MVA-based vaccine candidates expressing SARS-CoV-2 prefusion-stabilized spike proteins of the Wuhan, Beta or Omicron BA.1 variants protect transgenic K18-hACE2 mice against Omicron infection and elicit robust and broad specific humoral and cellular immune responses

Despite the decrease in mortality and morbidity due to SARS-CoV-2 infection, the incidence of infections due to Omicron subvariants of SARS-CoV-2 remains high. The mutations acquired by these subvariants, mainly concentrated in the receptor-binding domain (RBD), have caused a shift in infectivity and transmissibility, leading to a loss of effectiveness of the first authorized COVID-19 vaccines, among other reasons, by neutralizing antibody evasion. Hence, the generation of new vaccine candidates adapted to Omicron subvariants is of special interest in an effort to overcome this immune evasion. Here, an optimized COVID-19 vaccine candidate, termed MVA-S(3P_BA.1), was developed using a modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein from the Omicron BA.1 variant. The immunogenicity and efficacy induced by MVA-S(3P_BA.1) were evaluated in mice in a head-to-head comparison with the previously generated vaccine candidates MVA-S(3P) and MVA-S(3Pbeta), which express prefusion-stabilized S proteins from Wuhan strain and Beta variant, respectively, and with a bivalent vaccine candidate composed of a combination of MVA-S(3P) and MVA-S(3P_BA.1). The results showed that all four vaccine candidates elicited, after a single intramuscular dose, protection of transgenic K18-hACE2 mice challenged with SARS-CoV-2 Omicron BA.1, reducing viral loads, histopathological lesions, and levels of proinflammatory cytokines in the lungs. They also elicited anti-S IgG and neutralizing antibodies against various Omicron subvariants, with MVA-S(3P_BA.1) and the bivalent vaccine candidate inducing higher titers. Additionally, an intranasal immunization in C57BL/6 mice with all four vaccine candidates induced systemic and mucosal S-specific CD4+ and CD8+ T-cell and humoral immune responses, and the bivalent vaccine candidate induced broader immune responses, eliciting antibodies against the ancestral Wuhan strain and different Omicron subvariants. These results highlight the use of MVA as a potent and adaptable vaccine vector against new emerging SARS-CoV-2 variants, as well as the promising feature of combining multivalent MVA vaccine candidates.The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was supported by Fondo COVID-19 grant COV20/00151 (Spanish Health Ministry, Instituto de Salud Carlos III (ISCIII)), Fondo Supera COVID-19 grant (Crue Universidades-Banco Santander), CSIC grant 202120E079, grant CNS2022-135511 funded by the Spanish Ministry of Science, Innovation and Universities (MCIU)/Spanish Research Agency (AEI)/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR to promote the consolidation of research, and funds from Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC) co-financed with FEDER funds (to JG-A); CSIC grant 2020E84, la Caixa Banking Foundation grant CF01-00008, Ferrovial, and MAPFRE donations (to ME); and Spanish MCIU/AEI/10.13039/501100011033 grant PID2020-114481RB-I00 (to JG-A and ME). This research work was also funded by the European Commission-NextGenerationEU, through CSIC’s Global Health Platform (PTI Salud Global) (to JG-A and ME). JG-A also received grants from the European Commission HORIZON-HLTH-2023-DISEASE-03-18 (Project MARVAX: 101137183 and Project FLAVIVACCINE: 101137006). JG-A and ME also acknowledges financial support from the Spanish AEI/10.13039/501100011033, through the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2013-0347, SEV-2017-0712). JC acknowledges MCIU and CSIC support (project number 202020E079). RD received grants from ISCIII (FIS PI2100989), the European Commission Horizon 2020 Framework Programme (Project VIRUSCAN FETPROACT-2016: 731868 and Project EPIC-CROWN-2: 101046084), and Fundación Caixa-Health Research HR18-00469 (Project StopEbola). The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.Peer reviewe

Immunogenicity and efficacy of a novel multi-patch SARS-CoV-2/COVID-19 vaccine candidate

23 Pág.While there has been considerable progress in the development of vaccines against SARS-CoV-2, largely based on the S (spike) protein of the virus, less progress has been made with vaccines delivering different viral antigens with cross-reactive potential.This research was supported by La Caixa Banking Foundation grant CF01-00008, Spanish Ministry of Science and Innovation (MCIN)/Spanish Research Agency grant PID2020-117425RB-C22, Fondo COVID-19 grant COV20/00151 (Spanish Health Ministry and Instituto de Salud Carlos III), CSIC grant 2020E84 and Ferrovial donations (to ME). AcknowledgmentsPeer reviewe

Non-replicative antibiotic resistance-free DNA vaccine encoding S and N proteins induces full protection in mice against SARS-CoV-2

17 p.-8 fig.SARS-CoV-2 vaccines currently in use have contributed to controlling the COVID-19 pandemic. Notwithstanding, the high mutation rate, fundamentally in the spike glycoprotein (S), is causing the emergence of new variants. Solely utilizing this antigen is a drawback that may reduce the efficacy of these vaccines. Herein we present a DNA vaccine candidate that contains the genes encoding the S and the nucleocapsid (N) proteins implemented into the non-replicative mammalian expression plasmid vector, pPAL. This plasmid lacks antibiotic resistance genes and contains an alternative selectable marker for production. The S gene sequence was modified to avoid furin cleavage (Sfs). Potent humoral and cellular immune responses were observed in C57BL/6J mice vaccinated with pPAL-Sfs + pPAL-N following a prime/boost regimen by the intramuscular route applying in vivo electroporation. The immunogen fully protected K18-hACE2 mice against a lethal dose (105 PFU) of SARS-CoV-2. Viral replication was completely controlled in the lungs, brain, and heart of vaccinated mice. Therefore, pPAL-Sfs + pPAL-N is a promising DNA vaccine candidate for protection from COVID-19.This work was funded by PTI-Salud Global (CSIC), Center for Technological and Industrial Development (CDTI), REACT-ANTICIPA-UCM (Comunidad de Madrid), and European Research Council (Advanced Grant VERDI, ERC2015AdG grant number 694160).Peer reviewe

AG5 is a potent non-steroidal anti-inflammatory and immune regulator that preserves innate immunity [Oral]

III Jornadas Científicas PTI+ Salud Global, 20-22 noviembre 2023, Madrid[Background] An archetypal anti-inflammatory compound against cytokine storm would inhibit it without suppressing the innate immune response. [Methods] AG5, an anti-inflammatory compound, has been developed as synthetic derivative of andrographolide, which is highly absorbable and presents low toxicity. [Result]. We found that the mechanism of action of AG5 is through the inhibition of caspase-1. interestingly, we show with in vitro generated human monocyte derived dendritic cells that AG5 preserves innate immune response. AG5 minimizes inflammatory response in a mouse model of lipopolysaccharide (LPS)-induced lung injury and exhibits in vivo anti-inflammatory efficacy in the SARS-CoV-2-infected mouse model. Furthermore, AG5 showed interesting antiviral activity against SARS-CoV-2 in humanized mice. [Conclusions] AG5 opens up a new class of anti-inflammatories, since contrary to NSAIDs, AG5 is able to inhibit the cytokine storm, like dexamethasone, but, unlike corticosteroids, preserves adequately the innate immunity. This is critical at the early stages of any naïve infection, but particularly in SARS-CoV-2 infectionsN

AG5 is a potent non-steroidal anti-inflammatory and immune regulator that preserves innate immunity

12 pages, 5 figures.-- This is an open access article under the CC BY-NC-ND licenseAn archetypal anti-inflammatory compound against cytokine storm would inhibit it without suppressing the innate immune response. AG5, an anti-inflammatory compound, has been developed as synthetic derivative of andrographolide, which is highly absorbable and presents low toxicity. We found that the mechanism of action of AG5 is through the inhibition of caspase-1. Interestingly, we show with in vitro generated human monocyte derived dendritic cells that AG5 preserves innate immune response. AG5 minimizes inflammatory response in a mouse model of lipopolysaccharide (LPS)-induced lung injury and exhibits in vivo anti-inflammatory efficacy in the SARS-CoV-2-infected mouse model. AG5 opens up a new class of anti-inflammatories, since contrary to NSAIDs, AG5 is able to inhibit the cytokine storm, like dexamethasone, but, unlike corticosteroids, preserves adequately the innate immunity. This is critical at the early stages of any naïve infection, but particularly in SARS-CoV-2 infections. Furthermore, AG5 showed interesting antiviral activity against SARS-CoV-2 in humanized miceThis work has been supported by NextGenerationEU Recovery and Resilience Facility (RRF) through the PTI+ Global Health Platform of Spanish National Research Council, grants SGL2103023 (PBA), SGL2103053 (MMA) and SGL2103015 (MM); by Spanish National Research Council through the program “Ayudas extraodinarias a proyectos de investigacion en el marco de las medidas urgentes extraodinarias para hacer frente al impacto económico y social del COVID-19”, grants CSIC-COV19-093 (PBA) and CSIC-COV19-117 (MM); by Generalitat Valenciana through the program “Ayudas urgentes para proyectos de investigación, desarrollo tecnológico e innovación (I+D+i) por la COVID-19”, grant GVA-COVID19/2021/059 (PBA); by the Conference of Rectors of the Spanish Universities, Spanish National Research Council and Banco Santander through the FONDO SUPERA COVID-19, grant CAPriCORn (JSM, JMB); by Severo Ochoa center of excellence program (grant CEX2021-001230-S) (PBA)Peer reviewe

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

African Swine Fever Virus Infection and Cytokine Response In Vivo: An Update

34 Pág.African swine fever (ASF) is a hemorrhagic viral disease of domestic pigs and wild suids (all Sus scrofa) caused by the ASF virus (ASFV). The disease is spreading worldwide without control, threatening pig production due to the absence of licensed vaccine or commercially available treatments. A thorough understanding of the immunopathogenic mechanisms behind ASFV infection is required to better fight the disease. Cytokines are small, non-structural proteins, which play a crucial role in many aspects of the immune responses to viruses, including ASFV. Infection with virulent ASFV isolates often results in exacerbated immune responses, with increased levels of serum pro-inflammatory interleukins (IL-1α, IL-1β, IL-6), TNF and chemokines (CCL2, CCL5, CXCL10). Increased levels of IL-1, IL-6 and TNF are often detected in several tissues during acute ASFV infections and associated with lymphoid depletion, hemorrhages and oedemas. IL-1Ra is frequently released during ASFV infection to block further IL-1 activity, with its implication in ASFV immunopathology having been suggested. Increased levels of IFN-α and of the anti-inflammatory IL-10 seem to be negatively correlated with animal survival, whereas some correlation between virus-specific IFN-γ-producing cells and protection has been suggested in different studies where different vaccine candidates were tested, although future works should elucidate whether IFN-γ release by specific cell types is related to protection or disease development.Authors would like to thank the Italian Ministry of Health (grant IZS SA 06/21) for funding. Authors would like to thank the support received from the European project VACDIVA (EU-H2020-862874).Peer reviewe

Necropsy Procedures and Evaluation of Macroscopic Lesions of Pigs Infected with African Swine Fever Virus

15-49 ppPathology complements and provides a fundamental link to other disciplines for disease investigations supporting molecular biology, genetics, immunology, or virology as core basis of scientific research. Necropsies are an essential tool in veterinary pathology for disease investigation and should be conducted in a routine, systematic, and standard approach. An orderly necropsy procedure will allow the prosector (veterinary clinicians or veterinary pathologists) to determine macroscopically normal or altered structures and allow, through experience, to acquire dexterity, speed, and confidence in the technique. In conjunction with standardized macroscopic scoring protocols, necropsy is a powerful tool especially when using experimental animal models in research. Here, we describe a systematic necropsy protocol to be conducted on pigs infected with African swine fever virus (ASFV). The methodology described only requires rudimentary instruments, and it is not time-consuming. In addition to performing accurate tissue and organ assessment, the technique intends the prosector to carry out sampling of organs and tissues of interest in ASFV-infected pigs.This publication was financially supported by the European project EU H2020-862874-VACDIVA, VetBioNet project, funded by the European Commission Horizon 2020 INFRAIA program; the Department for Environment, Food and Rural Affairs (DEFRA); the Scottish Government; and the Welsh Government.Peer reviewe

Investigations of Pro- and Anti-Apoptotic Factors Affecting African Swine Fever Virus Replication and Pathogenesis

African swine fever virus (ASFV) is a large DNA virus that replicates predominantly in the cell cytoplasm and is the only member of the Asfarviridae family. The virus causes an acute haemorrhagic fever, African swine fever (ASF), in domestic pigs and wild boar resulting in the death of most infected animals. Apoptosis is induced at an early stage during virus entry or uncoating. However, ASFV encodes anti-apoptotic proteins which facilitate production of progeny virions. These anti-apoptotic proteins include A179L, a Bcl-2 family member; A224L, an inhibitor of apoptosis proteins (IAP) family member; EP153R a C-type lectin; and DP71L. The latter acts by inhibiting activation of the stress activated pro-apoptotic pathways pro-apoptotic pathways. The mechanisms by which these proteins act is summarised. ASF disease is characterised by massive apoptosis of uninfected lymphocytes which reduces the effectiveness of the immune response, contributing to virus pathogenesis. Mechanisms by which this apoptosis is induced are discussed

Running title: Regulatory T cells interfere protection against ASFV

21 p.-11 fig.Following short immunization protocols, naturally attenuated African swine fever virus (ASFV) isolate OURT88/3 and deletion mutant BeninDeltaMGF have previously been shown to induce high percentage of protection in domestic pigs against challenge with virulent virus. Results obtained in the present study showed that a single intramuscular immunization of domestic pigs with OURT88/3 or BeninDeltaMGF followed by a challenge with virulent Benin 97/1 isolate at day 130 post-immunisation did not trigger the necessary mechanisms to generate immunological memory able to induce long-term protection against disease. All pigs developed acute forms of ASF. IFNgamma producing cells peaked at day 24 post-immunisation, declining thereafter. Surprisingly, levels of T regulatory cells (Tregs) and IL-10 were elevated at the end of the experiment suggesting that regulatory components of the immune system may inhibit effective protection.Importance. 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 been proven protective following short immunization protocols in which pigs were challenged a few weeks after the first immunization. Here, duration of immunity and immune responses induced over a duration of 130 days were studied during pre-challenge and after challenge of pigs immunised with the naturally attenuated isolate OURT88/3 and an attenuated gene-deleted isolate BeninDeltaMGF. After a single intramuscular immunization of domestic pigs with the OURT88/3 isolate o BeninDeltaMGF virus, animals were not protected against challenge with virulent Benin 97/1 ASFV genotype I isolate at day 130 post-immunization. Levels of T regulatory cells and IL-10 were elevated at the end of the experiment, suggesting that regulatory components of the immune system may inhibit effective protection.This study was funded by the UK’s BBSRC grants BBS/E/I/00002014, BBS/E/1/00007031, BBS/E/1/00007034 and BB/L004267/1.Peer reviewe