P Griebel

University of Saskatchewan, Saskatoon, Saskatchewan, Canada

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Publications (19)61.8 Total impact

  • Molecular &amp Cellular Proteomics 01/2005; 4:S141. · 7.25 Impact Factor
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    ABSTRACT: Plasmid DNA continues to attract interest as a potential vaccine-delivery vehicle. However, the mechanisms whereby immune responses are elicited by plasmids are not fully understood. Although there have been suggestions regarding the importance of CpG motifs in plasmid immunogenicity, the molecular mechanisms by which CpG motifs enhance immune responses to DNA vaccines are not well understood. As Toll-like receptor 9-deficient (TLR9−/−) mice fail to respond to the adjuvant effects of CpG oligonucleotides, we used these mice to determine the effect of CpG motifs in plasmids used for DNA immunization. In the study described below, we report that DNA immunization was as effective in eliciting antigen-specific antibody and at stimulating antigen-specific interferon-γ (IFN-γ)-secreting cells in TLR9−/− mice as in TLR9+/+ mice. This study illustrates that DNA vaccines elicit immune responses by multiple mechanisms and demonstrates that TLR9 is not essential for the induction of immune responses following DNA immunization.
    Immunology 08/2004; 113(1):114 - 120. DOI:10.1111/j.1365-2567.2004.01938.x · 3.74 Impact Factor
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    ABSTRACT: Bacterial DNA contains a much higher frequency of CpG dinucleotides than are present in mammalian DNA. Furthermore, bacterial CpG dinucleotides are often not methylated. It is thought that these two features in combination with specific flanking bases constitute a CpG motif that is recognized as a "danger" signal by the innate immune system of mammals and therefore an immune response is induced when these motifs are encountered. These immunostimulatory activities of bacterial CpG DNA can also be achieved with synthetic CpG oligodeoxynucleotides (ODN). Recognition of CpG motifs by the innate immune system requires engagement of Toll-like receptor 9 (TLR-9), which induces cell signaling and subsequently triggers a pro-inflammatory cytokine response and a predominantly Th1-type immune response. CpG ODN-induced innate and adaptive immune responses can result in protection in various mouse models of disease. Based on these observations, clinical trials are currently underway in humans to evaluate CpG ODN therapies for cancer, allergy and infectious disease. However, potential applications for immunostimulatory CpG ODN in species of veterinary importance are just being explored. In this review, we will highlight what is presently known about the immunostimulatory effects of CpG ODN in domestic animals.
    Veterinary Immunology and Immunopathology 02/2003; 91(2):89-103. DOI:10.1016/S0165-2427(02)00246-5 · 1.75 Impact Factor
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    ABSTRACT: Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in particular sequence contexts (CpG ODN) are recognized as a danger signal by the innate immune system of vertebrates. For this reason, CpG ODNs have a potential application as both an adjuvant and nonspecific immune modulator and are currently being evaluated in a number of human and veterinary clinical trials. Given their potent immunostimulatory activity, CpG ODNs could possibly induce adverse reactions. As all adjuvants and immune modulators must be nontoxic to meet safety requirements, it was essential to address the safety aspects of CpG ODNs. The current review summarizes experiments carried out to date to establish the safety of CpG ODNs in animals.
    Antisense and Nucleic Acid Drug Development 02/2003; 13(3):157-67. DOI:10.1089/108729003768247628
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    ABSTRACT: Availability of effective oral vaccine delivery vehicles should contribute to the success of oral immunization in domestic animals. To achieve this goal, we evaluated alginate microspheres for their capacity to induce mucosal immune responses following oral and enteric immunizations. Mice were immunized with either live porcine rotavirus (PRV) or its recombinant VP6 protein, encapsulated in alginate microspheres or unencapsulated. VP6-specific IgG (but no IgA) antibodies were detected in the sera of mice after a single intraperitoneal (i.p.) immunization with either VP6 in Incomplete Freund's adjuvant (VP6-IFA), VP6 in alginate microspheres (VP6-MS) or with live PRV in incomplete Freund's adjuvant (PRV-IFA). In contrast, VP6-specific IgA (but no IgG) was detected in culture supernatants of mesenteric lymph nodes from mice immunized i.p. with either VP6-IFA or with PRV-IFA. Oral immunization with VP6-MS induced the highest level of VP6-specific fecal IgA antibody, similar to responses induced by oral immunization with live PRV. Furthermore, the VP6-specific fecal IgA could be boosted by a secondary i.p. immunization with VP6. Further experiments were performed in a sheep intestinal 'loop' model to evaluate uptake of microspheres by Peyer's patches. Microspheres containing colloidal carbon were specifically bound and transported by follicle-associated epithelium of Peyer's patches. Additionally, mucosal immune responses were detected following enteric immunization with porcine serum albumin (PSA) encapsulated in alginate microspheres. Our results confirm that alginate microspheres are an effective oral delivery vehicle for protein antigens and intestinal IgA antibody responses are induced by antigens encapsulated in alginate microspheres without any additional mucosal adjuvant. These investigations confirm that alginate microspheres have the potential as an effective delivery vehicle for oral immunization of ruminants.
    Journal of Controlled Release 01/2003; 85(1-3):191-202. DOI:10.1016/S0168-3659(02)00280-8 · 7.26 Impact Factor
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    ABSTRACT: Oral immunization is the most effective way of inducing immune responses in the intestinal tract. Biodegradable microspheres have been used extensively for the delivery of antigens to the Peyer's patches (PPs) within the gut-associated lymphoid tissue (GALT). We evaluated various formulations of alginate microspheres for their capacity to induce mucosal immune responses in vivo. Multiple intestinal "loops" each containing a single PP, were surgically prepared in lambs. We have previously showed that PP in individual intestinal loops function as independent sites for the induction of immune responses. This animal model provides a system for directly comparing different antigen formulations within the same animal. Individual intestinal loops were injected with a model antigen, porcine serum albumin (PSA) encapsulated in three different formulations of alginate micropsheres. Three weeks after immunization, PSA-specific immune responses were assayed with antibody secreting cell (ASC) ELISPOT, lymphocyte proliferative responses (LPRs), IFN-gamma production and antibody secreted into intestinal loops. PSA encapsulated in alginate micropsheres or in saline induced humoral immune responses as indicated by the presence of numerous ASC. However, PSA-specific T-cell responses (LPR and IFN-gamma production) were not induced.
    Veterinary Immunology and Immunopathology 10/2002; 87(3-4):269-76. DOI:10.1016/S0165-2427(02)00052-1 · 1.75 Impact Factor
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    ABSTRACT: The immunogenicity and protective efficacy of a bovine herpesvirus 1 (BHV-1) subunit vaccine formulated with Emulsigen (Em) and a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) was determined in cattle. A truncated, secreted version of BHV-1 glycoprotein D (tgD) formulated with Em and CpG ODN at concentrations of 25, 2.5, or 0.25 mg/dose produced a more balanced immune response, higher levels of virus neutralizing antibodies, and greater protection after BHV-1 challenge compared to tgD adjuvanted with either Em or CpG ODN alone. In contrast, tgD formulated with Em and either 25 mg of a non-CpG ODN or another immunostimulatory compound, dimethyl dioctadecyl ammonium bromide, induced similar immunity and protection compared to tgD formulated with Em alone, a finding which confirms the immunostimulatory effect of ODN to be CpG motif mediated. Our results demonstrate the ability of CpG ODN to induce a strong and balanced immune response in a target species.
    Journal of Virology 10/2002; 76(18):9002-10. DOI:10.1128/JVI.76.18.9002-9010.2002 · 4.65 Impact Factor
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    ABSTRACT: Bovine leukemia virus (BLV) is closely associated with the development of B-cell leukemia and lymphoma in cattle. BLV infection has also been studied extensively in an in vivo ovine model that provides a unique system for studying B-cell leukemogenesis. There is no evidence that BLV can directly infect ovine B cells in vitro, and there are no direct data regarding the oncogenic potential of the viral Tax transactivator in B cells. Therefore, we developed ovine B-cell culture systems to study the interaction between BLV and its natural target, the B cell. In this study, we used murine CD154 (CD40 ligand) and gamma-chain-common cytokines to support the growth of B cells isolated from ovine lymphoid tissues. Integrated provirus, extrachromosomal forms, and viral transcripts were detected in BLV-exposed populations of immature, rapidly dividing surface immunoglobulin M-positive B cells from sheep ileal Peyer's patches and also in activated mature B cells isolated from blood. Conclusive evidence of direct B-cell infection by BLV was obtained through the use of cloned B cells derived from sheep jejunal Peyer's patches. Finally, inoculation of sheep with BLV-infected cultures proved that infectious virus was shed from in vitro-infected B cells. Collectively, these data confirm that a variety of ovine B-cell populations can support productive infection by BLV. The development of ovine B-cell cultures permissive for BLV infection provides a controlled system for investigating B-cell leukemogenic processes and the pathogenesis of BLV infection.
    Journal of Virology 03/2001; 75(3):1095-103. DOI:10.1128/JVI.75.3.1095-1103.2001 · 4.65 Impact Factor
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    ABSTRACT: We investigated the antigen-specific mucosal and systemic immune responses of newborn lambs following enteric immunization, targeting jejunal Peyer's patches with a human adenovirus vector that expressed the glycoprotein D (gD) of bovine herpesvirus-1. Both humoral and cell-mediated gD-specific mucosal immune responses were detected in newborn lambs (1-4 days old) after a single immunization and these responses were qualitatively and quantitatively similar to those detected in 5-6-week-old lambs. Passively transferred gD-specific maternal antibody did not significantly alter either mucosal or systemic gD-specific immune responses. Furthermore, enteric immunization of newborn lambs primed mucosal immune responses in the lungs. These observations confirmed that gut-associated lymphoid tissue of a newborn ruminant is immune competent and that enteric immunization may be an effective approach for the induction of both mucosal and systemic immune responses in the neonate.
    Vaccine 01/2001; 19(9-10):1284-93. DOI:10.1016/S0264-410X(00)00230-9 · 3.49 Impact Factor
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    ABSTRACT: To study the biological relevance of using bovine herpesvirus-1 (BHV-1) as a vector for expressing cytokines, a BHV-1 virus that expressed bovine interferon-gamma (IFN-gamma) was constructed. This recombinant virus (BHV-1/IFNgamma) was then used to infect the natural host in a respiratory disease model. In vitro characterization of the recombinant interferon-gamma confirmed that the cytokine expressed in BHV-1-infected cells was biologically active. The in vivo effects of the recombinant IFN-gamma were then analysed during a primary infection and after reactivation of a latent infection. During the primary infection, similar body temperature, clinical responses and virus shedding were observed for calves infected with either recombinant BHV-1/IFNgamma or parental gC(-)/LacZ(+) virus. An analysis of cellular and humoral responses did not reveal any significant immunomodulation by BHV-1/IFNgamma during the primary infection. The stability and activity of recombinant IFN-gamma was also analysed following the establishment of a latent infection. The presence of recombinant IFN-gamma did not significantly alter virus shedding following reactivation. The isolation of reactivated BHV-1/IFNgamma virus confirmed that a functional IFN-gamma gene was retained during latency. Thus, herpesviruses may provide virus vectors that retain functional genes during latency and recrudescence.
    Journal of General Virology 12/2000; 81(Pt 11):2665-73. · 3.53 Impact Factor
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    ABSTRACT: CD40 signaling induces B cell proliferative and differentiation responses that can be modulated by many different cytokines. Cytokines in the IL-2 receptor gamma chain (gammac)-common family are known to play an integral role in B cell development. Therefore, we investigated the possibility that CD40 signaling induced B cell responsiveness to multiple gammac-common cytokines and that individual gammac-common cytokines induced distinct B cell responses. B cells were isolated from lymphoid follicles of sheep Peyer's patches (PP) and co-cultured with murine CD40 ligand (mCD40L). CD40 signaling induced PP B cell responsiveness to recombinant human IL-2, IL-4, IL-7 and IL-15. mCD40L-induced B cell growth was enhanced by combining IL-4 with a second gammac-common cytokine and sustained B cell growth required co-stimulation with IL-4 plus IL-2, IL-7 and IL-15. gammac-common cytokine responsiveness remained dependent upon CD40 signaling, and removal of mCD40L resulted in B cell differentiation and cell death. Similar proliferative responses to mCD40L and gammac-common cytokines were observed for both immature (ileal) and mature (jejunal) PP B cells. Finally, the capacity of CD40-activated B cells to respond to multiple gammac-common cytokines was analyzed with individual PP B cell clones. All B cell clones displayed similar proliferative responses to IL-2 but quantitatively different responses to IL-4, IL-7 and IL-15. The biological significance of B cell responsiveness to multiple gammac-common cytokines is discussed.
    International Immunology 08/1999; 11(7):1139-47. DOI:10.1093/intimm/11.7.1139 · 3.18 Impact Factor
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    ABSTRACT: The majority of pathogens enter the body through mucosal surfaces and it is now evident that mucosal immunity can provide effective disease protection. However, the induction of mucosal immunity will require efficient targeting of mucosal vaccines to appropriate mucosa-associated lymphoid tissue. An animal model, based upon the surgical preparation of sterile intestinal 'loops' (blind-ended segments of intestine), was developed to evaluate mucosal and systemic immune responses to enteric vaccines in ruminants. The effectiveness of end-to-end intestinal anastomoses was evaluated and fetal surgery did not disrupt normal intestinal function in lambs up to 6-7 months after birth. The immunological competence of Peyer's patches (PP) within the intestinal 'loops' was evaluated with a human adenovirus 5 vector expressing the gD gene of bovine herpesvirus-1. This vaccine vector induced both mucosal and systemic immune responses when injected into intestinal 'loops' of 5-6-week-old lambs. Antibodies to the gD protein were detected in the lumen of intestinal 'loops' and serum and PP lymphocytes proliferated in response to gD protein. The immune competence of ileal and jejunal PP was compared and these analyses confirmed that jejunal PP are an efficient site for the induction of mucosal immune responses. This was confirmed by the presence of gD-specific antibody-secreting cells in jejunal but not ileal PP. Systemic but not mucosal immune responses were detected when the vaccine vector was delivered to the ileal PP. In conclusion, this model provided an effective means to evaluate the immunogenicity of potential oral vaccines and to assess the immunological competence of ileal and jejunal Peyer's patches.
    Immunology 08/1999; 97(3):455-61. DOI:10.1046/j.1365-2567.1999.00791.x · 3.74 Impact Factor
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    ABSTRACT: To determine the potential of replication-competent (E3-deleted) bovine adenovirus-3 (BAV-3) as a delivery system for vaccine antigens in calves, we evaluated the ability of recombinant BAV-3 expressing different forms of of bovine herpesvirus-1 (BHV-1) glycoprotein gD to protect against BHV-1 infection in calves that had pre-existing BAV-3 specific antibodies. Three- to four-month-old calves, vaccinated intranasally with recombinant BAV-3 expressing full-length gD (BAV3.E3gD) or a truncated version of gD (gDt) (BAV3.E3gDt), or with E3-deleted BAV-3 (BAV3.E3d; control), were challenged with BHV-1 strain 108. Vaccination with BAV3.E3gD or BAV3.E3gDt induced gD-specific antibody responses in serum and nasal secretions, and primed calves for gD-specific lymphoproliferative responses. In addition, all calves developed complement-independent neutralizing antibodies against BHV-1. Protection against viral challenge was observed in calves vaccinated with recombinant BAV3.E3gD or BAV3.E3gDt as shown by a significant reduction in body temperature and clinical disease, and a partial reduction in the amount and duration of virus excretion in nasal secretions. These results indicate that replication-competent BAV-3-based vectors can induce protective immune responses in calves (the natural host) that have pre-existing BAV-3-specific antibodies.
    Journal of General Virology 06/1999; 80 ( Pt 5):1263-9. · 3.53 Impact Factor
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    ABSTRACT: Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in peripheral blood mononuclear cells and in bovine B lymphoma (BL-3) cells. Attachment but not penetration of BHV-1 is necessary to induce apoptosis in target cells, suggesting that one or more BHV-1 envelope glycoproteins could be involved in the activation of the apoptotic process. In the present study, we demonstrate that, although BHV-1 virions devoid of glycoprotein D (BHV-1 gD-/-) still bind to BL-3 cells, they are no longer able to induce apoptosis. In contrast, virions that contain glycoprotein D (gD) in the viral envelope but do not genetically encode gD (BHV-1 gD-/+) induce a level of apoptosis comparable to that produced by wild-type (wt) BHV-1. In addition, monoclonal antibodies directed against gD, but not against gB or gC, strongly reduced the high levels of apoptosis induced by BHV-1. These observations demonstrate that the induction of apoptosis is directly due to BHV-1 viral particles harboring gD in the viral envelope. Interestingly, binding of affinity-purified gD to BL-3 cells did not induce apoptosis but inhibited the ability of wt BHV-1 to induce apoptosis. Altogether, these results provide evidence for the direct or indirect involvement of gD in the mechanism by which BHV-1 induces apoptosis.
    Virology 05/1999; 257(1):191-7. DOI:10.1006/viro.1999.9620 · 3.28 Impact Factor
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    ABSTRACT: The lack of bovine leukemia virus (BLV) expression is a consistent finding in freshly isolated ovine tumor cells and in the B-cell lines derived from these tumors. In order to gain further insight into the mechanisms of BLV silencing in these tumors, we have used the YR2 B-cell line, which was derived from the leukemic cells of a BLV-infected sheep. This cell line contains a single, monoclonally integrated, silent provirus, which cannot be reactivated either by stimulation in vitro or by in vivo injection of the tumor cells or cloned proviral DNA in sheep. Sequence analysis of the tax gene from the YR2 cell line identified two G-to-A transitions (G7924 to A7924 and G8149 to A8149) that result in E-to-K amino acid changes at positions 228 and 303 in the Tax protein. Following retroviral vector-mediated transfer of a wild-type tax gene into YR2 cells, we showed that BLV mRNA, viral proteins, and virions were produced, demonstrating that the cellular factors required for virus expression were present in the original YR2 cell line. Injection of this transduced YR2 cell line in sheep led to the rescue of replication-competent BLV proviruses. The integrated competent proviruses exhibited unique chimeric tax genes, which arose from homologous recombination between the transduced wild-type tax and the YR2-derived tax sequences. Furthermore, in one of these functional recombinant proviruses, only the A8149-to-G8149 reversion was present, providing clear evidence that the defect underlying the silent phenotype in YR2 cells results from a single C-terminal E303-to-K303 amino acid substitution in the BLV Tax protein. Our observations suggest that a single strategically located mutation in tax provides a mechanism for BLV inactivation in B-cell tumors.
    Journal of Virology 03/1999; 73(2):1054-65. · 4.65 Impact Factor
  • Handbook of Vertebrate Immunology, Edited by Pastoret, P. P. and Griebel, P. and Bazin, H. and Govaerts, A, 01/1998: chapter XII: pages 421-438; Academic press, London, UK.
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    ABSTRACT: Immunization with naked plasmid DNA effectively induces both humoral and cell-mediated immunity to vaccine antigens and can confer protection against numerous infectious diseases. To explore the potential use of DNA immunization to induce rotavirus-specific immune responses, we used plasmid DNA encoding the VP4 gene of bovine rotavirus (BRV). Intrasmuscular injection of the plasmid encoding the VP4 gene into C57BI/6 mice induced cell-mediated immunity as measured by cytokine production. Although DNA immunization did not induce a detectable BRV-specific antibody response, DNA-immunized animals were primed for antibody production and a cellular immune response. Following viral inoculation, the immunized animals displayed an enhanced number of BRV-specific antibody-secreting cells and cytotoxic activity. The immune response induced by DNA immunization alone or followed by viral inoculation was biased toward IFN-gamma production (Th1-like). CD4+ lymphocytes were the major source of IFN-gamma production in the spleen following DNA immunization. In contrast, a balanced cytokine production was observed in the spleens of animals receiving whole virus. These experiments showed that DNA immunization with a gene encoding the VP4 protein of BRV stimulated a Th1-like immune response in mice, and this bias in the immune response persisted following exposures to whole virus.
    Viral Immunology 02/1997; 10(3):117-27. · 1.64 Impact Factor
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    ABSTRACT: Non-major histocompatibility complex (MHC) restricted cytotoxicity is an important part of the immune reaction mounted in response to bovine herpes virus type 1 (BHV-1) infection. In this study, we evaluated the effect of BHV-1 infection on the ability of lung parenchyma leucocytes (LPL), cranial tracheobronchial lymph node cells (BLNC) and peripheral blood mononuclear leucocytes (PBML) to mediate this function. While LPL from non-infected calves mediated cytotoxicity against BHV-1-infected cells, a similar activity could not be detected in PBML or BLNC. In contrast, both LPL and PBML from naive calves could mediate cytotoxicity against K562 target cells but only after activation with interleukin-2 (IL-2). BLNC were unable to kill K562 cells. Infection of calves with BHV-1 enhanced the ability of LPL and PBML to kill BHV-1-infected cells. This enhancement was detected as early as Day 1 after infection in LPL whereas it could only be detected in PBML 8 days after infection. The results demonstrate that the leucocyte population present at the site of infection was able to mediate a potentially important antiviral function and that this function was enhanced rapidly in response to infection. Thus LPL-mediated cytotoxicity may be an important mechanism for the recovery from BHV-1 infection.
    Immunology 02/1992; 75(1):47-52. · 3.74 Impact Factor