A phase I/II dose-escalation multi-center study to evaluate the safety of infusion of natural killer cells or memory T cells as adoptive therapy in coronavirus pneumonia and/or lymphopenia: RELEASE study protocol.

BACKGROUND: Moderate/severe cases of COVID-19 present a dysregulated immune system with T cell lymphopenia and a hyper-inflammatory state. This is a study protocol of an open-label, multi-center, double-arm, randomized, dose-finding phase I/II clinical trial to evaluate the safety, tolerability, alloreactivity, and efficacy of the administration of allogeneic memory T cells and natural killer (NK) cells in COVID-19 patients with lymphopenia and/or pneumonia. The aim of the study is to determine the safety and the efficacy of the recommended phase 2 dose (RP2D) of this treatment for patients with moderate/severe COVID-19. METHODS: In the phase I trial, 18 patients with COVID-19-related pneumonia and/or lymphopenia with no oxygen requirement or with an oxygen need of ≤ 2.5 liters per minute (lpm) in nasal cannula will be assigned to two arms, based on the biology of the donor and the patient. Treatment of arm A consists of the administration of escalating doses of memory T cells, plus standard of care (SoC). Treatment of arm B consists of the administration of escalating doses of NK cells, plus SoC. In the phase II trial, a total of 182 patients with COVID-19-related pneumonia and/or lymphopenia requiring or not oxygen supplementation but without mechanical ventilation will be allocated to arm A or B, considering HLA typing. Within each arm, they will be randomized in a 1:1 ratio. In arm A, patients will receive SoC or RP2D for memory T cells plus the SoC. In arm B, patients will receive SoC or RP2D for NK cells plus the SoC. DISCUSSION: We hypothesized that SARS-CoV-2-specific memory T-lymphocytes obtained from convalescent donors recovered from COVID-19 can be used as a passive cell immunotherapy to treat pneumonia and lymphopenia in moderate/severe patients. The lymphopenia induced by COVID-19 constitutes a therapeutic window that may facilitate donor engraftment and viral protection until recovery. TRIAL REGISTRATION: ClinicalTrials.gov NCT04578210 . First Posted : October 8, 2020.

Phase I dose-escalation single centre clinical trial to evaluate the safety of infusion of memory T cells as adoptive therapy in COVID-19 (RELEASE).

BACKGROUND: Effective treatments are still needed to reduce the severity of symptoms, time of hospitalization, and mortality of COVID-19. SARS-CoV-2 specific memory T-lymphocytes obtained from convalescent donors recovered can be used as passive cell immunotherapy. METHODS: Between September and November 2020 a phase 1, dose-escalation, single centre clinical trial was conducted to evaluate the safety and feasibility of the infusion of CD45RA- memory T cells containing SARS-CoV-2 specific T cells as adoptive cell therapy against moderate/severe cases of COVID-19. Nine participants with pneumonia and/or lymphopenia and with at least one human leukocyte antigen (HLA) match with the donor were infused. The first three subjects received the lowest dose (1 × 105 cells/kg), the next three received the intermediate dose (5 × 105 cells/kg) and the last three received the highest dose (1 × 106 cells/kg) of CD45RA- memory T cells. Clinicaltrials.gov registration: NCT04578210. FINDINGS: All participants' clinical status measured by National Early Warning Score (NEWS) and 7-category point ordinal scales showed improvement six days after infusion. No serious adverse events were reported. Inflammatory parameters were stabilised post-infusion and the participants showed lymphocyte recovery two weeks after the procedure. Donor microchimerism was observed at least for three weeks after infusion in all patients. INTERPRETATION: This study provides preliminary evidence supporting the idea that treatment of COVID-19 patients with moderate/severe symptoms using convalescent CD45RA- memory T cells is feasible and safe. FUNDING: Clinical Trial supported by Spanish Clinical Research Network PT17/0017/0013. Co-funded by European Regional Development Fund/European Social Fund. CRIS CANCER Foundation Grant to AP-M and Agencia Valenciana de Innovación Grant AVI-GVA COVID-19-68 to BS.

SARS-CoV-2-Specific Memory T Lymphocytes From COVID-19 Convalescent Donors: Identification, Biobanking, and Large-Scale Production for Adoptive Cell Therapy.

Syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a second outbreak significantly delaying the hope for the virus' complete eradication. In the absence of effective vaccines, we need effective treatments with low adverse effects that can treat hospitalized patients with COVID-19 disease. In this study, we determined the existence of SARS-CoV-2-specific T cells within CD45RA- memory T cells in the blood of convalescent donors. Memory T cells can respond quickly to infection and provide long-term immune protection to reduce the severity of COVID-19 symptoms. Also, CD45RA- memory T cells confer protection from other pathogens encountered by the donors throughout their life. It is of vital importance to resolve other secondary infections that usually develop in patients hospitalized with COVID-19. We found SARS-CoV-2-specific memory T cells in all of the CD45RA- subsets (CD3+, CD4+, and CD8+) and in the central memory and effector memory subpopulations. The procedure for obtaining these cells is feasible, easy to implement for small-scale manufacture, quick and cost-effective, involves minimal manipulation, and has no GMP requirements. This biobank of specific SARS-CoV-2 memory T cells would be immediately available "off-the-shelf" to treat moderate/severe cases of COVID-19, thereby increasing the therapeutic options available for these patients.

Genomic sequences of five novel HLA class I alleles: A*30:129, B*08:195, B*51:01:62, C*01:147 and C*12:195:02.

Five new HLA class I alleles are described, A*30:129, B*08:195, B*51:01:62, C*01:147 and C*12:195:02.

Genomic sequences of HLA-A*68:169, HLA-B*07:298 and HLA-B*39:129.

Three new HLA class I alleles were described in the Spanish population. HLA-A*68:169 and -B*39:129 show one amino acid replacement at the α1-domain, compared to A*68:02 (P47 > L47) and -B*39:06 (S11 > A11), respectively. HLA-B*07:298 presents one nucleotide mutation within exon 1, resulting in a new amino acid position -14, L>Q, which has not been previously described in any HLA protein. Prediction of the B*07:298 signal peptide cleavage did not show significant differences in comparison with that obtained for the rest of HLA-B genes.

Characterization of a novel HLA-DRB1*07 allele, DRB1*07:83.

A new DRB1*07 allele, DRB1*07:83, was described in a Caucasian Spanish donor.

HLA-A*30:125 shows a new antigen binding domain created from HLA-A*30:01 and HLA-A*30:02.

A new HLA-A allele, A*30:125, was characterized in a Spanish individual.

Three new HLA class I alleles with synonymous mutations: HLA-A*03:01:62, -C*07:02:82 and -C*12:03:42.

Three new HLA class I alleles with synonymous mutations were identified.

Characterization of two new HLA-B alleles, HLA-B*07:299 and HLA-B*35:350.

Two novel HLA-B alleles, B*07:299 and B*35:350, were characterized by genomic full-length sequencing.

Genomic sequences of two novel HLA-C alleles, HLA-C*15:143 and HLA-C*07:109:02.

Two novel HLA-C alleles, C*07:109:02 and C*15:143, were characterized in two Spanish individuals.

Four new HLA class I alleles, HLA-A*02:681, HLA-A*30:111, HLA-A*68:164 and HLA-B*35:01:46.

Four new HLA class I alleles, HLA-A*02:681, HLA-A*30:111, HLA-A*68:164 and HLA-B*35:01:46 were described in Spaniards.

The new HLA-B*50:51 allele was generated by intralocus recombination between B*50:01:01:02 and B*14:02:01.

The new B*50:51 allele was found in 3 Caucasians from Southern Spain.

Characterization of three new HLA Class I Alleles in Spanish Caucasians, HLA-A*02:620, HLA-B*27:150 and HLA-B*07:05:01:02.

Three new HLA class I alleles, HLA-A*02:620, HLA-B*27:150 and HLA-B*07:05:01:02, were described in the Spanish Caucasoid population.

Human leucocyte antigen (HLA)-DQB1*03:02 and HLA-A*02:01 have opposite patterns in their effects on susceptibility to HIV infection.

OBJECTIVES: The objective of this study was to seek correlates of immune protection in HIV infection. We sought to elucidate the association between the presence of human leucocyte antigen (HLA) alleles, as well as killer immunoglobulin receptor (KIR) genotypes, and the susceptibility to HIV infection in a Spanish cohort of HIV-exposed seronegative (HESN) individuals. METHODS: A total of 152 individuals were evaluated: 29 HESN individuals in stable heterosexual relationships with an HIV-infected partner admitting high-risk sexual intercourse for at least 12 months prior to inclusion in the study, 61 HIV-infected patients and 62 healthy controls. HLA class I and II alleles and KIR genotypes were assessed in genomic DNA from all individuals in the study by polymerase chain reaction-sequence-specific oligonucleotide (PCR-SSO) using bead array technology. RESULTS: HESN individuals showed a higher prevalence of HLA-A3 (62%) and HLA-B44 (83%) supertypes compared with HIV-infected individuals (42% and 66%, respectively). Regarding specific HLA alleles, HESN individuals had a higher prevalence of HLA-A*33:01, DRB1*04 and DQB1*03:02 alleles (14%, 34% and 31%, respectively) and a lower prevalence of the HLA-A*02:01 allele (27%) than HIV-infected patients (3%, 15%, 11% and 52%, respectively; P < 0.05). Interestingly, in a multivariate analysis, only the presence of DQB1*03:02 and the absence of A*02:01 alleles were independently associated with HESN status [odds ratio (OR) 3.4 (95% confidence interval (CI) 1.1-10.5) and 0.4 (95% CI: 0.1-0.9), respectively; P < 0.05]. No KIR genotype was associated with susceptibility to HIV infection. CONCLUSIONS: Our data showed that the presence of the HLA class II allele DQB1*03:02 was a correlate of immune protection against HIV infection, while the presence of the HLA class I allele A*02:01 was associated with being infected with HIV.

RNA processing and protein expression of HLA-B*07:44N.

BACKGROUND: The assignment of human leukocyte antigen (HLA) null alleles is clinically relevant in the setting of stem cell transplantation. Cell surface expression profiling and mRNA processing analysis of the HLA-B allele previously designated as B*07:44, have been performed. MATERIALS AND METHODS: Cell surface expression of HLA-B*07:44 was determined using flow cytometry. Genomic full-length and HLA-B*07-specific cDNA sequencing were carried out by Sanger procedure. RESULTS: Flow cytometric analysis confirmed previous serologic results and demonstrated a lack of cell membrane expression of the HLA-B protein. The mRNA processing, studied using direct HLA-B*07-specific cDNA sequencing, revealed the presence of a unique, aberrantly spliced mRNA, with a deletion of the last 43 bp on the 5'-end of exon 4. The substitution from T to G at genomic position 1799 compared to B*07:02:01 introduced a new and stronger splice donor site at exon 4. This alternative splicing produced an mRNA containing a premature stop codon at position 280, explaining the absence of mature HLA-B7 protein on the cell surface. CONCLUSION: These findings led us to consider this HLA-B variant as a HLA null allele. The World Health Organization (WHO) Nomenclature Committee has since renamed this variant B*07:44N .

Two new HLA class I alleles described in a Spanish individual, HLA-A*11:01:01:04 and HLA-B*35:330.

Two new HLA class I alleles, HLA-A*11:01:01:04 and HLA-B*35:330, were characterized in a Spanish patient.

Exon 2 sequencing of the new HLA-DRB1 allele, DRB1*13:216.

A novel HLA-DRB1*13:216 allele was characterized in a Spanish bone marrow donor.

HLA-A*30:99 shows a new amino acid position 156 within HLA-A*30 subtypes.

A novel HLA-A*30:99 allele was characterized in a Spanish individual.

Report From the First and Second Spanish Killer Immunoglobulin-Like Receptor Genotyping Workshops: External Quality Control for Natural Killer Alloreactive Donor Selection in Haploidentical Stem Cell Transplantation.

An important factor affecting the success in the setting of related haploidentical hematopoietic stem cell transplantation (HSCT) is the graft-versus-leukemia effect mediated by natural killer (NK) cells when the donor displays NK alloreactivity versus the recipient. NK cell function is regulated by killer immunoglobulin-like receptors (KIR) and it has been described that donor KIR genotype influences transplantation outcome. This has led to a requirement of laboratories to have a quality assurance program for validation and control of their KIR genotyping methods. The goal of the 1st and 2nd Spanish KIR Genotyping Workshops was to provide an external proficiency testing program in KIR genotyping for Spanish immunology and transplant laboratories. These workshops were conducted during the years 2014-2016 and consisted of 17 participating laboratories typing a set of 20 samples. The presence/absence of 16 mandatory KIR loci (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 2DP1, 3DL1, 3DL2, 3DL3, 3DS1, and 3DP1) was evaluated per sample. Methods for KIR genotyping included polymerase chain reaction with the use of sequence-specific primers and sequence-specific oligoprobes. Consensus typing was reached in all samples, and the performance of laboratories in external proficiency testing was satisfactory in all cases. The polymorphism detected in the small sample studied in both workshops is indicative of an ample variety of KIR gene profiles in the Spanish population.

Genomic full-length sequence of two new HLA-C alleles, HLA-C*04:239 and HLA-C*05:137.

Two novel HLA-C alleles, C*04:239 and C*05:137, were characterized in Spanish individuals.