Induction Of Specific Antibodies Research Articles

Comparison of 3 kinds of Toll-like receptor ligands for inactivated avian H5N1 influenza virus intranasal immunization in chicken

To evaluate the effects of co-administration of inactivated avian influenza H5N1 virus (IAIV) and different Toll-like receptor (TLR) ligands in chickens, 10-d-old chickens were immunized intranasally with IAIV and TLR ligand [Bacillus subtilis spores, polyinosinic-polycytidylic acid, and CpG oligodeoxynucleotides (CpG-ODN), respectively]. The results showed that both anti-avian influenza virus (AIV) specific secretory IgA level in respiratory tract and anti-AIV specific IgG level in serum significantly increased, as well as the expressions of IL-12, interferon-γ, IL-6, and TLR in the nasal cavity and trachea after intranasal immunization with IAIV and TLR ligand. Among the used TLR ligands, B. subtilis spores as the adjuvant for nasal IAIV had the strongest effect on the expression of IL-6 and IL-12 (P < 0.01), whereas the CpG-ODN could present an advantageous effect on the induction of anti-AIV specific IgG and neutralization antibodies (P < 0.01). The chickens that were previously co-administrated with IAIV and B. subtilis spores could survive at an improved rate upon challenge by live AIV H5N1 virus. Our study suggested that B. subtilis spores, polyinosinic-polycytidylic acid, or CpG-ODN all could effectively enhance the local and systemic immune responses to IAIV in chickens. Considering of the effects and cost of these TLR ligands, we prospected that B. subtilis spores might serve as a more affordable and efficacious mucosal adjuvant for nasal IAIV in chickens.

Salivary IgA enhancement strategy for development of a nasal-spray anti-caries mucosal vaccine

Dental caries remains one of the most common global chronic diseases caused by Streptococcus mutans, which is prevalent all over the world. The caries prevalence of children aged between 5-6 years old in China is still in very high rate. A potent and effective anti-caries vaccine has long been expected for caries prevention but no vaccines have been brought to market till now mainly due to the low ability to induce and maintain protective antibody in oral fluids. This review will give a brief historical retrospect on study of dental caries and pathogenesis, effective targets for anti-caries vaccines, oral immune system and immunization against dental caries. Then, salivary IgA antibodies and the protective responses are discussed in the context of the ontogeny of mucosal immunity to indigenous oral streptococcal. The methods and advancement for induction of specific anti-caries salivary sIgA antibodies and enhancement of specific anti-caries salivary sIgA antibodies by intranasal immunization with a safe effective mucosal adjuvant are described. The progress in the enhancement of salivary sIgA antibodies and anti-caries protection by intranasal immunization with flagellin-PAc fusion protein will be highlighted. Finally, some of the main strategies that have been used for successful mucosal vaccination of caries vaccine are reviewed, followed by discussion of the mucosal adjuvant choice for achieving protective immunity at oral mucosal membranes for development of a nasal-spray or nasal-drop anti-caries vaccine for human.

Induction of prominent Th1 response in C57Bl/6 mice immunized with an E.coli-expressed multi T-cell epitope EgA31 antigen against Echinococcus granulosus

First step in developing an epitope-based vaccine is to predict peptide binding to the major histocompatibility complex (MHC) molecules. We performed computational analysis of unique available EgA31 sequence to locate appropriate antigenic propensity positions. T-cell epitopes with best binding affinity values of < 50% inhibitory concentration were selected using different available servers (Propred and IEDB). Peptides with 100% population coverage were selected. A DNA fragment corresponding to the furin linker enriched in Golgi apparatus was inserted sequentially between each epitope sequences in a synthetic DNA in order to cleave the chimeric protein into four separated peptides. Subsequently, the synthetic DNA was cloned into the pGEX4T-1 and pEGFP-N1 vectors and GST-ChEgA31 was expressed in E. coli strain BL21-DE3. The recombinant protein was detected by western blotting using an HRP-conjugated polyclonal anti-GST antibody. Fusion protein purified by affinity chromatography was used to raise antisera in rabbits. Results in agar gel immunodiffusion assay indicated induction of specific antibodies against multiepitope antigen in the tested rabbits. Cytokine assay was carried out in C57Bl/6 mice and the levels of cytokines were analyzed by sandwich ELISA. Interestingly, production of specific IFN-gamma was prominently higher in mice immunized with GST-ChEgA31 and pEGFP-ChEgA31 (650-1300 pg/ml) compared to control groups. No difference was observed in the level of IL-10 and IL-4 in immunized and GST control group. Challenge study with 500 live protoscolices of Echinococcus granulosus on immunized mice demonstrated protectivity level (50-60%). Based on our results, it appeared that the chimeric protein in the study was able to stimulate T-helper cell-1 (Th1) development and high level of cell mediated immunity in mice.

A cucumber mosaic virus based expression system for the production of porcine circovirus specific vaccines.

Potential porcine circovirus type 2 (PCV2) capsid protein epitopes, suitable for expression on the surface of cucumber mosaic virus (CMV) particles were determined by a thorough analysis of the predicted PCV capsid protein structure. The ab initio protein structure prediction was carried out with fold recognition and threading methods. The putative PCV epitopes were selected on the basis of PCV virion models and integrated into the plant virus coat protein, after amino acid position 131. The recombinants were tested for infectivity and stability on different Nicotiana species and stable recombinant virus particles were purified. The particles were tested for their ability to bind to PCV induced porcine antibodies and used for specific antibody induction in mice and pigs. The results showed that PCV epitopes expressed on the CMV surface were recognized by the porcine antibodies and they were also able to induce PCV specific antibody response. Challenge experiment with PCV2 carried out in immunized pigs showed partial protection against the infection. Based on these results it was concluded that specific antiviral vaccine production for the given pathogen was feasible, offering an inexpensive way for the mass production of such vaccines.

Vector replication and expression of HIV-1 antigens by the HIV/AIDS vaccine candidate MVA-B is not affected by HIV-1 protease inhibitors

MVA-B is an attenuated poxvirus vector expressing human immunodeficiency virus type 1 Env, Gag, Pol, and Nef antigens from clade B, and is considered a promising HIV/AIDS vaccine candidate. Recently, a phase I clinical trial in human healthy volunteers has shown that MVA-B is safe and highly immunogenic, inducing broad, polyfunctional, and long-lasting CD4+ and CD8+ T cell responses to HIV-1 antigens, with preference for effector memory T cells; and it also triggers the induction of specific antibodies to Env in most of the vaccines. While MVA recombinants expressing HIV-1 antigens are being used or plan to use in therapeutic clinical trials, little is known on the effect of HIV-1 highly active antiretroviral therapy in MVA life cycle. To define this role, here we have evaluated in established cell cultures and human dendritic cells to what extent different HIV-1 protease inhibitors affect virus replication and expression of HIV-1 antigens during MVA-B infection. The results obtained revealed that the most commonly used HIV-1 protease inhibitors (atazanavir, ritonavir, and lopinavir) had no effect on MVA-B virus growth kinetics, even at higher concentrations than those normally used on HAART. Furthermore, expression of gp120 and the fused Gag-Pol-Nef polyprotein in permissive and non-permissive cells infected with MVA-B were also not affected. These findings are relevant information for the therapeutic use of MVA-B as an HIV-1/AIDS vaccine.

Anti-Group B streptococci Ib antibodies protect against invasive aspergillosis. (166.9)

Abstract Aspergillus fumigatus (A.f.) the cause of invasive Aspergillosis (IA) has become a leading cause of death in immunosuppressed populations, due to the lack of effective therapies or vaccines. Studies using anti-A.f. monoclonal antibodies indicate that they can protect against IA. However, the lack of detailed information on the antigenic targets of protective anti-A.f. antibodies has hampered the development of an effective anti-A.f. vaccine. Sialylated epitopes found on a variety of pathogens including fungi and Group B Streptococci (GBS) are thought to be major virulence factors and are involved in attachment to host cells, uptake by phagocytes, and complement activation. Sialylated epitopes on A.f. may therefore provide a potential target for new therapies. Our preliminary data show that a purified, calcium-dependent GBSIb type-specific antibody, SMB19 (IgM,κ) also binds to A.f. and prolongs survival in a murine model of IA. Similarly, immunization of wild type and SMB19 VH bearing immunoglobulin transgenic mice with a GBSIb vaccine also provides protection. These results support our hypothesis that induction of GBSIb specific antibodies, perhaps supplemented with antifungal drugs, will lead to new vaccines or passive antibody prophylaxis to prevent and treat IA.

Influence of immunization with non-genotoxic PAH–KLH conjugates on the resistance of organisms exposed to benzo( a)pyrene

Polycyclic aromatic hydrocarbons (PAH) are recognized as common environmental pollutants released into the environment from many natural as well as man-made sources, and some have been classified as potent carcinogens. The main representative of the carcinogenic PAH is benzo( a)pyrene (B( a)P) which is known to induce genotoxic effects in vitro and in vivo, detected as PAH–DNA adducts. Long-term PAH exposure may be accompanied by an immunological response with the formation of antibodies against PAH as well as against PAH–DNA adducts. This paper describes the use of four PAH–keyhole-limpet haemocyanin (KLH) conjugates for the induction of specific and cross-reactive anti-PAH antibodies and focuses on the potential protective effects of anti-PAH antibodies produced after immunization of mice. In the in vitro experiments with HepG-2 cells, the genotoxicity of the PAH–KLH conjugates and the neutralizing effect of induced anti-PAH antibodies were evaluated. The titer of specific anti-PAH antibodies in sera and the amounts of DNA adducts in liver homogenates from immunized mice were investigated in vivo. The results show that anti-PAH antibodies of class IgG were induced during immunization. All the PAH–KLH conjugates tested were non-genotoxic and did not induce detectable DNA adducts in HepG2 cells or in the liver of immunized mice. The results show that only B( a)P-specific and B( a)P cross-reactive antibodies are able to neutralize B( a)P or its activated metabolites, which was revealed by a sudden decrease in the titer of anti-B( a)P antibodies in mouse sera after exposure to B( a)P. Furthermore, the anti-B( a)P antibodies produced by immunization were effective in reducing the amount of DNA adducts in mouse livers after intraperitoneal ( i.p.) exposure to B( a)P. The results suggest that immunization with PAH–KLH conjugates can protect organisms against the adverse effects of carcinogenic PAH.

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6. Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15. The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity. Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method with some modifications. Compared with the lipid transfection method, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory. The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

Group B streptococci Ib monoclonal antibodies protect against invasive aspergillosis. (99.7)

Abstract Aspergillus fumigatus (A.f.) the cause of invasive Aspergillosis (IA) has become a leading cause of death in immunosuppressed populations, due to the lack of effective therapies or vaccines. Studies using anti-A.f. monoclonal antibodies indicate that they can protect against IA. However, the lack of detailed information on the antigenic targets of protective anti-A.f. antibodies has hampered the development of an effective anti-A.f. vaccine. It is known that sialylated epitopes found on a variety of pathogens including fungi and Group B Streptococci (GBS) are involved in attachment to host cells, uptake by phagocytes, and complement activation. A.f. sialylated epitopes may therefore provide a potential target for new therapies. Our preliminary data show that a purified, calcium-dependent GBSIb-specific antibody, SMB19 (IgM,κ) also binds to A.f. and provides a degree of protection in a murine model of IA. Likewise, immunization of wild type and SMB19 VH bearing immunoglobulin transgenic mice with a GBSIb vaccine also provides protection. These results support our hypothesis that induction of GBSIb specific antibodies, perhaps supplemented with antifungal drugs, will lead to new vaccines or passive antibody prophylaxis to prevent and treat IA.

LRET-Based Biodetection of DNA Release in Live Cells Using Surface-Modified Upconverting Fluorescent Nanoparticles

In this work, we demonstrate near-infrared-to-visible upconverting fluorescent nanoparticles as a promising platform for lanthanide-based or luminescence resonance energy transfer (LRET)-based biodetection of DNA release in live cells. Highly monodispersed, stable aqueous suspension of nanoparticles, surface-functionalized with amino groups for binding to DNA, were prepared and characterized. These amino-functionalized nanoparticles were able to electrostatically bind to DNA and protect it from DNaseI degradation as shown by gel electrophoresis. Attachment of DNA to the nanoparticles was also confirmed by LRET, which was observed to occur between the donor nanoparticle and acceptor POPO-3 dye intercalating the DNA. The intrinsic fluorescence property of upconverting fluorescent nanoparticles makes them useful for tracking their cellular localization without the use of any additional fluorescent tag. We were able to track the movement of these DNA-loaded nanoparticles into the cell cytoplasm where they successfully released their genetic cargo. Successful transfection of the loaded DNA material in vitro and in vivo was confirmed by expression of its encoded green fluorescent protein (GFP) in Hela cells and induction of specific antibody in mice, respectively.

The Role of TLR2 and Bacterial Lipoprotein in Enhancing Airway Inflammation and Immunity

Non-typeable Haemophilus influenzae (NTHI) colonizes the lower respiratory tract of patients with chronic obstructive pulmonary disease and also causes exacerbations of the disease. The 16-kDa lipoprotein P6 has been widely studied as a potential vaccine antigen due to its highly conserved expression amongst NTHI strains. Although P6 is known to induce potent inflammatory responses, its role in the pathogenesis of NTHI infection in vivo has not been examined. Additionally, the presence of an amino-terminal lipid motif on P6 serves to activate host Toll-like receptor 2 (TLR2) signaling. The role of host TLR2 and NTHI expression of the lipoprotein P6 on the induction of airway inflammation and generation of adaptive immune responses following chronic NTHI stimulation was evaluated with TLR2-deficient mice and a P6-deficient NTHI strain. Absence of either host TLR2 or bacterial P6 resulted in diminished levels of immune cell infiltration within lungs of mice exposed to NTHI. Pro-inflammatory cytokine secretion was also reduced in lungs that did not express TLR2 or were exposed to NTHI devoid of P6. Induction of specific antibodies to P6 was severely limited in TLR2-deficient mice. Although mice exposed to the P6-deficient NTHI strain were capable of generating antibodies to other surface antigens of NTHI, these levels were lower compared to those observed in mice exposed to P6-expressing NTHI. Therefore, cognate interaction between host TLR2 and bacterial P6 serves to enhance lung inflammation and elicit robust adaptive immune responses during NTHI exposure. Strategies to limit NTHI inflammation while simultaneously promoting robust immune responses may benefit from targeting the TLR2:P6 signaling axis.

Dendritic Cell Vaccination in Combination with Anti-CD25 Monoclonal Antibody Treatment: A Phase I/II Study in Metastatic Melanoma Patients

The success of cancer immunotherapy depends on the balance between effector T cells and suppressive immune regulatory mechanisms within the tumor microenvironment. In this study we investigated whether transient monoclonal antibody-mediated depletion of CD25(high) regulatory T cells (Treg) is capable of enhancing the immunostimulatory efficacy of dendritic cell vaccines. Thirty HLA-A2.1(+) metastatic melanoma patients were vaccinated with mature dendritic cells pulsed with tumor peptide and keyhole limpet hemocyanin (KLH). Half of the patients were pretreated with daclizumab, a humanized antibody against the interleukin-2 (IL-2) receptor α-chain (CD25), either four or eight days before dendritic cell vaccinations. Clinical and immunologic parameters were determined. Daclizumab efficiently depleted all CD25(high) immune cells, including CD4(+)FoxP3(+)CD25(high) cells, from the peripheral blood within four days of administration. Thirty days after administration, daclizumab was cleared from the circulation and all CD25(+) cells reappeared. The presence of daclizumab during dendritic cell vaccinations prevented the induction of specific antibodies in vivo but not the presence of antigen-specific T cells. Daclizumab, however, did prevent these CD25(+) T cells from acquiring effector functions. Consequently, significantly less patients pretreated with daclizumab developed functional, vaccine-specific effector T cells and antibodies compared with controls. Daclizumab pretreatment had no significant effect on progression-free survival compared with the control group. Although daclizumab depleted the CD4(+)FoxP3(+)CD25(high) Tregs from the peripheral circulation, it did not enhance the efficacy of the dendritic cell vaccine. Residual daclizumab functionally suppressed de novo induced CD25(+) effector cells during dendritic cell vaccinations. Our results indicate that for immunotherapeutic benefit of transient Treg depletion, timing and dosing as well as Treg specificity are extremely important.

Detoxified Haemophilus ducreyi cytolethal distending toxin and induction of toxin specific antibodies in the genital tract

Haemophilus ducreyi causes genital ulceration (chancroid), a sexually transmitted infection and still an important factor which contributes to the spread of HIV in developing countries. The bacterium produces a cytolethal distending toxin (HdCDT) causing cell cycle arrest and apoptosis/necrosis of human cells and contributes to the aggravation of ulcers. The aim of the study was to induce toxin-neutralizing antibodies in the genital tract of mice. Repeated subcutaneous (sc) immunisations with 5–10 μg active HdCDT induced low levels of serum anti-HdCDT IgG without neutralizing capacity. High levels of specific IgG1 antibodies in serum and genital tract were generated after sc immunisations with 10 μg formaldehyde detoxified HdCDT toxoid alone and the addition of aluminium salts or RIBI (based on the lipid A moiety) as adjuvant further increased the level of serum antibodies. A high correlation was found between elevated levels of anti-HdCDT IgG in sera, the level of neutralizing activity and the antibody level in genital tract ( r = 0.8). Thus, induction of high antibody levels specific to HdCDT in the genital tissue can be achieved by parenteral immunisation with the toxoid. The HdCDT toxoid can be considered as a candidate component in vaccine against chancroid.

Effects of the administration of cholera toxin as a mucosal adjuvant on the immune and protective response induced by Proteus mirabilis MrpA fimbrial protein in the urinary tract

Proteus mirabilis is commonly associated with complicated UTI and expresses several virulence factors, including MR/P fimbriae. In the present study mice were immunised nasally with MrpA, the structural subunit of MR/P, with or without CT as a mucosal adjuvant. The animals were then challenged with P. mirabilis and induction of specific serum and urine IgG and IgA, IFN-gamma production and bacterial kidney and bladder colonization were assessed. MrpA-immunised mice exhibited significant induction of serum IgA and urine IgA and IgG. MrpA/CT-immunised mice showed both significant serum and urine IgA and IgG production. Only this group showed significant IFN-y production. Both groups of animals had significant decrease in bacterial colonization of kidneys but not of bladders. No correlation between specific antibody induction in serum and CFU decrease was observed in any group of animals. Our results suggest that a mucosal adjuvant (CT) in the urinary tract enhanced humoral and cytokine response although it did not influence the degree of protection against UTI provided by MrpA. Further studies are necessary to understand immune modulation in the urinary tract.

HLA Class I–Associated Immunodominance Affects CTL Responsiveness to an ESO Recombinant Protein Tumor Antigen Vaccine

Vaccination with full-length human tumor antigens aims at inducing or increasing antitumor immune responses, including CD8 CTL in cancer patients across the HLA barrier. We have recently reported that vaccination with a recombinant tumor-specific NY-ESO-1 (ESO) protein, administered with Montanide and CpG resulted in the induction of specific integrated antibody and CD4 T cell responses in all vaccinated patients examined, and significant CTL responses in half of them. Vaccine-induced CTL mostly recognized a single immunodominant region (ESO 81-110). The purpose of the present study was to identify genetic factor(s) distinguishing CTL responders from nonresponders. We determined the HLA class I alleles expressed by CTL responders and nonresponders using high-resolution molecular typing. Using short overlapping peptides spanning the ESO immunodominant CTL region and HLA class I/ESO peptide tetramers, we determined the epitopes recognized by the majority of vaccine-induced CTL. CTL induced by vaccination with ESO protein mostly recognized distinct but closely overlapping epitopes restricted by a few frequently expressed HLA-B35 and HLA-Cw3 alleles. All CTL responders expressed at least one of the identified alleles, whereas none of the nonresponders expressed them. Expression of HLA-B35 and HLA-Cw3 is associated with the induction of immunodominant CTL responses following vaccination with recombinant ESO protein. Because recombinant tumor-specific proteins are presently among the most promising candidate anticancer vaccines, our findings indicate that the monitoring of cancer vaccine trials should systematically include the assessment of HLA association with responsiveness.

Expression of an Escherichia coli antigenic fusion protein comprising the heat labile toxin B subunit and the heat stable toxin, and its assembly as a functional oligomer in transplastomic tobacco plants

Enterotoxigenic Escherichia coli (ETEC) strains are important pathogens in developing countries. Some vaccine formulations containing the heat labile toxin B subunit (LTB) have been used in clinical trials; however, the induction of neutralizing antibodies against the heat-stable toxin (ST), a poor immunogenic peptide, is necessary, as most ETEC strains can produce both toxins. In this study, a plant optimized synthetic gene encoding for the LTB-ST fusion protein has been introduced into plastids of tobacco leaf tissues, using biolistic microprojectile bombardment, in an effort to develop a single plant-based candidate vaccine against both toxins. Transplastomic tobacco plants carrying the LTB-ST transgene have been recovered. Transgene insertion into the plastid was confirmed by both PCR and Southern blot analysis. GM1-ELISA revealed that the LTB-ST fusion protein retained its oligomeric structure, and displayed antigenic determinants for both LTB and ST. Western blot analysis, using LTB antisera, confirmed the presence of a 17-KDa protein in transplastomic lines, with the correct antigenicity of the fusion protein. Expression levels of this fusion protein in different lines reached up to 2.3% total soluble protein. Oral immunization of mice with freeze-dried transplastomic tobacco leaves led to the induction of both serum and mucosal LTB-ST specific antibodies. Following cholera toxin challenge, a decrease of intestinal fluid accumulation was observed in mice immunized with LTB-ST-containing tobacco. These findings suggest that tobacco plants expressing LTB-ST could serve as a plant-based candidate vaccine model providing broad-spectrum protection against ETEC-induced diarrhoeal disease.

Production of antibodies to Little cherry virus 1 coat protein by DNA prime and protein boost immunization

Little cherry, an economically important disease of cherry is caused by at least two different viruses. One of these is Little cherry virus 1 (LChV-1) for the detection of which no efficient serological tools are available, so that diagnosis is based on molecular methods. In this study, different immunization strategies for producing antibodies against the viral coat protein of LChV-1 were tried, using either purified virus preparations, or bacterially expressed protein, or a DNA vector that expressed the cloned coat protein (CP) gene in vivo. Effective induction of specific antibodies to LChV-1 CP was obtained using DNA intramuscular immunization followed by a single boost with the recombinant protein. The entire coat protein sequence was cloned in a mammalian expression vector and, after being coated by an amphiphilic non-toxic reagent was delivered into rabbit. A protein boost increased the specific immune response against the virus protein. The sensitivity of this antiserum is lower if compared with that of antisera raised conventionally against other viruses, thus it requires improvements for use for diagnostic purposes.

The role of the secretory immune response in the infection by Entamoeba histolytica

Intestinal infection with the protozoan parasite Entamoeba histolytica elicits a local immune response with rising of specific secretory IgA (sIgA) antibodies detectable in several compartments associated to mucosa. Anti-amoebic sIgA antibodies have been reported in faeces, saliva, bile and breast milk from dysenteric patients and research trying to elucidate their role in protection has recently intensified. IgA antibodies inhibit the in vitro adherence of E. histolytica trophozoites to epithelial cell monolayers by recognizing several membrane antigens, including the galactose-binding lectin (Gal-lectin), main surface molecule involved in adherence, and the serine and cystein-rich proteins, all of them potential vaccine candidates. In fact, the presence of sIgA anti-Gal lectin in faeces of patients recovered from amoebic liver abscess (ALA) was associated with immunity to E. dispar. Moreover, the combined nasal and intraperitoneal vaccination of C3H/HeJ mice with native and recombinant Gal-lectin protected mice against an intracecal challenge with virulent E. histolytica trophozoites, protection that seemed to be associated with the induction of specific intestinal sIgA antibodies. Therefore, the stimulation of intestinal secretory response by mucosal delivery of amoebic antigens has been positioned as a promising strategy for inducing protection against human amoebiasis.

Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming.

The use of recombinant tumor antigen proteins is a realistic approach for the development of generic cancer vaccines, but the potential of this type of vaccines to induce specific CD8(+) T cell responses, through in vivo cross-priming, has remained unclear. In this article, we report that repeated vaccination of cancer patients with recombinant NY-ESO-1 protein, Montanide ISA-51, and CpG ODN 7909, a potent stimulator of B cells and T helper type 1 (Th1)-type immunity, resulted in the early induction of specific integrated CD4(+) Th cells and antibody responses in most vaccinated patients, followed by the development of later CD8(+) T cell responses in a fraction of them. The correlation between antibody and T cell responses, together with the ability of vaccine-induced antibodies to promote in vitro cross-presentation of NY-ESO-1 by dendritic cells to vaccine-induced CD8(+) T cells, indicated that elicitation of NY-ESO-1-specific CD8(+) T cell responses by cross-priming in vivo was associated with the induction of adequate levels of specific antibodies. Together, our data provide clear evidence of in vivo cross-priming of specific cytotoxic T lymphocytes by a recombinant tumor antigen vaccine, underline the importance of specific antibody induction for the cross-priming to occur, and support the use of this type of formulation for the further development of efficient cancer vaccines.