Antigen-specific IgA B memory cell responses to Shigella antigens elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine candidates.

We studied the induction of antigen-specific IgA memory B cells (B(M)) in volunteers who received live attenuated Shigella flexneri 2a vaccines. Subjects ingested a single oral dose of 10(7), 10(8) or 10(9) CFU of S. flexneri 2a with deletions in guaBA (CVD 1204) or in guaBA, set and sen (CVD 1208). Antigen-specific serum and stool antibody responses to LPS and Ipa B were measured on days 0, 7, 14, 28 and 42. IgA B(M) cells specific to LPS, Ipa B and total IgA were assessed on days 0 and 28. We show the induction of significant LPS-specific IgA B(M) cells in anti-LPS IgA seroresponders. Positive correlations were found between anti-LPS IgA B(M) cells and anti-LPS IgA in serum and stool; IgA B(M) cell responses to IpaB were also observed. These B(M) cell responses are likely play an important role in modulating the magnitude and longevity of the humoral response.

Neonatal mucosal immunization with a non-living, non-genetically modified Lactococcus lactis vaccine carrier induces systemic and local Th1-type immunity and protects against lethal bacterial infection.

Safe and effective immunization of newborns and infants can significantly reduce childhood mortality, yet conventional vaccines have been largely unsuccessful in stimulating the neonatal immune system. We explored the capacity of a novel mucosal antigen delivery system consisting of non-living, non-genetically modified Lactococcus lactis particles, designated as Gram-positive enhancer matrix (GEM), to induce immune responses in the neonatal setting. Yersinia pestis LcrV, used as model protective antigen, was displayed on the GEM particles. Newborn mice immunized intranasally with GEM-LcrV developed LcrV-specific antibodies, Th1-type cell-mediated immunity, and were protected against lethal Y. pestis (plague) infection. The GEM particles activated and enhanced the maturation of neonatal dendritic cells (DCs) both in vivo and in vitro. These DCs showed increased capacities for secretion of proinflammatory and Th1-cell polarizing cytokines, antigen presentation and stimulation of CD4(+) and CD8(+) T cells. These data show that mucosal immunization with L. lactis GEM particles carrying vaccine antigens represents a promising approach to prevent infectious diseases early in life.

Safety, reactogenicity and immunogenicity of Francisella tularensis live vaccine strain in humans.

We evaluated the safety, reactogenicity and immunogenicity of escalating doses of a new Francisella tularensis Live Vaccine Strain (LVS) lot by scarification (SCAR) or subcutaneously (SQ) in humans. Subjects (N=10/group) received one dose of LVS via SCAR at 10(5),10(7) or 10(9)cfu/ml or SQ at 10(2), 10(3),10(4) or 10(5)cfu/ml; 14 subjects received placebo. All doses/routes were well tolerated. When compared to placebo, vaccination with 10(7) SCAR and 10(9) SCAR resulted in significantly higher serologic response frequencies, as measured by ELISA for IgG, IgM, IgA and microagglutination; whereas vaccination with 10(5) SCAR, 10(7) SCAR 10(9) SCAR and 10(5) SQ elicited a significantly higher interferon-gamma response frequency.

Heterologous prime-boost strategy to immunize very young infants against measles: pre-clinical studies in rhesus macaques.

Infants in developing countries are at high risk of developing severe clinical measles if they become infected during the "window of vulnerability" (age 4-9 months), when declining maternal antibodies do not protect against wild virus, yet impede successful immunization by attenuated measles vaccine. We developed two Sindbis replicon-based DNA vaccines expressing measles virus hemagglutinin and fusion protein with the goal of priming young infants to respond safely and effectively to subsequent boosting with attenuated measles vaccine. Intradermal prime with DNA vaccines by needle-free injection followed by aerosol or parenteral boost with licensed measles vaccine was well tolerated by juvenile and young infant rhesus macaques, and protected against clinical measles and viremia on wild-type virus challenge. A proteosome-measles vaccine administered alone (three doses) or as a boost following DNA vaccine priming was also safe and protective. These promising results pave the way for clinical trials to assess this prime-boost strategy.

Construction, genotypic and phenotypic characterization, and immunogenicity of attenuated DeltaguaBA Salmonella enterica serovar Typhi strain CVD 915.

A promising live attenuated typhoid vaccine candidate strain for mucosal immunization was developed by introducing a deletion in the guaBA locus of pathogenic Salmonella enterica serovar Typhi strain Ty2. The resultant DeltaguaBA mutant, serovar Typhi CVD 915, has a gene encoding resistance to arsenite replacing the deleted sequence within guaBA, thereby providing a marker to readily identify the vaccine strain. CVD 915 was compared in in vitro and in vivo assays with wild-type strain Ty2, licensed live oral typhoid vaccine strain Ty21a, or attenuated serovar Typhi vaccine strain CVD 908-htrA (harboring mutations in aroC, aroD, and htrA). CVD 915 was less invasive than CVD 908-htrA in tissue culture and was more crippled in its ability to proliferate after invasion. In mice inoculated intraperitoneally with serovar Typhi and hog gastric mucin (to estimate the relative degree of attenuation), the 50% lethal dose of CVD 915 (7.7 x 10(7) CFU) was significantly higher than that of wild-type Ty2 (1.4 x 10(2) CFU) and was only slightly lower than that of Ty21a (1.9 x 10(8) CFU). Strong serum O and H antibody responses were recorded in mice inoculated intranasally with CVD 915, which were higher than those elicited by Ty21a and similar to those stimulated by CVD 908-htrA. CVD 915 also elicited potent proliferative responses in splenocytes from immunized mice stimulated with serovar Typhi antigens. Used as a live vector, CVD 915(pTETlpp) elicited high titers of serum immunoglobulin G anti-fragment C. These encouraging preclinical data pave the way for phase 1 clinical trials with CVD 915.

Expression, extracellular secretion, and immunogenicity of the Plasmodium falciparum sporozoite surface protein 2 in Salmonella vaccine strains.

Deleting transmembrane alpha-helix motifs from Plasmodium falciparum sporozoite surface protein (SSP-2) allowed its secretion from Salmonella enterica serovar Typhimurium SL3261 and S. enterica serovar Typhi CVD 908-htrA by the Hly type I secretion system. In mice immunized intranasally, serovar Typhimurium constructs secreting SSP-2 stimulated greater gamma interferon splenocyte responses than did nonsecreting constructs (P = 0.04).

A comparison of immunogenicity and in vivo distribution of Salmonella enterica serovar Typhi and Typhimurium live vector vaccines delivered by mucosal routes in the murine model.

We evaluated the immune responses elicited by attenuated Salmonella enterica serovar Typhi vaccine strain CVD 908-htrA and serovar Typhimurium strain SL3261 alone or as live vectors carrying a plasmid encoding fragment C of tetanus toxin (pTETnir15) in mice immunized intranasally and orogastrically, as well as the in vivo distribution of vaccine organisms following immunization. Higher serologic and proliferative responses against both vector and the foreign antigen were elicited when vaccines were delivered by intranasal route. Whereas both Salmonella strains were detected in the nasal tissue, lungs, and Peyer's patches following intranasal and orogastric immunization, larger numbers of vaccine organisms were recovered from these tissues when the vaccines were delivered intranasally.

DeltaguaBA attenuated Shigella flexneri 2a strain CVD 1204 as a Shigella vaccine and as a live mucosal delivery system for fragment C of tetanus toxin.

The DeltaguaBA Shigella flexneri 2a vaccine candidate, CVD 1204, was evaluated as a delivery system for the non-toxic C-terminal of tetanus toxin (fragment C), either as a polypeptide expressed in the bacteria or as a DNA vaccine. CVD 1204 was transformed with plasmid pTETnir15 which encodes the fragment C gene (tetC) under the control of the inducible prokaryotic nir15 promoter or a DNA vaccine plasmid pcDNA3tetC which encodes tetC under the eukaryotic hCMV promoter. Guinea pigs immunised intranasally (i.n.) with either recombinant strain mounted a secretory immune response against S. flexneri 2a Lipopolysaccharide (LPS) and were protected against ocular challenge with wild-type S. flexneri 2a. Both strains were effective in eliciting a serum IgG response against fragment C in guinea pigs following i.n. immunisation. Furthermore, serum from guinea pigs immunised with CVD 1204(pTETnir15) contained tetanus toxin neutralising antibodies. These results demonstrate that this S. flexneri 2a vaccine candidate can serve as a vehicle for the delivery of foreign antigens to the systemic immune system while retaining its capacity to serve as a mucosal Shigella vaccine.

In vivo characterization of the murine intranasal model for assessing the immunogenicity of attenuated Salmonella enterica serovar Typhi strains as live mucosal vaccines and as live vectors.

Attenuated Salmonella enterica serovar Typhi live vector vaccine strains are highly immunogenic in mice following intranasal but not orogastric inoculation. To elucidate the relationship between organs within which vaccine organisms are found and the induction of specific serum immunoglobulin G (IgG) antibodies, we examined the in vivo distribution of serovar Typhi vaccine strain CVD 908-htrA following intranasal administration. Vaccine organisms were cultured from the nasal lymphoid tissue (NALT), lungs, and Peyer's patches 2 min after intranasal inoculation. Vaccine organisms persisted longer in NALT than in other organs. By decreasing the volume of intranasal inoculum containing 10(9) CFU (from a single 30- or 10-microl dose to four 2.5-microl doses given over the course of 1 h), we were able to significantly reduce the number of vaccine organisms isolated from the lungs (P < 0.05) without reducing the number of vaccine organisms in NALT. Reducing the number of vaccine organisms in the lungs resulted in a significant decrease in the serum tetanus antitoxin response elicited by CVD 908-htrA expressing tetanus toxin fragment C under the control of the redox-responsive nir15 promoter. In contrast, a similar construct expressing tetanus toxin fragment C under control of the constitutive lpp promoter stimulated a strong serum IgG tetanus antitoxin response with both inoculation regimens. The data suggest that following intranasal inoculation, NALT is a sufficient inductive site for elicitation of an immune response against both the live vector and heterologous antigen and, as occurs following oral inoculation of humans, attenuated serovar Typhi vaccine organisms elicit serum IgG responses.

Attenuated deltaguaBA Salmonella typhi vaccine strain CVD 915 as a live vector utilizing prokaryotic or eukaryotic expression systems to deliver foreign antigens and elicit immune responses.

Attenuated Salmonella typhi strain CVD 915, harboring a deletion in guaBA that interrupts the biosynthesis of guanine nucleotides, was evaluated as a live vector vaccine for delivering foreign antigens utilizing prokaryotic or eukaryotic expression systems. Plasmids pTETnir15 and pcDNA3tetC encoding fragment C (Frag C) of tetanus toxin under the control of prokaryotic or eukaryotic promoters, respectively, were introduced into CVD 915 and administered intranasally to mice. Purified pcDNA3tetC and Frag C were given intramuscularly. High titers of serum IgG1, IgG2a, and IgG2b antibodies against Frag C were elicited by CVD 915(pTETnir15) and CVD 915(pcDNA3tetC). These responses were significantly higher than those induced by pcDNA3tetC. Proliferative responses and IL-2 and IFN-gamma production were observed in splenocytes exposed to S. typhi antigens and Frag C. We conclude that CVD 915 is a highly efficient live vector to carry foreign genes under eukaryotic or prokaryotic control and elicit potent immune responses.

Structure and protective capacity of tetanus and diphtheria antibodies produced during human pregnancy and transferred to new-born.

PROBLEM: The structure and protective activity of antibodies against tetanus (anti-T) and diphtheria (anti-D), produced during human pregnancy and transferred to new-born, was studied. METHOD: Antibody levels were measured by ELISA in non-pregnant women (control group), primiparae, and multiparae, and in their children. The proportion of symmetric and asymmetric IgG molecules was determined and their respective protective capacity evaluated. RESULTS: The quantity of asymmetric anti-T and anti-D antibodies in mothers at the time of delivery was roughly four- and three-fold that of the control group, respectively, dropping significantly 1 month later. A similar proportion of these antibodies was observed in the new-born. The lower neutralizing capacity of asymmetric molecules was demonstrated in vivo. CONCLUSION: Results show that during pregnancy there is a modulation of the immune response with an increase in the production of asymmetric molecules of lower protective capacity.

Development of an alternative method for testing the immunogenicity of diphtheria vaccines.

The immunogenicity of the diphtheria component of 73 commercial vaccines from five different manufacturers was tested by the toxin neutralization test (TNT) and the enzyme-linked immunosorbent assay (ELISA) developed in our laboratory. A comparison of the antibody levels measured by both assays showed a very good correlation (r = 0.95, p < 0.001). The results suggest that the proposed ELISA is a reliable, simple and economical alternative to the TNT in guinea pigs. Also, the ELISA was found to measure IgG antibody levels as low as 5.5 x 10(-5) IU ml-1. To evaluate the possibility of accelerating the active immunization during the activity test of vaccines, an alternative schedule using one single human dose was assayed. A very good correlation was observed between the IgG antibody response obtained with this schedule and with the traditional programme. Therefore, the cost and the time required to perform the activity test may be considerably reduced when both the rapid immunization schedule and the ELISA are used.

Inhibition enzyme-linked immunosorbent assay for detection of Pseudomonas fluorescens on meat surfaces.

An inhibition enzyme-linked immunosorbent assay was developed for Pseudomonas fluorescens enumeration of meat surfaces. The assay detected contamination levels as low as 3 x 10(5) bacteria per ml and could be completed within 4 h. It could be used as a framework for a test system for quantifying P. fluorescens spoilage in meat products.

Development of an ELISA for measuring the activity of tetanus toxoid in vaccines and comparison with the toxin neutralization test in mice.

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure anti-tetanus toxoid antibody levels in immunized guinea-pig sera as a useful alternative to the currently used toxin neutralization test (TNT) in determining the activity of the tetanus toxoid in vaccines. The ELISA was found to measure antibody levels as low as 5.8 x 10(-5) IU/ml. Furthermore, a comparison of the results from ELISA and TNT involving 132 different commercial vaccines showed a very good correlation (r = 0.94, p < 0.001) between antibody levels measured by both methods. The results suggest that the proposed ELISA is a reliable, simple and economical alternative to the TNT in mice for assessing the activity of tetanus toxoids in vaccines.