Heterologous mRNA/MVA delivering trimeric-RBD as effective vaccination regimen against SARS-CoV-2: COVARNA Consortium.

Despite the high efficiency of current SARS-CoV-2 mRNA vaccines in reducing COVID-19 morbidity and mortality, waning immunity and the emergence of resistant variants underscore the need for novel vaccination strategies. This study explores a heterologous mRNA/Modified Vaccinia virus Ankara (MVA) prime/boost regimen employing a trimeric form of the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein compared to a homologous MVA/MVA regimen. In C57BL/6 mice, the RBD was delivered during priming via an mRNA vector encapsulated in nanoemulsions (NE) or lipid nanoparticles (LNP), followed by a booster with a replication-deficient MVA-based recombinant virus (MVA-RBD). This heterologous mRNA/MVA regimen elicited strong anti-RBD binding and neutralizing antibodies (BAbs and NAbs) against both the ancestral SARS-CoV-2 strain and different variants of concern (VoCs). Additionally, this protocol induced robust and polyfunctional RBD-specific CD4 and CD8 T cell responses, particularly in animals primed with mLNP-RBD. In K18-hACE2 transgenic mice, the LNP-RBD/MVA combination provided complete protection from morbidity and mortality following a live SARS-CoV-2 challenge compared with the partial protection observed with mNE-RBD/MVA or MVA/MVA regimens. Although the mNE-RBD/MVA regimen only protects half of the animals, it was able to induce antibodies with Fc-mediated effector functions besides NAbs. Moreover, viral replication and viral load in the respiratory tract were markedly reduced and decreased pro-inflammatory cytokine levels were observed. These results support the efficacy of heterologous mRNA/MVA vaccine combinations over homologous MVA/MVA regimen, using alternative nanocarriers that circumvent intellectual property restrictions of current mRNA vaccine formulations.

Modulating the immune response to SARS-CoV-2 by different nanocarriers delivering an mRNA expressing trimeric RBD of the spike protein: COVARNA Consortium.

Vaccines based on mRNA technology have revolutionized the field. In fact, lipid nanoparticles (LNP) formulated with mRNA are the preferential vaccine platform used in the fight against SARS-CoV-2 infection, with wider application against other diseases. The high demand and property right protection of the most potent cationic/ionizable lipids used for LNP formulation of COVID-19 mRNA vaccines have promoted the design of alternative nanocarriers for nucleic acid delivery. In this study we have evaluated the immunogenicity and efficacy of different rationally designed lipid and polymeric-based nanoparticle prototypes against SARS-CoV-2 infection. An mRNA coding for a trimeric soluble form of the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 was encapsulated using different components to form nanoemulsions (NE), nanocapsules (NC) and lipid nanoparticles (LNP). The toxicity and biological activity of these prototypes were evaluated in cultured cells after transfection and in mice following homologous prime/boost immunization. Our findings reveal good levels of RBD protein expression with most of the formulations. In C57BL/6 mice immunized intramuscularly with two doses of formulated RBD-mRNA, the modified lipid nanoparticle (mLNP) and the classical lipid nanoparticle (LNP-1) were the most effective delivery nanocarriers at inducing binding and neutralizing antibodies against SARS-CoV-2. Both prototypes fully protected susceptible K18-hACE2 transgenic mice from morbidity and mortality following a SARS-CoV-2 challenge. These results highlight that modulation of mRNAs immunogenicity can be achieved by using alternative nanocarriers and support further assessment of mLNP and LNP-1 prototypes as delivery vehicles for mRNA vaccines.

Expansion of the cytochrome C oxidase subunit I database and description of four new lobose testate amoebae species (Amoebozoa; Arcellinida).

Arcellinida is ascending in importance in protistology, but description of their diversity still presents multiple challenges. Furthermore, applicable tools for surveillance of these organisms are still in developing stages. Importantly, a good database that sets a correspondence between molecular barcodes and species morphology is lacking. Cytochrome oxidase (COI) has been suggested as the most relevant marker for species discrimination in Arcellinida. However, some major groups of Arcellinida are still lacking a COI sequence. Here we expand the database of COI marker sequences for Arcellinids, using single-cell PCR, transcriptomics, and database scavenging. In the present work, we added 24 new Arcellinida COI sequences to the database, covering all unsampled infra- and suborders. Additionally, we added six new SSUrRNA sequences and described four new species using morphological, morphometrical, and molecular evidence: Heleopera steppica, Centropyxis blatta, Arcella uspiensis, and Cylindrifflugia periurbana. This new database will provide a new starting point to address new research questions from shell evolution, biogeography, and systematics of arcellinids.

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

Introduction: 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. Methods: In an effort to develop an immunogen with the capacity to induce broad antigen presentation, we have designed a multi-patch synthetic candidate containing dominant and persistent B cell epitopes from conserved regions of SARS-CoV-2 structural proteins associated with long-term immunity, termed CoV2-BMEP. Here we describe the characterization, immunogenicity and efficacy of CoV2-BMEP using two delivery platforms: nucleic acid DNA and attenuated modified vaccinia virus Ankara (MVA). Results: In cultured cells, both vectors produced a main protein of about 37 kDa as well as heterogeneous proteins with size ranging between 25-37 kDa. In C57BL/6 mice, both homologous and heterologous prime/boost combination of vectors induced the activation of SARS-CoV-2-specific CD4 and CD8 T cell responses, with a more balanced CD8+ T cell response detected in lungs. The homologous MVA/MVA immunization regimen elicited the highest specific CD8+ T cell responses in spleen and detectable binding antibodies (bAbs) to S and N antigens of SARS-CoV-2. In SARS-CoV-2 susceptible k18-hACE2 Tg mice, two doses of MVA-CoV2-BMEP elicited S- and N-specific bAbs as well as cross-neutralizing antibodies against different variants of concern (VoC). After SARS-CoV-2 challenge, all animals in the control unvaccinated group succumbed to the infection while vaccinated animals with high titers of neutralizing antibodies were fully protected against mortality, correlating with a reduction of virus infection in the lungs and inhibition of the cytokine storm. Discussion: These findings revealed a novel immunogen with the capacity to control SARS-CoV-2 infection, using a broader antigen presentation mechanism than the approved vaccines based solely on the S antigen.

When ecological transitions are not so infrequent: independent colonizations of athalassohaline water bodies by Arcellidae (Arcellinida; Amoebozoa), with descriptions of four new species.

The salinity and humidity barriers divide biodiversity and strongly influence the distribution of organisms. Crossing them opens the possibility for organisms to colonize new niches and diversify, but requires profound physiological adaptations and is supposed to happen rarely in evolutionary history. We tested the relative importance of each ecological barrier by building the phylogeny, based on mitochondrial cytochrome oxidase gene (COI) sequences, of a group of microorganisms common in freshwater and soils, the Arcellidae (Arcellinida; Amoebozoa). We explored the biodiversity of this family in the sediments of athalassohaline water bodies (i.e. of fluctuating salinity that have non-marine origins). We found three new aquatic species, which represent, to the best of our knowledge, the first reports of Arcellinida in these salt-impacted ecosystems, plus a fourth terrestrial one in bryophytes. Culturing experiments performed on Arcella euryhalina sp. nov. showed similar growth curves in pure freshwater and under 20 g/L salinity, and long-term survival at 50 g/L, displaying a halotolerant biology. Phylogenetic analyses showed that all three new athalassohaline species represent independent transition events through the salinity barrier by freshwater ancestor, in contrast to the terrestrial species, which are monophyletic and represent a unique ecological transition from freshwater to soil environments.

Study on the approach to HIV: health management and the healthcare process in Spain.

INTRODUCTION: HIV continues to represent a problem of great relevance for public health in Spain. This study aims to carry out an analysis that will provide in-depth knowledge of the resources, clinical care, and management during the diagnosis, follow-up, and treatment phases of HIV infection in Spain. METHODS: In the first phase, a multidisciplinary Scientific Committee designed an information collection tool in the form of a survey. In the second phase, carried out in the autonomous communities of Andalusia, Catalonia, and La Rioja, a multidisciplinary group of 42 experts, representatives of the public administration, clinical profiles, and representatives of NGOs in the field of HIV answered the survey. RESULTS: The assessment of HIV resources is generally positive. As regards diagnosis, the experts considered that there was good coordination between Primary and Hospital care. Regarding treatment, the evaluations reflected good opinions on therapeutic conciliation and adherence, with a negative opinion in the evaluation of drug interactions with antiretroviral treatment. Regarding follow-up, the perception expressed was disparate concerning the coordination between Hospital and Primary Care as well as the adaptation of care to chronicity, aging, fragility, mental health, and oncological processes. CONCLUSION: There are certain processes that can be improved in the management of HIV infection in people with HIV in Spain, including protocols for follow-up and coordination between primary and hospital care in the treatment and follow-up of the disease.

Full protection from SARS-CoV-2 brain infection and damage in susceptible transgenic mice conferred by MVA-CoV2-S vaccine candidate.

Vaccines against SARS-CoV-2 have been shown to be safe and effective but their protective efficacy against infection in the brain is yet unclear. Here, in the susceptible transgenic K18-hACE2 mouse model of severe coronavirus disease 2019 (COVID-19), we report a spatiotemporal description of SARS-CoV-2 infection and replication through the brain. SARS-CoV-2 brain replication occurs primarily in neurons, leading to neuronal loss, signs of glial activation and vascular damage in mice infected with SARS-CoV-2. One or two doses of a modified vaccinia virus Ankara (MVA) vector expressing the SARS-CoV-2 spike (S) protein (MVA-CoV2-S) conferred full protection against SARS-CoV-2 cerebral infection, preventing virus replication in all areas of the brain and its associated damage. This protection was maintained even after SARS-CoV-2 reinfection. These findings further support the use of MVA-CoV2-S as a promising vaccine candidate against SARS-CoV-2/COVID-19.

Cyphoderia ampulla (Cyphoderiidae: Rhizaria), a tale of freshwater sailors: The causes and consequences of ecological transitions through the salinity barrier in a family of benthic protists.

The salinity barrier that separates marine and freshwater biomes is probably the most important division in biodiversity on Earth. Those organisms that successfully performed this transition had access to new ecosystems while undergoing changes in selective pressure, which often led to major shifts in diversification rates. While these transitions have been extensively investigated in animals, the tempo, mode, and outcome of crossing the salinity barrier have been scarcely studied in other eukaryotes. Here, we reconstructed the evolutionary history of the species complex Cyphoderia ampulla (Euglyphida: Cercozoa: Rhizaria) based on DNA sequences from the nuclear SSU rRNA gene and the mitochondrial cytochrome oxidase subunit I gene, obtained from publicly available environmental DNA data (GeneBank, EukBank) and isolated organisms. A tree calibrated with euglyphid fossils showed that four independent transitions towards freshwater systems occurred from the mid-Miocene onwards, coincident with important fluctuations in sea level. Ancestral trait reconstructions indicated that the whole family Cyphoderiidae had a marine origin and suggest that ancestors of the freshwater forms were euryhaline and lived in environments with fluctuating salinity. Diversification rates did not show any obvious increase concomitant with ecological transitions, but morphometric analyses indicated that species increased in size and homogenized their morphology after colonizing the new environments. This suggests adaptation to changes in selective pressure exerted by life in freshwater sediments.

Full efficacy and long-term immunogenicity induced by the SARS-CoV-2 vaccine candidate MVA-CoV2-S in mice.

Two doses of the MVA-CoV2-S vaccine candidate expressing the SARS-CoV-2 spike (S) protein protected K18-hACE2 transgenic mice from a lethal dose of SARS-CoV-2. This vaccination regimen prevented virus replication in the lungs, reduced lung pathology, and diminished levels of pro-inflammatory cytokines. High titers of IgG antibodies against S and receptor-binding domain (RBD) proteins and of neutralizing antibodies were induced against parental virus and variants of concern, markers that correlated with protection. Similar SARS-CoV-2-specific antibody responses were observed at prechallenge and postchallenge in the two-dose regimen, while the single-dose treatment does not avoid vaccine breakthrough infection. All vaccinated animals survived infection and were also protected to SARS-CoV-2 reinfection. Furthermore, two MVA-CoV2-S doses induced long-term memory S-specific humoral and cellular immune responses in C57BL/6 mice, 6 months after immunization. The efficacy and immunological benefits of the MVA-CoV2-S vaccine candidate against COVID-19 supports its consideration for human clinical trials.

Arcellinida testate amoebae as climate miner's canaries in Southern Spain.

Southern Spain is currently under threat of desertification as a consequence of global climate change, which pressures on fragile ecosystems such as caves. The organisms living in these extremely stable environments are particularly sensitive and prone to extinction, therefore they can be used as bioindicators for climate change. Cyanobacterial mats form peculiar and vulnerable micro-ecosystems at the entrance of caves and house a diversity of protists. Amongst them, Arcellinida testate amoebae have been traditionally used as bioindicators for environmental quality, notably because their narrow ecological tolerance and their key ecological position as top predators of the microbial foodwebs. We report here two new species of Arcellinida found in the cyanobacterial mats of cave Hundidero, in Sierra de Grazalema, Malaga province, whose traits suggest a narrow tolerance for changes in humidity. We provide a formal description for Difflugia alhadiqa sp. nov. and Heleopera baetica sp. nov. based on morphometrics and 18S rRNA gene data, and propose using the presence of these species to indicate the good health of the cyanobacterial mats, like miner's canaries for local climate.

COVID-19 vaccine candidates based on modified vaccinia virus Ankara expressing the SARS-CoV-2 spike induce robust T- and B-cell immune responses and full efficacy in mice.

Vaccines against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are urgently needed. We developed two COVID-19 vaccines based on modified vaccinia virus Ankara (MVA) vectors expressing the entire SARS-CoV-2 spike (S) protein (MVA-CoV2-S); their immunogenicity was evaluated in mice using DNA/MVA or MVA/MVA prime/boost immunizations. Both vaccines induced robust, broad and polyfunctional S-specific CD4+ (mainly Th1) and CD8+ T-cell responses, with a T effector memory phenotype. DNA/MVA immunizations elicited higher T-cell responses. All vaccine regimens triggered high titers of IgG antibodies specific for the S, as well as for the receptor-binding domain; the predominance of the IgG2c isotype was indicative of Th1 immunity. Notably, serum samples from vaccinated mice neutralized SARS-CoV-2 in cell cultures, and those from MVA/MVA immunizations showed a higher neutralizing capacity. Remarkably, one or two doses of MVA-CoV2-S protect humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2. In addition, two doses of MVA-CoV2-S confer full inhibition of virus replication in the lungs. These results demonstrate the robust immunogenicity and full efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic.IMPORTANCE The continuous dissemination of the novel emerging SARS-CoV-2 virus, with more than 78 million infected cases worldwide and higher than 1,700,000 deaths as of December 23, 2020, highlights the urgent need for the development of novel vaccines against COVID-19. With this aim, we have developed novel vaccine candidates based on the poxvirus modified vaccinia virus Ankara (MVA) strain expressing the full-length SARS-CoV-2 spike (S) protein, and we have evaluated their immunogenicity in mice using DNA/MVA or MVA/MVA prime/boost immunization protocols. The results showed the induction of a potent S-specific T-cell response and high titers of neutralizing antibodies. Remarkably, humanized K18-hACE2 mice immunized with one or two doses of the MVA-based vaccine were 100% protected from SARS-CoV-2 lethality. Moreover, two doses of the vaccine prevented virus replication in lungs. Our findings prove the robust immunogenicity and efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic.

A Single Dose of an MVA Vaccine Expressing a Prefusion-Stabilized SARS-CoV-2 Spike Protein Neutralizes Variants of Concern and Protects Mice From a Lethal SARS-CoV-2 Infection.

We generated an optimized COVID-19 vaccine candidate based on the modified vaccinia virus Ankara (MVA) vector expressing a full-length prefusion-stabilized SARS-CoV-2 spike (S) protein, termed MVA-CoV2-S(3P). The S(3P) protein was expressed at higher levels (2-fold) than the non-stabilized S in cells infected with the corresponding recombinant MVA viruses. One single dose of MVA-CoV2-S(3P) induced higher IgG and neutralizing antibody titers against parental SARS-CoV-2 and variants of concern than MVA-CoV2-S in wild-type C57BL/6 and in transgenic K18-hACE2 mice. In immunized C57BL/6 mice, two doses of MVA-CoV2-S or MVA-CoV2-S(3P) induced similar levels of SARS-CoV-2-specific B- and T-cell immune responses. Remarkably, a single administration of MVA-CoV2-S(3P) protected all K18-hACE2 mice from morbidity and mortality caused by SARS-CoV-2 infection, reducing SARS-CoV-2 viral loads, histopathological lesions, and levels of pro-inflammatory cytokines in the lungs. These results demonstrated that expression of a novel full-length prefusion-stabilized SARS-CoV-2 S protein by the MVA poxvirus vector enhanced immunogenicity and efficacy against SARS-CoV-2 in animal models, further supporting MVA-CoV2-S(3P) as an optimized vaccine candidate for clinical trials.

Salivary biomarkers in Alzheimer's disease.

OBJECTIVES: The objective of this study was to evaluate the changes that can occur in saliva components in patients with Alzheimer's disease (AD) of different severity and determine if any of these components could be a biomarker of this disease. Therefore, a panel of selected analytes related to the amyloid cascade, the immune and adrenergic systems, among others, were analyzed in the saliva of patients with Alzheimer's disease. METHODS: A total of 152 patients with AD and controls were included. The severity of the disease was established according to the Global Deterioration Scale. Unstimulated whole saliva was collected. RESULTS: Salivary amyloid-ß42 was significantly lower, and complement C4 was significantly higher in the patients with AD than in the controls (p < 0.05 in both cases). Only complement C4 maintained its significant effect in the multivariate regression analysis. However, the area under the receiver operating characteristic curve of C4 was 0.613. No changes were found in any analyte regarding the severity of the disease. CONCLUSIONS: A decrease in amyloid-ß42 and an increase in complement C4 were detected in the saliva of patients with AD, but the changes did not show a high diagnostic performance for the detection of AD and were not associated with its severity. CLINICAL RELEVANCE: Although some analytes showed significant differences in saliva in patients with AD, in our study conditions the salivary biomarkers analyzed were not of enough diagnostic utility for being used in routine.

Salivary adiponectin, but not adenosine deaminase, correlates with clinical signs in women with Sjögren's syndrome: a pilot study.

OBJECTIVES: To evaluate salivary adiponectin and adenosine deaminase (ADA) in women suffering from Sjögren's syndrome (SS). METHODS: Salivary adiponectin and ADA were measured in patients with SS (n = 17) and compared to their values in healthy controls (n = 13) and patients suffering from drug-induced xerostomia (non-SS sicca group; n = 19). A clinical history was made for each patient, patients were examined clinically, and xerostomia inventory (XI) was performed. RESULTS: Salivary adiponectin corrected by total protein was higher in patients with SS than in healthy individuals (P < 0.05) or patients with non-SS sicca (P < 0.01) and correlated with XI (r = 0.555; P < 0.05). Salivary ADA was higher in patients with SS and non-SS sicca compared to controls (P < 0.05 in both cases). CONCLUSION: The results of the present study indicate that adiponectin and ADA are increased in the saliva of patients with SS. CLINICAL RELEVANCE: Salivary adiponectin corrected by total protein can be a potential biomarker of SS. TRIAL REGISTRATION: NCT03156569.

HIV Rapid Testing Programs in Non-Clinical Settings have the Potential to Constitute a Major Diagnostic Option for MSM in Spain.

We analyze the impact of HIV rapid testing (RT) programs in non-clinical settings (NCS) by evaluating their contribution to new diagnoses reported to the Spanish HIV Surveillance System (SINIVIH) from 2007 to 2012. We estimate the proportion of new diagnoses reported to SINIVIH attributable to them and the maximum annual contribution (MAC). Of 95.575 rapid tests conducted, 2061 were reactive; 1582 in men who have sex with men (MSM). The contribution of RT in NCS increased from 3.4% in 2007 to 11.0% in 2012 (8.1%-16.6% in MSM). RT programs contributed 25.3% of the new diagnoses reported in Catalonia (MAC:30.6%), 15% in the Canary Islands (MAC:16.2%) and 13.7% in the Basque Country (MAC:21.0%). Among MSM, contribution was of 45.2% in Catalonia (MAC:60.7%), 20.2% in the Canary Islands (MAC:21.3%) and 16.6% in the Basque country (MAC:20.9%). Especially among MSM, RT in NCS contributed a large proportion of the new HIV cases diagnosed in regions with a very high HIV incidence.