Shigella spp. are food-and waterborne pathogens that cause severe diarrheal and dysenteric disease associated with high morbidity and mortality. Individuals most often affected are children under 5 years of age in the developing world. The existence of multiple Shigella serotypes and the heterogenic distribution of pathogenic strains, as well as emerging antibiotic resistance, require the development of a broadly protective vaccine. All Shigella spp. utilize a type III secretion system (TTSS) to initiate infection. The type III secretion apparatus (TTSA) is the molecular needle and syringe that form the energized conduit between the bacterial cytoplasm and the host cell to transport effector proteins that manipulate cellular processes to benefit the pathogen. IpaB and IpaD form a tip complex atop the TTSA needle and are required for pathogenesis. Because they are common to all virulent Shigella spp., they are ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We examined the immunogenicity and protective efficacy of IpaB and IpaD, alone or combined, coadministered with a double mutant heat-labile toxin (dmLT) from Escherichia coli, used as a mucosal adjuvant, in a mouse model of intranasal immunization and pulmonary challenge. Robust systemic and mucosal antibody-and T cell-mediated immunities were induced against both proteins, particularly IpaB. Mice immunized in the presence of dmLT with IpaB alone or IpaB combined with IpaD were fully protected against lethal pulmonary infection with Shigella flexneri and Shigella sonnei. We provide the first demonstration that the Shigella TTSAs IpaB and IpaD are promising antigens for the development of a cross-protective Shigella vaccine.

Studies have confirmed the key role of Bacillus anthracis protective antigen (PA) in the US and UK human anthrax vaccines. However, given the tripartite nature of the toxin, other components, including lethal factor (LF), are also likely to contribute to protection. We examined the antibody and T cell responses to PA and LF in human volunteers immunized with the UK anthrax vaccine (AVP). Individual LF domains were assessed for immunogenicity in mice when given alone or with PA. Based on the results obtained, a novel fusion protein comprising D1 of LF and the host cell-binding domain of PA (D4) was assessed for protective efficacy. Murine protection studies demonstrated that both full-length LF and D1 of LF conferred complete protection against a lethal intraperitoneal challenge with B. anthracis STI spores. Subsequent studies with the LFD1-PAD4 fusion protein showed a similar level of protection. LF is immunogenic in humans and is likely to contribute to the protection stimulated by AVP. A single vaccine comprising protective regions from LF and PA would simplify production and confer a broader spectrum of protection than that seen with PA alone.

This study was undertaken to evaluate the feasibility of using recombinant dengue proteins to discriminate between acute dengue infections versus uninfected dengue samples. Dengue virus proteins E, NS1, NS3, and NS4B were cloned as fusion proteins and expressed in Escherichia coli. Recombinant products were tested in 100 serum samples obtained from acute dengue fever cases collected from 3 states of Mexico where dengue is endemic. Sera from 75 healthy individuals living in nonendemic areas for dengue were used as a control group. In sera from the dengue patients group, antibody responses to E protein were demonstrated in 91% of cases and NS1 protein was recognized to various extents (99%) within the first 7 days of infection. The antibody responses to NS3 and NS4B were frequently of low magnitude. Consistent negative antibody responses to all proteins were found in sera from the control group. These data suggest that the glutathione-S-transferase (GST)-dengue fusion proteins may be feasible antigens for a sensitive and specific serological assay.

Transferon® is a complex drug based on a mixture of low molecular weight peptides. This biotherapeutic is employed as a coadjuvant in clinical trials of several diseases, including viral infections and allergies. Given that macrophages play key roles in pathogen recognition, phagocytosis, processing, and antigen presentation, we evaluated the effect of Transferon® on phenotype and function of macrophage-like cells derived from THP-1 monocytes. We determined the surface expression of CD80 and CD86 by flow cytometry and IL-1β, TNF-α, and IL-6 levels by ELISA. Transferon® alone did not alter the steady state of PMA-differentiated macrophage-like THP-1 cells. On the contrary, simultaneous stimulation of cells with Transferon® and LPS elicited a significant increase in CD80 (P≤0.001) and CD86 (P≤0.001) expression, as well as in IL-6 production (P≤0.05) compared to the LPS control. CD80 expression and IL-6 production exhibited a positive correlation (r=0.6, P≤0.05) in cells exposed to Transferon® and LPS. Our results suggest that the administration of Transferon® induces the expression of costimulatory molecules and the secretion of cytokines in LPS-activated macrophages. Further studies are necessary to determine the implication of these findings in the therapeutic properties of Transferon®.

Zika virus (ZIKV), an emerging mosquito-borne flavivirus, has quickly spread in many regions around the world where dengue virus (DENV) is endemic. This represents a major health concern, given the high homology between these two viruses, which can result in cross-reactivity. The aim of this study was to determine the cross-reacting antibody response of the IgM and IgG classes against the recombinant envelope protein of ZIKV (rE-ZIKV) in sera from patients with acute-phase infection of different clinical forms of dengue, i.e., dengue fever (DF) and dengue hemorrhagic fever (DHF) (before the arrival of ZIKV in Mexico 2010), as well as acute-phase sera of ZIKV patients, together with the implications in neutralization and antibody-dependent enhancement. Differences in IgM responses were observed in a number of DF and DHF patients whose sera cross-reacted with the rE-ZIK antigen, with 42% recognition between acute-phase DHF and ZIKV but 27% recognition between DF and ZIKV. Regarding IgG antibodies, 71.5% from the DF group showed cross-reactivity to rE-ZIKV in contrast with 50% and only 25% of DHF and ZIKV serum samples, respectively, which specifically recognized the homologous antigen. The DHF group showed more enhancement of ZIKV infection of FCRγ-expressing cells compared to the DF group. Furthermore, the DHF group also showed a higher cross-neutralizing ability than that of DF. This is the first report where DF and DHF serum samples were evaluated for cross-reactivity against Zika protein and ZIKV. Furthermore, DENV serum samples cross-protect against ZIKV through neutralizing antibodies but at the same time mediate antibody-dependent enhancement in the sequential ZIKV infection.

Live attenuated bacteria hold great promise as multivalent mucosal vaccines against a variety of pathogens. A major challenge of this approach has been the successful delivery of sufficient amounts of vaccine antigens to adequately prime the immune system without overattenuating the live vaccine. Here we used a live attenuated Salmonella enterica serovar Typhi strain to create a bivalent mucosal plague vaccine that produces both the protective F1 capsular antigen of Yersinia pestis and the LcrV protein required for secretion of virulence effector proteins. To reduce the metabolic burden associated with the coexpression of F1 and LcrV within the live vector, we balanced expression of both antigens by combining plasmid-based expression of F1 with chromosomal expression of LcrV from three independent loci. The immunogenicity and protective efficacy of this novel vaccine were assessed in mice by using a heterologous prime-boost immunization strategy and compared to those of a conventional strain in which F1 and LcrV were expressed from a single low-copy-number plasmid. The serum antibody responses to lipopolysaccharide (LPS) induced by the optimized bivalent vaccine were indistinguishable from those elicited by the parent strain, suggesting an adequate immunogenic capacity maintained through preservation of bacterial fitness; in contrast, LPS titers were 10-fold lower in mice immunized with the conventional vaccine strain. Importantly, mice receiving the optimized bivalent vaccine were fully protected against lethal pulmonary challenge. These results demonstrate the feasibility of distributing foreign antigen expression across both chromosomal and plasmid locations within a single vaccine organism for induction of protective immunity.T he process of engineering live attenuated organisms for mucosal delivery of protective foreign antigens has become a sophisticated enterprise, with powerful improvements in expression technologies occurring over the past 3 decades (1-5). To date, the most straightforward implementation of such expression technologies has involved the use of multicopy plasmids. Plasmids have been engineered to encode nonantibiotic selection markers which confer stable maintenance of these plasmids, both in vitro and after vaccination, thereby promoting optimum expression of sufficient levels of antigen to elicit protective immunity (6-8). Antigen export systems have also been devised to export antigens out of the cytoplasm and either onto the cell surface or out into the surrounding milieu (9-11). Export of foreign antigens is now appreciated to improve immune responses, possibly by avoiding proteolytic degradation of antigens within the cytoplasm or periplasmic space of the vaccine organism (10,(12)(13)(14)(15)(16)(17).However, there can be additional pitfalls introduced by stabilized expression plasmids. Sustained production of large amounts of foreign antigen can impose a metabolic burden upon the vaccine that overattenuates the strain and results in reduced immunogenicity (1,(18)(19)(20)(21)(22). T...

DNA priming-protein boosting is a strategy used to establish strong immunity to a specific pathogen by the use of two different antigens through sequential delivery systems. In this work, two recombinant plasmids were used, one encoding for the dengue virus E protein, which is know to induce neutralizing antibodies (pcDNA 3.1/E), and the other encoding for the Dengue virus nonstructural protein 1 (pcDNA 3.1/NS1), as a source of B- and T-cell epitopes possibly involved in protective immunity. We showed that immunization of BALB/c mice with three priming doses of both plasmids pcDNA 3.1/E and/or pcDNA 3.1/NS1 were able to induce antibody responses to E protein with a single plasmid; in contrast to the antibody response to NS1 protein we observed an additive effect in terms of antibody response. Moreover, using a prime-boost protocol in which both plasmid constructs were co-administrated followed by a boost of homologous GST-E and GST-NS1 recombinant proteins, we observed an increased antibody response to NS1 and to E protein compared to animals vaccinated with the proteins or with dengue constructs alone. If neutralizing antibodies play an important role in dengue infection, antibodies generated with this regimen was also significantly better than the administration of the mix of proteins alone. These results suggest that NS1 and E proteins together could be considered in a design of subunit recombinant vaccines.

A DENV-2 plasmid named pEII*EIII/NS1*,containing sequences encoding portions of the envelope protein that are potentially involved in the induction of neutralizing antibodies and a portion of the NS1 sequence that is involved in protection, is reported in this work. The synthesized subunit protein was recognized by human sera from infected patients and had the predicted size. The immunogenicity of this construct was evaluated using a mouse model in a prime-boost vaccination approach. The priming was performed using the plasmid pEII*EIII/NS1*, followed by a boost with recombinant full-length GST-E and GST-NS1 fusion proteins. The mice showed specific antibody responses to the E and NS1 proteins, as detected by ELISA, compared to the response of animals vaccinated with the parental plasmid. Interestingly, some animals had neutralizing antibodies. These results show that EII*, EIII and NS1* sequences could be considered for the design ofa recombinant subunit vaccine against dengue disease.