Comparative Characterization of Transfection- and Infection-Derived Simian Immunodeficiency Virus Challenge Stocks for In Vivo Nonhuman Primate Studies

Abstract

h Simian immunodeficiency virus (SIV) stocks for in vivo nonhuman primate models of AIDS are typically generated by transfection of 293T cells with molecularly cloned viral genomes or by expansion in productively infected T cells. Although titers of stocks are determined for infectivity in vitro prior to in vivo inoculation, virus production methods may differentially affect stock features that are not routinely analyzed but may impact in vivo infectivity, mucosal transmissibility, and early infection events. We performed a detailed analysis of nine SIV stocks, comprising five infection-derived SIVmac251 viral swarm stocks and paired infection-and transfected-293T-cell-derived stocks of both SIVmac239 and SIVmac766. Representative stocks were evaluated for (i) virus content, (ii) infectious titer, (iii) sequence diversity and polymorphism frequency by single-genome amplification and 454 pyrosequencing, (iv) virion-associated Env content, and (v) cytokine and chemokine content by 36-plex Luminex analysis. Regardless of production method, all stocks had comparable particle/infectivity ratios, with the transfected-293T stocks possessing the highest overall virus content and infectivity titers despite containing markedly lower levels of virion-associated Env than infection-derived viruses. Transfected-293T stocks also contained fewer and lower levels of cytokines and chemokines than infection-derived stocks, which had elevated levels of multiple analytes, with substantial variability among stocks. Sequencing of the infection-derived SIVmac251 stocks revealed variable levels of viral diversity between stocks, with evidence of stock-specific selection and expansion of unique viral lineages. These analyses suggest that there may be underappreciated features of SIV in vivo challenge stocks with the potential to impact early infection events, which may merit consideration when selecting virus stocks for in vivo studies.

CD4 + T Cells Support Production of Simian Immunodeficiency Virus Env Antibodies That Enforce CD4-Dependent Entry and Shape Tropism In Vivo

“…In contrast to D470N and E84K, G62N was enriched in CD4-depleted animals at both day 11 and day 42 but was not significantly associated with CD4-independent entry. G62N is present at an overall frequency of 4% to 5% among SIVmac sequences and occasionally represents the dominant sequence among some SIVmac251 swarms (33,34). Thus, additional selective forces in the CD4-depleted animals other than CD4 independence per se appear to be responsible for selection at this residue.…”

“…Only one day 42 Env from a CD4-depleted animal did not mediate appreciable CD4-independent entry (RZj5.D42.9.2) and did not carry D470N, E84K, or any previously described polymorphism(s) associated with CD4 independence. G62N, E84K, and D470N have not been explicitly characterized in previous studies, but an analysis of SIVmac sequences revealed that G62N has ϳ5% frequency within SIVmac251 viral stocks (31)(32)(33)(34), while D470N and E84K are extremely rare and present mainly in in vivo-passaged SIVmac251-related isolates (34)(35)(36)(37)(38). Of note, all instances of G62N, E84K, and D470N that arose in these animals were the result of G-to-A nucleotide changes (Fig.…”

“…E84K is present at a low frequency in some stocks of SIVmacBK28 and SIVmac251 (33,34,36), and D470N is present in a few brain and plasma sequences in a model of rapid disease progression (37). E84K is also present in an in vitro-derived CD4-independent variant of SIVmac239 (35,64), although its specific contribution to CD4 independence was not assessed.…”

Enhanced antagonism of BST-2 by a neurovirulent SIV envelope

“…Therefore, to assess if these 4 amino acid substitutions affected the incorporation of Env glycoproteins by virions, SIVsmE543-3 and SIVsmE543CT clones were purified, concentrated, and examined by SDS-PAGE using a dual-color fluorescent imaging method ( Figure 5A), and the ratio of virion p27 content against virion gp120 content was calculated ( Figure 5B and refs. 27,28). The levels of virion-incorporated Env protein between SIVsmE543-3 and SIVsmE543CT clones were not significantly different, indicating that the mutations we have identified as enhancing replication in macrophages do not influence the virion incorporation of Env.…”

“…Quantitative measurements of viral p27 capsid (CA) and gp120 protein in virions for determinations of Gag/Env ratio were determined using dual-color fluorescent protein gel analysis as previously described (27,28). Briefly, SIVsmE543-3 and SIVsmE543CT clones were expanded in CEM cells, concentrated and purified, and examined by SDS-PAGE.…”