ABSTRACT
Limited cellular levels of the HIV transcriptional activator Tat are one contributor to proviral latency that might be targeted in HIV cure strategies. We recently demonstrated that lipid nanoparticles containing HIV tat mRNA induce HIV expression in primary CD4 T cells. Here, we sought to further characterize tat mRNA in the context of several benchmark latency reversal agents (LRAs), including inhibitor of apoptosis protein antagonists (IAPi), bromodomain and extra-Terminal motif inhibitors (BETi), and histone deacetylase inhibitors (HDACi). tat mRNA reversed latency across several different cell line models of HIV latency, an effect dependent on the TAR hairpin loop. Synergistic enhancement of tat mRNA activity was observed with IAPi, HDACi, and BETi, albeit to variable degrees. In primary CD4 T cells from durably suppressed people with HIV, tat mRNA profoundly increased the frequencies of elongated, multiply-spliced, and polyadenylated HIV transcripts, while having a lesser impact on TAR transcript frequencies. tat mRNAs alone resulted in variable HIV p24 protein induction across donors. However, tat mRNA in combination with IAPi, BETi, or HDACi markedly enhanced HIV RNA and protein expression without overt cytotoxicity or cellular activation. Notably, combination regimens approached or in some cases exceeded the latency reversal activity of maximal mitogenic T cell stimulation. Higher levels of tat mRNA-driven HIV p24 induction were observed in donors with larger mitogen-inducible HIV reservoirs, and expression increased with prolonged exposure time. Combination LRA strategies employing both small molecule inhibitors and Tat delivered to CD4 T cells are a promising approach to effectively target the HIV reservoir.
Subject(s)
HIV Infections , HIV-1 , Histone Deacetylase Inhibitors , Nanoparticles , Virus Latency , tat Gene Products, Human Immunodeficiency Virus , Humans , Anti-HIV Agents/pharmacology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/virology , CD4-Positive T-Lymphocytes/metabolism , Histone Deacetylase Inhibitors/pharmacology , HIV Antigens/genetics , HIV Infections/drug therapy , HIV Infections/virology , HIV-1/drug effects , HIV-1/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , tat Gene Products, Human Immunodeficiency Virus/genetics , Virus Latency/drug effectsABSTRACT
A major barrier to HIV-1 cure is caused by the pool of latently infected CD4 T-cells that persist under combination antiretroviral therapy (cART). This latent reservoir is capable of producing replication-competent infectious viruses once prolonged suppressive cART is withdrawn. Inducing the reactivation of HIV-1 gene expression in T-cells harboring a latent provirus in people living with HIV-1 under cART may result in depletion of this latent reservoir due to cytopathic effects or immune clearance. Studies have investigated molecules that reactivate HIV-1 gene expression, but to date, no latency reversal agent has been identified to eliminate latently infected cells harboring replication-competent HIV in cART-treated individuals. Stochastic fluctuations in HIV-1 tat gene expression have been described and hypothesized to allow the progression into proviral latency. We hypothesized that exposing latently infected CD4+ T-cells to Tat would result in effective latency reversal. Our results indicate the capacity of a truncated Tat protein and mRNA to reactivate HIV-1 in latently infected T-cells ex vivo to a similar degree as the protein kinase C agonist: phorbol 12-myristate 13-acetate, without T-cell activation or any significant transcriptome perturbation.
Subject(s)
HIV Infections , HIV-1 , Virus Activation , tat Gene Products, Human Immunodeficiency Virus , Humans , CD4-Positive T-Lymphocytes , HIV Infections/genetics , HIV Infections/metabolism , Proviruses/genetics , Virus Latency , Virus Replication , tat Gene Products, Human Immunodeficiency Virus/genetics , tat Gene Products, Human Immunodeficiency Virus/metabolism , HIV-1/genetics , HIV-1/metabolismABSTRACT
A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4+ T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 µg and 10 µg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.
Subject(s)
Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , COVID-19 Vaccines/administration & dosage , COVID-19/prevention & control , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Synthetic/administration & dosage , Alphavirus/genetics , Alphavirus/immunology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/immunology , Animals , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/virology , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , COVID-19 Vaccines/biosynthesis , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Female , Gene Expression , Humans , Immunity, Cellular/drug effects , Immunity, Humoral/drug effects , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-4/genetics , Interleukin-4/immunology , Mice , Mice, Transgenic , Replicon/immunology , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Th1 Cells/drug effects , Th1 Cells/immunology , Th1 Cells/virology , Transgenes , Treatment Outcome , Vaccination/methods , Vaccines, Synthetic/biosynthesis , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology , mRNA VaccinesABSTRACT
Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4-6 h) that remains stable for up to 4-6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA-LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable.
Subject(s)
Drug Carriers/administration & dosage , Factor IX/pharmacokinetics , Hemophilia B/therapy , Nanoparticles/administration & dosage , RNA, Messenger/pharmacokinetics , Animals , Cholesterol/chemistry , Cytokines/metabolism , Disease Models, Animal , Drug Compounding/methods , Drug Evaluation, Preclinical , Factor IX/genetics , Factor IX/metabolism , Female , Genetic Therapy/methods , Hemophilia B/genetics , Hemophilia B/metabolism , Hemophilia B/pathology , Humans , Hydrogen-Ion Concentration , Injections, Intravenous , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Knockout , Phosphatidylcholines/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacokineticsABSTRACT
The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells' transcriptome and proteome profiles.
Subject(s)
Gene Products, tat , HIV-1 , Nanoparticles , RNA, Viral , Virus Latency , Virus Latency/drug effects , Virus Latency/genetics , Gene Products, tat/genetics , Gene Products, tat/metabolism , RNA, Viral/administration & dosage , RNA, Viral/genetics , RNA, Viral/metabolism , Nanoparticles/administration & dosage , Nanoparticles/chemistry , HIV Infections/drug therapy , HIV Infections/genetics , HIV Infections/virology , Panobinostat/pharmacology , Antiretroviral Therapy, Highly Active , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/virology , CD4 Antigens/genetics , CD4 Antigens/metabolism , HIV-1/drug effects , HIV-1/genetics , Proviruses/drug effects , Proviruses/genetics , Single-Cell Gene Expression Analysis , HIV Core Protein p24/genetics , HIV Core Protein p24/metabolism , RNA, Long Noncoding/metabolism , Cells, Cultured , Humans , Ionomycin/pharmacologyABSTRACT
Phenylketonuria (PKU) is a genetic disorder affecting around 1 in 12,000 live births (1), caused by a mutation in the phenylalanine hydroxylase (PAH) gene in the liver which facilitates the catabolism of phenylalanine (Phe). Without a functional copy of PAH, levels of Phe in the blood and tissues rise, resulting in potentially life-threatening damage to the central nervous system. (2) Treatment options for PKU are limited, and center around adherence to a strict PKU diet that suffers from poor patient compliance. There are two approved drugs available, one of which must be used in conjunction with the PKU diet and another that has serious immunological side effects. Here we demonstrate that the LUNAR® delivery technology is capable of delivering mRNA for a replacement enzyme, the bacterial phenylalanine ammonia lyase (avPAL), into the hepatic tissue of a PKU mouse, and that the enzyme is capable of metabolizing Phe and reducing serum levels of Phe for more than five days post-transfection. We further demonstrate the ability of LUNAR to deliver a plant-derived PAL protein with a similar impact on the level of serum Phe. Taken together these results demonstrate both the capability of LUNAR for the targeted delivery of PAL mRNA into hepatic tissue in vivo, replacing the defective PAH protein and successfully reducing serum Phe levels, thereby addressing the underlying cause of PKU symptoms. Secondly, that plant-based PAL proteins are a viable alternative to bacterial avPAL to reduce the immunogenic response.
ABSTRACT
Phenylketonuria (PKU) is an inborn error caused by deficiencies in phenylalanine (Phe) metabolism. Mutations in the phenylalanine hydroxylase (PAH) gene are the main cause of the disease whose signature hallmarks of toxically elevated levels of Phe accumulation in plasma and organs such as the brain, result in irreversible intellectual disability. Here, we present a unique approach to treating PKU deficiency by using an mRNA replacement therapy. A full-length mRNA encoding human PAH (hPAH) is encapsulated in our proprietary lipid nanoparticle LUNAR and delivered to a Pah enu2 mouse model that carries a missense mutation in the mouse PAH gene. Animals carrying this missense mutation develop hyperphenylalanemia and hypotyrosinemia in plasma, two clinical features commonly observed in the clinical presentation of PKU. We show that intravenous infusion of LUNAR-hPAH mRNA can generate high levels of hPAH protein in hepatocytes and restore the Phe metabolism in the Pah enu2 mouse model. Together, these data establish a proof of principle of a novel mRNA replacement therapy to treat PKU.
ABSTRACT
The use of nucleic acid as a drug substance for vaccines and other gene-based medicines continues to evolve. Here, we have used a technology originally developed for mRNA in vivo delivery to enhance the immunogenicity of DNA vaccines. We demonstrate that neutralizing antibodies produced in rabbits and nonhuman primates injected with lipid nanoparticle (LNP)-formulated Andes virus or Zika virus DNA vaccines are elevated over unformulated vaccine. Using a plasmid encoding an anti-poxvirus monoclonal antibody (as a reporter of protein expression), we showed that improved immunogenicity is likely due to increased in vivo DNA delivery, resulting in more target protein. Specifically, after four days, up to 30 ng/mL of functional monoclonal antibody were detected in the serum of rabbits injected with the LNP-formulated DNA. We pragmatically applied the technology to the production of human neutralizing antibodies in a transchromosomic (Tc) bovine for use as a passive immunoprophylactic. Production of neutralizing antibody was increased by >10-fold while utilizing 10 times less DNA in the Tc bovine. This work provides a proof-of-concept that LNP formulation of DNA vaccines can be used to produce more potent active vaccines, passive countermeasures (e.g., Tc bovine), and as a means to produce more potent DNA-launched immunotherapies.
Subject(s)
Nanoparticles/administration & dosage , Orthohantavirus/immunology , Poxviridae/immunology , Vaccines, DNA , Viral Vaccines/immunology , Zika Virus/immunology , Animals , Animals, Genetically Modified , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Cattle , Chlorocebus aethiops , Chromosomes, Artificial, Human/genetics , Dose-Response Relationship, Immunologic , Female , Genes, Immunoglobulin , Macaca fascicularis , Male , Neutralization Tests , Plasmids , Rabbits , Vero CellsABSTRACT
We explored an emerging technology to produce anti-Hantaan virus (HTNV) and anti-Puumala virus (PUUV) neutralizing antibodies for use as pre- or post-exposure prophylactics. The technology involves hyperimmunization of transchomosomic bovines (TcB) engineered to express human polyclonal IgG antibodies with HTNV and PUUV DNA vaccines encoding GnGc glycoproteins. For the anti-HTNV product, TcB was hyperimmunized with HTNV DNA plus adjuvant or HTNV DNA formulated using lipid nanoparticles (LNP). The LNP-formulated vaccine yielded fivefold higher neutralizing antibody titers using 10-fold less DNA. Human IgG purified from the LNP-formulated animal (SAB-159), had anti-HTNV neutralizing antibody titers >100,000. SAB-159 was capable of neutralizing pseudovirions with monoclonal antibody escape mutations in Gn and Gc demonstrating neutralization escape resistance. SAB-159 protected hamsters from HTNV infection when administered pre- or post-exposure, and limited HTNV infection in a marmoset model. An LNP-formulated PUUV DNA vaccine generated purified anti-PUUV IgG, SAB-159P, with a neutralizing antibody titer >600,000. As little as 0.33 mg/kg of SAB-159P protected hamsters against PUUV infection for pre-exposure and 10 mg/kg SAB-159P protected PUUV-infected hamsters post-exposure. These data demonstrate that DNA vaccines combined with the TcB-based manufacturing platform can be used to rapidly produce potent, human, polyclonal, escape-resistant anti-HTNV, and anti-PUUV neutralizing antibodies that are protective in animal models.
ABSTRACT
Zika virus (ZIKV) is associated with congenital malformations in infants born to infected mothers, and with Guillain-Barré syndrome in infected adults. Development of ZIKV vaccines has focused predominantly on the induction of neutralizing antibodies, although a suboptimal antibody response may theoretically enhance disease severity through antibody-dependent enhancement (ADE). Here, we report induction of a protective anti-ZIKV CD8+ T cell response in the HLA-B*0702 Ifnar1-/- transgenic mice using an alphavirus-based replicon RNA vaccine expressing ZIKV nonstructural protein NS3, a potent T cell antigen. The NS3 vaccine did not induce a neutralizing antibody response but elicited polyfunctional CD8+ T cells that were necessary and sufficient for preventing death in lethally infected adult mice and fetal growth restriction in infected pregnant mice. These data identify CD8+ T cells as the major mediators of ZIKV NS3 vaccine-induced protection and suggest a new strategy to develop safe and effective anti-flavivirus vaccines.