A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-γ secretion and protection experiments, mediated by CD4⁺ and CD8⁺ cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.     

Yeast-expressed hantavirus Dobrava nucleocapsid protein induces a strong, long-lasting, and highly cross-reactive immune response in mice

In Europe, Dobrava virus (DOBV) carried by the yellow-necked field mouse Apodemus flavicollis is one of the hantaviruses that can cause severe hemorrhagic fever with renal syndrome in humans. For several hantaviruses, the nucleocapsid (N) protein has proven to be very immunogenic in humans and rodents and even can protect rodents against a virus challenge. To investigate the immunogenicity of DOBV N protein, BALB/c and C57BL/6 mice were immunized three times with a DOBV recombinant N (rN) protein expressed in yeast Saccharomyces cerevisiae together with complete Freund's, with incomplete Freund's, and without adjuvant, respectively. Mice of both strains elicited N-specific antibodies with end-point titers being as high as 1:1,000,000 in C57BL/6 mice. The antibodies induced by DOBV rN protein were highly cross-reactive to the rN proteins of hantaviruses Puumala and Hantaan. In both mice strains, DOBV rN protein induced N-specific antibodies of all IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3), suggesting a mixed Th1/Th2 immune response. Taken together, yeast-expressed DOBV rN protein represents a promising vaccine candidate.     

A dengue-2 Envelope fragment inserted within the structure of the P64k meningococcal protein carrier enables a functional immune response against the virus in mice

A gene fragment encoding for the amino acids (aa) 286-426 from the dengue Envelope (E) protein was expressed in Escherichia coli as two forms of fusion proteins. In one case, the E fragment was fused to the first 45 aa of the P64k protein from Neisseria meningitidis (PD2) while, in the other, it was inserted within the lipoil-binding domain of the aforementioned bacterial protein (PD3). PD2 was obtained as insoluble form within the cytoplasm of the bacteria while PD3 was distributed equally as soluble and insoluble forms. The insoluble forms of each protein as well as the soluble fraction of PD3 were semipurified to test the antigenicity and the immunogenicity in mice. The forms containing the entire P64k protein exhibited the highest recognition with different polyclonal and monoclonal antibodies. Consequently, the neutralizing antibodies elicited by the recombinant proteins were higher in the case of PD3 forms than with PD2, independently of the solubility status. In addition, mice inoculated with the semipurified insoluble form of PD3 were partially protected against lethal challenge with dengue-2 virus, administered by intracerebral inoculation. The results suggested the folding and carrier capacity of the P64k protein over the E fragment, converting PD3 as an attractive vaccine candidate against dengue-2 virus.     

Nucleocapsid-like particles of dengue-2 virus enhance the immune response against a recombinant protein of dengue-4 virus

In this study, we evaluate in mice a novel formulation containing nucleocapsid-like particles of dengue-2 virus (recNLP) co-immunized with a chimeric protein composed of the dengue-4 envelope domain III fused twice within the meningococcal P64k protein of Neisseria meningitidis (PD24). The animals receiving the PD24–recNLP mixture showed the highest levels of antiviral antibodies. Similar results were obtained for IFNγ secretion levels, indicating a functional Th1 cellular response. Consistently, the percentage of mice surviving after viral challenge was significantly higher for those immunized with the mixture than for those inoculated with PD24 protein alone. In addition, in vivo depletion experiments demonstrated the decisive role of CD4⁺ and CD8⁺ cells in the protection conferred by immunization with PD24–recNLP. In conclusion, this report demonstrates for the first time the adjuvant capacity of dengue-2 virus recNLP. Additionally, the evidence presented highlights the potential of these particles for enhancing the immune response against heterologous recombinant proteins.     

Isolation of a capsid protein of bluetongue virus that induces a protective immune response in sheep

A method to purify the neutralization specific antigen of bluetongue virus P2 in large amounts has been developed. The purified protein is free from virus-specified or cellular contaminants and its immunological specificity has been preserved. The purification is based on the observation that protein P2 can be dissociated from the virion by treatment with monovalent or divalent salts. The salt concentration required to solubilize the outer capsid proteins is pH dependent and in general decreases with a decrease in pH. P2 purified by extraction from polyacrylamide gels does not induce immune-precipitating or neutralizing antibodies. The response against P5, on the other hand, is much less conformational dependent and P5 purified from gels readily induces P5-precipitating antibodies in rabbits. These antibodies do not neutralize the virus. Purified P2, immunoabsorbed with anticore serum to remove trace amounts of P7, was injected into sheep. An initial dose of 50 micrograms of P2 was sufficient to induce P2-precipitating antibodies as well as neutralizing and hemagglutination-inhibiting antibodies. These sheep were fully protected against challenge with a virulent strain of the same BTV serotype. Lower doses of P2 still provided a significant level of protection even though no neutralizing antibodies could be detected.     

A plasmid encoding parts of the dengue virus E and NS1 proteins induces an immune response in a mouse model

A DENV-2 plasmid named pEII*EIII/NS1*, containing sequences encoding portions of the envelope protein that are potentially involved in the induction of neutralizing antibodies and a portion of the NS1 sequence that is involved in protection, is reported in this work. The synthesized subunit protein was recognized by human sera from infected patients and had the predicted size. The immunogenicity of this construct was evaluated using a mouse model in a prime-boost vaccination approach. The priming was performed using the plasmid pEII*EIII/NS1*, followed by a boost with recombinant full-length GST–E and GST–NS1 fusion proteins. The mice showed specific antibody responses to the E and NS1 proteins, as detected by ELISA, compared to the response of animals vaccinated with the parental plasmid. Interestingly, some animals had neutralizing antibodies. These results show that EII*, EIII and NS1* sequences could be considered for the design of a recombinant subunit vaccine against dengue disease.     

Photodynamic therapy induces an immune response against a bacterial pathogen

Photodynamic therapy (PDT) employs the triple combination of photosensitizers, visible light and ambient oxygen. When PDT is used for cancer, it has been observed that both arms of the host immune system (innate and adaptive) are activated. When PDT is used for infectious disease, however, it has been assumed that the direct antimicrobial PDT effect dominates. Murine arthritis caused by methicillin-resistant Staphylococcus aureus in the knee failed to respond to PDT with intravenously injected Photofrin(?). PDT with intra-articular Photofrin produced a biphasic dose response that killed bacteria without destroying host neutrophils. Methylene blue was the optimum photosensitizer to kill bacteria while preserving neutrophils. We used bioluminescence imaging to noninvasively monitor murine bacterial arthritis and found that PDT with intra-articular methylene blue was not only effective, but when used before infection, could protect the mice against a subsequent bacterial challenge. The data emphasize the importance of considering the host immune response in PDT for infectious disease.     

Photodynamic therapy induces an immune response against a bacterial pathogen

Photodynamic therapy (PDT) employs the triple combination of photosensitizers, visible light and ambient oxygen. When PDT is used for cancer, it has been observed that both arms of the host immune system (innate and adaptive) are activated. When PDT is used for infectious disease, however, it has been assumed that the direct antimicrobial PDT effect dominates. Murine arthritis caused by methicillin-resistant Staphylococcus aureus in the knee failed to respond to PDT with intravenously injected Photofrin^®. PDT with intra-articular Photofrin produced a biphasic dose response that killed bacteria without destroying host neutrophils. Methylene blue was the optimum photosensitizer to kill bacteria while preserving neutrophils. We used bioluminescence imaging to noninvasively monitor murine bacterial arthritis and found that PDT with intra-articular methylene blue was not only effective, but when used before infection, could protect the mice against a subsequent bacterial challenge. The data emphasize the importance of considering the host immune response in PDT for infectious disease.     

An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive antibody response in mice

Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains.     

Segments of puumala hantavirus nucleocapsid protein inserted into chimeric polyomavirus-derived virus-like particles induce a strong immune response in mice

Insertion of a short-sized epitope at four different sites of yeast-expressed hamster polyomavirus major capsid protein VP1 has been found to result in the formation of chimeric virus-like particles. Here, we demonstrate that the insertion of 45 or 120 amino acid-long segments from the N-terminus of Puumala hantavirus nucleocapsid protein into sites 1 (amino acids 80-89) and 4 (amino acids 288-295) of VP1 allowed the highly efficient formation of virus-like particles. In contrast, expression level and assembly capacity of fusions to sites 2 (amino acids 222-225) and 3 (amino acids 243-247) were drastically reduced. Immunization of BALB/c mice with chimeric virus-like particles induced a high-titered antibody response against the hantavirus nucleocapsid protein, even in the absence of any adjuvant. The strongest response was observed in mice immunized with virus-like particles harboring 120 amino acids of hantavirus nucleocapsid protein. According to the immunoglobulin subclass distribution of nucleocapsid protein-specific antibodies a mixed Th1/Th2 response was detected. The VP1 carrier itself also induced a mixed Th1/Th2 response, which was found to be reduced in mice immunized with virus-like particles harboring 120 amino acid-long inserts. In conclusion, hamster polyomavirus VP1 represents a promising carrier moiety for future vaccine development.     

Immunization with the recombinant PorB outer membrane protein induces a bactericidal immune response against Neisseria meningitidis

Infections with Neisseria meningitidis are characterized by life-threatening meningitis and septicemia. The meningococcal porin proteins from serogroup B meningococci have been identified as candidates for inclusion in vaccines to prevent such infections. In this study, we investigated the vaccine potential of the PorB porin protein free of other meningococcal components. The porB gene from a strain of Neisseria meningitidis expressing the class 3 outer membrane porin protein (PorB3) was cloned into the pRSETB vector, and the protein was expressed at high levels in a heterologous host Escherichia coli. The recombinant protein was purified to homogeneity by affinity chromatography and used for immunization after incorporation into liposomes and into micelles composed either of zwitterionic detergent or nondetergent sulfobetaine. The immunogenicity of these preparations was compared to recombinant PorB protein adsorbed to Al(OH)(3) adjuvant as a control. Although sera raised against the protein adsorbed to Al(OH)(3) reacted with the purified recombinant protein, sera raised against liposomes and micelles showed greater activity with native protein, as measured by enzyme immunoassay with outer membranes and by whole-cell immunofluorescence. Reactivity with native protein was considerably enhanced by incorporation of the adjuvant monophosphoryl lipid A into the liposome or micelle preparations. Recognition of the native protein was in a serotype-specific manner and was associated with the ability of the antisera to promote high levels of serotype-specific complement-mediated killing of meningococci. These results demonstrate that the PorB protein should be considered as a component of a vaccine designed to prevent serogroup B meningococcal infection.     

Identification of Toxoplasma gondii protein fractions induce immune response against melanoma in mice

Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses against tumor cells. We recently found that protein components of Toxoplasma gondii (T. gondii) could mature DCs efficiently. Therefore, in this study, we aimed to find the most effective protein components of T. gondii which are able to mature DCs and consequently instruct immune responses in tumor‐bearing mice. Soluble tachyzoite antigens (STAgs) were fractionated by ammonium sulfate precipitation and subsequently by anion‐exchange HPLC. Immature DCs (iDCs) were treated by these protein fractions and were monitored for IL‐12p70 and IL‐10 production. Moreover, the capacity of mature DCs (mDCs) to induce lymphocyte proliferation was investigated. Ultimately, we analyzed the ability of mDCs in instructing immune responses in tumor‐bearing mice. We found that ammonium sulfate fraction one (A1) matured‐DCs produced higher IL‐12 level and IL‐12/IL‐10 ratio; therefore, this fraction was selected for further fractionation by anion‐exchange HPLC. The results showed that anion‐exchange HPLC fraction 14 (C14) matured‐DCs secrete higher levels of IL‐12p70 and IL‐12p70/IL‐10 ratio. Survival of the mice matured by A1 fraction increased significantly compared to other groups. Moreover, SDS‐PAGE electrophoresis showed that different obtained fractions have distinct proteins based on their size. These results demonstrate that two protein fractions of T. gondii are able to mature DCs more efficient.     

Respiratory syncytial virus recombinant F protein (residues 255-278) induces a helper T cell type 1 immune response in mice

We have developed and evaluated an immunodominant respiratory syncytial virus (RSV) F antigen in a mouse model. The antigenic region corresponding to amino acids 255-278 of the RSV F protein was cloned into a vector containing the ctxA(2)B gene of cholera toxin (CT). The recombinant protein was expressed in Escherichia coli and analyzed on sodium dodecyl sulfate-polyacrylamide gels. The purified protein was evaluated by immunoblot and ganglioside GM(1) enzyme-linked immunosorbent assay to confirm the expression of the RSV F protein and to correct association of the recombinant protein to form a holotoxin-like chimera, respectively. We hypothesized that genetic fusion of modified CT-based adjuvant with RSV F immunodominant epitopes (rRF-255) would induce protective humoral and cellular immune responses in mice. Intranasal immunization of mice with rRF-255 overall induced higher concentrations of anti-RSV F-specific antibodies in both serum and saliva as compared with mice immunized intranasally with RSV or phosphate-buffered saline (PBS). Antibody isotype analysis (IgA, IgG1, IgG2a, and IgG2b) was also performed. The predominant IgG2a antibody isotype response in combination with cytokine analysis of helper T cell type 1 (interferon-gamma, interleukin [IL]-2, IL-12 p70, and tumor necrosis factor-alpha) and helper T cell type 2 (IL-4 and IL-10) responses revealed that rRF-255 antigen induces a prominent helper T cell type 1 immune response in mice. The rRF-255 antigen also induced serum neutralizing antibodies in immunized mice. Analysis of RSV load in lungs showed that rRF-255 immunization provided significant protection compared with PBS control animals.     

抗2型登革病毒M蛋白单克隆抗体的制备和生物学特性的鉴定

目的 克隆表达2型登革病毒M蛋白,用纯化的重组蛋白免疫Balb/c小鼠,通过杂交瘤技术制备可用于胶体金快速检测试条的抗2型登革病毒M蛋白的单克隆抗体(mAb),并鉴定其特性.方法 利用登革热2型病毒全长基因重组质粒,经PCR方法扩增出prm/m基因片段,在pET-32a(+)表达系统中表达,表达产物用Ni柱亲和层析纯化后,用于免疫Balb/c小鼠,采用杂交瘤技术制备抗M蛋白的mAb,采用间接ELISA方法和Western blot进行mAb特异性鉴定;同时采用间接ELISA方法鉴定mAb相对亲和力.结果 获得2株可分泌特异性mAb的杂交瘤细胞Ⅲ1A6和Ⅲ3D2;相对亲和力均在105 以上.Westernblot显示2株mAb能特异识别重组M蛋白.结论成功地制备出抗登革病毒2型病毒M蛋白的2株mAb,为建立快速特异检测登革病毒的实验方法提供了有力的工具.     

DNA priming E and NS1 constructs--homologous proteins boosting immunization strategy to improve immune response against dengue in mice

DNA priming-protein boosting is a strategy used to establish strong immunity to a specific pathogen by the use of two different antigens through sequential delivery systems. In this work, two recombinant plasmids were used, one encoding for the dengue virus E protein, which is know to induce neutralizing antibodies (pcDNA 3.1/E), and the other encoding for the Dengue virus nonstructural protein 1 (pcDNA 3.1/NS1), as a source of B- and T-cell epitopes possibly involved in protective immunity. We showed that immunization of BALB/c mice with three priming doses of both plasmids pcDNA 3.1/E and/or pcDNA 3.1/NS1 were able to induce antibody responses to E protein with a single plasmid; in contrast to the antibody response to NS1 protein we observed an additive effect in terms of antibody response. Moreover, using a prime-boost protocol in which both plasmid constructs were co-administrated followed by a boost of homologous GST-E and GST-NS1 recombinant proteins, we observed an increased antibody response to NS1 and to E protein compared to animals vaccinated with the proteins or with dengue constructs alone. If neutralizing antibodies play an important role in dengue infection, antibodies generated with this regimen was also significantly better than the administration of the mix of proteins alone. These results suggest that NS1 and E proteins together could be considered in a design of subunit recombinant vaccines.     

Baculovirus-expressed nonstructural protein NS2 of bluetongue virus induces a cytotoxic T-cell response in mice which affords partial protection.

Virus-specific cytotoxic T lymphocytes were generated in two strains of mice (BALB/c and CBA/Ca) against baculovirus recombinant proteins (minor and nonstructural) derived from bluetongue virus serotype 10. Immunization of mice with recombinant baculovirus insect cell extracts expressing the nonstructural protein NS2 (Bac-NS2) conferred partial protection against infection with vaccinia virus expressing the NS2 protein. This protective immunity was mediated by CD8+ cells. In contrast, no protection was observed when mice were immunized with similarly expressed Bac-NS1 or -NS3 or the virion minor structural proteins (Bac-VP1, -VP4, or -VP6). Furthermore, the in vitro cytotoxicity activity of T cells derived from immunized animals did not correlate to the protective efficacy of baculovirus recombinant proteins. The implications of this work with regard to the design of noninfectious subunit vaccines are discussed.     

Mycobacterium indicus pranii protein MIP_05962 induces Th1 cell mediated immune response in mice

Utility of Mycobacterium indicus pranii (MIP) as a multistage vaccine against mycobacterial infections demands identification of its protective antigens. We explored antigenicity and immunogenicity of a candidate protein MIP_05962 that depicts homology to HSP18 of M. leprae and antigen1 of Mycobacterium tuberculosis. This protein elicited substantial antibody response in immunized mice along with modulation of cellular immune response towards protective Th1 type. Both CD4⁺ and CD8⁺ subsets from immunized mice produced hallmark protective cytokines, IFN-γ, TNF-α and IL-2. This protein also enhanced the CD4⁺ effector memory that could act as first line of defence during infections. These results point to MIP_05962 as a protective antigen that contributes, in conjunction with others, to the protective immunity of this live vaccine candidate.     

Analysis of a successful immune response against hepatitis C virus

To investigate the type of immunity responsible for resolution of hepatitis C virus (HCV) infection, we monitored antibody and intrahepatic cytotoxic T lymphocyte (CTL) responses during acute (<20 weeks) infection in chimpanzees. Two animals who terminated infection made strong CTL but poor antibody responses. In both resolvers, CTL targeted at least six viral regions. In contrast, animals developing chronic hepatitis generated weaker acute CTL responses. Extensive analysis of the fine specificity of the CTL in one resolver revealed nine peptide epitopes and restriction by all six MHC class I allotypes. Every specificity shown during acute hepatitis persisted in normal liver tissue more than 1 yr after resolution. These results suggest that CD8+CTL are better correlated with protection against HCV infection than antibodies.