Modifying the NSs gene to improve live-attenuated vaccine for Rift Valley fever

Abstract

Vaccines for exotic emerging diseases are considered for populations concerned about economic loss or social panic by natural or intentional introduction of those pathogens into their countries. In particular, zoonotic diseases transmitted by native mosquitoes may cause devastating economic loss and public health impact in the absence of effective countermeasures. A number of countries are at risk of potential spread of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) from endemic areas in Africa. RVFV establishes vertical transmission in floodwater Aedes species, a primary vector of RVFV, and is also maintained by horizontal transmission between mosquito vectors and animals [1]. RVFV infection results in more than 90% death in newborn lambs, 40–100% of abortion or fetal malformation in pregnant ewes, and causes a similar abortion in cattle, goat or other ruminants [2]. Most of the human patients of RVF typically show a biphasic febrile illness and less than 1% of patients suffer from lethal hemorrhagic fever, thrombosis or neurological disorder, while 1–10% of patients develop partial or complete blindness [3]. After 82 years since the first recognized RVF outbreak in Kenya in 1930, RVF is now endemic to most of sub-Saharan Africa and has spread into Egypt, Madagascar and Yemen [4]. In the USA, RVFV is classified as a Category A Priority Pathogen by the NIH/the National Institute of Allergy and Infectious Diseases, and an overlap select agent by the US Department of Agriculture and Health and Human Service. RVFV is a negative-stranded RNA virus with a tripartite genome named small (S)-, medium (M)- and large (L)-segments; S-segment encodes N and nonstructural NSs proteins in an ambisense manner, M-segment encodes envelope Gn and Gc proteins, nonstructural NSm proteins and the less-characterized 78-kD protein, and L-segment encodes L protein (RNA-dependant RNA polymerase) [4]. Effective vaccines will minimize the impact of RVFV introduction into nonendemic countries. Past studies demonstrated that neutralizing antibodies play an important role in protection from lethal RVFV challenge [5]. Considering that a large number of ruminants are infected during RVF outbreaks, RVF vaccines should rapidly induce high levels of protective neutralizing antibodies in both ruminant and humans with a single dose. Currently, only a formalin-inactivated The Salk Institute-Government Service Division (TSI-GSD)-200 vaccine is available with an Investigational New Drug status in the USA. TSI-GSD-200 is produced from the Entebbe strain (a wild-type [wt] RVFV isolate from mosquitoes in the 1940s) at high containment facilities, and there is a limitation in available doses, while at least three doses are required for inducing a protective level of neutralizing antibody [5,6]. A live-attenuated vaccine strain, Smithburn vaccine, generated by numerous intracerebral passages in suckling mice, has been used in endemic countries since the 1950s. Smithburn vaccine causes abortion in vaccinated pregnant ruminants, and recent sequencing analysis showed a potential reassortment with the wt RVFV strain [7]. A live-attenuated candidate vaccine, MP-12, was generated in the 1980s from an Egyptian isolate, ZH548 strain, by 12 serial passages in human diploid lung MRC-5 cells in the presence of a chemical mutagen, 5-fluorouracil [8]. Ruminants vaccinated with a single dose of MP-12 elicit high levels of neutralizing antibodies [5]. MP-12 has 23 mutations compared with the parental ZH548 strain, among which some of the 19 mutations in M- and L-segments are considered to be responsible for its attenuation, while the S-segment still has a virulent phenotype [5,9]. A study suggested that MP-12 retains residual virulence in pregnant ewes during early pregnancy [10], whereas the safety in pregnant ruminants at later stages and newborn lambs has been demonstrated [5]. Furthermore, more than 100 human volunteers were successfully vaccinated with MP-12 vaccine without notable adverse effects [5]. Thus, MP-12 is much safer than the Smithburn strain, and is considered one of the most promising candidate vaccines for RVF in humans and ruminants in the USA [5]. In addition, MP-12 is the RVFV strain exempted from select agent rule, and can be handled in a biosafety level 2 laboratory.