First Membrane Proximal External Region-Specific Anti-HIV1 Broadly Neutralizing Monoclonal IgA1 Presenting Short CDRH3 and Low Somatic Mutations.

Mucosal HIV-1-specific IgA have been described as being able to neutralize HIV-1 and to block viral transcytosis. In serum and saliva, the anti-HIV IgA response is predominantly raised against the envelope of HIV-1. In this work, we describe the in vivo generation of gp41-specific IgA1 in humanized α1KI mice to produce chimeric IgA1. Mice were immunized with a conformational immunogenic gp41-transfected cell line. Among 2300 clones screened by immunofluorescence microscopy, six different gp41-specific IgA with strong recognition of gp41 were identified. Two of them have strong neutralizing activity against primary HIV-1 tier 1, 2, and 3 strains and present a low rate of somatic mutations and autoreactivity, unlike what was described for classical gp41-specific IgG. Epitopes were identified and located in the hepted repeat 2/membrane proximal external region. These Abs could be of interest in prophylactic treatment to block HIV-1 penetration in mucosa or in chronically infected patients in combination with antiretroviral therapy to reduce viral load and reservoir.

Zinc-Stabilized Chitosan-Chondroitin Sulfate Nanocomplexes for HIV-1 Infection Inhibition Application.

Polyelectrolyte complexes (PECs) constituted of chitosan and chondroitin sulfate (ChonS) were formed by the one-shot addition of default amounts of polyanion to an excess of polycation. Key variables of the formulation process (e.g., degree of depolymerization, charge mixing ratio, the concentration, and pH of polyelectrolyte solutions) were optimized based on the PECs sizes and polydispersities. The PECs maintained their colloidal stability at physiological salt concentration and pH thanks to the complexation of polyelectrolytes with zinc(II) ion during the nanoPECs formation process. The PECs were capable of encapsulating an antiretroviral drug tenofovir (TF) with a minimal alteration on the colloidal stability of the dispersion. Moreover, the particle interfaces could efficiently be functionalized with anti-OVA or anti-α4ß7 antibodies with conservation of the antibody biorecognition properties over 1 week of storage in PBS at 4 °C. In vitro cytotoxicity studies showed that zinc(II) stabilized chitosan-ChonS nanoPECs were noncytotoxic to human peripheral blood mononuclear cells (PBMCs), and in vitro antiviral activity test demonstrated that nanoparticles formulations led to a dose-dependent reduction of HIV-1 infection. Using nanoparticles as a drug carrier system decreases the IC50 (50% inhibitory concentration) from an aqueous TF of 4.35 µmol·L(-1) to 1.95 µmol·L(-1). Significantly, zinc ions in this system also exhibited a synergistic effect in the antiviral potency. These data suggest that chitosan-ChonS nanoPECs can be promising drug delivery system to improve the antiviral potency of drugs to the viral reservoirs for the treatment of HIV infection.

Conserved domains and structure prediction of human cytomegalovirus UL27 protein.

BACKGROUND: The human cytomegalovirus (HCMV) nuclear UL27 protein (pUL27) could be involved at the stage of nuclear egress. Maribavir is a new anti-HCMV drug that targets nuclear egress through direct inhibition of the HCMV serine-threonine kinase, UL97 protein (pUL97). Because maribavir-resistance-related mutations are observed in both proteins, pUL27 is thought to interfere with pUL97 activity; however, its mechanism of action remains unclear. METHODS: As there is no available crystal structure for pUL27 or any known structures of its homologous proteins, we attempted to identify pUL27 functional domains by sequence analysis, identification of conserved domains, structure prediction and matching with previously known maribavir resistance mutations. RESULTS: The UL27 sequence analysis of 20 HCMV wild-type strains and 8 ganciclovir-resistant HCMV strains allowed us to describe four conserved domains, to localize the putative phosphorylation sites and to identify protein-protein interface domains, suggesting that pUL27 could interact with either pUL97 or itself. CONCLUSIONS: Although the function of pUL27 is still unknown in the HCMV replication cycle, our approach identified target domains that appeared to be essential to the function of pUL27. This work provides a better understanding on the relative importance of each pUL27 mutation and could form the basis of later comparison analyses, when a three-dimensional structure of a pUL27 homologue will be available.

Putative functional domains of human cytomegalovirus pUL56 involved in dimerization and benzimidazole D-ribonucleoside activity.

BACKGROUND: Benzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89 (the large and small subunits of the HCMV terminase, respectively) their mechanism of action is not yet fully understood. We aimed here to better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives. METHODS: The HCMV pUL56 protein was studied by sequence analysis of the HCMV UL56 gene and herpesvirus counterparts combined with primary structure analysis of the corresponding amino acid sequences. RESULTS: The UL56 sequence analysis of 45 HCMV strains and counterparts among herpesviruses allowed the identification of 12 conserved regions. Moreover, comparison with the product of gene 49 (gp49) of bacteriophage T4 suggested that the pUL56 zinc finger is localized close to the dimerization site of pUL56, providing a spatial organization of the catalytic site that allows recognition and cleavage of DNA. CONCLUSIONS: This study provides a basis to investigate the mechanism of concatemeric DNA cleavage and a biochemical basis for DNA packaging inhibition by benzimidazole derivatives.

New functional domains of human cytomegalovirus pUL89 predicted by sequence analysis and three-dimensional modelling of the catalytic site DEXDc.

INTRODUCTION: Benzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89, the large and small subunits of the HCMV terminase respectively, their mechanism of action is not yet fully understood. METHODS AND RESULTS: To better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives, we studied the HCMV pUL89 protein by a genetic approach combined with primary structure analysis. The pUL89 sequence analysis of 25 HCMV strains and counterparts among herpesviruses allowed identification of 12 conserved regions. We also built a three-dimensional model of the pUL89 ATPasic catalytic site, including ATPase motor motifs 1, II and III, that may facilitate the development of future antiviral drugs active against HCMV. Finally, we identified several putative functional domains in pUL89, such as pUL89 zinc finger (pUL89-ZF), DNA cutting sites and portal binding sites, that are probably involved in CMV DNA cleavage and packaging.

Radiolabelled polymeric IgA: from biodistribution to a new molecular imaging tool in colorectal cancer lung metastases.

By radiolabelling monomeric (m) and polymeric (p) IgA with technetium 99m (99mTc), this study assessed IgA biodistribution and tumour-targeting potency. IgA directed against carcinoembryonic antigen (CEA), a colorectal cancer marker, was selected to involve IgA mucosal tropism. Ig was radiolabelled with 99mTc-tricarbonyl after derivatisation by 2-iminothiolane. 99mTc-IgA was evaluated by in vitro analysis. The biodistributions of radiolabelled anti-CEA mIgA, pIgA and IgG were compared in normal mice. Anti-CEA pIgA tumour uptake was studied in mice bearing the WiDr caecal orthotopic graft. IgA radiolabelling was obtained with a high yield, was stable in PBS and murine plasma, and did not alter IgA binding functionality (Kd ≈ 25 nM). Biodistribution studies in normal mice confirmed that radiolabelled pIgA - and to a lesser extent, mIgA - showed strong and fast mucosal tropism and a shorter serum half-life than IgG. In caecal tumour model mice, evaluation of the anti-CEA-pIgA biodistribution showed a high uptake in lung metastases, confirmed by histological analysis. However, no radioactivity uptake increase in the tumoural caecum was discerned from normal intestinal tissue, probably due to high IgA caecal natural tropism. In microSPECT/CT imaging, 99mTc-IgA confirmed its diagnostic potency of tumour in mucosal tissue, even if detection threshold by in vivo imaging was higher than post mortem studies. Contribution of the FcαRI receptor, studied with transgenic mouse model (Tsg SCID-CD89), did not appear to be determinant in 99mTc-IgA uptake. Pre-clinical experiments highlighted significant differences between 99mTc-IgA and 99mTc-IgG biodistributions. Furthermore, tumoural model studies suggested potential targeting potency of pIgA in mucosal tissues.

Ternary polysaccharide complexes: Colloidal drug delivery systems stabilized in physiological media.

Chitosan-hyaluronan (HYA) polyelectrolyte complexes (PECs) were designed to maintain their colloidal stabilities in physiological ionic strength and pH, via a new concept of ternary complexes. This strategy relied on the formation of a binary PEC between chitosan and a strong polyacid, dextran sulphate (DS) or heparin (HEP), and further functionalization with HYA. The major parameter leading to stabilized colloids was a high ratio of the degrees of polymerization of chitosan versus the strong polyacid. The process afforded either positive or negative particles when HYA was used in default or in excess (vs. chitosan) for the functionalization of the binary complexes. The most stable formulations were loaded with an antiretroviral drug tenofovir (TF), and could be surface functionalized with targeting IgAs. In vitro, the cationic TF loaded ternary complexes exhibited an inhibition of infection of PBMCs by the HIV-1 virus, superior to the free drug.

A novel mutation in the UL54 gene of human cytomegalovirus isolates that confers resistance to foscarnet.

Foscarnet is currently licensed for the treatment of human cytomegalovirus (HCMV) infection. Mutations proven to confer resistance to foscarnet have mostly been mapped to regions II, III and VI of the HCMV UL54-encoded DNA polymerase. We previously showed that sequential foscarnet-resistant HCMV isolates recovered from a patient with lymphoma had change N495K in region delta-C of the DNA polymerase. To evaluate the impact of change N495K on HCMV sensitivity to foscarnet, a recombinant HCMV strain carrying the mutation was produced by homologous recombination. The recombinant virus showed a 3.4-fold increase in foscarnet resistance, and remained sensitive to ganciclovir and cidofovir. In addition, the recombinant strain showed a reduction of infectious virus yield compared with its parent strain. Change N495K should be added to the list of mutations conferring resistance to foscarnet and be taken into account in the genotypic diagnosis of antiviral resistance.

Zinc-stabilized colloidal polyelectrolyte complexes of chitosan/hyaluronan: a tool for the inhibition of HIV-1 infection.

Zinc(ii) stabilized polyelectrolyte nano-complexes (PECs) of chitosan and hyaluronan (HYA) were designed as safe and efficient drug delivery systems. HIV-1 reverse transcriptase inhibitor tenofovir (TF) was quantitatively encapsulated and the particle interface could be functionalized in PBS with targeting proteins such as anti-α4ß7 immunoglobulin A. Chitosan-HYA nanoPECs were non-cytotoxic on human peripheral blood mononuclear cells (PBMCs), within the investigated nanoparticle concentrations. A dose-dependent reduction of the HIV-1 infection of PBMCs co-cultured with the nanocarriers was observed. Even more interestingly, a synergistic effect was evidenced with the nanocarriers by comparing the IC50 (50% inhibitory concentration) value of the aqueous TF solution (4.35 µmol L-1) with that of TF loaded nanoPECs (1.71 µmol L-1) and anti-α4ß7 IgA functionalized TF/nanoPECs (1.01 µmol L-1). This effect could be attributed to the presence of zinc(ii) in the formulation of the colloids. All these data establish that the zinc(ii) stabilized chitosan-HYA nanoPECs can be potentially efficient and safe colloidal delivery system candidates for enhancing antiviral activities in the treatment of HIV infection and AIDS.

Similarity of fine specificity of IgA anti-gliadin antibodies between patients with celiac disease and humanized α1KI mice.

Gliadins, and primarily α-gliadins containing several sequences such as aa 31-49, aa 56-88 (33-mer), aa 57-68, and aa 69-82, are critical in the induction of immune response or toxic reaction leading to the development of celiac disease (CLD). The role of IgA anti-gliadin antibodies (IgA AGA) is unknown. To this end, we prepared several humanized monoclonal IgA AGA using transgenic α1KI mice. Employing Pepscan with overlapping decapeptides of α-gliadin we observed a robust similarity between the specificity of humanized mouse monoclonal IgA AGA and IgA AGA from patients with florid CLD. The common immunodominant region included several sequential epitopes localized in the N-terminal part of α-gliadin (QFQGQQQPFPPQQPYPQPQPFP, aa 29-50, and QPFPSQQPYLQL, aa 47-58). Notably, IgA AGA produced by clones 8D12, 15B9, 9D12, and 18E2 had significant reactivity against sequences localized in the 33-mer, LQLQPFPQPQ (aa 56-65) and PQLPYPQPQPFL (aa 69-80). Humanized mouse monoclonal IgA AGA that have a known specificity are suitable as standard in ELISAs to detect serum IgA AGA of CLD patients and for studying the AGA pathogenic role in CLD, especially for analyzing the translocation of complex of specific IgA antibodies and individual gliadin peptides through enterocyte barrier.

Early selection of a new UL97 mutant with a severe defect of ganciclovir phosphorylation after valaciclovir prophylaxis and short-term ganciclovir therapy in a renal transplant recipient.

We describe the emergence of a new ganciclovir resistance mutation in the UL97 gene of human cytomegalovirus, deletion of codon 601, after valaciclovir and short-term ganciclovir therapy following kidney transplantation. Its role in ganciclovir resistance was supported by decreased ganciclovir phosphorylation in a recombinant vaccinia virus system.

Natural polymorphism of cytomegalovirus DNA polymerase lies in two nonconserved regions located between domains delta-C and II and between domains III and I.

We described the natural polymorphism of cytomegalovirus DNA polymerase in 42 unrelated isolates susceptible to ganciclovir, foscarnet, and cidofovir. All variations, including an eight-amino-acid deletion, were located between domains delta-C and II and between domains III and I, suggesting that these specific residues are not involved in enzymatic functions.