Preliminary Evaluation of a Bunyavirus Vector for Cancer Immunotherapy.

Replicon particles of Rift Valley fever virus, referred to as nonspreading Rift Valley fever virus (NSR), are intrinsically safe and highly immunogenic. Here, we demonstrate that NSR-infected human dendritic cells can activate CD8(+) T cells in vitro and that prophylactic and therapeutic vaccinations of mice with NSR encoding a tumor-associated CD8 peptide can control the outgrowth of lymphoma cells in vivo. These results suggest that the NSR system holds promise for cancer immunotherapy.

Heparan sulfate facilitates Rift Valley fever virus entry into the cell.

Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.

Acid-activated structural reorganization of the Rift Valley fever virus Gc fusion protein.

The entry of the enveloped Rift Valley fever virus (RVFV) into its host cell is mediated by the viral glycoproteins Gn and Gc. We investigated the RVFV entry process and, in particular, its pH-dependent activation mechanism using our recently developed nonspreading-RVFV-particle system. Entry of the virus into the host cell was efficiently inhibited by lysosomotropic agents that prevent endosomal acidification and by compounds that interfere with dynamin- and clathrin-dependent endocytosis. Exposure of plasma membrane-bound virions to an acidic pH (

Mutations in the M-gene segment can substantially increase replication efficiency of NS1 deletion influenza A virus in MDCK cells.

Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene segment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction but rather is related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production.

Foot-and-Mouth Disease Seroprevalence in Cattle in Eritrea.

Information about seroprevalence of foot-and-mouth disease (FMD) and virus serotypes in Eritrea is unavailable, but is very important as it may guide the choice of intervention measures including vaccination to be implemented. We carried out a cross-sectional study from February to June 2011 in Eritrea with a two-stage cluster design, sampling cattle in 155 villages with the objective of determining the seroprevalence of FMD in four administrative regions of the country. We analysed cattle sera (n = 2429) for FMD virus antibodies using the non-structural ELISA (NS ELISA) and virus neutralization test (VNT). The overall seroprevalence was 26% and 30% for the NS ELISA and VNT, respectively. FMD virus serotypes O (14%) and A (11%) were the most prevalent. Gash Barka showed the highest (39%) seroprevalence both in NS ELISA and VNT compared to the other three administrative regions. Strategic FMD virus vaccination with type O and A (matching circulating strains) in combination of zoo-sanitary measures would be the best control option for Eritrea which could be started in areas where the disease is less endemic.

Foot-and-mouth Disease Transmission in Africa: Implications for Control, a Review.

In Africa, for the control of foot-and-mouth disease (FMD), more information is needed on the spread of the disease at local, regional and inter-regional level. The aim of this review is to identify the role that animal husbandry, trade and wildlife have on the transmission of FMD and to provide a scientific basis for different FMD control measures in Africa. Review of literature, published reports and databases shows that there is more long distance spread of FMD virus serotypes within North, West, Central and East Africa than in southern Africa. In North, West, Central and East Africa migratory animal husbandry systems often related with search for grazing and water as well as trade are practiced to a greater extent than in southern Africa. In southern Africa, the role of African buffalo (Syncerus caffer) is more extensively studied than in the other parts of Africa, but based on the densities of African buffalo in Central and East Africa, one would assume that buffalo should also play a role in the epidemiology of FMD in this part of Africa. More sampling of buffalo is necessary in West, Central and East Africa. The genetic analysis of virus strains has proven to be valuable to increase our understanding in the spread of FMD in Africa. This review shows that there is a difference in FMD occurrence between southern Africa and the rest of the continent; this distinction is most likely based on differences in animal husbandry and trade systems. Insufficient data on FMD in wildlife outside southern Africa is limiting our understanding on the role wildlife plays in the transmission of FMD in the other buffalo inhabited areas of Africa.

Serological evidence indicates that foot-and-mouth disease virus serotype O, C and SAT1 are most dominant in eritrea.

Foot-and-mouth disease (FMD) is endemic in Eritrea and in most parts of Africa. To be able to control FMD using vaccination, information on the occurrence of various foot-and-mouth disease serotypes in Eritrea is needed. In this cross-sectional study, 212 sera samples were collected from FMD infected and recovered animals in Eritrea. These samples were tested for the presence of antibodies against FMD non-structural proteins (NSP) and neutralizing antibodies against six of the seven (all but SAT 3) serotypes of FMD virus (FMDV). Of these, 67.0% tested positive to non-structural protein antibodies in the FMD NS ELISA. By virus neutralization, FMDV serotype O antibodies were shown to be the most dominant (approximately 50%). Virus neutralization test results indicate that infection with serotype C and SAT 1 might have occurred, although there are no reports of isolation of these two serotypes. Because the samples were not randomly selected, further random serological surveillance in all age group animals is necessary both to estimate the prevalence of FMD in the country and to confirm the serological results with serotype C and SAT 1.

Evaluation of nonspreading Rift Valley fever virus as a vaccine vector using influenza virus hemagglutinin as a model antigen.

Virus replicon particles are capable of infection, genome replication and gene expression, but are unable to produce progeny virions, rendering their use inherently safe. By virtue of this unique combination of features, replicon particles hold great promise for vaccine applications. We previously developed replicon particles of Rift Valley fever virus (RVFV) and demonstrated their high efficacy as a RVFV vaccine in the natural target species. We have now investigated the feasibility of using this nonspreading RVFV (NSR) as a vaccine vector using influenza virus hemagglutinin as a model antigen. NSR particles were designed to express either the full-length hemagglutinin of influenza A virus H1N1 (NSR-HA) or the respective soluble ectodomain (NSR-sHA). The efficacies of the two NSR vector vaccines, applied via either the intramuscular or the intranasal route, were evaluated. A single vaccination with NSR-HA protected all mice from a lethal challenge dose, while vaccination with NSR-sHA was not protective. Interestingly, whereas intramuscular vaccination elicited superior systemic immune responses, intranasal vaccination provided optimal clinical protection.

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.

Vertical transmission of Rift Valley fever virus without detectable maternal viremia.

Rift Valley fever virus (RVFV) is a zoonotic bunyavirus that causes abortions in domesticated ruminants. Sheep breeds exotic to endemic areas are reportedly the most susceptible to RVFV infection. Within the scope of a risk assessment program of The Netherlands, we investigated the susceptibility of a native breed of gestating sheep to RVFV infection. Ewes were infected experimentally during the first, second, or third trimester of gestation. Mortality was high among ewes that developed viremia. Four of 11 inoculated ewes, however, did not develop detectable viremia nor clinical signs and did not seroconvert for immunoglobulin G (IgG) or IgM antibodies. Surprisingly, these ewes were found to contain viral RNA in maternal and fetal organs, and the presence of live virus in fetal organs was demonstrated by virus isolation. We demonstrate that RVFV can be transmitted vertically in the absence of detectable maternal viremia.

Transplacental transmission of BTV-8 in sheep: BTV viraemia, antibody responses and vaccine efficacy in lambs infected in utero.

Bluetongue virus (BTV) is an insect vector transmitted virus which causes an economically important disease in ruminants. BTV infection during pregnancy can result in infection of the foetus, which may lead to the birth of persistently infected or immunotolerant offspring. Since persistently infected animals continuously produce large amounts of virus they could be a source of infection for the insect vector. This could significantly influence the epidemiology of the virus and hence might require additional measures to control a BTV outbreak. Therefore, we investigated the potential of BTV-8 to induce persistent infection or immunotolerance in lambs in an experimental setting. Infection of eighteen 70-75 days pregnant ewes with wild type BTV-8 led to the birth of 25 out of 44 BTV RNA positive lambs (foetal infected, FI). All 23 FI lambs born alive also had anti BTV antibodies at birth; infectious virus could be recovered from 5 out of 25 FI lambs. Viral RNA loads decreased rapidly after birth; 19 out of 20 FI lambs that remained in the experiment until week 14 after birth, were RNA negative at that time. Since persistence of BTV-8 infection could not be demonstrated, we investigated whether foetal infection had an effect on protection against a field virus infection and on efficacy of vaccination. To this end, 5 FI lambs and 5 foetal non-infected (FNI) lambs were vaccinated with the inactivated Bovilis(®) BTV-8 vaccine, five months after birth. Three weeks after the vaccination, all lambs were infected with wild type BTV-8. The foetal infection did not interfere with vaccination efficacy. In contrast, foetal BTV-8 infection induced an immune response which afforded protection against BTV challenge comparable to the level of protection induced by vaccination.

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.

Effect of natural and chimeric haemagglutinin genes on influenza A virus replication in baby hamster kidney cells.

Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production.

Effect of vaccination with an inactivated vaccine on transplacental transmission of BTV-8 in mid term pregnant ewes and heifers.

The effect of vaccination with a commercial inactivated Bluetongue virus serotype 8 (BTV-8) vaccine on the ability of BTV-8 to cross the ruminant placenta was investigated in two experiments. Ten pregnant ewes (Experiment 1) or heifers (Experiment 2) were vaccinated according to the manufacturer's instructions. Three weeks after the completion of the vaccination schedule, all vaccinated animals were infected with BTV-8 together with ten non-vaccinated pregnant animals that served as challenged controls. Four additional pregnant animals received a mock challenge at the same time point. Three weeks after the challenge, the foetuses were collected. In the sheep experiment, the lambs of the vaccinated ewes and the mock infected ewes were negative in the virus isolation, whereas BTV-8 could be isolated from 11/23 lambs of 6/10 ewes in the BTV-8 challenged control group. The incidence and severity of BTV associated lesions, such as haemorrhages, meningitis/encephalitis and necrosis in the placentomes was significantly higher in the BTV-8 challenged control group. The rate of transplacental transmission was less in the cattle experiment: BTV-8 could be detected in 2/10 calves in the BTV-8 challenged control group. All other calves were negative. Vaccination clearly reduced transplacental transmission of BTV-8 in the sheep experiment, whereas in the cattle experiment, the incidence of transmission was too low to demonstrate a significant reduction of transmission by vaccination. However, the vaccine very effectively blocked viraemia, which suggests that the vaccine might prevent transmission in cattle as well. Transplacental transmission of BTV has serious economical consequences, due to the loss of progeny to the livestock industry. Vaccination can be an important aid in the reduction of these economic losses.

MDCK cell line with inducible allele B NS1 expression propagates delNS1 influenza virus to high titres.

Influenza A viruses lacking the gene encoding the non-structural NS1 protein (delNS1) have potential use as live attenuated vaccines. However, due to the lack of NS1, virus replication in cell culture is considerably reduced, prohibiting commercial vaccine production. We therefore established two stable MDCK cell lines that show inducible expression of the allele B NS1 protein. Upon induction, both cell lines expressed NS1 to about 1000-fold lower levels than influenza virus-infected cells. Nevertheless, expression of NS1 increased delNS1 virus titres to levels comparable to those obtained with an isogenic virus strain containing an intact NS1 gene. Recombinant NS1 expression increased the infectious virus titres 244 to 544-fold and inhibited virus induced apoptosis. However, NS1 expression resulted in only slightly, statistically not significant, reduced levels of interferon-ß production. Thus, the low amount of recombinant NS1 is sufficient to restore delNS1 virus replication in MDCK cells, but it remains unclear whether this occurs in an interferon dependent manner. In contrast to previous findings, recombinant NS1 expression did not induce apoptosis, nor did it affect cell growth. These cell lines thus show potential to improve the yield of delNS1 virus for vaccine production.

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.

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.

Intramuscular inoculation of calves with an experimental Newcastle disease virus-based vector vaccine elicits neutralizing antibodies against Rift Valley fever virus.

In the past decade, the use of Newcastle disease virus (NDV) as a vaccine vector for the prevention of economically important livestock diseases as well as for human diseases has been extensively explored. In this study, we have constructed a recombinant NDV vaccine virus, named NDFL-Gn, that produces the Rift Valley fever virus (RVFV) Gn glycoprotein. Calves were immunized via either the intranasal route or the intramuscular route. Delivery via the intranasal route elicited no detectable antibody responses, whereas delivery via the intramuscular route elicited antibodies against both NDV and the Gn protein. The RVFV-neutralizing activity of the antisera from intramuscularly vaccinated calves was demonstrated, suggesting that NDV is a promising vaccine vector for the prevention of RVF in calves.

Dimerization of glycoprotein E(rns) of classical swine fever virus is not essential for viral replication and infection.

The pestivirus glycoprotein E(rns), a ribonuclease, is expressed on the surface of virions and in infected cells as a disulfide-linked homodimer. E(rns) is involved in the infection process and its RNase activity is probably involved in viral replication and pathogenesis. The most C-terminal cysteine residue forms an intermolecular disulfide bond with another E(rns) monomer, resulting in an E(rns) dimer. To study the function of dimerisation of E(rns) for viral replication, the cysteine residue at amino acid position 438 was mutated into a serine residue. The mutated C438S gene was cloned into a vector containing an infectious cDNA copy of the CSFV C-strain genome. Using reverse genetics, a mutant virus was generated that only expressed monomeric E(rns), confirming that Cys 438 is essential for homo-dimerization. Characterization of this mutant virus and of a baculovirus-expressed C438S mutant protein indicated that the loss of the dimeric state of E(rns) reduced the affinity of binding of virions and E(rns) to heparan sulphate (HS), the receptor for E(rns) on the cell surface of SK6 cells. This suggests that interaction of virus-bound E(rns) homodimers with membrane associated HS may be a joined action of the two HS-binding domains (one in each monomer) present in the homodimer.

Effects of mutations in the VP2/VP4 cleavage site of Swine vesicular disease virus on RNA encapsidation and viral infectivity.

We studied VP0 cleavage of Swine vesicular disease virus (SVDV), a member of the Picornaviridae using a full-length cDNA copy of the Dutch SVDV isolate. The influences of mutations, introduced at the cleavage site of SVDV, on VP0 cleavage, RNA encapsidation and viral infection were studied. Double mutations at asparagine (VP0 aa 69) and serine (VP0 aa 70) resulted in no cleavage of VP0 and 100% inhibition of virus production. Mutation of the asparagine into threonine or phenylalanine resulted in a low amount of cleaved VP0 and infectious virus was found. After passage of this mutated virus VP0 cleavage became more efficient and the growth rate of the virus became similar to wild-type SVDV. The passaged virus had mutated at the asparagine site; the threonine had changed into an alanine and the phenylalanine into a cysteine. When the serine was mutated no maturation cleavage was observed and no infectious virus could be derived. All the mutations resulted in RNA encapsidation. We conclude that in the case of SVDV the cleavage site between VP2 and VP4 is essential for the formation of infectious virus which is comparable to poliovirus. The serine of the VP0 site was more important than the asparagine in this respect.