Spatial And Temporal Expression of Herpes Simplex Virus Type 1 Amplicon-Encoded Genes: Implications for Their Use As Immunization Vectors

Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642, USA.
Human Gene Therapy (Impact Factor: 3.76). 03/2007; 18(2):93-105. DOI: 10.1089/hum.2006.082
Source: PubMed


There is great interest in developing new immunization vectors. Helper virus-free herpes amplicons, plasmid-based vectors that encode no viral gene products and have an extremely large coding capacity, are attractive viral vaccine candidates for expressing recombinant proteins in vivo for immunization. Earlier studies in mice, using amplicons encoding the gp120 protein of human immunodeficiency virus (HIV), resulted in strikingly robust cellular immune responses as measured by cytotoxicity and interferon gamma enzyme-linked immunospot assays. To begin to understand how such vectors function in vivo to generate an immune response, we used amplicons encoding reporter constructs including green fluorescent protein (GFP) and luciferase to examine the duration of expression after administration to mice. Luciferase expression, measured with the IVIS system from Xenogen/Caliper Life Sciences (Hopkinton, MA) and by enzymatic assays of tissue extracts, revealed that expression after injection of the HSVluc amplicons peaked earlier than 24 hr after injection into mice. HSVegfp injection resulted in peak accumulation of GFP 24 hr after administration in vivo. Thus, both reporter genes revealed a rather rapid and robust expression pattern of short duration. The short period of expression appears in part to be due to gene silencing. Examination of the cells transduced by amplicons encoding GFP and human B7.1 suggested that the amplicons transduce a variety of cells, including professional antigen-presenting cells. From this and previous work, we conclude that amplicons may engender a potent immune response by directly transducing dendritic cells as well as by cross-priming of antigen produced by other transduced host cells.

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    • "In a study conceived to investigate how amplicon vectors function in vivo to generate an immune response, amplicon vectors expressing either GFP or luciferase reporter proteins were used to examine the duration of expression after administration to mice [46]. Injection with amplicons expressing luciferase revealed that transgene expression peaked earlier than 24 hr after injection in mice. "
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    ABSTRACT: Amplicons are helper-dependent herpes simplex virus type 1 (HSV-1)-based vectors that can deliver very large foreign DNA sequences and, as such, are good candidates both for gene delivery and vaccine development. However, many studies have shown that innate constitutive or induced cellular responses, elicited or activated by the entry of HSV-1 particles, can play a significant role in the control of transgenic expression and in the induction of inflammatory responses. Moreover, transgene expression from helper-free amplicon stocks is often weak and transient, depending on the particular type of infected cells, suggesting that cellular responses could be also responsible for the silencing of amplicon-mediated transgene expression. This review summarizes the current experimental evidence underlying these latter concepts, focusing on the impact on transgene expression of very-early interactions between amplicon particles and the infected cells, and speculates on possible ways to counteract the cellular protective mechanisms, thus allowing stable transgene expression without enhancement of vector toxicity.
    The Open Virology Journal 04/2010; 4(1):96-102. DOI:10.2174/1874357901004030096
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    • "Helper-free HSV-1 amplicon vectors are replication-defective, " gutless " vectors that possess compelling advantages as potential vaccine delivery platforms (reviewed in [16]). These advantages include (i) their ability to elicit strong cellular immune responses to encoded antigens [19] [20] [21], (ii) their broad host cell tropism and ability to directly transduce antigen presenting cells, including dendritic cells (DC), both in vivo and in vitro [21] [22]; (iii) their favorable safety profile and lack of expression of virally-encoded immunomodulatory genes; and (iv) their very large transgene capacity (up to 150 kb) [23] [24]. "
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    ABSTRACT: HSV-1 amplicon vectors elicit strong T-cell responses to encoded antigens but the qualitative nature of these responses is poorly understood. Antigen-specific CD4(+) and CD8(+) T-cell responses to amplicon and adenovirus (rAd5) vectors encoding HIV-1 gp120 were assessed following immunization of mice, by performing intracellular cytokine staining for IFNgamma, IL2 and TNFalpha, following stimulation of splenocytes with a HIV-1 Env peptide pool. The quality of the primary T-cell response to amplicon and rAd5 vectors was strikingly similar, but there were qualitative differences in responses to amplicon vectors that incorporated different promoters upstream of gp120 - suggesting that promoters can significantly influence immune response quality. When prime-boost combinations were studied, a rAd5 prime and amplicon boost elicited the highest T-cell response. Furthermore, protocols that incorporated a rAd5 prime consistently elicited a greater proportion of polyfunctional CD4(+) T-cells-regardless of boost. This suggests that initial priming can shape immune response quality after a boost. Overall, these findings provide insight into effective vector combinations for HIV-1 vaccine development.
    Vaccine 11/2007; 25(42):7410-21. DOI:10.1016/j.vaccine.2007.08.015 · 3.62 Impact Factor
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    ABSTRACT: Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2007. Helper-free herpes simplex virus type-1 (HSV-1) amplicon vectors are replicationdefective, “gutless” plasmid-based gene transfer vectors that possess compelling advantages as potential vaccine delivery platforms. These advantages include: a favorable safety profile due to the lack of expression of virally-encoded immunomodulatory genes, the capacity to encode large amounts of foreign DNA and a broad host cell tropism - including the ability to directly transduce antigen presenting cells such as dendritic cells. Previous work from our laboratory has shown that a single injection of a helper-free HSV-1 amplicon vector can elicit potent and durable immune responses to an encoded HIV antigen, even in the face of preexisting immunity to HSV. The focus of this thesis was a three-pronged examination of the hypothesis that it might be possible to maximize HSV-1 amplicon vector-induced immune responses to encoded HIV-1 antigens by further optimizing antigen design and vector delivery strategies. As an initial focus, amplicons encoding a modified HIV-1 clade C Env antigen were generated and tested, in vivo, to determine whether addition of an immunodominant CTL tag had any effect on the antigen-specific CD8+ T cell response. This study showed that immunization of mice with an amplicon vector encoding the tagged envelope protein elicited potent and detectable cellular immune responses. Planned follow-up experiments on the immune response to amplicon vectors encoding multiple Env antigens were abandoned when other investigators published similar studies. The second focus of the thesis was a determination of whether the volume of buffer (and the associated route of delivery)in which a fixed dose of amplicon encoding a model HIV-1 antigen (gp120) is delivered might affect the CD8+T-cell immune response to amplicon encoded antigens. The underlying rationale was that a larger inoculum volume might result in higher levels of non-specific immune activation due to minor tissue damage, and more broad dissemination of the vector. This study revealed that the inoculum volume had a minimal effect on the measured immune response to either recombinant adenovirus serotype 5 (rAd5) or HSV-1 amplicon vectors encoding a model HIV-1 antigen. The third focus of this thesis was a multilayered examination of the magnitude and quality of CD4+ and CD8+ T-cell immune responses elicited by a heterologous prime boost regimen combining HSV-1 amplicon and rAd5 vectors that encoded the same HIV-1 antigen (gp120). A careful comparison of the magnitude and quality of the T cell response that was elicited, at acute and late time points, following immunization with HSV-1 amplicon and rAd5 vectors showed that HSV-1 amplicon vectors and rAd5 vectors that encoded gp120 from the same transcriptional regulatory element elicited primary T cell responses that were similar both in magnitude and quality. In contrast, HSV-1 amplicon vectors that incorporated different promoter elements upstream of gp120 elicited qualitatively and quantitatively distinct antigenspecific T cell response profiles. Further, the heterologous combination of a rAd5 vector prime and a HSV-1 amplicon vector boost was found to yield the best combination of cellular immune response magnitude and quality. Taken together, these findings point the way forward to novel and more effective use of the HSV-1 amplicon vector system for HIV-1 vaccine development.
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