SUMMARY Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a combination of epigenomic modifiers and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress toward iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenomic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.

Paclitaxel cytotoxicity in breast tumors depends on multipolar spindle maintenance and preexisting chromosomal instability.

A number of simian and simian human immunodeficiency viruses (SIV and SHIV, respectively) have been used to assess the efficacy of HIV-1 vaccine strategies. Among these, SIVmac239 is considered among the most stringent because, unlike SHIV models, its full genome has coevolved in its macaque host and its tier 3 envelope glycoprotein (Env) is exceptionally hard to neutralize. Here, we investigated the ability of eCD4-Ig, an antibody-like entry inhibitor that emulates the HIV-1 and SIV receptor and coreceptor, to prevent SIVmac239 infection. We show that rh-eCD4-IgI39N expressed by recombinant adeno-associated virus (AAV) vectors afforded four rhesus macaques complete protection from high-dose SIVmac239 challenges that infected all eight control macaques. However, rh-eCD4-IgI39N–expressing macaques eventually succumbed to serial escalating challenge doses that were 2, 8, 16, and 32 times the challenge doses that infected the control animals. Despite receiving greater challenge doses, these macaques had significantly lower peak and postpeak viral loads than the control group. Virus isolated from three of four macaques showed evidence of strong immune pressure from rh-eCD4-IgI39N, with mutations located in the CD4-binding site, which, in one case, exploited a point-mutation difference between rh-eCD4-IgI39N and rhesus CD4. Other escape pathways associated with clear fitness costs to the virus. Our data report effective protection of rhesus macaques from SIVmac239.

Adeno-associated virus (AAV) delivery of potent and broadly neutralizing antibodies (bNAbs is a promising approach for the prevention of HIV-1 infection. The immunoglobulin G (IgG)1 subtype is usually selected for this application, because it efficiently mediates antibody effector functions and has a somewhat longer half-life. However, the use of IgG1-Fc has been associated with the generation of anti-drug antibodies (ADAs) that correlate with loss of antibody expression. In contrast, we have shown that expression of the antibody-like molecule eCD4-Ig bearing a rhesus IgG2-Fc domain showed reduced immunogenicity and completely protected rhesus macaques from simian-HIV (SHIV)-AD8 challenges. To directly compare the performance of the IgG1-Fc and the IgG2-Fc domains in a prophylactic setting, we compared AAV1 expression of rhesus IgG1 and IgG2 forms of four anti-HIV bNAbs: 3BNC117, NIH45-46, 10-1074, and PGT121. Interestingly, IgG2-isotyped bNAbs elicited significantly lower ADA than their IgG1 counterparts. We also observed significant protection from two SHIV-AD8 challenges in macaques expressing IgG2-isotyped bNAbs, but not from those expressing IgG1. Our data suggest that monoclonal antibodies isotyped with IgG2-Fc domains are less immunogenic than their IgG1 counterparts, and they highlight ADAs as a key barrier to the use of AAV1-expressed bNAbs.