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Bordetella pertussis is a highly contagious bacterium that is the causative agent of whooping cough (pertussis). Currently, acellular pertussis vaccines (aP, DTaP, and Tdap) are used to prevent pertussis disease. However, it is clear that the aP vaccine efficacy quickly wanes, resulting in the reemergence of pertussis. Furthermore, recent work performed by the CDC suggest that current circulating strains are genetically distinct from strains of the past. The emergence of genetically diverging strains, combined with waning aP vaccine efficacy, calls for reevaluation of current animal models of pertussis. In this study, we used the rat model of pertussis to compare two genetically divergent strains Tohama 1 and D420. We intranasally challenged 7-week-old Sprague-Dawley rats with 108 viable Tohama 1 and D420 and measured the hallmark signs/symptoms of B. pertussis infection such as neutrophilia, pulmonary inflammation, and paroxysmal cough using whole-body plethysmography. Onset of cough occurred between 2 and 4 days after B. pertussis challenge, averaging five coughs per 15 min, with peak coughing occurring at day 8 postinfection, averaging upward of 13 coughs per 15 min. However, we observed an increase of coughs in rats infected with clinical isolate D420 through 12 days postchallenge. The rats exhibited increased bronchial restriction following B. pertussis infection. Histology of the lung and flow cytometry confirm both cellular infiltration and pulmonary inflammation. D420 infection induced higher production of anti-B. pertussis IgM antibodies compared to Tohama 1 infection. The coughing rat model provides a way of characterizing disease manifestation differences between B. pertussis strains.

Over two decades ago acellular pertussis vaccines (aP) replaced whole cell pertussis vaccines (wP) in several countries. Since then, a resurgence in pertussis has been observed, which is hypothesized to be linked to waning immunity. To better understand why waning immunity occurs, we developed a long-term outbred CD1 mouse model to conduct the longest murine pertussis vaccine studies to date, spanning out to 532 days post primary immunization. Vaccine-induced memory results from follicular responses and germinal center formation; therefore, cell populations and cytokines involved with memory were measured alongside protection from challenge. Both aP and wP immunization elicit protection from intranasal challenge and generation of pertussis specific antibody responses in mice. Responses to wP vaccination were characterized by a significant increase in T follicular helper cells in the draining lymph nodes and CXCL13 levels in sera compared to aP mice. In addition, a population of B. pertussis+ memory B cells was found to be unique to wP vaccinated mice. This population peaked post-boost, and was measurable out to day 365 post-vaccination. Anti-B. pertussis and anti-pertussis toxoid antibody secreting cells increased one day after boost and remained high at day 532. The data suggest that follicular responses, and in particular CXCL13 levels in sera, should be monitored in pre-clinical and clinical studies for the development of the next-generation pertussis vaccines.

Bordetella pertussis colonizes the respiratory mucosa of humans, inducing an immune response seeded in the respiratory tract. An individual, once convalescent, exhibits long-term immunity to the pathogen., Bordetella pertussis colonizes the respiratory mucosa of humans, inducing an immune response seeded in the respiratory tract. An individual, once convalescent, exhibits long-term immunity to the pathogen. Current acellular pertussis (aP) vaccines do not induce the long-term immune response observed after natural infection in humans. In this study, we evaluated the durability of protection from intranasal (i.n.) pertussis vaccines in mice. Mice that convalesced from B. pertussis infection served as a control group. Mice were immunized with a mock vaccine (phosphate-buffered saline [PBS]), aP only, or an aP base vaccine combined with one of the following adjuvants: alum, curdlan, or purified whole glucan particles (IRI-1501). We utilized two study designs: short term (challenged 35 days after priming vaccination) and long term (challenged 6 months after boost). The short-term study demonstrated that immunization with i.n. vaccine candidates decreased the bacterial burden in the respiratory tract, reduced markers of inflammation, and induced significant serum and lung antibody titers. In the long-term study, protection from bacterial challenge mirrored the results observed in the short-term challenge study. Immunization with pertussis antigens alone was surprisingly protective in both models; however, the alum and IRI-1501 adjuvants induced significant B. pertussis-specific IgG antibodies in both the serum and lung and increased numbers of anti-B. pertussis IgG-secreting plasma cells in the bone marrow. Our data indicate that humoral responses induced by the i.n. vaccines correlated with protection, suggesting that long-term antibody responses can be protective.

Bordetella pertussis(Bp) is a highly contagious bacterium that is the causative agent of whooping cough (pertussis). Currently, acellular pertussis vaccines (aP; DTaP; Tdap) are used to prevent pertussis disease. However, it is clear that the aP vaccine efficacy quickly wanes, resulting in the re-emergence of pertussis. Furthermore, recent work performed by the CDC suggest that current circulating strains are genetically distinct from strains of the past. Emergence of genetically diverging strains combined with waning aP vaccine efficacy call for re-evaluation of current animal models of pertussis. In this study, we used the rat model of pertussis to compare two genetically divergent strains Tohama 1 and D420. We intranasally challenged seven-week-old Sprague-Dawley rats with 108viable Tohama 1 and D420 and measured the hallmark signs/symptoms ofBpinfection such as neutrophilia, pulmonary inflammation, and paroxysmal cough using whole body plethysmography. Onset of cough occurred between 2-4 days afterBpchallenge averaging five coughs per fifteen minutes, with peak coughing occurring at day eight post infection averaging upward of thirteen coughs per fifteen minutes. However, we observed an increase of coughs in rats infected with clinical isolate D420 through 12 days post challenge. The rats exhibited increased bronchial restriction followingBpinfection. Histology of the lung and flow cytometry confirm both cellular infiltration and pulmonary inflammation. D420 infection induced higher production of anti-BpIgM antibodies compared to Tohama 1 infection. The coughing rat model provides a way of characterizing disease manifestation differences betweenBpstrains.

Bordetella pertussis colonizes the respiratory mucosa of humans, inducing an immune response seeded in the respiratory tract. An individual, once convalescent, exhibits long-term immunity to the pathogen. Current acellular pertussis (aP) vaccines do not induce the long-term immune response observed after natural infection in humans. In this study, we evaluated the durability of protection from intranasal (i.n.) pertussis vaccines in mice. Mice that convalesced from B. pertussis infection served as a control group. Mice were immunized with a mock vaccine (phosphate-buffered saline [PBS]), aP only, or an aP base vaccine combined with one of the following adjuvants: alum, curdlan, or purified whole glucan particles (IRI-1501). We utilized two study designs: short term (challenged 35 days after priming vaccination) and long term (challenged 6 months after boost). The short-term study demonstrated that immunization with i.n. vaccine candidates decreased the bacterial burden in the respiratory tract, reduced markers of inflammation, and induced significant serum and lung antibody titers. In the long-term study, protection from bacterial challenge mirrored the results observed in the short-term challenge study. Immunization with pertussis antigens alone was surprisingly protective in both models; however, the alum and IRI-1501 adjuvants induced significant B. pertussis-specific IgG antibodies in both the serum and lung and increased numbers of anti-B. pertussis IgG-secreting plasma cells in the bone marrow. Our data indicate that humoral responses induced by the i.n. vaccines correlated with protection, suggesting that long-term antibody responses can be protective.

Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe pulmonary infections associated with high morbidity and mortality in immunocompromised patients. The development of a vaccine against P. aeruginosa could help prevent infections caused by this highly antibiotic-resistant microorganism., Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe pulmonary infections associated with high morbidity and mortality in immunocompromised patients. The development of a vaccine against P. aeruginosa could help prevent infections caused by this highly antibiotic-resistant microorganism. We propose that identifying the vaccine-induced correlates of protection against P. aeruginosa will facilitate the development of a vaccine against this pathogen. In this study, we investigated the mechanistic correlates of protection of a curdlan-adjuvanted P. aeruginosa whole-cell vaccine (WCV) delivered intranasally. The WCV significantly decreased bacterial loads in the respiratory tract after intranasal P. aeruginosa challenge and raised antigen-specific antibody titers. To study the role of B and T cells during vaccination, anti-CD4, -CD8, and -CD20 depletions were performed prior to WCV vaccination and boosting. The depletion of CD4+, CD8+, or CD20+ cells had no impact on the bacterial burden in mock-vaccinated animals. However, depletion of CD20+ B cells, but not CD8+ or CD4+ T cells, led to the loss of vaccine-mediated bacterial clearance. Also, passive immunization with serum from WCV group mice alone protected naive mice against P. aeruginosa, supporting the role of antibodies in clearing P. aeruginosa. We observed that in the absence of T cell-dependent antibody production, mice vaccinated with the WCV were still able to reduce bacterial loads. Our results collectively highlight the importance of the humoral immune response for protection against P. aeruginosa and suggest that the production of T cell-independent antibodies may be sufficient for bacterial clearance induced by whole-cell P. aeruginosa vaccination.

Bordetella pertussis(B. pertussis) is the causative agent of pertussis (whooping cough). Since the 1990s, pertussis has re-emerged in the United States despite an estimated 95% vaccine coverage. Our goal was to characterize neutrophil responses and gene expression profiles of murine lungs in the context of vaccination andB. pertussischallenge. We utilized a bioluminescent neutrophil mouse model (NECre luc) to track neutrophil recruitment. NECre luc mice were immunized with whole cell vaccine (WCV), acellular vaccine (ACV), or a truncated adenylate cyclase toxoid (RTX) vaccine. Neutrophil recruitment was measured in live mice across time and corroborated by flow cytometry and other data. WCV immunized mice showed signs of neutrophilia in response toB. pertussischallenge. Mice immunized with either ACV or WCV cleared the challenge infection; however immunization with RTX alone was not protective. RNA sequencing revealed distinctive gene expression profiles for each immunization group. We observed an increase in expression of genes associated with responses to infection, and changes in expression of distinct genes in each vaccine group, providing a complex view of the immune response toB. pertussisinfection in mice. This study suggests that combination of immunological analysis with transcriptomic profiling can facilitate discovery of pre-clinical correlates of protection for vaccine development.

Pseudomonas aeruginosa is an opportunistic pathogen causing acute and chronic respiratory infections associated with morbidity and mortality, especially in patients with cystic fibrosis. Vaccination against P. aeruginosa before colonization may be a solution against these infections and improve the quality of life of at-risk patients. To develop a vaccine against P. aeruginosa, we formulated a novel peptide-based P. aeruginosa subunit vaccine based on the extracellular regions of one of its major siderophore receptors, FpvA. We evaluated the effectiveness and immunogenicity of the FpvA peptides conjugated to keyhole limpet hemocyanin (KLH) with the adjuvant curdlan in a murine vaccination and challenge model. Immunization with the FpvA-KLH vaccine decreased the bacterial burden and lung edema after P. aeruginosa challenge. Vaccination with FpvA-KLH lead to antigen-specific IgG and IgM antibodies in sera, and IgA antibodies in lung supernatant. FpvA-KLH immunized mice had an increase in recruitment of CD11b+ dendritic cells as well as resident memory CD4+ T cells in the lungs compared to non-vaccinated challenged mice. Splenocytes isolated from vaccinated animals showed that the FpvA-KLH vaccine with the adjuvant curdlan induces antigen-specific IL-17 production and leads to a Th17 type of immune response. These results indicate that the intranasal FpvA-KLH conjugate vaccine can elicit both mucosal and systemic immune responses. These observations suggest that the intranasal peptide-based FpvA-KLH conjugate vaccine with curdlan is a potential vaccine candidate against P. aeruginosa pneumonia.

Whole cell vaccines are frequently the first generation of vaccines tested for pathogens and can inform the design of subsequent acellular or subunit vaccines. For respiratory pathogens, administration of vaccines at the mucosal surface can facilitate the generation of a localized mucosal immune response. Here, we examined the innate and vaccine-induced immune responses to infection by two respiratory pathogens: Bordetella pertussis and Pseudomonas aeruginosa. In a model of intranasal administration of whole cell vaccines (WCVs) with the adjuvant curdlan, we examined local and systemic immune responses following infection. These studies showed that intranasal vaccination with a WCV led to a reduction of the bacterial burden in the airways of animals infected with the respective pathogen. However, there were unique changes in the cytokines produced, cells recruited, and inflammation at the site of infection. Both mucosal vaccinations induced antibodies that bind the target pathogen, but linear regression and principal component analysis revealed that protection from these pathogens is not solely related to antibody titer. Protection from P. aeruginosa correlated to a reduction in lung weight, blood lymphocytes and neutrophils, and the cytokines IL-6, TNF-&alpha, KC/GRO, and IL-10, and promotion of serum IgG antibodies and the cytokine IFN-&gamma, in the lung. Protection from B. pertussis infection correlated strongly with increased anti-B-pertussis serum IgG antibodies. These findings reveal valuable correlates of protection for mucosal vaccination that can be used for further development of both B. pertussis and P. aeruginosa vaccines.

Current acellular pertussis vaccines fall short of optimal protection against the human respiratory pathogen Bordetella pertussis resulting in increased incidence of a previously controlled vaccine- preventable disease. Natural infection is known to induce a protective mucosal immunity. Therefore, in this study, we aimed to use acellular pertussis vaccines to recapitulate these mucosal immune responses. We utilized a murine immunization and challenge model to characterize the efficacy of intranasal immunization (IN) with DTaP vaccine or DTaP vaccine supplemented with curdlan, a known Th1/Th17 promoting adjuvant. Protection from IN delivered DTaP was compared to protection mediated by intraperitoneal injection of DTaP and whole-cell pertussis vaccines. We tracked fluorescently labeled DTaP after immunization and detected that DTaP localized preferentially in the lungs while DTaP with curdlan was predominantly in the nasal turbinates. IN immunization with DTaP, with or without curdlan adjuvant, resulted in anti-B. pertussis and anti-pertussis toxin IgG titers at the same level as intraperitoneally administered DTaP. IN immunization was able to protect against B. pertussis challenge and we observed decreased pulmonary pro-inflammatory cytokines, neutrophil infiltrates in the lung, and bacterial burden in the upper and lower respiratory tract at day 3 post challenge. Furthermore, IN immunization with DTaP triggered mucosal immune responses such as production of B. pertussis-specific IgA, and increased IL-17A. Together, the induction of a mucosal immune response and humoral antibody-mediated protection associated with an IN administered DTaP and curdlan adjuvant warrant further exploration as a pertussis vaccine candidate formulation., Facultad de Ciencias Exactas, Instituto de Biotecnologia y Biologia Molecular

Hematopoietic stem and progenitor cell (HSPC) compartments are altered to direct immune responses to infection. Their roles during immunization are not well-described. To elucidate mechanisms for waning immunity following immunization with acellular vaccines (ACVs) against Bordetella pertussis (Bp), we tested the hypothesis that immunization with Bp ACVs and whole cell vaccines (WCVs) differ in directing the HSPC characteristics and immune cell development patterns that ultimately contribute to the types and quantities of cells produced to fight infection. Our data demonstrate that compared to control and ACV-immunized CD-1 mice, immunization with an efficacious WCV drives expansion of hematopoietic multipotent progenitor cells (MPPs), increases circulating white blood cells (WBCs), and alters the size and composition of lymphoid organs. In addition to MPPs, common lymphoid progenitor (CLP) proportions increase in the bone marrow of WCV-immunized mice, while B220+ cell proportions decrease. Upon subsequent infection, increases in maturing B cell populations are striking in WCV-immunized mice. RNAseq analyses of HSPCs revealed that WCV and ACV-immunized mice vastly differ in developing VDJ gene segment diversity. Moreover, gene set enrichment analyses demonstrate WCV-immunized mice exhibit unique gene signatures that suggest roles for interferon (IFN) induced gene expression. Also observed in naive infection, these IFN stimulated gene (ISG) signatures point toward roles in cell survival, cell cycle, autophagy, and antigen processing and presentation. Taken together, these findings underscore the impact of vaccine antigen and adjuvant content on skewing and/or priming HSPC populations for immune response.

Bordetella pertussis is the primary causative agent of pertussis (whooping cough), which is a respiratory infection that leads to a violent cough and can be fatal in infants. There is a need to develop more effective vaccines because of the resurgence of cases of pertussis in the United States since the switch from the whole-cell pertussis vaccines (wP) to the acellular pertussis vaccines (aP; diphtheria-tetanus-acellular-pertussis vaccine/tetanus-diphtheria-pertussis vaccine). Adenylate cyclase toxin (ACT) is a major virulence factor of B. pertussis that is (i) required for establishment of infection, (ii) an effective immunogen, and (iii) a protective antigen. The C-terminal repeats-in-toxin domain (RTX) of ACT is sufficient to induce production of toxin-neutralizing antibodies. In this study, we characterized the effectiveness of vaccines containing the RTX antigen against experimental murine infection with B. pertussis. RTX was not protective as a single-antigen vaccine against B. pertussis challenge, and adding RTX to 1/5 human dose of aP did not enhance protection. Since the doses of aP used in murine studies are not proportionate to mouse/human body masses, we titrated the aP from 1/20 to 1/160 of the human dose. Mice receiving 1/80 human aP dose had bacterial burden comparable to those of naive controls. Adding RTX antigen to the 1/80 aP base resulted in enhanced bacterial clearance. Inclusion of RTX induced production of antibodies recognizing RTX, enhanced production of anti-pertussis toxin, decreased secretion of proinflammatory cytokines, such as interleukin-6, and decreased recruitment of total macrophages in the lung. This study shows that adding RTX antigen to an appropriate dose of aP can enhance protection against B. pertussis challenge in mice.

Breast cancer is the second most common cause of death among women. However, it is not deadly if the cancerous cells remain in the breast. The life threat starts when cancerous cells travel to other parts of body like lung, liver, bone and brain. So, most breast cancer deaths derive from metastasis to other organs. In this study, we introduce novel proteins and cellular pathways that play important roles in brain and lung metastases of breast cancer using Protein-Protein Interaction (PPI) networks. Our topological analysis identified genes such as RPL5, MMP2 and DPP4 which are already known to be associated with lung or brain metastasis. Additionally, we found four and nine novel candidate genes that are specific to lung and brain metastases, respectively. The functional enrichment analysis showed that KEGG pathways associated with the immune system and infectious diseases, particularly the chemokine signaling pathway, are important for lung metastasis. On the other hand, pathways related to genetic information processing were more involved in brain metastasis. By enriching the traditional PPI network with protein structural data, we show the effects of mutations on specific protein-protein interactions. By using the different conformations of protein CXCL12, we show the effect of H25R mutation on CXCL12 dimerization.