The genome revolution in vaccine research

The conventional approach to vaccine development is based on dissection of the pathogen using biochemical, immunological and microbiological methods. Although successful in several cases, this approach has failed to provide a solution to prevent several major bacterial infections. The availability of complete genome sequences in combination with novel advanced technologies, such as bioinformatics, microarrays and proteomics, have revolutionized the approach to vaccine development and provided a new impulse to microbial research. The genomic revolution allows the design of vaccines starting from the prediction of all antigens in silico, independently of their abundance and without the need to grow the pathogen in vitro. This new genome-based approach, which we have named "Reverse Vaccinology", has been successfully applied for Neisseria meningitidis serogroup B for which conventional strategies have failed to provide an efficacious vaccine. The concept of "Reverse Vaccinology" can be easily applied to all the pathogens for which vaccines are not yet available and can be extended to parasites and viruses.     

Genome-wide screening and identification of antigens for rickettsial vaccine development

The capacity to identify immunogens for vaccine development by genome-wide screening has been markedly enhanced by the availability of microbial genome sequences coupled to proteomic and bioinformatic analysis. Critical to this approach is in vivo testing in the context of a natural host–pathogen relationship, one that includes genetic diversity in the host as well as among pathogen strains. We aggregate the results of three independent genome-wide screens using in vivo immunization and protection against Anaplasma marginale as a model for discovery of vaccine antigens for rickettsial pathogens. In silico analysis identified 62 outer membrane proteins (Omp) from the 949 predicted proteins in the A. marginale genome. These 62 Omps were reduced to 10 vaccine candidates by two independent genome-wide screens using IgG2 from vaccinates protected from challenge following vaccination with outer membranes (screen 1) or bacterial surface complexes (screen 2). Omps with broadly conserved epitopes were identified by immunization with a live heterologous vaccine, A. marginale ssp. centrale (screen 3), reducing the candidates to three. The genome-wide screens identified Omps that have orthologs broadly conserved among rickettsial pathogens, highlighted the importance of identifying immunologically subdominant antigens, and supported the use of reverse vaccinology approaches in vaccine development for rickettsial diseases.     

Advances in the development of Salmonella-based vaccine strategies for protection against Salmonellosis in humans

Salmonella spp. are important human pathogens globally causing millions of cases of typhoid fever and non-typhoidal salmonellosis annually. There are only a few vaccines licensed for use in humans which all target Salmonella enterica serovar Typhi. Vaccine development is hampered by antigenic diversity between the thousands of serovars capable of causing infection in humans. However, a number of attenuated candidate vaccine strains are currently being developed. As facultative intracellular pathogens with multiple systems for transporting effector proteins to host cells, attenuated Salmonella strains can also serve as ideal tools for the delivery of foreign antigens to create multivalent live carrier vaccines for simultaneous immunization against several unrelated pathogens. Further, the ease with which Salmonella can be genetically modified and the extensive knowledge of the virulence mechanisms of this pathogen means that this bacterium has often served as a model organism to test new approaches. In this review we focus on (1) recent advances in live attenuated Salmonella vaccine development, (2) improvements in expression of foreign antigens in carrier vaccines and (3) adaptation of attenuated strains as sources of purified antigens and vesicles that can be used for subunit and conjugate vaccines or together with attenuated vaccine strains in heterologous prime-boosting immunization strategies. These advances have led to the development of new vaccines against Salmonella which have or will soon be tested in clinical trials.     

Recombinant viruses as tools to induce protective cellular immunity against infectious diseases.

Infections by intracellular pathogens such as viruses, some bacteria and many parasites, are cleared in most cases after activation of specific T cellular immune responses that recognize foreign antigens and eliminate infected cells. Vaccines against those infectious organisms have been traditionally developed by administration of whole live attenuated or inactivated microorganisms. Nowadays, research is focused on the development of subunit vaccines, containing the most immunogenic antigens from the particular pathogen. However, when purified subunit vaccines are administered using traditional immunization protocols, the levels of cellular immunity induced are mostly low and not capable of eliciting complete protection against diseases caused by intracellular microbes. In this review, we present a promising alternative to those traditional protocols, which is the use of recombinant viruses encoding subunit vaccines as immunization tools. Recombinant viruses have several interesting features that make them extremely efficient at inducing immune responses mediated by T-lymphocytes. This cellular immunity has recently been demonstrated to be of key importance for protection against malaria and AIDS, both of which are major targets of the World Health Organization for vaccine development. Thus, this review will focus in particular on the development of new vaccination protocols against these diseases.     

A recombinant multivalent combination vaccine protects against Chlamydia and genital herpes

Chlamydia trachomatis and Herpes simplex virus type 2 (HSV-2) genital infections pose a considerable public health challenge worldwide. Considering the high incidence of coinfections by the two pathogens, a combination vaccine that can be administered as a single regimen would be highly desirable. Recombinant Vibrio cholerae ghosts (rVCG) offer an attractive approach for the induction of humoral and cellular immune responses against human and animal pathogens. In this study, we evaluated a bivalent combination vaccine formulation comprising rVCG expressing chlamydial MOMP and HSV-2 glycoprotein D in mice for immunogenicity and protective efficacy against genital challenge with either pathogen. Mice immunized with the combination vaccine elicited secretory IgA and IgG2a antibodies to both chlamydial and HSV-2 antigens in serum and vaginal secretions. Robust antigen-specific mucosal and systemic T helper type 1 responses were induced in mice as measured by increased interferon-gamma levels produced by immune T cells in response to restimulation with target antigen in vitro. In addition, mice immunized with the combination vaccine were prophylactically protected from genital challenge with high doses of live Chlamydia and HSV-2. Thus, the combination vaccine regimen delivered by rVCG elicited adequate immune effectors that simultaneously protected against the individual pathogens.     

Intranasal and oral vaccination with protein-based antigens: advantages,challenges and formulation strategies

pathogens initiate their infections at the human mucosal surface. Therefore, mucosal vaccination, especially through oral or intranasal administration routes, is highly desired for infectious diseases. Meanwhile, protein-based antigens provide a safer alternative to the whole pathogen or DNA based ones in vaccine development. However, the unique biopharmaceutical hurdles that intranasally or orally delivered protein vaccines need to overcome before they reach the sites of targeting, the relatively low immunogenicity, as well as the low stability of the protein antigens, require thoughtful and fine-tuned mucosal vaccine formulations, including the selection of immunostimulants, the identification of the suitable vaccine delivery system, and the determination of the exact composition and manufacturing conditions. This review aims to provide an up-to-date survey of the protein antigen-based vaccine formulation development, including the usage of immunostimulants and the optimization of vaccine delivery systems for intranasal and oral administrations.     

Recent advances in veterinary vaccine adjuvants

Next generation veterinary vaccines are going to mainly comprise of either subunit or inactivated bacteria/viruses. These vaccines would require optimal adjuvants and delivery systems to accord long-term protection from infectious diseases in animals. There is an urgent need for the development of new and improved veterinary and human vaccine adjuvants. Adjuvants can be broadly divided into two classes, based on their principal mechanisms of action: vaccine delivery systems and 'immunostimulatory adjuvants'. Vaccine delivery systems are generally particulate e.g. emulsions, microparticles, ISCOMS and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC). In contrast, immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns, e.g. LPS, MPL and CpG DNA, which activate cells of the innate immune system. Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses and the ways in which immunostimulatory adjuvants might enhance this process in animals and humans alike.     

Genetic engineering applied to the development of vaccines

The simplest application of the modern genetic manipulation methods to vaccine development is the expression in microbial cells of genes from pathogens that encode surface antigens capable of inducing neutralizing antibodies in the host of the pathogen involved. This procedure has been exploited successfully for development of a vaccine against hepatitis B virus (HBV) that is now widely used. Similar approaches have been directed towards formulations for immunization against several other animal and human diseases and some of these preparations are now presently in trials. Of no less importance is the impact of biotechnology in providing reagents for fundamental studies of topics such as the determination of virulence, antigenic variation, virus receptors and the immunological response to viral antigens. The core antigen of HBV is a good example of a product of genetic engineering that is a valuable diagnostic reagent, and that is finding important use in immunological studies of particular pertinence to vaccine development.     

Phase and antigenic variation — the impact on strategies for bacterial vaccine design

Many pathogens have the ability to vary the antigenic composition of surface-associated antigens. Often, this variation is mediated by the regulation of gene expression. By varying its antigenicity, the pathogen is able to avoid host immune responses more efficiently; however, this makes the design of vaccines against pathogens that exhibit antigenic variation difficult. In this review, we use the pathogenic Neisseria as an example of antigenically variable bacteria and discuss some attempts to overcome the problems of vaccine design posed by such organisms.     

Salmonella typhimurium harboring plasmid expressing interleukin-12 induced attenuation of infection and protective immune responses

IL-12 is known to be an essential cytokine which appears to provide protective immunity against intracellular bacteria, such as Salmonella. In this study, we investigated the possibility of developing a vaccine using IL-12 against virulent Salmonella. We used the host defense system activated by cytokine IL-12. The highly virulent Salmonella strain (Salmonella typhimurium UK-1) was transformed with cytokine-expressing plasmids. These live, wild-type pathogens were used as vaccine strains without undergoing any other biological or genetic attenuating processes. The newly developed strains induced partial protection from infections (30-40%). Of note, the interleukin-12-transformed pathogen was safe upon immunization with low doses (10(3) cfu), induced IgG responses, and stimulated protective immune responses against Salmonella typhimurium in mice (80-100%). These results suggest that IL-12 induced attenuation of wild-type Salmonella in the host infection stage and vaccine development using the wild-type strain harboring plasmid-secreting IL-12 may be considered as an alternative process for intracellular bacterial vaccine development without the inconvenience of time-consuming attenuation processes.     

How do antigenically varying pathogens avoid cross-reactive responses to invariant antigens?

Pathogens such as trypanosomes and malaria use antigenic variation to evade immune responses and prolong the duration of infections. As pathogens typically express more than one antigen, even relatively rare conserved antigens might be expected to trigger cross-reactive immune responses capable of clearing the infection. We use simple mathematical models that explicitly consider the dynamic interplay between the replicating pathogen, immune responses to different antigens and immune exhaustion to explore how pathogens can escape the responses to both variable and invariant (conserved) antigens. Our results suggest two hypotheses. In the first, limited quantities of invariant antigens on each pathogen may lead to saturation in killing by cross-reactive responses. In the second, antigenic variation of the dominant antigens prolongs the duration of infection sufficiently to allow for exhaustion of the cross-reactive responses to subdominant, invariant epitopes prior to their being able to control the infection. These hypotheses make distinct predictions: the former predicts that cross-reactive responses will always be ineffective while the latter predicts that appropriately timed treatment could, by preventing exhaustion, lead to the generation of long-lasting protective cross-reactive immunity and thus act similarly to a vaccine.     

Biotechnology offers revolution to fish health management

Biotechnology has many applications in fish health management. The application of monoclonal antibodies (mAbs) provides a rapid means of pathogen identification; antibodies to immunoglobulins from different fish species can be used to monitor the host response following vaccination; and mAbs also have the potential for screening broodstock for previous exposure to pathogens. Luminex technology exemplifies a novel antibody-based method that can be applied to both pathogen detection and vaccine development. Molecular technologies, such as the polymerase chain reaction (PCR), real time PCR and nucleic acid sequence-based amplification (NASBA), have enabled detection, identification and quantification of extremely low levels of aquatic pathogens, and microarray technologies offer a new dimension to multiplex screening for pathogens and host response. Recombinant DNA technology permits large-scale, low-cost vaccine production, moreover DNA vaccination, proteomics, adjuvant design and oral vaccine delivery will undoubtedly foster the development of effective fish vaccines in the future.     

Production of glycoprotein vaccines in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.     

Optimizing vaccine development

Optimizing the development of modern molecular vaccines requires a complex series of interdisciplinary efforts involving basic scientists, immunologists, molecular biologists, clinical vaccinologists, bioinformaticians and epidemiologists. This review summarizes some of the major issues that must be carefully considered. The intent of the authors is to briefly describe key components of the development process to give the reader an overview of the challenges faced from vaccine concept to vaccine delivery. Every vaccine requires unique features based on the biology of the pathogen, the nature of the disease and the target population for vaccination. This review presents general concepts relevant for the design and development of ideal vaccines protective against diverse pathogens.     

Helicobacter pylori vaccine development based on combined subproteome analysis

Effective vaccines could provide long-term solutions to many important infectious diseases, however, vaccine development has been hampered by the slow identification of protective antigens. Proteomics provides global information about relevant antigen properties and thus might be ideally suited for identifying promising vaccine antigen subsets. Helicobacter pylori proteomics data are stored in a proteomics database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). In this review, we describe how a combined Helicobacter subproteome analysis resulted in the rapid identification of novel, highly protective antigens. This illustrates the great potential of pathogen proteomics for vaccine development.     

Maximum antigen diversification in a lyme bacterial population and evolutionary strategies to overcome pathogen diversity

Natural populations of pathogens and their hosts are engaged in an arms race in which the pathogens diversify to escape host immunity while the hosts evolve novel immunity. This co-evolutionary process poses a fundamental challenge to the development of broadly effective vaccines and diagnostics against a diversifying pathogen. Based on surveys of natural allele frequencies and experimental immunization of mice, we show high antigenic specificities of natural variants of the outer surface protein C (OspC), a dominant antigen of a Lyme Disease-causing bacterium (Borrelia burgdorferi). To overcome the challenge of OspC antigenic diversity to clinical development of preventive measures, we implemented a number of evolution-informed strategies to broaden OspC antigenic reactivity. In particular, the centroid algorithm—a genetic algorithm to generate sequences that minimize amino-acid differences with natural variants—generated synthetic OspC analogs with the greatest promise as diagnostic and vaccine candidates against diverse Lyme pathogen strains co-existing in the Northeast United States. Mechanistically, we propose a model of maximum antigen diversification (MAD) mediated by amino-acid variations distributed across the hypervariable regions on the OspC molecule. Under the MAD hypothesis, evolutionary centroids display broad cross-reactivity by occupying the central void in the antigenic space excavated by diversifying natural variants. In contrast to vaccine designs based on concatenated epitopes, the evolutionary algorithms generate analogs of natural antigens and are automated. The novel centroid algorithm and the evolutionary antigen designs based on consensus and ancestral sequences have broad implications for combating diversifying pathogens driven by pathogen–host co-evolution.Subject terms: Population genetics, Bacterial genetics     

Group B Streptococcus: global incidence and vaccine development

An ongoing public health challenge is to develop vaccines that are effective against infectious diseases that have global relevance. Vaccines against serotypes of group B Streptococcus (GBS) that are prevalent in the United States and Europe are not optimally efficacious against serotypes common to other parts of the world. New technologies and innovative approaches are being used to identify GBS antigens that overcome serotype-specificity and that could form the basis of a globally effective vaccine against this opportunistic pathogen. This Review highlights efforts towards this goal and describes a template that can be followed to develop vaccines against other bacterial pathogens.     

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients

Background

Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence.

Methods

In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni.

Results

Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI–TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro.

Conclusion

We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients.

General significance

This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.     

Recent developments in adjuvants for vaccines against infectious diseases

New generation vaccines, particularly those based on recombinant proteins and DNA, are likely to be less reactogenic than traditional vaccines, but are also less immunogenic. Therefore, there is an urgent need for the development of new and improved vaccine adjuvants. Adjuvants can be broadly separated into two classes, based on their principal mechanisms of action; vaccine delivery systems and 'immunostimulatory adjuvants'. Vaccine delivery systems are generally particulate e.g. emulsions, microparticles, iscoms and liposomes, and mainly function to target associated antigens into antigen presenting cells (APC). In contrast, immunostimulatory adjuvants are predominantly derived from pathogens and often represent pathogen associated molecular patterns (PAMP) e.g. LPS, MPL, CpG DNA, which activate cells of the innate immune system. Once activated, cells of innate immunity drive and focus the acquired immune response. In some studies, delivery systems and immunostimulatory agents have been combined to prepare adjuvant delivery systems, which are designed for more effective delivery of the immunostimulatory adjuvant into APC. Recent progress in innate immunity is beginning to yield insight into the initiation of immune responses and the ways in which immunostimulatory adjuvants may enhance this process. However, a rational approach to the development of new and more effective vaccine adjuvants will require much further work to better define the mechanisms of action of existing adjuvants. The discovery of more potent adjuvants may allow the development of vaccines against infectious agents such as HIV which do not naturally elicit protective immunity. New adjuvants may also allow vaccines to be delivered mucosally.