Immunogenicity is unrelated to protective immunity when induced by soluble and particulate antigens from Nocardia brasiliensis in BALB/c mice

Cell-mediated immunity plays a major role in protection against intracellular microbes. Nocardia brasiliensis is a facultative intracellular pathogen that causes chronic actinomycetoma. In this work, we injected BALB/c mice with soluble P24 and particulate antigens from N. brasiliensis. A higher antibody titer and lymphocyte proliferation was induced by the particulate antigen than by the soluble antigen. However, five months after antigen injection, antibody concentration and lymphocyte proliferation were similar. An increase in CD45R and CD4 T cells was unrelated to protective immunity. Active immunization with soluble or particulate antigens induced complete protection during the primary immune response. This protective response was IgM mediated. The higher immunogenicity was not related to protective immunity since the particulate antigen induced protection similar to the soluble antigen. Using particulate antigens for vaccination guarantees a stronger immune response, local and systemic side effects, but not necessarily protection.     

Intratracheal immunization with pili protein protects against mortality associated with Pseudomonas aeruginosa pneumonia in mice

We examined the protective effect of intratracheal immunization with Pseudomonas aeruginosa pili protein against respiratory infection caused by P. aeruginosa. Mice were immunized intratracheally or subcutaneously with purified pili protein or bovine serum albumin as a control. Intratracheally but not subcutaneously pili protein-immunized mice showed significant improvement of survival after intratracheal challenge with the PAO1 strain. Furthermore, bacterial cell counts in pili protein-immunized murine lungs were significantly decreased compared to controls at 18 h after the challenge. Antipili protein antibody titers in bronchoalveolar lavage fluid of intratracheally pili protein-immunized mice were higher than in bovine serum albumin immunized mice. However, antipili antibody titers were not increased in bronchoalveolar lavage fluid of subcutaneously pili protein-immunized mice, despite the high serum antipili antibody titers. Inoculation of P. aeruginosa induced immediate increases in interleukin-12 and interferon-gamma in bronchoalveolar lavage fluid of pili protein-immunized mice, reflecting an adequate and rapid immune response against P. aeruginosa respiratory tract infection. Our findings suggest that intratracheal pili protein immunization is effective against respiratory tract infection caused by P. aeruginosa in mice.     

Combined oral/nasal immunization protects mice from Sendai virus infection

Based on the concept of a common mucosal immune system wherein mucosal associated lymphocytes traffic among the various mucous membranes, the murine gastrointestinal tract was immunized with Sendai virus antigens in order to elicit a virus-specific immune response in the respiratory tract. Multiple intragastric (oral) administration of live or killed Sendai virus induced IgA and IgG antiviral antibodies in both gastrointestinal secretions and serum. When cholera toxin as an adjuvant was included along with virus, gut IgA and IgG as well as serum IgA responses were enhanced. Antiviral antibodies induced in respiratory secretions by oral killed virus plus cholera toxin, however, were variable and protection from virus challenge was not demonstrated. Significantly higher levels of respiratory antiviral antibodies were induced if immunization with oral killed Sendai virus/cholera toxin was combined with intranasal administration of small amounts of killed virus. The combined immunization also resulted in protection of both the upper and lower respiratory tracts from virus infection. Protection of the upper respiratory tract was correlated with the presence of IgA antiviral antibodies in nasal washings. On the other hand, protection of the lower respiratory tract was correlated with IgG antiviral antibodies in bronchoalveolar lavage fluids. Immunization with intranasal killed virus alone conferred partial protection to the lower respiratory tract and no protection to the upper respiratory tract. Thus, oral immunization with killed virus antigen could prime for a protective immune response in the murine respiratory tract and this protective response included IgA antibodies.     

Experimental study of the immunological activity of a preparation made from the antigenic complexes of opportunistic microorganisms administered by different routes

The data obtained in the experimental study of the humoral factors of local and systemic immunity, as well as the morphofunctional changes of internal organs, after multiple subcutaneous and aerosol immunization with the combined preparation of Proteus, Staphylococcus, Klebsiella pneumoniae and Escherichia coli antigens (preparation nC-4) are presented. The subcutaneous and aerosol administration of preparation nC-4 has been found to induce an increase in the levels of antibodies to all components of the preparation in the blood serum and in respiratory tract secretions. The introduction of the preparation through the respiratory tract resulted in an earlier intensive accumulation of specific antibodies and IgA in respiratory tract secretions. The results of the quantitative cytological study of respiratory tract secretions and the morphofunctional changes of the lymphoid tissue associated with the lungs are indicative of an important role played by cell-mediated immunity factors in the formation of local postvaccinal immunity.     

The dynamics of the T-lymphocyte subpopulations and the peroxidase activity of the neutrophils in children immunized with a live mumps vaccine

The amount of T-helpers and T-suppressors and the T-helper/T-suppressor ratio were determined in 30 practically healthy children aged 1.5-2 years, immunized with live parotitis vaccine prepared from strain -3. One immunization dose of the vaccine contained 7,950 HADU50. In all children peroxidase activity in the cytoplasm of neutrophils was studied. This investigation revealed that in all examined children the formation of immune response to immunization with live parotitis vaccine was accompanied by the development of an imbalance of immunoregulatory T-cell subpopulations and by functional changes in neutrophils, characterized by the inhibition of peroxidase activity. The changes revealed in this study were most pronounced in children with a high level of antibody formation.     

The role of T-lymphocytes in the development of a humoral immune response to the corpuscular antigen of Staphylococcus aureus

The dependence of humoral immune response and the formation of immunological memory to corpuscular staphylococcal antigen (CSA) on the T-system of immunity was studied in experiments on B-mice and on mice with the congenital absence of the thymus (nude). Primary and secondary immune response to CSA in athymic mice was found to be considerably less than in normal animals. After the repeated immunization of genetically athymic mice the pronounced secondary reaction of the formation of antibodies to CSA was observed. As shown in this investigation carried out with the use of adoptive transfer techniques, the induction of memory B-cells to CSA may occur in animals with congenital or experimentally induced T-immunodeficiency. The conclusion was made on the T-dependence of humoral immune response to CSA, the formation of immunological memory to this antigen being relatively T-independent.     

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an appropriate immune response to a variety of immunogens^1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be administered intranasally^1,3,4, intratracheally⁵ or as aerosols⁶. Delivery by each route involves distinct technical skills and limitations that need to be considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)⁷, potentially complicating interpretation of the results. In addition, swallowing, sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.Download video file.^{(48M, mov)}     

Effectiveness of enteric immunization in the development of secretory immunoglobulin A response and the outcome of infection with respiratory syncytial virus.

Cotton rats were immunized via intranasal, intradermal, or enteric routes with respiratory syncytial virus (RSV) or a live recombinant vaccinia virus expressing the RSV F glycoprotein (vaccinia F). The animals were tested for the appearance of RSV-specific antibody responses in the serum, bronchoalveolar lavage, and nasal wash after immunization and for virus replication 4 days after intranasal challenge with RSV. RSV antibody response in the serum and respiratory tract was demonstrated in all immunization groups and was significantly increased after intranasal challenge with RSV. Immunoglobulin A (IgA) antibody response in bronchoalveolar lavage fluid after intranasal or enteric immunization was two- to threefold higher than that after intradermal immunization. Nasal-wash IgA antibody response was not significantly different among three immunization groups, although mean antibody titer was the highest in intranasal immunization group. Complete resistance to replication of RSV challenge was observed in the lungs of cotton rats immunized by the intranasal or enteric routes, whereas a low level of replication was detected in the lungs of rats immunized intradermally. Enteric or intradermal immunization conferred partial protection to the upper respiratory tract, but complete protection of the upper respiratory tract was observed in the intranasal immunization group. These observations suggest that while enteric immunization is quite effective in inducing antibody responses in the respiratory tract, the magnitude of antiviral immunity induced in the respiratory tract after intranasal immunization may be superior to that observed after enteric immunization.     

Induction of anti-chlamydial mucosal immunity by transcutaneous immunization is enhanced by topical application of GM-CSF

Transcutaneous immunization (TCI) involves the direct application of antigen plus adjuvant to skin, taking advantage of the large numbers of Langerhans cells and other resident skin dendritic cells, that process antigen then migrate to draining lymph nodes where immune responses are initiated. We have used this form of immunization to protect mice against genital tract and respiratory tract chlamydial infection. Protection was associated with local antibody responses in the vagina, uterus and lung as well as strong Th1 responses in the lymph nodes draining the reproductive tract and lungs respectively. In this study we show that topical application of GM-CSF to skin enhances the numbers and activation status of epidermal dendritic cells. Topical application of GM-CSF also increased the immune responses elicited by TCI. GM-CSF supplementation greatly increased cytokine (IFNgamma and IL-4) gene expression in lymph node and splenic cells compared to cells from animals immunized without GM-CSF. IgG responses in serum, uterine lavage and bronchoalveolar lavage and IgA responses in vaginal lavage were also increased by topical application of GM-CSF. The studies show that TCI induces protection against genital and respiratory tract chlamydial infections and that topical application of cytokines such as GM-CSF can enhance TCI-induced antibody and cell-mediated immunity.     

Determination of rate constants for the antigen-antibody reaction. Kinetic characteristics of interaction of soluble and corpuscular antigen with specific antibodies

Using the previously developed procedure, the rate and equilibrium constants for the interaction of antibodies with soluble or corpuscular antigens were determined. Cow milk casein was used as a soluble antigen, while heat-inactivated Staphylococcus aureus cells--as a corpuscular antigen. The values of kinetic constants for the above reactions are close to those for various antigen--antibody pairs obtained by other investigators.     

Induction of delayed-type hypersensitivity with proteins made insoluble by polymerization

Protein antigens, made particulate by polymerization with ethyl chloroformate, were incorporated in Freund's complete adjuvant and used for footpad immunization of rats and guinea pigs. A comparison was made with animals similarly immunized with the native, soluble protein. Two to three weeks after immunization of rats with polymerized bovine serum albumin (Pol-BSA) and up to 8 weeks after immunization of guinea pigs with polymerized diphtheria toxoid, in vivo and in vitro evidence of delayed-type hypersensitivity (DTH) was found without measurable serum antibodies. Ten times more polymerized than soluble BSA was needed to induce comparable levels of DTH. This was not, however, true in the case of serum antibodies, since soluble BSA induced higher titers than the 1000 times larger amount of Pol-BSA. In addition, the titers in polymer-immunized rats were consistently low or under detectable level when followed up to 5 months after priming. These findings encourage the belief that insolubilization of antigens by polymerization guides the immune response toward cell-mediated immunity, whereas antibody formation becomes weaker. However, boosting of polymer-primed animals with soluble antigen resulted in the production of high levels of antibody.     

Respiratory Dendritic Cell Subsets Differ in Their Capacity to Support the Induction of Virus-Specific Cytotoxic CD8+ T Cell Responses

Dendritic cells located at the body surfaces, e.g. skin, respiratory and gastrointestinal tract, play an essential role in the induction of adaptive immune responses to pathogens and inert antigens present at these surfaces. In the respiratory tract, multiple subsets of dendritic cells (RDC) have been identified in both the normal and inflamed lungs. While the importance of RDC in antigen transport from the inflamed or infected respiratory tract to the lymph nodes draining this site is well recognized, the contribution of individual RDC subsets to this process and the precise role of migrant RDC within the lymph nodes in antigen presentation to T cells is not clear. In this report, we demonstrate that two distinct subsets of migrant RDC - exhibiting the CD103⁺ and CD11b^hi phenotype, respectively - are the primary DC presenting antigen to naïve CD4⁺ and CD8⁺ T lymphocytes in the draining nodes in response to respiratory influenza virus infection. Furthermore, the migrant CD103⁺ RDC subset preferentially drives efficient proliferation and differentiation of naive CD8⁺ T cells responding to infection into effector cells, and only the CD103⁺ RDC subset can present to naïve CD8⁺ T cells non-infectious viral vaccine introduced into the respiratory tract. These results identify CD103⁺ and CD11b^hi RDC as critical regulators of the adaptive immune response to respiratory tract infection and potential targets in the design of mucosal vaccines.     

The immunological properties of the main outer membrane protein of Legionella

The immunogenic properties of Legionella outer membrane main protein (OMMP) were studied by its effect on the proliferative activity of lymphocytes in guinea pigs. Preliminary immunization with OMMP activated only the specific and nonspecific proliferation of spleen cells. After infection with Legionella, secondary immune response developed in the spleen and lungs of previously immunized animals, in contrast to intact ones, and the nonspecific proliferative activity of lymphocytes in the spleen and lungs of previously immunized animals considerably increased. These results are indicative of the fact that Legionella OMMP, similarly to other Legionella antigens and immunomodulators, may be used for the formation of protective immunity.     

Noncytolytic extraction of soluble antigens from leukemic blast cells (L2C-EN)

Summary Lithium 3,5 diiodosalicylate (LIS), a chemical utilized for the noncytolytic extraction of cell surface antigens, was used in this study to extract glycoproteins from the cell membranes of L₂C-EN leukemic blast cells. The crude soluble antigen (LIS-L₂C) preparation was found to confer immunoprotection in syngeneic guinea pigs against a lethal challenge of L₂C-EN. Titration of the crude LIS-L₂C soluble antigen extract revealed that 1 mg antigen gave 100% protection against a 2×10⁵ viable tumor cell challenge 2 weeks after immunization and that immunizing doses of 0.1 mg, 0.25 mg, and 0.5 mg soluble antigen afforded 17%, 66%, and 83% protection, respectively. The specificity of this immune response was demonstrated by the failure of guinea pigs immunized with 1 mg LIS extract prepared from another guinea pig tumor (line 10 hepatoma) to be refractory to a similar L₂C tumor cell challenge. A cell-mediated immune response to the LIS-L₂C soluble antigen was observed in animals, based on a positive delayed hypersensitivity response to the soluble antigen 5 weeks after immunization. Similarly, in vitro testing revealed a specific blastogenic recognition of the soluble antigen by immune leukocytes.     

Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy

Different variants of the popliteal lymph node (PLN) assay have been published. Here we describe the adjuvant popliteal lymph node assay, an immune response assay to study the adjuvant activity of soluble substances as well as particulate matter. The substance to be studied for adjuvant activity is injected into the hind footpad of mice or rats together with an antigen. Adjuvant activity is determined as the increase in PLN weight and cell numbers in animals receiving antigen together with the substance under study, compared with PLN weight and cell numbers in animals given the antigen without the substance in question, and animals given the putative adjuvant alone. Because lymph node weight and cell numbers are immunologically non-specific parameters, specific immune response assays like serum antibody responses or antibody-forming cell numbers should additionally be performed. Different antigens and immune response assays may be used, depending on the research question asked. In relation to respiratory (or food) allergy, the assays should as a minimum include determination of specific IgE in serum, and preferably also IgG1 (mouse). Serum specific IgG2a antibody determination may be added to get an indication of the Th1-Th2-balance of the response. The adjuvant PLN assay, with cellular response assays performed in the draining popliteal lymph node and antibody determinations in serum, requires small amounts of test material. The assay offers a practical, sensitive and reproducible method to determine the adjuvant activity of soluble substances as well as particulate material, with the possibility to also perform mechanistic studies.     

Phenotypic comparison of allergic airway responses to house dust mite in three rat strains

Brown Norway (BN) rats develop a robust response to antigens in the lung, characterized by a large increase in allergen-specific immune function and pulmonary eosinophilia. The objective of this study was to investigate alternative models by determining whether other rat strains could be sensitized to house dust mite (HDM) antigen and whether the allergic disease process could be worsened with repeated allergen exposure. In general, BN rats sensitized by either subcutaneous or intratracheal routes exhibited increased pulmonary allergy compared with Sprague-Dawley (SD) and Lewis (L) rats. Multiple intratracheal allergen exposures incrementally increased HDM-specific immune function in BN rats but progressively decreased eosinophil recruitment and markers of lung injury. SD rats had more moderate responses, whereas L rats were relatively unresponsive. Because BN rats developed stronger clinical hallmarks of allergic asthma under various immunization regimes compared with SD and L rats, we conclude that the BN is the most appropriate strain for studying allergic asthma-like responses in rats. Phenotypic differences in response to HDM were associated with differences in the Th1/Th2 cytokine balance and antioxidant capacity.     

Evaluation of Mucosal and Systemic Immune Responses Elicited by GPI-0100- Adjuvanted Influenza Vaccine Delivered by Different Immunization Strategies

Vaccines for protection against respiratory infections should optimally induce a mucosal immune response in the respiratory tract in addition to a systemic immune response. However, current parenteral immunization modalities generally fail to induce mucosal immunity, while mucosal vaccine delivery often results in poor systemic immunity. In order to find an immunization strategy which satisfies the need for induction of both mucosal and systemic immunity, we compared local and systemic immune responses elicited by two mucosal immunizations, given either by the intranasal (IN) or the intrapulmonary (IPL) route, with responses elicited by a mucosal prime followed by a systemic boost immunization. The study was conducted in BALB/c mice and the vaccine formulation was an influenza subunit vaccine supplemented with GPI-0100, a saponin-derived adjuvant. While optimal mucosal antibody titers were obtained after two intrapulmonary vaccinations, optimal systemic antibody responses were achieved by intranasal prime followed by intramuscular boost. The latter strategy also resulted in the best T cell response, yet, it was ineffective in inducing nose or lung IgA. Successful induction of secretory IgA, IgG and T cell responses was only achieved with prime-boost strategies involving intrapulmonary immunization and was optimal when both immunizations were given via the intrapulmonary route. Our results underline that immunization via the lungs is particularly effective for priming as well as boosting of local and systemic immune responses.     

Listeriolysin O-liposome-mediated cytosolic delivery of macromolecule antigen in vivo: enhancement of antigen-specific cytotoxic T lymphocyte frequency,activity, and tumor protection

Cytotoxic T lymphocytes (CTLs) are primed by peptide antigens that are endogenously processed in the cytosol and presented in the context of major histocompatibility complex I (MHC I) molecules of antigen-presenting cells (APCs). Exogenous soluble protein antigens do not gain efficient entry into the cytosol of APCs, and therefore requires a special cytosolic delivery method. We have developed such a delivery strategy adopting the well-elucidated cytosol-invading listerial endosomal escape mechanism, and report here an efficient delivery of exogenous whole protein antigen into the cytosol in a mouse model. Co-encapsulation of listeriolysin O (LLO) inside liposome (LLO-liposome) was required for delivery of ovalbumin (OVA) into the cytosol of APCs in primary cultures. LLO-liposome-mediated OVA immunization in mice engendered significantly higher OVA-specific CTL activity and increased antigenic peptide-specific CTL precursor (CTLp) frequency as compared to non-LLO-liposome or soluble OVA immunizations. Interferon-gamma (IFN-gamma) production upon specific stimulation by MHC I-restricted peptide was also significantly stronger by the inclusion of LLO in the liposomes. Rerouting of antigen into the cytosol by LLO-liposomes, however, did not reduce the extent of anti-OVA antibody responses. Moreover, LLO-liposome-antigen vaccination was robust in conferring protection to mice from lethal challenges with antigen-expressing tumor cells. Our study demonstrates a novel delivery system for efficient introduction of exogenous protein into the cytosol in vivo, priming cellular immune responses, which are protective in nature.     

Enzymatic activity in the lung cells to the intrapulmonary administration of antigen

Histochemical study of the activity of the redox and hydrolytic enzymes in the immunocompetent cells of the lungs of guinea pigs following intratracheal immunization with complete typhoid antigen demonstrated that following a single immunization at the initial periods elevation of the activity of AP, LDH, NAD- and NADP-diaphorases was seen in the macrophages; later GDH, G-6-PDH and AP was activated in the plasma cells. Double immunization was followed by an earlier and more intensive increase of the enzymatic activity in the cells under study.