1. Introduction

Malaria, even with advances in modern medical technology, remains a serious health concern for the global community. It is caused by Plasmodium parasites, transmitted by anopheline mosquitos to their hosts. Even with all the progress that has been made, 95 countries and territories are still impacted with ongoing malaria transmission. According to the most recent Malaria health report, approximately 214 million new clinical cases and 438,000 deaths occurred in 2015 as a result of the Malaria parasite (WHO, 2015). Current treatments include artemisinin-based combination therapy (ACT), which is the most reliable and effective malaria treatment on the market at this time. Other preventative treatments include indoor residual spraying of insecticides and insecticide-treated mosquito nets. Unfortunately, with the increased and more widespread use of these treatments they have become less effective. This is due to the emergence of multidrug-resistant parasites and insecticide-resistant mosquitos, as a result of these preventative treatments (Ranson & Lissenden, 2016). Currently, the only options on the market that are in use are used to decrease mosquito populations or treat the malaria disease, already infected in the host. Currently, effective antimalarial vaccines are not currently available and reducing the rate of disease transmission is one of the key steps to control and eventually eradicate malaria. There are still many technical challenges that present itself when developing the vaccine, which may require an integrated approach, combining various treatment methods among which safe and effective malaria vaccines could be a crucial tool in the eradication of the disease (MalERA, 2011).

2. Stages of Vaccine Development

Currently, there are three different vaccine targets in development focusing on various stages of the Plasmodium life cycle. The first is pre-erythrocytic vaccines which target the sporozoites and liver stages and are designed to protect residents in low-endemic areas from being infected (Richie & Saul, 2002). The next vaccine target is blood-stage malaria vaccines targeting the asexual blood stages which aim to induce immunity to reduce the severity of the disease (Goodman & Draper, 2010). The last vaccine, where my research is focused, is transmission-blocking vaccines (TBVs) that target the sexual stages and mosquito midgut antigens, which induce immunity to interrupt malaria transmission (Carter et al., 2000). 

Malaria transmission-blocking vaccines rely on functional antibodies present in the serum of the vertebrate host that are ingested by mosquitoes together with Plasmodium gametocytes. These antibodies interact with the proteins present on the surface of sexual and sporogonic stages of Plasmodium or on the surface of the mosquito midgut and disrupt molecular interactions, such as fertilization, critical for malaria transmission (Sinden, 2017).

A vaccine that is able to target both asexual blood stages and sexual stages would not only offer direct protection against Malaria, but also have the benefit of reducing transmission as well. Presently, the majority of vaccine candidates only target a specific stage of the Plasmodium life cycle, so vaccines providing broad and sustained protection against multiple stages of the disease are still lacking (Theisen et al., 2014).

3. Pb51 Protein Background

Pre-clinical studies have led to the development of several potential Plasmodium transmission blocking candidates, of which our research will be focused on the Pb51 protein. Previous research examined extensive omics data in the PlasmoD8 database using defined criteria, found the Pb51 protein to be highly conserved in Plasmodium. It is presumed that this protein is expressed in both asexual blood stages and sexual stages based on available transcriptomic data (Wang et al., 2017). To date, this protein has only been studied in Plasmodium berghei, yet no research has been done on the much deadlier Plasmodium falciparum. In this study, the protein localization patterns, expression levels, and antibodies’ ability to block transmission will be evaluated, identifying the protein’s ability to block Plasmodium falciparum transmission.

Previous research on Pb51 was conducted in Plasmodium berghei, but not the more prevalent and deadlier Plasmodium falciparum. It is still unknown whether the Pb51 protein will maintain its properties and surface localization in this new model. In addition, no research has been conducted to determine the expression level changes in the Pb51 protein when Pb51 antibodies are introduced. Lastly, the ability of the antibodies to block transmission is still unknown, especially in this new model. I hypothesize that, because of how conserved the protein is, it will remain localized around the plasma membrane of the cell. Additionally, I believe that the expression levels of Pb51 will greatly decrease when Pb51 antibody is introduced into the system. Based on preliminary research conducted, I also believe the transmission blocking activity (TBA) will greatly increase with the Pb51 antibody, making it an important target for future vaccine research.

To determine whether the Pb51 antibody prevents the formation of Plasmodium falciparum ookinetes, we plan to measure the expression levels of the Pb51 gene when exposed to its antibody. Our hypothesis predicts a significant decrease in expression when the Pb51 antibody is present, resulting in increased transmission-blocking capabilities. It is still unknown how well Pb51 antibody is able to block TBA. To test this ability, we will be conducting a standard membrane feeding assay in order to quantify the antibodies’ ability to inhibit oocyst formation and determine the TBA of the antibody. Our hypothesis predicts that we will see a significant decrease in oocyst formation and have a high transmission blocking ability, resulting in an effective candidate for a malaria vaccine. 

4. Experimental Proposal 

The first experiment proposed will be done to confirm the localization patterns of the Pb51 protein in Plasmodium falciparum. This was previously done in Wang et al., 2017, but as stated previously, it was conducted using Plasmodium berghei parasite. I would like to rerun this test in order to ensure that the protein is fully conserved between the two species. In Plasmodium berghei, Pb51 was almost exclusively localized around the plasma membrane, making it an ideal target for antibody attachment. If Pb51 is not as conserved as previously thought, and is localized more in the cytoplasm, this will make the parasite much more difficult to target with the use of the Pb51 protein. I will be running an indirect immunofluorescence assay (IFA) to detect Pb51 expression in asexual blood stages, gametocytes, zygotes, retorts, ookinetes, and sporozoites of Plasmodium falciparum. 

Ookinetes will be obtained from dissection of the midgut 24 hrs post infection. They will be air-dried on slides and then fixed with paraformaldehyde in PBS for 20 min. After this the slide will be blocked in 5% skim milk in PBS for an hour. Pooled antisera against Pb51 will be diluted with 5% skim milk in PBS for an hour. After washing the slides, FITC-labeled goat anti-mouse IgG will be used as the secondary antibody. Once this is finished and the Ookinetes, and other stages of the Plasmodium life cycle, are correctly labeled with the Pb51 antisera, they will then be placed under the confocal microscope with fluorescent lighting. Localization patterns of the Pb51 protein can then be assessed in the various stages of the Plasmodium falciparum life cycle to determine if the plasma membrane localization remains consistent among species. If we can conclude that the protein remains conserved between species and that Pb51 remains localized around the plasma membrane, then in further research we can determine the mechanisms of the protein, hopefully leading to a potential vaccine.

The second proposed experiment will be conducted using qRT-PCR expression analysis to determine the fold change differences in the Pb51gene. Total RNA will be isolated from the midgut of Plasmodium infected mosquitos. Conditions of the qPCR will vary depending on the cycle. The Plasmodium falciparum gene Pf10_0203 an ADP ribosylation factor will be used as an internal reference to normalize each sample (Molina-Cruz et al., 2015). To determine whether the Pb51 antibody prevents the formation of P. falciparum ookinetes, we plan to measure the Pb51 gene which codes for the surface protein Pb51 expressed on both the asexual blood stages, and sexual stages of Plasmodium falciparum. We will be calculating the fold-change differences in A. gambiae treated with Pb51 antibodies or control IgG antibody using the 2-ΔΔCt method. Gene expression in NF gametocytes will be measured as negative control, because of its high rate of gametogenesis (Gebru et al., 2017). If we are able to see significant decreases in fold-change difference of the Pb51 gene compared to the control, we will be able to make another step in the right direction of ultimately leading to a vaccine. If we have conclusive results, it will mean the antibody is working effectively in decreasing the amount of surface protein Pb51 produced, confirming a potential target for transmission blocking activity in Plasmodium falciparum. In previous studies, researchers have not run qRT-PCR on the Pb51 gene to quantify fold-change differences; only localization and impact of ookinete formation have been looked at (Wang et al., 2017). Additionally, these were only done with Plasmodium berghei, which is a less harmful species of Plasmodium. 

The third experiment I propose for this study is the enzyme-linked immunosorbent assay (ELISA). This is used to detect the presence of Pb51 in a liquid sample using antibodies directed against the protein to be measured. It involves the Pb51 protein from Plasmodium falciparum that attaches to the surface of a flat-bottom 96-well ELISA plate. The matching antibody is then applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme, and in the final step, a substance containing the enzyme’s substrate is added. This reaction produces a detectable signal, which changes the color of the substrate, allowing for the detection of the Pb51 antibody (Lequin, 2005). 

For the ELISA assay, a flat bottom 96-well ELISA plate will be used. 1 ug/ml of recombinant Pb51 protein diluted in coating buffer will be coated onto the surface of each well and placed at 4 ˚C overnight. After this incubation period, the plates will be washed three times with TBS and blocked with general ELISA blocking buffer for two hours. IgG will be used as the control, and Pb51 antibody will be used as the experimental group for this ELISA test. Both of these antibodies will be diluted in blocking buffer and TBST, added to the antigen-coated wells and incubated for two hours. After incubation, plates will be washed three more times with TBST and incubated with goat anti-mouse or anti-rabbit immunoglobulin G conjugated to alkaline phosphatase secondary antibodies for another two hours. This step is used for the detection of the antibodies attached to the antigens. Once the incubation period is over, plates will be washed one final time and placed into the ELISA plate reader to detect the absorbance levels of each well with the given treatment. With this test we can determine how easily the Pb51 antibody is able to bind to the antigen, disabling its properties.

The last experiment I plan to conduct is the standard membrane feeding assay (SMFA). This is the current gold standard in testing mosquito based confirmatory transmission blocking assay for human malaria. It will be used to look at how the Pb51 protein affects oocyst formation and its transmission blocking activity of Plasmodium falciparum. A portion of the mosquitos being tested will be fed with Pb51 antibody, and the other portion will be fed the control IgG antibodies. On the day of the feed, drug treated mature P. falciparum gametocyte cultures will be centrifuged and the supernatant, along with the compounds added for the treatment, will be removed. This will then be formulated as artificial mosquito blood meals. Prepared blood meals will be fed to overnight starved 4-6 days old female Anopheles gambiae mosquitoes for the duration of 30-40 minutes via parafilm membrane attached to glass feeders (Colmenarejo et al., 2018). Once fed, these mosquitoes will be maintained in the controlled environment of an incubator for 7-8 days post feeding. After the incubation period, we will determine which mosquitoes have fully formed ovaries so we can remove them for dissection of midguts. Total number of oocysts in individual midguts will be counted using a light microscope at 100x magnification (Colmenarejo et al., 2018). Both infection prevalence (percentage of mosquitoes with one or more oocyst) and mean oocyst intensity of infection will be defined in each treatment. The oocyst load is compared between the treated and un-treated control groups. This will then in turn allow us to determine the TBA of the Pb51 antibody when fed to mosquitoes by calculating TBA as percent inhibition of infection intensity. Upon completion of the SMFA, if our hypothesis is correct, we will see a decrease in oocyst production in the midgut of the mosquitoes treated with the Pb51 antibody. This will also increase the TBA of the antibody, showing the antibody to have high transmission blocking activity. If all this is shown to be true, it will further demonstrate Pb51 antibody to help modulate and inhibit the spread of Plasmodium, ultimately leading to a vaccine target in the future. Demonstrating that Pb51 antibody inhibits oocysts formation and proliferation, as well as the transmission between species, is a crucial step in developing a vaccine. This is because Pb51 is a relatively unique protein that can be targeted during multiple stages of the Plasmodium life cycle, making this an easier, and possibly a more effective, target to disrupt Plasmodium’s transmission. 

4. Conclusion

If the Pb51 protein can show that it remains localized around the plasma membrane, and that its immunosuppressive properties can be silenced in the host at numerous stages in the Plasmodium falciparum life cycle, then we will be able to show that Pb51 is a potential target for a transmission blocking vaccine. Even if these experiments are successful, there is still more research that needs to be done before a vaccine trial can be conducted. The mechanisms behind Pb51 are still not fully understood, as well as if there are other areas of the protein that can be targeted to increase the TBA. All this information still needs to be done before any true conclusions can be reached. Targeting Pb51 helps prevent the formation of ookinetes in the midgut of mosquitoes, which blocks transmission. If we were able to trigger an immune response toward Plasmodium falciparum and be able to block the Pb51 protein to help reduce transmission, I believe this would be the most effective form of treatment. This project is one step of many to further our knowledge of Malaria, which will one day hopefully lead to a cure.