195 research outputs found
Viral load, clinical disease severity and cellular immune responses in primary varicella zoster virus infection in Sri Lanka
Background
In Sri Lanka, varicella zoster virus (VZV) is typically acquired during adulthood with significant associated disease morbidity and mortality. T cells are believed to be important in the control of VZV replication and in the prevention of reactivation. The relationship between viral load, disease severity and cellular immune responses in primary VZV infection has not been well studied.
Methodology
We used IFNγ ELISpot assays and MHC class II tetramers based on VZV gE and IE63 epitopes, together with quantitative real time PCR assays to compare the frequency and phenotype of specific T cells with virological and clinical outcomes in 34 adult Sri Lankan individuals with primary VZV infection.
Principal Findings
Viral loads were found to be significantly higher in patients with moderate to severe infection compared to those with mild infection (p<0.001) and were significantly higher in those over 25 years of age (P<0.01). A significant inverse correlation was seen between the viral loads and the ex vivo IFNγ ELISpot responses of patients (P<0.001, r = −0.85). VZV-specific CD4+ T cells expressed markers of intermediate differentiation and activation.
Conclusions
Overall, these data show that increased clinical severity in Sri Lankan adults with primary VZV infection associates with higher viral load and reduced viral specific T cell responses
Seroprevalance of dengue viral infections and serotype specific t cell responses in healthy individuals in Colombo, Sri lanka
Objective: Although dengue viral infections cause dengue haemorrhagic fever and fatalities in some individuals, it is a mild /asymptomatic infection in the majority of infected individuals. Therefore, we set out to determine silent dengue transmission in the community. Methods: 236 healthy individuals aged 5-80 years recruited from community to test the presence of anti-dengue virus(DV)antibodies. Ex vivo and cultured ELISpot assays for serotype specific (SS)DV peptides, DEN3 NS3 and non-dengue viral peptides were done in 47/263 individuals. Cultured ELISpots for SS responses done in 3 individuals < 20 years, in 20 for 20-40years age and in 24 aged >40years. Results: The seropositivity rates for DV-specific antibodies were 40%,62.5%,100% at 10,20,40 years of age respectively. A significant(p=0.001) and positive correlation observed with age and DV- seropositivity (Spearman r =0.8365,95%CI 0.55-0.95). SS T cell responses detected in all seropositive individuals(n=44) but absent in all dengue seronegative (n=3) individuals. SS responses seen in only 1 person of the <20 age group who responded to SS peptides of DEN-2. 3/20(25%), 6/20(40%), 3/20(25%) and 5/20(30%) of individuals between 2-40 years responded to at least one SS peptide of DEN-1, DEN-2, DEN3 and DEN-4 peptides respectively. 12/24(50%), 5/24(33%), 12/24(50%) and 6/24(25%) of individuals aged >40 years responded to at least one SS peptide of DEN-1, DEN-2, DEN-3 and DEN-4 peptides respectively. Conclusions: Seropositivity rates to the DV significantly rises with age, almost 100% at 40 years of age. The SS T cell responses to DEN-1 and DEN-3 SS peptides were more frequent aged >40 years.
Escaping High Viral Load Exhaustion: CD8 Cells with Altered Tetramer Binding in Chronic Hepatitis B Virus Infection
Deletion, anergy, and a spectrum of functional impairments can affect virus-specific CD8 cells in chronic viral infections. Here we characterize a low frequency population of CD8 cells present in chronic hepatitis B virus (HBV) infection which survive in the face of a high quantity of viral antigen. Although they do not appear to exert immunological pressure in vivo, these CD8 cells are not classically “tolerant” since they proliferate, lyse, and produce antiviral cytokines in vitro. They are characterized by altered HLA/peptide tetramer reactivity, which is not explained by TCR down-regulation or reduced functional avidity and which can be reversed with repetitive stimulation. CD8 cells with altered tetramer binding appear to have a specificity restricted to envelope antigen and not to other HBV antigens, suggesting that mechanisms of CD8 cell dysfunction are differentially regulated according to the antigenic form and presentation of individual viral antigens
High Frequency of Skin-homing Melanocyte-specific Cytotoxic T Lymphocytes in Autoimmune Vitiligo
Vitiligo is an autoimmune condition characterized by loss of epidermal melanocytes. Using tetrameric complexes of human histocompatibility leukocyte antigen (HLA) class I to identify antigen-specific T cells ex vivo, we observed high frequencies of circulating MelanA-specific, A*0201-restricted cytotoxic T lymphocytes (A2–MelanA tetramer+ CTLs) in seven of nine HLA-A*0201–positive individuals with vitiligo. Isolated A2–MelanA tetramer+ CTLs were able to lyse A*0201-matched melanoma cells in vitro and their frequency ex vivo correlated with extent of disease. In contrast, no A2–MelanA tetramer+ CTL could be identified ex vivo in all four A*0201-negative vitiligo patients or five of six A*0201-positive asymptomatic controls. Finally, we observed that the A2–MelanA tetramer+ CTLs isolated from vitiligo patients expressed high levels of the skin homing receptor, cutaneous lymphocyte-associated antigen, which was absent from the CTLs seen in the single A*0201-positive normal control. These data are consistent with a role of skin-homing autoreactive melanocyte-specific CTLs in causing the destruction of melanocytes seen in autoimmune vitiligo. Lack of homing receptors on the surface of autoreactive CTLs could be a mechanism to control peripheral tolerance in vivo
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Orchestrated control of filaggrin-actin scaffolds underpins cornification
Epidermal stratification critically depends on keratinocyte differentiation and programmed death by cornification, leading to formation of a protective skin barrier. Cornification is dynamically controlled by the protein filaggrin, rapidly released from keratohyalin granules (KHGs). However, the mechanisms of cornification largely remain elusive, partly due to limitations of the observation techniques employed to study filaggrin organization in keratinocytes. Moreover, while the abundance of keratins within KHGs has been well described, it is not clear whether actin also contributes to their formation or fate. We employed advanced (super-resolution) microscopy to examine filaggrin organization and dynamics in skin and human keratinocytes during differentiation. We found that filaggrin organization depends on the cytoplasmic actin cytoskeleton, including the role for α- and β-actin scaffolds. Filaggrin-containing KHGs displayed high mobility and migrated toward the nucleus during differentiation. Pharmacological disruption targeting actin networks resulted in granule disintegration and accelerated cornification. We identified the role of AKT serine/threonine kinase 1 (AKT1), which controls binding preference and function of heat shock protein B1 (HspB1), facilitating the switch from actin stabilization to filaggrin processing. Our results suggest an extended model of cornification in which filaggrin utilizes actins to effectively control keratinocyte differentiation and death, promoting epidermal stratification and formation of a fully functional skin barrier
SARS-CoV-2 neutralizing antibodies in patients with varying severity of acute COVID-19 illness
In order to support vaccine development, and to aid convalescent plasma therapy, it would be important to understand the kinetics, timing and persistence of SARS-CoV-2 neutralizing antibodies (NAbs), and their association with clinical disease severity. Therefore, we used a surrogate viral neutralization test to evaluate their levels in patients with varying severity of illness, in those with prolonged shedding and those with mild/asymptomatic illness at various time points. Patients with severe or moderate COVID-19 illness had earlier appearance of NAbs at higher levels compared to those with mild or asymptomatic illness. Furthermore, those who had prolonged shedding of the virus, had NAbs appearing faster and at higher levels than those who cleared the virus earlier. During the first week of illness the NAb levels of those with mild illness was significantly less (p = 0.01), compared to those with moderate and severe illness. At the end of 4 weeks (28 days), although 89% had NAbs, 38/76 (50%) in those with > 90 days had a negative result for the presence of NAbs. The Ab levels significantly declined during convalescence (> 90 days since onset of illness), compared to 4 to 8 weeks since onset of illness. Our data show that high levels of NAbs during early illness associated with clinical disease severity and that these antibodies declined in 50% of individuals after 3 months since onset of illness
High Frequency of Cytomegalovirus-Specific Cytotoxic T-Effector Cells in HLA-A*0201-Positive Subjects during Multiple Viral Coinfections
How the cellular immune response copes with diverse antigenic competition is poorly understood. Responses of virus-specific cytotoxic T lymphocytes (CTL) were examined longitudinally in an individual coinfected with human immunodeficiency virus type 1 (HIV-1), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CTL responses to all 3 viruses were quantified by limiting dilution analysis and staining with HLA-A*0201 tetrameric complexes folded with HIV-1, EBV, and CMV peptides. A predominance of CMV-pp65-speciflc CTL was found, with a much lower frequency of CTL to HIV-1 Gag and Pol and to EBV-BMLF1 and LMP2. The high frequency of CMV-speciflc CTL, compared with HIV-1- and EBV-specific CTL, was confirmed in an additional 16 HLA-A*0201-positive virus-coinfected subjects. Therefore, the human immune system can mount CTL responses to multiple viral antigens simultaneously, albeit with different strength
Molecular epidemiology of AY.28 and AY.104 delta sub-lineages in Sri Lanka
Background: The worst SARS-CoV-2 outbreak in Sri Lanka was due to the two Sri Lankan delta sub-lineages AY.28 and AY.104. We proceeded to further characterize the mutations and clinical disease severity of these two sub-lineages.
Methods: 705 delta SARS-CoV-2 genomes sequenced by our laboratory from mid-May to November 2021 using Illumina and Oxford Nanopore were included in the analysis. The clinical disease severity of 440/705 individuals were further analyzed to determine if infection with either AY.28 or AY.104 was associated with more severe disease. Sub-genomic RNA (sg-RNA) expression was analyzed using periscope.
Results: AY.28 was the dominant variant throughout the outbreak, accounting for 67.7% of infections during the peak of the outbreak. AY.28 had three lineage defining mutations in the spike protein: A222V (92.80%), A701S (88.06%), and A1078S (92.04%) and seven in the ORF1a: R24C, K634N, P1640L, A2994V, A3209V, V3718A, and T3750I. AY.104 was characterized by the high prevalence of T95I (90.81%) and T572L (65.01%) mutations in the spike protein and by the absence of P1640L (94.28%) in ORF1a with the presence of A1918V (98.58%) mutation. The mean sgRNA expression levels of ORF6 in AY.28 were significantly higher compared to AY.104 (p < 0.0001) and B.1.617.2 (p < 0.01). Also, ORF3a showed significantly higher sgRNA expression in AY.28 compared to AY.104 (p < 0.0001). There was no difference in the clinical disease severity or duration of hospitalization in individuals infected with these sub lineages.
Conclusions: Therefore, AY.28 and AY.104 appear to have a fitness advantage over the parental delta variant (B.1.617.2), while AY.28 also had a higher expression of sg-RNA compared to other sub-lineages. The clinical implications of these should be further investigated
Genomic and epidemiological analysis of SARS-CoV-2 viruses in Sri Lanka
Background: In order to understand the molecular epidemiology of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Sri Lanka, since March 2020, we carried out genomic sequencing overlaid on available epidemiological data until April 2021.
Methods: Whole genome sequencing was carried out on diagnostic sputum or nasopharyngeal swabs from 373 patients with COVID-19. Molecular clock phylogenetic analysis was undertaken to further explore dominant lineages.
Results: The B.1.411 lineage was most prevalent, which was established in Sri Lanka and caused outbreaks throughout the country until March 2021. The estimated time of the most recent common ancestor (tMRCA) of this lineage was June 1, 2020 (with 95% lower and upper bounds March 30 to July 27) suggesting cryptic transmission may have occurred, prior to a large epidemic starting in October 2020. Returning travellers were identified with infections caused by lineage B.1.258, as well as the more transmissible B.1.1.7 lineage, which has replaced B.1.411 to fuel the ongoing large outbreak in the country.
Conclusions: The large outbreak that started in early October, is due to spread of a single virus lineage, B.1.411 until the end of March 2021, when B.1.1.7 emerged and became the dominant lineage
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