MALARIA LIVER STAGE CULTURE: IN VITRO VERITAS?

Seven decades passed between Laveran’s discovery of the blood stage malaria parasites in 1880 and the studies of Shortt, Garnham, and others that demonstrated the exo-erythrocytic phase of parasite development in the mammalian liver.^1,2 In part, this delay may be ascribed to Fritz Schaudinn’s famous and erroneous “observation” in 1903 of the Plasmodium vivax sporozoite directly invading the red blood cell,³ although as we now know the mammalian malaria parasites must develop in the liver for several days prior to initiating the erythrocytic phase of infection. Schaudinn’s model held sway for decades despite the fact that no one could reproduce his findings.

Shortt and Garnham made their landmark observation in rodent malaria parasites,¹ as well as malaria parasites of humans and nonhuman primates,^2,4 but subsequent morphologic and biologic studies of the liver stage have primarily focused on rodent malaria parasites.⁵ Inside hepatocytes, sporozoites transform into liver stage parasites, which are also called exo-erythrocytic forms (EEFs). Each liver stage trophozoite grows, undergoes multiple nuclear divisions as a schizont, and ultimately differentiates into tens of thousands of first-generation merozoites. These merozoites are released into the liver sinusoids where they infect red blood cells. Liver stage parasites cause no overt pathology and no detectable symptoms, and it is interesting that the parasite has selected an immunologically tolerogenic organ⁶ as its bridgehead into the mammalian host.

Despite some progress in describing liver stage development, EEFs have remained frustratingly recalcitrant to reveal their biologic secrets during the nearly six decades since their discovery. Thus, liver stage cell biology and molecular biology of especially human malaria parasite liver stages are still in their infancy. Notwithstanding the fact that whole genome sequences are now available for a number of malaria parasite species and that modern systems biology tools can now analyze microarrays and high throughput proteomic studies that have been applied to all other life cycle stages of malaria parasites,⁷ comprehensive gene and protein expression profiles of EEFs have yet to be established. Liver stage parasites may be the most promising target for a vaccine that completely prevents infection. Complete protection has been repeatedly demonstrated by vaccination studies using irradiation-attenuated live sporozoites, which induce complete sterile protection against challenge.⁸ The protective mechanisms appear to mainly act against the liver stage. Therefore, elucidating the antigenic composition of liver stage parasites is critically important.

Why are human malaria liver stages so hard to study? First, they are difficult to find, the parasitologic equivalent of the “needle in the haystack.” A typical mosquito may inoculate dozens of sporozoites into a host that if uniformly successful at invading hepatocytes develop into one liver stage each. That results in a few dozen liver stage parasites in a three-pound organ! Equally difficult to find are human volunteers willing to undergo a liver wedge biopsy, and for this reason C. H. Howard, who underwent open surgery and biopsy of the liver after receiving hundreds of infective mosquito bites for the purpose of discovering the liver stage parasite, may deserve equal billing with Shortt and others in the discovery of the exo-erythrocytic form of P. falciparum.²

Rodent malaria models have the invaluable advantage that in vivo infected livers are directly accessible for analysis, but even here the paucity of liver stage material and the technical challenge to isolate infected hepatocytes have made it difficult to study liver stages in vivo. In vitro systems such as the human hepatoma cell line HepG2 that supports development of P. berghei rodent malaria parasites⁹ have been a great advance, but unfortunately this cell line and others do not support P. falciparum liver stage development with the exception of one cell line that supports it with low efficiency.¹⁰ Thus, to date the only reliable in vitro system available for the culture of P. falciparum are primary human hepatocytes.¹¹

In this issue of the journal, Sattabongkot and others describe a new hepatocyte line, HC-04, which was derived from normal human hepatocytes.¹² By painstakingly assessing sporozoite invasion and liver stage development, these investigators isolated a continuous cell line that sufficiently supports liver stage development of P. falciparum and P. vivax to yield merozoites that can initiate the erythrocytic phase. Because HC-04 supports liver stage development of the two most prevalent human malaria parasites, it will allow detailed comparative analysis of their liver stage biology. This may, for example, shed light on the differential growth regulation that must occur in dormant liver stages known as hypnozoites, which are responsible for the relapses of P. vivax malaria. Development of both parasite species appeared asynchronous in HC-04, but the timing of first merozoite release and thus the time to reach liver stage maturity corresponded approximately to the duration of in vivo development. A main advantage of this cell line over previously tested cell lines and primary human hepatocyte cultures is the greatly improved infection rates that are critical for using in vitro liver stage cultures in downstream experimental applications.

Sattabongkot and others have taken a great step forward with HC-04, but improvements on the HC-04 system will be needed to realize the full potential of liver stage cultures. The generation of clonal HC-04 lines and selection of clones with superior sporozoite infection and liver stage development rates is but one avenue for improvement. The present advance lays the ground for a routine, standardized, high-efficiency in vitro culture of the liver stages of human malaria parasites to be developed. Such a culture system will accelerate progress in important areas of malaria research, for example the identification of protective liver stage antigens, screening of novel drugs that act against liver stage parasites, and safety testing of live-attenuated whole sporozoite vaccines.