Immunogenicity and protective effect of hemolysis mutants of Mycoplasma pneumoniae

Two attenuated strains of Mycoplasma pneumoniae, P24-S1 and P24-S11, were tested for their ability as a live vaccine to confer on hamsters immune resistance against challenge infection with a virulent strain of M. pneumoniae, FH-P24. Fifty percent protection was obtained by vaccination with the P24-S1 strain administered once or twice. In contrast, only 10% of the animals were protected by the P24-S11 vaccine even when it was given three times. Vaccination with the P24-S1 strain resulted in higher humoral and cellular immune responses than the P24-S11 did. These results suggest that the P24-S1 strain has the primary qualities a vaccine which may be used for protection against human mycoplasmal infection.     

A comparison of the immune response induced by DNA or by an inactivated vaccine against tick-borne encephalitis

BALB/c mice were immunized with recombinant plasmid DNA pSVK3-ENS1 and pcDNAI-NS3 containing, respectively, genes E-NS1 and NS3 of tick-borne encephalitis (TBE) virus. Antibodies to TBE virus proteins were detected in the blood sera of the immunized animals by the method of the enzyme immunoassay. Though the titers of virus-specific antibodies in the sera of mice immunized with protein vaccines exceeded those registered after immunization with DNA vaccines, essential protective immunity was observed after the use of both vaccines.     

Identification of circulating antigens, including an immunoglobulin binding protein, from Toxoplasma gondii tissue cyst and tachyzoites in murine toxoplasmosis

Here we describe the identification of Toxoplasma gondii circulating antigens in sera of BALB/c mice experimentally infected with either the virulent RH strain, or the cystogenic WTD1 strain or with an isolate from a human patient. The circulating antigens were identified by immunoblot in tachyzoite (RH strain) and in tissue cyst (ME-49 strain) crude antigens, using antibodies produced by immunisation of BALB/c mice with homologous sera from infected animals. The most relevant tachyzoite antigen identified are in the following four clusters of 109-94, 67-57, 35-31 and 28-21 kDa. Tissue cyst-specific circulating antigens, like the 18 kDa one, were detected in sera from mice infected with the cystogenic strains. These immune sera, after depletion of tachyzoite specific antibodies, recognised three tissue cysts antigens with Mr of 120, 79 and 48 kDa, and a cluster of antigens in the range of 68-53 kDa. We produced monoclonal antibodies by fusion of myeloma cells with lymphocytes from the mouse immunised with circulating antigens from the RH strain. One of the clones (3A11/H12) obtained, secretes IgG(1) and recognises a peptide epitope from a tachyzoite 67 kDa protein. This parasite protein also binds irrelevant mouse IgG(1) as well as immunoglobulins from other species. The reactivity with non-specific antibodies was inhibited by preincubation with 2% normal mouse and goat serum, while the reaction with the monoclonal antibody 3A11/H12 was not. Furthermore, a biotinylated F(ab')(2) of an irrelevant mouse IgG(1) did not show any reactivity while the F(ab')(2) of the monoclonal antibody 3A11/H12 reacts specifically with the 67 kDa antigen suggesting that this circulating antigen is a putative Fc binding protein.     

Characterization and Pathogenicity of Hemolysis Mutants of Mycoplasma pneumoniae

Hemolysis mutants were produced by treating Mycoplasma pneumoniae FH-P24 strain with N-methyl-N-nitro-nitrosoguanidine and were classified into three different groups. The first group of mutants, strains P24-L1, L2, and L11, showed wide and clear hemolytic zones. Their attachment ability to erythrocytes of various animals and to hamster lung cells were the same as those of the parent strain. The second group, strain P24-S1, showed non-hemolysis and non-hemadsorption, but retained the attachment ability to lung cells, although not to erythrocytes. The third group, strain P24-S11, was non-hemolytic, had completely lost the attaching ability, and did not proliferate in vivo. Strains in the first group produced significant microscopic pneumonic lesions in hamsters while strain P24-S1 produced milder lung lesions. Strain P24-S11 did not cause any lung lesions, and organisms were not recovered from the lungs of hamsters. The attachment of M. pneumoniae to respiratory epithelium as a cause of infection and the existence of a relationship between the hemolytic abilities of the organisms and histopathogenicity in the hamster lung tissue were further supported by the present data. It was also shown that the use of hemolysis mutants is useful for the elucidation of pathogenesis in mycoplasmal infections.     

Association between M. pneumoniae hemolysis, attachment, and pulmonary pathogenicity

Three different groups of hemolysis mutants were produced by treatment of the M. pneumoniae FH-P24 strain with N-methyl-N-nitro-nitrosoguanidine. The first group of mutants, strains P24-L1, L2, and L11, showed wide and clear hemolytic zones, and attaching ability to erythrocytes and to hamster lung cells were the same as the properties of the parent strain and produced significant microscopic lung lesions. Mutant P24-S1 showed non-hemolysis and non-hemadsorption, yet retained the attaching ability to lung cells and produced milder lung lesions. Mutant P24-S11 showed none of those activities, did not cause any lung lesion, and was never recovered from the lungs of hamsters. A close relationship between the hemolytic ability of M. pneumoniae and the histopathogenicity in the hamster lung is suggested in this study. The attaching ability of organisms seems to be an important factor at the initial stage of infection.     

Immune responses and protective efficacy induced by 85B antigen and early secreted antigenic target-6 kDa antigen fusion protein secreted by recombinant bacille Calmette-Guérin

In an attempt to improve immune responses and protective efficacy, we constructed two recombinant bacille Calmette-Guérin (rBCG) strains expressing an 85B antigen (Ag85B) and early secreted antigenic target-6 kDa antigen (ESAT6) of Mycobacterium tuberculosis (MTB) fusion protein. Both rBCG strains have the same protein insertion but in a different order (Ag85B-ESAT6 and ESAT6-Ag85B). The cultured supernatant of rBCG strains and the sera from the mice immunized with the fusion protein Ag85B-ESAT6 or ESAT6-Ag85B formed a band with a fraction size of 37 kDa, equalivalent to the sum of Ag85B and ESAT6. Six weeks after BALB/c mice were immunized with BCG or rBCG, spleen lymphocytes showed significant proliferation in response to culture filtrate protein of MTB. Compared with the BCG group, mice vaccinated with rBCG elicited a high level increase of immunoglobulin G antibodies to culture filtrate protein in the serum. The gamma-interferon levels in the lymphocyte culture medium supernatants increased remarkably in the rBCG1 group, significantly higher than that of the BCG immunized group (p<0.05). Four weeks after vaccination, mice were infected with M. tuberculosis H37Rv and a dramatic reduction in the numbers of MTB colony forming units in the spleens and lungs was observed in the two rBCG immunization groups. Although these rBCG strains were more immunogenic, their protective effect was comparable to the classical BCG strain, and there were no significant differences between two rBCG groups (p>0.05).     

Identification of Chironomus kiiensis allergens, a dominant species of non-biting midges in Korea.

Non-biting midges are known to contain potent inhalant allergens. IgE antibody responses to the crude extract of Chironomus kiiensis adults, a dominant chironomid species in Korea, were examined. With the IgE-ELISA or passive cutaneous anaphylaxis reactions, increased levels of chironomid-specific IgE were detected in the skin test positive human sera, or immunized BALB/c mouse sera with the crude extract adsorbed to alum. IgE-immunoblot analysis showed major IgE-reacting protein band patterns, which reacted with more than 50% of the skin test positive human sera, at 110, 80, 73, 46, 40, 37, 34, and 31 kDa. The reactive band patterns were largely similar between skin test positive humans and immune BALB/c mice. However, the bands of 55, 31, 27, 26, 24, and 23 kDa were found only in sensitized humans, but not in immunized mice.     

牛血清中27kDa蛋白的免疫学特性研究

为探讨牛血清中存在的HBsAg样蛋白的性质,给HBV的发病机制、治疗、预防等研究提供依据,我们采集93份牛血清,以SDS-PAGE、Westem Blot对血清中HBsAg样蛋白进行研究,发现其分子量约为27kDa：以SDS-PAGE分离牛血清中的HBsAg样蛋白,经皮下多点免疫小鼠,可产生与人HBV编码蛋白HBsAg反应的抗体。表明该27kDa蛋白可能存在与HBsAg相似的免疫学特性。     

Brugia malayi: antibody responses to larval antigens in infected and immunized jirds.

Vaccination with irradiated third stage Brugia malayi larvae (L3) has been reported to induce partial protective immunity to L3 challenge in jirds. The purpose of this study was to identify antigens that may be targets of protective immunity in this model. Jirds were immunized by s.c. injection of irradiated L3 and challenged either s.c. or i.p. Necropsy was performed 11 wk after challenge. Partial protection was achieved in s.c. challenged animals; worm recovery was only 41% of that observed in unvaccinated controls, and worms recovered from immunized animals were stunted. Worm recoveries in immunized animals that were challenged i.p. did not differ from those of unimmunized controls. Group differences in parasite antigen levels in sera collected 2-11 wk after larval challenge were consistent with parasitological findings obtained at necropsy. Antibody studies compared prechallenge sera from immunized animals to sera from infected (unimmunized) controls. Antibody responses to L3 surface antigens (assessed by IFA) were much stronger after immunization than after infection. Immunoblot studies showed preferential recognition of several L3 antigens (97, 54, 48, and 40 kDa) by antibodies in sera from immunized animals. Additional studies are needed to determine whether immunization with such preferentially recognized antigens can induce protection to larval challenge comparable to or better than that observed with live vaccines.     

Monoclonal, but not polyclonal, antibodies protect against Plasmodium yoelii sporozoites

One of the primary strategies for malaria vaccine development has been to design subunit vaccines that induce protective levels of antibodies against the circumsporozoite (CS) protein of malaria sporozoites. In the Plasmodium yoelii mouse model system such vaccines have been uniformly unsuccessful in protecting against sporozoite-induced malaria. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of Navy yoelii sporozoite 1 (NYS1), an IgG3 mAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, a synthetic peptide derived from the repeat region of the P. yoelii CS protein, indicating that the epitope on sporozoites recognized by this mAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by indirect fluorescent antibody test and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing (QGPGAP)2 but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not absolute concentration could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective mAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. Although the results of the conventional antibody assays did not correlate with protection, sera from the protected animals inhibited sporozoite development in mouse hepatocyte cultures significantly more than did the sera from the unprotected, subunit vaccine-immunized animals, correlating with protection. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective mAb and nonprotective polyclonal antibodies will be important in the further development of malaria vaccines.     

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.     

Use of I region-restricted, antigen-specific T cell hybridomas to produce idiotypically specific anti-receptor antibodies

Murine T cell hybridomas bearing receptors for antigen plus I region gene products were used as immunogens in mice in an effort to raise anti-receptor antisera. The antisera were assayed for anti-receptor activity by the ability to inhibit interleukin 2 production by the T cell hybridomas stimulated by antigen and I region expressing antigen-presenting cells. The T cell hybridomas used in these experiments were made by fusing antigen-specific, I region-restricted BALB/c T cell blasts to the AKR thymoma, BW5147. Three groups of mice were immunized with the T cell hybridomas: (BALB/c X AKR)F1 animals, syngeneic to the hybridoma; (BALB.B X aKR)F1 animals, differing from the hybridomas at H2; and (C.B20 X AKR)F1 animals, differing from the hybridomas at Igh. Mice were immunized multiple times and sera from individual animals were assayed for anti-receptor antibodies. In all groups, some mice produced anti-receptor antibodies by the criterion that they were inhibitory in the assay mentioned above. The frequency of mice producing these inhibitory antibodies varied considerably between groups, with the (BALB.B X AKR)F1 animals producing these antibodies most frequently, and the (BALB/c X AKR)F1 animals producing them least often. All inhibitory antisera were idiotypically specific; they inhibited the response of the immunizing T cell hybridomas, but not the responses of closely related hybridomas with different specificities. Moreover, when they could be absorbed, the inhibitory antibodies could only be absorbed by the immunizing hybridoma. It is hoped that these antisera, and B cell hybridomas prepared from the immunized animals, will be useful in the elucidation of the structure of the receptors for antigen plus I region products on T cells.     

Protection against lethal measles virus infection in mice by immune-stimulating complexes containing the hemagglutinin or fusion protein.

The importance of each of the two surface glycoproteins of measles virus in active and passive immunization was examined in mice. Infected-cell lysates were depleted of either the hemagglutinin (H) or fusion (F) glycoprotein by using multiple cycles of immunoaffinity chromatography. The products were used to prepare immune-stimulating complexes (iscoms) containing either F or H glycoprotein. Such complexes are highly immunogenic, possibly as a result of effective presentation of viral proteins to the immune system [B. Morein, B. Sundquist, S. H?glund, K. Dalsgaard, and A. Osterhaus, Nature (London) 308:457-460, 1984]. Groups of 3-week-old BALB/c mice were inoculated with the iscom preparations. All animals developed hemolysis-inhibiting antibodies, whereas only sera of animals immunized with the iscoms containing the H glycoprotein had hemagglutination-inhibiting antibodies. Sera from animals immunized with the H or F preparation only precipitated the homologous glycoprotein in radioimmune precipitation assays. The immunized animals were challenged with a lethal dose of the hamster neurotropic variant of measles virus. Of the 7-week-old animals in the nonimmunized control group, 50% died within 10 days after challenge. No animals in the immunized groups showed symptoms of disease throughout the observation period of 3 months. Passive administration of anti-H monoclonal antibodies gave full protection against the 100% lethal acute infection with the hamster neurotropic variant of measles virus in newborn mice, whereas anti-F monoclonal antibodies failed to protect the animals. This study emphasizes that both H and F glycoproteins need to be considered in the development of measles virus subunit vaccines.     

Protective Efficacy of a Human Endogenous Retrovirus Envelope-Coated,Nonreplicable, Baculovirus-Based Hemagglutin Vaccine against Pandemic Influenza H1N1 2009

Despite the advantages of DNA vaccines, overcoming their lower efficacy relative to that of conventional vaccines remains a challenge. Here, we constructed a human endogenous retrovirus (HERV) envelope-coated, nonreplicable, baculovirus-based HA vaccine against swine influenza A/California/04/2009(H1N1) hemagglutin (HA) (AcHERV-sH1N1-HA) as an alternative to conventional vaccines and evaluated its efficacy in two strains of mice, BALB/c and C57BL/6. A commercially available, killed virus vaccine was used as a positive control. Mice were intramuscularly administered AcHERV-sH1N1-HA or the commercial vaccine and subsequently given two booster injections. Compared with the commercial vaccine, AcHERV-sH1N1-HA induced significantly higher levels of cellular immune responses in both BALB/c and C57BL/6 mice. Unlike cellular immune responses, humoral immune responses depended on the strain of mice. Following immunization with AcHERV-sH1N1-HA, C57BL/6 mice showed HA-specific IgG titers 10- to 100-fold lower than those of BALB/c mice. In line with the different levels of humoral immune responses, the survival of immunized mice after intranasal challenge with sH1N1 virus (A/California/04/2009) depended on the strain. After challenge with 10-times the median lethal dose (MLD₅₀) of sH1N1 virus, 100% of BALB/c mice immunized with the commercial vaccine or AcHERV-sH1N1-HA survived. In contrast, C57BL/6 mice immunized with AcHERV-sH1N1-HA or the commercial vaccine showed 60% and 70% survival respectively, after challenge with sH1N1 virus. In all mice, virus titers and results of histological analyses of lung tissues were consistent with the survival data. Our results indicate the importance of humoral immune response as a major defense system against influenza viral infection. Moreover, the complete survival of BALB/c mice immunized with AcHERV-sH1N1-HA after challenge with sH1N1 virus suggests the potential of baculoviral vector-based vaccines to achieve an efficacy comparable to that of killed virus vaccines.     

Trials for the co-expression of the merozoite surface protein-1 and circumsporozoite protein genes of Plasmodium vivax

Merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen, and circumsporozoite protein (CSP), a component of sporozoites that includes a Plasmodium vivax B-cell epitope, are strong candidates for use in a malaria vaccine. A chimeric recombinant gene containing portions of both msp-1 and csp from P. vivax separated by Pro-Gly linker motif was generated. The construct gene was named mlc (msp-1, linker, and csp). The MLC chimeric recombinant protein had a molecular weight of approximately 25 kDa when expressed in Escherichia coli, as determined with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. The purified chimeric protein reacted with the sera of patients infected with P. vivax but not with the sera of uninfected patients according to western blot analysis. The chimeric protein reacted well with sera of malaria patients (109/115, 94.78%) as assessed with enzyme-linked immunosorbent assay (ELISA). BALB/c mice that were orally immunized with the MLC chimeric recombinant protein successfully produced antigen-specific antibodies. Additionally, levels of the Th1-associated cytokines IL-12(p40), TNF-α, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Therefore, the E. coli-expressed MLC chimeric recombinant protein might be used as a valuable vaccine candidate for oral immunization against vivax malaria.     

Vaginal candidosis

Enzyme linked immunosorbent assay was found to be a convenient method for the investigation of antibodies in mice immunized with Candida albicans ribosomes. Antibodies against the ribosomal antigen were detected in all the sera of mice (ICR and BALB/ c) immunized with ribosomes and incomplete Freund's adjuvant and in some of the sera of mice immunized with ribosomes only; the titer of antibodies varied from 1320 to 110 240. Vaccination of mice with ribosomal protein and IFA resulted in a high titer of antiribosomal antibodies. Treatment of ribosomes with pronase abrogated the capacity of the ribosomes to elicit anti ribosomal humoral responses, suggesting that the antibodies detected were directed against the protein moiety of the ribosomes. The presence of antibodies in sera of immunized mice could not be correlated with the protection afforded by the ribosomal vaccination.     

Synthesis and bioactivities of two multiple antigen peptides as potential vaccine against schistosoma

Based on the two antigenic peptides, 26-43 (P26) and 116-131 (P116), derived from 28 kDa glutathione S-transferase of Schistosoma mansoni (Sm28GST), two multiple antigenic peptides (MAPs), (P26)4-MAP and (P116)4-MAP with the same oligomeric lysine core, were synthesized by stepwise solid-phase peptide synthesis method. The antigenicities and protective effects of these two MAPs were examined on experimental animals. As shown in the dot-ELISA result, the synthetic MAPs could be recognized and bound by immunoglobins in both patient's and infected-rabbit's sera. After Kunming mice were immunized with (P26)4-MAP, the worm burden reduction rate and the liver egg reduction rate were 59.9% and 61.1%. In (P26)4-MAP or (P116)4-MAP immunized BALB/c mice, the worm burden reduction rates were 37.5% and 62.5%, respectively, and the liver egg reduction rates were 35.1% and 54.0%, respectively.     

Protective immunity of <Emphasis Type="Italic">E. coli</Emphasis>-synthesized NS1 protein of Japanese encephalitis virus

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.     

减毒沙门氏菌介导的小鼠肝炎病毒S1基因DNA疫苗的免疫特性分析

根据GenBank公开序列自行设计一对引物,通过RT-PCR扩增出小鼠肝炎病毒的全长S1基因,并将其插入真核表达质粒pVAX1中,构建出重组真核表达质粒pVAX1-S1。将重组质粒转染COS-7细胞,采用间接免疫荧光检测出S1蛋白的体外表达。将重组质粒转入减毒鼠伤寒沙门氏菌SL7207中,构建出运送DNA疫苗的重组沙门氏菌SL7207(pVAX1-S1)。分别以5×108CFU、1×109CFU、2×109CFU剂量的重组菌口服接种6周龄BALB/c小鼠,试验结果表明,重组菌对小鼠具有良好的安全性。以1×109CFU剂量的重组菌口服免疫小鼠,抗体检测结果显示,在二免后两周和三免后两周,重组菌免疫组的血清抗体水平与SL7207(pVAX1)空载体免疫组间分别存在显著性差异(P<0.05)和极显著性差异(P<0.01)。在三免后两周重组菌免疫组出现了较高水平的肠黏膜抗体。     

Generation and specificity of monoclonal anti-idiotypic antibodies against human HIV-specific antibodies. I. Cross-reacting idiotopes are expressed in subpopulations of HIV-infected individuals

In this study we have generated monoclonal anti-idiotypic antibodies against human monoclonal and polyclonal anti-HIV antibodies in seropositive sera. A human anti-gp41 mAb (H2, IgM kappa) was used to immunize BALB/c mice and to prepare hybridoma anti-antibodies that react with H2 and not with normal human IgM. Similar monoclonal anti-antibodies were made in BALB/c mice immunized with Ig fraction prepared from a pool of HIV-seropositive sera. Both kinds of anti-idiotypic antibodies reacted with antibodies in pools of seropositive sera and with individual seropositive sera but not with normal human Ig or seronegative sera. The Id-positive Ig from single donors were isolated on two different anti-Id immunoabsorbents and shown to bind to p24 and gp120, respectively. The detection and isolation of idiotypically cross-reactive human anti-HIV antibodies from seropositive donors demonstrated, for the first time, the existence of shared Id expressed by antibodies against HIV Ag. The utility of cross-reacting anti-idiotypic antibodies as tools to dissect the network regulation of the anti-viral immunity in AIDS is discussed.