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

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

Mechanistic Modeling of the Effects of Acidosis on Thrombin Generation

BACKGROUND: Acidosis, a frequent complication of trauma and complex surgery, results from tissue hypoperfusion and IV resuscitation with acidic fluids. While acidosis is known to inhibit the function of distinct enzymatic reactions, its cumulative effect on the blood coagulation system is not fully understood. Here, we use computational modeling to test the hypothesis that acidosis delays and reduces the amount of thrombin generation in human blood plasma. Moreover, we investigate the sensitivity of different thrombin generation parameters to acidosis, both at the individual and population level. METHODS: We used a kinetic model to simulate and analyze the generation of thrombin and thrombin–antithrombin complexes (TAT), which were the end points of this study. Large groups of temporal thrombin and TAT trajectories were simulated and used to calculate quantitative parameters, such as clotting time (CT), thrombin peak time, maximum slope of the thrombin curve, thrombin peak height, area under the thrombin trajectory (AUC), and prothrombin time. The resulting samples of parameter values at different pH levels were compared to assess the acidosis-induced effects. To investigate intersubject variability, we parameterized the computational model using the data on clotting factor composition for 472 subjects from the Leiden Thrombophilia Study. To compare acidosis-induced relative parameter changes in individual (“virtual”) subjects, we estimated the probabilities of relative change patterns by counting the pattern occurrences in our virtual subjects. Distribution overlaps for thrombin generation parameters at distinct pH levels were quantified using the Bhattacharyya coefficient. RESULTS: Acidosis in the range of pH 6.9 to 7.3 progressively increased CT, thrombin peak time, AUC, and prothrombin time, while decreasing maximum slope of the thrombin curve and thrombin peak height (P < 10(–5)). Acidosis delayed the onset and decreased the amount of TAT generation (P < 10(–5)). As a measure of intrasubject variability, maximum slope of the thrombin curve and CT displayed the largest and second-largest acidosis-induced relative changes, and AUC displayed the smallest relative changes among all thrombin generation parameters in our virtual subject group (1-sided 95% lower confidence limit on the fraction of subjects displaying the patterns, 0.99). As a measure of intersubject variability, the overlaps between the maximum slope of the thrombin curve distributions at acidotic pH levels with the maximum slope of the thrombin curve distribution at physiological pH level systematically exceeded analogous distribution overlaps for CT, thrombin peak time, and prothrombin time. CONCLUSIONS: Acidosis affected all quantitative parameters of thrombin and TAT generation. While maximum slope of the thrombin curve showed the highest sensitivity to acidosis at the individual-subject level, it may be outperformed by CT, thrombin peak time, and prothrombin time as an indicator of acidosis at the subject-group level

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