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Research is underway to develop a vaccine to prevent and cure infection from herpes simplex virus (HSV). It emphasizes the critical need for immunization to address public health issues and the shortcomings of existing treatment options. Furthermore, studies on the HSV vaccine advance the field of immunology and vaccine creation, which may help in the battle against other viral illnesses. The current lack of such a vaccine is, in part, due to herpes viral latency in sensory ganglions. Current vaccines rely on tissue-resident memory CD8+ T cells, which are known to provide protection against subsequent HSV reinfection and reactivation without correlating with other immune subsets. For that reason, there is no effective vaccine that can provide protection against latent or recurrent herpes infection. This review focuses on conventional methods for evaluating the efficacy of a herpes vaccine using differential CD8+ T cells and important unaccounted immune aspects for designing an effective vaccine against herpes.

This review provides the potential of intestinal microbiota in vaccine design and application, exploring the current insights into the interplay between the intestinal microbiota and the immune system, with a focus on its intermediary function in vaccine efficacy. It summarizes families and genera of bacteria that are part of the intestinal microbiota that may enhance or diminish vaccine efficacy and discusses the foundational principles of vaccine sequence design and the application of gut microbial characteristics in vaccine development. Future research should further investigate the use of multi-omics technologies to elucidate the interactive mechanisms between intestinal microbiota and vaccine-induced immune responses, aiming to optimize and improve vaccine design.

Molecular mimicry, a phenomenon in which microbial or environmental antigens resemble host antigens, has been proposed as a potential trigger for autoimmune responses. In this study, we employed a bioinformatics approach to investigate the role of molecular mimicry in Clostridioides difficile-caused infections and the induction of autoimmune disorders due to this phenomenon. Comparing proteomes of host and pathogen, we identified 23 proteins that exhibited significant sequence homology and were linked to autoimmune disorders. The disorders included rheumatoid arthritis, psoriasis, Alzheimer’s disease, etc., while infections included viral and bacterial infections like HIV, HCV, and tuberculosis. The structure of the homologous proteins was superposed, and RMSD was calculated to find the maximum deviation, while accounting for rigid and flexible regions. Two sequence mimics (antigenic, non-allergenic, and immunogenic) of ≥10 amino acids from these proteins were used to design a vaccine construct to explore the possibility of eliciting an immune response. Docking analysis of the top vaccine construct C2 showed favorable interactions with HLA and TLR-4 receptor, indicating potential efficacy. The B-cell and T-helper cell activity was also simulated, showing promising results for effective immunization against C. difficile infections. This study highlights the potential of C. difficile to trigger autoimmunity through molecular mimicry and vaccine design based on sequence mimics that trigger a defensive response.

Research is underway to develop a vaccine to prevent and cure infection from herpes simplex virus (HSV). It emphasizes the critical need for immunization to address public health issues and the shortcomings of existing treatment options. Furthermore, studies on the HSV vaccine advance the field of immunology and vaccine creation, which may help in the battle against other viral illnesses. The current lack of such a vaccine is, in part, due to herpes viral latency in sensory ganglions. Current vaccines rely on tissue-resident memory CD8 + T cells, which are known to provide protection against subsequent HSV reinfection and reactivation without correlating with other immune subsets. For that reason, there is no effective vaccine that can provide protection against latent or recurrent herpes infection. This review focuses on conventional methods for evaluating the efficacy of a herpes vaccine using differential CD8 + T cells and important unaccounted immune aspects for designing an effective vaccine against herpes.

Canine parvovirus (CPV-2) is one of the most important pathogens of dogs of all ages, causing pandemic infections that are characterized by fatal hemorrhagic enteritis. The CPV-2 vaccine is recommended as a core vaccine for pet animals. Despite the intensive practice of active immunization, CPV-2 remains a global threat. In this study, a multi-epitope vaccine against CPV-2 was designed, targeting the highly conserved capsid protein (VP2) via in silico approaches. Several immunoinformatics methods, such as epitope screening, molecular docking, and simulation were used to design a potential vaccine construct. The partial protein sequences of the VP2 gene of CPV-2 and protein sequences retrieved from the NCBI were screened to predict highly antigenic proteins through antigenicity, trans-membrane-topology screening, an allergenicity assessment, and a toxicity analysis. Homologous VP2 protein sequences typically linked to the disease were identified using NCBI BLAST, in which four conserved regions were preferred. Overall, 10 epitopes, DPIGGKTGI, KEFDTDLKP, GTDPDDVQ, GGTNFGYIG, GTFYFDCKP, NRALGLPP, SGTPTN, LGLPPFLNSL, IGGKTG, and VPPVYPN, were selected from the conserved regions to design the vaccine construct. The molecular docking demonstrated the higher binding affinity of these epitopes with dog leukocyte antigen (DLA) molecules. The selected epitopes were linked with Salmonella enterica flagellin FliC adjuvants, along with the PADRE sequence, by GGS linkers to construct a vaccine candidate with 272 nucleotides. The codon adaptation and in silico cloning showed that the generated vaccine can be expressed by the E. coli strain, K12, and the sequence of the vaccine construct showed no similarities with dog protein. Our results suggest that the vaccine construct might be useful in preventing canine parvoviral enteritis (CPE) in dogs. Further in vitro and in vivo experiments are needed for the validation of the vaccine candidate.

Intranasal vaccination using influenza vectors is a promising approach to developing vaccines against respiratory pathogens due to the activation of the mucosa-associated immune response. However, there is no clear evidence of a vector design that could be considered preferable. To find the optimal structure of an influenza vector with a modified NS genomic segment, we constructed four vector expressing identical transgene sequences inherited from the F protein of the respiratory syncytial virus (RSV). Two vectors were designed aiming at transgene accumulation in the cytosol. Another two were supplemented with an IgGκ signal peptide prior to the transgene for its extracellular delivery. Surprisingly, adding the IgGκ substantially enhanced the T-cell immune response to the CD8 epitope of the transgene. Moreover, this strategy allowed us to obtain a better protection of mice from the RSV challenge after a single intranasal immunization. Protection was achieved without antibodies, mediated by a balanced T-cell immune response including the formation of the RSV specific effector CD8+ IFNγ+/IL10+-producing cells and the accumulation of Treg cells preventing immunopathology in the lungs of infected mice. In addition to the presented method for optimizing the influenza vector, our results highlight the possibility of achieving protection against RSV through a respiratory-associated T-cell immune response alone.

Acute myeloid leukemia (AML) is a leading blood cancer subtype that can be caused by 27 gene mutations. Previous studies have explored potential vaccine and drug treatments against AML, but many were proven immunologically insignificant. Here, we targeted this issue and applied various clinical filters to improve immune response. KIT is an oncogenic gene that can cause AML when mutated and is predicted to be a promising vaccine target because of its immunogenic responses when activated. We designed a multi-epitope vaccine targeting mutations in the KIT oncogene using CD8+ and CD4+ epitopes. We selected the most viable vaccine epitopes based on thresholds for percentile rank, immunogenicity, antigenicity, half-life, toxicity, IFNγ release, allergenicity, and stability. The efficacy of data was observed through world and regional population coverage of our vaccine design. Then, we obtained epitopes for optimized population coverage from PCOptim-CD, a modified version of our original Java-based program code PCOptim. Using 24 mutations on the KIT gene, 12 CD8+ epitopes and 21 CD4+ epitopes were obtained. The CD8+ dataset had a 98.55% world population coverage, while the CD4+ dataset had a 65.14% world population coverage. There were five CD4+ epitopes that overlapped with the top CD8+ epitopes. Strong binding to murine MHC molecules was found in four CD8+ and six CD4+ epitopes, demonstrating the feasibility of our results in preclinical murine vaccine trials. We then created three-dimensional (3D) models to visualize epitope–MHC complexes and TCR interactions. The final candidate is a non-toxic and non-allergenic multi-epitope vaccine against KIT mutations that cause AML. Further research would involve murine trials of the vaccine candidates on tumor cells causing AML.

Currently available COVID vaccines are effective in reducing mortality and severity but do not prevent transmission of the virus or reinfection by the emerging SARS-CoV-2 variants. There is an obvious need for better and longer-lasting effective vaccines for various prevailing strains and the evolving SARS-CoV-2 virus, necessitating the development of a broad-spectrum vaccine that can be used to prevent infection by reducing both the transmission rate and re-infection. During the initial phases of SARS-CoV-2 infection, the nucleocapsid (N) protein is one of the most abundantly expressed proteins. Additionally, it has been identified as the most immunogenic protein of SARS-CoV-2. In this study, state-of-the-art bioinformatics techniques have been exploited to design novel multiple epitope vaccines using conserved regions of N proteins from prevalent strains of SARS-CoV-2 for the prediction of B- and T-cell epitopes. These epitopes were sorted based on their immunogenicity, antigenicity score, and toxicity. The most effective multi-epitope construct with possible immunogenic properties was created using epitope combinations. EAAAK, AAY, and GPGPG were used as linkers to connect epitopes. The developed vaccines have shown positive results in terms of overall population coverage and stimulation of the immune response. Potential expression of the chimeric protein construct was detected after it was cloned into the Pet28a/Cas9-cys vector for expression screening in Escherichia coli. The developed vaccine performed well in computer-based immune response simulation and covered a diverse allelic population worldwide. These computational findings are very encouraging for the further testing of our candidate vaccine, which could eventually aid in the control and prevention of SARS-CoV-2 infections globally.

Vaccination is the most cost-effective means in the fight against infectious diseases. Various kinds of vaccines have been developed since the outbreak of COVID-19, some of which have been approved for clinical application. Though vaccines available achieved partial success in protecting vaccinated subjects from infection or hospitalization, numerous efforts are still needed to end the global pandemic, especially in the case of emerging new variants. Safe and efficient vaccines are the key elements to stop the pandemic from attacking the world now; novel and evolving vaccine technologies are urged in the course of fighting (re)-emerging infectious diseases. Advances in biotechnology offered the progress of vaccinology in the past few years, and lots of innovative approaches have been applied to the vaccine design during the ongoing pandemic. In this review, we summarize the state-of-the-art vaccine strategies involved in controlling the transmission of SARS-CoV-2 and its variants. In addition, challenges and future directions for rational vaccine design are discussed.

Background: The main purpose of this article is to introduce a universal mathematics-aided vaccine design method against malignant melanoma based on neoantigens. The universal method can be adapted to the mutanome of each patient so that a specific candidate vaccine can be tailored for the corresponding patient. Methods: We extracted the 1134 most frequent mutations in melanoma, and we associated each of them to a vector with 10 components estimated with different bioinformatics tools, for which we found an aggregated value according to a set of weights, and then we ordered them in decreasing order of the scores. Results: We prepared a universal table of the most frequent mutations in melanoma ordered in decreasing order of viability to be used as candidate vaccines, so that the selection of a set of appropriate peptides for each particular patient can be easily and quickly implemented according to their specific mutanome and transcription profile. Conclusions: We have shown that the techniques that are commonly used for the design of personalized anti-tumor vaccines against malignant melanoma can be adapted for the design of universal rankings of neoantigens that originate personalized vaccines when the mutanome and transcription profile of specific patients is considered, with the consequent savings in time and money, shortening the design and production time.

This review provides the potential of intestinal microbiota in vaccine design and application, exploring the current insights into the interplay between the intestinal microbiota and the immune system, with a focus on its intermediary function in vaccine efficacy. It summarizes families and genera of bacteria that are part of the intestinal microbiota that may enhance or diminish vaccine efficacy and discusses the foundational principles of vaccine sequence design and the application of gut microbial characteristics in vaccine development. Future research should further investigate the use of multi-omics technologies to elucidate the interactive mechanisms between intestinal microbiota and vaccine-induced immune responses, aiming to optimize and improve vaccine design.

Crimean–Congo haemorrhagic fever (CCHF), caused by Crimean–Congo haemorrhagic fever virus (CCHFV), is a disease of worldwide importance (endemic yet not limited to Asia, Middle East, and Africa) and has triggered several outbreaks amounting to a case fatality rate of 10–40% as per the World Health Organization. Genetic diversity and phylogenetic data revealed that the Asia-1 genotype of CCHFV remained dominant in Pakistan, where 688 confirmed cases were reported between the 2012–2022 period. Currently, no approved vaccine is available to tackle the viral infection. Epitope-based vaccine design has gained significant attention in recent years due to its safety, timeliness, and cost efficiency compared to conventional vaccines. In the present study, we employed a robust immunoinformatics-based approach targeting the structural glycoproteins G1 and G2 of CCHFV (Asia-1 genotype) to design a multi-epitope vaccine construct. Five B-cells and six cytotoxic T-lymphocytes (CTL) epitopes were mapped and finalized from G1 and G2 and were fused with suitable linkers (EAAAK, GGGS, AAY, and GPGPG), a PADRE sequence (13 aa), and an adjuvant (50S ribosomal protein L7/L12) to formulate a chimeric vaccine construct. The selected CTL epitopes showed high affinity and stable binding with the binding groove of common human HLA class I molecules (HLA-A*02:01 and HLA-B*44:02) and mouse major histocompatibility complex class I molecules. The chimeric vaccine was predicted to be an antigenic, non-allergenic, and soluble molecule with a suitable physicochemical profile. Molecular docking and molecular dynamics simulation indicated a stable and energetically favourable interaction between the constructed antigen and Toll-like receptors (TLR2, TLR3, and TLR4). Our results demonstrated that innate, adaptive, and humoral immune responses could be elicited upon administration of such a potent muti-epitope vaccine construct. These results could be helpful for an experimental vaccinologist to develop an effective vaccine against the Asia-1 genotype of CCHFV.

The current study established a research mapping of the vaccine design using bibliometric indicators and network visualization. For an analysis of the result, the study retrieved a total of 5379 documents from Scopus from 1983 to 2021. The study used the VOS Viewer and the RStudio tools for data visualization. The findings revealed that there has been significant growth in literature on vaccine design in the last two decades; in the last ten years, the year with the most publications were 2020, with 477 publications, and the highest had a total of 14,145 citations. D.R. Burton was ranked as the most prolific author, with 86 publications and 18,449 total citations and was observed as the most frequently published author in the domain. The National Institute of Health (NIH) was the most productive organization in the domain, with 266 publications. The document entitled “Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae” received a total of 1398 citations, and was the most cited document in the field of vaccine design. In network visualization, an analysis of the co-occurrence of keywords showed that “vaccine” and “vaccine design” occurred the most, which was 761 and 335 times, respectively. The study also observed that there were five clusters of author collaboration with a maximum of 18 authors and a minimum of two authors. The findings of the study will aid scholarly coalitions in the domains of medicine and health, information science and bibliometric professionals to carry out further research in the area of vaccine design.

Dense bodies (DB) are complex, noninfectious particles produced during CMVinfection containing envelope and tegument proteins that may be ideal candidates as vaccines. Although DB were previously described in fibroblasts, no evidence of DB formation has been shown after propagating CMV in epithelial cells. In the present study, both fibroblast MRC-5 and epithelial ARPE-19 cells were used to study DB production during CMV infection. We demonstrate the formation of epithelial cell-derived DB, mostly located as cytoplasmic inclusions in the perinuclear area of the infected cell. DB were gradient-purified, and the nature of the viral particles was confirmed using CMV-specific immunelabeling. Epithelial cell-derived DB had higher density and more homogeneous size (200–300 nm) compared to fibroblast-derived DB (100–600 nm).In agreement with previous results characterizing DB from CMV-infected fibroblasts, the pp65 tegument protein was predominant in the epithelial cell-derived DB. Our results also suggest that epithelial cells had more CMV capsids in the cytoplasm and had spherical bodies compatible with nucleus condensation (pyknosis) in cells undergoing apoptosis that were not detected in MRC-5 infected cells at the tested time post-infection. Our results demonstrate the formation of DB in CMV-infected ARPE-19 epithelial cells that may be suitable candidate to develop a multiprotein vaccine with antigenic properties similar to that of the virions while not including the viral genome.

Molecular mimicry between viral antigens and host proteins can produce cross-reacting antibodies leading to autoimmunity. The coronavirus SARS-CoV-2 causes COVID-19, a disease curiously resulting in varied symptoms and outcomes, ranging from asymptomatic to fatal. Autoimmunity due to cross-reacting antibodies resulting from molecular mimicry between viral antigens and host proteins may provide an explanation. Thus, we computationally investigated molecular mimicry between SARS-CoV-2 Spike and known epitopes. We discovered molecular mimicry hotspots in Spike and highlight two examples with tentative high autoimmune potential and implications for understanding COVID-19 complications. We show that a TQLPP motif in Spike and thrombopoietin shares similar antibody binding properties. Antibodies cross-reacting with thrombopoietin may induce thrombocytopenia, a condition observed in COVID-19 patients. Another motif, ELDKY, is shared in multiple human proteins, such as PRKG1 involved in platelet activation and calcium regulation, and tropomyosin, which is linked to cardiac disease. Antibodies cross-reacting with PRKG1 and tropomyosin may cause known COVID-19 complications such as blood-clotting disorders and cardiac disease, respectively. Our findings illuminate COVID-19 pathogenesis and highlight the importance of considering autoimmune potential when developing therapeutic interventions to reduce adverse reactions.

Two lineages of influenza B viruses (IBV) co-circulating in human beings have been posing a significant public health burden worldwide. A substantial number of broadly neutralizing antibodies (bnAbs) have been identified targeting conserved epitopes on hemagglutinin (HA) stem domain, posing great interest for universal influenza vaccine development. Various strategies to design immunogens that selectively present these conserved epitopes are being explored. However, it has been a challenge to retain native conformation of the HA stem region, especially for soluble expression in prokaryotic systems. Here, using a structure prediction tool AlphaFold2, we rationally designed a stable stem antigen “B60-Stem-8071”, an HA stem vaccine derived from B/Brisbane/60/2006 grafted with a CR8071 epitope as a linker. The B60-Stem-8071 exhibited better solubility and more stable expression in the E. coli system compared to the naïve HA stem antigen. Immunization with B60-Stem-8071 in mice generated cross-reactive antibodies and protected mice broadly against lethal challenge with Yamagata and Victoria lineages of influenza B virus. Notably, soluble expression of B60-stem-8071 in the E. coli system showed the potential to produce the influenza B vaccine in a low-cost way. This study represents a proof of concept for the rational design of HA stem antigen based on structure prediction and analysis.

Mucormycosis is a group of infections, caused by multiple fungal species, which affect many human organs and is lethal in immunocompromised patients. During the COVID-19 pandemic, the current wave of mucormycosis is a challenge to medical professionals as its effects are multiplied because of the severity of COVID-19 infection. The variant of concern, Omicron, has been linked to fatal mucormycosis infections in the US and Asia. Consequently, current postdiagnostic treatments of mucormycosis have been rendered unsatisfactory. In this hour of need, a preinfection cure is needed that may prevent lethal infections in immunocompromised individuals. This study proposes a potential vaccine construct targeting mucor and rhizopus species responsible for mucormycosis infections, providing immunoprotection to immunocompromised patients. The vaccine construct, with an antigenicity score of 0.75 covering, on average, 92–98% of the world population, was designed using an immunoinformatics approach. Molecular interactions with major histocompatibility complex-1 (MHC-I), Toll-like receptors-2 (TLR2), and glucose-regulated protein 78 (GRP78), with scores of −896.0, −948.4, and −925.0, respectively, demonstrated its potential to bind with the human immune receptors. It elicited a strong predicted innate and adaptive immune response in the form of helper T (Th) cells, cytotoxic T (TC) cells, B cells, natural killer (NK) cells, and macrophages. The vaccine cloned in the pBR322 vector showed positive amplification, further solidifying its stability and potential. The proposed construct holds a promising approach as the first step towards an antimucormycosis vaccine and may contribute to minimizing postdiagnostic burdens and failures.

Classical swine fever (CSF) is a severe disease that has caused serious economic losses for the global pig industry and is widely prevalent worldwide. In recent decades, CSF has been effectively controlled through compulsory vaccination with a live CSF vaccine (C strain). It has been successfully eradicated in some countries or regions. However, the re-emergence of CSF in Japan and Romania, where it had been eradicated, has brought increased attention to the disease. Because the traditional C-strain vaccine cannot distinguish between vaccinated and infected animals (DIVA), this makes it difficult to fight CSF. The emergence of marker vaccines is considered to be an effective strategy for the decontamination of CSF. This paper summarizes the progress of the new CSF marker vaccine and provides a detailed overview of the vaccine design ideas and immunization effects. It also provides a methodology for the development of a new generation of vaccines for CSF and vaccine development for other significant epidemics.

Exosomes are small subtypes of extracellular vesicles (EVs) naturally released by different types of cells into their environment. Their physiological roles appear to be multiple, yet many aspects of their biological activities remain to be understood. These vesicles can transport and deliver a variety of cargoes and may serve as unconventional secretory vesicles. Thus, they play a crucial role as important vectors for intercellular communication and the maintenance of homeostasis. Exosome production and content can vary under several stresses or modifications in the cell microenvironment, influencing cellular responses and stimulating immunity. During infectious processes, exosomes are described as double-edged swords, displaying both beneficial and detrimental effects. Owing to their tractability, the analysis of EVs from multiple biofluids has become a booming tool for monitoring various pathologies, from infectious to cancerous origins. In this review, we present an overview of exosome features and discuss their particular and ambiguous functions in infectious contexts. We then focus on their properties as diagnostic or therapeutic tools. In this regard, we explore the capacity of exosomes to vectorize immunogenic viral antigens and their function in mounting adaptive immune responses. As exosomes provide interesting platforms for antigen presentation, we further review the available data on exosome engineering, which enables peptides of interest to be exposed at their surface. In the light of all these data, exosomes are emerging as promising avenues for vaccine strategies.

Coronavirus disease (COVID-19) pandemic caused by the coronavirus SARS-CoV-2 represents an enormous challenge to global public health, with thousands of infections and deaths in over 200 countries worldwide. The purpose of this study was to identify SARS-CoV-2 epitopes with potential to interact in silico with the alleles of the human leukocyte antigen class I (HLA I) and class II (HLA II) commonly found in the Colombian population to promote both CD4 and CD8 immune responses against this virus. The generation and evaluation of the peptides in terms of HLA I and HLA II binding, immune response, toxicity and allergenicity were performed by using computer-aided tools, such as NetMHCpan 4.1, NetMHCIIpan 4.0, VaxiJem, ToxinPred and AllerTop. Furthermore, the interaction between the predicted epitopes with HLA I and HLA II proteins frequently found in the Colombian population was studied through molecular docking simulations in AutoDock Vina and interaction analysis in LigPlot+. One of the promising peptides proposed in this study is the HLA I epitope YQPYRVVVL, which displayed an estimated coverage of over 82% and 96% for the Colombian and worldwide population, respectively. These findings could be useful for the design of new epitope-vaccines that include Colombia among their population target.

Capsid virus-like particles (cVLPs) are used as molecular scaffolds to increase the immunogenicity of displayed antigens. Modular platforms have been developed whereby antigens are attached to the surface of pre-assembled cVLPs. However, it remains unknown to what extent the employed cVLP backbone and conjugation system may influence the immune response elicited against the displayed antigen. Here, we performed a head-to-head comparison of antigen-specific IgG responses elicited by modular cVLP-vaccines differing by their employed cVLP backbone or conjugation system, respectively. Covalent antigen conjugation (i.e., employing the SpyTag/SpyCatcher system) resulted in significantly higher antigen-specific IgG titers compared to when using affinity-based conjugation (i.e., using biotin/streptavidin). The cVLP backbone also influenced the antigen-specific IgG response. Specifically, vaccines based on the bacteriophage AP205 cVLP elicited significantly higher antigen-specific IgG compared to corresponding vaccines using the human papillomavirus major capsid protein (HPV L1) cVLP. In addition, the AP205 cVLP platform mediated induction of antigen-specific IgG with a different subclass profile (i.e., higher IgG2a and IgG2b) compared to HPV L1 cVLP. These results demonstrate that the cVLP backbone and conjugation system can individually affect the IgG response elicited against a displayed antigen. These data will aid the understanding and process of tailoring modular cVLP vaccines to achieve improved immune responses.

Linear B-cell epitope prediction research has received a steadily growing interest ever since the first method was developed in 1981. B-cell epitope identification with the help of an accurate prediction method can lead to an overall faster and cheaper vaccine design process, a crucial necessity in the COVID-19 era. Consequently, several B-cell epitope prediction methods have been developed over the past few decades, but without significant success. In this study, we review the current performance and methodology of some of the most widely used linear B-cell epitope predictors which are available via a command-line interface, namely, BcePred, BepiPred, ABCpred, COBEpro, SVMTriP, LBtope, and LBEEP. Additionally, we attempted to remedy performance issues of the individual methods by developing a consensus classifier, which combines the separate predictions of these methods into a single output, accelerating the epitope-based vaccine design. While the method comparison was performed with some necessary caveats and individual methods might perform much better for specialized datasets, we hope that this update in performance can aid researchers towards the choice of a predictor, for the development of biomedical applications such as designed vaccines, diagnostic kits, immunotherapeutics, immunodiagnostic tests, antibody production, and disease diagnosis and therapy.

The current pandemic has highlighted the ever-increasing risk of human to human spread of zoonotic pathogens. A number of medically-relevant zoonotic pathogens are negative-strand RNA viruses (NSVs). NSVs are derived from different virus families. Examples like Ebola are known for causing severe symptoms and high mortality rates. Some, like influenza, are known for their ease of person-to-person transmission and lack of pre-existing immunity, enabling rapid spread across many countries around the globe. Containment of outbreaks of NSVs can be difficult owing to their unpredictability and the absence of effective control measures, such as vaccines and antiviral therapeutics. In addition, there remains a lack of essential knowledge of the host–pathogen response that are induced by NSVs, particularly of the immune responses that provide protection. Vaccines are the most effective method for preventing infectious diseases. In fact, in the event of a pandemic, appropriate vaccine design and speed of vaccine supply is the most critical factor in protecting the population, as vaccination is the only sustainable defense. Vaccines need to be safe, efficient, and cost-effective, which is influenced by our understanding of the host–pathogen interface. Additionally, some of the major challenges of vaccines are the establishment of a long-lasting immunity offering cross protection to emerging strains. Although many NSVs are controlled through immunisations, for some, vaccine design has failed or efficacy has proven unreliable. The key behind designing a successful vaccine is understanding the host–pathogen interaction and the host immune response towards NSVs. In this paper, we review the recent research in vaccine design against NSVs and explore the immune responses induced by these viruses. The generation of a robust and integrated approach to development capability and vaccine manufacture can collaboratively support the management of outbreaking NSV disease health risks.

Funding vaccine development research is more complicated than simply putting out an announcement of funds available. The funders must decide whether product development can be accomplished by purely applied research, or whether more fundamental knowledge is needed before product development can be started. If additional basic knowledge is needed, identifying the specific area of the knowledge gap can be a challenge. Additionally, when there appears to be a clear path of applied research sometimes obstacles are encountered that require a return to more basic work. After deciding on the work to be done, funders must attract the scientists with the broad range of needed skills to cover all the stages of development. Collaborations must be promoted and alliances with other funders and industry must be developed. Funders use multiple tools and strategies to accomplish these tasks with varying success.

Peptide-based vaccines can be safer and more cost effective than whole organism vaccines. Previous studies have shown that inorganic polystyrene nanoparticles (PSNPs) covalently conjugated to the minimal immunodominant peptide epitope from murine liver stage malaria (SYIPSAEKI) induced potent CD8+ T cell responses. Many pathogens, including malaria, have polymorphic T cell epitope regions. Amino acid changes in positions that are contact residues for the T cell receptor (TCR) often alter the specific cross-reactivity induced by the peptide antigen, and it is largely assumed that changes outside of these residues have little impact. Herein, each amino acid residue (except major histocompatibility complex (MHC) anchors) was systematically changed to an alanine. Peptide epitopes with altered amino acids outside T cell contact residues were still recognized by T cells induced by PSNPs-SYIPSAEKI (KI) vaccines, albeit at lower levels, except for the variant SYIPSAAKI (A7). PSNPs-SYIPSAAKI vaccines further elicited high responses to the index KI peptide. None of the epitopes displayed altered peptide ligand (APL) antagonism in vitro, and re-stimulating SYIPSAEKI and SYIPSAAKI together synergistically enhanced IFN-γ production by the T cells. These results show epitope variation in non-TCR recognition residues can have effects on T cell reactivity, suggesting that such natural variation may also be driven by immune pressure. Additionally, when re-modelling peptides to enhance the cross-reactivity of vaccines, both TCR recognition and non-recognition residues should be considered.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans; the second-largest and most deadly outbreak to date occurred in Saudi Arabia. The dromedary camel is considered a possible host of the virus and also to act as a reservoir, transmitting the virus to humans. Here, we studied evolutionary relationships for 31 complete genomes of betacoronaviruses, including eight newly sequenced MERS-CoV genomes isolated from dromedary camels in Saudi Arabia. Through bioinformatics tools, we also used available sequences and 3D structure of MERS-CoV spike glycoprotein to predict MERS-CoV epitopes and assess antibody binding affinity. Phylogenetic analysis showed the eight new sequences have close relationships with existing strains detected in camels and humans in Arabian Gulf countries. The 2019-nCov strain appears to have higher homology to both bat coronavirus and SARS-CoV than to MERS-CoV strains. The spike protein tree exhibited clustering of MERS-CoV sequences similar to the complete genome tree, except for one sequence from Qatar (KF961222). B cell epitope analysis determined that the MERS-CoV spike protein has 24 total discontinuous regions from which just six epitopes were selected with score values of >80%. Our results suggest that the virus circulates by way of camels crossing the borders of Arabian Gulf countries. This study contributes to finding more effective vaccines in order to provide long-term protection against MERS-CoV and identifying neutralizing antibodies.

Actinoporins (APs) are a family of pore-forming toxins (PFTs) from sea anemones. These biomolecules exhibit the ability to exist as soluble monomers within an aqueous medium or as constitutively open oligomers in biological membranes. Through their conformational plasticity, actinoporins are considered good candidate molecules to be included for the rational design of molecular tools, such as immunotoxins directed against tumor cells and stochastic biosensors based on nanopores to analyze unique DNA or protein molecules. Additionally, the ability of these proteins to bind to sphingomyelin (SM) facilitates their use for the design of molecular probes to identify SM in the cells. The immunomodulatory activity of actinoporins in liposomal formulations for vaccine development has also been evaluated. In this review, we describe the potential of actinoporins for use in the development of molecular tools that could be used for possible medical and biotechnological applications.

Antibodies and other new antibody-like formats have emerged as one of the most rapidly growing classes of biotherapeutic proteins. Understanding the structural features that drive antibody function and, consequently, their molecular recognition is critical for engineering antibodies. Here, we present the structural architecture of conventional IgG antibodies alongside other formats. We emphasize the importance of considering antibodies as conformational ensembles in solution instead of focusing on single-static structures because their functions and properties are strongly governed by their dynamic nature. Thus, in this review, we provide an overview of the unique structural and dynamic characteristics of antibodies with respect to their antigen recognition, biophysical properties, and effector functions. We highlight the numerous technical advances in antibody structure prediction and design, enabled by the vast number of experimentally determined high-quality structures recorded with cryo-EM, NMR, and X-ray crystallography. Lastly, we assess antibody and vaccine design strategies in the context of structure and dynamics.

Molecular mimicry, a phenomenon in which microbial or environmental antigens resemble host antigens, has been proposed as a potential trigger for autoimmune responses. In this study, we employed a bioinformatics approach to investigate the role of molecular mimicry in Clostridioides difficile -caused infections and the induction of autoimmune disorders due to this phenomenon. Comparing proteomes of host and pathogen, we identified 23 proteins that exhibited significant sequence homology and were linked to autoimmune disorders. The disorders included rheumatoid arthritis, psoriasis, Alzheimer's disease, etc., while infections included viral and bacterial infections like HIV, HCV, and tuberculosis. The structure of the homologous proteins was superposed, and RMSD was calculated to find the maximum deviation, while accounting for rigid and flexible regions. Two sequence mimics (antigenic, non-allergenic, and immunogenic) of ≥10 amino acids from these proteins were used to design a vaccine construct to explore the possibility of eliciting an immune response. Docking analysis of the top vaccine construct C2 showed favorable interactions with HLA and TLR-4 receptor, indicating potential efficacy. The B-cell and T-helper cell activity was also simulated, showing promising results for effective immunization against C. difficile infections. This study highlights the potential of C. difficile to trigger autoimmunity through molecular mimicry and vaccine design based on sequence mimics that trigger a defensive response.

Most human immunodeficiency virus (HIV) vaccine trials have lacked efficacy and empirical vaccine lead targets are scarce. Thus far, the only independent correlate of reduced risk of HIV-1 acquisition in humans is elevated levels of V2-specific antibodies identified in the modestly protective RV144 vaccine trial. Ten years after RV144, human and non-human primate vaccine studies have reassessed the potential contribution of V2-specific antibodies to vaccine efficacy. In addition, studies of natural HIV-1 infection in humans have provided insight into the development of V1V2-directed antibody responses and their impact on clinical parameters and disease progression. Functionally diverse anti-V2 monoclonal antibodies were isolated and their structurally distinct V2 epitope regions characterized. After RV144, a plethora of research studies were performed using different model systems, immunogens, protocols, and challenge viruses. These diverse studies failed to provide a clear picture regarding the contribution of V2 antibodies to vaccine efficacy. Here, we summarize the biological functions and clinical findings associated with V2-specific antibodies and discuss their impact on HIV vaccine research.

Nanobodies or VHH (variable domains of heavy-chain only antibodies) are derived from camelid species such as llamas and camels. Nanobodies isolated and selected through phage display can neutralize a broad range of human immunodeficiency virus type 1 (HIV-1) strains. Nanobodies fit into canyons on the HIV envelope that may not be accessible to IgG (immunoglobulin G) containing both heavy and light chains, and they tend to have long CDR3 (complementarity-determining region 3) loops that further enhance recognition of otherwise cryptic epitopes. Nanobodies are readily expressed at high levels in bacteria and yeast, as well as by viral vectors, and they form relatively stable, heat-resistant molecules. Nanobodies can be linked to human Fc chains to gain immune effector functions. Bivalent and trivalent nanobodies recognizing the same or distinct epitopes on the envelope glycoproteins, gp120 and gp41, greatly increase the potency of HIV-1 neutralization. Nanobodies have potential applications for HIV-1 diagnostics, vaccine design, microbicides, immunoprophylaxis, and immunotherapy.

Canine parvovirus (CPV-2) is one of the most important pathogens of dogs of all ages, causing pandemic infections that are characterized by fatal hemorrhagic enteritis. The CPV-2 vaccine is recommended as a core vaccine for pet animals. Despite the intensive practice of active immunization, CPV-2 remains a global threat. In this study, a multi-epitope vaccine against CPV-2 was designed, targeting the highly conserved capsid protein (VP2) via in silico approaches. Several immunoinformatics methods, such as epitope screening, molecular docking, and simulation were used to design a potential vaccine construct. The partial protein sequences of the VP2 gene of CPV-2 and protein sequences retrieved from the NCBI were screened to predict highly antigenic proteins through antigenicity, trans-membrane-topology screening, an allergenicity assessment, and a toxicity analysis. Homologous VP2 protein sequences typically linked to the disease were identified using NCBI BLAST, in which four conserved regions were preferred. Overall, 10 epitopes, DPIGGKTGI, KEFDTDLKP, GTDPDDVQ, GGTNFGYIG, GTFYFDCKP, NRALGLPP, SGTPTN, LGLPPFLNSL, IGGKTG, and VPPVYPN, were selected from the conserved regions to design the vaccine construct. The molecular docking demonstrated the higher binding affinity of these epitopes with dog leukocyte antigen (DLA) molecules. The selected epitopes were linked with Salmonella enterica flagellin FliC adjuvants, along with the PADRE sequence, by GGS linkers to construct a vaccine candidate with 272 nucleotides. The codon adaptation and in silico cloning showed that the generated vaccine can be expressed by the E. coli strain, K12, and the sequence of the vaccine construct showed no similarities with dog protein. Our results suggest that the vaccine construct might be useful in preventing canine parvoviral enteritis (CPE) in dogs. Further in vitro and in vivo experiments are needed for the validation of the vaccine candidate.

Currently available COVID vaccines are effective in reducing mortality and severity but do not prevent transmission of the virus or reinfection by the emerging SARS-CoV-2 variants. There is an obvious need for better and longer-lasting effective vaccines for various prevailing strains and the evolving SARS-CoV-2 virus, necessitating the development of a broad-spectrum vaccine that can be used to prevent infection by reducing both the transmission rate and re-infection. During the initial phases of SARS-CoV-2 infection, the nucleocapsid (N) protein is one of the most abundantly expressed proteins. Additionally, it has been identified as the most immunogenic protein of SARS-CoV-2. In this study, state-of-the-art bioinformatics techniques have been exploited to design novel multiple epitope vaccines using conserved regions of N proteins from prevalent strains of SARS-CoV-2 for the prediction of B- and T-cell epitopes. These epitopes were sorted based on their immunogenicity, antigenicity score, and toxicity. The most effective multi-epitope construct with possible immunogenic properties was created using epitope combinations. EAAAK, AAY, and GPGPG were used as linkers to connect epitopes. The developed vaccines have shown positive results in terms of overall population coverage and stimulation of the immune response. Potential expression of the chimeric protein construct was detected after it was cloned into the Pet28a/Cas9-cys vector for expression screening in Escherichia coli. The developed vaccine performed well in computer-based immune response simulation and covered a diverse allelic population worldwide. These computational findings are very encouraging for the further testing of our candidate vaccine, which could eventually aid in the control and prevention of SARS-CoV-2 infections globally.

Antibody therapies with high efficiency and low toxicity are becoming one of the major approaches in antibody therapeutics. Based on high-throughput sequencing and increasing experimental structures of antibodies/antibody-antigen complexes, computational approaches can predict antibody/antigen structures, engineering the function of antibodies and design antibody-antigen complexes with improved properties. This review summarizes recent progress in the field of in silico design of antibodies, including antibody structure modeling, antibody-antigen complex prediction, antibody stability evaluation, and allosteric effects in antibodies and functions. We listed the cases in which these methods have helped experimental studies to improve the affinities and physicochemical properties of antibodies. We emphasized how the molecular dynamics unveiled the allosteric effects during antibody-antigen recognition and antibody-effector recognition.

Vaccination is the most cost-effective means in the fight against infectious diseases. Various kinds of vaccines have been developed since the outbreak of COVID-19, some of which have been approved for clinical application. Though vaccines available achieved partial success in protecting vaccinated subjects from infection or hospitalization, numerous efforts are still needed to end the global pandemic, especially in the case of emerging new variants. Safe and efficient vaccines are the key elements to stop the pandemic from attacking the world now; novel and evolving vaccine technologies are urged in the course of fighting (re)-emerging infectious diseases. Advances in biotechnology offered the progress of vaccinology in the past few years, and lots of innovative approaches have been applied to the vaccine design during the ongoing pandemic. In this review, we summarize the state-of-the-art vaccine strategies involved in controlling the transmission of SARS-CoV-2 and its variants. In addition, challenges and future directions for rational vaccine design are discussed.

Background : The main purpose of this article is to introduce a universal mathematics-aided vaccine design method against malignant melanoma based on neoantigens. The universal method can be adapted to the mutanome of each patient so that a specific candidate vaccine can be tailored for the corresponding patient. Methods : We extracted the 1134 most frequent mutations in melanoma, and we associated each of them to a vector with 10 components estimated with different bioinformatics tools, for which we found an aggregated value according to a set of weights, and then we ordered them in decreasing order of the scores. Results : We prepared a universal table of the most frequent mutations in melanoma ordered in decreasing order of viability to be used as candidate vaccines, so that the selection of a set of appropriate peptides for each particular patient can be easily and quickly implemented according to their specific mutanome and transcription profile. Conclusions : We have shown that the techniques that are commonly used for the design of personalized anti-tumor vaccines against malignant melanoma can be adapted for the design of universal rankings of neoantigens that originate personalized vaccines when the mutanome and transcription profile of specific patients is considered, with the consequent savings in time and money, shortening the design and production time.

Crimean-Congo haemorrhagic fever (CCHF), caused by Crimean-Congo haemorrhagic fever virus (CCHFV), is a disease of worldwide importance (endemic yet not limited to Asia, Middle East, and Africa) and has triggered several outbreaks amounting to a case fatality rate of 10-40% as per the World Health Organization. Genetic diversity and phylogenetic data revealed that the Asia-1 genotype of CCHFV remained dominant in Pakistan, where 688 confirmed cases were reported between the 2012-2022 period. Currently, no approved vaccine is available to tackle the viral infection. Epitope-based vaccine design has gained significant attention in recent years due to its safety, timeliness, and cost efficiency compared to conventional vaccines. In the present study, we employed a robust immunoinformatics-based approach targeting the structural glycoproteins G1 and G2 of CCHFV (Asia-1 genotype) to design a multi-epitope vaccine construct. Five B-cells and six cytotoxic T-lymphocytes (CTL) epitopes were mapped and finalized from G1 and G2 and were fused with suitable linkers (EAAAK, GGGS, AAY, and GPGPG), a PADRE sequence (13 aa), and an adjuvant (50S ribosomal protein L7/L12) to formulate a chimeric vaccine construct. The selected CTL epitopes showed high affinity and stable binding with the binding groove of common human HLA class I molecules (HLA-A*02:01 and HLA-B*44:02) and mouse major histocompatibility complex class I molecules. The chimeric vaccine was predicted to be an antigenic, non-allergenic, and soluble molecule with a suitable physicochemical profile. Molecular docking and molecular dynamics simulation indicated a stable and energetically favourable interaction between the constructed antigen and Toll-like receptors (TLR2, TLR3, and TLR4). Our results demonstrated that innate, adaptive, and humoral immune responses could be elicited upon administration of such a potent muti-epitope vaccine construct. These results could be helpful for an experimental vaccinologist to develop an effective vaccine against the Asia-1 genotype of CCHFV.

The current study established a research mapping of the vaccine design using bibliometric indicators and network visualization. For an analysis of the result, the study retrieved a total of 5379 documents from Scopus from 1983 to 2021. The study used the VOS Viewer and the RStudio tools for data visualization. The findings revealed that there has been significant growth in literature on vaccine design in the last two decades; in the last ten years, the year with the most publications were 2020, with 477 publications, and the highest had a total of 14,145 citations. D.R. Burton was ranked as the most prolific author, with 86 publications and 18,449 total citations and was observed as the most frequently published author in the domain. The National Institute of Health (NIH) was the most productive organization in the domain, with 266 publications. The document entitled "Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae" received a total of 1398 citations, and was the most cited document in the field of vaccine design. In network visualization, an analysis of the co-occurrence of keywords showed that "vaccine" and "vaccine design" occurred the most, which was 761 and 335 times, respectively. The study also observed that there were five clusters of author collaboration with a maximum of 18 authors and a minimum of two authors. The findings of the study will aid scholarly coalitions in the domains of medicine and health, information science and bibliometric professionals to carry out further research in the area of vaccine design.

Dense bodies (DB) are complex, noninfectious particles produced during CMVinfection containing envelope and tegument proteins that may be ideal candidates as vaccines. Although DB were previously described in fibroblasts, no evidence of DB formation has been shown after propagating CMV in epithelial cells. In the present study, both fibroblast MRC-5 and epithelial ARPE-19 cells were used to study DB production during CMV infection. We demonstrate the formation of epithelial cell-derived DB, mostly located as cytoplasmic inclusions in the perinuclear area of the infected cell. DB were gradient-purified, and the nature of the viral particles was confirmed using CMV-specific immunelabeling. Epithelial cell-derived DB had higher density and more homogeneous size (200-300 nm) compared to fibroblast-derived DB (100-600 nm).In agreement with previous results characterizing DB from CMV-infected fibroblasts, the pp65 tegument protein was predominant in the epithelial cell-derived DB. Our results also suggest that epithelial cells had more CMV capsids in the cytoplasm and had spherical bodies compatible with nucleus condensation (pyknosis) in cells undergoing apoptosis that were not detected in MRC-5 infected cells at the tested time post-infection. Our results demonstrate the formation of DB in CMV-infected ARPE-19 epithelial cells that may be suitable candidate to develop a multiprotein vaccine with antigenic properties similar to that of the virions while not including the viral genome.

Mucormycosis is a group of infections, caused by multiple fungal species, which affect many human organs and is lethal in immunocompromised patients. During the COVID-19 pandemic, the current wave of mucormycosis is a challenge to medical professionals as its effects are multiplied because of the severity of COVID-19 infection. The variant of concern, Omicron, has been linked to fatal mucormycosis infections in the US and Asia. Consequently, current postdiagnostic treatments of mucormycosis have been rendered unsatisfactory. In this hour of need, a preinfection cure is needed that may prevent lethal infections in immunocompromised individuals. This study proposes a potential vaccine construct targeting mucor and rhizopus species responsible for mucormycosis infections, providing immunoprotection to immunocompromised patients. The vaccine construct, with an antigenicity score of 0.75 covering, on average, 92-98% of the world population, was designed using an immunoinformatics approach. Molecular interactions with major histocompatibility complex-1 (MHC-I), Toll-like receptors-2 (TLR2), and glucose-regulated protein 78 (GRP78), with scores of -896.0, -948.4, and -925.0, respectively, demonstrated its potential to bind with the human immune receptors. It elicited a strong predicted innate and adaptive immune response in the form of helper T (Th) cells, cytotoxic T (TC) cells, B cells, natural killer (NK) cells, and macrophages. The vaccine cloned in the pBR322 vector showed positive amplification, further solidifying its stability and potential. The proposed construct holds a promising approach as the first step towards an antimucormycosis vaccine and may contribute to minimizing postdiagnostic burdens and failures.

Classical swine fever (CSF) is a severe disease that has caused serious economic losses for the global pig industry and is widely prevalent worldwide. In recent decades, CSF has been effectively controlled through compulsory vaccination with a live CSF vaccine (C strain). It has been successfully eradicated in some countries or regions. However, the re-emergence of CSF in Japan and Romania, where it had been eradicated, has brought increased attention to the disease. Because the traditional C-strain vaccine cannot distinguish between vaccinated and infected animals (DIVA), this makes it difficult to fight CSF. The emergence of marker vaccines is considered to be an effective strategy for the decontamination of CSF. This paper summarizes the progress of the new CSF marker vaccine and provides a detailed overview of the vaccine design ideas and immunization effects. It also provides a methodology for the development of a new generation of vaccines for CSF and vaccine development for other significant epidemics.

CD8 + T-cell responses to pathogens are directed against infected cells that present pathogen-encoded peptides on MHC class-I molecules. Although natural responses are polyclonal, the spectrum of peptides that qualify for epitopes is remarkably small even for pathogens with high coding capacity. Among those few that are successful at all, a hierarchy exists in the magnitude of the response that they elicit in terms of numbers of CD8 + T cells generated. This led to a classification into immunodominant and non-immunodominant or subordinate epitopes, IDEs and non-IDEs, respectively. IDEs are favored in the design of vaccines and are chosen for CD8 + T-cell immunotherapy. Using murine cytomegalovirus as a model, we provide evidence to conclude that epitope hierarchy reflects competition on the level of antigen recognition. Notably, high-avidity cells specific for non-IDEs were found to expand only when IDEs were deleted. This may be a host's back-up strategy to avoid viral immune escape through antigenic drift caused by IDE mutations. Importantly, our results are relevant for the design of vaccines based on cytomegaloviruses as vectors to generate high-avidity CD8 + T-cell memory specific for unrelated pathogens or tumors. We propose the deletion of vector-encoded IDEs to avoid the suppression of epitopes of the vaccine target.

Coronavirus disease (COVID-19) pandemic caused by the coronavirus SARS-CoV-2 represents an enormous challenge to global public health, with thousands of infections and deaths in over 200 countries worldwide. The purpose of this study was to identify SARS-CoV-2 epitopes with potential to interact in silico with the alleles of the human leukocyte antigen class I (HLA I) and class II (HLA II) commonly found in the Colombian population to promote both CD4 and CD8 immune responses against this virus. The generation and evaluation of the peptides in terms of HLA I and HLA II binding, immune response, toxicity and allergenicity were performed by using computer-aided tools, such as NetMHCpan 4.1, NetMHCIIpan 4.0, VaxiJem, ToxinPred and AllerTop. Furthermore, the interaction between the predicted epitopes with HLA I and HLA II proteins frequently found in the Colombian population was studied through molecular docking simulations in AutoDock Vina and interaction analysis in LigPlot+. One of the promising peptides proposed in this study is the HLA I epitope YQPYRVVVL, which displayed an estimated coverage of over 82% and 96% for the Colombian and worldwide population, respectively. These findings could be useful for the design of new epitope-vaccines that include Colombia among their population target.

Glycoproteins are processed endosomally prior to presentation to T cells and subsequent induction of specific antibodies. The sugar part of glycoconjugate may be degraded while the type of the process depends on the features of the particular structure. The generated carbohydrate epitopes may differ from native structures and influence immunogenicity of the antigens. We have devised a model of endosomal-like pre-processing of Bordetella pertussis 186 oligosaccharides (OSs) to verify how it affects the immunogenicity of their conjugates. The glycoconjugates of structurally defined forms of the dodecasaccharide OS were synthesized and their immunogenicity was assessed using immunochemical methods. The structural features of the oligosaccharides and their sensitivity to deamination were analyzed by NMR spectroscopy. The distal trisaccharide-comprising pentasaccharide conjugated to a protein was the most effective in inducing immune response against the B. pertussis 186 LOS and the immune response to the complete OS conjugates was significantly lower. This could be explained by the loss of the distal trisaccharide during the in-cell deamination process suggesting that the native structure is not optimal for a vaccine antigen. Consequently, our research has shown that designing of new glycoconjugate vaccines requires the antigen structures to be verified in context of possible endosomal reactions beforehand.