Plasmodium GPI-anchored micronemal antigen is essential for parasite transmission through the mosquito host.

Plasmodium parasites, the eukaryotic pathogens that cause malaria, feature three distinct invasive forms tailored to the host environment they must navigate and invade for life cycle progression. One conserved feature of these invasive forms is the micronemes, apically oriented secretory organelles involved in egress, motility, adhesion, and invasion. Here we investigate the role of GPI-anchored micronemal antigen (GAMA), which shows a micronemal localization in all zoite forms of the rodent-infecting species Plasmodium berghei. ∆GAMA parasites are severely defective for invasion of the mosquito midgut. Once formed, oocysts develop normally, however, sporozoites are unable to egress and exhibit defective motility. Epitope-tagging of GAMA revealed tight temporal expression late during sporogony and showed that GAMA is shed during sporozoite gliding motility in a similar manner to circumsporozoite protein. Complementation of P. berghei knockout parasites with full-length P. falciparum GAMA partially restored infectivity to mosquitoes, indicating conservation of function across Plasmodium species. A suite of parasites with GAMA expressed under the promoters of CTRP, CAP380, and TRAP, further confirmed the involvement of GAMA in midgut infection, motility, and vertebrate infection. These data show GAMA's involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function.

A Plasmodium falciparum ATP-binding cassette transporter is essential for liver stage entry into schizogony.

Plasmodium sporozoites invade hepatocytes and transform into liver stages within a parasitophorous vacuole (PV). The parasites then grow and replicate their genome to form exoerythrocytic merozoites that infect red blood cells. We report that the human malaria parasite Plasmodium falciparum (Pf) expresses a C-type ATP-binding cassette transporter, Pf ABCC2, which marks the transition from invasive sporozoite to intrahepatocytic early liver stage. Using a humanized mouse infection model, we show that Pf ABCC2 localizes to the parasite plasma membrane in early and mid-liver stage parasites but is not detectable in late liver stages. Pf abcc2 - sporozoites invade hepatocytes, form a PV, and transform into liver stage trophozoites but cannot transition to exoerythrocytic schizogony and fail to transition to blood stage infection. Thus, Pf ABCC2 is an expression marker for early phases of parasite liver infection and plays an essential role in the successful initiation of liver stage replication.

Amended recombinant cells (ARCs) expressing bovine IFN-gamma: an economical and highly effective adjuvant system.

Interferon-gamma (IFN-gamma) has potent immune stimulatory activity, but purifying recombinant protein is expensive, soluble cytokine delivery is inefficient, and high doses of IFN-gamma cause adverse systemic effects. We've shown, however, that chemically fixed, amended recombinant Pseudomonas fluorescens cells (ARCs), expressing soluble bovine IFN-gamma (BGI/ARCs), provide an effective production and delivery vehicle for highly active cytokine. In this report we investigate the immune enhancing activity of BGI/ARCs in the presence or absence of two other adjuvants which enhance cell recruitment and protein uptake. Adjuvant activity and immune-mediated protection was evaluated using recombinant, truncated glycoprotein-D (tgD) and a bovine herpesvirus-1 (BHV-1) disease challenge. Our initial dose-titration study showed that 100 microg of recombinant IFN-gamma in BGI/ARCs significantly increased both IgG1 and IgG2 tgD-specific antibody titres and IgG1/IgG2 ratios were significantly reduced with as little as 1.0 microg IFN-gamma. Vaccine formulation studies, using 20 microg tgD/vaccine dose and 100 microg IFN-gamma delivered in BGI/ARCs, formulated in phosphate buffered saline (PBS), induced significantly increased antibody responses, following both primary and secondary immunization, and immune-mediated protection following a BHV-1 respiratory infection. Co-formulating BGI/ARCs with either an oil and water emulsion (Emulsigen) or a polyphosphazene polymer (PCPP) did not significantly enhance tgD-specific antibody titres or disease protection when compared with BGI/ARCs alone. Surprisingly, co-delivering a single dose of BGI/ARCs with tgD protein in PBS had optimal adjuvant activity and the dose of IFN-gamma delivered was four-fold less than the dose previously shown to induce adverse systemic responses.

Amended recombinant cells (ARCs): an economical and surprisingly effective production and delivery vehicle for recombinant bovine IFN-gamma.

Recombinant Pseudomonas fluorescens cells, expressing over 40% protein as bovine interferon-gamma (IFN-gamma), were chemically fixed to sterilize the culture and amend the bacterial cell wall. When killed and fixed recombinant cells, termed here amended-recombinant-cells (ARCs), were assayed for interferon activity, we obtained the following surprising results: 1) sterilization and fixation did not inactivate ARC-encapsulated IFN-gamma; 2) ARC-encapsulated IFN-gamma and soluble, recombinant IFN-gamma were equally active in vitro but proteolysis was required for release of the ARC cytokine; and 3) ARC-encapsulated IFN-gamma was active in vivo with optimal adjuvant activity at a dose about 1000-fold less than previously reported for soluble, recombinant IFN-gamma and 100-fold less than doses which induced adverse systemic effects. The mechanism by which ARC-encapsulation increased IFN-gamma activity in vivo remains uncertain. However, our in vitro results show that sustained release of soluble IFN-gamma is a likely factor. The ARC production and delivery system achieves enhanced adjuvant activity with reduced risk of systemic effects, and the low cost of IFN-gamma production offers new opportunities for the use of this important cytokine.