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Cellular and humoral immune responses to alphavirus replicon vaccines expressing cytomegalovirus pp65, IE1, and gB proteins.

ABSTRACT: Development of vaccines against cytomegalovirus (CMV) is an important public health priority. We used a propagation-defective, single-cycle RNA replicon vector system derived from an attenuated strain of an alphavirus, Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRP) expressing various combinations of pp65, IE1, or gB proteins of human CMV. Protein expression in VRP-infected cells was highest with single-promoter replicons expressing pp65, IE1, a pp65/IE1 fusion protein, or the extracellular domain of gB and with double-promoter replicons expressing pp65 and IE1. Protein expression was lower with double- and triple-promoter replicons expressing gB, especially the full-length form of gB. BALB/c mice immunized with VRP expressing gB developed high titers of neutralizing antibody to CMV, and mice immunized with VRP expressing pp65, IE1, or a pp65/IE1 fusion protein developed robust antigen-specific T-cell responses as measured by gamma interferon enzyme-linked immunospot assay. Three overlapping immunodominant pp65 peptides contained a nine-amino-acid sequence (LGPISGHVL) that matches the consensus binding motif for a major histocompatibility complex H2-D(d) T-cell epitope. These data provide the basis for further development and clinical evaluation of an alphavirus replicon vaccine for CMV expressing the pp65, IE1, and gB proteins.

SUBMITTER: Reap EA

PROVIDER: S-EPMC1951075 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Cellular and humoral immune responses to alphavirus replicon vaccines expressing cytomegalovirus pp65, IE1, and gB proteins.

Reap Elizabeth A EA Dryga Sergey A SA Morris John J Rivers Bryan B Norberg Pamela K PK Olmsted Robert A RA Chulay Jeffrey D JD

Clinical and vaccine immunology : CVI 20070418 6

Development of vaccines against cytomegalovirus (CMV) is an important public health priority. We used a propagation-defective, single-cycle RNA replicon vector system derived from an attenuated strain of an alphavirus, Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRP) expressing various combinations of pp65, IE1, or gB proteins of human CMV. Protein expression in VRP-infected cells was highest with single-promoter replicons expressing pp65, IE1, a pp65/IE1 fusi ...[more]

PMID: 17442845

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Project description:Abstract: We have identified a cytomegalovirus virion protein capable of modulating the rapid induction of an interferon-like response in cells that follows virus binding and penetration. Functional genomics revealed a role for the major cytomegalovirus structural protein, pp65 (ppUL83), in counteracting this response. The underlying mechanism involves a differential impact of this structural protein on the regulation of interferon response factor 3 (IRF-3). In contrast, NF-{kappa}B is activated independent of pp65, and neither STAT1 nor STAT3 becomes activated by either virus. pp65 is sufficient to prevent the activation of IRF-3 when introduced alone into cells. pp65 acts by inhibiting nuclear accumulation of IRF-3 and is associated with a reduced IRF-3 phosphorylation state. Thus, this investigation shows that the major structural protein of cytomegalovirus is committed to the modulation of the IRF-3 response, a primary mediator of the type I interferon response. By subverting IRF-3, the virus escapes throwing a central alarm devoted to both immediate antiviral control and regulation of the immune response. Polyadenylated RNA from 107 confluent HFs was purified by using Oligotex (QIAGEN, Valencia, Calif.) from Trizol (Invitrogen)-extracted total RNA. This RNA was prepared, reverse transcribed, labeled with Cy3-dUTP or Cy5-dUTP (Amersham, Little Chalfont, Buckinghamshire, United Kingdom) by random primed synthesis with DNA pol I Klenow (Amersham Life Science, Inc., Cleveland, Ohio), and hybridized to spotted, human cDNA microarrays as previously described. Sequence-verified human cDNA microarrays (HE and HG series, 31,000 spots; HD51 series, 17,000 spots) were produced at Stanford. Images were collected by using a GenePix 4000B microarray scanner, manually flagged to eliminate poor spots and analyzed by GenePix Pro 2.0 (Axon, Union City, Calif.). A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments. Keywords: disease_state_design, pathogenicity_design

Dataset Information

Cellular and humoral immune responses to alphavirus replicon vaccines expressing cytomegalovirus pp65, IE1, and gB proteins.

Publications

Cellular and humoral immune responses to alphavirus replicon vaccines expressing cytomegalovirus pp65, IE1, and gB proteins.

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