Expression of a 28-Kilodalton Glutathione S-Transferase Antigen of Schistosoma mansoni on the Surface of Filamentous Phages and Evaluation of Its Vaccine Potential

A cloning and expression system that allows display of proteins on the surface of filamentous phages was exploited to display a 28-kDa glutathione S-transferase (Sm28GST) antigen of the human parasite Schistosoma mansoni. The phage-displayed Sm28GST (pdGST) was immunoreactive and was recognized by immune sera, suggesting that the Sm28GST protein displayed on the surface of phages potentially maintains native conformation. Subsequent immunization studies showed that mice can develop high titers of antibodies against pdGST and do not require any additional adjuvant for immunization. Isotype analysis suggested that the pdGST immunization predominantly induced immunoglobulin G2b (IgG2b), IgG3, and IgM anti-GST antibodies in mice. Furthermore, the pdGST immunization was found to confer about 30% protection after a challenge infection with 100 cercariae of S. mansoni in BALB/c mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against infectious agents.     

Systemic and Mucosal Immune Responses after Intranasal Administration of Recombinant Mycobacterium bovis Bacillus Calmette-Guérin Expressing Glutathione S-Transferase from Schistosoma haematobium

A major goal of current vaccine development is the induction of strong immune responses against protective antigens delivered by mucosal routes. One of the most promising approaches in that respect relies on the use of live recombinant vaccine carriers. In this study, Mycobacterium bovis BCG was engineered to produce an intracellular glutathione S-transferase from Schistosoma haematobium (Sh28GST). The gene encoding Sh28GST was placed under the control of the mycobacterial hsp60 promoter on a replicative shuttle plasmid containing a mercury resistance operon as the only selectable marker. The recombinant Sh28GST produced in BCG bound glutathione and expressed enzymatic activity, indicating that its active site was properly folded. Both intraperitoneal and intranasal immunizations of BALB/c mice with the recombinant BCG resulted in strong anti-Sh28GST antibody responses, which were enhanced by a boost. Mice immunized intranasally produced a mixed response with the production of Sh28GST-specific immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgA in the serum. In addition, high levels of anti-Sh28GST IgA were also found in the bronchoalveolar lavage fluids, demonstrating that intranasal delivery of the recombinant BCG was able to induce long-lasting secretory and systemic immune responses to antigens expressed intracellularly. Surprisingly, intranasal immunization with the BCG producing the Sh28GST induced a much stronger specific humoral response than intranasal immunization with BCG producing the glutathione S-transferase from Schistosoma mansoni, although the two antigens have over 90% identity. This difference was not observed after intraperitoneal administration.     

Macrophage nitric oxide synthase (NOS) activation by Nocardia opaca fractions and 15- and 56-kD isolated antigens

The Gram-positive bacterium, Nocardia opaca, is a source of substances with adjuvant effect, ability to stimulate macrophages and natural killer cells for enhanced cytotoxity and cytokine production and B lymphocytes for polyclonal immunoglobulin secretion. We determined the immunogenicity of isolated N. opaca fractions and prepared MoAbs against immunogenic water-soluble mitogen (NWSM). Two main proteins of molecular mass 15 and 56 kD were detected in Western blot analysis and isolated by affinity chromatography using anti-NWSM MoAb B7/7. Both these isolated nocardial antigens were found to stimulate mouse peritoneal macrophage NOS. The effect of 5 μg NWSM was comparable to that of 5 μg lipopolysaccharide (LPS) or 20 U of interferon-gamma (IFN-γ) added to cell cultures. The MoAb B7/7 decreased NO₂⁻ production induced by NWSM or by isolated nocardial antigens, but did not significantly influence the production elicited by LPS or IFN-γ. On the other hand, NOS activation by NWSM was not affected by anti-IFN-γ MoAb. The possible independent pathway for IFN-γ and NWSM macrophage activation is discussed.     

gp63 in stable cationic liposomes confers sustained vaccine immunity to susceptible BALB/c mice infected with Leishmania donovani

Visceral leishmaniasis is deadly if not treated, and development of a vaccine with long-term immunity remains a challenge. In this study, we showed that cationic distearoyl phosphatidylcholine (DSPC) liposomes, when used as vaccine adjuvant with the immunodominant 63-kDa glycoprotein (gp63) of Leishmania donovani promastigotes, induced significant protection against progressive visceral leishmaniasis in susceptible BALB/c mice. gp63 used without adjuvant elicited partial protection but in association with liposomes exhibited marked resistance in both the livers and spleens of the mice challenged 10 days after the last vaccination. The protective efficacy of liposomal gp63 vaccination was dose dependent, with 2.5 μg of protein showing optimal protection. The immunity conferred by this vaccine formulation was durable, as mice challenged 12 weeks after immunization were still protected, and the infection was controlled for at least 3 months postchallenge. Production of gamma interferon (IFN-γ) and interleukin-4 (IL-4) by splenic T cells, and of serum immunoglobulin G1 (IgG1) and IgG2a following immunization, suggested that a mixed Th1/Th2 response had been induced following immunization. However, control of disease progression and parasitic burden in mice vaccinated with gp63 in cationic DSPC liposomes was associated with enhancement of antigen-specific IFN-γ and downregulation of IL-4, demonstrating a Th1 bias. Long-term immunity elicited by this vaccine corresponded to, in addition to the presence of antigen-specific Th1, CD8⁺ T-cell responses. Our results demonstrated that stable cationic liposomes containing gp63 acted as a potent adjuvant for protein antigen to induce long-term protection against L. donovani that represents an alternative to DNA vaccination.     

Protective T Cell and Antibody Immune Responses against Hepatitis C Virus Achieved Using a Biopolyester-Bead-Based Vaccine Delivery System

Hepatitis C virus (HCV) infection is a major worldwide problem. Chronic hepatitis C is recognized as one of the major causes of cirrhosis, hepatocellular carcinoma, and liver failure. Although new, directly acting antiviral therapies are suggested to overcome the low efficacy and adverse effects observed for the current standard of treatment, an effective vaccine would be the only way to certainly eradicate HCV infection. Recently, polyhydroxybutyrate beads produced by engineered Escherichia coli showed efficacy as a vaccine delivery system. Here, an endotoxin-free E. coli strain (ClearColi) was engineered to produce polyhydroxybutyrate beads displaying the core antigen on their surface (Beads-Core) and their immunogenicity was evaluated in BALB/c mice. Immunization with Beads-Core induced gamma interferon (IFN-γ) secretion and a functional T cell immune response against the HCV Core protein. With the aim to target broad T and B cell determinants described for HCV, Beads-Core mixed with HCV E1, E2, and NS3 recombinant proteins was also evaluated in BALB/c mice. Remarkably, only three immunization with Beads-Core+CoE1E2NS3/Alum (a mixture of 0.1 μg Co.120, 16.7 μg E1.340, 16.7 μg E2.680, and 10 μg NS3 adjuvanted in aluminum hydroxide [Alum]) induced a potent antibody response against E1 and E2 and a broad IFN-γ secretion and T cell response against Core and all coadministered antigens. This immunological response mediated protective immunity to viremia as assessed in a viral surrogate challenge model. Overall, it was shown that engineered biopolyester beads displaying foreign antigens are immunogenic and might present a particulate delivery system suitable for vaccination against HCV.     

Differential induction of lupus associated antinuclear antibodies in MRL mice by monoclonal anti-Sm antibodies.

The effect of monoclonal autoantibodies on immunoregulation was investigated in MRL/MpJ-lpr/lpr mice. Passive transfer of KSm2 (a monoclonal IgG2a antibody directed against the 16 kD polypeptide of Sm) induced IgG antibodies to the other major immunoreactive polypeptides of Sm (28 and 29 kD) in all mice studied, and to polypeptides of the closely related antigen nRNP/Sm in 63% of the mice. In addition an increment in IgG anti-dsDNA antibodies, and in IgA and IgM anti-Sm antibodies, over control levels was observed. These effects were not due to polyclonal activation since anti-histone antibody levels were unaffected. Two other IgG2a monoclonal antibodies: KSm5 (directed against the 28 and 29 kD Sm polypeptides) and OX 12 (directed against an irrelevant antigen) failed to modulate the autoimmune responses of the mice in any way. These results demonstrate specific antibody-mediated connectivity between B cell clones producing autoantibodies against three distinct antigens.     

Lupus autoantibodies discriminate between the highly homologous Sm polypeptides B/B' and SmN by binding an epitope restricted to B/B'.

The ubiquitous Sm polypeptides B/B' (28 and 29 kD) and the highly homologous tissue-specific Sm N polypeptide (29 kD) share several autoepitopes recognized by systemic lupus erythematosus (SLE) sera. Previous studies on the antigenicity of nuclear antigens recognized by human autoantibodies have not discriminated between ubiquitous and tissue-specific forms. We set out to examine whether a tissue-specific nuclear antigen, Sm N, is autoantigenic in SLE by comparing the immunoreactivity of the most unique sequences in this polypeptide. Synthetic peptides from the two regions of least sequence homology that occur between Sm N and Sm B/B', a dodecamer (amino acid residues 179-190 containing five substitutions) and an undecamer (residues 203-213 containing four substitutions) were coupled to a carrier protein. These conjugates were used to quantify IgG anti-peptide antibodies in sera from patients with SLE. Of 43 sera with anti-Sm specificity, six bound to the B/B' 179-190 peptide but not to the N version. None of 17 anti-Sm-negative SLE sera bound these peptides. The second region of least sequence homology between N and B/B' (203-213) was not antigenic. Our data suggest that a subset of SLE patients with anti-Sm reactivity have IgG autoantibodies capable of discriminating between Sm N and SmB/B' polypeptides by binding a previously unreported SmB/B'-specific autoepitope. The data also indicate that brain and heart-specific anti-Sm antibodies do not exist in SLE sera, suggesting that these tissues do not participate in the induction or maintenance of the autoimmune anti-Sm response.     

Mucosal Immunogenicity of a Holotoxin-Like Molecule Containing the Serine-Rich Entamoeba histolytica Protein (SREHP) Fused to the A2 Domain of Cholera Toxin

One strategy for the induction of mucosal immune responses by oral immunization is to administer the antigen in conjunction with cholera toxin. Cholera toxin consists of one A polypeptide (CTA) which is noncovalently linked to five B subunits (CTB) via the A₂ portion of the A subunit (CTA₂). Coupling of antigens to the nontoxic B subunit of cholera toxin may improve the immunogenicity of antigens by targeting them to GM1 ganglioside on M cells and intestinal epithelial cells. Here, we describe the construction of a translational fusion protein containing the serine-rich Entamoeba histolytica protein (SREHP), a protective amebic antigen, fused to a maltose binding protein (MBP) and to CTA₂. When coexpressed in Escherichia coli with the CTB gene, these proteins assembled into a holotoxin-like chimera containing MBP-SREHP-CTA₂ and CTB. This holotoxin-like chimera (SREHP-H) inhibited the binding of cholera toxin to GM1 ganglioside. Oral vaccination of mice with SREHP-H induced mucosal immunoglobulin A (IgA) and serum IgG antiamebic antibodies and low levels of mucosal anti-CTB antibodies. Our studies confirm that the genetic coupling of antigens to CTA₂ and their coexpression in E. coli can produce holotoxin-like molecules that are mucosally immunogenic without the requirement for supplemental cholera toxin, and they establish the SREHP-H protein as a candidate for evaluation as a vaccine to prevent amebiasis.     

Expression of a 28-kilodalton glutathione S-transferase antigen of Schistosoma mansoni on the surface of filamentous phages and evaluation of its vaccine potential

A cloning and expression system that allows display of proteins on the surface of filamentous phages was exploited to display a 28-kDa glutathione S-transferase (Sm28GST) antigen of the human parasite Schistosoma mansoni. The phage-displayed Sm28GST (pdGST) was immunoreactive and was recognized by immune sera, suggesting that the Sm28GST protein displayed on the surface of phages potentially maintains native conformation. Subsequent immunization studies showed that mice can develop high titers of antibodies against pdGST and do not require any additional adjuvant for immunization. Isotype analysis suggested that the pdGST immunization predominantly induced immunoglobulin G2b (IgG2b), IgG3, and IgM anti-GST antibodies in mice. Furthermore, the pdGST immunization was found to confer about 30% protection after a challenge infection with 100 cercariae of S. mansoni in BALB/c mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against infectious agents.     

Immunogenicity and Efficacy of Flagellin-Envelope Fusion Dengue Vaccines in Mice and Monkeys

The envelope (E) protein of flaviviruses includes three domains, EI, EII, and EIII, and is the major protective antigen. Because EIII is rich in type-specific and subcomplex-specific neutralizing epitopes and is easy to express, it is particularly attractive as a recombinant vaccine antigen. VaxInnate has developed a vaccine platform that genetically links vaccine antigens to bacterial flagellin, a Toll-like receptor 5 ligand. Here we report that tetravalent dengue vaccines (TDVs) consisting of four constructs, each containing two copies of EIII fused to flagellin (R3.2x format), elicited robust and long-lived neutralizing antibodies (geometric mean titers of 200 to 3,000), as measured with a 50% focus reduction neutralization test (FRNT₅₀). In an immunogenicity study, rhesus macaques (n = 2) immunized subcutaneously with 10 μg or 90 μg of TDV three or four times, at 4- to 6-week intervals, developed neutralizing antibodies to four dengue virus (DENV) serotypes (mean post-dose 3 FRNT₅₀ titers of 102 to 601). In an efficacy study, rhesus macaques (n = 4) were immunized intramuscularly with 16 μg or 48 μg of TDV or a placebo control three times, at 1-month intervals. The animals that received 48-μg doses of TDV developed neutralizing antibodies against the four serotypes (geometric mean titers of 49 to 258) and exhibited reduced viremia after DENV-2 challenge, with a group mean viremia duration of 1.25 days and 2 of 4 animals being completely protected, compared to the placebo-treated animals, which all developed viremia, with a mean duration of 4 days. In conclusion, flagellin-EIII fusion vaccines are immunogenic and partially protective in a nonhuman primate model.     

Local transient induction of inflammatory cytokines after intranasal administration of recombinantBordetella pertussis

Inflammatory cytokines have been described to play a critical role in the orientation and amplification of the IgA immune response. In this study, we show that the intranasal administration of aBordetella pertussisstrain expressing the protective antigen glutathione-S-transferase ofSchistosoma mansoni(Sm28GST) induced an inflammatory response in the lungs of mice, characterized by the production of inflammatory cytokines, such as Tumor Necrosis Factor α, Interleukin-6 and Transforming-Growth Factor β. The production and the secretion of these cytokines in lung tissues were early and transient. Their presence was observed only during the first week after administration despite the persistence of the bacteria for 1 month. Two weeks after inoculation, Interleukin-10 secretion was detected in the lungs, which could explain the decrease in the production of inflammatory cytokines. These inflammation-regulating cytokines, induced in the lungs by the presence of the bacterial vector, could be part of the process generating the local immune response, in particular the anti-Sm28GST IgA response.     

Comparison of a Multiplexed Fluorescent Covalent Microsphere Immunoassay and an Enzyme-Linked Immunosorbent Assay for Measurement of Human Immunoglobulin G Antibodies to Anthrax Toxins

Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 μg/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 μg of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 μg/ml, while the dynamic range was 0.06 to 1.7 μg/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 μg of anti-PA IgG per ml, the RDL was 0.016 μg/ml, and the whole-serum equivalent MDC was 1.5 μg/ml. The dynamic range was 0.006 to 6.8 μg/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r² = 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.     

Erythema nodosum leprosum is associated with up-regulation of polyclonal IgG1 antibody synthesis

Erythema nodosum leprosum (ENL) is a serious complication of lepromatous (L) disease in leprosy. We have previously shown that of the four IgG subclasses, IgG1 and IgG3 Mycobacterium leprae-specific antibodies are significantly lower in leprosy patients during ENL reaction compared with untreated L patients. To see if this decrease results from a down-regulation of antibody synthesis during ENL, the frequency of antibody-secreting B cells (ABSC) in the blood compartment was determined by ELISPOT and related to serum immunoglobulin concentrations (μg/ABSC). Control groups consisted of 16 patients with stable L disease and 32 healthy endemic controls (EC). Paired samples were analysed during acute ENLS (n = 13) and after the reaction had subsided to identify changes associated with ENL. Polyclonal (PC) IgG1 was elevated in L patients compared with EC (325 μg versus 180 μg). Interestingly, patients during acute ENL showed concentrations higher than L patients (419 μg), which decreased after the reaction had subsided (260 μg), indicating the transient nature of the antibody response. IgG2 antibodies showed the reverse trend and were lower during ENL and increased after the reaction had subsided. The mean concentrations for PC IgG3 and IgG4 antibodies were similar during ENL and after the reaction had subsided. Thus, decrease in M. leprae-specific IgG1 and IgG3 antibodies is not related to down-regulation of B cell responses. Identification of factors which regulate PC IgG1 antibody synthesis may provide additional insights into determinants of ENL reactions.     

Protection against Toxoplasma gondii in mice immunized with Toxoplasma cell fractions, RNA and synthetic polyribonucleotides

Mice immunized with fractions obtained by centrifugation of disrupted Toxoplasma gondii trophozoites as well as with 200 μg of Toxoplasma ribonucleic acid (RNA) were resistant (as measured by time to death and total mortality) to challenge with Toxoplasma 30 days later. When mice were challenged at 15 days no protection was noted. A dose of 50 μg of Toxoplasma RNA was effective in protecting mice against lethal challenge only when incorporated into Freund's incomplete adjuvant. In studies performed to determine the specificity of the resistance observed, resistance was also noted in mice immunized with 200 μg of RNA extracted from normal mouse peritoneal macrophages, as well as in mice immunized with 100 μg of the synthetic polyribonucleotide polycytidylic acid. Polyadenylicuridylic acid conferred protection only when incorporated into Freund's incomplete adjuvant and polyinosinic—cytidylic acid had no effect. The protection induced by Toxoplasma RNA was eliminated by prior treatment of the preparation with ribonuclease but not by treatment with pronase, suggesting that the moiety responsible for the protective effect was RNA. In experiments designed to explore the mechanism of resistance in the vaccinated mice, macrophages harvested from mice which had been injected with Toxoplasma RNA 15 days earlier were found to be activated in that they resisted challenge with Listeria monocytogenes.     

Analysis of Human Serum Immunoglobulin G against O-Acetyl-Positive and O-Acetyl-Negative Serogroup W135 Meningococcal Capsular Polysaccharide

The capsular polysaccharide of Neisseria meningitidis serogroup W135 is expressed in both O-acetyl-positive (OA⁺) and O-acetyl-negative (OA⁻) forms. This study investigates the impact of OA status (OA⁺ versus OA⁻) on serological measurements of anti-W135 immunoglobulin G (IgG) antibodies in immunized adults. W135-specific serum antibody assignments were made for 28 postimmunization sera from adults by enzyme-linked immunosorbent assay using the meningococcal standard reference serum CDC1992. The established IgG concentration in micrograms per milliliter ([IgG]μg/ml) for CDC1992 against OA⁺ antigen (16.2 μg/ml) was used as a reference to assign a concentration of 10.13 μg/ml IgG against OA⁻ antigen by cross-standardization. Overall, the IgG assignments for these sera were higher against OA⁺ antigen (geometric mean concentration [GMC] = 7.16 μg/ml) than against OA⁻ antigen (GMC = 2.84 μg/ml). However, seven sera showed higher specific [IgG]μg/ml values against the OA⁺ antigen than against the OA⁻ antigen. These sera were also distinguished by the inability of fluid-phase OA⁻ antigen to compete for antibody binding to OA⁺ solid-phase antigen. Although there was no overall difference in functional activity measured by complement-mediated serum bactericidal assay (SBA) against OA⁺ and OA⁻ target bacteria (geometric mean titers of 9,642 and 9,045, respectively), three serum specimens showed a large difference in SBA antibody titers against OA⁺ versus OA⁻ W135 target bacteria, which may reflect different epitope specificities for these sera. Our data indicate that, for some sera, the agreement in anti-OA⁺ versus anti-OA⁻ W135 IgG assignments is serum specific and does not reflect the functional (killing) activity in vitro.     

Infection with Salmonella typhimurium modulates the immune response to Schistosoma mansoni glutathione-S-transferase.

Immune response polarization is controlled by several factors, including cytokines, antigen-presenting cells, antigen dose, and others. We have previously shown that adjuvants and live vectors play a critical role in polarization. Thus, immunization with the Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28-GST) in aluminum hydroxide induced a type 2 cytokine profile and the production of immunoglobulin G1 (IgG1)- and IgE-specific antibodies. In contrast, mice infected with recombinant Salmonella typhimurium expressing Sm28-GST developed a type 1 cytokine profile and produced IgG2a-specific antibodies against Sm28-GST and Salmonella antigens. In this study, to determine if S. typhimurium not expressing Sm28-GST would still influence the type of the response against this antigen, we compared the profiles of the immune responses generated against Sm28-GST administered in alum in mice infected and not infected with S. typhimurium. Infected mice generated both IgG1 and IgG2a antibodies against Sm28-GST, while noninfected mice produced only IgG1 anti-Sm28-GST antibodies. Moreover, interleukin-4 (IL-4) mRNA expression in infected mice was near background levels, while gamma interferon (IFN-gamma) mRNA expression in coinfected mice was significantly higher than in mice immunized with Sm28-GST in alum only. However, after antigen-specific stimulation in vitro with Sm28-GST, levels of IL-4 and IFN-gamma cytokine production were similar in the two groups of mice. These results suggest that (i) the immune milieu produced during an infection may modify the response against an irrelevant antigen and (ii) isotype switching may be influenced by the cytokine environment of a bystander immune response, even though the specific antigen-driven cytokine production is not modified. Thus, the isotypic profile is not always an absolute reflection of the cytokines produced by antigen-specific Th cells.     

The formation of immunoglobulins by human tissues in vitro: II. Quantitative studies

The present paper deals with some of the quantitative aspects of immunoglobulin formation in vitro. The results of this study show that isolated spleen cells synthesize about 1.25 μg IgG per 10⁸ lymphoid cells during the first 6 hours of incubation. The initial rate of synthesis was not maintained throughout the 48 hours of incubation, but decreased markedly after 6 hours.

Comparison of the intensity of the autoradiographic line with the amount of radioactive immunoglobulin in the precipitation line revealed that a very distinct line (gradation +++) corresponds to about 0.0125 μg IgG. The limit of visibility (+) is reached at approximately 0.0002 μg radioactive IgG.

     

Evaluation of specific humoral immune response and cross reactivity against <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> antigens induced in mice immunized with liposomes composed of total lipids extracted from <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

The development of a new tuberculosis (TB) vaccine has become one of the main objectives of the scientific community. Protein antigens have been widely explored as subunit TB vaccines, however lipid antigens could be equally important to be used or included in such a vaccine. The aim of this study was to demonstrate the potential of a liposome formulation composed of an extract of lipids from Mycobacterium smegmatis (Ms) as a TB vaccine candidate. We evaluated the immunogenicity of this formulation as well as the cross reactive response against antigens from Mycobacterium tuberculosis (MTb) in BALB/c mice. We determined the anti-liposome IgG response in sera from TB patients and from healthy subjects who displayed a positive (PPD+) or negative (PPD-) tuberculin skin test. A significant increase in anti-liposome IgG (p<0.05) was detected in animals immunized with Bacille Calmette-Guérin (BCG) compared with all groups, and in the group immunized with liposomes from Ms (LMs) compared to animals immunized with either LMs adjuvanted with aluminium (LMs-A) or the negative control group (phosphate buffered saline, PBS) respectively. With respect to the cross reactive response against a cocktail of cell wall antigens (CWA) from MTb, significantly higher IgG levels were observed in animals immunized with BCG and LMs compared to negative controls and either, aluminium-adjuvanted liposomes (LMs-A) or montanide (LMs-M) (p<0.05). Furthermore, the anti-liposome IgG response was significantly superior in sera from pulmonary TB patients compared to PPD+ and PPD- healthy subjects (p<0.001) suggesting the expression of these antigens in vivo during active MTb infection. The results obtained provide some evidence for the potential use of liposomes containing total lipid extracts of Ms as a TB vaccine candidate.     

Influence of Host Factors on Immunoglobulin G Concentration in Oral Fluid Specimens

The influence of host factors (tobacco use, dentition, bleeding gums, oral rinsing, nasal medications, and time since the last meal) on immunoglobulin G (IgG) concentration in oral fluids (OF) was determined by univariate and multivariate analysis. Significant differences in IgG concentration were found to be associated with human immunodeficiency virus (HIV) status (HIV antibody positive, +16.60 μg/ml, P = 0.0001), sex (female, +1.23 μg/ml, P = 0.004), dentition (+2.83 μg/ml, edentulous versus dentulous, P = 0.0001), bleeding gums (+6.35 μg/ml, P = 0.0001), and time since the last meal (+3.55 μg/ml, >6 h, P = 0.0001). These factors could impact diagnostic methods that rely on the immunoglobulin concentration in OF specimens.     

Expression of a Schistosoma mansoni 28-kilodalton glutathione S-transferase in the livers of transgenic mice and its effect on parasite infection.

Schistosomiasis is a debilitating tropical disease for which an effective vaccine is needed. A 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) has been shown to induce protective immunity. Sm28GST possesses significant sequence identity to mammalian GST isoforms. In order to study self-reactivity in mice immunized with Sm28GST and the concomitant phenomena of immune tolerance and epitope suppression, as well as their consequences for the protective immunity induced by this vaccination, we developed transgenic (Tg) mice that express Sm28GST under the control of a part of the mouse transferrin gene promoter. A study of (P28)Tg mice showed that the expression of Sm28GST was strictly localized in pericentrolobular hepatocytes. No histological change, inflammatory infiltrates, or modification of seric L-aspartate: 2-oxoglutarate aminotransferase concentration was observed over an 18-month period, despite a cross-reactivity between Sm28GST and a mouse molecule of 30 kDa. The immunoglobulin G anti-Sm28GST response of (P28)Tg mice immunized with recombinant Sm28GST was lower (P < 0.001) than that observed in non-(P28)Tg littermates and inversely proportional of Sm28GST liver expression. The response of non-(P28)Tg mouse spleen cells to Sm28GST stimulation was greater (P < 0.01) than that observed with (P28)Tg mouse spleen cells. (P28)Tg mice infected with 40 S. mansoni furcocercariae harbored more worms (P < 0.05) than did non-(P28)Tg control mice. The increase in the level of infection in (P28)Tg mice was reflected in concomitant increases in the numbers of adult worms and schistosome eggs found in livers and intestines after whole-body perfusion at 56 days postinfection, but no relative increase in the fertility of individual female worms was observed. The results obtained argue for the involvement of Sm28GST in reducing levels of infection and support the view that this enzyme has a central role in the maintenance of parasite viability, at least during its migration through host tissues.