Rafael Ribeiro Almeida

A DNA Vaccine Encoding Multiple HIV CD4 Epitopes Elicits Vigorous Polyfunctional, Long-Lived CD4+ and CD8+ T Cell Responses

T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4+ T cells are important for the generation and maintenance of functional CD8+ cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18), capable of eliciting broad CD4+ T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4+/CD8+ T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4+ and CD8+ T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2) simultaneously in response to HIV-1 peptides. For CD4+ T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2). The vaccine also generated long-lived central and effector memory CD4+ T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4+ T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8+ T cells and antibody responses- elicited by other HIV immunogens.

Innate Sensing of the Gut Microbiota: Modulation of Inflammatory and Autoimmune Diseases.

The mammalian gastrointestinal tract harbors a diverse microbial community with which dynamic interactions have been established over millennia of coevolution. Commensal bacteria and their products are sensed by innate receptors expressed in gut epithelia and in gut-associated immune cells, thereby promoting the proper development of mucosal immune system and host homeostasis. Many studies have demonstrated that host–microbiota interactions play a key role during local and systemic immunity. Therefore, this review will focus on how innate sensing of the gut microbiota and their metabolites through inflammasome and toll-like receptors impact the modulation of a distinct set of inflammatory and autoimmune diseases. We believe that a better understanding of the fine-tuning that governs host–microbiota interactions will further improve common prophylactic and therapeutic applications.

IL-10-Producing Regulatory B Cells Are Decreased in Patients with Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency in adults. CVID patients often present changes in the frequency and function of B lymphocytes, reduced number of Treg cells, chronic immune activation, recurrent infections, high incidence of autoimmunity and increased risk for malignancies. We hypothesized that the frequency of B10 cells would be diminished in CVID patients because these cells play an important role in the development of Treg cells and in the control of T cell activation and autoimmunity. Therefore, we evaluated the frequency of B10 cells in CVID patients and correlated it with different clinical and immunological characteristics of this disease. Forty-two CVID patients and 17 healthy controls were recruited for this study. Cryopreserved PBMCs were used for analysis of T cell activation, frequency of Treg cells and characterization of B10 cells by flow cytometry. IL-10 production by sorted B cells culture and plasma sCD14 were determined by ELISA. We found that CVID patients presented decreased frequency of IL-10-producing CD24hiCD38hi B cells in different cell culture conditions and decreased frequency of IL-10-producing CD24hiCD27+ B cells stimulated with CpG+PIB. Moreover, we found that CVID patients presented lower secretion of IL-10 by sorting-purified B cells when compared to healthy controls. The frequency of B10 cells had no correlation with autoimmunity, immune activation and Treg cells in CVID patients. This work suggests that CVID patients have a compromised regulatory B cell compartment which is not correlated with clinical and immunological characteristics presented by these individuals.

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.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Modulating APOBEC expression enhances DNA vaccine immunogenicity.

DNA vaccines have failed to induce satisfactory immune responses in humans. Several mechanisms of double‐stranded DNA (dsDNA) sensing have been described, and modulate DNA vaccine immunogenicity at many levels. We hypothesized that the immunogenicity of DNA vaccines in humans is suppressed by APOBEC (apolipoprotein B (APOB) mRNA‐editing, catalytic polypeptide)‐mediated plasmid degradation. We showed that plasmid sensing via STING (stimulator of interferon (IFN) genes) and TBK‐1 (TANK‐binding kinase 1) leads to IFN‐β induction, which results in APOBEC3A mRNA upregulation through a mechanism involving protein kinase C signaling. We also showed that murine APOBEC2 expression in HEK293T cells led to a 10‐fold reduction in intracellular plasmid levels and plasmid‐encoded mRNA, and a 2.6‐fold reduction in GFP‐expressing cells. A bicistronic DNA vaccine expressing an immunogen and an APOBEC2‐specific shRNA efficiently silenced APOBEC2 both in vitro and in vivo, increasing the frequency of induced IFN‐γ‐secreting T cells. Our study brings new insights into the intracellular machinery involved in dsDNA sensing and how to modulate it to improve DNA vaccine immunogenicity in humans.

T Cells Target APOBEC3 Proteins in Human Immunodeficiency Virus Type 1-Infected Humans and Simian Immunodeficiency Virus-Infected Indian Rhesus Macaques

ABSTRACT APOBEC3 proteins mediate potent antiretroviral activity by hypermutating the retroviral genome during reverse transcription. To counteract APOBEC3 and gain a replicative advantage, lentiviruses such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) have evolved the Vif protein, which targets APOBEC3 proteins for proteasomal degradation. However, the proteasome plays a critical role in the generation of T cell peptide epitopes. Whether Vif-mediated destruction of APOBEC3 proteins leads to the generation and presentation of APOBEC3-derived T cell epitopes on the surfaces of lentivirus-infected cells remains unknown. Here, using peptides derived from multiple Vif-sensitive APOBEC3 proteins, we identified APOBEC3-specific T cell responses in both HIV-1-infected patients and SIV-infected rhesus macaques. These results raise the possibility that these T cell responses may be part of the larger antiretroviral immune response.

P19-44. Priming with a DNA vaccine enconding HIV CD4+ T cell epitopes enhances responses against subsequent immunization with plasmid encoding Vif

Background Since none of the developed candidate vaccines against HIV has been effective, novel vaccine concepts are needed. Our group has identified conserved HIV CD4+ T cell epitopes binding promiscuously to multiple HLA-DR molecules, and frequently recognized by HIV-1-infected patients. A DNA vaccine encoding such epitopes (pVAXHIVBr18) showed strong cellular responses in mice. An early CD8+ T cell response against Vif is associated with control of SIV infection in primates. A vaccine capable of stimulating both CD4+ and CD8+ T cells can be useful to establish an appropriate immune control of HIV. We aim to test whether preimmunization with pVAX-HIVBr18, which contains the CD4/CD8 Vif (144-158) epitope, can boost Vif-specific CD8+ T cell responses after immunization with a plasmid encoding Vif (pVAX-Vif).