In this study the effects of repeated antigen injections on total complement (C') and C'4 of guinea pig serum were investigated to determine if constant antigenic stimulation would show changes in the C' and C'4 titers. Attempts were also made to correlate any changes with variations in antibody titers during the repeated antigen injections.

Listeria monocytogenes is the etiological agent of a life-threatening, opportunistic infection caused by the ingestion of contaminated foods. Although L. monocytogenes is divided into 13 serotypes, 98% of human illness is caused by serotype 1/2a, 1/2b and 4b strains, with serotype 4b accounting for almost all the major outbreaks of human listeriosis. The principle objective of this work was to develop surface-binding monoclonal antibodies (MAbs) highly specific for serotype 4b, as well as characterize their antigen targets to aid in the detection and isolation of serotype 4b strains using an antibody based procedure. To create such antibodies, mice were immunized with formalin killed whole cells of L. monocytogenes serotype 4b strain LI0521. A total of 15 MAbs reactive to serotype 4b isolates were shown to recognize a ~77 kDa surface antigen subsequently identified by mass spectrometry as surface associated autolysin, IspC. Epitope mapping experiments further revealed that each of the 15 MAbs bound to the cell wall binding GW domain of IspC and can be essentially divided into 4 major groups based on epitope localization. ELISA analysis of the reactivity of each of the MAbs with various L. monocytogenes serotypes indicated that several MAbs were 100% specific for serotype 4b isolates. Surface plasmon resonance experiments showed that the affinity constants for each of these MAbs fell within the range of 1.0 x 10-7 to 6.4 x 10-9 M. To determine whether IspC, shown to be well conserved among various serotype 4b strains, is a useful diagnostic marker with antibody-based methods, the expression of IspC was assessed in L. monocytogenes cultured under normal and stress conditions. A functional promoter directing the transcription of ispC gene was identified immediately upstream of the ispC open reading frame by constructing the promoterless lacZ gene fusion with the putative ispC promoter region and by 5'RACE analysis. Data obtained with the lacZ reporter gene system and immunofluorescent microscopy revealed that IspC is expressed on the cell surface under all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source and enrichment media) that allow for cellular division, although the level of ispC gene expression varies. In addition, a significant effort were put into elucidating the hydrolytic bond specificity of IspC by HPLC and mass spectrometry analysis of muropeptides released from IspC-mediated hydrolysis of L. monocytogenes peptidoglycan (PG). The results demonstrated that IspC functions as an N-acetylglucosaminidase capable of cleaving the β-1,4-glycosidic bond of the PG glycan strand. Furthermore, IspC was more efficient at hydrolysing fully Nacetylated PG from a PG deacetylase gene (pgdA) deletion mutant of L. monocytogenes than partially de-N-acetylated wild-type PG, indicating that modification of PG by de-Nacetylation of GlcNAc residues renders PG resistant to IspC hydrolysis. In conclusion, the surface autolysin IspC with the N-acetylglucosaminidase activity is a novel diagnostic marker for the 4b serotype strains, which can be explored , in conjunction with specific MAbs developed here, for detection and isolation of L. monocytogenes serotype 4b strains directly from food, environmental and clinical samples with the need for minimal or no culture enrichment.

Several cellular and subcellular fractions were prepared from Pseudomonas aeruginosa strain PA-7. Those found to be immunogenic in rabbits included a heat-stable lipopolysaccharide, a protein-lipopolysaccharide complex, a cell wall preparation arid a formalin-killed whole cell vaccine. However, a lipopolysaccharide preparation extracted with phenol and water was found to be a poor immunogen in rabbits. The cell wall fraction proved to be the most effective immunogen in terms of the amount of antibody evoked, and of the duration of the serum antibody response. Hyperimmune sera produced against all four antigens were found to contain a mixed population of 2-mercaptoethanol sensitive and 2-mercaptdethanol resistant antibodies. Gel filtration and ion exchange chromatography studies established the presence of both IgM and IgG immunoglobulins in all four types of hyperimmune serum. Whole immune serum, as well as the IgM and IgG serum fractions, afforded passive protection to mice challenged with twenty or more LD₅₀ of viable organisms. There was an indication that the IgG fraction of two of the four serum types provided better protection than did the IgM fraction, but precipitation studies indicated that this may have been due to greater numbers of IgG immunoglobulins. In addition serum containing a high proportion of 2-mercaptoethanol resistant antibody-was found to promote faster clearance of injected bacteria than did serum taken earlier in the response. Immunodiffusion studies indicated that all four antigenic preparations contained at least one common immunogen; moreover, all serum types were able to react with sheep red blood cells coated with the heat-stable lipopolysaccharide preparation in passive hemagglutination and hemolysin tests. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

It was established that antibodies could be evoked in rabbits against ferredoxin purified from cultures of Clostridium pasteurianum and against its performic acid oxidized derivative. The extent of cross-reaction was studied between the two antisera and four related antigens: native ferredoxin, iron-sulfide free ferredoxin, performic acid oxidized ferredoxin, arid S-carboxymethylated ferredoxin. All combinations demonstrated cross reactivity by complement fixation, and in the case of oxidized ferredoxin antiserum, three preparations, native ferredoxin, iron sulfide free ferredoxin, and performic acid oxidized ferredoxin precipitated antibody. The data obtained with these cross-reactivity studies Indicated that the cysteine-containing regions of the ferredoxin molecule were not critically involved as antigenic determinants. The C-terminal region of the protein was considered for further study. This octapeptide was synthesized and tested for its ability to combine with antibody directed against both native ferredoxin and its performic acid oxidized derivative. The peptide exhibited specific binding to both antisera as demonstrated by inhibition of complement fixation and precipitation, and by equilibrium dialysis experiments. It is suggested that C. pasteurianum ferredoxin is antigenic in rabbits, that cysteine residues are not involved in at least two of the antigenic regions of the protein, and that the C-terminal octapeptide is one of the antigenic determinants of this molecule. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The antigenic variability of the Influenza virus hinders our ability to develop new therapeutic and vaccine strategies which provide a broad protection against all influenza strains. It has been previously suggested that a means to approach this challenge is to identify conserved sequences within viral proteins and use these for future therapeutic targets. Although such conserved sequences are plentiful amongst the internal viral proteins, their lack of exposure to the host immune system makes mounting an immune response against these regions difficult. Alternatively, the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) have been shown to provide host protection against a limited number of influenza strains when used as vaccine targets; however conserved regions within these proteins which are also antibody accessible are extremely rare. My Ph.D. thesis project is focused on investigating the functional role of a conserved region within the NA protein and to further determine the protection afforded by a monoclonal antibody to this region. In a comprehensive bioinformatics analysis, the only universally conserved sequence amongst all influenza A and B viral NA has been previously identified as being located between amino acids (a.a.) 222-230 (dubbed the HCA-2 region). However, the potential role of this region remains largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics and growth kinetics, I have found that substitutions in this sequence significantly affect viral replication by impairing the catalytic activity, substrate-binding and thermostability of NA. These findings prompted me to further investigate if antibody to this region may provide protection against influenza infection. Indeed, universal monoclonal antibody (HCA-2 MAb) against this peptide provided broad inhibition against all nine subtypes of NA in vitro and heterosubtypic protection in mice challenged with lethal doses of mouse-adapted viruses. I further demonstrated that residues within this peptide that are exposed on the surface of NA and located in close proximity to the active site, I222 and E227, are indispensable for antibody-mediated inhibition. These data are the first to demonstrate a monoclonal antibody against the NA protein which provides heterosubtypic protection. Since I observed that the HCA-2 antibody provided a broad inhibition against all nine subtypes of influenza A NA, I decided to investigate whether this inhibitory effect could be extended against Influenza B. Here, I have further reported that HCA-2 MAb provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. I also demonstrate that the growth and NA enzymatic activity of two drug resistant influenza B strains are also inhibited by the HCA-2 antibody. The findings of my Ph.D. thesis project have thus demonstrated that the HCA-2 region is paramount to optimal viral function. Additionally, my data show that antibodies generated against this region provide heterosubtypic protection both in vitro and in vivo and against drug resistant strains. These results indicate that this universally conserved epitope should be further explored as a potential target for future antiviral intervention and vaccine-induced immune responses.

The effect of continual antigenic stimulation on the immune system of mice was studied using two different experimental approaches. A GVHR was induced in Fi mice by the injection of parental spleen cells at weekly intervals. Several weeks later the spleen cells of mice undergoing a GVHR were shown to be immunosuppressed as their in vitro responses to the mitogens Con A and LPS were substancially lower than control animals. The serum from these treated mice was also immunosuppressive to normal spleen cells. The proliferative response to Con A and allogeneic cells of normal: syngeneic, allogeneic, and parental spleen cells was 90% suppressed when GVH serum was added in comparison to the addition of normal serum. Similarly, the in vitro antibody response to a T dependant antigen was impaired; however, the antibody response to a T independant antigen was not impaired. These results indicate that T cell functions are more sensitive than are B cell function to immunosuppressive factors in the serum of mice undergoing a GVHR. The serum was fractionated by gel filtration on a Bio-Gel P-200 column. The inhibitory material in GVH serum eluted in the immunoglobulin fraction of serum which indicate that it has a molecular weight of 150,000 or greater. The second approach studied involved continual allogeneic stimulation. Parental type mice were injected at five day intervals with Fi spleen cells in order to induce a HVG reaction. After several injections the spleen cells from these mice were tested in vitro. The spleen cells from HVG mice responded the same as normal spleen cells to the mitogens Con A and LPS. The spleen cells from HVG mice showed an enhanced in vitro antibody response as compared to normal spleen cells. This enhancement was attributed to the allogeneic effect. This series of experiments have shown that the induction of a GVHR in mice can later lead to immunosuppression and production of immunosuppressive factors in the serum of these mice. The induction of a short term HVG reaction has no adverse effects on the immune system except for enhancing an antibody response. It is possible that a more prolonged HVG reaction would parallel the immunosuppression observed in mice undergoing a GVHR. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Recent work has shown that in vitro, the cytotoxic immune response to cell surface antigens is enhanced if the antigen to which the immune response is directed, is on cells bearing major histocompatibility antigens identical to those of the responding cells. This 'H-2 restriction' has been demonstrated in the mouse using virally infected cells, haptenated cells, cells bearing the male Y antigen, and cells differing at the minor histocompatibility loci. Other investigations have shown that antigenic determinants coupled to tolerated antigens or isologous serum proteins, elicit a humoral response which is weaker than that to the same determinant coupled to a heterologous carrier. This and other evidence suggest an inverse relationship between humoral and cell mediated immunity. The purpose of this investigation was to explore the humoral response to antigens on cells which are syngeneic or allogeneic to the recipient, in order to determine the influence of a tolerated as opposed to allogeneic background. The approach used in this study was as follows: Mice were immunized with antigen (rabbit Fab' fragments) attached to syngeneic, allogeneic, or F₁ (semi syngeneic), irradiated spleen cells. Specific anti-rabbit Fab' plaque forming cell numbers were determined five days after the third, weekly injection of Fab' coated spleen cells. Some of the spleen cells taken from the responding animals, on the day of sacrifice, were incubated in vitro with soluble antigen (rabbit Fab' fragments not specific for mouse cells) for four days. The results showed that the humoral response to antigens attached to cells bearing 'self histocompatibility antigens (i.e. syngeneic or F₁ semi syngeneic cells) was significantly weaker than the humoral response to the same antigen on allogeneic cells. The effect of in vitro incubation of responder spleen cells for four days with soluble antigen was to reverse this difference. Those spleen cells exhibiting lowered plaque forming cell numbers initially (i.e. those cells from mice immunized with antigen on syngeneic or F₁ cell surfaces) showed, after incubation, a response equal to or greater than those cells which intially (before in vitro incubation) demonstrated a larger response (i.e. cells from those mice immunized with antigen on allogeneic cell surfaces). / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The innovation of multifunctional efficient, and safer treatments is a major challenge in nanomedicine. For example, the combination of magneto-mechanical and the photothermal strategies into one single therapeutic stage is one of the promising developments in cancer treatment. Without specificity, however, these therapies would target and harm both cancer and healthy cells. Therefore, the goal of precision medicine is to focus on delivering therapies to specific cells and minimize the side effects on healthy. Therefore, in this study, biocompatible, magnetic iron nanowires were functionalized with antibodies directed against CD44, a cell surface marker that is overexpressed in a large number of cancer cells. To test the functionality of the antibodies following conjugation to the iron nanowires, immunostaining and immunoprecipitation were performed and confirmed that the antigenicity of the antibodies was preserved following their conjugation to the nanowires. Indeed, the antibody coated nanowires were shown to play a major role in enhancing the accumulation and the internalization of nanowires to the cell surface in both adherent cells (e.g. colon cancer cells) and suspension cells (e.g., leukemia cells). Moreover, inductively coupled plasma mass spectrometry was used to quantify the attached and internalized nanowires. After only 1 h, the presence of antibodies enhanced the ability of the NWs to specifically target cancer cells, by more than 60% in both colon and leukemic cancers, compared to their negative controls. In addition, the presence of antibodies did not affect the magnetization of the nanowires. Therefore, the combination of both magneto-mechanical and photothermal strategies in the presence of the antibodies functionalized nanowires was applied to two types of cancer cells, colon cancer and leukemia. Strikingly, the targeted nanowires resulted in more than 76±3.5% and 45.5±0.4% cell death of colon cancer and leukemic target cells and less than 40% of cells died from the non-targeted NWs. These results represent a significant finding, as this is the first study which examines the role antibodies play in the internalization of iron nanowires, and more importantly, the efficacy to kill cancer cells. It also confirmed the possibility of targeting cancer cells with functionalized nanowires and destroying these cells utilizing combined strategies.

Induction of apoptosis in cancer cells has increasingly been the focus of many therapeutic approaches in oncology field. Since its identification as a TNF family member, TRAIL (TNF-related apoptosis-inducing ligand) paved a new path in apoptosis inducing cancer therapies. Its selective ability to activate extrinsic and intrinsic cell death pathways in cancer cells only, independently from p53 mutations responsible for conventional therapeutics resistance, spotted TRAIL as a potent cancer apoptotic agent. Many recombinant preparations of TRAIL and death receptor targeting monoclonal antibodies have been developed and being tested pre-clinically and clinically both as a single agent and in combinations. Of note, the monoclonal antibodies were not the only type of antibodies developed to target TRAIL receptors. Recent technology has brought forth several single chain variable domains (scFv) designs fused recombinantly to TRAIL as well. Also, it is becoming progressively more understandable that field of nanotechnology has revolutionized cancer diagnosis and therapy. The recent breakthroughs in materials science and protein engineering have helped considerably in strategically loading drugs into nanoparticles or conjugating drugs to their surface. In this review we aim to comprehensively highlight the molecular knowledge of TRAIL in the context of its pathway, receptors and resistance factors. We also aim to review the clinical trials that have been done using TRAIL based therapies and to review various scFv designs, the arsenal of nano-carriers and molecules available to selectively target tumor cells with TRAIL.

The immunologic relatedness of various cofactor-binding sites of enzymes requiring different nucleotide cofactors was examined. Chicken antibodies specific for NADPH- or CoA-binding domains were raised using an NADPH- or CoA-requiring enzyme as an immunogen. Antibodies specific for either NADPH- or CoA-binding domains were isolated by immunoaffinity chromatography of the respective antisera using unrelated NADPH- or CoA-requiring enzymes as affinity ligands. The reactivities of the NADPH- and CoA-binding-site-specific antibodies with a variety of enzymes that required different cofactors was shown on Western blots of SDS-PAGE of the enzymes. Variable cross-reactivities were observed among all nucleotide-cofactor requiring enzymes with each specific cofactor-domain-antibody population. Numerous proteins not physiologically associated with nucleotide cofactors, including acyl carrier protein, were completely unreactive. Proteins that bound phosphoryl compounds either as substrates or cofactors showed varying degrees of reactivity with each population of specific antibodies. These included aldolase, ribulose-1,5-bisphosphate carboxylase/oxygenase, ribonuclease A, carbonic anhydrase and triosephosphate isomerase. The immunologic cross-reactivity suggested that these proteins share a common structural feature, probably a primary structure epitope, since the proteins had been subjected to denaturing polyacrylamide gel electrophoresis. A candidate for this common structural feature is a glycine-rich sequence comprising a phosphate binding loop.