Selective transmission of R5 HIV-1 variants: where is the gatekeeper?

To enter target cells HIV-1 uses CD4 and a coreceptor. In vivo the coreceptor function is provided either by CCR5 (for R5) or CXCR4 (for X4 HIV-1). Although both R5 and X4 HIV-1 variants are present in body fluids (semen, blood, cervicovaginal and rectal secretions), R5 HIV-1 appears to transmit infection and dominates early stages of HIV disease. Moreover, recent sequence analysis of virus in acute infection shows that, in the majority of cases of transmission, infection is initiated by a single virus. Therefore, the existence of a “gatekeeper” that selects R5 over X4 HIV-1 and that operates among R5 HIV-1 variants has been suggested. In the present review we consider various routes of HIV-transmission and discuss potential gatekeeping mechanisms associated with each of these routes. Although many mechanisms have been identified none of them explains the almost perfect selection of R5 over X4 in HIV-1 transmission. We suggest that instead of one strong gatekeeper there are multiple functional gatekeepers and that their superimposition is sufficient to protect against X4 HIV-1 infection and potentially select among R5 HIV-1 variants. In conclusion, we propose that the principle of multiple barriers is more general and not restricted to protection against X4 HIV-1 but rather can be applied to other phenomena when one factor has a selective advantage over the other(s). In the case of gatekeepers for HIV-1 transmission, the task is to identify them and to decipher their molecular mechanisms. Knowledge of the gatekeepers‘ localization and function may enable us to enhance existing barriers against R5 transmission and to erect the new ones against all HIV-1 variants

SITC cancer immunotherapy resource document: a compass in the land of biomarker discovery.

Since the publication of the Society for Immunotherapy of Cancer\u27s (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients

Contrasting Roles for TLR Ligands in HIV-1 Pathogenesis

The first line of a host's response to various pathogens is triggered by their engagement of cellular pattern recognition receptors (PRRs). Binding of microbial ligands to these receptors leads to the induction of a variety of cellular factors that alter intracellular and extracellular environment and interfere directly or indirectly with the life cycle of the triggering pathogen. Such changes may also affect any coinfecting microbe. Using ligands to Toll-like receptors (TLRs) 5 and 9, we examined their effect on human immunodeficiency virus (HIV)-1 replication in lymphoid tissue ex vivo. We found marked differences in the outcomes of such treatment. While flagellin (TLR5 agonist) treatment enhanced replication of CC chemokine receptor 5 (CCR 5)-tropic and CXC chemokine receptor 4 (CXCR4)-tropic HIV-1, treatment with oligodeoxynucleotide (ODN) M362 (TLR9 agonist) suppressed both viral variants. The differential effects of these TLR ligands on HIV-1 replication correlated with changes in production of CC chemokines CCL3, CCL4, CCL5, and of CXC chemokines CXCL10, and CXCL12 in the ligand-treated HIV-1-infected tissues. The nature and/or magnitude of these changes were dependent on the ligand as well as on the HIV-1 viral strain. Moreover, the tested ligands differed in their ability to induce cellular activation as evaluated by the expression of the cluster of differentiation markers (CD) 25, CD38, CD39, CD69, CD154, and human leukocyte antigen D related (HLA)-DR as well as of a cell proliferation marker, Ki67, and of CCR5. No significant effect of the ligand treatment was observed on apoptosis and cell death/loss in the treated lymphoid tissue ex vivo. Our results suggest that binding of microbial ligands to TLRs is one of the mechanisms that mediate interactions between coinfected microbes and HIV-1 in human tissues. Thus, the engagement of appropriate TLRs by microbial molecules or their mimetic might become a new strategy for HIV therapy or prevention

HIV-1 expressing the envelopes of transmitted/founder or control/reference viruses have similar infection patterns of CD4 T-cells in human cervical tissue ex vivo.

Recently, it was found that 80% of sexual HIV-1 transmissions are established by a single virion/viral genome. To investigate whether the transmitted/founder (T/F) viruses have specific biological properties favoring sexual transmission, we inoculated human cervical tissue explants with isogenic HIV-1 viruses encoding Env sequences from T/F and control reference (C/R) HIV-1 variants as well as with full length T/F HIV-1 and compared their replication efficiencies, T cell depletion, and the activation status of infected cells. We found that all the HIV-1 variants were capable of transmitting infection to cervical tissue ex vivo and in this system preferentially replicate in activated CD4 T cells and deplete these cells. There was no difference in the biological properties of T/F and C/R HIV-1 variants as evaluated in ex vivo cervical tissue

CD4 T cell depletion in C/R and T/F HIV-1 variants infected human cervical tissue <i>ex vivo</i>.

<p>Blocks of human cervical tissue were infected with C/R or T/F viruses, and cultured <i>ex vivo</i>. 12 days post infection, the tissues were enzymatically digested and their cells were analyzed by polychromatic flow cytometry. T cells were identified by staining for CD3 and side-scatter. In this subset CD4 and CD8 were identified. The depletion of CD4 T cells was estimated by comparing the ratio of CD8⁺ to CD4⁺ (CD8⁻) T cells in infected and uninfected controls, and expressing this ratio in infected tissue as a percent of the same ratio in matched uninfected control. Data showing individual experiments and summary box plots for n = 19 and n = 14 (median, 25^th and 75^th percentiles, and range of CD4/CD8 ratios in tissues infected with C/R viruses (empty boxes) and in tissues infected with T/F viruses, (filled boxes) are presented.</p

Replication of various C/R and T/F HIV-1 variants in human cervical tissue <i>ex vivo</i>.

<p>Donor-matched human cervical tissue blocks were infected <i>ex-vivo</i> with C/R and T/F viruses in presence or absence of 3TC. Culture media were collected every 3 days up to day 12 and the amount of p24 was measured. In addition, 12 days post infection, the tissues were enzymatically digested and their cells were analyzed by polychromatic flow cytometry. T cells were identified by side-scatter and staining for CD3. The CD4 T cell subset was defined as CD8^– T cells since the down-regulation of CD4 in HIV-1 infected cells precludes use of staining for CD4. CD4 T cell, which were infected with HIV-1 were revealed by staining for p24.(a) For each virus, cumulative values for the net p24 production ([p24] in untreated − [p24] in 3TC-treated donor-matched tissues) were computed and plotted as box plots (median, 25^th and 75^th percentiles, and range) on a log scale (left Y-axis; empty boxes; C/R virus: n = 23; T/F viruses: n = 30). The fractions of p24⁺ positive cells among CD8⁻CD3⁺ cells were plotted on a linear axis (right Y-axis; filled boxes; n = 19, and n = 14, respectively) (b). Bivariate plots showing p24 and CD8 staining in T cells; the values represent the fraction of CD8⁻CD3⁺ cells expressing p24 as defined by the plotted gate. Plots illustrate a representative experiment.</p

CD4 T cell activation in C/R and T/F HIV-1 variants infected human cervical tissue <i>ex vivo</i>.

<p>Blocks of human cervical tissue were infected with C/R and T/F viruses and cultured <i>ex vivo</i>. 12 days post infection, the tissues were enzymatically digested and their cells were analyzed by polychromatic flow cytometry. Cells were stained for T cell markers CD3, CD4 and CD8 as well as for the activation markers CD25, CD38, CD69, CD95, and HLA-DR. Infected cells were identified with intracellular staining for HIV-1 p24. The fraction of infected (p24⁺) CD4 T cells expressing a given activation marker was expressed as % of the fraction of T cells expressing the same marker in matched uninfected control tissue. Data showing individual experiments and summary box plots for n = 9 to 17 (median, 25^th and 75^th percentiles, and range) of activation marker expression in tissues infected with C/R viruses (empty boxes) and in tissues infected with T/F viruses (filled boxes) are presented.</p

SITC cancer immunotherapy resource document: a compass in the land of biomarker discovery

Since the publication of the Society for Immunotherapy of Cancer’s (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients