Luís Carlos de Souza Ferreira

A CCR4 antagonist combined with vaccines induces antigen-specific CD8+ T cells and tumor immunity against self antigens.

Regulatory T cells (Tregs) may impede cancer vaccine efficacy in hematologic malignancies and cancer. CCR4 antagonists, an emergent class of Treg inhibitor, have been shown to block recruitment of Tregs mediated by CCL22 and CCL17. Our aim was to demonstrate the ability of a CCR4 antagonist (a small chemical molecule identified in silico) when combined with vaccines to break peripheral tolerance controlled by Tregs, a prerequisite for the induction of CD8(+) T cells against self Ags. Immunization of transgenic or normal mice expressing tumor-associated self Ags (Her2/neu, OVA, gp100) with a CCR4 antagonist combined with various vaccines led to the induction of effector CD8(+) T cells and partial inhibition of tumor growth expressing self Ags in both prophylactic and therapeutic settings. The CCR4 antagonist was more efficient than cyclophosphamide to elicit anti-self CD8(+) T cells. We also showed that the population of Tregs expressing CCR4 corresponded to memory (CD44(high)) and activated (ICOS(+)) Tregs, an important population to be targeted to modulate Treg activity. CCR4 antagonist represents a competitive class of Treg inhibitor able to induce functional anti-self CD8(+) T cells and tumor growth inhibition when combined with vaccines. High expression of CCR4 on human Tregs also supports the clinical development of this strategy.

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FliC), a Toll-like receptor 5 (TLR5) agonist. FliC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P. vivax MSP1(19) in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSP1(19)-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malarial antigens and the innate immunity agonist FliC. It contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants.

Production of recombinant proteins in Escherichia coli

Attempts to obtain a recombinant protein using prokaryotic expression systems can go from a rewarding and rather fast procedure to a frustrating time-consuming experience. In most cases production of heterologous proteins in Escherichia coli K12 strains has remained an empirical exercise in which different systems are tested without a careful insight into the various factors affecting adequate expression of the encoded protein. The present review will deal with E. coli as protein factory and will cover some of the aspects related to transcriptional and translational expression signals, factors affecting protein stability and solubility and targeting of proteins to different cell compartments. Based on the knowledge accumulated over the last decade, we believe that the rate of success for those dedicated to expression of recombinant proteins based on the use E. coli strains can still be significantly improved.

CD8+ T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (CS) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS(280-288) epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS(280-288) peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations.

Bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors

Bacillus subtilis and some of its close relatives have a long history of industrial and biotechnological applications. Search for antigen expression systems based on recombinant B. subtilis strains sounds attractive both by the extensive genetic knowledge and the lack of an outer membrane, which simplify the secretion and purification of heterologous proteins. More recently, genetically modified B. subtilis spores have been described as indestructible delivery vehicles for vaccine antigens. Nonetheless both production and delivery of antigens by B. subtilis strains face some inherent obstacles, as unstable gene expression and reduced immunogenicity that, otherwise, can be overcome by already available gene technology approaches. In the present review we present the status of B. subtilis-based vaccine research, either as protein factories or delivery vectors, and discuss some alternatives for a better use of genetically modified strains.

Induction of resident memory T cells enhances the efficacy of cancer vaccine

Tissue-resident memory T cells (Trm) represent a new subset of long-lived memory T cells that remain in tissue and do not recirculate. Although they are considered as early immune effectors in infectious diseases, their role in cancer immunosurveillance remains unknown. In a preclinical model of head and neck cancer, we show that intranasal vaccination with a mucosal vector, the B subunit of Shiga toxin, induces local Trm and inhibits tumour growth. As Trm do not recirculate, we demonstrate their crucial role in the efficacy of cancer vaccine with parabiosis experiments. Blockade of TFGβ decreases the induction of Trm after mucosal vaccine immunization, resulting in the lower efficacy of cancer vaccine. In order to extrapolate this role of Trm in humans, we show that the number of Trm correlates with a better overall survival in lung cancer in multivariate analysis. The induction of Trm may represent a new surrogate biomarker for the efficacy of cancer vaccine. This study also argues for the development of vaccine strategies designed to elicit them.

Prolonged Shedding of Zika Virus Associated with Congenital Infection

In this case report, Zika virus was detected in the bloodstream of an infant who was congenitally infected for at least 2 months after birth.

Paracoccidioides brasiliensis vaccine formulations based on the gp43-derived P10 sequence and the Salmonella enterica FliC flagellin.

ABSTRACT Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Anti-PCM vaccine formulations based on the secreted fungal cell wall protein (gp43) or the derived P10 sequence containing a CD4+ T-cell-specific epitope have shown promising results. In the present study, we evaluated new anti-PCM vaccine formulations based on the intranasal administration of P. brasiliensis gp43 or the P10 peptide in combination with the Salmonella enterica FliC flagellin, an innate immunity agonist binding specifically to the Toll-like receptor 5, in a murine model. BALB/c mice immunized with gp43 developed high-specific-serum immunoglobulin G1 responses and enhanced interleukin-4 (IL-4) and IL-10 levels. On the other hand, mice immunized with recombinant purified flagellins genetically fused with P10 at the central hypervariable domain, either flanked or not by two lysine residues, or the synthetic P10 peptide admixed with purified FliC elicited a prevailing Th1-type immune response based on lung cell-secreted type 1 cytokines. Mice immunized with gp43 and FliC and intratracheally challenged with P. brasiliensis yeast cells had increased fungal proliferation and lung tissue damage. In contrast, mice immunized with the chimeric flagellins and particularly those immunized with P10 admixed with FliC reduced P. brasiliensis growth and lung damage. Altogether, these results indicate that S. enterica FliC flagellin modulates the immune response to P. brasiliensis P10 antigen and represents a promising alternative for the generation of anti-PCM vaccines.

Host and Bacterial Factors Affecting Induction of Immune Responses to Flagellin Expressed by Attenuated Salmonella Vaccine Strains

ABSTRACT Previous observations demonstrated that the delivery of recombinant Salmonella enterica serovar Dublin strains to mice via mucosal routes did not efficiently activate systemic and secreted antibody responses to either type d flagellin or genetically fused heterologous B-cell epitopes, thus reducing the usefulness of the protein as a carrier of epitopes for vaccine purposes. In this work, we investigated murine systemic and mucosal flagellin immunogenicity after oral immunization with attenuated Salmonella strains. The reduced anti-type d flagellin antibody responses in mice immunized via mucosal routes with three doses of flagellated S. enterica serovar Dublin strains were not caused by oral tolerance and could not be restored by coadministration of a mucosal adjuvant. The induction of antibody responses to Salmonella flagellins was shown to differ according to the genetic background, but not the haplotype, of the mouse lineage. Moreover, BALB/c mice orally immunized with S. enterica serovar Typhimurium strains developed anti-type i flagellin sera and secreted antibody responses, which indicated that the serovar of the Salmonella vaccine strain also affected flagellin immunogenicity. Analyses of cytokine responses of BALB/c mice immunized with three oral doses of flagellated S. enterica serovar Dublin vaccine strains showed that, in spite of the lack of antibody responses, elevated type d flagellin-specific CD4-cell-activation-dependent gamma interferon (IFN-γ) and interleukin-10 responses were elicited after the administration of the vaccine strains via either parenteral or mucosal routes. Similar cytokine production patterns were detected to a T-cell heterologous epitope, derived from the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC), in mice orally immunized with a Salmonella vaccine strain expressing hybrid flagella. These results indicate that the immunogenicities of Salmonella flagellins can differ significantly, depending on the murine host and on the bacterial vector used, and demonstrate that the induction of CD4-cell-activation-dependent IFN-γ production represents a major immune response triggered by flagellin and in-frame fused heterologous T-cell epitopes after the oral administration of recombinant S. enterica serovar Dublin vaccine strains.

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A2 and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

ABSTRACT Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A1 peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A2 peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2ΔAB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A2 sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2ΔAB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.