Projects
Human Projects List
1. Innate immune response to current vaccines
Antigens in vaccines are currently always given with adjuvants which enhances the immune system’s response to the vaccination. The only adjuvant approved in North America is alum. It is possible though that the currently available vaccines contain adjuvant activity other than that attributable to alum. The aim of this project is to see if these vaccines can activate the innate immune arm of our immune system. The pilot project experiment is to use different concentrations of the vaccine in our lab’s innate immune platform. The platform involves incubating whole blood or peripheral blood mononuclear cells (PBMCs) with the different concentrations of the vaccine for 6 hours or for 18 hours. The 6-hour stimulation will be used in our multi-color intracellular cytokine cytometry assay, while the 18-hr stimulation will for assayed for bulk cytokine production using multiplex ELISA (Luminex) and upregulation of cell-surface costimulatory markers (via flow cytometry).
2. Response of neonatal dendritic cells (DCs) to TLR stimulation
Dendritic cells are the professional antigen-presenting cells (APCs) of the immune system. Our hypothesis is that the sub-optimal immune response to vaccines (and infections) by neonates is due to potential signaling pathway defects in their dendritic cells. Neonatal T cells can also be “defective” but certain vaccine and infectious disease epidemiological studies show otherwise; hence, DCs are more interesting to study. Our approach in this study is to use purified, primary DCs stimulated with different TLR ligands in a time-course fashion, and then assayed for changes in gene expression using Illumina’s microarray platform. We will always compare results from the neonatal samples with those from adult samples. The analysis involves bioinformatics work which will lead to a list of candidate signaling pathways that we need to validate. The validation of the array experiments can be done in many ways but can start with the very simple real-time PCR assays to check the gene expression levels as measured by the microarrays. The “real” validation is to done by doing genetic and biochemical experiments either with human cells or with mouse models, e.g. knocking down (via use of RNAi methods, knockout mice, pharmacologic inhibitors, etc.) or upregulating (using transgenic over-expression, agonists, etc.) signaling pathways and seeing if the innate immune response will fit the hypothesis/model that we gleamed from our microarrays.
3. B cell and T cell immune responses against the human papilloma virus (HPV) vaccine
The lab is involved in a BC government-funded study on whether a two-dose regimen of the HPV vaccine is as good as the currently approved and recommended three-dose schedule. Obviously, the economic implication of this is important to politicians. For public health officials, an effective two-dose regimen is better because compliance to the immunization protocol can increase, i.e. we expect that it is easier for vaccinees to get both doses than all three doses. Our interesting part in this project is to define the correlates of protection against HPV. Most vaccines are deemed effective based on immunogenicity as measure by increased antibody titers after immunization, and we use this as a surrogate for actual protection. Unfortunately, antibody responses are not always a good predictor of protection. We will check if cellular-mediated immunity (CMI) is an acceptable (or even better) correlate of protection. The CMI assays will be done for both T and B cells. We have frozen PBMCs from women pre- and post-vaccination that we will use in our CMI assays. For T cells, the assays include measurement of cellular proliferation, cytokine production, and memory marker expression. This assay is fully developed in the lab using non-specific antigens. We just need to validate it and extend it using HPV antigens. For B cells, it will primarily be done by counting HPV-specific antibody-producing cells via an ELISpot assay.
4. Genetic determinants of vaccine responses
Our largest funded project in the lab is the neonatal immune response to TLR stimulation; Project no. 2 is part of this project. We have applied our innate immune platform (described in Project no. 1) to cord blood cells from newborns, and their blood after their first and second birthdays. Complementary to this project, we are collecting and have access to similar samples through the Canadian Healthy Infant Longitudinal (CHILD) Study and from a cohort of South African children that were exposed to, but uninfected with, HIV while in the womb. We have done and will continue to do other assays on these samples (genotyping, innate immune cytokine responses, vaccine antibody response measurements, etc.). Specific to this project is the correlation of the subject’s genotype with their TLR or vaccine response. We want to see if we can find an association between a person’s genetic makeup (genotype) and their response to stimulation (phenotype). The genotyping data that we have is for single-nucleotide polymorphisms for only 700+ genes (not the whole genome). We limited these genotyping to immune pathways. This project will involve the use and development of several bioinformatics tools.
5. Development of a high-throughput anti-influenza peptide immunoassay
We want to develop a new approach to measuring vaccine efficacy which would be more robust than the current standard assay and that would be better suited for rapid, high-volume processing of samples in a pandemic setting. The influenza virus has 2 surface proteins: haemagglutinin (H) and neuraminidase (N). H facilitates entry of the virus to cells of the respiratory tract. H-specific antibodies are made in the body and bind to this surface protein, thus preventing it from entering and thus, infecting the cells. This is called viral neutralization. H constantly mutates and so, a new vaccine has to be made each year. The current gold standard of testing vaccine efficacy, which is called the haemagglutination inhibition (HAI) test, looks at levels of immunity by correlating viral neutralization. It is labour-intensive and costly. The recent availability of assays using novel devices is revolutionizing laboratory evaluation of immune responses. One such device, called the Luminex system, allows multiple simultaneous analyses using tiny volumes of serum. We propose to use this system to directly measure levels of antibodies to influenza.