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.

Delivery of antigen to nasal-associated lymphoid tissue microfold cells through secretory IgA targeting local dendritic cells confers protective immunity.

BACKGROUND: Transmission of mucosal pathogens relies on their ability to bind to the surfaces of epithelial cells, to cross this thin barrier, and to gain access to target cells and tissues, leading to systemic infection. This implies that pathogen-specific immunity at mucosal sites is critical for the control of infectious agents using these routes to enter the body. Although mucosal delivery would ensure the best onset of protective immunity, most of the candidate vaccines are administered through the parenteral route. OBJECTIVE: The present study evaluates the feasibility of delivering the chemically bound p24gag (referred to as p24 in the text) HIV antigen through secretory IgA (SIgA) in nasal mucosae in mice. RESULTS: We show that SIgA interacts specifically with mucosal microfold cells present in the nasal-associated lymphoid tissue. p24-SIgA complexes are quickly taken up in the nasal cavity and selectively engulfed by mucosal dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-positive dendritic cells. Nasal immunization with p24-SIgA elicits both a strong humoral and cellular immune response against p24 at the systemic and mucosal levels. This ensures effective protection against intranasal challenge with recombinant vaccinia virus encoding p24. CONCLUSION: This study represents the first example that underscores the remarkable potential of SIgA to serve as a carrier for a protein antigen in a mucosal vaccine approach targeting the nasal environment.

Prior COVID-19 Immunization Does Not Cause IgA- or IgG-Dependent Enhancement of SARS-CoV-2 Infection.

Antibody-dependent enhancement (ADE) can increase the rates and severity of infection with various viruses, including coronaviruses, such as MERS. Some in vitro studies on COVID-19 have suggested that prior immunization enhances SARS-CoV-2 infection, but preclinical and clinical studies have demonstrated the contrary. We studied a cohort of COVID-19 patients and a cohort of vaccinated individuals with a heterologous (Moderna/Pfizer) or homologous (Pfizer/Pfizer) vaccination scheme. The dependence on IgG or IgA of ADE of infection was evaluated on the serum samples from these subjects (twenty-six vaccinated individuals and twenty-one PCR-positive SARS-CoV-2-infected patients) using an in vitro model with CD16- or CD89-expressing cells and the Delta (B.1.617.2 lineage) and Omicron (B.1.1.529 lineage) variants of SARS-CoV-2. Sera from COVID-19 patients did not show ADE of infection with any of the tested viral variants. Some serum samples from vaccinated individuals displayed a mild IgA-ADE effect with Omicron after the second dose of the vaccine, but this effect was abolished after the completion of the full vaccination scheme. In this study, FcγRIIIa- and FcαRI-dependent ADE of SARS-CoV-2 infection after prior immunization, which might increase the risk of severe disease in a second natural infection, was not observed.

Antiviral Activities of HIV-1-Specific Human Broadly Neutralizing Antibodies Are Isotype-Dependent.

Broadly neutralizing antibodies (bNAbs) offer promising opportunities for preventing HIV-1 infection. The protection mechanisms of bNAbs involve the Fc domain, as well as their Fab counterpart. Here, different bNAb isotypes including IgG1, IgA1, IgA2, and IgA122 (IgA2 with the hinge of IgA1) were generated and then produced in CHO cells. Their ability to neutralize pseudovirus and primary HIV-1 isolates were measured, as well as their potential ADCC-like activity using a newly developed assay. In our work, gp41-specific IgA seems to be more efficient than IgG1 in inducing ADCC-like activity, but not in its virus neutralization effect. We show that either gp120-specific IgA or IgG1 isotypes are both efficient in neutralizing different viral strains. In contrast, gp120-specific IgG1 was a better ADCC-like inducer than IgA isotypes. These results provide new insights into the neutralization and ADCC-like activity of different bNAbs that might be taken into consideration when searching for new treatments or antibody-based vaccines.

Alteration of microbiota antibody-mediated immune selection contributes to dysbiosis in inflammatory bowel diseases.

Human secretory immunoglobulins (SIg) A1 and SIgA2 guide mucosal responses toward tolerance or inflammation, notably through reverse-transcytosis, the apical-to-basal transport of IgA2 immune complexes via M cells of gut Peyer's patches. As such, the maintenance of a diverse gut microbiota requires broad affinity IgA and glycan-glycan interaction. Here, we asked whether IgA1 and IgA2-microbiota interactions might be involved in dysbiosis induction during inflammatory bowel diseases. Using stool HPLC-purified IgA, we show that reverse-transcytosis is abrogated in ulcerative colitis (UC) while it is extended to IgA1 in Crohn's disease (CD). 16S RNA sequencing of IgA-bound microbiota in CD and UC showed distinct IgA1- and IgA2-associated microbiota; the IgA1+ fraction of CD microbiota was notably enriched in beneficial commensals. These features were associated with increased IgA anti-glycan reactivity in CD and an opposite loss of reactivity in UC. Our results highlight previously unknown pathogenic properties of IgA in IBD that could support dysbiosis.

Impact of Dextran-Sodium-Sulfate-Induced Enteritis on Murine Cytomegalovirus Reactivation.

(1) Background: Ulcerative colitis (UC) is an inflammatory bowel disease that causes inflammation of the intestines, which participates in human cytomegalovirus (HCMV) reactivation from its latent reservoir. CMV-associated colitis plays a pejorative role in the clinical course of UC. We took advantage of a model of chemically induced enteritis to study the viral reactivation of murine CMV (MCMV) in the context of gut inflammation. (2) Methods: Seven-week-old BALB/c mice were infected by 3 × 103 plaque-forming units (PFU) of MCMV; 2.5% (w/v) DSS was administered in the drinking water from day (D) 30 to D37 post-infection to induce enteritis. (3) Results: MCMV DNA levels in the circulation decreased from D21 after infection until resolution of the acute infection. DSS administration resulted in weight loss, high disease activity index, elevated Nancy index shortening of the colon length and increase in fecal lipocalin. However, chemically induced enteritis had no impact on MCMV reactivation as determined by qPCR and immunohistochemistry of intestinal tissues. (4) Conclusions: Despite the persistence of MCMV in the digestive tissues after the acute phase of infection, the gut inflammation induced by DSS did not induce MCMV reactivation in intestinal tissues, thus failing to recapitulate inflammation-driven HCMV reactivation in human UC.

NOD2 deficiency increases retrograde transport of secretory IgA complexes in Crohn's disease.

Intestinal microfold cells are the primary pathway for translocation of secretory IgA (SIgA)-pathogen complexes to gut-associated lymphoid tissue. Uptake of SIgA/commensals complexes is important for priming adaptive immunity in the mucosa. This study aims to explore the effect of SIgA retrograde transport of immune complexes in Crohn's disease (CD). Here we report a significant increase of SIgA transport in CD patients with NOD2-mutation compared to CD patients without NOD2 mutation and/or healthy individuals. NOD2 has an effect in the IgA transport through human and mouse M cells by downregulating Dectin-1 and Siglec-5 expression, two receptors involved in retrograde transport. These findings define a mechanism of NOD2-mediated regulation of mucosal responses to intestinal microbiota, which is involved in CD intestinal inflammation and dysbiosis.

New chimeric TLR7/NOD2 agonist is a potent adjuvant to induce mucosal immune responses.

BACKGROUND: PRR (Pattern Recognition Receptor) agonists have been widely tested as potent vaccine adjuvants. TLR7 (Toll-Like Receptor 7) and NOD2 (nucleotide-binding oligomerization domain 2) are key innate receptors widely expressed at mucosal levels. METHODS: Here, we evaluated the immunostimulatory properties of a novel hybrid chemical compound designed to stimulate both TLR7 and NOD2 receptors. FINDING: The combined TLR7/NOD2 agonist showed increase efficacy than TLR7L or NOD2L agonists alone or combined in different in vitro models. Dual TLR7/NOD2 agonist efficiently stimulates TLR7 and NOD2, and promotes the maturation and reprogramming of human dendritic cells, as well as the secretion of pro-inflammatory or adaptive cytokines. This molecule also strongly induces autophagy in human cells which is a major intracellular degradation system that delivers cytoplasmic constituents to lysosomes in both MHC class I and II-restricted antigen presentation. In vivo, TLR7/NOD2L agonist is a potent adjuvant after intranasal administration with NP-p24 HIV vaccine, inducing high-quality humoral and adaptive responses both in systemic and mucosal compartments. Use of TLR7/NOD2L adjuvant improves very significantly the protection of mice against an intranasal challenge with a vaccinia virus expressing the p24. INTERPRETATION: Dual TLR7/NOD2L agonist is a very potent and versatile vaccine adjuvant and promote very efficiently both systemic and mucosal immunity. FUNDING: This work was supported by Sidaction.

A high mucosal blocking score is associated with HIV protection.

BACKGROUND: Early steps of HIV infection are mediated by the binding of the envelope to mucosal receptors as α4ß7 and the C-type lectins DC-SIGN and langerin. Previously Env-specific B-cell responses have been reported in highly exposed seronegative individuals (HESN). METHOD: Here, we studied gp120-specific antibodies ability to block HIV interaction with α4ß7, DC-SIGN and/or langerinin HESN. New cell-based assays were developed to analyze whether antibodies that can alter gp120 binding to α4ß7, DC-SIGN and/or langerin are induced in HESN. A mucosal blocking score (MBS) was defined based on the ability of antibodies to interfere with gp120/α4ß7, gp120/DC-SIGN, and gp120/langerin binding. A new MBS was evaluated in a cohort of 86 HESN individuals and compared with HIV+ patients or HIV- unexposed healthy individuals. RESULTS: Antibodies reducing gp120 binding to both α4ß7 and DC-SIGN were present in HESN serum but also in mucosal secretions, whereas antibodies from HIV+ patients facilitated gp120 binding to DC-SIGN. Any correlation was observed between MBS and the capacity of antibodies to neutralize infection of α4ß7 CD4+ T cells with primary isolates. CONCLUSIONS: MBS is significantly associated with protection in HESN and might reflect altered HIV spreading to mucosal-associated lymphoid tissues.

Exploiting Natural Cross-reactivity between Human Immunodeficiency Virus (HIV)-1 p17 Protein and Anti-gp41 2F5 Antibody to Induce HIV-1 Neutralizing Responses In Vivo.

Anti-p17 antibodies are able to neutralize human immunodeficiency virus (HIV) entry in a mouse model. In this study, we identified a region of sequence similarity between the epitopes of anti-p17 neutralizing antibodies and anti-gp41 neutralizing 2F5 antibody and verified cross-reactivity between p17 and 2F5 in vitro. The p17 sequence was modified to increase sequence identity between the p17 and 2F5 epitopes, which resulted in enhanced cross-reactivity in vitro. Immunogenicity of wild-type and modified p17 was characterized in a rabbit model. Both wild-type and mutated p17 induced anti-gp41 responses in rabbits; sera from these animals reacted with gp41 from different HIV clades. Moreover, introduction of the 2F5 sequence in p17 resulted in induction of antibodies with partially neutralizing activity. Based upon these data, we suggest that the natural cross-reactivity between HIV-1 p17 protein and 2F5 antibody can be exploited to induce antibodies with neutralizing activity in an animal model.

Generation of HIV-1 potent and broad neutralizing antibodies by immunization with postfusion HR1/HR2 complex.

BACKGROUND: The envelope glycoproteins are major targets for HIV vaccines. The N-terminal and the C-terminal regions of the gp41 interact to form six helix bundles that are responsible for the fusion between the viral and the target cell membranes. Monoclonal antibodies (Abs) able to disrupt the formation of this complex or to interfere with it could inhibit HIV fusion. Most of the well described gp41-specific broadly neutralizing Abs target conformational epitopes within the membrane proximal region of gp41 (MPER) and recognize linear peptides. METHOD AND RESULTS: In this study, a stable human transfected cell line, expressing a well folded heptad repeat regions 1 (HR1)/HR2 postfusion complex was developed. Transfected cells were highly immunogenic in mice and allowed the generation of 40 complex specific B-cell clones. Three of them were able to neutralize efficiently both HIV-1 laboratory adapted virus and primary isolates from different clades. Two neutralizing Abs (Nabs) FC-2 and FC-3 bound to a recombinant folded gp140 and blocked with a high potency HR1/HR2 fusion complex formation in vitro. The conformational epitopes of the three antibodies are different to 2F5, 4E10, D5 or NC-1 and mainly located in the MPER region. Abs were capable of inhibiting syncytium formation by blocking spatial interactions between HR1 and HR2 regions. CONCLUSION: These findings suggest that immunogenicity of gp41 is achievable and that the use of a fusion complex expressing human cell line is a highly potent immunogen to generate neutralizing antibodies against gp41 envelope glycoprotein.