Anti-MPER antibodies with heterogeneous neutralization capacity are detectable in most untreated HIV-1 infected individuals

Background The MPER region of the HIV-1 envelope glycoprotein gp41 is targeted by broadly neutralizing antibodies. However, the localization of this epitope in a hydrophobic environment seems to hamper the elicitation of these antibodies in HIV infected individuals. We have quantified and characterized anti-MPER antibodies by ELISA and by flow cytometry using a collection of mini gp41-derived proteins expressed on the surface of 293T cells. Longitudinal plasma samples from 35 HIV-1 infected individuals were assayed for MPER recognition and MPER-dependent neutralizing capacity using HIV-2 viruses engrafted with HIV-1 MPER sequences. Results Miniproteins devoid of the cysteine loop of gp41 exposed the MPER on 293T cell membrane. Anti-MPER antibodies were identified in most individuals and were stable when analyzed in longitudinal samples. The magnitude of the responses was strongly correlated with the global response to the HIV-1 envelope glycoprotein, suggesting no specific limitation for anti-MPER antibodies. Peptide mapping showed poor recognition of the C-terminal MPER moiety and a wide presence of antibodies against the 2F5 epitope. However, antibody titers failed to correlate with 2F5-blocking activity and, more importantly, with the specific neutralization of HIV-2 chimeric viruses bearing the HIV-1 MPER sequence; suggesting a strong functional heterogeneity in anti-MPER humoral responses. Conclusions Anti-MPER antibodies can be detected in the vast majority of HIV-1 infected individuals and are generated in the context of the global anti-Env response. However, the neutralizing capacity is heterogeneous suggesting that eliciting neutralizing anti-MPER antibodies by immunization might require refinement of immunogens to skip nonneutralizing responses

Decreased myocardial Titin expression in chronic alcoholic cardiomyopathy

Aims: Cardiomyopathy (CMP) with a reduced ejection fraction develops in a dose-28 dependent manner in one- third of subjects with a long-term history of heavy daily alcohol consumption. Ethanol alters heart transduction signals including excitation- contraction sarcomeric coupling, causing diastolic and systolic left-ventricular (LV) dysfunction. Titin is a giant structural sarcomeric filament macro protein involved in contractile heart function and contributes to cardiac myocyte elastic recoil, a key factor for diastolic LV filling. We evaluated whether titin expression is affected by chronic high-dose ethanol 35 consumption in alcoholic CMP. Methods and Results: We analyzed a total of 30 heart samples from human organ 37 donors: 20 from high alcohol consumers (10 without CMP and 10 with CMP) and 10 healthy controls. Patient evaluation comprised daily and lifetime ethanol consumption, chest X ray, 2-D echocardiography and LV histology. CMP was assessed by functional 40 and histological criteria. Titin activity was evaluated by specific immunohistochemical 41 (IHC) and transcript expression (rtPCR) assays. Titin IHC expression was clearly present in sarcomere areas of myocytes. Compared to healthy donors (82.58±3.36), alcohol consumers showed a significantly lower cardiac titin expression (71.29±3.16; 13.67±3.83% decrease; p=0.04), being significantly lower in alcohol consumers with CMP (62.31±4.18; 24.54±5.06% decrease, p<0.0009), compared to both their counter-parts without CMP (80.27±2.62; 2.80±3.17% decrease 47 vs. controls; p<0.0030 vs. alcoholic CMP). Titin transcript levels confirmed similar patterns of expression

Citopaticitat de la glicoproteïna de l'embolcall del VIH-1 : mecanismes directes i indirectes i implicació en la progressió clínica /

La infecció pel VIH-1 es caracteritza per una dràstica davallada en el recompte de cèl·lules T CD4+ que condueix a un estat d'immunodeficiència de l'individu, afavorint l'aparició de manifestacions definitòries de sida com són les infeccions oportunistes. Tot i això, la dinàmica de la pèrdua de cèl·lules T CD4+ no és igual en tots els pacients. En un extrem, hi podem trobar individus en els quals la pèrdua de cèl·lules T CD4+ és accentuadament ràpida, de manera que presenten recomptes de cèl·lules T CD4+ inferiors a les 350 cèl·lules/μl en menys de 3 anys (pacients ràpid progressors, RP). A l'altre extrem hi trobem un grup molt reduït d'individus que mantenen els recomptes de cèl·lules T CD4+ elevats (>400 cèl·lules/μl) i constants tot i presentar una replicació vírica activa a nivells elevats (pacients virèmics no progressors, VNP). La glicoproteïna de l'embolcall víric (Env) representa un dels principals determinants virològics de la citopaticitat del VIH-1, afectant tant les cèl·lules infectades com les no infectades a través de diversos mecanismes directes i indirectes. Per una banda, la unió de gp120 a la molècula CD4 (receptor víric) i a les molècules CXCR4 o CCR5 (coreceptors vírics i determinants del tropisme) així com la capacitat fusogènica de gp41 contribueixen de manera directa a la destrucció de cèl·lules T CD4. Per altra banda, la capacitat de gp41 (a través de l'epítop 3S) d'induir l'expressió a la superfície de les cèl·lules T CD4 del lligand NKp44L contribueix de manera indirecta en la seva destrucció, ja que l'expressió del lligand les sensibilitza per ser destruïdes mitjançant cèl·lules NK activades. Els objectius d'aquesta tesi són, per una banda, el desenvolupament d'un model in vitro per a l'estudi del tropisme i la capacitat fusogènica de l'Env així com la seva relació amb la destrucció de cèl·lules T CD4 per apoptosi i autofàgia i, per altra banda, avaluar la contribució relativa dels diferents mecanismes citopàtics descrits anteriorment en la destrucció de cèl·lules T CD4 in vivo caracteritzant Env aïllades de pacients RP i VNP. En aquest cas, també s'ha avaluat el paper de la resposta humoral anti-Env en els dos grups de pacients.La infección por VIH-1 se caracteriza por una drástica caída en el recuento de células T CD4+ que conduce a un estado de inmunodeficiencia del individuo, favoreciendo la aparición de eventos definitorios de sida como son las infecciones oportunistas. Aun así, la dinámica de pérdida de células T CD4+ no es idéntica en todos los pacientes. En un extremo podemos encontrar individuos que presentan una pérdida de células T CD4+ acentuadamente rápida, de modo que, después de la seroconversión, sus recuentos de células T CD4+ llegan a valores inferiores a las 350 células/μl en un período de tiempo inferior a los 3 años (pacientes rápido progresores, RP). En el otro extremo, encontramos un grupo muy reducido de individuos que mantienen los recuentos de células T CD4+ elevados (>400 células/μl) y constantes aunque presentan una replicación viral activa y elevada (pacientes virémicos no progresores, VNP). La glicoproteína de la envuelta viral (Env) representa uno de los principales determinantes virológicos de la citopaticidad del VIH-1, afectando tanto las células infectadas como las no infectadas a través de distintos mecanismos directos e indirectos. Por un lado, la unión de gp120 a la molécula CD4 (receptor viral) y a las moléculas CXCR4 o CCR5 (coreceptores virales y determinantes del tropismo) así como la capacidad fusogénica de gp41 contribuyen directamente a la destrucción de células T CD4+. Por otro lado, la capacidad de gp41 (a través del epítopo 3S) de inducir la expresión en la superficie de las células T CD4+ del ligando NKp44L contribuye indirectamente en su destrucción, ya que la expresión del ligando provoca que las células T CD4+ sean susceptibles de ser destruidas mediante células NK activadas. Los objetivos de esta tesis fueron, por un lado, desarrollar un modelo in vitro para el estudio del tropismo y la capacidad fusogénica de la Env así como su relación con la destrucción de células T CD4 por apoptosis y autofagia y, por otro lado, determinar la contribución relativa de los distintos mecanismos citopáticos descritos anteriormente en la destrucción de células T CD4 in vivo caracterizando Env aisladas de pacientes RP y VNP. Adicionalmente, también se planteó el estudio de la respuesta humoral anti-Env en los pacientes RP y VNP.HIV-1 infection is characterized by an important decrease on CD4+ T cell counts, resulting in weakened immune responses that lead to AIDS-defining events such as opportunistic infections. However, the dynamics of the CD4+ T cell loss is highly heterogeneous among HIV-infected individuals. On the one hand, some patients show a markedly fast decay on the CD4+ T cell counts, reaching values lower than 350 CD4 T cell/μl within the first three years after seroconversion (rapid progressors, RP). On the other hand, a really limited group of HIV-infected individuals shows high and maintained CD4+ T cell counts (>400 cells/μl) despite an active and high viral replication (viremic non progressors, VNP). The envelope glycoprotein (Env) represents one of the main virological determinants of HIV-1 cytopaticity, affecting both infected and non-infected cells through direct and indirect mechanisms. By one side, the binding of gp120 to the CD4 molecule (viral receptor) and CXCR4 or CCR5 (viral coreceptors which determine the viral tropism) in combination with the fusogenic capacity of gp41 directly contribute to the destruction of the CD4+ T cells. By the other side, gp41 induces the expression of NK cells ligand NKp44L on the surface of the CD4+ T cells through its 3S epitope, indirectly contributing to its destruction since the expression of the ligand renders these cells highly sensitive to be killed by activated NK cells. The objectives of this thesis were, by one hand, to develop an in vitro method to evaluate the Env tropism and fusogenic ability and its relation with CD4 T cells destruction by apoptosis and autophagy, and, by the other hand, to evaluate the relative contribution of the different cytopatic mechanisms described above in the destruction of CD4+ T cells in vivo, characterizing Env isolated from RP and VNP individuals. Moreover, the role of the humoral immune responses against Env was also evaluated for both groups of HIV-infected individuals

Susceptibility of Human Lymphoid Tissue Cultured ex vivo to Xenotropic Murine Leukemia Virus-Related Virus (XMRV) Infection

Xenotropic murine leukemia virus-related virus (XMRV) was generated after a recombination event between two endogenous murine leukemia viruses during the production of a prostate cancer cell line. Although the associations of the XMRV infection with human diseases appear unlikely, the XMRV is a retrovirus of undefined pathogenic potential, able to replicate in human cells in vitro. Since recent studies using animal models for infection have yielded conflicting results, we set out an ex vivo model for XMRV infection of human tonsillar tissue to determine whether XMRV produced by 22Rv1 cells is able to replicate in human lymphoid organs. Tonsil blocks were infected and infection kinetics and its pathogenic effects were monitored XMRV, though restricted by APOBEC, enters and integrates into the tissue cells. The infection did not result in changes of T or B-cells, immune activation, nor inflammatory chemokines. Infectious viruses could be recovered from supernatants of infected tonsils by reinfecting DERSE XMRV indicator cell line, although these supernatants could not establish a new infection in fresh tonsil culture, indicating that in our model, the viral replication is controlled by innate antiviral restriction factors. Overall, the replication-competent retrovirus XMRV, present in a high number of laboratories, is able to infect human lymphoid tissue and produce infectious viruses, even though they were unable to establish a new infection in fresh tonsillar tissue. Hereby, laboratories working with cell lines producing XMRV should have knowledge and understanding of the potential biological biohazardous risks of this virus

HIV transfer between CD4 T cells does not require LFA-1 binding to ICAM-1 and is governed by the interaction of HIV envelope glycoprotein with CD4

<p>Abstract</p> <p>Background</p> <p>Cell-to-cell HIV transmission requires cellular contacts that may be in part mediated by the integrin leukocyte function antigen (LFA)-1 and its ligands intercellular adhesion molecule (ICAM)-1, -2 and -3. The role of these molecules in free virus infection of CD4 T cells or in transinfection mediated by dendritic cells (DC) has been previously described. Here, we evaluate their role in viral transmission between different HIV producing cells and primary CD4 T cells.</p> <p>Results</p> <p>The formation of cellular conjugates and subsequent HIV transmission between productively infected MOLT cell lines and primary CD4 T cells was not inhibited by a panel of blocking antibodies against ICAM-1, ICAM-3 and α and β chains of LFA-1. Complete abrogation of HIV transmission and formation of cellular conjugates was only observed when gp120/CD4 interactions were blocked. The dispensable role of LFA-1 in HIV transmission was confirmed using non-lymphoid 293T cells, lacking the expression of adhesion molecules, as HIV producing cells. Moreover, HIV transmission between infected and uninfected primary CD4 T cells was abrogated by inhibitors of gp120 binding to CD4 but was not inhibited by blocking LFA-1 binding to ICAM-1 or ICAM-3. Rather, LFA-1 and ICAM-3 mAbs enhanced HIV transfer. All HIV producing cells (including 293T cells) transferred HIV particles more efficiently to memory than to naive CD4 T cells.</p> <p>Conclusion</p> <p>In contrast to other mechanisms of viral spread, HIV transmission between infected and uninfected T cells efficiently occurs in the absence of adhesion molecules. Thus, gp120/CD4 interactions are the main driving force of the formation of cellular contacts between infected and uninfected CD4 T cells whereby HIV transmission occurs.</p

The HR2 polymorphism N140I in the HIV-1 gp41 combined with the HR1 V38A mutation is associated with a less cytopathic phenotype

<p>Abstract</p> <p>Background</p> <p>Resistance to the fusion inhibitor enfuvirtide (ENF) is achieved by changes in the gp41 subunit of the HIV envelope glycoprotein (Env). Specific ENF-associated mutational pathways correlate with immunological recovery, even after virological failure, suggesting that the acquisition of ENF resistance alters gp41 pathogenicity. To test this hypothesis, we have characterized the expression, fusion capability, induction of CD4⁺T cell loss and single CD4⁺T cell death of 48 gp41 proteins derived from three patients displaying different amino acids (N, T or I) at position 140 that developed a V38A mutation after ENF-based treatment.</p> <p>Results</p> <p>In all cases, intra-patient comparison of Env isolated pre- or post-treatment showed comparable values of expression and fusogenic capacity. Furthermore, Env with either N or T at position 140 induced comparable losses of CD4⁺T-cells, irrespective of the residue present at position 38. Conversely, Env acquiring the V38A mutation in a 140I background induced a significantly reduced loss of CD4⁺T cells and lower single-cell death than did their baseline controls. No altered ability to induce single-cell death was observed in the other clones.</p> <p>Conclusions</p> <p>Overall, primary gp41 proteins with both V38A and N140I changes showed a reduced ability to induce single cell death and deplete CD4⁺T cells, despite maintaining fusion activity. The specificity of this phenotype highlights the relevance of the genetic context to the cytopathic capacity of Env and the role of ENF-resistance mutations in modulating viral pathogenicity <it>in vivo</it>, further supporting the hypothesis that gp41 is a critical mediator of HIV pathogenesis.</p

Preclinical scenario of targeting myocardial fibrosis with chimeric antigen receptor (CAR) immunotherapy

Fibrosis is present in an important proportion of myocardial disorders. Injury activates cardiac fibroblasts, which deposit excess extracellular matrix, increasing tissue stiffness, impairing cardiac function, and leading to heart failure. Clinical therapies that directly target excessive fibrosis are limited, and more effective treatments are needed. Immunotherapy based on chimeric antigen receptor (CAR) T cells is a novel technique that redirects T lymphocytes toward specific antigens to eliminate the target cells. It is currently used in haematological cancers but has demonstrated efficacy in mouse models of hypertensive cardiac fibrosis, with activated fibroblasts as the target cells. CAR T cell therapy is associated with significant toxicities, but CAR natural killer cells can overcome efficacy and safety limitations. The use of CAR immunotherapy offers a potential alternative to current therapies for fibrosis reduction and restoration of cardiac function in patients with myocardial fibrosis

Viremic HIV Infected Individuals with High CD4 T Cells and Functional Envelope Proteins Show Anti-gp41 Antibodies with Unique Specificity and Function

BACKGROUND: CD4 T-cell decay is variable among HIV-infected individuals. In exceptional cases, CD4 T-cell counts remain stable despite high plasma viremia. HIV envelope glycoprotein (Env) properties, namely tropism, fusion or the ability to induce the NK ligand NKp44L, or host factors that modulate Env cytopathic mechanisms may be modified in such situation. METHODS: We identified untreated HIV-infected individuals showing non-cytopathic replication (VL>10,000 copies/mL and CD4 T-cell decay<50 cells/µL/year, Viremic Non Progressors, VNP) or rapid progression (CD4 T-cells<350 cells/µL within three years post-infection, RP). We isolated full-length Env clones and analyzed their functions (tropism, fusion activity and capacity to induce NKp44L expression on CD4 cells). Anti-Env humoral responses were also analyzed. RESULTS: Env clones isolated from VNP or RP individuals showed no major phenotypic differences. The percentage of functional clones was similar in both groups. All clones tested were CCR5-tropic and showed comparable expression and fusogenic activity. Moreover, no differences were observed in their capacity to induce NKp44L expression on CD4 T cells from healthy donors through the 3S epitope of gp41. In contrast, anti- Env antibodies showed clear functional differences: plasma from VNPs had significantly higher capacity than RPs to block NKp44L induction by autologous viruses. Consistently, CD4 T-cells isolated from VNPs showed undetectable NKp44L expression and specific antibodies against a variable region flanking the highly conserved 3S epitope were identified in plasma samples from these patients. Conversely, despite continuous antigen stimulation, VNPs were unable to mount a broad neutralizing response against HIV. CONCLUSIONS: Env functions (fusion and induction of NKp44L) were similar in viremic patients with slow or rapid progression to AIDS. However, differences in humoral responses against gp41 epitopes nearby 3S sequence may contribute to the lack of CD4 T cell decay in VNPs by blocking the induction of NKp44L by gp41

Susceptibility of Human Lymphoid Tissue Cultured ex vivo to Xenotropic Murine Leukemia Virus-Related Virus (XMRV) Infection

BACKGROUND: Xenotropic murine leukemia virus-related virus (XMRV) was generated after a recombination event between two endogenous murine leukemia viruses during the production of a prostate cancer cell line. Although the associations of the XMRV infection with human diseases appear unlikely, the XMRV is a retrovirus of undefined pathogenic potential, able to replicate in human cells in vitro. Since recent studies using animal models for infection have yielded conflicting results, we set out an ex vivo model for XMRV infection of human tonsillar tissue to determine whether XMRV produced by 22Rv1 cells is able to replicate in human lymphoid organs. Tonsil blocks were infected and infection kinetics and its pathogenic effects were monitored RESULTS: XMRV, though restricted by APOBEC, enters and integrates into the tissue cells. The infection did not result in changes of T or B-cells, immune activation, nor inflammatory chemokines. Infectious viruses could be recovered from supernatants of infected tonsils by reinfecting DERSE XMRV indicator cell line, although these supernatants could not establish a new infection in fresh tonsil culture, indicating that in our model, the viral replication is controlled by innate antiviral restriction factors. CONCLUSIONS: Overall, the replication-competent retrovirus XMRV, present in a high number of laboratories, is able to infect human lymphoid tissue and produce infectious viruses, even though they were unable to establish a new infection in fresh tonsillar tissue. Hereby, laboratories working with cell lines producing XMRV should have knowledge and understanding of the potential biological biohazardous risks of this virus