Philip J. Griebel

A road less travelled: large animal models in immunological research

The main advances in immunology have been forged or underpinned by animal experiments. However, animal research now focuses excessively on one laboratory species, and there is too much redundant repetition and too few transfers from basic discovery to successful clinical application. These features can be improved markedly by placing more emphasis on biological relevance when evaluating animal models and by taking greater advantage of the unique experimental opportunities that are offered by large animals.

Monocytes are required for optimum in vitro stimulation of bovine peripheral blood mononuclear cells by non-methylated CpG motifs

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs within certain flanking base pairs are recognized as a danger signal by the innate immune system of vertebrates. Using lymphocyte proliferative response (LPR) and IFN-gamma secretion assays, a panel of 38 ODN was screened for immunostimulatory activity on bovine peripheral blood mononuclear cells. ODN composed of a nuclease resistant phosphorothioate backbone and a leading 5-TCGTCGTT-3 motif with two 5-GTCGTT-3 motifs were highly stimulatory in both assays. Flow cytometric analysis and cell-specific surface marker labeling determined that B-cells (surface IgM(+)) were the primary cell population responding in the LPR assay. Depletion of T cells (CD3(+)) from the PBMC population did not affect IFN-gamma secretion or B-cell proliferation when cultured with CpG-ODN. However, depletion of monocytes (DH59B(+)) completely abrogated the ability of CpG-ODN to stimulate IFN-gamma secretion, and significantly reduced the B-cell proliferative response. These data establish the identity of an optimal immunostimulatory CpG motif for cattle and demonstrate that monocytes play a pivotal role in the ability of cell populations to respond to CpG-ODN. These data provide insight for future studies investigating the mechanism of CpG-ODN bioactivity and its application in novel vaccine formulations and immunotherapy.

Mucosal immune responses following oral immunization with rotavirus antigens encapsulated in alginate microspheres.

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 Freunds adjuvant (VP6-IFA), VP6 in alginate microspheres (VP6-MS) or with live PRV in incomplete Freunds 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 Peyers patches. Microspheres containing colloidal carbon were specifically bound and transported by follicle-associated epithelium of Peyers 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.

Ileal and jejunal Peyer’s patches play distinct roles in mucosal immunity of sheep

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.

Intranasal immunization with liposome-formulated Yersinia pestis vaccine enhances mucosal immune responses

The induction of mucosal immune responses by a liposome-formulated Y. pestis vaccine (formaldehyde-killed whole cell vaccine; KWC) was evaluated. We demonstrated that intranasal immunization of mice with Y. pestis KWC vaccine, formulated with liposomes, significantly enhanced mucosal immune responses in the lung when compared to the responses induced with KWC vaccine alone. These immune responses were characterized by increased titres of specific IgA and IgG in mucosal secretions (lung and nasal washes), and an increased frequency of specific antibody-secreting cells in the lungs. In addition, antigen-specific proliferative responses and IFN-gamma-secreting cells were also significantly enhanced in the spleens of mice immunized with the KWC vaccine formulated in liposomes. Animals that were immunized intranasally with the KWC vaccine showed significant protection against an intranasal challenge with Y. pestis. These results highlight the importance of mucosal administration of vaccine antigens to stimulate immunity in the respiratory tract and demonstrate that liposome formulations can improve the effectiveness of conventional vaccines.

Effects of intradermally administered plasmid deoxyribonucleic acid on ovine popliteal lymph node morphology.

In the last decade it has become apparent that bacterial deoxyribonucleic acid (DNA) is recognized as a “danger signal” by the mammalian immune system. To investigate this interaction, sheep were injected intradermally two centimeters distal to the lateral prominence of the fibular head with 400 μg of purified plasmid DNA. Over a 28‐day period ultrasound measurements indicated a progressive increase in size of both plasmid and saline (controls) treated popliteal lymph nodes and at Day 30 macroscopic and histological measurements of the lymph nodes were determined. Compared with the contralateral control lymph nodes, plasmid exposed lymph nodes were heavier (2.8 ± 0.1g vs. 2.0 ± 0.6 g) and displayed prominent histological changes in the cortex and medulla. Average medullary cord thickness (114.2 ± 25.2 μm) and the average distance across medullary sinuses (64.4 ± 2.5 μm) were significantly greater after plasmid exposure relative to contralateral controls (62.7 ± 14.9 μm and 36.5 ± 1.0 μm, respectively). Total number of germinal centers (71.4 ± 17.7) and the total area of germinal centers (4.0 ± 1.3 mm2) within the cortex of popliteal lymph nodes exposed to plasmid were also significantly greater than the controls (40.4 ± 11.4 and 1.6 ± 0.5 mm2, respectively). Our results demonstrate that a single exposure to plasmid DNA has long term effects on regional lymph node weight and morphology. Anat Rec 262:186–192, 2001.

Differential display analysis of gene expression during the induction of mucosal immunity.

Abstract. One approach to understanding the physiologically relevant events during the induction of an immune response is to identify genes that are expressed when the immune system first encounters antigen. Such an investigation requires a naive but fully functional immune system, and the fetal lamb provides these conditions during the last trimester of gestation. Intestinal segments, containing a jejunal Peyers patch, were surgically prepared in fetal lambs (>120xa0days gestation) and individual intestinal segments were injected with either culture medium or infectious bovine rotavirus. Peyers patch tissue was collected 18xa0h postinfection. Histology and virus culture confirmed that bovine rotavirus had infected the mucosal epithelium. RNA was extracted from jejunal Peyers patch tissue and mRNA differential display was used to identify genes expressed following rotavirus infection. Ten cDNAs were identified by differential display and these cDNAs were isolated, cloned, and sequenced. One of the cDNAs sequenced, displayed homology to the gene encoding the sperm surface protein Sp17. Differential expression of this gene in antigen-exposed jejunal Peyers patches was confirmed by Northern blot and RT-PCR. The complete sequence for sheep Sp17 mRNA was obtained from a λ cDNA library, prepared from the jejunal Peyers patch of a young lamb. Sp17 expression was detected by RT-PCR in a variety of mucosa-associated lymphoid tissues but not in primary or other secondary lymphoid tissues. Thus, the fetal lamb model may be appropriate for identifying genes relevant to mucosal immunity.