Specific Antibodies In Mice Research Articles

A nonadjuvanted HLA-restricted peptide vaccine induced both T and B cell immunity against SARS-CoV-2 spike protein

During COVID-19 pandemic, cases of postvaccination infections and restored SARS-CoV-2 virus have increased after full vaccination, which might be contributed to by immune surveillance escape or virus rebound. Here, artificial linear 9-mer human leucocyte antigen (HLA)-restricted UC peptides were designed based on the well-conserved S2 region of the SARS-CoV-2 spike protein regardless of rapid mutation and glycosylation hindrance. The UC peptides were characterized for its effect on immune molecules and cells by HLA-tetramer refolding assay for HLA-binding ability, by HLA-tetramer specific T cell assay for engaged cytotoxic T lymphocytes (CTLs) involvement, by HLA-dextramer T cell assay for B cell activation, by intracellular cytokine release assay for polarization of immune response, Th1 or Th2. The specific lysis activity assay of T cells was performed for direct activation of cytotoxic T lymphocytes by UC peptides. Mice were immunized for immunogenicity of UC peptides in vivo and immunized sera was assay for complement cytotoxicity assay. Results appeared that through the engagement of UC peptides and immune molecules, HLA-I and II, that CTLs elicited cytotoxic activity by recognizing SARS-CoV-2 spike-bearing cells and preferably secreting Th1 cytokines. The UC peptides also showed immunogenicity and generated a specific antibody in mice by both intramuscular injection and oral delivery without adjuvant formulation. In conclusion, a T-cell vaccine could provide long-lasting protection against SARS-CoV-2 either during reinfection or during SARS-CoV-2 rebound. Due to its ability to eradicate SARS-CoV-2 virus-infected cells, a COVID-19 T-cell vaccine might provide a solution to lower COVID-19 severity and long COVID-19.

Double-stranded RNAs are promising adjuvants for enhancing immunogenicity of vaccines

Background. The most effective way to prevent infectious diseases is vaccination. Adjuvants contribute to the optimization of the immune response of vaccines. Double-stranded ribonucleic acids (dsRNAs) from natural sources are promising, but insufficiently studied adjuvants.
 The aim of the work was to study the adjuvant activity of dsRNA obtained from the killer strain of Saccharomyces cerevisiae using two models of induction of a specific immune response.
 Materials and methods. In the experiments, the substance of the drug Ridostin containing dsRNA, 21.72% (produced by Institute of Medical Biotechnology of the State Research Center of Virology and Biotechnology Vector), was used. A specific immune response was modeled using ovalbumin (OVA) or the substance of the EpiVacCorona vaccine (EVC). The experiments were carried out in 200 female BALB/c mice. Mice of the experimental groups were injected twice with antigen and adjuvant together with a 28-day interval, mice of the comparison group with antigen only. On the 10th day after the second immunization, blood samples were collected to determine the level of specific antibodies using enzyme immunoassay. The results were evaluated by calculation of the average geometric titers of specific antibodies against OVA or EVC.
 Results. OVA or EVC administered twice induced the specific antibodies in mice in dose-dependent titers. The combined administration of antigen and dsRNA increased the strength of the immune response. The highest stimulating effect of dsRNA was observed in the dose of 100 g/mouse administered into mice immunized with OVA (1 g/mouse) or in the dose of 50 g/mouse in mice immunized with EVC substance (0.25 of a human dose per mouse).
 Conclusion. The data obtained indicate that the substance of dsRNA exerts adjuvant properties, which gives reason to consider dsRNA as a promising adjuvant for peptide vaccines.

Immunogenicity and protective potential of chimeric virus-like particles containing SARS-CoV-2 spike and H5N1 matrix 1 proteins.

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has posed a constant threat to human beings and the world economy for more than two years. Vaccination is the first choice to control and prevent the pandemic. However, an effective SARS-CoV-2 vaccine against the virus infection is still needed. This study designed and prepared four kinds of virus-like particles (VLPs) using an insect expression system. Two constructs encoded wild-type SARS-CoV-2 spike (S) fused with or without H5N1 matrix 1 (M1) (S and SM). The other two constructs contained a codon-optimized spike gene and/or M1 gene (mS and mSM) based on protein expression, stability, and ADE avoidance. The results showed that the VLP-based vaccine could induce high SARS-CoV-2 specific antibodies in mice, including specific IgG, IgG1, and IgG2a. Moreover, the mSM group has the most robust ability to stimulate humoral immunity and cellular immunity than the other VLPs, suggesting the mSM is the best immunogen. Further studies showed that the mSM combined with Al/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges with mouse-adapted strain. The vaccine based on mSM and Al/CpG adjuvant is a promising candidate vaccine to prevent the COVID-19 pandemic.

Oral Immunization with Lactobacillus casei Expressing the Porcine Circovirus Type 2 Cap and LTB Induces Mucosal and Systemic Antibody Responses in Mice.

Porcine circovirus type 2 (PCV2) causes many diseases in weaned piglets, leading to serious economic losses to the pig industry. This study investigated the immune response following oral administration of Lactobacillus casei ATCC393 (L. casei 393) expressing PCV2 capsid protein (Cap) fusion with the Escherichia coli heat-labile toxin B subunit (LTB) in mice. Recombinant L. casei strains were constructed using plasmids pPG611.1 and pPG612.1. The expression and localization of proteins from recombinant pPG611.1-Cap-LTB (pPG-1-Cap-LTB)/L. casei 393 and pPG612.1-Cap-LTB (pPG-2-Cap-LTB)/L. casei 393 were detected. All recombinant strains were found to be immunogenic by oral administration in mice and developed mucosal and systemic immune responses against PCV2. The titers of specific antibodies in mice administered pPG-2-Cap-LTB/L. casei 393 were higher than those in mice administered pPG-1-Cap-LTB/L. casei 393 in serum and the mucosal samples. The mucosal immune response was not only limited to the gastrointestinal tract but was also generated in other mucosal parts. Thus, the application of recombinant L. casei could aid in vaccine development for PCV2.

Development of a Vaccine against SARS-CoV-2 Based on the Receptor-Binding Domain Displayed on Virus-Like Particles.

The ongoing coronavirus disease (COVID-19) pandemic is caused by a new coronavirus (severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)) first reported in Wuhan City, China. From there, it has been rapidly spreading to many cities inside and outside China. Nowadays, more than 110 million cases with deaths surpassing 2 million have been recorded worldwide, thus representing a major health and economic issues. Rapid development of a protective vaccine against COVID-19 is therefore of paramount importance. Here, we demonstrated that the recombinantly expressed receptor-binding domain (RBD) of the spike protein can be coupled to immunologically optimized virus-like particles derived from cucumber mosaic virus (CuMVTT). The RBD displayed CuMVTT bound to ACE2, the viral receptor, demonstrating proper folding of RBD. Furthermore, a highly repetitive display of the RBD on CuMVTT resulted in a vaccine candidate that induced high levels of specific antibodies in mice, which were able to block binding of the spike protein to ACE2 and potently neutralize SARS-CoV-2 virus in vitro.

Identification of a potential neutralizing linear epitope of hemagglutinin-neuraminidase in Newcastle disease virus

BackgroundThe hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) is a major antigen that can induce protective antibodies in poultry. However, its antigenic epitopes have not been fully elucidated. Therefore, defining the linear epitopes of HN, especially neutralizing epitopes, will be useful for revealing its antigenic characterization.MethodsIn this study, we analyzed B-cell immunodominant epitopes (IDEs) of the HN protein from the vaccine strain LaSota using pepscan technology with LaSota-specific chicken hyperimmune antisera. We constructed IDEs-RFP plasmids and prepared anti-IDEs peptide mouse sera to identify IDEs through immunological tests. At last, the different diluted anti-IDE antisera were used in BHK-21 cells to perform the neutralization test.ResultsFive IDEs of the HN were screened and further verified by indirect immunofluorescence assays, dot blots and Western blots with NDV- and IDEs-specific antisera. All five IDEs showed good immunogenicity. IDE5 (328–342 aa) could recognize only class II NDV but did not react with the class I strain. Most of the IDEs are highly conserved among the different strains. A neutralization test in vitro showed that the peptide-specific mouse antisera of IDE4 (242–256 aa) and HN341-355, a reported neutralizing linear epitope, could partially neutralize avirulent LaSota as well as virulent strains at similar levels, suggesting that IDE4 might be a potential neutralizing linear epitope.ConclusionThe HN protein is a major protective antigen of NDV that can induce neutralizing antibodies in animals. We identified five IDEs of the HN using a pepscan approach with NDV-specific chicken hyperimmune antisera. The five IDEs could elicit specific antibodies in mice. IDE4 (242–256 aa) was identified as a novel potential neutralizing linear epitope. These results will help elucidate the antigenic epitopes of the HN and facilitate the development of NDV vaccines.

The effect of apolipoprotein A-I and perfluorocarbon emulsion on the production of specific antibodies in mice.

Adjuvants are often required to improve the potentially low immunogenicity of vaccines. In this study, it is proposed to use an emulsion based on fluorocarbons as an adjuvant. Since this emulsion adsorbs only a small range of proteins, apolipoprotein A-I (ApoAI) was used as an anchor. Antigen and ApoAI were combined by creating a fusion construct. Results showed that the combined use of a perfluorocarbon emulsion and ApoAI during immunization significantly increases the specific antibody titer in mice and in its effectiveness this system is close to the incomplete Freund's adjuvant.

Priming with FLO8-deficient Candida albicans induces Th1-biased protective immunity against lethal polymicrobial sepsis

The morphological switch between yeast and hyphae of Candida albicans is essential for its interaction with the host defense system. However, the lack of understanding of host–pathogen interactions during C. albicans infection greatly hampers the development of effective immunotherapies. Here, we found that priming with the C. albicans FLO8-deficient (flo8) mutant, locked in yeast form, protected mice from subsequent lethal C. albicans infection. Deficiency of Dectin-2, a fungus-derived α-mannan recognition receptor, completely blocked flo8 mutant-induced protection. Mechanistically, the flo8 mutant-induced Dectin-2/CARD9-mediated IL-10 production in DCs and macrophages to block thymus atrophy by inhibiting the C. albicans-induced apoptosis of thymic T cells, which facilitated the continuous output of naive T cells from the thymus to the spleen. Continuous recruitment of naive T cells to the spleen enhanced Th1-biased antifungal immune responses. Consequently, depletion of CD4+ T cells or blockade of IL-10 receptor function using specific antibodies in mice completely blocked the protective effects of flo8 mutant priming against C. albicans infection. Moreover, mannans exposed on the surface of the flo8 mutant were responsible for eliciting protective immunity by inhibiting the C. albicans-induced apoptosis of thymic T cells to sustain the number of naive T cells in the spleen. Importantly, priming with the flo8 mutant extensively protected mice from polymicrobial infection caused by cecal ligation and puncture (CLP) by enhancing Th1-biased immune responses. Together, our findings imply that targeting FLO8 in C. albicans elicits protective immune responses against polymicrobial infections and that mannans extracted from the flo8 mutant are potential immunotherapeutic candidate(s) for controlling infectious diseases.

Requirement for memory B-cell activation in protection from heterologous influenza virus reinfection

While two memory compartments, memory B cells and long-lived plasma cells, are thought to contribute to the successful establishment of memory recall responses, the unique roles of each cellular compartment are still unclear. Herein, by tracing influenza anti-hemagglutinin (HA)-specific antibodies in mice, we demonstrate that pre-existing antibodies secreted by long-lived plasma cells are essential for protection from reinfection with the same influenza virus, whereas protection from secondary infection with an antigenically distinct influenza virus requires memory B-cell activation. These activated memory B cells were largely specific for the conserved HA stem region, and generated sufficient levels of antibodies for protection from heterologous reinfection. Given that the anti-stem plasmablasts derived from the memory B cells were higher affinity than those from naive B cells, our results suggest that maturation of anti-stem memory B cells during primary influenza infection and their subsequent activation are required for protection from reinfection by mutant viruses.

A Live Salmonella Vaccine Delivering PcrV through the Type III Secretion System Protects against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common Gram-negative opportunistic pathogen that is intrinsically resistant to a wide range of antibiotics. The development of a broadly protective vaccine against P. aeruginosa remains a major challenge. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express P. aeruginosa antigens. A fusion between the S. enterica type III secretion effector protein SseJ and the P. aeruginosa antigen PcrV expressed under the control of the sseA promoter was translocated by Salmonella into host cells in vitro and elicited the generation of specific antibodies in mice. Mice immunized with attenuated Salmonella expressing this fusion had reduced bacterial loads in the spleens and lungs and lower serum levels of proinflammatory cytokines than control mice after P. aeruginosa infection. Importantly, immunized mice also showed significantly enhanced survival in this model. These results suggest that type III secretion effectors of S. enterica are appropriate carriers in the design of a live vaccine to prevent infections caused by P. aeruginosaIMPORTANCE The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic pathogen that causes infections in cystic fibrosis and hospitalized patients. Therapeutic treatments are limited due to the emergence and spread of new antibiotic-resistant strains. In this context, the development of a vaccine is a priority. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express and deliver the Pseudomonas antigen PcrV. This vaccine induced the generation of specific antibodies in mice and protected them from lethal infections with P. aeruginosa This is an important step toward the development of an effective vaccine for the prevention of infections caused by P. aeruginosa in humans.

Molecular epidemiology and expression of capsular polysaccharides in Staphylococcus aureus clinical isolates in the United States.

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors under evaluation as vaccine antigens. Clinical S. aureus isolates have the biosynthetic capability to express either CP5 or CP8 and an understanding of the relationship between CP genotype/phenotype and S. aureus epidemiology is valuable. Using whole genome sequencing, the clonal relatedness and CP genotype were evaluated for disease-associated S. aureus isolates selected from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T) to represent different geographic regions in the United States (US) during 2004 and 2009–10. Thirteen prominent clonal complexes (CC) were identified, with CC5, 8, 30 and 45 representing >80% of disease isolates. CC5 and CC8 isolates were CP type 5 and, CC30 and CC45 isolates were CP type 8. Representative isolates from prevalent CC were susceptible to in vitro opsonophagocytic killing elicited by anti-CP antibodies, demonstrating that susceptibility to opsonic killing is not linked to the genetic lineage. However, as not all S. aureus isolates may express CP, isolates representing the diversity of disease isolates were assessed for CP production. While approximately 35% of isolates (primarily CC8) did not express CP in vitro, CP expression could be clearly demonstrated in vivo for 77% of a subset of these isolates (n = 20) despite the presence of mutations within the capsule operon. CP expression in vivo was also confirmed indirectly by measuring an increase in CP specific antibodies in mice infected with CP5 or CP8 isolates. Detection of antigen expression in vivo in relevant disease states is important to support the inclusion of these antigens in vaccines. Our findings confirm the validity of CP as vaccine targets and the potential of CP-based vaccines to contribute to S. aureus disease prevention.

Molecular characterization of Babesia microti seroreactive antigen 5-1-1 and development of rapid detection methods for anti-B. microti antibodies in serum

Babesiosis has become a new global threat impacting human health, and most human babesiosis cases are caused by Babesia microti. Until now few antigens of B. microti have been described which can be used for the diagnosis of human babesiosis. In the present study, we report on the bioinformatic analysis, cloning and expression of the sequence encoding the B. microti seroreactive antigen 5-1-1 to investigate its potential incorporation in serologic diagnostic tools for babesiosis. Bioinformatic analysis and recombinant gene expression were performed to molecularly characterize seroreactive antigen 5-1-1. Enhanced chemiluminescence (ECL)-Western blot methods were used to detect specific antibodies in infected mice. Immunofluorescence antibody assays (IFA) were performed to detect the localization of BmSA5-1-1 in B. microti parasites. ELISA and immunochromatographic (ICT) tests were developed using recombinant BmSA5-1-1 to evaluate its potential use in rapid detection methods for B. microti antibodies and for the diagnosis of babesiosis. A recombinant expression plasmid was constructed by inserting the target gene fragment in the pET28a vector after double digestion with BamHI and XhoI restriction enzymes. The recombinant BmSA5-1-1 protein was expressed in Escherichia coli (rBmSA5-1-1) and purified by means of Ni-nitrilotriacetic acid (NTA) agarose columns. Polyclonal antibodies were generated against rBmSA5-1-1. Based on indirect immunofluorescence assay results, BmSA5-1-1 appeared to localize on the surface of B. microti. ELISA tests using the rBmSA5-1-1 antigen detected specific antibodies from infected mice as early as 4 days post-infection. Our results indicate that the two methods we developed can detect specific antibodies in mice at different stages of infection with sensitivities of 100% (rBmSA5-1-1 ELISA) and 90% (ICT). The specificity of the two methods was 100%. Sera of patients suffering from other closely related parasitic diseases, such as malaria and toxoplasmosis, produced negative results. In conclusion, seroreactive antigen 5-1-1, a member of the BMN1 protein family, is expressed on the outer surface of B. microti and is a promising candidate antigen for the early diagnosis of babesiosis. rBmSA5-1-1 ELISA and ICT methods show good potential for detecting specific antibodies in mice at different stages of infection.

Characterization of Pb51 in Plasmodium berghei as a malaria vaccine candidate targeting both asexual erythrocytic proliferation and transmission

BackgroundA vaccine that targets multiple developmental stages of malaria parasites would be an effective tool for malaria control and elimination.MethodsA conserved gene in Plasmodium, the Plasmodium berghei gene (PBANKA_020570) encoding a 51 kDa protein (pb51 gene), was identified through search of the PlasmoDB database using a combination of expression and protein localization criteria. A partial domain of the Pb51 protein was expressed in a prokaryotic expression system (rPb51) and used for immunization in mice. The protein expression profile and localization were studied by Western blot and indirect immunofluorescence assay (IFA), respectively. The inhibitory effect of the anti-rPb51 antibodies on parasite proliferation was evaluated in erythrocytes in vivo. The transmission-blocking activity of the immune sera was determined by in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA).ResultsThe rPb51 elicited specific antibodies in mice. Western blot confirmed Pb51 expression in schizonts, gametocytes and ookinetes. IFA showed localization of Pb51 on the outer membranes of schizonts, gametocytes, zygotes, retorts, ookinetes and sporozoites of P. berghei. Mice immunized with the rPb51 protein significantly reduced parasite proliferation and gametocyte conversion in vivo. Moreover, the rPb51 antisera also significantly reduced the in vitro ookinete conversion when added into the ookinete culture medium. In DFA, mice immunized with the rPb51 reduced the prevalence of mosquito infection by 21.3% and oocyst density by 54.8%.ConclusionsIn P. berghei, P51 was expressed in both asexual erythrocytic and sexual stages and localized on the surface of these stages with the exception of the ring stage. The anti-rPb51 antibodies inhibited both P. berghei proliferation in mice and transmission of the parasite to mosquitoes.

Evaluation of antibody response against recombinant domain III proteins of dengue virus type 1 and 2.

Dengue, a mosquito borne viral disease caused by dengue virus has emerged as a major health problem during the last few decades. The envelope domain III (DIII) protein of dengue virus is highly immunogenic and capable of inducing neutralizing antibodies against wild-type dengue virus. The envelope domain III protein is a potential subunit vaccine candidate as well as a diagnostic reagent for dengue. This report describes the high yield production and immunogenicity of recombinant DIII proteins of dengue virus type 1 and 2. The subunit DIII proteins were produced in Escherichia coli using batch and fed-batch fermentation process. Immobilized metal affinity chromatography was used to capture DIII proteins of dengue virus type 1 and 2. The purified proteins were refolded by diafiltration to achieve biologically active proteins. After fed-batch fermentation, the recombinant E. coli resulted in purified DIII proteins of about 10.06 mg and 47.70 mg per gram of dry cell weight for recombinant dengue virus type 1 and 2 respectively with more than 95% purity. Biological function of the purified DIII proteins were confirmed by their ability to generate DIII specific antibodies in mice. The DIII antigens in combination with adjuvant resulted antibody endpoint titers of 1:64,000 and 1:1,28,000 for recombinant dengue virus type 1 and 2 respectively. These findings establish that the DIII proteins in combination with adjuvant are immunogenic, which suggests that refolded and purified DIII proteins can be a potential vaccine candidates.

Humoral Immune Response in Mice Elicited by Combined Yeast-Derived Hepatitis B Virus Core, Surface, and Mosaic Surface Antigens

Background: Although successful, the second-generation hepatitis B vaccine programs around the world have a small group of immunized individuals that does not respond efficiently to the vaccination. Other issues of these vaccines are individuals that are low or nonresponders and/or have incomplete protection against heterologous hepatitis B virus (HBV) genotypes/subtypes and against HBV escape mutants. In addition, there are approximately 240 million people chronically infected with HBV worldwide and 620,000 deaths per year caused by the infection. Methods: In this study we developed three Hansenula polymorpha plasmids containing the following sequences: (a) HBsAg subtype ayw, (b) HBcAg sequence subtype adw2, and (c) chimeric HBsAg (adw4/ ayw) - preS1 (adw2) - 3 repetitions of preS2 (genotypes A, B, and C). The sequences were successfully expressed and the antigens purified. Using Balb/c mice the antigens were tested in different dosage combinations. Results: Three antigens were obtained at a high purity level and with high reproducibility. We also assessed their immunogenic properties, showing that the antigens, individually or in combination, generated anti-HBs, anti-preS1, anti-preS2, and anti-HBc antibodies efficiently in mice. Conclusions: The formulation tests showed that a combination of 0.02 μg of HBs, 0.2 μg of preS1-preS2-HBs, and 0.02 μg of HBc was effective in eliciting specific antibodies in mice.

Protective antibodies against a sphingomyelinase D from Loxosceles intermedia spider venom elicited in mice with different genetic background

In the present investigation we used a recombinant LiD1 toxin, named rLiD1his, from Loxosceles intermedia brown spider to elicit specific antibodies in mice carrying different Human Leukocyte Antigens class II (HLAII) {DRB1.0401 (DR4), DQB1.0601 (DQ6) and DQB1.0302 (DQ8)} as well as in BALB/C and C57BL/6 control mice. All mice strains produced high antibody titers against rLiD1his but DR4 mice antibodies (the lower responder mice) were not able to recognize L. intermedia crude venom. The anti-rLiD1his sera, except from DR4 mice, were able to neutralize dermonecrotic, hemorrhagic and edematogenic effects of rLiD1his in naïve rabbits. Overlapping peptides from the amino acid sequence of LiD1 toxin were prepared by SPOT method and differences in LiD1 epitope recognition were observed using different mice anti-rLiD1his sera. The region 160DKVGHDFSGNDDISDVGK177 was recognized by transgenic DQ8 and DQ6 mice sera. Other epitopes were recognized by at least two different animals’ sera including 10MGHMVNAIGQIDEFVNLG27, 37FDDNANPEYTYHGIP51, 70GLRSATTPGNSKYQEKLV87 and 259AAYKKKFRVATYDDN273. Among these epitopes, the epitopes 37–51 and 160–177 have already been shown in previously studies as good candidates to be used alone or combined with other peptides to induce protective immune response against Loxosceles venoms. The results presented here highlight the importance of HLAII in antibody response and recognition of specific B-cell epitopes of rLiD1his spider toxin according to HLAII type and impact in the epitopic vaccine development against this spider.

Characterization of an Alpha Type Carbonic Anhydrase from Paracentrotus lividus Sea Urchin Embryos

Carbonic anhydrases (CA) are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the sea urchin, CA has a role in the formation of the calcitic skeleton during embryo development. Here, we report a newly identified mRNA sequence from embryos of the sea urchin Paracentrotus lividus, referred to as Pl-can. The complete coding sequence was identified with the aid of both EST databases and experimental procedures. Pl-CAN is a 447 aa-long protein, with an estimated molecular mass of 48.5kDa and an isoelectric point of 6.83. The in silico study of functional domains showed, in addition to the alpha type CA-specific domain, the presence of an unexpected glycine-rich region at the N-terminal of the molecule. This is not found in any other species described so far, but probably it is restricted to the sea urchins. The phylogenetic analysis indicated that Pl-CAN is evolutionarily closer to human among chordates than to other species. The putative role(s) of the identified domains is discussed. The Pl-can temporal and spatial expression profiles, analyzed throughout embryo development by comparative qPCR and whole-mount in situ hybridization (WMISH), showed that Pl-can mRNA is specifically expressed in the primary mesenchyme cells (PMC) of the embryo and levels increase along with the growth of the embryonic skeleton, reaching a peak at the pluteus stage. A recombinant fusion protein was produced in E. coli and used to raise specific antibodies in mice recognized the endogenous Pl-CAN by Western blot in embryo extracts from gastrula and pluteus.

Negatively Charged Carbon Nanohorn Supported Cationic Liposome Nanoparticles: A Novel Delivery Vehicle for Anti-Nicotine Vaccine.

Tobacco addiction is the second-leading cause of death in the world. Due to the nature of nicotine (a small molecule), finding ways to combat nicotine's deleterious effects has been a constant challenge to the society and the medical field. In the present work, a novel anti-nicotine vaccine based on nanohorn supported liposome nanoparticles (NsL NPs) was developed. The nano-vaccine was constructed by using negatively charged carbon nanohorns as a scaffold for the assembly of cationic liposomes, which allow the conjugation of hapten conjugated carrier proteins. The assembled bio-nanoparticles are stable. Mice were immunized subcutaneously with the nano-vaccine, which induced high titer and high affinity of nicotine specific antibodies in mice. Furthermore, no evidence of clinical signs or systemic toxicity followed multiple administrations of NsL-based anti-nicotine vaccine. These results suggest that NsL-based anti-nicotine vaccine is a promising candidate in treating nicotine dependence and could have potential to significantly contribute to smoking cessation.

Expression and immunogenic characterization of recombinant gp350 for developing a subunit vaccine against Epstein-Barr virus.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is linked to the development of various malignancies. There is an urgent need for effective vaccines against EBV. EBV envelope glycoprotein gp350 is an attractive candidate for a prophylactic vaccine. This study was undertaken to produce the truncated (codons 1-443) gp350 protein (gp350(1-443)) in Pichia pastoris and evaluate its immunogenicity. The gp350(1-443) protein was expressed as a secretory protein with an N-terminal His-tag in P. pastoris and purified through Ni-NTA chromatography. Immunization with the recombinant gp350(1-443) could elicit high levels of gp350(1-443)-specific antibodies in mice. Moreover, gp350(1-443)-immunized mice developed strong lymphoproliferative and Th1/Th2 cytokine responses. Furthermore, the recombinant gp350(1-443) could stimulate CD4(+) and CD8(+) T cell responses in vaccinated mice. Collectively, these findings demonstrated that the yeast-expressed gp350(1-443) retained strong immunogenicity. This study will provide a useful source for developing EBV subunit vaccine candidates.

An Env-derived multi-epitope HIV chimeric protein produced in the moss Physcomitrella patens is immunogenic in mice.

The first report on the recombinant production of a candidate vaccine in the moss system. The need for economical and efficient platforms for vaccine production demands the exploration of emerging host organisms. In this study, the production of an antigenic protein is reported employing the moss Physcomitrella patens as an expression host. A multi-epitope protein from the Human Immunodeficiency Virus (HIV) based on epitopes from gp120 and gp41 was designed as a candidate subunit vaccine and named poly-HIV. Transgenic moss plants were generated carrying the corresponding poly-HIV transgene under a novel moss promoter and subsequently seven positive lines were confirmed by PCR. The poly-HIV protein accumulated up to 3.7µgg(-1) fresh weight in protonema cultures. Antigenic and immunogenic properties of the moss-produced recombinant poly-HIV are evidenced by Western blots and by mice immunization assays. The elicitation of specific antibodies in mice was observed, reflecting the immunogenic potential of this moss-derived HIV antigen. This is the first report on the production of a potential vaccine in the moss system and opens the avenue for glycoengineering approaches for the production of HIV human-like glycosylated antigens as well as other vaccine prototypes under GMP conditions in moss bioreactors.