An immunodominant NP105-113-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease.

NP105-113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105-113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105-113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105-113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105-113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.

Publisher Correction: Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.

In the version of this article initially published, the first affiliation lacked 'MRC'; the correct name of the institution is 'MRC Weatherall Institute of Molecular Medicine'. Two designations (SP110Y and ST110H) were incorrect in the legend to Fig. 6f,h,i. The correct text is as follows: for panel f, "...loaded with either the CdtB(105-125)SP110Y (DRB4*SP110Y) or the CdtB(105-125)ST110H (DRB4*ST110H) peptide variants..."; for panel h, "...decorated by the DRB4*SP110Y tetramer (lower-right quadrant), the DRB4*ST110H (upper-left quadrant)..."; and for panel i, "...stained ex vivo with DRB4*SP110Y, DRB4*ST110H...". In Fig. 8e, the final six residues (LTEAFF) of the sequence in the far right column of the third row of the table were missing; the correct sequence is 'CASSYRRTPPLTEAFF'. In the legend to Fig. 8d, a designation (HLyE) was incorrect; the correct text is as follows: "(HlyE?)." Portions of the Acknowledgements section were incorrect; the correct text is as follows: "This work was supported by the UK Medical Research Council (MRC) (MR/K021222/1) (G.N., M.A.G., A.S., V.C., A.J.P.),...the Oxford Biomedical Research Centre (A.J.P., V.C.),...and core funding from the Singapore Immunology Network (SIgN) (E.W.N.) and the SIgN immunomonitoring platform (E.W.N.)." Finally, a parenthetical element was phrased incorrectly in the final paragraph of the Methods subsection "T cell cloning and live fluorescence barcoding"; the correct phrasing is as follows: "...(which in all cases included HlyE, CdtB, Ty21a, Quailes, NVGH308, and LT2 strains and in volunteers T5 and T6 included PhoN)...". Also, in Figs. 3c and 4a, the right outlines of the plots were not visible; in the legend to Fig. 3, panel letter 'f' was not bold; and in Fig. 8f, 'ND' should be aligned directly beneath DRB4 in the key and 'ND' should be removed from the diagram at right, and the legend should be revised accordingly as follows: "...colors indicate the HLA class II restriction (gray indicates clones for which restriction was not determined (ND)). Clonotypes are grouped on the basis of pathogen selectivity (continuous line), protein specificity (dashed line) and epitope specificity; for ten HlyE-specific clones (pixilated squares), the epitope specificity was not determined...". The errors have been corrected in the HTML and PDF versions of the article.

Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.

To tackle the complexity of cross-reactive and pathogen-specific T cell responses against related Salmonella serovars, we used mass cytometry, unbiased single-cell cloning, live fluorescence barcoding, and T cell-receptor sequencing to reconstruct the Salmonella-specific repertoire of circulating effector CD4+ T cells, isolated from volunteers challenged with Salmonella enterica serovar Typhi (S. Typhi) or Salmonella Paratyphi A (S. Paratyphi). We describe the expansion of cross-reactive responses against distantly related Salmonella serovars and of clonotypes recognizing immunodominant antigens uniquely expressed by S. Typhi or S. Paratyphi A. In addition, single-amino acid variations in two immunodominant proteins, CdtB and PhoN, lead to the accumulation of T cells that do not cross-react against the different serovars, thus demonstrating how minor sequence variations in a complex microorganism shape the pathogen-specific T cell repertoire. Our results identify immune-dominant, serovar-specific, and cross-reactive T cell antigens, which should aid in the design of T cell-vaccination strategies against Salmonella.

Group A Streptococcus induces CD1a-autoreactive T cells and promotes psoriatic inflammation.

Group A Streptococcus (GAS) infection is associated with multiple clinical sequelae, including different subtypes of psoriasis. Such post-streptococcal disorders have been long known but are largely unexplained. CD1a is expressed at constitutively high levels by Langerhans cells and presents lipid antigens to T cells, but the potential relevance to GAS infection has not been studied. Here, we investigated whether GAS-responsive CD1a-restricted T cells contribute to the pathogenesis of psoriasis. Healthy individuals had high frequencies of circulating and cutaneous GAS-responsive CD4+ and CD8+ T cells with rapid effector functions, including the production of interleukin-22 (IL-22). Human skin and blood single-cell CITE-seq analyses of IL-22-producing T cells showed a type 17 signature with proliferative potential, whereas IFN-γ-producing T cells displayed cytotoxic T lymphocyte characteristics. Furthermore, individuals with psoriasis had significantly higher frequencies of circulating GAS-reactive T cells, enriched for markers of activation, cytolytic potential, and tissue association. In addition to responding to GAS, subsets of expanded GAS-reactive T cell clones/lines were found to be autoreactive, which included the recognition of the self-lipid antigen lysophosphatidylcholine. CD8+ T cell clones/lines produced cytolytic mediators and lysed infected CD1a-expressing cells. Furthermore, we established cutaneous models of GAS infection in a humanized CD1a transgenic mouse model and identified enhanced and prolonged local and systemic inflammation, with resolution through a psoriasis-like phenotype. Together, these findings link GAS infection to the CD1a pathway and show that GAS infection promotes the proliferation and activation of CD1a-autoreactive T cells, with relevance to post-streptococcal disease, including the pathogenesis and treatment of psoriasis.

Chemokine-cleaving Streptococcus pyogenes protease SpyCEP is necessary and sufficient for bacterial dissemination within soft tissues and the respiratory tract.

SpyCEP is a Streptococcus pyogenes protease that cleaves CXCL8/IL-8 and its activity is associated with human invasive disease severity. We investigated the role of SpyCEP in S. pyogenes necrotizing fasciitis and respiratory tract infection in mice using isogenic strains differing only in SpyCEP expression. SpyCEP cleaved human CXCL1, 2, 6 and 8 plus murine CXCL1 and 2 at a structurally conserved site. Mice were infected in thigh muscle with a strain of S. pyogenes that expresses a high level of SpyCEP, or with an isogenic non-SpyCEP expressing strain. SpyCEP expression by S. pyogenes hindered bacterial clearance from muscle, and enhanced bacterial spread, associated with cleavage of murine chemoattractant CXCL1. Mice were then infected with Lactococcus lactis strains that differed only in SpyCEP expression. In contrast to the parent L. lactis strain (lacks SpyCEP), which was avirulent when administered intramuscularly, infection with a strain that expressed SpyCEP heterologously led to dramatic systemic illness within 24 h, failure to clear bacteria from muscle and marked dissemination to other organs. In the upper airways, SpyCEP expression was required for survival of L. lactis but not S. pyogenes. However, dissemination of S. pyogenes to the lung was SpyCEP-dependent and was associated with evidence of chemokine cleavage. Taken together, the studies provide clear evidence that SpyCEP is necessary and sufficient for systemic bacterial dissemination from a soft tissue focus in this model and also underlies dissemination in the respiratory tract.

HLA-E-restricted, Gag-specific CD8+ T cells can suppress HIV-1 infection, offering vaccine opportunities.

Human leukocyte antigen-E (HLA-E) normally presents an HLA class Ia signal peptide to the NKG2A/C-CD94 regulatory receptors on natural killer (NK) cells and T cell subsets. Rhesus macaques immunized with a cytomegalovirus-vectored simian immunodeficiency virus (SIV) vaccine generated Mamu-E (HLA-E homolog)-restricted T cell responses that mediated post-challenge SIV replication arrest in >50% of animals. However, HIV-1-specific, HLA-E-restricted T cells have not been observed in HIV-1-infected individuals. Here, HLA-E-restricted, HIV-1-specific CD8 + T cells were primed in vitro. These T cell clones and allogeneic CD8 + T cells transduced with their T cell receptors suppressed HIV-1 replication in CD4 + T cells in vitro. Vaccine induction of efficacious HLA-E-restricted HIV-1-specific T cells should therefore be possible.

MAIT cell clonal expansion and TCR repertoire shaping in human volunteers challenged with Salmonella Paratyphi A.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that can detect bacteria-derived metabolites presented on MR1. Here we show, using a controlled infection of humans with live Salmonella enterica serovar Paratyphi A, that MAIT cells are activated during infection, an effect maintained even after antibiotic treatment. At the peak of infection MAIT cell T-cell receptor (TCR)ß clonotypes that are over-represented prior to infection transiently contract. Select MAIT cell TCRß clonotypes that expand after infection have stronger TCR-dependent activation than do contracted clonotypes. Our results demonstrate that host exposure to antigen may drive clonal expansion of MAIT cells with increased functional avidity, suggesting a role for specific vaccination strategies to increase the frequency and potency of MAIT cells to optimize effector function.

Salmonella Typhi Bactericidal Antibodies Reduce Disease Severity but Do Not Protect against Typhoid Fever in a Controlled Human Infection Model.

Effective vaccines against Salmonella Typhi, a major cause of febrile illness in tropical regions, can have a significant effect as a disease control measure. Earlier work has shown that immunization with either of two Salmonella Typhi vaccines, licensed Ty21a or candidate M01ZH09, did not provide full immunity in a controlled human infection model. Here, we describe the human humoral immune responses to these oral vaccines and their functional role in protection after challenge with S. Typhi. Serum, obtained from healthy volunteers before and after vaccination with Ty21a or M01ZH09 or placebo and before and after oral challenge with wild-type S. Typhi, was assessed for bactericidal activity. Single-dose vaccination with M01ZH09 induced an increase in serum bactericidal antibodies (p = 0.001) while three doses of Ty21a did not. No association between bactericidal activity and protection against typhoid after challenge was seen in either vaccine arm. Bactericidal activity after vaccination correlated significantly with delayed disease onset (p = 0.013), lower bacterial burden (p = 0.006), and decreased disease severity scores (p = 0.021). Depletion of antibodies directed against lipopolysaccharide significantly reduced bactericidal activity (p = 0.009). We conclude that antibodies induced after ingestion of oral live-attenuated typhoid vaccines or after challenge with wild-type S. Typhi exhibit bactericidal activity. This bactericidal activity is mediated by anti-O:LPS antibodies and significantly reduces clinical symptoms but does not provide sterile immunity. This directs future vaccine studies toward other antigens or mechanisms of protection against typhoid.

Protective efficacy of DNA vaccines encoding outer membrane protein A and OmpK36 of Klebsiella pneumoniae in mice.

The immunogenicity of DNA vaccines expressing outer membrane proteins as antigens was evaluated in this study. DNA vaccines consisting of vector pVAX1 expressing either outer membrane protein A or OmpK36 were injected into mice by either the intradermal or the intramuscular route. Antibodies elicited were shown to be specifically reactive to OmpA and OmpK36 by immunoblotting. The immunoglobulin G (IgG) antibodies elicited by both vaccines included IgG1, IgG2a, IgG2b, and IgG3. Immunized mice exhibited a predominance of IgG1 over IgG2a, therefore indicating a stronger humoral response. Mice receiving either of the DNA vaccines produced high levels of interleukin-12 (IL-12) and IL-10 and low levels of gamma interferon, suggesting the induction of a mixed Th1 and Th2 response. Sera from DNA vaccine-immunized mice had significantly higher opsonic activity in opsonophagocytic assays than did sera from the control mice. The level of protection afforded by pOmpK36 DNA injected intradermally into mice was the highest. These results suggest that both OmpA and OmpK36 are excellent candidates for use in future studies of vaccination against infections caused by Klebsiella pneumoniae. This is the first study which established the efficacy of protection afforded by DNA vaccines based on outer membrane proteins against K. pneumoniae infections.

Impact of immunization against SpyCEP during invasive disease with two streptococcal species: Streptococcus pyogenes and Streptococcus equi.

Currently there is no licensed vaccine against the human pathogen Streptococcus pyogenes. The highly conserved IL-8 cleaving S. pyogenes cell envelope proteinase SpyCEP is surface expressed and is a potential vaccine candidate. A recombinant N-terminal part of SpyCEP (CEP) was expressed and purified. AntiCEP antibodies were found to neutralize the IL-8 cleaving activity of SpyCEP. CEP-immunized mice had reduced bacterial dissemination from focal S. pyogenes intramuscular infection and intranasal infection. We also identified a functional SpyCEP-homolog protease SeCEP, expressed by the equine pathogen Streptococcus equi, which was able to cleave both human and equine IL-8. CEP-immunized mice also demonstrated reduced bacterial dissemination from S. equi intramuscular infection. Therefore immunization against SpyCEP may provide protection against other streptococci species with homologous proteases.

Emerging role of the interleukin-8 cleaving enzyme SpyCEP in clinical Streptococcus pyogenes infection.

Neutrophil chemoattractant interleukin (IL)-8 is cleaved and inactivated by the Streptococcus pyogenes cell envelope protease SpyCEP. A range of clinical S. pyogenes strains of differing emm type demonstrated SpyCEP activity, although transcription of the SpyCEP gene cepA differed 1000-fold between isolates. Disruption of the 2-component regulatory system covR/S in pharyngeal isolates increased cepA transcription 100-fold; this finding is consistent with endogenous CovR/S-mediated repression of cepA being responsible for low SpyCEP expression in some S. pyogenes strains associated with pharyngitis. Among patients with invasive S. pyogenes infection, disease severity and outcome were associated with the SpyCEP activity of the isolate. Lethal invasive isolate H292 (emm81) expressed more cepA than did other tested isolates. This strain carried a unique covR mutation that impaired binding to the cepA promoter. CovR/S sequence comparison in other clinical isolates revealed community-wide dissemination of covS mutations but not covR mutations. The results highlight a potential hazard and underline the importance of continuing molecular epidemiological surveillance for community-wide dissemination of CovR/S mutant hyperinvasive strains.

Inhibition of gene expression and growth by antisense peptide nucleic acids in a multiresistant beta-lactamase-producing Klebsiella pneumoniae strain.

Klebsiella pneumoniae causes common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance. The emergence and spread of beta-lactamase-producing K. pneumoniae strains highlight the need to develop new therapeutic strategies. In this study, we developed antisense peptide nucleic acids (PNAs) conjugated to the (KFF)(3)K peptide and investigated whether they could mediate gene-specific antisense effects in K. pneumoniae. No outer membrane permeabilization was observed with antisense PNAs when used alone. Antisense peptide-PNAs targeted at two essential genes, gyrA and ompA, were found to be growth inhibitory at concentrations of 20 muM and 40 muM, respectively. Mismatched antisense peptide-PNAs with sequence variations of the gyrA and ompA genes when used as controls were not growth inhibitory. Bactericidal effects exerted by peptide-anti-gyrA PNA and peptide-anti-ompA PNA on cells were observed within 6 h of treatment. The antisense peptide-PNAs specifically inhibited expression of DNA gyrase subunit A and OmpA from the respective targeted genes in a dose-dependent manner. Both antisense peptide-PNAs cured IMR90 cell cultures that were infected with K. pneumoniae (10(4) CFU) in a dose-dependent manner without any noticeable toxicity to the human cells.

Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis.

Klebsiella pneumoniae is an opportunistic pathogen which causes pneumoniae, urinary tract infections and septicemia in immunocompromised patients. Hospital outbreaks of multidrug-resistant K. pneumoniae, especially those in neonatal wards, are often caused by strains producing the extended-spectrum-beta-lactamases (ESBLs). An immunoproteome based approach was developed to identify candidate antigens of K. pneumoniae for vaccine development. Sera from patients with acute K. pneumoniae infections (n = 55) and a control group of sera from healthy individuals (n = 15) were analyzed for reactivity by Western blot against ESBL K. pneumoniae outer membrane proteins separated by 2-DE. Twenty highly immunogenic protein spots were identified by immunoproteomic analysis. The immunogenic proteins that are most frequently recognized by positive K. pneumoniae sera were OmpA, OmpK36, FepA, OmpK17, OmpW, Colicin I receptor protein and three novel proteins. Two of the vaccine candidate genes, OmpA (Struve et al. Microbiology 2003, 149, 167-176) and FepA (Lai, Y. C. et al.. Infect Immun 2001, 69, 7140-7145), have recently been shown to be essential in colonization and infection in an in vivo mouse model. Hence, these two immunogenic proteins could serve as potential vaccine candidates.

Direct identification of Pseudomonas aeruginosa from blood culture bottles by PCR-enzyme linked immunosorbent assay using oprI gene specific primers.

A PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed for direct identification of Pseudomonas aeruginosa from positive BACTEC blood culture bottles. PCR primers were designed to target a 249 bp sequence of the oprI gene in P. aeruginosa. Biotin-labeled probe (PA3) targeted to the species-specific motif were hybridized to the digoxigenin-labeled amplified products from P. aeruginosa and captured on streptavidin-coated microtiter plates. The specificity of the assay using the PA3 probe was investigated with a range of microorganisms, which are commonly isolated from blood culture bottles serving as negative controls. The PCR-ELISA assay was shown to be highly specific for the identification of P. aeruginosa and was 10-fold more sensitive than an agarose gel-based detection method using the same pair of primers, with a detection limit at 10 fg of template. The PCR-ELISA assay developed in this study is 100% sensitive and 100% specific as it correctly identified all 73 positive and 42 negative controls as well as 25 double blind clinical samples. It significantly reduces the time needed for the identification of P. aeruginosa from positive BACTEC blood cultures bottles from 2-3 days to 6-8h.

Rapid detection of Klebsiella pneumoniae from blood culture bottles by real-time PCR.

A LightCycler real-time PCR hybridization probe-based assay which detects a partial Klebsiella pneumoniae 16S rRNA gene was developed for the rapid identification of K. pneumoniae directly from growth-positive blood culture bottles (BACTEC 9240 system) within 2 h. No cross-reactivity was observed with 65 negative-control blood cultures that grew bacteria other than K. pneumoniae and 48 negative blood cultures from double-blind experiments, thus demonstrating 100% specificity when compared to results of conventional biochemical characterization. The assay also showed 100% sensitivity, as it correctly identified all 142 positive-control blood cultures and 4 from double-blind trials.