As respiratory syncytial virus (RSV) is a respiratory pathogen that causes significant morbidity and mortality in infants, there has always been great interest in the development of a vaccine. In the 1960s, children were immunized with formalin-inactivated (FI)-RSV vaccines. Not only did these vaccines fail to prevent infection, but in most cases they resulted in enhanced disease upon subsequent exposure to the virus. In the intervening years, studies in mice have led to the hypothesis that the enhanced disease is due to an aberrant Th2-biased immune response. Thus, we hypothesized that formulating FI-RSV vaccines with a Th1 promoting adjuvant, such as CpG oligoeoxynucleotides (ODN), would result in the induction of protective immunity against RSV without risk of deleterious effects. We observed in calves that parenterally delivered FI-bovine RSV (BRSV) formulated with CpG ODN resulted in a shift towards a Th1-biased or more balanced immune response that was protective against BRSV.<p> As RSV infects the lung mucosa, vaccines that induce mucosal immunity are desirable. Parenterally delivered vaccines typically induce systemic immunity with low mucosal immune response levels, whereas mucosally delivered vaccines induce systemic and mucosal immunity. However, upon mucosal delivery there is an increased chance of vaccine components being degraded or washed away prior to the induction of immunity. Thus, we added polyphosphazenes (PP) to our mucosal vaccine formulations. PP are synthetic polymers that form non-covalent complexes with other vaccine components, increasing their stability. Intranasally delivered FI-BRSV co-formulated with CpG ODN and PP performed better than FI-BRSV alone, or FI-BRSV formulated with either adjuvant individually, in terms of inducing protective immunity against BRSV in mice. Furthermore, mice that received intranasally-delivered FI-BRSV or BRSV F protein co-formulated with CpG ODN and PP developed higher levels of immunity and protection than mice that received parenterally delivered vaccines. Because of the similarities between BRSV and HRSV, co-formulation of intranasally delivered HRSV vaccines with CpG ODN and PP could prove important in the development of a safe vaccine against HRSV in humans.

As respiratory syncytial virus (RSV) is a respiratory pathogen that causes significant morbidity and mortality in infants, there has always been great interest in the development of a vaccine. In the 1960s, children were immunized with formalin-inactivated (FI)-RSV vaccines. Not only did these vaccines fail to prevent infection, but in most cases they resulted in enhanced disease upon subsequent exposure to the virus. In the intervening years, studies in mice have led to the hypothesis that the enhanced disease is due to an aberrant Th2-biased immune response. Thus, we hypothesized that formulating FI-RSV vaccines with a Th1 promoting adjuvant, such as CpG oligoeoxynucleotides (ODN), would result in the induction of protective immunity against RSV without risk of deleterious effects. We observed in calves that parenterally delivered FI-bovine RSV (BRSV) formulated with CpG ODN resulted in a shift towards a Th1-biased or more balanced immune response that was protective against BRSV.<p> As RSV infects the lung mucosa, vaccines that induce mucosal immunity are desirable. Parenterally delivered vaccines typically induce systemic immunity with low mucosal immune response levels, whereas mucosally delivered vaccines induce systemic and mucosal immunity. However, upon mucosal delivery there is an increased chance of vaccine components being degraded or washed away prior to the induction of immunity. Thus, we added polyphosphazenes (PP) to our mucosal vaccine formulations. PP are synthetic polymers that form non-covalent complexes with other vaccine components, increasing their stability. Intranasally delivered FI-BRSV co-formulated with CpG ODN and PP performed better than FI-BRSV alone, or FI-BRSV formulated with either adjuvant individually, in terms of inducing protective immunity against BRSV in mice. Furthermore, mice that received intranasally-delivered FI-BRSV or BRSV F protein co-formulated with CpG ODN and PP developed higher levels of immunity and protection than mice that received parenterally delivered vaccines. Because of the similarities between BRSV and HRSV, co-formulation of intranasally delivered HRSV vaccines with CpG ODN and PP could prove important in the development of a safe vaccine against HRSV in humans.

One of the fundamental questions in mucosal immunology is how the intestine maintains tolerance to food antigens and commensal flora, and yet it is capable of mounting immune responses to pathogens. Peyers patches (PP) are lymphoid aggregates that are found in the small intestine and are the primary sites where adaptive immune responses are initiated in the intestine. An understanding of how PP cells regulate innate immune responses may provide information on how immune responses are regulated in the intestine. The toll-like receptors (TLRs) are a family of pattern recognition receptors (PRR) which provide a sensory mechanism for the detection of infectious threats. TLR9 recognizes bacterial DNA or synthetic CpG oligodeoxynucleotides (ODN). Cells that express TLR9 when stimulated with CpG ODN proliferate and produce Th1-like pro-inflammatory cytokines and upregulate co-stimulatory molecules. Because the intestine is constantly exposed to bacterial DNA from commensal flora, immune cells from the gut must have evolved mechanisms to modulate responses to TLR9 stimulation to prevent responses to harmless bacteria. Our hypothesis is that innate immune responses to the TLR9 agonist CpG ODN in Peyers patches (PP) are attenuated compared to other tissues such as blood or lymph nodes. This is due to local regulatory mechanisms unique to the intestinal microenvironment.<p> We conducted a number of experiments to test this hypothesis. We initially assessed the immunostimulatory activity of three available classes of CpG ODN in lymph nodes (LN), peripheral blood mononuclear cells (PBMC) and PP since this had not been done in ruminants. We found that CpG ODN induced strong IFNá, IFN-gamma, IL-12, lymphocyte proliferation and NK-like activity in LN and PBMC. In contrast, these responses were significantly less in PP stimulated with CpG ODN. We wondered whether the reduced responses of PP cells to CpG ODN were unique to the TLR9 agonist. For this reason we tested responses of cells from these tissues to poly (I:C), LPS, and single-stranded RNA, which are agonists for TLR3, TLR4, and TLR7/8 respectively. Additionally, we tested combinations of TLRs since others have reported that multiple TLR agonists may induce synergistic responses. All TLR agonists or their combinations either failed to induce detectable responses or the responses were significantly less in PP compared to other tissues. Thus we concluded that PP cells responses to TLR stimulation were attenuated. In all tissues tested, there were no synergistic responses (IFN-alpha, IFN-gamma and lymphocyte proliferation) following stimulation with combinations of agonists. However, there was inhibition of PBMC responses when TLR7/8 agonists were combined with CpG ODN (TLR9 agonist). Importantly, TLR7/8 agonists reduced the CpG-induced proliferative responses in purified blood B cells. Interestingly, ovine B cells constitutively expressed TLR7/8 and TLR9 mRNA, suggesting the potential for cross-talk between the receptors.<p> Interestingly, cell from all isolated tissues [ileal PP (IPP), jejunal PP (JPP), mesenteric LN (mLN) and PBMC] expressed similar levels of TLR9 mRNA, suggesting that the reduced responsiveness to CpG ODN stimulation in PP was not due to a lack of TLR9 expression.<p> Surprisingly, we observed that PP cells spontaneously secreted significant amounts of the immunoregulatory cytokine IL-10. Furthermore, we confirmed that CD21+ B cells were the source of the IL-10. We then examined the role of IL-10 in regulating IFN and IL-12 responses in PP. Neutralization of IL-10 resulted in a significant increase in the numbers of CpG-induced IFNá-secreting cells detected and in IFN-gamma and IL-12 production by PP cells (both follicular and interfollicular lymphocytes). Similarly, depletion of the CD21+ B cells resulted in significant increases in IFNá, IFN-gamma and IL-12 responses. These observations support the conclusion that IL-10-secreting PP CD21+ B cells suppress innate immune responses in PP. Further characterization by flow cytometry revealed that these cells were CD1b-CD5-CD11c-CD72+CD21+ IgM+ B cells. We have proposed that these IL-10-secreting PP CD21+ B cells are a novel subset of regulatory B cells (Bregs).<p> Finally, we examined the capacity of IL-10 secreting B cells (Bregs) to respond to CpG ODN. To achieve this, we compared CD21+ B cells from blood and JPP. Unlike blood CD21+ B cells, CD21+ B cells from JPP proliferated poorly in response to CpG ODN. Moreover, PP CD21+ B cells, unlike blood CD21+ B cells, do not secrete IgM or IL-12 in response to CpG stimulation, although both PP and blood CD21+ B cells express similar level of TLR9 mRNA. Neutralization of IL-10 did not enhance CpG-induced proliferative responses in PP CD21+ B cells. Thus IL-10 does not play a direct role in the hyporesponsiveness of PP CD21+ B cells to CpG ODN. To further explore the mechanism by which PP Bregs fail to respond to CpG ODN stimulation, we used a kinome analysis to determine whether the TLR9 pathway was functional in PP Bregs compared to blood CD21+ B cells. We observed that peptides representing critical adaptor molecules downstream of TLR9 such as IRAK1, TAK1, Casp8, p-38 MAPK, JNK, FOS, IKKá, NF-KB-p65 were not phosphorylated in JPP CD21+ B cells following CpG ODN stimulation. However, in blood CD21+ B cells stimulated with CpG ODN, the same peptides on the array were all highly phosphorylated leading to a functional TLR9 signaling pathway. Thus PP Bregs have evolved mechanisms by which the TLR9 signaling pathway is not activated following exposure to the TLR9 agonist, CpG ODN.<p> In conclusion, we clearly demonstrated that TLR9-induced responses in cells from PP are significantly attenuated. This is a consequence of PP CD21+ B cells (Bregs) that spontaneously secrete IL-10, which in turn conditions an anti-inflammatory environment in this tissue leading to poor cytokine responses to the TLR9 agonist, CpG ODN. Additionally, we show that Bregs are unresponsiveness to TLR9 stimulation. This unresponsiveness is due to regulatory mechanisms in Bregs leading to a dysfunctional TLR9 signaling pathway. These may represent strategies by which PP dampen innate responses to pathogen-associated molecular patterns (PAMPs) in intestinal immune tissues to maintain intestinal immune homeostasis. These conclusions are consistent with our hypothesis that TLR responses in PP cells are attenuated, and this is due to B cell-mediated regulatory mechanisms that are unique to the intestinal microenvironment.

The objective of this investigation was to evaluate the effects of administering CpG-ODN to commercial strain chickens as a potential adjuvant to vaccination against Salmonella, Eimeria spp., and Newcastle disease virus, or immunization to bovine serum albumin (BSA). During Experiment 1, which evaluated the dual application of CpG-ODN and a Newcastle disease virus vaccine, in the first of three replicate trials, on day 28 of the experiment, animals in the Vaccine + CpG 1& 14 experimental group were observed to have the highest levels of (p<0.05) anti-NDV IgG in serum. These levels were elevated above levels in animals from all other experimental groups. This suggestion for an adjuvant effect associated with CpG-ODN administration was not supported in the remaining two trials of experiment 1. Experiment 2 evaluated the potential for CpG-ODN to adjuvant a commercial live oocyst coccidial vaccine when applied by an oral route to neonatal broiler chickens. Overall, when body weight gain during challenge, development of intestinal lesions, and anti-Eimeria IgG levels were evaluated, vaccine administration alone was demonstrated to provide the best measure of protection among animals in all experimental groups, including those receiving either CpG-ODN or Non CpG-ODN. Experiment 3 investigated the simultaneous administration of CpG-ODN or Non-CpG ODN and a commercially acquired Salmonella typhimurium vaccine to SCWL chickens. Similar to experiments 1 and 2, antigen specific IgG responses in serum and indices of protection against field strain Salmonella challenge were variable and inconsistent. Anti-BSA IgG levels were compared in broiler and SCWL chickens immunized against BSA by a drinking water route of administration alone, or in combination with two different concentrations of CpG-ODN or Non CpG-ODN in experiment 4. The only observation where CpG-ODN and BSA co-administration resulted in anti-BSA IgG levels that were elevated above BSA alone immunized chickens was measured in broilers at day 19 post-final immunization. Taken together, given the variable results reported in this investigation related to the co-administration of ODN and vaccine or protein antigen, these data are largely inconclusive for suggesting that CpG-ODN can effectively adjuvant humoral immune responses in commercial strain chickens.

Zugl.: Giessen, Univ., Diss., 2009

Zugl.: Giessen, Universiẗat, Diss., 2009.

Heterosexual intercourse now accounts for the majority of HIV transmission within sub-Saharan Africa. The generation of microbicides and vaccines, therefore, requires a better understanding of the mucosal correlates of protection, including the role of HIV-specific IgA. It is now accepted that not all individuals are equally susceptible to HIV-1 infection, as exemplified by the HIV Exposed Seronegative (HESN) women of the Pumwani Cohort in Nairobi, Kenya. To assess whether mucosal IgA responses contribute to this protection, 3 novel IgA variable genes were cloned from HESN cervical B-cell cDNA. Nine monoclonal IgA Abs were produced, two of which were properly produced from cell culture. The HESN-derived A6/30L and A9/30L variants had a greater specificity for gp120IIIB than their A6/4L and A9/4L counterparts, while the A6 variant recognizes a distinct gp120 epitope compared to the broadly neutralizing antibody IgGb12. Further characterization of these IgA chains may suggest their suitability for use in microbicides or mucosal vaccines.

Heterosexual intercourse now accounts for the majority of HIV transmission within sub-Saharan Africa. The generation of microbicides and vaccines, therefore, requires a better understanding of the mucosal correlates of protection, including the role of HIV-specific IgA. It is now accepted that not all individuals are equally susceptible to HIV-1 infection, as exemplified by the HIV Exposed Seronegative (HESN) women of the Pumwani Cohort in Nairobi, Kenya. To assess whether mucosal IgA responses contribute to this protection, 3 novel IgA variable genes were cloned from HESN cervical B-cell cDNA. Nine monoclonal IgA Abs were produced, two of which were properly produced from cell culture. The HESN-derived A6/30L and A9/30L variants had a greater specificity for gp120IIIB than their A6/4L and A9/4L counterparts, while the A6 variant recognizes a distinct gp120 epitope compared to the broadly neutralizing antibody IgGb12. Further characterization of these IgA chains may suggest their suitability for use in microbicides or mucosal vaccines.

The lungs and upper airways are mucosal surfaces that are common site for infection with an enormous variety of inhaled pathogens. Therefore, induction of immune responses in the respiratory tract is crucial for protection against respiratory diseases. One of the pathogens infecting the host via the respiratory tract is Mycobacterium Tuberculosis. The reported efficacy of the currently used Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis is highly variable, ranging from 50% against pulmonary tuberculosis to 80% against disseminated tuberculosis. Recently, the current route of vaccination (intradermal) has been considered as a possible factor influencing the protective capacity of the BCG vaccine. In this regard, intradermal route most likely induces protective systemic responses while it fails to induce optimal responses in the lungs. Therefore, our working hypothesis is that vaccination should be directed towards the respiratory mucosal immunity in order to improve the degree of host protection in the lungs. In this thesis we studied the effect of the route of immunization as well as of different mucosal adjuvants on the induction of mucosal immune responses against the mycobacterial surface antigen PstS-1. We found that, the intranasal (i.n.) route of immunization was a more favorable route inducing strong local immune responses, than intraperitoneal (i.p.) route. Indeed, i.n. route immunization, unlike the i.p. route, elicited strong IgA responses in the lungs accompanied by a major influx of CD4+ T cells and a significant local production of IFN-gamma. IgA, being the predominant Ig isotype at mucosal tissues, is considered a major effector molecule involved in defense mechanisms against viral and bacterial pathogens at these sites. Therefore, we investigated the possible role of IgA in the protection of the respiratory mucosa against mycobacterial infections, using mice deficient in IgA and in the polymeric Ig receptor. We show that, deficient mice are more susceptible to mycobacterial infections than wild type mice, thereby demonstrating a role for IgA in protection against mycobacteria. Importantly, our studies revealed a reduced production of protective factors, such as INF-gamma and TNF-alpha in the lungs of deficient mice that was associated with the higher susceptibility seen in these mice compared to wild-type mice. We also conducted challenge experiments against another respiratory pathogen, Chlamydia pneumoniae, using IgA deficient mice. Likewise to mycobacteria, our data support a role for IgA in the protection of the respiratory tract against C. pneumoniae infection. Finally, we investigated the possible mechanisms explaining the reduced pro-inflammatory responses in IgA deficient mice. Our data indicated that IgA deficient mice present a defective response to stimulation with LPS or 19kDa which appears to be both, essentially due to suboptimal stimulation of macrophages and restricted to the lungs.

Zugl.: Giessen, Univ., Diss., 2007