Helicobacter pylori-pulsed dendritic cells induce H. pylori-specific immunity in mice

Background: The growing concern over the emergence of antibiotic‐resistant Helicobacter pylori infection is propelling the development of an efficacious vaccine to control this highly adaptive organism. Aim: We studied the use of a dendritic cell (DC)‐based vaccine against H. pylori infection in mice. Methods: The cellular immune responses to murine bone marrow‐derived DCs pulsed with phosphate‐buffered saline (PBS‐DC) or live H. pylori SS1 (HP‐DC) were assessed in vitro and in vivo. The protective immunity against H. pylori SS1 oral challenge was compared between HP‐DC or PBS‐DC immunized mice. The effect of regulatory T‐cell (Treg) depletion by anti‐CD25 antibody on HP‐DC vaccine efficacy was also evaluated. Results: HP‐DC induced a Th1‐dominant response in vitro. In vivo, HP‐DC immunized mice were characterized by a mixed Th1/Th2 peripheral immune response. However, in the stomach, HP‐DC immunized mice expressed a higher level of IFN‐γ compared to PBS‐DC immunized mice; no difference was found for interleukin‐5 expressions in the stomach. A lower bacterial colonization post‐H. pylori challenge was observed in HP‐DC immunized mice compared to PBS‐DC immunized mice with no significant difference in gastritis severity. H. pylori‐specific Th1 response and protective immunity were further enhanced in vivo by depletion of Treg with anti‐CD25 antibody. Conclusion: DC‐based anti‐H. pylori vaccine induced H. pylori‐specific helper T‐cell responses capable of limiting bacterial colonization. Our data support the critical role of effector cellular immune response in the development of H. pylori vaccine.     

Dendritic cell‐targeting DNA‐based nasal adjuvants for protective mucosal immunity to Streptococcus pneumoniae

To develop safe vaccines for inducing mucosal immunity to major pulmonary bacterial infections, appropriate vaccine antigens (Ags), delivery systems and nontoxic molecular adjuvants must be considered. Such vaccine constructs can induce Ag‐specific immune responses that protect against mucosal infections. In particular, it has been shown that simply mixing the adjuvant with the bacterial Ag is a relatively easy means of constructing adjuvant‐based mucosal vaccine preparations; the resulting vaccines can elicit protective immunity. DNA‐based nasal adjuvants targeting mucosal DCs have been studied in order to induce Ag‐specific mucosal and systemic immune responses that provide essential protection against microbial pathogens that invade mucosal surfaces. In this review, initially a plasmid encoding the cDNA of Flt3 ligand (pFL), a molecule that is a growth factor for DCs, as an effective adjuvant for mucosal immunity to pneumococcal infections, is introduced. Next, the potential of adding unmethylated CpG oligodeoxynucleotide and pFL together with a pneumococcal Ag to induce protection from pneumococcal infections is discussed. Pneumococcal surface protein A has been used as vaccine for restoring mucosal immunity in older persons. Further, our nasal pFL adjuvant system with phosphorylcholine‐keyhole limpet hemocyanin (PC‐KLH) has also been used in pneumococcal vaccine development to induce complete protection from nasal carriage by Streptococcus pneumoniae . Finally, the possibility that anti‐PC antibodies induced by nasal delivery of pFL plus PC‐KLH may play a protective role in prevention of atherogenesis and thus block subsequent development of cardiovascular disease is discussed.

     

Identification and characterization of Cryptococcus neoformans protein fractions that induce protective immune responses

Cryptococcus neoformans, the main causative agent of cryptococcosis, is a fungal pathogen that causes life‐threatening meningoencephalitis in immunocompromised patients. To date, there is no vaccine or immunotherapy approved to treat cryptococcosis. Cell‐ and antibody‐mediated immune responses collaborate to mediate optimal protection against C. neoformans infections. Accordingly, we identified cryptococcal protein fractions capable of stimulating cell‐ and antibody‐mediated immune responses and determined their efficacy to elicit protection against cryptococcosis. Proteins were extracted from C. neoformans and fractionated based on molecular mass. The fractions were then evaluated by immunoblot analysis for reactivity to serum extracted from protectively immunized mice and in cytokine recall assays for their efficacy to induce pro‐inflammatory and Th1‐type cytokine responses associated with protection. MS analysis revealed a number of proteins with roles in stress response, signal transduction, carbohydrate metabolism, amino acid synthesis, and protein synthesis. Immunization with select protein fractions containing immunodominant antigens induced significantly prolonged survival against experimental pulmonary cryptococcosis. Our studies support using the combination of immunological and proteomic approaches to identify proteins that elicit antigen‐specific antibody and Th1‐type cytokine responses. The immunodominant antigens that were discovered represent attractive candidates for the development of novel subunit vaccines for treatment and/or prevention of cryptococcosis.     

Salmonella Typhimurium strain expressing OprF‐OprI protects mice against fatal infection by Pseudomonas aeruginosa

Pseudomonas aeruginosa poses a major threat to human health and to the mink industry. Thus, development of vaccines that elicit robust humoral and cellular immunity against P. aeruginosa is greatly needed. In this study, a recombinant attenuated Salmonella vaccine (RASV) that expresses the outer membrane proteins fusion OprF_190–342‐OprI_21–83 (F1I2) from P. aeruginosa was constructed and the potency of this vaccine candidate assessed by measuring F1I2‐specific humoral immune responses upon vaccination through s.c. or oral routes. S.C. administration achieved higher serum IgG titers and IgA titers in the intestine and induced stronger F1I2‐specific IgG and IgA titers in lung homogenate than did oral administration, which resulted in low IgG titers and no local IgA production. High titers of IFN‐γ, IL‐4, and T‐lymphocyte subsets induced a mixed Th1/Th2 response in mice immunized s.c., indicating elicitation of cellular immunity. Importantly, when immunized mice were challenged with P. aeruginosa by the intranasal route 30 days after the initial immunization, s.c. vaccination achieved 77.78% protection, in contrast to 41.18% via oral administration and 66.67% via Escherichia coli‐expressed F1I2 (His‐F1I2) vaccination. These results indicate that s.c. vaccination provides a better protective response against P. aeruginosa infection than do oral administration and the His‐F1I2 vaccine.     

Increased Humoral Immune Responses of Pigs to Foot‐and‐Mouth Disease Vaccine Supplemented with Ginseng Stem and Leaf Saponins

Vaccination is a conventional approach against foot‐and‐mouth disease (FMD) in pigs. However, failure to elicit an immune response to vaccine has been reported. Our previous investigation showed that ginseng stem and leaf saponins (GSLS) and mineral oil acted synergistically to promote Th1/Th2 immune responses to FMD vaccine in mice. This study was designed to i) find the optimal doses of GSLS in oil‐emulsified FMD vaccines to induce immune responses in mice and pigs and ii) to evaluate the effect of oil‐emulsified FMD vaccine supplemented with GSLS on the immune responses in pigs, by measuring the serum indirect hemagglutination (IHA) titer and IgG and IgG subclass levels. The GSLS‐enhanced immune response to FMD oil‐emulsion vaccine depended on the dose of GSLS added to the vaccine. Addition of GSLS at a dose of 40 μg to 2 ml of FMD oil‐emulsified vaccine significantly enhanced the humoral immune responses in pigs, when compared to the vaccine without GSLS (P<0.05). The increased antibodies included IgG1 and IgG2. Hence, GSLS and oil adjuvant synergistically promoted the immune responses to vaccination against FMD in pigs, and GSLS could be a promising vaccine additive to improve oil‐emulsified veterinary vaccines.     

Mutant presenilin‐1 deregulated peripheral immunity exacerbates Alzheimer‐like pathology

Mutations in the presenilin‐1 (PS1) gene are independent causes of familial Alzheimer's disease (AD). AD patients have dysregulated immunity, and PS1 mutant mice exhibit abnormal systemic immune responses. To test whether immune function abnormality caused by a mutant human PS1 gene (mhPS1) could modify AD‐like pathology, we reconstituted immune systems of AD model mice carrying a mutant human amyloid precursor protein gene (mhAPP; Tg2576 mice) or both mhAPP and mhPS1 genes (PSAPP mice) with allo‐geneic bone marrow cells. Here, we report a marked reduction in amyloid‐β (Aβ) levels, β‐amyloid plaques and brain inflammatory responses in PSAPP mice following strain‐matched wild‐type PS1 bone marrow reconstitution. These effects occurred with immune switching from pro‐inflammatory T helper (Th) 1 to anti‐inflammatory Th2 immune responses in the periphery and in the brain, which likely instructed microglia to phagocytose and clear Aβ in an ex vivo assay. Conversely, Tg2576 mice displayed accelerated AD‐like pathology when reconstituted with mhPS1 bone marrow. These data show that haematopoietic cells bearing the mhPS1 transgene exacerbate AD‐like pathology, suggesting a novel therapeutic strategy for AD based on targeting PS1 in peripheral immune cells.     

Plant‐produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice

The global Zika virus (ZIKV) outbreak and its link to foetal and newborn microcephaly and severe neurological complications in adults call for the urgent development of ZIKV vaccines. In response, we developed a subunit vaccine based on the ZIKV envelope (E) protein and investigated its immunogenicity in mice. Transient expression of ZIKV E (zE) resulted in its rapid accumulation in leaves of Nicotiana benthamiana plants. Biochemical analysis revealed that plant‐produced ZIKV E (PzE) exhibited specific binding to a panel of monoclonal antibodies that recognize various zE conformational epitopes. Furthermore, PzE can be purified to >90% homogeneity with a one‐step Ni²⁺ affinity chromatography process. PzE are found to be highly immunogenic, as two doses of PzE elicited both potent zE‐specific antibody and cellular immune responses in mice. The delivery of PzE with alum induced a mixed Th1/Th2 immune response, as the antigen‐specific IgG isotypes were a mixture of high levels of IgG1/IgG2c and splenocyte cultures from immunized mice secreted significant levels of IFN‐gamma, IL‐4 and IL‐6. Most importantly, the titres of zE‐specific and neutralizing antibodies exceeded the threshold that correlates with protective immunity against multiple strains of ZIKV. Thus, our results demonstrated the feasibility of plant‐produced ZIKV protein antigen as effective, safe and affordable vaccines against ZIKV.     

Improved protective efficacy of a chimeric Staphylococcus aureus vaccine candidate iron‐regulated surface determinant B (N126–P361)‐target of RNAIII activating protein in mice

The pathogen Staphylococcus aureus causes a wide range of serious infections, necessitating urgent development of a vaccine against this organism. However, currently developed vaccines are relatively ineffective because of the limited antigenic component that is contained in the vaccine formulations. To develop an effective S. aureus candidate vaccine, overlapping PCR was used to add the truncated immunodominant antigen iron‐regulated surface determinant B (IsdB)_{(N126–P361)} (tIsdB) to the N‐terminal of intact antigen target of RNAIII activating protein (TRAP) and thus construct a tIsdB‐TRAP chimera. The humoral and cellular immune responses against tIsdB‐TRAP were compared with those against single or combined formulations. tIsdB‐TRAP elicited significantly stronger humoral responses in mice (P < 0.05). As to cellular immune responses in mice, the tIsdB‐TRAP group resulted in a greater IL‐4 response than did other groups (P < 0.05). Greater amounts of IL‐2 and IFN‐γ were found in the tIsdB‐TRAP group. Mouse challenge also showed that tIsdB‐TRAP provided better protection against S. aureus than did the control groups. These results suggest that this chimeric protein may be a promising pathogen target for further vaccine development.     

AFM force spectroscopy reveals how subtle structural differences affect the interaction strength between Candida albicans and DC‐SIGN

The fungus Candida albicans is the most common cause of mycotic infections in immunocompromised hosts. Little is known about the initial interactions between Candida and immune cell receptors, such as the C‐type lectin dendritic cell‐specific intracellular cell adhesion molecule‐3 (ICAM‐3)‐grabbing non‐integrin (DC‐SIGN), because a detailed characterization at the structural level is lacking. DC‐SIGN recognizes specific Candida‐associated molecular patterns, that is, mannan structures present in the cell wall of Candida. The molecular recognition mechanism is however poorly understood. We postulated that small differences in mannan‐branching may result in considerable differences in the binding affinity. Here, we exploit atomic force microscope‐based dynamic force spectroscopy with single Candida cells to gain better insight in the carbohydrate recognition capacity of DC‐SIGN. We demonstrate that slight differences in the N‐mannan structure of Candida, that is, the absence or presence of a phosphomannan side chain, results in differences in the recognition by DC‐SIGN as follows: (i) it contributes to the compliance of the outer cell wall of Candida, and (ii) its presence results in a higher binding energy of 1.6 k_BT. The single‐bond affinity of tetrameric DC‐SIGN for wild‐type C. albicans is ~10.7 k_BT and a dissociation constant k_D of 23 μM, which is relatively strong compared with other carbohydrate–protein interactions described in the literature. In conclusion, this study shows that DC‐SIGN specifically recognizes mannan patterns on C. albicans with high affinity. Knowledge on the binding pocket of DC‐SIGN and its pathogenic ligands will lead to a better understanding of how fungal‐associated carbohydrate structures are recognized by receptors of the immune system and can ultimately contribute to the development of new anti‐fungal drugs. Copyright © 2015 John Wiley & Sons, Ltd.     

Poly(I:C)/Alum Mixed Adjuvant Priming Enhances HBV Subunit Vaccine-Induced Immunity in Mice When Combined with Recombinant Adenoviral-Based HBV Vaccine Boosting

Background

Virus-specific cellular immune responses play a critical role in virus clearance during acute or chronic HBV infection. Currently, the commercially available HBV vaccine is combined with alum adjuvant, which stimulates mainly Th2 immune responses. Therefore, development of new therapeutic HBV vaccine adjuvants and immune strategies that also promote Th1 and CTL responses is urgently needed.

Methodology/Principal findings

To improve the immunity induced by the novel HBSS1 HBV vaccine, we evaluated the ability of adjuvants, including alum, CpG and polyriboinosinic polyribocytidylic acid [poly(I:C)], to enhance the response when boosted with the recombinant adenoviral vector vaccine rAdSS1. The immune responses to different adjuvant combinations were assessed in C57BL/6 mice by enzyme-linked immunosorbent assay (ELISA), ELISpot and cytokine release assays. Among the combinations tested, a HBV protein particle vaccine with CpG/alum and poly(I:C)/alum priming combinations accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres with a Th1 bias. After boosting with recombinant adenoviral vector vaccine rAdSS1, both groups produced a strong multi-antigen (S and PreS1)-specific cellular immune response. HBSS1 immunisation with poly(I:C)/alum priming also generated high-level CD4⁺ and CD8⁺ T cell responses in terms of Th1 cytokines (IFN-γand IL-2).

Conclusions

The protein-vaccine HBSS1 with mixed poly(I:C)/alum adjuvant priming, followed by a rAdSS1 vaccine boost, maximises specific antibody and Th1-biased cellular immune responses. This regime might prove useful in the development of HBV therapeutic vaccines. Furthermore, this promising strategy might be applied to vaccines against other persistent infections, such as human immunodeficiency virus and tuberculosis.     

A chloroplast‐derived Toxoplasma gondii GRA4 antigen used as an oral vaccine protects against toxoplasmosis in mice

The parasitic protozoan Toxoplasma gondii, the causal agent of toxoplasmosis, can infect most mammals and birds. In human medicine, T. gondii can cause complications in pregnant women and immunodeficient individuals, while in veterinary medicine, T. gondii infection has economic importance due to abortion and neonatal loss in livestock. Thus, the development of an effective anti‐Toxoplasma vaccine would be of great value. In this study, we analysed the expression of T. gondii GRA4 antigen by chloroplast transformation (chlGRA4) in tobacco plants and evaluated the humoral and cellular responses and the grade of protection after oral administration of chlGRA4 in a murine model. The Western blot analysis revealed a specific 34‐kDa band mainly present in the insoluble fractions. The chlGRA4 accumulation levels were approximately 6 μg/g of fresh weight (equivalent to 0.2% of total protein). Oral immunization with chlGRA4 resulted in a decrease of 59% in the brain cyst load of mice compared to control mice. ChlGRA4 immunization elicited both a mucosal immune response characterized by the production of specific IgA, and IFN‐γ, IL‐4 and IL‐10 secretion by mesenteric lymph node cells, and a systemic response in terms of GRA4‐specific serum antibodies and secretion of IFN‐γ, IL‐4 and IL‐10 by splenocytes. Our results indicate that oral administration of chlGRA4 promotes the elicitation of both mucosal and systemic balanced Th1/Th2 responses that control Toxoplasma infection, reducing parasite loads.     

Membrane‐bound HSP70‐engineered myeloma cell‐derived exosomes stimulate more efficient CD8+ CTL‐ and NK‐mediated antitumour immunity than exosomes released from heat‐shocked tumour cells expressing cytoplasmic HSP70

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour‐derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T‐cell immune responses. To enhance EXO‐based antitumour immunity, we generated an engineered myeloma cell line J558_HSP expressing endogenous P1A tumour antigen and transgenic form of membrane‐bound HSP70 and heat‐shocked J558_HS expressing cytoplasmic HSP70, and purified EXO_HSP and EXO_HS from J558_HSP and J558_HS tumour cell culture supernatants by ultracentrifugation. We found that EXO_HSP were able to more efficiently stimulate maturation of DCs with up‐regulation of Ia^b, CD40, CD80 and inflammatory cytokines than EXO_HS after overnight incubation of immature bone‐marrow‐derived DCs (5 × 10⁶ cells) with EXO (100 μg), respectively. We also i.v. immunized BALB/c mice with EXO (30 μg/mouse) and assessed P1A‐specific T‐cell responses after immunization. We demonstrate that EXO_HSP are able to stimulate type 1 CD4⁺ helper T (Th1) cell responses, and more efficient P1A‐specific CD8⁺ cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXO_HS. In addition, we further elucidate that EXO_HSP‐stimulated antitumour immunity is mediated by both P1A‐specific CD8⁺ CTL and non‐P1A‐specific natural killer (NK) responses. Therefore, membrane‐bound HSP70‐expressing tumour cell‐released EXO may represent a more effective EXO‐based vaccine in induction of antitumour immunity.     

Enhancing effects of the chemical adjuvant levamisole on the DNA vaccine pVIR‐P12A‐IL18‐3C

DNA‐based vaccination is an attractive alternative for overcoming the disadvantages of inactivated virus vaccines; however, DNA vaccines alone often generate only weak immune responses. In this study, the efficacy of LMS as a chemical adjuvant on a DNA vaccine (pVIR‐P12A‐IL18‐3C) encoding the P1‐2A and 3C genes of the FMDV and swine IL‐18, which provides protection against FMDV challenge, was tested. All test pigs were administered booster vaccinations 28 days after the initial inoculation, and were challenged with 1000 ID₅₀ FMDV O/NY00 20 days after the booster vaccination. Positive and negative control groups were inoculated with inactivated virus vaccine and PBS respectively. The DNA vaccine plus LMS induced greater humoral and cell‐mediated responses than the DNA vaccine alone, as evidenced by higher concentrations of neutralizing and specific anti‐FMDV antibodies, and by higher concentrations of T‐lymphocyte proliferation and IFN‐γ production, respectively. FMDV challenge revealed that the DNA vaccine plus LMS provided higher protection than the DNA vaccine alone. This study demonstrates that LMS may be useful as an adjuvant for improving the protective efficiency of DNA vaccination against FMDV in pigs.     

Platycodin D2 Improves Specific Cellular and Humoral Responses to Hepatitis B Surface Antigen in Mice

Platycodin D2 ( 1 ), a less hemolytic saponin from the root of Platycodon grandiflorum than platycodin D ( 2 ), was evaluated for the potential to enhance specific cellular and humoral immune responses to hepatitis B surface antigen (HBsAg) in mice. It significantly increased the concanavalin A (Con A)‐, lipopolysaccharide (LPS)‐, and HBsAg‐induced splenocyte proliferation in HBsAg‐immunized mice (P<0.05, P<0.01, and P<0.001, resp.). HBsAg‐specific IgG, IgG1, IgG2a, and IgG2b antibody titers in the serum were also markedly enhanced by 1 compared to the HBsAg control group (P<0.01 or P<0.001). Moreover, 1 significantly promoted the production of Th1 (IL‐2 and IFN‐γ) and Th2 (IL‐4 and IL‐10) cytokines from splenocytes in the HBsAg‐immunized mice (P<0.001). The adjuvant potential of 1 on splenocyte proliferation, serum HBsAg‐specific IgG2a and IgG2b antibody response, as well as Th1‐cytokine secretion from splenocytes in the HBsAg‐immunized mice was higher than that of Alum. The results suggest that 1 could improve both cellular and humoral immune responses to HBsAg in mice. Hence, 1 might be a promising adjuvant for hepatitis B vaccine with dual Th1‐ and Th2‐potentiating activity.     

Captive and free‐living red knots Calidris canutus exhibit differences in non‐induced immunity that suggest different immune strategies in different environments

Experiments on captive animals, in which conditions can be controlled, are useful for examining complex biological phenomena such as immune function. Such experiments have increased our understanding of immune responses in the context of trade‐offs and pathogen pressure. However, few studies have examined how captivity itself affects immune function. We used microbial killing, leukocyte concentrations and complement‐natural antibody assays to examine non‐induced (constitutive) immunity in captive and free‐living red knots Calidris canutus. Univariate and multivariate analyses indicated that captive and free‐living birds differed in their immune strategies. Captive birds showed reduced S. aureus killing, C. albicans killing, heterophils, and eosinophils. In a principal component analysis, the affected variables fell onto a single axis, that reflected phagocytosis and inflammation based immunity. We discuss possible reasons for this result in an immune cost and protective benefit framework.     

Plant‐expressed Fc‐fusion protein tetravalent dengue vaccine with inherent adjuvant properties

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly‐immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor‐targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell‐mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen‐specific CD4⁺ and CD8⁺ T‐cell proliferation, IFN‐γ and antibody production. The purified polymeric fraction of dengue PIGS (D‐PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low‐affinity Fcγ receptors on antigen‐presenting cells. These results show that the plant‐expressed D‐PIGS have the potential for translation towards a safe and easily scalable single antigen‐based tetravalent dengue vaccine.