Fifteen Years of Sm-p80-Based Vaccine Trials in Nonhuman Primates: Antibodies From Vaccinated Baboons Confer Protection in vivo and in vitro From Schistosoma mansoni and Identification of Putative Correlative Markers of Protection.

Recent advances in systems biology have shifted vaccine development from a largely trial-and-error approach to an approach that promote rational design through the search for immune signatures and predictive correlates of protection. These advances will doubtlessly accelerate the development of a vaccine for schistosomiasis, a neglected tropical disease that currently affects over 250 million people. For over 15 years and with contributions of over 120 people, we have endeavored to test and optimize Sm-p80-based vaccines in the non-human primate model of schistosomiasis. Using RNA-sequencing on eight different Sm-p80-based vaccine strategies, we sought to elucidate immune signatures correlated with experimental protective efficacy. Furthermore, we aimed to explore the role of antibodies through in vivo passive transfer of IgG obtained from immunized baboons and in vitro killing of schistosomula using Sm-p80-specific antibodies. We report that passive transfer of IgG from Sm-p80-immunized baboons led to significant worm burden reduction, egg reduction in liver, and reduced egg hatching percentages from tissues in mice compared to controls. In addition, we observed that sera from Sm-p80-immunized baboons were able to kill a significant percent of schistosomula and that this effect was complement-dependent. While we did not find a universal signature of immunity, the large datasets generated by this study will serve as a substantial resource for further efforts to develop vaccine or therapeutics for schistosomiasis

Watershed urbanization alters the composition and function of stream bacterial communities.

Watershed urbanization leads to dramatic changes in draining streams, with urban streams receiving a high frequency of scouring flows, together with the nutrient, contaminant, and thermal pollution associated with urbanization. These changes are known to cause significant losses of sensitive insect and fish species from urban streams, yet little is known about how these changes affect the composition and function of stream microbial communities. Over the course of two years, we repeatedly sampled sediments from eight central North Carolina streams affected to varying degrees by watershed urbanization. For each stream and sampling date, we characterized both overall and denitrifying bacterial communities and measured denitrification potentials. Denitrification is an ecologically important process, mediated by denitrifying bacteria that use nitrate and organic carbon as substrates. Differences in overall and denitrifying bacterial community composition were strongly associated with the gradient in urbanization. Denitrification potentials, which varied widely, were not significantly associated with substrate supply. By incorporating information on the community composition of denitrifying bacteria together with substrate supply in a linear mixed-effects model, we explained 45% of the variation in denitrification potential (p-value<0.001). Our results suggest that (1) the composition of stream bacterial communities change in response to watershed urbanization and (2) such changes may have important consequences for critical ecosystem functions such as denitrification

Sm-p80-based schistosomiasis vaccine mediated epistatic interactions identified potential immune signatures for vaccine efficacy in mice and baboons.

Schistosomiasis is a neglected parasitic disease of major public health concern as it affects over 250 million people in developing countries. Currently there is no licensed vaccine available against schistosomiasis. The Schistosoma mansoni calpain protein, Sm-p80, is a leading vaccine candidate now ready to move to clinical trials. In order to better assess Sm-p80 vaccine immunogenicity; here we used a systems biology approach employing RNA-sequencing to identify gene signatures and epistatic interactions following Sm-p80 vaccination in mouse and baboon models that may predict vaccine efficacy. Recombinant Sm-p80 + CpG-oligodeoxynucleotide (ODN) vaccine formulation induced both cellular and humoral immunity genes with a predominant TH1 response as well as TH2 and TH17 gene signatures. Early gene responses and gene-network interactions in mice immunized with rSm-p80 + ODN appear to be initiated through TLR4 signaling. CSF genes, S100A alarmin genes and TNFRSF genes appear to be a signature of vaccine immunogenicity/efficacy as identified by their participation in gene network interactions in both mice and baboons. These gene families may provide a basis for predicting desirable outcomes for vaccines against schistosomiasis leading to a better understanding of the immune system response to vaccination

Workflow schematic to evaluate Sm-p80 vaccine efficacy in mouse and baboon models.

<p><b>(A)</b> Experimental design to evaluate five Sm-p80 vaccine formulations for protection against <i>Schistosoma mansoni</i> challenge using transcriptomics. Thirty mice for each vaccination strategy (n = 15 for control and experimental) were immunized with DNA based vaccine (VR1020-Sm-p80); prime-boost approach (primed with DNA vaccine pcDNA3-Sm-p80 and boosted with recombinant Sm-p80 in ODN adjuvant); or with recombinant protein based vaccine (rSm-p80) in different adjuvants (alum, ODN, or GLA). Vaccinated control animals received empty DNA vector or adjuvant alone. All vaccinated animals received two booster immunizations prior to challenge infection with <i>S</i>. <i>mansoni</i>. For control challenge mice, naïve animals were infected with <i>S</i>. <i>mansoni</i> (not shown in figure). Eight weeks post-infection, animals were euthanized and necropsied to assess worm burden protection and tissue collection. Splenocytes from individual mice were pooled for RNA extraction and construction of cDNA libraries for high-throughput sequencing. Bioinformatics and transcriptomics allowed Sm-p80 vaccine formulations comparisons to identify signature molecules and network pathways. <b>(B)</b> Experimental design to identify early-gene signatures during the immune response to rSm-p80 + ODN vaccine. Experimental mice (n = 20) were immunized with rSm-80 + ODN while control mice were injected with ODN alone (n = 20). Five animals per group were euthanized at 24 hours, 48 hours, 7 days, and 21 days post-immunization. Splenocyte RNA was isolated and individual mouse cDNA libraries constructed for high-throughput sequencing. Bioinformatics analysis identified gene-networks during the immune response over time to vaccination with rSm-p80 + ODN. <b>(C)</b> Experimental design to assess rSm-p80 + ODN vaccine efficacy in a non-human primate model using transcriptomics. Baboons immunized with rSm-p80 + ODN (n = 8) or adjuvant alone (n = 8) received two booster vaccinations at four week intervals each. Prior to <i>S</i>. <i>mansoni</i> challenge infection (week 12), peripheral blood mononuclear cells were collected to establish a baseline immune signature (distinguish between vaccinated and control). Eight weeks post-infection (week 20) animals were euthanized and necropsied to assess worm burden. Individual tissue samples (PBMCs, spleens, and mesenteric lymph nodes) from each baboon were processed for RNA extraction and library preparation for sequencing. Data analysis identified Sm-p80 induced baboon tissue specific gene networks.</p

Cellular functions and inflammatory response network.

<p>IPA identified cellular functions and inflammatory response network at 24 hours post-vaccination. Expression values for 21 days, rSm-p80 + ODN post-challenge, and <i>S</i>. <i>mansoni</i> control challenge datasets were overlaid with predicted activation scores.</p

Transcriptomic comparison for five different Sm-p80 vaccine formulations.

<p><b>(A)</b> Distribution of differentially expressed mouse splenocyte genes (y axis) according to ontology classification for mice immunized with different Sm-p80 vaccine formulations and challenged with <i>S</i>. <i>mansoni</i> compared to control challenge in naïve mice (x axis). <b>(B)</b> Comparative levels of differentially expressed genes between formulations. <b>(C)</b> Heat map for relative gene expression values of 59 common genes (rows) comparing <i>S</i>. <i>mansoni</i> challenged and Sm-p80 formulations (columns). Identified genes were statistically significant (<i>P</i> < 0.01, Student’s <i>t</i>-test and greater than 2-fold change with fold change in Log₂). <b>(D)</b>. Heat map showing expression differences of 24 identified genes (rows) unique for Sm-p80 formulations (columns). <b>(E)</b> Circular visualization (CIRCOS) plot showing shared canonical pathways across vaccine formulations and control challenged mice. Significant canonical pathways (<i>P</i>-value <0.05, right-tailed Fisher Exact Test) were identified and compared across each condition.</p

Gene networks in baboon PBMCs and secondary lymphoid tissues after rSm-p80 + ODN vaccination and S. <i>mansoni</i> challenge.

<p>Gene ontology classification identified genes related to an immune system process for each tissue. Immune system process genes were imported into IPA to connect molecules and identify biological activities triggered by the gene networks. Immune system functions were mapped to identify the overall interactions and predicted activation states (activated = orange, inhibited = blue). Differences in expression values among individual genes are color coded (red = increased, blue = decreased fold change).</p

Classical B cell response gene network.

<p>Classical B cell response related gene network with overlaid expression values and z-score of activation.</p

Early gene signatures of immune response to recombinant Sm-p80 + ODN vaccination in mice.

<p><b>(A)</b> Differences in DEGs and immune system related mapped genes (y axis) across time-points (x axis) for mouse splenocytes. <b>(B)</b> Venn diagram depicting the number and percentage of significant differentially expressed genes (<i>P</i> <0.01) at 24 hours (blue), 48 hours (yellow), 7 days (red), or 21 days (green) post-vaccination are shown. Genes that are common to multiple time points are shown by the overlap. <b>(C)</b> Heat map analysis for 5 differentially expressed splenocyte genes (rows) common to all time points examined from 24 hours to 21 days (columns) post-rSm-p80 immunization. <b>(D)</b> Immune system related networks derived from RNA transcripts at each time-point post-rSm-p80 + ODN immunization. Differentially expressed genes identified at each time point were imported into IPA, and the list of genes identified from top regulator effects were selected for molecular network interactions. The overall interaction of each network is described as different biological processes at each time point. Differences in expression values are represented as red for higher fold change or blue lower fold change.</p