Frances Bowe

Mucosal Vaccination against Serogroup B Meningococci: Induction of Bactericidal Antibodies and Cellular Immunity following Intranasal Immunization with NadA of Neisseria meningitidis and Mutants of Escherichia coli Heat-Labile Enterotoxin

ABSTRACT Conjugated polysaccharide vaccines protect against serogroup C meningococci. However, this approach cannot be applied to serogroup B, which is still a major cause of meningitis. We evaluated the immunogenicity of three surface-exposed proteins from serogroup B Neisseria meningitidis (App, NhhA, and NadA) identified during whole-genome sequencing. Mice were immunized intranasally with individual proteins in the presence of wild-type Escherichia coli heat-labile enterotoxin (LTwt), LTR72, a partially inactivated mutant, or LTK63, a completely nontoxic mutant, as the adjuvant. Each of the meningococcal proteins induced significant cellular responses; NhhA and NadA induced strong antibody responses, but only NadA induced bactericidal antibody when administered intranasally with mucosal adjuvants. In addition, immunoglobulin A and bactericidal antibodies were detected in the respiratory tract following intranasal delivery of NadA. Analysis of antigen-specific cytokine production by T cells from immunized mice revealed that intranasal immunization with NadA alone failed to generate detectable cellular immune responses. In contrast, LTK63, LTR72, and LTwt significantly augmented NadA-specific gamma interferon, interleukin-4 (IL-4), IL-5, and IL-10 production by spleen and lymph node cells, suggesting that both Th1 and Th2 cells were induced in vivo. The strongest cellular responses and highest bactericidal antibody titers were generated with LTR72 as the adjuvant. These findings demonstrate that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the antigen as well as the adjuvant and suggest that nasal delivery of NadA with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci.

Salmonella enterica Serovar Typhimurium surA Mutants Are Attenuated and Effective Live Oral Vaccines

ABSTRACT A previously described attenuated TnphoA mutant (BRD441) of Salmonella enterica serovar Typhimurium C5 (I. Miller, D. Maskell, C. Hormaeche, K. Johnson, D. Pickard, and G. Dougan, Infect. Immun. 57:2758–2763, 1989) was characterized, and the transposon was shown to be inserted in surA, a gene which encodes a peptidylprolyl-cis,trans-isomerase. A defined surA deletion mutation was introduced into S. enterica serovar Typhimurium C5 and the mutant strain, namedS. enterica serovar Typhimurium BRD1115, was extensively characterized both in vitro and in vivo. S. entericaserovar Typhimurium BRD1115 was found to be defective in the ability to adhere to and invade eukaryotic cells. Furthermore, S. enterica serovar Typhimurium BRD1115 was attenuated by at least 3 log units when administered orally or intravenously to BALB/c mice. Complementation of the mutation with a plasmid carrying the intactsurA gene almost completely restored the virulence of BRD1115. In addition, S. enterica serovar Typhimurium BRD1115 demonstrated potential as a vaccine candidate, since mice immunized with BRD1115 were protected against subsequent challenge withS. enterica serovar Typhimurium C5. S. entericaserovar Typhimurium BRD1115 also showed potential as a vehicle for the effective delivery of heterologous antigens, such as the nontoxic, protective fragment C domain of tetanus toxin, to the murine immune system.

Protection against tetanus toxin using a plant-based vaccine.

Plant‐expressed vaccines may provide a unique opportunity for generating anti‐pathogen immunity, especially in countries where cold storage is lacking. In the following study, we show that soluble protein from tobacco leaves expressing fragment C of tetanus toxin protected mice against a lethal tetanus toxin challenge. More importantly, we show that a single intranasal (i.n.) vaccination was as efficient as oral delivery, inducing high levels of activated CD4+ T cells and anti‐toxin antibody. Unlike the oral route, i.n. delivery did not require the presence of adjuvant (cholera toxin). Indeed, addition of cholera toxin induced bystander immune responses to plant proteins as well. This is the first study documenting protective immunity by a single i.n. dose of plant vaccine. Plant‐based vaccines are promising because they are more heat stable, are easy to produce, cheap and do not require needles.

Early interactions of Salmonella enterica serovar typhimurium with human small intestinal epithelial explants

Background:Salmonella enterica serovar typhimurium (Styphimurium) causes invasive gastroenteritis in humans, a disease involving significant penetration of the intestinal mucosa. However, few studies have been undertaken to investigate this interaction directly using differentiated human gut tissue. Aims: To investigate the early interactions of an enteropathogenic strain of S typhimurium with human intestinal mucosa using human intestinal in vitro organ culture (IVOC). Methods: Wild-type and mutant derivatives of S typhimurium TML were used to compare interactions with cultured human epithelial cells, bovine ligated loops, and human intestinal IVOC. Results:S typhimurium TML was shown to attach to cultured Caco-2 brush border expressing cells and cause tissue damage and fluid accumulation in a ligated bovine loop model.Styphimurium TML bound predominantly to the mucus layer of human IVOC explants during the first four hours of IVOC incubation. From four to eight hours of IVOC incubation, small but characteristic foci of attaching and invading Styphimurium TML were detected as clusters of bacteria interacting with enterocytes, although there was no evidence for large scale invasion of explant tissues. Ruffling of enterocyte membranes associated with adherent Salmonella was visualised using electron microscopy. Conclusions: Human IVOC can be used as an alternative model for monitoring the interactions between S typhimurium and human intestinal epithelium, thus potentially offering insight into the early stages of human Salmonella induced gastroenteritis.

Use of the stationary phase inducible promoters, spv and dps, to drive heterologous antigen expression in Salmonella vaccine strains

We have investigated the ability of the growth phase regulated promoters dps and spv, to drive expression of heterologous antigens in Salmonella vaccine strains. Reporter plasmids were constructed which directed beta-galactosidase expression from dps (pDpslacZ) or spv (pSpvlacZ) and these were introduced independently into the Salmonella typhimurium vaccine strain SL3261 (aroA(-)). beta-galactosidase expression was induced 20-fold and 100-fold when broth cultures of SL3261 (pDpslacZ) or SL3261 (pSpvlacZ) respectively, entered the stationary phase of growth. Within macrophages, beta-galactosidase expression was induced 3.5-fold with SL3261 (pDpslacZ) and 7-fold with SL3261 (pSpvlacZ). The spv and dps promoters were used to drive independent expression of the C fragment domain of tetanus toxin (TetC) from plasmids harboured in S. typhimurium SL3261. Levels of anti-TetC antibodies were significantly higher in the sera of BALB/c mice perorally inoculated with SL3261 (pSpvtetC) or SL3261 (pDpstetC) compared to unvaccinated controls. This suggests that these promoter systems may be used to drive foreign antigen expression in live oral Salmonella vaccines.

Vaccines against gut pathogens

Many infectious agents enter the body using the oral route and are able to establish infections in or through the gut. For protection against most pathogens we rely on immunity to prevent or limit infection. The expression of protective immunity in the gut is normally dependent both on local (mucosal) and systemic mechanisms. In order to obtain full protection against some pathogens, particularly non-invasive micro-organisms such as Vibrio cholerae, mucosal immunity may be particularly important. There is a need to take these factors into account when designing vaccines targeting gut pathogens. Conventional parenteral vaccines (injected vaccines) can induce a degree of systemic immunity but are generally poor stimulators of mucosal responses. Thus, a basic prerequisite for designing novel vaccines against gut associated pathogens may be the requirement to induce mucosal and potentially systemic immunity.1 The most effective way to induce local immunity against infectious agents has so far proved to be direct application of vaccine antigens to mucosal surfaces (oral or intranasal delivery).2 The fact that we have so few effective oral vaccines shows that the induction of protective immunity through oral immunisation is not an easy goal to achieve as many antigens are poor oral immunogens. Here we will focus, using bacterial pathogens as examples, on some recent approaches being used to generate novel oral vaccines. Oral vaccines can be based on either live or non-living antigens. The generation of modern live oral vaccines involves the construction of genetically defined attenuated micro-organisms capable of inducing immunity in a non-harmful way. The recent improved understanding of bacterial virulence associated gene function offers the possibility of introducing multiple, defined, attenuating and stable mutations into the genome of bacterial pathogens. Furthermore, the use of precisely attenuated bacterial vectors as carriers for recombinant heterologous antigens can lead to the generation of …

Yersinia enterocoliticaaroA mutants as carriers of the B subunit of the Escherichia coli heat-labile enterotoxin to the murine immune system

Plasmid p5F which directs the expression of the Escherichia coli heat-labile enterotoxin B subunit (LT-B) from the ptac promoter was introduced into the attenuated Yersinia enterocolitica O:8 aroA mutant strain YAM.1. YAM.1 (p5F) expressed high levels of cell-associated and secreted LT-B in a stable fashion when grown on normal laboratory medium. The strain was used as a live oral vaccine in BALB/c mice and vaccinated mice developed high levels of gut-associated and systemic antibodies to both LT-B and the lipopolysaccharide (LPS) of the vaccine strain. Anti-LT-B and anti-LPS responses in the sera were predominantly of the IgG class whereas gut-associated antibodies were predominantly IgA. ELISPOT assays carried out on selected tissues prepared from vaccinated mice showed significant numbers of cells synthesising IgG and IgA antibodies to LT-B. These results show that Y. enterocolitica aroA mutants can be used effectively as carriers of heterologous antigens to the murine immune system.

Development of Attenuated Salmonella Strains that Express Heterologous Antigens.

The ability of attenuated strains of Salmonella to induce humoral, secretory, and cellular immune responses following oral ingestion has made them attractive as a system for delivering foreign antigens to the mammalian immune system. DNA capable of driving the expression of heterologous antigens can be introduced into Salmonella vaccine strains using a variety of approaches. In general, there are two common methods of expressing a foreign antigen in salmonellae: from plasmid vectors or from the bacterial chromosome. Since there are many similarities in the cellular and molecular biology of Escherichia coli and Salmonella, most of the genetic manipulations required to construct expression cassettes can be carried out in E. coli. The resulting constructs can then be introduced into the vaccine strains using simple transformatron or other similar techniques. However, the laboratory manipulation of Salmonella strains should be undertaken using techniques that do not lead to the accumulation of undefined genetic lesions, which may compromise the immunogenicity of Salmonella growing in vivo. With this in mind, we will describe appropriate techniques for manipulating Salmonella with the aim of constructing effective oral vaccines.

A vaccination strategy incorporating DNA priming and mucosal boosting using tetanus toxin fragment C (TetC)

Intramuscular (i.m.) immunisation of BALB/c mice with a DNA vaccine, pcDNA3/tetC, encoding fragment C (TetC) from tetanus toxin, stimulated production of TetC specific IgG2a antibodies in the serum and release of IFN-gamma from TetC stimulated splenocytes. A similar pattern of immune response was detected if pcDNA3/tetC primed mice were boosted i.m. with purified TetC protein or TetC and cholera toxin (included as an adjuvant). In contrast, control mice primed with the empty DNA vector pcDNA3 and boosted i.m. with TetC or TetC and CT, generated a dominant IgG1 specific anti-TetC response in the sera and low or undetectable levels of IFN-gamma from stimulated splenocytes. Thus, i.m. priming with a DNA vaccine modulated the subsequent immune response to the same antigen administered as a protein boost. Similar observations were made when DNA primed mice were boosted using the intranasal mucosal route of immunisation. Interestingly, although mice immunised with pcDNA3/tetC and boosted mucosally with TetC and CT produced anti-TetC IgA in mucosal secretions, the titres were reproducibly lower than those detected in mice immunised with the pcDNA3 vector alone. The immunomodulatory effect of pcDNA3/tetC appeared to be antigen specific as mucosal boosting with an unrelated antigen (pertactin) revealed no significant modulation in terms of the anti-pertactin immune response.

Evaluation of the intranasal challenge route in mice as a mucosal model for Candida albicans infection

The intranasal route was used to study Candida albicans infections in mice. Mice from two different inbred strains were challenged intranasally with C. albicans and the level of local and systemic colonization was monitored. DBA/2 mice were highly susceptible to challenge and viable C. albicans disseminated from the lungs to deeper tissues, including kidneys, liver and spleen within 48 h. In contrast, in BALB/c mice challenged in the same manner, C. albicans were retained within the lungs and cleared. Local and systemic anti-C. albicans immune responses were investigated. BALB/c mice exhibited higher titres of serum and mucosal anti-C. albicans IgA than DBA/2 mice. Splenocytes from BALB/c mice, but not from DBA/2 mice, produced detectable levels of interleukin-4 and -5 following stimulation with C. albicans antigens. Both DBA/2- and BALB/c-derived splenocytes produced interferon-gamma and interleukin-10 in response to similar stimulation. In conclusion, the intranasal route provided a simple, non-invasive murine model for investigating C. albicans infection through mucosal surfaces.