Computer aided selection of candidate vaccine antigens

Immunoinformatics is an emergent branch of informatics science that long ago pullulated from the tree of knowledge that is bioinformatics. It is a discipline which applies informatic techniques to problems of the immune system. To a great extent, immunoinformatics is typified by epitope prediction methods. It has found disappointingly limited use in the design and discovery of new vaccines, which is an area where proper computational support is generally lacking. Most extant vaccines are not based around isolated epitopes but rather correspond to chemically-treated or attenuated whole pathogens or correspond to individual proteins extract from whole pathogens or correspond to complex carbohydrate. In this chapter we attempt to review what progress there has been in an as-yet-underexplored area of immunoinformatics: the computational discovery of whole protein antigens. The effective development of antigen prediction methods would significantly reduce the laboratory resource required to identify pathogenic proteins as candidate subunit vaccines. We begin our review by placing antigen prediction firmly into context, exploring the role of reverse vaccinology in the design and discovery of vaccines. We also highlight several competing yet ultimately complementary methodological approaches: sub-cellular location prediction, identifying antigens using sequence similarity, and the use of sophisticated statistical approaches for predicting the probability of antigen characteristics. We end by exploring how a systems immunomics approach to the prediction of immunogenicity would prove helpful in the prediction of antigens.     

Immunoinformatics and the prediction of immunogenicity

Immunoinformatics is the application of informatics techniques to molecules of the immune system. One of the key goals of immunoinformatics is the development of computer aided vaccine design (CAVD), or computational vaccinology, and its application to the search for new vaccines. Key to solving this challenge is the prediction of immunogenicity, be that at the level of epitope, subunit vaccine or attenuated pathogen. This paper reviews the current state of play in the prediction of immunogenicity and focuses on well developed methods for the prediction of peptide binding affinity to major histocompatibility complexes, which are the necessary preliminary to the in silico identification of T cell epitopes.     

Using Multivalent Adenoviral Vectors for HIV Vaccination

Adenoviral vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. For effective vaccine development it is often necessary to express or present multiple antigens to the immune system to elicit an optimal vaccine as observed preclinically with mosaic/polyvalent HIV vaccines or malaria vaccines. Due to the wide flexibility of Ad vectors they are an ideal platform for expressing large amounts of antigen and/or polyvalent mosaic antigens. Ad vectors that display antigens on their capsid surface can elicit a robust humoral immune response, the “antigen capsid-incorporation” strategy. The adenoviral hexon protein has been utilized to display peptides in the majority of vaccine strategies involving capsid incorporation. Based on our abilities to manipulate hexon HVR2 and HVR5, we sought to manipulate HVR1 in the context of HIV antigen display for the first time ever. More importantly, peptide incorporation within HVR1 was utilized in combination with other HVRs, thus creating multivalent vectors. To date this is the first report where dual antigens are displayed within one Ad hexon particle. These vectors utilize HVR1 as an incorporation site for a seven amino acid region of the HIV glycoprotein 41, in combination with six Histidine incorporation within HVR2 or HVR5. Our study illustrates that these multivalent antigen vectors are viable and can present HIV antigen as well as His₆ within one Ad virion particle. Furthermore, mouse immunizations with these vectors demonstrate that these vectors can elicit a HIV and His₆ epitope-specific humoral immune response.     

Inferring Epitopes of a Polymorphic Antigen Amidst Broadly Cross-Reactive Antibodies Using Protein Microarrays: A Study of OspC Proteins of Borrelia burgdorferi

Epitope mapping studies aim to identify the binding sites of antibody-antigen interactions to enhance the development of vaccines, diagnostics and immunotherapeutic compounds. However, mapping is a laborious process employing time- and resource-consuming ‘wet bench’ techniques or epitope prediction software that are still in their infancy. For polymorphic antigens, another challenge is characterizing cross-reactivity between epitopes, teasing out distinctions between broadly cross-reactive responses, limited cross-reactions among variants and the truly type-specific responses. A refined understanding of cross-reactive antibody binding could guide the selection of the most informative subsets of variants for diagnostics and multivalent subunit vaccines. We explored the antibody binding reactivity of sera from human patients and Peromyscus leucopus rodents infected with Borrelia burgdorferi to the polymorphic outer surface protein C (OspC), an attractive candidate antigen for vaccine and improved diagnostics for Lyme disease. We constructed a protein microarray displaying 23 natural variants of OspC and quantified the degree of cross-reactive antibody binding between all pairs of variants, using Pearson correlation calculated on the reactivity values using three independent transforms of the raw data: (1) logarithmic, (2) rank, and (3) binary indicators. We observed that the global amino acid sequence identity between OspC pairs was a poor predictor of cross-reactive antibody binding. Then we asked if specific regions of the protein would better explain the observed cross-reactive binding and performed in silico screening of the linear sequence and 3-dimensional structure of OspC. This analysis pointed to residues 179 through 188 the fifth C-terminal helix of the structure as a major determinant of type-specific cross-reactive antibody binding. We developed bioinformatics methods to systematically analyze the relationship between local sequence/structure variation and cross-reactive antibody binding patterns among variants of a polymorphic antigen, and this method can be applied to other polymorphic antigens for which immune response data is available for multiple variants.     

Epitope-driven DNA vaccine design employing immunoinformatics against B-cell lymphoma: a biotech's challenge

DNA vaccination has been widely explored to develop new, alternative and efficient vaccines for cancer immunotherapy. DNA vaccines offer several benefits such as specific targeting, use of multiple genes to enhance immunity and reduced risk compared to conventional vaccines. Rapid developments in molecular biology and immunoinformatics enable rational design approaches. These technologies allow construction of DNA vaccines encoding selected tumor antigens together with molecules to direct and amplify the desired effector pathways, as well as highly targeted vaccines aimed at specific epitopes. Reliable predictions of immunogenic T cell epitope peptides are crucial for rational vaccine design and represent a key problem in immunoinformatics. Computational approaches have been developed to facilitate the process of epitope detection and show potential applications to the immunotherapeutic treatment of cancer. In this review a number of different epitope prediction methods are briefly illustrated and effective use of these resources to support experimental studies is described. Epitope-driven vaccine design employs these bioinformatics algorithms to identify potential targets of vaccines against cancer. In this paper the selection of T cell epitopes to develop epitope-based vaccines, the need for CD4(+) T cell help for improved vaccines and the assessment of vaccine performance against tumor are reviewed. We focused on two applications, namely prediction of novel T cell epitopes and epitope enhancement by sequence modification, and combined rationale design with bioinformatics for creation of new synthetic mini-genes. This review describes the development of epitope-based DNA vaccines and their antitumor effects in preclinical research against B-cell lymphoma, corroborating the usefulness of this platform as a potential tool for cancer therapy. Achievements in the field of DNA vaccines allow to overcome hurdles to clinical translation. In a scenario where the vaccine industry is rapidly changing from a mostly empirical approach to a rational design approach, these new technologies promise to discover and develop high-value vaccines, creating a new opportunity for future markets.     

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e. intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 μl of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.     

Optimisation of peptide-based cytotoxic T-cell determinants using

In this brief review we describe the methods that our group and others have developed in incorporating non-natural amino acids into peptide antigens, principally to increase protease resistance, for potential use in peptide-based vaccines. Peptide-based vaccination has the potential to generate protective immunity without the need for in situ antigen synthesis or further proteolytic processing of the antigen. The ability to deliver minimal T cell epitopes to the effector cells of the immune system also minimises unwanted side effects and simplifies clinical monitoring. The major hurdle in designing successful peptide-based vaccines resides in issues surrounding the delivery and stability of the peptide immunogen, as it is the form in which the epitope is delivered that will determine how it will be processed by the immune system and ultimately whether it will be capable of inducing an appropriate immune response. Thus, one of the confounding issues with peptide-based vaccines is their poor bioavailability, which is predominantly due to proteolysis and oxidative damage of the `naked' peptide. Strategies that stabilise peptide epitopes promise to overcome the current problems and make peptide-based immunogens more applicable in vaccine design.     

Metadherin peptides containing CD4+ and CD8+ T cell epitopes as a therapeutic vaccine candidate against cancer

The concept of peptide‐based vaccines against cancer has made noteworthy progress. Metadherin (MTDH) overexpression and its role in the development of diverse cancers make it an attractive target for cancer immunotherapy. In the current study, six different T cell epitope prediction tools were run to identify MTDH peptides with multiple immunogenic regions. Further, molecular docking was performed to assess HLA‐peptide binding interactions. Nine and eleven peptides fragments containing multiple CD8 ⁺ and CD4 ⁺ T‐cell epitopes, ranging from 9 to 20 amino acids, respectively, were obtained using a consensus immunoinformatics approach. The three peptides that were finally identified as having overlapping CD4 ⁺ and CD8 ⁺ T‐ cell epitopes are ARLREMLSVGLGFLRTELG, FLLGYGWAAACAGAR, YIDDEWSGLNGLSSADP. These peptides were found to not only have multiple T cell epitopes but also to have binding affinity with wide HLA molecules. A molecular docking study revealed that the predicted immunogenic peptides (with single or multiple T cell epitopes) of MTDH have comparable binding energies with naturally bound peptides for both HLA classes I and II. Thus, these peptides have the potential to induce immune responses that could be considered for developing synthetic peptide vaccines against multiple cancers.     

Integrating T-cell epitope annotations with sequence and structural information using DAS

Immunoinformatics is an emerging new field that benefits from computational analyses and tools that facilitate the understanding of the immune system. A large number of immunoinformatics resources such as immune-related databases and analysis software are available through the World Wide Web for the benefit of the research community. However, immunoinformatics developments have sometimes remained isolated from mainstream bioinformatics. Therefore, there is clearly a need for integration, which will empower the exchange of data and annotations within the scientific community in a quick and efficient fashion. Here, we have chosen the Distributed Annotation System (DAS), for integrating in house annotations on experimental and predicted HLA I-restriction elements of CD8 T-cell epitopes with sequence and structural information.     

Identification and characterization of merozoite surface protein 1 epitope

Malaria is an important tropical infection which urgently requires intervention of an effective vaccine. Antigenic variations of the parasite and allelic diversity of the host are main problems in the development of an effective malaria vaccine. Cytotoxic T lymphocytes (CTL) directed against Plasmodium falciparum­derived antigens are shown to play an important role for the protection against malaria. The merozoite surface protein 1 (MSP1) is expressed in all the four life-cycle stages of Plasmodium falciparum and did not find any sequence similarity to human and mouse reference proteins. MSP1 is a known target of the immune response and a single CTL epitope binding to the HLA­A*0201 is available for merozoite form. Here, we report the results from the computational characterization of MSP1, precursor (1720 residue) and screening of highest scoring potential CTL epitopes for 1712 overlapping peptides binding to thirty four HLA class­I alleles and twelve HLA class­I supertypes (5 HLA­A and 7 HLA­B) using bioinformatics tools. Supertypes are the clustered groups of HLA class­I molecules, representing a sets of molecules that share largely overlapping peptide binding specificity. The prediction results for MSP1 as adhesin and adhesin-like in terms of probability is 1.0. Results also show that MSP1 has orthologs to other related species as well as having non allergenicity and single transmembrane properties demonstrating its suitability as a vaccine candidate. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage.     

Immune Responses to HBsAg Conjugated to Protein D of Non-Typeable Haemophilus influenzae in Mice

BackgroundHepatitis B vaccine that contains an aluminum hydroxide adjuvant induces apoptotic death of Hepa 1–6 cells. Difficult-to-degrade chemical additives in vaccines effectively enhance vaccine immunogenicity, but also affect the host tissue. Identification of bio-molecules that are readily degraded and compatible in vivo as an adjuvant is important for vaccine research. The hapten–carrier effect suggests that stimulation of helper T (Th) cells by carrier adjuvants is feasible. Protein D (PD) of non-typeable Haemophilus influenzae covalently conjugated to some polysaccharide vaccines has been confirmed to convert T-cell independent (TI) antigens into T-cell dependent (TD) antigens, and elicit strong T-cell responses ultimately. Herein, we would substitube PD for aluminum hydroxide adjuvant in Hepatitis B vaccine.ConclusionsRecombinant truncated PD covalently conjugated to HBsAg antigen enhanced the immunogenicity of the antigen in mice simultaneously by humoral and cellular immune response, which would facilitate therapeutic hepatitis B vaccines.     

Human CD8+ T Cells from TB Pleurisy Respond to Four Immunodominant Epitopes in Mtb CFP10 Restricted by HLA-B Alleles

CD8⁺ T cells are essential for host defense to Mycobacterium tuberculosis (Mtb) infection and identification of CD8⁺ T cell epitopes from Mtb is of importance for the development of effective peptide-based diagnostics and vaccines. We previously demonstrated that the secreted 10-KDa culture filtrate protein (CFP10) from Mtb is a potent CD8⁺ T cell antigen but the repertoire and dominance pattern of human CD8 epitopes for CFP10 remained poorly characterized. In the present study, we undertook to define immunodominant CD8 epitopes involved in CFP10 using a panel of CFP10-derived 13–15 amino acid (aa) peptides overlapping by 11 aa. Four peptides in CFP10 were observed to induce significant CD8⁺ T cell responses and we further determined the size of the epitopes involved in each individual peptide tested. Four 9 aa CD8 epitopes were finally identified and deleting a single amino acid from the N or C terminus of either peptide markedly reduced IFN-γ production, suggesting that they are minimum of CD8 epitopes. In the individuals tested, each epitope represented a single immunodominant response in CD8⁺ T cells. The epitope-specific CD8⁺ T cells displayed effector or effector memory phenotypes and could upregulate the expression of CD107a/b upon antigen stimulation. In addition, we found that epitope-specific CD8⁺ T cells shared biased usage of T cell receptor (TCR) variable region of β chain (Vβ) 12, 9, 7.2 or Vβ4 chains. As judged from HLA-typing results and using bioinformatics technology for prediction of MHC binding affinity, we found that the epitope-specific CD8⁺ T cells are all restricted by HLA-B alleles. Our findings suggest that the four epitopes in CFP10 recognized by CD8⁺ T cells might be of importance for the development of Mtb peptide-based vaccines and for improved diagnosis of TB in humans.     

Genome-wide analysis of Excretory/Secretory proteins in Trypanosoma brucei brucei: Insights into functional characteristics and identification of potential targets by immunoinformatics approach

Trypanosoma brucei brucei (T.b.brucei) is an extra-cellular parasite that causes Animal African Trypanosomiasis (AAT) disease in animals. Till day, this disease is more difficult to treat and control due to lack of efficient vaccines and early diagnosis of the parasite infection. T.b.brucei Excretory/Secretory (ES) proteins were involved in pathogenesis and key for understanding the host-parasite interactions. Functions of T.b.brucei's ES proteins were poorly investigated and experimental identification is expensive and time-consuming. Bioinformatics approaches are cost-effective by facilitating the experimental analysis of potential drug targets for parasitic diseases. Here we applied several bioinformatics tools to predict and functionalize the annotation of 1104 ES proteins and immunoinformatics approaches carried out to predict and evaluate the epitopes in T.b.brucei. Secretory information, functional annotations and potential epitopes of each ES proteins were available at http://tbb.insilico.in. This study provides functional information of T.b.brucei for experimental studies to identify potential targets for diagnosis and therapeutics development.     

Secret-AAR: a web server to assess the antigenic density of proteins and homology search against bacterial and parasite secretome proteins

The secretome refers to all the Excreted/Secreted (ES) proteins of a cell, and these are involved in critical biological processes, such as cell-cell communication, and host immune responses. Recently, we introduced the Abundance of Antigenic Aegions (AAR) value to assess the protein antigenic density and to evaluate the antigenic potential of secretomes. Here, to facilitate the AAR calculation, we implemented it as a user-friendly webserver. We extended the webserver capabilities implementing a sequence-based tool for searching homologous proteins across secretomes, including experimental and predicted secretomes of Mycobacterium tuberculosis and Taenia solium. Additionally, twelve secretomes of helminths, five of Mycobacterium and two of Gram-negative bacteria are also available. Our webserver is a useful tool for researchers working on immunoinformatics and reverse vaccinology, aiming at discovering candidate proteins for new vaccines or diagnostic tests, and it can be used to prioritize the experimental analysis of proteins for druggability assays. The Secret-AAR web server is available at http://microbiomics.ibt.unam.mx/tools/aar/.     

Alternative Inactivated Poliovirus Vaccines Adjuvanted with Quillaja brasiliensis or Quil-A Saponins Are Equally Effective in Inducing Specific Immune Responses

Inactivated polio vaccines (IPV) have an important role at the final stages of poliomyelitis eradication programs, reducing the risks associated with the use of attenuated polio vaccine (OPV). An affordable option to enhance vaccine immunogenicity and reduce costs of IPV may be the use of an effective and renewable adjuvant. In the present study, the adjuvant activity of aqueous extract (AE) and saponin fraction QB-90 from Quillaja brasiliensis using poliovirus antigen as model were analyzed and compared to a preparation adjuvanted with Quil-A, a well-known saponin-based commercial adjuvant. Experimental vaccines were prepared with viral antigen plus saline (control), Quil-A (50 µg), AE (400 µg) or QB-90 (50 µg). Sera from inoculated mice were collected at days 0, 28, 42 and 56 post-inoculation of the first dose of vaccine. Serum levels of specific IgG, IgG1 and IgG2a were significantly enhanced by AE, QB-90 and Quil-A compared to control group on day 56. The magnitude of enhancement was statistically equivalent for QB-90 and Quil-A. The cellular response was evaluated through DTH and analysis of IFN-γ and IL-2 mRNA levels using in vitro reestimulated splenocytes. Results indicated that AE and QB-90 were capable of stimulating the generation of Th1 cells against the administered antigen to the same extent as Quil-A. Mucosal immune response was enhanced by the vaccine adjuvanted with QB-90 as demonstrated by increases of specific IgA titers in bile, feces and vaginal washings, yielding comparable or higher titers than Quil-A. The results obtained indicate that saponins from Q. brasiliensis are potent adjuvants of specific cellular and humoral immune responses and represent a viable option to Quil-A.     

Immuno-informatics based approaches to identify CD8+ T cell epitopes within the Leishmania donovani 3-ectonucleotidase in cured visceral leishmaniasis subjects

Leishmaniases are vector-borne diseases for which no vaccine exists. These diseases are caused by the Leishmania species complex. Activation of the CD8⁺ T cell is crucial for protection against intracellular pathogens, and peptide antigens are attractive strategies for the precise activation of CD8⁺ T in vaccine development against intracellular infections. The traditional approach to mine the epitopes is an arduous task. However, with the advent of immunoinformatics, in silico epitope prediction tools are available to expedite epitope identification. In this study, we employ different immunoinformatics tools to predict CD8⁺ T cell specific 9 mer epitopes presented by HLA-A*02 and HLA-B40 within the highly conserved 3′-ectonucleotidase of Leishmania donovani. We identify five promiscuous epitopes, which have no homologs in humans, theoretically cover 85% of the world's population and are highly conserved (100%) among Leishmania species. Presentation of selected peptides was confirmed by T2 cell line based HLA-stabilization assay, and three of them were found to be strong binders. The in vitro peptide stimulation of peripheral blood mononuclear cells (PBMC) from cured HLA-A02⁺ visceral leishmaniasis (VL) subjects produced significantly higher IFN-γ, IL-2 and IL-12 compared to no peptide control healthy subjects. Further, CD8⁺ cells from treated VL subjects produced significantly higher intracellular IFN-γ, lymphocyte proliferation and cytotoxic activity against selected peptides from the PBMCs of treated HLA-A02⁺ VL subjects. Thus, the CD8⁺ T cell specific epitopes shown in this study will speed up the development of polytope vaccines for leishmaniasis.     

A multi-epitope fusion antigen candidate vaccine for Enterotoxigenic Escherichia coli is protective against strain B7A colonization in a rabbit model

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of children’s and travelers’ diarrhea. Developing effective vaccines against this heterologous group has proven difficult due to the varied nature of toxins and adhesins that determine their pathology. A multivalent candidate vaccine was developed using a multi-epitope fusion antigen (MEFA) vaccinology platform and shown to effectively elicit broad protective antibody responses in mice and pigs. However, direct protection against ETEC colonization of the small intestine was not measured in these systems. Colonization of ETEC strains is known to be a determining factor in disease outcomes and is adhesin-dependent. In this study, we developed a non-surgical rabbit colonization model to study immune protection against ETEC colonization in rabbits. We tested the ability for the MEFA-based vaccine adhesin antigen, in combination with dmLT adjuvant, to induce broad immune responses and to protect from ETEC colonization of the rabbit small intestine. Our results indicate that the candidate vaccine MEFA antigen elicits antibodies in rabbits that react to seven adhesins included in its construction and protects against colonization of a challenge strain that consistently colonized naïve rabbits.     

Immunological characterization of peptide mimetics of carbohydrate antigens in vaccine design strategies.

Targeting antigens which cannot be readily addressed by genetic vectors is a major challenge in vaccine design. The inter-conversion of carbohydrate antigens into peptide mimetic forms provides a means to broaden the immune response to carbohydrate antigens. Peptides that mimic carbohydrate antigens offer new possibilities to augment immune responses to such antigens that include inducing carbohydrate reactive T-cell responses. Peptide mimeotopes can be formulated in a variety of ways that include multiple antigen peptides (MAP) and as DNA vaccines that prime for different antibody isotypes. On the immunological side we observe that: (i) depending on the immunogen formulation peptide mimetics can be processed by either CD5+ or CD5-B cells; (ii) peptide mimeotope immunization can induce cross-reactive responses to multiple carbohydrate forms; (iii) priming with peptide mimeotopes can enhance carbohydrate immune responses upon boosting and (iv) immunization with peptide mimeotopes can induce carbohydrate reactive T cells.     

HIV Antigen Incorporation within Adenovirus Hexon Hypervariable 2 for a Novel HIV Vaccine Approach

Adenoviral (Ad) vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. However, in some cases these conventional Ad-based vaccines have had sub-optimal clinical results. These sub-optimal results are attributed in part to pre-existing Ad serotype 5 (Ad5) immunity. In order to circumvent the need for antigen expression via transgene incorporation, the “antigen capsid-incorporation” strategy has been developed and used for Ad-based vaccine development in the context of a few diseases. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The major capsid protein hexon has been utilized for these capsid incorporation strategies due to hexon''s natural role in the generation of anti-Ad immune response and its numerical representation within the Ad virion. Using this strategy, we have developed the means to incorporate heterologous peptide epitopes specifically within the major surface-exposed domains of the Ad capsid protein hexon. Our study herein focuses on generation of multivalent vaccine vectors presenting HIV antigens within the Ad capsid protein hexon, as well as expressing an HIV antigen as a transgene. These novel vectors utilize HVR2 as an incorporation site for a twenty-four amino acid region of the HIV membrane proximal ectodomain region (MPER), derived from HIV glycoprotein gp41 (gp41). Our study herein illustrates that our multivalent anti-HIV vectors elicit a cellular anti-HIV response. Furthermore, vaccinations with these vectors, which present HIV antigens at HVR2, elicit a HIV epitope-specific humoral immune response.