Early Adaptive Immune Responses Research Articles

A novel metric-based approach of scoring early host immune response from oro-nasopharyngeal swabs predicts COVID-19 outcome

Unpredictable fatal outcome of COVID-19 is attributed to dysregulated inflammation. Impaired early adaptive immune response leads to late-stage inflammatory outcome. The purpose of this study was to develop biomarkers for early detection of host immune impairment at first diagnosis from leftover RNA samples, which may in turn identify high risk patients. Leftover RNA samples of COVID-19 patients at first diagnosis were stored. Following prospective follow-up, the samples were shorted and categorized into outcome groups. Impaired adaptive T cell response (severity score) and Impaired IL-10 response (undetectable IL-10 in the presence of high expression of a representative interferon response gene) were determined by RT-PCR based assay. We demonstrate that a T cell response based ‘severity score’ comprising rational combination of Ct values of a target genes’ signature can predict high risk noncomorbid potentially critical COVID-19 patients with a sensitivity of 91% (95% CI 58.7–99.8) and specificity of 92.6% (95% CI 75.7–99) (AUC:0.88). Although inclusion of comorbid patients reduced sensitivity to 77% (95% CI 54.6–92.2), the specificity was still 94% (95% CI 79.8–99.3) (AUC:0.82). The same for ‘impaired IL-10 response’ were little lower to predict high risk noncomorbid patients 64.2% (95% CI 35.1–87.2) and 82% (95% CI 65.5–93.2) respectively. Inclusion of comorbid patients drastically reduce sensitivity and specificity51.6% (95% CI 33.1–69.8) and 80.5% (95% CI 64.0–91.8) respectively. As best of our knowledge this is the first demonstration of a metric-based approach showing the ‘severity score’ as an indicator of early adoptive immune response, could be used as predictor of severe COVID-19 outcome at the time of first diagnosis using the same leftover swab RNA. The work flow could reduce expenditure and reporting time of the prognostic test for an earliest clinical decision ensuring possibility of early rational management.

Early antiviral CD4 and CD8 T cell responses and antibodies are associated with upper respiratory tract clearance of SARS-CoV-2.

T cells are involved in protective immunity against numerous viral infections. Data regarding functional roles of human T cells in SARS-CoV-2 (SARS2) viral clearance in primary COVID-19 are limited. To address this knowledge gap, samples were assessed for associations between SARS2 upper respiratory tract viral RNA levels and early virus-specific adaptive immune responses for 95 unvaccinated clinical trial participants with acute primary COVID-19 aged 18-86 years old, approximately half of whom were considered high risk for progression to severe COVID-19. Functionality and magnitude of acute SARS2-specific CD4 and CD8 T cell responses were evaluated, in addition to antibody responses. Most individuals with acute COVID-19 developed SARS2-specific T cell responses within 6 days of COVID-19 symptom onset. Early CD4 T cell and CD8 T cell responses were polyfunctional, and both strongly associated with reduced upper respiratory tract SARS2 viral RNA, independent of neutralizing antibody titers. Overall, these findings provide evidence for protective roles for circulating SARS2-specific CD4 and CD8 T cells during acute COVID-19.

A Robust Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Specific T- and B-Cell Response Is Associated With Early Viral Clearance in SARS-CoV-2 Omicron-Infected Immunocompromised Individuals.

The immunological determinants of delayed viral clearance and intrahost viral evolution that drive the development of new pathogenic virus strains in immunocompromised individuals are unknown. Therefore, we longitudinally studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immune responses in relation to viral clearance and evolution in immunocompromised individuals. Among Omicron-infected immunocompromised individuals, we determined SARS-CoV-2-specific T- and B-cell responses, anti-spike immunoglobulin G (IgG) and IgG3 titers, neutralization titers, and monoclonal antibody (mAb) resistance-associated mutations. The 28-day post-enrollment nasopharyngeal specimen defined early (reverse-transcription polymerase chain reaction [RT-PCR] negative ≤28 days) or late (RT-PCR positive >28 days) viral clearance. Of 30 patients included (median age, 61.9 [interquartile range, 47.4-72.3] years; 50% females), 20 (66.7%) received mAb therapy. Thirteen (43.3%) demonstrated early and 17 (56.7%) late viral clearance. Patients with early viral clearance and patients without resistance-associated mutations had significantly higher baseline interferon-γ release, and patients with early viral clearance had a higher frequency of SARS-CoV-2-specific B cells at baseline. In non-mAb-treated patients, day 7 IgG and neutralization titers were significantly higher in those with early versus late viral clearance. An early robust adaptive immune response is vital for efficient viral clearance and associated with less emergence of mAb resistance-associated mutations in Omicron-infected immunocompromised patients. This emphasizes the importance of early SARS-CoV-2-specific T- and B-cell responses and thereby provides a rationale for development of novel therapeutic approaches.

Preliminary evidence of localizing CD8+ T-cell responses in COVID-19 patients with PET imaging.

The upper respiratory tract (URT) is the entry site for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), from where it further disseminates. Early and effective adaptive immune responses are crucial to restrict viral replication and limit symptom development and transmission. Current vaccines increasingly incorporate strategies to boost mucosal immunity in the respiratory tract. Positron emission tomography (PET) is a non-invasive technology that measures cellular responses at a whole-body level. In this case series, we explored the feasibility of [89Zr]Zr-crefmirlimab berdoxam PET to assess CD8+ T-cell localization during active COVID-19. Our results suggest that CD8+ T-cell distributions assessed by PET imaging reflect their differentiation and functional state in blood. Therefore, PET imaging may represent a novel tool to visualize and quantify cellular immune responses during infections at a whole-body level.

CTIM-20. SYSTEMIC IMMUNE CORRELATES ASSOCIATED WITH LONG-TERM SURVIVAL AFTER THERAPEUTIC ADENOVIRUS (DNX2401) PLUS INTERFERON GAMMA FOR RECURRENT GLIOBLASTOMA EFFECT TRIAL (TARGET): A RANDOMIZED PHASE 1 TRIAL

Abstract Anti-tumor immune response following oncolytic virus treatment of glioblastoma may be enhanced by immune activating therapies. Here, in this Phase Ib, multicenter, randomized, open-label study we evaluated the combination of intra-tumoral delivery of oncolytic virus DNX-2401 with subcutaneous interferon gamma (IFN-γ) a T-helper 1 response activator in 37 patients with recurrent glioblastoma. In part 1, 27 patients were randomized 2:1 into DNX-2401 with IFN-γ and DNX-2401 alone with intra-tumoral DNX-2401 administered via a standard biopsy needle. In part 2, 10 patients received intra-tumoral DNX-2401 via an FDA-approved Alcyone MEMS Cannula designed to prevent backflow at high infusion flowrates. DNX-2401 was well tolerated but the addition of IFN- γ was not well tolerated. The primary efficacy endpoint, objective clinical response, was not met. The secondary endpoint of 12-month overall survival for DNX-2401 with IFN-γ, DNX-2401 alone and DNX-2401 via Cannula were 33%, 22%, 0% respectively. The 18-month OS for DNX-2401 with IFN-γ, DNX-2401 alone and DNX-2401 via Cannula were 25%, 9%, 0% respectively. The DNX-2401 with IFN- γ cohort had the most long-term survivors (44.2, 23.7, 20.6 and 20.1 months). Systemic antibody and immune cell profiling following DNX-2401 treatment showed that long term survivors had significantly higher anti-adenovirus specific antibody reactivity and a higher activated CD8+ effector memory T-cell sub-population at 2 months post-treatment relative to baseline. This systemic immune response did not occur in short term survivors following DNX-2401 treatment. Overall, these data suggest that an early and robust systemic adaptive immune response could be used as a biomarker for long term survival following DNX-2401 treatment of recurrent glioblastoma independent of other salvage treatments administered following disease progression during the trial. (Clinical Trial.gov registration: NCT02197169).

Interleukin-12 induces IFN-γ secretion and STAT signaling implying its potential regulation of Th1 cell response in Nile tilapia.

As a pleiotropic cytokine consisting of IL-12p35 and IL-12p40, Interleukin-12 (IL-12) features in inflammation regulation and anti-bacterial immunity. While IL-12 homologs have been identified in non-mammalian species, the precise mechanisms by which IL-12 contributes to early adaptive immune responses in vertebrates remain incompletely understood. Herein, an evolutionary conserved Oreochromis niloticus IL-12 (defined as OnIL-12) was identified by synteny characterization, structural comparisons and phylogenetic pattern of IL-12p35b and IL-12p40a. IL-12p35b and IL-12p40a exhibited widespread expression in lymphoid-related tissues of tilapia, while their mRNA expression in head-kidney demonstrated a significant increase after Edwardsiella piscicida infection. Compared with other lymphocytes, recombinant OnIL-12 (rOnIL-12) displayed stronger affinity binding to T cells. Although stimulation of lymphocytes with the p35b or p40a subunit resulted in a significant induction of IFN-γ expression, rOnIL-12 showed stronger potential to promote IFN-γ expression than these subunits. rOnIL-12 not only elevated the mRNA expression level Th1 cell-associated transcription factor T-bet in lymphocytes, but also increased the proportion of CD4-1+IFN-γ+ lymphocytes. Moreover, the mRNA and phosphorylation levels of STAT1, STAT3, STAT4 and STAT5 were enhanced by rOnIL-12. These findings will offer previous evidence for further exploration into the regulatory mechanisms of Th1 cellular immunity in early vertebrates.

A systematic comparison reveals dynamic differences in early adaptive immune responses of acute-resolving versus chronic HBV replication.

Chronic hepatitis B virus (HBV) infection has been characterized by lack of effective adaptive immune responses which are vital for the viral clearance. However, very little is known about the dynamics of adaptive immune responses during the early phase of chronic HBV infection especially in spleen and liver. Here, we used the hydrodynamic injection (HDI) mouse model to kinetically characterize differences in the features of adaptive immunity, including the frequencies, phenotypes and function of antigen-presenting cells and T cells in the spleen, peripheral blood mononuclear cells (PBMCs) and liver, of chronic versus acute-resolving HBV replication (AR). We found that mice with AR miceand mice with chronic HBV replication (CH) miceshowed early splenomegaly accompanied by T cell expansion in spleen but not in liver after HDI. Interestingly, the early and continuous increase in HBV-specific CD8+ T cells in spleen of CH mice was comparable to that in the AR mice. However, the splenic T cells of CH mice showed no activation phenotype compared with those in AR mice. Besides, increases in activated effector CD8+ T cells in PBMCs and liver at later time points were only observed in AR mice but not CH mice. CH mice also showed insufficient expansion of dendritic cells (DCs) in spleen and increased programmed death-1expression in DCs of the liver compared to AR mice. The adoptive transfer of total splenocytes or splenic CD8+ T cells of AR mice to CH mice demonstrated that their ability to break HBV tolerance varies at different stages of HBV clearance. Moreover, the adoptive transfer of splenocytes from AR mice induce functional activation of endogenous HBV-specific CD8+ T cells of CH mice. Our results suggest that early T cell priming and expansion initially happens in the periphery after HBV antigen exposure in acute-resolving and chronic replication. The paucity of T cell activation, and subsequent migration and liver infiltration is a key feature of the adaptive immune responses during the early phase of CH, which is probably caused by the dysfunction of DCs.

Evaluation of early innate and adaptive immune responses to the TB vaccine Mycobacterium bovis BCG and vaccine candidate BCGΔBCG1419c

The vaccine Mycobacterium bovis Bacillus Calmette-Guérin (BCG) elicits an immune response that is protective against certain forms of tuberculosis (TB); however, because BCG efficacy is limited it is important to identify alternative TB vaccine candidates. Recently, the BCG deletion mutant and vaccine candidate BCGΔBCG1419c was demonstrated to survive longer in intravenously infected BALB/c mice due to enhanced biofilm formation, and better protected both BALB/c and C57BL/6 mice against TB-induced lung pathology during chronic stages of infection, relative to BCG controls. BCGΔBCG1419c-elicited protection also associated with lower levels of proinflammatory cytokines (i.e. IL6, TNFα) at the site of infection in C57BL/6 mice. Given the distinct immune profiles of BCG- and BCGΔBCG1419c-immunized mice during chronic TB, we set out to determine if there are early immunological events which distinguish these two groups, using multi-dimensional flow cytometric analysis of the lungs and other tissues soon after immunization. Our results demonstrate a number of innate and adaptive response differences between BCG- and BCGΔBCG1419c-immunized mice which are consistent with the latter being longer lasting and potentially less inflammatory, including lower frequencies of exhausted CD4+ T helper (TH) cells and higher frequencies of IL10-producing T cells, respectively. These studies suggest the use of BCGΔBCG1419c may be advantageous as an alternative TB vaccine candidate.

Pertussis toxin suppresses dendritic cell-mediated delivery of B. pertussis into lung-draining lymph nodes

The adenylate cyclase (ACT) and the pertussis (PT) toxins of Bordetella pertussis exert potent immunomodulatory activities that synergize to suppress host defense in the course of whooping cough pathogenesis. We compared the mouse lung infection capacities of B. pertussis (Bp) mutants (Bp AC− or Bp PT–) producing enzymatically inactive toxoids and confirm that ACT action is required for maximal bacterial proliferation in the first days of infection, whereas PT action is crucial for persistence of B. pertussis in mouse lungs. Despite accelerated and near complete clearance from the lungs by day 14 of infection, the PT− bacteria accumulated within the lymphoid tissue of lung-draining mediastinal lymph nodes (mLNs). In contrast, the wild type or AC− bacteria colonized the lungs but did not enter into mLNs. Lung infection by the PT− mutant triggered an early arrival of migratory conventional dendritic cells with associated bacteria into mLNs, where the PT− bacteria entered the T cell-rich paracortex of mLNs by day 5 and proliferated in clusters within the B-cell zone (cortex) of mLNs by day 14, being eventually phagocytosed by infiltrating neutrophils. Finally, only infection by the PT− bacteria triggered an early production of anti-B. pertussis serum IgG antibodies already within 14 days of infection. These results reveal that action of the pertussis toxin blocks DC-mediated delivery of B. pertussis bacteria into mLNs and prevents bacterial colonization of mLNs, thus hampering early adaptive immune response to B. pertussis infection.

Th1 and Th17 cells are critical for limiting the severity of Staphylococcus aureus craniotomy infection

Abstract A craniotomy is performed to access the brain for tumor resection, localization and resection of epileptogenic foci, and aneurysm clipping. Despite prophylaxis, infectious complications after craniotomy range from 1–3%, with approximately half caused by Staphylococcus aureus (S. aureus), which forms a biofilm on the bone flap that is recalcitrant to antibiotics. Using our novel mouse model of S. aureus craniotomy infection, Th1 and Th17 infiltrates were found to preferentially home to the brain but not the subcutaneous galea; however, their functional importance is unknown. RAG1 knockout (KO) mice displayed significant increases in bacterial burden in the brain, galea, and bone flap at days 3, 7, and 14 post-infection, which was negated following the adoptive transfer of either in vitro skewed Th1 or Th17 cells. Interestingly, in vitro skewed Th17 cells acquired Th1 characteristics upon migrating into the brain in vivo as revealed by the production of Th1-associated cytokines such as IFN-γ and TNF-α with minimal IL-17A. We established that the window of T cell protection was during acute infection, since delaying Th1 or Th17 adoptive transfer until day 7 post-infection was no longer able to attenuate S. aureus burden in RAG1 KO mice compared to T cell transfers at day −1 or 3 after infection that were effective. In vitro studies established that both Th1 and Th17 cells augmented microglial, macrophage, and neutrophil S. aureus bactericidal activity and IL-12p70 and CXCL10 production. Collectively, these findings highlight the importance of an early adaptive immune response for bacterial containment during S. aureus craniotomy infection by promoting the antimicrobial activity of infiltrating leukocytes and resident microglia.

Evidence for transfer of maternal antigen specific cellular immunity against Mycobacterium avium ssp. paratuberculosis via colostrum in a goat twin model

Colostrum intake is one of the most important factors in neonatal health in ruminants, mainly because of its unique immunological properties. Both in practice as well as in research, the attention of lactogenic immunity is focused on the importance of colostral antibodies and less attention is given to the functional role of maternal cells in colostrum. Here we study the transfer of maternal leukocytes via colostrum and the functionality in goat kids. In experiment 1, twenty twin pairs of goat kids from dams previously immunized with an inactivated Mycobacterium avium subsp. paratuberculosis (MAP) vaccine were fed maternal colostrum from their dam (kid 1) or pasteurized and frozen/thawed bovine colostrum (kid 2). The presence of cell mediated immune response (CMIR) against Mycobacterium avium antigens in the kids was assessed using intradermal skin testing with PPD-A tuberculin. Linear mixed effect models showed an increase in skin thickness in response to intradermal PPD-A injection in maternal colostrum fed kids compared to bovine colostrum fed kids. After intradermal PPD-A application, serum concentration of MAP specific antibodies increased in kids fed maternal colostrum, indicating antigen specific activation of the adaptive immune system. We did not detect a similar increase in antibodies in the kids fed bovine colostrum.In experiment 2, a more reductionistic approach was applied to specifically study the effects of the transfer of maternal colostral leukocytes on CMIR in goat kids. Similar to experiment 1, twin kids from MAP immunized dams were randomly divided over two groups. The experimental group received colostrum replacer supplemented with fluorescently labelled colostral cells of the dam and the control group received colostrum replacer only. No difference in skin response following intradermal PPD-A injection was observed between both groups of kids. Histologic examination of the skin at the intradermal injection site did not show fluorescently labelled cells.In conclusion, in our initial experiment we observed an antigen specific CMIR in goat kids fed fresh colostrum with colostral leukocytes from vaccinated dams. The lack of a DTH response in kids fed colostrum replacer supplemented with maternal colostrum derived leukocytes indicated that the complete colostral matrix is probably required for colostrum leukocytes to transfer across the intestinal epithelial barrier and modulate the neonatal immune response. In line with earlier studies, our results indicate that caprine maternal leukocytes present in colostrum can functionally contribute to the newborns’ early adaptive immune responses adding to the importance of colostrum feeding in ruminant neonates.

Characterization of immunoglobulin and cytokine responses in Burkholderia mallei infected equids

Burkholderia mallei causes a highly fatal infectious disease in equines known as glanders. It is one of the OIE listed notifiable diseases, which entails strict control policy measures once B. mallei infection is confirmed in the susceptible hosts. Humans, especially equine handlers, veterinary professionals and laboratory workers are at greater risk to acquire the B. mallei infection directly through prolonged contact with glanderous equines, and indirectly through unprotected handling of B. mallei contaminated materials. Further, natural resistance of B. mallei to multiple antibiotics, aerosol transmission, lack of effective vaccine and treatment make this organism a potential agent of biological warfare. Results of experimental B. mallei infection in mouse and non-human primates and immunization with live attenuated B. mallei strains demonstrated that activation of early innate and adaptive immune responses play a critical role in controlling B. mallei infection. However, the immune response elicited by the primary hosts (equids) B. mallei infection is poorly understood. Therefore, we aimed to investigate immune responses in glanders affected horses (n = 23) and mules (n = 1). In this study, chronically infected equids showed strong humoral responses (IgM, IgG and IgA) specific to B. mallei type 6 secretory proteins such as Hcp1, TssA and TssB. The infected equids also elicited robust cellular responses characterized by significantly elevated levels of IFN-γ, TNF-α, IL-12, IL-17 and IL-6 in PBMCs. In addition, stimulation of equine PBMCs by Hcp1 resulted in the further elevation of these cytokines. Thus, the present study indicated that antibody response and T helper cell (Th) type 1-associated cytokines were the salient features of chronic B. mallei infection in horses. The immune responses also suggest further evaluation of these proteins as potential vaccine candidates.

Effect of protein C activator from Agkistrodon acutus venom on early adaptive immune response of septic rats

To investigate the effect of protein C activator (PCA) from Agkistrodon acutus venom (AAV) in modulating early adaptive immune response of septic rats. Rat models of sepsis were established by intraperitoneal injection of lipopolysaccharide (LPS; 10 mg/kg) in 36 SD rats, which were divided into 6 groups (n=6) for sample collection at 4, 6, 8, 12, 16 and 24 h after LPS injection, with 6 rats injected with saline as the control group. Another 36 rats were divided into two groups, and 30 min after LPS injection, the rats were treated with SEW2871 (a sphingosine-1-phosphate receptor 1 agonist; 0.5 mg/kg) or PCA group (0.1 mg/kg), and each group was divided into 3 groups (n=6) for sample collection at 6, 12 and 24 h after LPS injection. Plasma IL-4, S1P, IL-12 and IFN-γ levels of the rats were detected using ELISA, and the expressions of S1PR1 and CD103 in the mesenteric lymph nodes were detected with immunofluorescence assay. The plasma levels of S1P, IL-12, IL-4 and IFN-γ (P < 0.05) and the expressions of S1PR1 and CD103 in the mesenteric lymph nodes (P < 0.05) all increased significantly in the rats 24 h after LPS injection; IFN-γ/IL-4 ratio increased progressively within 6 h after LPS injection and then subsided gradually. Compared with those in the corresponding sepsis model subgroups, the levels of S1P, IL-12 and IFN-γ increased while IL- 4 level decreased significantly (P < 0.05), and the expression of S1PR1 and CD103 were reduced significantly (P < 0.05) in SEW2871-treated rats; both the plasma level of IL-4 and the expression of S1PR1 in the mesenteric lymph nodes increased significantly in PCA-treated rats (P < 0.05). PCA can regulate the balance of inflammation and immune response in the early stage of sepsis in rats possibly through the S1P-S1PR1 pathway.

Novel TLR 7/8 agonists for improving NK cell mediated antibody-dependent cellular cytotoxicity (ADCC)

There is a significant interest in designing therapeutic agents that can enhance ADCC and thereby improve clinical responses with approved antibodies. We recently reported the combination of an imidazoquinoline-based TLR7/8 agonist (522) with a monoclonal antibody improved ADCC in vitro and in vivo. In the present study, we tested several new small molecule TLR7/8 agonists that induce significantly higher cytokines compared to both the FDA-approved TLR7 agonist, imiquimod, and 522. We evaluated these agonists in combination with monoclonal antibody therapy, with the main goal of enhancing ADCC. Our studies show these TLR7/8 agonists induce robust pro-inflammatory cytokine secretion and activate NK cells. Specifically, we found the agonists 574 and 558 significantly enhanced NK cell-mediated ADCC in vitro as well as enhanced the anti-cancer efficacy of monoclonal antibodies in two different in vivo mouse models. Additionally, we found the agonists were able to stimulate CD8 T cells, likely indicative of an early adaptive immune response.

Elucidating the Functional Roles of Helper and Cytotoxic T Cells in the Cell-Mediated Immune Responses of Olive Flounder (Paralichthys olivaceus).

In higher vertebrates, helper and cytotoxic T cells, referred to as CD4 and CD8 T lymphocytes, respectively, are mainly associated with adaptive immunity. The adaptive immune system in teleosts involves T cells equivalent to those found in mammals. We previously generated monoclonal antibodies (mAbs) against olive flounder (Paralichthys olivaceus) CD4 T cells, CD4-1 and CD4-2, and used these to describe the olive flounder’s CD4 Tcell response during a viral infection. In the present study, we successfully produced mAbs against CD8 T lymphocytes and their specificities were confirmed using immuno-blotting, immunofluorescence staining, flow cytometry analysis andreverse transcription polymerase chain reaction (RT-PCR). The results showed that these mAbs are specific for CD8 T lymphocytes. We also investigated variations in CD4 and CD8 T cells populations, and analyzed the expression of immune-related genes expressed by these cells in fish infected with nervous necrosis virus or immunized with thymus dependent and independent antigens. We found that both CD4 and CD8 T lymphocyte populations significantly increased in these fish and Th1-related genes were up-regulated compared to the control group. Collectively, these findings suggest that the CD4 and CD8 T lymphocytes in olive flounder are similar to the helper and cytotoxic T cells found in mammals, and Th1 and cytotoxic immune responses are primarily involved in the early adaptive immune response against extracellular antigens.

Early T Cell and Binding Antibody Responses are Associated with COVID-19 RNA Vaccine Efficacy Onset

RNA vaccines against Covid-19 have demonstrated ~95% efficacy in Phase III clinical trials. Although complete vaccination consisted of two-doses, the onset of protection for both licensed RNA vaccines was observed as early as 12 days after a single dose. The adaptive immune response that coincides with this onset of protection could represent the necessary elements of immunity against Covid-19. Herein, we tracked the early adaptive immune responses after Covid-19 RNA vaccination, in a cohort of 20 healthcare workers. Our findings suggest that early T cell and binding antibody responses, rather than either receptor-blocking or virus neutralizing activity, induced early protection against Covid-19.Funding: This study was partially funded through a generousdonation from The Hourglass to support Covid-19 research in ViREMiCS. SK receives salary support from the Transition Award, RdA receives funding from the Open Research Fund Young Investigator Award, JGL and EEO receive salary support from the Clinician Scientist Award, and AB receives salary support from the Singapore Translational Research Award, all administered by the National Medical Research Council of Singapore.Conflict of Interest: Duke-NUS Medical School is in partnership with Arcturus Therapeutics to develop a self-replicating RNA vaccine against Covid-19, with EEO as the principal investigator. No monetary or personal benefits are derived from this partnership.Ethical Approval: This study was approved by the SingHealth Centralized Institutional Review Board (CIRB/F2021/2014). Healthcare workers (HCWs) from the Singapore Health Services institutions whowere eligible for Covid-19 vaccination were invited to participate in this study, and written informed consent was obtained.

Transcriptome Analysis Shows That IFN-I Treatment and Concurrent SAV3 Infection Enriches MHC-I Antigen Processing and Presentation Pathways in Atlantic Salmon-Derived Macrophage/Dendritic Cells.

Type I interferons (IFNs) have been shown to play an important role in shaping adaptive immune responses in addition to their antiviral properties in immune cells. To gain insight into the impact of IFN-I-induced pathways involved in early adaptive immune responses, i.e., antigen-presenting pathways, in an Atlantic salmon-derived (Salmo salar L.) macrophage cell line (TO-cells), we used a comparative de novo transcriptome analysis where cells were treated with IFN-I or kept untreated and concurrently infected with salmonid alphavirus subtype 3 (SAV3). We found that concurrent treatment of TO-cells with IFN-I and SAV3 infection (SAV3/IFN+) significantly enriched the major histocompatibility complex class I (MHC-I) pathway unlike the non-IFN-I treated TO-cells (SAV3/IFN−) that had lower expression levels of MHC-I pathway-related genes. Genes such as the proteasomal activator (PA28) and β-2 microglobulin (β2M) were only differentially expressed in the SAV3/IFN+ cells and not in the SAV3/IFN− cells. MHC-I pathway genes like heat shock protein 90 (Hsp90), transporter of antigen associated proteins (TAPs) and tapasin had higher expression levels in the SAV3/IFN+ cells than in the SAV3/IFN− cells. There were no MHC-II pathway-related genes upregulated in SAV3/IFN+-treated cells, and cathepsin S linked to the degradation of endosomal antigens in the MHC-II pathway was downregulated in the SAV3/IFN− cells. Overall, our findings show that concurrent IFN-I treatment of TO-cells and SAV3 infection enriched gene expression linked to the MHC-I antigen presentation pathway. Data presented indicate a role of type I IFNs in strengthening antigen processing and presentation that may facilitate activation particularly of CD8+ T-cell responses following SAV3 infection, while SAV3 infection alone downplayed MHC-II pathways.

Formation of Human T Cell Mucosal Resident Memory Starts in Early Life with the Intestines

Abstract Memory T cells are essential for optimal protection from previously encountered pathogens. Immune responses in infants lack the benefit of memory and are intrinsically distinct from those in adults. Early life adaptive immune responses have been characterized by increased generation of terminal effector subsets with decreased production of long lived memory. Here we investigate the origin of memory T cells utilizing flow cytometry and immunohistochemistry to characterize proportions and localization of T cell subsets in mucosal (intestine/lung) tissue from infant organ donors (n=12). At birth lung T cells are predominantly naïve and localized to the interstitium of the airways with memory accumulating over the first year of life. In contrast, significant proportions of memory phenotype T cells can be found in the lamina propria and intraepithelial layer of the intestine shortly after birth along with naïve cells. Effector memory subsets could be detected in robust proportions, highest in the jejunum, from 3 organ donors all less than 8 days of age. Additionally these memory T cells displayed high levels of canonical resident memory markers (CD69 and CD103). We conclude that mucosal tissues are host to naïve cells which cannot be attributable to circulating populations and that memory subsets are generated within these mucosal sites early in life. Importantly T cells in the youngest donors (ages 0–8 days) displayed resident memory phenotypes raising the possibility of prenatal development of adaptive memory responses populating the intestine. These results warrant additional investigation to determine the persistence and functionality of these subsets and emphasize the importance of the intestine to early life adaptive immunity.

HIV Subtype and Nef-Mediated Immune Evasion Function Correlate with Viral Reservoir Size in Early-Treated Individuals.

The HIV accessory protein Nef modulates key immune evasion and pathogenic functions, and its encoding gene region exhibits high sequence diversity. Given the recent identification of early HIV-specific adaptive immune responses as novel correlates of HIV reservoir size, we hypothesized that viral factors that facilitate the evasion of such responses-namely, Nef genetic and functional diversity-might also influence reservoir establishment and/or persistence. We isolated baseline plasma HIV RNA-derived nef clones from 30 acute/early-infected individuals who participated in a clinical trial of early combination antiretroviral therapy (cART) (<6 months following infection) and assessed each Nef clone's ability to downregulate CD4 and human leukocyte antigen (HLA) class I in vitro We then explored the relationships between baseline clinical, immunological, and virological characteristics and the HIV reservoir size measured 48 weeks following initiation of suppressive cART (where the reservoir size was quantified in terms of the proviral DNA loads as well as the levels of replication-competent HIV in CD4+ T cells). Maximal within-host Nef-mediated downregulation of HLA, but not CD4, correlated positively with post-cART proviral DNA levels (Spearman's R = 0.61, P = 0.0004) and replication-competent reservoir sizes (Spearman's R = 0.36, P = 0.056) in univariable analyses. Furthermore, the Nef-mediated HLA downregulation function was retained in final multivariable models adjusting for established clinical and immunological correlates of reservoir size. Finally, HIV subtype B-infected persons (n = 25) harbored significantly larger viral reservoirs than non-subtype B-infected persons (2 infected with subtype CRF01_AE and 3 infected with subtype G). Our results highlight a potentially important role of viral factors-in particular, HIV subtype and accessory protein function-in modulating viral reservoir establishment and persistence.IMPORTANCE While combination antiretroviral therapies (cART) have transformed HIV infection into a chronic manageable condition, they do not act upon the latent HIV reservoir and are therefore not curative. As HIV cure or remission should be more readily achievable in individuals with smaller HIV reservoirs, achieving a deeper understanding of the clinical, immunological, and virological determinants of reservoir size is critical to eradication efforts. We performed a post hoc analysis of 30 participants of a clinical trial of early cART who had previously been assessed in detail for their clinical, immunological, and reservoir size characteristics. We observed that the HIV subtype and autologous Nef-mediated HLA downregulation function correlated with the viral reservoir size measured approximately 1 year post-cART initiation. Our findings highlight virological characteristics-both genetic and functional-as possible novel determinants of HIV reservoir establishment and persistence.

Induction of protective immunity by recombinant peptidoglycan associated lipoprotein (rPAL) protein of Legionella pneumophila in a BALB/c mouse model

Legionella pneumophila causes a severe form of pneumonia known as Legionnaires’ disease especially in patients with impaired cellular immune response. In order to prevent the disease, immunogenicity and the level of the induction of protective immunity from the recombinant peptidoglycan–associated lipoprotein (rPAL) against Legionella pneumophila in BALB/c mice was examined. Mice immunized with (rPAL) rapidly increased an antibody response in serum and also displayed a strong activation of both innate and adaptive cell-mediated immunity as determined by antigen-specific splenocyte proliferation, an early production of pro-inflammatory cytokines in the serum and in the splenocyte cultures. Infection with a primary sublethal does of Legionella pneumophila serogroup 1, strain paris, caused resistance to a lethal challenge infection in the animals with 100% survival rate. However, mice treated with rPAL survived with 60% rate in 10 days after a lethal i.v challenge with L. pneumophila. All of the control animals receiving PBS died within 24 h. The present study indicates that recombinant protein PAL of Legionella pneumophila is strongly immunogenic and capable to elicit early innate and adaptive immune responses and lasting immunity against a lethal dose of Legionella pneumophila challenge. Antigenic characterization and immune protection of recombinant protein PAL would be of considerable value in comprehension the immune-pathogenesis of the disease and in development possible vaccine against the Legionella.