Pulmonary Immunization Research Articles

Controlled analysis of nanoparticle charge on mucosal and systemic antibody responses following pulmonary immunization

Controlled analysis of nanoparticle charge on mucosal and systemic antibody responses following pulmonary immunization

Controlled analysis of nanoparticle charge on mucosal and systemic antibody responses following pulmonary immunization

Pulmonary immunization enhances local humoral and cell-mediated mucosal protection, which are critical for vaccination against lung-specific pathogens such as influenza or tuberculosis. A variety of nanoparticle (NP) formulations have been tested preclinically for pulmonary vaccine development, yet the role of NP surface charge on downstream immune responses remains poorly understood. We used the Particle Replication in Non-Wetting Templates (PRINT) process to synthesize hydrogel NPs that varied only in surface charge and otherwise maintained constant size, shape, and antigen loading. Pulmonary immunization with ovalbumin (OVA)-conjugated cationic NPs led to enhanced systemic and lung antibody titers compared with anionic NPs. Increased antibody production correlated with robust germinal center B-cell expansion and increased activated CD4(+) T-cell populations in lung draining lymph nodes. Ex vivo treatment of dendritic cells (DCs) with OVA-conjugated cationic NPs induced robust antigen-specific T-cell proliferation with ∼ 100-fold more potency than soluble OVA alone. Enhanced T-cell expansion correlated with increased expression of surface MHCII, T-cell coactivating receptors, and key cytokines/chemokine expression by DCs treated with cationic NPs, which were not observed with anionic NPs or soluble OVA. Together, these studies highlight the importance of NP surface charge when designing pulmonary vaccines, and our findings support the notion that cationic NP platforms engender potent humoral and mucosal immune responses.

Development of probiotic-based immunoparticles for pulmonary immunization against Hepatitis B

The present study was oriented towards the development of pulmonary vaccine for Hepatitis B using probiotic biomass as an adjuvant. The antigen was spray dried in presence of heat treated, formalin treated and live probiotic biomass. The results indicated that the biomass itself without any additional cryoprotectant is capable of protecting the structural integrity of the antigen. We were able to retain more than 80% of the antigenicity. The scanning electron microscopic images indicated that the formulation bearing live probiotic biomass have spherical size, while the formulations with heat and formalin treated biomass shows irregular shaped particles. The developed formulations were further evaluated for in-vivo immune response. Immunoglobulin G (IgG) titre results were found to be comparable with marketed (aluminium adsorbed) formulations while significantly higher secretory immunoglobulin A titre showed better mucosal immune response than marketed formulation. Therefore, the probiotic biomass can be utilized as a potential cryoprotectant as well as a potent immunomodulator.

Enhanced pulmonary immunization with aerosolized inactivated influenza vaccine containing delta inulin adjuvant

Vaccination is the primary intervention to contain influenza virus spread during seasonal and pandemic outbreaks. Pulmonary vaccination is gaining increasing attention for its ability to induce both local mucosal and systemic immune responses without the need for invasive injections. However, pulmonary administration of whole inactivated influenza virus (WIV) vaccine induces a Th2 dominant systemic immune response while a more balanced Th1/Th2 vaccine response may be preferred and only induces modest nasal immunity. This study evaluated immunity elicited by pulmonary versus intramuscular (i.m.) delivery of WIV, and tested whether the immune response could be improved by co-administration of delta (δ)-inulin, a novel carbohydrate-based particulate adjuvant. After pulmonary administration both unadjuvanted and δ-inulin adjuvanted WIV induced a potent systemic immune response, inducing higher serum anti-influenza IgG titers and nasal IgA titers than i.m. administration. Moreover, the addition of δ-inulin induced a more balanced Th1/Th2 response and induced higher nasal IgA titers versus pulmonary WIV alone. Pulmonary WIV alone or with δ-inulin induced hemagglutination inhibition (HI) titers>40, titers which are considered protective against influenza virus. In conclusion, in this study we have shown that δ-inulin adjuvanted WIV induces a better immune response after pulmonary administration than vaccine alone.

Mucosal vaccination against tuberculosis using Ag85A-loaded immunostimulating complexes

Tuberculosis (TB) is one of the major devastating diseases in the world, mainly caused by Mycobacterium tuberculosis. Furthermore, multi-drug resistant TB and extremely drug resistant TB are becoming big problems globally. Bacillus Calmette-Guerin (BCG) is the only available vaccine which provides protection against TB. The BCG vaccine is effective in children but not recommended in adults and elderly patients due to an associated low risk of infection with Mycobacterium tuberculosis and variable effectiveness of the vaccine. The main aim of this research study is to develop such a vaccine which will provide a better and safer profile in children and adults, as well as in elderly patients. In this present study, we prepared pulmonary tubercular vaccine by using an Antigen 85 complex (Ag85)-loaded nanocarrier such as the immunostimulating complex (ISCOM). Immunological outcomes clearly indicated significant improvement in humoral as well as cellular immune responses after pulmonary immunization with ISCOMs containing Quil A in mice.

Pulmonary immunization of chickens using non-adjuvanted spray-freeze dried whole inactivated virus vaccine completely protects against highly pathogenic H5N1 avian influenza virus

Highly pathogenic avian influenza (HPAI) H5N1 virus is a major threat to public health as well as to the global poultry industry. Most fatal human infections are caused by contact with infected poultry. Therefore, preventing the virus from entering the poultry population is a priority. This is, however, problematic in emergency situations, e.g. during outbreaks in poultry, as there are currently no mass application methods to effectively vaccinate large numbers of birds within a short period of time. To evaluate the suitability of needle-free pulmonary immunization for mass vaccination of poultry against HPAI H5N1, we performed a proof-of-concept study in which we investigated whether non-adjuvanted spray-freeze-dried (SFD) whole inactivated virus (WIV) can be used as a dry powder aerosol vaccine to immunize chickens. Our results show that chickens that received SFD-WIV vaccine as aerosolized powder directly at the syrinx (the site of the tracheal bifurcation), mounted a protective antibody response after two vaccinations and survived a lethal challenge with HPAI H5N1. Furthermore, both the number of animals that shed challenge virus, as well as the level of virus shedding, were significantly reduced. Based on antibody levels and reduction of virus shedding, pulmonary vaccination with non-adjuvanted vaccine was at least as efficient as intratracheal vaccination using live virus. Animals that received aerosolized SFD-WIV vaccine by temporary passive inhalation showed partial protection (22% survival) and a delay in time-to-death, thereby demonstrating the feasibility of the method, but indicating that the efficiency of vaccination by passive inhalation needs further improvement. Altogether our results provide a proof-of-concept that pulmonary vaccination using an SFD-WIV powder vaccine is able to protect chickens from lethal HPAI challenge. If the efficacy of pulmonary vaccination by passive inhalation can be improved, this method might be suitable for mass application.

Lung Deposition and Cellular Uptake Behavior of Pathogen‐Mimicking Nanovaccines in the First 48 Hours

Pulmonary immunization poses the unique challenge of balancing vaccine efficacy with minimizing inflammation in the respiratory tract. While previous studies have shown that mice immunized intranasally with F1-V-loaded polyanhydride nanoparticles are protected from a lethal challenge with Yersinia pestis, little is known about the initial interaction between the nanoparticles and immune cells following intranasal administration. Here, the deposition within the lung and internalization by phagocytic cells of polyanhydride nanovaccines encapsulating F1-V are compared with that of soluble F1-V alone or F1-V adjuvanted with monophosphoryl lipid A (MPLA). Encapsulation of F1-V into polyanhydride nanoparticles prolonged its presence while F1-V administered with MPLA is undetectable within 48 h. The inflammation induced by the polyanhydride nanovaccine is mild compared with the marked inflammation induced by the MPLA-adjuvanted F1-V. Even though F1-V delivered with saline is detected in the lung 48 h after administration, it is known that this regimen does not elicit a protective immune response. The prolonged F1-V presence in the lung in concert with the mild inflammatory response provided by the nanovaccine provides new insights into the development of protective immune responses with a single intranasal dose.

Generation of effector memory T cell-based mucosal and systemic immunity with pulmonary nanoparticle vaccination.

Many pathogens infiltrate the body and initiate infection via mucosal surfaces. Hence, eliciting cellular immune responses at mucosal portals of entry is of great interest for vaccine development against mucosal pathogens. We describe a pulmonary vaccination strategy combining Toll-like receptor (TLR) agonists with antigen-carrying lipid nanocapsules [interbilayer-crosslinked multilamellar vesicles (ICMVs)], which elicit high-frequency, long-lived, antigen-specific effector memory T cell responses at multiple mucosal sites. Pulmonary immunization using protein- or peptide-loaded ICMVs combined with two TLR agonists, polyinosinic-polycytidylic acid (polyI:C) and monophosphoryl lipid A, was safe and well tolerated in mice, and led to increased antigen transport to draining lymph nodes compared to equivalent subcutaneous vaccination. This response was mediated by the vast number of antigen-presenting cells (APCs) in the lungs. Nanocapsules primed 13-fold more T cells than did equivalent soluble vaccines, elicited increased expression of mucosal homing integrin α₄β₇⁺, and generated long-lived T cells in both the lungs and distal (for example, vaginal) mucosa strongly biased toward an effector memory (T(EM)) phenotype. These T(EM) responses were highly protective in both therapeutic tumor and prophylactic viral vaccine settings. Together, these data suggest that targeting cross-presentation-promoting particulate vaccines to the APC-rich pulmonary mucosa can promote robust T cell responses for protection of mucosal surfaces.

Lung-resident memory CD8 T cells (TRM) are indispensable for optimal cross-protection against pulmonary virus infection

Previous studies have shown that some respiratory virus infections leave local populations of tissue TRM cells in the lungs which disappear as heterosubtypic immunity declines. The location of these TRM cells and their contribution to the protective CTL response have not been clearly defined. Here, fluorescence microscopy is used to show that some CD103(+) TRM cells remain embedded in the walls of the large airways long after pulmonary immunization but are absent from systemically primed mice. Viral clearance from the lungs of the locally immunized mice precedes the development of a robust Teff response in the lungs. Whereas large numbers of virus-specific CTLs collect around the bronchial tree during viral clearance, there is little involvement of the remaining lung tissue. Much larger numbers of TEM cells enter the lungs of the systemically immunized animals but do not prevent extensive viral replication or damage to the alveoli. Together, these experiments show that virus-specific antibodies and TRM cells are both required for optimal heterosubtypic immunity, whereas circulating memory CD8 T cells do not substantially alter the course of disease.

Physical and immunogenic stability of spray freeze-dried influenza vaccine powder for pulmonary delivery: Comparison of inulin, dextran, or a mixture of dextran and trehalose as protectants

One of the advantages of dry influenza vaccines over conventional liquid influenza vaccines is that they can be used for alternative routes of administration. Previous studies showed that spray freeze-drying is an excellent technique to prepare vaccine containing powders for pulmonary delivery (J.P. Amorij, V. Saluja, A.H. Petersen, W.L.J. Hinrichs, A. Huckriede, H.W. Frijlink, Pulmonary delivery of an inulin-stabilized influenza subunit vaccine prepared by spray-freeze drying induces systemic, mucosal humoral as well as cell-mediated immune responses in BALB/c mice, Vaccine 25 (2007) 8707–8717; S.A. Audouy, G. van der Schaaf, W.L.J. Hinrichs, H.W. Frijlink, J. Wilschut, A. Huckriede. Development of a dried influenza whole inactivated virus vaccine for pulmonary immunization, Vaccine (2011)). The aim of this study was to investigate the physical and immunogenic stability of spray freeze-dried whole inactivated virus influenza vaccine prepared by using inulin, dextran, and a mixture of dextran and trehalose as protectants. Physical and biochemical characteristics of the vaccine powder were maintained at temperatures up to 30°C for 3months. In addition, in vivo data indicate that also, the immunogenic properties of the vaccine were maintained under these storage conditions. On the other hand, in vivo results also revealed that subtle changes in powder characteristics were induced during storage at 30°C. However, laser diffraction measurements showed that problems associated with these subtle changes can be overcome by using dry powder inhalers with an efficient powder dispersing capacity.

Airway CD8(+) T cells induced by pulmonary DNA immunization mediate protective anti-viral immunity.

Vaccination strategies for protection against a number of respiratory pathogens must induce T-cell populations in both the pulmonary airways and peripheral lymphoid organs. In this study, we show that pulmonary immunization using plasmid DNA formulated with the polymer polyethyleneimine (PEI-DNA) induced antigen-specific CD8+ T cells in the airways that persisted long after antigen local clearance. The persistence of the cells was not mediated by local lymphocyte proliferation or persistent antigen presentation within the lung or airways. These vaccine-induced CD8+ T cells effectively mediated protective immunity against respiratory challenges with vaccinia virus and influenza virus. Moreover, this protection was not dependent upon the recruitment of T cells from peripheral sites. These findings demonstrate that pulmonary immunization with PEI-DNA is an efficient approach for inducing robust pulmonary CD8+ T-cell populations that are effective at protecting against respiratory pathogens.

Pulmonary Immunization for TB With Live cell-based Vaccines: the Importance of the Delivery Route

Pulmonary Immunization for TB With Live cell-based Vaccines: the Importance of the Delivery Route

Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8(+) T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8(+) T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4(+) T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8(+) T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8(+) T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.

Development of a dried influenza whole inactivated virus vaccine for pulmonary immunization

Stabilization and ease of administration are two ways to substantially improve the use of current vaccines. In the present study an influenza whole inactivated virus (WIV) vaccine was freeze-dried or spray-freeze dried in the presence of inulin as a cryoprotectant. Only spray-freeze drying rendered powders compatible with administration to the lungs by insufflation. Pulmonary administration of the powder vaccine obtained by this method to BALB/c mice led to a transient influx of neutrophils and a concomitant decrease in the number of macrophages as did administration of liquid vaccine. Inflammatory reactions to both vaccines were mild and short-lived. Immunization studies showed that the immunogenic properties of WIV vaccine were not affected by drying. Pulmonary administration of the powder WIV vaccine induced a systemic immune response of the same magnitude as liquid vaccine while mucosal IgA responses were higher for powder WIV. In a challenge study where immunized mice were exposed to a lethal dose of live virus, two pulmonary doses of either liquid or powder WIV vaccine were equally effective as a single intramuscular injection of subunit vaccine in terms of reduction of the viral load in the lungs. To conclude, in the models employed for these studies the use of a dry powder WIV vaccine for pulmonary immunization was shown to be safe and efficient.

New developments in dry powder pulmonary vaccine delivery

Pulmonary immunization has gained increased recognition as a means of triggering both a mucosal and systemic immune response without the use of needles. The appropriate formulation of antigens in a dry, solid state can result in improved stability, thereby removing cold-chain storage complications associated with conventional liquid-based vaccines. The particulate nature of dry powder vaccines could also induce a better immune response. This review describes our current understanding of pulmonary immunization, including possible barriers facing the development of pulmonary vaccines, and discusses recent advances in spray-drying technologies applicable to the production of dry powder formulations for pulmonary vaccine delivery.

Pulmonary Immunization of Guinea Pigs with Diphtheria CRM-197 Antigen as Nanoparticle Aggregate Dry Powders Enhance Local and Systemic Immune Responses

This study establishes the immune response elicited in guinea pigs after pulmonary and parenteral immunizations with diphtheria CRM-197 antigen (CrmAg). Several spray-dried powders of formalin-treated/untreated CrmAg nanoaggregates with L-leucine were delivered to the lungs of guinea pigs. A control group consisting of alum with adsorbed CrmAg in saline was administered by intramuscular injection. Animals received three doses of powder vaccines containing 20 or 40 μg of CrmAg. The serum IgG titers were measured for 16 weeks after the initial immunization; IgA titers were measured at the time of sacrifice in the broncho-alveolar lavage fluid. Further, toxin neutralization tests in naïve guinea pigs were performed for a few select serum samples. Histopathology of the lung tissues was conducted to evaluate inflammation or injury to the lung tissues. While the highest titer of serum IgG antibody was observed in guinea pigs immunized by the intramuscular route, those animals immunized with dry powder formulation by the pulmonary route, and without the adjuvant alum, exhibited high IgA titers. A pulmonary administered dry powder, compared to parenteral immunization, conferred complete protection in the toxin neutralization test. Mild inflammation was observed in lung tissues of animals receiving dry powder vaccines by the pulmonary route. Thus, administering novel CrmAg as dry powders to the lungs may be able to overcome some of the disadvantages observed with the existing diphtheria vaccine which is administered by the parenteral route. In addition, these powders will have the advantage of eliciting a high mucosal immune response in the lungs without using traditional adjuvants.

Comparison of antibody responses induced by a virus-like particle-based vaccine upon intramuscular, pulmonary, and vaginal delivery. (48.14)

Abstract Virus-like particles (VLPs) can be used as platforms to increase the immunogenicity of antigens that are normally poorly immunogenic, such as self-antigens. We have developed a bacteriophage VLP-based vaccine that targets the HIV coreceptor CCR5 and shown that, following intramuscular administration, it induces high-titer serum antibodies against CCR5 that can inhibit HIV/SIV infection. We have also assessed the ability of this vaccine to induce mucosal immune responses, which is desirable as many pathogens, including HIV, infect at mucosal surfaces. In rats, both intramuscular and pulmonary immunization induced high titer IgG and IgA against the vaccine in the serum, but only the aerosolized vaccine induced CCR5-specific IgA locally in the lung and remotely in the genital tract. We will also present data comparing the magnitude of this "secondary" response in the genital tract to the IgA response invoked following direct immunization of the genital mucosa with different VLP platforms, including VLPs from a virus (human papillomavirus) that normally infects in the genital tract and bacteriophage Qβ VLPs. Our results provide a general method for inducing broad systemic and mucosal antibody responses using VLP-based immunogens.

Efficient Generation of Mucosal and Systemic Antigen-Specific CD8+T-Cell Responses following Pulmonary DNA Immunization

Although mucosal CD8(+) T-cell responses are important in combating mucosal infections, the generation of such immune responses by vaccination remains problematic. In the present study, we evaluated the ability of plasmid DNA to induce local and systemic antigen-specific CD8(+) T-cell responses after pulmonary administration. We show that the pulmonary delivery of plasmid DNA formulated with polyethyleneimine (PEI-DNA) induced robust systemic CD8(+) T-cell responses that were comparable in magnitude to those generated by intramuscular (i.m.) immunization. Most importantly, we observed that the pulmonary delivery of PEI-DNA elicited a 10-fold-greater antigen-specific CD8(+) T-cell response in lungs and draining lymph nodes of mice than that of i.m. immunization. The functional evaluation of these pulmonary CD8(+) T cells revealed that they produced type I cytokines, and pulmonary immunization with PEI-DNA induced lung-associated antigen-specific CD4(+) T cells that produced higher levels of interleukin-2 than those induced by i.m. immunization. Pulmonary PEI-DNA immunization also induced CD8(+) T-cell responses in the gut and vaginal mucosa. Finally, pulmonary, but not i.m., plasmid DNA vaccination protected mice from a lethal recombinant vaccinia virus challenge. These findings suggest that pulmonary PEI-DNA immunization might be a useful approach for immunizing against pulmonary pathogens and might also protect against infections initiated at other mucosal sites.

A comparison between spray drying and spray freeze drying to produce an influenza subunit vaccine powder for inhalation

The aim of this study was to investigate two different processes to produce a stable influenza subunit vaccine powder for pulmonary immunization i.e. spray drying (SD) and spray freeze drying (SFD). The formulations were analyzed by proteolytic assay, single radial immunodiffusion assay (SRID), cascade impactor analysis, and immunization studies in Balb/c mice. Proteolytic assay and SRID analysis showed that antigen integrity after SFD was best conserved when the formulation was buffered by Hepes buffer saline (HBS). Surprisingly, antigen integrity after SD was better conserved when the formulation was buffered by phosphate buffer saline (PBS) rather than by HBS. The dispersion from the dry powder inhaler, the Twincer ®, resulted in a fine particle fraction (aerodynamic particle size < 5 µm) of 37% and 23% for spray dried and spray freeze dried powders, respectively. Immunogenicity of both vaccine formulations (SFD/HBS and SD/PBS) was similar to conventional liquid formulation after i.m. immunization. In addition, compared to i.m. immunizations, the pulmonary immunization with the dry powders resulted in significantly higher IgG titers. Furthermore, both the formulations remained biochemically and physically stable for at least 3 years of storage at 20 °C. Our results demonstrate that both optimized formulations are stable and have good inhalation characteristics.

Pulmonary Immunization of Guinea Pigs with Diphtheria CRM-197 Antigen as Nanoparticle Aggregate Dry Powders Enhance Local and Systemic Immune Responses

Pulmonary Immunization of Guinea Pigs with Diphtheria CRM-197 Antigen as Nanoparticle Aggregate Dry Powders Enhance Local and Systemic Immune Responses