Ada M.B Alves

Targeting the Non-structural Protein 1 from Dengue Virus to a Dendritic Cell Population Confers Protective Immunity to Lethal Virus Challenge

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4+ and CD8+ T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205+ DC population with poly (I:C) opens perspectives for dengue vaccine development.

Epitope specificity of antibodies raised against enterotoxigenic Escherichia coli CFA/I fimbriae in mice immunized with naked DNA.

The cfaB gene, coding for the CFA/I fimbrial adhesin of enterotoxigenic Escherichia coli (ETEC), was cloned and expressed as a fusion peptide with the glycoprotein D (gD) from herpes simplex virus type 1 (HSV) in the pRE4 eukaryotic expression vector, resulting in the recombinant plasmid pRECFA. All BALB/c mice injected intramuscularly (i.m.) with a single dose (100 micrograms) of the purified plasmid developed antibodies against epitopes found on dissociated CFA/I subunits as well as other homologous ETEC fimbriae. Surface-exposed epitopes found on intact CFA/I fimbriae were also recognized by antibodies derived from DNA immunization, but they did not overlap with those generated with purified CFA/I fimbriae. None of the sera raised in mice immunizated with pRECFA were able to agglutinate bacterial cells or inhibit haemagglutination promoted by CFA/I bearing ETEC cells. These results show that pRECFA can elicit CFA/I-specific antibodies, which may have different epitope specificities and functional properties compared with those generated with purified bacterial protein.

DNA immunisation against the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC)

The CFA/I fimbria promotes the attachment of enterotoxigenic Escherichia coli (ETEC) to the surface of human enterocytes. The generation of a protective immune response requires the induction of antibodies able to block the CFA/I-mediated binding of ETEC to receptors located on the small intestine epithelium or on the surface of human red blood cells, in hemagglutination tests. An eukaryotic expression plasmid, pBLCFA, encoding the CFA/I gene under the control of the human cytomegalovirus major immediate-early promoter was constructed as a prototype DNA vaccine against ETEC. pBLCFA-tranfected BHK-21 cells secreted a peptide cross-reacting with a monoclonal antibody raised against CFA/I subunits. BALB/c mice immunized intramuscularly with one or two doses of purified pBLCFA developed CFA/I-specific serum antibodies for at least 52 weeks, composed predominantly of the IgG1 subclass. pBLCFA-induced antibodies bind mainly to epitopes exposed on the surface of intact CFA/I fimbriae and do not react with immune recessive epitopes found in other ETEC fimbra sharing amino acid homologies with CFA/I. Furthermore, pBLCFA-induced antibodies were able to block the adhesive properties of the CFA/I fimbriae, as evaluated by the ability to inhibit the hemagglutination promoted by CFA/I-expressing ETEC cells. These results suggest that secretion of CFA/I encoded by pBLCFA preserves important conformational epitopes required for the generation of protective antibodies against the adhesive properties of the CFA/I fimbriae and open new perspectives for the development of DNA vaccines against enteric bacterial pathogens.

Immunoglobulin G subclass responses in mice immunized with plasmid DNA encoding the CFA/I fimbria of enterotoxigenic Escherichia coli.

Balb/c mice immunized either with a plasmid vector (pRECFA), encoding the CFA/I fimbrial adhesin of enterotoxigenic Escherichia coli (ETEC), or purified CFA/I fimbriae induced similar antibody responses in regard to the kinetics of serum total immunoglobulins and IgG. Nonetheless, the IgG subclass responses in mice vaccinated with DNA were composed predominantly by IgG2a (ranging from 39 to 74% of the total anti-CFA/I IgG) and, to a lesser extent, by IgG (ranging from 15 to 24% of the total anti-CFA/I IgG) during a 12 week observation period. On the other hand, mice immunized with purified CFA/I presented mainly an IgG1 antibody response (ranging from 39 to 67% of the total anti-CFA/I IgG). These results indicated that, irrespective of the immunogenic properties and-or origin of the encoded antigen, immunization with pRECFA elicited an specific IgG subclass response which may affect the ability of DNA vaccines to induce a protective immune response against CFA/I mediated colonization of ETEC bacterial cells.

Changes in Trypanosoma cruzi Kinetoplast DNA Minicircles Induced by Environmental Conditions and Subcloning

ABSTRACT. Reversible changes in kinetoplast DNA (kDNA) minicircles sequences were observed in clones of Trypanosoma cruzi strain Y, following a number of passages during exponential growth phase or after subcloning in blood‐free medium. kDNA restriction patterns of clones were similar to those of the original uncloned strain, while subclones presented distinct kDNA restriction patterns. Homology experiments demonstrated strong hybridization between kDNA with the same electrophoretic mobility patterns while only weak signals were observed with kDNA of different patterns. The changes observed, which are unprecedented in T. cruzi clones, characterize transkinetoplastidy, and seem to be associated with similarly reversible changes both in zymodeme and in infectivity.

New vaccine strategies against enterotoxigenic Escherichia coli: II: Enhanced systemic and secreted antibody responses against the CFA/I fimbriae by priming with DNA and boosting with a live recombinant Salmonella vaccine

The induction of systemic (IgG) and mucosal (IgA) antibody responses against the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC) was evaluated in mice primed with an intramuscularly delivered CFA/I-encoding DNA vaccine followed by two oral immunizations with a live recombinant Salmonella typhimurium vaccine strain expressing the ETEC antigen. The booster effect induced by the oral immunization was detected two weeks and one year after the administration of the DNA vaccine. The DNA-primed/Salmonella-boosted vaccination regime showed a synergistic effect on the induced CFA/I-specific systemic and secreted antibody levels which could not be attained by either immunization strategy alone. These results suggest that the combined use of DNA vaccines and recombinant Salmonella vaccine strains can be a useful immunization strategy against enteric pathogens.

New vaccine strategies against enterotoxigenic Escherichia coli: I: DNA vaccines against the CFA/I fimbrial adhesin

Stimulation of the mammalian immune system by administration of plasmid DNA has been shown to be an important approach for vaccine development against several pathogens. In the present study we investigated the use of DNA vaccines to induce immune responses against an enteric bacterial pathogen, enterotoxigenic Escherichia coli (ETEC). Three plasmid vectors encoding colonization factor antigen 1 (CFA/I), an ETEC fimbrial adhesin, were constructed. Eukaryotic cells transfected with each of these plasmids expressed the heterologous antigen in different compartments: bound to the cytoplasmic membrane (pRECFA), accumulated in the cytoplasm (pPolyCFA) or secreted to the outside medium (pBLCFA). BALB/c mice were intramuscularly (i.m.) inoculated with purified plasmid DNA and the systemic, cellular and secreted CFA/I-specific immune responses were analyzed. The results showed that all three DNA vaccine formulations could elicit CFA/I-specific immune responses. Moreover, cellular location of the plasmid-encoded CFA/I seems to have an important role in the induced immune response. Taken together, these results indicate that DNA vaccines also represent a promising approach against enteric bacterial pathogens.

Genomic variation in Trypanosoma cruzi clonal cultures

Spontaneous changes in restriction DNA profiles and pulsed-field gel electrophoresis (PFGE) patterns, along with a concomitant loss of infectivity, were observed in infective clones of Trypanosoma cruzi strain Y either following a number of passages during the exponential growth phase or after subcloning in liver infusion tryptone (LIT) medium using as the probe a genomic fragment of the parasite (pMYP16), indicating naturally occurring rearrangements of DNA sequences. No variation could be detected when the genomic DNA was probed with conserved T. cruzi tubulin and actin genes. There was no correlation between such rearrangements and the life-cycle forms of the parasites, since trypomas-tigote forms showed the same karyotype and hybridization patterns as did epimastigote forms. The variations observed could be reverted and infectivity, recovered after inoculation of the parasites in newborn mice.

Aspects of T Cell-Mediated Immunity Induced in Mice by a DNA Vaccine Based on the Dengue-NS1 Antigen after Challenge by the Intracerebral Route

Dengue disease has emerged as a major public health issue across tropical and subtropical countries. Infections caused by dengue virus (DENV) can evolve to life-threatening forms, resulting in about 20,000 deaths every year worldwide. Several animal models have been described concerning pre-clinical stages in vaccine development against dengue, each of them presenting limitations and advantages. Among these models, a traditional approach is the inoculation of a mouse-brain adapted DENV variant in immunocompetent animals by the intracerebral (i.c.) route. Despite the historical usage and relevance of this model for vaccine testing, little is known about the mechanisms by which the protection is developed upon vaccination. To cover this topic, a DNA vaccine based on the DENV non-structural protein 1 (pcTPANS1) was considered and investigations were focused on the induced T cell-mediated immunity against i.c.-DENV infection. Immunophenotyping assays by flow cytometry revealed that immunization with pcTPANS1 promotes a sustained T cell activation in spleen of i.c.-infected mice. Moreover, we found that the downregulation of CD45RB on T cells, as an indicator of cell activation, correlated with absence of morbidity upon virus challenge. Adoptive transfer procedures supported by CFSE-labeled cell tracking showed that NS1-specific T cells induced by vaccination, proliferate and migrate to peripheral organs of infected mice, such as the liver. Additionally, in late stages of infection (from the 7th day onwards), vaccinated mice also presented reduced levels of circulating IFN-γ and IL-12p70 in comparison to non-vaccinated animals. In conclusion, this work presented new aspects about the T cell-mediated immunity concerning DNA vaccination with pcTPANS1 and the i.c. infection model. These insights can be explored in further studies of anti-dengue vaccine efficacy.