To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8 ؉ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated-or virus-infected-target cells. This CD8 ؉ class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells. CD8ϩ cytotoxic T lymphocytes (CTLs) play an important role in the elimination of cells infected by pathogens and in the regression of tumors. CTLs recognize antigen-derived peptides presented by major histocompatibility complex (MHC) class I molecules on the cell surface and are usually activated by peptides resulting from the processing of endogenous intracellular proteins (1). Because antigens have to gain access to the cytosol to enter the class I-restricted presentation pathway, exogenous soluble proteins are usually unable to stimulate CTL responses. Therefore, several strategies have been developed to deliver exogenous antigens into the cytosol. Protein or peptide antigens delivered in association with appropriate adjuvants [complete Freund's adjuvant (2), incomplete Freund's adjuvant (3), or saponin (4)], liposomes (5), ISCOMs (6), or in particulate form linked to latex microspheres (7) efficiently stimulate CTL responses. However, alum (aluminum salts) is still the only adjuvant currently licensed for use in human vaccines. Recombinant live vectors [such as attenuated virus, vaccinia virus (8), mengo virus (9)] or bacteria [bacillus Calmette-Guérin (10), Salmonella (11), or Listeria (12)] have also been shown to sensitize CTLs in vivo but are risk-prone. Recombinant canarypox virus expressing gp160 from HIV-1, which cannot replicate in mammalian species, was recently shown to induce CTL responses in humans but only in less than 40% of the volunteers (13). DNA vaccination may also represent a powerful strategy to activate CTL responses (14), but the safety of this method remains to be determined. Therefore, the development of a safe strategy to induce CTL responses with nonreplicating antigens is still an important prerequisite for the design of new efficient vaccines.Recently, we have developed an antigen delivery system based on ...
Two major genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) have been described, which correspond to the European and North American isolates. PRRSV nucleocapsid (N) protein has been identified as the most immunodominant viral protein. The N genes from two PRRSV isolates, Olot/91 (European) and Que! bec 807/94 (North American), were cloned and expressed in : (i) baculovirus under the control of the polyhedrin promoter and (ii) Escherichia coli using the pET3x system. The N protein from both isolates was expressed much more efficiently in E. coli as a fusion protein than in baculovirus. The antigenicity of the protein was similar in both systems and it was recognized by a collection of 48 PRRSV-positive pig sera. The antigenic structure of the PRRSV N protein
We have analysed the potential capacity of hybrid porcine parvovirus (PPV) capsids to present foreign epitopes to the immune system. Foreign sequences were introduced into the N and C termini of PPV VP2, which was previously shown to assemble spontaneously into parvovirus-like particles. The integrity of the C terminus was shown to be essential for preserving the structure of the capsid and therefore could not be used for epitope fusion. In contrast, insertion of sequences corresponding to T and B cell poliovirus epitopes in the N terminus did not alter the formation of particles. Moreover, the chimeric capsids containing the C3 : T epitope were able to induce a T cell response in vivo. However, hybrid particles containing the C3 :B epitope fused to the N terminus did not induce any peptide-specific antibody response, suggesting that the inserted B cell epitope was not exposed at the surface of the particles. These results show that the N terminus in PPV empty capsids is not an adequate site for insertion of B cell epitopes, but may be useful for T cell epitope presentation and suggest that the N terminus is located in an internal position.
Canine parvovirus capsids are composed of 60 copies of VP2 and 6 to 10 copies of VP1. To locate essential sites of interaction between VP2 monomers, we have analyzed the effects of a number of VP2 deletion mutants representing the amino terminus and the four major loops of the surface, using as an assay the formation of virus-like particles (VLPs) expressed by recombinant baculoviruses. For the amino terminus we constructed three mutants with progressively larger deletions, i.e., 9, 14, and 24 amino acids. Deletions of 9 and 14 amino acids did not affect the morphology and assembly capabilities of the mutants. However, the mutant with the 24-amino-acid deletion did not show hemagglutination properties or correct VLP morphology, stressing again the relevance of the RNER domain in canine parvovirus functionality. Three of the four mutants with deletions in the loops failed to make correct VLPs, indicating that these regions are essential for correct capsid assembly and morphology. Only the mutant with the deletion in loop 2 was able to assemble in regular VLPs, suggesting that this loop has little or no effect in capsid morphogenesis. Further research has demonstrated that this region can tolerate the insertion of foreign epitopes that are correctly exposed in the surface of the capsid. This result opens the door to the use of these VLPs for antigen delivery.
We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4+ and CD8+ T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8+ T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8+ T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8+T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a new arterivirus that has spread rapidly all around the world in the last few years. The genomic region containing open reading frames (ORFs) 2 to 7 of PRRSV Spanish isolate Olot/91 was cloned and sequenced. The genomic sequence shared 95% identity with Lelystad and Tübingen isolates and between 61-64% with the ORF7 region of the American isolates. ORFs 2 to 7 were inserted into recombinant baculoviruses downstream of the polyhedrin promoter. Only ORFs 2, 3 5 and 7 were expressed in insect cells as detected by PRRS-specific pig antisera. To analyze the immunogenicity of these proteins and their ability to confer protection, Sf9 cells infected with recombinant baculoviruses expressing ORFs 3, 5 and 7 gene products were used to immunize pregnant sows, either individually or in combination. The results obtained indicate that ORFs 3 and 5 gene products could be major candidates for the development of a vaccine against PRRS since they conferred 68.4 and 50% protection, respectively, as evaluated by the number of piglets born alive and healthy at the time of weaning. In addition, piglets born to sows immunized with ORFs 3 and 5 proteins were seronegative to PRRSV after weaning, indicating absence of viral replication. ORF7 is the most immunogenic protein of PRRSV, but the antibodies induced in sows are non-protective and may even interfere with protection.
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