Envelope C2-V3-C3-specific antibodies from HIV-1 infected patients from Angola correlate with neutralization activity in plasma

Poster presented at the 2015 Keystone Symposia Conference X5: HIV Vaccines. Banff, Alberta, Canada, 22-27 March 2015

Étude de l’infection lytique du Virus Epstein-Barr dans le développement de tumeurs post-greffe

Le virus Epstein-Barr (VEB) est un pathogène opportuniste qui a la capacité d’immortaliser les lymphocytes B et de provoquer une prolifération maligne, appelée syndrome lymphoprolifératif post-transplantation (SLP), chez les individus immunodéprimés. A l’intérieur de ce groupe, les personnes à plus haut risque sont les enfants, puisqu’ils sont à risque de développer une infection primaire par le VEB pendant leur régime d’immunosuppression post-greffe. Dans le but de développer un anticorps préventif, notre laboratoire s’est attardé au rôle du cycle lytique du VEB dans le développement du SLP. À cette fin, le premier objectif du présent projet vise à fournir la preuve expérimentale de l’existence ou non d’une phase réplicative productive pendant l’infection aiguë des lymphocytes B sanguins. Un examen des événements qui se déroulent au tout début de l’infection par le VEB tant au niveau de la réplication virale qu’au niveau de l’expression des gènes lytiques précoces et tardifs a révélé l’existence d’une phase réplicative productive pendant l’infection aiguë. Ceci a permis de justifier l’élaboration, dans notre laboratoire, d’un anticorps chimère (murin-humain) neutralisant, dirigé contre la protéine gp350 située sur l’enveloppe virale. Le deuxième objectif, quant à lui, vise à fournir la preuve expérimentale de la capacité neutralisante de cet anticorps chimère. Des essais de caractérisation in vitro ont démontré une capacité de reconnaissance de la protéine cible, notamment la gp350, et une capacité de neutralisation du virus par l’anticorps chimère. L’anticorps chimère anti-gp350 pourra faire l’objet d’essais précliniques in vivo en vue d’évaluer sa capacité à reconnaître le virus et à prévenir l’apparition de tumeurs de type SLP chez les souris SCID. Il pourrait être éventuellement utilisé, par la suite, comme traitement préemptif contre les tumeurs dans l’espoir de mieux gérer les patients à risque de développer un SLP.

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A(2) and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2 Delta AB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2 Delta AB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.

Functional and immunological characterization of a natural polymorphic variant of a heat-labile toxin (LT-I) produced by enterotoxigenic Escherichia coli (ETEC)

Heat-labile toxins (LT) encompass at least 16 natural polymorphic toxin variants expressed by wild-type enterotoxigenic Escherichia coli (ETEC) strains isolated from human beings, but only one specific form, produced by the reference ETEC H10407 strain (LT1), has been intensively studied either as a virulence-associated factor or as a mucosal/transcutaneous adjuvant. In the present study, we carried out a biological/immunological characterization of a natural LT variant (LT2) with four polymorphic sites at the A subunit (S190L, G196D, K213E, and S224T) and one at the B subunit (T75A). The results indicated that purified LT2, in comparison with LT1, displayed similar in vitro toxic activities (adenosine 3`,5`-cyclic monophosphate accumulation) on mammalian cells and in vivo immunogenicity following delivery via the oral route. Nonetheless, the LT2 variant showed increased adjuvant action to ovalbumin when delivered to mice via the transcutaneous route while antibodies raised in mice immunized with LT2 displayed enhanced affinity and neutralization activity to LT1 and LT2. Taken together, the results indicate that the two most frequent LT polymorphic forms expressed by wild ETEC strains share similar biological features, but differ with regard to their immunological properties.

Association of HIV-1 Envelope-Specific Breast Milk IgA Responses with Reduced Risk of Postnatal Mother-to-Child Transmission of HIV-1.

UNLABELLED: Infants born to HIV-1-infected mothers in resource-limited areas where replacement feeding is unsafe and impractical are repeatedly exposed to HIV-1 throughout breastfeeding. Despite this, the majority of infants do not contract HIV-1 postnatally, even in the absence of maternal antiretroviral therapy. This suggests that immune factors in breast milk of HIV-1-infected mothers help to limit vertical transmission. We compared the HIV-1 envelope-specific breast milk and plasma antibody responses of clade C HIV-1-infected postnatally transmitting and nontransmitting mothers in the control arm of the Malawi-based Breastfeeding Antiretrovirals and Nutrition Study using multivariable logistic regression modeling. We found no association between milk or plasma neutralization activity, antibody-dependent cell-mediated cytotoxicity, or HIV-1 envelope-specific IgG responses and postnatal transmission risk. While the envelope-specific breast milk and plasma IgA responses also did not reach significance in predicting postnatal transmission risk in the primary model after correction for multiple comparisons, subsequent exploratory analysis using two distinct assay methodologies demonstrated that the magnitudes of breast milk total and secretory IgA responses against a consensus HIV-1 envelope gp140 (B.con env03) were associated with reduced postnatal transmission risk. These results suggest a protective role for mucosal HIV-1 envelope-specific IgA responses in the context of postnatal virus transmission. This finding supports further investigations into the mechanisms by which mucosal IgA reduces risk of HIV-1 transmission via breast milk and into immune interventions aimed at enhancing this response. IMPORTANCE: Infants born to HIV-1-infected mothers are repeatedly exposed to the virus in breast milk. Remarkably, the transmission rate is low, suggesting that immune factors in the breast milk of HIV-1-infected mothers help to limit transmission. We compared the antibody responses in plasma and breast milk of HIV-1-transmitting and -nontransmitting mothers to identify responses that correlated with reduced risk of postnatal HIV-1 transmission. We found that neither plasma nor breast milk IgG antibody responses were associated with risk of HIV-1 transmission. In contrast, the magnitudes of the breast milk IgA and secretory IgA responses against HIV-1 envelope proteins were associated with reduced risk of postnatal HIV-1 transmission. The results of this study support further investigations of the mechanisms by which mucosal IgA may reduce the risk of HIV-1 transmission via breastfeeding and the development of strategies to enhance milk envelope-specific IgA responses to reduce mother-to-child HIV transmission and promote an HIV-free generation.

A monovalent chimpanzee adenovirus Ebola vaccine boosted with MVA

BACKGROUND The West African outbreak of Ebola virus disease that peaked in 2014 has caused more than 11,000 deaths. The development of an effective Ebola vaccine is a priority for control of a future outbreak. METHODS In this phase 1 study, we administered a single dose of the chimpanzee adenovirus 3 (ChAd3) vaccine encoding the surface glycoprotein of Zaire ebolavirus (ZEBOV) to 60 healthy adult volunteers in Oxford, United Kingdom. The vaccine was administered in three dose levels — 1×1010 viral particles, 2.5×1010 viral particles, and 5×1010 viral particles — with 20 participants in each group. We then assessed the effect of adding a booster dose of a modified vaccinia Ankara (MVA) strain, encoding the same Ebola virus glyco- protein, in 30 of the 60 participants and evaluated a reduced prime–boost interval in another 16 participants. We also compared antibody responses to inactivated whole Ebola virus virions and neutralizing antibody activity with those observed in phase 1 studies of a recombinant vesicular stomatitis virus–based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) to determine relative potency and assess durability. RESULTS No safety concerns were identified at any of the dose levels studied. Four weeks after immunization with the ChAd3 vaccine, ZEBOV-specific antibody responses were similar to those induced by rVSV-ZEBOV vaccination, with a geometric mean titer of 752 and 921, respectively. ZEBOV neutralization activity was also similar with the two vaccines (geo- metric mean titer, 14.9 and 22.2, respectively). Boosting with the MVA vector increased virus-specific antibodies by a factor of 12 (geometric mean titer, 9007) and increased glycoprotein-specific CD8+ T cells by a factor of 5. Significant increases in neutralizing antibodies were seen after boosting in all 30 participants (geometric mean titer, 139; P<0.001). Virus-specific antibody responses in participants primed with ChAd3 remained positive 6 months after vaccination (geometric mean titer, 758) but were significantly higher in those who had received the MVA booster (geometric mean titer, 1750; P<0.001). CONCLUSIONS The ChAd3 vaccine boosted with MVA elicited B-cell and T-cell immune responses to ZEBOV that were superior to those induced by the ChAd3 vaccine alone. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT02240875.)

Ocular distribution, spectrum of activity, and in vivo viral neutralization of a fully humanized anti-herpes simplex virus IgG Fab fragment following topical application.

Herpes simplex ocular infection is a major cause of corneal blindness. Local antiviral treatments exist but are associated with corneal toxicity, and resistance has become an issue. We evaluated the biodistribution and efficacy of a humanized anti-herpes simplex virus (anti-HSV) IgG FAb fragment (AC-8; 53 kDa) following repeated topical administration. AC-8 was found in the corneal epithelium, anterior stroma, subepithelial stromal cells, and retinal glial cells, with preferential entry through the ocular limbus. AC-8 was active against 13 different strains of HSV-1, with 50% and 90% mean effective concentrations (MEC(50) and MEC(90), respectively) ranging from 0.03 to 0.13 μg/ml, indicating broad-spectrum activity. The in vivo efficacy of AC-8 was evaluated in a mouse model of herpes-induced ocular disease. Treatment with low-dose AC-8 (1 mg/ml) slightly reduced the ocular disease scores. A greater reduction of the disease scores was observed in the 10-mg/ml AC-8-treated group, but not as much as with trifluridine (TFT). AC-8 treatment reduced viral titers but less than trifluridine. AC-8 did not display any toxicity to the cornea or other structures in the eye. In summary, topical instillation of an anti-HSV FAb can be used on both intact and ulcerated corneas. It is well tolerated and does not alter reepithelialization. Further studies to improve the antiviral effect are needed for AC-8 to be considered for therapeutic use.

N-terminal amino acids of bovine alpha interferons are relevant for the neutralization of their antiviral activity

The structure-function relationship of interferons (IFNs) has been studied by epitope mapping. Epitopes of bovine IFNs, however, are practically unknown, despite their importance in virus infections and in the maternal recognition of pregnancy. It has been shown that recombinant bovine (rBo)IFN-alphaC and rBoIFN-alpha1 differ only in 12 amino acids and that the F12 monoclonal antibody (mAb) binds to a linear sequence of residues 10 to 34. We show here that the antiviral activities of these two IFNs were neutralized by the F12 mAb to different extents using two tests. In residual activity tests the antiviral activity dropped by more than 99% with rBoIFN-alphaC and by 84% with rBoIFN-alpha1. In checkerboard antibody titrations, the F12 mAb titer was 12,000 with rBoIFN-alphaC and only 600 with rBoIFN-alpha1. Since these IFNs differ in their amino acid sequence at positions 11, 16 and 19 of the amino terminus, only these amino acids could account for the different neutralization titers, and they should participate in antibody binding. According to the three-dimensional structure described for human and murine IFNs, these amino acids are located in the alpha helix A; amino acids 16 and 19 of the bovine IFNs would be expected to be exposed and could bind to the antibody directly. The amino acid at position 11 forms a hydrogen bond in human IFNs-alpha and it is possible that, in bovine IFNs-alpha, the F12 mAb, binding near position 11, would disturb this hydrogen bond, resulting in the difference in the extent of neutralization observed.

Characterization of major zinc containing myonecrotic and procoagulant metalloprotease 'malabarin' from non lethal trimeresurus malabaricus snake venom with thrombin like activity: its neutralization by chelating agents

A major myonecrotic zinc containing metalloprotease 'malabarin' with thrombin like activity was purified by the combination of gel permeation and anion exchange chromatography from T. malabaricus snake venom. MALDI-TOF analysis of malabarin indicated a molecular mass of 45.76 kDa and its N-terminal sequence was found to be Ile-Ile-Leu- Pro(Leu)-Ile-Gly-Val-Ile-Leu(Glu)-Thr-Thr. Atomic absorption spectral analysis of malabarin raveled the association of zinc metal ion. Malabarin is not lethal when injected i.p. or i.m. but causes extensive hemorrhage and degradation of muscle tissue within 24 hours. Sections of muscle tissue under light microscope revealed hemorrhage and congestion of blood vessel during initial stage followed by extensive muscle fiber necrosis with elevated levels of serum creatine kinase and lactate dehydrogenase activity. Malabarin also exhibited strong procoagulant action and its procoagulant action is due to thrombin like activity; it hydrolyzes fibrinogen to form fibrin clot. The enzyme preferentially hydrolyzes A? followed by B subunits of fibrinogen from the N-terminal region and the released products were identified as fibrinopeptide A and fibrinopeptide B by MALDI. The myonecrotic, fibrinogenolytic and subsequent procoagulant activities of malabarin was neutralized by specific metalloprotease inhibitors such as EDTA, EGTA and 1, 10-phenanthroline but not by PMSF a specific serine protease inhibitor. Since there is no antivenom available to neutralize local toxicity caused by T. malabaricus snakebite, EDTA chelation therapy may have more clinical relevance over conventional treatment.

Site-specific mutations in the mature form of human IL-18 with enhanced biological activity and decreased neutralization by IL-18 binding protein

IL-18 can be considered a proinflammatory cytokine mediating disease as well as an immunostimulatory cytokine that is important for host defense against infection and cancer. The high-affinity, constitutively expressed, and circulating IL-18 binding protein (IL-18BP), which competes with cell surface receptors for IL-18 and neutralizes IL-18 activity, may act as a natural antiinflammatory as well as immunosuppressive molecule. In the present studies, the IL-18 precursor caspase-1 cleavage site was changed to a factor Xa site, and, after expression in Escherichia coli, mature IL-18 was generated by factor Xa cleavage. Mature IL-18 generated by factor Xa cleavage was fully active. Single point mutations in the mature IL-18 peptide were made, and the biological activities of the wild-type (WT) IL-18 were compared with those of the mutants. Mutants E42A and K89A exhibited 2-fold increased activity compared with WT IL-18. A double mutant, E42A plus K89A, exhibited 4-fold greater activity. Unexpectedly, IL-18BP failed to neutralize the double mutant E42A plus K89A compared with WT IL-18. The K89A mutant was intermediate in being neutralized by IL-18BP, whereas neutralization of the E42A mutant was comparable to that in the WT IL-18. The identification of E42 and K89 in the mature IL-18 peptide is consistent with previous modeling studies of IL-18 binding to IL-18BP and explains the unusually high affinity of IL-18BP for IL-18.

Neutralization of pharmacological and toxic activities of Bothrops snake venoms by Schizolobium parahyba (Fabaceae) aqueous extract and its fractions

The aqueous extract prepared from Schizolobium parahyba (Sp) leaves, a native plant from Atlantic Forest (Brazil), was tested to analyse its ability to inhibit some biological and enzymatic activities induced by Bothrops alternatus (BaltCV) and Bothrops moojeni (BmooCV) snake venoms. Sp inhibited 100% of lethality, blood incoagulability, haemorrhagic and indirect haemolytic activities at a 1:10 ratio (venom/extract, w/w), as well as coagulant activity at a 1:5 ratio (venom/extract, w/w) induced by both venoms. BaltCV fibrinogenolytic activity was also neutralized by Sp at a 1:10 ratio, resulting in total protection of fibrinogen B beta chain and partial protection of A alpha chain. Interaction tests have demonstrated that, at certain extract/proteins ratios, Sp precipitates proteins non-specifically suggesting the presence of tannins, which are very likely responsible for the excellent inhibiting effects of the analysed ophidian activities. Sp aqueous extract chromatography on Sephadex LH-20 was carried out aiming at the separation of these compounds that mask the obtained results. Thus, the fractionation of Sp resulted in three fractions: F1 (methanolic fraction); F2 (methanol:water fraction, 1:1 v/v); and F3 (aqueous fraction). These fractions were analysed for their ability to inhibit the BaltCV fibrinogenolytic activity. F1 inhibited 100% the venom fibrinogenolytic activity without presenting protein precipitation effect; F2 showed only partial inhibition of this venom activity. Finally, F3 did not inhibit fibrinogen proteolysis, but presented strong protein precipitating action. We conclude that Sp aqueous extract, together with tannins, also contains other compounds that can display specific inhibitory activity against snake venom toxins.

Heparin-antivenom association: differential neutralization effectiveness in Bothrops atrox and Bothrops erythromelas envenoming

Heparin, in some regions of Brazil has been used in the treatment of bothropic accidents, but the data found in the literature are inconclusive about its effectiveness. The venoms of Bothrops atrox and of B. erythromelas were characterized according to their biological activities. The capacity of heparin in neutralizing these activities was tested with doses of 3 and 6 IU in isolated form and associated to Antibothropic Serum (ABS). It was verified that heparin, in doses of 3 and 6 IU, was not effective in neutralizing the desfibrinating and edema-forming activities of B. atrox venom and the hemorrhagic and coagulant actions of both venoms. Heparin diminished the effectiveness of the ABS in the neutralization of the hemorrhagic and edema-forming activities of the B. atrox venom. However, heparin in the 6 IU dose was capable of neutralize the edema-forming of the B. erythromelas and increase the effectiveness of the ABS. Heparin also neutralized the phospholipasic A2 activity of B. atrox (14.3%) and B. erythromelas (28.0%) venoms. For B. erythromelas venom, the associated treatment, heparin and ABS, was more effective in the neutralization of its lethal activity.

Neutralization of bitis parviocula (Ethiopian mountain adder) venom by the south african institute of medical research (SAIMR) antivenom

BACKGROUND: The Ethiopian mountain adder (Bitis parviocula) is a viperid known only from a few locations in southwestern Ethiopia. METHODS: a total of 30 µg of B. arietans and B. parviocula venoms were run on a 10-20% Tricine gel. To assay lethality dose fifty (LD50), five groups of eight mice for each venom were used. Hemorrhagic activity for crude venom was tested. Fibrinogenolytic activity of crude venom was measured using (2.5 mg/mL) of fibrinogen solution and (0.03 mg/mL) of crude venom. Gelatinase activity of the venom was tested on a Kodak X-OMAT TM film. Crude venoms of B. parviocula and B. arietans were tested for their abilities to affect clotting time, clotting rate and platelet function on whole human blood. RESULTS: The (SAIMR) antivenom was confirmed in this study to neutralize the lethal activity of venom from Bitis parviocula. The ED50s of SAIMR antivenom on B. parviocula and B. arietans neutralized half of 18.2 and 66.7 mg of venom, respectively. The hemorrhagic activities (MHDs) of B. parviocula and B. arietans were 0.88 and 1.7 µg, respectively. Bitis arietans and B. parviocula venoms degradated α and β chains at different times. The γ chains remained unaffected. Bitis parviocula venom did not exhibit gelatinase activity, while B. arietans had a MGD of 6.9 µg. At 3 mg/mL, the crude venoms of B. parviocula and B. arietans did not significantly affect clotting time or clotting rate. CONCLUSIONS: The SAIMR antivenom is very effective in neutralizing the venom of B. parviocula and should be considered in treating envenomations by these snakes.

Neutralization of Macrophage Migration Inhibitory Factor (MIF) by Fully Human Antibodies Correlates with Their Specificity for the β-Sheet Structure of MIF.

The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that recently emerged as an attractive therapeutic target for a variety of diseases. A diverse panel of fully human anti-MIF antibodies was generated by selection from a phage display library and extensively analyzed in vitro. Epitope mapping studies identified antibodies specific for linear as well as structural epitopes. Experimental animal studies revealed that only those antibodies binding epitopes within amino acids 50-68 or 86-102 of the MIF molecule exerted protective effects in models of sepsis or contact hypersensitivity. Within the MIF protein, these two binding regions form a β-sheet structure that includes the MIF oxidoreductase motif. We therefore conclude that this β-sheet structure is a crucial region for MIF activity and a promising target for anti-MIF antibody therapy.

Anti-MPER antibodies with heterogeneous neutralization capacity are detectable in most untreated HIV-1 infected individuals

Background The MPER region of the HIV-1 envelope glycoprotein gp41 is targeted by broadly neutralizing antibodies. However, the localization of this epitope in a hydrophobic environment seems to hamper the elicitation of these antibodies in HIV infected individuals. We have quantified and characterized anti-MPER antibodies by ELISA and by flow cytometry using a collection of mini gp41-derived proteins expressed on the surface of 293T cells. Longitudinal plasma samples from 35 HIV-1 infected individuals were assayed for MPER recognition and MPER-dependent neutralizing capacity using HIV-2 viruses engrafted with HIV-1 MPER sequences. Results Miniproteins devoid of the cysteine loop of gp41 exposed the MPER on 293T cell membrane. Anti-MPER antibodies were identified in most individuals and were stable when analyzed in longitudinal samples. The magnitude of the responses was strongly correlated with the global response to the HIV-1 envelope glycoprotein, suggesting no specific limitation for anti-MPER antibodies. Peptide mapping showed poor recognition of the C-terminal MPER moiety and a wide presence of antibodies against the 2F5 epitope. However, antibody titers failed to correlate with 2F5-blocking activity and, more importantly, with the specific neutralization of HIV-2 chimeric viruses bearing the HIV-1 MPER sequence; suggesting a strong functional heterogeneity in anti-MPER humoral responses. Conclusions Anti-MPER antibodies can be detected in the vast majority of HIV-1 infected individuals and are generated in the context of the global anti-Env response. However, the neutralizing capacity is heterogeneous suggesting that eliciting neutralizing anti-MPER antibodies by immunization might require refinement of immunogens to skip nonneutralizing responses.