BACKGROUND: Cellular substrate (CS) composed of phytohemagglutinin (PHA)-stimulated PBMNCs is required for isolating primary human immunodeficiency virus Type 1 from plasma (PHIV) during early acute infection. The transmitted founder PHIV is neutralization-sensitive and uses chemokine-receptor 5 to infect CD4-positive PBMNCs. Therefore, PHIV cocultured with optimized CS would enable efficient virologic diagnosis, vaccine development, and tests for broadly neutralizing antibodies (bnAb). STUDY DESIGN AND METHODS: Fifteen leukapheresis donations were used to isolate CD4-enriched PBMNCs. Individual or three or four donors' pooled cells were stimulated with PHA, and both CSs were evaluated in parallel cocultures using five different PHIV isolates from nucleic acid testing-positive anti-HIV-negative donations. Feasibility of coculturing PHIV with pooled PBMNC-CS after dimethyl sulfoxide cryopreservation was investigated. RESULTS: The cell-free supernatants from individual CSs contained mean HIV-p24 antigen varying between 20.35 and 85.54 ng/mL, while that from pooled CSs ranged between 57.54 and 88.10 ng/mL. Thus, PBMNCs pooled from multiple donors provide an optimal CS for coculturing PHIV. Cryopreserved PBMNC-CS for PHIV cocultures is promising for systematic biosynthesis of PHIV. CONCLUSIONS: Pooled CSs after cryopreservation were functional for PHIV replication and enable on-demand diagnostic cocultures, preparing panels of PHIV stocks for bnAb testing and developing an envelope-subunit vaccine candidate analogous to hepatitis B surface antigen, proven successful in preventing hepatitis B virus infection.
BACKGROUND: Cellular substrate (CS) composed of phytohemagglutinin (PHA)-stimulated PBMNCs is required for isolating primary human immunodeficiency virus Type 1 from plasma (PHIV) during early acute infection. The transmitted founder PHIV is neutralization-sensitive and uses chemokine-receptor 5 to infect CD4-positive PBMNCs. Therefore, PHIV cocultured with optimized CS would enable efficient virologic diagnosis, vaccine development, and tests for broadly neutralizing antibodies (bnAb). STUDY DESIGN AND METHODS: Fifteen leukapheresis donations were used to isolate CD4-enriched PBMNCs. Individual or three or four donors' pooled cells were stimulated with PHA, and both CSs were evaluated in parallel cocultures using five different PHIV isolates from nucleic acid testing-positive anti-HIV-negative donations. Feasibility of coculturing PHIV with pooled PBMNC-CS after dimethyl sulfoxide cryopreservation was investigated. RESULTS: The cell-free supernatants from individual CSs contained mean HIV-p24 antigen varying between 20.35 and 85.54 ng/mL, while that from pooled CSs ranged between 57.54 and 88.10 ng/mL. Thus, PBMNCs pooled from multiple donors provide an optimal CS for coculturing PHIV. Cryopreserved PBMNC-CS for PHIV cocultures is promising for systematic biosynthesis of PHIV. CONCLUSIONS: Pooled CSs after cryopreservation were functional for PHIV replication and enable on-demand diagnostic cocultures, preparing panels of PHIV stocks for bnAb testing and developing an envelope-subunit vaccine candidate analogous to hepatitis B surface antigen, proven successful in preventing hepatitis B virus infection.
Authors: Abby K Wegehaupt; Ellen K Roufs; Cory R Hewitt; Marisela L Killian; Oxana Gorbatenko; Cynthia M Anderson; M Scott Killian Journal: Biologicals Date: 2017-07-31 Impact factor: 1.856