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The incidence of whooping cough (pertussis), the respiratory disease caused byBordetella pertussis(BP) has increased in recent years, and it is suspected that the switch from whole-cell pertussis (wP) to acellular pertussis (aP) vaccines may be a contributing factor to the rise in morbidity. While a growing body of evidence indicates that T cells play a role in the control and prevention of symptomatic disease, nearly all data on human BP-specific T cells is related to the four antigens contained in the aP vaccines, and data detailing T cell responses to additional non-aP antigens, are lacking. Here, we derived a full-genome map of human BP-specific CD4+ T cell responses using a high-throughputex vivoActivation Induced Marker (AIM) assay, to screen a peptide library spanning over 3000 different BP ORFs. First, our data show that BP specific-CD4+ T cells are associated with a large and previously unrecognized breadth of responses, including hundreds of targets. Notably, fifteen distinct non-aP vaccine antigens were associated with reactivity comparable to that of the aP vaccine antigens. Second, the overall pattern and magnitude of CD4+ T cell reactivity to aP and non-aP vaccine antigens was similar regardless of aP vs wP childhood vaccination history, suggesting that the profile of T cell reactivity in adults is not driven by vaccination, but rather is likely driven by subsequent asymptomatic or sub-clinical infections. Finally, while aP vaccine responses were Th1/Th2 polarized as a function of childhood vaccination, CD4+ T cell responses to non-aP BP antigens vaccine responses were not, suggesting that these antigens could be used to avoid the Th2 bias associated with aP vaccination. Overall, these findings enhance our understanding of human T cell responses against BP and suggest potential targets for designing next-generation pertussis vaccines.

Preexisting immune response to SARS-CoV-2 Robust T cell responses to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus occur in most individuals with coronavirus disease 2019 (COVID-19). Several studies have reported that some people who have not been exposed to SARS-CoV-2 have preexisting reactivity to SARS-CoV-2 sequences. The immunological mechanisms underlying this preexisting reactivity are not clear, but previous exposure to widely circulating common cold coronaviruses might be involved. Mateus et al. found that the preexisting reactivity against SARS-CoV-2 comes from memory T cells and that cross-reactive T cells can specifically recognize a SARS-CoV-2 epitope as well as the homologous epitope from a common cold coronavirus. These findings underline the importance of determining the impacts of preexisting immune memory in COVID-19 disease severity. Science , this issue p. 89

SUMMARYSARS-CoV-2 infection and COVID-19 vaccines elicit memory T cell responses. Here, we report the development of two new pools of Experimentally-defined T cell epitopes derived from the non-spike Remainder of the SARS-CoV-2 proteome (CD4RE and CD8RE). The combination of T cell responses to these new pools and Spike (S) were used to discriminate four groups of subjects with different SARS-CoV-2 infection and COVID-19 vaccine status: non-infected, non-vaccinated (I−V−); infected and non-vaccinated (I+V−); infected and then vaccinated (I+V+); and non-infected and vaccinated (I−V+). The overall classification accuracy based on 30 subjects/group was 89.2% in the original cohort and 88.5% in a validation cohort of 96 subjects. The T cell classification scheme was applicable to different mRNA vaccines, and different lengths of time post-infection/post-vaccination. T cell responses from breakthrough infections (infected vaccinees, V+I+) were also effectively segregated from the responses of vaccinated subjects using the same classification tool system. When all five groups where combined, for a total of 239 different subjects, the classification scheme performance was 86.6%. We anticipate that a T cell-based immunodiagnostic scheme able to classify subjects based on their vaccination and natural infection history will be an important tool for longitudinal monitoring of vaccination and aid in establishing SARS-CoV−2 correlates of protection.

Both SARS-CoV-2 infections and COVID-19 vaccines elicit memory T cell responses. Here, we report the development of 2 pools of experimentally defined SARS-CoV-2 T cell epitopes that, in combination with spike, were used to discriminate 4 groups of subjects with different SARS-CoV-2 infection and COVID-19 vaccine status. The overall T cell-based classification accuracy was 89.2% and 88.5% in the experimental and validation cohorts. This scheme was applicable to different mRNA vaccines and different lengths of time post infection/post vaccination and yielded increased accuracy when compared to serological readouts. T cell responses from breakthrough infections were also studied and effectively segregated from vaccine responses, with a combined performance of 86.6% across all 239 subjects from the 5 groups. We anticipate that a T cell-based immunodiagnostic scheme to classify subjects based on their vaccination and natural infection history will be an important tool for longitudinal monitoring of vaccinations and for establishing SARS-CoV-2 correlates of protection.

Background Characterization of allergic responses to cockroach (CR), a common aeroallergen associated with asthma, has focused mainly on IgE reactivity, but little is known about T cell responses, particularly in children. We conducted a functional evaluation of CR allergen‐specific T cell reactivity in a cohort of CR allergic children with asthma. Methods Peripheral blood mononuclear cells (PBMCs) were obtained from 71 children, with mild‐to‐moderate asthma who were enrolled in a CR immunotherapy (IT) clinical trial, prior to treatment initiation. PBMC were stimulated with peptide pools derived from 11 CR allergens, and CD4+ T cell responses assessed by intracellular cytokine staining. Results Highly heterogeneous responses in T cell reactivity were observed among participants, both in terms of the magnitude of cytokine response and allergen immunodominance. Reactivity against Bla g 9 and Bla g 5 was most frequent. The phenotype of the T cell response was dominated by IL‐4 production and a Th2 polarized profile in 54.9% of participants, but IFNγ production and Th1 polarization was observed in 25.3% of the participants. The numbers of regulatory CD4+ T cells were also highly variable and the magnitude of effector responses and Th2 polarization were positively correlated with serum IgE levels specific to a clinical CR extract. Conclusions Our results demonstrate that in children with mild‐to‐moderate asthma, CR‐specific T cell responses display a wide range of magnitude, allergen dominance, and polarization. These results will enable examination of whether any of the variables measured are affected by IT and/or are predictive of clinical outcomes.

The role of T cell immunity has been acknowledged in recent vaccine development and evaluation. We tested the humoral and cellular immune responses to Flucelvax®, a quadrivalent inactivated seasonal influenza vaccine containing two influenza A (H1N1 Singapore/GP1908/2015 IVR-180 and H3N2 North Carolina/04/2016) and two influenza B (Iowa/06/2017 and Singapore/INFTT-16-0610/2016) virus strains, using peripheral blood mononuclear cells stimulated by pools of peptides overlapping all the individual influenza viral protein components. Baseline reactivity was detected against all four strains both at the level of CD4 and CD8 responses and targeting different proteins. CD4 T cell reactivity was mostly directed to HA/NA proteins in influenza B strains, and NP/M1/M2/NS1/NEP proteins in the case of the Influenza A strains. CD8 responses to both influenza A and B viruses preferentially targeted the more conserved core viral proteins. Following vaccination, both CD4 and CD8 responses against the various influenza antigens were increased in day 15 to day 91 post vaccination period and maintained a Th1 polarized profile. Importantly, no vaccine interference was detected, with the increased responses balanced across all 4 included viral strains for both CD4 and CD8 T cells, and targeting HA and multiple additional viral antigens.

The emergence of SARS-CoV-2 variants with evidence of antibody escape highlight the importance of addressing whether the total CD4+ and CD8+ T cell recognition is also affected. Here, we compare SARS-CoV-2-specific CD4+ and CD8+ T cells against the B.1.1.7, B.1.351, P.1, and CAL.20C lineages in COVID-19 convalescents and in recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. The total reactivity against SARS-CoV-2 variants is similar in terms of magnitude and frequency of response, with decreases in the 10%–22% range observed in some assay/VOC combinations. A total of 7% and 3% of previously identified CD4+ and CD8+ T cell epitopes, respectively, are affected by mutations in the various VOCs. Thus, the SARS-CoV-2 variants analyzed here do not significantly disrupt the total SARS-CoV-2 T cell reactivity; however, the decreases observed highlight the importance for active monitoring of T cell reactivity in the context of SARS-CoV-2 evolution., Graphical abstract, Tarke et al. show that SARS-CoV-2-specific memory CD4 and CD8 T cells exposed to the ancestral strain by infection or vaccination effectively recognize the variants B.1.1.7, B.1.351, P.1, and CAL.20C. The majority of T cell epitopes are unaffected by mutations in these variant strains.

SUMMARYThe emergence of SARS-CoV-2 variants highlighted the need to better understand adaptive immune responses to this virus. It is important to address whether also CD4+ and CD8+ T cell responses are affected, because of the role they play in disease resolution and modulation of COVID-19 disease severity. Here we performed a comprehensive analysis of SARS-CoV-2-specific CD4+ and CD8+ T cell responses from COVID-19 convalescent subjects recognizing the ancestral strain, compared to variant lineages B.1.1.7, B.1.351, P.1, and CAL.20C as well as recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. Similarly, we demonstrate that the sequences of the vast majority of SARS-CoV-2 T cell epitopes are not affected by the mutations found in the variants analyzed. Overall, the results demonstrate that CD4+ and CD8+ T cell responses in convalescent COVID-19 subjects or COVID-19 mRNA vaccinees are not substantially affected by mutations found in the SARS-CoV-2 variants.

SUMMARY The emergence of SARS-CoV-2 variants highlighted the need to better understand adaptive immune responses to this virus. It is important to address whether also CD4+ and CD8+ T cell responses are affected, because of the role they play in disease resolution and modulation of COVID-19 disease severity. Here we performed a comprehensive analysis of SARS-CoV-2-specific CD4+ and CD8+ T cell responses from COVID-19 convalescent subjects recognizing the ancestral strain, compared to variant lineages B.1.1.7, B.1.351, P.1, and CAL.20C as well as recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. Similarly, we demonstrate that the sequences of the vast majority of SARS-CoV-2 T cell epitopes are not affected by the mutations found in the variants analyzed. Overall, the results demonstrate that CD4+ and CD8+ T cell responses in convalescent COVID-19 subjects or COVID-19 mRNA vaccinees are not substantially affected by mutations found in the SARS-CoV-2 variants.

Understanding the mechanisms of allergen-specific immune modulation in nonallergic individuals is key to recapitulate immune tolerance and to develop novel allergy treatments. Herein, we characterized mouse-specific T cell responses in nonallergic laboratory animal-care workers before and after reexposure to mice. PBMCs were collected and stimulated with developed peptide pools identified from high-molecular-weight fractions of mouse allergen extracts. Sizable CD4 T cell responses were noted and were temporarily decreased in most subjects upon reexposure, with the magnitude of decrease positively correlated with time of reexposure but not the duration of the break. Interestingly, the suppression was specific to mouse allergens without affecting responses of bystander antigens. Further, PBMC fractioning studies illustrated that the modulation is unlikely from T cells, while B cell depletion and exchange reversed the suppression of responses, suggesting that B cells may be the key modulators. Increased levels of regulatory cytokines (IL-10 and TGF-β1) in the cell culture supernatant and plasma mouse-specific IgG4 were also observed after reexposure, consistent with B cell–mediated modulation mechanisms. Overall, these results suggest that nonallergic status is achieved by an active, time-related, allergen-specific, B cell-dependent regulatory process upon reexposure, the mechanisms of which should be detailed by further molecular studies.

IgE sensitization to cockroach allergens is associated with development of allergic diseases, such as asthma. To understand the relevance of different cockroach allergens for diagnosis and immunotherapy, a comprehensive analysis of IgE antibody levels and T cell reactivity to an expanded set of cockroach allergens and their relationship to disease was performed in a cohort of USA cockroach sensitized patients. IgE antibody levels to recombinant chitinase and hemocyanin were measured for 23 subjects by custom-made ImmunoCAPs and compared with IgE levels to eight cockroach allergens we previously reported for the same cohort. Ex vivo T cell activation (Ox40/PDL-1 expression) of PBMCs stimulated with peptide pools derived from 11 German cockroach proteins, including nine official cockroach allergens, plus chitinase and vitellogenin, was determined by flow cytometry. IgE prevalences to chitinase (17%) and hemocyanin (44%) were comparable to values for the other eight allergens that we previously reported (21–57%). Hemocyanin (Bla g 3), was a major allergen (one to which more than 50% of patients with an allergy to its source react) for a sub-group of 15 highly cockroach-sensitized subjects (IgE > 3.5 kUA/L: 53%). Chitinase was officially named as new allergen Bla g 12. Cockroach-specific IgE levels in plasma showed excellent correlation with the sum of 10 allergen-specific IgE (r = 0.94, p < 0.001). T cell reactivity to 11 proteins was highly variable among subjects, the highest being for vitellogenin, followed by Bla g 3. The main finding was that cockroach allergen-specific IgE and T cell reactivity patterns were unique per subject, and lacked immunodominant allergens and correlation with clinical phenotype/disease severity in the studied cohort. Knowing the subject-specific B/T cell reactivity profiles to a comprehensive panel of cockroach allergens will contribute to diagnosis of cockroach allergy and will be important for planning and assessing allergen immunotherapy outcomes, according to the allergen content in therapeutic cockroach extracts.

Summary Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide ‘megapools’, circulating SARS-CoV-2−specific CD8+ and CD4+ T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively. CD4+ T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike and N proteins each accounted for 11-27% of the total CD4+ response, with additional responses commonly targeting nsp3, nsp4, ORF3a and ORF8, among others. For CD8+ T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2−reactive CD4+ T cells in ∼40-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating ‘common cold’ coronaviruses and SARS-CoV-2., Highlights • Measuring immunity to SARS-CoV-2 is key for understanding COVID19 and vaccine development • Epitope pools detect CD4+ and CD8+ T cells in 100 and 70% of convalescent COVID patients • T cell responses are focused not only on spike but also on M, N and other ORFs • T cell reactivity to SARS-CoV-2 epitopes is also detected in non-exposed individuals, An analysis of immune cell responses to SARS-CoV-2 from recovered patients identifies the regions of the virus that is targeted and also reveals cross-reactivity with other common circulating coronaviruses

The immune response elicited by the protective whole-cell pertussis (wP) versus the less-protective acellular pertussis (aP) vaccine has been well characterized; however, important clinical problems remain unsolved, as the inability of the currently administered aP vaccine is resulting in the reemergence of clinical disease (i.e., whooping cough). Strong evidence has shown that original, childhood aP and wP priming vaccines provide a long-lasting imprint on the CD4+ T cells that impacts protective immunity. However, aP vaccination might prevent disease but not infection, which might also affect the breadth of responses to Bordetella pertussis (BP) antigens. Thus, characterizing and defining novel targets associated with T cell reactivity are of considerable interest. Here, we compare the T cell reactivity of original aP and wP priming for different antigens contained or not contained in the aP vaccine and define the basis of a full-scale genomic map of memory T cell reactivity to BP antigens in humans. Our data show that the original priming after birth with aP vaccines has higher T cell reactivity than originally expected against a variety of BP antigens and that the genome-wide mapping of BP using an ex vivo screening methodology is feasible, unbiased, and reproducible. This could provide invaluable knowledge towards the direction of a new and improved pertussis vaccine design.

The emergence of SARS-CoV-2 variants with evidence of antibody escape highlight the importance of addressing whether the total CD4+ and CD8+ T cell recognition is also affected. Here, we compare SARS-CoV-2-specific CD4+ and CD8+ T cells against the B.1.1.7, B.1.351, P.1, and CAL.20C lineages in COVID-19 convalescents and in recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. The total reactivity against SARS-CoV-2 variants is similar in terms of magnitude and frequency of response, with decreases in the 10%-22% range observed in some assay/VOC combinations. A total of 7% and 3% of previously identified CD4+ and CD8+ T cell epitopes, respectively, are affected by mutations in the various VOCs. Thus, the SARS-CoV-2 variants analyzed here do not significantly disrupt the total SARS-CoV-2 T cell reactivity; however, the decreases observed highlight the importance for active monitoring of T cell reactivity in the context of SARS-CoV-2 evolution.

Current evaluation of influenza vaccines focuses mainly on humoral response to surface hemagglutinin (HA), although both CD4 and CD8 T cell responses have been reported, to both HA and other viral proteins. We conducted a comprehensive functional analysis on the pre-existing and vaccine-induced immune responses to Flucelvax, a tetravalent mammalian cell-based influenza vaccine which in addition to HA contains additional viral proteins (NA, M1, M2, NP, NEP, NS1, PA, PB1, and PB2). Longitudinal samples of peripheral blood mononuclear cells from a cohort of 10 participants, were stimulated by pools of peptides overlapping individual influenza viral protein components. We examined both humoral and cellular responses, intra-vaccine interference, type-specific immunodominance patterns of responses and cytokine polarization. In addition to serological responses, our results demonstrated vaccine-induced CD4 and CD8 responses that were still present after 90 days post vaccination, a balanced immunogenicity across all 4 included viral strains, as well as unchanged type-specific immunodominance patterns and cytokine polarization post-vaccination. Our results demonstrated distinct CD4 reactivities mostly directed to HA/NA (neuraminidase) proteins in Influenza B strains, but similar CD8 responses to both influenza A and B viruses preferentially targeting the more conserved core viral proteins. The present study suggests that Flucelvax induces, both humoral, CD4 and CD8 cellular immunity, balanced across four different influenza strains and targeting HA and multiple additional viral antigens.

Despite a growing consensus on the involvement of γδ T cells in allergy and other human immunological disorders, the detailed mechanisms remain hypothetical due to lack of investigative tools. Herein, we sought to develop functional assays to study the role of γδ T cells in allergy. Using an Activation-Induced Marker (AIM) assay, based on the upregulation of 4-1BB (CD137) and CD69, we were able to detect ex vivo allergen-specific responses from γδ T cells to multiple allergen extracts in human PBMCs, including mouse, cockroach (CR), house dust mite (HDM) and timothy grass (TG) allergens, which were reproducible and observed in multiple allergic cohorts. The magnitude of allergen-reactive γδ T cells differed between allergic and non-allergic cohorts with HDM and TG allergy, but not mouse or CR allergy. Further characterization showed that the γδ T cells reactive to mouse allergen were mostly the Vδ2 subset and from effector memory (Tem and Temra) compartments. Interestingly, in mouse-specific allergic donors we found differential polarized responses for γδ and ab T cells, with a Th1 and Th2 pattern, respectively. Using a complementary AIM assay for CD40L (CD154) combined with intracellular staining (ICS), we observed that Th1 polarization (INFg and TNFa) in responses to mouse extract was also common in non-allergic donors. Conversely, In the case of CR responses, Th1 polarization of Vδ2 T cells was only observed in the allergic cohort. Overall, these results suggest that allergen-specific Vδ2 T cells are potential to skew dysregulated cytokine responses in immunotherapy. In conclusion, we developed new functional assays that serve as tools for in-depth characterization and deciphering the roles of γδ T cells in allergy.

Background: cow&rsquo, s milk allergy (CM) is among the most common food allergies in young children and is often outgrown by adulthood. Prior to developing a tolerance to CM, a majority of CM-allergic children may tolerate extensively-heated CM. This study aims to characterize the IgE- and T cell-reactivity to unheated CM and the progressively more heated CM-containing foods. Methods: CM-containing food extracts from muffin, baked cheese, custard and raw, pasteurized CM commercial extract were tested for skin prick test reactivity, IgE binding and T cell reactivity as assessed by IL-5 and IFN&gamma, production. Results: the skin prick test (SPT) reactivity was significantly decreased to muffin extract compared to raw, pasteurized CM. Both IgE- and T-cell reactivity were readily detectable against food extracts from all forms of CM. Western blot analysis of IgE reactivity revealed variability between extracts that was protein-specific. T cell-reactivity was detected against all four extracts with no significant difference in IL-5 or IFN&gamma, production between them. Conclusion: our data indicate that despite reduced clinical reactivity, extracts from heated CM-containing foods retain immunogenicity when tested in vitro, particularly at the T cell level.

A knowledge based approach has been adopted to identify novel NOP receptor agonists with simplified hydrophobes. Substitution of the benzimidazol-2-one piperidine motif with a range of hydrophobic groups and pharmacophore guided bio-isosteric replacement of the benzimidazol-2-one moiety was explored. Compound 51 was found to be a high affinity, potent NOP receptor agonist with reduced affinity for the hERG channel.

Background Malignant bowel obstruction is a recognised complication of advanced malignancy. Surgery is not always suitable therefore antiemetics are the mainstay of symptomatic management. There is little evidence for any specific antiemetic. NWAG was initiated to co-ordinate audits across palliative care settings within the north-west. Aims To review the assessment and management of nausea and vomiting in medically managed malignant bowel obstruction. To audit against six standards set following a literature review. Method Retrospective, multi-centre review of case notes. Results Thirteen organisations returned 101 data collection sheets (5 hospices 8 hospitals). Ninety-nine of these were used (41 from hospices and 58 from hospitals). The presence or absence of nausea, vomiting and colic was documented at initial assessment in 80%, 91%, 80% respectively (n=99). Antiemetics were prescribed via non-oral routes in patients with nausea and/or vomiting regularly in 90% (n=252) and PRN in 97% (n=236). Regular or continuous medications via non-oral routes were prescribed within 48 h of a documented episode of nausea and/or vomiting secondary to malignant bowel obstruction in 68%. Consideration was given to reviewing management of nausea and vomiting in patients requiring two or more PRN medications for nausea and vomiting in the preceding 24 h. Day 2 82% (n=22), Day 4 88% (n=19). Metoclopramide was stopped if colic was present in 27% (n=11). 6. The co-prescription of cyclizine and metoclopramide should not occur – regular 100% (n=99) Regular +PRN 93% (n=99) Conclusion Regular assessment of symptoms is crucial in patients with medically managed bowel obstruction. Frequency and route of administration should be considered. Regular review is required to establish effectiveness of management and the need for change. PRN and regular medication combinations need assessment.