RTE and CTE mRNA export elements synergistically increase expression of unstable, Rev-dependent HIV and SIV mRNAs

Studies of retroviral mRNA export identified two distinct RNA export elements utilizing conserved eukaryotic mRNA export mechanism(s), namely the Constitutive Transport Element (CTE) and the RNA Transport Element (RTE). Although RTE and CTE are potent in nucleocytoplasmic mRNA transport and expression, neither element is as powerful as the Rev-RRE posttranscriptional control. Here, we found that whereas CTE and the up-regulatory mutant RTEm26 alone increase expression from a subgenomic gag and env clones, the combination of these elements led to a several hundred-fold, synergistic increase. The use of the RTEm26-CTE combination is a simple way to increase expression of poorly expressed retroviral genes to levels otherwise only achieved via more cumbersome RNA optimization. The potent RTEm26-CTE element could be useful in lentiviral gene therapy vectors, DNA-based vaccine vectors, and gene transfer studies of other poorly expressed genes

Rev-Independent Simian Immunodeficiency Virus Strains Are Nonpathogenic in Neonatal Macaques

The viral protein Rev is essential for the export of the subset of unspliced and partially spliced lentiviral mRNAs and the production of structural proteins. Rev and its RNA binding site RRE can be replaced in both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) by the constitutive RNA transport element CTE of the simian type D retroviruses. We used neonatal macaques as a sensitive animal model to evaluate the pathogenicity of a pair of SIV mutant strains generated from Rev-independent molecular clones of SIVmac239 which differ only in the presence of the nef open reading frame. After high primary viremia, all animals remained persistently infected at levels below the threshold of detection. All macaques infected as neonates developed normally, and none showed any signs of immune dysfunction or disease during follow-up ranging from 2.3 to 4 years. Therefore, the Rev-RRE regulatory mechanism plays a key role in the maintenance of high levels of virus propagation, which is independent of the presence of nef. These data demonstrate that Rev regulation plays an important role in the pathogenicity of SIV. Replacement of Rev-RRE by the CTE provides a novel approach to dramatically lower the virulence of a pathogenic lentivirus. These data further suggest that antiretroviral strategies leading to even a partial block of Rev function may modulate disease progression in HIV-infected individuals

Long-Lasting Decrease in Viremia in Macaques Chronically Infected with Simian Immunodeficiency Virus SIVmac251 after Therapeutic DNA Immunization

Rhesus macaques chronically infected with highly pathogenic simian immunodeficiency virus (SIV) SIVmac251 were treated with antiretroviral drugs and vaccinated with combinations of DNA vectors expressing SIV antigens. Vaccination during therapy increased cellular immune responses. After the animals were released from therapy, the virus levels of 12 immunized animals were significantly lower (P = 0.001) compared to those of 11 animals treated with only antiretroviral drugs. Vaccinated animals showed a persistent increase in immune responses, thus indicating both a virological and an immunological benefit following DNA therapeutic vaccination. Several animals show a long-lasting decrease in viremia, suggesting that therapeutic vaccination may provide an additional benefit to antiretroviral therapy

DNA vaccination in rhesus macaques induces potent immune responses and decreases acute and chronic viremia after SIVmac251 challenge

Optimized plasmid DNAs encoding the majority of SIVmac239 proteins and delivered by electroporation (EP) elicited strong immune responses in rhesus macaques. Vaccination decreased viremia in both the acute and chronic phases of infection after challenge with pathogenic SIVmac251. Two groups of macaques were vaccinated with DNA plasmids producing different antigen forms, “native” and “modified,” inducing distinct immune responses. Both groups showed significantly lower viremia during the acute phase of infection, whereas the group immunized with the native antigens showed better protection during the chronic phase (1.7 log decrease in virus load, P = 0.009). Both groups developed strong cellular and humoral responses against the DNA vaccine antigens, which included Gag, Pol, Env, Nef, and Tat. Vaccination induced both central memory and effector memory T cells that were maintained at the day of challenge, suggesting the potential for rapid mobilization upon virus challenge. The group receiving the native antigens developed higher and more durable anti-Env antibodies, including neutralizing antibodies at the day of challenge. These results demonstrate that DNA vaccination in the absence of any heterologous boost can provide protection from high viremia comparable to any other vaccine modalities tested in this macaque model