Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine... more
Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility.
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Research Interests: Immunology and Medicine
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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... more
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...
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In early 2020, the COVID-19 pandemic sparked a global crisis that continues to pose a serious threat to human health and the economy. Further advancement in research is necessary and requires the availability of quality molecular tools,... more
In early 2020, the COVID-19 pandemic sparked a global crisis that continues to pose a serious threat to human health and the economy. Further advancement in research is necessary and requires the availability of quality molecular tools, including monoclonal antibodies. Here, we present the development and characterization of a collection of over 40 new monoclonal antibodies directed against different SARS-CoV-2 proteins. Recombinant SARS-CoV-2 proteins were expressed, purified, and used as immunogens. Upon development of specific hybridomas, the obtained monoclonal antibody (mAb) clones were tested for binding to recombinant proteins and infected cells. We generated mAbs against structural proteins, the Spike and Nucleocapsid protein, several non-structural proteins (nsp1, nsp7, nsp8, nsp9, nsp10, nsp16) and accessory factors (ORF3a, ORF9b) applicable in flow cytometry, immunofluorescence, or Western blot. Our collection of mAbs provides a set of novel, highly specific tools that wi...
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<p>Infected cells in temporal bone marrow <b>(A)</b>, in spiral ganglion <b>(B,C)</b> and <b>(D-F)</b> in lateral wall of cochlea <b>(D-F)</b>. Panels A (x60), B,F (x40), C,D,E (x20)... more
<p>Infected cells in temporal bone marrow <b>(A)</b>, in spiral ganglion <b>(B,C)</b> and <b>(D-F)</b> in lateral wall of cochlea <b>(D-F)</b>. Panels A (x60), B,F (x40), C,D,E (x20) and boxed areas digitally zoomed in right lower corner. Panels D,E demonstrate infected cells in the spiral ligament and Panels E, F illustrate infected basal cells in the stria vascularis. Newborn mice infected with 200PFU of Smith strain MCMV were sacrificed on postnatal day (PNd) 11 and after exhaustive perfusion and fixation, the inner ears were removed, decalcified and embedded in paraffin as described in Materials and Methods. Sections were stained with anti-MCMV IE-1 (pp89) antibody CROMA 101 and visualized using a horseradish peroxidase (HRP) conjugated second antibody followed by development with diaminobenzidine (brown). Virus infected (IE-1+) cells were detected in inner ear sections from 5/8 animals examined. Scale bars indicating 50u (A), 100u (B, F,), and 200u (C,D,E) are shown at bottom left of figures.</p
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Congenital human cytomegalovirus (HCMV) infection is the most common viral cause of long-term neurodevelopmental sequelae, including mental retardation, microcephaly and sensorineural hearing loss. As HCMV does not cross the species... more
Congenital human cytomegalovirus (HCMV) infection is the most common viral cause of long-term neurodevelopmental sequelae, including mental retardation, microcephaly and sensorineural hearing loss. As HCMV does not cross the species barrier, we employed a mouse model in which newborn mice are infected by intraperitoneal (i.p.) inoculation of mouse cytomegalovirus (MCMV). Following infection the virus disseminates to the central nervous system (CNS) and replicates in the brain parenchyma. CNS infection leads to the activation of resident microglial cells and the recruitment of peripheral immune cells. In addition, the virus induces delay in cerebellar growth. In our model of congenital MCMV infection, the initial neuroimmune responses are dominated by activation of resident microglial cells and the influx of NK cells, whose appearance coincides with detection of the virus in the brain. The number of NK cells in the CNS peaked at day 8 post infection (p.i.). In addition, we also obser...
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Despite the considerable progress toward the eradication of meningococcal disease with the introduction of glycoconjugate vaccines, previously unremarkable serogroup X has emerged in recent years, recording several outbreaks throughout... more
Despite the considerable progress toward the eradication of meningococcal disease with the introduction of glycoconjugate vaccines, previously unremarkable serogroup X has emerged in recent years, recording several outbreaks throughout the African continent. Different serogroup X polysaccharide-based vaccines have been tested in preclinical trials, establishing the principles for further improvement. To elucidate the antigenic determinants of the MenX capsular polysaccharide, we generated a monoclonal antibody, and its bactericidal nature was confirmed using the rabbit serum bactericidal assay. The antibody was tested by the inhibition enzyme-linked immunosorbent assay and surface plasmon resonance against a set of oligosaccharide fragments of different lengths. The epitope was shown to be contained within five to six α-(1–4) phosphodiester mannosamine repeating units. The molecular interactions between the protective monoclonal antibody and the MenX capsular polysaccharide fragment...
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Murine cytomegalovirus (MCMV) is widely used as a model of human CMV (HCMV) infection. However, this model relies on strains of MCMV that have been serially passaged in the laboratory for over four decades. These laboratory strains have... more
Murine cytomegalovirus (MCMV) is widely used as a model of human CMV (HCMV) infection. However, this model relies on strains of MCMV that have been serially passaged in the laboratory for over four decades. These laboratory strains have been cloned as bacterial artificial chromosomes (BACs), which permits rapid and precise manipulation. Low-passage strains of MCMV add to the utility of the mouse model of HCMV infection but do not exist as cloned BACs. This study describes the first such low-passage MCMV BAC. This BAC-derived G4 was initially attenuated in vivo , with subsequent full genomic sequencing revealing a novel spliced transcript required for salivary gland tropism. These data suggest that MCMV, like HCMV, undergoes tissue culture adaptation that can limit in vivo growth and supports the use of BAC clones as a way of standardizing viral strains and minimizing interlaboratory strain variation.
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Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune... more
Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells (“missing-self”). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.
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Research Interests: Inflammation, Neurodevelopment, Innate immunity, Medicine, Hearing, and 15 moreSensorineural Hearing Loss, Mice, Female, Animals, Otology, Male, Cytomegalovirus, Hearing Loss, Neurons, Mouse models, Cytomegalovirus Infections, organ of Corti, Auditory Brainstem Response, Cochlea, and Spiral Ganglion
CMVs efficiently target MHC I molecules to avoid recognition by cytotoxic T cells. However, the lack of MHC I on the cell surface renders the infected cell susceptible to NK cell killing upon missing self recognition. To counter this,... more
CMVs efficiently target MHC I molecules to avoid recognition by cytotoxic T cells. However, the lack of MHC I on the cell surface renders the infected cell susceptible to NK cell killing upon missing self recognition. To counter this, mouse CMV (MCMV) rescues some MHC I molecules to engage inhibitory Ly49 receptors. Here we identify a new viral protein, MATp1, that is essential for MHC I surface rescue. Rescued altered-self MHC I molecules show increased affinity to inhibitory Ly49 receptors, resulting in inhibition of NK cells despite substantially reduced MHC I surface levels. This enables the virus to evade recognition by licensed NK cells. During evolution, this novel viral immune evasion mechanism could have prompted the development of activating NK cell receptors that are specific for MATp1-modified altered-self MHC I molecules. Our study solves a long-standing conundrum of how MCMV avoids recognition by NK cells, unravels a fundamental new viral immune evasion mechanism, and ...
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Natural killer (NK) cells are innate lymphocytes that efficiently eliminate cancerous and infected cells. NKp46 is an important NK activating receptor shown to participate in recognition and activation of NK cells against pathogens, tumor... more
Natural killer (NK) cells are innate lymphocytes that efficiently eliminate cancerous and infected cells. NKp46 is an important NK activating receptor shown to participate in recognition and activation of NK cells against pathogens, tumor cells, virally infected cells, and self‐cells in autoimmune conditions, including type I and II diabetes. However, some of the NKp46 ligands are unknown and therefore investigating human NKp46 activity and its critical role in NK cell biology is problematic. We developed a unique anti‐human NKp46 monocloncal antibody, denoted hNKp46.02 (02). The 02 mAb can induce receptor internalization and degradation. By binding to a unique epitope on a particular domain of NKp46, 02 lead NKp46 to lysosomal degradation. This downregulation therefore enables the investigation of all NKp46 activities. Indeed, using the 02 mAb we determined NK cell targets which are critically dependent on NKp46 activity, including certain tumor cells lines and human pancreatic bet...
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NK cells play a critical role in host defense against viruses. In this study, we investigated the role of NKG2D in the expansion of NK cells after mouse CMV (MCMV) infection. Wild-type and NKG2D-deficient (Klrk1−/−) Ly49H+ NK cells... more
NK cells play a critical role in host defense against viruses. In this study, we investigated the role of NKG2D in the expansion of NK cells after mouse CMV (MCMV) infection. Wild-type and NKG2D-deficient (Klrk1−/−) Ly49H+ NK cells proliferated robustly when infected with MCMV strains engineered to allow expression of NKG2D ligands, which enhanced the response of wild-type NK cells. Naive NK cells exclusively express NKG2D-L, which pairs only with DAP10, whereas NKG2D-S expressed by activated NK cells pairs with DAP10 and DAP12, similar to Ly49H. However, NKG2D alone was unable to drive robust expansion of Ly49H− NK cells when mice were infected with these MCMV strains, likely because NKG2D-S was only transiently expressed postinfection. These findings demonstrate that NKG2D augments Ly49H-dependent proliferation of NK cells; however, NKG2D signaling alone is inadequate for expansion of NK cells, likely due to only transient expression of the NKG2D–DAP12 complex.
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Global pandemics caused by influenza or coronaviruses cause severe disruptions to public health and lead to high morbidity and mortality. There remains a medical need for vaccines against these pathogens. CMV (cytomegalovirus) is a... more
Global pandemics caused by influenza or coronaviruses cause severe disruptions to public health and lead to high morbidity and mortality. There remains a medical need for vaccines against these pathogens. CMV (cytomegalovirus) is a β-herpesvirus that induces uniquely robust immune responses in which remarkably large populations of antigen-specific CD8+ T cells are maintained for a lifetime. Hence, CMV has been proposed and investigated as a novel vaccine vector for expressing antigenic peptides or proteins to elicit protective cellular immune responses against numerous pathogens. We generated two recombinant murine CMV (MCMV) vaccine vectors expressing hemagglutinin (HA) of influenza A virus (MCMVHA) or the spike protein of severe acute respiratory syndrome coronavirus 2 (MCMVS). A single injection of MCMVs expressing either viral protein induced potent neutralizing antibody responses, which strengthened over time. Importantly, MCMVHA-vaccinated mice were protected from illness foll...
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Cytomegalovirus (CMV) is the most significant infectious cause of congenital abnormalities of the central nervous system including retardation, microcephaly, cerebellar atrophy, hearing loss and blindness. The pathogenesis of a viral... more
Cytomegalovirus (CMV) is the most significant infectious cause of congenital abnormalities of the central nervous system including retardation, microcephaly, cerebellar atrophy, hearing loss and blindness. The pathogenesis of a viral infection has not been fully elucidate yet and it may arise either because of direct viral damage to neuronal cells or indirectly because of different humoral factors. We have established a model of murine CMV (MCMV) brain infection in newborn mice. This model was used to determine the effect of MCMV infection on neuronal development. We have focused our analysis on the cerebellum because the majority of its cells mature postnatally making it accessible for our examination. Moreover, cerebellum has distinct cytoarchitecture and migration pattern of its neuronal cells has been well defined therefore each developmental disruption is easy to follow. Newborn mice have been infected intraperitoneally with MCMV. The viral presence in the brain has been analys...
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BackgroundThe use of checkpoint inhibitors has revolutionized cancer therapy. Unfortunately, these therapies often cause immune-related adverse effects, largely due to a lack of tumor specificity.MethodsWe stained human natural killer... more
BackgroundThe use of checkpoint inhibitors has revolutionized cancer therapy. Unfortunately, these therapies often cause immune-related adverse effects, largely due to a lack of tumor specificity.MethodsWe stained human natural killer cells using fusion proteins composed of the extracellular portion of various tumor markers fused to the Fc portion of human IgG1, and identified Nectin4 as a novel TIGIT ligand. Next, we generated a novel Nectin4 blocking antibody and demonstrated its efficacy as a checkpoint inhibitor in killing assays and in vivo.ResultsWe identify Nectin4 to be a novel ligand of TIGIT. We showed that, as opposed to all other known TIGIT ligands, which bind also additional receptors, Nectin4 interacts only with TIGIT. We show that the TIGIT-Nectin4 interaction inhibits natural killer cell activity, a critical part of the innate immune response. Finally, we developed blocking Nectin4 antibodies and demonstrated that they enhance tumor killing in vitro and in vivo.Conc...
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Incomplete block of B cell development and immunoglobulin production in mice carrying the ¢ MT mutation on the BALB/c background
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Ly49P recognition of cytomegalovirusinfected cells expressing H2-D k and CMVencoded m04 correlates with the NK cell antiviral response
Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious... more
Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious pathogen. Here, we propose a strategy for an HCMV vaccine that aims at the simultaneous activation of innate and adaptive immune responses. An HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor was found to be susceptible to control by natural killer (NK) cells, and preserved the ability to stimulate HCMV-specific T cells. Infection with the ULBP2-expressing HCMV strain caused diminished cell surface levels of MHC class I molecules. While expression of the NKG2D ligand increased the cytolytic activity of NK cells, NKG2D engagement in CD8+ T cells provided co-stimulation and compensated for lower MHC class I expression. Altogether , our data indicate that triggering of both arms of the immune system is a promising approach applicable to the generation of a live attenuated HCMV vaccine. Human cytomegalovirus (CMV) is a major cause of morbidity and mortality in congenitally infected newborns and immunocompromised individuals, indicating an utmost need for a vaccine to protect these vulnerable groups. Recent experimental studies in animal models, including non-human primates, have shown that attenuated CMVs trigger a potent immune response and are attractive vaccine candidates. However, an effective CMV vaccine is still not available. Here, we demonstrate that rational engineering of a live attenuated human CMV vaccine candidate is feasible. We equipped a CMV strain with an immunostimulatory molecule that is a ligand for an activating receptor present on both Natural Killer cells and CD8+ T cells. Moreover, we deleted several immunoevasins
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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... more
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 ...
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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 regulation of efficient... more
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 regulation of efficient virus replication. In this study, we comprehen-sively 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 dur-ing productive infection. These miRNAs are predicted to regulate a great number of potential tar-gets involved in different cellular processes and have only 33 shared targets. Among these, mem-bers 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 fo...
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Human cytomegalovirus (HCMV) is the most common cause of viral infection acquired in utero. Even though the infection has been studied for several decades, immune determinants important for virus control and mechanisms of long-term... more
Human cytomegalovirus (HCMV) is the most common cause of viral infection acquired in utero. Even though the infection has been studied for several decades, immune determinants important for virus control and mechanisms of long-term sequelae caused by infection are still insufficiently characterized. Animal models of congenital HCMV infection provide unique opportunity to study various aspects of human disease. In this review, we summarize current knowledge on the role of immune system in congenital CMV infection, with emphasis on lessons learned from mouse model of congenital CMV infection.
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Research Interests: Immunology and Biology
The early detection of herpesvirus infection by pattern recognition receptors (PRRs) triggers the production of type I interferons (IFN), which exert potent antiviral effects. Whilst MCMV is recognized to induce strong innate immune... more
The early detection of herpesvirus infection by pattern recognition receptors (PRRs) triggers the production of type I interferons (IFN), which exert potent antiviral effects. Whilst MCMV is recognized to induce strong innate immune responses, UV inactivation of MCMV results in an enhanced type I IFN response, indicating that the virus actively downmodulates innate immune signaling. Using an unbiased luciferase-based assay to screen an MCMV cDNA library, we have identified the M35 protein as a negative regulator of PRR-dependent type I IFN signaling. Infection of primary bone marrow derived macrophages with an MCMV mutant lacking M35 (M35stop) results in increased secretion of type I IFN as compared to wild-type (WT) MCMV. To confirm the physiological role of M35, we infected BALB/c mice with MCMV M35stop to assess the subsequent type I IFN response. MCMV M35stop induces significantly higher type I IFN serum levels than WT MCMV 8 h post infection, which correlates well with expression of tegument M35 protein. MCMV M35stop replicates to significantly reduced titres compared to WT MCMV in the spleen at day 3 post infection. MCMV is then disseminated via secondary viremia to the salivary glands, a site of persistence and dissemination for MCMV. MCMV M35stop is undetectable from the salivary glands from as early as 7 days post infection. This exclusion of M35stop from the salivary glands indicates that M35 is a critical determinant of viral replication in vivo. Collectively, our data demonstrate the first characterisation of M35 as a novel antagonist of type I IFN signaling downstream of PRRs.
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Signal transducers and activators of transcription (STAT) 1 is a key transcription factor involved in innate and adaptive immune responses by governing the responses to interferons (IFNs) type I, II and III. In order to elucidate the cell... more
Signal transducers and activators of transcription (STAT) 1 is a key transcription factor involved in innate and adaptive immune responses by governing the responses to interferons (IFNs) type I, II and III. In order to elucidate the cell type specific contributions of STAT1 to the host response to viral infections our lab generated conditional Stat1 mice. Cytomegalovirus (CMV) can trigger severe disease in immune suppressed individuals, is a frequent cause of congenital birth defects and still represents a major problem in health care. Immunity to acute and persistent CMV infection is intensively studied with respect natural killer (NK) and T cell function. Systemic STAT1 is required for defence against CMV but little is known about the contribution of STAT1 and innate immune cell function. Stat1ΔLysz mice showed highly efficient Stat1 allele depletion efficiencies in the myeloid compartment. Challenges of Stat1ΔLysz with various amounts of MCMV revealed no apparent effect of myeloid STAT1 to survival compared to wildtype mice. However, Stat1ΔLysz mice were deficient to restrict viral load in various organs and showed a high persistence of MCMV in salivary glands. With respect to early infection and viral spread we investigate cytokine expression and response activities, including cell type specific STAT activation. With respect to MCMV persistence we test the requirement of myeloid STAT1 for the activation of CD4+ and CD8+ T cells. This work was funded by the Austrian Science Fund (FWF) IAI DK W1212 and FWF SFB F28.
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Data from both human and animal studies clearly demonstrate that sex steroid hormones influence components of both innate and adaptive immunity, resulting in differences of immune responses between females and males. Moreover hormonal... more
Data from both human and animal studies clearly demonstrate that sex steroid hormones influence components of both innate and adaptive immunity, resulting in differences of immune responses between females and males. Moreover hormonal changes associated with pregnancy modify the severity of some infections and diseases. The aim of this study was to test whether the pregnancy influence the course of cytomegalovirus (CMV) infection in female BALB/c mice. Mice used in all experiments were treated with pregnant mare serum gonadotropin (PMGS) and human chorionic gonadotropin (hCG) to trigger ovulation and luteinization. Virgin animals as well as pregnant ones were intravenously infected with murine CMV (MCMV) and sacrificed each day from 1st to 10th day post infection. Viral titers were analyzed in different organs using standard plaque assay, while viral spread was examinated using immunohistochemistry. Our results showed higher susceptibility of pregnant mice to MCMV infection in all investigated organs. Moreover ovaries of pregnant mice showed delay in clearance of productive infection as compared to nonpregnant animals. We observed numerous clusters of infected cells situated in luteal bodies, whereas no cells positive for viral antigens were found within follicles. Our study provides a new insight in the pathogenesis of MCMV infection in female reproductive tissue as well as its dependency of sex steroid environment. These findings could bring the evidence of CMV infection in mechanisms of gonadal dysfunction.