Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria. Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved

Humanized Mouse Models for the Study of Human Malaria Parasite Biology, Pathogenesis, and Immunity

Malaria parasite infection continues to inflict extensive morbidity and mortality in resource-poor countries. The insufficiently understood parasite biology, continuously evolving drug resistance and the lack of an effective vaccine necessitate intensive research on human malaria parasites that can inform the development of new intervention tools. Humanized mouse models have been greatly improved over the last decade and enable the direct study of human malaria parasites in vivo in the laboratory. Nevertheless, no small animal model developed so far is capable of maintaining the complete life cycle of Plasmodium parasites that infect humans. The ultimate goal is to develop humanized mouse systems in which a Plasmodium infection closely reproduces all stages of a parasite infection in humans, including pre-erythrocytic infection, blood stage infection and its associated pathology, transmission as well as the human immune response to infection. Here, we discuss current humanized mouse models and the future directions that should be taken to develop next-generation models for human malaria parasite research

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\u27s involvement in sporozoite motility, egress, and invasion, implicating GAMA as a regulator of microneme function

Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice

<div><p>Gammaherpesviruses encode proteins with homology to the cellular purine metabolic enzyme formyl-glycinamide-phosphoribosyl-amidotransferase (FGARAT), but the role of these viral FGARATs (vFGARATs) in the pathogenesis of a natural host has not been investigated. We report a novel role for the ORF75A vFGARAT of murine gammaherpesvirus 68 (MHV68) in infectious virion production and colonization of mice. MHV68 mutants with premature stop codons in <i>orf75A</i> exhibited a log reduction in acute replication in the lungs after intranasal infection, which preceded a defect in colonization of multiple host reservoirs including the mediastinal lymph nodes, peripheral blood mononuclear cells, and the spleen. Intraperitoneal infection rescued splenic latency, but not reactivation. The 75A.stop virus also exhibited defective replication in primary fibroblast and macrophage cells. Viruses produced in the absence of ORF75A were characterized by an increase in the ratio of particles to PFU. In the next round of infection this led to the alteration of early events in lytic replication including the deposition of the ORF75C tegument protein, the accelerated kinetics of viral gene expression, and induction of TNFα release and cell death. Infecting cells to deliver equivalent genomes revealed that ORF75A was required for initiating early events in infection. In contrast with the numerous phenotypes observed in the absence of ORF75A, ORF75B was dispensable for replication and pathogenesis. These studies reveal that murine rhadinovirus vFGARAT family members ORF75A and ORF75C have evolved to perform divergent functions that promote replication and colonization of the host.</p></div

Frequencies of cells harboring viral genomes in C57BL/6 mice.

<p>Frequencies of cells harboring viral genomes in C57BL/6 mice.</p

Divergence of the gammaherpesvirus vFGARAT family.

<p><b>(A)</b> Phylogenetic tree of the vFGARATs of the indicated γ-hepesviruses (Genebank ID located in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006843#ppat.1006843.s007" target="_blank">S1 Table</a>). <b>(B)</b> Phylogenetic alignment using BLOSUM62 with previously characterized domains of the vFGARATs. <b>(C)</b> Pairwise alignment showing degrees of identity (lower left corner of table) or similarity (upper right corner of table) between the respective N-terminal or C-terminal portions of the indicated vFGARATs. The division point for each vFGARAT (red vertical line in B) was based on dotplot analysis for each indicated vFGARATS compared to MHV68 ORF75C.</p

Frequencies of cell populations reactivating viral genomes in C57BL/6 mice.

<p>Frequencies of cell populations reactivating viral genomes in C57BL/6 mice.</p

Accelerated gene expression coupled with replication defect upon 75A.stop infection of primary cells.

<p><b>(A)</b> Single-step growth curve in primary BMDM at an MOI of 5 with 75A.stop1.2 and 75A.stop1MR. <b>(B)</b> Timecourse analysis of gene products upon a single-step infection of BMDMs. <b>(C)</b> Multi-step growth curve in primary MEFs at an MOI of 0.05 with 75A.stop1.2 and 75A.stop1MR. <b>(D)</b> Timecourse analysis of gene products upon a low MOI infection of MEFs. <b>(E)</b> Schematic of the ORF75 locus and the multicistronic transcripts with strand-specific probes to indicated ORFs. <b>(F)</b> Northern blot analysis of NIH 3T12 fibroblast cells infected with indicated viruses at an MOI of 5. Membrane was hybridized with strand-specific ³²P-labled cDNA of ORF75A, ORF75B, ORF75C, M3, or 18S. Longer exposures with ORF75C probe detects the <i>orf75A/B/C</i> transcript. <b>(G)</b> Viral DNA quantification of input genomes at 2 hpi and subsequent replication at 24 hpi. For A, C, and E, bars represent the mean of three independent biological replicates +/- SD.</p

Input of equivalent genomes reveals role for ORF75A in driving productive replication in cell culture and in mice.

<p><b>(A)</b> Multi-step growth curve in NIH 3T12 cells infected with equivalent PFU or equivalent genomes. <b>(B-D)</b> Expression of the YFP reporter gene in NIH 3T12 fibroblasts <b>(B)</b>, primary fibroblasts (MEFs) <b>(C)</b>, and primary bone marrow-derived macrophage cells (BMDM) <b>(D)</b> upon infection with indicated viruses at an equivalent PFU or with equivalent genomes. <b>(E)</b> Acute replication in the lungs of mice 7 dpi. Mice were infected with indicated viruses at 1000 PFU or adjusted to infect at equivalent FFU. * p ≤ 0.05, ** p ≤ 0.005 ***, p ≤ 0.0005, and **** p ≤ 0.00005; significance determined by two-way ANOVA for A and one-way ANOVA for B-E.</p