LUVs recovered with Chitosan: a new preparation for vaccine delivery

Chitosan, alpha-(1-4)-amino-2-deoxy-beta-D-glucan, is a deacetylated form of chitin, an abundant natural polysaccharide present in crustacean shells. Its unique characteristics such as positive charge, biodegradability, biocompatibility, nontoxicity, and rigid linear molecular structure make this macromolecule ideal as drug carrier. The association between chitosan and liposomes was carefully described, where REVs (reverse phase evaporation vesicles) were sandwiched by chitosan. The usage of these particles in vaccine formulation is here proposed for the first time in the literature. The Chitosan-REVs now stabilized by polyvinilic alcohol were the vehicle for Diphtheria toxoid (Dtxd). Round chitosan-sandwiched REVs (REVs-Chi) particles of 373 +/- 17 nm containing 65% Dtxd were obtained. After 200 min of incubation in a simulated gastric fluid, 70% of the Dtxd was liberated from REVs-Chi in comparison to 100% of Dtxd liberated from pure REVs. In PBS, the Dtxd liberation from REVS-Chi was about 60%. Mice were immunized with Dtxd encapsulated within REVs-Chi and with other REVs/Dtxd formulations adsorbed onto Freund adjuvant or alumen [AIF and Al(OH)(3)]. The response patterns and the immune maturity were measured by IgG(1) and IgG(2a) titrations. REVs-Chi containing Dtxd elicited both antibodies production giving the animals higher immune response and selectivity. It was interesting that the memory of those mice immunized with REVs-Chi containing Dtxd enhanced, after booster, antibody production by 47% in contrast with 17 and 7% in mice immunized with the antigen vehiculated in REVs-AIF or REVs-Al(OH)(3), respectively.     

Enhanced Liposomal Vaccine Formulation and Performance: Simple Physicochemical and Immunological Approaches

The Dtxd (Diphtheria toxoid) was the first antigen encapsulated within liposomes, their adjuvant properties were discovered (their capacity to enhance the vaccine immunogenicity). The point here is not to propose a new method to prepare this lipossomal vaccine. The central idea is to give new dresses for old vaccines by using classical and well-established liposome preparation method changing only the encapsulation pH and the immunization protocol.

The most appropriate method of Dtxd encapsulation within liposome was based on lipid film hydration in 100 mM citrate buffer, pH 4.0. This was accompanied by changes on protein hydrophobicity, observed by CD and fluorescence spectroscopies. Whenever the Dtxd exposed its hydrophobic residues at pH 4.0, it interacted better with the lipossomal (observed by electrophoretic mobility) film than when its hydrophobic residues were buried (pH 9.0). The Dtxd partition coefficient in Triton-X114 and the acrylamide fluorescence quenching were also pH dependent. Both were bigger at pH 4.0 than at pH 9.0. The relationship protein structure and lipid interaction was pH dependent and now it can be easily maximized to enhance encapsulation of antigens in vaccine development.

Mice were primed with formulations containing 5 μg of Dtxd within liposomes prepared in pH 4.0 or 7.0 or 9.0. The boosters were done 38 or 138 days after the first immunization. The IgM produced by immediate response of all lipossomal formulations were higher than the control (free protein). The response patterns and the immune maturity were measured by IgG₁ and IgG_2a titrations. The IgG₁ titers produced by both formulations at pH 4.0 and 7.0 were at least 2² higher than those produced by mice injected lipossomal formulation at pH 9.0. When the boosters were done, 138 days after priming the mice produced a IgG_2a titer of 2⁹ and the group that received the booster 30 days after priming produced a titer of 2⁵. The strongest antibody production was the neutralizing antibody (2⁴⁵ higher than the control) produced by those mice injected with lipossomal formulation at pH 4.0 with the booster done 138 days after priming. The simple change on lipossomal pH formulation and timing of the booster enhanced both antibody production and selectivity.     

Collagen-induced arthritis is exacerbated in IL-10-deficient mice

IL-10 is a potent immunoregulatory cytokine attenuating a wide range of immune effector and inflammatory responses. In the present study, we assess whether endogenous levels of IL-10 function to regulate the incidence and severity of collagen-induced arthritis. DBA/1 wildtype (WT), heterozygous (IL-10^+/-) and homozygous (IL-10^-/-) IL-10-deficient mice were immunized with type II collagen. Development of arthritis was monitored over time, and collagen-specific cytokine production and anticollagen antibodies were assessed. Arthritis developed progressively in mice immunized with collagen, and 100% of the WT, IL-10^+/-, and IL-10^-/- mice were arthritic at 35 days. However, the severity of arthritis in the IL-10^-/- mice was significantly greater than that in WT or IL-1^+/- animals. Disease severity was associated with reduced IFN-γ levels and a dramatic increase in CD11b-positive macrophages. Paradoxically, both the IgG₁ and IgG_2a anticollagen antibody responses were also significantly reduced. These data demonstrate that IL-10 is capable of controlling disease severity through a mechanism that involves IFN-γ. Since IL-10 levels are elevated in rheumatoid arthritis synovial fluid, these findings may have relevance to rheumatoid arthritis.     

Adjuvant and immunogenic properties of bacterial lipopolysaccharide in IgE and IgG1 antibody formation in mice

The ability of Gram-negative lipopolysaccharide (LPS) to function as an adjuvant and as an antigen in IgE and IgG₁ immune responses in mice was investigated. LPS failed to induce LPS-specific IgE or IgG₁ under a variety of experimental conditions. Both isolated LPS and whole heat-killed bacteria were capable of enhancing IgE and IgG₁ antibody formation to a protein antigen, egg albumin (EA). The LPS-induced anti-EA, IgE, and IgG₁ antibody titers exhibited a cycling phenomenon with time. In the presence of LPS, IgE, and IgG₁ antibodies specific for EA did not occur in athymic nude (BALB/c-nu/nu) mice, demonstrating the inability of LPS to substitute for the stringent requirement for T cells in homocytotropic antibody formation.     

Heligmosomoides polygrus (equals nematospiroides dubius): humoral and intestinal immunologic responses to infection in mice

Measurements of serum IgG₁, IgG_2a, IgM, and IgA levels and antibody titers in these immunoglobulin classes were made at intervals after initial infection and challenge infection of mice immunized by two or three previous infections. Identical measurements were made on the content of the small intestine in mice which had been exposed to the same infection schedule. Sections of small intestine taken after initial infection and challenge infection were examined by the fluorescent antibody technique for changes in populations of immunoglobulin-containing cells and by routine histologic procedures for histopathologic changes.In serum, only IgG₁ was consistently increased after initial infection, and antibody in IgG₁ was detected within the first 2 wk of infection. In immunized animals, only IgG₁ and antibody of this class always responded to challenge infection, although antibody in other immunoglobulin classes was detected.IgA concentration of the intestinal content did not differ significantly after initial infection or challenge infection of immunized mice. Immunized mice had about twice the IgG₁ concentration in intestinal content as singly infected animals. No intestinal antibody was detected after initial infection; only IgG₁ antibody was detected in the intestinal content of immunized and challenged mice.Cell infiltrates in the intestinal mucosa and submucosa of immunized animals contained numerous IgG₁-containing cells. Mast cells and globular leukocytes were observed in the intestine of immunized animals.     

Comparative studies of the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine against bovine mastitis using non-invasive mouse mastitis as a model system

This study was undertaken to compare the immunogenicity and protective potential of biofilm vs planktonic Staphylococcus aureus vaccine for the prevention of mastitis using the mouse as a model system. Mice immunized with formalin-killed whole cell vaccine of S. aureus residing in a biofilm when delivered via an intramammary route produced a cell mediated immune response. Mice immunized with this biofilm vaccine showed significant reductions in colonization by S. aureus in mammary glands, severity of clinical symptoms and tissue damage in mammary glands in comparison with the mice immunized with formalin-killed whole cells of planktonic S. aureus. The planktonic vaccine administered by a subcutaneous route produced a significantly higher humoral immune response (IgG₁ and IgG) than the biofilm vaccine. However, considering the host response, tissue damage, the clinical severity and colonization of S. aureus in mammary glands, the biofilm vaccine performed better in immunogenicity and protective potential when administered by the intramammary route.     

Modulation of mouse anti-SRBC antibody response by placental extracts

Mouse placental extracts (PE) and corresponding Sephadex G-200 fractions were administered to isogeneic CBA mice along with an optimal immunizing dose of SRBC. Spleen cells were harvested 8 days later and transferred to CBA recipients, subsequently immunized with SRBC. The immunoregulatory activity of spleen cells from PE-treated donors was compared to cells from liver extract (LE)-treated controls or from mice immunized with SRBC only, using Cunningham's PFC direct and indirect tests. Within the dose range used, selective modulatory activities were obtained with cells from PE, but not from LE, treated mice, the latter being comparable to cell transfer effects from donors immunized with SRBC only. Spleen cells from animals injected with low doses of PE (0.25 to 4 mg per mouse) added to immunizing SRBC had a suppressive effect on the primary IgM response of recipients immunized against SRBC. In contrast, when SRBC were given to donor animals with higher doses of PE (8 to 13 mg), transferred spleen cells potentiated the IgM response of the recipients. These opposite suppressive and potentiating activities were found in distinct Sephadex G-200 fractions of 40 and 60 kDa, respectively. When the effect of PE treatment was tested within the same animal, the indirect secondary PFC response following a challenge with SRBC was significantly modified. We observed an overall suppression of the different isotypes after treatment with lower doses of PE or with its 40-kDa fraction. PE doses of 0.5 to 2 mg resulted in a stronger inhibition of IgM than IgG₁ production. This phenomenon was also obtained with the 40 KDa fraction. IgG₂ responses were significantly reduced by all doses of this fraction. In contrast, all doses of the 60-kDa fraction gave a strong stimulation of IgG₂ and IgM responses and a constant suppression of the IgG₁ response. This shows a clear dissociation between IgG₁ and C'-fixing (IgM, IgG₂) antibody classes as far as the influence of placental substances is concerned in their regulation. These data emphasize the relevance of isogeneic placental products as a useful physiological material capable of modulating xenogeneic immune responses (as well as allogeneic systems).     

Dietary omega-3 fatty acids enhance the B1 but not the B2 cell immune response in mice with antigen-induced peritonitis

The effects of omega-3 fatty acids on the adaptive immune response have mainly been analysed in vitro with varying results. How omega-3 fatty acids affect the adaptive immune response in vivo is largely unknown. This study examined the effects of dietary fish oil on the adaptive immune response in antigen-induced inflammation in mice, focusing on its effects on B cells and B cell subsets. Mice were fed a control diet with or without 2.8% fish oil, immunized twice with methylated BSA (mBSA) and peritonitis induced by intraperitoneal injection of mBSA. Serum, spleen and peritoneal exudate were collected prior to and at different time points after induction of peritonitis. Serum levels of mBSA-specific antibodies were determined by ELISA and the number of peritoneal and splenic lymphocytes by flow cytometry. The levels of germinal center B cells and IgM⁺, IgG⁺ and CD138⁺ cells in spleen were evaluated by immunoenzyme staining. Mice fed the fish oil diet had more peritoneal B1 cells, more IgM⁺ cells in spleen and higher levels of serum mBSA-specific IgM antibodies compared with that in mice fed the control diet. However, dietary fish oil did not affect the number of peritoneal B2 cells, splenic IgG⁺ or CD138⁺ cells or serum levels of mBSA-specific IgG antibodies in mice with mBSA-induced peritonitis. These results indicate that dietary fish oil can enhance the adaptive immune response, specifically the B1 cell response, which may lead to better protection against secondary infection as well as improvement in reaching homeostasis following antigenic challenge.     

Induction of suppressor T cells and tolerant B cells in vitro by DNP-IgG

Intravenous injection at proper time of irradiated reticulum cell sarcoma cells into SJL mice immunized with dinitrophenylated (DNP) keyhole limpet hemocyanin inhibits the production of anti-DNP IgG₁ and IgG₂ antibodies.     

In vitro inhibition of the helper activity of GAT-specific T-cell lines by a syngeneic anti-idiotypic serum: preferential effect on the IgG response

We described in this paper the characteristics of a syngeneic anti-idiotypic serum made in BALB/c against BALB/c anti-poly (Glu⁶⁰ Ala³⁰ Tyr¹⁰) (GAT) antibodies. This serum recognizes idiotypic determinants present in all anti-GAT sera whatever the allotypic markers of the mice used to prepare the sera. The functional effect of this serum on two helper cell lines is also described. Cell line BDF₁/52 was obtained from GAT immunized lymph node cells (LNC). Cell line BDF₁/E₃ was selected from splenic T-cells educated in vitro on GAT-pulsed adherent cells. Both lines were propagated in presence of filler cells, antigen, and medium containing T-cell growth factor(s) from splenic cells activated with concanavalin A. Both cell lines exhibit a helper activity as measured by the plaque-forming cell (PFC) response they induce in vitro in the presence of DNP-GAT and DNP sensitized B cells. Their helper activity is specific and they require a hapten-carrier bridge to activate B cells. These lines are able to induce IgG₁, IgG_2a and IgG_2b anti-TNP PFC. Syngeneic anti-idiotypic serum B 658 inhibits specifically the function of these two lines but does not affect the helper activity of an OVA-specific T-cell line. The blocking activity of the serum can be adsorbed on a hybridoma protein with anti-GAT activity. This inhibition affects more dramatically the IgG₁ response than the IgG_2a and IgG_2b responses.     

Bystander suppression of collagen-induced arthritis in mice fed ovalbumin

We wanted to assess whether B-cell and/or T-cell responses to collagen and thereby the course of collagen-induced arthritis could be suppressed by regulatory mechanisms associated with oral tolerance to an unrelated protein. DBA/1 mice were fed ovalbumin (OVA)-containing pellets ad libitum for 1 week and subsequently coimmunized twice, with a mixture of bovine collagen type II (BCII) and OVA in Freund's complete adjuvant. Mice fed OVA before coimmunization with BCII and OVA had significantly lower arthritic scores than mice immunized with BCII only. Their body weight increased during the study period and their anti-BCII antibody activity was significantly IgG_2a lower. The frequency of spleen cells producing IgG anti-BCII antibody was also reduced. Coimmunization per se slightly ameliorated the development of arthritis, resulting in an early, transient reduction. It resulted in significantly higher IgG₁ anti-BCII antibody activity and increased splenocyte secretion of IFN-γ and IL-10 in response to BCII. Our findings demonstrate that OVA-specific regulatory events induced by feeding OVA, i.e. bystander suppression, reduced the severity of arthritis in animals immunized with BCII and OVA. Anti-BCII specific antibody responses and cytokine secretion by types 1 and 2 T helper cells were also decreased.     

Taenia saginata: Production and characterization of monoclonal antibodies against Taenia saginata metacestode antigens

Cysticercosis is a major cause of economic loss in bovine production due to meat condemnation. Chemotherapy is being used in Brazilian cattle and a diagnostic test to improve the treatment program is desired. We produced monoclonal antibodies against crude (TAEB) and cyst fluid (TAEF) Taenia saginata metacestode antigens using immunized BALB/c mice. After cell fusion, 10 TAEB and nine TAEF hybrids were selected and cloned resulting in 18 IgG₁ and 32 IgM TAEB clones, and 9 IgG₁ and 9 IgM TAEF clones. Ascites was produced and Western blot testing was performed resulting in reactivity to protein fractions of low molecular weight (<18 kDa), 43, 55, 66 and 100 kDa. The indirect immunofluorescence test, with one monoclonal antibody against crude and one against cyst fluid antigens, recognized antigenic fractions of both the scolex and the bladder wall of metacestodes from naturally infected bovine.     

2B4 Is Dispensable for T-Dependent B Cell Immune Responses,but Its Deficiency Leads to Enhanced T-Independent Responses Due to an Increase in Peritoneal Cavity B1b Cells

The signaling lymphocyte activation molecule (SLAM) family plays important roles in adaptive immune responses. Herein, we evaluated whether the SLAM family member 2B4 (CD244) plays a role in immune cell development, homeostasis and antibody responses. We found that the splenic cellularity in Cd244 ^-/- mice was significantly reduced due to a reduction in both CD4 T cells and follicular (Fo) B cells; whereas, the number of peritoneal cavity B cells was increased. These findings led us to examine whether 2B4 modulates B cell immune responses. When we examined T-dependent B cell responses, while there was no difference in the kinetics or magnitude of the antigen-specific IgM and IgG₁ antibody response there was a reduction in bone marrow (BM) memory, but not plasma cells in Cd244 ^-/- mice. When we evaluated T-independent immune responses, we found that antigen-specific IgM and IgG₃ were elevated in the serum following immunization. These data indicate that 2B4 dampens T-independent B cell responses due to a reduction in peritoneal cavity B cells, but has minimal impact on T-dependent B cell responses.     

Detection of immunoglobulin heavy chain IgG3 polymorphism in wild mice with xenogeneic monoclonal antibodies

We have generated four xenogeneic rat antimouse IgG₃ monoclonal antibodies recognizing at least three different antigenic determinants (epitopes) on BALB/c IgG₃ molecules. These antibodies were used in solid-phase blocking radioimmunoassays for detection of the epitopes in sera of 40 inbred strains and 134 wild mice. These antibodies detect genetic polymorphism of IgG₃ isotype among wild mice even though there is no polymorphism found among 40 inbred strains tested (except X-chromosome-linked immunodeficient CBA/N strain which lacks IgG₃ molecules). An IgG₃ variant was also isolated from hybridomas derived from Mus spretus.Abbreviations Igh-C immunoglobulin heavy chain constant region - PVC polyvinyl chloride - RIA radioimmune assay - ELISA enzyme linked immunosorbent assay     

Direct and indirect plaque forming cells in extrapulmonary lymphoid tissue following local vs systemic injection of soluble antigen

AKR strain mice were immunized with solubilized SRBC stroma either by direct injection into the lower respiratory tract or intravenously via the tail vein. The number of plaque forming cells (PFC) in the draining plumonary lymph node (tracheobronchial node) and spleen were determined by direct (IgM) and indirect IgG₁, IgG_2b, IgA) plaque assays.Intravenously administered antigen induced an initially strong IgM response in the spleen which was subsequently followed by antibody of the IgG₁, IgG_2b, and IgA classes of immunoglobulins. The tracheobronchial lymph node contained a minimal number PFC representing all four types of immunoglobulins studied. Conversely, following a single local injection of antigen directly into the lower respiratory tract, the tracheobronchial node responded with relatively high concentrations of PFC of all classes. The response in the spleen, although higher than background, was barely detectable. The splenic response to locally administered antigen was, however, considerably augmented as a result of a second local injection given 45 days after the initial stimulation. Under these conditions, IgG₁ IgG_2b, and IgA were represented in both tissue sites by sharp increases in the number and a decrease in the time of appearance of their respective antibody forming cells. Comparable changes were not noted for the case of IgM.Serum hemagglutination titres following a single injection by either route did not vary significantly during the time course of the experiment (28 days). The sera from locally immunized mice were treated with the reducing agent dithiothreitol and hemagglutination titres, before and after treatment, were compared. The major serum activity observed during the first 10 days following injection was affected by reduction and could therefore be assigned to high molecular weight antibody (19S, 13S). Subsequent titres (Days 13–26) were less susceptible to DTT and are considered to represent low molecular weight immunoglobulins (7S).     

Acid-induced aggregation propensity of nivolumab is dependent on the Fc

Nivolumab, an anti-programmed death (PD)1 IgG₄ antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG₁), panitumumab (IgG₂) and atezolizumab (aglyco-IgG₁) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG₁?1?2?4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG₄ backbone.     

Preparation and Efficacy of a Live Newcastle Disease Virus Vaccine Encapsulated in Chitosan Nanoparticles

Background

Newcastle disease (ND) is a highly contagious viral disease of poultry caused by pathogenic strains of the Newcastle disease virus (NDV). Live NDV vaccines are administered by drinking water, eyedrops or coarse aerosol spray. To further enhance mucosal immune responses, chitosan nanoparticles were developed for the mucosal delivery of a live NDV vaccine.

Methodology/Principal Findings

A lentogenic live-virus vaccine (strain LaSota) against NDV encapsulated in chitosan nanoparticles were developed using an ionic crosslinking method. Chitosan nanoparticles containing the lentogenic live-virus vaccine against NDV (NDV-CS-NPs) were produced with good morphology, high stability, a mean diameter of 371.1 nm, an encapsulation rate of 77% and a zeta potential of +2.84 mV. The Western blotting analysis showed that NDV structural proteins were detected in NDV-CS-NPs. The virus release assay results of NDV-CS-NPs indicated that NDV was released from NDV-CS-NPs. Chickens immunized orally or intranasally with NDV-CS-NPs were fully protected whereas one out of five chickens immunized with the LaSota live NDV vaccine and three out of five chickens immunized with the inactivated NDV vaccine were dead after challenge with the highly virulent NDV strain F48E9.

Conclusions/Significance

NDV-CS-NPs induced better protection of immunized specific pathogen free chickens compared to the live NDV vaccine strain LaSota and the inactivated NDV vaccine. This study lays a foundation for the further development of mucosal vaccines and drugs encapsulated in chitosan nanoparticles.     

Genetic control of the immune response of mice to type III pneumococcal polysaccharide

BALBc mice immunized with Type III pneumococcal polysaccharide (SIII) had higher numbers of IgM plaque-forming cells (PFC) in the spleen than similarly immunized C57BL/Ks mice. The F₁ hybrids of these two strains had intermediate numbers of SIII-specific PFC. Analysis of the responses of F₂ and backcross strains indicated that the observed responses were compatible with results expected for control of the immune response to SIII at a single autosomal locus.     

Incorporation of the Tat cell‐penetrating peptide into nanofibers improves the respective antitumor immune response

A major challenge for the development of anticancer vaccines is the induction of a safe and effective immune response, particularly mediated by CD8+ T lymphocytes, in an adjuvant‐free manner. In this respect, we present a simple strategy to improve the specific CD8+ T cell responses using KFE8 nanofibers bearing a Class I (Kb)‐restricted peptide epitope (called E. nanofibers) without the use of adjuvant. We demonstrate that incorporation of Tat, a cell‐penetrating peptide (CPP) of the HIV transactivator protein, into E. nanofibers remarkably enhanced tumor‐specific CD8+ T cell responses. E. nanofibers containing 12.5% Tat peptide (E.Tat_12.5 nanofiber) increased antigen cross‐presentation by bone marrow‐derived dendritic cells as compared with E. nanofibers, or E. nanofibers containing 25 or 50% the Tat peptide. Uptake of KFE8.Tat_12.5 nanofibers by dendritic cells (DCs) was significantly increased compared with KFE8 nanofiber lacking Tat. Peritoneal and lymph node DCs of mice immunized with E.Tat_12.5 nanofibers exhibited increased presentation of the H2kb‐epitope (reminiscent for cross‐presentation) compared with DCs obtained from E. nanofiber vaccinated mice. Tetrameric and intracellular cytokine staining revealed that vaccination with E.Tat_12.5 triggered a robust and specific CD8+ T lymphocyte response, which was more pronounced than in mice vaccinated with E. nanofibers alone. Furthermore, E.Tat_12.5 nanofibers were more potent than E. nanofiber to induce antitumor immune response and tumor‐infiltrating IFN‐γ CD8 T lymphocyte. In terms of cancer vaccine development, we propose that harnessing the nanofiber‐based vaccine platform with incorporated Tat peptide could present a simple and promising strategy to induce highly effective antitumor immune response.