Development of a Novel, Single-Cycle Replicable Rift Valley Fever Vaccine

Abstract

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is an arbovirus that causes severe disease in humans and livestock in sub-Saharan African countries. Although the MP-12 strain of RVFV is a live attenuated vaccine candidate, neuroinvasiveness and neurovirulence of MP-12 in mice may be a concern when vaccinating certain individuals, especially those that are immunocompromised. We have developed a novel, single-cycle replicable MP-12 (scMP-12), which carries an L RNA, M RNA mutant encoding a mutant envelope protein lacking an endoplasmic reticulum retrieval signal and defective for membrane fusion function, and S RNA encoding N protein and green fluorescent protein. The scMP-12 underwent efficient amplification, then formed plaques and retained the introduced mutation after serial passages in a cell line stably expressing viral envelope proteins. However, inoculation of the scMP-12 into naïve cells resulted in a single round of viral replication, and production of low levels of noninfectious virus-like particles. Intracranial inoculation of scMP-12 into suckling mice did not cause clinical signs or death, a finding which demonstrated that the scMP-12 lacked neurovirulence. Mice immunized with a single dose of scMP-12 produced neutralizing antibodies, whose titers were higher than in mice immunized with replicon particles carrying L RNA and S RNA encoding N protein and green fluorescent protein. Moreover, 90% of the scMP-12-immunized mice were protected from wild-type RVFV challenge by efficiently suppressing viremia and replication of the challenge virus in the liver and the spleen. These data demonstrated that scMP-12 is a safe and immunogenic RVFV vaccine candidate.

The Envelope Proteins of the Bunyavirales

“…These loops project a few nonpolar residues, some of which are conserved across phleboviruses, the mutation of which to alanine or to a polar residue abrogates the pGc fusion activity. RVFV pGc mutants in the cd loop have been tested for fusogenicity using an assay based on syncytium formation in cells expressing Gn and a variant of Gc with a deletion in the cytoplasmic tail (Murakami et al, 2014). Mutations of the nonpolar residues Trp821 (W821N), Phe826 (F826N) (Fig.…”

Single-cycle replicable Rift Valley fever virus mutants as safe vaccine candidates

“…The immunogenicity of scMP-12 was higher than that conferred by MP-12-based RRP, but was lower than MP-12 (Murakami et al, 2014). scMP-12 carrying Gc with F826N and N827A mutations and a deletion of the C-terminal ER retrieval signal produced very low levels of non-infectious particles from infected naïve cells.…”

“…To improve the safety of MP-12, we have developed an MP-12-based, single-cycle replicable particle, scMP-12, which undergoes single-cycle replication in infected cells and immunized hosts (Murakami et al, 2014). Unlike RRP, scMP-12 carries three viral RNA segments, L RNA, M RNA carrying a mutation in Gc, and S-GFP RNA encoding N protein and GFP (Fig.…”

“…Accordingly, we targeted Gn/Gc proteins to the plasma membrane by deleting the ER retrieval signal in Gc protein and screened for fusion-incompetent Gn/Gc heterodimers by using a cell-to-cell membrane fusion assay under low pH conditions. Treatment of BSR-T7/5 cells transiently expressing Gn protein, Gc protein lacking the C-terminal ER retrieval signal, and Venus protein with low pH medium induced membrane fusion (Murakami et al, 2014). To estimate the effects of introduced mutations in the fusion peptide on the structural alteration of Gn/Gc proteins, we also performed immunofluorescent staining of the cells expressing Gn and Gc mutants by using an anti-Gn monoclonal antibody and anti-Gc-monoclonal antibody.…”

“…To estimate the effects of introduced mutations in the fusion peptide on the structural alteration of Gn/Gc proteins, we also performed immunofluorescent staining of the cells expressing Gn and Gc mutants by using an anti-Gn monoclonal antibody and anti-Gc-monoclonal antibody. We identified 6 amino-acid residues that are important for the fusion activity of Gc, while some mutants were not recognized by the anti Gc monoclonal antibody, implying that a mutation altered the conformation of the Gc (Murakami et al, 2014). For subsequent studies, we used a Gc mutant carrying a deletion of the C-terminal ER retrieval signal and F826N and N827A mutations, which abolished membrane fusion and were recognized by anti-Gc and anti-Gn monoclonal antibodies.…”