Manipulation of the Rift Valley Fever Virus M-Segment for Vaccine Development

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Rift Valley fever virus (RVFV) is a phlebovirus of the virus family, Bunyaviridae. Since the early 1900s, RVFV has continually caused economically devastating outbreaks in livestock and severe disease in humans. In pregnant ewes, RVFV infection can cause abortions in up to 100% of animals and high mortality rates (up to 60%), and it can cause fetal abnormalities and a high rate of mortality in newborn lambs. Originally, endemic to sub-Saharan Africa, RVF outbreaks have been documented in Egypt, Madagascar, the Arabian Peninsula, and, most recently, the island of Mayotte in the Indian Ocean. The proven potential for RVFV to cause outbreaks in “virgin soils” is of grave concern for the European Union and the United States of America, where competent vectors and susceptible host species are located. Vaccination of livestock is paramount in the prevention of outbreaks. Unfortunately, inactivated vaccines that are safe for both young and pregnant animals require boosters, and live-attenuated vaccines are not considered safe in pregnant animals. Thus, the development of a live-attenuated vaccine that is safe in pregnant animals is invaluable to the prevention of future RVF outbreaks and ultimately, the eradication of RVFV.
The first aim of this work was to determine the N-glycosylation status of the RVFV glycoproteins, Gn and Gc, and determine the roles of the N-glycans in DC-SIGN-mediated infection. The results determined that the single N-glycan site within Gn, at nt. 438, was N-glycosylated and three sites within Gc, at nt. 794, 1035, and 1077; are N-glycosylated. We also noted that the doublet band of Gc is generated by two distinct glycoforms, Gc-large and Gc-small, which are due to heterogeneous N-glycosylation at N1077. Enhanced infection of RVFV by DC-SIGN occurs by the Gn/Gc N-glycans in a redundant manner, and the N-glycans present at N438 (Gn) and N1077 (Gc) play an important role in DC-SIGN mediated infection. This work will aid in the development of vaccines that target the DC-SIGN receptor towards the facilitation of dendritic cells acting in their antigen-presenting capacity instead of viral dissemination. The second aim of the work presented was to create rMP-12 vaccine candidates that expressed increased levels of Gn and Gc. Neutralizing antibodies to Gn and Gc have been demonstrated to be important for protection against lethal RVFV challenge. Thus, we sought to optimize the translation efficiency of the preglycoprotein region of the M-segment. Within the preglycoprotein regions, there are five initiation codons, and the first initiation codon generates the 78 kDa-Gc precursor, while the second initiation codon generates the NSm-Gn-Gc precursor. We generated rMP-12 mutants with altered levels of Gn expression due to point mutations and truncation of the initiation codons. Truncation of the preglycoprotein region up to the 2nd initiation codon resulted in relative Gn expression levels that were almost 3-fold greater than the parental plasmid. However, this increase in relative Gn expression did not result in an increase in viral titer. We also investigated the Gn expression strategy of other phleboviruses, Sandfly fever Sicilian virus (SFSV) and Toscana virus (TOSV). Interestingly, relative Gn expression from the preglycoprotein regions of these viruses was 3-fold and 1.5 fold higher than RVFV. The knowledge gained from this aim will aid in the development of subunit vaccines based on the RVFV Gn and Gc proteins.

Rift Valley fever virus, M-Segment, Vaccine, Glycosylation, DC-SIGN, AUG, initiation codon