Expression of Human Immunodeficiency Virus Type 1 Gag Protein Precursor and Envelope Proteins from a Vesicular Stomatitis Virus Recombinant: High-Level Production of Virus-like Particles Containing HIV Envelope

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Experimental vaccines based on recombinant vesicular stomatitis viruses (VSV) expressing foreign viral proteins are protective in several animal disease models. Although these attenuated viruses are nonpathogenic in nonhuman primates when given by nasal, oral, or intramuscular routes, they are pathogenic in mice when given intranasally, and further vector attenuation may be required before human trials with VSV-based vectors can begin. Mutations truncating the VSV glycoprotein (G) cytoplasmic domain from 29 to 9 or 1 amino acid (designated CT9 or CT1, respectively) were shown previously to attenuate VSV growth in cell culture and pathogenesis in mice. Here we show that VSV recombinants carrying either the CT1 or CT9 deletion and expressing the human immunodeficiency virus (HIV) Env protein are nonpathogenic in mice, even when given by the intranasal route. We then carried out a detailed analysis of the CD8 ؉ T-cell responses, including in vivo cytotoxic T-cell activity, induced by these vectors. When given by either the intranasal or intraperitoneal route, the VSV-CT9 vector expressing HIV Env elicited primary and memory CD8 ؉ T-cell responses to Env equivalent to those elicited by recombinant wild-type VSV expressing Env. The VSV-CT1 vector also induced potent CD8 ؉ T-cell responses after intraperitoneal vaccination, but was less effective when given by the intranasal route. The VSV-CT1 vector was also substantially less effective than the VSV-CT9 or wild-type vector at inducing antibody to Env. The VSV-CT9 vector appears ideal because of its lack of pathogenesis, propagation to high titers in vitro, and stimulation of strong cellular and humoral immune responses.Vesicular stomatitis virus (VSV) is a membrane-bound enveloped virus and the prototypic member of the Rhabdoviridae family. VSV has a single negative-stranded RNA genome that encodes five structural proteins: the nucleocapsid (N), the phosphoprotein (P), the matrix protein (M), the glycoprotein (G), and the RNA-dependent RNA polymerase (L). VSV causes a self-limiting disease in livestock, resulting in lesions on the gums, mouth, and teats. Infection of humans with VSV is rare and often asymptomatic, but can result in mild flu-like symptoms (reviewed in reference 24).Our laboratory established a system for recovery of recombinant VSVs (rVSVs) from plasmid DNA (14) and developed VSV as a vector for high-level expression of foreign genes (29). We have also explored the use of VSV recombinants expressing foreign genes as experimental vaccines (12,20,22,23,25,27). For example, VSV recombinants expressing human immunodeficiency virus (HIV) 89.6 Env and simian immunodeficiency virus (SIV) mac239 Gag have protected macaques from progression to AIDS for over 3 years following challenge with SHIV 89.6P (25).In addition to being effective, human vaccines must be safe for use in both healthy and immunocompromised people. Vaccines based on live viruses are typically highly effective but are normally attenuated by multiple mutations before they are considered safe f...

Section: Discussionmentioning

confidence: 58%

Characterization of Nonpathogenic, Live, Viral Vaccine Vectors Inducing Potent Cellular Immune Responses

Publicover

Ramsburg

Rose

2004

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“…We used as a control VSV-rwt, which is an analogous wildtype VSV that was also recovered from plasmid DNA (27). These recombinants express high levels of the appropriate HIV-1 proteins (15,21,22).…”

Section: Resultsmentioning

confidence: 99%

“…rVSVs expressing HIV-1 Env and Gag (VSVGag, VSV-Env, and VSV-GagEnv) have been described previously (15,16,21,22), as has the corresponding recovered wild-type VSV (VSV-rwt) control (27). Stocks of VSV and recombinants were grown on BHK-21 cells.…”

Section: Methodsmentioning

confidence: 99%

Robust Recall and Long-Term Memory T-Cell Responses Induced by Prime-Boost Regimens with Heterologous Live Viral Vectors Expressing Human Immunodeficiency Virus Type 1 Gag and Env Proteins

Haglund

Leiner

Kerksiek

et al. 2002

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We investigated long-term memory and recall cellular immune responses to human immunodeficiency virus type 1 (HIV-1) Env and Gag proteins elicited by recombinant vesicular stomatitis viruses (VSVs) expressing Env and Gag. More than 7 months after a single vaccination with VSV-Env, ϳ6% of CD8 ؉ splenocytes stained with major histocompatibility complex class I tetramers containing the Env p18-I10 immunodominant peptide and showed a memory phenotype (CD44 Hi ). The level of tetramer-positive cells in memory was about 14% of the peak primary response. Recall responses elicited in these mice 5 days after boosting with a heterologous recombinant vaccinia virus expressing HIV-1 Env showed that 40 to 45% of CD8 ؉ splenocytes were tetramer positive and activated (CD62L Lo ), and these cells produced gamma interferon after stimulation with Env peptide, indicating that they were functional. Five months after the boost, the long-term memory cell population (tetramer positive, CD44 Hi ) constituted 30% of the CD8 ؉ splenocytes. Recall responses to HIV-1 Gag were examined in mice primed with VSV recombinants expressing HIV-1 Gag protein and boosted with a vaccinia virus recombinant expressing Gag. Using this protocol, we found that ϳ40% of CD8 ؉ splenocytes were activated (CD62L Lo ) and specific for a Gag immunodominant peptide (tetramer positive). The high-level Gag recall response elicited by the vaccinia virus-Gag was greater than that obtained by boosting with a VSV-Gag vector with a different VSV glycoprotein. The corresponding levels of CD44 Hi memory cells were also higher long after boosting with vaccinia virus-Gag than after boosting with a glycoprotein exchange VSV-Gag. Our results show that VSV vectors elicit high-level memory CTL responses and that these can be amplified as much as six-to sevenfold using a heterologous boosting vector.

“…It synthesizes five subgenomic mRNAs encoding five structural proteins (28). VSV recombinants can be recovered from DNA copies, and foreign genes can be expressed at high levels from multiple sites in the genome (11,14,17,31).It has been reported that cells transiently expressing HCV E1 or E2 chimeric proteins containing the VSV G transmembrane and cytoplasmic domains generate VSV pseudotypes when infected with a VSV mutant (ts045) that is temperature sensitive for G protein transport to the cell surface (16). Pseudotypes formed with either the E1G or E2G constructs were reported to infect mammalian cell lines, including cell lines not derived from the liver.…”

mentioning

confidence: 99%

Characterization of Vesicular Stomatitis Virus Recombinants That Express and Incorporate High Levels of Hepatitis C Virus Glycoproteins

Buonocore

Blight

Rice

et al. 2002

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We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein was deleted and replaced by one or both of the E1G and E2G genes, together with a green fluorescent protein gene. These ⌬G viruses incorporated E1G and E2G proteins at levels approximately equivalent to the normal level of VSV G itself, or about 1,200 molecules of each protein per virion. Given the potency of VSV recombinants as vaccines in other studies, this high-level expression and incorporation of HCV proteins into virions could be very important for development of an HCV vaccine. Despite the presence of E1G and E2G proteins at high levels in the virions, these virions did not infect cell lines that have been reported to support at least a low level of HCV infection and replication.Hepatitis C virus (HCV), the major cause of non-A, non-B viral hepatitis, was first identified in 1989 (3) and has infected approximately 170 million people worldwide (34), including about 4 million in the United States (1). HCV infection leads to chronic hepatitis, cirrhosis, and hepatocellular carcinoma and is now the leading cause of liver transplantation in the United States (7). Current treatment for HCV infection leads to sustained viral clearance in only about 50% of patients (24), and no vaccine is available to prevent new infections.HCV is a positive-stranded RNA virus with a genome of 9.6 kb encoding a polyprotein precursor of 3,000 amino acids (19). The polyprotein is proteolytically cleaved into 10 distinct products: the structural proteins (C, E1, E2, and P7) and several nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). E1 and E2 are type I transmembrane proteins which are highly glycosylated and associate to form a noncovalently linked heterodimer (6). The C-terminal hydrophobic regions of both proteins contain signals that are responsible for retaining these proteins in the endoplasmic reticulum (4,5,8). Deletions in these C-terminal regions and replacement with foreign transmembrane and cytoplasmic domains result in the expression of both E1 and E2 at the cell surface. E2 binds with high affinity to CD81, a tetraspanin protein expressed on various cell types including hepatocytes and B lymphocytes (23). However, definitive evidence that CD81 is an HCV receptor has not yet been obtained. Vesicular stomatitis virus (VSV) is a nonsegmented negative-strand RNA virus and the prototype of the rhabdovirus family. VSV infection in animals induces strong cellular and humoral immunity to its own proteins and to additional proteins encoded by recombinant viruses (12,20,26,35). VSV has an 11-kb RNA genome of negative (noncod...