Anchoring a secreted plasmodium antigen on the surface of recombinant vaccinia virus-infected cells increases its immunogenicity.

We show that the subcellular location of foreign antigens expressed in recombinant vaccinia viruses influences their effectiveness as immunogens. Live recombinant viruses induced very poor antibody responses to a secreted repetitive plasmodial antigen (the S-antigen) in rabbits and mice. The poor response accords with epidemiological data suggesting that S-antigens are poorly immunogenic. Appending the transmembrane domain of a membrane immunoglobulin (immunoglobulin G1) to its carboxy terminus produced a hybrid S-antigen that was no longer secreted but was located on the surface of virus-infected cells. This recombinant virus elicited high antibody titers to the S-antigen. This approach will facilitate the use of live virus delivery systems to immunize against a wide range of foreign nonsurface antigens.

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Interaction between Vaccinia Virus Extracellular Virus Envelope A33 and B5 Glycoproteins

Journal of Virology ◽

10.1128/jvi.00598-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8763-8777 ◽

Cited By ~ 30

Author(s):

Beatriz Perdiguero ◽

Rafael Blasco

Keyword(s):

Vaccinia Virus ◽

Protein Interactions ◽

Transmembrane Domain ◽

Recombinant Vaccinia Virus ◽

Protein Protein Interactions ◽

Virus Envelope ◽

Extracellular Virus ◽

Vaccinia Viruses ◽

Extracellular Form ◽

Infected Cells

ABSTRACT The extracellular form of vaccinia virus acquires its outer envelope by wrapping with cytoplasmic membranes that contain at least seven virus-encoded proteins, of which four are glycoproteins. We searched for interactions between the vaccinia virus A33 glycoprotein and proteins A34, A36, B5, F12, and F13. First, when myc epitope-tagged A33 was expressed in combination with other envelope proteins, A33 colocalized with B5 and A36, suggesting that direct A33-B5 and A33-A36 interactions occur in the absence of infection. A recombinant vaccinia virus (vA33Rmyc) was constructed by introduction of the myc-tagged A33 version (A33myc) into A33-deficient vaccinia virus. A33myc partially restored plaque formation and colocalized with enveloped virions in infected cells. Coimmunoprecipitation experiments with extracts of vA33Rmyc-infected cells confirmed the existence of a physical association of A33 with A36 and B5. Of these, the A33-B5 interaction is a novel finding, whereas the interaction between A33 and A36 has been previously characterized. A collection of vaccinia viruses expressing mutated versions of the B5 protein was used to investigate the domain(s) of B5 required for interaction with A33. Both the cytoplasmic domain and most of the extracellular domain, but not the transmembrane domain, of the B5 protein were dispensable for binding to A33. Mutations in the extracellular portions of B5 and A33 that enhance extracellular virus release did not affect the interaction between the two. In contrast, substituting the B5 transmembrane domain with that of the vesicular stomatitis virus G glycoprotein prevented the association with A33. Immunofluorescence experiments on virus mutants indicated that B5 is required for efficient targeting of A33 into enveloped virions. These results point to the transmembrane domain of B5 as the major determinant of the A33-B5 interaction and demonstrate that protein-protein interactions are crucial in determining the composition of the virus envelope.

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Plasmodium falciparum:Heterologous Synthesis of the Transmission-Blocking Vaccine Candidate Pfs48/45 in Recombinant Vaccinia Virus-Infected Cells

Experimental Parasitology ◽

10.1006/expr.1998.4315 ◽

1998 ◽

Vol 90 (2) ◽

pp. 165-174 ◽

Cited By ~ 22

Author(s):

Richard L.B. Milek ◽

Antoine A.F. DeVries ◽

Will F.G. Roeffen ◽

Henk Stunnenberg ◽

Peter J.M. Rottier ◽

...

Keyword(s):

Vaccinia Virus ◽

Vaccine Candidate ◽

Recombinant Vaccinia Virus ◽

Recombinant Vaccinia ◽

Transmission Blocking ◽

Infected Cells ◽

Transmission Blocking Vaccine

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Vaccinia Virus H2 Protein Is an Essential Component of a Complex Involved in Virus Entry and Cell-Cell Fusion

Journal of Virology ◽

10.1128/jvi.79.8.4744-4754.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 4744-4754 ◽

Cited By ~ 70

Author(s):

Tatiana G. Senkevich ◽

Bernard Moss

Keyword(s):

Vaccinia Virus ◽

Cell Fusion ◽

Essential Role ◽

Transmembrane Domain ◽

Virus Entry ◽

Recombinant Vaccinia Virus ◽

Low Ph ◽

Microscopic Appearance ◽

Recombinant Vaccinia ◽

Virus Morphogenesis

ABSTRACT The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.

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Enhancement of mucosal IgA responses by interleukins 5 and 6 encoded in recombinant vaccine vectors

Reproduction Fertility and Development ◽

10.1071/rd9940389 ◽

1994 ◽

Vol 6 (3) ◽

pp. 389 ◽

Cited By ~ 16

Author(s):

AJ Ramsay ◽

KH Leong ◽

D Boyle ◽

J Ruby ◽

IA Ramshaw

Keyword(s):

Immune Responses ◽

Mucosal Immunity ◽

Recombinant Vaccinia Virus ◽

Mucosal Vaccine ◽

Vaccine Vectors ◽

Heterologous Antigen ◽

Recombinant Vaccinia ◽

Interleukin 5 ◽

Wide Range

The expression of the genes for murine interleukin-5 (IL-5) or IL-6 in recombinant vaccinia virus vectors markedly increased IgA reactivity to co-expressed heterologous antigen in the lungs of mice inoculated intranasally with the viruses. These elevated local IgA responses reached a peak four times higher than those elicited by control viruses 14 days after infection and these peak levels were maintained for at least four weeks. Elevated IgA responses, reaching a peak 3-4 weeks after immunization, were also observed in the lungs of mice inoculated with IL-6 expressed by another vector, fowlpox virus. The results indicate that these factors enhance the development of mucosal IgA reactivity in vivo and suggest that their expression in mucosal vaccine vectors may stimulate local immune responses. The approach described in this study may be useful in stimulating mucosal immunity to a wide range of vector-encoded antigens, not only for vaccination against disease but also for immunocontraception by the co-expression of antigens involved in reproduction.

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Oral Immunization with Recombinant Vaccinia Virus Prime and Intramuscular Protein Boost Provides Protection against Intrarectal Simian-Human Immunodeficiency Virus Challenge in Macaques

Clinical and Vaccine Immunology ◽

10.1128/cvi.00597-15 ◽

2015 ◽

Vol 23 (3) ◽

pp. 204-212 ◽

Cited By ~ 7

Author(s):

Rajesh Thippeshappa ◽

Baoping Tian ◽

Brad Cleveland ◽

Wenjin Guo ◽

Patricia Polacino ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Immune Responses ◽

Vaccinia Virus ◽

Protective Efficacy ◽

Recombinant Vaccinia Virus ◽

Oral Immunization ◽

Recombinant Vaccinia ◽

Immunodeficiency Virus ◽

Vaccinia Viruses ◽

Hiv 1

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) acquisition occurs predominantly through mucosal transmission. We hypothesized that greater mucosal immune responses and protective efficacy against mucosal HIV-1 infection may be achieved by prime-boost immunization at mucosal sites. We used a macaque model to determine the safety, immunogenicity, and protective efficacy of orally delivered, replication-competent but attenuated recombinant vaccinia viruses expressing full-length HIV-1 SF162 envelope (Env) or simian immunodeficiency virus (SIV) Gag-Pol proteins. We examined the dose and route that are suitable for oral immunization with recombinant vaccinia viruses. We showed that sublingual inoculation of two vaccinia virus-naive pigtailed macaques with 5 × 108PFU of recombinant vaccinia viruses was safe. However, sublingual inoculation with a higher dose or tonsillar inoculation resulted in secondary oral lesions, indicating the need to optimize the dose and route for oral immunization with replication-competent vaccinia virus vectors. Oral priming alone elicited antibody responses to vaccinia virus and to the SF162 Env protein. Intramuscular immunization with the SF162 gp120 protein at either 20 or 21 weeks postpriming resulted in a significant boost in antibody responses in both systemic and mucosal compartments. Furthermore, we showed that immune responses induced by recombinant vaccinia virus priming and intramuscular protein boosting provided protection against intrarectal challenge with the simian-human immunodeficiency virus SHIV-SF162-P4.

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Proteolytic processing of QSOX1A ensures efficient secretion of a potent disulfide catalyst

Biochemical Journal ◽

10.1042/bj20130360 ◽

2013 ◽

Vol 454 (2) ◽

pp. 181-190 ◽

Cited By ~ 20

Author(s):

Jana Rudolf ◽

Marie A. Pringle ◽

Neil J. Bulleid

Keyword(s):

Disulfide Bonds ◽

Mammalian Cells ◽

Secretory Pathway ◽

Transmembrane Domain ◽

Subcellular Location ◽

Proteolytic Processing ◽

Protein Substrates ◽

Wide Range ◽

Efficient Processing ◽

Quiescin Sulfhydryl Oxidase

QSOX1 (quiescin sulfhydryl oxidase 1) efficiently catalyses the insertion of disulfide bonds into a wide range of proteins. The enzyme is mechanistically well characterized, but its subcellular location and the identity of its protein substrates remain ill-defined. The function of QSOX1 is likely to involve disulfide formation in proteins entering the secretory pathway or outside the cell. In the present study, we show that this enzyme is efficiently secreted from mammalian cells despite the presence of a transmembrane domain. We identify internal cleavage sites and demonstrate that the protein is processed within the Golgi apparatus to yield soluble enzyme. As a consequence of this efficient processing, QSOX1 is probably functional outside the cell. Also, QSOX1 forms a dimer upon cleavage of the C-terminal domain. The processing of QSOX1 suggests a novel level of regulation of secretion of this potent disulfide catalyst and producer of hydrogen peroxide.

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Highly Immunogenic Human Immunodeficiency Viruslike Particles are Produced by Recombinant Vaccinia Virus-Infected Cells

AIDS Research and Human Retroviruses ◽

10.1089/aid.1991.7.29 ◽

1991 ◽

Vol 7 (1) ◽

pp. 29-36 ◽

Cited By ~ 20

Author(s):

ANDREI N. VZOROV ◽

MICHAEL I. BUKRINSKY ◽

VLADIMIR B. GRIGORIEV ◽

YURII YU. TENTSOV ◽

ALICE G. BUKRINSKAYA

Keyword(s):

Vaccinia Virus ◽

Recombinant Vaccinia Virus ◽

Recombinant Vaccinia ◽

Infected Cells ◽

Viruslike Particles

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Vaccinia Virus A34 Glycoprotein Determines the Protein Composition of the Extracellular Virus Envelope

Journal of Virology ◽

10.1128/jvi.01969-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2150-2160 ◽

Cited By ~ 19

Author(s):

Beatriz Perdiguero ◽

María M. Lorenzo ◽

Rafael Blasco

Keyword(s):

Vaccinia Virus ◽

Protein Composition ◽

Recombinant Vaccinia Virus ◽

Virus Envelope ◽

Enveloped Virus ◽

Extracellular Virus ◽

Transmembrane Glycoprotein ◽

C Terminus ◽

Vaccinia Viruses ◽

Infected Cells

ABSTRACT The outer envelope of the extracellular form of vaccinia virus contains five virus-encoded proteins, F13, A33, A34, A56, and B5, that, with the exception of A56, are implicated in virus egress or infectivity. A34, a type II transmembrane glycoprotein, is involved in the induction of actin tails, the release of enveloped virus from the surfaces of infected cells, and the disruption of the virus envelope after ligand binding prior to virus entry. To investigate interactions between A34 and other envelope proteins, a recombinant vaccinia virus (vA34RHA) expressing an epitope-tagged version of A34 (A34HA) was constructed by appending an epitope from influenza virus hemagglutinin to the C terminus of A34. Complexes of A34HA with B5 and A36, but not with A33 or F13, were detected in vA34RHA-infected cells. A series of vaccinia viruses expressing mutated versions of the B5 protein was used to investigate the domain(s) of B5 required for interaction with A34. Both the cytoplasmic and the transmembrane domains of B5 were dispensable for binding to A34. Most of the extracellular domain of B5, which contains four short consensus repeats homologous to complement control proteins, was sufficient for A34 interaction, indicating that both proteins interact through their ectodomains. Immunofluorescence experiments on cells infected with A34-deficient virus indicated that A34 is required for efficient targeting of B5, A36, and A33 into wrapped virions. Consistent with this observation, the envelope of A34-deficient virus contained normal amounts of F13 but decreased amounts of A33 and B5 with respect to the parental WR virus. These results point to A34 as a major determinant in the protein composition of the vaccinia virus envelope.

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