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SARS-CoV-2 continues to infect millions of people worldwide. The subvariants arising from the variant-of-concern (VOC) Omicron include BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4, and BA.5. All possess multiple mutations in their Spike glycoprotein, notably in its immunogenic receptor-binding domain (RBD), and present enhanced viral transmission. The highly mutated Spike glycoproteins from these subvariants present different degrees of resistance to recognition and cross-neutralisation by plasma from previously infected and/or vaccinated individuals. We have recently shown that the temperature affects the interaction between the Spike and its receptor, the angiotensin converting enzyme 2 (ACE2). The affinity of RBD for ACE2 is significantly increased at lower temperatures. However, whether this is also observed with the Spike of Omicron and sub-lineages is not known. Here we show that, similar to other variants, Spikes from Omicron sub-lineages bind better the ACE2 receptor at lower temperatures. Whether this translates into enhanced transmission during the fall and winter seasons remains to be determined. [ABSTRACT FROM AUTHOR]

Elimination of human immunodeficiency virus (HIV) reservoirs is a critical endpoint to eradicate HIV. One therapeutic intervention against latent HIV is "shock and kill." This strategy is based on the transcriptional activation of latent HIV with a latency-reversing agent (LRA) with the consequent killing of the reactivated cell by either the cytopathic effect of HIV or the immune system. We have previously found that the small molecule 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HODHBt) acts as an LRA by increasing signal transducer and activator of transcription (STAT) factor activation mediated by interleukin-15 (IL-15) in cells isolated from aviremic participants. The IL-15 superagonist N-803 is currently under clinical investigation to eliminate latent reservoirs. IL-15 and N-803 share similar mechanisms of action by promoting the activation of STATs and have shown some promise in preclinical models directed toward HIV eradication. In this work, we evaluated the ability of HODHBt to enhance IL-15 signaling in natural killer (NK) cells and the biological consequences associated with increased STAT activation in NK cell effector and memory-like functions. We showed that HODHBt increased IL-15-mediated STAT phosphorylation in NK cells, resulting in increases in the secretion of CXCL-10 and interferon gamma (IFN-g) and the expression of cytotoxic proteins, including granzyme B, granzyme A, perforin, granulysin, FASL, and TRAIL. This increased cytotoxic profile results in increased cytotoxicity against HIV-infected cells and different tumor cell lines. HODHBt also improved the generation of cytokine-induced memory-like NK cells. Overall, our data demonstrate that enhancing the magnitude of IL-15 signaling with HODHBt favors NK cell cytotoxicity and memory-like generation, and thus, targeting this pathway could be further explored for HIV cure interventions. [ABSTRACT FROM AUTHOR]

COVID-19 vaccine efficacy has been evaluated in large clinical trials and in real-world situation. Although they have proven to be very effective in the general population, little is known about their efficacy in immunocompromised patients. HIV-infected individuals' response to vaccine may vary according to the type of vaccine and their level of immunosuppression. We evaluated immunogenicity of an mRNA anti-SARS CoV-2 vaccine in HIV-positive individuals. HIV-positive individuals (n = 121) were recruited from HIV clinics in Montreal and stratified according to their CD4 counts. A control group of 20 health care workers naïve to SARS CoV-2 was used. The participants' Anti-RBD IgG responses were measured by ELISA at baseline and 3–4 weeks after receiving the first dose of an mRNA vaccine). Eleven of 121 participants had anti-COVID-19 antibodies at baseline, and a further 4 had incomplete data for the analysis. Mean anti-RBD IgG responses were similar between the HIV negative control group (n = 20) and the combined HIV+ group (n = 106) (p = 0.72). However, these responses were significantly lower in the group with <250 CD4 cells/mm3. (p < 0.0001). Increasing age was independently associated with decreased immunogenicity. HIV-positive individuals with CD4 counts over 250 cells/mm3 have an anti-RBD IgG response similar to the general population. However, HIV-positive individuals with the lowest CD4 counts (<250 cells/mm3) have a weaker response. These data would support the hypothesis that a booster dose might be needed in this subgroup of HIV-positive individuals, depending on their response to the second dose. [ABSTRACT FROM AUTHOR]

Viruses use many different strategies to evade host immune responses. In the case of SARS-CoV-2, its Spike mutates rapidly to escape from neutralizing antibodies. In addition to this strategy, ORF8, a small accessory protein encoded by SARS-CoV-2, helps immune evasion by reducing the susceptibility of SARS-CoV-2-infected cells to the cytotoxic CD8+ T cell response. Interestingly, among all accessory proteins, ORF8 is rapidly evolving and a deletion in this protein has been linked to milder disease. Here, we studied the effect of ORF8 on peripheral blood mononuclear cells (PBMC). Specifically, we found that ORF8 can bind monocytes as well as NK cells. Strikingly, ORF8 binds CD16a (FcγRIIIA) with nanomolar affinity and decreases the overall level of CD16 at the surface of monocytes and, to a lesser extent, NK cells. This decrease significantly reduces the capacity of PBMCs and particularly monocytes to mediate antibody-dependent cellular cytotoxicity (ADCC). Overall, our data identifies a new immune-evasion activity used by SARS-CoV-2 to escape humoral responses. [ABSTRACT FROM AUTHOR]

We assessed the COVID-19 pandemic's impact on treatment of latent tuberculosis, and of active tuberculosis, at 3 centers in Montreal and Toronto, using data from 10 833 patients (8685 with latent tuberculosis infection, 2148 with active tuberculosis). Observation periods prior to declarations of COVID-19 public health emergencies ranged from 219 to 744 weeks, and after declarations, from 28 to 33 weeks. In the latter period, reductions in latent tuberculosis infection treatment initiation rates ranged from 30% to 66%. At 2 centers, active tuberculosis treatment rates fell by 16% and 29%. In Canada, cornerstone measures for tuberculosis elimination weakened during the COVID-19 pandemic. [ABSTRACT FROM AUTHOR]

HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we have developed a cell-based 'gain of function' assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. We developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu; however, this assay may be slightly modified to include more significant Vpu strains in the future. [ABSTRACT FROM AUTHOR]

The activity of broadly neutralizing antibodies (bNAbs) targeting HIV-1 depends on pleiotropic functions, including viral neutralization and the elimination of HIV-1-infected cells. Several in vivo studies have suggested that passive administration of bNAbs represents a valuable strategy for the prevention or treatment of HIV-1. In addition, different strategies are currently being tested to scale up the production of bNAbs to obtain the large quantities of antibodies required for clinical trials. Production of antibodies in plants permits low-cost and large-scale production of valuable therapeutics; furthermore, pertinent to this work, it also includes an advanced glycoengineering platform. In this study, we used Nicotiana benthamiana to produce different Fc-glycovariants of a potent bNAb, PGT121, with near-homogeneous profiles and evaluated their antiviral activities. Structural analyses identified a close similarity in overall structure and glycosylation patterns of Fc regions for these plant-derived Abs and mammalian cell-derived Abs. When tested for Fc-effector activities, afucosylated PGT121 showed significantly enhanced FcgRIIIa interaction and antibody dependent cellular cytotoxicity (ADCC) against primary HIV-1-infected cells, both in vitro and ex vivo. However, the overall galactosylation profiles of plant PGT121 did not affect ADCC activities against infected primary CD41 T cells. Our results suggest that the abrogation of the Fc N-linked glycan fucosylation of PGT121 is a worthwhile strategy to boost its Fc-effector functionality. IMPORTANCE PGT121 is a highly potent bNAb and its antiviral activities for HIV-1 prevention and therapy are currently being evaluated in clinical trials. The importance of its Fc-effector functions in clearing HIV-1-infected cells is also under investigation. Our results highlight enhanced Fc-effector activities of afucosylated PGT121 MAbs that could be important in a therapeutic context to accelerate infected cell clearance and slow disease progression. Future studies to evaluate the potential of plant-produced afucosylated PGT121 in controlling HIV-1 replication in vivo are warranted. [ABSTRACT FROM AUTHOR]

The HIV-1 envelope glycoprotein (Env) trimer [(gp120/gp41)3] is a metastable complex expressed at the surface of viral particles and infected cells that samples different conformations. Before engaging CD4, Env adopts an antibody-resistant "closed" conformation (State 1). CD4 binding triggers an intermediate conformation (State 2) and then a more "open" conformation (State 3) that can be recognized by nonneutralizing antibodies (nnAbs) such as those that recognize the coreceptor binding site (CoRBS). Binding of antibodies to the CoRBS permits another family of nnAbs, the anti-cluster A family of Abs which target the gp120 inner domain, to bind and stabilize an asymmetric conformation (State 2A). Cells expressing Env in this conformation are susceptible to antibody-dependent cellular cytotoxicity (ADCC). This conformation can be stabilized by small-molecule CD4 mimetics (CD4mc) or soluble CD4 (sCD4) in combination with anti-CoRBS Ab and anti-cluster A antibodies. The precise stoichiometry of each component that permits this sequential opening of Env remains unknown. Here, we used a cell-based enzyme-linked immunosorbent assay (CBE) to evaluate each component individually. In this assay, we used a "trimer mixing" approach by combining wild-type (wt) subunits with subunits impaired for CD4 or CoRBS Ab binding. This enabled us to show that State 2A requires all three gp120 subunits to be bound by sCD4/CD4mc and anti-CoRBS Abs. Two of these subunits can then bind anti-cluster A Abs. Altogether, our data suggest how this antibody-vulnerable Env conformation is stabilized. IMPORTANCE Stabilization of HIV-1 Env State 2A has been shown to sensitize infected cells to ADCC. State 2A can be stabilized by a "cocktail" composed of CD4mc, anti- CoRBS, and anti-cluster A Abs. We present evidence that optimal State 2A stabilization requires all three gp120 subunits to be bound by both CD4mc and anti-CoRBS Abs. Our study provides valuable information on how to stabilize this ADCC-vulnerable conformation. Strategies aimed at stabilizing State 2A might have therapeutic utility. [ABSTRACT FROM AUTHOR]

Background: Phosphate is an essential plant macronutrient required to achieve maximum crop yield. Roots are able to uptake soil phosphate from the immediate root area, thus creating a nutrient depletion zone. Many plants are able to exploit phosphate from beyond this root nutrient depletion zone through symbiotic association with Arbuscular Mycorrhizal Fungi (AMF). Here we characterise the relationship between root architecture, AMF association and low phosphate tolerance in strawberries. The contrasting root architecture in the parental strawberry cultivars 'Redgauntlet' and 'Hapil' was studied through a mapping population of 168 progeny. Low phosphate tolerance and AMF association was quantified for each genotype to allow assessment of the phenotypic and genotypic relationships between traits. Results: A "phosphate scavenging" root phenotype where individuals exhibit a high proportion of surface lateral roots was associated with a reduction in root system size across genotypes. A genetic correlation between "root system size" traits was observed with a network of pleiotropic QTL found to represent five "root system size" traits. By contrast, average root diameter and the distribution of roots appeared to be under two discrete methods of genetic control. A total of 18 QTL were associated with plant traits, 4 of which were associated with solidity that explained 46% of the observed variation. Investigations into the relationship between AMF association and root architecture found that a higher root density was associated with greater AMF colonisation across genotypes. However, no phenotypic correlation or genotypic association was found between low phosphate tolerance and the propensity for AMF association, nor root architectural traits when plants are grown under optimal nutrient conditions. Conclusions: Understanding the genetic relationships underpinning phosphate capture can inform the breeding of strawberry varieties with better nutrient use efficiency. Solid root systems were associated with greater AMF colonisation. However, low P-tolerance was not phenotypically or genotypically associated with root architecture traits in strawberry plants. Furthermore, a trade-off was observed between root system size and root architecture type, highlighting the energetic costs associated with a "phosphate scavenging" root architecture. [ABSTRACT FROM AUTHOR]

To minimize immune responses against infected cells, HIV-1 limits the surface expression of its envelope glycoprotein (Env). Here we demonstrate that this mechanism is specific for the Env conformation and affects the efficiency of ADCC. Using flow cytometry and confocal microscopy we show that broadly neutralizing antibodies (bNAbs) targeting the "closed" conformation of Env induce its internalization from the surface. In contrast, non-neutralizing antibodies (nNAbs) are displayed on the cell surface for prolonged period of times. The bNAb-induced Env internalization can be decreased by blocking dynamin function, which translates into higher susceptibility of infected cells to antibody-dependant cellular cytotoxicity (ADCC). Our results suggest that antibody-mediated Env internalization is a mechanism used by HIV-1 to evade immune responses against the "closed" conformation of Env expressed on HIV-1-infected cells. Importance HIV-1 has evolved to acquire several strategies to limit the exposure of its envelope glycoproteins (Env) on the surface of infected cells. In this study, we show that antibody-induced Env internalization is conformation specific and reduces the susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). Thus a better understanding this mechanism might help develop antibodies with improved capacities to mediate ADCC. [ABSTRACT FROM AUTHOR]

HIV-1 envelope glycoprotein (Env) conformation substantially impacts antibody-dependent cellular cytotoxicity (ADCC). Envs from primary HIV-1 isolates adopt a prefusion "closed" conformation, which is targeted by broadly neutralizing antibodies (bnAbs). CD4 binding drives Env into more "open" conformations, which are recognized by non-neutralizing Abs (nnAbs). To better understand Env-Ab and Env-CD4 interaction in CD4+ T cells infected with HIV-1, we simultaneously measured antibody binding and HIV-1 mRNA expression using multiparametric flow cytometry and RNA flow fluorescent in situ hybridization (FISH) techniques. We observed that env mRNA is almost exclusively expressed by HIV-1 productively infected cells that already downmodulated CD4. This suggests that CD4 downmodulation precedes env mRNA expression. Consequently, productively infected cells express "closed" Envs on their surface, which renders them resistant to nnAbs. Cells recognized by nnAbs were all env mRNA negative, indicating Ab binding through shed gp120 or virions attached to their surface. Consistent with these findings, treatment of HIV-1-infected humanized mice with the ADCC-mediating nnAb A32 failed to lower viral replication or reduce the size of the viral reservoir. These findings confirm the resistance of productively infected CD4+ T cells to nnAbs-mediated ADCC and question the rationale of immunotherapy approaches using this strategy., Importance: Antibody-dependent cellular cytotoxicity (ADCC) represents an effective immune response for clearing virally infected cells, making ADCC-mediating antibodies promising therapeutic candidates for HIV-1 cure strategies. Broadly neutralizing antibodies (bNAbs) target epitopes present on the native "closed" envelope glycoprotein (Env), while non-neutralizing antibodies (nnAbs) recognize epitopes exposed upon Env-CD4 interaction. Here, we provide evidence that env mRNA is predominantly expressed by productively infected cells that have already downmodulated cell-surface CD4. This indicates that CD4 downmodulation by HIV-1 precedes Env expression, making productively infected cells resistant to ADCC mediated by nnAbs but sensitive to those mediated by bnAbs. These findings offer critical insights for the development of immunotherapy-based strategies aimed at targeting and eliminating productively infected cells in people living with HIV.

The majority of naturally elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs) because they are unable to recognize the Env trimer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC-mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly neutralizing antibodies and even showed activity against HIV-1-infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies.IMPORTANCEThe elimination of HIV-1-infected cells remains an important medical goal. Although current antiretroviral therapy decreases viral loads below detection levels, it does not eliminate latently infected cells that form the viral reservoir. Here, we developed a cocktail of non-neutralizing antibodies targeting highly conserved Env regions and combined it with a potent indoline CD4mc. This combination exhibited potent ADCC activity against HIV-1-infected primary CD4 + T cells as well as monocyte-derived macrophages, suggesting its potential utility in decreasing the size of the viral reservoir.

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation that occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here, we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site, and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in the presence of CD4mc., Importance: There are several reasons that make it difficult to target the HIV reservoir. One of them is the capacity of infected cells to prevent the recognition of HIV-1 envelope glycoproteins (Env) by commonly elicited antibodies in people living with HIV. Small CD4-mimetic compounds expose otherwise occluded Env epitopes, thus enabling their recognition by non-neutralizing antibodies (nnAbs). A better understanding of the contribution of these antibodies to eliminate infected cells in the presence of CD4mc could lead to the development of therapeutic cure strategies.

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. Structural and conformational analyses reveal that CJF-III-288, in combination with anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation, This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

The mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-week supplementation of antiretroviral therapy (ART) with metformin, an indirect mTOR inhibitor used in type-2 diabetes treatment, reduced mTOR activation and HIV transcription in colon-infiltrating CD4 + T cells, together with systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein, we investigated the antiviral mechanisms of metformin. In a viral outgrowth assay performed with CD4 + T cells from ART-treated PWH, and upon infection in vitro with replication-competent and VSV-G-pseudotyped HIV-1, metformin decreased virion release, but increased the frequency of productively infected CD4 low HIV-p24 + T cells. These observations coincided with increased BST2/tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression, and improved recognition of productively infected T cells by HIV-1 envelope antibodies. Thus, metformin exerts pleiotropic effects on post-integration steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

CD4-mimetics (CD4mcs) are small molecule compounds that mimic the interaction of the CD4 receptor with HIV-1 envelope glycoproteins (Env). Env from primary viruses normally samples a "closed" conformation which occludes epitopes recognized by CD4-induced (CD4i) non-neutralizing antibodies (nnAbs). CD4mcs induce conformational changes on Env resulting in the exposure of these otherwise inaccessible epitopes. Here we evaluated the capacity of plasma from a cohort of 50 people living with HIV to recognize HIV-1-infected cells and eliminate them by antibody-dependent cellular cytotoxicity (ADCC) in the presence of a potent indoline CD4mc. We observed a marked heterogeneity among plasma samples. By measuring the levels of different families of CD4i Abs, we found that the levels of anti-cluster A, anti-coreceptor binding site and anti-gp41 cluster I antibodies are responsible for plasma-mediated ADCC in presence of CD4mc.

Plasma RNAemia, delayed antibody responses and inflammation predict COVID-19 outcomes, but the mechanisms underlying these immunovirological patterns are poorly understood. We profile 782 longitudinal plasma samples from 318 hospitalized patients with COVID-19. Integrated analysis using k-means reveals four patient clusters in a discovery cohort: mechanically ventilated critically-ill cases are subdivided into good prognosis and high-fatality clusters (reproduced in a validation cohort), while non-critical survivors segregate into high and low early antibody responders. Only the high-fatality cluster is enriched for transcriptomic signatures associated with COVID-19 severity, and each cluster has distinct RBD-specific antibody elicitation kinetics. Both critical and non-critical clusters with delayed antibody responses exhibit sustained IFN signatures, which negatively correlate with contemporaneous RBD-specific IgG levels and absolute SARS-CoV-2-specific B and CD4 + T cell frequencies. These data suggest that the "Interferon paradox" previously described in murine LCMV models is operative in COVID-19, with excessive IFN signaling delaying development of adaptive virus-specific immunity., (© 2024. The Author(s).)

Background: Chronic inflammation persists in some people living with human immunodeficiency virus (HIV) during antiretroviral therapy and is associated with premature aging. The glycoprotein 120 (gp120) subunit of HIV-1 envelope sheds and can be detected in plasma, showing immunomodulatory properties even in the absence of detectable viremia. We evaluated whether plasma soluble gp120 (sgp120) and a family of gp120-specific anti-cluster A antibodies, linked to CD4 depletion in vitro, contribute to chronic inflammation, immune dysfunction, and subclinical cardiovascular disease in participants of the Canadian HIV and Aging Cohort Study with undetectable viremia., Methods: Cross-sectional assessment of sgp120 and anti-cluster A antibodies was performed in 386 individuals from the cohort. Their association with proinflammatory cytokines and subclinical coronary artery disease was assessed using linear regression models., Results: High levels of sgp120 and anti-cluster A antibodies were inversely correlated with CD4+ T cell count and CD4/CD8 ratio. The presence of sgp120 was associated with increased levels of interleukin 6. In participants with detectable atherosclerotic plaque and detectable sgp120, anti-cluster A antibodies and their combination with sgp120 levels correlated positively with the total volume of atherosclerotic plaques., Conclusions: This study showed that sgp120 may act as a pan toxin causing immune dysfunction and sustained inflammation in a subset of people living with HIV, contributing to the development of premature comorbid conditions., Competing Interests: Potential conflicts of interest. W. M., C. T., M. P., and A. F. received funding from ViiV Healthcare. A. C. is a full-time employee of ViiV Healthcare. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

We have previously shown that blood levels of B-cell Activating Factor (BAFF) rise relatively to disease progression status in the context of HIV-1 infection. Excess BAFF was concomitant with hyperglobulinemia and the deregulation of blood B-cell populations, notably with increased frequencies of a population sharing characteristics of transitional immature and marginal zone (MZ) B-cells, which we defined as marginal zone precursor-like" (MZp). In HIV-uninfected individuals, MZp present a B-cell regulatory (Breg) profile and function, which are lost in classic-progressors. Moreover, RNASeq analyses of blood MZp from classic-progressors depict a hyperactive state and signs of exhaustion, as well as an interferon signature similar to that observed in autoimmune disorders such as Systemic Lupus Erythematosus (SLE) and Sjögren Syndrome (SS), in which excess BAFF and deregulated MZ populations have also been documented. Based on the above, we hypothesize that excess BAFF may preclude the generation of HIV-1-specific IgG responses and drive polyclonal responses, including those from MZ populations, endowed with polyreactivity/autoreactivity. As such, we show that the quantity of HIV-1-specific IgG varies with disease progression status. In vitro, excess BAFF promotes polyclonal IgM and IgG responses, including those from MZp. RNASeq analyses reveal that blood MZp from classic-progressors are prone to Ig production and preferentially make usage of IGHV genes associated with some HIV broadly neutralizing antibodies (bNAbs), but also with autoantibodies, and whose impact in the battle against HIV-1 has yet to be determined.

Background: Self-reported height, weight and body mass index (BMI) data are widely used to monitor trends in malnutrition. However, several studies expressed concerns about its reliability-citing trends of over-reporting and underreporting anthropometric data. This study aims to: (1) identify the validity of self-reported height and weight and BMI as compared with measured values and (2) examine the potential recurrence of malnutrition in an urban-based population., Methods: Paired t-tests and Pearson's correlation coefficients were conducted to identify potential discrepancies between self-reported and measured anthropometric data. These values were collected among 255 male and 400 female participants in the Davao City., Results: Height overestimation in females and underestimation in males were observed to be statistically significant (P < 0.05). Researchers also note an alarming rise in malnutrition cases when the Asia-Pacific Index was applied to the BMI study data. A 40.79 and 22% increase in obese cases among male and female respondents were recorded., Conclusion: Modifying participant-gathered height and weight values is likely to result in discrepancies between self-reported and measured values. Identifying a person's height and weight status is crucial to understanding who among the population experience malnutrition. Thus, policymakers are called to strengthen educational support that trains respondents to report reliable and valid health data., (© The Author(s) 2023. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

HIV-1 evades antibody-dependent cellular cytotoxicity (ADCC) responses not only by controlling Env conformation and quantity at the cell surface but also by altering NK cell activation via the downmodulation of several ligands of activating and co-activating NK cell receptors. The signaling lymphocyte activation molecule (SLAM) family of receptors, which includes NTB-A and 2B4, act as co-activating receptors to sustain NK cell activation and cytotoxic responses. These receptors cooperate with CD16 (FcγRIII) and other activating receptors to trigger NK cell effector functions. In that context, Vpu-mediated downregulation of NTB-A on HIV-1-infected CD4 T cells was shown to prevent NK cell degranulation via an homophilic interaction, thus contributing to ADCC evasion. However, less is known on the capacity of HIV-1 to evade 2B4-mediated NK cell activation and ADCC. Here, we show that HIV-1 downregulates the ligand of 2B4, CD48, from the surface of infected cells in a Vpu-dependent manner. This activity is conserved among Vpu proteins from the HIV-1/SIVcpz lineage and depends on conserved residues located in its transmembrane domain and dual phosphoserine motif. We show that NTB-A and 2B4 stimulate CD16-mediated NK cell degranulation and contribute to ADCC responses directed to HIV-1-infected cells to the same extent. Our results suggest that HIV-1 has evolved to downmodulate the ligands of both SLAM receptors to evade ADCC. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) can contribute to the elimination of HIV-1-infected cells and HIV-1 reservoirs. An in-depth understanding of the mechanisms used by HIV-1 to evade ADCC might help develop novel approaches to reduce the viral reservoirs. Members of the signaling lymphocyte activation molecule (SLAM) family of receptors, such as NTB-A and 2B4, play a key role in stimulating NK cell effector functions, including ADCC. Here, we show that Vpu downmodulates CD48, the ligand of 2B4, and this contributes to protect HIV-1-infected cells from ADCC. Our results highlight the importance of the virus to prevent the triggering of the SLAM receptors to evade ADCC., Competing Interests: The authors declare no conflict of interest.

While HIV-1-mediated CD4 downregulation protects infected cells from antibody-dependent cellular cytotoxicity (ADCC), shed gp120 binds to CD4 on uninfected bystander CD4 + T cells, sensitizing them to ADCC mediated by HIV + plasma. Soluble gp120-CD4 interaction on multiple immune cells also triggers a cytokine burst. The small molecule temsavir acts as an HIV-1 attachment inhibitor by preventing envelope glycoprotein (Env)-CD4 interaction and alters the overall antigenicity of Env by affecting its processing and glycosylation. Here we show that temsavir also blocks the immunomodulatory activities of shed gp120. Temsavir prevents shed gp120 from interacting with uninfected bystander CD4 + cells, protecting them from ADCC responses and preventing a cytokine burst. Mechanistically, this depends on temsavir's capacity to prevent soluble gp120-CD4 interaction, to reduce gp120 shedding, and to alter gp120 antigenicity. This suggests that the clinical benefits provided by temsavir could extend beyond blocking viral entry., Competing Interests: Declaration of interests W.M. and A.F. received funding from ViiV Healthcare. A.C. is a full-time employee of ViiV Healthcare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Binding to the host cell receptors, CD4 and CCR5/CXCR4, triggers large-scale conformational changes in the HIV-1 envelope glycoprotein (Env) trimer [(gp120/gp41) 3 ] that promote virus entry into the cell. CD4-mimetic compounds (CD4mcs) comprise small organic molecules that bind in the highly conserved CD4-binding site of gp120 and prematurely induce inactivating Env conformational changes, including shedding of gp120 from the Env trimer. By inducing more "open," antibody-susceptible Env conformations, CD4mcs also sensitize HIV-1 virions to neutralization by antibodies and infected cells to antibody-dependent cellular cytotoxicity (ADCC). Here, we report the design, synthesis, and evaluation of novel CD4mcs based on an indoline scaffold. Compared with our current lead indane scaffold CD4mc, BNM-III-170, several indoline CD4mcs exhibit increased potency and breadth against HIV-1 variants from different geographic clades. Viruses that were selected for resistance to the lead indane CD4mc, BNM-III-170, are susceptible to inhibition by the indoline CD4mcs. The indoline CD4mcs also potently sensitize HIV-1-infected cells to ADCC mediated by plasma from HIV-1-infected individuals. Crystal structures indicate that the indoline CD4mcs gain potency compared to the indane CD4mcs through more favorable π-π overlap from the indoline pose and by making favorable contacts with the vestibule of the CD4-binding pocket on gp120. The rational design of indoline CD4mcs thus holds promise for further improvements in antiviral activity, potentially contributing to efforts to treat and prevent HIV-1 infection.

Small CD4-mimetic compound (CD4mc), which inhibits the interaction between gp120 with CD4, acts as an entry inhibitor and induces structural changes in the HIV-1 envelope glycoprotein trimer (Env) through its insertion within the Phe43 cavity of gp120. We recently developed YIR-821, a novel CD4mc, that has potent antiviral activity and lower toxicity than the prototype NBD-556. To assess the possibility of clinical application of YIR-821, we tested its antiviral activity using a panel of HIV-1 pseudoviruses from different subtypes. YIR-821 displayed entry inhibitor activity against 53.5% (21/40) of the pseudoviruses tested and enhanced neutralization mediated by coreceptor binding site (CoRBS) antibodies in 50% (16/32) of these. Furthermore, when we assessed the antiviral effects using a panel of pseudoviruses and autologous plasma IgG, enhancement of antibody-mediated neutralization activity was observed for 48% (15/31) of subtype B strains and 51% (28/55) of non-B strains. The direct antiviral activity of YIR-821 as an entry inhibitor was observed in 53% of both subtype B (27/51) and non-B subtype (40/75) pseudoviruses. Enhancement of antibody-dependent cellular cytotoxicity was also observed with YIR-821 for all six selected clinical isolates, as well as for the transmitted/founder (T/F) CH58 virus-infected cells. The sequence diversity in the CD4 binding site as well as other regions, such as the gp120 inner domain layers or gp41, may be involved in the multiple mechanisms related to the sensitive/resistant phenotype of the virus to YIR-821. Our findings may facilitate the clinical application of YIR-821. IMPORTANCE Small CD4-mimetic compound (CD4mc) interacts with the Phe43 cavity and triggers conformational changes, enhancing antibody-mediated neutralization and antibody-dependent cellular cytotoxicity (ADCC). Here, we evaluated the effect of YIR-821, a novel CD4mc, against clinical isolates, including both subtype B and non-B subtype viruses. Our results confirm the desirable properties of YIR-821, which include entry inhibition, enhancement of IgG-neutralization, binding, and ADCC, in addition to low toxicity and long half-life in a rhesus macaque model, that might facilitate the clinical application of this novel CD4mc. Our observation of primary viruses that are resistant to YIR-821 suggests that further development of CD4mcs with different structural properties is required.

HIV-1 envelope (Env) conformation determines the susceptibility of infected CD4 + T cells to antibody-dependent cellular cytotoxicity (ADCC). Upon interaction with CD4, Env adopts more "open" conformations, exposing ADCC epitopes. HIV-1 limits Env-CD4 interaction and protects infected cells against ADCC by downregulating CD4 via Nef, Vpu, and Env. Limited data exist, however, of the role of these proteins in downmodulating CD4 on infected macrophages and how this impacts Env conformation. While Nef, Vpu, and Env are all required to efficiently downregulate CD4 on infected CD4 + T cells, we show here that any one of these proteins is sufficient to downmodulate most CD4 from the surface of infected macrophages. Consistent with this finding, Nef and Vpu have a lesser impact on Env conformation and ADCC sensitivity in infected macrophages compared with CD4 + T cells. However, treatment of infected macrophages with small CD4 mimetics exposes vulnerable CD4-induced Env epitopes and sensitizes them to ADCC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV-2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro . Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients., Competing Interests: A.F. has filed a provisional patent application on the following monoclonal antibodies: CV3-1 and CV3-25. A.F., J.P., V.L. M.A.S., V.-P.L., and E.H. have filed a provisional patent application on the following monoclonal antibodies: EH3 and EH8., (© 2022 The Author(s).)

COVID-19 convalescent plasmas (CCPs) are chosen for plasma therapy based on neutralizing titers and anti-Spike immunoglobulin levels. However, CCP characteristics that promote SARS-CoV-2 control are complex and incompletely defined. Using an in vivo imaging approach, we demonstrate that CCPs with low neutralizing (ID 50  ≤ 1:250), but moderate to high Fc-effector activity, in contrast to those with poor Fc function, delay mortality and/or improve survival of SARS-CoV-2-challenged K18-hACE2 mice. The impact of innate immune cells on CCP efficacy depended on their residual neutralizing activity. Fractionation of a selected CCP revealed that IgG and Ig(M + A) were required during therapy, but the IgG fraction alone sufficed during prophylaxis. Finally, despite reduced neutralization, ancestral SARS-CoV-2-elicited CCPs significantly delayed Delta and Beta-induced mortality suggesting that Fc-effector functions contribute to immunity against VOCs. Thus, Fc activity of CCPs provide a second line of defense when neutralization is compromised and can serve as an important criterion for CCP selection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer on the virion surface interacts with the host receptors, CD4 and CCR5/CXCR4, to mediate virus entry into the target cell. CD4-mimetic compounds (CD4mcs) bind the gp120 Env, block CD4 binding, and inactivate Env. Previous studies suggested that a C(5)-methylamino methyl moiety on a lead CD4mc, BNM-III-170, contributed to its antiviral potency. By replacing the C(5) chain with differentially substituted pyrrolidine, piperidine, and piperazine ring systems, guided by structural and computational analyses, we found that the 5-position of BNM-III-170 is remarkably tolerant of a variety of ring sizes and substitutions, both in regard to antiviral activity and sensitization to humoral responses. Crystallographic analyses of representative analogues from the pyrrolidine series revealed the potential for 5-substituents to hydrogen bond with gp120 Env residue Thr 283. Further optimization of these interactions holds promise for the development of CD4mcs with greater potency., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

Non-neutralizing antibodies (nnAbs) can eliminate HIV-1-infected cells via antibody-dependent cellular cytotoxicity (ADCC) and were identified as a correlate of protection in the RV144 vaccine trial. Fc-mediated effector functions of nnAbs were recently shown to alter the course of HIV-1 infection in vivo using a vpu-defective virus. Since Vpu is known to downregulate cell-surface CD4, which triggers conformational changes in the viral envelope glycoprotein (Env), we ask whether the lack of Vpu expression was linked to the observed nnAbs activity. We find that restoring Vpu expression greatly reduces nnAb recognition of infected cells, rendering them resistant to ADCC. Moreover, administration of nnAbs in humanized mice reduces viral loads only in animals infected with a vpu-defective but not with a wild-type virus. CD4-mimetics administration, known to "open" Env and expose nnAb epitopes, renders wild-type viruses sensitive to nnAbs Fc-effector functions. This work highlights the importance of Vpu-mediated evasion of humoral responses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Due to the recrudescence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections worldwide, mainly caused by the Omicron variant of concern (VOC) and its sub-lineages, several jurisdictions are administering an mRNA vaccine boost. Here, we analyze humoral responses induced after the second and third doses of an mRNA vaccine in naive and previously infected donors who received their second dose with an extended 16-week interval. We observe that the extended interval elicits robust humoral responses against VOCs, but this response is significantly diminished 4 months after the second dose. Administering a boost to these individuals brings back the humoral responses to the same levels obtained after the extended second dose. Interestingly, we observe that administering a boost to individuals that initially received a short 3- to 4-week regimen elicits humoral responses similar to those observed in the long interval regimen. Nevertheless, humoral responses elicited by the boost in naive individuals do not reach those present in previously infected vaccinated individuals., Competing Interests: Declaration of interests A.R.G. is a consultant for Relation Therapeutics. E.J.W. is consulting for or is an advisor for Merck, Marengo, Janssen, Related Sciences, Synthekine, and Surface Oncology. E.J.W. is a founder of Surface Oncology, Danger Bio, and Arsenal Biosciences., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Binding to the host cell receptors CD4 and CCR5/CXCR4 triggers conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer that promote virus entry. CD4 binding allows the gp120 exterior Env to bind CCR5/CXCR4 and induces a short-lived prehairpin intermediate conformation in the gp41 transmembrane Env. Small-molecule CD4-mimetic compounds (CD4mcs) bind within the conserved Phe-43 cavity of gp120, near the binding site for CD4. CD4mcs like BNM-III-170 inhibit HIV-1 infection by competing with CD4 and by prematurely activating Env, leading to irreversible inactivation. In cell culture, we selected and analyzed variants of the primary HIV-1 AD8 strain resistant to BNM-III-170. Two changes (S375N and I424T) in gp120 residues that flank the Phe-43 cavity each conferred an ~5-fold resistance to BNM-III-170 with minimal fitness cost. A third change (E64G) in layer 1 of the gp120 inner domain resulted in ~100-fold resistance to BNM-III-170, ~2- to 3-fold resistance to soluble CD4-Ig, and a moderate decrease in viral fitness. The gp120 changes additively or synergistically contributed to BNM-III-170 resistance. The sensitivity of the Env variants to BNM-III-170 inhibition of virus entry correlated with their sensitivity to BNM-III-170-induced Env activation and shedding of gp120. Together, the S375N and I424T changes, but not the E64G change, conferred >100-fold and 33-fold resistance to BMS-806 and BMS-529 (temsavir), respectively, potent HIV-1 entry inhibitors that block Env conformational transitions. These studies identify pathways whereby HIV-1 can develop resistance to CD4mcs and conformational blockers, two classes of entry inhibitors that target the conserved gp120 Phe-43 cavity. IMPORTANCE CD4-mimetic compounds (CD4mcs) and conformational blockers like BMS-806 and BMS-529 (temsavir) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. Although CD4mcs and conformational blockers inhibit HIV-1 entry by different mechanisms, they both target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor CD4 and is highly conserved among HIV-1 strains. Our study identifies changes near this pocket that can confer various levels of resistance to the antiviral effects of a CD4mc and conformational blockers. We relate the antiviral potency of a CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate changes in Env conformation and to induce the shedding of the gp120 exterior Env from the spike. These findings will guide efforts to improve the potency and breadth of small-molecule HIV-1 entry inhibitors.

SARS-CoV-2 infection of host cells starts by binding the Spike glycoprotein (S) to the ACE2 receptor. The S-ACE2 interaction is a potential target for therapies against COVID-19 as demonstrated by the development of immunotherapies blocking this interaction. VE607 - a commercially available compound composed of three stereoisomers - was described as an inhibitor of SARS-CoV-1. Here, we show that VE607 broadly inhibits pseudoviral particles bearing the Spike from major VOCs (D614G, Alpha, Beta, Gamma, Delta, Omicron - BA.1, and BA.2) as well as authentic SARS-CoV-2 at low micromolar concentrations. In silico docking, mutational analysis, and smFRET revealed that VE607 binds to the receptor binding domain (RBD)-ACE2 interface and stabilizes RBD in its "up" conformation. Prophylactic treatment with VE607 did not prevent SARS-CoV-2-induced mortality in K18-hACE2 mice, but it did reduce viral replication in the lungs by 37-fold. Thus, VE607 is an interesting lead for drug development for the treatment of SARS-CoV-2 infection., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

The heavily glycosylated HIV-1 envelope glycoprotein (Env) is the sole viral antigen present at the surface of virions and infected cells, representing the main target for antibody responses. The FDA-approved small molecule temsavir acts as an HIV-1 attachment inhibitor by preventing Env-CD4 interaction. This molecule also stabilizes Env in a prefusion "closed" conformation that is preferentially targeted by several broadly neutralizing antibodies (bNAbs). A recent study showed that an analog of temsavir (BMS-377806) affects the cleavage and addition of complex glycans on Env. In this study, we investigated the impact of temsavir on the overall glycosylation, proteolytic cleavage, cell surface expression, and antigenicity of Env. We found that temsavir impacts Env glycosylation and processing at physiological concentrations. This significantly alters the capacity of several bNAbs to recognize Env present on virions and HIV-1-infected cells. Temsavir treatment also reduces the capacity of bNAbs to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC). Consequently, the impact of temsavir on Env glycosylation and antigenicity should be considered for the development of new antibody-based approaches in temsavir-treated individuals. IMPORTANCE FDA-approved fostemsavir, the prodrug for the active moiety small molecule temsavir (GSK 2616713 [formally BMS-626529]), acts as an attachment inhibitor by targeting the HIV-1 envelope (Env) and preventing CD4 interaction. Temsavir also stabilizes Env in its "closed," functional state 1 conformation, which represents an ideal target for broadly neutralizing antibodies (bNAbs). Since these antibodies recognize conformation-dependent epitopes composed of or adjacent to glycans, we evaluated the impact of temsavir treatment on overall Env glycosylation and its influence on bNAb recognition. Our results showed an alteration of Env glycosylation and cleavage by temsavir at physiological concentrations. This significantly modifies the overall antigenicity of Env and therefore reduces the capacity of bNAbs to recognize and eliminate HIV-1-infected cells by ADCC. These findings provide important information for the design of immunotherapies aimed at targeting the viral reservoir in temsavir-treated individuals.

The HIV-1 Nef and Vpu accessory proteins are known to protect infected cells from antibody-dependent cellular cytotoxicity (ADCC) responses by limiting exposure of CD4-induced (CD4i) envelope (Env) epitopes at the cell surface. Although both proteins target the host receptor CD4 for degradation, the extent of their functional redundancy is unknown. Here, we developed an intracellular staining technique that permits the intracellular detection of both Nef and Vpu in primary CD4 + T cells by flow cytometry. Using this method, we show that the combined expression of Nef and Vpu predicts the susceptibility of HIV-1-infected primary CD4 + T cells to ADCC by HIV+ plasma. We also show that Vpu cannot compensate for the absence of Nef, thus providing an explanation for why some infectious molecular clones that carry a LucR reporter gene upstream of Nef render infected cells more susceptible to ADCC responses. Our method thus represents a new tool to dissect the biological activity of Nef and Vpu in the context of other host and viral proteins within single infected CD4 + T cells. IMPORTANCE HIV-1 Nef and Vpu exert several biological functions that are important for viral immune evasion, release, and replication. Here, we developed a new method allowing simultaneous detection of these accessory proteins in their native form together with some of their cellular substrates. This allowed us to show that Vpu cannot compensate for the lack of a functional Nef, which has implications for studies that use Nef-defective viruses to study ADCC responses.

The SARS-CoV-2 virus is the causing agent of the coronavirus disease 2019 (COVID-19) pandemic responsible for millions of deaths worldwide. The development of the humoral response to the virus has been the subject of intensive research. A flow cytometry-based assay using native full-length SARS-CoV-2 Spike protein expressed in 293 T cells was recently proposed as a complementary seropositivity assay. The aim of our study was to further develop the flow cytometry assay and to standardize its parameters for reliable inter-laboratory use. We have optimized the protocol, established the Receiving Operating Characteristic (ROC) curve and tested reproducibility using pre-COVID and convalescent, SARS-CoV-2 individual plasma samples. The flow-based assay was simplified and standardized by cultivating the 293 T cells in suspension and expressing results in Mean Equivalent Soluble Fluorochrome (MESF) using an internal antibody positive control. The ROC curve was determined with an area under the curve (AUC) of 0.996 and the assay specificity and sensitivity were established at 100% and 97.7% respectively. Reproducibility was good as determined on multiple cytometers, on different days, and with data acquisition as far as 72 h post-staining. The standardized assay could be used as a high throughput confirmatory assay in flow cytometry laboratories involved in serological testing., Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-021-00511-1., (© The Author(s), under exclusive licence to Springer Nature B.V. 2021.)

The standard regimen of the BNT162b2 mRNA vaccine for SARS-CoV-2 includes two doses administered three weeks apart. However, some public health authorities spaced these doses, raising questions about efficacy. We analyzed longitudinal humoral responses against the D614G strain and variants of concern for SARS-CoV-2 in a cohort of SARS-CoV-2-naive and previously infected individuals who received the BNT162b2 mRNA vaccine with sixteen weeks between doses. While administering a second dose to previously infected individuals did not significantly improve humoral responses, these responses significantly increased in naive individuals after a 16-week spaced second dose, achieving similar levels as in previously infected individuals. Comparing these responses to those elicited in individuals receiving a short (4-week) dose interval showed that a 16-week interval induced more robust responses among naive vaccinees. These findings suggest that a longer interval between vaccine doses does not compromise efficacy and may allow greater flexibility in vaccine administration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The evaluation of humoral protective immunity against SARS-CoV-2 remains crucial in understanding both natural immunity and protective immunity conferred by the several vaccines implemented in the fight against COVID-19. The reference standard for the quantification of antibodies capable of neutralizing SARS-CoV-2 is the plaque-reduction neutralization test (PRNT). However, given that it is a laboratory-developed assay, validation is crucial in order to ensure sufficient specificity and intra- and interassay precision. In addition, a multitude of other serological assays have been developed, including enzyme-linked immunosorbent assay (ELISA), flow cytometry-based assays, luciferase-based lentiviral pseudotype assays, and commercially available human ACE2 receptor-blocking antibody tests, which offer practical advantages in the evaluation of the protective humoral response against SARS-CoV-2. In this study, we validated a SARS-CoV-2 PRNT to assess both 50% and 90% neutralization of SARS-CoV-2 according to guidelines outlined by the World Health Organization. Upon validation, the reference-standard PRNT demonstrated excellent specificity and both intra- and interassay precision. Using the validated assay as a reference standard, we characterized the neutralizing antibody response in specimens from patients with laboratory-confirmed COVID-19. Finally, we conducted a small-scale multilaboratory comparison of alternate SARS-CoV-2 PRNTs and surrogate neutralization tests. These assays demonstrated substantial to perfect interrater agreement with the reference-standard PRNT and offer useful alternatives to assess humoral immunity against SARS-CoV-2. IMPORTANCE SARS-CoV-2, the causal agent of COVID-19, has infected over 246 million people and led to over 5 million deaths as of October 2021. With the approval of several efficacious COVID-19 vaccines, methods to evaluate protective immune responses will be crucial for the understanding of long-term immunity in the rapidly growing vaccinated population. The PRNT, which quantifies SARS-CoV-2-neutralizing antibodies, is used widely as a reference standard to validate new platforms but has not undergone substantial validation to ensure excellent inter- and intraassay precision and specificity. Our work is significant, as it describes the thorough validation of a PRNT, which we then used as a reference standard for the comparison of several alternative serological methods to measure SARS-CoV-2-neutralizing antibodies. These assays demonstrated excellent agreement with the reference-standard PRNT and include high-throughput platforms, which can greatly enhance capacity to assess both natural and vaccine-induced protective immunity against SARS-CoV-2.

Different serological assays were rapidly generated to study humoral responses against the SARS-CoV-2 Spike glycoprotein. Due to the intrinsic difficulty of working with SARS-CoV-2 authentic virus, most serological assays use recombinant forms of the Spike glycoprotein or its receptor binding domain (RBD). Cell-based assays expressing different forms of the Spike, as well as pseudoviral assays, are also widely used. To evaluate whether these assays recapitulate findings generated when the Spike is expressed in its physiological context (at the surface of the infected primary cells), we developed an intracellular staining against the SARS-CoV-2 nucleocapsid (N) to distinguish infected from uninfected cells. Human airway epithelial cells (pAECs) were infected with authentic SARS-CoV-2 D614G or Alpha variants. We observed robust cell-surface expression of the SARS-CoV-2 Spike at the surface of the infected pAECs using the conformational-independent anti-S2 CV3-25 antibody. The infected cells were also readily recognized by plasma from convalescent and vaccinated individuals and correlated with several serological assays. This suggests that the antigenicity of the Spike present at the surface of the infected primary cells is maintained in serological assays involving expression of the native full-length Spike.

Despite advances in COVID-19 management, identifying patients evolving toward death remains challenging. To identify early predictors of mortality within 60 days of symptom onset (DSO), we performed immunovirological assessments on plasma from 279 individuals. On samples collected at DSO11 in a discovery cohort, high severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA), low receptor binding domain–specific immunoglobulin G and antibody-dependent cellular cytotoxicity, and elevated cytokines and tissue injury markers were strongly associated with mortality, including in patients on mechanical ventilation. A three-variable model of vRNA, with predefined adjustment by age and sex, robustly identified patients with fatal outcome (adjusted hazard ratio for log-transformed vRNA = 3.5). This model remained robust in independent validation and confirmation cohorts. Since plasma vRNA’s predictive accuracy was maintained at earlier time points, its quantitation can help us understand disease heterogeneity and identify patients who may benefit from new therapies.

In HIV-1 infection, many antibodies (Abs) are elicited to Envelope (Env) epitopes that are conformationally masked in the native trimer and are only available for antibody recognition after the trimer binds host cell CD4. Among these are epitopes within the Co-Receptor Binding Site (CoRBS) and the constant region 1 and 2 (C1-C2 or cluster A region). In particular, C1-C2 epitopes map to the gp120 face interacting with gp41 in the native, "closed" Env trimer present on HIV-1 virions or expressed on HIV-1-infected cells. Antibodies targeting this region are therefore nonneutralizing and their potential as mediators of antibody-dependent cellular cytotoxicity (ADCC) of HIV-1-infected cells diminished by a lack of available binding targets. Here, we present the design of Ab-CD4 chimeric proteins that consist of the Ab-IgG1 of a CoRBS or cluster A specificity to the extracellular domains 1 and 2 of human CD4. Our Ab-CD4 hybrids induce potent ADCC against infected primary CD4 + T cells and neutralize tier 1 and 2 HIV-1 viruses. Furthermore, competition binding experiments reveal that the observed biological activities rely on both the antibody and CD4 moieties, confirming their cooperativity in triggering conformational rearrangements of Env. Our data indicate the utility of these Ab-CD4 hybrids as antibody therapeutics that are effective in eliminating HIV-1 through the combined mechanisms of neutralization and ADCC. This is also the first report of single-chain-Ab-based molecules capable of opening "closed" Env trimers on HIV-1 particles/infected cells to expose the cluster A region and activate ADCC and neutralization against these nonneutralizing targets. IMPORTANCE Highly conserved epitopes within the coreceptor binding site (CoRBS) and constant region 1 and 2 (C1-C2 or cluster A) are only available for antibody recognition after the HIV-1 Env trimer binds host cell CD4; therefore, they are not accessible on virions and infected cells, where the expression of CD4 is downregulated. Here, we have developed new antibody fusion molecules in which domains 1 and 2 of soluble human CD4 are linked with monoclonal antibodies of either the CoRBS or cluster A specificity. We optimized the conjugation sites and linker lengths to allow each of these novel bispecific fusion molecules to recognize native "closed" Env trimers and induce the structural rearrangements required for exposure of the epitopes for antibody binding. Our in vitro functional testing shows that our Ab-CD4 molecules can efficiently target and eliminate HIV-1-infected cells through antibody-dependent cellular cytotoxicity and inactivate HIV-1 virus through neutralization.

The seasonal nature of outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. Accordingly, temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The receptor-binding domain (RBD) of the Spike glycoprotein is known to bind to its host receptor angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Using biochemical, biophysical, and functional assays to dissect the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike glycoprotein with the ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide (including the B.1.1.7 (α) lineage), bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Neutralizing antibodies (NAbs) are effective in treating COVID-19, but the mechanism of immune protection is not fully understood. Here, we applied live bioluminescence imaging (BLI) to monitor the real-time effects of NAb treatment during prophylaxis and therapy of K18-hACE2 mice intranasally infected with SARS-CoV-2-nanoluciferase. Real-time imaging revealed that the virus spread sequentially from the nasal cavity to the lungs in mice and thereafter systemically to various organs including the brain, culminating in death. Highly potent NAbs from a COVID-19 convalescent subject prevented, and also effectively resolved, established infection when administered within three days. In addition to direct neutralization, depletion studies indicated that Fc effector interactions of NAbs with monocytes, neutrophils, and natural killer cells were required to effectively dampen inflammatory responses and limit immunopathology. Our study highlights that both Fab and Fc effector functions of NAbs are essential for optimal in vivo efficacy against SARS-CoV-2., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)