three eIF2α kinases: IK1, IK2, and PK4. Like GCN2, IK1 regulates stress response to amino acid starvation. IK2 inhibits development of malaria sporozoites present in the mosquito salivary glands. Here we show that the phosphorylation by PK4 of the regulatory serine 59 of Plasmodium eIF2α is essential for the completion of the parasites erythrocytic cycle that causes disease in humans. PK4 activity leads to the arrest of global protein synthesis in schizonts, where ontogeny of daughter merozoites takes place, and in gametocytes that infect Anopheles mosquitoes. The implication of these findings is that drugs that reduce PK4 activity should alleviate disease and inhibit malaria transmission.

L-Arginine stimulates intestinal intraepithelial lymphocyte functions and immune response in chickens orally immunized with live intermediate plus strain of infectious bursal disease vaccine.

Abstract Infectious bursal disease (IBD) continues to pose potential threat to poultry industry all over the world. The disease can spell disaster not only through its infection but also by break of immunity in chickens vaccinated for other diseases. l-Arginine, a ubiquitous, semi-essential amino acid has emerged as an imunostimulant from variety of human and animal studies. In the present study, we demonstrate the stimulatory effects of l-arginine on intestinal intraepithelial lymphocyte (iIELs) functions as well as on systemic immune response in chickens orally vaccinated with live intermediate plus (IP) strain of IBD vaccine. Challenge studies with virulent IBDV revealed complete (100%) protection in IP+ l-arginine group compared with 80% protection recorded in IP strain vaccinated chickens. Functional activities of iIELs evaluated by cytotoxicity assay demonstrated significantly high percentage cytotoxicity in IP+ l-arginine groups compared with IP group (P <0.05). Proliferative response of iIELs against IBDV antigen and Con-A was also significantly higher in IP+ l-arginine group. Similar results were obtained with peripheral blood mononuclear cell blastogenic response to IBDV and Con-A analyzed as an indicator of systemic cell-mediated immune response. Orally administered IP strain vaccine elicited good antibody titres in both the groups, IP and IP+ l-arginine, however, the antibody titres were significantly higher in IP+ l-arginine group compared with IP vaccinated group (P <0.05). These results clearly demonstrate that l-arginine stimulates intestinal and systemic immune response against IBDV.

A bicomponent Plasmodium falciparum investigational vaccine composed of protein-peptide conjugates

There is yet no licensed vaccine against malaria, a serious human disease affecting mostly children, with an annual death rate of about one million. Plasmodia, the malaria-causing parasites, have two obligatory hosts: mammals or birds, in which they multiply asexually, and mosquitoes with sexual multiplication. The most common and serious type of malaria is caused by Plasmodium falciparum. The circumsporozoite protein (CSP), a major surface antigen of sporozoites, is a protective antigen. A unique feature of P. falciparum CSP is its large central domain composed of over 30 tetrapeptide repeats of Asn-Ala-Asn-Pro (NANP). Several NANP peptide-protein conjugates were tested clinically but elicited a low level of CSP antibodies for a short duration. To provide a CSP-based candidate vaccine, we investigated recombinant CSP and NANP conjugates of various peptide lengths, with different N-terminal amino acids, bound at different ratios to various carrier proteins. Injected into mice, CSP alone and CSP or NANP conjugates induced antibodies with booster responses and were positive by the sporozoite imunofluorescent assay. The use of the mosquito stage P. falciparum ookinete surface protein, Pfs25, cross-linked onto itself as a carrier for NANP, induced in mice high levels of uniquely long-lasting antibodies to both vaccine components with secondary biological activities, that will provide immunity to liver infection by sporozoites and block transmission by mosquitoes.

A host GPCR signaling network required for the cytolysis of infected cells facilitates release of apicomplexan parasites.

Following intracellular replication, the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii cause host cell cytolysis to facilitate parasite release and disease progression. Parasite exit from infected cells requires the interplay of parasite-derived proteins and host actin cytoskeletal changes; however, the host proteins underlying these changes remain obscure. We report the identification of a Gα(q)-coupled host-signaling cascade required for the egress of both P. falciparum and T. gondii. Gα(q)-coupled signaling results in protein kinase C (PKC)-mediated loss of the host cytoskeletal protein adducin and weakening of the cellular cytoskeleton. This cytoskeletal compromise induces catastrophic Ca(2+) influx mediated by the mechanosensitive cation channel TRPC6, which activates host calpain that proteolyzes the host cytoskeleton allowing parasite release. Reinforcing the feasibility of targeting host proteins as an antiparasitic strategy, mammalian PKC inhibitors demonstrated activity in murine models of malaria and toxoplasmosis. Importantly, an orally bioavailable PKC inhibitor prolonged survival in an experimental cerebral malaria model.

Identification of non-CSP antigens bearing CD8 epitopes in mice immunized with irradiated sporozoites

Immunization of BALB/c mice with irradiated sporozoites (IrSp) of Plasmodium yoelii can lead to sterile immunity. The circumsporozoite protein (CSP) plays a dominant role in protection. Nevertheless after hyper-immunization with IrSp, complete protection is obtained in CSP-transgenic BALB/c mice that are T-cell tolerant to the CSP and cannot produce antibodies [CSP-Tg/JhT(-/-)]. This protection is mediated exclusively by CD8(+) T cells [1]. To identify the non-CSP protective T cell antigens, we studied the properties of 34 P. yoelii sporozoite antigens that are predicted to be secreted and to contain strong Kd-restricted CD8(+) T cell epitopes. The synthetic peptides corresponding to the epitopes were used to screen for the presence of peptide-specific CD8(+) T cells secreting interferon-γ (IFN-γ) in splenocytes from CSP-Tg/JhT(-/-) BALB/c mice hyper immunized with IrSp. However, the numbers of IFN-γ-secreting splenocytes specific for the non-CSP antigen-derived peptides were 20-100 times lower than those specific for the CSP-specific peptide. When mice were immunized with recombinant adenoviruses expressing selected non-CSP antigens, the animals were not protected against challenge with P. yoelii sporozoites although large numbers of CD8(+) specific T cells were generated.

Antibodies to Plasmodium circumsporozoite protein (CSP) inhibit sporozoite's cell traversal activity.

Plasmodium sporozoites are deposited in the skin of the mammalian host by Anopheles mosquitoes. To continue the life cycle, the sporozoites have to invade the hosts hepatocytes, where they transform into exoerythrocytic forms (EEFs) inside a parasitophorous vacuole. During their route from the skin to the liver, the parasites traverse the capillary epithelium in the dermis to enter the blood circulation, and cross the endothelium of liver sinusoids to enter the parenchyma. Cell traversal by sporozoites is usually measured by quantifying dyes that enter or are released from cells during incubation with salivary gland sporozoites. These methods do not distinguish cell traversal from cell wounding. Here we validate an assay that quantifies cell traversal of sporozoites through monolayers of MDCK cells that form tight junctions. We compared cell traversal of wt sporozoites and of parasites lacking the Type I membrane protein TLP (TRAP-like protein) previously implicated in cell traversal. We provide direct evidence that TLP ko sporozoites are defective in cell traversal and that they are retained inside the MDCK cytoplasm. We then used the MDCK assay to study the effect of a monoclonal antibody (3D11) to the circumsporozoite protein (CSP) on the parasites cell traversal. We show that 3D11 inhibits cell traversal at nanomolar concentrations. We conclude that antibodies elicited by CSP-based vaccines are likely to inhibit the migration of sporozoites from the skin to the liver.

UIS2: A Unique Phosphatase Required for the Development of Plasmodium Liver Stages

Plasmodium salivary sporozoites are the infectious form of the malaria parasite and are dormant inside salivary glands of Anopheles mosquitoes. During dormancy, protein translation is inhibited by the kinase UIS1 that phosphorylates serine 59 in the eukaryotic initiation factor 2α (eIF2α). De-phosphorylation of eIF2α-P is required for the transformation of sporozoites into the liver stage. In mammalian cells, the de-phosphorylation of eIF2α-P is mediated by the protein phosphatase 1 (PP1). Using a series of genetically knockout parasites we showed that in malaria sporozoites, contrary to mammalian cells, the eIF2α-P phosphatase is a member of the PP2C/PPM phosphatase family termed UIS2. We found that eIF2α was highly phosphorylated in uis2 conditional knockout sporozoites. These mutant sporozoites maintained the crescent shape after delivery into mammalian host and lost their infectivity. Both uis1 and uis2 were highly transcribed in the salivary gland sporozoites but uis2 expression was inhibited by the Pumilio protein Puf2. The repression of uis2 expression was alleviated when sporozoites developed into liver stage. While most eukaryotic phosphatases interact transiently with their substrates, UIS2 stably bound to phosphorylated eIF2α, raising the possibility that high-throughput searches may identify chemicals that disrupt this interaction and prevent malaria infection.

Improved intervention of atherosclerosis and cardiac hypertrophy through biodegradable polymer-encapsulated delivery of glycosphingolipid inhibitor

D-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glycosphingolipid synthesis inhibitor, holds promise for the treatment of atherosclerosis and cardiac hypertrophy but rapid in vivo clearance has severely hindered translation to the clinic. To overcome this impediment, we used a materials-based delivery strategy wherein D-PDMP was encapsulated within a biodegradable polymer composed of poly ethylene glycol (PEG) and sebacic acid (SA). PEG-SA was formulated into nanoparticles that were doped with (125)I-labeled PEG to allow in vivo bio-distribution and release kinetics of D-PDMP to be determined by using γ-scintigraphy and subsequently, by mass spectrometry. Polymer-encapsulation increased the residence time of D-PDMP in the body of a treated mouse from less than one hour to at least four hours (and up to 48 h or longer). This substantially increased in vivo longevity provided by polymer encapsulation resulted in an order of magnitude gain in efficacy for interfering with atherosclerosis and cardiac hypertrophy in apoE-/- mice fed a high fat and high cholesterol (HFHC) diet. These results establish that D-PDMP encapsulated in a biodegradable polymer provides a superior mode of delivery compared to unconjugated D-PDMP by way of increased gastrointestinal absorption and increased residence time thus providing this otherwise rapidly cleared compound with therapeutic relevance in interfering with atherosclerosis, cardiac hypertrophy, and probably other diseases associated with the deleterious effects of abnormally high glycosphingolipid biosynthesis or deficient catabolism.

Overexpression of Plasmodium berghei ATG8 by Liver Forms Leads to Cumulative Defects in Organelle Dynamics and to Generation of Noninfectious Merozoites

ABSTRACT Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity. IMPORTANCE Malaria is responsible for more mortality than any other parasitic disease. Resistance to antimalarial medicines is a recurring problem; new drugs are urgently needed. A key to the parasite’s successful intracellular development in the liver is the metabolic changes necessary to convert the parasite from a sporozoite to a replication-competent, metabolically active trophozoite form. Our study reinforces the burgeoning concept that organellar changes during parasite differentiation are mediated by an autophagy-like process. We have identified ATG8 in Plasmodium liver forms as an important effector that controls the development and fate of organelles, e.g., the clearance of micronemes that are required for hepatocyte invasion and the expansion of the apicoplast that produces many metabolites indispensable for parasite replication. Given the unconventional properties and the importance of ATG8 for parasite development in hepatocytes, targeting the parasite’s autophagic pathway may represent a novel approach to control malarial infections. Malaria is responsible for more mortality than any other parasitic disease. Resistance to antimalarial medicines is a recurring problem; new drugs are urgently needed. A key to the parasite’s successful intracellular development in the liver is the metabolic changes necessary to convert the parasite from a sporozoite to a replication-competent, metabolically active trophozoite form. Our study reinforces the burgeoning concept that organellar changes during parasite differentiation are mediated by an autophagy-like process. We have identified ATG8 in Plasmodium liver forms as an important effector that controls the development and fate of organelles, e.g., the clearance of micronemes that are required for hepatocyte invasion and the expansion of the apicoplast that produces many metabolites indispensable for parasite replication. Given the unconventional properties and the importance of ATG8 for parasite development in hepatocytes, targeting the parasite’s autophagic pathway may represent a novel approach to control malarial infections.