ChAdOx1-S adenoviral vector vaccine applied intranasally elicits superior mucosal immunity compared to the intramuscular route of vaccination.

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.

The humoral pattern recognition molecule PTX3 is a key component of innate immunity against urinary tract infection.

Immunity in the urinary tract has distinct and poorly understood pathophysiological characteristics and urinary tract infections (UTIs) are important causes of morbidity and mortality. We investigated the role of the soluble pattern recognition molecule pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity, in UTIs. PTX3-deficient mice showed defective control of UTIs and exacerbated inflammation. Expression of PTX3 was induced in uroepithelial cells by uropathogenic Escherichia coli (UPEC) in a Toll-like receptor 4 (TLR4)- and MyD88-dependent manner. PTX3 enhanced UPEC phagocytosis and phagosome maturation by neutrophils. PTX3 was detected in urine of UTI patients and amounts correlated with disease severity. In cohorts of UTI-prone patients, PTX3 gene polymorphisms correlated with susceptibility to acute pyelonephritis and cystitis. These results suggest that PTX3 is an essential component of innate resistance against UTIs. Thus, the cellular and humoral arms of innate immunity exert complementary functions in mediating resistance against UTIs.

Composition of the immunoglobulin G glycome associates with the severity of COVID-19.

A large variation in the severity of disease symptoms is one of the key open questions in coronavirus disease 2019 (COVID-19) pandemics. The fact that only a small subset of people infected with severe acute respiratory syndrome coronavirus 2 develops severe disease suggests that there have to be some predisposing factors, but biomarkers that reliably predict disease severity have not been found so far. Since overactivation of the immune system is implicated in a severe form of COVID-19 and the immunoglobulin G (IgG) glycosylation is known to be involved in the regulation of different immune processes, we evaluated the association of interindividual variation in IgG N-glycome composition with the severity of COVID-19. The analysis of 166 severe and 167 mild cases from hospitals in Spain, Italy and Portugal revealed statistically significant differences in the composition of the IgG N-glycome. The most notable difference was the decrease in bisecting N-acetylglucosamine in severe patients from all three cohorts. IgG galactosylation was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing.

Antibody response and the clinical presentation of patients with COVID-19 in Croatia: the importance of a two-step testing approach.

According to anti-SARS-CoV-2 seroresponse in patients with COVID-19 from Croatia, we emphasised the issue of different serological tests and need for combining diagnostic methods for COVID-19 diagnosis. Anti-SARS-CoV-2 IgA and IgG ELISA and IgM/IgG immunochromatographic assay (ICA) were used for testing 60 sera from 21 patients (6 with severe, 10 moderate, and 5 with mild disease). The main clinical, demographic, and haemato-biochemical data were analysed. The most common symptoms were cough (95.2%), fever (90.5%), and fatigue and shortness of breath (42.9%). Pulmonary opacities showed 76.2% of patients. Within the first 7 days of illness, seropositivity for ELISA IgA and IgG was 42.9% and 7.1%, and for ICA IgM and IgG 25% and 10.7%, respectively. From day 8 after onset, ELISA IgA and IgG seropositivity was 90.6% and 68.8%, and for ICA IgM and IgG 84.4% and 75%, respectively. In general, sensitivity for ELISA IgA and IgG was 68.3% and 40%, and for ICA IgM and IgG 56.7% and 45.0%, respectively. The anti-SARS-CoV-2 antibody distributions by each method were statistically different (ICA IgM vs. IgG, p = 0.016; ELISA IgG vs. IgA, p < 0.001). Antibody response in COVID-19 varies and depends on the time the serum is taken, on the severity of disease, and on the type of test used. IgM and IgA antibodies as early-stage disease markers are comparable, although they cannot replace each other. Simultaneous IgM/IgG/IgA anti-SARS-CoV-2 antibody testing followed by the confirmation of positive findings with another test in a two-tier testing is recommended.

Activity of fosfomycin against nosocomial multiresistant bacterial pathogens from Croatia: a multicentric study.

AIM: To determine in vitro susceptibility of multiresistant bacterial isolates to fosfomycin. METHODS: In this prospective in vitro study (local non-random sample, level of evidence 3), 288 consecutively collected multiresistant bacterial isolates from seven medical centers in Croatia were tested from February 2014 until October 2016 for susceptibility to fosfomycin and other antibiotics according to Clinical and Laboratory Standards Institute methodology. Susceptibility to fosfomycin was determined by agar dilution method, while disc diffusion was performed for in vitro testing of other antibiotics. Polymerase chain reaction and sequencing were performed for the majority of extended spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) and carbapenem-resistant isolates. RESULTS: The majority of 288 multiresistant bacterial isolates (82.6%) were susceptible to fosfomycin. The 236 multiresistant Gram-negative isolates showed excellent susceptibility to fosfomycin. Susceptibility rates were as follows: Escherichia coli ESBL 97%, K. pneumoniae ESBL 80%, Enterobacter species 85.7%, Citrobacter freundii 100%, Proteus mirabilis 93%, and Pseudomonas aeruginosa 60%. Of the 52 multiresistant Gram-positive isolates, methicillin-resistant Staphylococcus aureus showed excellent susceptibility to fosfomycin (94.4%) and vancomycin-resistant enterococcus showed low susceptibility to fosfomycin (31%). Polymerase chain reaction analysis of 36/50 ESBL-producing K. pneumoniae isolates showed that majority of isolates had CTX-M-15 beta lactamase (27/36) preceded by ISEcp insertion sequence. All carbapenem-resistant Enterobacter and Citrobacter isolates had blaVIM-1 metallo-beta-lactamase gene. CONCLUSION: With the best in vitro activity among the tested antibiotics, fosfomycin could be an effective treatment option for infections caused by multiresistant Gram-negative and Gram-positive bacterial strains in the hospital setting.

1-ethyl-3-(6-methylphenanthridine-8-il) urea modulates TLR3/9 activation and induces selective pro-inflammatory cytokine expression in vitro.

We have previously demonstrated the nucleic acid binding capacity of phenanthridine derivatives (PHTs). Because nucleic acids are potent inducers of innate immune response through Toll-like receptors (TLRs), and because PTHs bear a structural resemblance to commonly used synthetic ligands for TLR7/8, we hypothesized that PHTs could modulate/activate immune response. We found that compound M199 induces secretion of IL-6, IL-8 and TNFα in human PBMCs and inhibits TLR3/9 activation in different cellular systems (PBMCs, HEK293 and THP-1 cell lines).

Antimicrobial Sensitivity Profile of Chlamydia trachomatis isolates from Croatia in McCoy Cell Culture System and Comparison with the Literature.

BACKGROUND: Chlamydia trachomatis (C. trachomatis) is the most common bacterial agent of sexually transmitted infections around the world, but susceptibility testing of this pathogen is rarely pursued due to its intracellular niche. The principal aims of this research were to determine in vitro sensitivity profile of urogenital chlamydial strains isolated from Croatian patients and to compare obtained concentration values of different antimicrobial drugs mutually and with the literature. METHODS: Forty strains of C. trachomatis isolated during 2010-2012 at the National Reference Laboratory for Chlamydia and two reference strains were subjected to susceptibility testing in 96-well microtiter plates containing McCoy cell monolayers. Minimal inhibitory concentration (MIC) and minimal chlamydicidal concentration (MCC) were determined for azithromycin, doxycycline, and levofloxacin. Briefly, strains were inoculated on McCoy cells, followed by addition of serially diluted antimicrobial drugs. Upon incubation, growth of C. trachomatis was detected using fluorescein-conjugated antibody to the lipopolysaccharide genus antigen under the inverted fluorescent microscope. RESULTS: All chlamydial strains were susceptible to the antibiotics tested (MIC < 4 pg/mL), thus the pattern of homotypic or heterotypic resistance has not been found. MCC values were equal or 1-5 dilutions higher than MIC values. Statistically significant differences in the effectiveness of antimicrobial agents in vitro have been proven. Significant correlation has been found for MCCs in the case of two antimicrobial pairs: azithromycin and levofloxacin, and doxycycline and levofloxacin. Comparison of medians for different clinical samples did not reveal any significant difference. CONCLUSIONS: Although resistant strains have not been found in this study, several literature reports of unsuccessfully treated genitourinary infections caused by C. trachomatis require our alertness for possible discovery of resistant strains. Considering the overall antibiotic burden worldwide, pursuing this kind of research is crucial in order to detect possible decreased susceptibility (or even resistance) of chlamydial strains, despite the laborious and time-consuming methodology.

Serum Concentrations of TIM-3, LAG-3, and PD-1 in Patients with Hemorrhagic Fever with Renal Syndrome.

Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne disease widespread in Europe and Asia. HFRS is caused by negative-sensed single-stranded RNA orthohantaviruses transmitted to humans through inhaling aerosolized excreta of infected rodents. Symptoms of HFRS include acute kidney injury, thrombocytopenia, hemorrhages, and hypotension. The immune response raised against viral antigens plays an important role in the pathogenesis of HFRS. Inhibitory co-receptors are essential in regulating immune responses, mitigating immunopathogenesis, and reducing tissue damage. Our research showed an increased soluble form of inhibitory co-receptors TIM-3, LAG-3, and PD-1 in HFRS patients associated with disease severity. Our study aimed to investigate the impact of HFRS on the concentrations of soluble forms of inhibitory receptors TIM-3, LAG-3, and PD-1 in the patient's serum and the potential correlation with key clinical parameters. Our study aimed to investigate the impact of HFRS on the concentrations of soluble forms of inhibitory receptors TIM-3, LAG-3, and PD-1 in the patient's serum and their possible association with relevant clinical parameters. Using multiplex immunoassay, we found elevated levels of TIM-3, LAG-3, and PD-1 proteins in the serum of HFRS patients. Furthermore, increased levels were associated with creatinine, urea, lactate dehydrogenase concentrations, and platelet count. These findings suggest that these proteins play a role in regulating the immune response and disease progression.

A Comprehensive Analysis of Renal and Endothelium Dysfunction Markers Fourteen Years after Hemorrhagic Fever with Renal Syndrome Contraction.

While the pathology of acute hemorrhagic fever with renal syndrome (HFRS) has been widely researched, details on the chronic HFRS sequelae remain mainly unexplored. In this study, we analyzed the clinical and laboratory characteristics of 30 convalescent HFRS patients 14 years after the disease contraction, mainly emphasizing several endothelial dysfunction parameters. Convalescent HFRS patients exhibited significantly higher serum levels of erythrocyte sedimentation rate, von Willebrand factor, uric acid, C-reactive protein and immunoglobulin A when compared to healthy individuals. Furthermore, 24 h urine analyses revealed significantly lower sodium and potassium urine levels, as well as significantly higher proteinuria, microalbumin levels and ß2-microglobulin levels when compared to healthy individuals. First morning urine analysis revealed significantly higher levels of hematuria in convalescent HFRS patients. None of the additional analyzed endothelium dysfunction markers were significantly different in post-HFRS patients and healthy individuals, including serum and urine P-selectin, E-selectin, soluble intercellular adhesion molecule 1, vascular intercellular adhesion molecule 1 (sVCAM-1) and vascular endothelial growth factor (VEGF). However, binary logistic regression revealed a weak association of serum sVCAM-1 and urine VEGF levels with HFRS contraction. Generally, our findings suggest mild chronic inflammation and renal dysfunction levels in convalescent HFRS patients 14 years after the disease contraction.

The association of ventricular tachycardia and endothelial dysfunction in the setting of acute myocardial infarction with ST elevation.

BACKGROUND: Ventricular tachycardia (VT) is frequently seen in ischemic settings like acute myocardial infarction with ST segment elevation (STEMI). Endothelial dysfunction (ED) represents inflammation and the loss of all protective features of the endothelium. We aimed to examine the association between VT and ED in patients with STEMI. MATERIAL/METHODS: The study included 90 subjects (30 with VT and acute STEMI, 30 with STEMI without VT, and 30 controls). Sera of all subjects were tested on ED markers by enzyme immunoassay: sICAM-1 (intracellular adhesive molecule-1), sVCAM-1 (vascular adhesive molecule-1), P- and E-selectins, and VEGF (vascular endothelial growth factor). In addition, CRP (C-reactive protein) was detected. RESULTS: Significantly increased values of low-density lipoprotein, triglycerides, leukocytes, creatinine, and the number of cigarettes smoked were observed among patients with VT+STEMI in comparison to controls. The levels of E-selectin were significantly lower in the VT+STEMI group than in the other groups, while the levels of VCAM-1 were significantly higher in the groups with STEMI and VT+STEMI compared to the controls. Lower levels of VEGF were recorded in STEMI and VT+STEMI groups compared to the control group. A significant correlation between CRP and VCAM-1 in patients with VT +STEMI was demonstrated. CONCLUSIONS: We showed that ED may have a role in the immunopathogenesis of VT in patients with STEMI. The role of sE- selectin and correlation of sVCAM-1 with CRP as possible ED predictive markers in patients with VT+STEMI should be further investigated in a large cohort of patients.

West Nile virus outbreak in humans in Croatia, 2012.

During August and September 2012, seven cases of West Nile neuroinvasive disease were identified in three north-eastern counties of Croatia. Four cases were reported in Osijek-Baranja County, two in Brod-Posavina County and one in Vukovar-Srijem County. The median age of the patients was 62.7 years. All patients were hospitalized for 2-5 weeks. The patients from Slavonski Brod had more severe clinical presentation of disease with prolonged hospitalization. Medical entomological research was carried out in 64 localities, where 1785 mosquitoes were captured. Among the analyzed mosquitoes, 114 were determined to be Culex pipiens and subjected to molecular characterization for the presence of virus. No viral RNA was detected in mosquitoes. Subsequent public health measures taken include mosquito control in all settlements where disease was detected.

What can neutralizing antibodies tell us about the quality of immunity in COVID-19 convalescents and vaccinees?

During the SARS-CoV-2 pandemic, the lack of standardized measurements of the immune response after vaccination or recovery from COVID-19 resulted in incomparable results and hindered correlation establishment. Prioritizing reliable and standardized methods to monitor pathogen-specific immunity is crucial, not only during the COVID-19 pandemic but also for future outbreaks. During our study of the humoral immune response, we used a SARS-CoV-2 wild-type neutralization assay, ensuring the measurement of the immune response directed to all SARS-CoV-2 antigens in their proper conformation. A head-to-head comparison of the neutralizing antibody (NAb) responses elicited by four vaccines used in Europe during 2021 (BNT162b2, mRNA-1273, ChAdOx nCoV-19, and Ad26.COV2.S) and their comparison to NAb responses in convalescents showed that while the amount was comparable, NAbs induced by natural infection were of higher quality. Namely, NAbs produced by disease were better activators of the complement system than NAbs induced by vaccination. Furthermore, the contribution of spike protein-specific IgGs to the SARS-CoV-2 neutralization was lower in convalescents compared to vaccinees, indicating that those who recovered from COVID-19 were armed with antibodies of additional specificities and/or classes that contributed to virus neutralization. These findings suggest that a higher stringency of public policy measures targeting individuals who have recovered from COVID-19, in comparison to those who have been vaccinated, may not have been fully justified.

Hemorrhagic Fever with Renal Syndrome Patients Exhibit Increased Levels of Lipocalin-2, Endothelin-1 and NT-proBNP.

Hemorrhagic fever with renal syndrome (HFRS) is an acute zoonotic disease caused by viruses of the Orthohantavirus genus. This syndrome is characterized by renal and cardiopulmonary implications detectable with different biomarkers. Here, we explored the role of serum and urine levels of lipocalin-2, endothelin-1 and N-terminal pro-brain natriuretic peptide (NT-proBNP) in HFRS pathology. A total of twenty-eight patients hospitalized due to a Puumala orthohantavirus infection were included, with serum and urine samples collected on patient admission (acute phase) and discharge (convalescent phase). In comparison to healthy individuals, patients exhibited significantly higher acute-phase serum and urine levels of lipocalin-2, serum levels of endothelin-1 and serum and urine levels of NT-proBNP. Patients in the convalescent phase showed a significant decrease in urine lipocalin-2, serum endothelin-1 and serum and urine NT-proBNP levels. We recorded a strong correlation between serum levels of lipocalin-2 and endothelin-1 and urine levels of lipocalin-2 with several kidney injury markers, such as serum creatinine, urea, urine white blood cell count and proteinuria. We also demonstrated an independent correlation of serum and urine lipocalin-2 levels with acute kidney injury in HFRS. All in all, our results show an involvement of NT-proBNP, lipocalin-2 and endothelin-1 in the renal and cardiac pathology of HFRS.

Evaluation of the Neutralizing Antibody STE90-C11 against SARS-CoV-2 Delta Infection and Its Recognition of Other Variants of Concerns.

As of now, the COVID-19 pandemic has spread to over 770 million confirmed cases and caused approximately 7 million deaths. While several vaccines and monoclonal antibodies (mAb) have been developed and deployed, natural selection against immune recognition of viral antigens by antibodies has fueled the evolution of new emerging variants and limited the immune protection by vaccines and mAb. To optimize the efficiency of mAb, it is imperative to understand how they neutralize the variants of concern (VoCs) and to investigate the mutations responsible for immune escape. In this study, we show the in vitro neutralizing effects of a previously described monoclonal antibody (STE90-C11) against the SARS-CoV-2 Delta variant (B.1.617.2) and its in vivo effects in therapeutic and prophylactic settings. We also show that the Omicron variant avoids recognition by this mAb. To define which mutations are responsible for the escape in the Omicron variant, we used a library of pseudovirus mutants carrying each of the mutations present in the Omicron VoC individually. We show that either 501Y or 417K point mutations were sufficient for the escape of Omicron recognition by STE90-C11. To test how escape mutations act against a combination of antibodies, we tested the same library against bispecific antibodies, recognizing two discrete regions of the spike antigen. While Omicron escaped the control by the bispecific antibodies, the same antibodies controlled all mutants with individual mutations.

Resequencing of the Puumala virus strain Sotkamo from the WHO Arbovirus collection.

RNA viruses exhibit a high mutation rate as the RNA-dependent RNA polymerase lacks proofreading and repair capabilities. It is known that serial passaging on cell culture leads to virus adaptation. Puumala virus (PUUV) strain Sotkamo is the prototype virus for the low-pathogenic hantavirus Puumala, family Bunyaviridae. A full-length sequence of the strain Sotkamos tripartite genome was made available more than 15 years ago, after at least 15 passages on Vero E6 cells. A distinct sample from the sequenced strain, with unknown passage history, was then included in the WHO Arbovirus collection. The genome sequence of this included sample was determined in this study and exhibited over 99 % identity in comparison to the previously published sequence. A total of 23 nucleotide changes across all genome segments were found. The small segment had the highest nucleotide variance without changes on the protein level. Within the extraviral domain of the glycoproteins, the majority of non-synonymous mutations were detected, whereas the large segment is most conserved on the nucleotide level. It seems possible that the PUUV strain Sotkamo adapted differently to serial passaging on cell culture in two different laboratories. In addition, a distinct passage number could exhibit itself within the nucleotide differences.

Serum levels of metalloproteinases and their inhibitors during infection with pathogens having integrin receptor-mediated cellular entry.

BACKGROUND: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes with numerous roles in the normal immune response to infection. However, excess MMP activity following infection may lead to immunopathological processes that cause tissue damage. Their activity in normal tissues is subject to tight control, which is regulated by its specific endogenous tissue inhibitors (TIMPs). It is known that MMPs bind to cell surface proteins (e.g. integrins) and that such interactions can have modulatory effects on MMP functionality. The objective of this study was to determine whether there are differences in MMP and TIMP production during the acute phase of infection with different pathogens that use ß-integrins as their receptors for cell entry. METHODS: We measured the total amounts of soluble MMP-2, MMP-9, TIMP-1, and TIMP-2 in the sera from patients infected with Dobrava virus (DOBV), Coxiella burnetii, or uropathogenic Escherichia coli. Statistical analyses were used to correlate MMP/TIMP serum levels with different clinical laboratory parameters. RESULTS: The results showed that both of the bacterial infections generally manifested the stronger effect on MMP production, while in contrast, viral infection introduced stronger changes to metalloproteinase inhibitors. MMPs and TIMPs were significantly correlated with some of the clinical laboratory parameters in both bacterial infections, but no correlations were found for DOBV infection. CONCLUSIONS: These findings suggest diverse mechanisms by which MMP activity could be implicated in the pathology of these 2 bacterial infections versus the viral DOBV infection, despite the type of their cellular entry receptors.

Mycoplasma pneumoniae in adult community-acquired pneumonia increases matrix metalloproteinase-9 serum level and induces its gene expression in peripheral blood mononuclear cells.

BACKGROUND: The objective of this study was to assess the concentration of metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) in peripheral circulation and their mRNA expression in peripheral blood mononuclear cells (PBMCs) in patients with CAP caused by M. pneumoniae. MATERIAL/METHODS: We prospectively analyzed MMPs in 40 hospitalized patients with M. pneumoniae CAP on admission, and in the convalescent phase. Twenty healthy men were used as controls. Quantitative real-time PCR and ELISA tests were used. RESULTS: MMP-9 mRNA expression in PBMCs was increased in the acute phase of illness compared to the control group as well as in convalescent phase in which case it was statistically significant (Mann-Whitney; p=0.028). The same was found for MMP-9 plasma levels (Mann-Whitney test; p<0.001; p=0.001). Circulating MMP-2 concentration in acute patients was significantly lower than in the control group and convalescent phase (Mann-Whitney test; p=0.012; p=0.001), while no MMP-2 mRNA expression was found in PBMCs. The plasma level of MMP-9 correlated with leukocyte count in peripheral circulation (r=0.67, p<0.001). CONCLUSIONS: We conclude that M. pneumoniae in adult CAP induces activity of MMP-9 in peripheral blood circulation.