Lymph node dendritic cells harbor inducible replication-competent HIV despite years of suppressive ART.

Although gut and lymph node (LN) memory CD4 T cells represent major HIV and simian immunodeficiency virus (SIV) tissue reservoirs, the study of the role of dendritic cells (DCs) in HIV persistence has long been limited to the blood due to difficulties to access lymphoid tissue samples. In this study, we show that LN migratory and resident DC subpopulations harbor distinct phenotypic and transcriptomic profiles. Interestingly, both LN DC subpopulations contain HIV intact provirus and inducible replication-competent HIV despite the expression of the antiviral restriction factor SAMHD1. Notably, LN DC subpopulations isolated from HIV-infected individuals treated for up to 14 years are transcriptionally silent but harbor replication-competent virus that can be induced upon TLR7/8 stimulation. Taken together, these results uncover a potential important contribution of LN DCs to HIV infection in the presence of ART.

Viral Replicon Systems and Their Biosafety Aspects.

Introduction: Viral RNA replicons are self-amplifying RNA molecules generated by deleting genetic information of one or multiple structural proteins of wild-type viruses. Remaining viral RNA is used as such (naked replicon) or packaged into a viral replicon particle (VRP), whereby missing genes or proteins are supplied via production cells. Since replicons mostly originate from pathogenic wild-type viruses, careful risk consideration is crucial. Methods: A literature review was performed compiling information on potential biosafety risks of replicons originating from positive- and negative-sense single-stranded RNA viruses (except retroviruses). Results: For naked replicons, risk considerations included genome integration, persistence in host cells, generation of virus-like vesicles, and off-target effects. For VRP, the main risk consideration was formation of primary replication competent virus (RCV) as a result of recombination or complementation. To limit the risks, mostly measures aiming at reducing the likelihood of RCV formation have been described. Also, modifying viral proteins in such a way that they do not exhibit hazardous characteristics in the unlikely event of RCV formation has been reported. Discussion and Conclusion: Despite multiple approaches developed to reduce the likelihood of RCV formation, scientific uncertainty remains on the actual contribution of the measures and on limitations to test their effectiveness. In contrast, even though effectiveness of each individual measure is unclear, using multiple measures on different aspects of the system may create a solid barrier. Risk considerations identified in the current study can also be used to support risk group assignment of replicon constructs based on a purely synthetic design.

Development of an imaging system for visualization of Ebola virus glycoprotein throughout the viral lifecycle.

Ebola virus (EBOV) causes severe EBOV disease (EVD) in humans and non-human primates. Currently, limited countermeasures are available, and the virus must be studied in biosafety level-4 (BSL-4) laboratories. EBOV glycoprotein (GP) is a single transmembrane protein responsible for entry into host cells and is the target of multiple approved drugs. However, the molecular mechanisms underlying the intracellular dynamics of GP during EBOV lifecycle are poorly understood. In this study, we developed a novel GP monitoring system using transcription- and replication-competent virus-like particles (trVLPs) that enables the modeling of the EBOV lifecycle under BSL-2 conditions. We constructed plasmids to generate trVLPs containing the coding sequence of EBOV GP, in which the mucin-like domain (MLD) was replaced with fluorescent proteins. The generated trVLP efficiently replicated over multiple generations was similar to the wild type trVLP. Furthermore, we confirmed that the novel trVLP system enabled real-time visualization of GP throughout the trVLP replication cycle and exhibited intracellular localization similar to that of wild type GP. In summary, this novel monitoring system for GP will enable the characterization of the molecular mechanism of the EBOV lifecycle and can be applied for the development of therapeutics against EVD.

Replication-competent HIV-1 in human alveolar macrophages and monocytes despite nucleotide pools with elevated dUTP.

BACKGROUND: Although CD4+ memory T cells are considered the primary latent reservoir for HIV-1, replication competent HIV has been detected in tissue macrophages in both animal and human studies. During in vitro HIV infection, the depleted nucleotide pool and high dUTP levels in monocyte derived macrophages (MDM) leads to proviruses with high levels of dUMP, which has been implicated in viral restriction or reduced transcription depending on the uracil base excision repair (UBER) competence of the macrophage. Incorporated dUMP has also been detected in viral DNA from circulating monocytes (MC) and alveolar macrophages (AM) of HIV infected patients on antiretroviral therapy (ART), establishing the biological relevance of this phenotype but not the replicative capacity of dUMP-containing proviruses. RESULTS: As compared to in vitro differentiated MDM, AM from normal donors had sixfold lower levels of dTTP and a sixfold increased dUTP/dTTP, indicating a highly restrictive dNTP pool for reverse transcription. Expression of uracil DNA glycosylase (UNG) was eightfold lower in AM compared to the already low levels in MDM. Accordingly, ~ 80% of HIV proviruses contained dUMP, which persisted for at least 14-days due to low UNG excision activity. Unlike MDM, AM expression levels of UNG and SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) increased over 14 days post-HIV infection, while dUTP nucleotidohydrolase (DUT) expression decreased. These AM-specific effects suggest a restriction response centered on excising uracil from viral DNA copies and increasing relative dUTP levels. Despite the restrictive nucleotide pools, we detected rare replication competent HIV in AM, peripheral MC, and CD4+ T cells from ART-treated donors. CONCLUSIONS: These findings indicate that the potential integration block of incorporated dUMP is not realized during in vivo infection of AM and MC due to the near absence of UBER activity. In addition, the increased expression of UNG and SAMHD1 in AM post-infection is too slow to prevent integration. Accordingly, dUMP persists in integrated viruses, which based on in vitro studies, can lead to transcriptional silencing. This possible silencing outcome of persistent dUMP could promote viral latency until the repressive effects of viral dUMP are reversed.

Immunological Correlates of the HIV-1 Replication-Competent Reservoir Size.

Understanding what shapes the latent human immunodeficiency virus type 1 (HIV-1) reservoir is critical for developing strategies for cure. We measured frequency of persistent HIV-1 infection after 5 years of suppressive antiretroviral therapy initiated during chronic infection. Pretreatment CD8+ T-cell activation, nadir CD4 count, and CD4:CD8 ratio predicted reservoir size.

TZA, a Sensitive Reporter Cell-based Assay to Accurately and Rapidly Quantify Inducible, Replication-competent Latent HIV-1 from Resting CD4+ T Cells.

The latent HIV-1 viral reservoir in resting CD4+ (rCD4+) T cells represents a major barrier to an HIV-1 cure. There is an ongoing effort to identify therapeutic approaches that will eliminate or reduce the size of this reservoir. However, clinical investigators lack an assay to determine whether or not a decrease in the latent reservoir has been achieved. Therefore, it is critical to develop assays that can reproducibly quantify the reservoir size and changes therein, in participant's blood during a therapeutic trial. Quantification of the latent HIV viral reservoir requires a highly sensitive, cost-effective assay capable of measuring the low frequency of rCD4+ T cells carrying functional provirus. Preferably, such an assay should be such that it can be adopted for high throughput and could be adopted under conditions for use in large-scale clinical trials. While PCR-based assays are commonly used to quantify pro-viral DNA or intracellular RNA transcript, they cannot distinguish between replication-competent and defective proviruses. We have recently published a study where a reporter cell-based assay (termed TZA or TZM-bl based quantitative assay) was used to quantify inducible replication-competent latent HIV-1 in blood. This assay is more sensitive, cost-efficient, and faster than available technology, including the quantitative viral outgrowth assay or the Q-VOA. Using this assay, we show that the size of the inducible latent HIV-1 reservoir in virally suppressed participants on ART is approximately 70-fold larger than previous estimates. We describe here in detail an optimized method to quantitate latently infected cells using the TZA.

Assessing cross-reactivity of Junín virus-directed neutralizing antibodies.

Arenaviruses cause several viral hemorrhagic fevers endemic to Africa and South America. The respective causative agents are classified as biosafety level (BSL) 4 pathogens. Unlike for most other BSL4 agents, for the New World arenavirus Junín virus (JUNV) both a highly effective vaccination (Candid#1) and a post-exposure treatment, based on convalescent plasma transfer, are available. In particular, neutralizing antibodies (nAbs) represent a key protective determinant in JUNV infection, which is supported by the correlation between successful passive antibody therapy and the levels of nAbs administered. Unfortunately, comparable resources for the management of other closely related arenavirus infections are not available. Given the significant challenges inherent in studying BSL4 pathogens, our goal was to first assess the suitability of a JUNV transcription and replication-competent virus-like particle (trVLP) system for measuring virus neutralization under BSL1/2 conditions. Indeed, we could show that infection with JUNV trVLPs is glycoprotein (GP) dependent, that trVLP input has a direct correlation to reporter readout, and that these trVLPs can be neutralized by human serum with kinetics similar to those obtained using authentic virus. These properties make trVLPs suitable for use as a proxy for virus in neutralization assays. Using this platform we then evaluated the potential of JUNV nAbs to cross-neutralize entry mediated by GPs from other arenaviruses using JUNV (strain Romero)-based trVLPs bearing GPs either from other JUNV strains, other closely related New World arenaviruses (e.g. Tacaribe, Machupo, Sabiá), or the distantly related Lassa virus. While nAbs against the JUNV vaccine strain are also active against a range of other JUNV strains, they appear to have little or no capacity to neutralize other arenavirus species, suggesting that therapy with whole plasma directed against another species is unlikely to be successful and that the targeted development of cross-specific monoclonal antibody-based resources is likely needed. Such efforts will be supported by the availability of this BSL1/2 screening platform which provides a rapid and easy means to characterize the potency and reactivity of anti-arenavirus neutralizing antibodies against a range of arenavirus species.

Measuring HIV Persistence on Antiretroviral Therapy.

In order to determine if an eradication strategy for HIV is effective, it will be important to measure persistent replication-competent virus, the current barrier to a cure. Various assays are available that measure persistent virus, each with advantages and disadvantages that must be balanced in order to select the best assay for the experimental aim. Assays of free virus do not measure the latent form of the virus but can be utilised in conjunction with other assays in order to better understand HIV persistence on ART. The quantitative viral outgrowth assay (QVOA) is the gold standard assay for measuring persistent replication-competent virus, but it, along with assays that vary the classical QVOA method, underestimates the frequency of latently infected cells in blood due to the presence of non-induced yet intact and replication-competent proviruses. Assays that quantify or sequence specific genomic regions of HIV overestimate the size of the reservoir as they are unable to distinguish between intact and defective virus. As an alternative, sequencing the full-length integrated genome can better distinguish replication-competent provirus, but these methods may be expensive and time-consuming. Novel assays, and the application of these assays to novel questions, will be key to the development of future curative therapies for HIV.

Blood CXCR3+ CD4 T Cells Are Enriched in Inducible Replication Competent HIV in Aviremic Antiretroviral Therapy-Treated Individuals.

We recently demonstrated that lymph nodes (LNs) PD-1+/T follicular helper (Tfh) cells from antiretroviral therapy (ART)-treated HIV-infected individuals were enriched in cells containing replication competent virus. However, the distribution of cells containing inducible replication competent virus has been only partially elucidated in blood memory CD4 T-cell populations including the Tfh cell counterpart circulating in blood (cTfh). In this context, we have investigated the distribution of (1) total HIV-infected cells and (2) cells containing replication competent and infectious virus within various blood and LN memory CD4 T-cell populations of conventional antiretroviral therapy (cART)-treated HIV-infected individuals. In the present study, we show that blood CXCR3-expressing memory CD4 T cells are enriched in cells containing inducible replication competent virus and contributed the most to the total pool of cells containing replication competent and infectious virus in blood. Interestingly, subsequent proviral sequence analysis did not indicate virus compartmentalization between blood and LN CD4 T-cell populations, suggesting dynamic interchanges between the two compartments. We then investigated whether the composition of blood HIV reservoir may reflect the polarization of LN CD4 T cells at the time of reservoir seeding and showed that LN PD-1+ CD4 T cells of viremic untreated HIV-infected individuals expressed significantly higher levels of CXCR3 as compared to CCR4 and/or CCR6, suggesting that blood CXCR3-expressing CD4 T cells may originate from LN PD-1+ CD4 T cells. Taken together, these results indicate that blood CXCR3-expressing CD4 T cells represent the major blood compartment containing inducible replication competent virus in treated aviremic HIV-infected individuals.

Retroviral and Lentiviral Safety Analysis of Gene-Modified T Cell Products and Infused HIV and Oncology Patients.

Replication-competent retrovirus/lentivirus (RCR/L) and insertional oncogenesis are potential safety risks with integrating viruses in gene-modified cell therapies. As such, the Food and Drug Administration guidances outline RCR/L-monitoring methods throughout the entire gene therapy treatment cycle. We present data for 17 vector lots, 375 manufactured T cell products, and 308 patients post-infusion across both HIV and oncology indications, showing no evidence of RCR/L. Given our data, a Poisson probability model estimates that a single patient, or a group of patients, would need to be followed for at least 52.8 years to observe one positive RCR/L event, highlighting the unlikelihood of RCR/L development. Additionally, we estimate the median time for lentivirus-modified T cell products to fall below the 1% vector sequence threshold in peripheral or whole blood that would trigger vector integration site analysis. These estimated times are 1.4 months in hematologic malignancies, 0.66 month in solid tumors, and 0.92 month in HIV. Based on these considerable safety data in HIV and oncology and recent Biologics License Applications filed for lentiviral-modified T cell products for hematologic malignancies, this may be an opportune time to re-evaluate the current guidelines for T cell gene therapy product testing and long-term patient monitoring.

Absence of Replication-Competent Lentivirus in the Clinic: Analysis of Infused T Cell Products.

Exposure to replication-competent lentivirus (RCL) is a theoretical safety concern for individuals treated with lentiviral gene therapy. For certain ex vivo gene therapy applications, including cancer immunotherapy trials, RCL detection assays are used to screen the vector product as well as the vector-transduced cells. In this study, we reviewed T cell products screened for RCL using methodology developed in the National Gene Vector Biorepository. All trials utilized third-generation lentiviral vectors produced by transient transfection. Samples from 26 clinical trials totaling 460 transduced cell products from 375 subjects were evaluated. All cell products were negative for RCL. A total of 296 of the clinical trial participants were screened for RCL at least 1 month after infusion of the cell product. No research subject has shown evidence of RCL infection. These findings provide further evidence attesting to the safety of third-generation lentiviral vectors and that testing T cell products for RCL does not provide added value to screening the lentiviral vector product.

HIV Infection of Macrophages: Implications for Pathogenesis and Cure.

Although CD4+ T cells represent the major reservoir of persistent HIV and SIV infection, accumulating evidence suggests that macrophages also contribute. However, investigations of the role of macrophages are often underrepresented at HIV pathogenesis and cure meetings. This was the impetus for a scientific workshop dedicated to this area of study, held in Cambridge, MA in January 2017. The workshop brought together experts in the fields of HIV/SIV immunology and virology, macrophage biology and immunology, and animal models of HIV/SIV infection to discuss the role of macrophages as a physiologically relevant viral reservoir, and the implications of macrophage infection for HIV pathogenesis and strategies for cure. While still controversial, there is an emerging theory that infected macrophages likely persist in the setting of combination antiretroviral therapy. These macrophages could then drive persistent inflammation and contribute to the viral reservoir, which indicates the importance of addressing macrophages as well as CD4+ T cells with future therapeutic strategies.

A novel approach for addressing diseases not yielding to effective vaccination? Immunization by replication-competent controlled virus.

Vaccination involves inoculation of a subject with a disabled disease-causing microbe or parts thereof. While vaccination has been highly successful, we still lack sufficiently effective vaccines for important infectious diseases. We propose that a more complete immune response than that elicited from a vaccine may be obtained from immunization with a disease-causing virus modified to subject replication-essential genes to the control of a gene switch activated by non-lethal heat in the presence of a drug-like compound. Upon inoculation, strictly localized replication of the virus would be triggered by a heat dose administered to the inoculation site. Activated virus would transiently replicate with an efficiency approaching that of the disease-causing virus and express all viral antigens. It may also vector heterologous antigens or control co-infecting microbes.

Replication-competent influenza A viruses expressing a red fluorescent protein.

Like most animal viruses, studying influenza A in model systems requires secondary methodologies to identify infected cells. To circumvent this requirement, we describe the generation of replication-competent influenza A red fluorescent viruses. These influenza A viruses encode mCherry fused to the viral non-structural 1 (NS1) protein and display comparable growth kinetics to wild-type viruses in vitro. Infection of cells with influenza A mCherry viruses was neutralized with monoclonal antibodies and inhibited with antivirals to levels similar to wild-type virus. Influenza A mCherry viruses were also able to lethally infect mice, and strikingly, dose- and time-dependent kinetics of viral replication were monitored in whole excised mouse lungs using an in vivo imaging system (IVIS). By eliminating the need for secondary labeling of infected cells, influenza A mCherry viruses provide an ideal tool in the ongoing struggle to better characterize the virus and identify new therapeutics against influenza A viral infections.