Shining a Spotlight on Malaria

Figure 1: Female Anopheles
mosquito taking a blood meal.

In September this year, Kenya made history by becoming the first country to launch the world's first ever malaria vaccine pilot program. This shines a ray of hope for countries endeavoring to eliminate malaria, an infectious disease that takes a child's life every two minutes. The World Health Organization (WHO) reports that malaria claimed 435,000 lives globally in 2017. Efforts to prevent and treat malaria have seen disease incidence fall by 20 million cases, with the number of malaria cases falling from 239 million in 2010 to 219 million in 2017. However, despite these improvements, the WHO soberly reports that progress has stalled, with no reduction in the number of malaria cases recorded between 2015 and 2017.

In this Vaccine Spotlight blog post, we take a closer look at malaria, the biggest parasitic killer in the world. We review the basics on malaria as well as highlight some of the challenges and important research advances made in developing a vaccine to fight and ultimately eradicate this devastating disease.

How Does Aging Modulate the Expression of SDF1?

Scientists found that older mice have enhanced recruitment of methyltransferase, EZH2, to the SDF1 promoter, which prevents SDF1 gene expression and thus inhibits scar formation. Interestingly, pharmacological inhibition of EZH2 in older mice induced SDF1 and promoted scar formation. Moreover, wounded human skin organoids exhibited age-dependent expression of SDF1 and EZH2 like in the mice, suggesting inhibition of SDF1 can be useful in reducing scar formation following surgery in humans. Currently, SDF1 inhibitors are on the market being utilized to mobilize hematopoietic stem cells. Future studies will focus on utilizing these inhibitors to determine their potential use in preventing scar formation in many types of human tissue injuries, including those suffering from epidermolysis bullosa or in burn victims.

• What is malaria?
  

  
  Figure 2: Example of blood film
  being taken to test for malaria

  While malaria is frequently associated as a disease caused by mosquitos, female Anopheles mosquitos are merely extremely competent vectors of the Plasmodium parasite, the true perpetrator of this disease. There are five species of this unicellular protozoan parasite (P. falciparum, P. vivax, P. ovale, P. knowlesi and P. malariae) that cause malaria in humans. P. falciparum, however, is the most infectious and responsible for severe forms of the disease. When a mosquito bites a person infected with malaria, the parasite is taken up together with its blood meal. The next time the mosquito bites a human, these parasites are injected into the unfortunate victim together with the mosquito's saliva (Fig. 3).

  Once infected, malaria symptoms typically emerge anywhere from 10 days to a month, and are manifested in the form of fever, chills, lethargy, nausea, vomiting, diarrhea and other flu-like symptoms. As an infected person's red blood cell count decreases, an effect of the disease, they might become anemic and jaundiced. If the infection remains untreated and continues to advance, it may result in seizures, mental confusion, kidney failure, coma, and even death. People with lowered immunity, such as pregnant women, children, infants, and travelers from countries with no previous exposure to malaria, are particularly susceptible to this disease.

• How P. falciparum wreaks havoc in the human body

  P. falciparum adopts different forms during its life cycle. Here, we focus primarily on the stages of the parasite's life within humans.

  

  Figure 3: Brief overview of how P. falciparum infection causes malaria in the human body.

  • During its blood meal, an infected mosquito transfers sporozoites, a form of P. falciparum, from its saliva into the human host's bloodstream

  • Sporozoites travel to the liver where they invade hepatocytes and reproduce to form merozoites

  • Merozoites released back into the blood and invade red blood cells (RBCs), commencing entry into the blood stage of malaria infection

  • Within red blood cells, they transform into trophozoites which enlarge as they mature within the RBC

  • Multiple rounds of asexual nuclear division take place to form a schizont

  • Merozoites bud from the schizont and rupture the RBC, releasing merozoites which go on to invade other RBCs.

  • Trophozoites within hijacked RBCs can also transform into gametocytes which are capable of sexual reproduction – they do not rupture the RBC and instead, when taken up during a mosquito blood meal, mate and reproduce within the gut of the mosquito to generate more sporozoites

  

  Figure 4: Different forms of P. falciparum in a blood smear (black arrows)

  (B) Schizont (C) Gametocyte
  Images courtesy of DPDx.

• Obstacles to the search for a perfect vaccine
  

  Figure 5: Child getting
  vaccinated

  With the landmark national RTS,S vaccine pilot program in Kenya underway, and soon to be launched in Malawi and Ghana, perhaps eliminating this disease is not an insurmountable endeavor. However, while significant progress has been made, the vaccine is still far from perfect. RTS,S has to be administered to young children in four separate doses, and offers just partial protection. WHO reported that Phase III clinical trial results showed that the vaccine prevented 39% of cases of malaria in children, and 29% of severe malaria cases. It has also been noted that the efficacy of the vaccine diminished over time.

  Unfortunately, the quest to develop an effective malaria vaccine is fraught with challenges. It is known that immunity acquired from previous malarial infection does not confer lifelong protection against the disease. Furthermore, unlike traditional pharmaceuticals, the general lack of a market for vaccines coupled with the sheer technical complexity and financial investment of developing a vaccine against this complex parasite makes this a complicated venture, and deters drug manufacturers from allocating resources to R&D and manufacturing.

• PfRH5: A promising target for vaccine development?

  An article published in the journal Cell earlier this year has provided new insight into the development of an effective vaccine targeting the malaria parasite at the critical blood stage of infection.

  Using single B-cell sorting, they isolated and identified 17 distinct monoclonal antibodies (mAbs) from patients that had been immunized against the P. falciparum protein PfRH5 in a Phase Ia clinical trial (Fig. 6, Fig. 7A). PfRH5 is a highly conserved protein that plays an essential role for the binding of basigin to the host erythrocyte. Aotus monkeys that were vaccinated with full-length PfRH5 were shown to be protected against P. falciparum infection, and pAbs in the serum generated by these monkeys were able to inhibit P. falciparum growth in vitro.

  

  Figure 6: mAbs were isolated, sequenced, recombinantly expressed, and characterized from human volunteers that had been immunized with a PfRH5-based vaccine.

  Figure from Alanine et al. (2019) [article]

  To assess whether these anti-PfRH5 mAbs could prevent merozoite entry into erythrocytes, the research group assayed for in vitro growth inhibition activity (GIA) against the P. falciparum 3D7 clone. They found that the two most potent neutralizing mAbs (nAbs), R5.016 and R5.004, yielded EC50 values similar to the most potent known anti-merozoite mAbs generated in mice (Fig 7B). Further investigation involving epitope-binning and structure-based analysis of PfRH5 led researchers to trace the binding site of these two nAbs to the internal disordered loop of the protein.

  

  Figure 7: mAbs were grouped according to epitope bin (A), and the corresponding GIA of each antibody group was measured (B). The mAbs with the most potent neutralizing activity were R5.016 (red bin), and R5.004 (blue bin).

  Selected panels from Figure 3, Alanine et al. (2019) [article]

  Scientists determined that a truncated PfRH5 construct, PfRH5∆NL, bore all the relevant epitopes required to elicit these neutralizing mAbs within humans. They confirmed this by measuring the ability of full length PfRH5 and PfRH5∆NL to reverse the GIA of polyclonal IgG from the sera of a vaccinated humans (D84 IgG; Fig. 8A). In addition, by using GenScript's custom rabbit polyclonal antibody service, they were able to conclude that immunization with either the full length PfRH5 or PfRH5∆NL could elicit antibodies of comparable potency (Fig. 8B, 8C). Subsequently, using X-ray crystallography, they were able to conclusively identify the epitopes for R5.016 and R5.004 binding to PfRH5.

  

  Figure 8: Immunization with PfRH5∆NL can elicit relevant anti-PfRH5 nAbs. (A) Full length PfRH5 (PfRH5FL) and PfRH5∆NL reverse GIA of polyclonal D84 IgG from seven immunized humans. (B) GIA measurements and (C) corresponding EC50 measurements demonstrating that rabbits immunized with either PfRH5FL or PfRH5∆NL yielded polyclonal IgGs of comparable potency.

  Selected panels from Figure 3, Alanine et al. (2019) [article]

  The researchers then highlighted the importance of evaluating the usefulness of using nAbs in combination. When testing mAb pairs, they found that in some cases, despite targeting distinct epitopes, the mAbs would actually compete, antagonize and ultimately diminish the overall neutralization activity than if the mAb was used on its own. Excitingly, they additionally identified a class of antibodies that could potentiate the neutralizing effect of other PfRH5-binding mAbs and other merozoite proteins despite possessing no neutralizing capacity itself. This novel non-neutralizing antibody, R5.011, was discovered slow down the invasion process of PfRH5, extending the time window for neutralizing antibodies to reach target epitopes on PfRH5.

  By meticulously characterizing antibody activity, and using evidence from structural biology, researchers have identified a new, revolutionary strategy to inform and evaluate malaria vaccine design. Perhaps a vaccine which is able to elicit nAbs and non-neutralizing mAbs against a blood-stage malaria protein may be the key to specific and long lasting immunity against the disease.

  

  Figure 9: Structure overlay depicting potent nAbs R5.016 (red) and R5.004 (blue), and non-neutralizing antibody R5.011 (green) bound to PfRH5∆NL (A).
  Selected panel from Figure 6, Alanine et al. (2019) [ article]

  

  Figure 10: Graphical depiction summarizing research findings in this paper. Neutralizing antibodies (blue) that inhibit merozoite (MZ) entry into red blood cells (RBC), as well as a class of non-neutralizing antibodies (green) that potentiates nAb activity, have been identified (top panel). Each class of mAbs bind distinct epitopes on PfRH5 (bottom left panel). The non-neutralizing antibody discovered likely operates by slowing down the invasion of MZ into RBCs, affording more time for nAbs to reach the target epitope on PfRH5 (bottom right panel).
  Figure from Alanine et al. (2019) [article]

  *Figures in this section were gratefully obtained from the Cell publication in discussion: Alanine et. al. (2019) under the Creative Commons Attribution 4.0 International (CC BY 4.0). Images represented have been extracted from their larger, original figures and relabeled for clarity in this post, with no other modifications made.

• Where things stand

  Malarial vaccines have been in development for the past 50 years with still no licensed product confirmed to date. Today, there are quite a number of malarial vaccine candidates in clinical development, including the blood stage PfRH5-based vaccine described in the study above. In addition to RTS,S and the PfRH5-based vaccine described above, Sanaria ^® also has a vaccine developed based on the whole P. falciparum sporozoite that has been irradiated to render it non-infectious. Ongoing studies in Western Kenya show that the Sanaria^® vaccine appears to be safe, well-tolerated, and confers protection against malaria.

  By leveraging advances in the fields of vaccinology and increased scientific understanding of the molecular mechanisms that govern P. falciparum's infectivity and persistence in human hosts, perhaps we can look forward to the development of vaccines that will eradicate malaria for good.

• Centers for Disease Control and Prevention – Malaria

• World Health Organization – Malaria (website),WHO World Malaria Report 2018

• Alanine DGW, Quinkert D, Kumarasingha R, et al. Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies. Cell. 178(1), 216-228.e21 (2019).

• Draper SJ, Sack BK, King CR, et al. Malaria Vaccines: Recent Advances and New Horizons. Cell Host Microbe. 24(1), 43–56 (2018).

• Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. Lancet. 386(9988), 31–45 (2015).