Rapid GMP-Compliant Expansion of SARS-CoV-2-Specific T Cells From Convalescent Donors for Use as an Allogeneic Cell Therapy for COVID-19.

COVID-19 disease caused by the SARS-CoV-2 virus is characterized by dysregulation of effector T cells and accumulation of exhausted T cells. T cell responses to viruses can be corrected by adoptive cellular therapy using donor-derived virus-specific T cells. One approach is the establishment of banks of HLA-typed virus-specific T cells for rapid deployment to patients. Here we show that SARS-CoV-2-exposed blood donations contain CD4 and CD8 memory T cells which recognize SARS-CoV-2 spike, nucleocapsid and membrane antigens. Peptides of these antigens can be used to isolate virus-specific T cells in a GMP-compliant process. The isolated T cells can be rapidly expanded using GMP-compliant reagents for use as an allogeneic therapy. Memory and effector phenotypes are present in the selected virus-specific T cells, but our method rapidly expands the desirable central memory phenotype. A manufacturing yield ranging from 1010 to 1011 T cells can be obtained within 21 days culture. Thus, multiple therapeutic doses of virus-specific T cells can be rapidly generated from convalescent donors for potential treatment of COVID-19 patients.

Jaagsiekte sheep retrovirus infects multiple cell types in the ovine lung.

Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer of sheep caused by Jaagsiekte sheep retrovirus (JSRV). The details of early events in the pathogenesis of OPA are not fully understood. For example, the identity of the JSRV target cell in the lung has not yet been determined. Mature OPA tumors express surfactant protein-C (SP-C) or Clara cell-specific protein (CCSP), which are specific markers of type II pneumocytes or Clara cells, respectively. However, it is unclear whether these are the cell types initially infected and transformed by JSRV or whether the virus targets stem cells in the lung that subsequently acquire a differentiated phenotype during tumor growth. To examine this question, JSRV-infected lung tissue from experimentally infected lambs was studied at early time points after infection. Single JSRV-infected cells were detectable 10 days postinfection in bronchiolar and alveolar regions. These infected cells were labeled with anti-SP-C or anti-CCSP antibodies, indicating that differentiated epithelial cells are early targets for JSRV infection in the ovine lung. In addition, undifferentiated cells that expressed neither SP-C nor CCSP were also found to express the JSRV Env protein. These results enhance the understanding of OPA pathogenesis and may have comparative relevance to human lung cancer, for which samples representing early stages of tumor growth are difficult to obtain.

Jaagsiekte sheep retrovirus is present at high concentration in lung fluid produced by ovine pulmonary adenocarcinoma-affected sheep and can survive for several weeks at ambient temperatures.

Jaagsiekte sheep retrovirus (JSRV) causes a fatal lung cancer of sheep known as ovine pulmonary adenocarcinoma (OPA). OPA is a significant disease in many sheep-rearing countries and there is no effective method of control. A unique feature of OPA is the overproduction of fluid in the lung of affected animals. This lung fluid contains JSRV and provides a means of transmission through the inhalation of virus. In this study we demonstrated that lung fluid from different OPA cases contained between 10(7) and 10(10) copies of JSRV RNA per ml. Examination of JSRV RNA survival under conditions that mimic natural conditions suggested that intact JSRV virions may persist for several weeks in the environment. These are the first quantitative data on JSRV in lung fluid and provide valuable information for implementing appropriate biosecurity measures to control the spread of JSRV in the field.

High levels of human immunodeficiency virus infection of CD8 lymphocytes expressing CD4 in vivo.

Human immunodeficiency virus (HIV)-infected CD8 lymphocytes have been reported in vivo, but the mechanism of infection remains unclear. Experiments using the thy/hu mouse model support export of intrathymically infected CD8 precursors, while recent in vitro data suggest that mature CD8 lymphocytes upregulate CD4 upon activation (generating a CD8bright CD4dim phenotype) and are susceptible to HIV infection. To determine whether these mechanisms operate in vivo and to assess their relative importance in the generation of circulating HIV-infected CD8 lymphocytes, we quantified HIV long terminal repeat (LTR) DNA in CD8+ CD4- and CD8bright CD4dim lymphocytes isolated from HIV-infected individuals by fluorescence-activated cell sorting. HIV infection of CD8 lymphocytes was demonstrated in 17 of 19 subjects, with a significant inverse relationship between level of infection and CD4 lymphocyte count (R = -0.73; P < 0.001). The level of HIV infection of CD8bright CD4dim lymphocytes was significantly higher (median, 1,730 HIV LTR copies/10(6) cells; n = 9) than that of CD8+ CD4- lymphocytes (undetectable in seven of nine individuals; P < 0.01) and approached that of CD4 lymphocytes from the same individuals (median, 3,660 HIV LTR copies/10(6) cells). CD8bright CD4dim lymphocytes represented 0.8 to 3.3% of total CD8 lymphocytes and were most prevalent in the memory subset. Thus, HIV-infected CD8 lymphocytes commonly circulate in HIV-infected individuals and are generated through infection of activated CD8 lymphocytes rather than through export of intrathymically infected precursors. The high level of infection of CD8bright CD4dim lymphocytes could have a direct role in the decline in CD8 lymphocyte function that accompanies HIV disease progression.