Susan Pereira Ribeiro

A Vaccine Encoding Conserved Promiscuous HIV CD4 Epitopes Induces Broad T Cell Responses in Mice Transgenic to Multiple Common HLA Class II Molecules

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, “promiscuous” (multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II- transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.

CD4+ T Cell Epitope Discovery and Rational Vaccine Design

T cell epitope-driven vaccine design employs bioinformatic algorithms to identify potential targets of vaccines against infectious diseases or cancer. Potential epitopes can be identified with major histocompatibility complex (MHC)-binding algorithms, and the ability to bind to MHC class I or class II indicates a predominantly CD4+ or CD8+ T cell response. Furthermore, an epitope-based vaccine can circumvent evolutionary events favoring immune escape present in native proteins from pathogens. It can also focus on only the most relevant epitopes (i.e. conserved and promiscuous) recognized by the majority of the target population. Mounting evidence points to the critical role of CD4+ T cells in natural antigen encounter and active immunization. In this paper the need for CD4+ T cell help in vaccine development, the selection of CD4+ T cell epitopes for an epitope-based vaccine, and how the approach can be used to induce a protective effect are reviewed.

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.

Distinct Outcomes of Trypanosoma cruzi Infection in Hamsters Are Related to Myocardial Parasitism, Cytokine/Chemokine Gene Expression, and Protein Expression Profile

BACKGROUNDnTrypanosoma cruzi-infected outbred hamsters reproduce the range of different outcomes of Chagas disease noted in humans. We tested whether myocarditis, its mediators, and myocardial protein expression are related to the severity of the acute phase of T. cruzi infection in the hamster model.nnnMETHODSnMyocardium left ventricles (LVs) obtained from Syrian hamsters infected with T. cruzi were collected 21 days after infection. Myocarditis and the T. cruzi nest/antigen area were analyzed by histological and morphometric analysis. Cytokine and chemokine messenger RNA (mRNA) expression was analyzed using real-time reverse-transcriptase polymerase chain reaction. Differentially expressed proteins were identified by 2-dimensional electrophoresis, followed by mass spectrometry.nnnRESULTSnWhile in the acute phase of infection, 50% of animals displayed weight loss and signs of acute-phase infection (hereafter referred to as acute-phase signs [APS]) (e.g., lethargy, vomiting, and diarrhea). Both the T. cruzi nest/antigen area and the expression of interferon-gamma, tumor necrosis factor-alpha, interleukin-10, and CCL3 mRNA were significantly increased in the LVs of animals with APS, compared with the LVs of animals without APS. Animals with APS, those without APS, and uninfected animals demonstrated distinct myocardial expression of contractile, stress response, and metabolism proteins.nnnCONCLUSIONSnThe distinct outcomes of acute T. cruzi infection in Syrian hamsters are related to cardiac parasitism, cytokine expression, and changes in the expression of structural/contractile and stress response proteins that may be associated with alterations in the cardiomyocyte cytoskeleton.

The Effect of Ursolic Acid on Leishmania (Leishmania) amazonensis Is Related to Programed Cell Death and Presents Therapeutic Potential in Experimental Cutaneous Leishmaniasis

Among neglected tropical diseases, leishmaniasis is one of the most important ones, affecting more than 12 million people worldwide. The available treatments are not well tolerated, and present diverse side effects, justifying the search for new therapeutic compounds. In the present study, the activity of ursolic acid (UA) and oleanolic acid (OA) were assayed in experimental cutaneous leishmaniasis (in vitro and in vivo). Promastigote forms of L. amazonensis were incubated with OA and UA for 24h, and effective concentration 50% (EC50) was estimated. Ultraestructural alterations in Leishmania amazonensis promastigotes after UA treatment were evaluated by transmission electron microscopy, and the possible mode of action was assayed through Annexin V and propidium iodide staining, caspase 3/7 activity, DNA fragmentation and transmembrane mitochondrial potential. The UA potential was evaluated in intracellular amastigotes, and its therapeutic potential was evaluated in L. amazonensis infected BALB/c mice. UA eliminated L. amazonensis promastigotes with an EC50 of 6.4 μg/mL, comparable with miltefosine, while OA presented only a marginal effect on promastigote forms at 100 μg/mL. The possible mechanism by which promastigotes were eliminated by UA was programmed cell death, independent of caspase 3/7, but it was highly dependent on mitochondria activity. UA was not toxic for peritoneal macrophages from BALB/c mice, and it was able to eliminate intracellular amastigotes, associated with nitric oxide (NO) production. OA did not eliminate amastigotes nor trigger NO. L. amazonensis infected BALB/c mice submitted to UA treatment presented lesser lesion size and parasitism compared to control. This study showed, for the first time, that UA eliminate promastigote forms through a mechanism associated with programed cell death, and importantly, was effective in vivo. Therefore, UA can be considered an interesting candidate for future tests as a prototype drug for the treatment of cutaneous leishmaniasis.

CTLA-4+PD-1− Memory CD4+ T Cells Critically Contribute to Viral Persistence in Antiretroviral Therapy-Suppressed, SIV-Infected Rhesus Macaques

&NA; Antiretroviral therapy (ART) suppresses viral replication in HIV‐infected individuals but does not eliminate the reservoir of latently infected cells. Recent work identified PD‐1+ follicular helper T (Tfh) cells as an important cellular compartment for viral persistence. Here, using ART‐treated, SIV‐infected rhesus macaques, we show that CTLA‐4+PD‐1− memory CD4+ T cells, which share phenotypic markers with regulatory T cells, were enriched in SIV DNA in blood, lymph nodes (LN), spleen, and gut, and contained replication‐competent and infectious virus. In contrast to PD‐1+ Tfh cells, SIV‐enriched CTLA‐4+PD‐1− CD4+ T cells were found outside the B cell follicle of the LN, predicted the size of the persistent viral reservoir during ART, and significantly increased their contribution to the SIV reservoir with prolonged ART‐mediated viral suppression. We have shown that CTLA‐4+PD‐1− memory CD4+ T cells are a previously unrecognized component of the SIV and HIV reservoir that should be therapeutically targeted for a functional HIV‐1 cure. Graphical Abstract Figure. No caption available. HighlightsCTLA‐4+PD‐1− memory CD4+ T cells are enriched in SIV DNA across multiple tissuesPersistently infected CTLA‐4+PD‐1− T cells localize outside the follicle on ARTCTLA‐4+PD‐1− T cells, which share Treg cell features, harbor replication‐competent virusSeeding of CTLA‐4+PD‐1− memory CD4+ T cells predicts viral persistence during ART &NA; HIV persists in T follicular helper cells within the lymph node during antiretroviral therapy, but decays with time. McGary et al. identify the persistence of replication‐competent SIV and HIV outside the lymph node follicle in a unique subset of CTLA‐4+PD‐1− memory CD4+ T cells that share features with regulatory T cells.

The CD8+ Memory Stem T Cell (TSCM) Subset Is Associated with Improved Prognosis in Chronic HIV-1 Infection

ABSTRACT Memory stem T cells (TSCM) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. The hallmarks of HIV-1 pathogenesis are CD4+ T cell depletion and abnormal cellular activation. We investigated the impact of HIV-1 infection on the TSCM compartment, as well as any protective role these cells may have in disease progression, by characterizing this subset in a cohort of 113 subjects with various degrees of viral control on and off highly active antiretroviral therapy (HAART). We observed that the frequency of CD8+ TSCM was decreased in all individuals with chronic, untreated HIV-1 infection and that HAART had a restorative effect on this subset. In contrast, natural controllers of HIV-1 had the highest absolute number of CD4+ TSCM cells among all of the infected groups. The frequency of CD4+ TSCM predicted higher CD8+ TSCM frequencies, consistent with a role for the CD4+ subset in helping to maintain CD8+ memory T cells. In addition, TSCM appeared to be progenitors for effector T cells (TEM), as these two compartments were inversely correlated. Increased frequencies of CD8+ TSCM predicted lower viral loads, higher CD4+ counts, and less CD8+ T cell activation. Finally, we found that TSCM express the mucosal homing integrin α4β7 and can be identified in gut-associated lymphoid tissue (GALT). The frequency of mucosal CD4+ TSCM was inversely correlated with that in the blood, potentially reflecting the ability of these self-renewing cells to migrate to a crucial site of ongoing viral replication and CD4+ T cell depletion. IMPORTANCE HIV-1 infection leads to profound impairment of the immune system. TSCM constitute a recently identified lymphocyte subset with stem cell-like qualities, including the ability to generate other memory T cell subtypes, and are therefore likely to play an important role in controlling viral infection. We investigated the relationship between the size of the CD8+ TSCM compartment and HIV-1 disease progression in a cohort of chronically infected individuals. Our results suggest that HAART restores a normal frequency of CD8+ TSCM and that the natural preservation of this subset in the setting of untreated HIV-1 infection is associated with improved viral control and immunity. Therefore, the CD8+ TSCM population may represent a correlate of protection in chronic HIV-1 infection that is directly relevant to the design of T cell-based vaccines, adoptive immunotherapy approaches, or the pharmacologic induction of TSCM.

Analysis of iron superoxide dismutase‐encoding DNA vaccine on the evolution of the Leishmania amazonensis experimental infection

The present work aimed to evaluate the immunogenicity of Leishmania amazonensis iron superoxide dismutase (SOD)‐encoding DNA experimental vaccine and the protective properties of this DNA vaccine during infection. The SOD gene was subcloned into the pVAX1 plasmid, and it was used to immunize BALB/c mice. Twenty‐one days after the last immunization, mice were sacrificed (immunogenicity studies) or subcutaneously challenged with L. amazonensis (studies of protection), and alterations in cellular and humoral immune responses were evaluated, as well as the course of infection. Mice only immunized with pVAX1‐SOD presented increased frequencies of CD4+IFN‐γ+, CD8+IFN‐γ+ and CD8+IL‐4+ lymphocytes; moreover, high levels of IgG2a were detected. After challenge, mice that were immunized with pVAX1‐SOD had increased frequencies of the CD4+IL‐4+, CD8+IFN‐γ+ and CD8+IL‐4+ T lymphocytes. In addition, the lymph node cells produced high amounts of IFN‐γ and IL‐4 cytokines. Increased IgG2a was also detected. The pattern of immunity induced by pVAX1‐SOD partially protected the BALB/c mice from a challenge with L. amazonensis, as the animals presented reduced parasitism and lesion size when compared to controls. Taken together, these results indicate that leishmanial SOD modulates the lymphocyte response, and that the elevation in IFN‐γ possibly accounted for the decreased skin parasitism observed in immunized animals.

Increase in cholinergic modulation with pyridostigmine induces anti-inflammatory cell recruitment soon after acute myocardial infarction in rats

We tested the hypothesis that an increase in the anti-inflammatory cholinergic pathway, when induced by pyridostigmine (PY), may modulate subtypes of lymphocytes (CD4+, CD8+, FOXP3+) and macrophages (M1/M2) soon after myocardial infarction (MI) in rats. Wistar rats, randomly allocated to receive PY (40 mg·kg(-1)·day(-1)) in drinking water or to stay without treatment, were followed for 4 days and then were subjected to ligation of the left coronary artery. The groups-denominated as the pyridostigmine-treated infarcted (IP) and infarcted control (I) groups-were submitted to euthanasia 3 days after MI; the heart was removed for immunohistochemistry, and the peripheral blood and spleen were collected for flow cytometry analysis. Noninfarcted and untreated rats were used as controls (C Group). Echocardiographic measurements were registered on the second day after MI, and heart rate variability was measured on the third day after MI. The infarcted groups had similar MI areas, degrees of systolic dysfunction, blood pressures, and heart rates. Compared with the I Group, the IP Group showed a significant higher parasympathetic modulation and a lower sympathetic modulation, which were associated with a small, but significant, increase in diastolic function. The IP Group showed a significant increase in M2 macrophages and FOXP3(+)cells in the infarcted and peri-infarcted areas, a significantly higher frequency of circulating Treg cells (CD4(+)CD25(+)FOXP3(+)), and a less extreme decrease in conventional T cells (CD25(+)FOXP3(-)) compared with the I Group. Therefore, increasing cholinergic modulation with PY induces greater anti-inflammatory cell recruitment soon after MY in rats.

Novel mechanisms to inhibit HIV reservoir seeding using Jak inhibitors

Despite advances in the treatment of HIV infection with ART, elucidating strategies to overcome HIV persistence, including blockade of viral reservoir establishment, maintenance, and expansion, remains a challenge. T cell homeostasis is a major driver of HIV persistence. Cytokines involved in regulating homeostasis of memory T cells, the major hub of the HIV reservoir, trigger the Jak-STAT pathway. We evaluated the ability of tofacitinib and ruxolitinib, two FDA-approved Jak inhibitors, to block seeding and maintenance of the HIV reservoir in vitro. We provide direct demonstration for involvement of the Jak-STAT pathway in HIV persistence in vivo, ex vivo, and in vitro; pSTAT5 strongly correlates with increased levels of integrated viral DNA in vivo, and in vitro Jak inhibitors reduce the frequency of CD4+ T cells harboring integrated HIV DNA. We show that Jak inhibitors block viral production from infected cells, inhibit γ-C receptor cytokine (IL-15)-induced viral reactivation from latent stores thereby preventing transmission of infectious particles to bystander activated T cells. These results show that dysregulation of the Jak-STAT pathway is associated with viral persistence in vivo, and that Jak inhibitors target key events downstream of γ-C cytokine (IL-2, IL-7 and IL-15) ligation to their receptors, impacting the magnitude of the HIV reservoir in all memory CD4 T cell subsets in vitro and ex vivo. Jak inhibitors represent a therapeutic modality to prevent key events of T cell activation that regulate HIV persistence and together, specific, potent blockade of these events may be integrated to future curative strategies.