Improved immunogenicity against HIV-1 clade C antigens by using NYVAC-C with restored replication competence

Background: In order to improve the immunogenicity of currently available non-replicating pox virus HIV vaccine vectors, NYVAC was genetically modified through re-insertion of two host range genes (K1L and C7L), resulting in restored replicative capacity in human cells. Methods: In the present study these vectors, expressing either a combination of the HIV-1 clade C antigens Env, Gag, Pol, Nef, or a combination of Gal, Pol, Nef were evaluated for safety and immunogenicity in rhesus macaques, which were immunized at weeks 0, 4 and 12 either by scarification (conventional poxvirus route of immunization), intradermal or by intramuscular injection (route used in previous vaccine studies). Results: Replication competent NYVAC-C-KC vectors induced higher HIV-specific responses, as measured by IFN-g ELISpot assay, than the replication defective NYVAC-C vectors. Application through scarification only required one immunization to induce maximum HIV-specific immune responses. This method simultaneously induced relatively lower anti-vector responses. In contrast, two to three immunizations were required when the NYVAC-C-KC vectors were given by intradermal or intramuscular injection and this method tended to generate slightly lower responses. Responses were predominantly directed against Env in the animals that received NYVAC-C-KC vectors expressing HIV-1 Env, Gag, Pol, Nef, while Gag responses were dominant in the NYVAC-C-KC HIV-1 Gag, Pol, Nef immunized animals. Conclusion: The current study demonstrates that NYVAC replication competent vectors were well tolerated and showed increased immunogenicity as compared to replication defective vectors. Further studies are needed to evaluate the most efficient route of immunization and to explore the use of these replication competent NYVAC vectors in prime/boost combination with gp120 proteinbased vaccine candidates. This study was performed within the Poxvirus T-cell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Chagas disease: current epidemiological trends after the interruption of vectorial and transfusional transmission in the Southern Cone countries

Chagas disease, named after Carlos Chagas who first described it in 1909, exists only on the American Continent. It is caused by a parasite, Trypanosoma cruzi, transmitted to humans by blood-sucking triatomine bugs and by blood transfusion. Chagas disease has two successive phases, acute and chronic. The acute phase lasts 6 to 8 weeks. After several years of starting the chronic phase, 20% to 35% of the infected individuals, depending on the geographical area will develop irreversible lesions of the autonomous nervous system in the heart, esophagus, colon and the peripheral nervous system. Data on the prevalence and distribution of Chagas disease improved in quality during the 1980's as a result of the demographically representative cross-sectional studies carried out in countries where accurate information was not available. A group of experts met in Brasília in 1979 and devised standard protocols to carry out countrywide prevalence studies on human T. cruzi infection and triatomine house infestation. Thanks to a coordinated multi-country program in the Southern Cone countries the transmission of Chagas disease by vectors and by blood transfusion has been interrupted in Uruguay in1997, in Chile in 1999, and in 8 of the 12 endemic states of Brazil in 2000 and so the incidence of new infections by T. cruzi in the whole continent has decreased by 70%. Similar control multi-country initiatives have been launched in the Andean countries and in Central America and rapid progress has been recorded to ensure the interruption of the transmission of Chagas disease by 2005 as requested by a Resolution of the World Health Assembly approved in 1998. The cost-benefit analysis of the investments of the vector control program in Brazil indicate that there are savings of US$17 in medical care and disabilities for each dollar spent on prevention, showing that the program is a health investment with good return. Since the inception in 1979 of the Steering Committee on Chagas Disease of the Special Program for Research and Training in Tropical Diseases of the World Health Organization (TDR), the objective was set to promote and finance research aimed at the development of new methods and tools to control this disease. The well known research institutions in Latin America were the key elements of a world wide network of laboratories that received - on a competitive basis - financial support for projects in line with the priorities established. It is presented the time line of the different milestones that were answering successively and logically the outstanding scientific questions identified by the Scientific Working Group in 1978 and that influenced the development and industrial production of practical solutions for diagnosis of the infection and disease control.

Ku-deficient yeast strains exhibit alternative states of silencing competence.

In Saccharomyces cerevisiae, efficient silencer function requires telomere proximity, i.e. compartments of the nucleoplasm enriched in silencing factors. Accordingly, silencers located far from telomeres function inefficiently. We show here that cells lacking yKu balance between two mitotically stable states of silencing competence. In one, a partial delocalization of telomeres and silencing factors throughout the nucleoplasm correlates with enhanced silencing at a non-telomeric locus, while in the other, telomeres retain their focal pattern of distribution and there is no repression at the non-telomeric locus, as observed in wild-type cells. The two states also differ in their level of residual telomeric silencing. These findings indicate the existence of a yKu-independent pathway of telomere clustering and Sir localization. Interestingly, this pathway appears to be under epigenetic control.

Improvement of the HIV/AIDS vaccine candidate NYVAC-C by deletion of the viral type I IFN inhibitor and/or acquisition of replication competence

Background: Recombinant viruses based on the attenuated vaccinia virus strain NYVAC are promising HIV vaccine candidates as phase I/II clinical trials have shown good safety and immunogenicity profiles. However, this NYVAC strain is non-replicating in most human cell lines and encodes viral inhibitors of the immune system. Methods: With the aim to increase the immune potency of the current NYVAC-C vector (expressing the codon optimized clade C HIV-1 genes encoding gp120 and Gag-Pol-Nef polyprotein), we have generated and characterized three NYVAC-C-based vectors by, 1) deletion of the viral type I IFN inhibitor gene (NYVAC-CdeltaB19R), 2) restoration of virus replication competence in human cells by re-inserting K1L and C7L host range genes (NYVAC-C-KC) and, 3) combination of both strategies (NYVACC- KC-deltaB19R). Results: Insertion of the KC fragment restored the replication competence of the viruses in human cells (HeLa cells and primary dermal fibroblasts and keratinocytes), increased the expression of HIV antigens by more than 3-fold compared to the non-replicating homologs, inhibited apoptosis induced by the parental NYVAC-C and retained attenuation in a newborn mouse model. In adult mice, replication-competent viruses showed a limited capacity to replicate in tissues surrounding the inoculation site (ovaries and lymph nodes). After infection of keratinocytes, PBMCs and dendritic cells these viruses induced differential modulation in specific host cell signal transduction pathways, triggering genes important in immune modulation. Conclusion: We have developed improved NYVAC-C-based vectors with enhanced HIV-1 antigen expression, with the ability to replicate in cultured human cells and partially in some tissues, with an induced expression of cellular genes relevant to immune system activation, and which trigger IFN-dependent and independent signalling pathways, while maintaining a safety phenotype. These new vectors are promising new HIV vaccine candidates. These studies were performed within the Poxvirus Tcell Vaccine Discovery Consortium (PTVDC) which is part of the CAVD program.

Preliminary characterization of a Rhodnius prolixus hemolymph trypanolytic protein, this being a determinant of Trypanosoma rangeli KP1(+) and KP1(-) subpopulations' vectorial ability

Rhodnius prolixus is the main Trypanosoma rangeli vector in several Latin-American countries and is susceptible to infection with KP1(+) strains; however, it presents an invasion-resistant response to KP1(-) strains. The present work has identified a trypanolytic protein against T. rangeli KP1(-) in the R. prolixus hemolymph which was fractioned with ammonium sulfate (following dialysis). The results revealed a protein component which did not depend on divalent cations for its biological function whilst keeping its trypanolytic activity at temperatures ranging from -20ºC to 37ºC, at 7.0 to 10.5 pH. The protein was partially purified by gel filtration chromatography and ionic exchange chromatography. The major component presented a molecular weight of around 79 kDa and an isoelectric point between 4.9 and 6.3 and may be directly related to hemolymph trypanolytic activity against T. rangeli KP1(-) populations.

Use of geoprocessing to define malaria risk areas and evaluation of the vectorial importance of anopheline mosquitoes (Diptera: Culicidae) in Espírito Santo, Brazil

In Brazil, introduced malaria occurs from the flat to the sloping hot areas, predominantly outside the Amazon Region, where endemic malaria has occurred in the past. This is a consequence of human migrations to other Brazilian states, including the state of Espírito Santo (ES). The objective of this study was to use geoprocessing to define the areas at risk of introduced malaria transmission and evaluate the vectorial importance of species of anophelines in ES. Anophelines were sampled from 1997-2005 in 297 rural localities identified or not identified as foci of malaria during the last 20 years. The geoclimatic variables temperature, relief and marine influence were obtained from a database of the ES Natural Units. The 14,663 anophelines captured belonged to 22 species. A significant association was found between the occurrence of malaria foci and the presence of hot, low-lying areas or gently undulating to undulating relief. The occurrence of the disease was associated with the presence of Anopheles darlingi and Anopheles aquasalis. Geoprocessing was determined to be a useful tool for defining areas at risk for malaria and vectors in ES.

Distribution of sibling species of Anopheles culicifacies s.l. and Anopheles fluviatilis s.l. and their vectorial capacity in eight different malaria endemic districts of Orissa, India

The study was undertaken in eight endemic districts of Orissa, India, to find the members of the species complexes of Anopheles culicifacies and Anopheles fluviatilis and their distribution patterns. The study area included six forested districts (Keonjhar, Angul, Dhenkanal, Ganjam, Nayagarh and Khurda) and two non-forested coastal districts (Puri and Jagatsingpur) studied over a period of two years (June 2007-May 2009). An. culicifacies A, B, C and D and An. fluviatilis S and T sibling species were reported. The prevalence of An. culicifacies A ranged from 4.2-8.41%, B from 54.96-76.92%, C from 23.08-33.62% and D from 1.85-5.94% (D was reported for the first time in Orissa, except for occurrences in the Khurda and Nayagarh districts). The anthropophilic indices (AI) were 3.2-4.8%, 0.5-1.7%, 0.7-1.37% and 0.91-1.35% for A, B, C and D, respectively, whereas the sporozoite rates (SR) were 0.49-0.54%, 0%, 0.28-0.37% and 0.41-0.46% for A, B, C and D, respectively. An. fluviatilis showed a similarly varied distribution pattern in which S was predominant (84.3% overall); its AI and SR values ranged from 60.7-90.4% and 1.2-2.32%, respectively. The study observed that the co-existence of potential vector sibling species of An. culicifacies (A, C and D) and An. fluviatilis S (> 50%) was responsible for the high endemicity of malaria in forested districts such as Dhenkanal, Keonjhar, Angul, Ganjam, Nayagarh and Khurda (> 5% slide positivity rate). Thus, the epidemiological scenario for malaria is dependent on the distribution of the vector sibling species and their vectorial capacity.

Vectorial capacity, basic reproduction number, force of infection and all that: formal notation to complete and adjust their classical concepts and equations

A dimensional analysis of the classical equations related to the dynamics of vector-borne infections is presented. It is provided a formal notation to complete the expressions for the Ross' Threshold Theorem, the Macdonald's basic reproduction "rate" and sporozoite "rate", Garret-Jones' vectorial capacity and Dietz-Molineaux-Thomas' force of infection. The analysis was intended to provide a formal notation that complete the classical equations proposed by these authors.

Single cell analysis reveals similar functional competence of dominant and nondominant CD8 T-cell clonotypes.

Immune protection from infectious diseases and cancer is mediated by individual T cells of different clonal origin. Their functions are tightly regulated but not yet fully characterized. Understanding the contribution of each T cell will improve the prediction of immune protection based on laboratory assessment of T-cell responses. Here we developed techniques for simultaneous molecular and functional assessment of single CD8 T cells directly ex vivo. We studied two groups of patients with melanoma after vaccination with two closely related tumor antigenic peptides. Vaccination induced T cells with strong memory and effector functions, as found in virtually all T cells of the first patient group, and fractions of T cells in the second group. Interestingly, high functionality was not restricted to dominant clonotypes. Rather, dominant and nondominant clonotypes acquired equal functional competence. In parallel, this was also found for EBV- and CMV-specific T cells. Thus, the nondominant clonotypes may contribute similarly to immunity as their dominant counterparts.

Vector biology prospects in dengue research

We argue that using more natural blood feeding methods to study mosquito vector competence for dengue viruses and exploring the effect of viral infection on other mosquito life-history traits that influence vectorial capacity will significantly advance our understanding of dengue epidemiology.

Exendin-(9-39) is an inverse agonist of the murine glucagon-like peptide-1 receptor: implications for basal intracellular cyclic adenosine 3',5'-monophosphate levels and beta-cell glucose competence.

The effect of exendin-(9-39), a described antagonist of the glucagon-like peptide-1 (GLP-1) receptor, was evaluated on the formation of cAMP- and glucose-stimulated insulin secretion (GSIS) by the conditionally immortalized murine betaTC-Tet cells. These cells have a basal intracellular cAMP level that can be increased by GLP-1 with an EC50 of approximately 1 nM and can be decreased dose dependently by exendin-(9-39). This latter effect was receptor dependent, as a beta-cell line not expressing the GLP-1 receptor was not affected by exendin-(9-39). It was also not due to the endogenous production of GLP-1, because this effect was observed in the absence of detectable preproglucagon messenger RNA levels and radioimmunoassayable GLP-1. Importantly, GSIS was shown to be sensitive to this basal level of cAMP, as perifusion of betaTC-Tet cells in the presence of exendin-(9-39) strongly reduced insulin secretion. This reduction of GSIS, however, was observed only with growth-arrested, not proliferating, betaTC-Tet cells; it was also seen with nontransformed mouse beta-cells perifused in similar conditions. These data therefore demonstrated that 1) exendin-(9-39) is an inverse agonist of the murine GLP-1 receptor; 2) the decreased basal cAMP levels induced by this peptide inhibit the secretory response of betaTC-Tet cells and mouse pancreatic islets to glucose; 3) as this effect was observed only with growth-arrested cells, this indicates that the mechanism by which cAMP leads to potentiation of insulin secretion is different in proliferating and growth-arrested cells; and 4) the presence of the GLP-1 receptor, even in the absence of bound peptide, is important for maintaining elevated intracellular cAMP levels and, therefore, the glucose competence of the beta-cells.