Double-blinded, randomised, placebo-controlled trial of convalescent plasma for COVID-19: analyses by neutralising antibodies homologous to recipients' variants.

INTRODUCTION: Convalescent plasma (CP) emerged as potential treatment for COVID-19 early in the pandemic. While efficacy in hospitalised patients has been lacklustre, CP may be beneficial at the first stages of disease. Despite multiple new variants emerging, no trials have involved analyses on variant-specific antibody titres of CP. METHODS: We recruited hospitalised COVID-19 patients within 10 days of symptom onset and, employing a double-blinded approach, randomised them to receive 200 ml convalescent plasma with high (HCP) or low (LCP) neutralising antibody (NAb) titre against the ancestral strain (Wuhan-like variant) or placebo in 1:1:1 ratio. Primary endpoints comprised intubation, corticosteroids for symptom aggravation, and safety assessed as serious adverse events. For a preplanned ad hoc analysis, the patients were regrouped by infused CP's NAb titers to variants infecting the recipients i.e. by titres of homologous HCP (hHCP) or LCP (hLCP). RESULTS: Of the 57 patients, 18 received HCP, 19 LCP and 20 placebo, all groups smaller than planned. No significant differences were found for primary endpoints. In ad hoc analysis, hHCPrecipients needed significantly less respiratory support, and appeared to be given corticosteroids less frequently (1/14; 7.1%) than those receiving hLCP (9/23; 39.1%) or placebo (8/20; 40%), (p = 0.077). DISCUSSION: Our double-blinded, placebo-controlled CP therapy trial remained underpowered and does not allow any firm conclusions for early-stage hospitalised COVID-19 patients. Interestingly, however, regrouping by homologous - recipients' variant-specific - CP titres suggested benefits for hHCP. We encourage similar re-analysis of ongoing/previous larger CP studies. TRIAL REGISTRATION: ClinTrials.gov identifier: NCT0473040.

Macrophage metalloelastase (MME) as adjuvant for intra-tumoral injection of oncolytic adenovirus and its influence on metastases development.

Oncolytic adenoviruses are a promising treatment alternative for many advanced cancers, including colorectal cancer. However, clinical trials have demonstrated that single-agent therapy in advanced tumor masses is rarely curative. Poor spreading of the virus through tumor tissue is one of the major issues limiting efficacy. As oncolytic viruses kill preferentially cancer cells, high extracellular matrix (ECM) content constitutes potential barriers for viral penetration within tumors. In this study, the ECM-degrading proteases relaxin, hyaluronidase, elastase and macrophage metalloelastase (MME) were tested for their antitumor efficacy alone and in combination with oncolytic adenovirus. MME improved the overall antitumor efficacy of oncolytic adenovirus in subcutaneous HCT116 xenografts. In a liver metastatic colorectal cancer model, intra-tumoral treatment of primary tumors from HT29 cells with MME monotherapy or with oncolytic adenovirus inhibited tumor growth. Combination therapy showed no increased mortality in comparison with either monotherapy alone. Contradictory results of effects of MME on tumorigenesis and metastasis formation have been reported in the literature. This study demonstrates for the first time in a metastatic animal model that MME, as a monotherapy or in combination with oncolytic virus, does not increase tumor invasiveness. Co-administration of MME and oncolytic adenovirus may be a suitable approach for further optimization aiming at clinical applications for metastatic colorectal cancer.

A natural variability in the proline-rich motif of Nef modulates HIV-1 replication in primary T cells.

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC). The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.

Inhibition of cellular functions of HIV-1 Nef by artificial SH3 domains.

SH3 domains regulate many normal and pathological cellular processes by guiding specific protein interactions. Studies on binding of HIV-1 Nef to the SH3 domain of the Hck tyrosine kinase have indicated an important role for the SH3 RT-loop region in ligand binding. Here we have tested the potential of artificial Hck-derived SH3 domains carrying tailored RT-loops providing high affinity for Nef as intracellular inhibitors of Nef. These artificial SH3 domains efficiently associated with Nef in cells and thereby potently inhibited SH3-dependent Nef functions, such as association with p21-activated kinase-2 and induction of the transcription factor NFAT. On the other hand, biochemical and functional data indicated that the Nef-targeted SH3 domains were not prone to compete with normal SH3-mediated processes. Thus, RT-loop-modified SH3 domains represent a novel approach for selectively interfering with cellular signaling events, which could be exploited in research as well as in therapeutic applications.

A homologous naturally occurring mutation in Duffy and CCR5 leading to reduced receptor expression.

Genetic variations in the CC chemokine receptor (CCR5) leading to reduced or absent expression are associated with resistance to human immunodeficiency virus infection and delayed onset of acquired immunodeficiency syndrome. Similarly, lack of the red-cell chemokine receptor Duffy confers protection against malarial infection by Plasmodium vivax. Investigators have previously described a missense mutation (R89C) in the first intracellular loop of Duffy that results in reduced protein expression. The present study shows that the lower Duffy expression is due to loss of the positive charge at this position, resulting in protein instability. Moreover, R60S, a mutation in the first intracellular loop of CCR5 noted in a recent cohort study, likewise results in reduced surface expression and function of CCR5. The presence of a homologous, naturally occurring mutation that may be protective against disease thus defines a novel mechanism accounting for the decreased expression of these receptors in some individuals. (Blood. 2001;97:3651-3654)

Human immunodeficiency virus type 1 Nef selectively associates with a catalytically active subpopulation of p21-activated kinase 2 (PAK2) independently of PAK2 binding to Nck or beta-PIX.

We have recently identified the Nef-associated serine-threonine kinase (NAK) as the p21-activated kinase 2 (PAK2). Here we have taken advantage of the possibility to manipulate the functional properties of NAK by transfecting PAK2 cDNA or its mutant derivatives in order to further characterize the Nef-NAK complex. To exclude the possibility that some Nef variants might interact with PAK1 instead of PAK2, we also examined the identity of NAK complexed with divergent human immunodeficiency virus type 1 HIV-1 Nef proteins. All tested Nef proteins, including SF2, NL4-3, BH10, and HAN-2, associated with PAK2 but not with PAK1. By exchanging different regions between these two PAK proteins, the selective ability of PAK2 to associate with Nef could be mapped to the carboxy-terminal part of its regulatory domain. Binding of PAK2 with the adapter protein Nck or beta-PIX was found to be dispensable for the assembly of the Nef-PAK2 complex, whereas an intact Cdc42-Rac1 interactive binding motif was required. Most importantly, we found that NAK represented a distinct subpopulation of the total cellular PAK2 characterized by a high specific kinase activity. Thus, although only a small fraction of cellular PAK2 could be found in complex with Nef, NAK represented a major part of cellular PAK2 activity.

Activation of NFAT-dependent gene expression by Nef: conservation among divergent Nef alleles, dependence on SH3 binding and membrane association, and cooperation with protein kinase C-theta.

Here we show that the potential to regulate NFAT is a conserved property of different Nef alleles and that Nef residues involved in membrane targeting and SH3 binding are critical for this function. Cotransfection of an activated protein kinase C-theta (PKC-theta) with Nef implicated PKC-theta as a possible physiological cofactor of Nef in promoting NFAT-dependent gene expression and T-cell activation.

Synergistic activation of NFAT by HIV-1 nef and the Ras/MAPK pathway.

Nef is a lentiviral protein involved in pathogenesis of AIDS, but its molecular mechanisms of action remain incompletely understood. Here we report a novel effect of Nef on lymphocyte signaling, which is mediated via a T cell receptor (TCR)-independent contribution of Nef to induction of nuclear factor of activated T cells (NFAT), a transcription factor that plays a central role in coordinating T cell activation. Expression of Nef did not significantly alter the basal level of NFAT activity in Jurkat cells nor the increased activity following T cell receptor stimulation by anti-CD3 or anti-CD3 + anti-CD28. We also mimicked NFAT induction by TCR triggering by simultaneous activation of the Ras and calcium signaling pathways with phorbol 12-myristate 13-acetate and ionomycin, respectively. Strikingly, whereas activation of either of these pathways individually did not induce NFAT activity in control cells, in Nef-expressing cells phorbol 12-myristate 13-acetate treatment alone resulted in a 100-fold increase in NFAT-directed gene expression. Experiments with different dominant negative mutant signaling proteins, inhibitory chemicals, and Lck-deficient Jurkat cells revealed that this effect was mediated via activation of calcineurin by Nef-induced changes in calcium metabolism, but was independent of TCR-associated signaling events. This ability of Nef to substitute for triggering of the calcium pathway in induction of NFAT could promote activation of human immunodeficiency virus (HIV)-infected T cells in response to stimuli mediated via TCR or other cell surface receptors under conditions when activation of Ras rather than calcium signaling would otherwise predominate.

Interactions of HIV-1 NEF with cellular signal transducing proteins.

Nef is a 27 - 34 kD myristoylated protein unique to primate lentiviruses. A functional Nef gene is important for development of high viremia and simian AIDS in SIV infected rhesus macaques (1). In a transgenic mouse model expression of Nef protein alone when expressed under a CD4-promoter is sufficient to cause an AIDS like disease (2). A critical role for Nef in development of AIDS in humans is suggested by the observation that some individuals with a long-term nonprogressive HIV-1 infection are infected with viruses carrying naturally occurring Nef deletions (3-5). The mechanism of Nef action remains incompletely understood, but multiple lines of evidence point out to a role in modulation of cellular signaling pathways via physical and functional interactions with host cell proteins.

Interleukin-4-induced transcriptional activation by stat6 involves multiple serine/threonine kinase pathways and serine phosphorylation of stat6.

Stat6 transcription factor is a critical mediator of IL-4-specific gene responses. Tyrosine phosphorylation is required for nuclear localization and DNA binding of Stat6. The authors investigated whether Stat6-dependent transcriptional responses are regulated through IL-4-induced serine/threonine phosphorylation. In Ramos B cells, the serine/threonine kinase inhibitor H7 inhibited IL-4-induced expression of CD23. Treatment with H7 did not affect IL-4R-mediated immediate signaling events such as tyrosine phosphorylation of Jak1, Jak3, insulin receptor substrate (IRS)-1 and IRS-2, or tyrosine phosphorylation and DNA binding of Stat6. To analyze whether the H7-sensitive pathway was regulating Stat6-activated transcription, we used reporter constructs containing different IL-4 responsive elements. H7 abrogated Stat6-, as well as Stat5-, mediated reporter gene activation and partially reduced C/EBP-dependent reporter activity. By contrast, IL-4-induced transcription was not affected by wortmannin, an inhibitor of the phosphatidyl-inositol 3'-kinase pathway. Phospho-amino acid analysis and tryptic phosphopeptide maps revealed that IL-4 induced phosphorylation of Stat6 on serine and tyrosine residues in Ramos cells and in 32D cells lacking endogenous IRS proteins. However, H7 treatment did not inhibit the phosphorylation of Stat6. Instead, H7 inhibited the IL-4-induced phosphorylation of RNA polymerase II. These results indicate that Stat6-induced transcription is dependent on phosphorylation events mediated by H7-sensitive kinase(s) but that it also involves serine phosphorylation of Stat6 by an H7-insensitive kinase independent of the IRS pathway. (Blood. 2000;95:494-502)

Identification of the Nef-associated kinase as p21-activated kinase 2.

The Nef protein of primate immunodeficiency viruses plays an important role in the pathogenesis of acquired immunodeficiency syndrome (AIDS) [1] [2]. The interaction of Nef with the Nef-associated kinase (NAK) is one of the most conserved properties of different human and simian immunodeficiency virus (HIV and SIV) Nef alleles. The role of NAK association is currently not known but it has been implicated in enhanced viral infectivity in cell culture and in disease progression in SIV-infected macaques [3]. Previous studies have indicated that NAK shares many features with the p21-activated kinases (PAKs) [3], but the molecular identity of NAK has remained unknown. We have generated specific antisera against PAKs 1-3, and expressed these kinases individually as epitope-tagged proteins. By using these reagents in experiments involving partial proteolytic mapping, and exploiting the unique ability of PAK2 to serve as a caspase substrate, we have positively identified NAK as PAK2. Interestingly, although ectopic PAK2 overexpression efficiently replaced endogenous PAK2 from the complex with Nef, the total Nef-associated PAK2 activity was not increased, indicating the abundance of another cellular factor(s) as the limiting factor in Nef-PAK2 complex formation. Identification of NAK as PAK2 should now facilitate elucidation of its role as a mediator of the pathogenic effects of Nef.

SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef.

The avid binding of HIV-1 Nef to the Src homology-3 (SH3) domain of Hck (KD 250 nM) has been shown to involve an interaction between the RT-loop of Hck-SH3 and residues in Nef outside of its prototypic polyproline type II (PPII) helix-containing SH3-ligand region. Such distinctive interactions are thought to provide specificity and affinity for other SH3/ligand protein complexes as well. Here, we have constructed and successfully displayed on the surface of M13 bacteriophage particles a complex library of SH3 domains, which are derived from Hck but carry a random hexapeptide substitution in their RT-loops (termed RRT-SH3). Using this strategy we have identified individual RRT-SH3 domains that can bind to Nef up to 40-fold more avidly than Hck-SH3. Some of these high-affinity RRT-SH3 domains resembled Hck-SH3 in that they bound much less well to a Nef variant containing an engineered F90R mutation that interferes with docking of the native Hck RT-loop. In addition, we could also select RRT-SH3 domains with an opposite specificity, which were dependent on the Arg90 residue for strong binding, and bound 100-fold less well to unmodified Nef. These results demonstrate the utility of phage-display in engineering of signaling protein interaction domains, and emphasize the importance of the RT-loop in SH3 ligand selection, thus suggesting a general strategy for creating SH3 domains with desired binding properties.

Induction of activator protein 1 (AP-1) in macrophages by human immunodeficiency virus type-1 NEF is a cell-type-specific response that requires both hck and MAPK signaling events.

Human immunodeficiency virus type 1 (HIV-1) Nef is important for viral infectivity and pathogenicity. HIV-1 infection is associated with inappropriate activation and defects in the function of monocytes/macrophages. We have studied the effects of HIV-1 Nef in the murine (RAW264.7) and human (THP-1) monocyte-macrophage cell lines. Investigation of the activator protein-1 (AP-1) transcription factor showed that Nef expression induced both its DNA binding and transcriptional activities. Increased AP-1 DNA binding activity in RAW264.7 cells was associated with raised levels of c-Fos expression and induction of mRNA for the AP-1 responsive tissue inhibitor of metalloproteinases-1 (TIMP-1) gene. Mutagenesis and kinase inhibition studies were employed to determine signaling pathways used by Nef to induce AP-1. Data from these studies indicated that induction of AP-1 by Nef is likely to be mediated through the MAPK (ERK1 and 2) signaling pathway and requires the proline-rich PxxP motif of Nef, suggesting the involvement of upstream protein kinases belonging to the Src family. Effects of Nef on AP-1 induction were cell lineage-specific, being stimulatory in macrophages, inhibitory in T cells and without effect in HeLa cells. These latter two observations led us to test the possibility that cell-specific interactions of Nef with Src family proteins may modulate AP-1 activity. To this end we demonstrated that a dominant-negative Hck mutant caused inhibition of Nef-mediated AP-1 DNA binding activity in RAW cells. In conclusion, induction of AP-1 by Nef is a specific feature of human and murine macrophage cell lines that requires signal transduction events involving Hck and MAPKs.

SH3-Domain binding function of HIV-1 Nef is required for association with a PAK-related kinase.

HIV-1 Nef has previously been shown to bind to Src homology-3 (SH3) domains of a subset of Src family tyrosine kinases. In addition, Nef has been reported to coprecipitate with a serine/threonine kinase activity termed NAK (for Nef-associated kinase). The identity of NAK remains uncertain, but it has been suggested to represent a novel member of the p21-activated kinase (PAK) family. We report here that NAK autophosphorylation is increased not only by an activated form of the p21-family GTPase cdc42 but also by a plasma membrane-targeted fragment of the adapter protein Nck, thus providing further evidence that NAK is related to PAKs. A detailed structure-based mutational analysis of Nef revealed that all amino acid changes that inhibited the Nef/Hck-SH3 interaction, as measured by surface-plasmon resonance, also abolished coprecipitation of NAK. As PAK family proteins do not contain SH3 domains, these observations are best explained by a protein complex in which Nef, NAK, and an SH3-protein all contact each other. In addition, a number of conserved amino acids in Nef that are not involved in SH3 binding were also found to be crucial for association with NAK. Molecular modeling suggests that these residues are involved in formation of an adjacent binding surface for NAK or another critical component of the NAK/Nef complex.

HIV-1 Nef and host cell protein kinases.

Nef is a 27-34 kD myristoylated protein unique to primate lentiviruses. A functional Nef gene is important for development of high viremia and simian AIDS in SIV infected rhesus macaques. Notably, animals infected with Nef-deleted attenuated viruses are resistant to subsequent challenge with pathogenic wild-type viruses. A critical role for Nef in development of AIDS in humans has been suggested by the observation that some individuals with a long-term nonprogressive HIV-1 infection (persons who show no clinical or immunological signs of immunodeficiency despite being HIV seropositive for over a decade) are infected with viruses carrying naturally occurring Nef deletions. The mechanism of Nef action remains incompletely understood, but multiple lines of evidence point out to a role in modulation of cellular signaling pathways via physical and functional interactions with host cell protein kinases. These findings will be discussed in the following, preceded by a short introduction into the role of Nef in cell biology of HIV infection, which is intended to serve as a critical review of our current understanding on this enigmatic issue rather than a comprehensive review of the literature.

Human immunodeficiency virus type-1 mRNA splicing pattern in infected persons is determined by the proportion of newly infected cells.

Plasma viremia during HIV-1 infection is regulated by a dynamic balance between viral replication and removal of infected cells and cell-free virus. Administration of novel potent antiretroviral drugs provides an opportunity to study the consequences of perturbing this equilibrium by blocking de novo infections. In this study, we examined the expression of differentially spliced forms of HIV-1 mRNA, unspliced (US) and multiply spliced (MS), in peripheral blood mononuclear cells (PBMCs) of patients treated with HIV protease inhibitors or combination therapy. In all nine patients studied, a significant reduction in the MS/US mRNA ratio was observed after 1 week of treatment, suggesting that the majority of HIV MS mRNA in the steady-state situation prior to therapy was expressed by cells which had been infected during the previous couple of days. This idea was supported by a detailed analysis of serial PBMC specimens collected from two of the patients during the first hours and days after initiation of therapy. In both cases, a substantial decrease in MS mRNA expression was evident already after 48 hr, whereas the expression of US mRNA at this time was virtually unaffected. These data indicate that the HIV mRNA splicing pattern in vivo is mainly determined by the relative proportion of newly infected cells and suggest that examination of this pattern could be useful in evaluating the potency of antiretroviral therapies and in studying dynamics of HIV-1 infection.

Decay characteristics of HIV-1-infected compartments during combination therapy.

Analysis of changes in viral load after initiation of treatment with potent antiretroviral agents has provided substantial insight into the dynamics of human immunodeficiency virus type 1 (HIV-1). The concentration of HIV-1 in plasma drops by approximately 99% in the first two weeks of treatment owing to the rapid elimination of free virus with a half-life (t1/2) of < or =6 hours and loss of productively infected cells with a t1/2 of 1.6 days. Here we show that with combination therapy this initial decrease is followed by a slower second-phase decay of plasma viraemia. Detailed mathematical analysis shows that the loss of long-lived infected cells (t1/2 of 1-4 weeks) is a major contributor to the second phase, whereas the activation of latently infected lymphocytes (t1/2 of 0.5-2 weeks) is only a minor source. Based on these decay characteristics, we estimate that 2.3-3.1 years of a completely inhibitory treatment would be required to eliminate HIV-1 from these compartments. To eradicate HIV-1 completely, even longer treatment may be needed because of the possible existence of undetected viral compartments or sanctuary sites.

Stability in controlling viral replication identifies long-term nonprogressors as a distinct subgroup among human immunodeficiency virus type 1-infected persons.

Long-term nonprogressors (LTNPs) of human immunodeficiency virus type 1 (HIV-1) infection are characterized by low levels of HIV-1 replication and viral load. However, it has not been established whether they differ in this regard from progressors from the very early stage of infection. By studying peripheral blood mononuclear cell (PBMC) specimens from a longitudinally monitored cohort of HIV-1-infected men, we found that HIV-1 proviral copy numbers and HIV-1 mRNA expression levels as low or lower than those seen in seven carefully selected LTNPs were commonly observed in specimens collected soon after seroconversion from 28 subjects who became infected while under observation. However, only the LTNPs were able to stably maintain such an efficient viral control over time. Because of the instability of the early control of HIV-1 replication, the predictive value of HIV-1 mRNA expression in PBMCs at postseroconversion was found to be limited but significantly increased during the first year of infection. Besides their diagnostic implications, these data support the idea that LTNPs may be a pathophysiologically distinct subgroup among persons infected with HIV-1.