Jet Kant

Rift Valley fever virus subunit vaccines confer complete protection against a lethal virus challenge

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus causing significant morbidity and mortality in livestock and humans. Rift Valley fever is endemic in Africa, but also outside this continent outbreaks have been reported. Here we report the evaluation of two vaccine candidates based on the viral Gn and Gc envelope glycoproteins, both produced in a Drosophila insect cell expression system. Virus-like particles (VLPs) were generated by merely expressing the Gn and Gc glycoproteins. In addition, a soluble form of the Gn ectodomain was expressed and affinity-purified from the insect cell culture supernatant. Both vaccine candidates fully protected mice from a lethal challenge with RVFV. Importantly, absence of the nucleocapsid protein in either vaccine candidate facilitates the differentiation between infected and vaccinated animals using a commercial recombinant nucleocapsid protein-based indirect ELISA.

Rift Valley fever virus immunity provided by a paramyxovirus vaccine vector.

Rift Valley fever virus (RVFV) causes recurrent large outbreaks among humans and livestock. Although the virus is currently confined to the African continent and the Arabian Peninsula, there is a growing concern for RVFV incursions into countries with immunologically naïve populations. The RVFV structural glycoproteins Gn and Gc are preferred targets in the development of subunit vaccines that can be used to control future outbreaks. We here report the production of Gn and Gc by a recombinant vaccine strain of the avian paramyxovirus Newcastle disease virus (NDV) and demonstrate that intramuscular vaccination with this experimental NDV-based vector vaccine provides complete protection in mice. We also demonstrate that a single intramuscular vaccination of lambs, the main target species of RVFV, is sufficient to elicit a neutralizing antibody response.

Efficacy of three candidate Rift Valley fever vaccines in sheep

Rift Valley fever virus (RVFV) is a mosquito-transmitted Bunyavirus that causes high morbidity and mortality among ruminants and humans. The virus is endemic to the African continent and the Arabian Peninsula and continues to spread into new areas. The explosive nature of RVF outbreaks requires that vaccines provide swift protection after a single vaccination. We recently developed several candidate vaccines and here report their efficacy in lambs within three weeks after a single vaccination. The first vaccine comprises the purified ectodomain of the Gn structural glycoprotein formulated in a water-in-oil adjuvant. The second vaccine is based on a Newcastle disease virus-based vector that produces both RVFV structural glycoproteins Gn and Gc. The third vaccine comprises a recently developed nonspreading RVFV. The latter two vaccines were administered without adjuvant. The inactivated whole virus-based vaccine produced by Onderstepoort Biological Products was used as a positive control. Five out of six mock-vaccinated lambs developed high viremia and fever and one lamb succumbed to the challenge infection. A single vaccination with each vaccine resulted in a neutralizing antibody response within three weeks after vaccination and protected lambs from viremia, pyrexia and mortality.

A Single Vaccination with an Improved Nonspreading Rift Valley Fever Virus Vaccine Provides Sterile Immunity in Lambs

Rift Valley fever virus (RVFV) is an important pathogen that affects ruminants and humans. Recently we developed a vaccine based on nonspreading RVFV (NSR) and showed that a single vaccination with this vaccine protects lambs from viremia and clinical signs. However, low levels of viral RNA were detected in the blood of vaccinated lambs shortly after challenge infection. These low levels of virus, when present in a pregnant ewe, could potentially infect the highly susceptible fetus. We therefore aimed to further improve the efficacy of the NSR vaccine. Here we report the expression of Gn, the major immunogenic protein of the virus, from the NSR genome. The resulting NSR-Gn vaccine was shown to elicit superior CD8 and CD4-restricted memory responses and improved virus neutralization titers in mice. A dose titration study in lambs revealed that the highest vaccination dose of 106.3 TCID50/ml protected all lambs from clinical signs and viremia. The lambs developed neutralizing antibodies within three weeks after vaccination and no anamnestic responses were observed following challenge. The combined results suggest that sterile immunity was achieved by a single vaccination with the NSR-Gn vaccine.

European ring trial to evaluate ELISAs for the diagnosis of infection with Rift Valley fever virus.

A ring trial was organized to evaluate Rift Valley fever virus (RVFV) ELISAs by European laboratories. A total of five ELISAs, two of which specific for IgM antibodies, were evaluated by six participants. Sera were derived from cattle or sheep and originated from either a RVFV endemic area, a RVFV-free area or from experimental infection studies. Cohens kappa analysis showed higher than 90% agreement of two commercially available ELISAs with the virus neutralization test, suggesting that primary screening as well as serological confirmation using these ELISAs is feasible. More extensive validations with sera of known IgM status are, however, required to determine agreement between IgM ELISAs.

Comparative efficacy of two next-generation Rift Valley fever vaccines.

Rift Valley fever virus (RVFV) is a re-emerging zoonotic bunyavirus of the genus Phlebovirus. A natural isolate containing a large attenuating deletion in the small (S) genome segment previously yielded a highly effective vaccine virus, named Clone 13. The deletion in the S segment abrogates expression of the NSs protein, which is the major virulence factor of the virus. To develop a vaccine of even higher safety, a virus named R566 was created by natural laboratory reassortment. The R566 virus combines the S segment of the Clone 13 virus with additional attenuating mutations on the other two genome segments M and L, derived from the previously created MP-12 vaccine virus. To achieve the same objective, a nonspreading RVFV (NSR-Gn) was created by reverse-genetics, which not only lacks the NSs gene but also the complete M genome segment. We have now compared the vaccine efficacies of these two next-generation vaccines and included the Clone 13 vaccine as a control for optimal efficacy. Groups of eight lambs were vaccinated once and challenged three weeks later. All mock-vaccinated lambs developed high fever and viremia and three lambs did not survive the infection. As expected, lambs vaccinated with Clone 13 were protected from viremia and clinical signs. Two lambs vaccinated with R566 developed mild fever after challenge infection, which was associated with low levels of viral RNA in the blood, whereas vaccination with the NSR-Gn vaccine completely prevented viremia and clinical signs.

Four-segmented Rift Valley fever virus induces sterile immunity in sheep after a single vaccination

Rift Valley fever virus (RVFV), a mosquito-borne virus in the Bunyaviridae family, causes recurrent outbreaks with severe disease in ruminants and occasionally humans. The virus comprises a segmented genome consisting of a small (S), medium (M) and large (L) RNA segment of negative polarity. The M-segment encodes a glycoprotein precursor (GPC) protein that is co-translationally cleaved into Gn and Gc, which are required for virus entry and fusion. Recently we developed a four-segmented RVFV (RVFV-4s) by splitting the M-genome segment, and used this virus to study RVFV genome packaging. Here we evaluated the potential of a RVFV-4s variant lacking the NSs gene (4s-ΔNSs) to induce protective immunity in sheep. Groups of seven lambs were either mock-vaccinated or vaccinated with 10(5) or 10(6) tissue culture infective dose (TCID50) of 4s-ΔNSs via the intramuscular (IM) or subcutaneous (SC) route. Three weeks post-vaccination all lambs were challenged with wild-type RVFV. Mock-vaccinated lambs developed high fever and high viremia within 2 days post-challenge and three animals eventually succumbed to the infection. In contrast, none of the 4s-ΔNSs vaccinated animals developed clinical signs during the course of the experiment. Vaccination with 10(5) TCID50 via the IM route provided sterile immunity, whereas a 10(6) dose was required to induce sterile immunity via SC vaccination. Protection was strongly correlated with the presence of RVFV neutralizing antibodies. This study shows that 4s-ΔNSs is able to induce sterile immunity in the natural target species after a single vaccination, preferably administrated via the IM route.

Four-segmented Rift Valley fever virus-based vaccines can be applied safely in ewes during pregnancy

Rift Valley fever virus (RVFV) causes severe and recurrent outbreaks on the African continent and the Arabian Peninsula and continues to expand its habitat. This mosquito-borne virus, belonging to the genus Phlebovirus of the family Bunyaviridae contains a tri-segmented negative-strand RNA genome. Previously, we developed four-segmented RVFV (RVFV-4s) variants by splitting the M-genome segment into two M-type segments each encoding one of the structural glycoproteins; Gn or Gc. Vaccination/challenge experiments with mice and lambs subsequently showed that RVFV-4s induces protective immunity against wild-type virus infection after a single administration. To demonstrate the unprecedented safety of RVFV-4s, we here report that the virus does not cause encephalitis after intranasal inoculation of mice. A study with pregnant ewes subsequently revealed that RVFV-4s does not cause viremia and does not cross the ovine placental barrier, as evidenced by the absence of teratogenic effects and virus in the blood and organs of the fetuses. Altogether, these results show that the RVFV-4s vaccine virus can be applied safely in pregnant ewes.

Co-housing of Rift Valley Fever Virus Infected Lambs with Immunocompetent or Immunosuppressed Lambs Does Not Result in Virus Transmission

Rift Valley fever virus (RVFV) is transmitted among susceptible animals by mosquito vectors. Although the virus can be isolated from nasal and oral swabs of infected animals and is known to be highly infectious when administered experimentally via oral or respiratory route, horizontal transmission of the virus is only sporadically reported in literature. We considered that immunosuppression resulting from stressful conditions in the field may increase the susceptibility to horizontally transmitted RVFV. Additionally, we reasoned that horizontal transmission may induce immune responses that could affect the susceptibility of contact-exposed animals to subsequent infection via mosquito vectors. To address these two hypotheses, viremic lambs were brought into contact with sentinel lambs. One group of sentinel lambs was treated with the immunosuppressive synthetic glucocorticosteroid dexamethasone and monitored for signs of disease and presence of virus in the blood and target organs. Another group of contact-exposed sentinel lambs remained untreated for three weeks and was subsequently challenged with RVFV. We found that none of the dexamethasone-treated contact-exposed lambs developed detectable viremia, antibody responses or significant increases in cytokine mRNA levels. Susceptibility of immunocompetent lambs to RVFV infection was not influenced by previous contact-exposure. Our results are discussed in light of previous findings.

Microencephaly in fetal piglets following in utero inoculation of Zika virus

Zika virus (ZIKV) is a mosquito-borne flavivirus that became associated with microcephaly in newborns and Guillain–Barré syndrome in adults after its emergence in the Pacific and the Americas in 2015. Newly developed rodent and nonhuman primate models have already revealed important insights into ZIKV-induced neuropathology. Nonhuman primates are phylogenetically closely related to humans and are therefore preferred human surrogates in ZIKV research. However, the use of nonhuman primates, particularly during gestation, raises ethical issues. Considering that pigs also share many anatomical and physiological features with humans, this species may be an attractive alternative human surrogate for ZIKV research. Here, we inoculated 20 porcine fetuses in utero and assessed the effect of ZIKV on brain development 4 weeks later. All inoculated fetuses presented mild to severe neuropathology, characterized by a depletion of neurons in the cerebral cortex. In most cases, neuronal depletion was confined to specific cerebral lobes without affecting brain size, whereas in severe cases a more generalized depletion resulted in microencephaly. Although the virus was widespread in the sows’ placenta at the time of necropsy only low levels of viral RNA were detected in fetal brain samples, thereby preventing the identification of primary target cells. Our findings suggest that pigs can be used to study ZIKV-induced neurodevelopmental defects as currently observed in human neonates, varying from stunted brain growth to localized cortical neuronal depletion in the absence of major macroscopic abnormalities.