Long-Lasting Protective Antiviral Immunity Induced by Passive Immunotherapies Requires both Neutralizing and Effector Functions of the Administered Monoclonal Antibody

Using FrCas^E retrovirus-infected newborn mice as a model system, we have shown recently that a long-lasting antiviral immune response essential for healthy survival emerges after a short treatment with a neutralizing (667) IgG2a isotype monoclonal antibody (MAb). This suggested that the mobilization of adaptive immunity by administered MAbs is key for the success in the long term for the MAb-based passive immunotherapy of chronic viral infections. We have addressed here whether the anti-FrCas^E protective endogenous immunity is the mere consequence of viral propagation blunting, which would simply give time to the immune system to react, and/or to actual immunomodulation by the MAb during the treatment. To this aim, we have compared viral replication, disease progression, and antiviral immune responses between different groups of infected mice: (i) mice treated with either the 667 MAb, its F(ab′)₂ fragment, or an IgM (672) with epitopic specificity similar to that of 667 but displaying different effector functions, and (ii) mice receiving no treatment but infected with a low viral inoculum reproducing the initial viral expansion observed in their infected/667 MAb-treated counterparts. Our data show that the reduction of FrCas^E propagation is insufficient on its own to induce protective immunity and support a direct immunomodulatory action of the 667 MAb. Interestingly, they also point to sequential actions of the administered MAb. In a first step, viral propagation is exclusively controlled by 667 neutralizing activity, and in a second one, this action is complemented by FcγR-binding-dependent mechanisms, which most likely combine infected cell cytolysis and the modulation of the antiviral endogenous immune response. Such complementary effects of administered MAbs must be taken into consideration for the improvement of future antiviral MAb-based immunotherapies.Although monoclonal antibodies (MAbs) principally have been considered for anticancer applications heretofore (62, 64), they now are increasingly being considered to treat severe acute and chronic viral infections (43, 63, 83). The best-studied antiviral MAbs are (i) pavalizumab, a humanized anti-respiratory syncytial virus (RSV) MAb approved by the FDA in 1998 for treating severe lower-respiratory-tract diseases in infants (45); (ii) several anti-human immunodeficiency virus (HIV) MAbs, which have been used in macaque preclinical infection models and in several human trials (4, 5, 19, 27-30, 32, 42, 50, 55, 57, 76-79); and (iii) a few anti-hepatitis C virus (HCV) MAbs, some of which currently are being tested in humans (9, 22, 40). However, other MAbs, some of them of human origin, also have been generated against other human viruses in recent years. Among them are antibodies against Ebola virus (75), West Nile virus (WNV) (48, 53, 54), cytomegalovirus (CMV) (11), avian and human influenza viruses (59, 60, 73, 74), severe acute respiratory syndrome coronavirus (SARS CoV) (81), hepatitis B virus (HBV) (31, 35), Hanta virus (80, 82), and Nipah virus (80, 82). These antiviral MAbs all have been selected on the basis of their neutralizing activity and the possibility that they interfere with the antiviral immune response of treated hosts, because their effector functions have been considered surprisingly little so far. Addressing this question in clinical settings currently is not possible for a variety of reasons that include ethical, technical, and cost concerns. Therefore, we have turned to the neonatal infection of mice by the lethal FrCas^E retrovirus as a model system. This model allowed us to show that a very short immunotherapy by a neutralizing MAb of the IgG2a isotype (667 MAb) can permit, in addition to an immediate direct effect on the viral load, the mounting of a long-lasting endogenous antiviral immunity, which is essential for viral control and healthy survival (23-25). Because of the broad therapeutic perspectives opened by this observation, it now is essential to elucidate the molecular and cellular mechanisms underlying this effect.FrCas^E is a simple chimeric mouse retrovirus in which the env gene of the leukemogenic Friend murine leukemia virus (F-MuLV) was replaced by that of the neurodegeneration-inducing CasBr retrovirus (58). When 5 × 10⁴ infectious particles are inoculated into newborn mice under the age of 5 to 6 days, FrCas^E can enter the central nervous system (CNS) and induces a neurodegeneration fatal within 1 to 2 months with 100% incidence (15, 23, 41, 58). However, upon infection at a later time, FrCas^E can no longer enter the CNS. Instead, it replicates only in the periphery and gives rise to a fatal erythroleukemia preceded by spleen enlargement and a dramatic drop of the hematocrit. Erythroleukemia incidence and incubation period, however, are variable, depending on the inoculum and the date of infection (46).667 is an IgG2a/κ (44) directed to the main viral receptor-binding site of CasBr Env (16). It displays both in vitro (44) and in vivo (56) neutralizing activities. When rapidly (<2 days) administered for a few days to neonatally FrCas^E-infected pups, viral propagation is rapidly blunted, which prevents virus entry in the brain and subsequent neurodegeneration (23). Moreover, all 667-treated mice develop a strong, long-lasting antiviral immune response, which is necessary for them to survive healthy and with no sign of neurodegeneration or of erythroleukemia (23-25) and to resist viral challenges carried out as long as 14 months after first infection (23). Protective antiviral immunity is of a typical TH1 type with humoral and cytotoxic T-cell (CTL) contributions. The anti-FrCas^E humoral contribution is high, sustained, and principally of the IgG2a type with both in vitro neutralization- and complement-dependent cytolysis activities (23). Interestingly, it shows typical secondary response characteristics in viral challenge experiments (23), and anti-FrCas^E antibodies are transmitted transplacentally and through breastfeeding by mothers to children, where they manifest the same properties as those of 667 in the perinatal infection setting, i.e., they prevent mice from developing neurodegeneration and permit the induction of an endogenous protective antiviral immune response (25). Finally, the CTL response directed to infected cells was shown to be necessary for the protection of FrCas^E-infected, 667-treated mice, as the depletion of CD8⁺ T cells leads to death by retrovirally induced erythroleukemia (24).At this stage, an important issue is the clarification of whether the anti-FrCas^E protective immunity seen in 667 MAb-treated mice is due to actual immunomodulation by the MAb owing to its effector function(s) and/or is the consequence of viral propagation blunting, which would prevent the immune system from being overwhelmed by an excess of antigen and, hence, would give it time to react optimally. To address these two nonexclusive possibilities, we compared here viral propagations, health statuses, and endogenous immune responses in four groups of mice. The first three groups were mice neonatally infected under standard conditions (5 × 10⁴ infectious particles) and treated with either the natural 667 MAb, the antibody effector function-lacking F(ab′)₂ fragment of 667, or a neutralizing IgM (672) with effector functions inherently different from those of 667. The last group consisted of mice neonatally infected with a low FrCas^E inoculum but not subjected to immunotherapy, which is a condition permitting early viral propagation kinetics similar to those of animals infected and 667 MAb treated under standard conditions. Taken together, our data indicate that the drastic reduction of viral propagation shortly after infection is not sufficient for the induction of protective adaptive immunity and, thereby, point to an immunomodulatory action of 667. Interestingly, they also point to two sequential actions of the administered MAb. In the immediate postinfection period, viral spread is controlled exclusively by 667 neutralizing activity, and later it involves the cytolysis of infected cells owing to FcγR-binding-dependent mechanisms. Finally, our work shows that not all antibody isotypes are equally efficient at protecting infected mice and favoring the mounting of protective immunity, as 672 IgM immunotherapy-treated animals died of erythroleukemia.     

Theoretical study of the hydroxylation of phenolates by the Cu2O2(N,N′-dimethylethylenediamine)2 2+ complex

Tyrosinase catalyzes the ortho hydroxylation of monophenols and the subsequent oxidation of the diphenolic products to the resulting quinones. In efforts to create biomimetic copper complexes that can oxidize C–H bonds, Stack and coworkers recently reported a synthetic μ-η²:η²-peroxodicopper(II)(DBED)₂ complex (DBED is N,N′-di-tert-butylethylenediamine), which rapidly hydroxylates phenolates. A reactive intermediate consistent with a bis-μ-oxo-dicopper(III)-phenolate complex, with the O–O bond fully cleaved, is observed experimentally. Overall, the evidence for sequential O–O bond cleavage and C–O bond formation in this synthetic complex suggests an alternative mechanism to the concerted or late-stage O–O bond scission generally accepted for the phenol hydroxylation reaction performed by tyrosinase. In this work, the reaction mechanism of this peroxodicopper(II) complex was studied with hybrid density functional methods by replacing DBED in the μ-η²:η²-peroxodicopper(II)(DBED)₂ complex by N,N′-dimethylethylenediamine ligands to reduce the computational costs. The reaction mechanism obtained is compared with the existing proposals for the catalytic ortho hydroxylation of monophenol and the subsequent oxidation of the diphenolic product to the resulting quinone with the aim of gaining some understanding about the copper-promoted oxidation processes mediated by 2:1 Cu(I)O₂-derived species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Monoclonal antibody 667 recognizes the variable region A motif of the ecotropic retrovirus CasBrE envelope glycoprotein and inhibits Env binding to the viral receptor

Monoclonal antibody (MAb) 667 is a neutralizing mouse monoclonal antibody recognizing the envelope glycoprotein (Env) of the ecotropic neurotropic murine retrovirus CasBrE but not that of other murine retroviruses. Since 667 can be used for preclinical studies of antiviral gene therapy as well as for studying the early events of retroviral infection, we have cloned its cDNAs and molecularly characterized it in detail. Spot technique-based experiments showed that 667 recognizes a linear epitope of 12 amino acids located in the variable region A of the receptor binding domain. Alanine scanning experiments showed that six amino acids within the epitope are critical for MAb binding. One of them, D(57), is not present in any other murine retroviral Env, which suggests a critical role for this residue in the selectivity of 667. MAb 667 heavy- and light-chain cDNAs were functionally characterized by transient transfection into Cos-7 cells. Enzyme-linked immunosorbent assays and Biacore studies showed that the specificities as well as the antigen-binding thermodynamic and kinetic properties of the recombinant 667 MAb (r667) produced by Cos-7 cells and those of the parental hybridoma-produced MAb (h667) were similar. However, h667 was shown to contain contaminating retroviral and/or retrovirus-like particles which interfere with both viral binding and neutralization experiments. These contaminants could successfully be removed by a stringent purification protocol. Importantly, this purified 667 could completely prevent retrovirus binding to target cells and was as efficient as the r667 MAb produced by transfected Cos-7 cells in neutralization assays. In conclusion, this study shows that the primary mechanism of virus neutralization by MAb 667 is the blocking of the retroviral receptor binding domain of CasBrE Env. In addition, the findings of this study constitute a warning against the direct use of hybridoma cell culture supernatants for studying the initial events of retroviral cell infection as well as for carrying out in vivo neutralization experiments and suggest that either recombinant antibodies or highly purified antibodies are preferable for these purposes.     

p120 Catenin-associated Fer and Fyn tyrosine kinases regulate beta-catenin Tyr-142 phosphorylation and beta-catenin-alpha-catenin Interaction

beta-Catenin has a key role in the formation of adherens junction through its interactions with E-cadherin and alpha-catenin. We show here that interaction of beta-catenin with alpha-catenin is regulated by the phosphorylation of beta-catenin Tyr-142. This residue can be phosphorylated in vitro by Fer or Fyn tyrosine kinases. Transfection of these kinases to epithelial cells disrupted the association between both catenins. We have also examined whether these kinases are involved in the regulation of this interaction by K-ras. Stable transfectants of the K-ras oncogene in intestinal epithelial IEC18 cells were generated which show little alpha-catenin-beta-catenin association with respect to control clones; this effect is accompanied by increased Tyr-142 phosphorylation and activation of Fer and Fyn kinases. As reported for Fer, Fyn kinase is constitutively bound to p120 catenin; expression of K-ras induces the phosphorylation of p120 catenin on tyrosine residues increasing its affinity for E-cadherin and, consequently, promotes the association of Fyn with the adherens junction complex. Yes tyrosine kinase also binds to p120 catenin but only upon activation, and stimulates Fer and Fyn tyrosine kinases. These results indicate that p120 catenin acts as a docking protein facilitating the activation of Fer/Fyn tyrosine kinases by Yes and demonstrate the role of these p120 catenin-associated kinases in the regulation of beta-catenin-alpha-catenin interaction.     

Agonist-induced activation releases peroxisome proliferator-activated receptor beta/delta from its inhibition by palmitate-induced nuclear factor-kappaB in skeletal muscle cells

The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood, but there is a strong correlation between insulin resistance and intramyocellular lipid accumulation in skeletal muscle. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. The aim of this work was to study whether the exposure of skeletal muscle cells to palmitate affected peroxisome proliferator-activated receptor (PPAR) beta/delta activity. Here, we report that exposure of C2C12 skeletal muscle cells to 0.75 mM palmitate reduced (74%, P<0.01) the mRNA levels of the PPARbeta/delta-target gene pyruvatedehydrogenase kinase 4 (PDK-4), which is involved in fatty acid utilization. This reduction was not observed in the presence of the PPARbeta/delta agonist L-165041. This drug prevented palmitate-induced nuclear factor (NF)-kappaB activation. Increased NF-kappaB activity after palmitate exposure was associated with enhanced protein-protein interaction between PPARbeta/delta and p65. Interestingly, treatment with the PPARbeta/delta agonist L-165041 completely abolished this interaction. These results indicate that palmitate may reduce fatty acid utilization in skeletal muscle cells by reducing PPARbeta/delta signaling through increased NF-kappaB activity.     

Viscoelasticity of human alveolar epithelial cells subjected to stretch

Alveolar epithelial cells undergo stretching during breathing and mechanical ventilation. Stretch can modify cell viscoelastic properties, which may compromise the balance of forces in the alveolar epithelium. We studied the viscoelasticity of alveolar epithelial cells (A549) subjected to equibiaxial distention with a novel experimental approach. Cells were cultured on flexible substrates and subjected to stepwise deformations of up to 17% with a device built on an inverted microscope. Simultaneously, cell storage (G') and loss (G') moduli were measured (0.1-100 Hz) with optical magnetic twisting cytometry. G' and G' increased with strain up to 64 and 30%, respectively, resulting in a decrease in G'/G' (15%). This stretch-induced response was inhibited by disruption of the actin cytoskeleton with latrunculin A. G' increased with frequency following a power law with exponent alpha = 0.197. G' increased proportionally to G' but exhibited a more marked frequency dependence at high frequencies. Stretching (14%) caused a fall in alpha (13%). At high stretching amplitudes, actual cell strain (14.4%) was lower than the applied substrate strain (17.3%), which could indicate a partial cell detachment. These data suggest that cytoskeletal prestress modulates the elastic and frictional properties of alveolar epithelial cells in a coupled manner, according to soft glassy rheology. Stretch-induced cell stiffening could compromise the balance of forces at the cell-cell and cell-matrix adhesions.     

Cell volume regulation in immune cell apoptosis

The loss of cell volume is an early and fundamental feature of programmed cell death or apoptosis; however, the mechanisms responsible for cell shrinkage during apoptosis are poorly understood. The loss of cell volume is not a passive component of the apoptotic process, and a number of experimental findings from different laboratories highlight the importance of this process as an early and necessary regulatory event in the signaling of the death cascade. Additionally, the loss of intracellular ions, particularly potassium, has been shown to play a primary role in cell shrinkage, caspase activation, and nuclease activity during apoptosis. Thus, an understanding of the role that ion channels and plasma membrane transporters play in cellular signaling during apoptosis may have important physiological implications for immune cells, especially lymphocyte function. Furthermore, this knowledge may also have an impact on the design of therapeutic strategies for a variety of diseases of the immune system in which apoptosis plays a central role, such as oncogenic processes or immune system disorders. The present review summarizes our appreciation of the mechanisms underlying the early loss of cell volume during apoptosis and their association with downstream events in lymphocyte apoptosis.