Multiple Vaccine-Elicited Nonneutralizing Antienvelope Antibody Activities Contribute to Protective Efficacy by Reducing both Acute and Chronic Viremia following Simian/Human Immunodeficiency Virus SHIV89.6P Challenge in Rhesus Macaques

We have shown that following priming with replicating adenovirus type 5 host range mutant (Ad5hr)-human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) recombinants, boosting with gp140 envelope protein enhances acute-phase protection against intravenous simian/human immunodeficiency virus (SHIV)_89.6P challenge compared to results with priming and no boosting or boosting with an HIV polypeptide representing the CD4 binding site of gp120. We retrospectively analyzed antibodies in sera and rectal secretions from these same macaques, investigating the hypothesis that vaccine-elicited nonneutralizing antibodies contributed to the better protection. Compared to other immunized groups or controls, the gp140-boosted group exhibited significantly greater antibody activities mediating antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated viral inhibition (ADCVI) in sera and transcytosis inhibition in rectal secretions. ADCC and ADCVI activities were directly correlated with antibody avidity, suggesting the importance of antibody maturation for functionality. Both ADCVI and percent ADCC killing prechallenge were significantly correlated with reduced acute viremia. The latter, as well as postchallenge ADCVI and ADCC, was also significantly correlated with reduced chronic viremia. We have previously demonstrated induction by the prime/boost regimen of mucosal antibodies that inhibit transcytosis of SIV across an intact epithelial cell layer. Here, antibody in rectal secretions was significantly correlated with transcytosis inhibition. Importantly, the transcytosis specific activity (percent inhibition/total secretory IgA and IgG) was strongly correlated with reduced chronic viremia, suggesting that mucosal antibody may help control cell-to-cell viral spread during the course of infection. Overall, the replicating Ad5hr-HIV/SIV priming/gp140 protein boosting approach elicited strong systemic and mucosal antibodies with multiple functional activities associated with control of both acute and chronic viremia.A major goal of human immunodeficiency virus (HIV) vaccine development is the elicitation of protective antibodies capable of neutralizing the diversity of isolates in the worldwide pandemic (6, 61). Indeed, passively administered neutralizing antibodies have been shown to protect against pathogenic HIV/simian immunodeficiency virus (SIV) challenge in rhesus macaque models (4, 44, 45, 57). However, the extent to which other antibody-mediated protective mechanisms impact HIV/SIV infection is still unclear. Whether these alternate biologic activities would augment vaccine-induced protection has not been definitively established.In HIV-infected individuals, as in SIV- or simian/human immunodeficiency virus (SHIV)-infected rhesus macaques, systemic nonneutralizing antibodies appear early during acute infection, often preceding a neutralizing antibody response (21, 55). Although neutralizing antibody activity is critical for sterilizing immunity, recent studies suggest that antibodies may contribute to protection by other functional activities, such as antibody-dependent cellular cytotoxicity (ADCC) (20, 29), antibody-dependent cell-mediated viral inhibition (ADCVI) (22, 23), and transcytosis inhibition (19, 35, 59). Antibodies in secretions may directly block viral entry into intestinal and endocervical tissues by inhibiting transcytosis across epithelium, whereas local or serum-derived antibodies that mediate ADCC or ADCVI may exert protective effects by eliminating small foci of infected cells during the brief window of time that exists between transmission of virus across an epithelial cell barrier to the lamina propria and subsequent systemic spread (32). In support of this notion, mutation of the Fc portion of the broadly neutralizing monoclonal antibody, IgGb12, thereby preventing interaction with the FcγR on effector cells, rendered the antibody less able to mediate protection upon subsequent passive transfer and challenge of rhesus macaques (34). Thus, neutralizing antibodies themselves may mediate protection by additional functional activities.ADCC bridges innate and adaptive immunity. Mechanistically, it involves FcγR-bearing effector cells, such as NK cells, macrophages, neutrophils, and γδ T cells, and antibodies specific for antigens expressed on the surface of target cells. Upon interaction of these three components, the target cells are killed. Since the effector cells are not major histocompatibility complex restricted, ADCC is broadly applicable to diverse populations. Because the antibody specificity need not be restricted to neutralizing epitopes, ADCC may increase the breadth of antibody reactivity. In fact, we have shown that an HIV clade B immunization regimen elicited antibodies that mediated ADCC across several HIV clades (28). Antibodies that mediate ADCC have been shown to arise early in infection, before neutralizing antibodies (55, 60). They are present in the majority of infected individuals, and they have been associated with slow disease progression following both HIV and SIV infection (5, 8).ADCVI is closely related to ADCC, also requiring antibody that forms a bridge between an infected target cell and an FcγR-bearing effector cell (24). However, ADCVI is a broader activity not restricted solely to target cell lysis but, rather, encompassing several mechanisms by which viral replication following infection of target cells is inhibited. Thus, it may include ADCC activity but also involve noncytotoxic mechanisms of virus control, such as the secretion of inhibitory chemokines or FcγR-mediated phagocytosis of immune complexes (24, 25).Most HIV infections occur via a mucosal route, including cervicovaginal and rectal tissues (39, 52). Several nonmutually exclusive mechanisms for HIV-1 transmission across mucosal epithelia have been proposed (13, 56). Transcytosis of infectious virus across polarized columnar epithelial cells following contact of virally infected cells with apical epithelial cell surfaces is one mechanism for mucosal HIV entry (12). Rather than fusion and infection, interactions between the viral envelope proteins and epithelial surface molecules, such as glycosphingolipid galactosyl-ceramide (GalCer) (13, 47), an important component of endocytotic “raft” membrane microdomains, lead to transcytosis of the virus across the epithelial barrier and its trapping by submucosal dendritic cells which disseminate it to their target CD4⁺ T cells. Studies have shown that mucosal immunoglobulin A (IgA) antibody, a major component of the mucosal immune response, could block mucosal HIV-1 entry via transcytosis in vitro (2, 19). Therefore, mucosal antibodies blocking adherence of virus to epithelial cells and preventing HIV-1 transcytosis across the epithelial barrier and subsequent CD4⁺ T cell infection may afford additional protection against HIV/SIV infection.We have been pursuing a replicating adenovirus (Ad)-HIV/SIV prime/protein subunit boost AIDS vaccine approach (30, 51), which has elicited strong, durable protection against HIV, SIV, and SHIV challenges (11, 18, 41, 42, 50). An underlying goal of these studies has been elucidation of immune responses that correlate with protective efficacy. Recently, we studied the contribution of novel protein boosts to immunogenicity and protective efficacy in a SHIV_89.6P model (49). Immunized rhesus macaques were primed with Ad type 5 host range mutant (Ad5hr)-HIV_89.6Pgp140, -SIV₂₃₉gag, and -SIV₂₃₉nef recombinants. One group was not boosted, one was boosted with HIV_89.6P gp140ΔCFI protein (gp140 envelope with deletions in the cleavage site, fusion peptide, and part of the interspace between the two heptad repeats) (40), and one was boosted with a novel HIV-1 polypeptide “peptomer” representing the CD4 binding site of the envelope (54). The best protection was seen in the gp140-boosted group, with significant reductions in both acute and chronic viremia. Although Env-specific antibody and cellular responses were readily detected, none directly correlated with the better protection. Furthermore, neutralizing antibodies against SHIV_89.6P did not develop until 4 weeks postchallenge. Therefore, we hypothesized that vaccine-elicited nonneutralizing anti-Env antibodies might have contributed to the better control of acute and/or chronic viremia in the gp140 group. Here, we report retrospective evaluations of sera and rectal secretions from macaques in this comparative study for serum binding antibody avidity, an important characteristic of functional antibodies (38, 58), and nonneutralizing activities of systemic and mucosal antibodies, including ADCC, ADCVI, and transcytosis inhibition.     

Neutrophils reduce the parasite burden in Leishmania (Leishmania) amazonensis-infected macrophages

Background

Studies on the role of neutrophils in Leishmania infection were mainly performed with L. (L) major, whereas less information is available for L. (L) amazonensis. Previous results from our laboratory showed a large infiltrate of neutrophils in the site of infection in a mouse strain resistant to L. (L.) amazonensis (C3H/HePas). In contrast, the susceptible strain (BALB/c) displayed a predominance of macrophages harboring a high number of amastigotes and very few neutrophils. These findings led us to investigate the interaction of inflammatory neutrophils with L. (L.) amazonensis-infected macrophages in vitro.

Methodology/Principal Findings

Mouse peritoneal macrophages infected with L. (L.) amazonensis were co-cultured with inflammatory neutrophils, and after four days, the infection was quantified microscopically. Data are representative of three experiments with similar results. The main findings were 1) intracellular parasites were efficiently destroyed in the co-cultures; 2) the leishmanicidal effect was similar when cells were obtained from mouse strains resistant (C3H/HePas) or susceptible (BALB/c) to L. (L.) amazonensis; 3) parasite destruction did not require contact between infected macrophages and neutrophils; 4) tumor necrosis factor alpha (TNF-α), neutrophil elastase and platelet activating factor (PAF) were involved with the leishmanicidal activity, and 5) destruction of the parasites did not depend on generation of oxygen or nitrogen radicals, indicating that parasite clearance did not involve the classical pathway of macrophage activation by TNF-α, as reported for other Leishmania species.

Conclusions/Significance

The present results provide evidence that neutrophils in concert with macrophages play a previously unrecognized leishmanicidal effect on L. (L.) amazonensis. We believe these findings may help to understand the mechanisms involved in innate immunity in cutaneous infection by this Leishmania species.     

Structural plasticity of single chromatin fibers revealed by torsional manipulation

Magnetic tweezers were used to study the mechanical response under torsion of single nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular arrays are extremely resilient and can reversibly accommodate a large amount of supercoiling without much change in length. This behavior is quantitatively described by a molecular model of the chromatin three-dimensional architecture. In this model, we assume the existence of a dynamic equilibrium between three conformations of the nucleosome, corresponding to different crossing statuses of the entry/exit DNAs (positive, null or negative, respectively). Torsional strain displaces that equilibrium, leading to an extensive reorganization of the fiber's architecture. The model explains a number of long-standing topological questions regarding DNA in chromatin and may provide the basis to better understand the dynamic binding of chromatin-associated proteins.Note: In the supplementary information initially published online to accompany this article, Supplementary Figure 2 was mistakenly replaced by Supplementary Equation 2. The error has been corrected online.     

Structural determinations by circular dichroism spectra analysis using coupled oscillator methods: an update of the applications of the DeVoe polarizability model

The exciton (coupled oscillator) model for optical activity is a very useful and powerful method which allows to analyze a circular dichroism (CD) spectrum in a nonempirical way, arriving at a safe assignment of the absolute configuration of organic and inorganic compounds. Usually in this model only the exciton coupling of two electrically allowed transitions (oscillators) is taken into account. This approach has the important advantage of an easy application but, sometimes, it may lead to wrong results. Thus, in this review article a more general treatment, which allows considering the simultaneous coupling of several oscillators, i.e., the DeVoe model, is presented and critically analyzed, discussing in detail the latest applications reported in the literature. In the authors opinion, since the DeVoe model joins generality and reliability requiring an almost negligible computational effort, it represents the method of choice for stereochemical assignments, even by nonspecialists.     

Widespread triazole pesticide use affects infection dynamics of a global amphibian pathogen

The sixth mass extinction is a consequence of complex interplay between multiple stressors with negative impact on biodiversity. We here examine the interaction between two globally widespread anthropogenic drivers of amphibian declines: the fungal disease chytridiomycosis and antifungal use in agriculture. Field monitoring of 26 amphibian ponds in an agricultural landscape shows widespread occurrence of triazole fungicides in the water column throughout the amphibian breeding season, together with a negative correlation between early season application of epoxiconazole and the prevalence of chytrid infections in aquatic newts. While triazole concentrations in the ponds remained below those that inhibit growth of Batrachochytrium dendrobatidis, they bioaccumulated in the newts' skin up to tenfold, resulting in cutaneous growth-suppressing concentrations. As such, a concentration of epoxiconazole, 10 times below that needed to inhibit fungal growth, prevented chytrid infection in anuran tadpoles. The widespread presence of triazoles may thus alter chytrid dynamics in agricultural landscapes.     

Molecular analysis of mutations in the gene FMR-1 segregating in fragile X families

Molecular genetic analysis of the transmission of mutations in 73 families with fragile X (one of the largest samples evaluated so far) has confirmed previous hypotheses that the fragile X syndrome results from two consecutive mutational steps, designated premutation and full fragile X mutation. These mutations give rise to expansions of restriction fragments, most probably by amplification of the FMR-1 CGG repeat. Premutations are identified by small expansions that apparently have no effect on either the clinical or the cellular phenotype. Full mutations are reflected by large expansions and hypermethylation of the expanded gene region. All males showing large expansions were affected. Individuals with full mutations also expressed the fragile X, with only one exception. An affected mosaic male, showing a predominance of premutated fragments in his leukocytes, was shown to be fragile-X-negative on different occasions. About 50% of heterozygotes with full mutations were reported by clinicians to be mentally retarded. Conversion of the premutation to the full mutation may occur at oogenesis, as previously suggested, or after formation of a zygote at an early transitional stage in development when the CGG repeat behaves as a mitotically unstable element on maternally derived/imprinted X chromosomes carrying a premutation of sufficient repeat length.     

Phase-locked responses in the Limulus lateral eye. Theoretical and experimental investigation.

The 1:1 phase locking of the neural discharge to sinusoidally modulated stimuli was investigated both theoretically and experimentally. On the theoretical side, a neural encoder model, the self-inhibited leaky integrator, was considered, and the phase of the locked impulse was computed for each frequency in the locking range by imposing the condition that the "leaky integral" u(t) of the driving signal should reach the threshold for the first time one stimulus period after the preceding impulse. As u(t) can be a nonmonotonic function, this approach leads to results that sometimes differ from those reported in the literature. It turns out that the phase excursion is often much smaller than the values of about 180 degrees predicted from previous analysis. Moreover, our analysis shows a peculiar effect; the phase locking frequency range narrows when the input modulation depth increases. The theoretical predictions are then compared with phase-locked discharge patterns recorded from visual cells of the Limulus lateral eye, stimulated by sinusoidally modulated light or depolarizing current. The phases of the locked spikes at each of a number of modulation frequencies have been measured. The predictions offered by the model fit the experimental data, although there are some difficulties in determining the effective driving signal.     

Enhancing Effect of Manganese on L-DOPA-Induced Apoptosis in PC12 Cells

L-DOPA and manganese both induce oxidative stress-mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10-20 microM L-DOPA neither affected cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L-DOPA (50 microM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 microM L-DOPA, a concentration-dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L-DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N-acetyl-L-cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L-DOPA. An increase in autoxidation of L-DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress-mediated apoptosis in catecholaminergic cells may act synergistically.