New approaches to the treatment of severe malaria

 Written by Professor Nicholas King

Malaria is caused by several species of the Plasmodium parasite and is spread by mosquitoes, principally in Africa and Asia. In 2015, the World Health Organization (WHO) estimated the number of global malaria cases to be in excess of 200 million.

More than 400,000 of these cases resulted in death due to complications of severe malaria, with children under five accounting for more than 70 percent of deaths. Of these, 99 percent were infected with P. falciparum.

In severe malaria, infection of the red cells by the malaria parasite makes them stick to the blood vessel wall in the brain and lungs. White blood cells accumulate around the sticky red cells, gradually blocking the flow of blood to these organs, causing seizures, respiratory distress, coma and death.

The WHO recommends parenteral artesunate for 24 hours to treat severe malaria, followed by two days of artemisinin-based combination therapy with quinine-based drugs.

Despite the use of this medication, overall mortality is still 10 to 20 percent, because this treatment does not reduce the accumulation of cells.

With increasing resistance to recommended malaria medications and no current new anti-malarial drug or vaccine candidates, adjunctive therapies, such as immune-modifying therapy, combined with anti-malarial drugs, offer a novel potential avenue to reduce mortality.

To investigate potential approaches, we first used high parameter flow cytometry to characterise in detail the white blood cell accumulation in the brain and lung in a mouse model of severe malaria using P. berghei ANKA (PbA), which replicates more than 25 features of the human disease.

Using clustering analysis, we uncovered a specific population of monocytes expressing low levels of a migration-associated molecule Ly6C, on the cell surface Ly6C^lo monocytes present in much higher numbers than any other cell type. Previously thought to be the housekeeping cells of the blood vessels, Ly6C^lo monocytes are actually a major contributor to the accumulating cells in both brain and lung and therefore a potential target for therapeutic intervention.

This unprecedented discovery was enabled using the cutting-edge equipment and analysis techniques available in Sydney Cytometry, a core facility of the University of Sydney.

Under homeostatic conditions, Ly6C^lo monocytes originate in the bone marrow, their precursors emigrating in a constant trickle into the blood stream as inflammatory monocytes expressing high levels of Ly6C, which is quickly down-regulated as they become homeostatic monocytes.

In response to acute inflammatory stimuli, however, Ly6C^hi inflammatory monocytes start to emigrate from the bone marrow in large numbers within a few days. Their increased manufacture here displaces that of B cells and neutrophils.

Homing via the blood to inflammatory foci, the presence of large numbers of inflammatory monocytes is a hallmark of many diseases caused by infection, autoimmunity and tissue damage. Indeed, these cells are responsible in large part for the associated morbidity and mortality of these diseases.

We have previously shown that intravenous administration of negatively-charged, immune-modifying particles (IMP) reduce inflammatory monocyte infiltration, markedly increasing survival and improving recovery in a range of monocyte-mediated inflammatory disease models.

To confirm that the Ly6C^lo monocytes in the cellular accumulations in the brain and lung could arise from Ly6C^hi monocytes, we intravenously transferred pure, marked populations of Ly6C^hi monocytes isolated from the bone marrow into PbA-infected mice 24 hours before the onset of cerebral malaria (CM).

Within this period, transferred cells had homed to the brain and lung and down-regulated their Ly6C expression, consistent with our hypothesis that bone marrow-derived inflammatory monocytes were the likely precursors of the accumulating Ly6C^lo cells in CM and acute respiratory distress syndrome (ARDS).

To show further proof-of-principle, we then infused IMP on day 4 post infection, the timepoint at which monocytes first begin to accumulate in the brain as Ly6C^hi monocytes, well before the appearance of disease signs, to see if we could prevent the onset of CM.

In these experiments, some 50 percent of the mice survived an otherwise 100 percent lethal infection, with reduced monocyte numbers in the brain and lungs and reduced clinical disease scores. Not surprisingly, however, as IMP have no anti-malarial action; survivors subsequently showed an increasing concentration of parasites in their blood, which we abolished with quinine treatment.

Since patients do not usually present in advance of disease signs or symptoms, we treated infected animals at the onset of CM signs with artesunate in accordance with WHO protocol, combining it with IMP.

Strikingly, survival in mice treated with combination therapy was almost 90%, while in mice treated with artesunate or IMP alone, survival was 56 percent and 10 percent respectively.

Survival statistics were reflected in the clinical scores in each group, but more importantly, there was markedly reduced vascular occlusion and interstitial infiltration respectively, in the brain and lung, further borne out by significantly reduced Ly6C^lo monocyte numbers in both organs.

Notably, the combination of IMP and chloroquine (quinine-based) also gave this result. This is interesting, as chloroquine alone is much less effective at this late stage than artesunate alone. More interestingly, on a PbA re‑challenge, all survivors quickly cleared the parasite without CM signs, suggesting a robust anti-malarial immunity not previously seen in this model.

We have yet to fully understand the mechanism of action of this combination treatment. For example, since IMP act by sequestering Ly6C^hi cells in the spleen, we do not understand how artesunate enables IMP to effectively clear Ly6C^lo monocytes already occluding the cerebral blood vessels at the onset of CM, when IMP alone are minimally effective.

Nor do we understand how protective anti-malarial immunity is enabled or what elements are involved.

Nevertheless, these results are a first in any pre-clinical CM or ARDS model. They emphasise the immunopathological nature of monocyte accumulation in severe malaria and are proof-of-principle of the potential efficacy of combining specific immune-modifying and anti-malarial therapy, which almost doubled the survival seen with artesunate treatment alone.

This approach could markedly improve human recovery as well as potentially curbing the spread of drug resistance.

This article is based on a collaboration between Nicholas King and Georges Grau. See the full report for sources.