Fusion Antigen Research Articles

Antibodies derived from an enterotoxigenic Escherichia coli (ETEC) adhesin tip MEFA (multiepitope fusion antigen) against adherence of nine ETEC adhesins: CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA

Diarrhea continues to be a leading cause of death in children younger than 5 years in developing countries. Enterotoxigenic Escherichia coli (ETEC) is a leading bacterial cause of children's diarrhea and travelers’ diarrhea. ETEC bacteria initiate diarrheal disease by attaching to host receptors at epithelial cells and colonizing in small intestine. Therefore, preventing ETEC attachment has been considered the first line of defense against ETEC diarrhea. However, developing vaccines effectively against ETEC bacterial attachment encounters challenge because ETEC strains produce over 23 immunologically heterogeneous adhesins. In this study, we applied MEFA (multiepitope fusion antigen) approach to integrate epitopes from adhesin tips or adhesive subunits of CFA/I, CS1, CS2, CS3, CS4, CS5, CS6, CS21 and EtpA adhesins and to construct an adhesin tip MEFA peptide. We then examined immunogenicity of this tip MEFA in mouse immunization, and assessed potential application of this tip MEFA for ETEC vaccine development. Data showed that mice intraperitoneally immunized with this adhesin tip MEFA developed IgG antibody responses to all nine ETEC adhesins. Moreover, ETEC and E. coli bacteria expressing these nine adhesins, after incubation with serum of the immunized mice, exhibited significant reduction in attachment to Caco-2 cells. These results indicated that anti-adhesin antibodies induced by this adhesin tip MEFA blocked adherence of the most important ETEC adhesins, suggesting this multivalent tip MEFA may be useful for developing a broadly protective anti-adhesin vaccine against ETEC diarrhea.

Coupling of HIV-1 Antigen to the Selective Autophagy Receptor SQSTM1/p62 Promotes T-Cell-Mediated Immunity.

Vaccines aiming to promote T-cell-mediated immune responses have so far showed limited efficacy, and there is a need for novel strategies. Studies indicate that autophagy plays an inherent role in antigen processing and presentation for CD4+ and CD8+ T cells. Here, we report a novel vaccine strategy based on fusion of antigen to the selective autophagy receptor sequestosome 1 (SQSTM1)/p62. We hypothesized that redirection of vaccine antigen from proteasomal degradation into the autophagy pathway would increase the generation of antigen-specific T cells. A hybrid vaccine construct was designed in which the antigen is fused to the C-terminus of p62, a signaling hub, and a receptor that naturally delivers ubiquitinated cargo for autophagic degradation. Fusion of the human immunodeficiency virus-1 antigen Gagp24 to p62 resulted in efficient antigen delivery into the autophagy pathway. Intradermal immunization of mice revealed that, in comparison to Gagp24 delivered alone, fusion to p62 enhanced the number of Gagp24-specific interferon-γ-producing T cells, including CD8+ T cells. The strategy may also have the potential to modulate the antigenic peptide repertoire. Because p62 and autophagy are highly conserved between species, we anticipate this strategy to be a candidate for the development of T-cell-based vaccines in humans.

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6% of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression.

Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) strains are the main cause of diarrhea in pigs. Pig diarrhea especially post-weaning diarrhea remains one of the most important swine diseases. ETEC bacterial fimbriae including K88, F18, 987P, K99 and F41 promote bacterial attachment to intestinal epithelial cells and facilitate ETEC colonization in pig small intestine. ETEC enterotoxins including heat-labile toxin (LT) and heat-stable toxins type Ia (porcine-type STa) and type II (STb) stimulate fluid hyper-secretion, leading to watery diarrhea. Blocking bacteria colonization and/or neutralizing enterotoxicity of ETEC toxins are considered effective prevention against ETEC diarrhea. In this study, we applied the MEFA (multiepitope fusion antigen) strategy to create toxoid MEFAs that carried antigenic elements of ETEC toxins, and examined for broad antitoxin immunogenicity in a murine model. By embedding STa toxoid STaP12F (NTFYCCELCCNFACAGCY), a STb epitope (KKDLCEHY), and an epitope of Stx2e A subunit (QSYVSSLN) into the A1 peptide of a monomeric LT toxoid (LTR192G), two toxoid MEFAs, ‘LTR192G-STb-Stx2e-STaP12F’ and ‘LTR192G-STb-Stx2e-3xSTaP12F’ which carried three copies of STaP12F, were constructed. Mice intraperitoneally immunized with each toxoid MEFA developed IgG antibodies to all four toxins. Induced antibodies showed in vitro neutralizing activities against LT, STa, STb and Stx2e toxins. Moreover, suckling piglets born by a gilt immunized with ‘LTR192G-STb-Stx2e-3xSTaP12F’ were protected when challenged with ETEC strains, whereas piglets born by a control gilt developed diarrhea. Results from this study showed that the toxoid MEFA induced broadly antitoxin antibodies, and suggested potential application of the toxoid MEFA for developing a broad-spectrum vaccine against ETEC diarrhea in pigs.

Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination

The cholera toxin B subunit (CTB) is secreted in its pentameric form from Escherichia coli if its leader peptide is replaced with one of E. coli origin. However, the secretion of the pentamer is generally severely impaired when the molecule is mutated or fused to a foreign peptide. Therefore, we attempted to regenerate pentameric CTB from the inclusion bodies (IBs) of E. coli. Stepwise dialysis of the IBs solubilized in guanidine hydrochloride predominantly generated soluble high-molecular-mass (HMM) aggregates and only a small fraction of pentamer. Three methods to reassemble homogeneous pentameric molecules were evaluated: (i) using a pentameric coiled-coil fusion partner, expecting it to function as an assembly core; (ii) optimizing the protein concentration during refolding; and (iii) eliminating contaminants before refolding. Coiled-coil fusion had some effect, but substantial amounts of HMM aggregates were still generated. Varying the protein concentration from 0.05mg/mL to 5mg/mL had almost no effect. In contrast, eliminating the contaminants before refolding had a robust effect, and only the pentamer was regenerated, with no detectable HMM aggregates. Surprisingly, the protein concentration at refolding was up to 5mg/mL when the contaminants were removed, with no adverse effects on refolding. The regenerated pentamer was indistinguishable in its biochemical and immunological characteristics from CTB secreted from E. coli or choleragenoid from Vibrio cholerae. This study provides a simple but very efficient strategy for pentamerizing CTB with a highly homogeneous molecular conformation, with which it may be feasible to engineer CTB derivatives and CTB fusion antigens.

Sipuleucel-T (sip-T)–induced proliferative CD8+ T-cell responses to immunizing and secondary antigens.

165 Background: Sip-T is an FDA-approved autologous immunotherapy for patients (pts) with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). Sip-T is manufactured from peripheral blood mononuclear cells cultured with PA2024, a fusion antigen of prostatic acid phosphatase (PAP) conjugated to granulocyte macrophage-colony stimulating factor (GM-CSF). In sip-T–treated pts, T cell and antibody responses to PA2024 or PAP as well as antibody responses to secondary antigens (i.e., antigen spread) correlate with improved overall survival. To explore the biology of this relationship, we further characterized the T cell subpopulations involved in the cellular immune responses to sip-T. Methods: In vitro proliferative CD8+ (cytotoxic T lymphocyte) and CD4+ (T helper) T cell responses to PA2024 and PAP as well as to secondary antigens (PSA, LGALS3, and KRAS) were evaluated using flow cytometry on pt samples from two sip-T–containing clinical trials (STAND [NCT01431391] and STRIDE [NCT01981122]). Results: PA2024-specific CD8+ and CD4+ responses were observed beginning 2 weeks after the first sip-T infusion through week 26 in most evaluable pts. CD8+ and CD4+ responses to PAP were also observed, although the magnitude of this response (to a self-antigen) was smaller when compared with PA2024 responses. Most pts with CD8+ responses to PA2024 also had a CD4+ response, which occurred more frequently than CD8+ responses. Both CD8+ and CD4+ responses to secondary antigens were amplified after sip-T, and these CD8+ proliferative responses to secondary antigens were greater in magnitude compared with CD4+ responses. Conclusions: Here,we report the first evidence of antigen-specific CD8+ responses in pts receiving sip-T, indicating CD8+ T cell involvement in sip-T–mediated immune responses that occur in concert with the expected CD4+ T cell responses to soluble antigens (i.e., PA2024). These CD8+ responses were durable, lasting up to 26 weeks post–sip-T treatment. Importantly, responses to secondary antigens and in particular, CD8+ responses, were also amplified after sip-T treatment, suggesting that antigen spread could be resulting from sip-T–mediated tumor cell lysis. Clinical trial information: NCT01431391 and NCT01981122.

Construction of a fusion plasmid containing the PSCA gene and cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and its anti-tumor effect in an animal model of prostate cancer.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a negative regulator of T cell activation, which competes with CD28 for B7.1/B7.2 binding, and which has a greater affinity. Fusion of specific antigens to extracellular domain of CTLA4 represents a promising approach to increase the immunogenicity of DNA vaccines. In this study, we evaluated this interesting approach for CTLA4 enhancement on prostate stem cell antigen (PSCA)-specific immune responses and its anti-tumor effects in a prostate cancer mouse model. Consequently, we constructed a DNA vaccine containing the PSCA and the CTLA-4 gene. Vaccination with the CTLA4-fused DNA not only induced a much higher level of anti-PSCA antibody, but also increased PSCA-specific T cell response in mice. To evaluate the anti-tumor efficacy of the plasmids, murine models with PSCA-expressing tumors were generated. After injection of the tumor-bearing mouse model, the plasmid carrying the CTLA4 and PSCA fusion gene showed stronger inhibition of tumor growth than the plasmid expressing PSCA alone. These observations emphasize the potential of the CTLA4-fused DNA vaccine, which could represent a promising approach for tumor immunotherapy.

Novel fusion antigen displayed-bacterial ghosts vaccine candidate against infection of Escherichia coli O157:H7.

Infection with Escherichia coli O157:H7 may develop into hemorrhagic colitis, or hemolytic uremic syndrome (HUS), which usually causes kidney failure or even death. The adhesion and toxins are the important virulent factors. In this study, a novel vaccine candidate rSOBGs was constructed based on the bacterial ghost (BG). rSOBGs maintained the integrity of cellular morphology and displayed the linear Stx2Am-Stx1B antigen on the surface of outer membrane. rSOBGs induced Stxs-specific IgA/IgG antibodies and stronger intimin-specific IgA/IgG antibodies effectively in sera in this study. In vivo, the rSOBGs provided the higher protection rate (52%) than native bacterial ghost-OBGs (12%) when challenged intragastricly with high dose (500 LD50) viable E. coli O157:H7. Meanwhile, the rSOBGs provided higher protection rate (73.33%) than OBGs when challenged with 2 LD50 even to 5 LD50 lysed E. coli O157:H7. In vitro, the rSOBGs-immunized sera possessed neutralizing activity to lysed pathogenic bacteria. Furthermore, the results of histopathology also displayed that the administration of rSOBGs have the ability to reduce or inhibit the adhesion lesions and toxins damages of organs. The novel vaccine candidate rSOBGs induced both anti-toxin and anti-adhesion immune protection, suggesting the possibility to prevent the infectious diseases caused by Escherichia coli O157:H7.

Multi-epitope proteins for improved serological detection of Trypanosoma cruzi infection and Chagas Disease

We previously reported that tandem repeat (TR) proteins from Trypanosoma cruzi could serve as targets of the antibody response and be useful as diagnostic indicators. To optimize reagents for detecting T. cruzi infection we evaluated individual TR proteins and identified several that were recognized by the majority of Chagas patient's sera collected from individuals form Brazil. We then produced novel, recombinant fusion proteins to combine the reactive TR proteins into a single diagnostic product. Direct comparison of the antibody response of serum samples that were readily detected by the established fusion antigen used in commercial detection of Chagas disease, TcF, revealed strong responses to TcF43 and TcF26 proteins. While the TcF43 and TcF26 antigens enhanced detection and strength of signal, they did not compromise the specificity of detection compared to that obtained with TcF. Finally, it was apparent by testing against a panel of 84 serum samples assembled on the basis of moderate or weak reactivity against TcF (mostly signal:noise <5) that TcF43 and TcF26 could more strongly detected by many of the sera that had low TcF antibody levels. Taken together, these data indicate that TcF43 and TcF26 could be used to enhance the detection of T. cruzi infection as well as supporting a diagnosis of Chagas disease.

A DNA vaccine encoding mutated HPV58 mE6E7-Fc-GPI fusion antigen and GM-CSF and B7.1.

BackgroundPersistent infection with high-risk human papillomavirus (HPV) is a predominant cause of cervical cancer, and HPV58 is the third most common virus detected in the patients with cervical cancer in Asia. E6 and E7 are the viral oncogenes which are constitutively expressed in HPV-associated tumor cells and can be used as target antigens for related immunotherapy. In this study, we modified the HPV58 E6 and E7 oncogenes to eliminate their oncogenic potential and constructed a recombinant DNA vaccine that coexpresses the sig-HPV58 mE6E7-Fc-GPI fusion antigen in addition to granulocyte-macrophage colony-stimulating factor (GM-CSF) and B7.1 as molecular adjuvants (PVAX1-HPV58 mE6E7FcGB) for the treatment of HPV58 (+) cancer.MethodsPVAX1-HPV58 mE6E7FcGB recombinant DNA vaccine was constructed to express a fusion protein containing a signal peptide, a modified HPV58 mE6E7 gene, and human IgG Fc and glycosylphosphatidylinositol (GPI)-anchoring sequences using the modified DNA vaccine vector PVAX1-IRES-GM/B7.1 that coexpresses GM-CSF, and B7.1. C57BL/6 mice were challenged by HPV58 E6E7-expressing B16-HPV58 E6E7 cells, followed by immunization by PVAX1-HPV58 mE6E7FcGB vaccine on days 7, 14, 21 after tumor challenge. The cellular immune responses in immunized mice were assessed by measuring IFN-γ production in splenocytes upon stimulation by HPV58 E6E7-GST protein and the lysis of B16-HPV58 E6E7 target cells by splenocytes after restimulation with HPV58 E6E7-GST protein. The antitumor efficacy was evaluated by monitoring the growth of the tumor.ResultsPVAX1-HPV58 mE6E7FcGB elicited varying levels of IFN-lsgdB58onn T-cell immune responses and lysis of target cell in mice in response to the recombinant antigen HPV58 E6E7-GST. Furthermore, the vaccine also induced antitumor responses in the HPV58 (+) B16-HPV58 E6E7 tumor challenge model as evidenced by delayed tumor development.ConclusionThe recombinant DNA vaccine PVAX1-HPV58 mE6E7FcGB efficiently generates cellular immunity and antitumor efficacy in immunized mice. These data provide a basis for the further study of this recombinant vaccine as a potential candidate vaccine.

Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery.

Virus-like particles (VLPs) have been extensively explored as nanoparticle vehicles for many applications in biotechnology (e.g., vaccines, drug delivery, imaging agents, biocatalysts). However, amino acid sequence plasticity relative to subunit expression and nanoparticle assembly has not been explored. Whereas the hepatitis B core protein (HBc) VLP appears to be the most promising model for fundamental and applied studies; particle instability, antigen fusion limitations, and intrinsic immunogenicity have limited its development. Here, we apply Escherichia coli-based cell-free protein synthesis (CFPS) to rapidly produce and screen HBc protein variants that still self-assemble into VLPs. To improve nanoparticle stability, artificial covalent disulfide bridges were introduced throughout the VLP. Negative charges on the HBc VLP surface were then reduced to improve surface conjugation. However, removal of surface negative charges caused low subunit solubility and poor VLP assembly. Solubility and assembly as well as surface conjugation were greatly improved by transplanting a rare spike region onto the common shell structure. The newly stabilized and extensively modified HBc VLP had almost no immunogenicity in mice, demonstrating great promise for medical applications. This study introduces a general paradigm for functional improvement of complex protein assemblies such as VLPs. This is the first study, to our knowledge, to systematically explore the sequence plasticity of viral capsids as an approach to defining structure function relationships for viral capsid proteins. Our observations on the unexpected importance of the HBc spike tip charged state may also suggest new mechanistic routes toward viral therapeutics that block capsid assembly.

Plant-made oral vaccines against human infectious diseases-Are we there yet?

Although the plant-made vaccine field started three decades ago with the promise of developing low-cost vaccines to prevent infectious disease outbreaks and epidemics around the globe, this goal has not yet been achieved. Plants offer several major advantages in vaccine generation, including low-cost production by eliminating expensive fermentation and purification systems, sterile delivery and cold storage/transportation. Most importantly, oral vaccination using plant-made antigens confers both mucosal (IgA) and systemic (IgG) immunity. Studies in the past 5 years have made significant progress in expressing vaccine antigens in edible leaves (especially lettuce), processing leaves or seeds through lyophilization and achieving antigen stability and efficacy after prolonged storage at ambient temperatures. Bioencapsulation of antigens in plant cells protects them from the digestive system; the fusion of antigens to transmucosal carriers enhances efficiency of their delivery to the immune system and facilitates successful development of plant vaccines as oral boosters. However, the lack of oral priming approaches diminishes these advantages because purified antigens, cold storage/transportation and limited shelf life are still major challenges for priming with adjuvants and for antigen delivery by injection. Yet another challenge is the risk of inducing tolerance without priming the host immune system. Therefore, mechanistic aspects of these two opposing processes (antibody production or suppression) are discussed in this review. In addition, we summarize recent progress made in oral delivery of vaccine antigens expressed in plant cells via the chloroplast or nuclear genomes and potential challenges in achieving immunity against infectious diseases using cold-chain-free vaccine delivery approaches.

Strategies to improve BCG vaccine efficacy.

Strategies to improve BCG vaccine efficacy.

Abstract 1313: Characterization and isolation of antigen-responding T cells in Sipuleucel-T treated patients

Abstract Sipuleucel-T is an FDA-approved autologous cellular therapy for men with asymptomatic or minimally symptomatic metastatic castrate resistant prostate (mCRPC). Sipuleucel-T is manufactured from peripheral blood mononuclear cells (PBMCs) cultured with PA2024, a fusion antigen composed of prostatic acid phosphatase (PAP) fused to granulocyte macrophage-colony stimulating factor (GM-CSF). We have demonstrated that following three infusions this therapy results in T cell antigen specific responses directed against both PA2024 and PAP in treated patients. In this study we utilized the cellular dye Carboxyfluorescein succinimidyl ester (CSFE) and flow cytometric analysis to further characterize antigen responding T cells in Sipuleucel-T treated patients. We observed that CD4+ T cells predominate the antigen-responding T cell population following treatment, confirming our previous studies. Importantly, this approach has allowed us to also visualize a previously undetected population of antigen responding CD8+ T cells. The frequency of both of these populations is enhanced following treatment relative to baseline responses and the responding T cells display increased surface expression of the prototypical T cell activation markers CD134 and CD137 relative to non-proliferating T cells. Additionally, we are exploiting the increased surface expression of these activation markers on antigen-responding T cells to isolate these cells in an HLA and epitope-independent manner. Using this methodology we have demonstrated Sipuleucel-T is capable of inducing both CD4+ and CD8+ antigen-specific T cell responses in treated patients, and that we are capable of isolating these antigen-responding T cells specifically and with minimal manipulation. Citation Format: Jeff Pufnock, Tuyen Vu, James Trager, Nadeem Sheikh. Characterization and isolation of antigen-responding T cells in Sipuleucel-T treated patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1313. doi:10.1158/1538-7445.AM2015-1313

Current Progress in Developing Subunit Vaccines against Enterotoxigenic Escherichia coli-Associated Diarrhea

Diarrhea continues to be a leading cause of death in children <5 years of age, and enterotoxigenic Escherichia coli (ETEC) is the most common bacterial cause of children's diarrhea. Currently, there are no available vaccines against ETEC-associated diarrhea. Whole-cell vaccine candidates have been under development but require further improvements because they provide inadequate protection and produce unwanted adverse effects. Meanwhile, a newer approach using polypeptide or subunit vaccine candidates focusing on ETEC colonization factor antigens (CFAs) and enterotoxins, the major virulence determinants of ETEC diarrhea, shows substantial promise. A conservative CFA/I adhesin tip antigen and a CFA MEFA (multiepitope fusion antigen) were shown to induce cross-reactive antiadhesin antibodies that protected against adherence by multiple important CFAs. Genetic fusion of toxoids derived from ETEC heat-labile toxin (LT) and heat-stable toxin (STa) induced antibodies neutralizing both enterotoxins. Moreover, CFA-toxoid MEFA polypeptides, generated by fusing CFA MEFA to an STa-LT toxoid fusion, induced antiadhesin antibodies that broadly inhibited adherence of the seven most important ETEC CFAs associated with about 80% of the diarrhea cases caused by ETEC strains with known CFAs. This same antigen preparation also induced antitoxin antibodies that neutralized both toxins that are associated with all cases of ETEC diarrhea. Results from these studies suggest that polypeptide or subunit vaccines have the potential to effectively protect against ETEC diarrhea. In addition, novel adhesins and mucin proteases have been investigated as potential alternatives or, more likely, additional antigens for ETEC subunit vaccine development.

Co-administration of antigen with chemokine MCP-3 or MDC/CCL22 enhances DNA vaccine potency.

We evaluated the utility of chemokine MCP-3 and MDC/CCL22 as molecular adjuvants of DNA vaccines for botulinum neurotoxin serotype A (BoNT/A) in a Balb/c mouse model. Notably, the immunogenicity of the DNA vaccine against BoNT/A was not enhanced using a fusion of the AHc-C antigen with the MCP-3 or MDC/CCL22. Nevertheless, the potency of the DNA vaccine was significantly modulated and enhanced by co-administration of the AHc-C antigen with MCP-3 or MDC/CCL22. This strategy elicited high levels of humoral immune responses and protection against BoNT/A. The enhanced potency was further boosted by co-administration of the AHc-C antigen with both MCP-3 and MDC/CCL22 in Balb/c mice, but not by co-administration of AHc-C antigen with the MCP-3-MDC/CCL22 fusion. Co-immunization with both the MCP-3 and MDC/CCL22 constructs induced the highest levels of humoral immunity and protective potency against BoNT/A. Our results indicated that MCP-3 and MDC/CCL22 are effective molecular adjuvants of the immune responses induced by the AHc-C-expressing DNA vaccine when delivered by co-administration of the individual chemokines, but not when delivered in the form of a chemokine/antigen fusion. Thus, we describe an alternative strategy to the design and optimization of DNA vaccine constructs based on co-administration of the antigen with the chemokine rather than in the form of a chemokine/antigen fusion.

Evaluation of the immunogenicity of recombinant replicative DNA vaccines expressing multiple antigens of hepatitis C virus in a mice model

Objective To investigate the immunogenicity and cross protective effects of two novel HCV DNA vaccines in a mice model. Methods Two self-replicating alphavirus vector-based HCV DNA vaccines, pSCK CE1E2Y and pSCK H155, were constructed based on the genes encoding the structural proteins (Core, E1 and E2) and structural and NS3 fusion proteins (Core, E1 , E2 and NS3) of a HCV strain isolated from a Chinese patient (genotype 1b, Hebei strain), respectively. Western blot analysis was performed to detect the expression of fusion antigens. The BALB/c mice were intradermally immunized with the recombinant DNA vaccines by using electroporation. The immune responses induced in mice and the cross protective effects of the recombinant DNA vaccines were evaluated. Results The DNA vaccines effectively expressed the target antigens in vitro. The antigen-specific antibody responses and specific T cell immune responses were induced in mice by the immunization of replicative DNA vaccines. However, no effective cross protection was provided by either of the DNA vaccines in the surrogate challenge model based on a recombinant heterologous HCV (JFH1, 2a) vaccinia virus strain. Conclusion Although no effective cross protection was observed, both of the two replicative DNA vaccines could induce strong humoral and cellular immune responses against multi-target antigens of HCV strains. This study has paved the way for further investigation on the development of novel HCV vaccines. Key words: Hepatitis C virus; Replicative DNA vaccine; Fusion antigen; T cell response

Genetic fusions of a CFA/I/II/IV MEFA (multiepitope fusion antigen) and a toxoid fusion of heat-stable toxin (STa) and heat-labile toxin (LT) of enterotoxigenic Escherichia coli (ETEC) retain broad anti-CFA and antitoxin antigenicity.

Immunological heterogeneity has long been the major challenge in developing broadly effective vaccines to protect humans and animals against bacterial and viral infections. Enterotoxigenic Escherichia coli (ETEC) strains, the leading bacterial cause of diarrhea in humans, express at least 23 immunologically different colonization factor antigens (CFAs) and two distinct enterotoxins [heat-labile toxin (LT) and heat-stable toxin type Ib (STa or hSTa)]. ETEC strains expressing any one or two CFAs and either toxin cause diarrhea, therefore vaccines inducing broad immunity against a majority of CFAs, if not all, and both toxins are expected to be effective against ETEC. In this study, we applied the multiepitope fusion antigen (MEFA) strategy to construct ETEC antigens and examined antigens for broad anti-CFA and antitoxin immunogenicity. CFA MEFA CFA/I/II/IV [CVI 2014, 21(2):243-9], which carried epitopes of seven CFAs [CFA/I, CFA/II (CS1, CS2, CS3), CFA/IV (CS4, CS5, CS6)] expressed by the most prevalent and virulent ETEC strains, was genetically fused to LT-STa toxoid fusion monomer 3xSTaA14Q-dmLT or 3xSTaN12S-dmLT [IAI 2014, 82(5):1823-32] for CFA/I/II/IV-STaA14Q-dmLT and CFA/I/II/IV-STaN12S-dmLT MEFAs. Mice intraperitoneally immunized with either CFA/I/II/IV-STa-toxoid-dmLT MEFA developed antibodies specific to seven CFAs and both toxins, at levels equivalent or comparable to those induced from co-administration of the CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT. Moreover, induced antibodies showed in vitro adherence inhibition activities against ETEC or E. coli strains expressing these seven CFAs and neutralization activities against both toxins. These results indicated CFA/I/II/IV-STa-toxoid-dmLT MEFA or CFA/I/II/IV MEFA combined with 3xSTaN12S-dmLT induced broadly protective anti-CFA and antitoxin immunity, and suggested their potential application in broadly effective ETEC vaccine development. This MEFA strategy may be generally used in multivalent vaccine development.

Fusion of Glioblastoma Tumor Antigens to Herpes Simplex Virus-1 Glycoprotein D Enhances Secondary Adaptive Immune Responses in a DNA Vaccine Strategy

Glioblastoma multiforme (GBM) is a recurrent and fatal cancer. EGFRvIII, MAGE-3 and GLEA-2 are antigens that are found in this highly heterogeneous tumor and that are absent in normal tissue. Usually, conventional GMB treatments do not prevent recurrence, reinforcing the need for new therapeutic strategies. Vaccination can be an alternative GMB therapy capable to induce long-lasting and specific immune responses to tumors antigens but requires the activation of strong cellular responses. Fusion of tumor antigens with microbial-derived proteins is a rather simple approach that can enhance the immunogenicity of vaccines, particularly, DNA vaccines. In this study, we constructed DNA vaccines encoding GBM tumor antigens fused to glycoprotein D from herpes simplex virus-1 and evaluated their immunogenicity in C57BL/6 mice. The tumor antigens were correctly expressed by the DNA vaccines and induced cell mediated immune responses under experimental conditions. The vaccines encoding antigens genetically fused with gD induced higher cellular immune responses, associated with IFN-γ and IL-10 production, than vaccines encoding non-fused GMB antigens. Therefore, DNA vaccinations induced a Th1-biased immune response. We concluded that the strategy could be an effective immunotherapeutic approach for GBM.

Influence of trehalose 6,6′-diester (TDX) chain length on the physicochemical and immunopotentiating properties of DDA/TDX liposomes

Linking physicochemical characterization to functional properties is crucial for defining critical quality attributes during development of subunit vaccines toward optimal safety and efficacy profiles. We investigated how the trehalose 6,6′-diester (TDX) chain length influenced the physicochemical and immunopotentiating properties of the clinically tested liposomal adjuvant composed of dimethyldioctadecylammonium (DDA) bromide and analogues of trehalose-6,6′-dibehenate (TDB). TDB analogues with symmetrically shortened acyl chains [denoted X: arachidate (A), stearate (S), palmitate (P), myristate (Myr) and laurate (L)] were incorporated into DDA liposomes and characterized with respect to size, polydispersity index, charge, thermotropic phase behavior and lipid–lipid interactions. Incorporation of 11mol% TDX into DDA liposomes significantly decreased the polydispersity index when TDA, TDS, TDP and TDMyr were incorporated, whereas both the initial size and the charge of the liposomes were unaffected. The long-term colloidal stability was only decreased when including TDL in DDA liposomes. The fatty acid length of TDX affected the phase transition of the liposomes, and for the DDA/TDP and DDA/TDS liposomes a homogeneous distribution of the lipids in the bilayer was indicated. The membrane packing was studied further by using the Langmuir monolayer technique. Incorporation of TDS improved the packing of the lipid monolayer, as compared to the other analogues, suggesting the most favorable stability. Finally, immunization of mice with the recombinant tuberculosis fusion antigen Ag85B-ESAT-6-Rv2660c (H56) and the physicochemically most optimal formulations (DDA/TDB, DDA/TDS and DDA/TDP) induced comparable T-cell responses. In conclusion, of the investigated TDB analogues, incorporation of 11mol% TDS or TDP into DDA liposomes resulted in an adjuvant system with the most favorable physicochemical properties and an immunological profile comparable to that of DDA/TDB.