The present invention relates generally to immunization and immunotherapy for the treatment or prevention of HIV. In particular, the methods relate to in vivo and ex vivo enrichment of HIV-specific CD4 T cells. In certain embodiments, the disclosed compositions and methods can incorporate therapy in order to further enhance the HIV-specific CD4 T cells.

Provided are polynucleotides containing a modified PRE having a variant X gene that includes one or more stop codons not present in an unmodified, such as wild-type, hepatitis X gene. Also provided are polynucleotides containing a modified PRE having a variant X gene that includes one or more degradation sequences not present in an unmodified, such as wild-type, hepatitis X gene. The modified PRE can be operably linked to a nucleic acid encoding a recombinant protein. Also provided are expression cassettes, viral vectors and cells containing the polynucleotides, and compositions and methods of use thereof.

In accordance with the present invention, a method for increasing the yield of rLV vector particles comprising a trans gene encoding a therapeutic protein or fragment thereof is disclosed. In one approach, cells are transfected with plasmids encoding the necessary components for rLV production using a calcium chloride transfection mix at pH 7.1 wherein the calcium chloride and plasmids form a complex which is added to the cells at a constant speed. The cells are then incubated for a suitable time period wherein virus particle media is collected at least twice during the incubation period and stored in a cold storage unit, thereby reducing virus inactivation.

The present disclosure provides a method of generating a stable producer cell line. The generation of stable producer cell lines, such as those provided in accordance with the present invention, increases the reproducibility and ease of creating high titer lentiviral stocks while easing biosafety concerns and the variation in expressed envelope proteins defines the tropism of the generated virus. The present disclosure also provides for a novel lentiviral transfer vector plasmid.

The present invention relates generally to immunization and immunotherapy for the treatment or prevention of HIV. In particular, the methods relate to in vivo and ex vivo enrichment of HIV-specific CD4 T cells. In certain embodiments, the disclosed compositions and methods can incorporate therapy in order to further enhance the HIV-specific CD4 T cells.

The present disclosure provides a method of generating a stable producer cell line. The generation of stable producer cell lines, such as those provided in accordance with the present invention, increases the reproducibility and ease of creating high titer lentiviral stocks while easing biosafety concerns and the variation in expressed envelope proteins defines the tropism of the generated virus. The present disclosure also provides for a novel lentiviral transfer vector plasmid.

The invention is directed to a system comprising a lentivirus vector particle which encodes at least one guide RNA sequence that is complementary to a first DNA sequence in a host cell genome, a Cas9 protein, and optionally a donor nucleic acid molecule comprising a second DNA sequence. The invention also is directed to a method of altering a DNA sequence in a host cell using such a system, where the host cell can be in a human and the altered DNA can be of the human -globin gene. The invention also is directed to a fusion protein comprising a Cas9 protein and a cyclophilin A (CypA) protein. The invention also is directed to sequences of vectors that can be used in the system and method.

An enveloped viral particle producer or packaging cell, wherein the cell is genetically engineered to decrease expression of MHC-I on the surface of the cell.

The present invention relates generally to immunization and immunotherapy for the treatment or prevention of HIV. In particular, the methods relate to in vivo and ex vivo enrichment of HIV-specific CD4 T cells. In certain embodiments, the disclosed compositions and methods can incorporate therapy in order to further enhance the HIV-specific CD4 T cells.

The present invention provides for novel compositions and methods for delivering genes of interest to stem cells using vectors that contain differentiation-specific transcriptional regulatory elements. For example, stem cells in the internal epithelia could be transfected with a vaccine construct, which has an epithelial cell differentiation-specific promoter driving the expression of viral envelope proteins. When the promoter used is specific for terminally differentiated epithelial cells, then the viral envelope proteins will be expressed only in the upper part of the epithelia and therefore, stimulate the immune response. The infected epithelial stem cells in the basal layer will continue to produce new antigen-expressing cells, without being eliminated by the immune response. This invention will be useful in the development of vaccines against viral agents that target the internal mucosa like HIV.