Vaccination with Ad5 Vectors Expands Ad5-Specific CD8+ T Cells without Altering Memory Phenotype or Functionality

Adenoviral (Ad) vaccine vectors represent both a vehicle to present a novel antigen to the immune system as well as restimulation of immune responses against the Ad vector itself. To what degree Ad-specific CD8(+) T cells are restimulated by Ad vector vaccination is unclear, although such knowledge would be important as vector-specific CD8(+) T cell expansion could potentially further limit Ad vaccine efficacy beyond Ad-specific neutralizing antibody alone.Here we addressed this issue by measuring human Adenovirus serotype 5 (Ad5)-specific CD8(+) T cells in recipients of the Merck Ad5 HIV-1 vaccine vector before, during, and after vaccination by multicolor flow cytometry. Ad5-specific CD8(+) T-cells were detectable in 95% of subjects prior to vaccination, and displayed primarily an effector-type functional profile and phenotype. Peripheral blood Ad5-specific CD8(+) T-cell numbers expanded after Ad5-HIV vaccination in all subjects, but differential expansion kinetics were noted in some baseline Ad5-neutralizing antibody (Ad5 nAb) seronegative subjects compared to baseline Ad5 nAb seropositive subjects. However, in neither group did vaccination alter polyfunctionality, mucosal targeting marker expression, or memory phenotype of Ad5-specific CD8(+) T-cells.These data indicate that repeat Ad5-vector administration in humans expands Ad5-specific CD8(+) T-cells without overtly affecting their functional capacity or phenotypic properties. This is a secondary analysis of samples collected during the 016 trial. Results of the Merck 016 trial safety and immunogenicity have been previously published in the journal of clinical infectious diseases [1].ClinicalTrials.gov NCT00849680[http://www.clinicaltrials.gov/show/NCT00849680]

Immunogenicity of a Prime-Boost Vaccine Containing the Circumsporozoite Proteins of Plasmodium vivax in Rodents

Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. in the case of the deadly parasite P. falciparum, the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I.C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund's adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus- protein) vaccine regimens. the antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. the vaccine formulations described in this work should be useful for the further development of an anti-P. vivax vaccine.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)PNPDCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol CTCMol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWistar Inst Anat & Biol, Philadelphia, PA 19104 USAMalaria Vaccine & Drug Dev Ctr, Cali, ColombiaUniv Fed Santa Catarina, Dept Microbiol Imunol & Parasitol, Florianopolis, SC, BrazilUniv São Paulo, Fac Ciencias Farmaceut, Dept Anal Clin & Toxicol, São Paulo, BrazilNYU, Sch Med, Dept Pathol, Michael Heidelberger Div, New York, NY USAUniversidade Federal de São Paulo, Escola Paulista Med, Ctr Terapia Celular & Mol CTCMol, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 2009/15432-4FAPESP: 2012/13032-5CNPq: 471087/2013-0Web of Scienc

Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D ▿

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8+ T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8+ T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4+ T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8+ T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8+ T-cell responses, measured by intracellular gamma interferon (IFN-γ) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2Db-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 × 105 TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors

Delivery of self-amplifying RNA vaccines in in vitro reconstituted virus-like particles.

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction

Caracterização fenotípica e molecular de genitores de feijão tipo carioca quanto à resistência a patógenos

The present study aimed at the phenotypic and molecular characterization of 31 genotypes of the carioca-type common bean regarding the resistance to anthracnose, rust and angular leaf spot (ALS) pathogens. Thirteen pathotypes of Colletotrichum lindemuthianum, two of Uromyces appendiculatus and seven of Pseudocercospora griseola were inoculated. In the molecular characterization, five molecular markers, identified in advance, and linked to the different alleles of pathogens' resistance, were used. Seven genotypes showed resistance to 12 pathotypes of C. lindemuthianum. Nine genotypes presented resistance to five pathotypes of P. griseola. Ten genotypes were resistant to the pathotypes of U. appendiculatus. The lines VC 2, VC 3 and VC 5, besides Rudá-R (line pyramided with five genes which confer resistance to some pathotypes of anthracnose, rust and ALS), were the stood out for multiple resistance to the pathogens above described. Molecular polymorphism was detected between the most genotypes with Rudá-R, which indicates the possibility of using the molecular markers SCARF10, SCARY20, SCARAZ20, SCARH13 and OPX11.O objetivo deste estudo foi a caracterização fenotípica e molecular de 31 genótipos de feijão do tipo carioca, quanto à resistência aos patógenos da antracnose, ferrugem e mancha-angular. Foram realizadas inoculações com 13 patótipos de Colletotrichum lindemuthianum, dois de Uromyces appendiculatus e sete de Pseudocercospora griseola. Na caracterização molecular, foram utilizados cinco marcadores moleculares previamente identificados, ligados a diferentes alelos de resistência aos patógenos. Sete genótipos apresentaram resistência a 12 patótipos de C. lindemuthianum. Nove genótipos apresentaram resistência a cinco patótipos de P. griseola. Dez genótipos foram resistentes aos patótipos de U. appendiculatus. As linhagens VC 2, VC 3 e VC 5, além da Rudá-R (linhagem piramidada com cinco genes que conferem resistência a alguns patótipos de antracnose, ferrugem e mancha-angular), foram as que se destacaram quanto à resistência múltipla aos patógenos acima citados. Foi detectado polimorfismo molecular entre a maioria dos genótipos com a Rudá-R, o que indica a possibilidade de uso dos marcadores moleculares SCARF10, SCARY20, SCARAZ20, SCARH13 e OPX11

Effect of Preexisting Immunity on an Adenovirus Vaccine Vector: In Vitro Neutralization Assays Fail To Predict Inhibition by Antiviral Antibody In Vivo▿

A major obstacle to the use of adenovirus vectors derived from common human serotypes, such as human adenovirus 5 (AdHu5), is the high prevalence of virus-neutralizing antibodies in the human population. We previously constructed a variant of chimpanzee adenovirus 68 (AdC68) that maintained the fundamental properties of the carrier but was serologically distinct from AdC68 and resisted neutralization by AdC68 antibodies. In the present study, we tested whether this modified vector, termed AdCDQ, could induce transgene product-specific CD8+ T cells in mice with preexisting neutralizing antibody to wild-type AdC68. Contrary to our expectation, the data show conclusively that antibodies that fail to neutralize the AdCDQ mutant vector in vitro nevertheless impair the vector's capacity to transduce cells and to stimulate a transgene product-specific CD8+ T-cell response in vivo. The results thus suggest that in vitro neutralization assays may not reliably predict the effects of virus-specific antibodies on adenovirus vectors in vivo