Safety, Immunogenicity, and Protective Efficacy of the Whole-Cell/Recombinant B Subunit (WC/rBS) Oral Cholera Vaccine Against Travelers' Diarrhea

Background : A prospective, randomized, double-blind, placebo-controlled trial of WC/rBS oral cholera vaccine was conducted in 502 U.S. college students attending summer educational programs in Mexico.
Methods : Two doses of vaccine (or placebo) were administered 10 days apart immediately after arrival in Mexico.
Results : The vaccine was free of significant adverse side effects. Anticholera toxin seroconversion was demonstrated in 86.7% of vaccinees compared to 8.2% of controls (p < .001). Postvaccination titers varied according to disease status (travelers' diarrhea) and enteropathogen isolated when disease developed. Protective efficacy (PE) against enterotoxigenic Escherichia coli (ETEC) diarrhea was 50% (95% Cl, 14–71 %), beginning 7 days after the second dose of WC/rBS. However, 74% of ETEC cases occurred within 7 days of the second dose, when no efficacy was demonstrated.
Conclusions : Vaccines employed to prevent travelers' diarrhea will likely need to be administered before arrival in a developing country to be predictably beneficial. An unexpected finding was that infection with LT-ETEC after primary oral cholera immunization appears to augment the antitoxin response to WC/rBS vaccine.     

人群口服霍乱rBS/WC疫苗的安全性及免疫效果评价

目的 进一步观察自然人群口服重组霍乱毒素B亚单位/灭活霍乱弧菌全菌体疫苗的安全性和免疫学效果,为制定霍乱免疫预防策略提供依据.方法 采用随机、双盲、对照试验,随访观察每次服苗后3 d内以及全程服苗后1、2、3月的不良反应,按比例随机抽取试验组和对照组受试者采血,用酶联免疫法检测抗霍乱毒素抗体,采用SPSS软件对数据进行相关分析.结果 每次服苗后3 d内试验组的总不良反应率为1.16%,主要是轻微胃肠道反应;试验组抗霍乱毒素抗体,全程接种后1～6月维持较高的阳性率,全程接种后12月有所降低.结论 口服霍乱疫苗具有良好的安全性和短期效果,可用于流行区的霍乱免疫预防.     

Effect of dietary whey protein concentrate on primary and secondary antibody responses in immunized BALB/c mice

Proteins derived from the whey fraction of bovine milk are known to modulate immune responses. We have previously described a rennet whey protein concentrate (WPC) that can boost intestinal tract antibody responses to orally administered T-dependent antigens. In the present study, we investigated the effects of feeding WPC to mice on specific antibody responses to several orally or parenterally administered antigens, including influenza vaccine, diphtheria and tetanus toxoids, poliomyelitis vaccine, ovalbumin and cholera toxin sub-unit. WPC-fed mice produced elevated levels of antigen-specific intestinal tract and serum antibodies against all tested antigens, compared to mice that were fed a standard chow diet. Both primary and secondary intestinal tract antibody responses were elevated by WPC feeding, while only secondary serum responses were increased in WPC-fed mice. Significant up-regulation of intestinal tract antibody was observed within 2 weeks of primary oral immunizations. A period of pre-feeding with WPC, prior to commencement of immunization, did not alter the kinetics or magnitude of immune enhancement. These results identify bovine WPC as a potentially important dietary protein supplement, capable of enhancing humoral immune responses to a range of heterologous antigens.     

Nasal immunization studies using liposomes loaded with tetanus toxoid and CpG-ODN.

To increase the systemic and mucosal immune responses against the nasally administered tetanus toxoid, liposomes as a drug delivery system and CpG-ODN as an adjuvant were evaluated. Rabbits were nasally immunized with entrapped tetanus toxoid (TT) and CpG-ODN in neutral liposomes and systemic and mucosal immune responses were determined. Liposomes containing TT and CpG-ODN were prepared by dehydration-rehydration method. The volume mean diameter of liposomes was 2.3+/-0.6 microm. Encapsulation efficiency of TT and CpG-ODN was determined as 54.0+/-8.8 and 60.1+/-7.4, respectively. The leakage of the encapsulated TT from liposomes reached 7.38% after 3 months. Encapsulated TT kept its intact structure, and its immunoreactivity was also completely preserved, as shown by SDS-PAGE and ELISA methods. The highest serum IgG and antitoxin titers were observed in groups immunized with solution formulations (P < 0.001). However the highest mucosal sIgA titers were achieved by liposomes encapsulated with TT. CpG-ODN as an adjuvant was able to increase the serum IgG and antitoxin titers when co-administered with TT solution (P < 0.05) or co-encapsulated with TT in liposomes (P < 0.01), but failed to increase the sIgA titers in nasal lavages. No hemolysis occurred on incubation of liposomes and human RBCs. Also after nasal administration of plain liposomes to human volunteers, no local irritation was seen. Intranasal administration of liposomes encapsulated with vaccines showed to be an effective way for inducing the mucosal immune responses.     

Multiple antigen peptide consisting of B- and T-cell epitopes of F1 antigen of Y. pestis showed enhanced humoral and mucosal immune response in different strains of mice

Yersinia pestis is a causative agent of plague. F1 and V antigen based vaccines have shown remarkable protection in experimental animals. In order to develop epitope based immunogen, three B and one T-cell epitopes of F1 antigen with palmitate residue at amino terminal were assembled on a lysine backbone as multiple antigen peptide (MAP or F1-MAP). MAP was characterized by SDS-PAGE, immunoblot and immunoreactivity with anti F1 sera. MAP was entrapped in PLGA (polylactide-co-glycolide) microparticles and humoral, mucosal immune responses were studied after intranasal immunization with/without CpG ODN 1826 (CpG)/murabutide in different strains of mice. Serum and mucosal washes were measured for MAP specific IgG, IgA, sIgA and IgG subclasses in three strains of mice. F1-MAP showed high serum antibody and mucosal IgG and IgA peak antibody titers. MAP with CpG showed significantly high (p < 0.001) peak antibody titer ranging from 102,400 to 204,800 for IgG and 6400 to 12,800 for IgA. High mucosal sIgA and its secretary component detection confirmed generation of mucosal response in intestinal and lung washes. MAP antisera also showed significant immunoreactivity with individual peptides. Moreover, antibody specific activity (IgG, IgA and sIgA) positively correlates with peak antibody titers. Predominantly IgG2a/IgG2b subclass was observed with CpG formulation but in other formulation a mixed IgG1 and IgG2a response was observed. The present study highlights the importance of multiple antigen peptide approach of F1-antigen with CpG as an alternative approach for subunit vaccine.     

A murine model of enterohemorrhagic Escherichia coli O157:H7 infection to assess immunopotentiating activity of drugs on mucosal immunity: effect of drugs

An enterohemorrhagic Escherichia coli (EHEC) O157 oral infection murine model was established to examine the potentiating activity of drugs on mucosal immune responses. Groups of ICR mice inoculated intragastrically with 10(11) CFU/kg EHEC O157 showed chronic intestinal infection with the pathogen that persisted over 3 weeks and resulted in the synthesis of relatively high levels of antigen specific fecal IgA antibody. Intraperitoneal administration of 80 NU/kg Neurotropin, an immunopotentiator, augmented the antigen specific mucosal immune responses to EHEC O157. On the other hand, FK506 clearly suppressed the response. To further document the augmenting effect of Neurotropin on mucosal immune responses, mice were immunized intranasally with a mixture of ovalbumin and cholera toxin. Co-administration of 80 NU/kg Neurotropin significantly potentiated the synthesis of fecal IgA and serum IgG antibodies. These results suggest that Neurotropin has potential as a mucosal adjuvant to promote secretory IgA antibody production and that the mice model of oral infection with EHEC O157 is useful for immunopharmacological studies of bacterial infection-defensive mucosal immune responses.     

Intestinal immunoglobulin a response to naturally acquired enterotoxigenic Escherichia coli in US travelers to an endemic area of Mexico

BACKGROUND: Simple methods for detecting secretory immunoglobulin A (sIgA) immune responses following natural enteric infection and oral immunization are needed. METHODS: Fourteen students from the United States acquiring enterotoxigenic Escherichia coli (ETEC) diarrhea in Mexico were studied for fecal immunoglobulin A (IgA) response to their homologous infecting ETEC and to heat-labile (LT) toxin of ETEC using Dot-Blot microfiltration and enzyme-linked immunosorbent assay (ELISA) methods. Paired stool samples were collected on the day of presentation and 5 days later. RESULTS: Twelve of 14 (86%) patients with ETEC diarrhea (5 heat-stable [ST]/LT positive, 4 LT-only, and 5 ST-only) developed sIgA antibodies directed against their homologous ETEC and 6 (66%) of the 9 patients harboring ST/LT or LT-only strains developed sIgA LT-antibody responses. Single fecal samples from 9 healthy controls were negative for ETEC specific antibodies. CONCLUSIONS: Patients with diarrhea due to noninvasive ST/LT ETEC and LT ETEC commonly produce a specific sIgA antibody response early in the illness. We feel that the methods employed will be useful to detect antibodies during natural infection by enteric pathogens and following oral enteric vaccine administration.     

Local and systemic antibody response to rotavirus WC3 vaccine in adult volunteers

An evaluation of the safety and immunogenicity of WC3 rotavirus vaccine was evaluated in adult volunteers. Pre- and post-vaccination titers of neutralizing antibody to WC3 and to the four human rotavirus serotypes as well as serum and stool rotavirus IgA levels were measured. Vaccination was safe and did not induce elevation of liver enzymes. None of the 12 volunteers receiving WC3 vaccine shed detectable amounts of virus although antibody rises were detected in 11 of 12 vaccines. Nine developed and increase in WC3 neutralizing antibody, one additional subject had a rise in Wa (human serotype 1) neutralizing antibody while another subject only developed a rise in stool rotavirus IgA. All of the vaccine recipients with a rise in WC3 neutralizing antibody also developed a rise in neutralizing antibody against at least one of the four most common human rotavirus serotypes. A stool IgA rotavirus antibody response was detected in 6 of 9 WC3 recipients with measurable stool antibody. None of the control subjects developed significant rises in any of the antibody titers measured. WC3 rotavirus vaccine appears to be safe and induces systemic and local immune responses in adults suggesting that further evaluation of WC3 should be considered in infants.     

The humoral immune responses of patients bitten by the snake Bothrops jararaca (jararaca)

The isotype and specificity of antibodies produced by patients bitten by B. jararaca and submitted to serum therapy were studied. The IgG anti-B. jararaca antibodies have large individual dispersion, starting to appear 10 days after the first bite and increasing to at least 80 days after the bite. IgM antibodies appeared sooner than IgG antibodies but disappeared about 20 days after the bite. Secondary responses induced by an additional bite were characterized by a fast and higher IgG antibody response with no apparent change in the IgM antibody. The immunoblotting tests showed that the specificity of human anti-B. jararaca antibodies is heterogeneous, each patient recognizing different fractions in the B. jararaca venom.     

Antibody pharmacokinetics and pharmacodynamics

The U.S. Food and Drug administration (FDA) has approved several polyclonal antibody preparations and at least 18 monoclonal antibody preparations (antibodies, antibody fragments, antibody fusion proteins, etc.). These drugs, which may be considered as a diverse group of therapeutic proteins, are associated with several interesting pharmacokinetic characteristics. Saturable binding with target antigen may influence antibody disposition, potentially leading to nonlinear distribution and elimination. Independent of antigen interaction, concentration-dependent elimination may be expected for IgG antibodies, due to the influence of the Brambell receptor, FcRn, which protects IgG from catabolism. Antibody administration may induce the development of an endogenous antibody response, which may alter the pharmacokinetics of the therapeutic antibody. Additionally, the pharmacodynamics of antibodies are also complex; these drugs may be used for a wide array of therapeutic applications, and effects may be achieved by a variety of mechanisms. This article provides an overview of many of the complexities associated with antibody pharmacokinetics and pharmacodynamics.     

Combination testing for antibodies in the diagnosis of coeliac disease: comparison of multiplex immunoassay and ELISA methods

Background  Tissue transglutaminase (TTG) antibodies and newly developed deamidated gliadin peptide (DGP) antibodies have better accuracy than native gliadin antibodies. Multiplex immunoassay (MIA) measures multiple antibodies simultaneously providing a complete antibody phenotype with reduced turnaround time and cost.
Aim  To evaluate the agreement between MIA and enzyme-linked immunosorbent assay (ELISA) test results for coeliac antibodies in biopsy-proven coeliac patients and controls and to model the diagnostic utility of combination testing.
Methods  We compared the sensitivity, specificity and accuracy of MIA and ELISA methods for TTG and DGP antibodies in mainly adult untreated coeliac patients ( n  = 92) and controls ( n  = 124).
Results  There was excellent agreement and a significant correlation between the results of MIA and ELISA methods (κ > 0.8, r  >   0.7) for all tests, except TTG IgG. Diagnostic indices of individual and combination tests measured by the MIA method did not differ significantly from those measured by ELISA. The combination tests slightly increased sensitivity (if any test was positive) and specificity (if all tests were positive) compared to the individual tests.
Conclusions  Multiplex immunoassay testing for antibodies is as accurate as ELISA for coeliac disease diagnosis and has practical advantages over ELISA method. Rational combination testing can help identify patients who need intestinal biopsy and may reduce unnecessary biopsies.     

Induction of high antitoxin titers against tetanus toxoid in rabbits by intranasal immunization with dextran microspheres

Poor absorption of protein antigens through the mucosal membranes necessitates the use of mucoadhesive delivery systems. Regarding the advantages of mucosal immunization and also the penetration enhancement potential of dextran microspheres, in this study the adjuvant potential of these microspheres was compared with CpG-ODN. Cross-linked dextran microspheres (CDMs) were loaded with tetanus toxoid (TT). In vitro release studies were performed in a model, simulating the nasal cavity. The immunoreactivity of encapsulated TT was assayed by ELISA. Membrane toxicity and local irritating potential of CDM was examined by erythrocyte hemolysis and nasal administration to human nose, respectively. The various formulations were nasally administered to rabbits (n=4). Alum-adsorbed TT (AATT) was injected as the positive control. The serum IgG and nasal lavage sIgA titers were determined by ELISA method. Serum antitoxin titers were determined by toxin neutralization (TN) bioassay method. Mean diameter of CDM was 128.1+/-25.8 microm. Mean encapsulation efficiency was 20.3+/-3.2% (n=3). Antigenicity of encapsulated TT was 90.5+/-1.8% (n=3) that of original TT. Hemolysis studies showed no membrane disruption by CDM and none of the human subjects reported nasal irritation. Among the nasally immunized animals, the highest antitoxin titers was seen in the group immunized with CDM+TT (P<0.0001). The serum IgG titers of the CDM+TT group was higher than the TT solution group (P<0.05). The adjuvant potentials of CDM and CpG-ODN in inducing IgG titers was not significantly different (P>0.05). The lowest sIgA titers in the bronchial lavage were seen in the group of animals received AATT parenterally. Considering the proper release characteristics, desirable preservation of the antigen activity of TT, good mucoadhesion properties and also safety of CDM+TT, these microspheres could be regarded as an efficient mucosal adjuvant and antigen delivery system. These microspheres could induce very high antitoxin titers following nasal administration, while the CpG-ODN could not induce such titers. The antitoxin titers induced by CDM+TT was 175 times higher than the protective levels.     

Safety and Immunogenicity of a High–Potency Inactivated Hepatitis A Vaccine

Background: In recent years, several hepatitis A vaccines have been developed. We wished to evaluate the safety, reactogenicity, and immunogenicity of an inactivated hepatitis A vaccine, containing 1440 EI.U., and to monitor the kinetics of the antibodies monthly for the first year after administration of a single dose of vaccine.
Methods: We conducted an open clinical trial, started in March 1992 and completed in July 1993, at two general hospitals and one pediatric hospital in Antwerp, Belgium, with 194 healthy adult healthcare volunteers. Each volunteer received a single dose hepatitis A vaccine, given intramuscularly at month 0 and a booster at month 12. We undertook serologic follow-up of antihepatitis A virus (antiHAV) antibodies and liver enzymes at day 15 and at months 1, 6, 9, 12, and 13. For a random subgroup of participants, blood samples were taken monthly. In addition, all participants noted local and general symptoms following administration of the vaccine.
Results: This single dose vaccine was well-tolerated; 2 weeks after the vaccination, 80% of the participants had seroconverted (antiHAV antibodies ≥ 20 mlU/mL); after 1 month, seroconversion reached 99% (geometric mean titer (GMT): 383 mlU/mL). One year after the single dose of vaccine, 94% still had antiHAV antibodies above 20 mlU/mL (GMT: 176 mlU/mL). One month afterthe booster dose, seroconversion was 100%, and GMT increased from 176 mlU/mL at month 12 to 4775 mlU/mL at month 13.
Conclusions: The single dose hepatitis A vaccine is safe and highly immunogenic; it gives a rapid antibody production and a rapid increase of GMT. The persistence of protective antibodies until month 12 allows a booster at month 12. This schedule will easily fit into existing travel or occupational health vaccination schedules and will improve vaccination compliance.     

Cellular and humoral systemic and mucosal immune responses stimulated by an oral polybacterial immunomodulator in patients with chronic urinary tract infections

An oral polybacterial immunomodulator Urostim (U), composed of killed cells and their lysates from E. coli expressing type 1 and Pili, E. coli Rc mutant, P. mirabilis, K. pneumoniae and E. faecalis was created for immunoprophylaxis and immunotherapy of urinary tract infections (UTIs). In experimental animal models, the stimulating effect of U on lymphocyte functional activity, macrophage phagocytosis and antibody producing cells, was established. In this study the immuno-modulating effects of U on the proliferating capacity and ultrastructural morphologic changes of lymphocytes, cytokine production and specific systemic humoral and mucosal immune responses in patients with UTIs have been evaluated. Patients enrolled in the study, received orally 50 mg U daily for a period of three months. On days 0, 30 and 90 a quantitative analysis was performed on lymphoproliferative responses to polyclonal mitogens, IL-2 and the specific antigen U, the production of specific serum and saliva IgA, IgM and IgG antibodies to all components of U and the concentration of pro-inflammatory cytokines. There was significant improvement of non-specific and specific lymphoproliferative responses on days 30 and 90 after the onset of treatment with U, confirmed by electronmicroscopic studies. The highest concentrations of serum proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 were registered at baseline followed by a decrease until the end of the observation period. This finding correlates with the gradual decrease of immune activation as measured by the spontaneous lymphocyte proliferation. Data from the production of specific antibacterial antibodies in serum and saliva show two types of reactions. The first type was registered in patients with low pre-treatment levels in whom the concentration of specific antibodies increased on days 30 and 90. The second type of reaction was observed in patients with high pre-treatment levels, which dropped on day 30 and were usually followed by an increase at the end of the study. These results provide evidence for the immuno-modulating effect of U. Our data show that the oral administration of the polybacterial immunomodulator Urostim stimulates adequate cellular and humoral systemic and mucosal immune responses in patients with chronic UTIs.     

Nasal immunization studies by cationic, fusogenic and cationic-fusogenic liposomes encapsulated with tetanus toxoid

Particulate antigens are more effective than soluble antigens in induction of systemic and mucosal immunity; possibly because they are more efficiently endocytosed by mucosal-associated lymphoid tissue (MALT) M cells. In this study, we determined the systemic and mucosal immune responses in rabbits following intranasal immunization with tetanus toxoid (TT) entrapped in cationic, fusogenic and cationic-fusogenic liposomes. Liposomes containing TT were prepared by dehydration-rehydration method. The volume mean diameter of cationic, fusogenic and cationic-fusogenic liposomes were 3.4 +/- 0.6, 4.3 +/- 2.3 and 3.4 +/- 1.5 microm, respectively. Encapsulation efficiency of TT in cationic, fusogenic and cationic-fusogenic liposomes was respectively determined as 49.1 +/- 8.4%, 48.5 +/- 2.1% and 50.8 +/- 4.9%. After 3 months, the leaking of encapsulated TT from liposomes ranged between 2.02 - 5.46%. Immunoreactivities of encapsulated TT in all kinds of liposomes were completely preserved, as studied by Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Enzyme-Linked Immunosorbent Assay (ELISA). The highest serum immunoglobulin G (IgG) and antitoxin titers were observed in groups immunized with solution formulation (P< 0.001). However, the highest mucosal secretory IgA (sIgA) titers were achieved by fusogenic liposomes (five times more titers compared with TT solution, and 15 times more titers compared with i.m. vaccine), followed by cationic-fusogenic liposomes. No hemolysis was occurred on incubation of liposomes and human erythrocytes. Also after nasal administration of plain liposomes to human volunteers, no local irritation was seen. This study suggests that intranasal administration of fusogenic and cationic-fusogenic liposomes encapsulated with vaccines could be an effective way for inducing mucosal immune responses.     

Induction of systemic and mucosal immune responses by intranasal administration of alginate microspheres encapsulated with tetanus toxoid and CpG-ODN

In the induction of systemic and mucosal immunity, particulate antigens are more effective than soluble antigens; possibly because they are more efficiently endocytosed by mucosal-associated lymphoid tissue (MALT) M cells. In this study, we determined the systemic and mucosal immune responses in rabbits following intranasal immunization with encapsulated tetanus toxoid (TT) and CpG-ODN in alginate microspheres. The microspheres were less than 4 microm in diameter. Encapsulation efficiency of TT and CpG-ODN was determined as 47.7+/-6.6 and 34.2+/-7.4, respectively. Release of TT and CpG-ODN in a simulated model with nasal cavity was 14.2+/-3.06 and 36.7+/-2.4% after 4 h. Encapsulated TT preserved its intact structure, but its immunoreactivity was decreased to about 91+/-5%. The highest serum IgG and antitoxin, and nasal lavage IgA titers were observed in groups immunized with microsphere formulations. CpG-ODN as an adjuvant could increase the serum IgG and antitoxin titers when co-administered with TT solution, but its co-encapsulation with TT in alginate microspheres failed to potentiate the systemic immune response while induced high IgA titers in nasal lavages. No hemolysis was occurred on incubation of alginate microspheres and human RBCs. Also after nasal administration of plain microspheres to human volunteers, no local irritation was observed. Intranasal administration of microspheres encapsulated with vaccines showed to be an effective way for inducing a variety of immune responses and that a strong systemic IgG and mucosal IgA responses can be induced in rabbits with intranasal administration of alginate microspheres encapsulated with TT.     

Effect of corticosteroids on serum antinuclear antibodies in man

The effect of prednisone on serum levels of IgG antibodies to viral and bacterial antigens was measured and compared to its effect on IgG antibodies to nuclear antigens in 8 patients with systemic lupus erythematosus. Prednisone at 15-80 mg/day (mean 55 mg/day) for 14-30 days (mean 19 days) lowered the serum IgG by an average of 22% (p less than 0.005). An even greater reduction in IgG antinuclear antibodies occurred (mean 43%, p less than 0.001) including responses to double stranded DNA, single stranded DNA, and the nucleosides, adenosine, guanosine, cytidine and thymine riboside. In contrast, there was no alteration in serum IgG antibody levels to influenza virus vaccine and pneumococcal vaccine antigens. These results suggest that prednisone has a selective effect on the expression of autoimmunity which may, in part, be responsible for its clinical efficacy in systemic lupus erythematosus.     

Comparison of the effect of oral sulphasalazine,sulphapyridine and 5-amino-salicylic acid on the in vivo antibody response to oral and systemic antigen.

1. Mice, whose drinking water contained sulphasalazine, sulphapyridine or 5-amino-salicylic acid, received an antigenic challenge by cholera toxin administered either orally or systemically. 2. Sulphasalazine treated mice made less specific antibody of IgA class provided the antigen also was administered orally (P = 0.009 for days 7-28). When the antigen was administered systemically, there was a vigorous anti-cholera toxin antibody response of IgG class, and a lesser IgM but only a weak IgA response. The effect of sulphasalazine in this case was confined to the IgG response, which was significantly suppressed on day 28 (P = 0.008). 3. Sulphapyridine and 5-amino salicylic acid had no significant effect on the anti-cholera toxin (CT) responses of all three classes. 4. It therefore appears that in this model, only sulphasalazine is capable of influencing the humoral immune system, the antibody class affected depending on the route of entry of antigen. This may have implications for conditions such as rheumatoid arthritis and chronic inflammatory bowel disease, for which sulphasalazine has been found useful.     

Immunogenicity of a West Nile Virus DIII-Cholera Toxin A2/B Chimera after Intranasal Delivery

West Nile virus (WNV) causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII) region of the WNV envelope protein contains epitopes that are the target of neutralizing antibodies. We have constructed a chimeric fusion of the non-toxic cholera toxin (CT) CTA₂/B domains to DIII for investigation as a novel mucosally-delivered WNV vaccine. Purification and assembly of the chimera, as well as receptor-binding and antigen delivery, were verified by western blot, GM1 ELISA and confocal microscopy. Groups of BALB/c mice were immunized intranasally with DIII-CTA₂/B, DIII, DIII mixed with CTA₂/B, or CTA₂/B control, and boosted at 10 days. Analysis of serum IgG after 14 and 45 days revealed that mucosal immunization with DIII-CTA₂/B induced significant DIII-specific humoral immunity and drove isotype switching to IgG2a. The DIII-CTA₂/B chimera also induced antigen-specific IgM and IgA responses. Bactericidal assays indicate that the DIII-CTA₂/B immunized mice produced DIII-specific antibodies that can trigger complement-mediated killing. A dose escalation resulted in increased DIII-specific serum IgG titers on day 45. DIII antigen alone, in the absence of adjuvant, also induced significant systemic responses after intranasal delivery. Our results indicate that the DIII-CTA₂/B chimera is immunogenic after intranasal delivery and merits further investigation as a novel WNV vaccine candidate.     

Chitosan nanoparticles encapsulated vesicular systems for oral immunization: preparation, in-vitro and in-vivo characterization

BSA-loaded chitosan nanoparticles were prepared and encapsulated in vesicles (liposomes and niosomes) to make them acid resistant upon oral administration. Prepared systems were characterized in-vitro for shape, size, entrapment efficiency and stability in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.5). The immune stimulating activity was studied by measuring serum IgG titre and secretory IgA (sIgA) levels in mucosal secretions following oral administration of various formulations in albino rats. Significantly higher (P < 0.05) serum IgG titres were achieved following oral administration of novel nanoparticulate vesicular formulations as compared with unmodified chitosan nanoparticles. Further, high sIgA levels in mucosal secretions advocated a possible application of chitosan nanoparticle encapsulated in vesicles as an oral vaccine delivery carrier-adjuvant system.