Antibody glycosylation correlates with disease progression in SIV-Mycobacterium tuberculosis coinfected cynomolgus macaques.

Objectives: Tuberculosis (TB) remains a substantial cause of morbidity and mortality among people living with human immunodeficiency virus (HIV) worldwide. However, the immunological mechanisms associated with the enhanced susceptibility among HIV-positive individuals remain largely unknown. Methods: Here, we used a simian immunodeficiency virus (SIV)/TB-coinfection Mauritian cynomolgus macaque (MCM) model to examine humoral responses from the plasma of SIV-negative (n = 8) and SIV-positive (n = 7) MCM 8-week postinfection with Mycobacterium tuberculosis (Mtb). Results: Antibody responses to Mtb were impaired during SIV coinfection. Elevated inflammatory bulk IgG antibody glycosylation patterns were observed in coinfected macaques early at 8-week post-Mtb infection, including increased agalactosylation (G0) and reduced di-galactosylation (G2), which correlated with endpoint Mtb bacterial burden and gross pathology scores, as well as the time-to-necropsy. Conclusion: These studies suggest that humoral immunity may contribute to control of TB disease and support growing literature that highlights antibody Fc glycosylation as a biomarker of TB disease progression.

The Influence of Human IgG Subclass and Allotype on Complement Activation.

Complement activation via the classical pathway is initiated when oligomeric Igs on target surfaces are recognized by C1 of the complement cascade. The strength of this interaction and activation of the complement system are influenced by structural variation of the Ab, including Ab isotype, subclass, and glycosylation profile. Polymorphic variants of IgG have also been described to influence Fc-dependent effector functions. Therefore, we assessed complement binding, deposition, and complement-dependent cytotoxicity (CDC) of 27 known IgG allotypes with anti-trinitrophenyl specificity. Differences between allotypes within subclasses were minor for IgG1, IgG3, and IgG4 allotypes, and more substantial for IgG2. Allelic variant IGHG2*06, containing a unique serine at position 378 in the CH3 domain, showed less efficient complement activation and CDC compared with other IgG2 polymorphisms. We also observed variable cell lysis between IgG1 and IgG3, with IgG3 being superior in lysis of human RBCs and Ramos cells, and IgG1 being superior in lysis of Raji and Wien133 cells, demonstrating that a long-standing conundrum in the literature depends on cellular context. Furthermore, we compared IgG1 and IgG3 under different circumstances, showing that Ag density and Ab hinge length, but not complement regulators, define the context dependency of Ab-mediated CDC activity. Our results point toward a variation in the capacity of IgG subclasses to activate complement due to single amino acid changes and hinge length differences of allotypes to activate complement, which might give new insights on susceptibility to infectious, alloimmune, or autoimmune diseases and aid the design of Ab-based therapeutics.

Robust immunity to influenza vaccination in haematopoietic stem cell transplant recipients following reconstitution of humoral and adaptive immunity.

Objectives: Influenza causes significant morbidity and mortality, especially in high-risk populations. Although current vaccination regimens are the best method to combat annual influenza disease, vaccine efficacy can be low in high-risk groups, such as haematopoietic stem cell transplant (HSCT) recipients. Methods: We comprehensively assessed humoral immunity, antibody landscapes, systems serology and influenza-specific B-cell responses, together with their phenotypes and isotypes, to the inactivated influenza vaccine (IIV) in HSCT recipients in comparison to healthy controls. Results: Inactivated influenza vaccine significantly increased haemagglutination inhibition (HAI) titres in HSCT recipients, similar to healthy controls. Systems serology revealed increased IgG1 and IgG3 antibody levels towards the haemagglutinin (HA) head, but not to neuraminidase, nucleoprotein or HA stem. IIV also increased frequencies of total, IgG class-switched and CD21loCD27+ influenza-specific B cells, determined by HA probes and flow cytometry. Strikingly, 40% of HSCT recipients had markedly higher antibody responses towards A/H3N2 vaccine strain than healthy controls and showed cross-reactivity to antigenically drifted A/H3N2 strains by antibody landscape analysis. These superior humoral responses were associated with a greater time interval after HSCT, while multivariant analyses revealed the importance of pre-existing immune memory. Conversely, in HSCT recipients who did not respond to the first dose, the second IIV dose did not greatly improve their humoral response, although 50% of second-dose patients reached a seroprotective HAI titre for at least one of vaccine strains. Conclusions: Our study demonstrates efficient, although time-dependent, immune responses to IIV in HSCT recipients, and provides insights into influenza vaccination strategies targeted to immunocompromised high-risk groups.

Immune profiling of SARS-CoV-2 infection during pregnancy reveals NK cell and γδ T cell perturbations.

Pregnancy poses a greater risk for severe COVID-19; however, underlying immunological changes associated with SARS-CoV-2 during pregnancy are poorly understood. We defined immune responses to SARS-CoV-2 in unvaccinated pregnant and nonpregnant women with acute and convalescent COVID-19, quantifying 217 immunological parameters. Humoral responses to SARS-CoV-2 were similar in pregnant and nonpregnant women, although our systems serology approach revealed distinct antibody and FcγR profiles between pregnant and nonpregnant women. Cellular analyses demonstrated marked differences in NK cell and unconventional T cell activation dynamics in pregnant women. Healthy pregnant women displayed preactivated NK cells and γδ T cells when compared with healthy nonpregnant women, which remained unchanged during acute and convalescent COVID-19. Conversely, nonpregnant women had prototypical activation of NK and γδ T cells. Activation of CD4+ and CD8+ T cells and T follicular helper cells was similar in SARS-CoV-2-infected pregnant and nonpregnant women, while antibody-secreting B cells were increased in pregnant women during acute COVID-19. Elevated levels of IL-8, IL-10, and IL-18 were found in pregnant women in their healthy state, and these cytokine levels remained elevated during acute and convalescent COVID-19. Collectively, we demonstrate perturbations in NK cell and γδ T cell activation in unvaccinated pregnant women with COVID-19, which may impact disease progression and severity during pregnancy.

Factors affecting IgG4-mediated complement activation.

Of the four human immunoglobulin G (IgG) subclasses, IgG4 is considered the least inflammatory, in part because it poorly activates the complement system. Regardless, in IgG4 related disease (IgG4-RD) and in autoimmune disorders with high levels of IgG4 autoantibodies, the presence of these antibodies has been linked to consumption and deposition of complement components. This apparent paradox suggests that conditions may exist, potentially reminiscent of in vivo deposits, that allow for complement activation by IgG4. Furthermore, it is currently unclear how variable glycosylation and Fab arm exchange may influence the ability of IgG4 to activate complement. Here, we used well-defined, glyco-engineered monoclonal preparations of IgG4 and determined their ability to activate complement in a controlled system. We show that IgG4 can activate complement only at high antigen and antibody concentrations, via the classical pathway. Moreover, elevated or reduced Fc galactosylation enhanced or diminished complement activation, respectively, with no apparent contribution from the lectin pathway. Fab glycans slightly reduced complement activation. Lastly, we show that bispecific, monovalent IgG4 resulting from Fab arm exchange is a less potent activator of complement than monospecific IgG4. Taken together, these results imply that involvement of IgG4-mediated complement activation in pathology is possible but unlikely.

Impact of structural modifications of IgG antibodies on effector functions.

Immunoglobulin G (IgG) antibodies are a critical component of the adaptive immune system, binding to and neutralizing pathogens and other foreign substances. Recent advances in molecular antibody biology and structural protein engineering enabled the modification of IgG antibodies to enhance their therapeutic potential. This review summarizes recent progress in both natural and engineered structural modifications of IgG antibodies, including allotypic variation, glycosylation, Fc engineering, and Fc gamma receptor binding optimization. We discuss the functional consequences of these modifications to highlight their potential for therapeutical applications.

Acquisition of antibodies to Plasmodium falciparum and Plasmodium vivax antigens in pregnant women living in a low malaria transmission area of Brazil.

BACKGROUND: Pregnant women have increased susceptibility to Plasmodium falciparum malaria and acquire protective antibodies over successive pregnancies. Most studies that investigated malaria antibody responses in pregnant women are from high transmission areas in sub-Saharan Africa, while reports from Latin America are scarce and inconsistent. The present study sought to explore the development of antibodies against P. falciparum and Plasmodium vivax antigens in pregnant women living in a low transmission area in the Brazilian Amazon. METHODS: In a prospective cohort study, plasma samples from 408 pregnant women (of whom 111 were infected with P. falciparum, 96 had infections with P. falciparum and P. vivax, and 201 had no Plasmodium infection) were used to measure antibody levels. Levels of IgG and opsonizing antibody to pregnancy-specific variant surface antigens (VSAs) on infected erythrocytes (IEs), 10 recombinant VAR2CSA Duffy binding like (DBL domains), 10 non-pregnancy-specific P. falciparum merozoite antigens, and 10 P. vivax antigens were measured by flow cytometry, ELISA, and multiplex assays. Antibody levels and seropositivity among the groups were compared. RESULTS: Antibodies to VSAs on P. falciparum IEs were generally low but were higher in currently infected women and women with multiple P. falciparum episodes over pregnancy. Many women (21%-69%) had antibodies against each individual VAR2CSA DBL domain, and antibodies to DBLs correlated with each other (r ≥ 0.55, p < 0.0001), but not with antibody to VSA or history of infection. Infection with either malaria species was associated with higher seropositivity rate for antibodies against P. vivax proteins, adjusted odds ratios (95% CI) ranged from 5.6 (3.2, 9.7), p < 0.0001 for PVDBPII-Sal1 to 15.7 (8.3, 29.7), p < 0.0001 for PvTRAg_2. CONCLUSIONS: Pregnant Brazilian women had low levels of antibodies to pregnancy-specific VSAs that increased with exposure. They frequently recognized both VAR2CSA DBL domains and P. vivax antigens, but only the latter varied with infection. Apparent antibody prevalence is highly dependent on the assay platform used.

Analysis of Antibody Reactivity to Malaria Antigens by Microsphere-Based Multiplex Immunoassay.

Protein multiplex assays enable serological analysis of multiple target proteins simultaneously, using relatively small volumes of patient sample per assay. Here we present a detailed protocol to analyze antibody reactivity to malaria antigens by microsphere-based multiplex assay (xMAP technology). This method involves coupling of recombinant proteins to fluorescently labeled microspheres; simultaneous exposure of all microspheres to plasma or sera, and detection of antigen-specific antibodies with a fluorescent labeled anti-human Fc region antibody. In addition to total IgG, this assay can be adapted to measure multiple properties of the antibody Fc region, including subclass, isotype, and Fc receptor or complement C1q binding.

Immunoassay for quantification of antigen-specific IgG fucosylation.

BACKGROUND: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (FcγR). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell FcγRs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. METHODS: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives FcγRIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (FcγR-IgG ELISA). FINDINGS: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R2=0.93, p < 0.0001). The FEASI method was then used to quantify IgG levels and fucosylation in COVID-19 convalescent plasma which was independently validated by LC-MS. INTERPRETATION: FEASI can be reliably implemented to measure relative and absolute IgG Fc fucosylation and quantify binding of antigen-specific IgG to FcγR in a high-throughput manner accessible to all diagnostic and research laboratories. FUNDING: This work was funded by the Stichting Sanquin Bloedvoorziening (PPOC 19-08 and SQI00041) and ZonMW 10430 01 201 0021.

Identifying Targets of Protective Antibodies against Severe Malaria in Papua, Indonesia, Using Locally Expressed Domains of Plasmodium falciparum Erythrocyte Membrane Protein 1.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a diverse family of multidomain proteins expressed on the surface of malaria-infected erythrocytes, is an important target of protective immunity against malaria. Our group recently studied transcription of the var genes encoding PfEMP1 in individuals from Papua, Indonesia, with severe or uncomplicated malaria. We cloned and expressed domains from 32 PfEMP1s, including 22 that were upregulated in severe malaria and 10 that were upregulated in uncomplicated malaria, using a wheat germ cell-free expression system. We used Luminex technology to measure IgG antibodies to these 32 domains and control proteins in 63 individuals (11 children). At presentation to hospital, levels of antibodies to PfEMP1 domains were either higher in uncomplicated malaria or were not significantly different between groups. Using principal component analysis, antibodies to 3 of 32 domains were highly discriminatory between groups. These included two domains upregulated in severe malaria, a DBLß13 domain and a CIDRα1.6 domain (which has been previously implicated in severe malaria pathogenesis), and a DBLδ domain that was upregulated in uncomplicated malaria. Antibody to control non-PfEMP1 antigens did not differ with disease severity. Antibodies to PfEMP1 domains differ with malaria severity. Lack of antibodies to locally expressed PfEMP1 types, including both domains previously associated with severe malaria and newly identified targets, may in part explain malaria severity in Papuan adults.

Targeting BMI-1 in B cells restores effective humoral immune responses and controls chronic viral infection.

Ineffective antibody-mediated responses are a key characteristic of chronic viral infection. However, our understanding of the intrinsic mechanisms that drive this dysregulation are unclear. Here, we identify that targeting the epigenetic modifier BMI-1 in mice improves humoral responses to chronic lymphocytic choriomeningitis virus. BMI-1 was upregulated by germinal center B cells in chronic viral infection, correlating with changes to the accessible chromatin landscape, compared to acute infection. B cell-intrinsic deletion of Bmi1 accelerated viral clearance, reduced splenomegaly and restored splenic architecture. Deletion of Bmi1 restored c-Myc expression in B cells, concomitant with improved quality of antibody and coupled with reduced antibody-secreting cell numbers. Specifically, BMI-1-deficiency induced antibody with increased neutralizing capacity and enhanced antibody-dependent effector function. Using a small molecule inhibitor to murine BMI-1, we could deplete antibody-secreting cells and prohibit detrimental immune complex formation in vivo. This study defines BMI-1 as a crucial immune modifier that controls antibody-mediated responses in chronic infection.

Robust and prototypical immune responses toward influenza vaccines in the high-risk group of Indigenous Australians.

Morbidity and mortality rates from seasonal and pandemic influenza occur disproportionately in high-risk groups, including Indigenous people globally. Although vaccination against influenza is recommended for those most at risk, studies on immune responses elicited by seasonal vaccines in Indigenous populations are largely missing, with no data available for Indigenous Australians and only one report published on antibody responses in Indigenous Canadians. We recruited 78 Indigenous and 84 non-Indigenous Australians vaccinated with the quadrivalent influenza vaccine into the Looking into InFluenza T cell immunity - Vaccination cohort study and collected blood to define baseline, early (day 7), and memory (day 28) immune responses. We performed in-depth analyses of T and B cell activation, formation of memory B cells, and antibody profiles and investigated host factors that could contribute to vaccine responses. We found activation profiles of circulating T follicular helper type-1 cells at the early stage correlated strongly with the total change in antibody titers induced by vaccination. Formation of influenza-specific hemagglutinin-binding memory B cells was significantly higher in seroconverters compared with nonseroconverters. In-depth antibody characterization revealed a reduction in immunoglobulin G3 before and after vaccination in the Indigenous Australian population, potentially linked to the increased frequency of the G3m21* allotype. Overall, our data provide evidence that Indigenous populations elicit robust, broad, and prototypical immune responses following immunization with seasonal inactivated influenza vaccines. Our work strongly supports the recommendation of influenza vaccination to protect Indigenous populations from severe seasonal influenza virus infections and their subsequent complications.

Developing a multivariate prediction model of antibody features associated with protection of malaria-infected pregnant women from placental malaria.

Background: Plasmodium falciparum causes placental malaria, which results in adverse outcomes for mother and child. P. falciparum-infected erythrocytes that express the parasite protein VAR2CSA on their surface can bind to placental chondroitin sulfate A. It has been hypothesized that naturally acquired antibodies towards VAR2CSA protect against placental infection, but it has proven difficult to identify robust antibody correlates of protection from disease. The objective of this study was to develop a prediction model using antibody features that could identify women protected from placental malaria. Methods: We used a systems serology approach with elastic net-regularized logistic regression, partial least squares discriminant analysis, and a case-control study design to identify naturally acquired antibody features mid-pregnancy that were associated with protection from placental malaria at delivery in a cohort of 77 pregnant women from Madang, Papua New Guinea. Results: The machine learning techniques selected 6 out of 169 measured antibody features towards VAR2CSA that could predict (with 86% accuracy) whether a woman would subsequently have active placental malaria infection at delivery. Selected features included previously described associations with inhibition of placental binding and/or opsonic phagocytosis of infected erythrocytes, and network analysis indicated that there are not one but multiple pathways to protection from placental malaria. Conclusions: We have identified candidate antibody features that could accurately identify malaria-infected women as protected from placental infection. It is likely that there are multiple pathways to protection against placental malaria. Funding: This study was supported by the National Health and Medical Research Council (Nos. APP1143946, GNT1145303, APP1092789, APP1140509, and APP1104975).

Antibody mediated activation of natural killer cells in malaria exposed pregnant women.

Immune effector responses against Plasmodium falciparum include antibody-mediated activation of innate immune cells, which can induce Fc effector functions, including antibody-dependent cellular cytotoxicity, and the secretion of cytokines and chemokines. These effector functions are regulated by the composition of immunoglobulin G (IgG) Fc N-linked glycans. However, a role for antibody-mediated natural killer (NK) cells activation or Fc N-linked glycans in pregnant women with malaria has not yet been established. Herein, we studied the capacity of IgG antibodies from pregnant women, with placental malaria or non-placental malaria, to induce NK cell activation in response to placental malaria-associated antigens DBL2 and DBL3. Antibody-mediated NK cell activation was observed in pregnant women with malaria, but no differences were associated with susceptibility to placental malaria. Elevated anti-inflammatory glycosylation patterns of IgG antibodies were observed in pregnant women with or without malaria infection, which were not seen in healthy non-pregnant controls. This suggests that pregnancy-associated anti-inflammatory Fc N-linked glycans may dampen the antibody-mediated activation of NK cells in pregnant women with malaria infection. Overall, although anti-inflammatory glycans and antibody-dependent NK cell activation were detected in pregnant women with malaria, a definitive role for these antibody features in protecting against placental malaria remains to be proven.

Hypoxia-induced synthesis of respiratory nitrate reductase 2 of Streptomyces coelicolor A3(2) depends on the histidine kinase OsdK in mycelium but not in spores.

The three nitrate reductases (Nar) of the saprophytic aerobic actinobacterium Streptomyces coelicolor A3(2) contribute to survival when oxygen becomes limiting. In the current study, we focused on synthesis of the Nar2 enzyme, which is the main Nar enzyme present and active in exponentially growing mycelium. Synthesis of Nar2 can, however, also be induced in spores after extended periods of anoxic incubation. The osdRK genes (oxygen stress and development) were recently identified to encode a two-component system important for expression of the nar2 operon in mycelium. OsdK is a predicted histidine kinase and we show here that an osdK mutant completely lacks Nar2 enzyme activity in mycelium. Recovery of Nar2 enzyme activity was achieved by re-introduction of the osdRK genes into the mutant on an integrative plasmid. In anoxically incubated spores, however, the osdK mutant retained the ability to synthesize NarG2, the catalytic subunit of Nar2. We could also demonstrate that synthesis of NarG2 in spores occurred only under hypoxic conditions; anoxia, as well as O2 concentrations significantly higher than 1 % in the gas-phase, failed to result in induction of NarG2 synthesis. Together, these findings indicate that, although Nar2 synthesis in both mycelium and spores is induced by oxygen limitation, different mechanisms control these processes and only Nar2 synthesis in mycelium is under the control of the OsdKR two-component system.

Role of IgG3 in Infectious Diseases.

IgG3 comprises only a minor fraction of IgG and has remained relatively understudied until recent years. Key physiochemical characteristics of IgG3 include an elongated hinge region, greater molecular flexibility, extensive polymorphisms, and additional glycosylation sites not present on other IgG subclasses. These characteristics make IgG3 a uniquely potent immunoglobulin, with the potential for triggering effector functions including complement activation, antibody (Ab)-mediated phagocytosis, or Ab-mediated cellular cytotoxicity (ADCC). Recent studies underscore the importance of IgG3 effector functions against a range of pathogens and have provided approaches to overcome IgG3-associated limitations, such as allotype-dependent short Ab half-life, and excessive proinflammatory activation. Understanding the molecular and functional properties of IgG3 may facilitate the development of improved Ab-based immunotherapies and vaccines against infectious diseases.

A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis.

BACKGROUND: In the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed. METHODS: We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen. RESULTS: Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3-0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57BL/6 mice. BCG vaccination in our hands led to a reduction in bacterial burden after challenge with Mycobacterium tuberculosis of approx. 0.7 log10 CFU in lung and approx. 1 log10 CFU in spleen. This effect was also seen when using Mycobacterium smegmatis as the target of growth inhibition. An increase in mycobacterial numbers was found when splenocytes from interferon gamma-deficient mice were used, compared to wild type controls, indicating that immune mechanisms may also be investigated using this assay. CONCLUSIONS: We believe that the ex vivo mycobacterial growth inhibition assay could be a useful tool to help assess vaccine efficacy in future, alongside other established methods. It could also be a valuable tool for determination of underlying immune mechanisms.