Rapid diagnostic test: a critical need for outbreak preparedness and response for high priority pathogens.

Rapid diagnostic tests (RDTs) are critical for preparedness and response against an outbreak or pandemic and have been highlighted in the 100 Days Mission, a global initiative that aims to prepare the world for the next epidemic/pandemic by driving the development of diagnostics, vaccines and therapeutics within 100 days of recognition of a novel Disease X threat.RDTs play a pivotal role in early case identification, surveillance and case management, and are critical for initiating deployment of vaccine and monoclonal antibodies. Currently available RDTs, however, have limited clinical sensitivity and specificity and inadequate validation. The development, validation and implementation of RDTs require adequate and sustained financing from both public and private sources. While the World Health Assembly recently passed a resolution on diagnostic capacity strengthening that urges individual Member States to commit resources towards this, the resolution is not binding and implementation will likely be impeded by limited financial resources and other competing priorities, particularly in low-income countries. Meanwhile, the diagnostic industry has not sufficiently invested in RDT development for high priority pathogens.Currently, vaccine development projects are getting the largest funding support among medical countermeasures. Yet vaccines are insufficient tools in isolation, and pandemic preparedness will be incomplete without parallel investment in diagnostics and therapeutics.The Pandemic Fund, a global financing mechanism recently established for strengthening pandemic prevention, preparedness and response, may be a future avenue for supporting diagnostic development.In this paper, we discuss why RDTs are critical for preparedness and response. We also discuss RDT investment challenges and reflect on the way forward.

MicroRNA-155 Reinforces HIV Latency.

The presence of a small number of infected but transcriptionally dormant cells currently thwarts a cure for the more than 35 million individuals infected with HIV. Reactivation of these latently infected cells may result in three fates: 1) cell death due to a viral cytopathic effect, 2) cell death due to immune clearance, or 3) a retreat into latency. Uncovering the dynamics of HIV gene expression and silencing in the latent reservoir will be crucial for developing an HIV-1 cure. Here we identify and characterize an intracellular circuit involving TRIM32, an HIV activator, and miR-155, a microRNA that may promote a return to latency in these transiently activated reservoir cells. Notably, we demonstrate that TRIM32, an E3 ubiquitin ligase, promotes reactivation from latency by directly modifying IκBα, leading to a novel mechanism of NF-κB induction not involving IκB kinase activation.

A flexible model of HIV-1 latency permitting evaluation of many primary CD4 T-cell reservoirs.

Latently infected cells form the major obstacle to HIV eradication. Studies of HIV latency have been generally hindered by the lack of a robust and rapidly deployable cell model that involves primary human CD4 T lymphocytes. Latently infected cell lines have proven useful, but it is unclear how closely these proliferating cells recapitulate the conditions of viral latency in non-dividing CD4 T lymphocytes in vivo. Current primary lymphocyte models more closely reflect the in vivo state of HIV latency, but they are limited by protracted culture periods and often low cell yields. Additionally, these models are always established in a single latently infected cell type that may not reflect the heterogeneous nature of the latent reservoir. Here we describe a rapid, sensitive, and quantitative primary cell model of HIV-1 latency with replication competent proviruses and multiple reporters to enhance the flexibility of the system. In this model, post-integration HIV-1 latency can be established in all populations of CD4 T cells, and reactivation of latent provirus assessed within 7 days. The kinetics and magnitude of reactivation were evaluated after stimulation with various cytokines, small molecules, and T-cell receptor agonists. Reactivation of latent HIV proviruses was readily detected in the presence of strong activators of NF-κB. Latently infected transitional memory CD4 T cells proved more responsive to these T-cell activators than latently infected central memory cells. These findings reveal potentially important biological differences within the latently infected pool of memory CD4 T cells and describe a flexible primary CD4 T-cell system to evaluate novel antagonists of HIV latency.

Abortive HIV infection mediates CD4 T cell depletion and inflammation in human lymphoid tissue.

The mechanism by which CD4 T cells are depleted in HIV-infected hosts remains poorly understood. In ex vivo cultures of human tonsil tissue, CD4 T cells undergo a pronounced cytopathic response following HIV infection. Strikingly, >95% of these dying cells are not productively infected but instead correspond to bystander cells. We now show that the death of these "bystander" cells involves abortive HIV infection. Inhibitors blocking HIV entry or early steps of reverse transcription prevent CD4 T cell death while inhibition of later events in the viral life cycle does not. We demonstrate that the nonpermissive state exhibited by the majority of resting CD4 tonsil T cells leads to accumulation of incomplete reverse transcripts. These cytoplasmic nucleic acids activate a host defense program that elicits a coordinated proapoptotic and proinflammatory response involving caspase-3 and caspase-1 activation. While this response likely evolved to protect the host, it centrally contributes to the immunopathogenic effects of HIV.

Failure to restore the Vgamma2-Jgamma1.2 repertoire in HIV-infected men receiving highly active antiretroviral therapy (HAART).

Gammadelta (gammadelta) T cells expressing the Vgamma2-Jgamma1.2Vdelta2 (Vgamma9-JPVdelta2, alternate nomenclature) T cell receptor (TCR) constitute the major peripheral blood population of gammadelta T cells in adult humans and are specifically depleted during human immunodeficiency virus (HIV) disease. Vgamma2-Jgamma1.2Vdelta2 T cells provide a convenient model for assessing the impact of antiretroviral therapy on cell populations that are not susceptible to direct infection because they do not express CD4 and depletion occurs by indirect mechanisms. We obtained longitudinal PBMC samples from 16 HIV-infected individuals who enrolled in the Multicenter AIDS Cohort Study (MACS) and were starting highly active antiretroviral therapy (HAART). Vgamma2-Jgamma1.2Vdelta2 T cells were depleted in these individuals as a result of HIV infection. Despite evidence for clinical benefits of HAART, the Vgamma2-Jgamma1.2Vdelta2 T cell repertoire did not recover after HAART initiation irrespective of treatment duration. These studies highlight important defects among cell subsets lost due to indirect effects of HIV.

Isopentenyl pyrophosphate-activated CD56+ {gamma}{delta} T lymphocytes display potent antitumor activity toward human squamous cell carcinoma.

PURPOSE: The expression of CD56, a natural killer cell-associated molecule, on alphabeta T lymphocytes correlates with their increased antitumor effector function. CD56 is also expressed on a subset of gammadelta T cells. However, antitumor effector functions of CD56(+) gammadelta T cells are poorly characterized. EXPERIMENTAL DESIGN: To investigate the potential effector role of CD56(+) gammadelta T cells in tumor killing, we used isopentenyl pyrophosphate and interleukin-2-expanded gammadelta T cells from peripheral blood mononuclear cells of healthy donors. RESULTS: Thirty to 70% of expanded gammadelta T cells express CD56 on their surface. Interestingly, although both CD56(+) and CD56(-) gammadelta T cells express comparable levels of receptors involved in the regulation of gammadelta T-cell cytotoxicity (e.g., NKG2D and CD94), only CD56(+) gammadelta T lymphocytes are capable of killing squamous cell carcinoma and other solid tumor cell lines. This effect is likely mediated by the enhanced release of cytolytic granules because CD56(+) gammadelta T lymphocytes expressed higher levels of CD107a compared with CD56(-) controls following exposure to tumor cell lines. Lysis of tumor cell lines is blocked by concanamycin A and a combination of anti-gammadelta T-cell receptor + anti-NKG2D monoclonal antibody, suggesting that the lytic activity of CD56(+) gammadelta T cells involves the perforin-granzyme pathway and is mainly gammadelta T-cell receptor/NKG2D dependent. Importantly, CD56-expressing gammadelta T lymphocytes are resistant to Fas ligand and chemically induced apoptosis. CONCLUSIONS: Our data indicate that CD56(+) gammadelta T cells are potent antitumor effectors capable of killing squamous cell carcinoma and may play an important therapeutic role in patients with head and neck cancer and other malignancies.

Innate-like gammadelta T cell responses to mycobacterium Bacille Calmette-Guerin using the public V gamma 2 repertoire in Macaca fascicularis.

The V gamma 2 V delta 2 T cell subset responds to Bacille Calmette-Guerin (BCG) immunization in macaques and may be a component of protective immunity against tuberculosis. We characterized the effects of BCG on the V gamma 2 V delta 2 T cell receptor repertoire by comparing the starting population of V gamma 2 chains in cynomolgus macaques with the repertoire found after priming or booster immunization with BCG. The starting repertoire was dominated by public V gamma 2 chain sequences that were found repeatedly among unrelated animals. Primary exposure to BCG triggered expansion of cells expressing public V gamma 2 chains and booster immunization was often associated with contraction of these same subsets. Thus, BCG-reactive V gamma 2 chains were present at high frequency in the repertoire of mycobacteria-naïve macaques and they comprised the major response to primary or booster immunization. Normal selection processes that created the naïve V gamma 2 repertoire in macaques, also encoded the capacity for rapid responses to mycobacteria. The unusual composition of a normal V gamma 2 repertoire helps to explain the powerful gammadelta T cell responses to BCG immunization.

Individual Vgamma2-Jgamma1.2+ T cells respond to both isopentenyl pyrophosphate and Daudi cell stimulation: generating tumor effectors with low molecular weight phosphoantigens.

Human Vgamma2Vdelta2 T cells exhibit T cell receptor-dependent, MHC-unrestricted recognition of antigen and play important roles in tumor and pathogen immunity. To characterize antigen recognition by the Vgamma2Vdelta2 TCR, we used the combined approach of spectratyping and CDR3 sequence analysis that measures changes in the TCR repertoire before and after stimulation with a phosphoantigen (isopentenyl pyrophosphate) or an irradiated tumor cell line (Daudi B lymphoma). Here we describe common Vgamma2 chains that are substantially involved in the response to both phosphoantigens and tumor cells. The recognition properties of common Vgamma2 chains explains the observation that Vgamma2Vdelta2 T cells expanded by phosphoantigen stimulation specifically recognize and kill some but not all tumor cell lines. Our studies further justify efforts to stimulate tumor immunity by administering low molecular weight phosphoantigens and boosting the frequency and tumor effector functions of circulating Vgamma2Vdelta2 T cells.

Vaccinia virus inhibits T cell receptor-dependent responses by human gammadelta T cells.

Vaccinia virus (VV) is an effective vaccine and vector but has evolved multiple mechanisms for evading host immunity. We characterized the interactions of VV (TianTan and New York City Board of Health strains) with human gammadelta T cells because of the role they play in immune control of this virus. Exposure to VV failed to trigger proliferative responses in gammadelta T cells from unprimed individuals, but it was an unexpected finding that VV blocked responses to model antigens by the Vgamma2Vdelta2 T cell subset. Infectious or ultraviolet light-inactivated VV inhibited proliferative Vgamma2Vdelta2 T cell responses to phosphoantigens and tumor cells, prevented cytolysis of Daudi B cells, and reduced cytokine production. Inhibiting Vgamma2Vdelta2 T cells may be a mechanism for evading host immunity and increasing VV virulence. Increased VV replication or expression in the absence of gammadelta T cell responses might contribute to its potency as a vaccine against poxvirus and recombinant antigens.

Gamma interferon secretion by human Vgamma2Vdelta2 T cells after stimulation with antibody against the T-cell receptor plus the Toll-Like receptor 2 agonist Pam3Cys.

Circulating Vgamma2Vdelta2 T-cell populations in healthy human beings are poised for rapid responses to bacterial or viral pathogens. We asked whether Vgamma2Vdelta2 T cells use the Toll-like receptor (TLR) family to recognize pathogen-associated molecular pattern molecules and to regulate cell functions. Analysis of expanded Vgamma2Vdelta2 T-cell lines showed the abundant presence of TLR2 mRNA, implying that these receptors are important for cell differentiation or function. However, multiple efforts to detect TLR2 protein on the cell surface or in cytoplasmic compartments gave inconsistent results. Functional assays confirmed that human Vgamma2Vdelta2 T cells could respond to the TLR2 agonist (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH trihydrochloride (Pam3Cys), but the response required coincident stimulation through the gammadelta T-cell receptor (TCR). Dually stimulated cells produced higher levels of cytoplasmic or cell-free gamma interferon and showed increased expression of the lysosome-associated membrane protein CD107a on the cell surface. A functional TLR2 that requires coincident TCR stimulation may increase the initial potency of Vgamma2Vdelta2 T-cell responses at the site of infection and promote the rapid development of subsequent acquired antipathogen immunity.

The Vgamma2/Vdelta2 T-cell repertoire in Macaca fascicularis: functional responses to phosphoantigen stimulation by the Vgamma2/Jgamma1.2 subset.

Circulating Vgamma2/Vdelta2 T cells in human and non-human primates respond to small molecular weight non-peptidic phosphoantigens in a major histocompatibility complex (MHC)-unrestricted manner. These responses are encoded by the Vgamma2/Jgamma1.2 chain of the T-cell receptor and are positively selected during early development to create a biased repertoire in adults. We characterized the Vgamma2 chain in cynomolgus macaques (Macaca fascicularis) to develop a non-human primate model for studying the effects of infection and therapy on the circulating Vgamma2/Vdelta2 T-cell subset. The cynomolgus macaque Vgamma2 chain was highly homologous to the Vgamma2 chain from human beings and rhesus macaques (Macaca mulatta), though we noted conserved substitutions in critical residues within the CDR3 for both macaque species. Despite these substitutions, Vgamma2/Vdelta2+ T cells from cynomolgus monkeys exhibited polyclonal responses to two different phosphoantigens. Proliferative responses were observed with both isopentenylpyrophosphate and alendronate, but stronger interferon-gamma secretory responses were observed with isopentenylpyrophosphate. In vitro stimulation and expansion led to selective outgrowth of the Vgamma2/Jgamma1.2 subset, with a marked shift in the Vgamma2 spectratype. As a result of the less biased starting repertoire for Vgamma2, the cynomolgus macaque constitutes a sensitive model for examining the effects of in vitro or in vivo treatments on the Vgamma2/Vdelta2 T-cell population. Our studies establish the value of cynomolgus macaques as a model for Vgamma2/Vdelta2 T-cell responses to non-peptidic antigens, and further evidence the remarkable evolutionary conservation of this unusual, phosphoantigen-responsive T-cell subset that is found only in primate species.