DNA-plasmids of HIV-1 induce systemic and mucosal immune responses

DNA-based immunization has been shown to induce protective immunity against several microbial pathogens including HIV-1. Several routes of DNA vaccination have been exploited. However, the properties of the immune responses seem to differ with the different routes used for DNA delivery, ultimately affecting the outcome of experimental challenge. We measured the primary immune response following one vaccination. This report presents differences associated with three different DNA delivery routes: intramuscular injection, intranasal application, and gene-gun based immunization. Induction of systemic humoral immune responses was achieved most efficiently by either intranasal or gene-gun mediated immunization, followed by intramuscular injection. Mucosal IgA was reproducibly induced by intranasal instillation of the DNA, and found in lung washings, faeces, and vaginal washings. Cytotoxic T cells were not induced by a single immunization, but were observed after three immunizations using intramuscular injections.     

Identification of a peptide enhancing mucosal and systemic immune responses against EGFP after oral administration in mice

Gangliosides are receptors for various peptides and proteins including neuropeptides, beta-amyloid proteins, and prions. Recently, the role of gangliosides in mucosal immunization has attracted attention due to the emerging interest in oral vaccination. Ganglioside GM1 exists in abundance on the surface of the M cells of Peyer's patch, a well-known mucosal immunity induction site. In the present study we identified a peptide ligand for GM1 and tested whether it played a role in immune induction. GM1-binding peptides were selected from a phage-displayed dodecapeptide library and one peptide motif, GWKERLSSWNRF, was fused to the C-terminus of enhanced green fluorescent protein (EGFP). The fusion protein, but not EGFP fused with a control peptide, was concentrated around Peyer's patch after incubation in the lumen of the intestine ex vivo. Furthermore, oral feeding of the fusion protein but not control EGFP induced mucosal and systemic immune responses against EGFP resembling Th2-type immune responses.     

Dendritic cells in innate immune responses against HIV

Dendritic cells (DCs) were recently found to be innate immunity effectors against tumoral cells and viruses. (i) In response to most viruses, including HIV, plasmacytoid DCs are responsible for most of the type I IFN secretion, which is strongly anti-viral and induces TH1 type responses. Myeloid DCs secrete IL-12, which is also important for TH1-type and cytotoxic responses. In HIV patient blood, both DC population numbers decrease as early as the primary stage. Plasmacytoid DC numbers correlate with type I IFN secretion, which is a prognosis predictor, particularly under treatment. IL-12 secretion is also defective. Immunotherapies to replace the defective cytokines or to restore a potentially defective DC-T lymphocyte feed-back might help patients restore their immune responses under antiviral therapy. (ii) After measles and other viral infections, or incubation with dsRNA, DCs become cytotoxic and consequently exhibit natural killer function, through upregulation of type I IFN secretion which enhances TRAIL expression. In HIV infection, this mechanism was not demonstrated yet, but it might a) be responsible for the massive apoptosis of uninfected lymphocytes, and b) increase specific immunity through cross-presentation of antigens from infected cells killed by DCs. (iii) DCs direct expansion and effector functions of NK cells in the absence of adaptive-type cytokines and modulate NKT cell IFN-gamma production. Reciprocally, NK activation triggers DC maturation. HIV-1 Tat inhibits NK cell cytotoxicity directly and probably through inhibition of IL-12 secretion by DC. Therefore, understanding the functions of DCs in innate immune responses and in pathogenesis will help obtain better HIV replication control.     

Secretory IgA possesses intrinsic modulatory properties stimulating mucosal and systemic immune responses

Secretory IgA (SIgA) is essential in protecting mucosal surfaces by ensuring immune exclusion. In addition, SIgA binds selectively to M cells in Peyer's patches (PP), resulting in transport across the epithelium and targeting of dendritic cells (DC) in the dome region. The immunological consequences of such an interaction are unknown. In this study, we find that oral delivery of SIgA comprising human secretory component and mouse IgA induces human secretory component-specific Ab and cellular responses in mucosal and peripheral tissues in mice. This takes place in the absence of co-addition of cholera toxin, identifying so far unraveled properties in SIgA. Specific immune responses are accompanied by sustained IL-10 and TGF-beta expression in draining mesenteric lymph nodes and spleen. SIgA also triggers migration of DC to the T cell-rich regions of PP, and regulates expression of CD80 and CD86 on DC in PP, mesenteric lymph nodes, and spleen. These results provide evidence that mucosal SIgA re-entering the body exerts a function of Ag delivery that contributes to effector and/or regulatory pathways characteristic of the intestinal mucosal compartment.     

Rectal immunization with rotavirus virus-like particles induces systemic and mucosal humoral immune responses and protects mice against rotavirus infection

To evaluate whether the rectal route of immunization may be used to provide appropriate protection against enteric pathogens such as rotaviruses (RV), we studied the antibody response and the protection induced by rectal immunization of mice with RV virus-like particles (VLP). For this purpose, 6-week-old BALBc mice were rectally immunized twice with RV 8-2/6/7-VLP derived from the bovine RV RF81 strain either alone or combined with various adjuvants including four toxins [cholera toxin (CT) and three attenuated Escherichia coli-derived heat-labile toxins (LTs), LT(R192G), LT(R72), and LT(K63)] and two Toll-like receptor-targeting adjuvants (CpG and resiquimod). Six weeks after the second immunization, mice were challenged with murine RV strain ECw. RV VLP administered alone were not immunogenic and did not protect mice against RV challenge. By contrast, RV VLP combined with any of the toxin adjuvants were immunogenic (mice developed significant titers of anti-RV immunoglobulin A [IgA] in both serum and feces and of anti-RV IgG in serum) and either efficiently induced complete protection of the mice (no detectable fecal virus shedding) or, for LT(K63), reduced the amount of fecal virus shedding after RV challenge. When combined with RV VLP, CpG and resiquimod failed to achieve protection, although CpG efficiently induced an antibody response to RV. These results support the consideration of the rectal route for the development of new immunization strategies against RV infection. Rectal delivery of a VLP-based vaccine might allow the use of adjuvants less toxic than, but as efficient as, CT.     

Novel AAV-based genetic vaccines encoding truncated dengue virus envelope proteins elicit humoral immune responses in mice

The envelope protein of dengue virus is involved in host cell attachment for entry and induction of protective immunity. Current efforts are focused on producing a tetravalent vaccine by mixing four monovalent vaccine components. In this work, we developed a genetic vaccine based on a novel adeno-associated viral (AAV) vector expressing the carboxy-terminal truncated envelope protein (79E) of dengue virus. The expression of the recombinant 79E protein in HEK 293 cells was confirmed by Western blot. Vectors packaged with novel AAV capsids (AAV2/8 or AAV2/rh32.33) were injected into C57BL/6 mice intramuscularly. Dengue virus antigen was produced in the mice and induced long-lasting antibody responses against the dengue virus still detectable 20 weeks after immunization. AAV2/8 vaccine induced higher anti-dengue virus antibody levels than AAV2/rh32.33 vaccine or AAV plasmid. Furthermore, the anti-dengue antibodies could neutralize homogeneous dengue virus. These results demonstrated that the AAV vaccines possessed appropriate immunogenicity and could be used for the development of an effective dengue vaccine.     

Genetic immunization with lung-targeting macroaggregated polyethyleneimine-albumin conjugates elicits combined systemic and mucosal immune responses

Genetic immunization is a novel form of vaccination in which transgenes are delivered into the host to produce the foreign protein within host cells. Although systemic immune responses have been relatively easy to induce by genetic immunization, the induction of regional and mucosal immunity has often been more challenging. To address the problem of eliciting mucosal immunity in the lung, we utilized macroaggregated albumin to target plasmid DNA to the lung. Macroaggregated albumin is trapped in the lung after i. v. injection, and it is routinely used in radiolabeled form as an imaging modality to evaluate pulmonary blood flow. To couple DNA to this targeting agent, polyethyleneimine (a polycation that binds DNA and enhances transfection) was conjugated to serum albumin, and the conjugate was aggregated by heating to produce particles of 25-100 microm. The resulting particles bound plasmid DNA avidly, and when injected i.v. in mice, the particles distributed in the peripheral lung tissue in the alveolar interstitium. Particle-bound luciferase plasmid transfected a variety of cell lines in vitro, and after i.v. injection, gene expression was detected exclusively in the lung. Using human growth hormone as the encoded foreign Ag for immunization, i.v. injection of the particle-bound plasmid elicited both pulmonary mucosal and systemic immune responses, whereas naked DNA injected either i.v. or i.m. elicited only systemic responses. Thus, particle-bound plasmid DNA may have utility for genetic immunization by intravascular delivery to the lung and potentially to other organs and tissues.     

Sulfated fucan oligosaccharides elicit defense responses in tobacco and local and systemic resistance against tobacco mosaic virus

Sulfated fucans are common structural components of the cell walls of marine brown algae. Using a fucan-degrading hydrolase isolated from a marine bacterium, we prepared sulfated fucan oligosaccharides made of mono- and disulfated fucose units alternatively bound by alpha-1,4 and alpha-1,3 glycosidic linkages, respectively. Here, we report on the elicitor activity of such fucan oligosaccharide preparations in tobacco. In suspension cell cultures, oligofucans at the dose of 200 microg ml(-1) rapidly induced a marked alkalinization of the extracellular medium and the release of hydrogen peroxide. This was followed within a few hours by a strong stimulation of phenylalanine ammonia-lyase and lipoxygenase activities. Tobacco leaves treated with oligofucans locally accumulated salicylic acid (SA) and the phytoalexin scopoletin and expressed several pathogenesis-related (PR) proteins, but they displayed no symptoms of cell death. Fucan oligosaccharides also induced the systemic accumulation of SA and the acidic PR protein PR-1, two markers of systemic acquired resistance (SAR). Consistently, fucan oligosaccharides strongly stimulated both local and systemic resistance to tobacco mosaic virus (TMV). The use of transgenic plants unable to accumulate SA indicated that, as in the SAR primed by TMV, SA is required for the establishment of oligofucan-induced resistance.     

Increasing M2 epitope density enhances systemic and mucosal immune responses to influenza A virus

Recombinant proteins bearing one (sM2) or three (3sM2) copies of M2 epitope from influenza virus were expressed in Escherichia coli. Mice were administrated with these two proteins and systemic and mucosal immune responses were analyzed. Compared with sM2, 3sM2 induced M2-specific serum IgG and mucosal IgA antibodies with neutralizing activity more efficiently in the whole immune course and the later mucosal immunization improved this advantage. MDCK cells pretreated with 3sM2 antisera showed less pathogenic morphological changes compared with that of pretreated with sM2. Together, our results demonstrated that high epitope density in one recombinant protein can enhance humoral immune responses in both systemic and mucosal immunization, and combination of systemic and mucosal immunization enhances this advantage.     

Effects of intestinal survival surgery on systemic and mucosal immune responses in SIV-infected rhesus macaques

Abstract: Evaluation of cellular immunity in the intestinal lamina propria of rhesus macaques has been used previously to assess protective immunity against mucosal simian immunodeficiency virus (SIV) challenges. As this technique requires survival surgery to obtain jejunal tissue, effects of surgical stress on the immune system were investigated. SIV-specific immune responses, including IgG and IgA binding antibodies in sera and mucosal secretions, IgG and IgA secreting cells in peripheral blood, IgG neutralizing antibodies, T-cell proliferative responses, and interferon-γ secretion by peripheral blood mononuclear cells, were evaluated pre- and post-surgery in macaques immunized with adenovirus-SIV recombinant vaccines and SIV envelope protein and in SIV-infected macaques. No differences in these immune parameters were observed in SIV-naïve, immunized macaques or healthy SIV-infected macaques with regard to surgery. A dramatic increase in total IgA antibody level following surgery in the rectal secretions of one SIV-infected macaque that was rapidly progressing to AIDS and failed to recover from surgery was attributed to an abscess that developed at the intestinal site. To date, nearly 30 other macaques have undergone the intestinal survival surgery, some on more than one occasion, without experiencing any clinical difficulty. Overall, our results suggest that in healthy macaques, intestinal resection survival surgery can be conducted safely. Further, the method can be used to reliably sample the intestinal mucosa without major or persistent impact on humoral or cellular immune responses.     

Superoxide dismutase A antigens derived from molecular analysis of sarcoidosis granulomas elicit systemic Th-1 immune responses

Background

Sarcoidosis is an idiopathic granulomatous disease with pathologic and immunologic features similar to tuberculosis. Routine histologic staining and culture fail to identify infectious agents. An alternative means for investigating a role of infectious agents in human pathogenesis involves molecular analysis of pathologic tissues for microbial nucleic acids, as well as recognition of microbial antigens by the host immune system. Molecular analysis for superoxide dismutase A (sodA) allows speciation of mycobacteria. SodA is an abundantly secreted virulence factor that generates cellular immune responses in infected hosts. The purpose of this study is to investigate if target antigens of the sarcoidosis immune response can be identified by molecular analysis of sarcoidosis granulomas.

Methods

We detected sodA amplicons in 12 of 17 sarcoidosis specimens, compared to 2 of 16 controls (p = 0.001, two-tailed Fisher''s exact test), and 3 of 3 tuberculosis specimens (p = 0.54). Analysis of the amplicons revealed sequences identical to M. tuberculosis (MTB) complex, as well as sequences which were genetically divergent. Using peripheral blood mononuclear cells (PBMC) from 12 of the 17 sarcoidosis subjects, we performed enzyme-linked immunospot assay (ELISPOT) to assess for immune recognition of MTB sodA peptides, along with PBMC from 26 PPD- healthy volunteers, and 11 latent tuberculosis subjects.

Results

Six of 12 sarcoidosis subjects recognized the sodA peptides, compared to one of 26 PPD- controls (p = 0.002), and 6/11 PPD+ subjects (p = .68). Overall, 10 of the 12 sarcoidosis subjects from whom we obtained PBMC and archival tissue possessed molecular or immunologic evidence for sodA.

Conclusion

Dual molecular and immunologic analysis increases the ability to find infectious antigens. The detection of Th-1 immune responses to sodA peptides derived from molecular analysis of sarcoidosis granulomas reveals that these are among the target antigens contributing to sarcoidosis granulomatous inflammation.     

The immune responses to bacterial antigens encountered in vivo at mucosal surfaces

Mammals have evolved a sophisticated immune system for handling antigens encountered at their mucosal surfaces. The way in which mucosally delivered antigens are handled influences our ability to design effective mucosal vaccines. Live attenuated derivatives of pathogens are one route towards the development of mucosal vaccines. However, some molecules, described as mucosal immunogens, are inherently immunogenic at mucosal surfaces. Studies on mucosal immunogens may facilitate the identification of common characteristics that contribute to mucosal immunogenicity and aid the development of novel, non-living mucosal vaccines and immunostimulators.     

滴鼻免疫有效的诱导粘膜及系统免疫反应

为了探讨粘膜疫苗滴鼻免疫对小鼠不同粘膜部位和系统免疫部位的影响,将BALB/c小鼠随机分为三组,每组15只,FSM-2117或FS-5416 4×107cfu/只经滴鼻途径免疫小鼠,间隔两周,4次免后7天活杀,收集鼻咽、肺、肠、生殖道冲洗液和血清,ELISA法检测其中特异性抗福氏、宋内氏LPSIg和AIgG;分离NALT、鼻通道、脾、小肠PP淋巴细胞,细胞中加入全菌破碎抗原共培养,于不同时间取出细胞,提取RNA做RT-PCR.两株菌苗经鼻内免疫均诱发了鼻咽、肺、胃肠道和生殖道等不同粘膜部位及血清中特异性抗福氏、宋内氏LPSIgA、IgG的显著增加(P＜0.01);免疫动物的NALT、NP、PP结淋巴细胞在体外经抗原刺激后均在不同时间出现IFN-γ和IL-4mRNA的表达,而TGF-β mRNA的表达在免疫前后无明显变化.疫苗经鼻粘膜免疫可诱导不同粘膜部位及系统免疫反应的发生,鼻粘膜是一个安全有效的免疫途径.     

Salmonella vaccines secreting measles virus epitopes induce protective immune responses against measles virus encephalitis

In the present study we describe a live vaccine against measles virus (MV) infection on the basis of attenuated Salmonella typhimurium aroA secreting MV antigens via the Escherichia coli alpha-hemolysin secretion system. Two well-characterized MV epitopes, a B-cell epitope of the MV fusion protein (amino acids 404-414) and a T-cell epitope of the MV nucleocapsid protein (amino acids 79-99) were fused as single or repeating units to the C-terminal secretion signal of the E. coli hemolysin and expressed in secreted form by the attenuated S. typhimurium aroA SL7207. Immunization of MV-susceptible C3H mice revealed that S. typhimurium SL7207 secreting these antigens provoked a humoral and a cellular MV-specific immune response, respectively. Mice vaccinated orally with a combination of both recombinant S. typhimurium strains showed partial protection against a lethal MV encephalitis after intracerebral challenge with a rodent-adapted, neurotropic MV strain.     

Human immune responses against sexual stages of malaria parasites: considerations for malaria vaccines

Studies on the natural immune responses to the sexual stages of malaria parasites have been reviewed in the context of human malaria transmission-blocking vaccines. Antibodies against the sexual stages of the malaria parasite, gametocytes and gametes, are readily evoked by natural malaria infections. These antibodies that suppress infectivity at high concentrations can, at low concentrations, enhance the development of the parasite in the mosquito; however, because enhancing antibodies are prevalent during natural malaria infections, it is likely that a vaccine would rapidly boost these antibodies to blocking levels. The immunogenicity of sexual stage antigens appears to be constrained in the human host, probably due to T epitope polymorphism and MHC restriction in humans. These constraints apply mainly to those antigens that are sensitive targets of host immunity such as the gamete surface antigens and not to internal gamete antigens, indicating that antigenic polymorphism may have evolved in response to immune selection pressure. Evidence for immunosuppression of the host by exposure to endemic malaria is presented and its consequences on vaccine development are discussed.     

Nasal administration of Lactococcus lactis improves local and systemic immune responses against Streptococcus pneumoniae

Lactococcus lactis NZ9000 is a non-pathogenic non-invasive bacterium extensively used for the delivery of antigens and cytokines at the mucosal level. However, there are no reports concerning the per se immunomodulatory capacity of this strain. The aim of the present study was to investigate the intrinsic immunostimulating properties of the nasal administration of L. lactis NZ9000 in a pneumococcal infection model. Mice were preventively treated with L. lactis (2, 5 or 7 days with 10(8) cells/day per mouse) and then challenged with Streptococcus pneumoniae. The local and the systemic immune responses were evaluated. Our results showed that nasal administration of L. lactis for 5 days (LLN5d) increased the clearance rate of S. pneumoniae from lung and prevented the dissemination of pneumococci into blood. This effect coincided with an upregulation of the innate and specific immune responses in both local and systemic compartments. LLN5d increased phagocyte activation in lung, blood and bone marrow, determined by NBT and peroxidase tests. Anti-pneumococcal immunoglobulin (Ig)A in bronchoalveolar lavages (BAL) and IgG in BAL and serum were increased in the LLN5d group. Lung tissue injury was reduced by LLN5d treatment as revealed by histopathological examination and albumin concentration and lactate dehydrogenase activity in BAL. The adjuvant effect of L. lactis in our infection model would be an important advantage for its use as a delivery vehicle of pneumococcal proteins and nasal immunization with recombinant L. lactis emerges as an effective route of vaccination for both systemic and mucosal immunity against pneumococcal infection.