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treatments, and neo-antigens are the targets of immune system in cancer patients who
respond to the treatments. The cancer vaccine field is focused on using neo-antigens from
unique point mutations of genomic sequence in the cancer patient for making
personalized cancer vaccines. However, we choose a different path to find frameshift
neo-antigens at the mRNA level and develop broadly effective cancer vaccines based on
frameshift antigens.
In this dissertation, I have summarized and characterized all the potential frameshift
antigens from microsatellite regions in human, dog and mouse. A list of frameshift
antigens was validated by PCR in tumor samples and the mutation rate was calculated for
one candidate – SEC62. I develop a method to screen the antibody response against
frameshift antigens in human and dog cancer patients by using frameshift peptide arrays.
Frameshift antigens selected by positive antibody response in cancer patients or by MHC
predictions show protection in different mouse tumor models. A dog version of the
cancer vaccine based on frameshift antigens was developed and tested in a small safety
trial. The results demonstrate that the vaccine is safe and it can induce strong B and T cell
immune responses. Further, I built the human exon junction frameshift database which
includes all possible frameshift antigens from mis-splicing events in exon junctions, and I
develop a method to find potential frameshift antigens from large cancer
immunosignature dataset with these databases. In addition, I test the idea of ‘early cancer
diagnosis, early treatment’ in a transgenic mouse cancer model. The results show that
ii
early treatment gives significantly better protection than late treatment and the correct
time point for treatment is crucial to give the best clinical benefit. A model for early
treatment is developed with these results.
Frameshift neo-antigens from microsatellite regions and mis-splicing events are
abundant at mRNA level and they are better antigens than neo-antigens from point
mutations in the genomic sequences of cancer patients in terms of high immunogenicity,
low probability to cause autoimmune diseases and low cost to develop a broadly effective
vaccine. This dissertation demonstrates the feasibility of using frameshift antigens for
cancer vaccine development.
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The DENV and the Zika (ZIKV) FVs frequently co-circulate and generally cause mild self-liming febrile illnesses. However, a secondary infection with a heterologous DENV serotype may lead to life threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF/DSS have been linked to antibody dependent enhancement of infection (ADE), a phenomenon that occurs when antibodies (Abs) formed against an initial infection with one serotype of DENV cross-reacts but does not neutralize a heterologous DENV serotype in a secondary infection. Furthermore, Abs raised against the ZIKV have been observed to cross-react with the DENV and vice versa, which can potentially cause ADE and lead to severe DENV disease. The ZIKV can be transmitted vertically and has been linked to devastating congenital defects such as microcephaly in newborns. FDA approved treatments do not exist for DENV and ZIKV illnesses. Thus, there is a need for safe and effective treatments for these co-circulating viruses. Here, a tetravalent bispecific antibody (bsAb) targeting the ZIKV and all four serotypes of the DENV was expressed in the Nicotiana benthamiana (N. benthamiana) plant. Functional assays of the DENV/ZIKV bsAb demonstrated binding, neutralization, and a significant reduction in ADE activity against both the DENV and the ZIKV.
A single chain variable fragment (scFv) and a diabody based on an antibody directed against the immune checkpoint inhibitor PD-L1, were also expressed in N. benthamiana leaves. The smaller sizes of the scFv and diabody confers them with the ability to penetrate deeper tissues making them beneficial in diagnostics, imaging, and possibly cancer therapy. The past few decades has seen long strives in recombinant protein production in plants with significant improvements in production, safety, and efficacy. These characteristics make plants an attractive platform for the production of recombinant proteins, biologics, and therapeutics.
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Previously, our group engineered a plant-derived monoclonal antibody (MAb) (pHu-E16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed several pHu-E16 variants to improve its efficacy. These variants included a single-chain variable fragment (scFv) of pHu-E16 fused to the heavy chain (HC) constant domains (CH1-3) of human IgG (pHu-E16scFv-CH1-3) and a tetravalent molecule (Tetra pHu-E16) assembled from pHu-E16scFv-CH1-3 with a second pHu-E16scFv fused to the light chain (LC) constant region. pHu-E16scFv-CH1-3 and Tetra pHu-E16 were efficiently expressed and assembled in plants. To assess the impact of differences in N-linked glycosylation on pHu-E16 variant assembly and function, we expressed additional pHu-E16 variants with various combinations of HC and LC components.
Our study revealed that proper pairing of HC and LC was essential for the complete N-glycan processing of antibodies in both plant and animal cells. Associated with their distinct N-glycoforms, pHu-E16, pHu-E16scFv-CH1-3 and Tetra pHu-E16 exhibited differential binding to C1q and specific Fcγ receptors (FcγR). Notably, none of the plant-derived Hu-E16 variants showed antibody-dependent enhancement (ADE) activity in CD32A+ human cells, suggesting the potential of plant-produced antibodies to minimize the adverse effect of ADE. Importantly, all plant-derived MAb variants exhibited at least equivalent in vitro neutralization and in vivo protection in mice compared to mammalian cell-produced Hu-E16. This study demonstrates the capacity of plants to express and assemble a large, complex and functional IgG-like tetravalent mAb variant and also provides insight into the relationship between MAb N-glycosylation, FcγR and C1q binding, and ADE. These new insights may allow the development of safer and cost effective MAb-based therapeutics for flaviviruses, and possibly other pathogens.
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Although emerging evidence indicates that deep-sea water contains an untapped reservoir of high metabolic and genetic diversity, this realm has not been studied well compared with surface sea water. The study provided the first integrated meta-genomic and -transcriptomic analysis of the microbial communities in deep-sea water of North Pacific Ocean. DNA/RNA amplifications and simultaneous metagenomic and metatranscriptomic analyses were employed to discover information concerning deep-sea microbial communities from four different deep-sea sites ranging from the mesopelagic to pelagic ocean. Within the prokaryotic community, bacteria is absolutely dominant (~90%) over archaea in both metagenomic and metatranscriptomic data pools. The emergence of archaeal phyla Crenarchaeota, Euryarchaeota, Thaumarchaeota, bacterial phyla Actinobacteria, Firmicutes, sub-phyla Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria, and the decrease of bacterial phyla Bacteroidetes and Alphaproteobacteria are the main composition changes of prokaryotic communities in the deep-sea water, when compared with the reference Global Ocean Sampling Expedition (GOS) surface water. Photosynthetic Cyanobacteria exist in all four metagenomic libraries and two metatranscriptomic libraries. In Eukaryota community, decreased abundance of fungi and algae in deep sea was observed. RNA/DNA ratio was employed as an index to show metabolic activity strength of microbes in deep sea. Functional analysis indicated that deep-sea microbes are leading a defensive lifestyle.
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Background: The use of culture-independent nucleic acid techniques, such as ribosomal RNA gene cloning library analysis, has unveiled the tremendous microbial diversity that exists in natural environments. In sharp contrast to this great achievement is the current difficulty in cultivating the majority of bacterial species or phylotypes revealed by molecular approaches. Although recent new technologies such as metagenomics and metatranscriptomics can provide more functionality information about the microbial communities, it is still important to develop the capacity to isolate and cultivate individual microbial species or strains in order to gain a better understanding of microbial physiology and to apply isolates for various biotechnological applications.
Results: We have developed a new system to cultivate bacteria in an array of droplets. The key component of the system is the microbe observation and cultivation array (MOCA), which consists of a Petri dish that contains an array of droplets as cultivation chambers. MOCA exploits the dominance of surface tension in small amounts of liquid to spontaneously trap cells in well-defined droplets on hydrophilic patterns. During cultivation, the growth of the bacterial cells across the droplet array can be monitored using an automated microscope, which can produce a real-time record of the growth. When bacterial cells grow to a visible microcolony level in the system, they can be transferred using a micropipette for further cultivation or analysis.
Conclusions: MOCA is a flexible system that is easy to set up, and provides the sensitivity to monitor growth of single bacterial cells. It is a cost-efficient technical platform for bioassay screening and for cultivation and isolation of bacteria from natural environments.
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The unicellular microalga Haematococcus pluvialis has emerged as a promising biomass feedstock for the ketocarotenoid astaxanthin and neutral lipid triacylglycerol. Motile flagellates, resting palmella cells, and cysts are the major life cycle stages of H. pluvialis. Fast-growing motile cells are usually used to induce astaxanthin and triacylglycerol biosynthesis under stress conditions (high light or nutrient starvation); however, productivity of biomass and bioproducts are compromised due to the susceptibility of motile cells to stress. This study revealed that the Photosystem II (PSII) reaction center D1 protein, the manganese-stabilizing protein PsbO, and several major membrane glycerolipids (particularly for chloroplast membrane lipids monogalactosyldiacylglycerol and phosphatidylglycerol), decreased dramatically in motile cells under high light (HL). In contrast, palmella cells, which are transformed from motile cells after an extended period of time under favorable growth conditions, have developed multiple protective mechanisms - including reduction in chloroplast membrane lipids content, downplay of linear photosynthetic electron transport, and activating nonphotochemical quenching mechanisms - while accumulating triacylglycerol. Consequently, the membrane lipids and PSII proteins (D1 and PsbO) remained relatively stable in palmella cells subjected to HL. Introducing palmella instead of motile cells to stress conditions may greatly increase astaxanthin and lipid production in H. pluvialis culture.