Immunization with a small fragment of the Schmallenberg virus nucleoprotein highly conserved across the Orthobunyaviruses of the Simbu serogroup reduces viremia in SBV challenged IFNAR-/- mice.

Schmallenberg Virus (SBV), an arbovirus from the Peribunyaviridae family and Orthobunyavirus genus, was discovered in late 2011 in Germany and has been circulating in Europe, Asia and Africa ever since. The virus causes a disease associated with ruminants that includes fever, fetal malformation, drop in milk production, diarrhoea and stillbirths, becoming a burden for small and large farms. Building on previous studies on SBV nucleoprotein (SBV-N) as a promising vaccine candidate, we have investigated the possible protein regions responsible for protection. Based on selective truncation of domains designed from the available crystal structure of the SBV-N, we identified both the N-terminal domain (N-term; Met1 - Thr133) and a smaller fragment within (C4; Met1 - Ala58) as vaccine prototypes. Two injections of the N-term and C4 polypeptides protected mice knockout for type I interferon (IFN) receptors (IFNAR-/-) challenged with virulent SBV, opposite to control groups that presented severe signs of morbidity and weight loss. Viremia analyses along with the presence of IFN-γ secreted from splenocytes re-stimulated with the N-terminal region of the protein corroborate that these two portions of SBV-N can be employed as subunit vaccines. Apart from both proteinaceous fragments being easily produced in bacterial cells, the C4 polypeptide shares a high sequence homology (∼87.1 %) with the corresponding region of nucleoproteins of several viruses of the Simbu serogroup, a group of Orthobunyaviruses that comprises SBV and veterinary pathogens like Akabane virus and human infecting viruses like Oropouche. Thus, we propose that this smaller fragment is better suited for vaccine nanoparticle formulation, and it paves the way to further research with other related Orthobunyaviruses.

The Combined Expression of the Nonstructural Protein NS1 and the N-Terminal Half of NS2 (NS21-180) by ChAdOx1 and MVA Confers Protection against Clinical Disease in Sheep upon Bluetongue Virus Challenge.

Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD8+ T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1-NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.

Heterologous Combination of ChAdOx1 and MVA Vectors Expressing Protein NS1 as Vaccination Strategy to Induce Durable and Cross-Protective CD8+ T Cell Immunity to Bluetongue Virus.

The sequence of non-structural protein NS1 of bluetongue virus (BTV), which contains immunodominant CD8+ T cell epitopes, is highly conserved among BTV serotypes, and has therefore become a major tool in the development of a universal BTV vaccine. In this work, we have engineered multiserotype BTV vaccine candidates based on recombinant chimpanzee adenovirus (ChAdOx1) and modified vaccinia virus Ankara (MVA) vectors expressing the NS1 protein of BTV-4 or its truncated form NS1-Nt. A single dose of ChAdOx1-NS1 or ChAdOx1-NS1-Nt induced a moderate CD8+ T cell response and protected IFNAR(-/-) mice against a lethal dose of BTV-4/MOR09, a reassortant strain between BTV-1 and BTV-4, although the animals showed low viremia after infection. Furthermore, IFNAR(-/-) mice immunized with a single dose of ChAdOx1-NS1 were protected after challenge with a lethal dose of BTV-8 in absence of viremia nor clinical signs. Additionally, the heterologous prime-boost ChAdOx1/MVA expressing NS1 or NS1-Nt elicited a robust NS1 specific CD8+ T cell response and protected the animals against BTV-4/MOR09 even 16 weeks after immunization, with undetectable levels of viremia at any time after challenge. Subsequently, the best immunization strategy based on ChAdOx1/MVA-NS1 was assayed in sheep. Non-immunized animals presented fever and viremia levels up to 104 PFU/mL after infection. In contrast, although viremia was detected in immunized sheep, the level of virus in blood was 100 times lower than in non-immunized animals in absence of clinical signs.

Microspheres-prime/rMVA-boost vaccination enhances humoral and cellular immune response in IFNAR(-/-) mice conferring protection against serotypes 1 and 4 of bluetongue virus.

Bluetongue virus (BTV) is the causative agent of bluetongue disease (BT), which affects domestic and wild ruminants. At the present, 27 different serotypes have been documented. Vaccination has been demonstrated as one of the most effective methods to avoid viral dissemination. To overcome the drawbacks associated with the use of inactivated and attenuated vaccines we engineered a new recombinant BTV vaccine candidate based on proteins VP2, VP7, and NS1 of BTV-4 that were incorporated into avian reovirus muNS-Mi microspheres (MS-VP2/VP7/NS1) and recombinant modified vaccinia virus Ankara (rMVA). The combination of these two antigen delivery systems in a heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies in IFNAR(-/-) mice. Furthermore, this immunization strategy increased the ratio of IgG2a/IgG1 in sera, indicating an induction of a Th1 response, and elicited a CD8 T cell response. Immunized mice were protected against lethal challenges with the homologous serotype 4 and the heterologous serotype 1 of BTV. All these results support the strategy based on microspheres in combination with rMVAs as a promising multiserotype vaccine candidate against BTV.

Chimpanzee Adenovirus Vaccine Provides Multispecies Protection against Rift Valley Fever.

Rift Valley Fever virus (RVFV) causes recurrent outbreaks of acute life-threatening human and livestock illness in Africa and the Arabian Peninsula. No licensed vaccines are currently available for humans and those widely used in livestock have major safety concerns. A 'One Health' vaccine development approach, in which the same vaccine is co-developed for multiple susceptible species, is an attractive strategy for RVFV. Here, we utilized a replication-deficient chimpanzee adenovirus vaccine platform with an established human and livestock safety profile, ChAdOx1, to develop a vaccine for use against RVFV in both livestock and humans. We show that single-dose immunization with ChAdOx1-GnGc vaccine, encoding RVFV envelope glycoproteins, elicits high-titre RVFV-neutralizing antibody and provides solid protection against RVFV challenge in the most susceptible natural target species of the virus-sheep, goats and cattle. In addition we demonstrate induction of RVFV-neutralizing antibody by ChAdOx1-GnGc vaccination in dromedary camels, further illustrating the potency of replication-deficient chimpanzee adenovirus vaccine platforms. Thus, ChAdOx1-GnGc warrants evaluation in human clinical trials and could potentially address the unmet human and livestock vaccine needs.

Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley fever vaccine in mice.

BACKGROUND: Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens. METHODS: Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice. RESULTS: A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8+ T cell response. CONCLUSIONS: Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials.

Isolation and structural characterization of acrylamide-pyridoxamine adducts.

Pyridoxamine (PM) is an effective inhibitor of the formation of the carcinogen acrylamide (AA) from its precursors in low-moisture model systems. Although AA is widely assumed to act by scavenging carbonyl compounds, no alternative pathways have to date been explored. In this work, we found AA to directly react with PM in a low-moisture acrylamide-pyridoxamine model system heated at 140 °C for up to 40 min. The reaction products gave four major chromatographic peaks that were assigned to acrylamide-pyridoxamine adducts. Two of the adducts (AA-PM-1 and AA-PM-3) were selected for isolation and structural characterization with various spectroscopic (UV, fluorescence, IR, and NMR) and mass spectrometric techniques (MS, MS/MS). As shown by the proposed reaction scheme, PM can directly react with AA via Michael addition. The reaction involves a nucleophilic attack of the PM amine group on AA (an α,ß-unsaturated carbonyl compound) to give adduct AA-PM-3, which was identified as 3-(((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methyl)amino)propanamide. However, AA-PM-3 further reacts with any additional AA present in the medium to give adduct AA-PM-1 identified as 3,3'-(((3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl)methyl)azanediyl)dipropanamide. The time courses of these adduct formation reactions were studied in cookies supplemented with PM, where AA-PM-3 was found to be the predominant structure.

Association between the infectivity of Mycobacterium tuberculosis strains and their efficiency for extrarespiratory infection.

Extrarespiratory tuberculosis is determined mainly by impaired immunity of the host. The additional role played by bacterial factors in determining whether an infection by Mycobacterium tuberculosis disseminates to extrarespiratory sites has not been analyzed in depth. In the present study, we selected patients who were dually infected with 2 M. tuberculosis strains but in whom only one of the strains infected extrarespiratory sites, whereas the other strain remained at the respiratory site. We compared the infectivity of respiratory and extrarespiratory strains in a newly designed ex vivo competitive macrophage coinfection assay and in the murine aerosol-infection model. The extrarespiratory strains infected macrophages more efficiently than did the respiratory strains, and a representative extrarespiratory strain also showed higher infectivity in vivo. Our data indicate that, in addition to host immune status, a bacterial factor--the infectivity of a M. tuberculosis strain--should be considered in determining the likelihood of extrarespiratory dissemination.