<p> <i>Candida albicans</i> is an opportunistic yeast pathogen and can cause life-threatening systemic candidiasis. The cell surface of <i>C. albicans</i> is enriched with mannan that is resistant to complement activation in the absence of antimannan antibodies. To better appreciate antimannan antibody functions in human immunity, our laboratory constructed the human recombinant antimannan Fc-free fragment M1 Fab. M1 Fab was subsequently converted to full-length human recombinant antimannan antibodies: M1g1 (IgG1), M1a1 (IgA1), M1a2 (IgA2). Each retains the identical M1 Fab binding region but differ in the isotype. Previously, our laboratory has established that M1 Fab can increase C3b deposition to <i>C. albicans </i> via the alternative pathway and that M1g1 activates the alternative and classical pathways of complement and increases phagocytosis of <i> C. albicans</i> by murine macrophages. The purpose of this study was to assess the influence of M1a1 and M1a2 on M1g1 mediated complement activation and phagocytosis of <i>C. albicans.</i> M1a1 or M1a2 was found unable to promote C3b-deposition to <i>C. albicans</i> as determined by flow cytometry and immunofluorescence microscopy. The formation of the alternative pathway convertase on <i>C. albicans</i> was promoted by M1 Fab but not by M1-Fab contained within M1a1 or M1a2. Additionally, M1g1 mediated C3b deposition was inhibited by M1a1 or M1a2 in a dose-dependent manner. Finally, M1a1 or M1a2 each significantly increased phagocytosis of <i> C. albicans</i> (<i>P</i> &lt;0.001) by human neutrophils independent of serum. The presence of M1a1 or M1a2 did not inhibit M1g1-mediated phagocytosis, indicating a redundant function of IgG1 and IgA antibodies in opsonophagocytosis. Thus, human antimannan IgA subclass variants hinder complement activation while increasing neutrophil phagocytosis of <i>C. albicans.</i> These results contribute to a more complete understanding of the role of serum IgA in host immunity.</p>

<p> The purpose of these studies was to determine the role of Interleukin-17A (IL-17A) in the immune response to respiratory mycoplasma infection. Serum levels of IL-17A increase in disease-susceptible BALB/c mice, but not disease-resistant C57BL/6 mice, infected with <i>Mycoplasma pulmonis</i>. Increased serum IL-17A was associated with mycoplasma pathology during infection in BALB/c mice, including: the presence of pulmonary neutrophils, progressive weight loss, and the development of inflammatory lung lesions. </p><p> Neutralizing the function of IL-17A using monoclonal anti-IL-17A antibodies during mycoplasma infection reduced disease severity in disease-susceptible BALB/c mice, but not disease-resistant C57BL/6 mice. Providing daily intra-peritoneal injections of anti-IL-17A antibodies to BALB/c mice infected with <i> M. pulmonis</i> was effective at reducing weight loss, the prevalence of clinical signs, and the incidence of gross lesions. Histological lesions, characterized by the presence of pulmonary neutrophils, were also lower in infected BALB/c mice receiving anti-IL-17A antibodies daily. Bacterial burden remained unaffected in mice regardless of treatment. Neutralizing IL-17A throughout infection was effective at reducing late mycoplasma pathology, a period influenced by the actions of adaptive immunity and this is supported by a reduction in disease severity when infected BALB/c mice were provided intra-peritoneal injections of anti-IL-17A antibodies only after T-cells infiltrate the lungs. </p><p> Pulmonary T-cells, specifically CD4⁺ T-helper (Th17) cells, were the primary source of IL-17A throughout infection with <i>M. pulmonis </i> in disease-susceptible BALB/c mice. Although Th17 cells increased in the lung after infection, the Th17 response did not reach its peak until the later stages of infection and coincided with when the neutralization of IL-17A started to reduce the severity of disease. IL-17A⁺ T-cells did not express Retinoic Acid Related (RAR) Orphan Receptor-&gamma;t (ROR&gamma;t), a signature Th17 transcription factor, after infecting BALB/c mice with <i> M. pulmonis</i> and suggests that ROR&gamma;t is not a suitable marker to identify the IL-17A⁺ T-cells worsening mycoplasma disease. </p><p> The effect of neutralizing IL-17A was mimicked in disease-susceptible BALB/c mice depleted of neutrophils during <i>M. pulmonis</i> infection. Depleting neutrophils in BALB/c mice infected with <i>M. pulmonis</i> abrogated weight loss while reducing the appearance of both clinical signs and gross lesions. IL-17A promotes pathology during disease utilizing various mechanisms, one of which is to mobilize and activate neutrophils; however, the IL-17A failed to worsen mycoplasma disease in the absence of neutrophils during <i>M. pulmonis</i> infection in BALB/c mice. These results suggest that IL-17A relies only upon neutrophil recruitment and activation to exacerbate mycoplasma disease. Supporting this, combining the neutralization of IL-17A with the depletion of neutrophils failed to lessen disease severity beyond what either treatment could achieve alone. These findings underscore IL-17A or neutrophils as targets for inhibition to reduce the severity of disease during mycoplasma infection. </p><p> Both IL-4 and IL-17A increase in the lungs of BALB/c mice infected with <i> M. pulmonis</i> and there are Th17 cells that secrete IL-4. In STAT6 KO mice that respond poorly to IL-4 and generate defective Th2-mediate immunity, neutralizing IL-17A also reduced inflammatory damage during <i> M. pulmonis</i> infection. Treating STAT6 KO mice with anti-IL-17A antibodies during <i>M. pulmonis</i> infection reduced weight loss, the prevalence of clinical signs, and incidence of inflammatory lesions. Like wild-type mice, the pathologic effect of IL-17A manifested during the later stages of <i> M. pulmonis</i> infection in STAT6 KO mice and coincided with the activation of adaptive immunity. Neutralizing IL-17A also failed to change mycoplasma numbers during infection in STAT6 KO mice. IL-17A is highlighted as an independent contributor to mycoplasma pathology with no impact on mycoplasma clearance; inhibiting the activation of Th2- and Th17-mediated immune responses could increase resistance by permitting the development of protective responses during infection. </p><p> This work emphasizes the importance of IL-17A and Th17 cells as an autonomous immune response worsening neutrophil-mediated pathology during late mycoplasma infection in susceptible mice. Monoclonal antibodies that neutralize the function of IL-17A could reduce the severity of disease during mycoplasma infection in man and animals. Directly targeting neutrophils may also lessen the negative impact IL-17A has on mycoplasma pathology. Vaccines that do not activate IL-17A-mediated immunity could reduce the susceptibility to mycoplasma infection and allow for the development of immune responses that lead to mycoplasma clearance. IL-17A functions to worsen disease severity without impacting mycoplasma clearance, and so IL-17A is identified as a contributor to pathology during infection. </p><p>

<p> <i>Listeria monocytogenes</i> is a Gram-positive intracellular pathogen that is readily amenable to genetic manipulation and for which there are excellent <i>in vitro</i> and <i>in vivo </i> virulence models. These attributes have allowed a thorough examination of the molecular underpinnings of <i>L. monocytogenes</i> pathogenesis, however, there are still a number of major unresolved questions that remain to be answered. For example, it has been known for many years that <i> L. monocytogenes</i> rapidly changes its transcriptional profile upon access to the host cytosol, however the host cues and bacterial components that are involved in driving this change have remained continually unanswered. One large piece of evidence came when the long-sought co-factor for the primary virulence regulator, PrfA, was discovered to be the antioxidant tripeptide, glutathione. Glutathione was demonstrated to play a crucial role in the activation of PrfA <i>in vivo</i>&mdash; a finding that has since led to two important discoveries that are described herein. First, the activation of PrfA <i>in vitro</i> requires both exogenous glutathione and a metabolic licensing step that can be recapitulated by a chemically defined synthetic media. Second, glutathione also functions as a post-translational regulator of the pore-forming virulence factor, Listeriolysin O (LLO), by reversibly binding via an S-glutathionylation reaction and preventing membrane association of the LLO monomers. These discoveries elucidate numerous regulatory roles for glutathione during infection and describe how <i>L. monocytogenes </i> is able to sense and respond to critical host compartments to mount a successful infection. </p><p> Upon entry to the host cell cytosol, the facultative intracellular pathogen <i> Listeria monocytogenes</i> coordinates the expression of numerous essential virulence factors by allosteric binding of glutathione (GSH) to the Crp-Fnr family transcriptional regulator, PrfA. Here we report that robust virulence gene expression can be recapitulated by growing bacteria in a synthetic medium (iLSM) containing GSH or other chemical reducing agents. Bacteria grown under these conditions were 45-fold more virulent in an acute murine infection model and conferred greater immunity to a subsequent lethal challenge compared to bacteria grown in conventional media. During cultivation <i>in vitro </i>, PrfA activation was completely dependent on intracellular levels of GSH, as a glutathione synthase mutant (&Delta;gshF) was activated by exogenous GSH but not reducing agents. PrfA activation was repressed in iLSM supplemented with oligopeptides, but suppression was relieved by stimulation of the stringent response. These data suggest that cytosolic <i>L. monocytogenes</i> interpret a combination of metabolic and redox cues as a signal to initiate robust virulence gene expression <i>in vivo</i>. </p><p> Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutating the cysteine residue to alanine has minor effects on overall protein function. Thus, the function of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC Listeriolysin O (LLO), secreted by the facultative intracellular pathogen <i>Listeria monocytogenes</i>, was post-translationally modified by a S-glutathionylation at this conserved cysteine residue, and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein <i>in vitro</i>, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation and retained full hemolytic activity. A mutant strain of <i>L. monocytogenes </i> expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from wild-type <i>in vitro</i>, yet was attenuated 4-6 fold in a competitive murine infection model <i>in vivo</i>. This study suggests that S-glutathionylation may represent a mechanism by which CDC family proteins are post-translationally modified and regulated, and help explain an evolutionary pressure behind the highly conserved undecapeptide cysteine.</p><p>

Simian virus 40 (SV40) entry through caveolae represents a novel form of endocytosis. SV40 translocation to caveolae was mainly examined by cell fractionation and electron microscopy (EM) in CV-1 African green monkey kidney fibroblast cells. SV40 translocated to the caveolin-enriched low density Triton-insoluble complexes as identified by Western blotting. SV40 partitioning into this fraction was inhibited by nystatin. EM analysis indicated that SV40 translocation to caveolae was prevented by the tyrosine kinase inhibitor genistein (thin sections), but SV40 was not blocked from entering the Triton-insoluble low density fraction (negative staining). Northern analysis suggested that an SV40 signal that upregulates c-myc is transmitted through caveolae. The role of the major histocompatibility complex (MHC) class I proteins was not clear in SV40 translocation to caveolae. This is partly because of the high background level of class I molecules in the low density fraction isolated by sodium carbonate sucrose gradients (LDF-C). Class I proteins were enriched in the LDF-C relative to the whole cell extract or to the plasma membrane isolated by silica coating. The clustering of class I molecules was independent of any induction. Indirect immunofluorescence labeling of class I proteins and caveolin indicated that class I molecules colocalize with caveolae. MHC class I signaling and its physiological relevance were analyzed using a monoclonal antibody (mAb) W6/32. By immunoprecipitation and thin layer chromatography, ras was found to be activated by W6/32. W6/32 did not seem to increase the tyrosine-phosphorylation level in phospholipase C-$\gamma1.$ Engagement of class I molecules did not seem to induce apoptosis, but resulted in a distinct physiological response, the adhesion strengthening in CV-1 cells to Jurkat cells. The adhesion strengthening seemed to be sensitive to the protein kinase C inhibitor calphostin C, but enhanced by the tyrosine kinase inhibitor genistein. Furthermore, the mAb-induced adhesion strengthening was transient, antibody-concentration dependent, but independent of the Fc portion of the mAb. A serum factor was found to inhibit the basal level of adhesion in CV-1 cells to Jurkat cells. These findings represent one of the first investigations of MHC class I signaling in non-lymphoid cell lines.

Viral infection of cells often requires many host cell factors. Despite the identification of numerous viral receptor molecules relatively little is known about the roles of these molecules in viral entry, and intracellular targeting. Major histocompatibility complex (MHC) class I proteins are components of the Simian Virus 40 (SV40) cell surface receptor. This interaction between virus and host cell is of interest since MHC class I proteins have typically been studied for their importance in cellular immune responses. MHC class I proteins acquire antigenic peptides in the endoplasmic reticulum (ER). SV40 is unique in its ability to target the ER from the cell surface. Thus, the possibility exists that surface MHC class I proteins may recycle and target the ER. A major question is do MHC class I proteins internalize and target SV40 to the ER. The goal of this thesis was to better characterize SV40 entry, and determine if MHC class I proteins are directly involved in viral internalization. Basic studies were therefore conducted to determine the fate of MHC class I proteins on fibroblasts. SV40 entry into cells was found to occur rather slowly. The majority of preadsorbed virions remain at the cell surface for up to 2.5 hours. Furthermore, three hours were required for preadsorbed SV40 to internalize and escape antiserum neutralization. This suggests that SV40 infectious entry is relatively slow. Cytosol acidification greatly reduced clathrin-dependent endocytosis, but had no inhibitory effect on SV40 infectious entry. Therefore, SV40 infectious entry does not occur by receptor-mediated endocytosis. Treatment of cells with PMA, nystatin, or filipin prevents internalization via caveolae. All three treatments prevented SV40 infectious entry. Thus, caveolae may be the sites of SV40 penetration into cells. To evaluate the role of MHC class I proteins in SV40 endocytosis, cell surface proteins were labeled with $\sp{125}$I. Protease treatment of cells was used to distinguish proteins that have internalized from those remaining at the cell surface. Protease resistant $\sp{125}$I-MHC class I proteins were not detected in the absence, or presence of SV40. Furthermore, a $\sp{125}$I-MHC class I protein-specific monoclonal antibody did not internalize into cells. These results suggest that surface MNC class I proteins are not internalized into cells, and SV40 does not induce their internalization. Truncated $\sp{125}$I-MHC class I proteins were detected in the media. 1,10-phenanthroline prevented accumulation of $\sp{125}$I-MHC class I proteins in the media. Thus, metalloprotease activity is involved in shedding of surface MHC class I proteins from CV-1 cells. The loss of the B$\sb2$-microglobulin subunit from the MHC class I heavy chain was found to precede metalloprotease cleavage of the heavy chain. Collectively, these results suggest that the fate of MHC class I proteins on fibroblast is shedding into the media, and these molecules are probably not directly involved in SV40 entry.

Staphylococcal Enterotoxin B (SEB) is one of the potent exotoxins synthesized by Staphylococcus aureus that causes toxic shock, is a primary cause of food poisoning and is a Class B bioterrorism agent. SEB, a superantigen, mediates antigen-independent activation of a major subset of the T-cell population by crosslinking TCRs of T-cells with MHC class II molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. This excessive secretion of cytokines, some of which are inflammatory, causes immune dysregulation, systemic inflammation and disease. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin’s interaction with the TCR or MHC class II and provide protection against the debilitating effects of this superantigen. In a series of experiments, we derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of noncrossreactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically-acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human PBMCs, or splenocytes derived from BALB/c or HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T cell activation and cytokine production. When tested in vivo in HLA-DR3 transgenic mouse TSS model, the two chimeric antibodies acted synergistically and provided full protection against SEB-mediated TSS symptoms and lethality of SEB. Furthermore, combination of chimeric anti-SEB, an extracellular inhibitor of SEB, and pharmacological agents (γ-secretase inhibitors, rapamycin, or lovastatin), an inhibitor of intracellular pathways recruited by SEB, provided significant reduction of SEB-induced T cell activation in cultures of mouse splenocytes and human PBMCs. Combination of chimeric anti-SEB antibody and lovastatin also provided in vivo protection against lethal doses of SEB in HLA-DR3 transgenic TSS model. In this study, we have developed a pair of chimeric anti-SEB antibodies (for the first time) that neutralize SEB efficiently in vitro as well as in vivo. In addition, we demonstrated that in vivo protection against lethal doses of SEB can be achieved by a statin of proven safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts. Both these findings have provided potential treatment options for diseases mediated by SEB, as there is no prophylaxis, or therapy against accidental or malicious exposure.

Chlamydia trachomatis is an obligate intracellular parasite which has a trophism for columnar epithelial cells. It is recognized as a major cause of endemic trachoma and conjunctivitis, as well as being the most prevalent sexually transmitted disease in the United States. The species can be subdivided into two biovars according to their host cell specificity and disease epidemiology. The trachoma biovar is associated with infections of ocular origin, or with the genital epithelia. The lymphogranuloma venereum (LGV) biovar is a sexually transmitted biovar causing a more invasive disease involving the lymphoid tissues. Inguinal and femoral lymph nodes become infected, producing swelling, necrosis and lymphocutaneous fistulae. The differences between biovar infectivity may in part be due to specific membrane antigens of the infectious elementary body and soluble antigens. Soluble antigens released during the infective cycle are likely to be crucial for the organism's survival. The identification of antigenic epitopes present on these molecules can aid in both serodiagnosis and selective immunization procedures which combat this pathogen. A soluble Chlamydia trachomatis esterase has been identified within the supernatant of 48 hour infected McCoy cell tissue cultures. The extraction procedure utilized a combination of ammonium sulfate precipitation, gel filtration, and immunoprecipitation techniques. The isolated protein has an approximate molecular weight of 26,000 to 30,000 M$\sb{\rm r}$ as demonstrated by gel filtration and SDS-PAGE size estimation. The esterase co-isolates with a chlamydial protein, and also contains serovar-specific epitopes. Use of rabbit anti-B serovar antibodies to deplete the esterase activity from the protein fraction confirms this molecule as being a chlamydial protein. Primates infected with B serovar C. trachomatis produce antibodies directed towards this antigen. This indicates that this molecule is immunogenic during infection, and perhaps plays a biological role in the pathogenesis of infection. Isoelectric focusing techniques has enabled the partial purification of a 26,000 M$\sb{\rm r}$ molecule, with a pI value of approximately 6.6, released during chlamydial infection. This molecule co-isolates with fractions containing esterase activity. This molecule is absent in mock-infected McCoy cell supernatants. Guinea pig antisera directed towards this protein is able to recognize specific epitopes present on the surface of infectious chlamydial elementary bodies.