Progress 05/01/12 to 04/30/16
Outputs
Target Audience:scientists, equine industry, animal production industry, professional students, veterinarians Changes/Problems:Overall, our study was delayed for 1 year due to a low number of equine offsprings in the early years of the award (due to the 2008 economic crisis), and a technical problem/personnel replacement in the Cornell Cell Sorting facility. All the proposed experiments were completed. The only change in the proposed experiments was in Aim 4, with the replacement of the transcriptome analysis for quantitative RT-PCR because stimulated dendritic cells in vitro yielded insufficient RNA for the transcriptome but quantity and quality were sufficient for quantitative RT-PCR of targeted genes relevant to antigen presentation and lymphocyte activation/modulation; final analyses are underway. Instead, peripheral blood samples from D3 and D42 foals were submitted for transcriptome and analyses of data are underway. Preliminary data indicates age-dependent expression of molecules that are involved in co-stimulation and activation of lymphocytes. What opportunities for training and professional development has the project provided?We have gained experience in performing, troubleshooting, and comparing cell-sorting methods (flow cytometry and magnetic cell sorting), antigen captureELISAs, immunoglobulin variable region gene sequencing andanalysis, transcriptome and geneanalysis.We learned about developmental humoral competence in the equine neonate. The individuals who obtained training and professional development through these studies were: Maria Julia Bevilaqua Felippe, MV, MS, PhD Rebecca Tallmadge, PhD Steven Miller, MS Understanding how vaccines work is relevant to humans and veterinary species because the neonatal phase is the first and likely the most important opportunity for providing health care and the administration of vaccines, both with life-long outcomes. Our project investigated the competence of neonatal antibody production in response to vaccination, in the presence or absence of circulating maternally-derived antibodies, using antigen-specific B cell antibody sequence. This innovative and sensitive method can be tailored and applied to any species, overcome the challenge of measuring the confounding levels of maternally-derived antibodies during early age, and reveal the effect of re-vaccination (booster) in the humoral immune response. In addition, the transcriptome analyses of equine neonate leukocytes and comparisons with equine adult leukocytes highlight key immune developmental characteristics in the neonatal phase that are relevant to vaccine response and immunity to pathogens. How have the results been disseminated to communities of interest?We reported our results to the scientific community in the annual USDA-NIFA AFRI Animal Health and Animal Welfare Project Director Workshops (from 2012-2015), and veterinary meetings: 2015 - Tallmadge RL, Felippe MJB. Assessing neonatal responses to vaccination at the protein and gene level: Ig and more Ig. Plant & Animal Genome Conference, PAG XXII, San Diego, CA. Abstract oral presentation on January 10, 2015, 5:05pm. 2015 - Tallmadge RL, Felippe MJB. Equine neonatal immunoglobulin production and diversity in response to vaccination. 5TH European Veterinary Immunology Workshop, Vienna, Austria. Abstract poster presentation,published in the programme and abstract book, page 113. Two manuscripts are being prepared with the data generated from this award. Their production comes after a delay in our project to obtainsufficient number of foals for the groups,and technical issues with and technician replacement for the cell sorter at the College. Once all the assays were processed (cell sorting and culture, monocyte differentiation into dendritic cells, lymphocyte proliferation) for all samples (foals and adult controls, different time points) in the third year of the award, subsequent analyses were performed and finalized for protein and gene expression (antigen-specific serum antibody ELISA, antigen-specific B cell IgM and IgG variable region sequencing and CDR3 length distribution, memory B cell quantification, cytokine expression of stimulated cells) in the forth year (extension) of the award. In addition, in the past year, we performed transcriptome of foal peripheral blood mononuclear cells in the first month of life and adult horse cells. A summary of findings is reported below: Rebecca L. Tallmadge, Steven C. Miller, Stephen Parry, M. Julia B. Felippe. Equine neonatal vaccination induces measureable humoral immune responses in the first month of life. This manuscript is being finalized for submission to Clinical and Vaccine Immunology for publication. Abstract: Despite great need, the value of preventative neonatal vaccination strategies is controversial in veterinary medicine: concerns exist with the interference of passively transferred maternal antibodies and the level of immaturity of the innate and adaptive immune system at birth. We hypothesized that equine neonates generate a diverse antigen-specific immunoglobulin (Ig) response in response to vaccination, even in the presence of maternal antibodies. To test this hypothesis, foals were vaccinated in the neonatal period with keyhole limpet hemocyanin (KLH) or equine influenza vaccine and humoral immune responses were assessed until 42 days of life. Vaccination with keyhole limpet hemocyanin (KLH) at 3 and 21 days yielded a robust KLH-specific antibody response at 28 days of life. A separate group of foals was vaccinated with KLH in the presence of maternal KLH-specific antibodies. Endogenous KLH-specific antibodies were produced in these foals at day 28 as well, although a 10-fold difference (p = 0.068) in antibody concentration was measured when compared to the group of non-vaccinated dams. When investigating the Ig sequencing of isolated KLH-specific B lymphocytes from the vaccinated foal group, we observed an increase in Ig sequence diversity at days 35 and 42, which was significantly more diverse than non-selected Ig sequences (p = 0.002). However, these foal Ig sequences encoded less diversity than adult KLH-specific Ig sequences (p = 0.018). Endogenous foal serum IgG production in response to influenza vaccination was difficult to distinguish from circulating maternal antibodies, however, influenza-specific Ig sequences provided an alternative readout and revealed IgM and IgG diversity similar to that of KLH-specific B cells from adult horses. This work provides evidence of neonatal foals' ability to generate antigen-specific humoral immune responses to vaccination in the first month of life, regardless of the presence of maternal antibodies. Rebecca L. Tallmadge, Qi Sun, Minhui Wang, M. Julia B. Felippe.Transcriptome and functional analyses of equine neonatal peripheral blood leukocytes.This manuscript in its final phase of analyses and will be submitted toClinical and Vaccine Immunologyfor publication. The capacity of developing immunity is often questioned in the neonatal period. This study was undertaken to assess how neonatal foal monocyte-derived dendritic cells responded to antigen, and gather information about developmental aspects of foal peripheral blood mononuclear cells in the first month of life when compared to adult horse cells. In vitro dendritic cell cultures were performed for foal cells from day 3 and day 42, and adult horses (n=3 per group). We hypothesize that neonatal cells would generate a better response to whole pathogens, such as R. equi and E. coli in contrast to purified components of the pathogens, such as the VapA protein of R. equi and lipopolysaccharide (LPS) from E. coli. Gene expression measured using quantitative RT-PCR demonstrated that CD40 mRNA expression was increased with either whole pathogen or a pathogen fragment in comparison to non-stimulated samples at each time point. MHC class II DRA mRNA expression was not detected in the non-stimulated cells for 2 foals at day 3. For the third foal, DRA expression was not up-regulated by stimulation with whole pathogen or a respective pathogen fragment. By day 42, DRA mRNA expression was detected in all non-stimulated cells for all 3 foals, but again stimulation with whole pathogen or a respective pathogen fragment failed to up-regulate expression, with the exception of E.coli stimulation in one foal. For adult mares, one mare showed very robust up-regulation with stimulation, however the other mares had negligible changes in DRA gene expression. Altogether, substantial variation was found between individuals within each group. As for peripheral blood isolated leukocytes, day 3 (before vaccination) and day 42 (after 3 doses of vaccination with keyhole limpet hemocyanin, KLH, and influenza vaccine) cells were stimulated in vitro with influenza neuraminidase N8, KLH, phytohemagglutinin (PHA), or not-stimulated (NS) for 72 hours, and the expression cytokines IL-4 and IFN-gamma by CD4 T cells measured by flow cytometry; results did not detect a difference in the expression of cytokines from cells collected before and after vaccination, indicating a lack of recall response in the first month of life. Finally, the transcriptome of peripheral blood mononuclear cells was sequenced from foals at day 1 of life, from the same foals at 42 days of life, and adult horses (n=4 per group). Multidimensional scaling was performed to measure the relationship of the transcriptome profiles among samples. The foal samples from day 1 clustered together, whereas the foal samples from day 42 were more dispersed but closer to the adult horse samples. Differential gene expression tests were performed between the age groups and detected up-regulated and down-regulated genes, with adjusted p-values of p < 0.05 and fold difference > 2. Enrichment analysis was performed to determine biological processes affected by differential gene expression over time between foal samples from days 1 and 42, and between foal samples from day 42 and adult horses. Between days 1 and 42, 760 transcripts were affected within the response to stimulus group, whereas only 275 were affected between day 42 foals and adult horses. Further, the immune system process showed 331 differentially expressed transcripts between days 1 and 42 foals, and 121 between day 42 foals and adult horses. These data emphasizethe marked changes underway in the immune system of foals during the first 6 weeks of life, and our paired data (same foals sampled at day 1 and 42) provide powerful tracking of gene expression over time. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The Impact of our study: The data showed that antigen-specific immune responses can be generated in the first month of life in foals, in the presence of maternal antibodies, with a diverse and polyclonal repertoire that undergo isotype switching. The antigen-specific IgG sequence diversity in vaccinated foals was equivalent to that of vaccinated adult horses. Importantly, we observed that serum immunoglobulin concentration is not a sensitive method to measure foal humoral immune competence, and antigen-specific immunoglobulin sequencing provided additional means to measure humoral maturation and function. These data added to the understanding of immune response to vaccines inneonatal animals, and better understanding of developmental immunology. This is the first time that antigen-specific antibody response was measured at this level of detail in the equine neonate (or perhaps the neonate of any species). Importantly, this methodology can be tailored and applied to any species. The goal of this project was to evaluate neonatal immune responses to vaccines, based on B cell immunoglobulin heavy chain variable region (VDJ) sequence analysis, serum antibody levels, development of plasma and memory cells, and the function of antigen presenting cells in early life. Specifically, the proposed experiments investigated: 1) neonatal antibody production in response to protein vaccines; 2) how the neonatal response is improved with a vaccine booster; 3) how maternally-derived antibodies affect the production and diversity of neonatal antibodies; 4) neonatal antibody repertoire diversity; 5) neonatal cellular immune system recognition of vaccine antigens; and 6) progressive development of antigen presentation cells. One outstanding innovation brought by our study is the measurement of the immune response in the neonate using antigen-specific B-cell immunoglobulin variable region sequence, designed to detect with high sensitivity low levels of antigen-specific B cells and gene recombination, not possible in the young using the traditional serum antigen-specific antibody concentrations. This approach overcomes the challenge of measuring the confounding levels of maternally-derived antibodies during early age, and allows understanding of the effect of re-vaccination (booster) in the humoral immune response. Another is the transcriptome analyses of equine neonate leukocytes and comparisons with equine adult leukocytes that highlight key immune developmental characteristics in the neonatal phase. The following was accomplished under each specific aim: Aim 1a) Determine the extent of antigen-specific immunoglobulin diversity generated in response to neonatal vaccination & Aim 1b) Determine the neonatal humoral response to a vaccine booster using the same assays as aim 1a. Progress: These experiments demonstrated that neonatal foals generated a measurable and diverse antigen-specific IgG antibody response to KLH vaccinations and boosters within the first 6 weeks of life. Impact: The robust antigen-specific serum IgG antibodies detected at 28 days of life documented the earliest response to intramuscular vaccination in postnatal foals. Efficacious neonatal vaccination would provide a major contribution to improving the health of young livestock as well as herd health, as disease reservoirs could be decreased. Percentage of completion: 100 Aim 2a) Define the effect of maternal antibodies on the diversity of neonatal immunoglobulin production in response to vaccination & Aim 2b) Define the effect of maternal antibodies on the diversity of neonatal immunoglobulin production in response to vaccine booster. Progress: These experiments revealed that neonatal foals generated antigen-specific serum IgG responses to vaccination in the presence of circulating maternal antibodies. Further, the antigen-specific immunoglobulin nucleotide diversity of these responses was not statistically different from that of adult mares, indicating immunologic competence. Impact: The clear demonstration of neonatal foals' ability to mount robust antigen-specific humoral immune responses in the presence of maternal antibodies removed a barrier from instituting neonatal vaccination as a common practice. Percentage of completion: 100 Aim 3a) Determine the neonatal immune response to a commercially available vaccine used against a relevant pathogen & Aim 3b) Measure the effect of a vaccine booster on influenza-specific humoral responses. Progress: These experiments revealed that although endogenous soluble IgG response to influenza vaccination was difficult to distinguish from passively-transferred maternal antibodies over 42 days of life, influenza-specific B cells expressing IgM or IgG were identified in these foals. Impact: Antigen-specific immunoglobulin sequencing was a more sensitive test to detect low levels of endogenously-produced antibodies, not possible previously. Percentage of completion: 100 Aim 3c) Assess the development of influenza-specific memory B cells in vaccinated neonates. Progress: These experiments indicated that detection of lymphocyte memory cell response in the peripheral blood using mRNA expression of CD38 and CD27 was limited at 42 days of life, and subsequent samples at older age would be informative of the effect of age and circulating maternal antibodies in immunologic memory development. Impact: Detection of antigen-specific memory B cell responses in the first 6 weeks of life was inconsistent and limited; longer period of study would be more informative. Percentage of completion: 100 Aim 3d) Investigate the neonatal cellular immune response to a vaccine booster using lymphocyte proliferation and cytokine production assays. Progress: In these experiments, no statistically significant differences were detected amongst the groups in response to antigenic stimulation in vitro, indicating that expansion of CD4 memory cells was limited at this age despite 3 vaccinations. Impact: Detection of antigen-specific CD4 lymphocyte memory cell responses in the first 6 weeks of life was limited; longer period of study would be more informative. Percentage of completion: 100 Aim 4a) Identify developmentally-regulated gene expression changes in APCs when exposed to inactivated whole influenza organism or its fragment & Aim 4b) Contrast the gene expression profiles identified in aim 4a with adult APC response to the same stimuli. Progress: Overall, the data indicated that individuals differed in their responses to pathogen stimulation (whole pathogen versus fragment, and type of pathogen/structure). Due to the extensive variation among individuals, more samples are needed in each group to increase statistical power. Transcriptome data illustrated the marked dynamics underway in the immune system of foals during the first 6 weeks of life, both at the level of individual genes and biological processes. Our paired data (same foals sampled at day 1 and 42) provided this powerful insight and allowed kinetic analysis of gene expression. Impact: The extensive variation in response to pathogen structure stimulation among individuals has relevance to developing vaccines that are effective across populations. Inclusion of multiple pathogen structures in vaccine products may increase the number of individuals that respond to vaccination. Detailed knowledge of immune gene expression kinetics during early life derived from transcriptome studies will further inform effective vaccine response and our understanding of the development of the immune system. Percentage of completion: 100
Publications
- Type:
Journal Articles
Status:
Other
Year Published:
2016
Citation:
Rebecca L. Tallmadge, Steven C. Miller, Stephen Parry, M. Julia B. Felippe. Equine neonatal vaccination induces measurable humoral immune responses in the first month of life. Clinical and Vaccine Immunology.
- Type:
Journal Articles
Status:
Other
Year Published:
2016
Citation:
Rebecca L. Tallmadge, Qi Sun, Minhui Wang, M. Julia B. Felippe. Transcriptome and functional analyses of equine neonatal peripheral blood leukocytes. Clinical and Vaccine Immunology.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Tallmadge RL, Felippe MJB. Assessing neonatal responses to vaccination at the protein and gene level: Ig and more Ig. Plant & Animal Genome Conference, PAG XXII, San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Tallmadge RL, Felippe MJB. Equine neonatal immunoglobulin production and diversity in response to vaccination. 5TH European Veterinary Immunology Workshop, Vienna, Austria.
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