Functional Recognition by CD8+ T Cells of Epitopes with Amino Acid Variations Outside Known MHC Anchor or T Cell Receptor Recognition Residues.

Peptide-based vaccines can be safer and more cost effective than whole organism vaccines. Previous studies have shown that inorganic polystyrene nanoparticles (PSNPs) covalently conjugated to the minimal immunodominant peptide epitope from murine liver stage malaria (SYIPSAEKI) induced potent CD8+ T cell responses. Many pathogens, including malaria, have polymorphic T cell epitope regions. Amino acid changes in positions that are contact residues for the T cell receptor (TCR) often alter the specific cross-reactivity induced by the peptide antigen, and it is largely assumed that changes outside of these residues have little impact. Herein, each amino acid residue (except major histocompatibility complex (MHC) anchors) was systematically changed to an alanine. Peptide epitopes with altered amino acids outside T cell contact residues were still recognized by T cells induced by PSNPs-SYIPSAEKI (KI) vaccines, albeit at lower levels, except for the variant SYIPSAAKI (A7). PSNPs-SYIPSAAKI vaccines further elicited high responses to the index KI peptide. None of the epitopes displayed altered peptide ligand (APL) antagonism in vitro, and re-stimulating SYIPSAEKI and SYIPSAAKI together synergistically enhanced IFN-γ production by the T cells. These results show epitope variation in non-TCR recognition residues can have effects on T cell reactivity, suggesting that such natural variation may also be driven by immune pressure. Additionally, when re-modelling peptides to enhance the cross-reactivity of vaccines, both TCR recognition and non-recognition residues should be considered.

Immunological effects among workers who handle engineered nanoparticles.

OBJECTIVE: To determine whether exposure of workers handling engineered nanoparticles (ENPs) may result in increased inflammation and changes in lung function. METHODS: A prospective panel study compared changes in several markers of inflammation for ENP handling and non-ENP handling control workers. Nanoparticle exposure was measured during ENP handling and for controls. Lung function, fraction of exhaled nitric oxide (FeNO), C-reactive protein (CRP), blood cell counts and several serum cytokines were measured at baseline, at the end of the shift and at the end of the working week. RESULTS: Nanoparticle exposure was not higher when ENPs were being handled; nanoparticle counts were higher in offices and in ambient air than in laboratories. There were no differences at baseline in lung function, FeNO, haemoglobin, platelet, white cell counts or CRP levels between those who handled nanoparticles and those who did not, with or without asthmatic participants. There were statistically significant increases in sCD40 and sTNFR2 over the working day for those who handled ENPs. The changes were larger and statistically significant over the working week and sCD62P also showed a statistically significant difference. The changes were slightly smaller and less likely to be statistically significant for atopic than for non-atopic participants. CONCLUSIONS: Even at low ENP exposure, increases in three cytokines were significant over the week for those who handled nanoparticles, compared with those who did not. However, exposure to low and transient levels of nanoparticles was insufficient, to trigger measurable changes in spirometry, FeNO, CRP or blood cell counts.

Differential uptake of nanoparticles and microparticles by pulmonary APC subsets induces discrete immunological imprints.

There is increasing interest in the use of engineered particles for biomedical applications, although questions exist about their proinflammatory properties and potential adverse health effects. Lung macrophages and dendritic cells (DC) are key regulators of pulmonary immunity, but little is known about their uptake of different sized particles or the nature of the induced immunological imprint. We investigated comparatively the immunological imprints of inert nontoxic polystyrene nanoparticles 50 nm in diameter (PS50G) and 500 nm in diameter (PS500G). Following intratracheal instillation into naive mice, PS50G were preferentially taken up by alveolar and nonalveolar macrophages, B cells, and CD11b(+) and CD103(+) DC in the lung, but exclusively by DC in the draining lymph node (LN). Negligible particle uptake occurred in the draining LN 2 h postinstillation, indicating that particle translocation does not occur via lymphatic drainage. PS50G but not PS500G significantly increased airway levels of mediators that drive DC migration/maturation and DC costimulatory molecule expression. Both particles decreased frequencies of stimulatory CD11b(+)MHC class II(hi) allergen-laden DC in the draining LN, with PS50G having the more pronounced effect. These distinctive particle imprints differentially modulated induction of acute allergic airway inflammation, with PS50G but not PS500G significantly inhibiting adaptive allergen-specific immunity. Our data show that nanoparticles are taken up preferentially by lung APC stimulate cytokine/chemokine production and pulmonary DC maturation and translocate to the lung-draining LN via cell-associated transport. Collectively, these distinctive particle imprints differentially modulate development of subsequent lung immune responses. These findings support the development of lung-specific particulate vaccines, drug delivery systems, and immunomodulators.

The effects of engineered nanoparticles on pulmonary immune homeostasis.

Engineered nanoparticles (ENP), which could be composed of inorganic metals, metal oxides, metalloids, organic biodegradable and inorganic biocompatible polymers, are being used as carriers for vaccine and drug delivery. There is also increasing interest in their application as delivery agents for the treatment of a variety of lung diseases. Although many studies have shown ENP can be effectively and safely used to enhance the delivery of drugs and vaccines in the periphery, there is concern that some ENP could promote inflammation, with unknown consequences for lung immune homeostasis. In this study, we review research on the effects of ENP on lung immunity, focusing on recent studies using diverse animal models of human lung disease. We summarize how the inflammatory and immune response to ENP is influenced by the diverse biophysical and chemical characteristics of the particles including composition, size and mode of delivery. We further discuss newly described unexpected beneficial properties of ENP administered into the lung, where biocompatible polystyrene or silver nanoparticles can by themselves decrease susceptibility to allergic airways inflammation. Increasing our understanding of the differential effects of diverse types of nanoparticles on pulmonary immune homeostasis, particularly previously underappreciated beneficial outcomes, supports rational ENP translation into novel therapeutics for prevention and/or treatment of inflammatory lung disorders.

Inert 50-nm polystyrene nanoparticles that modify pulmonary dendritic cell function and inhibit allergic airway inflammation.

Nanoparticles are being developed for diverse biomedical applications, but there is concern about their potential to promote inflammation, particularly in the lung. Although a variety of ambient, anthropogenic and man-made nanoparticles can promote lung inflammation, little is known about the long-term immunomodulatory effects of inert noninflammatory nanoparticles. We previously showed polystyrene 50-nm nanoparticles coated with the neutral amino acid glycine (PS50G nanoparticles) are not inflammatory and are taken up preferentially by dendritic cells (DCs) in the periphery. We tested the effects of such nanoparticles on pulmonary DC function and the development of acute allergic airway inflammation. Surprisingly, exposure to PS50G nanoparticles did not exacerbate but instead inhibited key features of allergic airway inflammation including lung airway and parenchymal inflammation, airway epithelial mucus production, and serum allergen-specific IgE and allergen-specific Th2 cytokines in the lung-draining lymph node (LN) after allergen challenge 1 mo later. PS50G nanoparticles themselves did not induce lung oxidative stress or cardiac or lung inflammation. Mechanistically, PS50G nanoparticles did not impair peripheral allergen sensitization but exerted their effect at the lung allergen challenge phase by inhibiting expansion of CD11c(+)MHCII(hi) DCs in the lung and draining LN and allergen-laden CD11b(hi)MHCII(hi) DCs in the lung after allergen challenge. PS50G nanoparticles further suppressed the ability of CD11b(hi) DCs in the draining LN of allergen-challenged mice to induce proliferation of OVA-specific CD4(+) T cells. The discovery that a defined type of nanoparticle can inhibit, rather than promote, lung inflammation via modulation of DC function opens the door to the discovery of other nanoparticle types with exciting beneficial properties.

Methods of effective conjugation of antigens to nanoparticles as non-inflammatory vaccine carriers.

It has recently become clear that nanoparticle size is a major determinant for how antigen presenting cells (APCs), and specifically dendritic cells (DC) recognize and handle particles, and hence a critical parameter for the formulation of particulate vaccines that aim to induce immunity by targeting DC. Our previous studies in mice and sheep have shown polystyrene nanoparticles of 40-50 nm (PSNPs) with covalently bound antigen offer a new class of vaccines, which contain only 2 elements, antigen and particle, and no added inflammatory stimuli, but evoke very potent combined CD8 T cell and antibody responses. Herein we have optimized the methods for antigen conjugation to PSNPs to controllably promote a single antigen (protein or peptide) layer coating on the nanoparticle. Surprisingly, these nanovaccines not only continued to induce high levels of CD8 T cells in vivo, but were further more potent antibody inducers than nanoparticles containing multiple antigen layers. Addressing the issue of antigen loading on PSNPs, we found an optimal range, above or below which immunogenicity is changed either for antibodies or CD8 T cells. The mechanism behind the induction of high levels of CD8 T cells was further explored by assessing the DC subset that takes up the PSNPs in vivo, and these were found to be preferentially CD8(+) CD11c(+) DC in the lymph node draining the injection site. Since the levels of induced antibodies were highly elevated, and CD8(+) DC do not traditionally induce antibodies, we further sought to find if, despite no detectable inflammation at the injection site, the PSNPs may perhaps induce inflammatory cytokines locally in the lymph node after injection, or systemically in sera, resulting in an adjuvant effect. The initial findings presented herein show no detectable induction of the key inflammatory cytokines such as TNF-α, IL-1 or IL-6, suggesting a novel "non-inflammatory" adjuvant mechanism.

The signalling imprints of nanoparticle uptake by bone marrow derived dendritic cells.

Nanoparticles (NP) possess remarkable adjuvant and carrier capacity, therefore are used in the development of various vaccine formulations. Our previous studies demonstrated that inert non-toxic 40-50 nm polystyrene NP (PS-NP) can promote strong CD8 T cell and antibody responses to the antigen, in the absence of observable inflammatory responses. Furthermore, instillation of PS-NP inhibited the development of allergic airway inflammation by induction of an immunological imprint via modulation of dendritic cell (DC) function without inducing oxidative stress in the lungs in mice. This is in contrast to many studies which show that a variety of ambient and man-made NP promote lung immunopathology, raising concerns generally about the safe use of NPs in biomedicine. Most NPs are capable of inducing inflammatory pathways in DC largely mediated by signalling via the extracellular signal-regulated kinase 1/2 (ERK). Herein, we investigate whether PS-NPs also activate ERK in DC in vitro. Our data show that PS-NP do not induce ERK activation in two different types of bone marrow derived (BM) DC cultures (expanded with GM-CSF or with GM-CSF together with IL-4). The absence of such signalling was not due to lack of PS-NP uptake by BM-DC as confirmed by confocal microscopy and flow cytometry. The process of NP uptake by DC usually initiates ERK signalling, suggesting an unusual uptake pathway may be engaged by PS-NPs. Indeed, data herein showns that uptake of PS-NP by BM-DC was substantially inhibited by phorbol myristate acetate (PMA) but not cytochalasin D (CCD), suggesting an uptake pathway utilising caveole for PS-NP. Together these data show that BM-DC take up PS-NP via a caveole-dependent pathway which does not trigger ERK signalling which may explain their efficient uptake by DC, without the concomitant activation of conventional inflammatory pathways.

Low-Temperature Synthesis of Hollow ß-Tricalcium Phosphate Particles for Bone Tissue Engineering Applications.

ß-Tricalcium phosphate (ß-TCP) has been extensively used in bone tissue engineering in the form of scaffolds, granules, or as reinforcing phase in organic matrices. Solid-state reaction route at high temperatures (>1000 °C) is the most widely used method for the preparation of ß-TCP. The high-temperature synthesis, however, results in the formation of hard agglomerates and fused particles which necessitates postprocessing steps such as milling and sieving operations. This, inadvertently, could lead to introducing unwanted trace elements, promoting particle shape irregularity as well as compromising the biodegradability and bioactivity of ß-TCP because of the solid microstructure of particles. In this study, we introduce a one-pot wet-chemical method at low temperatures (between 160 and 170 °C) to synthesize hollow ß-TCP (hß-TCP) submicron particles of an average size of 300 nm with a uniform rhombohedral shape. We assessed the cytocompatibility of the hß-TCP using primary human osteoblasts (HOB), adipose-derived stem cells (ADSC), and antigen-presenting cells (APCs). We demonstrate the bioactivity of the hß-TCP when cultured with HOB, ADSC, and APCs at a range of particle concentrations (up to 1000 µg/mL) for up to 7 days. hß-TCP significantly enhances osteogenic differentiation of ADSC without the addition of osteogenic supplements. These findings offer a new type of ß-TCP particles prepared at low temperatures, which present various opportunities for developing ß-TCP based biomaterials.

Pullulan-Coated Iron Oxide Nanoparticles for Blood-Stage Malaria Vaccine Delivery.

Vaccines against blood-stage malaria often aim to induce antibodies to neutralize parasite entry into red blood cells, interferon gamma (IFNγ) produced by T helper 1 (Th1) CD4+ T cells or interleukin 4 (IL-4) produced by T helper 2 (Th2) cells to provide B cell help. One vaccine delivery method for suitable putative malaria protein antigens is the use of nanoparticles as vaccine carriers. It has been previously shown that antigen conjugated to inorganic nanoparticles in the viral-particle size range (~40-60 nm) can induce protective antibodies and T cells against malaria antigens in a rodent malaria challenge model. Herein, it is shown that biodegradable pullulan-coated iron oxide nanoparticles (pIONPs) can be synthesized in this same size range. The pIONPs are non-toxic and do not induce conventional pro-inflammatory cytokines in vitro and in vivo. We show that murine blood-stage antigen MSP4/5 from Plasmodium yoelii could be chemically conjugated to pIONPs and the use of these conjugates as immunogens led to the induction of both specific antibodies and IFNγ CD4+ T cells reactive to MSP4/5 in mice, comparable to responses to MSP4/5 mixed with classical adjuvants (e.g., CpG or Alum) that preferentially induce Th1 or Th2 cells individually. These results suggest that biodegradable pIONPs warrant further exploration as carriers for developing blood-stage malaria vaccines.

A Synthetic Nanoparticle Based Vaccine Approach Targeting MSP4/5 Is Immunogenic and Induces Moderate Protection Against Murine Blood-Stage Malaria.

Malaria remains a significant health problem in many tropical and sub-tropical regions. The development of vaccines against the clinically active blood-stage of infection needs to consider variability and polymorphism in target antigens, and an adjuvant system able to induce broad spectrum immunity comprising both antibodies and helper T cells. Moreover, recent studies have shown some conventional pro-inflammatory adjuvants can also promote expansion of immunosuppressive regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC), both of which could negatively impact malaria disease progression. Herein, we explore the ability of a model nanoparticle delivery system (polystyrene nanoparticles; PSNPs), previously proven to not induce conventional inflammation, Treg or MDSC, to induce immunity to MSP4/5 from Plasmodium yoelii, a member of the MSP4 and MSP5 family of proteins which are highly conserved across diverse malaria species including P. falciparum. The results show PSNPs-MSP4/5 conjugates are highly immunogenic, inducing immune responses comprising both T helper 1 (Th1) and Th2 cellular immunity, and a spectrum of antibody subclasses including IgG1, IgG2a, and IgG2b. Benchmarked against Alum and Complete Freund's Adjuvant (CFA), the immune responses that were induced were of comparable or higher magnitude, for both T cell frequencies by ELISpot and antibody responses in terms of ELISA end titer. Importantly, immunization with PSNPs-MSP4/5 induced partial protection against malaria blood-stage infection (50-80%) shown to be mechanistically dependent on interferon gamma (IFN-γ) production. These results expand the scope of adjuvants considered for malaria blood-stage vaccine development to those that do not use conventional adjuvant pathways and emphasizes the critical role of cellular immunity and specifically IFN-γ producing cells in providing moderate protection against blood-stage malaria comparable to Freunds adjuvant.

Investigation of a novel approach to scoring Giemsa-stained malaria-infected thin blood films.

Daily assessment of the percentage of erythrocytes that are infected ('percent-parasitaemia') across a time-course is a necessary step in many experimental studies of malaria, but represents a time-consuming and unpopular task among researchers. The most common method is extensive microscopic examination of Giemsa-stained thin blood-films. This study explored a method for the assessment of percent-parasitaemia that does not require extended periods of microscopy and results in a descriptive and permanent record of parasitaemia data that is highly amenable to subsequent 'data-mining'. Digital photography was utilized in conjunction with a basic purpose-written computer programme to test the viability of the concept. Partial automation of the determination of percent parasitaemia was then explored, resulting in the successful customization of commercially available broad-spectrum image analysis software towards this aim. Lastly, automated discrimination between infected and uninfected RBCs based on analysis of digital parameters of individual cell images was explored in an effort to completely automate the calculation of an accurate percent-parasitaemia.

Design of Peptide-Based Nanovaccines Targeting Leading Antigens From Gynecological Cancers to Induce HLA-A2.1 Restricted CD8+ T Cell Responses.

Gynecological cancers are a leading cause of mortality in women. CD8+ T cell immunity largely correlates with enhanced survival, whereas inflammation is associated with poor prognosis. Previous studies have shown polystyrene nanoparticles (PSNPs) are biocompatible, do not induce inflammation and when used as vaccine carriers for model peptides induce CD8+ T cell responses. Herein we test the immunogenicity of 24 different peptides, from three leading vaccine target proteins in gynecological cancers: the E7 protein of human papilloma virus (HPV); Wilms Tumor antigen 1 (WT1) and survivin (SV), in PSNP conjugate vaccines. Of relevance to vaccine development was the finding that a minimal CD8+ T cell peptide epitope from HPV was not able to induce HLA-A2.1 specific CD8+ T cell responses in transgenic humanized mice using conventional adjuvants such as CpG, but was nevertheless able to generate strong immunity when delivered as part of a specific longer peptide conjugated to PSNPs vaccines. Conversely, in most cases, when the minimal CD8+ T cell epitopes were able to induce immune responses (with WT1 or SV super agonists) in CpG, they also induced responses when conjugated to PSNPs. In this case, extending the sequence around the CD8+ T cell epitope, using the natural protein context, or engineering linker sequences proposed to enhance antigen processing, had minimal effects in enhancing or changing the cross-reactivity pattern induced by the super agonists. Nanoparticle approaches, such as PSNPs, therefore may offer an alternative vaccination strategy when conventional adjuvants are unable to elicit the desired CD8+ T cell specificity. The findings herein also offer sequence specific insights into peptide vaccine design for nanoparticle-based vaccine carriers.

Sperm Protein 17 Expression by Murine Epithelial Ovarian Cancer Cells and Its Impact on Tumor Progression.

The cancer testis antigen sperm protein 17 (Sp17) is a promising antigenic target in epithelial ovarian cancer (EOC) vaccine development. However, its role in ovarian cancer is unclear. We isolated and expanded Sp17⁺ and Sp17- clones from the murine EOC cell line ID8, and compared their in-vitro cell growth characteristics and in-vivo tumorigenicity. We also examined the potential co-expression of molecules that may influence cancer cell survival and interaction with immune cells. These include stimulatory and immunosuppressive molecules, such as major histocompatibility class I molecules (MHC I), MHC II, cytotoxic T lymphocyte associated antigen-4 (CTLA-4), CD73, CD39, tumor necrosis factor receptor II (TNFRII), signal transducer and activator of transcription 3 (STAT3) and programmed death-ligand 1 (PD-L1). Whilst the presence of Sp17 was not correlated with the ID8 cell proliferation/growth capacity in vitro, it was critical to enable progressive tumor formation in vivo. Flow cytometry revealed that Sp17⁺ ID8 cells displayed higher expression of both STAT3 and PD-L1, whilst MHC II expression was lower. Moreover, Sp17high (PD-L1⁺MHCII-) cell populations showed significantly enhanced resistance to Paclitaxel-induced cell death in vitro compared to Sp17low (PD-L1-MHCII⁺) cells, which was associated in turn with increased STAT3 expression. Together, the data support Sp17 as a factor associated with in-vivo tumor progression and chemo-resistance, validating it as a suitable target for vaccine development.

Immunotherapeutic Interleukin-6 or Interleukin-6 Receptor Blockade in Cancer: Challenges and Opportunities.

Interleukin 6 (IL-6), a well-known pro-inflammatory cytokine with pleiotropic activity is a central player in chronic inflammatory diseases including cancers. Therefore, blockade of the IL-6 signalling pathway has become a target for the therapy of diverse cancers such as multicentric Castleman's disease (CD), multiple myeloma and solid tumours including renal, prostate, lung, colorectal and ovarian cancers. Monoclonal antibodies against IL-6 (Siltuximab) and the IL-6 receptor (IL-6R) (Tocilizumab) have emerged as potential immunotherapies, alone or in combination with conventional chemotherapy. Human trials have demonstrated the ability to block IL-6 activity and in multicentric CD lead to durable clinical response and longer disease stabilisation. However, the efficacy of these treatments is still debatable for other cancers. New generation therapeutics in development such as Clazakizumab, Sarilumab, and soluble gp130-Fc have the additional features of improved binding affinity, better specificity with reduced adverse effects. A deeper understanding of the immunological basis of these agents, as well as of the challenges that are faced by immunotherapy-based products in clinical trials, will help select the most promising anti-IL-6/IL-6R therapies for large scale use. Concurrently, current research efforts to personalize treatments may help in the treatment of patients that would greatly benefit from IL-6 blocking therapies.

Engineered Hydrogen-Bonded Glycopolymer Capsules and Their Interactions with Antigen Presenting Cells.

Hollow glycopolymer microcapsules were fabricated by hydrogen-bonded layer-by-layer (LbL) assembly, and their interactions with a set of antigen presenting cells (APCs), including dendritic cells (DCs), macrophages (MACs), and myeloid derived suppressor cells (MDSCs), were investigated. The glycopolymers were obtained by cascade postpolymerization modifications of poly(oligo(2-ethyl-2-oxazoline methacrylate)-stat-glycidyl methacrylate) involving the modification of the glycidyl groups with propargylamine and the subsequent attachment of mannose azide by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Multilayer assembly of the hydrogen-bonding pair (glycopolymer/poly(methacrylic acid) (PMA)) onto planar and particulate supports (SiO2 particles, d = 1.16 µm) yielded stable glycopolymer films upon cross-linking by CuAAC. The silica (SiO2) particle templates were removed yielding hollow monodisperse capsules, as demonstrated by fluorescence and scanning electron microscopy. Cellular uptake studies using flow cytometry revealed the preferential uptake of the capsules by DCs when compared to MACs or MDSCs. Mannosylated capsules showed a cytokine independent cis-upregulation of CD80 specifically on DCs and a trans-downregulation of PDL-1 on MDSCs. Thus, the glycopolymer capsules may have potential as vaccine carriers, as they are able to upregulate costimulatory molecules for immune cell stimulation on DCs and at the same time downregulate immune inhibitory receptors on suppressor APC such as MDSCs.

Understanding CD8+ T-cell responses toward the native and alternate HLA-A*02:01-restricted WT1 epitope.

The Wilms' tumor 1 (WT1) antigen is expressed in solid and hematological malignancies, but not healthy tissues, making it a promising target for cancer immunotherapies. Immunodominant WT1 epitopes, the native HLA-A2/WT1126-134 (RMFPNAPYL) (HLA-A2/RMFPNAPYL epitope (WT1A)) and its modified variant YMFPNAPYL (HLA-A2/YMFPNAPYL epitope (WT1B)), can induce WT1-specific CD8+ T cells, although WT1B is more stably bound to HLA-A*02:01. Here, to further determine the benefits of those two targets, we assessed the naive precursor frequencies; immunogenicity and cross-reactivity of CD8+ T cells directed toward these two WT1 epitopes. Ex vivo naive WT1A- and WT1B-specific CD8+ T cells were detected in healthy HLA-A*02:01+ individuals with comparable precursor frequencies (1 in 105-106) to other naive CD8+ T-cell pools (for example, A2/HIV-Gag77-85), but as expected, ~100 × lower than those found in memory populations (influenza, A2/M158-66; EBV, A2/BMLF1280-288). Importantly, only WT1A-specific naive precursors were detected in HLA-A2.1 mice. To further assess the immunogenicity and recruitment of CD8+ T cells responding to WT1A and WT1B, we immunized HLA-A2.1 mice with either peptide. WT1A immunization elicited numerically higher CD8+ T-cell responses to the native tumor epitope following re-stimulation, although both regimens produced functionally similar responses toward WT1A via cytokine analysis and CD107a expression. Interestingly, however, WT1B immunization generated cross-reactive CD8+ T-cell responses to WT1A and could be further expanded by WT1A peptide revealing two distinct populations of single- and cross-reactive WT1A+CD8+ T cells with unique T-cell receptor-αß gene signatures. Therefore, although both epitopes are immunogenic, the clinical benefits of WT1B vaccination remains debatable and perhaps both peptides may have separate clinical benefits as treatment targets.

Magnetic Nanovectors for the Development of DNA Blood-Stage Malaria Vaccines.

DNA vaccines offer cost, flexibility, and stability advantages, but administered alone have limited immunogenicity. Previously, we identified optimal configurations of magnetic vectors comprising superparamagnetic iron oxide nanoparticles (SPIONs), polyethylenimine (PEI), and hyaluronic acid (HA) to deliver malaria DNA encoding Plasmodium yoelii (Py) merozoite surface protein MSP119 (SPIONs/PEI/DNA + HA gene complex) to dendritic cells and transfect them with high efficiency in vitro. Herein, we evaluate their immunogenicity in vivo by administering these potential vaccine complexes into BALB/c mice. The complexes induced antibodies against PyMSP119, with higher responses induced intraperitoneally than intramuscularly, and antibody levels further enhanced by applying an external magnetic field. The predominant IgG subclasses induced were IgG2a followed by IgG1 and IgG2b. The complexes further elicited high levels of interferon gamma (IFN-γ), and moderate levels of interleukin (IL)-4 and IL-17 antigen-specific splenocytes, indicating induction of T helper 1 (Th1), Th2, and Th17 cell mediated immunity. The ability of such DNA/nanoparticle complexes to induce cytophilic antibodies together with broad spectrum cellular immunity may benefit malaria vaccines.

Exacerbation of Ventilation-Induced Lung Injury and Inflammation in Preterm Lambs by High-Dose Nanoparticles.

Mechanical ventilation of preterm neonates causes lung inflammation and injury, with potential life-long consequences. Inert 50-nm polystyrene nanoparticles (PS50G) reduce allergic inflammation in the lungs of adult mice. We aimed to confirm the anti-inflammatory effects of PS50G in a sheep asthma model, and investigate the effects of prophylactic administration of PS50G on ventilation-induced lung injury (VILI) in preterm lambs. We assessed lung inflammatory cell infiltration, with and without PS50G, after airway allergen challenge in ewes sensitised to house dust mite. Preterm lambs (0.83 gestation) were delivered by caesarean section for immediate tissue collection (n = 5) or ventilation either with (n = 6) or without (n = 5) prophylactic intra-tracheal administration of PS50G nanoparticles (3% in 2 ml). Ventilation was continued for a total of 2 h before tissue collection for histological and biomolecular assessment of lung injury and inflammation. In ewes with experimental asthma, PS50G decreased eosinophilic infiltration of the lungs. Ventilated preterm lambs showed molecular and histological signs of lung injury and inflammation, which were exacerbated in lambs that received PSG50G. PS50G treatment decreased established inflammation in the lungs of asthmatic sheep. However, prophylactic administration of PSG50 exacerbated ventilation-induced lung injury and lung inflammation in preterm lambs.

A Model to Study the Impact of Polymorphism Driven Liver-Stage Immune Evasion by Malaria Parasites, to Help Design Effective Cross-Reactive Vaccines.

Malaria parasites engage a multitude of strategies to evade the immune system of the host, including the generation of polymorphic T cell epitope sequences, termed altered peptide ligands (APLs). Herein we use an animal model to study how single amino acid changes in the sequence of the circumsporozoite protein (CSP), a major target antigen of pre-erythrocytic malaria vaccines, can lead to a reduction of cross reactivity by T cells. For the first time in any APL model, we further compare different inflammatory adjuvants (Montanide, Poly I:C), non-inflammatory adjuvants (nanoparticles), and peptide pulsed dendritic cells (DCs) for their potential capacity to induce broadly cross reactive immune responses. Results show that the capacity to induce a cross reactive response is primarily controlled by the T cell epitope sequence and cannot be modified by the use of different adjuvants. Moreover, we identify how specific amino acid changes lead to a one-way cross reactivity: where variant-x induced responses are re-elicited by variant-x and not variant-y, but variant-y induced responses can be re-elicited by variant-y and variant-x. We discuss the consequences of the existence of this one-way cross reactivity phenomenon for parasite immune evasion in the field, as well as the use of variant epitopes as a potential tool for optimized vaccine design.