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Untreated or reoccurring group A Streptococcus (GAS) infection can lead to a number of post-infection complications, including rheumatic heart disease. There is no licenced vaccine for the treatment or prevention of GAS infection. We identified that a cyclic decapeptide plays a significant positive influence on the adjuvant activity of several lipid-antigen mixtures. Here, three synthetic vaccine components were synthesised: (1) J8-PADRE represents the GAS B cell antigen (J8) conjugated to the universal T helper epitope (PADRE); (2) a synthetic toll like receptor 2 (TLR2) ligand based on a C16 alkyl chain lipid moiety; and (3) a cyclic carrier deca-peptide. Previously, through structure-immune activity investigations, it was observed that a physical mixture of these three components had significantly higher IgG immune responses when compared to a fully conjugated vaccine construct. Expanding the scope of this structure-activity investigation, we show that the presence of the cyclic peptide is required for the induction of a strong, balanced Th1/Th2 immune response when compared with lipid and antigen only, and cyclic lipopeptide plus B/T cell antigen physical mixtures.

Globo H is a tumor-associated carbohydrate antigen (TACA), which serves as a valuable target for antitumor vaccine or cancer immunotherapies. However, most TACAs are T-cell-independent, and they cannot induce powerful immune response due to their poor immunogenicity. To address this problem, herein, several Globo H analogues with modification on the N-acyl group were prepared through a preactivation-based glycosylation strategy from the non-reducing end to the reducing end. These modified Globo H derivatives were then conjugated with carrier protein CRM197 to form glycoconjugates as anticancer vaccine candidates, which were used in combination with adjuvant glycolipid C34 for immunological studies. The immunological effects of these synthetic vaccine candidates were evaluated on Balb/c mice. The results showed that the fluorine-modified N-acyl Globo H conjugates can induce higher titers of IgG antibodies that can recognize the naturally occurring Globo H antigen on the surface of cancer cells and can eliminate cancer cells in the presence of a complement, indicating the potential of these synthetic glycoconjugates as anticancer vaccine candidates.

We previously identified Alcaligenes spp. as a commensal bacterium that resides in lymphoid tissues, including Peyer’s patches. We found that Alcaligenes-derived lipopolysaccharide acted as a weak agonist of Toll-like receptor four due to the unique structure of lipid A, which lies in the core of lipopolysaccharide. This feature allowed the use of chemically synthesized Alcaligenes lipid A as a safe synthetic vaccine adjuvant that induces Th17 polarization to enhance systemic IgG and respiratory IgA responses to T-cell–dependent antigens (e.g., ovalbumin and pneumococcal surface protein A) without excessive inflammation. Here, we examined the adjuvant activity of Alcaligenes lipid A on a Haemophilus influenzae B conjugate vaccine that contains capsular polysaccharide polyribosyl ribitol phosphate (PRP), a T-cell–independent antigen, conjugated with the T-cell–dependent tetanus toxoid (TT) antigen (i.e., PRP-TT). When mice were subcutaneously immunized with PRP alone or mixed with TT, Alcaligenes lipid A did not affect PRP-specific IgG production. In contrast, PRP-specific serum IgG responses were enhanced when mice were immunized with PRP-TT, but these responses were impaired in similarly immunized T-cell—deficient nude mice. Furthermore, TT-specific—but not PRP-specific—T-cell activation occurred in mice immunized with PRP-TT together with Alcaligenes lipid A. In addition, coculture with Alcaligenes lipid A promoted significant proliferation of and enhanced antibody production by B cells. Together, these findings suggest that Alcaligenes lipid A exerts an adjuvant activity on thymus-independent Hib polysaccharide antigen in the presence of a T-cell–dependent conjugate carrier antigen.

Vaccines are one of the most effective strategies to fight infectious diseases. Reverse vaccinology strategies provide tools to perform in silico screening and a rational selection of potential candidates on a large scale before reaching in vitro and in vivo evaluations. Leishmania infection in humans produces clinical symptoms in some individuals, while another part of the population is naturally resistant (asymptomatic course) to the disease, and therefore their immune response controls parasite replication. By the identification of epitopes directly in humans, especially in those resistant to the disease, the probabilities of designing an effective vaccine are higher. The aim of this work was the identification of Leishmania epitopes in resistant humans. To achieve that, 11 peptide sequences (from Leishmania antigenic proteins) were selected using epitope prediction tools, and then, peripheral blood mononuclear cells (PBMCs) were isolated from human volunteers who were previously divided into four clinical groups: susceptible, resistant, exposed and not exposed to the parasite. The induction of inflammatory cytokines and lymphoproliferation was assessed using monocyte-derived dendritic cells (moDCs) as antigen-presenting cells (APCs). The response was evaluated after exposing volunteers’ cells to each peptide. As a result, we learned that STI41 and STI46 peptides induced IL-8 and IL-12 in moDCs and lymphoproliferation and low levels of IL-10 in lymphocytes differentially in resistant volunteers, similar behavior to that observed in those individuals to L. panamensis lysate antigens. We conclude that, in silico analysis allowed for the identification of natural Leishmania epitopes in humans, and also STI41 and STI46 peptides could be epitopes that lead to a cellular immune response directed at parasite control.

The aim of this study was to construct a vaccine peptide candidate against pandemic Influenza H1N1 hemagglutinin and to test its structure. With the help of bioinformatic algorithms we showed that the sequence encoding the second polypeptide of pandemic Influenza H1N1 hemagglutinin (HA2) is protected from nonsynonymous mutations better than the sequence encoding its first polypeptide (HA1). With the help of secondary and ternary structure predicting algorithms we found the fragment of HA2 with the most reproducible secondary structure and synthesized the NY25 peptide corresponding to the residues Asn117 - Tyr141 of HA2. According to the circular dichroism spectra analysis, the peptide has short helix and beta hairpin. According to the analysis of differential fluorescence quenching results, two tyrosine residues are situated on a long distance from each other. These facts taken together with the positive results of affine chromatography with the serum of a person immunized by full-length hemagglutinin confirm that the structure of the fragment of viral full-length protein has been reproduced in the synthetic NY25 peptide. Amino acid sequence of the NY25 peptide (NLYEKVRSQLKNNAKEIGNGCFEFY) is relatively conserved in 18 subtypes of Influenza A virus hemagglutinin.

A randomized, double-blind, controlled clinical trial was conducted to assess the safety and protective efficacy of the Plasmodium vivax circumsporozoite (CS) protein. A total of 35 healthy adults, either malaria-naïve (n=17) or P.vivax semi-immune (n=18), were enrolled and immunized intramuscularly (i.m.) at months 0, 2, and 6, with the PvCS (150 μg) formulated in Montanide ISA-51 adjuvant. Most volunteers developed PvCS specific antibody and T-cell responses and were subjected to a P. vivax sporozoite controlled human malaria infection (CHMI) 30 days after the last immunization. Sterile protection was observed in five of 11 naïve (42%) and four of 11 semi-immune (36%) volunteers by showing no parasitemia during the 60 days follow-up, as did a semi-immune control (1/5) volunteer. All non-protected volunteers developed malaria symptoms 10-19 days after CHMI and were immediately treated with antimalarial drugs. This is the first study of a P. vivax sub-unit vaccine demonstrating sterile protection against P. vivax infection.

Hepatitis C virus remains a global threat, despite the availability of highly effective direct-acting antiviral (DAA) drugs. With thousands of new infections annually, the need for a prophylactic vaccine is evident. However, traditional vaccine design has been unable to provide effective vaccines so far. Therefore, alternative strategies need to be investigated. In this work, a chemistry-based approach is explored towards fully synthetic peptide-based vaccines using epitope mimicry, by focusing on highly effective and conserved amino acid sequences in HCV, which, upon antibody binding, inhibit its bio-activity. Continuous and discontinuous epitope mimics were both chemically synthesized based on the HCV-E2 glycoprotein while using designed fully synthetic cyclic peptides. These cyclic epitope mimics were assembled on an orthogonally protected scaffold. The scaffolded epitope mimics have been assessed in immunization experiments to investigate the elicitation of anti-HCV-E2 glycoprotein antibodies. The neutralizing potential of the elicited antibodies was investigated, representing a first step in employing chemically synthesized epitope mimics as a novel strategy towards vaccine design.

Apical membrane antigen 1 is a microneme protein which plays an indispensable role during Apicomplexa parasite invasion. The detailed mechanism of AMA-1 molecular interaction with its receptor on bovine erythrocytes has not been completely defined in Babesia bovis. This study was focused on identifying the minimum B. bovis AMA-1-derived regions governing specific and high-affinity binding to its target cells. Different approaches were used for detecting ama-1 locus genetic variability and natural selection signatures. The binding properties of twelve highly conserved 20-residue-long peptides were evaluated using a sensitive and specific binding assay based on radio-iodination. B. bovis AMA-1 ectodomain structure was modelled and refined using molecular modelling software. NetMHCIIpan software was used for calculating B- and T-cell epitopes. The B. bovis ama-1 gene had regions under functional constraint, having the highest negative selective pressure intensity in the Domain I encoding region. Interestingly, B. bovis AMA-1-DI (100YMQKFDIPRNHGSGIYVDLG119 and 120GYESVGSKSYRMPVGKCPVV139) and DII (302CPMHPVRDAIFGKWSGGSCV321)-derived peptides had high specificity interaction with erythrocytes and bound to a chymotrypsin and neuraminidase-treatment sensitive receptor. DI-derived peptides appear to be exposed on the protein&rsquo, s surface and contain predicted B- and T-cell epitopes. These findings provide data (for the first-time) concerning B. bovis AMA-1 functional subunits which are important for establishing receptor-ligand interactions which could be used in synthetic vaccine development.

Self-amplifying mRNAs derived from the genomes of positive-strand RNA viruses have recently come into focus as a promising technology platform for vaccine development. Non-virally delivered self-amplifying mRNA vaccines have the potential to be highly versatile, potent, streamlined, scalable, and inexpensive. By amplifying their genome and the antigen encoding mRNA in the host cell, the self-amplifying mRNA mimics a viral infection, resulting in sustained levels of the target protein combined with self-adjuvanting innate immune responses, ultimately leading to potent and long-lasting antigen-specific humoral and cellular immune responses. Moreover, in principle, any eukaryotic sequence could be encoded by self-amplifying mRNA without the need to change the manufacturing process, thereby enabling a much faster and flexible research and development timeline than the current vaccines and hence a quicker response to emerging infectious diseases. This chapter highlights the rapid progress made in using non-virally delivered self-amplifying mRNA-based vaccines against infectious diseases in animal models. We provide an overview of the unique attributes of this vaccine approach, summarize the growing body of work defining its mechanism of action, discuss the current challenges and latest advances, and highlight perspectives about the future of this promising technology.

COVID-19, a global pandemic causing to date more than 50 million cases and more than a million deaths, has to be controlled. SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) was identified as the causative agent. Controversy about this virus origin and infectious mechanism for adapting to humans remains a matter for discussion. Among all strategies for obtaining safe and potent vaccines, approaches based on attenuated-killed virus and non-replicating RNA viral vectors are demonstrating promising results. However, specificity of viral components targeted by human antibodies so far has not been demonstrated. A consistent strategy for obtaining functional-active antigens from SARS-CoV-2 specific ligands lead us to propose and test a number of synthetic components. From hundreds of starting sequences only fifteen fulfilled the design requirements and were produced as monomer and polymer forms and immuno-chemically tested. The design was based on worldwide representative reported virus genomes. A bioinformatics scheme by conventional methods and knowledge on MHC-I and II antigen processing mechanisms and HLA haplotype-restriction was performed including sensitive and resistant human populations to virus infection. Covid-19 patients’ sera reactivity for synthetic SARS-CoV-2-designed components have proven a high recognition of specific molecules, as well as some evidence for a long-lasting humoral immune response.

Highlights • Genome-wide analysis of 566 Indian SARS-CoV-2 genomes. • Finding potential 23 consensus conserved regions. • 34, 37 and 29 best immunogenic and antigenic MHC-I, MHC-II T-cell and B-cell epitopes. • Physico-chemical properties of the epitopes are studied. • Docking with Ramachandran Plot and Z-score are performed., SARS-CoV-2 has a high transmission rate and shows frequent mutations, thus making vaccine development an arduous task. However, researchers around the globe are working hard to find a solution e.g. synthetic vaccine. Here, we have performed genome-wide analysis of 566 Indian SARS-CoV-2 genomes to extract the potential conserved regions for identifying peptide based synthetic vaccines, viz. epitopes with high immunogenicity and antigenicity. In this regard, different multiple sequence alignment techniques are used to align the SARS-CoV-2 genomes separately. Subsequently, consensus conserved regions are identified after finding the conserved regions from each aligned result of alignment techniques. Further, the consensus conserved regions are refined considering that their lengths are greater than or equal to 60nt and their corresponding proteins are devoid of any stop codons. Subsequently, their specificity as query coverage are verified using Nucleotide BLAST. Finally, with these consensus conserved regions, T-cell and B-cell epitopes are identified based on their immunogenic and antigenic scores which are then used to rank the conserved regions. As a result, we have ranked 23 consensus conserved regions that are associated with different proteins. This ranking also resulted in 34 MHC-I and 37 MHC-II restricted T-cell epitopes with 16 and 19 unique HLA alleles and 29 B-cell epitopes. After ranking, the consensus conserved region from NSP3 gene is obtained that is highly immunogenic and antigenic. In order to judge the relevance of the identified epitopes, the physico-chemical properties and binding conformation of the MHC-I and MHC-II restricted T-cell epitopes are shown with respect to HLA alleles.

Aims: The aim of this study was to create a new version of the PentaFOLD algorithm and to test its performance experimentally in several proteins and peptides. Background: Synthetic vaccines can cause production of neutralizing antibodies only in case if short peptides form the same secondary structure as fragments of full-length proteins. The PentaFOLD 3.0 algorithm was designed to check stability of alpha helices, beta strands, and random coils using several propensity scales obtained during analysis of 1730 3D structures of proteins. Objective: The algorithm has been tested in the three peptides known to keep the secondary structure of the corresponding fragments of full-length proteins: the NY25 peptide from the Influenza H1N1 hemagglutinin, the SF23 peptide from the diphtheria toxin, the NQ21 peptide from the HIV1 gp120; as well as in the CC36 peptide from the human major prion protein. Method: Affine chromatography for antibodies against peptides accompanied by circular dichroism and fluorescence spectroscopy were used to check the predictions of the algorithm. Result: Immunological experiments showed that all abovementioned peptides are more or less immunogenic in rabbits. The fact that antibodies against the NY25, the SF23, and the NQ21 form stable complexes with corresponding full-length proteins has been confirmed by affine chromatography. The surface of SARS CoV-2 spike receptor-binding domain interacting with hACE2 has been shown to be unstable according to the results of the PentaFOLD 3.0. Conclusion: The PentaFOLD 3.0 algorithm (http://chemres.bsmu.by/PentaFOLD30.htm) can be used with the aim to design vaccine peptides with stable secondary structure elements.

Liver resident-memory CD8+ T cells (TRM cells) can kill liver-stage Plasmodium-infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver TRM cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I-restricted peptide epitope (to stimulate Plasmodium-specific CD8+ T cells) and an adjuvant component, the NKT cell agonist α-galactosylceramide (α-GalCer). A single dose of this vaccine in mice induced substantial numbers of intrahepatic malaria-specific CD8+ T cells expressing canonical markers of liver TRM cells (CD69, CXCR6, and CD101), and these cells could be further increased in number upon vaccine boosting. We show that modifications to the peptide, such as addition of proteasomal-cleavage sequences or epitope-flanking sequences, or the use of alternative conjugation methods to link the peptide to the glycolipid improved liver TRM cell generation and led to the development of a vaccine able to induce sterile protection in C57BL/6 mice against Plasmodium berghei sporozoite challenge after a single dose. Furthermore, this vaccine induced endogenous liver TRM cells that were long-lived (half-life of ~425 days) and were able to maintain >90% sterile protection to day 200. Our findings describe an ideal synthetic vaccine platform for generating large numbers of liver TRM cells for effective control of liver-stage malaria and, potentially, a variety of other hepatotropic infections.

This paper continues a recent study of the spike protein sequence of the COVID-19 virus (SARS-CoV-2). It is also in part an introductory review to relevant computational techniques for tackling viral threats, using COVID-19 as an example. Q-UEL tools for facilitating access to knowledge and bioinformatics tools were again used for efficiency, but the focus in this paper is even more on the virus. Subsequence KRSFIEDLLFNKV of the S2' spike glycoprotein proteolytic cleavage site continues to appear important. Here it is shown to be recognizable in the common cold coronaviruses, avian coronaviruses and possibly as traces in the nidoviruses of reptiles and fish. Its function or functions thus seem important to the coronaviruses. It might represent SARS-CoV-2 Achilles' heel, less likely to acquire resistance by mutation, as has happened in some early SARS vaccine studies discussed in the previous paper. Preliminary conformational analysis of the receptor (ACE2) binding site of the spike protein is carried out suggesting that while it is somewhat conserved, it appears to be more variable than KRSFIEDLLFNKV. However compounds like emodin that inhibit SARS entry, apparently by binding ACE2, might also have functions at several different human protein binding sites. The enzyme 11β-hydroxysteroid dehydrogenase type 1 is again argued to be a convenient model pharmacophore perhaps representing an ensemble of targets, and it is noted that it occurs both in lung and alimentary tract. Perhaps it benefits the virus to block an inflammatory response by inhibiting the dehydrogenase, but a fairly complex web involves several possible targets.

This paper concerns study of the genome of the Wuhan Seafood Market isolate believed to represent the causative agent of the disease COVID-19. This is to find a short section or sections of viral protein sequence suitable for preliminary design proposal for a peptide synthetic vaccine and a peptidomimetic therapeutic, and to explore some design possibilities. The project was originally directed towards a use case for the Q-UEL language and its implementation in a knowledge management and automated inference system for medicine called the BioIngine, but focus here remains mostly on the virus itself. However, using Q-UEL systems to access relevant and emerging literature, and to interact with standard publically available bioinformatics tools on the Internet, did help quickly identify sequences of amino acids that are well conserved across many coronaviruses including 2019-nCoV. KRSFIEDLLFNKV was found to be particularly well conserved in this study and corresponds to the region around one of the known cleavage sites of the SARS virus that are believed to be required for virus activation for cell entry. This sequence motif and surrounding variations formed the basis for proposing a specific synthetic vaccine epitope and peptidomimetic agent. The work can, nonetheless, be described in traditional bioinformatics terms, and readily reproduced by others, albeit with the caveat that new data and research into 2019-nCoV is emerging and evolving at an explosive pace. Preliminary studies using molecular modeling and docking, and in that context the potential value of certain known herbal extracts, are also described., Highlights • Bioinformatics studies are carried out on the COVID-19 virus. • A sequence motif KRSFIEDLLFNKV is of particular interest. • Based on the above, synthetic peptides are designed. • Preliminary considerations are also given to non-peptide organic molecules.

Fully synthetic vaccine, in which one or multi-molecular antigens are conjugated to a synthetic carrier with well-defined chemical structure, is a new direction to develop carbohydrate-based vaccine against cancer and pathogens. Toll like receptor (TLR) agonists with the ability to stimulate immune response have been widely investigated and been applied as build-in adjuvants to construct fully synthetic vaccines. In particular, remarkable progress has been achieved in recent years in the development of vaccines constructed with the agonists of TLR1/2, TLR2/6 and TLR4 and tumor-associated carbohydrate antigens (TACAs). These di-, tri- or multi-component vaccine candidates showed attractive immunological properties. This review highlights recent advances in developing full synthetic carbohydrate antigen based vaccines, with an emphasis on the structure-activity relationships that provide a primary basis for future vaccine design and immunotherapy developing.

The surge of SARS-CoV-2 has created a wave of pandemic around the globe due to its high transmission rate. To contain this virus, researchers are working around the clock for a solution in the form of vaccine. Due to the impact of this pandemic, the economy and healthcare have immensely suffered around the globe. Thus, an efficient vaccine design is the need of the hour. Moreover, to have a generalised vaccine for heterogeneous human population, the virus genomes from different countries should be considered. Thus, in this work, we have performed genome-wide analysis of 10,664 SARS-CoV-2 genomes of 73 countries around the globe in order to identify the potential conserved regions for the development of peptide based synthetic vaccine viz. epitopes with high immunogenic and antigenic scores. In this regard, multiple sequence alignment technique viz. Clustal Omega is used to align the 10,664 SARS-CoV-2 virus genomes. Thereafter, entropy is computed for each genomic coordinate of the aligned genomes. The entropy values are then used to find the conserved regions. These conserved regions are refined based on the criteria that their lengths should be greater than or equal to 60 nt and their corresponding protein sequences are without any stop codons. Furthermore, Nucleotide BLAST is used to verify the specificity of the conserved regions. As a result, we have obtained 17 conserved regions that belong to NSP3, NSP4, NSP6, NSP8, RdRp, Helicase, endoRNAse, 2'-O-RMT, Spike glycoprotein, ORF3a protein, Membrane glycoprotein and Nucleocapsid protein. Finally, these conserved regions are used to identify the T-cell and B-cell epitopes with their corresponding immunogenic and antigenic scores. Based on these scores, the most immunogenic and antigenic epitopes are then selected for each of these 17 conserved regions. Hence, we have obtained 30 MHC-I and 24 MHC-II restricted T-cell epitopes with 14 and 13 unique HLA alleles and 21 B-cell epitopes for the 17 conserved regions. Moreover, for validating the relevance of these epitopes, the binding conformation of the MHC-I and MHC-II restricted T-cell epitopes are shown with respect to HLA alleles. Also, the physico-chemical properties of the epitopes are reported along with Ramchandran plots and Z-Scores and the population coverage is shown as well. Overall, the analysis shows that the identified epitopes can be considered as potential candidates for vaccine design.

Since 2004, when the first synthetic glycoconjugate vaccine against the pneumonia and meningitis causing bacterium Haemophilus influenza type b (Hib) approved for human use in Cuba was reported, 34 million doses of the synthetic vaccine have been already distributed in several countries under the commercial name of Quimi-Hib. However, despite the success of this product, no other synthetic glycoconjugate vaccine has been licensed in the following 13 years. As well as avoiding the need to handle pathogens, synthetic glycoconjugates offer clear advantages in terms of product characterization and the possibility to understand the parameters influencing immunogenicity. Nevertheless, large scale application of synthetic sugars has been perceived as challenging because of manufacturing costs and process complexity compared to natural polysaccharides. Chemoenzymatic approaches, one-pot protocols, and automated solid-phase synthesis are rendering carbohydrate production considerably more attractive for industrialization. Here we identify three areas where chemical approaches can advance this progress: (i) chemical or enzymatic methods enabling the delivery of the minimal polysaccharide portion responsible for an effective immune response; (ii) site-selective chemical or enzymatic conjugation strategies for the exploration of the conjugation point in immune responses against carbohydrate-based vaccines, and the consistent preparation of more homogeneous products; (iii) multicomponent constructs targeting receptors responsible for immune response modulation in order to control its quality and magnitude. We discuss how synthesis of bacterial oligosaccharides is useful toward understanding the polysaccharide portion responsible for immunogenicity, and for developing robust and consistent alternatives to natural heterogeneous polysaccharides. The synthesis of sugar analogues can lead to the identification of hydrolytically more stable versions of oligosaccharide antigens. The study of bacterial polysaccharide biosynthesis aids the development of in vitro hazard-free oligosaccharide production. Novel site-selective conjugation methods contribute toward deciphering the role of conjugation sites in the immunogenicity of glycoconjugates and prove to be particularly useful when glycans are conjugated to protein serving as carrier and antigen. The orthogonal incorporation of two different carbohydrate haptens enables the reduction of vaccine components. Finally, coordinated conjugation of glycans and small molecule immunopotentiators supports simplification of vaccine formulation and localization of adjuvant. Synergistic advancement of these areas, combined with competitive manufacturing processes, will contribute to a better understanding of the features guiding the immunological activity of glycoconjugates and, ultimately, to the design of improved, safer vaccines.

NH36 is a vital enzyme of the DNA metabolism and a specific target for anti-Leishmania chemotherapy. We developed second-generation vaccines composed of the FML complex or its main native antigen, the NH36 nucleoside hydrolase of Leishmania (L.) donovani and saponin, and a DNA vaccine containing the NH36 gene. All these vaccines were effective in prophylaxis and treatment of mice and dog visceral leishmaniasis (VL). The FML-saponin vaccine became the first licensed veterinary vaccine against leishmaniasis (Leishmune®) which reduced the incidence of human and canine VL in endemic areas. The NH36, DNA or recombinant protein vaccines induced a Th1 CD4+IFN-γ+ mediated protection in mice. Efficacy against VL was mediated by a CD4+TNF-α T lymphocyte response against the NH36-F3 domain, while against tegumentary leishmaniasis (TL) a CD8+ T lymphocyte response to F1 was also required. These domains were 36–41 % more protective than NH36, and a recombinant F1F3 chimera was 21% stronger than the domains, promoting a 99.8% reduction of the parasite load. We also identified the most immunogenic NH36 domains and epitopes for PBMC of active human VL, cured or asymptomatic and DTH+ patients. Currently, the NH36 subunit recombinant vaccine is turning into a multi-epitope T cell synthetic vaccine against VL and TL.

Brucellosis is primarily a zoonotic disease caused by members of the Brucella genus which consists of 11 recognized species based on pathogenicity and host preferences. Peptide vaccines are an alternative strategy to conventional vaccines based on the use of short peptide sequences and engineered to induce highly targeted immune responses, avoiding allergenic and/or reactogenic sequences. In this study, antigenic peptide sequence of Brucella abortus, WLAEIKQRSLMVHG, was chemically synthesized by adding tryptophan to the N-terminus of sequence, purified and characterized for the first time in the literature. Molecular weight of the peptide was determined by using LC-MS. A linear response (R-2 > 0.998) is observed for peptide in the range of 2-250 mu g/mL. The LOD and LOQ values are 0.08 and 0.27 mu g/kg respectively. Precision and accuracy ranges were found to be %RSD < 0.2 and 57.3-103.2%, respectively. Fluorescence property of the peptide was shown via fluorescence spectroscopy measurement. The 3D de novo structure of the peptide was predicted with PEP-FOLD server. According to the data obtained from the lipophilicity study (LogD(7.4) = - 3.093 +/- 0.195), the peptide can not cross the blood-brain barrier if applied via intravenously. This has shown us that peptide can be used as a peptide candidate at the vaccine studies. The production of antigenic peptides as in this study is the main component of the preparation of synthetic vaccine systems.

Glycoprotein MUC1 is an attractive target for anti-tumor vaccine development. However, the weak immunogenicity of MUC1 remains a significant problem. To solve this problem, several STn derivatives with N-acetyl modifications were synthesized and incorporated into a 20-amino acid MUC1 tandem repeat sequence. The modified STn-MUC1 glycopeptides were further connected to a carrier protein keyhole limpet hemocyanin (KLH). The immunological effects of these synthetic vaccine conjugates were evaluated using the BALB/c mouse model. The results showed that vaccine V2 elicited higher titers of antibodies which cross-reacted with the native STn-MUC1 antigen. Moreover, the elicited antisera reacted with the STn-MUC1 antigen-positive tumor cells, indicating that the carbohydrate antigen modification strategy may hold potential to overcome the weak immunogenicity of natural MUC1 glycopeptides.

Immunoinformatics has come by leaps and bounds to finding potent vaccine candidates against various pathogens. In the current study, a combination of different T (CD4+ and CD8+) and B cell epitope prediction tools was applied to find peptides containing multiple epitopes against Ebola nucleoprotein (NP) and the presentation of peptides to human leukocyte antigen (HLA) molecules was analyzed by prediction, docking and population coverage tools. Further, potential peptides were analyzed by ELISA for peptide induced IFN-γ secretion in peripheral blood mononuclear cells isolated from healthy volunteers. Six peptides were obtained after merging the overlapping multiple HLA I (CD8+) and II (CD4+) restricted T cell epitopes as well as B cell epitopes and eliminating the peptides liable to generate autoimmune and allergic response. All peptides displayed 100% conservancy in Zaire ebolavirus. In other Ebola virus species (Sudan, Bundibugyo and Tai forest) and Filoviridae members (Lloviuvirus and Margburgvirus), some peptides were found to be conserved with minor variations. Prediction tools confirmed the ability of predicted peptides to bind with diverse HLA (HLA-A, HLA-B, HLA-DP, HLA-DQ and HLA-DR) alleles. CABS-dock results displayed that the average root mean square deviation (RMSD) value was less than three in majority of cases representing strong binding affinity with HLA alleles. Population coverage analysis predicted high coverage (> 85%) for expected immune response in four continents (Africa, America, Asia and Europe). Nine out of ten blood samples exhibited enhanced IFN-γ secretion for two peptides (P2 and P3). Thus, the identified NP peptides can be considered as potential synthetic vaccine candidates against Ebola virus.

The design of synthetic vaccine peptides and other constructs (e.g., for developing immunodiagnostics) is informed by B-cell epitope prediction for antipeptide paratopes, which crucially depends on physicochemically and biologically meaningful interpretation of pertinent experimental data as regards paratope-epitope binding, with negative data being particularly problematic as they may be due to artefacts of immunization and immunoassays. Yet, the problem posed by negative data remains to be comprehensively addressed in a manner that clearly defines their role in the further development of B-cell epitope prediction. Hence, published negative data were surveyed and analyzed herein to identify key issues impacting on B-cell epitope prediction. Data were retrieved via searches using the Immune Epitope Database (IEDB) and review of underlying primary sources in literature to identify said issues, which include (1) inherent tendency toward false-negative data with use of solid-phase immunoassays and/or monoclonal paratopes, (2) equivocal data (i.e., both positive and negative data obtained from similar experiments), and (3) failure of antipeptide paratopes to cross-react with antigens of covalent structure and/or conformation different from that of the peptide immunogens despite apparent identity between curated epitope sequences. Analysis of experimental details thus focused on negative data from fluid-phase (e.g., immunoprecipitation) assays for detection of polyclonal paratope-epitope binding. Underlying literature references were reviewed to confirm the identification of negative data included for analysis. Furthermore, data from assays to detect cross-reaction of antipeptide antibody with protein antigen were included only if supported by positive data on either the corresponding reaction of the same antibody with peptide antigen or cross-reaction of said antibody with denatured protein antigen, to exclude the possibility that negative data on cross-reaction were due to absence of antipeptide paratopes in the first place (e.g., because of failed immunization due to insufficient immunogenicity and/or immune tolerance). Among currently available negative binding data on antipeptide antibodies, very few are on polyclonal responses yet also clearly attributable to conformational differences between peptide immunogens and native cognate proteins thereof. This dearth of negative data suitable for benchmarking B-cell epitope prediction conceivably could be addressed by generating positive data on binding of polyclonal antipeptide antibodies to cognate-protein sequences (e.g., in solid-phase immunoassays using unfolded protein antigen) to complement negative data on failure of the same antibodies to cross-react with native protein (e.g., in fluid-phase immunoassays, without artefactual covalent modification of antigens that tends to produce false-negative results). As regards cross-reactive binding of native cognate proteins by antipeptide antibodies (e.g., as mechanistic basis for novel vaccines and immunotherapeutics), negative data are most informative where attributable to conformational differences between peptide immunogens and target proteins. This is favored by careful peptide-immunogen design (e.g., avoiding covalent backbone and sidechain differences vis-a-vis target protein sequence) and positive data on antibody binding of the target protein sequence (e.g., in unfolded protein) paired with negative data on the same antibody using native protein antigen (e.g., from fluid- rather than solid-phase assays).

Streptococcus pneumoniae remains a deadly disease in small children and the elderly even though conjugate and polysaccharide vaccines based on isolated capsular polysaccharides (CPS) are successful. The most common serotypes that cause infection are used in vaccines around the world, but differences in geographic and demographic serotype distribution compromises protection by leading vaccines. The medicinal chemistry approach to glycoconjugate vaccine development has helped to improve the stability and immunogenicity of synthetic vaccine candidates for several serotypes leading to the induction of higher levels of specific protective antibodies. Here, we show that marketed CPS-based glycoconjugate vaccines can be improved by adding synthetic glycoconjugates representing serotypes that are not covered by existing vaccines. Combination (coformulation) of synthetic glycoconjugates with the licensed vaccines Prevnar13 (13-valent) and Synflorix (10-valent) yields improved 15- and 13-valent conjugate vaccines, respectively, in rabbits. A pentavalent semisynthetic glycoconjugate vaccine containing five serotype antigens (sPCV5) elicits antibodies with strong in vitro opsonophagocytic activity. This study illustrates that synthetic oligosaccharides can be used in coformulation with both isolated polysaccharide glycoconjugates to expand protection from existing vaccines and each other to produce precisely defined multivalent conjugated vaccines.

Mycobacterium tuberculosis is considered one of the most successful pathogens in the history of mankind, having caused 1.7 million deaths in 2016. The amount of resistant and extensively resistant strains has increased; BCG has been the only vaccine to be produced in more than 100 years though it is still unable to prevent the disease’s most disseminated form in adults; pulmonary tuberculosis. The search is thus still on-going for candidate antigens for an antituberculosis vaccine. This paper reports the use of a logical and rational methodology for finding such antigens, this time as peptides derived from the Rv3587c membrane protein. Bioinformatics tools were used for predicting mycobacterial surface location and Rv3587c protein structure whilst circular dichroism was used for determining its peptides’ secondary structure. Receptor-ligand assays identified 4 h igh a ctivity b inding p eptides (HABPs) binding specifically to A549 alveolar epithelial cells and U937 monocyte-derived macrophages, covering the region between amino acids 116 and 193. Their capability for inhibiting Mtb H37Rv invasion was evaluated. The recognition of antibodies from individuals suffering active and latent tuberculosis and from healthy individuals was observed in HABPs capable of avoiding mycobacterial entry to host cells. The results showed that 8 HABPs inhibited such invasion, two of them being common for both cell lines: 39265 (155VLAAYVYSLDNKRLWSNLDT173) and 39266 (174APSNETLVKTFSPGEQVTTY192). Peptide 39265 was the least recognised by antibodies from the individuals’ sera evaluated in each group. According to the model proposed by FIDIC regarding synthetic vaccine development, peptide 39265 has become a candidate antigen for an antituberculosis vaccine.

Toll like receptors (TLRs) are a family of transmembrane proteins which play a key role in innate immunity. When TLRs come into contact with a potential threat, they initiate a signaling cascade leading to release of cytokines and chemokines, maturation of antigen presenting cells and immune activation. Molecules which can activate TLRs may be utilized for vaccine development and act as vaccine adjuvants. Adjuvants can facilitate production of more effective vaccines based on antigens produced by recombinant techniques or by DNA vaccines, which are often poorly immunogenic since they lack the endogenous innate immunostimulatory components of the pathogen. In this paper the structural properties of such prospective compounds are thoroughly discussed.

Antitumor vaccine, which is promising for tumor therapy, has been extensively studied. Some encouraging results of chemically synthetic vaccine designs based on the tumor-associated antigen mucin 1 have been achieved. However, some shortcomings such as low efficiency and difficult purification restrict their clinical application. To overcome these difficulties, we designed a novel antitumor vaccine of glycopeptide nanoconjugates based on the multilayer self-assembly through the interaction of positive and negative charges. This vaccine formed the spherical structure and effectively activated the macrophage in vitro. Besides, it also induced high titer of antibodies against mucin 1 glycopeptide. The induced antibodies could highly bind to the tumor cells and effectively kill them by activation of the complement dependent cytotoxicity complex. This novel strategy provides a new way for the development of simple and effective antitumor vaccine.

Glycolipid–peptide conjugates designed to release vaccine components within target cells ensuring potent CD1d dependent T cell responses., It is known that T cells can eliminate tumour cells through recognition of unique or aberrantly expressed antigens presented as peptide epitopes by major histocompatibility complex (MHC) molecules on the tumour cell surface. With recent advances in defining tumour-associated antigens, it should now be possible to devise therapeutic vaccines that expand specific populations of anti-tumour T cells. However there remains a need to develop simpler efficacious synthetic vaccines that possess clinical utility. We present here the synthesis and analysis of vaccines based on conjugation of MHC-binding peptide epitopes to α-galactosylceramide, a glycolipid presented by the nonpolymorphic antigen-presenting molecule CD1d to provoke the stimulatory activity of type I natural killer T (NKT) cells. The chemical design incorporates an enzymatically cleavable linker that effects controlled release of the active components in vivo. Chemical and biological analysis of different linkages with different enzymatic targets enabled selection of a synthetic vaccine construct with potent therapeutic anti-tumour activity in mice, and marked in vitro activity in human blood.

Enterococcus faecalis is one of most important nosocomial and often multi-antibiotic resistant pathogens responsible for infections that are difficult to treat. Previously, a cell-wall polysaccharide termed diheteroglycan (DHG) was isolated and characterized as a promising vaccine candidate. However, the configuration of its lactic acid (LA) residue attached to the galactofuranoside unit was not assessed, although it influences conformation of DHG chain in terms of biological recognition and immune evasion. This study proves the R configuration of the LA residue by means of chemical analysis, investigation of intramolecular NMR nuclear Overhauser effects and molecular dynamics simulations of native DHG and corresponding R and S models, which were obtained by using pyranoside-into-furanoside rearrangement. As alternative treatment and prevention strategies for E. faecalis are desperately needed, this discovery may offer the prospect of a synthetic vaccine to actively immunize patients at risk.

Dengue virus (DV) is the etiologic agent of dengue fever, the most significant mosquito-borne viral disease in humans. Most DV vaccine approaches are focused on generating antibody mediated responses; one such DV vaccine is approved for use in humans but its efficacy is limited. While it is clear that T cell responses play important role in DV infection and subsequent disease manifestations, fewer studies are aimed at developing vaccines that induce robust T cells responses. Potent T cell based vaccines require 2 critical components: the identification of specific T cell stimulating MHC associated peptides, and an optimized vaccine delivery vehicle capable of simultaneously delivering the antigens and any required adjuvants. We have previously identified and characterized DV specific HLA-A2 and -A24 binding DV serotypes conserved epitopes, and the feasibility of an epitope based vaccine for DV infection. In this study, we build on those previous studies and describe an investigational DV vaccine using T cell epitopes incorporated into a calcium phosphate nanoparticle (CaPNP) delivery system. This study presents a comprehensive analysis of functional immunogenicity of DV CaPNP/multipeptide formulations in vitro and in vivo and demonstrates the CaPNP/multipeptide vaccine is capable of inducing T cell responses against all 4 serotypes of DV. This synthetic vaccine is also cost effective, straightforward to manufacture, and stable at room temperature in a lyophilized form. This formulation may serve as an effective candidate DV vaccine that protects against all 4 serotypes as either a prophylactic or therapeutic vaccine.

NKT cells are CD1d-restricted, glycolipid antigen-reactive, immunoregulatory T lymphocytes that can serve as a bridge between the innate and adaptive immunities. NKT cells have a wide range of therapeutic application in autoimmunity, transplant biology, infectious disease, cancer, and vaccinology. Rather than triggering "danger signal" and eliciting an innate immune response, αGalCer-based NKT-cell agonist act via a unique mechanism, recruiting NKT cells which play a T helper-like role even without peptide as Th epitope. Importantly, the non-polymorphism of CD1d render glycolipid a universal helper epitope, offering the potential to simplify the vaccine construct capable of eliciting consistent immune response in different individuals. This review details recent advances in the design of synthetic vaccines using NKT-cell agonist as adjuvant, highlighting the role of organic synthesis and conjugation technique to enhance the immunological actives and to simplify the vaccine constructs.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease in cloven-hoofed animals. A synthetic vaccine candidate consisting of dendrimeric peptides harbouring two copies of a B-epitope [VP1(136–154)] linked to a T-cell epitope [3A(21–35)] of FMDV confers protection to type O FMDV challenge in pigs. Herein we show in cattle that novel dendrimeric peptides bearing a T-cell epitope [VP1(21–40] and two or four copies of a B-cell epitope [VP1(135–160)] from type O1 Campos FMDV (termed BT and BT, respectively) elicited FMDV specific immune responses to similar levels to a commercial vaccine. Animals were challenged with FMDV and 100% of vaccinated cattle with BT or BT were protected to podal generalization. Moreover, bovines immunized with BT were completely protected (with no clinical signs) against FMDV challenge after three vaccine doses, which was associated with titers of viral neutralizing antibodies in serum higher than those of BT group (p< 0.05) and levels of opsonic antibodies similar to those of animals immunized with one dose of FMDV commercial vaccine. Bovines vaccinated with both dendrimeric peptides presented high levels of IgG1 anti FMDV in sera and in mucosa. When IgA in nasal secretions was measured, 20% or 40% of the animals in BT or BT groups respectively, showed anti-FMDV IgA titers. In addition, BT and BT peptides evoked similar consistent T cell responses, being recognized in vitro by lymphocytes from most of the immunized cattle in the proliferation assay, and from all animals in the IFN-γ production assay. Taken together, these results support the potential of dendrimers BT or BT in cattle as a highly valuable, cost-effective FMDV candidate vaccine with DIVA potential., National Institute of Agricultural Technology (grant number PNSA 1115052) and cooperation agreement INTA-INIA (Argentina-Spain SA 20938). Evaluation of vaccine immunogenicity and protection in cattle. Work at Universitat Pompeu Fabra and CBMSO was supported by MINECO, Spain (grant number AGL2014-52395-C2)

Breaking tolerance is crucial for effective tumor immunotherapy. We showed that vaccines containing tumor-associated human MUC1 glycopeptides induce strong humoral antitumor responses in mice. The question remained whether such vaccines work in humans, in systems where huMUC1 is a self-antigen. To clarify the question, mice transgenic in expressing huMUC1, mimicking the self-tolerant environment, and wild-type mice were vaccinated with a synthetic vaccine. This vaccine comprised STn and Tn antigens bound to a MUC1 tandem repeat peptide coupled to tetanus toxoid. The vaccine induced strong immune responses in wild-type and huMUC1-transgenic mice without auto-aggressive side effects. All antisera exhibited almost equivalent binding to human breast tumor cells. Similar increases of activated B-, CD4+ T-, and dendritic cells was found in the lymph nodes. The results demonstrate that tumor-associated huMUC1 glycopeptides coupled to tetanus toxoid are promising antitumor vaccines.

There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intratypic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-encoding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT1, SAT2, and SAT3 serotypes. Viruses of the seven serotype were rescued successfully. Each chimeric FMDV with a replacement of P1 showed serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Vaccination of pigs with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for a customized vaccine with challenge tools to protect against epizootic disease caused by specific serotypes or subtypes of FMDV. IMPORTANCE Foot-and-mouth disease (FMD) virus (FMDV) causes significant economic losses. For vaccine preparation, the selection of vaccine strains was complicated by high antigenic variation. In the present study, we suggested an effective strategy to rapidly prepare and evaluate mass-produced customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of viruses of seven representative serotypes. These chimeric viruses generally replicated readily in cell culture and had a particle size similar to that of the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses for all serotypes, especially for FMD-free countries, which have prohibited the import of FMDVs.

Human lymphatic filariasis, the parasitic disease caused by the filarial nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori, is ranked as the second most complex clinical condition leading to permanent and long-term disability. The multiple antigen peptide (MAP) approach is an effective method to chemically synthesize and deliver multiple T and B cell epitopes as the constituents of a single immunogen. Here, we report on the design, chemical synthesis, and immunoprophylaxis of three epitopes that have been identified from promising vaccine candidates reported in our previous studies, constructed as MAP on an inert lysine core for human lymphatic filariasis in Jird model. Two epitopes from Thioredoxin and one epitope from Transglutaminase were constructed as MAP in an inert lysine core. The immunoprophylaxis of the synthetic vaccine construct studied in Jird models showed protective antibody (1 in 64,000 titer) and cellular immune response. Thioredoxin-Transglutaminase MAP (TT MAP) conferred a significantly high protection of 63.04% compared to control (8.5%). Multi-antigen peptide vaccine is one best approach to provide immunity against multiple antigens delivered by the complex filarial parasite.

A major obstacle to enzyme replacement therapy (ERT) with recombinant human acid-α-glucosidase (rhGAA) for Pompe disease is the development of high titers of anti-rhGAA antibodies in a subset of patients, which often leads to a loss of treatment efficacy. In an effort to induce sustained immune tolerance to rhGAA, we supplemented the rhGAA therapy with a weekly intravenous injection of synthetic vaccine particles carrying rapamycin (SVP-Rapa) during the first 3 weeks of a 12-week course of ERT in GAA-KO mice, and compared this with three intraperitoneal injections of methotrexate (MTX) per week for the first 3 weeks. Empty nanoparticles (NP) were used as negative control for SVP-Rapa. Co-administration of SVP-Rapa with rhGAA resulted in more durable inhibition of anti-rhGAA antibody responses, higher efficacy in glycogen clearance in skeletal muscles, and greater improvement of motor function than mice treated with empty NP or MTX. Body weight loss was observed during the MTX-treatment but not SVP-Rapa-treatment. Our data suggest that co-administration of SVP-Rapa may be an innovative and safe strategy to induce durable immune tolerance to rhGAA during the ERT in patients with Pompe disease, leading to improved clinical outcomes., Highlights • SVP-Rapa induced durable rhGAA-specific immune tolerance to ERT in GAA-KO mice. • SVP-Rapa co-treatment reduced muscle glycogen more efficiently than methotrexate. • SVP-Rapa significantly improved muscle strength and motor function by ERT. • This study suggests an innovative and safe immunomodulatory ERT for Pompe disease.

The task of epitope discovery and vaccine design is increasingly reliant on bioinformatics analytic tools and access to depositories of curated data relevant to immune reactions and specific pathogens. The Immune Epitope Database and Analysis Resource (IEDB) was indeed created to assist biomedical researchers in the development of new vaccines, diagnostics, and therapeutics. The Analysis Resource is freely available to all researchers and provides access to a variety of epitope analysis and prediction tools. The tools include validated and benchmarked methods to predict MHC class I and class II binding. The predictions from these tools can be combined with tools predicting antigen processing, TCR recognition, and B cell epitope prediction. In addition, the resource contains a variety of secondary analysis tools that allow the researcher to calculate epitope conservation, population coverage, and other relevant analytic variables. The researcher involved in vaccine design and epitope discovery will also be interested in accessing experimental published data, relevant to the specific indication of interest. The database component of the IEDB contains a vast amount of experimentally derived epitope data that can be queried through a flexible user interface. The IEDB is linked to other pathogen-specific and immunological database resources.

Chemical conjugates comprising synthetic Toll-like receptor ligands (TLR-L) covalently bound to antigenic synthetic long peptides (SLP) are attractive vaccine modalities, which can induce robust CD8+ T-cell immune responses. Previously, we have shown that the mechanism underlying the power of TLR-L SLP conjugates is improved delivery of the antigen together with a dendritic cell activation signal. In the present study, we have expanded the approach to tumor-specific CD4+ as well as CD8+ T-cell responses and in vivo studies in two nonrelated aggressive tumor models. We show that TLR2-L SLP conjugates have superior mouse CD8+ and CD4+ T-cell priming capacity compared with free SLPs injected together with a free TLR2-L. Vaccination with TLR2-L SLP conjugates leads to efficient induction of antitumor immunity in mice challenged with aggressive transplantable melanoma or lymphoma. Our data indicate that TLR2-L SLP conjugates are suitable to promote integrated antigen-specific CD8+ and CD4+ T-cell responses required for the antitumor effects. Collectively, these data show that TLR2-L SLP conjugates are promising synthetic vaccine candidates for active immunotherapy against cancer. Cancer Immunol Res; 2(8); 756–64. ©2014 AACR.

Development of a broad-spectrum synthetic vaccine against TB would represent an important advance to the limited vaccine armamentarium against TB. It is believed that the esx family of TB antigens may represent important vaccine candidates. However, only 4 esx antigens have been studied as potential vaccine antigens. The challenge remains to develop a vaccine that simultaneously targets all 23 members of the esx family to induce enhanced broad-spectrum cell-mediated immunity. We sought to investigate if broader cellular immune responses could be induced using a multivalent DNA vaccine representing the esx family protein members delivered via electroporation. In this study, 15 designed esx antigens were created to cross target all members of the esx family. They were distributed into groups of 3 self-processing antigens each, resulting in 5 trivalent highly optimized DNA plasmids. Vaccination with all 5 constructs elicited robust antigen-specific IFN-γ responses to all encoded esx antigens and induced multifunctional CD4 Th1 and CD8 T cell responses. Importantly, we show that when all constructs are combined into a cocktail, the RSQ-15 vaccine, elicited substantial broad Ag-specific T cell responses to all esx antigens as compared with vaccination with BCG. Moreover, these vaccine-induced responses were highly cross-reactive with BCG encoded esx family members and were highly immune effective in a BCG DNA prime-boost format. Furthermore, we demonstrate the vaccine potential and immunopotent profile of several novel esx antigens never previously studied. These data highlight the likely importance of these novel immunogens for study as preventative or therapeutic synthetic TB vaccines in combination or as stand alone antigens.

In spite of the clear importance of protein O-mannosylation in brain glycobiology, tools are lacking for specific detection, enrichment, and identification of proteins containing these modifycations. We envisioned inducing antibodies that specifically recognize O-mannose glycans on proteins and peptides. With this in mind, we prepared a glycopeptide vaccine construct containing the N-acetyllactosamine-extended mannose motif Galβ1-4GlcNAcβ1-2ManαThr, found as a common core structure on almost all mammalian O-mannosyl glycoproteins identified. O-mannose glycosylated amino acid building blocks and the corresponding glycopeptides were prepared by chemical synthesis and then conjugated to an immune carrier protein. After administration of the synthetic vaccine into rabbits, strong immune responses were obtained. Further evaluation by ELISA neutralization experiments and glycopeptide microarrays showed that the induced antibodies were highly specific to the glycopeptide antigen.

Highly decorated: Tumor-associated MUC1 glycopeptide and tetanus toxoid T-cell epitope P2 can be attached to water-soluble poly(N-(2-hydroxypropyl)methacrylamide) carriers by orthogonal ligation techniques. Fully synthetic vaccine A with additional nanostructure-promoting domains induced antibodies that exhibit high affinity to tumor cells.

Background This manuscript describes the development of a novel synthetic immunotherapy (HIV-v) composed of four multi-epitope polypeptides targeting conserved regions in the Nef, Rev, Vif and Vpr viral proteins. Immunogenicity and cytotoxicity of HIV-v are discussed. Methods Short conserved T-cell multi-epitope regions were identified in silico in the HIV proteome. The immunogenicity of the identified HIV-v polypeptides was assessed in vivo by immunisation of C57BLK6 mice transgenic for HLA-A*0201. Splenocytes from immunised animals were exposed in vitro to soluble HIV-v polypeptides or to syngeneic (T1) or allogeneic (Jurkat) cells transfected with these polypeptides. Specific T-cell reactivity was assessed by cell-based IFN-γ ELISA. Virus specific CD3 + CD8+ IFN-γ+ recall responses were also determined by flow cytometry following in vitro exposure of splenocytes from immunised mice to syngeneic (T1) and allogeneic (H9) cells infected with HIV-1 strain IIIB. HIV-v specific antibodies were quantified by ELISA whilst antibody mediated anti-viral immunotherapeutic effect on T1 cells infected with a laboratory adapted and a primary isolate of the HIV-1 virus was assessed in a LDH-based complement mediated lysis assay. Results HIV-v elicited antigen-specific IgG and IFN−γ responses against the synthetic polypeptides in the formulation. HIV-v specific T cells recognised polypeptides presented either as soluble antigen or complexed to HLA-A*0201 following natural processing and presentation by syngeneic human T1 cells. Moreover, the CD3 + CD8+ component of the response recognised syngeneic T1 cells naturally infected with HIV-1 in a virus-specific and MHC restricted-manner. The HIV-v specific IgG response was also able to recognise human T1 cells naturally infected with HIV-1 and induce cell death through classic activation of complement. Conclusions HIV-v induces a vaccine-specific type I immune response characterised by activation of effector CD8+ T cell and antibody responses that recognise and kill human cell lines naturally infected with a laboratory adapted and a primary isolate of the HIV-1 virus. The data supports the hypothesis that alternative HIV protein targets can be effectively used to prime both cellular and antibody immune responses of clinical value in the prevention and treatment of HIV infection.

Brugia malayi is a threadlike nematode cause's swelling of lymphatic organs, condition well known as lymphatic filariasis; till date no invention made to effectively address lymphatic filariasis. In this analysis we a have predicted suitable antigenic peptides from Brugia malayi antigen protein for peptide vaccine design against lymphatic filariasis based on cross protection phenomenon as, an ample immune response can be generated with a single protein subunit. We found MHC class II binding peptides of Brugia malayi antigen protein are important determinant against the diseased condition. The analysis shows Brugia malayi antigen protein having 505 amino acids, which shows 497 nonamers. In this assay, we have predicted MHC-I binding peptides for 8mer_H2_Db (optimal score- 15.966), 9mer_H2_Db (optimal score- 15.595), 10mer_H2_Db (optimal score- 19.405), 11mer_H2_Dballeles (optimal score- 23.801). We also predicted the SVM based MHCII-IAb nonamers, 51-FQQIDPLDA, 442-FAAIACLVH, 206-YLNPFGHQF, 167-WYVIMAACY, 367-YAMIVIRLL, 434- LVITTAANF, 176-LDSYCLWKP, 435-VITTAANFA, 364-WPGYAMIVI (optimal score- 13.963); MHCII-IAd nonamers, 52-QQIDPLDAE, 171-MAACYLDSY, 239-QWRSVILCN, 168-YVIMAACYL, 3-QYLSVHSLS, 322-EILLHAKVV, 417- LGIIASFVS, 396-KAIFLAHFG, 167-WYVIMAACY, 269-LALHCINVI, 93-FINKAAPKQ, 259-NCIIVLKAF, 79- QGVLLIIPR, 22-TILQRSQAI, 63-RGFVYGNVS, 109-NISSLAFET,(optimal score- 16.748); and MHCII-IAg7 nonamers 171-MAACYLDSY, 73-KIVNGAQGV, 259-NCIIVLKAF, 209-PFGHQFSFE, 102-SCDTLLKNI, 25-QRSQAIRIV, 444- AIACLVHLF, 88-SLVNGFINK, 252-FPRHQLLNC, 471-RFVLANDNE, 52-QQIDPLDAE, 469-HRRFVLAND, 457- SNRHYFLAD, 362-KSWPGYAMI, 476-NDNEGEDFE, 370-IVIRLLQAL (optimal score- 19.847) which represents potential binders from Brugia malayi antigen protein. The method integrates prediction of MHC class I binding proteasomal C-terminal cleavage peptides and Eighteen potential antigenic peptides at average propensity 1.063 having highest local hydrophilicity. Thus a small antigen fragment can induce immune response against whole antigen. This approach can be applied for designing subunit and synthetic peptide vaccines.

Background A major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new “cluster-based” typing system of 175 M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines. Methods M protein sequences (AA 16–50) from the E4 cluster containing 17 emm types of GAS were analyzed using de novo 3-D structure prediction tools and the resulting structures subjected to chemical diversity analysis to identify sequences that were the most representative of the 3-D physicochemical properties of the M peptides in the cluster. Five peptides that spanned the range of physicochemical attributes of all 17 peptides were used to formulate synthetic and recombinant vaccines. Rabbit antisera were assayed for antibodies that cross-reacted with E4 peptides and whole bacteria by ELISA and for bactericidal activity against all E4G AS. Results The synthetic vaccine rabbit antisera reacted with all 17 E4 M peptides and demonstrated bactericidal activity against 15/17 E4G AS. A recombinant hybrid vaccine containing the same E4 peptides also elicited antibodies that cross-reacted with all E4 M peptides. Conclusions Comprehensive studies using structure-based design may result in a broadly protective M peptide vaccine that will elicit cluster-specific and emm type-specific antibody responses against the majority of clinically relevant emm types of GAS.

Cryptococcus neoformans is a fungal pathogen that can cause life-threatening infections in immunocompromised patients. The development of a vaccine based on the capsular polysaccharide of C. neoformans is still an open challenge due to the heterogeneity of the capsular polysaccharide and the difficulty of identifying protective epitopes. Therefore, construction of structurally defined part structures of the C. neoformans GXM capsule is in great demand. Herein is presented the synthesis of a 3-O-naphthalenylmethyl protected trisaccharide thioglycoside building block which is present in C. neoformans serotype C polysaccharide. Its property as a donor in a glycosylation reaction with a model acceptor has been evaluated together with its behaviour as an acceptor following removal of the temporary protecting group. The heavily branched hexasaccharide was obtained in good yields and excellent α-selectivity. The frame shifted octasaccharide structural triad motif for serotype C was also prepared following the same building block strategy. For the first time this structural motif, which is the most substituted amongst the four C. neoformans serotypes, was prepared. Three synthesized C. neoformans serotype C fragments of varying size, from penta-up to octasaccharide, were deprotected and will be included in unique glycoarrays to further investigate the possibility to develop a synthetic vaccine against this pathogen.

Human papilliomavirus (HPV) oncogene E7, essential for the transformation and maintenance of the malignancy of cervical cancer cells, represents an ideal tumor-specific antigen for vaccine development. However, due to the poor immunogenicity of E7 protein, an effective therapeutic E7 vaccine is still lacking. Dendritic cells (DCs) are probably the most potent antigen presenting cells for the induction of cytotoxic T lymphocyte (CTL) response, which is crucial for tumor control. In this study, we tested whether targeting the E7 antigen to DCs in vivo would elicit therapeutic antitumor CTL response. We generated the DEC205-specific single-chain variable fragment (scFv) and E7 long peptide fusion protein [scFv(DEC205)-E7] based on the novel method of protein assembly we recently developed. This fusion protein vaccine demonstrated highly efficient DC-targeting in vivo and elicited much stronger protective CTL response than non-DC-targeting control vaccine in naive mice. Furthermore, the scFv(DEC205)-E7 vaccine showed significant therapeutic antitumor response in TC-1 tumor bearing mice. Importantly, PD-L1 blockade further improved the therapeutic effect of the scFv(DEC205)-E7 vaccine. Thus, the current study suggests an efficient strategy for cervical cancer immunotherapy by combining the DC(DEC205)-targeting E7 vaccine and PD-L1 blockade.

Malignant transformations are often associated with aberrant glycosylation processes that lead to the expression of new carbohydrate antigens at the surface of tumor cells. Of these carbohydrate antigens, the Tn antigen is particularly highly expressed in many carcinomas, especially in breast carcinoma. We designed MAG-Tn3, a fully synthetic vaccine based on three consecutive Tn moieties that are O-linked to a CD4+ T cell epitope, to induce anti-Tn antibody responses that could be helpful for therapeutic vaccination against cancer. To ensure broad coverage within the human population, the tetanus toxoid-derived peptide TT830-844 was selected as a T-helper epitope because it can bind to various HLA-DRB molecules. We showed that the MAG-Tn3 vaccine, which was formulated with the GSK proprietary immunostimulant AS15 and designed for human cancer therapy, is able to induce an anti-Tn antibody response in mice of various H-2 haplotypes, and this response correlates with the ability to induce a specific T cell response against the TT830-844 peptide. The universality of the TT830-844 peptide was extended to new H-2 and HLA-DRB molecules that were capable of binding this T cell epitope. Finally, the MAG-Tn3 vaccine was able to induce anti-Tn antibody responses in cynomolgus monkeys, which targeted Tn-expressing tumor cells and mediated tumor cell death both in vitro and in vivo. Thus, MAG-Tn3 is a highly promising anticancer vaccine that is currently under evaluation in a phase I clinical trial. Electronic supplementary material The online version of this article (doi:10.1007/s00262-016-1802-0) contains supplementary material, which is available to authorized users.