Intranasal immunization with Yersinia enterocolitica O:8 cellular extract protects against local challenge infection

Yersinia enterocolitica is enteropathogenic for humans and rodents. Immune protection from oral and respiratory pathogens may be most effectively elicited following intranasal (i.n.) vaccination. An experimental murine intranasal challenge model was used to evaluate the immunogenicity of a Y. enterocolitica O:8 cellular extract (CE) in mucosa. This antigenic preparation has demonstrated to induce protection by subcutaneous immunization. Mice were immunized intranasally with two doses of CE. Immunized and nonimmunized animals were challenged with 5 x 10(6) colony-forming units (CFU) by nasal infection. Antibodies in serum and bronchoalveolar lavage (b.a.l.) fluid were assessed before and 48 hr after challenge. The CFU were determined by analysis of lung homogenate samples. The CE immunization induced significant b.a.l.-specific IgA and IgG, and serum-specific IgG, IgA and IgM. Histopathological studies 24 and 48 hr postchallenge demonstrated that immunization protected against progressive lesions resulting from Y. enterocolitica invasion of the pulmonary mucosa. The CFU in the lungs showed that CE immunization led to significant clearance as compared to the bacterial level in nonimmunized controls. From the results obtained, it can be concluded that CE can induce local and systemic immunity and protect against nasal infection.     

The pattern of inflammation in rat sepsis due to enterotoxin-producing Staphylococcus aureus: a comparison with ischemia-reperfusion injury

Sepsis and trauma have similarities in their immunopathologic profiles. Both conditions can result in multi-system organ failure which is sometimes associated with cytokine generation and inflammatory cell activation. Furthermore, decreases in peripheral blood monocyte expression of HLA-DR have been noted in both human sepsis and trauma. However, the magnitude, onset, and time course of such stimuli are often difficult to ascertain in human studies. Thus, to study a more detailed in vivo immunologic profile in these conditions, rat models were employed. Our aim was to describe and analyze cytokine and peripheral blood immunophenotype patterns in bacterially induced rat sepsis and to compare this to rat ischemia-reperfusion injury. Sprague-Dawley rats underwent either bacterial injection with enterotoxin producing Staphylococcus aureus or hind limb ischemia/ reperfusion. Two bacterial doses which were either lethal or sublethal at 24-48 hours were utilized. Peripheral blood neutrophils and B-lymphocytes were studied for expression of beta-integrins (CD11b and CD11b/c) and I-A, respectively, using flow cytometry. Corresponding plasma levels of TNF alpha and interferon gamma were measured by ELISA. At 24 hr, a lethal bacterial lethal bacterial dose injection resulted in significantly higher levels of neutrophil CD11b/c expression (p < 0.005) compared with ischemia-reperfusion treatment. B-cell I-A expression was also higher in lethal sepsis. Gamma interferon levels were significantly higher in lethal sepsis compared with ischemia-reperfusion (p = 0.005). Studies over time showed that CD11b expression and interferon gamma were both more marked at 6 hr than at 24 hr in lethal sepsis. This pattern was not observed in sublethal sepsis or in ischemia-reperfusion. CD11b/c expression on the other hand remained elevated at comparable levels at 6 and 24 hr in lethal sepsis. B-cell I-A expression in ischemia-reperfusion and sublethal sepsis decreased at 24 hr compared with baseline. Lethal sepsis in rats injected with enterotoxin producing staphylococcus results in phasic alterations in neutrophil CD11b and plasma interferon levels prior to death. In analogy to the findings of monocyte decreases in DR expression observed in human trauma and sepsis, rat B-cell I-A expression showed decreases in sublethal sepsis as well as in ischemia-reperfusion injury. However, this was not observed in lethal sepsis. These findings have implications in understanding the immunologic/inflammatory changes observed in human sepsis and trauma.     

IL-15 augments CD8+ T cell-mediated immunity against Toxoplasma gondii infection in mice

Cytokines of the Th1 profile are important mediators of protective host immunity against Toxoplasma gondii infection in mice. In this study we describe the effect of the recently identified cytokine, IL-15, on prevention of murine infection with T. gondii. Administration of exogenous rIL-15 with soluble Toxoplasma lysate Ag (TLA) provides complete protection against a lethal parasite challenge, whereas treatment with either rIL-15 or TLA alone is not protective. Following immunization with TLA/rIL-15, there is a significant proliferation of splenocytes expressing the CD8+ phenotype in response to TLA. A significant rise in the level of serum IFN gamma was observed in vaccinated mice. Adoptive transfer of CD8+ T cells, but not CD4+ T cells, from TLA/rIL-15-vaccinated mice protects naive mice from a lethal parasite challenge. These CD8+ T cells exhibit enhanced CTL activity against target macrophages infected with T. gondii. Mice that have been immunized are protected against lethal parasite challenge for at least 1 mo postvaccination. These observations demonstrate that TLA when administered with exogenous rIL-15 generates toxoplasmacidal Ag-specific CD8+ T cells. These T cells proliferate upon exposure to parasite Ag, exhibit long term memory CTL against infected target cells, and may be involved in host immune memory to this parasite.     

Mucosal DNA vaccine immunization against measles with a highly attenuated Shigella flexneri vector

An intranasal vaccine vector would elicit protective immunity at the respiratory mucosa, the portal of entry and the primary site for replication for measles virus (MV) and other respiratory viruses. In a murine model of pulmonary Shigella, we demonstrate here that a candidate-attenuated Shigella vaccine vector is safely tolerated in IFN-gamma deficient mice at an inoculum that is 1 million-fold higher than the inoculum of the wild-type parent strain that would be lethal for greater than 90% of these mice. Also, following intranasal inoculation, the Deltaasd Shigella harboring a DNA MV vaccine plasmid induces a vigorous MV-specific Th1-type (both CD8+ CTL and IFN-gamma) and, to a lesser degree, Th2-type responses among splenocytes in addition to low levels of IgG and IgA in the serum. Priming for MV-specific CTL responses was possible in mice that had prior infection with a wild-type Shigella of the same serotype. Remarkably, mice immunized by the intranasal route with attenuated Shigella harboring the DNA MV vaccine plasmid had a level of MV-specific CTL activity among splenocytes that was comparable with levels observed in mice immunized by the i.p. route with attenuated Salmonella typhi harboring the same DNA vaccine plasmid, despite the fact that Shigella remained localized to the lungs, yet Salmonella disseminated to the spleen following inoculation. Thus, Deltaasd Shigella represents a very useful vector for delivery of DNA vaccines to mucosal lymphoid tissues.     

Immunization against rabies with plant-derived antigen

We previously demonstrated that recombinant plant virus particles containing a chimeric peptide representing two rabies virus epitopes stimulate virus neutralizing antibody synthesis in immunized mice. We show here that mice immunized intraperitoneally or orally (by gastric intubation or by feeding on virus-infected spinach leaves) with engineered plant virus particles containing rabies antigen mount a local and systemic immune response. After the third dose of antigen, given intraperitoneally, 40% of the mice were protected against challenge infection with a lethal dose of rabies virus. Oral administration of the antigen stimulated serum IgG and IgA synthesis and ameliorated the clinical signs caused by intranasal infection with an attenuated rabies virus strain.     

Induction of a local anti-IpaC antibody response in mice by use of a Shigella flexneri 2a vaccine candidate: implications for use of IpaC as a protein carrier

The capacity of Shigella flexneri to act as a delivery system for stimulation of local immunity was investigated by use of an S. Flexneri 2a vaccine candidate (SC602). This vaccine strain was constructed by deletion of virulence genes responsible for dissemination (icsA) and survival (iuc:iut) of bacteria within the colonic mucosa. Among the most immunogenic S. flexneri 2a proteins inducing a local immunoglobulin A antibody response, the IpaC invasion was selected as a potential protein carrier. Overexpression of IpaC from a plasmid in trans within SC602 (SC602/pIpaC) was shown to be required for the induction of optimal anti-IpaC antibody response in the murine pulmonary infection model. A weak anti-IpaC antibody response was obtained after intranasal inoculations with SC602/pIpaC live bacteria. This response was enhanced by combining systemic priming with SC602/pIpaC killed bacteria and local boosting with SC602/pIpaC live bacteria. These results suggest that naive B cells primed systemically could account for local antibody production. This immunization protocol will allow further evaluation of the immunogenicity of IpaC hybrid proteins expresses in SC602.     

Neonatal DNA immunization with a plasmid encoding an internal viral protein is effective in the presence of maternal antibodies and protects against subsequent viral challenge

Conventional vaccines are remarkably effective in adults but are much less successful in the very young, who are less able to initiate a mature immune response and who may carry maternal antibodies which inactivate standard vaccines. We set out to determine whether DNA immunization might circumvent these problems. We have previously shown that intramuscular injection of plasmid DNA encoding the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) is capable of inducing immune responses and protecting 50% of adult mice against lethal and sublethal challenge with LCMV. Here we demonstrate that mouse pups injected with the same plasmid hours or days after birth produce major histocompatibility complex-restricted, NP-specific cytotoxic T lymphocytes (CTL) that persist into adulthood; 48% of vaccinated pups responded to subsequent sublethal viral challenge by the accelerated production of anti-NP LCMV-specific CTL, indicating that these animals had been successfully immunized by the plasmid DNA. In addition, these mice showed a >95% reduction in splenic viral titers 4 days postinfection compared to control mice, demonstrating a more rapid control of infection in vivo. Furthermore, pups born of and suckled on LCMV-immune dams (and therefore containing passively acquired anti-LCMV antibodies at the time of DNA inoculation) responded to the DNA vaccine in a similar manner, showing that maternally derived anti-LCMV antibodies do not significantly inhibit the generation of protective immune responses following DNA vaccination. These findings suggest that, at least in this model system, DNA immunization circumvents many of the problems associated with neonatal immunization.     

Treatment with anti-interleukin-10 monoclonal antibody enhances early resistance to but impairs complete clearance of Listeria monocytogenes infection in mice

Mice that received an anti-interleukin-10 (anti-IL-10) neutralizing monoclonal antibody (MAb) (SXC-1) prior to infection with Listeria monocytogenes initially demonstrated resistance to the infection, as indicated by reduced recovery of L. monocytogenes from their spleens and livers during the first 5 days after challenge. Anti-IL-10 MAb-treated mice then demonstrated reduced resistance during the later stage of infection, as indicated by persistent infection with L. monocytogenes in their livers 11 days after challenge. Aspartate aminotransferase (AST) levels (a measure of liver damage) in the sera of control mice increased between 1 and 5 days after challenge, while anti-IL-10 MAb-treated mice maintained lower AST levels. At 7 days after challenge, AST levels in the sera of control mice decreased as the numbers of organisms declined. In contrast, AST levels increased as the infections persisted in anti-IL-10 MAb-treated mice. The AST levels in serum reflected liver histopathology as anti-IL-10 MAb-treated mice exhibited fewer granulomatous lesions and less necrosis of liver tissue than the control mice during the first 5 days after challenge. Anti-IL-10 MAb treatment altered the expression of inflammatory cytokine mRNAs during L. monocytogenes infection. Control MAb-treated mice exhibited increased expression of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor mRNA in their lives during L. monocytogenes infection, but this increase did not occur in anti-IL-10 MAb-treated mice. Gamma interferon mRNA expression in the livers of the control MAb-treated mice was increased between 1 and 5 days after L. monocytogenes challenge and then decreased at 7 days after challenge. In contrast, gamma interferon mRNA expression in the livers of anti-IL-10 MAb-treated mice was not decreased until 7 days after challenge. These results indicate that endogenous IL-10 has both beneficial and detrimental effects on the host response to L. monocytogenes infection in mice.     

Blockade of endogenous TNF-alpha exacerbates primary and secondary pulmonary histoplasmosis by differential mechanisms

We investigated the mechanisms by which endogenous TNF-alpha modulates host defenses during experimental primary and secondary pulmonary infection with Histoplasma capsulatum (Hc). Neutralization of TNF-alpha in vivo resulted in increased CFU and 100% mortality in naive and immune mice challenged with Hc intranasally. Levels of IFN-gamma and granulocyte macrophage-CSF were elevated in TNF-alpha-neutralized naive mice, whereas IL-4, -6, -10 and TGF-beta did not differ from controls. In contrast, in secondary histoplasmosis, significant elevations of IL-4 and -10 were observed in TNF-alpha-depleted mice. Alveolar macrophages (Mphi) did not exert fungistatic activity against Hc after exposure to recombinant murine TNF-alpha, recombinant murine IFN-gamma, or both. The increase in susceptibility to primary Hc infection was associated with diminished production of reactive nitrogen intermediates by alveolar Mphi from TNF-alpha-depleted mice, whereas production of nitric oxide during secondary histoplasmosis was similar in both groups. Upon secondary challenge, TNF-alpha-depleted mice were rescued by concomitant neutralization of IL-4 and IL-10, but not either cytokine alone. Thus, TNF-alpha is critical for controlling primary and secondary infection with Hc, and the mechanisms that lead mice to succumb to primary or secondary infection when endogenous TNF-alpha is blocked are different.     

Release of interleukin-12 in experimental Escherichia coli septic shock in baboons: relation to plasma levels of interleukin-10 and interferon-gamma

Interleukin (IL)-12 is thought to be a key factor for the induction of interferon gamma (IFN-gamma), a cytokine essential for the lethal effects of endotoxin. We report here on the release of the nonfunctional subunit of IL-12, p40, as well as biologically active heterodimeric IL-12, p70, after administration of a lethal (n = 5) or sublethal (n = 8) dose of live Escherichia coli to baboons. Remarkably, on lethal challenge, peak levels of p40 were observed at 3 hours that were about twofold lower than those elicited after sublethal challenge (2,813 +/- 515 pg/mL v 4,972 +/- 732 pg/mL, P < .05). This disparity was also observed, although to a lesser extent, for IL-12 p70 antigen, of which maximum levels of 91 +/- 47 pg/mL and 151 +/- 41 pg/mL were measured 6 hours after a lethal or sublethal dose of E coli, respectively. Circulating p70 antigen correlated with IL-12 biologic activity (r = 0.869; P < .001). When comparing lethal to sublethal conditions, lower peak levels of IL-12 on lethal E coli sharply contrasted with higher levels of other proinflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, IL-1beta, IL-6, and IL-8 observed in these animals. Lower IL-12 concentrations in the lethal group may have resulted in part from the enhanced production of IL-10, a known inhibitor of IL-12 synthesis in vitro, as peak levels of this cytokine 3 hours postchallenge inversely correlated with peak levels of IL-12, in particular p40 (r = -0.802; P < .01). Contrary to what might be expected if IFN-gamma were solely induced by IL-12, lethally challenged baboons generated threefold more IFN-gamma at 6 hours than those receiving a sublethal dose (P < .05). Moreover, higher levels of IFN-gamma were associated with lower p40/p70 ratios, suggesting that, in agreement with observations in vitro, IFN-gamma may have preferentially upregulated the release of p70 over p40. These data show that IL-12 is released in experimental septic shock in nonhuman primates and suggest that IL-10 and IFN-gamma are involved in the regulation of this release. Furthermore, this study indicates that the systemic release of IL-12 might be essential, but is not likely sufficient, to promote lethal production of IFN-gamma in sepsis.     

In vivo effects of locally secreted IL-10 on the murine antitumor immune response

BACKGROUND: Interleukin-10 (IL-10) is a cytokine secreted by the TH2 class of murine lymphocytes that suppresses the secretion of interferon-gamma (IFN-gamma) by TH1 lymphocytes and inhibits macrophage-mediated T-cell stimulation and cytotoxicity. The observation that IL-10 is produced by human carcinomas in vitro and in vivo led to the hypothesis that this cytokine plays a role in the suppression of the human anti-tumor immune response. We tested this hypothesis in a murine model. METHODS: To evaluate the effect of IL-10 on the induction of an anti-tumor immune response, mice were immunized with tumor cells transfected with the IL-10 gene and then challenged with parental tumor. The effect of the local secretion of IL-10 on an established immune response was tested by immunizing mice with parental tumor and then challenging with IL-10-secreting tumors. RESULTS: IL-10-secreting tumors were as effective immunogens as control tumors. Immune mice rejected IL-10-secreting tumors as readily as control challenge tumors. In an in vitro assay, IL-10 did not inhibit CD8 lymphocyte secretion of IFN-gamma in response to tumor stimulation. One IL-10-secreting tumor clone regressed when injected into naive mice and induced an antigen-specific immune response capable of protecting mice from subsequent tumor challenge. CONCLUSIONS: The local secretion of IL-10 did not inhibit either the induction of an antitumor immune response or the ability of established effector cells to reject challenge tumors. In contrast to its effect on TH1 lymphocytes, IL-10 does not inhibit IFN-gamma secretion by CD8 lymphocytes.     

A nontoxic adjuvant for mucosal immunity to pneumococcal surface protein A

In this study, we demonstrated that pneumococcal surface protein A (PspA) nasally administered with a nontoxic A subunit mutant of cholera toxin (mCT) S61F elicited a protective immune response. Immunization with PspA and mCT elicited higher levels of PspA-specific IgG and IgA Abs in serum and of IgG and IgA anti-PspA Ab-forming cells in spleens, cervical lymph nodes (CLN), and lung tissue when compared to nonimmunized mice. Furthermore, significant PspA-specific IgA Abs were induced in saliva and nasal secretions. These responses were dependent on the use of mCT as a mucosal adjuvant. The PspA-specific Ab responses induced by mCT S61F were comparable with those induced by native CT (nCT). Analysis of cytokine responses showed that nasal PspA plus mCT S61F enhanced the induction of PspA-specific CD4+ T cells producing IL-4 but not IFN-gamma in CLN at both the protein and mRNA levels. Importantly, significant numbers of mice intranasally immunized with PspA plus mCT S61F were protected from lethal challenge with capsular serotype 3 Streptococcus pneumoniae A66. These results show that intranasal administration of PspA together with mCT S61F is an effective mucosal vaccine against pneumococcal infection and induces CD4+ Th2-type cells, which provide help for both mucosal and systemic Ab responses.     

Resistance to and recovery from lethal influenza virus infection in B lymphocyte-deficient mice

In the adaptive immune response to most viruses, both the cellular and humoral arms of the immune system play complementary roles in eliminating virus and virus-infected cells and in promoting recovery. To evaluate the relative contribution of CD4+ and CD8+ effector T lymphocytes in virus clearance and recovery, we have examined the host response to lethal type A influenza virus infection in B lymphocyte-deficient mice with a targeted disruption in the immunoglobulin mu heavy chain. Our results indicate that naive B cell-deficient mice have a 50- 100-fold greater susceptibility to lethal type A influenza virus infection than do wild type mice. However, after priming with sublethal doses of influenza, immune B cell-deficient animals show an enhanced resistance to lethal virus infection. This finding indicates that an antibody-independent immune-mediated antiviral mechanism accounts for the increased resistance to lethal virus challenge. To assess the contribution of influenza-specific CD4+ and CD8+ effector T cells in this process, defined clonal populations of influenza-specific CD4+ and CD8+ effector T cells were adoptively transferred into lethally infected B cell-deficient mice. Cloned CD8+ effectors efficiently promoted recovery from lethal infection, whereas cloned CD4+ T cells conferred only partial protection. These results suggest that memory T lymphocytes can act independently of a humoral immune response in order to confer resistance to influenza infection in immune individuals. The potential implications of these results for vaccination against human influenza infection are discussed.     

Short-chain fatty acids improve clinical, pathologic, and microbiologic features of experimental shigellosis

Because of the metabolic and antibacterial actions of short-chain fatty acids (SCFA), their roles in modifying the clinicopathologic features of shigellosis were evaluated in a rabbit model of shigellosis. Acute colitis was induced in adult rabbits by intracolonic administration of Shigella flexneri 2a. After 24 h, rabbits were given 6-h colonic infusions of SCFA (acetate, propionate, n-butyrate; 60:30:40 mM) or SCFA-free solution (control); groups of rabbits were killed in batches of 2 or 3 animals at 24, 48, 72, and 96 h after treatment, for histologic and bacteriologic assessment. SCFA significantly reduced fecal blood and mucus and improved clinical symptoms. Histologically, SCFA significantly (P<.01) reduced mucosal congestion, cellular infiltration, and necrotic changes. SCFA also significantly (P<.05) reduced the number of shigellae in the colon. No such improvements occurred in the control group. SCFA may be useful agents in improving clinicopathologic features of shigellosis and should be clinically evaluated.     

Polypyrimidine tract-binding protein positively regulates inclusion of an alternative 3''-terminal exon

A leading candidate for a vaccine targeted at the erythrocytic stages of plasmodial parasite development is the merozoite surface protein-1 (MSP-1). We have previously shown that the carboxyl-terminal region of MSP-1 derived from Plasmodium yoelii yoelii 17XL, expressed as a fusion protein with glutathione S-transferase (GST-PYC2), can immunize mice against an otherwise lethal homologous challenge infection. This protection has been shown to be predominantly mediated by antibodies. We report here on the efficacy of immunization with MSP-1 carboxyl regions when the challenge is a heterologous rodent parasite species. The course of parasitemia was not altered in mice immunized with GST-PYC2 and challenged with 10(4) heterologous Plasmodium chabaudi adami parasites, as both control and immunized mice developed infections that peaked at day 7 and then rapidly declined. Similarly, mice immunized with GST-PYC2 and challenged with 10(5) Plasmodium berghei ANKA parasites displayed virulence similar to that seen in infection control mice. The homologous region of the P. chabaudi adami MSP-1 gene was similarly expressed as a fusion protein with GST. Mice immunized with GST-PCC2 and challenged with 10(4) parasites showed significant protection against homologous P. chabaudi adami infection but no protection whatsoever against heterologous P. yoelii yoelii 17XL infection. These in vivo results correlate with the observation that sera generated by immunization with the carboxyl region of MSP-1 recognizes this protein from homologous, but not heterologous, radiolabeled parasite protein preparations.     

Modification of experimental Porphyromonas gingivalis murine infection by immunization with a polysaccharide-protein conjugate

To better understand the role of the capsular polysaccharide in the virulence of Porphyromonas gingivalis, the effect of immunization with a polysaccharide-protein conjugate on experimental murine infection was evaluated. The conjugate was prepared using polysaccharide isolated from P. gingivalis strain ATCC 53977 and bovine serum albumin. One group of 22 mice was immunized by intraperitoneal injection with the conjugate and a control group of 25 mice was similarly immunized with bovine serum albumin. Serum antibody reactive to the polysaccharide, as determined by enzyme-linked immunosorbent assay, was elevated in the group of mice immunized with the polysaccharide-protein conjugate but not in the mice immunized with bovine serum albumin. Both groups of mice were challenged with P. gingivalis strain ATCC 53977 (10(10) cells) administered subcutaneously on the dorsal surface. Following challenge, the mice immunized with the polysaccharide-protein conjugate appeared healthier and demonstrated less weight loss than did the control group of mice. Ulcerative lesions at secondary locations were smaller in mice immunized with the polysaccharide-protein conjugate. Thus, immunization of mice with a conjugate containing P. gingivalis polysaccharide could reduce the severity of but not prevent an invasive infection with P. gingivalis.     

Cytokine profiles of lesional and splenic T cells in Porphyromonas gingivalis infection in a murine model

T cell cytokine profiles in the spleens and Porphyromonas gingivalis-induced lesions of P. gingivalis-immunized mice were examined. BALB/c mice were immunized with P. gingivalis outer membrane (OM) antigens/mouse weekly for 3 weeks followed by challenge with live organisms 2 weeks after the final immunization. Control mice were immunized with PBS. Spleens were excised at 0 and 4 days and lesions at 1, 4, and 7 days after challenge. Splenic and lesional CD4 and CD8 cells were stained for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma, and IL-10. More than 50% of the T cells in the spleens of immunized mice were IFN-gamma positive at day 0 which was significantly higher than for IL-4 or IL-10, these levels decreasing significantly 4 days after challenge. Less than 6% of the T cells in sham immunized mice were cytokine positive at day 0, although at day 4, there was a significant increase in the percent IL-10 positive CD4 cells and IL-4 and IL-10 positive CD8 cells. There were no differences in the percent IL-4, IFN-gamma, or IL-10 positive T cells in the lesions of immunized mice, but there was a dramatic decrease at day 7 to very low levels in control mice. In conclusion, the results of the present study show a predominant Th1 response in the spleens of BALB/c mice after immunization with P. gingivalis OM antigens, suggesting that a protective immune response to P. gingivalis may involve a strong IFN-gamma response.     

Genetic vaccination against Coccidioides immitis: comparison of vaccine efficacy of recombinant antigen 2 and antigen 2 cDNA

Previous studies from our laboratory established that C-ASWS, an alkali-soluble, water-soluble extract from cell walls of Coccidioides immitis, protects mice against lethal challenge with this fungus. The C-ASWS extract contains a glycosylated protein, designated antigen 2 (Ag2), and a polysaccharide antigen. We recently cloned Ag2 cDNA and showed that the recombinant fusion protein elicited strong delayed-type hypersensitivity responses in immunized mice. This investigation was undertaken to determine if the recombinant Ag2 protein, expressed as an Ag2-glutathione S-transferase (GST) fusion protein, or Ag2 cDNA would protect mice against lethal challenge with C. immitis. The recombinant Ag2-GST protein protected BALB/c mice against intraperitoneal challenge with 250 arthroconidia, as assessed by a decrease in fungal CFU in tissues. The Ag2-GST-immunized mice did not show, however, an increased survival during a 30-day period postinfection. By contrast, immunization of mice with Ag2 cDNA ligated into the pVR1012 plasmid engendered protection against intraperitoneal challenge with 2,500 arthroconidia and against pulmonary challenge with 50 arthroconidia. Vaccine efficacy paralleled the development of delayed-type hypersensitivity responses to C. immitis antigen. Whereas mice vaccinated with the recombinant Ag2-GST protein did not mount footpad hypersensitivity to C-ASWS or the recombinant Ag2-GST protein, mice vaccinated with the pVR1012-Ag2 construct mounted a strong footpad hypersensitivity and their spleen cells secreted gamma interferon upon in vitro stimulation with the Ag2-containing C-ASWS extract. This is the first investigation to show that genetic immunization can protect against lethal challenge with C. immitis.     

Characterization of virulence genes of enteroinvasive Escherichia coli by TnphoA mutagenesis: identification of invX, a gene required for entry into HEp-2 cells

While enteroinvasive Escherichia coli (EIEC) and shigellae are genotypically nearly identical, a difference has been reported in the infective dose to humans: EIEC is 10,000-fold less infectious than shigellae. A possible basis for this difference lies in the inherent invasiveness of these bacteria toward epithelial cells. Thus, despite the high degree of homology between the invasion plasmids of EIEC and shigellae, substantial differences in genetic organization and/or sequence may exist. We have undertaken a systematic genetic analysis of the EIEC plasmid pSF204, using transposon mutagenesis. Congo red-negative TnphoA insertion mutants (Pcr- PhoA-) and TnphoA fusion mutants (PhoA+) were isolated and screened for the ability to invade cultured HEp-2 cells. Most invasion-negative (Inv-) mutations mapped to a 30-kb segment of the invasion plasmid, including homologs of the Shigella flexneri ipa, mxi, and spa genes. Inv- PhoA+ fusions in the EIEC ipaC, mxiG, mxiJ, mxiM, and mxiD homologs and in a proposed new gene, named invX, located downstream of the spa region were identified and characterized. This analysis indicates the presence of the ipaC, mxiG, mxiJ, mxiM, mxiD, and invX gene products in the EIEC cell envelope and demonstrates a strict requirement for these genetic loci in invasion. Overall, our results suggest a high degree of genetic, structural, and functional homology between the EIEC and S. flexneri large invasion plasmids.     

Transcutaneous immunization with cholera toxin protects mice against lethal mucosal toxin challenge

We recently reported that application of cholera toxin (CT) to the skin results in transcutaneous immunization and induces a systemic Ab response to both CT and coadministered Ags. In this paper, we demonstrate antitoxin IgG and IgA Abs in sera, lung washes, and stool samples from immunized mice as well as a broad spectrum of IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3) in the sera. Mice immunized with CT by the transcutaneous route exhibited significant protection from intranasal challenge with a lethal dose of CT. Thus, clinically relevant immunity against mucosal toxin challenge can be achieved via the transcutaneous route.