Immunoaffinity-isolated antigens induce protective immunity against larval Strongyloides stercoralis in mice

The objective of this study was to identify soluble protein antigens that would induce protective immunity against infective-stage larvae (L-3) of Strongyloides stercoralis in mice. Deoxycholate (DOC)-soluble proteins derived from L-3, adsorbed to aluminum hydroxide, induced protective immunity in BALB/c mice. The immunized mice generated parasite-specific IgG that could transfer passive immunity to na?ve animals. The protective antibodies bound to parasite antigens found in the muscles and nerve cords of the L-3. An IgG affinity chromatography column generated with IgG from the sera of DOC-immunized mice was used to purify specific larval antigens. Proteins were eluted from the affinity column with sizes of 80, 75, 61, 57, 43, and 32 kDa. This antigen pool stimulated both proliferation and IL-5 production by splenocytes recovered from mice immunized with live L-3. Vaccination of mice with the immunoaffinity-isolated antigens led to significant protective immunity, with 83% of challenge larvae killed. This study demonstrates that IgG-isolated proteins are candidate antigens for a vaccine against larval S. stercoralis.     

Reconstituted membranes of tumour cells (proteoliposomes) induce specific protection to murine lymphoma cells

Summary Antigens presented on cell membranes or on liposomes are usually more immunogenic than antigens in soluble form, this being one of the reasons for the weak immunogenicity of extracted tumour-associated transplantation antigens (TATA). The main objective of this study is to solubilize TATA from tumour cells and to present them on a membrane-like structure to the immune system. Crude tumour cell membranes of SL2 lymphosarcoma cells (a spontaneously arising, weakly immunogenic tumour) were solubilized with octylglucoside or sodium deoxycholate, and reconstituted membranes (proteoliposomes) were prepared by detergent removal. Mice immunized s.c. with reconstituted membranes were protected against an i. p. challenge with tumour cells. Although octylglucoside solubilized only 41% of the membrane proteins, the reconstituted membranes were as immunoprotective as crude membranes. (Glyco)proteins were probably the major membrane components in the reconstituted membranes that induce immunoprotection, as mice immunized with preparations constituted of (glyco)lipids from SL2 cells could not reject SL2 cells. If Freund's complete adjuvant was used with the first immunization injection, no potentiation of the elicited immune responses was observed. Besides the membrane TATA, SL2 cells contained an apparently non-membrane-bound TATA, which was found in the cytoplasm. It is concluded that detergent solubilization of membranes and subsequent preparation of reconstituted membranes can be used to obtain membrane tumour-associated antigens that retain activity for induction of protective tumour immunity. The major advantage of this method is that membrane proteins are solubilized and are subsequently presented on a membrane-like structure that resembles the tumour cell membrane. On theoretical and practical grounds it provides a promising alternative for whole-cell vaccines.     

Dendritic cells pulsed with fungal RNA induce protective immunity to Candida albicans in hematopoietic transplantation

Immature myeloid dendritic cells (DC) phagocytose yeasts and hyphae of the fungus Candida albicans and induce different Th cell responses to the fungus. Ingestion of yeasts activates DC for production of IL-12 and Th1 priming, while ingestion of hyphae induces IL-4 production and Th2 priming. In vivo, generation of antifungal protective immunity is induced upon injection of DC ex vivo pulsed with Candida yeasts but not hyphae. In the present study we sought to determine the functional activity of DC transfected with yeast or hyphal RNA. It was found that DC, from either spleens or bone marrow, transfected with yeast, but not hyphal, RNA 1) express fungal mannoproteins on their surface; 2) undergo functional maturation, as revealed by the up-regulated expression of MHC class II Ags and costimulatory molecules; 3) produce IL-12 but no IL-4; 4) are capable of inducing Th1-dependent antifungal resistance when delivered s.c. in vivo in nontransplanted mice; and 5) provide protection against the fungus in allogeneic bone marrow-transplanted mice, by accelerating the functional recovery of Candida-specific IFN-gamma-producing CD4(+) donor lymphocytes. These results indicate the efficacy of DC pulsed with Candida yeasts or yeast RNA as fungal vaccines and point to the potential use of RNA-transfected DC as anti-infective vaccines in conditions that negate the use of attenuated microorganisms or in the case of poor availability of protective Ags.     

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.     

Japanese encephalitis virus structural and nonstructural proteins expressed in Escherichia coli induce protective immunity in mice

Ectodomain of Japanese encephalitis virus (JEV) E protein [domains I through III (D1–3), domains I and II (D1–2) and domain III (D3)] and the nonstructural protein 1 (NS1) were expressed in Escherichia coli, and administered to BALB/c mice via the intranasal (i.n.) route. The E protein, but not the NS1, induced JEV-specific serum IgG with virus-neutralization capacity in vitro. When mice were lethally challenged with JEV, i.n. immunization with D1–3, D1–2, D3, or a mouse brain-derived formalin-inactivated JE vaccine conferred complete protection, while an 80% protection rate was observed in the NS1 immunized mice. Cytokine analysis of the cervical lymph nodes of mice i.n. immunized with D1–3 or NS1 revealed antigen-specific IL-2 and IL-17 responses, but no IFN-γ T cell response, were observed. This study demonstrates for the first time the i.n. vaccine efficacy of the E. coli-expressed recombinant JEV proteins.     

The generation of tumor-specific in vivo protective immunity in the tumor mass from mice rendered tolerant to tumor antigens

Summary C3H/He mice were inoculated i.v. with 10⁶ heavily X-irradiated syngeneic X5563 plasmacytoma cells 3 times at 4 day intervals. When these mice received an appropriate immunization procedure consisting of i. d. inoculation of viable tumor cells plus the surgical resection of the tumor which enables i.v. nonpresensitized mice to produce anti-X5563 immunity, they failed to develop tumor-specific immunity. This was demonstrated by the abrogation in potential of spleen and lymph node cells to generate in vivo protective immunity. In contrast, the tumor mass from X5563 tumor-bearing mice which had received the i.v. presensitization contained comparable anti-X5563 tumor neutralizing activity to that obtained from the tumor mass from nonpresensitized, X5563 tumor-bearing mice. Such an in vivo protective immunity was revealed to be mediated by tumor-specific T cells. These results demonstrate the differential generation and antitumor capability of tumor infiltrating T cells and T cells in lymphoid organs from mice which are in the tumor-specific tolerant state. The results are discussed in the context of potential utilization of tumor infiltrating in vivo protective T cells to enhance the local tumor-specific immunity in tumor-specific tolerant mice.This work was supported by Special Project Research-Cancer Bioscience from the Ministry of Education, Science and Culture     

Novel recombinant parapoxvirus vectors induce protective humoral and cellular immunity against lethal herpesvirus challenge infection in mice

Orf virus (ORFV; Parapoxvirus ovis) was used to develop a novel vector system for the generation of effective and safe live vaccines. Based on the attenuated ORFV strain D1701-V, recombinants were produced that express the glycoproteins gC (D1701-VrVgC) or gD (D1701-VrVgD) of the alphaherpesvirus of swine, pseudorabies virus (PRV). Expression of gC and gD was also demonstrated on the surface of recombinant virus-infected murine cells that do not produce infectious ORFV. Single or combined immunization with the ORFV recombinants protected different mouse strains of a host species nonpermissive for ORFV against a fulminant, lethal PRV challenge infection equal to immunization with PRV live vaccine. Most notably, even a single immunization with D1701-VrVgC was protective, whereas two applications of D1701-VrVgD were required for immune protection. The higher protective capacity of D1701-VrVgC correlated with the induction of a strong specific humoral immune response. This suggestion was supported by transfer experiments using sera from recombinant-immunized mice, which resulted in partial gC but not gD antibody-mediated protection of the na?ve recipients. Remarkably, immunization of different immune-deficient mice demonstrated that the application of the PRV gC-expressing recombinant controlled the challenge infection in the absence of either CD4(+) or CD8(+) T cells, B cells, or an intact perforin pathway. In contrast, D1701-VrVgD-immunized mice lacking CD4(+) T cells exhibited reduced protection, whereas animals lacking CD8(+) T cells, B cells, or perforin resisted the challenge infection. The present study demonstrates the potential of these new vector vaccines to efficiently prime both protective humoral and cell-mediated immune mechanisms in a host species nonpermissive for the vector virus.     

Divergent envelope E2 alphavirus sequences spanning amino acids 297 to 352 induce in mice virus-specific protective immunity and antibodies with complement-mediated cytolytic activity.

We have proposed a general algorithm for identification of potential immunoprotective domains (cassettes) on the envelope E2 polypeptide of alphaviruses (H. Grosfeld, B. Velan, M. Leitner, S. Cohen, S. Lustig, B.E. Lachmi, and A. Shafferman, J. Virol. 63:3416-3422, 1989). To assess the generality of our approach, we compared analogous E2 cassettes from Sindbis virus (SIN) and Semliki Forest virus (SFV), two alphaviruses which are philogenetically very remote. The antigenically distinct SFV E2 and SIN E2 cassettes exhibit comparable immunological characteristics. Most significantly, the SIN E2 LMN cassette cluster (E2 amino acids 297 to 352 fused to beta-galactosidase), like the analogous SFV E2 LMN cassettes, elicited high titers of antivirus antibodies in mice and proved to be highly effective in protection against lethal challenge. Mice immunized with SIN E2 LMN were completely protected against intracerebral challenge of 10 to 100 50% lethal doses of different neurovirulent SIN strains. Anti-SIN LMN antibodies, like anti-SFV LMN antibodies, lacked in vitro neutralizing activity, yet both exerted protection against homologous challenge upon transfer to mice. The two antibody preparations exhibited virus-specific complement-mediated cytolysis of cells infected with the homologous but not heterologous virus. These results suggest a possible mechanism for virus-specific E2 LMN-induced protection and demonstrate the generality of our methodology for deciphering immunogenic and protective domains in alphavirus systems. Results suggest also that the E2 LMN sequence of any given alphavirus should be considered as a component of a synthetic vaccine against that specific virus.     

Immunogenicity is unrelated to protective immunity when induced by soluble and particulate antigens from Nocardia brasiliensis in BALB/c mice

Cell-mediated immunity plays a major role in protection against intracellular microbes. Nocardia brasiliensis is a facultative intracellular pathogen that causes chronic actinomycetoma. In this work, we injected BALB/c mice with soluble P24 and particulate antigens from N. brasiliensis. A higher antibody titer and lymphocyte proliferation was induced by the particulate antigen than by the soluble antigen. However, five months after antigen injection, antibody concentration and lymphocyte proliferation were similar. An increase in CD45R and CD4 T cells was unrelated to protective immunity. Active immunization with soluble or particulate antigens induced complete protection during the primary immune response. This protective response was IgM mediated. The higher immunogenicity was not related to protective immunity since the particulate antigen induced protection similar to the soluble antigen. Using particulate antigens for vaccination guarantees a stronger immune response, local and systemic side effects, but not necessarily protection.     

Treml4, an Ig superfamily member, mediates presentation of several antigens to T cells in vivo, including protective immunity to HER2 protein

Members of the triggering expressed on myeloid cells (Trem) receptor family fine-tune inflammatory responses. We previously identified one of these receptors, called Treml4, expressed mainly in the spleen, as well as at high levels by CD8α(+) dendritic cells and macrophages. Like other Trem family members, Treml4 has an Ig-like extracellular domain and a short cytoplasmic tail that associates with the adaptor DAP12. To follow up on our initial results that Treml4-Fc fusion proteins bind necrotic cells, we generated a knockout mouse to assess the role of Treml4 in the uptake and presentation of dying cells in vivo. Loss of Treml4 expression did not impair uptake of dying cells by CD8α(+) dendritic cells or cross-presentation of cell-associated Ag to CD8(+) T cells, suggesting overlapping function between Treml4 and other receptors in vivo. To further investigate Treml4 function, we took advantage of a newly generated mAb against Treml4 and engineered its H chain to express three different Ags (i.e., OVA, HIV GAGp24, and the extracellular domain of the breast cancer protein HER2). OVA directed to Treml4 was efficiently presented to CD8(+) and CD4(+) T cells in vivo. Anti-Treml4-GAGp24 mAbs, given along with a maturation stimulus, induced Th1 Ag-specific responses that were not observed in Treml4 knockout mice. Also, HER2 targeting using anti-Treml4 mAbs elicited combined CD4(+) and CD8(+) T cell immunity, and both T cells participated in resistance to a transplantable tumor. Therefore, Treml4 participates in Ag presentation in vivo, and targeting Ags with anti-Treml4 Abs enhances immunization of otherwise naive mice.     

Antigen delivery to plasmacytoid dendritic cells via BST2 induces protective T cell-mediated immunity

Plasmacytoid dendritic cells (PDCs) are capable of presenting Ags to T cells in a tolerogenic or immunogenic manner depending on the formulation of the Ag and the mode of stimulation. It has not been investigated whether effective adaptive immune responses useful for vaccination can be induced by Ab-mediated Ag targeting to PDCs in vivo. In this study, we show that Ag delivered to murine PDCs via bone marrow stromal cell Ag 2 (BST2)/CD317 in combination with TLR agonists as adjuvants is specifically presented by PDCs in vivo and elicits strong cellular and humoral immune responses. These include IFN-γ production by CD4(+) T cells and high Ab titers with a broad range of IgG isotypes. In addition, BST2-mediated Ag delivery in the presence of polyinosinic-polycytidylic acid as adjuvant induces cytotoxic T lymphocytes that are functional in vivo. A single immunization with Ag-fused anti-BST2 Ab together with polyinosinic-polycytidylic acid as adjuvant is sufficient to trigger protective immunity against subsequent viral infection and tumor growth. We conclude that despite the potential tolerogenic properties of PDCs, Ag targeting to PDCs in combination with TLR agonists as adjuvants is an effective vaccination strategy.     

Critical role of preconceptional immunization for protective and nonpathological specific immunity in murine neonates

Expression of Th2 immunity against environmental Ags is the hallmark of the allergic phenotype and contrasts with the Th1-like pattern, which is stably expressed in healthy adults throughout life. Epidemiological studies indicate that the prenatal environment plays an important and decisive role in the development of allergy later in life. Since the underlying mechanisms were unclear, an animal model was developed to study the impact of maternal allergy on the development of an allergic immune response in early life. An allergic Th2 response was induced in pregnant mice by sensitization and aerosol allergen exposure. Both, IgG1 and IgG2a, but not IgE, Abs cross the placental barrier. Free allergen also crosses the placental area and was detected in serum and amniotic fluids of neonatal F(1) mice. These F(1) mice demonstrated a suppressed Th1 response, as reflected by lowered frequencies and reduced levels of IFN-gamma production. Development of an IgE response against the same allergen was completely prevented early in life. This effect was mediated by diaplacental transfer of allergen-specific IgG1 Abs. In contrast, allergic sensitization against a different allergen early in life was accelerated in these mice. This effect was mediated by maternal CD4 and OVA-specific Th2 cells induced by allergic sensitization during pregnancy. These data indicate a critical role for maternal T and B cell response in shaping pre- and postnatal maturation of specific immunity to allergens.     

Toxoplasma gondii: DNA vaccination with bradyzoite antigens induces protective immunity in mice against oral infection with parasite cysts

Infection of the host by Toxoplasma gondii leads to an acute systemic dissemination of tachyzoites, followed by a chronic phase, in which bradyzoites, enclosed in cysts, persist in the brain, the heart, and other tissues. Among putative vaccine candidates, the bradyzoite antigens BAG1 and MAG1 look promising since they are preferentially expressed during the chronic stage of the parasite. This work focused on studying the immunogenicity of bradyzoite antigens in a mouse model of chronic toxoplasmosis. A mixture of plasmids directing the cytoplasmic expression of MAG1 and BAG1 in mammalian cells was used to immunize mice. We show here that immunized mice developed, preferentially, specific anti-MAG1 and anti-BAG1 IgG2a subclass antibodies, indicating a shift towards a Th1-like response after DNA immunization. We then demonstrated that DNA immunization followed by challenge infection elicited effective protection in mice, suggesting that bradyzoite antigens should be considered in the design of vaccines against toxoplasmosis.     

Studies on the transfer of protective immunity with lymphoid cells from mice immune to malaria sporozoites.

In an effort to understand the mechanisms involved in the protective immunity to malarial sporozoites, an A/J mouse/Plasmodium berghei model was studied. Protective immunity could consistently be adoptively transferred only by using sublethal irradiation of recipients (500 R); a spleen equivalent (100 X 10(6))of donor cells from immune syngeneic mice; and a small booster immunization (1 X 10(4)) of recipients with irradiation-attenuated sporozoites. Recipient animals treated in this manner were protected from lethal challenge with 1 X 10(4) nonattenuated sporozoites. Immune and nonimmune serum and spleen cells from nonimmune animals did not protect recipient mice. Fewer immune spleen cells (50 X 10(6)) protected some recipients. In vitro treatment of immune spleen cells with anti-theta sera and complement abolished their ability to transfer protection. This preliminary study suggests that protective sporozoite immunity can be transferred with cells, and that it is T cell dependent.     

Polyclonal rabbit anti-murine plasmacytoma cell globulins induce myeloma cells apoptosis and inhibit tumour growth in mice

Multiple myelomas (MMs) are etiologically heterogeneous and there are limited treatment options; indeed, current monoclonal antibody therapies have had limited success, so more effective antibodies are urgently needed. Polyclonal antibodies are a possible alternative because they target multiple antigens simultaneously. In this study, we produced polyclonal rabbit anti-murine plasmacytoma cell immunoglobulin (PAb) by immunizing rabbits with the murine plasmacytoma cell line MPC-11. The isolated PAb bound to plasma surface antigens in several MM cell lines, inhibited their proliferation as revealed by MTT assay, and induce apoptosis as indicated by flow cytometry, microscopic observation of apoptotic changes in morphology, and DNA fragmentation on agarose gels. The cytotoxicity of PAb on MPC-11 cell lines was both dose-dependent and time-dependent; PAb exerted a 50% inhibitory effect on MPC-11 cell viability at a concentration of 200?μg/ml in 48?h. Flow cytometry demonstrated that PAb treatment significantly increased the number of apoptotic cells (48.1%) compared with control IgG (8.3%). Apoptosis triggered by PAb was confirmed by activation of caspase-3, -8, and -9. Serial intravenous or intraperitoneal injections of PAb inhibited tumour growth and prolonged survival in mice bearing murine plasmacytoma, while TUNEL assay demonstrated that PAb induced statistically significant apoptosis (P?相似文献   

Concomitant and protective immunity in mice exposed to repeated infections with Echinostoma malayanum

Concomitant immunity and its consequence against infection play roles in regulating worm burdens in helminthiasis. Under natural conditions, this immunity is generated by exposure to repeated low dose or trickle infection. In this study, concomitant immunity was induced in mice exposed repeatedly to infection with Echinostoma malayanum and its protective effect on a challenge infection evaluated. A profile of worm burden from exposure to 10 metacercariae/mouse/week rose rapidly during the first 2 weeks reaching a plateau from week 3 to 8 post infection. Based on a cumulative dose of infection, worm recoveries were around 75% in the first 2 weeks, dropped to 50% at week 3 and 19% at week 8. After week 2, adult worm burden was constant and no juvenile worms were found after week 3 of the experiment. To examine the effect of resistance against reinfection, mice in the experimental group were primarily infected with 10 metacercariae/week for 5 weeks, treated with praziquantel and were challenged with 75 metacercariae/animal. The number of worms recovered from the experimental groups was significantly lower than that from naïve control groups beginning from 24 h to 28 days post challenge. The worms in the experimental group showed growth retardation and the proportion of adult worms was lower than that in the control animals especially during the first 3 weeks of the experiment. Parasite fecundity was also suppressed compared with that in the control group. The selective effects of protective immunity on establishment, growth, and fecundity of challenged worms affected the population dynamics of E. malayanum which is a similar phenomenon to concomitant immunity in schistosomiasis.     

Effect of UV-irradiation, spleen cells and MLN cells on protective immunity against Angiostrongylus cantonensis infection in mice

The UV-irradiated larvae of Angiostrongylus cantonensis were used for inducing immunity in mice. A single oral dose of 100 infective UV-irradiated larvae exposed for 5 min or 15 min induced 91% and 97% protection against subsequent infection. Adoptive protection against A. cantonensis could also be transferred by spleen and mesenteric lymph node cells obtained from vaccinated mice.     

Th17 cells and IL-17 in protective immunity to vaginal candidiasis

Background

Th17 cells play a major role in coordinating the host defence in oropharyngeal candidiasis. In this study we investigated the involvement of the Th17 response in an animal model of vulvovaginal candidiasis (VVC).

Methods

To monitor the course of infection we exploited a new in vivo imaging technique.

Results

i) The progression of VVC leads to a strong influx of neutrophils in the vagina soon after the challenge which persisted despite the resolution of infection; ii) IL-17, produced by vaginal cells, particularly CD4 T cells, was detected in the vaginal wash during the infection, reaching a maximum 14 days after the challenge; iii) The amount and kinetics of IL-23 in vaginal fluids were comparable to those in vaginal cells; iv) The inhibition of Th17 differentiation led to significant inhibition of IL-17 production with consequent exacerbation of infection; v) An increased production of βdefensin 2 was manifested in cells of infected mice. This production was strongly reduced when Th17 differentiation was inhibited and was increased by rIL-17 treatment.

Conclusions

These results imply that IL-17 and Th17, along with innate antimicrobial factors, have a role in the immune response to vaginal candidiasis.     

Induction of protective immunity by primed B-1 cells in Toxoplasma gondii -infected B cell-deficient mice

We examined the role of B‐1 cells in protection against Toxoplasma gondii infection using B cell‐deficient mice (μMT mice). We found that primed but not naïve B‐1 cells from wild‐type C57BL/6 mice protected B cell‐deficient recipients from challenge infection. All μMT mice transferred with primed B‐1 cells survived more than 5 months after T. gondii infection, whereas 100% of μMT mice transferred with naïve B‐1 cells succumbed by 18 days after infection. Additionally, high expression of both T help (Th) 1‐ and Th2‐type cytokines and a high level of nitric oxide production were observed in T. gondii‐infected μMT mice transferred with primed B‐1 cells. Thus, it was clearly demonstrated that B‐1 cells play an important role in host protection against T. gondii infection in μMT mice.