Non-living Vaccines Research Articles

Attenuated vaccines for tropical theileriosis, babesiosis and heartwater: the continuing necessity

Overwhelming evidence has accumulated of the effectiveness of immunization with live attenuated vaccines to control tick-borne diseases of livestock. Despite several disadvantages, vaccination with live attenuated organisms against tropical theileriosis, babesiosis and possibly heartwater constitutes one of the most cost-effective intervention strategies. Although great advances have been made through genomics and proteomics research, this has not yet translated into effective non-living vaccines. As a result, there is a continuing necessity to use available live vaccines in tick and tick-borne disease-control strategies adapted to conditions prevailing in many parts of the world.

Immunodominant Francisella tularensis antigens identified using proteome microarray. ©Crown Copyright 2007 Dstl

Stimulation of protective immune responses against intracellular pathogens is difficult to achieve using non-replicating vaccines. BALB/c mice immunized by intramuscular injection with killed Francisella tularensis (live vaccine strain) adjuvanted with preformed immune stimulating complexes admixed with CpG, were protected when systemically challenged with a highly virulent strain of F. tularensis (Schu S4). Serum from immunized mice was used to probe a whole proteome microarray in order to identify immunodominant antigens. Eleven out of the top 12 immunodominant antigens have been previously described as immunoreactive in F. tularensis. However, 31 previously unreported immunoreactive antigens were revealed using this approach. Twenty four (50%) of the ORFs on the immunodominant hit list belonged to the category of surface or membrane associated proteins compared to only 22% of the entire proteome. There were eight hypothetical protein hits and eight hits from proteins associated with different aspects of metabolism. The chip also allowed us to readily determine the IgG subclass bias, towards individual or multiple antigens, in protected and unprotected animals. These data give insight into the protective immune response and have potentially important implications for the rational design of non-living vaccines for tularemia and other intracellular pathogens.

Prospects for recombinant vaccines against Babesia bovis and related parasites

Babesial parasites infect cattle in tropical and temperate regions of the world and cause significant morbidity and mortality. Discovery of protective antigens that could be used in a killed vaccine has been slow and to date there are few promising vaccine candidates for cattle Babesia. This review describes mechanisms of protective innate and adaptive immune responses to babesial parasites and different strategies to identify potentially protective protein antigens of B. bovis, B. bigemina, and B. divergens. Successful parasites often cause persistent infection, and this paper also discusses how B. bovis evades and regulates the immune response to promote survival of parasite and host. Development of successful non-living recombinant vaccines will depend on increased understanding of protective immune mechanisms and availability of parasite genomes.

B subunit of E. coli enterotoxin as adjuvant and carrier in oral and skin vaccination

Mucosal sites are one of the main natural ports of entry into the body. Stimulation of a local response by antibodies as the systemic protection may enhance the efficacy of non-living vaccines, and allow for vaccination by subunit vaccines without the need for injection. Mucosal or skin vaccination necessitates a suitable adjuvant and carrier. Escherichia coli heat-labile enterotoxin (LT) and its B subunit (LTB) have been found to be effective adjuvants. The aim of this study was to efficiently produce and purify recombinant LTB (brLTB), and examine its adjuvant and carrier properties. The gene encoding LTB was cloned and expressed in E. coli, and the product was found to have a pentameric form with the ability to bind the cell receptor, GM1 ganglioside. A one-step method for efficient purification and concentration of brLTB was developed. Both oral and intramuscular vaccination with purified brLTB yielded high antibody titers, which detected the whole toxin. In an attempt to test its adjuvant characteristics, brLTB was mixed with either BSA or a recombinant protein (rKnob of egg drop syndrome adenovirus) and delivered intramuscularly, orally or transcutaneously. The addition of brLTB significantly elevated the antibody response in groups vaccinated orally and transcutaneously, but had no influence in injected groups. Vaccination with another recombinant protein, (viral protein 2 of infectious bursal disease virus) supplemented with brLTB did not elevate the antibody response, as compared to vaccination with the antigen alone. These results demonstrate that the addition of brLTB makes oral and transcutaneous vaccination with protein antigens possible.

A Non-Living Nasal Influenza Vaccine Can Induce Major Humoral and Cellular Immune Responses in Humans without the Need for Adjuvants

Twenty-eight healthy adult volunteers were immunized intranasally with an inactivated whole-virus influenza vaccine based on the strain A/New Caledonia/20/99 (H1N1), either in saline or mixed with formaldehyde-inactivated Bordetella pertussis as a mucosal adjuvant, or in a thixotropic vehicle with mucoadhesive properties. After four doses, all groups of vaccinees developed significant IgG- and IgA-antibody responses, measured by ELISA, in respectively serum and nasal secretions. None of the volunteers had demonstrable hemagglutination inhibition (HAI) antibodies in serum before being immunized, whereas more than 80% of them reached HAI titers ? 40, considered protective, after immunizations. In addition, cellular immune responses, measured as significant increases in CD4+ T-cell proliferation and granzyme B-producing cytotoxic T-cells, were detected against the vaccine strain as well as against heterologous virus strains (H3N2). However, no additive effect on these responses could be demonstrated with use of B. pertussis or the thixotropic substance in the present vaccines. It appeared, actually, that the mucoadhesive vehicle containing the thixotropic substance was less efficient than were the two other formulations. An influenza vaccine made as a simple particulate formulation of inactivated virus, and given repeatedly onto the nasal mucosa, may thus be an attractive alternative to currently available vaccines.

Anthrax: an overview

Anthrax is a zoonotic disease that occurs in domesticated and wild animals. Humans become infected by contact with infected animals or contaminated products, but there are no cases of human-to-human transmission. Under natural circumstances, infection occurs by the cutaneous route and extremely rarely, by the inhalational or gastrointestinal routes. Cutaneous anthrax, the most common form is usually curable. A small percentage of cutaneous infections become systemic, and these can be fatal. An aerosol exposure to spores causes inhalational anthrax. This form of disease begins with non-specific symptoms followed in two to three days by the sudden onset of respiratory distress with dyspnea, cyanosis, and stidor. It is rapidly fatal. Treatment consists of massive doses of antibiotics and supportive care. Post exposure antibiotic prophylaxis is effective in experimental animals and should be instituted as soon as possible after exposure. A licensed nonliving vaccine is available for human use.

Vaccines against Infections Caused by Salmonella, Shigella, and Pathogenic Escherichia coli.

Infectious diseases represent one of the most common causes of death worldwide, with the enteropathogenic bacteria Salmonella and Shigella and pathogenic Escherichia coli being among the most detrimental. Currently, vaccination represents the preferred method of preventing such infections. For stimulating the adaptive immune response, immunizations are frequently based on formulations which include inactivated whole-cell vaccines, live attenuated vaccines, or subunit vaccines. These can be administered via a parenteral or mucosal route, the latter having the advantage that it most closely mimics the actual course of infection. In addition to the type of vaccine and method of application, important consideration needs to be paid to safety, efficacy, and cost, which are often major bottlenecks in the successful implementation of vaccines. In this chapter we take a limited look at the history surrounding vaccinations involving Salmonella, Shigella, and pathogenic E. coli. Salmonella infections, which can lead to typhoid fever, are becoming increasing difficult to treat with antibiotics due to multi-drug-resistant strains. At present, the parenteral Vi-based subunit vaccines and the live attenuated oral vaccine Ty21a have proven to be the vaccines of choice, with high levels of protective efficacy and limited side effects. Shigella infections are responsible for the diarrheal disease shigellosis. Various live and nonliving mucosal and parenteral vaccines have been tested, with the most promising candidates evolving around those that stimulate the production of O-antigen-specific antibodies. Pathogenic Escherichia coli infections can lead to severe diseases due to the bacterium's production of several specific toxins. Vaccines against this bacterium target its toxins, as well as surface-exposed antigens, all of which have been found to be effective as immunogens.

Approaches towards the development of a vaccine against tuberculosis: recombinant BCG and DNA vaccine

The last few years have witnessed intense research on vaccine development against tuberculosis. This has been driven by the upsurge of tuberculosis cases globally, especially those caused by multi-drug-resistant Mycobacterium tuberculosis strains. Various vaccine strategies are currently being developed which can be broadly divided into the so-called living and non-living vaccines. Examples are attenuated members of the M. tuberculosis complex, recombinant mycobacteria, subunit proteins and DNA vaccines. Given current developments, we anticipate that recombinant BCG and DNA vaccines are the most promising. Multiple epitopes of M. tuberculosis may need to be cloned in a vaccine construct for the desired efficacy to be achieved. The technique of assembly polymerase chain reaction could facilitate such a cloning procedure.

Rational approaches to immune regulation.

Our laboratory is interested in the properties of proteins that render them immunogenic, and how such immunogenicity may be modulated in vivo. We are attempting to enhance the immune response in the design of more effective vaccines against viral diseases, such as HIV, and against tumor antigens expressed on breast, ovarian, and cervical cancer and B cell lymphomas. Our main approach is to use a facultative intracellular bacterium, Listeria monocytogenes, which has the unusual ability to live and grow in the cytoplasm of the cell and is thus an excellent vector for targeting passenger antigens to the major histocompatibility complex (MHC) class I pathway of antigen processing with the generation of authentic cytotoxic T lymphocytes (CTL) epitopes. In the field of tumor immunotherapy, we are also developing nonliving vaccine vectors for tumor antigens.

Recombinant Vibrio cholerae ghosts as a delivery vehicle for vaccinating against Chlamydia trachomatis

An efficacious vaccine is needed to control the morbidity and burden of rising healthcare costs associated with genital Chlamydia trachomatis infection. Despite considerable efforts, the development of reliable chlamydial vaccines using conventional strategies has proven to be elusive. The 40 kDa major outer membrane protein (MOMP) of C. trachomatis is so far the most promising candidate for a subunit vaccine. The lack of satisfactory protective immunity with MOMP-based vaccine regimens to date would suggest that either MOMP alone is inadequate as a vaccine candidate or better delivery systems are needed to optimize the effect of MOMP. Recombinant Vibrio cholerae ghosts (rVCG) are attractive for use as non-living vaccines because they possess strong adjuvant properties and are excellent vehicles for delivery of antigens of vaccine relevance to mucosal sites. The suitability of the ghost technology for designing an anti-chlamydial vaccine was evaluated by constructing a rVCG vector-based candidate vaccine expressing MOMP (rVCG-MOMP) and assessing vaccine efficacy in a murine model of C. trachomatis genital infection. Intramuscular delivery of the rVCG-MOMP vaccine induced elevated local genital mucosal as well as systemic Th1 responses. In addition, immune T cells from immunized mice could transfer partial protection against a C. trachomatis genital challenge to naı̈ve mice. These results suggest that rVCG expressing chlamydial proteins may constitute a suitable subunit vaccine for inducing an efficient mucosal T cell response that protects against C. trachomatis infection. Altogether, the potency and relatively low production cost of rVCG offer a significant technical advantage as a chlamydial vaccine.

Anthrax vaccines.

The only impetus for the development of new anthrax vaccines is to protect humans against the intentional use of Bacillus anthracis as a bioterrorist or warfare agent. Live attenuated vaccines against anthrax in domesticated animals were among the very first vaccines developed. This was followed by the development of nonliving component vaccines leading to the eventual licensure of protein-based vaccines for human use in the 1970s. This chapter will review the recent advances in developing protein, live attenuated, and genetic vaccines against anthrax.

Vaccines against cysticercosis and hydatidosis

The recombinant vaccines that have been developed against cysticercosis and hydatidosis in sheep and cattle are remarkable for their effectiveness and are prominent as examples of the very few non-living vaccines against parasitic diseases. Their development has been through practical application of molecular parasitology, utilising immunochemical techniques in antigen identification and recombinant DNA methods in antigen production. This brief overview discusses the contribution of molecular techniques to the successful development of recombinant vaccines against Taenia ovis, Taenia saginata and Echinococcus granulosus as well as the immunological and genomic studies that have arisen from their development.

Anthrax.

Bacillus anthracis was shown to be the etiological agent of anthrax by R. Koch and L. Pasteur at the end of the nineteenth century. The concepts on which medical microbiology are based arose from their work on this bacterium. The link between plasmids and major virulence factors of B. anthracis was not discovered until the 1980s. The three toxin components are organized in two A-B type toxins, and the bacilli are covered by an antiphagocytic polyglutamic capsule. Structure-function analysis of the toxins indicated that the common B-domain binds to a ubiquitous cell receptor and forms a heptamer after proteolytic activation. One enzyme moiety is an adenylate cyclase and the other is a Zn(2+) metalloprotease, which is able to cleave MAPKKs. The capsule covers an S-layer sequentially composed of two distinct proteins. Knowledge of the toxins facilitates the design of safer veterinary vaccines. Spore-structure analysis could contribute to the improvement of human nonliving vaccines. The phylogeny of B. anthracis within the Bacillus cereus group is also reviewed.

Nouveaux modes d'administration des vaccins

Since the time of Louis Pasteur, immunization has rhymed almost exclusively with injection. For mass vaccination programs (e.g. in under industrialized countries and in the military), needleless systems of injection by air jets had their moment of glory in the 1970s. They have been abandoned because of the theoretical risk of cross contamination (with hepatitis B virus, hepatitis C virus, and HIV) Although a system that relied on individual injection heads/ampules might have been able to continue the mass vaccinations, most efforts, instead, are directed today at jet injectors intended for single use. Progress in immunology and advances in formulation have allowed exploration of the transcutaneous route and, especially, the mucosal routes, nasal and oral, which both show promise. Via the intranasal route, live attenuated and non-living adjuvanted vaccines are being developed, most notably for the influenza virus. Via the oral route, vaccine research has been particularly geared towards bacterial vaccines (e.g. Vibrio cholere, enterotoxigenic, Escherichia coli, and Shigella spp). Finally, will we be able to vaccinate ourselves one day by eating fruits or vegetables genetically modified to express vaccine antigens? A multi-valent vaccine that may be administered safely by a route other than injection, which would be available to everyone on Earth is not a utopia… but the road ahead remains long.

Characterization and immunogenicity of Vibrio cholerae ghosts expressing toxin-coregulated pili

Bacterial ghosts are attractive for use as non-living vaccines and as carriers of heterologous antigens of vaccine relevance. Ghosts were prepared from Vibrio cholerae strains of O1 or O139 serogroup after growth under culture conditions, which favor or repress the production of toxin-coregulated pili (TCP). Immunoblotting confirmed the TCP status of these V. cholerae ghosts (VCG), which retained the cellular morphology and envelope sub-component profile of viable bacteria. Rabbits were immunized with VCGs prepared from O139 bacteria with TCP-positive or TCP-negative phenotypes and the resulting sera assayed for antibodies to lipopolysaccharide (LPS) and to TCP. Regardless of the TCP status of the VCG preparations used for immunization, all animals produced antibodies to LPS as demonstrated in bactericidal assays. These antibodies were probably responsible for the capacity of the antisera to confer passive immunity to challenge with the homologous O139 strain in the infant mouse cholera model (IMCM). Only following immunization with TCP-positive VCG, however, were antibodies to TCP generated, as judged by the potential of antisera to mediate protection against a challenge strain of heterologous serogroup.

New tuberculosis vaccines based on attenuated strains of the Mycobacterium tuberculosis complex.

The world urgently needs a better tuberculosis vaccine. Bacille Calmette-Guerin (BCG), an attenuated strain of Mycobacterium bovis, has been very widely used as a vaccine for many years but has had no major effect on reducing the incidence of tuberculosis. A number of alternative living and non-living vaccines are being investigated. Live vaccine candidates include genetically modified forms of BCG, genetically attenuated strains of the Mycobacterium tuberculosis complex and genetically engineered vaccinia virus and Salmonella strains. Non-living vaccine candidates include killed mycobacterial species, protein subunits and DNA vaccines. One requirement for acceptance of any new vaccine will be a favourable comparison of the protection it induces relative to BCG in a range of animal models, some of which may need further development. Molecular genetic techniques are now available that enable production of live attenuated strains of the M. tuberculosis complex with vaccine potential. In the first of two broadly different approaches that are being used, large numbers of mutants are produced by transposon mutagenesis or illegitimate recombination and are screened for properties that correlate with attenuation. In the second approach, putative genes that may be required for virulence are identified and subsequently inactivated by allelic exchange. In both approaches, mutants that are attenuated need to be identified and subsequently tested for their vaccine efficacy in animal models. Many mutants of the M. tuberculosis complex have now been produced and the vaccine properties of a substantial number will be assessed in the next 3 years.

Extended recombinant bacterial ghost system

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new stategy for adjuvant free combination vaccines.

Special Section Future Trendsin Vaccination: Is cardiovascular disease preventable by vaccination?

The possibility of using vaccination as a tool in the prevention of atherosclerotic disease was opened by the findings that infection with Chlamydia pneumoniae was an independent risk factor for cardiovascular disease, including acute myocardial infarction. Since this finding, data have accumulated confirming the initial epidemiological association and demonstrating the presence of C. pneumoniae and/or its components in vascular lesions. Recent intervention trials with antimicrobial drugs have furthermore suggested a pathogenetic relationship. The role of C. pneumoniae needs, however, to be further confirmed before deciding on the use of a possible vaccine. At present, a vaccine for C. pneumoniae is not available but development is ongoing. The task is far from easy: the intracellular bacteria cannot be reached by antibodies, and the stimulation of CDS' T cells required for protection is difficult with a nonliving vaccine. On the other hand, recent advances in biotechnology, including the sequence of the full genome of C. pneumoniae, provide unique tools for the work. With enough interest in the development of a C. pneumoniae vaccine the first clinical trials could be expected in several years' time. They will, however, have to be extensive in order to ascertain the safety of such a new type of vaccine intended for use in populations in which many have already been infected with the bacteria and many are chronic carriers. Who should be vaccinated is a question to be considered at that point.

New strategies for combination vaccines based on the extended recombinant bacterial ghost system

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a great variety of bacteria and are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts do not need the addition of adjuvants to induce immunity in experimental animals they can also be used as carriers or targeting vehicles or as adjuvants in combination with subunit vaccines. Matrixes like dextran which can be used to fill the internal lumen of ghosts can be substituted with various ligands to bind the subunit or other materials of interest. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. As carriers of foreign antigens there is no limitation in the size of foreign antigens to be inserted and the capacity of all spaces including the membranes, periplasma and internal lumen of the ghosts can be fully utilized. Using the different building blocks and combining them into the recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

Duration of serum antibody responses following vaccination and revaccination of cattle with non-living commercial Pasteurella haemolytica vaccines

This study was designed to determine the duration of serum antibody responses to Pasteurella haemolytica whole cells (WC) and leukotoxin (LKT) in weanling beef cattle vaccinated with various non-living P. haemolytica vaccines. Serum antibodies to P. haemolytica antigens were determined periodically through day 140 by enzymelinked immunosorbent assays. At day 140, cattle were revaccinated, and antibody responses periodically determined through day 196. Three vaccines were used in two experiments (A and B), OneShot™, Presponse ® HP tK , and Septimune ® PH-K. In general, all three vaccines between 7 and 14 days induced antibody responses to WC after vaccination. Antibodies to LKT were induced with OneShot and Presponse. Revaccination at days 28 and 140 usually stimulated anamnestic responses. Serum antibodies to the various antigens remained significantly increased for up to 84 days after vaccination or revaccination. The intensity and duration of antibody responses were variable depending on the experiment and vaccines used. Vaccination with OneShot usually stimulated the greatest responses to WC. Vaccination with OneShot or Presponse resulted in equivalent primary anti-LKT responses. In experiment B, spontaneous seroconversion was found in numerous calves on day 112. Revaccination of those cattle at day 140 resulted in markedly variable antibody responses such that several groups had no increase in antibody responses.