Preerythrocytic Vaccine Antigens Enhance Sterile Protection in Mice Induced by Circumsporozoite Protein.

Preerythrocytic vaccines prevent malaria by targeting parasites in the clinically silent sporozoite and liver stages and preventing progression to the virulent blood stages. The leading preerythrocytic vaccine, RTS,S/AS01E (Mosquirix), entered implementation programs in 2019 and targets the major sporozoite surface antigen, circumsporozoite protein (CSP). However, in phase III clinical trials, RTS,S conferred partial protection with limited durability, indicating a need to improve CSP-based vaccination.

The Form of an Antigen and Its Molecular Context Do Matter: Infectious versus Attenuated Plasmodium Sporozoites.

The physicochemical properties of an antigen (Ag) influence the type, specificity, as well as duration of emerging immune responses. Like immune responses arising to nominal protein Ags, reactivities to protozoan parasites, Plasmodium falciparum and P. berghei, the causative agents of human and mouse malaria, respectively, are shaped by the form of the parasite.

Identification of a Novel CD8 T Cell Epitope Derived from Protective Liver-Stage Antigen.

We recently identified novel (Pb) liver stage (LS) genes that as DNA vaccines significantly reduce Pb LS parasite burden (LPB) in C57Bl/6 (B6) mice through a mechanism mediated, in part, by CD8 T cells. In this study, we sought to determine fine antigen (Ag) specificities of CD8 T cells that target LS malaria parasites. Guided by algorithms for predicting MHC class I-restricted epitopes, we ranked sequences of 32 Pb LS Ags and selected ~400 peptides restricted by mouse H-2K and H-2D alleles for analysis in the high-throughput method of caged MHC class I-tetramer technology.

Correction: Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein.

[This corrects the article DOI: 10.1371/journal.pone.

Identification of Novel Pre-Erythrocytic Malaria Antigen Candidates for Combination Vaccines with Circumsporozoite Protein.

Malaria vaccine development has been hampered by the limited availability of antigens identified through conventional discovery approaches, and improvements are needed to enhance the efficacy of the leading vaccine candidate RTS,S that targets the circumsporozoite protein (CSP) of the infective sporozoite. Here we report a transcriptome-based approach to identify novel pre-erythrocytic vaccine antigens that could potentially be used in combination with CSP. We hypothesized that stage-specific upregulated genes would enrich for protective vaccine targets, and used tiling microarray to identify P.

Detection of Plasmodium berghei and Plasmodium yoelii Liver-Stage Parasite Burden by Quantitative Real-Time PCR.

Direct detection and quantification of liver-stage Plasmodium parasites became possible with the development of quantitative real-time PCR (qPCR). Here we describe the measurement of parasite burden in the livers of mice infected with the rodent malaria species, Plasmodium berghei and Plasmodium yoelii. This method is based on detection of expression of parasite ribosomal 18S RNA and can serve as an endpoint to accurately evaluate the efficacy of vaccines targeting the preerythrocytic stages of malaria.

Characterization of Liver CD8 T Cell Subsets that are Associated with Protection Against Pre-erythrocytic Plasmodium Parasites.

Murine models of malaria, such as Plasmodium berghei (Pb) and Plasmodium yoelii (Py), have been used for decades to identify correlates of protection associated with immunization using radiation-attenuated sporozoites (RAS). To date, RAS is the only known immunization regimen to consistently deliver 100 % sterilizing immunity and is considered the "gold standard" of protection against malaria. The ability to isolate lymphocytes directly from the liver of immune mice has facilitated the identification of correlates of protection at the site of infection.

Plasmodium cellular effector mechanisms and the hepatic microenvironment.

Plasmodium falciparum malaria remains one of the most serious health problems globally. Immunization with attenuated parasites elicits multiple cellular effector mechanisms capable of eliminating Plasmodium liver stages. However, malaria liver stage (LS) immunity is complex and the mechanisms effector T cells use to locate the few infected hepatocytes in the large liver in order to kill the intracellular LS parasites remain a mystery to date.

Sequential Phase 1 and Phase 2 randomized, controlled trials of the safety, immunogenicity and efficacy of combined pre-erythrocytic vaccine antigens RTS,S and TRAP formulated with AS02 Adjuvant System in healthy, malaria naïve adults.

In an attempt to improve the efficacy of the candidate malaria vaccine RTS,S/AS02, two studies were conducted in 1999 in healthy volunteers of RTS,S/AS02 in combination with recombinant Plasmodium falciparum thrombospondin-related anonymous protein (TRAP). In a Phase 1 safety and immunogenicity study, volunteers were randomized to receive TRAP/AS02 (N=10), RTS,S/AS02 (N=10), or RTS,S+TRAP/AS02 (N=20) at 0, 1 and 6-months. In a Phase 2 challenge study, subjects were randomized to receive either RTS,S+TRAP/AS02 (N=25) or TRAP/AS02 (N=10) at 0 and 1-month, or to a challenge control group (N=8).

Memory T cells maintain protracted protection against malaria.

Immunologic memory is one of the cardinal features of antigen-specific immune responses, and the persistence of memory cells contributes to prophylactic immunizations against infectious agents. Adequately maintained memory T and B cell pools assure a fast, effective and specific response against re-infections. However, many aspects of immunologic memory are still poorly understood, particularly immunologic memory inducible by parasites, for example, Plasmodium spp.

First-in-human evaluation of genetically attenuated Plasmodium falciparum sporozoites administered by bite of Anopheles mosquitoes to adult volunteers.

Background: Immunization with genetically engineered, attenuated malaria parasites (GAP) that arrest during liver infection confers sterile protection in mouse malaria models. A first generation Plasmodium falciparum GAP (Pf p52(-)/p36(-) GAP) was previously generated by deletion of two pre-erythrocytic stage-expressed genes (P52 and P36) in the NF54 strain.

Methods: A first-in-human, proof-of-concept, safety and immunogenicity clinical trial in six human volunteers was conducted.

The effect of antiretrovirals on Plasmodium falciparum liver stages.

HIV and malaria overlap geographically, but the full impact of different antiretrovirals on malaria remains poorly understood. We examined the antimalarial activity of the HIV protease inhibitors lopinavir and saquinavir and the non-nucleoside reverse transcriptase inhibitor nevirapine on Plasmodium falciparum liver stages. Our results demonstrate that the HIV PI lopinavir inhibits liver stage parasites at clinically relevant concentrations, that is, at drug levels achieved in HIV-infected patients on standard dosing regimens.

The survival of memory CD8 T cells that is mediated by IL-15 correlates with sustained protection against malaria.

Ag-specific memory T cell responses elicited by infections or vaccinations are inextricably linked to long-lasting protective immunity. Studies of protective immunity among residents of malaria endemic areas indicate that memory responses to Plasmodium Ags are not adequately developed or maintained, as people who survive episodes of childhood malaria are still vulnerable to either persistent or intermittent malaria infections. In contrast, multiple exposures to radiation-attenuated Plasmodium berghei sporozoites (Pb γ-spz) induce long-lasting protective immunity to experimental sporozoite challenge.

Memory CD8 T cells specific for plasmodia liver-stage antigens maintain protracted protection against malaria.

Immunologic memory induced by pathogenic agents or vaccinations is inextricably linked to long-lasting protection. Adequately maintained memory T and B cell pools assure a fast, effective, and specific response against re-infections. Studies of immune responses amongst residents of malaria endemic areas suggest that memory responses to Plasmodia antigens appear to be neither adequately developed nor maintained, because persons who survive episodes of childhood malaria remain vulnerable to persistent or intermittent malaria infections.

HIV nonnucleoside reverse transcriptase inhibitors and trimethoprim-sulfamethoxazole inhibit plasmodium liver stages.

Background: Although nonnucleoside reverse transcriptase inhibitors (NNRTIs) are usually part of first-line treatment regimens for human immunodeficiency virus (HIV), their activity on Plasmodium liver stages remains unexplored. Additionally, trimethoprim-sulfamethoxazole (TMP-SMX), used for opportunistic infection prophylaxis in HIV-exposed infants and HIV-infected patients, reduces clinical episodes of malaria; however, TMP-SMX effect on Plasmodium liver stages requires further study.

Methods: We characterized NNRTI and TMP-SMX effects on Plasmodium liver stages in vivo using Plasmodium yoelii.

Quantification of sporozoite invasion, migration, and development by microscopy and flow cytometry.

There is an important role for in vitro assays to better understand the initial steps of malaria infection. In this section, we describe both microscopy-based and flow cytometry-based sporozoite invasion, migration and development assays with the rodent malaria parasites, Plasmodium berghei and Plasmodium yoelii, and the human malaria parasite, Plasmodium falciparum.

Immunization with the RTS,S/AS malaria vaccine induces IFN-γ(+)CD4 T cells that recognize only discrete regions of the circumsporozoite protein and these specificities are maintained following booster immunizations and challenge.

In a Phase 2a trial of the RTS,S/AS vaccine, we described significant association between protection against infection and vaccine-induced CD4 T cells. To determine whether processing of the circumsporozoite protein as a component of the RTS,S particulate antigen yields the same HLA-DR-restricted epitopes as those recognized by CD4 T cells from donors immunized by exposure to attenuated or infectious sporozoites we mapped the specificities of the RTS,S primed CD4 T cells by measuring IFN-γ cultured Elispot responses to pairs of overlapping 15 a.a.

Protective immunity induced with the RTS,S/AS vaccine is associated with IL-2 and TNF-α producing effector and central memory CD4 T cells.

A phase 2a RTS,S/AS malaria vaccine trial, conducted previously at the Walter Reed Army Institute of Research, conferred sterile immunity against a primary challenge with infectious sporozoites in 40% of the 80 subjects enrolled in the study. The frequency of Plasmodium falciparum circumsporozoite protein (CSP)-specific CD4(+) T cells was significantly higher in protected subjects as compared to non-protected subjects. Intrigued by these unique vaccine-related correlates of protection, in the present study we asked whether RTS,S also induced effector/effector memory (T(E/EM)) and/or central memory (T(CM)) CD4(+) T cells and whether one or both of these sub-populations is the primary source of cytokine production.

Disruption of the Plasmodium falciparum liver-stage antigen-1 locus causes a differentiation defect in late liver-stage parasites.

The malaria parasite Plasmodium falciparum infects humans and first targets the liver where liver-stage parasites undergo pre-erythrocytic replication. Liver-stage antigen-1 (LSA-1) is currently the only identified P. falciparum protein for which expression is restricted to liver stages.

A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes.

Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4(+) T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: human leukocyte antigen-DR1 (HLA-DR1), HLA-DM and cathepsins.

An early commitment to expression of a particular TCRVbeta chain on CD8(+) T cells responding to attenuated Plasmodium berghei sporozoites is maintained following challenge with infectious sporozoites.

Protection induced by irradiated Plasmodium berghei sporozoites (Pbgamma-spz) in mice is linked to CD8(+) T cells specific for exo-erythrocytic-stage Ags, and intrahepatic memory CD8(+) T cells are associated with protracted protection. However, the Ag specificity of the protective CD8(+) T cells remains largely unknown. In this study, we characterized the TCR Vbeta usage by intrahepatic CD8(+) T cells during gamma-spz immunization and after the challenge with infectious Pb sporozoites.