Impact of ChAdOx1 or DNA Prime Vaccination on Magnitude, Breadth, and Focus of MVA-Boosted Immunogen-Specific T Cell Responses.

The efficacy of anti-viral T-cell vaccines may greatly depend on their ability to generate high-magnitude responses targeting a broad range of different epitopes. Recently, we created the HIV T-cell immunogen HTI, designed to generate T-cell responses to protein fragments more frequently targeted by HIV controllers. In the present study, we aim to maximize the breadth and magnitude of the T-cell responses generated by HTI by combining different vaccine vectors expressing HTI. We evaluated the ability to induce strong and broad T-cell responses to the HTI immunogen through prime vaccination with DNA plasmid (D) or Chimpanzee Adenovirus Ox1 (ChAdOx1; C) vectors, followed by a Modified Virus Ankara (MVA; M) vaccine boost (DDD, DDDM, C, and CM). HTI-specific T-cell responses after vaccination were measured by IFN-γ-ELISpot assays in two inbred mice strains (C57BL/6 and BALB/c). CM was the schedule triggering the highest magnitude of the response in both mice strains. However, this effect was not reflected in an increase in the breadth of the response but rather in an increase in the magnitude of the response to specific immunodominant epitopes. Immunodominance profiles in the two mouse strains were different, with a clear dominance of T-cell responses to a Pol-derived peptide pool after CM vaccination in C57BL/6. Responses to CM vaccination were also maintained at higher magnitudes over time (13 weeks) compared to other vaccination regimens. Thus, while a ChAdOx1 prime combined with MVA booster vaccination generated stronger and more sustained T-cell responses compared to three DNA vaccinations, the ChAdOx1 primed responses were more narrowly targeted. In conclusion, our findings suggest that the choice of vaccine vectors and prime-boost regimens plays a crucial role in determining the strength, duration, breadth, and focus of T-cell responses, providing further guidance for selecting vaccination strategies.

Converging and evolving immuno-genomic routes toward immune escape in breast cancer.

The interactions between tumor and immune cells along the course of breast cancer progression remain largely unknown. Here, we extensively characterize multiple sequential and parallel multiregion tumor and blood specimens of an index patient and a cohort of metastatic triple-negative breast cancers. We demonstrate that a continuous increase in tumor genomic heterogeneity and distinct molecular clocks correlated with resistance to treatment, eventually allowing tumors to escape from immune control. TCR repertoire loses diversity over time, leading to convergent evolution as breast cancer progresses. Although mixed populations of effector memory and cytotoxic single T cells coexist in the peripheral blood, defects in the antigen presentation machinery coupled with subdued T cell recruitment into metastases are observed, indicating a potent immune avoidance microenvironment not compatible with an effective antitumor response in lethal metastatic disease. Our results demonstrate that the immune responses against cancer are not static, but rather follow dynamic processes that match cancer genomic progression, illustrating the complex nature of tumor and immune cell interactions.

Vagus nerve dysfunction in the post-COVID-19 condition: a pilot cross-sectional study.

OBJECTIVES: The post-COVID-19 condition (PCC) is a disabling syndrome affecting at least 5%-10% of subjects who survive COVID-19. SARS-CoV-2 mediated vagus nerve dysfunction could explain some PCC symptoms, such as dysphonia, dysphagia, dyspnea, dizziness, tachycardia, orthostatic hypotension, gastrointestinal disturbances, or neurocognitive complaints. METHODS: We performed a cross-sectional pilot study in subjects with PCC with symptoms suggesting vagus nerve dysfunction (n = 30) and compared them with subjects fully recovered from acute COVID-19 (n = 14) and with individuals never infected (n = 16). We evaluated the structure and function of the vagus nerve and respiratory muscles. RESULTS: Participants were mostly women (24 of 30, 80%), and the median age was 44 years (interquartile range [IQR] 35-51 years). Their most prevalent symptoms were cognitive dysfunction 25 of 30 (83%), dyspnea 24 of 30 (80%), and tachycardia 24 of 30 (80%). Compared with COVID-19-recovered and uninfected controls, respectively, subjects with PCC were more likely to show thickening and hyperechogenic vagus nerve in neck ultrasounds (cross-sectional area [CSA] [mean ± standard deviation]: 2.4 ± 0.97mm2 vs. 2 ± 0.52mm2 vs. 1.9 ± 0.73 mm2; p 0.08), reduced esophageal-gastric-intestinal peristalsis (34% vs. 0% vs. 21%; p 0.02), gastroesophageal reflux (34% vs. 19% vs. 7%; p 0.13), and hiatal hernia (25% vs. 0% vs. 7%; p 0.05). Subjects with PCC showed flattening hemidiaphragms (47% vs. 6% vs. 14%; p 0.007), and reductions in maximum inspiratory pressure (62% vs. 6% vs. 17%; p ≤ 0.001), indicating respiratory muscle weakness. The latter findings suggest additional involvement of the phrenic nerve. DISCUSSION: Vagus and phrenic nerve dysfunction contribute to the complex and multifactorial pathophysiology of PCC.

Vaccination with an HIV T-Cell Immunogen (HTI) Using DNA Primes Followed by a ChAdOx1-MVA Boost Is Immunogenic in Gut Microbiota-Depleted Mice despite Low IL-22 Serum Levels.

Despite the important role of gut microbiota in the maturation of the immune system, little is known about its impact on the development of T-cell responses to vaccination. Here, we immunized C57BL/6 mice with a prime-boost regimen using DNA plasmid, the Chimpanzee Adenovirus, and the modified Vaccinia Ankara virus expressing a candidate HIV T-cell immunogen and compared the T-cell responses between individuals with an intact or antibiotic-depleted microbiota. Overall, the depletion of the gut microbiota did not result in significant differences in the magnitude or breadth of the immunogen-specific IFNγ T-cell response after vaccination. However, we observed marked changes in the serum levels of four cytokines after vaccinating microbiota-depleted animals, particularly a significant reduction in IL-22 levels. Interestingly, the level of IL-22 in serum correlated with the abundance of Roseburia in the large intestine of mice in the mock and vaccinated groups with intact microbiota. This short-chain fatty acid (SCFA)-producing bacterium was significantly reduced in the vaccinated, microbiota-depleted group. Therefore, our results indicate that, although microbiota depletion reduces serum levels of IL-22, the powerful vaccine regime used could have overcome the impact of microbiota depletion on IFNγ-producing T-cell responses.

Animal models to study the neurological manifestations of the post-COVID-19 condition.

More than 40% of individuals infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have experienced persistent or relapsing multi-systemic symptoms months after the onset of coronavirus disease 2019 (COVID-19). This post-COVID-19 condition (PCC) has debilitating effects on the daily life of patients and encompasses a broad spectrum of neurological and neuropsychiatric symptoms including olfactory and gustative impairment, difficulty with concentration and short-term memory, sleep disorders and depression. Animal models have been instrumental to understand acute COVID-19 and validate prophylactic and therapeutic interventions. Similarly, studies post-viral clearance in hamsters, mice and nonhuman primates inoculated with SARS-CoV-2 have been useful to unveil some of the aspects of PCC. Transcriptomic alterations in the central nervous system, persistent activation of immune cells and impaired hippocampal neurogenesis seem to have a critical role in the neurological manifestations observed in animal models infected with SARS-CoV-2. Interestingly, the proinflammatory transcriptomic profile observed in the central nervous system of SARS-CoV-2-inoculated mice partially overlaps with the pathological changes that affect microglia in humans during Alzheimer's disease and aging, suggesting shared mechanisms between these conditions. None of the currently available animal models fully replicates PCC in humans; therefore, multiple models, together with the fine-tuning of experimental conditions, will probably be needed to understand the mechanisms of PCC neurological symptoms. Moreover, given that the intrinsic characteristics of the new variants of concern and the immunological status of individuals might influence PCC manifestations, more studies are needed to explore the role of these factors and their combinations in PCC, adding further complexity to the design of experimental models.

Plasma proteomic profiling identifies CD33 as a marker of HIV control in natural infection and after therapeutic vaccination.

BACKGROUND: Biomarkers predicting the outcome of HIV-1 virus control in natural infection and after therapeutic interventions in HIV-1 cure trials remain poorly defined. The BCN02 trial (NCT02616874), combined a T-cell vaccine with romidepsin (RMD), a cancer-drug that was used to promote HIV-1 latency reversal and which has also been shown to have beneficial effects on neurofunction. We conducted longitudinal plasma proteomics analyses in trial participants to define biomarkers associated with virus control during monitored antiretroviral pause (MAP) and to identify novel therapeutic targets that can improve future cure strategies. METHODS: BCN02 was a phase I, open-label, single-arm clinical trial in early-treated, HIV infected individuals. Longitudinal plasma proteomes were analyzed in 11 BCN02 participants, including 8 participants that showed a rapid HIV-1 plasma rebound during a monitored antiretroviral pause (MAP-NC, 'non-controllers') and 3 that remained off ART with sustained plasma viremia <2000 copies/ml (MAP-C, 'controllers'). Inflammatory and neurological proteomes in plasma were evaluated and integration data analysis (viral and neurocognitive parameters) was performed. Validation studies were conducted in a cohort of untreated HIV-1+ individuals (n = 96) and in vitro viral replication assays using an anti-CD33 antibody were used for functional validation. FINDINGS: Inflammatory plasma proteomes in BCN02 participants showed marked longitudinal alterations. Strong proteome differences were also observed between MAP-C and MAP-NC, including in baseline timepoints. CD33/Siglec-3 was the unique plasma marker with the ability to discriminate between MAPC-C and MAP-NC at all study timepoints and showed positive correlations with viral parameters. Analyses in an untreated cohort of PLWH confirmed the positive correlation between viral parameters and CD33 plasma levels, as well as PBMC gene expression. Finally, adding an anti-CD33 antibody to in vitro virus cultures significantly reduced HIV-1 replication and proviral levels in T cells and macrophages. INTERPRETATION: This study indicates that CD33/Siglec-3 may serve as a predictor of HIV-1 control and as potential therapeutic tool to improve future cure strategies. FUNDING: Spanish Science and Innovation Ministry (SAF2017-89726-R and PID2020-119710RB-I00), NIH (P01-AI131568), European Commission (GA101057548) and a Grifols research agreement.

Schlafen 12 restricts HIV-1 latency reversal by a codon-usage dependent post-transcriptional block in CD4+ T cells.

Latency is a major barrier towards virus elimination in HIV-1-infected individuals. Yet, the mechanisms that contribute to the maintenance of HIV-1 latency are incompletely understood. Here we describe the Schlafen 12 protein (SLFN12) as an HIV-1 restriction factor that establishes a post-transcriptional block in HIV-1-infected cells and thereby inhibits HIV-1 replication and virus reactivation from latently infected cells. The inhibitory activity is dependent on the HIV-1 codon usage and on the SLFN12 RNase active sites. Within HIV-1-infected individuals, SLFN12 expression in PBMCs correlated with HIV-1 plasma viral loads and proviral loads suggesting a link with the general activation of the immune system. Using an RNA FISH-Flow HIV-1 reactivation assay, we demonstrate that SLFN12 expression is enriched in infected cells positive for HIV-1 transcripts but negative for HIV-1 proteins. Thus, codon-usage dependent translation inhibition of HIV-1 proteins participates in HIV-1 latency and can restrict the amount of virus release after latency reversal.

Sirtuin-2, NAD-Dependent Deacetylase, Is a New Potential Therapeutic Target for HIV-1 Infection and HIV-Related Neurological Dysfunction.

The implementation and access to combined antiretroviral treatment (cART) have dramatically improved the quality of life of people living with HIV (PLWH). However, some comorbidities, such as neurological disorders associated with HIV infection still represent a serious clinical challenge. Soluble factors in plasma that are associated with control of HIV replication and neurological dysfunction could serve as early biomarkers and as new therapeutic targets for this comorbidity. We used a customized antibody array for determination of blood plasma factors in 40 untreated PLWH with different levels of viremia and found sirtuin-2 (SIRT2), an NAD-dependent deacetylase, to be strongly associated with elevated viral loads and HIV provirus levels, as well as with markers of neurological damage (a-synuclein [SNCA], brain-derived neurotrophic factor [BDNF], microtubule-associated protein tau [MAPT], and neurofilament light protein [NFL]). Also, longitudinal analysis in HIV-infected individuals with immediate (n = 9) or delayed initiation (n = 10) of cART revealed that after 1 year on cART, SIRT2 plasma levels differed between both groups and correlated inversely with brain orbitofrontal cortex involution. Furthermore, targeting SIRT2 with specific small-molecule inhibitors in in vitro systems using J-LAT A2 and primary glial cells led to diminished HIV replication and virus reactivation from latency. Our data thus identify SIRT2 as a novel biomarker of uncontrolled HIV infection, with potential impact on neurological dysfunction and offers a new therapeutic target for HIV treatment and cure. IMPORTANCE Neurocognitive disorders are frequently reported in people living with HIV (PLWH) even with the introduction of combined antiretroviral treatment (cART). To identify biomarkers and potential therapeutic tools to target HIV infection in peripheral blood and in the central nervous system (CNS), plasma proteomics were applied in untreated chronic HIV-infected individuals with different levels of virus control. High plasma levels of sirtuin-2 (SIRT2), an NAD+ deacetylase, were detected in uncontrolled HIV infection and were strongly associated with plasma viral load and proviral levels. In parallel, SIRT2 levels in the peripheral blood and CNS were associated with markers of neurological damage and brain involution and were more pronounced in individuals who initiated cART later in infection. In vitro infection experiments using specific SIRT2 inhibitors suggest that specific targeting of SIRT2 could offer new therapeutic treatment options for HIV infections and their associated neurological dysfunction.

Disruption of the HLA-E/NKG2X axis is associated with uncontrolled HIV infections.

The contribution of the HLA-E/NKG2X axis in NK-mediated control of HIV infection remains unclear. We have studied the relationship between HLA-E expression and phenotypical as well as functional characteristics of NK cells, in the context of chronic HIV infection and in an in vitro model of acute infection. High viremia in HIV+ individuals was related to increased HLA-E expression, and changes in NK subpopulations, especially a reduction of the CD56bright as well as an increase in adaptive NK subpopulation. Uncontrolled HIV infection was also characterized by a reversion of the NKG2A/NKG2C expression ratio and a loss of positive and negative regulation of NK mediated by HLA-E. This was reflected in a lower cytotoxic, degranulation and cytokine production capacity, especially in CD56bright and adaptive NK. In line with these results, HLA-E expression showed a positive correlation with viral growth inhibition in an in vitro model of acute infection at day 7, which was lost after 14 days of culture. Using HLA-E expressing K562 cells, we determined that only one out of 11 described HIV-derived HLA-E epitopes increased HLA-E surface stability. In spite of that, eight of the 11 epitopes were capable of increasing degranulation and three drove differences in NK-cell mediated cell lysis or cytokine secretion. In conclusion, our results indicate that HLA-E molecules presenting HIV-derived epitopes may sensitize target cells for NK lysis in early HIV infection. However, prolonged exposure to elevated HLA-E expression levels in vivo may lead to NK cell dysfunction and reduced viral control In chronic infection.

Vaccination with an HIV T-cell immunogen induces alterations in the mouse gut microbiota.

The gut microbiota is emerging as a crucial factor modulating vaccine responses; however, few studies have investigated if vaccines, in turn, can alter the microbiota and to what extent such changes may improve vaccine efficacy. To understand the effect of T-cell vaccination on the gut microbiome, we administered an HIV-1 T-cell immunogen (HTI arm) or PBS (control, Mock arm) to C57Bl/6 mice following a heterologous prime-boost scheme. The longitudinal dynamics of the mice gut microbiota was characterized by 16 S ribosomal RNA sequencing in fecal samples collected from cages, as well as from three gut sections (cecum, small and large intestine). Serum and spleen cells were obtained at the last time point of the study to assess immune correlates using IFNγ ELISPOT and cytokine Luminex® assays. Compared with Mock, HTI-vaccinated mice were enriched in Clostridiales genera (Eubacterium xylanophilum group, Roseburia and Ruminococcus) known as primary contributors of anti-inflammatory metabolites, such as short-chain fatty acids. Such shift was observed after the first HTI dose and remained throughout the study follow-up (18 weeks). However, the enriched Clostridiales genera were different between feces and gut sections. The abundance of bacteria enriched in vaccinated animals positively correlated with HTI-specific T-cell responses and a set of pro-inflammatory cytokines, such as IL-6. This longitudinal analysis indicates that, in mice, T-cell vaccination may promote an increase in gut bacteria known to produce anti-inflammatory molecules, which in turn correlate with proinflammatory cytokines, suggesting an adaptation of the gut microbial milieu to T-cell-induced systemic inflammation.

Kinetics of immune responses elicited after three mRNA COVID-19 vaccine doses in predominantly antibody-deficient individuals.

Mass vaccination campaigns reduced COVID-19 incidence and severity. Here, we evaluated the immune responses developed in SARS-CoV-2-uninfected patients with predominantly antibody-deficiencies (PAD) after three mRNA-1273 vaccine doses. PAD patients were classified based on their immunodeficiency: unclassified primary antibody-deficiency (unPAD, n = 9), common variable immunodeficiency (CVID, n = 12), combined immunodeficiency (CID, n = 1), and thymoma with immunodeficiency (TID, n = 1). unPAD patients and healthy controls (HCs, n = 10) developed similar vaccine-induced humoral responses after two doses. However, CVID patients showed reduced binding and neutralizing titers compared to HCs. Of interest, these PAD groups showed lower levels of Spike-specific IFN-γ-producing cells. CVID individuals also presented diminished CD8+T cells. CID and TID patients developed cellular but not humoral responses. Although the third vaccine dose boosted humoral responses in most PAD patients, it had limited effect on expanding cellular immunity. Vaccine-induced immune responses in PAD individuals are heterogeneous, and should be immunomonitored to define a personalized therapeutic strategies.

Administration of broadly neutralizing anti-HIV-1 antibodies at ART initiation maintains long-term CD8+ T cell immunity.

In simian-human immunodeficiency virus (SHIV)-infected non-human primates, broadly neutralizing antibodies (bNAbs) against the virus appear to stimulate T cell immunity. To determine whether this phenomenon also occurs in humans we measured HIV-1-specific cellular immunity longitudinally in individuals with HIV-1 starting antiviral therapy (ART) with or without adjunctive bNAb 3BNC117 treatment. Using the activation-induced marker (AIM) assay and interferon-γ release, we observe that frequencies of Pol- and Gag-specific CD8+ T cells, as well as Gag-induced interferon-γ responses, are significantly higher among individuals that received adjunctive 3BNC117 compared to ART-alone at 3 and 12 months after starting ART. The observed changes in cellular immunity were directly correlated to pre-treatment 3BNC117-sensitivity. Notably, increased HIV-1-specific immunity is associated with partial or complete ART-free virologic control during treatment interruption for up to 4 years. Our findings suggest that bNAb treatment at the time of ART initiation maintains HIV-1-specific CD8+ T cell responses that are associated with ART-free virologic control.

Safety, immunogenicity and effect on viral rebound of HTI vaccines in early treated HIV-1 infection: a randomized, placebo-controlled phase 1 trial.

HIVACAT T-cell immunogen (HTI) is a novel human immunodeficiency virus (HIV) vaccine immunogen designed to elicit cellular immune responses to HIV targets associated with viral control in humans. The AELIX-002 trial was a randomized, placebo-controlled trial to evaluate as a primary objective the safety of a combination of DNA.HTI (D), MVA.HTI (M) and ChAdOx1.HTI (C) vaccines in 45 early-antiretroviral (ART)-treated individuals (44 men, 1 woman; NCT03204617). Secondary objectives included T-cell immunogenicity, the effect on viral rebound and the safety of an antiretroviral treatment interruption (ATI). Adverse events were mostly mild and transient. No related serious adverse events were observed. We show here that HTI vaccines were able to induce strong, polyfunctional and broad CD4 and CD8 T-cell responses. All participants experienced detectable viral rebound during ATI, and resumed ART when plasma HIV-1 viral load reached either >100,000 copies ml-1, >10,000 copies ml-1 for eight consecutive weeks, or after 24 weeks of ATI. In post-hoc analyses, HTI vaccines were associated with a prolonged time off ART in vaccinees without beneficial HLA (human leukocyte antigen) class I alleles. Plasma viral load at the end of ATI and time off ART positively correlated with vaccine-induced HTI-specific T-cell responses at ART cessation. Despite limited efficacy of the vaccines in preventing viral rebound, their ability to elicit robust T-cell responses towards HTI may be beneficial in combination cure strategies, which are currently being tested in clinical trials.

Limited Humoral and Specific T-Cell Responses After SARS-CoV-2 Vaccination in PWH With Poor Immune Reconstitution.

BACKGROUND: We analyzed humoral and cellular immune responses induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) messenger RNA (mRNA) vaccines in people with human immunodeficiency virus (HIV; PWH) who had CD4+ T-cell counts <200/µL (HIV<200 group). METHODS: This prospective cohort study included 58 PWH in the HIV<200 group, 36 with CD4+ T-cell counts >500/µL (HIV>500 group), and 33 HIV-1-negative controls (control group). Antibodies against the SARS-CoV-2 spike protein (anti-S immunoglobulin [Ig] G) and the receptor-binding domain (anti-RBD IgG) were quantified before and 4 weeks after the first and the second doses of BNT162b2 or mRNA-1273 (at week 8). Viral neutralization activity and T-cell responses were also determined. RESULTS: At week 8, anti-S/anti-RBD IgG responses increased in all groups (P < .001). Median (interquartile range) anti-S and anti-RBD IgG levels at week 8 were 153.6 (26.4-654.9) and 171.9 (61.8-425.8) binding antibody units (BAU)/mL, respectively, in the HIV<200 group, compared with 245.6 (145-824) and 555.8 (166.4-1751) BAU/mL in the HIV>500 group and 274.7 (193.7-680.4) and 281.6 (181-831.8) BAU/mL in controls (P < .05). Neutralizing capacity and specific T-cell immune responses were absent or reduced in 33% of those in the HIV<200 group, compared with 3.7% in the HIV>500 group (P < .01). CONCLUSIONS: One-third of PWH with CD4+ T-cell counts <200/µL show low anti-S/anti-RBD IgG levels, reduced in vitro neutralization activity against SARS-CoV-2, and no vaccine-induced T cells after receiving coronavirus disease 2019 mRNA vaccines.

HIV T-cell immunogen design and delivery.

PURPOSE OF THE REVIEW: Not all T-cell responses against HIV are created equally and responses of certain epitope specificities have been associated with superior control of infection. These insights have spurred the development of a wide range of immunogen sequences, each with particular advantages and limitations. RECENT FINDINGS: We review some of the most advanced designs that have reached or are close to reaching human clinical trials, with a special focus on T-cell immunogen developed for therapeutic use. We also touch upon the importance of how immunogens are delivered and point out the lamentable fact that there is essentially no alignment between different designs and vaccine regimens, which is a major hindrance to accelerated advances in the field. SUMMARY: The design of an immunogen able to induce T-cell responses of adequate specificity and functionality is subject of a wide range of preclinical and clinical studies. Few designs have shown promise to date, but emerging data highlight the critical contribution of specificity to effective antiviral activity in vivo .

T-Follicular-Like CD8+ T Cell Responses in Chronic HIV Infection Are Associated With Virus Control and Antibody Isotype Switching to IgG.

T cell responses are considered critical for the in vivo control of HIV, but the contribution of different T cell subsets to this control remains unclear. Using a boosted flow cytometric approach that is able to differentiate CD4+ and CD8+ T cell Th1/Tc1, Th2/Tc2, Th17/Tc17, Treg and Tfh/Tfc-like HIV-specific T cell populations, we identified CD8+ Tfc responses that were related to HIV plasma viral loads and associated with rate of antibody isotype class switching to IgG. This favorable balance towards IgG responses positively correlated with increased virus neutralization, higher avidity of neutralizing antibodies and more potent antibody-dependent cell cytotoxicity (ADCC) in PBMCs from HIV controllers compared to non-controllers. Our results identified the CD8+ Tfc-like T-cell response as a component of effective virus control which could possibly be exploited therapeutically.

Discordance between HIV-1 Population in Plasma at Rebound after Structured Treatment Interruption and Archived Provirus Population in Peripheral Blood Mononuclear Cells.

Antiretroviral therapy (ART) can sustain the suppression of plasma viremia to below detection levels. Infected individuals undergoing a treatment interruption exhibit rapid viral rebound in plasma viremia which is fueled by cellular reservoirs such as CD4+ T cells, myeloid cells, and potentially uncharacterized cellular sources. Interrogating the populations of viruses found during analytical treatment interruption (ATI) can give insights into the biologically competent reservoirs that persist under effective ART as well as the nature of the cellular reservoirs that enable viral persistence under ART. We interrogated plasma viremia from four rare cases of individuals undergoing sequential ATIs. We performed next-generation sequencing (NGS) on cell-associated viral DNA and cell-free virus to understand the interrelationship between sequential ATIs as well as the relationship between viral genomes in circulating peripheral blood mononuclear cells (PBMCs) and RNA from rebound plasma. We observed population differences between viral populations recrudescing at sequential ATIs as well as divergence between viral sequences in plasma and those in PBMCs. This indicated that viruses in PBMCs were not a major source of post-ATI viremia and highlights the role of anatomic reservoirs in post-ATI viremia and viral persistence. IMPORTANCE Even with effective ART, HIV-1 persists at undetectable levels and rebounds in individuals who stop treatment. Cellular and anatomical reservoirs ignite viral rebound upon treatment interruption, remaining one of the key obstacles for HIV-1 cure. To further examine HIV-1 persistence, a better understanding of the distinct populations that fuel viral rebound is necessary to identify and target reservoirs and the eradication of HIV-1. This study investigates the populations of viruses found from proviral genomes from PBMCs and plasma at rebound from a unique cohort of individuals who underwent multiple rounds of treatment interruption. Using NGS, we characterized the subtypes of viral sequences and found divergence in viral populations between plasma and PBMCs at each rebound, suggesting that distinct viral populations appear at each treatment interruption.

Skewed Cellular Distribution and Low Activation of Functional T-Cell Responses in SARS-CoV-2 Non-Seroconvertors.

The role of T cells in the control of SARS-CoV-2 infection has been underestimated in favor of neutralizing antibodies. However, cellular immunity is essential for long-term viral control and protection from disease severity. To understand T-cell immunity in the absence of antibody generation we focused on a group of SARS-CoV-2 Non-Seroconvertors (NSC) recovered from infection. We performed an immune comparative analysis of SARS-CoV-2 infected individuals stratified by the absence or presence of seroconversion and disease severity. We report high levels of total naïve and low effector CD8+ T cells in NSC. Moreover, reduced levels of T-cell activation monitored by PD-1 and activation-induced markers were observed in the context of functional SARS-CoV-2 T-cell responses. Longitudinal data indicate the stability of the NSC phenotype over three months of follow-up after infection. Together, these data characterized distinctive immunological traits in NSC including skewed cellular distribution, low activation and functional SARS-CoV-2 T-cell responses. This data highlights the value of T-cell immune monitoring in populations with low seroconversion rates in response to SARS-CoV-2 infection and vaccination.

Evaluation of the Thermal Stability of a Vaccine Prototype Based on Virus-like Particle Formulated HIV-1 Envelope.

The long-term storage stability of vaccines has a major impact on the roll-out and success of global immunization programs. For the Human Immunodeficiency Virus type 1 (HIV-1) virus-like particle (VLP) vaccine prototype evaluated here, nanoparticle tracking analysis (NTA), and enzyme-linked immunoabsorbent assay (ELISA) results demonstrated a remarkable structural stability. VLPs maintained their integrity and the recognition of relevant B-cell epitopes for three months at 4 and -20 °C. Interestingly, most particles remained intact and preserved the recognition of relevant epitopes even after a week of storage at room temperature.

Gut microbiome signatures linked to HIV-1 reservoir size and viremia control.

BACKGROUND: The potential role of the gut microbiome as a predictor of immune-mediated HIV-1 control in the absence of antiretroviral therapy (ART) is still unknown. In the BCN02 clinical trial, which combined the MVA.HIVconsv immunogen with the latency-reversing agent romidepsin in early-ART treated HIV-1 infected individuals, 23% (3/13) of participants showed sustained low-levels of plasma viremia during 32 weeks of a monitored ART pause (MAP). Here, we present a multi-omics analysis to identify compositional and functional gut microbiome patterns associated with HIV-1 control in the BCN02 trial. RESULTS: Viremic controllers during the MAP (controllers) exhibited higher Bacteroidales/Clostridiales ratio and lower microbial gene richness before vaccination and throughout the study intervention when compared to non-controllers. Longitudinal assessment indicated that the gut microbiome of controllers was enriched in pro-inflammatory bacteria and depleted in butyrate-producing bacteria and methanogenic archaea. Functional profiling also showed that metabolic pathways related to fatty acid and lipid biosynthesis were significantly increased in controllers. Fecal metaproteome analyses confirmed that baseline functional differences were mainly driven by Clostridiales. Participants with high baseline Bacteroidales/Clostridiales ratio had increased pre-existing immune activation-related transcripts. The Bacteroidales/Clostridiales ratio as well as host immune-activation signatures inversely correlated with HIV-1 reservoir size. CONCLUSIONS: The present proof-of-concept study suggests the Bacteroidales/Clostridiales ratio as a novel gut microbiome signature associated with HIV-1 reservoir size and immune-mediated viral control after ART interruption. Video abstract.