Potential Subunit Vaccine Candidates Research Articles

Synthesis of an Immunologically Active Heptamannoside of Mycobacterium tuberculosis by the [Au]/[Ag]-Catalyzed Activation of Ethynylcyclohexyl Glycosyl Carbonate Donor.

Tuberculosis (TB) is one of the most dreadful diseases, killing more than 3 million humans annually. M. tuberculosis (MTb) is the causative agent for TB and has a thick and waxy cell wall, making it an attractive target for immunological studies. In this study, a heptamannopyranoside containing 1 → 2 and 1 → 6 α-mannopyranosidic linkages has been explored for the immunological evaluations. The conjugation-ready heptamannopyranoside was synthesized by exploiting the salient features of recently discovered [Au]/[Ag]-glycosidation of ethynylcyclohexyl glycosyl carbonate donors. The glycan was conjugated to the ESAT6, an early secreted protein of MTb for further characterization as a potential subunit vaccine candidate.

Leptospira Lipid A Is a Potent Adjuvant That Induces Sterilizing Immunity against Leptospirosis.

Leptospirosis is a globally significant zoonotic disease. The current inactivated vaccine offers protection against specific serovars but does not provide complete immunity. Various surface antigens, such as Leptospira immunoglobulin-like proteins (LigA and LigB), have been identified as potential subunit vaccine candidates. However, these antigens require potent adjuvants for effectiveness. Bacterial lipopolysaccharides (LPSs), including lipid A, are a well-known immunostimulant, and clinical adjuvants often contain monophosphoryl lipid A (MPLA). Being less endotoxic, we investigated the adjuvant properties of lipid A isolated from L. interrogans serovar Pomona (PLA) in activating innate immunity and enhancing antigen-specific adaptive immune responses. PLA activated macrophages to a similar degree as MPLA, albeit at a higher dose, suggesting that it is less potent in stimulation than MPLA. Mice immunized with a variable portion of LigA (LAV) combined with alum and PLA (LAV-alum-PLA) exhibited significantly higher levels of LAV-specific humoral and cellular immune responses compared to alum alone but similar to those induced by alum-MPLA. The adjuvant activity of PLA resembles that of MPLA and is primarily achieved through the increased recruitment, activation, and uptake of antigens by innate immune cells. Furthermore, like MPLA, PLA formulation establishes a long-lasting memory response. Notably, PLA demonstrated superior potency than MPLA formulation and provided sterilizing immunity against the leptospirosis in a hamster model. Overall, our study sheds light on the adjuvant properties of Leptospira lipid A and offers promising avenues for developing LPS-based vaccines against this devastating zoonotic disease.

Immunoinformatics-guided selection of immunogenic peptides from betanodavirus induce antigen-specific antibody production in hybrid grouper, Epinephelus fuscoguttatus x Epinephelus lanceolatus

Betanodavirus is a significant pathogen that causes viral nervous necrosis (VNN) in a wide range of fish species. It could cause up to 100% mortality in the infected farmed fish, especially at the larval stage. Red-Spotted Grouper Nervous Necrosis Virus (RGNNV), one of the betanodavirus genotypes, has been proven to have a wide range of host-compatibility across warm-water fish species. To date, there is no viable strategy to combat this virus infection, but vaccination approach is known to be effective against infectious diseases. Thus, current study aimed to identify immunogenic peptides as potential subunit vaccine candidates against grouper betanodavirus. Nucleotide sequences of the grouper nervous necrosis virus were retrieved from the National Center for Biotechnology Information database. B- and T-cell epitopes were predicted using the Immune Epitope Database analysis resource and the NetCTL 1.2 server, respectively. The predicted B- and T-cell epitopes were analysed for its respective antigenicity, immunogenicity, physico-chemical properties, and surface accessibility. Selected epitopes were then synthesized for immunization assay to examine antigen-specific antibody production in the immunized grouper. Five epitopes (one B-cell and four T-cell epitopes) were selected based on the immunogenicity score. The B-cell epitope has the highest immunogenicity score of 0.9333. Physico-chemical properties analysis of the B-cell epitope shown highest GRAVY score of 0.516 and was predicted to possess the longest estimated half-life of 30 h. Structural analysis has shown that the B-cell epitope possesses the largest accessible surface on the virus particle. Immunization of grouper with the selected B-cell epitope induced the highest antigen-specific antibody production as compared to grouper that was immunized with the selected T-cell epitope. This study demonstrated the feasibility of reverse vaccinology approach in the selection and design of immunogenic peptides against NNV in grouper to serve as the potential vaccine candidate.

Screening of immunogenic proteins and evaluation of vaccine candidates against Mycoplasma synoviae

Mycoplasma synoviae (M. synoviae) is a serious avian pathogen that causes significant economic losses to chicken and turkey producers worldwide. The currently available live attenuated and inactivated vaccines provide limited protection. The objective of this study was to identify potential subunit vaccine candidates using immunoproteomics and reverse vaccinology analyses and to evaluate their preliminary protection. Twenty-four candidate antigens were identified, and five of them, namely RS01790 (a putative sugar ABC transporter lipoprotein), BMP (a substrate-binding protein of the BMP family ABC transporter), GrpE (a nucleotide exchange factor), RS00900 (a putative nuclease), and RS00275 (an uncharacterized protein), were selected to evaluate their immunogenicity and preliminary protection. The results showed that all five antigens had good immunogenicity, and they were localized on the M. synoviae cell membrane. The antigens induced specific humoral and cellular immune responses, and the vaccinated chickens exhibited significantly greater body weight gain and lower air sac lesion scores and tracheal mucosal thicknesses. Additionally, the vaccinated chickens had lower M. synoviae loads in throat swabs than non-vaccinated chickens. The protective effect of the RS01790, BMP, GrpE, and RS00900 vaccines was better than that of the RS00275 vaccine. In conclusion, our study demonstrates the potential of subunit vaccines as a new approach to developing M. synoviae vaccines, providing new ideas for controlling the spread of M. synoviae worldwide.

A non-toxic recombinant Clostridium septicum α toxin induces protective immunity in mice and rabbits

Clostridium septicum alpha toxin (CSA) plays significant roles in ruminant's braxy. Genetically engineered CSA has been shown to function as a potential vaccine candidate in the prevention of the disease caused by Clostridium septicum. In the present study, we synthesized a non-toxic recombinant, rCSAm4/TMD by introducing four amino acid substitutions (C86L/N296A/H301A/W342A) and 11-amino-acid deletion (residues 212 to 222). Compared to recombinant CSA, rCSAm4/TMD showed no cytotoxicity to MDCK cells and was not fatal to mice. Moreover, rCSAm4/TMD could protect immunized mice against 5 × mouse LD100 (100% lethal dose) of crude CSA without obvious pathological change. Most importantly, rabbits immunized with rCSAm4/TMD produced high titers of neutralizing antibodies which protected the rabbits against crude CSA challenge. These data suggest that genetically detoxified rCSAm4/TMD is a potential subunit vaccine candidate against braxy.

An Immunoinformatics-Based Study of Mycobacterium tuberculosis Region of Difference-2 Uncharacterized Protein (Rv1987) as a Potential Subunit Vaccine Candidate for Preliminary Ex Vivo Analysis.

Mycobacterium tuberculosis (Mtb) is the pathogen that causes tuberculosis and develops resistance to many of the existing drugs.The sole licensed TB vaccine, BCG, is unable to provide a comprehensive defense. So, it is crucial to maintain the immunological response to eliminate tuberculosis. Our previous in silico study reported five uncharacterized proteins as potential vaccine antigens. In this article, we considered the uncharacterized Mtb H37Rv regions of difference (RD-2) Rv1987 protein as a promising vaccine candidate. The vaccine quality of the protein was analyzed using reverse vaccinology and immunoinformatics-based quality-checking parameters followed by an ex vivo preliminary investigation. In silico analysis of Rv1987 protein predicted it as surface localized, secretory, single helix, antigenic, non-allergenic, and non-homologous to the host protein. Immunoinformatics analysis of Rv1987 by CD4 + and CD8 + T-cells via MHC-I and MHC-II binding affinity and presence of B-cell epitope predicted its immunogenicity. The docked complex analysis of the 3D model structure of the protein with immune cell receptor TLR-4 revealed the protein's capability for potential interaction. Furthermore, the target protein-encoded gene Rv1987 was cloned, over-expressed, purified, and analyzed by mass spectrometry (MS) to report the target peptides. The qRT-PCR gene expression analysis shows that it is capable of activating macrophages and significantly increasing the production of a number of key cytokines (TNF-α, IL-1β, and IL-10). Our in-silico analysis and ex vivo preliminary investigations revealed the immunogenic potential of the target protein. These findings suggest that the Rv1987 be undertaken as a potent subunit vaccine antigen and that further animal model immuno-modulation studies would boost the novel TB vaccine discovery and/or BCG vaccine supplement pipeline.

Identification of a Novel Linear B-Cell Epitope of HbpA Protein from Glaesserella parasuis Using Monoclonal Antibody.

Glaesserella parasuis (G. parasuis.) is the etiological pathogen of Glässer's disease, which causes high economic losses to the pig industry. The heme-binding protein A precursor (HbpA) was a putative virulence-associated factor proposed to be potential subunit vaccine candidate in G. parasuis. In this study, three monoclonal antibodies (mAb) 5D11, 2H81, and 4F2 against recombinant HbpA (rHbpA) of G. parasuis SH0165 (serotype 5) were generated by fusing SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with the rHbpA. Indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA) demonstrated that the antibody designated 5D11 showed a strong binding affinity with the HbpA protein and was chosen for subsequent experiments. The subtypes of the 5D11 were IgG1/κ chains. Western blot analysis showed that mAb 5D11 could react with all 15 serotype reference strains of G. parasuis. None of the other bacteria tested reacted with 5D11. In addition, a linear B-cell epitope recognized by 5D11 was identified by serial truncations of HbpA protein and then a series of truncated peptides were synthesized to define the minimal region that was required for mAb 5D11 binding. The 5D11 epitope was located on amino acids 324-LPQYEFNLEKAKALLA-339 by testing the 5D11 monoclonal for reactivity with 14 truncations. The minimal epitope 325-PQYEFNLEKAKALLA-339 (designated EP-5D11) was pinpointed by testing the mAb 5D11 for reactivity with a series of synthetic peptides of this region. The epitope was highly conserved among G. parasuis strains, confirmed by alignment analysis. These results indicated that mAb 5D11 and EP-5D11 might potentially be used to develop serological diagnostic tools for G. parasuis. Three-dimensional structural analysis revealed that amino acids of EP-5D11 were in close proximity and may be exposed on the surface of the HbpA protein.

A non-toxic recombinant bivalent chimeric protein rETXm3CSAm4/TMD as a potential vaccine candidate against enterotoxemia and braxy.

Clostridium perfringens epsilon toxin (ETX) and Clostridium septicum alpha toxin (CSA) are lethal and necrotizing toxins, which play key roles in enterotoxemia and braxy of ruminants, respectively. In the present study, we synthesized a bivalent chimeric protein rETXm3CSAm4/TMD comprising ETXm3 (Y30A/H106P/Y196A) and CSAm4/TMD (C86L/N296A/H301A/W342A and a deletion of residues 212 to 222). Compared with recombinant ETX and recombinant CSA, rETXm3CSAm4/TMD showed no cytotoxicity in Madin-Darby Canine Kidney cells and was not fatal to mice. Moreover, rETXm3CSAm4/TMD could protect immunized mice against 10×mouse LD100 of crude ETX or 3×mouse LD100 of crude CSA without obvious histopathologic difference. Most importantly, both rabbits and sheep immunized with rETXm3CSAm4/TMD produced high titers of neutralizing antibody which protected the animals against the challenge with crude ETX or crude CSA. These data suggest that genetically detoxified rETXm3CSAm4/TMD is a potential subunit vaccine candidate against enterotoxemia and braxy.

Designing a novel E2-IFN-γ fusion protein against CSFV by immunoinformatics and structural vaccinology approaches

Subunit vaccines with high purity and safety are gradually becoming a main trend in vaccinology. However, adjuvants such as interferon-gamma (IFN-γ) are required to enhance immune responses of subunit vaccines due to their poor immunogenicity. The conjugation of antigen with adjuvant can induce more potent immune responses compared to the mixture of antigen and adjuvant. At the same time, the selection of linker, indispensable in the construction of the stable and bioactive fusion proteins, is complicated and time-consuming. The development of immunoinformatics and structural vaccinology approaches provides a means to address the abovementioned problem. Therefore, in this study, a E2-IFN-γ fusion protein with an optimal linker (E2-R2-PIFN) was designed by bioinformatics approaches to improve the immunogenicity of the classical swine fever virus (CSFV) E2 subunit vaccine. Moreover, the E2-R2-PIFN fusion protein was expressed in HEK293T cells and the biological effects of IFN-γ in E2-R2-PIFN were confirmed in vitro via Western blotting. Here, an alternative method is utilized to simplify the design and validation of the antigen-adjuvant fusion protein, providing a potential subunit vaccine candidate against CSFV. KEY POINTS: • An effective and simple workflow of antigen-adjuvant fusion protein design and validation was established by immunoinformatics and structural vaccinology. • A novel E2-IFN-γ fusion protein with an optimal linker was designed as a potential CSFV vaccine. • The bioactivity of the newly designed fusion protein was preliminarily validated through in vitro experiments.

Enhanced immunogenicity of the tuberculosis subunit Rv0572c vaccine delivered in DMT liposome adjuvant as a BCG-booster

COVID-19 has affected the progress made in the prevention and treatment of tuberculosis (TB); hence, the mortality of tuberculosis has risen. Different strategies-based novel TB vaccine candidates have been developed. This study identifies strategies to overcome the limitations of Bacille Calmette-Guérin (BCG) in preventing latent infection and reactivation of TB. The latency antigen Rv0572c was selected based on the mechanism of interaction between Mycobacterium tuberculosis and its host. The rRv0572c protein was used to stimulate whole blood samples derived from patients with clinically diagnosed active TB (ATBs) or latent TB infections (LTBIs) and healthy control (HCs) donors, confirming that this protein can be recognized by T cells in patients with TB, especially LTBIs. C57BL/6 mice were used to investigate the immunogenicity of the rRv0572c protein emulsified in the liposome adjuvant dimethyldioctadecylammonium [DDA], monophosphoryl lipid A [MPLA], trehalose-6, 6′-dibehenate [TDB] (DMT). The results demonstrated that rRv0572c/DMT could boost BCG-primed mice to induce antigen-specific CD4+ T cell production and generate functional T cells dominated by antigen-specific CD8+ T cells. The rRv0572c/DMT vaccine could also trigger limited Th2 humoral immune responses. These findings suggest that rRv0572c/DMT is a potential subunit vaccine candidate that can be used as a booster vaccine for BCG.

Computational discovery and ex-vivo validation study of novel antigenic vaccine candidates against tuberculosis

Tuberculosis (TB) is a complex infectious bacterial disease, which has evolved with highly successful mechanisms to interfere with host defenses and existing classes of antibiotics to resist eradication. The single obtainable TB vaccine, Bacille Calmette-Guerin (BCG) has failed to provide regular defense for respiratory TB in adults. In this study, a bioinformatics and immunoinformatics approach was applied on Mycobacterium tuberculosis (Mtb) H37Rv proteomes to discover the potential subunit vaccine candidates that elicit both tuberculosis-specific T-cells and B-cell immune response. A total of 4049 proteins of MtbH37RvMtbH37Rv were retrieved and subjected to in silico sequence-based analysis. Finally, five (P9WL69 (Rv2599), P9WIG1 (Rv0747), P9WLQ1 (Rv1987), O53608 (Rv0063), O06624 (Rv1566c)) novel putative proteins were selected. Among the five putative antigenic vaccine candidates, P9WL69 protein was selected for the ex-vivo validation study. The P9WL69 protein encoding gene was amplified and cloned on pET21b vector. The success of the recombinant clone (pET21b-RV2599) was confirmed by colony PCR, insert release test and sequencing. Furthermore, the identified epitopes of the P9WL69 protein were considered for in silico docking and molecular dynamics simulation study using Toll-like Receptors (TLRs) (TLR-2, TLR-4, TLR-9), Mannose receptor, and Myeloid differentiation 88 (MYD88) to understand their binding affinity towards the development of immunogenic vaccines against tuberculosis.

Hydrophobic Mycobacterial Antigens Elicit Polyfunctional T Cells in Mycobacterium bovis Immunized Cattle: Association With Protection Against Challenge?

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a chronic disease of cattle with a detrimental impact on food quality and production. Research on bTB vaccines has predominantly been focused on proteinaceous antigens. However, mycobacteria have a thick and intricate lipid outer layer and lipids as well as lipopeptides are important for immune-evasion and virulence. In humans, lipid extracts of M. tuberculosis have been shown to elicit immune responses effective against M. tuberculosis in vitro. Chloroform-methanol extraction (CME) was applied to M. bovis BCG to obtain a hydrophobic antigen extract (CMEbcg) containing lipids and lipopeptides. CMEbcg stimulated IFN-γ+IL-2+ and IL-17A+IL-22+ polyfunctional T cells and elicited T cell responses with a Th1 and Th17 cytokine release profile in both M. bovis BCG vaccinated and M. bovis challenged calves. Lipopeptides were shown to be the immunodominant antigens in CMEbcg, stimulating CD4 T cells via MHC class II. CMEbcg expanded T cells killed CMEbcg loaded monocytes and the CMEbcg-specific CD3 T cell proliferative response following M. bovis BCG vaccination was the best predictor for reduced pathology following challenge with M. bovis. Although the high predictive value of CMEbcg-specific immune responses does not confirm a causal relationship with protection against M. bovis challenge, when taking into account the in vitro antimycobacterial phenotype of CMEbcg-specific T cells (e.g. Th1/Th17 cytokine profile), it is indicative that CMEbcg-specific immune responses could play a functional role in immunity against M. bovis. Based on these findings we conclude that lipopeptides of M. bovis are potential novel subunit vaccine candidates and that further studies into the functional characterization of lipopeptide-specific immune responses together with their role in protection against bovine tuberculosis are warranted.

PCV cap proteins fused with calreticulin expressed into polymers in Escherichia coli with high immunogenicity in mice

BackgroundPorcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus diseases (PCVDs) which causes huge yearly economic losses in the swine industry. Capsid protein (Cap) is the major structural protein of PCV2 that can induce a protective immune response. Therefore, developing a novel and safe subunit vaccine against PCV2 infection is needed.ResultsIn this study, the Cap gene was bound to the truncated calreticulin (CRT) (120–250 aa/120–308 aa) at the N/C terminal, and then the CRT-Cap fusion genes were expressed in Escherichia coli (E.coli). The size-exclusion chromatography and dynamic light scattering (DLS) data showed that the purified recombinant CRT-Cap fusion protein (rP5F) existed in the form of polymers. Immunization with rP5F stimulated high levels of PCV2 specific antibody and neutralization antibody in mice, which were almost identical to those induced by the commercial subunit and inactivated vaccines. The lymphocyte proliferation and cytokine secretion were also detected in rP5F immunized mice. According to the results of PCV2-challenge experiment, the virus loads significantly decreased in mice immunized with rP5F. The data obtained in the current study revealed that rP5F had the potential to be a subunit vaccine candidate against PCV2 in the future.ConclusionsWe have successfully expressed Cap-CRT fusion proteins in E.coli and optimized rP5F could form into immunogenic polymers. Mice immunized with rP5F efficiently induced humoral and part of cellular immune responses and decreased the virus content against PCV2-challenge, which suggested that rF5P could be a potential subunit vaccine candidate.

Characterization of novel antigenic vaccine candidates for nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae infection

Streptococcus agalactiae is one of the important pathogens responsible for high mortality and economic losses of the tilapia industry worldwide. Based on ten serovars of S. agalactiae infection, subunit vaccine with conserved antigens is promising strategy corresponding stimulated long-term immunity and provides protection for animals against different serotypes of S. agalactiae. In the present study, eight proteins (AP, AL, LivK, ESAT6, essA, essB, essC and esaA) were selected from the S. agalactiae serotype Ia genome as immunogenic antigens with bioinformation and immune experiment assays. These recombinant proteins were successfully obtained through expression in Escherichia coli and the immunogenicity was assessed in tilapia challenge model. The results showed that the recombinant proteins caused high-level-specific antibodies production and high lysozyme activities, suggesting that the recombinant proteins induced specific humoral immune response and innate immune response of tilapia. The signficant increase were observed in the cytokines levels of TNF-α, IL-1β, IFN-γ, cc1, cc2 and immune-related genes levels of CD8α and MHC factors in the spleen and head kidney tissues, suggesting that the recombinant proteins induced immune response of tilapia through cytokines signal pathway and activated high cytotoxic T-lymphocyte (CTL) activity of tilapia. Furthermore, vaccinated tilapia conferred high levels of protection against challenge with a lethal dose of highly virulent serovar Ⅰa (highest RPS was 91.60% in AL and essC protein groups). Our results indicated that the eight recombinant proteins induced high level of immune responses and offered protection against S. agalactiae infection, could be potential subunit vaccine candidates.

Analysis of the Role of TpUB05 Antigen from Theileria parva in Immune Responses to Malaria in Humans Compared to Its Homologue in Plasmodium falciparum the UB05 Antigen.

Despite the amount of resources deployed and the technological advancements in molecular biology, vaccinology, immunology, genetics, and biotechnology, there are still no effective vaccines against malaria. Immunity to malaria is usually seen to be species- and/or strain-specific. However, there is a growing body of evidence suggesting the possibility of the existence of cross-strain, cross-species, and cross-genus immune responses in apicomplexans. The principle of gene conservation indicates that homologues play a similar role in closely related organisms. The homologue of UB05 in Theileria parva is TpUB05 (XP_763711.1), which has been tested and shown to be associated with protective immunity in East Coast fever. In a bid to identify potent markers of protective immunity to aid malaria vaccine development, TpUB05 was tested in malaria caused by Plasmodium falciparum. It was observed that TpUB05 was better at detecting antigen-specific antibodies in plasma compared to UB05 when tested by ELISA. The total IgG raised against TpUB05 was able to block parasitic growth in vitro more effectively than that raised against UB05. However, there was no significant difference between the two study antigens in recalling peripheral blood mononuclear cell (PBMC) memory through IFN-γ production. This study suggests, for the first time, that TpUB05 from T. parva cross-reacts with UB05 from P. falciparum and is a marker of protective immunity in malaria. Hence, TpUB05 should be considered for possible development as a potential subunit vaccine candidate against malaria.

In silico design of a vaccine candidate based on autotransporters and HSP against the causal agent of shigellosis, Shigella flexneri

Shigellosis is a diarrheal disease that causes high mortality every year, especially in children, elderly and immunocompromised patients. Recently, resistance strains to antibiotic therapy are in the rise and the World Health Organization prioritizes the development of a safe vaccine against the most common causal agent of shigellosis, Shigella flexneri. This pathogen uses autotransporter proteins such as SigA, Pic and Sap to increase virulence and some of them have been described as highly immunogenic proteins. In this study, we used immune-informatics analysis to identify the most antigenic epitope as a vaccine candidate on three passenger domains of auto-transporter proteins encoded on the pathogenic island SHI-1, to induce immunity against S. flexneri. Epitope identification was done using various servers such as Bepipred, Bcepred, nHLAPRED, NetMHCII, Rankpep and IEDB and the final selection was done based on its antigenicity using the VaxiJen server. Moreover, to enhance immunity, the GroEL adjuvant was added to the final construct as a Toll-like receptor 2 (TLR2) agonist. On the other hand, to predict the tertiary structure, the I-TASSER server was used, and the best model was structurally validated using the ProSA-web software and the Ramachandran plot. Subsequently, the model was refined and used for docking and molecular dynamics analyses with TLR2, which demonstrated an appropriate and stable interaction. In summary, a potential subunit vaccine candidate, that contains B and T cell epitopes with proper physicochemical properties was designed. This multiepitope vaccine is expected to elicit robust humoral and cellular immune responses and vest protective immunity against S. flexneri.

Genome-wide screening of lipoproteins in Actinobacillus pleuropneumoniae identifies three antigens that confer protection against virulent challenge

Actinobacillus pleuropneumoniae is an important veterinary pathogen that causes porcine pleuropneumonia. Lipoproteins of bacterial pathogens play pleiotropic roles in the infection process. In addition, many bacterial lipoproteins are antigenic and immunoprotective. Therefore, characterization of lipoproteins is a promising strategy for identification of novel vaccine candidates or diagnostic markers. We cloned 58 lipoproteins from A. pleuropneumoniae JL03 (serovar 3) and expressed them in Escherichia coli. Five proteins with strong positive signals in western blotting analysis were used to immunize mice. These proteins elicited significant antibody responses, and three of them (APJL_0922, APJL_1380 and APJL_1976) generated efficient immunoprotection in mice against lethal heterologous challenge with A. pleuropneumoniae 4074 (serovar 1), both in the active and passive immunization assays. Then immunogenicity of these three lipoproteins (APJL_0922, APJL_1380 and APJL_1976) were further tested in pigs. Results showed that these proteins elicited considerable humoral immune responses and effective protective immunity against virulent A. pleuropneumoniae challenge. Our findings suggest that these three novel lipoproteins could be potential subunit vaccine candidates.

Induction of Mycobacterium Tuberculosis Lipid-Specific T Cell Responses by Pulmonary Delivery of Mycolic Acid-Loaded Polymeric Micellar Nanocarriers.

Mycolic acid (MA), a major lipid component of Mycobacterium tuberculosis (Mtb) cell wall, can be presented by the non-polymorphic antigen presenting molecule CD1b to T cells isolated from Mtb-infected individuals. These MA-specific CD1b-restricted T cells are cytotoxic, produce Th1 cytokines, and form memory populations, suggesting that MA can be explored as a potential subunit vaccine candidate for TB. However, the controlled elicitation of MA-specific T cell responses has been challenging due to difficulties in the targeted delivery of lipid antigens and a lack of suitable animal models. In this study, we generated MA-loaded micellar nanocarriers (MA-Mc) comprised of self-assembled poly(ethylene glycol)-bl-poly(propylene sulfide; PEG-PPS) copolymers conjugated to an acid sensitive fluorophore to enhance intracellular delivery of MA to phagocytic immune cells. Using humanized CD1 transgenic (hCD1Tg) mice, we found these nanobiomaterials to be endocytosed by bone marrow-derived dendritic cells (DCs) and localized to lysosomal compartments. Additionally, MA-Mc demonstrated superior efficacy over free MA in activating MA-specific TCR transgenic (DN1) T cells in vitro. Following intranasal immunization, MA-Mc were primarily taken up by alveolar macrophages and DCs in the lung and induced activation and proliferation of adoptively transferred DN1 T cells. Furthermore, intranasal immunization with MA-Mc induced MA-specific T cell responses in the lungs of hCD1Tg mice. Collectively, our data demonstrates that pulmonary delivery of MA via PEG-PPS micelles to DCs can elicit potent CD1b-restricted T cell responses both in vitro and in vivo and MA-Mc could be explored as subunit vaccines against Mtb infection.

Efficacy of heat-labile enterotoxin B subunit-adjuvanted parenteral porcine epidemic diarrhea virus trimeric spike subunit vaccine in piglets.

Devastating outbreaks of porcine epidemic diarrhea (PED) started in China in late 2010 and rapidly spread to North America and Asia causing severe diarrhea and high mortality in neonatal piglets, indicating that a new generation of vaccine against porcine epidemic diarrhea virus (PEDV) is urgently needed. In the present study, to mimic the native spike (S) glycoprotein, a stable cell line producing the trimeric ectodomain of S glycoprotein of the PEDV Pintung-52 (PEDV-PT) strain was successfully established by incorporating T4 bacteriophage foldon sequence of fibritin trimerization domains at the C-terminal end and replacing the signal peptide of S protein with the tissue plasminogen activator signal peptide sequence at the N terminal end. The trimeric structure, bio-reactivity to PEDV-specific antibodies, and the N-glycosylation level of the recombinant S protein were characterized. To induce systemic and mucosal immunity, conventional 5-week-old piglets were immunized with the trimeric S glycoprotein combined with the B subunit of Escherichia coli heat-labile enterotoxin (LTB) by the intramuscular (IM) route. As compared with the control group, all piglets in the S protein-LTB immunized (IM PEDV S-LTB) group generated systemic PEDV S-specific IgG and neutralizing antibody in blood but a low level of fecal PEDV-specific IgA and limited protection against challenge of PEDV-PT strain. Our results suggest that the recombinant PEDV trimeric S glycoprotein could be a potential subunit vaccine candidate against PEDV, but IM immunization with LTB as the adjuvant provided insufficient protection. The development of a vaccine regimen for inducing mucosal immunity is an important task for generating a successful subunit vaccine against PEDVs.

Postgenomic Approaches and Bioinformatics Tools to Advance the Development of Vaccines against Bacteria of the Burkholderia cepacia Complex.

Bacteria of the Burkholderia cepacia complex (Bcc) remain an important cause of morbidity and mortality among patients suffering from cystic fibrosis. Eradication of these pathogens by antimicrobial therapy often fails, highlighting the need to develop novel strategies to eradicate infections. Vaccines are attractive since they can confer protection to particularly vulnerable patients, as is the case of cystic fibrosis patients. Several studies have identified specific virulence factors and proteins as potential subunit vaccine candidates. So far, no vaccine is available to protect from Bcc infections. In the present work, we review the most promising postgenomic approaches and selected web tools available to speed up the identification of immunogenic proteins with the potential of conferring protection against Bcc infections.