Antibody Subclasses Research Articles

Exploring the depths of IgG4: insights into autoimmunity and novel treatments.

IgG4 subclass antibodies represent the rarest subclass of IgG antibodies, comprising only 3-5% of antibodies circulating in the bloodstream. These antibodies possess unique structural features, notably their ability to undergo a process known as fragment-antigen binding (Fab)-arm exchange, wherein they exchange half-molecules with other IgG4 antibodies. Functionally, IgG4 antibodies primarily block and exert immunomodulatory effects, particularly in the context of IgE isotype-mediated hypersensitivity reactions. In the context of disease, IgG4 antibodies are prominently observed in various autoimmune diseases combined under the term IgG4 autoimmune diseases (IgG4-AID). These diseases include myasthenia gravis (MG) with autoantibodies against muscle-specific tyrosine kinase (MuSK), nodo-paranodopathies with autoantibodies against paranodal and nodal proteins, pemphigus vulgaris and foliaceus with antibodies against desmoglein and encephalitis with antibodies against LGI1/CASPR2. Additionally, IgG4 antibodies are a prominent feature in the rare entity of IgG4 related disease (IgG4-RD). Intriguingly, both IgG4-AID and IgG4-RD demonstrate a remarkable responsiveness to anti-CD20-mediated B cell depletion therapy (BCDT), suggesting shared underlying immunopathologies. This review aims to provide a comprehensive exploration of B cells, antibody subclasses, and their general properties before examining the distinctive characteristics of IgG4 subclass antibodies in the context of health, IgG4-AID and IgG4-RD. Furthermore, we will examine potential therapeutic strategies for these conditions, with a special focus on leveraging insights gained from anti-CD20-mediated BCDT. Through this analysis, we aim to enhance our understanding of the pathogenesis of IgG4-mediated diseases and identify promising possibilities for targeted therapeutic intervention.

Prevalence and Clinical Significance of Immunoglobulin G1 and Immunoglobulin G3 Subclasses of Anti-D Antibody in Rh D Alloimmunized Antenatal Women

ABSTRACT Background and Objectives: Hemolytic disease of the fetus and newborn (HDFN) occurs as a result of maternal sensitization to paternal antigens. Studies have shown that the immunoglobulin G (IgG) subclass in maternal serum – particularly the presence of IgG1 and IgG3 either singly or in combination, has an impact on the presence and severity of HDFN. The column agglutination technology is used for the identification of IgG1 and IgG3 subclasses. Therefore, the prevalence of IgG1 and IgG3 subclasses in a cohort of alloimmunized Rh (D) antenatal women at our center was studied and was correlated with severity of HDFN. The aims of this study were as follows: (1) to find out the prevalence of IgG1 and IgG3 subclasses among Rh (D) alloimmunized women and (2) to assess the clinical outcome of newborns born to mothers with respect to IgG subclass. Methodology: This was a descriptive study done on 75 antenatal Rh (D) alloimmunized women who fulfilled the inclusion criteria. Blood samples from study participants were subjected to IgG subtyping using the “DAT IgG1/IgG3 ID card” (Bio-Rad, Switzerland), and the outcome of pregnancy was assessed by categorizing the newborns into mild, moderate, and severe HDFN. The data obtained were analyzed using SPSS version 16. Prevalence was calculated and severity of HDFN was correlated with subclass. Results: Among the study population, 23% of mothers had IgG1 alone. Majority (72%) had both IgG1 + IgG3 subclasses and a few (5%) had IgG2 + IgG4. When the mother had both IgG1 + IgG3 subclasses in their body most newborns had only mild HDFN. Few cases of moderate-to-severe cases of HDFN were seen. Most newborns had only mild HDFN even though there were few moderate-to-severe cases of HDFN. No significant association could be demonstrated between severity of HDFN and type of IgG subclasses. Conclusion: Majority of maternal IgG found in HDFN was a combination of IgG1+IgG3.Such cases exhibited stronger grades of reaction in Indirect Coombs Test. A significant association could not be demonstrated between severity of HDFN and type of IgG subclasses.

Understanding Fc function for rational vaccine design against pathogens.

Antibodies represent the primary correlate of immunity following most clinically approved vaccines. However, their mechanisms of action vary from pathogen to pathogen, ranging from neutralization, to opsonophagocytosis, to cytotoxicity. Antibody functions are regulated both by antigen specificity (Fab domain) and by the interaction of their Fc domain with distinct types of Fc receptors (FcRs) present in immune cells. Increasing evidence highlights the critical nature of Fc:FcR interactions in controlling pathogen spread and limiting the disease state. Moreover, variation in Fc-receptor engagement during the course of infection has been demonstrated across a range of pathogens, and this can be further influenced by prior exposure(s)/immunizations, age, pregnancy, and underlying health conditions. Fc:FcR functional variation occurs at the level of antibody isotype and subclass selection as well as post-translational modification of antibodies that shape Fc:FcR-interactions. These factors collectively support a model whereby the immune system actively harnesses and directs Fc:FcR interactions to fight disease. By defining the precise humoral mechanisms that control infections, as well as understanding how these functions can be actively tuned, it may be possible to open new paths for improving existing or novel vaccines.

Hepatitis C virus modified sE2F442NYT as an antigen in candidate vaccine facilitates human immune cell activation.

The rational selection of hepatitis C virus (HCV) vaccine antigen will aid in the prevention of future chronic liver disease burden and associated healthcare costs. We have previously shown that HCV E2 glycoprotein is not highly immunogenic, and the modification of E2 reduced CD81 binding and displayed altered cytokine and protective immune responses in vitro and in a surrogate mouse model. Here, we compared the influence of a parental and a modified sE2F442NYT glycoprotein region from HCV genotype 1a for the activation of peripheral blood mononuclear cell (PBMC)-derived dendritic cells (DCs), CD4+T cells, and B cells. Modified sE2F442NYT, when incubated with DCs, induced a higher number of CD86-positive cells. The sE2F442NYT or parental sE2 encapsulated as mRNA-lipid nanoparticle (sE2F442NYT mRNA-LNP) primed DCs co-cultured with autologous CD4+T cells did not induce CD25 or forkhead box P3 expression. PBMC-derived CD4+T cells treated with sE2F442NYT exhibited enhanced signal transducer and activator of transcription (Stat)1/Stat4 phosphorylation in response to anti-CD3/CD28 stimulation in comparison to parental sE2 treatment and facilitated isotype switching in B cells, leading to the generation of a broader subclass of antibodies. Cells treated with modified sE2F442NYT displayed an increase in activated Stat3 and extracellular signal-regulated kinase (ERK). Likewise, PBMC-derived naïve B cells upon in vitro stimulation with sE2F442NYT induced an increased proliferation, Stat3 and ERK activation, and protein kinase B (Akt) suppression. Thus, the modified sE2F442NYT antigen from HCV facilitates improved DC, CD4+T, and B cell activation compared to parental sE2 to better induce a robust protective immune response, supporting its selection as an HCV candidate vaccine antigen for preclinical and clinical HCV vaccine trials.IMPORTANCEThe nature of an enhanced immune response induced by sE2F442NYT will help in the selection of a broad cross-protective antigen from hepatitis C virus genotypes, and the inclusion of relatively conserved sE1 with sE2F442NYT may further strengthen the efficacy of the candidate vaccine in evaluating it for human use.

Elizabethkingia anophelis outer membrane vesicles as a novel vaccine candidate against infection: insights into immune response and potential for passive immunity.

Elizabethkingia anophelis, a Gram-negative pathogen, causes infections such as bacteraemia, pneumonia, and neonatal meningitis. The pathogen resists most antimicrobial classes, making novel approaches urgently needed. In natural settings, Gram-negative bacteria secrete outer membrane vesicles (OMVs) that carry important molecules in the bacterial life cycle. These OMVs are enriched with proteins involved in virulence, survival, and carbohydrate metabolism, making them a promising source for vaccine development against the pathogen. This study investigated the efficacy of imipenem-induced OMVs (iOMVs) as a vaccine candidate against E. anophelis infection in a mouse pneumonia model. Mice immunized with iOMVs were completely protected during lethal-dose challenges. Passive immunization with hyperimmune sera and splenocytes conferred protection against lethal pneumonia. Further investigation is needed to understand the mechanisms underlying the protective effects of iOMV-induced passive immunity, such as the action on specific antibody subclasses or T cell subsets.

Containing anti-PLA2R IgG antibody induces podocyte injury in idiopathic membranous nephropathy

Background Idiopathic membranous nephropathy is widely recognized as an autoimmune kidney disease that is accompanied by the discovery of several autoantibodies, and the antibody subclass in the circulation of patients with iMN is mainly IgG. However, the direct pathogenic effect of the containing anti-PLA2R IgG antibody on podocytes is not clear. Method A protein G affinity chromatography column was used to purify serum IgG antibodies. Containing anti-PLA2R IgG antibodies from iMN patients and IgG from healthy controls were also obtained. Based on the established in vitro podocyte culture system, purified IgG antibodies from the two groups were used to stimulate podocytes, and the expression of essential podocyte proteins (podocin), the levels of inflammatory cytokines in the cell supernatant, cytoskeletal disorders, and podocyte apoptosis were analyzed. Results Compared with that in the normal IgG group, the expression of podocin and podocin mRNA was reduced (p = 0.016 and p = 0.005, respectively), the fluorescence intensity of podocin on the surface of podocytes was reduced, the cytoskeleton of podocytes was disordered and reorganized, and the ratio of podocyte apoptosis was increased in the iMN group (p = 0.008). Conclusion The containing anti-PLA2R IgG antibody might have a direct damaging effect on podocytes in idiopathic membranous nephropathy.

Analyzing features of measles immune response in adult patients

Measles is a highly contagious, vaccine-preventable anthroponotic infection. Despite the policy of active measles vaccination for entire global population, measles has not completely disappeared, it wanes and waxes in outbreaks. It has been shown that not only the unvaccinated but also the vaccinated, which is quite understandable, subjects are involved in measles progression. The study was aimed at assessing features of the immune response in adult measles patients at rise in disease incidence upon its eradication. 1.158 blood sera from measles patients aged 18 to 70 living in Moscow and the Moscow region were studied by ELISA (Euroimmun, Germany). The vast majority of subjects were aged 1840 years comprising 72.36% cases. 69 sera collected at a later timepoint (1020 days from rash onset) were excluded and analyzed separately. According to anti-measles IgG and avidity level, the data were stratified as follows: group 1 with a primary type of immune response 582 sera (53.44%), not vaccinated in childhood; group 2 (secondary response) 446 sera (40.96%), vaccinated in childhood, but lost anti-measles antibodies; group 3 with intermediate level of parameters 61 sera. To clarify the type of immune response in group 3, an additional analysis of the spectrum of anti-measles antibody subclasses was carried out. It was shown that a mixed type of response was detected in this group: 31 subjects mainly had primary immune response, and 30 subjects secondary immune response. At the same time, 40 out of 61 subjects were vaccinated against measles 13 months prior to disease onset, i.e., it was overlapped on arising post-vaccination immunity. Thus, the previously identified trend towards the loss of post-vaccination immunity among older schoolchildren and young adults vaccinated in childhood who are involved in the epidemic process continues to worsen.

Evaluation of isotype-based serology for diagnosis of Schistosoma mansoni infection in individuals living in endemic areas with low parasite burden

Intestinal schistosomiasis is a chronic and debilitating disease that affects public health systems worldwide. Control interventions to reduce morbidity primarily involve the diagnosis and treatment of infected individuals. However, the recommended Kato-Katz (KK) parasitological method shows low sensitivity in individuals with low parasite loads and is not useful for monitoring elimination of parasite transmission at later stages. In the current study, we evaluated the accuracy of serum reactivity levels of different immunoglobulin isotypes in an enzyme-linked immunosorbent assay (ELISA), utilizing Schistosoma mansoni crude extracts, with the aim to improve the diagnosis of infected individuals with low parasite loads. The serum reactivity of IgM and IgG subclass antibodies (IgG1, IgG3, and IgG4) against soluble adult worm and egg antigen preparations was evaluated in residents from a schistosomiasis-endemic area in northern Minas Gerais, Brazil. The parasitological status of the study population was determined through fecal examination with multiple parasitological tests to create a consolidated reference standard (CRS) plus a fecal DNA detection test (q-PCR). Twelve months after praziquantel treatment, a second serum sample was obtained from the population for reexamination. A two-graph receiver operating characteristic curve (TG-ROC) analysis was performed using the serum reactivity of non-infected endemic controls and egg-positive individuals, and the cut-off value was established based on the intersection point of the sensibility and specificity curves in TG-ROC analyses. The diagnostic accuracy of each serological test was evaluated in relation to the parasitological CRS and to the combination of CRS plus qPCR results. The data revealed that serum reactivity of IgM and IgG3 against S. mansoni antigens did not allow identification of infected individuals from the endemic area. In contrast, serum IgG1 and IgG4-reactivity against schistosome antigens could distinguish between infected and non-infected individuals, with AUC values ranging between 0.728–0.925. The reactivity of IgG4 anti-soluble egg antigen - SEA (sensitivity 79 %, specificity 69 %, kappa = 0.49) had the best diagnostic accuracy, showing positive reactivity in more than 75 % of the infected individuals who eliminated less than 12 eggs per gram of feces. Moreover, serum IgG4 reactivity against SEA and against soluble worm antigen preparation (SWAP) was significantly reduced in the serum of infected individuals after 12 months of confirmed parasitological cure and in the absence of re-infection. These results reinforce that the described IgG4 anti-SEA ELISA assay is a sensitive alternative for the diagnosis of active intestinal schistosomiasis in individuals from endemic areas, including in those with a very low parasite load.

Diagnostic probability classification in suspected borreliosis by a novel Borrelia C6-peptide IgG1- subclass antibody test.

The tick-borne multisystemic infection caused by Borrelia burgdorferi sensu lato, Lyme borreliosis, or Lyme disease, occurring in temperate regions of the northern hemisphere, continues to spread geographically with the expanding tick population. Despite the rising perceived risk of infection in the population, the clinical diagnosis of Borrelia infection is not always obvious and the most important laboratory test, antibody detection, has limited accuracy in diagnosing active disease. According to international guidelines, the primary serology test, which has a high sensitivity-low specificity, should, be verified using a high specificity confirmation test to improve the specificity. However, this enhancement in specificity comes at the cost of lower sensitivity. This two-step procedure is often omitted in everyday clinical practice. An optimal primary test would be one where no secondary tests for confirmation would be necessary. In the present study, the performance of a novel assay for quantitating IgG1-subclass antibodies to Borrelia C6-peptide was compared to a commercial reference assay of total IgG and IgM antibodies to Borrelia C6-peptide in the setting of a high endemic area for borreliosis. A derivation study on a retrospective clinical material was performed to compare the performance parameters and assess the discriminatory properties of the assays, followed by a prospective validation study. The IgG1-antibody assay achieved comparable summary performance parameters to those of the reference assay. The sensitivity was almost 100% while the specificity was about 50%. In a high-endemic setting, characterized by high background seropositivity of about 50% and disease prevalence of approximately 10%, antibody tests are unable to rule-in active Borrelia infection. The rule-out assessment of the methods revealed that of 1000 patients, 7 - 54 with negative results based on the reference method could have an active Borrelia infection. Such uncertainty was not found for the index test and may help improve the risk classification of patients.

Prospective assessment of pre-existing and de novo anti-HLA IgE in kidney, liver, lung and heart transplantation.

Antibody mediated rejection (ABMR) is a major factor limiting outcome after organ transplantation. Anti-HLA donor-specific antibodies (DSA) of the IgG isotype are mainly responsible for ABMR. Recently DSA of the IgE isotype were demonstrated in murine models as well as in a small cohort of sensitized transplant recipients. In the present study, we aimed to determine the frequency of pre-existing and de novo anti-HLA IgE antibodies in a cohort of 105 solid organ transplant recipients. We prospectively measured anti-HLA IgE antibodies in a cohort of kidney (n=60), liver, heart and lung (n=15 each) transplant recipients before and within one-year after transplantation, employing a single-antigen bead assay for HLA class I and class II antigens. Functional activity of anti-HLA IgE antibodies was assessed by an in vitro mediator release assay. Antibodies of the IgG1-4 subclasses and Th1 and Th2 cytokines were measured in anti-HLA IgE positive patients. Pre-existing anti-HLA IgE antibodies were detected in 10% of renal recipients (including 3.3% IgE-DSA) and in 4.4% of non-renal solid organ transplant recipients (heart, liver and lung cohort). Anti-HLA IgE occurred only in patients that were positive for anti-HLA IgG, and most IgE positive patients had had a previous transplant. Only a small fraction of patients developed de novo anti-HLA IgE antibodies (1.7% of kidney recipients and 4.4% of non-renal recipients), whereas no de novo IgE-DSA was detected. IgG subclass antibodies showed a distinct pattern in patients who were positive for anti-HLA IgE. Moreover, patients with anti-HLA IgE showed elevated Th2 and also Th1 cytokine levels. Serum from IgE positive recipients led to degranulation of basophils in vitro, demonstrating functionality of anti-HLA IgE. These data demonstrate that anti-HLA IgE antibodies occur at low frequency in kidney, liver, heart and lung transplant recipients. Anti-HLA IgE development is associated with sensitization at the IgG level, in particular through previous transplants and distinct IgG subclasses. Taken together, HLA specific IgE sensitization is a new phenomenon in solid organ transplant recipients whose potential relevance for allograft injury requires further investigation.

Class switch towards spike protein-specific IgG4 antibodies after SARS-CoV-2 mRNA vaccination depends on prior infection history

Vaccinations against SARS-CoV-2 reduce the risk of developing serious COVID-19 disease. Monitoring spike-specific IgG subclass levels after vaccinations may provide additional information on SARS-CoV-2 specific humoral immune response. Here, we examined the presence and levels of spike-specific IgG antibody subclasses in health-care coworkers vaccinated with vector- (Sputnik, AstraZeneca) or mRNA-based (Pfizer-BioNTech, Moderna) vaccines against SARS-CoV-2 and in unvaccinated COVID-19 patients. We found that vector-based vaccines elicited lower total spike-specific IgG levels than mRNA vaccines. The pattern of spike-specific IgG subclasses in individuals infected before mRNA vaccinations resembled that of vector-vaccinated subjects or unvaccinated COVID-19 patients. However, the pattern of mRNA-vaccinated individuals without SARS-CoV-2 preinfection showed a markedly different pattern. In addition to IgG1 and IgG3 subclasses presented in all groups, a switch towards distal IgG subclasses (spike-specific IgG4 and IgG2) appeared almost exclusively in individuals who received only mRNA vaccines or were infected after mRNA vaccinations. In these subjects, the magnitude of the spike-specific IgG4 response was comparable to that of the spike-specific IgG1 response. These data suggest that the priming of the immune system either by natural SARS-CoV-2 infection or by vector- or mRNA-based vaccinations has an important impact on the characteristics of the developed specific humoral immunity.

Detection of a natural antibody targeting the shed ectodomain of BP180 in mice

BackgroundPemphigoid diseases are characterized by subepidermal blister formation accompanied by autoantibodies targeting skin component molecules, such as BP180. It is suggested that an epitope–phenotype correlation exists among autoantibodies recognizing BP180. However, it is unclear which regions of BP180 are likely targets for autoantibodies. ObjectiveTo elucidate the portions of BP180 where antibodies tend to react under the breakdown of immune tolerance. MethodsWe immunized mice with full-length mouse BP180 (mBP180) to produce anti-mBP180 antibodies. Using the immunized mice, hybridoma cells were established to produce anti-mBP180 antibodies. We analyzed the characteristics of the anti-mBP180 antibodies that were produced in terms of epitopes, immunoglobulin subclasses, and somatic hypermutations. ResultsHybridoma cells derived from immunized mice with full-length mBP180 produced antibodies targeting the intracellular domain (IC) and the shed ectodomain (EC) of mBP180. Using the domain-deleted mBP180 recombinant protein, we revealed that monoclonal anti-mBP180 EC antibodies react to neoepitopes on the 13th collagenous region of cleaved mBP180, which corresponds to the epitopes of linear IgA bullous dermatosis antibodies in human BP180. Furthermore, the subclasses of these antibodies could be distinguished by epitope: The subclass of the anti-mBP180 IC monoclonal antibodies was IgG, whereas that of the anti-mBP180 EC antibodies was IgM. Of note, a clone of these IgM mBP180 EC antibodies was a germline antibody without somatic hypermutation, which is also known as a natural antibody. ConclusionThese data suggest that mice potentially have natural antibodies targeting the neoepitopes of cleaved mBP180 EC.

Structural delineation and computational design of SARS-CoV-2-neutralizing antibodies against Omicron subvariants

SARS-CoV-2 Omicron subvariants have evolved to evade receptor-binding site (RBS) antibodies that exist in diverse individuals as public antibody clones. We rationally selected RBS antibodies resilient to mutations in emerging Omicron subvariants. Y489 was identified as a site of virus vulnerability and a common footprint of broadly neutralizing antibodies against the subvariants. Multiple Y489-binding antibodies were encoded by public clonotypes and additionally recognized F486, potentially accounting for the emergence of Omicron subvariants harboring the F486V mutation. However, a subclass of antibodies broadly neutralized BA.4/BA.5 variants via hydrophobic binding sites of rare clonotypes along with high mutation-resilience under escape mutation screening. A computationally designed antibody based on one of the Y489-binding antibodies, NIV-10/FD03, was able to bind XBB with any 486 mutation and neutralized XBB.1.5. The structural basis for the mutation-resilience of this Y489-binding antibody group may provide important insights into the design of therapeutics resistant to viral escape.

Effect of posttranslational modifications and subclass on IgG activity: from immunity to immunotherapy.

Humoral immune responses are characterized by complex mixtures of polyclonal antibody species varying in their isotype, target epitope specificity and affinity. Posttranslational modifications occurring during antibody production in both the antibody variable and constant domain create further complexity and can modulate antigen specificity and antibody Fc-dependent effector functions, respectively. Finally, modifications of the antibody backbone after secretion may further impact antibody activity. An in-depth understanding of how these posttranslational modifications impact antibody function, especially in the context of individual antibody isotypes and subclasses, is only starting to emerge. Indeed, only a minute proportion of this natural variability in the humoral immune response is currently reflected in therapeutic antibody preparations. In this Review, we summarize recent insights into how IgG subclass and posttranslational modifications impact IgG activity and discuss how these insights may be used to optimize therapeutic antibody development.

Autoimmune nodo-paranodopathies 10 years later: Clinical features, pathophysiology and treatment.

Autoimmune neuropathies are classified, on the basis of pathophysiology, as demyelinating or axonal. The term nodo-paranodopathy, introduced in 2013 to better categorize the neuropathies with antiganglioside antibodies and later expanded to include neuropathies with antibodies to nodal and paranodal axoglial complexes, characterizes disorders in which the nodal region is critical in the pathogenesis. These neuropathies, although presenting electrophysiologic demyelinating features do not show pathologic evidence of segmental demyelination, or, although being classified as axonal, can show reversible nerve conduction failure and rapid recovery contrary with the communal concept of an axonal neuropathy. In this personal view is reported, with a splitting approach, an update on autoimmune nodo-paranodopathies, classified according to the domains of peripheral nerve fiber, the target antigens and the antibody class and subclass involved. The clinical features, the electrophysiologic findings, the results of the immunopathological and ultrastructural studies, the pathophysiology and treatment are also described. The nodo-paranodopathy category integrates the clinical classification of autoimmune neuropathies and expands the traditional dichotomous demyelinating and axonal classification. It helps to a better systematization pointing to the domain and target antigens of the autoimmune process, it resolves conflicting pathologic and electrophysiologic findings, reconciles the contradiction that axonal neuropathies may be rapidly reversible, avoids taxonomical confusion and possible misdiagnoses. Finally this categorization, through the identification of the specific antibody and its prevalent class and subclass, clarifies the pathophysiological mechanisms and addresses to a more targeted therapeutic approach.

Viral vector delivered immunogen focuses HIV-1 antibody specificity and increases durability of the circulating antibody recall response.

The modestly efficacious HIV-1 vaccine regimen (RV144) conferred 31% vaccine efficacy at 3 years following the four-shot immunization series, coupled with rapid waning of putative immune correlates of decreased infection risk. New strategies to increase magnitude and durability of protective immunity are critically needed. The RV305 HIV-1 clinical trial evaluated the immunological impact of a follow-up boost of HIV-1-uninfected RV144 recipients after 6-8 years with RV144 immunogens (ALVAC-HIV alone, AIDSVAX B/E gp120 alone, or ALVAC-HIV + AIDSVAX B/E gp120). Previous reports demonstrated that this regimen elicited higher binding, antibody Fc function, and cellular responses than the primary RV144 regimen. However, the impact of the canarypox viral vector in driving antibody specificity, breadth, durability and function is unknown. We performed a follow-up analysis of humoral responses elicited in RV305 to determine the impact of the different booster immunogens on HIV-1 epitope specificity, antibody subclass, isotype, and Fc effector functions. Importantly, we observed that the ALVAC vaccine component directly contributed to improved breadth, function, and durability of vaccine-elicited antibody responses. Extended boosts in RV305 increased circulating antibody concentration and coverage of heterologous HIV-1 strains by V1V2-specific antibodies above estimated protective levels observed in RV144. Antibody Fc effector functions, specifically antibody-dependent cellular cytotoxicity and phagocytosis, were boosted to higher levels than was achieved in RV144. V1V2 Env IgG3, a correlate of lower HIV-1 risk, was not increased; plasma Env IgA (specifically IgA1), a correlate of increased HIV-1 risk, was elevated. The quality of the circulating polyclonal antibody response changed with each booster immunization. Remarkably, the ALVAC-HIV booster immunogen induced antibody responses post-second boost, indicating that the viral vector immunogen can be utilized to selectively enhance immune correlates of decreased HIV-1 risk. These results reveal a complex dynamic of HIV-1 immunity post-vaccination that may require careful balancing to achieve protective immunity in the vaccinated population. Trial registration: RV305 clinical trial (ClinicalTrials.gov number, NCT01435135). ClinicalTrials.gov Identifier: NCT00223080.

Effect of tocilizumab in subarachnoid hemorrhage-induced cerebral vasospasm of experimental rats

Aim: This study aimed to evaluate the effects of tocilizumab (TCZ), a recombinant humanized, anti-human monoclonal antibody of the immunoglobulin G1k subclass, on vascular morphological changes, endothelial apoptosis, and the levels of pro-inflammatory and apoptotic cytokines, such as IL-6, tumor necrosis factor-alpha (TNF-α), caspase-3, Bcl-2 associated X-protein (BAX), and vascular endothelial growth factor (VEGF) in a rat SAH model.
 Material and Method: The rats were randomly assigned (animal study) to 4 groups KONÜDAM Experimental Animal Research Center, Necmettin Erbakan University, Meram Faculty of Medicine, Konya, Turkey; 15/03/2019): (1) normal control (without SAH); (2) SAH (without treatment); (3) SAH treated with saline (SAH + Sal.); and (4) SAH treated with TCZ (SAH + Toc.). The tissues were measured using enzyme-linked immunosorbent assay (ELISA) kits. A series of brain and basilar artery sections were categorized into several subgroups for hematoxylin and eosin (H&E) staining, immunohistochemistry, and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.
 Results: The levels of caspase, BAX, and IL-6 in the SAH + TOC group were significantly lower than in other groups. TCZ treatment significantly increased the lumen of the basilar artery compared with that in the SAH and SAH + SAL groups without treatment (p=0.002 and p=0.004 respectively). SAH increased the apoptotic index in the endothelium compared with TCZ treatment (p=0.027) groups.
 Conclusion: It can be concluded that TCZ is safe and effective for treating experimental SAH. The results reveal clearly experimental evidence for the potential clinical application of TCZ in SAH patients.

Subclass Effects on Self-Association and Viscosity of Monoclonal Antibodies at High Concentrations.

The effects of a subclass of monoclonal antibodies (mAbs) on protein-protein interactions, formation of reversible oligomers (clusters), and viscosity (η) are not well understood at high concentrations. Herein, we quantify a short-range anisotropic attraction between the complementarity-determining region (CDR) and CH3 domains (KCDR-CH3) for vedolizumab IgG1, IgG2, or IgG4 subclasses by fitting small-angle X-ray scattering (SAXS) structure factor Seff(q) data with an extensive library of 12-bead coarse-grained (CG) molecular dynamics simulations. The KCDR-CH3 bead attraction strength was isolated from the strength of long-range electrostatic repulsion for the full mAb, which was determined from the theoretical net charge and a scaling parameter ψ to account for solvent accessibility and ion pairing. At low ionic strength (IS), the strongest short-range attraction (KCDR-CH3) and consequently the largest clusters and highest η were observed with IgG1, the subclass with the most positively charged CH3 domain. Furthermore, the trend in KCDR-CH3 with the subclass followed the electrostatic interaction energy between the CDR and CH3 regions calculated with the BioLuminate software using the 3D mAb structure and molecular interaction potentials. Whereas the equilibrium cluster size distributions and fractal dimensions were determined from fits of SAXS with the MD simulations, the degree of cluster rigidity under flow was estimated from the experimental η with a phenomenological model. For the systems with the largest clusters, especially IgG1, the inefficient packing of mAbs in the clusters played the largest role in increasing η, whereas for other systems, the relative contribution from stress produced by the clusters was more significant. The ability to relate η to short-range attraction from SAXS measurements at high concentrations and to theoretical characterization of electrostatic patches on the 3D surface is not only of fundamental interest but also of practical value for mAb discovery, processing, formulation, and subcutaneous delivery.

Neurologic sequelae of COVID-19 are determined by immunologic imprinting from previous coronaviruses.

Coronavirus disease 2019 (COVID-19), which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health emergency. Although SARS-CoV-2 is primarily a respiratory pathogen, extra-respiratory organs, including the CNS, can also be affected. Neurologic symptoms have been observed not only during acute SARS-CoV-2 infection, but also at distance from respiratory disease, also known as long-COVID or neurological post-acute sequelae of COVID-19 (neuroPASC). The pathogenesis of neuroPASC is not well understood, but hypotheses include SARS-CoV-2-induced immune dysfunctions, hormonal dysregulations and persistence of SARS-CoV-2 reservoirs. In this prospective cohort study, we used a high throughput systems serology approach to dissect the humoral response to SARS-CoV-2 (and other common coronaviruses: 229E, HKU1, NL63 and OC43) in the serum and CSF from 112 infected individuals who developed (n = 18) or did not develop (n = 94) neuroPASC. Unique SARS-CoV-2 humoral profiles were observed in the CSF of neuroPASC compared with serum responses. All antibody isotypes (IgG, IgM, IgA) and subclasses (IgA1-2, IgG1-4) were detected in serum, whereas CSF was characterized by focused IgG1 (and absence of IgM). These data argue in favour of compartmentalized brain-specific responses against SARS-CoV-2 through selective transfer of antibodies from the serum to the CSF across the blood-brain barrier, rather than intrathecal synthesis, where more diversity in antibody classes/subclasses would be expected. Compared to individuals who did not develop post-acute complications following infection, individuals with neuroPASC had similar demographic features (median age 65 versus 66.5 years, respectively, P = 0.55; females 33% versus 44%, P = 0.52) but exhibited attenuated systemic antibody responses against SARS-CoV-2, characterized by decreased capacity to activate antibody-dependent complement deposition (ADCD), NK cell activation (ADNKA) and to bind Fcγ receptors. However, surprisingly, neuroPASC individuals showed significantly expanded antibody responses to other common coronaviruses, including 229E, HKU1, NL63 and OC43. This biased humoral activation across coronaviruses was particularly enriched in neuroPASC individuals with poor outcome, suggesting an 'original antigenic sin' (or immunologic imprinting), where pre-existing immune responses against related viruses shape the response to the current infection, as a key prognostic marker of neuroPASC disease. Overall, these findings point to a pathogenic role for compromised anti-SARS-CoV-2 responses in the CSF, likely resulting in incomplete virus clearance from the brain and persistent neuroinflammation, in the development of post-acute neurologic complications of SARS-CoV-2 infection.

IgG1-Dominant Antibody Response Induced by Recombinant Trimeric SARS-CoV-2 Spike Protein with PIKA Adjuvant.

Recombinant trimeric SARS-CoV-2 Spike protein with PIKA (polyI:C) adjuvant induces potent and durable neutralizing antibodies that protect against multiple SARS-CoV-2 variants. The immunoglobulin subclasses of viral-specific antibodies remain unknown, as do their glycosylation status on Fc regions. In this study, we analyzed immunoglobulins adsorbed by plate-bound recombinant trimeric SARS-CoV-2 Spike protein from serum of Cynomolgus monkey immunized by recombinant trimeric SARS-CoV-2 Spike protein with PIKA (polyI:C) adjuvant. The results showed that IgG1 was the dominant IgG subclass as revealed by ion mobility mass spectrometry. The average percentage of Spike protein-specific IgG1 increased to 88.3% as compared to pre-immunization. Core fucosylation for Fc glycopeptide of Spike protein-specific IgG1 was found to be higher than 98%. These results indicate that a unique Th1-biased, IgG1-dominant antibody response was responsible for the effectiveness of PIKA (polyI:C) adjuvant. Vaccine-induced core-fucosylation of IgG1 Fc region may reduce incidence of severe COVID-19 disease associated with overstimulation of FCGR3A by afucosylated IgG1.