Refined analytical pipeline for the pharmacodynamic assessment of T-cell responses to vaccine antigens.

Pharmacodynamic assessment of T-cell-based cancer immunotherapies often focus on detecting rare circulating T-cell populations. The therapy-induced immune cells in blood-derived clinical samples are often present in very low frequencies and with the currently available T-cell analytical assays, amplification of the cells of interest prior to analysis is often required. Current approaches aiming to enrich antigen-specific T cells from human Peripheral Blood Mononuclear Cells (PBMCs) depend on in vitro culturing in presence of their cognate peptides and cytokines. In the present work, we improved a standard, publicly available protocol for T-cell immune analyses based on the in vitro expansion of T cells. We used PBMCs from healthy subjects and well-described viral antigens as a model system for optimizing the experimental procedures and conditions. Using the standard protocol, we first demonstrated significant enrichment of antigen-specific T cells, even when their starting frequency ex vivo was low. Importantly, this amplification occurred with high specificity, with no or neglectable enrichment of irrelevant T-cell clones being observed in the cultures. Testing of modified culturing timelines suggested that the protocol can be adjusted accordingly to allow for greater cell yield with strong preservation of the functionality of antigen-specific T cells. Overall, our work has led to the refinement of a standard protocol for in vitro stimulation of antigen-specific T cells and highlighted its reliability and reproducibility. We envision that the optimized protocol could be applied for longitudinal monitoring of rare blood-circulating T cells in scenarios with limited sample material.

mRNA Splicing of UL44 and Secretion of Alphaherpesvirinae Glycoprotein C (gC) Is Conserved among the Mardiviruses.

Marek's disease (MD), caused by gallid alphaherpesvirus 2 (GaAHV2) or Marek's disease herpesvirus (MDV), is a devastating disease in chickens characterized by the development of lymphomas throughout the body. Vaccine strains used against MD include gallid alphaherpesvirus 3 (GaAHV3), a non-oncogenic chicken alphaherpesvirus homologous to MDV, and homologous meleagrid alphaherpesvirus 1 (MeAHV1) or turkey herpesvirus (HVT). Previous work has shown most of the MDV gC produced during in vitro passage is secreted into the media of infected cells although the predicted protein contains a transmembrane domain. We formerly identified two alternatively spliced gC mRNAs that are secreted during MDV replication in vitro, termed gC104 and gC145 based on the size of the intron removed for each UL44 (gC) transcript. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized GaAHV3 (strain 301B/1) and HVT also secrete gC due to mRNA splicing. To address this, we collected media from 301B/1- and HVT-infected cell cultures and used Western blot analyses and determined that both 301B/1 and HVT produced secreted gC. Next, we extracted RNAs from 301B/1- and HVT-infected cell cultures and chicken feather follicle epithelial (FFE) skin cells. RT-PCR analyses confirmed one splicing variant for 301B/1 gC (gC104) and two variants for HVT gC (gC104 and gC145). Interestingly, the splicing between all three viruses was remarkably conserved. Further analysis of predicted and validated mRNA splicing donor, branch point (BP), and acceptor sites suggested single nucleotide polymorphisms (SNPs) within the 301B/1 UL44 transcript sequence resulted in no gC145 being produced. However, modification of the 301B/1 gC145 donor, BP, and acceptor sites to the MDV UL44 sequences did not result in gC145 mRNA splice variant, suggesting mRNA splicing is more complex than originally hypothesized. In all, our results show that mRNA splicing of avian herpesviruses is conserved and this information may be important in developing the next generation of MD vaccines or therapies to block transmission.

The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation.

Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

The complete Kaposi sarcoma-associated herpesvirus genome induces early-onset, metastatic angiosarcoma in transgenic mice.

Kaposi sarcoma (KS) is the most common cancer in persons living with HIV. It is caused by KS-associated herpesvirus (KSHV). There exists no animal model for KS. Pronuclear injection of the 170,000-bp viral genome induces early-onset, aggressive angiosarcoma in transgenic mice. The tumors are histopathologically indistinguishable from human KS. As in human KS, all tumor cells express the viral latency-associated nuclear antigen (LANA). The tumors transcribe most viral genes, whereas endothelial cells in other organs only transcribe the viral latent genes. The tumor cells are of endothelial lineage and exhibit the same molecular pattern of pathway activation as KS, namely phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR, interleukin-10 (IL-10), and vascular endothelial growth factor (VEGF). The KSHV-induced tumors are more aggressive than Ha-ras-induced angiosarcomas. Overall survival is increased by prophylactic ganciclovir. Thus, whole-virus KSHV-transgenic mice represent an accurate model for KS and open the door for the genetic dissection of KS pathogenesis and evaluation of therapies, including vaccines.

Heritability and genome-wide association study of vaccine-induced immune response in Beagles: A pilot study.

Both genetic and non-genetic factors contribute to individual variation in the immune response to vaccination. Understanding how genetic background influences variation in both magnitude and persistence of vaccine-induced immunity is vital for improving vaccine development and identifying possible causes of vaccine failure. Dogs provide a relevant biomedical model for investigating mammalian vaccine genetics; canine breed structure and long linkage disequilibrium simplify genetic studies in this species compared to humans. The objective of this study was to estimate the heritability of the antibody response to vaccination against viral and bacterial pathogens, and to identify genes driving variation of the immune response to vaccination in Beagles. Sixty puppies were immunized following a standard vaccination schedule with an attenuated combination vaccine containing antigens for canine adenovirus type 2, canine distemper virus, canine parainfluenza virus, canine parvovirus, and four strains of Leptospira bacteria. Serum antibody measurements for each viral and bacterial component were measured at multiple time points. Heritability estimations and GWAS were conducted using SNP genotypes at 279,902 markers together with serum antibody titer phenotypes. The heritability estimates were: (1) to Leptospira antigens, ranging from 0.178 to 0.628; and (2) to viral antigens, ranging from 0.199 to 0.588. There was not a significant difference between overall heritability of vaccine-induced immune response to Leptospira antigens compared to viral antigens. Genetic architecture indicates that SNPs of low to high effect contribute to immune response to vaccination. GWAS identified two genetic markers associated with vaccine-induced immune response phenotypes. Collectively, these findings indicate that genetic regulation of the immune response to vaccination is antigen-specific and influenced by multiple genes of small effect.

Establishment and application of a gold nanoparticle-based immunochromatographic test strip for the detection of avian leukosis virus P27 antigen in egg white samples.

Avian leukemia is an infectious tumorous disease of chickens caused by subgroup A of the avian leukemia virus (ALV-A), which mainly causes long-term viremia, slow growth, immune suppression, decreased production performance, multi-tissue tumors, and even death. The infection rate of this disease is very high in chicken herds in China, causing huge economic losses to the poultry industry every year. We successfully expressed the specific antigen protein of ALV (P27) through recombinant protein technology and screened a pair of highly sensitive monoclonal antibodies (mAbs) through mouse immunity, cell fusion, and antibody pairing. Based on this pair of antibodies, we established a dual antibody sandwich ELISA and gold nanoparticle immunochromatographic strip (AuNP-ICS) detection method. In addition, the parameters of the dual antibody sandwich ELISA and AuNP-ICS were optimized under different reaction conditions, which resulted in the minimum detection limits of 0.2 ng mL-1 and 1.53 ng ml-1, respectively. Commonly available ELISA and AuNP-ICS products on the market were compared, and we found that our established immune rapid chromatography had higher sensitivity. This established AuNP-ICS had no cross-reactivity with Influenza A (H1N1), Influenza A (H9N2), respiratory syncytial virus (RSV), varicella-zoster virus (VZV), Listeria monocytogenes listeriolysin (LLO), and Staphylococcal enterotoxin SED or SEC. Finally, the established AuNP-ICS was used to analyze 35 egg samples, and the results showed 5 positive samples and 30 negative samples. The AuNP-ICS rapid detection method established by our group had good specificity, high sensitivity, and convenience, and could be applied to the clinical sample detection of ALV-A.

Advances in rapid point-of-care virus testing.

Outbreaks of viral diseases seriously jeopardize people's health and cause huge economic losses. At the same time, virology provides a new perspective for biology, molecular biology and cancer research, and it is important to study the discovered viruses with potential applications. Therefore, the development of immediate and rapid viral detection methods for the prevention and treatment of viral diseases as well as the study of viruses has attracted extensive attention from scientists. With the continuous progress of science and technology, especially in the field of bioanalysis, a series of new detection techniques have been applied to the on-site rapid detection of viruses, which has become a powerful approach for human beings to fight against viruses. In this paper, the latest research progress of rapid point-of-care detection of viral nucleic acids, antigens and antibodies is presented. In addition, the advantages and disadvantages of these technologies are discussed from the perspective of practical application requirements. Finally, the problems and challenges faced by rapid viral detection methods and their development prospects are discussed.

Evaluation of a Revised Point-of-Care Test for the Detection of Feline Leukaemia p27 Antigen and Anti-p15E Antibodies in Cats.

The first point-of-care (PoC) test (v-RetroFel®; modified version 2021) determining the presence of FeLV p27 antigen and FeLV anti-p15E antibodies has become recently commercially available to identify different feline leukaemia virus (FeLV) infection outcomes. This study aimed to assess this PoC test's performance concerning FeLV p27 antigen and FeLV anti-p15E antibody detection. Sensitivity, specificity, positive and negative predictive values (PPV, NPV) were assessed after ten minutes (recommended) and 20 min (prolonged) incubation times. The test results were evaluated as either positive or negative. Serum samples from 934 cats were included, originating from Italy (n = 269), Portugal (n = 240), Germany (n = 318), and France (n = 107). FeLV p27 antigen and anti-p15E antibodies were measured by reference standard ELISAs and compared to the PoC test results. The PoC test was easy to perform and the results easy to interpret. Sensitivity and specificity for FeLV p27 antigen were 82.8% (PPV: 57.8%) and 96.0% (NPV: 98.8%) after both, ten and 20 minues of incubation time. Sensitivity and specificity for anti-p15E antibodies were 31.4% (PPV: 71.6%) and 96.9% (NPV: 85.1%) after ten minutes incubation time; sensitivity was improved by a prolonged incubation time (20 min) to 40.0% (PPV: 76.3%), while specificity remained the same (96.9%, NPV: 86.7%). Despite the improved sensitivity using the prolonged incubation time, lower than ideal sensitivities for both p27 antigen and especially anti-p15E antibodies were found, indicating that the PoC test in its current version needs further improvement prior to application in the field.

Identification and genetic characterization of a recently identified enterovirus C116 in China.

Enterovirus C116 (EV-C116) is a new member of the enterovirus C group which is closely associated with several infectious diseases. Although sporadic studies have detected EV-C116 in clinical samples worldwide, there is currently limited information available. In this study, two EV-C-positive fecal specimens were detected in apparently healthy children, which harbored low abundance, through meta-transcriptome sequencing. Based on the prototypes of several EV-Cs, two lineages were observed. Lineage 1 included many types that could not cause EV-like cytopathic effect in cell culture. Three genogroups of EV-C116 were divided in the maximum likelihood tree, and the two strains in this study (XZ2 and XZ113) formed two different lineages, suggesting that EV-C116 still diffuses worldwide. Obvious inter-type recombination events were observed in the XZ2 strain, with CVA22 identified as a minor donor. However, another strain (XZ113) underwent different recombination situations, highlighting the importance of recombination in the formation of EV-Cs biodiversity. The EV-C116 strains could propagate in rhabdomyosarcoma cell cultures at low titer; however, EV-like cytopathic effects were not observed. HEp-2, L20B, VERO, and 293T cell lines did not provide an appropriate environment for EV-C116 growth. These results challenge the traditional recognition of the uncultured nature of EV-C116 strains and explain the difficulty of clinical detection.

Cordycepin Inhibits Enterovirus A71 Replication and Protects Host Cell from Virus-Induced Cytotoxicity through Adenosine Action Pathway.

Enterovirus A71 (EV-A71) infection typically causes mild illnesses, such as hand-foot-and-mouth disease (HFMD), but occasionally leads to severe or fatal neurological complications in infants and young children. Currently, there is no specific antiviral treatment available for EV-A71 infection. Thus, the development of an effective anti-EV-A71 drug is required urgently. Cordycepin, a major bioactive compound found in Cordyceps fungus, has been reported to possess antiviral activity. However, its specific activity against EV-A71 is unknown. In this study, the potency and role of cordycepin treatment on EV-A71 infection were investigated. Results demonstrated that cordycepin treatment significantly reduced the viral load and viral ribonucleic acid (RNA) level in EV-A71-infected Vero cells. In addition, EV-A71-mediated cytotoxicity was significantly inhibited in the presence of cordycepin in a dose-dependent manner. The protective effect can also be extended to Caco-2 intestinal cells, as evidenced by the higher median tissue culture infectious dose (TCID50) values in the cordycepin-treated groups. Furthermore, cordycepin inhibited EV-A71 replication by acting on the adenosine pathway at the post-infection stage. Taken together, our findings reveal that cordycepin could be a potential antiviral candidate for the treatment of EV-A71 infection.

Wastewater-Based Epidemiology for Viral Surveillance from an Endemic Perspective: Evidence and Challenges.

Wastewater-based epidemiology (WBE) is currently used to monitor not only the spread of the viral SARS-CoV-2 pandemic but also that of other viruses in endemic conditions, particularly in the absence of syndromic surveillance. The continuous monitoring of sewage requires high expenditure and significant time investments, highlighting the need for standardized methods and structured monitoring strategies. In this context, we conducted weekly wastewater monitoring in northwestern Tuscany (Italy) and targeted human adenovirus (HAdV), norovirus genogroup II (NoVggII), enterovirus (EV), and SARS-CoV-2. Samples were collected at the entrances of treatment plants and concentrated using PEG/NaCl precipitation, and viral nucleic acids were extracted and detected through real-time reverse transcription qPCR. NoVggII was the most identified target (84.4%), followed by HAdV, SARS-CoV-2, and EV. Only HAdV and EV exhibited seasonal peaks in spring and summer. Compared with data that were previously collected in the same study area (from February 2021 to September 2021), the results for SARS-CoV-2 revealed a shift from an epidemic to an endemic pattern, at least in the region under investigation, which was likely due to viral mutations that led to the spreading of new variants with increased resistance to summer environmental conditions. In conclusion, using standardized methods and an efficient monitoring strategy, WBE proves valuable for viral surveillance in pandemic and epidemic scenarios, enabling the identification of temporal-local distribution patterns that are useful for making informed public health decisions.

Disease-Modifying Treatments for Multiple Sclerosis Affect Measures of Cellular Immune Responses to EBNA-1 Peptides.

BACKGROUND AND OBJECTIVES: Epstein-Barr virus (EBV) has been strongly implicated in the pathogenesis of multiple sclerosis (MS). Despite this, there are no routinely used tests to measure cellular response to EBV. In this study, we analyzed the cellular response to EBV nuclear antigen-1 (EBNA-1) in people with MS (pwMS) using a whole blood assay. METHODS: This cross-sectional study took place in a dedicated MS clinic in a university hospital. We recruited healthy controls, people with epilepsy (PWE), and pwMS taking a range of disease-modifying treatments (DMTs) including natalizumab, anti-CD20 monoclonal antibodies (mAbs), dimethyl fumarate (DMF), and also treatment naïve. Whole blood samples were stimulated with commercially available PepTivator EBNA1 peptides and a control virus-cytomegalovirus (CMV) peptide. We recorded the cellular response to stimulation with both interferon gamma (IFN-γ) and interleukin-2 (IL-2). We also compared the cellular responses to EBNA1 with IgG responses to EBNA1, viral capsid antigen (VCA), and EBV viral load. RESULTS: We recruited 86 pwMS, with relapsing remitting MS, in this group, and we observed a higher level of cellular response recorded with IFN-γ (0.79 IU/mL ± 1.36) vs healthy controls (0.29 IU/mL ± 0.90, p = 0.0048) and PWE (0.17 IU/mL ± 0.33, p = 0.0088). Treatment with either anti-CD20 mAbs (0.28 IU/mL ± 0.57) or DMF (0.07 IU/mL ± 0.15) resulted in a cellular response equivalent to control levels or in PWE (p = 0.26). The results of recording IL-2 response were concordant with IFN-γ: with suppression also seen with anti-CD20 mAbs and DMF. By contrast, we did not record any differential effect of DMTs on the levels of IgG to either EBNA-1 or VCA. Nor did we observe differences in cellular response to cytomegalovirus between groups. DISCUSSION: This study demonstrates how testing and recording the cellular response to EBNA-1 in pwMS may be beneficial. EBNA-1 stimulation of whole blood samples produced higher levels of IFN-γ and IL-2 in pwMS compared with controls and PWE. In addition, we show a differential effect of currently available DMTs on this response. The functional assay deployed uses whole blood samples with minimal preprocessing suggesting that employment as a treatment response measure in clinical trials targeting EBV may be possible.

Modified streptavidin-biotin based lateral flow test strip for rapid detection of SARS-CoV-2 S1 antigen in saliva samples.

Compared to other infectious diseases, for which LFT development can take years, SARS-CoV-2 antigen LFTS were developed and deployed within months. LFTS for antigen detection were adopted on an unprecedented scale during the COVID-19 pandemic, but many of them lack the sensitivity especially for samples with low viral load. In our previous work, we developed an enhanced signal strip for detection of SARS CoV-2 SI antigens in saliva. Here we introduce some modification to improve the sensitivity, and specificity, and to lower the cost of the strip, by using biotin streptavidin (BS) system. In the modified BS strip, gold-streptavidin and biotinylated Nanobodies (Nbs) against S1 antigen were externally mixed with the tested samples (saliva or nasopharyngeal swab) before their application on the sample pad of the test strip containing angiotensin converting enzyme (ACE-2), as the capturing probe. The study included 320 individuals, with 180 being positively confirmed by RT-PCR and 140 confirmed negative, as well as, 45 health care workers, who were responsible for screening and handling of surgical cases in General Surgery Department and COVID clinic of TBRI. Our results proved that modified BS strip improved the overall sensitivity and specificity of S1antigen detection in saliva samples (95.21% and 99.29% respectively) compared to our previously developed enhanced LFTS (91.66% and 98.57% respectively). Also, the sensitivity of cases with Ct ≤ 30, Ct ≤ 35, and Ct ≤ 40 using the modified BS strip showed higher values (98.54%, 95.38%, and 88.89% respectively), compared to the corresponding results of our previously developed enhanced LFTS (95.86%, 92.31%, and 82.22% respectively). There were no cross-reactions with either Middle East respiratory syndrome corona virus MERS-CoV or SARS-CoV antigens. Furthermore, we found that the lower viral detection limit (LVD) of BS strip was obviously lower than our previous LVD limit of the enhanced LFTS (0.2 × 104 copies/ml vs. 0.4 × 104 copies/ml, respectively). Our developed BS strip showed that saliva samples gave better results than nasopharyngeal swabs of the same patients. The fact of using smaller amounts of Nbs, and ACE2, as well as the dispensing off of conjugate pad when applying BS strip modifications, justified the expected reduction in the costs of the strip. The implementation of BS strips on saliva samples of 45 health co-workers, who were tested 4 and 6 days after exposure to infection, showed an increase in the sensitivity, starting from the 4th day and reaching its highest level on the 6th day in both high risk and paramedic groups (90.9%, and 80.0%, respectively). This study provides evidence that employment of the modified BS system could increase the sensitivity of the strips, lower their cost, and render them an effective screening tool for early detection of the virus in saliva of suspected Covid-19 patients.

Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development.

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Oral administration of a recombinant modified RBD antigen of SARS-CoV-2 as a possible immunostimulant for the care of COVID-19.

BACKGROUND: Developing effective vaccines against SARS-CoV-2 that consider manufacturing limitations, equitable access, and acceptance is necessary for developing platforms to produce antigens that can be efficiently presented for generating neutralizing antibodies and as a model for new vaccines. RESULTS: This work presents the development of an applicable technology through the oral administration of the SARS-CoV-2 RBD antigen fused with a peptide to improve its antigenic presentation. We focused on the development and production of the recombinant receptor binding domain (RBD) produced in E. coli modified with the addition of amino acids extension designed to improve antigen presentation. The production was carried out in shake flask and bioreactor cultures, obtaining around 200 mg/L of the antigen. The peptide-fused RBD and peptide-free RBD proteins were characterized and compared using SDS-PAGE gel, high-performance chromatography, and circular dichroism. The peptide-fused RBD was formulated in an oil-in-water emulsion for oral mice immunization. The peptide-fused RBD, compared to RBD, induced robust IgG production in mice, capable of recognizing the recombinant RBD in Enzyme-linked immunosorbent assays. In addition, the peptide-fused RBD generated neutralizing antibodies in the sera of the dosed mice. The formulation showed no reactive episodes and no changes in temperature or vomiting. CONCLUSIONS: Our study demonstrated the effectiveness of the designed peptide added to the RBD to improve antigen immunostimulation by oral administration.

Features of Cytokine and VEGFA Gene Expression Modified with SARS-CoV-2 Virus in an In Vitro Experiment (Using the Example of the SARS-CoV-2 Vaccine Antigen).

The influence of SARS-CoV-2 antigen on the cytokine-producing function of immune cells was studied. We observed suppression of the production of proinflammatory cytokines by 11-46% relative to the spontaneous level under the influence of SARS-CoV-2 antigen vaccine simulator, as well as when it was co-administered with cortisol (IL-6 by 1.8 times and IFNγ by 1.57 times) compared with control samples. IL-8 production was reduced by 1.72 times relative to its spontaneous level. IL-8 production was reduced by 1.72 times relative to its spontaneous level. Under conditions of SARS-CoV-2 stimulation with the vaccine antigen in vitro, an increase in the relative scaled expression of the VEGFA gene by 2.16 times relative to the spontaneous level was observed, which can be regarded as a model "cytokine storm" scenario. The obtained experimental data verify the ideas about the pathogenetic mechanisms of the COVID-19 and can contribute to the development of new approaches to the correction of its complications.

Seroprevalence of Hepatitis B virus and human immunodeficiency virus co-infection in pregnant women from Osun State, Nigeria.

INTRODUCTION: Hepatitis B virus and human immunodeficiency virus (HBV/HIV) co-infection is a global health concern due to its significant impact on morbidity and mortality. Reports of HBV/HIV co-infections are increasing in Nigeria, but information on the disease burden in pregnant women and its implications on the fetus is scarce. This study aimed to determine the prevalence of HBV/HIV co-infection in pregnant women. In addition, the study identified the risk factors for the disease in pregnant women attending antenatal clinics in Osun State, Nigeria. METHODOLOGY: We collected plasma samples from 303 consenting pregnant women and used enzyme-linked immunosorbent assay (ELISA) to test for HBV (HBsAg) and HIV I/II antigens. We obtained demographic and risk factor data on HBV and HIV transmission using a structured questionnaire. RESULTS: Our analysis revealed a prevalence of 3.96% for HBV/HIV co-infection in pregnant women. Bivariate analysis indicated a history of blood transfusion, oral or anal sex, and multiple sexual partners may be associated with an increased likelihood of HBV/HIV co-infection in pregnant women. After adjusting for other variables in multivariate analysis, none of these risk factors were significant at the 5% level. In contrast, formal education was a potential preventive factor in this population. CONCLUSIONS: Our study provides valuable information on the disease burden of HBV/HIV co-infection in pregnant women in Osun State, Nigeria, highlighting the importance of routine screening for HBV and HIV during antenatal care and emphasizing the importance of implementing preventive measures to reduce the morbidity and mortality associated with HBV/HIV co-infection.

Respiratory viral detection in the plasma and cerebrospinal fluid (CSF) of young febrile infants.

Background: Respiratory viral infections are common in febrile infants ≤90 days. However, the detection of viruses other than enterovirus in the blood and cerebrospinal fluid (CSF) of young infants is not well defined. We sought to quantify the occurrence of respiratory viruses in the blood and CSF of febrile infants ≤90 days. Methods: We conducted a nested cohort study examining plasma and CSF samples from febrile infants 15-90 days via rtPCR. The samples were tested for respiratory viruses (respiratory syncytial virus, influenza, enterovirus, parechovirus, adenovirus, bocavirus). Clinical and laboratory data were also collected to determine the presence of serious bacterial infections (SBI). Results: Twenty-four percent (30 of 126) of infants had plasma/CSF specimens positive for a respiratory virus. Enterovirus and parechovirus were the most commonly detected respiratory viruses. Viral positivity was highest in plasma samples at 25% (27 of 107) compared with CSF samples at 15% (nine of 62). SBIs (specifically urinary tract infections) were less common in infants with a sample positive for a respiratory virus compared to those without a virus detected (3% vs. 26%, p = 0.008). Conclusions: Our findings support the use of molecular diagnostics to include the identification of parechovirus in addition to enterovirus in febrile infants ≤90 days. Additionally, these data support the utilization of blood specimens to diagnose enterovirus and parechovirus infections in febrile infants ≤90 days.

Molecular Amplification and Cell Culturing Efficiency for Enteroviruses' Detection in Cerebrospinal Fluids of Algerian Patients Suffering from Meningitis.

Enteroviruses (EVs) represent a major cause of viral meningitis, being responsible for nearly 1 billion infections each year worldwide. Several techniques were developed to obtain better diagnostic results of EV infections. Herein, we evaluated the efficiency of EV detection through isolation on both Rhabdomyosarcoma (RD) and Vero cell line cultures, conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR. Thus, 50 cerebrospinal fluid (CSF) samples belonging to patients suspected to have viral meningitis in northern Algeria were collected, anonymously numbered from 1 to 50 and subjected to the above-mentioned techniques for EV detection. Using real-time RT-PCR, 34 CSF samples were revealed to be positive for viral origin of meningitis (68%). Thirteen of them were positive when the conventional RT-PCR was used (26%), and only three samples gave positive results when the cell culture technique was used (6%). Surprisingly, two cell culture-positive CSF samples, namely, 31 and 39, were negative using RT-PCR directly on the original samples. However, they turned to be positive when amplification was carried out on their corresponding cell culture supernatant. The cell-cultured viral isolates were then identified by sequencing their viral genome's VP1 regions. All of them were revealed to belong to the echovirus 27 strain. This investigation demonstrates that RT-PCR techniques are often more sensitive, accurate and much faster, providing reliable results within a clinically acceptable timeframe. However, viral isolation on cell cultures remains crucial to obtain enough viral load for serological tests or even to avoid the rare, but existing, false negative PCR.

Enhancer-promoter activation by the Kaposi sarcoma-associated herpesvirus episome maintenance protein LANA.

Higher-order genome structure influences the transcriptional regulation of cellular genes through the juxtaposition of regulatory elements, such as enhancers, close to promoters of target genes. While enhancer activation has emerged as an important facet of Kaposi sarcoma-associated herpesvirus (KSHV) biology, the mechanisms controlling enhancer-target gene expression remain obscure. Here, we discover that the KSHV genome tethering protein latency-associated nuclear antigen (LANA) potentiates enhancer-target gene expression in primary effusion lymphoma (PEL), a highly aggressive B cell lymphoma causally associated with KSHV. Genome-wide analyses demonstrate increased levels of enhancer RNA transcription as well as activating chromatin marks at LANA-bound enhancers. 3D genome conformation analyses identified genes critical for latency and tumorigenesis as targets of LANA-occupied enhancers, and LANA depletion results in their downregulation. These findings reveal a mechanism in enhancer-gene coordination and describe a role through which the main KSHV tethering protein regulates essential gene expression in PEL.