Genetic manipulation of Anopheles stephensi immunity to increase plasmodium falciparum salivary gland sporozoite infection levels

Human malaria is responsible for over 700,000 deaths a year. To stay abreast of the threat posed by the parasite, a constant stream of new drugs and vector control methods are required. This study focuses on a vaccine that has the potential to protect against parasite infection, but has been hindered by developmental challenges. In malaria prevention, live, attenuated, aseptic, Plasmodium falciparum sporozoites (PfSPZ) can be administered as a highly protective vaccine. PfSPZ are produced using adult female Anopheles stephensi mosquitoes as bioreactors. Production volume and cost of a PfSPZ vaccine for malaria are expected to be directly correlated with Plasmodium falciparum infection intensity in the salivary glands. The sporogonic development of Plasmodium falciparum in A. stephensi to fully infected salivary gland stage sporozoites is dictated by the activities of several known components of the mosquito’s innate immune system. Here I report on the use of genetic technologies that have been rarely, if ever, used in Anopheles stephensi Sda500 to increase the yield of sporozoites per mosquito and enhance vaccine production. By combining the Gal4/UAS bipartite system with in vivo expression of shRNA gene silencing, activity of the IMD signaling pathway downstream effector LRIM1, an antagonist to Plasmodium development, was reduced in the midgut, fat body, and salivary glands of A. stephensi. In infection studies using P. berghei and P. falciparum these transgenic mosquitoes consistently produced significantly more salivary gland stage sporozoites than wildtype controls, with increases in P. falciparum ranging from 2.5 to 10 fold. Using Plasmodium infection assays and qRT-PCR, two novel findings were identified. First, it was shown that 14 days post Plasmodium infection, transcript abundance of the IMD immune effector genes LRIM1, TEP1 and APL1c are elevated, in the salivary glands of A. stephensi, suggesting the salivary glands may play a role in post midgut defense against the parasite. Second, a non-pathogenic IMD signaling pathway response was observed which could suggest an alternative pathway for IMD activation. The information gained from these studies has significantly increased our knowledge of Plasmodium defense in A. stephensi and moreover could significantly improve vaccine production.

Effects of Aging and Moderate Calorie Restriction on the Reproductive Axis of the Male Rhesus Macaque (Macaca mulatta)

Calorie restriction (CR) has been established as the only non-genetic method of altering longevity and attenuating biological changes associated with aging. This nutritional paradigm has been effective in nematodes, flies, rodents, dogs and possibly non-human primates. Its long history notwithstanding, little is known regarding the exact mechanism(s) of CR action or its potential impact on the hypothalamic-pituitary-gonadal (HPG) axis. The objectives of this project were to: 1) analyze neuroendocrine changes to the HPG axis that occur with aging and 2) evaluate the effects of moderate CR on reproductive function in male rhesus macaques. Pituitary gene expression profiling, semi-quantitative RT-PCR (sqRT-PCR) and immunohistochemistry showed circadian clock mechanism components present in three age categories of macaques, demonstrated age differences in expression for Per2, indicated differential expression of Per2 and Bmal1 at opposing time points and revealed daily rhythmic expression of REV-ERBα protein. These data indicate the ability of the macaque pituitary to express core-clock genes, their protein products, and to do so in a 24-hour rhythm. Young Adult CON and CR pituitary gene expression profiles detected potential differential expression in <150 probesets. A decline in>TSHR and CGA was detected in CR macaques as measured by sqRT-PCR. Other genes investigated showed no diet-induced changes. Young Adult CON and CR testicular gene expression profiles detected potential differential expression in <300 probesets although mRNA expression was not altered based on sqRT-PCR and real-time RT-PCR. Age-related>and/or diet-induced changes in HSD17β3, INSL3, CSNK1E and CGA were observed in a separate experiment with CGA in Old Adult CR subjects returning to youthful levels. Semen samples were collected from Young Adult CON and CR macaques. Normal spermiogram measures, ZP-binding, AR assay and SCSA^® were conducted and indicated no differences between CON and CR-treated animals. Both groups exhibited similar daily testosterone profiles with no differences in mean or maximum levels; however, daily minimum testosterone levels were lower in CON animals. It appears that moderate CR had limited impact on neuroendocrine or reproductive function in male rhesus macaques based on our selected endpoints. Thus, advantageous CR health benefits can be achieved without obvious negative consequences to the HPG axis.