Polymeric nanostructure vaccines: applications and challenges.

INTRODUCTION: The use of biocompatible polymers, from natural or synthetic sources, opened the door for a new era in vaccine research. These polymers offer the possibility to develop nanostructured antigen carriers that can be easily internalized by antigen-presenting cells, due to their nanometric size. Besides, the incorporation of an adjuvant allows increasing and modulating the immune response for both, polymers with or without self-adjuvant properties. AREAS COVERED: The historical background and the state-of-the-art in the use of polymers as antigen carriers are addressed in the first part of this review. Then, an overview of the immunology of vaccination is provided. Finally, the main advances in the field, based on the prototypes that are licensed or undergoing clinical trials, but also the challenges that limit the translation of many polymer-based nanostructure vaccines with promising preclinical results, are discussed. EXPERT OPINION: Polymeric nanostructured vaccines have a great potential in modern vaccinology. However, the translation into the market is hampered due to several limitations. Studies on correlates of protection to provide suitable biomarkers, new and better methods of synthesis to produce more reproducible nanovaccines, a deeper knowledge in the immune system and in the physiopathology of the infectious diseases will surely improve and boost the field in the next years.

Multi-parameter flow cytometry immunophenotyping distinguishes different stages of tuberculosis infection.

OBJECTIVES: To identify new potential host biomarkers in blood to discriminate between active TB patients, uninfected (NoTBI) and latently infected contacts (LTBI). METHODS: A blood cell count was performed to study parent leukocyte populations. Peripheral blood mononuclear cells (PBMCs) were isolated and a multi-parameter flow cytometry assay was conducted to study the distribution of basal and Mycobacterium tuberculosis (Mtb)-stimulated lymphocytes. Differences between groups and the area under the ROC curve (AUC) were investigated to assess the diagnostic accuracy. RESULTS: Active TB patients presented higher Monocyte-to-lymphocyte and Neutrophil-to-lymphocyte ratios than LTBI and NoTBI contacts (p<0.0001; AUC>0.8). Lymphocyte subsets with differences (p >0.05; AUC >0.7) between active TB and both contact groups include the basal distribution of Th1/Th2 ratio, Th1-Th17, CD4+ Central Memory (TCM) or MAIT cells. Expression of CD154 is increased in Mtb-activated CD4+ TCM and Effector Memory T cells in active TB and LTBI compared to NoTBI. In CD4+T cells, expression of CD154 showed a higher accuracy than IFNγ to discriminate Mtb-specific activation. CONCLUSIONS: We identified different cell subsets with potential use in tuberculosis diagnosis. Among them, distribution of CD4 TCM cells and their expression of CD154 after Mtb-activation are the most promising candidates.

The size and composition of polymeric nanocapsules dictate their interaction with macrophages and biodistribution in zebrafish.

Macrophages are pivotal cells of the innate immune system specialized in the phagocytosis of foreign elements. Nanoparticles intentionally designed to target macrophages and modulate their response are of especial interest in the case of chronic inflammatory diseases, cancer and for vaccine development. This work aimed to understand the role of size and shell composition of polymeric nanocapsules (NCs) in their interaction with macrophages, both in vitro and in vivo. A systematic study was performed using two different sizes of inulin and chitosan NCs, negatively and positively charged, respectively, small (≈ 70 nm) and medium (170-250 nm). The in vitro results showed that small NCs interacted more efficiently with macrophages than their larger counterparts. Inulin NCs were significantly less toxic than chitosan NCs. Finally, following in vivo administration (intravenous/intramuscular) to zebrafish, small NCs, regardless of their composition, disseminated considerably faster and further than their medium size counterparts. These results emphasize how small changes in the nanometric range can lead to a remarkably different interaction with the immune cells and biodistribution profile.

Protamine Nanocapsules for the Development of Thermostable Adjuvanted Nanovaccines.

One of the main challenges in the development of vaccine has been to improve their stability at room temperature and eliminate the limitations associated with the cold chain storage. In this paper, we describe the development and optimization of thermostable nanocarriers consisting of an oily core with immunostimulating activity, containing squalene or α tocopherol surrounded by a protamine shell. The results showed that these nanocapsules can efficiently associate the recombinant hepatitis B surface antigen (rHBsAg) without compromising its antigenicity. Furthermore, the freeze-dried protamine nanocapsules were able to preserve the integrity and bioactivity of the associated antigen upon storage for at least 12 months at room temperature. In vitro studies evidenced the high internalization of the nanocapsules by immunocompetent cells, followed by cytokine secretion and complement activation. In vivo studies showed the capacity of rHBsAg-loaded nanocapsules to elicit protective levels upon intramuscular or intranasal administration to mice. Overall, our data indicate that protamine nanocapsules are an innovative thermostable nanovaccine platform for improved antigen delivery.

Polymeric Nanocapsules for Vaccine Delivery: Influence of the Polymeric Shell on the Interaction With the Immune System.

The use of biomaterials and nanosystems in antigen delivery has played a major role in the development of novel vaccine formulations in the last few decades. In an effort to gain a deeper understanding of the interactions between these systems and immunocompetent cells, we describe here a systematic in vitro and in vivo study on three types of polymeric nanocapsules (NCs). These carriers, which contained protamine (PR), polyarginine (PARG), or chitosan (CS) in the external shell, and their corresponding nanoemulsion were prepared, and their main physicochemical properties were characterized. The particles had a mean particle size in the range 250-450 nm and a positive zeta potential (~30-40 mV). The interaction of the nanosystems with different components of the immune system were investigated by measuring cellular uptake, reactive oxygen species production, activation of the complement cascade, cytokine secretion profile, and MAP kinases/nuclear factor κB activation. The results of these in vitro cell experiments showed that the NC formulations that included the arginine-rich polymers (PR and PARG) showed a superior ability to trigger different immune processes. Considering this finding, protamine and polyarginine nanocapsules (PR and PARG NCs) were selected to assess the association of the recombinant hepatitis B surface antigen (rHBsAg) as a model antigen to evaluate their ability to produce a protective immune response in mice. In this case, the results showed that PR NCs elicited higher IgG levels than PARG NCs and that this IgG response was a combination of anti-rHBsAg IgG1/IgG2a. This work highlights the potential of PR NCs for antigen delivery as an alternative to other positively charged nanocarriers.

Highly versatile immunostimulating nanocapsules for specific immune potentiation.

AIM: To develop a new core-shell type (nanocapsules) adjuvant system composed of squalene and polyglucosamine (PG) and to evaluate its immunostimulant capacity. RESULTS: The defined PG nanocapsules exhibited the capacity to efficiently associate the selected antigens (recombinant hepatitis B surface antigen and hemagglutinin of influenza virus) onto their polymeric surface (70-75%), and the immunostimulant imiquimod within the oily core. The resulting nanovaccines, with a particle size of 200-250 nm and a positive zeta-potential (∼+60 mV), were able to significantly potentiate and modulate the immune response to the selected antigens upon intramuscular administration to mice. Their efficacy as novel adjuvants was attributed to their enhanced cell internalization and effective intracellular imiquimod/antigen delivery, together with their prolonged residence time at the injection site. CONCLUSION: The nanocapsules described herein have the capacity to enhance, prolong and modulate the immune response of subunit antigens and, therefore, they could be proposed as a platform for the codelivery of different antigens and immunostimulators.

Co-delivery of viral proteins and a TLR7 agonist from polysaccharide nanocapsules: a needle-free vaccination strategy.

Here we report a new nanotechnology-based nasal vaccination concept intended to elicit both, specific humoral and cellular immune responses. The concept relies on the use of a multifunctional antigen nanocarrier consisting of a hydrophobic nanocore, which can allocate lipophilic immunostimulants, and a polymeric corona made of chitosan (CS), intended to associate antigens and facilitate their transport across the nasal mucosa. The Toll-like receptor 7 (TLR7) agonist, imiquimod, and the recombinant hepatitis B surface antigen (HB), were selected as model molecules for the validation of the concept. The multifunctional nanocarriers had a nanometric size (around 200 nm), a high positive zeta potential (+45 mV) and a high antigen association efficiency (70%). They also exhibited the ability to enter macrophages in vitro and to effectively deliver the associated imiquimod intracellularly, as noted by the secretion of pro-inflammatory cytokines (i.e. IL-6 and TNF-α). However, the nanocarriers did not induce the in vitro activation of the complement cascade. Finally, the positive effect of the co-delivery of HB and imiquimod from the nanocapsules was evidenced upon intranasal administration to mice. The nanocapsules containing imiquimod elicited a protective immune response characterized by increasing IgG levels over time and specific immunological memory. Additionally, the levels of serum IgG subclasses (IgG1 and IgG2a) indicated a balanced cellular/humoral response, thus suggesting the capacity of the nanocapsules to modulate the systemic immune response upon nasal vaccination.

A polymer/oil based nanovaccine as a single-dose immunization approach.

The recognized necessity for new antigen delivery carriers with the capacity to boost, modulate and prolong neutralizing immune responses prompted our approach, in which we describe a multifunctional nanocarrier consisting of an oily nanocontainer protected by a polymeric shell made of chitosan (CS), named CS nanocapsules (CSNC). The CS shell can associate the antigen on its surface, whereas the oily core might provide additional immunostimulating properties. In this first characterization of the system, we intended to study the influence of different antigen organizations on the nanocarrier's surface (using the recombinant hepatitis B surface antigen -rHBsAg- as a model antigen) on their long-term immunopotentiating effect, without any additional immunostimulant. Thus, two prototypes of antigen-loaded CSNC (CSNC+ and CSNC-), exhibiting similar particle size (200 nm) and high antigen association efficiency (>80%), were developed with different surface composition (polymer/antigen ratios) and surface charge (positive/negative, respectively). The biological evaluation of these nanovaccines evidenced the superiority of the CSNC+ as compared to CSNC- and alum-rHBsAg in terms of neutralizing antibody responses, following intramuscular vaccination. Moreover, a single dose of CSNC+ led to similar IgG levels to the positive control. The IgG1/IgG2a ratio suggested a mixed Th1/Th2 response elicited by CSNC+, in contrast to the typical Th2-biased response of alum. Finally, CSNC+ could be freeze-dried without altering its physicochemical properties and adjuvant effect in vivo. In conclusion, the evaluation of CSNC+ confirms its interesting features for enhancing, prolonging and modulating the type of immune response against subunit antigens, such as rHBsAg.