SIV antigen immunization induces transient antigen-specific T cell responses and selectively activates viral replication in draining lymph nodes in retroviral suppressed rhesus macaques

<p>Abstract</p> <p>Background</p> <p>HIV infection causes a qualitative and quantitative loss of CD4⁺T cell immunity. The institution of anti-retroviral therapy (ART) restores CD4⁺T cell responses to many pathogens, but HIV-specific responses remain deficient. Similarly, therapeutic immunization with HIV antigens of chronically infected, ART treated subjects results in poor induction of HIV-specific CD4 responses. In this study, we used a macaque model of ART treatment during chronic infection to study the virologic consequences of SIV antigen stimulation in lymph nodes early after immunization. Rhesus CMV (RhCMV) seropositive, Mamu A*01 positive rhesus macaques were chronically infected with SIVmac251 and treated with ART. The immune and viral responses to SIV gag and RhCMV pp65 antigen immunization in draining lymph nodes and peripheral blood were analyzed. Animals were immunized on contralateral sides with SIV gag and RhCMV pp65 encoding plasmids, which allowed lymph nodes draining each antigen to be obtained at the same time from the same animal for direct comparison.</p> <p>Results</p> <p>We observed that both SIV and RhCMV immunizations stimulated transient antigen-specific T cell responses in draining lymph nodes. The RhCMV-specific responses were potent and sustained (50 days post-immunization) in the periphery, while the SIV-specific responses were transient and extinguished quickly. The SIV antigen stimulation selectively induced transient SIV replication in draining lymph nodes.</p> <p>Conclusions</p> <p>The data are consistent with a model whereby viral replication in response to SIV antigen stimulation limits the generation of SIV antigen-specific responses and suggests a potential mechanism for the early loss and poor HIV-specific CD4⁺T cell response observed in HIV-infected individuals.</p

Matrix Metalloproteinases in Tuberculosis-Immune Reconstitution Inflammatory Syndrome and Impaired Lung Function Among Advanced HIV/TB Co-infected Patients Initiating Antiretroviral Therapy

AbstractBackgroundHIV-infected patients with pulmonary TB (pTB) can have worsening of respiratory symptoms as part of TB-immune reconstitution inflammatory syndrome (TB-IRIS) following antiretroviral therapy (ART) initiation. Thus, reconstitution of immune function on ART could drive incident lung damage in HIV/TB.MethodsWe hypothesized that increases in matrix metalloproteinases (MMPs), which can degrade lung matrix, on ART are associated with TB-IRIS among a cohort of advanced, ART naïve, HIV-infected adults with pTB. Furthermore, we related early changes in immune measures and MMPs on ART to lung function in an exploratory subset of patients post-TB cure. This study was nested within a prospective cohort study. Rank sum and chi-square tests, Spearman's correlation coefficient, and logistic regression were used for analyses.ResultsIncreases in MMP-8 following ART initiation were independently associated with TB-IRIS (p=0.04; adjusted odds ratio 1.5 [95% confidence interval: 1.0–2.1]; n=32). Increases in CD4 counts and MMP-8 on ART were also associated with reduced forced expiratory volume in one-second post-TB treatment completion (r=−0.7, p=0.006 and r=−0.6, p=0.02, respectively; n=14).ConclusionsART-induced MMP increases are associated with TB-IRIS and may affect lung function post-TB cure. End-organ damage due to TB-IRIS and mechanisms whereby immune restoration impairs lung function in pTB deserve further investigation

Systematic development of ionizable lipid nanoparticles for placental mRNA delivery using a design of experiments approach

Ionizable lipid nanoparticles (LNPs) have gained attention as mRNA delivery platforms for vaccination against COVID-19 and for protein replacement therapies. LNPs enhance mRNA stability, circulation time, cellular uptake, and preferential delivery to specific tissues compared to mRNA with no carrier platform. However, LNPs are only in the beginning stages of development for safe and effective mRNA delivery to the placenta to treat placental dysfunction. Here, we develop LNPs that enable high levels of mRNA delivery to trophoblasts in vitro and to the placenta in vivo with no toxicity. We conducted a Design of Experiments to explore how LNP composition, including the type and molar ratio of each lipid component, drives trophoblast and placental delivery. Our data revealed that utilizing C12-200 as the ionizable lipid and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as the phospholipid in the LNP design yields high transfection efficiency in vitro. Analysis of lipid molar composition as a design parameter in LNPs displayed a strong correlation between apparent pKa and poly (ethylene) glycol (PEG) content, as a reduction in PEG molar amount increases apparent pKa. Further, we present one LNP platform that exhibits the highest delivery of placental growth factor mRNA to the placenta in pregnant mice, resulting in synthesis and secretion of a potentially therapeutic protein. Lastly, our high-performing LNPs have no toxicity to both the pregnant mice and fetuses. Our results demonstrate the feasibility of LNPs as a platform for mRNA delivery to the placenta, and our top LNP formulations may provide a therapeutic platform to treat diseases that originate from placental dysfunction during pregnancy

Declines in Lung Function After Antiretroviral Therapy Initiation in Adults With Human Immunodeficiency Virus and Tuberculosis: A Potential Manifestation of Respiratory Immune Reconstitution Inflammatory Syndrome

End-organ impairment has received relatively little research attention as a possible manifestation of tuberculosis immune reconstitution inflammatory syndrome (TB-IRIS). In this prospective cohort study, one-half of adults with human immunodeficiency virus and pulmonary tuberculosis experienced meaningful declines in lung function on antiretroviral therapy, suggesting a role for lung function in TB-IRIS definitions

Increased Erythropoiesis in Mice Injected With Submicrogram Quantities of Pseudouridine-containing mRNA Encoding Erythropoietin

Advances in the optimization of in vitro-transcribed mRNA are bringing mRNA-mediated therapy closer to reality. In cultured cells, we recently achieved high levels of translation with high-performance liquid chromatography (HPLC)-purified, in vitro-transcribed mRNAs containing the modified nucleoside pseudouridine. Importantly, pseudouridine rendered the mRNA non-immunogenic. Here, using erythropoietin (EPO)-encoding mRNA complexed with TransIT-mRNA, we evaluated this new generation of mRNA in vivo. A single injection of 100 ng (0.005 mg/kg) mRNA elevated serum EPO levels in mice significantly by 6 hours and levels were maintained for 4 days. In comparison, mRNA containing uridine produced 10–100-fold lower levels of EPO lasting only 1 day. EPO translated from pseudouridine-mRNA was functional and caused a significant increase of both reticulocyte counts and hematocrits. As little as 10 ng mRNA doubled reticulocyte numbers. Weekly injection of 100 ng of EPO mRNA was sufficient to increase the hematocrit from 43 to 57%, which was maintained with continued treatment. Even when a large amount of pseudouridine-mRNA was injected, no inflammatory cytokines were detectable in plasma. Using macaques, we could also detect significantly-increased serum EPO levels following intraperitoneal injection of rhesus EPO mRNA. These results demonstrate that HPLC-purified, pseudouridine-containing mRNAs encoding therapeutic proteins have great potential for clinical applications

Ionizable lipid nanoparticles for in utero mRNA delivery.

Clinical advances enable the prenatal diagnosis of genetic diseases that are candidates for gene and enzyme therapies such as messenger RNA (mRNA)-mediated protein replacement. Prenatal mRNA therapies can treat disease before the onset of irreversible pathology with high therapeutic efficacy and safety due to the small fetal size, immature immune system, and abundance of progenitor cells. However, the development of nonviral platforms for prenatal delivery is nascent. We developed a library of ionizable lipid nanoparticles (LNPs) for in utero mRNA delivery to mouse fetuses. We screened LNPs for luciferase mRNA delivery and identified formulations that accumulate within fetal livers, lungs, and intestines with higher efficiency and safety compared to benchmark delivery systems, DLin-MC3-DMA and jetPEI. We demonstrate that LNPs can deliver mRNAs to induce hepatic production of therapeutic secreted proteins. These LNPs may provide a platform for in utero mRNA delivery for protein replacement and gene editing

Spots & stripes: pleomorphic patterning of stem cells via p-ERK-depenendent cell chemotaxis shown by feather morphogenesis & mathematical simulation

A key issue in stem cell biology is the differentiation of homogeneous stem cells towards different fates which are also organized into desired configurations. Little is known about the mechanisms underlying the process of periodic patterning. Feather explants offer a fundamental and testable model in which multi-potential cells are organized into hexagonally arranged primordia and the spacing between primordia. Previous work explored roles of a Turing reaction–diffusion mechanism in establishing chemical patterns. Here we show that a continuum of feather patterns, ranging from stripes to spots, can be obtained when the level of p-ERK activity is adjusted with chemical inhibitors. The patterns are dose-dependent, tissue stage-dependent, and irreversible. Analyses show that ERK activity-dependent mesenchymal cell chemotaxis is essential for converting micro-signaling centers into stable feather primordia. A mathematical model based on short-range activation, long-range inhibition, and cell chemotaxis is developed and shown to simulate observed experimental results. This generic cell behavior model can be applied to model stem cell patterning behavior at large