Nose-associated Lymphoid Tissue Research Articles

Polymeric epitope-based vaccine induces protective immunity against group A Streptococcus

Group A Streptococcus (Strep A) is a life-threatening human pathogen with no licensed vaccine. Here, we used a biopolymer particle (BP) approach to display repeats of Strep A vaccine candidate peptides p*17 and K4S2 derived from M and non-M protein, respectively. BPs densely displaying both peptides (BP-p*17-S2) were successfully assembled in one-step inside an engineered endotoxin-free Escherichia coli strain. Purified BP-p*17-S2 showed a spherical core-shell morphology with a biopolymer core and peptide shell. Upon formulation with aluminum hydroxide as adjuvant, BP-p*17-S2 exhibited a mean diameter of 2.9 µm and a positive surface charge of 22 mV. No cytotoxicity was detected when tested against HEK-293 cells. Stability studies showed that BP-p*17-S2 is ambient-temperature stable. Immunized mice showed no adverse reactions, while producing high titers of peptide specific antibodies and cytokines. This immune response could be correlated with protective immunity in an animal model of infection, i.e. intranasal challenge of mice with Strep A, where a significant reduction of >100-fold of pathogen burden in nose-associated lymphoid tissue, lung, and spleen was obtained. The cost-effective scalable manufacture of ambient-temperature stable BPs coated with Strep A peptides combined with their immunogenic properties offer an attractive alternative strategy to current Strep A vaccine development.

Two MP2CL5 Antigen Vaccines from Naegleria fowleri Stimulate the Immune Response against Meningitis in the BALB/c Model.

Naegleria fowleri is an etiological agent that generates primary amoebic meningoencephalitis; unfortunately, no effective treatment or vaccine is available. The objective of this work was to determine the immunoprotective response of two vaccine antigens, as follows: (i) the polypeptide band of 19 kDa or (ii) a predicted immunogenic peptide from the membrane protein MP2CL5 (Smp145). Both antigens were administered intranasally in mice using cholera toxin (CT) as an adjuvant. The survival rate and immune response of immunized mice with both antigens and challenged with N. fowleri trophozoites were measured in the nose-associated lymphoid tissue (NALT) and nasal passages (NPs) by flow cytometry and enzyme-linked immunosorbent assay (ELISA). We also determined the immunolocalization of both antigens in N. fowleri trophozoites by confocal microscopy. Immunization with the polypeptide band of 19 kDa alone or coadministered with CT was able to confer 80% and 100% of protection, respectively. The immunization with both antigens (alone or coadministered with CT) showed an increase in T and B lymphocytes. In addition, there was an increase in the expression of integrin α4β1 and IgA in the nasal cavity of protected mice, and the IgA, IgG, and IgM levels were increased in serum and nasal washes. The immunolocalization of both antigens in N. fowleri trophozoites was observed in the plasma membrane, specifically in pseudopod-like structures. The MP2CL5 antigens evaluated in this work were capable of conferring protection which would lead us to consider them as potential candidates for vaccines against meningitis caused by N. fowleri.

A 250-kDa glycoprotein of Naegleria fowleri induces protection and modifies the expression of α4β1 and LFA-1 on T and B lymphocytes in mouse meningitis model.

The aims of this work were to evaluate the protective role of the 250-kDa polypeptide band of Naegleria fowleri. We designed an immunization strategy in Balb/c mice which were inoculated by i.n. route with an electrocuted 250-kDa polypeptide band of N.fowleri. We observed that the 250-kDa band induced 80% of protection, whereas the coadministration with Cholera Toxin induced 100% of protection. Moreover, high levels of IgA- and IgG-specific antibodies were detected by ELISA assay. We also analysed migration molecules (α4β1 and LFA-1) on T and B lymphocytes in nose-associated lymphoid tissue (NALT), cervical lymph nodes (CN) and nasal passages (NP) by flow cytometry. We observed that the percentage of B cells (B220/α4β1) and T cells (CD4/α4β1) in NP were higher in all immunized groups compared with the other compartments analysed. Finally, we detected by immunohistochemistry ICAM-1 and V-CAM-1 in the nasal cavity. The immunization with the 250-kDa polypeptide band, protect mice against N.fowlerichallenge and modifies migration molecules and their ligands.

Co-localization of lymphoid aggregates and lymphatic networks in nose- (NALT) and lacrimal duct-associated lymphoid tissue (LDALT) of mice

BackgroundThe lymphatic vascular pattern in the head of mice has rarely been studied, due to problems of sectioning and immunostaining of complex bony structures. Therefore, the association of head lymphoid tissues with the lymphatics has remained unknown although the mouse is the most often used species in immunology.ResultsHere, we studied the association of nasal and nasolacrimal duct lymphatics with lymphoid aggregates in 14-day-old and 2-month-old mice. We performed paraffin sectioning of whole, decalcified heads, and immunostaining with the lymphatic endothelial cell-specific antibodies Lyve-1 and Podoplanin. Most parts of the nasal mucous membrane do not contain any lymphatics. Only the region of the inferior turbinates contains lymphatic networks, which are connected to those of the palatine. Nose-associated lymphoid tissue (NALT) is restricted to the basal parts of the nose, which contain lymphatics. NALT is continued occipitally and can be found at both sides along the sphenoidal sinus, again in close association with lymphatic networks. Nasal lymphatics are connected to those of the ocular region via a lymphatic network along the nasolacrimal duct (NLD). By this means, lacrimal duct-associated lymphoid tissue (LDALT) has a dense supply with lymphatics.ConclusionsNALT and LDALT play a key role in the immune system of the mouse head, where they function as primary recognition sites for antigens. Using the dense lymphatic networks along the NLD described in this study, these antigens reach lymphatics near the palatine and are further drained to lymph nodes of the head and neck region.NALT and LDALT develop in immediate vicinity of lymphatic vessels. Therefore, we suggest a causative connection of lymphatic vessels and the development of lymphoid tissues.

Naegleria fowleri immunization modifies lymphocytes and APC of nasal mucosa.

We investigated whether intranasal immunization with amoebic lysates plus cholera toxin modified the populations of T and B lymphocytes, macrophages and dendritic cells by flow cytometry from nose-associated lymphoid tissue (NALT), cervical lymph nodes (CN), nasal passages (NP) and spleen (SP). In all immunized groups, the percentage of CD4 was higher than CD8 cells. CD45 was increased in B cells from mice immunized. We observed IgA antibody-forming cell (IgA-AFC) response, mainly in NALT and NP. Macrophages from NP and CN expressed the highest levels of CD80 and CD86 in N.fowleri lysates with either CT or CT alone immunized mice, whereas dendritic cells expressed high levels of CD80 and CD86 in all compartment from immunized mice. These were lower than those expressed by macrophages. Only in SP from CT-immunized mice, these costimulatory molecules were increased. These results suggest that N.fowleri and CT antigens are taking by APCs, and therefore, protective immunity depends on interactions between APCs and T cells from NP and CN. Consequently, CD4 cells stimulate the differentiation from B lymphocytes to AFC IgA-positive; antibody that we previously found interacting with trophozoites in the nasal lumen avoiding the N.fowleri attachment to nasal epithelium.

Formulation of the respiratory syncytial virus fusion protein with a polymer-based combination adjuvant promotes transient and local innate immune responses and leads to improved adaptive immunity

Respiratory syncytial virus (RSV) causes serious upper and lower respiratory tract infections in newborns and infants. Presently, there is no licensed vaccine against RSV. We previously reported the safety and efficacy of a novel vaccine candidate (ΔF/TriAdj) in rodent and lamb models following intranasal immunization. However, the effects of the vaccine on the innate immune system in the upper and lower respiratory tracts, when delivered intranasally, have not been characterized. In the present study, we found that ΔF/TriAdj triggered transient production of chemokines, cytokines and interferons in the nasal tissues and lungs of BALB/c mice. The types of chemokines produced were consistent with the populations of immune cells recruited, i.e. dendritic cells, macrophages and neutrophils, in the nose-associated lymphoid tissue (NALT), lung and their draining lymph nodes of the ΔF/TriAdj-immunized group. In addition, ΔF/TriAdj stimulated cellular activation with generation of mucosal and systemic antibody responses, and conferred complete protection from viral infection in the lungs upon RSV challenge. The effect of ΔF/TriAdj was short-lived in the nasal tissues and more prolonged in the lungs. In addition, both innate and adaptive immune responses were lower when mice were immunized with ΔF alone. These results suggest that ΔF/TriAdj modulates the innate mucosal environment in both upper and lower respiratory tracts, which contributes to robust adaptive immune responses and long-term protective efficacy of this novel vaccine formulation.

75. Effect of forced swimming on B and CD4+ T cells of NALT

The nose-associated lymphoid tissue (NALT), a compartment located at the base of the nose of some rodents, induces an immune response. Stress causes an increase in the secretion of glucocorticoid and catecholamine hormones by activation of the hypothalamic–pituitary–adrenal axis. The aim of this study was to determine the effects of stress by forced swimming on cell populations of B and T lymphocytes, the secretion of IgA, and the production of corticosterone. We used 10-week-old male BALB/c mice divided into a control group and an experimental group. The secretion of IgA in the nasal wash was determined by ELISA, the cell populations of B and T lymphocytes were quantified by flow cytometry, and the corticosterone levels were measured by an enzyme immunoassay. A significant difference of the secretion of IgA in nasal wash was found in stressed mice. Flow cytometry showed that CD4+ T cells increased in the stressed animals in both the NALT and the spleen, the latter organ representing the control system. We conclude that in response to the acute stressor applied in this study, the NALT showed itself to be sensitive, both in its cellular immune response represented by T lymphocytes as well as its humoral immune response represented by IgA, possibly as a result of the influence of hormones secreted during stress. This work was supported by SIP-COFAA-IPN.

Infection with 2009 H1N1 influenza virus primes for immunological memory in human nose-associated lymphoid tissue, offering cross-reactive immunity to H1N1 and avian H5N1 viruses.

Influenza is a highly contagious mucosal infection in the respiratory tract. The 2009 pandemic H1N1 (pH1N1) influenza virus infection resulted in substantial morbidity and mortality in humans. Little is known on whether immunological memory develops following pH1N1 infection and whether it provides protection against other virus subtypes. An enzyme-linked immunosorbent spot assay was used to analyze hemagglutinin (HA)-specific memory B cell responses after virus antigen stimulation in nose-associated lymphoid tissues (NALT) from children and adults. Individuals with serological evidence of previous exposure to pH1N1 showed significant cross-reactive HA-specific memory B cell responses to pH1N1, seasonal H1N1 (sH1N1), and avian H5N1 (aH5N1) viruses upon pH1N1 virus stimulation. pH1N1 virus antigen elicited stronger cross-reactive memory B cell responses than sH1N1 virus. Intriguingly, aH5N1 virus also activated cross-reactive memory responses to sH1N1 and pH1N1 HAs in those who had previous pH1N1 exposure, and that correlated well with the memory response stimulated by pH1N1 virus antigen. These memory B cell responses resulted in cross-reactive neutralizing antibodies against sH1N1, 1918 H1N1, and aH5N1 viruses. The 2009 pH1N1 infection appeared to have primed human host with B cell memory in NALT that offers cross-protective mucosal immunity to not only H1N1 but also aH5N1 viruses. These findings may have important implications for future vaccination strategies against influenza. It will be important to induce and/or enhance such cross-protective mucosal memory B cells.

Antigen‐dependent rescue of nose‐associated lymphoid tissue (NALT) development independent of LTβR and CXCR5 signaling

Nose-associated lymphoid tissue (NALT) in the rodent upper respiratory tract develops postnatally and is considered to be independent of several factors known to be involved in the organogenesis of LN and Peyer's patches (PP). In this study we demonstrate that at least two different pathways result in NALT development. Following NALT anlage formation the intrinsic pathway relies on a signaling cascade including those mediated through the chemokine receptor CXCR5 and the lymphotoxin beta receptor (LTbetaR). This allows for the formation of high endothelial venules and thereby the recruitment of lymphocytes into NALT. Alternatively, high endothelial venule formation and lymphocyte recruitment can be induced in the NALT anlage by environmental signals, which are independent of LT-betaR and chemokine receptor CXCR5 signaling but in part rely on CD40 ligand. Thus, our study identifies a novel mechanism that facilitates the rescue of NALT development at late stages in adult life independent of the canonical LTbetaR-CXCR5 signaling axis.

Coincidence of different structures of mucosa-associated lymphoid tissue (MALT) in the respiratory tract of children: no indications for enhanced mucosal immunostimulation in sudden infant death syndrome (SIDS)

Mucosa-associated lymphoid tissue (MALT) is the principal inductive site for mucosal immune responses that are capable of T and B cell responses and antigen-specific responses. In previous independent studies different structures of MALT, e.g. bronchus-, larynx- and nose-associated lymphoid tissue (BALT, LALT, NALT) have been described separately in various frequencies in the human respiratory tract over life spans. Because upper respiratory tract infections are common in infants, dysregulations of mucosal immune responses might be seriously involved in the aetiology of sudden infant death syndrome (SIDS). In the present study the coincidental occurrence of the three different MALT structures in the respiratory tract within the same patients were studied, and cases of SIDS and children who had died from different traumatic and natural causes of death (non-SIDS) were compared. First, the frequency of BALT and LALT in 46 children (35 SIDS, 11 non-SIDS) with or without NALT were examined. A tendency was found of a coincidence of respiratory MALT structures. In 50 additional cases of infant death (30 SIDS, 20 non-SIDS) from the multi-centric German Study on Sudden Infant Death Syndrome (GeSID) where death had occurred in the first year of life, the coincidence was evaluated. A coincidental occurrence of BALT, LALT and NALT or BALT and LALT (each about 30%) was found in both groups, whereby the coincidence in SIDS and the control patients did not differ. Interestingly, the children with coincidental MALT were strikingly older, supporting the hypothesis of respiratory MALT formation via environmental stimulation over time.

Immune response to Mycoplasma pulmonis in nasal mucosa is modulated by the normal microbiota.

The impact of commensal bacteria on lymphocyte responses in the upper airways was studied in rat nasal mucosa after infection with the pathogen Mycoplasma pulmonis. Phenotyping was performed in situ by paired immunofluorescence staining in germ-free (GF) and conventional (CV) rats before and 3 wk after the monoinfection. Intraepithelial lymphocytes had expanded significantly in GF (P = 0.02) but not in CV rats. Furthermore, a striking proportional increase of T-cell receptor (TCR)alphabeta(+)CD4(+) cells was observed both in the lamina propria and epithelium of GF (P < 0.01) but not of CV rats. Notably, in contrast to the pre-infection state, both mucosal compartments showed a percentage of TCRalphabeta(+)CD4(+) cells that was significantly higher in GF (P = 0.03-P < 0.01) than in CV rats after the monoinfection. In parallel, both compartments displayed a percentage of TCRalphabeta(+) CD8(+) cells that was decreased in GF (P < 0.01) but not in CV rats. The small fraction of TCRgammadelta(+) T cells observed (< 5%) did not change quantitatively or phenotypically after infection. The size of organized nose-associated lymphoid tissue was, on average, increased 5.2-fold in GF rats versus 2.6-fold in CV rats. Collectively, our results demonstrated that the normal microbiota modulated markedly the nasal immune response elicited by monoinfection with M. pulmonis.

Intranasal immunization with Toxoplasma gondii SAG1 induces protective cells into both NALT and GALT compartments.

Intranasal (i.n.) immunization with the SAG1 protein of Toxoplasma gondii plus cholera toxin (CT) provides protective immunity. The aim of this study was to analyze the cellular activation of several mucosal compartments after i.n. immunization. Cervical and mesenteric lymph node (CLN and MLN, respectively) lymphoid cell and intraepithelial lymphocyte (IEL) passive transfer experiments were performed with CBA/J mice immunized i.n. with SAG1 plus CT. CLN and MLN cells and IEL isolated 42 days after immunization conferred protective immunity on naive recipient mice challenged with strain 76K T. gondii, as assessed by the reduction in the number of brain cysts. There were proliferative specific responses in nose-associated lymphoid tissue and the CLN and MLN cells from mice immunized with SAG1 plus CT, but no cytokine was detectable. Thus, protective immunity is associated with a specific cellular response in the nasal and mesenteric compartments after i.n. immunization.

Nose-associated lymphoid tissue (NALT) in the rat

Nose-associated lymphoid tissue (NALT) in the rat