Seronegative Donors Research Articles

Genetic diversity of the human immunodeficiency virus of type 1 in Gabonese transfusional settings

BackgroundThe high endemicity of transfusion-transmissible infections (TTIs) in sub-Saharan Africa is a real public health problem. To reduce the risk of HIV transmission through blood donation, the NBTC of Gabon has launched in recent years a reorganization of its blood transfusion system. This study aims to characterize the molecular strains of HIV-1 circulating in donors and to estimate the risk of viral transmission.Materials and methodsA cross-sectional study was carried out during the period from August 2020 to August 2021 among 381 donors who had agreed to donate blood at the National Blood Transfusion Center (NBTC). Viral load was determined by Abbott Real-Time (Abbott m2000®, Abbott) and sequencing by the Sanger method (ABI 3500 Hitachi®). The phylogenetic tree was constructed by MEGA X software. Data were checked, entered, and analyzed using SPSS version 21.0 software, with p ≤ 0.05 considered statistically significant.ResultsA total of 381 donors were enrolled in the study. Among the 359 seronegative donors, five (5) seronegative donors were detected positive for HIV-1 using Real-Time PCR. The residual risk was 648 per 1,000,000 donations. The prevalence of residual infection was 1.4% [0,01; 0,03]. Sixteen (16) samples were sequenced. The strains obtained were CRF02_AG (50%), subtype A1 (18.8%), subtype G (12.5%), CRF45_cpx (12.5%) and subtype F2 (6.2%). Six sequences clustered with A1, G, CRF02_AG, and CRF45_cpx subtypes.ConclusionThe residual risk of HIV-1 transmission by blood transfusion remains a concern in the Gabonese transfusional settings. A policy based on improving the current screening strategy would involve the implementation of the nucleic acid test (NAT) in order to optimize the safety of the donation by detecting the HIV-1 subtypes in circulation in the donors.

Effectiveness of Using Nucleic Acid Amplification Test to Screen Blood Donors for Hepatitis B, Hepatitis C, and HIV: A Tertiary Care Hospital Experience From Pakistan.

Background Ensuring blood safety is the primary goal of transfusion medicine. Despite extensive serological tests and strict safety measures, the risk of transfusion-transmitted infections (TTIs) still exists. As applied to blood screening, Nucleic Acid Amplification Test (NAT)offers much higher sensitivity for detecting viral infections. It is, however, currently available to a handful of centers due to the high cost. This study aims to establish the Effectiveness of NAT by assessing the NAT yield and residual risk of transmission of Hepatitis B virus (HBV), hepatitis C virus (HCV)and HIV with and without NAT testing. Material and method This prospective cross-sectional study recruited blood donors from January 2020 to November 2022. All donors underwent routine serologic screening. Only serologically negative donors were tested for HBV, HCV, and HIV by NAT. The NAT yield and residual risk (RR) per million donors were computed for viral infections in seronegative blood donors and calculated using the incidence/window period model. Result A total of 59708 donors were included during the study period. The overall prevalence of TTI's were: For HCV 1.7% (n = 1018), HBV 1.5% (n = 918), HIV 0.07% (n = 47), Syphilis 1.2% (n = 758) and malaria 0.3% (n = 218). Out of 57759 seronegative donors, thirty-four NAT-reactive samples were identified, with 3 cases of HCV, 31 cases of HBV, and Nil HIV cases. NAT yield of HBV was 1 in 1863 with an RR of 8.6 per million, followed by HCV with a NAT yield of 1 in 19253 and RR of 0.8 per million donations. NAT testing reduced RR for HBV by 48.9% and HCV by 94.5%. Conclusion Our study showed that NAT detected 34 out of 57759 cases initially missed by serological tests. The study suggests that the parallel use of serology and NAT screening of donated blood would be beneficial.

1589. Long term hematopoietic cell transplant outcomes in patients at high risk for cytomegalovirus infections in the era of letermovir

Abstract Background Letermovir (LTV) primary prophylaxis (PP) has reduced the incidence of clinically significant cytomegalovirus infection (CS-CMVi) in allogeneic hematopoietic cell transplant (allo-HCT) recipients. However, it is not clear if these are similar to low-risk recipients (seronegative donor [D-] and recipient [R-]). Prior studies have reported that D-/R- allo-HCT recipients have reduced relapse rates, graft versus host disease (GVHD), and overall mortality. We compared the clinical outcomes of R+ allo-HCT recipients with and without LTV PP to D-/R- allo-HCT recipients. Methods This is a single-center study of allo-HCT recipients who underwent transplantation between March 2016 and December 2018. Data on baseline and transplant characteristics were collected. Outcomes included incidence of CS-CMVi, GVHD, all-cause mortality, relapse and NRM. Univariate analysis was performed to compare outcomes in R+ with and without LTV PP cohorts to D-/R- cohort. Categorical and continuous variables were compared using Fisher’s exact test and Wilcoxon rank sum respectively. Survival curves were compared with the log-rank test. Patients who relapsed after transplant were excluded from NRM analysis. Results A total of 616 patients underwent an allo-HCT; 124 R+ were on LTV PP, 415 were R+ on no LTV PP as well as the 77 who were CMV D-/R-. When compared to D-/R- group, R+ group who received or not LTV PP were more likely to have AML and ALL, and underwent myeloablative conditioning. The incidence of CS-CMVi was the lowest in the D-/R- group (4%), followed by the R+ on LTV PP (17%), and the highest in the R+ group on no LTV PP (53%) (table 2). Kaplan-Meier survival analysis for NRM 1 year after transplant showed trends towards lower survival in R+ allo-HCT recipients who did not receive LTV PP when compared to the D-/R- group at day 200 and day 360 (p=0.07 vs. p=0.08, respectively). In addition, R+ allo-HCT on LTV PP had similar NRM at day 360 compared to D-/R- (p=0.77) and lower NRM at day 360 compared to R+ allo-HCT on no LTV PP (p=0.0192). Conclusion Our study showed a reduction in the rate of CS-CMVi within 48 weeks post HCT in high risk allo-HCT recipients on LTV. NRM was lower in R+ allo-HCT recipients on LTV PP when compared to those without LTV. No difference in NRM was seen between D-/R- allo-HCT and R+ allo-HCT on LTV PP. Disclosures Terri Lynn Shigle, PharmD, BCOP, Takeda: Advisor/Consultant Gabriella Rondon, MD, Omeros: Advisor/Consultant Elizabeth Shpall, MD, Adaptimmune: Advisor/Consultant|Affimed: License agreement|Axio: Advisor/Consultant|Bayer Helathcare Pharmaceuticals: Honoraria|Fibroblasts and FibrioBiologics: Advisor/Consultant|Navan: Advisor/Consultant|NY Blood Center: Advisor/Consultant|Takeda: License agreement Ella J. Ariza Heredia, MD, Merck & Co. Inc: Grant/Research Support Roy F. Chemaly, MD/MPH, Karius: Advisor/Consultant|Karius: Grant/Research Support.

2121. The Impact of Donor CMV Serostatus on Outcomes of CMV Infections in the Era of Letermovir

Abstract Background Cytomegalovirus (CMV) infection is a frequent complication after allogeneic hematopoietic cell transplant (allo-HCT) and may increase the risk of other viral infections through its immunomodulatory effects. Donor CMV serology (seropositive [D+] or seronegative donor [D-]) in CMV-seropositive (R+) or seronegative recipients (R-) may impact outcomes post allo-HCT. We analyzed the significance of donor CMV serostatus in a large cohort of alloHCT recipients. Methods This is a single-center, retrospective cohort study of 651 allo-HCT recipients cared for at our institution between March 2016 and December 2018. Data on baseline demographics, transplant characteristics, preventive strategies, CMV infection characteristics, and transplant-related outcomes (development of graft versus host disease (GVHD), all-cause mortality, and non-relapse mortality) were collected. A univariate analysis was performed for outcomes of interest using CMV serostatus D-/R- as a control group. Results Out of the 651 allo-HCT recipients, 77 were D-/R-, 43 D+/R-, 290 D+/R+, and 241 D-/R+ (table 1). Most patients underwent HCT for AML (40%), received myeloablative conditioning (51%), and had a matched unrelated donor (MUD) HCT (46%). In 2018, letermovir was used in 27% of the D+/R+, 18% of the D-/R+ allo-HCT recipients (table 1) for a total of 116 (55%) allo-HCT recipients. Compared to the CMV D-/R- group, D+/R+ and D-/R+ groups (table 2) had a greater incidence of clinically signicant CMV infections (CS-CMVi) (3.9% vs. 40% vs. 50.6%; all p< 0.01, respectively), CMV end organ disease (0% vs. 14.8% vs. 19.1%; all p< 0.001, respectively), and refractory/resistant (R/R) CMV infections (0% vs. 5.5% vs. 12.4%; all p< 0.03, respectively) with 48 weeks of allo-HCT. CS-CMVi and R/R CMV was more common in D-/R+ allo-HCT when compared to D+/R+ group (50.6% vs. 40.0%, p< 0.001). D-/R+ allo-HCT had worse non-relapse mortality at day 100 compared to D-/R- (3.9% vs. 10.8%, p=0.07). Table 1:Baseline characteristicsTable 2:Outcome analysis Conclusion Allo-HCT recipients with CMV seronegative donor and recipient had less CMV related complications and a trend towards better survival when compared to D-/R+ allo-HCT. CMV D-/R+ HCT recipients had greater CMV related complications when compared to CMV D+/R+ HCT recipients, possibly due to the protective affect of donor seropositivity. Disclosures Terri Lynn Shigle, PharmD, BCOP, Takeda: Advisor/Consultant Gabriella Rondon, MD, Omeros: Advisor/Consultant Elizabeth Shpall, MD, Adaptimmune: Advisor/Consultant|Affimed: License agreement|Axio: Advisor/Consultant|Bayer Helathcare Pharmaceuticals: Honoraria|Fibroblasts and FibrioBiologics: Advisor/Consultant|Navan: Advisor/Consultant|NY Blood Center: Advisor/Consultant|Takeda: License agreement Ella Ariza-Heredia, MD, MERCK: Grant/Research Support Roy F. Chemaly, MD/MPH, Karius: Advisor/Consultant|Karius: Grant/Research Support.

Sars-Cov-2-Specific CD4+ and CD8+ T Cell Responses Can Originate from Cross-Reactive CMV-Specific T Cells

Introduction: SARS-CoV-2-specific CD4+ and CD8+ T cells play an important role in COVID-19 pathogenesis. In addition to de novo SARS-CoV-2-specific T cell responses in infected individuals, SARS-CoV-2-specific T cells have also been identified in samples frozen down before the pandemic. So far, pre-existing SARS-CoV-2 specific T cell responses has mostly been ascribed to cross-reactive T cells that were initially primed by other coronaviruses. The presence of these cross-reactive T cells is not surprising considering these human coronaviruses share a relatively high sequence homology. However, T cells are also able to cross-react towards dissimilar pathogens, called heterologous immunity. Given the high prevalence and magnitudes of pre-pandemic SARS-CoV-2-specific T cells, we hypothesized that pre-pandemic SARS-CoV-2 T cell immunity could be explained by cross-reactive cytomegalovirus (CMV)-specific T cells. Methods & Results: PBMCs from CMV seropositive and seronegative donors that were cryopreserved before the pandemic were incubated with SARS-CoV-2 peptide pools. To detect SARS-CoV-2-specific T cells using flow cytometry, CD4+ T cell responses were identified by upregulation of CD154 and CD137 whereas CD8+ T cell responses were identified by upregulation of CD137 and IFN-γ production. In the CMV seropositive group, clear CD4+ T cell responses against SARS-CoV-2 membrane and CD8+ T cell responses against SARS-CoV-2 spike were detected whilst this was not observed in CMV seronegative donors. To investigate whether these T cells were reactive against both SARS-CoV-2 and CMV, the SARS-CoV-2 reactive T cells were single cell sorted, clonally expanded, and tested in co-culture assays in which stimulator cells expressing the relevant human leukocyte antigen (HLA) alleles were pulsed with overlapping peptide pools, peptide libraries or single peptides. The results demonstrated that the pre-pandemic SARS-CoV-2 membrane-specific CD4+ T cells recognized both SARS-CoV-2 membrane and CMV-pp65 peptide pools in HLA-DRB3*02:02, and specifically recognized AGILRNLVPM peptide of CMV-pp65. The pre-pandemic SARS-CoV-2 spike-specific CD8+ T cells recognized spike peptide FVSNGTHWF (FVS) and, interestingly, dissimilar CMV-pp65 peptide IPSINVHHY (IPS) both presented by HLA-B*35:01. Importantly, these FVS/IPS-cross-reactive T cells displayed a higher avidity for CMV compared to SARS-CoV-2, which illustrates that these cells were originally primed against CMV since repeated antigen exposure results in a selection for high avidity T cell clonotypes. This selection cannot have occurred in response to SARS-CoV-2-specific peptides in SARS-CoV-2-unexposed individuals. PBMCs from pre-pandemic CMV-seropositive donors that express HLA-B*35:01 were screened for the presence of these dual IPS/FVS-specific CD8+ T cells by peptide-HLA tetramer technology. In multiple donors these cross-reactive T cells were detected, and isolation followed by direct T cell receptor (TCR) sequencing of the IPS/FVS-tetramer positive cells showed that these T cells express an amino acid-identical TCR, illustrating that this SARS-CoV-2/CMV cross-reactivity of the IPS/FVS T cells is caused by a public TCR. To investigate the potential of the IPS/FVS-specific T cells to kill virus-infected cells, and thereby reducing spreading of SARS-CoV-2, the T cells were incubated with SARS-CoV-2-infected airway epithelial Calu-3 cells. Intracellular SARS-CoV-2 RNA was reduced at low virus titers in IPS/FVS-specific T cell co-cultures, indicating that the cross-reactive T cells are killing SARS-CoV-2-infected cells and thereby reduce spreading of SARS-CoV-2 during initial infection. Circulating IPS/FVS-specific T cells were not phenotypically activated in two patients with advanced COVID-19, as measured by flow cytometry analysis of CD38 and HLA-DR expression, indicating that cross-reactive T cells may more likely play a role during early stage of infection. Conclusion: CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-pandemic immunity against SARS-CoV-2. Pre-print link: https://www.biorxiv.org/content/10.1101/2022.07.31.502203v1

Letermovir Prophylaxis for High-Risk Allogenic Transplantation Recipients, Real- World Israeli Experience

Introduction: Cytomegalovirus (CMV) infection is a common complication after allogeneic hematopoietic-cell transplantation (alloHCT) causing significant morbidity and mortality. The standard approach for preventing clinically significant CMV infection (CS-CMVi) and CMV disease (CMVd), is preemptive therapy (PET) upon detection of CMV reactivation. In 2017 letermovir was approved for CMV prophylaxis in CMV-seropositive allogeneic HCT recipients from transplantation through day 100 after alloHCT. At 2020, letermovir was approved in Israel for prophylaxis in CMV-seropositive alloHCT recipients who were at higher risk for CMV reactivation (seronegative donor, HLA mismatched unrelated donor, haploidentical family donor, ex-vivo T cell depleted graft or recipient suffering from acute Graft Versus Host Disease (aGVHD) requiring >1mg/kg steroid therapy). There is paucity of real-world data regarding the efficacy and outcomes of letermovir prophylaxis in the post-transplant setting. The aim of our study was to report the real-world Israeli experience with letermovir prophylaxis in terms of incidence of CS-CMVi, CMVd and aGVHD, chronic Graft Versus Host Disease (cGVHD), survival and all-cause mortality. Methods: We retrospectively analyzed a cohort of 45 alloHCT recipients at high risk for CMV reactivation, from 4 Israeli transplant centers, who underwent alloHCT between January 2020 and -December 2021 and received letermovir prophylaxis (study group). A comparison was performed, to a matched historical cohort of 45 alloHCT recipients, who underwent alloHCT between 2014 and 2020 and were treated according to PET policy (control group). Matching was guided by the risk for CMV reactivation. Results: Demographics (recipient's age, gender, ethnicity), disease and pre-transplant characteristics (disease status at transplantation, donor match, conditioning intensity, graft source) of the two groups were comparable accept for a higher percentage of ABO minor mismatch in the control group versus the study group (37% compared to 6%, respectively; p=0.028). There was a higher rate of CS-CMVi during the first 100 days post-transplant in the control group versus the study group (53/3% compared to 35.6%, respectively; p=0.09). There was no difference in CMVd rate between the two groups as well as in relapse rate and overall survival. Acute and chronic GVHD incidence was higher in the control group compared to the study group (71.1% vs 60%; p=0.186 and 57.8% vs 33.3%; p= 0.02, respectively). Conclusions: Our real -world Israeli experience cohort of letermovir prophylaxis in high risk alloHCT recipients demonstrates a significant reduction in GVHD occurrence in patients receiving letermovir prophylaxis. GVHD is a significant cause of morbidity and mortality in alloHCT recipients. Our real-world experience is in line with the well-established contiguity and causal relation between CMV infection and GVHD, showing a reduction in CS-CMVi during the prophylaxis period with a corresponding reduction in acute and chronic GVHD. Our cohort is limited by its retrospective nature and a relatively small sample size. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Seroprevalence and systemic immune biomarkers associated with Toxoplasma gondii infection in blood donors from Southern Brazil

The high seroprevalence of Toxoplasma gondii infection in Blood Banks could be a potential risk for contamination of blood recipients. The discovery of new biomarkers may help to distinguish between seropositive and seronegative donors. This study determined the seroprevalence and profile of systemic immune biomarkers associated with Toxoplasma gondii infection among blood donors from Southern Brazil. Peripheral blood was collected from 510 blood donors (52.2 % male; mean age: 36.61), 310, and 200 from Erechim, and Chapecó municipalities, respectively. Specific Toxoplasma gondii IgG and IgM antibodies were detected by Eletrochemioluminescence. Nested PCR and qPCR were performed to detectToxoplasma gondii DNA. Twenty-seven inflammatory factors were analyzed using a high-performance Luminex assay. Among 310 blood donors from Erechim, 44.5 % (138/310) were IgM(−)/IgG(+), and 1.3 % (4/310) were IgM(+)/IgG(+), while out of 200 blood donors from Chapeco, 42.5 % (85/200) were IgM(−)/IgG(+), and 2 % (4/200) were IgM(+)/ IgG(+). We did not find Toxoplasma gondii DNA in the samples analyzed by Nested PCR and qPCR.Additionally, IgM(−)/IgG(+) donors presented higher levels ofdistinct systemic mediators, and were indicated to be high producers of several systemic mediators (CCL11, CCL2, CCL3, CCL4, CXCL10, IL-1β, IL-17, IFN-γ, IL-4, IL-9, IL-13, IL-10, IL-1Ra, vascular endothelial growth factor/VEGF, platelet-derived growth factor/PDGF, granulocyte–macrophage colony-stimulating factor/GM-CSF, and IL-7). However, IgM(+)/IgG(+) donors were found as high producers of CXCL8, CXCL10, CCL4, IL-1β, IL-1Ra, IL-9, IL-13, and PDGF, while IgM(−)/IgG(−) donors showed unaltered levels for the most soluble mediators evaluated. These distinct biomarker signatures might help identify potential factors to distinguish between IgM(−) and IgM(+) donors.

Risk factor analysis for cytomegalovirus reactivation under prophylaxis with letermovir after allogeneic hematopoietic stem cell transplantation.

Letermovir has been approved as a novel cytomegalovirus (CMV) prophylactic agent after allogeneic hematopoietic stem cell transplantation (HSCT). However, there are still insufficient data to properly evaluate the real-world role of letermovir, and the risk factors for CMV reactivation under letermovir prophylaxis have not been clarified. We performed a single-institution retrospective analysis of patients under prophylaxis with or without letermovir who underwent allogeneic HSCT between March 2012 and December 2019. In August 2018, letermovir was added to the clinical practice at our institution for the prophylaxis of CMV reactivation in allogeneic HSCT recipients. Patients who underwent HSCT without prophylactic letermovir from March 2012 until September 2018 served as a historical control. The cumulative incidence of clinically significant CMV infection (CS-CMVi) was significantly lower in the letermovir group than in the historical control group not receiving letermovir (30.2%vs. 71.6%, p < .05, at 100 days). In addition, the cumulative incidence of non-relapse mortality (NRM) at day 500 was significantly lower in the letermovir group (4.7%vs. 19.8%, p < .05). We then performed a risk factor analysis for developing CS-CMVi in the letermovir group. The only significant factor identified by this multivariable analysis was transplantation from a CMV seronegative donor to a seropositive recipient (Hazard ratio = 2.76, 95% confidence interval 1.14-6.68, p < .05). Our study showed that letermovir prophylaxis significantly reduced the incidence of CS-CMVi and NRM in a real-world setting and that the CMV serostatus of the donor remained as a risk factor for CS-CMVi even under letermovir prophylaxis.

Functional Restoration of Exhausted CD8 T Cells in Chronic HIV-1 Infection by Targeting Mitochondrial Dysfunction.

CD8 T cell exhaustion is a hallmark of HIV-1 infection, characterized by phenotypic and functional CD8 T cell abnormalities that persist despite years of effective antiretroviral treatment (ART). More recently, the importance of cellular metabolism in shaping T cell antiviral function has emerged as a crucial aspect of immunotherapeutics aimed at re-invigorating exhausted CD8 T cells but remains under-investigated in HIV-1 infection. To gain a better insight into this process and identify new targets for effective CD8 T cell restoration we examined the metabolic profile of exhausted CD8 T cells in HIV-1 infection. We show that relative to HIV-1 elite controllers (EC) and HIV-1 seronegative donors, CD8 T cells from HIV-1 viraemic individuals are skewed toward a PD-1hiEOMEShiT-betlowTIGIT+ phenotype that is maintained during ART. This exhausted signature is enriched in HIV-specific CD8 T cells, compared to CMV-specific CD8 T cell populations, and further delineated by higher expression of the glucose transporter, Glut-1, impaired mitochondrial function and biogenesis, reflecting underlying metabolic defects. A notable improvement in antiviral HIV-specific CD8 T cell function was elicited via mitochondrial antioxidant treatment in combination with pharmacological modulation of mitochondrial dynamics and IL-15 treatment. These findings identify mitochondria as promising targets for combined reconstitution therapies in HIV-1 infection.

Anatomy of Omicron BA.1 and BA.2 neutralizing antibodies in COVID-19 mRNA vaccinees

SARS-CoV-2 vaccines, administered to billions of people worldwide, mitigate the effects of the COVID-19 pandemic, however little is known about the molecular basis of antibody cross-protection to emerging variants, such as Omicron BA.1, its sublineage BA.2, and other coronaviruses. To answer this question, 276 neutralizing monoclonal antibodies (nAbs), previously isolated from seronegative and seropositive donors vaccinated with BNT162b2 mRNA vaccine, were tested for neutralization against the Omicron BA.1 and BA.2 variants, and SARS-CoV-1 virus. Only 14.2, 19.9 and 4.0% of tested antibodies neutralize BA.1, BA.2, and SARS-CoV-1 respectively. These nAbs recognize mainly the SARS-CoV-2 receptor binding domain (RBD) and target Class 3 and Class 4 epitope regions on the SARS-CoV-2 spike protein. Interestingly, around 50% of BA.2 nAbs did not neutralize BA.1 and among these, several targeted the NTD. Cross-protective antibodies derive from a variety of germlines, the most frequents of which were the IGHV1-58;IGHJ3-1, IGHV2-5;IGHJ4-1 and IGHV1-69;IGHV4-1. Only 15.6, 20.3 and 7.8% of predominant gene-derived nAbs elicited against the original Wuhan virus cross-neutralize Omicron BA.1, BA.2 and SARS-CoV-1 respectively. Our data provide evidence, at molecular level, of the presence of cross-neutralizing antibodies induced by vaccination and map conserved epitopes on the S protein that can inform vaccine design.

Broadly recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles.

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can affect T cell response in COVID-19. We studied T cell responses to SARS-CoV-2 and HCoVs in convalescent COVID-19 donors and identified a highly conserved SARS-CoV-2 sequence, S811-831, with overlapping epitopes presented by common MHC class II proteins HLA-DQ5 and HLA-DP4. These epitopes are recognized by low-abundance CD4 T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. T cell cross-reactivity is driven by the high conservation across human and animal coronaviruses of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC class II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly conserved CD4 T cell epitope broadly recognized across human populations.

Identification of CPAF as the immunoprevalent antigen of Chlamydia trachomatis

Abstract Chlamydia trachomatis (CT) is a common sexually transmitted bacterial infection, that in women can cause pelvic inflammatory disease and infertility. No preventative vaccine has been developed against CT. Immunity to CT is primarily mediated by Th1 CD4+ T cells. We are defining immunoprevalent CT proteins in a well-defined cohort of CT seropositive women with the goal of defining novel vaccine immunogens. We screened 30 women one month after a CT-positive test by cultured IFN-γ ELISpot. Ten-day short-term cell lines (STCL) were generated against overlapping peptides spanning 21 CT antigens. The threshold for a positive CT-specific T cell response (≥ 300 spot-forming cells, SFU, per 106 cells) was defined following a screening of 12 CT seronegative donors. CT− specific T cell responses were detected in 27/30 CT-seropositive women. On average, women harbored T cell responses to two CT proteins (range 0–6). Strikingly, CT858 (CPAF) elicited a T cell response in 16/30 women with an average of 966 IFN-g SFU/106 cells (range 300–3,633 SFU/106 cells). Data to date suggest CPAF-specific T cell responses are predominantly CD4-restricted. In preliminary studies, we have also detected CT-specific T cell responses in men with documented CT infection, including a T cell response to CPAF. We are currently mapping CPAF T cell epitopes and expanding our screen to other CT secreted proteins. In summary, CT858 (CPAF) is an immunoprevalent antigen in women and a promising vaccine immunogen. Supported by UNC Chlamydia Vaccine Initiative (U19AI144181)

Primary Cytomegalovirus Infection in a Low-Risk Lung Transplant Recipient Manifesting as a Pleural Effusion

Introduction Community-acquired Cytomegalovirus (CMV) infection in a seronegative transplant recipient (R) from a seronegative donor (D) is a rare occurrence that carries significant clinical and prognostic implications. Few case reports exist describing this entity in lung transplant recipients. Case Report A 58-year-old man with bilateral lung transplant for sarcoidosis presented with three days of diarrhea and dyspnea. He underwent an uneventful bilateral lung transplantation (CMV D-/R-) six weeks prior, receiving basiliximab and methylprednisolone for induction. He was discharged two weeks later on tacrolimus, mycophenolate motefil, and prednisone taper as maintenance immunosuppression. He was receiving acyclovir for herpes viruses prophylaxis. He was seen weekly post-discharge and continued to have clear chest radiographs and unremarkable bloodwork. On presentation, his physical examination was notable for decreased breath sounds at the right base. His laboratory values revealed creatinine of 2.4 mg/dL. His chest radiograph showed new right pleural effusion. He was admitted for hydration and diarrhea work up. Abdominal computed tomography (CT) revealed mild diverticulitis with no colitis and his stool studies were positive for Clostridium difficile. Chest CT showed hazy and linear markings with thin-walled cysts in right lower lobe, adjacent to a moderate pleural effusion. CMV by polymerase chain reaction resulted at 318,200 copies/mL. He was treated with intravenous ganciclovir and underwent a thoracenthesis. Half a liter of clear pleural fluid was removed and was notable for lymphocytic predominance of 72% as well as polytypic plasma cells and a small number of B lymphocytes with no surface immunoglobulins on flow cytometry. Subsequent radiograph showed completely re-expanded lung. Within two days, the effusion re-accumulated and additional half a liter were drained, revealing of 95% lymphocytes, with complete re-expansion of the lung. Concomitant viral load remained elevated at 150,328 copies/mL. He was discharged on valganciclovir, his viral load decreased to an undetectable level, and his radiographs have remained free of effusion. Summary While primary CMV infection is rare in low-risk lung transplant recipients, CMV disease should be considered in the differential diagnosis of early post-operative pleural effusion. Community-acquired Cytomegalovirus (CMV) infection in a seronegative transplant recipient (R) from a seronegative donor (D) is a rare occurrence that carries significant clinical and prognostic implications. Few case reports exist describing this entity in lung transplant recipients. A 58-year-old man with bilateral lung transplant for sarcoidosis presented with three days of diarrhea and dyspnea. He underwent an uneventful bilateral lung transplantation (CMV D-/R-) six weeks prior, receiving basiliximab and methylprednisolone for induction. He was discharged two weeks later on tacrolimus, mycophenolate motefil, and prednisone taper as maintenance immunosuppression. He was receiving acyclovir for herpes viruses prophylaxis. He was seen weekly post-discharge and continued to have clear chest radiographs and unremarkable bloodwork. On presentation, his physical examination was notable for decreased breath sounds at the right base. His laboratory values revealed creatinine of 2.4 mg/dL. His chest radiograph showed new right pleural effusion. He was admitted for hydration and diarrhea work up. Abdominal computed tomography (CT) revealed mild diverticulitis with no colitis and his stool studies were positive for Clostridium difficile. Chest CT showed hazy and linear markings with thin-walled cysts in right lower lobe, adjacent to a moderate pleural effusion. CMV by polymerase chain reaction resulted at 318,200 copies/mL. He was treated with intravenous ganciclovir and underwent a thoracenthesis. Half a liter of clear pleural fluid was removed and was notable for lymphocytic predominance of 72% as well as polytypic plasma cells and a small number of B lymphocytes with no surface immunoglobulins on flow cytometry. Subsequent radiograph showed completely re-expanded lung. Within two days, the effusion re-accumulated and additional half a liter were drained, revealing of 95% lymphocytes, with complete re-expansion of the lung. Concomitant viral load remained elevated at 150,328 copies/mL. He was discharged on valganciclovir, his viral load decreased to an undetectable level, and his radiographs have remained free of effusion. While primary CMV infection is rare in low-risk lung transplant recipients, CMV disease should be considered in the differential diagnosis of early post-operative pleural effusion.

COVID-19 and humoral immune response in convalescent plasma donors in Pakistani cohort.

Current COVID-19 pandemic has affected the entire globe. While there was no vaccine neither any specific treatment, investigational use of convalescent plasma has been explored in clinical trials. A prospective multicenter study of convalescent plasma was conducted. Donors were tested for total Anti-SARS-CoV-2 antibodies by electrochemiluminescence (ECLIA) and RT-PCR for COVID-19. Enzyme Linked Immunosorbent Assay (ELISA) was used to detect semi-quantitative and quantitative IgG anti-SARS-COV-2 antibodies. IgG Immunofluorescence-based lateral flow immunoassay (LFIA) was used to recheck seronegative donors. A total of 400 donors were enrolled. Twelve donors were SARS-CoV-2 positive by RT-PCR. Nine of 12 donors had developed SARS-CoV-2 IgG antibodies, while in 3 donors antibodies were not developed. A total of 70 donors (17.5%) were deferred due to seronegative status; 64 (16%) of them did not develop antibodies when plasma collection was planned. The IgG semiquantitative ELISA was positive in 282 and quantitative in 284 of 330 donors with a mean value of &gt;1:160 and 44.10±39.22 IU/ml respectively. A total of 116 (29%) donors did not show IgG humoral response to COVID-19 even 28 days from the onset of illness. Subsequently, LFIA method was able to detect IgG antibodies in 20 of 48 (41.6%) seronegative donors and in 20 of 34 (58.8%) ECLIA positive ELISA negative donors. Viral RNA detection in recovered asymptomatic patients with concomitant IgG antibodies indicates recovery. Inability to detect antibodies by different testing kits may be due to their different antigenic targets or sensitivity. Significance of a positive COVID-19 RT-PCR in asymptomatic recovered patients is yet to be determined.

Magnetic Enrichment of SARS-CoV-2 Antigen-Binding B Cells for Analysis of Transcriptome and Antibody Repertoire

The ongoing COVID-19 pandemic has had devastating health impacts across the globe. The development of effective diagnostics and therapeutics will depend on the understanding of immune responses to natural infection and vaccination to the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While both B-cell immunity and T-cell immunity are generated in SARS-CoV-2-infected and vaccinated individuals, B-cell-secreted antibodies are known to neutralize SARS-CoV-2 virus and protect from the disease. Although interest in characterizing SARS-CoV-2-reactive B cells is great, the low frequency of antigen-binding B cells in human blood limits in-depth cellular profiling. To overcome this obstacle, we developed a magnetic bead-based approach to enrich SARS-CoV-2-reactive B cells prior to transcriptional and antibody repertoire analysis by single-cell RNA sequencing (scRNA-seq). Here, we describe isolation of SARS-CoV-2 antigen-binding B cells from two seropositive donors and comparison to nonspecific B cells from a seronegative donor. We demonstrate that SARS-CoV-2 antigen-binding B cells can be distinguished on the basis of transcriptional profile and antibody repertoire. Furthermore, SARS-CoV-2 antigen-binding B cells exhibit a gene expression pattern indicative of antigen experience and memory status. Combining scRNA-seq methods with magnetic enrichment enables the rapid characterization of SARS-CoV-2 antigen-binding B cells.

Knowledge and Practice of Hepatitis B and C Prevention among Seropositive and Seronegative Prospective Blood Donors in a Tertiary Care Hospital in Eastern Nigeria

Introduction: Hepatitis B and C infections are endemic in Nigeria and the major causes of chronic liver diseases, including hepatocellular carcinoma. One of the major routes of transmission is by transfusion of infected blood or blood products. Awareness of the disease is important, and this study seeks to compare the knowledge and practice of the disease among the seropositive and seronegative prospective blood donors. Aim: To assess the difference in the level of knowledge and preventive practice of blood borne hepatitis among hepatitis B and hepatitis C seropositive and seronegative prospective blood donors. Materials and Methods: The present study was a descriptive survey carried out between June 2019 to January 2020 in the Blood Donor’s Department of the University of Nigeria Teaching Hospital, Enugu, Nigeria. A pretested self/interviewer’s administered questionnaire was used to assess the knowledge and practice among 100 hepatitis B and hepatitis C seropositive and 305 seronegative prospective blood donors. The hepatitis B and C seropositive subjects were screened using Enzyme Immunosorbent Assay (ELISA) technique. The findings and scores were analysed using IBM Corporation 2019 Statistical Package for the Social Sciences (SPSS) software version 21.0. Results: Total of 305 respondents were seronegative while 82 and 18 were hepatitis B and C seropositive, respectively. The mean age of the study participants was 27.78±7.17 years, whereas it was 28.09±6.36 years and 27.46±7.968 years among seropositive and seronegative respondents, respectively. In the study, 98 (98%) males and 2 (2%) females were in the seropositive group while, 268 (87.9%) males and 37 (12.1%) females were in seronegative group. The overall knowledge score and practice score were 56.3% and 28.1%, respectively. In the seropositive group, the average knowledge score was 27.3% and the average practice score was 17%. While in the seronegative group, the average knowledge score was 65.7% and the average score on practice score was 31.8%. There was a significant association between seropositive and being a male (p-value=0.001), less educated (p-value=0.002), currently unmarried (p-value=0.029), knowledge above average (p-value=0.001), and practice below average (p=0.002). None of the respondents had hepatitis B vaccination. Conclusion: Based on the results obtained from the present study, it is evident that the level of knowledge and practice among the seropositive respondent is significantly low when compared with the seronegative groups. There is a need for a serious awareness campaign on the transmission and prevention of hepatitis B and C, and the possibility of extending hepatitis B immunisation coverage in the country owing to the increasing burden of the disease.

Abstract 12091: Incidence and Predictors of Progression to Chagas Cardiomyopathy: Long-Term Follow-Up of a Large Cohort of Trypanosoma cruzi- Infected Individuals

Introduction: There are few contemporary prospective cohorts of Trypanosoma cruzi ( T.cruzi )-infected individuals. This study aimed to determine the incidence of new-onset cardiomyopathy and death attributable to T. cruzi infection. Methods: We enrolled 1088 individuals: 499 T. cruzi -seropositive blood donors identified in routine donor screening, 488 age- and sex-matched T. cruzi - seronegative blood donors, and 101 patients with established Chagas cardiomyopathy recruited from a tertiary center. Subjects underwent cardiovascular evaluation with ECG and echocardiogram at the baseline (2008-2010) and follow-up (2018-2019) visits. Cardiomyopathy was diagnosed based on ECG and echocardiographic findings at baseline and follow-up. Vital status was determined by linkage with the national Brazilian mortality system. Results: Mortality was 80.9 deaths/1000 person-years (py) in established cardiomyopathy and 15.1 deaths/1000py in T. cruzi -seropositive donors with cardiomyopathy at baseline. The mortality rate among T. cruzi -seropositive blood donors without cardiomyopathy at baseline and T. cruzi -seronegative controls was similar, with mortality rates of 3.67 and 3.62 events/1000 py, respectively. In T. cruzi -seropositive donors, age (hazard ratio [HR] = 1.05 per year), anti-T.cruzi antibody level (HR = 1.40 per unit increase in serology assay signal/cut off) and the presence of cardiomyopathy at baseline (HR = 3.01) were independent predictors of mortality. The incidence of new-onset cardiomyopathy in T. cruzi -seropositive donors was 13.8 events/1000 py (95% CI 9.5-19.6) compared with 4.6 events/1000 py (95% CI 2.3-8.3) in controls, with an absolute incidence difference attributable to T. cruzi infection of 9.2 (95% CI 3.6 - 15.0) events/1000py. T. cruzi antibody level at baseline was the main independent predictor of subsequent new-onset cardiomyopathy with an adjusted odds ratio of 1.37 (95% CI 1.07-1.76) per unit increase in antibody level. Conclusions: We present a comprehensive description of the natural history of T. cruzi infection in a contemporary patient population. The results highlight the central importance of total anti- T. cruzi antibody titer as a marker of Chagas disease activity and risk of progression.

Incidence and Outcomes of Toxoplasma Reactivation in Patients with Hematologic Diseases after Allogeneic Hematopoietic Stem Cell Transplantation

Incidence and Outcomes of Toxoplasma Reactivation in Patients with Hematologic Diseases after Allogeneic Hematopoietic Stem Cell Transplantation

Cytomegalovirus and Epstein Barr Virus Reactivation after Allogeneic Stem Cell Transplantation in Pre- and Post-Letermovir Era

Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) reactivation are two of the most frequent infections affecting patients receiving allogeneic stem cell transplantation (HSCT). Up to 80% of seropositive patients might experience CMV reactivation in absence of prophylaxis, expecially when a seropositive patient receive stem cell graft from seronegative donor. The introduction of Letermovir notably reduced the risk of CMV infection also in high risk patients. EBV is a herpesvirus widely spread in common population with a prevalence of approximatively 80%. As a consequence, many recipient and donor have EBV seropositive status. We would evaluate if a difference occurred in the CMV and EBV incidence after Letermovir introduction, which casually coincided in our center with the transition to a single graft-versus host disease (GvHD) prophylaxis schedule including cyclophosphamide post (Cy-post).We enrolled patient submitted to allogeneic stem cell transplant in our centre from January 2015 to december 2020. Patients were divided into two groups. The pre-letermovir group (group A, n=230) included patients who had received prophylaxis with ciclosporine A (CSA) plus micophenolic acid (MFA) (N=17) or CSA plus short course methotrexate (MTX) (N=96) or CSA plus MFA plus Cy-post (n=117). In the post-letermovir group (group B, n=135) all patients received the same GvHD prophylaxis with CSA plus MFA plus Cy-post. Sixty-eight of them received letermovir in addition to acyclovir as CMV prophylaxis.In the group A, CMV infection rate was affected by HLA mismatch (HR 1.70, p=0.003), Cy-post use (HR1.49, p=0.02), anti-thymocyte globulin (ATG) use (HR 0.62, p=0.01), bone marrow or cord blood source (HR 1.46, p=0.03), CMV seropositive recipient (HR 10, p=0.0001) and CMV seronegative donor for a seropositive recipient (HR 2.2, p<0.0001). Multivariate analysis confirmed CMV seropositive recipient (HR 9, p=0.002), CMV seronegative donor for seropositive recipient (HR 1.67, p=0.008) and HLA mismatch (HR 3.58, p=0.006) as independent variables for CMV infection. EBV infection appeared to be affected by underlying disease remission status at transplant (HR 1.64, p=0.01), HLA mismatch (HR 0.42, p<0.0001), GvHD prophylaxis other than Cy-post (HR 2.60, p<0.0001), ATG use (HR 2.60, p<0.0001), unrelated donor (MUD) (HR 2.01, p=0.0005) and aGvHD occurrence (HR 1.62, p=0.01). Multivariate analysis confirmed aGvHD occurrence (HR 1.74, p=0.007) and underlying disease remission at transplant (HR 1.90, p=0.002) as independent variables for EBV infection.In the group B, reduced intensity conditioning (HR 5.94, p=0.007), letermovir use (HR 0.23, p=0.0009), and aGvHD occurrence (HR 3.13, p=0.007) were independent risk factors for CMV infection in multivariate analysis. The 100-day CI of CMV infection was of 3% (95% CI 1-12) in patients receiving letermovir vs. 26% (95% CI 17-39) in the others (p=0.02, figure 1A). In the same cohort of patients EBV infection appeared to be more frequent for patients receiving ATG (57% vs. 24%, p=0.05), MUD donor (32% vs. 17%, p=0.04) and who experienced CMV concomitant infection (42% vs. 28%, p=0.03). However, these data were not confirmed in multivariate analysis.According to GvHD prophylaxis, CMV infection appeared to be more frequent in patients receiving Cy-post as GvHD prophylaxis (1-yr CI 68% vs. 54.6%, p=0.02, figure 1B), contrarily to EBV infection which appeared to be less frequent in the same patients (1-yr CI 41% vs. 62%, p<0.0001, figure 1C). However, CMV infection rate was notably reduced by letermovir introduction, while EBV infection rate not appeared to be affected by that.Figure 1: A) Day 100 cumulative incidence of CMV according to letermovir prophylaxis administration; B) One year cumulative incidence of CMV according to cyclophosphamide post transplant administration; C) One year cumulative incidence of EBV according to cyclophosphamide post transplant administration. [Display omitted] DisclosuresMetafuni: Jazz: Other: Invited Clinical case presentation at meeting. Laurenti: Roche: Honoraria; Gilead: Honoraria; Abbvie: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; AstraZeneca: Honoraria, Speakers Bureau. Sica: Alexion: Consultancy; Jazz Pharma: Consultancy.

Early Administration of Partially HLA Matched Third Party Virus-Specific T-Cells in Conjunction with Antiviral Treatment for Initial Viral Infection after Allogeneic Stem Cell Transplant Is Safe and Leads to High Rates of Viral Control

Introduction:Reactivation of viruses such as cytomegalovirus (CMV) or Epstein Barr virus (EBV) after allogeneic hemopoietic stem cell transplant (aHSCT) is associated with increased non relapse mortality and a requirement for antivirals with mainly hemopoietic and renal toxicities that can further compromise transplant outcomes and increase health care costs. The use of 3 rd party virus specific T cells (VSTs) has been effective in treating recurrent and refractory viral reactivation after transplant and leads to rapid restoration of viral immunity. We investigated whether early administration of 3 rd party VSTs together with antiviral therapy could safely enhance immune recovery and improve viral control in patients requiring treatment for their initial CMV and EBV infection after aHSCT.Methods:We performed a single arm phase 1 clinical trial in which aHSCT patients requiring treatment for their first CMV or EBV reactivation (or EBV driven malignancy) received infusions of partially HLA matched 3 rd party VSTs within 7 days of commencing standard antiviral treatment. Patients were required to have a viral copy number of at least 1,000 copies/mL for CMV, 10,000 copies/mL of blood for EBV, or proven tissue infection irrespective of copy number for treatment initiation. T cell products were expanded from G-CSF stimulated aphereses from normal donors following peptide stimulation and CD137 magnetic bead selection. T cells were cultured for up to 12 days before specificity testing and cryopreservation. Patients were eligible to receive up to 4 doses of VSTs at 4 week intervals with products selected with a minimum of 1 of 6 HLA matches at HLA-A, -B and -DRB1 with antiviral activity demonstrated through the shared HLA molecule. The primary endpoint of the study was infusion safety.Results:Thirty aHSCT patients were treated with 1-4 VST infusions (27 CMV, 3 EBV) commencing at a median of 4 days after initiation of antiviral treatment. 27 patients were transplanted for hematological malignancies, 3 for immune deficiencies. Conditioning was myeloablative in 12 patients and the majority of patients (22/30) received in vivo T cell depletion. 7/27 CMV seropositive recipients were transplanted from CMV seronegative donors. A total of 41 infusions were given, most frequently targeting antigens presented through shared HLA molecules A2 and/or B7. All infusions were administered at a cell dose of 2 x 10 7/m 2. VST products were CD3 + (median 97.9%, range 96.2 - 99.4%), with median percentage of CD3 +CD8 + 85.8% (range 23.2 - 95.5%). There was one infusion related adverse event consisting of fever that resolved rapidly after admission and antibiotics. Overall viral response rate was 100% with a complete response rate of 94% (Figure 1). Of the 28 patients who achieved a CR (after either 1 or 2 infusions), 22 remained virus PCR negative (n = 8) or below the limit of quantitation (n = 14) for the duration of follow up. 3 patients had brief episodes of quantifiable reactivation not requiring additional therapy and 3 patients required a second infusion following initial CR. All remained PCR negative after their 2 nd CR. All 3 patients treated for EBV PTLD achieved sustained CR. Overall rates of acute and chronic GVHD post-infusion were 33% (10/30) and 20% (6/30) respectively (grade IIII/IV aGVHD 10%, severe cGVHD 7%). VST infusion was associated with a reduction in activation and inhibitory marker expression on CD4 + and CD8 + lymphocytes within the first 30 days and recovery of CD8 + (and more slowly CD4 +) CD45RA -CD62L - effector memory cells. Within the first 100 days after infusion there was an increase in interferon-γ responsiveness of blood lymphocytes. CMV and EBV specific tetramer positive T cells were detected comprising up to 13% of CD8 + cells for up to 6 months post infusion. At 1 year post transplant, non relapse mortality was 10%, cumulative incidence of relapse was 7% and overall survival was 87%.Conclusion:The combination of traditional antiviral treatment and early administration of 3 rd party VSTs is safe and achieves high rates of viral control without evidence of increased acute or chronic GVHD and with evidence of enhanced immune responses to viral antigens. 1 year overall survival was high with low rates of non relapse mortality and relapse. These encouraging results require confirmation in a prospective randomized study comparing best available therapy with best available therapy combined with early VST administration. [Display omitted] DisclosuresRitchie: Novartis: Honoraria; BMS: Research Funding; Takeda: Research Funding; CRISPR Therapeutics: Research Funding; Amgen Inc: Honoraria, Research Funding; CSL: Honoraria.