Overview: Novel approaches to vaccine delivery remain one of the greatest challenges in the pharmaceutical and vaccine industries, and have been cited as one of the top 10 technologies that will greatly impact global health. Subunit and killed vaccines require the addition of adjuvants: substances that increase immune responses induced by the vaccine. Conventional adjuvants included in commercially available vaccines often provide only partial protection and induce severe tissue reactions at the injection site. There is a great need for effective and safe adjuvants to improve available vaccines and the chances of bringing to market some of the candidate vaccines made available by recent advances in biotechnology.
We conduct research and development of novel adjuvants, formulations and delivery systems such as needle-free, with the overall goal of improving vaccines. The CpG Immune Modulation project is designed to capitalize on the discovery that specific DNA sequences (CpG) can stimulate the mammalian innate immune response. This is because specific CpG immunostimulatory sequences are found at their highest concentrations in bacteria and are rare in the genome of higher animals. Vertebrate immune systems therefore “perceive” CpGs as a danger signal and respond through the induction of an innate immune response.
Polyphosphazenes are synthetic biodegradable polymers that have a high safety profile as indicated by clinical trials in humans. These polymers appear to have a range of biological activities. We describe below our development of polyphosphazenes as adjuvants and delivery systems for vaccines and therapeutics.
CpG Immune Modulation
Background:
With increasing concerns regarding the use of antibiotics in livestock production, it is estimated that economic losses due to infectious disease will increase. As of 1998, the EU prohibited use for animal growth promotion of all antibiotics important in the treatment of human disease - only four antibiotics not used in human medicine are available for general agricultural use. Thus, we will need to develop better ways of controlling disease if we hope to maintain a competitive livestock industry in Canada. One way of achieving this goal is by developing safer and more effective vaccines, for example by boosting the host’s immune response to respond better to vaccines or to clear the infection naturally.
This project is designed to capitalize on the discovery that specific DNA sequences (CpG) can stimulate the mammalian innate immune response. This is because specific CpG immunostimulatory sequences are found at their highest concentrations in bacteria and are rare in the genome of higher animals. Vertebrates therefore “perceive” CpGs as a danger signal and respond through the induction of the innate immune system. CpGs have been shown to protect mice and chickens against a variety of infectious agents and are also potent adjuvants when incorporated into vaccine formulations. Thus, CpGs have shown promise as stand-alone therapeutic compounds as well as components of vaccines.
CpGs are being evaluated in humans for the treatment of cancer, allergy and infectious disease. However, the potential of CpGs in veterinary medicine has only recently been systematically explored, partly because suitable immunostimulatory motifs for domestic species had not been identified. Our research has focussed on the use of CpGs as therapeutic compounds for poultry, cattle and swine and as components of animal vaccines.
Objectives:
To identify and characterize DNA motifs that stimulate the immune system of domestic animals with the intent to:
There is overwhelming evidence from laboratory animal models that CpG oligodeoxynucleotides (ODN) can protect against infectious diseases (viral, bacterial and protozoal) and cancer, and that ODN are potent vaccine adjuvants. We have confirmed adjuvant activity of CpG ODN in cattle and demonstrated that CpG can reduce virus shedding in ruminants. For the first time, we have also shown a clear significant response to inclusion of CpG in an equine vaccine.
Additionally, we have shown that CpG ODN as “stand alone” immunotherapy can protect against infection in some animal models.
Further results are detailed below.
Effective CpG ODN sequences differ between species. Analyses of various proteins after treatment with class A (2216) or class B (2007) ODN showed that there are important qualitative differences between sheep and cattle in their cellular responses to CpG. Additional results support this and show that while CpG ODN have effects in many animal species, there may be some qualitative and quantitative differences between species that need to be investigated.
Differences in the TLR9 receptor are probably responsible for the specificity of CpG ODN. To date, TLR9 is the only known receptor for CpG ODN and TLR9 is required for CpG-mediated responses. We found that the TLR9 sequences of several mammalian species show regions of significant homology. However, significant structural differences in the extracellular domain of TLR9 may account for species-specific recognition of CpG ODN sequences.
Knowing which tissues express TLR9 is valuable in deciding where to target the CpG. These studies revealed that TLR9 expression was over 100-fold higher in lymphoid tissues (tonsils/lymph node) than blood. Also, TLR9 expression was over 10-fold higher in alveolar macrophages than in blood.
Characterization of immune response induced by CpG: Identification of the cells and the cytokines produced at the injection site will allow us to understand the mechanism of immunostimulation by CpG ODNs. We found that Emulsigen and CpG in combination produced the most potent effects in terms of obvious perivascular, leukocytic infiltration of the injection site. The majority of the infiltrating cells were of the monocyte/macrophage lineage, with some CD1+ dendritic cells and T cells present. IFN-alpha/gamma were also induced in large amounts, and were associated with dendritic cells (DC) and natural killer (NK) cells.
We hypothesized that the strong in vivo response to Class B (2007) ODN is brought about by immune cells that are not present in the peripheral blood, but perhaps are found in lymph nodes or other immune compartments. We confirmed that lymph node cells and PBMC respond differently to CpG ODN and therefore need to be considered separately.
Cells from prescapular and mesenteric lymph nodes, as well as cells isolated from ileal and jejunal Peyer’s patches were stimulated with CpG ODNs representative of the three classes (A, B and C). Interestingly, the lymph nodes had far better responses compared to the Peyer’s patches.
We compared the immunostimulatory responses of the class C CpG ODN with that of class A and B in sheep peripheral blood mononuclear cells (PBMC) and lymph node cells (LNC). The three classes of CpG ODN induced responses in PBMC from newborn lambs, suggesting that CpG can stimulate the innate immune system in newborn animals.
Similar studies in pigs indicate that all three classes of ODN induce substantial amounts of IL-1, TNF-alpha, and IL-6, but very little IFN-gamma in PBMC.
Optimization of CpG as an adjuvant: CpGs are most effective as immunostimulants when they are combined with a lipid-based adjuvant such as VTA-M or Emulsigen. We found in cattle that CpG ODN/Emulsigen combinations had the strongest adjuvant capacity and produced a more balanced Th1/Th2 immune response.
In separate experiments, we found that class B and C ODNs stimulate innate immunity in vivo in sheep. Administration of CpG ODN in Emulsigen to the bronchial mucosa stimulated the innate immune system and enhanced CpG-specific effects.
A 100-µg/kg dose of CpG ODN/Emulsigen gave the largest and most consistent responses in sheep and is therefore the optimum dose that will be used in subsequent studies. In pigs, our results indicate that 50 micrograms of CpG in VTA formulations may be close to the minimum lowest effective dose of CpG under the test conditions.
Knowing that CpGs have strong synergistic effects with adjuvants such as Emulsigen, we also investigated in mice whether CpGs and polyphosphazines would act synergistically, and found they did.
Protection against disease challenge: A major objective of our research is to develop CpG for activating innate immunity to provide protection at mucosal sites, particularly in animals exposed to respiratory pathogens. Therefore, we compared the protective effects of CpG treatment given by intra-tracheal or by subcutaneous (SC) injection in a parainfluenza-3 virus challenge experiment. This experiment showed treatment with CpG ODN 7909 can control PI-3 virus replication and that the route of treatment is important, with SC being more effective.
Another major objective of our research is to develop CpG for enhancing efficacy to a number of vaccines against important diseases of animals. The aim of this vaccination trial was to evaluate a four-way vaccine containing modified live BVDV, BHV-1 and BPIV3 vaccine and killed BRSV vaccine with or without CpG ODN (ODN 2007), by vaccination and challenge in cattle. Results indicate that all vaccines provided protection from viral infection. Although there were differences among the vaccines with respect to some read-outs, no one vaccine group was consistently better than the others.
Background:
Given the need for effective and safe adjuvants, VIDO is studying polyphosphazenes as a platform technology for animal and human vaccine formulation and delivery, and this area forms a component of larger projects at VIDO. Polyphosphazenes are synthetic biodegradable polymers that have a high safety profile as indicated by clinical trials in humans. These polymers appear to have a range of biological activities and can be made in microspheres, indicating that they have potential for diverse applications.
In this regard, we have identified three broad areas for polyphosphazene applications in the vaccine field:
a) Adjuvants
b) Delivery systems (microspheres)
c) Stimulation of innate immunity
For example, VIDO is using polyphosphazenes in conjunction with CpG in our Neonatal Immunization program.
We have tested several related polyphosphazenes in animals. Our results showed that: i) polymers dramatically increase the level of immune responses and these immune responses are maintained for a long time; (ii) they induce a balanced immune response, which is more likely to be protective against disease; and (iii) polymers are effective adjuvants in domestic animals (sheep) and do not cause tissue reactions at the site of injection.
We have been investigating the potential of polyphosphazenes as mucosal adjuvants, and have found that antibody titers increased significantly. Challenge experiments showed adjuvant activity similar to CpG ODN.
Test mucosal adjuvant activity of polyphosphazenes (PCPP). Mice immunized with antigen and PCPP had serum antibody titers approximately 1,000-fold higher than the mice immunized with antigen alone. The PCPP did not appear to enhance the immune responses to this antigen when given intranasally. In a separate experiment, mice immunized with vaccine formulated in PCPP had significantly reduced virus levels in the lungs compared to mice immunized with vaccine in phosphate-buffered saline.
Select dose of polymer for adjuvant activity in sheep. We previously observed that polymers enhanced immune responses to a model antigen, porcine serum albumin (PSA) in sheep. We investigated the optimal dose for this observed antigen. Two doses (0.5 and 1.0 mg) clearly had an adjuvant effect as they significantly enhanced PSA-specific antibody responses.
Project Leader:
