The progress made in research and dissemination of vaccines since Pasteur’s time is astounding today. In the 1880s, it was not suspected that the vaccine would trigger such a revolution in medicine and pharmacy. The first Pasteur biopreparations were four vaccines: against cholera, anthrax, swine erysipelas and rabies. These vaccines were simple products with a short shelf life, containing weakened or killed pathogens preserved in alcohol. They were produced initially in small laboratories in quantities depending on the demand. In the following years, the production of vaccines moved to large laboratories of pharmaceutical companies, which allowed a large proliferation of antigens in a variety of vaccines produced and massive rise in the number of doses in human and veterinary medicine. Currently, there are over 100 times more veterinary vaccines on the market in Poland than in the time of Louis Pasteur. Only a hundred years after Pasteur created the first vaccine, there was another scientific breakthrough of great importance in this field,
Parallel development of HTS technology and vaccinology
At the end of the 20th century, the first instruments for automated DNA sequencing by capillary electrophoresis were introduced, the so-called “first generation”. The technique was the Sanger method, which allows the reading of DNA sequences. It involves the process of copying the DNA molecule
In 2005, it was time for the "next generation" instruments giving the possibility of sequencing from 84 kb per run to 1 gigabase (Gb) per run, a short read, mass technique and parallel sequencing - high-throughput sequencing (HTS) previously more commonly referred to as next-generation sequencing (NGS). It is used in various fields of science. HTS yields much larger datasets at a much lower cost than the Sanger method. The current progressive platforms give highly parallel sequencing, with a result of a higher order of magnitude (5) (Fig. 2). The development of genome sequencing technology has high momentum. After the first-generation Sanger technology, the second-generation technologies have found uptake (of which the dominant examples are the Genome Analyzer from Illumina, e.g. MiSeq or HiSeq (2); Roche 454 (33); and SOLiD from Applied Biosystems (44)) and even the third generation technologies have appeared (for example MinIon from Oxford Nanopore Technologies and PacBio RS from Pacific Biosciences). The Sanger method was used to sequence the first human genome for 15 years and the result was published at the beginning of the 21st century, while the HiSeq X Ten System introduced in 2014 can sequence approximately 45 human genomes in 24 hours. It is worth adding that in addition to the time of sequencing being much shorter, its cost is now nearly 3,000,000 times lower (18).
Comparison of the Sanger method with the HTS technique (17, 36)
HTS has also been introduced into vaccine research to swift and conspicuous effect. This technology provides a means to broaden knowledge of the complexity of the genomes of vaccine pathogens and their biological characteristics and interaction with the host organism, as well as immunogenetics in the broad sense. Besides the scientific benefits, HTS has also made it possible to design vaccines against certain pathogens for the first time (31). Systems of such a large bandwidth are the future of personalised creation of vaccines and understanding of the genotype – phenotype interaction (11).
A vaccine is an immunotherapeutic preparation of biological origin containing antigens that stimulate the immune system of humans or animals to create immunity after their administration, in other words acquired immunity. The main task of immunotherapy is to protect public health. Vaccines are used for imparting controlled immunity comparable to natural immunity acquired after infection with wild-type organisms. The ability to stimulate the immune system is referred to as immunogenicity (24). There are many different vaccines and also many different methods for classifying them. Primarily they are divided into two groups. The first groups is live attenuated vaccines that contain a pathogen devoid of pathogenic properties. They cause a natural but non-pathogenic infection and confer lifelong immunity
A new branch of medicine dealing with protective vaccinations - vaccinology is a scientific discipline that deals with the vaccine in its concept. It extends not only over scientific research into the development of new vaccines, but also over their subsequent use (38), and hence encompasses post-vaccination immunology, administration of the vaccine, legal regulations, and vaccination methods, programmes and monitoring. Also in the realm of vaccinology is assessment of a vaccine’s effectiveness as well as its impact on the environment (13). Immunisations have indisputably reduced the cost of treating clinical disease, but also the mortality from infectious diseases. The development of vaccinology has given the ability to control many deadly diseases, such as smallpox, diphtheria, polio, tuberculosis, measles and whooping cough. The flagship example is the eradication of smallpox (6). At the end of the eighteenth century, Edward Jenner proved his hypothesis correct that the pus in milkmaids’cowpox blisters could protect against smallpox. Jenner’s finding was impactful by launching a global vaccination campaign in 1979, the total elimination of smallpox was achieved (30). It is one of many examples showing that vaccines have undoubtedly contributed to improving public health in the world.
Despite this, there are still many diseases that do not have an effective vaccine. The design of 21st century vaccines is complicated because vaccinologists are currently dealing with rapidly evolving pathogens, and the help of HTS molecular biology tools is vital for this reason.
The vast majority of vaccines contain strains originating from the field, which lose their initial virulence as a result of various physical or chemical treatments (i.e. multiple passage or temperature treatment). Phenotypic changes observed as non-virulence are reflected in the genotype of a given pathogen. However, occasionally, vaccination-related side effects are observed and sometimes they are connected with the genomes of the vaccine strain. Examples of these side effects are those from vaccines against poliomyelitis caused by poliovirus (PV). The attenuation of vaccine strains involves the alteration of various nucleotides in the genome of PV, among other locations in the coding part of the VP1 or VP3 proteins. It was revealed that vaccine-associated paralytic poliomyelitis noted in recipients of the vaccine could be caused by vaccine strain revertants. That is why it is important to control the strains at the level of their genomes. Recently, the French Official Medicines Control Laboratory (OMCL) of the Agence Nationale de Sécurité du Médicament et des Produits de Santé (ANSM) reported the possibility of using the next generation sequencing (NGS) method as a vaccine quality control test. ANSM implemented a high-throughput sequencing method instead of the mutant analysis by polymerase chain reaction and restriction enzyme cleavage method for monitoring genetic mutations as a quality control test for inactivated poliovirus (PV) vaccines. This allows the determination of neurovirulent viral repertoires in a shorter time, reduces the cost of research and, above all, eliminates the need to use radioactive isotopes (1). Scientists have proven that in the Sabin 3 strain, a mutation at nucleotide 472 of VP1 (
Montmayeur
The control of the genetic stability of microorganisms in vaccines is another example of HTS application in vaccinology. Liu
The main application of HTS in medical research is the analysis of the genomes of pathogenic microorganisms. Additionally, with HTS host–pathogen interaction can be studied and differences in the immune response of the host with respect to B and T cells can be analysed (31). All these HTS possibilities have found application in vaccine research. Recently, a new term in vaccinology has appeared - vaccinomics. This is a multidisciplinary field taking elements from immunology and genetics, with the creation of an individual vaccination therapy as its main goal. Vaccinomics allows the designer of vaccines to achieve faster results in the production of vaccines than traditional vaccinology can deliver. One of the parts of vaccinomics is reverse vaccinology, the reverse engineering of vaccines (20).
Reverse vaccinology is for the prediction of the most immunogenic epitope. A whole genome elucidated using HTS is then screened by very specialised and effective bioinformatic tools to identify such epitopes (20, 42). Reverse vaccinology has enabled the design of vaccines against diseases such as anthrax, malaria, and meningitis (20). The bacterium
Reverse vaccinology recently focused on the identification of vaccine antigens by characterisation of the interactions between antigens and neutralising monoclonal antibodies (mAbs). Guo
Thanks to the HTS technique, it is possible to determine the nucleotide sequence of a given test material from a specific organism. Then the obtained DNA sequences of different organisms are compared to discover the mechanisms of their evolution (14). To exploit this possibility most fully it is essential to make the discovered nucleotide sequences available
The increasing availability of sequencing results requires careful analysis using sophisticated IT tools, which allow the information contained in the genotype to be translated into what is observable at the phenotype level and then be used in vaccinology. In this aspect immunoinformatics is very useful. Urrutia-Baca
HTS is also increasingly used in veterinary vaccinology, both in the development and control of animal vaccines and in recognising the mechanism of virus strain attenuation. This method of learning how attenuation takes place was applied in the development of the vaccine against Marek’s disease. The virus contained in this vaccine, gallid herpesvirus 2 (GaHV-2), was sequenced and it was revealed that serial passage causes the formation of mixed virus populations, which differ in the number and size of genetic changes after attenuation (45). Another example of the use of HTS in animal vaccinology is the search for genetic markers of vaccine strains. Knowledge of such markers would make it possible to quickly distinguish between vaccine and field strains. Such an attempt was made with the vaccine against infectious laryngotracheitis (ILT) of chickens (7). Moreover, a subsequent study on infectious laryngotracheitis virus (ILTV) showed the risk of using different attenuated vaccines in the same population of birds. It revealed that recombinations between vaccine strains led to the creation of virulent recombinant viruses responsible for the disease of Australian poultry flocks (26).
HTS is also used in virulence recovery studies on veterinary vaccines. It may happen that a given disease is effectively controlled by vaccines but despite this, regains of virulence by the disease’s viral agent in these products are reported. This was the case with the vaccine against avian metapneumovirus (aMPV), which infects the respiratory tracts of domestic poultry. From the sequencing studies, a single nucleotide mutation in the vaccine was found that was consistent with the consensus sequence of the virulent adherent virus. Franzo
Another problem in veterinary vaccinology is the distinction between the vaccine and the field strain. The strains included in veterinary vaccines, similarly to human ones, come from field strains that lose their original virulence due to various treatments. The loss of this virulence has consequences at the molecular level but very often they are unknown. However, it should be remembered that strains similar to the vaccine strain still circulate in the field. When a vaccine-like strain is detected in individuals with clinical symptoms of the disease, it raises the question of whether it is actually a vaccine strain or a vaccine-like wild-type one. This duality of possibility was raised by scientists from an Anglo–Italian research group in their publication on infectious bronchitis virus (IBV) vaccines. They appealed to producers to introduce a vaccine and a method (test) to distinguish it from wild-type strains at the same time (27). It seems that the HTS technique could be very useful in finding a suitably distinctive molecular marker.
A significant improvement in the quality of work with the HTS platform would be achieved through the introduction of validated reference standards. Interpretation of the HTS data depends on many factors, such as the complexity of the genome or technical errors resulting from both sequencing and sample preparation. Reference standards would help alleviate these errors by providing accurate data analysis (16). Deep sequencing makes it possible to identify foreign genetic material in a sample, even if we have no information on the probable contamination of that sample. HTS techniques have already been used to detect accidental factors in vaccines as well as cell lines or sera, so it seems that the creation and distribution of HTS reference standards is an extremely important issue for vaccine manufacturers and control laboratories (34). Currently, there are a considerable number of viral metagenomics methods and many options for creating sequencing libraries, but also many scientific and commercial bioinformatic platforms. These databases are constantly evolving. Therefore, it is important to have reliable reference materials to confirm that these different methods give comparable test results. Mee
At the end of 2019, a global pandemic broke out caused by a coronavirus which emerged first in Wuhan, Hubei Province, China. The World Health Organization (WHO) announced the standard format for referring to it, coronavirus disease-2019 (COVID-19), and the International Committee on the Taxonomy of Viruses (ICTV) named this novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (19). Coronaviruses (CoVs) are a group of many different viruses that have the potential to cause an epidemic because of their ability to cross the species barrier and spread rapidly in a new host species. There are several vaccines containing coronaviruses to immunise animals (especially against IBV caused by gammacoronaviruses), but until recently no vaccines existed to immunise humans, despite the severe public health threat of diseases caused by the CoVs (43). Therefore, the scientific world has recently been looking for safe and effective immunoproducts for SARS-CoV-2 which could be quickly put into production at scale (8, 49, 21). HTS platforms became a helpful tool in the scientific race against time. Recent HTS analysis showed that SARS-CoV-2 shares overall genomic similarity with SARS-CoV (79%) and some even with Middle East respiratory syndrome coronavirus (MERS-CoV) (50%). Precise sequence comparison of each gene region may give a better answer as to how SARS-CoV-2 interferes with the host immune response. It seems that SARS-CoV-2 utilises strategies to modulate the host innate immune response similar to those of MERS-CoV (37). HTS was also used to identify SARS-CoV-2 tropism and revealed that the spike protein receptor-binding domain of the virus uses angiotensin-converting enzyme 2 as a cell receptor (37). The COVID-19 situation made the necessity to protect human life the most exigent obligation and many scientific centres began to work on vaccines aimed at minimising the clinical manifestations of the disease. Various ideas have been used in the development of these vaccines: inactivated virus, nucleic acid, adenovirus-based vectors, and recombinant subunits. Studies carried out at the Gamaleya Research Institute of Epidemiology and Microbiology (Russia) developed a vaccine (Sputnik V) which consists of two components, recombinant adenovirus type 5 and recombinant adenovirus type 26 vectors, and both of them contain the gene for SARS-CoV-2 spike glycoprotein (29). A similar but single adenovirus-based vaccine was developed by the Oxford-AstraZeneca and Johnson & Johnson teams. After more than twenty years of research on the development of vaccines based on messengerRNA (mRNA) technology, they are commercialised by two companies, Pfizer/ BioNTech and Moderna. It should be added that the first vaccine against COVID-19 approved for use in the European Union was the Comirnaty vaccine from Pfizer/BioNTech. Soon after, sanctioned by the positive evaluation the European Medicines Agency, the European Commission approved the mRNA-1273 vaccine from Moderna. A similar idea of mRNA will be the basis of a vaccine being developed by the German CureVac company. A protein-based coronavirus vaccine is also under development, as well as several which contain inactivated virus. All the vaccines in use are highly effective, ranging from 70–95% depending on the dose used. However, there is concern over how effective these vaccines will be against the emerging new SARS-CoV-2 variants. Their detection is only possible exploiting HTS technology.