Specific Serum Antibody Titers Research Articles

Methanol extract of Nyctanthes arbortristis seeds enhances non-specific immune responses and protects Oreochromis mossambicus (Peters) against Aeromonas hydrophila infection

Immunostimulation using medicinal plant extracts is a promising approach for prevention and control of diseases with reference to sustainable fish farming. Oreochromis mossambicus, dubbed as aquatic chicken is a cultured fish worldwide and a laboratory model organism. Aeromonas hydrophila is one of the major bacterial pathogens in fish farming that causes huge loss to aquaculture industries. In this study, we investigated the efficacy of methanol extract of Nyctanthes arbortristis seeds on disease resistance of O. mossambicus against live virulent A. hydrophila. We also investigated its effect on the non-specific immune parameters such as serum lysozyme, myeloperoxidase, antiprotease and specific immune parameters in terms of specific serum antibody titres assayed by bacterial agglutination test. Our studies indicate that intra-peritoneal administration of 20mg/kg methanol extract increases the Relative Percent Survival (RPS) of O. mossambicus challenged with LD80 of A. hydrophila. Further, both non-specific and specific immune parameters were enhanced by the methanol extract. Further experiments at molecular levels in the laboratory and also efficacy testing at field level are essential before applying this plant product in aquaculture industry.

Recent Advances in the Diagnosis of Pneumocystis Pneumonia.

Pneumocystis jirovecii is a prototypical opportunistic pathogen, causing an asymptomatic or mild infection in normal hosts and fulminating pneumonia (Pneumocystis pneumonia, PCP) in immunocompromised hosts. PCP is a leading cause of morbidity and mortality in immunocompromised patients such as AIDS patients. Microscopic detection of cysts and trophic forms of P. jirovecii in respiratory secretions is simple and useful but may underestimate the P. jirovecii infection. Conventional polymerase chain reaction (PCR) and nested PCR increase the sensitivity and specificity to identify PCP and provide an approach to discriminate PCP from pulmonary P. jirovecii colonization, but the targeted genes and cut-off value from quantitative real-time PCR remain to be determined. Serum (1-3)-β-D-glucan level and the specific serum antibody titer are ancillary indicators for PCP diagnosis. The successful cultivation of P. jirovecii in vitro is an important progress for PCP research. The diagnosis of PCP relies on the combination of these laboratory examinations as well as the clinical presentations.

B subunits of cholera toxin and thermolabile enterotoxin of Escherichia coli have similar adjuvant effect as whole molecules on rotavirus 2/6-VLP specific antibody responses and induce a Th17-like response after intrarectal immunization

The purpose of this study was to evaluate the adjuvant effect of the B subunits of cholera toxin (CT) and the thermolabile enterotoxin of Escherichia coli (LT) by the intrarectal route of immunization and compare them to the whole molecules CT and LT-R192G, a non toxic mutant of LT, using 2/6-VLP as an antigen, in mice.All molecules induced similar antigen specific antibody titers in serum and feces, whereas different T cell profiles were observed. CTB and LTB, conversely to CT and LT-R192G, did not induce detectable production of IL-2 by antigen specific T cells. Moreover, CTB, conversely to LT-R192G, CT and LTB, did not induce antigen specific CD4+CD25+Foxp3− and Foxp3+ T cells, thus showing different effects between the B subunits themselves. However, all molecules induced an antigen specific Th17 response. In conclusion, B subunits are potent adjuvants on B cell responses by the intrarectal route. Although their impact on T cell responses are different, all molecules induce a 2/6-VLP-specific Th17 T cell response that may play a major role in helping B cell responses and thus in adjuvanticity and protection.

Over-Expression and Immunogenicity of Outer Membrane Protein L (OprL) of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a notorious pathogen that causes opportunistic infections in humans and animals. Despite high number of patients who may develop P. aeruginosa infections and the threat of antibiotic treatment failure due to fast development of antimicrobial resistance, surprisingly no vaccine is currently available in the market against the bacterium. The present study is aimed to produce recombinant outer membrane protein L (rOprL) of P. aeruginosa using suitable prokaryotic expression system and to evaluate its immunogenicity in experimental animals so that it can be considered as a vaccine candidate in future. OprL was successfully expressed and purified as ~19 kDa protein. Immunization of mice with rOprL resulted in increased antigen specific serum antibody titer and protected the mice against P. aeruginosa challenge. This preliminary study suggests that OprL may prove to be a potential subunit vaccine candidate against P. aeruginosa infections.

Evaluation of recombinant leukocidin domain of VvhA exotoxin of Vibrio vulnificus as an effective toxoid in mouse model

Vibrio vulnificus hemolysin A (VvhA) is a pore forming toxin and plays an important role in the pathogenesis. The hemolytic and cytotytic property of VvhA toxin is associated with N-terminal leukocidin domain which triggers apoptotic signaling cascade in epithelial cells. The present study was undertaken to assess the protective efficacy of recombinant VvhA leukocidin domain (rL/VvhA) against VvhA toxin challenge using in vitro and in vivo assays. The rL/VvhA protein was found to be non-toxic with no significant hemolytic or cytotoxic effects. Intraperitoneal (I.P.) immunization of BALB/c mice with rL/VvhA protein elicited significantly higher specific serum antibody titer with mixed Th1/Th2 mediated immune responses. HeLa cell monolayer supplemented with anti-rL/VvhA antibodies were effectively protected (viability 86.69%) against lethal 5 LD50 toxin challenge. An effective in vitro proliferation of lymphocyte was observed upon re-stimulation of rL/VvhA primed splenocytes with formalin inactivated VvhA toxin (fVvhA). Co-expression of Th1/Th2 polarized cytokines (IFN-γ, IL-12 and IL-4), were seen in the cell culture supernatant. In contrast to sham immunized mice, rL/VvhA immunized mice demonstrated significant protection (90% survival) against native toxin challenge in vitro and in vivo infection models. These results suggested leukocidin domain of the VvhA toxin as protective immunogen for possible protection against V. vulnificus VvhA.

CCR10 expression is required for immunogenicity of a HIV-1env DNA vaccine encoding CCL28 to enhance HIV-1env-specific IgG and IgA at relevant mucosal sites (VAC7P.986)

Abstract Development of a vaccine that drives anti-viral mucosal B cell responses is critical for protection against HIV-1 infection. It is currently unknown whether triggering CCR10/CCL28 pathways in a DNA-based vaccine results in induction of HIV-1env specific B cell immunity at mucosal sites of infection. We hypothesized that co-immunization with HIV-1env/CCL28 molecular adjuvant would augment B cell responses at gastrointestinal and vaginal sites and require CCR10. CCL28 co-immunized WT mice displayed a significant enhancement of HIV-1env specific antibody titers in serum, feces and vaginal washes, and enhanced HIV-1 specific IgA responses were abrogated in CCR10KO mice. CCL28 co-immunization did not increase the breadth of linear B cell epitopes in WT mice, but augmented the dominant epitopes elicited by antigen immunization alone. The frequency of splenic and intestinal IgA+CD19+B220+CD138+CCR10+ plasmablasts was augmented in the CCL28 co-immunized WT mice over antigen-only immunized WT controls. The physiological relevance of these findings was confirmed in a NHP model of intravaginal SIVsmE660 challenge in which CCL28 co-immunization resulted in significant increases in serum/vaginal IgG/IgA, decrease in peak viral loads, significant suppression of viral titers over 120 days, and recovery of CD4 T cells. These data support a role for CCL28 in targeting protective anti-viral B cells to mucosal sites when delivered as molecular adjuvants for HIV-1env DNA-based vaccines.

Evidence of Airborne Excretion of Pneumocystis carinii during Infection in Immunocompetent Rats. Lung Involvement and Antibody Response

To better understand the role of immunocompetent hosts in the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected Sprague Dawley rats was quantified by means of a real-time PCR assay, in parallel with the kinetics of P. carinii loads in lungs and specific serum antibody titres. Pneumocystis-free Sprague Dawley rats were intratracheally inoculated at day 0 (d0) and then followed for 60 days. P. carinii DNA was detected in lungs until d29 in two separate experiments and thereafter remained undetectable. A transient air excretion of Pneumocystis DNA was observed between d14 and d22 in the first experiment and between d9 and d19 in the second experiment; it was related to the peak of infection in lungs. IgM and IgG anti-P. carinii antibody increase preceded clearance of P. carinii in the lungs and cessation of airborne excretion. In rats receiving a second challenge 3 months after the first inoculation, Pneumocystis was only detected at a low level in the lungs of 2 of 3 rats at d2 post challenge and was never detected in air samples. Anti-Pneumocystis antibody determinations showed a typical secondary IgG antibody response. This study provides the first direct evidence that immunocompetent hosts can excrete Pneumocystis following a primary acquired infection. Lung infection was apparently controlled by the immune response since fungal burdens decreased to become undetectable as specific antibodies reached high titres in serum. This immune response was apparently protective against reinfection 3 months later.

The immune complex CTA1-DD/IgG adjuvant specifically targets connective tissue mast cells through FcγRIIIA and augments anti-HPV immunity after nasal immunization.

We have previously reported that CTA1-DD/IgG immune complexes augment antibody responses in a mast cell-dependent manner following intranasal (IN) immunizations. However, from a safety perspective, mast cell activation could preclude clinical use. Therefore, we have extended these studies and demonstrate that CTA1-DD/IgG immune complexes administered IN did not trigger an anaphylactic reaction. Importantly, CTA1-DD/IgE immune complexes did not activate mast cells. Interestingly, only connective tissue, but not mucosal, mast cells could be activated by CTA1-DD/IgG immune complexes. This effect was mediated by FcγRIIIA, only expressed on connective tissue mast cells, and found in the nasal submucosa. FcγRIIIA-deficient mice had compromised responses to immunization adjuvanted by CTA1-DD/IgG. Proof-of-concept studies revealed that IN immunized mice with human papillomavirus (HPV) type 16 L1 virus-like particles (VLP) and CTA1-DD/IgG immune complexes demonstrated strong and sustained specific antibody titers in serum and vaginal secretions. From a mast cell perspective, CTA1-DD/IgG immune complexes appear to be safe and effective mucosal adjuvants.

Enhancement of humoral immunity in mice by coupling pUCpGs10 and aluminium to the HCV recombinant immunogen

AimTo investigate the enhancement of humoral immunity when CpG ODN (cytidine phosphate guanosine oligodeoxynucleotides) and aluminium adjuvants are complexed with the HCV (Hepatitis C virus) recombinant immunogen in mice.MethodsAfter immunizing Balb/c mice with the recombination HCV antigen adjuvanted with pUCpGs10 and/or aluminium(antigen+CpG+alum, antigen+CpG, antigen+alum, antigen+PBS), enzyme-linked immunosorbent assay (ELISA) was used to measure the specific serum antibody titers of IgG, to determine the neutralization response to various peptide genotypes, and to determine the concentration of IL-6 and IL-10 in supernatants of in vitro cultured splenic lymphocytes. Enzyme-linked immunospot assay (ELISPOT) was used to quantify the non-specific and specific splenic antibody-secreting cells (ASCs), and flow cytometry (FCM) determined the ratio of different splenic lymphocytes. The serum of rabbits immunized with the recombinant pBVGST/HVR1 antigen immunoprecipitated the HCV isolated from 12 patients' serum.ResultsThe sera antibody titers were 1:51200, 1:9051, 1:18102, 1:6400 respectively after the final immunization and demonstrated good neutralization responses to the six gene peptide containing 1a, 1b, 2a, 3a, 4a and 6a. The aluminum adjuvant increased the population of both specific ASCs (P < 0.01) and total ASCs(P < 0.05), with a proportional rise in concentrations of CD19+CD27+ (P < 0.05), as well as levels of IL-6, IL-10 (P < 0.05) in splenic lymphocytes. The results clearly indicated a significantly higher number of CD19+CD38+ splenic lymphocytes with the aluminum and pUCpGs10 adjuvant present compared to the control group(P < 0.05). Anti-HVR1 antibody in induced mice can cross-reactively capture HCV particles (10/12).Conclusions1. The aluminum adjuvant induces a potent Th2-biased immune response by increasing both the populations of specific and total ASCs and the ratio of CD19+CD27+ cells. 2. The pUCpGs10 complexed with the aluminum adjuvant boosts the population of plasma cells and increase the efficiency of the immune response. 3. The two adjuvants have synergistic effects on humoral immunity. 4. The recombinant HVR1 protein has the possibility of generating broadly reactive anti-HVR1 antibody.

Heterologous Epitope-Scaffold Prime∶Boosting Immuno-Focuses B Cell Responses to the HIV-1 gp41 2F5 Neutralization Determinant

The HIV-1 envelope glycoproteins (Env) gp120 and gp41 mediate entry and are the targets for neutralizing antibodies. Within gp41, a continuous epitope defined by the broadly neutralizing antibody 2F5, is one of the few conserved sites accessible to antibodies on the functional HIV Env spike. Recently, as an initial attempt at structure-guided design, we transplanted the 2F5 epitope onto several non-HIV acceptor scaffold proteins that we termed epitope scaffolds (ES). As immunogens, these ES proteins elicited antibodies with exquisite binding specificity matching that of the 2F5 antibody. These novel 2F5 epitope scaffolds presented us with the opportunity to test heterologous prime∶boost immunization strategies to selectively boost antibody responses against the engrafted gp41 2F5 epitope. Such strategies might be employed to target conserved but poorly immunogenic sites on the HIV-1 Env, and, more generally, other structurally defined pathogen targets. Here, we assessed ES prime∶boosting by measuring epitope specific serum antibody titers by ELISA and B cell responses by ELISpot analysis using both free 2F5 peptide and an unrelated ES protein as probes. We found that the heterologous ES prime∶boosting immunization regimen elicits cross-reactive humoral responses to the structurally constrained 2F5 epitope target, and that incorporating a promiscuous T cell helper epitope in the immunogens resulted in higher antibody titers against the 2F5 graft, but did not result in virus neutralization. Interestingly, two epitope scaffolds (ES1 and ES2), which did not elicit a detectable 2F5 epitope-specific response on their own, boosted such responses when primed with the ES5. Together, these results indicate that heterologous ES prime∶boost immunization regimens effectively focus the humoral immune response on the structurally defined and immunogen-conserved HIV-1 2F5 epitope.

Stemucronatoside K, a novel C 21 steroidal glycoside from Stephanotis mucronata, inhibited the cellular and humoral immune response in mice

Stephanotis mucronata (Blanco) Merr. has been used for rheumatoid arthritis in Chinese folk herb medicine. Guided by bioactive test, a novel potent immunosuppressive C 21 steroidal glycoside stemucronatoside K (SMK) was isolated from this plant. Its structure was elucidated on the basis of the chemical evidence and extensive spectroscopic methods. We investigated the immunosuppressive effects of SMK in vitro and in vivo. SMK significantly suppressed concanavalin A (Con A)- and lipopolysaccharide (LPS)-stimulated splenocyte proliferation in vitro in a concentration-dependent manner. ICR mice were immunized subcutaneously with OVA on the first day and administered intraperitoneally with SMK at the doses of 2.5, 5, and 10 mg/kg once daily for 10 days. At 24 h after the last administration, mitogen- and OVA-stimulated splenocyte proliferation, the levels of cytokines from splenocytes, and specific antibody titers in serum were measured. SMK significantly inhibited Con A-, LPS- and OVA-induced splenocyte proliferation in the immunized mice in a dose-dependent manner. OVA-specific IgG, IgG1, and IgG2b antibody titers were significantly reduced by SMK compared with the control group. SMK also significantly decreased OVA-induced interleukin-2 (IL-2), interferon-γ (IFN-γ), and IL-4 production from splenocytes in the OVA-immunized mice. These results demonstrated that SMK could suppress the cellular and humoral immune response in mice. This study provided evidence to understand the therapeutic effects of S. mucronata and an immunosuppressive natural product compound to further researches to be developed as immunosuppressant.

Development of a tripartite vector system for live oral immunization using a Gram-negative probiotic carrier

The mucosa represents the primary target site and thus the first barrier for most microbial pathogens. Nevertheless, nearly all present-day vaccines are applied by an invasive route, target the systemic immune system, and do not confer efficient mucosal protection. Currently, mucosal immunity can only be achieved by the delivery of antigens via the mucosal route. Therefore, multiple efforts are under way to develop mucosal vaccines and particularly live oral vaccines as these would confer considerable advantages. We have engineered the AIDA autotransporter system for the surface presentation and/or release of heterologous polypeptides. This study is focused on the development and evaluation of a tripartite live bacterial vector system for oral vaccination based on the AIDA autotransporter, heterologous virulence factor-derived (poly-)peptides, and the apathogenic Escherichia coli Nissle 1917 strain as a live carrier. Potentially with this system also attenuated Salmonella or Shigella strains might be employed as carriers. Model antigens included e.g. the p60 antigen of Listeria monocytogenes, the OspA/OspG antigens of B. burgdorferi, the LT-B subunit of E. coli, and Stx-B subunits of enterohemorrhaghic E. coli (EHEC), all representing crucial virulence factors of important bacterial pathogens. Exemplary oral immunization studies were conducted using different regimes in BALB/c mice with candidate vaccines expressing Stx B-subunits and OspA and OspG proteins. To monitor the induction of immune responses, specific antibody titers in serum as well as secreted mucosal antibodies of local and distal mucosal surfaces were determined. Antigen-specific mucosal as well as systemic antibodies could be induced; however, thus far the response turned out to be heterogeneous and appeared not to be sufficient to mediate protection.

High Incidence of Actinobacillus Actinomycetemcomitans Infection in Acute Coronary Syndrome

Recent epidemiological studies suggest that periodontitis is an important risk factor for coronary heart disease (CHD). The aim of this study was to evaluate the association between periodontitis and CHD, particularly acute coronary syndrome (ACS), focusing on microbiological and immunological features. Twenty-eight CHD patients, 15 with ACS and 13 with chronic CHD, were included in this study. Coronary angiography, periodontal examination, and dental radiography were performed in all patients. Subgingival plaque, saliva, and blood samples were analyzed for the periodontopathogens Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythensis, Treponema denticola, and Prevotella intermedia using polymerase chain reaction. Specific serum antibody titers to the 5 periodontal pathogens were determined by enzyme-linked immunosorbent assay. It was found that 33% of the ACS patients (5/15) harbored A. actinomycetemcomitans in oral samples, whereas no A. actinomycetemcomitans (0/13) was found in the chronic CHD patients (P < 0.05). Furthermore, ACS patients showed significantly higher serum IgG titers to A. actinomycetemcomitans (P < 0.05) compared with chronic CHD. More tooth loss and alveolar bone loss were noted in ACS patients than in chronic CHD patients, although the differences were not statistically significant. Periodontal pathogens, particularly A. actinomycetemcomitans, may play a role in the development of ACS.

Nasal Immunization with a Malaria Transmission-Blocking Vaccine Candidate, Pfs25, Induces Complete Protective Immunity in Mice against Field Isolates of Plasmodium falciparum

Malaria transmission-blocking vaccines based on antigens expressed in sexual stages of the parasites are considered one promising strategy for malaria control. To investigate the feasibility of developing noninvasive mucosal transmission-blocking vaccines against Plasmodium falciparum, intranasal immunization experiments with Pichia pastoris-expressed recombinant Pfs25 proteins were conducted. Mice intranasally immunized with the Pfs25 proteins in the presence of a potent mucosal adjuvant cholera toxin induced robust systemic as well as mucosal antibodies. All mouse immunoglobulin G (IgG) subclasses except IgG3 were found in serum at comparable levels, suggesting that the immunization induced mixed Th1 and Th2 responses. Consistent with the expression patterns of the Pfs25 proteins in the parasites, the induced immune sera specifically recognized ookinetes but not gametocytes. In addition, the immune sera recognized Pfs25 proteins with the native conformation but not the denatured forms, indicating that mucosal immunization induced biologically active antibodies capable of recognizing conformational epitopes of native Pfs25 proteins. Feeding Anopheles dirus mosquitoes with a mixture of the mouse immune sera and gametocytemic blood derived from patients infected with P. falciparum resulted in complete interference with oocyst development in mosquito midguts. The observed transmission-blocking activities were strongly correlated with specific serum antibody titers. Our results demonstrated for the first time that a P. falciparum transmission-blocking vaccine candidate is effective against field-isolated parasites and may justify the investigation of noninvasive mucosal vaccination regimens for control of malaria, a prototypical mucosa-unrelated mosquito-borne parasitic disease.

Mucosal immune responses are related to reduction of bacterial colonization in the stomach after therapeutic Helicobacter pylori immunization in mice

The aim of this study was to investigate the capacity of oral and parenteral therapeutic immunization to reduce the bacterial colonization in the stomach after experimental Helicobacter pylori infection, and to evaluate whether any specific immune responses are related to such reduction. C57BL/6 mice were infected with H. pylori and thereafter immunized with H. pylori lysate either orally together with cholera toxin or intraperitoneally (ip) together with alum using immunization protocols that previously have provided prophylactic protection. The effect of the immunizations on H. pylori infection was determined by quantitative culture of H. pylori from the mouse stomach. Mucosal and systemic antibody responses were analyzed by ELISA in saponin extracted gastric tissue and serum, respectively, and mucosal CD4 + T cell responses by an antigen specific proliferation assay. Supernatants from the proliferating CD4 + T cells were analyzed for Th1 and Th2 cytokines. The oral, but not the parenteral therapeutic immunization induced significant decrease in H. pylori colonization compared to control infected mice. The oral immunization resulted in markedly elevated levels of serum IgG+M as well as gastric IgA antibodies against H. pylori antigen and also increased H. pylori specific mucosal CD4 + T cell proliferation with a Th1 cytokine profile. Although the parenteral immunization induced dramatic increases in H. pylori specific serum antibody titers, no increases in mucosal antibody or cellular immune responses were observed after the ip immunization compared to control infected mice. These findings suggest that H. pylori specific mucosal immune responses with a Th1 profile may provide therapeutic protection against H. pylori.

Immune tolerance: DNA vaccine as strategy in autoimmune diseases

Autoimmune diseases (AID) affect 5–8% of occidental populations. Theses AID are characterized by the destruction of self-tissue following activation of autoimmune responses mediated by T cells or B cells, or both. Manipulations of immune responses to protect against autoimmunity as potential immunotherapeutic implications using DNA vaccination could help to treat and/or prevent AID. Recently, we have generated an animal model to study the role of the molecular mimicry in breaking tolerance to a neo-antigen in the liver. Transgenic (Tg) mice expressing the nucleoprotein (NP) gene from lymphocytic choriomingitis virus (LCMV) as neo-self antigen in their hepatocytes develop an autoimmune hepatitis after immunization with plasmids coding for intracellular or secreted LCMV-NP forms [1]. Four groups of transgenic mice (Tg-NP) were immunized via the intramuscular route with both these plasmids (pNP) alone or co-injected with IL-12-expressing plasmid (pIL-12). NP-specific serum antibody titres were detected in an ELISA test. All animals injected with different plasmid-based gene expression vectors showed different anti-NP antibody profiles. The IgG1 subclass is an indication of Th2 immune response, whereas IgG2 subclass production reflects a Th1 immune response. Mice vaccinated with intracellular form-coding plasmid alone or with pIL-12 developed an antibody response characterized by more pronounced Th2 profiles (IgG1 isotype). In contrast, those injected with the secreted form injections alone or with pIL-12 produced exclusively IgG2 isotypes indicative of a Th1 response. These results suggest that immune response profiles provoked by DNA vaccine seem to be dependent on the protein form rather than on the cytokine adjuvant or on the administration route of the DNA vaccine. Even if cytokine (IL-12) driving B cells and T cells to a Th1 phenotype response was added, specific serum antibody titres were significantly higher in mice co-injected with the intracellular form compared with those co-immunized with the secreted form. The form of autoantigen used in DNA vaccination may induce protection from autoimmunity by inducing a cytokine shift in the immune response to the antigens responsible for the disease. Moreover, the induction of shift strategies could orient the profile of T cells responding to disease target antigens from Th1 to Th2 or Th2 to Th1 according to the autoimmune process involved in each AID in order to induce a selective protective immune response.

Oral immunization of animals with transgenic cherry tomatillo expressing HBsAg.

To investigate the expression of recombinant HBsAg (rHBsAg) in transgenic cherry tomatillo in order to explore the feasibility of producing HBV oral vaccine with cherry tomatillo by animal immune tests. The recombinant plant expression vector containing HBsAg gene was constructed. Mediated with Agrobacterium tumefaciens, HBsAg gene was transferred into cotyledons of cherry tomatillo. Transformed cherry tomatillos were obtained through hygromycin delay-selection. Integrated DNA in transgenic cherry tomatillo was confirmed by hygromycin resistance selection, Gus detection, polymerase chain reaction (PCR) and dot blotting analysis. Antigenicity of rHBsAg was examined by ELISA and the immunogenicity of rHBsAg derived from transgenic cherry tomatillo tissues was confirmed by oral feed of transformed tissues to BALB/c mice primed with commercial HBV vaccines. Specific antibody titers in mice's serum were examined by ELISA every week. By far, 10 positive lines of transgenic cherry tomatillos containing HBsAg gene were obtained. Among different organs of the same transgenic cherry tomatillo, level of rHBsAg expressed in leaves was the highest with the yield up to 300ng/g fresh weight. And the rHBsAg expression level in fruits was about 10 ng/g fresh weight. In animal immune tests, oral delivery with transgenic tissues to mice primed with commercial vaccine instead of naive mice resulted in significant immune response. The result of this animal immune test indicated the rHBsAg derived from transgenic cherry tomatillo possessed normal immunogenicity. This work demonstrated the feasibility to generate oral immunogenic rHBsAg in transgenic cherry tomatillo, and would provide some experimental approach for the production of low-cost oral vaccines using transgenic cherry tomatillo in large scale.

Effect of Experimental Zinc Deficiency on Immunological Responses in Salmonella -infected Guinea-pigs

Cellular and humoral immune responses to Salmonella dublin infection were studied in guinea-pigs given zinc-deficient (ZnD) or zinc-adequate (ZnA) diet, containing 1·03 or 52·4 mg Zn/kg, respectively. On day 30, each animal of each dietary group was inoculated intraperitoneally with 106viable cells ofS. dublin strain 493. On the 15th day after infection, the immune responses were assessed by skin hypersensitivity (SH) tests and enzyme-linked immunosorbent assay (ELISA) for serum antibody with Salmonella -extract antigen. ZnD animals had significantly decreased ability to produce Salmonella -specific immediate and delayed type SH responses. These results were further substantiated by histological examination of skin sections. The ELISA indicated significantly lowerSalmonella -specific serum antibody titres in ZnD animals than in ZnA animals. Mean viable counts of S. dublin in the liver and spleen of ZnD animals were significantly higher than those in ZnA animals.

Increased lymphoproliferative response to human herpesvirus type 6A variant in multiple sclerosis patients

Several reports have suggested an association of human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) based on immunohistochemical demonstration of HHV-6 antigens in inflammatory lesions, detection of increased HHV-6 specific serum antibody titers, and amplification of HHV-6 DNA from sera and cerebrospinal fluid of MS patients but not in controls. Characterization of the cellular immune response of MS patients to HHV-6 may further clarify the role of HHV-6 in MS and provide insight into the pathogenesis of this immune-mediated disease. We have compared lymphoproliferative responses to HHV-6A (U1102)-, HHV-6B (Z29)-, and HHV-7 (H7SB)-infected cell lysates in healthy controls and patients with MS. Most healthy controls (71%) proliferated to HHV-6B lysate, and fewer (33%) responded to the HHV-6A lysate. In contrast, 67% of MS patients had a lymphoproliferative response to HHV-6A, which is a significant increase in comparison with healthy controls. A similar frequency of lymphoproliferative response (78%) to HHV-6B was demonstrated in MS patients. Lymphoproliferation to HHV-7 lysate was demonstrated in 23% of healthy controls and 28% of MS patients. These results indicate that the lymphoproliferative response to the HHV-6A variant, which was recently reported to have greater neurotropism, is increased in MS patients.

Serum antibody response to oral infection precedes but does not prevent Porphyromonas gingivalis-induced alveolar bone loss in mice.

The purpose of this study was to determine whether humoral immunity prevents bacterially induced alveolar bone loss. BALB/cByJ mice were orally infected with the human periodontopathic bacterium Porphyromonas gingivalis, and were compared with sham-infected mice. Specific serum antibody titers to P. gingivalis were measured by enzyme-linked immunosorbent assay. Alveolar bone levels were measured as the distance from the cementoenamel junction to the alveolar bone crest and bone loss was defined as a change in bone levels over time or between infected and sham-infected animals. The specific humoral response was predominantly of the IgG isotype, although low levels of specific serum IgA were also present. Antibody titers in the infected animals were significantly different from those in the sham-infected animals by 18 days and remained at maximal levels at 47 days. Bone loss became significant by 26 days and continued to progress at 47 days. Thus the serum antibody response to oral infection with P. gingivalis preceded detectable bone loss and remained elevated while bone loss increased. The presence of specific antibody did not prevent the onset or progression of bone loss.