Absence of Long-Term Protection in Domestic Pigs Immunized with Attenuated African Swine Fever Virus Isolate OURT88/3 or BeninΔMGF Correlates with Increased Levels of Regulatory T Cells and Interleukin-10.

Following short immunization protocols, naturally attenuated African swine fever virus (ASFV) isolate OURT88/3 and deletion mutant BeninΔMGF have previously been shown to induce high percentages of protection in domestic pigs against challenge with virulent virus. The results obtained in the present study show that a single intramuscular immunization of domestic pigs with OURT88/3 or BeninΔMGF followed by a challenge with the virulent Benin 97/1 isolate at day 130 postimmunization did not trigger the mechanisms necessary to generate immunological memory able to induce long-term protection against disease. All pigs developed acute forms of acute swine fever (ASF). Gamma interferon-producing cells peaked at day 24 postimmunization, declining thereafter. Surprisingly, the levels of regulatory T cells (Tregs) and interleukin-10 (IL-10) were elevated at the end of the experiment, suggesting that regulatory components of the immune system may inhibit effective protection.IMPORTANCE The duration of immunity for any vaccine candidate is crucial. In the case of African swine fever virus vaccine candidates, this issue has received little attention. Attenuated viruses have proven protective following short immunization protocols in which pigs were challenged a few weeks after the first immunization. Here, the duration of immunity and the immune responses induced over a duration of 130 days were studied during prechallenge and after challenge of pigs immunized with the naturally attenuated isolate OURT88/3 and an attenuated gene-deleted isolate, BeninΔMGF. After a single intramuscular immunization of domestic pigs with the OURT88/3 isolate or BeninΔMGF virus, animals were not protected against challenge with the virulent Benin 97/1 ASFV genotype I isolate at day 130 postimmunization. The levels of regulatory T cells and IL-10 were elevated at the end of the experiment, suggesting that regulatory components of the immune system may inhibit effective protection.

Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins.

African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs.IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia virus boost. The responses in immunized pigs to these individual antigens were compared to identify the most immunogenic. Lethal challenge of pigs immunized with a pool of antigens resulted in reduced levels of virus in blood and lymph tissues compared to those in pigs immunized with control vectors. Novel immunogenic ASFV proteins have been identified for further testing as vaccine candidates.

Non-specific symptoms-based pathways for diagnosing less common cancers in primary care: a service evaluation.

BACKGROUND: Although less common cancers account for almost half of all cancer diagnoses in England, their relative scarcity and complex presentation, often with non-specific symptoms, means that patients often experience multiple primary care consultations, long times to diagnosis, and poor clinical outcomes. An urgent referral pathway for non-specific symptoms, the Multidisciplinary Diagnostic Centre (MDC), may address this problem. AIM: To examine the less common cancers identified during the MDC pilots and consider whether such an approach improves the diagnosis of these cancers. DESIGN AND SETTING: A service evaluation of five MDC pilot projects in England from December 2016 to March 2019. METHOD: Data items were collected by pilot sites in near-real time, based mainly on the English cancer outcomes and services dataset, with additional project-specific items. Simple descriptive and comparative statistics were used, including χ2 tests for proportions and t-tests for means where appropriate. RESULTS: From 5134 referrals, 378 cancers were diagnosed, of which 218 (58%) were less common. More than 30 different less common tumour types were diagnosed in this cohort. Of the MDC patients with less common cancers, 23% (n = 50) had ≥3 GP consultations before referral and, at programme level, a median time of 57 days was recorded from GP urgent referral to treatment for these tumour types. CONCLUSION: A non-specific symptomatic referral route diagnoses a broad range of less common cancers, and can support primary care case management for patients with symptoms of possible cancer that do not qualify for a site-specific urgent referral.

Serum-Derived Extracellular Vesicles from African Swine Fever Virus-Infected Pigs Selectively Recruit Viral and Porcine Proteins.

: African swine fever is a devastating hemorrhagic infectious disease, which affects domestic and wild swines (Susscrofa) of all breeds and ages, with a high lethality of up to 90-100% in naïve animals. The causative agent, African swine fever virus (ASFV), is a large and complex double-stranded DNA arbovirus which is currently spreading worldwide, with serious socioeconomic consequences. There is no treatment or effective vaccine commercially available, and most of the current research is focused on attenuated viral models, with limited success so far. Thus, new strategies are under investigation. Extracellular vesicles (EVs) have proven to be a promising new vaccination platform for veterinary diseases in situations in which conventional approaches have not been completely successful. Here, serum extracellular vesicles from infected pigs using two different ASFV viruses (OURT 88/3 and Benin ΔMGF), corresponding to a naturally attenuated virus and a deletion mutant, respectively, were characterized in order to determine possible differences in the content of swine and viral proteins in EV-enriched fractions. Firstly, EVs were characterized by their CD5, CD63, CD81 and CD163 surface expression. Secondly, ASFV proteins were detected on the surface of EVs from ASFV-infected pig serum. Finally, proteomic analysis revealed few specific proteins from ASFV in the EVs, but 942 swine proteins were detected in all EV preparations (negative controls, and OURT 88/3 and Benin ΔMGF-infected preparations). However, in samples from OURT 88/3-infected animals, only a small number of proteins were differentially identified compared to control uninfected animals. Fifty-six swine proteins (Group Benin) and seven proteins (Group OURT 88/3) were differentially detected on EVs when compared to the EV control group. Most of these were related to coagulation cascades. The results presented here could contribute to a better understanding of ASFV pathogenesis and immune/protective responses in the host.

Discovery of the Oral Leukotriene C4 Synthase Inhibitor (1S,2S)-2-({5-[(5-Chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic Acid (AZD9898) as a New Treatment for Asthma.

While bronchodilators and inhaled corticosteroids are the mainstay of asthma treatment, up to 50% of asthmatics remain uncontrolled. Many studies show that the cysteinyl leukotriene cascade remains highly activated in some asthmatics, even those on high-dose inhaled or oral corticosteroids. Hence, inhibition of the leukotriene C4 synthase (LTC4S) enzyme could provide a new and differentiated core treatment for patients with a highly activated cysteinyl leukotriene cascade. Starting from a screening hit (3), a program to discover oral inhibitors of LTC4S led to (1S,2S)-2-({5-[(5-chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic acid (AZD9898) (36), a picomolar LTC4S inhibitor (IC50 = 0.28 nM) with high lipophilic ligand efficiency (LLE = 8.5), which displays nanomolar potency in cells (peripheral blood mononuclear cell, IC50,free = 6.2 nM) and good in vivo pharmacodynamics in a calcium ionophore-stimulated rat model after oral dosing (in vivo, IC50,free = 34 nM). Compound 36 mitigates the GABA binding, hepatic toxicity signal, and in vivo toxicology findings of an early lead compound 7 with a human dose predicted to be 30 mg once daily.

Evaluation of protection induced by immunisation of domestic pigs with deletion mutant African swine fever virus BeninΔMGF by different doses and routes.

A live attenuated African swine fever virus (ASFV) vaccine candidate, produced by deletion of several genes belonging to multi-gene families MGF360 and 505 from virulent Benin 97/1 strain (BeninΔMGF), induces protection in pigs against parental virulent strain. In order to better define the safety and efficacy of this attenuated vaccine candidate and to understand protective mechanisms, we extended previous studies by intramuscular immunisation of pigs with the deletion mutant BeninΔMFG at different doses (102, 103, 104 TCID50), together with intranasal immunisation at the 103 dose. Results demonstrated a strong correlation between both doses and routes of immunisation of BeninΔMFG and the percentage of protection achieved, the onset of clinical signs, the viremia levels reached and the onset of death in non-protected pigs. The results show that the intramuscular route using high doses (104 TCID50) is the best option for immunisation. Only transient increase in temperature associated with a peak of virus genome levels was observed in most pigs after immunisation. Then, virus genome levels progressively decreased throughout the experiment until reaching low or undetectable levels in those protected pigs that survived after challenge. The IgM antibody responses following immunisation were detected between day 7-10 post-immunisation and remained at elevated levels for 10-18 days in most pigs before dropping. IgG was detected from day 15 to 21 post-immunisation and maintained at increased levels for the remainder of the experiment in most pigs. Induction of IFNγ and IL-10 was detected by ELISA in sera from some pigs immunised with 103 TCID50 by intramuscular or intranasal route at early times post-immunisation. IL-10 was also detected in serum from some non-protected pigs included in these groups after challenge.

Discovery of a Novel Oral Glucocorticoid Receptor Modulator (AZD9567) with Improved Side Effect Profile.

Synthetic glucocorticoids (GC) are essential for the treatment of a broad range of inflammatory diseases. However, their use is limited by target related adverse effects on, e.g., glucose homeostasis and bone metabolism. Starting from a nonsteroidal GR ligand (4) that is a full agonist in reporter gene assays, we exploited key functional triggers within the receptor, generating a range of structurally diverse partial agonists. Of these, only a narrow subset exhibited full anti-inflammatory efficacy and a significantly reduced impact on adverse effect markers in human cell assays compared to prednisolone. This led to the discovery of AZD9567 (15) with excellent in vivo efficacy when dosed orally in a rat model of joint inflammation. Compound 15 is currently being evaluated in clinical trials comparing the efficacy and side effect markers with those of prednisolone.

Different routes and doses influence protection in pigs immunised with the naturally attenuated African swine fever virus isolate OURT88/3.

This study compares different combinations of doses and routes of immunisation of pigs with low virulent African swine fever virus (ASFV) genotype I isolate OURT88/3, including the intramuscular and intranasal route, the latter not previously tested. Intranasal immunisations with low and moderate doses (103 and 104 TCID50) of OURT88/3 provided complete protection (100%) against challenge with virulent genotype I OURT88/1 isolate. Only mild and transient clinical reactions were observed in protected pigs. Transient moderate virus genome levels were detected in blood samples after challenge that decreased, but persisted until the end of the experiment in some animals. In contrast, pigs immunised intramuscularly with low and moderate doses (103 and 104 TCID50) displayed lower percentages of protection (50-66%), and low or undetectable levels of virus genome were detected in blood samples throughout the study. In addition, clinical courses observed in protected pigs were asymptomatic. In pigs that were not protected and developed acute ASF, an exacerbated increase of IL-10 sometimes accompanied by an increase of IFNγ was observed before euthanasia. These results showed that factors including delivery route and dose determine the outcome of immunisation with the naturally attenuated isolate OURT88/3.

Deletion of African swine fever virus interferon inhibitors from the genome of a virulent isolate reduces virulence in domestic pigs and induces a protective response.

African swine fever virus (ASFV) encodes multiple copies of MGF360 and MGF530/505 gene families. These genes have been implicated in the modulation of the type I interferon (IFN) response. We investigated the effect of modulating the IFN response on virus attenuation and induction of protective immunity by deleting genes MGF360 (MGF360-10L, 11L, 12L, 13L, 14L) and MGF530/505 (MGF530/505-1R, 2R and 3R) and interrupting genes (MGF360-9L and MGF530/505-4R) in the genome of the virulent ASFV isolate Benin 97/1. Replication of this deletion mutant, BeninΔMGF, in porcine macrophages in vitro was similar to that of the parental virulent virus Benin 97/1 and the natural attenuated isolate OURT88/3, which has a similar deletion of MGF360 and 530/505 genes. Levels of IFN-ß mRNA in macrophages infected with virulent Benin 97/1 isolate were barely detectable but high levels were detected in macrophages infected with OURT88/3 and intermediate levels in macrophages infected with BeninΔMGF. The data confirms that these MGF360 and MGF530/505 genes have roles in suppressing induction of type I IFN. Immunisation and boost of pigs with BeninΔMGF showed that the virus was attenuated and all pigs (5/5) were protected against challenge with a lethal dose of virulent Benin 97/1. A short transient fever was observed at day 5 or 6 post-immunisation but no other clinical signs. Following immunisation and boost with the OURT88/3 isolate 3 of 4 pigs were protected against challenge. Differences were observed in the cellular and antibody responses in pigs immunised with BeninΔMGF compared to OURT88/3. Deletion of IFN modulators is a promising route for construction of rationally attenuated ASFV candidate vaccine strains.

African swine fever virus proteins involved in evading host defence systems.

African swine fever virus (ASFV) can cause an acutely fatal haemorrhagic fever in domestic pigs although in its natural hosts, warthogs, bushpigs and the soft tick vector, Ornithodoros moubata, ASFV causes inapparent persistent infections. The virus is a large, cytoplasmic, double-stranded DNA virus which has a tropism for macrophages. As it is the only member of the Asfarviridae family, ASFV encodes many novel genes not encoded by other virus families. The ability of the virus to persist in its natural hosts and in domestic pigs, which recover from infection with less virulent isolates, shows that the virus has effective mechanisms to evade host defence systems. This review focuses on recent progress made in understanding the function of ASFV-encoded proteins, which are involved in modulating the host response to infection. Growing evidence suggests that a major strategy used by the virus is to modulate signalling pathways in infected macrophages, thus interfering with the expression of a large number of immunomodulatory genes. One potent immunomodulatory protein, A238L, inhibits both activation of the host NFkappaB transcription factor and inhibits calcineurin phosphatase activity. Calcineurin-dependent pathways, including activation of the NFAT transcription factor, are therefore inhibited. Another ASFV-encoded protein, CD2v, resembles the host CD2 protein, which is expressed on T cells and NK cells. This virus protein causes the adsorption of red blood cells around virus-infected cells and extracellular virus particles. Expression of the CD2v protein aids virus dissemination in pigs and the protein also has a role in impairing bystander lymphocyte function. This may be mediated either by a direct interaction of CD2v extracellular domain with ligands on lymphocytes or by an indirect mechanism involving interaction of the CD2v cytoplasmic tail with host proteins involved in signalling or trafficking pathways. Two ASFV proteins, an IAP and a Bcl2 homologue, inhibit apoptosis in infected cells and thus facilitate production of progeny virions. The prediction is that half to two-thirds of the approximately 150 genes encoded by ASFV are not essential for replication in cells but have an important role for virus survival and transmission in its hosts. These genes provide an untapped repository, and will be valuable tools for deciphering not only how the virus manipulates the host response to infection to avoid elimination, but also useful for understanding important host anti-viral mechanisms. In addition, they may provide leads for discovery of novel immunomodulatory drugs.

Correlation of cell surface marker expression with African swine fever virus infection.

The expression of surface markers on African swine fever virus (ASFV) infected cells was evaluated to assess their involvement in infection. Previous findings indicated CD163 expression was correlated with ASFV susceptibility. However, in this study the expression of porcine CD163 on cell lines did not increase the infection rate of these cells indicating other factors are likely to be important in determining susceptibility to infection. On adherent porcine bone marrow (pBM) cells the expression of CD45 was strongly correlated with infection. CD163 and CD203a expression correlated at intermediate levels with infection, indicating cells expressing these markers could become infected but were not preferentially infected by the virus. Most of the cells expressing MHCII were infected, indicating that they may be preferentially infected although expression of MHCII was not essential for infection and a large percentage of the infected cells were MHCII negative. CD16 showed a marked decrease in expression following infection and significantly lower levels of infected cells were shown to express CD16. Altogether these results suggest CD163 may be involved in ASFV infection but it may not be essential; the results also highlight the importance of other cell markers which requiring further investigation.

Protection of European domestic pigs from virulent African isolates of African swine fever virus by experimental immunisation.

African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs.

Domains involved in calcineurin phosphatase inhibition and nuclear localisation in the African swine fever virus A238L protein.

The African swine fever virus A238L protein inhibits calcineurin phosphatase activity and activation of NF-kappaB and p300 co-activator. An 82 amino acid domain containing residues 157 to 238 at the C-terminus of A238L was expressed in E. coli and purified. This purified A238L fragment acted as a potent inhibitor of calcineurin phosphatase in vitro with an IC50 of approximately 70 nM. Two putative nuclear localisation signals were identified between residues 80 to 86 (NLS-1) and between residues 203 to 207 overlapping with the N-terminus of the calcineurin docking motif (NLS-2). Mutation of these motifs independently did not reduce nuclear localisation compared to the wild type A238L protein, whereas mutation of both motifs significantly reduced nuclear localisation of A238L. Mutation of the calcineurin docking motif resulted in a dramatic increase in the nuclear localisation of A238L provided an intact NLS was present. We propose that binding of calcineurin to A238L masks NLS-2 contributing to the cytoplasmic retention of A238L.