Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.CNPqINCTVFAPESPUS

Protective immunity to DENV2 after immunization with a recombinant NS1 protein using a genetically detoxified heat-labile toxin as an adjuvant

Submitted by Sandra Infurna ([email protected]) on 2016-08-25T10:36:11Z No. of bitstreams: 1 ada_alves_etal_IOC_2012.pdf: 817391 bytes, checksum: 7aa356e7eacf65a8a014b1d9c58c833a (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-08-25T10:48:19Z (GMT) No. of bitstreams: 1 ada_alves_etal_IOC_2012.pdf: 817391 bytes, checksum: 7aa356e7eacf65a8a014b1d9c58c833a (MD5)Made available in DSpace on 2016-08-25T10:48:19Z (GMT). No. of bitstreams: 1 ada_alves_etal_IOC_2012.pdf: 817391 bytes, checksum: 7aa356e7eacf65a8a014b1d9c58c833a (MD5) Previous issue date: 2012Universidade de São Paulo. Departamento de Microbiologia. Laboratório de Desenvolvimento de Vacinas. São Paulo, SP, Brasil.Universidade de São Paulo. Departamento de Microbiologia. Laboratório de Desenvolvimento de Vacinas. São Paulo, SP, Brasil.Universidade de São Paulo. Departamento de Microbiologia. Laboratório de Desenvolvimento de Vacinas. São Paulo, SP, Brasil.Universidade de São Paulo. Departamento de Microbiologia. Laboratório de Desenvolvimento de Vacinas. São Paulo, SP, Brasil.Universidade de São Paulo. Departamento de Microbiologia. Laboratório de Desenvolvimento de Vacinas. São Paulo, SP, Brasil / Universidade Estadual de Santa Cruz. Ilhéus, BA, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biotecnologia e Fisiologia de Infecções Virais. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biotecnologia e Fisiologia de Infecções Virais. Rio de Janeiro, RJ, Brasil.Universidade de São Paulo. Departamento de Microbiologia. Laboratório de Desenvolvimento de Vacinas. São Paulo, SP, Brasil.The dengue virus non-structural 1 (NS1) protein contributes to evasion of host immune defenses and represents a target for immune responses. Evidences generated in experimental models, as well as the immune responses elicited by infected individuals, showed that induction of anti-NS1 immunity correlates with protective immunity but may also result in the generation of cross-reactive antibodies that recognize platelets and proteins involved in the coagulation cascade. In the present work, we evaluated the immune responses, protection to type 2 dengue virus (DENV2) challenges and safety parameters in BALB/c mice vaccinated with a recombinant NS1 protein in combination with three different adjuvants: aluminum hydroxide (alum), Freund's adjuvant (FA) or a genetically detoxified derivative of the heat-labile toxin (LT(G33D)), originally produced by some enterotoxigenic Escherichia coli (ETEC) strains. Mice were subcutaneously (s.c.) immunized with different vaccine formulations and the induced NS1-specific responses, including serum antibodies and T cell responses, were measured. Mice were also subjected to lethal challenges with the DENV2 NGC strain. The results showed that maximal protective immunity (50%) was achieved in mice vaccinated with NS1 in combination with LT(G33D). Analyses of the NS1-specific immune responses showed that the anti-virus protection correlated mainly with the serum anti-NS1 antibody responses including higher avidity to the target antigen. Mice immunized with LT(G33D) elicited a prevailing IgG2a subclass response and generated antibodies with stronger affinity to the antigen than those generated in mice immunized with the other vaccine formulations. The vaccine formulations were also evaluated regarding induction of deleterious side effects and, in contrast to mice immunized with the FA-adjuvanted vaccine, no significant hepatic damage or enhanced C-reactive protein levels were detected in mice immunized with NS1 and LT(G33D.) Similarly, no detectable alterations in bleeding time and hematological parameters were detected in mice vaccinated with NS1 and LT(G33D). Altogether, these results indicate that the combination of a purified recombinant NS1 and a nontoxic LT derivative is a promising alternative for the generation of safe and effective protein-based anti-dengue vaccine

Construction of bicistronic DNA vaccines encoding HPV, HIV and HSV antigens for expression in mammalian cells.

<p>(A) Schematic linear representation of the trivalent DNA vaccines. pIRES I and pIRES II contain gDp24 and gDE7 chimeric gene fusions, which are inverted with regard to the CMV promoter and IRES sequence. The empty vector pIRES Ø was used as a control. The nucleotide numbers corresponding to the IRES sequence and the cloned chimeric genes are indicated. (B) In vitro expression of the chimeric proteins encoded by pIRES I (left panels) and pIRES II (right panels). Non-permeabilized pIRES I- or pIRES II-transfected COS-7 cells were labeled with antigen-specific antibodies for the simultaneous detection of the HSV-1 protein gD and the HIV-1 protein p24 or the HPV-16 oncoprotein E7. Green, gD; red, p24 or E7; yellow, co-localization of gD with p24 or E7; blue, DAPI nuclear staining.</p

Induction of <i>in vivo</i> E7- and p24-specific cytolytic CD8⁺ T cell responses in immunized mice.

<p>(A–B) <i>In vivo</i> antigen-specific CD8⁺ T cell-dependent cytotoxic responses in the vaccinated mice was measured two weeks after the last immunization dose. Spleen cells from BALB/c or C57BL/6 mice were labeled with CFSE and pulsed with synthetic peptides representing the immunodominant MHC-I-restricted epitopes of p24 (A) or E7 (B). Data shown in A and B represent the compilation of two independent experiments, encompassing four and five mice per group (n = 9) with results based on the response of each animal. (C) The protective immunity elicited in BALB/c mice immunized with pIRES I or pIRES II was measured after challenge with a recombinant vaccinia virus expressing the HIV-1 protein Gag. Female BALB/c mice were challenged with 2×10⁶ P.F.U. of rVV-Gag, and 5 days later, the level of viable vaccinia virus in the ovaries was determined after titration in Vero cells. Data shown in C represent the compilation of two independent experiments carried out with pooled samples from five mice per group. *p<i><</i>0.05. (D) Prophylactic and (E) therapeutic anti-tumor immunity in C57BL/6 mice immunized with pIRES I or PIRES II. The prophylactic anti-tumor effects were determined in five vaccinated female mice after the s.c. transplantation of 7.5×10⁴TC-1 cells two weeks after the last vaccination. The therapeutic anti-tumor effects induced by the vaccines were determined after transplantation of 7.5×10⁴TC-1 cells one day before the administration of the first vaccine dose. Data shown in D and E represent the compilation of two independent experiments, with five mice per group. The survival curves D and E raised <i>p</i> values of 0.0001 and 0.0006, respectively, in the Logrank test for trend. pIRES is the empty vector used as immunization control.</p

Activation of antigen-specific IFN-γ-producing CD8+ T cell precursors in mice immunized with pIRES I or pIRES II.

<p>(A-B) Spleen cells from BALB/c mice spleen cells were stimulated with the MHC-I-restricted p24-specific peptide, and the p24-specific IFN-γ-producing CD8⁺ T cells were detected by intracellular cytokine staining (A) or ELISPOT assay (B). (C–D) Spleen cells from C57BL/6 mice were stimulated with the MHC-I-restricted E7-specific peptide, and the E7-specific IFN-γ-producing CD8⁺ T cells were detected by IFN-γ intracellular staining (C) or ELISPOT assay (D). Mice were i.m. immunized with three doses of the DNA vaccines with one week intervals between doses (100 µg/dose). The CD8⁺ T-cell responses were analyzed two weeks after the last dose. *p<0.05. Data represent the compilation of two independent experiments with four mice per immunization group (n = 8) and results expressed by each animal analyzed. pIRES is the empty vector used as immunization control.</p