Reverse Vaccinology- Definition, Principle, Production, Uses

Against a particular disease, vaccines are biological compositions that help in enhancing the immunity of a person. The term vaccine was introduced by Edward Jenner in 1796. Later, Pasteur proposed the concept of vaccinology, followed by Jonas Salk, who invented the poliovirus vaccine using formaldehyde treatment along with Albert Sabin. With the start of the genomic era, new revolutions have been taking place in the field of vaccines.

The application of shotgun sequencing has been introduced in giving the whole genomic sequences of several pathogens. With the completion of the sequence of the first living organism, the genomic data was used for the preparation of the vaccines against the organism. The complete genomic sequence of an organism is the reservoir of genes encoding the proteins that can act as potential antigens that can be used as vaccine candidates. This technique of identifying the proteins that are exposed on the surface by using genome instead of the microorganism, this novel approach is known as “reverse vaccinology”.

Reverse Vaccinology
Reverse Vaccinology

Definition of reverse vaccinology

Reverse vaccinology (RV) is defined as a computational approach that aims to identify putative vaccine candidates in the protein-coding genome (proteome) of pathogens.

The first pathogen addressed by the reverse vaccinology approach was Meningococcus B (MenB), a pathogen that causes 50% of meningococcal meningitis worldwide. This bacterium had been refractory to vaccine development because its capsular polysaccharide is identical to a human self-antigen, whereas the bacterial surface proteins are extremely variable.

History of reverse vaccinology

1995- Craig Venter published the genome of a free-living organism i,e, Haemophilus influenza which served as a tool for initiation of reverse vaccinology.

In the 1990s- the development of the serotype B meningitides vaccine started.

2000- companies Chiron vaccine, TIGR, and oxford led by Rino Rappuoli published a whole-genome sequence of MenB (MC58) strain authored by Tettelin et al and Pizza et al. and identified several surface antigens.

2013- after successful completion, the vaccine got approval in the UK 

Definition of reverse vaccinology 2.0

  • Reverse vaccinology 2.0 in modification to reverse vaccinology which utilizes high throughput protein expression, animal models, and genomics, uses human monoclonals, B cell repertoire deep sequencing, proteomics, and structure-based antigen design.
  • In this, Genomics is used not only for antigen discovery, but also for antigen expression, conservation, and epidemiology.
  •  Human monoclonals are used to identify protective antigens/epitopes. 
  • Structural characterization of the Ab–antigen complex is used to instruct antigen design.

Principle of reverse vaccinology

An epitope is an antigenic determinant that plays an important role in the immunity of an organism. These are present on the surface of organisms that can be detected by the antibody. Reverse vaccinology deals with computational analysis of the genome that can be used for the prediction of the epitopes that are surface proteins. So the epitopes play an important role in the development of a candidate vaccine. The major role played in the immune system is B and T lymphocytes.

  • B cells are important in recognizing the epitopes of the antigens that can be identified by the paratopes of antibodies. 
  • T cells play a role in cell-mediated immunity as the processed antigenic peptides interact with the T cell when they are presented in the context of the T cell. 

So the prediction of the epitopes of T and B cells plays an important role in the determination of the candidate vaccine. The epitope prediction plays an important role in designing the epitope-based vaccine.

Figure: A schematic pipeline of reverse vaccinology. Image Source: Wenzhen Liao et al. 2017.

T-cell epitope mapping and prediction

  • Peptide-based vaccines require the application of dominant Epitopes, which elicit a greater immune response than other Epitopes. 
  • It overtone the disadvantages of the traditional vaccine approach which fails to activate a greater cell-mediated immune response against variable pathogens, which is required for clearance of hypervariable viral infections such as HCV and HIV.
  • T cell recognizes the antigenic peptides only when they are presented by MHC I or II, with the help of the CD4 and CD8 molecule. Given the importance of T-cell responses in controlling viral infections, the larger number of T-cell epitope mapping and prediction algorithms available today comes as no surprise. 
  • One of the more comprehensive programs seems to be EpiMatrix from EpiVax Inc
  • EpiMatrix tool: set can predict epitopes against over 100 different MHC class I and class II alleles. 
  • The EpiMatrix platform is also closely tied with additional computational tools such as ClustiMer (scans EpiMatrix results for T-cell epitope ‘clusters’), BlastiMer (automated BLAST tool), OptiMatrix (involved in de-immunizing sequences), Conservatrix (involved in finding conserved epitopes) and Vaccine CAD ( an in silico vaccine design algorithm).

B-Cell epitope prediction and mapping

  • The antigen-antibody interaction plays an important role in immunity, binding takes place at antigenic determinants also known as B-cell epitopes. The B-cell epitopes are defined by a specific surface region of an antigenic protein.
  • They are divided into two different types of epitopes: 
    • linear epitopes 
    • conformational epitopes.
  • The linear epitopes are short peptides while conformational epitopes are composed of amino acids folded in 3- a dimensional protein structure. 
  • The mapping of the B cell epitopes can be done by various techniques. The focus of the scientist is only on the determination of linear B cell epitope. The propensity value of amino acid plays an important role in the determination of its position in B cell epitopes. It was introduced by Hopp and Woods. They utilized the Levitt hydrophobicity scale for the determination of the propensity value for each amino acid. 
  • Several tools are available for the prediction of linear B cell epitopes:
    • PREDITOPE: it uses a multiparametric algorithm based on hydrophilicity, accessibility, flexibility, and secondary structure properties of the amino acids.
    • PEOPLE: it uses the same parameters as PREDITOPE and in addition includes the assessment of beta turns.
    • BepiPred: it is based on random forests trained on B cell epitopes obtained from the 3D structure of antibody-antigen complexes.
    • ABC pred: uses the machine learning-based method for the prediction of the linear B cell epitopes. 

The conformational B cell epitope prediction can be done by following:

  1. Sequence-based prediction method
  • It does not require the target antigen structure to be known. 
  •  It depends on the determination of the antigen-antibody complexes using X-ray crystallography. 
  • DiscoTope: used for the determination of the conformational B cell epitope prediction. PEPITOPE:  uses a combination of propensity value and half-sphere exposure value of amino acid residues.
  1. Mimotpoe analysis based prediction method
  • It combines both computational and experimental techniques for B cell epitope mapping. 
  • It determines the organization of the genuine epitopes.
  •  Examples of B-cell epitope-mapping algorithms include 3DEX (3D-Epitope-Explorer), CEP (conformational epitope predictor), and DiscoTope
  • 3DEX software is designed to allow the localization of linear peptide sequences within the three-dimensional structure of a protein.
  •  CEP predicts epitopes of proteins with known structures using the accessibility of residues and spatial distance cutoffs to predict antigenic determinants, conformational epitopes, and sequential epitopes. 
  • DiscoTope was designed specifically for the prediction of conformational B-cell epitopes. 
  1. Developments in B cell epitope prediction 
  • Prediction of the protective linear B cell epitopes,
  • hybrid and consensus prediction of B cell epitopes, 
  • improved conformational B cell epitope prediction,
  • critical assessment of B cell epitope prediction,
  • immune epitope database and analysis resources.
Schematic diagram of the reverse vaccinology process
Figure: Schematic diagram of the reverse vaccinology process. The structure of the leptospiral cell and associated proteins is shown. Overview of the RV process: (1) selection of proteins from the genome sequence; (2) production of the recombinant subunit vaccines; and (3) evaluation of the RV candidates: protein-related humoral and cellular responses and protection against lethal challenge. LPS, lipopolysaccharide. Image Source: Odir A. Dellagostin et al. 2017.

Production of reverse vaccinology

  1. First, computer analysis of the whole genome identifies the genes coding for predicted antigens and eliminates antigens with homologies to human proteins. 
  2. Then the identified antigens are screened for expression by the pathogen and immunogenicity during infection.
  3. The selected antigens are then used to immunize animals and test whether immunization induces a protective response.
  4. Protective antigens are then tested for their presence and conservation in a collection of strains representative of the species (molecular epidemiology).
  5.  Finally, selected antigens are manufactured on large scale for clinical trials, and candidate vaccines are tested for safety and protective immunity in humans using established correlates of protection or efficacy studies.
  6. Scientific, clinical, and technical information is then analyzed and approved by regulatory agencies, such as the Food and Drug Administration (FDA) or the European Medicinal Agency (EMA).
  7. Policy-making bodies, such as the ACIP and equivalent bodies from other nations, make a recommendation on how the vaccine should be used.
  8. The approved vaccine is then commercialized and used on large scale. At this point, phase IV clinical studies confirm safety.

Applications of reverse vaccinology


  • First reverse vaccine: against serogroup B Neisseria meningitidis. 
  • Because of the hypervariable nature of the pathogen and its protein similarity to humans, the vaccine failed.
  • The specific surface protein of N. meningitidis was selected as a vaccine candidate using computer algorithms. The expression of this protein in E.coli is confirmed using ELISA. 
  • These proteins are outer membrane proteins, surface proteins, and surface-associated lipoproteins, that were potential vaccine candidates against Men B. The analysis of many protein sequences was done to check their antigenicity but only the few could act as good vaccine candidate which has a bactericidal activity that can induce the protective immunity against Men B strains. The successful vaccine was then introduced to human volunteers under phase III trials.


  • Signal peptides, LPXTG motifs, transmembrane helices, and many surface proteins can be easily identified from the whole genome sequence of the L. monocytogenes with the help of various web-based tools.
  • Various tools are used that have their specific role. 
  1. SignalP 3.0: The tool was used for checking the position and the presence of the signal peptide cleavage positions in protein with the reference to Gram-positive bacteria.
  2. TMHMM: The tool was used for determining the number of the transmembrane helices in proteins with the help of the hydrophobic amino acid.
  3. Tipo: The tool was used for determining the no. of the lipoproteins and it distinguishes between the lipoprotein signal peptides, other signal peptides, and n terminal membrane helices by the gram-positive bacteria.
  4. PSORTb: The subcellular localization of the proteins can be done by PSORTb, a valuable tool for genome analysis. 
  • By the above tools, one can easily identify the surface proteins which can be easily used as antigenic epitopes against which the vaccine was produced.


  • Bacillus anthracis is the causative agent of anthrax which infects animals and humans. The organism was used as a weapon for bioterrorism. So the development of a vaccine is an excellent approach to the prevention of the spread of the disease. 
  • Until the 20th century, many people and animals had been killed by anthrax. The first vaccine was prepared against anthrax by Pasteur. 
  • The reverse vaccinology approach was used for the formulation of a candidate vaccine. The antigenic determinants were found by using EMBOSS
  • The antigenic determinant with a greater LCV value was used for designing a molecule for the vaccine candidate. Docking was also done with MHC I molecule showing stable interaction.

Traditional vaccinology vs Reverse vaccinology

CharactersTraditional VaccinologyReverse Vaccinology
Antigens availableUsing biochemical and genetic tools, only a limited number of antigens can be used.All antigens can be identified using genetic tools
Property of antigensThe most abundant and immunogenic antigens of cultivable microbes can be identified.All antigens of all types of microbes can be identified. 
Immunology of the antigensVariable antigens, that elicit a greater immune response. Since some mimic self-antigens, thus cause autoimmunity.Conserved antigens can be detected, which are not very immunogenic. Self-antigens can be negatively selected. 
Polysaccharide antigensImportant vaccine candidateCannot be used in reverse vaccinology. To discover carbohydrate antigens, operons coding for polysaccharides can be detected. 
T cell epitopesKnown epitopes are limited to the known antigens.Virtually every single T cell epitope is available. Screening of the total T cell immunity can be done by overlapping peptides.


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  6. Singh H, Ansari HR, Raghava GPS (2013) Improved Method for Linear B-Cell Epitope Prediction Using Antigen’s Primary Sequence. PLoS ONE 8(5): e62216
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  8. Rappuoli, Rino et al. “Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design.” The Journal of Experimental Medicine 213 (2016): 469 – 481.

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