Lassa Fever: a research perspective Speaker: Maria S. Salvato, PhD University of Maryland Biotechnology Institute audio presentation

Highlights

	Lassa virus is a bisegmented, negative-strand RNA virus with ambisense coding.
	The replication cycle has many targets for antivirals, although ribavirin is the only one presently in use.
	The virus has effects on host chemokines, mostly associated with suppression of innate immunity leading to suppression of acquired immunity.
	The monkey model for pathogenesis using LCMV-WE (lymphocytic choriomeningitis virus WE) has been useful for early diagnosis via transcriptome analysis, and for showing interferon-responsive genes, repair proteins, and translation factors that are affected at the transcription level.
	Vaccine efforts have demonstrated that live vaccines work, but non-infectious vaccines are in the pipeline.

Molecular structure

"Lassa fever virus was first identified as a member of the arenaviruses in the late 1960s," said Maria S. Salvato of the University of Maryland Biotechnology Institute as she laid the groundwork for her presentation on Lassa fever research. "The arenaviruses are named after the Latin word Arenosus, meaning 'sandy,' because of their granular appearance in electron micrographs." Lassa fever virus is closely related to the prototype arenavirus, lymphocytic choriomeningitis virus (LCMV). The benign relative of Lassa virus, Mopeia, is used in many studies comparing virulent and benign viruses.

Besides their granular appearance, the second unique feature of the arenaviruses is that their single-stranded RNA is in two genome segments, L (for large) and S (for small), and the four genes of the arenaviruses are encoded in an ambisense manner, Z [zinc-binding] and GP [glycoprotein] genes in the positive sense and the L and NP [nucleocapsid protein] genes in the negative sense, Salvato continued.

The Lassa fever virus is like other arenaviruses in its structure, with the most abundant molecule being the NP that associates with the viral RNA. It has a bimolecular genome structure, with the NP and polymerase forming the protein part of the ribonucleoprotein particle. The envelope glycoprotein spikes are comprised of membrane-embedded GP2 ionically bound to extracellular GP1. The Z protein is a small zinc-binding protein that functions as a matrix protein.

Viral life cycle and antiviral targets

Studies of the Lassa virus life cycle reveal potential antiviral targets at each step. The currently available treatment, ribavirin, is very effective (See Lassa fever: a clinical perspective). Salvato noted that IV ribavirin administered within three days of disease onset can reduce mortality from 50% to 2%-5%. "However, ribavirin treatments using sterile iv drips are not economical in West Africa."

The first step in the virus life cycle is adsorption to the cell surface. Earlier studies showed that Lassa virus infectivity is inactivated by exposure of virions to acid pH or high salt, which explains why the infectious dose of Lassa virus is so much higher when given orally than intravenously, said Salvato. "Polysulfates such as dextransulfate and heparin have been shown to block viral adsorption for some South American arenaviruses, but these have not yet been explored for Old World arenaviruses such as Lassa."

The next step is receptor-mediated endocytosis. Early work showed that neutralizing antibodies bind the outer portion of the envelope glycoprotein (GP1). Later, an assay was developed to help identify the cellular receptor for arenaviruses. Subsequent studies showed that a soluble protein, a-dystroglycan (a-DG) could block virus entry into cells for certain strains of Lassa virus and LCMV. Although subsequent experiments with an a-DG peptide linked to an Fc receptor failed to block entry of LCMV or Lassa viruses, "this may still be a promising approach if a larger portion of a-DG is used," Salvato said.

Blocking virus entry into cells with an a-DG peptide is a promising approach.

Another way to block virus entry is to use neutralizing antibodies. Convalescent serum from individuals who survived Lassa fever should be useful for this purpose. However, Salvato pointed out that this approach has worked only on rare occasions; its failure has been attributed to low neutralizing titers as a result of the immunosuppressant activities of the Lassa virus. "If a proper reagent were made that could neutralize in high titers, it's possible that this could be a therapy for Lassa fever," she said.

In the second stage of endocytosis, virus particles are surrounded by endocytic vesicles, Salvato continued. A peptide within the GP2 glycoprotein is responsible for viral fusion with endosomal cell membranes, a step that was shown to be sensitive to lysomotropic compounds. These compounds, which include ammonium chloride, chloroquin, and carboxylic ionophores such as monensin work by raising the endosomal pH and preventing endosomal acidification, she explained. "The anesthetic protein procaine and the pinocytotic agent caffeine block this step for some South American viruses, but it's not yet known whether this would work for Lassa virus."

Virus particles are uncoated at the surface of the endosome, ejecting the ribonucleoprotein from the virion and beginning the task of virus transcription. It is not clear what type of antiviral can block the uncoating step. However, Salvato suspects that some of the zinc-reactive compounds that have been shown to block arenavirus replication in cell culture could be working by blocking the function of the viral zinc-binding proteins NP and Z in uncoating.

Several zinc-reactive antivirals also seem to inhibit arenavirus replication.

Several zinc-reactive antivirals also seem to inhibit arenavirus replication. These are thought to act on the zinc-binding regions of the Z protein, inhibiting its function, and there is also a zinc-binding region of NP that might be affected. "The biological consequence of inhibiting Z function is that you may abort virus budding and therefore slow down replication," she explained.

Another important step in the virus life cycle is the translation and maturation cleavage of the viral envelope glycoprotein. "Although there are no inhibitors of cleavage available, the current definition of the cleavage site and the knowledge that cleavage must occur to obtain infectious particles opens the door to rational design of cleavage inhibitors."

Finally, myristic acid analogues can act as antivirals by inhibiting viral assembly and budding, Salvato said.

Lassa fever pathogenesis

Salvato then described several aspects of Lassa fever pathogenesis. Defining the earliest events in the disease can help researchers link those events to outcomes, which could improve diagnosis, she said. Early gene expression changes might also be useful as diagnostics, in that they might identify a disease before its pathogen is detectable.

Studies have shown that in contrast to Ebola, Lassa virus seems to suppress, rather than induce, pro-inflammatory chemokines. In one study, for example, interleukin-8 (IL-8) was produced in uninfected cultures and in cultures infected with Mopeia virus, but was suppressed in cultures infected with Lassa fever virus. The finding, based on this and other studies, "contradicted the view that Lassa fever was a disease caused by raging pro-inflammatory cytokines and promoted the view that the pro-inflammatory response of the host is an early protective mechanism and that its suppression is a pathogenic event," said Salvato.

Lassa suppresses innate immune responses and acquired adaptive immunity.

The finding was corroborated by the work of the Centers for Disease Control team in West Africa looking at Lassa patients who survived or succumbed to fatal Lassa fever. "The fatalities had lower levels of IL-8 and IP-10 (another chemokine) than the survivors so they probably succumbed due to an absence of neutrophils and lymphocytes for systemic defense."

"The view that Lassa suppressed some host cytokine responses evolved to the view that Lassa suppressed innate immune responses that were essential for the development of acquired adaptive immunity," Salvato continued. "This is probably an accurate view of the pathogenesis of Lassa fever. We've shown in the monkey model (macaques infected with LCMV-WE) that surviving animals develop high cell-mediated immune responses earlier than animals that succumb, so an adequate cell-mediated immune response is probably critical for survival.

Genetic studies are also helping to elucidate Lassa fever pathogenesis. Recent experiments suggest that of the 40,000 human genes, roughly 3% are differentially regulated during the course of the disease-approximately 600 in the liver, 446 in the spleen, and 185 in the blood. Eighteen of the genes are regulated in the same way in all three tissues. "We're proceeding to validate these genes by real-time PCR and other types of experiments," Salvato noted.

A gene that is down-regulated by the second day of infection is the proline-rich homeobox (PRH) gene, which is needed for repair of hematopoietic and hepatic systems. This gene expresses a transcription factor that ordinarily resides in the nucleus, but is in the cytoplasm when it is inactive. The effects on this gene by the virulent viral infection are obvious both at the transcription level and at the level of subcellular localization. In benign infection the PRH protein does not change its subcellular distribution, but in the virulent infection the nuclei become depleted of PRH.

Vaccine efforts

Salvato concluded her presentation with an overview of studies by numerous groups involved in Lassa fever vaccine research. Key points included the following:

	Monkeys inoculated with live Mopeia virus (a Lassa fever-related non-pathogenic virus) were fully protected from Lassa virus.
	An inactivated Lassa fever virus induced a high titer of antibodies against viral proteins in vaccinated macaques, but did not protect against a lethal challenge.
	VV NPLFV protected guinea pigs from a challenge by the Lassa fever virus, but failed to protect macaques.
	An attenuated reassortant vaccine (MOP/LAS) protected mice and guinea pigs from Lassa fever challenge. "In a rather remarkable experiment with the guinea pigs, it was shown that this vaccine does not need a great deal of time to develop protection before the lethal challenge," Salvato commented. "It can be delivered on the day of lethal challenge and still manages to protect seven of nine guinea pigs. This indicates that the MOP vaccine is probably eliciting protective innate responses and that innate immunity is an essential component of the protective mechanism."
	A recombinant Salmonella (delivered orally) expressing the NP of Lassa fever virus protected 30% of mice from heterologous lethal intracerebral challenge with LCMV
	Vaccinia expressing the GP genes of the Lassa fever virus protected both guinea pigs and macaques. Both GP1 and GP2 were necessary to induce protection; however, immune protection was not correlated with antibody levels, suggesting that the cell-mediated immune response is critical in protecting against Lassa fever

In summary, the critical point for Lassa vaccines is that innate and cell-mediated immunity are most important for protection, said Salvato. "So far, only live vaccines are protective. But non-infectious vaccines are in the pipeline," she concluded.