Multi-functional nanocomplex codelivery of Trp2 and R837 to activate melanoma-specific immunity.

Antigen-adjuvant combination could induce a protective and long-lasting anti-tumor immune response. However, exploiting system which could co-deliver melanoma antigen peptide Trp2 (Tyrosinase-related protein 2) and Toll-like-receptor-7 (TLR7) agonists imiquimod (R837) both are poor aqueous solubility is still challenging. Our new nanocomplex was explored for specific delivery of Trp2 and R837 into antigen-presenting cells (APCs). R837 was loaded into mannosylated-ß-cyclodextrin (Man-CD) to target dendritic cells (DCs) by binding mannose receptors (MR) on DCs. A fusion peptide (WT) was constructed by incorporating the amino acid region of TAT (cell-penetrating peptide) into Trp2 to improve the TAT-mediated intracellular efficiency. Negatively charged sodium alginate (SA), a biocompatible polymer, which can induce adjuvant responses by affecting the functions of DCs, was complexed with Man-CD/R837 and WT via physical adsorption. The optimized nanocomplex promoted the cellular uptake and showed remarkable efficacy to enhance the secreting of Th1-cytokines. This multi-functional nanocomplex system may allow effective targeting-codelivery of multi-hydrophobic drugs and be a promising subunit vaccine candidate as a potential prevention action of tumor.

Rapamycin-Loaded Biomimetic Nanoparticles Reverse Vascular Inflammation.

RATIONALE: Through localized delivery of rapamycin via a biomimetic drug delivery system, it is possible to reduce vascular inflammation and thus the progression of vascular disease. OBJECTIVE: Use biomimetic nanoparticles to deliver rapamycin to the vessel wall to reduce inflammation in an in vivo model of atherosclerosis after a short dosing schedule. METHODS AND RESULTS: Biomimetic nanoparticles (leukosomes) were synthesized using membrane proteins purified from activated J774 macrophages. Rapamycin-loaded nanoparticles were characterized using dynamic light scattering and were found to have a diameter of 108±2.3 nm, a surface charge of -15.4±14.4 mV, and a polydispersity index of 0.11 +/ 0.2. For in vivo studies, ApoE-/- mice were fed a high-fat diet for 12 weeks. Mice were injected with either PBS, free rapamycin (5 mg/kg), or rapamycin-loaded leukosomes (Leuko-Rapa; 5 mg/kg) once daily for 7 days. In mice treated with Leuko-Rapa, flow cytometry of disaggregated aortic tissue revealed fewer proliferating macrophages in the aorta (15.6±9.79 %) compared with untreated mice (30.2±13.34 %) and rapamycin alone (26.8±9.87 %). Decreased macrophage proliferation correlated with decreased levels of MCP (monocyte chemoattractant protein)-1 and IL (interleukin)-b1 in mice treated with Leuko-Rapa. Furthermore, Leuko-Rapa-treated mice also displayed significantly decreased MMP (matrix metalloproteinases) activity in the aorta (mean difference 2554±363.9, P=9.95122×10-6). No significant changes in metabolic or inflammation markers observed in liver metabolic assays. Histological analysis showed improvements in lung morphology, with no alterations in heart, spleen, lung, or liver in Leuko-Rapa-treated mice. CONCLUSIONS: We showed that our biomimetic nanoparticles showed a decrease in proliferating macrophage population that was accompanied by the reduction of key proinflammatory cytokines and changes in plaque morphology. This proof-of-concept showed that our platform was capable of suppressing macrophage proliferation within the aorta after a short dosing schedule (7 days) and with a favorable toxicity profile. This treatment could be a promising intervention for the acute stabilization of late-stage plaques.

Cancer Vaccines: Adjuvant Potency, Importance of Age, Lifestyle, and Treatments.

Although the discovery and characterization of multiple tumor antigens have sparked the development of many antigen/derived cancer vaccines, many are poorly immunogenic and thus, lack clinical efficacy. Adjuvants are therefore incorporated into vaccine formulations to trigger strong and long-lasting immune responses. Adjuvants have generally been classified into two categories: those that 'depot' antigens (e.g. mineral salts such as aluminum hydroxide, emulsions, liposomes) and those that act as immunostimulants (Toll Like Receptor agonists, saponins, cytokines). In addition, several novel technologies using vector-based delivery of antigens have been used. Unfortunately, the immune system declines with age, a phenomenon known as immunosenescence, and this is characterized by functional changes in both innate and adaptive cellular immunity systems as well as in lymph node architecture. While many of the immune functions decline over time, others paradoxically increase. Indeed, aging is known to be associated with a low level of chronic inflammation-inflamm-aging. Given that the median age of cancer diagnosis is 66 years and that immunotherapeutic interventions such as cancer vaccines are currently given in combination with or after other forms of treatments which themselves have immune-modulating potential such as surgery, chemotherapy and radiotherapy, the choice of adjuvants requires careful consideration in order to achieve the maximum immune response in a compromised environment. In addition, more clinical trials need to be performed to carefully assess how less conventional form of immune adjuvants, such as exercise, diet and psychological care which have all be shown to influence immune responses can be incorporated to improve the efficacy of cancer vaccines. In this review, adjuvants will be discussed with respect to the above-mentioned important elements.

Antigenic Epitope Analysis and Efficacy Evaluation of GRA41 DNA Vaccine Against T. gondii Infection.

BACKGROUND: Toxoplasma gondii has a comprehensive impact on a great range of warm-blood mammals, in which one-third of the population all over the world is involved. Dense granular proteins, regarded as GRA family, mediating substantial interface between host cell cytoplasm and parasite, are widely studied for preventing the infection of T. gondii. PURPOSE: As is handled in our study, the effect of intramuscularly injecting the genetic vaccine pEGFP-C1/GRA41 encoding a novel dense granule protein, GRA41, was evaluated. METHODS: At the beginning, bioinformatics analysis was used to evaluate epitopes of both B cells and T cells on the GRA41 protein of T. gondii. Afterwards, recombinant plasmids (pEGFP-C1/GRA41) were injected into BALB/c mice and the quantity of IgG and its subclass IgG2a remarkably increased. IFN-γ, distinctive from the other cytokines (IL-4, and IL-10), was significant in growth. Afterwards, the intraperitoneal challenge was executed for recording survival time with tachyzoites with high virulence (in RH strain) and counting the number of brain cysts was carried out after the infection of PRU strain (low virulence). RESULTS: In pEGFP-C1/GRA41 group, the survival period was significantly longer (13.3 ± 3.37 days) after tachyzoites attack with the RH strain in high virulence, compared with the other groups (less than 8 days). Additionally, the cyst quantity is remarkably lower and the rate of reduction could reach 59.34%. CONCLUSION: All the results indicated effective protection of DNA vaccine encoding GRA41 against T. gondii.

A novel therapeutic vaccine composed of a rearranged human papillomavirus type 16 E6/E7 fusion protein and Fms-like tyrosine kinase-3 ligand induces CD8+ T cell responses and antitumor effect.

The development of cervical cancer is mainly caused by infection with high risk genotypes of human papillomavirus, particularly type 16 (HPV16), which accounts for more than 50% of cervical cancer. The two early viral oncogenes, E6 and E7, are continuously expressed in cervical cancer cells and are necessary to maintain the malignant cellular phenotype, thus providing ideal targets for immunotherapy of cervical cancer. In this study, a novel vaccine strategy was developed based on a rationally shuffled HPV16 E6/E7 fusion protein, the addition of Fms-like tyrosine kinase-3 ligand (Flt3L) or the N domain of calreticulin (NCRT), and the usage of a CpG adjuvant. Four recombinant proteins were constructed: m16E6E7 (mutant E6/E7 fusion protein), rm16E6E7 (rearranged mutant HPV16 E6/E7 fusion protein), Flt3L-RM16 (Flt3L fused to rm16E6E7), and NCRT-RM16 (NCRT fused to rm16E6E7). Our results suggest that Flt3L-RM16 was the most potent of these proteins in terms of inducing E6- and E7-specific CD8+ T cell responses. Additionally, Flt3L-RM16 significantly induced regression of established E6/E7-expressing TC-1 tumors. Higher doses of Flt3L-RM16 trended toward higher levels of antitumor activity, but these differences did not reach statistical significance. In summary, this study found that Flt3L-RM16 fusion protein is a promising therapeutic vaccine for immunotherapy of HPV16-associated cervical cancer.

Virus-Mimetic Fusogenic Exosomes for Direct Delivery of Integral Membrane Proteins to Target Cell Membranes.

An efficient system for direct delivery of integral membrane proteins is successfully developed using a new biocompatible exosome-based platform. Fusogenic exosomes harboring viral fusogen, vascular stomatitis virus (VSV)-G protein, can fuse with and modify plasma membranes in a process called "membrane editing." This can facilitate the transfer of biologically active membrane proteins into the target cell membranes both in vitro and in vivo.

Twin-screw extruded lipid implants containing TRP2 peptide for tumour therapy.

Much effort has been put in the development of specific anti-tumour immunotherapies over the last few years, and several studies report on the use of liposomal carriers for tumour-associated antigens. In this work, the use of lipid implants, prepared using two different extruders, was investigated for sustained delivery in tumour therapy. The implants consisted of cholesterol, soybean lecithin, Dynasan 114, trehalose, ovalbumin (OVA) or a TRP2 peptide, and Quil-A. Implants were first produced on a Haake Minilab extruder, and then a scale-down to minimal quantities of material on a small scale ZE mini extruder was performed. All formulations were characterised in terms of extrudability, implant properties and in vitro release behaviour of the model antigen ovalbumin. The type of extruder used to produce the implants had a major influence on implant properties and the release behaviour, demonstrating that extrusion parameters and lipid formulations have to be individually adapted to each extrusion device. Subsequently, lipid implants containing TRP-2 peptide were extruded on the ZE mini extruder and investigated in vitro and in vivo. The in vivo study showed that mice having received TRP2 loaded implants had delayed tumour growth for 3days compared to groups having received no TRP2.

In vitro assessment of antibody-conjugated gold nanorods for systemic injections.

BACKGROUND: The interest for gold nanorods in biomedical optics is driven by their intense absorbance of near infrared light, their biocompatibility and their potential to reach tumors after systemic administration. Examples of applications include the photoacoustic imaging and the photothermal ablation of cancer. In spite of great current efforts, the selective delivery of gold nanorods to tumors through the bloodstream remains a formidable challenge. Their bio-conjugation with targeting units, and in particular with antibodies, is perceived as a hopeful solution, but the complexity of living organisms complicates the identification of possible obstacles along the way to tumors. RESULTS: Here, we present a new model of gold nanorods conjugated with anti-cancer antigen 125 (CA125) antibodies, which exhibit high specificity for ovarian cancer cells. We implement a battery of tests in vitro, in order to simulate major nuisances and predict the feasibility of these particles for intravenous injections. We show that parameters like the competition of free CA125 in the bloodstream, which could saturate the probe before arriving at the tumors, the matrix effect and the interference with erythrocytes and phagocytes are uncritical. CONCLUSIONS: Although some deterioration is detectable, anti-CA125-conjugated gold nanorods retain their functional features after interaction with blood tissue and so represent a powerful candidate to hit ovarian cancer cells.

Multifunctional nanoparticles co-delivering Trp2 peptide and CpG adjuvant induce potent cytotoxic T-lymphocyte response against melanoma and its lung metastasis.

Immunotherapy has shown the potential to become an essential component of the successful treatment of various malignancies. In many cases, such as in melanoma, however, induction of a potent and specific T-cell response against the endogenous antigen or self-antigen still remains a major challenge. To induce a potent MHC I-restricted cytotoxic T-lymphocyte (CTL) response, cytosol delivery of an exogenous antigen into dendritic cells is preferred, if not required. Lipid-calcium-phosphate (LCP) nanoparticles represent a new class of intracellular delivery systems for impermeable drugs. We are interested in exploring the potential of LCP NPs for use as a peptide vaccine delivery system for cancer therapy. To increase the encapsulation of Trp2 peptide into the calcium phosphate precipitate core of LCP, two phosphor-serine residues were added to the N-terminal of the peptide (p-Trp2). CpG ODN was also co-encapsulated with p-Trp2 as an adjuvant. The NPs were further modified with mannose to enhance and prolong the cargo deposit into the lymph nodes (LNs), which ensured persistent antigen loading and stimulation. Compared with free Trp2 peptide/CpG, vaccination with LCP encapsulating p-Trp2 and CpG resulted in superior inhibition of tumor growth in both B16F10 subcutaneous and lung metastasis models. An IFN-γ production assay and in vivo CTL response study revealed that the improved efficacy was a result of a Trp2-specific immune response. Thus, encapsulation of phospho-peptide antigens into LCP may be a promising strategy for enhancing the immunogenicity of poorly immunogenic self-antigens for cancer therapy.

Lethality in a murine model of pulmonary anthrax is reduced by combining nuclear transport modifier with antimicrobial therapy.

BACKGROUND: In the last ten years, bioterrorism has become a serious threat and challenge to public health worldwide. Pulmonary anthrax caused by airborne Bacillus anthracis spores is a life-threatening disease often refractory to antimicrobial therapy. Inhaled spores germinate into vegetative forms that elaborate an anti-phagocytic capsule along with potent exotoxins which disrupt the signaling pathways governing the innate and adaptive immune responses and cause endothelial cell dysfunction leading to vascular injury in the lung, hypoxia, hemorrhage, and death. METHODS/PRINCIPAL FINDINGS: Using a murine model of pulmonary anthrax disease, we showed that a nuclear transport modifier restored markers of the innate immune response in spore-infected animals. An 8-day protocol of single-dose ciprofloxacin had no significant effect on mortality (4% survival) of A/J mice lethally infected with B. anthracis Sterne. Strikingly, mice were much more likely to survive infection (52% survival) when treated with ciprofloxacin and a cell-penetrating peptide modifier of host nuclear transport, termed cSN50. In B. anthracis-infected animals treated with antibiotic alone, we detected a muted innate immune response manifested by cytokines, tumor necrosis factor alpha (TNFα), interleukin (IL)-6, and chemokine monocyte chemoattractant protein-1 (MCP-1), while the hypoxia biomarker, erythropoietin (EPO), was greatly elevated. In contrast, cSN50-treated mice receiving ciprofloxacin demonstrated a restored innate immune responsiveness and reduced EPO level. Consistent with this improvement of innate immunity response and suppression of hypoxia biomarker, surviving mice in the combination treatment group displayed minimal histopathologic signs of vascular injury and a marked reduction of anthrax bacilli in the lungs. CONCLUSIONS: We demonstrate, for the first time, that regulating nuclear transport with a cell-penetrating modifier provides a cytoprotective effect, which enables the host's immune system to reduce its susceptibility to lethal B. anthracis infection. Thus, by combining a nuclear transport modifier with antimicrobial therapy we offer a novel adjunctive measure to control florid pulmonary anthrax disease.

Long term stability of a recombinant Plasmodium falciparum AMA1 malaria vaccine adjuvanted with Montanide(®) ISA 720 and stabilized with glycine.

Plasmodium falciparum apical membrane antigen 1 (AMA1) is an asexual blood-stage vaccine candidate against the malaria parasite. AMA1-C1/ISA 720 refers to a mixture of recombinant AMA1 proteins representing the FVO and 3D7 alleles in 1:1 mass ratio, formulated with Montanide(®) ISA 720 as a water-in oil emulsion. In order to develop the AMA1-C1/ISA 720 vaccine for human use, it was important to determine the shelf life of this formulation. Previously it was found 267 mM glycine stabilized the proteins in Montanide(®) ISA 720 formulations for a short period of time at 2-8°C [25]. We now test the long term stability of AMA1-C1 at 10 and 40 µg/mL formulated with Montanide(®) ISA 720 with 50mM glycine as a stabilizer. Stability of AMA1-C1/ISA 720 at different time points following formulation (0, 5, 12 or 18 months) was evaluated by determining the mean particle size (diameter of the mean droplet volume), total protein content by a Modified Lowry assay, identity and integrity using western blot and SDS-PAGE. Our results showed that the mean particle size of these emulsions increased over time, whereas protein content, as determined by an ELISA method using a monoclonal antibody against penta-his, decreased over time. For the 10 µg/mL AMA1-C1/ISA 720 vaccine, the protein content was 6.5±2.2 µg/mL, and for the 40 µg/mL AMA1-C1/ISA 720 vaccine, the protein content was only 8.2±2.3 µg/mL after 18 months of storage at 2-8°C. These results suggest that the integrity of the protein was affected by long-term storage. The results of the present study indicate that the AMA1-C1/ISA 720 emulsion was unstable after 12 months of storage, after which AMA1-C1 proteins were partially degraded.

Listeria-derived ActA is an effective adjuvant for primary and metastatic tumor immunotherapy.

Tumor immunotherapy is currently at the cusp of becoming an important aspect of comprehensive cancer treatment in the clinic. However, the need for improved adjuvants to augment immune responses against tumor antigens is always present. In this paper, we characterize the Listeria monocytogenes-derived actin-nucleating protein, ActA, as a novel adjuvant for use in tumor immunotherapy. ActA is a virulence factor that is expressed on the cell surface of L. monocytogenes and facilitates the production of actin tails that propel Listeria throughout the cytosol of an infected host cell. It is believed that this ActA-dependent cytosolic motility allows Listeria to evade adaptive host cell defenses and facilitates its invasion into a proximal uninfected host cell. However, there is evidence that ActA fused to a tumor antigen and delivered by L. monocytogenes can perform a beneficial function in tumor immunotherapy as an adjuvant. Our investigation of this adjuvant activity demonstrates that ActA, either fused to or administered as a mixture with a tumor antigen, can augment anti-tumor immune responses, break immune tolerance and facilitate tumor eradication, which suggests that ActA is not only an effective adjuvant in tumor immunotherapy but can also be applied in a number of therapeutic settings.

A single nasal dose of fms-like tyrosine kinase receptor-3 ligand, but not peritoneal application, enhances nontypeable Haemophilus influenzae-specific long-term mucosal immune responses in the nasopharynx.

Nasal vaccination is an effective therapeutic regimen for preventing otitis media. In the development of nasal vaccine, an appropriate adjuvant is required. In the present study, we examined the efficacy of fms-like tyrosine kinase receptor-3 ligand (Flt3L) as a mucosal adjuvant. Flt3L was administered intranasally or peritoneally to mice, which were then immunized intranasally with P6 protein of nontypeable Haemophilus influenzae (NTHi), and P6-specific immune responses were examined. In addition, NTHi challenges were performed and the level of NTHi was quantified in nasal washes. Nasal application of Flt3L induced an increase in the number of dendritic cells in nasal-associated lymphoid tissue. P6-specific nasal wash immunoglobulin (Ig)A and serum IgG titers were elevated significantly after nasal immunization. Enhanced NTHi clearance from the nasopharynx was also observed. The effect of nasal vaccination with P6 combined with nasal Flt3L application was prolonged. These results indicate the potential of Flt3L as an effective mucosal adjuvant and suggest that nasal vaccination with P6 in combination with nasal Flt3L might be an effective regimen for the induction of NTHi-specific protective immunity.

Jagged1 suppresses collagen-induced arthritis by indirectly providing a negative signal in CD8+ T cells.

Distinct Notch ligands possess a characteristic ability in terms of functional T cell differentiation. However, the precise role or the therapeutic potential of each Notch ligand in autoimmune diseases is largely unknown. In this study, we examined whether Jagged1 modulates a collagen-induced rheumatoid arthritis (CIA) model by altering T cell responses. The injection of a soluble Jagged1-encoding plasmid, sJag1-P, before or even after initial type II collagen (CII) immunization suppressed the disease severity of CIA. However, this treatment did not suppress CII-specific CD4(+) T cell proliferation and CII-specific Ab production. Depletion of either CD4(+) or CD8(+) T cells ameliorated CIA severity and sJag1-P further improved CIA in CD4(+) but not CD8(+) T cell-depleted mice. Injection of OVA and Jagged1-encoding plasmids inhibited proliferation of OVA-specific granzyme B-producing CD8(+) T cells, although Jagged1 could not directly inhibit CD8(+) T cell proliferation in vitro. The blockade of Jagged1 by an anti-Jagged1 Ab exacerbated CIA, whereas this effect was not observed in the absence of CD8(+) T cells. These data indicate that Jagged1 is able to deliver an indirect negative signal into CD8(+) T cells in vivo, which suggests its therapeutic potential in the treatment of CD8(+) T cell-mediated diseases, including rheumatoid arthritis.

Cytokines in combination to treat radiation-induced myelosuppresssion: evaluation of SCF + glycosylated EPO + pegylated G-CSF as an emergency treatment in highly irradiated monkeys.

Multicytokine therapy may be useful to counteract radiation-induced myelosuppression. We assessed the stem cell factor + glycosylated erythropoietin + pegylated granulocyte colony-stimulating factor combination (SEG) as an emergency treatment. SEG in highly irradiated monkeys efficacy appeared to be restricted to granulopoiesis. Early administration of Erythropoietin did not prevent radiation-induced anemia.

Prophylactic treatment with fms-like tyrosine kinase-3 ligand after burn injury enhances global immune responses to infection.

Severely burned patients are susceptible to infections with opportunistic organisms due to altered immune responses and frequent wound contamination. Immunomodulation to enhance systemic and local responses to wound infections may be protective after burn injury. We previously demonstrated that pretreatments with fms-like tyrosine kinase-3 (Flt3) ligand (Flt3L), a dendritic cell growth factor, increase the resistance of mice to a subsequent burn injury and wound infection by a dendritic cell-dependent mechanism. This study was designed to test the hypothesis that Flt3L administration after burn injury decreases susceptibility to wound infections by enhancing global immune cell activation. Mice were treated with Flt3L after burn injury and examined for survival, wound and systemic bacterial clearance, and immune cell activation after wound inoculation with Pseudomonas aeruginosa. To gain insight into the local effects of Flt3L at the burn wound, localization of Langerhans cells was examined. Mice treated with Flt3L had significantly greater numbers of CD25-expressing T cells and CD69-expressing T and B cells, neutrophils, and macrophages after, but not before, infection. Overall leukocyte apoptosis in response to infection was decreased with Flt3L treatment. Survival and local and systemic bacterial clearance were enhanced by Flt3L. Langerhans cells appeared in the dermis of skin bordering the burn wound, and further increased in response to wound infection. Flt3L augmented the appearance of Langerhans cells in response to both injury and infection. These data suggest that dendritic cell enhancement by Flt3L treatments after burn injury protects against opportunistic infections through promotion of local and systemic immune responses to infection.

Inhibition of natural type I IFN-producing and dendritic cell development by a small molecule receptor tyrosine kinase inhibitor with Flt3 affinity.

In vivo steady-state type I natural IFN-producing and dendritic cell (DC) development is largely dependent on Flt3 signaling. Natural IFN-producing and DC progenitors and their respective downstream cell populations express the flt3 receptor, and Flt3 ligand (Flt3L)(-/-) mice have reduced while Flt3L-injected mice develop markedly increased numbers of both cell types. In the present study, we show that SU11657, a small multitargeted receptor tyrosine kinase inhibitor with Flt3 affinity, suppressed in vitro natural IFN-producing and DC development in Flt3L-supplemented mouse whole bone marrow cell cultures in a dose-dependant manner, while DC development in GM-CSF-supplemented cultures was not affected. In vivo SU11657 application led to a significant decrease of both natural IFN-producing and DCs, comparable to the reduction observed in Flt3L(-/-) mice. Conversely, Flt3L plasma levels increased massively in inhibitor-treated animals, likely via a regulatory feedback loop, without being able to compensate for pharmacological Flt3 inhibition. No obvious toxicity was observed, and hemopoietic progenitor cell and stem cell function remained intact as assessed by myeloid colony-forming unit activity and in vivo bone marrow repopulation assays. Furthermore, upon treatment discontinuation, IFN-producing and DCs recovered to normal levels, proving that treatment effects were transient. Given the importance of IFN-producing and DCs in regulation of immune responses, these findings might lead to new pharmacological strategies in prevention and treatment of autoimmune diseases and complications of organ or blood cell transplantation.

PppA, a surface-exposed protein of Streptococcus pneumoniae, elicits cross-reactive antibodies that reduce colonization in a murine intranasal immunization and challenge model.

The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the present conjugate vaccine may still cause pneumococcal disease. To address these serotypes, and the remaining otitis media due to Streptococcus pneumoniae, efforts have been devoted to identifying protective protein antigens. Immunity to conserved surface proteins important for adhesion, nutrient acquisition, or other functions could result in a reduction of colonization and a lower disease potential. We have been searching for conserved surface-exposed proteins from S. pneumoniae that may be involved in pathogenesis to test as vaccine candidates. Here, an approximately 20-kDa protein that has significant homology to a nonheme iron-containing ferritin protein from Listeria innocua and other bactoferritins was identified as pneumococcal protective protein A (PppA). We expressed and purified recombinant PppA (rPppA) and evaluated its potential as a vaccine candidate. The antibodies elicited by purified rPppA were cross-reactive with PppA from multiple strains of S. pneumoniae and were directed against surface-exposed epitopes. Intranasal immunization of BALB/c mice with PppA protein and either a synthetic monophosphoryl lipid A analog, RC529AF, or a cholera toxin mutant, CT-E29H, used as an adjuvant reduced nasopharyngeal colonization in mice following intranasal challenge with a heterologous pneumococcal strain. PppA-specific systemic and local immunoglobulin G (IgG) and IgA antibody responses were induced. The antisera reacted with whole cells of a heterologous S. pneumoniae type 3 strain. These observations indicate that PppA may be a promising candidate for inclusion in a vaccine against pneumococcal otitis media.

Fusion of two malaria vaccine candidate antigens enhances product yield, immunogenicity, and antibody-mediated inhibition of parasite growth in vitro.

A Plasmodium falciparum chimeric protein 2.9 (PfCP-2.9) was constructed consisting of the C-terminal regions of two leading malaria vaccine candidates, domain III of apical membrane ag-1 (AMA-1) and 19-kDa C-terminal fragment of the merozoite surface protein 1 (MSP1). The PfCP-2.9 was produced by Pichia pastoris in secreted form with a yield of 2600 mg/L and approximately 1 g/L of final product was obtained from a three-step purification process. Analysis of conformational properties of the chimeric protein showed that all six conformational mAbs interacted with the recombinant protein were reduction-sensitive, indicating that fusion of the two cysteine-rich proteins retains critical conformational epitopes. PfCP-2.9 was found to be highly immunogenic in rabbits as well as in rhesus monkeys (Macaca mulatta). The chimeric protein induced both anti-MSP1-19 and anti-AMA-1(III) Abs at levels 11- and 18-fold higher, respectively, than individual components did. Anti-PfCP-2.9 sera from both rabbits and rhesus monkeys almost completely inhibited in vitro growth of the P. falciparum FCC1/HN and 3D7 lines when tested at a 6.7-fold dilution. It was shown that the inhibition is dependent on the presence of Abs to the chimeric protein and their disulfide bond-dependent conformations. Moreover, the activity was mediated by a combination of growth-inhibitory Abs generated by the individual MSP1-19 and AMA-1(III) of PfCP-2.9. The combination of the extremely high yield of the protein and enhancement of its immune response provides a basis to develop an effective and affordable malaria vaccine.

A single intratracheal dose of the growth factor Fms-like tyrosine kinase receptor-3 ligand induces a rapid differential increase of dendritic cells and lymphocyte subsets in lung tissue and bronchoalveolar lavage, resulting in an increased local antibody production.

Repetitive doses of the growth factor Fms-like tyrosine kinase receptor-3 ligand (Flt3L) have resulted in increased numbers of dendritic cells (DC) in various organs, and the effect on protective or tolerogeneic responses in the gut wall has been documented in the literature. In this study, for the first time, Flt3L was locally applied in the trachea of rats using a single dose only. A dose-dependent increase not only of DC, but also of T lymphocytes (CD4(+) and CD8(+)), was seen with a maximum on day 3. The effects on the cells in the lung interstitium and the bronchoalveolar space showed some differences. The use of tetanus toxoid as a model Ag applied intratracheally after the local Flt3L stimulation resulted in increased levels of specific IgA and IgG in the lung. Thus, this novel approach of locally stimulating APCs by topical application of a DC growth factor before applying the Ag offers a new vaccination strategy.