IgA quantification as a good predictor of the neutralizing antibodies levels after vaccination against SARS-CoV-2.

Background: Vaccination against COVID-19 was implemented very quickly, but the emergence of new variants that can evade the previous acquired immunological protection highlights the importance of understanding the mechanisms involved in the immune response generated after SARS-CoV-2 infection or vaccination. Objectives: Since most of our knowledge on the humoral immunity generated against SARS-CoV-2 has been obtained from studies with infected patients before vaccination, our goal here was to evaluate seroconversion and its correlation with the titers of neutralizing antibodies (NAbs) in individuals who received the complete initial recommended vaccination schedule with three different vaccines. Study design: We analyzed serum IgG, IgA and total NAbs against the trimeric SARS-CoV-2 Spike (S) protein or its receptor binding domain (RBD) in blood samples collected from 118 healthy individuals without known previous infection, before and after receiving the first and the second dose of CoronaVac (n = 18), ChAdOx-1 (n = 68) or BNT162b2 (n = 32) vaccines. Results: We found that although IgG titers were high in all sera collected after the two doses of these vaccines, NAbs amounts varies among the groups. In contrast, serum NAbs concentrations were much more comparable to the IgA levels, indicating that these antibodies would have a major neutralizing capacity against SARS-CoV-2. Conclusions: Altogether our data suggest that quantification of serum anti-S or anti-RBD IgA, rather than IgG, may be a valuable tool to screen NAbs and may be considered for surveillance of vaccine coverage.

Combination of E- and NS1-Derived DNA Vaccines: The Immune Response and Protection Elicited in Mice against DENV2.

The occurrence of dengue disease has increased radically in recent decades. Previously, we constructed the pE1D2 and pcTPANS1 DNA vaccines encoding the DENV2 envelope (E) and non-structural 1 (NS1) proteins, respectively. To decrease the number of plasmids in a tetravalent candidate vaccine, we constructed a bicistronic plasmid, pNS1/E/D2, encoding these two proteins simultaneously. We evaluated the protective immunity induced in mice vaccinated with the pNS1/E/D2 candidate and compared to the responses elicited by immunization with the former vaccines isolated or in combination. We transfected BHK-21 cells with the different plasmids and detected recombinant proteins by immunofluorescence and mass spectrometry assays to confirm antigen expression. BALB/c mice were inoculated with the DNA vaccines followed by a lethal DENV2 challenge. ELISA, PRNT50, and IFN-gamma ELISPOT assays were performed for the investigation of the humoral and cellular responses. We observed the concomitant expression of NS1 and E proteins in pNS1/E/D2-transfected cells. All E-based vaccines induced anti-E and neutralizing antibodies. However, anti-NS1 antibodies were only observed after immunization with the pcTPANS1 administered alone or combined with pE1D2. In contrast, splenocytes from pNS1/E/D2- or pcTPANS1 + pE1D2-vaccinated animals responded to NS1- and E-derived synthetic peptides. All the DNA vaccines conferred protection against DENV2.

Contact System Activation in Plasma from Dengue Patients Might Harness Endothelial Virus Replication through the Signaling of Bradykinin Receptors.

Since exacerbated inflammation and microvascular leakage are hallmarks of dengue virus (DENV) infection, here we interrogated whether systemic activation of the contact/kallikrein-kinin system (KKS) might hamper endothelial function. In vitro assays showed that dextran sulfate, a potent contact activator, failed to generate appreciable levels of activated plasma kallikrein (PKa) in the large majority of samples from a dengue cohort (n = 70), irrespective of severity of clinical symptoms. Impaired formation of PKa in dengue-plasmas correlated with the presence of cleaved Factor XII and high molecular weight kininogen (HK), suggesting that the prothrombogenic contact system is frequently triggered during the course of infection. Using two pathogenic arboviruses, DENV or Zika virus (ZIKV), we then asked whether exogenous BK could influence the outcome of infection of human brain microvascular endothelial cells (HBMECs). Unlike the unresponsive phenotype of Zika-infected HBMECs, we found that BK, acting via B2R, vigorously stimulated DENV-2 replication by reverting nitric oxide-driven apoptosis of endothelial cells. Using the mouse model of cerebral dengue infection, we next demonstrated that B2R targeting by icatibant decreased viral load in brain tissues. In summary, our study suggests that contact/KKS activation followed by BK-induced enhancement of DENV replication in the endothelium may underlie microvascular pathology in dengue.

Sofosbuvir shows a protective effect against vertical transmission of Zika virus and the associated congenital syndrome in rhesus monkeys.

The outbreaks of Zika virus (ZIKV) infection in Brazil, 2015-2016, were associated with severe congenital malformations. Our translational study aimed to test the efficacy of the antiviral agent sofosbuvir (SOF) against vertical transmission of ZIKV and the associated congenital syndrome (CZS), using a rhesus monkey model. Eight pregnant macaques were successfully infected during the organogenesis phase with a Brazilian ZIKV strain; five of them received SOF from two to fifteen days post-infection. Both groups of dams showed ZIKV-associated clinical signals, detectable ZIKV RNA in several specimens, specific anti-ZIKV IgM and IgG antibodies, and maternal neutralizing antibodies. However, malformations occurred only among non-treated dam offspring. Compared to non-treated animals, all SOF-treated dams had a shorter ZIKV viremia and four of five neonates had undetectable ZIKV RNA in blood and tissue samples. These results support further clinical evaluations aiming for the prevention of CZS.

T Cell Responses Induced by DNA Vaccines Based on the DENV2 E and NS1 Proteins in Mice: Importance in Protection and Immunodominant Epitope Identification.

The importance of the cellular immune response against DENV has been increasingly highlighted in the past few years, in particular for vaccine development. We have previously constructed two plasmids, pE1D2, and pcTPANS1, encoding the envelope (E) ectodomain (domains I, II, and III) and the non-structural 1 (NS1) protein of dengue virus serotype 2 (DENV2), respectively. In the present work, we analyzed the induction of the cellular response in mice immunized with these DNA vaccines and identified the immunogenic peptides. Vaccinated BALB/c mice became protected against a lethal challenge of DENV2. Depletion of CD4+ cells in vaccinated animals almost completely abolished protection elicited by both vaccines. In contrast, a significant number of pE1D2- and pcTPANS1-immunized mice survived virus challenge after depletion of CD8+ cells, although some animals presented morbidity. To identify immunogenic peptides recognized by T cells, we stimulated splenocytes with overlapping peptide libraries covering the E and NS1 proteins and evaluated the production of IFN-γ by ELISPOT. We detected two and three immunodominant epitopes in the E and NS1 proteins, respectively, and four additional NS1-derived peptides after virus challenge. Characterization by intracellular cytokine staining (ICS) revealed that both CD4+ and CD8+ T cells were involved in IFN-γ and TNF-α production. The IFN-γ ICS confirmed reaction of almost all E-derived peptides before challenge and identified other epitopes after infection. All NS1-derived peptides were able to elicit IFN-γ production in CD4+ cells, while only a few peptides induced expression of this cytokine in CD8+ T lymphocytes. Interestingly, we observed an increase in the frequency of either CD4+ or CD8+ T cells producing TNF-α after immunization with the pE1D2 and challenge with DENV2, while lymphocytes from pcTPANS1-vaccinated animals maintained ordinary TNF-α production after virus infection. We also assessed the recognition of E and NS1 immunogenic peptides in C57BL/6 mice due to the difference in MHC haplotype expression. Two NS1-derived epitopes featured prominently in the IFN-γ response with cells from both animal strains. Overall, our results emphasize the importance of the T cell response involved in protection against dengue induced by E and NS1 based DNA vaccines.

BALB/c mice infected with DENV-2 strain 66985 by the intravenous route display injury in the central nervous system.

Dengue is a mild flu-like arboviral illness caused by dengue virus (DENV) that occurs in tropical and subtropical countries. An increasing number of reports have been indicating that dengue is also associated to neurological manifestations, however, little is known regarding the neuropathogenesis of the disease. Here, using BALB/c mice intravenously infected with DENV-2 strain 66985, we demonstrated that the virus is capable of invading and damaging the host's central nervous system (CNS). Brain and cerebellum of infected animals revealed histological alterations such as the presence of inflammatory infiltrates, thickening of pia matter and disorganization of white matter. Additionally, it was also seen that infection lead to altered morphology of neuroglial cells and apoptotic cell death. Such observations highlighted possible alterations that DENV may promote in the host's CNS during a natural infection, hence, helping us to better understand the neuropathological component of the disease.

Dataset of proteins mapped on HepG2 cells and those differentially abundant after expression of the dengue non-structural 1 protein.

The data supplied in this article are related to the research article entitled "The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach" (K. Rabelo, M.R. Trugillo, S.M. Costa, B.A. Pereira, O.C. Moreira, A.T. Ferreira et al., 2016) [1]. The present article provides the inventory of peptides and proteins mapped in a hepatocyte cell line (HepG2) by mass spectrometry in the presence of the non-structural protein 1 (NS1) of Dengue 2 virus (DENV2). Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) or pMAXGFP, encoding the green fluorescent protein (GFP), a protein unrelated to dengue. Differentially abundant protein lists were obtained by comparing cells transfected with pcENS1 and controls.

The effect of the dengue non-structural 1 protein expression over the HepG2 cell proteins in a proteomic approach.

Dengue is an important mosquito borne viral disease in the world. Dengue virus (DENV) encodes a polyprotein, which is cleaved in ten proteins, including the non-structural protein 1 (NS1). In this work, we analyzed the effect of NS1 expression in one hepatic cell line, HepG2, through a shotgun proteomic approach. Cells were transfected with pcENS1 plasmid, which encodes the DENV2 NS1 protein, or the controls pcDNA3 (negative control) and pMAXGFP (GFP, a protein unrelated to dengue). Expression of NS1 was detected by immunofluorescence, western blot and flow cytometry. We identified 14,138 peptides that mapped to 4,756 proteins in all analyzed conditions. We found 41 and 81 differentially abundant proteins when compared to cells transfected with plasmids pcDNA3 and pMAXGFP, respectively. Besides, 107 proteins were detected only in the presence of NS1. We identified clusters of proteins involved mainly in mRNA process and viral RNA replication. Down regulation expression of one protein (MARCKS), identified by the proteomic analysis, was also confirmed by real time PCR in HepG2 cells infected with DENV2. Identification of proteins modulated by the presence of NS1 may improve our understanding of its role in virus infection and pathogenesis, contributing to development of new therapies and vaccines. BIOLOGICAL SIGNIFICANCE: Dengue is an important viral disease, with epidemics in tropical and subtropical regions of the world. The disease is complex, with different manifestations, in which the liver is normally affected. The NS1 is found in infected cells associated with plasma membrane and secreted into the circulation as a soluble multimer. This protein is essential for virus viability, although its function is not elucidated. Some reports indicate that the NS1 can be used as a protective antigen for the development of a dengue vaccine, while others suggest its involvement in viral pathogenesis. In this work, we report an in-depth comprehensive proteomic profiling resulting from the presence of NS1 in HepG2 cells. These results can contribute to a better understanding of the NS1 role during infection.

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.

Cooperation between CD4+ T Cells and Humoral Immunity Is Critical for Protection against Dengue Using a DNA Vaccine Based on the NS1 Antigen.

Dengue virus (DENV) is spread through most tropical and subtropical areas of the world and represents a serious public health problem. At present, the control of dengue disease is mainly hampered by the absence of antivirals or a vaccine, which results in an estimated half worldwide population at risk of infection. The immune response against DENV is not yet fully understood and a better knowledge of it is now recognized as one of the main challenge for vaccine development. In previous studies, we reported that a DNA vaccine containing the signal peptide sequence from the human tissue plasminogen activator (t-PA) fused to the DENV2 NS1 gene (pcTPANS1) induced protection against dengue in mice. In the present work, we aimed to elucidate the contribution of cellular and humoral responses elicited by this vaccine candidate for protective immunity. We observed that pcTPANS1 exerts a robust protection against dengue, inducing considerable levels of anti-NS1 antibodies and T cell responses. Passive immunization with anti-NS1 antibodies conferred partial protection in mice infected with low virus load (4 LD50), which was abrogated with the increase of viral dose (40 LD50). The pcTPANS1 also induced activation of CD4+ and CD8+ T cells. We detected production of IFN-γ and a cytotoxic activity by CD8+ T lymphocytes induced by this vaccine, although its contribution in the protection was not so evident when compared to CD4+ cells. Depletion of CD4+ cells in immunized mice completely abolished protection. Furthermore, transfer experiments revealed that animals receiving CD4+ T cells combined with anti-NS1 antiserum, both obtained from vaccinated mice, survived virus infection with survival rates not significantly different from pcTPANS1-immunized animals. Taken together, results showed that the protective immune response induced by the expression of NS1 antigen mediated by the pcTPANS1 requires a cooperation between CD4+ T cells and the humoral immunity.

The pathology of severe dengue in multiple organs of human fatal cases: histopathology, ultrastructure and virus replication.

Dengue is a public health problem, with several gaps in understanding its pathogenesis. Studies based on human fatal cases are extremely important and may clarify some of these gaps. In this work, we analyzed lesions in different organs of four dengue fatal cases, occurred in Brazil. Tissues were prepared for visualization in optical and electron microscopy, with damages quantification. As expected, we observed in all studied organ lesions characteristic of severe dengue, such as hemorrhage and edema, although other injuries were also detected. Cases presented necrotic areas in the liver and diffuse macro and microsteatosis, which were more accentuated in case 1, who also had obesity. The lung was the most affected organ, with hyaline membrane formation associated with mononuclear infiltrates in patients with pre-existing diseases such as diabetes and obesity (cases 1 and 2, respectively). These cases had also extensive acute tubular necrosis in the kidney. Infection induced destruction of cardiac fibers in most cases, with absence of nucleus and loss of striations, suggesting myocarditis. Spleens revealed significant destruction of the germinal centers and atrophy of lymphoid follicles, which may be associated to decrease of T cell number. Circulatory disturbs were reinforced by the presence of megakaryocytes in alveolar spaces, thrombus formation in glomerular capillaries and loss of endothelium in several tissues. Besides histopathological and ultrastructural observations, virus replication were investigated by detection of dengue antigens, especially the non-structural 3 protein (NS3), and confirmed by the presence of virus RNA negative strand (in situ hybridization), with second staining for identification of some cells. Results showed that dengue had broader tropism comparing to what was described before in literature, replicating in hepatocytes, type II pneumocytes and cardiac fibers, as well as in resident and circulating monocytes/macrophages and endothelial cells.

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.

The synergistic effect of combined immunization with a DNA vaccine and chimeric yellow fever/dengue virus leads to strong protection against dengue.

The dengue envelope glycoprotein (E) is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2) and a chimeric yellow fever/dengue 2 virus (YF17D-D2). The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.

Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver chemokines in dengue fever.

Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1ß but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed.

Induction of a protective response in mice by the dengue virus NS3 protein using DNA vaccines.

The dengue non-structural 3 (NS3) is a multifunctional protein, containing a serino-protease domain, located at the N-terminal portion, and helicase, NTPase and RTPase domains present in the C-terminal region. This protein is considered the main target for CD4+ and CD8+ T cell responses during dengue infection, which may be involved in protection. However, few studies have been undertaken evaluating the use of this protein as a protective antigen against dengue, as well as other flavivirus. In the present work, we investigate the protective efficacy of DNA vaccines based on the NS3 protein from DENV2. Different recombinant plasmids were constructed, encoding either the full-length NS3 protein or only its functional domains (protease and helicase), fused or not to a signal peptide (t-PA). The recombinant proteins were successfully expressed in transfected BHK-21 cells, and only plasmids encoding the t-PA signal sequence mediated protein secretion. Balb/c mice were immunized with the different DNA vaccines and challenged with a lethal dose of DENV2. Most animals immunized with plasmids encoding the full-length NS3 or the helicase domain survived challenge, regardless of the presence of the t-PA. However, some mice presented clinical signs of infection with high morbidity (hind leg paralysis and hunched posture), mainly in animal groups immunized with the DNA vaccines based on the helicase domain. On the other hand, inoculation with plasmids encoding the protease domain did not induce any protection, since mortality and morbidity rates in these mouse groups were similar to those detected in the control animals. The cellular immune response was analyzed by ELISPOT with a specific-CD8+ T cell NS3 peptide. Results revealed that the DNA vaccines based on the full-length protein induced the production of INF-γ, thus suggesting the involvement of this branch of the immune system in the protection.

DNA vaccines against dengue virus type 2 based on truncate envelope protein or its domain III.

Two DNA vaccines were constructed encoding the ectodomain (domains I, II and III) of the DENV2 envelope protein (pE1D2) or only its domain III (pE2D2), fused to the human tissue plasminogen activator signal peptide (t-PA). The expression and secretion of recombinant proteins was confirmed in vitro in BHK cells transfected with the two plasmids, detected by immunofluorescence or immunoprecipitation of metabolically labeled gene products, using polyclonal and monoclonal antibodies against DENV2. Besides, results reveal that the ectodomain of the E protein can be efficiently expressed in vivo, in a mammalian system, without the prM protein that is hypothesized to act as a chaperonin during dengue infection. Balb/c mice were immunized with the DNA vaccines and challenged with a lethal dose of DENV2. All pE1D2-vaccinated mice survived challenge, while 45% of animals immunized with the pE2D2 died after infection. Furthermore, only 10% of pE1D2-immunized mice presented some clinical signs of infection after challenge, whereas most of animals inoculated with the pE2D2 showed effects of the disease with high morbidity degrees. Levels of neutralizing antibodies were significantly higher in pE1D2-vaccinated mice than in pE2D2-immunized animals, also suggesting that the pE1D2 vaccine was more protective than the pE2D2.

New chemical method of viral inactivation for vaccine development based on membrane fusion inhibition.

Membrane fusion is an essential step in the entry of enveloped viruses into their host cells. This process is triggered by conformational changes in viral surface glycoproteins. We have demonstrated previously that modification of vesicular stomatitis virus (VSV) with diethylpyrocarbonate (DEPC) abolished the conformational changes on VSV glycoprotein and the fusion reaction induced by the virus. Moreover, we observed that viral treatment with DEPC inactivates the virus, preserving the conformational integrity of its surface proteins. In the present work, we evaluated the potential use of DEPC as a viral inactivating chemical agent for the development of useful vaccines. Pathogenicity and viral replication in Balb/c mice were abolished by viral treatment with 0.5mM DEPC. In addition, antibodies elicited in mice after intraperitoneal immunization with DEPC-inactivated VSV mixed with adjuvants were able to recognize and neutralize the native virus and efficiently protected animals against the challenge with lethal doses of VSV. These results together suggest that viral inactivation with DEPC seems to be a suitable method for the development of safe vaccines.

Protection against dengue type 2 virus induced in mice immunized with a DNA plasmid encoding the non-structural 1 (NS1) gene fused to the tissue plasminogen activator signal sequence.

Dengue is one of the most important arboviral diseases in humans, and although efforts over the last decades have dealt with the development of a vaccine, this vaccine is not available yet. In order to evaluate the potential of a DNA vaccine based on the non-structural 1 (NS1) protein against dengue virus (DENV), we constructed the pcTPANS1 plasmid which contains the secretory signal sequence derived from human tissue plasminogen activator (t-PA) fused to the full length of the DENV-2 NS1 gene. Results indicate that pcTPANS1 promotes correct expression of NS1 in eukaryotic cells and drives secretion of the recombinant protein to the surrounding medium in a dimeric form. Balb/c mice, intramuscularly inoculated with this plasmid, presented high levels of antibodies, recognizing mainly surface-exposed conformational epitopes present in the NS1 protein expressed by insect cells. Long-term antibody response was observed in animals 56 weeks after the first plasmid inoculation, and a rapid, efficient secondary response was observed after a DNA boost. Vaccinated animals were challenged against DENV-2 in two murine models, based on intracerebral (i.c.) and intraperitoneal (i.p.) virus inoculations, and in both cases, pcTPANS1-immunized mice were protected. Overall, these results provide further support for the use of such a plasmid in a possible approach for the development of a vaccine against DENV.

Antibody-inducing properties of a prototype bivalent herpes simplex virus/enterotoxigenic Escherichia coli DNA vaccine.

The antibody-inducing properties of a bacterial/viral bivalent DNA vaccine (pRECFA), expressing a peptide composed of N- and C-terminal amino acid sequences of the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) fused with an inner segment encoding the major structural subunit of enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae (CFA/I), was evaluated in BALB/c mice following intramuscular immunization. The bivalent pRECFA vaccine elicited serum antibody responses, belonging mainly to the IgG2a subclass, against both CFA/I and HSV gD proteins. pRECFA-elicited antibody responses cross-reacted with homologous and heterologous ETEC fimbrial antigens as well as with type 1 and type 2 HSV gD proteins, which could bind and inactivate intact HSV-2 particles. On the other hand, CFA/I-specific antibodies could bind but did not neutralize the adhesive functions of the bacterial CFA/I fimbriae. In spite of the functional restriction of the antibodies targeting the bacterial antigen, the present evidence suggests that fusion of heterologous peptides to the HSV gD protein represents an alternative for the design of bivalent DNA vaccines able to elicit serum antibody responses.