THE TRANSMISSION EFFICIENCY OF PLASMODIUM YOELII INFECTED MOSQUITOES

Malaria is a life-threatening infectious disease caused by the Plasmodium parasite. Nearly half of the global population is at risk of acquiring malaria and there are approximately 500,000 deaths and 200 million cases annually. The infective form of the parasite, the sporozoite, is transmitted by the female Anopheles mosquito as she probes on a human host in search of a blood meal. Although it has been over 100 years since Ronald Ross discovered that Anopheles mosquitoes are the vector for the parasite, we still do not fully understand the early transmission dynamics of Plasmodium. One aspect that is poorly described is the probability of developing a blood stage infection after the bite of an infected mosquito. The entomological inoculation rate estimates the number of infected bites that an individual receives, but at present there is no understanding of the likelihood that sporozoites inoculated by a bite will successfully infect the host. This work provides the first laboratory estimate of the proportion of infected bites to a naïve host that result in a blood stage infection. In addition, four factors that may influence the transmission efficiency—the intensity of salivary gland infection, the duration of probing, the anatomical location on the host exposed to the mosquito bite, and the success of the mosquito in acquiring a blood meal—are considered. Using the rodent parasite Plasmodium yoelii in Anopheles stephensi mosquitoes, we determined that the transmission efficiency of a single mosquito bite is 21%. Further, the proportion of bites that result in an infection is not dependent on probe time, probe location, or acquisition of a blood meal; however a significantly greater probability of blood stage infection is present when the salivary glands of the probing mosquito are heavily infected

Creation and preclinical evaluation of genetically attenuated malaria parasites arresting growth late in the liver.

Whole-sporozoite (WSp) malaria vaccines induce protective immune responses in animal malaria models and in humans. A recent clinical trial with a WSp vaccine comprising genetically attenuated parasites (GAP) which arrest growth early in the liver (PfSPZ-GA1), showed that GAPs can be safely administered to humans and immunogenicity is comparable to radiation-attenuated PfSPZ Vaccine. GAPs that arrest late in the liver stage (LA-GAP) have potential for increased potency as shown in rodent malaria models. Here we describe the generation of four putative P. falciparum LA-GAPs, generated by CRISPR/Cas9-mediated gene deletion. One out of four gene-deletion mutants produced sporozoites in sufficient numbers for further preclinical evaluation. This mutant, PfΔmei2, lacking the mei2-like RNA gene, showed late liver growth arrest in human liver-chimeric mice with human erythrocytes, absence of unwanted genetic alterations and sensitivity to antimalarial drugs. These features of PfΔmei2 make it a promising vaccine candidate, supporting further clinical evaluation. PfΔmei2 (GA2) has passed regulatory approval for safety and efficacy testing in humans based on the findings reported in this study

Analysis of the diverse antigenic landscape of the malaria protein RH5 identifies a potent vaccine-induced human public antibody clonotype

The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria

THE TRANSMISSION EFFICIENCY OF PLASMODIUM YOELII INFECTED MOSQUITOES

Malaria is a life-threatening infectious disease caused by the Plasmodium parasite. Nearly half of the global population is at risk of acquiring malaria and there are approximately 500,000 deaths and 200 million cases annually. The infective form of the parasite, the sporozoite, is transmitted by the female Anopheles mosquito as she probes on a human host in search of a blood meal. Although it has been over 100 years since Ronald Ross discovered that Anopheles mosquitoes are the vector for the parasite, we still do not fully understand the early transmission dynamics of Plasmodium. One aspect that is poorly described is the probability of developing a blood stage infection after the bite of an infected mosquito. The entomological inoculation rate estimates the number of infected bites that an individual receives, but at present there is no understanding of the likelihood that sporozoites inoculated by a bite will successfully infect the host. This work provides the first laboratory estimate of the proportion of infected bites to a naïve host that result in a blood stage infection. In addition, four factors that may influence the transmission efficiency—the intensity of salivary gland infection, the duration of probing, the anatomical location on the host exposed to the mosquito bite, and the success of the mosquito in acquiring a blood meal—are considered. Using the rodent parasite Plasmodium yoelii in Anopheles stephensi mosquitoes, we determined that the transmission efficiency of a single mosquito bite is 21%. Further, the proportion of bites that result in an infection is not dependent on probe time, probe location, or acquisition of a blood meal; however a significantly greater probability of blood stage infection is present when the salivary glands of the probing mosquito are heavily infected

Experimental determination of the force of malaria infection reveals a non-linear relationship to mosquito sporozoite loads.

Plasmodium sporozoites are the infective stage of the malaria parasite. Though this is a bottleneck for the parasite, the quantitative dynamics of transmission, from mosquito inoculation of sporozoites to patent blood-stage infection in the mammalian host, are poorly understood. Here we utilize a rodent model to determine the probability of malaria infection after infectious mosquito bite, and consider the impact of mosquito parasite load, blood-meal acquisition, probe-time, and probe location, on infection probability. We found that infection likelihood correlates with mosquito sporozoite load and, to a lesser degree, the duration of probing, and is not dependent upon the mosquito's ability to find blood. The relationship between sporozoite load and infection probability is non-linear and can be described by a set of models that include a threshold, with mosquitoes harboring over 10,000 salivary gland sporozoites being significantly more likely to initiate a malaria infection. Overall, our data suggest that the small subset of highly infected mosquitoes may contribute disproportionally to malaria transmission in the field and that quantifying mosquito sporozoite loads could aid in predicting the force of infection in different transmission settings

An optimized messenger RNA vaccine candidate protects non-human primates from Zika virus infection

Abstract Zika virus (ZIKV), an arbovirus transmitted by mosquitoes, was identified as a cause of congenital disease during a major outbreak in the Americas in 2016. Vaccine design strategies relied on limited available isolate sequence information due to the rapid response necessary. The first-generation ZIKV mRNA vaccine, mRNA-1325, was initially generated and, as additional strain sequences became available, a second mRNA vaccine, mRNA-1893, was developed. Herein, we compared the immune responses following mRNA-1325 and mRNA-1893 vaccination and reported that mRNA-1893 generated comparable neutralizing antibody titers to mRNA-1325 at 1/20th of the dose and provided complete protection from ZIKV challenge in non-human primates. In-depth characterization of these vaccines indicated that the observed immunologic differences could be attributed to a single amino acid residue difference that compromised mRNA-1325 virus-like particle formation

Optimal expression of TCRβ is associated with coexpression of both CD14 and F4/80 on CD11b^high cells.

<p><i>A</i>: C57BL/6 (n = 6) and Balb/c (n = 6) mice were infected with 10⁶<i>Plasmodium berghei</i> ANKA parasites. CD14 and F4/80 expression was then measured on gated CD11b^high splenocytes on days 2, 4, and 6 post-infection and in naïve mice (data not shown) and the absolute number of CD11b^high, CD11b^highCD14⁺, CD11b^highF4/80⁺, and CD11b^highCD14⁺F4/80⁺ cells was enumerated at each time point. <i>B</i>: The effect of CD14 and F4/80 on TCRβ expression on CD11b^high splenocytes was also determined in C57BL/6 and Balb/c mice by comparing the expression of TCRβ on CD11b^high, CD11b^highCD14⁺, CD11b^highF4/80⁺, and CD11b^highCD14⁺F4/80⁺ cells. The percentage of cellular subsets that were TCRβ⁺CD3ε⁻ and the TCRβ-FITC mean fluorescence intensity (MFI) were calculated on days 2, 4, and 6 post-<i>Pb−A</i> infection. Data presented is representative of three independent experiments. The absolute number of cellular subsets that were TCRβ⁺CD3ε⁻ can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201043#pone.0201043.s003" target="_blank">S3 Fig</a>.</p

TCRβ expression by the macrophage correlates with <i>Plasmodium berghei</i> ANKA parasite burden.

<p>On day 3 post-infection, the correlation between the percentage of Ly6G⁻CD11b^highCD14⁺F4/80⁺ macrophages that are TCRβ⁺CD3ε⁻ and peripheral parasitemia (parasitized erythrocytes/total erythrocytes x 100) in five individual mice was determined. Results shown are representative of four independent experiments. Pearson r = 0.96, P<0.01.</p

TCRβ-expressing macrophages induced by a pathogenic murine malaria correlate with parasite burden and enhanced phagocytic activity

<div><p>Macrophages express a wide array of invariant receptors that facilitate host defense and mediate pathogenesis during pathogen invasion. We report on a novel population of CD11b^highCD14⁺F4/80⁺ macrophages that express TCRβ. This population expands dramatically during a <i>Plasmodium berghei</i> ANKA infection and sequesters in the brain during experimental cerebral malaria. Importantly, measurement of TCRβ transcript and protein levels in macrophages in wildtype versus nude and <i>Rag1</i> knockout mice establishes that the observed expression is not a consequence of passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells or nonspecific antibody staining to an Fc receptor or cross reactive epitope. We also demonstrate that TCRβ on brain sequestered macrophages undergoes productive gene rearrangements and shows preferential Vβ usage. Remarkably, there is a significant correlation in the proportion of macrophages that express TCRβ and peripheral parasitemia. In addition, presence of TCRβ on the macrophage also correlates with a significant increase (1.9 fold) in the phagocytosis of parasitized erythrocytes. By transcriptional profiling, we identify a novel set of genes and pathways that associate with TCRβ expression by the macrophage. Expansion of TCRβ-expressing macrophages points towards a convergence of the innate and adaptive immune responses where both arms of the immune system cooperate to modulate the host response to malaria and possibly other infections.</p></div

TCRβ expression correlates with enhanced phagocytosis of parasites by macrophages.

<p><i>A</i>: Splenocytes and parasitized red blood cells (pRBCs) were isolated from C57BL/6 mice on day 3 post-infection with <i>Plasmodium berghei</i> ANKA. A 1:1 ratio of splenocytes (labeled with macrophage and T lymphocyte markers) and pRBCs (labeled with CellTrace^®) were then incubated for 90 minutes to assess the effect of TCRβ expression by Ly6G⁻CD11b^highF4/80⁺ macrophages on phagocytosis in an <i>in vitro</i> assay. <i>B</i>: Expression of TCRβ correlates with enhanced phagocytosis of pRBCs by macrophages. n = 3 for each phagocytosis assay. Results are presented as mean ± standard deviation and are a replicate of three independent experiments. Significant difference between TCRβ⁺ versus TCRβ⁻ macrophages is indicated as **P < 0.01. P−value was calculated using the Mann Whitney <i>U</i> test.</p