Objective(s): Despite antiretroviral therapy (ART), there is an unmet need for therapies to mitigate immune activation in HIV infection. The goal of this study is to determine whether the apoA-I mimetics 6F and 4F attenuate macrophage activation in chronic HIV. Design: Preclinical assessment of the in-vivo impact of Tg6F and the ex-vivo impact of apoA-I mimetics on biomarkers of immune activation and gut barrier dysfunction in treated HIV. Methods: We used two humanized murine models of HIV infection to determine the impact of oral Tg6F with ART (HIV+ART+Tg6F+) on innate immune activation (plasma human sCD14, sCD163) and gut barrier dysfunction [murine I-FABP, endotoxin (LPS), LPS-binding protein (LBP), murine sCD14]. We also used gut explants from 10 uninfected and 10 HIV-infected men on potent ART and no morbidity, to determine the impact of ex-vivo treatment with 4F for 72 h on secretion of sCD14, sCD163, and I-FABP from gut explants. Results: When compared with mice treated with ART alone (HIV+ART+), HIV+ART+Tg6F+ mice attenuated macrophage activation (h-sCD14, h-sCD163), gut barrier dysfunction (m-IFABP, LPS, LBP, and m-sCD14), plasma and gut tissue oxidized lipoproteins. The results were consistent with independent mouse models and ART regimens. Both 4F and 6F attenuated shedding of I-FABP and sCD14 from gut explants from HIV-infected and uninfected participants. Conclusion: Given that gut barrier dysfunction and macrophage activation are contributors to comorbidities like cardiovascular disease in HIV, apoA-I mimetics should be tested as therapy for morbidity in chronic treated HIV.

Studies of two SARS-CoV-2 mRNA vaccines suggested that they yield ∼95% protection from symptomatic infection at least short-term, but important clinical questions remain. It is unclear how vaccine-induced antibody levels quantitatively compare to the wide spectrum induced by natural SARS-CoV-2 infection. Vaccine response kinetics and magnitudes in persons with prior COVID-19 compared to virus-naı̈ve persons are not well-defined. The relative stability of vaccine-induced versus infection-induced antibody levels is unclear. We addressed these issues with longitudinal assessments of vaccinees with and without prior SARS-CoV-2 infection using quantitative enzyme-linked immunosorbent assay (ELISA) of anti-RBD antibodies. SARS-CoV-2-naı̈ve individuals achieved levels similar to mild natural infection after the first vaccination; a second dose generated levels approaching severe natural infection. In persons with prior COVID-19, one dose boosted levels to the high end of severe natural infection even in those who never had robust responses from infection, increasing no further after the second dose. Antiviral neutralizing assessments using a spike-pseudovirus assay revealed that virus-naı̈ve vaccinees did not develop physiologic neutralizing potency until the second dose, while previously infected persons exhibited maximal neutralization after one dose. Finally, antibodies from vaccination waned similarly to natural infection, resulting in an average of ∼90% loss within 90 days. In summary, our findings suggest that two doses are important for quantity and quality of humoral immunity in SARS-CoV-2-naı̈ve persons, while a single dose has maximal effects in those with past infection. Antibodies from vaccination wane with kinetics very similar to that seen after mild natural infection; booster vaccinations will likely be required.

Mouse somatic cells can be reprogrammed into induced pluripotent stem cells by defined factors known to regulate pluripotency, including Oct4, Sox2, Klf4, and c-Myc. Together with Oct4, Sox2 plays a major role as a master endogenous pluripotent genes trigger in reprogramming. It has been reported that Sirtuin 1 (Sirt1), a member of the Sirtuin family of NAD 1 -dependent protein deacetylases, is involved in embryonic stem cell antioxidation, differentiation, and individual development. However, as a deacetylation enzyme, whether Sirt1 influences reprogramming through its post-translational modification function remains unknown. In this study, we provide evidence that deacetylation of Sox2 by Sirt1 is required for reprogramming. We found that a low level of Sox2 acetylation could significantly increase reprogramming efficiency. Furthermore, we found that Sox2 can be deacetylated by Sirt1 in an Oct4-mediated manner. Compared with wild-type cells, Sirt1-null mouse embryonic fibroblasts exhibit decreased reprogramming efficiency, and overexpression of Sirt1 rescues this defect. In addition, Sirt1 functions in the regulation of reprogramming through deacetylating Sox2. Taken together, we have identified a new regulatory role of Sirt1 in reprogramming and provided a link between deacetylation events and somatic cell reprogramming.

Due to the durability and persistence of reservoirs of HIV-1-infected cells, combination antiretroviral therapy (ART) is insufficient in eradicating infection. Achieving HIV-1 cure or sustained remission without ART treatment will require the enhanced and persistent effective antiviral immune responses. Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy and show promise in treating HIV-1 infection. Persistence, trafficking, and maintenance of function remain to be a challenge in many of these approaches, which are based on peripheral T cell modification. To overcome many of these issues, we have previously demonstrated successful long-term engraftment and production of anti-HIV CAR T cells in modified hematopoietic stem cells (HSCs) in vivo. Here we report the development and in vivo testing of second generation CD4-based CARs (CD4CAR) against HIV-1 infection using a HSCs-based approach. We found that a modified, truncated CD4-based CAR (D1D2CAR) allows better CAR-T cell differentiation from gene modified HSCs, and maintains similar CTL activity as compared to the full length CD4-based CAR. In addition, D1D2CAR does not mediate HIV infection or stimulation mediated by IL-16, suggesting lower risk of off-target effects. Interestingly, stimulatory domains of 4-1BB but not CD28 allowed successful hematopoietic differentiation and improved anti-viral function of CAR T cells from CAR modified HSCs. Addition of 4-1BB to CD4 based CARs led to faster suppression of viremia during early untreated HIV-1 infection. D1D2CAR 4-1BB mice had faster viral suppression in combination with ART and better persistence of CAR T cells during ART. In summary, our data indicate that the D1D2CAR-41BB is a superior CAR, showing better HSC differentiation, viral suppression and persistence, and less deleterious functions compared to the original CD4CAR, and should continue to be pursued as a candidate for clinical study.

The HIV reservoir remains to be a difficult barrier to overcome in order to achieve a therapeutic cure for HIV. Several strategies have been developed to purge the reservoir, including the “kick and kill” approach, which is based on the notion that reactivating the latent reservoir will allow subsequent elimination by the host anti-HIV immune cells. However, clinical trials testing certain classes of latency reactivating agents (LRAs) have so far revealed the minimal impact on reducing the viral reservoir. A robust immune response to reactivated HIV expressing cells is critical for this strategy to work. A current focus to enhance anti-HIV immunity is through the use of chimeric antigen receptors (CARs). Currently, HIV-specific CARs are being applied to peripheral T cells, NK cells, and stem cells to boost recognition and killing of HIV infected cells. In this review, we summarize current developments in engineering HIV directed CAR-expressing cells to facilitate HIV elimination. We also summarize current LRAs that enhance the “kick” strategy and how new generation and combinations of LRAs with HIV specific CAR T cell therapies could provide an optimal strategy to target the viral reservoir and achieve HIV clearance from the body.

Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness.

Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore, Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion, the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.

SIRT1, a mammalian ortholog of yeast silent information regulator 2 (Sir2), is an NAD + -dependent protein deacetylase that plays a critical role in the regulation of vascular function. The current study aims to investigate the functional significance of deacetylase activity of SIRT1 in heart. Here we show that the early postnatal hearts expressed the highest level of SIRT1 deacetylase activity compared to adult and aged hearts. We generated transgenic mice with cardiac-specific expression of a dominant-negative form of the human SIRT1 (SIRT1H363Y), which represses endogenous SIRT1 activity. The transgenic mice displayed dilated atrial and ventricular chambers, and died early in the postnatal period. Pathological, echocardiographic and molecular phenotype confirmed the presence of dilated cardiomyopathy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling analysis revealed a greater abundance of apoptotic nuclei in the hearts of transgenic mice. Furthermore, we show that cardiomyocyte apoptosis caused by suppression of SIRT1 activity is, at least in part, due to increased p53 acetylation and upregulated Bax expression. These results indicate that dominant negative form of SIRT1 (SIRT1H363Y) overexpression in mouse hearts causes cardiomyocyte apoptosis and early-onset heart failure, suggesting a critical role of SIRT1 in preserving normal cardiac development during the early postnatal period. deacetylase, SIRT1, apoptosis, heart failure Citation: