The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut ...is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.
The PhoB/PhoR-dependent response to inorganic phosphate (Pi)-starvation in Vibrio cholerae O1 includes the expression of vc0719 for the response regulator PhoB, vca0033 for an alkaline phosphatase ...and vca1008 for an outer membrane protein (OMP). Sequences with high identity to these genes have been found in the genome of clinical and environmental strains, suggesting that the Pi-starvation response in V. cholerae is well conserved. VCA1008, an uncharacterized OMP involved in V. cholerae pathogenicity, presents sequence similarity to porins of Gram-negative bacteria such as phosphoporin PhoE from Escherichia coli. A three-dimensional model shows that VCA1008 is a 16-stranded pore-forming β-barrel protein that shares three of the four conserved lysine residues responsible for PhoE anionic specificity with PhoE. VCA1008 β-barrel apparently forms trimers that collapse into monomers by heating. Properties such as heat modifiability and resistance to denaturation by sodium dodecyl sulfate at lower temperatures permitted us to suggest that VCA1008 is a classical porin, more precisely, a phosphoporin due to its Pi starvation-induced PhoB-dependent expression, demonstrated by electrophoretic mobility shift assay and promoter fusion-lacZ assays.
Individuals with potential exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) do not necessarily develop PCR or antibody positivity, suggesting that some individuals may clear ...subclinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections
. Here we hypothesize that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-2 (refs.
), would expand in vivo to support rapid viral control, aborting infection. We measured SARS-CoV-2-reactive T cells, including those against the early transcribed replication-transcription complex (RTC)
, in intensively monitored healthcare workers (HCWs) who tested repeatedly negative according to PCR, antibody binding and neutralization assays (seronegative HCWs (SN-HCWs)). SN-HCWs had stronger, more multispecific memory T cells compared with a cohort of unexposed individuals from before the pandemic (prepandemic cohort), and these cells were more frequently directed against the RTC than the structural-protein-dominated responses observed after detectable infection (matched concurrent cohort). SN-HCWs with the strongest RTC-specific T cells had an increase in IFI27, a robust early innate signature of SARS-CoV-2 (ref.
), suggesting abortive infection. RNA polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and SARS-CoV-2 clades. RNA polymerase was preferentially targeted (among the regions tested) by T cells from prepandemic cohorts and SN-HCWs. RTC-epitope-specific T cells that cross-recognized HCoV variants were identified in SN-HCWs. Enriched pre-existing RNA-polymerase-specific T cells expanded in vivo to preferentially accumulate in the memory response after putative abortive compared to overt SARS-CoV-2 infection. Our data highlight RTC-specific T cells as targets for vaccines against endemic and emerging Coronaviridae.
The Omicron, or Pango lineage B.1.1.529, variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) carries multiple spike mutations with high transmissibility and partial neutralizing ...antibody (nAb) escape. Vaccinated individuals show protection against severe disease, often attributed to primed cellular immunity. We investigated T and B cell immunity against B.1.1.529 in triple BioNTech BNT162b2 messenger RNA-vaccinated health care workers (HCWs) with different SARS-CoV-2 infection histories. B and T cell immunity against previous variants of concern was enhanced in triple-vaccinated individuals, but the magnitude of T and B cell responses against B.1.1.529 spike protein was reduced. Immune imprinting by infection with the earlier B.1.1.7 (Alpha) variant resulted in less durable binding antibody against B.1.1.529. Previously infection-naïve HCWs who became infected during the B.1.1.529 wave showed enhanced immunity against earlier variants but reduced nAb potency and T cell responses against B.1.1.529 itself. Previous Wuhan Hu-1 infection abrogated T cell recognition and any enhanced cross-reactive neutralizing immunity on infection with B.1.1.529.
The aim of this study was to perform a clinical validation of the defining characteristics of impaired memory (IM) in elderly patients at a long-term care institution.
A sample of 123 elderly ...patients was evaluated with a questionnaire designed to identify IM according to the NANDA-I taxonomy. Accuracy measures were calculated for the total sample and for males and females separately.
Sensitivity and specificity values indicated that: (1) inability to learn new skills is useful in screening IM, and (2) forgets to perform a behavior at a scheduled time, forgetfulness, inability to learn new information, inability to recall events, and inability to recall factual information are confirmatory indicators.
Specific factors can affect the manifestation of IM by elderly patients. The results may be useful in improving diagnostic accuracy and efficiency of the IM nursing diagnosis.
Understanding the nature of immunity following mild/asymptomatic infection with SARS-CoV-2 is crucial to controlling the pandemic. We analyzed T cell and neutralizing antibody responses in 136 ...healthcare workers (HCW) 16-18 weeks after United Kingdom lockdown, 76 of whom had mild/asymptomatic SARS-CoV-2 infection captured by serial sampling. Neutralizing antibodies (nAb) were present in 89% of previously infected HCW. T cell responses tended to be lower following asymptomatic infection than in those reporting case-definition symptoms of COVID-19, while nAb titers were maintained irrespective of symptoms. T cell and antibody responses were sometimes discordant. Eleven percent lacked nAb and had undetectable T cell responses to spike protein but had T cells reactive with other SARS-CoV-2 antigens. Our findings suggest that the majority of individuals with mild or asymptomatic SARS-CoV-2 infection carry nAb complemented by multispecific T cell responses at 16-18 weeks after mild or asymptomatic SARS-CoV-2 infection.
The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut ...is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.
SARS‐CoV‐2 infection results in different outcomes ranging from asymptomatic infection to mild or severe disease and death. Reasons for this diversity of outcome include differences in challenge ...dose, age, gender, comorbidity and host genomic variation. Human leukocyte antigen (HLA) polymorphisms may influence immune response and disease outcome. We investigated the association of HLAII alleles with case definition symptomatic COVID‐19, virus‐specific antibody and T‐cell immunity. A total of 1364 UK healthcare workers (HCWs) were recruited during the first UK SARS‐CoV‐2 wave and analysed longitudinally, encompassing regular PCR screening for infection, symptom reporting, imputation of HLAII genotype and analysis for antibody and T‐cell responses to nucleoprotein (N) and spike (S). Of 272 (20%) HCW who seroconverted, the presence of HLA‐DRB1*13:02 was associated with a 6·7‐fold increased risk of case definition symptomatic COVID‐19. In terms of immune responsiveness, HLA‐DRB1*15:02 was associated with lower nucleocapsid T‐cell responses. There was no association between DRB1 alleles and anti‐spike antibody titres after two COVID vaccine doses. However, HLA DRB1*15:01 was associated with increased spike T‐cell responses following both first and second dose vaccination. Trial registration: NCT04318314 and ISRCTN15677965.
SARS‐CoV‐2 infection results in different outcomes ranging from asymptomatic infection, to mild or severe disease and death. We investigated the association of HLAII alleles with case definition symptomatic COVID‐19, virus‐specific antibody and T cell immunity. Presence of HLA‐DRB1*13:02 was associated with increased risk of symptomatic COVID‐19, while, after vaccination, HLA DRB1*15:01 was associated with increased spike T cell responses.
We hypothesised that host-response biomarkers of viral infections might contribute to early identification of individuals infected with SARS-CoV-2, which is critical to breaking the chains of ...transmission. We aimed to evaluate the diagnostic accuracy of existing candidate whole-blood transcriptomic signatures for viral infection to predict positivity of nasopharyngeal SARS-CoV-2 PCR testing.
We did a nested case-control diagnostic accuracy study among a prospective cohort of health-care workers (aged ≥18 years) at St Bartholomew's Hospital (London, UK) undergoing weekly blood and nasopharyngeal swab sampling for whole-blood RNA sequencing and SARS-CoV-2 PCR testing, when fit to attend work. We identified candidate blood transcriptomic signatures for viral infection through a systematic literature search. We searched MEDLINE for articles published between database inception and Oct 12, 2020, using comprehensive MeSH and keyword terms for “viral infection”, “transcriptome”, “biomarker”, and “blood”. We reconstructed signature scores in blood RNA sequencing data and evaluated their diagnostic accuracy for contemporaneous SARS-CoV-2 infection, compared with the gold standard of SARS-CoV-2 PCR testing, by quantifying the area under the receiver operating characteristic curve (AUROC), sensitivities, and specificities at a standardised Z score of at least 2 based on the distribution of signature scores in test-negative controls. We used pairwise DeLong tests compared with the most discriminating signature to identify the subset of best performing biomarkers. We evaluated associations between signature expression, viral load (using PCR cycle thresholds), and symptom status visually and using Spearman rank correlation. The primary outcome was the AUROC for discriminating between samples from participants who tested negative throughout the study (test-negative controls) and samples from participants with PCR-confirmed SARS-CoV-2 infection (test-positive participants) during their first week of PCR positivity.
We identified 20 candidate blood transcriptomic signatures of viral infection from 18 studies and evaluated their accuracy among 169 blood RNA samples from 96 participants over 24 weeks. Participants were recruited between March 23 and March 31, 2020. 114 samples were from 41 participants with SARS-CoV-2 infection, and 55 samples were from 55 test-negative controls. The median age of participants was 36 years (IQR 27–47) and 69 (72%) of 96 were women. Signatures had little overlap of component genes, but were mostly correlated as components of type I interferon responses. A single blood transcript for IFI27 provided the highest accuracy for discriminating between test-negative controls and test-positive individuals at the time of their first positive SARS-CoV-2 PCR result, with AUROC of 0·95 (95% CI 0·91–0·99), sensitivity 0·84 (0·70–0·93), and specificity 0·95 (0·85–0·98) at a predefined threshold (Z score >2). The transcript performed equally well in individuals with and without symptoms. Three other candidate signatures (including two to 48 transcripts) had statistically equivalent discrimination to IFI27 (AUROCs 0·91–0·95).
Our findings support further urgent evaluation and development of blood IFI27 transcripts as a biomarker for early phase SARS-CoV-2 infection for screening individuals at high risk of infection, such as contacts of index cases, to facilitate early case isolation and early use of antiviral treatments as they emerge.
Barts Charity, Wellcome Trust, and National Institute of Health Research.
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