Simian Immunodeficiency Virus Vector Research Articles

Rev-Independent Expression of Syntheticgag-polGenes of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus: Implications for the Safety of Lentiviral Vectors

The safety of lentiviral vectors for clinical applications is still a major concern. The gag-pol expression plasmids and the lentiviral vectors used in previous studies contain homologous regions, which constitute a risk for recombination events. Synthetic gag-pol genes of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were therefore constructed, in which the codon usage was optimized for expression in human cells without altering the amino acid sequences. The synthetic gag-pol genes allowed efficient expression of these genes in the absence of Rev and the 5' untranslated leader region. Both the HIV-1 and the SIV synthetic gag-pol expression plasmids could mediate transduction of an SIV vector into nondividing human cells with titers of about 10(6) transducing units/ml. Similar titers were obtained with a four-plasmid vector-packaging system based on HIV-1. Using a biological assay, homologous recombination events between the synthetic gag-pol expression plasmids and an SIV vector were undetectable and in comparison with a previously used gag-pol expression plasmid at least approximately 100-fold less frequent. By eliminating regions of homology and sequences involved in packaging, synthetic gag-pol genes should improve the safety profile of lentiviral vectors.

Development of novel simian immunodeficiency virus vectors carrying a dual gene expression system.

The development of highly efficient and safe gene transfer methods suitable for clinical use is required for human gene therapies. We have developed a novel lentiviral vector system, based on the nonpathogenic simian immunodeficiency virus from African green monkeys (SIVagm), that carries a unique dual gene expression system. This system utilizes the lentivirus Rev responsive element (RRE). Self-inactivating vectors were also developed by deleting a U3 region in the 3' long terminal repeat (3' LTR) of the virus. When pseudotyped with a vesicular stomatitis virus envelope glycoprotein G (VSV-G), the SIVagm-based vectors could transduce both growth-arrested human cells and terminally differentiated neuronal cell lines. Using these vectors, two reporter genes could be expressed simultaneously at equal levels, and expression levels of both genes could be altered by modifying the length of the RRE sequence. These SIVagm-based vectors might offer safety advantages over other lentivirus-based vectors. Furthermore, the novel dual gene expression system described here could increase the usefulness and value of both viral and nonviral vectors in gene therapy.

Expression of gamma interferon by simian immunodeficiency virus increases attenuation and reduces postchallenge virus load in vaccinated rhesus macaques.

Simian immunodeficiency virus (SIV) infection of macaques is a model for human immunodeficiency virus (HIV) infection. We have previously reported the construction and characterization of an SIV vector with a deletion in the nef gene (SIV(delta nef)) and expressing gamma interferon (SIV(HyIFN)) (L. Giavedoni and T. Yilma, J. Virol. 70:2247-2251, 1996). We now show that rhesus macaques vaccinated with SIV(HyIFN) have a lower viral load than a group similarly immunized with SIV(delta nef). Viral loads remained low in the SIV(HyIFN)-vaccinated group even though SIV expressing gamma interferon could not be isolated after 6 weeks postimmunization in these animals. All immunized and two naive control macaques became infected when challenged with virulent SIV(mac251), at 25 weeks postvaccination. In contrast to the two naive controls that died by 12 and 18 weeks postchallenge, all vaccinated animals remained healthy for more than 32 weeks. In addition, postchallenge cell-associated virus load was significantly lower in SIV(HyIFN)-immunized animals than in the group vaccinated with SIV(delta nef). These findings indicate that cytokine-expressing viruses can provide a novel approach for development of safe and efficacious live attenuated vaccines for AIDS.

Rev/RRE-Independent Mason–Pfizer Monkey Virus Constitutive Transport Element-Dependent Propagation of SIVmac239 Vectors Using a Single Round of Replication Assay

In a step toward creating live-attenuated or DNA subunit vaccines for AIDS, the replication of simian immunodeficiency virus (SIV) was studied independently of the Rev and RRE (Rev-responsive element) regulatory system, over a single round. To accomplish this, theenvgene of an SIV vector was made defective by the insertion of a SV40 promoter/enhancerhygromycin B phosphotransferasegene cassette. Using this vector as the backbone, molecular clones of SIV were generated that contained a mutated Rev, Rev(−), a deleted RRE, RRE(−), or both, Rev(−)RRE(−). It has been shown recently that human immunodeficiency virus type 1 (HIV-1) Rev and RRE functions can be replacedin vitroby acis-acting sequence, constitutive transport element (CTE), from simian type D retroviruses. To determine whether such acis-acting element from Mason–Pfizer monkey virus (MPMV) would substitute for SIV Rev and RRE functions, the MPMV CTE was inserted either into the Nef ORF or at the junction ofvpxandvprof our Rev(−), RRE(−), and Rev(−)RRE(−) SIV molecular clones. Cell-free viral stocks harvested from Cos cells following transfections of these molecular clones revealed that these stocks were infectious over a single round of replication; however, their replication was attenuated 16-fold compared to that of wild-type virus. In addition, our experiments revealed that CTE functions in a position-dependent manner such that its insertion at the junction ofvpxandvprattenuated SIV replication 8- to 12-fold compared to the attenuation observed when it was inserted in thenefregion. Our results demonstrate that MPMV CTE is capable of substituting for SIV Rev and RRE functions, resulting in an attenuated ability to produce infectious virus.

Construction and characterization of replication-competent simian immunodeficiency virus vectors that express gamma interferon

We report the construction and characterization of several replication-competent simian immunodeficiency virus (SIV) vectors with a deletion in the viral nef gene (SIV(delta nef)) that express gamma interferon (IFN-gamma). The expression of the cytokine gene was controlled either by the simian virus 40 early promoter or by the SIV 5' long terminal repeat regulatory sequences, utilizing the nef gene splice signals. To enhance the expression of IFN-gamma, the two in-frame nef start codons were mutated without altering the Env amino acid sequence (SIV(HyIFN)). Plasmids containing full-length proviral genomes were used to obtain high-titer stocks of each recombinant virus in cell cultures. Expression of IFN-gamma by SIV(HyIFN) reached levels as high as 10(6) U/ml after 11 days in culture. The IFN-gamma gene was unstable and sustained deletions after serial passage of SIV(delta nef) vectors in CEM-X-174 cells. The degree of instability appears to depend on size and orientation of the insert and the expression of IFN-gamma. Only one virus, SIV(HyIFN), expressed detectable levels of IFN-gamma up to the sixth passage. Prospects for the use of IFN-gamma and other lymphokines to enhance the safety and efficacy of live attenuated vaccines are discussed.

Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles.

Packaging of retroviral RNA is attained through the specific recognition of a cis-acting encapsidation site (located near the 5' end of the viral RNA) by components of the Gag precursor protein. Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) are two lentiviruses that lack apparent sequence similarity in their putative encapsidation regions. We used SIV vectors to determine whether HIV-1 particles can recognize the SIV encapsidation site and functionally propagate SIV nucleic acid. SIV nucleic acid was replicated by HIV-1 proteins. Thus, efficient lentivirus pseudotyping can take place at the RNA level. Direct examination of the RNA contents of virus particles indicated that encapsidation of this heterologous RNA is efficient. Characterization of deletion mutants in the untranslated leader region of SIV RNA indicates that only a very short region at the 5' end of the SIV RNA is needed for packaging. Comparison of this region with the corresponding region of HIV-1 reveals that both are marked by secondary structures that are likely to be similar. Thus, it is likely that a similar higher-order RNA structure is required for encapsidation.

Simian immunodeficiency virus vectors: Replication and pseudotyping

We studied a single round of replication of Simian immunodeficiency virus (SIV) through the use of a replication defective vector that expresses the hygromycin resistance gene. It was possible to pseudotype SIV particles by complementation with the env gene from a murine amphotropic retrovirus. Moreover, SIV RNA was packaged and propagated by core particles of the heterologous lentivirus, HIV-1. These results indicate that coinfection of cells with SIV and other retroviruses could lead to infection of new cell types in nature.