Vaccinia virus induces endoplasmic reticulum stress and activates unfolded protein responses through the ATF6α transcription factor.

BACKGROUND: Cell responses to different stress inducers are efficient mechanisms that prevent and fight the accumulation of harmful macromolecules in the cells and also reinforce the defenses of the host against pathogens. Vaccinia virus (VACV) is an enveloped, DNA virus, belonging to the Poxviridae family. Members of this family have evolved numerous strategies to manipulate host responses to stress controlling cell survival and enhancing their replicative success. In this study, we investigated the activation of the response signaling to malformed proteins (UPR) by the VACV virulent strain-Western Reserve (WR)-or the non-virulent strain-Modified Vaccinia Ankara (MVA). METHODS: Through RT-PCR RFLP and qPCR assays, we detected negative regulation of XBP1 mRNA processing in VACV-infected cells. On the other hand, through assays of reporter genes for the ATF6 component, we observed its translocation to the nucleus of infected cells and a robust increase in its transcriptional activity, which seems to be important for virus replication. WR strain single-cycle viral multiplication curves in ATF6α-knockout MEFs showed reduced viral yield. RESULTS: We observed that VACV WR and MVA strains modulate the UPR pathway, triggering the expression of endoplasmic reticulum chaperones through ATF6α signaling while preventing IRE1α-XBP1 activation. CONCLUSIONS: The ATF6α sensor is robustly activated during infection while the IRE1α-XBP1 branch is down-regulated.

Zoonotic vaccinia virus strains belonging to different genetic clades exhibit immunomodulation abilities that are proportional to their virulence.

BACKGROUND: The vaccinia virus (VACV) isolates, Guarani P1 virus (GP1V) and Passatempo virus (PSTV), were isolated during zoonotic outbreaks in Brazil. Each one of them belongs to two different VACV clades, defined by biological aspects that include virulence in mice and phylogenetic analysis. Considering that information about how vaccinia viruses from different groups elicit immune responses in animals is scarce, we investigated such responses in mice infected either by GP1V (group 2) or PSTV (group 1), using VACV Western Reserve strain (VACV-WR) as control. METHODS: The severity of the infections was evaluated in BALB/c mice considering diverse clinical signs and defined scores, and the immune responses triggered by GP1V and PSTV infections were analysed by immune cell phenotyping and intra-cytoplasmic cytokines detection. RESULTS: We detected a reduction in total lymphocytes (CD3 +), macrophages (CD14 +), and NK cells (CD3-CD49 +) in animals infected with VACV-WR or GP1V. The VACV-WR and GP1V viruses, belonging to the most virulent group in a murine model, were able to down-modulate the cell immune responses upon mice infection. In contrast, PSTV, a virus considered less virulent in a murine model, showed little ability to down-modulate the mice immune responses. Mice infected with VACV-WR and GP1V viruses presented significant weight loss and developed lesions in their spleens, as well as damage to liver and lungs whereas mice infected with PSTV developed only moderate clinical signs. CONCLUSIONS: Our results suggest that VACV immunomodulation in vivo is clade-related and is proportional to the strain's virulence upon infection. Our data corroborate the classification of the different Brazilian VACV isolates into clades 1 and 2, taking into account not only phylogenetic criteria, but also clinical and immunological data.

The Investigation of Promoter Sequences of Marseilleviruses Highlights a Remarkable Abundance of the AAATATTT Motif in Intergenic Regions.

Viruses display a wide range of genomic profiles and, consequently, a variety of gene expression strategies. Specific sequences associated with transcriptional processes have been described in viruses, and putative promoter motifs have been elucidated for some nucleocytoplasmic large DNA viruses (NCLDV). Among NCLDV, the Marseilleviridae is a well-recognized family because of its genomic mosaicism. The marseilleviruses have an ability to incorporate foreign genes, especially from sympatric organisms inhabiting Acanthamoeba, its main known host. Here, we identified for the first time an eight-nucleotide A/T-rich promoter sequence (AAATATTT) associated with 55% of marseillevirus genes that is conserved in all marseilleviruses lineages, a higher level of conservation than that of any giant virus described to date. We instigated our prediction about the promoter motif by biological assays and by evaluating how single mutations in this octamer can impact gene expression. The investigation of sequences that regulate the expression of genes relative to lateral transfer revealed that the promoter motifs do not appear to be incorporated by marseilleviruses from donor organisms. Indeed, analyses of the intergenic regions that regulate lateral gene transfer-related genes have revealed an independent origin of the marseillevirus intergenic regions that does not match gene-donor organisms. About 50% of AAATATTT motifs spread throughout intergenic regions of the marseilleviruses are present as multiple copies. We believe that such multiple motifs are associated with increased expression of a given gene or are related to incorporation of foreign genes into the mosaic genome of marseilleviruses.IMPORTANCE The marseilleviruses draw attention because of the peculiar features of their genomes; however, little is known about their gene expression patterns or the factors that regulate those expression patterns. The limited published research on the expression patterns of the marseilleviruses and their unique genomes has led us to study the promoter motif sequences in the intergenic regions of the marseilleviruses. This work is the first to analyze promoter sequences in the genomes of the marseilleviruses. We also suggest a strong capacity to acquire foreign genes and to express those genes mediated by multiple copies of the promoter motifs available in intergenic regions. These findings contribute to an understanding of genomic expansion and plasticity observed in these giant viruses.

The Large Marseillevirus Explores Different Entry Pathways by Forming Giant Infectious Vesicles.

UNLABELLED: Triggering the amoebal phagocytosis process is a sine qua non condition for most giant viruses to initiate their replication cycle and consequently to promote their progeny formation. It is well known that the amoebal phagocytosis process requires the recognition of particles of >500 nm, and most amoebal giant viruses meet this requirement, such as mimivirus, pandoravirus, pithovirus, and mollivirus. However, in the context of the discovery of amoebal giant viruses in the last decade, Marseillevirus marseillevirus (MsV) has drawn our attention, because despite its ability to successfully replicate in Acanthamoeba, remarkably it does not fulfill the >500-nm condition, since it presents an ∼250-nm icosahedrally shaped capsid. We deeply investigated the MsV cycle by using a set of methods, including virological, molecular, and microscopic (immunofluorescence, scanning electron microscopy, and transmission electron microscopy) assays. Our results revealed that MsV is able to form giant vesicles containing dozens to thousands of viral particles wrapped by membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggested that these giant vesicles are able to stimulate amoebal phagocytosis and to trigger the MsV replication cycle by an acidification-independent process. Also, we observed that MsV entry may occur by the phagocytosis of grouped particles (without surrounding membranes) and by an endosome-stimulated pathway triggered by single particles. Taken together, not only do our data deeply describe the main features of MsV replication cycle, but this is the first time, to our knowledge, that the formation of giant infective vesicles related to a DNA virus has been described. IMPORTANCE: Triggering the amoebal phagocytosis process is a sine qua non condition required by most giant viruses to initiate their replication cycle. This process requires the recognition of particles of >500 nm, and many giant viruses meet this requirement. However, MsV is unusual, as despite having particles of ∼250 nm it is able to replicate in Acanthamoeba Our results revealed that MsV is able to form giant vesicles, containing dozens to thousands of viral particles, wrapped in membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggest that these giant vesicles are able to stimulate phagocytosis using an acidification-independent process. Our work not only describes the main features of the MsV replication cycle but also describes, for the first time to our knowledge, the formation of huge infective vesicles in a large DNA viruses.

A reduction of viral mRNA, proteins and induction of altered morphogenesis reveals the anti-HTLV-1 activity of the labdane-diterpene myriadenolide in vitro.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) has been associated with leukemia/lymphoma (ATL) and myelopathy/tropical spastic paraparesis (HAM/TSP), in addition to other inflammatory diseases as well as infection complications. Therapeutic approaches for HTLV-1-related pathologies are limited. The labdane diterpene myriadenolide (AMY) is a natural product that exhibit biological activities, such as anti-inflammatory and antiviral activity as reported for HIV and herpesvirus. RESULTS: We demonstrated that this natural product was able to inhibit the expression of gag-pol mRNA and substantially reduced the expression of the structural proteins p19 and gp46. Comparison of treated and untreated cells shows that AMY alters both the morphology and the release of viral particles. The Atomic Force Microscopy assay showed that the AMY treatment reduced the number of particles on the cell surface by 47%. CONCLUSION: We demonstrated that the labdane diterpene myriadenolide reduced the expression of the structural proteins and the budding of viral particles, besides induces altered morphogenesis of HTLV-1, conferring on AMY a new antiviral activity that may be useful for the development of new compounds with specific anti-HTLV-1 activity.

Poxvirus A51R proteins regulate microtubule stability and antagonize a cell-intrinsic antiviral response.

Numerous viruses alter host microtubule (MT) networks during infection, but how and why they induce these changes is unclear in many cases. We show that the vaccinia virus (VV)-encoded A51R protein is a MT-associated protein (MAP) that directly binds MTs and stabilizes them by both promoting their growth and preventing their depolymerization. Furthermore, we demonstrate that A51R-MT interactions are conserved across A51R proteins from multiple poxvirus genera, and highly conserved, positively charged residues in A51R proteins mediate these interactions. Strikingly, we find that viruses encoding MT interaction-deficient A51R proteins fail to suppress a reactive oxygen species (ROS)-dependent antiviral response in macrophages that leads to a block in virion morphogenesis. Moreover, A51R-MT interactions are required for VV virulence in mice. Collectively, our data show that poxviral MAP-MT interactions overcome a cell-intrinsic antiviral ROS response in macrophages that would otherwise block virus morphogenesis and replication in animals.

Design, development, and validation of multi-epitope proteins for serological diagnosis of Zika virus infections and discrimination from dengue virus seropositivity.

Zika virus (ZIKV), an arbovirus from the Flaviviridae family, is the causative agent of Zika fever, a mild and frequent oligosymptomatic disease in humans. Nonetheless, on rare occasions, ZIKV infection can be associated with Guillain-Barré Syndrome (GBS), and severe congenital complications, such as microcephaly. The oligosymptomatic disease, however, presents symptoms that are quite similar to those observed in infections caused by other frequent co-circulating arboviruses, including dengue virus (DENV). Moreover, the antigenic similarity between ZIKV and DENV, and even with other members of the Flaviviridae family, complicates serological testing due to the high cross-reactivity of antibodies. Here, we designed, produced in a prokaryotic expression system, and purified three multiepitope proteins (ZIKV-1, ZIKV-2, and ZIKV-3) for differential diagnosis of Zika. The proteins were evaluated as antigens in ELISA tests for the detection of anti-ZIKV IgG using ZIKV- and DENV-positive human sera. The recombinant proteins were able to bind and detect anti-ZIKV antibodies without cross-reactivity with DENV-positive sera and showed no reactivity with Chikungunya virus (CHIKV)- positive sera. ZIKV-1, ZIKV-2, and ZIKV-3 proteins presented 81.6%, 95%, and 66% sensitivity and 97%, 96%, and 84% specificity, respectively. Our results demonstrate the potential of the designed and expressed antigens in the development of specific diagnostic tests for the detection of IgG antibodies against ZIKV, especially in regions with the circulation of multiple arboviruses.

Algorithms for predicting COVID outcome using ready-to-use laboratorial and clinical data.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging crisis affecting the public health system. The clinical features of COVID-19 can range from an asymptomatic state to acute respiratory syndrome and multiple organ dysfunction. Although some hematological and biochemical parameters are altered during moderate and severe COVID-19, there is still a lack of tools to combine these parameters to predict the clinical outcome of a patient with COVID-19. Thus, this study aimed at employing hematological and biochemical parameters of patients diagnosed with COVID-19 in order to build machine learning algorithms for predicting COVID mortality or survival. Patients included in the study had a diagnosis of SARS-CoV-2 infection confirmed by RT-PCR and biochemical and hematological measurements were performed in three different time points upon hospital admission. Among the parameters evaluated, the ones that stand out the most are the important features of the T1 time point (urea, lymphocytes, glucose, basophils and age), which could be possible biomarkers for the severity of COVID-19 patients. This study shows that urea is the parameter that best classifies patient severity and rises over time, making it a crucial analyte to be used in machine learning algorithms to predict patient outcome. In this study optimal and medically interpretable machine learning algorithms for outcome prediction are presented for each time point. It was found that urea is the most paramount variable for outcome prediction over all three time points. However, the order of importance of other variables changes for each time point, demonstrating the importance of a dynamic approach for an effective patient's outcome prediction. All in all, the use of machine learning algorithms can be a defining tool for laboratory monitoring and clinical outcome prediction, which may bring benefits to public health in future pandemics with newly emerging and reemerging SARS-CoV-2 variants of concern.

FEAR antiviral response pathway is independent of interferons and countered by poxvirus proteins.

The human facilitates chromatin transcription (FACT) complex is a chromatin remodeller composed of human suppressor of Ty 16 homologue (hSpt16) and structure-specific recognition protein-1 subunits that regulates cellular gene expression. Whether FACT regulates host responses to infection remained unclear. We identify a FACT-mediated, interferon-independent, antiviral pathway that restricts poxvirus replication. Cell culture and bioinformatics approaches suggest that early viral gene expression triggers nuclear accumulation of SUMOylated hSpt16 subunits required for the expression of E26 transformation-specific sequence-1 (ETS-1)-a transcription factor that activates virus restriction programs. However, biochemical studies show that poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting structure-specific recognition protein-1 binding to SUMOylated hSpt16 and by tethering SUMOylated hSpt16 to microtubules. Furthermore, A51R antagonism of FACT enhances poxvirus replication in human cells and virulence in mice. Finally, we show that FACT also restricts rhabdoviruses, flaviviruses and orthomyxoviruses, suggesting broad roles for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 antiviral response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

New anti-SARS-CoV-2 aminoadamantane compounds as antiviral candidates for the treatment of COVID-19.

Here, the antiviral activity of aminoadamantane derivatives were evaluated against SARS-CoV-2. The compounds exhibited low cytotoxicity to Vero, HEK293 and CALU-3 cells up to a concentration of 1,000 µM. The inhibitory concentration (IC50) of aminoadamantane was 39.71 µM in Vero CCL-81 cells and the derivatives showed significantly lower IC50 values, especially for compounds 3F4 (0.32 µM), 3F5 (0.44 µM) and 3E10 (1.28 µM). Additionally, derivatives 3F5 and 3E10 statistically reduced the fluorescence intensity of SARS-CoV-2 protein S from Vero cells at 10 µM. Transmission microscopy confirmed the antiviral activity of the compounds, which reduced cytopathic effects induced by the virus, such as vacuolization, cytoplasmic projections, and the presence of myelin figures derived from cellular activation in the face of infection. Additionally, it was possible to observe a reduction of viral particles adhered to the cell membrane and inside several viral factories, especially after treatment with 3F4. Moreover, although docking analysis showed favorable interactions in the catalytic site of Cathepsin L, the enzymatic activity of this enzyme was not inhibited significantly in vitro. The new derivatives displayed lower predicted toxicities than aminoadamantane, which was observed for either rat or mouse models. Lastly, in vivo antiviral assays of aminoadamantane derivatives in BALB/cJ mice after challenge with the mouse-adapted strain of SARS-CoV-2, corroborated the robust antiviral activity of 3F4 derivative, which was higher than aminoadamantane and its other derivatives. Therefore, aminoadamantane derivatives show potential broad-spectrum antiviral activity, which may contribute to COVID-19 treatment in the face of emerging and re-emerging SARS-CoV-2 variants of concern.

Manipulation of unfolded protein response by zoonotic vaccinia virus strains Guarani P1 and Passatempo.

The replicative success of vaccinia virus (VACV) depends on its ability to subvert host functions. Poxviruses multiplication and maturation are closely associated with the endoplasmic reticulum (ER) and its membranes. This organelle responds to disturbances caused by the accumulation of misfolded proteins, leading to processing of these proteins or even programmed cell death through the unfolded protein response (UPR). Several studies show that different viruses can activate UPR pathway components and negatively modulate others. Here, we investigate the effects of infections by zoonotic VACV strains from Brazil, Guarani P1 virus (GP1V) and Passatempo virus (PSTV), in the activation of UPR pathway sensors. We observed translocation of ATF6 to the nucleus as well as transcriptional increase after GP1V, PSTV, and reference strain Western Reserve (WR) infection. XBP1 processing appears to be negatively modulated after VACV infection; however, inhibition of the inositol-requiring enzyme 1 (IRE1) kinase domain led to a reduction in plaque sizes for these viruses. The absence of PKR-like endoplasmic reticulum kinase (PERK) has an impact on the plaque phenotype of GP1V, PSTV viruses, as well as for the prototypical strain WR. These results indicate that the VACV manipulates the three arms of the UPR path differently to ensure replicative success.

Seroprevalence of SARS-CoV-2 in hospital workers in the southern region of Minas Gerais state in Brazil: An analysis of the pre-vaccine period.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection seroprevalence can be performed by detecting anti-SARS-CoV-2 antibodies. The survey is essential to understand the disease transmission's dynamic in the studied population. This study aimed to carry out a seroepidemiological survey of SARS-CoV-2 in three hospitals located in the south of Minas Gerais state, Brazil. 859 samples were collected from August to December 2020 when SARS-CoV-2 vaccines were still not available and Enzyme-linked immunosorbent assays (ELISA) were performed on participants sera. The average age of participants was 38 years, and most were women (71.4%). Likewise, most participants were classified as health professionals with direct or indirect contact with patients with COVID-19 (74.5%). The other participants tested belonged to other sectors, such as the administrative one (11,6%). Considering clinical symptoms, 15.8% of participants reported diarrhoea, 6.4% fever, 5.8% respiratory distress, and 7.0% loss of smell and taste. Many participants reported contact with infected patients (63.35%). Regarding the ELISA tests, 21.6% of the participants had positive results and hospital 3 had the highest positivity (21.7%), followed by hospital 2 (21.6%) and hospital 1 (20.3%). The prevalence was higher in women compared to men (22,8% and 18,7%, respectively). Regarding the area of expertise, the highest positivity (20.9%) was observed among health professionals. However, professionals who worked exclusively with COVID-19 had lower positivity when compared to professionals who did not work directly with COVID-19 (22.0% and 21.5%, respectively). When analysing the correlation between the ELISA tests with the other variables, a significant association was detected with these previous serological variables, previous contact with COVID-19 and the presence of fever symptoms, loss of smell and taste. Clinical symptoms associated with serological tests are important tools for monitoring the disease among health professionals.

A FACT-ETS-1 Antiviral Response Pathway Restricts Viral Replication and is Countered by Poxvirus A51R Proteins.

The FACT complex is an ancient chromatin remodeling factor comprised of Spt16 and SSRP1 subunits that regulates specific eukaryotic gene expression programs. However, whether FACT regulates host immune responses to infection was unclear. Here, we identify an antiviral pathway mediated by FACT, distinct from the interferon response, that restricts poxvirus replication. We show that early viral gene expression triggers nuclear accumulation of specialized, SUMOylated Spt16 subunits of FACT required for expression of ETS-1, a downstream transcription factor that activates a virus restriction program. However, poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting SSRP1 for binding to SUMOylated Spt16 in the cytosol and by tethering SUMOylated Spt16 to microtubules. Moreover, we show that A51R antagonism of FACT enhances both poxvirus replication in human cells and viral virulence in mice. Finally, we demonstrate that FACT also restricts unrelated RNA viruses, suggesting a broad role for FACT in antiviral immunity. Our study reveals the F ACT- E TS-1 A ntiviral R esponse (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

Surveillance of SARS-CoV-2 immunogenicity: loss of immunodominant HLA-A*02-restricted epitopes that activate CD8+ T cells.

Introduction and methods: In this present work, coronavirus subfamilies and SARS-CoV-2 Variants of Concern (VOCs) were investigated for the presence of MHC-I immunodominant viral peptides using in silico and in vitro tools. Results: In our results, HLA-A*02 haplotype showed the highest number of immunodominant epitopes but with the lowest combined prediction score. Furthermore, a decrease in combined prediction score was observed for HLA-A*02-restricted epitopes when the original strain was compared to the VOCs, indicating that the mutations on the VOCs are promoting escape from HLA-A2-mediated antigen presentation, which characterizes a immune evasion process. Additionally, epitope signature analysis revealed major immunogenic peptide loss for structural (S) and non-structural (ORF8) proteins of VOCs in comparison to the Wuhan sequence. Discussion: These results may indicate that the antiviral CD8+ T-cell responses generated by original strains could not be sufficient for clearance of variants in either newly or reinfection with SARS-CoV-2. In contrast, N epitopes remain the most conserved and reactive peptides across SARS-CoV-2 VOCs. Overall, our data could contribute to the rational design and development of new vaccinal platforms to induce a broad cellular CD8+ T cell antiviral response, aiming at controlling viral transmission of future SARS-CoV-2 variants.

33rd Brazilian Society for Virology (SBV) 2022 Annual Meeting.

Each year, the Brazilian Society for Virology promotes a national meeting during the second semester of the year. In October 2022, the 33rd meeting took place at Arraial da Ajuda, Porto Seguro, Bahia, in-person:.this was the first in-person meeting since 2019, as the 2020 and 2021 events occurred online due to the issues imposed by COVID-19. It was a great pleasure for the whole audience to return to an in-person event, which certainly improved the interactions between the attendees in all ways. As usual, the meeting involved massive participation of undergraduate, graduate, and postdoc students, and several noteworthy international researchers were present. During five afternoons and evenings, attendees could discuss and learn about the most recent data presented by distinguished scientists from Brazil and other countries. In addition, young virology researchers from all levels could present their latest results as oral presentations and posters. The meeting covered all virology areas, with conferences and roundtables about human, veterinary, fundamental, environmental, invertebrate, and plant virology. The costs associated with attending the in-person event caused a slight reduction in the number of attendees compared to the two online events. However, even with this issue, the attendance was impressive. The meeting successfully achieved its most important goals: inspiring young and senior scientists and discussing high-quality, up-to-date virology research.

Diversity of HLA-A2-Restricted and Immunodominant Epitope Repertoire of Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Protein: Novel Insights among N-Terminal, Central and C-Terminal Regions.

The present study sought to search for the immunodominance related to the N-terminal, Central and C-terminal regions of HTLV-1 Tax using novel, cutting-edge peptide microarray analysis. In addition, in silico predictions were performed to verify the presence of nine amino acid peptides present along Tax restricted to the human leukocyte antigen (HLA)-A2.02*01 haplotype, as well as to verify the ability to induce pro-inflammatory and regulatory cytokines, such as IFN-γ and IL-4, respectively. Our results indicated abundant dose-dependent reactivity for HLA-A*02:01 in all regions (N-terminal, Central and C-terminal), but with specific hotspots. Furthermore, the results of fold-change over the Tax11-19 reactivity obtained at lower concentrations of HLA-A*02:01 reveal that peptides from the three regions contain sequences that react 100 times more than Tax11-19. On the other hand, Tax11-19 has similar or superior HLA-A*02:01 reactivity at higher concentrations of this haplotype. The in silico analysis showed a higher frequency of IFN-γ-inducing peptides in the N-terminal portion, while the C-terminal portion showed a higher frequency of IL-4 inducers. Taken together, these results shed light on the search for new Tax immunodominant epitopes, in addition to the canonic Tax11-19, for the rational design of immunomodulatory strategies for HTLV-1 chronic diseases.

Taming the SARS-CoV-2-mediated proinflammatory response with BromAc®.

Introduction: In the present study, the impact of BromAc®, a specific combination of bromelain and acetylcysteine, on the SARS-CoV-2-specific inflammatory response was evaluated. Methods: An in vitro stimulation system was standardized using blood samples from 9 healthy donors, luminex assays and flow cytometry were performed. Results and discussion: BromAc® demonstrated robust anti-inflammatory activity in human peripheral blood cells upon SARS-CoV-2 viral stimuli, reducing the cytokine storm, composed of chemokines, growth factors, and proinflammatory and regulatory cytokines produced after short-term in vitro culture with the inactivated virus (iSARS-CoV-2). A combined reduction in vascular endothelial growth factor (VEGF) induced by SARS-CoV-2, in addition to steady-state levels of platelet recruitment-associated growth factor-PDGFbb, was observed, indicating that BromAc® may be important to reduce thromboembolism in COVID-19. The immunophenotypic analysis of the impact of BromAc® on leukocytes upon viral stimuli showed that BromAc® was able to downmodulate the populations of CD16+ neutrophils and CD14+ monocytes observed after stimulation with iSARS-CoV-2. Conversely, BromAc® treatment increased steady-state HLA-DR expression in CD14+ monocytes and preserved this activation marker in this subset upon iSARS-CoV-2 stimuli, indicating improved monocyte activation upon BromAc® treatment. Additionally, BromAc® downmodulated the iSARS-CoV-2-induced production of TNF-a by the CD19+ B-cells. System biology approaches, utilizing comprehensive correlation matrices and networks, showed distinct patterns of connectivity in groups treated with BromAc®, suggesting loss of connections promoted by the compound and by iSARS-CoV-2 stimuli. Negative correlations amongst proinflammatory axis and other soluble and cellular factors were observed in the iSARS-CoV-2 group treated with BromAc® as compared to the untreated group, demonstrating that BromAc® disengages proinflammatory responses and their interactions with other soluble factors and the axis orchestrated by SARS-CoV-2. Conclusion: These results give new insights into the mechanisms for the robust anti-inflammatory effect of BromAc® in the steady state and SARS-CoV-2-specific immune leukocyte responses, indicating its potential as a therapeutic strategy for COVID-19.

Quality attributes of CTVad1, a nanoemulsified adjuvant for phase I clinical trial of SpiN COVID-19 vaccine.

Aim: To develop, characterize and evaluate an oil/water nanoemulsion with squalene (CTVad1) to be approved as an adjuvant for the SpiN COVID-19 vaccine clinical trials. Materials & methods: Critical process parameters (CPPs) of CTVad1 were standardized to meet the critical quality attributes (CQAs) of an adjuvant for human use. CTVad1 and the SpiN-CTVad1 vaccine were submitted to physicochemical, stability, in vitro and in vivo studies. Results & conclusion: All CQAs were met in the CTVad1 production process. SpiN- CTVad1 met CQAs and induced high levels of antibodies and specific cellular responses in in vivo studies. These results represented a critical step in the process developed to meet regulatory requirements for the SpiN COVID-19 vaccine clinical trial.

Immune modulation in primary vaccinia virus zoonotic human infections.

In 2010, the WHO celebrated the 30th anniversary of the smallpox eradication. Ironically, infections caused by viruses related to smallpox are being increasingly reported worldwide, including Monkeypox, Cowpox, and Vaccinia virus (VACV). Little is known about the human immunological responses elicited during acute infections caused by orthopoxviruses. We have followed VACV zoonotic outbreaks taking place in Brazil and analyzed cellular immune responses in patients acutely infected by VACV. Results indicated that these patients show a biased immune modulation when compared to noninfected controls. Amounts of B cells are low and less activated in infected patients. Although present, T CD4(+) cells are also less activated when compared to noninfected individuals, and so are monocytes/macrophages. Similar results were obtained when Balb/C mice were experimentally infected with a VACV sample isolated during the zoonotic outbreaks. Taking together, the data suggest that zoonotic VACVs modulate specific immune cell compartments during an acute infection in humans.