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

ABSTRACT 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 109 CFU (from a single 30- or 10-μl dose to four 2.5-μl 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.

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Antibiotic-Free Plasmid Stabilization by Operator-Repressor Titration for Vaccine Delivery by Using Live Salmonella enterica Serovar Typhimurium

Infection and Immunity ◽

10.1128/iai.73.4.2005-2011.2005 ◽

2005 ◽

Vol 73 (4) ◽

pp. 2005-2011 ◽

Cited By ~ 38

Author(s):

Helen S. Garmory ◽

Matthew W. Leckenby ◽

Kate F. Griffin ◽

Stephen J. Elvin ◽

Rosa R. Taylor ◽

...

Keyword(s):

Salmonella Enterica ◽

Vaccine Development ◽

Antibiotic Resistance Gene ◽

Vaccine Delivery ◽

Marker Genes ◽

Antigen Delivery ◽

Protein Vaccine ◽

Heterologous Antigen ◽

Serovar Typhimurium

ABSTRACT Live, attenuated bacteria are effective vectors for heterologous antigen delivery. However, loss of heterologous gene-bearing plasmids is problematic, and antibiotics and their resistance genes are not desirable for in vivo DNA vaccine delivery due to biosafety and regulatory concerns. To solve this problem, we engineered the first vaccine delivery strain that has no requirement for antibiotics or other selectable marker genes to maintain the recombinant plasmid. This model strain of Salmonella enterica serovar Typhimurium, SLDAPD, uses operator-repressor titration (ORT) technology, which requires only the short, nonexpressed lacO sequence for selection and maintenance. SLDAPD, recovered from the spleens and Peyer's patches of mice following oral inoculation, was shown to maintain a plasmid that, in contrast, was lost from parental strain SL3261. We also demonstrated successful application of this technology to vaccine development, since SLDAPD carrying a plasmid without an antibiotic resistance gene that expressed the Yersinia pestis F1 antigen was as efficacious in protecting vaccinated mice against plague as the parental SL3261 strain carrying an antibiotic-selected version of this plasmid. Protection of mice against plague by immunization with Salmonella expressing F1 has previously required two or more doses; here we demonstrated for the first time protective immunity after a single oral immunization. This technology can easily be used to convert any suitable attenuated strain to an antibiotic-free ORT strain for recombinant protein vaccine delivery in humans.

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Prior Immunity to Homologous and HeterologousSalmonella Serotypes Suppresses Local and Systemic Anti-Fragment C Antibody Responses and Protection from Tetanus Toxin in Mice Immunized with Salmonella Strains Expressing Fragment C

Infection and Immunity ◽

10.1128/iai.67.8.3810-3815.1999 ◽

1999 ◽

Vol 67 (8) ◽

pp. 3810-3815 ◽

Cited By ~ 33

Author(s):

Mark Roberts ◽

Andrew Bacon ◽

Jingli Li ◽

Steven Chatfield

Keyword(s):

Tetanus Toxin ◽

Geometric Mean ◽

Lethal Dose ◽

Oral Immunization ◽

Secretory Iga ◽

Heterologous Antigen ◽

Mammalian Immune System ◽

Heterologous Antigens ◽

Antigen Fragment

ABSTRACT We have investigated the effect of preexisting immunity to homologous (Salmonella typhimurium) or heterologous (S. dublin) serotypes of Salmonella on the ability of an attenuated S. typhimurium aroA aroD vector (BRD509) to immunize mice against the heterologous antigen fragment C (FrgC). We studied two strains, BRD847 and BRD937, expressing FrgC carried on plasmids that differ only with respect to the promoter controlling FrgC expression, the nirB promoter in the case of BRD847 and the htrA promoter in the case of BRD937. Mice were preimmunized orally with S. typhimurium BRD509,S. dublin aroA aroD (BRD620), or saline. Forty-four days later, they were immunized orally with BRD847 or BRD937. Prior immunity to S. typhimurium severely depressed the serum immunoglobulin G (IgG) and IgA anti-FrgC response in both BRD847- and BRD937-immunized mice. Mice with existing immunity to S. dublin also had lower IgG anti-FrgC geometric mean titers (GMTs) than did mice preimmunized with saline, but this difference was significant only in the case of mice immunized with BRD937. However, in nonimmune mice or in mice preimmunized with S. typhimuriumor S. dublin, the anti-FrgC IgG GMTs were always higher in mice in the BRD937 groups than in the equivalent BRD847 groups. This is reflected in the effect of prior immunity on the ability of oral immunization with BRD847 or BRD937 to protect mice from challenge with a lethal dose of tetanus toxin. All of the mice preimmunized with saline and then immunized with BRD847 or BRD937 survived challenge. Only 20% of the animals immunized with BRD847 and 60% of the mice in the BRD937 group survived tetanus toxin challenge if they were preimmunized with BRD509. Preexisting immunity to S. dublindid not affect the ability of BRD937 to immunize mice against tetanus, but it did reduce the efficiency of BRD847: only 60% percent of the mice survived challenge. The intestinal secretory IgA responses to FrgC were very similar in the BRD847 and BRD937 groups. Prior immunity did depress the IgA anti-FrgC titers but only significantly so in the mice preimmunized with BRD509. These results show that preexistingSalmonella immunity, particularly to homologous serotypes, can severely compromise the ability of live Salmonellavectors to deliver heterologous antigens to the mammalian immune system. However, the results also indicate that this may be overcome by the design of more powerful in vivo expression systems.

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Optimization of Salmonella enterica Serovar Typhi ΔaroC ΔssaV Derivatives as Vehicles for Delivering Heterologous Antigens by Chromosomal Integration and In Vivo Inducible Promoters

Infection and Immunity ◽

10.1128/iai.73.1.362-368.2005 ◽

2005 ◽

Vol 73 (1) ◽

pp. 362-368 ◽

Cited By ~ 17

Author(s):

Richard Stratford ◽

Nicola D. McKelvie ◽

Nicky J. Hughes ◽

Emma Aldred ◽

Claire Wiseman ◽

...

Keyword(s):

Salmonella Enterica ◽

Aromatic Amino Acids ◽

Luria Bertani ◽

Core Antigen ◽

Human Macrophage ◽

U937 Cells ◽

Heterologous Antigen ◽

Luria Bertani Broth ◽

Heterologous Antigens

ABSTRACT Novel candidate live oral vaccines based on a Salmonella enterica serovar Typhi ZH9 (Ty2 ΔaroC ΔssaV) derivative that directed the expression of either the B subunit of Escherichia coli heat-labile toxin or hepatitis B virus core antigen from the bacterial chromosome using the in vivo inducible ssaG promoter were constructed. The levels of attenuation of the two S. enterica serovar Typhi ZH9 derivatives were similar to that of the parent as assessed by measuring the replication of bacteria within human macrophage-like U937 cells. The expression of heterologous antigen in the respective S. enterica serovar Typhi ZH9 derivatives was up-regulated significantly within U937 cells compared to similar S. enterica serovar Typhi ZH9 derivative bacteria grown in modified Luria-Bertani broth supplemented with aromatic amino acids. Immunization of mice with these S. enterica serovar Typhi ZH9 derivatives stimulated potent antigen-specific serum immunoglobulin G responses to the heterologous antigens.

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The Immunogenicity of the C Fragment of Tetanus Neurotoxin in Production of Tetanus Antitoxin

BioMed Research International ◽

10.1155/2018/6057348 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 8

Author(s):

Rui Yu ◽

Chong Ji ◽

Junjie Xu ◽

Denghai Wang ◽

Ting Fang ◽

...

Keyword(s):

Tetanus Toxin ◽

Low Cost ◽

Horse Serum ◽

Tetanus Antitoxin ◽

Tetanus Neurotoxin ◽

Group 4 ◽

Antibody Titers ◽

Group 5 ◽

Two Stages

The demand of tetanus antitoxin (TAT) as tetanus treatment in developing and underdeveloped countries is still great since it is relatively easy to achieve and affordable. However, there are still issues in the preparation of highly effective TAT with tetanus toxoid (TT) as the immunogen. The tetanus toxin native C-fragment (TeNT-Hc) retains many properties and it is a very promising candidate for the development of tetanus human vaccine. In this study, we tested the immunogenicity of TeNT-Hc in the preparation of tetanus antibodies, by TeNT-Hc alone or in different combinations with TT. The antibody titers and components in horse serum or plasma in different groups were analyzed and compared with those immunized by the conventional TT and it showed comparability with the results of traditional methods. The plasma efficacy and in vivo tetanus toxin neutralization were also tested. After two stages of immunizations, the average potency in plasma of all groups reached more than 1,000 IU / mL except that in group 4. In group 5, the first two basic immunizations with TT and the subsequent immunizations with TeNT-Hc, it showed slightly higher antibody titers and potency. This study demonstrated that TeNT-Hc is a safe, effective, and yet easy-to-produce low-cost immunogen and suitable for TT replacement in tetanus antitoxin production.

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Mucosal and Systemic Immune Responses to Chimeric Fimbriae Expressed by Salmonella enterica Serovar Typhimurium Vaccine Strains

Infection and Immunity ◽

10.1128/iai.68.6.3129-3139.2000 ◽

2000 ◽

Vol 68 (6) ◽

pp. 3129-3139 ◽

Cited By ~ 22

Author(s):

Huaiqing Chen ◽

Dieter M. Schifferli

Keyword(s):

Immune Responses ◽

Vaccine Strain ◽

Salmonella Enterica ◽

Genetic Manipulation ◽

Genetically Engineered ◽

Spike Protein ◽

Transmissible Gastroenteritis Virus ◽

Heterologous Antigen ◽

Serovar Typhimurium

ABSTRACT Recombinant live oral vaccines expressing pathogen-derived antigens offer a unique set of attractive properties. Among these are the simplicity of administration, the capacity to induce mucosal and systemic immunity, and the advantage of permitting genetic manipulation for optimal antigen presentation. In this study, the benefit of having a heterologous antigen expressed on the surface of a live vector rather than intracellularly was evaluated. Accordingly, the immune response of mice immunized with a Salmonella enterica serovar Typhimurium vaccine strain expressing the Escherichia coli 987P fimbrial antigen on its surface (Fas+) was compared with the expression in the periplasmic compartment (Fas−). Orally immunized BALB/c mice showed that 987P fimbriated Salmonella serovar Typhimurium CS3263 (aroA asd) with pCS151 (fas+asd +) elicited a significantly higher level of 987P-specific systemic immunoglobulin G (IgG) and mucosal IgA than serovar Typhimurium CS3263 with pCS152 (fasD mutant,asd +) expressing 987P periplasmic antigen. Further studies were aimed at determining whether the 987P fimbriae expressed by serovar Typhimurium χ4550 (cya crp asd) could be used as carriers of foreign epitopes. For this, the vaccine strain was genetically engineered to express chimeric fimbriae carrying the transmissible gastroenteritis virus (TGEV) C (379-388) and A (521-531) epitopes of the spike protein inserted into the 987P major fimbrial subunit FasA. BALB/c mice administered orally serovar Typhimurium χ4550 expressing the chimeric fimbriae from thetet promoter in pCS154 (fas+asd +) produced systemic antibodies against both fimbria and the TGEV C epitope but not against the TGEV A epitope. To improve the immunogenicity of the chimeric fimbriae, the in vivo inducible nirB promoter was inserted into pCS154, upstream of the fas genes, to create pCS155. In comparison with the previously used vaccine, BALB/c mice immunized orally with serovar Typhimurium χ4550/pCS155 demonstrated significantly higher levels of serum IgG and mucosal IgA against 987P fimbria. Moreover, mucosal IgA against the TGEV C epitope was only detected with serovar Typhimurium χ4550/pCS155. The induced antibodies also recognized the epitopes in the context of the full-length TGEV spike protein. Hence, immune responses to heterologous chimeric fimbriae onSalmonella vaccine vectors can be optimized by using promoters known to be activated in vivo.

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Protective Antigen-Mediated Antibody Response against a Heterologous Protein Produced In Vivo by Bacillus anthracis

Infection and Immunity ◽

10.1128/iai.68.10.5731-5734.2000 ◽

2000 ◽

Vol 68 (10) ◽

pp. 5731-5734 ◽

Cited By ~ 15

Author(s):

Fabien Brossier ◽

Martine Weber-Levy ◽

Michèle Mock ◽

Jean-Claude Sirard

Keyword(s):

Antibody Response ◽

Bacillus Anthracis ◽

Tetanus Toxin ◽

Protective Antigen ◽

Lethal Factor ◽

Specific Response ◽

Lethal Challenge ◽

Heterologous Antigen

ABSTRACT Bacillus anthracis secretes a lethal toxin composed of two proteins, the lethal factor (LF) and the protective antigen (PA), which interact within the host or in vitro at the surfaces of eukaryotic cells. Immunization with attenuated B. anthracisstrains induces an antibody response against PA and LF. The LF-specific response is potentiated by the binding of LF to PA. In this study, we investigated the capacity of PA to increase the antibody response against a foreign antigen. We constructed a chimeric gene encoding the PA-binding part of LF (LF254) fused to the C fragment of tetanus toxin (ToxC). The construct was introduced by allelic exchange into the locus encoding LF. Two recombinant B. anthracis strains secreting the hybrid protein LF254-ToxC were generated, one in a PA-producing background and the other in a PA-deficient background. Mice were immunized with spores of the strains, and the humoral response and protection against tetanus toxin were assessed. The B. anthracis strain producing both PA and LF254-ToxC induced significantly higher antibody titers and provided better protection against a lethal challenge with tetanus toxin than did its PA-deficient counterpart. Thus, PA is able to potentiate protective immunity against a heterologous antigen, demonstrating the potential of B. anthracis recombinant strains for use as live vaccine vehicles.

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