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The distribution of the functional subsets of porcine T cells, the cytolytic/suppressor (Tc/s) and the helper/inducer (Th/i) cells was studied in cryostat sections of thymus, lymph nodes, tonsils, Peyer's patches, spleen and liver using the indirect immunoperoxidase technique. Three murine monoclonal antibodies (mAb) were used. The mAb 8/1 reacts with an antigen present on all T cells and on cells of the myeloid lineage; the antigen has not yet been characterized biochemically. The mAb 295/33 (anti- T8) binds to the porcine T8 antigen and defines the Tc/s subset, while mAb PT-4 (anti-T4) detects the porcine T4 antigen and defines the Th/i subset. Practically all thymocytes were stained by inAb 8/1. The majority of cortical thymocytes apparently co-expressed T8 and T4, whereas distinct fractions of medullary cells were labelled by either anti-T8 or anti-T4. In peripheral lymphoid organs all three mAb reacted with cells in the thymus-dependent areas and with cells scattered in the lymphoid follicles. In lymph nodes, tonsils and Peyer's patches, anti-T8 and anti-T4 each labelled approximately half of the cells stained by mAb 8/1. in the periarteriolar lymphoid sheath of the spleen, anti-T4 labelled more cells than did anti-T8. The reactivity of mAb 8/1 with the Kupffer cells of the liver demonstrated the expression of the 8/1 antigen on cells of the monocyte lineage. The T8 and T4 antigens could not be detected in acetone-fixed and paraffin-embedded sections, while the antigen recognized by mAb 8/1 remained preserved. Altogether, despite an inverted microanatomical structure of porcine lymph nodes, the frequency and distribution of T8+ and T4+ cells in thymus-dependent areas proved to be similar to those found in other species. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV), a representative member of the Betaherpesvirinae subfamily of herpesviruses, is common in the human population, but immunocompetent individuals are generally asymptomatic when infected with this virus. However, in immunocompromised individuals and immunologically immature fetuses and newborns, CMV can cause a wide range of often long-lasting morbidities and even death. CMV is not only widespread throughout the population but it is also widespread in its hosts, infecting and establishing latency in nearly all tissues and organs. Thus, understanding the pathogenesis of and immune responses to this virus is a prerequisite for developing effective prevention and treatment strategies. Multiple arms of the immune system are engaged to contain the infection, and general concepts of immune control of CMV are now reasonably well understood. Nonetheless, in recent years, tissue-specific immune responses have emerged as an essential factor for resolving CMV infection. As tissues differ in biology and function, so do immune responses to CMV and pathological processes during infection. This review discusses state-of-the-art knowledge of the immune response to CMV infection in tissues, with particular emphasis on several well-studied and most commonly affected organs., (© 2024. The Author(s).)

Current vaccines against COVID-19 elicit immune responses that are overall strong but wane rapidly. As a consequence, the necessary booster shots have contributed to vaccine fatigue. Hence, vaccines that would provide lasting protection against COVID-19 are needed, but are still unavailable. Cytomegaloviruses (CMVs) elicit lasting and uniquely strong immune responses. Used as vaccine vectors, they may be attractive tools that obviate the need for boosters. Therefore, we tested the murine CMV (MCMV) as a vaccine vector against COVID-19 in relevant preclinical models of immunization and challenge. We have previously developed a recombinant MCMV vaccine vector expressing the spike protein of the ancestral SARS-CoV-2 (MCMV S ). In this study, we show that the MCMV S elicits a robust and lasting protection in young and aged mice. Notably, spike-specific humoral and cellular immunity was not only maintained but also even increased over a period of at least 6 months. During that time, antibody avidity continuously increased and expanded in breadth, resulting in neutralization of genetically distant variants, like Omicron BA.1. A single dose of MCMV S conferred rapid virus clearance upon challenge. Moreover, MCMV S vaccination controlled two variants of concern (VOCs), the Beta (B.1.135) and the Omicron (BA.1) variants. Thus, CMV vectors provide unique advantages over other vaccine technologies, eliciting broadly reactive and long-lasting immune responses against COVID-19., Competing Interests: LČ-Š, SJ, and YK are applicants for a patent based on MCMV as a vaccine vector. LČ-Š served as advisor to SANOFI for COVID vaccines, unrelated to this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Metzdorf, Jacobsen, Kim, Teixeira Alves, Kulkarni, Brdovčak, Materljan, Eschke, Chaudhry, Hoffmann, Bertoglio, Ruschig, Hust, Šustić, Krmpotić, Jonjić, Widera, Ciesek, Pöhlmann, Landthaler and Čičin-Šain.)

The aim of this study was to characterize the systemic cytokine signature of critically ill COVID-19 patients in a high mortality setting aiming to identify biomarkers of severity, and to explore their associations with viral loads and clinical characteristics. We studied two COVID-19 critically ill patient cohorts from a referral centre located in Central Europe. The cohorts were recruited during the pre-alpha/alpha (November 2020 to April 2021) and delta (end of 2021) period respectively. We determined both the serum and bronchoalveolar SARS-CoV-2 viral load and identified the variant of concern (VoC) involved. Using a cytokine multiplex assay, we quantified systemic cytokine concentrations and analyzed their relationship with clinical findings, routine laboratory workup and pulmonary function data obtained during the ICU stay. Patients who did not survive had a significantly higher systemic and pulmonary viral load. Patients infected with the pre-alpha VoC showed a significantly lower viral load in comparison to those infected with the alpha- and delta-variants. Levels of systemic CTACK, M-CSF and IL-18 were significantly higher in non-survivors in comparison to survivors. CTACK correlated directly with APACHE II scores. We observed differences in lung compliance and the association between cytokine levels and pulmonary function, dependent on the VoC identified. An intra-cytokine analysis revealed a loss of correlation in the non-survival group in comparison to survivors in both cohorts. Critically ill COVID-19 patients exhibited a distinct systemic cytokine profile based on their survival outcomes. CTACK, M-CSF and IL-18 were identified as mortality-associated analytes independently of the VoC involved. The Intra-cytokine correlation analysis suggested the potential role of a dysregulated systemic network of inflammatory mediators in severe COVID-19 mortality., (© 2024. The Author(s).)

Innate lymphoid cells (ILCs) are largely tissue-resident cells that participate in the maintenance of tissue homeostasis and react early to inflammatory events. Mature ILCs are divided into three major groups based on the transcription factors required for their development and function. Under physiological conditions, ILCs are present within the choroid plexus and meninges while the CNS parenchyma is almost devoid of these cells. However, pathological conditions such as autoimmune neuroinflammation and viral infections of the CNS result in the infiltration of ILCs into parenchyma. In this article, we provide an overview of the involvement and function of the ILCs within the CNS during physiological conditions and in infections, autoimmune diseases, neurodegeneration, and injury., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kveštak, Mihalić, Jonjić and Brizić.)

Despite the availability of currently approved antiviral drugs, infections with human cytomegalovirus (HCMV) still cause clinically challenging, sometimes life-threatening situations. There is an urgent need for enhanced anti-HCMV drugs that offer improved efficacy, reduced dosages and options for long-term treatment without risk of the development of viral drug resistance. Recently, we reported the pronounced anti-HCMV efficacy of pharmacological inhibitors of cyclin-dependent kinases (CDKs), in particular, the potential of utilizing drug synergies upon combination treatment with inhibitors of host CDKs and the viral CDK-like kinase pUL97 (vCDK/pUL97). Here, we expand this finding by further assessing the in vitro synergistic antiviral interaction between vCDK and CDK inhibitors towards HCMV as well as non-human cytomegaloviruses. An extension of this synergy approach was achieved in vivo by using the recombinant MCMV-UL97/mouse model, confirming the high potential of combination treatment with the clinically approved vCDK inhibitor maribavir (MBV) and the developmental CDK7 inhibitor LDC4297. Moreover, mechanistic aspects of this synergistic drug combination were illustrated on the levels of intracellular viral protein transport and viral genome replication. The analysis of viral drug resistance did not reveal resistance formation in the case of MBV + LDC4297 combination treatment. Spanning various investigational levels, these new results strongly support our concept, employing the great potential of anti-HCMV synergistic drug treatment.

Infections in early life can elicit substantially different immune responses and pathogenesis than infections in adulthood. Here, we investigate the consequences of murine cytomegalovirus infection in newborn mice on NK cells. We show that infection severely compromised NK cell maturation and functionality in newborns. This effect was not due to compromised virus control. Inflammatory responses to infection dysregulated the expression of major transcription factors governing NK cell fate, such as Eomes, resulting in impaired NK cell function. Most prominently, NK cells from perinatally infected mice have a diminished ability to produce IFN-γ due to the downregulation of long non-coding RNA Ifng-as1 expression. Moreover, the bone marrow's capacity to efficiently generate new NK cells is reduced, explaining the prolonged negative effects of perinatal infection on NK cells. This study demonstrates that viral infections in early life can profoundly impact NK cell biology, including long-lasting impairment in NK cell functionality., (© 2023. Springer Nature Limited.)

Human cytomegalovirus (HCMV) frequently causes congenital infections, resulting in birth defects and developmental disorders. A vaccine is needed, but unavailable. We analyzed the potential of CMV mutants, lacking their STAT2 antagonists to serve as live attenuated vaccine viruses in mice. Infections with attenuated viruses elicited strong ELISA-reactive binding IgG responses and induced neutralizing antibodies as well as antibodies stimulating cellular Fcγ receptors, including the antibody-dependent cellular cytotoxicity (ADCC)-eliciting receptors FcγRIII/CD16 and FcγRIV. Accordingly, vaccinated mice were fully protected against challenge infections. Female mice vaccinated prior to gestation transmitted CMV-specific IgG to their offspring, which protected the progeny from perinatal infections in a mouse model for congenital CMV disease. To define the role of maternal antibodies, female mice either capable or incapable of producing antibodies were vaccinated and subsequently bred to males of the opposite genotype. Challenge infections of the genotypically identical F1 generation revealed the indispensability of maternal antibodies for vaccine-induced protection against cytomegaloviruses., (© 2023. The Author(s).)

Morphogenesis of herpesvirus particles is highly conserved; however, the capsid assembly and genome packaging of human cytomegalovirus (HCMV) exhibit unique features. Examples of these include the essential role of the small capsid protein (SCP) and the existence of the β-herpesvirus-specific capsid-associated protein pp150. SCP and pp150, as well as the UL77 and UL93 proteins, are important capsid constituents, yet their precise mechanism of action is elusive. Here, we analyzed how deletion of the open reading frames (ORFs) encoding pUL77, pUL93, pp150, or SCP affects the protein composition of nuclear capsids. This was achieved by generating HCMV genomes lacking the respective genes, combined with a highly efficient transfection technique that allowed us to directly analyze these mutants in transfected cells. While no obvious effects were observed when pUL77, pUL93, or pp150 was missing, the absence of SCP impeded capsid assembly due to strongly reduced amounts of major capsid protein (MCP). Vice versa, when MCP was lacking, SCP became undetectable, indicating a mutual dependence of SCP and MCP for establishing appropriate protein levels. The SCP domain mediating stable MCP levels could be narrowed down to a C-terminal helix known to convey MCP binding. Interestingly, an SCP-EGFP (enhanced green fluorescent protein) fusion protein which also impaired the production of infectious progeny acted in a different manner, as capsid assembly was not abolished; however, SCP-EGFP-harboring capsids were devoid of DNA and trapped in paracrystalline nuclear structures. These results indicate that SCP is essential in HCMV because of its impact on MCP levels and reveal SCP as a potential target for antiviral inhibitors. IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen causing life-threatening disease in immunocompromised individuals. Virus-specific processes such as capsid assembly and genome packaging can be exploited to design new antiviral strategies. Here, we report on a novel function of the HCMV small capsid protein (SCP), namely, ensuring stable levels of major capsid protein (MCP), thereby governing capsid assembly. Furthermore, we discovered a mutual dependence of the small and major capsid proteins to guarantee appropriate levels of the other respective protein and were able to pin down the SCP domain responsible for this effect to a region previously shown to mediate binding to the major capsid protein. In summary, our data contribute to the understanding of how SCP plays an essential part in the HCMV infection cycle. Moreover, disrupting the SCP-MCP interface may provide a starting point for the development of novel antiviral drugs.

Studies assessing the dynamics and duration of antibody responses following SARS-CoV-2 infection or vaccination are an invaluable tool for vaccination schedule planning, assessment of risk groups and management of pandemics. In this study, we developed and employed ELISA assays to analyze the humoral responses to Nucleocapsid and Spike proteins in vaccinated health-care workers (HCW) and critically ill COVID-19 patients. Sera of more than 1000 HCWs and critically ill patients from the Clinical Hospital Center Rijeka were tested across a one-year period, encompassing the spread of major SARS-CoV-2 variants of concern (VOCs). We observed 97% of seroconversion in HCW cohort as well as sustained anti-Spike antibody response in vaccinees for more than 6 months. In contrast, the infection-induced anti-Nucleocapsid response was waning significantly in a six-month period. Furthermore, a substantial decrease in vaccinees' anti-Spike antibodies binding to Spike protein of Omicron VOC was also observed. Critically ill COVID-19 patients had higher levels of anti-Spike and anti-Nucleocapsid antibodies compared to HCWs. No significant differences in anti-Spike and anti-Nucleocapsid antibody levels between the critically ill COVID-19 patients that were on non-invasive oxygen supplementation and those on invasive ventilation support were observed. However, stronger anti-Spike, but not anti-Nucleocapsid, antibody response correlated with a better disease outcome in the cohort of patients on invasive ventilation support. Altogether, our results contribute to the growing pool of data on humoral responses to SARS-CoV-2 infection and vaccination.

This article describes procedures for infecting adult mice with murine cytomegalovirus (MCMV) and for infecting newborn mice to model congenital CMV infection. Methods are included for propagating MCMV in cell cultures and preparing a more virulent form of MCMV from the salivary glands of infected mice. A plaque assay is provided for determining MCMV titers of infected tissues or virus stocks. Also, methods are described for preparing the murine embryonic fibroblasts used for propagating MCMV, and for the plaque assay. © 2022 Wiley Periodicals LLC., (© 2022 Wiley Periodicals LLC.)

Herpes simplex virus 1 (HSV-1) expresses a large number of miRNAs, and their function is still not completely understood. In addition, HSV-1 has been found to deregulate host miRNAs, which adds to the complexity of the regulation of efficient virus replication. In this study, we comprehensively addressed the deregulation of host miRNAs by massive-parallel sequencing. We found that only miRNAs expressed from a single cluster, miR-183/96/182, are reproducibly deregulated during productive infection. These miRNAs are predicted to regulate a great number of potential targets involved in different cellular processes and have only 33 shared targets. Among these, members of the FoxO family of proteins were identified as potential targets for all three miRNAs. However, our study shows that the upregulated miRNAs do not affect the expression of FoxO proteins, moreover, these proteins were upregulated in HSV-1 infection. Furthermore, we show that the individual FoxO proteins are not required for efficient HSV-1 replication. Taken together, our results indicate a complex and redundant response of infected cells to the virus infection that is efficiently inhibited by the virus.

While SARS-CoV-2 detection in sputum and swabs from the upper respiratory tract has been used as a diagnostic tool, virus quantification showed poor correlation to disease outcome and thus, poor prognostic value. Although the pulmonary compartment represents a relevant site for viral load analysis, limited data exploring the lower respiratory tract is available, and its association to clinical outcomes is relatively unknown. Using bronchoalveolar lavage (BAL) and serum samples, we quantified SARS-CoV-2 copy numbers in the pulmonary and systemic compartments of critically ill patients admitted to the intensive care unit of a COVID-19 referral hospital in Croatia during the second and third pandemic waves. Clinical data, including 30-day survival after ICU admission, were included. We found that elevated SARS-CoV-2 copy numbers in both BAL and serum samples were associated with fatal outcomes. Remarkably, the highest and earliest viral loads after initiation of mechanical ventilation support were increased in the non-survival group. Our results imply that viral loads in the lungs contribute to COVID-19 disease severity, while blood titers correlate with lung virus titers, albeit at a lower level. Moreover, they suggest that BAL SARS-CoV-2 copy number quantification at ICU admission may provide a predictive parameter of clinical COVID-19 outcomes.

COVID-19 vaccines prevent severe forms of the disease, but do not warrant complete protection against breakthrough infections. This could be due to suboptimal mucosal immunity at the site of virus entry, given that all currently approved vaccines are administered via the intramuscular route. In this study, we assessed humoral and cellular immune responses in BALB/c mice after intranasal and intramuscular immunization with adenoviral vector ChAdOx1-S expressing full-length Spike protein of SARS-CoV-2. We showed that both routes of vaccination induced a potent IgG antibody response, as well as robust neutralizing capacity, but intranasal vaccination elicited a superior IgA antibody titer in the sera and in the respiratory mucosa. Bronchoalveolar lavage from intranasally immunized mice efficiently neutralized SARS-CoV-2, which has not been the case in intramuscularly immunized group. Moreover, substantially higher percentages of epitope-specific CD8 T cells exhibiting a tissue resident phenotype were found in the lungs of intranasally immunized animals. Finally, both intranasal and intramuscular vaccination with ChAdOx1-S efficiently protected the mice after the challenge with recombinant herpesvirus expressing the Spike protein. Our results demonstrate that intranasal application of adenoviral vector ChAdOx1-S induces superior mucosal immunity and therefore could be a promising strategy for putting the COVID-19 pandemic under control., (© 2022 Wiley-VCH GmbH.)