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.     

Antibody Response and Protection Induced by Immunization with Smooth and Rough Strains in Experimental Salmonellosis

The antibody response of mice to a smooth strain of Salmonella typhimurium was shown previously to be extremely rapid and potent. As measured by the complement-mediated bactericidal reaction, it was also found to be highly specific as well as reproducible. Experiments which studied the effects of antigen type (live or heat-killed), antigen dose, and the route of immunization indicated that the most rapid and highest antibody response was achieved with live, smooth organisms injected by the intraperitoneal route. Living vaccines of rough strains of either S. typhimurium or S. enteritidis induced antibodies directed against the corresponding smooth organisms. The response to the rough strains was apparently due to antibody production rather than to the simple release of preformed natural antibody. The duration of protection conferred by the rough strain vaccines was closely correlated with the endotoxic content of the immunizing strain. Smooth heat-killed vaccines and a rough live vaccine protected against homologous but not heterologous challenge. In contrast, immunization with a smooth live vaccine protected mice against both homologous and heterologous challenge infections. Protection was not due to a local effect in the peritoneal cavity, since mice were also protected against subcutaneous challenge. The secondary antibody response, induced in immunized animals by the virulent challenge infection, was demonstrated to be rapid and potent, and hence a factor to be considered in protection.     

A recombinant multivalent combination vaccine protects against Chlamydia and genital herpes

Chlamydia trachomatis and Herpes simplex virus type 2 (HSV-2) genital infections pose a considerable public health challenge worldwide. Considering the high incidence of coinfections by the two pathogens, a combination vaccine that can be administered as a single regimen would be highly desirable. Recombinant Vibrio cholerae ghosts (rVCG) offer an attractive approach for the induction of humoral and cellular immune responses against human and animal pathogens. In this study, we evaluated a bivalent combination vaccine formulation comprising rVCG expressing chlamydial MOMP and HSV-2 glycoprotein D in mice for immunogenicity and protective efficacy against genital challenge with either pathogen. Mice immunized with the combination vaccine elicited secretory IgA and IgG2a antibodies to both chlamydial and HSV-2 antigens in serum and vaginal secretions. Robust antigen-specific mucosal and systemic T helper type 1 responses were induced in mice as measured by increased interferon-gamma levels produced by immune T cells in response to restimulation with target antigen in vitro. In addition, mice immunized with the combination vaccine were prophylactically protected from genital challenge with high doses of live Chlamydia and HSV-2. Thus, the combination vaccine regimen delivered by rVCG elicited adequate immune effectors that simultaneously protected against the individual pathogens.     

Murine melanoma-specific tumor rejection activity elicited by a purified, melanoma-associated antigen

B700 is a melanoma-specific glycoprotein antigen, with a m.w. of 65,000 and an isoelectric point of 4.5; this antigen has been shown to bear significant sequence homology to a normally occurring protein, serum albumin. The production of B700 is apparently restricted to all the murine melanomas tested, since a variety of other transformed and untransformed cell lines do not contain detectable levels of this antigen. The capacity of B700 to function as a tumor-specific transplantation antigen (TSTA) is demonstrated in this study. This activity has been titrated, and it is shown that mice immunized with B700 are able to significantly inhibit the growth of B16 F10 melanomas after subcutaneous challenge; immunized mice can also inhibit the establishment and growth of experimental metastases in the lungs after i.v. challenge with B16 melanoma cells. The TSTA was found to cross-protect also against challenge with two other murine melanoma lines, JB/RH and K1735, but was specific in that the growth of two nonmelanoma lines (RBL-5 leukemia and MCA-105 sarcoma) was not affected. B700 is also shown in this study to be unrelated to other known murine tumor antigens, or to murine leukemia virus antigens. It is further shown that mice immunized with B700 produced antibodies specific to B700 that were not cross-reactive with albumins from various mammalian sources.     

Immune response of turbot, Psetta maxima (L.) (Pisces: Teleostei), to formalin-killed scuticociliates (Ciliophora) and adjuvanted formulations

The increasing frequency of scuticociliatosis in turbot culture has stressed the need of knowledge on the immune responses to these parasites, for further developing of prevention and control strategies. The immune response of turbot to killed parasites, alone (Ag) or in combination with Montanide ISA 763A (MON), was studied in a laboratory-scale experiment. The variations of several innate immune factors and the antibody response were analysed in immunized vs. non-immunized fish at different times after immunization, and also after a challenge with live ciliates. Amongst innate immune factors, serum lysozyme increased progressively in all inoculated groups. Differences in innate immune factors in Ag and Ag-MON fish with respect to controls were mainly evidenced after challenge, especially for serum complement. Serum antibody levels increased in immunized fish after booster and particularly after challenge. In addition, certain protection was obtained for immunized groups compared to controls or to fish receiving MON alone, and the levels of specific antibodies were also the highest in immunized groups. The obtained information could be useful for further design of immunoprophylactic formulations against scuticociliatosis.     

Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope “tags”

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.     

Role of IL-4 in aversion induced by food allergy in mice

To ascertain the role of IL-4 in aversion to antigen induced by food allergy, wild type and IL-4 deficient BALB/c mice were sensitized with ovalbumin and challenged orally with egg white. Sensitized wild type mice had increased production of IL-4 by spleen and mesenteric lymph node cells in vitro, higher levels of serum anti-ovalbumin IgE and IgG1, aversion to ingestion of the antigen and loss of body weight after continuous oral challenge. Intestinal changes in wild type sensitized mice included eosinophil infiltration and increased mucus production. The IL-4 deficiency impaired the development of food allergy and the aversion to antigen, suggesting the involvement of the antigen specific antibodies. When IL-4 deficient mice received serum from sensitized wild type donors, the aversion was restored. These results indicate that production of IL-4 and specific IgE/IgG1 antibodies correlate with aversion to antigen induced by food allergy in mice.     

Mucosal adjuvant activity of oligomannose-coated liposomes for nasal immunization

In the present study, we investigated the effectiveness of liposomes coated with a neoglycolipid consisting of mannotriose and dipalmitoylphosphatidylcholine (Man3-DPPE) as an adjuvant for induction of mucosal immunity. Immunization of BALB/c mice with ovalbumin (OVA)-encapsulated Man3-DPPE-coated liposomes (oligomannose-coated liposomes; OMLs) by a nasal route produced high levels of OVA-specific IgG and IgA antibodies in serum of immunized mice 1 week after the last nasal immunization, whereas no significant serum antibody responses were observed in mice that received OVA in uncoated liposomes or OVA alone. Seven weeks after the last nasal immunization, nasal challenge with an excess amount of OVA in mice that had received OVA/OMLs led to an anamnestic response to the antigen that resulted in 5- to 10-fold increases of antigen-specific serum IgG and IgA antibodies. Only mice immunized nasally with OML/OVA secreted antigen-specific secretory IgA in nasal washes and produced interferon-gamma secreting cells in nasopharyngeal-associated lymphoreticular tissue. Taken together, these results show that nasal administration of OMLs induces mucosal and systemic immunity that are specific for the entrapped antigen in the liposomes. Thus, liposomes coated with synthetic neoglycolipids might be useful as adjuvants for induction of mucosal immunity.     

Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope “tags”

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system''s ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.     

Antibodies to the strain-specific and cross-reactive determinants of the haemagglutinin of influenza H3N2 virus. 3. Selection of antigenic variations in vitro and in vivo

Antigenic variants of influenza A/Hong Kong/1/68 (H3N2) were obtained in vitro by letting virus multiply in the allantosis-on-shell system in the presence of anti-haemagglutinin antibodies, prepared from immune goat serum to purified haemagglutinin antigen, and in vivo by giving mice antibody intraperitoneally one day before challenge with a sublethal dose of live virus. In both systems it was shown that the most narrowly reacting strain-specific antibodies selected antigenic variants at an apparently higher rate than a more cross-reactive preparation of antibodies.     

Comparison of simian virus 40 large T antigen recombinant protein and DNA immunization in the induction of protective immunity from experimental pulmonary metastasis

In this report we examine the ability of a recombinant tumor antigen preparation to prevent the establishment of experimental pulmonary metastasis. Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was injected into BALB/c mice followed by challenge with an intravenous injection of syngeneic SV40-transformed tumorigenic cells. The experimental murine pulmonary metastasis model allows for the accurate measurement of metastatic lessions in the lungs at various times after the challenge, using computer-assisted video image analysis. Following challenge, lung metastasis and survival data for the groups of mice were obtained. Animals immunized with recombinant SV40 T-Ag showed no detectable sign of lung metastasis and survived for more than 120 days after challenge. Antibodies specific for SV40 T-Ag were detected in the serum of immunized mice by enzyme-linked immunosorbent assay. Splenocytes obtained from mice immunized with recombinant SV40 T-Ag did not lyse syngeneic tumor cells, indicating that no cytotoxic T lymphocyte response was induced. Control mice developed extensive lung metastasis and succumbed to lethal tumor within 4 weeks after challenge. These data indicate that immunization with the recombinant SV40␣T-Ag induces protective, T-Ag-specific immunity in an experimental pulmonary tumor metastasis model. Received: 20 August 1998 / Accepted: 25 November 1998     

Trypanosoma cruzi specific antibody response in immunized C3H(He) mice during infection

C3H(He) mice previously immunized with live culture derived Corpus Christi strain T. cruzi are significantly protected (up to 100% survival) against challenge by Brazil strain blood trypanosomes. The antibody response, directed against the Brazil strain or the Corpus Christi strain, in these mice has been observed by comparing sera from mice immunized only, infected only, or immunized and infected. The anti- T. cruzi titers determined by both direct agglutination (DA) and indirect fluorescence (IFA) were routinely found to be highest for immunized and infected mice with immunized mice and infected mice following in decreasing order. The use of mercaptoethanol treatment of sera (DA) and isotope specific second antibody (IFA) showed that IgG is the major parasite specific immunoglobulin response through infection. Evidence of cross-reacting antigens on the two parasite strains was found. By both DA and IFA, 11 of 18 anti-Brazil strain monoclonal antibodies were found to react (IFA titers of 320 or greater) with both parasite strains. No evidence of localization of cross-reacting antigens (using mouse antisera) or antigenic determinants (using monoclonal antibodies) was found in that uniform fluorescence over the parasite was observed in all IFA tests.     

Preexisting immunity to poliovirus does not impair the efficacy of recombinant poliovirus vaccine vectors

Recombinant viruses are attractive candidates for the development of novel vaccines. A number of viruses have been engineered as vaccine vectors to express antigens from other pathogens or tumors. Inoculation of susceptible animals with this type of recombinant virus results in the induction of both humoral and cellular immune responses directed against the foreign antigens. A general problem to this approach is that existing immunity to the vector can diminish or completely abolish the efficacy of the viral vector. In this study, we investigated whether poliovirus recombinants are capable of inducing effective immunity to the foreign antigen in previously vaccinated animals. Antipoliovirus immunity was induced in susceptible mice by intraperitoneal immunization with live poliovirus. Immunized mice developed antibodies directed against capsid proteins that effectively neutralized poliovirus in vitro and protected animals from a lethal challenge with a high dose of pathogenic poliovirus. To test whether preexisting immunity reduces the efficacy of vaccination with recombinant poliovirus, immunized mice were inoculated with a recombinant poliovirus expressing the C-terminal half of chicken ovalbumin (Polio-Ova). Animals developed ovalbumin-specific antibodies and cytotoxic T lymphocytes (CTL). While the antibody titers observed in preimmune and naive mice were similar, the overall CTL response appeared to be reduced in preimmune mice. Importantly, vaccination with Polio-Ova was able to effectively protect preimmune mice against lethal challenge with a tumor expressing the antigen. Thus, preexisting immunity to poliovirus does not compromise seriously the efficacy of replication-competent poliovirus vaccine vectors. These results contrast with those observed for other viral vaccine vectors and suggest that preexisting immunity does not equally affect the vaccine potential of individual viral vectors.     

Factors Affecting Formation of Incomplete Vi Antibody in Mice.

Gaines, Sidney (Walter Reed Army Institute of Research, Washington, D.C.), Julius A. Currie, and Joseph G. Tully. Factors affecting formation of incomplete Vi antibody in mice. J. Bacteriol. 90:635-642. 1965.-Single immunizing doses of purified Vi antigen elicited complete and incomplete Vi antibodies in BALB/c mice, but only incomplete antibody in Cinnamon mice. Three of six other mouse strains tested responded like BALB/c mice; the remaining three, like Cinnamon mice. Varying the quantity of antigen injected or the route of administration failed to stimulate the production of detectable complete Vi antibody in Cinnamon mice. Such antibody was evoked in these animals by multiple injections of Vi antigen or by inoculating them with Vi-containing bacilli or Vi-coated erythrocytes. The early protection afforded by serum from Vi-immunized BALB/c mice coincided with the appearance of incomplete Vi antibody, 1 day prior to the advent of complete antibody. Persistence of incomplete as well as complete antibody in the serum of immunized mice was demonstrated for at least 56 days after injection of 10 mug of Vi antigen. Incomplete Vi antibody was shown to have blocking ability, in vitro bactericidal activity, and the capability of protecting mice against intracerebral as well as intraperitoneal challenge with virulent typhoid bacilli. Production of incomplete and complete Vi antibodies was adversely affected by immunization with partially depolymerized Vi antigens.     

用重组腺病毒表达A组轮状病毒主要中和抗原VP7可获得良好的免疫学效果

为探索利用重组腺病毒表达轮状病毒的结构抗原以制备轮状病毒基因工程疫苗的可行性,构建了一株可表达A组轮状病毒主要中和抗原VP7的重组腺病毒AdEasyCVP7.AdEasyCVP7感染293细胞后,RT－PCR证明VP7基因有转,Western blotting试验可检测到VP7的表达。随后,用AdEasyCVP7通过灌胃和滴鼻两种不同途径免疫小鼠,并对免疫后小鼠的血清抗体和粘膜抗体进行了比较。初次免疫后,两组小鼠均有应答,但血清抗体滴度及阳转率不同。再次免疫后,滴鼻组小鼠显示出明显的加强效果。对肺灌洗液中的sIgA及肺、肠粘膜组织匀浆中的IgA进行检测发现滴鼻组的免疫学效果明显优于灌胃组。对血清中和抗体的检测表明,初次和再次免疫后,两组小鼠血清中均有中和抗体产生。该研究为轮状病毒基因工程疫苗的免疫方案、免疫途径及免疫保护作用等的进一步研究奠定了基础。     

Protection of mice against Clostridium chauvoei infection by anti-idiotype antibody to a monoclonal antibody to flagella

Abstract Polyclonal rabbit anti-idiotypic antibody (anti-Id) against the protective monoclonal antibody specific to the flagella of Clostridium chauvoei was produced, purified, and characterized. Anti-Id inhibited the binding of its related monoclonal antibody to the flagellar antigen, suggesting that the anti-Id bore an internal image of the flagellar antigen. When mice were immunized with anti-Id intraperitoneally, the survival rate increased significantly, compared with mice immunized with normal rabbit IgG ( P < 0.01), and specific anti-flagellar antibodies were induced.     

The gH-gL Complex of Herpes Simplex Virus (HSV) Stimulates Neutralizing Antibody and Protects Mice against HSV Type 1 Challenge

The herpes simplex virus type 1 (HSV-1) gH-gL complex which is found in the virion envelope is essential for virus infectivity and is a major antigen for the host immune system. However, little is known about the precise role of gH-gL in virus entry, and attempts to demonstrate the immunologic or vaccine efficacy of gH and gL separately or as the gH-gL complex have not succeeded. We constructed a recombinant mammalian cell line (HL-7) which secretes a soluble gH-gL complex, consisting of gH truncated at amino acid 792 (gHt) and full-length gL. Purified gHt-gL reacted with gH- and gL-specific monoclonal antibodies, including LP11, which indicates that it retains its proper antigenic structure. Soluble forms of gD (gDt) block HSV infection by interacting with specific cellular receptors. Unlike soluble gD, gHt-gL did not block HSV-1 entry into cells, nor did it enhance the blocking capacity of gD. However, polyclonal antibodies to the complex did block entry even when added after virus attachment. In addition, these antibodies exhibited high titers of complement-independent neutralizing activity against HSV-1. These sera also cross-neutralized HSV-2, albeit at low titers, and cross-reacted with gH-2 present in extracts of HSV-2-infected cells. To test the potential for gHt-gL to function as a vaccine, BALB/c mice were immunized with the complex. As controls, other mice were immunized with gD purified from HSV-infected cells or were sham immunized. Sera from the gD- or gHt-gL-immunized mice exhibited high titers of virus neutralizing activity. Using a zosteriform model of infection, we challenged mice with HSV-1. All animals showed some evidence of infection at the site of virus challenge. Mice immunized with either gD or gHt-gL showed reduced primary lesions and exhibited no secondary zosteriform lesions. The sham-immunized control animals exhibited extensive secondary lesions. Furthermore, mice immunized with either gD or gHt-gL survived virus challenge, while many control animals died. These results suggest that gHt-gL is biologically active and may be a candidate for use as a subunit vaccine.     

Cellular mechanisms involved in protection and recovery from influenza virus infection in immunodeficient mice.

We investigated the role of different lymphocyte subpopulations in the host defense reaction against influenza virus infection, taking advantage of various immunodeficient mouse strains. Whereas, following immunization, wild-type animals showed complete protection against challenge with a lethal dose of A/PR8/34 (PR8) virus, mice that lack both B and T cells but not NK cells (namely, scid and RAG2(-/-) mice) did not display any protective effect in similar conditions. By contrast, J(H)D(-/-) mice devoid of B cells and immunized with virus showed a protective response after challenge with a lethal dose. The immunized J(H)D(-/-) mice that survived completely recovered from the influenza virus infection. Immunized J(H)D(-/+) mice exhibited a more complete protection, suggesting the role of specific antibodies in resistance to infection. To assess the role of natural immunity in the host defense against influenza virus, we carried out experiments with scid mice challenged with lower but still lethal doses of PR8 virus. While an increased NK activity and an increased number of NK1.1+ cells in lungs of scid mice infected with PR8 virus were noted, in vivo depletion of the NK1.1+ cells did not affect the overall survival of the mice. Our results show that specific T cells mediate protection and recovery of J(H)D(-/-) mice immunized with live virus and challenged with lethal doses of influenza virus.     

Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F protein induces a systemic immune response

Respiratory syncytial virus (RSV) is one of the most important pathogens of infancy and early childhood. Here a fruit-based edible subunit vaccine against RSV was developed by expressing the RSV fusion (F) protein gene in transgenic tomato plants. The F-gene was expressed in ripening tomato fruit under the control of the fruit-specific E8 promoter. Oral immunization of mice with ripe transgenic tomato fruits led to the induction of both serum and mucosal RSV-F specific antibodies. The ratio of immunoglobulin subclasses produced in response to immunization suggested that a type 1 T-helper cell immune response was preferentially induced. Serum antibodies showed an increased titer when the immunized mice were exposed to inactivated RSV antigen.     

Protective effect of Bacillus anthracis surface protein EA1 against anthrax in mice

Bacillus anthracis spores germinate to vegetative forms in host cells, and produced fatal toxins. A toxin-targeting prophylaxis blocks the effect of toxin, but may allow to grow vegetative cells which create subsequent toxemia. In this study, we examined protective effect of extractable antigen 1 (EA1), a major S-layer component of B. anthracis, against anthrax. Mice were intranasally immunized with recombinant EA1, followed by a lethal challenge of B. anthracis spores. Mucosal immunization with EA1 resulted in a significant level of anti-EA1 antibodies in feces, saliva and serum. It also delayed the onset of anthrax and remarkably decreased the mortality rate. In addition, the combination of EA1 and protective antigen (PA) protected all immunized mice from a lethal challenge with B. anthracis spores. The numbers of bacteria in tissues of EA1-immunized mice were significantly decreased compared to those in the control and PA alone-immunized mice. Immunity to EA1 might contribute to protection at the early phase of infection, i.e., before massive multiplication and toxin production by vegetative cells. These results suggest that EA1 is a novel candidate for anthrax vaccine and provides a more effective protection when used in combination with PA.