A novel SARS-CoV-2 Beta RBD DNA vaccine directly targeted to antigen-presenting cells induces strong humoral and T cell responses.

Throughout the COVID-19 pandemic, several variants of concern (VoC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have evolved, affecting the efficacy of the approved COVID-19 vaccines. To address the need for vaccines that induce strong and persistent cross-reactive neutralizing antibodies and T cell responses, we developed a prophylactic SARS-CoV-2 vaccine candidate based on our easily and rapidly adaptable plasmid DNA vaccine platform. The vaccine candidate, referred to here as VB2129, encodes a protein homodimer consisting of the receptor binding domain (RBD) from lineage B.1.351 (Beta) of SARS-CoV-2, a VoC with a severe immune profile, linked to a targeting unit (human LD78ß/CCL3L1) that binds chemokine receptors on antigen-presenting cells (APCs) and a dimerization unit (derived from the hinge and CH3 exons of human IgG3). Immunogenicity studies in mice demonstrated that the APC-targeted vaccine induced strong antibody responses to both homologous Beta RBD and heterologous RBDs derived from Wuhan, Alpha, Gamma, Delta, and Omicron BA.1 variants, as well as cross-neutralizing antibodies against these VoC. Overall, preclinical data justify the exploration of VB2129 as a potential booster vaccine that induces broader antibody- and T cell-based protection against current and future SARS-CoV-2 VoC.

Redirector of Vaccine-induced Effector Responses (RoVER) for specific killing of cellular targets.

BACKGROUND: In individuals with malignancy or HIV-1 infection, antigen-specific cytotoxic T lymphocytes (CTLs) often display an exhausted phenotype with impaired capacity to eliminate the disease. Existing cell-based immunotherapy strategies are often limited by the requirement for adoptive transfer of CTLs. We have developed an immunotherapy technology in which potent CTL responses are generated in vivo by vaccination and redirected to eliminate target cells using a bispecific Redirector of Vaccine-induced Effector Responses (RoVER). METHODS: Following Yellow fever (YF) 17D vaccination of 51 healthy volunteers (NCT04083430), single-epitope YF-specific CTL responses were quantified by tetramer staining and multi-parameter flow cytometry. RoVER-mediated redirection of YF-specific CTLs to kill antigen-expressing Raji-Env cells, autologous CD19+ B cells or CD4+ T cells infected in vitro with a full-length HIV-1-eGFP was assessed in cell killing assays. Moreover, secreted IFN-γ, granzyme B, and TNF-α were analyzed by mesoscale multiplex assays. FINDINGS: YF-17D vaccination induced strong epitope-specific CTL responses in the study participants. In cell killing assays, RoVER-mediated redirection of YF-specific CTLs to autologous CD19+ B cells or HIV-1-infected CD4+ cells resulted in 58% and 53% killing at effector to target ratio 1:1, respectively. INTERPRETATION: We have developed an immunotherapy technology in which epitope-specific CTLs induced by vaccination can be redirected to kill antigen-expressing target cells by RoVER linking. The RoVER technology is highly specific and can be adapted to recognize various cell surface antigens. Importantly, this technology obviates the need for adoptive transfer of CTLs. FUNDING: This work was funded by the Novo Nordisk Foundation (Hallas Møller NNF10OC0054577).

Strategies for enhancing CAR T cell expansion and persistence in HIV infection.

Chimeric Antigen Receptor (CAR) T cell therapies are tremendously successful in hematological malignancies and show great promise as treatment and curative strategy for HIV. A major determinant for effective CAR T cell therapy is the persistence of CAR T cells. Particularly, antigen density and target cell abundance are crucial for the engagement, engraftment, and persistence of CAR T cells. The success of HIV-specific CAR T cells is challenged by limited antigen due to low cell surface expression of viral proteins and the scarcity of chronically infected cells during antiretroviral therapy. Several strategies have been explored to increase the efficacy of CAR T cells by enhancing expansion and persistence of the engineered cells. This review highlights the challenges of designing CAR T cells against HIV and other chronic viral infections. We also discuss potential strategies to enhance CAR T cell expansion and persistence in the setting of low antigen exposure.

Development of HIV-Resistant CAR T Cells by CRISPR/Cas-Mediated CAR Integration into the CCR5 Locus.

Adoptive immunotherapy using chimeric antigen receptor (CAR) T cells has been highly successful in treating B cell malignancies and holds great potential as a curative strategy for HIV infection. Recent advances in the use of anti-HIV broadly neutralizing antibodies (bNAbs) have provided vital information for optimal antigen targeting of CAR T cells. However, CD4+ CAR T cells are susceptible to HIV infection, limiting their therapeutic potential. In the current study, we engineered HIV-resistant CAR T cells using CRISPR/Cas9-mediated integration of a CAR cassette into the CCR5 locus. We used a single chain variable fragment (scFv) of the clinically potent bNAb 10-1074 as the antigen-targeting domain in our anti-HIV CAR T cells. Our anti-HIV CAR T cells showed specific lysis of HIV-infected cells in vitro. In a PBMC humanized mouse model of HIV infection, the anti-HIV CAR T cells expanded and transiently limited HIV infection. In conclusion, this study provides proof-of-concept for developing HIV-resistant CAR T cells using CRISPR/Cas9 targeted integration.

CD169 (Siglec-1) as a Robust Human Cell Biomarker of Toll-Like Receptor 9 Agonist Immunotherapy.

Immunotherapy is a promising therapeutic area in cancer and chronic viral infections. An important component of immunotherapy in these contexts is the activation of innate immunity. Here we investigate the potential for CD169 (Siglec 1) expression on monocytes to serve as a robust biomarker for activation of innate immunity and, particular, as a proxy for IFN-α production. Specifically, we investigated the effects of Toll-like receptor 9 agonism with MGN1703 (lefitolimod) across experimental conditions ex vivo, in humanized mice, and in clinical trial participants. Ex vivo we observed that the percentage of classical monocytes expressing CD169 increased dramatically from 10% pre-stimulation to 97% 24 hrs after MGN1703 stimulation (p<0.0001). In humanized NOG mice, we observed prominent upregulation of the proportions of monocytes expressing CD169 after two doses of MGN1703 where 73% of classical monocytes were CD169 positive in bone marrow following MGN1703 treatment vs 19% in vehicle treated mice (p=0.0159). Finally, in a clinical trial in HIV-infected individuals receiving immunotherapy treatment with MGN1703, we observed a uniform upregulation of CD169 on monocytes after dosing with 97% of classical monocytes positive for CD169 (p=0.002). Hence, in this comprehensive evaluation ex vivo, in an animal model, and in a clinical trial, we find increases in the percentage of CD169 positive monocytes to be a reliable and robust biomarker of immune activation following TLR9 agonist treatment.

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS-CoV-2 infection.

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS-CoV-2 infection is critical for developing treatments for severe COVID-19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID-19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL-6. Using an in vitro stem cell-based human pDC model, we further demonstrate that pDCs, while not supporting SARS-CoV-2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL-6, IL-8, CXCL10) cytokines that protect epithelial cells from de novo SARS-CoV-2 infection. Via targeted deletion of virus-recognition innate immune pathways, we identify TLR7-MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll-like receptor (TLR)2 is responsible for the inflammatory IL-6 response. We further show that SARS-CoV-2 engages the receptor neuropilin-1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL-6 response, suggesting neuropilin-1 as potential therapeutic target for stimulation of TLR7-mediated antiviral protection.

Inflammation and Platelet Activation After COVID-19 Vaccines - Possible Mechanisms Behind Vaccine-Induced Immune Thrombocytopenia and Thrombosis.

Introduction of vaccines against COVID-19 has provided the most promising chance to control the world-wide COVID-19 pandemic. However, the adenovirus-vector based Oxford/AstraZeneca [ChAdOx1] (AZ) and Johnson & Johnson [Ad26.CoV2.S] COVID-19 vaccines have been linked with serious thromboembolic events combined with thrombocytopenia, denominated Vaccine-induced Immune Thrombocytopenia and Thrombosis (VITT). The pathogenesis of COVID-19 VITT remain incompletely understood; especially the initial events that trigger platelet activation, platelet factor (PF)4 release, complex formation and PF4 antibody production are puzzling. This is a prospective study investigating the impact of different COVID-19 vaccines on inflammation (CRP, TNF-α, IL-1ß, IL-6, IL-8, IL-10), vascular endothelial activation (syndecan-1, thrombomodulin, E-selectin, ICAM-1, ICAM-3, VCAM-1), platelet activation (P-selectin, TGF-ß, sCD40L) and aggregation (Multiplate® impedance aggregometry), whole blood coagulation (ROTEM®), thrombin generation and PF4 antibodies to reveal potential differences between AZ and mRNA vaccines in individuals without VITT. The study included 80 (55 AZ and 55 mRNA) vaccinated individuals and 55 non-vaccinated age- and gender matched healthy controls. The main findings where that both vaccines enhanced inflammation and platelet activation, though AZ vaccination induced a more pronounced increase in several inflammatory and platelet activation markers compared to mRNA vaccination and that post-vaccination thrombin generation was higher following AZ vaccination compared to mRNA vaccination. No difference in neither the PF4 antibody level nor the proportion of individuals with positive PF4 antibodies were observed between the vaccine groups. This is the first study to report enhanced inflammation, platelet activation and thrombin generation following AZ vaccination compared to mRNA vaccination in a head-to-head comparison. We speculate that specific components of the AZ adenovirus vector may serve as initial trigger(s) of (hyper)inflammation, platelet activation and thrombin generation, potentially lowering the threshold for a cascade of events that both trigger complications related to excessive inflammation, platelet and coagulation activation as observed in epidemiological studies and promote development of VITT when combined with high-titer functionally active PF4 antibodies.

Persistent Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Immunocompromised Host Displaying Treatment Induced Viral Evolution.

We report a coronavirus disease 2019 case with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persisting beyond 333 days in an immunocompromised patient with chronic lymphocytic leukemia, asymptomatically carrying infectious SARS-CoV-2 at day 197 postdiagnosis. In addition, viral sequencing indicates major changes in the spike protein over time, temporally associated with convalescent plasma treatment.

HIV Antibody Fc N-Linked Glycosylation Is Associated with Viral Rebound.

Changes in antibody glycosylation are linked to inflammation across several diseases. Alterations in bulk antibody galactosylation can predict rheumatic flares, act as a sensor for immune activation, predict gastric cancer relapse, track with biological age, shift with vaccination, change with HIV reservoir size on therapy, and decrease in HIV and HCV infections. However, whether changes in antibody Fc biology also track with reservoir rebound time remains unclear. The identification of a biomarker that could forecast viral rebound time could significantly accelerate the downselection and iterative improvement of promising HIV viral eradication strategies. Using a comprehensive antibody Fc-profiling approach, the level of HIV-specific antibody Fc N-galactosylation is significantly associated with time to rebound after treatment discontinuation across three independent cohorts. Thus virus-specific antibody glycosylation may represent a promising, simply measured marker to track reservoir reactivation.

Comparable human reconstitution following Cesium-137 versus X-ray irradiation preconditioning in immunodeficient NOG mice.

Humanized mouse models are used extensively in research involving human pathogens and diseases. However, most of these models require preconditioning. Radio-active sources have been used routinely for this purpose but safety issues have motivated researchers to transition to chemical or X-ray based preconditioning. In this study, we directly compare 350 kV X-ray and Cs-137 low-dose precondition of NOG mice before human stem cell transplantation. Based on flow cytometry data, we found that engraftment of human cells into the mouse bone marrow was similar between radiation sources. Likewise, human engraftment in the peripheral blood was comparable between Cs-137 and three different X-ray doses with equal chimerization kinetics. In primary lymphoid organs such as the thymus and lymph nodes, and spleen, liver and lung, human-to-mouse chimerization was also comparable between irradiation sources. Development of different CD4 and CD8 T cells as well as these cells' maturation stages, i.e. from naïve to effector and memory subsets were generally analogous. Based on our results, we conclude that there are no discernable differences between the two sources in the low-dose spectrum investigated. However, while we encourage the transition to X-ray-based sources, we recommend all research groups to consider technical specifications and dose-finding studies.

The Use of Toll-Like Receptor Agonists in HIV-1 Cure Strategies.

Toll-like receptors (TLRs) are a family of pattern recognition receptors and part of the first line of defense against invading microbes. In humans, we know of 10 different TLRs, which are expressed to varying degrees in immune cell subsets. Engaging TLRs through their specific ligands leads to activation of the innate immune system and secondarily priming of the adaptive immune system. Because of these unique properties, TLR agonists have been investigated as immunotherapy in cancer treatment for many years, but in recent years there has also been growing interest in the use of TLR agonists in the context of human immunodeficiency virus type 1 (HIV-1) cure research. The primary obstacle to curing HIV-1 is the presence of a latent viral reservoir in transcriptionally silent immune cells. Due to the very limited transcription of the integrated HIV-1 proviruses, latently infected cells cannot be targeted and cleared by immune effector mechanisms. TLR agonists are very interesting in this context because of their potential dual effects as latency reverting agents (LRAs) and immune modulatory compounds. Here, we review preclinical and clinical data on the impact of TLR stimulation on HIV-1 latency as well as antiviral and HIV-1-specific immunity. We also focus on the promising role of TLR agonists in combination strategies in HIV-1 cure research. Different combinations of TLR agonists and broadly neutralizing antibodies or TLRs agonists as adjuvants in HIV-1 vaccines have shown very encouraging results in non-human primate experiments and these concepts are now moving into clinical testing.

IL-1R3 blockade broadly attenuates the functions of six members of the IL-1 family, revealing their contribution to models of disease.

Interleukin (IL)-1R3 is the co-receptor in three signaling pathways that involve six cytokines of the IL-1 family (IL-1α, IL-1ß, IL-33, IL-36α, IL-36ß and IL-36γ). In many diseases, multiple cytokines contribute to disease pathogenesis. For example, in asthma, both IL-33 and IL-1 are of major importance, as are IL-36 and IL-1 in psoriasis. We developed a blocking monoclonal antibody (mAb) to human IL-1R3 (MAB-hR3) and demonstrate here that this antibody specifically inhibits signaling via IL-1, IL-33 and IL-36 in vitro. Also, in three distinct in vivo models of disease (crystal-induced peritonitis, allergic airway inflammation and psoriasis), we found that targeting IL-1R3 with a single mAb to mouse IL-1R3 (MAB-mR3) significantly attenuated heterogeneous cytokine-driven inflammation and disease severity. We conclude that in diseases driven by multiple cytokines, a single antagonistic agent such as a mAb to IL-1R3 is a therapeutic option with considerable translational benefit.

TLR9 agonist MGN1703 enhances B cell differentiation and function in lymph nodes.

BACKGROUND: TLR9 agonists are being developed as immunotherapy against malignancies and infections. TLR9 is primarily expressed in B cells and plasmacytoid dendritic cells (pDCs). TLR9 signalling may be critically important for B cell activity in lymph nodes but little is known about the in vivo impact of TLR9 agonism on human lymph node B cells. As a pre-defined sub-study within our clinical trial investigating TLR9 agonist MGN1703 (lefitolimod) treatment in the context of developing HIV cure strategies (NCT02443935), we assessed TLR9 agonist-mediated effects in lymph nodes. METHODS: Participants received MGN1703 for 24 weeks concurrent with antiretroviral therapy. Seven participants completed the sub-study including lymph node resection at baseline and after 24 weeks of treatment. A variety of tissue-based immunologic and virologic parameters were assessed. FINDINGS: MGN1703 dosing increased B cell differentiation; activated pDCs, NK cells, and T cells; and induced a robust interferon response in lymph nodes. Expression of Activation-Induced cytidine Deaminase, an essential regulator of B cell diversification and somatic hypermutation, was highly elevated. During MGN1703 treatment IgG production increased and antibody glycosylation patterns were changed. INTERPRETATION: Our data present novel evidence that the TLR9 agonist MGN1703 modulates human lymph node B cells in vivo. These findings warrant further considerations in the development of TLR9 agonists as immunotherapy against cancers and infectious diseases. FUND: This work was supported by Aarhus University Research Foundation, the Danish Council for Independent Research and the NovoNordisk Foundation. Mologen AG provided study drug free of charge.

Non-covalent hitchhiking on endogenous carriers as a protraction mechanism for antiviral macromolecular prodrugs.

Albumin is a highly successful tool of drug delivery providing drastically extended body and blood residence time for the associated cargo, but it only traffics single drug copies at a time. In turn, macromolecular prodrugs (MP) are advantaged in carrying a high drug payload but offering only a modest extension of residence time to the conjugated drugs. In this work, we engineer MP to contain terminal groups that bind to albumin via non-covalent association and reveal that this facile measure affords a significant protraction for the associated polymers. This methodology is applied to MP of acyclovir, a successful drug against herpes simplex virus infection but with poor pharmacokinetics. Resulting albumin-affine MP were efficacious agents against herpes simplex virus type 2 (HSV-2) both in vitro and in vivo. In the latter case, sub-cutaneous administration of MP resulted in local (vaginal) antiviral effects and a systemic protection. Presented benefits of non-covalent association with albumin are readily transferrable to a wide variety of MP in development for drug delivery as anticancer, anti-inflammatory, and anti-viral measures.

Characterization of the HIV-1 transcription profile after romidepsin administration in ART-suppressed individuals.

OBJECTIVES: Reversing HIV-1 latency has been suggested as a strategy to eradicate HIV-1. We investigated the effect of romidepsin on the HIV transcription profile in participants from the REDUC part B clinical trial. DESIGN: Seventeen participants on suppressive antiretroviral therapy were vaccinated with six doses of the therapeutic vaccine Vacc-4x followed by treatment with three doses of romidepsin. Samples from nine study participants were available for HIV transcription profile analysis. METHODS: Read-through, total (TAR), elongated (longLTR), polyadenylated (polyA) and multiply-spliced (Tat-Rev) HIV transcripts and total HIV DNA were quantified at baseline (visit 1) and 4 h after the second (visit 10b) and third (visit 11b) romidepsin infusions. RESULTS: Read-through, total, elongated, and polyadenylated HIV transcripts increased after romidepsin infusion (P = 0.020, P = 0.0078, P = 0.0039, P = 0.027, respectively), but no changes were observed in multiply-spliced HIV RNA or HIV DNA. No change was observed in the ratio of read-through/total HIV transcripts. The ratio of elongated/total HIV RNA increased after romidepsin (P = 0.016), whereas the ratio of polyadenylated/elongated HIV decreased. Both elongated HIV transcripts and total HIV DNA correlated negatively with the time to viral rebound after interruption of ART. CONCLUSIONS: In these patients, romidepsin increased early events in HIV transcription (initiation and especially elongation), but had less effect on later stages (completion, multiple splicing) that may be required for comprehensive latency reversal and cell killing. Without cell death, increased HIV transcription before or after latency reversal may hasten viral rebound after therapy interruption.

Treatment of HIV-Infected Individuals with the Histone Deacetylase Inhibitor Panobinostat Results in Increased Numbers of Regulatory T Cells and Limits Ex Vivo Lipopolysaccharide-Induced Inflammatory Responses.

Histone deacetylase inhibitors (HDACi) modulate the transcriptional activity of all cells, including innate and adaptive immune cells. Therefore, we aimed to evaluate immunological effects of treatment with the HDACi panobinostat in HIV-infected patients during a clinical phase IIa latency reversal trial. Using flow cytometry, we investigated changes in T cell activation (CD69, CD38, HLA-DR) and the expression of CD39 and CTLA4 on regulatory T cells (Tregs). Whole-blood stimulations were performed and cytokine responses measured using Luminex. Gene expression in purified peripheral blood mononuclear cells (PBMCs) was evaluated using an Affymetrix HTA 2.0 gene chip. We found that proportions of CD4+ and CD8+ T cells expressing CD69 increased 24 h after initial panobinostat administration (P < 0.01), followed by an increase in the proportions of CD38+ HLA-DR+-coexpressing CD4+ T cells on day 4 (P = 0.02). Concurrently, proportions of Tregs increased by 40% (P = 0.003). Treg CTLA4 median fluorescent intensity (MFI) increased by 25% (P = 0.007), and CD39 MFI on CD39+ Treg increased by 12% (P = 0.02). Lipopolysaccharide (LPS)-induced inflammatory responses (interleukin-1ß [IL-1ß], IL-6, IL-12p40, and tumor necrosis factor alpha [TNF-α]) in whole blood were significantly downregulated 4 days after initial dosing. Lastly, panobinostat induced significant changes in the overall gene expression pattern (fold change, >1.5; false-discovery-rate [FDR]-corrected P, <0.05). Importantly, measures of immune function returned to baseline after panobinostat treatment and follow-up revealed no sustained effect on overall gene expression. IMPORTANCE The effect of treatment with histone deacetylase inhibitors on the immune system in HIV-infected individuals is not clear. Analysis of results from a clinical trial in which 15 HIV-infected individuals received 12 doses of panobinostat identified a significant impact on both T cell activation status and regulatory T cell suppressive marker expression and a reduced level of monocytic responsiveness to inflammatory stimuli. These changes were substantiated by global gene expression analysis. Collectively, the results suggest that panobinostat has multiple effects on innate and adaptive immune responses. Importantly, all the effects were transient, and further panobinostat treatment did not cause persistent long-term changes in gene expression patterns in HIV-infected individuals.

Macromolecular prodrugs of ribavirin: Polymer backbone defines blood safety, drug release, and efficacy of anti-inflammatory effects.

Macromolecular (pro)drugs hold much promise as broad-spectrum antiviral agents as either microbicides or carriers for intracellular delivery of antiviral drugs. Intriguing opportunity exists in combining the two modes of antiviral activity in the same polymer structure such that the same polymer acts as a microbicide and also serves to deliver the conjugated drug (ribavirin) into the cells. We explore this opportunity in detail and focus on the polymer backbone as a decisive constituent of such formulations. Fourteen polyanions (polycarboxylates, polyphosphates and polyphosphonates, and polysulfonates) were analyzed for blood pro/anti coagulation effects, albumin binding and albumin aggregation, inhibitory activity on polymerases, cytotoxicity, and anti-inflammatory activity in stimulated macrophages. Ribavirin containing monomers were designed to accommodate the synthesis of macromolecular prodrugs with disulfide-exchange triggered drug release. Kinetics of drug release was fast in all cases however enhanced hydrophobicity of the polymer significantly slowed release of ribavirin. Results of this study present a comprehensive view on polyanions as backbone for macromolecular prodrugs of ribavirin as broad-spectrum antiviral agents.

Estimating Initial Viral Levels during Simian Immunodeficiency Virus/Human Immunodeficiency Virus Reactivation from Latency.

Human immunodeficiency virus (HIV) viremia rebounds rapidly after treatment interruption, and a variety of strategies are being explored to reduce or control viral reactivation posttreatment. This viral rebound arises from reactivation of individual latently infected cells, which spread during ongoing rounds of productive infection. The level of virus produced by the initial individual reactivating cells is not known, although it may have major implications for the ability of different immune interventions to control viral rebound. Here we use data from both HIV and simian immunodeficiency virus (SIV) treatment interruption studies to estimate the initial viral load postinterruption and thereby the initial individual reactivation event. Using a barcoded virus (SIVmac239M) to track reactivation from individual latent cells, we use the observed viral growth rates and frequency of reactivation to model the dynamics of reactivation to estimate that a single reactivated latent cell can produce an average viral load equivalent to ∼0.1 to 0.5 viral RNA (vRNA) copies/ml. Modeling of treatment interruption in HIV suggests an initial viral load equivalent of ∼0.6 to 1 vRNA copies/ml. These low viral loads immediately following latent cell reactivation provide a window of opportunity for viral control by host immunity, before further replication allows viral spread. This work shows the initial levels of viral production that must be controlled in order to successfully suppress HIV reactivation following treatment interruption.IMPORTANCE Current treatment for HIV is able to suppress viral replication and prevent disease progression. However, treatment cannot eradicate infection, because the virus lies silent within latently infected cells. If treatment is stopped, the virus usually rebounds above the level of detection within a few weeks. There are a number of approaches being tested aimed at either eradicating latently infected cells or controlling the virus if it returns. Studying both the small pool of latently infected cells and the early events during viral reactivation is difficult, because these involve very small levels of virus that are difficult to measure directly. Here, we combine experimental data and mathematical modeling to understand the very early events during viral reactivation from latency in both HIV infection of humans and SIV infection of monkeys. We find that the initial levels of virus are low, which may help in designing therapies to control early viral reactivation.

Cellular immunogenicity of human papillomavirus vaccines Cervarix and Gardasil in adults with HIV infection.

Human papillomavirus (HPV) infection is a frequent cause of malignant and non-malignant disease, in particular among persons with HIV. HPV serotype-specific anti L1 antibodies protect against HPV infection but little is known about prophylactic HPV vaccine-induced cell-mediated immunity against HPV in high-risk individuals. We recently showed that both HPV vaccines (Gardasil® and Cervarix®) induce solid, serological immune responses in HIV-infected persons. This study aimed to characterize HPV-specific CD4 T cells in HIV-infected HPV-vaccine recipients, T cell responses being critical for B cell activation and antibody-isotype switching. Thirty HIV-infected patients on long-term antiretroviral treatment (ART) received 3 doses of either Cervarix (n = 15) or Gardasil (n = 15) vaccine at month 0, 1.5 and 6. Cryopreserved peripheral blood mononuclear cells (PBMC) from baseline, 7 and 12 months were subjected to 24-hour stimulation with specific pools of HPV L1-peptides (HPV6, 11, 16, 18, 31 and 45) and HPV E6/E7-peptide pools (HPV6/11 and HPV16/18). Fluorescence-activated cell sorting with intracellular staining (IC-FACS) against CD4, CD154, IL-2, and IFNγ was performed. Frequencies (%) of HPV-antigen specific CD4+ T cells (CD154+/IL-2+ or CD154+/ IFNγ+) were determined. Both HPV-vaccines significantly and comparably enhanced cell-mediated vaccine L1 antigen-specific immunity in HIV-positive adults receiving ART therapy at month 7 and 12 after first vaccine dose. This suggests that the vaccines induce CD4 T cellular memory despite HIV-induced immune compromisation.

Romidepsin-induced HIV-1 viremia during effective antiretroviral therapy contains identical viral sequences with few deleterious mutations.

OBJECTIVE: To investigate the origin of the HIV-1 viremia induced by the latency-reversing agent romidepsin. DESIGN: Six individuals on suppressive antiretroviral therapy received romidepsin administered intravenously once weekly for 3 consecutive weeks. CD4 T cells were obtained at baseline, following the second and third romidepsin infusion, and 10 weeks after the final romidepsin treatment. Plasma samples were collected 24 and 72 h after romidepsin infusions. METHODS: Single-genome sequencing of the env and p24-RT region was used to genetically characterize the virus from proviral DNA, the transcribed cell-associated RNA and the plasma RNA pool. RESULTS: In three of six participants with available plasma samples we identified plasma HIV-1 RNA sequences that were identical to DNA and/or cell-associated RNA sequences from peripheral blood CD4 T cells. In two participants, plasma RNA sequences contained expansions of identical sequences, corresponding to 62 and 100% of the total sequences, respectively. Plasma HIV-1 RNA had very low amounts of defective viruses compared to cell-associated RNA (odds ratio 20.85, P < 0.001) and to DNA (odds ratio 7.07, P = 0.011) during romidepsin therapy. CONCLUSIONS: Romidepsin induced transcription from proviruses in peripheral blood cells, which contributed to viremia in patients on suppressive therapy. The intermingling of these cell-associated HIV-1 RNA with DNA sequences indicates transcription from a diverse range of proviruses, but the expansions of identical viral plasma sequences with few defects indicate that the romidepsin-induced viremia arises from intact proviruses with highly similar or identical genetic backgrounds.