Cytokine responses to major human Cytomegalovirus antigens in mouse model.

Human cytomegalovirus (CMV) continues to be a source of severe complications in immunologically immature and immunocompromised hosts. Effective CMV vaccines that help diminish CMV disease in transplant patients and avoid congenital infection are essential. Though the exact roles of defense mechanisms are unidentified, virus-specific antibodies and cytokine responses are known to be involved in controlling CMV infections. Identifying the CMV antigens that trigger these protective immune responses will help us choose the most suitable CMV-related proteins for future vaccines. CMV envelope glycoprotein B (UL55/gB), matrix proteins (UL83/pp65, UL99/pp28, UL32/pp150), and assembly protein UL80a/pp38 are known to be targets for antiviral immune responses. We immunized mice intraperitoneally with these five CMV-related proteins for their ability to induce specific antibody responses and cytokine production in a mouse model. We observed a significant CMV-antigen-specific antibody response to UL80a/pp38 and UL83/pp65 (E/C>2.0). Mice immunized with UL80a/pp38 had significantly higher concentrations of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, and IL-17A (p<0.05). Mice immunized with UL83/pp65 showed significantly higher concentrations of GM-CSF, IFN-γ, IL-2 IL-4, IL-10, IL-12, IL-17A, and TNF-α. Ratios of Th1 to Th2 cytokines revealed a Th1 cytokine bias in mice immunized with UL80a/pp38, UL83/pp65, UL32/pp150, and UL55/gB. We suggest that stimulation with multiple CMV-related proteins, which include UL80a/pp38, UL83/pp65, UL32/pp150, and UL55/gB antigens, will allow both humoral and cellular immune responses to be efficiently activated, thus serving as appropriate CMV antigens for future novel vaccines and immune-based therapeutic design.

Influence of SARS-COV-2 Infection on Cytokine Production by Mitogen-Stimulated Peripheral Blood Mononuclear Cells and Neutrophils in COVID-19 Intensive Care Unit Patients.

We sought to investigate the influence of SARS-CoV-2 infection on the cytokine profiles of peripheral blood mononuclear cells (PBMCs) and neutrophils from coronavirus disease 2019 (COVID-19) intensive care unit (ICU) patients. Neutrophils and PBMCs were separated and stimulated with the mitogen phytohemagglutinin. Culture supernatants of mitogen-stimulated PBMCs and neutrophils from 88 COVID-19 ICU patients and 88 healthy controls were evaluated for levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-α, IFN-γ, interleukin (IL)-2, -4, -5, -6, -9, -10, -12, -17A, and tumor necrosis factor (TNF)-α using anti-cytokine antibody MACSPlex capture beads. Cytokine profiles of PBMCs showed significantly lower levels of GM-CSF, IFN-γ, IL-6, IL-9, IL-10, IL-17A, and TNF-α (p < 0.0001) in COVID-19 ICU patients. In contrast, COVID-19 ICU patients showed higher median levels of IL-2 (p < 0.001) and IL-5 (p < 0.01) by PBMCs. As for neutrophils, COVID-19 ICU patients showed significantly lower levels of GM-CSF, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-17A, IL-12, TNF-α (p < 0.0001), and IFN-α (p < 0.01). T-helper (Th)1:Th2 cytokine ratios revealed lower inflammatory cytokine for PBMCs and neutrophils in COVID-19 ICU patients. Cytokine production profiles and Th1:Th2 cytokine ratios suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has an immunomodulatory effect on PBMCs and neutrophils. This study also suggests that the increased levels of several cytokines in the serum are not sourced from PBMCs and neutrophils.

Strain Variation Based on Spike Glycoprotein Gene of SARS-CoV-2 in Kuwait from 2020 to 2021.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which was first identified in Wuhan, China, in December 2019. With the global transmission of the virus, many SARS-CoV-2 variants have emerged due to the alterations of the spike glycoprotein. Therefore, the S glycoprotein encoding gene has widely been used for the molecular analysis of SARS-Co-2 due to its features affecting antigenicity and immunogenicity. We analyzed the S gene sequences of 35 SARS-CoV-2 isolates in Kuwait from March 2020 to February 2021 using the Sanger method and MinION nanopore technology to confirm novel nucleotide alterations. Our results show that the Kuwaiti strains from clade 19A and B were the dominant variants early in the pandemic, while clade 20I (Alpha, V1) was the dominant variant from February 2021 onward. Besides the known mutations, 21 nucleotide deletions in the S glycoprotein in one Kuwaiti strain were detected, which might reveal a recombinant SARS-CoV-2 with the defective viral genome (DVG). This study emphasizes the importance of closely perceiving the emerging clades with these mutations during this continuous pandemic as some may influence the specificity of diagnostic tests, such as RT-PCR and even vaccine design directing these positions.

Modulation of Production of Th1/Th2 Cytokines in Peripheral Blood Mononuclear Cells and Neutrophils by Hepatitis C Virus Infection in Chronically Infected Patients.

This study investigated the influence of Hepatitis C virus (HCV) infection on the cytokine production profiles of the peripheral blood monoculear cells (PBMC) and neutrophils in chronically naïve HCV-infected patients. Seventy-five genotype-4 naïve HCV-infected patients (HCV+) and healthy subjects (HCV-) were enrolled. The neutrophils and the PBMC were separated by density gradient sedimentation and stimulated with a mitogen. The culture supernatants were evaluated for levels of IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, and TNF-α using anti-cytokine antibody MACSPlex capture beads. The PBMC cytokine profiles of HCV+ patients showed significantly lower mean values for IFN-γ, IL-2, IL-6, IL-9, and IL-10 (p < 0.0001) as compared to HCV- subjects. In contrast, HCV+ patients showed higher mean levels of PBMC cytokine values for IL-5 and TNF-α (p < 0.0001). As for neutrophils, HCV+ patients showed significantly lower mean levels of IFN-α, IFN-γ, IL-2, IL-4, IL-6, IL-9, and IL-10 (p < 0.0001). In contrast, the neutrophils from HCV+ patients showed higher mean levels of IL-5, IL-12, and TNF-α (p < 0.0001). Th1-Th2 cytokine ratios suggested a lower Th1 bias in HCV+ subjects as compared to HCV- subjects. Our results suggest that chronic HCV infection brings about an immunomodulatory effect not only on neutrophils, but also to a lower extent on PBMCs.

Modulation of Immune Cell Subsets by Hepatitis C Virus and Antiviral Therapy in Early Virological Response HCV Genotype 4-Infected Patients with Compensated Liver Disease.

BACKGROUND: Resolution of chronic hepatitis C virus (HCV) infection requires a complicated interaction between immune cell subsets. The effect of antiviral therapy on immune cell subsets remains to be defined. This study aimed to investigate the absolute count of certain immune cell subsets during therapy with pegylated interferon-α and ribavirin (PegIFN/RBV). MATERIALS AND METHODS: Sixty HCV genotype 4-infected patients with compensated liver disease were treated with PegIFN/RBV therapy for 52 weeks. Efficacy was measured by studying the early virological response (EVR) at post-therapy week 12. Absolute counts of mature T cells, T helper cells, T cytotoxic cells, activated T cells, natural killer cells, natural killer/T (NKT) cells, B cells, and T regulatory cells (Treg), and the ratio of T helper to T cytotoxic cells were longitudinally analyzed by flow cytometry throughout the treatment and follow-up course. RESULTS: Of the 60 genotype 4-infected subjects, 39 (65%) had EVR and 21 (35%) were non-EVR patients. In the first part of this study, there were significantly lower mean absolute count values of mature T, T cytotoxic, B, and NKT cells. Also, we detected statistically significantly lower mean values for the percentages of T cytotoxic, NKT, Treg, and activated T cells of HCV-infected patients at baseline values when compared with healthy subjects. After the initiation of PegIFN/RBV therapy, frequencies of T helper cells, activated T cells, Treg cells, B cells, and T helper:T cytotoxic ratio were found to be significantly lower in EVR patients than in non-EVR patients (p < 0.05). In contrast, frequencies of T cytotoxic and NKT cells were significantly increased in EVR patients when compared to non-EVR patients (p < 0.05). CONCLUSION: These results suggest a pattern of higher levels of T cytotoxic and NKT cells, and lower levels of T helper, activated T, Treg, and B cell populations in patients who respond favorably to PegIFN/RBV therapy.

Spectrum of Viral Infections Among Primary Immunodeficient Children: Report From a National Registry.

Objective: To present the frequency and spectrum of viral infections in primary immunodeficient children. Methods: The data was obtained from the Kuwait National Primary Immunodeficiency Disorders (PIDs) Registry during the period of 2004-2018. Results: A total of 274 PID children were registered in KNPIDR during the study period with predominance of immunodeficiencies affecting cellular and humoral immunity, followed by combined immunodeficiencies with associated syndromic features and diseases of immune dysregulation. Overall infectious complications affected 82.4% of the patients, and viral infections affected 31.7% of the registered patients. Forty-five patients (16.4%) developed viral infections caused by at least 2 organisms, among those 20 patients were affected by three or more viral infections. There was a statistically significant association between viral infections and PID category. However, there was no statistically significant association between viral infections and gender or the patients' onset age. There was a total of 170 viral infections during the study period and the causes of these infections were predominated by CMV (22.2%), adenovirus (11.7%), EBV (11.1%), and enteroviruses (7.4%). CMV and parainfluenza infections were more common in the group of immunodeficiencies affecting cellular and humoral immunity while EBV and human papilloma virus (HPV) were more common in the immune dysregulation group and combined immunodeficiencies with associated syndromic features, respectively. The most common presentation was viremia (28.8%) followed by pneumonia (28.2%) and skin infections (17.6%). The most common causes of viremia were CMV followed by adenovirus and EBV, while the most common organisms causing pneumonia were CMV followed by rhinovirus and parainfluenza. There were 80 deaths among the registered patients, 10% were caused by viral infections. Conclusions: Viral infections are common in PIDs and result into a wide-range of clinical manifestations causing significant morbidity and mortality.

Hepatitis B virus, hepatitis C virus and human immunodeficiency virus infections among people who inject drugs in Kuwait: A cross-sectional study.

Injection drug use (IDU) is one of the most significant risk factors for viral hepatitis (B and C) and human immunodeficiency virus (HIV) infections. This study assessed seroprevalence rates of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) in people who inject drugs (PWID) in Kuwait. We conducted a cross-sectional study from April to September 2017. A total of 521 consecutive subjects, admitted at Al-Sabah Hospital. The serological and virological markers of HBV, HCV, and HIV were tested  using automated platforms. The mean age of the participants was 32.26 yrs, and the sex ratio (Male/Female) was 15.28. The prevalence rates of HBsAg, anti-HCV, and anti-HIV antibodies were 0.38% (95% CI: 0.07-1.53%), 12.28% (95% CI: 9.65-15.48), and 0.77% (95% CI: 0.25-2.23%), respectively. HCV-RNA was evident in 51.72% (95% CI: 38.34-64.87%) among anti-HCV positive participants. Multivariate analysis showed that the high prevalence of HCV infection amongst PWID is associated with age. Whereas, multivariate analysis revealed no significant associations with age and gender regarding HIV and HBV infections. The results suggest that high rates of HBV, HCV, and HIV infections among injecting drug users than the general population. These findings emphasize the importance of introducing interventions and harm reduction initiatives that have a high impact on reducing needle sharing.

Human Washington University Polyomavirus in Patients with Respiratory Tract Infection in Kuwait.

OBJECTIVE: To determine Washington University (WU) polyomavirus strains circulating among hospitalized patients with respiratory tract infections (RTI) in Kuwait. MATERIALS AND METHODS: Samples from 459 hospitalized children and adult RTI patients were screened for respiratory viruses by polymerase chain reaction from April 2013 to April 2016. The VP2 gene from WU virus (WUV)-positive samples was sequenced and subjected to phylogenetic analysis. RESULTS: Of the 459 hospitalized RTI patients, 18 (3.9%) patients were positive for WUV infection. WUV infection was common among children aged ≤11 years (9 patients, 50%). Among the 18 WUV-infected hospitalized patients, viral co-infection was detected in 9 patients (50%). The most common viruses associated with mixed infection were respiratory syncytial virus and human rhinovirus (2 patients, 11.1% each). Of the 18 WUV-infected patients, 4 were sequenced and subjected to phylogenetic analysis. The circulating strains belong to type Ia and IIIb. CONCLUSION: This study enabled us to detect WUV among hospitalized RTI patients. Co-infection with other respiratory viruses was notable. Two circulating WUV genotypes (Ia and IIIb) were identified among hospitalized RTI patients in Kuwait.

The potential influence of human parainfluenza viruses detected during hospitalization among critically ill patients in Kuwait, 2013-2015.

BACKGROUND: The four types of human parainfluenza viruses (PIV) are important causes of community-acquired pneumonia, particularly in children; however, limited information exists about the incidence of PIV in critically ill patients. The aim of this study is to describe the spectrum, incidence and clinical features of PIV-associated infections diagnosed during the hospital stay of patients admitted to pediatric intensive care unit (PICU) and intensive care unit (ICU) of 5 medical centers across Kuwait. METHODS: This was a population-based, retrospective study from 2013 to 2015. Specimens were analyzed by molecular methods. This analysis was performed using the database of Virology Unit, Mubarak Al-Kabeer Hospital. Data from 1510 admitted patients with suspected respiratory viral infections was extracted. RESULTS: The database contained a total of 39 (2.6%) patients infected with PIV (53.8% male and 46.2% females) and 20 (51.3%) were under 1 year of age. The most frequently isolated type was type 3 (28, 71.8%) followed by type 1 (9, 23.1%). At admission the most common clinical diagnosis was pneumonia in 12 patients (30.8%, p < 0.05) followed by bronchiolitis in 10 patients (25.6%). CONCLUSION: PIV plays an important yet unrecognized role in the outcomes of PIUC and ICU patients. Our results contribute to the limited epidemiologic data of PIV in PIUC and ICU in this region.

The Prevalence of Human Bocavirus, Human Coronavirus-NL63, Human Metapneumovirus, Human Polyomavirus KI and WU in Respiratory Tract Infections in Kuwait.

OBJECTIVE: The aim of this study was to investigate the prevalence of human coronavirus (HCoV)-NL63, human metapneumovirus (hMPV), human bocavirus (Boca), human polyomavirus KI (KIV) and human polyomavirus WU (WUV) in respiratory tract infections (RTI) in Kuwait. MATERIALS AND METHODS: Respiratory samples from 735 hospitalized patients with RTI from September 2010 to April 2013 were evaluated for the presence of HCoV-NL63, hMPV, Boca, KIV and WUV using molecular assays, polymerase chain reaction (PCR) and reverse-transcription PCR. RESULTS: Of the 735 patients, 285 (38.8%) were diagnosed with viral RTI. The distribution of respiratory viruses was hMPV: 15 (5.3%), Boca: 14 (4.9%), WUV: 10 (3.5%) and KIV: 4 (1.4%). HCoV-NL63 was not detected in any of the samples. CONCLUSIONS: These newly discovered viruses were associated with the development of RTI in Kuwait. The rapid identification of these viral infections could aid in the control of nosocomial transmission, reduce the use of antibiotics and improve treatment and management strategies.

Mixed viral infections circulating in hospitalized patients with respiratory tract infections in kuwait.

The aim of this study was to determine the frequency of viral mixed detection in hospitalized patients with respiratory tract infections and to evaluate the correlation between viral mixed detection and clinical severity. Hospitalized patients with respiratory tract infections (RTI) were investigated for 15 respiratory viruses by using sensitive molecular techniques. In total, 850 hospitalized patients aged between 3 days and 80 years were screened from September 2010 to April 2014. Among the 351 (47.8%) patients diagnosed with viral infections, viral mixed detection was identified in 49 patients (14%), with human rhinovirus (HRV) being the most common virus associated with viral mixed detection (7.1%), followed by adenovirus (AdV) (4%) and human coronavirus-OC43 (HCoV-OC43) (3.7%). The highest combination of viral mixed detection was identified with HRV and AdV (2%), followed by HRV and HCoV-OC43 (1.4%). Pneumonia and bronchiolitis were the most frequent reason for hospitalization with viral mixed detection (9.1%). There were statistical significance differences between mixed and single detection in patients diagnosed with bronchiolitis (P = 0.002) and pneumonia (P = 0.019). Our findings might indicate a significant association between respiratory virus mixed detection and the possibility of developing more severe LRTI such as bronchiolitis and pneumonia when compared with single detection.

CD4(+) T cell levels are decreased during active CMV infection in kidney transplant recipients.

The numbers of T lymphocytes and T cell subsets (CD2(+), CD3(+), CD4(+), CD8(+)), activated T cells (CD26(+)), B cells (CD19(+)), granulocytes (CD15(+)) and natural killer cells (CD16/56) were monitored by flow cytometry in 79 kidney transplant recipients, 35 of whom had cytomegalovirus infection. The percentages of these cells were correlated with viral load, as determined by cytomegalovirus antigenemia. Development of cytomegaloviral infection coincided with a significant reduction in the percentages of CD4(+) (P < 0.005) and CD3(+) (P < 0.05) cells. Monitoring of lymphocyte subsets may provide useful information on immunological events during cytomegaloviral infection.

Changes in cell-mediated immunity in kidney transplant recipients with active CMV infection.

This study was aimed at determining (a) the extent of proliferation of peripheral blood mononuclear cells (PBMC) in response to stimulation by cytomegalovirus (CMV)-infected fibroblasts and (b) the levels of Th1 and Th2 cytokine production in kidney transplant recipients with and without active CMV infection. Thirty patients with, and 39 without active CMV infection, diagnosed by a CMV antigenemia assay (AA), were studied. PBMC of patients with active CMV infection showed significantly lower proliferation than those without ongoing CMV infection (P<0.0001). The levels of Th2-type cytokines (interleukin (IL-) 4 and IL-10) in AA-negative and AA-positive kidney transplant recipients were similar but the levels of the Th1-type cytokines interferon-gamma, tumor necrosis factor-alpha (P<0.05) and IL-2 were significantly lower in AA-positive kidney transplant recipients (P<0.0005).