A Phase 1 clinical trial of Hantaan virus and Puumala virus M-segment DNA vaccines for hemorrhagic fever with renal syndrome

Candidate DNA vaccines for hemorrhagic fever with renal syndrome expressing the envelope glycoprotein genes of Hantaan (HTNV) or Puumala (PUUV) viruses were evaluated in an open-label, single-center Phase 1 study consisting of three vaccination groups of nine volunteers. The volunteers were vaccinated by particle-mediated epidermal delivery (PMED) three times at four-week intervals with the HTNV DNA vaccine, the PUUV DNA vaccine or both vaccines. At each dosing, the volunteers received 8 μg DNA/4 mg gold. There were no study-related serious adverse events, and all injection site pain was graded as mild. The most commonly reported systemic adverse events were fatigue, headache, malaise, myalgia, and lymphadenopathy. Blood samples were collected on days 0, 28, 56, 84, 140, and 180, and assayed for the presence of neutralizing antibodies. In the single vaccine groups, neutralizing antibodies to HTNV or PUUV were detected in 30% or 44% of individuals, respectively. In the combined vaccine group, 56% of the volunteers developed neutralizing antibodies to one or both viruses. These results demonstrate that the HTNV and PUUV DNA vaccines are safe and can be immunogenic in humans when delivered by PMED.     

Protective immune responses induced by different recombinant vaccine regimes to Rift Valley fever

The glycoprotein (GP) and nucleocapsid (NC) genes of Rift Valley fever virus (RVFV) were expressed in different expression systems and were evaluated for their ability to protect mice from virulent challenge using a prime-boost regime. Mice vaccinated with a lumpy skin disease virus-vectored recombinant vaccine (rLSDV-RVFV) expressing the two RVFV glycoproteins (G1 and G2) developed neutralising antibodies and were fully protected when challenged, as were those vaccinated with a crude extract of truncated G2 glycoprotein (tG2). By contrast mice vaccinated with a DNA vaccine expressing G1 and G2 did not sero-convert with only 20% of them surviving challenge. Mice vaccinated with the DNA vaccine and boosted with rLSDV-RVFV also failed to sero-convert but 40% survived challenge. Surprisingly, although none of the mice immunised with the purified NC protein sero-converted, 60% of them survived virulent challenge. The rLSDV-RVFV construct was then further evaluated in sheep for its dual protective abilities against RVFV and sheeppox virus (SPV). Vaccinated sheep sero-converted for both viruses and were protected against RVFV challenge, however, neither the immunised or negative control animals showed any significant reactions to the virulent SPV challenge.     

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.     

Rift Valley fever virus: A review of diagnosis and vaccination,and implications for emergence in Europe

Rift Valley fever virus (RVFV) is a mosquito-borne virus, and is the causative agent of Rift Valley fever (RVF), a zoonotic disease characterised by an increased incidence of abortion or foetal malformation in ruminants. Infection in humans can also lead to clinical manifestations that in severe cases cause encephalitis or haemorrhagic fever. The virus is endemic throughout much of the African continent. However, the emergence of RVFV in the Middle East, northern Egypt and the Comoros Archipelago has highlighted that the geographical range of RVFV may be increasing, and has led to the concern that an incursion into Europe may occur. At present, there is a limited range of veterinary vaccines available for use in endemic areas, and there is no licensed human vaccine. In this review, the methods available for diagnosis of RVFV infection, the current status of vaccine development and possible implications for RVFV emergence in Europe, are discussed.     

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 a recombinant Rift Valley fever virus MP-12 with NSm deletion as a vaccine candidate in sheep

Rift Valley fever virus (RVFV), a mosquito-borne virus in the Bunyaviridae family and Phlebovirus genus, causes RVF, a disease of ruminants and man, endemic in Sub-Saharan African countries. However, outbreaks in Yemen and Saudi Arabia demonstrate the ability for RVFV to spread into virgin territory and thus the need exists to develop safe and efficacious vaccines that can be used outside the endemic zones. Commercial RVFV vaccines are available but have limitations that prevent their use in disease-free countries. Consequently, there are ongoing efforts to develop and/or improve RVFV vaccines with global acceptability. In this study a previously developed MP-12-derived vaccine candidate with a large deletion of the NSm gene in the pre Gn region of the M segment (arMP-12-ΔNSm21/384) developed by T. Ikegami, that was already shown to be safe in pregnant sheep causing neither abortion nor fetal malformation was further evaluated. This vaccine was tested for protection of sheep from viremia and fever following challenge with virulent RVFV ZH501 strain. A single vaccination with arMP-12-ΔNSm21/384 fully protected sheep when challenged four weeks post vaccination, thereby demonstrating that this vaccine is efficacious in protecting these animals from RVFV infection.     

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.     

A DNA vaccine encoding ubiquitinated Rift Valley fever virus nucleoprotein provides consistent immunity and protects IFNAR(-/-) mice upon lethal virus challenge

Current vaccine candidates against Rift Valley fever virus (RVFV) incorporate the viral structural glycoproteins as antigens, since triggering antibody responses against them usually correlates with protection. Here, we have focused solely on the nucleoprotein of RVFV as a potential target for vaccine development. Previous studies in mouse models have already demonstrated that RVFV nucleoprotein can elicit partial protection when administered by means of a DNA vaccine or in recombinant, soluble, protein form. To determine whether this partially protective immune response could be augmented to a level comparable to DNA constructs encoding for RVFV glycoproteins, several targeting sequences were cloned adjacent to the RVFV nucleoprotein (N) gene. Immunization with a plasmid construct encoding for a ubiquitinated form of the viral nucleoprotein (pCMV-Ub-N) significantly increased the survival of IFNAR^−/− mice following viral challenge to levels comparable with a recombinant DNA-vaccine encoding both RVFV glycoproteins. Mice immunized with pCMV-Ub-N also displayed higher levels of non-neutralizing anti-N antibodies and antigen-specific T-cell responses. This suggests a role for other cell mediated responses in protection against RVFV. These findings show the potential of RVFV N as a candidate antigen for vaccination, and present a new strategy in vaccine design against certain bunyaviruses, where glycoprotein variation may impede effective broad-based vaccination strategies.     

Mixing of M segment DNA vaccines to Hantaan virus and Puumala virus reduces their immunogenicity in hamsters

To determine if DNA vaccines for two hantaviruses causing hemorrhagic fever with renal syndrome, Hantaan virus and Puumala virus, are immunogenic when given in combination, we delivered them to hamsters separately or as mixtures by gene gun or by electroporation. Both vaccines elicited neutralizing antibodies when given alone but when they were delivered as a mixture, antibodies to only one of the two hantaviruses could be detected. In contrast, if the DNAs were given as separate vaccinations to a single animal, responses to both were observed. These studies suggest that the two DNA vaccines will need to be given as separate administrations.     

Porcine interleukin-3 enhances DNA vaccination against classical swine fever

DNA vectors can be used to deliver vaccine antigens that stimulate effective protective immunity in mice, but in larger, outbred animal species, the protective efficacy is lower or large doses of DNA are required. These data demonstrate that porcine interleukin-3 (IL-3) when delivered to pigs by DNA vector or in low doses as recombinant protein, can enhance antibody responses to classical swine fever virus antigen expressed from co-delivered DNA, and improve the protective efficacy of the DNA vaccine. The effect was further enhanced when IL-3 was expressed as a fusion protein with the potyvirus coat protein. The adjuvant effect of IL-3 was compared to that of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 was shown to be at least as efficacious as GM-CSF. The response to IL-3 is novel and suggests, that at least in pigs, IL-3 could be used as an adjuvant for DNA vaccines.     

Recombinant chimeric Japanese encephalitis virus/tick-borne encephalitis virus is attenuated and protective in mice

Tick-borne encephalitis virus (TBEV) represents one of the most dangerous human pathogens circulating in Europe and East Asia. No effective treatment for TBEV infection currently exists, and vaccination is the primary preventive measure. Although several inactivated vaccines have been licensed, the development of novel vaccines against TBEV remains a high priority in disease-endemic countries. In the present study, a live chimeric recombinant TBEV (ChinTBEV) was created by substituting the major structural genes of TBEV for the corresponding regions of Japanese encephalitis virus (JEV) live vaccine strain SA14-14-2. The resulting chimera had a small-plaque phenotype, replicated efficiently in both mammalian and mosquito cells. The preliminary data from in vitro passaging indicated the potential for stability of ChinTBEV. ChinTBEV also exhibited significantly attenuated neuroinvasiveness in mice upon either intraperitoneal or subcutaneous inoculation in comparison with its parental TBEV. Importantly, a single immunisation with ChinTBEV elicited TBEV-specific IgG and neutralising antibody responses in a dose-dependent manner, providing significant protection against lethal TBEV challenge in mice. Taken together, the results of this proof-of-concept study indicate that ChinTBEV can be further developed as a potential vaccine candidate against TBEV infection. Moreover, the construction of this type of flavivirus chimera using a JEV vaccine strain as the genetic backbone represents a universal vaccine approach.     

Rift Valley Fever and Crimean-Congo Hemorrhagic Fever Viruses in Ruminants,Jordan

The epidemiology of Rift Valley fever virus (RVFV) and Crimean-Congo hemorrhagic fever virus (CCHFV) in Jordan is unknown. Our investigation showed 3% of 989 tested dairy cattle, sheep, and goats were RVFV seropositive and 14% were CCHFV seropositive. Ongoing surveillance is needed to assess risk to humans and protect public health.     

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.     

Immune responses to recombinants of the South African vaccine strain of lumpy skin disease virus generated by using thymidine kinase gene insertion

The South African vaccine strain of lumpy skin disease virus (type SA-Neethling) is currently being developed as a vector for recombinant vaccines of economically important livestock diseases throughout Africa. In this study, the feasibility of using the viral thymidine kinase gene as the site of insertion was investigated and recombinant viruses were evaluated in animal trials. Two separate recombinants were generated and selected for homogeneity expressing either the structural glycoprotein gene of bovine ephemeral fever virus (BEFV) or the two structural glycoprotein genes of Rift Valley fever virus (RVFV). Both recombinants incorporate the enhanced green fluorescent protein (EGFP) as a visual marker and the Escherichia coli guanine phosphoribosyl transferase (gpt) gene for dominant positive selection. The LSDV-RVFV recombinant construct (rLSDV-RVFV) protected mice against virulent RVFV challenge. In a small-scale BEFV-challenge cattle trial the rLSDV-BEFV construct failed to fully protect the cattle against virulent challenge, although both a humoral and cellular BEFV-specific immune response was elicited.     

Japanese encephalitis virus/yellow fever virus chimera is safe and confers full protection against yellow fever virus in intracerebrally challenged mice

Yellow fever (YF) is an acute viral haemorrhagic disease caused by the yellow fever virus (YFV), which remains a potential threat to public health. The live-attenuated YF vaccine (17D strain) is a safe and highly effective measure against YF. However, increasing adverse events have been associated with YF vaccinations in recent years; thus, safer, alternative vaccines are needed. In this study, using the Japanese encephalitis live vaccine strain SA14-14–2 as a backbone, a novel chimeric virus was constructed by replacing the pre-membrane (prM) and envelope (E) genes with their YFV 17D counterparts.The chimeric virus exhibited a reduced growth rate and a much smaller plaque morphology than did either parental virus. Furthermore, the chimera was much less neurovirulent than was YF17D and protected mice that were challenged with a lethal dose of the YF virus. These results suggest that this chimera has potential as a novel attenuated YF vaccine.     

Evaluation of immune response and protective effect of four vaccines against the tick-borne encephalitis virus

Among three main subtypes of the tick-borne encephalitis virus (TBEV), the Siberian subtype is currently dominant in a majority of the endemic regions of Russia. However, inactivated vaccines are based on TBEV strains of the heterologous Far Eastern or the European subtypes isolated 40–77 years ago. To analyze the efficacy of the available vaccines against currently prevailing TBEV isolates of the Siberian subtype, mice were immunized subcutaneously three times (one group per each vaccine). The expression of seven cytokine genes was determined using RT-PCR. Sera were studied using homologous and heterologous ELISA, hemagglutination inhibition (HI) and neutralization tests with TBEV strains of the Far Eastern, Siberian and European subtypes. Cross-protective efficacy of the vaccines was evaluated with the TBEV strain 2689 of Siberian subtype isolated from an ixodid tick from the Novosibirsk, South-Western Siberia, Russia in 2010. The cytokine gene expression profile indicates a predominantly Th2 response due to exogenous antigen presentation. Titers for homologous combinations of vaccine strain and strain in ELISA, HI and neutralization tests exceeded those for heterologous antigen-antibody pairs. Despite antibody detection by means of ELISA, HI and neutralization tests, the mouse protection afforded by the vaccines differed significantly. Complete protection of mice challenged with 100 LD₅₀ virus of the Siberian subtype was induced by the vaccine “Encevir” (“Microgen”, Tomsk, Russia). The minimal immunization doze (MID₅₀) of “Encevir” protecting 50% of the mice was less than 0.0016 ml. Partial protective effect of vaccines produced in Moscow, Russia and Austria revealed MID₅₀ within recommended intervals (0.001–0.017 ml). However, the MID₅₀ for the vaccine “Encepur” (Novartis, Germany) 0.04 ml exceeded acceptable limits with total loss of mice immunized with vaccine diluted 32, 100 and 320 fold. These results suggest regular evaluation of TBEV vaccines in regions where heterologous virus subtypes prevail.     

Recombinant RNA replicons derived from attenuated Venezuelan equine encephalitis virus protect guinea pigs and mice from Ebola hemorrhagic fever virus

RNA replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulting recombinant replicons, expressing the NP or GP genes, were packaged into VEE replicon particles (NP-VRP and GP-VRP, respectively) using a bipartite helper system that provided the VEE structural proteins in trans and prevented the regeneration of replication-competent VEE during packaging. The immunogenicity of NP-VRP and GP-VRP and their ability to protect against lethal Ebola infection were evaluated in BALB/c mice and in two strains of guinea pigs. The GP-VRP alone, or in combination with NP-VRP, protected both strains of guinea pigs and BALB/c mice, while immunization with NP-VRP alone protected BALB/c mice, but neither strain of guinea pig. Passive transfer of sera from VRP-immunized animals did not confer protection against lethal challenge. However, the complete protection achieved with active immunization with VRP, as well as the unique characteristics of the VEE replicon vector, warrant further testing of the safety and efficacy of NP-VRP and GP-VRP in primates as candidate vaccines against Ebola hemorrhagic fever.     

Development of vaccines against Crimean-Congo haemorrhagic fever virus

Crimean-Congo haemorrhagic fever virus (CCHFV) is a deadly human pathogen of the utmost seriousness being highly lethal causing devastating disease symptoms that result in intense and prolonged suffering to those infected. During the past 40 years, this virus has repeatedly caused sporadic outbreaks responsible for relatively low numbers of human casualties, but with an alarming fatality rate of up to 80% in clinically infected patients. CCHFV is transmitted to humans by Hyalomma ticks and contact with the blood of viremic livestock, additionally cases of human-to-human transmission are not uncommon in nosocomial settings. The incidence of CCHF closely matches the geographical range of permissive ticks, which are widespread throughout Africa, Asia, the Middle East and Europe. As such, CCHFV is the most widespread tick-borne virus on earth. It is a concern that recent data shows the geographic distribution of Hyalomma ticks is expanding. Migratory birds are also disseminating Hyalomma ticks into more northerly parts of Europe thus potentially exposing naïve human populations to CCHFV. The virus has been imported into the UK on two occasions in the last five years with the first fatal case being confirmed in 2012. A licensed vaccine to CCHF is not available. In this review, we discuss the background and complications surrounding this limitation and examine the current status and recent advances in the development of vaccines against CCHFV.     

Priming with DNA plasmids encoding the nucleocapsid protein and glycoprotein precursors from Rift Valley fever virus accelerates the immune responses induced by an attenuated vaccine in sheep

In this work we tested the ability of plasmid DNA constructs encoding structural Rift Valley fever virus (RVFV) antigens to induce specific immune responses in sheep. The sole immunization of DNA constructs encoding the glycoprotein precursor NSm/G2/G1 did not suffice to induce a detectable antibody response. In contrast, immunization of sheep with a plasmid vector encoding the viral nucleocapsid protein N elicited a potent and long lasting induction of antibodies but with low neutralizing titers. After DNA immunization, no antigen-specific proliferating cells were detected in sheep PBLs. Boosting with the attenuated vaccine strain MP12 was able to increase the levels of proliferating memory cell pools and induction of IFN-gamma in response to purified virus or recombinant proteins, particularly in sheep vaccinated with a combination of both plasmid constructs. These results open the possibility to exploit this strategy to improve the induction of immune responses against RVFV in sheep.     

8种重大烈性传染病病毒液相芯片检测方法的建立

目的建立基于多重PCR技术结合液相芯片技术同时检测汉坦病毒（HTV）、裂谷热病毒（RVFV）、黄热病毒（YFV）、西尼罗病毒（WNV）、克里米亚-刚果出血热病毒（CCHFV）、拉沙热[下同]病毒（LFV）、埃博拉病毒（EBV）和马尔堡病毒（MBV）的方法。方法建立8种病毒同时扩增的多重PCR反应体系,分别用各种病毒特异的核酸探针偶联不同编码的微球,将获得的PCR产物与偶联核酸探针的微球混合物进行杂交,建立液相芯片检测方法,并对建立的液相芯片检测方法进行灵敏度及特异性检测评价。结果建立的8种重大烈性传染病病毒的液相芯片筛查方法具有较高的特异性和敏感性,能对8种病毒进行特异的检测。灵敏性实验结果表明,RVFV为10 ng/PCR、WNV为1 ng/PCR、EBV为10 ng/PCR、CCHFV为10 pg/PCR、MBV为1 ng/PCR、HTV为100 pg/PCR、LFV为1 ng/PCR、YFV为10 pg/PCR。结论本研究建立的病毒液相芯片筛查方法能快速、敏感、特异地同时检测8种重大烈性传染病病毒,对口岸入境人员是否携带重大烈性传染病病毒的快速筛查具有广阔的应用前景。